Plant Cells I. A Water Balloon in a Box This is a good model for a plant cell.

Thus, there are two major components to a plant cell: A. The Box This part of the cell is analogous to the cell wall. Like a cardboard box, the cell wall is relatively rigid, it is non-living and highly structured. Its more obvious functions are to support and protect the cell. It is produced by the protoplast (see below). If you stack up a bunch of boxes you generally cant make a very large tower before it comes crashing down. Since a plant is essentially constructed of numerous small boxes, why dont they fall apart, too? The answer is glue! Plants glue their cells together with pectic polysaccharides. These carbohydrates, which make up the outermost layer of the plant cell wall (called the middle lamella), bind adjacent cells together. Cooks use pectins extracted from the middle lamella to solidify jams and jellies. Pores or air spaces (called intercellular spaces) exist between adjacent cells because of the difficulty of packing of cells with rigid walls. Try and squeeze a bunch of irregularly boxes into a room without leaving any space between them! The intercellular spaces are important for gas exchange and water transport, some movements (i.e., sensitive plants - water moves into/out of theses spaces; nyctinastic movements - sleep movements) and freezing protection (i.e., water moves out of cells into the spaces to minimize cellular damage on freezing. Trivia note: prized ginseng roots have a translucent appearance - apparently obtained by freezing). Putting a cell in a box presents one major problem - how do cells talk to one another since they are now effectively isolated in their own small compartment? Plants solved this problem by putting windows in the box! Or in other words, there are lots of specialized pores through the wall called plasmodesmata that provide a cytoplasmic connection ("cytoplasmic bridges") between adjacent plant cells. Thus, the cytoplasm of a plant is essentially contiguous throughout the entire plant. Imagine hopping on board a tiny submarine like in the films Fantastic Voyage or Innerspace. You can essentially travel from cell-to-cell throughout the plant without ever leaving the cytoplasm nor crossing any cell membranes. Cool! The plasmodesmata are 40-50 nm in diameter. The maximum sized object that can pass through as a molecular mass of 700-1000 daltons, which is equivalent to a molecule 1.5 - 2.0 nm. The plasmodesmata have a unique structure. The plasma membranes from adjacent cells are continuous through the pore. In addition, the ER (see below) is also continuous between adjacent cells. The narrow tubule of ER is called the desmotubule and in the center there may be dark staining rod (called then "central rod"). The cytoplasmic channel between desmotubule and membrane is called the "cytoplasmic sleeve." Protein fibers radiate from the desmotubule to the membrane like spokes on a bicycle. These fibers may act like a sieve or screen. Plasmodesmata are aggregated in pits and there are 1-15 per mm2. They can make up as much as 1% of the cell surface. Plasmodesmata are formed during cell division (see cell notes) or secondarily after division. They may or may not be branched, too. B. The water balloon - this is analogous to the protoplast. The protoplast is everything inside the cell wall. It is the "living" part of the cell and includes:

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cytosol - matrix in which the other cell components are embedded; nucleus - the cell brain; vacuole - membrane bound storage pool; assorted organelles; and

ergastic (non-living) substances which includes crystals ("far out, man"), tannins, and starch grains. II. Separating the balloon from the box.

Just as you can remove a balloon from a box, so too can a plant physiologist liberate the protoplast from the cell wall. This is accomplished by slicing up a leaf (or other tissue) and then floating it in a solution containing wall-digesting enzymes like pectinase, cellulase, and hemicellulase suspended in an inert osmotic agent (0.7 M mannitol or sorbitol) in a buffered solution. The enzymes degrade the components of the cell wall releasing the protoplasts. This will be the focus of at least one or our lab sessions. III. Organelles in Both Plant and Animal Cells First, we will study the components of the protoplast, but will especially concentrate on those structures unique to plants. In the next lecture well tackle the cell wall. A. Plasma or Cell membrane Cell boundary; selectively permeable; bilayer of phospholipids with inserted protein. Phospholipids are unique molecules - they are amphipathic, meaning that they have both hydrophilic and hydrophobic regions. They have a glycerol backbone; one of the hydroxyls is bonded to a phosphate and another charged group, the other two hydroxyls are esterified to fatty acids. The fatty acids range in length from C14 - C24. One fatty acid is usually unsaturated and the other is saturated. The unsaturated fatty acid is kinked which helps to keep plant cell membranes fluid at cool temperatures. As a result plant phospholipids usually have a higher degree of unsaturation than animals. Hydrophobic interactions between the tail regions of the phospholipids hold the membrane together. Some proteins are found: (1) just on the outside or inside surfaces of the membrane (peripheral proteins - non-covalent interactions and anchored proteins - covalently bound to lipids, etc); or (2) embedded in the membrane (integral protein), many of which span the membrane (transmembrane proteins). Hydrophilic regions of the integral proteins are oriented to the outside of the membrane whereas hydrophobic regions are embedded within the phospholipid bilayer. Lipid soluble materials can readily pass through but charged or ionized substances (hydrophilic) pass through very slowly, if at all. The function of the membrane is to: (1) regulate traffic; (2) separate the internal from external environment; (3) serve as a platform on which some reactions can occur; (4) participate in some reactions (i.e., the membrane components are important intermediates or enzymes); and (5) provide some structural integrity for the cell. B. Nucleus The cell "brain". Surrounded by a double membrane (two phospholipid bilayers) - the nuclear membrane. Has pores. The structure of the pores is complex comprised of a more than 100 proteins. The pore opening is surrounding by a series of proteins and these are attached to a series radial spokes. Nucleoplasm - matrix within nucleus. DNA, which is found in the nucleus, may be condensed into chromosomes or not (chromatin). There may be one or more nucleolus (site of ribosome production). The nucleus is 5-20 mm in diameter. There is a layer of intermediate filaments (see below) just inside the nuclear envelope; called the nuclear lamina. C. Cytoplasm/cytosol The cytosol is the gel-like matrix within the cell in which the other structures are embedded. The cytoplasm refers to the cell materials inside the membrane. D. Mitochondria These organelles, like the nucleus and plastids, are double-membrane bound. They vary in shape from tubular (like sausages) to spherical. They reproduce by fission, have their own ribosomes and DNA (a circular loop like prokaryotic cells). The inner membrane has a larger surface area so it must be folded into finger-like projections (called cristae) to fit inside the outer membrane. Mitochondria are found in all eukaryotic cells. They are the sites of cellular respiration - process by which energy is

released from fuels such as sugar. The mitochondria are the power plant of the cell. They are small (15 mm) and generally numerous (500-2000 per cell). A popular misconception is that "plants have chloroplasts, animals have mitochondria." Plant cells, at least green plant cells (i.e., leaf cells), have both. Root cells only have mitochondria. Mitochondrial DNA which comprises about 200 kbases, codes for some of the genes required for cellular respiration including the 70S ribosomes and components of the electron transport system. The inner membrane differs from the plasma membrane in that it has a higher protein content (70%) and unique phospholipids (i.e., cardiolipin). E. Ribosome Sites of protein synthesis (translation). Two subunits; one large and the other small. Made in the nucleus from rRNA and protein. Ribosomes are tiny (0.25 mm) and numerous (5 - 50 X 1010 per cell). Since ribosomes are not surrounded by a membrane, they are not considered to be "true" organelles. Some ribosomes are 'free' (produce proteins that remain in the cell) while others are attached to the ER (produce proteins for export). To export a protein, the mRNA and subunits of the ribosome bind together. A signal recognition particle (SRP) binds to specific amino acids in the newly forming protein. The SRP, which is bound to the protein/mRNA/ribosome, then binds to a receptor in the ER membrane. As the protein is made it is released into the lumen of the ER and the SRP sequence of the protein is snipped off. F. Endoplasmic reticulum A series of membranous tubes and sacs (cisternae) that run throughout the cell. Rough ER has ribosomes associated with it and is laminar while smooth ER lacks ribosomes and is tubular. Totally man. The ER has several functions including: (1) synthesis of lipids and membranes (smooth ER); (2) serving as a site for the synthesis of proteins by the ribosomes (rough ER); (3) transport (a type of cell 'highway' system); and (4) support. G. Peroxisomes Membrane sac containing enzymes for metabolizing waste products from photosynthesis, fats and amino acids. Hydrogen peroxide is a product of metabolism in peroxisomes. Catalase, which breaks down the peroxide is also present and serves as a marker enzyme for these organelles. H. Glyoxisomes Membrane sac containing enzymes for fat metabolism. Especially common in seeds. Also contain catalase. I. Golgi apparatus Pancake- or pita bread-like stack of membranes. Particularly important in cells that produce materials for export (secretion). They have a polarity (cis - imports vesicles from ER; trans - exports vesicles). The Golgi is the site of processing and packaging cellular components. Vesicles containing proteins, lipids and other materials, fuse with the Golgi (cis side), release contents, which then get processed, sorted, packaged and re-released from the other side (trans face). The Golgi also is active in synthesizing many cell components, especially carbohydrates and is involved in tagging proteins with carbohydrates and other side chains for sorting them to their final destination. There are two models for the movement of materials thru the Golgi: (1) Vesicle Migration Model - in this case a vesicle fuses with the cis side, then ultimately a new vesicle pinch off this stack and fuses with teh next one, and so on, until the vesicle reaches the trans side; and (2) Escalator Model - a vesicle fuses with the cis side and never leaves this stack. Rather, the stack on the trans side releases vesicles and then disintegrates while a new stack forms on the cis side. The original vesicle is now in the "second" stack, and so on until it reaches the trans side. Vesicles are tagged with various proteins to direct them to the appropriate locations. J. Microtubules Hollow tubes made of a mix of alpha and beta tubulin, which are globular proteins. There are essentially 13 columns of proteins. The tubes are about 25 mm in diameter. Microtubules are involved

in the cell cytoskeleton (for support), cell movements (cilia, flagella) and cell division (spindle). Assembly of microtubules is prevented by colchicine, an inhibitor derived from Crocus bulbs. Low calcium concentration favors the formation of microtubules K. Microfilaments Protein strands. Solid. Made from G-actin. Involved with the cell cytoskeleton. Main function is support. They are about 7 nm in diameter. L. Intermediate filaments These are similar to microfilaments. They are also made of protein in the keratin family; about 10 nm diameter. M. Cilia/flagella For cellular movements. Cilia = many, short; flagella = few, long. Have a 9+2 arrangement of microtubules. Prongs on the tubules are ATPases (dynein) to hydrolyze ATP to provide energy for movement. These are not particularly common in plants. N. The Cytomembrane system The membranous organelles (ER, vesicles, golgi, cell membrane) comprise a group of organelles that cooperate and function together. For example, imagine the synthesis of cellulose in the cell wall of a plant. Cellulose synthesis requires the enzyme cellulose synthase. Ribosomes (rough ER) makes enzyme passes through RER to smooth ER packaged into a vesicle pinches off to golgi (cis face) processed repackaged into vesicle pinches off (trans face) cell membrane fuses releases contents cellulose synthase makes cellulose. O. Others

Microbodies - a general term for any single membrane bound organelle typically derived from the ER that contain catalase and/or hydrogen peroxide producing enzymes. This includes the peroxisomes and glyoxisomes; Microsomes - a "biochemical" term for the fraction that is obtained from high speed centrifugation of cell homogenates. It includes membrane fragments and ribosomes. Oleosome (spherosomes) - these are lipid bodies. The coolest thing about them is that they are encased by one-half of a cell membrane; in other words, just a single phospholipid layer.

IV. Organelles Unique to Plants - Plastids Plastids are double membrane-bound organelles in plants. They contain their own DNA (in nucleoid region) and ribosomes. They are semi-autonomous and reproduce by fission similar to the division process in prokaryotes. If plastids only arise from other plastids and cant be built "from scratch", then where do they come from? The egg. Plastids are inherited cytoplasmically, primarily through the female - however, there are examples of paternal inheritance of plastids. The plastid DNA carries several genes including the large subunit of rubisco and those for resistance to some herbicides. The chemistry of the membranes differs from the plasma membrane - plastid membranes are comprised of glycosylglycerides rather than phospholipids (the phosphate in the polar head group in glycosylglycerides is replaced with galactose or a related sugar). There are several types of plastids including:

1 2

Proplastids - small, precursors to the other plastid types, found in young cells, actively growing tissues; Chloroplasts - sites of photosynthesis (energy capture). They contain photosynthetic pigments including chlorophyll, carotenes and xanthophylls. The chloroplast is packed with membranes,

3 4 5 6

called thylakoids. The thylakoids may be stacked into pancake- like piles called grana (granum, singular). The "liquidy" material in the chloroplast is the stroma. A chloroplast is from 5-20 m in diameter and there are usually 50-200 per cell. The chloroplast genome has about 145 Kbase pairs, it is smaller than that of the mitochondria (200 kbases). About 1/3 of the total cell DNA is extranuclear (in the chloroplasts and mitochondria); Chromoplasts - non-photosynthetic, colored plastids; give some fruits (tomatoes, carrots) and flowers their color; Amyloplasts - colorless, starch-storing plastids; Leucoplast - another term for amyloplast; Etioplast - plastid whose development into a chloroplast has been arrested (stopped). These contain a dark crystalline body, prolamellar body, which is essentially a cluster of thylakoids in a somewhat tubular form.

Plastids can dedifferentiate and convert from one form into another. For example, think about the ripening processing in tomato. Initially, green tomatoes have oodles of chloroplasts which then begin to accumulate lycopene (red) and become chromoplasts. Usually you find only chromoplasts or chloroplasts in a cell, but not both. V. Organelles Unique to Plants - Vacuoles This is the large, central cavity containing fluid, called cell sap, found in plant cells. he vacuole is surrounded by a membrane (tonoplast). Back to the water balloon in the box model - imagine the vacuole to be analogous to another water balloon inside our protoplast balloon. This water balloon is a separate entity that can be physically removed from the cell. The vacuole is penetrated by strands of cytoplasm - transvacuolar strands. The tonoplast and plasma membrane have different properties such as thickness (tonoplast thicker). Virtually every plant cell has a large, well-developed vacuole that makes up to 90% or more of the cell volume. Wow! Meristematic and embryonic cells are exceptions. Young tissues have many small vacuoles. As the cell grows the vacuoles expand and eventually coalesce. These small vacuoles appear to be derived from the Golgi. The central vacuole contains water, ions, organic acids, sugars, enzymes, and a variety of secondary metabolites. Among the hydrolytic enzymes are proteases (digest protein), ribonucleases (digest RNA) and glycosidases (break links between monosaccharides). These enzymes are typically not used for recycling cellular components but rather leak out on cell senescence. There are smaller lytic vacuoles, which contain digestive enzymes, that are used for this purpose. Another type of vacuole, protein bodies, are vacuoles that store proteins. Why do plants cells have a large central vacuole? Important roles of the vacuoles are: A. Energetically efficient means to increase surface to volume ratio in the dendritic growth form Since 90% of the cell volume is vacuole, therefore 90% of the cell is water, which is relatively cheap in metabolic terms. Thus, it allows plants to get big with a minimal energy investment. Plants are particularly 'smart,' since, the cell wall, which comprises much of the remaining 10% or so of the cell is a polymer of glucose. Cellulose is a great bargain! It is stronger and cheaper than comparable polymers. Lets compare:

polymer

compound/production

tensile strength

value (weight product/weight glucose to make) cellulose collagen silk 0.83 0.40 0.40

(newton m2 x 109) 30 2 10

B. Water storage Probably a minor role; mostly in succulents C. Waste disposal The vacuole can be considered the cell cesspool. It contains many secondary metabolites including a variety of hydrolytic enzymes like the "marker enzyme" alpha mannosidase. In this regard, the vacuole is analogous to the lysosome. Lets consider differences between plants and animals in terms of wastes. Plants have little waste. Their nutrients are in dilute form, they use them efficiently and hence, there is little left over. Plants remind me of my Dad who was a Marine during WWII. He said that the Marine philosophy at dinner was to "eat all you want, but all that you take." Plants do just that - most everything they "ingest" they use. The minimal wastes plants produce can be stored in the cells in the vacuoles (or disposed of in other ways - released as a gas into the air, leached out of roots or leaves). In contrast, animals rely on nutrients in a concentrated form. They are rarely selective about what they eat; much un-usable material is ingested along with the "good stuff". Thus, they have a large quantity of waste and needed to evolve specialized digestive/excretory systems to get rid of it. Further, animals dont have the luxury of having bulky vacuoles and internally storing wastes because these adaptations would limit motility. Some related ideas: Heterotrophic plants (like carnivorous plants) have excessive wastes. The plant strategy is usually to discard the structure with the wastes (i.e., insect remains) and produce a new one. For example, pitcher plant leaves fill up with insect parts and the plant produces a new batch of leaves the next season. Plants are able to do this because of their architectural design; 2 Humans start out life very much like a plant. Newborns are non-motile and breast-fed. Breast milk is dilute and nutritious. And, it is highly digestible leaving relatively few leftovers. Hence, newborn diapers are relatively innocuous. However, as babies become more animal-like and begin solid food, diapers are best left to the strong of stomach; 3 Cell walls - have been suggested that the cell wall first evolved as a site for waste disposal for excess carbon. Whatdayathink? D. pH regulation

The vacuole is a pool to dump excess protons. There is an active proton pump in the tonoplast. The cell sap has a pH of 2-5.7, whereas the cytosol is ca. 7.0. E. Storage of essential ions Ions are pumped into the vacuole for water balance. Potassium and calcium. in particular, are stored in the vacuole. F. Cell enlargement

Cell growth requires some force to allow for the cell to increase in size. Water pressure provides the force and it moves into the vacuole. For example, root hair enlargement is due entirely to vacuolar enlargement. G. Facilitates diffusion The cytosol essentially forms a thin coating around the large vacuole which in effect, increases the surface-to-volume ratio of the cytoplasm. It provides easier access and shorter diffusion distances between any part of the cytoplasm and the external environment of the cell. This can be particularly important for chloroplasts. H. Structural support The vacuole helps to maintain turgor pressure in plant cells due to the opposing forces of tensile strength of the wall vs. compression strength of water. Cell Walls - Structure & Function I. Functions of the cell wall: The cell wall serves a variety of purposes including:

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maintaining/determining cell shape (analogous to an external skeleton for every cell). Since protoplasts are invariably round, this is good evidence that the wall determines the shape of plant cells. Support and mechanical strength (allows plants to get tall, hold out thin leaves to obtain light) prevents the cell membrane from bursting in a hypotonic medium (i.e., resists water pressure) controls the rate and direction of cell growth and regulates cell volume ultimately responsible for the plant architectural design and controlling plant morphogenesis since the wall dictates that plants develop by cell addition has a metabolic role (i.e., some of the proteins in the wall are enzymes for transport, secretion) physical barrier to: (a) pathogens; and (b) water in suberized cells. However, remember that the wall is very porous and allows the free passage of small molecules, including proteins up to 60,000 MW. The pores are about 4 nm (Tepfert & Taylor 1987) carbohydrate storage - the components of the wall can be reused in other metabolic processes (especially in seeds). Thus, in one sense the wall serves as a storage for carbohydrates signaling - fragments of wall, called oligosaccharins, act as hormones. Oligosaccharins, which can result from normal development or pathogen attack, serve a variety of functions including: (a) stimulate ethylene synthesis; (b) induce phytoalexin synthesis; (c) induce chitinase and other enzymes; (d) increase cytoplasmic calcium levels and (d) cause an "oxidative burst". This burst produces hydrogen peroxide, superoxide and other active oxygen species that attack the pathogen directly or cause increased cross-links in the wall making the wall harder to penetrate.

Let's look at how this system works. Consider a pathogenic fungus like Phytophthora. In contact with the host plant the fungus releases enzymes such as pectinase that break down plant wall components into oligosaccharins. The oligosaccharins stimulate the oxidative burst and phytoalexin synthesis, both which will deter the advance of the fungus. In addition, the oligosaccharins stimulate chitinase and glucanase production in the plant. These are released and begin to digest the fungal wall. The fragments of fungal wall also act as oligosaccharins in the plant to further induce phytoalexin synthesis. Cool! 10 recognition responses - for example: (a) the wall of roots of legumes is important in the nitrogen-fixing bacteria colonizing the root to form nodules; and (b) pollen-style interactions at mediated by wall chemistry. 11 economic products - cell walls are important for products such as paper, wood, fiber, energy, shelter, and even roughage in our diet. II. Wall Components - Chemistry

The main ingredient in cell walls are polysaccharides (or complex carbohydrates or complex sugars) which are built from monosaccharides (or simple sugars). Eleven sugars are common in these polysaccharides including like glucose and galactose. Carbohydrates are good building blocks because they can produce a nearly infinite variety of structures. There are a variety of other components in the wall including protein, and lignin. Let's look at these wall components in more detail: A. Cellulose 1,4-glucan (structure provided in class). Made of as many as 25,000 individual glucose molecules. Every other molecule (called residues) is "upside down". Cellobiose (glucose-glucose disaccharide) is the basic building block. Cellulose readily forms hydrogen bonds with itself (intra-molecular H-bonds) and with other cellulose chains (inter-molecular H-bonds). A cellulose chain will form hydrogen bonds with about 36 other chains to yield a microfibril. This is somewhat analogous to the formation of a thick rope from thin fibers. Microfibrils are 5-12 nm wide and give the wall strength - they have a tensile strength equivalent to steel. Some regions of the microfibrils are highly crystalline while others are more "amorphous". B. Cross-linking glycans (=Hemicellulose) Diverse group of carbohydrates that used to be called hemicellulose. Characterized by being soluble in strong alkali. They are linear (straight), flat, with a -1,4 backbone and relatively short side chains. Two common types include xyloglucans and glucuronarabinoxylans. Other less common ones include glucomannans, galactoglucomannans, and galactomannans. The main feature of this group is that they dont aggregate with themselves - in other words, they dont form microfibrils. However, they form hydrogen bonds with cellulose and hence the reason they are called "cross-linking glycans". There may be a fucose sugar at the end of the side chains which may help keep the molecules planar by interacting with other regions of the chain. C. Pectic polysaccharides These are extracted from the wall with hot water or dilute acid or calcium chelators (like EDTA). They are the easiest constituents to remove from the wall. They form gels (i.e., used in jelly making). Another diverse group of polysaccharides that are particularly rich in galacturonic acid (galacturonans = pectic acids). Polymers of primarily 1,4 galacturonans (=polygalacturonans) are called homogalacturons (HGA) and are particularly common. These are helical in shape. Divalent cations, like calcium, also form cross-linkages to join adjacent polymers creating a gel. Pectic polysaccharides can also be cross-linked by dihydrocinnamic or diferulic acids. The HGA's (galacturonans) are initially secreted from the golgi as methylated polymers; the methyl groups are removed by pectin methylesterase to initiate calcium binding. Other pectic acids include Rhamnogalacturonan II (RGII) which features rhamnose and galacturonic acid in combination with a large diversity of other sugars in varying linkages. Dimers of RGII can be cross-linked by boron atoms linked to apiose sugars in a side chain. Although most pectic polysaccharides are acidic, others are composed of neutral sugars including arabinans and galactans. The pectic polysaccharides serve a variety of functions including determining wall porosity, providing a charged wall surface for cell-cell adhesion (middle lamella), cell-cell recognition, pathogen recognition and others. D. Protein Wall proteins are typically glycoproteins (polypeptide backbone with carbohydrate sidechains). The proteins are particularly rich in the amino acids hydroxyproline (hydroxyproline-rich glycoprotein, HPRG), proline (proline-rich protein, PRP), and glycine (glycine-rich protein, GRP). These proteins form rods (HRGP, PRP) or beta-pleated sheets (GRP). Extensin is a well-studied HRGP. HRGP is induced by wounding and pathogen attack. The wall proteins also have a structural role since: (1) the amino acids are characteristic of other structural proteins such as collagen and gelatin; and (2) to extract the protein from the wall requires destructive conditions. Protein appears to be cross-linked to

pectic substances and may have sites for lignification. The proteins may serve as the scaffolding used to construct the other wall components. Another group of wall proteins are heavily glycosylated with arabinose and galactose. These arabinogalactan proteins, or AGP's, seem to be tissue specific and may function in cell signaling. They may be important in embryogenesis and growth and guidance of the pollen tube. E. Lignin Polymer of phenolics, especially phenylpropanoids. Lignin is primarily a strengthening agent in the wall. It also resists fungal/pathogen attack. F. Suberin, wax, cutin A variety of lipids are associated with the wall for strength and waterproofing. G. Water The wall is largely hydrated and comprised of between 75-80% water. This is responsible for some of the wall properties. For example, hydrated walls have greater flexibility and extensibility than nonhydrated walls. III. Morphology of the Cell Wall - there are three major regions of the wall: Middle lamella - outermost layer, glue that binds adjacent cells, composed primarily of pectic polysaccharides. 2 Primary wall - wall deposited by cells before and during active growth. The primary wall of cultured sycamore cells is comprised of pectic polysaccharides (ca. 30%), cross-linking glycans (hemicellulose; ca 25%), cellulose (15-30%) and protein (ca. 20%) (see Darvill et al, 1980). The actual content of the wall components varies with species and age. All plant cells have a middle lamella and primary wall. 3 Secondary Wall - some cells deposit additional layers inside the primary wall. This occurs after growth stops or when the cells begins to differentiate (specialize). The secondary wall is mainly for support and is comprised primarily of cellulose and lignin. Often can distinguish distinct layers, S1, S2 and S3 - which differ in the orientation, or direction, of the cellulose microfibrils. IV. Tire analogy for the cell wall

The wall is similar to a tire that has a series of steel belts or cords embedded in an amorphous matrix of rubber. In the plant cell wall, the "cords" are analogous to the cellulose microfibrils and they provide the structural strength of the wall. The matrix of the wall is analogous to the rubber in the tire and is comprised of non-cellulosic wall components. How are the various wall polymers assembled? It appears that: cross-linking glycans (hemicellulosic polysaccharides) are hydrogen bonded to the cellulose microfibrils 2 cross-linking glycans may also be entrapped inside cellulose microfibrils as they form 3 the different types of pectic polysaccharides are covalently bonded to one another 4 calcium bridges link pectic acids 5 connections between the protein and other wall polymers are still not clear 6 pectic polysaccharides and cross-linking glycans interact 7 cross-linking glycans are linked by ferulic acid bridges or boron V. Wall Formation

The cell wall is made during cell division when the cell plate is formed between daughter cell nuclei. The cell plate forms from a series of vesicles produced by the golgi apparatus. The vesicles migrate along the cytoskeleton and move to the cell equator. The vesicles coalesce and dump their

contents. The membranes of the vesicle become the new cell membrane. The golgi synthesizes the non-cellulosic polysaccharides. At first, the golgi vesicles contain mostly pectic polysaccharides that are used to build the middle lamella. As the wall is deposited, other non-cellulosic polysaccharides are made in the golgi and transported to the growing wall. Cellulose is made at the cell surface. The process is catalyzed by the enzyme cellulose synthase that occurs in a rosette complex in the membrane. Cellulose synthase, which is initially made in by the ribosomes (rough ER) and move from the ER vesicles golgi vesicle cell membrane. The enzyme apparently has two catalytic sites that transfer two glucoses at a time (i.e., cellobiose) from UDPglucose to the growing cellulose chain. Sucrose may supply the glucose that binds to the UDP. Wall protein is presumably incorporated into the wall in a similar fashion. Remember that the wall is made from the outside in. Thus, as the wall gets thicker the lumen (space within the wall) gets smaller. Exactly how the wall components join together to form the wall once they are in place is not completely understood. Two methods seem likely: self assembly. This means that the wall components spontaneously aggregate; and enzymatic assembly various enzymatic reactions (XET) are designed for wall assembly. For example, one group of enzymes "stitches" xylans together in the wall to form long chains. Oxidases may catalyze additional cross-linking between wall components and pectin methyl esterase may play an important role (see below). VI. Strong Wall/Cell Expansion Paradox (dont you just love a good paradox?)

