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International Journal of Agricultural Science
and Research (IJASR)
ISSN 2250-0057
Vol. 3 Issue 2, Jun 2013, 217-232
© TJPRC Pvt. Ltd.
CATALASE-DEFICIENT MUTANTS IN LENTIL (Lens culinaris MEDIK.):
PERTURBATIONS IN MORPHO-PHYSIOLOGY, ANTIOXIDANT REDOX AND
CYTOGENETIC PARAMETERS
DIBYENDU TALUKDAR1 & TULIKA TALUKDAR
2
1Department of Botany, R. P. M. College, Uttarpara, West Bengal, India
2Department of Botany, Krishnagar Government College, Krishnanagar, Nadia, West Bengal, India
ABSTRACT
Two catalase (CAT)-deficient mutants namely, catLc1 and catLc2 were isolated in EMS-induced (0.15% and
0.5%, 6 h) M2 population of lentil (Lens culinaris Medik.) var. VL 125. Leaf and root CAT activity was only about 22.53%
and 9.16%, respectively, in catLc1, and was nearly 11.22% and 30.84% of mother variety in catLc2. Sharp differences
were observed between the two mutants for morpho-physiological, antioxidant status and cytogenetic parameters.
Compared to mother variety, root growth was affected in catLc1, while shoot growth was markedly reduced in catLc2
mutant. Significantly low redox status of ascorbate and glutathione under CAT-deficiency presumably crippled the H2O2-
scavenging capacity, resulting in abnormal accumulation of H2O2 and concomitantly, high rate of membrane lipid
peroxidation and electrolyte leakage as the marks of oxidative stress in the two mutants with more severe effect in roots of
catLc1 and leaves of catLc2. At controlled pot experiment under a range of irradiance (100-400 µmol m-2
s-1
), CAT-
deficiency coupled with membrane damage increased in catLc2 plants, while catLc1 mutant was largely unaffected. Under
CAT-deficiency, mitotic disruptions were severe in catLc1 roots while meiotic anomalies were very high in catLc2. CAT-
deficiency in lentil originated as recessive mutations by the action of two different non-allelic loci.
KEYWORDS: Lentil, Catalase-Deficient Mutant, Genetic Control, Antioxidant Defense, Meiotic Associations
INTRODUCTION
Isolation and characterization of stable mutants exhibiting alterations in different antioxidant defense components
are valuable approach towards better understanding of plants‟ response to abiotic stresses. Mutational strategy provides a
powerful tool to study the genetic, physiological and molecular mechanisms protecting plants against metal toxicity
(Tsyganov et al., 2007). Among the common edible legumes, this tool has been successfully used in Pisum sativum L. and
Lathyrus sativus L. to decipher metal tolerance and accumulation (Tsyganov et al., 2007; Talukdar, 2012a, b), role of
glutathione in metal tolerance (Talukdar, 2012b, e), over-production of thiol compounds (Talukdar, 2012c), to assess gene-
dosage effect on antioxidant defense (Talukdar, 2011c), and tolerance to salinity stress (Talukdar, 2011a, b). Like peas,
lentil is a cool-season edible pulse crop grown widely in the Indian subcontinent, West Asia, Ethiopia, North Africa and
parts of southern Europe, Oceania and North America (Erskine et al., 2011; Talukdar, 2013 a) and has tremendous health
benefits (Erskine et al., 2011; Talukdar, 2013e). Most of the lentil varieties in India have been developed mainly through
pure line selection and intraspecific hybridization, inadvertently leading to the narrowing-down of genetic base. This
makes them vulnerable to a number of biotic and abiotic factors besides reducing their realized genetic potential due to
lesser hidden variability (Ferguson et al., 1998). Lentil experiences diverse types of abiotic stresses, of which salinity,
drought, and metal toxicity reportedly have detrimental effects on its growth and yield (Talukdar, 2012f, 2013a). Despite a
protein rich pulse crop with high nutritional values, genetic improvement of this crop has not reached its desirable peak
218 Dibyendu Talukdar & Tulika Talukdar
mainly due to non-availability of reliable cytogenetic, genomic and biochemical tools. In grass pea, a close relative of
lentil, induced mutagenic technique has been used successfully to develop diverse types of cytogenomic tools (Talukdar,
2009, 2010, 2011d, 2012a,d), the potential of which is now being exploited to ascertain the intrinsic biochemical defense
response and their genetic/physiological stability under abiotic stress (Talukdar, 2012a-c). To augment physiological
understanding and genetic mechanism of stress tolerance, similar possibilities is being explored in lentil (Wani and Khan,
2003).
Catalase (CAT) is a tetrameric iron porphyrin that catalyzes the dismutation of two molecules of H2O2 to water
and O2. While plants contain several types of H2O2-metabolizing proteins, catalases are highly active enzymes that do not
require cellular reductants as they primarily catalyse a dismutase reaction (Mhamdi et al., 2010). The first plant CAT
mutants, isolated in the C4 plant maize, did not show obvious phenotypes (Willekens et al. 1997). Subsequently, however,
a photorespiratory screen of a mutant collection in the C3 plant barley identified a stable line with only about 10% wild-
type leaf CAT activity (Kendall et al., 1983). In model plant Arabidopsis, no photorespiratory CAT mutants were identified
by using a forward genetics approach, although several knockout mutants have been constructed through insertional
mutagenesis (Kendall et al., 1983; Bueso et al., 2007). The response of plant antioxidant defense to excess H2O2 under
CAT deficiency has been demonstrated in barley mutant (Queval et al., 2007). Among the non-enzymatic components of
this defense system, ascorbate (AsA) and glutathione (GSH) play pivotal role in diverse types of stress responses including
high irradiance (Noctor et al., 2002; Queval et al., 2007). To the best of my knowledge, no CAT-deficient mutant was
studied in grain legumes. As part of a broad strategy to develop novel and desirable mutants for stress response in lentil,
induced mutagenic technique has been adopted and progeny with variant phenotype was screened for antioxidant capacity.
