Non enzymatic defense systems a. -tocopherol Vitamin E

Kaur Nayyar : Heavy metal toxicity to food legumes: effects, antioxidative defense and tolerance mechanisms 9 and guaiacol peroxidase activities when treated with CdCl 2 0-50 mM. No significant changes in the glutathione reductase activity were shown by the treated plants Sandalio et al. 2001. Dixit et al. 2001 also reported the effects of 4 and 40uM cadmium on the antioxidants and antioxidant enzymes in the pea roots and leaves, separately. The results indicated that the levels of lipid peroxidation and H 2 O 2 increased in both the roots and leaves. Activities of SOD, APX, GST and GR were more at 40 uM concentration while GPOX decreased in the roots. Aluminium phytotoxicity causes oxidative stress in developing green gram seedlings and a significant increase in lipid peroxidation, peroxide content accompanied by a decrease in catalase activity Panda et al. 2003. However, superoxide dismutase, peroxidase and glutathione reductase activities increased with increasing aluminium concentrations. Both the contents of glutathione and ascorbate decreased with the elevated metal concentrations. In another experiment the effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean Glycine max were investigated Cakmak and Horst 1991. Soybean seedlings treated with Al AlCI 3 concentrations ranging from 10 to 75 µM showed the enhancement of lipid peroxidation in the crude extracts of the root tips, the activities of SOD and peroxidase increased while catalase decreased. The effects of cadmium 5 uM and zinc 100 uM on the antioxidant enzyme activities in bean Phaseolus vulgaris were reported Chaoni et al.1997. Lipid peroxidation was enhanced in all plant organs of the plant and the catalase activity was decreased in both roots and leaves but not in stems. Mercury toxicity in alfaalfa Medicago sativa by treating the plants with 0–40 µM HgCl 2 for 7 d resulted in oxidative stress Zhou 2007. It was observed that treatment with Hg 2+ increased the activities of NADH oxidase and lipoxygenase LOX and damaged the biomembrane lipids. There was enhancement in the total activities of APX, POD and CAT. Several antioxidative metabolites such as ascorbate and glutathione GSH differentially accumulated in leaves. Table:1 Location and function of nonenzymatic antioxidants in plant cell Antioxidant Location in the plant Function α –tocopherol Chloroplast envelope, thylakoid membranes and plastoglobuli. Deactivating the photosynthesis-derived ROS and scavenging lipid peroxyl radicals in thylakoid membranes Ascorbic Acid Usually higher in photosynthetic cells and meristems and some fruits and highest in mature leaves with fully developed chloroplast Powerful water soluble antioxidant, prevent or minimize the damage caused by ROS. Glutathione Cell compartments like cytosol, ER, vacuole, mitochondria, chloroplasts, peroxisomes and in apoplast Role in antioxidative defense, regulation of sulfate transport, signal transduction, detoxification of xenobiotics and the expression of stress-responsive genes Carotenoids Leaves, fruits and floral parts Photoprotective role in addition to scavenging of ROS. Proline Cytosol Osmoregulation, seed germination, membrane integrity, inhibition of water loss and an antioxidant Salicylic Acid Cytosol Seed germination, stomatal closure, an antioxidant Flavonoids Leaves, floral parts, and pollens. Role as an antioxidant, flowers, fruits, and seed pigmentation, protection against UV light, defence against phytopathogens role in plant fertility and germination of pollen.

4.2 Non enzymatic defense systems

The non enzymatic defense systems of the plants include various molecules such as glutathione, proline, -tocopherols, carotenoids and flavonoids. Various antioxidants like cysteine, proline, ascorbic acid and non- protein thiols also play an important role in detoxification of toxic metal ions Singh and Sinha 2005. Their involvement in defense against metals in described below in legumes.

