Effect Of Water Deficit Stress On Types And Activity Of Endoproteases In Petals Of Cut Rose

This work was aimed to investigate induction of the activity and types of endoproteases (EP) related to tolerance to water deficit stress (WDS) in cut rose (Rosa hybrida) during WDS. Two cultivars, 'Samantha' with the strong tolerance to WDS, and 'Belami', with the medium one, were used as materials. To make different process of WDS, the two cultivars were treated separately with two manners, namely flowers with or without branches, and the activity and types of EP were determined based on the spectrophotometer method. 'Samantha' was furthermore used to probe into the induction of the activity and types of EP by repeated WDS treatment through gelatin-SDS-PAGE method.1. Based on comparison of the two cultivars under WDS:1) A turning point, in which recovery rate of flowers was going to be decreasing rapidly, was observed in flowers with branches, and the occurrence of the turning point was later in 'Samantha'(30 h) than in 'Belami'(24 h). The turning point of quick falling of water potential (WP) of flower to slow falling was also observed in the flowers of both cultivar, these points were high identical with the turning points of their recovery rate.2) WDS-induced increase of EP activity was later in 'Samantha' than in 'Belami'. And the turning points of quick increase of EP activity induced by WDS in both cultivars were consistent with the turning points of their recovery rates. Changes of both Metallo-proteinase and serine proteinase activities of petals induced by WDS were significantly smaller in 'Samantha' than in 'Belami'.The experiments with the flowers without branches showed substantially the same results as those described above with the flowers with branches.These results above suggested that the difference of tolerance to WDS between different cultivars might be related to the different changes of metallo-proteinase and serine proteinase activities in petals induced by WDS.2. Based on the results of'Samantha' under repeated WDS treatment:1) WR for 6 h after WDS for 24 h, the bend states of flower neck could recover to the normal, at the same time, the WP of flower could come back to the level of no stress. But, the fresh weight of branch and the EP activity of petals could not get back to a basal low level as that of before WDS treatment until WR for 12 h. When WDS being given again, the similar changes took on in flower appearance, flower and branch water conditions, while more acutely change presented in EP activity of petals.2) The quick increase of serine proteinase activity in petals was correlative with senescence. The activity was induced strongly by WDS. WDS for 18 h, the activity was the same as that of senescence (stage 6) and did not decreased significantly until WR for 6 h. WDS for 6 h was given again, the activity came to the level of senescence. The WDS-inducible appearance of flower was weaken by ABA; the falling of water potential, the loss of fresh weight and the hoist of EP activity were controlled.3) The determining method of gelatin-containing polyacrylamide gels in EP from petals of cut rose was optimized. The result showed that removing salts and pigments from crude extracts through column of Sephadex G-25 was vital to the preparation of protein sample. The prepared samples were treated for 10min at 40 ℃, loaded in 12 % separating gel containing 0.15 % gelatin, following electrophoresis, incubated for 16 h, Clear bands were observed on the dark blue background.4) The results of gelatin-SDS-PAGE showed that the number of bands induced by WDS was the same as that of senescence-related EPs. According to the molecular weight from large to small, named EP1(200 kDa), EP2(123.5 kDa), EP3(97.4 kDa) respectively. Among them, EP2 and EP3 were most correlative with senescence. The optimum activation temperature of EPl, EP2 and EP3 was 42 ℃; As for the optimum activation pH, EPl had obvious activity from acidic to neutral, EP2 and EP3 had clear activity from neutral to alkali, and all the three had obvious activity was pH 7; EP2 and EP3 were serine proteinases, while EP1 was metallo-proteinase.