In the process, six plants exhibiting CAT-deficiency were isolated at M2 generation, and advanced to next generation to
perform a detail study. The objective of the present study was, therefore, framed to 1) measure the CAT activity in leaves
and roots, 2) characterize the morpho-physiological and antioxidant metabolism of CAT-deficient mutants, 3) assess the
mitotic and meiotic consequences of CAT-deficiency, and 4) ascertain the genetic control of the CAT-deficiency in the
advanced selfing and inter-crossed progenies.
MATERIALS AND METHODS
Induction and Detection of CAT-Deficient Mutants
Fresh and healthy seeds of lentil (Lens culnaris Medik. cv. VL 125) were collected from Pulses and Oilseed
Research Station, Berhampore, West Bengal, India and grown for two seasons (2008 and 2009) in a private farm at
Kalyani (22°59' N/88° 29' E), West Bengal, India. After ascertaining uniformity of seed age and homozygosity, fresh seeds
presoaked in water for 5 h were treated with freshly prepared 0.15%, 0.25% and 0.5% aqueous solution of ethylmethane
sulfonate (EMS) for 6 h with intermittent shaking at 25 °C ± 2° C keeping a control (distilled water). After the stipulated
period, seeds were thoroughly washed with running tap water and sown in the field treatment wise (300 seeds treatment-1
)
along with untreated seeds as control in triplicate during November, 2009 (Temperature 20 °C /18 °C, day/night, RH 72%
± 4%, photoperiod 10 h, irradiance 180- 200 µmol m-2
s-1
). Selfed seeds of individual M1 plants were harvested separately
and were grown in next season in a randomized block design keeping a distance of 30 cm between rows and 20 cm
between plants to raise M2 progeny. Standard agronomic practices were followed to grow healthy plant progeny.
Phenotypes of about 3200 M2 individuals were screened during winter of 2009 and 2010 in an agricultural farm at Kalyani,
West Bengal, India. Antioxidant enzyme activities of all the variant types obtained from mutagenized population were
measured as pilot screening. Four variant plants showing normal growth but with leaf-bleaching in 0.15% EMS-treated
progeny and two plants with stunted habit in 0.5% EMS-treated population were initially found to be extremely deficient
Catalase-Deficient Mutants in Lentil (Lens culinaris Medik.): Perturbations in 219 Morpho-Physiology, Antioxidant Redox and Cytogenetic Parameters
(10-20% of normal level) in leaf catalase activity. These six plants (M2) were completely separated from rest of the
mutants; their seeds were separately harvested and field-grown in the next season (10 seeds plant-1
) to develop M3 progeny.
The mutants were maintained through self-pollination in the above mentioned field condition of West Bengal, India.
Morpho-physiological and yield traits were recorded from M3 plants at harvest (Table 1).
On the basis of phenotypic differences with catalase deficiency, progenies of these six plants were tentatively
grouped under two types of mutants in lentil: a) catLc 1(catalase deficient Lens culinaris mutant 1, leaf bleaching) and
catLc 2 (catalase deficient Lens culinaris mutant 2, stunted shoot).
Test of CAT-Deficient Mutants in Controlled Conditions
Seeds of M3 plants (after confirming M2 result of CAT activity level in leaves and roots) were allowed to
germinate at 25 °C in Petri dishes. Germinated seedlings (four plants pot-1
) were allowed to grow in 12 inches earthen
porous pots for 7 d. Number of seedlings pot-1
were thinned to one and pots were arranged in completely randomized
block design in three replicates, imposing growth irradiance of a) 100 µ mol m-2
s-1
, b) 200 µ mol m-2
s-1
, c) 300 µ mol m-2
s-1
, and d) 400 µ mol m-2
s-1
in 10/14 h day/night regime and CO2 concentration of 400 µL L-1
(air) on CAT-deficient plants
and control plant (variety VL-125). The seedlings were allowed to grow for another 14 d. CAT activity, photosynthetic
rate, pigment contents, H2O2 concentration, lipid peroxidation level, and redox status of ascorbate and glutathione were
determined in leaves of 21 d old plant (harvest). Fresh and dry weights of shoots (leaves+ stems) were measured after
harvest.
Catalase Activity Assay
CAT (EC 1.11.1.6) activity was measured following the procedure of Aebi (1984). CAT (EC 1.11.1.6) was
extracted in 50 mM K-phosphate buffer (pH 7.0) and 0.5 % PVP, and its activity was assayed by measuring the reduction
of H2O2 at 240 nm (ε = 39.4 M-1
cm-1
) for 1 min [23]. Enzyme activity was expressed as nmol H2O2 min-1
mg-1
protein.
Measurement of Physiological Parameters
Leaf chlorophyll and carotenoid contents were determined by the method of Lichtenthaler (1987). Leaf tissue (50
mg) was homogenized in 10 ml chilled acetone (80%). The homogenate was centrifuged at 4000 g for 12 min. Absorbance
of the supernatant was recorded at 663, 647 and 470 nm for chlorophyll a, chlorophyll b and carotenoids, respectively. The
contents were expressed as mg chlorophyll or carotenoids g-1
FW.
Leaf photosynthetic rate was assayed following earlier methods (Coombs et al., 1985)using a portable
photosynthesis system (LI-6400XT, LI-COR, Inc, USA).
Reduced (GSH) and oxidized (GSSG) glutathione contents were estimated following the method of Griffith
(1985). Reduced (AsA) and oxidized (DHA) ascorbate contents were determined by the method of Law et al. (1983).
