Transcriptome analysis reveals that cytokinins inhibit adventitious root formation through the MdRR12-MdCRF8 module in apple rootstock

Adventitious root (AR) formation is great significance for apple rootstock breeding. Transcriptome analyses were performed with cytokinins (CTKs) signal treatments to analyze the mechanism of AR formation. The results showed that 6-benzyadenine (6-BA) treatment inhibited AR formation. Histological analysis also observed that AR primordium cell formation was significantly suppressed by 6-BA treatment; the ratio of auxin/cytokinins exhibited the lowest values at 1 and 3 day (d) in the 6-BA treatment group. Furthermore, the differentially expressed genes were divided into five categories, including auxin, cytokinins, other hormones, cell cycle, and carbohydrate metabolism pathways. Due to the study of cytokinins signal treatment, it is important to understand the particular module mediated by the cytokinins pathway. The expression level of MdRR12 (a family member of B-type cytokinins-responsive factors) was significantly upregulated at 3 d by 6-BA treatment. Compared to the wild type, the 35S::MdRR12 transgenic tobaccos suppressed AR formation. The promoter sequence of MdCRF8 contains AGATT motif elements that respond to MdRR12. RNA-seq and RT-qPCR assays predicted cytokinins response factor (MdCRF8) to be a downstream gene regulated by MdRR12. The activity of the pro-MdCRF8-GUS promoter was obviously induced by 6-BA treatment and inhibited by lovastatin (Lov) treatment. Yeast one-hybrid, dual-luciferase reporter, and GUS coexpression assays revealed that MdRR12 could directly bind to the MdCRF8 promoter. Additionally, 35S::MdCRF8 transgenic tobaccos also blocked AR growth. Compared to the wild type, 35S::MdRR12 and 35S::MdCRF8 transgenic tobaccos enhanced sensitivity to cytokinins. Thus, we describe that MdRR12 and MdCRF8 function as integrators of cytokinins signals that affect cell cycle- and carbohydrate metabolism-related genes to regulate cell fate transition during AR formation. On the basis of these results, we concluded that the MdRR12-MdCRF8 module is involved in the negative regulation of AR formation in apple rootstock and can potentially be applied in agriculture using genetic approaches.