Glycolate Induces Redox Tuning Of Photosystem II in Vivo: Study of a Photorespiration Mutant

Glycolate accumulated in a photorespiration mutant replaces the bicarbonate on the acceptor-side iron of PSII, lowering the yield of singlet oxygen by redox tuning the primary quinone.

Abstract

Bicarbonate removal from the nonheme iron at the acceptor side of photosystem II (PSII) was shown recently to shift the midpoint potential of the primary quinone acceptor Q_A to a more positive potential and lowers the yield of singlet oxygen (¹O₂) production. The presence of Q_A⁻ results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against ¹O₂ in vivo under photorespiratory conditions. Here, we compared the properties of Q_A in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant deficient in peroxisomal HYDROXYPYRUVATE REDUCTASE1 (hpr1-1), which accumulates glycolate in leaves, with the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between Q_A and Q_B in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of Q_A to a more positive value. The yield of ¹O₂ production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of Q_A/Q_A⁻. In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities, consistent with diminished electron transfer out of PSII. This study indicates that replacement of bicarbonate at the nonheme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the nonheme iron by glycolate may represent a regulatory mechanism that protects PSII against photooxidative stress under low-CO₂ conditions.

PSII, the water/plastoquinone photooxidoreductase, is at the heart of the major energy cycle that powers the biosphere. Chlorophyll-based photochemistry drives charge separation followed by electron transfer reactions that result in the reduction of quinones on one side of the thylakoid membrane and the oxidation of water on the other. The electron acceptor side of PSII contains a nonheme ferrous iron that is flanked by the two quinones, Q_A and Q_B (Rutherford, 1987; Michel and Deisenhofer, 1988; Cardona et al., 2012; Müh et al., 2012). This structure is homologous to that of the purple bacterial reaction center (Deisenhofer et al., 1985). Like the situation in the purple bacterial reaction center, in PSII the nonheme iron is coordinated by four His residues, two from D1 and two from D2, and a bidentate carboxylic acid, a Glu in purple bacteria (Deisenhofer et al., 1985), and a bicarbonate (HCO₃⁻) in PSII (Rutherford, 1987; Michel and Deisenhofer, 1988; Hienerwadel and Berthomieu, 1995; Umena et al., 2011). The bicarbonate binding is responsible for important differences between the two types of reaction centers (Rutherford, 1987; Cardona et al., 2012; Müh et al., 2012; Brinkert et al., 2016). Bicarbonate is thought to play a role in the Q_B protonation pathway (Blubaugh and Govindjee, 1988; van Rensen et al., 1988; Sedoud et al., 2011). It has been shown that bicarbonate can be displaced by other ligands, some of which have physiological relevance and, thus, may play a regulatory role in the activity of PSII (Roach et al., 2013).

The redox potential of the first quinone acceptor, Q_A, of PSII has been the subject of research for decades, with the problem once thought solved (Johnson et al., 1995; Krieger et al., 1995) but becoming ambiguous again in recent years (Shibamoto et al., 2009; Ido et al., 2011). The ambiguity on the midpoint potential (E_m) of Q_A/Q_A⁻ was resolved recently by Brinkert et al. (2016), who demonstrated that two different values of E_m (Q_A/Q_A⁻) exist in PSII with an active water-splitting complex depending on the presence of bicarbonate bound to the nonheme iron. The values of E_m (Q_A/Q_A⁻) have been determined to be −145 mV in the presence of bicarbonate and −70 mV when bicarbonate had been released from the nonheme iron. The increase of the E_m (Q_A/Q_A⁻) in the absence of bicarbonate decreased the generation of singlet oxygen (¹O₂) by favoring a direct charge recombination pathway (Johnson et al., 1995; Brinkert et al., 2016). This increase of the E_m (Q_A/Q_A⁻) may be physiologically important to protect PSII against ¹O₂-induced photodamage in conditions where a plant is exposed to excess light and especially under drought conditions, when CO₂ inside the chloroplasts becomes more limiting. Consequently, such low CO₂ concentrations considerably promote the oxygenation of ribulose-1,5-bisphosphate by Rubisco and, thus, further elevate the production of 2-phosphoglycolate and glycolate inside the chloroplast (Zelitch and Barber, 1960; Bowes et al., 1971; Lorimer and Andrews, 1981; Wingler et al., 2000; for review, see Tolbert, 1997; Obata et al., 2016).

