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).