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Photosynth Res DOI 10.1007/s11120-013-9885-3

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Photosynthetic electron transport and specific photoprotective responses in wheat leaves under drought stress Marek Zivcak • Marian Brestic • Zuzana Balatova • Petra Drevenakova • Katarina Olsovska • Hazem M. Kalaji Xinghong Yang • Suleyman I. Allakhverdiev



Received: 18 March 2013 / Accepted: 3 July 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract The photosynthetic responses of wheat (Triticum aestivum L.) leaves to different levels of drought stress were analyzed in potted plants cultivated in growth chamber under moderate light. Low-to-medium drought stress was induced by limiting irrigation, maintaining 20 % of soil water holding capacity for 14 days followed by 3 days without water supply to induce severe stress. Measurements of CO2 exchange and photosystem II (PSII) yield (by chlorophyll fluorescence) were followed by simultaneous measurements of yield of PSI (by P700

See Table 1 for other symbols representing chlorophyll fluorescence and P700 parameters apart from the abbreviations listed.

Electronic supplementary material The online version of this article (doi:10.1007/s11120-013-9885-3) contains supplementary material, which is available to authorized users. M. Zivcak  M. Brestic (&)  Z. Balatova  P. Drevenakova  K. Olsovska Department of Plant Physiology, Slovak Agricultural University, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic e-mail: [email protected] M. Zivcak e-mail: [email protected] Z. Balatova e-mail: [email protected] P. Drevenakova e-mail: [email protected]

absorbance changes) and that of PSII. Drought stress gradually decreased PSII electron transport, but the capacity for nonphotochemical quenching increased more slowly until there was a large decrease in leaf relative water content (where the photosynthetic rate had decreased by half or more). We identified a substantial part of PSII electron transport, which was not used by carbon assimilation or by photorespiration, which clearly indicates activities of alternative electron sinks. Decreasing the fraction of light absorbed by PSII and increasing the fraction absorbed by PSI with increasing drought stress (rather than assuming equal absorption by the two photosystems) support a proposed function of PSI cyclic electron flow to generate a proton-motive force to activate nonphotochemical dissipation of energy, and it is consistent with the observed accumulation of oxidized P700 which causes a decrease in PSI electron acceptors. Our results X. Yang State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian 271018, China S. I. Allakhverdiev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, Moscow 127276, Russia e-mail: [email protected] S. I. Allakhverdiev Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia

K. Olsovska e-mail: [email protected] H. M. Kalaji Department of Plant Physiology, Faculty of Agriculture and Biology, Warsaw Agricultural University SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland e-mail: [email protected]

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support the roles of alternative electron sinks (either from PSII or PSI) and cyclic electron flow in photoprotection of PSII and PSI in drought stress conditions. In future studies on plant stress, analyses of the partitioning of absorbed energy between photosystems are needed for interpreting flux through linear electron flow, PSI cyclic electron flow, along with alternative electron sinks. Keywords Drought stress  Wheat  Photosynthetic electron transport  Cyclic electron transport around PSI  Photosystem stoichiometry  Chlorophyll fluorescence  Alternative electron sinks Abbreviations ACO2 CO2 assimilation rate Cyt b6/f Cytochrome b6/f gm Mesophyll conductance gs Stomatal conductance LED Light emitting diode LHC Light harvesting complex NPQ Nonphotochemical quenching P700 Primary electron donor of PSI (reduced form) P700? Primary electron donor of PSI (oxidized form) PAR Photosynthetic active radiation PQ Plastoquinone PSI Photosystem I PSII Photosystem II QA Primary PSII acceptor qE pH-dependent energy dissipation RuBP Ribulose 1,5-bisphosphate RWC Relative water content DpH Transthylakoid pH gradient WW Water potential

Introduction Drought is one of the main factors negatively affecting the productivity of agricultural or natural ecosystems (Passioura 2007; Ciais et al. 2005) and the diversity of plant species (Engelbrecht et al. 2007). It has also a global impact on carbon gain (Buermann et al. 2007). Drought mostly affects plants through its effects on photosynthesis. However, photosynthetic responses to drought stress are complex, involving the interplay of different structural levels at different time scales in relation to plant development (Chaves et al. 2009). The effects of drought stress on assimilation can be direct or indirect. Direct effects are through decreased CO2 availability to chloroplasts due to a decrease in stomatal and mesophyll conductances (Chaves 1991; Lal et al. 1996; Chaves et al. 2002; Flexas et al. 2004, 2012), or are a

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consequence of changes in photosynthetic metabolism (Tezara et al. 1999; Lawlor and Cornic 2002; Maroco et al. 2002; Parry et al. 2002; etc.). In conditions of limited CO2 diffusion, photorespiration lowers the energetic efficiency of photosynthesis in C3 plants (Ogren 1984). Indirect or secondary effects are through oxidative stress, which can contribute to the nonstomatal limitation of photosynthesis. This can seriously affect the leaf’s photosynthetic machinery, mostly as a result of the interaction between drought and excessive light or under multiple stress conditions (Ort 2001; Chaves and Oliveira 2004; Foyer and Noctor 2009). Photosynthetic responses to water deficit are influenced by stress intensity, duration, and rate of progression—these factors determining whether mitigation processes associated with acclimation will be initiated (Chaves et al. 2009). The photosynthetic machinery is necessarily flexible, utilizing several mechanisms to prevent the harmful effects of highly reactive intermediates during the conversion of light into usable forms of energy. These mechanisms also need to insure that the output ratio of ATP/NADPH matches the demands of plant metabolism (Kramer et al. 2004; Cruz et al. 2005; Kramer and Evans 2011). The central process in higher plants is nonphotochemical quenching, by which excess light energy is harmlessly dissipated as heat (Muller et al. 2001). Nonphotochemical quenching typically dominates a rapidly reversible component called qE, resulting in the thermal dissipation of excess absorbed light energy in the light-harvesting antenna of PSII. qE is induced by a low thylakoid lumen pH and a high DpH that are generated by photosynthetic electron transport under excess light conditions. These processes represent a form of feedback regulation of the light-dependent reactions of photosynthesis (Niyogi et al. 2005). The low pH of thylakoid lumen activates qE, by protonating the protein PsbS (Li et al. 2000) and by activating violaxanthin deepoxidase, which converts violaxanthin to antheraxanthin and zeaxanthin in xanthophyll cycle (Demmig-Adams 1990). There is also evidence for dissipation of excess energy in the reaction center of PSII where electron transfer is initiated (Ivanov et al. 2008). In addition, under low CO2 supply, energy from linear electron flow can in part be redirected to photorespiration (Kozaki and Takeba 1996) or to some alternative pathways—mainly the water–water cycle (Asada 1999). In photosystem I, the cyclic electron flow is activated (Golding and Johnson 2003; Golding et al. 2004; reviewed in Johnson 2005, 2011; Miyake 2010). There is controversy over the role and contribution of this process to photoprotection and balancing ATP/NADPH production ratio. Munekage and coworkers demonstrated the essential role of cyclic electron flow both for photoprotection and producing additional ATP for Calvin cycle (Munekage

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et al. 2002, 2004). However, the contribution of fast cyclic electron transport to the buildup of DpH on thylakoid membrane and ATP synthesis has been strongly questioned by researchers that still support the idea of photoprotective role in stress conditions (Laisk et al. 2007, 2010). Cruz et al. (2005) have argued for the important role of mechanisms (e.g., by changing the proton conductivity of the ATP-synthase) that modulate qE sensitivity to DpH; such mechanisms can efficiently modulate ATP/NADPH output ratio by interacting with linear or alternative electron flows. Drought stress has also been observed to induce longterm changes in the structure, content, and activity of individual photosynthetic components (Kohzuma et al. 2009). Specifically, drought stress has been associated with a decrease in leaf absorbance i.e., a decrease of chlorophyll content (Balaguer et al. 2002; Maroco et al. 2002), an increase of the xanthophyll pool (Maroco et al. 2002), a decrease of expression of ATP synthase (Tezara et al. 1999; Kohzuma et al. 2009), a decrease in expression of cyt b6/f (Kohzuma et al. 2009) or expression of functional proteins (Zadraznik et al. 2013), etc. In this article, we present data on the effects of drought stress on photosynthesis in wheat (T. aestivum L.). Wheat is one of the most important crops worldwide (Bonjean and Angus 2001). It is also interesting because, as grasses are evolutionary younger than dicots, they may differ in physiological responses to drought (e.g., Flexas et al. 2012). There are many studies showing the high acclimation capacity of wheat photosynthetic apparatus to environmental stresses (e.g., Gray et al. 1996; Mehta et al. 2010; Mathur et al. 2011; Vassileva et al. 2012; Brestic et al. 2012). Moreover, wheat and its relatives (e.g., barley) are frequently used in photosynthesis research, providing a broad baseline for comparisons as well as numerical inputs to photosynthetic models—although these are mostly valid only for non-stressed (NS) conditions (e.g., Yin et al. 2009). Our study focuses on functional changes at the level of photosynthetic electron transport and related photosynthetic processes affected by different levels of prolonged drought stress. We use a relatively long duration of drought stress during moderate light intensity, allowing us to observe physiological changes that can be attributed to acclimation rather than to injury (e.g., by photoinhibition or by oxidative stress). Based on our results, we discuss the roles of alternative electron sinks and cyclic electron flow in the protection of PSII and PSI in conditions of limited water supply, and the effects of drought stress on the distribution of absorbed light energy between photosystems I and II.