In vitro HCO₃⁻ at the nonheme iron can be replaced by small carboxylic acids like glycolate, glyoxylate, and formate (Wydrzynski and Govindjee, 1975; Vermaas and Rutherford, 1984; Petrouleas et al., 1994; Hienerwadel and Berthomieu, 1995). In vivo, acetate affects the energetics of PSII in Chlamydomonas reinhardtii when grown under mixotrophic conditions in acetate-containing medium (Roach et al., 2013). Experimental evidence for bicarbonate replacement under physiologically relevant conditions is lacking so far. High acetate concentrations inside the chloroplast do not occur naturally in photoautotrophically grown algae or in higher plants. By contrast, it is known that glycolate accumulates in some mutants of the photorespiratory pathway. Timm et al. (2008, 2013) reported an Arabidopsis (Arabidopsis thaliana) T-DNA insertional knockout mutant of NADH-dependent HYDROXYPYRUVATE REDUCTASE1 (HPR1) that is unable to catalyze the reduction of hydroxypyruvate to glycerate in the peroxisomes. However, due to the presence of a cytosolic bypass using NADPH-dependent HPR2 (Timm et al., 2008), hpr1-1 is still able to complete photorespiratory carbon recycling but with a slightly lower efficiency. Accordingly, several upstream metabolites, particularly glycolate, accumulate in this mutant to moderate levels (Timm et al., 2013).

While glycolate concentrations in specific cellular compartments of the mutant were not determined in these earlier studies, features of a double mutant line deficient in both known glycolate transporters and other data indicate that glycolate and glycolic acid can diffuse through the chloroplast envelope to some extent (Takabe and Akazawa, 1981; Pick et al., 2013; Walker et al., 2016; South et al., 2017). However, elevated leaf contents indicate correspondingly higher glycolate contents mainly in the stroma of hpr1-1 chloroplasts. Compared with the more strongly impaired double transporter or other photorespiratory mutants (Timm et al., 2012), hpr1-1 shows only minor metabolic and physiological alterations and, therefore, may serve as a suitable model to test whether the accumulation of glycolate affects the E_m (Q_A/Q_A⁻) in vivo. Since such mutants present a clear phenotype, it seems likely that the study of such photorespiratory mutants would be advantageous compared with drought-stressed wild-type plants, for which reproducible states are more difficult to achieve due to the numerous other factors (light stress, temperature, wilting, and senescence induction) that are liable to vary with time during the generation of drought stress.

In this work, we have addressed the question of the physiological relevance of bicarbonate replacement by glycolate in vivo in Arabidopsis. We used the hpr1-1 mutant to test whether electron transport at the acceptor side of PSII is modified and whether the yield of ¹O₂ generation is affected, as predicted from the recent model of bicarbonate-mediated regulation of PSII electron transfer (Brinkert et al., 2016).

RESULTS

The accumulation of glycolate in leaves of the hpr1-1 mutant depended on the developmental stage of the plants (Fig. 1). Three-week-old mutant plants showed a significant but relatively low increase in glycolate levels. In the next weeks of growth, the accumulation of glycolate increased and reached a maximum level (about 12-fold compared with the wild type) a few days before flowering started. Five-week-old plants were chosen to investigate the effect of glycolate on the PSII electron acceptor side. The quantum yield of PSII of dark-adapted plants was not affected in hpr1-1 (F_v/F_m [wild type] = 0.757 ± 0.009 and F_v/F_m [hpr1-1] = 0.759 ± 0.010; n = 11), showing that the photosynthetic apparatus was not damaged even though the leaves were significantly smaller than those of the wild type (Supplemental Fig. S1).

Figure 1.

Alteration in leaf glycolate content in the hpr1-1 mutant compared with wild-type Columbia-0 (Col-0) during five stages of plant development. Arabidopsis plants were grown in normal air, and leaves of at least four individual plants per genotype were harvested in the middle of the photoperiod after 3, 4, 5, 6, and 7 weeks of germination. Mutant-to-wild-type ratios ± sd of glycolate contents (relative amounts, arbitrary units mg⁻¹ fresh weight) are shown, with the mean wild-type value at 3 weeks post germination arbitrarily set to 1 (wild type, solid line; hpr1-1, dashed line). Gray shading indicates the developmental stage of the onset of flowering. Asterisks show significant changes compared with the corresponding wild-type time point, and plus signs show them compared with the wild type at 3 weeks post germination according to Student’s t test (P < 0.05).

