Stable Isotope Labeling and Quantification of Photosynthetic Metabolites.

Stable isotope labeling with 13CO2 coupled with mass spectrometry allows monitoring the incorporation of 13C into photosynthetic intermediates and is a powerful technique for the investigation of the metabolic dynamics of photosynthesis. We describe here a protocol for 13CO2 labeling of large leaved plants and of Arabidopsis thaliana rosette, and a method for quantitative mass spectrometry analyses to uncover the labeling pattern of Calvin-Benson cycle sucrose, and starch synthesis as well as carbon-concentrating mechanism metabolites.

NTRC and thioredoxins m1/m2 underpin the light acclimation of plants on proteome and metabolome levels.

During photosynthesis, plants must manage strong fluctuations in light availability on different time scales, leading to long-term acclimation and short-term responses. However, little is known about the regulation and coordination of these processes and the modulators involved. In this study, we used proteomics, metabolomics, and reverse genetics to investigate how different light environmental factors, such as intensity or variability, affect long-term and short-term acclimation responses of Arabidopsis (Arabidopsis thaliana) and the importance of the chloroplast redox network in their regulation. In the wild type, high light, but not fluctuating light, led to large quantitative changes in the proteome and metabolome, accompanied by increased photosynthetic dynamics and plant growth. This finding supports light intensity as a stronger driver for acclimation than variability. Deficiencies in NADPH-thioredoxin reductase C (NTRC) or thioredoxins m1/m2, but not thioredoxin f1, almost completely suppressed the re-engineering of the proteome and metabolome, with both the induction of proteins involved in stress and redox responses and the repression of those involved in cytosolic and plastid protein synthesis and translation being strongly attenuated. Moreover, the correlations of protein or metabolite levels with light intensity were severely disturbed, suggesting a general defect in the light-dependent acclimation response, resulting in impaired photosynthetic dynamics. These results indicate a previously unknown role of NTRC and thioredoxins m1/m2 in modulating light acclimation at proteome and metabolome levels to control dynamic light responses. NTRC, but not thioredoxins m1/m2 or f1, also improves short-term photosynthetic responses by balancing the Calvin-Benson cycle in fluctuating light.

Natural variation in metabolism of the Calvin-Benson cycle.

The Calvin-Benson cycle (CBC) evolved over 2 billion years ago but has been subject to massive selection due to falling atmospheric carbon dioxide, rising atmospheric oxygen and changing nutrient and water availability. In addition, large groups of organisms have evolved carbon-concentrating mechanisms (CCMs) that operate upstream of the CBC. Most previous studies of CBC diversity focused on Rubisco kinetics and regulation. Quantitative metabolite profiling provides a top-down strategy to uncover inter-species diversity in CBC operation. CBC profiles were recently published for twenty species including terrestrial C3 species, terrestrial C4 species that operate a biochemical CCM, and cyanobacteria and green algae that operate different types of biophysical CCM. Distinctive profiles were found for species with different modes of photosynthesis, revealing that evolution of the various CCMs was accompanied by co-evolution of the CBC. Diversity was also found between species that share the same mode of photosynthesis, reflecting lineage-dependent diversity of the CBC. Connectivity analysis uncovers constraints due to pathway and thermodynamic topology, and reveals that cross-species diversity in the CBC is driven by changes in the balance between regulated enzymes and in the balance between the CBC and the light reactions or end-product synthesis.

Multi-omics quantitative data of tomato fruit unveils regulation modes of least variable metabolites.

