Increased α-ketoglutarate links the C3-C4 intermediate state to C4 photosynthesis in the genus Flaveria.

As a complex trait, C4 photosynthesis has multiple independent origins in evolution. Phylogenetic evidence and theoretical analysis suggest that C2 photosynthesis, which is driven by glycine decarboxylation in the bundle sheath cell, may function as a bridge from C3 to C4 photosynthesis. However, the exact molecular mechanism underlying the transition between C2 photosynthesis to C4 photosynthesis remains elusive. Here, we provide evidence suggesting a role of higher α-ketoglutarate (AKG) concentration during this transition. Metabolomic data of 12 Flaveria species, including multiple photosynthetic types, show that AKG concentration initially increased in the C3-C4 intermediate with a further increase in C4 species. Petiole feeding of AKG increases the concentrations of C4-related metabolites in C3-C4 and C4 species but not the activity of C4-related enzymes. Sequence analysis shows that glutamate synthase (Fd-GOGAT), which catalyzes the generation of glutamate using AKG, was under strong positive selection during the evolution of C4 photosynthesis. Simulations with a constraint-based model for C3-C4 intermediate further show that decreasing the activity of Fd-GOGAT facilitated the transition from a C2-dominant to a C4-dominant CO2 concentrating mechanism. All these results provide insight into the mechanistic switch from C3-C4 intermediate to C4 photosynthesis.

The Evolution of C4 Photosynthesis in Flaveria (Asteraceae): Insights from the Flaveria linearis Complex.

Flaveria is a leading model for C4 plant evolution due to the presence of a dozen C3-C4 intermediate species, many of which are associated with a phylogenetic complex centered around Flaveria linearis. To investigate C4 evolution in Flaveria, we updated the Flaveria phylogeny and evaluated gas exchange, starch δ13C, and activity of C4 cycle enzymes in 19 Flaveria species and 28 populations within the F. linearis complex. A principal component analysis identified six functional clusters: (1) C3, (2) sub-C2, (3) full C2, (4) enriched C2, (5) sub-C4, and (6) fully C4 species. The sub-C2 species lacked a functional C4 cycle, while a gradient was present in the C2 clusters from little to modest C4 cycle activity as indicated by δ13C and enzyme activities. Three Yucatan populations of F. linearis had photosynthetic CO2 compensation points equivalent to C4 plants but showed little evidence for an enhanced C4 cycle, indicating they have an optimized C2 pathway that recaptures all photorespired CO2 in the bundle sheath (BS) tissue. All C2 species had enhanced aspartate aminotransferase activity relative to C3 species and most had enhanced alanine aminotransferase activity. These aminotransferases form aspartate and alanine from glutamate and in doing so could help return photorespiratory nitrogen (N) from BS to mesophyll cells, preventing glutamate feedback onto photorespiratory N assimilation. Their use requires upregulation of parts of the C4 metabolic cycle to generate carbon skeletons to sustain N return to the mesophyll, and thus could facilitate the evolution of the full C4 photosynthetic pathway.

Evolution of gene regulatory network of C4 photosynthesis in the genus Flaveria reveals the evolutionary status of C3-C4 intermediate species.

C4 photosynthesis evolved from ancestral C3 photosynthesis by recruiting pre-existing genes to fulfill new functions. The enzymes and transporters required for the C4 metabolic pathway have been intensively studied and well documented; however, the transcription factors (TFs) that regulate these C4 metabolic genes are not yet well understood. In particular, how the TF regulatory network of C4 metabolic genes was rewired during the evolutionary process is unclear. Here, we constructed gene regulatory networks (GRNs) for four closely evolutionarily related species from the genus Flaveria, which represent four different evolutionary stages of C4 photosynthesis: C3 (F. robusta), type I C3-C4 (F. sonorensis), type II C3-C4 (F. ramosissima), and C4 (F. trinervia). Our results show that more than half of the co-regulatory relationships between TFs and core C4 metabolic genes are species specific. The counterparts of the C4 genes in C3 species were already co-regulated with photosynthesis-related genes, whereas the required TFs for C4 photosynthesis were recruited later. The TFs involved in C4 photosynthesis were widely recruited in the type I C3-C4 species; nevertheless, type II C3-C4 species showed a divergent GRN from C4 species. In line with these findings, a 13CO2 pulse-labeling experiment showed that the CO2 initially fixed into C4 acid was not directly released to the Calvin-Benson-Bassham cycle in the type II C3-C4 species. Therefore, our study uncovered dynamic changes in C4 genes and TF co-regulation during the evolutionary process; furthermore, we showed that the metabolic pathway of the type II C3-C4 species F. ramosissima represents an alternative evolutionary solution to the ammonia imbalance in C3-C4 intermediate species.

