A deficiency in chloroplastic ferredoxin 2 facilitates effective photosynthetic capacity during long-term high light acclimation in Arabidopsis thaliana

Authors

  • Jun Liu,

    1. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat–sen University, Guangzhou, China
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    • These authors contributed equally to this work.
  • Peng Wang,

    1. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat–sen University, Guangzhou, China
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    • These authors contributed equally to this work.
  • Bing Liu,

    1. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat–sen University, Guangzhou, China
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  • Dongru Feng,

    1. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat–sen University, Guangzhou, China
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  • Jie Zhang,

    1. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat–sen University, Guangzhou, China
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  • Jianbin Su,

    1. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat–sen University, Guangzhou, China
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  • Yang Zhang,

    1. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat–sen University, Guangzhou, China
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  • Jin–Fa Wang,

    1. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat–sen University, Guangzhou, China
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  • Hong–Bin Wang

    Corresponding author
    1. State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat–sen University, Guangzhou, China
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Summary

Photosynthetic electron transport is the major energy source for cellular metabolism in plants, and also has the potential to generate excess reactive oxygen species that cause irreversible damage to photosynthetic apparatus under adverse conditions. Ferredoxins (Fds), as the electron-distributing hub in the chloroplast, contribute to redox regulation and antioxidant defense. However, the steady-state levels of photosynthetic Fd decrease in plants when they are exposed to environmental stress conditions. To understand the effect of Fd down-regulation on plant growth, we characterized Arabidopsis thaliana plants lacking Fd2 (Fd2–KO) under long-term high light (HL) conditions. Unexpectedly, Fd2–KO plants exhibited efficient photosynthetic capacity and stable thylakoid protein complexes. At the transcriptional level, photoprotection-related genes were up-regulated more in the mutant plants, suggesting that knockout Fd2 lines possess a relatively effective photo-acclimatory responses involving enhanced plastid redox signaling. In contrast to the physiological characterization of Fd2–KO under short-term HL, the plastoquinone pool returned to a relatively balanced redox state via elevated PGR5-dependent cyclic electron flow during extended HL. fd2 pgr5 double mutant plants displayed severely impaired photosynthetic capacity under HL treatment, further supporting a role for PGR5 in adaptation to HL in the Fd2–KO plants. These results suggest potential benefits of reducing Fd levels in plants grown under long-term HL conditions.

Introduction

Higher plants grown under natural conditions inevitably face fluctuating light intensity. Absorption of excess light intensity under high light (HL) induces an imbalance in the distribution of excitation energy between the two photosystems and results in photo-oxidative damage to photosynthetic pigments and proteins, which in turn decreases the photosynthetic capacity, plant growth and productivity (Li et al., 2009). Photosynthetic protein complexes are connected in series to create the photosynthetic electron transport chain (PETC) in the thylakoid membranes, are responsible for the capture of light, and mediate the primary reactions of photosynthesis (Nelson and Yocum, 2006; Rochaix, 2011). Among these complexes, photosystem II (PSII) is particularly prone to damage by exposure to HL; this damage must be rapidly and effectively repaired to avoid photo-inhibition (Keren et al., 2005). In plants, HL is sensed by multiple signals, which are subsequently transduced into photoprotective and photo-acclimatory responses, including dissipation of absorbed light energy as heat (qE), scavenging systems for reactive oxygen species (ROS), and electron transfer flow alteration via cyclic electron flow (CEF) around photosystem I (PSI) (Li et al., 2009; Joliot and Johnson, 2011). CEF is considered to contribute to generation of a transmembrane proton gradient that is involved in the synthesis of ATP, which supplies energy to repair the photodamaged PSII subunits during recovery of PSII activity. CEF occurs via two pathways: the antimycin-sensitive proton gradient regulation 5 (PGR5)/PGR5-like photosynthetic phenotype 1 (PGRL1)-mediated pathway and the NAD(P)H dehydrogenase-dependent pathway. It is the PGR5/PGRL1-mediated pathway that is mainly responsible for CEF in C3 plants and the induction of PGR5/PGRL1-mediated CEF is facilitated by HL (Shikanai, 2007; Okegawa et al., 2008). In addition, the redox regulation of gene expression is also an important acclimatory response to HL (Yao et al., 2011; Foyer et al., 2012). As the electron acceptor of PSII, the plastoquinone (PQ) pool is generally considered to be an ideal redox sensor to ensure balanced excitation of PSI and PSII (Pfannschmidt et al., 2001; Li et al., 2009). The increasing number of identified regulatory components has revealed a complex redox regulatory network that enables chloroplasts to respond to flexible environmental conditions (Dietz and Pfannschmidt, 2011).

