This study presents an integrated multivariate and univariate analysis of circadian metabolomic signatures in Ilex paraguariensis (yerba mate) clones cultivated under semi-hydroponic conditions. Using repeated measures ANOVA-Simultaneous Component Analysis (RM-ASCA+) and hierarchical clustering on principal components (HCPC), we explored clone-specific and photoperiod-dependent metabolic responses across light and dark phases, complemented by high-resolution timepoint sampling (HRS) and targeted screening. Clone EC21 exhibited elevated levels of sugars, amino acids, and organic acids, suggesting a metabolic strategy adapted to saline stress and nocturnal energy demands. Photoperiod effects revealed circadian regulation of central carbon metabolites (e.g., glucose, fructose, maltose) and phenylpropanoid intermediates linked to bioactive compounds such as caffeoyl-quinic acids. Interaction effects highlighted metabolic plasticity, particularly in nitrogen assimilation, with compounds like 2-oxo-glutaric acid, glutamine, and ornithine showing clone-specific temporal patterns. Caffeine, a heritable and physiologically relevant metabolite, displayed distinct circadian profiles. EC24 accumulated caffeine during the day, while EC21 peaked at night. This dynamic distribution, supported by allantoin patterns in caffeine catabolism, suggests divergent nitrogen turnover strategies between clones. These findings underscore the importance of genotype selection and temporal regulation in optimizing yerba mate performance under semi-hydroponic systems. The combined use of RM-ASCA+ and univariate analysis proved to be a powerful approach for profiling metabolomic rhythms, offering valuable insights for breeding programs targeting bioactive compound enhancement, stress resilience, and metabolic efficiency.

Starch branching enzyme IIb (OsSBEIIb) plays a key role in determining amylopectin structure and starch digestibility. In this study, CRISPR/Cas9 was used for splice site disruption of OsSBEIIb in the elite indica cultivar IR64, generating a truncated protein with impaired function and reduced relative expression levels. The result showed extensive remodeling of starch fine structure, including reduced short-chain amylopectin chains, increased long-chain glucan fractions, higher apparent amylose content up to 39% and elevated resistant starch (RS) levels up to 10%. These modifications were accompanied with substantial 23-unit decreased predicted glycemic index (pGI), from 80 ± 1 in SBE2b_WT to 57 ± 1 in sbe2b mutant lines. Biophysical analyses showed reduced relative crystallinity but higher gelatinization temperatures, indicating more thermally stable starch structures. Beyond starch, sbe2b mutants displayed improved nutritional profiles, with crude protein content increasing by up to 10% and elevated levels of several essential amino acids, including lysine, methionine, threonine and histidine. Metabolite and lipid profiling revealed broader endosperm metabolic shifts affecting carbohydrates, amino acids, phenylpropanoids, fatty acids and triacylglycerols. However, these nutritional benefits were offset by grain quality and agronomic trade-offs, notably increased chalkiness and reduced seed weight. Overall, OsSBEIIb disruption effectively lowers glycemic properties while enhancing protein quality through coordinated metabolic changes in rice endosperm.

Photoautotrophic organisms fix inorganic carbon (Ci) by RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO) and PHOSPHOENOLPYRUVATE CARBOXYLASE (PEPC). Monitoring Ci assimilation rates in vivo is a major challenge in analyzing photoautotrophic metabolism and engineering improved photosynthesis, as conventional methods do not distinguish between these two fluxes. We explored widely applied gas chromatography mass spectrometry (GC-MS) metabolite profiling for C-positional fractional 13C enrichment (E13C) analyses of aspartate to differentiate within one molecule between PEPC, RUBISCO, and CBB cycle activities by 13C pulse-labeling. We validated this method using two GC-MS instruments and two prevailing chemical derivatization methods. We selectively determined E13C at each carbon position of aspartate with accuracy < 1% and precision < 2.5%. In combination with dynamic 13CO2 labeling of Synechocystis cultures, we determined PEPC activity in vivo alongside assessments of RUBISCO and CBB cycle activities. We demonstrate that RUBISCO is inactive in the dark, whereas PEPC remains active but at a lower rate than during the day. Accurate quantifications of aspartate concentrations and positional E13Cs provide molar Ci assimilation rates of photoautotrophic Synechocystis cultures. This technology can be combined with C-positional analyses of other metabolites, for example 3-phosphoglycerate, and may be adapted to characterize natural and biosynthetically engineered Ci-assimilation.