4.2. a. -tocopherol Vitamin E

It is the major vitamin E compound found in leaf chloroplasts, where it is primarly located in the chloroplast envelope, thylakoid membranes and plastoglobuli. Table.1. It is responsible for deactivating the photosynthesis-derived reactive oxygen species and thus preventing the propagation of lipid peroxidation by scavenging lipid peroxyl radicals in thylakoid membranes. The alpa-tocopherol levels are very sensitive to environmental stresses and change differentially depending on the magnitude of the stress and species- sensitivity to stress. Recently, it has been found that oxidative stress activates the expression of genes responsible for the synthesis of tocopherols in higher plants Wu et al. 2007. Srivastava et al. 2005 reported a general induction in -tocopherol content in Anabaena doliolum under NaCl and Cu2+ stress. The effects of lead, copper, cadmium and mercury on the content of chlorophyll, proline, retinol, -tocopherol and ascorbic acid were investigated in 17-day-old bean seedlings Phaseolus vulgaris L. A significant increase in the levels of -tocopherol was indicated, also increase in proline and ascorbic acid was reported Zengin and Munzuroglu 2005. 4.2.b. Ascorbic acid Vitamin C Ascorbic acid ASC is the most abundant, powerful and water soluble antioxidant acts to prevent or in minimizing the damage caused by ROS in plants. It also occurs in all plant 1 0 Journal of Food Legumes 263 4, 2013 tissues, usually being higher in photosynthetic cells and meristems in and some fruits. Its concentration is reported to be highest in mature leaves with fully developed chloroplast and with highest chlorophyll. Usually, ASC remains available in reduced form in leaves and chloroplast under normal conditions Smirnoff 2000. Studies report that the ASC content of the roots and shoots of two cultivars of pigeonpea Cajanus cajan decreased with an increasing concentration Madhava Rao and Sresty 2000 of Zn and Ni. The effect of ascorbic acid on soybean seedlings grown on medium containing a high concentration of copper were investigated Golan-Goldhirsh et al. 1995 and reported that ascorbic acid prevented the entry of Cu into the plant roots and did not let copper toxicity signs. There are some reports where a decrease in the ascorbic acid in the roots and nodules of Glycine max under Cd stress has also been observed Balestrasse et al. 2004. Cd also decreases the ascorbic acid content in the leaves of A. thaliana and P. sativum Romero-Puertas et al. 2007. 4.2.c. Glutathione Glutathione is one of the crucial metabolites in plants, which is considered as most important intracellular defense against ROS induced oxidative damage. It generally occurs abundantly in reduced form GSH in plant tissues and is mostly localized in all cell compartments like cytosol, endoplasmic reticulum, vacuole, mitochondria, chloroplasts, peroxisomes as well as in apoplast and plays a central role in several physiological processes, including regulation of sulfate transport, signal transduction, conjugation of metabolites, detoxification of xenobiotics and the expression of stress-responsive genes Mullineaux et al. 2006. GSH plays a key role in the antioxidative defense system by regenerating another potential water soluble antioxidant like ASC, via the ASC-GSH cycle Foyer and Halliwell 1976. With increase in stress, GSH concentrations usually decline and redox state becomes more oxidized, leading to degradation of the plant system Tausz et al. 2004. During heavy metal stress, GSH concentration in the cell elevates. For example, increased concentration of GSH has been observed with the increasing Cd concentration in P. sativum Metwally et al.2005 and V. mungo Molina et al. 2008. On the other hand, Srivastava et al. 2005 reported an appreciable decline in GR activity and GSH pool under Cu stress in Anabaena dolicum and significantly higher increase under salt stress. 4.2.d. Carotenoids Carotenoids are the protective pigments that are found in plants and microorganisms. These are lipid soluble antioxidants playing many functions in plant metabolism including oxidative stress tolerance. A decrease in carotenoid and chlorophyll contents in V. mungo plants with increasing Cd concentration was observed Rai et al. 2004, Singh et al. 2008. In H. vulgare seedlings also, a reduction in carotenoids content was observed under Cd-stress Demirevska-Kepova et al. 2006. 