Molecular Plant

Dendrobium officinale is a typical epiphytic orchid. We report the telomere-to-telomere (T2T) genome assembly for D. officinale, representing the first T2T reference genome within the Orchidaceae family. The assembly is anchored to 19 chromosomes and contains 38 complete telomeres and 15 characterized centromeres. We further generated haplotype-resolved assemblies of the autotetraploid genome, identifying 12,761 sets of tetra-allelic genes. Based on synonymous substitution analysis, we inferred that the autotetraploidization event occurred approximately 0.86 million years ago. A systematic analysis of the SWEET gene family across the genus Dendrobium revealed that the gene family size is shaped primarily by epiphytic types and environmental factors. In D. officinale from Langshan, eight SWEET genes were specifically expressed in roots, suggesting they may play specialized roles in the root mycorrhizal system, potentially contributing to the D. officinales ability to recruit and maintain fungal partners. Together, these resources provide valuable foundations for studies of orchid evolution, functional genomics, and molecular breeding.

Virus-induced genome editing (VIGE) using compact RNA-guided endonucleases is a transformational new approach in plant biotechnology, enabling tissue-culture-independent and transgene-free genome editing (Hu et al. 2025; Liu et al. 2025; Weiss et al. 2025). We recently established a VIGE approach for heritable editing at single loci in Arabidopsis by delivering the compact genome editor ISYmu1 TnpB (Ymu1) and its guide RNA (gRNA) via Tobacco Rattle Virus (TRV) (Weiss et al. 2025). Here, we greatly improved this approach by devising a multiple gRNA expression system and by utilizing an engineered high-activity Ymu1 variant (Ymu1-WFR) (Zhou et al. 2026) to develop an efficient multiplexed genome editing platform.

In many flowering plants, the transition from vegetative growth to reproductive development is regulated by seasonal changes in photoperiod. Under inductive photoperiods, leaves produce the florigen FT (FLOWERING LOCUS T), which is transported to the shoot apex to promote flowering. The photoperiod is known to have a major effect on the flowering of chrysanthemum. In the perennial short-day (SD) plant Chrysanthemum seticuspe, the expression of CsFTL3 (FT-like gene) does not increase immediately after shifting from long-day (LD) to SD conditions but gradually accumulates under continuous SD conditions, peaking during inflorescence development. However, the underlying mechanism remains elusive. We show that CsFDL1 (an ortholog of FD) and CsFTL3 exhibit a significant inverse expression pattern in leaves during the initial stage of short-day inductions. Furthermore, the expression of CsFTL3 is upregulated in the leaves of CsFDL1-knockdown transgenic lines. CsFDL1 is expressed in leaves and forms a complex with CsFTL3 to recognize several TCGA- and ACGT-containing motifs in the CsFTL3 promoter. The CsFTL3-CsFDL1 complex downregulates CsFTL3 expression, thereby preventing its excessive induction by SD signals and inhibiting precocious floral transition. This study reveals that CsFDL1 acts as a key early repressor in the photoperiodic flowering pathway of chrysanthemum leaf, mediating negative feedback regulation by forming a complex with CsFTL3 to achieve precise temporal control of short-day-dependent flowering responses.

Wheat grain weight and flour quality largely depend on starch biosynthesis, yet the mechanisms by which transcription factors coordinate this process remain poorly understood. In this study, using an integrative strategy that combines genome-wide association analysis with yeast two-hybrid library screening, we identify TaNF-YC10, a Nuclear Factor Y transcription factor, as a positive regulator of starch accumulation in the wheat endosperm. Loss of TaNF-YC10 reduces starch content and alters starch granule size distribution, whereas overexpression enhances starch accumulation and increases grain weight. TaNF-YC10 binds and activates core starch biosynthetic-related genes, including AGPL1, GBSS1, YUC11, and NF-YB7, and forms higher-order transcriptional complexes with TaNF-YB1 and TabHLH95 to coordinate multiple regulatory pathways. TaNF-YC10-A1-Hap2 is associated with higher starch content and thousand grain weight and has been selected during wheat breeding in China. Collectively, our findings establish TaNF-YC10 as a pivotal transcriptional hub in starch regulation and highlight its potential as a target for genetic improvement of grain yield in wheat.

