MicroRNA166 Monitors SPOROCYTELESS/NOZZLE for Building of the Anther Internal Boundary

The internal boundary between inner and outer microsporangia within anthers is essential for male fertility of vascular plants. Dehiscence zones embedded in the boundary release pollen for fertilization. However, the molecular mechanism underlying boundary formation in anthers remains poorly understood. Here, we report that microRNA166 (miR166) and its target PHABULOSA ( PHB ) regulate SPOROCYTELESS/NOZZLE ( SPL/NZZ ), which controls microsporogenesis. In developing anthers of Arabidopsis ( Arabidopsis thaliana ), the expression domains of miR165/6 and SPL/NZZ are overlapped and rearranged synchronously. Dominant mutation of PHB suppresses SPL/NZZ expression on the adaxial sides of stamens, resulting in a thickened boundary, whereas activation of MIR166g up-regulates SPL/NZZ expression, leading to ectopic microsporogenesis in the boundary. PHB limits the expression domains of SPL/NZZ to facilitate construction of the boundary, while miR166 preserves the expression domains of SPL/NZZ by inhibiting PHB to allow the inner microsporangia to take shape. Subsequently, PHB activates the key stem cell maintainer WUSCHEL in anthers to restrict the stomium cells to the boundary so that dehiscence zones develop and release pollen properly. These findings link adaxial/abaxial polarity to microsporogenesis in building of the internal boundary of anthers and thus advance the concepts underlying the establishment of the internal structure of male organs.