The evolution of stomatal traits along the trajectory toward C4 photosynthesis.

C4 photosynthesis optimizes plant carbon and water relations, allowing high photosynthetic rates with low stomatal conductance. Stomata have long been considered a part of the C4 syndrome. However, it remains unclear how stomatal traits evolved along the path from C3 to C4. Here, we examined stomata in the Flaveria genus, a model used for C4 evolutionary study. Comparative, transgenic, and semi-in vitro experiments were performed to study the molecular basis that underlies the changes of stomatal traits in C4 evolution. The evolution from C3 to C4 species is accompanied by a gradual rather than an abrupt change in stomatal traits. The initial change appears near the Type I intermediate stage. Co-evolution of the photosynthetic pathway and stomatal traits is supported. On the road to C4, stomata tend to be fewer in number but larger in size and stomatal density dominates changes in anatomical maximum stomatal conductance (gsmax). Reduction of FSTOMAGEN expression underlies decreased gsmax in Flaveria and likely occurs in other C4 lineages. Decreased gsmax contributes to the increase in intrinsic water-use efficiency in C4 evolution. This work highlights the stomatal traits in the current C4 evolutionary model. Our study provides insights into the pattern, mechanism, and role of stomatal evolution along the road toward C4.

Overexpression of cytoplasmic C4 Flaveria bidentis carbonic anhydrase in C3 Arabidopsis thaliana increases amino acids, photosynthetic potential, and biomass.

An important method to improve photosynthesis in C3 crops, such as rice and wheat, is to transfer efficient C4 characters to them. Here, cytosolic carbonic anhydrase (CA: ßCA3) of the C4 Flaveria bidentis (Fb) was overexpressed under the control of 35 S promoter in Arabidopsis thaliana, a C3 plant, to enhance its photosynthetic efficiency. Overexpression of CA resulted in a better supply of the substrate HCO3- for the endogenous phosphoenolpyruvate carboxylase in the cytosol of the overexpressers, and increased its activity for generating malate that feeds into the tricarboxylic acid cycle. This provided additional carbon skeleton for increased synthesis of amino acids aspartate, asparagine, glutamate, and glutamine. Increased amino acids contributed to higher protein content in the transgenics. Furthermore, expression of FbßCA3 in Arabidopsis led to a better growth due to expression of several genes leading to higher chlorophyll content, electron transport, and photosynthetic carbon assimilation in the transformants. Enhanced CO2 assimilation resulted in increased sugar and starch content, and plant dry weight. In addition, transgenic plants had lower stomatal conductance, reduced transpiration rate, and higher water-use efficiency. These results, taken together, show that expression of C4 CA in the cytosol of a C3 plant can indeed improve its photosynthetic capacity with enhanced water-use efficiency.

A scheme for C4 evolution derived from a comparative analysis of the closely related C3, C3-C4 intermediate, C4-like, and C4 species in the genus Flaveria.

KEY MESSAGE: A comparative analysis of the genus Flaveria showed a C4 evolutionary process in which the anatomical and metabolic features of C4 photosynthesis were gradually acquired through C3-C4 intermediate stages. C4 photosynthesis has been acquired in multiple lineages of angiosperms during evolution to suppress photorespiration. Crops that perform C4 photosynthesis exhibit high rates of CO2 assimilation and high grain production even under high-temperature in semiarid environments; therefore, engineering C4 photosynthesis in C3 plants is of great importance in the application field. The genus Flaveria contains a large number of C3, C3-C4 intermediate, C4-like, and C4 species, making it a good model genus to study the evolution of C4 photosynthesis, and these studies indicate the direction for C4 engineering. C4 photosynthesis was acquired gradually through the C3-C4 intermediate stage. First, a two-celled C2 cycle called C2 photosynthesis was acquired by localizing glycine decarboxylase activity in the mitochondria of bundle sheath cells. With the development of two-cell metabolism, anatomical features also changed. Next, the replacement of the two-celled C2 cycle by the two-celled C4 cycle was induced by the acquisition of cell-selective expression in addition to the upregulation of enzymes in the C4 cycle during the C3-C4 intermediate stage. This was supported by an increase in cyclic electron transport activity in response to an increase in the ATP/NADPH demand for metabolism. Suppression of the C3 cycle in mesophyll cells was induced after the functional establishment of the C4 cycle, and optimization of electron transport by suppressing the activity of photosystem II also occurred during the final phase of C4 evolution.

