A Balanced PGR5 Level is Required for Chloroplast Development and ...

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mulating PGR5 in the thylakoid membrane, chloroplast development was delayed, especially in the cotyledons. Although photosynthetic electron transport was ...
Plant Cell Physiol. 48(10): 1462–1471 (2007) doi:10.1093/pcp/pcm116, available online at www.pcp.oxfordjournals.org ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

A Balanced PGR5 Level is Required for Chloroplast Development and Optimum Operation of Cyclic Electron Transport Around Photosystem I Yuki Okegawa 1, Terri A. Long 2, 5, Megumi Iwano 3, Seiji Takayama 3, Yoshichika Kobayashi 1, Sarah F. Covert 4 and Toshiharu Shikanai 1, * 1

Graduate School of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashiku, Fukuoka, 812-8581 Japan Department of Genetics, University of Georgia, Athens, GA 30602, USA 3 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, 630-0101 Japan and 4 Warnell School of Forestry and Natural Resources, University of Georgia. Athens, GA 30602-2152, USA 2

PSI cyclic electron transport contributes markedly to photosynthesis and photoprotection in flowering plants. Although the thylakoid protein PGR5 (Proton Gradient Regulation 5) has been shown to be essential for the main route of PSI cyclic electron transport, its exact function remains unclear. In transgenic Arabidopsis plants overaccumulating PGR5 in the thylakoid membrane, chloroplast development was delayed, especially in the cotyledons. Although photosynthetic electron transport was not affected during steady-state photosynthesis, a high level of nonphotochemical quenching (NPQ) was transiently induced after a shift of light conditions. This phenotype was explained by elevated activity of PSI cyclic electron transport, which was monitored in an in vitro system using ruptured chloroplasts, and also in leaves. The effect of overaccumulation of PGR5 was specific to the antimycin A-sensitive pathway of PSI cyclic electron transport but not to the NAD(P)H dehydrogenase (NDH) pathway. We propose that a balanced PGR5 level is required for efficient regulation of the rate of antimycin A-sensitive PSI cyclic electron transport, although the rate of PSI cyclic electron transport is probably also regulated by other factors during steady-state photosynthesis. Keywords: Antimycin A — Arabidopsis — Ferredoxin — NPQ — PGR5 — PSI cyclic electron transport. Abbreviations: AL, actinic light; ETR, electron transport rate; Fd, ferredoxin; FR, far-red; FQR, ferredoxin-plastoquinone reductase; ML, measuring light; NDH, NAD(P)H dehydrogenase; NPQ, non-photochemical quenching; PAM, pulse amplitude modulation; PGR5, PROTON GRADIENT REGULATION 5; PQ, plastoquinone; qE, energization-dependent quenching.

Introduction Photosynthetic electron transport consists of two main routes: linear electron transport and PSI cyclic electron transport. Linear electron transport from water to NADPþ

is driven by two photosystems and results in O2 evolution at PSII and generation of NADPH. Electron transport between PSII and PSI is mediated by the Cyt b6f complex and coupled with translocation of protons across the thylakoid membranes from the stroma to the lumen. The resulting pH is utilized in ATP synthesis. In PSI cyclic electron transport, however, electrons are recycled from either NAD(P)H or ferredoxin (Fd) to plastoquinone (PQ), generating pH without accumulation of reduced species (Shikanai 2007). Both linear and PSI cyclic electron transport have been shown to be essential for photosynthesis and photoprotection in flowering plants (Munekage et al. 2002, Munekage et al. 2004). PSI cyclic electron transport was first discovered from its coupling with ATP synthesis approximately 50 years ago (cyclic phosphorylation), before the establishment of a concept of linear electron transport (Arnon et al. 1954, Arnon 1959, Arnon et al. 1967). In flowering plants, PSI cyclic electron transport consists of two pathways, both of which partly share the route taken by electrons with linear electron transport (Joe¨t et al. 2001, Munekage et al. 2004). The NAD(P)H dehydrogenase (NDH)-dependent pathway was originally discovered in cyanobacteria (Ogawa 1991, Mi et al. 1995) and was also shown to operate PSI cyclic electron transport in chloroplasts (Burrows et al. 1998, Shikanai et al. 1998). Although the NDH complex is a major player in PSI cyclic electron transport in cyanobacteria, its contribution is minor in flowering plants. Instead, the Fd-dependent route of PSI cyclic electron transport, which was discovered by Arnon and co-workers (Arnon et al. 1954, Arnon 1959, Arnon et al. 1967), contributes markedly to pH generation in flowering plants. Despite the long history of interest (Bendall and Manasse 1995) and the documented physiological significance (Munekage et al. 2002, Munekage et al. 2004), our knowledge of the machinery of Fd-dependent electron transport is very limited. Fd-dependent PQ reduction activity has been observed in earlier studies and was shown to be sensitive to antimycin A (Tagawa et al. 1963). Numerous studies suggest that antimycin A inhibits PSI

