Novel microRNAs controlling the anthocyanin biosynthesis pathway in tomato

Abstract

The anthocyanins are compounds from the secondary metabolism of plants and belong to the flavonoids group. They are responsible for the blue, red and purple pigmentation in many plant tissues, as leaves, flowers, seeds and fruits. For plants they are mainly responsible for the growth and development, defense against pathogens, drought tolerance and protection against UV radiation. In addition, the anthocyanin are extremely beneficial to human health, they fight and prevent a range of chronic diseases. Among these benefits, antioxidant effects, prevention against cardiovascular diseases and cancer stand out. The main vegetables and fruits present in our diet accumulate anthocyanin just in small quantities, and in most cases being restricted to the epidermal layers of the edible parts. Since the peel usually represents less than 5% of the total mass of edible parts of the plant, a high anthocyanin accumulation is desirable, and the tomato is a great candidate for this improvement, since it is the principal vegetable in worldwide. microRNAs (miRNAs) are a class of small non-coding RNAs that contain 21-24 nucleotides and regulate gene expression post transcriptionally, by mRNA cleavage or suppressing translation. In tomato, the miRNAs role in the anthocyanin biosynthesis pathway is still not well characterized. Aiming for a better understanding of the action of the miRNAs in this pathway, we performed the prediction, validation and knockout of some miRNAs as well as the expression analyses for some of the anthocyanin pathway related genes. The miRNA target prediction identified the genes Anthocyanidin synthase (ANS), SlAN1 (bHLH) and Chalcone Synthase (CHS) being possible regulated by the miR5368, miR6024-3p and miR9471b-3p, respectively. We analyzed the expression of these miRNAs and anthocyanin related genes in Micro-Tom triple mutant Aft/atv/hp2 and Micro-Tom Wild-Type by RT-qPCR. Afterwards, we performed the knockout of each miRNA precursor in Micro-Tom Wild-Type by CRISPR-Cas9. As a result, we observed transformed explants with a purple phenotype in leaf veins and stem, indicating that the miR5368, miR6024 and miR9471 promote negative regulation in the anthocyanin biosynthesis pathway acting in the genes ANS, SlAN1 and CHS respectively.