The Plant Journal

While the core transcription factor TAF4b is known to regulate meiotic transcription and recombination in model plant Arabidopsis thaliana, its functional specialization and evolutionary dynamics remain poorly understood in paleopolyploid crops. To address this, we investigated how the unique, single-copy TAF4b gene modulates early meiotic progression and reproductive success in Brassica rapa. We characterized multiple independent taf4b TILLING lines for fertility and meiotic behavior, then used this information to select a mutant line for further investigation using a pipeline combining whole-genome background sequencing (DNA-seq), stage-specific anther transcriptomics (RNA-seq), yeast two-hybrid assays, and quantitative cytogenetics. Disruption of the meiosis-enriched TAF4b gene on chromosome A7 universally reduced crossover frequency and depleted HEI10 foci, triggering occasional univalent formation. Global RNA-seq and structural modeling indicate that this phenotype is driven by a precise transcriptional dysregulation and altered molecular interactions restricted to the interphase/leptotene transition. In conclusion, TAF4b acts as a stage-specific upstream gatekeeper of the early prophase I expression program in B. rapa, thereby playing an essential, non-redundant role in its meiotic stability.

Premise:: Conifers comprise one-sixth of global forests and landscape genomic studies are urgently needed to understand their biology, yet relatively few conifer species have reference genomes. Targeted exon-capture is a practical alternative to whole-genome sequencing, but selecting loci without a reference genome remains a barrier. We developed a multi-tissue reference transcriptome for pi non pine (Pinus edulis) to ask how multi-tissue sampling improves probe design, and what is lost under a conventional single-tissue approach. Methods: We assembled a reference transcriptome from four diploid tissues (needle, phloem, root, vegetative bud) and one haploid tissue (megagametophyte), using the latter as a pseudo-guide. We designed a target-capture probe-set by prioritizing highly expressed P. edulis transcripts and transcripts matching a conifer exon database. Results: We recovered 76% of conserved plant genes with few duplicated transcripts. Filtering yielded 15,664 transcripts totaling 31.4 Mb, ~0.1% of the genome. Tissue-specific expression was substantial: 27.4% of transcripts occurred exclusively in non-needle tissues. Functional annotation confirmed complementary contributions, including seed-desiccation and germination genes from megagametophytes, and defense- and stress-associated genes from roots and phloem. Discussion: We built a transcriptome and efficient probe set using a roadmap suitable for other non-model plants with large genomes; keys to success were prioritizing complementary tissues and using expression-based filtering.

Plastidial phosphorylase (Pho1) regulates starch metabolism and also modulates photosystem I (PSI) activity. In rice, Pho1 contains an 80 amino acid intrinsically disordered region (IDR), L80, that acts as a negative regulator. While total removal of the IDR boosts seedling growth, flowering time, overall biomass, and grain yield and alters PSI redox properties, the precise underlying regulatory basis remains unclear. To pinpoint IDR regulatory elements, four Pho1 partial-IDR deletion variants ({delta}N, {delta}C, {delta}M, and {delta}N{delta}C) were evaluated. Lines carrying {delta}M and {delta}C exhibited enhanced starch metabolism, yielding larger, heavier grains, and flowered 5 to10 days earlier than wild type. Both d{delta}M and {delta}C share an acidic motif, (E/V)SEEV, identifying it as a primary negative regulator of starch synthesis and floral timing. By contrast, no partial deletion fully mirrored the PSI redox modifications of Pho1{delta}L80. These results demonstrate that starch biosynthesis and flowering are governed by one or more discrete sequence motifs within the middle and C-terminal regions, whereas PSI modulation likely requires the structural integrity of the intact IDR.