Alterations of the PSII acceptor side in the hpr1-1 mutant compared with the wild type were followed by chlorophyll fluorescence measurements. Figure 2 shows that the fluorescence induction was delayed in hpr1-1 leaves compared with the wild type. Modification of the PSII acceptor side by the binding of glycolate to the nonheme iron is expected to slow the electron transfer between Q_A and Q_B (Petrouleas et al., 1994). The fluorescence decay also is slowed in the mutant, both in the presence and absence of the herbicide dichlorophenyl dimethylurea (DCMU), an inhibitor that binds to the Q_B-binding site (Fig. 3; Table I). In the presence of DCMU, the fluorescence decay is attributed to the charge recombination reaction S₂Q_A⁻, with S₂ being an oxidation state of the manganese cluster of the water-splitting complex. The decay was slower in the mutant compared with the wild type, indicating a stabilization of the charge pair and a modification of the midpoint potential of the redox couple Q_A/Q_A⁻. In the absence of DCMU, the decay was fitted with a sum of two exponential decay functions.

Figure 2.

Chlorophyll fluorescence induction curves. Detached leaves of 5-week-old Arabidopsis plants were dark incubated for 15 min at 5°C. Variable fluorescence was obtained by illuminating the leaf with actinic red light (37 µmol quanta m⁻² s⁻¹). The arrow indicates the onset of actinic light. Representative curves for the wild type (wt; black) and hpr1-1 (red) are shown. The variable fluorescence (F_m − F₀) was normalized (F₀ wild type, 0.176; F₀ hpr1-1, 0.185; F_m wild type, 0.5; F_m hpr1-1, 0.465). r.u., Relative units.

Figure 3.

Chlorophyll fluorescence decay. Detached leaves of 5-week-old Arabidopsis plants were dark incubated for 15 min at 5°C. Variable fluorescence was induced by a saturating single-turnover flash. At top, leaves were infiltrated with 20 µm DCMU prior to the measurements. At bottom, no DCMU was added. Representative curves for the wild type (wt; black) and hpr1-1 (red) are shown. The variable fluorescence (F_m − F₀) was normalized. Top F_v (at 0.8 s) wild type, 0.0822; F_v (at 0.8 s) hpr1-1, 0.0441; F_v (at 12 s) wild type, 0.0441; F_v (at 12 s) hpr1-1, 0.0363; bottom F_v (at 0.8 ms) wild type, 0.4558; F_v (at 0.8 ms) hpr1-1, 0.4526; F_v (at 16 ms) wild type, 0.1701; F_v (at 16 ms) hpr1-1, 0.2108. r.u., Relative units.

Table I. Fitted parameters for fluorescence decay in the absence and presence of DCMU (Fig. 3).

Lifetimes (t1 and t2) were obtained after fitting a biexponential decay function to the fluorescence yield decay of leaves of 5-week-old wild-type and hpr1-1 mutant plants. In the presence of DCMU, the decay was fitted with a monoexponential decay function. Measurements were performed on detached leaves at 5°C. If indicated, leaves were vacuum infiltrated with 20 µm DCMU prior to the measurements. Leaves from different plants were measured (means ± sd; n = 4).

Plant	Fluorescence Decay
	+DCMU	No Addition
		t1	t2
Wild type	0.584 ± 0.052 s	0.470 ± 0.021 ms	5.13 ± 0.54 ms
hpr1-1	0.730 ± 0.004 s	0.748 ± 0.096 ms	13.33 ± 1.87 ms

As shown in Table I, both phases were slower in the mutant, indicating decreased electron transfer between Q_A and Q_B in hpr1-1. According to de Wijn and van Gorkom (2001), different phases of Q_A⁻ reoxidation are assigned to electron transfer to Q_B (or Q_B⁻), which is bound to the Q_B site at the time of the flash (fast phase), and to electron transfer to Q_B but rate limited by the exchange of a reduced quinone (Q_BH₂) in the Q_B site for a PQ from the pool (slow phase). Three-week-old hpr1-1 plants, which accumulated much less glycolate compared with 5-week-old plants (Fig. 1), showed a small deceleration of fluorescence induction and no difference in fluorescence decay kinetics compared with wild-type plants of the same age (Supplemental Fig. S2). Therefore, we attribute the changes in the fluorescence kinetics of 5-week-old hpr1-1 plants to a specific effect of glycolate accumulation; thus, a pleiotropic effect of the mutation on photosynthetic electron transport properties likely can be excluded.

