探討水稻 (Oryza sativa L.) 幼苗脂肪酸組成與低溫逆境耐受性之關係

Abstract

脂肪酸於植物正常生長及逆境適應方面，在生理、生化及代謝上皆扮演許多重要角色。不僅可作為不同能量儲存的形式、膜系的構成，更是植物適應外界環境溫度變化的關鍵因子。為了解水稻之脂肪酸組成及其不飽和程度是否亦與低溫逆境耐受性相關，本研究先利用 GC-FID 建立水稻脂肪酸分析平台，再進一步利用對低溫敏感之臺中在來一號 (TCN1) 及耐低溫之臺農67號 (TNG67) 水稻品種為材料 ，以 GC-MS (Gas chromatography–mass spectrometer) 技術平台分析兩品種地上部及地下部於低溫處理下脂肪酸組成之變化情形。結果顯示，TCN1水稻幼苗於短時間低溫處理下，地上部飽和脂肪酸 (C18:0、C20:0、C22:0、C24:0及C26:0) 含量有下降之趨勢，而地下部之不飽和脂肪酸 (C18:2及C18:3) 於長時間及短時間低溫處理下皆呈顯著性下降；TNG67 水稻幼苗於短時間及長時間低溫處理，不論地上部或地下部之脂肪酸組成並無太大的變化，僅C26:0於地上部短時間低溫處理後呈顯著性上升而長時間處理下則下降；相反的於地下部長時間處理時，C26:0則呈顯著性上升。另外分析兩品種於低溫逆境下葉綠體脂肪酸組成之變化情形，發現TNG67之 LCFAs (Long chain fatty acids)含量變化程度較TCN1大，且具有顯著性差異。若以判斷不飽和脂肪酸比例及程度之參數：不飽和脂肪酸比 (Un./Sa.) 與不飽和指數 (Double bond index, DBI) 分析地上部、地下部之細胞膜及葉綠體類囊膜脂肪酸組成變化。發現TNG67地上部及地下部可藉由維持較高的不飽和程度以維持細胞膜性的穩定性來抵禦低溫逆境，且與Fv/Fm值具有顯著性的正相關。而TNG67葉綠體之不飽和程度也高於TCN1，是由於TNG67具有較高含量的C18:3，且飽和脂肪酸C16:0及C18:0含量也較低於TCN1。此外利用qRT-PCR分析與催化不飽和脂肪酸生合成的關鍵酵素OsFADs 之基因表現，發現低溫可誘導TNG67地上部OsFAD7及OsFAD8基因之大量表現，推測其可能有助於細胞內JAs之生合成。此外，TNG67地上部α-Tocopherol 及 Campesterol 於低溫逆境下相較TCN1之下降卻仍可維持一定的含量。α-Tocopherol為植物體內重要的抗氧化物，且Campesterol為細胞內生合成BRs (Brassinosteroids) 之前驅物，而C24:0、C26:0及其衍生物則有助於蠟質及角質層之生合成，以上皆有助於TNG67抵禦低溫逆境所造成的傷害。未來，將可利用以上結果提供育種者作為篩選出耐低溫逆境品種之代謝產物標誌 (Metabolite biomarkers)。

Fatty acids participate in numerous physiological, biochemical and metabolic processes of plants and play important roles in plant growth, development and adaption in the environmental stresses. Fatty acids are not only used for energy storage, membrane composition but also serve as key factors in the adaptation of environmental temperature change. To address the issue that whether changes of fatty acids and unsaturation levels are correlated with cold stress tolerance in rice, we initially used GC-FID to establish the analysis platform of fatty acid profiles. Then, we used two different varieties of rice, TCN1 (cold-sensitive cultivar) and TNG67 (cold-tolerance cultivar), as experimental materials to compare the corresponding fatty acid compositional changes under cold stress by GC-MS. The results showed that the percentage of saturated fatty acids (C18:0, C20:0, C22:0, C24:0 and C26:0) was declined after short-term cold stress treatment in shoot of TCN1 rice seedlings. Moreover, the levels of C18:2 and C18:3 in TCN1 root were also significantly decreased under short- and long- term cold stress. Whereas, in the cold-tolerant cultivar TNG67, the composition of fatty acids was maintained as the same except C26:0 amount was increased in shoot under short-term cold stress but reduced with long-term cold treatment. We were also interested in analyzing the alteration of fatty acids profile in TCN1 and TNG67 chloroplasts. The accumulation of long chain fatty acids (LCFAs) was obviously higher in TNG67 compared to that of TCN1. We further took advantage of other two parameters, Unsaturated fatty acids/ Saturated fatty acids (Un./Sa.) and Double Bond Index (DBI) to analyze the portion and degree of unsaturated fatty acids in the plasma and the thylakoid membranes. The results indicated that the maintenance of higher content of unsaturated fatty acids could keep membrane stability either in the shoot or root of TNG67 than in TCN1. These two parameters were positively correlated with Fv/Fm value in TNG67. Compared with TCN1, the thylakoid membrane in the chloroplast of TNG67 had higher level of C18:3 and lower content of C16:0 and C18:0. These results suggested that the the cold stress tolerance of TNG67 may be due to maintenance of unsaturated level in the plasma and chloroplast thylakoid membranes, which provides a way for keeping membrane stability under cold stress. In addition, the real-time PCR technique was applied to monitor the gene expression changes of rice fatty acid desaturase gene family. We found that the gene expressions of ω-3 fatty acid desaturase-related genes, OsFAD7 and OsFAD8, were highly induced under short-term cold stress in the shoot of TNG67. This may lead to the gain of C18:3 which enhances JA biosynthesis and increases cold stress tolerance. Interestingly, TNG67 could keep fixed amounts of α-Tocopherol and campesterol under cold stress. α-Tocopherol is well known as an antioxidant material and campesterol was the precursor of brassinosteroids. Also, the increase of C24:0, C26:0 and its derivatives could be used for the the biosynthesis of wax and cutin in TNG67. From this study in the future, we can take advantage of these metabolites as biomarkers for the selection and breeding of cold stress tolerance-realted rice varieties