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Present address: Department of Biology, Duke University, Durham, NC 27708, USA. *Correspondence author: E-mail, [email protected]; Fax, þ81-92-642-2882. 1462

Function of PGR5 in PSI cyclic electron transport

Results PGR5 can overaccumulate in the thylakoid membrane To investigate the effect of PGR5 overexpression, wildtype Arabidopsis plants were transformed with PGR5 under

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cyclic electron transport (Heber et al. 1978, Mills et al. 1978, Mills et al. 1979, Moss and Bendall 1984, Miyake et al. 1995, Joe¨t et al. 2001). An Arabidopsis mutant, pgr5, which is defective in PSI cyclic electron transport, was isolated on the basis of its high Chl fluorescence at high light intensities (Shikanai et al. 1999). pgr5 is impaired in antimycin A-sensitive PSI cyclic electron transport (Munekage et al. 2002). PROTON GRADIENT REGULATION 5 (PGR5) is a small protein without any metal-binding motifs. Therefore, it is unlikely that PGR5 mediates the electron transport from Fd to PQ as a direct electron carrier. The pgr5 defect may destabilize or inactivate the putative enzyme complex of Fd-dependent PQ reductase (FQR). From the first discovery of PSI cyclic electron transport, the idea that Fd donates electrons to PQ via the Q cycle of the Cyt b6f complex has been discussed. Recent findings that Fd-NADPþ reductase (FNR) associates with the Cyt b6f complex (Zhang et al. 2001) and that the Cyt b6f complex contains an unexpected heme, heme x (also referred to as heme ci), which is not conserved in the Cyt bc1 complex in mitochondria (Kurisu et al. 2003, Stroebel et al. 2003), prompted us to reconsider this classical idea. Consequently, we characterized PSI cyclic activity in an Arabidopsis mutant, pgr1, which is conditionally impaired in Q cycle activity (Munekage et al. 2001, Jahns et al. 2002). This analysis did not provide any evidence to support the involvement of the Cyt b6f complex in PGR5dependent, Fd-dependent PQ reduction (Okegawa et al. 2005). Furthermore, from the phenotype of the AtLFNR knockout lines, the membrane-bound FNR is unlikely to be involved in PGR5-dependent PSI cyclic electron transport (Lintala et al. 2007). Nor has there been any other experimental evidence that the Cyt b6f complex is involved in PGR5-dependent PQ reduction. Despite its physiological significance, the molecular mechanism underlying PSI cyclic electron transport remains unknown and PGR5 is the only molecule known to be involved in the process. Thus further study of PGR5 should provide important clues about the molecular identity of this puzzling electron transport. In this study, we show that overaccumulation of PGR5 delayed chloroplast development, especially in the cotyledons. The characterization of electron transport indicated that higher level of PGR5 elevated the activity of PGR5-dependent PSI cyclic electron transport, which disturbs the redox balance in chloroplasts under fluctuating light conditions. Here, we discuss the function of PGR5 as a regulator of PSI cyclic electron transport.

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Fig. 1 Protein blot analysis of PGR5 in the 35S::PGR5 lines. PGR5 and subunits of the Cyt b6f complex and RubisCO were immunodetected using specific antibodies. Chloroplast preparations were further fractionated into the thylakoid membrane and stromal fractions. Lanes were loaded with protein samples corresponding to 0.5 mg Chl.

the control of the cauliflower mosaic virus 35S promoter. In many lines, the level of PGR5 protein was lower than in the wild type, probably due to co-suppression. However, protein blot analysis using PGR5 antibody identified two independent lines (#1 and #2) in which the PGR5 level was higher than in the wild type (Fig. 1). Overexpression of PGR5 was also confirmed by RNA blot analysis in these lines (data not shown). Protein blot analysis with serial diluted samples indicated that the overexpressors accumulated approximately five times more PGR5 than the wild type (data not shown). In the same way as PGR5 was encoded by the endogenous gene in the wild type, excessively accumulated PGR5 in the overexpressors was localized to the thylakoid fraction of the chloroplasts but not to the stromal fraction, suggesting that overexpression does not affect protein localization (Fig. 1). The accumulation of PGR5 was stably transmitted to the T3 generation, which was used for further analysis.