Sacha inchi (Plukenetia volubilis L.) is an emerging woody oilseed crop prized for its high alpha-linolenic acid (ALA) content. Despite its nutritional and economic value, the lack of high-quality genomic resources has hindered genetic improvement and the elucidation of its unique polyunsaturated fatty acid and lipid biosynthetic pathways. In this study, we report a high-quality, chromosome-scale genome assembly of sacha inchi with a total length of 710.62 Mb, integrated from Illumina, PacBio, and chromosome conformation capture (Hi-C) technology. The genome harbors 37,570 protein-coding genes, and 379.86 Mb (53.45%) of repetitive sequences. Phylogenomic analysis reveals that sacha inchi diverged from its closest relative Ricinus communis, [~] approximately 36.2 million years ago. Comparative genomics indicates that sacha inchi experienced only ancient whole genome duplication events. To elucidate the mechanisms governing ALA biosynthesis and triacylglycerol (TAG) accumulation in sacha inchi seeds, we performed temporal transcriptome profiling across six seed development stages. Our findings demonstrate that high TAG content is primarily driven by the sustained expression of biosynthetic genes and low activity of degradation genes during mid-to-late seed development. Notably, while genes encoding stearoyl-ACP desaturases (SADs) maintain the precursor pool, the expression of genes encoding fatty-acid desaturase 2 (FAD2) and fatty-acid desaturase 3 (FAD3) is positively correlated with the final accumulation of C18:2 and C18:3 fatty acids. We also identified lncRNAs as potential epigenetic regulators of these key pathways. This high-quality genome provides a critical foundation for elucidating the molecular mechanisms of seed growth and development in sacha inchi.

Polyploidization plays a fundamental role in plant evolution and crop domestication. However, due to the high similarity of genomic sequences between some homologous or homeologous chromosomes, the assembly of some polyploid genomes is extremely difficult, frequently resulting in erroneous assemblies, such as sequence chimeras and sequence collapse. The genus Brachypodium is an important model system for the study of polyploidy in grasses. However, high-quality reference genomes are still lacking for its complex polyploid perennial species. In this study, we developed a bioinformatic pipeline for the accurate assembly of high-quality reference genomes at the chromosomal level for two representative perennial Brachypodium species with conflicting collapsed segments, the allotetraploid B. phoenicoides (2n = 4x = 28) and the autohexaploid B. boissieri (2n = 6x = 48). We developed an innovative methodology (CollapsedChrom) that uses depth-of-read profiling and relies on prior karyotypic information to systematically detect and rescue collapsed regions. This depth-sensitive curation strategy successfully recovered 328.9 Mb and 195.8 Mb of previously collapsed sequences in the genomes of B. phoenicoides and B. boissieri, respectively. Comprehensive quality assessments demonstrated the high quality of our final assemblies. Our chromosomal-level assemblies fully capture the genomic architectures of these species. These robust genomic resources overcome long-standing challenges in polyploid assembly and provide an essential foundation for future research on the evolutionary dynamics, subgenomic interactions, and functional biology of complex polyploid plant genomes.

Plant amino acids function as both pathogen nutrients and essential drivers of systemic immunity. The regulation of amino acid homeostasis through transporters is a essential for mounting a robust and coordinated immune response in plants during pathogen infection. Using systems biology and integrative network science, we investigated bacterial virulence in Arabidopsis. By comparing gene coexpression networks of effector-triggered susceptibility (ETS) and pattern-triggered immunity (PTI), we uncovered a plant amino acid-related processes specifically linked to ETS. Integrating time-series transcriptomics, protein-DNA interactions, and mathematical simulations, we identified ANAC046 as a transcriptional regulator of amino acid processes during ETS. Single-cell RNA-Seq revealed that amino acid transporters are primarily expressed in companion and mesophyll cells, while functional validation confirmed ANAC046s roles in promoting susceptibility. Further integration of transcriptome and interactome data showed that amino acid-related genes interact with key immune hub proteins. Network topology analysis enabled the characterization of seven additional genes involved in plant defense. To support community-wide research, we developed MIData, an open-access platform for pre-analyzed Arabidopsis networks. Together, our findings demonstrate the power of systems-level approaches in uncovering hierarchical regulatory mechanisms underlying plant susceptibility to bacterial pathogens.

