bs_bs_banner Plant, Cell and Environment (2013) 36, 1296–1310 doi: 10.1111/pce.12059 Plastid terminal oxidase (PTOX) has the potential to act as a safety valve for excess excitation energy in the alpine plant species Ranunculus glacialis L. CONSTANCE LAUREAU1, ROSINE DE PAEPE2, GWENDAL LATOUCHE1, MARIA MORENO-CHACÓN1, GIOVANNI FINAZZI3,4,5,6, MARCEL KUNTZ3,4,5,6, GABRIEL CORNIC1 & PETER STREB1 1Ecologie, Systématique et Evolution, Université Paris-Sud 11, UMR-CNRS 8079, Bâtiment 362, 91405 Orsay cedex, France, 2Institut de Biologie des Plantes, Université Paris-Sud 11, UMR-CNRS 8618, Bâtiment 630, 91405 Orsay cedex, France, 3Unité Mixte Recherche 5168, Laboratoire Physiologie Cellulaire et Végétale, Centre National Recherche Scientifique, F-38054 Grenoble, France, 4Commissariat à l’Energie Atomique et Energies Alternatives, l’Institut de Recherches en Technologies et Sciences pour le Vivant, F-38054 Grenoble, France, 5Université Grenoble 1, F-38041 Grenoble, France and 6Institut National Recherche Agronomique, UMR1200, F-38054 Grenoble, France ABSTRACT PSII; PFD, photon flux density; PQ, plastoquinone; PSI, pho- tosystem I; PSII, photosystem II; PTOX, plastid terminal Ranunculus glacialis leaves were tested for their plastid ter- oxidase; qL, fraction of open PSII centres; RD, dark respira- minal oxidase (PTOX) content and electron flow to pho- tion, RL, light respiration; ROS, reactive oxygen species; torespiration and to alternative acceptors. In shade-leaves, Rubisco, ribulose 1·5-bisphosphate carboxylase/oxygenase; the PTOX and NAD(P)H dehydrogenase (NDH) content SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel were markedly lower than in sun-leaves. Carbon assimilation/ electrophoresis. light and Ci response curves were not different in sun- and shade-leaves, but photosynthetic capacity was the highest in INTRODUCTION sun-leaves. Based on calculation of the apparent specificity factor of ribulose 1·5-bisphosphate carboxylase/oxygenase Alpine plants growing at high altitude have to cope regularly (Rubisco), the magnitude of alternative electron flow unre- with extreme climatic events such as very high photon flux lated to carboxylation and oxygenation of Rubisco correlated densities (PFDs) combined with either low or high tempera- to the PTOX content in sun-, shade- and growth chamber- tures (Körner 1999; Laureau, Bligny & Streb 2011; Streb & leaves. Similarly, fluorescence induction kinetics indicated Cornic 2012). Combinations of high PFDs with temperature more complete and more rapid reoxidation of the plastoqui- extremes can lead to high rates of chloroplastic electron none (PQ) pool in sun- than in shade-leaves. Blocking elec- transport, which may induce the formation of reactive tron flow to assimilation, photorespiration and the Mehler oxygen species (ROS) and possible cellular destructions reaction with appropriate inhibitors showed that sun-leaves (Asada 1999; Ort & Baker 2002; Suzuki & Mittler 2006). were able to maintain higher electron flow and PQ oxidation. Numerous mechanisms to balance electron transport with The results suggest that PTOX can act as a safety valve in light absorption and carbon assimilation were demonstrated R. glacialis leaves under conditions where incident photon in several plant species, including the dissipation of excess flux density (PFD) exceeds the growth PFD and under con- absorbed energy as heat (Li et al. 2009) as well as the con- ditions where the plastoquinone pool is highly reduced. Such sumption of excess electrons by photorespiration (Ort & conditions can occur frequently in alpine climates due to Baker 2002; Streb et al. 2005; Streb & Cornic 2012), by the rapid light and temperature changes. Mehler reaction (Asada 1999; Ort & Baker 2002), by the malate valve (Scheibe & Dietz 2012) and electron cycling Key-words: alternative electron flow; photorespiration; pho- around photosystem I (PSI) to increase the proton gradient tosynthesis; stress tolerance. (Cornic et al. 2000). All these mechanisms can interact to keep the plastoquinone (PQ) pool oxidized. Their impor- Abbreviations: AN, net carbon assimilation; Ca (Ci), external tance probably depends on the physiological state of the (internal) CO2 partial pressure; DBMIB, 2,5-dibromo-3- plants and on the plant species. methyl-6-isopropyl-p-benzoquinone; EDTA, ethylenediami- Furthermore, several scavenging systems can keep the netetraacetic acid; ETRC (ETRO), electron flow to the ROS accumulation under control to prevent ROS-induced carboxylation (oxygenation) reaction of Rubisco; Fo (Fm), damage (Noctor & Foyer 1998;Asada 1999).While several of minimum (maximum) fluorescence yield; JT, total electron these protective mechanisms increase in response to low- flux at PSII; NDH, NAD(P)H dehydrogenase; NPQ, non- temperature