JXB Advance Access originally published online on March 28, 2008
Journal of Experimental Botany 2008 59(7):1789-1798; doi:10.1093/jxb/erm373
RESEARCH PAPER |
The effects of Rubisco activase on C4 photosynthesis and metabolism at high temperature
1ARC Centre for Excellence in Plant Energy Biology, Research School of Biological Sciences, Australian National University, Canberra, ACT 2601, Australia
2Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, ACT 2601, Australia
3School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, WA 6009, Australia
* To whom correspondence should be addressed. E-mail: Susanne.Caemmerer{at}anu.edu.au
Received 11 October 2007; Revised 25 November 2007 Accepted 18 December 2007
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Abstract |
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The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme facilitates the release of sugar phosphate inhibitors from Rubisco catalytic sites thereby influencing carbamylation. T1 progeny of transgenic Flaveria bidentis (a C4 dicot) containing genetically reduced levels of Rubisco activase were used to explore the role of the enzyme in C4 photosynthesis at high temperature. A range of T1 progeny was screened at 25 °C and 40 °C for Rubisco activase content, photosynthetic rate, Rubisco carbamylation, and photosynthetic metabolite pools. The small isoform of F. bidentis activase was expressed and purified from E. coli and used to quantify leaf activase content. In wild-type F. bidentis, the activase monomer content was 10.6±0.8 µmol m–2 (447±36 mg m–2) compared to a Rubisco site content of 14.2±0.8 µmol m–2. CO2 assimilation rates and Rubisco carbamylation declined at both 25 °C and 40 °C when the Rubisco activase content dropped below 3 µmol m–2 (125 mg m–2), with the status of Rubisco carbamylation at an activase content greater than this threshold value being 44±5% at 40 °C compared to 81±2% at 25 °C. When the CO2 assimilation rate was reduced, ribulose-1,5-bisphosphate and aspartate pools increased whereas 3-phosphoglycerate and phosphoenol pyruvate levels decreased, demonstrating an interconnectivity of the C3 and C4 metabolites pools. It is concluded that during short-term treatment at 40 °C, Rubisco activase content is not the only factor modulating Rubisco carbamylation during C4 photosynthesis.
Key words: C4 photosynthesis, Flaveria bidentis, high temperature, Rubisco, Rubsico activase
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Introduction |
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High temperature can lead to a rapid reduction in C3 photosynthetic rate, the mechanism of which is often attributed to the inactivation of Rubisco (EC 4.1.1.3 [EC] 9) following multimeric dissociation and/or denaturation of Rubisco activase (Crafts-Brandner and Salvucci, 2004; Salvucci and Crafts-Brandner, 2004). Rubisco activase (RA) facilitates carbamylation and the maintenance of Rubisco activity in vitro by removing tight-binding sugar phosphates from Rubisco catalytic sites in an ATP-dependent manner (Portis, 2003; Robinson and Portis, 1988; Streusand and Portis, 1987). It has been hypothesized that at high temperature, the rate of Rubisco inactivation is faster than the ability of RA to reactivate the Rubisco enzyme (Crafts-Brandner and Salvucci, 2002). Kurek et al. (2007), using gene shuffling technology to create Arabidopsis RA isoforms with improved thermal stability, showed that photosynthesis and growth were improved under moderate heat stress in transgenic Arabidopsis expressing these thermotolerant RA isoforms. This provides evidence that manipulation of activase properties can improve C3 photosynthesis although no detailed studies relating increased photosynthesis with improved Rubisco activity have so far been carried out with these plants.
The C4 photosynthetic pathway is a biochemical CO2 concentrating mechanism that provides elevated CO2 partial pressure, between 10–100-fold greater than in air, at the site of Rubisco carboxylation in the bundle sheath (Furbank and Hatch, 1987; Jenkins et al., 1989). At high temperatures, C4 plants are hypothesized to have an advantage over C3 plants by largely eliminating oxygenation of Rubisco and allowing Rubisco to operate close to its maximal rate (Berry and Bjorkman, 1980). C4 species are known to have RA in bundle sheath tissue (Salvucci et al., 1987) and an earlier study demonstrated that RA is still essential for maintaining C4 photosynthesis at 25 °C even though Rubisco operates in a high CO2 environment (von Caemmerer et al., 2005).
