4.2 PTOX as a safety valve in SA under salt stress to protect photosystems from over-reduction
PTOX is an interfacial membrane protein (Berthold & Stenmark, 2003) attached to the stromal-side of the thylakoid membrane (Lennon et al., 2003). PTOX is involved in the carotenoid biosynthesis (Carol & Kuntz, 2001) and has been implicated in the oxidation of the plastoquinol pool, PQH2 (Joët et al., 2002). Similar to the increase of PTOX under salt conditions in SA, the PTOX levels have been found to increase in plants exposed to abiotic stress such as high temperatures, high light and drought (Quiles, 2006; Díaz et al., 2007; Ibáñez et al., 2010), low temperatures and high light (Ivanov et al., 2012), salinity (Stepien & Johnson, 2009) and in alpine plants at low temperature and high UV exposure (Streb et al., 2005; Laureau et al., 2013), implying a generic role of PTOX under stress.
Data from this study provide new evidence for the protective role of PTOX under salt stress. Fo of Chl a fluorescence (OJIP) was found to increase in SV but was not changed or changed little for SA (data not shown). After exposure to salt stress, the J-step of OJIP curves was significantly enhanced for SVcompared to SA (data not shown). 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 . In this regard, similar results have been reported by Shahbazi et al. (2007). These authors proved similar effect of high light treatment on the mutant of tomato ghost (gh ) defective in PTOX compared to the control San Marzano (SM) (Shahbazi et al., 2007). The data from this study, together with these earlier studies, suggests that PTOX can oxidize over-reduced PQ pool and hence provides protective roles.
As a reflection of the protective role, SA plants grew normally at a moderate salt stress and even survived under NaCl concentrations up to 550 mM NaCl without significant mortality. The Chl content of leaves did not drop significantly, particularly at NaCl concentrations below 250 mM (Figure 4B) and both stomatal conductance (gs ) and assimilation at atmospheric CO2 concentrations (A) were maintained (Essemine et al., unpublished data). By comparison, SVwas unable to survive at NaCl level higher than 100 mM for two weeks; even at NaCl concentrations lower than 100 mM, the Chl content ofSV dropped drastically by about 42 and 58% after 12 days exposure of SV to 50 and 100 mM NaCl, respectively (Figure 4A), concurrent with a dramatic decline in both gs and A(Essemine et al., unpublished data).
The protective role is clearly shown by changes in the linear electron transfer rates under NaCl treatments. In SV, under salt stress, we observed a decrease in linear electron transfer rate (LEF) inSV, as shown by the decrease in the g ETCat saturating CO2, which has a concentration of 2000 μL L-1 at either 21% or 2% O2 levels (Figure 5). Such decrease is common among C3 species under stress, e.g. drought (Golding & Johnson, 2003), salt (Stepien & Johnson, 2009), and anaerobiosis (Haldimann & Strasser, 1999). InSA , in contrast, there was no apparent decrease in LET under salt (Figure 5B); which suggests that the photosystem II in SA under stress was well protected. Consistent with these differential capacities to protect photosystem under salt, we observed much higher accumulation of ROS in SV compared to SA , even though the salt concentration used to treat SV was 50 mM, while that used to treat SA was 250 mM (Figure 9, 10). The ROS detected here may include highly reactive singlet oxygen (Kearns, 1971), the superoxide anion radical and hydrogen peroxide (Fridovich, 1997). The severe damage of salt to photosystem in SV is also reflected by a swelling in the chloroplast structure for SV after exposure to salt (Essemine et al., unpublished data). Altogether, these data suggest that having higher PTOX activity under salt (Figure 8) may contributed to the protection of chloroplast structure and function, as shown by maintenance of the photosynthetic linear electron transfer, chlorophyll a content, and less accumulation of ROS in leaves.
It is worth mentioning here that the protective function of PTOX has been studied earlier through transgenic approaches. However, the data obtained so far from transgenic experiments are still not conclusive. When PTOX from Chlamydomonas reinhardii was transferred into tobacco (Ahmad et al., 2012), it resulted in growth retardation; furthermore, instead of inducing increased resistance to high light, it led to increased vulnerability to high light for tobacco. The ortholog of PTOX in Arabidopsis has also been studied using both mutant and over-expression lines; which however, did not provide evidence for a role of PTOX in the regulation of PQ redox status (Rosso et al., 2006). In tobacco, however, over-expression of PTOX led to increased photoprotection under low light but increased vulnerability under high light, or which the authors suggest that the PTOX can only provide a sufficient photoprotection when the reactive oxygen species generated by PTOX can be effectively detoxified (Heyno et al., 2009). However, the enhanced sensitivity of plant growth to high light was not shown in tobacco over-expressing PTOX from Arabidopsis (Joët et al., 2002). In high mountain species Ranunculus glacialis , the rate of the linear electron transfer far exceeds the rate of consumption of electrons for carbon assimilation rate under different temperature and light levels; especially under 21% O2 and high Ci, suggesting a major role of PTOX in photoprotection (Streb et al., 2005).