Journal of Experimental Botany

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Journal of Experimental Botany, Vol. 51, No. 90001, pp. 357-368, February 2000
© 2000 Oxford University Press

Photosynthetic electron sinks in transgenic tobacco with reduced amounts of Rubisco: little evidence for significant Mehler reaction

Sari A. Ruuska^1,2, Murray R. Badger¹, T. John Andrews¹ and Susanne von Caemmerer^1,2,3

¹ Molecular Plant Physiology Group, Research School of Biological Sciences, The Australian National University, GPO Box 475, Canberra ACT 2601, Australia
² Photobioenergetics Group, Research School of Biological Sciences, The Australian National University, GPO Box 475, Canberra ACT 2601, Australia

Received 26 March 1999; Accepted 14 October 1999

	Abstract

Top Abstract Introduction Materials and methods Results Discussion Conclusion References

 
Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of the small subunit of Rubisco were used to investigate the role of O₂ as an electron acceptor during photosynthesis. The reduction in Rubisco has reduced the capacity for CO₂-fixation in these plants without a similar reduction in electron transport capacity. Concurrent measurements of chlorophyll fluorescence and CO₂ assimilation at different CO₂ and O₂ partial pressures showed close linear relationships between chloroplast electron transport rates calculated from chlorophyll fluorescence and those calculated from CO₂-fixation. These relationships were similar for wild-type and transgenic plants, indicating that the reduced capacity for CO₂ fixation in the transgenic plants did not result in extra electron transport not associated with the photosynthetic carbon reduction (PCR) or photorespiratory carbon oxidation (PCO) cycle. This was further investigated with mass spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ exchange made concurrently with measurements of chlorophyll fluorescence. In all tobacco lines the rates of ¹⁸O₂ uptake in the dark were similar to the ¹⁸O₂ uptake rates at very high CO₂ partial pressures in the light. Rates of oxygenase activity calculated from ¹⁸O₂ uptake at the compensation point were linearly related to the Rubisco content of leaves. The ratios of oxygenase to carboxylase rates were calculated from measurements of ¹⁶O₂ evolution and ¹⁸O₂ uptake at the compensation point. These ratios were lower in the transgenic plants, consistent with their higher CO₂ compensation points. It is concluded that although there may be some electron transport to O₂ to balance conflicting demands of NADPH to ATP requirements, this flux must decrease in proportion with the reduced demand for ATP and NADPH consumption in the transgenic lines. The altered balance between electron transport and Rubisco capacity, however, does not result in rampant electron transport to O₂ or other electron transport acceptors in the absence of PCR and PCO cycle activity.

Key words: Rubisco, tobacco, chlorophyll fluorescence, Mehler reaction, oxygen exchange.

	Introduction

Top Abstract Introduction Materials and methods Results Discussion Conclusion References

 
Linear electron transport in chloroplasts produces reduced ferredoxin. The reducing equivalents can then be transferred to several other acceptors. Most of the electrons are used to reduce NADP⁺ to NADPH, the majority of which is used in the photosynthetic carbon reduction (PCR) or photorespiratory carbon oxidation (PCO) cycles. There are additional pathways in chloroplasts that use either reduced ferredoxin or NADPH (Genty and Harbinson, 1996). One of the alternative sinks is the oxaloacetic acid–malate shuttle, which can transport reducing equivalents out of the chloroplast (Scheibe, 1987). Other alternative sinks in the chloroplasts include nutrient (especially nitrate) assimilation and biosynthetic activities, as well as cyclic electron flow around PSI (Heber et al., 1978; Furbank and Horton, 1987). PSI can also transfer electrons, either directly or via reduced ferredoxin, to O₂ in a process termed the Mehler reaction (Mehler, 1957). This photoreduction of O₂ produces highly reactive superoxide radicals which are rapidly detoxified by the ascorbate–peroxidase pathway which further consumes reducing equivalents (NADPH or reduced ferredoxin) (Badger, 1985; Asada and Takahashi, 1987).

Of these alternate electron sinks, the photoreduction of O₂ has attracted the most attention. Nevertheless, at the moment the quantitative significance of O₂ photoreduction in vivo is unclear. Mass-spectrometric measurements of isolated chloroplasts, mesophyll cells (Furbank et al., 1982) or whole leaves (Canvin et al., 1980; Gerbaud and Andre, 1980) have reported O₂ uptake in light, which could not be accounted for by Rubisco oxygenation or mitochondrial respiration (reviewed by Osmond and Grace, 1995). On the other hand, combined measurements of leaf gas exchange and chlorophyll fluorescence in vivo have not always found evidence of significant extra electron transport (Genty et al., 1989; Cornic and Briantais, 1991; Loreto et al., 1994). It is thought that O₂ photoreduction occurs especially when the ferredoxin pool is highly reduced, thus allowing linear electron flow to continue when NADP is scarce (Neubauer and Yamamoto, 1992; Wiese et al., 1998). It has been suggested that O₂ photoreduction can assist in developing and maintaining a high transthylakoid pH gradient, which in turn enhances non-radiative dissipation of light energy and protects light reactions from photodamage (Neubauer and Yamamoto, 1992; Björkman and Demmig-Adams, 1995). In addition, the stoichiometries of NADPH and ATP production and consumption in chloroplasts are different and the alternative sinks are considered necessary to enable ATP production without NADP reduction (reviewed by Noctor and Foyer, 1998).

