3.6 Plastid terminal oxidase (PTOX) as a plastohydroquinone:oxygenoxidoreductase
The improved efficiency and/or the additional turnover of PSII under salt treatment in spartina at the presence of 21% oxygen, compared to either control with 21% O2 or 250 mM NaCl with 2% O2, is very likely attributed to electron transfer directly to molecular oxygen (O2). Since experiments were conducted under a saturating CO2 concentration of 2000 μL L-1, we exclude the contribution of photorespiration to this effect. Usually, the photo-reduction of O2 may occur at the acceptor side of PSI via the Mehler reaction; however, the lack of a sensitivity of PSI parameters to oxygen suggests that this is unlikely the reason, or at least not the only reason. So here we test the possibility that the putative quinone-oxygen oxidoreductase, the plastid terminal oxidase (PTOX) or IMMUTANS protein (Shahbazi et al., 2007; Heyno et al., 2009) might have played a role as well for SA .
To determine whether the PTOX may play a role in electron transfer from PSII to O2, measurements of ETRII were performed on leaves obtained from control and salt-treated SA andSV which were vacuum infiltrated with either water or a solution of the PTOX inhibitor n -propyl gallate (n -PG; 3,4,5-trihydroxy-benzoic acid-n -propyl ester; Joët et al., 2002; Josse et al., 2003; Kuntz, 2004; Rosso et al., 2006; Houille-Vernes et al., 2011; Sun & Wen, 2011; Trouillard et al., 2012; Shirao et al., 2013; Nawrocki et al., 2015). In SV , PSII quantum yield (ɸPSII) was insensitive to n -PG, regardless whether the plants have been exposed to NaCl treatment or not (Figure 7A). This was also the case for control SA . In SA exposed to 250 mM NaCl, ɸPSII was insensitive to n -PG (Figure 7B). ɸPSII measured 12 days after initiating NaCl treatment was reduced by about 32 and 45%, in leaves infiltrated with 5 mM n -PG, in the presence of 21 and 2% O2, respectively (Figure 7B), falling thereby to the control level or even slightly lower (Figure 7B). Interestingly, at low O2 in salt-stressed plants, we observed a decrease in the ɸPSII. This suggests strongly that molecular oxygen (O2) may act as a terminal electron acceptor by oxidizing the plastoquinol (PQH2).
The effect of n -PG suggests a potential activity of plastid terminal oxidase (PTOX) located on the stromal side of the membrane inSA though this does not exclude a potential contribution of the Mehler reaction to electron transport. To measure electron flow to oxygen excluding any contribution of the Mehler reaction, leaves were infiltrated with the cytochrome b6/f (Cytb6/f) inhibitor dibromothymoquinone or 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), a specific inhibitor of the Qo-binding site (Malkin, 1981, 1982; Rich et al., 1991; Schoepp et al., 1999). In SV , this almost completely abolished the ɸPSII and thereby the electron flow beyond the cytb6/f, regardless the NaCl treatment (Figure 7C). In control SA leaves, DBMIB also strongly inhibited ɸPSII, though a residual ɸPSII and also electron transfer remained. In salt-stressed SA leaves, DBMIB only partially inhibited ɸPSII with decreasing O2 concentration resulting in greater inhibition of ɸPSII. The extent of DBMIB insensitive, oxygen-sensitive ɸPSII decrease was similar to that ofn -PG-sensitive electron transport in the same leaves (Figure 7B and D).
The dramatic decline in ɸPSII in the presence of DBMIB at low O2 in salt treated Spartina leaves (Figure 7D) might be explained as a double restriction in the electrons flux beyond PSII. First limitation due to the blockage (or shortage) in the electrons flow towards PSI due to the presence of DBMIB and the second curtailment is tightly linked to the drop in the O2level (2%).
Western-blot analyses of thylakoid membrane extracts of SA andSV using antibodies raised against Zea mays PTOX revealed the presence of a 35-kDa band in both species (Figure 8). For untreated plants, SA showed higher protein abundance than in SV . In the latter (SV ), salt treatments resulted in a slight increase in the PTOX abundance (Figure 8A and inset), though the expression level of PTOX transcript insignificantly decreased (Figure 8B). In SA , treatment with 250 mM NaCl elevated PTOX abundance by 3~4 times compared to the control (Figure 8A and inset). Similarly, the transcript abundance of PTOX was also elevated under NaCl treatment by the same amount (Figure 8A-B).