1 2

How can the wall be strong (it must withstand pressures of 100 MPa!), yet still allow for expansion? Good question, eh? The answer requires that the wall: A. Be capable of expansion In other words, only cells with primary walls are capable of growth since the formation of the secondary wall precludes further expansion of the cell. The sequence of microfibril orientation changes during development. Initially the microfibrils are laid down somewhat randomly (isotropically). Such a cell can expand in any direction. As the cell matures, most microfibrils are laid down laterally, like the hoops of a barrel, which restricts lateral growth but permits growth in length. As the cell elongates the microfibrils take on an overlapping cross-hatched pattern, similar to fiberglass. This occurs because the cell expands like a slinky - the width of the cell doesn't change by the microfibrils become aligned in the direction of growth just like the spring. This overlapping of microfibrils, which is strong and lightweight, prohibits further expansion. But, what determines the orientation of the microfibrils? They are correlated with the direction of the microtubules in the cell. Evidence: treating a cell with colchicine or oryzalin (which inhibit microtubule formation) destroys the orientation of the microfibrils. The microtubules apparently direct the cellulose synthesizing enzymes to the plasma membrane. In addition to cellulose microfibril orientation, mature walls apparently loose their ability to expand because the wall components become resistant to loosening-activities. This would occur if there were increased cross-linking between wall components during maturation. This would result from:

1 2 3

producing wall polysaccharides in a form that makes tighter complexes with cellulose or other materials increasing the lignin in the wall would increase cross-links between polymers de-esterifying the pectic acids would increase calcium bridges;

B. Loosening (or Stress relaxation) the wall at the appropriate time Even though the microfibrils may be in the proper position to permit loosening, the wall is still rather strong. Recall that our wall model proposed strong and weak links between the wall components. When the wall is loosened bonds (i.e., H-bonds) are temporarily broken to allow the wall components to slide or creep past one another. So, how is the wall temporarily loosened? 1. Protons are the primary wall loosening factor (Acid Growth Hypothesis). This idea was first proposed by David Rayle and R. Cleland in 1970. Some evidence:

acid buffers stimulate elongation and rapid responses 5-15 min even in non-living tissues (Evans,1974); acid secretion is associated with sites of cell elongation (see Evans & Mulkey, 1981) Fusicoccin, a diterpene glycoside extracted from a fungus, stimulates proton secretion (activates a H+/K+ pump) and stimulates elongation.

2. Mechanism of proton action: Protons stimulate wall loosening by:

disrupting acid-labile bonds such as H-bonds and calcium bridges; and enhancing the activity of enzymes that break wall cross-links including H-bonds and calcium bridges. Evidence for the enzyme involvement includes: (1) when primary walls are heated or treated with protein denaturing agents they can't be "loosened" by acid; and (2) adding proteins extracted from growing walls to heat-treated walls restores the acid response. Expansins appear to be the primary wall-loosening enzymes. This class of proteins are activated by low pH and break the hydrogen bonds between cellulose and the cross-linking glycans. Other candidates for enzymes involved include: (1) pectin methyl esterase which would break the calcium bridges between pectins by esterifying the carboxyl groups; and (2) hydrolases which would hydrolyze the cross-linking glycans (hemicelluloses). For example, xyloglucan endotransglycosylase (XET) has been shown to cleave cross-linking glycans that could allow slippage of the wall components

3. The acid effect is induced by indole-3-acetic acid (IAA, auxin), one of the major plant hormones. IAA stimulates proton excretion and cell growth/elongation. Evidence:

peeled coleoptiles + IAA medium acidic; peeled coleoptiles + water not acidic; and flooding auxin-treated tissue with neutral buffers prevents the growth response.

4. Mechanism of Auxin Action How does auxin stimulate proton excretion and wall elongation? There are two ideas: Hypothesis 1: Auxin activates pre-existing H+-ATPase pump proteins in the cell membrane. These proteins transport protons from the protoplast into the wall. Auxin probably first binds to a receptor molecule and this complex then actives the pump. This process is active - thus the pump requires ATP. Evidence: ATP stimulated acidification is observed soon after auxin treatment. Hypothesis 2: Auxin stimulates transcription and translation. Transcription/translation (protein synthesis) would be required to produce proton pump proteins (a wonderful alliteration), respiratory enzymes to provide ATP to power the process; and even enzymes for the synthesis of wall components and cell solutes (see C. & D. below). Evidence for the involvement of transcription/translation:

Nooden (1968) found that artichoke disks increased in size when incubated with IAA but that the addition of antimycin (a protein synthesis inhibitor) prevented this response; soybean hypocotyls incubated with 2,4-D (an analog of IAA) produce at least 3 new polypeptides within three hours (Zurfluh & Guilfoyle, 1980); in vitro translation of mRNA occurs within 15 minutes of IAA treatment The proton effect is short-lived. Cell elongation stops 30-60 minutes after acidification. Continuous elongation requires longer term metabolic changes such as protein synthesis. C. Wall synthesis occurs As the cell grows, wall synthesis needs to occur. Think about the color of a balloon as it is blown up it gets lighter in color as the balloon gets larger because the thickness of the balloon decreases as it expands and stretches. Using this logic, we expect that plant cells should become thinner as they expand. Right? Wrong - cell walls remain a relatively uniform thickness throughout cell growth. Thus, we can conclude that new wall material must be made during cell elongation. D. Enhanced solute synthesis The solute concentration of the cell remains constant during cell enlargement. This suggests that solutes are being synthesized since the volume of the cell is increasing. Maintaining a high solute concentration is necessary to allow for water uptake. E. Lock wall in place after expansion is complete Once wall elongation is completed, the cell needs to "lock it" in place. This likely happens as the temporary bonds that were broken reform, and due to increased interactions (including enzymatic) between wall molecules. F. Water Uptake/Pressure Water, Diffusion and Osmosis Water, of its very nature, as it occurs automatically in the process of cosmic evolution, is fit, with a fitness no less marvelous and varied than that fitness of the organism which has been won by the process of adaptation in the course of organic evolution. L. J. Henderson The Fitness of the Environment, 1913 I. Water is absolutely essential for all living organisms The evidence:

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Most organisms are comprised of at least 70% or more water. Some plants, like a head of lettuce, are made up of nearly 95% water; When organisms go dormant, they loose most of their water. For example, seeds and buds are typically less than 10% water, as are desiccated rotifers, nematodes and yeast cells; Earth is the water planet (that's why astronomers get so excited about finding water in space). Water is the limiting resource for crop productivity in most agricultural systems (see text for supporting data)

Tom Robbins, author of Even Cowgirls Get the Blues, has eloquently stated the importance of water: "Water - the ace of elements. Water dives from the clouds without parachute, wings or safety net. Water runs over the steepest precipice and blinks not a lash. Water is buried and rises again; water walks on fire and fire gets the blisters. Stylishly composed in any situation - solid, gas or liquid -

speaking in penetrating dialects understood by all things - animal, vegetable or mineral - water travels intrepidly through four dimensions, sustaining, destroying, and creating. Always in motion, ever-flowing (whether at stream rate or glacier speed), rhythmic, dynamic, ubiquitous, changing and working its changes, a mathematics wrong side out, a philosophy in reverse, the ongoing odyssey of water is virtually irresistible." Now, let's change our perspective for a minute and put ourselves in the place of water. According to Robbins, water is so important that "[i]t has even been suggested that life evolved as a means to transport water." Well, this is certainly good fodder for a late night discussion, but I doubt that anyone would question Frank Salisburys and Cleon Rosss (1992) statement that "Plant physiology is ... the study of water." II. Water is important because it is polar and readily forms hydrogen bonds A. Water is Polar In other words, the water molecule has a positively-charged (hydrogen side) and negatively-charged side (oxygen). This occurs because: the hydrogen atoms are arranged at an angle of about 105 degrees; the covalent bond between O-H is polarized. This is caused by an unequal sharing of electrons between these atoms which, in turn, results in a slight negative charge on the oxygen atom (electronegative) and slight positive charge on the hydrogen; 3 oxygen has an unshared pair of electrons (the molecule is tetrahedral-shaped). B. Hydrogen Bonds

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The 'fancy' definition of a hydrogen bond is that it is a weak bond that forms between a hydrogen atom that is covalently bonded to an electronegative atom (like oxygen) and another electronegative atom. In other words, a positively-charged hydrogen atom is attracted to a negatively-charged oxygen. The end result is that water readily forms hydrogen bonds with itself and other polar molecules. When likes attract it is termed cohesion (i.e., hydrogen bonds between water molecules). When unlikes attract, it is called adhesion (i.e., when a paper towel absorbs water, water and cellulose adhere to one another). Cohesion and adhesion are responsible for capillary action, the movement of water up a thin tube. In liquid water, hydrogen bonds between water molecules are continuously made and broken. The molecules can even form temporary "quasi-crystalline" areas. Individually, each hydrogen bond is weak (20 kJ mol-1), but collectively they give water many unique properties (a Marxist molecule!). III. The Properties of Water A. Water is a liquid at physiological temperatures (i.e., between 0-100 C). In other words, water has a high boiling point and a high melting point when compared to other similar-sized molecules such as ammonia, carbon dioxide, hydrogen sulfide. These other molecules are gases at room temperature. This is important because if life exists anywhere, we predict that it occurs between approx. 0 and 100 C. Temperatures much below 0 are too cold to permit significant chemistry for metabolism; temperatures above 100 tend to disrupt bonds. B. Water has a high heat of vaporization. In other words, it takes a lot of energy (ca. 44 kJ mol-1) to convert water from a liquid to a gas; or stated another way, Water resists evaporation. This property is responsible for water's use in evaporative cooling systems, hence the reason dog's pant, people perspire, and leaves transpire.

C. Water has a high specific heat (heat capacity). It takes a lot of energy (4.184 J g-1 C-1, or the non-SI unit is the calorie where 1 cal = 4.184 J) to raise the temperature of water (because it requires a lot of energy to break/make hydrogen bonds). Thus, water is slow to heat up and cool down, or stated another way, water resists temperature changes. This is why you can swim reasonably comfortably in the Sag in late fall but not the spring. In contrast, a sidewalk has low specific heat - it heats up quickly (try walking barefoot on a sidewalk in summer on a sunny day), but cools down quickly. This property is important in water's role as a thermal buffer. It's not surprising that desert plants are succulent - to help resist temperature fluctuations. D. Water has a high heat of fusion. It takes a lot of energy to convert water from a solid to a liquid, or put another way, water resists freezing. Energy is required to break the collective hydrogen bonds holding water in its solid configuration. Conversely, a lot of energy (6 kJ mol-1) must be released by water to freeze. This property is used by citrus growers - prior to a light freeze they spray fruit with water; ice forms releasing the heat of fusion which will help protect the crop from serious damage. E. Water has a high surface tension. It takes a lot of energy to break through the surface of water, because water molecules at the surface are attracted (cohesion) to others within the liquid much more than they are to air. Thus, water acts as though it has a skin. This phenomenon is important at air/water interfaces and explains why: (1) water rises up a thin tube (capillary action); (2) raindrops are round (the molecules at the surface attract one another); (3) water striders and other bugs can "walk on water"; (4) a meniscus forms; and (5) a belly-whopper into a pool of ammonia would not hurt nearly as much as one into water. F. The density of water decreases on crystallization. Good thing too, or else ice fishing would be a moist business. This occurs because when ice forms each water molecule is hydrogen bonded to exactly four others. At four degrees, water is it's densest, and each water molecule is attracted to slightly more than four others. Thus, as water cools it gets denser and denser until it reaches 4 C, then, it gets less dense. And ice floats. G. Water is a universal solvent. Water dissolves more different kinds of molecules than any other solvent. Hydrophilic (water-loving) molecules dissolve readily in water (likes dissolve likes), hydrophobic (water-fearing) ones do not. H. Water has high tensile strength and incompressibility. In other words, if you put water in a tube and put a piston on either end, you wont be able to push the pistons together. Thus, water is good for hydraulic systems because when it is squeezed it doesn't compress and produces positive pressures (hydrostatic pressures). This pressure provides the driving force for cell growth and other plant movements. The pressure is measured in units of Pascals (or actually MegaPascals, MPa). One MPa is approximately equal to ten atmospheres or 10 bars. In a similar fashion, if you fill the tube with water, remove any air bubbles, and then pull the pistons away from one another, the water column resists breaking. This will result in a suction on the water column - just like putting your finger on the end of a syringe and pulling back the plunger. Negative pressures (tensions) can develop in the water column. Very sizable tensions can be generated in a thin water column. However, cavitation, when air comes out of solution at negative pressures, can be a problem. I. Water is transparent to light. This is important because chloroplasts (inside a cell) are obviously surrounded by water. If water were opaque, plants couldn't photosynthesize. From an ecological perspective, the penetration of in water determines the distribution of aquatic plants.

J. Water is chemically inert. It doesn't react unless it is enzymatically designed to do so. K. Water dissociates into protons and hydroxide ions. This serves as the basis for the pH system (see below). L. Water affects the shape, stability & properties of biological molecules. For example, many ions (such as sodium) and molecules (such as DNA and wall components) are normally hydrated. This means that water is hydrogen bonded to them and in some cases (i.e., sodium) forms a hydration shell around them. IV. Functions of Water. In addition to the functions mentioned above, water: is a major component of cells is a solvent for the uptake and transport of materials is a good medium for biochemical reactions is a reactant in many biochemical reactions (i.e., photosynthesis) provides structural support via turgor pressure (i.e., leaves) is the medium for the transfer of plant gametes (sperms swim to eggs in water, some aquatic plants shed pollen underwater) 7 offspring (propagule) dispersal (think "coconut") 8 plant movements are the result of water moving into and out of those parts (i.e., diurnal movements, stomatal opening, flower opening) 9 cell elongation and growth 10 thermal buffer 11 perhaps most importantly, water has directed the evolution of all organisms. You can think of morphological features of organisms as a consequence of water availability. For example, consider organisms growing in xeric (dry), mesic (moderate) and hydric (aquatic) environments. V. Acids and Bases (a review from introductory biology)

1 2 3 4 5 6

Water ionizes to a small degree to form a hydrogen ion (or proton) and hydroxide ion (OH-). In reality, two water molecules form a hydronium ion (H 30+) and a hydroxide ion (OH-).

In pure water, [H+] = [OH-] This solution is neutral [H+] > [OH-] Then, the solution is an acid (acidic) [H+] < [OH-] Then the solution is a base (alkaline) Thus: An acid is a substance that increases the [H+], or as the chemists say, is a proton donor. eg. HCl H+ + Cl2 A base is a substance that increases the [OH-]; or from the perspective of a proton, a base is a substance that decreases the proton concentration; it is a proton acceptor. e.g. NaOH Na+ + OH- (accepts protons to make water) e.g. NH3 (ammonia) + H+ NH4+ (ammonium ion) The pH Scale:

pH is the scale to express the degree of acidity (or alkalinity) of a solution. The scale ranges from 0 to 14 where 1 is highly acidic, 7 is neutral, and 14 is highly alkaline.

As the pH increases, the [H+] decreases and the [OH-] increases As the pH decreases, the [H+] increases and the [OH-] decrease pH = - log[H+] Points to note:

1 2

the pH scale is based on proton concentration; and the pH scale is logarithmic, there is a 10-fold difference in concentration between each pH unit.

at pH 7: [H+] = [OH-] 0.0000001 mol H liter = 10 H+ = 0.0000001 OH- liter-1

+ -1 -7

pH = log [H+] = -log[10-7] = - (-7) = 7 The products of [H ] x [OH ] always equals 10-14. Thus, you can always determine concentration of one if you know the other. For example, if the [H +] = 10-2, then the [OH-] is 10-12. VI. Living systems are very sensitive to pH Organisms must maintain pH within tolerable ranges. This is a good example of homeostasis. A buffer is a solution that resists fluctuations in pH when additional OH- or H+ are added. They maintain a constant pH and usually consist of a proton donor and a proton acceptor. [e.g., blood pH must be between 7.36 (venous) and 7.41 (arterial). The carbonic acid/bicarbonate buffer helps to maintain pH: H2CO3 (carbonic acid; proton donor) H+ + HCO3- (bicarbonate ion; proton acceptor) VII. Water Movement - There are two major ways to move molecules: A. Bulk (or Mass) Flow. This is the mass movement of molecules in response to a pressure gradient. The molecules move from hi low pressure, following a pressure gradient. A good example would be a faucet. When you turn a faucet on, water comes out. This occurs because the water in the tap is under pressure relative to the air outside the faucet. A toilet is another example; high pressure in the tank/bowl but lower pressure in the sewer system. Some molecular movements rely on bulk flow which requires a mechanism to generate the pressure gradient. For example, animals have evolved a pump (i.e., heart) that is designed for the bulk flow of molecules through the circulatory system. B. Diffusion The net, random movement of individual molecules from one area to another. The molecules move from [hi] [low], following a concentration gradient. Another way of stating this is that the molecules move from an area of high free energy (higher concentration) to one of low free energy (lower concentration). The net movement stops when a dynamic equilibrium is achieved.
+ -

Imagine opening a bottle of perfume containing volatile essential oils in a very, very still room. Initially, the essential oils are concentrated in a corner of the room. As the molecules move randomly, in every different direction, over time they will eventually appear throughout the room. Ultimately the essential oils will reach a point, dynamic equilibrium, at which they are evenly distributed throughout the room. At this point the molecules are still moving. They continue to move randomly in every direction. The only difference is that there is no net change in the overall distribution of the perfume in the room. Now imagine that the room is divided by a partition with holes (which is analogous to a membrane). If we place a drop of perfume on one side of the partition and then count at intervals the number of essential oil molecules on either side of the partition and graph the results: insert: plot # molecules vs. time on both sides of the partition. (not included) We will observe that the number of molecules on one side will decrease while the other will increase until they reach dynamic equilibrium. At equilibrium the molecules continue to move randomly, back and forth from one side of the partition to the other. Hence the number of molecules on either side of the partition at any given time is simply chance. The number oscillates about the midpoint. A caveat Although this theoretical example can help us to better understand the nature of diffusion, it is technically wrong. The molecular movement attributed to diffusion in this example is really due to air movements in the room, or convection. True examples of diffusion are hard to come by (see Vogel, 1994; Wheatley, 1993). Nevertheless, it serves our purpose to illustrate the general concept of diffusion. C. Osmosis This is a specialized case of diffusion; it represents the diffusion of a solvent (typically water) across a membrane. D. Dialysis Another specialized case of diffusion; it is the diffusion of solute across a semi-permeable membrane. Example consider a cell containing a sugar dissolved in water. If water (the solvent) moves out of the cell into the surroundings it moves osmotically; if the sugar (solute) moves into the surroundings, it is an example of dialysis. VII. Factors influencing the rate of diffusion - Several factors influence the rate of diffusion. These include: A. Concentration Gradient. As previously stated, solutes move from an area of high concentration to one of lower concentration; in other words, in response to a concentration gradient. Although this is true for most solutes, it is NOT important for water. The concentration of water (55.2 - 55.5 mol L-1) is nearly constant under all conditions (i.e., MW = 18 g/mol, and 1000 g/liter; thus, 1000/18 = 55.5 mol/L). Ficks Law - is an equation that relates the rate of diffusion to the concentration gradient (C1 C2) and resistance (r). Diffusion rate, also called flux density (J s, in units of mol m-2 s-1) can be expressed in the simplified version of Fick's equation as: Js = (C1 - C2) / r The take-home-lessons from this equation are that the rate of diffusion are:

the rate of diffusion is directly proportional to the concentration gradient. The greater the difference in concentration between two areas, the greater the rate of diffusion. Thus, when the gradient is zero, there will be no net diffusion, diffusion will only occur so long as a concentration gradient exists; the rate of diffusion is indirectly proportional to resistance. In other words, the greater the resistance to diffusion, the lower the rate of diffusion. Resistance refers to anything that reduces the rate of diffusion such as the partition in our perfume example. The width of the partitions is a resistance; the wider the partitions, the lower the resistance. And, the membrane is a resistance to the movement of ions and other charged substances in or out of cells; and the rate of diffusion is inversely proportional to distance traveled (also a function of resistance). For example, some typical diffusion rates for water are 10 m - 0.1 sec; 100 m -1 sec; and 1 mm - 100 sec. As the text demonstrates nicely, diffusion is effective over short distances, but is pathetically slow over long distances.

B. Molecular Speed. According to kinetic theory, particles like atoms and molecules are in always in motion at temperatures above absolute zero (0 K = -273 C). The take-home-lesson is that molecular movement is:

1 2

directly proportional to temperature; and indirectly related to molecular weight (heavier particles move more slowly than lighter, smaller ones). At room temperature, the average velocity of a molecule is fast - about 2 km/sec (=3997 mph!).

C. Temperature - increases the rate of molecular movement, therefore, increases the rate of diffusion D. Pressure - increases speed of molecules, therefore, increase the rate of diffusion E. Solute effect on the chemical potential of the solvent. Solute particles decrease the free energy of a solvent. The critical factor is the number of particles, not charge or particle size. Essentially solvent molecules, such as water in a biological system, move from a region of greater mole fraction to a region where it has a lower mole fraction. The mole fraction of solvent = # solvent molecules/ total (# solvent molecules + # solute molecules). This is particularly important in the movement of water. Water moves from an area of higher mole fraction or higher energy to an area of lower mole fraction or lower energy.

Measure of the energy state of water. This is a particularly important concept in plant physiology because it determines the direction and movement of water. A. First, some definitions:

1 2 3 4

Free energy of water - energy available to do work (J = n m) Chemical potential () - free energy/unit quantity (usually per mole) (J mol-1) Water potential (w) - chemical potential of water, compared to pure water at the same temperature and pressure. The units are in pressure because: (a) plant cells under pressure (remember the wall?); and (b) it is easier to measure pressure. Derivation of units - Water potential is official defined as the chemical potential of water divided by the partial molar volume. divide chemical potential (J mol-1) by the partial molar volume of water (L mol-1). This can be simplified in two ways: J mol-1/ L mol-1 = J / liter = energy / volume = (weight x distance)/area x distance = force/area = pressure

or looking at it another way J mol-1/ L mol-1 = n x m mol-1/ m3 mol-1 = n m-2 = MPa Pressure is measured in MPa (megapascals). 1 MPa = 10 bars = 10 atm. (as an aside, 1 atm = 760 mm Hg = 14. 7 lbs sq in-1)

B. Equation for water potential (must account for the factors that influence the diffusion of water and other substances): w = p + s + g where w = water potential; p = pressure potential; s = solute or osmotic potential; and g = gravity potential. 1. Solute (or osmotic) potential (s) This is the contribution due to dissolved solutes. Solutes always decrease the free energy of water, thus there contribution is always negative. The solute potential of a solution can be calculated with the vant Hoff equation: s = - miRT where m = molality (moles/1000 g); i = ionization constant (often 1.0); R = gas constant (0.0083 liter x MPa/mol deg); and T = temperature (K). 2. Pressure (or Pressure Potential; p) Due to the pressure build up in cells thanks to the wall. It is usually positive, although may be negative (tension) as in the xylem. Pressure can be measured with an osmometer. 3. Matric potential This is the contribution to water potential due to the force of attraction of water for colloidal, charged surfaces. It is negative because it reduces the ability of water to move. In large volumes of water it is very small and usually ignored. However, it can be very important in the soil, especially when referring to the root/soil interface. 4. Gravity (g) Contributions due to gravity which is usually ignored unless referring to the tops of tall trees. C. The water potential of pure water is zero. Water potentials in intact plant tissue are usually negative (because of the large quantities of dissolved solutes in cells). D. Water potential is the sum of the contributions of the various factors that influence water potential E. Measuring Water Potential - we will discuss the following techniques in class/lab: 1. Pressure bomb - a steel chamber that can be pressurized, usually with nitrogen. The sample is placed in the chamber with the petiole or surface exposed through a hole in the lid. The sample is pressurized and the pressure that is required to force water to appear on the cut surface is assumed to be equivalent to the water potential of the tissue. 2. Chardakov Method - Dye Drop method 3. Gravimetric method F. Measuring Solute Potential Solute potential can be measured by:

Freezing Point Depression - dissolved solutes lower the freezing point of a liquid (think salt and MN roads in the winter). A 1 molal solution with an osmotic potential of -2.27 MPa lowers the freezing point (fp) by -1.86 degrees. Thus: s = -2.27/1.86 and rearranging, s (MPa) = -1.22 (fp); 2 Incipient Plasmolysis - a tissue is incubated in a series of solutions of known water potential. The point at which membrane just pulls away from tissue is "incipient plasmolysis" and considered to the equivalent to the osmotic potential of the tissue. 3 Vapor pressure osmometer - dissolved solutes increase the boiling point or decrease the vapor pressure of a liquid. A thermocouple hooked to a recorder is placed in an airtight chamber with the tissue sample or standard. The thermocouple is also linked to a reference junction. Thermocouples are made of two different metals (constantan and chromel) and a current will flow if there is a difference in temperature between the reference junction and thermocouple. A water droplet or KCl (aq) solutions of known osmolarity are placed on the thermocouple. Depending on the osmotic potential of the solution, water will either evaporate from or condense on the droplet. This is turn causes a current change in the thermocouple which can be detected by a meter. The cooling rate is plotted vs. s or [KCl] to yield a standard curve from which the s of the tissue is determined. IX. The Movement of water across a membrane is a combination of diffusion and bulk flow

Individual water molecules diffuse across the membrane. In addition, there are integral proteins in the membrane that form a channel or pore through which water moves. These pores are important and water molecules essentially move through these pores by bulk flow. The proteins are called aquaporins and are essentially water transport channels. Water is moving passively (following a gradient of free energy). Water Transport I. Soil-plant-air continuum. The movement of water follows the pathway: soil uptake root stem leaf transpiration air The driving force for water movement is the water potential gradient that exists from soil to air. Or in other words: soil > root > stem > leaf > air Some typical values water potential values (in MPa) for a tree are: trunk -0.7; twig -2.3, leaf -2.5. In class, we may also look at some data for ivy. II. Soil Plant A. What is soil? Soil is a mixture of organic (dung, decayed organic materials, decomposers) and inorganic (weathered rock) materials, gases (oxygen, carbon dioxide, ethylene), and liquid. B. Soil type - determined by: (1) composition; (2) texture or particle size ( sand > silt > clay. A loam is a soil with 10-25% clay and equal parts of sand and silt); and (3) structure (i.e., compaction) C. Water and soil

Saturated - soil before drained. Gravitational water - water that drains and is not tightly bound; = 0 MPa

Field capacity - soil that holds all the water it can against gravity. Capillary water -water held by capillary action, water at field capacity; = -0.015 MPa 3 Permanent wilting percentage - soil moisture content at which plants can't get enough water. For most, = -1.5 MPa 4 Graphic relationship of soil water potential vs. water content (%). Take-home lessons 1 between PWP and FC is the water available for a plant to use; 2 clay holds more water than sand at any ; and 3 clay holds water more tightly (i.e., @10% water Ysand > clay). This is essentially a s/v problem, since smaller particles in clay they have a larger total surface and hence, has more charged surfaces that will bind water tightly. 5 Soil water potential is a function of osmotic potential (which is usually near zero except in saline soils) and mostly pressure (used to call it matrix potential; this refers to the tension generated because of the attraction of colloidal particles; i.e., adhesion). The pressure in the soil can be calculated from the equation: p = -2T/r where T = surface tension (7.28 x 10-8 MPa) and r = radius of curvature of the meniscus). Water movement through soil - mostly due to bulk flow as a result of pressure gradients, with some diffusion. 6 Spuds McSaupe plays with sponges III. Plant Air (= Transpiration)

Or more simply stated, the movement of water from plant to air occurs via transpiration. Air has a very high capacity for holding water. For example at 20 C, the water potential of water in air at 100% RH = 0 MPa; 98% RH -2.7 MPa; 50% RH = -93.5 MPa. Conclusion - there is a very steep water potential gradient from soil to air. Essentially, the plant just inserts itself between the two and takes advantage of passive transport.