Polyamines are small, polycationic molecules with amino groups that are present in most living organisms. Studies indicate that polyamines increase general protein synthesis and are essential for efficient translation. While progress has been made in understanding the role of polyamines in translation in bacteria and mammals, their contribution and mode of action in plants remain largely unexplored. In a previous study, we found that putrescine (Put) and the pathogen-associated molecular pattern (PAMP) from bacterial flagellin (flg22) transcriptionally induced ribosome biogenesis in plants. Here we examined the impact of polyamines (Put and spermine, Spm) and flg22 on ribosome complex formation in Arabidopsis. Our results indicate that polyamines, flg22 and their combinations increase the abundance of actively translating polysomes. Riboproteomic analyses revealed that polyamines and flg22 trigger differential changes in the accumulation of ribosomal proteins, which are structurally confined in response to Put. Importantly, Put was found binding to non-translating and actively translating ribosomes, suggesting that this polyamine has a role in functional aspects of translation, such as stabilization and/or remodeling of polysomal complexes. Additional global proteomics analyses in polyamine biosynthesis mutants revealed that lower Put availability triggers changes in proteins associated with ribonucleoprotein complex binding and biogenesis. Overall, our findings highlight the effect of polyamines and flg22 on shaping the ribosomal protein composition of actively translating ribosomes in plants.

Understanding crop responses to drought stress is crucial for securing future agricultural productivity. Guard cells regulate transpiration and thus the yield burden under drought conditions. Therefore, the influence of repeated drought stress on the guard cell metabolome of Zea mays L. was investigated to improve our understanding of crop resilience mechanisms. A controlled greenhouse experiment with physiological evaluation and a non-targeted metabolomics approach was used to analyse unprimed and primed guard cells. Primed and unprimed maize plants showed similar overall physiological and metabolic responses to drought, with gas exchange and general metabolic patterns largely unaffected by priming. However, distinct priming effects emerged in specific metabolites. Metabolites of the alanine and aspartate pathway, as well as those of the glycine, serine and threonine pathway were less impacted by drought stress in guard cells than in mesophyll cells, suggesting the emphasis of plants to maintain stable guard cell metabolomes for functional integrity. In contrast, the increase in sugar concentrations in guard cells was similar to that in mesophyll cells, suggesting a pivotal role of sugars in guard cells during drought conditions. New insights into cell type-specific metabolic responses to drought stress will contribute to a better understanding of stress memory in maize. Enhancing guard cell resilience could help optimise water use efficiency for sustainable agricultural production under climate change conditions.

INTRODUCTION:Since the early 2000s, metabolomics has grown rapidly, becoming integral to fields like life sciences, health, and environmental research. This expansion has led to the formation of national and international societies, such as Germany's DGMet, to tackle emerging challenges. One of DGMet's goals is to improve measurement quality by assessing community needs for harmonization and standardization. A recent survey within the German-speaking community aimed to identify current practices and gaps in the use of chemical standards and reference materials, to guide future standardization efforts and collaborative initiatives. METHODS:An online survey was conducted between June 2023 and April 2024. The survey consisted of 38 key questions and was open to research institutions from Germany, Austria, and Switzerland. RESULTS:The survey was accessed by 68 laboratories, with 23 institutes providing complete or partial responses (34% response rate), which is comparable to rates reported in similar surveys within the metabolomics and lipidomics communities. Respondents were mainly experienced researchers from Germany, focusing mainly on health-related ("red") metabolomics, as indicated by 78% of the respondents, followed by microbial ("grey", 48%) and plant ("green", 39%) metabolomics (multiple answers possible). The use of targeted methods was reported more frequently (91%) than that of non-targeted methods (78%), whereas metabolite fractions studied were equally split between polar, midpolar and lipid fractions (83% each). Human (74%), mouse (61%) and Arabidopsis (30%) were the most frequently studied organisms. Most participants used synthetic chemical standards for instrument qualification (83%), calibration (78%), and metabolite identification (74%), while matrix reference materials were mainly applied for quality control (52%) and method validation (44%). There was a strong demand for more standards, especially for metabolite identification and quantification, with cost being a major barrier, particularly for isotopically labelled standards and certified reference materials. CONCLUSIONS:Valuable insights into the use of standards and reference materials within the German-speaking metabolomics community were obtained. Moving forward, the community should address critical gaps in metabolomics standardization. To achieve this, it must share its knowledge, articulate its needs clearly, and actively engage in joint efforts with national metrology institutes and international standardization initiatives.