Materials and methods Plant material After vernalisation at 6 °C for 2 months, plants of winter wheat (T. aestivum L., cv. Ilona) were transplanted and cultivated individually in pots (0.5 l) containing standard peat substrate Klassman TS1. The pots were regularly irrigated and occasionally fertilized using standard liquid fertilizer with micronutrients. Before and during the drought stress protocol, plants were grown in a growth chamber with artificial light provided by fluorescent tubes (Osram Fluora) with maxima in red and blue spectral regions; the incident PAR at leaf level was app. 200 lmol photons m-2 s-1. The photoperiod was 14-h light/10-h dark cycle with light intensity reduced by half during the first and the last hours of light period. Temperature ranged between 18 °C at night and 23 °C during the light period. Drought stress treatment and measuring protocol Drought stress in wheat plants was induced after the seventh leaf from the base appeared. Plants within the experiment were divided into two groups: ‘‘non-stressed’’ and ‘‘droughtstressed.’’ Before the start of the experiment, all pots were fully irrigated to achieve the maximum water capacity. All pots were then weighed. The soil of some pots was dried to get the percentage of soil dry mass in a water saturated soil; this was used to calculate dry mass of soil in each experimental pot. The weight of the empty pot with the dry mass was then subtracted from each actual pot weight to get the maximum water capacity (from fully irrigated pots) or actual water content. Soil water content (expressed as % of soil water capacity) in each pot was calculated as the ratio of actual to maximum water content. All pots were weighed daily, and water was supplied to 70 % of soil water capacity in control plants and 20 % of soil water capacity in stressed plants—the soil water content below *15 % of soil water capacity caused wilting. After 14 days, the water supply for drought-stressed plants was stopped for 3 days. Measurements were taken from the leaves of drought-stressed plants during the 7th–14th day period (before the full withholding of irrigation). Leaves from this period are referred to as ‘‘initial drought stress’’ (ID) or ‘‘moderate drought stress’’ (MD), depending on leaf water status—water availability decreased during the day, see Fig. 1 for respective leaf water content. Measurements after full withholding of irrigation were taken from the 14th to 16th day of the experiment, referred to as the ‘‘severe drought’’ (SD) period. In addition to measurements on stressed plants, leaves of control plants were also tested and are labeled as ‘‘non-stressed.’’

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The RWC was determined using the fresh weight (FW) measured immediately after taking the sample. After that, the leaf sample was saturated in distilled water for *12 h at a low temperature (4 °C) in the dark to obtain the saturated weight (SW). Finally, the sample was dried at 80 °C for 12 h to obtain dry weight (DW). RWC was calculated according to Turner (1981) as RWC (%) = (FW - DW)/ (SW - DW)*100. Leaf water potential (WW) of fresh leaf segments was measured by the psychrometric method using microvoltmeter Wescor HR-33 with measuring chamber C-52 (Wescor, USA). Three segments of each sample were measured, and the average value was used in the analyses. Simultaneous measurements of gas exchange and chlorophyll fluorescence

Fig. 1 Definition of drought stress levels based on the values of leaf relative water content (RWC), leaf water potential, and stomatal conductance. a Relationship between the leaf water potential (WW) and the leaf relative water content (RWC) measured in wheat leaves during drought stress. b Relationship between the leaf RWC and stomatal conductance (gs) measured in wheat leaves during drought stress. The four levels of drought stress were defined as follows (points within squares): NS no stress, ID initial drought, MD moderate drought, and SD severe drought

All measurements were taken from the last appearing leaf on the plant. The simultaneous measurements of CO2 assimilation with chlorophyll fluorescence, and simultaneous measurements of P700 and chlorophyll fluorescence were performed on the same leaf. Between the different measurements, the plants were exposed to ambient light for at least half an hour to eliminate the effect of previous measurements. The same leaf was then used for destructive determination of water potential and relative water content (RWC). Measurements of the leaf water status Leaf water status was measured both as the RWC and as the leaf water potential (WW). The samples were taken from the middle part of the leaf blade; this part was previously used also in all other measurements.

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The induction curve was recorded using a Licor 6400 gasometer (Licor, USA) with simultaneous measurement of chlorophyll fluorescence. Before the measurements, plants were exposed to ambient light in the growth chamber for at least 30 min. Immediately before the measurements, plants were dark adapted for 20 min in dark box and for app. 3 min in the measuring head. The F0 and FM values were then determined using saturation flash (6,500 lmol photons m-2 s-1), and the actinic light provided by LED light unit (1,000 lmol photons m-2 s-1) was switched on. Within the measuring head, the following conditions were maintained: leaf temperature 20 °C, reference CO2 content 380 ppm, and ambient air humidity. Every 120 s, the gas exchange rates were measured followed by saturation pulse and far-red pulse for F00 determination. The duration of the induction curve was at least 30 min—in some cases longer if it took more time to reach the steady state. The curves, as well as the steady-state values of photosynthetic parameters, were used in the analyses described below. A range of measured and calculated fluorescence parameters were used in the analysis (Table 1). We also calculated electron transport fluxes based on gas exchange measurements. The rate of linear electron transport based on requirements to support photosynthesis was calculated according to Harley et al. (1992); gm was considered to be infinite: Jg ¼ 4ðACO2 þ RL Þðci þ 2C Þ=ðci  C Þ, or where gm equal to gs was used: Jg ¼ 4½ACO2 þ RL ½ðci  ACO2 =gm Þþ 2C =½ðci  ACO2 =gm Þ  C , where ci represents intercellular content of CO2, ACO2 represents measured CO2 assimilation (both values obtained from gas exchange measurements), RL represents mitochondrial respiration in light (the values reported by Yin et al. 2009 were used), and C* represents CO2 compensation point measured in the absence of respiration. We used for calculation the values of

Photosynth Res Table 1 Measured and calculated chlorophyll fluorescence parameters Parameters

Name and basic physiological interpretation

Measured or computed inputs for calculation of fluorescence and P700 parameters F, F0

Fluorescence emission from dark- or light-adapted leaf, respectively

F0 Fm, Fm

Minimum fluorescence from dark-adapted leaf, (PS II centers open) 0

Maximum fluorescence from dark- or light-adapted leaf, respectively (PS II centers closed)

FV = Fm - F0

Maximum variable fluorescence from dark-adapted leaf

F00

Minimum fluorescence from light-adapted leaf

Fs0

Steady state fluorescence at any light level

P

P700 absorbance at given light intensity

Pm, Pm0

Maximum P700 signal measured using saturation light pulse following after short far-red pre-illumination in dark (Pm) or light-adapted state

Chlorophyll fluorescence parameters derived from the saturation pulse analysis FV/Fm = 1 - (F0/Fm)

Estimated maximum quantum efficiency (yield) of PSII photochemistry (Kitajima and Butler 1975; Krause and Weis 1991; Schreiber et al. 1995)

UPSII = (Fm - F0 )/Fm0 JPSII = aII  PAR  UPSII

Estimated effective quantum yield (efficiency) of PSII photochemistry at given PAR (Genty et al. 1989) Rate of linear electron transport in PSII at given PAR, and portion of PAR absorbed by PSII (aII) (Bjo¨rkman and Demmig 1987; Genty et al. 1989)

NPQ = (Fm - Fm0 )/Fm0 SVo = F0/F00 - 1

Nonphotochemical quenching of Fm (Schreiber et al. 1988; Walters and Horton 1991) Nonphotochemical quenching of F0 (Ha¨rtel and Lokstein 1995)

qP = (Fm0 - Fs0 )/(Fm0 F00 )

Coefficient of photochemical quenching based on the ‘‘puddle’’ model (i.e., unconnected PS II units) (Schreiber 1986; Bjo¨rkman and Demmig 1987; Bilger and Bjo¨rkman 1990)

qL = qP(F0/Fs0 )

Coefficient of photochemical quenching based on the ‘‘lake’’ model (i.e., fully connected PS II units) (Kramer et al. 2004)

UNO = 1/ [NPQ ? 1 ? qL(Fm/F0 1)]

Quantum yield of nonregulated energy dissipation in PSII (Kramer et al. 2004)

UNPQ = 1 - UPSII - UNO

Quantum yield of pH-dependent energy dissipation in PSII (Kramer et al. 2004)

PSI parameters derived from the saturation pulse analysis of P700 absorbance UPSI = (Pm0 - P)/Pm

Estimated effective quantum yield (efficiency) of PSI photochemistry at given PAR (Klughammer and Schreiber 1994)

UND = P/Pm

Fraction of overall P700 that is oxidized in given state i.e., a lack of electrons coming from electron donors (Klughammer and Schreiber 1994)

UNA = (Pm - Pm0 )/Pm

Fraction of overall P700 that is oxidized in given state by saturation pulse i.e., a lack of electron acceptors (Klughammer and Schreiber 1994)

C* 37 lmol mol-1 in NS and ID leaves, 42 lmol mol-1 in MD leaves, and 64 lmol mol-1 in SD leaves as published by Galmes et al. (2006). This is in accordance with our unpublished measurements in wheat. The ACO2 and RL values are used also for calculation of the rate of electron transport used in photorespiration: JPR ¼ 2=3½JPSII  4ðACO2 þ RL Þ (Epron et al. 1995; Valentini et al. 1995); the JPSII is the electron transport calculated using the data obtained by chlorophyll fluorescence measurements: JPSII = aII  PAR  UPSII (PAR—photosynthetic active radiation incident on the surface of the leaf; UPSII—estimated effective quantum yield (efficiency) of PS II photochemistry at given PAR; aII, portion of incident PAR absorbed by PSII; the equal absorbance (0.84) of PAR by leaf was assumed in all samples; see also Table 1).

