小分子熱休克蛋白質是植物體內種類相當豐富的一群蛋白質，能與變性蛋白質結合，防止其沉澱而減緩對細胞的傷害。實驗室先前發現OsHSP16.9A與OsHSP101可能在高溫下產生交互作用，也發現OsHSP16.9A與OsHSP101會累積於胚中，並在泡水後兩天開始減少。為了更進一步證實OsHSP16.9A與OsHSP101之交互作用，我們利用比例式雙螢光分子互補系統（rBiFC）來偵測彼此間在細胞內的交互作用。結果顯示OsHSP16.9A與OsHSP101可以在正常及高溫條件下具有交互作用。我們也發現當OsHSP16.9A第74個胺基酸從Glu突變成Asp時，其與OsHSP101的交互作用就會大幅下降。由此可知，OsHSP16.A的第74個胺基酸對於與OsHSP101形成交互作用十分重要。此外，我們也分析了TNG67、CO39、N22、IR64及IR50等五種水稻在種子充實期間OsHSP16.9A與OsHSP101的累積情形。結果顯示OsHSP16.9A及OsHSP101的累積趨勢相當一致，從乳熟期開始累積，並在黃熟期、完熟期達到高峰。綜上所述，水稻會在種子發育期間累積大量OsHSP16.9A及OsHSP101，可能藉此使種子獲得高溫耐受性，直到萌芽後開始衰退，因此，我們認為OsHSP16.9A與OsHSP101間可能有著類似於OsHSA32與OsHSP101間的正回饋關係。;Small heat shock proteins represent the most abundant heat shock proteins in plants. These proteins have chaperon activity, which can prevent cellular proteins from thermal-induced irreversible denaturation under heat stress. By the proteomic approach, we found that OsHSP101 is a potential candidate interacting with OsHSP16.9A during high temperature. In addition, our previous study found the abundance of OsHSP16.9A and OsHSP101 was in the embryo and vanished after imbibition for two days. To clear up the interplay between OsHSP16.9A and OsHSP101, we used ratiometric bimolecular fluorescence complementation (rBiFC) to investigate the interaction between OsHSP16.9A and OsHSP101. The results show that OsHSP16.9A interacts with OsHSP101 under non-stress and heat stress conditions. Besides, we found that mutation of the 74th amino acid residue, Glu to Asp, of the OsHSP16.9A leads to abolish the interaction of OsHSP16.9A and OsHSP101. Our results indicate that the 74th amino acid of OsHSP16.9A is critical for OsHSP16.9A–OsHSP101 interaction under thermal stress. Besides, we also analyzed OsHSP16.9A and OsHSP101 accumulation level during seed maturation in 5 different rice cultivar, including TNG67, CO39, N22, IR50 and IR28. Accumulation levels of OsHSP16.9A and OsHSP101 showed similar patterns during seed maturation. OsHSP16.9A and OsHSP101 start to accumulate at the milk stage and reach the peak at the hard dough stage or mature stage. These results indicate that accumulation of OsHSP16.9A and OsHSP101 during seed development may be important for seeds to gain thermotolerance. When a suitable environment manifests for seed germination, OsHSP16.9A and OsHSP101 will start to decline. Therefore, we suppose there has a positive feedback loop between OsHSP16.9A and OsHSP101, which similar to OsHSA32 and OsHSP101.
