PLANT GENE & TRAIT

2014, Vol. 5, No.5, 33-39 http://pgt.biopublisher.ca

Research Report                                                                                                          Open Access 
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
Correspondings author, dibyendutalukdar9@gmail.com; Authors
Plant Gene and Trait, 2014, Vol.5, No.5 doi: 10.5376/pgt.2014.05.0005
Copyright © 2014 Talukdar, 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.

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

Background productivity and quality of grain in legume crops

Mutational strategy provides a powerful tool to study (Wirtz and Hell, 2006; Tabe et al., 2010; Khan and
the genetic, physiological and molecular mechanisms Mazid, 2011; Liao et al., 2012). Plants generally take

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
PLANT GENE AND TRAIT 
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

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)
Received: 17 Mar., 2014 | Accepted: 25 Mar., 2014 | Published: 28 Mar., 2014
Differential Response of Cysteine-deficient Lentil (Lens culinaris Medik.) Mutants Impaired in Foliar O-acetylserine(thiol)-lyase
Expression 34

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
Traits L 414 cysLc1 cysLc2
Shoot height (cm) 30.15±1.2a 12.06±1.0b 12.11±1.2b
Root length (cm) 15.9±0.90a 3.98±0.78b 4.01±0.82b
Shoot dry weight/plant (g) 4.38±0.87a 1.43±0.37b 1.46±0.29b
Root dry weight/plant (g) 10.11±1.21a 2.26±0.32b 2.35±0.28b
Seed yield/plant (g) 0.98±0.17a 0.28±0.17b 0.33±0.19b
Seed cysteine content (nmol/g fresh weight) 22.8±1.9a 5.71±1.1c 8.48±1.4b
Serine acetyl transferase activity (U/mg protein) 0.66±0.08a 0.69±0.12a 0.70±0.10a

PLANT GENE AND TRAIT 
O-acetylserine (thiol) lyase (nmol cysteine/ min/ mg protein) 23.2±1.97a 4.94±0. 98c 7.06±0.78b
GSH content (nmol/g fresh weight) 139.6±2.87a 31.82±2.63c 50.03±3.18b
GSSG (nmol/g fresh weight) 20.4±0.91b 40.11±1.23a 38.19±1.18a
GSH redox [GSH/(GSH+GSSG)] 0.875±0.11a 0.443±0.06c 0.567±0.07b
Note: Data are means±SE of at least four replicates. Means followed by similar alphabets at superscript are not significantly (P>0.05)
different by ANOVA followed by Duncan’s Multiple Range Test. GSH-Reduced glutathione, GSSG-glutathione disulfide or oxidized
glutathione

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).
35
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
Note: Lentil EF1-α was used as housekeeping gene. Lane
1-control variety L 414, lane 2-cysLc1 mutant, lane 3-cysLc2
mutant, lane 4-F1 plant of control×cysLc1 mutant, lane 5-F1
plant of control×cysLc2 mutant, lane 6, 7-F2-recessive
homogygous plant (cysLc1 cysLc1) recovered from control×
cysLc1 mutant (OAS-TL 1 transcripts not detected), lane 8 and
9-F2-recessive homogygous (cysLc2 cysLc2) plant recovered
from control×cysLc2 mutant (OAS-TL2 transcripts absent),
lane 10-F1 plant (normal) of cysLc1×cysLc2 mutant, lane 11,
12-F2 plant with normal OAS-TL expressions, lane 13-F2
progeny plant with mutant phenotype (cysLc1, no OAS-TL 1
transcript), 14-F2 plant with mutant phenotype (cysLc2,
OAS-TL2 transcript absent), M- 100bp DNA marker (→200bp)
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/g fresh weight). The trait was
segregated in F2 and corresponding test crosses
(F1×mutant). The segregating traits exhibited good fit
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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/g
fresh weight) ratio in F2 (χ2 = 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/g
fresh weight) but segregated into normal (69 plants,
cysteine level mean 21.6±2.1 nmol/g fresh weight) and
mutant phenotype (43 plants, cysteine level mean
6.1±1.1 nmol/g fresh 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
PLANT GENE AND TRAIT 
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,
Differential Response of Cysteine-deficient Lentil (Lens culinaris Medik.) Mutants Impaired in Foliar O-acetylserine(thiol)-lyase
Expression
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
36
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.
PLANT GENE AND TRAIT 
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
37
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.
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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).
PLANT GENE AND TRAIT 
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 F2 generation
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).
Differential Response of Cysteine-deficient Lentil (Lens culinaris Medik.) Mutants Impaired in Foliar O-acetylserine(thiol)-lyase
Expression
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 analyzed by quantitative reverse
transcription polymerase chain reaction (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
Table 2 Oligonucleotide primers used in qRT-PCR analysis of the expression of selected target genes in lentil genotypes, a F-forward,
R-reverse
Target genes Primers (5ʹ → 3ʹ)a
LcOAS-TL 1 F- CTCACAAGATTCAAGGGATAGGA-R- GTCATGGCTTCCGCTTCTTTC-
LcOAS-TL 2 F-GGATCCGCAGTGTCTGTACCAACGAAA-R-GACGTCTCACAATTCTGGCTTCAT-
LcSAT1;1 F–AGCCATACTTCCTTTATCTCTGAGTG-R-AACAGTATATGTACTCTCAGCAGTAAC-
LcSAT1;2 F-GGACCATACTTCCTTTATCTCTGAGTG-R-CTACTAGCAATAATCAACCTTTTCATC-
EF1-α (House keeping) F- TGTCGACTCTGGGAAGTCAA-R-CTCTTTCCCTTTCAGCCTTG-
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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