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).