Ferredoxins (Fds) are a group of small, soluble electron carriers that distribute electrons from PSI to diverse acceptors involved in chloroplast metabolic processes (Arnon, 1988). At least four Fd isoforms have been identified in plants, including leaf-type Fds and root-type Fds. Fd1 and Fd2 are considered to be leaf-type Fds, which share high sequence identity but accumulate at different abundances. In Arabidopsis thaliana, Fd1 and Fd2 constitute 10 and 90% of the total leaf-type Fds, respectively (Hanke et al., 2004). Knockout and RNA interference (RNAi) Arabidopsis plants lacking Fd2 showed a lower rate of linear electron flow (LEF) and an over-reduced PETC, while fd1-RNAi plants showed an enhanced LEF rate during light induction (Hanke and Hase, 2008; Voss et al., 2008). The results support the notion that Fd1 and Fd2 are, at least partially, functional divided between CEF and LEF, respectively (Hanke et al., 2004). As the major mobile electron carrier in plants, Fds play an important role in the regulation of multiple metabolic pathways and transduction of redox signals into the regulatory network (Scheibe and Dietz, 2012). Down-regulation of Fd in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum), and knockout or knockdown of Fd2 in Arabidopsis, resulted in plants showing growth arrest and inactivation of photosynthesis (Holtgrefe et al., 2003; Hanke and Hase, 2008; Voss et al., 2008; Blanco et al., 2011). The results of the study in potato also implied that a decrease in Fd below 40% of wild-type (WT) levels would fatally affect plant growth (Holtgrefe et al., 2003). However, genome-wide analysis of transcription profiles indicates that the level of Fd-specific transcripts decreases when plants are grown under hostile environmental conditions (Zimmermann et al., 2004). The study in tobacco also showed a decrease in Fd protein content under stress conditions such as low temperature and water deprivation (Tognetti et al., 2006). Also, Fd levels decrease under stress conditions in many photosynthetic micro-organisms, which share a common ancestor with chloroplasts (Falk et al., 1995; Mazouni et al., 2003). It is worth noting that a compensatory mechanism exists in the micro-organisms to cope with the consequences of Fd decrease under adverse conditions by inducing synthesis of flavodoxin, an isofunctional electron carrier that is also involved in most electron transfer processes, but the gene encoded flavodoxin is not found in plants (Tognetti et al., 2006; Zurbriggen et al., 2007).

At present, the mechanism of Fd down-regulation in plants under adverse conditions is unclear. In this study, we aimed to investigate the physiological significance of decreased Fd levels with regard to plant acclimation to HL stress conditions in Arabidopsis. Interestingly, Fd2 transcript and protein levels gradually decreased with extended HL irradiance. Fd2 knockout (Fd2–KO) plants exhibited unexpected tolerance to prolonged HL treatment compared with WT plants. Furthermore, the biochemical and molecular analyses in vivo revealed that the deficiency of Fd2 stimulated photoprotective mechanisms involving up-regulation of photoprotection-related genes and elevation of PGR5-dependent CEF under long-term HL conditions. This maintained photosynthetic capacity and stabilized the photosynthetic apparatus in Fd2–KO plants. Collectively, our results indicate that down-regulation of the major Fd in Arabidopsis may serve as a protective strategy for plants grown under long-term HL stress conditions.

Results

Phenotypic response of WT and Fd2–KO plants to prolonged HL treatment

Experimental analysis has shown that Fd protein abundance decreased in tobacco when plants are exposed to oxidative stress conditions (Tognetti et al., 2006). We wished to assess the changes in Fd expression under long-term adverse conditions in Arabidopsis. To this end, the relative transcript levels of Fd1 and Fd2 in WT (Nossen) plants grown under continuous moderate HL were determined by quantitative real-time PCR. The data showed that the Fd2 transcript levels in the WT plants remained stable for 24 h of HL relative to that under GL, but were down-regulated to a lower level (approximately 10% of that in unstressed plants) after an extended period of HL treatment (up to 120 h HL; Figure 1a). In contrast to Fd2, the Fd1 transcript levels exhibited a more than twofold increase under 48 h HL, before decreasing to a lower level during prolonged HL treatment. To confirm a similar decrease in protein levels, immunoblot analysis was performed using an antibody raised against plant Fd (Method S1). The results showed that the Fd2 protein levels in WT plants decreased gradually during HL treatment to approximately 13% of that in unstressed WT plants after 120 h HL (Figure 1b,c). The results suggest that down-regulation of Fd2, the major photosynthetic Fd, occurred in a stepwise manner in response to long-term HL stress in Arabidopsis.

Figure 1.

Relative mRNA transcript and protein abundance of Fds in WT Arabidopsis during long-term HL treatment. (a) Changes in relative mRNA transcript levels of AtFd1 and AtFd2 in WT plants during long-term HL treatment. Expression levels were normalized to the expression of UBQ4. Values are means ± SE from three independent quantitative real-time PCR experiments. (b) Immunoblot analysis of Fd2. Total proteins from WT plants at various HL time points were probed using anti-Fd and anti-RbcL antisera. (c) Levels of Fd2 (normalized against RbcL) are shown relative to the amounts measured in WT plants under GL, for which the value was set to 1. Values are means ± SD of three biological replicates. r.u., relative units of densitometry intensity.

To dissect the effects of decreasing Fd2 expression on long-term stress, we used a knockout mutant lacking Fd2 (Fd2–KO) in Arabidopsis. Fd2–KO plants showed retarded growth and reduction of photosynthetic characteristics compared to the corresponding WT plants when grown under growth light conditions (GL) (Figure S1a and Table S1). However, the plastid ultrastructure, as determined by transmission electron microscopy (TEM), was not significantly different between WT and Fd2–KO plants (Figure S1b), indicating that lack of the major Fd does not affect plastid development. We then investigated the performance of Fd2–KO plants during long-term HL stress (up to 120 h). Plants grown under GL for 4 weeks were exposed to continuous HL. The leaves of WT plants gradually became shriveled during extended HL treatment, but the Fd2–KO plants remained relatively vigorous and green (Figure 2a). This was verified by using trypan blue staining to detect cell death in the leaves (Figure S2 and Method S2). Clusters of dead cells were clearly observed only in the leaves of WT after 48 h HL, while cell death was significantly attenuated in the Fd2–KO plants. After 10 days of continuous HL, the Fd2–KO mutant seedlings still grew better than WT plants (Figure S3). These results provide a preliminary indication that Fd2–KO plants adapt well to long-term HL stress.

Figure 2.