BACKGROUND: The composition of ripe fruits depends on various metabolites which content evolves greatly throughout fruit development and may be influenced by the environment. The corresponding metabolism regulations have been widely described in tomato during fruit growth and ripening. However, the regulation of other metabolites that do not show large changes in content have scarcely been studied. RESULTS: We analysed the metabolites of tomato fruits collected on different trusses during fruit development, using complementary analytical strategies. We identified the 22 least variable metabolites, based on their coefficients of variation. We first verified that they had a limited functional link with the least variable proteins and transcripts. We then posited that metabolite contents could be stabilized through complex regulations and combined their data with the quantitative proteome or transcriptome data, using sparse partial-least-square analyses. This showed shared regulations between several metabolites, which interestingly remained linked to early fruit development. We also examined regulations in specific metabolites using correlations with individual proteins and transcripts, which revealed that a stable metabolite does not always correlate with proteins and transcripts of its known related pathways. CONCLUSIONS: The regulation of the least variable metabolites was then interpreted regarding their roles as hubs in metabolic pathways or as signalling molecules.

CP12 fine-tunes the Calvin-Benson cycle and carbohydrate metabolism in cyanobacteria.

The regulatory protein CP12 can bind glyceraldehyde 3-phosphate dehydrogenase (GapDH) and phosphoribulokinase (PRK) in oxygenic phototrophs, thereby switching on and off the flux through the Calvin-Benson cycle (CBC) under light and dark conditions, respectively. However, it can be assumed that CP12 is also regulating CBC flux under further conditions associated with redox changes. To prove this hypothesis, the mutant Δcp12 of the model cyanobacterium Synechocystis sp. PCC 6803 was compared to wild type and different complementation strains. Fluorescence microscopy showed for the first time the in vivo kinetics of assembly and disassembly of the CP12-GapDH-PRK complex, which was absent in the mutant Δcp12. Metabolome analysis revealed differences in the contents of ribulose 1,5-bisphosphate and dihydroxyacetone phosphate, the products of the CP12-regulated enzymes GapDH and PRK, between wild type and mutant Δcp12 under changing CO2 conditions. Growth of Δcp12 was not affected at constant light under different inorganic carbon conditions, however, the addition of glucose inhibited growth in darkness as well as under diurnal conditions. The growth defect in the presence of glucose is associated with the inability of Δcp12 to utilize external glucose. These phenotypes could be complemented by ectopic expression of the native CP12 protein, however, expression of CP12 variants with missing redox-sensitive cysteine pairs only partly restored the growth with glucose. These experiments indicated that the loss of GapDH-inhibition via CP12 is more critical than PRK association. Measurements of the NAD(P)H oxidation revealed an impairment of light intensity-dependent redox state regulation in Δcp12. Collectively, our results indicate that CP12-dependent regulation of the CBC is crucial for metabolic adjustment under conditions leading to redox changes such as diurnal conditions, glucose addition, and different CO2 conditions in cyanobacteria.

Point mutations that boost aromatic amino acid production and CO2 assimilation in plants.

Aromatic compounds having unusual stability provide high-value chemicals and considerable promise for carbon storage. Terrestrial plants can convert atmospheric CO2 into diverse and abundant aromatic compounds. However, it is unclear how plants control the shikimate pathway that connects the photosynthetic carbon fixation with the biosynthesis of aromatic amino acids, the major precursors of plant aromatic natural products. This study identified suppressor of tyra2 (sota) mutations that deregulate the first step in the plant shikimate pathway by alleviating multiple effector-mediated feedback regulation in Arabidopsis thaliana. The sota mutant plants showed hyperaccumulation of aromatic amino acids accompanied by up to a 30% increase in net CO2 assimilation. The identified mutations can be used to enhance plant-based, sustainable conversion of atmospheric CO2 to high-energy and high-value aromatic compounds.

13CO2 labeling kinetics in maize reveal impaired efficiency of C4 photosynthesis under low irradiance.