Simultaneous measurements of P700 redox state and chlorophyll fluorescence The P700 redox state was measured using a Dual PAM-100 with a dual wavelength (830/875 nm) unit (following Klughammer and Schreiber 1994). Saturation pulses (10,000 lmol photons m-2 s-1), intended primarily for determination of chlorophyll fluorescence parameters, were also used for the assessment of P700 parameters. As in case of gas exchange measurements, analyzed plants were first exposed to ambient light in growth chamber for at least 30 min; immediately before the measurements, plants were dark adapted for 20 min in a dark box and for app. 2 min in the measuring head. After determination of F0, FM, and PM, the induction curve at light intensity similar to ambient

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(174 lmol photons m-2 s-1) was used for induction of photosynthesis. After a steady state was reached, a rapid light curve was initiated (light intensities 14, 30, 61, 103, 134, 174, 224, 347, 539, 833, 1036, 1295, 1602, and 1960 lmol photons m-2 s-1; duration of illumination at each light intensity was 30 s). After this, a light intensity of 174 lmol photons m-2 s-1 was applied again for a few minutes. The measuring protocol was finished by initiating another induction curve at moderate light intensity (539 lmol photons m-2 s-1) for 5 min, followed by measurements of dark recovery kinetics for 5 min, with saturation flashes every 20 s. In all chlorophyll fluorescence records, the correction for PSI fluorescence was done according to the method of Pfundel (1998).

Results Effect of drought stress: water relations and stomatal responses To impose water stress, the soil water content in pots was initially maintained at 20 % of water capacity. The water supply was sufficient for plants to survive. Thus, it was possible to observe the long-term effects of drought stress including acclimation of photosynthetic structures and processes. This treatment led to decreases of leaf RWC and water potential (WW) compared with NS plants (Fig. 1a) in which leaf water status was kept in a narrow range (RWC from 86 to 92 %; WW from -0.6 to -1.2 MPa). As irrigation was performed in the late afternoon (after all measurements had been taken), leaf water status varied considerably during the day. During the period with limited water supply, leaf RWC ranged between 85 and 63 % and WW fluctuated from -1.2 to -2.2 MPa. In the last period, water was not supplied to plants, and the value of these parameters fell below -2.2 MPa, leading to almost full stomatal closure. Thus, we can consider this as a critical leaf water potential value (Van den Berg et al. 2002). Both the soil and leaf water statuses affected the activity of stomata; there was an exponential relationship between RWC and stomatal conductance (gs) values measured after 30 min of illumination by strong actinic light (Fig. 1b). In NS plants, the gs values ranged between 0.5 and 1.2 mol m-2 s-1, indicating that the stomata were fully open. During the period of limited irrigation, two different conditions were distinguished depending on water availability. In early measurements, the leaf water content was sufficient to maintain a relatively high maximum of gs (0.3–0.5 mol m-2 s-1); such measurements were denoted as ‘‘initial drought’’ (ID). After plants had used the

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available water, leaf water content and stomatal conductance decreased indicating ‘‘moderate drought’’ (MD) conditions; the maximum of gs during 30 min of illumination in these plants was in a relatively narrow range (0.1 and 0.3 mol m-2 s-1). After the water supply was stopped, in addition to a decrease of water content below the critical level, leaf stomata were almost fully closed. Measurements taken during this period were denoted as ‘‘severe drought’’ (SD). Simultaneous measurements of gas exchange and chlorophyll fluorescence The photosynthetic induction in dark-adapted leaves was monitored by saturation pulse method of chlorophyll fluorescence measured together with CO2 and water vapor exchange (Fig. 2). Recordings were continued as long as necessary to obtain the steady state as indicated by stable values of ACO2 and gs—the minimum time used was 30 min. Recording began after 20 min of dark adaptation and F0, Fm measurements. These measurements were necessary for calculation of quenching parameters by the saturation pulse method (Schreiber 1986). In addition to different steady-state values of stomatal conductance, leaves subjected to different drought stress levels also differed in the times required for stomata to start opening (Fig. 2a, b). Moreover, there was clear shift in the amount of time needed to achieve the steady state (\10 min in NS vs. more than 30 min in SD). While the electron transport rate calculated from gas exchange data (JG) closely followed the trend of ACO2, JPSII values (calculated using chlorophyll fluorescence data) were substantially higher under drought stress than the JG, especially under the SD (Fig. 2c vs d). The higher values of JPSII may indicate the presence of alternative electron flow and/or imprecise estimates of electron transport rates; both cases are discussed below. The distribution of electrons within linear electron transport between RuBP carboxylation and photorespiration (estimated using the formula of Epron et al. 1995 and Valentini et al. 1995) reached the steady state very quickly in NS and ID (Fig. 2e), but stabilized much later in MD and SD. After 30 min of photosynthetic induction at high light, significantly higher photorespiration remained in MD and SD. In severely stressed plants, the proportion of electrons consumed in oxygenation was two times higher than in NS plants. Surprisingly, high stress levels did not lead to a progressive increase in nonphotochemical quenching (Fig. 2f). No significant differences in regulated PSII dissipation in high light were observed between the leaves of NS, ID, and MD plants. However, NPQ was significantly enhanced in SD leaves: NPQ increased after *12 min, and this increase was apparent until the end of recording.

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Fig. 2 The values of selected photosynthetic parameters derived from gas exchange and chlorophyll fluorescence measurement during photosynthetic induction following dark-adapted state (20 min in dark before the measurements) toward the steady state. The measurements were recorded in air CO2 concentration of 380 lmol mol-1, leaf temperature 20 °C, and PAR 1,000 lmol photons m-2 s-1. a CO2 assimilation rate (ACO2 ). b Leaf stomatal conductance (gs). c PSII electron transport rate (JPSII) calculated based on measurements of PSII quantum yields, assuming the equal distribution of absorbed

light between PSI and PSII. d Rate of electron transport consumed by carboxylation plus oxygenation of RuBP (Jg), calculated by the data from gas exchange measurements. e Ratio of electron transport consumed by photorespiration (JPR) to the total PSII electron transport. f Nonphotochemical quenching of maximum fluorescence (NPQ). The average values ± standard errors from 8 to 10 plants are presented. NS non-stressed samples, ID initial drought, MD moderate drought, SD severe drought

The steady-state values of key photosynthetic parameters derived from induction curves together with leaf water status parameters (Supplementary Table 1) reflect the effects of different level of drought stress, with the exception of Fv/Fm parameter that was almost unaffected even in SD, as was demonstrated also in our previous studies (e.g., Zivcak et al. 2008). Leaf water status (RWC) and selected photosynthetic parameters measured in the steady state (Fig. 3) showed linear correlations with ACO2 , JPSII, and proportion of total electron flow associated with photorespiration (JPR/JPSII).

Nonphotochemical quenching grew exponentially with water loss (in the range of our observations). The intercellular CO2 content linearly decreased with decreasing RWC (WW). However, below 65 % of RWC or (-2 MPa of WW) the ci values significantly increased. Using leaf stomatal conductance (gs) as a reference parameter, a clear logarithmic relationship with ACO2 and JPSII as well as an exponential decay of the JPR/JPSII ratio can be observed. The relationship between gs and ci is more complicated, with some bifurcation of the trend at low gs related to leaf water status. This can be attributed to

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Photosynth Res Fig. 3 Relationships between leaf water status (relative water content, RWC and water potential, WW) or stomatal conductance (gs), and the steady-state values of photosynthetic parameters derived from simultaneous measurements of gas exchange and chlorophyll fluorescence: CO2 assimilation (ACO2 ), intercellular CO2 content (ci), PSII electron transport (JPSII), ratio of electron transport consumed by photorespiration (JPR) to the total PSII electron transport, and nonphotochemical quenching (NPQ). The best-fit curves are presented

nonstomatal effects in SD plants such as a decrease in mesophyll conductance (cf. Flexas et al. 2012 for review) or changes in Rubisco enzymatic activities (Lawlor and Cornic 2002), and ATP synthesis (Tezara et al. 1999). Simultaneous measurements of chlorophyll fluorescence and P700 absorbance Measurements based on the saturation pulse method allow for the calculation of PSII quantum yields (Schreiber 1986) and PSI quantum yields (Klughammer and Schreiber 1994). On each leaf, the induction kinetics at moderate light, the rapid light curve, recovery at moderate light, the induction curve at high light, and dark recovery kinetics were recorded subsequently. The same leaves were also used for gasometric measurements and for measurements of leaf water status (see ‘‘Materials and methods’’ for details).

