Elongation Characters Of Wheat Coleoptiles And Effects Of Cell Wall Loosening Reagents

For a growing plant cell to expand, it must breakthrough its cell wall's restrict firstly. Although many researches have already showed that maybe expansin is the key factor to control cell wall extension, relationships among expansin, hydrolases and cell wall extension, how does the expansin regulate cell wall extension and the relationship between xylanase and extension of type II cell wall etc are still unclear. In this paper, the extension character of wheat coleoptiles, the relationship among expansin hydrolase, especially xylanase, and cell wall extension were studied. Results are as follows.-The elongation of coleoptiles from Graminean was believed exclusively based on cell expansion, not on cell division. However, divided cells were first found in etiolated wheat (Triticum aestivum L.) coleoptiles in this paper and they covered all the areas except the tip but most frequent in the middle region when length of etiolated wheat coleoptiles changed from 1 mm to 7 mm, and disappeared when they reached and exceeded 9 mm. Therefore, elongation of coleoptiles was due to both cell division and cell elongation when coleoptiles were from 1 mm to 7 mm long, although it became exclusively depended on cell elongation when coleoptiles were 9 mm or more long. KCN was first found which could inhibit plant cell wall extensions and this inhibition was probably due to its inhibition of expansin; This inhibition was pH-dependant and cell wall extension was more sensitive to KCN at pH 5.0. KCN could inhibit both the xyloglucan (XG) hydrolytic activity and the cell wall extension activity of Cell2A (an endo-glucanase from Trichoderma reesei), and the inhibitory degrees of KCN on these two activities were different. These implied that Cell2A's cell wall extension activity was not come from its XG hydrolytic activity, and the crucial factor to regulate extension of type I cell wall was still α-expansin. A β-expansin-like protein, which could recover the deactivated cell wall extensions ofwheat coleoptiles, was purified from the cellulase compound (Cytolase CL) of T. reesei by the chromatography method of CM-cellulase and CM-300 HPLC. Protein sequencing and blasting results illustrated that this p-expansin-like protein might be a new protein. This purified protein could hydrolyze xylan and arabinoxylan. The relativities beween coleoptiles' extension and xylanase from wheat aleuronic layers, or, and xynlll (xylanase III) from the same T. reesei were studied for probing whether xylanase participate in the extension of plant cell. Results showed that: both the crude and the purified xylanase from wheat aleuronic layers just could promote the cell wall extension rate of wheat coleoptiles continuously increase; xylanase from wheat aleuronic layers and P-expansin had the very strong synergistic effect on the deactivated cell wall extensions of wheat coleoptiles; low concentration of recombinant xynlll had no evident effect on the deactivated cell wall extensions of wheat coleoptiles, but high concentration of it could break the cell walls of coleoptiles; there were also evident synergistic effects of xynlll with p-expansin or with Cell2A on cell wall extensions. All the above results demonstrated that the effect of xylanase on coleoptiles' cell wall was through hydrolyzation; the cell wall extension activity of p-expansin-like protein might not come from its xylan-hydrolytic effect, and the xylan-hydrolytic activity of the purified p-expansin-like protein might come from its minim miscellaneous protein. These results also implied that in cell walls in vivo, there would be synergistic effects among xylanase, EG and P-expansin etc. too, and the function of p-expansin might be dominant and the functions of xylanase and EG etc. might be assistant. Non-elongating cell walls of coleoptiles, first treated with hemicellulases (xynlll or/and Cell2A) and then treated with p-expansin, could not extend. These results implied that the disappearance of cell wall hydrolytic enzymes and P-expansin was not the key cause of the disappearance of cell wall extension activity. Phenolic compounds accumulating evidently in non-elongating cell walls showed that phenolic compounds accumulation might be one of the causes of the rigidification of coleoptiles' cell walls.