The biochemical mechanism behind photosynthetic inactivation at high temperature in C4 plants has not been studied in detail (Sage and Kubien, 2007). Crafts-Brandner and Salvucci (2002) concluded that Rubisco inactivation of maize leaves (a C4 plant) by a moderate heat stress treatment was underpinned by the inability of RA to keep pace with the increased rate of Rubisco inactivation. However, other studies highlighted that stress-induced perturbations to electron transport capacity should not be ignored (Sage and Kubien, 2007; Schrader et al., 2004).
In vitro experiments have demonstrated that misprotonated inhibitors are rapidly released at high temperature from Rubisco active sites and that the relative extent of fallover (the slow inactivation of Rubisco activity in vitro) is reduced at high temperature and high CO2 in leaves of C3 species (Kim and Portis, 2006; Schrader et al., 2006). It is not known how important activase-alleviated inhibitor binding to Rubisco catalytic sites is at high temperature in the CO2-enriched bundle sheath environment of C4 leaves.
In this study, a series of Flaveria bidentis (L.) Kuntze transformants with an antisense construct targeted at the mRNA of the RA gene, rca (von Caemmerer et al., 2005), was used to examine the relationship between RA content and C4 photosynthesis at high temperature. The relationships between leaf gas exchange, activase content, Rubisco activity, and leaf metabolite pools are explored and it was demonstrated that activase content is not a limiting factor for C4 photosynthesis under our experimental conditions.
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Materials and methods |
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Plant growth
T1 generation of selected anti-activase primary transformants of F. bidentis (L.) Kuntze, described by von Caemmerer et al. (2005) were grown in a growth cabinet under approximately 10 mbar CO2, 75% relative humidity, 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 fertilized with a slow release fertilizer (Osmocote, Scotts Australia, Castle Hill, Australia).
Isolation of a cDNA encoding F. bidentis Rubisco activase
A cDNA fragment encoding the 3'-end of RA was amplified from an adaptor-ligated F. bidentis leaf cDNA library (Tetu et al., 2007) using an oligonucleotide primer that encoded the conserved nucleotide-binding domain at positions 163–170 of the spinach Rubisco activase (Werneke et al., 1988) and the AP1 primer (Clontech, Palo Alta, CA, USA). AmpliTaq Gold Buffer II, and 1 unit AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) were used with other PCR components and touchdown cycling conditions as described in the Marathon cDNA Amplification Kit manual (Clontech). The rca fragment encoding the 5'-sequence end of the RA cDNA was subsequently isolated from the cDNA library using the AP1 primer and a F. bidentis rca-specific primer (5'-CTCACGGTACCGCTGTCTGATGAGC).
The open reading frame (ORF) encoding F. bidentis RA (GenBank accession number EU202926) and its plastid targeting sequence was amplified using the MasterTaq Kit (Eppendorf, Hamburg, Germany) with 200 nM forward (5'-CCATTCTCAGCCTCCCACCTTC) and reverse (5'-AATATATAAATACAGCTTTCACCACC) primers, 200 µM dNTPs, and 0.1 vol. of a reverse transcription (RT) reaction from mRNA isolated from F. bidentis leaves (Tetu et al., 2007). The veracity of the ORF sequence was confirmed by determining the sequence of several RT-PCR products derived from the mRNA of several individual F. bidentis plants.
Purifying recombinant F. bidentis activase
The mature coding sequence of the 43 kDa small isoform of F. bidentis RA was amplified using primers 5'SacII FAct (5'-TCCGCGGTGGAATGGAGAAAGAGATCGAAGAGACC-3', SacII site in italics) and 3'FAct (5'-CTATTCTTTTCTGGCAAAG-3', complement of the stop codon in bold) and the product cloned into pGEM-T-Easy and sequenced. The 1166 bp SacII-EcoRI rca fragment was cloned in-frame downstream of a sequence coding a 6x-histidine-tagged ubiquitin (H6Ub) peptide in plasmid pHue (Baker et al., 2005). The H6Ub-RA fusion was expressed in E. coli Bl2L (DE3) cells, purified by immobilized metal affinity chromatography and the H6Ub removed using ubiquitin protease as previously described (Baker et al., 2005). Protein content was standardized relative to BSA using a dye binding assay (Pierce).