The H2O2 content was estimated following the earlier methods (Wang et al., 2007). Fresh tissue of 0.1 g was
powdered and blended with 3 ml acetone for 30 min at 4 °C. Then the sample was filtered through eight layers of gauze
cloth. After addition of 0.15 g active carbon, the sample was centrifuged twice at 3000 g for 20 min at 4 °C, then 0.2 ml
20% TiCl4 in HCl and 0.2 ml ammonia was added to 1 ml of the supernatant. After reaction, the compound was
centrifuged at 3000 g for 10 min, the supernatant was discarded and the pellet was dissolved in 3 ml of 1 M H2SO4 and
spectrum measurement was taken at 410 nm. H2O2 content was measured from the absorbance at 410 nm using a standard
curve.
220 Dibyendu Talukdar & Tulika Talukdar
Lipid peroxidation rates were determined by measuring the malondialdehyde (MDA) equivalents following the
earlier adopted method (Hodges et al., 1999). About 0.5 g of fresh tissue was homogenized in a mortar with 80% ethanol.
The homogenate was centrifuged at 3000 g for 12 min at 4°C. The pellet was extracted twice with the same solvent. The
supernatants were pooled and 1 ml of this sample was added to a test tube with an equal volume of either the solution
comprised of 20% TCA and 0.01% butylated hydroxy toluene (BHT) or solution of 20% TCA, 0.01% BHT and 0.65%
TBA. Samples were heated at 95 °C for 25 min and cooled to room temperature. Absorbance was measured at 450, 532
and 600 nm. Level of lipid peroxides was calculated and expressed as nmol MDA g-1
fresh weight.
Electrolyte leakage (EL) was assayed by measuring the ions leaching from tissue into deionised water (Dionisio-
Sese & Tobita 1998). The EL was expressed as a percentage by the formula, EL (%) = (EC1) / (EC2) ×100.
Cytogenetic Study
Root-tip mitosis and flower bud meiosis were studied following the procedures employed earlier (Talukdar and
Biswas, 2007; Talukdar, 2008, 2010, 2012d]. For mitotic preparations, fresh and healthy roots were pretreated with 2 mM
8-hydroxyquinoline for 2 h at room temperature followed by fixation in 45% acetic acid for 15 minutes at 4°C. These were
then hydrolyzed in a mixture of 1N HCL and 45% acetic acid (2:1) at 60°C for 10s. The root tips were stained and
squashed in 1% aceto-orcein. The mitotic index (MI %) was calculated by dividing cells among the examined total cells.
For meiosis, suitable sized flower buds were fixed between 9.00 A.M and10.00 A.M in propionic acid-alcohol (1:2) for 6
h, and then were preserved in 70 % alcohol for future studies. After washing the fixed buds in distilled water, anthers were
smeared in 1 % propiono-carmine solution to analyze meiosis in the microsporocytes. Photomicrographs were taken from
well scattered plants. Sterility of pollen grains was studied following staining of randomly selected anthers with 1 %
acetocarmine solution (Talukdar, 2013b) and expressed as percentage.
To trace the mode of inheritance and allelic relationship of CAT-deficiency, both catLc1 and catLc2 were crossed
with each other and also with their mother control cultivar VL-125. The F1 seeds were harvested from respective line with
utmost care and sown in next season to grow F2 progeny. F2 plants showing recessive phenotype were advanced to F3 to
test the homozygosity for the concerned phenotype. CAT activity was assayed in leaves of segregating population. Chi-
square test was employed to test the goodness of fit between observed and expected values for all crosses.
Statistical Analysis
The results presented are the mean values ± standard errors of at least four replicates. Statistical significance (P <
0.05) between mean values of control and treated plants was estimated by t test, using „Microsoft Excel data analysis tool
pack, 2007‟.
RESULTS
Growth Performance and CAT Activity in Lentil Mutants
Both the mutants exhibited reduction in growth parameters, but growth of the aboveground portions (stem height,
leaf length, shoot fresh and dry weight) was significantly reduced in the mutant catLc2 (Figure 1a-c). Barring stem height
and irregular patches of leaf-bleaching, shoot growth was nearly normal in catLc1 mutant (Figure 1b). On the other hand,
length of roots and its fresh as well as dry weight showed higher rate of decrease in catLc1 mutant than those in catLc2
plants. Compared to control and catLc1, the catLc2 mutant exhibited growth of short duration with early flowering and
maturations. Leaf chlorophyll a content, chlorophyll a/b ratio, rate of photosynthesis and seed yield plant-1
were also
decreased significantly in catLc2 plants, while these were nearly normal in catLc1 mutant. Pollen sterility increased over
Catalase-Deficient Mutants in Lentil (Lens culinaris Medik.): Perturbations in 221 Morpho-Physiology, Antioxidant Redox and Cytogenetic Parameters
control plants by about 3.6-fold in catLc1 and >18-fold in catLc2 (Table 1). Marginal changes were observed in
chlorophyll b and carotenoid contents of the two mutants.
In both leaves and roots of two lentil mutants, CAT activity decreased significantly in comparison to their control
(Table 1). This was tested in both field conditions and in controlled environment. In field-grown catLc1, leaf and root CAT
activity was only about 22.53% and 9.16%, respectively, of its control variety VL 125. In the same condition, catLc2
mutant showed leaf CAT activity nearly 11.22% and root activity about 30.84% of VL 125 (Table 1). Under controlled
growth condition, where only leaf samples were assayed, similar results were obtained when the two mutants and their
control were exposed up to 200 µ mol m-2
s-1
growth irradiance (Figure 2a). In increasing irradiance, CAT activity was
largely unchanged in catLc1 mutant but it decreased significantly (~1.7-2.5-fold from its value in 100 µ mol m-2
s-1
) in
catLc2. By contrast to both the mutants, CAT activity was nearly doubled in the control at the higher irradiance levels
(Figure 2a).
