College of Horticulture, Yangling Sub-Center of National Center for Apple Improvement, Northwest A&F University, Yangling 712100, Shannxi, P.R. China.
Plant Sci. 2022 May;318:111220. doi: 10.1016/j.plantsci.2022.111220. Epub 2022 Feb 16.
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.
不定根（AR）的形成对于苹果砧木的培育具有重要意义。本研究通过细胞分裂素（CTK）信号处理进行转录组分析，以分析 AR 形成的机制。结果表明，6-苄基腺嘌呤（6-BA）处理抑制了 AR 的形成。组织学分析还观察到，6-BA 处理显著抑制了 AR 原基细胞的形成；在 6-BA 处理组中，生长素/细胞分裂素的比值在第 1 天和第 3 天（d）最低。此外，差异表达基因分为 5 类，包括生长素、细胞分裂素、其他激素、细胞周期和碳水化合物代谢途径。由于对细胞分裂素信号处理的研究，了解细胞分裂素途径介导的特定模块非常重要。6-BA 处理后，MdRR12（B 型细胞分裂素反应因子家族成员）的表达水平在第 3 天显著上调。与野生型相比，35S::MdRR12 转基因烟草抑制了 AR 的形成。MdCRF8 的启动子序列包含 AGATT 基序元件，该元件响应 MdRR12。RNA-seq 和 RT-qPCR 分析预测，细胞分裂素应答因子（MdCRF8）是 MdRR12 调控的下游基因。pro-MdCRF8-GUS 启动子的活性明显受 6-BA 处理诱导，受 lovastatin（Lov）处理抑制。酵母单杂交、双荧光素酶报告基因和 GUS 共表达实验表明，MdRR12 可以直接结合到 MdCRF8 启动子上。此外，35S::MdCRF8 转基因烟草也阻断了 AR 的生长。与野生型相比，35S::MdRR12 和 35S::MdCRF8 转基因烟草对细胞分裂素的敏感性增强。因此，我们描述了 MdRR12 和 MdCRF8 作为细胞分裂素信号的整合因子，影响 AR 形成过程中的细胞周期和碳水化合物代谢相关基因，从而调节细胞命运的转变。基于这些结果，我们得出结论，MdRR12-MdCRF8 模块参与了苹果砧木不定根形成的负调控，并且可以通过遗传方法在农业中应用。
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