Hi-C data is commonly used for reference-free de novo scaffolding. However, with the rapid increase in high-quality reference genomes, reference-guided workflows are now more practical for assembling large numbers of target genomes without relying on costly and labor-intensive Hi-C sequencing. Recently, a pangenome graph-based haplotype sampling algorithm was introduced to generate personalized graphs for target genomes. Such graphs have strong potential as references for reference-guided contig scaffolding. Here, we present noHiC, a reference-guided scaffolding pipeline supporting key steps of plant contig scaffolding. A distinctive feature of noHiC is the nohic-refpick script, generating a best-fit synthetic reference (synref) from a pangenome graph that is genetically close to the target contigs. This enables the integration of genetic information from many references (up to 48 in our tests) without using them separately during scaffolding. Synrefs showed advantages over highly contiguous conventional references in reducing false contig breaking during reference-based correction. Additionally, nohic-refpick can be combined with fast scaffolders (ntJoin) to rapidly produce highly contiguous assemblies using synrefs derived from pangenome graphs. The noHiC pipeline, used alone or in combination with ntJoin, can generally produce assemblies that are structurally consistent with public Hi-C-based or manually curated genomes. The pipeline is publicly available at https://github.com/andyngh/noHiC. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/712436v1_ufig1.gif" ALT="Figure 1"> View larger version (9K): org.highwire.dtl.DTLVardef@40bd8forg.highwire.dtl.DTLVardef@5d2bbborg.highwire.dtl.DTLVardef@e214a3org.highwire.dtl.DTLVardef@b90b06_HPS_FORMAT_FIGEXP M_FIG C_FIG

High-throughput sequencing technologies have enabled the generation of high-quality reference genomes for numerous rice cultivars. However, inferring gene functions, associated phenotypes, and causal variants from these sequences remains challenging. The Rice Annotation Project Database (RAP-DB; https://rapdb.dna.affrc.go.jp) is a curated genomic resource that provides comprehensive gene annotations for the reference genome of Oryza sativa ssp. japonica cv. Nipponbare. Since its major update in 2013, gene models and functional annotations have been continuously revised through expert manual curation of newly published literature related to rice genes. As of March 2025, a total of 6,631 transcripts corresponding to 6,371 loci have been curated based on 4,699 peer-reviewed publications. These curated genes are functionally characterized and are frequently associated with agronomic traits, including yield components, stress tolerance, and disease resistance. To support molecular breeding, RAP-DB now provides a curated catalogue of 904 agronomically important loci, including gene symbols, functional descriptions, and associated traits, together with more than 1,000 functionally characterized alleles compiled from the literature. In addition to in-house expert curation, RAP-DB integrates community-curated datasets for major gene families, such as WRKY transcription factors, S-domain receptor-like kinases, and leucine-rich repeat-containing receptors, thereby expanding coverage of key regulatory and defense-related genes. RAP-DB also incorporates reanalyzed RNA sequencing expression profiles alongside microarray-based expression data and co-expression networks, offering gene-centric views of expression patterns across tissues, conditions, and developmental stages. Furthermore, RAP-DB is linked to genome-wide variation datasets from diverse rice varieties through the TASUKE+ genome browser, enabling exploration of allelic diversity across varieties. To enhance annotation quality and long-term sustainability, AI-assisted literature screening and a web-based feedback system have been introduced, allowing users to submit corrections to gene models and report newly characterized genes or relevant publications. Together, these developments strengthen RAP-DB as a primary, literature-based gene annotation resource and provide a practical foundation for molecular breeding in rice.

O_LIModerate leaf rolling in rice is crucial for plant architecture and stress adaptation, but its molecular regulation remains unclear. We investigated the role of RLC3/OsBLH4, a BELL-type homeobox transcription factor, in controlling leaf rolling and drought tolerance, addressing gaps in lignin biosynthesis and cell wall development mechanisms. C_LIO_LIWe used gene map-based cloning (rlc3-1, rlc3-2), CRISPR/Cas9 knockout lines (rlc3-ko#11, rlc3-ko#12), and allelic complementation to validate RLC3s function. Additionally, we employed biochemical assays, gene expression analysis, and protein interaction studies to explore its regulatory network. C_LIO_LIRLC3 mutations impaired lignin biosynthesis and secondary cell wall formation, reducing bulliform cells area and causing midrib defects. These structural abnormalities accelerated water loss, leading to excessive leaf rolling and compromised drought tolerance. Mechanistically, RLC3 directly activates lignin synthesis genes (OsPAL5, OsCOMT5, OsCCR4, OsCAld5H1) and interacts with KNOX transcription factors (OSH1, OSH45, OSH71) to form a KNOX-BELL complex, further regulating lignin content and cell wall development. C_LIO_LIRLC3 orchestrates lignin deposition and secondary cell wall development to control leaf rolling, water transport, and drought tolerance. This study reveals a novel KNOX-BELL-lignin regulatory module governing leaf morphology and stress adaptation, offering targets for crop improvement under drought conditions. C_LI