Physiological Parameters of the Mutants
Changes in physiological and biochemical characteristics observed in the two mutants compared with the control
were summarized in table 1 and 2. Both reduced and oxidized forms of ascorbate were largely normal in leaves of field-
grown catLc1 mutant (Table 2). Root DHA level, however, increased significantly over control in the mutant. In both
organs of catLc2 plants, AsA content decreased significantly with concomitant rise of DHA. The redox state of AsA was
quite normal in catLc1, but it declined sharply in both parts of catLc2 plants (Table 2).
Although total glutathione (GSH+GSSG) contents increased substantially in both the mutants over their control,
GSH concentration varied significantly between the two mutants and also between two organs of the same mutant (Table
2). GSH level in leaves of catLc1 mutant increased approximately 1.2-fold, while it decreased nearly 2.06-fold in leaves of
catLc2 plants. Leaf GSSG level was quite normal in catLc1 mutant but it increased significantly (6.6-fold) in catLc2 plants.
Root GSH and GSSG level, on the other hand, was nearly normal in catLc2 plants but GSH content reduced more than 2-
fold and GSSG increased nearly 3-fold in roots of catLc1 plants. GSH redox, thus, declined below 0.4 in leaves of catLc2
and roots of catLc1 plants, otherwise it was quite normal (Table 2).
Foliar H2O2 content increased over control by about 2-fold in catLc1 mutant and >8.0-fold in catLc2 plants (Table
2). Root H2O2 level exhibited nearly 4-fold enhancement in catLc1 but was quite normal in catLc2 plants. Similar trend
was noticed in case of MDA and membrane electrolyte leakage percentage of both the mutants (Table 2). Comparing
control, both the parameters increased sharply in roots of catLc1 mutant and in leaves of catLc2 plants. Marginal variations
were observed in rest of the cases (Table 2).
Growth Traits and Oxidative Metabolism in Leaves of Two Mutants at High Irradiance
Shoot dry weight increased significantly in control plants exposed to 300 and 400 µmol m-2
s-1
growth irradiance
(Figure 2b). However, there was no remarkable change in shoot dry weight as well as leaf bleaching phenotype of catLc1
across 100-400 µ mol m-2
s-1
irradiance. Shoot dry weight in catLc2 plants gradually declined as the doses increased, and
compared to control it became significant since 200 µ mol m-2
s-1
irradiation (Figure 2b). Similar trend was noticed in leaf
chl a/b ratio and rate of photosynthesis (Figure 2c, d). Like control, both AsA and GSH redox state varied slightly in
catLc1 plants while it declined sharply in leaves of catLc2 plants at elevated irradiance (Figure 2e, f). Both H2O2 and
MDA content were marginally increased in leaves of catLc1 plants as the irradiation enhanced. However, it increased
significantly in catLc2 plants from 200 µ mol m-2
s-1
onwards, and about 13-14-fold increase of both parameters was
observed at 400 µ mol m-2
s-1
(Figure 2g, h). Similar trend was noticed in case of membrane electrolyte leakage (Figure 2i).
222 Dibyendu Talukdar & Tulika Talukdar
Mitotic and Meiotic Consequences Associated with CAT-Deficiency
In control, the 14 chromosomes were clearly visible in root-tip mitosis (Figure 3a). However, in both the mutants
mitotic and meiotic abnormalities were observed in varying frequencies (Table 3). Root mitosis and mitotic index was
quite normal in catLc2 mutant, barring occurrence of chromosome breaks and laggard in low frequency. However,
occurrence of sticky metaphase, chromosome breakage, laggard and sticky bridge formation were significantly higher in
root tip mitosis of catLc1 mutant (Figure 3b-d). Mitotic index was also significantly reduced in catLc1 (Table 3). In
meiosis, the 14 chromosomes were arranged in 7 bivalents during metaphase I and showed equal 7-7 separation at
anaphase I in flower bud PMCs (Figure 3e, f). Chromosomal aberrations were manifested by the formation of univalents (6
II + 2I) at metaphase I and unequal 8-6 and 6-2-6 separations, anaphase bridge formation and laggard chromosomes at
anaphase I (Figure 3 g-k). Development of micronucleus was observed only in catLc2 mutant during telophase II (Figure
3l). Meiotic anomalies were significantly higher in catLc2 mutant compared with catLc1 (Table 3).
Inheritance of CAT-Deficiency in Lentil Mutants
Crosses between catLc1and control and between catLc2 and control yielded F1 plants which showed completely
normal CAT activity in both organs. The F1 segregated for the traits in F2 and back crosses, and segregation of normal vs
deficient CAT level exhibited good fit to 3:1 in F2 and 1:1 in corresponding back crosses (Table 4). Allelism of two CAT-
deficient mutants was tested in reciprocal crosses between catLc1 and catLc2 mutants (Table 4). In F1, all the plants
showed normal phenotype accompanied with quite normal CAT activity. In F2, four types of plants- plant with normal
growth and CAT activity, catLc1 phenotype (leaf bleaching and reduced CAT), catLc2 (dwarf and reduced CAT) and a
completely new variant plant showing extremely short height with necrotic lesions in leaves and stems were isolated. The
frequency of these four types fit well (χ2= 1.70, at 3df) with 9:3:3:1 ratio (Table 4). CAT activity level was extremely low
in this variant plant, containing only 3% of control plant in both organs. One or two flowers appeared in this plant with
greatly reduced shoot and root growth, very high leaf bleaching, shortened growth period (29 d ± 4.5) and very poor seed
yield (0.04 g plant-1
) and bred true in F3 generation. Considering phenotypes of both catLc1 and catLc2, this group of
variant plants was tentatively designated as double mutant (catLc1 catLc2) for CAT-deficiency.
