SEARCH

SEARCH BY CITATION

Keywords:

  • NAD(P)H dehydrogenase;
  • cyclic electron transport;
  • temperature response;
  • photosynthesis;
  • CO2 assimilation;
  • light response

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

The role of NAD(P)H dehydrogenase (NDH)-dependent cyclic electron flow around photosystem I in photosynthetic regulation and plant growth at several temperatures was examined in rice (Oryza sativa) that is defective in CHLORORESPIRATORY REDUCTION 6 (CRR6), which is required for accumulation of sub-complex A of the chloroplast NDH complex (crr6). NdhK was not detected by Western blot analysis in crr6 mutants, resulting in lack of a transient post-illumination increase in chlorophyll fluorescence, and confirming that crr6 mutants lack NDH activity. When plants were grown at 28 or 35°C, all examined photosynthetic parameters, including the CO2 assimilation rate and the electron transport rate around photosystems I and II, at each growth temperature at light intensities above growth light (i.e. 800 μmol photons m−2 sec−1), were similar between crr6 mutants and control plants. However, when plants were grown at 20°C, all the examined photosynthetic parameters were significantly lower in crr6 mutants than control plants, and this effect on photosynthesis caused a corresponding reduction in plant biomass. The Fv/Fm ratio was only slightly lower in crr6 mutants than in control plants after short-term strong light treatment at 20°C. However, after long-term acclimation to the low temperature, impairment of cyclic electron flow suppressed non-photochemical quenching and promoted reduction of the plastoquinone pool in crr6 mutants. Taken together, our experiments show that NDH-dependent cyclic electron flow plays a significant physiological role in rice during photosynthesis and plant growth at low temperature.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

Plants capture light energy through light-harvesting systems, including chlorophylls and carotenoids, and the absorbed light energy drive photosynthetic electron transport through the thylakoid membranes of the chloroplasts. Electrons released from water in photosystem II (PSII) are ultimately transferred to NADP+ via photosystem I (PSI), resulting in production of NADPH. Electrons also pass through the cytochrome b6/f complex, generating a proton gradient across the thylakoid membrane. Together with the protons deposited in the thylakoid lumen by the water-splitting complex associated with PSII, these protons translocated through the cytochrome b6/f complex into the lumen enable ATP production by the ATP synthase complex. ATP and NADPH generated by light reactions are utilized primarily in the Calvin–Benson cycle and the photorespiratory cycle. Cyclic electron transport also occurs, which solely depends on the PSI photochemical reaction; these electrons also pass through the cytochrome b6/f complex. Cyclic electron transport can generate a proton gradient (ΔpH) and drives ATP synthesis by ATP synthase without concomitant generation of NADPH (Heber et al., 1978; Heber and Walker, 1992).

In higher plants, two alternative pathways have been demonstrated for PSI cyclic electron transport (Shikanai, 2007). The main pathway is mediated by the ferredoxin-dependent PQ reductase (FQR), and two proteins, PROTON GRADIENT REGULATION 5 (PGR5) (Munekage et al., 2002) and PGR5-LIKE 1 (PGRL1) (DalCorso et al., 2008) are essential for the electron transport, although it is still unclear how these proteins are required (Nandha et al., 2007). Another pathway is mediated by the chloroplast NADH dehydrogenase-like complex (NDH) (Burrows et al., 1998; Shikanai et al., 1998), which has been recently shown to form a supercomplex with PSI (Peng and Shikanai, 2011; Peng et al., 2011). PSI cyclic electron transport is suggested to be essential for balancing the ATP/NADPH production ratio and/or for protecting photosystems from damage via stromal over-reduction (Shikanai, 2007; Miyake, 2010; Takahashi and Badger, 2011), as it can generate an additional proton gradient, inducing non-photochemical quenching (NPQ) to dissipate excessive absorbed light energy (Niyogi, 1999).

The role of NDH-dependent cyclic electron flow (CEF) in photosynthetic regulation in response to various environmental stresses has been studied extensively, as NDH-defective mutants of tobacco (Nicotiana tabacum) do not show any decrease in photosynthetic activity compared to the wild-type under non-stress conditions (Burrows et al., 1998; Kofer et al., 1998; Shikanai et al., 1998; Horvath et al., 2000). The photosynthetic activity in the NDH-defective mutants is sensitive to short-term severe stress, including strong-light stress (Endo et al., 1999; Takabayashi et al., 2002), low-humidity stress (Horvath et al., 2000), drought stress (Munne-Bosch et al., 2005) and extreme-temperature stress (treatment at 4°C, Li et al., 2004; treatments at 4 or 42°C, Wang et al., 2006). As inhibition of CO2 assimilation induced by strong light, heat, chilling or water stress could lead to over-reduction of the electron transport chain, NDH-dependent CEF has been proposed to prevent over-reduction of the stroma, especially under stress conditions (Rumeau et al., 2007; Shikanai, 2007). However, in previous studies, the plants were exposed to extremely severe conditions that are rarely seen in nature. Moreover, no direct experimental evidence has demonstrated the physiological relevance of NDH-dependent pathways in the response of photosynthesis and plant growth to long-term environmental changes.

Photosynthesis has long been recognized as one of the most temperature-sensitive processes in plants (Berry and Björkman, 1980). Plants are frequently exposed to environmental stress, and it is common for more than one abiotic stress to occur at a given time, for instance low temperature (or high temperature) and strong light. Environmental stress limits crop productivity and is also a major determining factor in the distribution of plants across different environments. Therefore, understanding the physiological mechanisms of plant response to environmental stress will benefit both agriculture and the environment. The temperature response of the CO2 assimilation rate is described by a parabolic curve with an optimum temperature, and thus CO2 assimilation is inhibited at both low and high temperatures (Yamori et al., 2005, 2006, 2008, 2010a,b). Many studies often concentrate on the important role of PSI cyclic electron transport at high temperature (e.g. Havaux, 1996; Bukhov et al., 1999; Schrader et al., 2004; Quiles, 2006; Zhang and Sharkey, 2009). As low temperature often causes over-reduction of the stroma and subsequent photoinhibition (Huner et al., 1998), PSI cyclic electron transport may also be important for photosynthetic regulation at low temperature. Here, the role of NDH-dependent cyclic electron transport in the regulation of photosynthesis and plant growth under both low and high temperatures was examined in NDH-defective mutants of rice (Oryza sativa). The growth analysis was performed at growth temperatures of 20/15°C, 28/23°C or 35/30°C. Measurements of gas exchange, chlorophyll fluorescence and the P700+ reduction rate were made over a range of light intensities under each growth temperature conditions. Moreover, the effect of NDH-dependent cyclic electron transport on alleviation of photoinhibition after strong light under each growth condition was evaluated.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

