REVIEW OF LITERATURE

2.1

Brassica juncea L.
Brassica juncea, also known as Indian mustard or mustard greens or leaf mustard,

is perennial herb, usually grown as an annual or biennial. 2.1.1 Etymology The English word mustard is derived from the Middle English moustarde, a combination of the Old French words moust which means must and ardens meaning burning (Antol, 1999). Moust is derived from the Latin mustum, meaning new wine. Romans were the first to experiment with the preparation of mustard as a condiment. They mixed unfermented grape juice, known as must with ground mustard seeds to make burning must, mustum ardens-hence must ard (Hazen, 1993). 2.1.2 Indian mustard: Classification, habit and habitat Kingdom: Plantae Division: Angiospermae Class: Order: Family: Genus: Dicotyledons Brassicales Brassicaceae Brassica

Species: juncea Variety: PBR 91 2.1.3 Description B. juncea is a herbaceous plant with an erect, branched stem up to 1.0 m tall, with a taproot reaching 60-80 cm in depth, lower leaves petioled, green, sometimes with a whitish bloom, ovate to obovate, variously lobed with toothed or frilled edges; upper leaves subentire, short and petioled, constricted at intervals, sessile. The flowers consist of 4 (four) yellow petals arranged in a cruciform manner, 4 (four) yellowish green sepals, a short green pistil with a knobby stigma, and tetradynamous stamens with yellow anthers. They are pollinated by bees that soon develop into sickle-shaped green seed pods. Seeds are sown in very early spring. Plants are generally harvested before fruits are

fully ripe to reduce shattering. The growing period is from 4060 days depending on the variety and weather conditions. Indian mustard is a cool-season vegetable, growing well at monthly average temperatures ranging from 15 to 18C. It can tolerate annual precipitation of about 500 to 4,200 mm, annual temperature of 6 to 27C and pH of 4.3 to 8.3. 2.1.4 Origin and Distribution The primary center of origin of Indian mustard is thought to be central Asia while secondary centers in central and western China. Major Indian mustard producing countries include Canada, China, Germany, France, Australia, Pakistan, Poland and India. B. juncea is an amphidiploid and second most important edible oilseed crop in India after groundnut and accounts for about 30% of the total oilseeds produced in the country. Indian mustard is cultivated in the states of Punjab, Rajasthan, Uttar Pradesh (UP) Assam, Gujarat, Haryana, Madhya Pradesh (MP), and West Bengal (WB) as a Rabi crop (Duhoon et al., 1998; Dutta et al., 2008; Misra et al., 2010) (Fig. 1).
UP WB MP Punjab Haryana Rajasthan Uttar Pradesh West Bengal Madhya Pradesh

UP ASSAM

Gujarat

MP WB

Fig 1. Map of India showing Brassica juncea growing regions (Source: Duhoon et al., 1998; Dutta et al., 2008) 7

2.1.5 Economic importance of Brassica juncea L. B. juncea is valued for its intense flavours and healing properties. This plant is cultivated mainly as an oil crop. It is a good bee plant (Fomina, 1962). All over the world, mustard is used for its appetizing flavor and preservative value and the seeds are used largely for tempering food. Mustard is available in the form of seeds, powders and oil. Recently, B. juncea has been explored for its biodiesel potential (Jham et al., 2009). Its economic importance is listed below: 2.1.5.1 Nutritional importance The plant appears in some form in African, Indian, Chinese, Japanese, and Soul food cuisine. The leaves, the seeds, and the stem of Indian mustard are edible. Seeds of B. juncea contain 25-30% fatty non-drying oil and glycoside sinigrine. The leaves of young plants are used as green vegetables as they supply enough sulphur and minerals in the diet. B. juncea is used to make the Indian pickle called Achar, and the Chinese pickle known as Zha cai (Everitt, 2007). Young tender leaves of mustard greens are used in salads or mixed with other salad greens. Older leaves with stems may be eaten fresh, canned or frozen, for potherbs, and to a limited extent in salads. Its basal leaves are eaten raw and used in salads or cooked like spinach. Leaves and stems are also added to soups and stews. Mustard greens are often cooked with ham or salt pork, and may be used in soups and stews. Seed residue is used as cattle feed and in fertilizers. In Asia, some kinds of mustard are pickled (called hum choy and sajur asin) (Grubben and Denton, 2004). The seeds are very pungent and used to season meats and other dishes. Although widely and extensively grown as a vegetable, it is being grown more for its seeds which yield an essential oil and condiment. Oil is used to pickle foods in Kashimiri and Bengali cooking. It is used as cooking oil in parts of India and Bangladesh. Mustard oil is one of the healthiest edible oils. Mustard oil is healthier than olive oil because it has no trans fats, has low saturated fats, high mono-unsaturated fats and polyunsaturated fatty acids such as omega-3. It is stable at high temperatures, which makes it ideal for Indian cooking and even deep frying. Mustard is also a cheaper alternative of edible oil and makes the food tastier. In very small amounts, it is often used by the food industry for flavoring. Mustard seed meal is good source of protein (2836%) and phenolic

antioxidants such as sinapine and sinapic acid (Das et al., 2009). Mustard oil is also used as hair oil, lubricants and as a substitute for olive oil in Russia. Oilseed cake is used for cattle feed and manure. The chemical compositions of main parts of B. juncea are tabulated as below in Tables 1(a), 1(b) and 1(c). Table 1(a): Chemical composition of main parts of B. juncea (per 100 g) (Source: Leung, 1980) S.No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Chemical components Protein Fat Total carbohydrate Water Fiber Ash Ca P Fe K Ascorbic acid Riboflavin Niacin Thiamine -carotene equivalent Leaf 2.4 g 0.4 g 4.3 g 91.8 g 1.0 g 1.1 g 160 mg 48 mg 2.7 mg 297 mg 73 mg 0.14 mg 0.8 mg 0.06 mg 1825 g Root 1.9 g 0.3 g 8.8 g 85.2 g 2.0 g 3.8 g 111 mg 65 mg 1.6 mg 447 mg 0.21 mg 0.12 mg 0.7 mg 0.05 mg 45 g

Table 1(b): Chemical composition of B. juncea seeds (per 100 g) (Source: Leung, 1980) S.No. 1. 2. 3. 4. 5. 6. Chemical components Protein Fat Total carbohydrate Water Fiber Ash Seed 24.6 g 35.5 g 28.4 g 6.2 g 8.0 g 5.3 g

Table 1(c): Sterol composition of B. juncea seed (%) (Source: Leung, 1980) S.No. 1. 2. 3. 4. Sterols Brassicasterol Free campasterol Sitosterol -7-stigmasterol Seed 19.2 23.6 57.2 trace amounts

2.1.5.2 Medicinal value Indian mustard is reported to be anodyne, apertif, emetic, diuretic, rubefacient, and stimulant, it is also a folk remedy for arthritis, footache and rheumatism (Duke and Wain, 1981). Seeds are used for curing tumors in China whereas roots are used as a galactagogue in Africa. Ingestion may impart a body odor repellent against mosquitoes (Burkill, 1966). Believed to be aperient and tonic, the oil is used as a counter-irritant and stimulant. In Java, the plant is used as an antisyphilitic emmenagogue. Leaves applied to the forehead are known to relieve headache (Burkill, 1966). In Korea, the seeds are used for abscesses, colds, rheumatism, lumbago and stomach disorders. Chinese eat the leaves in soups for bladder inflammation or hemorrhage. Mustard oil is used for skin eruptions and ulcers (Perry, 1980). The seeds, crushed in honey, are known to cure coryza. Swallowing mustard seeds soaked in mustard oil, cures stomachache. Massaging the body with mustard oil is very beneficial as it cures flatulence and makes the body strong. Massage with the oil is thought to improve blood circulation, muscular development and good texture to human skin. The oil is also antibacterial. In skin diseases, the local application of seed oil is beneficial as it is antiseptic and antiinflammatory. The oil, with salt is an effective gargle in dental infections and pyorrhea (Hemingway et al., 1961). Derivatives of the mustard constituent i.e. allyl isothiocyanate, forms the basis for toxic agents such as mustard gases of warfare and the antineoplastic nitrogen mustard. Mustard also has antioxidant activity and pharmacological effects on cardiovascular disease, cancer and diabetes. The dried, ripe seeds are used commercially. Mustard and its oil have been used as a topical treatment for rheumatisms and arthritis. Mustard seeds have been used as appetite stimulants, emetics, and diuretics. The oil has a strong smell, a hot nutty taste, and is mainly used for cooking in Bengal, Bihar and other areas of India and Bangladesh. The oil makes up about 30% of the mustard seeds. For North Indians, mustard oil is not just a cooking medium but it is very much intricately interwoven with their culture. Mustard oil is beneficial to human health because of its low content of saturated fats, ideal ratio of omega-3 and omega 6 fatty acids, content of antioxidants such as vitamin E and also of the fact that it is cold pressed (extracted at 45-500C). The pungent taste of the mustard condiment results when ground mustard seeds are mixed with water, vinegar, or other liquids (or chewed). Under these

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conditions, a chemical reaction between the enzyme myrosinase and a glucosinolate known as sinigrin from the seeds leads to the production of allyl isothiocyanate. The main component of mustard oil is allyl isothiocyanate (Yu et al., 2003). Allyl isothiocyanate serves the plant as a defence against herbivores. Since it is harmful to the plant itself, it is stored in the harmless form of a glucosinolate, separate from the myrosinase enzyme. Once the herbivore chews the plant, the noxious allyl isothiocyanate is produced. Seeds of this plant are widely used in America, Japan, China and other countries and regions as a traditional pungent spice, a source of edible oil and protein and a kind of medicine. B. juncea, an amphidiploid species, is grown as an oilseed crop in India. Although, oilseed Brassicas are grown over 15% of arable land in India but their productivity is considerably hindered by various biotic and abiotic stresses (Shah, 2002; Purty et al., 2008; Sirhindi et al., 2009; Yusuf et al., 2010), like drought, chilling, pesticides and heavy metals etc. The soil in which plants grow may contain phytotoxic levels of the heavy metals including Cr, Cu, Hg and Ni, Zn etc.

2.2

Heavy Metals
Heavy metals are defined as metals with density higher than 5 g cm-3. 53 of the 90

naturally occurring elements are heavy metals (Weast, 1984), but not all of them are of biological importance. Heavy metal toxicity to plants vary with plant species, specific metal, concentration, chemical form, soil composition and pH, as many heavy metals are considered to be essential for plant growth. Though some heavy metals are essential as micronutrients, their higher concentrations are toxic for plants (Panda et al., 2003; Sharma and Agarwal, 2005). Heavy metal contamination of the biosphere has increased sharply for the last few years and poses major environmental and human problems worldwide (Ensley, 2000). Heavy metals are ubiquitous and persistent environmental pollutants that are introduced into the environment through anthropogenic activities, such as smelters, mining, power station industry, and the application of metal-containing pesticides, fertilizers, and sewage sludge (Malik, 2004). Heavy metal contamination of soils and waterways is a serious environmental problem with potentially harmful consequences to agriculture and human health (Sanita di Toppi and Gabbrielli, 1999, Hegedus et al., 2001; Arvind and

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Prasad, 2005). Metals like Cd, Pb, Zn, Cr, etc. when present in high concentration in soil show potential toxic effects on overall growth and metabolism of plants (Agrawal and Sharma, 2006). According to Schutzendubel and Polle (2002), metals such as Ni, Cu, V, Co, W and Cr are highly toxic to plants when they exceed trace levels. It proves detrimental to living organisms even at low concentrations (Gallego et al., 2002). The effect of their toxic influence on plants is largely a strong and fast inhibition of growth processes of various plant parts, as well as the degradation of photosynthetic apparatus, often correlated with progressing senescence processes (Molas, 2002; Sobkowiak and Dekert 2003; Alaoui-Sosse et al., 2004; Lin et al., 2005). Heavy metal toxicity may severely interfere with physiological and biochemical functions (El-Sheekh et al., 2003; Parmar and Chanda, 2005). Roots are usually shortened and thickened or poorly developed (Casella et al., 1988). Growth inhibition and senescence stimulation, caused by heavy metals in excess, are intriguing effects as the knowledge of their mechanisms can have a great significance in ecophysiology and medicine. Problem of heavy metal contamination in agricultural lands is responsible for limiting the crop productivity (Qadir et al., 2004). They can directly interact and suppress functioning of various essential biological components, in particular the electron transport system in mitochondria as well as chloroplast and enzymes like Rubisco, nitrogenase and nitrate reductase (Alia et al., 2001) either by directly interacting or replacing essential nutrients that are required for their functioning (Atal et al., 1991). Heavy metals are known to promote incomplete reduction of molecular oxygen leading to the generation of free radicals due to inhibition in photosynthetic electron transport. Oxidative damage involving lipid peroxidation has been observed in higher plants in the presence of toxic levels of Cu, Cd, Zn and Fe (Atal et al., 1991). 2.2.1 Cobalt (Co) Cobalt, a transition element, is an essential component of numerous enzymes and co-enzymes. It has been shown to affect growth and metabolism of plants depending on the concentration and status of cobalt in soil (Palit et al., 1994; Collins et al., 2010). In higher plants, it probably participates in chlorophyll b formation. Even though this metal is less frequently encountered in metalloenzymes than copper, iron, manganese or zinc, it is an important cofactor in vitamin B12-dependent enzymes and in some non-corrin