The occurrence of O₂ photoreduction in vivo was investigated using transgenic tobaccos with reduced amounts of Rubisco. The reduction in Rubisco reduced the capacity for CO₂ fixation in these plants without a similar reduction in electron transport capacity (Hudson et al., 1992). These plants have high non-photochemical quenching, indicating high delta pH, because ATP and NADPH consumption is inhibited. It has been suggested that O₂ photoreduction is particularly important under these conditions to allow linear electron transport to continue (Osmond and Grace, 1995). To give these surmises a quantitative context, electron transport rates were compared with measurements of CO₂ uptake, chlorophyll fluorescence and mass spectrometric measurements of ¹⁶O₂ evolution and gross ¹⁸O₂ uptake.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion Conclusion References

 
Plant material and growth conditions
Transgenic tobacco with reduced amounts of Rubisco was grown from seeds collected from the selfed T1 progeny of a tobacco (Nicotiana tabacum cv. W38) transformant with a single insertion of an antisense gene directed against the Rubisco small subunit (Hudson et al., 1992). Seedlings segregate in a 1:2:1 ratio into homozygous, hemizygous or null type (wild type). Homozygous plants (referred to as anti-SSu2 plants) have two copies of the rbcS antisense gene and typically 10–15% of the wild-type Rubisco content. Hemizygous plants (referred to as anti-SSu1 plants) have one copy of the antisense gene and 30–40% of the wild-type Rubisco content. Untransformed cv. W38 tobaccos were used as controls. All plants were grown in 5 l pots in garden soil, in an air-conditioned glasshouse under natural illumination. The peak irradiance was 900–700 µmol m^-2 s^-1. Plants were given Hewitt's complete nutrient solution three times a week (Hewitt and Smith, 1975). Plants were used 6–8 weeks after germination, depending on the growth rate.

Measurements of CO₂ assimilation and chlorophyll fluorescence
Gas exchange and chlorophyll fluorescence was measured simultaneously with a LI-6400 portable system (Li-Cor, Lincoln, Nebraska). The system was fitted with a special cuvette that could hold a polyfurcated fibre optic connecting different light sources and measuring beams to ensure even distribution of illumination and detection of light over a leaf area of 4.15 cm² (Siebke et al., 1997). CO₂ response curves were measured at 2, 20 or 40% O₂ at an average irradiance of 830 µmol quanta m^-2 s^-1 and leaf temperature of 25 °C. Chlorophyll fluorescence was measured using a pulse-modulated fluorometer (PAM 101, Walz, Effeltrich, Germany ). At each CO₂ partial pressure, after gas exchange had reached steady-state, 1–2 saturating flashes (800 ms) were given to determine the quantum efficiency of PSII (Genty et al., 1989). Mitochondrial respiration rates not associated with photorespiration were estimated in two ways. They were either measured directly prior to experiments as CO₂ release, while the leaves were kept in darkness at 350 µbar CO₂, 200 mbar O₂ and 25 °C (R_d dark). Or the respiratory CO₂ release was estimated from CO₂ response curves as the (negative) of CO₂ assimilation rate at _* (R_d*), where _* is the CO₂ partial pressure where the rate of photorespiratory CO₂ release equals the rate of carboxylation. _*(=0.5O/S_c/o, where S_c/o is Rubisico's CO₂ to O₂ specificity) was determined previously for tobacco as 38.6 µbar at 200 mbar O₂ (von Caemmerer et al., 1994) and calculated at other O₂ partial pressures assuming a direct proportionality with O₂ partial pressure.

Mass spectrometric measurements
O₂ and CO₂ exchange were measured from wild-type and anti-SSu tobacco leaf discs using a closed leaf chamber attached to a mass spectrometer (MM6: VG, Winsford, UK) (as described by Maxwell et al., 1998). Discs were taken from illuminated leaves. The chamber, containing the leaf disc, was first darkened and then flushed with nitrogen. A known volume ¹⁸O₂ was added to give an atmosphere of 2, 10 or 20% O₂, and then a known volume of pure CO₂ was injected into the chamber to create an atmosphere of 1% or 2% CO₂. Following a dark period of about 10 min, the light (970 µmol m^-2 s^-1 at the leaf surface) was turned on and photosynthesis was allowed to proceed until CO₂ was depleted to a low, steady-state value which corresponded to the CO₂ compensation point. This took approximately 20 min and the size of the leaf discs of the transgenic tobacco was increased so that all experiments took approximately the same time. The gas exchange was measured with the mass spectrometer by continuously monitoring ¹⁶O₂ (mass 32), ¹⁸O₂ (mass 36) and CO₂ (mass 44). Net CO₂ assimilation was measured from the reduction in the CO₂ concentration, and gross O₂ evolution, gross O₂ uptake and net O₂ exchange were calculated from the changes in ¹⁶O₂ and ¹⁸O₂ (Canvin et al., 1980). During some of the measurements, fluorescence was continuously monitored and saturating pulses (600 ms) were given at 2–3 min intervals.

Measurements of Rubisco content
Rubisco catalytic site concentration was determined by the stoichiometric binding of [¹⁴C] 2'-carboxy-D-arabinitol1,5-bisphosphate (as described by Butz and Sharkey, 1989, and Ruuska et al., 1998). Measurements were made on leaf discs taken from the same leaf that mass spectrometric measurements were made on.