C4 photosynthesis allows faster photosynthetic rates and higher water and nitrogen use efficiency than C3 photosynthesis, but at the cost of lower quantum yield due to the energy requirement of its biochemical carbon concentration mechanism. It has also been suspected that its operation may be impaired in low irradiance. To investigate fluxes under moderate and low irradiance, maize (Zea mays) was grown at 550 µmol photons m-2 s-l and 13CO2 pulse-labeling was performed at growth irradiance or several hours after transfer to 160 µmol photons m-2 s-1. Analysis by liquid chromatography/tandem mass spectrometry or gas chromatography/mass spectrometry provided information about pool size and labeling kinetics for 32 metabolites and allowed estimation of flux at many steps in C4 photosynthesis. The results highlighted several sources of inefficiency in low light. These included excess flux at phosphoenolpyruvate carboxylase, restriction of decarboxylation by NADP-malic enzyme, and a shift to increased CO2 incorporation into aspartate, less effective use of metabolite pools to drive intercellular shuttles, and higher relative and absolute rates of photorespiration. The latter provides evidence for a lower bundle sheath CO2 concentration in low irradiance, implying that operation of the CO2 concentration mechanism is impaired in this condition. The analyses also revealed rapid exchange of carbon between the Calvin-Benson cycle and the CO2-concentration shuttle, which allows rapid adjustment of the balance between CO2 concentration and assimilation, and accumulation of large amounts of photorespiratory intermediates in low light that provides a major carbon reservoir to build up C4 metabolite pools when irradiance increases.

Interactions Between Carbon Metabolism and Photosynthetic Electron Transport in a Chlamydomonas reinhardtii Mutant Without CO2 Fixation by RuBisCO.

A Chlamydomonas reinhardtii RuBisCO-less mutant, ΔrbcL, was used to study carbohydrate metabolism without fixation of atmospheric carbon. The regulatory mechanism(s) that control linear electron flow, known as "photosynthetic control," are amplified in ΔrbcL at the onset of illumination. With the aim to understand the metabolites that control this regulatory response, we have correlated the kinetics of primary carbon metabolites to chlorophyll fluorescence induction curves. We identify that ΔrbcL in the absence of acetate generates adenosine triphosphate (ATP) via photosynthetic electron transfer reactions. Also, metabolites of the Calvin Benson Bassham (CBB) cycle are responsive to the light. Indeed, ribulose 1,5-bisphosphate (RuBP), the last intermediate before carboxylation by Ribulose-1,5-bisphosphate carboxylase-oxygenase, accumulates significantly with time, and CBB cycle intermediates for RuBP regeneration, dihydroxyacetone phosphate (DHAP), pentose phosphates and ribose-5-phosphate (R5P) are rapidly accumulated in the first seconds of illumination, then consumed, showing that although the CBB is blocked, these enzymes are still transiently active. In opposition, in the presence of acetate, consumption of CBB cycle intermediates is strongly diminished, suggesting that the link between light and primary carbon metabolism is almost lost. Phosphorylated hexoses and starch accumulate significantly. We show that acetate uptake results in heterotrophic metabolism dominating phototrophic metabolism, with glyoxylate and tricarboxylic acid (TCA) cycle intermediates being the most highly represented metabolites, specifically succinate and malate. These findings allow us to hypothesize which metabolites and metabolic pathways are relevant to the upregulation of processes like cyclic electron flow that are implicated in photosynthetic control mechanisms.

Rising rates of starch degradation during daytime and trehalose 6-phosphate optimize carbon availability.

Many plants, including Arabidopsis (Arabidopsis thaliana), accumulate starch in the light and remobilize it to support maintenance and growth at night. Starch synthesis and degradation are usually viewed as temporally separate processes. Recently, we reported that starch is also degraded in the light. Degradation rates are generally low early in the day but rise with time. Here, we show that the rate of degradation in the light depends on time relative to dawn rather than dusk. We also show that degradation in the light is inhibited by trehalose 6-phosphate, a signal for sucrose availability. The observed responses of degradation in the light can be simulated by a skeletal model in which the rate of degradation is a function of starch content divided by time remaining until dawn. The fit is improved by extension to include feedback inhibition of starch degradation by trehalose 6-phosphate. We also investigate possible functions of simultaneous starch synthesis and degradation in the light, using empirically parameterized models and experimental approaches. The idea that this cycle buffers growth against falling rates of photosynthesis at twilight is supported by data showing that rates of protein and cell wall synthesis remain high during a simulated dusk twilight. Degradation of starch in the light may also counter over-accumulation of starch in long photoperiods and stabilize signaling around dusk. We conclude that starch degradation in the light is regulated by mechanisms similar to those that operate at night and is important for stabilizing carbon availability and signaling, thus optimizing growth in natural light conditions.

