Aluminium (Al) toxicity is a major constraint on crop productivity in acidic soils (pH < 5.5), severely impairing root growth and function. While organic acid (OA)-mediated chelation and DNA damage response (DDR)-mediated control of cell division are recognized as key tolerance mechanisms, their integration at the cellular level remains poorly understood. Here, we employed Translating Ribosome Affinity Purification (TRAP) to achieve cell-type-specific gene expression profiling of Arabidopsis thaliana roots, enabling the dissection of molecular responses in whole roots, the quiescent centre, and cortical tissues under Al stress. Our results reveal a spatially resolved reprogramming in which mitochondrial metabolism, particularly tricarboxylic acid (TCA) cycle activity, is preferentially induced in differentiated tissues, whereas meristematic regions display limited metabolic adjustment. In parallel, genes associated with cell cycle regulation and DNA damage checkpoints exhibit pronounced, cell-type-dependent modulation, indicating a coordinated DDR-driven control of root growth dynamics. These findings uncover a previously underexplored functional interplay between mitochondrial metabolic pathways and DDR signaling in mediating intracellular Al detoxification. By linking metabolic reprogramming with cell cycle regulation in a spatially resolved manner, this study provides new mechanistic insights into plant tolerance to Al toxicity. Such knowledge advances our understanding of plant adaptation to hazardous soil conditions and offers potential targets for developing crops with enhanced resilience to Al stress.

Cowpea severe mosaic virus (CPSMV) represents a major constraint for cowpea (Vigna unguiculata (L.) Walp.) cultivation. However, the metabolic basis underlying CPSMV resistance remains poorly understood. Given the importance of metabolism for plant defence against pathogens, we hypothesized that cowpea resistance to CPSMV is associated to the activation of defence-related pathways from both primary and secondary metabolisms. We investigated the metabolic dynamics associated with CPSMV infection in the resistant cowpea genotype Macaibo using a time-resolved, network-based metabolomics approach. Conventional RT-PCR suggests that the intensity of the CPSMV coat protein band increased in the first eight Hours After CPSMV Inoculation (HAI) but appeared reduced in the following 72 HAI. Metabolomic analyses revealed that CPSMV infection had little impact on secondary metabolism, whereas several primary metabolites were significantly altered over time. Transient increases in fumarate, pyruvate, and several amino acids, including alanine, asparagine, glutamine, glutamate, isoleucine, leucine, proline, serine, threonine, and valine, were observed at 48 HAI compared with mock-treated leaves. Network analysis indicated that overall metabolic network density and heterogeneity remained relatively stable during infection. However, a highly connected metabolic module composed of the branched-chain amino acids (BCAAs) isoleucine, leucine, and valine emerged in infected leaves. Our results indicate that CPSMV infection reshapes primary metabolic dynamics without extensive alterations in secondary metabolism during early infection in cowpea leaves. Despite these metabolic adjustments, the overall metabolic network structure remained stable, suggesting that a robust metabolic reprograming, including the activation of BCAA-associated pathways, may help buffer viral perturbation in cowpea leaves.

Aluminium (Al) toxicity is a critical factor that limits plant growth in acidic soils (pH < 5.5) worldwide. Intracellular Al can bind to various biomolecules and significantly affect gene expression, protein biosynthesis, and cell membrane integrity, ultimately inhibiting nutrients and water uptake. This leads to the arrest of root cell divisions and growth. Organic acids (OAs) produced in mitochondrial-related reactions are strongly linked to Al tolerance, as they bind to Al and neutralize its toxic effects. Recently, the manipulation of root cell divisions through DNA checkpoints has been proposed as an alternative to increase Al tolerance. In this context, molecular interactions between these two mechanisms could potentially enhance plant tolerance to Al toxicity. Here, we investigate this topic using a translatome approach, which enables specific analyses of root cell types (namely, whole root, the quiescent centre region, and the cortical region). This approach offers a promising tool for unravelling how mitochondrial metabolism, particularly the TCA cycle, and the DNA damage response (DDR) pathway are interconnected in Al-mitigation process. Our results revealed that the TCA cycle is induced in differentiated cells, with no significant alteration in meristematic cells, while genes associated with cell cycle progression and DNA checkpoints show differential expression across the distinct cell types. Collectively, our data contribute to understand cell-specific internal detoxification mechanism related to Al tolerance in plants. This knowledge could assist crop breeding by providing a better understanding of the cellular physiological responses to Al toxicity. ### Competing Interest Statement The authors have declared no competing interest.

