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Another "green revolution" is needed for crop yields to meet demands for food. The international C(4) Rice Consortium is working toward introducing a higher-capacity photosynthetic mechanism--the C(4) pathway--into rice to increase yield. The goal is to identify the genes necessary to install C(4) photosynthesis in rice through different approaches, including genomic and transcriptional sequence comparisons and mutant screening.

Summary Ribulose bisphosphate carboxylase (Rubisco) activase catalyzes the activation of Rubisco in vivo. Activase antisense DNA mutants of tobacco have been generated to explore the control that activase exerts on the photosynthetic process. These mutants have up to 90% reductions in activase protein levels as a consequence of an inhibition of activase mRNA accumulation. It is shown that photosynthesis, measured as the rate of CO2 exchange (CER), is modestly decreased in plants exposed to high irradiances. The decreases in CER in the transgenic plants are accompanied by corresponding decreases in Rubisco activation, indicating that activase has a direct effect on photosynthetic rates in the antisense plants by influencing the activation state of Rubisco. It is concluded that in high light conditions, control of photosynthesis is largely shared between Rubisco and activase. Plant growth is also impaired in mutant plants that have severe reductions in activase. The inhibition of activase in the antisense plants does not have an impact on the accumulation of Rubisco large subunit or small subunit mRNAs or proteins. This indicates that the concerted expression of the genes for activase (Rca) and Rubisco (rbcL and rbcS) in response to light, developmental factors and circadian controls is not due to feedback regulation of rbcL or rbcS by the amount of activase protein.

When leaves are exposed to low temperature, sugars accumulate and transcription factors in the C-repeat binding factor ( CBF ) family are expressed, which, together with CBF independent pathways, are known to contribute to the cold acclimation process and an increase in freezing tolerance. What is not known, however, is whether expression of these cold-regulated genes can be induced systemically in response to a localized cold treatment. To address this, pre-existing, mature leaves of warm-grown Arabidopsis thaliana were exposed to a localized cold treatment (near 10 ° C) whilst conjoined newly developing leaves continued only to experience warmer temperatures. In initial experiments on wild-type A. thaliana (Col-0) using real-time reverse transcription–PCR (RT–PCR) we observed that some genes— including CBF genes, certain downstream cold-responsive ( COR ) targets and CBF -independent transcription factors— respond to a direct 9 ° C treatment of whole plants. In subsequent experiments, we found that the treatment of expanded leaves with temperatures near 10 ° C can induce cold-associated genes in conjoined warm-maintained tissues. CBF1 showed a particularly strong systemic response, although CBF -independent transcription factors also responded. Moreover, the localized cold treatment of A. thaliana (C24) plants with a luciferase reporter fused to the promoter region of KIN2 indicated that in warmmaintained leaves, KIN2 might respond to a systemic signal from remote, directly cold-treated leaves. Collectively, our study provides strong evidence that the processes involved in cold acclimation are partially mediated by a signal that acts systemically. This has the potential to act as an earlywarning system to enable developing leaves to cope better with the cold environment in which they are growing.

Cells associated with veins of petioles of C(3) tobacco possess high activities of the decarboxylase enzymes required in C(4) photosynthesis. It is not clear whether this is the case in other C(3) species, nor whether these enzymes provide precursors for specific biosynthetic pathways. Here, we investigate the activity of C(4) acid decarboxylases in the mid-vein of Arabidopsis, identify regulatory regions sufficient for this activity, and determine the impact of removing individual isoforms of each protein on mid-vein metabolite profiles. This showed that radiolabelled malate and bicarbonate fed to the xylem stream were incorporated into soluble and insoluble material in the mid-vein of Arabidopsis leaves. Compared with the leaf lamina, mid-veins possessed high activities of NADP-dependent malic enzyme (NADP-ME), NAD-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). Transcripts derived from both NAD-ME, one PCK and two of the four NADP-ME genes were detectable in these veinal cells. The promoters of each decarboxylase gene were sufficient for expression in mid-veins. Analysis of insertional mutants revealed that cytosolic NADP-ME2 is responsible for 80% of NADP-ME activity in mid-veins. Removing individual decarboxylases affected the abundance of amino acids derived from pyruvate and phosphoenolpyruvate. Reducing cytosolic NADP-ME activity preferentially affected the sugar content, whereas abolishing NAD-ME affected both the amino acid and the glucosamine content of mid-veins.

