MIR172d Is Required for Floral Organ Identity and Number in Tomato

MicroRNA172 (miR172) functions as a central regulator of flowering time and flower development by post-transcriptional repression of APETALA2-LIKE transcription factors. In the model crop Solanum lycopersicum (tomato), the miR172 family is still poorly annotated and information about the functions of specific members is lacking. Here, de-novo prediction of tomato miR172 coding loci identified seven genes (SlMIR172a-g), that code for four unique species of miR172 (sly-miR172). During reproductive development, sly-miR172s are differentially expressed, with sly-miR172c and sly-miR172d being the most abundant members in developing flowers, and are predicted to guide the cleavage of eight APETALA2-LIKE transcription factors. By CRISPR-Cas9 co-targeting of SlMIR172c and SlMIR172d we have generated a battery of loss-of-function and hypomorphic mutants (slmir172c-d(CR)). The slmir172c-d(CR) plants developed normal shoot but their flowers displayed graded floral organ abnormalities. Whereas slmir172c(CR) loss-of-function caused only a slight greening of petals and stamens, hypomorphic and loss-of-function slmir172d(CR) alleles were associated with the conversion of petals and stamens to sepaloids, which were produced in excess. Interestingly, the degrees of floral organ identity alteration and proliferation were directly correlated with the reduction in sly-miR172d activity. These results suggest that sly-miR172d regulates in a dose-dependent manner floral organ identity and number, likely by negatively regulating its APETALA2-like targets.