DISCUSSIONS
The two lentil mutants exhibiting severe deficiency in CAT activity in the present study were isolated in EMS-
induced mutagenic population of lentil variety VL 125 during M2 generation, and the trend was maintained in M3
generation. While root growth was affected in catLc1 mutant, shoot growth was severely perturbed in catLc2 mutant.
Reduced root growth in catLc1 seedlings has been manifested by significant decrease in root length, and root fresh as well
as dry weight. On the other hand, low shoot dry weight in catLc2 mutant was mainly due to marked reduction in leaf length
and shoot height, giving a characteristic stunted habit of the mutant. Perturbation in shoot growth of this mutant was,
presumably, due to significant decline in leaf photosynthesis rate which might be attributed to substantial loss in
chlorophyll a and concomitant decline in chlorophyll a/b ratio. Degradation of photosynthetic apparatus is the single most
determining factor for yield reduction in plants (Zhu et al., 2010), and might lead to substantial yield loss in the present
catLc2 mutant. Grain yield plant-1
in catLc1 plants was also low compared to control. However, the normal level of
chlorophyll a and chlorophyll a/b ratio might facilitate considerably higher yield in catLc1 plants in comparison to catLc2
mutant.
CAT deficiency in the two lentil mutants triggered different phenotypic symptoms; while leaf bleaching was
conspicuous in catLc1, stunted dwarf growth was manifested in catLc2 under normal irradiance (150 μmol m−2
s−1
) and
Catalase-Deficient Mutants in Lentil (Lens culinaris Medik.): Perturbations in 223 Morpho-Physiology, Antioxidant Redox and Cytogenetic Parameters
field conditions (10 h day/14 h night). In agreement with this observation, CAT-deficient barley reportedly showed leaf
bleaching (Kendall et al., 1983). In Arabidopsis cat2 mutant, dwarf phenotype was found under short day conditions with
spreading of necrotic lesions under long day conditions (Mhamdi et al., 2010). Remarkably enough, both the present
mutants despite grown uniformly in normal short day winter field conditions exhibited different phenotypes, and also, there
was no increase in bleaching area under increasing irradiance in controlled growth condition. The results indicated origin
of CAT-deficient phenotypes in catLc1 mutant irrespective of day length and irradiance levels. On the other hand, lower
shoot growth in catLc2 mutant at higher irradiance might be due to declining levels of CAT activity accompanied with low
chlorophyll content and reduced photosynthetic rate. The result suggested sensitivity of the mutant to elevated irradiance,
demonstrating a close link to the rate of photorespiratory H2O2 production, consistent to the earlier studies in tobacco and
Arabidopsis (Queval et al., 2007; Mhamdi et al., 2010).
The differential morpho-physiological manifestation of CAT-deficiency between catLc1 and catLc2 in the present
study was also observed in mitotic and meiotic stages of cell divisions. Barring presence of chromosome breakage at
metaphase and laggard at anaphase, root mitosis was otherwise normal in catLc2 mutant. However, occurrence of sticky
metaphase, chromosome breakage, laggard and bridge formation accompanied by reduction in mitotic index in root tip
mitosis of catLc1 mutant at much higher frequency than that in catLc2 strongly indicated severity of mitotic disruption in
the former case. Completely opposite scenario was observed in flower bud meiosis. Compared to control, both mutants
exhibited meiotic anomalies, indicating perturbation of reproductive growth under CAT-deficiency. However, meiotic
aberrations were significantly higher in catLc2 compared to catLc1, suggesting differential effect of CAT-deficiency in the
cell division process of the two mutants. The formation of univalent suggested synaptic disturbances at meiosis I, and
through unequal separation of chromosomes during anaphase I it may lead to the formation of unbalanced gametes
(Talukdar and Biswas, 2007; Talukdar, 2008, 2012d). The unequal 8 to 6 separation was quite high in catLc2, and as an
obvious consequence of this disturbance, percentage of sterile pollens increased significantly in catLc2 mutant.
Furthermore, high frequency of univalent as observed in the present catLc2 mutant may result in laggard and is usually
eliminated through cell division process through micronuclei formation (Talukdar, 2008, 2012d). Micronuclei results from
chromosome fragments or whole chromosome lagging during cell division, and is an index of cytogenetic damage (Duan et
al., 2000). Micronucleus formation was not observed in catLc1 mutant. Obviously, inhibition of normal root growth in
catLc1 mutant was linked with impediment with mitotic cycle in growing root tips, whereas catLc2 suffered cytotoxic
damage and loss of genetic material in its reproductive organ under CAT-deficiency.
Alteration in morpho-physiological parameters and disruption in cell divisional phases in CAT-deficient mutants
of lentils was accompanied by intracellular redox perturbation. This was strongly evidenced by the abnormal accumulation
of DHA (oxidized ascorbate) and GSSG (oxidized glutathione). Significant reduction of AsA redox state in leaves and
roots of catLc2 mutant could be attributed to marked drop in AsA (reduced ascorbate) content with concomitant rise in
DHA level. On the other hand, decrease in AsA redox in roots of catLc1 mutant was mainly due to significant increase in
DHA content while AsA content was largely normal. Significant accumulation of GSSG level accompanied with reduction
in GSH content resulted in decrease of GSH redox values in roots of catLc1 and leaves of catLc2. GAH redox was quite
normal in leaves of catLc1 and roots of catLc2 mutant. Interestingly, growth inhibition was not apparent in catLc1 shoot
and in roots of catLc2 plants in field. Similarly, under increasing irradiance in controlled pot experiment, foliar redox of
both compounds decreased abnormally in catLc2 plants, suggesting redox balance in favor of oxidative state in catLc2
plants. GSH plays pivotal role in shoot and root growth by maintaining proper progression of cell cycle in growing tissues,
and its vital role in this regard was observed in AsA and GSH-deficient mutants of grass pea (Talukdar, 2012b, e), a
224 Dibyendu Talukdar & Tulika Talukdar
legume closely related to lentil. Similar situation was encountered in Arabidopsis, where application of BSO, an inhibitor
of GSH synthesis, led to nearly rootless phenotypes (Vernoux et al., 2000). Certainly, decrease in GSH-redox has more
dramatic effect compared to AsA-redox on growth of present lentil mutants under CAT-deficiency. Furthermore, this redox
perturbation occurs in a condition where there was marginal change in total ascorbate and glutathione content in leaves and
roots of both the mutants (both field and pot experiments). The result is in agreement with earlier reports in CAT-deficient
barley mutant (Smith et al., 1984), and strongly confirmed the importance of redox state over total contents of antioxidant
molecules in regulation of plant growth (Foyer and Noctor, 2009; Talukdar, 2013c) . Decline in both GSH and ASA levels
in the present lentil mutants suggests continuous consumption of these molecules to compensate CAT deficiency, leading
to more oxidized cellular redox state, notably reflected in their redox status.