IV. Soil Root

1 2 3 4

6 7

Root anatomy We will go over structure of the root including epidermis, cortex, endodermis, Casparian strip, stele, phloem, xylem, pericycle. Root Formation Roots develop from the pericycle; film loop Apoplast vs. symplast Recall that the apoplast refers to the "non-living" regions of the plant and the symplast is the "living" areas. Region of Water Absorption Most of the water is absorbed near the tip of the root. The further from the tip, the less water that is taken up by the root. This roughly correlates to the region of the root that is suberized. Route of Water Movement There are three routes water can follow: (a) Apoplastic water follows an apoplastic route from soil through cortex. However, it must enter the stele symplast because of the Casparian strip. Once inside, it leaks back out and enters the apoplast (xylem) where it is transported to the apex of the plant. This appears to be the major route of transport; (b) Symplastic: Transmembrane in other words, the water moves from cell-to-cell crossing membranes as it goes; and (c) Symplastic: Plasmodesmata the water moves from cell-to-cell via the plasmodesmata. Root as an osmometer Analogy - allows for the development of root pressures in the stem. These can be measured and are about 0.2 - 0.3 MPa. Guttation and hydathodes

V. Root Leaves A. What is the transport tissue for water? Xylem. Evidence comes from various tracer studies where xylem is loaded with dyes. Ill bet youve done the classic "celery stalk in food coloring" experiment. Well see a film loop and maybe play with some celery B. In which cells does water move? Vessels & Tracheids There are four major types of cells in the xylem: (a) tracheids - long, tapered ends, thick secondary wall; (b) vessel elements, - shorter, ends attached; (c) fibers - long and skinny with thick secondary wall, mostly for support; and (d) parenchyma - alive, thin, store starch and other materials, lateral transport. The primary water transport cells are tracheids and vessels. Note that gymnosperms only have tracheids whereas angiosperms have both and primarily rely on vessels for water transport. Both tracheids and vessels have pits, thin circular regions, in the walls. C. How much pressure is required to move water to the top of a tall tree, that is say, 100 meters tall? Let's calculate. We can measure the velocity of flow in the xylem to be 4 - 13 mm s-1 in vessels with a diameter of 100 - 200 mm. For our calculations, let's use a flow rate of 4 mm s -1 (= 4 x 10-3 m s-1) and a vessel radius of 40 m (= 0.00004 m). According to Poiseuille's Law - flow rate is directly proportional to the pressure gradient and the cross sectional area of the pipe but inversely proportional to the viscosity of the fluid. Thus, this is mathematically expressed as the Poiseuille equation: Jv = (()(r4)( P))/8 () where = viscosity of water (assume it is the same as in a cell, 10 -3 Pa s) Now, divide the equation by the cross-sectional area of a vessel ( r2). Thus, the equation simplifies to: Jv = (( r2 )( P ))/8 () Substituting the values for flow and vessel diameter: 4 x 10-3 m/sec = (0.00004 m )2(P)/ 8 (10-3 Pa s) P = 20,000 Pa m-1 P = 0.02 MPa m-1 If the tree is 100 meters, then: 0.02 MPa m-1 x 100 m = 2 MPa However, we must also take into account the effects of gravity (0.01 MPa m-1). Thus, for a 100 m tree: 0.01 MPa m-1 x 100 m = 1 MPa for gravity Finally, the total pressure required to move water to the top of a 100 meter tree equals: 2.0 + 1.0 = 3 MPa D. How is Water Moved to the top of trees? 1. Is water moved to the tops of trees by a "push from the bottom" pump? - NO

Several lines of evidence show that this type of pump doesn't exist: (a) dissections showed there is no anatomical area in the stem or root that could serve as a pump; (b) when German physiologists cut off a tree in a vat of picric acid it continued to transport water. This suggested that a stem pump was not involved since the picric acid should have killed living cells stopping the pump; (c) a root pump isn't involved or else when a plant is decapitated, the stump should continue to gush water; and (d) recall that root pressures only generate 0.2-0.3 MPA but that a pressure of at least 3 MPa is required to move water to the tops of tall trees. To summarize, root pressure doesn't have nearly enough power. 2. Is water moved to the tops of trees by "capillary action" - NO Capillary action is the movement of water up a thin tube due to surface tension and the cohesive and adhesive properties of water. Essentially the meniscus "pulls" the water up the tube. Without worrying about the derivation of the equation, the height to which a column of water can move is inversely related to the radius of the pipe and is mathematically expressed as: h = 14.87/r (where r = radius in m; and h = height in meters). Let's look at some actual data

Table 1: Capillary Heights of Water Movement Tube Radius (m) 10 40 (tracheid) 100 0.005 (size of pores in wall) Column Height (m) 1.4877 0.37 0.148 2975 (ca. 3 kilometers)

Vessels are too wide to support movement very high and obviously capillarity cannot be responsible for water movement. Further, even it could, it would only move to the top of the plant once; capillary action can't continually pull the water up. 3. Cohesion-Tension Theory - YES! This idea was first proposed by HH Dixon (Transpiration and the Ascent of Sap in Plants, 1914). According to this hypothesis, water is drawn up and out of the plant by the force of transpiration. Because of the cohesive/adhesive properties of water, as one water molecule evaporates at the opening it pulls the other molecules and sends this pull all the way down the column. If this is true them, water transport in plants must meet the following criteria:

The system must have little resistance. The vessels and tracheids are hollow at maturity. Imagine how difficult it would be to move water through a clogged pipe. Let's calculate how much pressure would be necessary if the water transport cells were "alive." We'll use Fick's Law: Jv = Lp (Lp = hydraulic conductance which is the inverse of resistance)

If we assume that water movement occurs at the rate we used earlier (Jv = 4 x 10 -3 m s-1) and we use a typical value of 4 x 10-7 m s-1 MPa-1 for Lp, then: -3 4 x 10 m s-1 = (4 x 10-7 m s-1 MPa-1) () = 4 x 10-3 m s-1/4 x 10-7 m s-1 MPa-1

 = 104 MPa (this is the pressure required to move the water across just one membrane! Compare to the value we calculated above) if, the cell length is 100 m (10-4 m), then we can calculate the force required per meter: 104 MPa/10-4 m = 108 MPa m-1

The columns of water must be continuous from the leaves to the soil. If not, it would be analogous to having a chain with a single broken link it would be impossible to pull anything attached to the other end. The tracheids and vessels form a continuous water column. If there are gaps - or air bubbles - water must be routed around these bubbles. Cavitation, or vacuum boiling, is the fancy term for air coming out of solution when the water columns break. By the way, this is one reason why you don't want to go outside and beat on the trunk of a tree on a hot sunny day...it could cause many of the columns to break so that a plant may have a difficult time transporting water. Check out the Per Scholander stories written by Dr. V Berg. If cavitation does occurs, the plant responds by: (a) transporting water around the blocked cell; or (b) redissolving the air bubble, which usually occurs at night; and/or (3) forming new xylem cells; in other words, xylem is disposable. Only the most recent cells in the latest seasons growth are actually functional. The remainder of wood in a tree is non-functional because it has cavitated and/or filled with other waste materials. In addition, it is thought that one function of bordered pits is to stop the movement of air bubbles from a cavitated cell to another thereby isolating the impact of cavitation. There must be sufficient pulling force. Even though ca. 3 MPa are required to move water to the top of a tall tree, the water potential gradient from soil to air is considerably steeper (on the order of -100 MPa.) The xylem should be under a tension. Several lines of evidence support this prediction: (a) Cut a stem and the water will snap up into the top and accumulate at the cut surface on the bottom; (b) Dendrometer studies - this device is essentially a band wrapped around a tree that is hooked to a pressure transducer. As the tree transpires the diameter of the tree is measured. These experiments show that the diameter of the stem is smallest during the day when transpiration is occurring and largest at night, as we would. Imagine putting your finger on the end of a straw and then sucking on the other end. The straw will get thinner (collapse) as you apply tension to the air in the straw - just like a plant stem; (c) Puncturing the xylem of an actively transpiring tree with an ice pick may result in a hissing sound as air is sucked into the stem (see Dr. Berg's water stories); and (d) Dye solutions are rapidly sucked into a tree trunk when punctured with a knife and then transported in both upward and downward directions. Since the pressure in the stem is lower than atmospheric the dye solution is quickly sucked in. We will see a demonstration of this in a video that a previous class made (BIOL327- Spring 2001) made. The tensile strength of water must be able to withstand the pull. In other words, the columns of water must not snap as they are being pulled. As the water is pulled up the tree the water column puts up a resistance, much like stretching a rubber band. Just like a rubber band will snap if pulled too hard, so too will a water column break or cavitate. This occurs because the reduced pressures in the water column cause gases to come out of solution and form a vapor lock in that cell. Cavitation can be heard by placing a sensitive microphone on the plant. Tiny popping noises can be heard, a little like a bowl of rice krispies. The fact that water has a very high tensile strength, more than sufficient to withstand the pulling forces necessary to move water to the top of tall trees, was demonstrated by an elegant

experiment in which water was centrifuged in Z-shaped tubes. As an aside, the tensile strength of water may be one of the factors that limits the height of trees - the tensions in the stems of taller trees would be too great and the water columns would snap. It's perhaps not a surprise that the tallest trees, California redwoods, grow along the fog enshrouded coast. This helps to minimize the rate of water loss and ultimately reduce the tensions in the xylem (Zimmer, 2000). Tracheids and vessels must be able to withstand tensions without imploding. Hence the reason that they have thick cell walls with circular thickenings. It's no surprise that wood is hard. 4. Pressure Compensation Theory - Controversial. Recent work by Martin Canny and others have challenged the validity of the Cohesion-Tension Theory (s Gas Exchange/Transpiration I. Definitions Transpiration - evaporation of water from a plant surface Evapotranspiration - evaporation of water from a plant surface and the soil (and abiotic surroundings. Take-home-lesson: Plants loose a lot of water by transpiration. See text for some specific examples. II. Photosynthesis/Transpiration Paradox (or perhaps more accurately, a "Compromise" or "Dilemma") Recall the equation for photosynthesis where: CO2 + H2O (CH2O) n + O2 This equation suggests that:

1 2 3

Gases, such as carbon dioxide and oxygen, are important in the overall energy metabolism of plants; Plants must exchange gases with the environment; and In order to obtain carbon dioxide plants will necessarily loose water (transpire). In other words, transpiration is a necessary evil of photosynthesis.

III. Theoretical considerations

1 2

A large surface area is required for efficient gas exchange. Thus, animals have lungs or gills; plants have leaves and within the leaf - spongy mesophyll. It's time to review your leaf anatomy. A large surface area for exchanging gases offers a large surface area for desiccation. Animals solve this problem by placing the absorptive surface inside a humid cavity (lung) opened with a small exit pore(s). Plants put the absorptive surface (spongy mesophyll) inside the leaf and cover it with a water impermeable layer (cuticle) peppered with a series of pores (stomata). The cuticle is comprised of waxes, which are an assortment of long chain hydrocarbons and, in particular, cutin (C16-C18 hydroxylated fatty acids). Note that even though these strategies minimize desiccation from the absorptive surfaces of plants and animals, they dont completely eliminate it.

Placing the absorptive surfaces inside the organism to reduce desiccation presents a problem getting the gases to the absorptive surface. Animals use an active pumping mechanism (e.g., lungs/diaphragm) to move gases inside the organism by bulk flow. The gases are circulated by another pumping system (heart). The distances needed to move the gases are too great to be accounted for by simple diffusion. Plants do not have a pumping mechanism for moving gases; they rely on diffusion (and bulk flow). In either case, plants do not actively move gases. This is one reason why leaves are so thin (recall our previous discussion) - diffusion is not efficient over long distances (i.e., diffusion is inversely related to the square of distance). For example, it would take about 2.5 seconds for glucose to diffuse the distance across a cell membrane (0.5 m) but approximately 32 years to go one meter! 4 Paradox and Compromise. In order to obtain carbon dioxide for photosynthesis plants needed to evolve a large, thin absorptive surface (leaves with spongy layer) and then protect it from desiccation. Thus we can consider this the photosynthesis/transpiration "paradox". Actually, it might better be considered a "compromise" since that is what a plant needs to do - strike a compromise or balance between the amount of carbon dioxide absorbed and the amount of water lost by transpiration. IV. Further complications

Not only is water loss a "necessary evil" of photosynthesis, but to make matters worse, the tendency to loose water is greater than the tendency of carbon dioxide to diffuse into the plant. As evidence, let's calculate the transpiration ratio, which is a measure of the the amount of water loss relative to the amount of carbon fixation. transpiration ratio = mol water transpired / mol CO2 fixed If carbon dioxide uptake (or fixation) and water loss are equal, this ratio should be one. In reality, experiments show that this ratio is closer to 200! Thus, for every 200 kg of water transpired, 1 kg of dry matter is fixed by a plant. Lets see why: A. Diffusion and gradients. Recall that during diffusion molecules move from an area of higher chemical potential (or concentration or chemical energy) to an area of lower chemical potential (or concentration or free energy) and that the driving force for diffusion is the gradient from one area to another. We can express this relationship mathematically using Ficks Law: Jv = (c1 - c2)/r where Jv = flux density, (mol m-2s-1); c1 - c2 = concentration gradient, and r = resistance (a function of distance, medium viscosity, membrane permeability, etc.). We can simplify this equation to: diffusion = gradient/resistance (distance) Now lets compare the rates of diffusion for both water and carbon dioxide. Since resistance, or the distance that either carbon dioxide or water must diffuse into/out of the leaf is the same, then diffusion is directly proportional to the gradient.

Carbon dioxide - has a very shallow, or small, gradient from inside to outside of the leaf. Ambient carbon dioxide concentrations are approximately 0.03% (= 0.34 mmol mol -1) and the internal concentration cannot be less than zero. Thus, the gradient is no larger than 0.34 mmol mol-1 (0.34 - 0), a relatively small difference. Water - has a very steep gradient from inside to outside. At a relative humidity of 50% and 25 C the water potential of water in air is ca. -100 MPa (=32 mmol mol-1). The air in the substomatal cavity of a leaf is typically fully saturated, with a RH near 100%, and has a water potential near zero. Thus, the water potential gradient is 100 MPa (or 32 mmol mol-1), which is considerably steeper than that for carbon dioxide.

Conclusion: based on gradient alone, the water has a much greater tendency to diffuse out of the leaf than carbon dioxide to diffuse into the leaf. B. Diffusion and molecular weight. Recall that diffusion is inversely related to molecular weight. Simply put, the heavier the molecule the more slowly it will diffuse. No big surprise. Or, to express this mathematically: rate = 1 / sq rt MW The relationship between the rate of diffusion of two molecules can be summarized by the following relationship: Rate A / Rate B = sq rt B / sq rt A Thus to calculate the ratio of water loss to carbon dioxide uptake: H20 loss/CO2 uptake = sq rt 44 / sq rt 18 = 1.56 Conclusion: Based on molecular weight alone, the tendency for water to diffuse out of the leaf is 1.56 times greater than the tendency for carbon dioxide to diffuse into the leaf. V. The photosynthesis/transpiration compromise revisited Although it seems as though water loss is a serious, intractable problem for a plant, it is NOT. The reason - plants continually compromise between the amount of carbon dioxide absorbed and the amount of water loss. This compromise is mediated by the stomata, whose function is to regulate gas exchange. VI. Stomatal Structure Anatomy of a stoma (stomata, plural) - guard cells, subsidiary cells, substomatal cavity, cuticle, ledge (or lip), stomatal apparatus. The subsidiary cells are epidermal cells that may be specialized and different from the other epidermal cells. The function of the ledge is to prevent liquid water from seeping into the pore. Interestingly, cutin covers most of the cells in the substomatal cavity; only regions near the actual opening are free of cuticle and most water is lost from this area. For images of various guard cells/stoma click here. 2 Types of guard cells: (1) elliptical or kidney-shaped. These are characteristic of dicots and other non-grasses; and (2) dumb-bell or dog-bone shaped - characteristic of grasses (called graminaceous type) 3 Common features - (1) thickened inner walls; and (2) radial micellation - the cellulose microfibrils radiate out around the circumference of the pore; (3) chloroplasts - these are the only epidermal cells with chloroplasts; and (4) connected end-to-end. VII. The beauty of stomata

The evolution of a water-impermeable covering of the absorptive surface that was peppered with oodles of pores was a great idea. The stomata are ideal structures for regulating gas exchange because:

There are lots of them on any plant surface. In fact, there can be as many as 1000 mm-2. Obviously, the larger the number of pores, the greater the amount of total diffusion that can occur (see accompanying data. Plot: total diffusion (g hr -1) vs. total pore number, and total diffusion (g hr-1) vs. pores sq cm-1. A large number of pores is necessary so that plants are able to absorb enough carbon dioxide for photosynthesis. The pores comprise a large area of the surface of a plant. I n fact, stomata occupy as much as 2-3% of the total leaf surface area. As expected, the greater the pore area the greater the rate of diffusion [plot total diffusion vs. total pore area (cm-2) see accompanying data] which is advantageous for maximizing carbon dioxide uptake.

The pores are small. On average, stomata are about 14 m in diameter. Although more total diffusion occurs through large pores [total diffusion (g hr-1) vs. pore dia. (m)], small pores are more efficient than larger ones [diffusion rate (g hr-1 cm-2) vs. pore dia (m) see accompanying data]. This is due to the edge effect (related to surface-to-volume ratio). Smaller pores have a greater proportion of edge. As molecules reach the edge they "spill over" and in effect have a shorter diffusion distance to get away from the pore. 4 The pores are optimally spaced. A significant boundary layer of humid air forms around leaf surfaces. This humid area reduces the rate of further transpiration. It occurs because diffusion shells from adjacent pores fuse. If the pores were much farther separated, they wouldn't form a nice boundary layer. The thickness of the boundary layer is further affected by: (1) wind speed; (2) presence of hairs; and (3) sunken chambers. 5 The pores are optimally located. In grasses, the stomata are distributed approximately equally on both sides of the leave whereas in herbaceous dicots there are generally more on the underside (abaxial) than the upper (adaxial) side. In woody dicots there are usually few stomata in the upper surface, whereas aquatic plants with floating leaves have most stomata in the upper surface. These modifications are important to minimize/control water loss. Conifers and xeric plants often put the stoma in sunken chambers. 6 The degree of opening/closing of the pore is closely regulated by the plant in response to the environment. In effect, any factor that can impact the rate of photosynthesis or overall water status of the plant will influence the action of the guard cells (see below). I like our textbook description that stomata are "multisensory hydraulic valves." 7 Test these ideas by studying the data supplied (click here for Diffusion from a standard leaf) VIII. Mechanics of Guard Cell Action

Guard cells open because of the osmotic entry of water into the GC. In turn, this increases the turgidity (water pressure) in the GC and causes them to elongate. The radial orientation of cellulose microfibrils prevents increase in girth. Since GC are attached at the ends and because the inner wall is thicker, the guard cells belly out with the outer wall moving more pulling open the guard cell. Guard cell closure essential involves reversing this process. In class we'll see a great, old, film loop about this process. We can summarize the mechanics of GC action as follows: stoma closed (GC flaccid) water uptake (osmosis) increase pressure stoma open (GC turgid) IX. Physiology of Guard Cell Action. Part I. Since water is the driving force for GC action, this means that there must be a gradient in water potential between the GC and the surrounding cells (subsidiary cells). Thus, to open a stoma, there must be a mechanism to generate a water potential gradient. A. Hypotheses for how the water potential gradient is established include:

2 3

There is a rapid decrease in pressure in surrounding cells (i.e., subsidiary cells shrink which would result in the GC expanding and taking up water). Using a fine needle transducer, Mary Edwards and Meidner showed that there is some decrease in surrounding cell P, but this is not a major factor. There is an increase in the stretchability of the GC cell wall. This would result in an expansion of the GC with a concomitant uptake of water. Little evidence exists for this idea. There is a decrease in the osmotic potential in the GC - Much evidence supports this hypothesis. For example, Humble and Raschke (1971) showed that the solute potential of turgid GC (open stoma) of broad bean is -3.5 MPA but when the guard cells are flaccid (stoma closed), the osmotic potential is -1.9 MPa. Thus, the solute potential of the GC decreases (becomes more negative) when open. Since the GC volume increases, this must mean that there is an accumulation or synthesis of solute.

Thus, we can modify our schematic diagram: stoma closed (GC flaccid) add solute lower s decrease w water uptake (osmosis) increase pressure stoma open (GC turgid) B. What is the solute and where does it come from?

Carbohydrates, such as sucrose Since guard cells are the only epidermal cells with chloroplasts, plant physiologists have long hypothesized that sucrose and related carbohydrates are osmotic regulators of guard cells. For example, the starch content of guard cell chloroplasts decreases as the stomata open. This idea, the "starch-sugar hypothesis", was the first postulated mechanism for guard cell activity. It lost popularity after the role of potassium ions was discovered, but most now agree that both sugar and potassium ions play a role in guard cell regulation. Sucrose seems to be especially important in closing guard cells. see graph in text/class Where does the sucrose come from? (a) hydrolysis of starch in the GC chloroplasts. In other words, an indirect product of photosynthesis (evidence: starch grains disappear during opening); or (b) a direct product of carbon fixation (photosynthesis).

3 4

Malate Malate is an organic acid (C4). You may already be familiar with its role in the Kreb's cycle in the mitochondria. In plants, malate is also derived from the hydrolysis of starches. The enzyme phosophoenolpyruvate carboxylase (PEPase) binds carbon dioxide (actually bicarbonate ions) to (PEP, 3-carbons, an intermediate in glycolysis) to produce oxaloacetate (C4) which is then reduced to malate and stored in the vacuole. Chloride ions Chloride ions are transported into the cell from the apoplast via a Cl -/H+ symport in which a proton gradient is used to "drag" the chloride into the cell. Potassium ions This appears to be the primary osmotic agent, especially for opening the GC in the morning. The potassium comes comes from surrounding cells. Evidence: (a) if you strip epidermis from a leaf, which breaks many epidermal cells but not the more resistant GC, the GC will only open if K+ is provided in the medium; (b) potassium concentrations increase in the guard cells upon opening (see Table 1)

Table 1: Potassium in the stomatal aperture of Commelina communis Open K+ (mol) in GC K+ (mol) in epidermal cells Thus, we can modify our original scheme: stoma closed (GC flaccid) sucrose/potassium/malate/chloride ions lower s decrease w water uptake (osmosis) increase pressure stoma open (GC turgid) 0.45 0.07 Closed 0.10 0.45

To close the stoma, the reverse process occurs. However, time course studies indicate that potassium uptake is associated with opening of the stomata in the morning, but sucrose loss is more closely associated with closure in the afternoon. Thus, the final modification to our scheme: stoma closed (GC flaccid) potassium and chloride ion uptake, malate synthesis lower s decrease w water uptake (osmosis) from subsidiary cells pressure increases stoma open (GC turgid) ||||| sucrose (potassium, chloride, malate) decreases s increases w increases water loss pressure decreases stoma closed (GC flaccid) X. Environmental Control of GC Action Whatever physiological mechanism we finally postulate for the GC, it must also be compatible with the action of various environmental factors that are known to regulate stomatal activity. Since guard cells respond to their environment, especially any factors that impact the photosynthesis/transpiration compromise. We expect any factor important in photosynthesis to exert regulatory control on GC. And, we expect water to have the final word on control since if a plant dries out too much it's as good as dead! A. Light - exerts strong control. In general: light = open; dark = closed (except CAM plants). What kind of light is important?

Red & blue light The action spectrum for the process suggests that both red and blue light are important regulators of guard cell activity and that their action is, at least partially, mediated by photosynthesis (recall red and blue light are used in photosynthesis). Further evidence that photosynthesis is important - DCMU (an inhibitor of PS) prevents stomatal action. So, what is photosynthesis doing? (a) provides sugars (sucrose and glucose) for osmotic regulation; (b) provides ATP (via photophosphorylation) to power ion pumps (see below); (c) reduces internal CO2 levels which stimulates opening (see below); and (d) reduces the pH in the lumen of the thylakoid that stimulates the synthesis of the blue light receptor pigment (see below).

