Ectopic Overexpression of bol-miR171b Increases Chlorophyll Content and Results in Sterility in Broccoli (Brassica oleracea L var. italica)

MiR171 plays pleiotropic roles in the growth and development of several plant species. However, the mechanism underlying the miR171-mediated regulation of organ development in broccoli remains unknown. In this study, bol-miR171b was characterized and found to be differentially expressed in various broccoli organs. The ectopic overexpression of bol-miR171b in Arabidopsis affected the leaf and silique development of transgenic lines. In particular, the chlorophyll content of leaves from overexpressed bol-miR171b transgenic Arabidopsis was higher than that of the vector controls. The fertility and seed yield of Arabidopsis with overexpressed bol-miR171b were markedly lower than those of the vector controls. Similarly, overexpressed bol-miR171b transgenic broccoli exhibited dark green leaves with high chlorophyll content, and nearly all of the flowers were sterile. These results demonstrated that overexpression of bol-miR171b could increase the chlorophyll content of transgenic plants. Degradome sequencing was conducted to identify the targets of bol-miR171b. Two members of the GRAS gene family, BolSCL6 and BolSCL27, were cleaved by bol-miR171b-3p in broccoli. In addition to the genes targeted by bol-miR171b-3p, adenylylsulfate reductase 3 (APSR3), which played important roles in plant sulfate assimilation and reduction, was speculated to be cleaved by bol-miR171b-5p, suggesting that the star sequence of bol-miR171b may also have functions in broccoli. Comparative transcriptome analysis further revealed that the genes involved in chloroplast development and sulfate homeostasis should participate in the bol-miR171b-mediated regulatory network. Taken together, these findings provided new insights into the function and regulation of bol-miR171b in broccoli and indicated the potential of bol-miR171b as a small RNA molecule that increased leaf chlorophyll in plants by genetic engineering.