acclimation (Ensminger, Berninger & Streb photochemical fluorescence quenching; FCO2, quantum 2012), their abundance is variable in different alpine species efficiency of CO2 assimilation; FPSII, quantum efficiency of and they are also widely distributed in plant species incapa- ble to survive alpine climates (Streb & Cornic 2012). Moreo- Correspondence: P. Streb. E-mail: [email protected] ver, the artificial decline of glutathione in two alpine plant 1296 © 2013 John Wiley & Sons Ltd PTOX in alpine plants 1297 species, one with strong (Soldanella alpina) and the other investigated: (1) did the electron flow through PTOX corre- with weak (Ranunculus glacialis) antioxidative protection late with PTOX content and could this activity contribute capacity, does not affect their susceptibility to low significantly to electron consumption? and (2) did PTOX temperature-induced photoinhibition (Laureau et al. 2011). content correlate with the sensibility to photoinhibition? The These findings raise the question whether alpine plants function of PTOX as a safety valve in relation to photores- balance excess excitation energy with an alternative, more piration under high-mountain stress conditions is discussed. specific safety valve, which is quantitatively less important in non-alpine plants. According to previous investigations, all MATERIAL AND METHODS alpine plant species investigated for plastid terminal oxidase (PTOX) contain high amounts of this protein. In addition, the Plant material and growing conditions PTOX protein content increases with the altitude where these plants were collected from (Streb et al. 2005; Laureau et al. R. glacialis (L) plants were collected in the French Alps at 2011).In lowland plants,the amount of PTOX is generally low approximately 2400 m altitude as described by Laureau et al. (Lennon, Prommeenate & Nixon 2003; Streb et al. 2005) and (2011).Whole plants were transferred into pots with soil from their capacity to accept excess photosynthetically generated the growing site in the first year and either further cultivated electrons was estimated to be insignificant (Ort & Baker 2002; at 2400 m altitude in full sunlight (sun-leaves), protected by Trouillard et al. 2012). However, in plant species acclimated to green nylon (high-density polyethylene) to reduce the inci- stress conditions like cold, heat, drought salinity and high dent PFD by 50% (shade-leaves) or transferred to a growing -2 -1 light,the amount of PTOX protein increases strongly,suggest- chamber at low altitude at 6 °C and 100 mmol m s PFD ing a potential involvement in stress tolerance (reviewed by (24 h). Measurements of mature leaves started the second McDonald et al. 2011; Sun & Wen 2011). In salt-stressed Thel- year and were repeated for at least two subsequent years. lungiella, up to 30% of photosynthetic electron flow was esti- Only leaves fully developed under the described conditions mated to be attributed to PTOX (Stepien & Johnson 2009). were used for the experiments. The PTOX protein can transfer electrons from PQ to molecular oxygen, thus forming H2O (Carol & Kuntz 2001). Microclimatic conditions PTOX is involved in chloroplast development. During caro- tenoid synthesis, desaturase reactions require PQ and are The leaf temperature and the PFD arriving at the leaf level driven by a redox chain in which PTOX is likely to reoxidize were recorded every 30 s during the first week after leaf reduced PQ (Carol & Kuntz 2001; Kuntz 2004). The lack of expansion for shade- and sun-leaves with a Campbell data PTOX leads to the variegated leaf phenotype of the Arabi- logger (CR23X; Campbell Scientific Inc., Utah, UT, USA) as dopsis mutant immutans and the tomato mutant ghost,in described by Laureau et al. (2011). For shade- and sun-leaves, which the bleached sectors originate early during chloroplast temperature and PFD were measured on three independent biogenesis as a consequence of a deficit in carotenoid content leaves. From these measurements, hourly mean leaf tempera- (Carol & Kuntz 2001). In mature leaves, PTOX is no longer ture and PFD during the day (0800–1800 h) and night (1800– needed for carotenoid synthesis, but it may act as a safety 0800 h) were calculated and shown in addition to the highest valve to keep the PQ pool oxidized and to prevent photooxi- and lowest measured values. dative damage under stress conditions (McDonald et al. 2011; Sun & Wen 2011; Streb & Cornic 2012). In Chlamydomonas Gas exchange and fluorescence measurements reinhardtii cells, two different PTOX proteins have been recently described, one is assumed to be involved in carote- Net CO2 uptake by leaves in combination with chlorophyll noid synthesis, while the second is supposed to play a role in fluorescence emission was measured using a LI-6400 (Li-Cor