JXB Advance Access originally published online on August 31, 2006
Journal of Experimental Botany 2006 57(12):3195-3207; doi:10.1093/jxb/erl083
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RESEARCH PAPER |
The lack of mitochondrial complex I in a CMSII mutant of Nicotiana sylvestris increases photorespiration through an increased internal resistance to CO2 diffusion
1Laboratoire d'Ecologie, Systématique et Evolution, CNRS, UMR 8079, UFR IFR 87, Université Paris XI, Orsay, France
2Laboratoire Mitochondries et Métabolisme, Institut de Biotechnologie des Plantes, Université Paris XI, Orsay, France
3Department of Biochemistry, Mahatma Phule Krishi Vidyapeeth, Rahuri-413 722, Maharashtra, India
*To whom correspondence should be addressed. E-mail: peter.streb{at}ese.u-psud.fr
Received 24 February 2006; Accepted 15 June 2006
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Abstract |
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The cytoplasmic male sterile II (CMSII) mutant lacking complex I of the mitochondrial electron transport chain has a lower photosynthetic activity but exhibits higher rates of excess electron transport than the wild type (WT) when grown at high light intensity. In order to examine the cause of the lower photosynthetic activity and to determine whether excess electrons are consumed by photorespiration, light, and intercellular CO2, molar fraction (ci) response curves of carbon assimilation were measured at varying oxygen molar fractions. While oxygen is the major acceptor for excess electrons in CMSII and WT leaves, electron flux to photorespiration is favoured in the mutant as compared with the WT leaves. Isotopic mass spectrometry measurements showed that leaf internal conductance to CO2 diffusion (gm) in mutant leaves was half that of WT leaves, thus decreasing the cc and favouring photorespiration in the mutant. The specificity factor of Rubisco did not differ significantly between both types of leaves. Furthermore, carbon assimilation as a function of electrons used for carboxylation processes/electrons used for oxygenation processes (JC/JO) and as a function of the calculated chloroplastic CO2 molar fraction (cc) values was similar in WT and mutant leaves. Enhanced rates of photorespiration also explain the consumption of excess electrons in CMSII plants and agreed with potential ATP consumption. Furthermore, the lower initial Rubisco activity in CMSII as compared with WT leaves resulted from the lower cc in ambient air, since initial Rubisco activity on the basis of equal cc values was similar in WT and mutant leaves. The retarded growth and the lower photosynthetic activity of the mutant were largely overcome when plants were grown in high CO2 concentrations, showing that limiting CO2 supply for photosynthesis was a major cause of the lower growth rate and photosynthetic activity in CMSII.
Key words: Complex I-deficient CMS mutant, internal conductance, mitochondria, Nicotiana sylvestris, photorespiratory metabolism
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Mitochondrial function is commonly thought to interact with the photosynthetic activity of plant cells (Krömer, 1995; Padmasree et al., 2002). Unsurprisingly, the cytoplasmic male sterile II (CMSII) mutant of Nicotiana sylvestris lacking mitochondrial complex I (Pineau et al., 2005) and suffering from a decreased efficiency of NADH oxidation (Gutierres et al., 1997) has a lower photosynthetic activity than wild-type (WT) leaves when measured under ambient atmospheric conditions (380 ppm CO2, 21% O2) at different photon flux densities (Sabar et al., 2000; Dutilleul et al., 2003a). Nevertheless, this difference is not constant and depends on plant growth light conditions, decreasing at low and increasing at high growth irradiances (Priault et al., 2006). Furthermore, photosynthetic capacity, as determined by oxygen evolution at a high CO2 molar fraction, is identical in CMSII and WT leaves (Dutilleul et al., 2003a). This correlates with similar total Rubisco and maximum sucrose phosphate synthase (SPS) activities in both types of leaf (Dutilleul et al., 2003a; Priault et al., 2006). However, initial Rubisco and limiting SPS activities are lower in CMSII than in WT leaves when plants are grown under high light, suggesting that mutant leaves are somehow impaired in the activation of photosynthetic enzymes (Priault et al., 2006). Furthermore, stomatal resistance to CO2 diffusion was found to be similar in WT and CMSII leaves (Dutilleul et al., 2003a). In this contribution, the interaction of mitochondrial function with photosynthetic activity is further characterized in order to evaluate the role of photorespiration as an alternative electron acceptor suppressing photosynthetic activity of CMSII leaves.
