大豆幼苗粒腺體顯微構造的探討以及其與熱休克蛋白質結合後之熱保護

Abstract

本研究以生長兩天的大豆白化幼苗為材料， 探討植物幼苗及粒腺體呼吸作用耐熱性的獲得．其中包括檢查植物幼苗的呼吸速率，粒腺體的呼吸活性，及其對鈣離子的吸收能力．同時，在TEM下觀察大豆幼苗根尖細胞，和分離出粒腺體的顯微構造． 實驗結果顯示，無論是植物幼苗或是分離出的粒腺體，都有形成耐熱性的能力．大豆幼苗在45℃(2hr)下呼吸速率極低，但40℃(2hr)仍有正常的呼吸速率．若在45℃前先給與40℃(2hr)處理，則呼吸速率可提高一倍，且在28℃下恢復的能力極佳．分離出的粒腺體在42.5℃的水浴中幾乎沒有呼吸活性，但若事先給與植物幼苗38℃(2hr) - 42.5℃(0.5hr)的處理，則可使其在42.5℃的水浴中仍具有呼吸活性．若在38℃(2hr) - 42.5℃(0.5hr)處理後再將幼苗種回28℃溫度下4小時，此時，粒腺體又喪失了在42.5℃水浴中的呼吸活性． TEM下觀查的情形，也有類似的結果．45℃(2hr)處理後的大豆幼苗根間的細胞，其中的微細構造完全瓦解，但在40℃(2hr)則保存了幾乎所有的完整性，若45℃前給與—40℃(2hr)的處理，細胞內遭受破壞的情形不若直接以45℃處理的嚴重，且比較45℃(2hr)及40℃(2hr)-45℃(2hr)兩種處理之後在28℃下恢復的情形，只有後者顯示了極佳的恢復潛能，並在28℃(12hr)之後，細胞內的膜狀構造就幾乎完全恢復原先的完整狀態． 無論在生體內(in vivo)或試管中(in vitro)的研究結果，均支持了植物細胞若要獲得耐熱性(thermotolerance)，首先細胞內要先累積足夠量的HSPs(40℃(2hr)或38℃(2hr))，其次，這些HSPs要選擇性地結合至膜狀胞器上(45℃(2hr)或42.5℃(0.5hr))，以建立各個胞器，整個細胞，以至整株植物體的耐熱性．

Two-day-old etiolated soybean seedlings were used to study the thermotolerance of their respiration functions including seedling respiration rate, isolated mitochondria 02 consumption and Ca2+ uptake activity. Also, we investigated their ultrastructures under TEM. Following the elevation of temperature, the respiration rate of seedlings decreased, and it was not possible to recover after a 45℃(2h) treatment. Whereas, a 40℃(2h) treatment or 40℃ (2h) before shifted to 45℃(2h) had potential to recover when grown in 28℃. Mitochondria isolated which had been subjected to various heat shock(HS) treatments i.e., A.28℃(2h), B.38℃(2h), C.38℃(2h) - 42.5℃(30'), and D.38℃(2h) - 42.5℃(30') - 28℃(4h), were monitored for 02 consumption using an Oxygen electrode. Mitochondria isolated after all four HS treatments were active in 02 consumption at 28℃ in response to succinate and ADP (derived P/O ratios were 1.4, 1.3, 1.2 and 1.2, respectively). However, only mitochondria isolated after treatment C. were active in O2 uptake at 42.5℃ (derived P/O ratio was 1.1). Our data demonstrate that association of HS proteins with mitochondria by treatment C. enables them to continue to function at 42.5℃ (i.e., they become thermotolerant). Mitochondria from which associated HS proteins were allowed to dissociate (treatment D) showed no significant O2 consumption at 42.5℃. Isolated mitochondria from treatment C. and treatment A. were compared by TEM. They appeared to be very similar, and no significant ultrastructural differences were noted.