Uncovering the Molecular Link Between miR156.SPL15 and Carotenoid Accumulation in Arabidopsis

Carotenoid Cleavage Dioxygenases (CCDs) are an enzyme family that cleaves specific double bonds in carotenoids. MicroR156 in Arabidopsis regulates a network of genes by repressing 10 SPL genes, among which, SPL15 was found to regulate shoot branching and carotenoid accumulation. The expression of CCD1, CCD4, CCD7, CCD8, NCED2, NCED3, NCED5, NCED6, NCED9 and SPL15 was evaluated in siliques at 10 days post anthesis and in 10-day-old roots in Arabidopsis wild type, sk156 (miR156 overexpression mutant), RS105 (miR156 overexpression line), spl15 (SPL15 knockout mutant) and two 35S:SPL15 lines. Results showed that most of CCD/NCED genes were affected both in the roots and siliques by function of miR156. In addition transcript levels of four CCD/NCED genes were up regulated in 35S:SPL15 lines. To test binding of SPL15 to affected CCD/NCED genes, 35S:SPL15-GFP transgenic lines were generated and three independent lines were subjected to ChIP-qPCR. The results revealed strong and selective occupancy of SPL15 on the promoter regions of candidate CCD/NCED genes. In addition, sk156 and RS105 had larger size of elaioplasts and plastoglobules compared to wild type. My results indicate that miR156/SPL15 control carotenoid accumulation by regulating expression of CCD/NCED genes and by increasing the cell’s capacity to store carotenoids