The Plant Journal

Biolistic transformation is a versatile tool in plant science, yet high equipment costs and tissue damage from high-pressure gas remain significant barriers. Building on our previously developed "TSGMAC", a low-cost, helium-free biolistic system, we report three major advancements to enhance its throughput, delivery quality, and quantitative capability. First, a "guide barrel" assembled from commercial DIY fittings was developed; it effectively eliminates physical tissue damage and ensures uniform particle distribution, even in soft tissues like bok choy (Brassica rapa subsp. chinensis). Second, a rapid gene expression platform using PCR products was characterized. Results demonstrate that linear DNA constructs are efficiently circularized via non-homologous end joining (NHEJ) in plant cells, and protein expression is robust regardless of the relative positions of the promoter, coding sequence, and terminator. This system bypasses time-consuming cloning. Third, a cost-effective, highly sensitive dual-luciferase assay system utilizing teal Luc (teLuc) and inexpensive firefly luciferase (FLuc) inhibitors was established. This integrated workflow enables rapid, quantitative molecular biology using supermarket-obtained materials and standard PCR reagents. Our findings provide a practical foundation for plant scientists, synergistically accelerating gene functional analysis and genetic tool development.

Arabidopsis thaliana naturally occurs across a wide geographic range and displays extensive natural variation in several traits including adaptive responses to the abiotic environment (e.g. temperature, drought, salt). Quantitative techniques like Genome Wide Association Studies (GWAS) enable mapping the genetic basis of such environmental responses and benefits from extensive genetic variation, but the size of the chosen diversity panel is often limited by phenotyping capacity. Most studies therefore use subpanels, often based on maximization of genetic diversity. However, this type of selection may overrepresent cosmopolitan alleles and underrepresent rare environment-specific alleles. Here, we demonstrate that the genetic variation in a GWAS subpanel of Arabidopsis thaliana accessions depends almost entirely on the number of accessions in the panel and very little on the composition of the panel. We present the EcoCore panel designed by grouping accessions of the 1001 genomes (1001G; 1135 accessions) collection, based on their native collection environment and selecting an equal number of accessions from each environment. We assessed hypocotyl lengths of plants grown at control and ambient high temperatures (20{degrees}C and 28{degrees}C) for 913 accessions of the 1001G and mapped these traits with the full 1001G panel versus the EcoCore panel. The EcoCore panel revealed novel genetic associations with hypocotyl length which is attributed to enrichment of alleles from rare environments. We present the EcoCore panel as a manageable resource for studying phenotypic plasticity and the genetic basis of plant-environment interactions.

Efforts to boost crop productivity have focused on improving photosynthesis. However, plant respiration consumes over 90% of fixed carbon, fueling growth, stress responses, and resource acquisition. Despite its metabolic centrality, respiratory regulation remains underexplored as a yield target. Here, we identify AtMLSP1 (At4g17085), a 57-amino-acid mitochondrial inner membrane peptide in Arabidopsis thaliana. Loss-of-function atmlsp1 mutants show impaired growth, while AtMLSP1 overexpression boosts biomass, seed yield, and high light tolerance. No induction of alternative oxidase protein abundance or activity was observed in atmlsp1 plants, a response normally triggered by respiratory perturbation. Mitochondria isolated from atmlsp1 lines had an approximately 90% reduction in the amount of the intermembrane space protein Cytochrome c and a 30% reduction in all subunits of complex III, while other respiratory complexes remain unaffected. As expected, loss-of-function plants atmlsp1 reveal a significant decrease in total respiratory capacity and membrane potential, but mitochondrial integrity, and abundance of complex I, II and complex IV were unchanged. Overexpression of the orthologues of this gene in Oryza sativa (rice) and Glycine max (soybean) results in a significant increases in yield (seed number and size) under field conditions across different locations.