Rice crr6 mutants do not accumulate the NDH complex

CRR6 is a stromal protein that is required for the assembly of NDH sub-complex A in Arabidopsis (Peng et al., 2010), and is encoded by a single-copy gene (Os08g0167500) in rice. The Tos17 retrotransposon was inserted into the exon of this gene (OsCRR6) (Figure 1a). Homozygous Tos17 insertion plants (crr6; −/−) were identified by PCR analysis (Figure 1b) from progeny of a heterozygous plant provided by the National Institute of Agrobiological Sciences of Japan. As Tos17 mutant lines may include multiple insertions in a single genome, the crr6 mutant plant was crossed with the wild-type, and the resulting F2 plants containing the homozygous wild-type CRR6 allele were used as control plants (+/+) in the experiments described below (Figure 1b). As the control plants behaved like wild-type plants in all experiments, we describe differences between crr6 mutants (−/−) and the control plants (+/+). The fact that the TOS17 insertion caused the mutant phenotype was further confirmed in the F2 generation (see below).

image

Figure 1.  Isolation and characterization of the Oryza sativa Tos17 insertion mutant OsCRR6 by PCR and immunoblot analysis. (a) Structure of the OsCRR6 gene (Os08g0167500). The exons and introns are indicated by boxes and lines, respectively. The insertion site of Tos17 is indicated. Arrows indicate the positions of primers LP1, LP2 and RP used for PCR for genotype determination. Primer LP1 was designed on the basis of the Tos17 sequence, while primers LP2 and RP were designed on the basis of the OsCRR6 gene sequence. (b) Determination of genotype [homozygous for Tos17 insertion (−/−), control homozygous (+/+)] in Tos17 mutant lines and wild-type plants by nested PCR. Primer pairs are indicated. (c) Immunoblot analysis in OsCRR6 mutants. Leaf extracts were separated by SDS-PAGE and immunodetected using specific antibodies. The extracted proteins were loaded on an equal fresh weight basis; the series of dilutions in wild-type plants is indicated. CRR6 is a stromal protein required for accumulation of NDH sub-complex A, while NdhK is a subunit of sub-complex A and cytochrome f is a subunit of the cytochrome b6/f complex.

Download figure to PowerPoint

Immunoblot analysis showed that CRR6 was not present in crr6 mutants (Figure 1c). The chloroplast NDH complex is divided into four parts (the A, B, membrane and lumen sub-complexes; Peng et al., 2008, 2011), and CRR6 is required for accumulation of sub-complex A in Arabidopsis (Peng et al., 2008). To study the effect of disruption of CRR6 on accumulation of the NDH complex in rice, we assessed the level of NdhK, a subunit of sub-complex A of the NDH complex, in crr6 mutants (Figure 1c). The NdhK level was drastically reduced to below the detection limit in crr6 mutants. Thus, consistent with observations in Arabidopsis (Peng et al., 2010), accumulation of NDH sub-complex A was impaired in rice crr6 mutants.

NDH activity is monitored by a transient post-illumination increase in chlorophyll fluorescence, as a result of NDH-dependent reduction of the plastoquinone pool in darkness (Shikanai et al., 1998). This transient increase in chlorophyll fluorescence was detected in control plants at all temperatures (Figure 2), and both the rate of the increasing phase and the amplitude of the post-illumination increase in chlorophyll fluorescence were enhanced in control plants at higher temperatures. However, the transient increase in chlorophyll fluorescence was not observed in crr6 mutants at any temperature (Figure 2). Thus, consistent with the absence of NdhK (Figure 1c), NDH activity is completely absent in crr6 mutants.

image

Figure 2.  Monitoring of NDH activity by chlorophyll fluorescence analysis. The curve shows a typical trace of chlorophyll fluorescence in the wild-type (WT). Leaves were exposed to actinic light (AL: 200 μmol photons m−2 sec−1) after the measuring light was turned on (Fo level: minimum yield of Chl fluorescence). The AL was turned off and the subsequent change in the chlorophyll fluorescence level was monitored as an indicator of NDH activity. Leaves were dark-adapted in a temperature-controlled chamber at the indicated temperatures for at least 10 min, and fluorescence was measured at the same temperature. Insets are magnified traces from the boxed area. The fluorescence levels were normalized against the Fm levels (maximum yield of Chl fluorescence).

Download figure to PowerPoint

Effect of NDH-dependent CEF on plant growth

In wild-type plants, the relative growth rate (RGR) was maximal at 28°C and showed a temperature response similar to that of the final dry weight (Figures 3 and 4). The net assimilation rate (NAR) showed a similar temperature response to RGR in wild-type plants (Figure 4). On the other hand, the leaf area ratio (LAR) was similar between 28 and 35°C, and was greater than at 20°C (Figure 4). These results indicate that the reduction in RGR at high temperature was solely associated with the reduction in NAR, whereas reductions in both NAR and LAR led to the large reduction in RGR at low temperature.

image

Figure 3.  Effect of NDH-dependent CEF on plant biomass production. Effects of growth temperature on final dry weight were analyzed in plants grown at the indicated temperatures. Values are means ± SE (= 4–6). Significant differences among wild-type, the control and crr6 mutants were examined using the Tukey–Kramer multiple comparison test (< 0.05). Asterisks indicate a significant difference in the crr6 mutant only compared to the control plants and wild-type.

Download figure to PowerPoint

image

Figure 4.  Effect of NDH-dependent CEF on plant growth analysis parameters. Effects of growth temperature on relative growth rate (RGR), net assimilation rate (NAR) and leaf area ratio (LAR) were analyzed in plants grown at the indicated temperatures. Values are means ± SE (= 4–6). Significant differences among wild-type, the control and crr6 mutants were examined using the Tukey–Kramer multiple comparison test (< 0.05). Asterisks indicate a significant difference in the crr6 mutant only compared to the control plants and wild-type.

Download figure to PowerPoint

To examine the effect of NDH-dependent CEF on plant growth at various temperatures, the effect of the crr6 defect on the plant growth at 20, 28 and 35°C was examined. When plants were grown at 28 and 35°C, plant growth was similar between the control plants and crr6 mutants (Figures 3 and 4). However, when plants were grown at 20°C, the crr6 defect caused reductions in the final dry weight (Figure 3). Growth analyses showed decreases in RGR and NAR (Figure 4). As LAR did not decrease (Figure 4), the reduction in RGR at low temperature in crr6 mutants was solely attributable to reductions in NAR.

To confirm the link between the Tos17 insertion into CRR6 and the reduced plant growth at low temperature, the genotypes of F2 plants were determined by PCR (Figure S1). F2 plants with a homozygous wild-type CRR6 allele (WT*, +/+) and plants with a heterozygous Tos17 insertion (+/−) showed similar dry weight to wild-type plants (WT, +/+). In contrast, F2 plants with a homozygous Tos17 insertion (−/−) showed a significant reduction in dry weight compared to the other plants (Tukey–Kramer method, < 0.05; Figure S1). The result indicates that the growth phenotype at low temperature is linked to the Tos17 insertion into CRR6, which results in the absence of NDH in thylakoid membranes.

Photosynthetic components in crr6 mutants

Growth temperature had a large effect on photosynthetic components in wild-type plants. The contents of leaf nitrogen, Rubisco and cytochrome f per fresh weight or per unit leaf area were greater in wild-type plants grown at lower temperatures (Figure 1c and Table 1). The contents of CRR6 and NdhK per fresh weight or per unit leaf area were also greater in wild-type plants grown at lower temperatures (Figure 1c). Contents of chlorophyll per unit of leaf area were similar irrespective of growth temperature, but the chlorophyll a/b ratio was greater in wild-type plants grown at a lower temperature.