Blue light There is an additional effect of blue light on stomatal activity that is irrespective of its role in photosynthesis. The evidence: (a) blue light is 10x more effective than red light; (b) in saturating levels of red light, treatment with blue light will cause additional GC opening; (c) the action spectrum for blue light is similar to other "blue light responses", a "three-finger pattern"; (d) photosynthesis inhibitors block the red light effect but not the blue light effect.

What is blue light doing?

Blue light activates a H+-ATPase in the membrane (recall the proton pump for cell elongation?). Evidence: (a) potassium accumulates in isolated GC protoplasts treated with blue light and causes them to swell; (b) blue light causes the acidification of the medium of GC protoplasts under saturating red light conditions; (c) fusicoccin, which stimulates proton pumping, also stimulates stomatal action; (d) vanadate (VO3-, blocks the proton pump) and CCCP (carbonyl cyanide m-chlorophenylhydrazone, an ionophore that makes the membrane leaky to protons) both inhibit stomatal opening. Blue light stimulates starch breakdown and malate synthesis. Blue light stimulates cellular respiration (which among other things may be required to produce ATP for the proton pump.

What is the receptor for the blue light? The action spectrum for the blue light response shows a "three finger pattern," which is characteristic of other blue light responses (i.e., phototropism). Absorption spectra of potential receptor pigments show a good match between zeaxanthin, a carotenoid pigment (C40) that occurs in the chloroplast thylakoid, and the action spectrum. Further zeaxanthin levels are directly correlated with stomatal aperture. How does blue light cause stomatal closure? Photosynthetically active radiation (red and blue light) cause an acidification of the chloroplast lumen. This activates the synthesis of zeaxanthin, which in turn, zeaxanthin activates a calcium-ATP pump in the chloroplast membrane that decreases calcium concentrations in the cytosol. This, in turn activates the proton pump in the cell membrane. B. Low oxygen levels GC open C. Carbon dioxide - intracellular level is an important regulatory control.

lo CO2 (i.e., during the day, used by photosynthesis) = open hi CO2 (i.e., at night, produced during respiration) = closed

D. pH effect

hi pH open lo pH closed

This effect seems mediated by: 1. Carbon dioxide Concentration. Recall the interaction of carbon dioxide and water: CO2 + H2O CO2 (aq) H2CO3 (aq) H+ + HCO3- + + CO3H therefore: low CO2 = hi pH = open hi CO2 = lo pH = closed 2. H+/ATPase proton pump. The pump is required for stomatal opening (see above). Protons are transferred from the cytosol into the apoplast. As protons are removed from the cytosol, the pH increases. E. Water - protects against excessive water loss. This is the prevailing and overriding control mechanism. There are two mechanisms by which water loss regulates stomatal closure, one of which is active and the other passive.

Hydropassive Control - simply put, as the plant looses water, the turgidity of the leaf cells, including guard cells, decreases and this results in stomatal closure. The plant is not "intentionally" closing the stoma, it is simply a consequence of drying out.

Hydroactive Control - this mechanism is one in which the plant actually seems to monitor its water status. When the water potential drops below some critical level, it engages a cascade of events that close the stomata. Presumably the plant is measuring pressure (turgor) and then synthesizes or releases an anti-transpirant that is translocated (moved) to the GC to cause closure.

The anti-transpirant is abscisic acid (ABA), one of the major plant growth regulators. It is active in very low concentration (10-6 M) and appears very rapidly after water stress (within 7 minutes). After 48 hours, the [ABA] increases nearly 50x. ABA comes from two sources: (a) root in response to water stress, the xylem sap pH increases which in turn stimulates the release of ABA into the xylem sap for transport to the leaves. This seems to be a root signal to the leaves that "water stress is coming"; and (b) leaves water stress stimulates a synthesis of new ABA and redistribution of existing ABA. Mechanism of Action: Treatment with ABA results in decrease of potassium, chloride and malate levels in the guard cells which in turn increase the water potential resulting in water efflux. Evidence suggests that there is an ABA receptor in the cell membrane. The receptor: (a) activates calcium channels in the membrane causing calcium uptake from the apoplast; (b) activates calcium channels in the tonoplast causing calcium release from the vacuole into the cytosol; (c) activates chloride (and malate) efflux channels; (d) inactivates potassium ion "in" channels; (e) inactivates the cell membrane proton pump; and (f) causes an increase in pH that activates potassium efflux channels. Thus, in short, ABA treatment causes an increase in cellular calcium levels which in turn results in decreases potassium and chloride levels and turns off the proton pump. F. Temperature Increased temperatures usually increase stomatal action, presumably to open them for evaporative cooling. If the temperature becomes too high the stomata close due to water stress and increased CO 2 that results from respiration. G. Wind Often causes closure because it: (a) brings CO2 enriched air; and (b) increases the rate of transpiration that causes water stress which causes the stomata to close. In some cases, wind causing stomatal opening to increase transpiration for cooling. XI. Physiology of Guard Cell Action - Part II Now, let's pull everything we've learned together to hypothesize a mechanism of action. First, there are a couple of other observations that we also need to reconcile with our mechanism:

1 2

Starch disappears in open stomata. Alkaline conditions favors the starch hydrolysis and acidic conditions favors starch synthesis. Starch hydrolysis is activated by blue light; and PEPcase activity is high in GC. Phosphoenolpyruvate carboxylase catalyzes the reaction: phosphoenolpyruvate (PEP) + HCO 3oxaloacetate (OAA).

XII. Grand Model. (see diagram; we will discuss in class) XIII. Why does transpiration occur?

1 2 3

Transport in plants. This is important to a small degree. Transpiration is certainly not a necessity. Heat loss (latent heat of vaporization) Carry nutrients in the soil to the plant

Perhaps plant cells need to maintain some optimal level of turgidity and this helps them do so. Solute Transport: Phloem Structure & Function

I. Definition Solute transport in plants, translocation, primarily occurs in the phloem, but it can occur in the xylem. II. Solute Transport in the Xylem

Some solutes are transported in the xylem Water and dissolved ions are the main substances in vessels/tracheids These materials are transported via transpiration stream Xylem sap may also contain organic materials, usually in relatively low concentration (with a notable exception being maple sap in the spring which is comprised of 2% or more sucrose). See table on overhead. Substances move at different rates depending on matrix effects, metabolic needs, etc.

III. Solute Transport in the Phloem

Phloem is difficult to study in plants because: (1) the transport cells/tissue in plants are small (microscopic) in comparison to the transport structures in animals; (2) there is a very rapid response of the phloem to wounding (contents under pressure); (3) transport in plants is intracellular (vs. extracellular in animals); and (4) the transport cells are alive. Phloem is the primary transport tissue for photosynthates (photoassimilates, or simply stated organic materials). Radiotracer studies in which leaves are briefly exposed to 14C-labeled carbon dioxide show that radioactive photosynthates are localized in the phloem. Aphids Don't Suck Kennedy & Mittler (1953) first noted that aphids could be used as a direct pipeline to the phloem. Phloem-feeding aphids stick their hollow, syringe-like stylet directly into phloem cells. Surprisingly, the phloem doesnt seal itself in response. Aphids don't suck; rather, the phloem contents are forced into the aphid (thus the phloem is under pressure) and the excess oozes out the anus (honeydew). Thus, aphid studies demonstrate that the phloem is under pressure. Further, the honeydew can be collected and we can identify its composition. Better yet, after anaesthetizing the aphid with CO2 the body is severed from the stylet leaving a miniature spile tapped directly into the phloem. Phloem Content (see table on overhead) Analysis - early studies to determine the content of the phloem involved cutting into the plant and analyzing the contents of the sap that was recovered. The problem is that you couldn't be sure that your sample wasn't contaminated by xylem exudates or other materials. Aphid studies described above helped to solve this problem. Phloem is rich in: 1. Carbohydrates - make up 16-25% of sap. The major organic transport materials are sucrose, stachyose (sucrose-gal), raffinose (stachyose-gal). These are excellent choices for transport materials for two reasons: (a) they are non-reducing sugars (the hydroxyl group on the anomeric carbon, the number one carbon, is tied up) which means that they are less reactive and more chemically stable; and (b) the linkage between sucrose and fructose is a "high-energy" linkage similar to that of ATP. Thus, sucrose is a good transport form that provides a high energy, yet stable packet of energy; 2. Amines/amides (0.04-4%) such as asparagine, glutamine, aspartic acid, ureides like ureas, citrulline, allantoin and allantoic acid. These compounds serve to transport "nitrogen";

3. ATP, hormones, sugar alcohols like sorbitol (apple, pear, prune) and mannitol (mangrove, olive), and an assortment of other organic materials; and 4. Inorganic substances including magnesium and potassium. Direction of phloem transport Information derived from several experiments; check out the Phloem Case Studies. (1) Classic girdling experiments (removing the bark of a woody plant) by Malphigi (1675) and Hales (1725) provided some of the earliest evidence. These experiments showed the accumulation of material above the girdle, and that carbohydrates were not translocated below the girdle. Thus, plants transport substances in the phloem downward toward the roots. (2) Sophisticated girdling experiments, using tracers like 32P, 13C, and 14C demonstrate that substances in the phloem are transported downward towards the roots OR upwards toward the shoot meristem. See data on overheads. (3) Aphids and tracers (see overhead) Conclusions - phloem transports organic materials from sites of production (called a source) to a site of need (called a sink). Thus, the typical direction of transport is downward from the primary source (leaves) to the major sink (roots). Rate of phloem transport Aphid experiments once again provide an answer...translocation rates average about 30 cm hour-1 or even faster. The phloem is under pressure Studies with aphids showed that the sap was "pushed" out of the plant suggesting the phloem is under pressure. More recent studies with sophisticated pressure probes have shown a pressure gradient from source to sink.

6 7

IV. Phloem Anatomy A. Cell types.

Sieve tube members or sieve elements. These cells are joined end to end to make a sieve tube. Theye are called sieve cells in gymnosperms. At maturity, these cells: (a) are alive, (b) have a functional plasma membrane and therefore are osmotically active/responsive; (c) no tonoplast or vacuole; (d) no nucleus, thus no DNA-directed protein synthesis, (e) few mitochondria or plastids; (f) the ER is primarily beneath plasma membrane and it is mostly smooth. Sieve elements are joined by sieve plates. These have numerous pores lined with callose ( 1-3 glucan). Callose forms rings around the pore, like a grommet. The wall region in the middle of the grommet hollows out and the membranes from the two adjacent cells are connected. Callose can plug the pore if the cell is damaged. The amount of callose observed varies with season, age, metabolism. Callose synthase is in the cell membrane.

Companion cells (angiosperms; albuminous cells - gymnosperms) These cells have a dense cytoplasm, mitochondria, nucleus, golgi, ER, chloroplasts - the standard goodies. Although their function is not well understood, they can be considered "nurse cells" to the sieve tube members. These cells are derived from the same cambial initial cell as the sieve tube members. There are three types of companion cells:

(a) "ordinary" with chloroplasts, few plasmodesmata connections to other cells except sieve elements, smooth inner walls, normal chloroplasts; (b) transfer more plasmodesmata, ingrowths in the wall to increase the S/V ratio; and (c) intermediary many plasmodesmata, vacuoles, undeveloped chloroplasts. The transfer and "ordinary" companion cells likely function to remove solutes from the apoplast Parenchyma cells These are vacuolated, storage cells. They help in lateral conduction and may help in transferring material to/from sieve cells. Transfer cells are specialized parenchyma cells. Fibers - primarily for support. Side note: Mesophyll cells in a leaf are close (perhaps 1-3 cells away) to a minor vein.

3 4 5

V. P protein

MW 14,000-158,000 Originally thought to be a carbohydrate and was called slime because it gelled when exposed to the air Various forms, bundles of fibers or amorphous areas or even crystalline Appear early in development of sieve elements Only in angiosperms at least two proteins, PP1 and PP2 Once the sieve pores form, the P-protein disperses through the pore. The protein is fibrous P protein plugs the pore when the cell is damaged. synthesized in companion cells

VI. Mechanism for phloem transport

Requirements The model must account for: (1) speed of transport. The process is much faster than simple diffusion. For example, a conservative estimate of the mass transfer rate in phloem is 15 g cm-2 hr-1. If the rate was based solely on diffusion is would be predicted to be 200 g cm -2 hr-1; (2) bidirectional flow - recall that substances can be transported down or up in the phloem; and (3) pressures in the phloem Pressure flow (or Bulk Flow) hypothesis of Munch. This is the best model that fits the data. The Model: Phloem transport is analogous to the operation of a double osmometer (see diagram). If solute is added to bulb A osmotic potential decreases osmotic uptake of water pressure increases bulk flow of water and solute to bulb B pressures increases in bulb water potential in B greater than in beaker osmotic flow of water into the beaker water returns to side A via the connection. This system could be maintained indefinitely if there is a mechanism to remove solute (sucrose) at the end (sink) and a mechanism to add solute (source). Sinks include young leaves, roots, developing fruits. Sources include mature leaves, cotyledons, endosperm, and bulbs and storage roots in spring. Sinks and sources can change depending upon the nutritional need of the plant. Thus, roots can be a source in the spring but are sinks for the majority of the growing season.

3 1

Plants as osmometers. If this model is valid then..... Sieve tubes should be continuous pipes...they are.

Sieve tubes should provide minimal resistance to flow. In other words, the sieve tubes shouldnt be clogged by P-protein. This is true in specimens that are rapidly prepared. However, this was a major concern in early experiments because the phloem always appeared clogged up in TEM pictures. Further, this explains why sieve tube members have few "typical" cellular structures - they would "get in the way." 3 The phloem should be under pressure. As the aphid experiments suggest....it is. In fact, mini-pressure gauges can be attached to a severed aphid stylet and the pressure can be measured. It varies from 0.1-2.5 MPa. Further, there should be a pressure gradient from source to sink (driving force for movement). There is...see overhead. 4 Sieve elements must have a membrane (for development of pressure gradients) - they do. 5 There should be an osmotic potential gradient from source to sink (there is...see overhead). The source region of the phloem has a considerably lower osmotic potential than the sink regions. 6 There must be a mechanism to load solutes from the source into sieve cells. This process must be active since the solutes (usually sucrose) are being loaded against a concentration gradient. Evidence - respiratory inhibitors block the process. The loading mechanism should be: Selective - it should only load the materials that are transported. This is supported by radiotracer studies; abraded leaves have been shown to only load materials that are normally transported; Allow for apoplastic (from protoplast to wall to protoplast) or symplastic (from protoplasts to protoplast via plasmodesmata) transport. In some species, sucrose transport is symplastic - from mesophyll protoplast to cc-se protoplast via plasmodesmata. In others, sucrose loading into the cc-se complex involves an apoplastic step (mesophyll protoplasts to apoplast to cc-se protoplast. Provide a mechanism to transport sucrose across the membrane - the sucrose/proton cotransport system. According to this model, protons are pumped out of the sieve cells into the apoplast by a membrane-bound H+-ATPase the proton concentration increases in the apoplast pH decreases K+ is brought into the sieve cell to balance the charge the proton gradient provides the driving force for transporting sucrose against a gradient the sucrose and protons bind to a carrier protein in the membrane and are released in the sieve tube member. Evidence: the pH is high in sieve tubes; if the pH of the apoplast is increased there will be no sucrose uptake; there is a hi potassium conc. in sieve tube members. A membrane carrier is likely involved since PCMBS (p-chloromercuribenzene sulfonic acid), an inhibitor of membrane proteins, interferes with sucrose uptake. 7 There must be a mechanism to unload solute at the sink. Sucrose is unloaded into the apoplast in some tissues (i.e., ovules) and into the symplast of others (growing/respiring tissues like young leaves, meristems). 8 Apoplastic transport and unloading can occur via two methods: (a) sucrose is hydrolyzed by acid invertase to glucose and fructose upon reaching the sink. This maintains the gradient for transport. The glucose and fructose are taken up by the sink cells and stored or further metabolized as in maize; or (b) sucrose is unloaded into the sink by a carrier co-transport system like in sucrose loading. 9 The empty ovule technique has been useful in these studies. 10 Some metabolism is required (for loading/unloading) and to maintain sucrose against a concentration gradient. This explains the response to respiratory inhibitors. Phloem transport is also inhibited by anoxia and cold temperatures - both thought to exert their effect through energy metabolism. VII. Problems with the model

Bidirectionality - how can phloem translocate materials in two different directions at once? It cant, at least not within the same sieve tube. However, presumably sieve tubes within a single vascular bundle could be transporting in opposite directions assuming each is acting appropriately.

Plant Growth Substances/Hormones I. Plant Hormones - do they exist? You betcha. In class we will provide some case studies, including early experiments by Darwin (1880), Boysen-Jensen (1913), Paal (1918) and Went (1923) involving phototropic curvature in canary grass coleoptiles and more recent work concerning fruit set in soybean. Conclusion: from these studies, and countless others, we conclude that plants possess a welldeveloped system of chemical messengers that induce (inhibit or promote) growth and developmental responses. These chemical messengers are termed "hormones". II. Plant hormones - What are they? They are defined as:

1 2 3 4 5 6

small organic compounds; synthesized by the plant; active in low concentration (<10-6); promote or inhibit growth and developmental responses; often show a separation of the site of production and the site of action (although this isnt as clear a distinction as in animals).

Based on these criteria, would the following substances be considered hormones? Ca2+, sucrose, 2 4 - D, glycine, or K+? III. Plants vs. animals There are a few significant differences in the nature of hormones found in plants and animals. These are summarized in the following table:

Plants number of hormones specificity of action work together site action/production separation fewer non-specific yes no

Animals many specific no yes

Thus, there are comparatively few plant hormones, each elicits a variety of responses and often works together with other hormones. In contrast, animals have numerous different kinds of hormones, each with a specific function, and it works alone to induce a response. Why the differences? Good question. It may be partly a function of our ignorance; in other words, there are likely to be many more plant hormones that simply havent yet been identified. Nevertheless, I suspect that at least part of the reason for the differences is related to body design. Recall that plants have an architectural design with a limited number of parts that are repeated. It follows that only a limited number of hormones would be necessary to induce growth/developmental responses. With a mechanical design and numerous separate parts, animals required unique chemical messengers to interact with each one. IV. Plant hormones

There are five major groups, based on chemical structure. With the exception of the latter two, each group represents a family of related compounds. These groups are: (1) auxins; (2) gibberellins; (3) cytokinins; (4) abscisic acid; and (5) ethylene. In addition, there are a variety of other plant "hormones" including the brassinosteroids, oligosaccharides, polyamines, jasmonic acid, salicylic acid, systemin, and putative hormones like those involved in flowering (florigen). V. How do we know a substance is a hormone? In the past, physiologists simply applied the substance to a plant or excised part to see if it caused a response. If there was a response, then we assumed that the substance was a hormone involved in the response. However, responding to an exogenous application of a hormone doesnt necessarily mean that the substance has any endogenous (in vivo) action. Thus, weve become a little more picky and only accept that a substance is a hormone if:

1 2 3 4

Exogenous application causes the response; Lowering endogenous levels prevents the response; Lowering endogenous levels followed by exogenous application restores the response; Endogenous levels should be related to the response (i.e., increase before the response occurs).

Check out the case study concerning bolting in Agrostemma lithago (Jones and Zeevart, 1980, Planta 149:269). VI. Mechanism of hormone action Hormones act on target tissues to activate a receptor. The general mechanism is: hormone target tissue/cell receptor signal amplification response Thus, for a response to occur:

1 2 3 4 5

the hormone must be present in sufficient quantity; the target tissue must be sensitive (sensitized) to the hormone; the target tissue recognizes the hormone (i.e., there must be a receptor to which the hormone can bind); the binding of the hormone/receptor should initiate a change in the receptor (amplification). Calcium is often involved and its interaction is mediated by the protein calmodulin; and the activated receptor initiates a physiological response

VII. Techniques to study hormones A. Bioassays A bioassay examines the effect of a test substance on a plant tissue. To perform a hormone bioassay, a test plant is chosen that lacks the hormone for a response. Known amounts of hormone are added to the plant, the response is measured, and a "standard curve" is produced. To determine if a sample contains the hormone, the test plant is treated in a similar fashion. If present the hormone can be quantified by comparing its response to the samples of known concentration. For example, a variety of rice (Tauginbozu) lacks GA and is often used in bioassay studies. After treatment with various concentrations of GA, leaf length vs. [GA] is plotted.

Table: Comparison of the advantages and disadvantages of bioassays Advantages simple & easy inexpensive

Disadvantages sensitive to impurities false positive tests can result lower sensitivity than other methods

B. Immunological studies Antibodies are made against the plant hormones and then used as specific probes to localize and quantify. The antibodies are usually coupled to radioisotopes or fluorescent dyes to make it easier to trace. This technique is very sensitivity and specific. C. Instrumental Methods: GC-MS; HPLC; high specificity and sensitivity VIII. Methods for regulating endogenous levels The internal levels of plant hormones must be tightly controlled so that the response occurs only at the appropriate time. Regulation of hormonal action is achieved by: (1) controlling the rate of hormone synthesis; (2) forming conjugates, which are inactive storage forms where the hormone is covalently bonded to a sugar, amino acid or other molecule; (3) enzyme degradation; (4) transporting the hormone away/toward the site; and (5) compartmentalizing the substance in an organelle such as the chloroplast. IX. Hormone Studies We will study each of the major groups of hormones. For each we will focus on:

1 2 3 4 5 6 7 8 9 10

general structure/chemistry biosynthesis (pathway, site, precursors) regulation of endogenous levels bioassays mode of transport (tissue, direction) physiological actions/roles (especially important) antagonists/inhibitors mechanism of action,, receptors commercial applications (if any) cool stuff Plant Hormone Case Studies - The Experimental Evidence

I. Case Study: Phototropism and Coleoptiles. The phototropic response of coleoptiles to the light has long fascinated biologists. The following diagram show a summary of some of the experiments that have been performed? List at least four conclusions that can be made from these experiments.

II. Case Study: Soybeans and Fruit Set. Soybeans (Clark E1t)produce racemes of several flowers beneath a leaf (see diagram). Flowers were removed from some to investigate factors that influenced which pods set (mature seeds form inside) and which ones abort (fall off). The data are provided below. List at least three conclusions from these data.

Treatment none remove flowers 1 - 3 remove flowers 1 - 3, add extract of these to their "holes"

Position 1 - 3 most set -----

Position 6 - 8 most abort most set most abort

III. Case Study - Flowering in Corn-cockle Corn-cockle (Agrostemma githago) is a weedy plant that "bolts" when it flowers. In other words, it produces a flowering stem that rapidly elongates. Corn-cockle only bolts/flowers under long day (LD) conditions. Which hormone(s) is involved in this rapid growth response? Jones and Zeevaart (1980) hypothesized that gibberellic acid (GA) causes the bolting response. They grew corn-cockle under long day (LD) and short day (SD) treatments. Some plants were treated with GA20, one of the gibberellins. Other plants were also treated with with a GA synthesis inhibitor, Amo-1618. Their data

are summarized below. Stem length was measured 25 days after the first long day. GA was applied six times, every other day, 1.2 l.

Treatment

Stem Length (cm)

SD 2.2 SD + GA20 11.2 SD + GA20 + Amo-1618 10.2 LD 36.7 LD + Amo-1618 2.9 LD + GA20 + Amo-1618 30.2 They also measured GA levels and stem height for several days after long day treatment. These data are reproduced below:

Analysis: Study the data/experiment. Do these data suggest that GA is involved in bolting in corncockle? Cite data to support each of the four major criteria for proving a chemical is a hormone. Exogenous applications of the chemical to the target tissue brings about the developmental change. 1. Which data show that exogenous application of hormone stimulates the response? Be specific. Lowering the endogenous levels of the chemical (using chemicals or genetic manipulation) presents the developmental change from occurring under conditions that would normally bring about the change. 2. Which data show that reducing the endogenous levels of hormone prevents the response? Be specific. If the endogenous levels are lowered, adding the chemical exogenously will cause the developmental change. 3. Which data show that reducing the endogenous levels and adding back the hormone exogenously restores the response?

During normal development the endogenous levels of the chemical rise in the appropriate tissue before the developmental effect is observed. 4. Which data show that the hormone levels appear and peak before the response? Plant Hormones - Auxin I. Introduction Auxin is a general name for a group of hormones that are involved with growth responses (i.e., elongate cells, stimulate cell division in callus). Not surprisingly, the term "auxin" is derived from the Greek word "to increase or grow". This was the first group of plant hormones discovered. II. Chemistry/Structure A. Naturally Occurring Auxins The most important auxin found in plants is indole-3-acetic acid (IAA). IAA is comprised of an indole ring linked to acetic acid (see overhead). Other auxins that have been isolated from plants include indole ethanol, indole acetaldehyde, indole acetonitrile, phenylacetic acid (PAA), and 4-chloroindoleacetic acid. These are probably converted to IAA in vivo. B. Synthetics with Auxin Activity There are a variety of substances that are not known to occur in plants that have auxin activity. These include indolebutyric acid (IBA); naphthalene acetic acid (NAA); 2,4- dichloro-phenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The exact mechanism of action of these compounds is not known, but they may inhibit nucleic acid synthesis. C. Chemistry of action Although a variety of molecular structures have auxin activity (i.e., derivatives of indole, benzene and naphthalene), these molecules seem to share a few features that appear to be required for activity. Auxin activity seems to be correlated with a flat, hydrophobic ring system that separates negatively-charged (acidic, carboxyl group) side chain and positively charged region. There is a charge separation of about 0.5 nm. Note that the indole unit isn't required for activity, but a planar ring system is. D. Conjugated forms Auxins, as do other hormones, occur in a free or conjugated (bound to sugars, alcohols or other molecules) form. In fact, up to 98% of the auxin may be bound. Auxins may be conjugated with inositol, coenzyme A or glucosides (sugars). E. Anti-auxins PCIB (p-chlorophenoxyisobutryic acid) is a compound that competes with auxin for binding sites. However, it doesn't cause any growth response. III. Biosynthesis A. Site Auxin is made in actively growing tissue which includes young leaves, fruits, and especially the shoot apex. Made in cytosol of cells B. Routes There are two major routes to the production of IAA.

Tryptophan-dependent Pathways. The similarity of chemical structure of IAA and tryptophan suggested a connection between these. Considerable research has shown that tryptophan, one of the protein amino acids, is a precursor of auxin biosynthesis. Overall, the conversion of tryptophan to IAA can occur by: (1) a transamination followed by a decarboxylation; (2) a decarboxylation followed by a transamination; or (3) formation of IAA via an oxime (C=NOH) and nitrile (CN). Tryptophan-independent Pathway - this route doesn't involve tryptophan directly as an intermediate to the formation of auxin.