To obtain further evidence that the acceptor side of PSII, and especially Q_A, was altered in the mutant, we performed thermoluminescence measurements in the presence of DCMU. To obtain sufficient resolution of emission originating from S₂Q_A⁻ recombination (Q-band), the sample needed to be excited at low temperatures. This was not possible with Arabidopsis leaves. Freezing of Arabidopsis leaves leads to the leakage of phenolic compounds that quench the light emission. Exciting leaves at −5°C indicated a shift in the temperature maximum of the Q-band in hpr1-1 toward a higher temperature; however, most of the emission was lost rapidly when leaves were excited at this temperature and the Q-band could not be fully resolved (Supplemental Fig. S3). Therefore, we decided to work with isolated spinach (Spinacia oleracea) thylakoid membranes that had been preincubated for 5 min in the light with 40 mm glycolate. This treatment inactivated linear electron transport in thylakoid membranes by about 50% as measured by oxygen evolution (Supplemental Fig. S4). When thylakoid membranes were incubated with glycolate in a bicarbonate-containing buffer, less inhibition at the same glycolate concentrations was observed, showing a competition between HCO₃⁻ and glycolate for the same binding site (Supplemental Fig. S4). In thermoluminescence measurements, the temperature maximum of the Q-band was shifted by glycolate from about 3°C to 11°C (Fig. 4; Table II), indicating a stabilization of the charge pair S₂Q_A⁻.

Figure 4.

Effect of glycolate on the thermoluminescence Q-band in spinach thylakoid membranes. Thermoluminescence (relative units [r.u.]) is shown as a function of temperature. Black circles, absence of glycolate; white circles, thylakoid membranes preincubated with 40 mm glycolate in room light. All samples contained 20 μm DCMU added after the incubation. Samples were excited by a single-turnover flash at −15°C.

Table II. Temperature maximum of the Q-band.

Thermoluminescence measurements were performed with isolated spinach thylakoid membranes in the presence of 20 µm DCMU and in the absence or presence of 40 mm glycolate (means ± sd; n = 6). Samples were preincubated for 5 min in room light with glycolate in a degassed buffer or in a buffer containing 4 mm NaHCO₃⁻.

Treatment	Temperature Maximum
No addition	3.3°C ± 0.9°C
40 mm glycolate	12.0°C ± 1.4°C
40 mm glycolate + 4 mm NaHCO3−	8.2°C ± 1.0°C

The delay of fluorescence induction, fluorescence decay, and the increase of the temperature maximum of the Q-band indicated that glycolate indeed affects the midpoint potential of Q_A/Q_A⁻ and shifts it to a more positive value. According to previous work (Johnson et al., 1995; Cser and Vass, 2007; Sugiura et al., 2014; Brinkert et al., 2016), a shift of E_m (Q_A/Q_A⁻) to a more positive value alters the charge recombination pathways in PSII, favoring direct charge recombination reactions to the ground state and leading to less generation of triplet chlorophyll and ¹O₂. Figure 5 shows light-induced ¹O₂ generation in isolated thylakoids from the Arabidopsis wild type and hpr1-1 and in isolated spinach thylakoids in the presence of 40 mm glycolate. Less ¹O₂ was generated (a decrease of ∼30%) in the mutant than in the wild type and after incubation of spinach thylakoids with glycolate. The presence of bicarbonate in the incubation medium of thylakoid membranes diminished the effect of glycolate. In this assay, thylakoids were illuminated in the absence of an artificial electron acceptor. In the absence of an artificial electron acceptor, oxygen evolution is negligible and charge recombination reactions in PSII are favored. Under physiological conditions in vivo, a similar situation occurs when forward linear electron transport is saturated and the PQ pool is highly reduced. Thus, the glycolate-induced difference in oxygen evolution seen in spinach thylakoids in the presence of an artificial acceptor and uncoupler (Supplemental Fig. S4) is not relevant to ¹O₂ generation.

Figure 5.