Table 1.   Physiological components of photosynthesis
Growth temperatureGenotypeTotal nitrogen (mmol m−2)Rubisco (μmol m−2)Cytochrome f (%)Chlorophyll (mmol m−2)Chlorophyll a/b
  1. Contents of total nitrogen, Rubisco, cytochrome f and chlorophyll were quantified. The cytochrome f content is shown as a percentage relative to the wild-type (WT). Values are means ± SE (= 4–6). Different letters indicate significant differences in the photosynthetic components among WT, control and crr6 mutants at each growth temperature (Tukey–Kramer multiple comparison test; < 0.05).

Growth at 20°CWT146 ± 3a7.68 ± 0.29a100.0 ± 4.6a0.65 ± 0.02a4.15 ± 0.05a
Control148 ± 4a7.50 ± 0.30a96.0 ± 3.6a0.67 ± 0.03a4.23 ± 0.05a
crr6132 ± 4a6.54 ± 0.26b85.4 ± 3.3b0.61 ± 0.02a4.21 ± 0.06a
Growth at 28°CWT115 ± 2a4.91 ± 0.31a70.0 ± 2.5a0.63 ± 0.02a3.71 ± 0.03a
Control118 ± 5a5.01 ± 0.40a68.0 ± 3.3a0.60 ± 0.03a3.60 ± 0.04a
crr6113 ± 4a5.25 ± 0.45a65.8 ± 3.5a0.59 ± 0.02a3.55 ± 0.05a
Growth at 35°CWT108 ± 3a4.01 ± 0.19a48.5 ± 6.3a0.60 ± 0.02a3.48 ± 0.04a
Control98 ± 5a4.11 ± 0.22a50.2 ± 5.8a0.60 ± 0.03a3.40 ± 0.05a
crr6100 ± 3a4.41 ± 0.30a44.9 ± 2.0a0.62 ± 0.02a3.30 ± 0.05a

The effect of mutations of crr6 on the photosynthetic components was examined at growth temperatures of 20, 28 or 35°C (Table 1). When plants were grown at 28 and 35°C, the levels of all photosynthetic components were similar between the control plants and crr6 mutants. However, when plants were grown at 20°C, the contents of Rubisco and cytochrome f were significantly lower in crr6 mutants than in the control plants, and contents of leaf nitrogen and chlorophyll content tended to be lower in crr6 mutants than in the control plants.

Effect of NDH-dependent CEF on the regulation of photosynthesis

The light-intensity response of several photosynthetic parameters was measured at the same temperature as used for plant growth experiments (Figure 5). In wild-type plants, the electron transport rate around PSI (ETRI), that around PSII (ETRII) and the CO2 assimilation rate at a CO2 concentration of 390 μmol mol−1 (A390) were highest at 28°C at the same light intensities, showing a similar temperature response to NAR. On the other hand, NPQ was similar between 28 and 35°C, but lower than at 20°C. If CEF is functioning, ETRI will be larger than ETRII, i.e. the CEF (calculated as ETRI − ETRII) will show positive values and the relative CEF (calculated as ETRI/ETRII) will exceed 1. In wild-type plants, the CEF calculated as ETRI − ETRII was similar irrespective of temperature (data not shown). However, the ETRI/ETRII ratio did not differ between 28 and 35°C, but was lower than at 20°C, indicating that the contribution of CEF estimated from ETRI/ETRII was higher at low temperature. In the present study, the ETRI/ETRII ratio was used to evaluate the relative importance of NDH-dependent CEF at various temperatures.

image

Figure 5.  Effect of NDH-dependent CEF on in vivo electron transport activity and CO2 assimilation. The light response of chlorophyll fluorescence, P700 redox state and gas exchange were determined simultaneously. The electron transport rate around PSI (ETRI), the electron transport rate around PSII (ETRII), non-photochemical quenching (NPQ), the proportion of PSII centers in the open state (with QA oxidized) (qL), the CO2 assimilation rate (A390), stomatal conductance (gs) and intercellular CO2 concentration (Ci) at a CO2 concentration of 390 μmol mol−1 were analyzed as described in Experimental procedures. Values are means ± SE (= 4–6). Significant differences among wild-type, the control and crr6 mutants were examined using the Tukey–Kramer multiple comparison test (< 0.05). Asterisks indicate a significant difference in the crr6 mutant only compared to the control plants and wild-type.

Download figure to PowerPoint

The effect of the crr6 defect on photosynthetic parameters was also examined at each temperature (Figure 5). In plants grown at 28 and 35°C, ETRI, ETRII and A390 at light intensities above growth light (800 μmol photons m−2 sec−1) were similar between crr6 mutants and control plants. In contrast, ETRI and A390 below 800 μmol photons m−2 sec−1 were significantly lower in crr6 mutants than in the control plants (Figure 5). ETRII below 800 μmol photons m−2 sec−1 was similar between the control plants and crr6 mutants. As a result, the ETRI/ETRII ratio below 800 μmol photons m−2 sec−1 was lower in crr6 mutants than in control plants. Below 800 μmol photons m−2 sec−1, the reductions in ETRI with no effect on ETRII in crr6 mutants resulted in a concomitant reduction of the plastoquinone pool (1−qL, the proportion of closed PS II center, is high) and a low trans-thylakoid proton gradient (low NPQ), because of the reduced capacity of ETRI relative to ETRII. NPQ and 1 − qL at strong light of 2000 μmol photons m−2 sec−1 tended to be lower in crr6 mutants than in control plants.

When plants were grown at 20°C, ETRI and A390 at all light intensities were lower in crr6 mutants than in the control plants, whereas ETRII above 800 μmol photons m−2 sec−1 was lower in crr6 mutants than in control plants but did not differ between crr6 mutants and the control plants below 800 μmol photons m−2 sec−1 (Figure 5). The ETRI/ETRII ratio below 800 μmol photons m−2 sec−1 was lower in crr6 mutants than in control plants, but was similar above 800 μmol photons m−2 sec−1. The 1 − qL was higher in crr6 mutants than in control plants at all light intensities analyzed, and the NPQ was lower in crr6 mutants than in the control plants, especially at light intensities higher than 1000 μmol photons m−2 sec−1. The stomatal conductance (gs) and intercellular CO2 concentration (Ci) were not significantly affected in crr6 mutants at any light intensity or at any growth temperature. These results suggest that the reduction in plant growth at 20°C in crr6 mutants could be attributed to reductions in photosynthetic capacities as a result of the lack of NDH-dependent CEF.

The dark respiration rate (Rd) was also lower in crr6 mutants than in the control plants when plants were grown at 20°C (Figure 6). However, there were no differences in Rd between crr6 mutants and the control plants when plants were grown at 28 or 35°C. These effects of growth temperature on Rd are consistent with its effects on A390 at the growth light intensity (i.e., 800 μmol photons m−2 sec−1) (Figure 6).

image

Figure 6.  Effect of NDH-dependent CEF on the dark respiration rate (Rd) and the CO2 assimilation rate (A390) at a light intensity of 800 μmol photons m−2 sec−1. Values are means ± SE (= 4–6). Significant differences among wild-type, the control and crr6 mutants were examined using the Tukey–Kramer multiple comparison test (< 0.05). Asterisks indicate a significant difference in the crr6 mutant only compared to the control plants and wild-type.