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cobalt-dependent enzymes such as [Co]-nitrile hydratase and some other enzymes in bacteria, yeast and plants (Kobayashi and Shimizu, 1999). The beneficial effects of cobalt include retardation of growth, increase in drought resistance in seeds and inhibition of ethylene biosynthesis. This trace element can be a contaminant in soil due to agricultural additives, metal refineries and coal ash (Bakkaus et al., 2005). Toxic concentrations (exceeding permissible limits) inhibit tetrapyrrole biosynthesis and active ion transport in plants. Cobalt is known to cause irreversible damage to a number of fundamental metabolic components and plant cell and membrane. It also modifies the structure and number of chloroplasts per unit area of leaf. Toxicity of cobalt causes inhibition of PS2 activity and Hill reaction. Co inhibits either the reaction centre or component of PS2 acceptor by modifying secondary quinone electron acceptor Qb site. Co2+ reduces the export of photoassimilates in leaves and dark fixation of CO2 (Palit et al., 1994). In CAM and C4 plants, it hampers fixation of CO2 by inhibiting the activity of enzymes involved. High concentrations of metal impedes RNA synthesis and decrease the amounts of DNA and RNA by modifying the activity of a large number of exo- and endo nucleases. Toxic effects of Co include leaf fall, discolored veins and reduced shoot weight. Being a component of vitamin B12 and cobamide coenzyme, it helps in the fixation of molecular nitrogen in root nodules of leguminosae. But in cyanobacteria, Co inhibits ammonia uptake, formation of heterocyst, and nitrate reductase activity (Table 2). 2.2.2 Chromium (Cr) Cr is the seventh most abundant metal on earths crust (Katz and Salem, 1994). It was first discovered in the Siberian red lead ore (crocoite) in 1798 by the French chemist Vauquelin and is a transition element located in the group VI-B of the periodic table. Cr is associated with some biological pathways, mainly with glucose tolerance (Felcman and Bragana, 1987). It is an important environmental contaminant released into the atmosphere due to its huge industrial usage. Cr is extensively used in both the trivalent and hexavalent forms in industries like steel, leather, textile etc. (Dixit et al., 2002). In nature, chromium exists in two different stable oxidation states, Cr (III) and Cr (VI). Both of these forms differ in terms of mobility, bioavailability and toxicity. Cr (VI) is more toxic than Cr (III) (Han et al., 2004; Panda and Choudhary, 2005). Cr (VI) forms chromate and dichromate and is highly soluble in water. Cr is phytotoxic above certain

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threshold levels. Cr stress causes inhibition of seed germination leading to retardation of the growth in plants. This can be attributed to the inhibition of cell division (Liu et al., 1993). During seed germination, hydrolysis of proteins and starch takes place, providing amino acids and sugars. Under Cr stress, a decrease in both and amylases has been reported, which is one of the important factors for germination inhibition in many plants in view of the impaired supply of sugar to developing embryo axis (Zied, 2001). Cr phytotoxicity can also result in degradation of plant pigments, nutrient imbalance, alteration of antioxidative enzyme activities (Table 2) and induction of oxidative stress in plants (Barcelo and Poschenrieder et al., 1997; Panda and Patra, 2000). Beside, Cr can alter chloroplast and membrane ultrastructure in plants (Panda et al., 2003; Shanker et al., 2005). Accumulation of this metal by plants can induce chlorosis in young leaves, reduce pigment content, damage root cells and cause ultrastuctural modifications of the chloroplast and cell membrane (Choudhury and Panda, 2004; Bluskov et al., 2005; Panda, 2007; Kumar and Joshi, 2008; Sinha et al., 2009). 2.2.3 Nickel (Ni) Nickel is a major environmental contaminant and one of the widespread heavy metals, defined as ultramicronutrient. It is delivered into the environment as factory waste of high-temperature technologies of ferrous and non-ferrous metallurgy, burning of solid and liquid fuels, field irrigation with water contaminated with heavy metals, transfer of sewage leftovers into soil and application of high rates of organic and mineral fertilizers and pesticides contaminated with heavy metals (Orlov et al., 2002). The explicit role of Ni in metabolism is its essentiality as a component of the enzyme urease, where two Ni ions are in stoichometric quantities. Therefore, deficiency of Ni results in a wide array of physiological alterations in plants which include slowing down of urea metabolism. This, in turn, leads to leaf-tip necrosis or leaf-burn disease and mouse-ear disease. However, at an elevated level, it generates a number of toxic symptoms in plants (Bal and Kasprzak, 2002). These include plant growth retardation, changes in water relations, decreased uptake of mineral nutrients, disruption of photosynthetic machinery, inhibition of enzyme activity, degradation of photosynthetic pigments, causing stomatal closure, thus leading to inhibition of photosynthesis (Freeman et al., 2004; Ali et al., 2009; Chen et al., 2009). Ni toxicity also generates

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oxidative stress and alters antioxidative defence system of plants (Maheshwari and Dubey, 2009) (Table 2). 2.2.4 Zinc (Zn) Zinc is a major industrial pollutant of the terrestrial and aquatic environment (Barak and Helmke, 1993). Although Zn is an essential element, which belongs to Group-II of the periodic table, but at high concentrations, it is strongly toxic and hamper plant growth (Marschner, 1995). Microelements, such as Zn, are essential and are involved in numerous physiological processes (Rengel, 2000). Zn is a constituent of metalloenzyme or a cofactor for several enzymes such as peroxidase, dehydrogenase, anhydrase and oxidase. It plays an important role in regulation of photosynthesis, nitrogen metabolism, cell multiplication and auxin synthesis in plants (Vaillant et al., 2005). Further, it also plays a vital role in the synthesis of nucleic acid and proteins and helps in the utilization of nitrogen and phosphorous during seed formation. Zn is a fundamental component of many important enzymes (Cu/Zn SOD), a structural stabilizer for proteins, membrane and DNA-binding proteins (Zn-fingers). It is known to have a stabilizing and protective effect on the biomembranes against oxidative and peroxidative damage, loss of plasma membrane integrity and also alteration of the permeability of the membrane. Zn acts as a plant nutrient but at higher concentrations, it is toxic (Baccio et al., 2005). An excess of Zn is indicated by a decrease in growth and development, metabolic activity and an induction of oxidative damage in various plant species (Dong et. al., 2006) (Table 2). The general symptoms are stunting of shoot, curling and rolling of young leaves, chlorosis, retardation of root growth, inhibition of Fe translocation, disintegration of cell organelles, disruption of biomembranes, condensation of chromatin material and increase in number of nucleoli (Rout and Das, 2003; Broadley et al., 2007). Zn toxicity leads to chlorosis in young leaves, and inhibits photosynthesis at various steps and through different mechanisms. It shows a specific effect on the Calvin cycle and photosystem activities (Sagardoy et al., 2009; Auda et al., 2010). 2.2.5 Heavy metal stress and antioxidative defence system Heavy metals such as Mn, Fe, Ni, Cu and Zn are intrinsic components of enzymes and are therefore essential to plant survival. However, at higher concentrations, metal

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ions may become extremely toxic as they can inhibit enzyme function and cause oxidative damage in plants (Anderson et al., 2001) due to alteration in biochemical parameters. Heavy metals stress induce oxidative stress by generating free radicals and toxic reactive oxygen species (ROS) such as superoxide radical (O2.-), hydrogen peroxide (H2O2), hydroxyl radical (OH.) and alkoxyl radical (RO.). In plants, ROS are continuously produced predominantly in chloroplast, mitochondria and peroxisomes. (Aravind and Prasad, 2003; Hegedus et al., 2001). These ROS are partially reduced forms of atmospheric oxygen and under normal conditions their production in cells is low and tightly controlled (Dat et al., 2000; Apel and Hirt, 2004). Heavy metal toxicity enhances the production of ROS up to 30-folds (Mittler, 2002). The enhanced titers of ROS in stressed and senescing tissues increase the prospect of oxidative damage to a range of essential macromolecules such as lipids, proteins, polysaccharides, nucleic acids and inactivation of enzymes activities thus affecting the cell viability (Gratao et al., 2005). These species react with lipids, proteins, pigments and nucleic acids and cause lipid peroxidation, membrane damage Ultimately, these ROS might lead to the death of plant cell by enhancing the expression of the ROS-dependent and cell death related genes. Other factors like MAPK-driven phosphorylation cascades, regulatory post

transcriptional modifications such as protein oxidation and nitrosylation causing ROSdependent cell death (Breusegem and Dat, 2006). It is important for cells to control the concentration of ROS tightly, but not to eliminate them completely (Schutzendubel and Polle, 2002), which suggests the existence of a dual concentration-dependent role of ROS in plants (Kovalchuk et al., 2005). One of the mechanisms that makes a plant species tolerant to heavy metal stress is the presence of strong antioxidative defence system (Pandey et al., 2005; Israr et al., 2006) (Fig. 2) comprising of both antioxidative enzymes and non-enzymatic antioxidants (Table 3). Among antioxidative enzymes, superoxide dismutase (SOD, EC 1.15.1.1) plays a vital role in the protection of cells against oxidative injury. This enzyme catalyzes disproportionation of O2- yielding O2 and H2O2, the latter being subsequently scavenged by peroxidase ans catalase. In the reaction catalyzed by ascorbate peroxidase (APOX, EC 1.11.1.11), H2O2 is reduced to H2O and ascorbate (AA) is oxidized to

monodehydroascorbate (MDHA), which is the product of univalent oxidation of AA. In

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chloroplasts, MDHA may be rapidly reduced to ascorbate by reduced ferredoxin. Besides, monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) reduces MDHA to AA using NAD(P)H. When MDHA is not directly reduced to AA it is spontaneously disproportionated to AA and dehydroascorbate (DHA), the divalently oxidized product. DHA is reduced to AA by dehydroascorbate reductase (DHAR, EC 1.8.5.1) using GSH (reduced glutathione) as an electron donor. This reaction generates glutathione disulphide (GSSG), which is in turn re-reduced to GSH in the reaction catalyzed by glutathione reductase (GR, EC 1.6.4.2). This enzyme participates not only in H2O2 scavenging, but also in redox signalling and activation of the protective mechanisms under stress through compartment-specific regulation of the GSH/GSSG ratio (Foyer et al., 1997; Kocsy et al., 2001). AA may also be regenerated through the non-enzymatic reduction of DHA by GSH. APOX together with ascorbate-regenerating enzymes, GR as well as AA and glutathione constitute the ascorbate-glutathione cycle, also known as the Asada-FoyerHalliwell pathway, which is considered to be the principal way of removing H2O2 in plants (Noctor and Foyer, 1998; Asada, 2006). Catalase (CAT, EC 1.11.1.6) catalyzes disproportionation of H2O2 to H2O and molecular oxygen. Apart from CAT, non-specific peroxidases such as guaiacol peroxidase (POD, EC 1.11.1.7) utilizing H2O2 for oxidation of a wide array of substrates, are involved in the removal of this ROS in plants. In addition to antioxidative enzymes, low-molecular weight antioxidants are involved in the regulation of ROS levels in plant cells. They include metabolites such as ascorbate, glutathione, various phenolic compounds, certain amino acids, as well as lipid soluble compounds, such as -tocopherol and -carotene (Polle, 2001; Foyer et al., 2006). AA and -tocopherol are extremely effective antioxidants as they are relatively poor electron donors and efficiently scavenge OH., O2. and singlet oxygen. AA takes part in a variety of growth processes, electron transport, photoprotection and regulation of photosynthesis, in preserving the activities of enzymes which contain prosthetic transition metal ions. It is also active in regeneration of the lipophilic antioxidant viz. -tocopherol and in antioxidant processes, reacting directly with OH., superoxide, singlet oxygen and H2O2. Carotenoids protect chlorophyll by absorbing surplus of excitation energy and by quenching singlet oxygen. (Vanacker et al., 2000; Arora et al., 2002). Glutathione (GluCysGly) is a cellular protectant and is also the chief pool of non-protein reduced

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sulfur in plants. Its protective role is threefold. First, reduced glutathione (GSH) is an important antioxidant. Second, conjugation of glutathione to electrophilic molecules by glutathione-S-transferases (GSTs) plays a protective role in detoxification of xenobiotics. Third, both glutathione and phytochelatins (polymers of -GluCys) chelate heavy metals facilitating their sequestration in to the vacuole (Cobett, 1998; 2000; 2002). It also affects expression of defence-related genes and is associated with stress resistance. GSH may also be involved in redox regulation of the cell cycle. Further, it may act as an imperative buffer, preserving enzyme inactivation (Noctor et al., 1998). A major function of glutathione in protection against oxidative stress is the re-reduction of AA in the ascorbate-glutathione cycle (Noctor et al., 1998). Glutathione also reacts nonenzymatically with singlet oxygen species, superoxide and hydroxyl radical (Kuzniak, and Sklodowska, 2001). As a component of the ascorbate-glutathione cycle, GSH is involved in the control of H2O2 concentration in plant cells (Kocsy et al., 2001). The ascorbate-glutathione cycle enzyme activities are present in root and leaf cell organelles such as chloroplasts, mitochondria, plastids and peroxisomes (Tausz et al., 2004). Several plant hormones like abscisic acid, ethylene, salicylic acid jasmonates and brassinosteroids (BRs) play a determinant role in plant defence signaling pathways, implicating oxidative stress (Bajguz and Hayat, 2009; Bari and Jones, 2009).

2.3

Brassinosteroids
Brassinosteroids (BRs), plant-specific steroid hormones, affect many aspects of

plant growth and development (Sasse, 2003; Kim et al., 2009; Guo et al., 2010; Ryu et al., 2010). BRs are common plant-produced compounds that can function as growth regulators (Bishop et al., 2006). In addition, it has been suggested that BRs could be included in the category of phytohormones (Haubrick and Assmann, 2006). 2.3.1 Discovery These hormones were discovered in 1970s, when Mitchell et al. (1970) reported promotion in stem elongation and cell division by the treatment of organic extracts of Brassica napus pollen. First BR was isolated in a crystalline form from rape pollen by Grove et al. (1979) and was named as brassinolide (BL) with an emperical formula of C28H48O6 (MW = 480). In 1982, another steroidal substance with growth-promoting nature was isolated from the insect galls of chestnut (Castenea crenata) and named as

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castasterone (Yokota et al., 1982). The discovery of brassinolide and castasterone gave an impetus to the idea of the presence of steroidal hormones with growth-promoting characteristics in the plant kingdom. As the first steroidal hormone with growthpromoting nature was obtained from B. napus, the name brassinosteroids was given to this new class of substances. Since then, a number of related steroidal compounds have been isolated from a variety of plant sources. Recently, BRs have been isolated from tea leaves and seeds and from Aegle leaves by Gupta et al. (2004), Bhardwaj et al. (2007a) and Sondhi et al. (2008). Natural BRs have a common 5-cholestan skeleton, and their structural variations come from the kind and orientation of functionalities on the skeleton. The compounds can be classified as C27, C28 or C29 BRs depending on the alkylsubstitution pattern of the side chain. Brassinolide, 24-epibrassinolide (24-EBL) and 28homobrassinolide (28-HBL) are the three biologically active BRs being widely used in physiological studies (Fig. 3). 2.3.2 Distribution BRs are ubiquitously distributed throughout the plant kingdom (Bhardwaj et al., 2006, 2007a; Bajguz and Hayat, 2009) (Table 4) and play essential role in modulating the growth and differentiation at nanomolar to micromolar concentrations (Clouse and Sasse, 1998). The occurrence of BRs has been demonstrated in almost every part of plants (Table 5), such as pollen, flower buds, seeds, fruits, vascular cambium, leaves, shoots and roots (Bajguz and Hayat, 2009). The highest measured concentration of BRs has been observed in the pollens of Brassica napus and Vicia faba (10-1 nmol g-1 fresh weight, brassinolide) and the lowest in immature seeds and sheaths of Brassica campestris var. pekinensis (10-7 nmol g-1 fresh weight, homocastasterone) (Khripach et al., 2000). These steroidal compounds occur in free form and conjugated to sugars and fatty acids. 70 compounds belonging to the class of BRs were fully characterized (65 in free form and 5 conjugated) in plants by spectrometric methods in 2003. Presumably, there are a number BRs and their conjugates in plants yet to be described (Bajguz and Tretyn, 2003). BRs control several important agronomic traits such as flowering time, plant architecture, seed yield and stress tolerance (Li et al., 2008; Divi and Krishna, 2009). Exogenous application of BRs may influence a range of diverse processes of plant

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growth and development (Cao et al., 2005; Ozdemir et al., 2004; Yu et al., 2004; Fu et al., 2008).

