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BIOENERGETICS AND PHOTOSYNTHESIS

Reductions of Rubisco Activase by Antisense RNA in the C₄ Plant Flaveria bidentis Reduces Rubisco Carbamylation and Leaf Photosynthesis

Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia (S.v.C., L.H., V.Q.); Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia (N.V.); and Commonwealth Scientific and Industrial Research Organisation, Division of Plant Industry, Canberra, Australian Capital Territory 2601, Australia (A.G.M., R.T.F.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION CONCLUSIONS MATERIALS AND METHODS LITERATURE CITED

 
To function, the catalytic sites of Rubisco (EC 4.1.1.39) need to be activated by the reversible carbamylation of a lysine residue within the sites followed by rapid binding of magnesium. The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme is thought to aid the release of sugar phosphate inhibitors from Rubisco's catalytic sites, thereby influencing carbamylation. In C₃ species, Rubisco operates in a low CO₂ environment, which is suboptimal for both catalysis and carbamylation. In C₄ plants, Rubisco is located in the bundle sheath cells and operates in a high CO₂ atmosphere close to saturation. To explore the role of Rubisco activase in C₄ photosynthesis, activase levels were reduced in Flaveria bidentis, a C₄ dicot, by transformation with an antisense gene directed against the mRNA for Rubisco activase. Four primary transformants with very low activase levels were recovered. These plants and several of their segregating T₁ progeny required high CO₂ (>1 kPa) for growth. They had very low CO₂ assimilation rates at high light and ambient CO₂, and only 10% to 15% of Rubisco sites were carbamylated at both ambient and very high CO₂. The amount of Rubisco was similar to that of wild-type plants. Experiments with the T₁ progeny of these four primary transformants showed that CO₂ assimilation rate and Rubisco carbamylation were severely reduced in plants with less than 30% of wild-type levels of activase. We conclude that activase activity is essential for the operation of the C₄ photosynthetic pathway.

Rubisco of C₄ species operate in a high CO₂ environment. Bundle sheath CO₂ concentrations have not been measured directly but have been estimated to be between 10- and 100-fold greater than in the ambient air (Furbank and Hatch, 1987; Jenkins et al., 1989). One might expect that the high pCO₂ occurring in the bundle sheath may aid in promoting full carbamylation; however, C₄ species are also known to have activase (Salvucci et al., 1987), and the requirement for the removal of RuBP and other phosphorylated inhibitors apparently remains. However, not all C₄ species show light-modulated variations in Rubisco carbamylation (Sage and Seemann, 1993), and in some C₄ species (e.g. Zea mays) the degree of dark inactivation is much less than that of C₃ species (Usuda, 1985; Sage and Seemann, 1993).

We have transformed Flaveria bidentis L. Kuntze (a C₄ dicot) with an antisense construct targeted at the mRNA of Rubisco activase and generated F. bidentis plants with a range of activase levels. In this paper, we explore the relationship between leaf activase content and C₄ photosynthesis and show that, although activase is present in saturating amounts for steady-state photosynthesis at high light in wild-type plants, it is essential for C₄ photosynthesis.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSIONS MATERIALS AND METHODS LITERATURE CITED

 

Characterization of Primary Transformants

To ensure the survival of primary transformants with low activase contents, plants were raised in growth chambers in an atmosphere containing 2.5 kPa pCO₂ (see "Materials and Methods" for details). Primary transformants were initially screened with gas exchange measurements at ambient pCO₂ of 40 Pa and high light (Fig. 1). Out of the 19 primary transformants measured, 4 (A, SS, Q, and RR) exhibited very low photosynthetic rates under these conditions but had higher rates when measurements were made on leaf discs with a mass spectrometer at 1.5 kPa pCO₂ (Table I).

View larger version (26K): [in this window] [in a new window]   Figure 1. CO2 assimilation rates of wild type (black columns) and a number of kanamycin-resistant primary transformants (white columns). Gas exchange measurements were made at an ambient pCO2 of 40 Pa, irradiance of 1,500 µmol m–2 s–1, and leaf temperature of 25°C. Activase amounts of primary transformants A, SS, Q, RR, and Aa are shown in Figure 2.