Calculations of electron transport rates
The rate of ATP consumption by the PCR and PCO cycles (following Farquhar and von Caemmerer, 1982) can be given by

(1)

and the rate of NADPH consumption of the PCR and PCO cycles by

(2)

where V_c and V_o are the rates of Rubisco carboxylation and oxygenation and _* is the CO₂ compensation point in the absence of mitochondrial respiration and C_c is the chloroplast CO₂ partial pressure. The ratio V_o/V_c is also given by:

(3)

Two electrons are required per NADPH such that the rate of electron transport required to satisfy the rate of NADPH consumption of the PCR and PCO cycle, J_NADPH is

(4)

The whole chain electron transport required to satisfy the ATP consumption of the PCR and PCO cycles was calculated in two ways. First, a proton requirement of 3H⁺/ATP was assumed and no Q-cycle operation (2H⁺/e) thus multiplying equation (1) by 3/2

(5)

Secondly, a proton requirement of 4H⁺/ATP was assumed and a Q-cycle operation (3H⁺/e) thus multiplying equation (1) by 4/3

(6)

The net rate of CO₂ assimilation, A, is given by

(7)

and using equation (3)

(8)

To calculate the rate of electron transport required satisfying the NADPH consumption of the PCR and PCO cycles from gas exchange measurements of CO₂ assimilation rates, equation (4) and (8) are combined and

(9)

(von Caemmerer and Farquhar, 1981). This equation was used to calculate J_g from gas exchange measurements of CO₂ assimilation in Figs 1 and 3. The chloroplast CO₂ partial pressure, C_c, was obtained from intercellular CO₂ partial pressure, C_i, and A as C_c = C_i – A/g_i with a transfer conductance for CO₂, g_i=0.3 mol m^-2 s^-1 bar^-1 (von Caemmerer et al., 1994). The electron transport rate required for the ATP consumption of the PCR and PCO cycle can be calculated in a similar manner from equations (5) or (6) and (8) (Fig .7).

View larger version (27K): [in this window] [in a new window]   Fig. 1. Electron transport rate calculated from gas exchange (Jg) and fluorescence (Jf) during steady-state photosynthesis at different CO2 and O2 concentrations in wild-type (WT) and anti-SSu (anti-SSu 1, anti-SSu 2) tobaccos. The measurements were conducted at 830 µmol quanta m-2 s-1 and leaf temperature of 25 °C. Jg was calculated from equation (9) with Rd estimated at * for each leaf (Table 1). Jf was calculated with equation (10) and absorptance (Abs) was measured for each leaf (Table 1). The regression parameters are given in Table 2.

 
The electron transport rate through PSII (J_f) was calculated from chlorophyll fluorescence as

(10)

where (F '_m-F)/F '_m is the quantum yield of electron flow to PSII (Genty et al., 1989) and I is the incident irradiance. Leaf absorptance, Abs, was measured for each leaf using a Taylor integrating sphere. The fraction of absorbed light distributed to PSII was assumed to be 0.48, and this resulted in a good fit for measurements made on wild-type tobacco at 2% O₂ and different CO₂ partial pressures and irradiance (data not shown).

Whole-chain electron transport rate was also calculated from the mass spectrometric measurements either from O₂ evolution as 4x¹⁶O₂ evolution, or from gross CO₂ uptake at 2% O₂ as 4x(A+R_d), where A is the CO₂ uptake and R_d the rate of CO₂ evolution in darkness.

Calculation of V_o, V_c and C_c from mass spectrometric measurements
These calculations are similar to those outlined previously (Renou et al., 1990). Rubisco oxygenase activity is a major component of the leaf's oxygen uptake processes. One mol of O₂ is consumed per mol of RuBP oxygenated and a further mol of O₂ is consumed in the PCO cycle by glycolate oxidase in the peroxisomes (Badger, 1985). In these calculations no O₂ uptake was assumed at PSI via the Mehler reaction and it was assumed that the respiratory O₂ uptake by mitochondria was the same in the light and dark and therefore:

(11)

If it is assumed that all whole chain electron transport results in NADPH production for PCR and PCO cycle activity then

(12)

The ratio V_o/V_c of oxygenation to carboxylation is dependent on chloroplast CO₂, C_c, and O₂ partial pressure and with _* known C_c can be calculated from equation (3).

	Results

Top Abstract Introduction Materials and methods Results Discussion Conclusion References

 
CO₂ assimilation rate and chlorophyll fluorescence
The CO₂ response of gas exchange and PSII were measured at three different O₂ partial pressures and the corresponding electron transport rates J_g and J_f were calculated as described in the Materials and methods from equations (9) and (10). For this set of measurements, a system that measures both the gas exchange and chlorophyll fluorescence over a leaf area of 4.15 cm² was used. It was found that, in these plants, the difference between respiration measured in the dark and estimated in the light at _* was small (Table 1). The O₂ partial pressure did not have any significant effect on the estimate of R_d at _* and the mean estimate given in Table 1 was used in equation (9). Two wild-type, anti-SSu 1 and anti-SSu 2 plants were analysed (Fig. 1). The linear regression for individual genotypes and for the combined data are given in Table 2. There was a good correlation between estimates of electron transport rates made from gas exchange and fluorescence measurements and the relationship between J_f and J_g was similar at all O₂ partial pressures. However, there are variations in the slopes and it is suspected that this may partly be because of technical problems of measuring incident light.

