Dynamics of Leaf Photosynthesis: Rapid Response Measurements and Their Interpretations

Preface

The evolution of new scientific approaches is analogous to general principles of biological evolution. Evolution of a new species occurs with the highest probability under stress conditions in a relatively isolated population, it needs an occasional strong mutation to take place and favourable conditions for the mutant to develop. These conditions were present in the former Soviet Estonia during the 1960s and 1970s, where biological science developed under the conditions of limiting supplies, and in relative isolation from the West.

A group of geophysicists and actinometrists in the Institute of Astrophysics and Atmospheric Physics of the Academy of Science of the former Soviet Estonia in Tartu, under the leadership of Dr Juhan Ross, had developed sophisticated instruments for the measurement of radiation reflected from the plant canopy. These studies caught the interest of Professor Anatoli Aleksandrovitch Nichiporovich, who suggested cooperation with the Moscow Timiryazev Institute of Plant Physiology to measure photosynthetic radiation in plant canopies and to work out a theory of canopy productivity. We joined the group after we had both graduated from the Department of Physics, University of Tartu (Agu Laisk in 1961 and Vello Oja in 1966), as did most of our colleagues. It was pure chance that the first objects of our investigation were plants. We were not biologists but just educated physicists who were applying their skills in plant science. A mutation had to take place to convert physicists into biologists.

For Agu Laisk, this mutation happened at a field station where the group was measuring light response curves of photosynthesis of maize leaves for inputs to productivity theory. (Nikita Khruschev had seen high-productivity maize crops in Idaho and wanted to have the same all over the Soviet Union, including Estonia, where we have only three months of summer). A lower canopy leaf was left in the instrument while we went to lunch. When we returned, the low maximum photosynthesis rate of the leaf had increased by about a factor of three. The realisation dawned that a leaf is not a constant physical object like a crystal but is able to adjust its responses to environmental conditions. The moment we asked, ‘What determines the rate of leaf photosynthesis?’ we became biologists, and this simple question has led our research ever since.

Of course we had discovered the already well known stomatal regulation of photosynthesis. However, we soon became one of the first groups (simultaneously with Olle Bjorkman) to base photosynthetic data on estimates of intercellular CO2 concentration. Our team of two — Vello Oja creating instruments and carrying out the most sophisticated experiments and Agu Laisk experimenting, interpreting, writing and promoting our program abroad — has survived over 30 years. We like to think of it as an efficient form of research, something like the cooperation of the C4 and C3 cycles in C4 photosynthesis. Most recently Bakhtijor Rassoulov from Dushanbe, Tajik Republic, our most successful student, has joined us, to apply his experience and make our gas system commercially available.

How did these mutants survive in Soviet Estonia? The late Aksel Kipper, a famous Estonian astronomer and Director of the Institute of Astrophysics and Atmospheric Physics decided that good science should survive, even if it was biology in an institute of astronomy. It took about 15 years to prove the quality of our research. Among other things, we pioneered an understanding of the effects of uneven stomatal opening on calculations of intercellular CO2 concentrations, the relationships of photosynthesis and photorespiration, the fast CO2 binding to ribulosebisphosphate carboxylase in vivo, and the limitations caused by regeneration of the CO2 acceptor ribulose bisphosphate and the adaptation of mesophyll cell photosynthesis to the light profile in the leaf. Our gas exchange measurement system was very different from conventional systems: the leaf chamber was very small and the leaf was attached to the glass window. We had two gas systems and one leaf chamber, contrary to the common approaches where many leaf chambers were monitored by one system. Evidently, the gas system was too different from others and our results were little known in the West because they were mostly published in Russian.

Our international recognition and collaboration developed slowly. In 1966 Agu Laisk was allowed to visit his father Heino Laisk who then lived in Cleveland, Ohio, and whom he had not seen since 1944. Before departure, A.A. Nichiporovitch recommended that he also visit Ed Lemon in Cornell University. The visit was a success. The next year Ed Lemon visited us in Tartu and in 1976 Laisk was invited to a Gordon Conference in New Hampton, NH. These contacts were not always successful. Although Ed Lemon arranged an invitation to participate in the first experiments in a new phytotron opened in New Zealand in 1971, it was strongly recommended to Laisk that he refuse the invitation on the grounds that he was too busy with sensitive research contracts in Estonia (we were never involved in military-oriented contracts!).

