JXB Advance Access originally published online on May 19, 2006
Journal of Experimental Botany 2006 57(9):2075-2085; doi:10.1093/jxb/erj161
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RESEARCH PAPER |
The mitochondrial CMSII mutation of Nicotiana sylvestris impairs adjustment of photosynthetic carbon assimilation to higher growth irradiance
1Laboratoire d'Ecophysiologie Végétale, UFR Scientifique d'Orsay, Université Paris XI, Orsay, France
2Laboratoire Signalisation Redox, Institut de Biotechnologie des Plantes, Université Paris XI, Orsay, France
3Laboratoire Mitochondries et Métabolisme, CNRS, UMR 8618, Institut de Biotechnologie des Plantes, Université Paris XI, Orsay, France
*To whom correspondence should be addressed. E-mail: peter.streb{at}ese.u-psud.fr
Received 11 January 2005; Accepted 10 February 2006
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Abstract |
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The CMSII mutant of Nicotiana sylvestris, which lacks a functional mitochondrial complex I, was used to investigate chloroplast–mitochondria interactions in light acclimation of photosynthetic carbon assimilation. CMSII and wild-type (WT) plants were grown at 80 µmol m–2 s–1 photosynthetic active radiation (PAR; 80) and 350 µmol m–2 s–1 PAR (350). Carbon assimilation at saturating PFD was markedly higher in WT 350 leaves as compared with WT 80 leaves, but was similar in CMS 80 and CMS 350 leaves, suggesting that the mutant is unable to adjust photosynthesis to higher growth irradiance. WT 350 leaves showed several general characteristic light acclimation responses [increases in leaf specific area (LSA), total chlorophyll content, and chlorophyll a/b ratio, and a higher light compensation point]. In contrast, a similar chlorophyll content and chlorophyll a/b ratio were measured for both CMS 80 and CMS 350 leaves, while LSA and the light compensation point acclimated as in the WT. The failure of CMSII to adjust photosynthesis to growth PFD did not result from lower quantum efficiency of PSII, lower whole-chain electron transport rates (ETRs), or lower ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) and sucrose phosphate synthase (SPS) capacities. Excess ETR not used for carbon assimilation was even higher in CMS 350 than in WT 350. Since photochemical fluorescence quenching and the initial activity of NADP malate dehydrogenase (NADP-MDH) were identical in WT 350 and CMS 350 leaves but the activation state of NADP-MDH was different, redox signals from primary ETR are not involved in the signal transduction of light acclimation, while a contribution of stromal redox state cannot be excluded. When mature plants were transferred between 350 and 80 conditions, the mutant showed acclimatory tendencies, although adjustments were not as rapid or as marked as in the WT, and the response of the initial activities of Rubisco and NADP-MDH was impaired or altered. Initial activities of Rubisco and SPS at limiting concentration were also affected in CMS 350 as compared with WT plants when compared at growth irradiance or after in situ activation at 1000 µmol m–2 s–1 PAR. The data demonstrate that chloroplast–mitochondria interactions are important in light acclimation, and modulation of the activation state of key photosynthetic enzymes could be an important mechanism in this cross-talk.
Key words: NADP-malate dehydrogenase, net carbon assimilation, photosynthetic light acclimation, primary photosynthetic reactions, Rubisco, SPS
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Introduction |
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Mitochondria contribute to the photosynthetic metabolism of leaves. In the photorespiratory pathway they are responsible for the conversion of glycine to serine (Douce and Neuburger, 1999), and they also support the cytosol with ATP required for sucrose synthesis (Krömer, 1995). Mitochondria may contribute to cellular redox balance by electron consumption mediated by the malate valve (Hoefnagel et al., 1998; Padmasree et al., 2002). The inhibition of mitochondrial metabolism by oligomycin decreases sucrose synthesis and, in turn, limits photosynthesis (Krömer and Heldt, 1991; Hurry et al., 1995). Hence, mitochondrial function is necessary for optimal photosynthetic activity (Krömer, 1995; Padmasree et al., 2002).
