MicroRNA534a control of BLADE-ON-PETIOLE 1 and 2 mediates juvenile-to-adult gametophyte transition in Physcomitrella patens.

The Arabidopsis thaliana BLADE-ON-PETIOLE genes encode a pair of transcriptional coactivators that regulate lateral organ architecture by promoting cell differentiation in their proximal regions. To gain insight into the roles of BOP genes early in land plant evolution, we characterized the functions of Physcomitrella patens BOP1 and BOP2 and their negative regulator Pp-miR534a. We show that in ΔPpMIR534a mutants lacking mature Pp-miR534a, cleavage of PpBOP1/2 is abolished, leading to elevated PpBOP1/2 transcript levels. These loss-of-function mutants display an accelerated gametophore development thus correlating elevated levels of PpBOP1/2 with premature bud formation. This is further supported by our finding that exposure to cytokinin, which is known to induce bud formation on caulonema, downregulates PpMIR534a transcription and increases the accumulation of PpBOP1 in apical caulonema cells. Reporter gene fusions showed that PpMIR534a is ubiquitously expressed in protonema whereas PpBOP1/2 accumulation is restricted almost exclusively to potent caulonema apical cells and their side branch initials, but absent from differentiated cells. Together, our data propose that PpBOP1/2 act as positive regulators of protonema differentiation and that Pp-miR534a is required to control the timing of the juvenile-to-adult gametophyte transition by spatially restricting their expression to caulonema stem cells. As protonemata develop, increased cytokinin levels downregulate Pp-MIR534a transcription in these cells until a threshold level of PpBOP1/2 is reached that triggers cell differentiation and bud formation.