Download figure to PowerPoint

To study the function of NDH-dependent CEF on the short-term response of photosynthetic parameters to the temperature shift, the photosynthetic parameters were examined at low (15°C) and high (40°C) leaf temperatures in plants grown at 28°C (Figure S2). The results showed similar trends to the effect of NDH-dependent CEF on the long-term temperature response (Figure 5), indicating that lack of NDH-dependent CEF affects photosynthetic activity at low temperature. On the other hand, there were no differences in Rd at any leaf temperature between crr6 mutants and the control plants when plants were grown at 28°C (Figure S3).

We estimated the rate of linear electron transport at PSII (Jf) using chlorophyll fluorescence data. However, due to a number of uncertainties, such estimates may not be strictly related to the actual rate of linear electron transport at PSII (ETRII). To overcome this limitation of chlorophyll fluorescence and obtain quantitative estimates, we analyzed the relationship between linear electron transport rates estimated from chlorophyll fluorescence (Jf = ETRII) and gas-exchange data (Jg). There was a strong positive relationship between Jf and Jg (Figure S4; R2 > 0.98 for all plants at all temperatures), indicating that use of chlorophyll fluorescence data for estimation of ETRII is acceptable in the present study (von Caemmerer, 2000).

Effects of NDH-dependent CEF on the alleviation of photoinhibition

The effect of the crr6 mutation on the decrease in Fv/Fm was measured after exposure to strong light at 2000 μmol photons m−2 sec−1 for 90 min at each growth temperature. Before the strong light treatment, Fv/Fm was similar between crr6 mutants and the control plants at any growth temperature (Figure 7). The crr6 mutants showed a decrease in Fv/Fm after incubation for 15 min in darkness after strong light treatment compared to control plants. The effect was more pronounced in plants grown at low temperature. However, this effect was diminished after 30 min dark incubation (Figure 7), indicating that NDH-dependent CEF is involved in the recovery of Fv/Fm after strong light treatment, but the effect is small.

image

Figure 7.  Effect of NDH-dependent CEF on alleviation of photoinhibition. The effect of the crr6 mutation on photoinhibition was examined. Leaves were exposed to strong light at 2000 μmol photons m−2 sec−1 at the indicated temperature for 90 min. The proportion of active PSII (Fv/Fm) was measured after dark incubation for 15 or 30 min at the indicated temperature. Values are means ± SE (= 4–6). Significant differences among wild-type, the control and crr6 mutants were examined using the Tukey–Kramer multiple comparison test (< 0.05). Asterisks indicate a significant difference in the crr6 mutant only compared to the control plants and wild-type.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

NDH-dependent CEF in rice plays a more important role in photosynthetic regulation at low temperature than high temperature

The physiological relevance of NDH-dependent CEF has not been elucidated (Shikanai, 2007). Since it was reported that the role of NDH in photoprotection at low temperature is less important (Savitch et al., 2001; Wang et al., 2006), the significance of NDH-dependent CEF at low temperature has been questioned (Rumeau et al., 2007). The ETRI/ETRII ratio was greater in plants grown at lower growth temperature, indicating that PSI CEF is accelerated at lower temperature (Figure 5), as has been suggested in tobacco and cucumber (Cucumis sativus) (Kim et al., 2001; Barth and Krause, 2002; Miyake et al., 2004). Moreover, the present study has shown that the lack of NDH-dependent CEF in crr6 mutants decreased photosynthesis (ETRI, ETRII and CO2 assimilation) at both low growth temperature and low leaf temperature compared with control plants (Figures 5 and S2), and this effect on photosynthesis caused a corresponding reduction in plant biomass (Figures 3 and 4). Taken together, the present study clearly shows the importance of NDH-dependent CEF during CO2 assimilation and plant growth at low temperature in rice. This is supported by reports that substantial up-regulation of the NDH complex occurs in several species in response to photoinhibitory irradiation at low temperatures (Teicher et al., 2000; Streb et al., 2005).

Field-grown plants typically experience low temperature and high light conditions for extensive time periods. Low temperature slows down the Calvin cycle and induces over-reduction of photosynthetic electron transport carriers under high light, which ultimately causes photoinhibition (Demmig-Adams and Adams, 1992; Huner et al., 1998; Asada, 1999). Moreover, it has been reported that the CO2 assimilation rate at low temperatures is often limited by ribulose 1,5-bisphosphate (RuBP) regeneration which would be in turn limited by the production rate of ATP and NADPH, or by triose phosphate utilization (Hikosaka et al., 2006; Sage and Kubien, 2007; Yamori et al., 2010b). The present study also confirmed that low temperature and high light conditions induced the reduction of photosynthetic electron transport carriers (higher 1 − qL; Figures 5 and S2) and that the lack of NDH-dependent CEF in crr6 mutants increased such reduction of photosynthetic electron transport carriers compared with control plants (Figure 5). Impairment of NDH-dependent CEF it tended to not cause pronounced photoinhibition after strong light for 90 min at any temperature, but it tended to decrease the Fv/Fm ratio, especially at low temperature (Figure 7). It has been reported that high light treatment induces over-reduction of the stroma in NDH-deficient mutants of tobacco (Endo et al., 1999; Takabayashi et al., 2002) and Arabidopsis (Ishikawa et al., 2008). Thus it is possible that NDH in C3 plants normally functions to prevent over-reduction of the stroma at strong light under stressed conditions.

However, it is mostly not known how NDH alleviates the over-reduction of electron transport carriers. NDH-mediated CEF may directly balance ATP and NADPH production, as chloroplast NDH is probably the proton pump, similar to bacterial and mitochondrial complex I. On the other hand, as the rate of NDH-dependent CEF is estimated to be very low and its contribution to proton gradient formation is not significant (Munekage et al., 2002; Okegawa et al., 2008), it is possible that NDH may regulate the rate of PGR5-dependent CEF by poising the redox level of intersystem electron carriers (Peltier and Cournac, 2002; Munekage et al., 2004). Plants have developed adaptive mechanisms to control the efficiency of light energy utilization and photosynthetic electron transport. Dissipation of extra light energy and/or extra ATP production via NDH-dependent CEF at low temperature may confer a selective advantage of sufficient magnitude to explain the conservation of plastid ndh genes during the course of evolution, especially in rice, which is known to be a cold-sensitive plant.

Many researchers have extensively studied the role of CEF in photosynthetic reactions at high temperature (e.g. Havaux, 1996; Pastenes and Horton, 1996; Bukhov et al., 1999). The CEF calculated from ETRI − ETR II in wild-type plants was similar irrespective of growth temperatures (Figure 5), but tended to be greater at higher leaf temperature in wild-type plants grown at 28°C (Figure S2). However, the ETRI/ETRII ratio was lowest at high temperature, suggesting less contribution of CEF at higher temperatures. Moreover, photosynthetic capacity and plant growth were indistinguishable between crr6 mutants and control plants at high temperature, indicating that NDH-dependent CEF does not play an important role in the regulation of photosynthesis and electron transport at high temperature (Figures 5 and S2). As rice is tolerant to heat (Yamori et al., 2009, 2010b), it may acclimatize to high temperatures below 40°C. In contrast, in Arabidopsis, which is sensitive to heat, lack of NDH-dependent CEF slightly reduced photosynthetic capacity at 40°C (Zhang and Sharkey, 2009). The results for NDH-defective mutants of tobacco, which is tolerant to heat, are complicated, as Wang et al. (2006) showed that NDH-dependent CEF had an important role in alleviation of oxidative damage caused by high temperature, whereas other groups obtained contradictory results (Sazanov et al., 1998; Yamane et al., 2000). Thus the role of NDH-dependent CEF at high temperature may differ depending on plant species.

