Abstract Glomerella leaf blotch (GLB), a severe fungal disease caused by Colletotrichum gloeosporioides, threatens the apple (Malus domestica Borkh.) industry worldwide. The susceptibility of apple to GLB is controlled by a single dominant locus. Although several markers for GLB susceptibility have been developed, the causal genes and their functional variations have not been explored. In this study, we used MapQTL and fine mapping to identify SNP544 C/G in the MdWRKY11 promoter as the major factor underlying the variation in GLB susceptibility in apple. The SNP544 G allele generates a W-box motif that is directly bound by MdWRKY29 and activates MdWRKY11 expression during C. gloeosporioides infection. In turn, MdWRKY11 binds to the MdDMR6.1 promoter and activates its expression, leading to salicylic acid degradation and leaf necrosis. The MdWRKY11 SNP544 G allele was strongly associated with GLB susceptibility, with a prediction accuracy of 96.2% in 767 Malus accessions. In addition to SNP544, we identified nine allelic variations in MdWRKY11 across 29 Malus accessions, with a combined prediction accuracy for GLB susceptibility of 99.6%. These findings shed light on the genetic control of GLB susceptibility in apple and provide an efficient molecular marker for breeding GLB-resistant cultivars.
Abstract The corolla is a primary determinant of floral architecture and reproductive success. However, the processes that translate localized growth patterns into species-specific morphologies remain unclear. The complex inflorescences (capitula) of Asteraceae consist of different types of flowers. In Chrysanthemum × morifolium, anemone-type varieties represent a distinct floral form characterized by dramatically elongated corolla tubes in bisexual disc florets. Using the anemone-type chrysanthemum, characterized by dramatically elongated disc florets, we demonstrate that sustained corolla formation depends on localized auxin enrichment. Two auxin response factors, CmARF6 and CmARF8.1, have been identified as positive regulators of this process. These ARFs are post-transcriptionally regulated by miR167 in a dose-dependent manner, establishing a regulatory module that directly activates the auxin-biosynthesis gene CmYUC2 and auxin-transport gene CmPILS1. Concurrently, CmARF6 and CmARF8.1 stimulate localized tissue proliferation by activating CmSAUR64 and CmSAUR76. Comparative analyses across diverse Asteraceae species suggest that auxin-associated regulatory mechanisms may contribute to region-specific corolla growth. Our findings demonstrate that auxin regulation contributes to region-specific corolla growth in the bisexual disc florets of chrysanthemum, providing a framework for investigating related morphological variation in other Asteraceae species.
Abstract Serine/arginine (SR) splicing factors can alter downstream precursor messenger RNA (pre-mRNA) splicing patterns and influence gene expression. However, the influences of different isoforms generated by alternative splicing of SR proteins themselves on the transcription and splicing of downstream target genes in plants remain to be elucidated. Here, we demonstrate that the interaction between arginine/serine-rich zinc knuckle-containing protein 32 (RS2Z32) splicing isoforms and the Arabidopsis thaliana ortholog of human splicing factor SC35 (SC35) leads to the formation of distinct complexes, through which the recognition of splice sites within intron 3 of the transcription factor circadian clock associated 1 (CCA1) gene is regulated, ultimately resulting in an altered ratio of the CCA1 splice variants. Competitive binding of CCA1α and CCA1β to the flowering locus C (FLC) gene promoter can either promote or inhibit FLC transcription and flowering. This study elucidates the molecular mechanism by which RS2Z32 regulates FLC expression independently of its canonical splicing function, enhancing our understanding of transcriptional regulation mediated by splicing factors in plants.
Abstract Plants are multicellular systems composed of highly specialized cell types, whose distinct transcriptomes, proteomes, and metabolomes collectively shape growth, development, defense, and environmental adaptation. While single-cell transcriptomics has transformed our understanding of plant cellular heterogeneity, metabolites provide a direct and integrative readout of cellular biochemical activity and physiological state and can offer valuable insights into phenotype. They provide essential information that gene expression alone does not capture. Recent advances in mass spectrometry-based single-cell metabolomics (scMet) have enabled direct measurement of metabolic variation across individual cells and subcellular domains. In parallel, mass spectrometry imaging (MSI) has emerged as a powerful approach for mapping the spatial organization of metabolites within intact plant organs, offering new opportunities to connect chemical diversity with anatomy, cell identity, and biological functions. This review summarizes current technologies for plant single-cell sampling, scMet, and MSI for spatial metabolomics. It further highlights emerging efforts to illuminate the plant’s cellular and spatial dark metabolome through spectral libraries, computational metabolomics, and AI-assisted annotation. A long-term objective is to build quantitative, spatially resolved, and biologically interpretable plant signaling and metabolic networks at single-cell resolution.
Abstract Prime editing enables precise modification of plant genomes and provides a means to recreate beneficial alleles underlying agronomically important traits. However, efficient and multiplexable prime editing systems remain limited in dicot crops. Here, we developed optimized prime editing systems for tomato (Solanum lycopersicum) and applied them to engineer and stack disease-resistance alleles. Using three tomato prime editors, TomPE2, TomPE6c, and TomPE7, we recreated a naturally occurring resistance-associated deletion identified in pepper by precisely deleting two leucine residues from the CYSTM-domain proteins SlBs5 and SlBs5L. The resulting tomato lines exhibited enhanced resistance to Xanthomonas, demonstrating that a resistance allele can be functionally recreated across related crop species. In parallel, introduction of premature stop codons into the susceptibility gene SlDMR6 conferred resistance to both Xanthomonas and Pseudomonas syringae. Transcriptome profiling revealed distinct defense states: Slbs5/Slbs5L plants predominantly exhibited enhanced pathogen-induced defense responses, whereas Sldmr6 plants showed elevated basal defense. Comparison of prime editor architectures and culture temperatures revealed differences in editing activity and precision, with TomPE6c producing the highest proportion of precise edits. We further developed a modular multiplex prime editing system capable of simultaneously modifying multiple loci, enabling resistance alleles and regulatory variants to be combined within a single generation. The engineered alleles were stably inherited, and resistance phenotypes were maintained under elevated temperature conditions. Together, these results establish prime editing as an effective platform for precise recreation, engineering, and multiplex stacking of disease-resistance alleles and provide a framework for accelerating resistance breeding in crops.
