利用不同水稻品種探討糖類對水稻低溫逆境及耐受性之關係

Abstract

碳水化合物(Carbohydrate)在植物逆境生理中扮演許多重要角色，如：養分提供、保護膜系、滲透壓調節及訊息傳導分子，碳水化合物可存在於細胞質、液胞及細胞壁。而根據結構可以單糖、雙糖、寡糖及多醣構造存在，越來越多研究指出碳水化合物與各種非生物性逆境間具有相關連性。本論文以細胞內可溶性多醣為萃取目標，首先了解台農67號地上部在低溫下多醣變化，包含多醣產率、多醣分子量及單糖組成。多醣分子量上有一低分子量720~770 Da多醣體隨低溫處理其含量隨之增加從6%升至13%，單糖組成上葡萄糖及果糖隨低溫處理其含量分別由20及36 μmol/g PS增加至32及54 μmol/g PS，而阿拉伯糖、岩藻糖、山梨醇及肌醇隨低溫處理其量則由102，9，2及0.6 μmol/g PS減少至50，5，0.94及0.43 μmol/g PS。除此之外，本試驗也進行台農67號於低溫處理下基因表現之microarray分析，其中特別針對碳水化合物代謝相關基因進行探討。最後更結合糖類變化與碳水化合物代謝相關基因之資料數據模擬水稻於低溫下碳水化合物代謝途徑之改變，結果顯示基因表現及代謝物之變化兩者相關程度可達八至九成。此外以定量real-time PCR確認該些基因之表現。為了更深入探討糖類在植物耐冷性之角色，以兩個品種台中在來1號(不耐冷)及台農67號(耐冷)為實驗材料進行低溫、回溫處理，觀察水稻地上部及地下部糖類變化。地上部及地下部糖類變化中，葡萄糖及果糖含量最可能在TCN1及TNG67扮演耐冷性之差異，而阿拉伯糖在不同水稻品種及組織上具有相反的變化趨勢，TNG67半乳糖含量則都會因低溫處理含量上升。 另外為了進一步瞭解低溫對於多醣組成之變化，本試驗比較大量表現AtICE1轉殖株及TNG67水稻間多醣之差異，本實驗室先前研究顯示大量表現AtICE1轉殖株可提升轉殖水稻耐冷性。本論文也針對AtICE1轉殖水稻進行多方生理分析，闡述AtICE1轉殖株在生理上之耐冷機制。AtICE1轉殖株其過氧化氫及丙二醛含量都比非轉殖株低。抗氧化酵素SOD、APX、CAT及GR活性分析都顯示低溫下轉殖株比非轉殖株高。抗氧化物抗壞血酸鹽含量也顯示低溫下轉殖株比非轉殖株高。以上結果皆顯示轉殖株抗氧化能力比非轉殖株高。此外全可溶性糖類、胺基氮及蛋白質含量之測定，顯示轉殖株在胺基氮及蛋白質含量與非轉殖株上有差異。最後，測定地上部糖類變化，在常溫及低溫下轉殖株其葡萄糖及果糖含量都比非轉株高。 本論文對於多醣與水稻耐冷性間關係上有了初步認識，並且將Microarray基因表現與糖類含量變化進行綜合解釋，瞭解糖類含量之變化可能受轉錄層次之基因表現所調控。而在大量表現AtICE1之基因表現轉殖株上推測AtICE1提升轉殖株抗氧化能力，特別葡萄糖及果糖含量轉殖株都比非轉殖株高。

Carbohydrates play important roles in plant stress physiology, such as nutrient supplementation, osmotic adjustment, maintain of membranes stability and singaling molecule. Carbohydrates can be present in cytosol, vacuole and cell wall. Depend on the composition and structures, carbohydrates can be grouped into monosaccharide, disaccharide, oligosaccharide and polysaccharide. Many studies had shown the correlation of carbohydrates change under various abiotic stress in plant. In this study, we are interesting in understanding the complexity of intracellular water-soluble polysaccharide (PS) changes in rice under cold stress. High-performance anion-exchange chromatography was used to analyze the effect of cold stress on polysaccharide pattern changes in TNG67, including total PS production, the molecular weight distribution and sugar composition. The accumulation of PS fraction (0.72~0.74 kDa) was increased from 6% to 13% in TNG67 rice shoot under cold treatment. The compositional analysis of PS showed that glucose and fructose level increased from 20 and 36 μmol/g PS to 32 and 54 μmol/g PS during cold treatment, however; arabinose, fucose, sorbitol and myo-inositol level decreased from 102, 9, 2 and 0.6 μmol/g PS to 50, 5, 0.94 and 0.43 μmol/g PS. In addition, we analyzed gene expression of TNG67 under cold treatment with microarray, especially for those carbohydrate metabolism related gene. Finally, we tried to integrate sugar expression profiling data and microarray gene analysis to reveal the correlation of gene and sugar change under cold treatment in rice. The correlation between carbohydrate metabolite and biosynthesis related genes change was high as 80~90%. We also confirmed the gene expression by real-time PCR. To further revel in the role of polysaccharide in rice cold tolerance, we used different rice cultivars TCN1 (cold sensitive) and TNG67 (cold tolerant) as experimental materials and monitored the carbohydrate profiling change in shoot and root of TCN1 and TNG67 under cold and rewarm condition. The content of various sugars in shoot of TNG67 was higher than TCN1 under rewarm condition, and the those sugar contents was also higher TNG67 root under cold. This result suggested that the glucose and fructose may play important role in rice cold stress tolerance.The dynamic change of arabinose is opposite in different cultivars and tissue.The content of galactose was increased in TNG67 under cold treatment. Meanwhile, to investigate the role of PS change under cold stress, we compared the PS expression profiling between AtICE overexpressed transgenic rice and TNG67. The overexpression of AtICE1 with 35S promoter in rice had been shown to enhance cold stress tolerance (Hsin-Hsiu Fang, 2008).Here, we further confirmed the cold stress tolerance of AtICE1 overexpressed transgenic rice by various physiological assays. The content of MDA and H2O2 in transgenic rice was lower than WT. The superoxide dismutase (SOD), ascobate peroxidase (APX) and catalase (CAT) activity of transgenic rice was higher than those of WT. The level of ascobate/dehydroascobate (ASC/DHA) was higher in transgenic rice. Taken together, the above results indicated antioxidant activity was higher in transgenic rice. The amino nitrogen and protein content of transgenic rice was higher than WT under normal and cold condition. By analysis of PS expression profiling HPAEC showed that the glucose and fructose content of transgenic rice was higher than WT either at normal or cold condition. In this study, we established the correlation between intracellular PS and rice under cold treatment. We also integrated sugar contents and microarray gene expression to demonstrate that levels of carbohydrate may regulate at transcription level. We also characterized the physiologic response, especially antioxidant activity in AtICE1 overexpressed transgenic rice and showed that carbohydrates, particularly glucose and fructose increased in AtICE1-OX compared to wild type.