IV. Transport A. Basipetal (or Polar) Transport Auxin is transported in a basipetal (towards the base, base-seeking) direction. In other words, auxin moves from the shoot tip towards the roots and from the root tip towards the shoot. Evidence - (1) Seedling vs. [auxin]; and (2) 14C labeled IAA applied to the top of a stem section is recovered only from the bottom of the stem section. When auxin is applied to the end of stem segments, it is only transported from the "top" of the section to the "bottom" as demonstrated in these data:

Uprig ht
14

Upside down 10,000 300

C-IAA in donor block (dpm)C-IAA in donor block (dpm)

14

10,00 0 7,500

C-IAA in receiver block (dpm)C-IAA in receiver block (dpm) B. Mechanism of polar transport

1 2 3

Transport Rate IAA is not transported through the transpiration stream or phloem because the rate of movement is too slow. The rate of transport is consistent with diffusion. Tissue of transport - appears to occur in parenchyma cells associated with the vascular tissue. Model for polar transport: (a) Protons are moved out of the cell by a proton pump that requires ATP; (b) IAA is protonated at low pH; (c) IAA-H passes through the lipid membrane. It can enter or leave anywhere; (d) once inside the cell, the IAA-H ionizes in response to the higher pH; (e) IAA- requires a permease to pass through the membrane; (f) histochemical studies have shown that a permease is only located at the bottom of the cell - resulting in a net movement of auxin out of the bottom of the cells. Evidence for polar transport model: (a) transport is blocked by respiratory poisons (i.e., demonstrates the need for ATP and a proton pump); (b) unlabeled IAA competes with C 14 labeled IAA for uptake in to the cells - this suggests that a carrier of some sort is required ; (c) fluorescein-labeled antibodies show that the permease is localized at bottom of cells; and (d) the transport of auxin is blocked by NPA (napthylthalamic acid), TIBA (triiodobenzoic acid) and flavanoids. These inhibitors appear to block the permease. Proton-Auxin CoTransport Mechanism - In addition to the passive pH-dependent mechanism described, there a membrane transport protein seems involved that cotransports protons and IAA into the cell. This tranport protein is localized in the upper side of the cells.

V. Bioassays

There are four classic bioassays for auxin. These tests, which are all based on the ability of auxin to stimulate shoot growth (or inhibit root growth) are: A. Avena coleoptile curvature test Pioneered by F. Went. The angle of curvature of a decapitated oat coleoptile is measured after placing an agar block containing auxin on one side. Then, angle of curvature vs. [IAA] is plotted. B. Avena coleoptile elongation The ratio of final length/original length for oat coleoptiles or pea stem sections is measured after the tissues are floated in solutions containing different concentrations of IAA. Elongation vs. [IAA] is plotted. C. Split pea curvature test A section of pea hypocotyl is obtained and split halfway down the middle. After incubating the sections in solutions of known IAA concentration, the angle of curvature is measured. Note that only the epidermal cells are responding to the treatment. D. Cress root inhibition This bioassay is based on the ability of auxin to stop root growth. A ratio of treatment/control growth is plotted vs. log [IAA]. VI. Auxin responses IAA is involved in the following responses: A. Cell elongation and wall relaxation Discussed earlier in the semester. Basis of several bioassays and the discovery of auxin. NOTE: Normally exogenous application of IAA has little effect on plants.

B. Cell differentiation Promotes differentiation of vascular tissue (i.e., xylem & phloem).

C. Ethylene production IAA apparently stimulates the production of ethylene.

D. Inhibition of root growth [IAA] > 10-6 M inhibit root elongation. However, very low (>10 -8 M) favor root elongation.

E. Stimulate root initiation (lateral roots, adventitious roots) Roots always form at the basal end of cutting F. Flowering Although most plants dont initiate the production of flowers after auxin treatment, pineapple and its relatives (Bromeliaceae) do. Once flowers are initiated, in many species, IAA promotes the formation of female flowers, especially in cucurbits (gourd family).

G. Parthenocarpic fruit development - as per RCBr Lab H. Apical dominance The apical meristem (apex) controls or dominates the development of the lateral buds. (PS - a bud is an embryonic shoot with immature leaves and stem). Apical dominance also occurs in roots. It is responsible for the Xmas tree shape of many trees and prevents an individual from becoming too topheavy.

1 2

Function: (a) maintain upward growth; (b) maximize light absorption; (c) prevent top-heaviness; and (d) provide mechanism to replace the apical bud if it is removed by grazing or damage. Thimann & Skoog (1934) - first suggested a correlation between [IAA] and apical dominance. This idea was further developed by Cholodny-Went who proposed that plant tissues responded to different concentrations of IAA (see figure). For example, hi [IAA] stimulates shoot growth but inhibits bud and roots. Thus, IAA is produced at the tip of the plant and is transported downward. The high concentrations near the apex inhibit lateral buds. As the concentration decreases it frees the buds from the inhibition and they develop.

Evidence:

1 2 3
But:

every gardener knows that removing the apical meristem results in bushier plants. This is consistent with the Cholodny-Went hypothesis. If IAA is applied to a de-tipped plant it prevents lateral bud development IAA stimulates the formation of ethylene which is known to inhibit lateral bud formation. branching mutants of tomato don't export 14C-IAA

1 2 3 4 3

[IAA] in buds after the tip is removed should decrease, and they don't [IAA] that act to replace the tip are much higher than levels in the intact plant 14 C-IAA doesnt enter the lateral buds when applied at tip Some plants dont respond to exogenous IAA Nutrient diversion hypothesis There is little development of vascular tissue to the lateral buds. This has lead some to propose that the lateral buds fail to develop because they get no nutrients because the nutritional traffic is being diverted to the apex. Evidence: (a) application of cytokinins stimulates lateral bud development; cytokinins known to direct nutritional traffic, especially during senescence; (b) tomato mutants that exhibit strong apical dominance have lower levels of cytokinin; (c) there is a good correlation between cytokinin level and bud development. Bud Inhibition Another possible reason why buds fail to develop is because they have an inhibitor. It has been shown that ABA inhibits bud and seed development in many species. The buds of decapitated plants have a lower [ABA] than intact plants. High [ABA] in the bud are maintained by applying IAA to decapitated apex.

I. Tropic responses - such as gravitropism and phototropism J. Abscission Formation of the abscission layer is correlated with the [IAA] in leaf. As long as the [IAA] in the leaf is high relative to the stem, then the abscission layer doesnt form. When the [IAA] in the leaf drops, which occurs normally during the growing season, it stimulates the formation of the abscission layer

forms and the leaf falls. The IAA presumably works by decreasing the sensitivity of the cells to ethylene which is the primary hormone involved in initiating leaf drop. VII. Regulating Endogenous Levels All of the general methods (i.e., degradation, conjugates, regulate rate of synthesis, sequestering in different regions) of regulation are probably operative. In particular, IAA is probably shuffled back and forth between the two main areas, or pools, where it is stored the cytosol and chloroplast. IAA oxidase, a type of peroxidase, converts IAA to inactive metabolites. There are several degradative pathways. PLANT HORMONES - GIBBERELLINS I. General

"foolish seedling" disease (bakanae) - rice plants tall, weak and spindly with little grain - was observed early this century by Japanese farmers. associated with the fungus, Gibberella fujikuroi. liquid from the culture medium applied to plants caused symptoms (Kurosawa, 1926). three gibberellins were isolated and identified from the medium & fungus independent confirmation in labs in Europe and US. gibberellins known from angiosperms, gymnosperms, ferns, mosses, algae, fungi and even a few bacteria.

II. Chemistry

diterpenes (C20, though some have lost a carbon and are C19) based on isoprene skeleton characterized by having a complex system of 4-5 rings (ent-kaurene ring system) and a carboxyl (acidic) side chain more than 110 different gibberellins are known - abbreviated GA1...GAn. No more than 12 or so different gibberellins occur in any one species only a few (about 15) of the GA's have biological activity; the rest are likely breakdown products of, or precursors to, the active ones GA1 is the most active GA GA3 was the first one discovered and is readily available commercially (produced by G. fujikuroi cultures). C19 GA's are more active than C20 GA's can occur in a free or conjugated form (i.e., glucosides)

III. Biosynthesis A. Site - young leaves, roots, and developing seeds (developing endosperm) and fruits. B. Pathway

terpene pathway basic terpene building block is isoprene (isopentenylpryophosphate, IPP), a five carbon unit five major steps: (1) synthesis of IPP; (2) condensation of 4 isoprene units to form geranylgeranylpyrophosphate (GGPP); (3) GGPP cyclizes to form the kaurene ring system; (4) a methyl group of kaurene is oxidized to a carboxyl group to form GA12-aldehyde; (5) GA12aldehyde is the precursor to the other GA's two routes to the production of IPP (isoprene): (1) Mevalonate-dependent pathway - occurs in cytosol; mevalonate is especially important for sterol biosynthesis; mevalonate is derived from

acetyl CoA; and (2) Mevalonate independent pathway especially in plastids, important for carotenoid biosynthesis C. GA synthesis inhibitors

Kaurene synthesis inhibitors - Phosphon D, CCC (cycocel), and Amo1618. Kaurene oxidation inhibitors - Ancymidol (A-rest) and paclobutural Another inhibitor is B-Nine (alar).

IV. Bioassays/Analysis A. Three common bioassays used for gibberellin are:

Lettuce hypocotyl elongation Dwarf rice (var. Tanginbozu rice) leaf sheath elongation alpha-amylase production in barley

B. Instrumental methods now the method of choice, especially GC-MS V. Transport

made in the tissue in which it is used transport occurs through xylem, phloem, or cell-to-cell. phloem seems to be most important transport route transport is not polar, as it is for auxin. not purely diffusion.

VI. Disposal/Regulation of endogenous levels

Formation of conjugates Slow hydrolysis

VII. Actions A. Promotes stem elongation When applied to intact plants, GA usually causes an increase, unlike auxin. It overcomes dwarfism in mutants that have a mutation in the GA synthesis pathway. dwarf = short; wild type = tall; dwarf + GA = tall. Thus, GA application: (1) stimulates elongation; and (2) acts on intact plants. GA stimulates stem elongation by: stimulating cell division. Specifically, GA increases the transition from G1 S phase of the cell cycle; 2 increasing amylase (and other hydrolytic enzymes) production increases hydrolysis of starch provides glucose and other sugars that: (a) lower water potential which provides driving force for water uptake; (b) provide energy through cell respiration; and (c) provide materials for building cell walls; and 3 increase cell wall plasticity (by a mechanism other than how auxin works). B. Overcomes dormancy in seeds and buds

Treating dormant seeds with GA stimulates germination (see below) C. Involved in parthenocarpic fruit development - remember lab D. Flowering Recall the hormone exercise we did? To summarize: LD = tall (bolts); SD = short; SD + GA = tall. Thus, GA stimulates bolting in Long Day plants and can substitute for long days or cold treatments that are necessary for flowering. E. Mobilization of food reserves in grass seed germination GA is produced by the scutellum (cotyledon) of the embryo stimulates the production of amylase by the aleurone layer amylase hydrolyzes starch to simple sugars absorbed by scutellum and translocated to embryo for growth. The production of amylase occurs de novo. That is, gibberellin stimulates transcription. In short: GA binds to membrane receptor interacts with a protein complex (heterotrimeric G protein) that activates a GA signaling intermediate turns off a repressor transcription of GA-MYB mRNA translated in cytosol to make GA-MYB protein returns to nucleus to bind to alpha-amylase gene promoter region activates transcription of alpha-amylase mRNA translated in ribosomes on RER transported to golgi secretory vesicles release alpha-amylase. This last step is apparently regulated by a calcium dependent mechanism that was also activated by the heterotrimeric G protein complex. Brewers take advantage of GA's ability to stimulate germination and enzymes which are important in the brewing process. F. Juvenility Plants exist in a juvenile and adult form. As in humans, the main difference is whether the plants are able to flower (reproduce). In some plants there is little morphological difference between juvenile and adult forms, whereas in others, the two forms are very distinct. For example, in beans, the first (juvenile) leaves are entire (heart-shaped) while the adult leaves are trifoliate. Lancewood is a New Zealand plant that has very distinctive juvenile and adult forms. In fact, they are so different that botanists originally mistook the two forms for different species. The juvenile form is unbranched with long (ca. 12 in) linear, drooping leaves that look a little like the ribs of a folded umbrella. When the plant reaches about 15 foot it switches to the adult form which is branched and has smaller, ovate leaves. It has been suggested that this is a modification to prevent predation by moa, a large, formerly common but now extinct bird. Gibberellin stimulates the transition between the juvenile and adult forms. In ivy, the adult form (unlobed leaves, shorter internodes) is converted to the juvenile form (lobed leaves, longer internodes) by GA treatment. G. Sex expression In plants with separate male and female flowers, GA application can determine sex. For example, in cucumber, hemp and spinach, GA treatment increases the proportion of male flowers. In maize, GA treatment causes female flower development. IX. Commercial Applications increase size of grapes (spray at time of blooming and fruit set stage) increase distance between grapes in a cluster to minimize fungi/disease Breweries - increase starch digestion for malting process Delay senescence - spray on fruit like naval oranges

celery stalk elongation (but must use carefully because of increased storage problems) sugar cane increased growth and yields Minimize lodging Plant Hormones - Cytokinins I. General

Called "cytokinins" because they stimulate cell division (i.e., cytokinesis) Haberlandt (1913) noted that non-dividing potato parenchyma cells would revert to actively dividing ones in the presence of phloem sap. This observation suggested a soluble material was responsible for cell division. Folke Skoog (1940's) and colleagues at Univ. of Wisconsin found that cultured tobacco pith tissue explants would proliferate only if they were supplemented with various substances such as autoclaved herring sperm or coconut milk. Miller (1956) identified the first cytokinin, called kinetin, in the herring sperm. Cytokinins occur in most plants including mosses, ferns, conifers, algae and diatoms

II. Chemistry A. General

adenine derivatives (amino purines) occur as: (a) the free nitrogenous base; (b) a nucleoside (base + ribose); (c) a nucleotide (base + ribose + phosphate); or (d) glycosides The free base is the active form. approximately 40 different structures known. Zeatin (Z), which was first isolated from maize (Zea mays) is the most common cytokinin. Other naturally occurring cytokinins include, dihydrozeatin (DHZ) and isopentenyladenosine (IPA).

B. Synthetic cytokinins

kinetin (as above) probably byproduct of zeatin degradation there are several other substances with cytokinin activity such as benzyl adenine (benzylaminopurine; BA).

C. Cytokinins and nucleic acids

can occur as a modified base in tRNA, but the bases exist in the cis form, rather than the typical trans form. These modified bases that are found in all organisms from bacteria to plants to humans. The function of the tRNA cytokinins is not clear, but after hydrolysis of the tRNA the products can act as a cytokinin. The importance of the tRNA derived cytokinins in overall growth and development is not clear, either. Interestingly plants have different sets of tRNAs with different cytokinins that participate in protein synthesis in the cytoplasm and the plastids.

III. Synthesis

Site: synthesized primarily in the meristematic region of the roots. This is known in part because roots can be cultured (grown in artificial medium in a flask) without added cytokinin, but stem cells cannot. Cytokinins are also produced in developing embryos and crown gall tissues first major precursor to the cytokinin is AMP (adenosine monophosphate) side chains of the cytokinins are made by the terpene pathway (IPP isoprene) and added to the AMP by cytokinin synthase there is some speculation that surface and endophytic bacteria (i.e., Methylobacterium) may be the actual source of plant cytokinins. Rationale: (1) haven't isolated some of the putative genes for cytokinin synthesis; (2) remove bacteria show impaired cytokinin production. tRNA nucleotides are modified to cytokinins after the tRNA is transcribed (post-transcriptional processing)

IV. Transport

via xylem (transpiration stream) in peas, a signal from the leaves may signal/regulate transport of cytokinins from the roots zeatin ribosides are the main transport form; converted to the free base or glucosides in the leaves some cytokinin also moves in the phloem.

V. Bioassays/Analysis A. Bioassay

1 2 3

Callus culture cell proliferation - not used too much because it takes too long Expansion of radish or cocklebur cotyledons Inhibition of chlorophyll loss by detached oat leaves during senescence

B. Methods of Analysis liquid chromatography, mass spectroscopy; radioimmunoassays VI. Disposal

forms conjugates with glucosides. major storage form of cytokinin, inactive cytokinin oxidase may be an important route of disposal, too.

VII. Actions A. Control morphogenesis

in plant tissue cultures, cytokinin is required for the growth of a callus (an undifferentiated, tumor-like mass of cells):

callus + auxin + no cytokinin little growth of callus callus + auxin + cytokinin

callus grows well, undifferentiated

ratio of cytokinin and auxin are important in determining the fate of the callus:

callus + low [cytokinin/auxin] callus grows well, forms roots callus + high [cytokinin/auxin]

callus grows well, forms meristem & shoots

some tissues become habituated during repeated cell culture loose the requirement for cytokinin in the growth medium

B. Crown Gall

tumor-like mass of undifferentiated cells that typically occurs near the crown (junction of root and stem) of the plant caused by the bacterium Agrobacterium tumefaciens carries a plasmid (Ti plasmid; a plasmid is a small loop of non-chromosomal DNA) with loci/genes for auxin production (tms), zeatin production (tmr) and opines (are nitrogen-containing molecules that provide food for the bacteria). upon infection, the plasmid is incorporated into the plant cell genome which begins to overproduce auxin and cytokinin stem forms an undifferentiated tumor (crown gall) as predicted, if the tms genes (auxin production) are deleted from the plasmid, which would increase the cytokinin/auxin ratio, the resultant crown gall is "shooty". If the tmr genes are deleted the gall is "rooty".

C. Regulates the cell cycle/cell division (hence, the name "cytokinins) especially by controlling the transition from G2 mitosis. This effect is moderated by cyclin-dependent protein kinases (CDK's) and their subunits, cyclins. D. Delay senescence

senescence is the programmed aging process that occurs in plants (and other organisms for that matter). loss of chlorophyll, RNA, protein and lipids. cytokinin application to an intact leaf markedly reduces the extent and rate of chlorophyll and protein degradation and leaf drop correlation between cytokinin levels and senescence. For example, as detached leaves senesce the cytokinin levels drop. And, when these leaves are treated with auxin to stimulate rooting, when roots form the senescence process stops and cytokinin levels rise. Tobacco plants + senescence promoter sequence + ipt gene (cytokinin gene) causes gene to become active on senescence no senescence

The exact mechanism by which this occurs is unclear but likely involves the ability of cytokinins to mobilize nutrients. Application of cytokinin to a leaf will cause it to act as a sink and nutrients will be directed towards it. E. Greening Promotes the light-induced formation of chlorophyll and conversion of etioplasts to chloroplasts (greening process). F. Promote lateral bud development Cytokinin application to dormant buds will cause them to develop. A witches broom is caused by a pathogen such as the bacterium Corynebacterium fascians (or A. tumefaciens) that produces cytokinin which, in turn, causes stimulates lateral bud development (branching). These results suggest that apical dominance may be related to cytokinin, too. For example, when tobacco cells are infected with the Ti-plasmid that has been modified to possess the heat shock promoter, a heat treatment stimulates the cells to produce increased amounts of cytokinin. These plants exhibit less apical dominance and remain green longer than non-heat treated

controls. Thus, these results support the conclusion that senescence and apical dominance are related to cytokinin levels. G. Promote cell expansion Cytokinins stimulate the expansion of cotyledons. This is the basis for the classical bioassay. The mechanism is associated with increased plasticity of the cell wall, not associated with acidification. VIIII. Mechanism of action - Specific binding sites (receptor) for cytokinin are known. These may be ribosomal proteins. Thus, it is not too surprising that cytokinins have been shown to regulate protein synthesis. Abscisic Acid (ABA) I. General

Bennet-Clark and Kefford (1953) described the presence of an inhibitor of coleoptile elongation in oats about 10 years later, Addicott et al. found a substance that stimulated abscission of fruits in cotton and they named it abscisin II about the same time, Wareing found a substance in sycamore leaves that promoted dormancy in buds and called it dormin it was soon clear that these were the same substance a conference in 1967 straightened out the name and it was decided to call the hormone abscisic acid (ABA).

II. Chemistry

a single structure, not a family of related structures like the gibberellins sesquiterpene (i.e., terpenoid) - C15 - made from 3 isoprene units occurs as cis form; S-enantiomer is natural and active form found in all green plants, also in some mosses, algae, and fungi related to lunularic acid which is found in liverworts.

III. Biosynthesis

plastids most tissues, especially leaves and seeds terpene pathway IPP is the first intermediate; not derived from mevalonate (mevalonate-independent); comes from intermediates of glycolysis ABA derived from the breakdown of carotenoids. Evidence: maize mutants blocked in carotene synthesis low levels of ABA vivipary (see below) Pathway: IPP farnesyl pyrophosphate carotenoids (C40; violaxanthin) xanthoxin ABA

IV. Bioassays/Analysis A. Bioassays there are several including:

inhibition of seed germination inhibition of GA induced alpha-amylase production

B. Analysis Gas chromatography, HPLC, and immunoassay

V. Disposal/Regulation

rapid changes in endogenous levels (up to 100x within a few days) ABA levels can be regulated by: (a) degradation; (b) compartmentalization; (c) transport; (d) conjugation to a sugar or other molecule; and (e) conversion (oxidation) into phaseic acid and dihydrophaseic acid.

VI. Transport - xylem and phloem (greater amounts) VII. Actions A. Growth Inhibitor Widespread growth inhibitor; often antagonistic of GA actions B. Maintains or "seals in" bud and seed dormancy (i.e., prevents germination) In fact, ABA is made during the terminal stages of embryo development. Among it's roles in seed dormancy is to: (1) provide desiccation tolerance of the embryo by promoting synthesis of proteins involved in the process; and (b) promote accumulation of seed storage proteins. C. Prevents vivipary Development of the embryo without a dormant period. Some evidence: viviparous mutants have reduced [ABA]; and fluridone stimulates treatment stimulates vivipary (fluridone is an inhibitor of carotenoid biosynthesis that blocks ABA production) D. Inhibits auxin induced growth (seems to block the H+ pump) E. Stomatal closure under water stress (remember our unit on gas exchange?) F. Abscission & senescence Involved, though perhaps only a minor role

VIII. Mechanism of action A. Effects on plasma membrane B. Inhibits protein synthesis C. Regulation of genes (transcription)

Ethylene I. General

the Chinese may have been the first to observe the effects of ethylene when they noted that burning incense increased fruit ripening in 1864 leaks in gas lights in street lamps were reported to stunt plant growth and defoliate trees in 1901, D. Neljubow realized that his dark-grown pea seedlings were short, fat and negatively gravitropic (the triple response) because of a component in "laboratory air" which he subsequently identified as ethylene Cousins (1910) first reported that ethylene occurred in plants.

II. Chemistry

single compound (like ABA) and is not a family of related ones (i.e., gibberellins) CH2=CH2 ethylene (MW 28) is similar in size/shape as water a gaseous plant hormone

III. Biosynthesis A. General:

made by most plants including angiosperms, gymnosperms, ferns, mosses, liverworts also synthesized by fungi and bacteria made by all parts of the plant meristematic regions (shoot apex) and senescing tissues are rich sources nodes make more ethylene than internodes ethylene production is stimulated by physiological stresses including wounding, anaerobic conditions, flooding, chilling, disease and drought. during the climacteric which is the sudden surge of respiratory activity that occurs at the peak of ripening in many fruits - lots of ethylene is made

B. Pathway of synthesis

1 2 3

the first precursor is methionine (one of the protein amino acids) methionine + ATP S-adenosyl-methionine (SAM) ACC synthase amino cyclopropane carboxylic acid (ACC) ACC oxidase CH2=CH2 + HCN (hydrogen cyanide) + CO2. To summarize the highlights: ATP reacts with methionine to form SAM SAM is essentially a carrier form of methionine (it is involved in other reactions in the cell) ACC synthase is the most crucial enzyme in the pathway. It is the rate limiting step and induced by: (a) fruit ripening; (b) flower senescence; (c) in response to IAA; (d) under wounding; (e) chilling injury; (f) drought; (g) flooding; (h) in response to ethylene ("one bad apple spoils the whole bunch"). ACC synthase is cytosolic and coded by a multi-gene family. methionine is re-claimed via the Yang cycle.

5 6

ACC oxidase was formerly called "ethylene forming enzyme, abbreviated EFE." It requires Fe2+ and ascorbate for activity which explains why it took awhile to characterize this enzyme. It is also the product of a multigene family. One of the products of ACC oxidase activity is HCN which explains why most plants have enzyme systems for detoxifying/metabolizing cyanide. ACC oxidase is inhibited by anaerobic conditions and cobalt ions but stimulated by ripening.

IV. Inhibitors

silver ions (Ag+), CO2 and KMnO4 inhibit ethylene actions. These bind to ethylene receptors or otherwise interfere with the mechanism of ethylene action. Aminovinylglycine (AVG) and aminooxyacetic acid (AOA) block the action of ACC synthase. Since these compounds are knows to block pyridoxal enzymes, it suggested that they participate in the process.

V. Disposal

probably not a concern since it is a gas in addition, malonate will bind to ACC forming N-malonyl ACC. Unlike most conjugates that can be hydrolyzed to produce the original compound, this one is NOT converted back to ethylene. a conjugate with glutamic acid (GACC) may be an important regulator of ethylene levels

VI.Actions A. Fruit ripening Ethylene triggers fruit ripening (i.e., climacteric) - Flavr-Savr tomato. B. Abscission This is the shedding of plant parts. Occurs at a specialized layer of cells the abscission layers. Auxin apparently prevents leaf abscission by maintaining cells in the abscission zone insensitive to ethylene. When auxin levels in the leaf decline, the tissues become sensitive to ethylene that promotes abscission by producing and secreting cellulases, etc. C. Epinasty Downward bending of leaves - common response to flooding or waterlogged soils. D. Triple Response Pea seedlings treated with ethylene are short (inhibits internode elongation), fat (increase stem thickness) and "stupid" (horizontal growth, no positive gravitropism). Further, they show little leaf expansion and possess an apical hook. E. Thigmomorphogenesis The change in growth form in response to a mechanical stimulation such as touch. F. Stimulates germination in cereals, peanuts; promotes sprouting in potato tubers and other bulbs. G. Flower senescence Stimulated by ethylene. Adding AVG to carnation can keep them fresh for weeks. VII. Commercial applications Ethrel (Ethephon) liquid sprayed onto plants. It contains a dilute solution of 2-chloroethylphosphonic acid that breaks down to give off ethylene. Among others things, it is used to synchronize flowering

and fruit set in pineapples. Commercial fruits are usually stored in low O2 to inhibit ethylene biosynthesis or under high CO2 to prevent ethylene action as a ripening promoter.

