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