Débora Gonçalves Gouveia

Lysine metabolism connects amino acid biosynthesis with mitochondrial respiration through coordinated synthesis and degradation pathways that interface with the tricarboxylic acid (TCA) cycle and the mitochondrial electron transport chain (mETC). The critical role of this pathway is underscored by the embryonic lethality of the Arabidopsis L,L-diaminopimelate aminotransferase mutant (dapat), which is defective in a key step of lysine biosynthesis. Hypomorphic dapat mutant plants exhibit profound metabolic reprogramming and a constitutive energy-limited phenotype under non-stress conditions, suggesting that lysine biosynthesis contributes to basal metabolic homeostasis. We therefore hypothesized that impaired lysine biosynthesis restricts metabolic flexibility, but that changes in CO2 availability may partially compensate through carbon and nitrogen remodeling. To test this, we performed comprehensive metabolite profiling of wild-type and dapat mutant plants under low-, ambient-, and elevated-CO2 conditions. Elevated CO2 (eCO2) induced broad reorganization of carbon and nitrogen metabolism in both genotypes, reducing photorespiratory and stress-associated amino acids and modulating key metabolic ratios, including glycine/serine and malate/aspartate, in a genotype- and time-dependent manner. Proteomics revealed DAPAT photosynthetic compensation under ambient CO2, with RuBisCO over-accumulation and suppressed TOR activity, as indicated by decreased RPS6 phosphorylation, which eCO2 restored in parallel with phenotypic rescue. These adjustments were associated with partial attenuation of the DAPAT metabolic imbalance. Our findings reveal functional connections among lysine biosynthesis, photorespiration, and CO2-responsive metabolic flexibility, demonstrating that lysine metabolism serves as a regulatory node linking amino acid homeostasis to carbon availability and respiratory adjustment. This provides mechanistic insight into how plants maintain energy balance under fluctuating CO2 environments.

Global warming is increasing the frequency and intensity of high-temperature episodes, limiting plant productivity. However, the molecular mechanisms integrating primary metabolism with the heat stress response remains poorly understood. Here, we show that lysine biosynthesis contributes to the coordination of physiological, metabolic and transcriptional responses to heat stress in Arabidopsis thaliana. We compared wild-type, the lysine-biosynthesis mutant dapat, and the salicylic acid (SA)-biosynthesis and signaling mutants sid2-1 and npr1-3 under prolonged warming (6C above control for 7 days) and heat shock (38C for 6 h), followed by recovery. We assessed growth, gas exchange, photosynthetic performance, free SA, salicylic acid glucoside (SAG), salicylic acid glucose ester (SGE), and total SA content, primary metabolite profiles, heat-stress-responsive gene expression and transcriptome-wide changes by RNA sequencing. Before heat stress, dapat mutant presented a distinct metabolic state, marked by amino-acid accumulation, altered organic-acid profiles, reduced soluble sugars and elevated endogenous SA. This metabolic configuration persisted during prolonged warming, whereas WT and SA-pathway mutants underwent more dynamic reprogramming. Heat shock, by contrast, elicited a more convergent response across genotypes. Despite reduced basal PSII efficiency, dapat maintained photosynthetic performance during prolonged warming and recovered. Its transcriptional response, however, differed from that of WT and SA-pathway mutants: selected heat-responsive genes were constitutively or more strongly expressed, whereas some canonical heat-stress regulators showed weaker induction after heat shock. RNA-seq further revealed a largely conserved core heat-shock response but genotype-dependent regulation of defense, hormone and amino-acid-metabolism programs, particularly during recovery. Together, these findings indicate that impaired DAPAT activity establishes a metabolically primed but energetically constrained state that reshapes gas exchange, photosynthetic acclimation and heat-responsive transcription. Lysine homeostasis therefore emerges as a regulatory node linking primary metabolism and SA accumulation with SA-dependent and SA-independent components of heat-stress acclimation.