Tar spot, caused by the obligate fungus Phyllachora maydis, significantly threatens maize (Zea mays L.) production across the Americas. Host genetic resistance offers the most viable long-term management strategy. Building on observed differential tar spot tolerance in parents B73 and Mo17, we utilized a strategic core subset of 94 intermated B73 x Mo17 (IBM-94) population to characterize the genetic architecture of resistance. Phenotypic analysis of a panel of 96 recombinant inbreds including the susceptible parent, Mo17, and the moderately resistant parent, B73 confirmed stable differences in susceptibility, with B73 consistently demonstrating moderate resistance compared to Mo17. Analysis of variance revealed highly significant genetic variation within the population (F = 12.96; p < 0.001). High Pearson correlation (r = 0.8706, p < 0.0001) and coefficient of determination (R2 = 0.7579) across environments indicated that 76% of the phenotypic variance is attributable to genetic factors. Linkage mapping identified a novel, consistent major QTL cluster on chromosome 1. This cluster comprises five regions (qTAR_1.1 through qTAR_1.5) exceeding the significance threshold (LOD 3.8) in both years. We identified 74 candidate genes including bZIP, and RING/U-box proteins at significant SNP peaks. Additionally, gene annotation revealed a high concentration of wall-associated kinases and S-locus lectin protein kinases within the qTAR_1.4 and qTAR_1.5 regions, alongside potential defense-related transcription factors (MYB, bZIP, and C2H2 zinc fingers).These findings provide a framework for high-resolution mapping and functional validation to accelerate the development of tar spot-resistant maize cultivars.

Pine needles contain two vascular cell types unique to gymnosperms: Transfusion parenchyma (tp) and tracheids (tt). Since they form the only connections between vascular bundles and bundle sheath, we hypothesised that they are involved in regulating the needles water import and photoassimilate export. Synchrotron-based tomography enabled us to quantify volume changes of tp and tt cells in Pinus pinea needles systematically along the needle and throughout a diurnal day cycle, as well as under rehydration. As a physiological indicator of tps carbohydrate status served their starch content. Segmentation of the comprehensive data uncovered dramatic volume changes during dehydration and showed a diurnal course of starch formation and degradation. These changes suggest a yet unknown osmotic water flux between tp and tt, balanced by the formers carbohydrate status. Confirming our hypothesis, excess of photoassimilates in tp cells went into starch synthesis during the day. Starch mobilisation during the night increased the osmotic potential in tp and led to water intake. According to the decreasing starch fraction from base to needle tip, this mechanism is predominant in the upper needle segments, particularly after rehydration of dehydrated needles. Mechanistically, osmolytes in tp cells maintain tension in tt for the needles water import. HighlightSynchrotron tomographic microscopy uncovers diurnal starch fluctuations and osmotic water pumping in inner tissues of pine needles that are utilised at night and when recovering from dehydration

Fitness costs of plant disease defence are often subtle and difficult to quantify. In this study, we therefore used comparative high-throughput phenotyping in two independent facilities to assess growth, morphology and physiology of potato (cv. Desiree) with high time-resolution monitoring different defence mechanisms under pathogen-free conditions. Plants were either treated weekly with the resistance inducers {beta}-aminobutyric acid (BABA; 10 mM) or potassium phosphite (KPhi; 36 mM) or comprised six transgenic lines expressing late blight resistance genes (single Rpi genes or a three-gene stack) or reduced jasmonate perception (StCOI1-RNAi). Over four weeks, image-derived traits revealed consistent cross-facility effects for plant height and colour: BABA treatment increased plant height but reduced canopy area and induced a paler greenness signature, whereas KPhi caused minimal and transient growth effects. Chlorophyll fluorescence at the NaPPI facility indicated reduced vitality (Rfd_Lss) in BABA-treated plants and increased Rfd_Lss following KPhi, while maximum PSII efficiency was largely unchanged. Several transgenic lines showed somewhat reduced above-ground biomass. Enzyme activity profiling produced distinct treatment and genotype signatures, but was strongly modulated by facility conditions that overrode these specificities. Overall, high-throughput phenotyping robustly detected subtle growth-defence trade-offs across platforms. HighlightHigh-throughput optical phenotyping validated across two independent research facilities reveals that stacked resistance genes and resistance inducers in potato trigger subtle growth trade-offs. Graphical abstracts O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/713143v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@89df47org.highwire.dtl.DTLVardef@1a1ce64org.highwire.dtl.DTLVardef@1f52f0dorg.highwire.dtl.DTLVardef@1e41c35_HPS_FORMAT_FIGEXP M_FIG C_FIG Experimental timeline for high-throughput plant phenotyping platforms. Created in BioRender. Poque, S. (2026) https://BioRender.com/nmkve7g