Phenotype and photosynthetic characteristics of Fd2–KO and WT plants under GL and long-term HL exposure. (a) Phenotype of Fd2–KO and WT plants during long-term HL treatment. Scale bar = 1 cm. (b) Fluorescence images for measurement of Fv/Fm. False color codes range from black (0,) via red, orange, yellow, green, blue and violet to purple (1). (c–f) Changes in the values for Fv/Fm (c), Y(II) (d), Pm (e) and Y(I) (f) for Fd2–KO and WT plants under long-term HL conditions. Values are means ± SE from six independent biological replicates. Asterisks indicate significant differences for Fd2–KO plants compared with the respective WT plants (**< 0.01).

Fd2 deficiency enhances the recovery of photosynthetic efficiency following long-term HL treatment

To investigate the physiological basis for the HL-resistant phenotype of Fd2–KO, the maximum quantum yield of PSII (Fv/Fm) was evaluated at a series of time points. The Fv/Fm values were similar (approximately 0.82) for both WT and Fd2–KO plants under GL, which is a typical value for uninhibited Arabidopsis plants (Bjorkman and Demmig, 1987; Baker, 2008). After transfer to HL conditions, WT plants displayed a slightly decreased Fv/Fm of 0.75 ± 0.04, whereas mutants had a markedly lower value of 0.65 ± 0.03 at 12 h HL (Figure 2b,c), indicating that Fd2–KO plants experienced severe photo-inhibition in the early stages of HL treatment. However, the Fv/Fm value recovered in the Fd2–KO plants, and there was no significant difference between WT and Fd2–KO plants at 48 h HL treatment. Surprisingly, when the HL treatment was extended, the Fv/Fm ratio in the Fd2–KO plants increased to 0.81 ± 0.02, compared with 0.67 ± 0.08 in WT plants, after 120 h HL (Figure 2b,c). In agreement with the observed Fv/Fm values, the operating PSII photosynthetic efficiency, Y(II), showed similar changes during HL treatment in the Fd2–KO plants. The Y(II) value in the Fd2–KO plants decreased rapidly from 0.42 ± 0.02 to 0.07 ± 0.01 during the first 12 h of HL, and recovered to 0.3 ± 0.02 (a 70% higher value than that for WT) after 120 h HL (Figure 2d). The data imply that the Fd2–KO mutants have the ability to recover PSII photosynthetic capacity during long-term HL exposure.

As Fd2–KO plants showed a markedly decreased P700 oxidation level under short-term HL treatment (Voss et al., 2008), but the PSII capacity cannot be recovered when PSI is severely damaged by HL (Huang et al., 2010), we assessed the effects of long-term HL stress on PSI activity in the Fd2–KO mutants. To do so, the maximum photo-oxidizable P700 (Pm), an indicator of the functional intactness of PSI, and the photochemical quantum yield of PSI (Y[I]) were measured. The Pm value in Fd2–KO plants dramatically decreased to 0.15 ± 0.01 after 24 h HL, but returned to 0.56 (close to the initial value in untreated mutant plants) after 120 h of HL. In contrast, Pm gradually decreased from a higher level (approximately 0.69) to 0.39 ± 0.05 in equivalently treated WT plants (Figure 2e). Similarly, Y(I) showed a much lower initial value in Fd2–KO plants as the acceptor side of PSI was limited for electron transfer under GL, but this value increased from 0.18 ± 0.03 to 0.39 ± 0.04, approximately 195% of the WT value, after 120 h HL (Figure 2f). A possible interpretation is that the higher PSI activity facilitates maintenance of higher levels of PSII efficiency for Fd2–KO plants under long-term HL exposure.

The composition and abundance of photosynthetic complexes are stably maintained in Fd2–KO plants after long-term HL

We hypothesized that the efficient photosynthetic capacity in mutants following long-term HL stress may be associated with stable protein levels in the photosynthetic complexes. To address this possibility, we compared thylakoid proteins levels in WT and Fd2–KO plants. Immunoblot analysis was performed using antibodies raised against specific subunits of the photosynthetic protein complexes. After 120 h of HL treatment, the levels of plastid-encoded PSII and cytochrome b6/f subunits were decreased in WT plants, but remained stable or even increased in the Fd2–KO plants (Figure 3a,b). The amounts of the light-harvesting complex (LHC) proteins Lhcb1 and Lhca1, the PSI reaction center protein PsaA and the ATPase β–subunit (Atpβ) were similar in both WT and Fd2–KO (Figure 3a,b). Chlorophyll levels were significantly lower in the Fd2–KO plants prior to HL treatment (Table S1 and Method S3), suggesting that the total thylakoid protein amounts loaded into the gel may be higher in Fd2–KO plants than in WT plants. Therefore, we determined the chlorophyll levels after HL treatment. The chlorophyll content in WT plants decreased dramatically after 120 h of HL to a level that was lower than that of Fd2–KO plants (Figure 3c). As a result, the higher levels of thylakoid proteins in Fd2–KO relative to WT were pronounced when normalized to the fresh weight after 120 h HL treatment (Figure 3d).

Figure 3.

Immunoblot analysis of representative thylakoid proteins from WT and Fd2–KO plants. (a) Immunoblot analysis of thylakoid proteins isolated from leaves of HL-treated and GL-grown WT and Fd2–KO plants using 15% SDS-urea-PAGE. Aliquots of 0.5 μg (¼ Fd2–KO), 1 μg (½ Fd2–KO), 2 μg (Fd2–KO) and 2 μg (WT) chlorophyll were loaded. Blots were probed with antibodies against the individual subunits of PSII (D1, D2, CP43 and CP47), PSI (PsaA), cytochrome b6f (Cyt f) and the nuclear-encoded proteins Lhcb1, Lhca1, subunit of oxygen evolving system of PSII (PsbO) and Atpβ. For each protein, a representative example of at least three biological replicates is shown. (b) Densitometry of photosynthetic proteins from the immunoblots in (a). The signal intensity of the immunoblots for WT under GL was set to 1. (c) Chlorophyll content of 4-week-old seedlings estimated based on leaf fresh weight under GL or 120 h HL. Values are means ± SE of three biological replicates. (d) Protein quantification from the immunoblots in (a) normalized by leaf fresh weight based on the chlorophyll content of plants under GL or HL conditions. The protein abundance is relative to the amount present in WT under GL, for which the value is set to 1. Values are means ± SD of three biological replicates. Asterisks indicate significant differences for Fd2–KO plants compared with the respective WT plants (**< 0.01).

