Kaur Nayyar : Heavy metal toxicity to food legumes: effects, antioxidative defense and tolerance mechanisms 3
the application of zinc Fontes and Cox 1998. Prolonged exposure to zinc causes chlorosis in the younger leaves,
which can extend to older leaves Ebbs and Kochian 1997.
3. Effects on Legumes
Legumes belonging to the family Fabaceae are responsible for substantial part of the global flux of nitrogen
from atmospheric nitrogen to the fixed forms such as ammonia, nitrate and organic nitrogen. Most of these are agriculturally
important food crops and are a rich source of proteins to the animals and human beingsGupta 1987. Most of the
leguminous crops are affected by metal stress present in the soil mainly due to contaminated agrochemicals and sewage
sludge. Due to this, various plant physiological activities like seed germination, nutrition distribution, enzymes activity,
alternation of the membrane permeability, nitrogen fixation, photosynthesis, transport of the assimilates and respiration
are adversely affected.
3.1. a. Cytology:
Heavy metals adversely affect the cytology of the plant cells. The genotoxicity of heavy metals influences the DNA
synthesis, duplication of DNA and chromosomes causing many chromosomal aberrations Cheng 2003. They are known
to cause deleterious effect on the cell division of the plants, which depend upon the concentration and the intensity of
the exposure of the same.
Fig: 1 Heavy metal induced chromosomal abnormalities in plant cells
The effects of Cd on the cell division of the root tips in beans were studied by Mo and Li 1992. The treatment of beans
by Cd, Pb , and Hg at concentrations 0.01,1.0 and 10 ppm, respectively shortened the cell division but extended the cell
cycle. The chromosomes of beans exposed to Cd, Pd, Hg got injured and revealed polyploidy, C-karyokinesis and various
chromosomal abnormalities like formation of chromosomal bridges, chromosomal rings and chromosome fragmentation,
chromosome micro-nuclei and nuclear decomposition Mo and Li 1992, Liu et al.1992, Duan and Wang 1995. Fig. 1. Gomez-
Arroyo et al. 2001 reported that the salts of nickel, cobalt and cadmium increased the frequency of the sister chromatid
exchanges SCE in Vicia faba. It has been observed in the some plants that mercury poisoning leads to the disturbance
in the spindle activity resulting in the formation of polyploid and aneuploid cells and c-tumours. Kumar 2007 studied the
mutagenic potential of lead 25, 50, 100, 200, and 300 ppm in Lathyrus sativus
and showed that chromosomal abnormalities increased with the lead nitrate concentration. These
abnormalities included condensed bivalents, laggards, bridges, cytomixis and stickiness of the chromosomes.
Siddiqui et al.2009 studied the effect of cadmium on the root tips of Pisum sativum L. Seeds of P. sativum were treated with
a series of concentrations ranging from 0.125, 0.250, 0.500 and 1 mM Cd for 6 h. It was reported that the overall percentage of
aberrations generally increased with increasing concentrations of Cd. The most common chromosomal
abnormalities were laggards, bridges, stickiness, precocious separation and fragments. Zhang et al. 2009 investigated
the effects of different concentrations of Cd 1-50µM on cell division and nucleoli in root tip cells of Vicia faba. Results
revealed that Cd induced c-mitosis, chromosome bridges, chromosome stickiness and lagging chromosomes. Small
amounts of nucleolus materials were extruded from the nucleus into the cytoplasm at 1µM Cd when the root tips were exposed
for 24 hours. Zhang et al. 2009 studied the effects of different concentrations of Al 10 µM, 50 µM, 100 µM on nucleoli in
root tip cells in hydroponically grown Vicia faba L. It was revealed that aluminum significantly inhibited root growth of
V. faba
treated with 50 µM and 100 µM concentrations. In the nucleolus of root tip cells, some particulates containing
agyrophilic proteins were extruded from the nucleus into the cytoplasm, and some were scattered in the nucleus after Al
stress in the plants. In the root tips of green gram Vigna radiata.
L, arsenic and manganese induced chromosomal stickiness at higher concentrations. However it was reported
that arsenic had more toxic effect than manganese on the root tip cells of greengram during mitosis Mumthaz 2010.
Studies were carried out by Muneer et al. 2011 on root
tips and leaves of Vigna radiata, where 15 days old plantlets grown in nutrient Hoagland media were exposed to various
levels of cadmium chloride 0.05, 0.10 and 0.50 mM for 48 and 72 hours. It was shown that Cd exhibited inhibitory effect on
cytological studies namely, mitotic index and chromosome number. Chromosomal studies showed various chromosomal
abnormalities such as laggard chromosomes, anaphasic bridges, and uni-distribution of chromosomes.