Cells associated with veins of C(3) species often contain significant amounts of chlorophyll, and radiotracer analysis shows that carbon present in the transpiration stream may be used for photosynthesis in these cells. It is not clear whether CO2 is also supplied to these cells close to veins via stomata, nor whether this veinal photosynthesis supplies carbon skeletons to particular metabolic pathways. In addition, it has not been possible to determine whether photosynthesis in cells close to veins of C(3) plants is quantitatively important for growth or fitness. To investigate the role of photosynthesis in cells in and around the veins of C(3) plants, we have trans-activated a hairpin construct to the chlorophyll synthase gene (CS) using an Arabidopsis thaliana enhancer trap line specific to veins. CS is responsible for addition of the phytol chain to the tetrapyrolle head group of chlorophyll, and, as a result of cell-specific trans-activation of the hairpin to CS, chlorophyll accumulation is reduced around veins. We use these plants to show that, under steady-state conditions, the extent to which CO2 is supplied to cells close to veins via stomata is limited. Fixation by minor veins of CO2 supplied to the xylem stream and the amount of specific metabolites associated with carbohydrate metabolism and the shikimate pathway were all reduced. In addition, an abundance of transcripts encoding components of pathways that generate phosphoenolpyruvate were altered. Leaf senescence, growth rate and seed size were all reduced in the lines with lower photosynthetic ability in veins and in cells close to veins.

The characteristics of sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity in leaves of Phaseolus vulgaris L. cv. Linden was studied in plants subjected to water stress and various CO2 and light treatments. When water was withheld for 3 days causing mild water stress (–0.9 MPa), the activity of SPS measured in crude extracts was reduced ca 50%. The effect of water stress was most evident when the enzyme was assayed with saturating amounts of its substrates fructose 6-phosphate and UDP glucose. Placing a water-stressed plant in an atmosphere containing 1% CO2 reversed the effect of water stress on SPS activity over 5 h even though the water stress was not relieved. Holding unstressed leaves in low CO2 partial pressure reduced the extractable activity of SPS. After 1 h of low CO2 treatment the effect of low CO2 could be reversed by 20 min of 5% CO2. However, after 24 h of low CO2 treatment, less SPS activity was recovered by the 20 min treatment. The cytosolic protein synthesis inhibitor cycloheximide prevented the slow recovery of SPS activity, but did not affect the rapid recovery of SPS. We conclude that the effect of water stress on SPS activity was a consequence of the inhibition of photosynthesis caused by stomatal closure. Responses of Phaseolus vulgaris SPS to light were similar to the response to low CO2 in that the effects were most pronounced under Vmax assay conditions. This is the first report of this type of light response of SPS in a dicotyledonous species.

The oxidative pentose phosphate pathway (OPPP) provides plants with important substrates for both primary and secondary metabolism via the oxidation of glucose-6-phosphate. The OPPP is also thought to generate large amounts of reducing power to drive various anabolic processes. In animals this major pathway is located within the cytoplasm of cells, but in plants its subcellular compartmentation is far from clear. Although several enzymes of the OPPP were demonstrated to have both cytosolic and plastidic counterparts, there is yet no evidence for a full set of functional enzymes in each compartment. We report here the isolation of two coding sequences from tomato (Lycopersicon esculentum L.) which encode phylogenetically distant sequences (ToTal1 and ToTal2) that putatively encode distinct plastidic TA isoforms. The kinetic characterization of ToTal1 revealed that, unlike other enzymes of the non-oxidative branch of the OPPP, ToTal1 does not follow a Michaelis-Menten mode of catalysis which has implications for its role in regulating carbon flux between primary and secondary metabolism. TA genes appear to be differentially regulated at the level of gene expression in plant tissues and in response to environmental factors which suggests that TA isoforms have a non-overlapping role for plant metabolism.