The importance of sulfate fertilization in plant production has becoming increasingly relevant due to the decline in atmospheric sulfur inputs. Moreover, high-intensity cropping systems are increasingly facing drought scenarios. The role of stomata is crucial during drought stress and is linked to sulfate metabolism. This study investigates the impact of sulfate application on the drought response of grapevine and maize guard cells. Both plant species may serve as crop model for analysing underlying physiological processes in a low fertilizer demanding crop such as grapevine and a high fertilizer demanding crop such as maize. Increased sulfate concentration in leaves was triggered by drought in maize and grapevine, but in grapevine only when additional sulfate was applied. Additional sulfate application improved sulfur availability under drought conditions, which enhanced drought stress response in grapevine and maize. This was characterized by enhanced metabolic acclimation under drought conditions. The effect of sulfate on the drought stress response was markedly diminished in guard cells, indicating enhanced metabolic stability of guard cells against external influences. These results underscore the significance of adequate sulfate supply to crops for optimal drought stress response and suggest that sulfate fertilisation may serve as a potential option to enhance drought acclimation.

Light drives plant life through photosynthesis, a process that takes place in the thylakoid membrane of the chloroplast, an organelle of cyanobacterial origin. The formation of thylakoid membranes within the chloroplast involves the eukaryote-specific factor CHLOROPLAST SEC14 LIKE PROTEIN 1 (CPSFL1), which shares strong sequence homology with the vesicle trafficking regulator SEC14. CSPFL1 is essential for vesicle formation, yet its specific molecular function in this process has remained unclear. In this study, we characterized CSPFL1 functions both in vitro and in vivo. Using a minimal membrane system of giant unilamellar vesicles (GUVs), we show that CPSFL1 alone can induce vesiculation. This process is mediated by lipid binding and membrane deformation, driven by curvature sensing and lipid-protein electrostatics. When expressed in the prokaryote E. coli, the eukaryote-specific CSPFL1 induces membrane curvature and vesicle formation. Plastid CPSFL1 co-purifies with vesicular structures. Lipid compositional analysis of CPSFL1-induced vesicles from bacteria reveals the presence of quinone precursors as cargo, linking CSPFL-mediated vesicle formation to prenylquinone transport. Together, our data suggest that during plant evolution, the eukaryotic vesicle formation system was co-opted for the transport of membrane integral metabolites from the inner envelope to the thylakoid membrane. ### Competing Interest Statement The authors have declared no competing interest.

Rising atmospheric CO2 levels are driving climate change, increasing temperatures, and impacting plant metabolism. In this study, we analyzed the effect of high CO2 concentrations (eCO2) and elevated temperatures on primary and specialized metabolism in two Brassica oleracea genotypes: a wild-type accession (Winspit) and a commercial variety (Parthenon). Metabolic profiling revealed common patterns of changes in several primary metabolites, including amino acids (glutamate, aspartate) and organic acids (citrate, glycerate, ascorbate), under high temperature and/or eCO₂. Interestingly, these changes were organ-specific. In shoots, aspartate and glutamate decreased under elevated temperature when combined with eCO₂, while in roots, their levels increased. Citrate increased in roots of both genotypes under high temperatures, but decreased in shoots. However, ascorbate levels dropped in roots and rose in shoots under combined stress.Glucosinolate content and profiles also showed consistent yet organ-dependent responses. The increase in CO2 and temperature had a greater effect on total glucosinolate content in roots compared to shoots, particularly due to the increase in indolic glucosinolates, while aliphatic glucosinolates mainly increased in shoots. This led to a decreased aliphatic/indolic glucosinolates ratio in roots. As a consequence of alterations in primary and specialized metabolites, nitrogen and sulfur homeostasis were also disrupted. Our findings highlight that plant acclimation to eCO2 and temperatures is highly coordinated, involving primary and specialized metabolism at the whole-plant level, and that not only genotype or environmental specific changes but also organ specific patterns of response must be considered.

Plant acclimation occurs through system-wide mechanisms that include proteome shifts, some of which occur at the level of protein synthesis. All proteins are synthesized by ribosomes. Rather than being monolithic, transcript-to-protein translation machines, ribosomes can be selective and cause proteome shifts. In this study, we use apical root meristems of germinating seedlings of the monocotyledonous plant barley as a model to examine changes in protein abundance and synthesis during cold acclimation. We measured metabolic and physiological parameters that allowed us to compare protein synthesis in the cold to optimal rearing temperatures. We demonstrated that the synthesis and assembly of ribosomal proteins are independent processes in root proliferative tissue. We report the synthesis and accumulation of various macromolecular complexes and propose how ribosome compositional shifts may be associated with functional proteome changes that are part of successful cold acclimation. Our study indicates that translation initiation is limiting during cold acclimation while the ribosome population is remodelled. The distribution of the triggered ribosomal protein heterogeneity suggests that altered compositions may confer 60S subunits selective association capabilities towards translation initiation complexes. To what extent selective translation depends on heterogeneous ribo-proteome compositions in barley proliferative root tissue remains a yet unresolved question.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.

RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO) is the most abundant enzyme and CO2 bio-sequestration system on Earth. Its in vivo activity is usually determined by 14CO2 incorporation into 3-phosphoglycerate (3PGA). However, the radiometric analysis of 3PGA does not distinguish carbon positions. Hence, RUBISCO activity that fixes carbon into the 1-C position of 3PGA and Calvin-Benson-Bassham (CBB) cycle activities that redistribute carbon into its 2-C and 3-C positions are not resolved. This study aims to develop technology that differentiates between these activities. In source fragmentation of gas chromatography-mass spectrometry (GC-MS) enables paired isotopologue distribution analyses of fragmented substructures and the complete metabolite structure. GC-MS measurements after dynamic photosynthetic 13CO2 labeling allowed quantification of the 13C fractional enrichment (E13C) and molar carbon assimilation rates (A13C) at carbon position 1-C of 3PGA by combining E13C from carbon positions 2,3-C2 and 1,2,3-C3 with quantification of 3PGA concentrations. We validated the procedure using two GC-time of flight-MS instruments, operated at nominal or high mass resolution, and tested the expected 3PGA positional labeling by in vivo glycolysis of positional labeled glucose isotopomers. Mutant analysis of the highly divergent GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASEs (GAPDH1 and 2) from Synechocystis sp. PCC 6803 revealed full inactivation of the CBB cycle with maintained RUBISCO activity in Delta gapdh2 and a CBB cycle modulating role of GAPDH1 under fluctuating CO2 supply. RUBISCO activity in the CBB-deficient Delta gapdh2 can re-assimilate CO2 released by catabolic pathways. We suggest that RUBISCO activity in Synechocystis can scavenge carbon lost through the pentose phosphate pathway or other cellular decarboxylation reactions. An integrative approach enables the analysis of RUBISCO-mediated carbon assimilation and reveals continued RUBISCO activity in cyanobacteria following inactivation of the Calvin-Benson-Bassham cycle.

Root traits significantly shape rhizosphere microbiomes, yet their interaction with microbes is often overlooked in plant breeding programs. Here, we propose that selecting modern cultivars based on microbiome interactive trait (MIT), such as root biomass, exudate patterns and the rhizosphere microbiome, can enhance agricultural sustainability by interacting effectively with soil microbiomes, which in turn, promotes plant growth and resistance to stress, thereby reducing reliance on synthetic crop protectants. Through a stepwise selection process (in silico and in vitro) that started with approximately 1000 potato genotypes, we chose 51 potato cultivars based on known phenotypical properties and distinct root exudate patterns. We conducted a greenhouse experiment to evaluate their capacity to interact with the soil microbiome and to assess their MIT scores. Our findings revealed that cultivars significantly influence plant growth, metabolite profiles, and rhizosphere fungal community composition. Moreover, we observed a positive correlation between microbial community diversity and root biomass. Additionally, leaf metabolites were correlated with rhizosphere bacterial composition, supporting the plant holobiont framework. Utilising z-scores, we aggregated all data related to plant growth, metabolomes, and microbiomes, creating a classification of 51 cultivars based on a gradient of MIT scores. By examining the distribution of low, intermediate, and high MIT, we identified a group of 11 potato cultivars suitable for further studies to assess their resilience and productivity under low-input production systems. This study provides an in-depth correlation between microbiome and several plant traits across 51 cultivars, offering tools to facilitate and expedite the incorporation of microbiome traits into breeding goals to support sustainable agriculture.

