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.

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.

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.

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.

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.

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.

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.

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.

The Photorespiratory Metabolite 2-Phosphoglycolate Regulates Photosynthesis and Starch Accumulation in Arabidopsis.

The Calvin-Benson cycle and its photorespiratory repair shunt are in charge of nearly all biological CO2 fixation on Earth. They interact functionally and via shared carbon flow on several levels including common metabolites, transcriptional regulation, and response to environmental changes. 2-Phosphoglycolate (2PG) is one of the shared metabolites and produced in large amounts by oxidative damage of the CO2 acceptor molecule ribulose 1,5-bisphosphate. It was anticipated early on, although never proven, that 2PG could also be a regulatory metabolite that modulates central carbon metabolism by inhibition of triose-phosphate isomerase. Here, we examined this hypothesis using transgenic Arabidopsis thaliana lines with varying activities of the 2PG-degrading enzyme, 2PG phosphatase, and analyzing the impact of this intervention on operation of the Calvin-Benson cycle and other central pathways, leaf carbohydrate metabolism, photosynthetic gas exchange, and growth. Our results demonstrate that 2PG feeds back on the Calvin-Benson cycle. It also alters the allocation of photosynthates between ribulose 1,5-bisphosphate regeneration and starch synthesis. 2PG mechanistically achieves this by inhibiting the Calvin-Benson cycle enzymes triose-phosphate isomerase and sedoheptulose 1,7-bisphosphate phosphatase. We suggest this may represent one of the control loops that sense the ratio of photorespiratory to photosynthetic carbon flux and in turn adjusts stomatal conductance, photosynthetic CO2 and photorespiratory O2 fixation, and starch synthesis in response to changes in the environment.

Purification and functional characterization of the vacuolar malate transporter tDT from Arabidopsis.

The exact transport characteristics of the vacuolar dicarboxylate transporter tDT from Arabidopsis are elusive. To overcome this limitation, we combined a range of experimental approaches comprising generation/analysis of tDT overexpressors, 13CO2 feeding and quantification of 13C enrichment, functional characterization of tDT in proteoliposomes, and electrophysiological studies on vacuoles. tdt knockout plants showed decreased malate and increased citrate concentrations in leaves during the diurnal light-dark rhythm and after onset of drought, when compared with wildtypes. Interestingly, under the latter two conditions, tDT overexpressors exhibited malate and citrate levels opposite to tdt knockout plants. Highly purified tDT protein transports malate and citrate in a 1:1 antiport mode. The apparent affinity for malate decreased with decreasing pH, whereas citrate affinity increased. This observation indicates that tDT exhibits a preference for dianion substrates, which is supported by electrophysiological analysis on intact vacuoles. tDT also accepts fumarate and succinate as substrates, but not α-ketoglutarate, gluconate, sulfate, or phosphate. Taking tDT as an example, we demonstrated that it is possible to reconstitute a vacuolar metabolite transporter functionally in proteoliposomes. The displayed, so far unknown counterexchange properties of tDT now explain the frequently observed reciprocal concentration changes of malate and citrate in leaves from various plant species. tDT from Arabidopsis is the first member of the well-known and widely present SLC13 group of carrier proteins, exhibiting an antiport mode of transport.

Relationship between irradiance and levels of Calvin-Benson cycle and other intermediates in the model eudicot Arabidopsis and the model monocot rice.