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.

Change in expression levels of NAD kinase-encoding genes in Flaveria species.

Nicotinamide adenine dinucleotides (NAD(H)) and NAD phosphates (NADP(H)) are electron carriers involved in redox reactions and metabolic processes in all organisms. NAD kinase (NADK) is the only enzyme that phosphorylates NAD+ into NADP+, using ATP as a phosphate donor. In NADP-dependent malic enzyme (NADP-ME)-type C4 photosynthesis, NADP(H) are required for dehydrogenation by NADP-dependent malate dehydrogenase (NADP-MDH) in mesophyll cells, and decarboxylation by NADP-ME in bundle sheath cells. In this study, we identified five NADK genes (FbNADK1a, 1b, 2a, 2b, and 3) from the C4 model species Flaveria bidentis. RNA-Seq database analysis revealed higher transcript abundance in one of the chloroplast-type NADK2 genes of C4F. bidentis (FbNADK2a). Comparative analysis of NADK activity in leaves of C3, C3-C4, and C4Flaveria showed that C4Flaveria (F. bidentis and F. trinervia) had higher NADK activity than the other photosynthetic-types of Flaveria. Taken together, our results suggest that chloroplastic NAD kinase appeared to increase in importance as C3 plants evolved into C4 plants in the genus Flaveria.

Dynamic changes of genome sizes and gradual gain of cell-specific distribution of C4 enzymes during C4 evolution in genus Flaveria.

C4 plants are believed to have evolved from C3 plants through various C3 -C4 intermediate stages in which a photorespiration-dependent CO2 concentration system known as C2 photosynthesis operates. Genes involved in the C4 cycle were thought to be recruited from orthologs present in C3 species and developed cell-specific expression during C4 evolution. To understand the process of establishing C4 photosynthesis, we performed whole-genome sequencing and investigated expression and mesophyll- or bundle-sheath-cell-specific localization of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), pyruvate, orthophosphate dikinase (PPDK) in C3 , C3 -C4 intermediate, C4 -like, and C4 Flaveria species. While genome sizes vary greatly, the number of predicted protein-coding genes was similar among C3 , C3 -C4 intermediate, C4 -like, and C4 Flaveria species. Cell-specific localization of the PEPC, NADP-ME, and PPDK transcripts was insignificant or weak in C3 -C4 intermediate species, whereas these transcripts were expressed cell-type specific in C4 -like species. These results showed that elevation of gene expression and cell-specific control of pre-existing C4 cycle genes in C3 species was involved in C4 evolution. Gene expression was gradually enhanced during C4 evolution, whereas cell-specific control was gained independently of quantitative transcriptional activation during evolution from C3 -C4 intermediate to C4 photosynthesis in genus Flaveria.

A single serine to alanine substitution decreases bicarbonate affinity of phosphoenolpyruvate carboxylase in C4Flaveria trinervia.

Phosphoenolpyruvate (PEP) carboxylase (PEPc) catalyzes the first committed step of C4 photosynthesis generating oxaloacetate from bicarbonate (HCO3-) and PEP. It is hypothesized that PEPc affinity for HCO3- has undergone selective pressure for a lower KHCO3 (Km for HCO3-) to increase the carbon flux entering the C4 cycle, particularly during conditions that limit CO2 availability. However, the decrease in KHCO3 has been hypothesized to cause an unavoidable increase in KPEP (Km for PEP). Therefore, the amino acid residue S774 in the C4 enzyme, which has been shown to increase KPEP, should lead to a decrease in KHCO3. Several studies reported the effect S774 has on KPEP; however, the influence of this amino acid substitution on KHCO3 has not been tested. To test these hypotheses, membrane-inlet mass spectrometry (MIMS) was used to measure the KHCO3 of the photosynthetic PEPc from the C4Flaveria trinervia and the non-photosynthetic PEPc from the C3F. pringlei. The cDNAs for these enzymes were overexpressed and purified from the PEPc-less PCR1 Escherichia coli strain. Our work in comparison with previous reports suggests that KHCO3 and KPEP are linked by specific amino acids, such as S774; however, these kinetic parameters respond differently to the tested allosteric regulators, malate and glucose-6-phosphate.

