miR156 regulates somatic embryogenesis by modulating starch accumulation in citrus

Somatic embryogenesis (SE)is a major regeneration approach for in vitro cultured tissues of plants, including citrus. However, SE capability is difficult to maintain, and recalcitrance to SE has become a major obstacle to plant biotechnology. We previously reported that miR156-SPL modules regulate SE in citrus callus. However, the downstream regulatory pathway of the miR156-SPL module in SE remains unclear. In this study, we found that the transcription factors (TFs)CsAGL15 and CsFUS3 bind to the CsMIR156A promoter and activate its expression. Suppression of csi-miR156a function leads to up-regulation of four target genes CsSPLs and reduction of SE efficiency. In the STTM-miR156a overexpressed callus (MIM156), the number of amyloplasts and starch content were significantly reduced, and genes involved in starch synthesis and transport were down-regulated; csi-miR172d was down-regulated, whereas the target genes CsTOE1.1 and CsTOE1.2, which inhibit the expression of starch biosynthesis genes, were up-regulated. In our working model, CsAGL15 and CsFUS3 activate csi-miR156a, which represses CsSPLs and further regulates csi-miR172d and CsTOEs, thus to alter starch accumulation level in callus cells and regulate SE in citrus. This study elucidates the pathway of miR156-SPLs and miR172-TOEs mediated regulation of SE, and provides clues to enhancing SE capability in citrus.