NDH-dependent CEF participates in alleviation of stromal over-reduction

The Fv/Fm ratio did not differ between crr6 mutants and control plants before strong light treatment at low temperature (Figure 7), indicating that there was no photoinhibitory effect during plant growth. Moreover, impairment of NDH-dependent CEF did not cause exacerbation of photoinhibition in rice after strong light for 90 min and dark incubation for 30 min (Figure 7), as has been reported for NDH-defective mutants of tobacco (Barth and Krause, 2002) and Arabidopsis (Ishikawa et al., 2008). Thus, NDH-dependent CEF would not be essential for photoprotection, but our results suggest the possibility that NDH-dependent CEF participates in alleviation of reduction of the plastoquinone pool, because its absence increased 1 − qL at low temperature (Figures 5 and S2).

There was a difference in Fv/Fm between crr6 mutants and control plants after strong light treatment and dark incubation for 15 min at lower temperatures (Figure 7). This suggests that NDH-dependent CEF participates in relaxation of the proton gradient after strong light, and/or that the NDH complex may be involved in regulation of state transition under strong light conditions. Further studies are necessary to elucidate a role for the NDH complex in the regulation of photosynthesis after rapid changes of light intensity.

Role of NDH-dependent CEF in the regulation of photosynthesis at low light intensity

At low light intensity, crr6 mutants decreased the ETRI and CO2 assimilation rate, although the ETRII was similar to that of the control plants (Figures 5 and S2), suggesting that NDH-dependent CEF plays an important role in the regulation of photosynthesis under low light conditions. Many researchers have examined the physiological role of CEF at high light intensity, and believe that CEF is important in balancing the ratio of generation of ATP and NADPH to prevent over-reduction of the stroma under high light intensity (for a review, see Shikanai, 2007). Experimentally, it has been reported that NDH-defective mutants of Arabidopsis (e.g. Munekage et al., 2002, 2004; Okegawa et al., 2008) and tobacco (e.g. Burrows et al., 1998; Shikanai et al., 1998; Ishikawa et al., 2008) show no differences in photosynthetic characteristics under low light intensity. Thus there are inter-specific differences in the functions of NDH-dependent CEF between rice and these species.

The cytochrome b6/f complex showed much tighter control of electron transport capacity and photosynthesis at high light intensity than the ATP synthase complex did, suggesting that it is the rate of NADPH production that limits whole-chain electron transport and thus the CO2 assimilation rate at high light intensity, rather than the ATP production rate (von Caemmerer, 2000; Yamori et al., 2011b). However, the present study suggests that, at low light intensity, NDH-dependent CEF may be important for ATP synthesis via an additional proton gradient across the thylakoid membrane, and the CO2 assimilation rate may be limited by the rate of ATP production rather than the rate of NADPH production.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

The NDH complex has recently been shown to form the supercomplex with PSI (Peng et al., 2010, 2011; Peng and Shikanai, 2011). However, the physiological function of NDH-dependent CEF around PSI is still unclear. It was considered that NDH-dependent CEF is less important in the regulation of photosynthesis at low temperature (for a review, see Rumeau et al., 2007). However, in our studies with rice, NDH-dependent CEF performs a significant physiological role during photosynthesis and plant growth at low temperature, and impairment of NDH led to decreases in ETR and CO2 assimilation at low light as well as at low temperature. In natural environments, irradiance and temperatures often fluctuate according to the time of day and the season of the year. NDH-dependent CEF contributes to photosynthetic regulation and plant growth especially at low light and/or low temperature (e.g. cloudy weather, early morning and/or fall/winter season).

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

Plant material

The rice OsCRR6 mutant (line H0198; Os08g0167500) created by insertion of the Tos17 retrotransposon and its wild-type (Oryza sativa ssp. japonica cv. Hitomebore) were obtained from the National Institute of Agrobiological Sciences of Japan. We performed PCR experiments to isolate homozygous mutants, heterozygous mutants and control plants after crossing the homozygous mutant and the wild-type (primer LP1: 5′-ATTGTTAGGTTGCAAGTTAGTTAAGA-3′; primer LP2: 5′-AGGTGCGCAACAATGAAAGT-3′, primer RP: 5′-TCATGAAGGACCAGCAGGAT-3′) (Figure 1). Seeds including wild-type rice (cv. Nipponbare), the homozygous line containing the Tos17 insertion at the crr6 gene (−/−) and control progeny of OsCRR6 that have no Tos17 insertion in the crr6 gene but have the same genetic background as the homozygous lines (+/+) were examined.

The plants were grown hydroponically in an environmentally controlled growth chamber (Yamori et al., 2011a) at a photosynthetic photon flux density of 800 μmol photons m−2 sec−1, a 12 h photoperiod (between 8:00 am and 8:00 pm) and an ambient CO2 concentration of 390 μmol mol−1. During the first 21 days, the seedlings were grown at day/night growth temperatures of 28/23°C, and then transferred to temperatures of 20/15, 28/23 or 35/30°C. The hydroponic solution used for this study was previously described by Makino et al. (1988). These solutions were renewed once a week, and were continuously aerated.

Analyses of gas exchange, chlorophyll fluorescence and P700 measurements

Light-response curves for gas exchange, chlorophyll fluorescence and P700 redox state were obtained simultaneously using GFS-3000 and DUAL-PAM-100 measuring systems (Walz, http://www.walz.com) between 9:00 am and 4:00 pm in the uppermost, fully expanded new leaves of 60–80-day-old plants. After leaves had dark-adapted for 15 min (to obtain open reaction centers), a saturating pulse was applied to obtain maximal fluorescence and maximal P700 changes. Then, leaves were exposed to high light (i.e. 1200–1500 μmol photons m−2 sec−1) for 30 min to obtain a steady state before measurement of several photosynthetic parameters every 5 min at a CO2 concentration of 390 μmol mol−1 at each photosynthetic photon flux density. The quantum yield of PSI (ΦPSI) is defined by the proportion of overall P700 that is reduced in a given state and not limited by the acceptor side. It is calculated from the complementary PSI quantum yields of non-photochemical energy dissipation Y(ND) and Y(NA), i.e. ΦPSI = 1 − Y(ND) − Y(NA), where Y(ND) = 1 − P700red and Y(NA) = (Pm − Pm′)/Pm (Klughammer and Schreiber, 1994). Y(ND) represents the proportion of overall P700 that is oxidized in a given state, which is enhanced by a trans-thylakoid proton gradient (photosynthetic control at the cytochrome b6/f complex as well as down-regulation of PSII) and photodamage to PSII. Y(NA) represents the proportion of overall P700 that cannot be oxidized by a saturation pulse in a given state due to lack of acceptors, and is enhanced by dark adaptation (deactivation of key enzymes of the Calvin–Benson cycle) and damage at the site of CO2 fixation. The quantum yield of PSII [ΦPSII = (Fm′ − F ′)/Fm′], photochemical quenching [qP = (Fm′ − F ′)/(Fm′ − Fo′)], non-photochemical quenching [NPQ = (Fm − Fm′)/Fm′] and the proportion of PSII centers in the open state (with oxidized primary quinone accepter, QA) [qL = qP × (Fo′/F ′)] were calculated from the measurement of chlorophyll fluorescence, as described by Baker (2008). The electron transport rate (ETRI or ETRII) was calculated as 0.5 × abs I × ΦPSI or 0.5 × abs I × ΦPSII, where 0.5 is the proportion of absorbed light reaching PSI or PSII, and abs I is absorbed irradiance taken as 0.84 of incident irradiance.