Genetically encoded biosensors are central to plant cell biology, but low reporter signal can limit their use. Here, we developed modular intronised fluorophores to improve reporter output in transient and stable plant expression systems. Matched nuclear-localised triple mCitrine and mScarlet3 reporters were compared with and without introduced introns in Nicotiana benthamiana and Arabidopsis thaliana. Intronisation increased fluorescence, with the magnitude of enhancement depending on the fluorophore and expression context. Immunoblot analyses supported increased reporter accumulation. In Arabidopsis, intronised reporters driven by the tissue-specific SCARECROW promoter, produced stronger fluorescence signals that remained detectable farther from the root tip than non-intronised versions. Intronised versions of the abscisic acid (ABA) biosensor ABACUS2 retained ABA responsiveness and showed a lower frequency of reduced fluorescence between generations. These modular components provide a practical toolbox for improving fluorescent reporter detection and maintaining usable biosensor expression, supporting their application in plant cell biology and quantitative imaging.

The apoplast is a primary location for interactions between plants and invasive pathogens and is the site where many pathogen-secreted effectors are perceived by cell surface immune receptors to activate defence responses. However, our understanding of apoplastic interactions remains limited because protein interactions are difficult to investigate in this harsh extracellular environment. TurboID-based proximity labelling (PL) has emerged as a powerful approach for studying protein interactions in plants, though its application has to date been restricted to intracellular proteins. Here, we designed and validated a TurboID-based PL strategy for investigating protein interactions in the leaf apoplast using the well-characterised interaction between the Phytophthora infestans elicitor INF1 and the receptor-like protein (RLP) REL in Nicotiana benthamiana. Transient expression of SP-INF1-TurboID (INF1-T) induced a cell death (CD) response comparable to that triggered by native INF1, demonstrating that fusion of the TurboID tag did not impair INF1 recognition by REL. Apoplastic localisation of both INF1-T and the control construct SP-eGFP-TurboID (eGFP-T) was confirmed, validating their suitability for PL experiments. Efficient TurboID-mediated biotinylation was achieved in the apoplast using co-infiltration of biotin, ATP and magnesium acetate. Streptavidin-HRP immunoblotting revealed distinct biotinylation profiles for INF1-T and eGFP-T. Furthermore, co-immunoprecipitation demonstrated specific biotinylation of REL by INF1-T, but not by eGFP-T, in wild-type, bak1 and sobir1/sobir1-like N. benthamiana. These findings demonstrate that TurboID-based PL is functional in the apoplast and provides a proof-of-concept for investigating elicitor-receptor interactions in this compartment.

Light is essential for plant growth and development. Sustainably feeding a constantly-growing human population will likely involve adapting crop plants to intercropping and high planting density by rational manipulation of light signaling. Here, we edited the tomato (Solanum lycopersicum) genome to generate lines with a light-stable version of ELONGATED HYPOCOTYL 5 (HY5), a master transcription factor involved in the integration of light and hormone signaling. Removing the tomato HY5 N-terminal domain required for interaction with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) prevented light-dependent protein degradation and resulted in a gain-of-function phenotype of short seedlings. Elongation growth was also compromised under proximity shade conditions either simulated by enriching white light (W) with far-red light (W+FR) or achieved by growing plants at a higher density. Transcriptomic analysis of gene expression changes after exposure to W+FR for 24h revealed a reduced number of shade-responsive genes in edited lines compared to unedited, wild-type controls, many of which are related to growth and hormone (notably auxin) biosynthesis and signaling. The reduced elongation observed in edited lines correlated with enhanced resistance to infection by viral, bacterial and fungal pathogens, both under low and high density conditions. These results indicate that our editing approach allows the generation of gain-of-function tomato plants in which HY5 is camouflaged to avoid COP1 recognition and eventual degradation. Our findings therefore provide a biotechnological tool to create more compact and pathogen-resistant plants amenable to high planting densities.

