Reductions of Rubisco Activase by Antisense RNA ... - Plant Physiology

Reductions of Rubisco Activase by Antisense RNA in the C4 Plant Flaveria bidentis Reduces Rubisco Carbamylation and Leaf Photosynthesis Susanne von Caemmerer*, L. Hendrickson, V. Quinn, N. Vella, A.G. Millgate, and R.T. Furbank 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.)

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.

The C4 photosynthetic pathway is a biochemical CO2-concentrating mechanism that provides elevated CO2 partial pressure (pCO2) at the site of Rubisco carboxylation in the bundle sheath. This suppresses photorespiration and allows Rubisco (EC 4.1.1.39) to operate close to its maximal rate, such that CO2 assimilation in C4 plants is effectively CO2 saturated in air (Hatch, 1987). Similar to photosynthesis in C3 species, there is a strong correlation between Rubisco content of leaves and CO2 assimilation rate at high irradiance, demonstrating that Rubisco carboxylation is among the rate-limiting steps of C4 photosynthesis (Usuda et al., 1984; Hunt et al., 1985; Sage et al., 1987; Furbank et al., 1996; von Caemmerer et al., 1997). For CO2 fixation to take place, Rubisco’s catalytic sites must first be activated. This requires the carbamylation of a Lys residue within Rubisco’s catalytic sites to allow the binding of a Mg21 (for review, see Andrews and Lorimer, 1987). Sugar phosphates can also bind to Rubisco catalytic sites, and this interferes with both the process of carbamylation and full activity of the carbamylated enzyme (Badger and Lorimer, 1981; Jordan et al., 1983; Edmondson et al., 1990a; Zhu and * Corresponding author; e-mail [email protected]; fax 61–2–61255075. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.056077.

Jensen, 1991). In vivo, the activation and maintenance of Rubisco activity are facilitated by a second protein called Rubisco activase. Activase was first identified by analysis of the rca mutant of Arabidopsis (Arabidopsis thaliana), which required high CO2 for growth (Somerville et al., 1982; Salvucci et al., 1985). In vitro studies revealed that activase requires ATP to function (Streusand and Portis, 1987) and removes sugar phosphates from Rubisco catalytic sites (Robinson and Portis, 1988; Portis, 1990). The precise mechanism by which activase promotes Rubisco carbamylation remains unclear but must involve the interaction of activase with Rubisco sugar phosphate complexes (Portis, 2003). Mate et al. (1996) and Salvucci and Ogren (1996) proposed models for the mechanism of activase action. They suggested a mechanistic model in which activase functions to open closed polypeptide loops within the protein structure at the catalytic site that normally enclose tight binding ligands. It is hypothesized that when activase is activated by ATP hydrolysis it can recognize Rubisco with a closed-loop protein structure and bind selectively to these catalytic sites. This causes the loops to open, releasing the ligand and returning activase to its inactive form. Mate et al. (1996) formulated this in a mathematical model and showed that full carbamylation could result at subsaturating CO2 from the ability of activase to promote the release of ribulose 1,5-bisphosphate

Plant Physiology, February 2005, Vol. 137, pp. 747–755, www.plantphysiol.org Ó 2005 American Society of Plant Biologists

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(RuBP) from the carbamylated and uncarbamylated forms. 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 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). Immunoblotting with tobacco (Nicotiana tabacum) activase antibody showed that the transformants with low photosynthetic rates also had low concentrations of Rubisco activase (Fig. 2). Unlike tobacco (Mate et al., 1993, 1996) F. bidentis has two activase polypetides of similar Mr to those of Arabidopsis (Fig. 2). To assess Rubisco carbamylation levels, leaf discs were collected under growth conditions (2.5 kPa pCO2, 500 mmol quanta m22 s21, 25°C) and snap frozen in liquid nitrogen. Rubisco content and carbamylation were measured by stoichiometric binding of 14 C-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 con748

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 mmol m22 s21, and leaf temperature of 25°C. Activase amounts of primary transformants A, SS, Q, RR, and Aa are shown in Figure 2.

tents of all primary transformants were similar to wild type (Fig. 3). The maximum carbamylation measured under this relatively low irradiance levels was approximately 50%. Leaf discs were also collected under similar growth conditions after plants had been exposed to ambient CO2 (40 Pa) for several hours. This reduced carbamylation levels even further in plants with the lowest activase content but had no effect on carbamylation in wild type and with intermediate activase content. The effect was significant at the 0.05% level (Table I). CO2 response curves of CO2 assimilation showed that the reduction of CO2 assimilation rate at ambient CO2 correlated primarily with a reduction in the CO2saturated 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-toleaf variation. Relationship between Activase Content and CO2 Assimilation Rate

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. Plant Physiol. Vol. 137, 2005

Rubisco Activase Is Essential for C4 Photosynthesis

Table I. Comparison of CO2 assimilation rates and Rubisco carbamylation levels measured at ambient CO2 and high CO2 (1.5 or 2.5 kPa pCO2) of selected primary transformants and wild-type plants Measurements of CO2 assimilation rate were made with LI-COR gas exchange system at 40 Pa CO2, 1,500 mmol quanta m22 s21, and 25°C or in a closed mass spectrometric system at 1.5 kPa pCO2. For measurements of Rubisco after carbamylation leaf samples were collected in the growth cabinet at either 2.5 kPa or 40 Pa pCO2 at an irradiance of 500 mmol quanta m22 s21 at 25°C. Primary Transformants

CO2 Assimilation Rate 22

mmol m

A Q SS RR Aa C PP TT NN Wild type Wild type

At 40 Pa pCO2 20.63 5.0 1.9 1.7 21.6 22.6 30.0 28.0 20.2 31.6 24.1

Rubisco Carbamylation

21

s

At 1.5 kPa pCO2 15.3 21.6 19.6 13.3

Activase levels were quantified by immunoblotting against a dilution series of a particular wild-type standard, which was taken as 100%. We observed a range of activase levels among wild-type plants. The T1 generations of the four primary transformants were segregating populations with respect to activase levels, and the relationship between CO2 assimilation rate and activase content showed that activase was saturating for wild type under these steady-state gas exchange conditions. There was, however, a strong relationship between activase content and CO2 assimilation rates once activase levels were reduced below 30% of wild type. Plants with very low activase content had very low CO2 assimilation rates, replicating what had been observed for the primary transformants.