Sucrose synthases are not involved in starch synthesis in Arabidopsis leaves.

Many plants accumulate transitory starch reserves in their leaves during the day to buffer their carbohydrate supply against fluctuating light conditions, and to provide carbon and energy for survival at night. It is universally accepted that transitory starch is synthesized from ADP-glucose (ADPG) in the chloroplasts. However, the consensus that ADPG is made in the chloroplasts by ADPG pyrophosphorylase has been challenged by a controversial proposal that ADPG is made primarily in the cytosol, probably by sucrose synthase (SUS), and then imported into the chloroplasts. To resolve this long-standing controversy, we critically re-examined the experimental evidence that appears to conflict with the consensus pathway. We show that when precautions are taken to avoid artefactual changes during leaf sampling, Arabidopsis thaliana mutants that lack SUS activity in mesophyll cells (quadruple sus1234) or have no SUS activity (sextuple sus123456) have wild-type levels of ADPG and starch, while ADPG is 20 times lower in the pgm and adg1 mutants that are blocked in the consensus chloroplastic pathway of starch synthesis. We conclude that the ADPG needed for starch synthesis in leaves is synthesized primarily by ADPG pyrophosphorylase in the chloroplasts.

Metabolic profiles in C3, C3-C4 intermediate, C4-like, and C4 species in the genus Flaveria.

C4 photosynthesis concentrates CO2 around Rubisco in the bundle sheath, favouring carboxylation over oxygenation and decreasing photorespiration. This complex trait evolved independently in >60 angiosperm lineages. Its evolution can be investigated in genera such as Flaveria (Asteraceae) that contain species representing intermediate stages between C3 and C4 photosynthesis. Previous studies have indicated that the first major change in metabolism probably involved relocation of glycine decarboxylase and photorespiratory CO2 release to the bundle sheath and establishment of intercellular shuttles to maintain nitrogen stoichiometry. This was followed by selection for a CO2-concentrating cycle between phosphoenolpyruvate carboxylase in the mesophyll and decarboxylases in the bundle sheath, and relocation of Rubisco to the latter. We have profiled 52 metabolites in nine Flaveria species and analysed 13CO2 labelling patterns for four species. Our results point to operation of multiple shuttles, including movement of aspartate in C3-C4 intermediates and a switch towards a malate/pyruvate shuttle in C4-like species. The malate/pyruvate shuttle increases from C4-like to complete C4 species, accompanied by a rise in ancillary organic acid pools. Our findings support current models and uncover further modifications of metabolism along the evolutionary path to C4 photosynthesis in the genus Flaveria.

Carbon flux through photosynthesis and central carbon metabolism show distinct patterns between algae, C3 and C4 plants.

Photosynthesis-related pathways are regarded as a promising avenue for crop improvement. Whilst empirical studies have shown that photosynthetic efficiency is higher in microalgae than in C3 or C4 crops, the underlying reasons remain unclear. Using a tailor-made microfluidics labelling system to supply 13CO2 at steady state, we investigated in vivo labelling kinetics in intermediates of the Calvin Benson cycle and sugar, starch, organic acid and amino acid synthesis pathways, and in protein and lipids, in Chlamydomonas reinhardtii, Chlorella sorokiniana and Chlorella ohadii, which is the fastest growing green alga on record. We estimated flux patterns in these algae and compared them with published and new data from C3 and C4 plants. Our analyses identify distinct flux patterns supporting faster growth in photosynthetic cells, with some of the algae exhibiting faster ribulose 1,5-bisphosphate regeneration and increased fluxes through the lower glycolysis and anaplerotic pathways towards the tricarboxylic acid cycle, amino acid synthesis and lipid synthesis than in higher plants.

Topology of the redox network during induction of photosynthesis as revealed by time-resolved proteomics in tobacco.