Grain oil content and composition are complex quantitative traits that shape cereal grain quality and nutritional value. Sorghum (Sorghum bicolor), a heat- and drought-adapted C crop essential for global food and feed security, remains insufficiently characterized with respect to grain lipidome diversity and its genetic architecture. Here, we integrated population-scale whole-grain lipidomics with genome-wide association studies (GWAS) in 266 sorghum accessions. Lipidome profiling revealed extensive natural variation in triacylglycerols (TAGs), accompanied by coordinated shifts in phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs), explaining 87% of population-level differences in total grain oil. Lipidome-wide GWAS identified approximately 1.6 million significant variant-trait associations and resolved 55 loci linked to plastidial fatty acid synthesis, TAG assembly, lipid transport, and membrane remodeling. These loci, many undetected in previous GWAS of bulk oil content, demonstrated the increased mapping resolution achieved through lipidomics. Integration with metabolic gene clusters revealed significant enrichment of lipid-associated variants within terpene and saccharide-terpene biosynthetic clusters, indicating coordinated genetic regulation between central lipid metabolism and specialized metabolic pathways. Variants within these clusters explained more than 50% of the variance in measured grain oil content and exhibited additive effects of favorable alleles. Haplotype analyses further identified 27 elite sorghum accessions and 12 linked markers for marker-assisted improvement of sorghum grain oil. These findings elucidate the multilayered genetic architecture of sorghum grain lipid diversity and showcase the value of large-scale lipidomics integrated with GWAS for accelerating C crop grain quality improvement.

Flexible developmental programs enable plants to customize their organ size and cellular composition. In leaves of eudicots, the stomatal lineage produces two essential cell types, stomata and pavement cells, and plants can adjust the total numbers and ratios of these cell types in response to external cues. Central to this flexibility is the stomatal lineage-initiating transcription factor, SPEECHLESS (SPCH). Here we explore the mechanisms underlying SPCHs involvement in environmental response. Using multiplexed CRISPR/Cas9 editing of SlSPCH cis-regulatory sequences in tomato, we identified variants with altered stomatal development responses to drought, light and temperature cues. By creating and live-cell tracking translational reporters of SlSPCH and its paralogues SlMUTE and SlFAMA, we revealed the corresponding cellular events that lead to the environmental change-driven responses in stomatal production and leaf form. Plants bearing the novel reporters and SlSPCH variants are powerful resources for fundamental and applied studies of tomato resilience in response to climate change.

Brassinosteroids (BRs) are key regulators of plant growth and have been implicated in suppressing glucosinolates (GSLs) biosynthesis in Brassicaceae species, including Arabidopsis thaliana. However, the molecular mechanism linking BR signaling to transcriptional control of the aliphatic GSL pathway remains unclear. Here, we provide genetic and molecular evidence that the BR-responsive transcription factor BZR1 negatively regulates aliphatic GSL biosynthesis through interaction with TOPLESS (TPL) family corepressors and the histone deacetylase HISTONE DEACETYLASE 19 (HDA19). The gain-of-function mutant bzr1-1D exhibited reduced accumulation of aliphatic glucosinolates (GSLs), whereas disruption of TPL family genes (tpl, tpr1, tpr4) or HDA19 resulted in elevated GSL levels, supporting a repressive role for the BZR1-associated complex in the regulation of GSL biosynthetic gene expression. Furthermore, we uncover a functional connection between ABA and BR signaling in this process. The ABA-responsive transcription factor ABI5 reduces the expression of UBP12 and UBP13, which encode deubiquitinases known to influence BZR1 stability. Genetic and transcriptional analyses indicate that ABI5-mediated attenuation of the UBP12/13-BZR1 pathway contributes to enhanced aliphatic GSL accumulation under ABA treatment. Collectively, our findings delineate a regulatory framework linking ABA and BR signaling pathways and suggest that modulation of the UBP12/13-BZR1 module by ABI5 integrates growth and defense responses by fine-tuning aliphatic GSLs biosynthesis in Arabidopsis. One sentence summaryan ABA signaling factor, ABI5 acts to suppress expression of brassinosteroids (BR) hormone signaling genes like UBP12, UBP13, and BZR1 to promote biosynthesis of defensive secondary metabolites, glucosinolates (GSLs) in Arabidopsis.