Quantification of leaf Rubisco activase content
RA was detected in whole leaf extracts using immunoblotting essentially as described by Mate et al. (1996). RA polypeptides were detected with a GSH S-transferase/spinach activase fusion protein antiserum and with an anti-IgG alkaline phosphatase conjugated secondary antibody (Bio-Rad Laboratories, Hercules, CA, USA). Immunoreactive peptides were identified using the AttoPhos fluorescence substrate system (Promega, Madison, Wisconsin) and quantified by densitometry (ImageJ software; http://rsb.info.nih.gov/ij/) relative to a dilution series of known amounts of purified recombinant F. bidentis small isoform RA (Fig. 1).
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Gas exchange analysis
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 1500 µmol quanta m–2 s–1 and a leaf temperature of 25 °C or 40 °C, and the CO2 was controlled at 400 µbar for standard measurements. Leaves were allowed to acclimate to the gas exchange conditions for at least 20 min. The high evapotranspirational cooling by the F. bidentis leaves limited the maximum achievable temperature for gas exchange to 40 °C. Immediately after completing the gas exchange measurements, leaf discs were taken from the analysed regions for measurements of Rubisco content and carbamylation.
Measurement of total and carbamylated Rubisco active site content
The total content of Rubisco catalytic sites was measured by stoichiometric binding of 14C-carboxy-arabinitol-P2. The carbamylated site content was determined by exchanging 14C-carboxy-arabinitol-P2 loosely bound at uncarbamylated sites with a 500 M excess of unlabelled carboxy-arabinitol-P2 as initially described by Butz and Sharkey (1989), and modified by von Caemmerer et al. (2005). The carbamylation state of Rubisco was calculated from the ratio of carbamylated to total sites. The extraction buffer consisted of 50 mM EPPS (pH 8.1), 1 mM EDTA, 10 mM DTT, 0.01% (v/v) Triton X-100, 1% (w/v) PVPP, and 20 µl of protease inhibitor cocktail (Sigma, St Louis, Missouri) per 600 µl extraction.
Rapid freeze clamping of leaves
Leaf gas exchange was measured with a Li-Cor 6400 in a custom-built leaf chamber attached to a rapid-kill apparatus (Badger et al., 1984; Ruuska et al., 1998). Illumination was provided by 150 W slide projector light, which was passed via a mirror to the leaf chamber. Leaves were acclimated in the chamber at 400 µbar pCO2, a leaf temperature of 25 °C or 40 °C, and an irradiance of 1500 µmol quanta m–2 s–1 for 40 min before the leaf was rapidly freeze clamped between two liquid nitrogen-cooled copper rods. The frozen 5.3 cm2 leaf disc was stored at –80 °C prior to extraction for metabolite assays.
Metabolite measurements
Leaf discs were ground to a fine powder in liquid nitrogen and extracted with 1 ml of frozen 1 M HCLO4 and metabolites assayed as described previously (Furbank and Leegood, 1984; Leegood and von Caemmerer, 1988) using a diode array spectrophotometer (8452A; Hewlett-Packard). Malate, aspartate, pyruvate, phosphoenol pyruvate (PEP), triose phosphate (Triose-P), 3-phosphoglycerate (PGA), and ribulose-1,5-bisphosphate (RuBP) were measured immediately after extraction. Triose-P, PGA and RuBP were measured consecutively in the same assay as described previously (He et al., 1997), using Rubisco purified from tobacco (Nicotiana tabacum) as described by Schrader et al. (2006).
Statistical analysis
Where statistical means comparison was necessary, two-sample independent t tests and one-way and two-way ANOVAs were undertaken using a P cut-off of 0.05. These tests were undertaken using Origin v7.0220 (OriginLab, Northampton, MA, USA).
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Quantification of leaf activase in F. bidentis
F. bidentis was shown to have two Rubisco activase (RA) isoforms (von Caemmerer et al., 2005). The short form of F. bidentis (RA) was cloned to generate a recombinant RA protein that was used to measure the RA content in F. bidentis leaves using quantitative immunoblotting (Fig. 1). The method of Baker et al. (2005) was employed to express and purify RA protein (Fig. 1A). Although the 6x-histidine-tagged ubiquitin-RA (H6Ub-RA) fusion was not entirely soluble, H6Ub-RA was abundantly expressed in E. coli and the H6Ub-fusion technique provided an efficient means to obtain highly purified RA protein (final purified RA concentration of 2.2 mg ml–1) (Fig. 1A). Some free ubiquitin was detected in the lysate in both the soluble and affinity-purified fractions because the H6Ub-RA fusion is cleaved in E. coli at the peptide bond between the ubiquitin and the fused protein (Catanzariti et al., 2004).