A real breakthrough came in 1983 when we organized an international meeting on photosynthesis in Tallinn, after the International Photosynthesis Congress in Brussels. We had an unexpected number of foreigners, some of them, for example Ulrich Heber from Wiirzburg and David Walker from Sheffield, later became our close friends. Many subsequent developments in our research were suggested and initiated by these people. For example, David introduced us to the primary processes of photosynthesis and fluorescence measurement during Agu’s visits to Sheffield. Ulrich has sponsored a long and fruitful cooperation on the measurements of CO2 solubility in leaves during our visits to Wiirzburg. The gas system in Wiirzburg University, which senses CO2 fluxes through changes in O2 concentration, is Vello’s most sophisticated creation.

Some experiments, first published in this book, were carried out in cooperation with Dick Peterson from Connecticut Agricultural Experiment Station (800 nm absorbance measurements) and Louise Anderson from Illinois State University, Chicago (dark-light induction of photosynthesis) who visited our laboratory in Tartu and with whom we had a very pleasant and creative cooperation. A fruitful cooperation has developed with Gerry Edwards (Washington State University), who has introduced me to the complex biochemistry of C4 plants. Other original experiments reported here were carried out by our former student Olavi Kiirats and by our present co-worker Hillar Eichelmann, to whom we are indebted.

An outstanding position among our friends belongs to Barry Osmond. He came to the Tallinn conference with several colleagues (a visit that attracted the attention of, and led to several interviews with, the Australian Security Intelligence Organisation). Our subsequent visits to Canberra opened the way, facilitated by a quirk of Soviet bureaucracy. In the former Soviet Union it was a rule that only those who had already been on scientific work abroad were allowed to go again. Since Estonia regained independence in 1991, so many of these problems seem like a bad dream. The publication of this book is due to support and hard work by Barry, whose contribution is by far more than that of an editor. We have experienced several false starts, and every rewriting has changed the character of the book.

The present version is a rather condensed description of the principles and of the design of some rapid-response gas exchange measurement systems, plus the description of an extensive series of applications. However, a good scientific instrument alone is not much use. Just as a Steinway piano can be an awkward piece of furniture in one’s living room until it comes alive in the hands of a good pianist, so outstanding results can be obtained with a scientific instrument only after long training. Different applications of the fast-response gas exchange measurements described in this book are meant to guide those who wish to become more skilled in dynamic leaf gas exchange measurements to more sophisticated levels of investigation. The interpretation of results needs even more experience, and a willingness to integrate in vivo and in vitro approaches. Therefore, our examples of applications are followed by interpretations. This way the book, initially intended to be a guide to methods, now almost completely reviews our contributions to the understanding of leaf photosynthesis. It is a guide to new approaches in the construction of equipment and to the scientific conclusions that can be drawn from rapid-response measurements.

We are especially indebted to our wives Tiiu Laisk and Malle Oja, because they have done their best to support our efforts on the frontier of science by creating stress-free homes with a lovely atmosphere.

Agu Laisk

Vello Oja

Tartu

1

Conceptual background

A plant leaf is a complex organ that has evolved to carry out photosynthesis in air and to feed the products to other organs of the plant. On the one hand, this complexity can make the investigation of photosynthesis of leaves difficult. On the other hand, intact leaves contain all the natural regulatory systems and the cell environment is relatively undisturbed by experimental procedures. The fear of using leaves for detailed mechanistic studies of photosynthesis is often exaggerated. In fact, there is no better organization of photosynthesizing cells for experimental purposes than that of intact leaves. Cells in leaves are organized in a thin layer in a porous way, making possible the fastest diffusion of gases, while still maintaining good water and nutrient supplies. Therefore, the new insights into the process of photosynthesis obtained using intact leaves are the most reliable biologically, provided that complexities introduced by the structure of the leaf are correctly considered in the data interpretation.

Considering the complexities means first of all considering the physical processes of gas transport and light propagation in the leaf tissue, but also the possible internal limitations to photosynthesis caused by assimilate transport and secondary metabolic processes. True, the presence of the epidermis and the frequently unpredicted stomatal reactions need to be considered, but this can be done by parallel measurements of CO2 exchange and transpiration. The high concentration of chlorophyll in leaves can be a problem for the optical measurements, causing steep gradients of light in the leaf cross-section and reabsorption of chlorophyll fluorescence. However, in many types of leaves the photosynthetic characteristics seem to be adapted to the average light intensity in the given layer of cells, which makes the leaf responses very close to the response of a single cell. There is also the option of using optically thinner leaves.

