Biology subjects

Arrivault, S.

Publications and source records attributed to Arrivault, S..

12 recordsLinked to original sources

bioRxiv2026

C4 photosynthetic pathway fluxes in transgenic rice plants

Most land plants photosynthesize using the C3 pathway, in which ribulose bisphosphate carboxylase/oxygenase (Rubisco) fixes CO2 into 3-carbon acids. The C4 pathway, a biochemical CO2-concentrating mechanism that operates in the context of specialized leaf anatomy to concentrate CO2 around Rubisco, is more efficient. Introduction of the C4 pathway into the C3 crop rice could increase yield by 50%. Expression of five C4 enzymes in transgenic rice previously led to flux through the first step. However, there was no evidence for flux later in the cycle. Here we developed new transgenic rice lines and novel protocols to detect C4 cycle activity: CO2 fixation into C4 acids by carboxylation of a C3 compound, decarboxylation, refixation of CO2 by Rubisco, and regeneration of the C3 donor. We demonstrate that these four core C4 reactions are operating in rice, establishing the in vivo flux framework needed to progress towards a functional carbon-concentrating mechanism

plant biology↗

bioRxiv2026

Intercellular Concentration Gradients of 3-Phosphoglycerate and Triose-Phosphate Demonstrate Operation of an Energy Shuttle in NAD-Malic Enzyme and Phosphoenolypyruvate Carboxykinase C4 Subtypes

In C4 photosynthesis, incoming CO2 is incorporated in mesophyll cells (MC) into 4-carbon acids that diffuse to bundle sheath cells (BSC) and decarboxylated to generate a high CO2 concentration that suppresses the oxygenation reaction of Rubisco. Decarboxylation can occur by NADP-malic enzyme, (NADP-ME), NAD-malic enzyme (NAD-ME) or phosphoenolpyruvate carboxykinase (PEPCK). NADP-ME generates NADPH in the BSC chloroplast and species that use it as the major route for decarboxylation typically have dimorphic BSC chloroplasts with little or no photosystem II. They operate an energy shuttle: much of the 3-phosphoglycerate formed in the Calvin-Benson cycle diffuses to the MC, enters the chloroplasts and is reduced to triose phosphates that return to the BSC. In species where carboxylation occurs mainly via NAD-ME or PEPCK, BSC chloroplasts possess photosystem II. Indirect evidence indicates they nevertheless have the capacity to operate an energy shuttle. We show here that NAD-ME and PEPCK species possess large pools of 3PGA and triose phosphates and, for two examples of each subtype, opposed concentration gradients of 3-phosphoglycerate and triose phosphates to drive rapid exchange between the BSC and MC. Reasons for and consequences of the widespread operation of the intercellular energy shuttle in C4 plants are discussed. Highlight StatementAn intercellular energy shuttle in which 3-phosphoglycerate moves from the bundle sheath to the mesophyll and triose phosphates return to the bundle sheath is a general feature of C4 photosynthesis.

plant biology↗

bioRxiv2026

Variance in Calvin-Benson cycle intermediate levels between closely-related species in the tomato clade

Published studies have reported species-variance between profiles of Calvin-Benson cycle (CBC) intermediates, not only between C4 species and C3 species, but also within C3 species (Arrivault et al., 2019, Borghi et al. 2019). It was proposed that this variance reflects lineage-dependent changes in the balance between different reactions, or poising, of the CBC. These earlier studies investigated phylogenetically-unrelated C3 species. In the current study, CBC intermediates were profiled in five closely-related species from Solanum sect. lycopersicon subsect. Lycopersicum. The levels of individual CBC intermediates showed many significant differences. In a principal component analysis, whilst three species (Solanum lycopersicum, Solanum cheesmaniae, Solanum neorickii) overlapped, Solanum pimpinellifolium and especially Solanum pennellii grouped separately, and were at opposing ends of the distribution. When combined with published data, whilst the separation between Solanum species was retained, they formed a group that was separated from five other C3 species, as well as two C4 species. It is discussed that the observed variation in CBC metabolites profiles within Solanum, together with their separation from other C3 species, supports the idea that CBC evolution is shaped both by phylogenetic relatedness and lineage-specific adaptation. HighlightVariance of intermediate levels points to poising of the Calvin-Benson cycle varying between closely-related species in the tomato clade Solanum sect. lycopersicon subsect. Lycopersicum

plant biology↗

bioRxiv2025

TPS Proteins coordinate plant growth with sugar availability via the SnRK1 Kinase

