先前研究發現，阿拉伯芥及水稻幼苗可藉由HSA32和HSP101的交互作用延長後天耐熱性：HSP101促進HSA32的表現，而HSA32減緩HSP101的降解。在分子層次上，HSA32如何作用仍不清楚，高等植物的HSA32和古生菌的2-磷酸-3-磺基乳酸合成酶 ((2R)-phospho-3-sulfolactate synthase, PSL synthase) 蛋白質序列相似，並含有一個結合3''-磷酸腺苷-5''-磷酸硫酸 (3''-phosphoadenosine-5''-phosphosulfate, PAPS) 的高度保守結構基序 (motif) 。由於PAPS是亞硫酸基轉移酶 (sulfotransferase) 催化硫酸化反應 (sulfonation reaction) 的磺酸基 (sulfonate group) 供給者，因此，我們推測HSA32可能是一種亞硫酸基轉移酶 (sulfotransferase)，參與一種已知或未知含硫化合物之合成，例如，硫酸膽鹼 (choline sulfate)。硫酸膽鹼是一種兼容滲透劑，由未知的亞硫酸基轉移酶催化膽鹼及PAPS反應而成。我假設HSA32會在熱逆境下催化硫酸膽鹼之生成，用以調節HSP101的降解。然而，UPLC-MS/MS分析結果發現硫酸膽鹼的含量不會受到加熱或缺少HSA32影響，顯示HSA32並不參與硫酸膽鹼合成。由於HSA32的同源蛋白在陸生植物普遍地存在，為了解該蛋白的結構與功能的演化程度，我們將來自高等和低等植物的五種HSA32同源蛋白表達於阿拉伯芥hsa32-1突變株，以進行功能互補測試。結果顯示只有高等植物的HSA32可以減緩HSP101的降解以及恢復hsa32-1的耐熱性。總結我的實驗結果，高等及低等植物的HSA32具有不同的分子功能，可能分別合成不同的含硫化合物。

It has been shown that the effect of acquired thermotolerance is extended by the interplay between Heat-Stress-Associated 32-kD protein (HSA32) and Heat Shock Protein 101 (HSP101) in Arabidopsis and rice. HSP101 promotes the production of HSA32, and HSA32 reduces the degradation of HSP101. The molecular action of HSA32 is unknown. It had been proposed that HSA32 may participate in sulfur metabolism due to sequence similarity with archaea (2R)-phospho-3-sulfolactate synthase (PSL synthase). Moreover, this study identified found a highly conserved 3''-phosphoadenosine-5''-phosphosulfate (PAPS)-binding motif sequence of HSA32 in higher plants. PAPS is an activated sulfur donor for the sulfation reaction catalyzed by sulfotransferases. Thus, HSA32 probably acts as a sulfotransferase. Choline sulfate is synthesized by choline sulfotransferase whose activity had been detected but had not been cloned yet. Therefore, I hypothesized that HSA32 catalyzes the production of choline sulfate under heat stress to suppress the degradation of HSP101. However, the result of UPLC-MS/MS analysis showed that choline sulfate can be detected in hsa32-1 to a level as high as in wild type under both normal and heat stress condition. The result indicates that HSA32 is not involved in choline sulfate biosynthesis. To further examine the crosstalk between HSA32 and HSP101 of higher and lower plant origins, Arabidopsis hsa32-1 knockout mutant was complemented with HSA32 from higher and lower plants. The results showed that only higher plant HSA32 rescued the defect of hsa32-1 under heat stress by retarding the degradation of HSP101. Taken together, the results suggest that higher and lower plant HSA32s have distinct molecular functions, probably by synthesizing different sulfur-containing compounds.

謝誌 i
摘要 ii
Abstract iv
Abbreviations vi
Chapter 1 Introduction 1
1.1 Plant heat shock response 1
1.2 Thermotolerance and heat stress memory in plants 2
1.3 The function of HSA32 and its homologs 3
1.4 Sulfotransferases in Arabidopsis 6
1.5 Specific aims 8
Chapter 2 Materials and Methods 10
2.1 Bioinformatic analysis 10
2.2 Plant materials and thermotolerance assays 11
2.3 Total RNA extraction, synthesis of cDNA, and quantitative PCR 13
2.4 Genomic DNA extraction 15
2.5 Cloning and building binary vectors for Arabidopsis transformation 15
2.6 Selection of homozygous transgenic lines with single T-DNA insertion event 18
2.7 Protein extraction and immunoblotting 18
2.8 Analysis of 35S-labeled metabolites in plant extract 19
2.9 Detection and measurement of choline sulfate in plant extract 20
Chapter 3 Results 22
3.1 Phylogenetic analysis of HSA32 homologs 22
3.2 Choline sulfate content is not affected in hsa32-1 25
3.3 Functional analysis of HSA32 homologs by complementation assay 27
3.3.1 Cloning of PpHSA32 and plant transformation 27
3.3.2 Thermotolerance assay and expression analysis of HSA32 homologs and AtHSP101 in transgenic plants 29
3.4 Functional analysis of HSP101 homologs by complementation assay 31
3.4.1 Cloning of HSP101 homologs and plant transformation 31
3.4.2 Thermotolerance assay and expression analysis of HSP101 homologs in transgenic plants 33
Chapter 4 Discussion 35
4.1 The origin of HSA32 homologs 35
4.2 The function of HSA32 in higher plants is different from that in lower plants 36
4.3 N-terminal HA-tagged HSP101 cannot complement the defect of hsp101 under heat stress 37
Chapter 5 Future work 39
Tables and Figures 41
Appendix 70
References 91

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