Calcium (Ca2+) is a universal second messenger that orchestrates plant growth, development, and stress responses. Plant calcineurin B-like (CBL) proteins sense and relay Ca2+; signals in response to pathogens to activate downstream defense signalling pathways. Early blight disease, caused by the necrotrophic fungal pathogen Alternaria solani, affects tomato crops and severely limits their productivity. In the present study, we found that the tolerant tomato cultivar (Arka Rakshak) restricts A. solani infection and disease progression compared to the susceptible cultivar (PKM-1). Comparative transcriptome and qRT-PCR analysis of A. solani-infected tomato leaves showed that SlCBL1-2 (Solyc08g007160.3), a Ca2+; sensor, is up-regulated in the tolerant cultivar, both relative to its own uninfected control and the infected susceptible cultivar, whereas it was not significantly induced in the uninfected susceptible cultivar. Protein sequence analysis indicates that SlCBL1-2 contains four EF-hand motifs, an N-terminal N-myristoylation/S-acylation module and a C-terminal FPSF motif, and it is predicted to interact with several CBL-interacting serine/threonine protein kinases (CIPKs). However, the evolutionary analysis, conformational transitions upon binding to Ca2+, and the Ca2+-binding affinity of SlCBL1-2 have not been previously characterized. Molecular Dynamics (MD) simulation of the AlphaFold structure of SlCBL1-2 indicates a well-folded structure and binding of two Ca2+ ions at EF-hands 3 and 4. Further, far-UV circular dichroism and tryptophan fluorescence spectroscopy of recombinant SlCBL1-2 indicate a natively folded structure in the absence of Ca2+ and undergo minimal secondary and tertiary structural changes upon Ca2+-binding, in contrast to several Ca2+-binding proteins. Of note, SlCBL1-2 binds Ca2+ with apparent KD values of 526 {+/-} 113 {micro}M and 28.7 {+/-} 9.47 {micro}M, as evidenced by isothermal titration calorimetry. Together, our study provides an experimental description of the Ca2+-binding properties of tomato CBL protein and defines the molecular basis by which pathogen-induced SlCBL1-2 may act as a Ca2+ sensor in defense signalling.

Sporadically, all plastids produce dynamic tubular extensions in response to myriad stresses. Although the use of fluorescent proteins revealed these transient tubules to be stroma-filled and led to their being called stromules, the mechanism underlying tubule extension from a spheroidal plastid remained unknown. As plastids in viridiplantae have two bounding membranes the fluid stroma remains confined within the inner envelope membrane (IEM) while the outer envelope membrane (OEM) around the IEM forms the interface with the rest of the cytoplasm. Here, using a sucrose treatment for inducing tubules in the dark in transgenic Arabidopsis plants expressing different stroma and OEM-targeted fluorescent proteins we carried out comparative molecular-genetic and lipidomics analyses. Our data strongly suggest that plastid tubule formation occurs due to increase in bilayer-forming galactolipids such as digalactosyldiacylglycerol (DGDG) on the OEM. While the dynamic behaviour of stromules occurs within an external encasing OEM, stresses such as phosphate starvation and high light intensity cause the galactolipid-rich OEM-tubule projecting beyond the stromule to be shed into the cytoplasm. OEM shedding from long tubules with extended plastid outreach into the cell suggests their important role in dispersing plastid-derived, membrane-stabilizing, galactolipids to the rest of the cell to augment its resilience to stress.

Mitochondrial genomes of angiosperms are unusual due to their large size, low gene content, and high structural variation. In the palm family (Arecaceae), mitochondrial architecture is highly dynamic, characterized by expansive sizes, fragmentation, and extensive recombination activity. This biological complexity presents a significant bioinformatic challenge, especially since only three complete palm mitochondrial genomes are publicly available to date. Here, we present the complete mitochondrial genome sequence for the Amazonian palm Euterpe precatoria, assembled de novo using PacBio HiFi long-read sequencing data. The assembly required extensive manual curation and graph tracing to resolve a complex recombination topology. The final resolved sequence spans 1,268,308 base pairs and is supported by a mean read coverage depth estimated at 746X. Annotation revealed a high degree of structural complexity, including 177 total gene features corresponding to 76 unique genes. This gene set includes 41 unique protein-coding genes (56 features), 32 unique tRNA genes (118 features), and three unique rRNA genes, reflecting the characteristic gene duplication and fragmentation documented in Arecaceae. The complete mitochondrial genome sequence is publicly available and provides a high-quality resource for understanding the mechanisms of plant mitochondrial genome expansion and structural evolution.