Obviously, ascorbate and glutathione level is markedly and reproducibly disturbed in CAT-deficient lentil
mutants. The reduction in AsA-redox in the present case is not in total agreement with common perceptions that Ascorbate
redox state is much less perturbed than that of glutathione in CAT-deficient plants (Mhamdi et al., 2010due to more
positive (less reducing) redox potential of AsA/DHA couple than the GSH/GSSG couple (Foyer and Noctor, 2009). Rather,
it can be partially supported if we consider the change in organ-specific way in the present mutant. In roots of catLc1 and
leaves of catLc2 mutant, the GSH redox value was measured far lower than the AsA redox. Completely opposite scenario
was manifested in roots of catLc2 plants. Presumably, regeneration potential of AsA and GSH by AsA-GSH cycle
enzymes particularly, dehydroascorbate reductase and glutathione reductase hold the key in maintaining redox balance
between thiol and non-thiol antioxidant molecules under CAT-deficiency. This scenario was previously observed in a
double cat2 gr1 mutant of Arabidopsis (Mhamdi et al., 2010) and in an AsA-deficient mutant of grass pea (Talukdar,
2012b).
The down-regulation of CAT is a serious challenge for reductive pathways that metabolize H2O2 (Mhamdi et al.,
2010; Talukdar, 2012b; 2013b, c). In the present study, abnormal accumulation of measurable H2O2 in roots of catLc1 and
leaves of catLc2 strongly suggested specific impact of CAT-deficiency on ROS metabolism of lentil. With increasing
irradiance in controlled pot experiment where only leaf sample were analyzed in a fixed CO2 concentration, catLc1 mutant
were capable to maintain H2O2 in nearly normal level but H2O2 level showed steep rise in catLc2 leaves. This suggested
capability of catLc1 but failure of catLc2 mutant to contain excess H2O2 generated by photorespiration. H2O2 is a
prominent ROS within cell, but its dual roles in induction of oxidative stress and as a signaling molecule in particular
cellular concentration has been observed in lentil seedlings exposed to cadmium (Talukdar, 2012f). In the present study,
high H2O2 content was accompanied with significant rise in MDA, a cytotoxic aldehyde resulting from membrane lipid
peroxidation, and percentage of electrolyte leakage in roots of catLc1 and leaves of catLc2. This, along with increase in all
the three parameters in catLc2 leaves and their more or less normal levels in catLc1 plants under pot experiment, strongly
indicated role of H2O2 as stress inducer to trigger oxidative damage in the present material. High lipid peroxidation has the
potential to damage photosynthetic pigment apparatus (Jin et al., 2010; Talukdar, 2012f, 2013c) , and is probably
responsible for lower pigment levels in catLc2 leaves in field as well as pot material. Significant increase in MDA content
and loss of photosynthetic apparatus has been recognized as the marks of oxidative stress (Jin et al., 2010; Talukdar,
2013d) and may be one of the prime reasons for severe growth inhibition of catLc2 mutant. The extent of oxidative damage
indicated that CAT-deficiency was not compensated by other H2O2-scavenging machinery in the lentil mutants, more
specifically in roots of catLc2 and leaves of catLc1. Normal levels of H2O2 accompanied with normal MDA and EL % in
other cases confirmed possible involvement of other ROS-scavenging mechanisms. To what extent CAT activity has been
complimented / compensated by enzymatic defense in CAT-deficient lentil mutant further studies in this regard are needed.
Catalase-Deficient Mutants in Lentil (Lens culinaris Medik.): Perturbations in 225 Morpho-Physiology, Antioxidant Redox and Cytogenetic Parameters
Mode of inheritance of CAT-deficiency was traced in self-pollinated as well as in intercrossed population,
involving two mutants and their control. In all parents, marginal variation in CAT activity was found in advanced
generations, suggesting true breeding nature of the mutant traits. Inheritance studies revealed monogenic recessive nature
of the low CAT activity in both the mutant types with the dominant allele was always with control. The result is in
agreement with monogenic recessive nature of symptoms related to CAT-deficiency in plants (Acevado et al., 2001).