2.4

Physiological Roles of Brassinosteroids

Brassinosteroids are plant hormones with pleiotropic effects as they influence

diverse physiological processes such as seed germination, growth, rhizogenesis, senescence, leaf abscission and elongation, cell division, pollen tube growth, leaf bending and epinasty, induced synthesis of ethylene, promote plant tropisms by modulating polar auxin transport, reproductive and vascular development, membrane polarization and proton pumping, synthesis of nucleic acids and proteins, activation of enzymes and affect source/sink relationships (Geuns, 1978, 1983; Kaur and Thukral, 1991, 1993; Dogra and Thukral, 1995a, 1995b, 1996a and 1996b; Bhardwaj and Thukral, 2000b; Hayat and Ahmad, 2003; Sasse, 2003; Yu et al., 2004; Xu, 2006; Deng et al., 2007; Oh et al., 2009). It is proposed that the changes induced by BRs are mediated through repression and/or depression of specific genes (Felner, 2003). Besides, BRs are also recognized to have ameliorative role in plants, subjected to various kinds of biotic and abiotic stresses (Sharma and Bhardwaj, 2007a; Ali et al., 2008a, 2008b; Arora et al., 2008b; Bajguz and Hayat, 2009; Xia et al., 2009b; Kim et al., 2009; Arora et al., 2010; Bhardwaj et al., 2010; Hayat et al., 2010). Some physiological attributes of BRs are as follows: 2.4.1 Germination The application of BL, 24-EBL and 28-HBL caused enhancement in seed germination of Arachis hypogea, Lepidum sativus, Oryza sativa, Triticum aestivum, Lycopersicum esculentum and Orabanchae minor (Sasse, 1995; Steber and McCourt; 2001; Rao et al., 2002). Sasse (1995) reported that the application of 24-EBL resulted in substantial improvement in the seed germination and seedling growth of Eucalyptus camaldulensis under salinity stress. Seed germination in the presence of 150 mM of NaCl was enhanced by 24-EBL treatment but when seedlings were grown hydroponically under salt stress, uptake of 24-EBL through roots caused greater damage. Steber and Mc Court (2001) studied the GA mutant Arabidopsis plants and found that 24-EBL and BL treatment promoted the seed germination. They also found that germination of both BR-biosynthetic and BR-insensitive mutant (det 2-1 and bri 1-1) was

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inhibited more strongly in the presence of ABA than the wild type. Endogenous BRs as well as GA was needed to overcome this ABA-induced dormancy. Hayat and Ahmad (2003) reported that 10-10 M and 10-8 M concentrations of HBL were most effective in increasing the percent germination of wheat grains. It was observed that seeds soaked in 28-HBL for 8 hrs showed significant improvement in percent germination by enhancing the activities of -amylase, catalase, peroxidase, soluble sugars and proteins. Chen et al. (2004) found that GCR1 could act independently of heterotrimeric Gprotein in response to BRs and GAs in Arabidopsis during seed germination. Signal is recognized by seven-transmembrane (7TM) cell-surface receptors associated with heterotrimeric G-proteins. Plants use heterotrimeric G-protein signaling in the regulation of growth and development, particularly in hormonal control of seed germination, but it is not yet clear which of these responses utilize a 7TM receptor. Arabidopsis GCR1 had a predicted 7TM spanning domain and other features characteristic of 7TM receptors. GCR1 played a positive role in GA and BR-regulated seed germination. But GCR1, unlike a typical 7TM receptor acted independently of the heterotrimeric G-protein during seed germination. Moreover, Ali et al. (2005) reported that exogenous application of 28HBL (10-8 M) to Cicer arietinum seeds enhanced their percentage of germination. Nomura et al. (2007) investigated the levels of endogenous BRs and the expression of the biosynthesis/metabolism/perception genes involved during the development and germination of Pisum sativum seeds. The levels of BL, CS and the transcript levels of two BR C-6 oxidases (CYP85A1 and CYP85A6) reached a maximum suggesting the significance involvement of BL and CS in seed development Divi and Krishna (2010) reported that the BRs in overcame the inhibitory effects of ABA in seed germination and promoted cold stress tolerance in Arabidopsis seedlings. 2.4.2 Cell division Plant growth via cell elongation and cell division requires the co-ordination of several processes, some of which appears to be influenced by BRs (Haubrick and Assmann, 2006). In cultured parenchyma cells of Helianthus tuberosus, application of nanomolar concentrations of BR stimulated cell division by 50% in the presence of auxin and

21

cytokinin (Clouse and Zurek, 1991). The promotion of cell division by BRs (24-EBL and BL) had also been reported in Chinese cabbage and in Petunia protoplasts in the presence of auxin and cytokinin (Nakajima et al., 1996; Oh and Clouse, 1998). It was further suggested that mechanism of promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin. 24-EBL also up-regulated transcription of CycD3, a D-type cyclin in det2 suspension cultures (Hu et al., 2000). Miyazawa et al. (2003) found that BL-promoted cell division of tobacco BY-2 cell lines in the absence of exogenous auxin and during the early phase of culture. The promotion of cell division was confirmed by RNA gel blot analyses using cell-cyclerelated gene probes. Cell division in BY-2 cell lines was correlated with dose-dependent increases in mitotic indices and transcript accumulation of the B-type cyclin. The effect of 28-HBL alone or in combination with auxins on induction and elongation of shoots regenerated from apical meristems of banana cultured in vitro was studied by Nassar (2004). When 28-HBL was added simultaneously or sequentially with auxins, induction and elongation of shoots was markedly improved as compared to each of them alone, which showed an additive effect of the two growth regulators. 28-HBL had a remarkable stress protective effect and greatly reduced the percentage of heat injury. Itoh et al. (2005) found that BL facilitated both the elongation and expansion of Solenogyne mikadoi leaves. Kartal et al. (2009) reported that 28-HBL treated barley roots revealed greater mitotic activity and significant enlargements at the root tips. 2.4.3 Cell expansion The plant cell wall forms a highly cross-linked, rigid matrix that opposes cell expansion and differentiation. In order for elongation and other morphogenetic processes to occur, the cellwall must be modified, i.e. by wall relaxation or loosening and by incorporation of new polymers into the extending wall to maintain wall integrity. Several proteins with possible roles in cellwall modification processes have been identified, including glucanases, xyloglucan endotransglycosylases (XETs). It was observed by Bates and Goldsmith (1983) and Barbier-Brygoo et al. (1991) that elongation induced by auxins was also associated with hyperpolarization of PM by proton pump activity as it was observed in case of BRs. Plasticity of cell wall is increased when proton extrusion by H+-ATPases acidifies the apoplast, thereby activating cell wall loosening enzymes. These

22

enzymes helped to synthesize new cell wall and membrane materials. Studies carried out by Cerana et al. (1983, 1984) reported that treatment with BRs increases ATPase activity in Azuki bean epicotyls and maize roots, leading to proton extrusion and induced the cell wall relaxation. It was reported that BRs may increase the abundance of mRNA transcripts for wall-modifying proteins such as XETs (Xu et al., 1995). Cosgrove (1997) is of the view that expansins are primarily responsible for wall relaxation. Glucanases and XETs affect the extent of expansin activity by altering the viscosity of the hemi-cellulose matrix. Furthermore, XETs may function to incorporate new xyloglucan into the growing wall, and cellulose biosynthesis may also occur. BRs are reported to cause pronounced elongation of hypocotyls, epicotyls and peduncle of dicots, as well as coleoptiles and mesocotyls of monocots (Clouse, 1996). BR-induced expansion is accompanied by proton extrusion and hyperpolarization of cell membranes and these effects have also been observed in the asymmetric expansion in Chlorella vulgaris, an alga (Bajguz and Czerpak, 1996). The growth induced by BRs is due to both cell division and elongation (Clouse and Sasse, 1998). BRs up-regulate expression of the BRU1, TCH4, LeBR1, OsXTR1 and OsXTR3 genes, which encode xyloglucan endotrans-glycosylases or hydrolases (XTHs or XETs) in soybean, Arabidopsis, tomato and rice, respectively (Xu et al., 1995; Uozo et al., 2000). These enzymes are involved in cell wall biosynthesis and modification. Morillon et al. (2001) reported that BRs impact turgor-driven cell expansion by affecting the activity of aquaporins, the water channels that help the plant cell osmoregulation. Microarray analysis has also shown that BL treatment up-regulates several additional genes related to cell expansion and cell wall organization, (Goda et al., 2002). It was further proposed that BL may affect cell shape and expansion via regulation of microtubule dynamics. HBL was found to re-organize the disordered microtubules in BR-deficient Arabidopsis mutant and induced cell elongation (Catterou et al., 2001a). In internode cells of the BL insensitive rice mutant d61 (Osbri1), microtubules either could not be detected or were disorganized (Yamamuro et al., 2000). Later on, Hong et al. (2002) suggested that internode cells of the BR-deficient dwarf 1 (brd1) rice mutant also had a failure in microtubule formation and arrangement, based on the fact that brd1 cell shape and arrangement is similar to that of OsBri1 mutants.

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Yamagami et al. (2009) reported that BRs bind to leucine-rich repeat kinase Brassinosteroid-Insensitive 1 (BRI1) localized to the plasma membrane, activate transcription factors in collaboration with cytosolic kinases and phosphatases, and regulate BR-responsive gene expression. A dominant mutation, bil4-1D, showed cell elongation in the presence of the BR-specific inhibitor Brz. Brz suppresses expression of the BIL4 gene in wild-type plants, and overexpression of BIL4 in bil4-1D suppresses the BR deficiency caused by Brz thus indicating that BIL4 mediates cell elongation. Zhang et al. (2009) observed that BRs induce cell elongation at the adaxial side of the lamina joint to promote leaf bending. They identified a rice mutant (ili1-D) which revealed an increased lamina inclination phenotype similar to that caused by BR treatment. The ili1-D mutant over-expresses an HLH protein homologous to Arabidopsis thaliana Paclobutrazol Resistance1 (PRE1) and the human Inhibitor of DNA binding proteins. ILI1 and PRE1 interact with basic helix-loop-helix (bHLH) protein IBH1 (ILI1 binding bHLH), whose overexpression causes erect leaf in rice and dwarfism in Arabidopsis. Overexpression of ILI1 or PRE1 increased cell elongation and suppressed dwarf phenotypes caused by overexpression of IBH1 in Arabidopsis. Thus, ILI1 and PRE1 may inactivate inhibitory bHLH transcription factors through heterodimerization. BR increased the RNA levels of ILI1 and PRE1 but repressed IBH1 through the transcription factor BZR1. The spatial and temporal expression patterns support roles of ILI1 in laminar joint bending and PRE1/At IBH1 in the transition from growth of young organs to growth arrest. These results suggested that BR regulated plant development through a pair of antagonizing HLH/bHLH transcription factors that acted downstream of BZR1 in Arabidopsis and rice. 2.4.4 Vegetative Growth Steroid hormones are conserved between animals and plants as signaling molecules to control growth and development (Yamagami et al., 2009). The use of inhibitors of BR biosynthesis and exogenous application of BRs to whole plants effectively explain the importance of BRs in plant growth. Franck-Duchenne et al. (1998) tested 24-EBL for in vitro stem elongation of sweet pepper in the culture medium. In vitro regeneration of sweet pepper (Capsicum annuum L.) was performed via direct organogenesis. The resulting shoot-buds were placed on media containing 0.1 M 24-

24

EBL in the presence or absence of 9.1 M zeatin and 5.2 M GA3 for further stem elongation. It was observed that 24-EBL does not always act directly on stem elongation but may be an elicitor and/or an enhancer of elongation in concert with endogenous and other exogenously added growth regulators. Application of BL enhanced the leaf sheath lengths of rice when grown in light, whereas pretreatment of seeds enhanced mesocotyl elongation in the dark. But at higher concentration it showed inhibitory effects (Chon et al., 2000). Hayat et al. (2001a) compared the effects of different concentrations of indole-3-acetic acid, GA, kinetin, ABA and BRs. It was found that BR was most effective even in M concentration to enhance the chlorophyll levels. However, Hunter (2001) observed the inhibitory effects of 24-EBL (0.1-10 M) on root and shoot length, dry weight, and nodule and lateral root numbers in case of soybean seedlings. Time and duration of exposure of BRs to plants may also play an important role in determining the growth of plants. In case of rice, shoot lengths of seedlings were appreciably promoted for seven days after continuous and first day treatments of the seeds but not after treatment on days 3 (three) and 4 (four) of germination (Fujii and Saka, 2001). Yin et al. (2002) proposed that BRs promote stem elongation by regulating gene expression and by enhancing the rate of accumulation of BES1. BRs signal through a plasma membrane localized receptor kinase BRI1. Application of 24-EBL to wheat seedlings enhanced the content of agglutinin in the most elongated roots, without a concomitant increase in ABA concentration (Shakirova et al., 2002). The effects of two BR analogues, BB-6 and BB-16 were evaluated in cladodes of Opuntia ficus-indica in concentration ranging from 0.00001 to 10 mg l-1. Both BR analogues stimulated larger number of vegetative buds under both green house and field conditions thereby increasing the number of harvested cladodes and total harvested fresh weight. These BR analogues have also been reported to accelerate growth during the first stage of vegetative bud development (Cortes et al., 2003). Mazorra et al. (2004) studied by Effects of structural analogs of BRs on the recovery of growth inhibition by a specific BR biosynthesis inhibitor Brz2001. BR inhibitor blocked the growth of roots, hypocotyls and epicotyls of soybean seedlings, but the application of 24-EBL completely reversed the inhibitory effects of Brz2001. MH5