Analyses of photoinhibition

The leaves were placed in a temperature-controlled chamber at 390 μmol mol−1 CO2 concentration and 60% relative humidity in a portable gas exchange system (LI-6400; Li-COR, http://www.licor.com). The leaves were exposed to high light at 2000 μmol photons m−2 sec−1 using a cool light source (PCS-HRX; Nippon Pl, http://www.npinet.co.jp) at the required temperature for 90 min. The proportion of active PSII (Fv/Fm) was measured in high light-treated leaves after dark incubation for 15 or 30 min at the required temperature using a chlorophyll fluorescence measuring device (Walz).

Quantification of photosynthetic components and immunoblot analysis

Immediately after the gas exchange measurements, leaf samples were taken, immersed in liquid nitrogen and stored at −80°C. The frozen leaf samples were ground in liquid nitrogen and homogenized in an extraction buffer (Yamori et al., 2011a). The contents of leaf nitrogen, chlorophyll and Rubisco were quantified as described by Yamori et al. (2011a). Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The contents of CRR6, cytochrome f of the cytochrome b6/f complex, and NdhK, which is a subunit of sub-complex A of the NDH complex, were quantified by immunoblotting with each antibody (Peng et al., 2010). The leaf extract of one wild-type leaf was selected as a standard (100%), and diluted for use as a dilution series on gels for protein quantifications relative to wild-type. Signals were detected using an ECL Plus Western blotting detection kit (GE Healthcare, http://www.gehealthcare.com) and visualized using an LAS3000 chemiluminescence analyzer (Fujifilmhttp://fujifilm.com). Immunoblots were quantified using Scion Image software (Scion Corp, http://www.scioncorp.com). Chlorophyll was extracted in 80% v/v acetone, and chlorophyll content was determined as described by Porra et al. (1989).

Growth analysis

Growth analysis was performed in plants sampled at 21, 42 and 63 days after germination (Nagai and Makino, 2009). Leaf area was determined using an AMM-8 leaf area metre (Hayashi-denko, http://www.hayashidenko.co.jp), and leaf blades, leaf sheaths and roots were oven-dried at 80°C for more than a week (e.g., 7-10 days), and their dry weight was measured. Growth analysis parameters, including relative growth rate (RGR), net assimilation rate (NAR) and leaf area ratio (LAR), were calculated from total dry weight and leaf area, using the equations below:

  • image(1)

where W1 and W2 are total dry weights of the whole plant (including roots) at times t1 and t2,

  • image(2)

where L1 and L2 are total leaf areas of the whole plant at times t1 and t2, and

  • image(3)

Statistical analyses

The Tukey–Kramer multiple comparison test was performed using statview version 4.58 (SAS Institute Inc., http://www.statview.com).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

