Title: How does cyclic electron flow alleviate photoinhibition in Arabidopsis?

33 Pages·2008·0.36 MB·English

Plant Physiology Preview. Published on December 31, 2008, as DOI:10.1104/pp.108.134122
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Title: How does cyclic electron flow alleviate photoinhibition in Arabidopsis?
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Shunichi Takahashi*, Sara E Milward, Da-Yong Fan, Wah Soon Chow and Murray R
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Badger
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Molecular Plant Physiology Group and ARC Center of Excellence in Plant Energy
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Biology, Research School of Biological Sciences, The Australian National University,
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Canberra, Australian Capital Territory, 0200 Australia (S.T., S.E.M., M.R.B.);
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Photobioenergetics Group, Research School of Biological Sciences, The Australian
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National University, Canberra, Australian Capital Territory, 0200 Australia (D.-Y.F.,
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W.S.C.); and State Key Laboratory of Vegetation and Environmental Change, Institute
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of Botany, The Chinese Academy of Sciences, Beijing 100093, China (D.-Y.F.)
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Corresponding author:
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*Dr. Shunichi Takahashi; Address: Molecular Plant Physiology Group and ARC Center
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of Excellence in Plant Energy Biology, Research School of Biological Sciences, The
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Australian National University, Canberra, Australian Capital Territory, 0200 Australia;
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Tel: [phone]; Fax: [phone]; Email: [email protected]
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Manuscript information: Text pages: 29 p; Figures: 5 figures (plus 3 supplemental
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figures).
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Word and character Counts: The number of words in the abstract: 217 words; the
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total number of characters in the paper: 39932.
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Abbreviations Footnote: CEF, cyclic electron flow; NPQ, non-photochemical
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Copyright 2008 by the American Society of Plant Biologists
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quenching of chlorophyll fluorescence; PSI, photosystem I; PSII, photosystem II; qE,
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thermal energy dissipation; VDE, violaxanthin de-epoxidase.
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ABSTRACT
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Cyclic electron flow (CEF) around photosystem I (PSI) has a role in avoiding
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photoinhibition of photosystem II (PSII), which occurs under conditions where the rate
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of photodamage to PSII exceeds the rate of its repair. However, the molecular
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mechanism underlying how CEF contributes to photoprotection is not yet well
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understood. We examined the effect of impairment of CEF and thermal energy
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dissipation (qE) on photoinhibition using CEF (pgr5) and qE (npq1 and npq4) mutants
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of Arabidopsis thaliana exposed to strong light. Impairment of CEF by mutation of
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pgr5 suppressed qE and accelerated photoinhibition. We found that impairment of qE,
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by mutations of pgr5, npq1 and npq4, caused inhibition of the repair of photodamaged
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PSII at the step of the de novo synthesis of the D1 protein. In the presence of the
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chloroplast protein synthesis inhibitor chloramphenicol, impairment of CEF, but not
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impairment of qE, accelerated photoinhibition and a similar effect was obtained when
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leaves were infiltrated with the protonophore, nigericin. The results suggest that
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CEF-dependent generation of pH across the thylakoid membrane helps to alleviate
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photoinhibition by at least two different photoprotection mechanisms: one is linked to
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qE generation and prevents the inhibition of the repair of photodamaged PSII at the step
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of protein synthesis; and the other is independent of qE and suppresses photodamage to
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PSII.
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INTRODUCTION
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Light damages the photosynthetic machinery, primarily photosystem II (PSII),
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during photosynthesis, thereby causing photoinhibition (Takahashi and Murata, 2008).
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Since photoinhibition decreases photosynthetic efficiency, plants need to manage
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photoinhibition for optimal growth and productivity. Mutation of genes encoding
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proteins involved in cyclic electron flow (CEF) around photosystem I (PSI) increases
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the sensitivity of plants to photoinhibition of PSII (and also PSI) under strong light
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(Munekage et al., 2002). Thus, CEF has been implicated as one of the mechanisms
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responsible for protecting PSII from photoinhibition in conditions where the absorption
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of light energy for photosynthesis exceeds its rate of consumption in chloroplasts.