The Nicotiana sylvestris mutant cytoplasmic male sterile (CMS)II lacks the NAD7 protein of complex I of the mitochondrial electron transport chain and is functionally defective in NADH oxidation (Gutierres et al., 1997). Since the CMSII mutant is viable, NAD(P)H oxidation in the mitochondria proceeds through alternative NAD(P)H-dehydrogenases with less respiratory efficiency (Sabar et al., 2000). Accordingly, CMSII has lower assimilation rates in ambient air (21% oxygen, 380 ppm CO2) compared with the wild type (WT) (Sabar et al., 2000; Dutilleul et al., 2003a). However, WT and CMSII leaves had the same photosynthetic capacity, as indicated by maximum oxygen evolution at saturating CO2, and similar amounts and activities of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), suggesting that the photorespiratory pathway is involved in depression of photosynthesis under ambient air conditions in the mutant (Dutilleul et al., 2003a). This previous analysis suggested that decreased photosynthesis in the mutant was not caused by stomatal limitations to CO2 diffusion or insufficient mitochondrial ATP supply for sucrose synthesis (Dutilleul et al., 2003a).
Previous data suggest that effects on photosynthetic activity in CMSII are linked to alteration of the cellular redox balance. This is shown by modified expression of antioxidative genes and increased stress tolerance, accumulation of leaf NAD and NADH, and adjustment of interactions between carbon and nitrogen assimilation (Dutilleul et al., 2003b, 2005). Similarly, the FRO1 mutation of Arabidopsis, which is also affected in mitochondrial complex I function, has altered nuclear gene expression, probably mediated by reactive oxygen species (Lee et al., 2002). It is now established that light-dependent changes in the redox state of chloroplast electron transport components such as plastoquinone and cytochrome b6/f, as well as redox-active soluble compounds such as thioredoxin and glutathione, control gene expression in the chloroplast and the nucleus (Pfannschmidt, 2003). Among the encoded products are the reaction centre proteins of photosystem I (PSI) and PSII as well as light-harvesting complex (LHCII) proteins and the large subunit of Rubisco (Pfannschmidt, 2003), proteins clearly indispensable for photosynthetic function and capacity.
Irrespective of the signals involved, photosynthetic activity and capacity depend on the developmental history of a leaf (Anderson and Osmond, 1987). Shade- or low-light-grown plants have thinner leaves and lower photosynthetic capacity, and photosynthesis is saturated at lower photon flux density (PFD) than in sun- or high-light-grown plants (Bailey et al., 2001; Murchie et al., 2002; Akoumianaki-Ioannidou et al., 2004). The acclimation of leaves to higher or lower irradiance is accompanied by the adjustment of pigment contents and chlorophyll (Chl) a/b ratios, photosystem antenna size, Rubisco content, and sucrose synthesis (Anderson and Osmond, 1987; Bailey et al., 2001; Murchie et al., 2002). Finally, light acclimation of leaves serves to maintain the chloroplastic electron transport chain in a preferentially oxidized state in order to prevent over-reduction and maintain efficient photosynthetic performance (Bailey et al., 2004). Hence, it is tempting to speculate that light acclimation of carbon assimilation is triggered by the chloroplastic redox state, in the electron transport chain and/or in the soluble fraction. If so, acclimation could be dependent on mitochondrial function via its interference with cellular redox status. Up to now, however, little experimental evidence has been presented to show that chloroplast composition depends on redox signals (Walters, 2005).
To analyse the importance of mitochondrial electron transport status in light acclimation, this study compared the ability of CMSII and WT leaves to adjust photosynthesis to low or high growth light intensity. As key parameters accompanying acclimation, leaf thickness, Chl content, and Chl a/b ratios were measured. Furthermore, the influence of light growth irradiance on primary as well as on secondary photosynthetic reactions was investigated, while the relative redox state of the primary electron acceptor QA (qP) and of the soluble chloroplast fraction was estimated.
The aims of the present study were: (i) to examine whether the absence of mitochondrial complex I has any effect on light acclimation of carbon assimilation; and (ii) if so, to investigate whether this influence is mediated by changes in chloroplast redox state.
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Materials and methods |
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Plant material and growing conditions
Nicotiana sylvestris mutant plants (CMSII) and WT were used for the experiments (Gutierres et al., 1997). Plants were grown in a greenhouse under controlled conditions (16 h light/8 h dark) at either 80 or 350 µmol m–2 s–1 photosynthetic active radiation (PAR) produced by sodium lamps (SON-T AGRO Philips Electronics) at 23/15 °C day/night temperature. The plants were regularly irrigated with nutrient solution (Hydrokani C2 Hydro Agri Spécialités, France). WT and CMSII were grown for 6–18 weeks (Table 1) until they had reached a similar developmental stage as decided by the time before shoot expansion preceding flowering. For measurements, the youngest fully developed leaves were taken after the first hour in the light period. For some experiments, plants were transferred for several days from low light to high light conditions, and vice versa.
