T. John Andrews

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.

Correlations between oxygen isotope ratios of wood constituents of Quercus and Pinus samples from around the world

The oxygen isotope compositions of three wood constituents (the solvent-extractable portion, lignin and α-cellulose) were measured for samples collected from Quercus and Pinus trees around the world. Among Pinus samples all wood constituents were positively related to modelled δ18O of source water at the site, while among Quercus samples whole wood, lignin and α-cellulose showed positive relationships. The data support the hypothesis that many oxygen atoms in lignin exchanged with unenriched stem water during synthesis, rather than retaining the full isotopic signal from the molecular oxygen added during hydroxylation of the aromatic ring. The data also suggest that extraction of α-cellulose from wood samples is unnecessary for isotope studies looking at correlations with site parameters.

Faster Rubisco Is the Key to Superior Nitrogen-Use Efficiency in NADP-Malic Enzyme Relative to NAD-Malic Enzyme C4 Grasses

In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (kcat; 3.8 versus 5.7 s−1 at 25°C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)−1 in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster kcat.

Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants.

Transgenic manipulation of the photosynthetic CO2-fixing enzyme, ribulose bisphosphate carboxylase/oxygenase (Rubisco) in higher plants provides a very specific means of testing theories about photosynthesis and its regulation. It also encourages prospects for radically improving the efficiencies with which photosynthesis and plants use the basic resources of light, water, and nutrients. Manipulation was once limited to variation of the leafs total content of Rubisco by transforming the nucleus with antisense genes directed at the small subunit. More recently, technology for transforming the small genome of the plastid of tobacco has enabled much more precise manipulation and replacement of the plastome-encoded large subunit. Engineered changes in Rubiscos properties in vivo are reflected as profound changes in the photosynthetic gas-exchange properties of the leaves and the growth requirements of the plants. Unpredictable expression of plastid transgenes and assembly requirements of some foreign Rubiscos that are not satisfied in higher-plant plastids provide challenges for future research.

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.

The Gene for the Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Small Subunit Relocated to the Plastid Genome of Tobacco Directs the Synthesis of Small Subunits That Assemble into Rubisco

To assess the extent to which a nuclear gene for a chloroplast protein retained the ability to be expressed in its presumed preendosymbiotic location, we relocated the RbcS gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to the tobacco plastid genome. Plastid RbcS transgenes, both with and without the transit presequence, were equipped with 3′ hepta-histidine–encoding sequences and psbA promoter and terminator elements. Both transgenes were transcribed abundantly, and their products were translated into small subunit polypeptides that folded correctly and assembled into the Rubisco hexadecamer. When present, either the transit presequence was not translated or the transit peptide was cleaved completely. After assembly into Rubisco, transplastomic small subunits were relatively stable. The hepta-histidine sequence fused to the C terminus of a single small subunit was sufficient for isolation of the whole Rubisco hexadecamer by Ni2+ chelation. Small subunits produced by the plastid transgenes were not abundant, never exceeding ∼1% of the total small subunits, and they differed from cytoplasmically synthesized small subunits in their N-terminal modifications. The scarcity of transplastomic small subunits might be caused by inefficient translation or assembly.

Substrate isomerization inhibits ribulosebisphospate carboxylase‐oxygenase during catalysis

The inhibition of purified spinach ribulosebisphosphate carboxylase‐oxygenase which occurs progressively during catalysis in vitro is caused by accumulation of at least two tight‐binding inhibitors at the catalytic site. Reduction of these inhibitors with NaB3H4, followed by dephosphorylation, produced a mixture of xylitol and arabinitol, thus identifying one of them as D‐xylulose 1,5‐bisphosphate. It was formed during carboxylation, presumably by a stereochemically incorrect reprotonation of the 2,3‐enediolate intermediate bound at the catalytic site. Under the conditions used, this epimerization occurred approximately once for every 400 carboxylation turnovers. Another inhibitor may be 3‐keto‐D‐arabinitol 1,5‐bisphosphate which would also be formed by misprotonation of the enediolate intermediate, but at C‐2 rather than at C‐3.

Rubisco: Assembly and Mechanism

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco, E.C. 4.1.1.39) is unique to photosynthetic metabolism. Two intensively studied aspects of Rubisco physiology are covered in this chapter, its post-translational assembly and its mechanism of action. Bacterial Rubisco can be assembled in vitro and in bacterial hosts but, as yet, assembly in vitro of higher-plant Rubiscos has not been reported. This focuses attention on the assembly pathway for higher plant Rubisco, which has been known for some time to be related to the presence of molecular chaperones in chloroplasts. Analysis of mutants, transformation of plants and bacteria with chloroplast chaperones, and the development of in vitro translation and assembly systems based on chloroplast extracts, have been directed at resolving this problem. It appears from these data that certain bacterial chaperones do not interfere with the assembly of higher plant Rubisco. As in cyanobacterial systems, the absence of S subunits leads to the accumulation of L8-like particles whose subunits can later be recruited to form Rubisco. Subtle differences between the way S subunits assemble with higher-plant and cyanobacterial L8-like particles suggest that this process may be concerted with assembly of L8 in the case of the higher-plant enzyme. The catalytic mechanism of Rubisco depends on two co-factors; a divalent metal ion, usually Mg2+ and a CO2 molecule that carbamylates a specific lysyl residue, K201, in the active site. This carbamate plays a crucial role in initiating catalysis by abstracting the C3 proton of ribulose bisphosphate and it may also act as a general-base catalyst for succeeding steps. Sofar, Rubisco’s use of a carbamate as a base appears to be unique among enzymes. The catalytic sequences of both the carboxylation reaction, and the oxygenation reaction that competes with it, proceed through multiple steps, each of a complexity rivaling that of the complete reaction of many other enzymes. The structure of the active site must change subtly between steps. Selectivity between CO2 and O2, of paramount importance to photosynthetic efficiency, is determined by the relative reactivity of the enediol(ate) form of the substrate for the two gases.