Photosynthetically produced electrons provide energy for various metabolic pathways, including carbon reduction. Four Calvin-Benson cycle enzymes and several other plastid proteins are activated in the light by reduction of specific cysteines via thioredoxins, a family of electron transporters operating in redox regulation networks. How does this network link the photosynthetic chain with cellular metabolism? Using a time-resolved redox proteomic method, we have investigated the redox network in vivo during the dark­to­low light transition. We show that redox states of some thioredoxins follow the photosynthetic linear electron transport rate. While some redox targets have kinetics compatible with an equilibrium with one thioredoxin (TRXf), reduction of other proteins shows specific kinetic limitations, allowing fine-tuning of each redox-regulated step of chloroplast metabolism. We identified five new redox-regulated proteins, including proteins involved in Mg2+ transport and 1O2 signaling. Our results provide a system-level functional view of the photosynthetic redox regulation network.

Targeted metabolite profiling as a top-down approach to uncover interspecies diversity and identify key conserved operational features in the Calvin-Benson cycle.

Improving photosynthesis is a promising avenue to increase crop yield. This will be aided by better understanding of natural variance in photosynthesis. Profiling of Calvin-Benson cycle (CBC) metabolites provides a top-down strategy to uncover interspecies diversity in CBC operation. In a study of four C4 and five C3 species, principal components analysis separated C4 species from C3 species and also separated different C4 species. These separations were driven by metabolites that reflect known species differences in their biochemistry and pathways. Unexpectedly, there was also considerable diversity between the C3 species. Falling atmospheric CO2 and changing temperature, nitrogen, and water availability have driven evolution of C4 photosynthesis in multiple lineages. We propose that analogous selective pressures drove lineage-dependent evolution of the CBC in C3 species. Examples of species-dependent variation include differences in the balance between the CBC and the light reactions, and in the balance between regulated steps in the CBC. Metabolite profiles also reveal conserved features including inactivation of enzymes in low irradiance, and maintenance of CBC metabolites at relatively high levels in the absence of net CO2 fixation. These features may be important for photosynthetic efficiency in low light, fluctuating irradiance, and when stomata close due to low water availability.

Assessing Protein Synthesis and Degradation Rates in Arabidopsis thaliana Using Amino Acid Analysis.

Plants continually synthesize and degrade proteins, for example, to adjust protein content during development or during adaptation to new environments. In order to estimate global protein synthesis and degradation rates in plants, we developed a relatively simple and inexpensive method using a combination of 13 CO2 labeling and mass spectrometry-based analyses. Arabidopsis thaliana plants are subjected to a 24-hr 13 CO2 pulse followed by a 4-day 12 CO2 chase. Soluble alanine and serine from total protein and glucose from cell wall material are analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and their 13 C enrichment (%) is estimated. The rate of protein synthesis during the 13 CO2 pulse experiment is defined as the rate of incorporation of labeled amino acids into proteins normalized by a correction factor for incomplete enrichment in free amino acid pools. The rate of protein degradation is estimated as the difference between the rate of protein synthesis and the relative growth rate calculated using the 13 C enrichment of glucose from cell wall material. Degradation rates are also estimated from the 12 CO2 pulse experiment. The following method description includes setting up and performing labeling experiments, preparation and measurement of samples, and calculation steps. In addition, an R script is provided for the calculations. 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Setting up the 13 CO2 labeling system and stable isotope labeling of Arabidopsis thaliana rosette leaves Basic Protocol 2: Extraction of soluble amino acids for GC-TOF-MS analysis Basic Protocol 3: Preparation of amino acids from total protein for GC-TOF-MS analysis Basic Protocol 4: Preparation of sugars from cell wall material for GC-TOF-MS analysis Basis Protocol 5: GC-TOF-MS analysis of 13 C-labeled samples and estimation of 13 C enrichment (%) Basis Protocol 6: Estimation of protein synthesis and degradation rates.

Prunus Hexokinase 3 genes alter primary C-metabolism and promote drought and salt stress tolerance in Arabidopsis transgenic plants.