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Gas exchange and chlorophyll fluorescence measurements
CO2 gas exchange was measured together with Chl fluorescence emission on attached leaves with a Li-Cor 6400-40 infrared gas analysis system (Li-Cor Corp., Lincoln, NE, USA) equipped with a leaf chamber fluorometer. Leaves were dark adapted for at least 30 min to determine dark respiration, Fo, and Fm. During actinic illumination, Chl fluorescence measurements were taken continuously (Ft). After stabilization, gas exchange was determined followed by a saturating flash of 2 s duration to measure F'm and a short period of far red light to measure F'o. From these measurements, several fluorescence parameters were calculated according to Schreiber et al. (1986) and Genty et al. (1989): qP=(F'm–Ft)/(F'm–F'o) and
PSII=F'm–Ft/F'm. The whole-chain electron transport rate (ETR) was calculated according to Ghashghaie and Cornic (1994). Therefore, the quantum yield of PSII (PSII) was calibrated against the quantum yield of CO2 assmilation (CO2) in an atmosphere containing 0.5% oxygen (Fig. 2), where CO2 is the quantum yield of gross photosynthesis as approximated by net carbon assimilation+dark respiration. For CO2 calculation, leaf absorbance was measured for each plant type with a UV-VIS spectrometer (Lambda 18; Perkin Elmer, Shelton, CT, USA). ETR in air was recalculated as described by Ghashghaie and Cornic (1994) (ETR=CO2xPARx4). The advantage of this method using a calibration of PSII against CO2 is a better approximation of linear ETR over the whole leaf as compared with the classical method of Krall and Edwards (1992), in particular if leaf thickness and different Chl contents may influence light penetration, usually resulting in slightly lower ETR under atmospheric conditions (Ghashghaie and Cornic 1994). In order to express the excess of transported electrons not used for carbon assimilation, carbon assimilation and dark respiration were subtracted from the recalculated ETR [ETR–(4An+Rd)] assuming that four electrons are necessary for the assimilation of one CO2 (Streb et al., 2005).
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Enzyme and chlorophyll measurements
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco: EC 4.1.1.39):
Two leaf discs (9.62 cm2, 0.25 g fresh weight) were frozen in liquid nitrogen and stored at –80 °C. Frozen material was ground to a fine powder with a mortar and a pestle in liquid nitrogen and extracted in 5 ml of 100 mM HEPES-KOH (pH 7.5), 5 mM MgCl2, 5 mM EGTA, 6% (w/w) polyvinylpyrrolidone-25, 7% (w/w) polyethylene glycol 20000, 2 mM dithiothreitol (DTT), 10% (v/v) glycerol, 20 µM 4-amidinophenylmethanesulphonyl fluoride, 1 µM pepsttin, and 1 µM leupeptin. Polyvinylpolypyrrolidone (50%, w/w) was added during grinding. The homogenate was centrifuged for 30 min at 4 °C in a microcentrifuge (5804 R, Eppendorf, Germany) at 12 500 g. The supernatant was desalted with Sephadex G25TM columns (Pharmacia, AMERSHAM, Sweden) before enzyme assays.
Both initial and total activities were measured in 3 ml cuvettes containing 100 mM Bicine-KOH (pH 8), 25 mM NaHCO3, 20 mM MgCl2, 5 mM creatine phosphate, 0.25 mM NADH, 3.5 mM ATP, 5 U ml–1 glyceraldehyde 3-phosphate dehydrogenase, 5 U ml–1 phosphoglycerokinase, and 5 U ml–1 creatine phosphokinase. The reaction was initiated by the addition of 0.5 mM ribulose-1,5-bisphosphate. For total Rubisco activity, extracts were preincubated for 12 min at 25 °C in the measuring buffer before starting the reaction. The activities were determined by following NADH oxidation at 340 nm. The results were divided by 2 in order to present Rubisco activity as CO2 consumption.
