PRC2 regulates cytokinin and HD-ZIP III pathways to orchestrate vascular tissue pattern formation in Arabidopsis

Polycomb repressive complex 2 (PRC2) regulates plant development, but systemic PRC2 mutants have pleiotropic defects that complicate investigation of its role in regulating secondary growth. Here, we used CRISPR-TSKO to generate a vascular tissue-specific knockout of the PRC2 component FERTILIZATION INDEPENDENT ENDOSPERM (FIE) driven by the promoter of the vascular tissue-specific gene WUSCHEL HOMEOBOX RELATED 14 (WOX14), termed WOX14pro:FIE-KO, in Arabidopsis (Arabidopsis thaliana). WOX14pro:FIE-KO plants showed ectopic vascular bundles, vascular cylinders within the phloem parenchyma, and inhibited differentiation of reticulate or pitted vessels and fibers in xylem. RNA-seq and ChIP analyses revealed that FIE directly mediates H3K27me3 deposition on genes encoding isopentenyltransferase (IPT) cytokinin biosynthesis enzymes, repressing their transcription and modulating cytokinin's spatial distribution and responses in hypocotyls. Genetic manipulation of cytokinin biosynthesis or signal transduction partially rescued vascular defects in FIE-knockout hypocotyls. Furthermore, we identified HOMEODOMAIN LEUCINE ZIPPER CLASS III (HD-ZIPIII) transcription factors as downstream targets of the FIE-IPT module, showing reduced expression in WOX14pro:FIE-KO and restoration in WOX14pro:FIE/IPT3/IPT5/IPT7-KO. Overexpression of ARABIDOPSIS HOMEOBOX GENE 8 (ATHB8) in WOX14pro:FIE-KO also partially rescued the vascular defects. Collectively, our findings expand the application of tissue-specific knockout technology in plants and establish PRC2 and its downstream signaling cascade as regulators of vascular tissue patterning.