小分子量熱休克蛋白質是植物體內種類最豐富的一群蛋白質，能與變性蛋白質結合，防止其沉澱而對細胞造成傷害。目前已知OsHSP16.9A與OsHSP18.0皆為水稻第一族群小分子量熱休克蛋白質的成員，分別位於第一和第三對染色體上，皆會在高溫41℃ 下被誘導表現。本研究中發現OsHSP16.9A在種子成熟時期表現於胚中，而在萌芽六天內其表現量逐漸下降。大量表現OsHSP16.9A的轉殖株（OsHSP16.9A-OE）種子具有耐熱性，且其種子的白堊質成分較少，有助於稻米品質的提升。與其胺基酸序列相似度高的基因-OsHSP18.0，其大量表現的轉殖株（OsHSP18.0-OE）種子則不具有耐熱性。我們利用OsHSP16.9A-OE之cDNA微陣列發現，參與在澱粉合成途徑中之關鍵酶的腺苷二磷酸葡萄糖焦磷酸化酶基因-OsAPL3，顯著受OsHSP16.9A誘導表現，並在電顯下觀察得知轉殖株種子osapl3的澱粉粒比野生型小且圓，OsHSP16.9A-OE+osapl3的澱粉粒則比osapl3的澱粉粒大，代表OsHSP16.9A和OsAPL3沒有直接的關聯性。最後我們利用蛋白質質譜技術與蛋白質交互作用實驗結果顯示，OsHSP101對於OsHSP16.9A幫助提升種子耐熱的功能是必須的。;Small heat shock proteins (sHSPs) represent the most abundant HSPs in plants, showing molecular chaperone activity to prevent thermal-induced irreversible denaturation of protein. Previous results revealed that OsHSP16.9A and OsHSP18.0, were members of the sHSP-CI gene family in rice (Oryza sativa Tainung No.67) on chromosome 1 and 3. And they were highly homologous to each other. Our study showed OsHSP16.9A accumulates during embryo maturation and persist at high levels during the 6 day following seed imbibition in the absence of heat stress. OsHSP16.9A-OE showed not only increasing thermotolerance in seeds but grain quality as compared with the WT and OsHSP18.0-OE. By cDNA microarray expression profiling, we identified OsAPL3, a enzyme (AGPase) for starch metabolism, was upregulated in OsHSP16.9A-OE seed with control and high temperature treatment. SEM analyses showed that starch granules in the OsHSP16.9A-OE+osapl3 mutants are larger in size and more angular in shape compared to the WT and osapl3 mutants by plant transformation. The results revealed that OsHSP16.9A does not directly interact with OsAPL3 in starch granule arrangement. In addition, we also found OsHSP101 interacted with OsHSP16.9A in rice seeds by Proteomic approach. Therefore, OsHSP101 is necessary for OsHSP16.9A function in thermotolerance of rice seeds.