View this table: [in this window] [in a new window]   Table 1. The rates of CO2 assimilation, mitochondrial respiration and maximal Rubisco carboxylation, and leaf absorptance in wild-type and anti-SSu tobaccos The respiration rates were estimated in two ways. Firstly, they were either measured directly prior to experiments as CO2 release in darkness (Rd dark). Secondly, the respiratory CO2 release was estimated from measurements of CO2 assimilation rate at * at 2, 20 and 40% O2 and the mean value is given (Rd*) (Materials and methods). The estimated maximal carboxylation rate of Rubisco (Vcmax) in the different plant types is taken from Ruuska et al. (Ruuska et al., 1998). The CO2 assimilation rate was measured at 300 µbar CO2, 20% O2, an irradiance of 830 µmol m-2 s-1 and leaf temperature of 25 °C. Leaf absorptance was measured using Taylor integrating sphere. Two plants per genotype were analysed.

 

View this table: [in this window] [in a new window]   Table 2. Linear regression parameters for the comparison between electron transport rates calculated from fluorescence and gas exchange The number of replicates for each plant genotype is given in the brackets, and the values of the slope and intercept are the mean±SD of the regression. The PSII absorption coefficient was determined to be 0.48 (Materials and methods). The data are shown in Fig. 1.

 

Measurements of ¹⁶O₂ evolution and ¹⁸O₂ uptake coupled with measurements of chlorophyll fluorescence
CO₂ and O₂ exchange:
Net CO₂ uptake, ¹⁶O₂ evolution, ¹⁸O₂ uptake, and net O₂ exchange of the wild-type and anti-SSu tobacco leaf discs in a closed system attached to a mass spectrometer were also measured (Maxwell et al., 1998). This measuring system allowed the monitoring of chlorophyll fluorescence from the leaf disc at the same time. Leaf discs were exposed to an atmosphere containing 1% CO₂ and 20, 10 or 2% O₂. The O₂ uptake and CO₂ evolution were first recorded in the dark. Then the light was turned on and photosynthesis was allowed to proceed in light until the CO₂ compensation point was reached. Figure 2 shows examples of O₂ exchange and CO₂ uptake patterns of wild-type, anti-SSu 1 and anti-SSu 2 plants measured at 20% O₂ as a function of the CO₂ partial pressure inside the leaf chamber. A summary of all data at the different O₂ partial pressures is given in Table 3.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Net CO2 and O2 exchange, 16O2 evolution and 18O2 uptake of wild-type (A) and anti-SSu tobacco (B, C) measured with a mass spectrometer as a function of the prevailing CO2 concentration. Leaf discs were in a closed chamber coupled to the mass spectrometer. Actinic light was provided via fibre optics through a transparent window. The starting CO2 concentration was approximately 1–2% within the chamber, and the measurements were carried out during depletion as a result of the CO2 assimilation. The measurements were conducted at 20% O2, irradiance at the leaf surface of 970 µmol m-2 s-1, and the chamber was thermostated to 25 °C. The dashed line indicates the rate of 18O2 uptake measured in the dark. The average atmospheric pressure in Canberra is 955 mbar.

 

View this table: [in this window] [in a new window]   Table 3. Summary of the CO2 and O2 exchange data of the wild-type and anti-SSu tobaccos measured with a mass spectrometer as described in Fig. 2 Measurements were made at three O2 concentrations. The rates of Rubisco carboxylation, Vc and oxygenation, V0 and chloroplast CO2 partial pressure were calculated from the CO2/O2 exchange data as described in the Material and methods.

 
At high CO₂ and 20% O₂, the rates of ¹⁸O₂ uptake in light were slightly greater than the ¹⁸O₂ uptake measured in darkness in all plants (Fig. 2; Table 3). There was no difference at 10% O₂ and ¹⁸O₂ uptake at high CO₂ was less than dark measurements at 2% O₂ (Table 3). However, dark measurements were made for only 10 min on leaf discs that had previously been exposed to light. There was little difference in mass spectrometric measurements of CO₂ evolution in darkness at the different O₂ concentrations and these were similar to gas exchange measurements of CO₂ evolution. Transgenic plants had lower dark respiration rates than wild-type plants (Tables 1, 3).

In the light, as the CO₂ partial pressure decreased, the ¹⁸O₂ uptake increased indicating the onset of the photorespiratory O₂ uptake by Rubisco (Fig. 2). In all plants the net O₂ evolution closely matched the CO₂ uptake (Table 3). In wild-type plants the CO₂ uptake usually saturated at chamber CO₂ of about 0.5% CO₂ indicating the transition from Rubisco to electron transport limitation (von Caemmerer and Farquhar, 1981). This is a very high CO₂ concentration, but the leaf disc in the chamber had a very high boundary layer resistance to gaseous diffusion. Furthermore, the shapes of the photosynthetic response curves are dependent on stomatal conductance, which could not be measured in this system. Anti-SSu plants exhibited more Michaelis-Menten like kinetics, consistent with the notion that the CO₂ assimilation in these plants is limited by the Rubisco capacity. V_cmax was reached at 1% CO₂ in the anti-SSu plants: the average V_cmax were 27 for SSu1 plants and 6 µmol m^-2 s^-1 for SSu2 plants, which are similar to the V_cmax estimations made earlier (von Caemmerer et al., 1994; Ruuska et al., 1998). (No in vivo estimates of V_cmax can be made for the wild-type leaves, since Rubisco does not limit CO₂ assimilation rates at high CO₂ concentrations.)