25

and BB6 (growth promoting spirostane analogs of BR) were found to partially overcome Brz2001-induced growth inhibitory effects. MH5 was found to be more effective than BB6 to reverse the Brz2001-induced growth defects in soybean seedlings. Malabadi and Nataraja (2007) highlighted the role of 24-EBL in

micropropagation of orchids. In vitro regeneration and initiation of protocorm like bodies (PLBs) in Cymbidium elegans were achieved using shoot tip sections and 24-EBL supplemented basal medium. The highest percentage of explants (91.0%) producing PLBs (24.0) was recorded in 4.0 M 24-EBL supplemented basal medium. Nieves et al. (2007) studied the effect of two spirostanic BRs analogs BB-6 and MH-5 (0.001 and 0.01 mg l-1) on protein metabolism in sugarcane during somatic embryogenesis. It was observed that treatment of BRs analogs reduced the proline level and enhanced the content of soluble protein and storage protein. Changes in free-proline levels and content of soluble protein and storage protein could be an indication that BRs might be involved in stress responses. Pereira-Netto et al., (2009) reported that brassinolide differentially affected elongation and formation of main and primary lateral shoots in Marubakaido apple rootstock (Malus prunifolia (Willd.) Borkh cv. Marubakaido, which resulted in reduced apical dominance. Treatment of shoots with increasing doses of Brz220 led to a progressive inhibition of main shoot elongation Swami and Rao (2010) reported that 24-EBL and 28-HBL promoted rooting and early vegetative growth of Coleus (Plectranthus forskohlii (Wild.) Briq. stem cuttings. 2.4.5 Vascular differentiation BRs are also found to play a significant role in vascular differentiation (Clouse and Sasse, 1998). Exogenous application of BL at nM concentration enhanced the rate of differentiation of tracheary elements by 10 folds. (Clouse and Zurek, 1991). Uniconazole, an inhibitor of both gibberellins (GAs) and BR biosynthesis, prevented differentiation of Zinnia elegans mesophyll cells in to tracheary elements, and this inhibition was overcome by BR but not by gibberellins application (Iwasaki and Shibaoka, 1991). BRs regulated expression of BRU1 gene encoding a xyloglucan endotransglycosylase (XET) in soybean, also strengthened the role of BRs in xylem differentiation (Zurek et al.,

26

1994). XETs were involved in cell wall modification, expansion, vascular differentiation and fruit ripening (Fry et al., 1992). Modification of cambium division in BR deficient mutant further suggested the involvement of endogenous BRs in xylem differentiation in vivo (Szekeres et al., 1996). Role of BRs in vascular differentiation was also supported by isolation of BRs from cambial region of Pinus silvestris (Kim et al., 1990). The role of BRs in vascular differentiation was further confirmed by using inhibitor of BR biosynthesis. It was observed that inhibition of secondary xylem development by brassinazole (Brz) in Lepidum sativum got alleviated by the application of BL (Nagata et al., 2001). In Arabidopsis, a provascular/ procambial cell-specific gene, VH1, which marked the transition to the procambial state, had been identified. Although it encoded a leucine-rich repeat receptor kinase, it did not respond to EBL (Clay and Nelson, 2002). Ohashi-Ito and Fukuda (2003) found that transcription factors were positively regulated by BRs in Zinnia members. Sterols have been implicated in the establishment of the vascular pattern, in auxin transport and distribution, and more recently in leaf senescence (Willemsen et al., 2003). Cano-Delgado et al., (2004) found that BRI1 is ubiquitously expressed in growing cells while the expression of BRL1 and BRL3 is restricted to non-overlapping subsets of vascular cells in Arabidopsis. Loss-of-function of brl1 caused abnormal phloem:xylem differentiation ratios and enhanced the vascular defects of a weak bri1 mutant. bri1 brl1 brl3 triple mutants enhance bri1 dwarfism and exhibited abnormal vascular

differentiation. Thus, BR receptors were observed to have specific functions in cell growth and vascular differentiation. 2.4.6 Reproductive Biology The development of male and female reproductive organs is coordinately regulated by many external and internal cues. These include hormones and external stimuli. The application of BRs results in enhanced fertilization. Grove et al. (1979) reported that pollens are the richest source of endogenous BRs. Hewitt et al. (1985) studied the pollen tube growth in vitro and compared the response of pollen tubes between applications of different concentrations of GA, auxin and BRs. It was observed that 1.0 nM BR induced maximum elongation of the pollen tube. Further the role of BRs,

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in reproduction was strengthened by the observations made on BR-deficient dwf4 mutant and cpd mutant where filament failed to elongate such that pollens although viable fail to reach stigma (Szekeres et al., 1996). BRs are also found to affect the flowering of different plants. Subcellular localization of BRs was explored in pollen of Brassica napus and Lolium temulentum, using polyclonal antibodies generated against castasterone. It was observed that BRs might be stored in developing starch granules and released on imbibition (Sasse et al., 1992). The relative distribution of BRs has also been explored in pollen grains and it was observed that conjugated testasterone was present at the microspore stage. The level of conjugated testasterone got decreased as the pollens developed and levels of free BRs were increased (Asakawa et al., 1996). In case of grape, spraying of BRs in autumn increased the number of flowers, while such application in late winter reduced flower production (Rao et al., 2002). Singh and Shono (2003) observed that in-vitro pollen germination was more tolerant to high temperature in tomato pollen when treated with 24-EBL. A significantly higher in-vitro pollen germination, enhanced tube growth and low pollen bursting were observed in the presence of epibrassinolide at 35C. Kesy et al. (2004) had observed the inhibitory effects of BRs on the flowering of short-day plant Pharbitis nil. The degree of flowering inhibition was depended on the concentration and method of BR application as well as the length of the inductive period. In Pharbitis nil, plants regenerated from sub-induced apices treated with BL at concentration of 1 and 10 M, the flower formation was inhibited completely. Using real-time quantitative RT-PCR and microarray experiments, Ye et al. (2010) found that the expression of many key genes required for anther and pollen development were suppressed in BR biosynthetic and signaling mutants. ChIP analysis further demonstrated that BES1, an important transcription factor for BR signaling, directly bind to the promoter regions of genes encoding transcription factors required for anther and pollen development (SPL/NZZ, AMS, MS1,TDF1 and MS2). 2.4.7 Photosynthesis Photosynthesis plays an important role in the crop growth. Environmental factors are also involved in the disturbance of this characteristic. Green leaves and stem are the sites for photosynthesis and its rate also depends on chlorophyll bearing surface area

28

(Edwards & Walkers, 1983). The role of BRs in vegetative growth was further studied by their capacity to enhance the rate of photosynthesis. The spray application of 24-EBL was carried out to study gas-exchange, chlorophyll fluorescence characteristics, rubisco activity, and carbohydrate metabolism in cucumber (Cucumis sativus L.) plants grown in a greenhouse. 0.1 mg l-1 concentration of 24-EBL was found to be the most effective in increasing light-saturated net CO2 assimilation rate accompanied by maximum rate of rubisco carboxylation and RuBP regeneration. 24-EBL-treated leaves also showed the higher quantum yield of PS II electron transport than the controls. There was also increase in activity of rubisco and sucrose, soluble sugars, and starch contents (Yu et al., 2004). Fariduddin et al. (2006) studied effect of 28-HBL on the nitrate reductase, carbonic anhydrase activities and net photosynthetic rate in Vigna radiate. The leaves of the plants treated with 28-HBL not only photosynthesize at a faster rate, with higher chlorophyll content but also possessed an extended period of metabolic activity because 28-homobrassinolide delayed senescence. Xia et al. (2009c) showed that treatment with 24-EBL improved growth of cucumber (Cucumis sativus) plants. This improvement was associated with increased CO2 assimilation and quantum yield of PSII (Phi(PSII)). Brassinazole treatment reduced plant growth, CO2 assimilation and Phi (PSII). Thus, the growth-promoting activity of BRs may be attributed to enhanced plant photosynthesis. The effects of 24-EBL and Brz on a number of photosynthetic parameters and their affecting factorssuch as contents and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was studied. Northern and Western blotting revealed that 24-EBL up-regulated, while Brz downregulated, the expressions of certain photosynthetic genes i.e. rbcL, rbcS. In addition, 24-EBL had a positive effect on the activation of Rubisco based on increased maximum Rubisco carboxylation rates and total Rubisco activity. The accumulation patterns of Rubisco activase (RCA) based on immunogold-labeling experiments This suggested a role of RCA in BR-regulated activation state of Rubisco. It was observed that enhanced expression of genes encoding other Calvin cycle genes after 24-EBL treatment might play a positive role in RuBP regeneration thereby increasing maximum carboxylation rate of Rubisco. Thus, BRs promoted growth and photosynthesis in

29

cucumber by positively regulating synthesis and activation of a variety of photosynthetic enzymes such as Rubisco. 2.4.8 Senescence BRs play a crucial role in regulating the process of senescence. In contrast to cytokinins, BRs promoted senescence in Xanthium, Rumex explants and in detached cotyledons of cucumber seedlings and leaves of mung bean seedlings (He et al., 1996; Rao et al., 2002). Under the influence of BRs, altered activities of antioxidative enzymes (POD, SOD and CAT) and increased level of malondialdehyde (MDA) content were observed and it was suggested that BRs might regulate these effects via activated oxygen. Various studies suggested that 24-EBL and 28-HBL inhibited the oxidative degradation, decreased MDA levels and acted as membrane protectant thereby delaying senescence (Ershova and Khripach, 1996; Anuradha and Rao, 2007). Further, the role of BRs in senescence was confirmed by Li et al. (1996) in case of BR-deficit Arabidopsis mutants. These BR-deficit Arabidopsis mutants showed delayed chloroplast senescence. A model for leaf senescence regulating network in Arabidopsis was developed by He et al. (2001). Out of 147 senescence-associated enhancer trap lines, 24-EBL could activate only two and till now associated genes have not been cloned. Saglam-Cag (2007) studied the effects of 24-EBL on senescence of wheat leaves and it was observed that 24-EBL activated senescence at higher concentrations. Zhu et al. (2010) investigated the effects of brassinosteroids (BRs) against blue mould rot caused by Penicillium expansum and on senescence of harvested jujube fruit. Brassinosteroids at a concentration of 5.0 M effectively inhibited development of blue mould rot and enhanced the activities of defence-related enzymes such as polyphenoloxidase, phenylalanine ammonia lyase, superoxide dismutase and catalase. BRs significantly delayed senescence of jujube fruit by reducing ethylene production and maintained fruit quality. Therefore, the effects of BRs on reducing decay caused by P. expansum may be associated with induction of disease resistance and delay of senescence injujube fruit.

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2.5

Cross-talk

between

brassinosteroids

and

other

plant

hormones/metabolites
Synchronized regulation of plant growth and development in response to environment requires a cross-talk between hormones and the signaling pathways initiated by the external stimuli. (Bajguz and Hayat, 2009). In recent years it has become apparent that plant growth regulators rarely act alone, but rather their signaling pathways are intermingled in complex networks (Dettmer et al., 2009). At the molecular level, the perception of extracellular stimuli and activation of appropriate responses require a complex interplay of signaling cascades. Phytohormones such as abscisic acid, salicylic acid, and jasmonic acid regulate the protective responses of plants to various kinds of stresses independently and through additive, synergistic or antagonistic cross talks (Bostock, 2005; Lorenzo and Solano, 2005; Xia et al., 2009b). Molecular approaches also present the idea that there exists a cross-talk between BRs and other phytohormones (Bouquin et al., 2001; Vert et al., 2008). Analysis of the growth promoting activities and stress-ameliorative properties of BRs revealed that BRs must be interacting with other hormones (Krishna, 2003; Sasse, 2003; Haubrick and Assmann et al., 2006). The interaction of BRs with auxin produced synergistic effects while ABA showed an antagonistic effect of BR action (Abraham et al., 2003). With GA the effects were additive. Cross talk between BRs and auxins, GA, ethylene ABA, JA and polyamines included alteration in the expression of hormone biosynthetic genes and/or signaling intermediates (Goda et al., 2002; Lin et al., 2003). 2.5.1 BRs and auxin Brassinosteroids (BRs) act synergistically with auxin to regulate a variety of plant developmental and physiological processes. BRs activated the expression of auxinresponsive genes and this had been confirmed by RNA blots and microarray experiments (Mussig et al., 2002). BRs have been shown to synergistically promote cell elongation when supplied with auxin (Mandava, 1988). Tanaka et al. (2003) reported that coapplication of auxins and BL to Arabidopsis seedlings resulted in synergistic increase in hypocotyl elongation. Bao et al. (2004) observed that BRs interacted synergistically with auxins to promote lateral root growth in Arabidopsis. BRs application promoted lateral root development by stimulating acropetal auxin transport (from base to tip) in the root. 31