Bhat 2011 reported that heavy metals around automobile refining shops induce synergistic cytogenetic
effects in Trifolium repens and noticed a significant increase in micronucleus MN, chromosomal aberrations CAs,
percentage of nuclei with comet tails NCTs, the relative comet
4 Journal of Food Legumes 263 4, 2013
tail length CTL, comet tail DNA CT, DNA, and tail moment TM with increased concentration of three heavy metals, like
Cd, Pb and Hg. The most prominent abnormalities induced by heavy metals were micronucleus, precocious separation, and
laggard formation. Ritambhara et al.2010 carried out some cytogenetic studies to evaluate the genotoxic effect of lead
Pb and zinc Zn on the gametic cells of grass pea Lathyrus sativus
. A severe chromosome stickiness in meiosis impairing normal chromosome segregation was reported, which
persisted upto microspore stage, ultimately leading to chromosome degeneration. Recently, Oladele et al. 2013
reported thatincreased metal pollution can lead to some irreversible cytogenetic effects in plants and higher organisms.
Their group investigated the effects of lead and zinc nitrates at different concentrations: 25, 50 and 100 mgL on the
chromosomes of bambara groundnut Vigna subterranean. The results show the most frequent chromosomal anomalies
induced by these heavy metals as stickiness and bridges.
3.1.b. Anatomy and Ultrastructure
Han et al. 2004 have reported that uptake and accumulation of metals at higher concentrations cause ultra-
structural and anatomical changes in the plant cells. These include structural modifications in the chloroplast, thylakoid
membranes, deposition of electron dense globules in vacuoles, increase or decrease in the size of cells, reduction of the
intercellular spaces and also changes in the turgor pressure of the plant cells. In the thylakoids, modifications include
swelling and curling of the thylakoidal membranes. Also, it has been reported that heavy metal stress causes degradation
of polypeptide compositions of the thylakoid membrane, reduction, disappearance or swelling of the grana cascade of
chloroplast mitochondria Cheng 2003. Heavy metal induced cellular and ultrastructural modifications have been illustrated
in Fig.2. There was accumulation of callus on the sieve plates of
phloem of Phaseolus vulgaris seedlings exposed to excess of Co, Ni and Zn Peterson and Rauser 1979. In case of beans,
toxicity of chromium has also found to decrease the diameter of the treachery vessels, which resulted in reduced
longitudinal water movement Vazques et al.1987. Reduced turgor pressure and plasmolysis in the epidermal and cortical
cells of bush bean plants, which were exposed to chromium was also observed Vazques et al.1987. Formation of wall
ingrowths in hypodermal cells of the cadmium treated Phaseolus vulgaris
roots was reported Vazques et al.1989. Cadmium treatments to Arachis hypogea plants induced
xerophyte anatomic features of leaves i.e. thick lamina, upper epidermis, palisade mesophyll, high palisade to spongy
thickness ratio, as well as abundant and small stomata Shi and Cai 2008. In 5µgml cadmium treated roots of bush bean
plants Phaseolus vulgaris L. cv. Contender grown on perlite, plastid ultrastructure was hardly affected, while in the upper
parts of the plant the chloroplasts showed severe alterations. Younger trifoliate leaves showed greater disruption of
chlorophyll synthesis and plastid ultrastructure Barcelo et al.
1987. In the experiments of Baszynski et al. 1980 and Stoyanova and Chakalova 1990, it was established that
cadmium, applied in toxic concentrations, disturbs the chloroplast envelope and the integrity of the membrane system
and leads to increased plastoglobule number, changing the lipid composition and the ratios of the main structural
components of thylakoid membranes. Heavy metals effects on the structure and functions of the photosynthetic
membranes of the higher plants showed that the sub- microstructure of the chloroplast was changed Yang 1991.
Ultra structural alternations in the cortical root cells of pigeon pea in response to zinc and nickel both at cellular and the
organelle level were observed Sresty et al. 1999. Unusual deposition of electron dense globules in the vacuoles of the
root cortical cells and appearance of unusual two nucleoli occurred. The ultra structural analysis of the leaves of pea
plants grown with 50µM CdCl
2
showed the internal cell disturbances characterized by an increase of mesophyll cell
size, a reduction of the intercellular spaces and severe disturbances in the chloroplast structure Sandalio et al. 2001.
In the pea leaves treated with Cd, there was disorganization of the chloroplast structure, with an increase in the number of
plastoglobuli and formation of vesicles in the vacuoles Mc Carthy et al. 2001. The effect of Cu
2+
at concentrations 50 and 75uM on the ultra-structure of the chloroplasts of
the bean seedlings revealed that excess of copper induced changes in the ultra-structure of chloroplasts visible in form
of deterioration in the grana structure and the accumulation and swelling of starch grains in the stroma Bouazizi
et al
. 2010.
Fig: 2: Heavy metal induced ultrastructural modifications in the cell and chloroplast
Kaur Nayyar : Heavy metal toxicity to food legumes: effects, antioxidative defense and tolerance mechanisms 5
3.2 Germination of legume seeds