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CEF generates a pH across the thylakoid membrane through increased
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electron transfer from PSI back to plastoquinone. The flow of electrons during CEF
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occurs via two pathways: the NAD(P)H dehydrogenase-dependent and antimycin
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A-sensitive pathways (Shikanai, 2007). However, the majority of CEF induced pH is
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associated with the antimycin A-sensitive pathway (Munekaga et al., 2004). Recent
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studies have demonstrated that the antimycin A-sensitive pathway is dependent on a
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PGR5-PGRL1 complex that interacts with PSI (DalCorso et al., 2008). It is likely that
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the antimycin A-sensitive pathway is regulated by the redox state of the NADPH pool
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and is facilitated under excess light conditions (Okegawa et al., 2008).
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Photoinhibition is due to net photodamage of PSII. In previous photodamage
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models, light energy absorbed by photosynthetic pigments was assumed to cause
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photodamage to PSII through acceptor- and donor-side photoinhibition. However,
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recent studies have demonstrated that photodamage is attributable to light absorbed
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directly by manganese in the oxygen-evolving complex (Hakala et al., 2005; Ohnishi et
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al., 2005; Nishiyama et al., 2006; Tyystjärvi, 2008). After photodamage to the
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oxygen-evolving complex, the reaction centre of PSII is subsequently damaged by light
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absorbed by photosynthetic pigments (Hakala et al., 2005; Ohnishi et al., 2005). The
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photodamaged PSII is rapidly and effectively repaired through the replacement of
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photodamaged PSII proteins with newly synthesized proteins, primarily the D1 protein
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(Ohad et al., 1984; Mattoo and Edelman, 1987; Aro et al., 1993; Aro et al., 2005).
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Thus, photoinhibition occurs only under conditions where the rate of photodamage
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exceeds the rate of its repair (Aro et al., 1993; Murata et al., 2007; Takahashi and
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Murata, 2008). To prevent photoinhibition, photoprotective mechanisms are used by
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the plant to both suppress the photodamage to PSII (eg., chloroplast avoidance
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movement (Kasahara et al., 2002)) and to minimise oxidative inhibition of the repair of
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photodamaged PSII (eg., photorespiratory pathway and Calvin cycle (Takahashi et al.,
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2007) and ROS scavenging systems (Nishiyama et al., 2001)).
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Under conditions of excess light, plants dissipate unused absorbed light energy
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harmlessly as heat in the antenna proteins of PSII (see review (Niyogi, 1999)). This
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mechanism is called energy-dependent thermal dissipation (qE) and is measured as a
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component of non-photochemical quenching of chlorophyll fluorescence (NPQ). NPQ
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can be subdivided into three components, qE, photoinhibition (qI), and state transitions
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(qT). Under strong light conditions, qE is the major component of NPQ and quenches
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up to 80% of the excited chlorophyll in plants. qE is associated with the conversion of
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violaxanthin to zeaxanthin, via the intermediate antheraxanthin, by the catalyst,
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violaxanthin de-epoxidase (VDE) (Niyogi et al., 1997; Niyogi et al., 1998) and
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protonation of PsbS (Li et al., 2002), which is an integral membrane subunit of PSII.
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Both reactions are regulated by low lumenal pH, which is accompanied by the
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generation of pH (Munekage et al., 2002; Shikanai, 2007). Thus, generation of
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increased pH through CEF is important for the activation of qE. Impairment of qE
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by the mutation of genes coding for the proteins VDE (npq1) and PsbS (npq4) in
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Arabidopsis causes acceleration of photoinhibition of PSII under strong light (Niyogi et
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al., 1998; Li et al., 2002). Since, in the previous photoinhibition model, photodamage
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to PSII was proposed to be attributable to light absorbed by photosynthetic pigments,
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energy dissipation through qE was assumed to help avoid photodamage to PSII.