The ability to sense and respond to nutrients determines adaptation and survival in all organisms. In plants, sucrose stimulates growth and developmental progression via the signalling sugar trehalose 6-phosphate (T6P) which reflects sucrose availability. T6P acts, at least partly, by inhibiting the protein kinase SUCROSE NON-FERMENTING 1 (SNF1)-RELATED KINASE 1 (SnRK1) but the underlying mechanisms are poorly understood. Here, we identify a group of catalytically inactive T6P synthase (TPS) proteins, TPS5/6/7, as important factors for coupling the T6P signal to SnRK1 activity. In Arabidopsis thaliana, lack of TPS5/6/7 causes severe growth defects, particularly in roots. This is accompanied by a metabolic signature that is suggestive of T6P insensitivity and impaired sucrose utilization. Using a combination of genetics, SnRK1 activity assays, and imaging, we demonstrate that the growth defects of the tps5/6/7 mutant are due to SnRK1 misregulation and are reverted by knocking-down SnRK1 in this background. Co-immunoprecipitation assays further show that T6P promotes the interaction of TPS proteins with SnRK1 in a highly specific and dose-dependent manner. Our results support a model where TPS proteins act as T6P sensors, inhibiting non-nuclear SnRK1 activity when sucrose is abundant to promote biosynthetic processes and growth.

plant biology↗

bioRxiv2025

Exploring the diversity of the CO2-concentrating mechanism (CCM) in different C4 subtypes

C4 plants have traditionally been classified into NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME) and PEP carboxykinase (PEPCK) subtypes based on the predominant C4-acid decarboxylating enzyme. To investigate the relative contributions of malate and aspartate to C4-pathway fluxes in each subtype, we performed 13CO2 pulse and pulse-chase labelling experiments on four C4 grass species: Zea mays and Setaria viridis (NADP-ME), Panicum miliaceum (NAD-ME) and Megathyrsus maximus (PEPCK). Only a proportion (8-50%) of the total malate pool in the leaves is photosynthetically active whereas essentially all of the aspartate pool is photosynthetically active. Estimates of metabolic fluxes indicate that approximately two thirds of the C4 pathway flux is via malate in Z. mays and the remaining third via aspartate, while in S. viridis 50% of the flux is via malate and 50% via aspartate. In P. miliaceum and M. maximus, 91% and 85% of the flux is via aspartate and the remaining 5% and 15% via malate, respectively. The results reveal greater complexity of C4 pathway fluxes than is usually represented in textbook diagrams, and demonstrate the feasibility of using non-radioactive 13CO2 in pulse-chase labelling experiments to study C4 photosynthesis and to detect C4 pathway fluxes in C3 plants engineered to perform C4 photosynthesis. Highlight StatementPhotosynthetic fluxes in C4 species are more complex than most textbook models show, with malate and aspartate both carrying C4 cycle fluxes in all three subtypes (NADP-ME, NAD-ME and PEPCK).

plant biology↗

bioRxiv2025

In vivo RUBISCO activity in Synechocystis is regulated by RuBP availability

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is the main CO2-fixing enzyme on earth and entry point of carbon into the Calvin-Benson-Bassham cycle. Fueled by photosynthesis, C-assimilation by RUBISCO must be tightly controlled. RUBISCO regulation upon transition from light to darkness is not fully understood in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Synechocystis does not have a RUBISCO activase that regulates RUBISCO activity in vascular plants by removing intrinsic sugar phosphate inhibitors from its active site. Instead, the regulatory CP12 protein of Synechocystis inactivates glyceraldehyde 3-phosphate dehydrogenase (GAPDH2) and phosphoribulokinase (PRK) during darkness. This mechanism indicates metabolic regulation of RUBISCO. We investigated C-assimilation in vivo at the transition to darkness by dynamic 13CO2 labeling experiments. We monitored RUBISCO activity by 13C-incorporation into 1-C position of 3PGA. Other than the wild type, the{Delta} cp12 mutant continued to assimilate 13CO2 into 3PGA in darkness. RUBISCO abundances and specific activities were not altered in{Delta} cp12 and upon light to dark transition. CP12 was required to shut down the CBB cycle during the night. Complementation of{Delta} cp12 by native CP12 ({Delta}cp12::cp12) and CP12 with mutated conserved cysteines in its GAPDH2- and PRK-binding domains ({Delta}cp12::cp12{Delta}Cys) showed that both native binding domains are required to fully inactivate the CBB cycle in the night. RuBP levels were highly elevated in{Delta} cp12 upon transition to darkness. Complementation with mutated and native CP12 variants gradually reduced RuBP to wild type levels and revealed highly significant correlation between RuBP concentration and the time-shifted 13C-uptake into 3PGA. We propose that RUBISCO activity in Synechocystis at day-night transition is regulated through depletion and blocked regeneration of RuBP. 13C-positional analyses of aspartate suggest regeneration of RuBP in{Delta} cp12 via dysregulated gluconeogenesis and the oxidative pentose phosphate path. We demonstrate that RUBISCO activity of Synechocystis is present throughout diurnal growth and depends on the availability of its substrate.