Overexpression of PGR5 affects chloroplast development One of the prominent phenotypes of the 35S::PGR5 seedlings cultured in soil was a delay in greening, especially in the cotyledons (Fig. 2). They appeared almost albino or yellow green, and gradually turned green during seedling growth (Fig. 2A). Greening started from the edge of the leaf, and the cells along the vascular tissues often remained yellow. Consistently with the delay in cotyledon greening, the initial growth of the 35S::PGR5 seedlings was retarded, although the final seedling size was identical to that of the wild type. Compared with that in the wild type, Chl content

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Fig. 2 Developmental phenotypes of plants overexpressing PGR5. (A) Three-week-old seedlings cultured under long day conditions (16 h light/8 h dark). In the 35S::PGR5 lines, cotyledons appeared almost albino (red arrow) and turned to green later, although the cells along the vascular tissues remained yellow (yellow arrow). (B) Three-week-old 35S::PGR5 seedling cultured under continuous light conditions. In addition to the presence of albino-like cotyledons (red arrow), true leaves were variegated (yellow arrow). (C) Plants were grown for 2 months under short day conditions (8 h light/16 h dark). Although there was no distinct phenotype in the cotyledons of the 35S::PGR5 lines, their Chl content was lower and their petioles were white. Bars ¼ 10mm.

Table 1 Chl contents of leaves from the wild type and the 35S::PGR5 lines 1 week

2 weeks

3 weeks

4 weeks

Wild type 0.74  0.072 1.00  0.070 1.05  0.18 1.28  0.10 35S::PGR5 0.60  0.071 0.77  0.015 0.88  0.039 1.00  0.043

Values are means  SD (n ¼ 5). Chl contents were expressed as mg per mg fresh weight.

was reduced to 80% in seedlings of the 35S::PGR5 lines cultured for either 1 or 4 weeks (Table 1). Interestingly, the greening phenotype was markedly influenced by day length. The cotyledon phenotype was evident when the seedlings were cultured under long day conditions (16 h light/8 h dark). Under continuous light conditions, the 35S::PGR5 lines produced almost albino cotyledons. Furthermore, the rosette leaves were variegated (Fig. 2B) and the growth of seedlings was also severely

Fig. 3 Ultrastructure of plastids from the wild type (WT) and the 35S::PGR5 lines. Cotyledons of the WT and the 35S::PGR5 seedlings cultured under constant light conditions were used for analysis by transmission electron microscopy. Plastids from the WT (A) and the 35S::PGR5 lines (B, C and D). Bars indicate 1 mm.

delayed. Under short day conditions (8 h light/16 h dark), however, the 35S::PGR5 lines produced green cotyledons and the growth rate was similar to that of the wild type, although the petiole and stem of the 35S::PGR5 lines remained white even in mature seedlings (Fig. 2C). These results indicate that overexpression of PGR5 affects greening, not only of cotyledons but also of rosette leaves, and that day length influences the extent of the phenotype, although the exact physiological mechanism is unclear. How is chloroplast development influenced in leaves exhibiting the strong phenotype? To analyze the ultrastructure of the chloroplasts, cotyledons from seedlings cultured under continuous light conditions were subjected to analysis under a transmission electron microscope (Fig. 3). In the wild type, the chloroplasts in the cotyledons showed stacking of thylakoid membranes and the presence of several starch granules, as did the chloroplasts in the mature leaves (Fig. 3A). However, the cotyledons of the 35S::PGR5 lines contained undeveloped plastids, which were similar to proplastids. The plastids contained some large vesicles and also small plastoglobule-like vesicles (Fig. 3B). Some plastids contained a single layer of inner membranes (Fig. 3C) that probably develop into the thylakoid membranes with thinner grana stacking (Fig. 3D). These immature chloroplasts did not contain any starch granules, suggesting that photosynthetic activity was low (Fig. 3C, D). In the rosette leaves of the 35S::PGR5 lines, almost all chloroplasts contained normal thylakoid membranes,

Function of PGR5 in PSI cyclic electron transport

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Fig. 4 In vivo analysis of electron transport activity. (A) Light intensity dependence of NPQ of Chl fluorescence. Values are means  SD (n ¼ 5) for the wild type (WT) and pgr5. (B) Time courses of induction and relaxation of NPQ. NPQ was monitored for 4^min at a light intensity of 50 mmol photons m–2 s–1 (white bar) and subsequently for 2 min in the dark (black bar) in the WT, pgr5 and the 35S::PGR5 lines. Values are means  SD (n ¼ 5) for the WT and pgr5. The data for the 35S::PGR5 lines are an average of both #1 and #2 (n ¼ 10). (C) Time courses of induction and relaxation of NPQ after a shift of light intensity. NPQ was monitored for 4^min at each light intensity of 13 and 58 mmol photons m–2 s–1 (white bars) and in the dark (black bar). Values are means  SD (n ¼ 5) for the WT and pgr5. (D) Light intensity dependence of ETR. ETR was depicted relative to PSII  light intensity (mmol photons m–2 s–1). ETR was represented as relative values of the maximum ETR in the WT (100%). Values are means  SD (n ¼ 5) for the WT and pgr5. In (A) and (B), representative results for two seedlings are shown for each line of the 35S::PGR5 (#1 and #2). Before the analysis the plants were dark adapted for 41 h.

but occasionally some chloroplasts contained thinner thylakoid membranes than in the wild type (data not shown). We did not find any alteration in the chloroplast ultrastructure in pgr5 (data not shown). We concluded that overaccumulation of PGR5 markedly affected the development of chloroplasts, especially in the cotyledons.

