臺灣博碩士論文加值系統

果膠是植物初級細胞壁的主要成份之一，在高基氏體中形成，並以高度甲基酯化的形態被送至細胞壁中。在細胞壁中的果膠會受到甲基酯化酶 (PME) 的作用，去除所擁有的甲基酯結構。根據研究，甲基酯化酶在植物生長以及抗蟲害機制中扮演著重要的角色，但是在植物耐熱機制中所扮演的角色卻少有研究。我們以53個阿拉伯芥PME的T-DNA插入突變株進行耐熱性狀測試，發現二個PME34 (At3g49220) 相同基因座的突變株皆出現存活率下降的性狀。經過實驗後發現，PME34基因的受損並不會影響到熱休克蛋白基因的表現量，同時PME34的轉錄會受到ABA的誘導，並會在保衛細胞中會大量表現。所以我們推斷，PME34的突變會使得植株失去控制氣孔開關的能力，導致植株出現不耐熱的性狀。因此、在阿拉伯芥熱休克反應機制當中，PME34會藉由本身的酵素功能改變保衛細胞的細胞壁結構，影響細胞壁的彈性，從而改變氣孔的孔徑大小以調節蒸散作用的速率達到散熱的效果，增加植株對熱休克的耐受性。

Pectin, a major component of the primary cell wall, is synthesized in the Golgi apparatus and exported to the cell wall in a highly methylesterified form, then de-methylesterified by pectin methylesterase (PME). The effect of PME on the pectin methylesterification status plays a key role in plant development and plant–pathogen interactions, but its role under heat stress (HS) was poorly studied. Thermotolerance assay of Arabidopsis 53 PME homologous-T-DNA insertion lines revealed 2 null-mutant alleles of PME34 (At3g49220) both consistently showed reduced thermotolerance; nevertheless their impairment was independently associated with the expression of HS-related genes. PME34 transcript induction depended on abscisic acid and was highly expressed in guard cells. We showed PME34 mutation has a defect in the control of stomatal movement resulted in a heat-sensitive phenotype. Hence, PME34 has a role in the regulation of transpiration by controlling the degree of stomatal aperture that was achieved by enzymatic actives during HS response. PME34 is required for regulating guard cell-wall flexibility to mediate HS tolerance in Arabidopsis.

摘要 1
Abstract 2
Abbreviations 3
Chapter 1 Introduction 4
1.1 Pectin Structure and Biosynthesis 4
1.2 Heat Shock Response 6
1.3 Pectin methylesterase 8
1.4 Purpose for this Research 12
Chapter 2 Materials and Methods 14
2.1 Plant Materials and Growth Conditions 14
2.2 Leaf Surface Temperature Measurement 14
2.3 Water Loss Measurement 15
2.4 Stomatal Aperture Measurement 15
2.5 Constructs 15
2.6 Protoplast Preparation, Transfection and Protease Treatment 16
2.7 Subcellular Localization in Onion Epidermal Cells 16
2.8 Drought Experiment and Pigment Content Determination 17
2.9 RNA Isolation, cDNA Synthesis and Real-Time Quantitative PCR (qRT-PCR) 17
2.10 HSPs Quantitation 18
2.11 Stomatal Density and Vein Pattern Analysis 18
Chapter 3 Results 19
3.1 Leaf Surface Temperature Measurement 19
3.2 Water Loss Measurement and Stomatal Aperture Measurement 19
3.3 PME34 Promoter::GUS Assay and Photosynthesis Pigment Content Determination 21
3.4 PME34 Expression Level in ABA-Related Mutants 21
3.5 HS-related Genes Expression in Response to Heat Shock in pme34 22
3.6 Membrane Topology of PME34 22
3.7 Stomatal Density and Cotyledon Vein Pattern 24
Chapter 4 Discussion 25
4.1 PME34 Contributes to the Control of Stomatal Movement 25
4.2 PME34 Specifically Expressed in the Vascular Tissues and Guard Cells 28
4.3 Drought Experiment and Pigment Content Determination 28
4.4 PME34 Expression Level Is Regulated by ABA 29
4.5 HS-related Genes Expression in Response to HS in pme34 29
4.6 The C-terminal PME Activity Domain Is Localized in the Extracellular Space 30
4.7 PME34 Has No Effect on Stomatal Density and Cotyledon Vein Networks 31
Chapter 5 Conclusions and Prospects 33
Figures 35
References 47
Supplemental Data 55
Appendix 68

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