Differential Response of Cysteine-deficient Lentil (Lens culinaris Medik.) Mutants Impaired in Foliar O-acetylserine(thiol)-lyase Expression  

Dibyendu Talukdar
Department of Botany, R.P.M. College (University of Calcutta), Uttarpara, Hooghly 712258, West Bengal, India
Author    Correspondence author
Plant Gene and Trait, 2014, Vol. 5, No. 5   doi: 10.5376/pgt.2014.05.0005
Received: 17 Mar., 2014    Accepted: 25 Mar., 2014    Published: 28 Mar., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Talukdar, 2014, Differential Response of Cysteine-deficient Lentil (Lens culinaris Medik.) Mutants Impaired in Foliar O-acetylserine(thiol)-lyase Expression, Plant Gene and Trait, Vol.5, No.5 33-39 (doi: 10.5376/pgt.2014.05.0005)

Abstract

Lentil is a cool-season pulse crop, rich in protein but deficient in two sulphur-containing amino acids cysteine and methionine. Due to low genetic variability in existing germplasm, induced mutagenic technique has been adopted in lentil, and two mutant lines exhibiting poor growth and low dry weight were isolated in M2-mutagenized (0.10% and 0.15% EMS, 6 h) population of variety L 414. Further analysis revealed that plants from both mutant lines were highly deficient in seed cysteine (Cys) content, and thus, were tentatively designated as cysLc1 and cysLc2 mutants. Mutant plants were advanced to M3 generation. Biochemical analysis through cysteine synthesizing pathway in leaves revealed that activity of serine acetyl transferase (SAT) was normal in both the mutant progenies but both were highly deficient in foliar O-acetylserine(thiol)-lyase (OAS-TL) activity. Transcriptomic analysis by qRT-PCR confirmed normal expression of SAT in both mutants but revealed differential expressions of two OAS-TL isoforms; OAS-TL 1 isoform was not detectable in cysLc1 mutant while expression of OAS-TL 2 isoform was totally repressed in leaves of cysLc2 mutant. Genetic studies and test of allelism pointed out that both the mutants were recessive and were complementing with each other to produce normal in F1 and normal along with mutant plants in F2 progeny. The progeny plants exhibiting normal phenotype showed normal mRNA transcripts of both OAS-TL isoforms. Being stable and self-fertile, the mutants will give vital clues in genetic basis of thiol-metabolic network of lentil crops.

Keywords
EMS-Mutagenesis; Gene expression analysis; Glutathione; Lentil; OAS-TL isoforms

Mutational strategy provides a powerful tool to study the genetic, physiological and molecular mechanisms of plant metabolism. This technique has been successfully used to develop cytogenetic and breeding tools in different legume crops including lentil (Fazal Ali et al., 2010; Talukdar, 2009; 2013), the potential of which is now being exploited to ascertain the intrinsic metabolic events in grain legumes (Talukdar, 2012a; 2012b; Tsyganov et al., 2013). Lentil is a cool-season edible pulse crop grown widely in the Indian subcontinent, West Asia, North Africa and parts of Europe, Oceania and North America (Erskine et al., 2011) and has tremendous health benefits (Erskine et al., 2011; Talukdar, 2012c). Despite a protein rich pulse crop with high nutritional values, improvement of this crop has not reached its desirable peak due to low genetic variability.

