Andreas Peter Michael Weber

Whole-genome duplications (WGDs) have played a central role in plant evolutionary innovation, yet their genome-wide influence on complex physiological traits such as carbon-concentrating mechanisms (CCMs) remains poorly resolved. Here, we present high-quality genome assemblies and annotations for 35 species within the Heliantheae alliance (Asteraceae), spanning the complex photosynthetic spectrum from C3 to C4. Ks-based analyses combined with gene tree-species tree reconciliation identified a shared paleopolyploid event (WGD-2) ~30 million years ago. Using phylogenetically informed association mapping with these species, we detected 451 functional MapMan BINs with copy number variation significantly associated with interspecific variation in the CO2 compensation point. Notably, 303 of these BINs contain greater than 10% WGD-2-derived genes, indicating a strong enrichment of duplicated loci within photosynthetically relevant functional modules. Effect-size analysis revealed that the C3-to-C4 transition is governed by threshold-dependent genomic architecture rather than gradual additive change. Substantial expansions in key transcription factor modules, including HD-ZIP, HSF, LOB, and TCP, were associated with strong non-linear physiological shifts, consistent with genomic tipping points. By integrating comparative genomics, physiological trait variation, and evolutionary analysis, this study establishes a mechanistic link between ancient polyploidy and contemporary photosynthetic diversification. These findings provide a framework for understanding the genetic basis of carbon concentrating mechanism evolution and inform future optimization of photosynthetic efficiency in Asteraceae crops such as sunflower.

Cellular energy homeostasis relies on the precise exchange of nucleotides across subcellular compartments. The transport of ADP and ATP is essential for maintaining cellular energy balance. Transporters of these molecules are abundant proteins in the mitochondrial membrane, mediating their exchange between the mitochondrial matrix and the cytosol. In this study, we investigated the function of ADP/ATP Carrier 1 (AAC1) in plants to understand whether its regulation and function are similar to those described in animal and yeast systems. Our results revealed that AAC1 specifically transports ADP and ATP, while aac1 mutants displayed lower O2 consumption without evident decreases in cytosolic ATP levels. However, total adenylate profiling revealed altered adenylate balance and reduced adenylate energy charge in aac1 roots, indicating that reduced AAC1 function perturbs energy homeostasis beyond changes in ATP content alone. AAC1 was also required for proper mitochondrial structure, as aac1 mutant mitochondria exhibited a less electron-dense matrix and occasional absence of cristae. Suppressing AAC1 expression in plants altered vegetative and reproductive growth, reducing leaf number and root elongation while increasing silique size and seed weight. These findings provide novel insight into the role of AAC1 in mitochondrial energy dynamics. Biochemical analyses show a high specificity of AAC1 for adenine nucleotides, facilitating ADP/ATP exchange driven by the mitochondrial membrane potential, while excluding monophosphates and divalent ATP complexes. By correlating these biochemical characteristics with impaired mitochondrial respiration, altered adenylate balance, ultrastructural defects, and altered energy allocation, our study highlights the broader physiological impacts of disrupted adenylate transport mediated by AAC1.

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.

Mitochondrial transport processes are critical for oxidative phosphorylation; however, the physiological roles of many members of the plant mitochondrial carrier family remain poorly characterized. In Arabidopsis thaliana, three PHOSPHATE TRANSPORTER 3 (PHT3) proteins are identified as potential mitochondrial phosphate carriers, but only PHT3;1 and PHT3;2 have been investigated. Here, we examined the least studied member of this family, AtPHT3;3, which exhibits elevated expression in reproductive tissues. After confirming the subcellular localization of PHT3;3 to mitochondria, we performed investigations on two T-DNA insertion alleles, pht3;3-1 and pht3;3-2 by integrating physiological, metabolomics, and phylogenetics analysis. Both mutants exhibited higher rates of aborted and missing seeds, embryonic arrest at the early torpedo stage, and impaired seedling growth. These phenotypes were partially restored through complementation with a constitutively expressed AtPHT3;3 transgene. Metabolite profiling of pre-anthesis and post-anthesis flowers revealed a significant accumulation of glycolytic intermediates and altered pools of amino acids and organic acids in the pht3;3 mutants, indicating that the loss of AtPHT3;3 is associated with changes in primary metabolism. Phylogenetic and protein sequence analyses showed that AtPHT3;3 diverged from AtPHT3;1 and AtPHT3;2, sharing ancestry with yeast phosphate inorganic carrier 2 (Pic2) and human solute carrier family 25 member 3 (SLC25A3). However, the transport substrate and biochemical activity of AtPHT3;3 remain to be established. Overall, these findings identified AtPHT3;3 as a mitochondrial carrier protein required for late embryo development, seed formation, seedling performance, and reproductive-tissue metabolism. These results highlight the importance of mitochondrial transport processes in plant reproductive development.