Metabolite profiles provide a top-down overview of the balance between the reactions in a pathway. We compared Calvin-Benson cycle (CBC) intermediate profiles in different conditions in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) to learn which features of CBC regulation differ and which are shared between these model eudicot and monocot C3 species. Principal component analysis revealed that CBC intermediate profiles follow different trajectories in Arabidopsis and rice as irradiance increases. The balance between subprocesses or reactions differed, with 3-phosphoglycerate reduction being favoured in Arabidopsis and ribulose 1,5-bisphosphate regeneration in rice, and sedoheptulose-1,7-bisphosphatase being favoured in Arabidopsis compared with fructose-1,6-bisphosphatase in rice. Photosynthesis rates rose in parallel with ribulose 1,5-bisphosphate levels in Arabidopsis, but not in rice. Nevertheless, some responses were shared between Arabidopsis and rice. Fructose 1,6-bisphosphate and sedoheptulose-1,7-bisphosphate were high or peaked at very low irradiance in both species. Incomplete activation of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase may prevent wasteful futile cycles in low irradiance. End-product synthesis is inhibited and high levels of CBC intermediates are maintained in low light or in low CO2 in both species. This may improve photosynthetic efficiency in fluctuating irradiance, and facilitate rapid CBC flux to support photorespiration and energy dissipation in low CO2.

Metabolite profiles reveal interspecific variation in operation of the Calvin-Benson cycle in both C4 and C3 plants.

Low atmospheric CO2 in recent geological time led to the evolution of carbon-concentrating mechanisms (CCMs) such as C4 photosynthesis in >65 terrestrial plant lineages. We know little about the impact of low CO2 on the Calvin-Benson cycle (CBC) in C3 species that did not evolve CCMs, representing >90% of terrestrial plant species. Metabolite profiling provides a top-down strategy to investigate the operational balance in a pathway. We profiled CBC intermediates in a panel of C4 (Zea mays, Setaria viridis, Flaveria bidentis, and F. trinervia) and C3 species (Oryza sativa, Triticium aestivum, Arabidopsis thaliana, Nicotiana tabacum, and Manihot esculenta). Principal component analysis revealed differences between C4 and C3 species that were driven by many metabolites, including lower ribulose 1,5-bisphosphate in C4 species. Strikingly, there was also considerable variation between C3 species. This was partly due to different chlorophyll and protein contents, but mainly to differences in relative levels of metabolites. Correlation analysis indicated that one contributory factor was the balance between fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, and Rubisco. Our results point to the CBC having experienced different evolutionary trajectories in C3 species since the ancestors of modern plant lineages diverged. They underline the need to understand CBC operation in a wide range of species.

Efficient 2-phosphoglycolate degradation is required to maintain carbon assimilation and allocation in the C4 plant Flaveria bidentis.

Photorespiration is indispensable for oxygenic photosynthesis since it detoxifies and recycles 2-phosphoglycolate (2PG), which is the primary oxygenation product of Rubisco. However, C4 plant species typically display very low rates of photorespiration due to their efficient biochemical carbon-concentrating mechanism. Thus, the broader relevance of photorespiration in these organisms remains unclear. In this study, we assessed the importance of a functional photorespiratory pathway in the C4 plant Flaveria bidentis using knockdown of the first enzymatic step, namely 2PG phosphatase (PGLP). The isolated RNAi lines showed strongly reduced amounts of PGLP protein, but distinct signs of the photorespiratory phenotype only emerged below 5% residual PGLP protein. Lines with this characteristic were stunted in growth, had strongly increased 2PG content, exhibited accelerated leaf senescence, and accumulated high amounts of branched-chain and aromatic amino acids, which are both characteristics of incipient carbon starvation. Oxygen-dependent gas-exchange measurements consistently suggested the cumulative impairment of ribulose-1,5-bisphosphate regeneration with increased photorespiratory pressure. Our results indicate that photorespiration is essential for maintaining high rates of C4 photosynthesis by preventing the 2PG-mediated inhibition of carbon utilization efficiency. However, considerably higher 2PG accumulation can be tolerated compared to equivalent lines of C3 plants due to the differential distribution of specific enzymatic steps between the mesophyll and bundle sheath cells.

Leaf Starch Turnover Occurs in Long Days and in Falling Light at the End of the Day.