Relaxation of transiently induced NPQ was slow in the 35S::PGR5 lines under low light conditions Unexpectedly, overaccumulation of PGR5 delayed chloroplast development (Figs. 2, 3). To study the casual relationship between the PGR5 overaccumulation and

chloroplast development, we characterized the photosynthetic electron transport in developed mature leaves in detail (Fig. 4). Although there were no differences in the Chl fluorescence parameters of the wild type and the 35S::PGR5 lines during steady-state photosynthesis, we observed a clear phenotype in the 35S::PGR5 lines transiently after a shift of light coditions. Fig. 4 shows the light intensity dependence of Chl fluorescence parameters. In the analysis of light intensity dependence, Chl fluorescence parameters were recorded 2 min after a shift to each light intensity. In the wild type, the parameters of Chl fluorescence are close to those during the steady state.

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The non-photochemical quenching (NPQ) of chlorophyll fluorescence consists of several components. In flowering plants, NPQ depends largely on the qE (energization-dependent quenching) component, which reflects the size of the thermal dissipation of excessive absorbed light energy from PSII. The qE component is characterized by its relatively fast induction and relaxation kinetics on a physiological time scale of seconds to several minutes (Horton et al. 1996). qE induction is triggered by acidification of the thylakoid lumen, a process in which PGR5-dependent PSI cyclic electron transport is essential (Fig. 4A; Munekage et al. 2002). In the wild type, qE was induced at light intensities of 4100 mmol photons m–2 s–1 (Fig. 4A). In the 35S::PGR5 lines, however, a high level of NPQ was induced even at the very low light intensity of 20 mmol photons m–2 s–1 (Fig. 4A). To characterize this high-level NPQ, the time course of NPQ induction and relaxation was analyzed at a light intensity of 50 mmol photons m–2 s–1 and subsequently in the dark (Fig. 4A). Even in the wild type, NPQ was transiently induced within 2 min after a shift from dark to light. This NPQ consists mainly of qE, since its induction is impaired in the Arabidopsis npq4 (non-photochemical quenching 4) mutant (Li et al. 2000, Kalituho et al. 2006), which is defective in the essential machinery for thermal dissipation (Munekage et al. 2002). PGR5 is essential for this transient qE induction (Fig. 4B; Munekage et al. 2002). In the 35S::PGR5 lines, NPQ remained high for longer, probably because of the slow relaxation kinetics of qE (Fig. 4B). However, high NPQ was relaxed to the wild-type level during steady-state photosynthesis (Fig. 4B). Thus, the NPQ phenotype was observed transiently but not during steady-state photosynthesis. Notably, this transient induction of NPQ was not relaxed for longer during the step-wise increase in light intensity every 2 min (Fig. 4A). This result suggests that transient qE is induced not only during a shift from dark to light but also by fluctuations of light intensity. To test this possibility, we examined the effect of a change in light intensity on NPQ. Once plants were adapted to the light intensity of 13 mmol photons m–2 s–1, the NPQ level was not drastically affected after a shift of light intensity to 58 mmol photons m–2 s–1 in the wild type (Fig. 4A). This result suggests that the Calvin cycle activity was fully induced during 4 min in the light. The slight increase in NPQ is unlikely to be due to qE, since the NPQ level was similar to that in pgr5 and the NPQ was not relaxed in the dark for 4 min (Fig. 4C). In 35S::PGR5 lines, however, a shift of light intensity from 13 to 58 mmol photons m–2 s–1 caused transient induction of high-level NPQ (Fig. 4C). This NPQ was also relaxed to the wild-type level during steady-state photosynthesis. This result indicates that the overaccumulation of PGR5 disturbs the redox balance in chloroplasts not