View larger version (39K): [in this window] [in a new window]   Figure 2. Immunodetection of Rubisco activase in leaf extracts of F. bidentis wild type (Wt) and primary transformants A, SS, Q, RR, and Aa and of Arabidopsis separated by SDS-Page. Samples were loaded on gels on an equal-leaf area basis, and Rubisco activase polypeptides were detected with a reduced glutathione S-transferase/spinach activase fusion protein antiserum followed by a horseradish peroxidase conjugated secondary antibody and chemiluminescence.

To assess Rubisco carbamylation levels, leaf discs were collected under growth conditions (2.5 kPa pCO₂, 500 µmol quanta m^–2 s^–1, 25°C) and snap frozen in liquid nitrogen. Rubisco content and carbamylation were measured by stoichiometric binding of ¹⁴C-carboxy-arabinitol-P₂. Several of the primary transformants had low Rubisco carbamylation states, particularly the four primary transformants with the lowest photosynthetic rates; however, Rubisco contents of all primary transformants were similar to wild type (Fig. 3). The maximum carbamylation measured under this relatively low irradiance levels was approximately 50%.

View larger version (23K): [in this window] [in a new window]   Figure 3. Rubisco carbamylation and Rubisco catalytic site content of leaves of selected primary transformants and wild-type plants. Leaf samples were collected in the growth cabinet under growth conditions of 2.5 kPa pCO2 at an irradiance of 500 µmol quanta m–2 s–1 at 25°C.

CO₂ response curves of CO₂ assimilation showed that the reduction of CO₂ assimilation rate at ambient CO₂ correlated primarily with a reduction in the CO₂-saturated rate of CO₂ assimilation (Fig. 4). There was no perceptible increase in CO₂ assimilation between 5 and 100 Pa CO₂ in the primary transformants SS and Q, although they exhibited higher photosynthetic rates in the mass spectrometric gas exchange system (Table I). However, measurements were not made on the same leaves, and there may have been some leaf-to-leaf variation.

View larger version (21K): [in this window] [in a new window]   Figure 4. CO2 assimilation rate as a function of intercellular pCO2 measured on leaves of selected primary transformants (black symbols) and a wild-type plant (white symbols). Measurements were made at an irradiance of 1,500 µmol quanta m–2 s–1 and a leaf temperature of 25°C.

The T₁ progeny of the four primary transformants with the lowest activase levels (A, SS, Q, and RR) were grown in a growth cabinet at 1 kPa pCO₂. However, we noticed that plants with very low activase levels were growing slowly and had chlorotic leaves. We therefore grew a second set of the T₁ generations in a growth cabinet that could be elevated to 2.5 kPa pCO₂. From both sets of plants, we obtained a similar relationship between the CO₂ assimilation rate measured at high light and ambient CO₂ and activase levels, and the data were combined in Figure 5.

View larger version (20K): [in this window] [in a new window]   Figure 5. CO2 assimilation rate as a function of Rubisco activase content. Measurements were made on wild-type leaves (white symbols) and leaves of plants of the T1 generation of primary transformants A, SS, Q, and RR (black symbols). Gas exchange measurements were made at an ambient pCO2 of 38 Pa, irradiance of 1,500 µmol m–2 s–1, and leaf temperature 25°C. Activase content of leaves gas exchange measurements were made on was quantified by immunodetection as a percentage of wild-type levels. Further details are given in "Materials and Methods."