Plants are continuously exposed to a myriad of DNA-damaging agents, including environmental cues such as sunlight. At the cellular level, plants respond to DNA damage by activating DNA damage response (DDR) pathways, in which chromatin remodelers play an important role. Among them, the evolutionary conserved INO80 complex (INO80c) has been shown in Arabidopsis to play a key role in DDR, notably by positively regulating Homologous Recombination (HR). Arabidopsis EIN6 ENHANCER (EEN) is the homolog of Yeast INO EIGHTY SUBUNIT 6 and interacts with the N-terminal region of INO80 in the INO80c. Using plant phenotyping, cellular and molecular biology, and third-generation sequencing technology we investigated how INO80 and EEN regulate plant development and genome integrity. We uncovered new roles for INO80 and EEN in plant growth and for INO80 in fine tuning endoreduplication. In addition, linear genome analysis revealed an important and unexpected function for the INO80-EEN complex in preventing Protein Coding Genes (PCGs) from structural rearrangements in somatic tissue and upon exposure to UV-B. Therefore, our results shed new light on the previously overlooked roles of INO80 and EEN in protecting genome integrity at PCGs.

As an important ornamental crop, Phalaenopsis orchids exhibit extraordinary trait diversity with unclear genetic bases. Here, we present a haplotype-resolved, chromosome-level genome for an aneuploid cultivar Santiago. We reveal pervasive variation in the number and sequence among homoeologous genes. When serving as a reference, this genome facilitates the genetic understanding of trait variation in a hybrid population that is highly representative in trait polymorphisms. Specifically, nucleotide polymorphisms in a potential long-distance enhancer and the consequential expression variation of PsAGL6-2, presence/absence of PsMYB12 and b-type homoeologous gene of PsMYB2 determine lip morphology variation, presence/absence of venation-associated stripes and background pink color, respectively. Diverse functions of PsMYBx1 in repressing anthocyanin accumulation across different cultivars further enhances the color patterning diversity in Phalaenopsis. Our study provides a practical framework for using a highly heterogeneous, haplotype-resolved genome to decode phenotypic diversity and has the potential to promote marker-assisted breeding in Phalaenopsis.

MicroRNAs (miRNAs) are key post-transcriptional regulators in plants, yet the evolutionary dynamics of the entire miRNA-mediated regulatory system remain poorly understood. We performed small RNA sequencing and comparative genomics analyses across all eight AA-genome species of Oryza within a well-resolved phylogenetic framework. We found that miRNA gene (MIRNA) families evolve with low birth-death rates, while individual paralogous MIRNAs turn over rapidly. We identified a novel mechanism in which transposable element (TE) insertions de novo generates functional miRNA target sites. miRNA target genes are characterized by distinct evolutionary signatures, including greater sequence length, lower GC content, moderately highly expression, reduced expression variability, and higher evolutionary conservation, which is consistent with their role as a conserved kernel regulatory subsystem. In contrast, the majority of recently evolved target genes that have acquired their miRNA binding sites through TE insertions tend to exhibit the opposite set of features. Furthermore, we uncovered co-evolutionary signatures between duplicated MIRNAs and their target genes, as well as between MIRNAs and phased siRNAs (phasiRNAs). Taken together, our study reveals multi-level evolutionary dynamics driven by TEs that rapidly generate new regulatory circuit and proposes a generalizable TE-MIRNA co-option model for regulatory network expansion in plants.

Together with biosynthesis and transport, inactivation regulates the concentration of indole-3-acetic acid (IAA), a key auxinic compound with a myriad of functions in plant development. Main inactive IAA metabolites are categorised into oxidised forms and ester- or amide-linked conjugates. DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) and DAO2, 2-oxoglutarate and iron-dependent dioxygenases, contribute to IAA oxidative inactivation in collaboration with group II GRETCHEN HAGEN3 (GH3) IAA-amido synthetases, while a group of UDP-glycosyltransferases (UGTs) conjugate IAA to sugars. To study the IAA inactivation routes, we generated combinatorial mutants between all group II GH3s (gh3oct) and DAO1 or DAO2, as well as between the DAOs and main UGTs. In vivo [13C6]IAA feeding experiments traced the metabolic fate of the exogenously applied IAA, supporting the main IAA inactivation pathway, in which DAO acts downstream of GH3s. They also indicated that UGT-mediated IAA glycosylation is more important than previously assumed for modulating IAA levels and plant development. Our metabolic and transcriptomic data further revealed that gh3oct may still produce some GH3 activity, explaining previous reported phenotypic inconsistencies. Our data additionally suggest that other not yet identified metabolic activities might play a role in IAA overproducing plants, and that the premature downregulation of flowering time integrators like FLOWERING LOCUS C (FLC) likely underlies the early flowering of gh3oct and gh3oct dao1 plants.