Wild-type F. bidentis grown under the experimental conditions described here had high leaf RA monomer contents of 10.4±0.8 µmol m–2 (447±36 mg m–2), assuming a molecular mass of 43 kDa for the RA monomer. The average Rubisco site content was 14.2±0.8 µmol m–2, giving a ratio of 1.5±0.1 RA tetramers to each Rubisco hexadecamer. Immunoblot analyses based on the densitometry of the RA isoform bands following SDS–PAGE showed the relative abundance of both isoforms in F. bidentis leaves were comparable (data not shown).
CO2 assimilation rate and Rubisco carbamylation state at high temperature
As the T1 progeny of four anti-activase F. bidentis lines with low RA levels require high CO2 partial pressures (pCO2) for growth (von Caemmerer et al., 2005), all the plants were grown in air supplemented with approximately 10 mbar pCO2. Gas exchange measurements of selected T1 progeny and wild-type controls were made at an ambient pCO2 of 400 µbar and 1500 µmol quanta m–2 s–1 at 25 °C or 40 °C. Similar levels of Rubisco were measured in the leaves of both wild-type F. bidentis and the anti-activase lines sampled (overall mean 14.3±0.4 µmol sites m–2).
The influence of variant leaf RA content on net photosynthetic CO2 uptake (A) and Rubisco carbamylation state in leaves incubated at 25 °C and 40 °C is presented in Fig. 2. The photosynthetic rate of the transformants was reduced at both temperatures when the RA content was less than 25% of wild-type levels and this correlated with reduced Rubisco carbamylation states (Fig. 2). In the wild-type leaves, net photosynthetic rates at 40 °C (39.7±0.6 µmol m–2 s–1) were significantly higher than at 25 °C (34.0±0.3 µmol m–2 s–1; P < < 0.0001) as determined by a two sample independent t test. However, the Rubisco carbamylation state was significantly reduced from 81±2% at 25 °C to 44±5% at 40 °C (P <0.0001) (Fig. 2).
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The in vivo catalytic turnover rate (kcat) was calculated from measurements of CO2 assimilation rate, dark respiration, and the carbamylated site concentration of Rubisco (see Materials and methods). The respiration rates used were 2 and 4 µmol m–2 s–1 at 25 °C and 40 °C, respectively, and had been measured by Dwyer et al. (2007). The in vivo kcat was not correlated with RA content at either temperature and the average in vivo kcat was significantly higher for 40 °C treated leaves (7.6±0.4 s–1) compared to 25 °C treated leaves (3.5±0.2 s–1).
Photosynthetic metabolite content in anti-activase plants
Using rapid (<0.1 s) freeze-clamping and spectrophotometric assays, changes in select photosynthetic metabolites could be detected in leaves from which gas exchange measurements were made at 25 °C and 40 °C (Figs 3, 4, 5
). Since RA content can be decreased substantially without an effect on CO2 assimilation rate, only the mean metabolite levels of the anti-activase lines with photosynthetic rates less than 10 µmol m–2 s–1 were included in the summary (Table 1).
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In wild-type leaves, there were no differences in metabolite levels between the 25 °C and 40 °C except that PGA and pyruvate concentrations were significantly less at 40 °C than at 25 °C when comparison was made with a 2-way ANOVA (Table 1). The mean RuBP levels of wild-type leaves were significantly lower in 40 °C-treated leaves compared with 25 °C-treated leaves when compared on an individual means basis (t test; P <0.05) but not when compared by a 2-way ANOVA.
In the transgenic plants, all C3 metabolites mimicked to varying extents constraint of photosynthetic CO2 assimilation by RA (Fig. 2). At both temperatures, RuBP concentration was significantly increased when the photosynthetic rate was reduced by low activase contents (
25% wild-type RA levels; Fig. 3; Table 1). PGA levels showed the reverse trend, which resulted in similar RuBP/PGA ratios at both 25 °C and 40 °C and an abrupt increase in the ratio in leaves with low levels of RA (Fig. 3). The mean triose-P/PGA ratio increased at both temperatures in a hyperbolic relationship similar to the RuBP/PGA ratio at RA levels below 125 mg m–2 RA (data not shown). However, the phosphate pool contained in RuBP, PGA, and triose-P was similar for all leaves, irrespective of temperature and RA content (Table 1).