An rbcL mRNA-binding protein is associated with C3 to C4 evolution and light-induced production of Rubisco in Flaveria.

Nuclear-encoded RLSB protein binds chloroplastic rbcL mRNA encoding the Rubisco large subunit. RLSB is highly conserved across all groups of land plants and is associated with positive post-transcriptional regulation of rbcL expression. In C3 leaves, RLSB and Rubisco occur in all chlorenchyma cell chloroplasts, while in C4 leaves these accumulate only within bundle sheath (BS) chloroplasts. RLSB's role in rbcL expression makes modification of its localization a likely prerequisite for the evolutionary restriction of Rubisco to BS cells. Taking advantage of evolutionarily conserved RLSB orthologs in several C3, C3-C4, C4-like, and C4 photosynthetic types within the genus Flaveria, we show that low level RLSB sequence divergence and modification to BS specificity coincided with ontogeny of Rubisco specificity and Kranz anatomy during C3 to C4 evolution. In both C3 and C4 species, Rubisco production reflected RLSB production in all cell types, tissues, and conditions examined. Co-localization occurred only in photosynthetic tissues, and both proteins were co-ordinately induced by light at post-transcriptional levels. RLSB is currently the only mRNA-binding protein to be associated with rbcL gene regulation in any plant, with variations in sequence and acquisition of cell type specificity reflecting the progression of C4 evolution within the genus Flaveria.

Shared characteristics underpinning C4 leaf maturation derived from analysis of multiple C3 and C4 species of Flaveria.

Most terrestrial plants use C3 photosynthesis to fix carbon. In multiple plant lineages a modified system known as C4 photosynthesis has evolved. To better understand the molecular patterns associated with induction of C4 photosynthesis, the genus Flaveria that contains C3 and C4 species was used. A base to tip maturation gradient of leaf anatomy was defined, and RNA sequencing was undertaken along this gradient for two C3 and two C4 Flaveria species. Key C4 traits including vein density, mesophyll and bundle sheath cross-sectional area, chloroplast ultrastructure, and abundance of transcripts encoding proteins of C4 photosynthesis were quantified. Candidate genes underlying each of these C4 characteristics were identified. Principal components analysis indicated that leaf maturation and the photosynthetic pathway were responsible for the greatest amount of variation in transcript abundance. Photosynthesis genes were over-represented for a prolonged period in the C4 species. Through comparison with publicly available data sets, we identify a small number of transcriptional regulators that have been up-regulated in diverse C4 species. The analysis identifies similar patterns of expression in independent C4 lineages and so indicates that the complex C4 pathway is associated with parallel as well as convergent evolution.

A MEM1-like motif directs mesophyll cell-specific expression of the gene encoding the C4 carbonic anhydrase in Flaveria.

The first two reactions of C4 photosynthesis are catalysed by carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) in the leaf mesophyll (M) cell cytosol. Translatome experiments using a tagged ribosomal protein expressed under the control of M and bundle-sheath (BS) cell-specific promoters showed transcripts encoding CA3 from the C4 species Flaveria bidentis were highly enriched in polysomes from M cells relative to those of the BS. Localisation experiments employing a CA3-green fluorescent protein fusion protein showed F. bidentis CA3 is a cytosolic enzyme. A motif showing high sequence homology to that of the Flaveria M expression module 1 (MEM1) element was identified approximately 2 kb upstream of the F. bidentis and F. trinervia ca3 translation start sites. MEM1 is located in the promoter of C4 Flaveria ppcA genes, which encode the C4-associated PEPC, and is necessary for M-specific expression. No MEM1-like sequence was found in the 4 kb upstream of the C3 species F. pringlei ca3 translation start site. Promoter-reporter fusion experiments demonstrated the region containing the ca3 MEM1-like element also directs M-specific expression. These results support the idea that a common regulatory switch drives the expression of the C4 Flaveria ca3 and ppcA1 genes specifically in M cells.