The lower rate of net CO2 uptake observed in CMSII as compared with WT leaves may result from higher rates of CO2 loss by respiration in the light, since rates of dark respiration are higher in mutant when compared with WT leaves (Dutilleul et al., 2003a; Priault et al., 2006). Alternatively, photosynthetic activity may be affected by mitochondrial ATP supply for cytosolic sucrose synthesis (Krömer, 1995; Hoefnagel et al., 1998; Padmasree et al., 2002). However, previous investigations at saturating CO2 where the effect of limiting ATP supply should be most pronounced indicated that ATP supply for sucrose synthesis was not the primary cause for the lower photosynthetic activity of CMSII leaves (Dutilleul et al., 2003a).
Mitochondria may also interact with photosynthetic activity as a sink of excessive reducing equivalents generated during linear photosynthetic electron transport, in particular during photosynthetic induction (Hurry et al., 1995; Igamberdiev et al., 1998). Excess electrons can be exported from the chloroplast via the malate valve to support mitochondrial electron oxidation (Krömer, 1995; Padmasree et al., 2002; Scheibe, 2004). During photosynthetic induction, the initial activity of the chloroplastic NADP-malate dehydrogenase is higher in CMSII leaves than in WT leaves (Dutilleul et al., 2003a), although both are very similar during steady-state photosynthesis (Priault et al., 2006). In addition, high light increased the initial activity of NADP-malate dehydrogenase in the WT to higher levels than in the mutant leaves (Priault et al., 2006). Hence a limited capacity to transfer electrons from chloroplasts to mitochondria for oxidation cannot be ruled out as a contribution to the lower steady-state photosynthetic activity of the mutant plants grown at high photon flux density (PFD).
In addition to the lower photosynthetic activity, photosynthetic electron transport rates not used for carbon assimilation, as estimated by chlorophyll fluorescence and gas exchange, are slightly higher in high-light-grown mutant plants than in the corresponding WT leaves (Priault et al., 2006). This suggests additional electron-consuming processes in CMSII. However, the difference in excess electron transport between CMSII and WT leaves, as well as the difference in photosynthetic activity, disappear when plants are grown at a low light intensity (Priault et al., 2006).
Excess electrons are often transferred to oxygen (Ort and Baker, 2002). In addition to mitochondrial respiration, oxygen acts as an electron acceptor during photorespiration, the Mehler reaction, and chlororespiration mediated by the plastid terminal oxidase (PTOX) (Carol and Kuntz, 2001; Ort and Baker, 2002). The importance of these different pathways for electron consumption in photosynthesis is still a matter of debate, but photorespiration and the Mehler reaction are assumed to consume the majority of excess electrons (Asada, 1999; Ort and Baker, 2002). Electron consumption by the Mehler reaction and by the PTOX may alter the NADPH/ATP ratio generated by primary photochemistry in favour of ATP (Asada, 1999; Streb et al., 2005). Thus, this could theoretically compensate for the lower efficiency of ATP production in mitochondria of CMSII leaves if one assumes that ATP produced in the chloroplast would be available to other cell compartments. On the other hand, the Mehler reaction produces reactive oxygen species, putting an additional burden on cell metabolism (Kaiser, 1979). Since the antioxidative scavenging capacity is higher in mutant leaves when compared with the WT (Dutilleul et al., 2003b), reactive oxygen production might be sufficiently compensated without inhibition of carbon metabolism. Furthermore, according to Backhausen et al. (2000) and König et al. (2002), the malate valve is activated at a lower electron pressure than the Mehler reaction, but under steady-state conditions the NADP-malate dehydrogenase activity is not elevated in CMSII as compared with WT leaves (Priault et al., 2006).
The consumption of excess electrons by photorespiration would directly affect photosynthetic carbon assimilation by consuming both NADPH and ATP and by producing CO2 (Douce and Heldt, 2000). Nevertheless, previous investigations showed that (i) leaf internal CO2 molar ratios (ci) were identical in WT and mutant leaves under varying conditions; (ii) serine:glycine ratios did not change during photosynthesis in the mutant; and (iii) the initial Rubisco activity was lower in the mutant. Such observations suggest that mutant leaves do not show an increased photorespiratory activity (Dutilleul et al., 2003a; Priault et al., 2006).
The present investigation aimed to clarify the role of different electron-consuming pathways, in particular that of photorespiration, as possible electron sinks affecting the photosynthetic activity in CMSII leaves. Since the rate of oxygen consumption and the amount of electrons allocated to oxygen reduction had not been measured previously, it remained plausible that a lower internal conductance (gm) in mutant plants mediates a lower cellular CO2 molar fraction, enhancing photorespiratory oxygen reduction. Furthermore, although unlikely, the CO2/O2 specificity factor of Rubisco may be different in CMSII and WT leaves. Therefore, gas exchange and chlorophyll fluorescence measurements at varying internal CO2 and O2 molar fractions, as well as mass spectrometry were applied to evaluate the role of photorespiration in CMSII plants. It is shown that the CMSII mutation did not lead to an altered specificity factor of Rubisco, but rather it gave rise to a decrease in the internal CO2 conductance and, consequently, a decline of photosynthetic activity.