Sesame (Sesamum indicum) is one of the earliest domesticated oilseed crops and is valued for antioxidant lignans that stabilize oil quality. However, the genomic and evolutionary history of the genus Sesamum, including the origin of its allotetraploid relative S. radiatum and the diversification of lignan metabolism, remains poorly understood owing to limited chromosome-scale genomic resources. Here we present chromosome-level genome assemblies for three wild Sesamum species, two Ceratotheca species and a Japanese sesame cultivar to reconstruct genome and karyotype evolution across the Sesamum-Ceratotheca complex. Comparative analyses show that the derived x=16 lineage originated from an ancestral x=13 karyotype through chromosome fission, fusion and translocation, whereas another x=13 lineage underwent extensive restructuring associated with retrotransposon expansion. Phylogenomics places Ceratotheca within the x=16 Sesamum clade and reveals that S. radiatum originated through hybridization involving a C. sesamoides-like ancestor. The antioxidative lignan gene CYP92B14 was reintroduced via the BB progenitor, linking hybridization with restoration of oil-stabilizing metabolism during sesame evolution.

Seedling emergence is a pivotal step of plant survival, requiring rapid hypocotyl elongation for soil penetration. This energy-demanding process necessitates active mitochondrial respiration, which inevitably induces oxidative damage. Therefore, plants evolved a quality control mechanism that selectively removes dysfunctional mitochondria through the mitophagy pathway. Here, we identified SPL2, a mitochondrial E3 ligase which is essential for hypocotyl elongation and seedling emergence through degrading mitochondrial outer membrane proteins, such as TRB1 and FIS1A. Intriguingly, these proteins also interact with an ER protein, VAP27-1, forming a complex at the ER-mitochondria contact sites, which is essential for mitophagy initiation. The spl2 mutant exhibits enhanced ER-mitochondrial tethering and mitophagy activation, whereas its overexpression has the opposite effects. The expression of SPL2 increases after light perception, in agreement with the reduced mitophagy. Collectively, our findings reveal novel mechanistic insights into seedling emergence, which are coordinated through protein ubiquitination, ER-mitochondrial interaction, and mitophagy.

Fruit development is a typical angiosperm feature that greatly facilitates seed dispersal. Despite extensive studies on the gene regulatory network underlying pod shattering in the dry Arabidopsis fruit and the ripening process in the fleshy tomato fruit, it is yet unclear if a conserved regulatory network acts in early fruit development. Here, we investigated the roles of Petunia x hybrida (petunia) FRUITFULL (FUL), SHATTERPROOF (SHP) and APETALA 2 (AP2) homologs, three types of transcription factors repeatedly associated with fruit development and/or ripening. Petunia is closely related to tomato but produces dry dehiscent fruits like Arabidopsis. Our functional analysis revealed that the three petunia FUL-like genes, PETUNIA FLOWERING GENE (PFG), FLORAL BINDING PROTEIN 26 (FBP26) and FBP29, redundantly regulate endocarp development. They promote the formation of regularly shaped inner endocarp cells, probably via auxin/brassinosteroid signalling and cell wall modification. Furthermore, we discovered that the SHP-like gene FLORAL BINDING PROTEIN 6 (FBP6) has an opposite role, promoting more mesocarp-shaped endocarp cells, indicating that the FUL-like and SHP-like genes act antagonistically in early pericarp development. Finally, we show that the AP2-like genes REPRESSOR OF B-FUNCTION 1 (ROB1), ROB2 and ROB3 are crucial factors in petunia fruit development. rob1 rob2 rob3 mutants completely fail to dehisce and show major defects in pericarp patterning. The ROB transcription factors repress the activity of the FUL-like genes, and have, together with FBP6, an opposite effect on auxin and brassinosteroid signalling genes. Our study suggests that a module consisting of antagonistically acting TFs, including co-orthologs of AP2, FUL and SHP, regulates early pericarp patterning, at least partially via auxin and brassinosteroids.