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).