College of Food Science and Nutritional Engineering, China Agricultural University, Qinghua East Road No 17, Haidian District, Beijing, 100083, China.
Plant Mol Biol. 2017 May;94(1-2):61-78. doi: 10.1007/s11103-017-0593-9. Epub 2017 Feb 28.
Hexokinase (HXK, EC 2.7.1.1) is a multifunctional protein that both is involved in catalyzing the first step of glycolysis and plays an important role in sugar signaling. However, the supporting genetic evidence on hexokinases (CsHXKs) from grape (Vitis vinifera L. cv. Cabernet Sauvignon) berries has been lacking. Here, to investigate the role of CsHXK isoforms as glucose (Glc) and abscisic acid (ABA) sensors, we cloned two hexokinase isozymes, CsHXK1 and CsHXK2 with highly conserved genomic structure of nine exons and eight introns. We also found adenosine phosphate binding, substrate recognition and connection sites in their putative proteins. During grape berry development, the expression profiles of two CsHXK isoforms, sucrose synthases (SuSys) and cell wall invertase (CWINV) genes increased concomitantly with high levels of endogenous Glc and ABA. Furthermore, we showed that in wild type grape berry calli (WT), glucose repressed the expression levels of sucrose synthase (SuSy) and cell wall invertase (CWINV) genes, while ABA increased their expression levels. ABA could not only effectively improve the expression levels of SuSy and CWINV, but also block the repression induced by glucose on the expression of both genes. However, after silencing CsHXK1 or CsHXK2 in grape calli, SuSy and CWINV expression were enhanced, and the expressions of the two genes are insensitive in response to Glc treatment. Interestingly, exogenous ABA alone could not or less increase SuSy and CWINV expression in silencing CsHXK1 or CsHXK2 grape calli compared to WT. Meantime, ABA could not block the repression induced by glucose on the expression of SuSy and CWINV in CsHXK1 or CsHXK2 mutants. Therefore, Glc signal transduction depends on the regulation of CsHXK1 or CsHXK2. ABA signal was also disturbed by CsHXK1 or CsHXK2 silencing. The present results provide new insights into the regulatory role of Glc and ABA on the enzymes related to sugar metabolism in grape berry.
己糖激酶（HXK，EC 2.7.1.1）是一种多功能蛋白，既能催化糖酵解的第一步，又在糖信号中发挥重要作用。然而，葡萄（Vitis vinifera L. cv. Cabernet Sauvignon）浆果中己糖激酶（CsHXKs）的遗传证据支持一直缺乏。在这里，为了研究 CsHXK 同工酶作为葡萄糖（Glc）和脱落酸（ABA）传感器的作用，我们克隆了两个己糖激酶同工酶，CsHXK1 和 CsHXK2，它们具有高度保守的九个外显子和八个内含子的基因组结构。我们还在它们的假定蛋白中发现了腺苷酸磷酸结合、底物识别和连接位点。在葡萄浆果发育过程中，两个 CsHXK 同工酶（蔗糖合酶（SuSys）和细胞壁转化酶（CWINV）基因的表达谱与高水平的内源性 Glc 和 ABA 同时增加。此外，我们表明在野生型葡萄浆果愈伤组织（WT）中，葡萄糖抑制蔗糖合酶（SuSy）和细胞壁转化酶（CWINV）基因的表达水平，而 ABA 增加它们的表达水平。ABA 不仅可以有效提高 SuSy 和 CWINV 的表达水平，还可以阻断葡萄糖对这两个基因表达的抑制作用。然而，在葡萄愈伤组织中沉默 CsHXK1 或 CsHXK2 后，SuSy 和 CWINV 的表达增强，且两个基因对 Glc 处理的反应不敏感。有趣的是，与 WT 相比，外源 ABA 单独处理不能或较少增加沉默 CsHXK1 或 CsHXK2 的葡萄愈伤组织中 SuSy 和 CWINV 的表达。同时，ABA 不能阻断 CsHXK1 或 CsHXK2 突变体中葡萄糖对 SuSy 和 CWINV 表达的抑制作用。因此，Glc 信号转导依赖于 CsHXK1 或 CsHXK2 的调节。ABA 信号也被 CsHXK1 或 CsHXK2 沉默所干扰。本研究结果为葡萄浆果中 Glc 和 ABA 对糖代谢相关酶的调控作用提供了新的见解。
