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Differential Response of Cysteine-deficient Lentil (
Lens culinaris
Medik.) Mutants Impaired in Foliar
O
-acetylserine(thiol)-lyase
Expression
36
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 F
1
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
F
2
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
PLANT GENE AND TRAIT