Strigolactones (SLs) modulate multiple aspects of plant development and stress physiology. This study investigated their role in maize response to abiotic stress by comparing an SL-biosynthesis mutant (zmccd8) with wild-type (WT) seedlings grown for 4 weeks in vermiculite under nutrient and water limitation. Plant growth, time-course pigment accumulation, targeted gene expression, and root transcriptomic profiles were analyzed. Our results showed that zmccd8 plants were largely unable to induce leaf senescence and efficient nutrient remobilization toward younger tissues under nitrogen (N) deficiency, a response previously associated with maize adaptation to low N availability. In parallel, the mutant developed a smaller root system, mainly due to limited adventitious root formation, particularly under N shortage. Root transcriptomic profiling revealed that N deficiency strongly affected WT plants, inducing extensive regulation of pathways involved in nitrogen metabolism and transport, secondary metabolism, ethylene and MAPK signaling, oxidative stress responses, and major transcription factor families. These responses were largely absent in the zmccd8 mutant, suggesting reduced transcriptional plasticity and compromised capacity to cope with stress-associated oxidative imbalance. Conversely, despite inducing substantial physiological and molecular responses, water stress elicited only modest SL-dependent regulation, with limited and heterogeneous changes between genotypes. Overall, our findings demonstrate that in maize, SLs act in a stress-specific manner, playing a predominant role in acclimatisation to nitrogen deficiency through coordinated regulation of senescence, nutrient remobilization, root architecture, and gene expression, while contributing more marginally to water-stress acclimatisation. These results provide new insights into SLs' role in shaping maize physiological plasticity under abiotic stress conditions.

Photorespiration is a costly cellular process that reduces photosynthetic efficiency. While mitigating photorespiratory losses could boost crop yields, the interconnection of photorespiration with other processes is increasingly recognized. Its high carbon turnover generates mitochondrial one-carbon (C1) metabolites, including formate, but their contribution to cellular C1 metabolism has remained unclear. DNA methylation is an important epigenetic modification that depends on methyl groups provided by folate-mediated C1 metabolism. Here we show that photorespiration supplies C1 units for DNA methylation in Arabidopsis. We demonstrate that carbon from formate is incorporated into 5-methylcytosine through the C1-tetrahydrofolate synthase pathway, which operates predominantly during the day. Elevated CO2 that suppresses photorespiration alters the methylome, especially when the serine-derived C1 supply, which compensates for a blocked formate-derived supply, is compromised. These findings establish a metabolic link between photorespiration and epigenome stability and provide a framework for understanding methylome dynamics under rising CO2 levels and other environmental influences on photorespiration.

The tight coordination of sulfur metabolism and growth regulation is predicated upon nutrient availability. Central to this balancing act is the utilization of cysteine (Cys) for the formation of methionine (Met) and S-adenosylmethionine (SAM). Plants that are severely deficient in regenerating Met due to reduced methylthioadenosine (MTA) nucleosidase activity experience numerous developmental abnormalities. Here, we assess the developmental, metabolic, and regulatory effects associated with decreased MTA recycling. We show that MTA over-accumulation predominantly occurs in reproductive tissues and leads to reduced levels of Cys, Met, and SAM, as well as elevated S-adenosylhomocysteine. These disruptions of primary sulfur utilization also lead to the misregulation of energy metabolism and altered cell cycle progression. RNA-seq experiments show a general down-regulation of many developmental and reproductive genes. Targeted metabolite analyses demonstrate clear impacts on the methyl index which are reflected in the results of bisulfite-sequencing experiments including global alterations in CG gene-body methylation levels and decreases of CHG and CHH methylation in transposable elements. Our findings demonstrate the broad impacts of MTA metabolism on plant development, sulfur utilization and the maintenance of the methyl index.

Stomatal closure allows plants to conserve water by reducing transpiration during drought. Surprisingly, the assimilation of the macronutrient sulfur is intimately connected to the drought stress response. This Tansley insight will only briefly touch on the general impact of sulfate assimilation on the production of drought-response metabolites. Instead, the emphasis will be on the unexpected role of cysteine in triggering guard cell-autonomous abscisic acid biosynthesis in response to diverse drought-associated stresses. A particular focus will be on identifying the chloroplast-localized cysteine synthase complex as a sensor hub that integrates long-distance soil-drying signals and local high-light signals to mediate stress-induced stomatal closure. Furthermore, we will discuss the emerging role of cysteine-derived sulfide as a signal in stomatal closure.