Sulphur metabolism is fundamental to agricultural productivity and quality of grain in legume crops (Wirtz and Hell, 2006; Tabe et al., 2010; Khan and Mazid, 2011; Liao et al., 2012). Plants generally take sulphur from soil as sulphate. After reduction of sulphate to sulphide, the sulphide combined with O-acetylserine (OAS) forms cysteine in a reaction catalyzed by O-acetylserine (thiol) lyase (OAS-TL) either in free active homodimer or in association with serine acetyl transferase (SAT) as an inactive subunit of the cysteine synthase (CS) complex (Takahashi et al., 2011). Cysteine is the first committed molecule in plant metabolism that contains both sulphur and nitrogen, and, thus, the regulation of its biosynthesis is of utmost importance for the synthesis of a number of essential metabolites in plant pathways (Wirtz and Hell, 2006). Cysteine is incorporated into proteins and glutathione (GSH) directly (Wirtz and Hell, 2006). Several studies including mutants of Arabidopsis indicate that decreased activity of CS ultimately compromise cysteine level and GSH synthesis in plants. Plant cells contain different SAT and OAS-TL enzymes that are localized in the cytosol, plastids, and mitochondria, resulting in a complex variety of isoforms and different subcellular cysteine pools (Wirtz and Hell, 2006; Lo´pez-Martı´n et al., 2008). Recently, two novel catalase-deficient mutants differing in antioxidant defence response have been isolated and genetically characterized in lentil (Talukdar and Talukdar, 2013a) and coordinated expression of genes involved in sulphur metabolisms during stress tolerance has been revealed (Talukdar and Talukdar, 2014). In this on-going investigation to identify novel biochemical mutants, two plants exhibiting poor growth potential and very low seed cysteine content have been isolated in M2-progeny of lentil variety L 414. Biochemical, molecular and genetic analysis of the two mutations in the backdrop of seed cysteine content, cysteine synthesizing enzymes, gene expression of their isoforms and GSH level in leaves was carried out, a description of which is being presented in this communication.
1. Results
1.1 Growth and yield of lentil mutants
Compared to control variety L 414, the two lentil mutants exhibited significant (P<0.05) retardation of growth; while shoot height was reduced by about 2.5-fold, root length was decreased by about 4-fold (Table 1). Shoot and root dry weight was also declined by nearly 3-fold and 4.5-fold, respectively (Table 1). Per plant seed yield was decreased by 3-3.5-fold in relation to control (Table 1).


Table 1 Growth traits, seed yield and biochemical characteristics of cysLc1 and cysLc2 mutants (M3) and control variety L 414 in Lens culinaris Medik. at harvest


1.2 Seed cysteine content and enzyme activity
Compared to control variety, seed cysteine content was significantly (P<0.05) lower in both the mutants but the degree of reduction was different (Table 1), based on which the M3 progeny plants of two M2 variants were primarily designated as cysLc1 (cysteine-deficient Lens culinaris mutant 1) and cysLc2 (cysteine-deficient Lens culinaris mutant 2). Cysteine content was reduced by 4-fold in cysLc1 but by nearly 2.7-fold in cysLc2. In order to ascertain the possible reason behind thiol deficiency, activities of foliar SAT and OAS-TL were assayed. Compared to control, SAT activity was non-significantly (P>0.05) changed in both the mutants, but OAS-TL level was 4.2-fold low in cysLc1 and was 3.3-fold reduced in cysLc2 roots (Table 1).
1.3 Analysis of mRNA gene expression
In cysLc1, OAS-TL1 transcript was not detected at all while expression of OAS-TL2 isoform was close to control plants (Figure 1). Contrastingly, genes controlling OAS-TL1 isoform exhibited normal expression but OAS-TL2 expression was not detectable in roots of cysLc2 mutant by qRT-PCR study (Figure 1). Expression of two SAT isoforms namely LcSAT1 and LcSAT2 was also detected (Figure 1). mRNA trans- cripts of both isoforms changed non-significantly (P>0.05) between the mutant lines and control variety.


Figure 1 mRNA gene expression of OAS-TL1, OAS-TL2, SAT1 and SAT2 isoforms of variety L 414, lentil mutants and their segregating progenies