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 Δcp12 mutant continued to assimilate 13CO2 into 3PGA in darkness. RUBISCO abundances and specific activities were not altered in Δcp12 and upon light to dark transition. CP12 was required to shut down the CBB cycle during the night. Complementation of Δcp12 by native CP12 ( Δcp12::cp12 ) and CP12 with mutated conserved cysteines in its GAPDH2- and PRK-binding domains ( Δcp12::cp12ΔCys ) showed that both native binding domains are required to fully inactivate the CBB cycle in the night. RuBP levels were highly elevated in Δcp12 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 Δcp12 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. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, FOR2816, KO 2329/7-2, HA 2002/23-2, MA 4918/4-2

Hyaluronic acid (HA) is a glycosaminoglycan composed of alternating units of N-acetylglucosamine and glucuronic acid. High moisture retention, viscoelasticity and biocompatibility are unique features that make HA polymers attractive compounds for medical applications and aesthetic purposes. Current synthesis of HA polymers relies on microorganisms and requires supply of glucose in bioreactors to produce glucose-6-phosphate and fructose-6-phosphate as precursors for HA biosynthesis. By contrast, photosynthetic organisms generate glucose-6-phosphate and fructose-6-phosphate as autotrophic products of CO2 fixation via the Calvin-Benson-Bassham (CBB) cycle. Here we explored the possibility to harness chloroplast metabolism for the light-driven production of HA in the model organism tobacco (Nicotiana tabacum). An operon of five streptococcal genes were introduced into the plastid genome of tobacco to drive HA-synthesis by expression elements that confer low, medium or high expression levels. Photoautotrophic growth over the entire life cycle was only achieved in transplastomic lines with low transgene expression levels. Surprisingly, accumulation of HA polymers was observed only under heterotrophic growth conditions. Proteomic analysis revealed low accumulation levels of the first pathway enzyme in the transplastomic lines, and low contents of the final pathway enzyme (HA synthase) upon autotrophic growth. Altered abundances of proteins involved in photosynthesis and central metabolism were observed under autotrophic growth conditions, and metabolite profiling confirmed that photoautotrophic HA biosynthesis depleted CBB cycle derivatives and triggered plastid-associated stress responses. Our work demonstrated the feasibility of tapping the CBB cycle for HA synthesis and identified bottlenecks for plant-based production of carbohydrate polymers.

The increasing popularity of “superseeds” such as flax, sesame, amaranth and quinoa as functional foods raises the need for robust analytical methods for authentication purposes. In this work, a standardized workflow for the extraction, characterization and identification of unique peptides that may be used as markers to distinguish superseed species was investigated. Ammonium bicarbonate/urea (Ambi/urea) extraction, sodium dodecyl sulfate (SDS) buffer and trichloroacetic acid (TCA) precipitation were initially implemented and, based on the level and composition of the extracted proteins, the SDS buffer protocol was selected. Electrophoresis analysis revealed consistent protein profiles between biological replicates from each of the eleven seed species, confirming the reproducibility of the SDS buffer protocol. Targeted mass spectrometry successfully identified species-specific peptide markers for six of eleven superseeds investigated, including peptides from conlinins in flaxseed (WVQQAK), 11S globulins in sesame (LVYIER), oleosin in quinoa (DVGQTIESK), agglutin-like lectins in amaranth (CAGVSVIR), as well as cupin-like proteins in poppy seeds (INIVNSQK) and edestins in hemp seeds (FLQLSAER). Moreover, proteome cross-analysis allowed us to disqualify the isomeric peptide LTALEPTNR from 11S globulins present in amaranth and quinoa. However, no reliable markers were identified for chia, canihua, basil, black cumin, and psyllium seeds under current conditions. While this targeted proteomics approach shows promise for superseed authentication, comprehensive method validation and alternative strategies for marker-deficient species are required before routine implementation.

The seed-to-seedling transition represents a key developmental and metabolic switch in plants. Catabolism of seed storage reserves fuels germination and early seedling emergence until photosynthesis is established. The seed-to-seedling developmental transition is controlled by Polycomb repressive complex 2 (PRC2). However, the coordination of PRC2 activity and its contribution to transcriptional reprogramming during seedling establishment remain unknown. By analyzing H3K27me3 re-distribution and changes in gene transcription in the shoot and root tissues of heterotrophic and photoautotrophic Arabidopsis (Arabidopsis thaliana) seedlings, we reveal 2 phases of PRC2-mediated gene repression. The first phase is independent of light and photosynthesis and results in the irreversible repression of the embryo maturation program, marked by heterotrophy and reserve storage molecule biosynthesis. The second phase is associated with the repression of metabolic pathways related to germination and early seedling emergence, and H3K27me3 deposition in this phase is sensitive to photosynthesis inhibition. We show that preventing the transcription of the PRC2-repressed glyoxylate cycle gene ISOCITRATE LYASE promotes the vegetative phase transition in PRC2-depleted plants. Our findings underscore a key role of PRC2-mediated transcriptional repression in the coordinated metabolic and developmental switches that occur during seedling emergence and emphasize the close connection between metabolic and developmental identities.