NADP-malate dehydrogenase (NADP-MDH: EC. 1.1.1.82):
Initial and maximal NADP-MDH assays were measured as described in Dutilleul et al. (2003a), except that 5 mM DTT was combined with 20 µM thioredoxin in order to confirm maximum activity. The activities were determined by following the NADPH oxidation at 340 nm over 5 min, suggesting that side activity by NAD-MDH is very low (Scheibe and Stitt, 1988).
Sucrose phosphate synthase (SPS: EC 2.4.1.14):
SPS activities under limiting (SPSlim) and saturating (SPSmax) substrate conditions were determined spectrophotometrically at 620 nm by comparison with sucrose standard, suggesting that limiting conditions approach physiological substrate and inhibitor concentrations, as described by Pelleschi et al. (1997).
Chlorophyll and protein contents:
Chl content and the Chl a/b ratio were determined spectrophotometrically from 80% acetone extracts using the absorption coefficients of Porra et al. (1989).
Soluble proteins were extracted from frozen material ground in liquid nitrogen into a buffer containing 0.1 M TRIS–HCl (pH 8.1), 10% sucrose, and 0.5% ß-mercaptoethanol. The extract was centrifuged for 5 min at 15 000 g, and protein content was determined according to Bradford (1976).
If not indicated otherwise, all experiments were repeated independently at least three times and the standard error is shown. Analysis of variance (ANOVA) statistical analysis was performed with STATISTICA software (STATSOFT Inc., Tulsa, USA) in order to estimate statistically significant differences at the P <0.05 level.
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Results |
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Characterization of basic parameters in WT and CMSII plants grown at different irradiance
WT plants and the CMSII mutant were grown at either low light (80 µmol m–2 s–1 PAR: WT 80 and CMS 80) or high light (350 µmol m–2 s–1 PAR: WT 350 and CMS 350). Plants were investigated at the same developmental stage, as decided by the time of shoot expansion before flowering. At this time, 6–9 leaves were completely developed. WT plants grew faster than CMSII plants and expanded leaves had a larger size (Table 1). High irradiance accelerated growth as compared with low irradiance. The leaf area and the leaf specific area (LSA) were clearly lower in WT 350 than in WT 80 (Table 1). A decrease in LSA was also observed in CMS 350 as compared with CMS 80, but total leaf size was similar in both growth conditions (Table 1). Soluble protein content on a leaf area basis was higher in high-light-grown plants compared with low-light-grown plants and in CMSII compared with WT leaves. A decrease of LSA and an increase of protein content, total Chl content, and of the Chl a/b ratio in WT 350 leaves compared with WT 80 leaves altogether are characteristic responses of plants grown under high light conditions in comparison with low-light-grown plants (Table 1). In contrast, the Chl content was very similar in CMS 80 and CMS 350, in both cases being much higher than in WT 80 but lower than in WT 350. In CMSII, the Chl a/b ratios remained similar in differently grown leaves (Table 1).
Net CO2 uptake
Light response curves of photosynthetic carbon assimilation showed a marked acclimation response to growth irradiance in the WT but not in the mutant (Fig. 1). Net carbon uptake in WT 350 leaves was much higher at light intensities >250 µmol m–2 s–1 PAR as compared with WT 80 leaves, while the light compensation point was shifted to higher PFD and the initial slope of carbon assimilation was steeper (Table 1). In the CMSII mutant, maximum net carbon uptake and the initial slope of carbon assimilation increased only slightly in CMS 350 leaves as compared with CMS 80 leaves. However, similar to the WT, the light compensation point was also higher in CMS 350 than in CMS 80 (Table 1). Furthermore, maximum applied PFD did not completely saturate photosynthesis in CMS 350. In summary, maximum net carbon uptake was comparable in WT 80 and the mutant leaves grown under both light conditions, but the light compensation point was elevated in mutant leaves (Fig. 1, Table 1). The lower net carbon uptake of CMSII as compared with the WT described previously (Sabar et al., 2000; Dutilleul et al., 2003a) was therefore dependent on light growth conditions and was almost absent when the WT and the mutant were compared after growth at low light (Fig. 1).