We investigated whether starch degradation occurs at the same time as starch synthesis in Arabidopsis (Arabidopsis thaliana) leaves in the light. Starch accumulated in a linear fashion for about 12 h after dawn, then accumulation slowed and content plateaued. Following decreases in light intensity, the rate of accumulation of starch declined in proportion to the decline in photosynthesis if the decrease occurred <10 h after dawn, but accumulation ceased or loss of starch occurred if the same decrease in light intensity was imposed more than 10 h after dawn. These changes in starch accumulation patterns after prolonged periods in the light occurred at both high and low starch contents and were not related to time-dependent changes in either the rate of photosynthesis or the partitioning of assimilate between starch and Suc, as assessed from metabolite measurements and 14CO2 pulse experiments. Instead, measurements of incorporation of 13C from 13CO2 into starch and of levels of the starch degradation product maltose showed that substantial starch degradation occurred simultaneously with synthesis at time points >14 h after dawn and in response to decreases in light intensity that occurred >10 h after dawn. Starch measurements in circadian clock mutants suggested that the clock influences the timing of onset of degradation. We conclude that the propensity for leaf starch to be degraded increases with time after dawn. The importance of this phenomenon for efficient use of carbon for growth in long days and for prevention of starvation during twilight is discussed.

Impaired Malate and Fumarate Accumulation Due to the Mutation of the Tonoplast Dicarboxylate Transporter Has Little Effects on Stomatal Behavior.

Malate is a central metabolite involved in a multiplicity of plant metabolic pathways, being associated with mitochondrial metabolism and playing significant roles in stomatal movements. Vacuolar malate transport has been characterized at the molecular level and is performed by at least one carrier protein and two channels in Arabidopsis (Arabidopsis thaliana) vacuoles. The absence of the Arabidopsis tonoplast Dicarboxylate Transporter (tDT) in the tdt knockout mutant was associated previously with an impaired accumulation of malate and fumarate in leaves. Here, we investigated the consequences of this lower accumulation on stomatal behavior and photosynthetic capacity as well as its putative metabolic impacts. Neither the stomatal conductance nor the kinetic responses to dark, light, or high CO2 were highly affected in tdt plants. In addition, we did not observe any impact on stomatal aperture following incubation with abscisic acid, malate, or citrate. Furthermore, an effect on photosynthetic capacity was not observed in the mutant lines. However, leaf mitochondrial metabolism was affected in the tdt plants. Levels of the intermediates of the tricarboxylic acid cycle were altered, and increases in both light and dark respiration were observed. We conclude that manipulation of the tonoplastic organic acid transporter impacted mitochondrial metabolism, while the overall stomatal and photosynthetic capacity were unaffected.

Inhibition of plastid PPase and NTT leads to major changes in starch and tuber formation in potato.

The importance of a plastidial soluble inorganic pyrophosphatase (psPPase) and an ATP/ADP translocator (NTT) for starch composition and tuber formation in potato (Solanum tuberosum) was evaluated by individual and simultaneous down-regulation of the corresponding endogenous genes. Starch and amylose content of the transgenic lines were considerably lower, and granule size substantially smaller, with down-regulation of StpsPPase generating the most pronounced effects. Single-gene down-regulation of either StpsPPase or StNTT resulted in increased tuber numbers per plant and higher fresh weight yield. In contrast, when both genes were inhibited simultaneously, some lines developed only a few, small and distorted tubers. Analysis of metabolites revealed altered amounts of sugar intermediates, and a substantial increase in ADP-glucose content of the StpsPPase lines. Increased amounts of intermediates of vitamin C biosynthesis were also observed. This study suggests that hydrolysis of pyrophosphate (PPi) by action of a psPPase is vital for functional starch accumulation in potato tubers and that no additional mechanism for consuming, hydrolysing, or exporting PPi exists in the studied tissue. Additionally, it demonstrates that functional PPi hydrolysis in combination with efficient ATP import is essential for tuber formation and development.

The Interplay between Carbon Availability and Growth in Different Zones of the Growing Maize Leaf.