Production of ¹O₂. Thylakoid membranes isolated from Arabidopsis wild type (wt) and hpr1-1 (black bars) or from spinach (white bars) were illuminated for 2 min at 670 µmol quanta m⁻² s⁻¹ in the presence of 100 mm 2,2,6,6-tetramethyl-4-piperidone hydrochloride. In the case of Arabidopsis, two preparations from material grown independently for 5 weeks were used. In the case of spinach thylakoid membranes, samples were preincubated for 5 min in room light in the presence of 40 mm glycolate either in a degassed buffer or in a buffer containing 4 mm NaHCO₃. At left, typical electron paramagnetic resonance (EPR) spectra: hpr1-1 (top) and the wild type (wt; bottom). At right, EPR signal size (means ± sd; n = 4). **, P < 0.5 and *, P < 0.10 according to Student’s t test.

A slowing of electron transport on the acceptor side of PSII slows the reduction of the PQ pool and may affect electron donation to PSI. Under steady-state conditions, donation to PSI is normally limited by plastoquinol oxidation by the cytochrome b₆f complex and not by electron donation to the PQ pool (Stiehl and Witt, 1969; for review, see Tikhonov, 2014). Figure 6 shows the light dependency of the limitation of electron donation to PSI [donor-side limitation; Y(ND)] and from PSI [acceptor-side limitation; Y(NA)] measured by absorption changes of P₇₀₀. Under the experimental conditions used here, there was very little PSI acceptor-side limitation observed for both genotypes, wild type and hpr1-1, while donor-side limitation was increased in hpr1-1 compared with the wild type at light intensities higher than 200 µmol quanta m⁻² s⁻¹. This shows that, at higher light intensities, a slowing of electron transport at the PSII acceptor side indeed affects electron donation to PSI. When the electron transport rate was calculated from chlorophyll fluorescence parameters, a lower rate was seen in hpr1-1 at light intensities higher than 200 µmol quanta m⁻² s⁻¹ (Fig. 7).

Figure 6.

Activity of PSI in wild-type (wt) and hpr1-1 leaves. The redox state of the PSI primary donor P₇₀₀ was monitored through the changes in absorbance at 830 versus 875 nm. Five-week-old Arabidopsis plants were kept in the dark for 5 min prior to the measurements. Following the initial determination of maximal oxidation of P₇₀₀ with far-red light, actinic light of the indicated intensities was given for 60 s. PSI donor-side limitation [Y(ND)] and acceptor-side limitation [Y(NA)] are based on saturating pulse analyses. Black circles, the wild type; white circles, hpr1-1 (means ± sd; n = 8; measurements with three biological replicates grown separately).

Figure 7.

Apparent photosynthetic electron transport rate (ETR) in the wild type and hpr1-1 versus light intensity. Five-week-old Arabidopsis plants were exposed for 60 s to the indicated light intensities before the chlorophyll fluorescence parameters were determined. Mature leaves were chosen for the measurements. Black circles, the wild type; white circles, hpr1-1 (means ± sd; n = 8; measurements with three biological replicates grown separately).

Saturation of electron transport was achieved at lower light intensities in hpr1-1 compared with the wild type, and the electron transport rate even decreased in hpr1-1 at light intensities higher than 400 µmol quanta m⁻² s⁻¹, indicating negative effects of glycolate and possibly other metabolite accumulation on the overall photosynthetic process. The hpr1-1 mutant showed reduced growth (Supplemental Fig. S1), which was already reported by Timm et al. (2008) and indicates some systemic effects of the mutation on photosynthetic-photorespiratory carbon metabolism. These could include, for example, the reported inhibition of Rubisco activity at a very high glycolate concentration in vitro (Gonzalez Moro et al., 1997) but most likely are unrelated to the modification of the acceptor side of PSII studied here. Accordingly, the susceptibility of hpr1-1 to high light was unaltered compared with the wild type (Supplemental Fig. S5).

DISCUSSION

It is well known that small carboxylate anions can replace bicarbonate as ligand to the nonheme iron in PSII in vitro: electron transfer from Q_A to Q_B is slowed down in bicarbonate-depleted PSII or in PSII in which bicarbonate was replaced with formate, acetate, glyoxylate, or glycolate (Vermaas and Rutherford, 1984; Petrouleas et al., 1994; Berthomieu and Hienerwadel, 2001). Acetate or glycolate shifts the midpoint potential of the redox couple Q_A/Q_A⁻ to a more positive potential also in vivo. This was shown by thermoluminescence measurements of C. reinhardtii grown in medium containing 17.5 mm acetate (Roach et al., 2013) and in this study with the Arabidopsis photorespiration mutant hpr1-1, which accumulates glycolate compared with the wild type (Fig. 1; Timm et al., 2008, 2013). In the light, when Q_A is in its reduced state, the replacement of bicarbonate by carboxylates become more likely (Petrouleas et al., 1994; Brinkert et al., 2016).

