Graham S. Hudson

The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco

Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO2 assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO2 partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO2 assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO2, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, kcat, of 3.5+0.2 mol CO2·(mol sites)−1·s−1 at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO2·(mol sites)−1·−1 in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO2 and O2, the rate of CO2 assimilation was measured at 25° C at different intercellular partial pressures of CO2 and O2. These measurements were combined with carbon-isotope analysis (13C/12C) of CO2 in the air passing over the leaf to estimate the conductance for transfer of CO2 from the substomatal cavities to the sites of carboxylation (0.3 mol·m−2·s−1·bar−1) and thus the partial pressure of CO2 at the sites of carboxylation. The calculated Michaelis-Menten constants for CO2 and O2 were 259 ±57 μbar (8.6±1.9μM) and 179 mbar (226 μM), respectively, and the effective Michaelis-Menten constant for CO2 in 200 mbar O2 was 549 μbar (18.3 μM). From measurements of the photocompensation point (Γ* = 38.6 ubar) we estimated Rubiscos relative specificity for CO2, as opposed to O2 to be 97.5 in vivo. These values were dependent on the size of the estimated CO2-transfer conductance.

Transfer conductance and leaf anatomy in transgenic tobacco with a reduced content of rubisco

The CO2 transfer conductance in leaves quantifies the ease with which CO2 can diffuse from sub-stomatal cavities to sites of carboxylation within the chloroplast. The aim of this work was to test the hypothesis that the CO2 transfer conductance is proportional to the surface area of chloroplasts exposed to intercellular airspaces. We compared two genotypes, wild-type and transgenic tobacco, that had been transformed with an antisense gene directed at the mRNA of the Rubisco small subunit. Transgenic tobacco had lower rates of CO2 assimilation than wild-type but similar chlorophyll contents. Leaf anatomy was altered by growing plants in two different environments: a high daily irradiance in a growth cabinet (12 h photoperiod of 1 mmol quanta m-2 s-1) and a sunlit glasshouse. The growth cabinet gave at least twice the daily irradiance compared to the glasshouse. The CO2 transfer conductance was calculated from combined measurements of gas exchange and carbon isotope discrimination measured in 2% oxygen. Following gas exchange measurement, leaves were sampled for biochemical and anatomical measure- ment. In transgenic tobacco plants, Rubisco content was 35% of that found in the wild-type tobacco, the CO2 assimilation rate was 50% of the wild-type rate and the chlorophyll content was unaltered. While leaf mass per unit leaf area of transgenic tobacco was 82% of that of the wild-type, differences in leaf thickness and surface area of mesophyll cells exposed to intercellular airspace per unit leaf area (Smes) were small (92 and 87% of wild-type, respectively). Leaves grown in the growth cabinet under high daily irradiance were thicker (63%), had a greater Smes (41%) due to the development of thicker palisade tissue, had higher photosynthetic capacity (27%) and contained more chlorophyll (58%) and Rubisco (77%), than leaves from plants grown in the glasshouse. Irrespective of genotype or growth environment, CO2 transfer conductance varied in proportion to surface area of chloroplasts exposed to intercellular airspaces. While the method for calculating CO2 transfer conductance could not distinguish between limitations due to the gas or liquid phases, there was no reduction in CO2 transfer conductance associated with more closely packed cells, thicker leaves, nor with increasing chloroplast thickness in tobacco.

Effects of Ambient CO2 Concentration on Growth and Nitrogen Use in Tobacco (Nicotiana tabacum) Plants Transformed with an Antisense Gene to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

Growth of the R1 progeny of a tobacco plant (Nicotiana tabacum) transformed with an antisense gene to the small subunit of ribulose-1,5-carboxylase/oxygenase (Rubisco) was analyzed under 330 and 930 [mu]bar of CO2, at an irradiance of 1000 [mu]mol quanta m-2 s-1. Rubisco activity was reduced to 30 to 50% and 13 to 18% of that in the wild type when one and two copies of the antisense gene, respectively, were present in the genome, whereas null plants and wild-type plants had similar phenotypes. At 330 [mu]bar of CO2 all antisense plants were smaller than the wild type. There was no indication that Rubisco is present in excess in the wild type with respect to growth under high light. Raising ambient CO2 pressure to 930 [mu]bar caused plants with one copy of the DNA transferred from plasmid to plant genome to achieve the same size as the wild type at 330 [mu]bar, but plants with two copies remained smaller. Differences in final size were due mostly to early differences in relative rate of leaf area expansion (m2 m-2 d-1) or of biomass accumulation (g g-1 d-1): within less than 2 weeks after germination relative growth rates reached a steady-state value similar for all plants. Plants with greater carboxylation rates were characterized by a higher ratio of leaf carbon to leaf area, and at later stages, they were characterized also by a relatively greater allocation of structural and nonstructural carbon to roots versus leaves. However, these changes per se did not appear to be causing the long-term insensitivity of relative growth rates to variations in carboxylation rate. Nor was this insensitivity due to feedback inhibition of photosynthesis in leaves grown at high partial pressure of CO2 in the air (pa) or with high Rubisco activity, even when the amount of starch approached 40% of leaf dry weight. We propose that other intrinsic rate-limiting processes that are independent of carbohydrate supply were involved. Under plentiful nitrogen supply, reduction in the amount of nitrogen invested in Rubisco was more than compensated for by an increase in leaf nitrate. Nitrogen content of organic matter, excluding Rubisco, was unaffected by the antisense gene. In contrast, it was systematically lower at elevated pa than at normal pa. Combined with the positive effects of pa on growth, this resulted in the single-dose antisense plants growing as fast at 930 [mu]bar of CO2 as the wild-type plants at 330 [mu]bar of CO2 but at a lower organic nitrogen cost.