only during induction of photosynthesis but also by a shift of light intensity. A Chl fluorescence parameter, ETR (electron transport rate), represents the relative rate of photosynthetic electron transport through PSII. At low light intensities, ETR was only slightly lower in the 35S::PGR5 lines than in the wild type, probably because of transient qE induction under light-limiting conditions (Fig. 4A, D). In contrast, ETR was not affected at higher light intensities. Consistently with the phenotype of NPQ, there was no difference in the ETR during steady-state photosynthesis of the wild type and the 35S::PGR5 lines. This result indicated that the initial delay in growth of the 35S::PGR5 seedlings is ascribable mainly to the low level of photosynthetic activity in the cotyledons, in which chloroplast development is delayed, rather than to low photosynthetic activity in the green leaves. Activity of PSI cyclic electron transport is elevated in the 35S::PGR5 lines In the 35S::PGR5 lines, qE induction was enhanced under fluctuating light conditions (Fig. 4). qE induction depends on the build-up of pH, suggesting that the activity of PSI cyclic electron transport was elevated. To assess this possibility, electron donation to PQ from Fd was compared using ruptured chloroplasts isolated from leaves of the wild type, pgr5 and the 35S::PGR5 lines. Fd-dependent PQ reduction was monitored as an increase in Chl fluorescence emitted from PSII upon exposure to a measuring light (ML) of relatively high intensity (1.0 mmol photons m–2 s–1). At this light intensity, the fluorescence increase mostly reflects the reduction in PQ by PSI cyclic electron transport (Endo et al. 1998). In this assay system, NADPH is essential for electron donation to Fd via the reverse reaction of FNR in the dark (Miyake and Asada 1994). Application of NADPH did not reduce PQ since NDH also requires Fd in ruptured chloroplasts (Endo et al. 1998, Munekage et al. 2002). However, PQ was rapidly reduced by the subsequent addition of Fd in wild-type chloroplasts (Fig. 5). In pgr5, Fd-dependent PQ reduction was significantly suppressed, as reported previously (Munekage et al. 2002). The pgr5 defect in PQ reduction was mimicked in wild-type chloroplasts by adding antimycin A, which inhibits PGR5-dependent PSI cyclic electron transport. Unlike the case in pgr5, the rate of PQ reduction and the final level of reduction of PQ were higher in the 35S::PGR5 lines than in the wild type. Although our assay does not provide exact quantitative information on the ETR, it is significantly higher in the 35S::PGR5 lines compared with that in the wild type. Addition of antimycin A to the 35S::PGR5 chloroplasts impaired Fd-dependent PQ reduction to the level of wild-type chloroplasts treated with antimycin A. This result indicates that the ability to

Function of PGR5 in PSI cyclic electron transport

reduce PQ depended on PGR5 but not on the NDH complex. This is consistent with the fact that the transient increase in Chl fluorescence after the actinic light (AL) had been turned off was not affected in either pgr5 (Munekage et al. 2004) or the 35S::PGR5 lines (data not shown). We conclude that overaccumulation of PGR5 specifically enhances antimycin A-sensitive PSI cyclic electron transport but not NDH-dependent PSI cyclic electron transport. In vivo analysis of PSI cyclic activity in the 35S::PGR5 lines In the ruptured chloroplasts, PGR5-dependent PSI cyclic activity was enhanced in the 35S::PGR5 lines (Fig. 5). To test whether the same is true in leaves, changes in Chl

Chl fluorescence

35S::PGR5 WT 35S::PGR5+A.A WT+A.A pgr5

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Fig. 5 Electron donation to PQ in ruptured chloroplasts. Increases in Chl fluorescence by the addition of NADPH (0.25 mM) and Fd (5 mM) under a measuring light (intensity of 1.0 mmol photons m–2 s–1) were monitored in osmotically ruptured chloroplasts (10 mg Chl ml–1) of the wild type (WT), pgr5 and the 35S::PGR5 lines. Ruptured chloroplasts were incubated with 10 mM antimycin A (WT þ A.A and 35S::PGR5 lines þ A.A) before the measurement.

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fluorescence and P700þ (oxidized PSI reaction center Chl dimer) absorbance at a wavelength of 830 nm were simultaneously monitored using a pulse amplitude modulation (PAM) Chl fluorometer (Fig. 6). When the wild-type plant was irradiated with ML of relatively high intensity (1.3 mmol photons m–2 s–1), the apparent Fo level (Fo0 ) gradually increased (Fig. 6A). Since no increase in Fo0 level was detected using ML with lower light intensities (0.05–0.1 mmol photons m–2 s–1), which are commonly used in PAM analysis (data not shown), and the Fo0 decreased to the original level when we turned on far-red (FR) light, which preferentially activates PSI, this increase in Fo0 depended on PQ reduction. The increase in Fo0 level was enhanced in the 35S::PGR5 lines compared with the wild type, suggesting that PQ is reduced to a greater extent in the 35S::PGR5 lines. In contrast, the increase in Fo0 was minimal in pgr5. These results suggest that even at very low light intensity of 1.3 mmol photons m–2 s–1, overaccumulation of PGR5 enhances the electron donation to the PQ pool probably from Fd. Even under the background of ML of relatively high intensity, P700 was almost fully oxidized to P700þ by exposure to FR light (Fig. 6A, B). In the wild type, FR light oxidized P700 with relatively fast kinetics (Fig. 6B; t ¼ 10–15 s: t is the time required to reach the maximum P700þ level). Compared with the wild type, the rate of oxidation of P700 was slightly faster in pgr5 (t ¼ 7 s). It was essential to use ML of this intensity to detect the difference between the wild type and pgr5, implying that an electron input from PSII is essential for the operation of PGR5dependent PSI cyclic electron transport. In contrast, P700 oxidation was slower in the 35S::PGR5 lines (t ¼ 20–30 s).