 
1.4 GSH, GSSG and GSH-redox state
Compared to control, GSH content was reduced by 4.4-fold in cysLc1 and by 2.8-fold in cysLc2 but GSSG level was enhanced by nearly 2-fold in both cases (Table 1). Low GSH level but increasing GSSG content led to decline in GSH-redox state in roots of both the mutants (Table 1).
1.5 Genetic basis of cysteine-deficient mutants
All F1 progeny plants (190) obtained from control×mutants exhibited normal (control like) growth accompanied by usual seed cysteine level (mean 19.3±1.9 nmol/gfresh weight). The trait was segregated in F2 and corresponding test crosses (F1×mutant). The segregating traits exhibited good fit to 3 (151 plants, normal growth and cysteine level mean 20.8±1.9 nmol/g fresh weight): 1 (52 plants, poor growth, deficient seed cysteine 5.8±1.0 nmol/gfresh weight) ratio in F22 = 0.04, 1 df, P<0.05) and 1:1 (41 normal: 37 mutant, χ2=0.20, 1 df, P<0.05) in back crosses. However, all F1s (178 plants) derived from cysLc1×cysLc2 exhibited normal growth and usual level of seed cysteine (mean 22.3±1.2 nmol/gfresh weight) but segregated into normal (69 plants, cysteine level mean 21.6±2.1 nmol/gfresh weight) and mutant phenotype (43 plants, cysteine level mean 6.1±1.1 nmol/gfresh weight), exhibiting good fit (χ2=1.31, P<0.05) to 9 (normal phenotype):7 (mutant phenotype) ratio in F2. Gene expression analysis confirmed down-regulation of either OAS-TL1 or OAS-TL2 isoform transcripts in plants showing mutant phenotype but normal expressions of both isoforms in plants exhibiting control like phenotype in segregating F2 progeny (Figure 1).
2 Discussion
2.1 cysLc1 and cysLc2: two unique biochemical mutants isolated in lentil
Induced mutagenic techniques have earlier been successfully used to isolate novel biochemical mutants exhibiting modulations in antioxidant defense components in edible legumes such as lentil, common beans, and grass pea (Talukdar, 2012a; 2012b; Talukdar and Talukdar, 2013a; 2013b). In present case, both the mutants are unique in legumes, containing very low seed cysteine level and deficiency in a major cysteine-synthesizing enzyme in photosynthetic organ. Biochemical mutants with altered carbohydrate and reducing sugar content, protein and amino acid methionine content, amylase inhibitor deficient and anti-nutritional contents like phytic acid have been reported in pea, black gram, pigeon pea, winged bean and soybean (Chougule et al., 2004; Gandhi et al., 2012; Bhalerao and Kothekar, 2013; Kumari et al., 2014), but none of the mutants were studied in respect of sulphur metabolisms. A sulphur deficiency-induced gene, sdi1 has been characterized in wheat (Howarth et al., 2009), but this type of work has not been carried out in grain legumes. Along with severe deficiency in seed cysteine content, both the present mutants exhibited significant retardation in growth habits, manifested by substantial decrease in stem and root growth. Growth retardation is a common phenomenon orchestrated through mutagenesis, as also observed in other legumes (Talukdar, 2009; Fazal Ali et al., 2010; Kozgar et al., 2012). In lentil, similar phenomenon was observed in EMS-induced two mutant lines catLc1 and catLc2, impaired in catalase activities, but the mutants differed in magnitude of growth retardation of shoots and roots (Talukdar and Talukdar, 2013a).
2.2 Reduced OAS-TL activity has cascading effects on foliar GSH-redox and seed cysteine level in the mutant
Growth retardation in both the mutants was associated with severe deficiency in seed cysteine content. The current model of cysteine formation proposes that the OAS formed in the SAT-OAS-TL complex decreases the binding affinity of both enzymes, and OAS-TL is released to convert OAS to cysteine (Saito et al., 1994; Tabe et al., 2010). In the present study, significantly low level of OAS-TL activity may, thus, jeopardize the prospect of conversion of OAS to cysteine, despite normal level of SAT, and might be responsible for reduced level of cysteine in both the mutants. The results also suggested that foliar cysteine synthesis is an important event in maintaining proper cysteine level in edible sink organs. The deficiency of OAS-TL level in the two lentil mutants was confirmed by qRT-PCR based transcriptomic analysis, and gene expressions of two isoforms of the enzyme was detected. Interestingly, absence of OAS-TL1 isoform expression was presumably responsible for reduced OAS-TL activity in cysLc1 mutant whereas crippled expressions of OAS-TL2 isoform resulted in decreased enzyme activity in cysLc2 mutant. In Arabidopsis, OAS-TL1 represents cytosolic isoform, and has immense importance in cysteine and GSH biosynthesis and plant’s tolerance to metal stresses (Lo´pez-Martı´n et al., 2008). Present results strongly suggested that knocking down of respective isoforms resulted in severe reduction in OAS-TL activity in both the mutants, and impeded cysteine biosynthesis. Also, regulation of gene expression occurred mainly at transcriptional level. The non-significant changes in SAT expressions in both the mutants strongly indicated constitutive expressions of both isoforms and resulted in marginal variation in SAT activity in both the mutants.
