4.1 Salt stress induced up-regulation of electron flow through the PTOX activity in Spartina
There is a huge difference between SV and SA regarding their physological response to salt. Here, we found that in SA,with time, either a stable or an increase in the K+/Na+ was observed (Figure 3E-F). This maintenance or increase in the K+/Na+ is a major trait associated with salt tolerance (Shabala & Pottosin, 2014). Na+tolerance is associated with SOS1 antiporter localized to the root epidermis (Shi et al., 2002). Mostly, halophytes exhibit higher SOS1 abundance (Oh et al., 2009). Therefore, exclusion of Na+ should also be a mechanism involved in salt-tolerance in SA. In addition to this known mechanism of salt tolerance, here our data suggest that under salt, SA gained increased salt tolerance through increased electron flow through PTOX.
First, under normal growth conditions, i.e. when there was no salt stress, the NDH-dependent CEF activity was more than two times higher inSA than in SV under normal condition (Figure 6); however, after NaCl treatment, the NDH activity was enhanced by 2.36 times inSV but decreased by about 25% in SA , compared to their respective control (Figure 6). After exposure to salt stress, the J-step of OJIP curves was significantly enhanced for SV compared toSA (data not data). The increased J level is an indicator of a more reduced PQ pool and a more pronounced Q-A (primary electron acceptor of PSII) accumulation under salt stress (Haldimann & Strasser, 1999). This leads to a strong PSII acceptor side limitation and a high PQ pool over-reduction in SV compared to SA . Furthermore, we found that under salt stress, the level of NPQ was similar between SAand SV, i.e. the incident light energy was not more dissipated in the form of heat in SA , as compared to SV. There must be a major source of electron which accept electron in SA under salt stress.
Second, experiments using inhibitors suggest that PTOX is a major sink of electrons in SA under salt. To test this, we examined the PSII photoinhibition following salt stress in presence of n -PG (PTOX inhibitor) or DBMIB (Qo-binding site of Cytb6f inhibitor) at atmospheric CO2(390 μL L-1 CO2) and in presence of 2 or 21% O2 (Figure 7). Our results revealed that the restriction in electrons flow towards PTOX (n-PG ) has little effect on the PSII in SV (Figure 7A) but significantly decreased PSII in SA under both normal and even more severely under low O2 (Figure 7B). This reflects that a proportion of electrons from PSII is sensitive to both to n -PG and O2 (13%, Figure 7B). This provides an evidence that an efficiently operating PTOX in SA but not in SV under salt stress. In fact, even under non-salt condition, there is a proportion of electron from PSI flow into PTOX driven reactions.
Thirdly, using DBMIB, we observed that in SA, as compared toSV , under high NaCl treatment, the PSII was less photoinhibited, especially at the presence of 21% O2 (Fig 7C, D); this is possibly because under severe salt stress, electrons can be used to reduce O2 in SA through PTOX without passing through Cytb6f. Consistent with this possibility, we observed an enhancement in the primary PSII electron transfer rate under salt in the presence of 21% O2 and saturating CO2, 2000 μL L-1 (Figure 5B). Under 2000 ml·L−1 CO2, the flux through photorespiration minimizes, hence the photorespiration as a major sink for reducing power is minimized. This provides further evidence that PTOX may functions as a major electron sink in SA under salt stress. Furthermore, in line with this notation, this enhancement of electron transfer rate was not observed under low O2(2%) under salt stress (Figure 5A, B). The gene expression and Westernblot analysis (Figure 8) also showed that under salt stress, there were increased amount of PTOX RNA and protein abundance inSA , but not in SV (Figure 8). Therefore, upon salt stress, the SA shows drastically increased electron flow into TPOX. The increase of PTOX levels have also been reported earlier in plants under stress, e.g. exposure of tomato to high light (Shahbazi et al., 2007) or thellungiella to salt stress (Stepien &Johnson, 2009).