Mesophyll Chloroplast Investment in C3, C4 and C2 Species of the Genus Flaveria.

The mesophyll (M) cells of C4 plants contain fewer chloroplasts than observed in related C3 plants; however, it is uncertain where along the evolutionary transition from C3 to C4 that the reduction in M chloroplast number occurs. Using 18 species in the genus Flaveria, which contains C3, C4 and a range of C3-C4 intermediate species, we examined changes in chloroplast number and size per M cell, and positioning of chloroplasts relative to the M cell periphery. Chloroplast number and coverage of the M cell periphery declined in proportion to increasing strength of C4 metabolism in Flaveria, while chloroplast size increased with increasing C4 cycle strength. These changes increase cytosolic exposure to the cell periphery which could enhance diffusion of inorganic carbon to phosphenolpyruvate carboxylase (PEPC), a cytosolic enzyme. Analysis of the transcriptome from juvenile leaves of nine Flaveria species showed that the transcript abundance of four genes involved in plastid biogenesis-FtsZ1, FtsZ2, DRP5B and PARC6-was negatively correlated with variation in C4 cycle strength and positively correlated with M chloroplast number per planar cell area. Chloroplast size was negatively correlated with abundance of FtsZ1, FtsZ2 and PARC6 transcripts. These results indicate that natural selection targeted the proteins of the contractile ring assembly to effect the reduction in chloroplast numbers in the M cells of C4 Flaveria species. If so, efforts to engineer the C4 pathway into C3 plants might evaluate whether inducing transcriptome changes similar to those observed in Flaveria could reduce M chloroplast numbers, and thus introduce a trait that appears essential for efficient C4 function.

Insights into the regulation of C4 leaf development from comparative transcriptomic analysis.

C4 photosynthesis is more efficient than C3 photosynthesis for two reasons. First, C4 plants have evolved a repertoire of C4 enzymes to enhance CO2 fixation. Second, C4 leaves have Kranz anatomy with a high vein density in which the veins are surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. The BS and M cells are not only functionally well differentiated, but also well-coordinated for rapid transport of photo-assimilates between the two types of photosynthetic cells. Recent comparative transcriptomic and anatomical analyses of C3 and C4 leaves have revealed early onset of C4-related processes in leaf development, suggesting that delayed mesophyll differentiation contributes to higher C4 vein density, and have identified some candidate regulators for the higher vein density in C4 leaves. Moreover, comparative transcriptomics of maize husk (C3) and foliar leaves (C4) has identified a cohort of candidate regulators of Kranz anatomy development. In addition, there has been major progress in the identification of transcription factor binding sites, greatly increasing our knowledge of gene regulation in plants.

Transcriptome comparisons shed light on the pre-condition and potential barrier for C4 photosynthesis evolution in eudicots.

C4 photosynthesis evolved independently from C3 photosynthesis in more than 60 lineages. Most of the C4 lineages are clustered together in the order Poales and the order Caryophyllales while many other angiosperm orders do not have C4 species, suggesting the existence of biological pre-conditions in the ancestral C3 species that facilitate the evolution of C4 photosynthesis in these lineages. To explore pre-adaptations for C4 photosynthesis evolution, we classified C4 lineages into the C4-poor and the C4-rich groups based on the percentage of C4 species in different genera and conducted a comprehensive comparison on the transcriptomic changes between the non-C4 species from the C4-poor and the C4-rich groups. Results show that species in the C4-rich group showed higher expression of genes related to oxidoreductase activity, light reaction components, terpene synthesis, secondary cell synthesis, C4 cycle related genes and genes related to nucleotide metabolism and senescence. In contrast, C4-poor group showed up-regulation of a PEP/Pi translocator, genes related to signaling pathway, stress response, defense response and plant hormone metabolism (ethylene and brassinosteroid). The implications of these transcriptomic differences between the C4-rich and C4-poor groups to C4 evolution are discussed.

Promotion of Cyclic Electron Transport Around Photosystem I with the Development of C4 Photosynthesis.

