Citrus polyembryony gene CitRWP activates alternative NAD(P)H dehydrogenase and triggers H2O2 accumulation

Citrus exhibits polyembryony, a form of sporophytic apomixis, which involves development of nucellar cells into embryos. Recent genetic mapping identified CitRWP, an RWP-RK transcription factor as a key regulator of polyembryony, however, there is lack of homozygous genotype of CitRWP and its promoter in nature. The mechanism of this phenomenon remains unclear. Here, we demonstrate that overexpression of CitRWP induces somatic embryogenesis in Arabidopsis roots and leads to the accumulation of reactive oxygen species (ROS) and cell death in vegetative tissues of Fortunella hindsii. Transcriptomic analysis of CitRWP-overexpressing leaves revealed enrichment of differentially expressed genes involved in ROS metabolism, redox regulation, and antioxidant defense pathways. Analysis of the CitRWP promoter with miniature inverted-repeat transposable element (MITE) insertion revealed that no homozygous genotypes were detected among the artificially generated hybrid progeny, which is indicative of lethality of the CitRWP-overexpressing genotype. Luciferase assay, Electrophoretic Mobility Shift Assay, and CUT&Tag-qPCR confirmed that CitRWP directly binds to and activates the alternative NAD(P)H dehydrogenase (NDA) promoter. Overexpression of NDA in F. hindsii resulted in H2O2 accumulation, leaf chlorosis, and growth inhibition. Our results indicate that CitRWP promotes H2O2 production via NDA activation, providing new insights into its regulatory role in citrus polyembryony.