The Capsicum genus shows remarkable phenotypic diversity, making it an excellent system to study non‐climacteric fruit ripening. Unlike climacteric model species, such as tomato (Solanum lycopersicum), the regulatory networks linking transcriptome and metabolome to fruit quality traits remain poorly understood in non‐climacteric crop species. To address this gap, we selected four contrasting C. chinense accessions and performed integrated transcriptomic and metabolomic analyses to investigate the regulation of total soluble solids (TSS) accumulation. We profiled 16 922 genes and 63 metabolic features across two fruit developmental stages (immature and mature), including sugars, organic acids, capsaicinoids, and other secondary metabolites. We identified more than 3800 differentially expressed genes and detected strong correlations between gene expression and metabolite levels. Some metabolites, including chlorophylls, carotenoids, and starch, showed consistent temporal trends across genotypes, while others showed genotype‐dependent variation. Our results demonstrate that pepper fruit ripening involves a transcriptional shift toward soluble sugar accumulation, characterized by upregulation of starch‐hydrolyzing enzymes (CaAMY1/2, CaBAM1), invertases (CaINV1, CaCWINV3), sucrose synthase (CaSUS2), and the sugar transporter CaSWEET10, alongside downregulation of the starch biosynthetic gene CaSBE1. Among these, CaSUS2, CaSWEET10, and CaBAM1 emerged as key candidate regulators. These results suggest that coordinated starch degradation and sucrose transport primarily drive TSS increase, while secondary metabolism undergoes independent shifts that characterize other aspects of the ripening process.

Lysine represents a metabolic hub linking mitochondrial energy status with amino acid catabolism, yet its role in coordinating plant growth and development remains poorly characterised. The dapat mutant, which is deficient in L,L ‐diaminopimelate aminotransferase (DAPAT), exhibits severe growth inhibition associated with broad metabolic imbalance. This metabolic reprogramming is characterised by simultaneous depletion of carbohydrates (sugars and starch), and organic acids, accompanied by elevated amino acid pools, reflecting a profound disruption of carbon–nitrogen balance. These metabolic constraints coincide with altered expression of gibberellin (GA) biosynthetic genes ( CPS , KS , GA3ox1 ), suggesting a potential link between lysine metabolism, energy status, and GA‐mediated growth regulation. We therefore hypothesised that aberrant developmental defects in dapat arise from impaired GA metabolism. To test this hypothesis, we treated mutant plants with exogenous GA₃. Multivariate metabolomic analysis revealed a striking disconnect: GA restored the normal morphological phenotype of dapat but failed to correct the underlying metabolic dysfunction. GA‐treated dapat plants maintain amino acid remobilisation despite growth restoration, as indicated by pronounced DIN6 induction, a marker of catabolic stress following energy starvation. These findings establish that lysine biosynthesis represents a critical metabolic constraint on plant development, and that growth hormone signalling can decouple morphological rescue from metabolic homoeostasis. This work exposes a fundamental mechanistic uncoupling between hormone‐induced growth and energy balance in lysine‐deficient plants, highlighting the critical role of lysine metabolism in integrating developmental and energetic cues.

Lysine metabolism has been studied for almost 40 years and is clearly involved in plant response to stresses; however, questions regarding the importance of lysine remain open. Here, we investigated the metabolic and physiological impacts of exogenous application of gibberellic acid (GA 3 ) on the development of a mutant with drastically reduced activity of the lysine biosynthesis enzyme L,L-DAPAT (L,L-diaminopimelate aminotransferase). The DAPAT mutation culminated in reduced growth by impacting carbon and nitrogen metabolism, likely associated with apparent putative stress, even under optimal growth conditions. In general, the deficiency in lysine synthesis resulted in a lower content of reducing sugars and starch, especially at the end of the night. Furthermore, a substantial increase in amino acid levels was observed in dapat mutant plants, indicating a metabolic adjustment and consequent imbalance in carbon and nitrogen metabolisms, possibly as- sociated with continued growth in plants deficient in lysine biosynthesis. Mutant plants exhibited a differential expression of CPS, KS, and GA3ox genes, involved in the biosyn- thesis of Gibberellins (GA). Notably, the DAPAT mutation did not eliminate the respon- siveness to exogenous GA application of this mutant, promoting partial recovery of the characteristic dwarf phenotype. Although GA 3 treatment does not seem to affect the met- abolic pattern of the mutant plants, it is possibly responsible for the C/N imbalance, evi- denced by the increased expression of the sugar-starvation marker gene DIN6. Taken together, the results obtained indicate that lysine metabolism is a key point in the balance and allocation of soluble and reserve carbohydrates, and highlight the importance of L,L- DAPAT in controlling growth, primary metabolism, and hormone regulation. Keywords: Amino acids. Lysine biosynthesis. L,L-diaminopimelate aminotransferase. Gibberellin. Metabolic reprogramming.