Understanding stomatal response to fluctuations in atmospheric carbon dioxide concentration ([CO2]) and water deficit is important to predict the overall plant performance under these two major environmental stressing factors. In Arabidopsis, slow-type anion channel (SLAC1) plays a central role in the control of stomatal closure with impacts on water use efficiency. Here we investigated whether the stomatal component also plays a central role in limiting photosynthesis in plants showing constitutive higher stomatal conductance (gs), such as slac1 mutants, under high CO2 levels and water restriction. Under these conditions, slac1 plants showed similar photosynthetic performance and growth when compared to wild-type (WT) plants. Furthermore, our results suggest that the constitutive increased stomatal aperture in slac1 plants is associated with a reorganization of primary metabolism in guard cells, in addition to the ion transport impairment, previously observed in slac1 mutants. Altogether, our results indicate that higher gs in slac1 plants are not translated into enhanced photosynthetic performance and growth, independently of CO2 levels and watering conditions.

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.

Although significant efforts to produce carotenoid-enriched foods either by biotechnology or traditional breeding strategies have been carried out, our understanding of how changes in the carotenoid biosynthesis might affect overall plant performance remains limited. Here, we investigate how the metabolic machinery of well characterized tomato carotenoid mutant plants [namely crimson (old gold-og), Delta carotene (Del) and tangerine (t)] adjusts itself to varying carotenoid biosynthesis and whether these adjustments are supported by a reprogramming of photosynthetic and central metabolism in the source organs (leaves). We observed that mutations og, Del and t did not greatly affect vegetative growth, leaf anatomy and gas exchange parameters. However, an exquisite metabolic reprogramming was recorded on the leaves, with an increase in levels of amino acids and reduction of organic acids. Taken together, our results show that despite minor impacts on growth and gas exchange, carbon flux is extensively affected, leading to adjustments in tomato leaves metabolism to support changes in carotenoid biosynthesis on fruits (sinks). We discuss these data in the context of our current understanding of metabolic adjustments and carotenoid biosynthesis as well as regarding to improving human nutrition.

L-serine (Ser) and L-glycine (Gly) are critically important for the overall functioning of primary metabolism. We investigated the interaction of the phosphorylated pathway of Ser biosynthesis (PPSB) with the photorespiration-associated glycolate pathway of Ser biosynthesis (GPSB) using Arabidopsis thaliana PPSB-deficient lines, GPSB-deficient mutants, and crosses of PPSB with GPSB mutants. PPSB-deficient lines mainly showed retarded primary root growth. Mutation of the photorespiratory enzyme Ser-hydroxymethyltransferase 1 (SHMT1) in a PPSB-deficient background resumed primary root growth and induced a change in the plant metabolic pattern between roots and shoots. Grafting experiments demonstrated that metabolic changes in shoots were responsible for the changes in double mutant development. PPSB disruption led to a reduction in nitrogen (N) and sulfur (S) contents in shoots and a general transcriptional response to nutrient deficiency. Disruption of SHMT1 boosted the Gly flux out of the photorespiratory cycle, which increased the levels of the one-carbon (1C) metabolite 5,10-methylene-tetrahydrofolate and S-adenosylmethionine. Furthermore, disrupting SHMT1 reverted the transcriptional response to N and S deprivation and increased N and S contents in shoots of PPSB-deficient lines. Our work provides genetic evidence of the biological relevance of the Ser-Gly-1C metabolic network in N and S metabolism and in interorgan metabolic homeostasis.

Plants encounter various environmental stresses throughout development, including shade, high light, drought, hypoxia, extreme temperatures, and metal toxicity, all of which adversely affect growth and productivity. Organic acids (OAs), besides serving as intermediates in the tricarboxylic acid (TCA) cycle, play crucial roles in multiple metabolic pathways and cellular compartments, including mitochondrial metabolism, amino acid metabolism, the glyoxylate cycle, and the photosynthetic mechanisms of C4 and CAM plants. OAs contribute to stress tolerance by acting as root chelating agents, regulating ATP production, and providing reducing power for detoxifying reactive oxygen species (ROS). They also participate in the biosynthesis of solutes involved in stress signaling and osmoregulation, particularly during stomatal movements. This review explores how OAs regulate plant metabolism in response to specific abiotic stresses, emphasizing the increased production of malate, citrate, and succinate, which enhance resilience to water deficits, metal toxicity, and flooding. Since these mechanisms involve intricate metabolic networks, changes in OA metabolism present promising and underexplored potential for agriculture. Understanding these mechanisms could lead to innovative strategies for developing crops with greater resilience to climate change, whether through genetic manipulation or by selecting varieties with favorable metabolic responses to stress.