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Materials and methods |
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Plant material and growing conditions
Nicotiana sylvestris mutant (CMSII) and WT plants were grown in a greenhouse under controlled conditions at a PFD of 350 µmol m–2 s–1 for 6–10 weeks until they reached a similar developmental stage of 6–8 leaves preceding shoot expansion and flowering, as described in Priault et al. (2006). For measurements, the youngest fully developed leaves were taken after the first hour in the light period.
Gas exchange and chlorophyll fluorescence measurements
Responses of net carbon assimilation (An) and chlorophyll fluorescence to PFD (light curves) and internal CO2 molar fraction (ci: An/ci curves) under ambient (21%) and low (2% and 0.5%) oxygen content were measured simultaneously on attached leaves with an open infrared gas analysis system equipped with a leaf chamber fluorometer (Li-Cor 6400-40; Li-Cor Inc., Lincoln, NE, USA). Leaves were dark adapted for at least 30 min to determine dark respiration, Fo and Fm. During actinic illumination, chlorophyll fluorescence measurements were taken continuously (Ft). After stabilization of gas exchange, CO2 assimilation 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):
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The whole chain electron transport rate (ETR) was recalculated from the 
PSII/CO2 calibration curve according to Ghashghaie and Cornic (1994) as described in Priault et al. (2006) assuming that four electrons are used for the fixation of one molecule of CO2:
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Maximal carboxylation rate (Vc max) determination based on Michaelis–Menten kinetics
Vc max was obtained after redrawing the An/ci curves as a Lineweaver–Burk diagram at limiting ci 1/An=f(1/ci–
). It was then determined as the ‘inverse’ of the intersection point of this relationship with the y-axis. The regression coefficient of the diagram used for Vmax determination was 0.96 for WT and 0.86 for mutant leaves.
Calculations based on gas exchange and chlorophyll fluorescence measurements
Respiration in the light was estimated according to the method of Laisk (1977) as described in von Caemmerer (2000). An/ci curves were measured at three different PFDs (100, 500, and 800 µmol m–2 s–1) at six different CO2 levels ranging from 120 to 50 ppm ca (Fig. 1). The intersection point of the three An/ci curves was used to determine C* (x-axis) and the day respiratory rate, Rl (y-axis). The value of C* was used to calculate the apparent specificity factor of Rubisco according to Laing et al. (1974) using the relationship:
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Stomatal limitation to CO2 diffusion was calculated according to Farquhar and Sharkey (1982) as:
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Electron fluxes to the carboxylation (Jc) and oxygenation (Jo) reaction of Rubisco were calculated from measured total electron flux (Jt) at 800 µmol m–2 s–1 PFD, net carbon assimilation (An), and day respiration (Rl) according to Epron et al. (1995) assuming that photorespiration and carbon assimilation are the only reactions competing for electrons as:
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Carbon isotope discrimination (
obs) and internal conductance measurements
The carbon isotope discrimination associated with photosynthesis was measured using the system already described by Tcherkez et al. (2005). Briefly, an assimilation chamber was connected in parallel to the sample air hose of the gas-exchange system Li-6400 (Li-Cor Inc.). The outlet air of the chamber was regularly shunted and sent to a glass bulb to collect a gas sample. The latter was introduced (off-line) in the chromatographic column of the elemental analyser NA-1500 (Carlo-Erba, Milan, Italy) using a helium stream, and the carbon isotope composition (
13C) of the CO2 peak was measured with the isotope ratio mass spectrometer, IRMS (VG Optima Micromass, Villeurbanne, France). The carbon isotope discrimination was calculated using the equation of Evans et al. (1986):
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is the ratio ce/ce–co where ce and co are the CO2 molar fractions in the inlet and outlet air, respectively, and e and o are the isotope composition values of inlet (empty chamber) and outlet air, respectively. If the theoretical discrimination is denoted as i, the Farquhar et al. (1982) equation is obtained:
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) and b is the fractionation by Rubisco (29). ci and ca are the molar fractions of CO2 in intercellular spaces and in the leaf-surrounding air, respectively.
The internal conductance (gm) was obtained as described by von Caemmerer and Evans (1991), by the slope obtained when plotting the deviation of obs from the theoretical value (i–obs) against An/ca. In order to avoid any variation in the intercept, the CO2 molar fraction pa was maintained constant at 400 µl l–1. Different levels of An were obtained with different light levels. Temperature was fixed at 22 °C with a water bath, and the oxygen fraction was kept at 21%. Inlet CO2 was obtained from a gas cylinder (Air Liquide, Grigny, France) with a fixed 13C value of –51.2±0.2.