The Relationship between Side Reactions and Slow Inhibition of Ribulose-bisphosphate Carboxylase Revealed by a Loop 6 Mutant of the Tobacco Enzyme

The first directed mutant of a higher plant ribulose-bisphosphate carboxylase/oxygenase (Rubisco), constructed by chloroplast transformation, is catalytically impaired but still able to support the plants photosynthesis and growth (Whitney, S. M., von Caemmerer, S., Hudson, G. S., and Andrews, T. J. (1999) Plant Physiol. 121, 579–588). This mutant enzyme has a Leu to Val substitution at residue 335 in the flexible loop 6 of the large subunit, which closes over the substrate during catalysis. Its active site was intact, as judged by its barely impaired competency in the initial enolization step of the reaction sequence, and its ability to bind tightly the intermediate analog, 2′-carboxy-d-arabinitol-1,5-bisphosphate. Prompted by observations that the mutant enzyme displayed much less slow inhibition during catalysis in vitro than the wild type, its tendency to catalyze side reactions and its response to the slow inhibitor d-xylulose-1,5-bisphosphate were studied. The lessening in slow inhibition was not caused by reduced production of inhibitory side products. Except for pyruvate production, these reactions were strongly enhanced by the mutation, as was the ability to catalyze the carboxylation of d-xylulose-1,5-bisphosphate. Rather, reduced inhibition was the result of lessened sensitivity to these inhibitors. The slow isomerization phase that characterizes inhibition of the wild-type enzyme by d-xylulose-1,5-bisphosphate was completely eliminated by the mutation, and the mutant was more adept than the wild type in catalyzing the benzylic acid-type rearrangement of d-glycero-2,3-pentodiulose-1,5-bisphosphate (produced by oxidation of the substrate, d-ribulose-1,5-bisphosphate). These observations are consistent with increased flexibility of loop 6 induced by the mutation, and they reveal the underlying mechanisms by which the side reactions cause slow inhibition.

Characterisation of inorganic carbon fluxes, carbonic anhydrase(s) and ribulose-1,5-biphosphate carboxylase-oxygenase in the green unicellular alga Coccomyxa

Processes involved in the uptake and fixation of dissolved inorganic carbon (DIC) were characterised for Coccomyxa, the green algal primary photobiont of the lichen Peltigera aphthosa and compared with the freeliving alga Chlamydomonas reinhardtii Dangeard (WT cc 125+). A mass-spectrometer disequilibrium technique was used to quantify fluxes of both HCOinf3sup−and CO2 in the two algae, while activities of carbonic anhydrases (CAs) were examined in intact cells by measuring 18O exchange from doubly labelled CO2 (13C18O18O) to water and by using CA inhibitors. The CO2-fixation kinetics of intact Coccomyxa cells were also compared with the carboxylation efficiency of its isolated and purified primary carboxylating enzyme, ribulose-1,5-bisphosphate carboxylaseoxygenase (Rubisco). The two algae were found to be significantly different in their modes of acquiring CO2 for photosynthesis. Chlamydomonas was able to actively transport both HCOinf3sup−and CO2 from the external medium, while Coccomyxa clearly favoured CO2 as a substrate. Both algae were found to possess external as well as internal CAs, but the relative amounts of these as well as their overall significance for the functioning of photosynthesis differed. In Coccomyxa, the internal CA activity was significantly higher than in Chlamydomonas and also predominated over the external activity. In Chlamydomonas, both transport and fixation of DIC were severely inhibited by ethoxyzolamide, an inhibitor of external and internal CAs as well as the DIC-transporting system, while this inhibitor only caused a minor inhibition of photosynthesis in Coccomyxa. These results thus give strong support for earlier indirect observations of the absence of a CO2concentrating mechanism in Coccomyxa. In addition, Coccomyxa was found to possess a Rubisco with a higher carboxylation efficiency than Chlamydomonas, having a Km (CO2) of 12 +3 μM CO2 and a CO2/O2 specificity factor (Sc/o) of 83 +2, and it may hence be concluded that the absence of the CO2-concentrating mechanism is positively correlated with a more efficient Rubisco in this alga.