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Fig. 6 In vivo analysis of PSI cyclic electron transport activity. (A) Irradiation with measuring light (ML) of relatively high intensity (1.3 mmol photons m–2 s–1) gradually increased the apparent Fo level (Fo0 ) of Chl fluorescence. FR light and pulses of saturating light were applied as indicated. The P700þ level was monitored during the same time courses as absorbance changes at a wavelength of 830 nm. (B) Close-up of oxidation kinetics of P700 by FR light in a background of relatively high-intensity ML.

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These results suggest that more electrons are donated from the stromal electron pool to PQ during FR light illumination in the 35S::PGR5 lines than in the wild type, although the phenotype is likely to be a result of complex events caused by an alteration in electron transport (see Discussion for more details). Consistently with the result of the in vitro assay using ruptured chloroplasts (Fig. 5), the in vivo analysis indicated that the activity of PGR5-dependent PSI cyclic electron transport was elevated in the 35S::PGR5 lines at least under the conditions used.

Discussion Since the discovery of PGR5 (Munekage et al. 2002), it has been a critical question how PGR5 is involved in PSI cyclic electron transport. A hypothesis is that PGR5 is an accessory subunit of the FQR complex, since PGR5 is unlikely to mediate the electron transport directly. Therefore, it was somewhat unexpected that overexpressed PGR5 accumulated stably in the thylakoid membrane (Fig. 1). Since PGR5 does not contain any transmembrane domains, it is likely to associate with other thylakoid proteins. The PGR5 expression may limit the level of this putative protein complex. It is also possible that PGR5 interacts with thylakoid proteins which accumulate in excess even in the absence of PGR5. Unexpectedly, overaccumulation of PGR5 severely affected chloroplast development, especially in the cotyledons. It is puzzling that this phenotype depended on day length. In flowering plants, Fd plays a central role in redox regulation and donates electrons to a variety of reactions (Hanke et al. 2004). Enhancement of PSI cyclic electron transport may pleiotrophically affect redox homeostasis in chloroplasts. In cyanobacteria, introduction of a maize Fd that favors the cyclic route of electrons enhances PSI cyclic electron transport activity, resulting in competition for Fd oxidation between PSI cyclic electron transport and nitrite reduction (Kimata-Ariga et al. 2000). As a result, the cyanobacteria exhibit a nitrogen-deficient phenotype. However, we could not find any tight link between the cotyledon phenotype and the presence of alternative nitrogen sources of ammonium or nitrate (data not shown). Reduced Fd is also required for desaturation of fatty acids in plastids as an electron donor (Lyle et al. 2003). However, no difference was detected in the desaturation levels of fatty acids between the 35S::PGR5 lines and the wild type (Supplementary Table S1). Taken together with the results of Chl fluorescence analysis, activation of PSI cyclic electron transport is transient and it is unlikely that the elevated activity of PGR5-dependent PSI cyclic electron transport competes with other reactions oxidizing Fd during steady-state photosynthesis.

