Introduction

The respiratory activity of plants in light conditions has been debated for a long time, particularly with regard the level of respiration that occurs in photosynthetic cells in the presence of light, with several early studies suggesting the inhibition of respiration during the daytime (Budde and Randall 1990; Krömer 1995; Villar et al. 1995; Tcherkez et al. 2005). During the in vivo respiratory metabolism of illuminated leaves, the down-regulation of several metabolic pathways [e.g., glycolysis and tricarboxylic acid (TCA) cycle] accompanies the light/dark transition and emphasizes the decrease of the decarboxylations in the TCA cycle as a metabolic basis of the light-dependent inhibition of mitochondrial respiration (Krömer 1995; Tcherkez et al. 2005). In contrast, it has been reported that mitochondrial respiration is not only active in the light but that certain respiratory pathways (especially the electron transport chain) are also required for maximal rates of photosynthetic carbon assimilation (Krömer 1995; Dutilleul et al. 2003; Raghavendra and Padmasree 2003). Mitochondrial electron transport and ATP synthesis play an important role in the optimization of photosynthesis by reoxidizing the excess redox equivalents generated by chloroplasts, thus protecting against photoinhibition of photosynthesis (Sasadadevi and Raghavendra 1992; Noctor et al. 2007) and providing the ATP required for sucrose synthesis in the cytosol (Hoefnagel et al. 1998). This indicates that mitochondrial electron transport and ATP synthesis processes function in light conditions. However, the nature of day respiration in illuminated leaves has yet to be fully clarified and, in particular, the regulation mechanisms that operate during mitochondrial respiration remain poorly understood.

Previous studies have shown that the respiratory activity of plants in the light is between 25 and 100% of the levels observed in the dark, which suggests that respiration is inhibited during photosynthesis (Krömer 1995). Other enzymes may also be inhibited, including pyruvate kinase, the pyruvate dehydrogenase complex, TCA cycle enzymes, and mitochondrial isocitrate dehydrogenase (Lin et al. 1989; Budde and Randall 1990; Krömer 1995; Igamberdiev and Gardeström 2003; Tovar-Mendez et al. 2004; Tcherkez et al. 2005), and inhibition of malic acid decarboxylase (Hill and Bryce 1992). Photoinhibition is probably also involved in the inhibition of pyruvate dehydrogenase, as this enzyme is down-regulated by NH3, which is a byproduct of photorespiratory glycine decarboxylation (Krömer 1995). Light can directly influence the respiratory electron transport chain via photoreceptor-mediated transcriptional control, which may be necessary to support the photosynthetic metabolism (Escobar et al. 2004; Islam et al. 2009). Respiration in the light may also affect cellular carbon and nitrogen assimilation (Foyer and Noctor 2000; Yoshida and Noguchi 2009). The physiological changes would result from severe alterations in mitochondrial dynamics including mitochondrial swelling, transmembrane loss, and the cessation of mitochondrial movement (Gao et al. 2008; Zhang and Xing 2008), probably by production of reactive oxygen species (ROS) (Gao et al. 2008; Zhang et al. 2009).

The chlorophyll-deficient rice mutant OsCHLH, which lacks the largest subunit of Mg2+-chelatase, was isolated by T-DNA gene trapping (Jung et al. 2003). This mutant exhibits dysfunctional chloroplast activity (Jung et al. 2003), yet similar growth rates and somewhat greater respiratory O2 uptake were observed in continuously dark-grown seedlings of the oschlh mutant when compared with wild-type seedlings (Goh et al. 2007). However, the oschlh mutant showed a light-dependent decrease in CO2 uptake and O2 evolution in intact leaves in response to light intensity (Goh et al. 2004). Therefore, this chlorina-type plant is of great interest to investigate the respiratory activity of mitochondria, particularly with respect to the state of illumination, as the photosynthetic electron transport rates do not occur in light conditions.

In this study, we examined mitochondrial activity in chlorophyll-deficient rice mutant seedlings in order to elucidate the regulatory mechanisms that underlie the inhibition of mitochondrial electron transport in the light. We found that high H2O2 levels may be involved in the light-mediated inhibition of mitochondrial activity in this mutant.