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Primary photosynthetic reactions
In order to investigate whether primary photosynthetic reactions may limit carbon assimilation in the mutant leaves, ETR was calculated after plotting the
PSII against the CO2 in an atmosphere containing 0.5% oxygen (Fig. 2). Both mutant and WT leaves grown under either light condition showed the same relationship between quantum efficiency of PSII and carbon assimilation in an oxygen-depleted atmosphere, indicating that PSII function is not impaired in the mutant. In WT as well as mutant leaves, nine photons were necessary for the assimilation of one molecule of CO2 (Fig. 2). The relationship shown in Fig. 2 was used to recalculate the ETR under atmospheric conditions as described by Ghashghaie and Cornic (1994). As shown in Fig. 3A, ETR in high-light-grown leaves exceeded ETR in low-light-grown leaves. At light intensities >500 µmol m–2 s–1 PAR, ETR in WT 350 and CMS 350 leaves was similar though slightly higher in WT 350 leaves. ETR in WT 80 and CMS 80 leaves differed only marginally at light intensities between 250 and 500 µmol m–2 s–1 PAR.
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In order to examine whether ETR might become a limiting factor in CMSII leaves, ETR was compared with net carbon uptake (Fig. 1). ETR not used for carbon assimilation is higher in CMS 350 than in WT 350 and therefore is in excess (Fig. 3B). At all light intensities applied, ETR exceeded carbon assimilation markedly in WT and mutant leaves, irrespective of growth conditions, suggesting that carbon assimilation is not limited by ETR in either condition. Furthermore, in CMS 350, excess electron transport is highest, necessitating additional alternative electron sinks (Fig. 3B).
Photochemical fluorescence quenching was higher in high-light-grown than in low-light-grown leaves and there was virtually no major difference between the WT and the mutant leaves, in agreement with ETR measurements (Fig. 3C).
Secondary photosynthetic reactions
Acclimation of photosynthetic activity in mutant leaves may also be affected by secondary photosynthetic reactions. As key enzymes, initial and total Rubisco, and SPSlim and SPSmax activities were investigated as well as the activation state of these enzymes. Initial Rubisco activity, when measured after 1 h at growth irradiance, was highest in WT 350 leaves and significantly lower in all other leaves, but the activation state was lowest in CMS 350. The initial and total activity of Rubisco was higher in leaves grown at high light than in leaves grown at low light (Fig. 4A). Total Rubisco activity was similar in CMS 350 and WT 350 leaves and in CMS 80 and WT 80 leaves. In order to examine whether CMS 350 leaves are affected in Rubisco activation as compared with the WT and with CMS 80, leaf discs were illuminated for 3 h at 1000 µmol m–2 s–1 PAR. As shown in Fig. 4B, initial Rubisco activity and the activation state increased in all varieties, but initial Rubisco activity was still highest in WT 350 leaves.
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Similarly to Rubisco, SPSlim and SPSmax activities of WT leaves depended on growth light conditions (Fig. 4C). However, SPSlim and SPSmax activities were similar in CMSII leaves grown at either light intensity. Furthermore, the SPSmax activity in both mutant leaves nearly corresponded to the maximum activity in WT 350 leaves (Fig. 4C). The SPSlim activity of CMS 80 and CMS 350 leaves was intermediate to that of high-light-grown and low-light-grown WT leaves (Fig. 4C). It should be noted that, on the basis of total leaf Chl content, SPSmax activities were identical in all varieties but SPSlim activity was still highest in WT 350 leaves (not shown) as was the SPS activation state (Fig. 4C).
In summary, enzyme capacities of Rubisco and SPS are nearly identical in WT 350 and CMS 350 leaves and therefore obviously not the primary cause for lower photosynthetic activity in CMS 350 as compared with WT 350 leaves, but initial and limiting activities of both enzymes were affected in CMS 350 leaves.
NADP-MDH activity
NADP-MDH activity was investigated as a possible electron sink in the chloroplast, which may support mitochondrial respiration with electrons via the malate valve and as an indicator of the chloroplast stroma redox state. Initial NADP-MDH at growth irradiance was higher in CMS 80 leaves than in WT 80, WT 350, and CMS 350 leaves, but the difference was not significant (Table 2). The NADP-MDH activation state was highest in low-light-grown WT plants and lowest in high-light-grown WT plants. Maximum NADP-MDH activity was, however, highest in WT 350 leaves, intermediate in CMSII grown under both light conditions, and lowest in WT 80 leaves (Table 2). On the basis of the Chl content, maximum NADP-MDH activities did not differ in all cultivars (not shown). During 3 h exposure of leaves to 1000 µmol m–2 s–1 PAR, the initial NADP-MDH activity increased strongly in WT leaves grown at both light conditions but remained almost constant in the mutant leaves (Table 2). This is also shown by a much stronger increase of the NADP-MDH activation state in WT as compared with CMSII leaves, while maximum NADP-MDH activity was not changed by light activation in all leaf types (Table 2).