The relationship between CO2-assimilation rate, Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO2-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r1 progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO2 supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO2-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO2-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO2-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase Deficiency Delays Senescence of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase but Progressively Impairs Its Catalysis during Tobacco Leaf Development

Transgenic tobacco (Nicotiana tabacum L. cv W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) activase grew more slowly than wild-type plants in a CO2-enriched atmosphere, but eventually attained the same height and number of leaves. Compared with the wild type, the anti-activase plants had reduced CO2 assimilation rates, normal contents of chlorophyll and soluble leaf protein, and much higher Rubisco contents, particularly in older leaves. Activase deficiency greatly delayed the usual developmental decline in Rubisco content seen in wild-type leaves. This effect was much less obvious in another transgenic tobacco with an antisense gene directed against chloroplast-located glyceraldehyde-3-phosphate dehydrogenase, which also had reduced photosynthetic rates and delayed development. Although Rubisco carbamylation was reduced in the anti-activase plants, the reduction was not sufficient to explain the reduced photosynthetic rate of older anti-activase leaves. Instead, up to a 10-fold reduction in the catalytic turnover rate of carbamylated Rubisco in vivo appeared to be the main cause. Slower catalytic turnover by carbamylated Rubisco was particularly obvious in high-CO2-grown leaves but was also detectable in air-grown leaves. Rubisco activity measured immediately after rapid extraction of anti-activase leaves was not much less than that predicted from its degree of carbamylation, ruling out slow release of an inhibitor from carbamylated sites as a major cause of the phenomenon. Nor could substrate scarcity or product inhibition account for the impairment. We conclude that activase must have a role in vivo, direct or indirect, in promoting the activity of carbamylated Rubisco in addition to its role in promoting carbamylation.

Effects of mutations at residue 309 of the large subunit of ribulosebisphosphate carboxylase from Synechococcus PCC 6301

Previous studies [G. S. Hudson et al. (1989) J. Biol. Chem. 265, 808-814] showed that the faster turnover rates and lower affinities for CO2 of ribulosebisphosphate carboxylase/oxygenases from C4 plants, compared to C3 and C3/C4 plants, were specified by the chloroplast-encoded large subunits. In pairs of closely related C3 and C4 species from three genera, these kinetic changes were accompanied by only three to six amino acid residue substitutions, depending on the genus. None of these substitutions occurred near the active site and only one, 309Met (C3) to Ile (C4), was common to all three genera. Unlike the plant carboxylases, the highly homologous enzyme from the cyanobacterium Synechococcus PCC 6301 folds and assembles properly when its rbcL and rbcS genes are coexpressed in Escherichia coli. Furthermore, the cyanobacterial enzyme has Ile at position 309 of the large subunit, a high turnover number, and a poor affinity for CO2. 309Ile was replaced with Met and several other residues by site-directed mutagenesis of the cyanobacterial rbcL. Met and Leu were tolerated at this position with no alteration in the kinetic or structural properties of the assembled holoenzyme. However, substitution with Val, Gly, Trp, or Arg prevented the assembly of the subunits. The indifference to Met or Ile at this position, as well as the tolerance for Leu which is not observed with any natural ribulosebisphosphate carboxylase, leads to the conclusion that either the 309Met/Ile substitution has no effect on the kinetic properties of the plant enzyme, despite the correlation apparent in previous studies, or the cyanobacterial enzyme is sufficiently different from the plant enzyme in other respects that the influence of residue 309 is masked.

Two promoters control the aroH gene of Escherichia coli

The aroH gene from Escherichia coli encodes 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (Trp), one of three isoenzymes which catalyse the first committed step in the biosynthesis of aromatic amino acids and vitamins. S1 mapping and primer extension analysis of in vivo transcripts revealed the presence of two nonoverlapping promoters for aroH. The more distal of these has been described previously and is negatively regulated by the trp repressor. The second promoter is active under conditions of growth in rich medium, and may be involved in ensuring sufficient levels of precursors for the biosynthesis of aromatic vitamins under these growth conditions.

Rubisco Catalysis In Vitro and In Vivo

Studies of the mechanisms and regulation of photosynthesis in higher plants can benefit from detailed comparisons between the properties of isolated proteins and inferences about them derived from physiological measurements with whole leaves. This approach allows the increasing understanding of molecular details provided by the reductionist approach to be tested and integrated so that the relative importance of the various details can be assessed.

Directed Mutagenesis of the Large Subunit of Tobacco Rubisco Assessed In Vivo

Mutational analyses of the CO2-fixing enzyme Rubisco from higher plants has been hindered by the inability to fold and assemble the enzyme correctly in heterologous expression systems. Nevertheless, a wealth of information exists about Rubisco’s structure and function (1,2). Development of a method for transforming the chloroplast genome (plastome) (3) provides an avenue for using this knowledge to manipulate the higher plant enzyme. Chloroplast transformation occurs via homologous recombination, thereby allowing site directed mutagenesis of chloroplast genes, such as the rbcL gene for the large subunit of Rubisco.

The Chloroplast Genes Encoding CF0 Subunits of ATP Synthase

The H+-ATP synthase of the chloroplast thylakoid membranes is essential for electron transport and photophosphorylation during photosynthesis. It has an F1F0 structure analogous to the proton-translocating ATP synthases of bacteria and mitochondria, that is, a proton pore sector (CF0) within the thylakoid membrane and an ATPase moiety (CF1) protruding into the stroma. In this paper, we examine the composition and predicted structure of the CF0 as deduced from three genes for CF0 subunits and from data for the equivalent proteins from E.coli and mitochondria.