Total metabolite pools of the C4 cycle were also invariant with RA content (Table 1). The levels of the individual metabolites, namely pyruvate, alanine, PEP, and aspartate, making up this pool, were more variable than the C3 metabolite levels (cf Figs 3 and 4), however, similar trends were detected. Perturbing the C3 cycle by limiting RA concentration and thereby CO2 uptake led to a reduction in PEP, pyruvate, and alanine contents in the leaves of anti-activase plants and an increase in leaf aspartate levels (Table 1; Fig. 4). Leaf PEP and PGA contents were positively correlated, with a similar relationship observed at both temperatures (Fig. 5).
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Discussion |
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Rubisco activase content does not constrain C4 photosynthesis at high temperature
Using a transgenic approach, data are presented that strongly suggests that RA is not the primary constraint on photosynthetic rate or carbamylation state at high temperature in F. bidentis (Fig. 2; Table 1). Similar to the results of previous studies, F. bidentis RA concentrations could be reduced to less than 25% of wild-type levels at 25 °C before a decrease in steady-state CO2 assimilation rates and Rubisco carbamylation were observed (Jiang et al., 1994; Mate et al., 1996; Eckardt et al., 1997; von Caemmerer et al., 2005). At 40 °C, F. bidentis leaves exhibited a hyperbolic relationship with a similar initial slope to that at 25 °C (Fig. 2). This indicates that, regardless of temperature, RA content limited carbamylation state and photosynthetic rate below an effective ratio of one RA tetramer to three Rubisco hexadecamers. This critical threshold ratio is in a similar range as that of C3 plants (1:5 for high-light grown Arabidopsis; Eckardt et al., 1997), suggesting that high-light acclimated C3 and C4 plants have a similar in vivo requirement for activase relative to Rubisco protein content (Salvucci et al., 2006). It should be noted that for low-light-grown Arabidopsis thaliana, presumably operating under RuBP regeneration-limited conditions, the amount of RA is significantly reduced and closer to a higher critical threshold of 1:2 (Eckardt et al., 1997). Future experiments investigating photosynthetic responses to activase should consider growth condition as a source of variation and possible sensitivity to activase-mediated heat stress.
It has been suggested that the temperature-induced decrease in Rubisco activation in C3 plants results largely from the inability of activase to keep pace with a faster rate of Rubisco inactivation as temperatures increase and leaf RA content would therefore be expected to be important in a high-temperature response (Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004). The models of activase action proposed by Mate et al. (1996) and Kim and Portis (2006) both predict a hyperbolic response between Rubisco carbamylation and RA activity with a lower intial slope at 40 °C compared to 25 °C if it is assumed that the rate of Rubisco deactivation increases relative to RA activity and this is not what was observed here (Fig. 2). At 40 °C Rubisco carbamylation state is significantly reduced compared to 25 °C at absolute RA contents well above the critical threshold of 1:3 discussed above (Fig. 2). Similarly, Kim and Portis (2005) also observed no difference in the temperature response of transgenic Arabidopsis with 40% of wild-type levels of RA compared to wild type. These results suggest that either activase activity is limited by another process, such as, for example, the rate of ATP production, or that there is another activase-independent mechanism limiting Rubisco carbamylation at higher temperature.
It is intriguing that the low Rubisco carbamylation at 40 °C is not dependent on the amount of activase and raises the question of what could be the determinants of carbamylation state. In vitro Rubisco carbamylation state is CO2 and magnesium-dependent and increases with increasing CO2 concentration (Laing and Christeller, 1976; Lorimer et al., 1976). It may be that under CO2-saturation in C4 leaves, Mg2+ becomes a limiting factor for carbamylation in vivo. The in vitro data from Kim and Portis (2006) demonstrated that low Mg2+ does exacerbate a high temperature decarbamylation of Rubisco in spinach extracts. The in vivo physiological free Mg2+ concentrations are unknown for C4 bundle-sheath chloroplasts. However altered CO2 and Mg2+ concentration relative to the Michaelis constants for activation, which are likely to increase with temperature, would also predict a hyperbolic response with decreased initial slope (Mate et al., 1996).