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Reversibility of light acclimation
Low-light-grown plants were transferred for 3 d and 7 d to high light growth conditions, and vice versa. As can be seen from Figs 5 and 6, nearly all measured parameters in leaves after transfer to another light condition corresponded to values measured in leaves grown at the respective irradiance within 7 d after transfer. In particular, net carbon assimilation, taken here as an indicator for light acclimation, and dark respiration started rapidly, within 3 d after the transfer to adjust values to changing growth irradiance. No major difference in the response of WT and mutant leaves was observed (Fig. 5). Similarly, whole-chain ETR changed rapidly and markedly in response to altered light growth conditions in mutant and WT leaves. However, ETR in leaves transferred from low to high light did not completely correspond to ETR in leaves grown at high irradiance within the investigation period (Fig. 5). Photochemical quenching was identical in WT and mutant leaves, before, during, and after the transfer to another growth irradiance (Fig. 5). Similarly, total Rubisco activity was adjusted to changing growth irradiance in the mutant and the WT leaves, but adjustment of initial Rubisco and NADP-MDH activity was impaired in the mutant (Fig. 6). While maximum NADP-MDH activity acclimated to changing growth conditions in WT leaves, initial NADP-MDH activity decreased within the first 3 d after transfer from high- to low-light growth condition but remained nearly unaffected in low-light-grown leaves transferred to high light (Fig. 6). In mutant leaves, initial and maximum NADP-MDH activity did not respond within the first 3 d to changing light conditions. After 7 d of transfer to another growth light condition, initial NADP-MDH activity was intermediate to that in leaves grown at either high or low light (Fig. 6).
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Discussion |
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Carbon assimilation is only affected after growing CMSII in high light
The CMSII mutant of N. sylvestris, lacking the functional complex I of the mitochondrial electron transport chain, has a lower photosynthetic activity than the WT, despite similar photosynthetic capacity (Sabar et al., 2000; Dutilleul et al., 2003a). The present investigation shows that decreased photosynthesis is partially caused by the inability of the mutant to adjust photosynthetic carbon assimilation to higher growth irradiance. As shown in Fig. 1, carbon assimilation is very similar in CMSII and WT leaves when grown at low light but significantly different when grown at high light.
Light acclimation of chlorophyll content and ratio is affected in the CMSII mutant
The inability to adjust photosynthetic carbon assimilation to higher growth irradiance in the mutant raises the question of whether light acclimation in general is impaired in CMSII leaves. Acclimation to high growth light involves decreases in LSA and, usually, a higher Chl content on a leaf area basis, an increase of the Chl a/b ratio reflecting a relative lower ratio of LHCs to reaction centres, a higher photosynthetic activity at growth irradiance and at saturating PFD, and a higher light compensation point (Anderson and Osmond, 1987; Bailey et al., 2001; Murchie et al., 2002; Walters, 2005). Nicotiana sylvestris WT plants show all these characteristic light acclimation responses, but CMSII leaves were affected in light acclimation of Chl content and Chl a/b ratio (Table 1; Fig. 1). However, similarly to WT leaves, the LSA was even more decreased in CMS 350 as compared with CMS 80 leaves, and a higher protein content, fresh weight, and dry weight in CMS as compared with the WT indicated slightly thicker leaves. Furthermore, the light compensation point also acclimated to growth light. In conclusion, mutant leaves were only affected in light acclimation of Chl content and Chl a/b ratio, indicating altered composition of LHCs in comparison with the WT.