Abstract The N-terminal acetyltransferase B (NatB) acetylates ~20% of the eukaryotic proteome. However, the role of NatB-mediated N-terminal acetylation (NTA) for the regulation of the proteome fate remains unclear in eukaryotes. In this study, we demonstrate that CRISPR-Cas9-mediated deletion of NatB activity in plants results in significantly lowered global protein turnover due to decreased ubiquitin-proteasome system (UPS) activity and protein translation. Quantitative proteomics uncovers that NatB substrates are significantly enriched in the fraction of stabilized proteins in natb mutants. We provide direct evidence that the absent NTA of KIN11, a subunit of the autophagy-controlling energy sensor SnRK1, protects it from UPS-mediated destruction. The resulting accumulation of KIN11 is responsible for the increased resistance of natb mutants to energy limitation induced by prolonged darkness. Our findings establish NatB as a central regulator of UPS–autophagy interplay and highlight its role in maintaining proteome stability and enabling dynamic stress responses in plants.

Cysteine biosynthesis is the entry point of reduced sulfur into plant metabolism and underlies the formation of numerous sulfur-containing compounds essential for stress adaptation. Cysteine is produced by the consecutive action of serine acetyltransferase (SERAT) and O-acetylserine(thiol)lyase (OAS-TL), which assemble into the cysteine synthase complex (CSC). CSC formation is reversible and regulated by the cysteine precursors O-acetylserine (OAS) and sulfide, linking cysteine production to the cellular status of carbon, nitrogen, and sulfur. Traditionally, the CSC has been hypothesized as a metabolic sensor of the carbon/nitrogen and sulfur supply for cysteine biosynthesis. However, recent studies reveal a broader role. The CSC is present in multiple subcellular compartments and shows functional diversity across plant species. Emerging evidence shows that CSC dynamics are tightly integrated with environmental signaling pathways, enabling plants to coordinate sulfur metabolism with responses to stress conditions such as high light, drought, heavy metals, and pathogen challenge. In this review, we synthesize recent advances in the characterization of SERAT and OAS-TL proteins and highlight the CSC as a regulatory hub that integrates metabolic status with stress signaling to respond to specific environmental stimuli.

OBJECTIVE:Metabolic inflexibility has been shown to be associated with type 2 diabetes (T2D) and diabetic nephropathy (DN). However, data are lacking, proving that reconstitution of metabolic flexibility by using a 6-month periodic fasting (PF) regimen may improve albuminuria. METHODS:In this post hoc analysis of a randomized-controlled trial, we investigated whether the PF regimen enhanced metabolic flexibility in individuals with T2D and DN showing improvement of albuminuria (responders) compared to non-responders. Participants followed every month either a 5-day fasting-mimicking diet or a Mediterranean diet for 6 months. LC-MS/MS-based comprehensive metabolic profiling was performed in plasma samples before, during, and after the intervention. Changes in metabolomic patterns and enriched signalling pathways were analysed between study groups. RESULTS:PF induced a sustained shift toward enhanced fatty acid oxidation, lipid utilization, and amino acids turnover, particularly in responders. Responders exhibited persistent elevations in short-chain acylcarnitines and cholesteryl esters, indicating more efficient lipid oxidation and tighter integration of lipid metabolism with the tricarboxylic acid cycle. Increased glycine and serine levels suggested enhanced cellular maintenance, a protein-sparing effect, and a metabolic shift favouring lipid over carbohydrate. In contrast, non-responders demonstrated only transient and limited metabolic shifts. Unsupervised clustering identified distinct metabolic response patterns, reinforcing the potential of personalized dietary interventions. CONCLUSIONS:These findings demonstrate that diet-induced restoration of metabolic flexibility is associated with improved albuminuria in T2D, suggesting broader implications for precise nutritional strategies in diabetes management.

The tight coordination of sulfur metabolism and growth regulation is predicated upon nutrient availability. Central to this balancing act is the utilization of cysteine (Cys) for the formation of methionine (Met) and S-adenosylmethionine (SAM). Plants that are severely deficient in regenerating Met due to reduced methylthioadenosine (MTA) nucleosidase activity experience numerous developmental abnormalities. Here, we assess the developmental, metabolic, and regulatory effects associated with decreased MTA recycling. We show that MTA over-accumulation predominantly occurs in reproductive tissues and leads to reduced levels of Cys, Met, and SAM, as well as elevated S-adenosylhomocysteine. These disruptions of primary sulfur utilization also lead to the misregulation of energy metabolism and altered cell cycle progression. RNA-seq experiments show a general down-regulation of many developmental and reproductive genes. Targeted metabolite analyses demonstrate clear impacts on the methyl index which are reflected in the results of bisulfite-sequencing experiments including global alterations in CG gene-body methylation levels and decreases of CHG and CHH methylation in transposable elements. Our findings demonstrate the broad impacts of MTA metabolism on plant development, sulfur utilization and the maintenance of the methyl index. ### Competing Interest Statement The authors have declared no competing interest.

