- 學位論文
高溫下水稻充實期多元胺與水稻品質形成的關係
Relationship between polyamines and the formation of grain quality during grain-filling stage under high temperature
摘要
水稻穎果充實期遭遇高溫增加白堊質粒比例，降低完整米率，碾米品質及米粒外觀劣化為現今稻米市場一大問題。本研究室前人研究顯示高溫下外加氮素可減少糙米白堊質發生，且高溫處理之穎果內生多元胺含量下降，但仍須釐清高溫下穎果內多元胺的功能及其與氮素的關係 。為瞭解高溫下氮素與多元胺影響水稻穎果產量及品質的機制，本研究選擇台灣良質米品種TK9為材料，於穎果充實期進行常溫(25/20 oC, 日/夜溫)及高溫處理 (35/30 oC, 日/夜溫)，並於溫度處裡下外加氮肥，分析各發育階段之水稻穎果及成熟穀粒之外觀品質、儲存性蛋白含量、穎果果皮葉綠素含量及氧化逆境相關生理指標，且進一步利用高壓液相層析 (HPLC)設備分析多元胺含量，並以即時定量聚合酶連鎖反應 (Real-time Quantitative PCR)分析多元胺代謝相關基因及逆境反應相關基因之表現。 結果顯示高溫降低穎果產量及品質，高溫下外加氮素處理之單粒重及儲存性蛋白質含量較高溫處理高，且外加氮素降低白堊質發生率。穎果相關生理分析指出高溫加氮減緩高溫對穎果之生理傷害，如延緩果皮葉綠素降解，減少H2O2及膜系過氧化指標malondialdehyde (MDA)含量。多元胺含量分析結果顯示，穎果內主要多元胺為spermidine，其次為putrescine及spermine，其比例不受溫度處理影響；而高溫下外加氮素可恢復高溫導致之多元胺含量降低。基因表現結果顯示高溫下GBSS、Pro7、Glu等乾物質合成相關基因被抑制，而無氧呼吸相關基因pyruvate decarbox-ylase (PDC)與alchol dehydrogenase (ADH)表現上升，顯示高溫下能量供應效率較低且乾物質合成也被抑制；此外，高溫下提升polyamine oxidase (PAO)表現，可能為導致穎果多元胺含量較少且提升H2O2含量之主因。反之高溫外加氮素處理之GBSS、Pro7、Glu較高,且降低PDC、ADH及PAO表現，顯示氮素可恢復穎果乾物質合成能力，且維持多元胺含量及減少H2O2產生，推測高溫下加氮處理可透過抑制PAO表現緩解高溫對穎果之過氧化傷害。為確認PAO對穎果生理之影響，另於高溫下外加spermidine及1,8-diaminooctane (1,8-DO, PAO抑制劑)處理，結果顯示外加spermidine或抑制PAO活性可增加果皮葉綠素、降低H2O2及MDA含量。推測高溫下氮素可藉由抑制PAO表現，有助於維持多元胺含量，也減少H2O2產生避免過氧化逆境的發生。
並列摘要
High temperature during the gain-filling stage can increase chalky appear-ance and reduce weight of rice grains. Our previous studies showed that high temperature can reduce polyamine level of rice grains, and application of nitrogen fertilizer can reduce the chalkiness of rice grains. However, the relationship between polyamines level and nitrogen fertilizer during rice grain filling stage at high temperature is still unclear. In order to understand the role of polyamine and its relationship with nitrogen in developing rice grains under high temperature, rice cultivar TK 9 was used as material in this experiment. After flowering, TK 9 was exposed to high temperature for 15 days with or without nitrogen addition during grain filling stage. Grain quality, major storage protein and physiolocal responses were determined after harvest. Polyamine levels of developing rice caryopsis were analyzed by HPLC. Real-time quantitative PCR was further utilized to determine the gene expression of key genes in response to high temperature and nitrogen application. The results showed that high temperature caused deleterious effects on the yield and quality of rice grains. Applying nitrogen increased dry matter and major storage proteins content, while reduced chalkiness grain ratio. Physiological analysis showed that nitrogen moderated the deleterious ef-fects of high temperature by delaying chlorophyll degradation of pericarp and reducing Malondialdehyde (MDA) concentration. In developing caryopsis, the major polyamine was spermidine (Spd), followed by putrescine (Put) and Spermine (Spm), regardless of temperature treatments. Nitrogen application recovered the decreased level of polyamine under high temperature. In further gene expression analysis, high temperature repressed genes expression related to biosynthesis of starch and protein, such as GBSS, Pro7 and Glu, whereas elevated the expression of hypoxia fermentation-related genes, such as pyruvate decar-boxylase (PDC) and alchol dehydrogenase (ADH). In addition, high temperature enhanced expression of PAO gene, which led to reduction of Spd level and release of hydrogen peroxide (H2O2). High temperature also enhanced expression of the cell death related gene PBZ. On the other hand, nitrogen application repressed expression of PAO gene under high temperature, suggesting that nitrogen prevented degradation of spd and alleviated oxidative stress. Under high temperature, external applycation of spd or 1, 8-diaminooctane (1, 8-DO, PAO inhibitor) sustained caryopsis chlorophyll content and grain weight, but reduced accumulation of H2O2 and MDA. The present findings suggest that the decreased level of polyamines may be involved in high temperature induced defective caryopsis traits, and the nitrogen application can ameliorate the deleterious effects of high temperature by inhibiting the expression of PAO gene.