Chromatin organization regulates genome stability and gene expression by controlling DNA accessibility to transcription factors and regulatory complexes. DNA-protein interactions are commonly investigated using chromatin immunoprecipitation (ChIP), which relies on specific antibodies often involving technically demanding protocols. CRISPR-Cas technologies have enabled sequence-specific targeting of genomic loci using catalytically inactive Cas9 (dCas9), but most CRISPR-based chromatin capture approaches in plants require transient or stable transformation to express the CRISPR machinery, limiting their applicability across species, tissues and physiological contexts. Here, we present GRASP (Genomic Region Affinity Sequestration by CRISPR-Purification), a transformation-independent strategy for sequence-specific chromatin isolation operating directly on purified plant nuclei. In GRASP, dCas9-gRNA ribonucleoprotein complexes are used to capture predefined genomic regions from chromatin under native conditions, bypassing the need for transgene expression. Using grapevine and tomato as model systems, we demonstrate efficient and highly specific enrichment of target loci, including telomeric repeats as well as low-copy and single-copy genomic regions, with qPCR and NGS validation. These results establish GRASP as a robust and broadly applicable platform for locus-specific chromatin isolation in plants. Beyond sequence-specific DNA isolation, GRASP establishes a versatile platform for potential downstream analyses of locus-associated chromatin components, including protein complexes, distal DNA-DNA interactions and chromatin-associated RNAs, providing new opportunities to investigate regulatory architecture in plant genomes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/712347v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@13758e8org.highwire.dtl.DTLVardef@adfd82org.highwire.dtl.DTLVardef@de81f4org.highwire.dtl.DTLVardef@25c2d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Starch synthesis in wheat endosperm involves the initiation of large A-type starch granules during early grain development, followed by small B-type granules in later grain development. It is established that MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1) plays an important role in granule initiation in Arabidopsis chloroplasts, but how it influences A- and B-type initiations in wheat amyloplasts is not known. We discovered that due to a gene duplication in cereals, wheat contains two MFP1 paralogs, MFP1.1 and MFP1.2, which are both expressed in the developing endosperm. We generated a series of durum wheat mutants defective in all homoeologs of either MFP1.1 or MFP1.2, or both. While starch granule size distributions and granule morphology of mfp1.1 and mfp1.2 mutants were identical to those of the wild-type, the mfp1.1 mfp1.2 mutants had fewer, but larger B-type granules - suggesting that the two paralogs play redundant roles in B-type granule initiation. Consistent with this, both paralogs interacted with B-GRANULE CONTENT 1 (BGC1), a key protein required for proper B-type granule initiation in wheat, and both paralogs could partially complement defects in starch initiation in the Arabidopsis mfp1 mutant. Our work demonstrates that MFP1 is required for establishing correct starch granule number in non-photosynthetic amyloplasts, but its role in wheat is limited to B-type granule initiation. One-sentence summaryWheat has two MFP1 paralogs that interact with the granule initiation protein, BGC1 and influence B-type granule initiation in non-photosynthetic amyloplasts of endosperm.

Long-read sequencing has shown a rapid technological development during the last years. It has been established as the standard method for the sequencing of plant genomes and has also gained importance for full plasmid sequencing. As Sanger sequencing has a limited read length of about 1 kb, long read sequencing offers a great advantage, as the full plasmid can be sequenced in one read. Here, we present a cost-effective workflow to sequence full plasmids and compare the results against an expectation. The per plasmid cost of this workflow is determined by the number of plasmids investigated simultaneously, but can be lower than the price of a single Sanger sequencing reaction. We developed a workflow for automatic data processing, which allows us to complete sequencing and data analysis within a day.

The Nucleotide-Binding Domain Leucine-Rich Repeat (NLR) gene family is a central component of plant immune systems, yet its diversity and evolutionary dynamics remain poorly characterized in long-lived tree species. Here, we performed a genome-wide analysis of the NLR gene family in Quercus suber (cork oak) using InterNLR, a new annotation tool, and explored their expression regulation in response to biotic and abiotic stresses. A total of 918 NLR and NLR-like genes were identified, encompassing both canonical and non-canonical members. Phylogenetic analyses based on the NB-ARC domain highlighted the distinct evolutionary trajectory of RNL proteins, which function as helper NLRs and show evidence of clade-specific gene duplication. Transcriptomic analyses revealed pronounced tissue-specific expression patterns, with RNLs exhibiting significantly elevated expression in xylem, suggesting a specialized role in this tissue. Under drought stress, seven NLR genes were differentially expressed and shared orthology with known abiotic stress-responsive genes. Notably, a CNL gene (LOC111996439) responded to both biotic and abiotic stresses, indicating a potential role as an integrative regulator of early defence responses, while an ADR1 orthologue (LOC112022539) suggests molecular crosstalk between stress signaling pathways. Population genetic analyses further revealed signatures of both positive and balancing selection acting on NLR genes. Together, these results provide new insights into the evolution, expression, and functional diversification of NLRs in cork oak. This work advances our understanding of immune gene architecture in an ecologically and economically important forest tree species.