Forward genetics is a powerful approach for gene discovery, but identifying causal mutations becomes difficult when mutants are maintained in heterogeneous populations with uncertain pedigrees. This is exemplified by classical tasselseed (ts) mutants, which have long served as a genetic model for studying sex determination and carpel suppression. Decades of repeated outcrossing to diverse inbred lines have created substantial genetic heterogeneity, limiting the effectiveness of conventional bulked-segregant analysis sequencing (BSA-Seq). To address this, we developed a BSA-Seq framework that integrates flexible experimental designs, multiple reference genomes, and complementary statistical methods tailored for genetically heterogeneous populations. Applying this framework revealed that reference genome selection is critical for mapping success and that Euclidean distance raised to the fourth power (ED4) outperformed homozygosity mapping (HM). Furthermore, the framework enables simultaneous mapping of multiple mutations within a single population, eliminating the need for additional mapping populations. Applying this framework to 26 ts mutant stocks from the Maize Genetics Cooperation Stock Center, we successfully mapped 24 mutants to genomic intervals containing known ts genes, while the remaining mutants mapped to distinct genomic intervals, defining novel candidate regions underlying carpel suppression. Together, these results demonstrate that historical mutant collections represent an underutilized resource for gene discovery and establish a generalizable mapping strategy for unlocking their genetic potential across diverse species.

In many flowering plants, self-incompatibility (SI) is a genetically controlled recognition system that prevents self-fertilization, thereby promoting outcrossing and maintaining genetic diversity within populations. The gametophytic SI system in grasses (Poaceae) is governed by two multiallelic loci, S and Z, each locus comprising of three genes governing the initial recognition between pollen and stigma. Identifying these genes establishes the molecular basis of SI in grasses, but how its unique two-locus architecture has shaped the evolution of the underlying loci and recognition genes remains largely unexplored. Here, we report targeted sequence capture of SI genes in 362 genotypes across nine forage grass species, revealing extensive allelic richness and sequence diversity. The SI genes exhibited elevated nucleotide diversity ({pi}) (0.134 < {pi} < 0.403) and great amino acid sequence divergence, ranging between 48.2-58.0% for the female determinants (sS and sZ) and 66.5-83.4% for the male determinants (SI-DUF247). The SI-DUF247 proteins also displayed trans-generic polymorphism, indicating that alleles predate speciation. Phylogenetic analysis of SI determinants showed congruence at both S- and Z-locus, consistent with co-evolution of recognition partners. Our results demonstrate that the unique two-locus SI system in grasses shares evolutionary characteristics with other SI systems, suggesting that their reproductive function is shaping their evolution. These findings also indicate that the grass SI loci are under long-term balancing selection to maintain recognition specificity while creating sufficient allelic diversity. Our study provides comprehensive sequence resources for SI genes and new insights into the evolution of the unique two-locus SI system of grasses.

Photosynthetic electron transport is tightly regulated to balance ATP and NADPH production and prevent over-reduction of the photosynthetic machinery. How this balance is maintained under severe ATP limitation, particularly under anoxia, is not fully resolved. Here, we used several deletion mutants of chloroplast ATP synthase subunits from Chlamydomonas reinhardtii to examine how electron flow is reorganized under anoxic conditions through the interplay between linear electron flow, cyclic electron flow, and alternative electron sinks. Using a combination of optical spectroscopy methods and membrane inlet mass spectrometry, we characterized changes in photosynthetic electron transport, proton motive force relaxation and gas exchange. Under oxic conditions, the {Delta}atpH mutant (lacking the c-subunit) showed strong acceptor-side limitation at photosystem I (PSI), whereas atpC1-2 (lacking the {gamma}-subunit) resembled the wild type. Under anoxia, this limitation was partially relieved, coinciding with sustained electron transport and prolonged hydrogen production. In particular, the {Delta}atpH mutant exhibited increased cyclic electron flow under oxic conditions and maintained PSI activity under anoxia despite impaired ATP synthesis. Prolonged hydrogen production was also observed in atpC1-2, which did not share the increased respiration of {Delta}atpH. Hydrogenase activity under anoxia is consistent with a role as an electron sink that may help alleviate PSI acceptor-side limitation and rebalance the redox state of the electron transport chain. These findings show that ATP synthase deficiency reorganizes photosynthetic electron flow and promotes prolonged hydrogen production, and point to hydrogenase as a candidate electron sink under energy-constrained conditions.