Interestingly, leaf bleaching and stunted habit appeared unmodified with catLc1 and catLc2 phenotypes, respectively,
confirming their homozygosity in the present material. A completely different type of result, however, was encountered
when the two mutants were crossed reciprocally. Occurrence of F1 plants with normal level of CAT activity and absence of
either leaf bleaching or stunted habit and its segregations into four different plant types: normal, catLc1, catLc 2, and a
double-mutant type, consistent with the ratio of 9 : 3 : 3 : 1in F2, suggested involvement of two independent non-allelic loci
(designated as CatLc1/catLc1 and CatLc2/catLc2) in controlling CAT deficiency in the two mutant types under study. Both
the loci exhibited dominance over their respective recessive alleles (catLc1 and catLc2). In presence of both the genes in
dominant form (CatLc1–CatLc 2-), normal phenotype appeared whereas presence of catLc1 gene in double recessive form
(catLc1catLc1 CatLc1-) produced phenotypes characteristic of catLc1 type. On the other hand, catLc2 phenotype occurred
in the presence of double recessive nature of catLc2 gene (CatLc2-catLc2 catLc2). In homozygous recessive condition of
both the genes (catLc1 catLc1 catLc2 catLc2) a completely different phenotype showing measurable CAT activity only 3%
in both organs and extreme reduction in growth parameters was obtained, indicating origin of double mutant for CAT
deficiency in lentil. Occurrence of leaf-bleaching in catLc1 and stunted habit in catLc2 along with CAT-deficiency in
advanced generations suggested pleiotropic effect of CAT-deficiency on morphological and yield components in lentil
mutants.
Over expressing antioxidative response is one of the strategies to enhance stress resistance in plants under
agronomically relevant conditions (Mhamdi et al., 2010). Within this objective, plant systems that allow inducible,
endogenous increases in ROS availability may be useful tools. The present catLc1 and catLc2 mutants in lentils are first of
its kind in any food legumes, exhibiting sharp differences in manifestations of CAT-deficiency in morpho-physiological,
antioxidant status, cytological and genetic control mechanisms, and may be used conveniently to study the physiological
role of photorespiratory H2O2 in ROS signaling in planta without involving any exogenous induction to modulate H2O2
levels.
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APPENDICES
Table 1: Morphological, Yield-Related and Physiological Characteristics of catLc1 and catLc2 (M3) and Their
Control Variety VL-125 in Lens culinaris Medik. At Harvest
Traits A CatLc1 CatLc 2 Control
Plant height (cm) 28.87 ± 1.34 17.56 ± 0.67* 33.16 ± 1.58
Leaf length (cm) 13.76 ± 1.09 8.89 ± 0.58* 17.76 ± 1.09
Root length (cm) 4.56 ± 0.51* 9.18 ± 0.33 10.9 ± 0.27
Shoot FW plant-1
(g) 21.96 ± 10 11.78 ± 5.9* 23.15 ± 8.78
Shoot DW plant-1
(g) 2.46 ± 1.9 1.44 ± 2.1* 3.19 ± 3.3
Root FW plant-1
(g) 39.3 ± 4.4* 63.7 ± 3.0 72.2 ± 4.8
Root DW plant-1
(g) 3.89 ± 0.26* 7.89 ± 0.53 8.83 ± 0.62
Days to flowering 51.7 ± 5.1 23.6 ± 3.8* 43.3 ± 7.9
Days to 50% flowering 60.5 ± 8.9 34.6 ± 4.9* 61.6 ± 11.5
Days to maturity 125.5 ± 11 93.5 ± 5.8* 133.3 ± 9.0
Catalase-Deficient Mutants in Lentil (Lens culinaris Medik.): Perturbations in 229 Morpho-Physiology, Antioxidant Redox and Cytogenetic Parameters
Table 1: Cont’d
Pollen sterility (%) 7.56 ± 0.98* 38.6 ± 1.67* 2.12 ± 0.67
Seed yield plant-1
(g) 0.61 ± 0.09 0.22 ± 0.25 0.89 ± 0.17
Chlorophyll a (mg g-1
FW) 2.06 ± 0.29 1.09 ± 0.41* 2.41 ± 0.67
Chlorophyll b (mg g-1
FW) 1.22 ± 0.16 1.23 ± 0.21 1.67 ± 0.19
Carotenoids (mg g-1
FW) 1.19 ± 0.21 1.35 ± 0.32 1.29 ± 0.43
Photosynthesis rate (µM CO2 m-2
s-1
) 10.18 ± 0.05 6.20 ± 0.11* 13.28 ± 0.09
Values are Means ± SE of Four Replicates A
FW-fresh weight, DW-dry weight. * Significantly different from control At P < 0.05.
Table 2: Activity of Catalases (CAT), Reduced, Oxidized and Redox State of Ascorbate and Glutathione, H2O2,
MDA (Lipid Peroxidation) and Electrolyte Leakage in Leaves (L) and Roots (R) of catLc1, catLc2 Mutants (M3) and
their Control at Harvest
Traits a CatLc1 CatLc2 Control
CAT activity (nmol H2O2
min-1
mg-1
protein) (L) 17.89 ± 1.1* 8.91± 2.3* 79.4 ± 5.7
CAT activity (nmol H2O2
min-1
mg-1
protein) (R) 4.01 ± 2.5* 20.51 ± 1.8* 43.8 ± 3.3
AsA (nmol g-1
FW) (L) 902.0 ± 11 656.8 ± 10* 852.0 ± 15
DHA (nmol g-1
FW) (L) 111.1 ± 5.9 298.6 ± 8.9* 102.1 ± 7.6
AsA redox
(AsA/AsA+DHA) (L) 0.889 ± 0.09 0.689 ± 0.12* 0.895 ± 0.10
AsA (nmol g-1
FW) (R) 691.8 ± 9.0 518.7 ± 8.2* 717.8 ± 5.0
DHA (nmol g-1
FW) (R) 191.0 ± 7.7* 393.3 ± 5.9* 97.3 ± 11.2
AsA redox
(AsA/AsA+DHA) (R) 0.788 ± 0.13 0.565 ± 0.11* 0.879 ± 0.08
GSH (nmol g-1
FW) (L) 211.2 ± 4.8* 87.9 ± 3.5* 181.1 ± 1.9
GSSG (nmol g-1
FW) (L) 30.8 ± 1.5 139.9 ± 5.0* 21.2 ± 1.1
GSH redox
(GSH/GSH+GSSG) (L) 0.877 ± 2.1 0.385 ± 2.0* 0.895 ± 0.17
GSH (nmol g-1
FW) (R) 109.6 ± 1.0* 218.9 ± 1.1 265 ± 5.7
GSSG (nmol g-1
FW) (R) 216.4 ± 2.8* 79.8 ± 1.0 70.8 ± 4.0
GSH redox
(GSH/GSH+GSSG) (R) 0.340 ± 1.5* 0.730 ± 0.09 0.790 ± 0.10
MDA (nmol g-1
FW)(L) 3.88 ± 2.2 12.9 ± 4.2* 3.23 ± 1.9
MDA (nmol g-1
FW)(R) 24.15 ± 1.9* 4.6 ± 2.6 3.89 ± 3.1
H2O2 (µmol g-1
FW) (L) 2.16 ± 0.09 8.27 ± 1.1* 1.03 ± 0.06
H2O2 (µmol g-1
FW) (R) 8.61 ± 0.11* 2.54 ± 2.3 2.19 ± 0.36
Electrolyte leakage% (L) 5.93 ± 1.2 23.8 ± 1.5* 6.6 ± 0.92
Electrolyte leakage% (R) 19.5 ± 1.6* 9.9 ± 2.1 8.94 ± 2.2
Values are Means ± SE of Three Independent Experiments a FW-fresh weight, DW-dry weight, AsA-reduced ascorbate, DHA-dehydroascorbate (oxidized ascorbate), GSH-
reduced glutathione, GSSG-glutathione disulfide (oxidized glutathione), MDA-malondealdehyde. * Significantly different
from control variety VL 125 at P < 0.05.
