Salinity-induced alterations in growth morphology, gas exchange, chlorophyll fluorescence and ion accumulation patterns of P. pinnata
In order to assess salt tolerance capacity in P. pinnata , 30 days old seedlings were exposed to 300 and 500 mM NaCl concentrations for 1, 4 and 8 days. After 8 days of salt treatment (DAS), leaves of 300 mM NaCl treated plants remained healthy and green as that of controls, while 500 mM NaCl stress exposure caused tip burns at 8DAS (Figure 1A). Reason behind selection of the specific time points for this study was that plants were treated with two different salt concentrations (300 mM NaCl and 500 mM NaCl) and kept under observation each day for induction of salt-induced morphological symptoms including yellowing of leaves, necrosis, wilting and shedding of old leaves. However, we couldn’t observe any morphological changes till 7DAS (Days After Salt-treatment) in 500 mM NaCl treated plants. Further, at 8DAS, 500 mM NaCl treated plants started to show wilting symptoms (which was shown in Supplementary Figure S1) and with increasing treatment time further (After 10DAS) these plants were continued to shed off its leaves (Supplementary Figure S1). Interestingly, at this point of time, 300 mM NaCl treated plants did not display any salt-induced morphological symptoms. With the above observation, we concluded that the 8DAS plants could probably give us possible clues regarding salt tolerance mechanisms. We observed that carbon exchange rate (CER) was changed with salt concentration and treatment time (Supplementary Figure 2A). Upon 300 mM NaCl stress, the CER was not changed significantly at 1 and 4DAS, while a significant reduction of ~50% was observed at 8DAS. In 500 mM NaCl treated plants, the CER values were progressively decreased with the treatment time. However, the levels of chlorophylls pigments were not changed significantly in both 300 and 500 mM salt treated plants at all-time points (Supplementary Figure 2B). Further, 300 mM NaCl treated plants could maintain a constant Fv/Fm values like control plants at all-time points (Supplementary Figure 2C). Conversely, 500 mM NaCl treated plants showed a little decrease in Fv/Fm values at 4 and 8DAS. Similar trend was observed with relative water content (RWC) in leaves and roots of Pongamia. The LRWC was maintained equal to that of controls in 300 mM NaCl treated plants, whereas in 500 mM NaCl treated plants these values tend to decrease progressively with the treatment time (Supplementary Figure 2D). In contrast, the RRWC values did not change significantly in both 300 and 500 mM NaCl treated plants.
Differences in photosynthetic and morphological responses to salt stress showed in Figure 1A and Supplementary Figure 2A-D might result of difference in their Na+ accumulation patterns across the plant, we tested the following hypothesis. As shown in the Supplementary Figure 2E-G, ions such as Na+, Cl-, and Ca2+ content were increased dose dependently in both 300 and 500 mM NaCl treated plants with treatment time. In detail, root showed a significant increase in Na+ content when compared to leaves. The peak value of root Na+ ion content was about 65 mg. g-1 DW in 500 mM NaCl treated plants at 8DAS. Similarly, root Cl- levels were enhanced by ~2.7, ~2.7, and ~1.4-fold in 300 mM NaCl treated plants, and ~3.8, ~4.0 and ~4.3-fold up-regulation was observed in roots of 500 mM NaCl treated plants at 1, 4 and 8DAS, respectively. Further, Ca2+ levels were slightly increased in roots of 300 mM NaCl treated plants at 1, 4 and 8DAS. While, in roots of 500 mM NaCl treated plants, Ca2+ levels were increased significantly at 1 and 4DAS, while these levels were unchanged at 8DAS.