Trehalose 6-phosphate (Tre6P) is a signalling metabolite that maintains sucrose homeostasis and links plant growth and development to the availability of sucrose. Most of our knowledge of the nexus between Tre6P and sucrose comes from studies on arabidopsis (Arabidopsis thaliana), and it is unclear whether this close relationship is generally conserved across other species. To address this question, we investigated the diel changes in sucrose and Tre6P in leaves from a phylogenetically diverse set of angiosperms with different phloem loading and carbohydrate storage strategies: arabidopsis, Alchemilla molis, strawberry (Fragaria×ananassa), Plantago major, melon (Cucumis melo), and wheat (Triticum aestivum). Despite large differences in their sucrose and Tre6P levels, there were positive correlations between sucrose and Tre6P across all species. Network analysis confirmed a strong association between Tre6P and sucrose in all species, and also revealed a common link with malate, consistent with positive regulation of malate synthesis by Tre6P. In combination with previous observations that Tre6P is synthesized in and around the leaf vasculature, our findings suggest that Tre6P primarily reflects the vascular transport pool of sucrose in leaves. We conclude that the sucrose-Tre6P nexus is widespread among angiosperms, with a conserved role in regulation of sucrose metabolism and transport.

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

Summary In C 4 photosynthesis, incoming CO 2 is incorporated in mesophyll cells (MC) into 4-carbon acids that diffuse to bundle sheath cells (BSC) and decarboxylated to generate a high CO 2 concentration that suppresses the oxygenation reaction of Rubisco. Decarboxylation can occur by NADP-malic enzyme, (NADP-ME), NAD-malic enzyme (NAD-ME) or phospho enol pyruvate 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 C 4 plants are discussed. Highlight Statement An 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 C 4 photosynthesis.

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the first irreversible reaction of the CO2 concentrating mechanism (CCM) in C4 photosynthesis. Engineering C4 photosynthesis into the C3 plant rice (Oryza sativa) to increase photosynthetic efficiency requires sufficient activity and proper regulation of PEPC. In previous studies, in vivo PEPC activity remains low despite substantial in vitro activity. Here, we tested the activity and regulation of PEPC expressed in rice using either the coding (cDNA) or genomic (gDNA) sequences of the C4 photosynthetic isoform of PEPC from maize (Zea mays) driven by the ZmPEPC promoter, termed cDNAlines and gDNAlines, respectively. We quantified PEPC enzyme characteristics in vitro, estimated in planta PEPC activity using 13CO2 labeling, and assessed diel phosphorylation status. Both cDNAlines and gDNAlines showed higher PEPC activities than wild-type rice, but in planta activity was <2.1% of in vitro activity. PEPC from gDNAlines had similar affinity to HCO3- but decreased affinity to phosphoenolpyruvate (PEP) in the absence of the positive effector glucose-6-phosphate. In contrast to maize, PEPC in rice lines was primarily phosphorylated in the dark. Estimated PEP concentrations in rice were higher than those required for half-maximal activity of maize PEPC, suggesting that PEP availability was not limiting. The estimated malate pool was higher than required to inhibit phosphorylated and especially dephosphorylated maize PEPC, consistent with malate inhibiting activity in vivo and compounded by a mismatch in light activation. These results indicate that relieving inhibitory and regulatory constraints, rather than further increasing extractable PEPC capacity, is likely required to increase PEPC-dependent CO2 capture in engineered rice.

Crop breeding faces the challenge of balancing yield with resource efficiency and stress tolerance. The key regulatory metabolite trehalose 6-phosphate (T6P) integrates carbon status with growth and stress responses. Manipulating T6P levels through genetic modification or chemical intervention provides a powerful tool to tackle the growing challenges of crop production.

Over 2 decades ago, antisense rbcS tobacco lines with a progressive decrease in Rubisco abundance allowed network analysis of the regulation of photosynthesis, metabolism, and whole plant allocation. In the 1970 and 1980s, the study of the regulation of metabolism and growth was largely descriptive. Conceptual frameworks had been formulated that would allow a more rigorous approach, for example, to generate a small decrease in enzyme abundance and measure the resulting change in pathway flux. The lack of suitable mutants, however, made this approach practically impossible. This changed drastically when Agrobacterium-mediated transformation made it possible to alter expression of enzymes and other proteins at will. (Quick et al. in Planta 183:542–554, 1991a) and subsequent papers used antisense lines with a progressive decrease in Rubisco abundance to show that the contribution of Rubisco to the control of photosynthesis varies greatly, depending on the conditions in which photosynthesis is occurring and the conditions in which the plants had been grown. Analogous experiments by us and others on other Calvin–Benson cycle enzymes showed that they could also exert control and that the distribution of control depended on the conditions. We also used the rbcS antisense lines to, first, show that small decrease in Rubisco abundance is often accommodated by the photosynthetic apparatus to minimize the inhibition of photosynthesis and, second, explore how a larger decrease in Rubisco abundance and the resulting inhibition of photosynthesis impacts on central carbon and nitrogen metabolism, specialized metabolism, and whole plant architecture. This approach anticipated future developments like network analysis and system biology, is still relevant to designing strategies to improve crop photosynthesis, and can provide insights into photosynthetic performance and trade-offs in the field in a fluctuating environment.