Plants assimilate carbon in their photosynthetic tissues in the light. However, carbon is required during the night and in nonphotosynthetic organs. It is therefore essential that plants manage their carbon resources spatially and temporally and coordinate growth with carbon availability. In growing maize (Zea mays) leaf blades, a defined developmental gradient facilitates analyses in the cell division, elongation, and mature zones. We investigated the responses of the metabolome and transcriptome and polysome loading, as a qualitative proxy for protein synthesis, at dusk, dawn, and 6, 14, and 24 h into an extended night, and tracked whole-leaf elongation over this time course. Starch and sugars are depleted by dawn in the mature zone, but only after an extension of the night in the elongation and division zones. Sucrose (Suc) recovers partially between 14 and 24 h into the extended night in the growth zones, but not the mature zone. The global metabolome and transcriptome track these zone-specific changes in Suc. Leaf elongation and polysome loading in the growth zones also remain high at dawn, decrease between 6 and 14 h into the extended night, and then partially recover, indicating that growth processes are determined by local carbon status. The level of Suc-signaling metabolite trehalose-6-phosphate, and the trehalose-6-phosphate:Suc ratio are much higher in growth than mature zones at dusk and dawn but fall in the extended night. Candidate genes were identified by searching for transcripts that show characteristic temporal response patterns or contrasting responses to carbon starvation in growth and mature zones.

Spatially resolved metabolic analysis reveals a central role for transcriptional control in carbon allocation to wood.

The contribution of transcriptional and post-transcriptional regulation to modifying carbon allocation to developing wood of trees is not well defined. To clarify the role of transcriptional regulation, the enzyme activity patterns of eight central primary metabolism enzymes across phloem, cambium, and developing wood of aspen (Populus tremula L.) were compared with transcript levels obtained by RNA sequencing of sequential stem sections from the same trees. Enzymes were selected on the basis of their importance in sugar metabolism and in linking primary metabolism to lignin biosynthesis. Existing enzyme assays were adapted to allow measurements from ~1 mm3 sections of dissected stem tissue. These experiments provided high spatial resolution of enzyme activity changes across different stages of wood development, and identified the gene transcripts probably responsible for these changes. In most cases, there was a clear positive relationship between transcripts and enzyme activity. During secondary cell wall formation, the increases in transcript levels and enzyme activities also matched with increased levels of glucose, fructose, hexose phosphates, and UDP-glucose, emphasizing an important role for transcriptional regulation in carbon allocation to developing aspen wood. These observations corroborate the efforts to increase carbon allocation to wood by engineering gene regulatory networks.

Metabolite pools and carbon flow during C4 photosynthesis in maize: 13CO2 labeling kinetics and cell type fractionation.

Worldwide efforts to engineer C4 photosynthesis into C3 crops require a deep understanding of how this complex pathway operates. CO2 is incorporated into four-carbon metabolites in the mesophyll, which move to the bundle sheath where they are decarboxylated to concentrate CO2 around RuBisCO. We performed dynamic 13CO2 labeling in maize to analyze C flow in C4 photosynthesis. The overall labeling kinetics reflected the topology of C4 photosynthesis. Analyses of cell-specific labeling patterns after fractionation to enrich bundle sheath and mesophyll cells revealed concentration gradients to drive intercellular diffusion of malate, but not pyruvate, in the major CO2-concentrating shuttle. They also revealed intercellular concentration gradients of aspartate, alanine, and phosphenolpyruvate to drive a second phosphoenolpyruvate carboxykinase (PEPCK)-type shuttle, which carries 10-14% of the carbon into the bundle sheath. Gradients also exist to drive intercellular exchange of 3-phosphoglycerate and triose-phosphate. There is rapid carbon exchange between the Calvin-Benson cycle and the CO2-concentrating shuttle, equivalent to ~10% of carbon gain. In contrast, very little C leaks from the large pools of metabolites in the C concentration shuttle into respiratory metabolism. We postulate that the presence of multiple shuttles, alongside carbon transfer between them and the Calvin-Benson cycle, confers great flexibility in C4 photosynthesis.