Lower net CO₂ uptake and O₂ evolution rates were noted frequently at very high compared to intermediate CO₂ partial pressures. Leaf discs were taken from illuminated leaves, which were subjected to a brief dark period to measure respiration rates, but it is unlikely that the lower rates at very high CO₂ concentrations are the result of a time-dependent photosynthetic induction. The inhibition was particularly pronounced at 20% O₂ and less so at low O₂ partial pressures. The authors have no explanation of what causes the inhibition of CO₂ and O₂ exchange at very high CO₂. Ögren and Evans observed similar effects and suggested that lowered chloroplast pH due to dissolved CO₂ may be responsible (Ögren and Evans, 1993).

Estimates of electron transport rates
In some instances chlorophyll fluorescence was measured concurrently with mass spectrometric O₂ and CO₂ exchange. Figure 3a compares whole-chain electron transport rate based on the mass-spectrometric ¹⁶O₂ evolution rate, with that calculated from chlorophyll fluorescence, J_f. The measurements were conducted at three different O₂ concentrations on wild-type or anti-SSu plants. The correlation between the two estimates is good regardless of the O₂ partial pressure or plant genotype and passes close to the origin. The measured slopes were less than 1. It is again suspected that this may partly be because of technical problems in measuring incident light. The chamber designs of the Li-Cor and the mass spectrometer chambers are different, which may explain the disparity in slopes between Fig. 1 and Fig. 3A, but serves to highlight the technical difficulties in comparing chlorophyll fluorescence and gas exchange measurements.

View larger version (21K): [in this window] [in a new window]   Fig. 3. (A) Comparison between the electron transport rates calculated from gross 16O2 evolution and electron transport rates calculated from the chlorophyll fluorescence (Jf) in wild-type and anti-SSu tobaccos at different O2 concentrations. The 16O2 evolution was measured from leaf discs with a mass spectrometer as described in Fig. 2, and fluorescence was determined simultaneously and Jf was calculated with equation (10) as described in Fig. 1. The irradiance was 970 µmol m-2 s-1 and leaf chamber temperature was 25 °C. (B) Relationship between electron transport rates calculated from gross CO2 uptake and from gross 16O2 evolution in wild-type and anti-SSu tobaccos at 2% O2. The measurements were made concomitantly with the measurements shown in (A).

 
At low O₂ partial pressure, the electron transport rate can also be estimated from CO₂ uptake as 4x(A+R_d) (Fig. 3B). There was also good correlation between electron transport estimated from ¹⁶O₂ and CO₂ exchange at 2% O₂.

Estimates of the ratio of Rubisco oxygenation to carboxylation, V_o/V_c, at the compensation point
Total ¹⁶O₂ evolution and ¹⁸O₂ uptake were used to estimate the rates of Rubisco carboxylation and oxygenations as outlined in Materials and methods (Tourneaux and Peltier, 1995). This was done by subtracting the dark O₂ uptake from the values in the light. Thus this calculation ignores the possibility of differences in mitochondrial O₂ uptake in the light and the dark or a variable contribution of Mehler ascorbate peroxidase reaction to O₂ uptake processes (see later discussion). With the closed system used it was easy to measure O₂ and CO₂ exchange at the compensation point and V_o/V_c has been estimated for this condition (Table 3). At all O₂ partial pressures this ratio was less in the transgenic tobacco with reduced amounts of Rubisco than in wild-type leaves. The reduced ratio of V_o/V_c is explained by the fact that the CO₂ compensation point is greater in transgenic compared to wild-type plants (Fig. 4). The increase in the compensation point in turn is the result of an increase in the ratio of R_d to V_cmax resulting from the reduced Rubisco content of the anti-SSu tobaccos. The measured values of the compensation point agree with model predictions (Fig. 4; Table 1). Since there is no net flux at the compensation point ambient CO₂ partial pressures are equal to those in the chloroplast. The lines in Fig. 5 show the predicted relationship between V_o/V_c and chloroplast CO₂ partial pressure at the three O₂ partial pressures using Rubisco kinetic constants (as determined by von Caemmerer et al., 1994). At _*, V_o/V_c=2 at all O₂ partial pressures, however mitochondrial CO₂ evolution leads to higher actual CO₂ compensation points, , and this reduces the V_o/V_c. The ratio of V_o/V_c was used to estimate chloroplast CO₂ partial pressure (Table 3; Fig. 5). The authors do not have very precise estimates of CO₂ at low ambient CO₂ partial pressures in the mass spectrometric system, but the estimates of are similar to those obtained from gas exchange measurements (Fig. 4; Table 3).

View larger version (26K): [in this window] [in a new window]   Fig. 4. CO2 compensation point, , at different O2 partial pressures in wild-type (WT) and transgenic tobacco with reduced amounts of Rubisco (anti-SSu1, anti-SSu2). Measurements were either made with a Li-Cor gas exchange system (closed symbols) or calculated from mass spectrometric data (open symbols, Table 3). The lines depict the theoretical relationship between and O2 partial pressure: where *O=*=38.6 at 200 mbar O2, O stands for O2 partial pressure, Kc=259 µbar, and Ko=178 mbar are the Michaelis–Menten constants of Rubisco carboxylation and oxygenations (von Caemmerer et al., 1994). Rd/Vcmax=0.045(WT),=0.069 (anti-SSu1) and=0.15 (anti-SSu2) (Table 1).

