The lineage-specific CitmiRn23-CitXS20 module regulates apomixis in citrus through modulation of H₂O₂ level

Most citrus species exhibit polyembryony, a mode of sporophytic apomixis that asexual embryos develop in the nucellus. Polyembryony can propagate clonal seedlings but impedes crossbreeding. A 21-nt novel miRNA, CitmiRn23, and target gene CitXS20, encoding a XS domain protein, antagonistically express between polyembryonic and monoembryonic (sexual reproductive) ovules. CitmiRn23 overexpression and RNA interference (RNAi) of CitXS20 was conducted in minicitrus (Fortunella hindsii). The protein interacted with CitXS20 was identified, and exogenous treatments validated the effect of H₂O₂ on polyembryony. CitmiRn23 overexpression and CitXS20 RNAi both repressed nucellar embryogenesis (NE) and thus increased monoembryonic seed rate and hybrid rate. CitXS20 interacted with CitCAT2 and impaired its catalase activity. In ovaries with downregulated CitXS20, catalase activity was upregulated, while H₂O₂ level was decreased, indicating that interaction with CitXS20 might retard CitCAT2 activity in H₂O₂ scavenging. H₂O₂ preferentially accumulated in polyembryonic ovules. The treatment with H₂O₂ increased polyembryony level, whereas treatment with diphenyliodonium chloride reduced H₂O₂ level and polyembryony level, and thus increased hybrid rate. The citrus-specific CitmiRn23-CitXS20 module regulates NE through CitCAT2-mediated modulation of H₂O₂ level in ovule, which provides insights into apomixis mechanisms and promising approaches to regulate polyembryony, and thus facilitates crossbreeding in citrus.