Pangenome-level gene family identification often applies sequence similarity clustering without phylogenetic or synteny information, which risks biologically misleading evolutionary inferences. Using five transcription factor families (bHLH, MYB, NAC, WRKY, MADS-box) across 401 rice pangenome accessions, we compared clustering strategies: OrthoFinder alone, cd-hit alone, MMseqs2 alone, and OrthoFinder-informed refinement by cd-hit or MMseqs2. Methods solely based on sequence similarity merged distinct orthogroups and generated fewer orthogroups than approaches incorporating graph-based orthology. Conflicting cluster assignments, measured against OrthoFinder, varied strongly among families, from approximately 14% in MADS-box to approximately 57% in MYB, and were associated with protein length differences. Core, shell, and cloud gene classifications shifted substantially depending on the method, especially in MYB, NAC, and WRKY families. Critically, Ka/Ks distributions for core genes were highly method-sensitive, with orthology-aware methods yielding more convergent and less variable estimates of selective pressure, whereas noncore gene estimates remained robust. These findings demonstrate that neglecting graph-based orthogroup inference inflates methodological artifacts. We recommend a two-step strategy: initial graph-based orthogroup delineation followed by sequence similarity refinement to balance evolutionary accuracy and resolution in pangenome-scale gene family studies.

AbstractEarly sowing is a key strategy to improve maize productivity and resilience under climate change, but it exposes plants to prolonged chilling stress that can severely compromise seedling establishment. While previous genetic studies have focused on germination or very early stages, tolerance to long-term chilling during the autotrophic transition remains poorly characterized. Here, we combined genome-wide association studies (GWAS) and transcriptome analysis on QTL near-isogenic lines (NILs) to dissect the genetic architecture of early vigor under chilling in maize. We identified a major genomic region on chromosome 4 (LD_COL4), harboring two QTLs within a 2.7 Mb interval, that were consistently associated with early vigor under long-term chilling conditions. Transcriptomic analysis of contrasted NILs revealed a cluster of differentially expressed genes co-localizing with LD_COL4, pointing to two strong candidate genes, Zm00001d048582, an ortholog of the Arabidopsis OPS gene that regulates the brassinosteroid (BR) signaling pathway upstream of the key transcription factors BES1 and BZR1, and Zm00001d048612, a brassinosteroid-signaling kinase (BSK). Multiple orthologs of BES1/BZR1 modulators were differentially expressed between genotypes under chilling, supporting the involvment of brassinosteroid signaling in this response. These findings highlight both genes as promising targets for marker-assisted breeding and gene editing to improve maize adaptation to early sowing.

T-DNA insertion mutants are widely used to disrupt genes and infer their functions, yet the insertions can also trigger unintended genomic changes that confound phenotypic interpretation. Here, we used T-DNA insertion mutants affecting the major mitochondrial malate dehydrogenase (MDH1) and the heterodimeric NAD-dependent malic enzymes (ME1 and ME2) to examine their functional coordination across photoperiods and irradiance regimes. Under short days, especially at low light intensity, mdh1xme2 mutants were markedly smaller than wild type and, unexpectedly, than the mdh1xme1xme2 triple mutant, and they showed a more pronounced reduction in photosynthetic capacity. ME1 was undetectable in mdh1xme2, implying that the double and triple mutants effectively lack heterodimeric ME and should therefore behave similarly, contrary to what we observed. Whole-genome analysis resolved this discrepancy by revealing that the MDH1 T-DNA insertion in mdh1xme2 is accompanied by a major rearrangement, a 137-kbp duplication downstream of the insertion site, which was absent in the mdh1xme1xme2 triple mutant. This duplication increased gene dosage and elevated transcript abundance across the duplicated interval, while proteomics detected 5 of the 38 encoded proteins, including PEPC1. mdh1xme2 accumulated oxaloacetate-derived amino acids and displayed an altered carbon/nitrogen balance, making PEPC1 a plausible contributor to the exacerbated mdh1xme2 phenotype. Together, our data indicate that a T-DNA-linked structural variant can amplify expression of dozens of genes and intensify phenotypes at specific conditions, thereby affecting the interpretation of genotype-phenotype relationships. Because Agrobacterium-mediated DNA transfer also underpins many genome-editing workflows, our findings argue that structural validation around insertion/editing loci should be considered essential when interpreting T-DNA-derived plant lines.