Materials and methods

Plant materials and growth conditions

Rice (Oryza sativa var. japonica cv. Dongjin) seedlings were grown and mutants were screened on media containing 0.5× Murashige and Skoog basal salts with 0.2% (w/v) phytagel for 8–10 days, unless otherwise stated. Plants were cultured in a temperature-controlled growth chamber (28 ± 1°C) using a 16-h light/8-h dark regime with an intensity of 100 μmol m−2 s−1, unless otherwise specified.

Measurement of mesophyll cell membrane potential (V m)

Changes in membrane potential of mesophyll cells were measured using the method of Kari et al. (2003). Briefly, the secondary leaf was cut and floated on a solution (pH 6.0) of 10 mM KCl and 1 mM CaCl2 overnight in the dark. Leaf strips were then secured to a Plexiglas strip using Terostat (Teroson Werke, Heidelberg, Germany), placed into a perfusion chamber holding a reference electrode and mounted onto a microscope stage. The leaf strip was continuously bathed with solution throughout the experiment with a gravity-fed perfusion system. A microelectrode was inserted under microscopic control using perpendicular green light of less than 1 μmol m−2 s−1. Microelectrodes were pulled from borosilicate glass capillaries (Kwik-Fil; World Precision Instrument, Sarasota, FL, USA), backfilled with 0.3 M KCl and used if tip potentials were less than ±10 mV. The V m of the second or third cell encountered upon insertion of the microelectrode was recorded continuously through light-on/light-off transitions. White light (150 μmol m−2 s−1) was supplied directly to a leaf tissue with a projector lamp through a fiberglass light guide.

Total ATP assays

The levels of ATP were measured by the luciferin–luciferase method using the ENLITEN ATP bioluminescence detection kit (Promega, USA), according to the manufacturer’s instructions (Goh et al. 2004). The luminescence was integrated for 5 s using a luminometer (Luminoskan Ascent 2.1 Int., Germany).

Determination of pyruvate and l-lactate concentrations and of the NADH/NAD+ ratio

To determine the NADH/NAD+ ratio, pyruvate and lactate concentrations were determined using a dual wavelength spectrophotometer (Sigma ZFP 22; Eppendorf, Germany), as described by Planchet et al. (2005). Frozen samples (1 g of fresh weight) were extracted using 2 ml of 7.5% perchloric acid. The extract was centrifuged (12,000g for 10 min) and the supernatant was transferred to a new tube. Twenty microliters of 2 M Tris was added to each 1 ml of supernatant and the solution was neutralized to pH 7.0 with 5 M K2CO3. After centrifugation (12,000g for 10 min), the supernatant was collected and stored at −80°C until further use. Enzymatic determination of the l-lactate concentration was performed using the glutamate pyruvate transaminase (GPT)–lactate dehydrogenase (LDH) system. Fifty microliters of extract was added to 1 ml of buffer solution (100 mM CHES, pH 10) containing glutamate (10 μM), GPT (5 U), and NAD+ (2 mM). The reaction was initiated by the addition of LDH (3 U). To determine the concentration of pyruvate, 50 μl of extract was added to 1 ml of buffer solution (100 mM Hepes, pH 7.6) containing NADH (0.5 mM) and LDH (0.15 U). After each reaction, an appropriate internal standard was added to the mixture for the determination of the concentration of these two metabolites.

Determination of internal NADH oxidation

Mitochondria were isolated from the leaves of 10-day-old plants, using a self-generating Percoll gradient in combination with a linear gradient of 0–10% (w/v) polyvinylpyrrolidone (PVP) K-30 in a single step, essentially as described by Day et al. (1985). Glycine (2 mM) was added to the gradient and wash media to maintain maximal glycine decarboxylase activity. The isolated mitochondria had a high degree of membrane intactness, as judged by the latency of cytochrome c in the presence or absence of 0.025% (w/v) Tritone X-100 (Møller et al. 1987). The percentage latency of both enzymes was calculated by the previous methods (Møller et al. 1987; Rasmusson and Møller 1991). For determination of internal NADH oxidation, mitochondria were osmotically burst in 1 mM MOPS pH 7.2, 0.1 mM EGTA for 8 min at room temperature in order to permeabilize the inner membrane. The suspension was then supplemented to make up assay medium [10 mM MOPS pH 7.2, 2.5 mM MgCl2, 0.1 mM EGTA, 0.4 μM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone], and the reaction was started by the addition of NADH. The oxidation of NADH (0.1 mM) was measured on intact mitochondria at 340 nm in assay medium. The internal rotenone-insensitive NADH oxidation was assayed in the presence of 15 μM rotenone. Rotenone-sensitive activity was determined as the difference with and without rotenone. This osmotic treatment disrupted approximately 80% of the inner mitochondrial membranes, as determined by mitochondrial matrix enzyme malate dehydrohenase latency (Møller et al. 1987).