Plastid terminal oxidase content
Leaf material was ground in liquid nitrogen and protein was extracted as described by Dutilleul et al. (2003b). A 60 µg aliquot of total leaf protein was separated on a 12% SDS–polyacrylamide gel and transferred to nitrocellulose membranes as described by Gutierres et al. (1997).
The PTOX protein content was determined with polyclonal antibodies against the protein of Arabidopsis thaliana as described by Cournac et al. (2000). The antibody concentration was used at a final dilution of 1/10 000. Samples from leaves of transgenic Solanum lycopersicum (Lycopersicon esculentum Mill.) cv. Micro-Tom (PTOX+) overexpressing the PTOX from A. thaliana and cultivated as described elsewhere (Josse, 2003) were used as a positive control.
Enzyme measurements
Glycolate oxidase (EC 1.1.3.1):
Glycolate oxidase activity was extracted and measured as described in Streb et al. (2005).
Rubisco activation/deactivation
Leaves grown under normal atmospheric CO2 and O2 molar fractions were illuminated for 1 h at a PFD of 350 µmol m–2 s–1 at reduced or increased ca of 280 ppm for WT leaves and 510 ppm for CMSII leaves. These ca molar fractions correspond to an expected cc value of CMSII and WT leaves at 400 ppm ca as calculated from An/ci curves considering measured gs and gm values. Net carbon assimilation was measured during, and initial Rubisco activity directly after, exposure to altered ca. Initial and total Rubisco activity was determined as described by Priault et al. (2006), expressing activity as CO2 consumption.
Growth at high CO2 molar fraction
Plants cultivated for 2 weeks under normal growth conditions as described above were transferred to six different closed chambers (PFD, 150 µmol m–2 s–1, 16 h light period, day/night temperature of 23/15 °C). The CO2 concentration of three chambers was maintained at 400±50 ppm ca and the other three chambers at higher (1000±100 ppm) ca. One chamber of each treatment was filled with either WT or CMSII plants, while the third chamber was filled with WT and CMSII plants. Each chamber contained eight different plants. Thus, in total, 12 different plants of each type were grown under each condition.
The leaf number was counted every 6 d on each plant. Glycolate oxidase activity and light-saturated net carbon assimilation at 1000 µmol m–2 s–1 PFD were measured as described previously on the first fully developed leaf after 5–6 weeks at the respective CO2 treatment.
Statistical analyses
If not indicated otherwise, all experiments were independently repeated at least three times and the standard error is given. Analysis of variance (ANOVA) statistical analysis was performed with STATISTICA software (STATSOFT Inc., Tulsa, OK, USA) in order to estimate the statistically significant difference at the P <0.05 level.
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Carbon assimilation at different O2 molar fractions
Light response curves of photosynthetic carbon assimilation are shown in Fig. 2A and B. Net carbon assimilation in ambient air (21% oxygen, 380 ppm CO2) was higher in WT than in mutant leaves. In order to estimate to what extent oxygen suppresses net carbon assimilation, light response curves were repeated in 2% and 0.5% oxygen at 380 ppm CO2. As can be seen from Fig. 2A and B, carbon assimilation increased in both CMSII and WT leaves in an oxygen-depleted atmosphere. While in the WT leaves carbon assimilation increased nearly to the same extent in 2% and 0.5% oxygen, 2% oxygen was not sufficient to attain a maximum stimulation of carbon assimilation in CMSII leaves. It is noteworthy that low oxygen stimulated carbon assimilation more in CMSII than in WT leaves, as shown in Fig. 2D. The difference in carbon assimilation between WT and mutant leaves decreased at low oxygen molar fractions (Fig. 2C). However, maximum photosynthesis at a low oxygen molar fraction remained lower in the mutant (23.1 µmol m–2 s–1 as compared with 27.4 µmol m–2 s–1 in the WT). Interestingly, the lower net carbon assimilation in CMSII was accompanied by higher rates of excess electron transport, ranging from 6 to 18 µmol m–2 s–1 electrons at different PFDs as compared with WT leaves.
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Day respiration, stomatal limitation, and PTOX protein content
In order to examine whether enhanced day respiration (Rl) may contribute to the lower net carbon assimilation observed in CMSII leaves, Rl was estimated by the Laisk (1977) method using An/ci curves at low ci and varying PFD, as described in von Caemmerer (2000) (for methods, see Fig. 1). Dark respiration (Rn) was higher in CMSII than in the WT leaves. However, in this experiment, the difference was only significant at the P <0.06 level, but a significant difference was confirmed previously (Sabar et al., 2000). By contrast Rl was similar in both types of leaves (Table 1). The stomatal limitation to CO2 diffusion, Ls, calculated according to Farquhar and Sharkey (1982), was also identical for WT and CMSII leaves (Table 1), accounting for 16–17% in both types of leaf.