Test Yourself: Plant Growth Substances For each of the following, indicate the appropriate hormone (auxin, gibberellin, ABA, cytokinin, ethylene). There may be more than one correct answer for some questions.

Phytochrome and Photomorphogenesis I. Plant way of life revisited Plants, like all organisms, must be able to monitor and respond to environmental conditions. For example, a dark-grown seedling "knows" that it has a limited time to get to light until it runs out of energy. In response, an "etiolated" plant exhibits a variety of features in response to darkness including expanded internodes for rapid growth, an apical hook (in dicots), unexpanded leaves, no chlorophyll. A brief exposure to light causes internode elongation to slow, the hook to uncurl, leaves to expand and chlorophyll synthesis to begin. Thus, light has an obvious impact on the form of the plant (photomorphogenesis).

II. The signal: red & far-red light In many photomorphogenetic responses, red and far-red light are important environmental signals. For example, from studies of light sensitive lettuce seed germination (see Table 1), Borthwick and Hendricks concluded that: (1) germination is dependent upon which wavelength of light is received last; and that (2) this response is the product of a photo-reversible pigment.

Table 1: Germination of Grand Rapids Lettuce Seeds after brief exposures to red or far-red light Treatment none red red, far red r, fr, r r, fr, r, fr r, fr, r, fr, r r, fr, r, fr, r, fr r, fr, r, fr, r, fr, r Percent Germination 8.5 98 54 100 43 99 54 98

III. The receptor: Phytochrome The best characterized, and most important receptor for light-induced growth responses is phytochrome. The absorption spectrum of phytochrome closely matches the action spectrum implicating it in these processes. see action/absorption spectra

IV. Nature of phytochrome A. Chemistry Phytochrome is a pigment-protein complex (called a holoprotein = chromophore + apoprotein)

1 2

Pigment (chromophore) blue-green open chain tetrapyrolle; called phytochromobilin (overhead) made in the plastids Protein (apoprotein) glycoprotein soluble dimer (MW 240,000 D = 240 kD); each of the two peptides are identical with a MW ca. 124,000 D and comprised of ca. 1128 amino acids. gene(s) have been cloned and the amino acid sequence is known; large proportion of hydrophobic amino acids; suggests phytochrome is associated with membranes. tetrapyrolle is covalently-bonded to the protein via a thioether linkage involving a cysteine. one chromophore per dimer. holochrome apparently "self assembles" (autocatalytic) after the individual components are synthesized. For example, the protein is made by ribosomes associated with the ER and the tetrapyrolle is synthesized in the plastids, which is not too surprising considering the similarity it has to the structure of chlorophyll. coded by phy genes Types of phytochrome - phytochrome in dark-grown seedlings is distinctly different than that in light-grown plants. These two forms are:

Type I - found in dark grown, etiolated seedlings; most studied (MW 124 kD; maximum absorption - Pr 666 nm, Pfr 730 nm) Type II - found in light grown plants; (118 kD, Pr 654; Pfr 724) Conclusions phytochrome in etiolated plants (type I) is slightly larger and absorbs light maximally at a longer wavelength than phytochrome from light-grown plants (type II). differ mainly in apoprotein. at least five different proteins that can be identified immunologically; proteins are about 50% similar to one another. proteins are coded by 5 different genes called phy A - phy E. The phytochrome they make is called PHY A etc. PHY A is found only in dark-grown seedlings (Type 1); the other four types occur in both etiolated and green (light-grown) plants. Interestingly, PHY A is unstable. The PHY A is probably important for detecting the presence of light while the others monitor its quality. B. Photoreversibility The unique feature of phytochrome is that it exhibits photoreversibility; it exists in two forms that are interchangeable. Pr - red light absorbing form and Pfr - far red light absorbing form. When Pr absorbs red light (ca. 660 nm) it is converted into Pfr. When Pfr absorbs far red light (ca. 730 nm) it is converted into Pr. In short, phytochrome acts like a light switch. This can be depicted:

Pr Pfr The absorption spectrum for phytochrome will be provided. Note that there is some overlap in the spectra and also note that there is some absorption of blue light.

C. Chemical Changes during photoreversibility The main difference between the two forms is a cis-trans isomerization that occurs between one pair of tetrapyrolles. This changes has the effect of extending or opening up the chromophore. The protein also undergoes a conformation change. One piece of evidence that supports this is that the protein is more readily digested in the Pfr form. D. Efficiency of photoconversion Phytochrome acts like a weird light switch that only turns off/on a portion of the lights. In other words, red light treatment of Pr results in about 85% Pfr + 15% Pr; far red light treatment of Pfr results in 97% Pr + 3% Pfr. Thus, at photo-equilibrium not all the phytochrome is interconverted. The reason for this is because the absorption spectra for the two pigments overlap and they are essentially competing reactions. A measure of the efficiency conversion is the ratio of the Pfr to the total which is expressed as follows efficiency = Pfr/(Pr+Pfr = phytochrome total) In red light = 0.85; far red 720 nm = 0.03; this varies with the environment (see text and overhead) E. Intermediates There are a series of intermediates in the conversion from Pr to Pfr. The intermediates are unstable but there is probably always a small pool of them available. There are apparently three intermediate stages in conversion of Pr to Pfr; and 2 stages in conversion of Pfr to Pr. Thus we can modify the original equation: Pr Pr* [1] [2] [3] Pfr Pfr Pfr* [1] [2] Pr (The "*" refers to initial excited stage of phytochrome when it absorbs a photon.) V. Regulation of Phytochrome levels A. Synthesis Phytochrome makes up about 0.2% of the total protein in a dark grown plant. And, there is about 50x more phytochrome in an etiolated plant than a green one. Pr is the form synthesized by the plant; only form in the dark; light inhibits synthesis of Pr. Thus: phy gene mRNA Pr Pfr B. Destruction Both are degraded in vivo. Pfr is more labile (unstable). Protease digestion is an important route of hydrolysis; Pfr is more accessible to hydrolysis, perhaps because it gets tagged by proteins like ubiquitin for disposal. Thus: phy gene mRNA Pr Pfr destruction

VI. Pfr is the active form The first hint of this came from lettuce seed germination experiments. Since the seeds only germinated in the light and since Pr is only found in dark grown plants, Pfr must be the active form. Thus:

phy gene mRNA Pr Pfr destruction or physiological action

VII. Localization

wide distribution (including angiosperms, gymnosperms, algae, bryophytes) meristems and leaves found throughout cell (in cytoplasm and associated with organelles) Pr seems to be dispersed through cytosol and in nuclei and plastids Pfr seems to be sequestered in clumps - granules about 300 nm in size with no membranes

VIII. Actions - there are many phytochrome-mediated responses (see listing). These can be roughly grouped into three major categories: A. Induction-Reversion Responses or, Low Fluence (LF) Responses These are the "classic" responses that are induced by red light and reversed by far red. These responses are sensitive to 1 mol m-2 , are photoreversible, and saturate at 1000 mol m -2. Examples include:

Light-sensitive seed germination. Note that many seeds require light for germination (like lettuce), whereas others are inhibited by light (wild oats, Phacelia, Royal Paulownia). Phytochrome presumably stimulates GA synthesis/release which in turn, stimulates the mobilization of stored reserves (recall the GA lecture). This provides energy for the germinating seed and also decreases the solute potential for water uptake necessary for providing the force for the radicle to push its way through the seed. Reversal of etiolation Recall that etiolated plants are those that have been grown in the dark and exhibit a series of characteristics including elongated internodes, no chlorophyll, apical hook or unopened coleoptile, unexpanded or coiled leaves. These are all adaptations to save energy and get to the light asap. Light stimulates increased [cytokinin] which stimulates cell division and greening; increases [GA] which presumably stimulates IAA oxidase production to get rid of excess IAA that causes the long spindly growth. Also, IAA stimulates ethylene synthesis which maintains the apical hook. Treating the apical hook with red light + ethylene maintains the hook. Change surface charge In a classic experiment, Tanada (1968) observed that red light-treated barley root tips adhered to the sides of a beaker (with a negatively charged surface) but released after far red treatment. It was subsequently show that red light caused the surface to become positively charged and far red light negatively charged. Red light causes a depolarization of membranes and far-red reverses or even causes a slight hyperpolarization. Chloroplast rotation in Mougeotia (Haupt)

B. Very Low Fluence (VLF) Responses These are extremely sensitive to low levels of light (0.1 nmol m -2. Less than 0.02% is Pfr). These are not photo-reversible and saturate at 50 nmol m-2. Some examples include mesocotyl elongation in cereals; and Arabidopsis seed germination. C. High fluence (HF) or High Irradiance Response (HIR)

1 2

Prolonged or continuous light (10 mmol m-2); fluence rate is important; and not fully photoreversible. Apparently what is important is maintaining a low level of Pfr over time. Another pigment is likely involved - a blue-UVA receptor (cryptochrome?). Examples of phenomena involved in this reaction include:

anthocyanin synthesis in seedlings and apple skins inhibition of hypocotyl elongation in mustard, lettuce and petunia plume hook opening in lettuce cotyledon enlargement in lettuce ethylene production in sorghum.

VI. Mechanism of Action A. Stimulates transcription Jaffee (1969) found RNA levels in pea increased after red light treatment. Rubisco is lightstimulated, other genes inhibited. Phytochrome involved at the transcriptional level. The mechanism of action is not clear, but the Pfr may activate an inactive regulatory protein that moves into the nucleus to bind to a site on the DNA near the genes of interest to promote their transcription. B. Calmodulin This calcium binding protein may be involved in phytochrome action. Pfr has been shown to stimulate calcium uptake in Mougeotia. Calcium complexes with calmodulin and can stimulate enzymes. Predictions:

1 2 3 4 5

Calcium should increase after red light treatment - it does (Mougeotia) Calcium ionophores like A23187 can substitute for red light - it does Valinomycin - a potassium ionophore has no effect Calmodulin treatment should stimulate activity in absence of red light Calmodulin inhibitors should prevent the respones - they do Flowering

I. An overview The process of flowering requires the vegetative meristem (buds) to change into a reproductive meristem. This process, which is termed evocation, involves the following sequence of events: juvenile vegetative phase adult vegetative phase adult reproductive phase flowering A. Juveniles vs. adults Juvenile plants cannot flower; they are capable of only vegetative growth. Thus, like in humans and other animals, the ability to reproduce marks the transition from the juvenile phase to adulthood. As we previously discussed (GA section), juvenile plants often differ in appearance from the adult. For example, leaves may change shape (i.e., ivy adult - simple leaves; juvenile - lobed leaves) or degree of compounding (i.e., beans: adult compound leaf; juvenile simple leaf). Juvenile tissues are produced first, near the base of the plant. The adult phase is usually stable and can be propagated from plant to plant. B. Ripe to Flower.

The adult can flower and is said to be "ripe-to-respond (or flower)" or "competent". In other words, it has the potential to flower when the conditions are appropriate. This may be a mechanism to insure that there is a sufficient vegetative mass (i.e., leaves, roots) to support the reproductive output. C. Juvenile to Adult Transformation. The maturation of the juvenile into the adult may be mediated by a variety of factors including:

1 2 3 4 5

size (more important than age) age (i.e., century plants, bamboo); leaf number; growth conditions (conditions that favor growth promote the transition to adult phase; poor conditions, such as water stress, lack of light, low temp, prolong the juvenile phase) other factors.

Ultimately, one or more of these factors likely induce changes in (1) hormones (such as GA; for example, recall that GA application stimulates the adult phase in conifers but in ivy promotes juvenility), (2) nutrient levels (i.e., lack of a carbohydrate supply to the meristematic region), or (3) other chemicals, that in turn trigger the developmental switch to adulthood. Thus we can modify our original scheme: juvenile vegetative phase transition factors (i.e., size, age) induce hormonal or other changes adult vegetative phase (competent) adult reproductive phase flowering D. Adult Vegetative-to-Reproductive Transition The transition from the vegetative to reproductive buds is usually triggered by an environmental signal, typically photoperiod or temperature. This signal synchronizes flowering to environmental events. Thus, this is a type of "timing mechanism" that plants use to coordinate actions with the season. If flowers are produced at the wrong time of the year the pollinator may not be available, or it may be too dry (or wet), or there may not be enough time before winter to allow time for successful seed set. Once the inductive signal has been received then the plant meristem is said to be "determined". In other words, it is now committed to flower. Thus, we can modify the diagram again: juvenile vegetative phase transition factors (i.e., size, age) induce hormonal or other changes adult vegetative phase (competent) environmental signal (i.e., photoperiod, temperature) adult reproductive phase (determined) flowering expressed In some plants, an environmental signal is not necessary to trigger the transition to the determined state. These plants move directly in the reproductive phase after becoming competent. The two major signals for inducing flowering are light (photoperiod) and temperature (cold treatment)

II. Light, or more specifically, photoperiod and flowering A. A brief history.

Tournois (1914) was one of the first to report the influence of photoperiod on hops and hemp. Garner's and Allard's classic studies showed that a tobacco mutant, Maryland Mammoth, which failed to flower under field conditions, did so in the greenhouse in the winter in response to photoperiod. B. The flowering response to day length varies with the species: Short day plants (SDP) - require one or more days with less than a certain amount of daylight. Or, the critical day length to induce flowering must be less than some maximum. These species usually flower in the spring or fall. 2 Long day Plants (LDP) - require one or more days with more than a minimum day length to flower. The critical day length must be longer than a minimum. 3 Day neutral plants (DNP) - ambivalent to day length 4 Plants exhibit a variety of intermediate responses and combinations. For example, there are long-short day plants. After an inductive long-day photoperiod, these plants require short days to flower. This is a good strategy to insure flowering in the late summer. 5 One inductive photoperiod may suffice to induce flowering (i.e., cocklebur, Japanese morning glory); or, flowering many require several days, with a cumulative effect. 6 Light may have a quantitative effect on flowering - in other words, SD may stimulate the percentage of plants that flower. C. The night period is more important than the day.

Using cocklebur, a SDP, Bonner & Hamner (data provided in class) showed that it flowers if it received one critical photoperiod with less than 8 hours of light (or, > 16 h darkness). The proportion of light/dark is not important in flowering. A light break during the night interrupts the flowering response, but a dark period during the day has little effect on flowering. The timing of the night break is important (see data). Conclusion: long day plants can be called "short night plants" and short day plants can be called "long night plants". D. Receptor. 1. Location. The receptor is located in the leaves. Evidence: (a) defoliated plants are insensitive to photoperiod; (b) plants with a single leaf in the inductive photoperiod will bloom (e.g., Perilla; Chailakhyan, 1961); (c) the receptor doesn't seem to reside in meristem since treating the meristem with inductive photoperiod doesn't initiate flowering (Chailakhyan). 2. Nature of the receptor. Since light is involved, it suggests that a pigment plays a role in photoperiodism. Phytochrome is a likely candidate because: (a) the light break shows red/far red sensitivity; (b) the action spectrum for the light break in cocklebur is consistent with phytochrome (data provided in class). In fact, there is evidence for the participation of two forms of phytochrome. E. Transducing mechanism

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A diffusible substance plays a role. This is clear since the leaves are the receptors, but the meristem is converted to reproductive stage. Something must be translocated from the reception site (leaves) to the action site (meristem). Grafting experiments provide further evidence (see data). Rate of transport consistent with phloem movement. The diffusible substance is probably similar in most plants. If a SDP is grafted to a LDP and they are placed in a short-day photoperiod (that can induce flowering in the SDP), they will both flower, even though the LDP is not in its inductive photoperiod.

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Florigen - name given to the proposed flowering "hormone". There is little direct chemical evidence for its existence. Most evidence is from physiological experiments as described above. An extract of induced cocklebur has weak floral-inducing ability. GA may be involved (recall our hormone discussion?). In brief, exogenous application of GA can substitute for photoperiod, especially in LDP's Ethylene, IAA or cytokinin are associated with flowering in some species. One problem with many of these studies is that the concentration of hormone used was much larger than the amounts that are naturally-occurring and could stimulate abnormal processes. Other substances may be involved in flowering, too. Anthesin is a hypothetical hormone proposed by Chailakhyan that may stimulate flowering in SDP when associated with GA. This existence of this substance is dubious.

III. Timing Mechanisms in plants - a small, but necessary tangent - youll soon see how it relates A. The plant way of life revisited. Recall that a stationary organism like a plant must be able to accurately predict when the environment is going to change. Since plants cant move they must respond to these changes by relatively slow growth responses/movements that take time. Thus, plants must have an accurate sense of time to distinguish daily and seasonal changes and respond to them. B. Examples of timing mechanisms in plants: Timing mechanisms include: (1) Flowering - recall that plants must flower at the appropriate time of the season; (2) Bud break and seed germination - timing mechanism to determine when spring has arrived; develop too soon and they risk freeze injury, develop too late and they may not have enough time to complete the life cycle. C. Requirements for a biological clock. The clock must be: accurate (keep good time or be reset each day); insensitive to capricious events in the environment (those that are not predictable such as temperature, wind speed, precipitation); and 3 have a transducing mechanism to couple the clock to a physiological response. D. Hourglass (or cumulative) Timers.

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Egg timers and water clocks are good examples of hourglass timers. These clocks measure time by monitoring the interval of time required for a certain event (i.e., sand to run out, or water to drip) to occur. They measure a single interval and then need to be reset. Hourglass timers in plants include: ripeness-to-flower. Essentially the life of the plant can be considered to be an hourglass timer that isnt reset; 2 induction/reversion phenomena due to phytochrome (i.e., seed germination); and 3 seed germination/bud break - many seeds and buds measure the amount of inhibitor present and develop when the level drops below some critical value. E. Oscillating or rhythmic timers

These measure time intervals between regular oscillations, such as the sweeps of a pendulum. Many events in plants show rhythmic oscillations such as the opening/closing of flowers, leaf movements (nyctinasty), and even growth rates.

Anatomy of an oscillation Period refers to the time between repeating points of the cycle, or in other words, the time it takes to complete one cycle. Period is symbolized by tau. Most biological rhythms have a

period of ca. 24 hours though they vary from 21-27. Hence, they are called circadian, because they last approximately (circa) one day (diem). Other types of rhythms include lunar, annual, ultradian (less than a day). Amplitude - intensity of oscillations, or in other words, the difference between the peaks and troughs. Oscillating timers. These are free running - in other words they dont have to be restarted at each period. And they will continue under constant conditions though the response will eventually damp out. This is evidence that there is an endogenous oscillator; an hourglass timer would not keep running without an external stimulus to reset it. This is analogous to a spring-loaded watch. Just like we need to coordinate our clocks with the season (i.e., "spring ahead or fall back"), plants must be able to set their oscillating timer to correspond to environmental changes. This is called entrainment and the signal that synchronizes the rhythm is the zeitgeber. Light is the zeitgeber. Both blue and red light are important. The red light sensitive species show red/farred reversibility suggesting that phytochrome is the receptor for the response.

The nucleus is the site of an endogenous, oscillating timer (see data). Now back to our regularly scheduled program.......

IV. Transducing mechanism for photoperiodism A. Photoperiodism as an hourglass timer. According to this hypothesis, plants measure the ratio of Pfr/Pr. An LDP would flower when the ratio is high (i.e., more Pfr) and but SDP would flower when the ratio is low (more Pr). Since Pfr is labile and is broken down at night or reverts back to Pr - the longer the night, the lower the phytochrome (Pfr) content. Thus, phytochrome is like the sand in an egg timer; the relative amount of Pfr remaining at the end of the night would be an indication of the day length. To "reset" an egg timer, you simply turn it over. Similarly, the flowering timer would be reset during the day when Pfr levels are re-established. Problems with this hypothesis: (a) the half-life of Pfr breakdown is too short; and (b) if phytochrome degradation is involved, the process should be temperature sensitive (i.e., Q10 should increase), but it is not. B. Photoperiodism as an oscillating timer. A more recent idea, and one that has greater support, is that flowering is controlled by an oscillating timer. Evidence: (1) Chenopodium rubrum (SDP) seedlings maintained in continuous light will flower if exposed to single dark period. The length of the dark period shows a rhythmic effect on flowering; (2) Duckweed (Lemna) studies also show an oscillating timer to exist. C. Photoperiodic Control of Plant Growth. Other aspects of plant growth and development are affected by photoperiod including:

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seed germination (some like long days, others short days); stem growth (promoted by long days; root and storage organ formation (induced by short days in potato, dahlia and radish, long days in onion); vegetative reproduction (long days - strawberry runners, Bryophyllum plantlets); sexual reproduction (flower induction and development); autumn response; and flowering.

D. Photoperiodism summary.

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plants are affected by photoperiod in many ways; there is a wide diversity of responses of flowering to photoperiodism (i.e., LDP, SDP); Plants must be ripe-to-flower before they will respond; dark period is important; an hourglass timer may be involved; an oscillating timer is surely involved; a flowering hormone is involved; flowering can be induced by exogenous hormone application.

V. Temperature and flowering A. Overview. Many plants require a cold treatment to induce flowering. This is termed vernalization and is a "smart" way to time when winter is over (type of hourglass timer). Vernalization is common in biennials and winter annuals (such as winter wheat). The effect can be qualitative or quantitative. Vernalization usually works in concert with photoperiod - in other words, vernalization is required to make the plants sensitive to photoperiod. Thus, this acts as a "fail-safe" system to insure flowering at the appropriate time of year (after winter!). The term was coined by Lysenko (remember him - inheritance of acquired characteristics?). B. Signal. Cold, actual temperature varies from -5 to 15 C. C. Receptor.

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Some seeds can be vernalized (data for rye). However, they must be hydrated (dry, unimbibed seeds are insensitive). Biennials not responsive as seeds. The meristem perceives cold treatment - grafting experiments and tissue culture experiments with rye. Some plants (henbane) need to reach a certain size to be responsive to cold treatment, whereas others (rye) can be treated as seeds.

D. Transducing mechanism

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Plants can "remember" the signal. In other words, cold-treated plants will grow vegetatively for quite awhile before flowering. This suggests that the induced state must be permanent, or at the least be relatively stable, in many species. A hormone doesn't seem to be absolutely required since the meristem is the source of the receptor and it could pass the permanent change on to future cells. A chemical signal may be involved - the transmission of a signal through grafts has been noted with some. This hormone has been termed vernalin, but not yet isolated. Plants can be de-vernalized if the cold treatment is followed by a heat. In rye, 30 C for 3-5 days will do the job. Rye can also be devernalized by drying and anaerobic conditions. GA can substitute for cold in some cold-requiring species. But, GA may primarily affect bolting (stem elongation). Vernalization of isolated embryos requires oxygen and carbohydrate - suggests an energydependent process. hotosynthesis: Light Dependent Reactions

(or, Life is a photochemical phenomenon)

I. Overview of photosynthesis: Photosynthesis can be defined as the light-driven synthesis of carbohydrate. The equation for this reaction, that youve seen many times is: CO2 + H2O + light + chloroplast (CH20)n + O2 From this simple equation we can make some elegant conclusions: A. Photosynthesis is a redox reaction.

Some definitions: (a) Reduction - gain of electrons; (b) Oxidation - loss of electrons; (c) helpful mnemonics to remember: "oil rig" - oxidation is loss, reduction is gain, or "Leo says grrrr" - loss equals oxidation, gain reduction; (d) Redox reaction - reaction in which one component is oxidized and the other is reduced. Obviously, electrons must come from somewhere and go somewhere. The reduction sequence of carbon: carbon dioxide (most oxidized form of carbon) carboxyl (organic acid) carbonyl (aldehydes, ketones) hydroxyl (alcohols) methyl methane (most reduced form of carbon). Note: each step requires the addition (or removal) of two electrons and two protons for reduction (oxidation). Two steps also require the addition/removal of water. How can you tell if a molecule has been oxidized or reduced? (1) look for a change in valence (i.e., Fe2+ Fe3+ represents an oxidation because an electron was lost, increasing the total positive charge); (2) In many biological redox reactions, oxidation is usually accompanied by a loss of protons (hydrogen ions) and reduction is accompanied by a gain of protons; and (3) look for a decrease in the number of oxygen atoms. Biological redox reactions may require electron donors and/or acceptors. These include: (1) NAD+; (2) NADP+; and (3) FAD; which are coenzymes (organic compounds, other than the substrate, required by an enzyme for activity): NAD(P) + (ox) + 2e- + 2H+ NAD(P)H (red) + H+ FAD(ox) + 2e- + 2H+ FADH2 (red)

Reducing Potential - potential for components to participate in a redox reaction; to predict the direction and tendency of electrons to flow between two electron carriers. The take-homelessons are: (1) the more negative the reducing potential the better the electron donor; (2) the more positive the reducing potential the better the electron acceptor; (3) spontaneous electron passage occurs from a carrier with more negative reducing potential to one with a more positive reducing potential.

B. CO2 is reduced to a carbohydrate. C. Water is oxidized (to oxygen). D. Water supplies the electrons for the reduction; water is cleaved in the process yielding oxygen as a byproduct. E. Light provides the energy for the reduction.

