Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China; Fruit and Tea Research Institute, Hubei Academy of Agricultural Sciences, Wuhan 430064, China.
Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China; Center of Applied Biotechnology, Wuhan University of Bioengineering, Wuhan 430415, China.
Plant Sci. 2018 Dec;277:121-131. doi: 10.1016/j.plantsci.2018.10.015. Epub 2018 Oct 21.
In citrus, genetic improvement via biotechnology is challenging due to insufficient understanding of molecular barriers that prevent regeneration by somatic embryogenesis (SE). Our previous study indicated that LEC genes were involved in SE in citrus, but their regulatory roles remain to be elucidated. Here, we cloned one of the LEC genes, CsFUS3, and show that it is preferentially expressed during SE and in the embryogenic callus (EC) derived from citrus varieties with strong embryogenic competence. The overexpression of CsFUS3 in recalcitrant citrus callus restored embryogenic competence. Complementation of the loss-of-function Arabidopsis fus3 mutant with the CsFUS3 gene restored normal late embryogenesis, which is consistent with the CsFUS3 and AtFUS3 proteins contributing to the same regulatory network in Arabidopsis. Transcriptome profiling revealed that the expression of particular TFs that promote SE was up-regulated in the citrus overexpression (OE) line. The 104 differentially expressed genes associated with hormone biosynthesis, catabolism, and signaling are particularly noteworthy. The dynamic change in the ratio of ABA to GA during SE in wild-type callus mirrored the expression pattern of CsFUS3. In contrast, in the OE line, the ratio of ABA to GA was higher and the capacity for SE was greater when the OE line was separately treated with ABA and GA biosynthesis inhibitors. Taken together, our results demonstrate that the overexpression of CsFUS3 appears to establish a cellular environment favorable to SE, at least in part by promoting a high ABA to GA ratio and by regulating the expression of TFs that promote SE.
在柑橘中，由于对体细胞胚胎发生（SE）再生所阻止的分子障碍的理解不足，生物技术的遗传改良具有挑战性。我们之前的研究表明，LEC 基因参与了柑橘中的 SE，但它们的调控作用仍有待阐明。在这里，我们克隆了一个 LEC 基因 CsFUS3，并表明它在 SE 期间以及在具有强胚胎发生能力的柑橘品种中衍生的胚胎发生愈伤组织（EC）中优先表达。CsFUS3 在难以再生的柑橘愈伤组织中的过表达恢复了胚胎发生能力。用 CsFUS3 基因补充拟南芥 fus3 功能丧失突变体恢复了正常的晚期胚胎发生，这与 CsFUS3 和 AtFUS3 蛋白在拟南芥中参与相同的调控网络一致。转录组分析显示，在柑橘过表达（OE）系中，促进 SE 的特定 TF 的表达上调。与激素生物合成、分解代谢和信号转导相关的 104 个差异表达基因尤其值得注意。在野生型愈伤组织中，ABA 与 GA 比值在 SE 过程中的动态变化与 CsFUS3 的表达模式相吻合。相比之下，在 OE 系中，当单独用 ABA 和 GA 生物合成抑制剂处理 OE 系时，ABA 与 GA 的比值更高，SE 的能力更强。总之，我们的结果表明，CsFUS3 的过表达似乎建立了有利于 SE 的细胞环境，至少部分是通过促进高 ABA 与 GA 比值和调节促进 SE 的 TF 的表达来实现的。