C4 plants have traditionally been classified into NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate 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-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 9% and 15% via malate, respectively. The results 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.

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. 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. Levels of individual CBC intermediates showed many significant differences. In a principal component analysis, whilst three species (Solanum lycopersicum, Solanum  cheesmaniae, and 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 by phylogenetic relatedness and, by implication, lineage-specific adaptation.

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 noninvasive measurements and metabolic profiling to study the efficiency of C4 photosynthesis in maize (Zea mays) under 2 conditions that can lead to decoupling between electron transport and carbon assimilation: fluctuating light and suboptimal temperature. Measurements were performed under 3 fluctuating light regimes and at 3 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 suboptimal 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 greater 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 at room temperature, as well as avoid oxidative damage by strongly downregulating electron transfer under short-term exposure to low temperatures.

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.

In the field, plants continually experience changes in irradiance. Research in C3 species has revealed that while the Calvin-Benson cycle (CBC) adjusts rapidly to changing irradiance, there is substantial loss of photosynthetic 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 subjected maize (Zea mays) to a sudden increase or decrease of irradiance in the nonsaturating range and performed time-resolved measurement of photosynthetic rate and profiling of metabolites from the CBC, the CO2-concentrating mechanism (CCM), the energy shuttle, photorespiration, and end-product biosynthesis. After a decrease in irradiance, photosynthesis is transiently buffered by energy delivered from transformations in the large metabolite pools in the energy shuttle and CCM. During the subsequent decline in photosynthesis, metabolism transitions to a suboptimal state for photosynthesis in low irradiance, from which it takes several minutes to recover. One reason is that end-product synthesis depletes the metabolite pools that drive intercellular shuttles and time is required to replenish these pools. After an increase in irradiance, there is an initial rapid rise of photosynthesis linked to build-up of CBC intermediates and, probably, activation of enzymes in the CBC and the CCM. This is followed by a further slow rise of photosynthesis linked to gradual accumulation of the large metabolite pools that drive intercellular shuttles. In addition, in both transitions, transient imbalances between pumping and utilization of CO2 lead to further losses in photosynthetic efficiency.

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. ### Competing Interest Statement The authors have declared no competing interest. Fundação para a Ciência e Tecnologia, https://ror.org/00snfqn58, UIDB/04551/2020, UIDP/04551/2020, LA/P/0087/2020, PTDC/BIA-FBT/4942/2020, 2023.06360.CEECIND/CP2836/CT0009, PD/BD/150239/2019 Max Planck Society John Fell Fund, 0013575

Nonstructural carbohydrate (NSC) concentrations might reflect the strategies described in the leaf economic spectrum (LES) due to their dependence on photosynthesis and respiration. We examined if NSC concentrations correlate with leaf structure, chemistry, and physiology traits for 114 species from 19 sites and 5 biomes around the globe. Total leaf NSC concentrations varied greatly from 16 to 199 mg g-1 dry mass and were mostly independent of leaf gas exchange and the LES traits. By contrast, leaf NSC residence time was shorter in species with higher rates of photosynthesis, following the fast-slow strategies in the LES. An average leaf held an amount of NSCs that could sustain one night of leaf respiration and could be replenished in just a few hours of photosynthesis under saturating light, indicating that most daily carbon gain is exported. Our results suggest that NSC export is clearly linked to the economics of return on resource investment.