 

View larger version (27K): [in this window] [in a new window]   Fig. 5. The relationship between chloroplast CO2 partial pressure, Cc, and the ratio of the Rubisco oxygenation and carboxylation (Vo/Vc). The solid lines are the modelled responses, predicted from equation (3). The data points plotted are calculated mean values of Vo/Vc and Cc from the mass-spectrometric measurements at the compensation point (see Materials and methods) for wild-type (WT) and transgenic tobacco with reduced amounts of Rubisco (SSu1). Conditions were as in Fig. 2. Arrows indicate the value of * at 2, 10 or 20% O2.

 

Estimates of V_o at the compensation point
There was a close correlation between the oxygenase rates calculated from ¹⁸O₂ uptake and the Rubisco content of leaves (Fig. 6). The lines in Fig. 6 depict the theoretical relationship predicted between Rubisco content and Rubisco oxygenase rate (Farquhar and von Caemmerer, 1982) using the kinetic constants determined by von Caemmerer et al. (von Caemmerer et al., 1994). There was good agreement obtained at all O₂ partial pressures.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Rubisco oxygenase rates, Vo, at the compensation point, , calculated from mass-spectrometric measurements of 18O2, uptake as a function of Rubisco site concentration in wild-type and anti-SSu plants. Measurements were made at 20%, 10% and 2% O2 and a leaf chamber temperature of 25 °C and the calculation of Vo is described by equation (11) in Materials and methods. The lines are the oxygenase rates predicted from (Farquhar and von Caemmerer, 1982) with the kinetic constants defined in the legend to Fig. 4. Vomax=1.15xRubisco site content and was taken as 55, 37, and 15 µbar at 200, 100, and 20 mbar O2, respectively.

 

Extra electron transport to balance ATP and NADPH requirements
The different rates of ATP and NADPH consumption by the PCR and PCO cycle have led to the suggestion that alternative sinks (other than NADPH) may be necessary to balance the conflicting requirements. The ratio of the rates of ATP consumption to that of NADPH consumption varies between 1.75 at low CO₂ partial pressures to 1.5 at high CO₂ partial pressures. Estimates of the extra whole chain electron transport needed to balance the ATP and NADPH requirement at low compared to high CO₂ partial pressure are shown in Fig. 7. The estimates are strongly dependent on the assumption made for the proton stoichiometry of ATP synthesis and operation of a Q-cycle. Two possible scenarios are shown in Fig. 7. It has been common to assume a stoichiometry of 3H⁺/ATP and no Q-cycle operation (Farquhar and von Caemmerer, 1982; Noctor and Foyer, 1998). If this extra electron transport requirement where to be met by electron transport to O₂ this would result in 23% of electron flow to O₂ at the compensation point and 13% at high CO₂ partial pressure. Recent studies of the energy coupling of the chloroplast ATP synthase favour stoichiometry of 4H⁺/ATP (Haraux and Kouchkovsky, 1998) and the possible operation of the Q-cycle around the cytochrome bf complex is now also widely accepted (Mitchell, 1977; Rich, 1988). In this case only 9% is required at the compensation point and almost none at high CO₂ partial pressure. Large electron flows to O₂ would be required if the stoichiometry of 4 protons per ATP were to occur in the absence of Q-cycle function. If 3H⁺/ATP were required and the Q-cycle operated ATP consumption would require less electron transport than NADPH consumption.

View larger version (23K): [in this window] [in a new window]   Fig. 7. (A) Modelled rates of electron transport required for PCR and PCO cycles at different chloroplast CO2 partial pressures, Cc, considering NADPH and ATP requirements. For the calculations of the electron transport required for ATP consumption, either 3H+/ATP and no Q-cycle (dashed line) or 4H+/ATP and Q-cycle activity (dotted line) was assumed. The difference between the electron transport rates required to satisfy the ATP (JATP) and NADPH (JNADPH) consumption is also shown. The curves were generated from a modelled CO2 response curve of CO2 assimilation rate with Vcmax=80 µmol m-2 s-1 and J=126 µmol m-2 s-1 (Farquhar and von Caemmerer, 1982). (B) The difference between the electron transport rates required to satisfy the ATP (JATP) and NADPH (JNADPH) consumption as a fraction of total electron transport. The calculations are described in Materials and methods section.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion Conclusion References

 
Transgenic tobacco lines with reduced amounts of Rubisco were used to investigate the role of O₂ as an alternate electron acceptor during photosynthesis. It has previously been established that the reduction in Rubisco has reduced the capacity for CO₂ fixation in these plants without a similar reduction in electron transport capacity, which results in an increase in non-photochemical quenching (Quick et al., 1991; Hudson et al., 1992; Ruuska et al., 1998). The molecular manipulation has also led to other imbalances: for example, stomatal conductance is unaffected by the reduction in photosynthetic capacity (Hudson et al., 1992). These measurements show that mitochondrial respiration rates, measured as O₂ uptake (Table 3) or CO₂ evolution (Tables 1, 3), have also not been reduced in proportion with the reduced CO₂ fixation capacity, which is evident in an increase in the CO₂ compensation point in the transformants (Fig. 4). Because of their high capacity of electron transport, it was reasoned that these plants could be used to examine the role that extra electron transport to O₂ may have in dissipating superfluous energy (for review see Osmond and Grace, 1995). This question was addressed with two different types of experiment. In the first set of experiments, combined measurements were made of CO₂ assimilation rate and chlorophyll fluorescence at different CO₂ and O₂ partial pressures. In a second set of measurements, O₂ uptake was measured directly in a mass spectrometric system and compared with predicted rates of Rubisco oxygenase.