plant biology↗

bioRxiv2025

Metabolite profiling reveals slow and uncoordinated adjustment of C4 photosynthesis to sudden changes in irradiance

In the field plants continually experience changes in irradiance. Research in C3 species has revealed that whilst the Carbon-Benson cycle (CBC) adjusts rapidly to changing irradiance, there is substantial loss of photosynthesis efficiency due to slow adjustment of energy dissipation and stomatal conductance. Less is known about the impact of changing irradiance on photosynthetic efficiency in C4 species. We have subjected maize to a sudden increase or decrease of irradiance in the non-saturating range and performed time-resolved measurement of photosynthetic rate and profiling of metabolites from the CBC, the CO2-concentrating mechanism (CCM), photorespiration and end-product biosynthesis. After a decrease in irradiance, photosynthesis is transiently buffered by energy delivered by transformations in the large pools of metabolites that are involved in intercellular shuttles. During the subsequent decline in photosynthesis, metabolism transitions to a sub-optimal state for photosynthesis in low irradiance, from which it takes several minutes to recover. One reason is that the large pools of metabolites that facilitate intercellular shuttles run down too far and it takes time to build them up again. After an increase in irradiance, there is a long delay until a new steady rate of photosynthesis is achieved. The delay is partly due to regulation of enzymes in the CBC and CCM, but also to the need to build up large pools of metabolites to drive intercellular shuttles. In addition, in both transitions, transient imbalances between the pumping and utilization of CO2 lead to increased back-leakage of CO2 or to photorespiration, further decreasing photosynthetic efficiency.

plant biology↗

bioRxiv2025

Sub-optimal temperature leads to tighter coupling between photosynthetic electron transport and CO2 assimilation under fluctuating light in maize.

The C4 carbon concentrating pathway promotes high CO2 assimilation rates. To keep C4 photosynthesis energetically efficient, electron transport reactions and downstream biochemistry need to be carefully balanced. Here we use a combination of non-invasive measurements and metabolic profiling to study the efficiency of C4 photosynthesis in maize under two conditions that can lead to decoupling between electron transport and carbon assimilation: fluctuating light and suboptimal temperature. Measurements were performed for three fluctuating light regimes and three temperatures, providing the most detailed study to date of the interaction between fluctuating light and suboptimal temperature on the photosynthetic performance of maize, an important global crop. At room temperature, CO2 assimilation rates were decoupled from photosynthetic electron transport under fluctuating light regimes, in contrast to tight coordination observed under constant light. This decoupling was underpinned by metabolic flexibility and buffering by large pools of C4 transfer metabolites. Surprisingly, at sub-optimal temperatures, CO2 assimilation rates became more tightly coupled to photosynthetic electron transport rates under fluctuating light regimes. This appeared to be caused by strong feedback downregulation of electron transport and a stronger degree of light-saturation of CO2 assimilation at low temperature. Low temperature impacted carbon assimilation rates more strongly than metabolite pools or intercellular metabolite distribution, which could reflect negative effects on diffusional metabolite transfer through plasmodesmata. Altogether, these results show that maize is able to maintain energetic efficiency by buffering light transitions under room temperature, as well as avoid oxidative damage by strongly downregulating electron transfer under short-term exposure to low temperature. One-sentence summaryAnalysis of maize CO2 assimilation under fluctuating light shows significant decoupling from photosynthetic electron transport at room temperature, supported by metabolic flexibility and buffering by large pools of C4 transfer metabolites, but tight coordination is restored under suboptimal temperature due to enhanced feedback regulation of electron transport and a stronger degree of light saturation of CO2 assimilation.