Rubisco Carbamylation, Content, and in Vivo Catalytic Turnover Rate

The relationship between Rubisco carbamylation and activase content was similar to that observed for CO₂ assimilation rate and acitvase content (Figs. 5 and 6). In wild-type plants, Rubisco carbamylation was between 70% and 80% at 1,500 µmol quanta m^–2 s^–1 and between 40% and 50% at 500 µmol quanta m^–2 s^–1 (Fig. 6; Table I). Carbamylation of Rubisco was less than 20% in plants with very low activase levels, similar to what was observed for primary transformants with low activase levels. These carbamylation levels were considerably less than carbamylation levels observed in the dark for the wild type (66% ± 3% at 1.5 kPa pCO₂ and 44% ± 2% at 40 Pa pCO₂) and plants with very low activase content (70% ± 2% at 1.5 kPa pCO₂ and 60% at 40 Pa pCO₂). There were no significant differences in Rubisco content when plants were raised at 2.5 kPa pCO₂; however, when plants were raised at 1 kPa pCO₂, transformants with the lowest activase content failed to thrive and had lower Rubisco levels (Fig. 6). There were also no significant differences in phosphoenolpyruvate (PEP) carboxylase content quantified with immunoblotting for transformants raised at 2.5 kPa pCO₂ (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 6. Rubisco carbamylation (A) and Rubisco content (B) as a function of Rubisco activase content in leaves of wild-type plants (white symbols) and leaves of plants of the T1 generation of primary transformants A, SS, Q, and RR (black symbols). Triangles depict plants grown at 1 kPa pCO2 and circles depict plants grown at 2.5 kPa pCO2. For plants grown at 1 kPa pCO2, leaf discs were sampled in the growth cabinet at approximately 40 pCO2 and irradiance of 1,000 µmol m–2 s–1 and 30°C the day after gas exchange measurements. For plants grown at 2.5 kPa pCO2, leaf discs were sampled immediately after gas exchange from the area had been measured at 38 Pa pCO2, 1,500 µmol quanta m–2 s–1, and leaf temperature of 25°C.

View larger version (15K): [in this window] [in a new window]   Figure 7. In vivo catalytic turnover rate of Rubisco, Kcat at 25°C, for F. bidentis leaves with different activase content. Kcat was calculated from gross CO2 assimilation rates measured as described in Figure 5, and Rubisco carbamylated site content for leaves where leaf discs were sampled directly after gas exchange measurements (see Fig. 6 legend). Symbols are defined in Figure 6 legend. Average dark respiration rates measured were 2 µmol m–2 s–1, and this value was added to net CO2 assimilation rate to obtain gross CO2 assimilation rate.

Mate et al. (1996) formulated a mathematical model of activase action using the sequence of carbamylation and catalytic reactions originally proposed by Farquhar (1979) and showed that full carbamylation could result at ambient CO₂ concentrations from the ability of activase to promote the release of RuBP from carbamylated and uncarbamylated Rubisco sites. Because bundle sheath concentrations can be 10 to 100 times greater than ambient CO₂, we have used the model of Mate et al. (1996) to explore the interaction between activase action and CO₂ concentration on carbamylation status (Fig. 8). Below we give a brief description of the model.

View larger version (19K): [in this window] [in a new window]   Figure 8. Modeled carbamylation status of Rubisco as a function of pCO2 at RuBP saturation and different ratios of Kf/Kr' using the mathematical model of activase function developed by Mate et al. (1996). The model assumes that activase activity increases Kf/Kr', where Kr' is the apparent Michaelis Menten constant for RuBP and Kf defines the ratio of uncarbamylated free Rubisco sites to uncarbamylated Rubisco site RuBP complexes in the steady state. The curves shown were calculated using equation (A14) from the appendix of Mate et al. (1996): where C denotes the CO2 concentration, M the free magnesium concentration (5 mM), Ke and Kd are the dissociation constants of the Rubisco site CO2 and Rubisco site CO2 and magnesium complexes. Kd = 1,600 µM and KeKd = 160,000 µM2 (Laing and Christeller, 1976; Lorimer et al., 1976).