Plants are negatively affected by aluminum (Al) in acidic soils (pH ≤ 5.0), which impairs root growth and ultimately plant yield. Organic acids are closely related to Al neutralization improving the metal tolerance with an expansive metabolic cost. Here, we investigated phenotypic, metabolic, and genetic responses of three Arabidopsis thaliana ecotypes, Columbia (Col-0), Wassilewskija (Ws) and Landsberg erecta (Ler) in response to Al. By comparing the respective control plants with Al-exposed plants, the ecotypes Col-0 and Ws displayed stronger reductions in root growth and reproductive yield than Ler. All ecotypes presented high expression of ALMT1, an Al-resistance associated gene. Further analyses revealed a large accumulation of diverse amino acids, carbohydrates and organic acids following Al stress in both shoot and root tissues of Col-0 and Ws plants, but not in Ler. Altogether, our results suggest that a higher capacity for using and translocating reduced carbon molecules seems crucial to overcoming Al stress in the Ler ecotype. In addition, lower expenses in carbon molecules might be linked to a higher capacity to deal with Al stress, supporting a tight relationship between primary metabolism and Al stress responses. Finally, novel insights on the influence of Al over plant growth and primary metabolism in both shoots and roots are described.

Among the adenylate carriers identified in Arabidopsis thaliana, only the AMP/ATP transporter ADNT1 shows increased expression in roots under waterlogging stress conditions. Here, we investigated the impact of a reduced expression of ADNT1 in A. thaliana plants submitted to waterlogging conditions. For this purpose, an adnt1 T-DNA mutant and two ADNT1 antisense lines were evaluated. Following waterlogging, ADNT1 deficiency resulted in a reduced maximum quantum yield of PSII electron transport (significantly for adnt1 and antisense Line 10), indicating a higher impact caused by the stress in the mutants. In addition, ADNT1 deficient lines showed higher levels of AMP in roots under nonstress condition. This result indicates that the downregulation of ADNT1 impacts the levels of adenylates. ADNT1-deficient plants exhibited a differential expression pattern of hypoxia-related genes with an increase in non-fermenting-related-kinase 1 (SnRK1) expression and upregulation of adenylate kinase (ADK) under stress and non-stress conditions. Together, these results indicated that the lower expression of ADNT1 is associated with an early "hypoxic status" due to the perturbation of the adenylate pool caused by reduced AMP import by mitochondria. This perturbation, which is sensed by SnRK1, results in a metabolic reprogramming associated with early induction of the fermentative pathway in ADNT1 deficient plants.

Deschampsia antarctica is one of the only two native vascular plants in Antarctica, mostly located in the ice-free areas of the Peninsula's coast and adjacent islands. This region is characterized by a short growing season, frequent extreme climatic events, and soils with reduced nutrient availability. However, it is unknown whether its photosynthetic and stress tolerance mechanisms are affected by the availability of nutrients to deal with this particular environment. We studied the photosynthetic, primary metabolic, and stress tolerance performance of D. antarctica plants growing on three close sites (<500 m) with contrasting soil nutrient conditions. Plants from all sites showed similar photosynthetic rates, but mesophyll conductance and photobiochemistry were more limiting (~25%) in plants growing on low-nutrient availability soils. Additionally, these plants showed higher stress levels and larger investments in photoprotection and carbon pools, most probably driven by the need to stabilize proteins and membranes, and remodel cell walls. In contrast, when nutrients were readily available, plants shifted their carbon investment towards amino acids related to osmoprotection, growth, antioxidants, and polyamines, leading to vigorous plants without appreciable levels of stress. Taken together, these findings demonstrate that D. antarctica displays differential physiological performances to cope with adverse conditions depending on resource availability, allowing it to maximize stress tolerance without jeopardizing photosynthetic capacity.

Cassava's storage roots represent one of the most important sources of nutritional carbohydrates worldwide. Particularly, smallholder farmers in sub-Saharan Africa depend on this crop plant, where resilient and yield-improved varieties are of vital importance to support steadily increasing populations. Aided by a growing understanding of the plant's metabolism and physiology, targeted improvement concepts already led to visible gains in recent years. To expand our knowledge and to contribute to these successes, we investigated storage roots of eight cassava genotypes with differential dry matter content from three successive field trials for their proteomic and metabolic profiles. At large, the metabolic focus in storage roots transitioned from cellular growth processes toward carbohydrate and nitrogen storage with increasing dry matter content. This is reflected in higher abundance of proteins related to nucleotide synthesis, protein turnover, and vacuolar energization in low starch genotypes, while proteins involved in sugar conversion and glycolysis were more prevalent in high dry matter genotypes. This shift in metabolic orientation was underlined by a clear transition from oxidative- to substrate-level phosphorylation in high dry matter genotypes. Our analyses highlight metabolic patterns that are consistently and quantitatively associated with high dry matter accumulation in cassava storage roots, providing fundamental understanding of cassava's metabolism as well as a data resource for targeted genetic improvement.

