摘要
用离体细胞壁伸展活性重组法，对大豆（Ｇｌｙｃｉｎｅｍａｘ（Ｌ．）Ｍｅｒ．）幼苗下胚轴细胞壁特异性扩张蛋白的活性鉴定和特性研究结果表明，大豆幼苗下胚轴的延伸生长，既与扩张蛋白的活性升高有关，也与其细胞壁对扩张蛋白的敏感性增加有关；热钝化大豆下胚轴细胞壁的伸展活性，一旦被外源扩张蛋白所恢复，用酸性缓冲液（ｐＨ４．５）代替扩张蛋白提取液，伸展活性不受影响；但若换用中性缓冲液（ｐＨ６．８），伸展活性丧失殆尽，且与活细胞壁一样，随缓冲液的交替更换而反复逆转；大豆和黄瓜幼苗扩张蛋白可以与其热钝化的细胞壁相互交叉重组。另外，重组的细胞壁伸展活性对扩张蛋白浓度和ｐＨ的依赖性，符合一般酶的催化特征，说明扩张蛋白不仅在植物细胞的延伸生长过程中起着极为重要的作用，而且还暗示植物细胞壁的内源伸展就是该蛋白介导的一种生物化学过程。
Expansins, a newly discovered class of cell wall proteins, were the only proteins that, to date, have been shown to have the ability to restore the “acid growth' response of the heatinactivated cell wall in an in vitro assay. In order to characterize these proteins, an automatic extensometer had been previously constructed by modification of an equalarm mechanical balance with a linear variable differential transformer (LVDT) and with some easily available laboratory equipment. The objective of this study was to confirm and complement the work on expansin in cucumber (Cucumis sativus L.) seedlings carried out in the expansindiscoverers' laboratory and in addition, to further examination of the extensometer built in the authors' laboratory. It was reported that, firstly, expansin activity was maximal in cell wall from the growing region of soybean (Glycine max L.) hypocotyls but was negligible or lacking in that from mature, basal regions and cotyledons. Correspondingly, walls from the growing tissue had a strong susceptibility to the action of expansin, whereas the nongrowing tissues became insensitive to the expansin action. It was concluded that the growth of soybean hypocotyl was associated with an increase in both expansin activity and wall susceptibility to the expansin action. Secondly, the heatinactivated wall extension could be induced by cross reconstitution with crude expansin extract between soybean and cucumber species. Thirdly, once the heatinactivated wall has been pretreated with the exogenous expansin, the reconstituted wall required no further expansin for extension indicating that exogenous expansin could specifically bind to cell wall and be enough to repeatedly exert its action without releasing from the cell wall into the external solution, i.e., a single expansin molecule could gradually break a series of loadbearing bonds one by one while moving along the cell wall, and thereby permitting the wall to extend. Fourthly, reconstitution of the wall extension activity was evidently dependent on the expansin concentration and the pH of the bathing solution, which was consistent with the catalytic characteristics of classical enzymes. Finally, endogenous and reconstituted wall extension could be significantly induced in 50 mmol/L sodium acetate at pH 4.5 and completely inhibited in 50 mmol/L Hepes at pH 6.8, especially these phenomena could continuously be caused by switching incubation buffer from one to the other alternately, suggesting that change in pH of bathing solution could only affect the conformation of expansin (thus leading to denaturation or renaturation of it) but not the affinity of it for cell wall. In summary, these observations lend further support to the fact that expansin could mediate the acidinduced extension of the isolated wall, probably through a biochemical or enzymatic process exerting directly to the cell wall. This protein may play an essential role in the control of plant cell growth in vivo.
基金
国家自然科学基金