The putative structural state of the photosynthetic protein complexes was analyzed using BN/SDS–PAGE. Six major photosynthetic complexes were detected and labeled I–VI (Figure 4a) (Peng et al., 2008). The levels of thylakoid protein complexes in Fd2–KO plants were similar to WT when grown under GL, but were markedly higher in the Fd2–KO mutants after 120 h HL (Figure 4a). Two-dimensional SDS-urea-PAGE gels showed that the subunits of PSII (D1, D2, CP43 and CP47), especially in the PSII super-complexes and dimeric PSII complex, were markedly reduced in WT plants, but were stable in the mutant plants after 120 h of HL (Figure 4b). Taken together, the results indicate that the steady level of thylakoid protein complexes correlates well with the efficiency of the photosynthetic capacity in Fd2–KO under HL conditions.

Figure 4.

Analysis of thylakoid protein complexes from WT and Fd2–KO plants. (a) BN-PAGE analysis of thylakoid photosynthetic complexes. Thylakoids were isolated from equal amounts of chlorophyll (8 μg) obtained from WT and Fd2–KO plants under GL and 120 h HL. The bands detected were identified as specific protein complexes in accordance with previously published profiles (Peng et al., 2008). (b) Two-dimensional separation of protein complexes in the thylakoid membranes. Individual lanes from the BN gels in (a) were analyzed by 15% SDS-urea-PAGE, and stained using CBB. The experiment was repeated three times with similar results.

Fd2-deficient plants exhibit an altered PETC redox state

Depletion of Fd2 has been reported to cause an over-reduction of the PETC (Voss et al., 2008), but the photosynthetic capacity was restored in the mutants after prolonged HL. To investigate the effect of Fd2 deficiency on the redox changes in the PETC during long-term HL treatment, the redox state of the PQ pool was evaluated. It is widely accepted that a positive correlation exists between reduction of QA, the primary quinone electron acceptor of PSII, and reduction of the PQ pool (QA reduction is calculated as 1 – qP or 1 – qL; Kramer et al., 2004). The value of 1 – qP was higher in the Fd2–KO plants during the first 24 h of HL, but subsequently decreased from 0.62 ± 0.07 at 24 h of HL to 0.46 ± 0.06 after 120 h HL treatment, which was lower than that in WT (0.68 ± 0.05; Figure 5a). The value of 1 – qL showed a similar trend (Figure 5b). As HL represents a non-steady state condition, 1 – qP or 1 – qL may not be the right parameter to describe the QA redox state. Consequently, we used F′/Fm, the raw signal of the chlorophyll fluorescence yield, as an indicator of the relative QA redox state (Suorsa et al., 2012). F′/Fm was markedly higher in the Fd2–KO plants than in WT during 72 h of HL treatment, indicating an imbalance in the energy excitation between the two photosystems. However, upon continued HL treatment for 120 h, the QA reduction state gradually decreased to 0.26 ± 0.02, which is appreciably lower than the WT value (0.43 ± 0.07; Figure 5c). The results confirm that the redox state of the PQ pool became relatively rebalanced during long-term HL treatment in the Fd2–KO mutants.

Figure 5.

Changes in the relative QA redox state in plants during HL treatment. Changes in 1 – qP (a), 1 – qL (b) and F′/Fm (c) for WT and Fd2–KO plants during HL treatment. Values are means and SE for three biological replicates. Asterisks indicate statistically significant differences for Fd2–KO compared with the respective WT plants (**< 0.01).

Changes in nuclear-encoded gene expression in the Fd2–KO plants

The redox states of components of the PETC, primarily plastoquinone (PQ), may release signals to regulate expression of nuclear genes that are responsible for stress acclimation (Fernandez and Strand, 2008). To investigate the effect of Fd2 mutations on the regulation of gene expression under HL, quantitative real-time PCR was performed to examine relative transcript levels of HL-responsive genes related to the PQ redox state (Li et al., 2009). The expression profiles for most of the genes assayed were similar for WT and Fd2–KO plants following the shift to HL and exhibit HL-induced accumulation; however, the degree of elevation for some of these genes was much higher in the Fd2–KO mutants (Figure 6). Specifically, the transcript levels of Early-light induced protein 2 (ELIP2) (Hutin et al., 2003) in Fd2–KO under GL were approximately 0.17 ± 0.003. Upon induction of HL for 12 and 24 h, these levels were increased to 77 ± 9.6 and 85 ± 13.8, respectively. This increase (approximately 400-fold) is much larger than the 30-fold increase observed in identically treated WT plants at 12 h HL. Two members of a zinc-finger transcription factor family, ZAT10 and ZAT12 (Iida et al., 2000; Rossel et al., 2007), showed 1.6- and 2.2-fold reductions, respectively, in expression levels in Fd2–KO plants compared to WT under GL conditions. However, these two genes were highly up-regulated in the mutants between 12 and 48 h of HL. ZAT12 was especially up-regulated, with levels five times higher than in WT after 24 h HL treatment. Also, expression of cytosolic ascorbate peroxidase 2 (APX2) was five times lower in the mutants than WT levels under GL conditions, but 3.3 times higher after 24 h HL treatment. In addition, studies have shown that HL treatment rapidly inhibits Lhcb gene transcription (Nott et al., 2006; Li et al., 2009). In this study, the Lhcb2.4 expression level was slightly higher in Fd2–KO under GL, but decreased rapidly to a level similar to that in WT after plants were exposed to HL conditions (Figure 6).