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Complementary quantum yields of PSII (graphs left) and PSI (graphs right) were recorded within rapid light curves (Fig. 4). The sum of three complementary quantum yields of PSII (UPSII, UNO, and UNPQ) or PSI (UPSI, UNA, and UND) is defined as unity. As anticipated, the PSII and PSI effective quantum yields (Fig. 4a, b) show a similar trend, both in accordance with previously presented results. The curves of quantum yield of regulated dissipation processes in PSII (UNPQ) are also in accordance with previously presented results. While NS, ID, and MD leaves show very similar trends, a steeper increase in UNPQ as well as higher maximum values were observed in SD leaves (Fig. 4c). The curves of nonregulated dissipation (UNO) are not significantly different among drought stress levels. The UND (Fig. 4d) represents the fraction of total P700 which is oxidized in given state (P700?/P700T). Hence, it is a measure of the PSI donor-side limitation causing

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Fig. 4 Light responses of complementary quantum yields of PSII and PSI recorded in plants subjected to different levels of drought stress. a The effective quantum yield of PSII (UPSII); b the effective quantum yield of PSI (UPSI); c the quantum yield of regulated nonphotochemical quenching in PSII (UNPQ); d the quantum yield of the PSI nonphotochemical quenching caused by the donor-side limitation, i.e., the fraction of overall P700 that is oxidized in a given state (UND); e the fraction of energy captured by PSII passively dissipated in form of heat and fluorescence (UNO); f the quantum yield of the PSI

nonphotochemical quenching caused by the acceptor-side limitation, i.e., the fraction of overall P700 that cannot be oxidized in a given state (UNA). The small plots within each graph show the initial phase of each light curve. The rapid light curves were obtained after previous induction at moderate light; the duration of each interval with a given light intensity was 30 s (see ‘‘Materials and methods’’ for details). The average values ± standard errors from 8 to 10 plants are presented. NS non-stressed samples, ID initial drought, MD moderate drought, SD severe drought

nonphotochemical energy dissipation in PSI (Klughammer and Schreiber 1994). The trend of this parameter is very similar to (UNPQ). However, this parameter was sensitive to moderate (and insignificantly also to initial) drought, which was not seen in UNPQ. Furthermore, there was a fraction of P700 that was not reduced by saturation flash in a given state i.e., PSI acceptors (UNA) that are not fully oxidized (Fig. 4f). The acceptor-side limitation was generally low, becoming negligible at moderate-to-high light intensities, while donor-side limitation shows an exponential increase. In general, the major differences between observed drought stress levels were apparent in the initial part of the

light response for the assessed parameters. At low-tomoderate light intensities, the PSII quantum yield (UPSII) continuously decreased with increasing light. In contrast, the PSI quantum yield (UPSI) temporarily stopped decreasing, leading to a significant increase in the UPSI to UPSII ratio at low light intensities (Fig. 5). This phenomenon can be seen in all plants. However, in drought-stressed plants, it appears at lower light intensities (e.g., at 100 lmol photons m-2 s-1 in SD leaves, while only at 220 lmol photons m-2 s-1 in NS leaves). The initial trends of light dependencies of UPSI to UPSII ratio are very similar to those observed for NPQ (Fig. 5). As the

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Specifically, an increase in NPQ associated with a decrease in the UPSI to UPSII ratio (small graph in Supplementary Fig. 1a) as well as with a slowly decreasing PSI donor-side limitation (small graph in Supplementary Fig. 1b). Using the dark relaxation kinetics, we estimated the energydependent (pH-dependent) fraction of nonphotochemical quenching (qE). We found that qE values increased with severity of drought stress. However, the differences were not fully proportional to the differences in the NPQ values; this indicates that there can be also differences in slowrelaxing components of nonphotochemical quenching, which also contribute to values of parameter NPQ. Quantification of PSII electron transport to alternative electron sinks

Fig. 5 Light responses of a nonphotochemical quenching in PSII (NPQ) and b the ratio of the effective quantum yield of PSI and PSII (UPSI/UPSII). The small plots within each graph show the initial phase of each light curve. The rapid light curves were performed after previous induction at moderate light; duration of each interval with a given light intensity was 30 s (see ‘‘Materials and methods’’ for details). The average values ± standard errors from 8 to 10 plants are presented. NS non-stressed samples, ID initial drought, MD moderate drought, SD severe drought

increase of UPSI to UPSII ratio can be considered to be a symptom of enhancement of cyclic electron transport, this suggests that cyclic flow has a role in nonphotochemical energy dissipation (Heber and Walker 1992; Munekage et al. 2002; Livingston et al. 2010). To estimate correctly the rate of cyclic electron flow using PSII and PSI quantum yields, the values need to be corrected by the coefficient of absorbed energy distribution between PSI and PSII (Huang et al. 2010). Therefore, the decrease of UPSI to UPSII ratio that we observed does not necessarily mean that the rate of cyclic electron flow decreases, as the uncorrected ratio itself does not always represent the true estimate of cyclic electron flow, especially in high light intensities (see discussion below). Additional information about photoprotective responses can be obtained from dark recovery kinetics after reaching the steady state (Supplementary Fig. 1). Here, the trends of parameters were similar to previous observations; however, some surprising contradictory trends were also observed.

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To estimate the fraction of linear electron transport that is not used for carboxylation or oxygenation of RuBP, the difference between electron transport calculated using the quantum yields of PSII photochemistry from chlorophyll fluorescence measurements were compared, and the electron transport calculated from gas exchange measurements, both measured simultaneously (data presented in Fig. 2). Hence, the area between the curves represents the measure of electrons released by PSII that must be utilized by alternative electron sinks (Fig. 6). In this figure, we present the data assuming equal distribution of absorbed light between PSII and PSI. The data indicate that in the NS plants outside of photochemistry associated with photosynthesis, there is little additional electron sinks, while with increasing drought stress, there is significant increase in additional electron sinks.

Discussion Stomatal and nonstomatal effects at different level of drought stress A decrease in leaf water content due to drought stress mainly affects photosynthesis through stomatal closure, which causes a shortage in CO2 supply leading to a decrease in linear electron transport (Lawlor and Cornic 2002). Our results indicate that stomatal responses vary in degree, becoming more pronounced with increasing severity of stress. The effect of CO2 shortage on linear electron flow was partly ameliorated by an increase in photorespiration rate. Photorespiration may itself be part of the dissipatory mechanisms adopted by plants to mitigate oxidative damage resulting from insufficient utilization of electrons (Kozaki and Takeba 1996). However, there are also experimental results showing a negligible protective

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Fig. 6 The values of linear electron transport rate calculated from PSII effective quantum yields (JF = JPSII) calculated assuming equal distribution of electrons between PSI and PSII and the electron transport rate used either for carboxylation or oxygenation of RuBP calculated using data obtained by gas exchange measurements (JG). Both JF and JG were calculated using records of simultaneous measurements of gas exchange and chlorophyll fluorescence within

photosynthetic induction curve (see ‘‘Materials and methods’’ for details). The difference JF - JG represents a common way to estimate additional electron flux in other pathways besides photosynthesis and photorespiration; hence, the color fill between JF and JG illustrates the total sum of electrons used in alternative electron pathways. The average values from 8 to 10 plants are presented. NS non-stressed samples, ID initial drought, MD moderate drought, SD severe drought

effect of photorespiration in conditions of drought (Brestic et al. 1995). In addition to RuBP, there are several alternative acceptors of electrons from linear electron transport, such as oxygen in the Mehler reaction (Asada 1999) or nitrite (Eichelmann et al. 2011). Nevertheless, the most efficient way to prevent oxidative damage is through the precise control of electron transport rate associated with wellregulated dissipation of excessive light energy in the process of nonphotochemical quenching (reviewed in Mu¨ller et al. 2001). We observed direct correlations between leaf water status and CO2 assimilation, PSII electron transport rate, photorespiration rate, and nonphotochemical quenching (Fig. 3). However, all these parameters were correlated with stomatal conductance, which decreased with the increasing stress, resulting in a decrease in ci until severe water stress. This finding supports the view that the dominant limitation of photosynthesis is through stomatal effects (Cornic and Massacci 1996). Particularly interesting is the relationship between the steady-state values of NPQ and stomatal conductance. NPQ appears to remain constant until some threshold value of gs is reached, after which there is a large increase in NPQ. It should be noted that the measurements were done at high light intensity which would induce maximum capacity for NPQ. However, NPQ gradually increased with

decreasing RWC, suggesting it is more associated with leaf water status. The threshold level of low gs which induced a sharp rise in NPQ was *0.12 mmol m-2 s-1 when CO2 assimilation rate was reduced to about 50 % of its NS value (*12 lmol CO2 m-2 s-1); leaf RWC was below 70 % and the leaf water potential was below -1.5 MPa. Such values of RWC and WW are considered to be threshold levels at which the major nonstomatal limitation of photosynthesis occurs (Lawlor and Cornic 2002). The most frequently reported processes contributing to a decrease of photosynthesis not caused by stomata are impaired ATP synthesis (Tezara et al. 1999; Cruz et al. 2005), decrease in Rubisco activity (Holaday et al. 1992; Wingler et al. 1999), RuBP availability (Flexas et al. 2004), and impairment of photochemistry (Bjo¨rkman and Powles 1984). The typical symptom of nonstomatal limitation is the occurrence of a ci inflection point. This is clearly visible in our measurements (Fig. 3). Lal et al. (1996) attributed the occurrence of ci inflection points either to a decrease of mesophyll conductance or to stomatal patchiness. The interaction between stomatal conductance and leaf water status (which we documented by divergence of ci values at low gs) has not been commonly described in research articles (Flexas and Medrano 2002). Nevertheless, it supports the idea of the direct effect of low water potential on metabolic processes, independent of stomatal closure (Ortiz-Lopez et al. 1991).

