Liu Yi-Hsin, Tseng Tong-Seung, Wu Ching-Rong, Cho Shu-Ting, Kuo Chih-Horng, Huang Xin-Jie, Cheng Jung-Yi, Hsu Kuo-Hsuan, Lin Kung-Fu, Liu Chia-Chin, Yeh Ching-Hui
Department of Life Sciences, National Central University, Taoyuan, Taiwan.
Department of BioAgricultural Science, National Chiayi University, Chiayi, Taiwan.
Plant Sci. 2023 May;330:111634. doi: 10.1016/j.plantsci.2023.111634. Epub 2023 Feb 11.
Class I small heat shock proteins (CI sHSPs), OsHsp16.9A and OsHsp18.0, share 74% identity in amino acid sequences and accumulate in response to heat shock treatments. Individual rice transformants overexpressing OsHsp16.9A and OsHsp18.0 exhibit distinct thermoprotection/thermotolerance modes. Under high temperature stress, OsHsp16.9A-overexpressing lines showed higher seed germination rate, seedling survival, and pollen germination than wild-type controls, while OsHsp18.0 overexpression provided higher thermoprotection/thermotolerance for seedling survival. To elucidate the functional roles of OsHsp16.9A, mass spectrometry was used to identify OsHsp16.9A-interacting proteins. OsHsp101 was consistently identified in the OsHsp16.9A protein complex in several mass spectrometry analyses of seed proteins from OsHsp16.9A-overexpressing lines. Both OsHsp16.9A and OsHsp101 proteins accumulated during similar developmental stages of rice seeds and formed a heat-stable complex under high temperature treatments in in vitro assays. Co-localization of OsHsp16.9A and OsHsp101 was observed via ratiometric bimolecular fluorescence complementation analyses. Amino acid mutation studies revealed that OsHsp16.9A glutamate residue 74 and amino acid residues 23-36 were essential for OsHsp16.9A-OsHsp101 interaction. Moreover, overexpressing OsHsp16.9A in OsHsp101 knockdown mutants did not increase the seed germination rate under heat stress, which further confirmed the functional roles of OsHsp16.9A-OsHsp101 interaction in conferring thermotolerance to rice plants.
I类小分子热激蛋白（CI sHSPs），即OsHsp16.9A和OsHsp18.0，其氨基酸序列的一致性为74%，并且在热激处理后会积累。分别过表达OsHsp16.9A和OsHsp18.0的水稻转化体表现出不同的热保护/耐热模式。在高温胁迫下，过表达OsHsp16.9A的株系比野生型对照表现出更高的种子萌发率、幼苗存活率和花粉萌发率，而过表达OsHsp18.0则为幼苗存活提供了更高的热保护/耐热性。为了阐明OsHsp16.9A的功能作用，采用质谱法鉴定与OsHsp16.9A相互作用的蛋白。在对过表达OsHsp16.9A株系种子蛋白进行的多次质谱分析中，OsHsp101始终在OsHsp16.9A蛋白复合物中被鉴定出来。在水稻种子的相似发育阶段，OsHsp16.9A和OsHsp101蛋白都会积累，并且在体外试验的高温处理下形成热稳定复合物。通过比率型双分子荧光互补分析观察到OsHsp16.9A和OsHsp101的共定位。氨基酸突变研究表明，OsHsp16.9A的谷氨酸残基74以及氨基酸残基23 - 36对于OsHsp16.9A与OsHsp101的相互作用至关重要。此外，在OsHsp101基因敲除突变体中过表达OsHsp16.9A并不会提高热胁迫下的种子萌发率，这进一步证实了OsHsp16.9A与OsHsp101相互作用在赋予水稻植株耐热性方面的功能作用。
