College of Life Sciences, Fujian Agricultural and Forestry University, Fuzhou, Fujian, China; Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China.
Key Laboratory for Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China; Subtropical Agriculture Research Institute, Fujian Academy of Agricultural Sciences, Zhangzhou, Fujian, China.
Protein Expr Purif. 2021 Jul;183:105875. doi: 10.1016/j.pep.2021.105875. Epub 2021 Mar 16.
The poor grain filling of inferior spikelets (IS) situated on the lower secondary rachis branch leads to a remarkable decrease in rice yield and quality. The AGPase small subunit 2 (AGPS2) encodes a small subunit of adenosine diphosphate-glucose pyrophosphorylase (AGPase) enzyme, which plays an important role in sucrose-starch conversion and starch biosynthesis in the grain filling of rice. In the present study, qPCR analysis showed low expression abundance of AGPS2 in IS, compared to the superior spikelets (SS), which was consistent with the lower grain weight of IS. To evaluate the molecular mechanism of AGPS2, we first identified the AGPS2 interaction network through Co-immunoprecipitation (Co-IP). In total, 29 proteins of AGPS2 interaction network were characterized by LC-MS/MS. Bioinformatics analysis revealed that, the characterized proteins in the interaction network are likely to be related to starch synthesis, sugar conversion, energy pathway, and folding/modification, and most of them were involved in the grain filling of rice. The sequent Co-IP analysis showed that AGPS2 can bind to starch branching enzyme (SBE), pullulanase (PUL) and starch debranching enzyme (DBE) and assemble into starch synthesizing protein complex (SSPC). In addition, the 14-3-3 protein GF14e was also found to interact with AGPS2. Further analysis by qPCR showed that the expression of GF14e was much higher on IS than on SS. The qPCR results also showed that the expression of GF14e was relatively stable in SS, but changed significantly in IS under alternate wetting and moderate soil drying (WMD), which is consistent with the AGPS2 expression pattern. Our present work provides direct molecular evidence for the different expression patterns of AGPS2 in SS and IS, which could be greatly helpful for the molecular amelioration of the poor grain filling of IS in rice.
劣质小穗（IS）的灌浆不良导致水稻产量和品质显著下降。AGPase 小亚基 2（AGPS2）编码的是二磷酸腺苷-葡萄糖焦磷酸化酶（AGPase）的小亚基，该酶在水稻灌浆过程中蔗糖-淀粉转化和淀粉生物合成中发挥重要作用。在本研究中，qPCR 分析表明，AGPS2 在 IS 中的表达丰度较低，与 SS 相比，IS 的谷粒重量较低。为了评估 AGPS2 的分子机制，我们首先通过免疫共沉淀（Co-IP）鉴定了 AGPS2 的互作网络。通过 LC-MS/MS 共鉴定到 29 个 AGPS2 互作网络蛋白。生物信息学分析表明，互作网络中的蛋白可能与淀粉合成、糖转化、能量途径和折叠/修饰有关，且大部分蛋白与水稻灌浆有关。随后的 Co-IP 分析表明，AGPS2 可以与淀粉分支酶（SBE）、普鲁兰酶（PUL）和淀粉脱支酶（DBE）结合，并组装成淀粉合成蛋白复合物（SSPC）。此外，还发现 14-3-3 蛋白 GF14e 与 AGPS2 相互作用。进一步的 qPCR 分析表明，GF14e 在 IS 上的表达明显高于 SS。qPCR 结果还表明，GF14e 在 SS 上的表达相对稳定，但在交替湿润和适度土壤干燥（WMD）下 IS 中的表达变化显著，与 AGPS2 的表达模式一致。本研究为 SS 和 IS 中 AGPS2 不同表达模式提供了直接的分子证据，对水稻 IS 灌浆不良的分子改良有很大帮助。