It is possible that the change in the redox state of the PQ pool may disturb chloroplast development even though the effect is transient. The PQ reduction level is sensed by plants to control a variety of reactions (Pfannschmidt et al. 1999, Bellafiore et al. 2005, Bonardi et al. 2005). In a variegated Arabidopsis immutans mutant, the activity of PTOX (plastid terminal oxidase), which is involved in PQ oxidation, is impaired (Carol et al. 1999, Wu et al. 1999). PTOX is essential for chloroplast development via PQ oxidation, which is probably required for the activity of phytoene desaturase. In the 35S::PGR5 lines, the PQ pool is more reduced by electrons in mature leaves compared with that in the wild type (Fig. 6). The phosphorylation level of the light-harvesting complex II (LHCII) in the dark in the 35S::PGR5 lines was slightly higher than in the wild type, although the level was identical to that of the wild type in the light (data not shown). Although we are not sure whether this minor difference in the PQ reduction level markedly affects chloroplast development, it is possible that the difference is more evident in the chloroplasts in leaf primordial cells or in embryonic cells developing into cotyledons. Overaccumulation of PGR5 induced a high level of qE during the induction period of photosynthesis and after a shift of light conditions (Fig. 4B, C). qE induction is triggered by acidification of the thylakoid lumen, which occurs as a consequence of balance between the generation and relaxation of pH. After a shift of light conditions, enhanced PSI cyclic electron transport might increase pHgenerating activity. Simultaneously, Fd-dependent PSI cyclic electron transport may transiently compete with the Fd–thioredoxin system, which activates the Calvin cycle enzymes for the initiation of CO2 fixation (Buchanan and Balmer 2005). This idea was supported by the fact that the NPQ phenotype of the 35S::PGR5 lines was milder when the seedlings were analyzed after insufficient dark adaptation (data not shown). We consider that the effect of an imbalance between linear and PSI cyclic electron transport may be somehow minimized during steady-state photosynthesis in the chloroplasts. It is possible that the NADPþ/ NADPH ratio regulates the rate of PGR5-dependent PSI cyclic electron transport, as suggested in vivo (Breyton et al. 2006). We also clarified that NADPþ can compete with PGR5-dependent PSI cyclic electron transport in isolated thylakoids (Y. Okegawa et al. unpublished). Even in the 35S::PGR5 lines, the electron flow may be mainly in the linear mode in the presence of NADPþ. It is also possible that the slight modification in balance of linear and PSI cyclic electron transport is exaggerated due to the low relaxation of pH after a shift of light conditions. In the Fd-dependent PQ reduction assay, the rate of PQ reduction and the final reduction levels were elevated in the overexpressors (Fig. 5). This effect was completely

Function of PGR5 in PSI cyclic electron transport

impaired by antimycin A to the level of the wild type treated with the same inhibitor. This result strongly suggests that the overaccumulation of PGR5 strictly enhanced the antimycin A-sensitive pathway but not the NDH-dependent pathway. This is consistent with the phenotype of pgr5, in which NDH activity is not affected (Munekage et al. 2004). Although our results still cannot specify the function of PGR5, apparently it is specifically related to antimycin A-sensitive PSI cyclic electron transport. The results of simultaneous measurement of Chl fluorescence and P700 oxidation also showed the elevated activity of PSI cyclic electron transport in vivo in the 35S::PGR5 lines. For technical reasons we had to use an ML of relatively high intensity, and we discovered differences in Fo0 levels among the wild type, pgr5 and 35S::PGR5 lines (Fig. 6A). Even low-intensity light led to increased reduction of the PQ pool, the extent of which depended on the PGR5 dosage in different genotypes. PQ reduction in the dark was recently reported in tobacco which overaccumulated Fd (Yamamoto et al. 2006). This accumulation of Fd consequently activated PSI cyclic electron transport. Consistent with the phenotype of PQ reduction at low light intensity, P700 oxidation was delayed in the 35S::PGR5 lines and accelerated in pgr5 during FR light illumination (Fig. 6B). This delay of P700 oxidation in the 35S::PGR5 lines can be explained by enhanced electron donation to PQ via the PSI cyclic pathway compared with the wild type. It is also possible that the phenotype was affected by reduced activity of PQ oxidation via limited electron acceptance from Fd via FNR. Taken together with the nature of the qE phenotype (Fig. 4), both mechanisms are plausible explanations for this result. The phenotype was more evident when the plants were dark adapted for at least 1 h before analysis, as observed in the case of NPQ (Fig. 4). In conclusion, overaccumulation of PGR5 enhanced the activity of PGR5-dependent PSI cyclic electron transport. Taken together with the results from the pgr5 mutant (Munekage et al. 2002, Munekage et al. 2004), the level of accumulation of PGR5 seemed to be correlated with the activity of PSI cyclic electron transport, suggesting that the level of PGR5 drastically modifies the mode of electron transport. However, this effect is transient during fluctuations of light conditions, and the electron transport may be regulated by other factors such as the NADPþ/NADPH ratio during steady-state photosynthesis. Even though the effect of PGR5 overaccumulation is transient, it influences chloroplast development. These results suggest that the PGR5 level is strictly regulated. Since Fd plays crucial roles as a redox regulatory center in flowering plants, it may not be easy to modify the activity of PGR5-dependent PSI cyclic electron transport drastically to cope with fluctuations in environments. Alternatively, plants may have

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selected the strategy of regulating the activity of NDHdependent PSI cyclic electron transport to adapt to environmental stresses (Lascano et al. 2003). Plants may also respond to additional requirements for ATP to energize C4 photosynthesis (Takabayashi et al. 2005).