Hexokinases (HXKs) and fructokinases (FRKs) are the only two families of enzymes in plants that have been identified as able to phosphorylate Glucose (Glc) and Fructose (Fru). Glc can only be phosphorylated in plants by HXKs, while Fru can be phosphorylated by either HXKs or FRKs. The various subcellular localizations of HXKs in plants indicate that they are involved in diverse functions, including anther dehiscence and pollen germination, stomatal closure in response to sugar levels, stomatal aperture and reducing transpiration. Its association with modulating programmed cell death, and responses to oxidative stress and pathogen infection (abiotic and biotic stresses) also have been reported. To extend our understanding about the function of HXK-like genes in the response of Prunus rootstocks to abiotic stress, we performed a detailed bioinformatic and functional analysis of hexokinase 3-like genes (HXK3s) from two Prunus rootstock genotypes, 'M.2624' (Prunus cerasifera Ehrh × P. munsoniana W.Wight & Hedrick) and 'M.F12/1' (P. avium L.), which are tolerant and sensitive to hypoxia stress, respectively. A previous large-scale transcriptome sequencing of roots of these rootstocks, showed that this HXK3-like gene that was highly induced in the tolerant genotype under hypoxia conditions. In silico analysis of gene promoters from M.2624 and M.F12/1 genotypes revealed regulatory elements that could explain differential transcriptional profiles of HXK3 genes. Subcellular localization was determinates by both bioinformatic prediction and expression of their protein fused to the green fluorescent protein (GFP) in protoplasts and transgenic plants of Arabidopsis. Both approaches showed that they are expressed in plastids. Metabolomics analysis of Arabidopsis plants ectopically expressing Prunus HXK3 genes revealed that content of several metabolites including phosphorylated sugars (G6P), starch and some metabolites associated with the TCA cycle were affected. These transgenic Arabidopsis plants showed improved tolerance to salt and drought stress under growth chamber conditions. Our results suggest that Prunus HXK3 is a potential candidate for enhancing tolerance to salt and drought stresses in stone fruit trees and other plants.

Regulation of Central Carbon and Amino Acid Metabolism in Plants.

Fluctuations in the prevailing environmental conditions, including light availability and intensity, CO2/O2 ratio, temperature, and nutrient or water supply, require rapid metabolic switches to maintain proper metabolism [...].

Installation of C4 photosynthetic pathway enzymes in rice using a single construct.

Introduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two-cell metabolic prototype for an NADP-malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP-malate dehydrogenase, pyruvate orthophosphate dikinase and NADP-malic enzyme from Zea mays, driven by cell-preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13 CO2 labelling demonstrated a 10-fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP-malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.

A Partial C4 Photosynthetic Biochemical Pathway in Rice.

Introduction of a C4 photosynthetic pathway into C3 rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2 at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously accumulates four of the core C4 photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME), and pyruvate phosphate dikinase (ZmPPDK). This led to enhanced enzyme activity and mild phenotypic perturbations but was largely neutral in its effects on photosynthetic rate. Measurements of the flux of 13CO2 through photosynthetic metabolism revealed a significant increase in the incorporation of 13C into malate, consistent with increased fixation of 13CO2 via PEP carboxylase in lines expressing the maize PEPC enzyme. However, there was no significant differences in labeling of 3-phosphoglycerate (3PGA) indicating that there was no carbon flux through NADP-ME into the Calvin-Benson cycle. There was also no significant difference in labeling of phosphoenolpyruvate (PEP) indicating that there was no carbon flux through PPDK. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein (OsGDCH) abundance led to a photosynthetic phenotype characteristic of the reduced OsGDCH line and higher labeling of malate, aspartate and citrate than in the quintuple line. There was evidence of 13C labeling of aspartate indicating 13CO2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C4 cycle, these results demonstrate for the first-time a partial flux through the carboxylation phase of NADP-ME C4 metabolism in transgenic rice containing two of the key metabolic steps in the C4 pathway.