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The content of PTOX, as an alternative electron sink possibly affecting carbon assimilation, was estimated by western blotting (Fig. 3). Clearly, the amount of the PTOX protein was similar in both types of N. sylvestris leaf, and low compared with tomato (S. lycopersicum) leaves overexpressing the PTOX protein. This excludes the possibility of higher rates of electron consumption by the PTOX in CMSII mutant leaves.
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Photorespiration as an electron sink affecting carbon assimilation
In order to evaluate the contribution of photorespiration to oxygen consumption, carbon assimilation curves were measured as a function of the internal molar CO2 ratio (ci) in 21% and 0.5% oxygen (Fig. 4A). Carbon assimilation at low ci (up to 500 ppm) was higher in WT than in mutant leaves when measured in 21% oxygen, but this difference was nearly absent in 0.5% oxygen. This is particularly evident when the initial slope of carbon assimilation (between 50 and 200 ppm ca) was calculated. Despite the fact that carbon assimilation in 21% oxygen was not completely saturated at high ci, it was very similar in mutant and WT leaves (
26 µmol m–2 s–1). However, the maximum carbon assimilation in 21% oxygen was lower than in 0.5% oxygen (28 µmol m–2 s–1) in both WT and mutant leaves, thus showing that O2 still suppresses carbon assimilation at high CO2 molar fractions. Nevertheless, the difference in An between ambient and low oxygen molar fractions was small, suggesting that photorespiration was largely suppressed in 21% oxygen at high ci. A similar photosynthetic capacity of mutant and WT leaves in 21% oxygen was also obvious when the maximum carboxylation velocity (Vc max), as estimated from An/ci curves, was compared between both leaf types (Table 1).
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As shown previously (Priault et al., 2006), ETR in excess was higher in CMS compared with WT leaves. This difference is highest at ambient CO2 and declines at higher ci (not shown), suggesting that electrons may be used for photorespiration. In agreement with this, rates of calculated electron transport to carboxylation (Jc) as compared with oxygenation (Jo) Jc/Jo were higher in WT (2.0) than in CMSII leaves (1.5) under ambient air at a PFD of 800 µmol m–2 s–1 (Table 1).
Internal leaf conductance to CO2 diffusion and the specificity factor of Rubisco
Higher rates of photorespiration at the same ci in the mutant as compared with the WT may result from a different leaf internal conductance to CO2 diffusion (gm) and/or a lower specificity factor of Rubisco. The specificity factor of Rubisco, as calculated on the basis of an equal ci, was nearly identical in WT and CMSII leaves (Table 1). The gm was measured by isotope ratio mass spectrometry at 400 ppm CO2 and 21% oxygen. Mass spectrometry data are shown in Fig. 5 for WT and mutant leaves. As shown in Table 1, gm was approximately twice as high in the WT (0.23 mol m–2 s–1) as in the mutant leaves (0.1 mol m–2 s–1). As a consequence, cc values are different in CMSII and WT leaves at the same ci. Therefore, the specificity factor of Rubisco was recalculated on the basis of the same cc, which was calculated using gm as:
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Figure 1 was redrawn replacing ci by cc and assuming that the measured gm values did not change with carbon assimilation (not shown). However, it should be noted that gm values given here are only sufficiently accurate for An at around 400 ppm ca, at which gm was measured. The apparent specificity factor of Rubisco was then calculated replacing C* by
* in Equation 4. Table 1 shows that the specificity factor of Rubisco as calculated on the basis of an equal cc is still not significantly different in WT and CMSII leaves, confirming that gm is the major factor modulating photorespiration in CMSII leaves. The apparent specificity factor of Rubisco as calculated on the basis of ci was used to calculate the ratio of the carboxylation to the oxygenation velocity (Vc/Vo). As expected, Vc/Vo was lower in CMSII than in WT leaves (Table 1). In accordance, a slightly higher glycolate oxidase activity was measured in the mutant when compared with WT leaves (Table 1).