Proline is an important component of salt-stress responses of plants. In this study the role of proline as part of salt-stress signalling in the desert plant Pancratium maritimum L. was examined. The data showed that salt-stress brought about a reduction of the growth and protein content, particularly at 300 mM NaCl, that was significantly increased by exogenous proline. In the leaves, salt-stress up-regulated ubiquitin, a small protein targeting damaged proteins for degradation via the proteasome, up to 5-fold as detected by western blotting. This change was also affected by proline even in non-stressed leaves. However, salt-stress resulted in a decrease in the amount of ubiquitin-conjugates, particularly in the roots, and this effect was reversed by exogenous proline. Severe salt-stress resulted in an inhibition of the antioxidative enzymes catalase and peroxidase as revealed by spectrophotometric assays and activity gels, but the activity of these enzymes was also maintained significantly higher in the presence of proline. Salt-stress also up-regulated several dehydrin proteins, analysed by western blotting, even in non-stressed plants. It is concluded that proline improves the salt-tolerance of Pancratium maritimum L. by protecting the protein turnover machinery against stress-damage and up-regulating stress protective proteins.

Another “green revolution” is needed for crop yields to meet demands for food. The international C 4 Rice Consortium is working toward introducing a higher-capacity photosynthetic mechanism—the C 4 pathway—into rice to increase yield. The goal is to identify the genes necessary to install C 4 photosynthesis in rice through different approaches, including genomic and transcriptional sequence comparisons and mutant screening.

Photorespiration results from the oxygenase reaction catalysed by ribulose–1,5–bisphosphate carboxylase/oxygenase. In this reaction glycollate–2–phosphate is produced and subsequently metabolized in the photorespiratory pathway to form the Calvin cycle intermediate glycerate–3–phosphate. During this metabolic process, CO 2 and NH 3 are produced and ATP and reducing equivalents are consumed, thus making photorespiration a wasteful process. However, precisely because of this inefficiency, photorespiration could serve as an energy sink preventing the overreduction of the photosynthetic electron transport chain and photoinhibition, especially under stress conditions that lead to reduced rates of photosynthetic CO 2 assimilation. Furthermore, photorespiration provides metabolites for other metabolic processes, e.g. glycine for the synthesis of glutathione, which is also involved in stress protection. In this review, we describe the use of photorespiratory mutants to study the control and regulation of photorespiratory pathways. In addition, we discuss the possible role of photorespiration under stress conditions, such as drought, high salt concentrations and high light intensities encountered by alpine plants.

The importance of subzero temperature interactions with elevated CO2 on plant carbon metabolism has received rather little attention, despite their likely role in influencing future vegetation productivity and dynamics. Here we focused on the critical issues of CO2-enrichment effects on leaf-freezing temperatures, subsequent membrane damage, and recovery of the photosynthetic system. We show that growth in elevated CO2 (70 Pa) results in a substantial and significant (P < 0.01) increase (up to 4°C) in the ice nucleation temperature of leaves of Maidenhair tree (Ginkgo biloba), which was observed consistently throughout the 1999 growing season relative to their ambient CO2 (35 Pa) counterparts. We suggest that increased sensitivity of leaves to ice damage after growth in elevated CO2 provides an explanation for increased photoinhibition observed in the field early and late in the growing season when low nighttime temperatures are experienced. This new mechanism is proposed in addition to the earlier postulated explanation for this phenomenon involving a reduction in the rate of triose-P utilization owing to a decrease in the rate of carbohydrate export from the leaf.

A broad overview of the physiology and biochemistry of Rubisco is presented with a comparison of information obtained in vitro and in vivo. First a brief background to kinetic properties of Rubisco is given and Rubisco’s influence on photosynthetic metabolism is reviewed for C3, C4, CAM and C3-C4 species. The effect of environmental variables such as light and CO2 are considered for both short and longer term effects on the activity and abundance of Rubisco protein. Over the past few years experiments with transgenic plants with antisense RNA constructs to Rubisco, Rubisco activase and several of the PCR cycle enzymes have added new insights into Rubisco physiology. For example, transgenic tobacco with reduced amount of Rubisco has allowed the identification of environmental conditions where Rubisco exerts maximal control on photosynthesis. These plants have also been used to determine Rubisco kinetic constants in vivo. Transgenic plants with reduced amounts of Rubisco activase have been used to elucidate the role of activase in vivo.

The apparent Km value for inorganic phosphate (Pi) of the chloroplastic ATPase has been measured under different conditions of light intensity, uncoupler concentration and energy-transfer inhibitor concentration. Systematic changes in the values were observed which were similar to but not identical with reported changes in the Km for ADP under similar conditions (Quick, W.P. and Mills, J.D. (1987) Biochim. Biophys. Acta 893, 197–207). Both sets of experimental data could be simulated using a mathematical model based on: (a) Michaelis-Menten kinetics of the ATPase with respect to ADP and Pi; and (b) delocalised chemiosmotic energy coupling. We conclude that the changes observed in the Km are a result of systematic changes in the protonmotive force during the Km analysis.

