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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 M
2
-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 (M
3
) and control variety L 414 in
Lens culinaris
Medik. at harvest
Traits
L 414
cysLc1
cysLc2
Shoot height (cm)
30.15±1.2
a
12.06±1.0
b
12.11±1.2
b
Root length (cm)
15.9±0.90
a
3.98±0.78
b
4.01±0.82
b
Shoot dry weight/plant
(g)
4.38±0.87
a
1.43±0.37
b
1.46±0.29
b
Root dry weight/plant
(g)
10.11±1.21
a
2.26±0.32
b
2.35±0.28
b
Seed yield/plant (g)
0.98±0.17
a
0.28±0.17
b
0.33±0.19
b
Seed cysteine content (nmol/g fresh weight)
22.8±1.9
a
5.71
±1.1
c
8.48
±1.4
b
Serine acetyl transferase activity (U/mg protein)
0.66±0.08
a
0.69±0.12
a
0.70±0.10
a
O-acetylserine (thiol) lyase (nmol cysteine/ min/ mg protein)
23.2±1.97
a
4.94±0. 98
c
7.06±0.78
b
GSH content (nmol/g fresh weight)
139.6±2.87
a
31.82±2.63
c
50.03±3.18
b
GSSG (nmol/g fresh weight)
20.4±0.91
b
40.11±1.23
a
38.19±1.18
a
GSH redox [GSH/(GSH+GSSG)]
0.875±0.11
a
0.443±0.06
c
0.567±0.07
b
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 M
3
progeny plants of two M
2
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).
PLANT GENE AND TRAIT