We wish to thank Dr W. S. Chow and Dr S. Takahashi (Australian National University, Canberra) and Dr C. Miyake (Kobe University, Japan) for their generous advice, and Dr T. Kazama (Tohoku University, Sendai, Japan) for his help in crossing rice plants. This work was supported by grants from the Japan Society for the Promotion of Science (postdoctoral fellowships to W.Y. and scientific research grant number 20380041 to A.M.), the Ministry of Education, Culture, Sports, Science and Technology, Japan (Scientific Research on Innovative Area grant number 21114006 to A.M.) and the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation: grant numbers GPN0007 to A.M. and GPN0008 to T.S.).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information
  • Asada, K. (1999) The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 601639.
  • Baker, N.R. (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu. Rev. Plant Biol. 59, 89113.
  • Barth, C. and Krause, G.H. (2002) Study of tobacco transformants to assess the role of chloroplastic NAD(P)H dehydrogenase in photoprotection of photosystems I and II. Planta, 216, 273279.
  • Berry, J.A. and Björkman, O. (1980) Photosynthetic response and adaptation to temperature in higher plants. Annu. Rev. Plant Physiol. 31, 491543.
  • Bukhov, N.G., Wiese, C., Neimanis, S. and Heber, U. (1999) Heat sensitivity of chloroplasts and leaves: leakage of protons from thylakoids and reversible activation of cyclic electron transport. Photosynth. Res. 59, 8193.
  • Burrows, P.A., Sazanov, L.A., Svab, Z., Maliga, P. and Nixon, P.J. (1998) Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes. EMBO J. 17, 868876.
  • von Caemmerer, S. (2000) Biochemical Models of Leaf Photosynthesis. Collingwood, Australia: CSIRO Publishing.
  • DalCorso, G., Pesaresi, P., Masiero, S., Aseeva, E., Schunemann, D., Finazzi, G., Joliot, P., Barbato, R. and Leister, D. (2008) A complex containing PGRL1 and PGR5 is involved in the switch between linear and cyclic electron flow in Arabidopsis. Cell, 132, 273285.
  • Demmig-Adams, B. and Adams, W.W. III (1992) Photoprotection and other responses of plants to high light stress. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 599626.
  • Endo, T., Shikanai, T., Takabayashi, A., Asada, K. and Sato, F. (1999) The role of chloroplastic NAD(P)H dehydrogenase in photoprotection. FEBS Lett. 457, 58.
  • Havaux, M. (1996) Short-term responses of photosystem I to heat stress. Photosynth. Res. 47, 8597.
  • Heber, U. and Walker, D.A. (1992) Concerning a dual function of coupled cyclic electron transport in leaves. Plant Physiol. 100, 16211626.
  • Heber, U., Egneus, H., Hank, U., Jensen, M. and Koster, S. (1978) Regulation of photosynthetic electron transport and phosphorylation in intact chloroplasts and leaves of Spinacia olereacea. Planta, 143, 4149.
  • Hikosaka, K., Ishikawa, K., Borjigidai, A., Muller, O. and Onoda, Y. (2006) Temperature acclimation of photosynthesis: mechanisms involved in the changes in temperature dependence of photosynthetic rate. J. Exp. Bot. 57, 291302.
  • Horvath, E.M., Peter, S.O., Joet, T., Rumeau, D., Cournac, L., Horvath, G.V., Kavanagh, T.A., Schafer, C., Peltier, G. and Medgyesy, P. (2000) Targeted inactivation of the plastid ndhB gene in tobacco results in an enhanced sensitivity of photosynthesis to moderate stomatal closure. Plant Physiol. 123, 13371349.
  • Huner, N.P.A., Öquist, G. and Sarhan, F. (1998) Energy balance and acclimation to light and cold. Trends Plant Sci. 3, 224230.
  • Ishikawa, N., Endo, T. and Sato, F. (2008) Electron transport activities of Arabidopsis thaliana mutants with impaired chloroplastic NAD(P)H dehydrogenase. J. Plant. Res. 121, 521526.
  • Kim, S.J., Lee, C.H., Hope, A.B. and Chow, W.S. (2001) Inhibition of photosystems I and II and enhanced back flow of photosystem I electrons in cucumber leaf discs chilled in the light. Plant Cell Physiol. 42, 842848.
  • Klughammer, C. and Schreiber, U. (1994) An improved method, using saturating light pulses, for the determination of photosystem I quantum yield via P700+-absorbance changes at 830 nm. Planta, 192, 261268.
  • Kofer, W., Koop, H.U., Wanner, G. and Steinmuller, K. (1998) Mutagenesis of the genes encoding subunits A, C, H, I, J and K of the plastid NAD(P)H-plastoquinone-oxidoreductase in tobacco by polyethylene glycol-mediated plastome transformation. Mol. Gen. Genet. 258, 166173.
  • Li, X.-G., Duan, W., Meng, Q.-W., Zou, Q. and Zhao, S.-J. (2004) The function of chloroplastic NAD(P)H dehydrogenase in tobacco during chilling stress under low irradiance. Plant Cell Physiol. 45, 103108.
  • Makino, A., Mae, T. and Ohira, K. (1988) Differences between wheat and rice in the enzymic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase and the relationship to photosynthetic gas exchange. Planta, 174, 3038.
  • Miyake, C. (2010) Alternative electron flows (water–water cycle and cyclic electron flow around PSI) in photosynthesis: molecular mechanisms and physiological functions. Plant Cell Physiol. 51, 19511963.
  • Miyake, C., Shinzaki, Y., Miyata, M. and Tomizawa, K.I. (2004) Enhancement of cyclic electron flow around PSI at high light and its contribution to the induction of non-photochemical quenching (NPQ) of Chl fluorescence in intact leaves of tobacco plants. Plant Cell Physiol. 45, 14261433.
  • Munekage, Y., Hojo, M., Meurer, J., Endo, T., Tasaka, M. and Shikanai, T. (2002) PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis. Cell, 110, 361371.
  • Munekage, Y., Hashimoto, M., Miyake, C., Tomizawa, K., Endo, T., Tasaka, M. and Shikanai, T. (2004) Cyclic electron flow around photosystem I is essential for photosynthesis. Nature, 429, 579582.
  • Munne-Bosch, S., Shikanai, T. and Asada, K. (2005) Enhanced ferredoxin-dependent cyclic electron flow around photosystem I and α-tocopherol quinone accumulation in water-stressed ndhB-inactivated tobacco mutants. Planta, 222, 502511.
  • Nagai, T. and Makino, A. (2009) Differences between rice and wheat in temperature responses of photosynthesis and plant growth. Plant Cell Physiol. 50, 744755.
  • Nandha, B., Finazzi, G., Joliot, P., Hald, S. and Johnson, G.N. (2007) The role of PGR5 in the redox poising of photosynthetic electron transport. Biochim. Biophys. Acta, 1767, 12521259.
  • Niyogi, K.K. (1999) Photoprotection revisited: genetic and molecular approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 333359.
  • Okegawa, Y., Kagawa, Y., Kobayashi, Y. and Shikanai, T. (2008) Characterization of factors affecting the activity of photosystem I cyclic electron transport in chloroplasts. Plant Cell Physiol. 49, 825834.
  • Pastenes, C. and Horton, P. (1996) Effect of high temperature on photosynthesis in beans. 1. Oxygen evolution and chlorophyll fluorescence. Plant Physiol. 112, 12451251.
  • Peltier, G. and Cournac, L. (2002) Chlororespiration. Annu. Rev. Plant Biol. 53, 523550.
  • Peng, L. and Shikanai, T. (2011) Supercomplex formation with photosystem I is required for the stabilization of the chloroplast NADH dehydrogenase-like complex in Arabidopsis. Plant Physiol. 155, 16291639.
  • Peng, L., Shimizu, H. and Shikanai, T. (2008) The chloroplast NAD(P)H dehydrogenase complex interacts with photosystem I in Arabidopsis. J. Biol. Chem. 283, 3487334879.
  • Peng, L., Cai, W. and Shikanai, T. (2010) Chloroplast stromal proteins, CRR6 and CRR7, are required for assembly of the NAD(P)H dehydrogenase subcomplex A in Arabidopsis. Plant J. 63, 203211.
  • Peng, L., Yamamoto, H. and Shikanai, T. (2011) Structure and biogenesis of the chloroplast NAD(P)H dehydrogenase complex. Biochim. Biophys. Acta, 1807, 945953.
  • Porra, R.J., Thompson, W.A. and Kriedemann, P.E. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta, 975, 384394.
  • Quiles, M.J. (2006) Stimulation of chlororespiration by heat and high light intensity in oat plants. Plant Cell Environ. 29, 14631470.
  • Rumeau, D., Peltier, G. and Cournac, L. (2007) Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response. Plant Cell Environ. 30, 10411051.
  • Sage, R.F. and Kubien, D.S. (2007) The temperature response of C3 and C4 photosynthesis. Plant Cell Environ. 30, 10861106.
  • Savitch, L.V., Barker-Astrom, J., Ivanov, A.G., Hurry, V., Oquist, G., Huner, N.P. and Gardestrom, P. (2001) Cold acclimation of Arabidopsis thaliana results in incomplete recovery of photosynthetic capacity, associated with an increased reduction of the chloroplast stroma. Planta, 214, 295303.
  • Sazanov, L.A., Burrows, P.A. and Nixon, P.J. (1998) The chloroplast Ndh complex mediates the dark reduction of the plastoquinone pool in response to heat stress in tobacco leaves. FEBS Lett. 429, 115118.
  • Schrader, S.M., Wise, R.R., Wacholtz, W.F., Ort, D.R. and Sharkey, T.D. (2004) Thylakoid membrane responses to moderately high leaf temperature in Pima cotton. Plant Cell Environ. 27, 725735.
  • Shikanai, T. (2007) Cyclic electron transport around photosystem I: genetic approaches. Annu. Rev. Plant. Biol. 58, 199217.
  • Shikanai, T., Endo, T., Hashimoto, T., Yamada, Y., Asada, K. and Yokota, A. (1998) Directed disruption of the tobacco ndhB gene impairs cyclic electron flow around photosystem I. Proc. Natl Acad. Sci. USA, 95, 97059709.
  • Streb, P., Josse, E.-M., Gallouet, E., Baptist, F., Kuntz, M. and Cornic, G. (2005) Evidence for alternative electron sinks to photosynthetic carbon assimilation in the high mountain plant species Ranunculus glacialis. Plant Cell Environ. 28, 11231135.
  • Takabayashi, A., Endo, T., Shikanai, T. and Sato, F. (2002) Post-illumination reduction of the plastoquinone pool in chloroplast transformants in which chloroplastic NAD(P)H dehydrogenase was inactivated. Biosci. Biotechnol. Biochem. 66, 21072111.
  • Takahashi, S. and Badger, M.R. (2011) Photoprotection in plants: a new light on photosystem II damage. Trends Plant Sci. 16, 5360.
  • Teicher, B.H., Moller, B.L. and Scheller, H.V. (2000) Photoinhibition of photosystem I in field-grown barley (Hordeum vulgare L.): induction, recovery and acclimation. Photosynth. Res. 64, 5361.
  • Wang, P., Duan, W., Takabayashi, A., Endo, T., Shikanai, T., Ye, J. and Mi, H. (2006) Chloroplastic NAD(P)H dehydrogenase in tobacco leaves functions in alleviation of oxidative damage caused by temperature stress. Plant Physiol. 141, 465474.
  • Yamane, Y., Shikanai, T., Kashino, Y., Koike, H. and Satoh, K. (2000) Reduction of QA in the dark: another cause of fluorescence Fo increases by high temperatures in higher plants. Photosynth. Res. 63, 2334.
  • Yamori, W., Noguchi, K. and Terashima, I. (2005) Temperature acclimation of photosynthesis in spinach leaves: analyses of photosynthetic components and temperature dependencies of photosynthetic partial reactions. Plant Cell Environ. 28, 536547.
  • Yamori, W., Suzuki, K., Noguchi, K., Nakai, M. and Terashima, I. (2006) Effects of Rubisco kinetics and Rubisco activation state on the temperature dependence of the photosynthetic rate in spinach leaves from contrasting growth temperatures. Plant Cell Environ. 29, 16591670.
  • Yamori, W., Noguchi, K., Kashino, Y. and Terashima, I. (2008) The role of electron transport in determining the temperature dependence of the photosynthetic rate in spinach leaves grown at contrasting temperatures. Plant Cell Physiol. 49, 583591.
  • Yamori, W., Noguchi, K., Hikosaka, K. and Terashima, I. (2009) Cold-tolerant crop species have greater temperature homeostasis of leaf respiration and photosynthesis than cold-sensitive species. Plant Cell Physiol. 50, 203215.
  • Yamori, W., Evans, J.R. and von Caemmerer, S. (2010a) Effects of growth and measurement light intensities on temperature dependence of Co2 assimilation rate in tobacco leaves. Plant Cell Environ. 33, 332343.
  • Yamori, W., Noguchi, K., Hikosaka, K. and Terashima, I. (2010b) Phenotypic plasticity in photosynthetic temperature acclimation among crop species with different cold tolerances. Plant Physiol. 152, 388399.
  • Yamori, W., Nagai, T. and Makino, A. (2011a) The rate-limiting step for CO2 assimilation at different temperatures is influenced by the leaf nitrogen content in several C3 crop species. Plant Cell Environ. 34, 764777.
  • Yamori, W., Takahashi, S., Makino, A., Price, G.D., Badger, M.R. and von Caemmerer, S. (2011b) The roles of ATP synthase and the cytochrome b6/f complexes in limiting chloroplast electron transport and determining photosynthetic capacity. Plant Physiol. 155, 956962.
  • Zhang, R. and Sharkey, T.D. (2009) Photosynthetic electron transport and proton flux under moderate heat stress. Photosynth. Res. 100, 2943.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