Figure 6.

HL-inducible nuclear gene expression analysis in plants after long-term HL treatment. Expression levels were normalized to those for the UBQ4 gene, and were then normalized to expression in WT plants grown under GL. Error bars indicate SE obtained from three independent quantitative real-time PCR experiments. Asterisks indicate statistically significant differences for Fd2–KO compared with the respective WT plants (**< 0.01).

It has been reported that all antioxidant enzyme genes are nuclear-encoded, and their expression is controlled by retrograde signaling mechanisms (Baier and Dietz, 2005). We compared the transcript regulation of four genes encoding the thylakoid-bound and stromal ascorbate peroxidases, dehydroascorbate reductase and copper/zinc superoxide dismutase (tAPX, sAPX, DHAR and CuZnSOD, respectively) (Oelze et al., 2012). During continuous HL treatment, the relative expression levels of these genes were all up-regulated to a much larger extent in the Fd2-deficient plants, especially after 24 and 48 h of HL (Figure 7a), indicating enhanced radical-scavenging abilities in the mutant plants.

Figure 7.

Changes in expression of antioxidant genes and Fd2 homologs during long-term HL treatment. Relative expression of antioxidant genes (a) and Fd homologs (b) during HL exposure in WT and Fd2–KO plants. Expression levels were normalized to those for the UBQ4 gene, and were then normalized to expression in WT plants grown under GL. Error bars indicate SE obtained from three independent quantitative real-time PCR experiments. Asterisks indicate statistically significant differences for Fd2–KO compared with the respective WT plants (**< 0.01).

As the majority of Fds are depleted in Fd2–KO plants, we investigated the effects of the redox signals on transcription of Fd2 homologs in mutant plants. The results showed that Fd1 and the ferredoxin gene FdC1 (Voss et al., 2011) were both up-regulated in Fd2–KO plants to levels higher than in WT plants after 120 h HL treatment (Figure 7b). The increased minor Fd isoforms are suggested to serve in alleviation of excess electron pressure between PSI and PSII in the mutants (Hanke and Hase, 2008; Voss et al., 2011; Blanco et al., 2013).

Induction of PGR5-dependent CEF in Fd2-deficient plants

Apart from nuclear gene regulation via redox signaling, non-photochemical quenching (NPQ) is considered to be the most important strategy to dissipate the excess energy in the PETC (Niyogi et al., 1998; Muller et al., 2001). WT and Fd2–KO plants were both capable of inducing NPQ under HL conditions, but the induction of NPQ was much higher (2.35 ± 0.09) in the Fd2–KO plants than WT plants (1.51 ± 0.10) after 120 h HL treatment (Figure 8a). As measurement of NPQ is also used to monitor the efficiency of CEF under HL conditions (Shikanai, 2007), we investigated whether the mutants maintained stromal redox status after prolonged HL by altering the electron transfer flow between PSI and PSII. Changes in Y(I)/Y(II) were used to estimate the induction of CEF (Harbinson and Foyer, 1991; Grieco et al., 2012). The value of Y(I)/Y(II) was significantly lower in the mutants (approximately 0.4) than in WT (approximately 1.3) under GL, but WT and mutants exhibited a similar value (approximately 1.8) after 120 h HL (Figure S4a), indicating that the CEF was markedly increased in the Fd2–KO plants. In addition, Y(NA), the quantum yield of non-photochemical energy dissipation of reaction centers due to PSI acceptor side limitation, was significantly higher in mutant plants under GL, but decreased dramatically from 0.82 to 0.2 after 120 h HL (Figure S4b), indicating that over-reduction of the PSI acceptor side was prevented in Fd2–KO after long-term HL stress.

Figure 8.

Changes in NPQ and immunodetection of CEF-related proteins in plants during long-term HL treatment. (a) Changes in NPQ of WT and Fd2–KO plants during continuous HL. Values are means ± SE of three biological replicates. (b) Immunoblots of PGR5 and PGRL1 proteins in WT and Fd2–KO plants during long-term HL treatment. Aliquots of 1.5 μg (one-half Fd2–KO), 3 μg (Fd2–KO) and 3 μg (WT) chlorophyll were loaded. A CBB-stained gel of the samples (shown below) was used to estimate quantities loaded. (c) Quantification of band intensities for the blots shown in (b). Signal intensities of each protein were expressed relative to GL-grown WT plants using means ± SD from three biological replicates. r.u., relative units of densitometry intensity. Asterisks indicate statistically significant differences for Fd2–KO compared with the respective WT plants (**< 0.01).

PGR5 and PGRL1 have been reported to be involved in the primary CEF pathway (Munekage et al., 2002; DalCorso et al., 2008). We attempted to assess the regulation of PGR5 and PGRL1 at the protein level (Method S1). Immunoblots showed that the mutants contained similar amounts of PGR5 and PGRL1 as WT under GL conditions, but both of these proteins accumulated to much higher levels (230 and 156%, respectively) in the leaves of Fd2–KO plants than in WT plants after 120 h HL (Figure 8b,c). PGR5 has recently been shown to play a regulatory role in partitioning electrons between LEF and CEF, which is essential in the protection of PSI against photodamage (Suorsa et al., 2012). To further analyze the contribution of PGR5 to the photo-acclimation process in the Fd2–KO mutant, we created two fd2 pgr5 double mutant lines (Figure 9a,b), named fd2 pgr5-1 and fd2 pgr5-2. The two fd2pgr5 lines both showed a reduction in growth and lower chlorophyll content compared to Fd2–KO and the pgr5 single mutant (Figure 9a and Table S1). Consistent with this phenotype, the light intensity dependence of the electron transport rate and NPQ were much lower in the double mutants than in the Fd2–KO plants (Figure 9c,d). Furthermore, Y(II) was significantly lower, and the reduction of the PQ pool (1 – qP) was dramatically increased in double mutant plants under GL conditions (Table S1). This suggests a much more severe defect in photosynthesis in the fd2 pgr5 double mutants. Fv/Fm was also measured, and in contrast to the trend observed with Fd2–KO plants, fd2 pgr5 plants showed a consistent lower level in Fv/Fm from 0.8 ± 0.02 to approximately 0.5 ± 0.09 after HL treatment (Figure 9e), and Y(II) decreased to 0. In addition to Fv/Fm, Y(NO) is also a good indicator of photodamage, and the much higher value of Y(NO) in fd2 pgr5 and pgr5 plants following long-term HL is due to the defect in induction of NPQ (Figure 9f). This suggests inefficient photochemical energy conversion and protective regulatory mechanisms in the fd2 pgr5 double mutant lines.