Comparisons between estimates of electron transport rates
It was shown that measurements of chlorophyll fluorescence could be used to calculate electron flow through PSII per unit quantum flux (Genty et al., 1989). Together with a measure of light absorbed by the leaf, this provides estimates of whole chain electron transport rate through PSII. It has been shown frequently that a close linear relationship exists between these estimates of electron transport and CO₂ fixation rates of C₃ leaves under non-photorespiratory conditions (Genty et al., 1989; Cornic and Briantais, 1991; Ghashghaie and Cornic, 1994; Laisk and Loreto, 1996). Under photorespiratory conditions energy consumption by the PCO cycle needs to be considered (von Caemmerer and Farquhar, 1981; Laisk and Loreto, 1996). Electron transport rates calculated from fluorescence and gas exchange measurements have been used (together with various assumptions) to estimate the CO₂/O₂ specificity of Rubisco (Peterson, 1989, 1990); or the mesophyll diffusion conductance to CO₂ (Evans and von Caemmerer, 1996, and references therein) or for the estimation of electron transport to alternate electron sinks (Loreto et al., 1994; Laisk and Loreto, 1996). From previous experiments with transgenic tobacco plants with reduced amounts of Rubisco, independent estimates of the internal conductance to CO₂ diffusion had already been made (Evans et al., 1994) and of the kinetic parameters of tobacco Rubisco (von Caemmerer et al., 1994). Using these, good quantitative agreement was found between electron transport estimated from chlorophyll fluorescence and that calculated from CO₂ fixation rates under various O₂ and CO₂ partial pressures (Fig. 1; Table 2). This indicates that the in vivo estimate of tobacco relative specificity (von Caemmerer et al., 1994) correctly partitions the energy use between PCO and PCR activity.

Since electron transport estimates from gas exchange only calculate the electron transport required for PCR and PCO cycle activity, the difference between it and the fluorescence estimate has been taken as an indication of extra electron transport to other sinks such as O₂ (Loreto et al., 1994; Laisk and Loreto, 1996). Although it is unreasonable to assume that the amount of extra electron transport should be constant across the various CO₂ and O₂ partial pressures, such extra electron transport would be particularly apparent at low fluxes and therefore be seen as a positive intercept in Fig. 1 (Table 2). The NADPH requirement of the PCR and PCO cycle was used to calculate the electron transport rate (equation 9). More electron transport would be required if the ATP requirement was met solely by whole chain electron transport (Fig. 7) and this should also be apparent as extra electron transport in these calculations. The largest intercept was observed in wild-type plants (10% of maximum electron transport, Table 2), which is similar to previous estimates using this technique (Loreto et al., 1994). The intercept was less in the transgenic plants (Table 2) and a disproportionate larger electron transport rate could not be detected in leaves of transgenic plants by this technique.

There are technical difficulties that can affect the comparison between estimates obtained from fluorescence and gas exchange measurements. For example, the accurate estimate of incident irradiance is essential and can be problematic with different chamber designs. Furthermore, chlorophyll fluorescence measures a different population of chloroplasts than gas exchange and this can also lead to small differences making it impossible confidently to conclude about extra electron transport fluxes of low magnitudes. Therefore, mass spectrometric measurements were also made of ¹⁶O₂ and ¹⁸O₂ exchange on leaf discs together with fluorescence measurements. ¹⁶O₂ evolution is derived solely from water splitting at PSII (Canvin et al., 1980; Badger, 1985) and therefore also measures all whole chain electron flow through PSII. Measurements at various O₂ and CO₂ concentrations showed excellent correlations between the two estimates (Fig. 3A). Similar comparisons between chlorophyll fluorescence and ¹⁶O₂ evolution have been made (Genty et al., 1992; Maxwell et al., 1998). There were also excellent correlations between ¹⁶O₂ and net CO₂ exchange at 2% O₂ low enough to suppress photorespiration almost completely (Fig. 3A). This O₂ concentration should still permit substantial Mehler reaction, which has been reported to have a K_m(O₂) in the range of 0.15–5% O₂ (Badger, 1985). Nevertheless, there was no evidence for extra electron transport in the transgenic plants with reduced amounts of Rubisco under these conditions.

Measurements of O₂ uptake
Rubisco oxygenase activity is a major component of the leaf's oxygen uptake processes. One mol of O₂ is consumed per mol of RuBP oxygenated and a further 0.5 mol of O₂ is consumed in the PCO cycle by glycolate oxidase in the peroxisomes (Badger, 1985). Added to this are the mitochondrial O₂ uptake associated with the oxidation of NADPH and FADH₂ and the O₂ uptake associated with the Mehler ascorbate peroxidase pathway. The contributions of each of these processes to the total O₂ uptake are difficult to disentangle. However, it is known that mitochondrial respiration is the only O₂-consuming process in darkness and that photorespiration is suppressed completely at 1% CO₂. Thus it is significant that, for both wild-type and transgenic tobacco, the O₂ uptake under these conditions was similar (Table 3). This equivalence leaves little scope for O₂ uptake by the Mehler/ascorbate peroxidase pathway unless mitochondrial O₂ uptake is suppressed substantially by light. Although little is known about the effect of light on mitochondrial respiration (Hoefnagel et al., 1998), the general concordance between these measurements of R_d in darkness and in the light at _* (Table 1) provides little evidence for such light suppression.