Goda et al. (2004) reported the interaction between BRs and auxins by using an affymetrix gene chip representing approximately 8,300 Arabidopsis genes. They studied comprehensive transcript profiles over 24 h in response to indole-3-acetic acid (IAA) and brassinolide (BL). They identified 409 genes as induced by BL, 276 genes as IAA inducible, and 637 genes in total. Out of these, 48 were regulated by both IAA and BL. The SAUR, GH3, and IAA gene families were the largest group of genes regulated by both IAA and BL. A number of the early auxin-inducible genes were not specifically regulated by auxin but were regulated by these two hormones in common. A promoter had been identified which was responsive to both auxin and BR (Nemhauser et al., 2004). BRs such as 24-epicastasterone and 24-EBL promoted root elongation in Arabidopsis. The growth-stimulating effect of exogenous BRs was not reduced by the auxin transport inhibitor (2,3,5-triidobenzoic acid). Simultaneous administration of 24-EBL and 2,4-dichlorophenoxyacetic acid resulted in additive effects. Exogenous GAs did not promote root elongation in the BR-deficient mutants. Thus, the root growth-stimulating effect of BRs appeared to be largely independent of auxin and GA action. BRs also interacted with other phytohormones on the gene expression level. Only a limited set of auxin- and ethylene-related genes showed altered expression levels. Genes related to other phytohormones barely showed changes, providing further evidence for an autonomous stimulatory effect of BR on root growth (Mussig and Altmann, 2003). Grauwe et al. (2005) described the involvement of BRs in auxin and ethylenecontrolled processes in the hypocotyls of both light and dark grown seedlings of Arabidopsis. They found that BR biosynthesis was necessary for the development of an apical hook. Moreover, either application of BRs or disruption of BR synthesis altered auxin response, most probably by affecting auxin transport, which ultimately resulted in the disappearance of the apical hook. BRs enhanced plant tropism responses by enhancing the expression of PIN2 and ROP2 from root tip to the elongation zone, which ultimately increased the gravitropism (Li et al., 2005). Similarly, the relationship between BL and auxin in the promotion of stem elongation was studied by Sasse (2006) using hook and subhook zone of the stem of six day old etiolated Pisum sativum seedlings. It was found that BL-induced elongation was significantly reduced by low concentrations of the 2, 6

32

dichlorobenzonitrole. But antiauxin (2 p-chlorophenoxy isobutyric acid) did not affect BL induced elongation. This showed interdependency of BL on auxin. Vert et al. (2008) that BR-regulated BIN2 enhanced expression of auxin-induced genes by directly inactivating repressor ARFs (members of auxin response factor family of transcriptional regulators) leading to synergistic increase in transcription. Nakamura et al. (2009) conducted lamina joint tests to elucidate the mechanism of cross-talk between BR and auxin signaling in lamina joint bending. In BR biosynthetic mutants d2 and brd1, which were defective in C-23 hydroxylase and C-6 oxidase, the lamina joint response to auxin was significantly higher than that of wild-type plants. The other BR-biosynthetic mutants, brd2, osdwarf4 and d11, which were defective in C-22hydroxylated BRs, showed less or no response to auxin. These results suggest that C-22hydroxylated BRs are involved in auxin-induced lamina joint bending. 2.5.2 BRs and GA BRs have been found to have additive effects with GA (gibbrellic acid). Tanaka et al. (2003) reported that the synergism observed between BL and GA might be related to the fact that both hormones increased expression of MERI5, an XET which is involved in loosening of the cell wall. BRs interacted with GA to promote seed germination. Chen et al. (2004) revealed that BRs and GA both regulate the expression of GPA1 (alpha subunit of the G protein in Arabidopsis) during seed germination. Yang et al. (2004) carried out the microarray analysis of BRs and GA regulated gene expression in rice seedlings and it was observed that both BR and GA influenced growth and development by coordinately regulating the expression of specific groups of genes. Wang et al. (2009) demonstrated that OsGSR1, a member of the GAST (GAstimulated transcript) gene family, plays important roles in both BR and GA pathways, and also mediated an interaction between the two signaling pathways. The OsGSR1 RNAi transgenic rice shows a reduced level of endogenous BR, and the dwarf phenotype could be rescued by the application of brassinolide. The yeast two-hybrid assay revealed that OsGSR1 interacts with DIM/DWF1, an enzyme that catalyzes the conversion from 24-methylenecholesterol to campesterol in BR biosynthesis. These results suggest that

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OsGSR1 activates BR synthesis by directly regulating a BR biosynthetic enzyme at the post-translational level. 2.5.3 BRs and ABA BR-deficient sax1 mutant showed enhanced stomatal closure suggesting antagonistic relationship between BRs and ABA (Ephritikhine et al., 1999). Steber and McCourt (2001) reported that BR signals are needed for overcoming the dormancy of seeds caused by ABA as BR biosynthesis and signaling mutants are ABA hypersensitive. The interactions between ABA and BRs had been studied in Arabidopsis by Friedrichsen et al. (2002) where three genes, BEE1, BEE2, and BEE3, were positively regulated by BRs and negatively regulated by ABA. These genes encoded transcription factors important in several developmental programs such as floral organogenesis, light signaling and hormone responses. Karnachuk et al.(2002) reported that pretreatment of Arabidopsis seeds with 24-EBL enhanced the level of free and bound auxin and of free ABA, a slight enhancement in free cytokinin and decrease in level of bound ABA. BR treatment also inhibited the expression of the ABA-induced gene P5CS1 which is important for the synthesis of the proline (Abraham et al., 2003). The interaction of BL and ABA was further documented by Kurepin et al. (2008) in heat stressed Brassica napus seedlings. The seedlings exposed to the heat stress (45 0C) and 10-6 M solution of BL one hour prior to the beginning of thermal stress revealed nearly double amount of endogenous ABA levels in comparison to only heat-stressed seedlings. So this study suggested that the heat tolerance provided by BRs might be mediated by BRs-induced higher level of endogenous ABA. 2.5.4 BRs and Jasmonates BRs also interact with jasmonates and it was reported by Schaller et al. (2000) that BRs treatment altered the expression of an important enzyme (12-oxophytodienoate reductase 3 (OPR3)) required for the biosynthesis of JA. BR treatment enhanced the level of OPR3 transcript level. In addition to OPR3, BRs also enhanced the expression of OPR1 and OPR2. But the microarray analysis of BR regulated gene expression was not able to provide clear image of function of BRs in JA-mediated signaling (Goda et al., 2002; Mussig and Altmann, 2003). 34

Campos et al. (2009) observed that the combination of brassinosteroids and jasmonates, have an effect on trichome density and allelochemical content, in an opposite mode. Defence traits were promoted by jasmonates whereas BR reduces them. BR deficient mutant dpy increases pubescence, proteinase inhibitor expression and zingiberene biosynthesis whereas JA-insensitive jai1-1mutant oppositely regulated these processes. 2.5.5 BRs and Ethylene BRs stimulated the ethylene production via regulation of genes involved in ethylene synthesis. They enhanced the production of ethylene in case of mung bean when it was applied to the epicotyl segments (Arteca and Arteca, 2001). Chang et al. (2004) studied the interaction of BL and ethylene in the gravitropic response mechanism of maize primary roots. BL was involved in the gravitropic response in primary roots via ethylene production, but it acted in a way that differed somewhat from that of ethylene. There was enhancement in gravitropic curvature in a dose-dependent manner when ethylene was applied exogenously. This effect of ethylene was confirmed by the fact that AVG, a specific action inhibitor of ACC synthase, reduced the gravitropic curvature in the presence and absence of BL. Since AVG did not inhibit BL-increased gravitropic curvature completely, it was proposed that BL might act on the gravitropic response by ways other than simply through enhanced ethylene production. BL might exhibit some of its stimulatory effect in the absence of ethylene. In addition, BL reduced the presentation time and lag period for the gravitropic response, whereas ethylene increased them. One possible mechanism of such action was that BL affects protein kinase activity, since the protein kinase inhibitors, staurosporine and H89, reduced BL-increased gravitropic curvature. Grauwe et al (2005) showed that brassinosteroids are involved in auxin and ethylene controlled processes Arabidopsis hypocotyls (both, light- and dark-grown). Brassinosteroid biosynthesis play vital role in the formation of an exaggerated apical hook and either application or disruption of BR synthesis altered auxin response by affecting auxin transport and finally causing disappearance of apical hook. It was also

35

found that hypocotyl elongation, which was stimulated by ethylene in the light, is regulated by same mechanism as apical hook formation in dark. Arteca and Arteca (2008) studied the effects of BRs, auxin, and cytokinin on ethylene production in Arabidopsis thaliana plants. It was observed that inflourescence stalks produced the highest amount of ethylene in response to IAA as compared with other plant parts tested. But the Inflourescences treated with BL alone had no effect on ethylene production. However, when BL was used in combination with IAA there was a significant enhancement in ethylene production as compared to the level of ethylene produced by application of IAA alone. 2.5.6 BRs and Salicylic acid Nakashita et al. (2003) revealed that the BL (brassinolide) mediated resistance does not require salicylic acid (SA) in tobacco. In order to study the potentialities of BL activity on stress responding systems, they analyzed its ability to induce disease resistance in tobacco and rice plants. The experimental analysis revealed that wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with brassinolide and the pathogen infection assays using NahG transgenic plants indicated that brassinolide-induced resistance did not require SA biosynthesis. 2.5.7 BRs and Polyamines Cavusoglu and Kabar (2007) studied the comparative effects of gibberellic acid, kinetin, benzyladenine, ethylene, 24-EBL triacotanol and polyamines (cadaverine, putrescine, spermidine, spermine), alone or in combinations, on germination and early seedling growth under high temperature conditions in barley and radish seeds. High temperature delayed and inhibited the germinations of both the species (barley and radish). Only three of the single applications of gibberellic acid, kinetin and 24-EBL alleviated the effects of high temperature on germination of barley seeds. All the combinations composed of these three growth regulators removed more successfully the adverse effect of high temperature on germination. Choudhary et al. (2009a) reported that 24-EBL regulated the synthesis of polyamines and auxins in Raphanus sativus L. cv. Pusa Chetki seedlings under Cu metal

36

stress. The effects of various concentrations of 24-EBL (10-11, 10-9 and 10-7 M) on polyamines and auxins was analyzed in 7-days old seedlings of R. sativus under Cu stress. Putrescine, Cadaverine and spermidine got enhanced in 10-11, 10-9 and 10-7 M respectively of 24-EBL treatment when applied alone to `R. sativus seedlings. However, metal treatment supplemented with Cu (0.2 mM) revealed small increase in polyamines thereby indicating the modulation of stress responses by BRs via regulating the contents of polyamines. 2.5.8 BRs and Amino acids Morera-Boado et al. (2009) studied the interaction of the most active natural brassinosteroid, BL, with the twenty natural amino acids utilizing the multiple minima hypersurface method to model the molecular interactions explicitly. Resulting thermodynamic data gave useful information about the amino acids with the greatest association for brassinolide and the stabilities of such complexes. Density functional theory (DFT) method was performed to explore the formation of molecular complexes. The semiempirical geometries and stability order of these complexes were in good agreement with the DFT calculations. Each group of amino acids possessed a preferential zone of interaction with BL, forming the polar-charged amino acids the most stable complexes. This study could contribute to future investigations of the interaction of brassinosteroids with the receptor protein in plants.

2.6

Role of BRs in Agriculture

Since the discovery of BL in 1970, field experiments were conducted to ensure its

practical application in the agriculture. Field trials made in USA (1970), in Japan and USSR showed the beneficial effects of BRs and confirmed their usefulness as agricultural chemicals (Mitchell and Gregory, 1972; Gregory, 1981; Fujita, 1985; Takeuchi, 1992; Khripach et al., 1993, 2000). In addition to growth alterations, BRs also influence plant development, enhance yield and provide tolerance to plants under stressed conditions. Under field trials better results were obtained with 24-EBL and 28-HBL than BL, even though the activities of BL were higher in bioassays. It promoted the use of 24-EBL and 28-HBL in agricultural applications. Now days, 24-EBL is used as the active ingredient in field preparations and these are officially registered and have large scale application in agriculture. 24-EBL is the active ingredient of the plant growth promoting preparation

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Epin which was recommended for treatment of agricultural plant such as tomato, potato, cumber, pepper and barley to enhance their yield and quality in Russia, Belarus and Japan (Moiseev, 1998). Another chemical Tianfengsu was developed in China, which is a mixture of 24-EBL and its unnatural 22S, 23S isomer. It is widely used in china to increase yield of crops such as rice, maize, wheat, tobacco, vegetables and fruits (Ikekawa and Zhao, 1991) 2.6.1 Biosafety of BRs Before utilizing BRs commercially in agriculture, scientists have studied their biosafety aspects. Being the normal constituent of all plants, BRs are consumed by mammals, so no additional harmful effects can be expected from their use in agriculture. The confirmation of their safety had been obtained from toxicological studies made in the Sanitary-Hygienic Institute of Belarus for 24-EBL. It was observed that LD50 (orally) in mice (female) was more than 1000 mg kg-1 and LD50 (orally and dermally) in rats (male/female) was more than 2000 mg kg-1. In addition, 0.2 % 24-EBL did not irritate mucous membranes of rabbit eyes. Further Ames test for mutagenic activity carried out at Scientific Research Center of Toxicologic and Hygienic Regulation of Biopreparations of Russia, was negative. Studies on fish toxicity also showed no negative effects, but showed toxico- protective properties (Khripach et al., 2000). 2.6.2 Effect of BRs on Yield and Quality of Crops, Vegeatbles and Fruits Brassinolide, 24-EBL and 28-HBL and some other BRs have been tested in field trials to determine their effect on plant growth and development and on crop yield in natural conditions. Soaking tobacco seed in 0.01-0.05 mg l-1 solution of 24-EBL significantly enhanced percent seed germination. Also the growth of the leaves, roots and leaf nicotine content increased by foliar application of 24-EBL (Han et al., 1987; Ikekawa and Zhao, 1991). Further Zhang (1987, 1989) noted that the foliar spray of 24-EBL, prior to the tassel emergence, significantly decreased the kernel abortion of its ear tips and increased the corn yield. Enhanced corn yield was associated with increased weight of 1000 grains, number of grains per ear, enhanced photosynthetic rate and acceleration in elongation of pollen tube. There was 10% increase in yield of corn by spraying 0.01 mg l-1 24-EBL.