Figure 9.

Characterization of the fd2pgr5 double mutants. (a) Phenotypic appearance of the two fd2 pgr5 double mutant lines (fd2 pgr5–1 and fd2 pgr5–2). Seedlings were grown under GL for 3 weeks after germination. (b) Immunodetection of Fd2 and PGR5 in fd2 pgr5 plants. A CBB-stained gel (shown at the bottom) was used to estimate gel loading. (c,d) Light intensity dependence of the relative electron transport rate (ETR) (c) and NPQ (d) in WT and fd2 pgr5 plants. The electron transport rate is relative to the maximum electron transport rate in the WT (100%). Values are means ± SE of three biological replicates. (e,f) Changes in the Fv/Fm (e) and Y(NO) (f) in WT and fd2 pgr5 plants under HL conditions. Values are mean ± SE of three biological replicates.

Moreover, we assayed the putative structural states of the photosynthetic protein complexes in the double mutants using BN/SDS–PAGE. Strikingly, PSII super-complexes were almost absent and other protein complexes were also dramatically reduced in both pgr5 and fd2 pgr5 mutants after 120 h HL treatment (Figure 10a). The results obtained with 2D SDS-urea-PAGE gels after Coomassie brilliant blue staining confirmed that the amounts of PSII subunits in PSII super-complexes were reduced in pgr5 mutants, whereas both PSI and PSII subunits in thylakoid protein complexes were severely decreased in fd2 pgr5 plants after 120 h HL (Figure 10b). These results further show that the protective PGR5-dependent CEF is triggered in Fd2–KO mutant plants to protect the photosynthetic apparatus and alleviate inhibition of PSII under prolonged HL stress conditions.

Figure 10.

Analysis of thylakoid protein complexes from fd2 pgr5 plants. (a) BN-PAGE analysis of thylakoid protein complexes. Thylakoids were isolated from equal amounts of chlorophyll (8 μg) obtained from Fd2–KO, pgr5 and fd2 pgr5 plants grown under GL and 120 h HL. (b) Two-dimensional separation of protein complexes in the thylakoid membranes. Individual lanes from the BN gels shown in (a) were analyzed by 15% SDS-urea-PAGE and stained using CBB. The experiment was repeated three times with similar results.

Discussion

Arabidopsis lacking Fd2 exhibited an unexpectedly efficient photosynthetic capacity under long-term HL conditions

Fd2 is the major photosynthetic Fd in Arabidopsis, and its loss leads to the typical symptoms of Fd reduction reported previously in other species such as potato and tobacco (Holtgrefe et al., 2003; Blanco et al., 2011), mainly growth arrest, photosynthesis inhibition and electron transport reduction (Figure S1a and Table S1). Previous experiments using Arabidopsis Fd2 knockout or knockdown lines focused on their performance under normal growth or short-term stress conditions, which revealed an important role of Fd2 in the LEF (Hanke and Hase, 2008; Voss et al., 2008, 2011). In this study, Fd2–KO mutant plants exhibited unexpectedly robust photo-acclimation under long-term HL conditions. After 120 h HL treatment, PSI and PSII photochemical activities returned to normal unstressed WT levels in the mutants, in contrast to the decreased photosynthetic capacity of identically treated WT plants (Figure 2b–f). Moreover, proteins in the PSII reaction center, primarily the D1 protein, are damaged under HL irradiation and need to be rapidly repaired and reassembled to enable efficient photosynthetic electron transport (Baena-Gonzalez and Aro, 2002; Lu et al., 2011). There was clear stability of various PSII complexes in the Fd2–KO mutants after 120 h HL treatment (Figure 4). Therefore, maintenance of the structural and functional integrity of the photosynthetic protein complexes facilitates efficient photosynthetic capacity in the Fd2-deficient plants during long-term HL treatment.

Impaired photochemistry and photosynthetic complexes are probably a consequence of over-excitation pressure (1 – qP, 1 – qL or F′/Fm) in the PETC of Fd2–KO plants under GL and short-term HL conditions (Figure 5). Intriguingly, Fd2–KO exhibited a stepwise decrease in the relative QA reduction state under the extended HL treatment (Figure 5), indicating a gradual easing of the over-excitation pressure. Therefore, rebalancing of the PETC and retention of stable PSII complexes allowed Fd2–KO plants to recover and maintain their photosynthetic capacity much better than WT plants.