If one attempts to avoid these difficulties by using protoplasts, chloroplasts or thylakoid preparations from leaves, one only introduces other difficulties. A mixture of the cells or chloroplasts with different photosynthetic parameters makes the system even more inhomogenous than the original leaf, where the same cells or chloroplasts were arranged in a certain way, determined by genetic and environmental factors. Probably the greatest limitation of leaves as experimental objects is the self-protecting capacity of living cells against the invasion of external chemicals. This makes the application of inhibitors to leaves rather uncertain because one never knows how effectively or uniformly they penetrate to the site of action. However, the modern techniques of molecular biology can create transgenic plants with changed levels of specific proteins at specific locations, an approach which is biologically cleaner than the use of inhibitors.

In this book we show how rapid-response techniques can be used to investigate mechanisms of photosynthesis using intact leaves. We focus on the use of gas exchange measurements of CO2 and O2 to analyse metabolic biochemistry, and on optical measurements of chlorophyll fluorescence and 800 nm absorbance, the signals that carry information about the functioning of electron transport and photobiochemistry in the photosystems. We start by describing the physical principles of these signals and the limitations these impose on the measurement methods. We then proceed to describe some advanced experimental systems for simultaneous gas exchange and optical measurements on leaves. A large part of the book is dedicated to the description of specific applications and the interpretation of the data. The examples of interpretation are meant not only as tutorial lessons for repeating these experiments with different leaf systems and environments, but are presented as steps that have advanced scientific understanding of the leaf photosynthetic processes. Our interpretations may be provocative, but we hope readers will be stimulated to design their own experiments and contribute further to changing the way we think about photosynthesis. A specific feature of our approach is that not only steady-state but transient processes are considered as a source of information. It is noteworthy that only transients can reveal the information about the internal pools of metabolites that are active in photosynthetic metabolism.

1.1 WHY RAPID RESPONSE?

We are accustomed to thinking in terms of steady-state situations because these seem to be established points of reference. However, life is dynamic and all of its sub-processes are transient. Much the same can be said of our understanding of key life processes, such as photosynthesis. New techniques continually alter our ability for both reductionist insights and integrative, holistic interpretation. The concept of steady state is relevant only to relaxation times (Fig. 1.1). Actually, processes with slower relaxation are defined as steady state (or, better, quasi-steady state).

During conventional measurements of the CO2 or light response curves of leaf photosynthesis we usually observe transients after a change in light intensity or CO2 concentration. These transients finally fade out and the signals approach levels which are considered to be steady state. In data analysis the rapid transients are usually ignored, although they contain information about processes that have shorter relaxation times than those which we thought to define as the steady state. If we put aside our patience as a limiting factor, the objective limits for measurements of slow processes are set by the drifts of the measuring instrument when these approach the magnitude of drifts in the measured process itself.

If we take a wider approach and are interested in photosynthetic processes with different relaxation times, then we should record the transients and extract information from them. The maximum speed of processes which we can record is limited by the relaxation time of our measuring instrument. In the hierarchy of the photosynthetic processes, the window which we are able to observe is set by the instrument response time on the one hand and by the instrument stability on the other. Widening the window towards rapid processes considerably increases the amount of information which can be revealed per unit of experimentation time. In this book we will discuss the principles of construction of fast-response instruments for measurements of leaf gas exchange and optical properties. Most of the components used in these instruments are similar to those used to construct the more familiar steady-state gas exchange measurement systems (Eckardt 1968a; Mooney et al. 1971; Sěsták et al. 1971; Pearce et al. 1976; Field et al. 1989). The principles of measurement do not differ, but for fast-response studies, special attention has to be given to volume and flow relationships of the system.

The word ‘system’ is widely used to describe the gas exchange measurement apparatus since there is no single sensor of photosynthesis available. The rate of photosynthesis is calculated from several measurements (concentrations, flow rates etc.) which are made by the different instruments which compose a system (Field et al. 1989). At the outset it is important to consider leaf areas and shapes, the necessary threshold sensitivity and response time, plus the characteristics of available gas analysers. In addition to the range of measurements needed one must also consider requirements for leaf preconditioning, and the range of external parameters to which the leaf is exposed, such as light intensity and spectral quality, CO2 concentration, O2 concentration, water vapour concentration, leaf temperature, and the possibility of rapid killing of the leaf. Studies of transients in leaves under these controlled conditions give insights which help us to build a logical (or mathematical) model of photosynthesis. We have developed this approach extensively, describing it as ‘a kinetic method for studying leaf photosynthesis’ (Laisk 1977, 1991), and with it the corresponding mathematical models (Laisk et al. 1989a; Laisk and Walker 1989; Laisk and Eichelmann 1989; Laisk 1993; Laisk et al. 1997).

