The miR159a‐DUO1 module regulates pollen development by modulating auxin biosynthesis and starch metabolism in citrus

• Published in: Journal of integrative plant biology 2024-07, Vol.66 (7), p.1351-1369
• Main Authors: Xu, Yanhui, Tian, Wenxiu, Yin, Minqiang, Cai, Zhenmei, Zhang, Li, Yuan, Deyi, Yi, Hualin, Wu, Juxun
• Format: Article
• Language: English
• Subjects: auxin; Auxins; Biosynthesis; citrus; Citrus fruits; DNA sequencing; Fortunella hindsii; Fruits; Gene expression; Gene sequencing; Indoleacetic acid; Male sterility; Males; Metabolism; miRNA; Modules; Phenotypes; Plant breeding; Pollen; pollen development; Pollination; Regulatory mechanisms (biology); seedlessness; Starch; starch metabolism
• ISSN: 1672-9072; 1744-7909; 1744-7909
• DOI: 10.1111/jipb.13656

ABSTRACT Achieving seedlessness in citrus varieties is one of the important objectives of citrus breeding. Male sterility associated with abnormal pollen development is an important factor in seedlessness. However, our understanding of the regulatory mechanism underlying the seedlessness phenotype in citrus is still limited. Here, we determined that the miR159a‐DUO1 module played an important role in regulating pollen development in citrus, which further indirectly modulated seed development and fruit size. Both the overexpression of csi‐miR159a and the knocking out of DUO1 in Hong Kong kumquat (Fortunella hindsii) resulted in small and seedless fruit phenotypes. Moreover, pollen was severely aborted in both transgenic lines, with arrested pollen mitotic I and abnormal pollen starch metabolism. Through additional cross‐pollination experiments, DUO1 was proven to be the key target gene for miR159a to regulate male sterility in citrus. Based on DNA affinity purification sequencing (DAP‐seq), RNA‐seq, and verified interaction assays, YUC2/YUC6, SS4 and STP8 were identified as downstream target genes of DUO1, those were all positively regulated by DUO1. In transgenic F. hindsii lines, the miR159a‐DUO1 module down‐regulated the expression of YUC2/YUC6, which decreased indoleacetic acid (IAA) levels and modulated auxin signaling to repress pollen mitotic I. The miR159a‐DUO1 module reduced the expression of the starch synthesis gene SS4 and sugar transport gene STP8 to disrupt starch metabolism in pollen. Overall, this work reveals a new mechanism by which the miR159a‐DUO1 module regulates pollen development and elucidates the molecular regulatory network underlying male sterility in citrus. Overexpression of the microRNA miR159a and knockout of the MYB transcription factor gene DUO1 in citrus led to pollen development defects and abnormal starch metabolism. The miR159a–DUO1 module acts on auxin and starch metabolism to regulate pollen development in citrus.