F. Photosynthesis is an energy conversion process that ultimately converts light energy to chemical energy (carbohydrate). In a broad sense, it is an example of the 1st Law of Thermodynamics - energy cannot be created nor destroyed, but it can be changed from one form to another. G. BLACK BOX summary model for photosynthesis. Diagram in class that shows two boxes (light dependent & light independent reactions). This model further shows that during the light-dependent and light-independent reactions that there are three major types of energy conversions during photosynthesis: Conversion 1: Radiant energy (sunlight) electrical energy (passage of electrons via a series of carrier). This reaction series is part of the light-dependent reactions (z-scheme, non-cyclic electron flow) Conversion 2: Electrical energy "Labile" chemical energy (ATP, NADPH; unstable, not readily stored). During this step, ATP and NADPH are produced as the end result of non-cyclic electron flow. Conversion 3: "Labile" chemical energy Stable chemical energy (carbohydrate). This last step is the light-independent reactions or Calvin-Benson cycle. This process requires ATP and NADPH. II. Chloroplasts - specialized organelles that carry out the process of photosynthesis A. Structure. Remember the cell unit? To jog your memory, reread Chapter 1. Terms that you should know are thylakoid (or lamellae), lumen (intermembrane space), envelope, double membrane, stroma, granum, granal thylakoids (or lamellae), stromal thylakoids (lamellae), and starch grains. Chloroplasts may contain fat globules (plastoglobuli). Stacked (or appressed) regions - portion of granum in which thylakoids are adjacent to one another. Non-stacked (non-appressed) regions - regions of the chloroplast where the thylakoids are not adjacent to another. B. Ontogeny and phylogeny - recall the cell unit? C. Chemistry - Chloroplasts contain:

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DNA - circular loop; 120-160 kilobases that code for about 120 proteins; RNA; ribosomes; proteins - some are coded by the nuclear genome, others by the chloroplastic genome. For example, rubisco, an important enzyme, has 2 different subunits, one from each source. The nuclear genes are essential for chloroplast function; pigments - make up about 7% of the chloroplast. These are molecules with a color that absorb light. Two major groups of pigments in higher plants, chlorophylls and carotenoids/xanthophylls. These occur in the thylakoids because they are highly hydrophobic (fat soluble)

D. Pigments 1. Chlorophylls These molecules look like a tennis racket. The head of the racket is a porphyrin ring system, made of four pyrolle units linked together (tetrapyrolle). It has a long hydrocarbon tail, called phytol (C20), that is derived from the terpene pathway (diterpene), built from the isoprene skeleton. Magnesium is chelated in the ring. The tail is important for orienting the molecule in the membrane. The interaction of the chlorophyll with the membrane is non-covalent and is important because it ultimately determines the physical properties of the chlorophyll.

chlorophyll a - methyl group chlorophyll b - formyl group phaeophytin - chlorophyll without the magnesium chlorophyllide - chlorophyll without the tail 2. Carotene/xanthophylls Both are terpenoid pigments, tetraterpenoids (C-40). Carotenes are hydrocarbons, xanthophylls are oxygenated. These pigments are orange and yellow in color. 3. Chlorophyll biosynthesis - Some take-home-lessons:

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4. Light and

ALA ( -aminolevulinic acid) is the first well-established precursor ALA is derived from -ketoglutarate (or glutamate) (a Kreb's cycle intermediate, from the mitochondrion) 2 ALA condense to form a unit of pyrolle 4 pyrolles condense to form porphyrin (tetrapyrolle) Magnesium is inserted A photoreduction step occurs (converts protochlorophyllide chlorophyllide) the tail is added the Greening Process

Recall that etiolated plants (grown in the dark) are yellowish but turn green rapidly when placed in the light. Light is required, among reasons, to:

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convert etioplasts chloroplasts; photo-reduce protochlorophyllide to chlorophyllide; and activate enzymes for ALA synthesis.

III. Conversion 1: Photons to electrons A. Nature of light Light is part of the electromagnetic spectrum - radiation emitted by sun. Acts as discrete particles (called photons) traveling as waves. Wavelength - distance between any two crests (or troughs). Symbolized by lambda (); frequency - number of waves passing a point in one second (). Frequency is inversely related to wavelength = c/ where c = speed of light (3 x 10 10 cm sec-1). The energy of a photon is a quantum. B. Which photons are important in photosynthesis? Run an action spectrum (plot of a physiological process vs. wavelength). insert action spectrum of photosynthesis here Conclusion: radiations between 400-700 nm are photosynthetically active (termed PAR). Specifically, red (600s) and blue (400s) light are important. C. Photons must be absorbed to be used in a photochemical reaction. In other words, only those molecules that absorb quanta participate in photosynthesis. So, which molecules absorb the red and blue light? Run an absorption spectrum of potential pigment candidates (plot of light absorption vs. wavelength) and compare it to the action spectrum.

insert absorption spectrum of photosynthetic pigments here Chlorophyll a & b absorb light in the red and blue regions of the visible spectrum. Note that the absorption spectra match the action spectrum of photosynthesis and hence, implicates (though doesnt prove) that they are involved in the process. (Subsequent work has shown the chlorophylls to be the major photosynthetic pigments). D. Quantity vs. Quality Light quality - refers to the wavelengths of light that are important. Photosynthetically active radiations (PAR) range from 400 - 700 nm with peaks in the red and blue. 2 Light quantity - refers to the amount of light (PAR) received; units of mol m-2 s-1 , called the photon fluence rate; or units of energy, J m-2 s-1. E. What happens when chlorophyll absorbs light?

The chlorophyll molecule becomes excited (this takes only 10 -15 sec = femptosec) and an electron moves to an outer energy level. This is diagrammed: CHL (ground state) CHL* (excited state) Blue light excites an electron to a higher energy level than red light. Imagine the "bell ringer" at a carnival. The electrons change spin at the first (S1) and second (S2) excited singlet states. Electrons dont stay excited long (10-9 sec), because they either: return to the ground state and release their absorbed energy as heat (thermal deactivation); 2 return to ground state and release their extra energy as light (fluorescence); 3 transfer their energy to another molecule; kind of like hitting pool balls (resonance transfer); or 4 change spin and revert to a triplet state (same spin as ground state) and be used in a photochemical reaction (photochemistry). F. Why excite electrons?

The ultimate purpose of exciting electrons from chlorophyll is to provide the energy needed to transfer electrons from water to NADP+. Recall that spontaneous electron transfers proceed from a carrier with a more negative redox potential to a more positive one. The redox potential of water/oxygen = +0.82 eV while for NADP/H = -0.32 eV. Thus, photosynthetic electron flow is not a spontaneous process and requires an energy. G. How much energy is required to transfer electrons from water to NADP +? First, let's calculate the actual redox difference (Em) between water and NADPH: Em = Em (acceptor) - Em (donor). Or, Em = -0.320 - (0.820) = -1.14 = ca. -1.2 eV. The actual amount of energy involved is calculated from the equation: G = -n F Em where F = Faraday constant = 96,000 J/coulombs, and n = number of electrons involved in the reaction (which equals one for each photon). Substituting in the equation: G = - (1) x 96000 x (-1.14) = 109440 J mol-1 (=109.4 kJ mol-1 )

To summarize, approx. 110 kJ mol-1 is required to reduce NADPH from water. H. Do red and blue photons have enough energy? Let's calculate the energy in red photons. Assume red photons have a wavelength of 660 nm = 6.6x10-5 cm. The energy of a photon is expressed by the following equation: E = h where h = Plancks constant which relates energy to frequency of oscillation and is 6.6255 x 10 -34 J sec photon-1; and = pulses sec-1. Since = c/ (see A above), we can substitute back in original equation: E = hc/ Take home lesson #1: the energy of a photon is inversely proportional to its wavelength. Thus, blue light has more energy than red light. E = ((6.625x10-34 j sec photon-1)(3x1010 cm sec-1))/6.6x10-5 cm = 3.01x10-19 j photon-1 multiply by Avogadros number = 3.01x10-19 j photon-1 x 6.02 x 1023 photon mol-1 = 181,000 j mol-1 = 181 kj mol-1 Take home lesson #2: Red light has more than enough energy to do the job. IV. Chloroplast complexes: There are four major complexes in the chloroplast. These are physically distinct from one another and can be isolated from the chloroplast by electrophoresis and ultracentrifugation. A. Photosystem II (PSII) Complex

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large multi-subunit protein complex occurs in the stacked regions of the granal thylakoids integral proteins - coded by the chloroplast genome; including D1 (33 k) and D2 (31 KD) peripheral proteins - coded by nuclear genome; bind Ca2+ and ClP680 reaction center - a unique chlorophyll a, maximum red light absorption at 680nm; maybe two chlorophyll a molecules; this is the chlorophyll that "looses" electrons manganese ions (Mn2+ ) phaeophytin, plastoquinone LHCII - Light harvesting pigment complex associated with PSII. It is comprised of (a) 250 chlorophyll a & b, in approximately equal amounts; (b) several carotenoids; (c) proteins -

each pigment is associated with protein (ca. 15 pigments/protein); the protein is coded by the nuclear genome B. Cytochrome b/f Complex 1. occurs in stacked and non-stacked regions 2. cytochrome b (b-type cytochrome, not associated with protein) 3. cytochrome f (c-type cytochrome, associated with protein) 4. non heme iron-sulfur protein (Fe-SR) C. Photosystem I (PSI) Complex

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occurs in non-stacked regions (stromal thylakoids) about 11 polypeptides - including 1a & 1b that are coded by a single operon in the chloroplast genome, bind p700 50-100 chl a electron carriers LHCI - contains about 100 chlorophylls; 4:1 ratio of chl a: chl b.; the protein is encoded by nuclear genome P700 reaction center chlorophyll a

D. ATP synthase/Coupling Factor Complex 1. occurs in non-stacked regions 2. stalk - CFo (4 polypeptides) 3. head - CF1 (5 polypeptides) 4. nine polypeptides, some nuclear, some chloroplastic V. The Z-Scheme (Or, the Light-Dependent Reactions; Or, Non-cyclic photophosphorylation). A. Overview During the light-dependent reactions of photosynthesis, electrons are transferred from water to NADP+. This reaction is depicted as follows: H2O NADP+ As the electrons move from water to NADP+, they pass through three of the four complexes described above - Photosystem II (PSII), a cytochrome b/f complex (cyt b/f), and Photosystem I (PSI). After electrons are removed from water, they are sequentially shuttled from PSII to the cyto b-f complex to PSI and then finally to NADP+. Thus: H2O PSII Cytb/f PSI NADP+ Since PSII, cyt b/f, and PSI are physically separated from one another, there must be a means to transfer electrons between the complexes. A mobile form of plastoquinone (PQ) transfers electrons from PSII to cyt b-f. A copper-containing protein, plastocyanin (PC), transfers electrons from the cytochrome b-f complex to PSI. Thus, the reaction sequence is modified as follows: H2O PSII PQ Cytb/f PC PSI NADP+ The transfer of electrons from PSI to NADP+ is mediated by a soluble complex found in the stroma, ferredoxin (Fd). Thus our revised equation:

H2O PSII PQ Cytb/f PC PSI Fd NADP+ The transfer of electrons from water to PSII involves an "oxygen evolving complex" (OEC), part of PSII, that is rich in chloride and manganese ions. Thus, H2O OEC PSII PQ Cytb/f PC PSI Fd NADP+ B. Origin of the name Derived from the zig-zag arrangement of components with regard to redox potential. But, why dont we call it the N-scheme? C. Oxygen evolving complex The energy of a single photon is not sufficient to split water. Experiments suggest that 4 photons are required to split two water molecules. Since only one electron can be excited at a time (Einstein Law of Photochemical equivalents), this presents a minor problem. The solution a water oxidizing "clock". Single electrons are transferred through a series of intermediate stages sequentially increasing the electron deficit to a total of four. At this point the original oxidation state is restored by extracting four electrons from water. Diagram of the water-oxidizing clock - in class Take-Home-Lessons:

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Evidence:

A series of five intermediate states, S0 - S4 are postulated; Initially the clock is in the So state, and may be associated with Mn II S1, which may be associated with Mn III, is the most stable form; S2 may be associated with Mn IV; S3 may be associated with a histidine (one of the amino acids in the D1 protein); The nature of S4 isnt clear; Conversion from one state to the next requires one photon and results in the loss of one electron to P680; and the loss of 4 total electrons generates a strong enough potential to split water.

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after a dark equilibration period, oxygen is released after the third light flash and then after every fourth flash; explains occurrence of Mn in photosystem II.

D. PQ Shuttle (Q cycle)

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PQH2 is reduced on the stromal side of the thylakoid in PSII PQH2 shuttles over to the lumen side of thylakoid and gets oxidized when it transfers its electrons to the cyt b/f complex One electron is given to an Fe-S protein, which in turn, passes it to cyt f and then to PC. The other electron is given to cyt b which then partially reduces another PQ. The "leftover" protons are dumped into the lumen A second PQH2 from PSII shuttles to the cyto b/f complex and essentially repeats step 3 one electron is passed to Fe-S then to cyt f and to PC. The other electron from PQH 2 is given to cyt b and then to PQ to fully reduce it to PQH2 which must also grab two protons from the stroma.

Take-Home-Lessons: for every two electrons shuttled to PSI, four protons are moved across the membrane! The stoichiometry requires more than 2 protons per electron pair to account for ATP synthesis. E. Herbicides and electron transport

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Urea derivatives - DCMU (diuron) blocks electron flow at a point after Q. They bind to the Qb binding site, preventing PQ from doing so. Interestingly, there are resistant varieties, that have a single amino acid substitution in the D1 binding protein. Viologen dyes - paraquat/diquat - accept electrons from the reducing side of PSI - thus, they interrupt electron flow and also convert oxygen to superoxide - which causes damage to membranes, etc.

VI. Photophosphorylation Literal translation - "the light driven synthesis of ATP" Occurs by the same mechanism, Chemiosmotic hypothesis, that occurs during ATP synthesis in the mitochondria A. Non-cyclic Electrons are passed in a single direction, one-way, no backtracking, from water to NADP+. B. Cyclic Electrons cycle through PSI. This occurs when carriers get backed up with electrons but still want to get rid of electrons. This is a mechanism for generating additional ATP. Features:

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asymmetric distribution of electron carriers in the thylakoids; some carriers require only electrons (i.e., cytochromes, chlorophyll), others both (i.e., PQ); at the transitions, protons will be extracted or expelled; the transitions occur at the surfaces of the membrane; PQ is the major source of the proton gradient; reduction of PQ occurs at the stroma side of the thylakoid and removes 2 protons from the stroma; PQ shuttles electrons to the cyt b/f complex on the lumen side of the thylakoid. Only electrons are passed to the cyt b/f complex, the protons are expelled into the lumen. Thus, PQ is oxidized on the lumen side of the membrane; the Q cycle shuttles additional protons across the membrane; the thylakoid is impermeable to protons; a pH gradient is generated across the membrane, the stoma pH is ca. 8 while the lumen is ca. 5; the pH gradient provides the energy for the synthesis of ATP; ATP is synthesized by an ATPase associated with the CF complex; and protons escaping through the channel drives ATP synthesis something like water turning a mill.

Evidence: lots!

a pH gradient exists in the chloroplast. Discharging the gradient with buffers prevents ATP synthesis;

artificially creating a gradient allows for ATP synthesis; uncouplers like DNP "poke holes" in the thylakoid making it "leaky" and discharging the gradient preventing ATP synthesis. Note that this doesnt stop electron flow - in fact, it usually increases the rate. VII. How many photosystems? Two

2 3

Emerson observed that the rate of photosynthesis was greater than the sum of the rates when red light (660 nm) and far red light (710 nm) were given separately. This synergistic effect, called the Emerson enhancement Effect, suggested two cooperating systems which has been the conventional wisdom for a long time. utotrophism: Carbon Reactions (Calvin Cycle, C4, CAM) I. The final frontier - Photosynthetic Carbon Reduction (step 3)

called "dark reactions" because reactions dont require light - however, note that these reactions can (and normally do) occur in the light. In one sense they can be considered "light-dependent" since they require the ATP and NADPH generated during the Z scheme. called the Calvin cycle - after the fellow and his colleagues who worked out most of the reactions. If you had done it, you too, would own a Nobel Prize. occurs in the stroma there are three major steps: fixation reduction rearrangement/recharging/release

A. Carbon dioxide fixation Carbon dioxide is fixed (trapped, bound) to form an organic compound (phosphoglyceric acid, PGA) carbon dioxide condenses with RuBP (ribulose bisphosphate; C5) to form 2 molecules of PGA (C3) first product of carbon fixation is PGA (Calvins experiments) catalyzed by the enzyme ribulose bisphosphate carboxylase (rubisco). rubisco is the most abundant protein on earth; it makes up 50% of leaf protein the reaction mechanism is diagrammed in the text (on overhead) B. Reduction Step in which the temporary chemical (ATP) and reducing (NADPH) potentials that were generated in the light-dependent reactions are used to reduce the PGA (an acid) to a carbonyl (glyceraldehyde 3phosphate; abbreviated G3P or GAP) PGA is reduced to G3P this is a two-step reaction sequence first, PGA is phosphorylated with ATP to 1,3-bisphosophoglycerate which is subsequently reduced to G3P (note a phosphate is lost during this reaction). NADPH provides the electrons for the reduction energy requirements - at this point in the cycle, for each carbon dioxide fixed, two ATP and two NADPH are required (one for each of the two PGAs) C. Rearrangement/Recharging/Release Complex series of reactions (rearrangment) that result in the net removal of a C3 carbohydrate from the cycle (release) and the production of the precursor to the starting material (recharging):

see overhead and diagram in text for details the cycle must turn 3 times for the production of one net triose

the end product of the cycle is ribulose-5-P (RuP) ATP converts ribulose-5-P to RuBP ATP comes from the Z scheme E. Summary The fixation of 1 carbon dioxide requires: 3 ATP and 2 NADPH.

II. Regulation of the Calvin Cycle We will not cover this in class except to say that regulation of the cycle is obviously important. There are several regulatory controls:

1 2 3 4
III. C3 Plants

rubisco - light activated; allosteric regulation - rubisco has a binding site for CO2; rubisco activase - protein that "activates" rubisco; and Fd/thioredoxin - several enzymes require a reduction to become activated.

Plants that exhibit the type of photosynthetic carbon reduction that we described above are termed C3 plants. In other words, the first product of carbon dioxide fixation is a 3-carbon compound (PGA). Thus, when radioactively labeled carbon dioxide is fed to a plant, the first place that it shows up is PGA. IV. Photorespiration Light stimulated production of carbon dioxide in the presence of oxygen

not associated with mitochondrial respiration requires light not accompanied by ATP synthesis wastes energy (i.e., ATP, NADPH)

A. Observations on photorespiration Not all plants photorespire - see plot of carbon dioxide production (= respiration rate) vs. time for tobacco and maize. Note the dark rates are the same, but the light rate is much greater in tobacco. 2 Plants that photorespire typically show light saturation - points to note: (a) plot of carbon dioxide uptake (=Ps rate) vs. fluence for tobacco and maize in ambient oxygen (21%); (b) light saturation point - point at which increasing fluence yields a constant amount of photosynthesis; (c) light compensation point - fluence at which the amount of photosynthesis just equals the amount of respiration; and (d) note that plants that photorespire (like tobacco) have a higher light compensation point and light saturate. 3 Plants that photorespire have a higher CO2 compensation point. In other words, it takes a greater amount of carbon dioxide to break even. 4 Oxygen inhibits photosynthesis in plants that photorespire (called the Warburg effect) - plot carbon dioxide uptake vs. fluence for maize and tobacco at 1.5% oxygen 5 Carbon dioxide is limiting to plants that photorespire - for evidence see: (a) plot of carbon dioxide fixation vs. carbon dioxide concentration for maize and red clover (photorespires); and (b) plot of carbon dioxide uptake vs. fluence for tobacco and maize at ambient oxygen at varying carbon dioxide levels. B. Making sense of the data

The data cited above suggest that carbon dioxide and oxygen have antagonistic (opposite) actions in photosynthesis and act in a competitive manner. C. The problem - rubisco Unlike most enzymes, rubisco is not substrate specific - it also has an oxygenase function. In addition to its normal substrate (carbon dioxide) rubisco also binds oxygen to RuBP. Although rubisco has a higher affinity for binding carbon dioxide (Km = 9 M), if enough oxygen is present, it acts as a competitive inhibitor (the Km for oxygen is 535 M). D. The reaction catalyzed by ribulose bisphosphate carboxylase/ oxygenase When rubisco binds oxygen to RuBP, the RuBP is essentially split in half to a 3 carbon piece and a 2 carbon fragment according to the following reaction: RuBP + oxygen + rubisco PGA (C3)+ phosphoglycolate (C2) Compare this to the normal reaction: RuBP + oxygen + rubisco 2 PGA (C3) Thus, rubisco has oxygenase activity as well as a carboxylase. E. What determines which process will occur? Oxygenase activity occurs when:

1 2

carbon dioxide levels are low - during periods of active photosynthesis; and oxygen levels are high - due to the activity of PSII; high light intensity.

The ratio of [carbon dioxide]/[oxygen] ultimately determines the product of the rubisco reaction. if [carbon dioxide/oxygen] = high; then it favors normal Calvin cycle if [carbon dioxide/oxygen] = low; then it favors oxygenase activity

V. Photosynthetic carbon oxidation (PCO), or, Glycolate Cycle The purpose of this pathway is to metabolize and reclaim the carbon in phosphoglycolate A. Overview of the major steps:

1 2 3 4 5 6 7 8 9

The products of rubisco oxygenase activity are phosphoglycolate and PGA; PGA enters the Calvin cycle as normal; Phosphoglycolate is dephosphorylated to glycolate and is then shuttled out of the chloroplast into the peroxisome; Recall that peroxisomes are single membrane-bound organelles that contain catalase. They also have the marker enzyme glycolate oxidase; In the peroxisome, the glycolate is oxidized to glyoxylate by glycolate oxidase. This is a redox reaction. Oxygen gets reduced to hydrogen peroxide; Catalase converts the potentially destructive hydrogen peroxide to oxygen and water; Glyoxylate is converted to glycine (an amino acid) by a transamination reaction. Glycine is transported out of the peroxisome into the mitochondrion. Two glycine molecules condense to form serine releasing carbon dioxide. This process requires NADH; Serine is further metabolized in the peroxisome to glycerate; Glycerate enters the chloroplast, is phosphorylated and enters the Calvin cycle;

B. The Highlights - The glycolate cycle: is oxidative; occurs in three organelles; reclaims some (75%), but not all, of the carbon from glycolate; carbon dioxide is released in the mitochondria and is hence the reason this is a type of "respiration". C. Why do plant photorespire? From a Darwinian perspective, wed expect that this process would have been selected against. However, the fact that so many plants do it, suggests that it may have an unappreciated function. Possibilities include: (a) salvage the carbon lost during rubisco oxygenase action; (b) mechanism to help prevent destruction by excess light.

VI. C4 Photosynthesis, or, How maize avoids photorespiration Plants that avoid photorespiration have a unique modification of photosynthesis. They are called C4 plants because the first product of carbon dioxide fixation is a 4-carbon compound, not PGA as it is in C3 plants. Examples: There are many plants that have this specialized modification. Found in many different and unrelated groups of plants which indicates that it apparently evolved independently several times. Even within a genus, some members can be C4 others C3. C4 photosynthesis is common in grasses like maize, sorghum, crabgrass and members of the Centrospermae (a closely related group of plants that includes Chenopodiaceae, Amaranthaceae, Aizoaceae, Nyctaginaceae, Portulaceae, Zygophyllaceae). Not all grasses are C4; for example, Kentucky blue grass (Poa pratensis; common lawn grass) is C3. A. How do C4 plants avoid photorespiration? The answer is simple - C4 plants separate the site of oxygen production (PSII) from rubisco (Calvin cycle). But how? PSII and rubisco are placed in different:

Cells. In typical C3 plants the chloroplasts are dispersed throughout the mesophyll. Usually there is a well-defined palisade and spongy layer. In contrast, C4's have a more or less uniform mesophyll layer with a well-developed bundle sheath around each vein. This is called Kranz anatomy, because the bundle sheaths appears like a wreath surrounding the vein. In C4 plants, the Calvin cycle activity occurs primarily in the bundle sheath cells, whereas PSII activity occurs in the mesophyll cells. Chloroplasts - The chloroplasts of C4 are dimorphic. Bundle sheath cell (BSC) chloroplasts are agranal. Recall that PSII occurs in the appressed regions of the chloroplasts. Thus, agranal chloroplasts have little PSII activity; but, they do have hi PSI activity. The mesophyll cell (MC) chloroplasts have typical granal stacking, but low rubisco activity. Thus, most carbon fixation (carbohydrate production) occurs in the bsc. Smart, eh?

B. Since C4 plants have separated the Calvin cycle PSII, there must be a mechanism to get carbon dioxide into the BSC since:

1 2

there is relatively slow diffusion to deep, interior regions of the leaf, especially considering; the ambient level of carbon dioxide is low.

In order to solve this problem, plants required a mechanism to:

fix carbon dioxide in regions of the leaf where it occurs in high concentration (i.e., MC). The enzyme that catalyzes this reaction is phosphoenolpyruvate carboxylase (PEPcase). This enzyme binds carbon dioxide (actually bicarbonate) to PEP to form oxaloacetate (reaction diagram). This reaction occurs in the cytoplasm. Note that OAA is a C4 compound. Hence these plants are called C4 - because the first product of carbon fixation is a four carbon compound. transport the fixed carbon dioxide (which is in the form of a C4 compound like malate or aspartate) from the MC to the BSC. OAA is converted to another C4 compound that, in turn, migrates to the BSC where it is decarboxylated and used in the Calvin cycle. The "leftover" C3 shuttles back to the MC to pick up another carbon dioxide and repeat the process.

C. General scheme - on overhead, covered in class D. Details Note that there are at least three different types of C4 plants. They differ in specific form in which carbon dioxide is transported. E. Advantages of C4 metabolism Plants that exhibit this type of photosynthesis are characteristic of hot, tropical environments that have a high light fluence. The advantage of C4 in these circumstances is that C4 metabolism:

1 2 3 4

avoids the photorespiratory loss of carbon improves the water use efficiency of the plants results in higher rates of photosynthesis at high temperatures improves the efficiency of nitrogen utilization (because C3 require lots of rubisco)

VII. Crassulacean Acid Metabolism - CAM plants A. Origin of the name Crassulacean refers to the Stonecrop family (Crassulaceae) and related succulents in which this process is common. To date, plants in more than 18 different families including Cactaceae (Cactus family) and Bromeliaceae (Pineapple family) have been shown to carry out CAM metabolism. Acid is derived from the observation that these plants accumulate large amounts of organic acids in the dark. Plants with CAM metabolism evolved in dry, hot, high light environments. This is largely a mechanism to conserve water. Recall the photosynthesis-transpiration compromise (paradox)? Plants in dry environments cant afford to compromise - they loose too much water opening their stomates during the day. CAM plants solved this problem by opening up the stomates at night to obtain carbon dioxide. This strategy is just the reverse of "normal" plants. But, this presents another problem - ATP & NAPDH, which are products of the light dependent reactions, are not available when the carbon dioxide is fixed. The solution to this problem was to store the carbon dioxide during the night until ATP and NADPH were available the following day. Thus, there is a temporal separation of initial carbon fixation via PEPcase and the Calvin cycle (C4 plants have a spatial separation). B. PEPcase This is the initial enzyme that fixes carbon dioxide. The product is ultimately malate which accumulates in the vacuole during the night (hence the "acid" term). C. Sequence of events. Night stomates open nocturnal transpiration (lower than diurnal) and carbon fixation by PEPcase OAA produced reduced with NADPH to malate shuttled into vacuole acid content of vacuole increases starch depleted to provide PEP for carboxylation day stomates close

transpiration decreased acid content decreases malate decarboxylated to provide carbon dioxide for Calvin cycle starch content increases

VIII. Comparison of C3, C4 and CAM Photosynthesis

Feature Leaf anatomy

C3 no distinct bundle sheath rubisco PGA (C3) one type 1: 3: 2 450-950 15 - 30 2.8 No 50 - 150 (Hi) Light saturation easily achieved Yes Yes 15-25 Low (26 soybean; 30 wheat)

C4 Kranz anatomy

CAM Usually no palisade cells, large vacuoles PEPcase OAA (C4) one type 1: 6.5: 2 18-125 (low) 2.5 - 3.0 No 0-5 (in dark) Yes Late afternoon 35 low, variable

Initial carboxylating enzyme Product of CO2 fixation Chloroplasts Theoretical energy requirements (CO2: ATP: NADPH) Transpiration ratio (g H2O/g dry wt) Photosynthesis rate (mg CO2 fixed dm-2 h-1) chl a/b ratio Requirement for sodium as a micronutrient? Carbon dioxide compensation point (ppm) Response to light Photosynthesis inhibited by oxygen? Photorespiration detectable? Temperature optimum for photosynthesis Dry matter production (bushels/acre)

PEPcase OAA (C4) dimorphic 1: 5 : 2 250-350 40 - 80 3.9 Yes 0-10 (low) No light saturation No Only in bundle sheath 30-47 High (87 maize; 50 sorghum)

Alternative Conventions for Components of Water Potential Students planning further study of plant water relations should note that the components of water potential defined in the text are sometimes given different names and symbols. In particular, the equation

(textbook Equation 3.6) is often replaced by the following equivalent equation:

In this alternative convention, P is the same as p. It is the hydrostatic pressure of the solution, and may be positive, as in turgid cells, or negative, as in xylem water. The symbol is called osmotic pressure and is the negative of s. That is, has positive values, and s has negative values. "Osmotic pressure" is the term that physical chemists, zoologists, and many others use to denote the effect of dissolved solutes on the free energy of water. Most handbooks of physics and chemistry use the term "osmotic pressure" and the symbol . The negative sign in front of in the equation above accounts for the reduction in water potential (w) by dissolved solutes. Thus s = . A very interesting, if somewhat unconventional, account of the history and physical meaning of osmotic pressure is given by Hammel and Scholander (1976). Unfortunately, some authors have mixed the conventions for s and , leading to unnecessary confusion about what is meant by the symbol . Thus, is sometimes incorrectly called osmotic

potential instead of osmotic pressure, and it may be used either as a positive quantity or as a negative quantity. Regarding matric potential, it is usually designated by the symbol t.