Primary photosynthetic reactions are not affected in the mutant
The inability of CMSII to adjust light absorption properties to growth light may affect the efficiency of primary photosynthetic reactions. This was investigated after comparing the PSII with the CO2 in an oxygen-depleted atmosphere (0.5% oxygen). As can be concluded from Fig. 2, the PSII and CO2 were identical in mutant and WT leaves grown under either growth irradiance. However, whole-chain ETR under atmospheric conditions was slightly lower in CMS 350 than in WT 350 but very similar in CMS 80 and WT 80 leaves (Fig. 3A), raising the question of whether ETR might limit carbon assimilation in CMSII leaves. The difference in whole-chain ETR between WT 350 and CMS 350 is, however, insufficient to explain lower net carbon assimilation in CMS 350 (Fig. 1). In contrast, comparing ETR with carbon assimilation shows that excess ETR is higher in CMS 350 than in WT 350 leaves (Fig. 3B). The possible sink for excess electrons in CMS 350 is presently unclear and remains to be examined. According to the fact that initial Rubisco activity is lower in CMS 350 than in WT 350 and leaf internal CO2 concentrations are similar (Dutilleul et al., 2003a; unpublished results), enhanced activities of photorespiration in the mutant leaves seem to be unlikely. Moreover, previous measurements of glycine:serine ratios provide little evidence for increased photorespiration in the mutant (Dutilleul et al., 2003a). Current studies in our laboratory investigate the possibility of an altered specificity factor of Rubisco and different mesophyll conductance in order to clarify the role of photorespiration in CMSII. Furthermore, NADP-MDH total and initial activities were not elevated in CMS 350 compared with WT 350 leaves, either under different growth conditions or after in situ activation at 1000 µmol m–2 s–1 PAR, and the activation state was very similar, excluding the possibility that the malate valve balances excess electrons in the mutant (Table 2). In contrast, initial NADP-MDH activity strongly increased in the WT but not in the mutant during light activation.
Initial activities of Rubisco and SPS are affected in the mutant leaves
In accordance with previous reports (Anderson and Osmond, 1987; Bailey et al., 2001; Walters, 2005), total Rubisco activity acclimated to growth light. This response was consistently observed in WT and CMSII leaves, suggesting a similar capacity for carbon assimilation, as reported previously (Dutilleul et al., 2003a). Savitch et al. (2000) have shown that SPS, catalysing the last step in sucrose synthesis, also acclimated to growth light intensity. This was also observed in SPSlim and SPSmax activity of the WT but not in the mutant leaves when activities were calculated on the basis of leaf area. The SPSmax activity was very similar in CMS 80 and CMS 350, suggesting that low-light-grown leaves already have elevated SPS activities (Fig. 4). In conclusion, total Rubisco activity and SPSmax did not correlate with lower photosynthetic activity of high-light-grown CMSII in comparison with high-light-grown WT leaves.
In contrast, the lower initial activity of Rubisco and SPSlim in CMS 350 as compared with WT 350 leaves is in good agreement with lower net carbon assimilation rates in the mutant. In order to investigate whether acclimation to higher light intensity impairs activation of Rubisco in the mutant, leaf discs were illuminated with 1000 µmol m–2 s–1 PAR for 3 h. Initial Rubisco activity was increased in mutant and WT leaves and both were higher in high-light-grown plants than in low-light-grown plants (Fig. 4). The activation state of Rubisco after light activation was similar in all varieties, but the initial activity was still lower in CMS 350 as compared with WT 350.
Redox signalling in WT and CMSII leaves
Previous studies suggest that the CMSII mutation changes the cellular redox balance as compared with the WT, causing increased expression of antioxidative enzymes and enhanced stress tolerance associated with accumulation of NAD and NADH (Dutilleul et al., 2003b, 2005). The redox state of components of the photosynthetic electron transport chain and of the soluble stroma fraction of chloroplasts, as well as reactive oxygen species, control the transcription of photosynthetic genes (Pfannschmidt, 2003). Among these redox-controlled gene products are the LHC of PSII and the large subunit of Rubisco (Pfannschmidt, 2003). Since total Chl contents were higher in CMS 80 than in WT 80 leaves but Chl a/b ratios were significantly lower in CMS 350 than in WT 350 leaves (Table 1), redox signals may be involved in the acclimation response of the WT but not of the mutant. According to Bailey et al. (2004), acclimation to growth light intensity serves to keep the photosynthetic electron transport chain oxidized and to guarantee efficient photosynthesis.
Two indicators for the redox state of the chloroplast were investigated, qP and NADP-MDH. qP was used to estimate the relative proportion of open and closed PSII reaction centres, whereas 1–qP is also known as the excitation pressure of PSII (Huner et al., 1998). However, qP may be valid only as a relative measure of the PSII reduction state, if the connectivity of PSII reaction centres is not known (Manuel et al., 1999). Nevertheless, qP was very similar in WT and mutant plants under all investigated conditions and higher in plants grown at high light than in plants grown at low light (Fig. 3). Furthermore, qP acclimated rapidly in WT as well as in mutant leaves to changing growth irradiance when plants were transferred from low- to high-light growth conditions, or vice versa (Fig. 5). Hence, a different redox state of the plastoquinone pool in WT and mutant plants under the respective growth irradiance is very unlikely, and could not serve as a signal in the WT as it is missing in the mutant.
