Differential expression of genes associated with ion transport
and membrane potential in leaves and roots of P. pinnata
To assess the effect of salinity stress on the gene expression profile
of Pongamia, we monitored the expression profile of several key
salt-responsive genes including SOS pathway components (SOS1, SOS2 and
SOS3), transporters (NHX1, HKT1:1, CLC1, V-CHX1, CCX1,
V-H+ATPaseB subunit, V-H+ATPaseE
subunit, PM-H+ATPase1,
PM-H+ATPase4.1, PM-H+ATPase4.1-like,
CNGC5 and CNGC17), and calcium-dependent protein kinases (CDPK3 and
CDPK32) (Figure 6).
Sodium proton exchanger 1 (NHX1) was significantly up-regulated by
~2.6 and ~3.6-fold in leaves of 500 mM
NaCl treated plants at 1 and 4DAS respectively, while these levels
remained unchanged in leaves of 300 mM NaCl treated plants at all-time
points and 500 mM NaCl at 8DAS respectively (Figure 6A). Further, the
NHX1 gene expression was significantly increased only in 500 mM NaCl
treated plants at 1DAS (Figure 6B). Interestingly, high affinity
transporter 1:1 (HKT1:1) levels significantly increased by
~3.6 and ~2.0-folds in both leaves and
roots of 300 mM NaCl treated plants at 1DAS. The SOS (Salt Overly
Sensitive) 2 levels showed increase/ decrease in leaves of 300 and 500
mM NaCl treated plants, while these levels were unchanged in the roots
of salt treated plants. SOS3 levels showed a significant up-regulation
by ~3.9, ~2.0, and
~3.7-fold in leaves of 300 mM NaCl treated plants as
well as ~5.9, ~6.0 and
~6.0-fold increase was observed in laves of 500 mM NaCl
treated plants respectively. However, we did not observe much changes in
expression levels of chloride channel 1 (CLC1) in both leaves and roots
of salt treated plants. The vacuolar cation proton exchanger 1 (V-CHX1)
levels were marginally induced in both leaves and roots of salt treated
plants. Under salt stress, cation calcium exchanger 1 (CCX1) levels were
induced significantly by ~2.0-foldin 300 mM NaCl treated
plants only at 8DAS, while these levels significantly increased by
~3.0, ~2.5 and 1.9-fold in roots of 300
mM NaCl treated plants, though these levels remained unchanged in 500 mM
NaCl treated plants. Vacuolar proton ATPaseB subunit
(V-H+ATPaseB subunit) and
V-H+ATPaseE subunit expression levels were slightly
induced in both leaves and roots of salt treated plants. We also
monitored expression levels of three plasma membrane proton ATPase
(PM-H+ATPase) isoforms PM-H+ATPase1,
4.1, 4.1-like in both leaves and roots of salt treated plants. The
expression levels of PM-H+ATPase1, 4.1, and 4.1-like
genes were similar to those of control levels in leaves of salt treated
plants, while PM-H+ATPase4.1-like levels were
marginally increased in roots of 300 mM NaCl treated plants only at
4DAS. Cyclic nucleotide-gated ion channel 5 (CNGC5) levels were
decreased or increased in leaves of treated plants while these levels
were significantly induced in roots of treated plants at 1, 4 and 8DAS.
Furthermore, the expression level of CNGC17 gene was decreased/
unchanged in both leaves and roots of treated plants. Calcium-dependent
protein kinase3 (CDPK3) and CDPK32 levels were increased/ unchanged in
both leaves and roots salt treated plants.
Gene primers for NHX3, NHX6, NHX6-like, SOS1, SOS1-like,
H+-ATPase4, H+-ATPase4-like,
CNGC17-like, and V-H+ppase exhibited with multiple
banding patterns their expression pattern significantly varied in leaves
and roots of both control and salt-treated plants (given in
Supplementary Figures 7 and 8, primers were given in the Supplementary
Table 7).