Can Negative Turgor Pressures Exist in Living Cells? One of the challenging aspects of understanding plant water relations is the remarkable range of pressures, both positive and negative, that occur within the bodies of plants. Negative pressures (or tensions), which depend upon the cohesive strength of water coupled with the strength of lignified cell walls to resist deformation, play an important role in water transport through the xylem. Positive pressures, which depend upon the semipermeable nature of the plasma membrane and the elastic nature of primary cell walls, occur in all hydrated living plants cells but can be especially large in sieve tubes and guard cells. Living plant cells are typically assumed to have only positive pressures (e.g., Textbook Figure 3.10). However, there appears to be no reason that negative pressures could not also occur within the cytoplasm of living plant cells. This web topic explores the existence and potential role of negative turgor pressures in plants. When a cell loses water in air, turgor declines and solute concentrations increase. At the turgor loss point (p = 0), the hydrostatic pressure in the cell sap is equal to atmospheric pressure, meaning that no net force is exerted on the cell wall. If water continues to be lost from the cell, the pressure within the cytoplasm drops below atmospheric pressure, resulting in a force imbalance that collapses the cell wall. The deformation of living cells upon desiccation is called cytorrhysis. Note that the plasma membrane is pressed against the cell wall throughout desiccation (i.e., plasmolysis does not occur) because the hydrostatic pressure in cytoplasm remains greater than the hydrostatic pressure in apoplast (see also Textbook Figure 3.9). In the living cells described above, a decrease in cell water potential below the turgor loss point is balanced by an increase in solute concentration as the volume of the cell decreases. Thus, true negative pressures do not develop. In contrast, xylem conduits have rigid cell walls that resist deformation, allowing them to sustain negative pressures without imploding. This raises the question of what happens when water is lost from living cells that have thick walls or are embedded in a rigid matrix of cells (e.g., xylem parenchyma). Might these cells resist deformation and thus develop negative turgor pressures? It is important to emphasize that this is a controversial area, due in large part to the absence of direct measurements of negative turgor pressures within cells (Tyree 1976). However, before reviewing the evidence for negative turgor pressures, it is worthwhile to consider what physiological effects might result from the development of such negative pressures in living cells. The major outcome of negative turgor is that it allows stiff-walled cells to decrease in water potential without undergoing major changes in cell volume or osmotic concentration. Because cytorrhysis might cause physical damage to the wall and/or cell membranes, while the high concentrations of solutes resulting from the reduction in cell volume might adversely affect the conformation of membranes and proteins, it is possible to imagine a physiological role or benefit for the development of negative turgor pressures in plant cells. We now turn this around and ask if there are any downsides to the generation of negative turgor pressures in living cells? In the xylem, the primary issue associated with negative pressures is cavitation; could this also be a concern for living cells? The primary mechanism for cavitation in plants is air seeding (see Chapter 4), reflecting the fact that the probability of the de novo formation of gas voids in water (either by homogeneous or heterogeneous nucleation) is extremely low (Pickard 1983). In air seeding, air is sucked in through the cell wall, where it then nucleates the transition to the vapor phase. For air seeding to occur in a living cell experiencing negative pressures, air would have to

be pulled through the very small pores of the cell wall. While one can imagine this happening, the movement of air across the cell wall would result in plasmolysis and thus the immediate release of any tension in the cytoplasm because the plasma membrane is not capable of withstanding any significant pressure. Other costs associated with the ability of living cells to generate negative pressures include the metabolic costs of producing rigid cell walls, as well as any limitations lignified walls might place on physiological function. In addition, a strategy for avoiding cell damage due to desiccation via the generation of negative pressures might impose limitations on cell size. The strength of a cell to withstand collapse (and thus generate negative pressures) is inversely proportional to cell size. Evidence for negative turgor pressure in plants is limited, in part reflecting the fact that few researchers have devoted much attention to this issue. Living cells with flexible (unlignified) cell walls deform relatively easily upon desiccation and thus do not appear to support negative pressures. However, measurements of the forces needed to collapse cell walls suggest that living cells with thick walls can withstand forces in the range of 1.0 MPa (Oertli et al. 1990). Visual examination of tissues adapted to withstand very cold temperatures also provides indirect evidence for negative turgor. For example, frozen ray parenchyma cells often do not exhibit significant deformation, despite the very strong desiccatory effects (low water potential) of extracellular ice. One can hypothesize that the existence of negative pressures within these cells acts to balance the water potential gradient across the cell membrane (i.e., between the cytoplasm and ice formed within the apoplast). In summary, negative turgor pressure remains an intriguing but little-studied area of plant water relations. While it does not appear to form in cells such as the leaf mesophyll, that can deform easily, its existence in living cells with either lignified cell walls or where the cell is embedded in a matrix of lignified tissues (e.g., living cells within wood) cannot be ruled out. The potential benefits of negative turgor pressures in terms of preventing mechanical and osmotic damage associated with severe desiccation makes this topic worthy of further study.

Measuring Water Potential Plant scientists have expended considerable effort in devising accurate and reliable methods for evaluating the water status of a plant. Four instruments that have been used extensively to measure w , s , and p are described here: psychrometer, pressure chamber, cryoscopic osmometer, and pressure probe. Psychrometer (w measurement) Psychrometry (the prefix "psychro-" comes from the Greek word psychein, "to cool") is based on the fact that the vapor pressure of water is lowered as its water potential is reduced. Psychrometers measure the water vapor pressure of a solution or plant sample, on the basis of the principle that evaporation of water from a surface cools the surface.

Web Figure 3.5.A Diagram illustrating the use of isopiestic psychrometry to measure the water potential of a plant tissue. (Click image to enlarge.) One psychrometric technique, known as isopiestic psychrometry, has been used extensively by John Boyer and coworkers (Boyer and Knipling 1965) and is illustrated in Web Figure 3.5.A. Investigators make a measurement by placing a piece of tissue sealed inside a small chamber that contains a temperature sensor (in this case, a thermocouple) in contact with a small droplet of a standard solution of known solute concentration (known s and thus known w). If the tissue has a lower water potential than that of the droplet, water evaporates from the droplet, diffuses through the air, and is absorbed by the tissue. This slight evaporation of water cools the drop. The larger the difference in water potential between the tissue and the droplet, the higher the rate of water transfer and hence the cooler the droplet. If the standard solution has a lower water potential than that of the sample to be measured, water will diffuse from the tissue to the droplet, causing warming of the droplet. Measuring the change in temperature of the droplet for several s olutions of known w makes it possible to calculate the water potential of a solution for which the net movement of water between the droplet and the tissue would be zero (Web Figure 3.5.B) signifying that the droplet and the tissue have the same water potential.

Web Figure 3.5.B The temperature of the sensor in the psychrometer shown in Web Figure 3.5.A depends on the water potential of the solution relative to the water potential of the tissue sample. (Click image to enlarge.) Psychrometers can be used to measure the water potentials of both excised and intact plant tissue. Moreover, the method can be used to measure the s of solutions. This can be particularly useful with plant tissues. For example, the w of a tissue is measured with a psychrometer, and then the tissue is crushed and the s value of the expressed cell sap is measured with the same instrument. By combining the two measurements, researchers can estimate the turgor pressure that existed in the cells before the tissue was crushed (p = w s). A major difficulty with this approach is the extreme sensitivity of the measurement to temperature fluctuations. For example, a change in temperature of 0.01C corresponds to a change in water potential of about 0.1 MPa. Thus, psychrometers must be operated under constant temperature conditions. For this reason, the method is used primarily in laboratory settings. There are many variations in psychrometric technique; int erested readers should consult Brown and Van Haveren 1972, Slavik 1974, and Boyer 1995. Pressure chamber (w measurement) A relatively quick method for estimating the water potential of large pieces of tissues, such as leaves and small shoots, is by use of the pressure chamber . This method was pioneered by Henry Dixon at Trinity College, Dublin, at the beginning of the twentieth century, but it did not come into widespread use until P. Scholander and coworkers at the Scripps Institution of Oceanography improved the instrument design and showed its practical use (Scholander et al. 1965). In this technique, the organ to be measured is excised from the plant and is partly sealed in a pressure chamber (Web Figure 3.5.C). Before excision, the water column in the xylem is under tension. When the water column is broken by excision of the organ (i.e., its tension is relieved allowing its p to rise to zero), water is pulled rapidly from the xylem into the surrounding living cells by osmosis. The cut surface consequently appears dull and dry. To make a measurement, the investigator pressurizes the chamber with compressed gas until the distribution of water between the living cells and the xylem conduits is returned to its initial, pre-excision, state. This can be detected visually by observing when the water returns to the open ends of the xylem conduits that can be seen in the cut surface. The pressure needed to bring the water back to its initial distribution is called the balance pressure and is

readily detected by the change in the appearance of the cut surface, which becomes wet and shiny when this pressure is attained.

Web Figure 3.5.C The pressure chamber method for measuring plant water potential. The diagram at left shows a shoot sealed into a chamber, which may be pressurized with compressed gas. The diagrams at right show the state of the water columns within the xylem at three points in time: (A) The xylem is uncut and under a negative pressure, or tension. (B) The shoot is cut, causing the water to pull back into the tissue, away from the cut surface, in response to the tension in the xylem. (C) The chamber is pressurized, bringing the xylem sap back to the cut surface. (Click image to enlarge.) The pressure chamber is often described as a tool to measure the tension in the xylem. However, this is only strictly true for measurements made on a non-transpiring leaf or shoot (for example, one that has been previously enclosed in a plastic bag). When there is no transpiration, the water potential of the leaf cells and the water potential in the xylem will come into equilibrium. The balancing pressure measured on such a non-transpiring shoot is equal in magnitude but opposite in sign to the pressure in the xylem (p). Because the water potential of our non-transpiring leaf is equal to the water potential of the xylem, one can calculate the water potential of the leaf by adding together p and s of the xylem, provided one collects a sample of xylem sap for determination of s. Luckily s of the xylem is usually small (> -0.1 MPa) compared to typical midday tensions in the xylem (p of 1 to 2 MPa). Thus, correction for the s of the xylem sap is frequently omitted. Balancing pressure measurements of transpiring leaves are more difficult to interpret. The fact that water is flowing from the xylem to the leaf means that differences in water potential must exist. When the transpiring leaf or shoot is cut off, the tension in the xylem is instantly relieved and water is drawn into the leaf cells until the water potentials of the xylem and the leaf cells come into equilibrium. Because the total volume of the leaf cells is much larger than the volume of sap in the xylem, this equilibrium water potential will be heavily weighted towards that of the leaf. Thus, any measurement of the balancing pressure on such a leaf or shoot will result in a value that is approximately the water potential of the leaf, rather than the tension of the xylem. (To be exact, one would have to add the s of the xylem sap to the negative of the balancing pressure to get the leaf water potential.) One can explore the differences between the water potential of the xylem and the water potential of a transpiring leaf by comparing balancing pressures measured on covered (i.e., non-transpiring) versus uncovered (transpiring) leaves. Pressure chamber measurements provide a quick and accurate way of measuring leaf water potential. Because the pressure chamber method does not require delicate instrumentation or temperature control, it has been used extensively under field conditions (Tyree and Hammel 1972). For a more complete description of the theory and operation of the pressure chamber see Boyer, 1995. Cryoscopic osmometer (s measurement)

The cryoscopic osmometer measures the osmotic potential of a solution by measuring its freezing point. Solutions have colligative properties that collectively depend on the number of dissolved particles and not on the nature of the solute. For example, solutes reduce the vapor pressure of a solution, raise its boiling point, and lower its freezing point. The specific nature of the solute does not matter. One of the colligative properties of solutions is the decrease in the freezing point as the solute concentration increases. For example, a solution containing 1 mol of solutes per kilogram of water has a freezing point of 1.86C, compared with 0C for pure water. Various instruments can be used to measure the freezing-point depression of solutions (for two examples, see Prager and Bowman 1963, and Bearce and Kohl 1970). With a cryoscopic osmometer, solution samples as small as 1 nanoliter (10 9 L) are placed in an oil medium located on the temperature-controlled stage of a microscope (Web Figure 3.5.D). The very small sample size allows sap from single cells to be measured and permits rapid thermal equilibration with the stage. To prevent evaporation, the investigator suspends the samples in oil-filled wells in a silver plate (silver has high thermal conductivity). The temperature of the stage is rapidly decreased to about 30 C, which causes the sample to freeze. The temperature is then raised very slowly, and the melting process in the sample is observed through the microscope. When the last ice crystal in the sample melts, the temperature of the stage is recorded (note that the melting and freezing points are the same). It is straightforward to calculate the solute concentration from the freezing-point depression; and from the solute concentration (cs), s is calculated as RTcs .This technique has been used to measure droplets extracted from single cells (Malone and Tomos 1992).

Web Figure 3.5.D A cryoscopic osmometer measures the concentration of total dissolved solutes by measuring the freezing-point depression of a solution. (A) Very small liquid samples are loaded onto

the temperature-controlled stage of a microscope. (B) When the temperature is quickly reduced, the samples supercool and freeze. (C) Slowly warming the stage causes the samples to thaw. The temperature at which the last ice crystal melts provides a measure of the melting point of the sample. (Click image to enlarge.) Pressure probe (p measurement) If a cell were as large as a watermelon or even a grape, measuring its hydrostatic pressure would be a relatively easy task. Because of the small size of plant cells, however, the development of methods for direct measurement of turgor pressure has been slow. Using a micromanometer, Paul Green at the University of Pennsylvania developed one of the first direct methods for measuring turgor pressure in plant cells (Green and Stanton 1967). In this technique, an air-filled glass tube sealed at one end is inserted into a cell (Web Figure 3.5.E). The high pressure in the cell compresses the trapped gas, and from the change in volume one can readily calculate the pressure of the cell from the ideal gas law (pressure volume = constant). This method works only for cells of relatively large volume, such as the giant cell of the filamentous green alga Nitella. For smaller cells, the loss of cell sap into the glass tube is sufficient to deflate the cell and this yields artifactually low pressures.

Web Figure 3.5.E Use of the micromanometer, a pressure probe, to measure cell turgor pressure. Nitella cells (which are particularly largeabout 100 mm in diameter and many centimeters long)

were used for these measurements. (After Green 1968.) For higher plant cells, which are several orders of magnitude smaller in volume than Nitella, a more sophisticated device, the pressure probe , was developed by Ernest Steudle, Ulrich Zimmermann, and their colleagues in Germany (Husken et al. 1978). This instrument is similar to a miniature syringe (Web Figure 3.5.F). A glass microcapillary tube is pulled to a fine point and is inserted into a cell. The microcapillary is filled with silicone oil, a relatively incompressible fluid that can be readily distinguished from cell sap under a microscope. When the tip of the microcapillary is first inserted into the cell, cell sap begins to flow into the capillary because of the initial low pressure of that region. Investigators can observe such movement of sap under the microscope and counteract it by pushing on the plunger of the device, thus building up a pressure. In such fashion the boundary between the oil and the cell sap can be pushed back to the tip of the microcapillary. When the boundary is returned to the tip and is held in a constant position, the initial volume of the cell is restored and the pressure inside the cell is exactly balanced by the pressure in the capillary. This pressure is measured by a pressure sensor in the device. Thus the hydrostatic pressure of individual cells may be measured directly.

Web Figure 3.5.F Diagram of the simplest pressure probe (not to scale). The primary advantage of this method over the one shown in Web Figure 3.5.E is that cell volume is minimally disturbed. Minimal disturbance is of great importance for the tiny cells that are typical of higher plants, in which loss of even a few picoliters (1012 L) of fluid can substantially reduce turgor pressure. (Click image to enlarge.)

This method has been used to measure p and other parameters of water relations in cells of both excised and intact tissues of a variety of plant species (Steudle 1993). The primary limitation of this method is that some cells are too small to measure. Furthermore, some cells tend to leak after being stabbed with the capillary, and others plug up the tip of the capillary, thereby preventing valid measurements. The pressure probe has also been adapted to measure positive and negative values of p in the xylem (Heydt and Steudle 1991). However, technical problems with cavitation (see textbook Chapter 4) limit the measurement of negative p by this technique.

Wilting and Plasmolysis Plasmolysis is the separation of plant cell cytoplasm from the cell wall as a result of water loss. It is unlikely to occur in nature, except in severe conditions. Plasmolysis is induced in the laboratory by immersing a plant cell in a strongly saline or sugary solution, so that water is lost by osmosis. If onion epidermal tissue is immersed in a solution of calcium nitrate, cells rapidly lose water by osmosis and the protoplasm of the cells shrinks (Web Figure 3.7.A). This occurs because the calcium and nitrate ions freely permeate the cell wall and encounter the selectively permeable plasma membrane. The large vacuole in the center of the cell originally contains a dilute solution with much lower osmotic pressure than that of the calcium nitrate solution on the other side of the membrane. The vacuole thus loses water and becomes smaller. The space between the cell membrane and the cell wall enlarges and the plasma membrane and the protoplasm within it contract to the center of the cell. Strands of cytoplasm extend to the cell wall because of plasma membrane-cell wall attachment points. Plasmolysed cells die unless they are transferred quickly from the salt or sugar solution to water.

Web Figure 3.7.A Plasmolysis of an Epidermis Cell of Allium cepa after Addition of Calcium Nitrate. Both chloroplast and vacuole become unevenly shaped. Strands of cytoplasm remain between the shrinking protoplasm and the neighboring cells. Courtesy of Dr. Peter v. Sengbusch, University of Hamburg.

Soil Hydraulic Conductivity and Water Potential Soil hydraulic conductivity is a function of the water potential of the soil. Conductivity measures the ease with which water moves through the soil. As water content (and hence the water potential) decreases, the hydraulic conductivity decreases drastically (Web Figure 4.2.A, note the logarithmic scales).

Web Figure 4.2.A Soil hydraulic conductivity as a function of the water potential of the soil. Conductivity measures the ease with which water moves through the soil. The decrease in conductivity as the soil dries is due primarily to the movement of air into the soil to replace the water. As air moves in, the pathways for water flow between soil particles become smaller and more tortuous, and flow becomes more difficult. The overall shape of this curve is representative of many soils, but the shape for a particular soil may be influenced by the size distribution of its particles and by its organic matter content. The field capacity is the amount of water the soil is able to retain against gravitational forces. The permanent wilting point is the soil water potential value at which plants cannot regain turgor pressure even at night, in the absence of transpiration. The decrease in conductivity as the soil dries is due primarily to the movement of air into the soil to replace the water. As air moves in, the pathways for water flow between soil particles become smaller and more tortuous, and flow becomes more difficult.

The overall shape of this curve is representative of many soils, but the shape for a particular soil may be influenced by the size distribution of its particles and by its organic matter content. The field capacity (labeled on the curve) is the maximum amount of water the soil is able to retain against gravitational forces. The permanent wilting point (labeled on the curve) is the soil water potential value at which plants cannot regain turgor pressure even at night, in the absence of transpiration. Leaf Transpiration and Water Vapor Gradients Transpiration from the leaf depends on two major factors: the difference in water vapor concentration between the leaf air spaces and the the external air and, the diffusional resisitance (r) of this pathway. This concept of transpiration is analogous to the flow of electrons in an electric circuit. Indeed, an electrical analog is commonly used as a model for water vapor loss from the leaf (Web Figure 4.4.A). In this analog, resistances are associated with each part of the pathway; the major ones are the resistance at the stomatal pore (rs) and the resistance due to the layer of unstirred air at the surface of the leaf (rb) (the so-called boundary layer). Transpiration rate (E, in mol m2 s1) may be related to diffusional resistances (r, in s m1) by the following equation (Web Equation 4.5.1):

Let's examine the factors in Equation 4.5.1 in greater detail. The difference in water vapor concentration is expressed as cwv (leaf) cwv (air). (Sometimes vapor pressures are used instead of concentrations, and the difference is called the water vapor pressure deficit. Water vapor pressure (pwv) is measured in kilopascals (kPa) and is proportional to water vapor concentration (Web Table 4.5.1) where cwv (leaf) is the water vapor concentration inside the leaf and cwv (air) is the water vapor concentration of the air outside the leaf, both expressed in moles per cubic meter (mol m 3). Resistance (r) is the inverse of conductance; that is, resistance = 1/conductance. Thus, a high resistance is the same as a low conductance. Expression of this value in terms of resistances is preferred over expression in terms of conductances in some instances because resistances in series may be summed to calculate a total resistance, as in the above equation, whereas a similar calculation of conductances in series is more complicated. In the leaf, the total resistance is due mostly to the diffusion limitation imposed by the stomatal pore, but other parts of the pathway for water vapor loss, such as the boundary layer (which we will discuss shortly), may contribute significantly to r. Vapor pressures and concentrations are equivalent; in our analysis we will use the latter. The water vapor concentration of bulk air (cwv (air)) can be readily measured, but that of the leaf (cwv (leaf)) is more difficult to assess. We can estimate it by assuming that the air space in the leaf is close to water potential equilibrium with the cell wall surfaces. This approximation is not strictly true, because water is diffusing away from these surfaces. However, it introduces little error because the major resistance to vapor loss is at the stomatal pore. Moreover, the volume of air space inside the leaf is small, whereas the wet surface from which water evaporates is comparatively large. Air space volume is about 5% of the total leaf volume for pine needles, 10% for corn leaves, 30% for barley, and 40% for tobacco leaves. The internal surface area from which water evaporates may be from 7 to 30 times the external leaf area. This high ratio of surface area to volume makes for rapid vapor equilibration inside the leaf.

Making the assumption of equilibrium, we can calculate the water vapor concentration in the leaf air spaces if we know (1) the water potential of the leaf (which is the same as the water potential of the wall surfaces from which water is evaporating) and (2) the leaf temperature. Let's take as an example a leaf with a water potential of 1.0 MPa. To reach vapor equilibrium, water evaporates from the cell wall surfaces until the water potential of the air inside the leaf equals the water potential of the leaf. The water potential of the air is given by the following equation (Web Equation 4.5.2):

where R is the gas constant, T is temperature (in degrees Kelvin), Vw is the partial molar volume of liquid water, and RH is the relative humidity of the air. Relative humidity is the water vapor concentration expressed as a fraction of the saturation water vapor concentration, cwv(sat.) (Web Equation 4.5.3):

RH varies between 0 and 1; RH multiplied by 100 is the percentage relative humidity. Web Table 4.5.1 shows RH values as a function of water potential, calculated from Web Equation 4.5.2. This table shows that the air spaces of living leaves must have a high RH, a value of nearly 1 (100%), when water potentials are in the physiological range. Moreover, outside air, with RH of, for example, 0.5 (50%), has a remarkably low water potential.

To convert from RH to cwv, we need to know cwv(sat.). The saturation water vapor concentration is strongly dependent on temperature. As the air temperature rises, the water-holding capacity of air increases sharply, (see textbook Figure 4.11). In the range of 10 to 35C, an increase in air temperature of 12C doubles the water vapor concentration of saturated air. This is an important observation. If our leaf with a water potential of 1.0 MPa warms up abruptly from 20 to 32C, the relative humidity in the leaf air space drops abruptly from 99.3 to almost 50%. This drop in RH results because the water-holding capacity of the air, cwv(sat.), doubles. As a result of the drop in RH, water will evaporate in the air space until RH returns to a value of 99.3% and the air is again in water potential equilibrium with the leaf. As a consequence of this change in temperature, cwv(leaf) (the water vapor concentration of the leaf air space) increases from 0.95 to 1.87 mol m3, which makes for a steeper concentration difference driving the diffusional loss of water from the leaf. For this reason, leaf temperature is an important determinant of the transpiration rate. Textbook Table 4.2 illustrates how RH, cwv, and water potential change at various points in the transpiration pathway. We see that cwv decreases along each step of the pathway from the cell wall surface to the bulk air outside the leaf. Keep in mind that RH can increase along part of this pathway because the external air temperature may be lower than the temperature of the leaf. The important points to remember are (1) that the driving force for water loss from the leaf is the absolute concentration difference (difference in cwv, not in RH), and (2) that this difference depends on leaf temperature.

Consequences of autotrophic nutrition - Motility is no longer required; Or possible. One of the main reasons for motility is to obtain food. Since the nutrients required by plants are "omnipotent" there was never an evolutionary pressure for "motility." Lets quickly compare the nutrients used by plants and animals:

Table 1: Comparison of Plant & Animal Nutrition

Nutrient form of uptake concentration distribution

Plant inorganic (CO2, water, ions) dilute (i.e., CO2 = 0.03%) omnipotent

Animal organic (proteins, carbohydrates, fats) concentrated localized