三種酚酸化合物對水稻葉綠素生合成及根部與葉部細胞微細構造之影響

Abstract

本研究選用稻桿在土壤中分解後產生最多量的三種酚酸（p-coumaric、ferulic及o-hydroxyphenylacetic acid）（Chou and Lin，1976），以研究它們對水稻葉綠素生合成及對根及葉部細胞構造之影響，並探討其作用機制。 取上述三種酚酸分別以0（只加水耕液）、25、50及100 ppm序列處理以水耕方式栽培在黑暗中生長二週的水稻黃化苗後予以12/12小時光/暗處理，於1、3、6、12、24、48小時後，測量葉綠素生合成終產物（葉綠素a、葉綠素b）及三種中間代謝物protochllorophyllide（PChlide）、Mg-protoporphyrin IX（MGPP）及protoporphyrin IX（PPIX）的含量變化。其結果指出各代謝物含量隨處理濃度的增加而減少。在這三種酚酸中以ferulic acid的抑制效果最明顯，在處理後24小時內各代謝物含量極少，於24小時後才快速增加；其次?p-coumaric acid；而以o-hydroxyphenylacetic acid處理時影響較小。經由酚酸處理一週後，植株已有明顯的差異尤其是植株轉綠不完全及葉片枯萎等，其中以p-coumaric acid 50 ppm處理最明顯，其次?ferulic acid，再其次?o-hydroxyphenylacetic acid。自葉片分離的葉綠體類囊膜（thylakoids）總蛋白質含量減少。葉綠體類囊膜蛋白質經由SDS—PAGE電泳結果，各色帶的含量變化以不加酚酸的對照組?最多，其次?o-hydroxyphenylacetic acid，以ferulic acid及p-coumaric acid處理時最少，此結果與植株枯萎程度、葉綠體類囊膜總蛋白質含量及三種葉綠素生合成過程中間代謝物（包括PChlide、MGPP及PPIX）與終產物（葉綠素a及葉綠素b）的含量變化大致上相符合。由上述結果指出葉綠素的生合成受酚酸的影響而減少。推測酚酸在葉綠素生合成途徑中的可能抑制位置?合成porphyrin結構的相關部位。 以穿透式電子顯微鏡觀察水稻根及葉部的微細構造（ultrastructure），結果指出根部細胞膜系被破壞，並有部份細胞瓦解；葉部方面則因所取樣之葉片的葉綠體構造已發育完全受酚酸影響較小而未有顯著差異。 由本研究結果可知，以水耕栽培在黑暗中生長的水稻黃化苗，在處理酚酸後並照光（12/12小時，光/暗）的情況下葉綠素的生合成受酚酸影響而減少。並由本研究結果推測酚酸在葉綠素生合成途徑中的可能抑制位置位於與合成porphyrin結構相關的部位。水稻根部因直接接觸到水耕液中的酚酸在細胞構造上已有明顯不同；葉部方面則因葉綠體構造已發育完全故未觀察到有顯著差異。

Three phenolic acids p-coumaric, ferulic and o-hydroxyphenylacetic acids were selected to observe the effects of the common phytotoxins in paddy soil on the chlorophyll biosynthesis and the ultrastructure of root and leaf cells of rice plants. Two weeks old etiolated rice seedlings were treated with the three phenolic acids in a series of concentration, 0 (as control), 25,50,100 ppm in Kimura's culture solution. Under the light/dark (12/12 hr) photoperiods, five intermediates, such as chlorophyll a, chlorophyll b, protochlorophyllide (PChlide), Mg-protoporphyrin IX (MGPP), protoporphyrin IX (PPIX) in the process of chlorophyll biosynthesis, were measured at the time elapse of 1, 3, 6, 12, 24 and 48 hr after treatment. Results indicated that the contents of these intermediates decreased as the concentration of phenolics increased. Of these three phenolic acids, ferulic acid revealed the most remarkable effect then p-coumaric acid and o-hydroxyphenylacetic acid the least effect. In the first 24 hours treated with ferulic acid, the contents of these five intermediates were very low; however, that of intermediates increased rapidly 24 hr after treatment. The growth of rice plants was significantly retarded after one week of treatment with these three phenolic acids, showing that leaves were wilt and less green in color. In particularly, p-coumaric acid revealed significant effect then did ferulic acid and o-hydroxyphenylacetic acid. The total protein contents of chloroplast thylakoids from leaves were lower as compared with that of control plants. The results of SDS-PAGE indicated that the contents of chloroplast thylakoid proteins treated with these three phenolic acids, o-hydroxyphenylacetic, ferulic and p-coumaric aicds were significantly lower than that of control. The total protein contents of chloroplast thylakoid of rice leaves decreased as well as the contents of the five intermediates of chlorophyll biosynthesis, concluding that these three phenoilc acids may inhibit the processes of chlorophyll biosynthesis at the steps of porphyrin structure biosynthesis. In the observation of ultrastrucrutes of roots of rice plants treated with tree phenolic acids, some of the the cells were destroyed. But there were not siginificantly different in the cells of leaves.