The miR399- CsUBC24 module regulates reproductive development and male fertility in citrus.

MicroRNA399 (miR399) regulates phosphate homeostasis in plants by downregulating the expression of PHOSPHATE2 (PHO2, or UBC24 encoding the ubiquitin-conjugating E2 enzyme) . We previously identified CsmiR399a.1 in a small RNA sequencing (sRNA-seq) screen of a male-sterile somatic cytoplasmic hybrid (or cybrid) of pummelo (Citrus grandis). Here, we report that miR399 affects reproductive development and male fertility in citrus. Downregulation of CsmiR399a.1 using a short tandem target mimic (STTM) led to abnormal floral development, inhibition of anther dehiscence and decreased pollen fertility. When grown in inorganic phosphate (Pi)-sufficient conditions, CsmiR399a.1-STTM plants had lower total phosphorus content in their leaves than the wild type (WT) and showed typical symptoms of Pi deficiency. In CsmiR399a.1-STTM plants, the expression of genes involved in starch metabolism and Pi-homeostasis was significantly different than in the WT. Thus, we conclude that miR399-STTM mimicked Pi deficiency, disturbed starch metabolism, and was responsible for pollen grain collapse in the transgenic lines. We identified CsUBC24, a citrus homolog of Arabidopsis thaliana AtUBC24 (PHO2), as a target of CsmiR399a.1 that physically interacts with the floral development regulators SEPALLATA family (CsSEP1.1, CsSEP1.2, CsSEP3), and the anther dehiscence regulator INDUCER OF CBF EXPRESSION 1 (CsICE1). We hypothesize that CsUBC24 downregulates the CsSEPs, which disrupts the floral meristem identity regulatory network and leads to developmental abnormalities in flowers. By interacting with CsICE1, CsUBC24 disturbs stomate function on the anther surface, which inhibits anther dehiscence. These findings indicate that a miR399-based mechanism influences both reproductive development and male fertility in citrus.