Castilleja (Orobanchaceae) is a diverse, facultatively hemiparasitic plant genus characterized by a complex evolutionary history of reticulate evolution and significant taxonomic ambiguity. High-quality genomic resources have historically been limited, hindering robust macro-evolutionary and macro-ecological inquiries. Here, we present the first high-quality, phased, chromosome-level reference genome for the diploid species Castilleja foliolosa. Utilizing a hybrid assembly strategy, we integrated long-read PacBio HiFi sequencing with Omni-C proximity ligation data to generate a highly contiguous nuclear assembly, complemented by reconstructed mitochondrial and chloroplastic genomes. The primary nuclear haplotype spans approximately 510 Mbp, containing 39,417 predicted genes and a substantial repetitive landscape covering ~66% of the genome. Our organellar assemblies reveal notable structural complexity: the chloroplast maintains heteroplasmy via two primary structural haplotypes, while the 611 kbp mitogenome adopts a circular master topology existing in two isomeric forms. Comparative genomic analyses demonstrate a largely conserved chromosomal architecture relative to the closely related Pedicularis cranolopha, punctuated by the presence of lineage-specific genes. Functional enrichment of Castilleja-specific orthogroups identifies a consistent association with telomere maintenance, DNA integration, and zinc ion binding, likely stemming from the substantial repeat content. This reference genome provides a robust framework for dissecting the genomic drivers of taxonomic diversification and adaptation within the Orobanchaceae family. This resource significantly enhances our capacity to untangle reticulate evolutionary patterns, offering a definitive foundation for future comparative studies of genome evolution in the Orobanchaceae lineage of parasitic plants.

The functional roles of glycans attached to the proline (Pro) rich region of arabinogalactan proteins are largely elusive. We have tested the possibility of type II arabinogalactans acting as protein fate determinants for the FASCICLIN LIKE ARABINOGALACTAN PROTEIN 4 (FLA4). Pro-to-Ala substitutions in the carboxy-terminal region caused in planta protein destabilization in an ECHIDNA-dependent trans-Golgi compartment and plasma membrane shedding in an aspartate protease-dependent manner. The introduction of diverse glycomodules to the FLA4 carboxy-terminus counteracted the stability loss, with AGP type glycosylation motifs more efficient than N-glycosylation signals and extensin structures. The resistance of FLA4 stability and localization to genetic interference with O-glycosylation suggested that additional protein properties, such as intrinsic disorder and secondary structure were crucial for FLA4 stability. Consistently, disorder-maintaining site directed mutants maintained FLA4 stability in a glycosylation-independent manner. Our data support a role for proline-rich regions in determining protein fate by generating intrinsically disordered domains in the peptide chain and providing acceptor sites for O-glycosylation.

Poplar (Populus spp.) is a model for tree biology and a platform for engineering woody biomass, biofuels, biomaterials, and bioproducts. Many relevant traits depend on tissue position, developmental stage, and cell type, yet these spatial relationships are difficult to recover from bulk or single-cell transcriptomes. Here, we generated a spatial transcriptome atlas of Populus tremula x P. alba INRA 717-1B4 across the shoot apex, axillary bud, stem, and petiole. After quality control, the atlas retained 29,687 spatial spots from 45 tissue sections and detected 58,748 genes. Histology-guided clustering and marker analysis resolved meristematic, epidermal, cortical, vascular, and organ-specific domains. Cross-organ comparisons assessed whether published markers retained tissue-associated expression across different anatomical contexts and developmental stages, while de novo analysis identified additional domain-enriched candidates. As case studies of the utility of the atlas, we examined the emergence of trichome-associated programs in the shoot apex and adaxial - abaxial expression differences in petioles. A trichome identity score based on poplar markers from the single-cell shoot atlas peaked along the inferred meristem-to-primordium trajectory, revealing spatially localized expression of trichome-associated programs during early leaf development. Petiole expression differences were concentrated in the epidermis and cortex and involved polarity-associated, auxin-responsive, and cell-wall-remodeling genes, with distinct expression profiles across leaf positions. Together, these data provide a spatial reference for investigating tissue differentiation and developmental patterning in a transformable poplar genotype.