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However, recent studies have demonstrated that impairment of qE had no significant
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effect on the process of photodamage per se to PSII which is consistent with the new
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photoinhibition model (Nishiyama et al., 2006; Sarvikas et al., 2006). Therefore,
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mechanisms of photoprotection associated with CEF and qE are still undefined.
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To further understand the role of CEF in photoprotection, we examined the
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effect of impairment of CEF and qE on photoinhibition of PSII under strong light using
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Arabidopsis thaliana mutants impaired in CEF (pgr5) and qE development (npq1 and
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npq4). The results clearly suggest that reduction in both CEF and qE resulted in
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inhibition of the synthesis of the D1 protein under strong light but only reduction in
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CEF caused an increase in direct photodamage to PSII.
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RESULTS
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Characteristics of pgr5 mutant
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All plants used in the present study were grown in medium light at 150 mol
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photons m-2 s-1. The pgr5 mutant, but not the npq1 or the npq4 mutants, grew slightly
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slower than the wild type. However, there was no significant difference in phenotype
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of mature leaves between the wild type and all mutants used. The photosynthetic CO
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fixation rate in mature leaves of the wild type was 6.2, 17.0 and 18.5 mol CO m-2 s-1
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in light at 100, 500 and 1000 mol photons m-2 s-1, respectively (Fig. 1A). The CO
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fixation rates were suppressed by 10-20% in the pgr5 mutants at all light intensities (Fig.
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1A). The photosynthetic O evolution rate in the wild type was 6.7, 18.4 and 21.1
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mol O m-2 s-1 in light at 100, 500 and 1000 mol photons m-2 s-1, respectively (Fig.
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1B). The effect of mutation of pgr5 on the O evolution rate was similar to that on the
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CO fixation rate (Fig. 1B). The CO fixation rate and the O evolution rate were
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suppressed by 10-20% in the npq4 mutant, but not significantly in the npq1 mutant, at
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any light intensity tested (Fig. 1A and B). Results indicate that there was no
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significant difference in the effect of mutations of pgr5, npq1 and npq4 on the
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photosynthetic activity at all light intensities, suggesting that reductions of CEF and qE
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had no direct effect on the photosynthetic activity.
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Impairment of CEF accelerated photoinhibition in both presence and absence of
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chloramphenicol
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The effect of impairment of CEF by the mutation of pgr5 on qE was examined
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by the measurement of NPQ (Fig. 2A). When the wild type was exposed to light at
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1000 mol photons m-2 s-1 for 10 min, the level of NPQ was increased to 2.5.
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However, in the pgr5 mutant, the level of NPQ increased to only 40% of wild type.
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When leaf disks from wild type and pgr5 mutant were infiltrated with 250 M
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antimycin A, which inhibits PGR5-dependent CEF, the level of NPQ was strongly
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suppressed in both the wild type and the pgr5 mutant, and the effect of pgr5 mutation
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on the level of NPQ was almost abolished (Supplemental Fig. 1A).
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To examine the effect of impairment of CEF on photoinhibition of PSII, we
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measured the maximal quantum yield of PSII (F /F ) in the wild type and the pgr5
v m
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mutant after exposure to strong light at 1000 mol photons m-2 s-1 (Fig. 2B). In the
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wild type, the F/F declined to 60% of initial level after exposure for 3h. However, in
v m
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the pgr5 mutant, the F /F declined to 40% of initial level. The presence of antimycin
v m
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A accelerated the decrease in F /F in both the wild type and the pgr5 mutant and
v m
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completely abolished the effect of pgr5 mutation on the decrease in the F/F
v m
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(Supplemental Fig. 1B). The results indicate that impairment of the CEF causes
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acceleration of photoinhibition. Antimycin A accelerated photoinhibition in the pgr5
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mutant, suggesting that acceleration of photoinhibition caused by antimycin A was not
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only due to inhibition of antimycin A (PGR5)-dependent CEF but also inhibition of
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other reactions (i.e., the alternative pathway in mitochondrial respiration).