Table 3: Mitotic and Meiotic Consequences (% of Total Scored Cells and PMCs) in catLc1 and catLc2 (M3) Mutants
and their Control Line VL-125 in Lens culinaris Medik (2n=2x=14)
Genotypes
(Mitosis)
Total Cells
Scored
Mitotic
Index
%
Sticky
Metaphase
Chromosome
Breaks Laggard Bridge
Bridge
with
Laggard
Unoriented
Chromosomes
catLc1 400 11.00* 55.66* 26.11* 5.01* 6.73* 5.30* 3.11*
catLc2 450 28.44 0.01 3.45* 1.23 0.00 0.18* 0.001
Control 450 31.33 0.002 0.005 0.08 0.002 0.03 0.001
Genotypes
(meiosis)
Total PMCs
scored 7 II 6II + 2 I 7-7 6-2-6 8-6 Bridge Micronucleus
catLc1 500 45.0* 0.05 0.04 0.09 2.2* 0.002 0.00
catLc2 500 28.7* 43.3* 31.5* 10.5* 6.5* 4.39* 3.90*
Control 500 82.0 0.00 0.02 0.001 0.004 0.001 0.001
Data represented are from four replicates per genotype, Asterisk denotes significant differences with control at P < 0.05.
230 Dibyendu Talukdar & Tulika Talukdar
Table 4: Inheritance and Test of Allelism in CAT-Deficiency of catLc1, catLc2 Mutants and Their Control Variety
VL-125 of Lentil (Lens culinaris Medik)
Cross Locus Phenotype
(F1)
F2/Back Cross Segregation ×
2 (3:1/1:1)
Normal CAT-Deficient
catLc1 ×
control catLc1
Normal CAT
activity 141 - - 44 0.15*
F1 ×
mutant catLc1 - 63 - - 57
0.30**
catLc2 ×
control catLc2
Normal CAT
activity 91 28 0.14*
F1 ×
mutant catLc2 - 76 - - 59
2.14**
catLc1×
catLc2
catLc1
catLc2
Normal CAT
activity
Normal
catLc1
phenotype
catLc2
phenotype
Double
mutant ×2 (9:3:3:1)
1.70 169
169 57 46 17
*, and **, consistent with 3:1 and 1:1, ratios, respectively at 5% level
Figure 1: Field Photo of Control Variety VL-125 (a), catLc1 (b) and catLc2 (c) Mutants of Lentil (Lens culinaris
Medik.) in Blooming Stage, Showing Normal Growth of Control, Irregular Patches of Leaf Bleaching but Otherwise
Normal Shoot of catLc1 Mutant, and Stunted Habit of catLc2 Mutant in Comparison to Control (Con), Respectively
Catalase-Deficient Mutants in Lentil (Lens culinaris Medik.): Perturbations in 231 Morpho-Physiology, Antioxidant Redox and Cytogenetic Parameters
Figure 2: Catalase (CAT) Activity (a), Shoot Dry Weight (b), Chlorophyll a/b Ratio (c), Photosynthesis Rate (d),
Ascorbare (AsA) Redox (e), Glutathione (GSH) Redox (f), H2O2 Content (g), Malondealdehyde (MDA) Level (h),
and Percentage Electrolyte Leakage (i) in Mother Control Variety and CatLc1 and CatLc2 Mutants of Lentil Under
Increasing Irradiance. Data are Means ± Standard Error of Four Replicates. * Significantly Different from Mother
Control at P < 0.05
Figure 3: Normal 2n=2x=14 Chromosomes at Metaphase of Mother Control (a), Chromosome Breaks
(Representative →) at Metaphase (b), Sticky Metaphase (c), and Sticky Bridge with Laggard (d) During Anaphase
of Root-Tip Mitosis of CatLc1 Mutant, and Normal 7 Bivalents at Metaphase I (e), Usual 7-7 Separation at
Anaphase I of Mother Control (f), 6 Bivalents+ 2 Univalents (→) at Metaphase I (g), 6-2-6 (h) and 8-6 Separation at
Anaphase I (i), Anaphase Bridge (→ )(j), Lagging Chromosome (→) (k), and Micronuclei Formation at Telophase
II (l) During Flower bud Meiosis of CatLc2 Mutant of Lentil. Scale 1SD = 10 µm