Photosynthetic organisms protect themselves from excess light through non-photochemical quenching (NPQ), a process that safely dissipates excess light energy as heat. Current understanding of NPQ is largely drawn from a few model systems, leaving it unclear how universal NPQ mechanisms are across the green lineage. We characterize the slow, sustained NPQ of the green alga, Auxenochlorella protothecoides x symbiontica (UTEX 250-A). The Auxenochlorella genome lacks Light-Harvesting Complex Stress Related (LHCSR), one of the two proteins required for the fast energy-dependent quenching (qE) component of NPQ. Mutants lacking the Photosystem II Subunit S (PSBS)-like gene (psbsl1) in Auxenochlorella had no effect on NPQ, indicating this alga lacks canonical qE. We identified two candidate enzymes associated with zeaxanthin-dependent quenching (qZ), which has slower kinetics: a plant-type Violaxanthin De-Epoxidase (VDE) and an algal-type Chlorophycean Violaxanthin De-Epoxidase (CVDE). High light did not trigger the expected conversion of violaxanthin to zeaxanthin, a pigment change that is a hallmark of qZ. Mutants lacking VDE (vde1), CVDE (cvde1), or both enzymes (vde1 cvde1) still displayed slow, reversible NPQ. However, when the Auxenochlorella VDE1 and CVDE1 were expressed in a VDE-deficient mutant of Nicotiana benthamiana, both enzymes restored zeaxanthin production and NPQ, confirming they are catalytically functional de-epoxidases. Together, these results reveal an alga that retains two functional, evolutionarily distinct xanthophyll-cycle enzymes yet does not rely on typical xanthophyll pigment dynamics for photoprotection. This points to a biological function that is decoupled from de-epoxidase activity, highlighting unexplored diversity in green algal photoprotection.

The chloroplast is a semi-autonomous organelle with a compact genome encoding ~100 expressed genes. Numerous and widespread promoters coupled with inefficient transcription termination leads to a highly complex primary transcriptome that undergoes extensive RNA quality control orchestrated by RNA binding proteins and ribonucleases. Among the ribonucleases, only RNase J (RNJ) is absolutely required for plant germination and survival, likely because its deficiency leads to massive accumulation of antisense RNA which inhibits translation. As one approach to understanding the selectivity of RNJ for antisense RNA and noncoding regions, we created Arabidopsis lines overexpressing catalytically inactive versions of the enzyme designed to trap its substrates. Initially, transgenic plants appeared phenotypically normal. However, crosses used to generate homozygous transgenic lines yielded progeny that developed varying degrees of chlorosis within 2-3 weeks. Concomitantly, these plants exhibited chloroplast transcriptome alterations similar to RNJ-silenced lines, along with mRNA changes consistent with retrograde signaling. This chlorosis could be correlated to the likely decay of RNJ protein, but not its endogenous transcript. The result could be replicated by attempting to overexpress a wild-type version of RNJ, in which case even one transgene copy was sufficient to induce chlorosis. Our results suggest that RNJ is under protein expression level surveillance to prevent wholesale destruction of the plastid transcriptome.