Low cysteine level was accompanied with reduced level of total foliar glutathione (GSH+GSSG) content in both the mutants. Substantial reduction of GSH level with concomitant rise in GSSG level led to reduction in GSH-redox in the mutant lines. GSH is a multipurpose thiol peptide, and functions as an efficient thiol-buffer to maintain delicate redox balance in favor of plant growth and development (Noctor et al., 2011; Talukdar, 2012a; 2012b). However, the peptide exclusively requires cysteine as one of its building blocks, and thus, it seems likely that apart of constant consumption by usual cellular processes low availability of cysteine also resulted in GSH-deficiency and concomitant fall in GSH-redox in both the mutants. In Arabidopsis knock-out mutant for cytosolic OAS-TL isoforms total intracellular cysteine and GSH concentrations were reduced, and the GSH redox state was shifted in favor of oxidized form (Lo´pez-Martı´n et al., 2008). GSH plays important role in plant growth through progression of cell cycle (Noctor et al., 2011). Thus, low GSH-redox in leaves of the present lentil mutants might be responsible for inhibition of growth and concomitantly, low root as well as shoot dry weight.
2.3 Genetic basis of cysteine-deficient mutations in lentils
Inheritance studies pointed out recessive nature of both the cysLc1 and cysLc2 mutations in lentil. Allelism test involving both the mutant lines indicated involvement of digenic mode of inheritance in controlling two mutant features which are complementing with each other to produce normal phenotype in F1 but absence of any of the alleles in dominant form resulted in mutant phenotype. Transcriptomic analysis using qRT-PCR revealed down-regulations of either OAS-TL 1 or OAS-TL 2 isoforms in origin of mutant phenotype in segregating F2 progenies. By contrast, the normal phenotype in segregating progeny might have been originated through normal (control like) expressions of both OAS-TL isoforms. Functional complementation has also been reported between two mutants, deficient in superoxide dismutase isoforms, in Phaseolus vulgaris L., leading to tolerance against arsenic stress (Talukdar and Talukdar, 2013b).
In conclusion, two mutants with huge cysteine deficiency in seed were isolated through EMS-mutagenesis in lentil. Results revealed disturbances in OAS-TL enzyme system in leaves of both the mutants which was confirmed by differential gene expressions of its isoforms in two mutants. Transcriptomic analysis and inheritance study revealed involvement of recessive mutations in OAS-TL loci, and isoforms of OAS-TL were complementing each other to provide normal activity of OAS-TL enzyme, to maintain thiol pool and subsequently, normal plant growth, and seed yield in lentil.
3 Materials and Methods
3.1 Induction and detection of mutants
Fresh and healthy seeds of lentil (Lens culnaris Medik. cv. L 414), collected from Pulses and Oilseed Research Station, Berhampore, West Bengal, India, were presoaked in water for five hours and treated with freshly prepared 0.10%, 0.15% and 0.5% aqueous solution of ethylmethane sulfonate (EMS) for six hours 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 to raise M1 progeny, following an earlier protocol (Taukdar and Talukdar, 2013a). 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. Out of about 1050 M2 individuals screened during winter of 2012 and 2013, two mutant plants showing poor plant growth and dry weight was isolated in 0.10% and 0.15% EMS-treated progeny. Further study revealed that the two plants were highly deficient in seed cysteine content. The two plants were self-pollinated in separate fields, and advanced to M3 generation. Progeny plants were harvested, and growth traits and yield was recorded. Plant parts were oven-dried at 60°C for two days and dry weight was then taken. Further biochemical and molecular analysis was done in leaves. Variety L 414 was used as control throughout the experiment.
3.2 Assay of cysteine synthesizing enzymes and measurement of cysteine content