Figure 1.1 An arbitrary scaling of relevant states, defined by approximate dimensions, and processes, defined by approximate relaxation times (Osmond and Chow 1988).

Tremendous developments in non-intrusive optically based measurements of photosynthetic processes have characterized the last decade of photosynthetic research (Schreiber et al. 1986, 1988). When these are combined with gas exchange systems one has a very powerful tool for the understanding of the interactions between the light and dark reactions of photosynthesis. Variations of light intensity and spectral quality are important in kinetic studies of photosynthesis. Single and multiple turnover pulses produce additional information. Furthermore, optical signals from the leaf surface, such as chlorophyll fluorescence and absorption changes in different spectral regions, can be measured in parallel with gas exchange. Multiple simultaneous measurements of optical signals, like chlorophyll fluorescence and 800 nm transmission, and of both O2 and CO2 exchange, give new insights into photosynthetic mechanisms (Walker and Osmond 1989). It is very difficult to achieve these measurements with conventionally projected light sources. The principal difficulties are uniformity of the primary beam, and coincidence of measuring beams. These difficulties are largely overcome by using fibre optic light guides.

In the rapid-response system we have had to abandon efforts to maintain near-natural conditions in the chamber. Instead, the system has been designed for laboratory studies of leaf responses to various experimental conditions created in the leaf chamber, which are precisely known. Such compromises are often necessary if we are to understand the fundamental mechanisms in complex systems. For example, the leaf disc O2 electrode system described by Walker (1987) uses a leaf chamber with a cut leaf section and a small volume of saturating CO2 (1–10%), and this system has permitted significant advances in our understanding of photosynthetic regulation, often through the analysis of oscillations (transients) in gas exchange and fluorescence.

It is a common belief that the more data points we measure the better. If we are dealing with a physical system, such as a crystal, then increasing the number of measurements would certainly increase the reliability of the results. But the plant is a living organism that changes gradually during the experiment and often one has to compromise the number of observations. Rapid-response techniques can reduce the time taken for experimental observations, thereby minimizing the biological changes that take place in the leaf, compared to the changes that occur during the prolonged steady-state measurements. For example, measurements done at the end of a 6-hour experiment are not comparable with those done on the same leaf at the beginning of the experiment, because those slower relaxing processes, such as carbohydrate accumulation and diurnal rhythms, significantly alter the metabolic context of photosynthesis. Furthermore, after-effects caused by the experimental procedures (such as altered gas composition or saturating light pulses) accumulate during the experiment. This means that the number of points measured on a curve must be a reasonable compromise between higher experimental accuracy and the effects of the measurements on biological stability.

Increasing the accuracy of the measurement system, rather than the number of measurements themselves, is the best way to reduce experimental error in a dynamic system. With confidence that each measured data point is a fact and not an artefact, the number of points measured may be reduced to the theoretical minimum. This shortens the experimental procedure and decreases the likelihood of errors caused by biological drifts. Of course, one point cannot represent a curve, but two points permit a straight line to be drawn, three points can identify a hyperbola or a second-order parabola, four points can identify a third-order parabola, etc. For most experimental purposes, curve-shape recognition can be based on four to five measured points, provided they are appropriately positioned.

Biological processes like photosynthesis usually saturate with respect to substrate (CO2) and energy (light), and, with respect to temperature, usually curve to a maximum. The information which is most valuable is not the curve as such, but some of its parameters, such as the initial slope, the maximum (plateau) and the convexity. In a CO2 curve then (as well as a light curve), at least two points must be on the ascending part of the curve to determine the slope and the intercept (respiration), and at least one should be on the plateau, to establish the maximum rate. One or two additional points in the region where the curve is bending from initial slope to saturation are necessary if the convexity is important or there is a suspicion that the plateau may have a slope. With temperature, the curve bends down and it is good to have the farthest point close to where the rate declines to zero again. Because experimental perturbation of the stabilized state induces an after-effect which could interfere with the next experimental treatment, it is always advisable to return to the stabilized state between consecutive experimental treatments. Slow drifts in the stabilized state, if they take place, indicate the changing biological status of the leaf.

Table 1.1 Parameters used in calculations of gas flow (Vargaftik, 1972)

1.2 BASIC PHYSICS OF GAS FLOW

In most gas exchange systems little attention is given to the physics of gas flow in tubes, orifices and capillaries of which the system is composed. Understanding the physics of gas flow is important for the design of any gas exchange system, but particularly so in rapid-response systems, where we use capillaries and orifices to control resistances in critical parts of the system. In the following sections we