Endopeptidases And Its Biochemistry Characters Of Cucumis Sativus During Leaf Senescence

Leaf senescence is an important event in plant development.It associates with protein degradation and the mobilization of the nitrogen to other vegetative tissues or to developing fruits.In the other hand,the degradation of protein would influence the rate of photosynthesis.So this was negative to plant products.Consequently,it is necessary to elucidate proteolytic mechanism and the characters of senescence-related proteases.Cucumber (Cucumis sativus L.) is a very important vegetable crop in China and it can be parthenocarpy and everbearing fruits.The yield of cucumber fruits will be increased highly if leaves senescence is delayed.To our knowledge,there are few reports about cucumber leaves senescence and its senescence-related proteases.The characterization and physiological roles of foliar endopeptidases remain unknown.In this experiment we employed cucumber for studying the leaf senescence.The changes and characters of cucumber endopeptidases were primarily concluded.The results showed that during the whole process of leaf growth and senescence,changes of photosynthesis rate and contents of chlorophyll in leaves were decline with leaves age. MDA content increase in senescing leaves.Meanwhile,SOD activity and protein content decrease during leaf senescence.Chloroplast ultrastructure showed the shape of chloroplast changed gradually from long ellipses to circles. The starch globule in chloroplast altered gradually from more and larger size to fewer and smaller, but the oil globule from fewer and smaller to more and larger.The gram lamellae varied progressively:from thinness and length to thickness and shortness;Form ranking along the long axle direction of chloroplast to arranging disorderly and blurring finally,consequently,this study suggested degradation of protein might correlate with the changes of chloroplast.In order to study the degradation of protein and changes of endopeptidase(EP) in cucumber (Cucumis sativus L.) leaves during natural senescence,the changes of endopeptidases isoenzymes were also showed by activity staining after gradient-polyacrylamide gel electrophoresis (G-PAGE) co-polymerized gelatin as a substrate.The results showed that both of chlorophyll and protein content decreased until 15 days after leaf unfolding.It was just the time that leaf expansion has ceased. Meanwhile,the degradation of protein was correlated with the increasing of proteolytic activities during leaf senescence.The optimum pH of endopeptidases was found at 7.0 and the optimum temperature at 40℃.besides,Ca²⁺ and cystine could increase its activities.Inhibition analysis suggested that four endopeptidases isoenzymes were serine" endopeptidases and the changes of them were different during leaf senescence.It implied that various endopeptidases might have its own physiological functions in cucumber leaf senescence.To further study the characters of endopeptidase (EP) isoenzymes in cucumber leaves, we employed leaves by dark-induced senescence.The results showed nine cucumber endopeptidases isoenzymes (CEP1-9) were detected during dark-induced process. Furthermore,EP appeared in natural senescence were also detected in dark-induced senescence.Four of which (CEP2,3,4 and CEP9) appeared all the time,however,the changes of the activities were different.Four CEPs (CEP5,6,7 and CEP8) were only detected in senescing leaves whose activities increased with darkness time.The biochemical properties of these nine cucumber endopeptidases isoenzymes in leaves were also characterized.All the CEP isoenzymes had high activities at 35-45^C, the optimum temperature was found to be 40℃.The major CEPs (CEP2,3,4,9) had no activities below 25℃or over 60℃and the bands appeared at a wide range ofpH from 5.0 to 9.0, but the optimum pH was 7.0.The CEP3 had' the highest thermal stability and its activity still existed even after incubation at 60℃.Inhibition analysis suggested that CEP2,3,4,9 were type of serine endopeptidases and CEP6 was a kind of cysteine protease.Consequently, serine endopeptidases might play an important roles in cucumber leaf senescence.To know about the role of serine-endopeptidase in leaf senescence, the effect of AEBSF on senescence was studied using detached cucumber leaves.The results showed that the senescence of cucumber leaves were delayed by AEBSF treated.The protein degradation of leaves declined slowly than control or ABA treatment, partly because the activity of.endopeptidases was lower in leaves.In addition, SOD activity increased and POD activity decreased in course of senescence.It suggested that the AEBSF delayed leaf senescence though inhibition the endopeptidase activity and influence the SOD and POD activity.Native PAGE with gelatin as substance in gel showed the activities of isoenzyme 3 and 4 were inhibited partly by AEBSF treatment on leaves.It implies that the two isoenzymes are type of serine endopeptidase and may play an important role in cucumber leaves senescence. Degradation of protein and changes of endopeptidase(EP) isoenzymes in cucumber (Cucumis sativus L.) leaves under different N supplied were also studied.The results indicate that nitrogen supply involves the realignment of constituent proteases and its activities.The protein concentration of the leaves can be manipulated by altering the N supply.Nitrogen supplied in cucumber leaves involves changes in protease activity and composition.It showed a positive correlation between nitrate supply and foliar protein concentration.Protein profiles,revealed by coomassie-stained SDS-PAGE,were changed between treatments and the relative contribution of the proteases was determined by Nitrogen supply.