The carbamylation state of Rubisco is also influenced by the binding of inhibitors at Rubisco uncarbamylated or carbamylated active sites. It is thought that activase interacts with Rubisco by opening closed loops at the catalytic site thus promoting carbamylation and catalysis (Andrews et al., 1995; Portis, 2003; Salvucci and Ogren, 1996). One activase-independent mechanism which could perhaps explain the reduced carbamylation at 40 °C compared to 25 °C at intermediate to high activase content, would be an increase of inhibitors binding to Rubisco sites that did not involve loop closure. It has been shown that activase has mixed specificity for some sugar phosphate bound forms of Rubisco and has no activity with others (Lilley and Portis, 1990).
CO2 assimilation rates in wild type and anti-activase plants with intermediate activase levels were greater at 40 °C compared to 25 °C, despite the reduced Rubisco carbamylation at 40 °C (Fig. 2). At 25 °C our calculated in vivo Rubisco catalytic turnover rates (kcat) of 3.5±0.2 s–1 matches well the in vitro kcat of 3.9 s–1 for F. bidentis (Kubien et al., 2003). This indicates that at 25 °C bundle-sheath pCO2 is already close to saturating. However, at 40 °C, the in vivo kcat of 7.6±0.4 s–1 is well below the in vitro 14 s–1 measured by Kubien et al. (2003). There could be several reasons for the lower in vivo kcat at 40 °C. It could mean that bundle-sheath pCO2 is not saturating at 40 °C under these conditions because of reduced C4 cycle activity relative to bundle sheath C3 cycle activity, or that the regeneration of RuBP is limiting. Since the carbamylated and total Rubisco site content were measured with the tight binding inhibitor carboxy-arabinitol-P2, which displaces other inhibitors bound to Rubisco sites, it could also be that there are inhibitors bound to the active site of Rubisco. This was suggested for tobacco anti-activase plants although no inhibitors could be identified on Rubisco sites in tobacco after rapid extraction of Rubisco from leaves (He et al., 1997).
Our metabolite data for wild type leaves at 25 °C agrees well in absolute terms with previous measurements (Leegood and von Caemmerer, 1994). The leaf RuBP contents and RuBP/PGA ratios did not increase at 40 °C relative to 25 °C and this argues against an increased Rubisco limitation at 40 °C and suggests that high temperature-treated leaves could be RuBP regeneration limited (Table 1). However, the RuBP pool sizes were 5.8 and 4.6 times the Rubisco site content at 25 °C and 40 °C, respectively. At 25 °C this is very similar to ratios observed in wild-type tobacco and found to be saturating at 25 °C (Price et al., 1995). It is difficult to know whether this is saturating for both temperatures given uncertainties in the in vivo Km (RuBP). In vitro the Km (RuBP) has a Q10 of 1.9 and is approximately 28 µM at 25 °C therefore the Km (RuBP) possibly increases to 80 µM at 40 °C. In vivo other phosphorylated compounds which act as competitive inhibitors to RuBP could further increase the apparent Km (RuBP) at both temperatures (Badger and Collatz, 1977; Badger and Lorimer, 1981). Thus our RuBP pool size measurements offer no obvious explanation for the lower than expected increases in CO2 assimilation rate from 25 °C to 40 °C. It is possible that bundle-sheath pCO2 is less saturating at 40 °C relative to 25 °C. The Km (CO2) of Rubisco more than doubles between 25 °C and 40 °C (von Caemmerer, 2000). If the capacity of the C4 cycle does not increase to the same extend, then bundle-sheath pCO2 could be less and this could explain the lower than expected catalytic turnover rate at 40 °C.
