CATALASE-DEFICIENT MUTANTS IN LENTIL (Lens …pakacademicsearch.com/pdf-files/agr/226/217-232 Vol. 3 Issue 2, Jun... · Significantly low redox status of ascorbate and glutathione

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.


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


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).


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


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


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.


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


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.


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


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

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