Enhanced plastid redox signaling facilitates HL acclimation in Fd2–KO plants

Optimal regulation of the photosynthetic capacity during long-term adaptation relies on changes in gene expression by plastid redox signaling (Dietz, 2003). It is likely that the chloroplast redox status serves as an enhanced pivotal signal to regulate gene expression in Fd2–KO plants in response to long-term HL. This possibility is supported by the observation of enhanced transcript levels of photoprotection-related genes in mutant plants compared to WT after HL exposure (Figures 6 and 7). Specifically, the observed up-regulation of ZAT12 may further affect acclimation-responsive genes, conferring tolerance to high levels of irradiance (Iida et al., 2000), whilst Early-light induced proteins (ELIPs) are also thought to have a photoprotective function, via binding of chlorophylls released from pigment-binding proteins and stabilization of the assembly of these proteins during protein turnover under fluctuating light conditions (Hutin et al., 2003).

Several lines of evidence indicate that the electron carriers between PSI and PSII, primarily the PQ pool, are possible original sites for redox sensing of environmental stress (Koussevitzky et al., 2007; Pogson et al., 2008). The changes in the QA reduction state and the results for gene regulation shown in Figures 5-7 support a signaling role for the PQ pool. Additionally, redox signals are also sensed at the acceptor side of PSI through thioredoxin- or ROS-mediated regulation (Kim and Mayfield, 1997). The obstructed electron transfer in the stroma of Fd2-deficient plants is expected to lower the reduction state of thioredoxin, suggesting that thioredoxin may not serve as a redox site for the Fd2–KO mutants. The ROS-scavenging potential appeared to be improved in the Fd2–KO plants via up-regulation of antioxidant genes during HL treatment (Figure 7a), indicating that ROS may also trigger signaling to the nucleus in Fd2–KO plants to avoid photo-oxidative damage. Thus the possibility of cross-talk between the PQ pool and ROS cannot be excluded.

PGR5-dependent CEF is required for photo-acclimation in Fd2–KO plants

Plants over-expressing PGR5, an important protein that participates in the primary CEF pathway, share interesting phenotypic similarities with Fd2–KO plants (Munekage et al., 2004). Arabidopsis plants constitutively over-expressing PGR5 show electron alterations in LEF and CEF that lead to decreased growth, but increased tolerance to long-term HL compared with WT plants (Long et al., 2008); resembling the HL-responsive phenotype of Fd2–KO in this study (Figure 2a and Figure S3). This suggests that the PGR5-dependent CEF pathway may be regulated in the Fd2–KO plants under long-term HL conditions. The hypothesis was further supported by two observations: (i) the abundance of PGR5 and PGRL1 proteins was increased in HL-treated Fd2–KO mutants (Figure 8b,c) and (ii) in contrast to the Fd2–KO single mutant, the photosynthetic efficiency and state of photosynthetic protein complexes were severely impaired in the fd2 pgr5 double mutant plants after long-term HL treatment (Figures 9e,f and 10).

Previous observations showed that RNAi and antisense RNA plants deficient in Fd exhibited growth arrest, decreased photosynthetic capacity and severe chlorosis (Holtgrefe et al., 2003; Hanke and Hase, 2008; Blanco et al., 2011). However, the techniques used in this study differ from those used by Holtgrefe et al. (2003) in potato, Hanke and Hase (2008) in Arabidopsis, and Blanco et al. (2011) in tobacco. The approaches used in those previous studies may be unable to discriminate between Fd isoforms, and RNAi probable down-regulated both Fd1 and Fd2 in Arabidopsis. Although long-term performance under HL was not tested in those studies, it is unlikely that the plants in the previous studies exhibited the photosynthetic recovery observed in Fd2–KO plants. Recent genetic analysis on the minor pea Fd isoform (PsFd1) showed that over-expression of PsFd1 in tobacco may alter electron partitioning and promote CEF around PSI (Blanco et al., 2013). Expression of the Fd homologs, Fd1 and FdC1, was slightly up-regulated in Fd2–KO plants after prolonged HL conditions (Figure 7b), implying that at least partial Fd complementation via transcriptional regulation compensates for the loss of Fd2; the increased level of Fd1 may function with PGR5 to elevate CEF. These results suggest that although Fd2–KO plants were subjected to more severe oxidative stress than WT plants, the excess reducing power in the PETC was dissipated via induction of PGR5-dependent CEF in response to an extensive photo-inhibitory process under long-term ambient conditions.

Effects of Fd2 down-regulation in Arabidopsis on photo-acclimation responses

It is worth noting that chloroplast Fd is down-regulated by environmental stress in plants (Zimmermann et al., 2004; Tognetti et al., 2006). In this study, Fd2 protein levels gradually decrease in Arabidopsis WT plants after exposure to HL (Figure 1). However, the excess electrons in the PETC lead to severe inhibition of photosynthesis when electron acceptors are limited at the reducing site of PSI. Down-regulation of Fd in several species has been reported to compromise cell survival in the normal growth environment (Holtgrefe et al., 2003; Hanke and Hase, 2008; Voss et al., 2008; Blanco et al., 2011). Therefore, there appears to be a contradiction regarding this phenomenon, and the mechanisms underlying this effect of Fd decrease are not yet clear. Based on our characterization of Fd2–KO plants, redox signaling and PGR5-dependent CEF are both enhanced in Fd2-deficient plants under HL conditions. These results show that the signaling and physiological changes brought about by a reduction in Fd appear to be a novel mode of adaptation in response to long-term HL. Additionally, expression of Fd2 showed a dramatic reduction in WT plants after continuous long-term HL, but remained stable during the early phase of HL treatment (Figure 1), which illustrates that loss of Fd2 does indeed severely affect the growth of plants under short-term stress as shown previously (Voss et al., 2008).

To conclude, although Fd2 plays a predominant role in LEF under GL, the evidence showed here suggests that loss of Fd2 in Arabidopsis plants appears to be beneficial for long-term adaptation to HL stress. This may provide a novel strategy for plant growth and survival under HL conditions, and further studies are required to investigate this phenomenon under other adverse environmental conditions.