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24-EBL also improved the tolerance of rape plants to cold injury and their by improving its seed yield by 10%. The spraying of 0.01 mg l-1 of 24-EBL at flowering stage, reduced premature abscission of flowers and young fruits of orange and grapes by decreasing the activities of cellulase in abscission zone and fruit attained maturity early (Hu et al., 1990; Xu et al., 1994b). 24-EBL also enhanced the yield and sugar content of sugar beet. Beet seeds soaked in 0.04 mg l-1 solution of in this brassinolide revealed maximum germination. Foliar spray of 0.04 mg l-1 solution of 24-EBL improved the growth of seedlings and the roots (Zhao and Chen, 2003). EBL showed the significant growth promoting effects on vegetables (celery, cabbage, onion and lettuce) and watermelon (Wang et al., 1998; Huang and Li, 1998). Foliar application of synthetic BR (TNZ 303) promoted the growth of tomato under stressed conditions (Zhao, 2000). Application of BRs not only enhanced the yield but also improved the quality of crops. The application of 28-HBL and 24-EBL to potato plants in a dose of 10-20 mg ha-1 enhanced the yield by 20% and improved the quality by decreasing nitrate content and enhancing starch and vitamin C content (Khripach et al., 1996a). BRs also reduced the accumulation of heavy metals and radioactive elements when plants are grown in areas that were contaminated by these (Khripach, 1995, 1996b; Sharma and Bhardwaj, 2007a, 2007b). BRs also stimulated the growth of mushrooms (Alexeeva et al., 1999). Application of 28-HBL (0.05 ppm) either as seed treatment or foliar spray to two wheat varieties (C306, drought tolerant and HD 2329, drought susceptible) increased their biomass production and grain yield even under irrigated and moisture-stress/rainfed conditions. This enhanced yield was associated with increased relative water content, nitrate reductase activity, chlorophyll content and photosynthesis (Sairam, 1994). Takeuchi et al. (1995) studied the effects of BR on conditioning and germination of clover broomrape (Orobance minor) seeds. BL applied at early stages of conditioning shortened the conditioning period required before clover broomrape seeds would germinate after exposure to germination stimulants such as dl-strigol and natural stimulants from red clover (Trifolium pratense L.) root exudates. Ramraj et al. (1997) studied yield responses of some economically important crop plants like wheat, rice, groundnut, mustard, potato and cotton to foliar application of low concentration of 28-

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HBL (0.25-0.5 mg l-1). The 28-HBL treatment significantly increased grain yield in wheat, rice and mustard, pod yield in groundnut, tuber yield in potato and seed cotton yield as compared to control. Korableva et al. (1999) reported that the treatment with 24EBL prolonged dormancy of potato tubers and increased their resistance to sprouting and diseases, changes that are associated with enhancement of ABA and ethylene levels, and also the presence of terpenoid and phenolic protective substances. Zhao and Chen (2003) studied the role of 24-EBL in agriculture. 24-EBL treatment enhanced the yield of wheat, tobacco, corn, rape orange, grape and sugar beet. Large scale field trials, over a period of 10 years have shown that percentage of grain setting, number of caryopsis per ear and weight of 1000 grains of wheat increased by the application of 24-EBL. This enhancement in yield was associated with increased rate of photosynthesis and direct translocation of photosynthates to the ears. tranc et al. (2008) observed the positive influence of BRs and Lexin preparation (fulvic and humic acids mixture and auxins) on physiological state and yield of soybean. Plants treated with these preparations were found to be more resistant to short term drought, showed better physiological state and energy balance of photosynthesis and higher seed yield. Song et al. (2009) suggested that the expression of OsBAK1, a potential gene to alter rice architecture, changed important agricultural traits of rice such as plant height, leaf erectness, grain morphologic features, and disease resistance responses. OsBAK1 is considerably influenced by BRs treatment. The erect-leaf is a significant morphological trait partially regulated by BRs in rice plants. Based on rice genome sequences, four closely related homologs of Arabidopsis BAK1 (AtBAK1) genes were amplified. Phylogenetic analysis and suppression of a weak Arabidopsis mutant bri1-5 indicated that OsBAK1 (Os08g0174700) is the closest relative of AtBAK1. Genetic, physiological, and biochemical analyses all suggested that the function of OsBAK1 is conserved with AtBAK1. Overexpression of a truncated intracellular domain of OsBAK1, but not the extracellular domain of OsBAK1, resulted in a dwarfed phenotype, similar to the rice BR-insensitive mutant plants. Therefore, OsBAK1 may be a potential molecular breeding tool for improving rice grain yield by modifying rice architecture.

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Wu et al. (2008) observed genes controlling hormone levels have been used to increase grain yields in wheat (Triticum aestivum) and rice (Oryza sativa) created transgenic rice plants expressing maize (Zea mays), rice, or Arabidopsis thaliana genes encoding sterol C-22 hydroxylases that control BRs levels using a promoter that is active only in the stems, leaves, and roots. The transgenic plants produced more tillers and more seed than wild-type plants. The seeds were heavier as well, specially the seed at the bases of the spikes that fill the least. BRs stimulate the flow of assimilate from the source to the sink. Microarray and photosynthesis analysis of transgenic plants revealed evidence of enhanced CO2 assimilation, enlarged glucose levels in the flag leaves, and increased assimilation of glucose to starch in the seed. These results further suggested that BRs stimulated the flow of assimilate. Hola et al. (2010) studied the the application of 1081014 M solutions of 24EBL or synthetic androstane analogue of castasterone in two field-grown inbred lines of maize (Zea mays) and their F1 hybrid in V3/4 and V6/7 developmental stages followed during the vegetative and early reproductive phases of plant development. BRs significantly affected (depending on the genotype and the developmental stage they were applied) the height of plants during the early weeks after their application, but not the final plant height nor the number of leaves. Spraying of Zea mays plants with BRs in V3/4 developmental stage increased the length leaf, whereas the application in V6/7 developmental stage had the opposite effect. The beginning of the reproductive phase of plant development and the flowering period were strongly influenced by the BRs application. Treatment of plants in V3/4 stage delayed and treatment of plants in V6/7 stage progressed the dates of anthesis and silking, irrespective of the type of BR used, its concentration or plant genotype. Various yield parameters were also affected by treatment of plants with BRs, but this effect depended on the developmental stage during which the BRs were applied, the plant genotype, the type of BR and its concentration. 2.6.3 Micropropagation/Tissue Culture Franck-Duchenne et al. (1998) explored the role of BRs in the area of tissue culture. EBL was tested for in vitro stem elongation of sweet pepper (Capsicum annuum L.) in the culture medium. It was observed that 24-EBL enhanced stem elongation might be through an elicitor or an enhancer of elongation.

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Bieberach et al. (2000) suggested that micropropagation of Cassava (Manihot esculenta Crantz), Yam (Dioscorea alata L.) and pineapple (Ananas comosus L. Merril) could be improved by the application of 28-homocastasterone and 3-acetyl-28homoteasterone. Schaefer et al. (2002) reported that application of 5-fluoro-28- homocastasterone to shoot apices of apple root stock (Mallus prunifolia (Wild.) Borkh) increased the multiplication rate of root stocks by 112 %. Pereira-Netto et al. (2006) further studied the role of BRs in tissue culture. Their findings proposed the role of BRs in micropropagation techniques for clonal propagation of woody angiosperms. 28-Homocastasterone (28-homoCS) was used to treat in vitrogrown shoots of a hybrid between Eucalyptus grandis and E. urophylla. Treated shoots showed enhanced elongation and formation of new main shoots at low doses but there was reduced elongation and formation of primary lateral shoots. The effect of 5monofluoro derivative of 28-homoCS (5F-28-homoCS) was also studied and it was observed that 5F-28-homoCS was unable to either stimulate elongation or formation of new main shoots, although it did stimulate elongation of primary lateral shoots. 2.6.4 Fruit ripening Fruit ripening is a unique plant development process with direct implications on our food supply, health and nutrition. The role of various phytohormones like auxins, cytokinins, GAs, ethylene and jasmonates (JA) has been studied in fruit ripening. BRs are recently implicated for their role in fruit ripening (Deluc et al., 2007). BRs are not only involved in fruit ripening but also decrease physiological drop of fruits (citrus, peach, apple, pear) from trees (Susumu et al., 1991). 24-EBL, 28-HBL, BL and BR analogue, BB-16 were found to accelerate the process of fruit ripening in case of tomato, rice, yellow passion fruit, grapes and cucumber (Vardhini and Rao, 2002; Montoya et al., 2005; Gomes et al., 2006; Deluc et al., 2007; Fu et al., 2008). Susumu et al. (1991) proposed a method of decreasing physiological drop from fruit trees using brassinolide. The treatment of overground portion of a growing citrus, peach, apple or pear with a liquid agricultural composition comprising BL in a concentration of about 110-5 ppm to about 5.0 ppm reduced the fruit drop successfully.

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In rice, BL treatment before and during flowering accelerated ripening and significantly increased starch content in hulled grains (Fujii and Saka, 2001). The effect of 24-EBL and 28-HBL on ripening of tomato pericarp discs was studied by Vardhini and Rao (2002). Application of BRs to pericarp discs resulted in elevated levels of lycopene and lowered chlorophyll levels. In addition, BR-treated pericarp discs exhibited decreased ascorbic acid and increased carbohydrate contents. Fruit ripening in tomato as induced by BRs was associated with increase in ethylene production. The study also revealed the ability of BRs in accelerating fruit-senescence. Montoya et al. (2005) revealed the importance of BR synthesis during fruit development in tomato. Their results indicated that endogenous BRs showed intense biosynthesis in developing tomato fruits, which were found to contain high amounts of BL. Symons et al. (2006) studied the role of BRs in ripening of non-climacteric fruit, grape (Vitis vinifera). In contrast to climacteric fruit, where ethylene is essential, BRs have played important role in ripening of non-climacteric fruit, such as grape (Vitis vinifera). It was observed that increase in endogenous BR levels but not indole-3-acetic acid (IAA) or GA levels have been associated with ripening of grapes. The function of the BR biosynthesis enzyme gene was confirmed by transgenic complementation of the tomato extreme dwarf (dx/dx) mutant. Expression analysis of these genes during berry development revealed transcript accumulation patterns that were consistent with a dramatic increase in endogenous BR levels observed at the onset of fruit ripening. Moreover, it was observed that application of BRs to grape berries significantly promoted ripening, while application of Brz, an inhibitor of BR biosynthesis, significantly delayed fruit ripening. Deluc et al. (2007) has also confirmed the role of BRs in grape berry development. The concentration of castasterone is low during the early stage of development and then increase at onset of ripening. It was revealed that there is enhancement in BRH 1 RING finger protein transcript, which is known to be downregulated by exogenous application of BR, decreased during early stage of development, but increased in fully matured berries.

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Further, Fu et al. (2008) showed that BRs promoted early fruit development in cucumber. The effect of 24-EBL and Brz on early fruit development, cell division, and expression of cyclin and cyclin-dependent kinases (CDKs) genes in two cucumber cultivars that differ in parthenocarpic capacity had been studied. The application of 24EBL induced parthenocarpic growth accompanied by active cell division in Jinchun No. 4, a cultivar without parthenocarpic capacity, whereas Brz treatment inhibited fruit set in Jinchun No. 2, a cultivar with natural parthenocarpic capacity. This inhibitory effect had been rescued by the application of 24-EBL. RT-PCR analysis showed that both pollination and EBL induced expression of cell cycle-related genes (CycA, CycB, CycD3;1, CycD3;2, and CDKB) after anthesis. BR6ox1 and SMT transcripts, two genes involved in BR synthesis, exhibited feedback regulation. This study strongly suggested that BRs play an important role during early fruit development in cucumber. Clouse (2008) and Yu et al. (2008) proposed the role of BRs in growth and flowering by molecular intersection in Arabidopsis. They observed a connection between BR signal transduction and pathways controlling floral initiation. A critical transcription factor required for BR dependent gene expression directly interacts with two transcription regulators having divergent roles in modulating time of flowering in Arabidopsis.

2.7

Prospects of Brassinosteroids in Stress Management

BRs are unique in their activities for not only regulating the diverse physiological

and morphogenetic responses in plants, but also having a significant role in amelioration of various biotic and abiotic stresses at nanomolar to micromolar concentrations (Krishna, 2003; Mussig et al., 2005). These are thermal, drought, heavy metals, infection, pesticides, salt and even viruses (Dhaubhadel et al., 1999; 2002; Wachsman et al., 2000, 2002, 2004a, 2004b; Krishna, 2003; Haubrick and Assmann, 2006; Sirhindi et al., 2009). Numerous reports have indicated that BRs application resulted in the enhancement of antioxidative enzymes activities (SOD, CAT, POD, APOX, GR, MDHAR, DHAR) and antioxidants under various kinds of stresses (Shahbaz et al., 2008; Bajguz and Hayat, 2009) to provide protection to the plants (Hayat et al., 2007a, 2007b; Arora et al., 2008a, Choudhary et al., 2009a) (Table 6). The protective effects of BRs and their structural analogs were studied on growth, lipid peroxidation and antioxidative system of rice, soyabean, tomato, potato, chickpea and maize under various stresses (Table 7) and it was

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observed that application of these hormones lowered the oxidative stress and promoted plant growth (Mazorra et al., 2002; Ozdemir et al., 2004; Almeida et al., 2005; Bhardwaj et al., 2007b; Ali et al., 2008a). 2.7.1 Antifungal Properties Exogenous application of BRs stimulated inner potentials of plants that is helpful not only in better survival in stress conditions, but also in diminishing disease damage. The potential of BRs to enhance plant resistance against fungal pathogen infection was documented in several studies (Khripach et al., 2000). Vasyukova et al. (1994) studied the interaction between Phytophthora infestans and potato tubers. The treatment of potato plants with BRs essentially reduced the incidence of Phytophthora infection. The increase in resistance in BRs treated potato tubers was associated with enhancement of ABA and ethylene levels and the presence of phenolic and terpenoid substances. BRs induced disease resistance was also noted in cucumber and barley plants. Spraying barley plants at tillering phase with 24-EBL decreased an extent of leaf disease induced by Helminthosporium teres Sacc. and increased grain yield even at a dose of 5 mg ha-1 (Pshenichnaya et al., 1997; Volynets et al., 1997b). The stress-protective effects of 24-EBL in cucumber against fungi were studied by Churikova and Vladimirova (1997). The increased activities of peroxidase and polyphenoloxidase enzymes, which are involved in the metabolism of polyphenols, was suggested as a factor leading to BR induced disease resistance in cucumber plants. 2.7.2 Antibacterial and antiviral properties One of the important characteristics of the protective action of BRs in plants is related to their ability to stimulate resistance to virus infections (Rodkin et al., 1997). It has been reported that BR treatment reduced virus infection in the starting plant material, various stages of plant development, and the first and second tuber generations of potato. The plants obtained from BR treated sowing material increased the crop yield by 56% and significantly reduced virus infection. The tobacco plants when given treatment of BRs against tobacco mosaic virus (TMV), the bacterial pathogens Pseudomonas syringae, and the fungal pathogen Odium species, showed lowered infection and better growth. Similarly in rice, the infection caused by Magnaporthe grisea and Xanthomonas oryzae which caused rice blast and bacterial blight respectively, was significantly reduced