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Alternative ways of PSII protection in drought stress Chloroplasts have evolved significant flexibility in mechanisms to cope with changes in demands for energy (Kramer and Evans 2011), which are activated under specific environmental and metabolic challenges. Their role is to prevent the production of deleterious reactive oxygen species and to optimize the ATP to NADPH ratio to cover the demand of metabolic processes, especially CO2 carboxylation (Kramer and Evans 2011). The chloroplast must balance the production and consumption of ATP and NADPH by augmenting production of intermediates or preventing the accumulation of excess intermediates. Mismatches in regulation will limit photosynthesis (Cruz et al. 2005; Kramer and Evans 2011). The excitation of photosystems must be well regulated, and this is achieved by several mechanisms. During drought stress and relatively high light conditions, substantial structural rearrangements for light protection are anticipated to be a major mechanism affecting photosystem stoichiometry (Bailey et al. 2001). Degradation of D1/D2 proteins of PSII has been shown to increase under water stress (Yuan et al. 2005). Moreover, Eichelmann and Laisk (2000) observed that stress conditions tend to decrease light harvesting antenna of PSII more than PSI. Such changes must be considered when converting photochemical efficiency of both photosystems estimated from chlorophyll fluorescence or P700 absorbance records into a rate of electron transport (Kramer and Evans 2011). Measurements of the quantum yield of PSII are frequently utilized to predict rates of linear electron transport. However, in most cases an equal distribution of light between photosystems is assumed (Genty et al. 1989; Krall and Edwards 1992). The PSI and PSII electron transport rates calculated assuming equal distribution of the absorbed light between PSI/PSII are shown in Fig. 7a, c. The difference between electron transport rates of PSI and PSII (Fig. 7e) represents an estimate of the electron transport rate of cyclic electron flow (Yamori et al. 2011). Hence, calculation assuming an equal distribution between photosystems would suggest that the highest cyclic electron transport will be in NS plants and that there will be a decrease or complete cessation of cyclic electron flow in drought-stressed plants at high light levels. Low or moderate drought stress effects on PSII effective quantum yields seemed to be insignificant, supporting the existence of an intact PSII electron transport in initial phases of drought stress (Brestic et al. 1995; Lawlor and Cornic 2002). The obtained results also support the claim of an essential role of cyclic electron transport for ATP formation (Heber and Walker 1992), which is needed more in plants running the carboxylation (NS) than in plants with closed stomata (discussed in Laisk et al. 2010). However,

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these results contradict reports showing high cyclic electron flow during drought stress (Golding and Johnson 2003; Huang et al. 2012) and an increase of cyclic electron flow at light intensities exceeding saturation level (Clarke and Johnson 2001; Brestic et al. 2008; Huang et al. 2012). A possible explanation of this contradiction is that an incorrect energy distribution factor was used in calculation, as we used a default value of 0.5, i.e., equal distribution of absorbed energy between PSII and PSI. Therefore, rather than using the default value of 0.5 assuming equal distribution, we adjusted the partitioning of absorbed energy between the photosystems with increasing drought to determine if the results would be more consistent with data and conclusions which could be drawn. We hypothesized that the rate of cyclic electron flow increases with increasing light and reaches a maximum, constant rate. We then recalculated the distribution factors for each treatment and the rates of electron transport (Fig. 7b, d, f). By decreasing the fraction of absorbance by PSII with increasing drought, the differences between PSII electron transport rates between the NS and drought stress treatments became greater. In contrast, the PSI electron transport appeared to be less affected by drought, resulting in the highest predicted cyclic electron flow in severely stressed plants (SD). Besides the fact that such results on cyclic electron flow are in concordance with many reports (Golding and Johnson 2003; Rumeau et al. 2007; Lehtima¨ki et al. 2010; Huang et al. 2012; etc.), this change in partitioning between photosystems is also supported by the fact that the new estimated values of PSII electron transport rate (JPSII) are much closer to the values of JG calculated using the gas exchange measurements (Supplementary Fig. 2); this issue will be discussed later. Therefore, we suggest that adjustment of light harvesting (what we see as an alteration of PSII/PSI energy distribution factor) can be considered as an important protective response, as illustrated by a decrease in the total pool of electrons, which are in excess of the needs of carboxylation or oxygenation (Supplementary Fig. 2). This can avoid the overreduction of PQ pool and, thus, it can prevent the PSII damage. The logical question is whether the proposed altered distribution of energy between photosystems is due to changes in protein complexes (i.e., state-transitions or longterm responses, etc.) or it is the spillover of energy. It is broadly accepted that the state transitions are associated with low light conditions (Dietzel et al. 2008). However, there are several recent studies suggesting that the activity of protein kinases in leaves (mainly STN7) can function even in high light conditions which could result in disconnection of LHCII from PSII, providing thus the protection against high light (Fristedt and Vener 2011). Specifically, in

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Fig. 7 Calculated values of electron transport rate through PSII and PSI as well as the difference between electron transport rates in wheat subjected to different level of drought stress. The values in graphs left (a, c, e) were calculated assuming equal distribution of absorbed energy between PSII and PSI. Graphs right (b, d, f) represent hypothetical values of electron fluxes calculated assuming unequal

distribution, where the ratios between PSII and PSI absorbance was recalculated to obtain stable difference (JPSI - JPSII) at high light intensities. The average values of PSII and PSI quantum yields were used for calculation, and the ratios of total PAR absorbed by PSII (aII) and PSI (aI) are presented in legend

monocots (such as wheat) exposed to drought or salt stress, it has been observed that the CP29 protein of LHCII is phosphorylated, with its subsequent lateral migration from grana stacks to stroma lamellae; in addition, the LHCII can be disconnected (Liu et al. 2009; Chen et al. 2013). Therefore, kinases may contribute to alteration of PSI/PSII stoichiometry in high light conditions. Another possible explanation for the observed decrease of JPSII–JPSI with increasing water stress is that there are some alternative auxiliary electron sinks on PSII acceptor

side. In this respect, Ivanov et al. (2012), in high light and low-temperature conditions, documented the important role of the plastid terminal oxidase (PTOX) as an alternative O2-dependent electron sink that can accept electrons directly from PSII (Cournac et al. 2000). The PTOX is involved also in chloroplast redox signaling pathways (Aluru and Rodermel 2004; McDonald et al. 2011) and in chlororespiration (Rumeau et al. 2007). The increase of PTOX was observed in conditions of stress, e.g., salinity (Stepien and Johnson 2009), high temperature (Diaz et al.

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2007) or low temperature (Savitch et al. 2010; Ivanov et al. 2012). PTOX activity avoids the overreduction of plastoquinone pool by its oxidation (Joe¨t et al. 2002; Josse et al. 2003). Moreover, Ivanov et al. (2012) also suggest the involvement of PTOX in modulation of cyclic electron flow and regulation of cyclic electron pathway in stress conditions. The activity of PTOX (or other auxiliary electron acceptor) as an electron sink as well as its regulatory function can well explain some of our observations; however, to explain fully the faster increase in JPSII compared to JPSI with increasing light (Fig. 7a, c, e), the rate of electron uptake in high light must be rather high (30 lmol e- m-2 s-1 or more). Our measurements, indeed, indicated in drought-stressed leaves such a high electron transport that needs to be utilized by alternative electron sinks (Fig. 6). On other hand, some studies on the function of PTOX as PSII electron acceptor challenges such high rates (discussed well in Stepien and Johnson 2009). However, we can not exclude that this, or some similar mechanism may be contributing as an important alternative sink in wheat when water supply is limiting. There are also other alternative sinks that can accept the electrons released by PSII. But it was shown that in C3 leaves the water–water cycle contributed \5 % to linear electron flow, even when CO2 fixation was inhibited (Ruuska et al. 2000; Clarke and Johnson 2001). Similarly, the published data on the contribution of nitrite reduction in high light (Eichelmann et al. 2011) does not explain the high alternative electron flow, especially in drought stress conditions with low nitrogen uptake. Even with a decrease in the fraction of energy absorbed by PSII (Fig. 7b, d, f), there is still an excess pool of electron that can be potentially harmful and need to be utilized by alternative electron sinks. Therefore we suggest that both adjustment of light harvesting and alternative electron flows (including auxiliary sinks) contribute to protection of PSII against photoinhibition. In addition to mechanistic issues, our analyses may have practical and technical implications. The commonly used approach of calculating electron transport rate by assuming equal distribution between PSI and PSII may overestimate rate of linear electron flow under drought stress conditions. This could result in overestimation of electron flow to alternative electron acceptors and underestimation of cyclic electron flow. Furthermore, the PSII electron transport is used in some photosynthetic models, e.g., for estimation of mesophyll conductance. The potential errors due to inaccurate estimates of PSII electron transport has been highlighted in several technical or methodical articles (e.g., Yin et al. 2009). Our analyses show how the use of a default value assuming equal distribution of energy absorption between the photosystems, versus a reduction in energy absorption by PSII with increasing drought can affect rates