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The effect of impairment of CEF on the process of photodamage to PSII was
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examined by the measurement of the F/F after exposure to strong light in the presence
v m
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of chloramphenicol (Fig. 2B). Leaf discs from the wild type and the pgr5 mutant were
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vacuum infiltrated with chloramphenicol and then exposed to strong light at 1000 mol
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photons m-2 s-1 for 3h. Chloramphenicol accelerated the decrease in the level of F /F
v m
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in both the wild type and the pgr5 mutant, and the extent of decrease was significantly
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faster in the pgr5 mutant (Fig. 2B). However, the effect of pgr5 mutation on the
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decrease in the level of F/F was completely abolished in the presence of antimycin A
v m
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(Supplemental Fig. 1). These results indicate that impairment of CEF caused
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acceleration of the photodamage to PSII.
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Impairment of qE accelerated photoinhibition only in the absence of
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chloramphenicol
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To examine whether the acceleration of photoinhibition caused by impairment
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of CEF is attributable to impairment of qE, the effect of mutation of genes encoding
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npq1 and npq4 on a decrease in the level of the F /F was measured in the absence or
v m
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presence of chloramphenicol. When npq1 and npq4 mutants were exposed to strong
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light at 1000 mol photons m-2 s-1 for 10 min, the level of NPQ was induced to 40% of
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the wild type in both mutants and indistinguishable to that in the pgr5 mutant (Fig 2A).
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When leaf discs were exposed to strong light in the absence of chloramphenicol,
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mutations of npq1 and npq4 caused acceleration of a decrease in the level of the F/F
v m
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(Fig. 2B). However, in the presence of chloramphenicol, there was no significant
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effect of npq1 and npq4 mutations on the decrease in the level of the F /F (Fig. 2B).
v m
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Results indicate that impairment of qE by mutations of npq1 and npq4 caused
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acceleration of photoinhibition through inhibition of the repair of photodamaged PSII
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but not acceleration of the photodamage to PSII. Thus, acceleration of photodamage
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due to impairment of CEF by the mutation of pgr5 was not attributable to suppression
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of qE.
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Methyl viologen accelerated photoinhibition only in the absence of
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chloramphenicol
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We examined whether the production of hydrogen peroxide caused acceleration
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of photoinhibition of PSII in Arabidopsis. Leaf discs from the wild type and the pgr5
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mutant were vacuum infiltrated with 200 M methyl viologen that generates superoxide
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and hydrogen peroxide through the electron transfer to oxygen at PSI. In the absence
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of chloramphenicol, methyl viologen accelerated a decrease in the level of F/F in both
v m
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the wild type (Fig. 3B) and the pgr5 mutant (supplemental figure 2B). However, in the
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presence of chloramphenicol, there was no significant effect of methyl viologen on the
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decrease in the level of the F/F in both the wild type (Fig. 3B) and the pgr5 mutant
v m
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(supplemental figure 2B). Results indicate that the production of reactive oxygen
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species, such as hydrogen peroxide and superoxide, accelerated photoinhibition through
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inhibition of the repair of photodamaged PSII but not acceleration of the photodamage
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to PSII. These results were consistent with previous report in cyanobacteria
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(Nishiyama et al., 2001). Thus, acceleration of photodamage caused by impairment of
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CEF is not attributable to the production of reactive oxygen species.
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Nigericin accelerated photoinhibition in both the presence and absence of
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chloramphenicol
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We examined whether inhibition of the generation of pH across the thylakoid
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membrane causes acceleration of photoinhibition of PSII using a protonophore nigericin.
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Leaf discs from the wild type and the pgr5 mutant were vacuum infiltrated with 1 mM
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nigericin. The effect of nigericin as a protonophore was examined by suppression of
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the NPQ development under strong light in both the wild type (Fig. 3A) and the pgr5
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mutant (supplemental figure 2A). When leaf discs from the wild type and the pgr5
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mutant were exposed to strong light, nigericin accelerated the decrease in the level of
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