Determination of H2O2 content

Whole leaf H2O2 was extracted according to the method of Park et al. (2004). The levels of H2O2 were determined using a colorimetric assay (Sigma, Product Number CS0270, USA). Absorbance was recorded at 550 nm.

Isolation of mesophyll protoplasts

Mesophyll protoplasts were enzymatically isolated from the leaves of 10-day-old plants, according to the procedure as described previously (Goh et al. 2004). The isolated protoplasts were kept in a solution of 0.6 M sorbitol and 1 mM CaCl2 on ice until use.

Catalase activity assay

Catalase activity was assayed by monitoring the decrease in absorbance at 240 nm caused by the decomposition of H2O2 (Park et al. 2004). Protein concentration was determined using the Bradford assay (Bradford 1976).

RNA isolation, PCR conditions, and quantification of PCR products

Total RNA was extracted from 8-day-old plants using the TRIzol kit, according to the manufacturer’s instructions (Invitrogen, Valencia, USA). The optimum number of cycles for RT-PCR was established for each primer set and serial dilutions were used to ensure linear amplification. The intensity of each band was quantified using the NIH Image J software, version 1.37 (NIH, Bethesda, Maryland, USA: http://rsb.info.nih.gov/nih-image/) and was normalized to that of OsACTIN1. PCR with Taq DNA polymerase (Life Technologies) was carried out basically for 30 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1.5 min, and finally 72°C for 15 min. For the specific primers, the optimal cycling parameters were 24 cycles for OsACTIN1 (LOC_Os03g50890; forward primer (F), 5′-ACGGCGATAACAGCTCCTCTT-3′, reverse primer (R), 5′-CCTCTTCCAGCCTTCCTTCA T-3′); 28 cycles for OsMT2b (accession no. NE7013; F, 5′-CAATTCTTGAGCTCAATCACC-3′; R, 5′-ACACACGCACACACTGACAAC-3′), OsTRX-h (accession no. D21836; F, 5′-CTGCCACAACAAGGACGAGTTCGAC-3′; R, 5′-CGATTTCGCATGATATTCGAGGACA-3′), OsGRX (accession no. X77150; F, 5′-CGTCTACAGCAAGTCTTACTGTCCT-3′; R, 5′-CACACGATACTAGACGGCAACAAGT-3′), OsAPX1 (accession no. D45423; F, 5′-ATCAAGGAGGAGATACCCACCATCT-3′; R, 5′-CTCACAGTGGTAGTCTGCTGGTTCA-3′), and OsFeSOD (F, 5′-GTGGATAGCTTGAATAAGCAGCTTG-3′; R, 5′-AAGCAGATTTCCACTATTTCACCGT-3′); and 32 cycles for OsAPX2 (accession no. AB53297; F, 5′-GTGGCACTCTGCTGGCACCTTCGAT-3′; R, 5′-CACCAGTGGACGGAAGGCTGG GTC A-3′), OsRAC1 (accession no. NE7013; F, 5′-AGATAGGGCCTATCTTGCTGATCA TC-3′; R, 5′-CTAGAGTTTCCTAGCTGCAAGC-3′), and OsAOXc (F, 5′-CTGAAGAAATCTTACGGCGG-3′; R, 5′-CCAAACAGATAACAGGACGC-3′) (Saika et al. 2002). For DNA gel blot analysis, 20 μL of the reaction mixture was separated on a 1.0% agarose gel. The PCR products were quantified using EDAS 120 system (Eastman Kodak, Rochester, NY, USA). OsACTIN1 was used as an internal control to normalize all data.

Statistical analysis

All data obtained were subjected to a one-way analysis of variance (ANOVA), and the mean differences were compared by lowest standard deviations test. Comparisons with P < 0.05 were considered significantly different.