plant biology↗

bioRxiv2024

Intercellular compartmentation of trehalose 6-phosphate metabolism in Setaria viridis leaves

Trehalose 6-phosphate (Tre6P) is a signal metabolite that links carbon metabolism with plant development. Our current understanding of Tre6P metabolism and signalling is predominantly based on studies in Arabidopsis thaliana. Some features could be adapted to the specific physiology, anatomy, and life cycle of this C3 eudicot model species, and thus might not be representative of other angiosperms. To better understand Tre6P metabolism in monocot C4 species we used Setaria viridis, which has been widely adopted as a model for the major C4 NADP-malic enzyme subtype crop species, such as maize (Zea mays), sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum). In this work, we analysed the levels of transcripts encoding Tre6P-related enzymes in different tissues and cell types from S. viridis. The TREHALOSE-6-PHOSPHATE SYNTHASE1 transcript, its encoded protein (TPS1, the enzyme responsible for Tre6P synthesis) and Tre6P were mainly located in bundle sheath cells of S. viridis. Our results show that Tre6P is predominately synthesized and located in bundle sheath and associated cells. HighlightTrehalose 6-phosphate, a sugar signalling metabolite, is mainly present in the bundle sheath cells of Setaria viridis leaves.

plant biology↗

bioRxiv2022

Operation of Carbon-Concentrating Mechanisms in Cyanobacteria and Algae requires altered poising of the Calvin-Benson cycle

Cyanobacteria and eukaryotic algae make a major contribution to global photosynthetic productivity. To cope with the low availability of CO2 in aqueous systems they deploy inorganic carbon-concentrating mechanisms (CCMs). These concentrate CO2 in microcompartments that contain Rubisco (carboxysomes in cyanobacteria; pyrenoids in green algae). The rest of the Calvin-Benson cycle (CBC) is located outside these microcompartments. We hypothesized that this physical separation requires modified poising of the CBC. Hence, Rubisco is physically separated from the other CBC enzymes outside these microcompartments. To test the hypothesis that this physical separation requires appropriate poising of the CBC, we profiled CBC metabolites under ambient CO2 in the cyanobacterium Synechocystis sp. PCC 6803 and three eukaryotic algae (Chlamydomonas reinhardtii, Chlorella sorokiniana, Chlorella ohadii). Comparison with recently reported profiles for a large set of terrestrial plants revealed that cyanobacteria and green algae have very distinctive CBC metabolite profiles, with low levels of pentose phosphates and, especially, high levels of ribulose 1,5-bisphosphate and 3-phosphoglycerate. We propose that large pools of the substrate and product of Rubisco are required to generate concentration gradients that drive movement into and out of the microcompartments. These observations raise questions about how CBC regulation was modified during the evolution of algal CCMs and their subsequent loss in terrestrial plants, and highlight that operation of CCMs requires co-evolution of the CBC. HighlightCBC metabolite profiles in the cyanobacterium Synechocystis and in three eukaryotic green algae at ambient CO2 concentration are very different to those in terrestrial plants, probably reflecting the operation of a carboxysome- or pyrenoid-based carbon concentrating mechanism.

biochemistry↗

bioRxiv2021

The pathway of starch synthesis in Arabidopsis thaliana 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 classical pathway of starch synthesis. We conclude that the ADPG needed for starch synthesis in leaves is synthesized primarily by ADPG pyrophosphorylase in the chloroplasts. Significance statementMutant analysis shows that sucrose synthase makes no significant contribution to transitory starch synthesis in Arabidopsis leaves, resolving a 20-year old controversy about one of the most important pathways of photosynthetic metabolism.

plant biology↗

bioRxiv2020

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 expresses 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), in a cell-specific manner. This led to enhanced enzyme activity but was largely neutral in its effects on photosynthetic rate and growth. 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. We also showed 13C labelling of aspartate indicating additional 13CO2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. However, there were no significant differences in labelling of 3-phosphoglycerate (3PGA) or phosphoenolpyruvate (PEP) indicating limited carbon flux through C4 enzymes into the Calvin-Benson cycle. 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 labelling of malate, aspartate and citrate. 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 flux through the carboxylation phase of C4 metabolism in transgenic rice containing the key metabolic steps in the C4 pathway.

plant biology↗