4.2.e. Proline Proline accumulation, accepted as an indicator of environmental stress, is also considered to have important protective roles.Some authors have suggested that proline acts as an antioxidant in Cd-stressed cells and thereby improves metal tolerance Sharma and Dietz 2006, Siripornadulsil et al. 2002. Others have reported that many plants have been reported to accumulate proline Pro when exposed to heavy metals Alia and Saradhi 1991, Talanova et al. 2000. However, the precise mechanism and the functional significance of proline accumulation in plants under heavy metal stress have not been elucidated to date. Costa and Morel 1994 have suggested that proline accumulation in plants under Cd stress is induced by a Cd imposed decrease of the plant water potential. However, proline maintains the water balance under Cd stress thereby suggesting that proline- mediated alleviation of water deficit stress could substantially contribute to the Cd tolerance of the plant. But the direct conclusive evidence as to the role for the water potential in heavy metal-induced proline accumulation is still lacking. A recent study has shown that proline alleviates Cd toxicity by detoxifying ROS, and increasing the activity of SOD and CAT and glutathione content Xu et al. 2009. Rai 2002 suggests that the possible role of proline against heavy metals is by forming chelates with the metals. There are indications that proline forms a proline-metal complex and protects the activity of glucose-6-phosphate dehydrogenase and nitrate reductase against inhibition by Cd and Zn Sharma et al.1998. Apart from acting as an heavy metal alleviator, proline also acts as an important cytoplasmic osmoticum, a scavenger of free radicals, source of nitrogen and carbon for post stress growth, a stabilizer of membranes, machinery for protein synthesis and a sink for energy to regulate redox potential Rai et al. 2004. Muneer et al. 2011 reported that proline content increase at all concentrations of cadmium exposure in Vigna radiata and maximum increase was found at 0.50 mM which was about 119 to 120. In Cajanus cajanand Vigna mungo there was an accumulation of proline under heavy metals Co, Cd. Zn and Pb treatment Alia and Saradhi 1991. Similar results were observed in case of Cajanus cajan when given aluminium treatment Bhamburdekar and Chavan 2011. 4.2.f. Phenolics Salicylic acid is an important signal molecule mediating many biotic and environmental stress-induced physiological responses in plants. The role of SA in regulating Hg-induced oxidative stress was investigated in the roots of alfalfa Medicago sativa by Zhou et al. 2006. It was seen that the plants pretreated with 0.2mM SA for 12 h and subsequently exposed to 10 µM Hg 2+ for 24 h attenuated toxicity to the root and also decreased lipid peroxidation in root cells. This suggests that exogenous SA may improve the tolerance of the plant to the Hg toxicity. Flavonoids, a related group of phenolics, occur widely in the plant kingdom, and are Kaur Nayyar : Heavy metal toxicity to food legumes: effects, antioxidative defense and tolerance mechanisms 1 1 commonly found in leaves, floral parts, and pollens. They usually accumulate in the plant vacuole as glycosides, but they also occur as exudates on the surface of leaves and other aerial plant parts. Flavonoids are suggested to have many functions in flowers, fruits, and seed pigmentation, protection against UV light, defence against phytopathogens pathogenic microorganisms, insects, animals, role in plant fertility and germination of pollen and, molecules in plant- microbe interaction. Apart from the above roles, flavonoids have as antioxidative activity Brown et al.1998. Besides having the function of ROS scavenging , flavonoids are able to function as chelators for metals, depending on the molecular structure Brown et al.1998 and hence can take part in plant defence. In Arabidopsis thaliana, the relation between flavonoids and heavy metal tolerance were investigated. Both Arabidopsis wild type and mutant lines with a defect in flavonoid biosynthesis were grown on media containing different heavy metals. Results revealed that root length and seedling weight were reduced in mutants more than in the wild type when grown on cadmium, while on zinc only root length was affected Keilig and Muller 2009.

5. Tolerance mechanisms