Experimental procedures

Plant materials and growth conditions

Arabidopsis thaliana Fd2–KO plants (a knockout line with a Ds T–DNA insertion in the Fd2 gene, At1 g60950; Voss et al., 2008) and the corresponding wild-type (Nossen) were kindly provided by Renate Scheibe (Department of Botany, University of Osnabrück, Germany). pgr5 mutants (Munekage et al., 2002) and the corresponding wild-type (ecotype Columbia gl1) were kindly provided by Toshiharu Shikanai (Plant Molecular Genetics, University of Kyoto, Japan). The fd2 pgr5 double mutant plants were generated by crossing Fd2–KO as the female parent with pgr5 mutant, and two double mutant lines were used in this work (fd2 pgr5–1 and fd2 pgr5–2). Plants were grown under normal growth light (GL) (120 μmol photons m−2 sec−1) with a 16 h light/8 h dark cycle at 2°C/18°C day/night, relative air humidity of 50–70%.

For HL treatment, plants were grown for 4 weeks under GL conditions, and then transferred to continuous HL (approximately 500 μmol photons m−2 sec−1) at 18°C. A lower temperature (18°C) was used to avoid increased leaf surface temperature caused by HL. Only fully expanded leaves were used for analysis.

Chlorophyll fluorescence measurements

Chlorophyll fluorescence parameters and images were monitored using a Maxi-Imaging-PAM Chlorophyll Fluorometer (Heinz Walz GmbH, http://www.walz.com/) as described by Zeng et al. (2010). The maximum quantum yield of PSII (Fv/Fm), the quantum yield of PSII (Y[II]), photochemical fluorescence quenching coefficients in PSII (qP and qL), non-photochemical quenching (NPQ) and quantum yield of non-regulated non-photochemical energy loss in PS II (Y[NO]) were recorded for dark-adapted or light-adapted WT and Fd2–KO plants.

The DUAL-PAM-100 measuring system (Heinz Walz GmbH) was used for determining both PSI (Pm, Y[I] and Y[NA]) and PSII photosynthetic parameters in vivo using detached leaves, according to the manufacturer's instructions. The relative QA redox state under non-steady-state conditions was measured as F′/Fm for dark-adapted or light-adapted leaves during a saturation pulse (6000 μmol photons m−2 sec−1 for 300 msec) as described previously (Grieco et al., 2012; Suorsa et al., 2012).

BN/SDS–PAGE

For first-dimension BN-PAGE, thylakoid membrane preparations equivalent to 8 μg of chlorophyll were solubilized by mixing with 1.5% w/v n–dodecyl-β–d–maltopyranoside. Samples were incubated at 4°C for 10 min, centrifuged at 12 000 g for 10 min, and then loaded onto a 1 mm thick separation gel with a 5–12% acrylamide gradient (Peng et al., 2008; Pribil et al., 2010). For the second-dimension SDS–PAGE, excised BN-PAGE lanes were soaked in SDS sample buffer (100 mm Tris/HCl pH 6.8, 2% SDS, 15% glycerol, 2.5% β–mercaptoethanol) for 20 min at room temperature, then layered onto 1.5 mm thick 15% SDS–PAGE gels containing 6 m urea (Peng et al., 2008). The gels were stained with Coomassie brilliant blue (CBB) G250 and scanned using a GS–800 densitometer (Bio–Rad, http://www.bio-rad.com/).

Quantitative real-time PCR

Total RNA was extracted from plants using a Plant RNA Kit (Omega, http://www.omegabiotek.com/). Five micrograms of total RNA were used for cDNA synthesis using the PrimeScript® RT reagent kit (Takara, http://www.takara-bio.com/) according to the manufacturer's instructions. Quantitative real-time PCR was performed using SYBR® Premix Ex Taq™ (Takara), and real-time amplification was monitored by the LightCycler480 system (Roche, https://www.roche-applied-science.com/). The expression of UBQ4 was relatively stable during HL treatment compared to the relative gene expression of Actin2/7, thus UBQ4 was used as an internal control in this study. Primer sequences are listed in Table S2.

Statistical analysis

Statistical analysis was performed either using GraphPad Prism 5.0 (http://www.graphpad.com/scientific-software/prism/) or SPSS 20.0 (http://www-01.ibm.com/software/analytics/spss/). Differences were analyzed by one-way anova followed by Tukey's multiple comparison test. Significance was accepted at the level of P < 0.01.

Accession numbers

Sequence data referred to in this article may be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: At3 g09640 (APX2), At2 g28190 (CuZnSOD), At5 g16710 (DHAR), At3 g22840 (ELIP1), At4 g14690 (ELIP2), At1 g10960 (Fd1), At1 g60950 (Fd2), At1 g32550 (FdC1), At3 g27690 (Lhcb2.4), At2 g05620 (PGR5), At4 g22890 (PGRL1A), At4 g08390 (sAPX), At1 g77490 (tAPX), At4 g36800 (UBQ4), At1 g27730 (ZAT10) and At5 g59820 (ZAT12).

Acknowledgments

We gratefully acknowledge Renate Scheibe (Department of Botany, University of Osnabrück, Germany) for kindly providing the Fd2–KO mutant seeds. We thank Toshiharu Shikanai (Plant Molecular Genetics, Kyoto University, Japan) for the kind gift of pgr5 mutant seeds and the PGR5 antibody. We thank Bernhard Grimm (Institute of Biology, Humboldt University Berlin, Germany) for critical reading of the manuscript. This research was supported by the National Natural Science Foundation of China (grant numbers 31171173 and 31371237), Guangdong Provincial Natural Science Foundation of China (grant number S2012010010533), the Fundamental Research Funds for the Central Universities (grant number 10lgpy34) and the Open Research Fund Program of Guangdong Key Laboratory of Plant Resources (grant number plant01k18).

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