In absolute terms, stroma concentrations of about 0.5 mm glycolate were reported (Takabe and Akazawa, 1981). Photosynthesizing spinach chloroplasts produce glycolate at high rates of 0.5 to 1 µmol min⁻¹ mg⁻¹ chlorophyll (Heldt et al., 1977; Kow et al., 1977). These values were obtained when intact chloroplasts were fed with radioactively labeled substrates that were used in glycolate synthesis (measured over a few minutes). Calculating the glycolate concentration accumulated over 5 min and using a stromal volume of 46 µL mg⁻¹ chlorophyll (Winter et al., 1994), chloroplasts are able to synthesize up to 100 mm glycolate, at least transiently. Hence, it is feasible that glycolate may accumulate in the stroma, at least temporarily, to levels that significantly alter the redox tuning of PSII in vivo. The shift in the temperature maximum of the Q-band in spinach thylakoid membranes in the presence of glycolate strongly supports this model (Fig. 4). Future studies will need to address the quantitative binding parameters.

It has been proposed by Brinkert et al. (2016) that the increase of the E_m (Q_A/Q_A⁻) induced by bicarbonate release may be an important regulation mechanism and protects PSII against ¹O₂-induced photodamage. Hence, small carboxylate anions like glycolate may further facilitate the reversible redox tuning of the midpoint potential of Q_A. Under physiologically relevant conditions like drought, the stomata are closed, the internal CO₂ concentration and accordingly the HCO₃⁻ concentration decrease, oxygenation becomes the increasingly dominant reaction of Rubisco, and glycolate concentrations inside the chloroplast may rise transiently to levels that allow the liberation of HCO₃⁻ from the nonheme iron in exchange for glycolate. The increase of glycolate may allow the replacement of HCO₃⁻ at the nonheme iron at HCO₃⁻ concentrations that are higher than those needed for bicarbonate to exchange for water ligands at the nonheme iron. However, the effect of glycolate on the E_m (Q_A/Q_A⁻) may be smaller than the effect of water binding to the nonheme iron. In HCO₃⁻-depleted PSII, the E_m (Q_A/Q_A⁻) was shifted by 75 mV toward a more positive value, while in the presence of formate it was shifted only by 50 mV (Brinkert et al., 2016). Nevertheless, ¹O₂ production is decreased in the presence of glycolate. In spinach thylakoids, the yield of ¹O₂ production was lowered by 50% in the presence of glycolate (Fig. 5), as reported previously for HCO₃⁻-depleted PSII (Brinkert et al., 2016). In thylakoids isolated from hpr1-1, the yield of ¹O₂ production was lowered only by 30%. This could indicate that a fraction of the glycolate may have been replaced by HCO₃⁻ during the thylakoid preparation. When spinach thylakoids were incubated with glycolate in the presence of HCO₃⁻, the yield of ¹O₂ production increased slightly compared with the samples incubated in degassed buffer.

The photorespiratory pathway potentially contributes to the protection of PSII against photoinhibition (Osmond, 1981; Wingler et al., 2000). It has been assumed that the consumption of ATP and NADPH in the photorespiratory pathway helps keep the electron transport chain oxidized, which, in turn, reduces charge recombination reactions in PSII, including the probability of chlorophyll triplet and ¹O₂ formation. Previous studies with mutants exhibiting a complete block in the photorespiratory carbon recycling indicated that photoinhibition is accelerated due to an inhibition of the repair of PSII in these plants (Takahashi et al., 2007). Different from hpr1-1, which can complete the photorespiratory carbon flow due to a cytosolic bypass (Timm et al., 2008), these mutants suffer from a pleiotropic phenotype and are unable to thrive in ambient air. In contrast to the increased susceptibility to light of the photorespiratory mutants studied by Takahashi et al. (2007), no significant acceleration of photoinhibition was found in the hpr1-1 mutant when exposed to photoinhibitory light conditions both in the absence and presence of the protein synthesis inhibitor lincomycin, which prevents the synthesis of the D1 protein and the repair of photodamaged PSII (Supplemental Fig. S5). The lower yield of ¹O₂ production in hpr1-1 may compensate for the negative effects of impaired photorespiration and help the plants to cope with light stress. In addition, a lower yield of ¹O₂ allows an efficient repair of the D1 protein, since reactive oxygen species have been shown to inhibit PSII repair (Nishiyama et al., 2006). Furthermore, one has to keep in mind that, in our study, plants were grown at ambient CO₂, while Takahashi et al. (2007) grew plants in high-CO₂ conditions and exposed them during the photoinhibition treatment to ambient CO₂. In addition, a slowing of electron donation to PSI, as observed in hpr1-1 (Fig. 6), may allow the photoaccumulation of P₇₀₀⁺, which acts as an efficient quencher of excess excitation energy (Bar-Eyal et al., 2015).