The cc dependence of carbon assimilation and Rubisco activity
When the distribution of electron flux to either the carboxylation or the oxygenation reaction of Rubisco (Jc/Jo), which was calculated independently of cc by chlorophyll fluorescence measurements, was considered, the difference in carbon assimilation between CMSII and WT leaves disappeared. As seen in Fig. 6A, carbon assimilation was identical in WT and CMSII leaves as a function of the Jc/Jo ratio. This ratio was previously used as an estimation of cc according to the equation:
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Since a lower initial Rubisco activity in CMSII leaves correlated with lower carbon assimilation as compared with WT leaves (Priault et al., 2006) and since cc may trigger carbon assimilation via Rubisco activation, leaves were treated on the basis of the same cc. Therefore, CMSII leaves were illuminated for 1 h at an increased ca which was calculated to match the cc of WT leaves exposed to the atmospheric CO2 molar fraction. Conversely, WT leaves were treated at a lower ca in order to match the calculated cc of CMSII leaves at ambient CO2. As shown in Fig. 7, initial Rubisco activity increased in CMSII leaves illuminated at higher ca and decreased in WT leaves illuminated at a lower ca. Furthermore, carbon assimilation correlated with the initial Rubisco activity in all treatments. Finally, initial Rubisco activities in WT and CMSII leaves treated at the same cc were not significantly different, although Rubisco activation in CMSII by a higher cc was more pronounced than Rubisco deactivation in WT by the lower cc (Fig. 7).
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Growth at high CO2
Assuming that photorespiratory activity in the mutant leaves decreases carbon gain and limits productivity and growth, it should be possible to alleviate these effects by growing mutant and WT leaves at increased CO2 molar fractions. Table 2 shows that photosynthetic activity was indeed similar in mutant and WT leaves when grown at 1000 ppm CO2. Furthermore, estimating growth by the increase of the number of leaves per day showed significant increases in CMSII, while growth of WT leaves was nearly unaffected by high CO2. The enzyme glycolate oxidase decreased under non-photorespiratory growing conditions in both plants, showing that the activity of this enzyme responded to photorespiratory activity.
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Discussion |
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When grown at high PFD, the CMSII mutant of N. sylvestris, which lacks mitochondrial complex I activity, has an impaired photosynthetic activity in ambient air (380 ppm CO2, 21% oxygen) when compared with WT plants (Pineau et al., 2005; Priault et al., 2006; Fig. 2). This lower photosynthetic activity is not caused by lower capacities of the primary photosynthetic reactions (Priault et al., 2006). Interestingly, photosynthetic electron transport exceeded carbon assimilation more in mutant than in WT leaves (Priault et al., 2006), implying a higher activity of additional alternative electron sinks in the mutant. Excess electrons originating from photosynthesis may also contribute to an altered redox homeostasis and higher cellular NADH contents in mutant leaves (Dutilleul et al., 2003b, 2005). The lower net carbon assimilation in CMSII is not caused by higher respiration rates in the light or by a larger stomatal limitation to CO2 diffusion (Table 1; Dutilleul et al., 2003a). Furthermore, the photosynthetic capacity determined at saturating CO2 as well as maximum activities of Rubisco and SPS are nearly identical in WT and CMSII leaves (Dutilleul et al., 2003a; Priault et al., 2006). Thus, the mitochondrial mutation of CMSII affects photosynthesis at limiting CO2 supply and is not caused by a decline in the capacity of primary metabolism associated with CO2 fixation.
Photorespiration is higher in the CMSII mutant
Oxygen acts as an electron acceptor competing with carbon assimilation (Fig. 2). In 2% and 0.5% oxygen, net carbon assimilation increased in WT and mutant leaves, but this stimulation was higher in the CMSII leaves. Accordingly, an atmosphere of 2% oxygen was sufficient to reach the maximum carbon assimilation rate in WT leaves but an atmosphere of 0.5% oxygen was required in the mutant leaves (Fig. 2). This may indicate a lower sensitivity to oxygen in mutant leaves. Furthermore, the difference in carbon assimilation between both types of leaf observed under normal oxygen molar fractions decreased at a low oxygen molar fraction, thus underlining the role of oxygen to suppress carbon assimilation in CMSII leaves.
Photorespiration and the Mehler reaction are quantitatively the major sinks for excess electrons with oxygen as the acceptor (Asada, 1999; Ort and Baker, 2002). Chlororespiration appears not to be a significant alternative route for oxygen reduction in the chloroplast (Carol and Kuntz, 2001). The content of the PTOX protein was similar in CMSII and WT leaves and therefore this pathway does not appear to contribute to reducing An in CMSII plants (Fig. 3). To date, exceptionally high contents of this protein have been observed only in the alpine plant species Ranunculus glacialis, where part of the CO2-insensitive electron consumption can be explained by PTOX activity (Streb et al., 2005). Enhanced rates of photorespiration in CMSII leaves are, however, questionable, since the glycine decarboxylation capacity is lower than in WT leaves and glycine:serine ratios in the light are similar (Dutilleul et al., 2003a). Furthermore, initial Rubisco activity is lower in CMSII than in WT leaves (Priault et al., 2006). Nevertheless, the glycine decarboxylase activity of CMSII leaves may be sufficiently high to cope with an enhanced photorespiratory activity. The uncertainty about whether or not photorespiration is involved in the additional electron flux is challenged by the observation that the difference in photosynthesis between mutant and WT leaves decreased in the presence of high CO2 or low oxygen molar fractions (Dutilleul et al., 2003a; Fig. 4). This was investigated in more detail in the present study by measuring carbon assimilation as a function of ci in atmospheres containing 21% and 0.5% oxygen (Fig. 4).
