Analyses of the early salt-induced changes of chemical composition in cultured ice plant cells

中文摘要 為探討植物在鹽逆境下之初期反應，本實驗以耐鹽植物冰花（Mesembryanthemum crystallinum L.; ice plant）的培養細胞為材料，將冰花細胞施以不同濃度的鈉離子、鈉/鉀離子、鈉/鈣離子溶液處理後，以傅立葉轉換紅外光譜儀（FT-IR）為測量工具，利用全反射衰減測量法（ATR），來及時偵測冰花細胞在加入上述溶液後，其表面化學成分例如蛋白質、酸性物質及碳氫化合物，在1800秒的偵測時間內變動的情形。冰花細胞在突然施予高濃度氯化鈉溶液，細胞內鈉離子含量會上升，造成鉀鈉比值下降，amide-II（1550 cm-1）吸收峰面積會增加，顯示鹽逆境下胞外蛋白質累積量增加，測定胞外總蛋白之含量，亦獲得與紅外光譜實驗類似的結果。進一步利用SDS/PAGE及Con A affinoblotting來分析胞外蛋白質之組成，結果發現數個特定蛋白質累積量有增加。此外，從紅外光譜的偵測結果顯示酸類物質（~1410 cm-1）及碳氫化合物（~1100 cm-1）之變化不大，顯示鹽逆境初期此二類物質可能扮演較為次要的角色。而在鹽逆境下外加入對高鈉離子的傷害具有拮抗作用之鉀或鈣離子時，amide-II（1550 cm-1）吸收峰面積會減少，顯示鹽逆境下胞外蛋白質累積量下降。由上述結果推測冰花細胞在鹽逆境初期，會經由特定蛋白質的作用，減緩細胞延長的速度，以適應外界突然增加之滲透逆境，待體內滲透調節機制啟動後，細胞之生長再行恢復。Abstract This thesis is aimed at the early responses to salt stress in halophyte ice plant (Mesembryanthemum crystallinum L.). Tissue culture-grown ice plant cells were used in this study. Two-week-old cells were stressed with different concentrations of NaCl, NaCl plus KCl, or NaCl plus CaCl2 and in situ monitoring the changes of chemical composition using FT-IR. Within 1800 seconds, IR signal detected the rapid changes of proteins, acids and carbohydrates on the surface of ice plant cells. When cells were salt-stressed, the Na+ content inside the cells increased in accompanied with the decrease of K/Na ratio. The area of amide-II (1550 cm-1) band increased as the NaCl concentration increased suggesting the accumulation of extracellular proteins during salt stress. Colorimetric measurement of total extracellular protein content yielded the similar result as the IR measurement. SDS/PAGE and ConA affinoblotting analyses showed the accumulation of certain polypeptides as well as glycoproteins in the extracellular space of ice plant cells upon salt stressed. IR measurement also detected the changes of acids (around 1410 cm-1) and carbohydrates (around 1100 cm-1) but the alterations of these two cellular components were not as significant as that of proteins suggesting these two may not play immediate roles in the initial responses to salt stress. Under high NaCl condition, addition of K+ or Ca2+, the antagonists of Na+, decreased the area of amide-II band suggesting the accumulation of extracellular protein was slowed down. The results suggest that certain proteins may act on the decrease of cell extension rate at the early stage of salt stress to accommodate with the sudden increase of high Na+ in the environment and the growth of cell resume after the mechanism of osmotic adjustment has launched.目錄： 中文摘要……………………...………………………………………...Ⅰ 英文摘要…………………...…………………………………………..Ⅱ 壹、前言：……………………………………………………................1 冰花耐鹽機制……………………………………………………3 （1）轉為CAM代謝……………………………………….3 （2）合成相容性溶質………………………………………3 （3）調控及區隔鈉離子進出細胞…………………………4 （4）提昇水分吸收及利用效率……………………………4 紅外光測量原理介紹……………………………………………6 紅外光漸逝波測量法原理簡介…………………………………9 光學測量系統說明……………………………………………..10 貳、材料與方法………………………………………………………..12 實驗材料………………………………………………………..12 （1）無菌播種……………………………………………..12 （2）癒傷組織誘導………………………………………..12 （3）配製不同濃度鈉、鉀、鈣離子溶液………………..12 實驗方法………………………………………………………..12 （1）紅外光譜測量………………………………………..12 （2）冰花細胞在氯化鈉溶液化學成分變化分析………..13 （3）冰花細胞中Na、K元素之測定……………………14 （4）SDS-PAGE…………………………………………...15 （5）醣蛋白的觀察………………………………………..15 參、結果………………………………………………………………..17 冰花細胞中主要化學成分之鑑定……………………………..17 冰花細胞對不同濃度鈉離子的反應的定量研究……………..20 （1）蛋白質變動…………………………………………..20 （2）酸類物質變動………………………………………..22 （3）碳水化合物變動……………………………………..22 （4）細胞內鈉、鉀離子在不同濃度氯化鈉鹽水處理 後之變化……………………………………………..23 （5）不同濃度氯化鈉溶液處理冰花細胞後胞外蛋白 質累積量之變化……………………………………..24 冰花細胞對不同濃度鈉/鉀離子混和溶液反應的定量研究….26 （1）蛋白質變動…………………………………………..26 （2）酸類物質變動………………………………………..28 （3）碳水化合物之變動…………………………………..29 冰花細胞對不同濃度鈉/鈣離子混和溶液反應的定量研究…29 （1）蛋白質變動………………………………………….30 （2）酸類物質變動……………………………………….30 （3）碳水化合物之變動………………………………….31 肆、討論……………………………………………………………….33 紅外光偵測系統探討………………………………………….33 冰花細胞對不同濃度鈉離子之初始反應…………………….34（1）蛋白質變動分析…………………………………….34 （2）酸類物質變動分析………………………………….36 （3）碳水化合物變動分析……………………………….37 鹽逆境下鉀離子對冰花細胞的效應………………………….38 鹽逆境下鈣離子的影響……………………………………….39 伍、參考文獻………………………………………………………….4