Materials and Methods Plant materials and growth conditions Arabidopsis thaliana wild type (ecotype Columbia gl1), pgr5 and 35S::PGR5 plants were grown in soil under growth chamber conditions (50 mmol photons m–2 s–1, 16 h light/8 h dark cycles, 238C) for 3–4 weeks. For simultaneous measurement of Chl fluorescence and P700 absorbance, seedlings were cultured under short day conditions (8 h light/16 h dark) for 8 weeks. Transformation of Arabidopsis thaliana The coding region of the PGR5 gene was cloned in pBI121 and introduced into A. thaliana wild type (ecotype Columbia gl1) via Agrobacterium tumefaciens MP90 by the floral dip procedure (Clough and Bent 1998). Transgenic seedlings were selected in terms of resistance to kanamycin on a selection medium. The medium consisted of a full-strength MS medium with 1% sucrose. Analysis of Chl content For pigment analysis, leaves (30 mg fresh weight) were harvested from 1- to 4-week-old seedlings. The materials were immediately frozen in liquid nitrogen and ground in 80% acetone, and then washed three times by centrifugation (15,00 0  g, 5 min). The Chl contents were determined spectrophotometrically by the method of Arnon (1949). Chl fluorescence analysis Chl fluorescence was measured with a MINI-PAM portable Chl fluorometer (Walz, Effeltrich, Germany). Minimum fluorescence at open PSII centers in the dark-adapted state (Fo) was excited by a weak ML (wavelength 650 nm) at a light intensity of 0.05–0.1 mmol photons m–2 s–1. A saturating pulse of white light (800 ms, 3,000 mmol photons m–2 s–1) was applied to determine the maximum fluorescence at closed PSII centers in the dark-adapted state (Fm) and during AL illumination (Fm0 ). The steady-state fluorescence level (Fs) was recorded during AL illumination (20– 1,000 mmol photons m–2 s–1). NPQ was calculated as (Fm–Fm0 )/Fm0 . The quantum yield of PSII (PSII) was calculated as (Fm0 Fs)/Fm0 (Genty et al. 1989). The relative ETR through PSII was calculated as PSII  light intensity (mmol photons m–2 s–1). For the analysis of the light intensity dependence of fluorescence parameters, the intensity of actinic light was increased in a step-wise manner every 2 min after applying a saturating pulse. Simultaneous measurements of Chl fluorescence and absorbance change of P700 The redox change of P700 was monitored by absorbance at a wavelength of 830 nm, and Chl fluorescence was monitored by using a PAM Chl fluorometer (Walz, Effeltrich, Germany) equipped with an emitter-detector unit (ED800T), as previously described (Schreiber et al. 1988). FR light (4700 nm, 36 W m–2) was applied to activate PSI preferentially.

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Function of PGR5 in PSI cyclic electron transport

Isolation of chloroplasts and protein extraction Leaves of 4- to 5-week-old plants were homogenized in a medium containing 330 mM sorbitol, 20 mM Tricine/NaOH (pH 8.4), 5 mM EGTA, 5 mM EDTA, 10 mM NaHCO3, 0.1% (w/v) BSA (bovine serum albumin) and 330 mg l–1 ascorbate. After centrifugation for 5 min at 3,000  g, the pellet was resuspended in 300 mM sorbitol, 20 mM HEPES/KOH (pH 7.6), 5 mM MgCl2 and 2.5 mM EDTA. For immunodetection, intact chloroplasts were further purified through 40% Percoll. Isolated intact chloroplasts were suspended in 20 mM HEPES/KOH (pH 7.6), 5 mM MgCl2 and 2.5 mM EDTA, followed by centrifugation at 10,000  g for 3 min. The pellet (thylakoid membrane fraction) and the soluble protein (stromal fraction) were dissolved in SDS sample buffer. Immunoblot analysis The proteins were separated by 15% SDS–PAGE using the conventional Tris-glycine buffer system for immunodetection of subunits of the Cyt b6f complex and RubisCO, and by 16.5% SDS–PAGE using a Tris-tricine buffer system for PGR5 (Schagger and von Jagow 1987), and transferred onto a PVDF (polyvinylidene fluoride) membrane. Immunodetecetion with antibodies against PGR5 and subunits of the Cyt b6f complex and RubisCO was performed as described in Munekage et al. (2002). In vitro assay of Fd-dependent PQ reduction Fd-dependent PQ reduction activity was measured in ruptured chloroplasts as previously described (Endo et al. 1998). As electron donors, 5 m M maize Fd (Sigma Aldrich) and 0.25 mM NADPH (Sigma Aldrich) were used. Antimycin A (Sigma Aldrich) at 10 m M was added before the measurement. Electron microscopy Chloroplast ultrastructure was observed as described previously (Watanabe et al. 1998). Supplementary material Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp. oxfordjournals.org.

Acknowledgments The authors are grateful to A. Makino and A. Yokota for the antibodies. T.S. was supported by a grant-in-aid for Creative Scientific Research (17GS0316) from JSPS and from Scientific Research on Priority Areas (16085206) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Y.O. was supported by a grant-in-aid (19-8015) from JSPS.

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(Received July 5, 2007; Accepted August 23, 2007)