JrATHB-12介导JrMYB113和JrMYB27调控不同类型红核桃

doi:10.1016/j.jia.2024.03.015

摘要/Abstract

摘要：

红核桃因其比普通核桃富含花青苷而具有广阔市场前景。然而，红核桃花青苷生物合成机制仍是未知的。我们研究了两种类型红核桃，R1型带有红色的果皮和种皮，R2型仅有红色的种皮。R1型红色部分主要为矢车菊素-3-O-半乳糖苷，而R2型中矢车菊素-3-O-半乳糖苷，矢车菊素-3-O-阿拉伯糖苷，矢车菊素-3-O-葡萄糖苷均占有一定比例。编码无色花青素双加氧酶/花青素合成酶的基因LDOX-2和LDOX-3被初步鉴定为分别是R1和R2型红核桃花青苷生物合成关键基因。MYB差异基因分析发现MYB27和MYB113分别在R1和R2型红色部位特异表达，被当做花青苷生物合成候选调控基因。拟南芥异源表达和核桃果实瞬时注射表明细胞核定位的MYB27和MYB113均能启动花青苷的积累，并且MYB27能促进LDOX-2的表达，MYB113能促进LDOX-3和UAGT-3的表达。酵母单杂交和EMSA分析进一步验证了MYB27仅能结合LDOX-2的启动子，而MYB113能结合LDOX-3和UAGT-3的启动子。此外，我们还鉴定到一个果皮特异表达的HD-Zip转录因子ATHB-12，在果皮中沉默它的表达后，仅有R2型红核桃果皮变红，同时MYB113表达增加，进一步实验表明ATHB-12能特异的与MYB113互作并结合其启动子。这些结果表明MYB27能通过调控LDOX-2表达来控制R1型红核桃着色，而MYB113能通过调控LDOX-3和UAGT-3表达来控制R2型红核桃着色，但ATHB-12能特定结合并抑制R2型果皮中MYB113的表达，从而使R2型红核桃果皮不着色。本研究揭示了不同类型红核桃花青苷生物合成机制，为未来选育红核桃品种提供了科学依据。

Abstract:

Red walnut has broad market prospects because it is richer in anthocyanins than ordinary walnut. However, the mechanism driving anthocyanin biosynthesis in red walnut is still unknown. We studied two types of red walnut, called red walnut 1 (R1), with a red pericarp and seed coat, and red walnut 2 (R2), with a red seed coat only. R1 mostly contained cyanidin-3-O-galactoside, while R2 contained a various amounts of cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, and cyanidin-3-O-glucoside. The LDOX-2 (LOC109007163) and LDOX-3 (LOC109010746) genes, which encode leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/ANS), were preliminarily indicated as the crucial genes for anthocyanin biosynthesis in R1 and R2, respectively. The MYB differential genes analysis showed that MYB27 and MYB113 are specifically expressed in the red parts of R1 and R2, respectively, and they are regarded as candidate regulatory genes. Ectopic expression in Arabidopsis and transient injection in walnut showed that both MYB27 and MYB113 were located in the nucleus and promoted anthocyanin accumulation, while MYB27 promoted the expression of LDOX-2, and MYB113 promoted the expression of LDOX-3 and UAGT-3. Yeast one-hybrid and electrophoretic mobility shift assays showed that MYB27 could only bind to the LDOX-2 promoter, while MYB113 could bind to the promoters of both LDOX-3 and UAGT-3. In addition, we also identified an HD-Zip transcription factor, ATHB-12, which is specifically expressed in the pericarp. After silencing the expression of ATHB-12, the R2 pericarp turned red, and MYB113 expression increased. Further experiments showed that ATHB-12 could specifically interact with MYB113 and bind to its promoter. This suggests that MYB27 controls R1 coloration by regulating LDOX-2, while MYB113 controls R2 coloration by regulating LDOX-3 and UAGT-3, but ATHB-12 can specifically bind to and inhibit the MYB113 of the R2 pericarp so that it becomes unpigmented. This study reveals the anthocyanin biosynthetic mechanisms in two different types of red walnut and provides a scientific basis for the selection and breeding of red walnut varieties.

Key words: JrATHB-12 , JrMYB113 , JrMYB27 , red walnut , anthocyanin biosynthesis

Haifeng Xu, Guifang Wang, Xinying Ji, Kun Xiang, Tao Wang, Meiyong Zhang, Guangning Shen, Rui Zhang, Junpei Zhang, Xin Chen. JrATHB-12介导JrMYB113和JrMYB27调控不同类型红核桃[J]. Journal of Integrative Agriculture, 2024, 23(8): 2649-2661.

Haifeng Xu, Guifang Wang, Xinying Ji, Kun Xiang, Tao Wang, Meiyong Zhang, Guangning Shen, Rui Zhang, Junpei Zhang, Xin Chen. JrATHB-12 mediates JrMYB113 and JrMYB27 to control the anthocyanin levels in different types of red walnut[J]. Journal of Integrative Agriculture, 2024, 23(8): 2649-2661.

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