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 carried out a13C-HCO3 labelling experiment in 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 altered the metabolic network structure of guard cells and 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 and leads to more drastic reductions in the content of 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 indicate that several metabolic pathways are redirected following PEPc-mediated CO2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed 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.

In cellular circumstances where carbohydrates are scarce, plants can use alternative substrates for cellular energetic maintenance. In plants, the main protein reserve is present in the chloroplast, which contains most of the total leaf proteins and represents a rich source of nitrogen and amino acids. Autophagy plays a key role in chloroplast breakdown, a well-recognised symptom of both natural and stress-induced plant senescence. Remarkably, an autophagic-independent route of chloroplast degradation associated with chloroplast vesiculation (CV) gene was previously demonstrated. During extended darkness, CV is highly induced in the absence of autophagy, contributing to the early senescence phenotype of atg mutants. To further investigate the role of CV under dark-induced senescence conditions, mutants with low expression of CV (amircv) and double mutants amircv1xatg5 were characterised. Following darkness treatment, no aberrant phenotypes were observed in amircv single mutants; however, amircv1xatg5 double mutants displayed early senescence and altered dismantling of chloroplast and membrane structures under these conditions. Metabolic characterisation revealed that the functional lack of both CV and autophagy leads to higher impairment of amino acid release and differential organic acid accumulation during starvation conditions. The data obtained are discussed in the context of the role of CV and autophagy, both in terms of cellular metabolism and the regulation of chloroplast degradation.

Plants are constantly exposed to environmental changes that affect their performance. Metabolic adjustments are crucial to controlling energy homoeostasis and plant survival, particularly during stress. Under carbon starvation, coordinated reprogramming is initiated to adjust metabolic processes, which culminate in premature senescence. Notwithstanding, the regulatory networks that modulate transcriptional control during low energy remain poorly understood. Here, we show that the WRKY45 transcription factor is highly induced during both developmental and dark-induced senescence. The overexpression of Arabidopsis WRKY45 resulted in an early senescence phenotype characterized by a reduction of maximum photochemical efficiency of photosystem II and chlorophyll levels in the later stages of darkness. The detailed metabolic characterization showed significant changes in amino acids coupled with the accumulation of organic acids in WRKY45 overexpression lines during dark-induced senescence. Furthermore, the markedly upregulation of alternative oxidase (AOX1a, AOX1d) and electron transfer flavoprotein/ubiquinone oxidoreductase (ETFQO) genes suggested that WRKY45 is associated with a dysregulation of mitochondrial signalling and the activation of alternative respiration rather than amino acids catabolism regulation. Collectively our results provided evidence that WRKY45 is involved in the plant metabolic reprogramming following carbon starvation and highlight the potential role of WRKY45 in the modulation of mitochondrial signalling pathways.

Auxin is an important hormone playing crucial roles during fruit growth and ripening; however, the metabolic impact of changes in auxin signalling during tomato (Solanum lycopersicum L.) ripening remains unclear. Here, we investigated the significance of changes in auxin signalling during different stages of fruit development by analysing changes in tomato fruit quality and primary metabolism using mutants with either lower or higher auxin sensitivity [diageotropica (dgt) and entire mutants, respectively]. Altered auxin sensitivity modifies metabolism, through direct impacts on fruit respiration and fruit growth. We verified that the dgt mutant plants exhibit reductions in fruit set, total fruit dry weight, fruit size, number of seeds per fruit, and fresh weight loss during post-harvest. Sugar accumulation was associated with delayed fruit ripening in dgt, probably connected with reduced ethylene levels and respiration, coupled with a lower rate of starch degradation. In contrast, despite exhibiting parthenocarpy, increased auxin perception (entire) did not alter fruit ripening, leading to only minor changes in primary metabolism. By performing a comprehensive analysis, our results connect auxin signalling and metabolic changes during tomato fruit development, indicating that reduced auxin signalling led to extensive changes in sugar concentration and starch metabolism during tomato fruit ripening.