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by BR treatments (Nakashita et al., 2003). Michelini et al. (2004) reported the in vitro and in vivo antiherpetic activity of three new synthetic BRs analogues. Chemical synthesis of three new synthetic BRs analogues like (22S,23S)-3-bromo-5,22,23trihydroxystigmastan-6-one, (22S, 23S)- 5-fluoro-3-22,23-trihydroxystigmastan-6one, (22S, 23S)-3-5,22,23-trihydroxystigmastan-6-one and their antiherpetic activity both in human conjunctive cell lines (IOBA-NHC) and in the murine herpetic stromal keratitis (HSK) experimental model were tested. All compounds prevented HSV1multiplication in NHC cells in a dose dependent manner when added after infection with no cytotoxicity. Significantly, in vitro studies had shown that EBL is capable of reducing or even arresting the growth of the HIV in cultured infected cells. BRs and their synthetic derivatives were found to be good inhibitors of herpes simplex virus type 1(HSV-1) and arena virus replication in cell culture. The arena virus was susceptible to the compounds throughout its replication cycle, and the HSV-1 was likely affected at a late step in multiplication (Wachsman et al., 2000, 2004a, 2004b). Khripach et al. (2005) reported that 24-EBL may be used in the prevention and cure of HIV infection and related conditions (AIDS related complex), both symptomatic and asymptomatic, or when exposure to HIV virus was suspected. Romanutti et al. (2007) reported the antiviral effects of a synthetic BR ((22S, 23S)-3-bromo-5,22,23-trihydroxystigmastan-6-one) against replication of vesicular stomatitis virus (VSV) in vero cells. Synthetic BR affected the late event of the virus growth cycle and inhibited virus protein synthesis and viral mature particle formation. Some synthetic BRs were tested against herpes simplex virus type 1(HSV-1) by Michelini et al. (2008) which induced an ocular chronic immunoinflammatory syndrome named herpetic stromal keratitis that might lead to vision impairment and blindness in mice. 2.7.3 Anticancer/Antiproliferative properties The effect of 24-EBL at subnanomolar concentrations was studied by Franek et al. (2003) in mouse hybridoma. 24-EBL was observed to modulate growth and production characteristics of a mouse hybridoma. A mouse hybridoma was cultured either in standard serum-free medium, or in medium diluted to 30%, in which the cells underwent nutritional stress. Steady-state parameters of semicontinuous cultures

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conducted at 24-EBL concentrations from 1016 to 109 mol l1 were evaluated. Typical effects of the 24-EBL found both in standard and in diluted media were increase in the value of mitochondrial membrane potential, drop of intracellular antibody level, increase in the fraction of the cells in the G0/G1 phase, and decrease in the fraction of the cells in the S phase. Viable cell density was significantly higher as compared to control at 24EBL concentrations ranging from 1013 and 1012 mol l1. So 24-EBL might induce perturbations in the cell division mechanism, in mitochondria performance, and in secreted protein synthesis in a mammalian cell line. Swaczynova et al. (2006) studied the anticancer properties of BRs. Natural types of BRs affected the viability, proliferation, differentiation, apoptosis and expression of some cell cycle related proteins in cancer cell lines. Cytotoxic activity of BRs were tested in vitro by Calcein AM assay. IC50 values were estimated for human breast adenocarcinoma cell lines (MCF-7estrogen-sensitive, MDA-MB-468estrogeninsensitive), human acute lymphoblastic leukemia cell line (CEM) and human myeloma cell line (RPMI 8226). TUNEL, DNA ladder assay and immunoblotting techniques were used for the analysis of changes of cell viability, proliferation, differentiation and apoptosis. 28-Homocastasterone inhibited the viability of cancer cell lines and significantly reduced or induced the expression of p21, p27, p53, cyclins, proteins of Bcl2 family and ER-alpha. The antiproliferative properties could be used for development of new brassinosteroid-derived generation of anticancer drugs. Malkova et al. (2008) studied the anticancer and antiproliferative activity of natural BRs. They tested the 28-homocastasterone and 24-EBL on the viability, proliferation, and cycling of hormone-sensitive/insensitive (MCF-7/MDA-MB-468) breast and (LNCaP/DU-145) prostate cancer cell lines. Both BRs inhibited cell growth in a dose dependent manner in the cancer cell lines. Flow cytometry analysis showed that BR treatment arrested MCF-7, MDA-MB-468 and LNCaP cells in G1 phase of the cell cycle and induced apoptosis in MDA-MB-468, LNCaP, and slightly in the DU-145 cells. 2.7.4 Antigenotoxic Properties 24-EBL was found to reveal antigenotoxic properties and this was evaluated through Allium cepa chromosomal aberration bioassay. Howell et al. (2007) investigated the effect of 24-EBL on the mitotic index and growth of onion (Allium cepa) root tips.

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Low doses of 24-EBL (0.005 ppm) nearly doubled the mean root length and the number of mitosis over that of controls. Intermediate doses of 24-EBL (0.05 ppm) also produced mean root lengths and number of mitosis that were significantly greater than those of the controls. But the highest dose of 24-EBL (0.5 ppm) produced mean root lengths and number of mitoses that were less than control values. Sondhi et al. (2008) isolated the 24-EBL from leaves of Aegle marmelos Correa. (Rutaceae) which was evaluated for the antigenotoxicity against maleic hydrazide (MH) induced genotoxicity in Allium cepa chromosomal aberration assay. It was observed that percentage of chromosomal aberrations induced by MH (0.01%) declined significantly with 24-EBL treatment. 10-7 M of 24-EBL was found to be most effective with 91.8% inhibition. 2.7.5 Effects on insect development BRs have been found to exhibit striking structural similarities to the ecdysteroids. Two triterpenoids isolated from seeds of cruciferous plants, cucurbitacins B and D, were found to be insect steroid hormone antagonists acting at the ecdysteroid receptor (Dinan et al., 1997). Zullo and Adam (2002) reviewed the effects of BRs on insect development, particularly on molting, Exploration of the effects of BRs in model systems has provided insights into the metabolism of these compounds. 24-epicastasterone or 24-EBL did not affect the evagination of imaginal wing discs, nor was there any effect on intact last instar larvae of the cotton leaf worm, Spodoptera littoralis after oral feeding (Smagghe et al., 2002). Ohri et al. (2002) reported that the treatment of root knot nematodes (Meloidogyne incognita) with BL revealed comparitively higher percentage of hatching in treated egg masses in comparison to control. Ohri et al. (2005) further revealed enhanced juvenile emergence of M. incognita by BRs treatments. Antioxidative defence system of root knot nematodes (M. incognita) was also stimulated by the treatment of BRs (Ohri et al., 2007). Besides, Ohri et al. (2008) studied the influence of 24-EBL on development of Meloidogyne incognita. 24-EBL augmented the percentage of hatching in treated egg masses as compared to control. 24-EBL treated juveniles stimulated more gall numbers and larger size of galls in roots of tomato plants.

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2.7.5.1 BRs and abiotic stresses

2.7.5.1.1 BRs and Drought Stress Drought stress in sugar beet, wheat and gram was alleviated by treatment with BRs (Sairam, 1994). A wheat variety sensitive to water stress on treatment with 28-HBL showed an increase in grain yield, relative water content and soluble protein contents, while exhibiting reduced ion leakage. The stress-protective properties of BRs had been attributed for enhancing the membrane stability. Xu et al. (1994a) reported that 24-EBL treatment decreased stomatal transpiration and improved the performance of plants under stress. Application of 24-EBL to different varieties of spring wheat under normal and stress conditions (drought soil) was studied by Prusakova et al. (2000). The plants sprayed with 24-EBL solutions in the beginning of booting stage of flowering resulted in higher water content in leaves. BRs have been established to increase plant resistance to drought and increase yield in different crops growing under drought conditions (Nilovskaya et al., 2001). Foliar spray of BRs at flowering stage was also found to enhance the root nodulation, cytokinin trans-zeatin riboside (ZR) content, activity of nitrogenase and yield, by ameliorating the water stress in French bean (Upreti and Murti, 2004). 24-EBL treatments also increased drought tolerance in A. thaliana and B. napus seedlings by changing expression of drought responsive genes (Kagale et al., 2007). In A. thaliana, the transcripts of rd29A, ERD10 and rd22 accumulated to higher level in 24EBL treated seedlings to ameliorate the drought stress. Hnilicka et al. (2007) reported that transcripts of an aquaporin gene BNPIP1, encoding plasma membrane intrinsic protein in B. napus, were present at higher levels in 24-EBL treated seedlings to ameliorate the drought stress. 24-EBL also helped to mitigate the negative impacts of drought and high temperature in case of three winter wheat varieties (Ebi, Estica, Samanta) thereby increasing its dry matter and yield of grain and straw. Li et al. (2008) studied the effects of BL on the survival, growth and drought resistance of 1-year-old Robinia pseudoacacia seedlings under water-stress. Seedling roots were soaked in BL solutions (0-0.4 mg l-1) before planting. Survival and growth of the seedlings were determined 8 months later. It was observed that soaking roots in BL (0.2 mg l-1) prior to planting significantly increased the survival and growth of seedlings.

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Treatment with 0.2 mg l-1 BL alleviated the water stress by decreasing the transpiration rate, stomatal conductance and malondialdehyde (MDA) content of seedlings when compared to untreated seedlings. Leaf water content, soluble sugar and free proline content and activities of antioxidative enzymes (CAT, SOD, POD) also increased in water stressed seedlings treated with 0.2 mg l-1 BL over the control to overcome the stress. Further, Jager et al. (2008) studied the endogenous level of BRs and ABA in wild type (WT) and BR-deficient mutant (lkb) and BR-perception mutant (lka) pea plants exposed to water stress. There was enhancement in level of ABA in water stressed plants but no alteration in level of BR. It was proposed that changes in endogenous BR levels were not normally part of the plants response to water stress. Behnamnia et al. (2009) reported that the foliar spray treatments of 24-EBL reduced peroxidation of lipids and H2O2 content but increased the activities of antioxidative enzymes (CAT, SOD, POD and APOX) and content of antioxidant compounds (ascorbate, carotenoids and proline) in drought stressed plants. Hence, BR treatment may be ameliorating the damage caused by drought stress. 2.7.5.1.2 BRs and Chilling Stress He et al. (1991) found that treatment with BL promoted the growth of maize seedlings following chilling treatment. Similarly, cucumber seedlings germinated from seeds soaked in BR solutions showed an enhanced growth as compared to the controls under cold conditions (50C for 3 days). Katsumi (1991) revealed that the chlorophyll content could be maintained in BR-treated seedlings during the cold stress. Kamuro and Takatsuto (1991) observed higher fruit settings in tomato plants sprayed with BRs under winter conditions. The application of 24-EBL on rice improved the resistance against chilling stress (1-50C) and the tolerance was associated with increased ATP, SOD activity and proline level. It indicated the involvement of BRs in membrane stability and osmoregulation (Rao et al., 2002). Huang et al. (2006) carried out BR regulated proteomics study of mung bean epicotyl exposed to chilling stress. Growth of mung bean epicotyls, suppressed by chilling stress were recovered by the application of 24-EBL. Seventeen proteins involved in metionine assimilation, ATP synthesis, cell wall construction and the stress responses, down-regulated by chilling stress were re-up-regulated by 24-EBL application. It was

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further observed by Kagale et al. (2007) that 24-EBL altered the expression of cold responsive genes in A. thaliana and B. napus seedlings thereby increasing their tolerance to lower temperature. 24-EBL increased the transcripts of three cold responsive structural genes-rd29a, a BN115 homolog and COR47 in A. thaliana and B. napus seedlings subjected to 2 0C for 3 days as compared with untreated seedlings in Cucumis sativus. Xia et al. (2009a) found that BR levels were positively correlated with the tolerance to photo-oxidative and cold stresses and resistance to cucumber mosaic virus. BR treatment enhanced NADPH oxidase activity and elevated H2O2 levels in apoplast. BR-induced H2O2 accumulation was accompanied by increased tolerance to oxidative stress. Inhibition of NADPH oxidase and chemical scavenging of H2O2 reduced BRinduced oxidative and cold tolerance and defence gene expression. Treatment of BR induced expression of both regulatory genes, such as RBOH, MAPK1, and MAPK3, and genes involved in defence and antioxidant responses. Thus, it was concluded that elevated level of H2O2 resulting from enhanced NADPH oxidase activity was involved in the BR-induced stress tolerance in cucumber. Honnerova et al. (2010) reported that the exogenous application of BRs to Zea mays plants under long term chilling stress did not affect the activities of photosystem I or II. 2.7.5.1.3 BRs and Thermal Stress Ultra structure of tomato leaf discs treated with BB6 (brassinosteroids analogue with spirostanic structure as active ingredient) under high temperature increased the rate of production of heat shock proteins, which protected mRNA from stress-induced degradation (Sam et al., 2001). Treatment of B. napus seedlings with 24-EBL under thermal stress significantly increased the thermotolerance of seedlings by accumulation of specific heat shock proteins (Dhaubhadel et al., 2002). The effect of 24-EBL and MH5 (polyhydroxylated spirostanic analogue of brassinosteroids) was analyzed by Mazorra et al. (2002) on POD, SOD and CAT activity in tomato leaf discs at 25-400C. BRs altered the activities of these enzymes, suggesting a role of 24-EBL and MH5 in the reduction of cell damage caused by heat stress. Tomato plants treated with 24-EBL were more tolerant to high temperature than the untreated plants. Singh and Shono (2005) reported that 24-