In summary, it appears that the photorespiratory metabolite glycolate interacts with the acceptor side of PSII in vivo. Hence, in addition to averting a too highly reduced state of the electron transport chain by the consumption of NADPH and ATP, photorespiration has a specific direct effect on electron transport: the glycolate-dependent modification of the redox properties of Q_A. This process favors the direct charge recombination pathway between P₆₈₀⁺ and Q_A⁻ and, in that way, lowers ¹O₂ generation in the reaction center of PSII. Such regulation may become physiologically relevant when the CO₂ concentration is temporarily low and glycolate concentrations are high in the chloroplast. At increasing CO₂, glycolate would leave the nonheme iron, bicarbonate would rebind, and full PSII activity would be reestablished. Reversible inactivation of PSII by glycolate thus represents one more way that elevated photorespiration signals low-CO₂ conditions in order to protect plants from photoinhibition in high light.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) wild-type (ecotype Columbia-0) and hpr1-1 (Supplemental Fig. S1) plants were grown in ambient air (390 µL L⁻¹ CO₂ and 21% oxygen) for 3 to 5 weeks in a growth cabinet in short-day conditions: 10 h of light (22°C), 14 h of dark (18°C), and light intensity of 170 µmol quanta m⁻² s⁻¹ (light source, fluorescent light tubes; Osram Dulux L 55W/840). Plants of each genotype were grown in four independent sets. Spinach (Spinacia oleracea) leaves were harvested at a local farm (Ferme de Viltain, Saclay, France).

Preparation of Thylakoid Membranes

Leaves were homogenized in a blender for 10 s in a buffer containing 0.33 m sorbitol, 60 mm KCl, 10 mm EDTA, 1 mm MgCl₂, and 25 mm MES, pH 6.1. The slurry was filtered through four layers of cheesecloth, and the filtrate was centrifuged at 3,000g for 3 min at 4°C. The supernatant was discarded, and the pellet was resuspended in 0.33 m sorbitol, 10 mm KCl, 10 mm EDTA, 1 mm MgCl₂, and 25 mm HEPES, pH 6.7 The suspension was centrifuged at 3,000g for 3 min at 4°C. This step was repeated once. Finally, the pellet was resuspended in 0.3 m sorbitol, 50 mm KCl, 1 mm MgCl₂, and 25 mm HEPES, pH 7.6. Before the measurements, chloroplasts were incubated for 20 s in a buffer containing 50 mm KCl, 1 mm MgCl₂, and 25 mm HEPES, pH 7.6, then the double volume of the buffer containing 0.6 m sorbitol, 50 mm KCl, 1 mm MgCl₂, and 25 mm HEPES, pH 7.6, was added.

Oxygen Evolution

Oxygen evolution of spinach thylakoid membranes was measured using a Clark electrode (Hansatech) at 20°C and saturating light intensity (white light, 2,000 µmol quanta m⁻² s⁻¹, using a halogen lamp; Osram XENOPHOT 15V 150W). One millimolar K₃[Fe(CN)₆] as electron acceptor (Anderson and Boardman, 1966) and 25 mm NH₄Cl as uncoupler (Krogmann et al., 1959) were used. Samples were preincubated for 5 min at room light with the given glycolate concentrations.

Chlorophyll Fluorescence

Chlorophyll fluorescence was measured on whole plants of Arabidopsis wild type and hpr1-1 with an imaging system (Imaging-PAM; Walz). Prior to the measurements, plants were dark adapted for 5 min to obtain the maximum fluorescence (F_m). The ETR was measured from the fluorescence parameters and calculated according to ETR = 0.5 × Y(II) × light intensity × 0.84 microequivalents m⁻² s⁻¹. The PSII quantum yield, Y(II), is defined as (F_m′ − F)/F_m′, with F being the fluorescence in actinic light and F_m′ being the maximum fluorescence obtained under illumination.

