The miR3367–lncRNA67–GhCYP724B module regulates male sterility by modulating brassinosteroid biosynthesis and interacting with Aorf27 in <i>Gossypium hirsutum</i>

Cytoplasmic male sterile (CMS) lines play a crucial role in utilization of heterosis in crop plants. However, the mechanism underlying the manipulation of male sterility in cotton by long non-coding RNA (lncRNA) and brassinosteroids (BRs) remains elusive. Here, using an integrative approach combining lncRNA transcriptomic profiles with virus-induced gene silencing experiments, we identify a flower bud-specific lncRNA in the maintainer line 2074B, lncRNA67, negatively modulating with male sterility in upland cotton (Gossypium hirsutum). lncRNA67 positively regulates cytochrome P274B (GhCYP724B), which acted as an eTM (endogenous target mimic) for miR3367. The suppression of GhCYP724B induced symptoms of BR deficiency and male semi-sterility in upland cotton as well as in tobacco, which resulted from a reduction in the endogenous BR contents. GhCYP724B regulates BRs synthesis by interacting with GhDIM and GhCYP90B, two BRs biosynthesis proteins. Additionally, GhCYP724B suppressed a unique chimeric open reading frame (Aorf27) in 2074A mitochondrial genome. Ectopic expression of Aorf27 in yeast inhibited cellular growth, and over expression of Aorf27 in tobacco showed male sterility. Overall, the results proved that the miR3367-lncRNA67-GhCYP724B module positively regulates male sterility by modulating BRs biosynthesis. The findings uncovered the function of lncRNA67-GhCYP724B in male sterility, providing a new mechanism for understanding male sterility in upland cotton.