Trehalose 6-phosphate (Tre6P) is an essential signal metabolite that reports and regulates the level of sucrose, linking growth and development to the metabolic status. We hypothesized that Tre6P plays a role in mediating the regulation of gene expression by sucrose. To test this, we performed transcriptomic profiling on Arabidopsis plants that expressed a bacterial trehalose-6-phosphate synthase (TPS) under the control of an ethanol-inducible promoter. Induction led to a 4-fold rise in Tre6P levels, a concomitant decrease in sucrose, and significant changes of over 13,000 transcripts and two-fold or larger changes of over 5000 transcripts. Comparison with nine published responses to sugar availability allowed some of these changes to be linked to the rise in Tre6P, while others were probably due to lower sucrose or other indirect effects. Changes linked to Tre6P included repression of photosynthesis and induction of many growth-related processes including ribosome biogenesis. About 500 starvation-related genes are known to be induced by SUCROSE-NON-FERMENTING-1-RELATED KINASE 1 (SnRK1). They were largely repressed by Tre6P in a manner consistent with Tre6P acting to inhibit SnRK1. SnRK1 also represses many genes that are involved in biosynthesis and growth. These responded to Tre6P in a more complex manner, pointing to Tre6P also interacting with further C-signaling pathways. In addition, elevated Tre6P modified expression of genes encoding regulatory subunits of the SnRK1 complex and TPS class II and FLZ proteins that are thought to modulate SnRK1 function, and genes involved in the circadian clock and in TOR, light, abscisic acid and other hormone signaling. One sentence summary An induced increase in trehalose 6-phosphate levels has direct effects on gene expression via inhibition of SUCROSE-NON-FERMENTING-1-RELATED KINASE 1 and interactions with light, circadian clock and phytohormone signaling, and widespread indirect effects on gene expression from reciprocal changes in sucrose levels.

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 the regulation of carbon metabolism by Tre6P 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, where it could play a fundamental role in the regulation of sucrose levels by modulating phloem loading.

Sucrose-nonfermenting 1 (SNF1)-related kinase 1 (SnRK1) is a central hub in carbon and energy signaling in plants, and is orthologous with SNF1 in yeast and the AMP-activated protein kinase (AMPK) in animals. Previous studies of SnRK1 relied on in vitro activity assays or monitoring of putative marker gene expression. Neither approach gives unambiguous information about in vivo SnRK1 activity. We have monitored in vivo SnRK1 activity using Arabidopsis (Arabidopsis thaliana) reporter lines that express a chimeric polypeptide with an SNF1/SnRK1/AMPK-specific phosphorylation site. We investigated responses during an equinoctial diel cycle and after perturbing this cycle. As expected, in vivo SnRK1 activity rose toward the end of the night and rose even further when the night was extended. Unexpectedly, although sugars rose after dawn, SnRK1 activity did not decline until about 12 h into the light period. The sucrose signal metabolite, trehalose 6-phosphate (Tre6P), has been shown to inhibit SnRK1 in vitro. We introduced the SnRK1 reporter into lines that harbored an inducible trehalose-6-phosphate synthase construct. Elevated Tre6P decreased in vivo SnRK1 activity in the light period, but not at the end of the night. Reporter polypeptide phosphorylation was sometimes negatively correlated with Tre6P, but a stronger and more widespread negative correlation was observed with glucose-6-phosphate. We propose that SnRK1 operates within a network that controls carbon utilization and maintains diel sugar homeostasis, that SnRK1 activity is regulated in a context-dependent manner by Tre6P, probably interacting with further inputs including hexose phosphates and the circadian clock, and that SnRK1 signaling is modulated by factors that act downstream of SnRK1.

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.

Cyanobacteria and eukaryotic algae make a major contribution to global photosynthetic productivity. To cope with the low availability of CO 2 in aqueous systems they deploy inorganic carbon-concentrating mechanisms (CCMs). These concentrate CO 2 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 CO 2 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. Highlight CBC 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.

Starch mobilization robustly paces to dawn despite severe disruption of the transcriptional circadian oscillator. Many plants, including Arabidopsis (Arabidopsis thaliana), accumulate starch in the daytime and remobilize it to support maintenance and growth at night. Starch accumulation is increased when carbon is in short supply, for example, in short photoperiods. Mobilization is paced to exhaust starch around dawn, as anticipated by the circadian clock. This diel pattern of turnover is largely robust against loss of day, dawn, dusk, or evening clock components. Here, we investigated diel starch turnover in the triple circadian clock mutant lhy cca1 elf3, which lacks the LATE ELONGATED HYPOCOTYL and the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) dawn components and the EARLY FLOWERING3 (ELF3) evening components of the circadian clock. The diel oscillations of transcripts for the remaining clock components and related genes like REVEILLE and PHYTOCHROME-INTERACING FACTOR family members exhibited attenuated amplitudes and altered peak time, weakened dawn dominance, and decreased robustness against changes in the external light-dark cycle. The triple mutant was unable to increase starch accumulation in short photoperiods. However, it was still able to pace starch mobilization to around dawn in different photoperiods and growth irradiances and to around 24 h after the previous dawn in T17 and T28 cycles. The triple mutant was able to slow down starch mobilization after a sudden low-light day or a sudden early dusk, although in the latter case it did not fully compensate for the lengthened night. Overall, there was a slight trend to less linear mobilization of starch. Thus, starch mobilization can be paced rather robustly to dawn despite a major disruption of the transcriptional clock. It is proposed that temporal information can be delivered from clock components or a semi-autonomous oscillator.