Fluorescence induction curves were measured on dark-adapted leaves at 5°C with a Dual-PAM-100 (Walz) using actinic red light (intensity of 37 µmol quanta m⁻² s⁻¹). Fluorescence decay was measured at 5°C after excitation with a saturating single-turnover flash. When indicated, leaves were vacuum infiltrated with 20 µm DCMU prior to the measurement. A temperature of 5°C was chosen to slow down slightly electron transfer reactions.

P₇₀₀ Absorption

The redox state of the primary donor of PSI, P₇₀₀, was monitored by following the changes in absorbance of 2-min dark-adapted leaves at 830 versus 875 nm using the Dual-PAM-100. To probe the maximum extent of P₇₀₀ oxidation, leaves were illuminated with far-red light superimposed on the saturating pulse of red light. Oxidation of P₇₀₀ was measured in leaves of the wild type and hpr1-1 as a function of the intensity of the actinic light. Y(NA) = (P_m − P_m′)/P_m is a measure of the limitation of electron transport at the PSI acceptor side. P_m represents the maximum oxidation of P₇₀₀ and P_m′ the maximum oxidation at a given light intensity after the application of a saturating light pulse. Y(ND) is a measure of the donor side limitation and is calculated as Y(ND) = 1 − P_700red, where P_700red represents the fraction of reduced P₇₀₀ at a given state (Klughammer and Schreiber, 2008).

Thermoluminescence

Thermoluminescence was measured using a home-built apparatus. Thylakoid membranes, at a concentration of 100 μg chlorophyll mL⁻¹, were incubated for 5 min at 20°C in room light in the presence or absence of 40 mm glycolate. Ten micromolar DCMU was added prior to the thermoluminescence measurement. Thermoluminescence was charged by single-turnover flashes with a xenon flash lamp at −15°C. Leaves were vacuum infiltrated with 20 µm DCMU just before the thermoluminescence measurements. The thermoluminescence signal was recorded during warming of the sample to 70°C at a heating rate of 0.4°C s⁻¹.

Detection of ¹O₂ by Room-Temperature EPR Spectroscopy

¹O₂ was trapped using the water-soluble spin probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride (Hideg et al., 2011) and measured with a Bruker e-scan (Bruker Biospin). Thylakoids (20 μg chlorophyll mL⁻¹) were illuminated for 2 min with red light (Schott filter RG 630) at 670 µmol quanta m⁻² s⁻¹ in 0.3 M sorbitol, 50 mM KCl, 1 mM MgCl₂ and 25 mM HEPES (pH 7.6) . Spectra were recorded using a flat cell containing 70 μL of sample. The microwave power was 9.77 GHz and 14.07 mW, with a modulation frequency of 86 kHz and amplitude of 1.01 G. Each spectrum was an average of eight scans each with a sweep time of 10.5 s.

Glycolate Measurements

For glycolate determination, the hpr1-1 mutant (Timm et al., 2008) was grown next to the corresponding wild type (Columbia-0) in normal air (390 µL L⁻¹ CO₂) as stated above. Leaf material (∼50 mg) was harvested in the middle of the photoperiod (5 h of illumination) at five different time points (3, 4, 5, 6, and 7 weeks after germination) during plant development and from at least four plants per genotype grown separately. Glycolate levels were quantified by gas-chromatography coupled to mass spectrometry as described previously (Engel et al., 2007).

Accession Number

The accession number for hpr1-1 is At1g68010.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Representative images of wild-type and hpr1-1 plants.

• Supplemental Figure S2. Chlorophyll fluorescence analysis of leaves of 3-week-old hpr1-1 and wild-type plants.

• Supplemental Figure S3. Thermoluminescence of hpr1-1 and wild-type leaves.

• Supplemental Figure S4. Inhibition of oxygen evolution by glycolate.

• Supplemental Figure S5. Photoinhibition of wild-type and hpr1-1 leaves.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• ATP CHEBI: CHEBI:15422

• HEPES CHEBI: CHEBI:46756

• NADPH CHEBI: CHEBI:16474

• acetate CHEBI: CHEBI:30089

• chlorophyll CHEBI: CHEBI:28966

• leaf AmiGo: PO:0025034

• oxygen CHEBI: CHEBI:25805