miR156-targeted and nontargeted SBP-box transcription factors act in concert to secure male fertility in Arabidopsis.

The SBP-box transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE8 (SPL8) is required for proper development of sporogenic tissues in Arabidopsis thaliana. Here, we show that the semisterile phenotype of SPL8 loss-of-function mutants is due to partial functional redundancy with several other members of the Arabidopsis SPL gene family. In contrast with SPL8, the transcripts of these latter SPL genes are all targeted by miR156/7. Whereas the introduction of single miR156/7-resistant SPL transgenes could only partially restore spl8 mutant fertility, constitutive overexpression of miR156 in an spl8 mutant background resulted in fully sterile plants. Histological analysis of the anthers of such sterile plants revealed an almost complete absence of sporogenous and anther wall tissue differentiation, a phenotype similar to that reported for sporocyteless/nozzle (spl/nzz) mutant anthers. Expression studies indicated a functional requirement for miR156/7-targeted SPL genes limited to early anther development. Accordingly, several miR156/7-encoding loci were found expressed in anther tissues at later stages of development. We conclude that fully fertile Arabidopsis flowers require the action of multiple miR156/7-targeted SPL genes in concert with SPL8. Either together with SPL/NZZ or independently, these SPL genes act to regulate genes mediating cell division, differentiation, and specification early in anther development. Furthermore, SPL8 in particular may be required to secure fertility of the very first flowers when floral transition-related miR156/7 levels might not have sufficiently declined.