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Plastid Terminal Oxidase Requires Translocation to the Grana Stacks to Act As a Sink for Electron Transport

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Plastid Terminal Oxidase Requires Translocation to the Grana Stacks to Act As a Sink for Electron Transport Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport Piotr Stepiena,1 and Giles N. Johnsonb,1 aWroclaw University of Environmental and Life Sciences, Department of Plant Nutrition, 50-357 Wroclaw, Poland; and bSchool of Earth and Environmental Sciences, University of Manchester, M13 9PT Manchester, United Kingdom Edited by Pierre A. Joliot, Institut de Biologie Physico-Chimique, Paris, France, and approved July 30, 2018 (received for review November 1, 2017) The plastid terminal oxidase (PTOX) has been shown to be an im- in response to stress (15). Evidence that PTOX can act as a portant sink for photosynthetic electron transport in stress-tolerant significant sink for electron transport under stress conditions was plants. However, overexpression studies in stress-sensitive species first put forward by Streb et al. (9), who measured significant have previously failed to induce significant activity of this protein. levels of PTOX protein and presented indirect evidence for its Here we show that overexpression of PTOX from the salt-tolerant activity in the high-montane species Ranunculus glacialis. This brassica species Eutrema salsugineum does not, alone, result in ac- was questioned, however, by Rosso et al. (16), who examined tivity, but that overexpressing plants show faster induction and a plants of Arabidopsis overexpressing PTOX (PTOX-OE) and greater final level of PTOX activity once exposed to salt stress. This failed to find evidence for a role either in acting as a significant implies that an additional activation step is required before activity electron sink or in protecting plants from high light stress. In is induced. We show that that activation involves the translocation other studies, PTOX activity has only been seen under non- of the protein from the unstacked stromal lamellae to the thylakoid equilibrium conditions with a very low estimated flux (17, 18). grana and a protection of the protein from trypsin digestion. This In wild-type Arabidopsis, PTOX is only present at around 1% represents an important activation step and opens up possibilities in of the other electron transport components and seems unlikely the search for stress-tolerant crops. to contribute to overall electron flux (18). In salt-stressed alternative electron transport | plant stress | photosynthesis | Eutrema, PTOX-dependent electron transport accounts for up chlororespiration | photoprotection to 30% of the total linear electron flow (8). This could sub- stantially reduce ROS production under stress, protecting leaves from photooxidative damage, and may play a crucial role in ost crop species are, to a greater or lesser extent, stress- Msensitive, and optimizing stress tolerance pathways may determining stress tolerance of Eutrema. If we could transfer this play a key role in securing food production (1). The greatest PTOX activity to crop species, it might be possible to signifi- potential for identifying stress-tolerance mechanisms comes cantly increase abiotic stress tolerance. However, previous at- from studies of extremophiles. During the last decade, the hal- tempts at overexpressing PTOX protein have not resulted in a – ophyte brassica Eutrema salsugineum (previously Thellungiella significant increase in electron transport capacity (16 18) and halophila) has emerged as a stress-tolerant model system (2–4). have in fact been reported to increase oxidative stress (19). This close relative of the widely studied model plant Arabidopsis Given the contradictions in previous data, we hypothesized thaliana is not only highly tolerant of salt but also of a variety of that the differences in PTOX activity between species are due to other abiotic stresses, including drought and extreme tempera- differences in the properties of the polypeptide in those species. tures (5–7). A better understanding of this tolerance will open To test this hypothesis, we overexpressed the Eutrema PTOX possibilities for developing stress-tolerant crops. Importantly, gene in Eutrema and Arabidopsis. We observed, however, that unlike many extremophiles, Eutrema is amenable to molecular the high activity in this plant is not simply related to protein studies, providing an excellent model system. content or differences in sequence from other species. Rather, Previously, we provided evidence that a protein termed the we present data that the localization of the PTOX protein is Plastid Terminal Oxidase (PTOX—a plastoquinone–oxygen ox- altered to allow for its increase activity. This opens research idoreductase) acts as a significant sink for photosynthetic elec- avenues for work on improving stress tolerance. tron transport in salt-stressed Eutrema (8). In Eutrema, we observed a strong, salt-induced up-regulation of PTOX protein, Significance which correlated with the induction of a substantial oxygen sensitivity of Photosystem II (PSII) electron transport. Electron Growing concerns