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EBL induced expression of MT-sHSP (mitochondrial small heat shock proteins), which possibly induced thermotolerance in tomato plants at high temperature (38 0C). Kagale et al. (2007) extended the studies of Dhaubhadel et al. (2002) in A. thaliana and BR-deficient mutant seedlings to determine if BR is vital for heat shock proteins synthesis. Similar to its effect on B. napus, 24-EBL (1.0 M) enhanced thermotolerance in A. thaliana seedlings exposed to the temperature stress of 43 0C was observed. But in contrast to B. napus where a considerable increase in heat shock protein (hsp) accumulation occurred in 24-EBL-treated seedlings, the level of hsp was comparable in both 24-EBL treated and untreated seedlings. BL solution (10-6 M) revealed thermotolerance against the heat stress (450C) to B. napus seedlings by enhancing the ABA level. Applied BL had no effect on endogenous ABA in plants maintained at normal temperatures. But ABA concentration was significantly elevated by heat stress alone and doubled by application of BL and heat stress (Kurepin et al., 2008). Ogweno et al. (2008) reported that 24-EBL pretreatment significantly alleviated high-temperature (400C/300C) induced inhibition of photosynthesis in Lycopersicon esculentum L. plants by increasing carboxylation efficiency (net photosynthetic rate, stomatal conductance, maximum carboxylation rate of rubisco, the maximum potential rate of electron transport contributed to ribulose-1,5-bisphosphate (RuBP), as well as the relative quantum efficiency of PS II photochemistry) and the activities of antioxidant enzymes (CAT, SOD, APOX, POD). 24-EBL application also reduced total hydrogen peroxide (H2O2) and malonaldehyde (MDA) contents, while significantly increasing shoot weight following heat stress. 2.7.5.1.4 BRs and Photomorphogenesis Light affects many developmental and physiological responses of plants throughout their lifetime. Several hormones have been implicated in promoting or antagonizing light responses (Nemhauser and Chory, 2002). It was suggested that light may alter either the concentration of BRs or the responsivity of cells to these steroids (Fankhauser and Chory, 1997). BR-insensitive mutant and Arabidopsis seedlings treated with a BR biosynthesis inhibitor (Brz) exhibited light-grown phenotypes when grown in the dark (Nagata et al., 2000). Goda et al. (2002) stated that BRs may act as regulators of the light signaling pathway rather than

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functioning as downstream mediators of light signal transduction. Symons et al. (2002) reported that BRs level was slightly increased in pea epicotyls in response to light. Turk et al. (2005) reported that Arabidopsis cytochrome P450 monooxygenase encoded by the BAS1 gene inactivates BRs and modulates photomorpho-

genesis. BAS1 was identified as the overexpressed gene responsible for a dominant, BRdeficient mutant, bas1-D. BAS1 and SOB7 act redundantly with respect to light promotion of cotyledon expansion, repression of hypocotyl elongation and flowering time. Application of BRs to overexpression lines of BAS1 or SOB7 results in enhanced metabolism of BRs, though only BAS1 overexpression lines confer enhanced conversion to 26-OHBL thus suggesting that SOB7 and BAS1 convert BL and CS into unique products. Whippo and Hangarter (2005) reported that auxin and brassinosteroid signaling function interdependently and the interactions between brassinosteroid and auxin signaling modulate phototrophic responses in Arabidopsis thaliana. Song et al. (2009) observed that BL treatment or light stimuli altered hormone biosynthesis and signaling-related genes, especially those of auxin indicating that BR may modulate photomorphogenesis through synergetic regulation with other hormones. 2.7.5.1.5 BRs and Salt Stress BRs treatment has been found to alleviate the negative impacts of salt stress. Exogenous application of BRs is effective in seed germination and growth of rice plants (Anuradha & Rao, 2001), chickpea (Ali et al., 2007) and maize (Arora et al., 2008a) under salt stress. Shahbaz and Ashraf (2007) assessed the influence of foliar spray application of BRs on wheat (Triticum aestivum L.) growth and nutrient accumulation under salinity stress. BRs improved the growth of wheat plants under salt stress. Zeng et al. (2009) investigated the involvement of endogenous BRs under salinity stress in Arabidopsis sp. using BR mutants (det2-1 and bin2-1). Seedling growth and germination of det2-1 and bin2-1 mutants were more sensitive to salt stress than that of Columbia wild type (WT). The transcript levels of salt-and ABA-induced genes P5CS1 and COR78 were less provoked in det2-1 than in WT under salt (NaCl) stress of 200 mM. Also, the basal proline level and the proline level induced by 200 mM of NaCl or 50 M ABA in both det2-1 and bin2-1 was increased resulting in decreased accumulation

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of proline. But, the exogenous application of 24-EBL could enhance proline accumulation, promote root elongation of WT and partially rescue the growth of det21 mutant under salt stress. Thus, endogenous BR treatment was positively involved in the plant response to salt stress in Arabidopsis sp. 2.7.5.1.6 BRs and Pesticide Stress Farmers use about 2.5 million tons of pesticides each year worldwide. Scientists have been inquiring about new ways of minimizing pesticide residues that remain in food crops after harvest. Recent research has suggested that BRs might be an answer to the problem (Xia et al., 2009a, 2009b). BRs have been found to be effective in overcoming the damage caused by the application of pesticides. Sasse (2003) reported that BRs treatment reduced the damage occurring from treatment of simazine, butachlor, or pretilachlor in rice thus proving effective in reducing damage caused by pesticides. Xia et al. (2006) examined the phytotoxic effect of nine pesticides, including three herbicides (paraquat, fluazifop-p-butyl and haloxyfop), three fungicides (cuproxat, flusilazole and cyazofamid) and three insecticides (abamectin, imidacloprid and chlorpyrifos) on leaves of Cucumis sativus. Paraquat treated plants showed the severest phytotoxic symptom with the highest reduction in net photosynthetic rate (Pn) while other pesticides except flusilazole inhibited net photosynthetic rate to various degrees. The inhibition of Pn by cuproxat was accompanied by decline in intercellular CO2 concentration and stomatal conductance whereas in plants treated with cyazofamid, the decreased Pn was associated with increased intercellular CO2 concentration. 24-EBL pre-treatment alleviated the inhibitions of Pn for the pesticides except flusilazole and paraquat. Pesticides application impairs the photosynthesis of cucumber seedlings as observed by decreased intercellular CO2 concentration and stomatal conductance. However, 24-EBL pre-treatment could enhance the activities of CO2 assimilation in cucumber plants thus providing resistance to pesticides. Xia et al. (2009b) reported that chlorpyrifos, a widely used insecticide, caused significant reductions of net photosynthetic rate (Pn) and quantum yield of PSII (PSII) in cucumber leaves. However, 24-EBL pretreatment alleviated the declines of Pn and PSII caused by chlorpyrifos application. This effect of 24-EBL was associated with reductions of chlorpyrifos residues. Application of 24-EBL was correlated with increased

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expression of pesticide detoxification genes such as the P450 monooxygenase and glutathione S-transferase genes thus suggesting that BRs enhanced plant tolerance to pesticides by modulating the metabolic process of these pesticides.

2.8

Heavy metal stress: BRs and their antioxidative potential

Plant growth and productivity is adversely affected by natures wrath in the form

of various biotic and abiotic stress factors such as bacterial, fungal, viral, drought, low temperature, salt, flooding, heat, oxidative stress and heavy metal toxicity, while growing in nature (Jaleel et al., 2009). Due to anthropogenic activities, mainly in the 20th century, heavy metal pollution has emerged as one of the major threat to agricultural crops. The metals like Zn, Ni, Cu, V, Co, W and Cr etc. when exceed trace levels are highly toxic to plants (Weast, 1984; Schutzendubel and Polle, 2002). Antioxidant mechanisms may provide a strategy to improve metal tolerance in plants (Jaleel et al., 2008). BRs are common plant-produced steroidal compounds with antioxidative characteristics. They are known to alter the antioxidant capacity of plants under stress conditions (Behnamnia et al., 2009). Arora et al. (2008a) reported that treatment with 24EBL increased the activity of some antioxidative enzymes causing mitigation of oxidative burst. Mazorra et al. (2002) reported that BRs act as secondary messengers for the induction of antioxidative defence system in stressed plants thereby effectively scavenging ROS in plants under stress. Ability of BRs to regulate cell membrane permeability and ion transport find their applications in the areas polluted with heavy metals and radioactive elements. Application of BRs to crops and plants lowered the uptake and accumulation of such elements. Treatment of sugarbeet, barley, tomato and radish with 24-EBL significantly reduced the absorption of heavy metals (Khripach et al., 1996b). Barley plants (cv. Zazersky) treated with 24-EBL in the booted stage at a dose of 10 mg ha-1 showed that the reduction of metal content in the plant was 40-98% in comparison with control. Soaking tomato seeds for 12 h in 10-8 M solution of 24-EBL before sowing was more efficient in decreasing the content of Zn and Cd in tomato fruits than spraying treatment. 24-EBL (10-8 M) blocked the heavy metals (cadmium, zinc, copper and lead) accumulation in Chlorella vulgaris cells (Bajguz, 2000a). 24-EBL at the concentration of 108 M in combination with heavy metals blocked metal accumulation in

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algal cells. It had been observed that BRs in combination with Pb induced the phytochelatins synthesis in Chlorella vulgaris (Bajguz, 2002). Exogenous application of 24-EBL in field (515 mg ha-1) to barley plants significantly decreased the degree of leaf diseases induced by mixed fungal infection along with an increase in crop yield. A comparison of the results with those obtained after application of Bayleton (fungicide) (0.5 kg ha-1) revealed the higher capacity of 24-EBL as a protective factor against fungi. An ameliorative effect of 24-EBL against fungi was also observed in field trials with cucumber which was manifested in terms of increased activities of polyphenoloxidase and peroxidase enzymes in the leaves of cucumber plants (Korableva et al., 2002). Ozdemir et al. (2004) showed that BR treatment increased activities of antioxidative enzymes in soybean and rice plants under stress conditions. 28-HBL protected Cicer arietinum from cadmium toxicity by enhancing the activities of nitrate reductase and carbonic anhydrase and also by increasing the plant growth, leghemoglobin content, nodule number, nitrogen and carbohydrates content in the nodules and chlorophyll content, which were decreased proportionately with the increasing concentrations of cadmium. The effect of 24-EBL on photosystem II (PS II) of winter rape plants under cadmium stress was examined by Janeczko et al. (2005). Cao et al. (2005) reported that BRs can induce the expression of some antioxidative genes and enhance the activities of antioxidative enzymes such as SOD, POD, APX and CAT. Anuradha and Rao (2007) studied the effects of 24-EBL and 28-HBL on seed germination, seedling growth, free proline levels and the activities of antioxidative enzymes (SOD, CAT, POD, APOX) of radish (Raphanus sativus) grown under cadmium stress. The effect of BRs on the activity of ascorbic acid oxidase (AAO) and lipid peroxidation in radish seedlings given Cd stress was also investigated. It was observed that BRs supplementation alleviated the toxic effect of the heavy metal and increased the percentage of seed germination and seedling growth. Out of the two BRs, 28-HBL (3.0 M) was found to be more effective than 24-EBL in stress mitigation. Amelioration of metal stress by BRs was associated with enhanced level of free proline and increased activities of CAT, SOD and APOX. BRs treatment reduced the activity of POD and AAO in heavy metal stressed R. sativus seedlings. 24-EBL and 28-HBL were further explored

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for their metal stress ameliorative properties by their application to Al stressed mung bean (Vigna radiata) seedlings. By the application of 24-EBL and 28-HBL, the sharp reduction in growth, activity of carbonic anhydrase, relative water content, chlorophyll content and rate of photosynthesis were alleviated. They also enhanced the activities of antioxidative enzymes (SOD, CAT, POD) and proline content to overcome the oxidative stress generated by Al (Ali et al., 2008a). Ill effects generated by Cd were further overcome by enhanced activities of antioxidative enzymes (SOD, CAT, POD and proline content (Hasan et al., 2008). Wang et al. (2009) investigated the effects of 24-EBL on the growth, photosynthesis, water status, lipid peroxidation, accumulation of reactive oxygen species, and activities or contents of antioxidant defence system in maize plants under Mn stress. With increasing Mn concentrations (150-750 mg kg1 soil), the growth of plants was inhibited. Foliage application with 0.1 mg l1 of 24-EBL significantly reduced the decrease in dry mass, chlorophyll content, photosynthetic rate, leaf water content, and water potential of plants grown under Mn stress. The oxidative stress caused by excess Mn, as reflected by the increase in MDA content and lipoxygenase (LOX) activity, accumulation of superoxide radical and H2O2, was greatly decreased by 24-EBL treatment. Further, 24-EBL application enhanced the activities of SOD, POD, CAT, APOX, DHAR and GR and the contents of reduced ascorbate and glutathione compared with the plants without 24-EBL treatment. Thus, up-regulation of antioxidative capacity in maize under Mn stress may be due to stress-ameliorative effects of 24-EBL. Kroutil et al. (2010) noted a decrease in heavy metal (Cu, Cd, Pb and Zn) uptake in spring wheat var. Vanek after the application of 24-EBL, 24-epicastasterone and 4154 during two different growth stages i.e. 2931 DC (off shooting) and 5960 DC (beginning of anthesis). In our earlier studies it had been observed that 24-EBL and 28-HBL treatments (presoaking) reduced the heavy metal (Cu, Zn, Mn, Co and Ni) uptake and accumulation in B. juncea seedlings and plants. The mechanism involved for reducing toxicity might be the chelation of the metal ions by the ligands. Such ligands include organic acids, amino acids, peptides or polypeptides (Sharma and Bhardwaj, 2007a, 2007b; Sharma et al., 2007, 2008; Bhardwaj et al., 2007b, 2008). Further our studies on heavy metal stress

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indicated that 28-HBL alleviated the Cu, Ni and Zn toxicity in maize seedlings by increasing the activities of SOD, CAT, POD, APOX and GR antioxidative enzymes (Bhardwaj et al., 2007b; Arora et al., 2008b, 2008c). Choudhary et al. (2009a) reported that 24-EBL regulated the synthesis of polyamines and auxins in Raphanus sativus L. cv. Pusa Chetki seedlings under copper metal stress.

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