Gian Luca Borghi

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.

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.

Summary: 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. ¹³CO₂ 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.

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.

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.

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.

During photosynthesis of higher plants, absorbed light energy is converted into chemical energy that, in part, is accumulated in the form of transitory starch within chloroplasts. In the following night, transitory starch is mobilized to sustain the heterotrophic metabolism of the plant. β-amylases are glucan hydrolases that cleave α-1,4-glycosidic bonds of starch and release maltose units from the non-reducing end of the polysaccharide chain. In Arabidopsis, nocturnal degradation of transitory starch involves mainly β-amylase-3 (BAM3). A second β-amylase isoform, β-amylase-1 (BAM1), is involved in diurnal starch degradation in guard cells, a process that sustains stomata opening. However, BAM1 also contributes to diurnal starch turnover in mesophyll cells under osmotic stress. With the aim of dissecting the role of β-amylases in osmotic stress responses in Arabidopsis, mutant plants lacking either BAM1 or BAM3 were subject to a mild (150mM mannitol) and prolonged (up to one week) osmotic stress. We show here that leaves of osmotically-stressed bam1 plants accumulated more starch and fewer soluble sugars than both wild-type and bam3 plants during the day. Moreover, bam1 mutants were impaired in proline accumulation and suffered from stronger lipid peroxidation, compared with both wild-type and bam3 plants. Taken together, these data strongly suggest that carbon skeletons deriving from BAM1 diurnal degradation of transitory starch support the biosynthesis of proline required to face the osmotic stress. We propose the transitory-starch/proline interplay as an interesting trait to be tackled by breeding technologies aimingto improve drought tolerance in relevant crops.