Results

Characterization of knock-out mutant seedlings of rice OsCHLH gene

The growth of wild-type seedlings was significantly greater than that of oschlh mutant seedlings (Fig. 1a). The fresh weight of 8-day-old seedlings was 0.193 ± 0.006 and 0.075 ± 0.003 g for wild-type and oschlh mutant plants, respectively, in the absence of sucrose (Fig. 1b). The addition of 3% sucrose, which stimulates lateral root formation and the maintenance of root-to-shoot ratios in plants (Macgregor et al. 2008), led to a greater enhancement of the growth rates of the oschlh mutant (77.7%) when compared with the wild-type (42.3%) seedlings. On the other hand, white light delivered to an intact wild-type rice leaf induced immediate depolarization of the membrane potential (V m), followed by rapid recovery (Fig. 2a). Specifically, initial depolarization/repolarization was followed by slow hyperpolarization of V m. Upon switching the light off, we observed depolarization after a transient hyperpolarization. A leaf of the albino oschlh mutant displayed no electrical responses to light (Fig. 2b). However, changes of V m in both wild-type and oschlh mutant were observed, although the magnitude of the response was different when the light was off. We contend therefore that dark responsive electric currents in theses mutant plants are due to mitochondrial respiration, indicating that the maintenance of growth rate is associated with a high efficiency of respiratory ATP production in dark condition.

Fig. 1
figure 1

Growth phenotype of the oschlh mutant seedlings. a Whole 8-day-old seedlings of wild-type and oschlh mutant plants in the presence or absence of 3% sucrose in 100 μmol m−2 s−1 light. Bar 1.4 cm. b Growth of oschlh mutant seedlings. The weight of fresh seedlings was measured from (a). Values are expressed as mean ± SE (n = 11 for wild-type and n = 8 for oschlh plants, P < 0.05)

Full size image
Fig. 2
figure 2

Light-induced changes of membrane potential (E m) in the oschlh mutant mesophyll cell. a WT and b oschlh mutant seedlings. The leaf strips were submerged and perfused in a flow-through chamber, and a conventional microelectrode was inserted into the first or second layer of mesophyll. After reading a steady value in a mesophyll cell for more than 15 min in the dark, the leaf strip was illuminated. The experimental conditions are described in "Materials and methods"

Full size image

ATP content of oschlh mutant leaves in dark and light conditions

We examined the levels of ATP in oschlh mutant leaves after an 8-h dark adaptation. Interestingly, a large drop in the ATP concentration was observed in the oschlh mutant 1 h after initiating light treatment (Fig. 3b), whereas a gradual increase in ATP levels was observed in wild-type plants over time in 100 μmol m−2 s−1 light (Fig. 3a). Moreover, the addition of sucrose induced a significant increase in ATP levels in wild-type and chlorina mutants in the dark, indicative of higher mitochondrial respiration. In light with an intensity of 100 μmol m−2 s−1, the ATP content increased by approximately 18% in the wild-type plants, but decreased by about 52% in the oschlh mutant (Fig. 3c). Under a higher light intensity of 400 μmol m−2 s−1, the ATP content decreased in wild-type and oschlh mutants, 2 h after the initiation of light treatment, resulting in 33.5 and 74.3% decreases, respectively (Fig. 3d), and suggesting an inhibition of mitochondrial respiration. In wild-type plants, this decrease could result from photoinhibition of photosynthetic ATP synthesis due to the actinic light treatment for 2 h at 400 μmol m−2 s−1 or it could be partially due to the photorespiratory metabolic consumption. In fact, the photosynthetic electron transport rates decreased in wild-type during the shift to higher light intensity (400 μmol m−2 s−1) from 55 μmol m−2 s−1 of actinic light (data not shown). These data therefore indicate that light influences the ATP production in oschlh mutant plants, suggesting that mitochondrial electron transport is inhibited in illuminated mutant leaves.