Mass spectrometry (MS)-based metabolomics has emerged as a powerful tool to address multifaceted biological questions. Commercial solutions like the ones developed at biocrates allow reliable and quantitative targeted metabolic profiling, including the conversion of the raw MS spectra into absolute concentrations of metabolites. These results can be exported for further analysis under several formats with varying levels of human-vs. machine-readability. The default output format is an Excel spreadsheet that favours human readability and therefore requires extra preparation steps for downstream bioinformatic analysis and data exploration. To streamline this next step for users of this platform, we developed MetAlyzer, an R package ( https://github.com/Lu-Group-UKHD/MetAlyzer ) specifically designed to handle the spreadsheets generated by WebIDQ, the biocrates workflow manager software. MetAlyzer converts WebIDQ-generated spreadsheets into flexible SummarizedExperiment objects and provides functions for data preprocessing, statistical testing, and visualization of differential metabolites. To further support data exploration and hypothesis generation by users without coding experience, we also developed an interactive and intuitive Shiny app ( https://metalyzer.shinyapps.io/MetAlyzer_ShinyApp/ ) that interfaces with MetAlyzer’s core functionality, enabling users to execute the complete analysis workflow without writing code. This combination can help scientists deepen their understanding of metabolomics results, supporting the broader adoption of metabolomics in the life sciences community.

Global warming intensifies drought and high light stress periods, causing severe water loss and decreased crop yield. The phytohormone abscisic acid (ABA) is the dominant signal governing stomatal closure and water loss. Here, we uncover three signaling axes triggered by soil dehydration and high light stress converging on the dynamic assembly of the cysteine-synthase-complex in chloroplasts (pCSC). We show that pCSC assembly triggers ABA biosynthesis and stomatal closure in response to the soil-drying signals, sulfate (axis 1) and CLE25 (axis 2), and the high light-induced oxylipin OPDA (axis 3). Loss of the pCSC increases sensitivity to soil-drying and impairs high light-induced stomatal closure. Our findings uncover that the dynamic assembly of the pCSC acts as a sensor hub, integrating local and long-distance stress signals to promote stomatal closure by supplying cysteine for ABA biosynthesis in guard cells. We applied this knowledge to generate a soil-drying resilient plant showing no growth penalty.

The ribosome-tethered N-terminal acetyltransferase A (NatA) acetylates 52% of soluble proteins in Arabidopsis thaliana. This co-translational modification of the N terminus stabilizes diverse cytosolic plant proteins. The evolutionary conserved Huntingtin yeast partner K (HYPK) facilitates NatA activity in planta, but in vitro, its N-terminal helix α1 inhibits human NatA activity. To dissect the regulatory function of HYPK protein domains in vivo, we genetically engineer CRISPR-Cas9 mutants expressing a HYPK fragment lacking all functional domains (hypk-cr1) or an internally deleted HYPK variant truncating helix α1 but retaining the C-terminal ubiquitin-associated (UBA) domain (hypk-cr2). We find that the UBA domain of HYPK is vital for stabilizing the NatA complex in an organ-specific manner. The N terminus of HYPK, including helix α1, is critical for promoting NatA activity on substrates starting with various amino acids. Consequently, deleting only 42 amino acids inside the HYPK N terminus causes substantial destabilization of the plant proteome and higher tolerance toward drought stress.

In humans and plants, 40% of the proteome is cotranslationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex is comprised of the catalytic subunit Nα-acetyltransferase 10 (NAA10) and the ribosome-anchoring subunit NAA15. The regulatory subunit Huntingtin Yeast Partner K (HYPK) and the acetyltransferase NAA50 join this complex in humans. Even though both are conserved in Arabidopsis (Arabidopsis thaliana), only AtHYPK is known to interact with AtNatA. Here we uncover the AtNAA50 interactome and provide evidence for the association of AtNAA50 with NatA at ribosomes. In agreement with the latter, a split-luciferase approach demonstrated close proximity of AtNAA50 and AtNatA in planta. Despite their interaction, AtNatA/HYPK and AtNAA50 exerted different functions in vivo. Unlike NatA/HYPK, AtNAA50 did not modulate drought tolerance or promote protein stability. Instead, transcriptome and proteome analyses of a novel AtNAA50-depleted mutant (amiNAA50) implied that AtNAA50 negatively regulates plant immunity. Indeed, amiNAA50 plants exhibited enhanced resistance to oomycetes and bacterial pathogens. In contrast to what was observed in NatA-depleted mutants, this resistance was independent of an accumulation of salicylic acid prior to pathogen exposure. Our study dissects the in vivo function of the NatA interactors HYPK and NAA50 and uncovers NatA-independent roles for NAA50 in plants.