At 40 Pa pCO2 4 8 4 43 53 47

43.6 43.6

41

% At 2.5 kPa pCO2 9.0 14 14 16 26 40 43 49 53 54 44

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). We calculated the in vivo catalytic turnover rate form measurements of CO2 assimilation rate and dark respiration and the carbamylated active site concentration of Rubisco (Fig. 7). For wild-type plants, the mean in vivo catalytic turnover rate was 3.5 s21 and was similar for most antiactivase plants, except perhaps for the plants with very low activase levels where it was lower. However, it is difficult to accurately estimate in vivo catalytic turnover for plants with very low assimilation rates. Modeled CO2 Dependence of Rubisco Carbamylation

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 mmol quanta m22 s21 and between 40% and 50% at 500 mmol quanta m22 s21 (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% 6 3% at 1.5 kPa pCO2 and 44% 6 2% at 40 Pa pCO2) and plants with very low activase content (70% 6 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 Plant Physiol. Vol. 137, 2005

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

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. 749


activity. In the model, activase activity is modeled through an increase in the ratio Kf/Kr#, where Kr# is the apparent Michaelis Menten constant for RuBP. Kf is an activase-mediated dissociation constant of the uncarbamylated Rubisco site RuBP complexes and defines the ratio uncarbamylated Rubisco sites to uncarbamylated Rubisco site RuBP complexes in the steady state. It can be seen from Figure 8 that at high activase activity (high Kf/Kr# 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 Kf/Kr#). The curve shown in Figure 8 where Kf/Kr# 5 1 is almost identical to the carbamylation predicted when RuBP concentration is 0.

DISCUSSION Rubisco Activase Is Essential for C4 Photosynthesis

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 mmol quanta m22 s21 at 25°C.

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. The model treats all Rubisco enzyme sites independently. It assumes that activase recognizes the specific conformation when several polypeptide loops at the catalytic site close over tight-binding ligands such as RuBP. It is assumed that activase binds to that conformation and in so doing causes the loops to retract, releasing the ligand and activase itself. The closedloop complexes can occur with both carbamylated and uncarbamylated Rubisco sites, and the unassisted rate of ligand release is very slow. However, at carbamylated sites, when the ligand is RuBP, catalysis provides another rapid means of opening loops through catalytic conversion of the RuBP to loosely binding products. The model demonstrated that, because of this, activase induces a much larger increase in the rate of opening of uncarbamylated Rubisco site RuBP complexes than in the rate of opening of the carbamylated catalytically competent Rubisco site RuBP complexes. This differential is the reason Rubisco carbamylation status responds to concentrations of both RuBP and CO2. Figure 8 shows the predicted CO2 dependence of Rubisco carbamylation at various levels of activase 750

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

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 mmol quanta m22 s21 and a leaf temperature of 25°C. Plant Physiol. Vol. 137, 2005


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 mmol m22 s21, 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 (Figs. 6 and 7). The measured in vivo values of 3.5 s21 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.

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 Plant Physiol. Vol. 137, 2005

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 mmol m22 s21 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 mmol quanta m22 s21, and leaf temperature of 25°C. 751


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 mmol m22 s21, and this value was added to net CO2 assimilation rate to obtain gross CO2 assimilation rate.

than at ambient CO2 (66% 6 3% compared to 44% 6 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 Kf/Kr#), 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

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 Kf/Kr# 5 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 (Ma¨chler and No¨sberger, 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 752

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): Carbamylated Rubisco 5

CM ; CM 1 Ke Kd ðKr# =Kf Þ

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 5 1,600 mM and KeKd 5 160,000 mM2 (Laing and Christeller, 1976; Lorimer et al., 1976). Plant Physiol. Vol. 137, 2005


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; Kf/Kr# 5 0.01) to uncarbamylated compared to carbamylated Rubisco sites. This fits with in vitro measurements of Kf for spinach (Spinacia oleracea; 20 nM) by Jordan and Chollet (1983), which shows it to be at least 2 orders of magnitude smaller than Kr# (20 mM or 2 mM 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, Ke, 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 mmol m22. With a catalytic turnover rate of 3.5 s21, this should have resulted in net CO2 assimilation rates of 3 to 8 mmol m22 s21 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 wildtype 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 Plant Physiol. Vol. 137, 2005

with low concentrations of functional catalytic sites but also with subsaturating CO2 concentrations. CONCLUSIONS

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 steadystate CO2 assimilation rates and Rubisco carbamylation were observed.

MATERIALS AND METHODS 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-GlyLys-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 mmol quanta m22 s21. 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 mmol quanta m22 s21. 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 LICOR 6400 portable gas exchange system (LI-COR, Lincoln, NE). Measurements were made at an irradiance of 1,500 mmol quanta m22 s21 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 LICOR CO2 injection system. First measurements were made at a pCO2 of 40 kPa

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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 14 C-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 mL 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 mM 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 HEPPSKOH, 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.

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