Figure S1. Characterization of Tos17 insertion mutants of Oryza sativa OsCRR6 mutant grown at 20°C. (a) Structure of the OsCRR6 gene (Os08g0167500). The exons and introns are indicated by boxes and lines, respectively. The insertion site of Tos17 is indicated. Horizontal half arrows show the positions of primers (LP1, LP2 and RP) for PCR for the genotype determination. The primer LP1 was designed from the Tos17 sequence, while LP2 and RP were designed from the OsCRR6 gene sequence. (b) Determination of genotypes [homozygous for Tos17 insertion (−/−), heterozygous for Tos17 insertion (+/−), the control homozygous (WT*: +/+)] in Tos17 mutant lines and the WT plants by PCR. Primer pairs used are indicated. (c) Dry mass (g plant−1) at the 42nd day after germination in plants grown at 20°C. To determine F2 genotypes, 100 F2 seeds from the F1 plant were planted. The F2 segregation ratio was 1:2:1 for homozygous for the wild-type allele (+/+), heterozygous for the Tos17 insertion (+/−) and homozygous for the Tos17 insertion (−/−), respectively. Eight seedlings of each genotype and eight wild-type plants were grown at 28/23°C for the first 21 days, and then transferred to 20/15°C. At the 42nd day after seed germination, the dry mass (g) was examined to determine their phenotypes. The average dry mass (±SD) was 0.735 ± 0.043 g, 0.733 ± 0.043 g, 0.0681 ± 0.037 g and 0.517 ± 0.030 g for WT (+/+), WT* (+/+), Hetero (+/−) and Homo (−/−), respectively.

Figure S2. Effect of NDH-dependent CEF on in vivo electron transport activity and CO2 assimilation in plants grown at 28°C. Light response of chlorophyll fluorescence, P700 redox state and gas exchange at indicated leaf temperature was simultaneously determined. The electron transport rate around photosystem I (ETR I), electron transport rate around photosystem II (ETR II), non-photochemical quenching (NPQ), and the fraction of PSII centers in the open state (with QA oxidized) (qL), CO2 assimilation rate (A390), stomatal conductance (gs), and intercellular CO2 concentration (Ci) at CO2 concentration of 390 μmol mol−1 were analyzed as described in Materials & Methods. Data represent means ± SE, n = 4–6. Significant differences among wild-type, the control and crr6 mutants are examined by Tukey–Kramer multiple comparison test (< 0.05). When there is a significant difference only in the crr6 mutant compared to the control plants and wild-type, * is indicated.

Figure S3. Effect of NDH-dependent CEF on dark respiration rate (Rd). Data represent means ± SE, n = 4–6. Significant differences among wild-type, the control and crr6 mutants are examined by Tukey–Kramer multiple comparison test (< 0.05). No significant differences between the control and crr6 mutants, and between WT and crr6 mutants were observed, at each measurement leaf temperature.

Figure S4. Relationship between linear electron transport rates estimated from chlorophyll fluorescence (Jf = ETR II) and gas-exchange (Jg). The relationship between Jf and Jg was determined at a range of PPFD and at several leaf temperatures. The Jf was determined from chlorophyll fluorescence as described in Experimental procedures. The Jg was determined from gas-exchange [i.e. Jg = (R d)(4Ci+8Γ*)/(Ci-Γ*)], where A (μmol m−2 s−1) is CO2 assimilation rate, Rd (μmol m−2 s−1) is the measured rates of dark respiration, Ci (μmol mol−1) is intercellular CO2, and Γ* (μmol mol−1) is the CO2 compensation point in the absence of day respiration (Bernacchi et al., 2001).

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
TPJ_4747_sm_FigS1-S4.pdf655KSupporting info item
TPJ_4747_sm_SupportingInformationlegends.doc31KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.