C4 photosynthesis is present in approximately 7,500 species classified into 19 families, including monocots and eudicots. In the majority of documented cases, a two-celled CO2-concentrating system that uses a metabolic cycle of four-carbon compounds is employed. C4 photosynthesis repeatedly evolved from C3 photosynthesis, possibly driven by the survival advantages it bestows in the hot, often dry, and nutrient-poor soils of the tropics and subtropics. The development of the C4 metabolic cycle greatly increased the ATP demand in chloroplasts during the evolution of malic enzyme-type C4 photosynthesis, and the additional ATP required for C4 metabolism may be produced by the cyclic electron transport around PSI. Recent studies have revealed the nature of cyclic electron transport and the elevation of its components during C4 evolution. In this review, we discuss the energy requirements of C3 and C4 photosynthesis, the current model of cyclic electron transport around PSI and how cyclic electron transport is promoted during C4 evolution using studies on the genus Flaveria, which contains a number of closely related C3, C4 and C3-C4 intermediate species.

RNA-Seq based phylogeny recapitulates previous phylogeny of the genus Flaveria (Asteraceae) with some modifications.

BACKGROUND: The genus Flaveria has been extensively used as a model to study the evolution of C4 photosynthesis as it contains C3 and C4 species as well as a number of species that exhibit intermediate types of photosynthesis. The current phylogenetic tree of the genus Flaveria contains 21 of the 23 known Flaveria species and has been previously constructed using a combination of morphological data and three non-coding DNA sequences (nuclear encoded ETS, ITS and chloroplast encoded trnL-F). RESULTS: Here we developed a new strategy to update the phylogenetic tree of 16 Flaveria species based on RNA-Seq data. The updated phylogeny is largely congruent with the previously published tree but with some modifications. We propose that the data collection method provided in this study can be used as a generic method for phylogenetic tree reconstruction if the target species has no genomic information. We also showed that a "F. pringlei" genotype recently used in a number of labs may be a hybrid between F. pringlei (C3) and F. angustifolia (C3-C4). CONCLUSIONS: We propose that the new strategy of obtaining phylogenetic sequences outlined in this study can be used to construct robust trees in a larger number of taxa. The updated Flaveria phylogenetic tree also supports a hypothesis of stepwise and parallel evolution of C4 photosynthesis in the Flavaria clade.

C2 photosynthesis generates about 3-fold elevated leaf CO2 levels in the C3-C4 intermediate species Flaveria pubescens.

Formation of a photorespiration-based CO2-concentrating mechanism in C3-C4 intermediate plants is seen as a prerequisite for the evolution of C4 photosynthesis, but it is not known how efficient this mechanism is. Here, using in vivo Rubisco carboxylation-to-oxygenation ratios as a proxy to assess relative intraplastidial CO2 levels is suggested. Such ratios were determined for the C3-C4 intermediate species Flaveria pubescens compared with the closely related C3 plant F. cronquistii and the C4 plant F. trinervia. To this end, a model was developed to describe the major carbon fluxes and metabolite pools involved in photosynthetic-photorespiratory carbon metabolism and used quantitatively to evaluate the labelling kinetics during short-term (14)CO2 incorporation. Our data suggest that the photorespiratory CO2 pump elevates the intraplastidial CO2 concentration about 3-fold in leaves of the C3-C4 intermediate species F. pubescens relative to the C3 species F. cronquistii.

The role of photorespiration during the evolution of C4 photosynthesis in the genus Flaveria.

C4 photosynthesis represents a most remarkable case of convergent evolution of a complex trait, which includes the reprogramming of the expression patterns of thousands of genes. Anatomical, physiological, and phylogenetic and analyses as well as computational modeling indicate that the establishment of a photorespiratory carbon pump (termed C2 photosynthesis) is a prerequisite for the evolution of C4. However, a mechanistic model explaining the tight connection between the evolution of C4 and C2 photosynthesis is currently lacking. Here we address this question through comparative transcriptomic and biochemical analyses of closely related C3, C3-C4, and C4 species, combined with Flux Balance Analysis constrained through a mechanistic model of carbon fixation. We show that C2 photosynthesis creates a misbalance in nitrogen metabolism between bundle sheath and mesophyll cells. Rebalancing nitrogen metabolism requires anaplerotic reactions that resemble at least parts of a basic C4 cycle. Our findings thus show how C2 photosynthesis represents a pre-adaptation for the C4 system, where the evolution of the C2 system establishes important C4 components as a side effect.