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of photochemistry and the potential contribution of cyclic photophosphorylation. Likewise, activity of auxiliary electron acceptors directly from PSII, such as PTOX could affect the relative ratio of cyclic and linear electron flow. Activity of the cyclic electron flow in drought stress PSI cyclic electron flow has been proposed to function under drought stress, which could function to induce qE by increased proton motive force and NPQ (Figs. 4, 5, Supplementary Fig. 1). Both the down-regulation of linear electron flow and up-regulation of cyclic electron flow avoid the excessive reduction of PSI electron acceptors. Such a reduction can result in the production of reactive species and therefore photo-oxidation of protein components (Tikkanen et al. 2006). Our results indicate that even at PAR up to levels equivalent to full sunlight (2,000 lmol photons m-2 s-1; ten times higher than that used during growth of the plants) the acceptor side was not overreduced—as indicated by parameter UNA (Fig. 4f). In such extreme conditions, it seems improbable that a low acceptor-side reduction can be maintained without high cyclic electron flow in drought-stressed leaves, as shown in Fig. 7b, d, f. The high UPSI/UPSII ratio which occurs under lower light, particularly under SD, is consistent with function of cyclic electron flow (Fig. 4f). With increasing light, as soon as this ratio started to increase (indicating an increase of cyclic electron flow), the UNA decreased. This result is in agreement with the recent identification of the direct role of cyclic electron flow in photoprotection (Laisk et al. 2010), as it ameliorates the excess of electrons on PSI acceptor side. In addition to a decrease of UNA, the initiation of cyclic electron flow (suggested by an increase in UPSI/UPSII ratio) was also associated with (i) an increase of nonphotochemical quenching (NPQ); (ii) the accumulation of oxidized P700? (indicated by the parameter UND); and (iii) the increase of the donor-side limitation as a result of down-regulation of electron transport between PSI and PSII. This phenomenon is also related to cyclic electron flow building the proton motive force, resulting in lumen acidification and subsequent slowing of electron transfer at the cyt b6f complex (Kramer and Evans 2011). The drought-induced decrease of electron transport rate between PSII and PSI was found also in dark-adapted samples (Goltsev et al. 2012). Although the increase in donor-side limitation was observed in both control and drought-stressed samples, the relationship of PSII electron transport rate with P700?/P700tot (Fig. 8a) indicates that the demand for electrons at PSI (redox poise of PSI) resulted in reduced electron transport from PSII in droughtstressed plants. The trend of QA /QAtot versus JPSII gave similar results (not shown here). Hence, the restriction on

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Fig. 8 a Relationship between the fraction of overall P700 that is oxidized in a given state (P700?/P700T, UND) and the electron transport rate through PSII (JPSII). b Relationships between the fraction of overall P700 that is oxidized in a given state (P700?/ P700T, UND) and the rate of cyclic electron transport around PSI (estimated as JPSI - JPSII). The parameters were calculated from the data obtained by simultaneous measurements of chlorophyll fluorescence and P700 in light curve records (see ‘‘Materials and methods’’); the data with unequal absorbance ratios (aI, aII) are used as shown in Fig. 7. The average values ± standard errors from 8 to 10 plants are presented. NS non-stressed samples, ID initial drought, MD moderate drought, SD severe drought

donation of electrons from PSII to PSI was increasing with increasing stress. The rate of cyclic electron flow calculated as a difference between JPSI and JPSII (from the adjusted partitioning of absorbance between photosystems, Fig. 8b) linearly correlated with the accumulation of oxidized P700? until the maximum rate of cyclic electron flow was reached. The initial slope of the correlation was similar in all groups; this close relationship also supports the role of cyclic electron transport in PSII protection through a buildup of DpH in thylakoids. This process enhances protonation of the lumen thereby limiting electron transport and triggering NPQ (Rumeau et al. 2007). By regulating electron transport into PSI via the cytb6f complex, cyclic electron flow also minimizes the probability of the formation of reactive oxygen species (ROS) such as

superoxide on the acceptor side of PSI (Foyer and Noctor 2009) and induces the expression of stress response genes (Tikkanen et al. 2006). The PSII electron transport rate at high light intensities surprisingly increases more quickly than donor-side limitation (Fig. 8a). We hypothesize that this is not a true increase, but this may be a result of further changes of PSI/ PSII energy distribution due to disconnection of PSII antennae in high light or other processes leading to overestimation of PSII electron transport (and hence underestimation of the cyclic electron transport rate) at high light intensities. In conclusion, the results of this study indicate that photosynthesis in wheat is limited mainly by stomatal effects in conditions of low-to-moderate drought stress. Enhancement of the nonstomatal effects in severely stressed plants seemed to be induced by low leaf water potential rather than the very low stomatal conductance. Drought stress gradually decreased the PSII electron transport, but the capacity for nonphotochemical quenching increased only in severely stressed plants where photosynthetic rate was decreased by half or more. Our results indicate that in drought-stressed leaves the electron balance can be partially equalized through alteration of energy distribution between PSII and PSI; in this case, the calculated PSII electron transport can be overestimated, and the real pool of nonutilized electrons would decrease compared with the model assuming the same energy distribution. Thus, in future studies, it will be important to utilize methods to measure the distribution of the absorbed energy between the photosystems using the methods of Laisk and Loreto (1996), Cardol et al. (2008), or other reliable method. It is evident that in drought-stressed plants, there is a relatively large portion of electrons that need to be utilized by alternative electron sinks. In addition to water–water cycle and nitrite reduction, we suggest also activity of auxiliary electron sinks on acceptor side of PSII (such as PTOX), which was previously shown to play a role as a safety valve protecting the photosynthetic apparatus. This would prevent overreduction of the PQ pool and protect the PSII against oxidative damage. In parallel, we propose that, with drought stress, the fraction of the absorbed light by PSII is decreased, with an increase in the ratio of the yields of PSI/ PSII, which would coincide with an increase of nonphotochemical quenching, an increase in the donor-side limitation and a decrease in the acceptor-side limitation of PSI. In summary, our results demonstrate that in drought stress conditions, there is a complex of mutually interconnected and precisely regulated photoprotective responses. Acknowledgments The authors thank Dr Richard J. Ladle (School of Geography and the Environment, Oxford University, UK, and Institute of Biological and Health Sciences, Federal University of

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Photosynth Res Alagoas, Prac¸a Afraˆnio Jorge, s/n, Prado, Maceio´, AL, Brazil) for reviewing and improving the English of the manuscript. The research described here has been supported by grant APVV-0197-10 and APVV-0661-10. This study was also supported by grants from the Russian Foundation for Basic Research and Molecular and Cell Biology Programs of the Russian Academy of Sciences to SIA.

References Aluru MR, Rodermel SR (2004) Control of chloroplast redox by the IMMUTANS terminal oxidase. Physiol Plant 120:4–11 Asada K (1999) The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50:601–639 Bailey S, Walters RG, Jansson S, Horton P (2001) Acclimation of Arabidopsis thaliana to the light environment: the existence of separate low light and high light responses. Planta 213:794–801 Balaguer L, Punaire FI, Martı´nez-Ferri E, Armas C, Valladares F, Manrique E (2002) Ecophysiological significance of chlorophyll loss and reduced photochemical efficiency under extreme aridity in Stipa tenacissima L. Plant Soil 240:343–352 Bilger W, Bjo¨rkman O (1990) Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynth Res 25:173–185 Bjo¨rkman O, Demmig B (1987) Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plant of diverse origins. Planta 170:489–504 Bjo¨rkman O, Powles SB (1984) Inhibition of photosynthetic reactions under water stress: interaction with light level. Planta 161:490–504 Bonjean AP, Angus WJ (2001) The world wheat book—a history of wheat breeding. Lavoisier Publishing, Paris Brestic M, Cornic G, Fryer MJ, Baker NR (1995) Does photorespiration protect the photosynthetic apparatus in French bean leaves from photoinhibition during drought stress? Planta 196:450–457 Brestic M, Zivcak M, Olsovska K, Repkova J (2008) Functional study of PS II and PSI energy use and dissipation mechanisms in barley wild type and chlorina mutants under high light conditions. In: Allen JF, Gantt E, Goldbeck JH, Osmond B (eds) Photosynthesis. Energy from the sun: 14th International congress on photosynthesis, Springer, Dordrecht, pp 1407–1411 Brestic M, Zivcak M, Kalaji HM, Allakhverdiev SI, Carpentier R (2012) Photosystem II thermostability in situ: environmentally induced acclimation and genotype-specific reactions in Triticum aestivum L. Plant Physiol Biochem 57:93–105 Buermann W, Lintner BR, Koven CD, Angert A, Pinzon JE, Tucker CJ et al (2007) The changing carbon cycle at Mauna Loa Observatory. Proc Natl Acad Sci USA 104:4249–4254 Cardol P, Bailleul B, Rappaport F, Derelle E, Be´al D, Breyton C, Bailey S, Wollman FA, Grossman A, Moreau H, Finazzi G (2008) An original adaptation of photosynthesis in the marine green alga Ostreococcus. Proc Natl Acad Sci USA 105:7881–7886 Chaves MM (1991) Effects of water deficits on carbon assimilation. J Exp Bot 42:1–16 Chaves MM, Oliveira MM (2004) Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture. J Exp Bot 55:2365–2384 Chaves MM, Pereira JS, Maroco J, Rodrigues ML, Ricardo CP, Osorio ML, Carvalho I, Faria T, Pinheiro C (2002) How plants cope with water stress in the field. Photosynthesis and growth. Ann Bot 89:907–916 Chaves MM, Flexas J, Pinheiro C (2009) Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann Bot 103:551–560