Leaf tissue was homogenized in buffers specific for each enzyme under chilled conditions. The homogenate was squeezed through four layers of cheese cloth and centrifuged at 12 000 g for 15 min at 4°C. The protein content of the supernatant was measured following Bradford (1976). The assay of serine acetyltransferase (SAT; EC 2.3.1.30) activity was performed following Blaszczyk et al. (2002). An enzyme unit was considered as the amount of enzyme catalyzing the acetylation of 1 pmol of L-serine per minute. The OAS-TL (EC 2.5.1.47) activity was assayed by measuring the production of L-cysteine. Assay was started by the addition of 5 μl crude extract (1 μg/μl total protein in 50 mM phosphate buffer, pH 8.0). Reactions were conducted in 50 mM phosphate buffer (pH 8.0) in the presence of 5 mM dithiotreitol (DTT), 12.5 mM O-acetyl L-serine (OAS), and 4 mM sodium sulfide (Na2S) in a total volume of 100 μl assay mixture and allowed to proceed for 30 min at 30°C. The reaction was terminated by the addition of 0.1 ml of 7.5% trichloroacetic acid (Saito et al., 1994). Amount of cysteine synthesized was determined following Gaitonde (1967).
3.3 Estimation of reduced and oxidized glutathione
Reduced (GSH) and oxidized glutathione (GSSG) content in lentil roots was measured following Griffith (1985).
3.4 Genetic control and allelism test of OAS-TL deficient mutations
Inheritance of mutations controlling OAS-TL deficiency was traced in segregating populations of F2generation derived from control variety L 414×mutants. For allelism test, intercrosses were made between mutants. Chi-square test was employed to test the goodness of fit between observed and expected values for all crosses.
3.5 Statistical analysis
Data are means±standard error (SE) of at least four replicates. Variance analysis was performed on all experimental data, and statistical significance (P<0.05) of means was determined by Duncan’s multiple range tests using SPSS software (SPS Inc., USA v. 10.0).
3.6 Relative gene expression analysis through quantitative RT-PCR
Gene expression levels of SAT and OAS-TL of control, two mutant lines, and segregating progeny plants were analyzedby quantitative reverse transcription polymerase chainreaction (qRT-PCR) technique. Total RNA was isolated using the RNA isolation kit (Chromous Biotech, Bangalore, India) and treated with DNaseI (Chromous Biotech, Bangalore, India) at 37°C for 30 min. The quality of total RNA samples was determined spectrophotometrically (Systonic, Kolkata, India) from A260/280 ratio and by 1% agarose gel electrophoresis. First strand cDNA was synthesized from DNA-free intact RNA with oligo-dT primers and with MmuLV reverse transcriptase enzyme kit (Chromous Biotech, Bangalore, India) following manufacturer’s instructions. Quantitative RT-PCR of first stand cDNA was run on ABI Step-One (Applied Biosystems, Foster City, CA) Real Time PCR machine. Amplification was done in a total reaction volume of 50 µl, containing template (first strand cDNA) 2.0 µl, forward primer 2.0 µl (100 ng), reverse primer 2.0 µl (100 ng), 2X PCR SYBR green ready mixture (Fast Q-PCR Master Mix, Chromous Biotech, India), 25.0 µl, and DEPC water 19.0 µl. Primers for selected genes (Table 2) were constructed by Primer ExpressTM V. 3.0 software (Applied Biosystems, Foster City, CA, USA) withthe search of available sequence databases (http://www.ncbi.nlm.nih.gov/Report=GenBank) and reports on lentil (Talukdar and Talukdar, 2014), common beans (Talukdar and Talukdar, 2013b) and Arabidopsis thaliana (Han and Kim, 2006). TheqRT-PCR cycling stages consisted of initial denaturation at94°C (3 min), followed by 35 cycles of 94°C (5 s), 62°C (10s),72°C (10 s) and a final extension stage at 72°C (2 min). A melting curve analysis was performed after every PCR reaction to confirm the accuracy of each amplified product. Samples for qRT- PCR were run in four biological replicates with each biological replicate contained the average of three technical replicates. DEPC water for the replacement of template was used as negative control. RT-PCR reaction mixtures were loaded onto 2% agarose gels in TAE buffer. A 100 bp DNA ladder was run on every gel. The mRNA levels were normalized against a Lens culinaris EF1- α as the housekeeping gene and the relative (to control) expression of target genes was calculated as 2−ΔΔCt (Livak and Schmittgen, 2001).


Table 2 Oligonucleotide primers used in qRT-PCR analysis of the expression of selected target genes in lentil genotypes, a F-forward, R-reverse

 
Authors' contributions
Sole author DT designed both the field and lab experiments, measured biochemical and molecular parameters, conducted statistical analysis, read and approved the final manuscript.
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