Fig. 3
figure 3

ATP content of oschlh mutant seedlings. a WT and b oschlh Time course of ATP production in the absence or presence of 3% sucrose in the growth medium. c,d Inhibition of ATP production by light in intact seedlings of wild-type and oschlh mutant. Leaves were harvested 2 h after the application of 100 μmol m−2 s−1 (a–c) and 400 μmol m−2 s−1 (d) light following an 8-h dark cycle. Other experimental conditions are as described in Fig. 1. Data are expressed as the mean ± SE (n = 3)

Full size image

Determination of the cytosolic NADH/NAD+ ratio in oschlh mutant leaves in dark and light conditions

To determine whether cytosolic nicotinamide adenine dinucleotide (NADH) is also regulated by light in the oschlh mutant leaves, we attempted to estimate the cytosolic NADH/NAD+ ratio by measuring the lactate/pyruvate ratio in the leaves of wild-type and oschlh mutants; this ratio is coupled to the cytosolic NADH/NAD+ ratio via LDH (Table 1). In illuminated leaves, the cytosolic NADH/NAD+ ratio was 1.33 × 10−4; this ratio was higher than that observed in leaves kept in the dark (0.68 × 10−4). As expected, the ratio was significantly decreased in oschlh mutants, which further indicates that light inhibits the mitochondrial metabolism in oschlh mutant leaves.

Table 1 Pyruvate and l-lactate production and cytosolic NADH/NAD+ ratio in leaves of WT and oschlh mutant seedlings
Full size table

On the other hand, induction of internal type II NADH dehydrogenase genes suggests that alternative respiratory chain may be specifically activated in the light (Amirsadeghi et al. 2007). It was proposed that this alternative respiratory pathway might be required to accommodate the increased levels of matrix NADH generated by the glycine oxidation step of photorespiration (Svensson and rasmusson 2001). We compared the content of matrix NADH in the oschlh mutant leaves in dark and 100 μmol m−2 s−1 light conditions (Fig. 4). The internal rotenone-sensitive NADH oxidation (via complex I) in isolated mitochondria (wild-type) was approximately 33% lower after plants were exposed to light for 2 h. In the oschlh mutant leaves, NADH oxidation via complex I decreased by approximately 80%. The internal rotenone-insensitive NADH oxidation activity was 61% higher in wild-type after plants were exposed to light for 2 h, whereas it was 21% lower in the oschlh mutant leaves. Our results indicate that NADH oxidation by the main respiratory chain (complex I) is significantly inhibited in illuminated oschlh mutant leaves.

Fig. 4
figure 4

NADH oxidation activities of mitochondria purified from leaves of oschlh mutant seedlings. Internal rotenone-sensitive NADH oxidation (white boxes). Internal rotenone-insensitive NADH oxidation (black boxes). Asterisks above the columns indicate values that are significantly different from control values (P < 0.05). Data represent the mean ± SE (n = 8 for wild-type and n = 6 for oschlh plants). The experimental conditions are described in "Materials and methods"

Full size image

H2O2 content in leaves of oschlh mutant seedlings

We hypothesized that the inhibition of mitochondrial activity by light results in elevated levels of reactive oxygen species (ROS), which include H2O2, in oschlh mutant leaves in the light. To examine the production of ROS in oschlh and wild-type plants in vivo, we examined the leaf H2O2 content over the course of a diurnal cycle, as the contribution of leaf mitochondria to whole cell H2O2 production varies as a function of light (Fig. 5a). In both genotypes, global leaf H2O2 increased significantly during the 16-h light period, but decreased over the 8-h dark period. At all times, however, the H2O2 content was higher in oschlh mutant plants than in wild-type plants, with this difference being most significant in the light. Our findings therefore imply that internal H2O2 levels may play a significant role in mitochondrial activity in oschlh mutant leaves.

Fig. 5
figure 5

Diurnal changes in H2O2 content (a) and associated catalase activity (b) in oschlh mutant leaves. Plants were grown in the presence of 3% sucrose for 10 days using a 16-h light/8-h dark cycle with 100 μmol m−2 s−1 light and were then exposed to a light intensity of 100 μmol m−2 s−1. Leaf samples were harvested at the indicated times during a diurnal cycle. The white and black horizontal bars indicate the light and dark periods, respectively. The open and closed columns represent oschlh mutant and wild-type plants, respectively. Values represent the mean ± SE from three independent experiments. Asterisks above the columns indicate values that are significantly different from the wild-type values after 2-h treatment in the dark in each box (P < 0.05)

Full size image

Catalases (CATs) are the major H2O2 scavengers and remove the bulk of cellular H2O2. The alteration of the levels of expression of these enzymes allows the in planta modulation of H2O2 concentration (Vandenabeele et al. 2004). We examined catalase abundance in oschlh and wild-type plant leaves during the diurnal cycle (Fig. 5b). The catalase activity patterns were similar in both genotypes and were enhanced by light in both cases. These results indicate that catalase operates normally but is not able to effectively reduce H2O2 levels in the mutant cells, which suggests that alternative H2O2-scavenging enzymes are necessary to regulate H2O2 levels in the oschlh mutant.

