熱效應對葉綠體光反應活性、形態及蛋白質合成之影響

Abstract

以七?十天大綠色豌豆(Pisum Sativum L. cv. Taichung No. 9)幼苗為材料，用Fish及Jagendorf (1982)的方法分離葉綠體，並經不連續的Percoll梯度離心純化之。經Lilley等人(1975)用氧氣偵測器分析葉綠體完整度的方法並配合電子顯微鏡(TEM)的觀察，證明其為完整度達90%以上的純化葉綠體，進而藉以探討熱壓迫對葉綠體的影響。 有關耐熱性(thermotolerance)的一些實驗顯示，37.5℃下植株所產生的蛋白質，可能與提供熱保護有關，使幼苗生長不再受40℃高溫的嚴重抑制；且從經過37.5℃兩小時處理的幼苗枝葉中所分離出的葉綠體，其釋氧能力比對照組（來自25℃兩小時處理的幼苗枝葉）更能忍受較高的溫度（35℃以上）。活體內3H-白胺酸標記的葉綠體蛋白質，經SDS-膠體電泳分析及自動放射顯像術顯示，37.5℃下的類型(Pattern)的確與25℃的不同，其中經80S核糖體合成的一群70-90-KD的高分子量蛋白質和一個約21-KD的低分子量蛋白質，以及經70S核糖體合成的一個33-KD蛋白質，可能是高溫下新出現或合成增多的。在試管內用3H-白胺酸標記分離葉綠體的蛋白質，經SDS-膠體電泳分析及自動放射顯像術所得的結果顯示，在35℃的熱壓迫下，葉綠體膜上的22.5及24-KD蛋白質及基質(Stroma)中的30及33-KD蛋白質，可能是25℃下所所沒有或合成較少的。

Chloroplasts were isolated from 7 to 10-day-old green seedlings of peas (Pisum sativum L. cv. Taichung No. 9) according to the method of Fish and Jagendorf (23) and were further purified by discontinuous Percoll density gradient centrifugation. The resulting chloroplasts were assayed by the method of Lilley (40) and observed under TEM, and proved to be over 90% intact chloroplasts. The purified, intact chloroplasts were then used to study their responses to heat stress. Experiments about thermotolerance showed that some proteins synthesized at 37.5°C could provide thermo-protection on growth of pea seedlings so as not to be seriously inhibited by the following treatment at 40°C for 2 hours. The FeCN-dependent O2 evolution of chloroplasts isolated from pea shoots shocked at 37.5°C for 2 hours were more thermotolerant than that of control (2 hours at 25°C). The autoradiogram of the SDS-PAGE of in vivo 3H-leucine labelled chloroplast proteins showed that some proteins (70 to 90-KD and 21-KD) were synthesized on 80S ribosomes and localized to chloroplasts and some (33-KD) was synthesized on 70S ribosomes of chloroplasts under 37.5°C but not or less under 25°C. The autoradiograms of the SDS-PAGE of in organello 3H-leucine-labelled chloroplast proteins showed that some proteins (22.5 and 24-KD) were synthesized to be membrane proteins and some (30 and 33-KD) were synthesized to be stromal proteins by isolated chloroplasts under 35°C but not or less under 25°C.