The ci response curves in 21% oxygen show that below 500 ppm, net carbon assimilation is markedly lower in CMSII than in WT leaves whereas in 0.5% oxygen, this difference becomes negligible. The initial slope of carbon assimilation is significantly lower in CMS as compared with WT leaves in 21% oxygen but not in 0.5% oxygen (Fig. 4B). Furthermore, when the electron fluxes to carboxylation (Jc) and oxygenation (Jo) were estimated according to Epron et al. (1995) (Table 1), assuming that the electrons are only used for carbon assimilation and photorespiration in both cases, it is obvious that more electrons are allocated to photorespiration in CMSII leaves when compared with the WT. The same result was obtained when calculating carboxylation and oxygenation velocities with the apparent specificity factor of Rubisco (Table 1). Net carbon assimilation in WT and CMSII leaves was identical when measured as a function of Jc/Jo (Fig. 6A) and very similar when measured as a function of calculated cc (Fig. 6B). This latter observation further suggests that Jc/Jo ratios give a good estimation of cc in the chloroplast, if the specificity factor of Rubisco is identical (Cornic and Massacci, 1996). Finally, the photorespiratory enzyme glycolate oxidase had a slightly higher activity in mutant leaves, thus mirroring leaf photorespiratory activity. It is shown in Table 2 that glycolate oxidase activity responds to photorespiratory growth conditions.
Internal leaf conductance is lower in the CMSII mutant
The higher photorespiration rate of mutant leaves as compared with the WT counterparts may result from a different specificity factor for Rubisco and/or a different mesophyll conductance for CO2 diffusion. Variable mesophyll conductance may occur in salt- and drought-stressed leaves (Flexas et al., 2004). In addition, the leaf anatomy is important for internal CO2 diffusion, and this in turn affects photosynthetic carbon assimilation (Morison et al., 2005). Mesophyll conductance, as estimated by carbon isotope discrimination, was remarkably different between the two leaf types, gm being half as high in mutant leaves when compared with WT leaves (Table 1). Similar results were obtained when mesophyll conductance was calculated according to Epron et al. (1995) using An/ci and An/cc curves (results not shown). Consequently, cc at the same ci is lower in CMSII leaves when compared with WT leaves, thus favouring photorespiration in the mutant. Since mesophyll conductance may be variable and dependent on photosynthetic activity (Loreto et al., 2003), calculations are only precise at 400 ppm ca where gm was measured. Despite this inaccuracy, fixed gm values were used to recalculate cc in order to replace ci. When carbon assimilation curves in 21% oxygen were redrawn as a function of cc (Fig. 6B), the difference between mutant and WT leaf photosynthetic activities largely disappeared, at least in the range of net carbon assimilation which was markedly different when measured on the basis of the same ci and which corresponded to the range which was used to determine gm. This confirmed that a different CO2 availability in the chloroplast was the major factor influencing photosynthetic activity in CMSII leaves. Duranceau et al. (2001) also explained the lower isotope discrimination in CMSII compared with WT leaves by a difference in mesophyll conductance.
The specificity factor of Rubisco was calculated on the basis of ci as well as cc. While the specificity factor of Rubisco was generally lower on the basis of cc than on the basis of ci and corresponded to values reported in the literature for tobacco leaves (Jordan and Ögren, 1981; Kane et al., 1994), it was not significantly different between WT and mutant leaves (Table 1).
The lower cc also correlated with the lower initial Rubisco activity in CMSII as compared with WT leaves in ambient air (Priault et al., 2006). When the initial Rubisco activity was measured in CMSII leaves after light activation at an increased ca, calculated to correspond to the cc in WT leaves in ambient air, initial Rubisco activity increased to the same value as in WT leaves. Conversely, initial Rubisco activity in WT leaves decreased when ca was reduced to achieve the cc in mutant leaves in ambient air. The fact that this latter reduction in WT leaves was less pronounced than the increase in mutant leaves may be explained by a longer lag time for Rubisco deactivation than for Rubisco activation. However, the current literature shows examples of the opposite response of Rubisco activation to higher or reduced CO2 levels. Initial Rubisco activity in vivo declined in several plant species at high CO2 (Sage et al., 1990; von Caemmerer and Quick, 2000; Cen and Sage, 2005). Nevertheless, this effect depends on light intensity and was measured when Rubisco activation was already very high. Therefore, variation of CO2 may affect the balance between Rubisco activity and ribulose-1,5-bisphosphate regeneration (Sage et al., 1990; Cen and Sage, 2005). In the present study, however, Rubisco activation was lower (Priault et al., 2006) and experiments were done at growth light, suggesting that Rubisco activity and ribulose-1,5-bisphosphate regeneration are well balanced.