Understanding the interplay of the proteome and the metabolome helps to understand cellular regulation and response. To enable robust inferences from such multi-omics analyses, we introduced and evaluated a workflow for combined proteome and metabolome analysis starting from a single sample. Specifically, we integrated established and individually optimized protocols for metabolomic and proteomic profiling (EtOH/MTBE and autoSP3, respectively) into a unified workflow (termed MTBE-SP3), and took advantage of the fact that the protein residue of the metabolomic sample can be used as a direct input for proteome analysis. We particularly evaluated the performance of proteome analysis in MTBE-SP3, and demonstrated equivalence of proteome profiles irrespective of prior metabolite extraction. In addition, MTBE-SP3 combines the advantages of EtOH/MTBE and autoSP3 for semi-automated metabolite extraction and fully automated proteome sample preparation, respectively, thus advancing standardization and scalability for large-scale studies. We showed that MTBE-SP3 can be applied to various biological matrices (FFPE tissue, fresh-frozen tissue, plasma, serum and cells) to enable implementation in a variety of clinical settings. To demonstrate applicability, we applied MTBE-SP3 and autoSP3 to a lung adenocarcinoma cohort showing consistent proteomic alterations between tumour and non-tumour adjacent tissue independent of the method used. Integration with metabolomic data obtained from the same samples revealed mitochondrial dysfunction in tumour tissue through deregulation of OGDH, SDH family enzymes and PKM. In summary, MTBE-SP3 enables the facile and reliable parallel measurement of proteins and metabolites obtained from the same sample, benefiting from reduced sample variation and input amount. This workflow is particularly applicable for studies with limited sample availability and offers the potential to enhance the integration of metabolomic and proteomic datasets.

Rice grains typically contain relatively high levels of toxic arsenic (As) but low levels of essential micronutrients. Biofortification of essential micronutrients while decreasing As accumulation in rice would benefit human nutrition and health. We generated transgenic rice expressing a gain-of-function mutant allele astol1 driven by the OsGPX1 promoter. astol1 encodes a plastid-localized O-acetylserine (thiol) lyase (OAS-TL) with Ser189Asn substitution (OsASTOL1S189N), which enhances cysteine biosynthesis by forming an indissociable cysteine synthase complex with its partner serine acetyltransferase (SAT). The effects on growth, As tolerance, and nutrient and As accumulation in rice grain were evaluated in hydroponic, pot and field experiments. The expression of OsASTOL1S189N in pOsGPX1::astol1 transgenic lines enhanced SAT activity, sulphate uptake, biosynthesis of cysteine, glutathione, phytochelatins and nicotianamine, and enhanced tolerance to As. The expression of OsASTOL1S189N decreased As accumulation while increased the accumulation of multiple macronutrients (especially sulphur, nitrogen and potassium) and micronutrients (especially zinc and selenium) in rice grain in a pot experiment and two field experiments, and had little effect on plant growth and grain yield. Our study provides a new strategy to genetically engineer rice to biofortify multiple essential nutrients, reducing As accumulation in rice grain and enhancing As tolerance simultaneously.

AbstractHypoglycemia triggers autonomic and endocrine counter-regulatory responses to restore glucose homeostasis, a response that is impaired in patients with diabetes and its long-term complication hypoglycemia-associated autonomic failure (HAAF). We show that insulin-evoked hypoglycemia is severely aggravated in mice lacking the cation channel proteins TRPC1, TRPC4, TRPC5, and TRPC6, which cannot be explained by alterations in glucagon or glucocorticoid action. By using various TRPC compound knockout mouse lines, we pinpointed the failure in sympathetic counter-regulation to the lack of the TRPC5 channel subtype in adrenal chromaffin cells, which prevents proper adrenaline rise in blood plasma. Using electrophysiological analyses, we delineate a previously unknown signaling pathway in which stimulation of PAC1 or muscarinic receptors activates TRPC5 channels in a phospholipase-C-dependent manner to induce sustained adrenaline secretion as a crucial step in the sympathetic counter response to insulin-induced hypoglycemia. By comparing metabolites in the plasma, we identified reduced taurine levels after hypoglycemia induction as a commonality in TRPC5-deficient mice and HAAF patients.