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.

Diurnal changes in light availability are a defining feature of life on Earth. Photoautotrophic organisms therefore store reduced carbon during the day to sustain energy metabolism at night. In cyanobacteria, glycogen is the primary carbon storage compound and supports both energy homeostasis and stress responses. Although glycogen-deficient Synechocystis strains have been studied previously, how these mutants cope with the loss of the major daytime carbon sink and can sustain themselves during the night remains unclear. Using single-cell microfluidics, transcriptomics, and metabolomics, we show that ΔglgC mutants exhibit pronounced light sensitivity. At sublethal light intensities, daytime transcriptional responses are dominated by downregulation of photosynthesis-related genes, likely preventing NADPH overaccumulation in the absence of a carbon sink. During the night, mutants display severe energy limitation, characterized by reduced ATP levels, altered redox balance, and depletion of central carbon intermediates. In contrast, fumarate and malate accumulate, indicating enhanced respiratory flux through succinate dehydrogenase. These metabolic constraints lead to extended lag phases and delayed cell divisions after the onset of light, demonstrating that glycogen-deficient cells fail to efficiently reinitiate growth after dawn. Overall, our results as a snapshot of the initial response to diurnal regimes highlight glycogen as a central integrator of diurnal physiology in Synechocystis, coordinating energy metabolism, redox balance, and cell division, with implications for metabolic robustness and the evolutionary constraints shaping (endo)symbiosis.

Lysine metabolism is a central determinant of plant growth, coupling amino acid biosynthesis to mitochondrial electron transport and the tricarboxylic acid (TCA) cycle to sustain cellular energy homeostasis. However, whether perturbation of lysine biosynthesis reshapes developmental transitions through epigenetic regulatory pathways remains unknown. Here, we show that reduced activity of L,L -diaminopimelate aminotransferase (DAPAT), a key enzyme in lysine biosynthesis, markedly reprograms growth and developmental timing in Arabidopsis thaliana . The lysine biosynthesis-deficient mutant dapat exhibits severe development constraints, displaying a pronounced flowering delay under long-day conditions accompanied by reduced shoot branching, fewer siliques, lower seed number per silique, decreased seed biomass, and reduced leaf production, a comprehensive developmental phenotype that underscores the critical role of lysine metabolism in orchestrating plant growth. Strikingly, neutral-day conditions partially rescue the flowering delay in dapat plants, revealing an unexpected environmental plasticity that positions lysine biosynthesis at the intersection of metabolic regulation, epigenetic remodeling, and developmental timing. Gene expression analysis of key flowering-time regulators that control the photoperiodic and vernalization pathways in Arabidopsis revealed upregulation of CONSTANS ( CO ) and FLOWERING LOCUS C ( FLC ), downregulation of FRIGIDA ( FRI ), and unaltered FLOWERING LOCUS T ( FT ), suggesting that lysine deficiency modulates flowering through a CO / FLC -dependent regulatory framework. Using whole-genome bisulfite sequencing (WGBS) we identified 957 genes with altered methylation patterns across the plant's five chromosomes. These changes occurred in three different DNA sequence contexts: 749 genes showed differences in CpG sites, while 104 genes each showed changes in CHG and CHH contexts. These epigenetic changes coincide with altered expression of genes involved in S -adenosyl-methionine metabolism and chromatin regulation, including upregulation of S -adenosyl-methionine synthase 2 ( MAT2 ), S -adenosyl-methionine synthase 4 ( MAT4 ), and histone methyltransferase SU(VAR)3–9 HOMOLOG 4 ( SUVH4 ), whereas SUVH5 is downregulated and SUVH6 remains unchanged. These findings uncover a previously unrecognized role for lysine biosynthesis via DAPAT enzyme in coordinating plant growth, flowering time, and reproductive output through metabolic-epigenetic regulation.