Early sowing of maize (Zea mays L.) is increasingly required to mitigate summer drought under climate change, making the acquisition of chilling tolerance a major agronomic challenge. Here, we investigated the molecular and physiological bases of cold tolerance using two maize near-isogenic lines (NILs) differing at two major chilling tolerance quantitative trait loci (QTLs) located on chromosome 4. Plants were exposed to low temperature (14{degrees}C day/10{degrees}C night) for 20 days and analyzed using an integrated multi-omics approach combining transcriptomics, soluble and cell wall proteomics, and metabolomics (primary and specialized metabolites), together with physiological measurements. Univariate and multivariate analyses revealed significant chilling-induced variability across all molecular layers, affecting [~]0.2% of genes, [~]6% of proteins, and a subset of specialized metabolites, while primary metabolites were largely stable. Integrative statistical analyses demonstrated that the soluble and cell wall proteomes contributed most strongly to the genotype effect, highlighting protein-level regulation as a major determinant of chilling tolerance. A restricted 5.15 Mb divergence region on chromosome 4 was sufficient to drive contrasting physiological responses, including differences in photosynthetic charge separation efficiency and leaf development, favoring the chilling-tolerant NIL. Notably, several components of the benzoxazinoid pathway located within the divergence region, including BX1 and associated specialized metabolites (BZX-like glucoside, DIBOA-glucoside-2, HBOA-glucoside-2), were specifically associated with chilling tolerance, suggesting a role in stress signaling and hormonal crosstalk. Overall, this study demonstrates that integrative multi-omics analyses provide a powerful framework to resolve genotype-specific regulatory mechanisms underlying chilling tolerance in maize and to identify candidate molecular targets for breeding. HighlightsO_LIFirst organ-resolved multi-omics dissection of chilling responses in maize NILs. C_LIO_LIA 5.1Mb divergence on chromosome 4 drives major physiological and molecular differences. C_LIO_LIChilling tolerance is linked to more robust photochemical homeostasis and sustained leaf development. C_LIO_LISoluble and cell-wall proteomes dominate the genotype-discriminating -omics signal. C_LIO_LIBenzoxazinoids and defense-related transcriptional modules are differentially activated. C_LIO_LICell wall remodeling enzymes and apoplastic peroxidases emerge as key tolerance players. C_LI

Plant hormones regulate almost every aspect of plant growth and development. KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling, which is thought to transduce signals derived from an unidentified ligand known as the KAI2 ligand (KL), regulates numerous traits, including seed germination in angiosperms and vegetative reproduction in bryophytes. The origin of KAI2 is believed to be ancient, and the evolution of its signaling pathways remains of significant interest. Ferns represent critical lineages for elucidating the evolution of land plant traits and growth mechanisms that enabled adaptation to terrestrial environments. Therefore, functional studies of key components of this pathway in ferns are essential for understanding the evolutionary trajectory of KAI2-dependent signaling during vascular plant diversification. However, experimental platforms for the CRISPR/Cas9 system, a powerful tool for investigating gene function, remain undeveloped in ferns. Here, we report an efficient CRISPR/Cas9 system based on ribozyme-gRNA-ribozyme (RGR) technology in the model fern, Ceratopteris richardii (C. richardii). We generated loss-of-function mutants of the KAI2 ortholog in C. richardii (CrKAI2), as well as the signaling components CrMAX2 and CrSMXL. We demonstrate that exogenous application of an artificial KL agonist increases the expression of KAI2-dependent signaling responsive genes in wild type plants; this response is abolished in Crkai2 mutants. These findings indicate that KAI2-dependent signaling is conserved in C. richardii. Furthermore, this study proposes an efficient CRISPR/Cas9 method that will facilitate genetic studies in ferns.

In nature, plants are subjected to multiple environmental stress factors simultaneously or sequentially. Recent studies revealed that when three or more stress factors impact a plant simultaneously (termed multifactorial stress combination; MFSC), plant survival declines, even if the intensity of each individual stress involved in the MFSC is low. We previously identified RAP2.3 as a key transcription factor (TF) required for Arabidopsis thaliana survival, specifically under a MFSC of salt+excess light+heat stress (i.e., S+EL+HS). Here we report that RAP2.3 is required for the expression of SIGMA3, a nuclear-encoded factor that directs plastid RNA polymerase to specific plastid promoters, and MYB51, a key stress response TF involved in glucosinolate metabolism and oxidative stress responses, specifically during a MFSC of S+EL+HS. Like rap2.3 mutants, myb51 and sig3 mutants display significantly low survival rate specifically under the MFSC of S+EL+HS. Based on MYB51 gene regulatory network analysis and characterization of jasmonic acid (JA) mutants, we further reveal that suppression of JA signaling could play an important role in promoting plant survival under conditions of S+EL+HS. Our findings uncover an additional layer of the response of plants to MFSC, as well as identify potential targets for breeding crops with enhanced tolerance to climate change.