Since the initial activity of NADP-MDH depends on activation by thioredoxin and, concomitantly, linear electron flux in the chloroplast (Miginiac-Maslow et al., 2000; Scheibe, 2004), it was taken as an indicator of chloroplast stroma redox state. Initial NADP-MDH activity was almost identical in WT 350, WT 80, and CMS 350 when leaves were taken after 1 h in the respective growth light, but slightly higher in CMS 80 leaves (Table 2). Therefore, under steady-state growth light conditions, the redox state of chloroplast stroma as estimated by initial NADP-MDH activity gave no indication that light acclimation in the WT is somehow triggered by this parameter nor that light acclimation in CMS 350 leaves might be affected. In fact, the differences in NADP-MDH activity in high- versus low-light-grown leaves are too low to conclude that there are different redox states of the chloroplast stroma, in particular since NAD-MDH activity might also contribute to the measured NADP-MDH activity (Scheibe and Stitt, 1988). Nevertheless, it was shown previously that NADP-MDH activity in CMSII exceeds that in the WT leaves during transients from darkness to light (Dutilleul et al., 2003a). Similar differences were observed during transfer from low- to high-light growth conditions (Fig. 6). Interestingly, maximum NADP-MDH activity showed no marked acclimatory response in CMS leaves in contrast to WT leaves (Fig. 6). Although changes in Rubisco and photosynthesis were still evident (Figs 5, 6), the extent of increase in these parameters in CMS 80 plants transferred to higher light was less than that observed in the WT under comparable conditions. Thus, some impairment of light acclimation in the mutant was also observed when plants were transferred between different growth irradiances. The present data indicate that this is not linked to changes in the redox state of photosynthetic electron transport, but effects mediated via changes in stromal redox state or redox cycling capacity cannot be discounted. The difference in carbon assimilation of high-light-grown WT and CMSII leaves may modify the composition of cellular sugar contents, raising the possibility that sugar signalling rather than different redox states is involved in light acclimation (Walters, 2005).
Reversibility of light acclimation
Finally, light acclimation with respect to photosynthetic carbon assimilation, dark respiration primary photosynthetic parameters, as well as total and initial enzyme activities were rapidly reversible in leaves of N. sylvestris and differences between mutant and WT leaves were small (Figs 5, 6). Within 3–7 d after changing growth light conditions, leaves of both CMSII and WT acclimated with respect to the measured parameters, suggesting that signal transduction for light acclimation is not affected in the mutant. However, contrary to results described by Murchie et al. (2005), Rubisco activity in fully developed leaves of N. sylvestris WT and CMSII acclimated to changing light conditions (Fig. 6) while Chl a/b ratios remained unchanged (not shown).
In conclusion, the decreased acclimation of carbon assimilation in CMS 350 indicates that the chloroplast–mitochondria interaction is important in photosynthetic light acclimation. The failure of the mutant to acclimate fully to higher irradiance is mainly manifested by the lower net carbon assimilation rate as compared with the WT, and this is correlated with lower initial Rubisco activity and with an unaltered Chl a/b ratio. Intriguing questions remain, however; notably, it remains to be established why Rubisco and SPS activities are less stimulated by high light growth in the mutant than in the WT, and which reactions consume excess electrons in CMS 350.
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Acknowledgements |
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We thank M Miginiac-Maslow for the generous gift of thioredoxin. The research was funded by the French MENRT and the CNRS.
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Abbreviations |
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Chl, chlorophyll; CMS, cytoplasmic male sterile; ETR, electron transport rate; LSA, leaf specific area; LHC, light-harvesting complex; NADP-MDH, NADP malate dehydrogenase; PAR, photosynthetic active radiation; PFD, photon flux density;
CO2/PSII, quantum yield of carbon assimilation/photosystem II; qP, photochemical chlorophyll fluorescence quenching; Rubisco, ribulose-1,5-bisphosphate carboxylase oxygenase; SPSlim/SPSmax, sucrose phosphate synthase at limiting/saturating substrate concentrations; WT, wild type. |
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