Adaptation of crops to recurrent drought stress is crucial for maintaining agricultural productivity and achieving food security under changing climate. Guard cells, pivotal regulators of plant water usage and assimilation, are central to this adaptation process. However, the metabolic dynamics of guard cells under drought stress remain poorly understood, particularly in grapevine, a prominent crop grown in arid regions, and maize, a staple crop with substantial water requirements. In this study, differences in guard cells metabolism during drought stress of grapevine and maize were investigated by performing physiological and metabolomic analyses. Metabolomic analysis highlighted differential responses in amino acids and sugars, with grapevine guard cells displaying greater stability in amino acid and sugar signatures, while maize showed marked increases in sugar levels. These findings suggest two distinct adaptive strategies, a vigorous acclimation of guard cells, as observed in maize, and an attenuated acclimation of guard cells, shown in grapevine. Understanding these metabolic adjustments is helpful for enhancing drought resilience in agricultural systems.

The cytochrome b559 heterodimer is a conserved component of photosystem II whose physiological role in photosynthetic electron transfer is enigmatic. A particularly puzzling aspect of cytochrome b559 has been its presence in etiolated seedlings, where photosystem II is absent. Whether or not the cytochrome has a specific function in etioplasts is unknown. Here, we have attempted to address the function of cytochrome b559 by generating transplastomic tobacco (Nicotiana tabacum) plants that overexpress psbE and psbF, the plastid genes encoding the two cytochrome b559 apoproteins. We show that strong overaccumulation of the PsbE apoprotein can be achieved in etioplasts by suitable manipulations of the promoter and the translation signals, while the cytochrome b559 level is only moderately elevated. The surplus PsbE protein causes striking ultrastructural alterations in etioplasts; most notably, it causes a condensed prolamellar body and a massive proliferation of prothylakoids, with multiple membrane layers coiled into spiral-like structures. Analysis of plastid lipids revealed that increased PsbE biosynthesis strongly stimulated plastid lipid biosynthesis, suggesting that membrane protein abundance controls prothylakoid membrane biogenesis. Our data provide evidence for a structural role of PsbE in prolamellar body formation and prothylakoid biogenesis, and indicate that thylakoid membrane protein abundance regulates lipid biosynthesis in etioplasts.

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we used 13C-HCO3 labelling of tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling into this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results collectively suggest that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells. Highlight PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis and the TCA cycle, whilst previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.

Photoautotrophic organisms fix inorganic carbon (Ci) by two enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoenolpyruvate carboxylase (PEPC). RUBISCO assimilates Ci (CO2) into the 1-C position of 3-phosphoglycerate (3PGA). The Calvin-Benson-Basham (CBB) cycle redistributes fixed carbon atoms into 2,3-C2 of the same molecule. PEPC uses phosphoenolpyruvate (PEP) derived from 3PGA and assimilates Ci (HCO3-) into 4-C of oxaloacetate (OAA). 1,2,3-C3 of OAA and of its transaminase product aspartate originate directly from 1,2,3-C3 of 3PGA. Positional isotopologue analysis of aspartate, the main downstream metabolite of OAA in the model cyanobacterium Synechocystis sp. PCC 6803 ( Synechocystis ), allows differentiation between PEPC, RUBISCO, and CBB cycle activities within one molecule. We explored in source fragmentation of gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) at nominal mass resolution and GC-atmospheric pressure chemical ionization-MS (GC-APCI-MS) at high mass resolution. This enabled the determination of fractional [13][1]C enrichment (E[13][1]C) at each carbon position of aspartate. Two prevailing GC-MS derivatization methods, i.e. trimethylsilylation and tert-butyldimethylsilylation, were evaluated. The method was validated by [13][1]C-isotopomer mixtures of positional labeled aspartic acid. Combination with dynamic [13][1]CO2 labeling of Synechocystis cultures allowed direct measurements of PEPC activity in vivo alongside analyses of RUBISCO and CBB cycle activities. Accurate quantification of aspartate concentration and positional E[13][1]C provided molar Ci assimilation rates during the day and night phases of photoautotrophic Synechocystis cultures. The validated method offers several applications to characterize the photosynthetic Ci fixation in different organisms.### Competing Interest StatementThe authors have declared no competing interest. [1]: #ref-13