Global leaf H2O2 concentrations were higher in the oschlh mutant in light condition, showing a 2- to 3-fold increase over time (Fig. 6a). The content of H2O2 in the leaves of both genotypes fluctuated over time, suggesting the activity of enzymes responsible for antioxidant defense. Furthermore, H2O2 was produced in high amounts in oschlh mutants in the dark. To establish the further role of H2O2 in mesophyll protoplasts of the chlorina type mutants, we examined the effects of ascorbic acid (AsA), a natural antioxidant, and CAT on the ATP content in light condition over time. As shown in Fig. 3b, ATP content of chlorina-type mutants was decreased in light condition over time (Fig. 6b). However, pre-incubating the protoplasts with AsA (Fig. 6c) or CAT (Fig. 6d) maintained the ATP content over time. Our findings therefore suggest that internal H2O2 levels inhibit the mitochondrial activity in oschlh mutant leaves.

Fig. 6
figure 6

H2O2 content (a) and effects of antioxidants on ATP content (b–d) in mesophyll protoplasts of wild-type and chlorina mutants. a H2O2 content in leaves of intact seedlings. Plants were grown with 3% sucrose in the medium for 10 days using a 16-h light/8-h dark cycle with 100 μmol m−2 s−1 light and were then exposed to light intensities of 100 μmol m−2 s−1. Leaf samples were harvested at the indicated times from the seedlings after 8-h dark adaptation. b–d ATP content in mesophyll protoplasts. The protoplasts (40 μg protein/ml) incubated in incubation medium (0.6 M sorbitol, 1 mM CaCl2, 10 mM KCl, 10 mM Mes-NaOH, pH 6.2) without (b) or with AsA at 1 mM (c) or with CAT at 100 Uml−1 (d) for 30 min in the dark and then exposed to light intensities of 100 μmol m−2s−1. Protoplast samples were harvested at the indicated times after light treatment at RT. Values are the mean ± SE from three independent experiments

Full size image

Analysis of H2O2-scavenging enzymes in leaves of oschlh mutant seedlings

From a typical microarray experiment, we identified significant changes in the expression levels of genes involved in oxygen and ROS metabolism (3.4 and 18.5% for genes up- and down-regulated, respectively) and in the response to oxidative stress (3.7 and 20.4% for genes up- and down-regulated, respectively) (data not shown). We therefore performed a PCR experiment to confirm the expression of H2O2-scavenging enzymes (Fig. 7a). Ascorbate peroxidase (APX) is a class I peroxidase that is localized in the cytosol (Teixeira et al. 2004). OsAPX1 was highly expressed in plants in the dark and its expression was further induced by light in wild-type plants, but not in chlorina-type plants (oschlh-1 and oschlh-2) (Fig. 7b-a). The expression of OsAPX2 (which is a class II peroxidase) was induced by high levels of light in the two chlorina-type plants (Fig. 7b-b). Light induced an increase in the cytosolic expression of thioredoxin (OsTRX-h) (Santos and Rey 2006); this gene was also up-regulated in the dark in the chlorina mutants when compared with wild-type plants (Fig. 7b-c). Interestingly, the promoters of OsTRX-h, glutaredoxin (OsGRX) (Minakuchi et al. 1994) (Fig. 7b-d), and cytosolic superoxide dismutase (OsFeSOD) (Feng et al. 2006) (Fig. 7b-f) contain a novel cis-element that regulates expression in response to methyl violgen, but does not respond to H2O2 (Santos and Rey 2006). The iron-associated superoxide dismutase gene OsFeSOD produced two protein isoforms of different sizes (Fig. 7a). In contrast with a previous report that indicated that the accumulation of both isoforms increases in response to light (Feng et al. 2006), our results showed that OsFeSODb (708 bp) appears to act antagonistically to OsFeSODa (859 bp) in the light. Metallothioneins are small, ubiquitous Cys-rich proteins involved in H2O2 scavenging and metal homeostasis (Wong et al. 2004). The expression of the metallothionein gene OsMT2b was significantly induced in all genotypes studied, in both dark and light conditions (Fig. 7b-e). In the genotypes examined here, OsRAC1 functioned in response to light as a suppressor of H2O2 scavengers, assuming that its enzyme activity is 27% lower in mutants in the light. Taken together, these results suggest that H2O2-scavenging enzymes would function normally except for OsAPX1 and OsRAC1 genes in the mutant. The expression of the alternative oxidase c (OsAOXc) gene, which encodes a mitochondrial antioxidant, was markedly induced in response to light in wild-type but not in mutant plants (Fig. 7b-i). The expression levels of this gene in the dark are negligible. Hence, low expression of light-inducible OsAOXc transcripts may also be responsible for the high levels of H2O2 detected in the illuminated leaves of the mutant. For this reason, it is possible that the inhibition of mitochondrial activity by light in oschlh mutant plants results in the accumulation of high levels of H2O2.