Growth of eukaryotic cells is regulated by the target of rapamycin (TOR). The strongest activator of TOR in metazoa is amino acid availability. The established transducers of amino acid sensing to TOR in metazoa are absent in plants. Hence, a fundamental question is how amino acid sensing is achieved in photo-autotrophic organisms. Here we demonstrate that the plant Arabidopsis does not sense the sulfur-containing amino acid cysteine itself, but its biosynthetic precursors. We identify the kinase GCN2 as a sensor of the carbon/nitrogen precursor availability, whereas limitation of the sulfur precursor is transduced to TOR by downregulation of glucose metabolism. The downregulated TOR activity caused decreased translation, lowered meristematic activity, and elevated autophagy. Our results uncover a plant-specific adaptation of TOR function. In concert with GCN2, TOR allows photo-autotrophic eukaryotes to coordinate the fluxes of carbon, nitrogen, and sulfur for efficient cysteine biosynthesis under varying external nutrient supply.

Over the past few decades, a close relationship between sulfur (S) and iron (Fe) in terms of functionality and nutrition was demonstrated in the tomato. However, very little is known about the regulatory mechanisms underlying S/Fe interactions. Recently, the potential role of citrate in plant adaptation to Fe deficiency and combined S and Fe deficiency has been described. It is known that an impaired organic acid metabolism may stimulate a retrograde signal, which has been proven to be linked to the Target of Rapamycin (TOR) signaling in yeast and animal cells. Recent reports provided evidence of TOR involvement in S nutrient sensing in plants. This suggestion prompted us to investigate whether TOR may play a role in the cross-talk of signaling pathway occurring during plant adaptation to combined nutrient deficiency of Fe and S. Our results revealed that Fe deficiency elicited an increase of TOR activity associated with enhanced accumulation of citrate. In contrast, S deficiency resulted in decreased TOR activity and citrate accumulation. Interestingly, citrate accumulated in shoots of plants exposed to combined S/Fe deficiency to values between those found in Fe- and S-deficient plants, again correlated with TOR activity level. Our results suggest that citrate might be involved in establishing a link between plant response to combined S/Fe deficiency and the TOR network.

Cysteine biosynthesis is essential for translation and represents the entry point of reduced sulfur into plant metabolism. The two consecutively acting enzymes serine acetyltransferase (SAT) and O-acetylserine-thiol-lyase catalyse cysteine production and form the cysteine synthase complex, in which SAT is activated. Here we show that tobacco (Nicotiana tabacum) expressing active SAT in plastids (referred to as PSA lines) shows substantial cysteine accumulation in plastids. Remarkably, enhanced cysteine production in plastids entirely abolished granal stack formation, impaired photosynthesis capacity, and decreased the number of chloroplasts in mesophyll cells of the PSA lines. A transgenic tobacco line expressing active SAT in the cytosol accumulated comparable amounts of thiols but displayed no phenotype. To dissect the consequences of cysteine synthase complex formation from enhanced SAT activity in tobacco plastids, we expressed an enzymatically inactive SAT that can still form the cysteine synthase complex in tobacco plastids (PSI lines). The PSI lines were indistinguishable from the PSA lines, although the PSI lines displayed no increase in plastid-localized SAT activity. Neither PSA lines nor PSI lines suffered from an oxidized redox environment in plastids that could have been causative for the disturbed photosynthesis. From these findings, we infer that the association of the plastid cysteine synthase complex itself triggers a signaling cascade controlling sulfur assimilation and photosynthetic capacity in leaves.

Plants, unlike animals, respond to environmental challenges with comprehensive developmental transitions that allow them to cope with these stresses. Here we discovered that antagonistic activation of the Target of Rapamycin (TOR) kinase in Arabidopsis thaliana roots and shoots is essential for the nutrient deprivation-induced increase in the root-to-shoot ratio to improve foraging for mineral ions. We demonstrate that sulfate limitation-induced downregulation of TOR in shoots activates autophagy, resulting in enhanced carbon allocation to the root. The allocation of carbon to the roots is facilitated by the specific upregulation of the sucrose-transporter genes SWEET11/12 in shoots. SWEET11/12 activation is indispensable for enabling sucrose to act as a carbon source for growth and as a signal for tuning root apical meristem activity via glucose-TOR signaling. The sugar-stimulated TOR activity in the root suppresses autophagy and maintains root apical meristem activity to support root growth to enhance mining for new sulfate resources in the soil. We provide direct evidence that the organ-specific regulation of autophagy is essential for the increased root-to-shoot ratio in response to sulfur limitation. These findings uncover how sulfur limitation controls the central sensor kinase TOR to enable nutrient recycling for stress-induced morphological adaptation of the plant body.