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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 M
1
progeny, following
an earlier protocol (Taukdar and Talukdar, 2013a).
Selfed seeds of individual M
1
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 M
2
progeny. Out of about 1050 M
2
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 M
3
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 (Na
2
S) 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 F
2
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).
PLANT GENE AND TRAIT