It can be seen from Figure 8 that at high activase activity (high K_f/K_r' values) carbamylation is dependent on CO₂ concentration only at CO₂ concentrations below 10 Pa. However, the CO₂ dependence is predicted to increase with decreasing activase activity (decreasing ratio of K_f/K_r'). The curve shown in Figure 8 where K_f/K_r' = 1 is almost identical to the carbamylation predicted when RuBP concentration is 0.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSIONS MATERIALS AND METHODS LITERATURE CITED

 

Rubisco Activase Is Essential for C₄ Photosynthesis

Using a Rubisco activase antisense construct to transform F. bidentis plants, we have isolated several primary transformants and T₁ plants with very low activase content in leaves (Figs. 1 and 5). Low activase content resulted in low net CO₂ assimilation rates at ambient pCO₂ (Table I; Figs. 1, 4, and 5). Our results demonstrate that activase is equally important for high photosynthetic rates during C₄ photosynthesis as it is for the C₃ photosynthetic system (Somerville et al., 1982; Mate et al., 1993, 1996; Eckardt et al., 1997) or for optimal photosynthesis in the green algae Chlamydomonas reinhardtii (Pollock et al., 2003). In primary transformants with very low activase content, CO₂ assimilation rates and Rubisco carbamylation were very low (Table I), showing that the most obvious effect of activase deficiency is to reduce the Rubisco carbamylation state in the light as had been observed for C₃ species (Mate et al., 1993, 1996; Eckardt et al., 1997). The CO₂ response curves of CO₂ assimilation showed reductions primarily in the CO₂-saturated rates of CO₂ assimilation (Fig. 4). C₄ photosynthetic models generally relate the CO₂-saturated assimilation rate at high light to Rubisco activity (Berry and Farquhar, 1978; Collatz et al., 1992; von Caemmerer and Furbank, 1999). Transgenic F. bidentis plants with reduced amounts of Rubisco due to an antisense construct to the small subunit of Rubisco also showed reductions in the CO₂-saturated rate of CO₂ assimilation (Furbank et al., 1996; von Caemmerer et al., 1997).

The Relationship between Activase Content, CO₂ Assimilation Rate, and Rubisco Carbamylation

In F. bidentis, Rubisco activase concentrations could be decreased to less than 30% of wild type before a decrease in steady-state CO₂ assimilation rates (measured at high irradiance and ambient pCO₂) were observed. These results are similar to results obtained in transgenic tobacco and Arabidopsis plants where activase levels could also be reduced to between 5% and 25% of wild-type levels before reductions in steady-state CO₂ assimilation rates were observed (Mate et al., 1993, 1996; Jiang et al., 1994; Eckardt et al., 1997). Thus, it appears that the requirement for activase is very similar in C₄ and C₃ photosynthetic systems despite the different CO₂ environment of the bundle sheath compared to C₃ mesophyll cells. In vivo catalytic turnover rates for Rubisco were similar for wild type and most anti-activase plants, showing that the observed decrease in CO₂ assimilation rate was almost completely accounted for by the decrease in Rubisco carbamylation (Figs. 6 and 7). The measured in vivo values of 3.5 s^–1 are similar to in vitro values measured previously for F. bidentis (Sage, 2002; Kubien et al., 2003).

In vitro, the catalytic activity of Rubisco exhibits a slow decline with time, often called fallover. It has been found that this decline in activity is the result of inhibitors binding tightly to carbamylated Rubisco sites (Edmondson et al., 1990b, 1990c). Rubisco catalyses a number of side reactions, and several products of these side reactions are candidates for such tight binding inhibitors (Kim and Portis, 2004). Activase activity has been shown to eliminate fallover in vitro, and the potential for tight binding inhibitors at carbamylated Rubisco sites exists in activase-deficient plants (He et al., 1997; Portis, 2003). Mate et al. (1993) and He et al. (1997) observed a reduction in in vivo catalytic turnover rates in transgenic tobacco with very low activase content. They suggested that this could be caused by inhibitors binding to the carbamylated sites in vivo but were unable to isolate these inhibitors. Three F. bidentis plants with very low activase content also appeared to show reductions in calculated in vivo turnover rate; however, there is uncertainty attached to these estimates because of uncertainties in respiration rates. Furthermore, in the C₄ photosynthetic pathway the reduction in in vivo catalytic turnover could also be the result of reduced bundle sheath concentration as discussed below.