123

Chen YE, Zhang ZI, Zhang HY, Zeng YX, Yuan S (2013) The significance of CP29 reversible phosphorylation in thylakoids of higher plants under environmental stresses. J Exp Bot 64:1167–1178 Ciais P, Reichstein M, Viovy N, Granier A, Ogee J, Allard V et al (2005) Europe-wide reduction in primary productivity caused by the heat and drought in 2003. Nature 437:529–533 Clarke JE, Johnson GN (2001) In vivo temperature dependence of cyclic and pseudocyclic electron transport in barley. Planta 212:808–816 Cornic G, Massacci A (1996) Leaf photosynthesis under drought stress. In: Baker NR (ed) Photosynthesis and the environment. Kluwer Academic Publishers, Dordrecht Cournac L, Redding K, Ravenel J, Rumeau D, Josse E-M, Kuntz M, Peltier G (2000) Electron flow between photosystem II and oxygen in chloroplasts of photosystem I-deficient algae is mediated by a quinol oxidase involved in chlororespiration. J Biol Chem 275:17256–17262 Cruz JA, Avenson TJ, Kanazawa A, Takizawa K, Edwards GE, Kramer DM (2005) Plasticity in light reactions of photosynthesis for energy production and photoprotection. J Exp Bot 56:395–406 Demmig-Adams B (1990) Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. Biochim Biophys Acta 1020:1–24 Diaz M, De Haro V, Munoz R, Quiles MJ (2007) Chlororespiration is involved in the adaptation of Brassica plants to heat and high light intensity. Plant Cell Environ 30:1578–1585 Dietzel L, Bra¨utigam K, Pfannschmidt T (2008) Photosynthetic acclimation: state transitions and adjustment of photosystem stoichiometry–functional relationships between short-term and long-term light quality acclimation in plants. FEBS J 275:1080– 1088 Eichelmann H, Laisk A (2000) Cooperation of photosystems II and I in leaves as analysed by simultaneous measurements of chlorophyll fluorescence and transmittance at 800 nm. Plant Cell Physiol 41:138–147 Eichelmann H, Oja V, Peterson RB, Laisk A (2011) The rate of nitrite reduction in leaves as indicated by O2 and CO2 exchange during photosynthesis. J Exp Bot 62:2205–2215 Engelbrecht BMJ, Comita LS, Condit R, Kursar TA, Tyree MT, Turner BL et al (2007) Drought sensitivity shapes species distribution patterns in tropical forests. Nature 447:80–82 Epron D, Godard G, Cornic G, Genty B (1995) Limitation of net CO2 assimilation rate by internal resistances to CO2 transfer in the leaves of two tree species (Fagus sylvatica and Castanea sativa Mill.). Plant Cell Environ 18:43–51 Flexas J, Medrano H (2002) Energy dissipation in C3 plants under drought. Funct Plant Biol 29:1209–1215 Flexas J, Bota J, Loreto F, Cornic G, Sharkey TD (2004) Diffusive and metabolic limitations to photosynthesis under drought and salinity in C3 plants. Plant Biol 6:269–279 Flexas J, Barbour MM, Brendel O, Cabrera HM, Carriquı´ M, Dı´azEspejo A et al (2012) Mesophyll diffusion conductance to CO2: an unappreciated central player in photosynthesis. Plant Sci 193–194:70–84 Foyer CH, Noctor G (2009) Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications. Antioxid Redox Signal 11:861–905 Fristedt R, Vener AV (2011) High light induced disassembly of photosystem II supercomplexes in Arabidopsis requires STN7dependent phosphorylation of CP29. PLoS ONE 6:e24565 Galmes J, Medrano H, Jaume F (2006) Acclimation of Rubisco specificity factor to drought in tobacco: discrepancies between in vitro and in vivo estimations. J Exp Bot 57:3659–3667 Genty B, Briantais JM, Baker NR (1989) The relationship between quantum yield of photosynthetic electron transport and

Photosynth Res quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87–92 Golding AJ, Johnson GN (2003) Down-regulation of linear and activation of cyclic electron transport during drought. Planta 218:107–114 Golding AJ, Finazzi G, Johnson GN (2004) Reduction of the thylakoid electron transport chain by stromal reductants: evidence for activation of cyclic electron transport upon dark adaptation or under drought. Planta 202:356–363 Goltsev V, Zaharieva I, Chernev P, Kouzmanova M, Kalaji MH, Yordanov I, Krasteva V, Alexandrov V, Stefanov D, Allakhverdiev SI, Strasser RJ (2012) Drought-induced modifications of photosynthetic electron transport in intact leaves: analysis and use of neural networks as a tool for a rapid non-invasive estimation. Biochim Biophys Acta 1817:1490–1498 Gray GR, Savitch LV, Ivanov AC, Huner NPA (1996) Photosystem II excitation pressure and development of resistance to photoinhibition. 2. Adjustment of photosynthetic capacity in winter wheat and winter rye. Plant Physiol 110:61–71 Harley PC, Loreto F, Di Marco G, Sharkey TD (1992) Theoretical considerations when estimating the mesophyll conductance to CO2 flux by analysis of the response of photosynthesis to CO2. Plant Physiol 98:1429–1436 Ha¨rtel H, Lokstein H (1995) Relationship between quenching of maximum and dark-level chlorophyll fluorescence in vivo: dependence on photosystem II antenna size. Biochim Biophys Acta 1228:91–94 Heber U, Walker D (1992) Concerning a dual function of coupled cyclic electron-transport in leaves. Plant Physiol 100:1621–1626 Holaday AS, Martindale W, Alred R, Brooks AL, Leegood RC (1992) Changes in activities of enzymes of carbon metabolism in leaves during exposure of plants to low temperature. Plant Physiol 98:1105–1114 Huang W, Zhang SB, Cao KF (2010) Stimulation of cyclic electron flow during recovery after chilling-induced photoinhibition of PSII. Plant Cell Physiol 51:1922–1928 Huang W, Yang SJ, Zhang SB, Zhang JL, Cao KF (2012) Cyclic electron flow plays an important role in photoprotection for the resurrection plant Paraboea rufescens under drought stress. Planta 235:819–828 ¨ quist G, Huner NPA (2008) Ivanov AG, Sane PV, Hurry V, O Photosystem II reaction center quenching: mechanisms and physiological role. Photosynth Res 98:565–574 Ivanov AG, Rosso D, Savitch LV, Stachula P, Rosembert M, Oquist G et al (2012) Implications of alternative electron sinks in increased resistance of PSII and PSI photochemistry to high light stress in cold-acclimated Arabidopsis thaliana. Photosynth Res 113:191–206 Joe¨t T, Genty B, Josse E-M, Kuntz M, Cournac L, Peltier G (2002) Involvement of a plastid terminal oxidase in plastoquinone oxidation as evidenced by expression of the Arabidopsis thaliana enzyme in tobacco. J Biol Chem 277:31623–31630 Johnson GN (2005) Cyclic electron transport in C3 plants: fact or artefact? J Exp Bot 56:407–416 Johnson GN (2011) Physiology of PSI cyclic electron transport in higher plants. Biochim Biophys Acta 1807:384–389 Josse E-M, Alcaraz J-P, Laboure´ A-M, Kuntz M (2003) In vitro characterization of a plastid terminal oxidase (PTOX). Eur J Biochem 270:3787–3794 Kitajima M, Butler WL (1975) Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochim Biophys Acta 376:105–115 Klughammer C, Schreiber U (1994) An improved method, using saturating light pulses, for the determination of photosystem I quantum yield via P700?-absorbance changes at 830 nm. Planta 192:261–268

