CO2-responsive CCT protein interacts with 14-3-3 proteins and controls the expression of starch synthesis-related genes
CO2-responsive CCT protein interacts with 14-3-3 proteins and controls the expression of starch synthesis-related genes
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DOI：
10.1111/pce.14084
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发表时间：
2021-05-22
影响因子：
7.3
通讯作者：
Morita, Ryutaro
中科院分区：
文献类型：
--
作者：
Fukayama, Hiroshi;Miyagawa, Fumihiro;Morita, Ryutaro
CO2响应CCT蛋白(CRCT)是淀粉合成相关基因的正调控基因，如ADP-葡萄糖焦磷酸化酶大亚基1和淀粉分支酶I，特别是在水稻叶鞘中。启动子GUS分析表明，CRCT仅在维管束中表达，而淀粉合成相关基因在叶肉细胞和淀粉储存薄壁细胞等不同部位表达。然而，使用FLAG-CRCT过表达系的染色质免疫沉淀(CHIP)和随后的qPCR分析表明，这些淀粉合成相关基因的5‘-侧翼区域有被CHIP富集化的趋势，表明CRCT可以与这些基因的启动子区域结合。CRCT的单体大小为34.2 kDa，凝胶过滤层析检测到大小为270 kDa的CRCT，说明CRCT在体内形成了复合体。免疫沉淀和随后的MS分析提取了几个类似14-3-3的蛋白质。酵母双杂交分析和双分子荧光互补分析证实了CRCT与14-3-3类蛋白的相互作用。尽管在表达位置上存在不一致，但这项研究提供了关于CRCT控制关键淀粉合成相关基因表达的分子功能的重要发现。
CO2-responsive CCT protein (CRCT) is a positive regulator of starch synthesis-related genes such as ADP-glucose pyrophosphorylase large subunit 1 and starch branching enzyme I particularly in the leaf sheath of rice (Oryza sativa L.). The promoter GUS analysis revealed that CRCT expressed exclusively in the vascular bundle, whereas starch synthesis-related genes were expressed in different sites such as mesophyll cell and starch storage parenchyma cell. However, the chromatin immunoprecipitation (ChIP) using a FLAG-CRCT overexpression line and subsequent qPCR analyses showed that the 5 '-flanking regions of these starch synthesis-related genes tended to be enriched by ChIP, suggesting that CRCT can bind to the promoter regions of these genes. The monomer of CRCT is 34.2 kDa; however, CRCT was detected at 270 kDa via gel filtration chromatography, suggesting that CRCT forms a complex in vivo. Immunoprecipitation and subsequent MS analysis pulled down several 14-3-3-like proteins. A yeast two-hybrid analysis and bimolecular fluorescence complementation assays confirmed the interaction between CRCT and 14-3-3-like proteins. Although there is an inconsistency in the place of expression, this study provides important findings regarding the molecular function of CRCT to control the expression of key starch synthesis-related genes.