High Bundle Sheath CO₂ Concentrations and the CO₂ Dependence of Rubisco Carbamylation

In contrast to C₃ species, Rubisco in C₄ species operates in a high CO₂ environment in the light, and bundle sheath CO₂ concentrations have been estimated to be between 10- and 100-fold greater than in the ambient air (Furbank and Hatch, 1987; Jenkins et al., 1989). In vitro Rubisco carbamylation state is CO₂ and magnesium dependent and increases with increasing CO₂ concentration (Laing and Christeller, 1976; Lorimer et al., 1976). In the simulations shown in Figure 8, the line for K_f/K_r' = 1 is almost identical to the CO₂ dependence predicted by Lorimer et al. (1976) with 5 mM free magnesium in the absence of RuBP. It predicts a carbamylation of approximately 30% at ambient CO₂ and close to full carbamylation at 2.5 kPa pCO₂. In vivo, such a strong CO₂ dependence of carbamylation has been observed only in the dark in the absence of RuBP in Triticum aestivum (Mächler and Nösberger, 1980) and Arabidopsis wild type and the rca mutant that lacks activase (Salvucci et al., 1986). In T. aestivum, Rubisco activation increased in the dark up to 10 kPa pCO₂. In F. bidentis wild-type plants, carbamylation in the dark was also greater at 1.5 kPa pCO₂ than at ambient CO₂ (66% ± 3% compared to 44% ± 2%), but Rubisco was not fully carbamylated at 1.5 kPa pCO₂. In the light, Rubisco carbamylation of wild-type plants and primary transformants with intermediate activase levels did not appear to be CO₂ dependent, whereas primary transformants with very low activase content showed a significant increase in carbamylation from 40 Pa to 2.5 kPa pCO₂ (Table I). The lack of a strong CO₂ dependence in the light has been observed previously in C₃ species. In Rhaphanus sativus and Arabidopsis Rubisco activation state was CO₂ dependent only below ambient CO₂ concentrations (Salvucci et al., 1986; von Caemmerer and Edmondson, 1986). These observed CO₂ dependencies in the light fit well with the predictions of the model of activase function constructed by Mate et al. (1996). The model shows that at RuBP saturation and high activase activity (high ratios of K_f/K_r'), a CO₂ dependence is expected only at very low CO₂ concentrations (Fig. 8). The model also predicts the CO₂ dependence to increase with increasing activase deficiency and this fits with our observations for F. bidentis. Neither Mate et al. (1993) nor Salvucci et al. (1986) found a CO₂ dependence of carbamylation in tobacco antiactivase plants or in the Arabidopsis activase-deficient rca mutant. However, it may be that the range of CO₂ concentrations examined (from ambient to 200 Pa in case of the Arabidopsis rca mutant) were not large enough to detect small increases in Rubisco activation, and further studies of the CO₂ dependence of Rubisco in the Arabidopsis rca mutants would be interesting.

Rubisco catalytic properties are known to differ between C₃ and C₄ species (Yeoh et al., 1981; Jordan and Ogren, 1983; Seemann et al., 1984; Badger and Andrews, 1987), and kinetic properties for Rubisco carbamylation have not been examined for C₄ species. We have thus assumed in Figure 8 that they are similar to those of C₃ species. To simulate the very low Rubisco carbamylations observed at high pCO₂ in transgenic F. bidentis with low activase content, the model predicts that RuBP should bind very tightly (100 times more tightly; K_f/K_r' = 0.01) to uncarbamylated compared to carbamylated Rubisco sites. This fits with in vitro measurements of K_f for spinach (Spinacia oleracea; 20 nM) by Jordan and Chollet (1983), which shows it to be at least 2 orders of magnitude smaller than K_r' (20 µM or 2 µM if chelation of RuBP by magnesium is taken into account; Yeoh et al., 1981; Roach and McFadden, 1983). Alternatively, the low carbamylation levels could be explained with a larger Michaelis Menten value, K_e, for carbamylation (see equation in Fig. 8 legend). It is clear that to understand species-specific differences in activase function, knowledge of the in vitro kinetics of carbamylation is also required.