Kohzuma K, Cruz JA, Akashi K, Hoshiyasu S, Munekage YN, Yokota A, Kramer DM (2009) The long-term responses of the photosynthetic proton circuit to drought. Plant Cell Environ 32:209–219 Kozaki A, Takeba G (1996) Photorespiration protects C3 plants from photooxidation. Nature 384:557–560 Krall JP, Edwards GE (1992) Relationship between photosystem II activity and CO2 fixation in leaves. Physiol Plant 86:180–187 Kramer DM, Evans JR (2011) The importance of energy balance in improving photosynthetic productivity. Plant Physiol 155:70–78 Kramer DM, Johnson G, Kiirats O, Edwards GE (2004) New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynth Res 79:209–218 Krause GH, Weis E (1991) Chlorophyll fluorescence and photosynthesis: the basics. Ann Rev Plant Physiol Plant Mol Biol 42:313–349 Laisk A, Loreto F (1996) Determining photosynthetic parameters from leaf CO2 exchange and chlorophyll fluorescence: ribulose1,5-bisphosphate carboxylase oxygenase specificity factor, dark respiration in the light, excitation distribution between photosystems, alternative electron transport rate, and mesophyll diffusion resistance. Plant Physiol 110:903–912 Laisk A, Eichelmann H, Oja V, Talts E, Scheibe R (2007) Rates and roles of cyclic and alternative electron flow in potato leaves. Plant Cell Physiol 48:1575–1588 Laisk A, Talts E, Oja V, Eichelmann H, Peterson R (2010) Fast cyclic electron transport around photosystem I in leaves under far-red light: a proton-uncoupled pathway? Photosynth Res 103:79–95 Lal A, Ku MSB, Edwards GE (1996) Analysis of inhibition of photosynthesis due to water-stress in the C3 species Hordeum vulgare and Vicia faba—electron-transport, CO2 fixation and carboxylation capacity. Photosynth Res 49:57–69 Lawlor DW, Cornic G (2002) Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants. Plant Cell Environ 25:275–294 Lehtima¨ki N, Lintala M, Allahverdiyeva Y, Aro EM, Mulo P (2010) Drought stress-induced upregulation of components involved in ferredoxin-dependent cyclic electron transfer. J Plant Physiol 167:1018–1022 Li XP, Bjo¨rkman O, Shih C, Grossman AR, Rosenquist M, Jansson S, Niyogi KK (2000) A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 403:391– 395 Liu WJ, Chen YE, Tian WJ, Du JB, Zhang ZW, Xu F, Zhang F, Yuan S, Lin HH (2009) Dephosphorylation of photosystem II proteins and phosphorylation of CP29 in barley photosynthetic membranes as a response to water stress. Biochim Biophys Acta 1787:1238–1245 Livingston AK, Cruz JA, Kohzuma K, Dhingra A, Kramer DM (2010) An Arabidopsis mutant with high cyclic electron flow around photosystem I (hcef) involving the NADPH dehydrogenase complex. Plant Cell 22:221–233 Maroco JP, Rodrigues ML, Lopes C, Chaves MM (2002) Limitations to leaf photosynthesis in grapevine under drought: metabolic and modelling approaches. Funct Plant Biol 29:1–9 Mathur S, Allakhverdiev SI, Jajoo A (2011) Analysis of high temperature stress on the dynamics of antenna size and reducing side heterogeneity of photosystem II in wheat leaves (Triticum aestivum). Biochim Biophys Acta Bioenerg 1807:22–29 McDonald AE, Ivanov AG, Bode R, Maxwell DP, Rodermel SR, Hu¨ner NPA (2011) Flexibility in photosynthetic electron transport: the physiological role of plastoquinol terminal oxidase (PTOX). Biochim Biophys Acta 1807:954–967 Mehta P, Allakhverdiev SI, Jajoo A (2010) Characterization of photosystem II heterogeneity in response to high salt stress in wheat leaves (Triticum aestivum). Photosynth Res 105:249–255

123

Photosynth Res Miyake C (2010) Alternative electron flows (water–water cycle and cyclic electron flow around PSI) in photosynthesis: molecular mechanisms and physiological functions. Plant Cell Physiol 51:1951–1963 Muller P, Li XP, Niyogi KK (2001) Non-photochemical quenching. A response to excess light energy. Plant Physiol 125:1558–1566 Munekage Y, Hojo M, Meurer J, Endo T, Tasaka M, Shikanai T (2002) PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis. Cell 110:361–371 Munekage Y, Hashimoto M, Miyake C, Tomizawa KI, Endo T, Tasaka M, Shikanai T (2004) Cyclic electron flow around photosystem I is essential for photosynthesis. Nature 429:579– 582 Niyogi KK, Li X-P, Rosenberg V, Jung H-S (2005) Is PsbS the site of non-photochemical quenching in photosynthesis? J Exp Bot 56:375–382 Ogren WL (1984) Photorespiration: pathways, regulation, and modification. Annu Rev Plant Physiol 35:415–442 Ort DR (2001) When there is too much light. Plant Physiol 125:29–32 Ortiz-Lopez A, Ort DR, Boyer JS (1991) Photophosphorylation in attached leaves of Helianthus annuus at low water potentials. Plant Physiol 96:1018–1025 Parry M, Andraloje PJ, Khan S, Lea PJ, Keys A (2002) Rubisco activity: effect of drought stress. Ann Bot 89:833–839 Passioura J (2007) The drought environment: physical, biological and agricultural perspectives. J Exp Bot 58:113–117 Pfundel EE (1998) Estimating the contribution of photosystem I to total leaf chlorophyll fluorescence. Photosynth Res 56:185–195 Rumeau D, Peltier G, Cournac L (2007) Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response. Plant Cell Environ 30:1041–1051 Ruuska SA, Badger MR, Andrews TJ, von Caemmerer S (2000) Photosynthetic electron sinks in transgenic tobacco with reduced amounts of Rubisco: little evidence for significant Mehler reaction. J Exp Bot 51:357–368 ¨ quist G, Huner NPA Savitch LV, Ivanov AG, Krol M, Sprott DP, O (2010) Regulation of energy partitioning and alternative electron transport pathways during cold acclimation of lodgepole pine is oxygen dependent. Plant Cell Physiol 51:1555–1570 Schreiber U (1986) Detection of rapid induction kinetics with a new type of high frequency modulated chlorophyll fluorescence. Photosynth Res 9:261–272 Schreiber U, Bilger W, Klughammer C, Neubauer C (1988) Application of the PAM fluorometer in stress detection. In: Lichtenthaler HK (ed) Applications of chlorophyll fluorescence. Kluwer, Dordrecht, pp 151–155 Schreiber U, Hormann H, Neubauer C, Klughammer C (1995) Assessment of photosystem II photochemical quantum yield by chlorophyll fluorescence quenching analysis. Aust J Plant Physiol 22:209–220 Stepien P, Johnson GN (2009) Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis thaliana and

123

the halophyte Tellungiella halophila. Role of the plastid terminal oxidase as an alternative electron sink. Plant Physiol 149:1154– 1165 Tezara W, Mitchell VJ, Driscoll SD, Lawlor DW (1999) Water stress inhibits plant photosynthesis by decreasing coupling factor and ATP. Nature 401:914–917 Tikkanen M, Piippo M, Suorsa M, Sirpio S, Mulo P, Vainonen J, Vener AV, Allahverdiyeva Y, Aro EM (2006) State transitions revisited—a buffering system for dynamic low light acclimation of Arabidopsis. Plant Mol Biol 62:779–79371 Turner NC (1981) Techniques and experimental approaches for the measurement of plant water status. Plant Soil 58:339–366 Valentini R, Epron D, De Angelis P, Matteucci G, Dreyer E (1995) In situ estimation of net CO2 assimilation, photosynthetic electron flow and photorespiration in Turkey oak (Q. cerris L.) leaves: diurnal cycles under different levels of water supply. Plant Cell Environ 18:631–640 Van den Berg M, Driessen PM, Rabbinge R (2002) Water uptake in crop growth models for land use systems analysis: II. Comparison of three simple approaches. Ecol Model 148:233–250 Vassileva V, Demirevska K, Simova-Stoilova L, Petrova T, Tsenov N, Feller U (2012) Long-term field drought affects leaf protein pattern and chloroplast ultrastructure of winter wheat in a cultivar-specific manner. J Agron Crop Sci 198:104–117 Walters R, Horton GP (1991) Resolution of components of nonphotochemical chlorophyll fluorescence quenching in barley leaves. Photosynth Res 27:121–133 Wingler A, Quick WP, Bungard RA, Bailey KJ, Lea PJ, Leegood RC (1999) The role of photorespiration during drought stress: an analysis utilizing barley mutants with reduced activities of photorespiratory enzymes. Plant Cell Environ 22:361–373 Yamori W, Sakata N, Suzuki Y, Shikanai T, Makino A (2011) Cyclic electron flow around photosystem I via chloroplast NAD(P)H dehydrogenase (NDH) complex performs a significant physiological role during photosynthesis and plant growth at low temperature in rice. Plant J 68:966–976 Yin X, Struik PC, Romero P, Harbinson J, Evers JB, Van der Putten PEL, Vos J (2009) Using combined measurements of gas exchange and chlorophyll fluorescence to estimate parameters of a biochemical C3 photosynthesis model: a critical appraisal and a new integrated approach applied to leaves in a wheat (Triticum aestivum) canopy. Plant Cell Environ 32:448–464 Yuan S, Liu WJ, Zhang NH, Wang MB, Liang HG, Lin HH (2005) Effects of water stress on major PSII gene expression and protein metabolism in barley leaves. Physiol Plant 125:464–473 Zadraznik T, Hollung K, Egge-Jacobsen W, Meglic V, Sustar-Vozlic J (2013) Differential proteomic analysis of drought stress response in leaves of common bean (Phaseolus vulgaris L.). J Proteomics 78:254–272 Zivcak M, Brestic M, Olsovska K, Slamka P (2008) Performance index as a sensitive indicator of water stress in Triticum aestivum. Plant Soil Environ 54:133–139

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