Effect of reduced Rubisco activity on photosynthetic metabolite content
Historically, varying light and CO2 has been utilized to observe the interaction between C4 and C3 cycle metabolites (Leegood and von Caemmerer, 1988, 1989, 1994). In this study, a transgenic approach was used to vary photosynthetic capacity in a C4 plant and to contrast C4 and C3 metabolism. Figure 6 is an illustration of the C4 metabolic pathway and its interaction with the C3 metabolic cycle in F. bidentis, a typical NADP-ME C4 dicot which can utilize both malate and aspartate (Meister et al., 1996). The co-ordinated function of C4 photosynthesis in F. bidentis requires a very specific leaf anatomy consisting of photosynthetic cells arranged in concentric rings around the leaf vasculature. In the outer mesophyll cells CO2 is initially fixed by PEP carboxylase into malate and aspartate which both diffuse into the bundle sheath cell compartment where they are subsequently decarboxylated to supply CO2 for Rubisco. The resultant pyruvate and alanine are then returned to the mesophyll cells where regeneration of pyruvate to PEP occurs (Fig. 6).
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The reduction in bundle sheath CO2-fixing capacity in anti-activase plants caused by the decrease in Rubisco carbamylation resulted in an increase in the RuBP pool and a decrease in PGA and triose-P content (Fig. 3; Table 1). This is very similar to results obtained with transgenic tobacco with antisense reductions in Rubisco content (Quick et al., 1991). During C4 photosynthesis some of the PGA is reduced in the mesophyll chloroplasts and triose-P is returned to the bundle sheath (Fig. 6), it is therefore interesting to note that there is no large decrease in the phosphate pool contained in RuBP, PGA, and triose-P (Table 1). A similar conservation of phosphate was noted when large variations in RuBP and PGA pools were induced by variation in pCO2 in radish leaves (von Caemmerer and Edmondson, 1986).
Furbank et al. (2000) suggested two ways in which the C3 and C4 cycles could be in regulatory co-ordination: The first is through the traffic of metabolites, particularly the interconversion of PEP and PGA, and the second is through the regulation of C4 acid decarboxylation. These data provide some evidence for both regulatory mechanisms (Table 1; Fig. 5). Interconversion of PGA and PEP by phosphoglycerate mutase and enolase occurs in the mesophyll cytosol and results in metabolic communication between C3 and C4 cycles. It has been shown that a close correlation exists between PGA and PEP pool sizes under different environmental conditions (Furbank and Leegood, 1984; Leegood and von Caemmerer, 1989). A positive correlation between leaf PGA and PEP was observed, indicating that the reduction in PGA pools caused by reduced Rubisco activity feeds back on to the PEP pools (Fig. 5). Secondly, when low RA content reduces CO2 assimilation rates, aspartate pools increase and alanine and pyruvate pools decrease, indicating a reduction in the decarboxylation rate (Table 1). This reduction may be forced by the reduced consumption of NADPH by the C3 cycle in the bundle sheath (Fig. 6). Thus our transgenic manipulation of bundle-sheath C3 cycle activity has served to demonstrate the metabolic interconnection between the C3 and C4 cycles in F. bidentis. It is, however, not known at present how this interconnection of metabolite pools affects the relative fluxes through the C4 and C3 cycles. Carbon isotope measurements indicated that the bundle-sheath CO2 leakiness increased in transgenic F. bidentis with reduced amounts of Rubisco (von Caemmerer et al., 1997). This would suggest that the two cycles are not necessarily tightly linked when C3 cycle activity is reduced in the bundle sheath.
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Conclusions |
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Our analysis of the role of RA using transgenic F. bidentis containing an antisense construct to the enzyme shows that, although Rubisco of C4 species operates in a high pCO2 environment (which may in itself promote carbamylation of Rubisco active sites), Rubisco is dependent on RA for catalysis. However, it was also found that the reduction in Rubisco carbamylation at 40 °C was not related to the amount of activase present, suggesting that other factors are involved in the modulation of Rubisco carbamylation during C4 photosynthesis. Measurements of key metabolites of the C3 and C4 cycles in these transgenics highlight the influence of Rubisco on the integrated function of the cycles during C4 photosynthesis.
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Acknowledgements |
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We gratefully acknowledge the technical assistance of Drs Yuoshi Tazoe and Asaph Cousins and we thank Dr Wataru Yamori for helpful discussion on the manuscript.
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Abbreviations |
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A, CO2 assimilation rate; PEP, phosphoenolpyruvate; PGA, 3-phosphoglycerate; pCO2, partial pressure of CO2; triose-P, triose phosphate; RuBP, D-ribulose-1,5-bisphosphate; Rubisco, RuBP carboxylase/oxygenase; RA, Rubisco activase.
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References |
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