High CO₂ Requirement for Growth of Antiactivase Plants with Very Low Activase Content

We were surprised to find that plants with severe activase deficiency required CO₂ concentrations above 1 kPa pCO₂ for growth. Plants with the lowest activase content had Rubisco active site content between 1.5 and 3 µmol m^–2. With a catalytic turnover rate of 3.5 s^–1, this should have resulted in net CO₂ assimilation rates of 3 to 8 µmol m^–2 s^–1 at CO₂ saturation depending on respiration rates. These rates are sufficient to allow antiactivase tobacco plants to grow at ambient or twice ambient concentrations (Mate et al., 1993; He et al., 1997). Experiments with PEP carboxylase inhibitor have shown that C₄ photosynthesis is not CO₂ saturated at 1 kPa pCO₂ when reliant on CO₂ diffusion from the atmosphere to the bundle sheath, emphasizing the gas tightness of the bundle sheath (Jenkins, 1989; Brown and Byrd, 1993). Dever et al. (1995), who isolated Amaranthus edulis mutants that lacked PEP carboxylase activity, were, however, able to grow their plants at 0.7 kPa pCO₂. In our case, it seemed surprising that an increase in CO₂ concentration from 1 to 2.5 kPa was able to rescue the growth of antiactivase plants with severe activase deficiency. There were no significant differences in the amount of PEP carboxylase content between transformants and wild-type plants. But there may have been some inhibition of the C₄ cycle in plants with the lowest activase content, such that these plants had to cope not only with low concentrations of functional catalytic sites but also with subsaturating CO₂ concentrations.

	CONCLUSIONS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSIONS MATERIALS AND METHODS LITERATURE CITED

 
Transgenic F. bidentis plants with low activase content demonstrate that acitvase is essential for the operation of the C₄ photosynthetic pathway, even though Rubisco in C₄ species operates in a high CO₂ environment. Although plants with severe activase deficiency required very high CO₂ concentration for growth, wild-type levels of activase are in apparent excess. Activase content could be reduced to less than 30% of wild-type levels before a reduction in steady-state CO₂ assimilation rates and Rubisco carbamylation were observed.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSIONS MATERIALS AND METHODS LITERATURE CITED

 

Construction of the Binary Plasmid

A partial cDNA for Flaveria. bidentis L. Kuntze was isolated from F. bidentis RNA by PCR technique using a specific 5' primer and a general 3' primer (Hudson et al., 1992). The specific primer 5'-GGGAGGCAAGGGTCAAGGTAA-3' was based on the codons for the amino acid sequence Gly-Gly-Gly-Lys-Gly-Gln-Gly-Lys located at the putative ATP-binding site of spinach (Spinacia oleracea) and Arabidopsis (Arabidopsis thaliana) Rubisco activase and had been used previously by Mate et al. (1993) to isolate a partial cDNA of tobacco (Nicotiana tabacum) rca. The 1-kb F. bidentis cDNA fragment was subcloned into pBin19 (cauliflower mosaic virus-nos), which is a derivative of pB121 (Jefferson et al., 1987) without the uidA gene. The resulting plasmid pBCFACT3 had the cDNA in the antisense orientation with respect to the cauliflower mosaic virus 35S promoter.

Plant Transformation and Regeneration

F. bidentis L. Kuntze was transformed and regenerated using the Agrobacterium method, as described by Chitty et al. (1994), except that shoots, selected on kanamycin, were immediately placed in a high CO₂ environment (approximately 2.5 kPa pCO₂). Three separate transformation experiments were done and approximately 20 transformants were regenerated and transferred to soil.