Estimation of electron and ATP consumption by photorespiration and carbon assimilation
In order to confirm that photorespiration was the major electron sink in CMSII leaves, measured ETRs were checked to match electron and ATP consumption by photorespiration and carbon assimilation in both plants. Results shown in Table 1 (Vc/Vo) and Fig. 6 (net assimilation) were combined to calculate the photorespiratory rate at a ca of 400 ppm CO2 with the relationship
 |
30% higher in the mutant than in the WT leaves. However, linear electron transport nearly compensates for all of the ATP consumption of the Calvin cycle and of photorespiration [(assuming that three protons are translocated for every electron transported in photosynthesis (Kobayashi et al., 1995) and that the ATPase consumes 4.67 protons/ATP (Avenson et al., 2005)] in both types of leaf (Table 3). The deficit of 15 µmol m–2 s–1 electrons in both calculations for ATP consumption might be due to sugar export for sucrose synthesis which decreases the ATP demand in the chloroplast, or to some additional cyclic electron flow around photosystem I (PSI) which increases the ATP production (Avenson et al., 2005). Nevertheless, linear electron transport produces more electrons than those that are consumed by NADPH/ferredoxin needed to support photorespiration and the Calvin cycle. The excess of 30 µmol m–2 s–1 electrons in CMSII and WT leaves may be explained by electron consumption for reduction processes and by the Mehler reaction. Our data show that both types of leaf did not differ in these activities during illumination. The malate valve may only partially contribute to this excess electron consumption (Priault et al., 2006). According to our calculations, <20% of total linear electron transport is consumed by alternative reduction reactions including the Mehler reaction and chlororespiration.
|
, where x is the photorespiratory activity in µmol m–2 s–1. Results are summarized in
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The importance of enhanced photorespiration in suppressing carbon assimilation in CMSII is further documented by growing plants at higher CO2 molar fractions. The retarded leaf growth and the lower photosynthetic activity were largely overcome when plants were grown under such non-photorespiratory conditions (Table 2).
In conclusion, photorespiration is higher in CMSII leaves, despite the fact that initial Rubisco and glycine decarboxylation activities are lower and the glycine:serine ratio is not markedly changed when compared with the WT (Dutilleul et al., 2003a; Priault et al., 2006). Considering calculated rates for photorespiration, ATP consumption matched photosynthetic electron transport rates in WT and CMSII leaves, and in both genotypes the amount of excess electrons was similar. The enhanced photorespiratory activity of CMSII is caused by a lower mesophyll conductance that reduces the cc. However, it remains to be elucidated how mitochondrial function or structure modulates mesophyll conductance and by which mechanism mesophyll conductance is increased in CMSII leaves.
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Acknowledgements |
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The research was funded by the French MENRT and the CNRS. The financial support of the European project NETCARB (contract HPRN-CT 1999-00059) is greatly appreciated. We thank Marcel Kuntz and Eve-Marie Josse for the generous gift of PTOX antibodies and protein samples of tomato PTOX+ plants. We acknowledge Michael Hodges for proofreading of the manuscript.
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Abbreviations |
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An, net CO2 assimilation rate; Ao, CO2 assimilation rate in the absence of stomatal resistance; C* and
*, intercellular and chloroplastic CO2 compensation point, respectively; ca, air CO2 molar fraction; cc, chloroplastic CO2 molar fraction; ce and co, CO2 molar fractions in the inlet and outlet air, respectively; ci, intercellular CO2 molar fraction; CMS, cytoplasmic male sterile; ETR, electron transport rate; Jc, electrons used for carboxylation processes; Jo, electrons used for oxygenation processes; Ls, relative limitation of net CO2 uptake by stomatal resistance; O, oxygen concentration; PFD, photon flux density; PTOX, plastid terminal oxidase; CO2, quantum yield of gross CO2 assimilation; PSII, quantum yield of electron flow through photosystem II; Rl, mitochondrial respiration in the light; Rn, mitochondrial night respiration; Sapp, Rubisco apparent specificity factor; SPS, sucrose phosphate synthase; Vc, carboxylation rate; Vo, oxygenation rate; Vc max, maximal carboxylation rate; WT, wild type. |
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