Departamento de Biología Vegetal y Ciencia del Suelo, Universidad de Vigo, Campus Universitario, 36310, Vigo, Spain.
Present Address; Department of Plant Pathology, Citrus Research and Education Center, UF-IFAS, 700 Experiment Station Rd, Lake Alfred, FL, 33850, USA.
BMC Plant Biol. 2020 Oct 23;20(1):487. doi: 10.1186/s12870-020-02701-z.
Somatic embryogenesis in grapevines is a complex process that depends on many physiological and genetic factors. One of its main limitations is the process of precocious germination of the somatic embryos in differentiation medium. This process lowers plant conversion rates from the somatic embryos, and it is probably caused by a low endogenous abscisic acid (ABA) content.
Precocious germination of the somatic embryos was successfully avoided by culturing grapevine cv. Mencía embryogenic aggregates over a semipermeable membrane extended on top of the differentiation medium. The weekly analysis of the endogenous ABA and ABA-glucosyl ester (ABA-GE) contents in the aggregates showed their rapid accumulation. The expression profiles of 9-cis-epoxycarotenoid dioxygenase (VvNCED1), 8'-hydroxylase (VvHyd2), UDP-glucosyltransferase (VvUGT) and β-glucosidase (VvBG2) genes in grapevine revealed that the occurrence of a first accumulation peak of endogenous ABA in the second week of culture over the semipermeable membrane was mainly dependent on the expression of the VvNCED1 gene. A second increase in the endogenous ABA content was observed in the fourth week of culture. At this point in the culture, our results suggest that of those genes involved in ABA accumulation, one (VvNCED1) was repressed, while another (VvBG2) was activated. Similarly, of those genes related to a reduction in ABA levels, one (VvUGT) was repressed while another (VvHyd2) was activated. The relative expression level of the VvNCED1 gene in embryogenic aggregates cultured under the same conditions and treated with exogenous ABA revealed the significant downregulation of this gene.
Our results demonstrated the involvement of ABA metabolism in the control of the maturation of grapevine somatic embryos cultured over a semipermeable membrane and two important control points for their endogenous ABA levels. Thus, subtle differences in the expression of the antagonistic genes that control ABA synthesis and degradation could be responsible for the final level of ABA during the maturation of grapevine somatic embryos in vitro. In addition, the treatment of somatic embryos with exogenous ABA suggested the feedback-based control of the expression of the VvNCED1 gene by ABA during the maturation of grapevine somatic embryos.
葡萄体细胞胚胎发生是一个复杂的过程，依赖于许多生理和遗传因素。其主要限制之一是体细胞胚胎在分化培养基中过早萌发。这个过程降低了体细胞胚胎的植物转化率，可能是由于内源脱落酸（ABA）含量低所致。
通过在分化培养基上培养葡萄 cv. Mencía 胚性聚集体在半透膜上，可以成功避免体细胞胚胎过早萌发。每周对半透膜上培养的聚集体中内源 ABA 和 ABA-葡萄糖酯（ABA-GE）含量的分析表明，它们迅速积累。葡萄 9-顺式环氧类胡萝卜素双加氧酶（VvNCED1）、8'-羟化酶（VvHyd2）、UDP-葡萄糖基转移酶（VvUGT）和β-葡萄糖苷酶（VvBG2）基因的表达谱表明，在半透膜上培养的第二周，内源 ABA 的第一个积累高峰的发生主要依赖于 VvNCED1 基因的表达。在培养的第四周观察到内源 ABA 含量的第二次增加。在这个培养点，我们的结果表明，在参与 ABA 积累的基因中，一个（VvNCED1）被抑制，而另一个（VvBG2）被激活。同样，在与 ABA 水平降低相关的基因中，一个（VvUGT）被抑制，而另一个（VvHyd2）被激活。在相同条件下培养的胚性聚集体中 VvNCED1 基因的相对表达水平显示该基因显著下调。
我们的结果表明，ABA 代谢参与了在半透膜上培养的葡萄体细胞胚胎成熟的调控，以及它们内源 ABA 水平的两个重要控制点。因此，控制 ABA 合成和降解的拮抗基因表达的细微差异可能是葡萄体细胞胚胎在体外成熟过程中 ABA 最终水平的原因。此外，用外源 ABA 处理体细胞胚胎表明，在葡萄体细胞胚胎成熟过程中，ABA 对 VvNCED1 基因表达的反馈控制。