Although iron (Fe) is abundant in soils, its low solubility, particularly under alkaline conditions, often limits plant Fe acquisition. Under these conditions, coumarin-mediated Fe mobilization becomes increasingly important. Arabidopsis thaliana FE DEFICIENCY-INDUCED KELCH REPEAT PROTEIN 1 (FDK1, AT3G07720) is strongly induced by Fe deficiency and regulated by the central transcription factor FIT, but its function is unknown. We investigated FDK1 using genetic, physiological, metabolite, and transcriptomic analyses. Loss of FDK1 altered root growth, whereas this phenotype was restored in the FDK1-Ox overexpression line. Under alkaline Fe deficiency, growth medium from FDK1-Ox contained strongly increased levels of the Fe-mobilizing coumarins fraxetin and esculetin compared with wild type, whereas their induction was attenuated in fdk1-1. RNA-seq analysis linked FDK1 abundance to coumarin biosynthesis and iron-homeostasis gene expression. FIT and many FIT-associated genes were highly expressed in FDK1-Ox, whereas several upstream iron-homeostasis regulators, including bHLH Ib factors, were highest in fdk1-1. These findings identify FDK1 as a component of the alkaline Fe-deficiency response that promotes iron-mobilizing coumarin accumulation and is associated with coordinated regulation of iron-homeostasis genes.

Groundnut (Arachis hypogaea) is an important oilseed crop, and its production is severely affected by drought worldwide. Improving the drought-tolerance of groundnut without compromising its agronomic traits is a major challenge for researchers. Here, a meta-transcriptome analysis identified tandemly duplicated G{beta} subunits (AhG{beta}1 and AhG{beta}3) as early signaling genes under water deficit conditions. Heterotrimeric G-protein {beta}-subunit is a major signaling element that can regulate diverse responses in plants. Heterologous complementation studies in Arabidopsis G{beta} mutant (agb1-2) demonstrated evolutionary conservation of AhG{beta} function, while the transgenic overexpression and RNAi knockdown in groundnut established AhG{beta}1 as a positive regulator of plant growth, yield, and drought tolerance. Integrated transcriptomic, transcription factor regulatory network, and metabolomic analyses demonstrated that AhG{beta}1 coordinates drought-responsive transcriptional and metabolic reprogramming characterized by suppression of cell wall-associated processes and activation of phenylpropanoid, flavonoid, and isoflavonoid biosynthesis. This reprogramming resulted in enhanced accumulation of antioxidant flavonoids, including quercetin and rutin, reduced ROS accumulation, and improved drought tolerance. Furthermore, exogenous application of these flavonoids enhanced drought recovery, highlighting their role as protective downstream metabolites. Collectively, our findings establish AhG{beta}1 as a conserved regulator linking early G-protein signaling with transcriptional and metabolic reprogramming to maintain redox homeostasis during drought stress and identify promising molecular and metabolic targets for improving drought resilience in groundnut.

{beta}-Cyclocitral ({beta}-cc), a {beta}-carotene oxidation product, has been proposed as an early retrograde signal that primes protection against photooxidative stress. Its temporal relationship with photosynthetic acclimation, however, remains unresolved. Exogenous {beta}-cc elicited cytosolic calcium changes within minutes and induced the {beta}-cc-responsive pAER::LUC detoxification reporter within 40 min. We exposed Arabidopsis thaliana plants to 1500 mol photons m-2 s-1 under ventilation creating a high-irradiance regime without concomitant heat stress and with a relatively mild photooxidative outcome (RT-EL). Non-photochemical quenching increased, whereas PSII maximum quantum yield progressively decreased during RT-EL, with both responses approaching a plateau by 16 h. Volatile {beta}-cc pretreatment did not improve photosynthetic performance or NPQ dynamics, whereas endogenous {beta}-cc and {beta}-cyclocitric acid accumulated only after prolonged RT-EL exposure (16 h). By contrast, hormone profiling and RNA-seq revealed extensive {beta}-cc-dependent reprogramming that was largely opposite to the RT-EL transcriptional response. Together, these results reveal a kinetic separation between rapid {beta}-cc signaling and its endogenous production. Under RT-EL major photosynthetic adjustments precede detectable {beta}-cc accumulation, whereas {beta}-cc triggers cellular responses within minutes once present. We therefore propose that the timing of {beta}-cc action during excess light is determined primarily by the onset of substantial {beta}-carotene oxidation.