Self-incompatibility (SI), a reproductive mechanism that prevents self-pollen from fertilizing the ovule, is widespread in flowering plants, including the Brassicaceae family, where it promotes outcrossing, genetic diversity, and hybrid vigor. Although prevalent in Brassica rapa, an economically vital crop, it remains poorly characterized in widely grown varieties, such as toria and yellow sarson, with prior studies primarily focused on Brassica napus. Given its potential for hybrid breeding and crop improvement in rapeseed (B. rapa), we characterized key SI-regulatory genes, analyzing their phylogenetic relationships, structure-function dynamics, and expression patterns. Our results indicate sequence, structural, and functional homology as well as conservation with previously known candidates. This study identifies SRK, FER, and ARC1 as essential, while MLPK plays a minor role in SI for the varieties under study. Furthermore, we identified that SRK, FER, and MLPK activate ROS during the SI response, while ARC1 does not. Our findings establish a foundation for harnessing this natural system to integrate agriculturally important traits and sustain them across generations via outcrossing.

While CRISPR/Cas-based gene editing technologies have the potential to greatly advance crop breeding endeavours, intellectual property-related challenges can hinder the ability to move such varieties to the market. Recently, an open-access Cas enzyme derived from large language models (OpenCRISPR-1) was developed and shown to function effectively in human cells. In this study, we demonstrate the successful use of this nuclease in a polyploid plant species (Medicago sativa), with mono- or bi-allelic editing observed in 30% of genotypes bearing OpenCRISPR-1. These findings indicate that OpenCRISPR-1 holds promise to expand the use of gene editing technology in the breeding of polyploid crops.

Malate and fumarate constitute a significant transient carbon stock that is dynamically synthesized during the photoperiod. These organic acids are diurnally stored and remobilised from the vacuole, and they have a key role in the cellular metabolic regulation. This function is well known in C4 and CAM plants. However, in C3 species that are the majority of terrestrial plants, the importance of the vacuolar accumulation/release and its influence on plant growth is still an open question. In Here we addressed this issue generating multiple knockout mutants in Arabidopsis thaliana lacking vacuolar anion channels of the Aluminium-Activated Malate Transporter (ALMT) family, to impair malate and fumarate transport to the vacuole. We show that in these mutants reducing vacuolar transport of malate and fumarate in mesophyll cells leads to a dramatic growth impairment. Metabolic and fluxomic analysis revealed that vacuolar malate and fumarate transport influences plant carbon and nitrogen metabolism as well as cellular pH and ionic homeostasis. In conclusion, our results show that the transport organic acids like malate and fumarate across the vacuolar membrane is essential for plant growth in a C3 plant too. These results establish the importance of the vacuolar pools of malate and fumarate in plant metabolism.

Grain legumes such as pea (Pisum sativum L.) accumulate large amounts of seed storage proteins without nitrogen fertilization due to their symbiosis with nitrogen-fixing bacteria, making them a key source of plant-based proteins. Seed growth and the accumulation of seed storage proteins are tightly regulated by complex gene networks; however, the mechanisms governing these processes in pea remain poorly understood. In this study, we generated a comprehensive seed expression atlas covering six developmental stages in pea (cv Cameor), including the key transition stage from embryogenesis to early seed filling, providing a detailed temporal resolution of transcriptional dynamics throughout seed development in this species. Co-expression network analysis highlighted several candidate transcription factors potentially involved in the transition towards seed filling. Among them, we characterized the seed-specific NF-YB transcription factor PsLEC1-like (PsL1L), the major LEC1-type factor expressed during early pea seed development. Functional analyses using TILLING mutants demonstrated that loss of PsL1L function reduces seed size and seed nitrogen content and impairs early embryo growth from the end of embryogenesis. Finally, we show that the expression of the B3-domain transcription factor PsFUS3, but not that of PsLEC2 or PsABI3, is reduced in the loss-of-function l1l mutant, suggesting that PsL1L acts upstream of PsFUS3 to control seed size.