The steady-state binding of [2- 3 H]ADP to thylakoid membrane protonmotive ATPase (CF 0 -CF 1 ) was studied using a quench technique similar to that developed by Strotmann, Bickel-Sandkotter and Shoshan (FEBS Lett. 101 (1979) 316–320). The amount of adenine nucleotide bound in the light and post-illumination dark period is shown to depend on the efficaency of uncoupling when the reaction is quenched. Previous observations that post-illumination binding of [2- 3 H]ADP displays a fast and a slow phase of binding were confirmed only when uncoupling efficiency was relatively low during quenching. When uncoupling efficiency is increased, the fast phase of dark [2- 3 H]ADP binding is abolished, and correspondingly more nucleotide appears to be bound upon quenching in the light. The previous assignment of the observed kinetic phases to different species of enzyme-nucleotide complex has been reassessed in the light of this new data.

The apparent Michaelis constant ( K m ) for ADP has been measured under various conditions of steady-state photophosphorylation in isolated thylakoid membranes. In addition, the steady-state ΔpH has been simultaneously estimated from the fluorescence quenching of 9-aminoacridine. The following results were obtained. (1) The standard procedure for estimating K m , by increasing the concentration of ADP, progressively lowered the steady-state ΔpH, thereby introducing an uncontrolled system variable into the K m analysis. This has the effect of lowering the apparent K m measured. (2) Lowering the light intensity lowered the observed K m , and addition of uncouplers increased the observed K m . The ability of uncouplers to increase K m was enhanced at lowered light intensities. In contrast, the effect of lowered light intensity on the observed K m was diminished and then reversed under progressively more uncoupled conditions. (3) The addition of energy-transfer inhibitors caused an increase in the observed K m for ADP. (4) All of the observations are qualitatively predicted by a mathematical model based on simple delocalised chemiosmotic energy coupling and Michaelis-Menten kinetics for the chloroplast ATPase with respect to ADP. It is concluded that the complex behaviour of the apparent K m for ADP under different conditions arises because ΔpH is an uncontrolled variable during the K m analysis and that the results are entirely consistent with a model of delocalised chemiosmotic energy coupling.

(1) The extent of thiol-modulation of chloroplastic CF0-CF1 in isolated barley protoplasts was determined by their capacity to hydrolyse ATP in a subsequently lysed assay system and was measured under conditions that vary the rate of CO2 fixation. CF0-CF1 activity could be separated from other ATPases by using the specific inhibitors triphenyltin and tentoxin. (2) Disruption of dark-adapted protoplasts resulted in little or no ATPase activity associated with CF0-CF1. Preillumination of the protoplasts induced CF0-CF1-dependent ATPase activity which was reversible in the dark. (3) Experiments using methyl viologen established that induction of CF0-CF1-dependent ATPase activity resulted from thiol-modulation of oxidised enzyme complexes by the endogenous thioredoxin system. (4) CF0-CF1 was shown to be rapidly thiol-modulated upon illumination ( t 1 2 ≈ 15 s ) well before steady-state rates of CO2-dependent O2 evolution were achieved. (5) The steady-state extent of light-induced thiol-modulation was shown to be light-saturated at 10 W · m−2, a light intensity far below that required to saturate the rate of CO2-dependent O2 evolution. (6) These results are consistent with the idea that thiol modulation of CF0-CF1 acts primarily as an on-off switch in vivo to prevent ATP hydrolysis in the dark.

There have been several reports that the apparent Michaelis Menton constant (Km) of the chloroplastic ATPase (CF0-CF1) for its substrate ADP varies according to the experimental conditions (1–3); this has also been observed in liver submitochondrial particles (4,5). Reduction of the rate of ATP synthesis by lowering the light intensity (1–3) or by adding an electron transport inhibitor (2) was shown to decrease the apparent Km for ADP. However, reduction of the rate of ATP synthesis using an uncoupler elevated the apparent Km for ADP (1,3). These results have been interpreted in several ways and used as evidence to support models of ATP synthesis involving a direct activation of CF0-CF1 by ΔμH+ (2,4,5) or by electron transport (1,3).