If electron transport to O₂ via the Mehler reaction is quantitatively insignificant, then a question arises about how the differing stoichiometries between ATP and NADPH consumption in the PCR and PCO cycle are balanced. There have been suggestions that diversion of electrons to O₂ instead of NADP serves this purpose. However, the magnitude of the diversion required depends on assumptions about the number of protons required for the synthesis of a molecule of ATP. If it is three (with no Q-cycle), 13% of the electron flow would need to be diverted to O₂ (or other electron acceptors unrelated to CO₂) at high CO₂ concentrations. However, if there is some Q-cycle activity and it is four, as some have favoured (Haraux and Kouchkovsky, 1998; Rich, 1988), very little diversion of electron flow to alternative acceptors would be required (Fig. 7).

These results differ from those of Canvin et al. (Canvin et al., 1980) who reported considerable O₂ uptake at high CO₂ but it is suspected that the CO₂ concentrations used in that study were not sufficient to suppress photorespiration completely.

Estimates of oxygenase activity at the compensation point
It has been suggested that photorespiratory O₂ uptake via Rubisco and O₂ uptake via Mehler can both promote non-assimilatory electron flow and stimulate photon utilization during CO₂-limited photosynthesis near the compensation point (Osmond and Grace, 1995, and references therein). Transgenic tobacco plants with reduced amounts of Rubisco are not only deprived of CO₂ fixation capacity, but the lack of Rubisco also precludes photorespiratory photon dissipation and thus these plants should particularly need to dissipate light energy via the Mehler ascorbate peroxidase reaction. Furthermore if electron transport to O₂ is important in balancing the differential requirements of ATP and NADPH consumption this would be particularly apparent at low CO₂ concentrations (Fig. 7).

Two calculations were made to assess O₂ uptake at the compensation point. It was assumed that there was no extra O₂ uptake by Mehler reaction and oxygenase rates were calculated from O₂ uptake as described in the Materials and methods section. Figure 6 shows a good correlation between the estimates of oxygenase activity at the compensation point and the amount of Rubisco in these leaves. If there had been large, extra electron flow to O₂ at the compensation point in the transgenic plants, an overestimate of oxygenase activity would have been expected. This study's Rubisco kinetic constants derived for tobacco from CO₂ assimilation measurements (von Caemmerer et al., 1994) were also used to predict oxygenase activity from measurements of Rubisco site concentration (Fig. 6). The close agreement between model prediction and estimates of oxygenase rates made from ¹⁸O₂ uptake measurements at the different O₂ concentrations creates confidence in these in vivo estimates of the Michaelis–Menten constants for Rubisco oxygenase (Fig. 6; von Caemmerer et al., 1994).

Both the ¹⁸O₂ and ¹⁶O₂ exchange measurements were also used to calculate the ratio of V_o/V_c. This ratio was less in plants with reduced amounts of Rubisco than in wild-type plants (Table 3). Model predictions in Fig. 5 show that this ratio is strongly dependent on chloroplast CO₂ partial pressure. Estimates of chloroplast CO₂ partial pressures made from this ratio, together with previous estimates of _*, predict compensation points similar to other gas exchange measurements (Table 3; Fig. 4). It is concluded that the O₂ uptake measured at the compensation point can be solely accounted for by Rubisco oxygenase activity.

O₂ photoreduction cannot proceed in the absence of ATP consumption
It appears from these measurements with the transgenic tobacco with reduced amounts of Rubisco that photoreduction of O₂ cannot proceed in the absence of ATP consumption associated with chloroplast carbon metabolism. This could be caused by the strong controlling influence of the thylakoid lumenal pH on regulation of electron flow through the bf complex (Laisk et al., 1997). This would seem to place strong limitations on any mechanisms proposed to allow oxygen to act as an alternate electron sink for the purposes of energy dissipation (Osmond and Grace, 1995). Plant mitochondrial electron transport on the other hand has developed at least two mechanisms to allow electron transport to oxygen to proceed without net proton partitioning. These are the alternative oxidase reaction (Day and Wiskich, 1995) and the presence of fatty acid cycling uncoupler proteins that allow proton movement through the membrane (Jezek et al., 1996). Similar mechanisms that would allow electron flow, unregulated by proton accumulation, have not as yet been identified in chloroplast membranes. The only degree of flexibility that may currently be invoked is the variable operation of the Q-cycle of the bf complex, changing the e/H⁺ ratio.

	Conclusion

Top Abstract Introduction Materials and methods Results Discussion Conclusion References

 
There has been continued interest in quantifying the electron transport flux to O₂. For example, estimates of this flux impinge on the ability to explain the isotopic composition of atmospheric oxygen (Yakir et al., 1994; Bender et al., 1994). However, it remains difficult to obtain accurate in vivo estimates. Several different approaches have been tried. The initial hope was that the transgenic plants with reduced amounts of Rubisco might prove useful to these enquiries because of their large electron transport capacity relative to Rubisco oxygenase activity. However, it was found that if there was electron transport rate to O₂ in these plants then it was scaled to the requirements of the PCR and PCO cycle activity. This suggests that, in tobacco at least, whole chain electron transport rate to O₂ is not possible in the absence of ATP consumption. This appears possible without any signs of chronic photoinhibition (Quick et al., 1991; Hudson et al., 1992). It also suggests that energy dissipation via the photorespiratory cycle is not essential in the protection against photohinibition.

	Acknowledgements

 
We thank Dr JR Evans and RT Furbank for helpful discussions of the manuscript.

	Footnotes

 
³ To whom correspondence should be addressed. Fax: +61 2 62495075. E-mail:susanne{at}rsbs.anu.edu.au

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