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Abstract Single-cell genomics is rapidly reshaping plant biology, yet broader adoption is limited by plant-specific technical constraints, fragmented tools, and inconsistent analytical practices. Here we report outcomes from the 2025 Summer Workshop for Plant Single-Cell Analysis, which convened researchers to define community needs and design shared solutions. Participants identified five priority challenge areas: (i) improving data quality through imputation, simulation, and deep generative modeling; (ii) developing automated, phylogenetically aware cell type annotation frameworks; (iii) reconstructing developmental trajectories and gene regulatory networks from single-cell and single-nucleus profiles; (iv) creating visualization approaches that embed transcriptional states into anatomically grounded plant organ contexts; and (v) using artificial intelligence (AI) agents and foundation models to orchestrate end-to-end single-cell workflows. In response, we established PlantSCHub, a community-curated web portal that aggregates protocols, datasets, and tutorials to support reproducible plant single-cell analysis. We outline conceptual roadmaps for cross-species integration, inference of multimodal trajectory and gene regulatory networks (GRNs), spatially anchored visualization, and AI scientist agents that dynamically coordinate analytical tools and literature. We discuss both scRNA-seq and scATAC-seq that are important for regulatory inference and cross-species analysis. Together, these efforts aim to transform isolated plant single-cell studies into an interoperable, evolving ecosystem that accelerates discovery and crop improvement. 

Organ initiation from the shoot apical meristem first gives rise to leaves during vegetative development and then flowers during reproductive development.LEAFY(LFY) is activated after floral induction and together with other factors promotes the floral program. LFY functions redundantly with APETALA1 (AP1) to activate the class B genesAPETALA3(AP3) andPISTILLATA(PI), the class C geneAGAMOUS(AG), and the class E geneSEPALLATA3, which leads to the specification of stamens and carpels, the reproductive organs of flowers. Molecular and genetic networks that control the activation ofAP3,PI,andAGin flowers have been well studied; however, much less is known about how these genes are repressed in leaves and how their repression is lifted in flowers. Here, we showed that two genes encodingArabidopsisC2H2 ZINC FINGER PROTEIN (ZFP) transcription factors, ZP1 and ZFP8, act redundantly to directly repressAP3,PI,andAGin leaves. AfterLFYandAP1are activated in floral meristems, they down-regulateZP1andZFP8directly to lift the repression onAP3,PI,andAG. Our results reveal a mechanism for how floral homeotic genes are repressed and derepressed before and after floral induction. 

Cellulose, the main component of the plant cell wall, is synthesized by the multimeric cellulose synthase (CESA) complex (CSC). In plant cells, CSCs are assembled in the endoplasmic reticulum or Golgi and transported through the endomembrane system to the plasma membrane (PM). However, how CESA catalytic activity or conserved motifs around the catalytic core influence vesicle trafficking or protein dynamics is not well understood. Here, we used yellow fluorescent protein (YFP)-tagged AtCESA6 and created 18 mutants in key motifs of the catalytic domain to analyze how they affected seedling growth, cellulose biosynthesis, complex formation, and CSC dynamics and trafficking in Arabidopsis thaliana. Seedling growth and cellulose content were reduced by nearly all mutations. Moreover, mutations in most conserved motifs slowed CSC movement in the PM as well as delivery of CSCs to the PM. Interestingly, mutations in the DDG and QXXRW motifs affected YFP-CESA6 abundance in the Golgi. These mutations also perturbed post-Golgi trafficking of CSCs. The 18 mutations were divided into 2 groups based on their phenotypes; we propose that Group I mutations cause CSC trafficking defects, whereas Group II mutations, especially in the QXXRW motif, affect protein folding and/or CSC rosette formation. Collectively, our results demonstrate that the CESA6 catalytic domain is essential for cellulose biosynthesis as well as CSC formation, protein folding and dynamics, and vesicle trafficking. 

Cellulose, the main component of the plant cell wall, provides a stable environment for cells and is the most abundant source of biomass on Earth. Endosidin20 (ES20) is a recently identified cellulose biosynthesis inhibitor (CBI) that targets the catalytic site of plant cellulose synthase (CESA). Here, we screened over 600 ES20 analogs for their inhibitory effects on plant growth and identified nine active analogs named ES20-1 to ES20-9. Among these, Endosidin20-1 (ES20-1) had stronger inhibitory effects on plant growth and cellulose biosynthesis than ES20. Previously identified Arabidopsis thaliana cesa6 alleles that reduce plant sensitivity to ES20 also caused reduced sensitivity to ES20-1 in terms of plant growth. At the biochemical level, we demonstrated that ES20-1, like ES20, directly interacts with CESA6. At the cellular level, this molecule, like ES20, induced the accumulation of cellulose synthase complexes (CSCs) at the Golgi apparatus and inhibited their secretion to the plasma membrane. Like ES20, ES20-1 likely targets the catalytic site of CESA. However, through molecular docking analysis using modeled full-length CESA6 structure, we found that both ES20 and ES20-1 might have another target site at the transmembrane regions of CESA6. Besides ES20, other CBIs such as Isoxaben, C17 and Flupoxam are widely used tools to dissect the mechanism of cellulose biosynthesis and are valuable resources for the development of herbicide. Multiple CESA mutants which are insensitive to these CBIs have been identified. Here, based on mutant genetic analysis and molecular docking analysis, we have identified the potential target sites of these CBIs on modeled CESA structure. Some bacteria also produce cellulose, and both ES20 and ES20-1 inhibited bacterial cellulose biosynthesis. Therefore, we conclude that ES20-1 is a more potent analog of ES20 that inhibits intrinsic cellulose biosynthesis in plants and both ES20 and ES20-1 show inhibitory effect in bacterial growth and cellulose synthesis, making them excellent tools for exploring the mechanisms of cellulose biosynthesis across kingdoms.