Plant Growth

Primary transformants were grown to seed in a growth cabinet under 2.5% CO₂ and an irradiance of 500 µmol quanta m^–2 s^–1. Air temperature was 25°C during a 14-h day and 18°C at night. Plants were watered daily and twice weekly with a complete nutrient solution. Primary transformants were allowed to grow to seed. One set of the T₁ generation of the primary transformants A, Q, SS, and RR was grown in a growth cabinet at 1 kPa pCO₂ and an irradiance of 500 µmol quanta m^–2 s^–1. Air temperature was 30°C during a 14-h day and 20°C at night, and the relative humidity was 70%. A second set of plants from the T₁ generation was grown in the same growth conditions as the primary transformants with 2.5 kPa pCO₂ except that day and night temperatures were approximately 30°C and 20°C.

Gas Exchange Measurements

Plants were brought from the high CO₂ growth cabinet to the laboratory, and gas exchange by young fully expanded leaves was measured using the LI-COR 6400 portable gas exchange system (LI-COR, Lincoln, NE). Measurements were made at an irradiance of 1,500 µmol quanta m^–2 s^–1 and a leaf temperature of 25°C, and the CO₂ was controlled at 40 or 38 Pa for standard measurements. Leaves were allowed to acclimate to the gas exchange conditions for at least 30 min. CO₂ response curves were made using the LI-COR CO₂ injection system. First measurements were made at a pCO₂ of 40 kPa then pCO₂ was lowered to 3 Pa and increased in steps up to a final value of 200 Pa.

Measurements of CO₂ exchange at 1.5 kPa pCO₂ were made on leaf discs of selected primary transformants with a mass spectrometric system described by Ruuska et al. (2000).

Measurement of Rubisco Activase Content

Rubisco activase was detected in whole-leaf extracts using immunoblotting essentially as described by Mate et al. (1996). Rubisco activase polypeptides were detected with a reduced glutathione S-transferase/spinach activase fusion protein antiserum followed by a horseradish peroxidase-conjugated secondary antibody and chemiluminescence. Activase content of transformants was quantified relative to wild-type amounts by loading a dilution series of wild-type leaf extract on each gel.

Measurement of Rubisco Content and Carbamylation

Content of Rubisco catalytic sites was measured by stoichiometric binding of ¹⁴C-carboxy-arabinitol-P₂ and its carbamylation status by exchanging ¹⁴C-carboxy-arabinitol-P₂ loosely bound at uncarbamylated sites with an excess of unlabeled C-carboxy-arabinitol-P₂ essentially as described by Butz and Sharkey (1989) and Ruuska et al. (1998). However, the following changes were made to the extraction procedure: for the primary transformants and corresponding wild type, 1-cm² leaf discs were extracted in 700 µL CO₂ free extraction buffer (50 mM HEPES-NaOH, pH 7.8) containing 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1% (w/v) polyvinylpolypyrrolidone, and 0.1% Triton containing 20 µM ¹⁴C-carboxy arabinitol-P₂ reduced to minimize changes in Rubisco activation states during extraction. Aliquots of the extract were removed to determine initial carbamylation of Rubisco sites after which MgCl₂ and NaHCO₃ were added to final concentrations of 20 mM and 15 mM, respectively, to determine total Rubisco sites. The extraction buffer used for the analysis of leaf discs of the T₁ generation contained 50 mM HEPPS-KOH, pH 8.1, rather than 50 mM HEPES-NaOH, pH 7.8, and protease inhibtor cocktail (P-9599, Sigma-Aldrich, St. Louis) instead of 1 mM phenylmethylsulfonyl fluoride.

Received November 4, 2004; returned for revision December 12, 2004; accepted December 13, 2004.

	FOOTNOTES

 
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.056077.

^* Corresponding author; e-mail susanne.caemmerer{at}anu.edu.au; fax 61–2–61255075.

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TOP ABSTRACT RESULTS DISCUSSION CONCLUSIONS MATERIALS AND METHODS LITERATURE CITED

 
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