Rapid population growth and increasing demand for food, feed, and bioenergy in these times of unprecedented climate change require breeding for increased biomass production on the world's croplands. To accelerate breeding programs, knowledge of the relationship between biomass features and underlying gene networks is needed to guide future breeding efforts. To this end, large-scale multiomics datasets were created with genetically diverse maize lines, all grown in long-term organic and conventional cropping systems. Analysis of the datasets, integrated using regression modeling and network analysis revealed key metabolites, elements, gene transcripts, and gene networks, whose contents during vegetative growth substantially influence the build-up of plant biomass in the reproductive phase. We found that S and P content in the source leaf and P content in the root during the vegetative stage contributed the most to predicting plant performance at the reproductive stage. In agreement with the Gene Ontology enrichment analysis, the cis-motifs and identified transcription factors associated with upregulated genes under phosphate deficiency showed great diversity in the molecular response to phosphate deficiency in selected lines. Furthermore, our data demonstrate that genotype-dependent uptake, assimilation, and allocation of essential nutrient elements (especially C and N) during vegetative growth under phosphate starvation plays an important role in determining plant biomass by controlling root traits related to nutrient uptake. These integrative multiomics results revealed key factors underlying maize productivity and open new opportunities for efficient, rapid, and cost-effective plant breeding to increase biomass yield of the cereal crop maize under adverse environmental factors.

The gene coding for the P200 protein of the bacterium, Mycoplasma pneumoniae (Mp), was cloned and sequenced. The sequence-derived data and biochemical data indicated that P200 has several features in common with the well characterized cytadherence-associated proteins, HMW1 and HMW3. These features consist of abnormal migration in SDS-PAGE, a central acidic domain with a high Pro content, repeated peptide blocks within the Pro-rich domain and P200 partitioning similar to HMW1 and HMW3 in the insoluble fraction after extraction of Mp with the detergent Triton X-100.

Among the many factors affecting soybean resistance against Asian soybean rust (ASR), the nutritional status of plants plays a pivotal role. Even though the beneficial effects of nickel (Ni) and glyphosate (Gl) on ASR control have been reported, the mechanism(s) involved in disease control promoted by Gl or its combination with Ni remains to be elucidated. Therefore, this study investigated the effects of Ni, Gl, and their combination on both defense mechanisms and antioxidative metabolism of soybean plants from a Gl-resistant cultivar infected by Phakopsora pachyrhizi. Severity of ASR was significantly reduced by 37, 68, and 77%, respectively, for Ni, Gl, and Ni + Gl sprayed plants compared to water-sprayed plants (control treatment). Infected and water-sprayed plants showed earlier and higher levels of reactive oxygen species and malondialdehyde than plants sprayed with Ni or Gl. The antioxidative metabolism of infected plants from control, Ni, Gl, and Ni + Gl treatments was not affected. For inoculated plants sprayed with Ni or Gl, beta-1,3-glucanase (GLU) activity and phenolics level were greater. Additionally, Ni-sprayed and infected plants showed greater phenylalanine ammonia-lyase (PAL) activity and earlier lignin production than water-sprayed and infected plants. Polyphenoloxidase activity was higher at 5 days after inoculation for Gl-sprayed plants regardless of P. pachyrhizi infection. The findings of this study shed light on an intrinsic interplay between Ni and Gl to decrease ASR symptoms, but changes in host defense responses were barely potentiated by Gl. Independent of Gl, Ni reduced ASR symptoms due to modulation of soybean defense mechanisms (higher GLU and PAL activities and great production of phenolics and lignin) against ASR.

Abstract Altering plant water use efficiency (WUE) is a promising approach for achieving sustainable crop production in changing climate scenarios. Here, we show that WUE can be tuned by alleles of a single gene discovered in elite maize (Zea mays) breeding material. Genetic dissection of a genomic region affecting WUE led to the identification of the gene ZmAbh4 as causative for the effect. CRISPR/Cas9-mediated ZmAbh4 inactivation increased WUE without growth reductions in well-watered conditions. ZmAbh4 encodes an enzyme that hydroxylates the phytohormone abscisic acid (ABA) and initiates its catabolism. Stomatal conductance is regulated by ABA and emerged as a major link between variation in WUE and discrimination against the heavy carbon isotope (Δ13C) during photosynthesis in the C4 crop maize. Changes in Δ13C persisted in kernel material, which offers an easy-to-screen proxy for WUE. Our results establish a direct physiological and genetic link between WUE and Δ13C through a single gene with potential applications in maize breeding.