Diurnal changes in light availability are a defining feature of life on Earth. Photoautotrophic organisms therefore store reduced carbon during the day to sustain energy metabolism at night. In cyanobacteria, glycogen is the primary carbon storage compound and supports both energy homeostasis and stress responses. Although glycogen-deficient Synechocystis strains have been studied previously, how these mutants cope with the loss of the major daytime carbon sink and can sustain themselves during the night remains unclear. Using single-cell microfluidics, transcriptomics, and metabolomics, we show that ΔglgC mutants exhibit pronounced light sensitivity. At sub-lethal light intensities, daytime transcriptional responses are dominated by downregulation of photosynthesis-related genes, likely preventing NADPH overaccumulation in the absence of a carbon sink. During the night, mutants display severe energy limitation, characterized by reduced ATP levels, altered redox balance, and depletion of central carbon intermediates. In contrast, fumarate and malate accumulate, indicating enhanced respiratory flux through succinate dehydrogenase. These metabolic constraints lead to extended lag phases and delayed cell divisions after the onset of light, demonstrating that glycogen-deficient cells fail to efficiently reinitiate growth after dawn. Overall, our results as a snapshot of the initial response to diurnal regimes highlight glycogen as a central integrator of diurnal physiology in Synechocystis, coordinating energy metabolism, redox balance, and cell division, with implications for metabolic robustness and the evolutionary constraints shaping (endo)symbiosis.

Pre-anthesis inflorescence greening (PAIG) is a distinctive developmental feature in members of the Triticeae such as barley, wheat, and rye. In barley (Hordeum vulgare L.), floral survival and fertility are major determinants of grain yield, yet the physiological processes supporting early inflorescence development remain poorly understood. Here, we investigated PAIG, a light-dependent chlorophyll accumulation occurring while immature inflorescences are still enclosed by leaf sheaths, and its role in overall inflorescence development. Using chlorophyll autofluorescence imaging and chlorophyll quantification, we found that PAIG in barley initiates at a surprisingly early developmental stage, when developing spikes are still enclosed by leaf sheaths. PAIG first appears in the developing central rachis and then progressively spreads to the spikelet primordia and other floral organs. Using a non-destructive dark treatment that prevents light exposure to developing inflorescences, we showed that inhibiting PAIG does not impact floral initiation. Yet, the dark treatment significantly decreases floral survival and pollen viability, especially at the tip of the inflorescence. This highlights the essential role of light-mediated PAIG in determining floral fate. Finally, analysis of natural variation for PAIG revealed a strong positive correlation between the extent of PAIG and floral survival. Our findings establish PAIG as an underappreciated hidden trait that supports floral viability and reproductive success, with implications for enhancing grain yield potential in cereals.

C4 photosynthesis evolved from the ancestral C3 pathway through coordinated leaf anatomical and metabolic reorganization that concentrates CO ₂ to reduce photorespiration. Quantitative understanding of these structure-function relationships remains limited. Here we used anatomy-aware metabolic modeling of a mesophyll-bundle sheath cell system to analyze the interdependence between leaf anatomy and photosynthetic metabolism on the C3-C4 spectrum. Our model faithfully recapitulates the transitory steps from C3 to C4 photosynthesis, reveals a crucial role for plasmodesmata in enabling the C3 to C4 transition, and points at potential pre-C2 metabolic states that provide benefits under conditions that promote elevated photorespiration. Incorporating bundle cell suberisation with our model predicts reduction of PSII activity and dominance of the NADP-ME C4 subtype in leaves with suberized bundle sheath cells and proposes a role for oxygen evolution at PSII as a potential driver for this mechanism. Varying bundle sheath leakage and photorespiratory conditions along the C3-C4 spectrum identify conditions under which C3-C4 intermediacy provides energetic benefits and underlines the notion of intermediate photosynthesis as a stable evolutionary state. Overall, our study sheds new light on the quantitative relationship between leaf anatomy and metabolism and its interaction with the environment and suggests targets for climate-adaptation in C3 plants.