Fig. 7
figure 7

RT-PCR analysis of the comparative expression levels of antioxidant genes in oschlh mutant leaves. After an 8-h dark adaptation period, leaves were exposed to light (100 μmol m−2 s−1) for 2 h and were then harvested. Total RNA from each sample (20 μg) was subjected to RNA gel blot analysis on the same membrane; the bands shown are the products of RT-PCR. a Gene expression was assessed using probes for the indicated genes and using OsACTIN1 as a loading control. Representative RNA gel blots are shown. b Quantitative analysis of expressed genes. Relative transcript levels were given by normalizing expression to the highest value for each gene. These experiments were repeated at least three times. a OSAPX1; b OsAPX2; c OsTRX-h; d OsGRX; e OsMT2b; f OsRAC1; g OsFeSOD (859 bp); h OsFeSOD (708 bp); i OsAOXc. L light, D dark. Black boxes dark treatment, white boxes light treatment. Asterisks above the columns indicate values that are significantly different from the wild-type values after 2-h treatment in the dark in each box (P < 0.05)

Full size image

Discussion

The results described here revealed the mechanisms underlying the regulation of mitochondrial respiration by light in chlorophyll-deficient rice mutant plants. The growth rate of the OsCHLH knockout mutant was considerably enhanced by incubation in sucrose (Fig. 1b). As the addition of sucrose to the growth medium increases the rate of mitochondrial respiration (Journet et al. 1986), the growth of the mutant seems to be closely associated with the respiratory metabolism. These results imply that mitochondrial function supports the cellular energy requirements of the oschlh mutant. On the other hand, aleaf of the albino oschlh mutant had no electrical responses to light, but did when the light was off (Fig. 2b). This light response indicates that ATP produced photosynthetically affects membrane potential (Spalding and Goldsmith 1993). However, changes of V m in both wild-type and oschlh mutant were observed, although the magnitude of the response was different when the light was off. These dark-induced changes to the membrane potential (V m) were not inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthetic electron transport inhibitor (Stahlberg et al. 2000). We contend therefore that dark responsive electric currents in theses mutant plants are due to mitochondrial respiration, indicating that the maintenance of growth rate is associated with a high efficiency of respiratory ATP production. These results imply that they were supported by the mitochondrial functions for oschlh mutants’ cellular energy requirements.

The main function of the mitochondria is the generation of ATP by oxidative phosphorylation (Fernie et al. 2004). In the current study, the mitochondrial activity displayed comparative levels of ATP in the oschlh plant leaves was inhibited by light (Fig. 3b). The ATP concentration in oschlh mutants were largely affected by light treatment, which implies that the respiratory metabolism is regulated by light. Some studies on leaf respiration have reported that the rate of mitochondrial respiration in the light is less than that in darkness (Brooks and Farquhar 1985; Krömer 1995; Atkin et al. 2002), with the degree of inhibition ranging from 16 to 77%. Under illumination, mitochondrial respiration may be down-regulated because photosynthesis supplies sufficient amounts of ATP and NADPH or the enzymes of the TCA are potentially inactivated (Loreto et al. 2001; Pinelli and Loreto 2003). Some reports based on O2 consumption suggest an inhibition of respiration in light conditions (Canvin et al. 1980; Bate et al. 1988), whereas others suggest that the rate of respiration is invariant, regardless of illumination conditions (Gerbaud and Andre 1980). Based on our current analysis, we hypothesized that this phenomenon is due to the inhibition of mitochondrial respiration by light in the mutant, as chlorina-type plant leaves have no PS II activity.

