22.Catalase Deficient.full

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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 culi narisMEDIK.):

PERTURBATIONS IN MORPHO-PHYSIOLOGY, ANTIOXIDANT REDOX AND

CYTOGENETIC PARAMETERS

DIBYENDU TALUKDAR1

& TULIKA TALUKDAR2

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 H 2O2-

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 mol 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 s tresses. 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


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218 Dibyendu Talukdar & Tulika Talukdarmainly 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 physiologicalunderstanding 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 plantArabidopsis, 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 M 2 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 leavesand 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 (2259' 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 mol 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


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Catalase-Deficient Mutants in Lentil (Lens culinarisMedik.): Perturbations in 219Morpho-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) catLc1(catalase deficient Lensculinaris mutant 1, leaf bleaching) and

catLc2 (catalase deficientLensculinaris 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 mol m-2 s-1, b) 200 mol m-2 s-1, c) 300 mol m-2

s-1

, and d) 400 mol m-2

s-1

in 10/14 h day/night regime and CO2 concentration of 400 L 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 ( = 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 gfor 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 portablephotosynthesis 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 gfor 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 gfor 10 min, the supernatant was discarded and the pellet was dissolved in 3 ml of 1 M H 2SO4 andspectrum measurement was taken at 410 nm. H2O2 content was measured from the absorbance at 410 nm using a standard

curve.


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220 Dibyendu Talukdar & Tulika TalukdarLipid 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 gfor 12 min at 4C. 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 solutioncomprised 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 (%) = (EC 1) / (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 4C. These were

then hydrolyzed in a mixture of 1N HCL and 45% acetic acid (2:1) at 60C 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 F 1 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

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22.Catalase Deficient.full