about food security and changing climates transport became sensitive to the PTOX inhibitor n-propyl gal- make the identification of stress-tolerance traits a priority. late, while the O2-sensitive portion of electron transport was Here we show that the activation of the plastid terminal oxi- shown to be insensitive to the Cyt b6f inhibitor DNP-INT. In dase, via its relocation within the thylakoid membrane, can contrast, expression of PTOX protein in Arabidopsis was in- induce a protective sink for electron transport. We have thus sensitive to salt treatment, and we found no evidence that it identified an unexpected mechanism for protein activation and contributed to electron flux under steady-state conditions. Based opened avenues for engineering plant stress tolerance. on our results and previous published work, we proposed that PTOX protects Eutrema leaves from oxidative stress under salt- Author contributions: P.S. and G.N.J. designed research; P.S. performed research; P.S. and stress conditions (8–10). G.N.J. analyzed data; and P.S. and G.N.J. wrote the paper. PTOX was originally identified as a defective protein in var- The authors declare no conflict of interest. iegated mutants (IMMUTANS, ghost) and was shown to play a This article is a PNAS Direct Submission. – critical role in carotenoid biosynthesis in green tissues (11 13). Published under the PNAS license. Biochemical evidence has pointed to PTOX being localized on 1To whom correspondence may be addressed. Email: [email protected] or giles. the stromal face of the unstacked regions of the thylakoid [email protected]. membrane (14). Binding of PTOX to the membrane has been This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. suggested to be regulated by light-induced changes in stroma pH, 1073/pnas.1719070115/-/DCSupplemental. which may provide a dynamic mechanism for activation of PTOX Published online September 4, 2018. 9634–9639 | PNAS | September 18, 2018 | vol. 115 | no. 38 www.pnas.org/cgi/doi/10.1073/pnas.1719070115 Downloaded by guest on October 1, 2021 Results Overexpression of Eutrema PTOX Protein Does Not Result in an Increase in Electron Transfer to Oxygen. Previously, we observed that, in unstressed plants of Eutrema and Arabidopsis, electron transport through PSII was largely insensitive to the oxygen concentration under conditions of saturating CO2 (8). This is consistent with a low rate of electron flow to oxygen. In plants of Eutrema, but not Arabidopsis, salt stress resulted in an increase in electron transport through PSII. This additional capacity was shown to be sensitive to oxygen and to the PTOX inhibitor n- propyl gallate. Salt stress induced an increase in PTOX protein. To test whether this additional electron transport was a simple consequence of the presence of the PTOX protein, we produced plants which overexpressed the native protein under control of the 35S promoter (SI Appendix, Fig. S1). Plants were produced with an up to eightfold increase in PTOX protein, as estimated using Western blot analysis (SI Appendix, Fig. S1). Examination of the relative expression of other thylakoid membrane proteins revealed no significant differences in stoichiometry between wild-type plants and their respective transgenic lines (SI Ap- pendix, Fig. S1). PTOX overexpression alone did not result in any change in PSII capacity (Fig. 1B) or in PSII electron trans- port becoming sensitive to oxygen (SI Appendix, Fig. S2). We Fig. 2. The effect of n-propyl gallate (A) and DBMIB (B) on PSII photo- chemical efficiency measured in the leaves of Arabidopsis thaliana and conclude therefore that expression of PTOX protein alone is not Eutrema salsugineum subjected to 0 and 100 and 0 and 250 mM NaCl, re- sufficient for electron transport to oxygen to occur. Additionally, spectively. Measurements were carried out 10 d after initiating salt treat- −1 we overexpressed the Eutrema PTOX protein in Arabidopsis. ment at 25 °C in the presence of 385 μLL CO2. Leaves were vacuum- Again, no evidence of activity of this protein was seen (Fig. 1B infiltrated with water in the presence of 21% (white bars) and 2% (light- and SI Appendix, Fig. S2). gray bars) oxygen or with 1 mM n-propyl gallate or 30 μM DBMIB in the presence of 21% (dark-gray bars) and 2% (black bars) oxygen. Data points PSII Activity Is Induced by Salt More Rapidly and to a Greater Extent represent the means ± SE of at least six replicates. in PTOX-OE Plants of Eutrema. WT and PTOX-OE plants of Eutrema and Arabidopsis were then exposed to salt stress for up consistent with our previous observations (8). In Arabidopsis to 14 d. Salt concentrations used were such as had previously plants, exposure to salt resulted in a progressive and marked been shown to increase leaf salt content without causing plant decline in PSII efficiency (ΦPSII) and a corresponding increase death (150 mM NaCl in Arabidopsis, 250 mM NaCl in Eutrema; in nonphotochemical quenching (Fig. 1 A and C). This re- see ref. 8 for details). In all plants, salt treatment resulted in a + + sponse was largely unaffected by heterologous expression of the marked increase in leaf Na content, with a reduction in K , Eutrema PTOX protein.
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