Mitochondria are the main target of oxidative damage in illuminated leaves (Foyer and Noctor 2000; Bartoli et al. 2004). Mitochondrial activity in the dark includes the oxidation of nicotinamide adenine dinucleotide phosphate (NAD(P)H) in the mitochondrial electron transport chain (Nunes-Nesi et al. 2007). A biochemical measurement revealed a drop in the cytosolic NADH/NAD+ ratio in oschlh mutant leaves, which was verified at the level of the cellular NAD(H) pool in the cytosol (Table 1). On the other hand, we compared the level of matrix NADH generated by the glycine oxidation step of photorespiration in the oschlh mutant leaves in dark and 100 μmol m−2 s−1 light conditions (Fig. 4). Our results indicate that NADH oxidation by the main respiratory chain (complex I) is significantly inhibited in illuminated oschlh mutant leaves.

The bulk of cellular H2O2 seems to be formed during substrate oxidation at the level of the mitochondrial complex II (Braidot et al. 1999). Because the addition of mitochondrially-targeted antioxidants prevents the inhibition of respiration (Murphy 2004) and the contribution of leaf mitochondria to whole cell H2O2 production varies between light and dark conditions, we further compared leaf H2O2 production throughout a diurnal cycle (Fig. 5a). The H2O2 content was under light and diurnal regulation in plants of both genotypes. The oschlh mutants produced high levels of H2O2 in the light and these levels decreased in the dark, which seems to indicate that mitochondria are affected by light. In contrast, abscisic acid (ABA), which mediates H2O2 production in cells, is partly involved in this phenomenon because of the impairment of ABA signal transduction mechanisms in chlorina Arabidopsis (Shen et al. 2006). As O2 consumption by mitochondria is significantly decreased after H2O2 treatment (Sweetlove et al. 2002), the activity of CAT may be induced to minimize oxidative damage in these experimental conditions (Figs. 5b and 6d). The treatment of protoplasts with ascorbic acid (AsA) also recovered ATP content that might be inhibited by internal H2O2 levels (Fig. 6c). Therefore, the generation of H2O2 in the cytosol results in the inhibition of mitochondrial activity in the mutant.

Changes in the respiratory metabolism in illuminated leaves may be indicative of changes in oxygen and ROS metabolism. The genes investigated, particularly OsAPX1 and OsRAC1, which are expressed at comparatively low levels in oschlh mutant plants (Fig. 7b). OsAOXc mitochondrial antioxidant transcripts were also poorly induced by light in the leaves of mutant plants and thus failed to lower H2O2 levels in this organelle. Increased levels of H2O2 result in ATP depletion in plant cells (Tiwari et al. 2002). Therefore, our results indicate that illumination leads to the accumulation of high H2O2 levels in cells and thereby inhibits mitochondrial electron transport in oschlh mutant plant leaves, which ultimately suppresses plant growth during the day. Furthermore, we consider this result to be a strong indication that the activity of mitochondria in the illuminated leaves of wild-type plants is closely related to the chloroplast activity.

On the other hand, complex I and III of the mitochondrial respiratory chain are the major site of intracellular ROS production. Mitochondria are the sensitive target of ROS attack and play a primary role in triggering and/or executing ROS-dependent apoptotic cell death in animals (Dat et al. 2003; Apel and Hirt 2004). In plants, mitochondria may also serve as first relay stations where the initial alteration in ROS homeostasis is triggered (Gao et al. 2008). Our results have shown that the contribution of leaf mitochondria to whole cell H2O2 production varied as a function of light. In the light regulation of plant respiratory activity, we have here questioned how mitochondria are governed by the light-associated processes. Many photoreceptors reside in the nucleus, cytosol or plasma membrane, with some photoreceptors communicating between the cellular compartments upon exposure to light (Chen et al. 2004), especially to blue light (Islam et al. 2009). In contrast, cryptochrome 3 is localized to chloroplasts and mitochondria (Kleine et al. 2003), indicating that these organelles may directly perceive light. We need to further investigate the photoreceptor-mediated control of mitochondria elsewhere.