Mechanism Of Putrescine Regulating Stomatal Opening Of Cucumber Leaves Under Salt Stress Mediated By H₂O₂ Signal

Cucumber is the main vegetable crop under protected cultivation in China and even in the world.It is favored by the majority of consumers because of its rich nutrition and various eating methods.The regeneration ability of cucumber roots is very weak and sensitive to salt.When cucumber plants were in a saline environment,their photosynthetic efficiency and biomass accumulation were decreased,which seriously affects the sustainable development of greenhouse cucumbers.Stomata are important factors affecting plant photosynthesis.In this study,the cucumber of‘Jinyou NO.4’used as the material,and application of Put on stomatal opening,endogenous polyamine（PAs）metabolism,and abscisic acid（ABA）content under salt stress,the effect of dependence on PAs was explores.of the hydrogen peroxide（H₂O₂）signal of polyamine degradation metabolism is involved in stomatal regulation under salt stress.Ccombined genetic transformation methods,the internal mechanism of Put regulating stomatal opening of cucumber under salt stress was analyzed.The research results have important theoretical value for enriching photosynthetic stress physiology of cucumber and breeding salt tolerant varieties.The main results are as follows:1.The effects of exogenous Put on the biomass,photosynthesis,stomatal opening,leaf temperature,endogenous PAsand H₂O₂content of cucumber seedlings under salt stress were studied.The mechanism of Put on the change of stomatal in cucumber leaves was discussed.The results showed that Put alleviated the inhibition of photosynthesis induced by salt stress and promoted plant growth.The plant height,dry weight and fresh weight increased by 1.05 times,1.57 times and 1.35 times respectively compared with salt treatment on the 7th day.Put increased the stomatal opening of cucumber leaves under salt stress,resulting in a significant increase in leaf surface temperature,which reached the maximum value at 6 h,with a maximum temperature increase of 2.3℃.Under salt stress,Put significantly increased the total content of endogenous PAs in cucumber leaves,and the total content of endogenous Put was the highest and reached the peak at 6 h,which was18.9 times higher than that of the control.Put significantly increased the content of H₂O₂in the salt stressed-cucumber leaves and stomata,showing a trend of first increasing and then decreasing,and reached the peak at 6 h,which was 27.97%higher than that in salt treatment.The above results indicated that exogenous Put could increase the photosynthetic capacity of cucumber seedlings by inducing changes of endogenous polyamine content in cucumber leaves under short-term salt stress（5 d）,promoting the production of H₂O₂signal molecules and alleviating the decrease of stomatal opening.2.In order to further study the regulatory mechanism of exogenous Put on stomatal opening of cucumber leaves under salt stress,H₂O₂production inhibitors and scavengers,reduced glutathione donors and scavengers were used.The results showed that Put increased the DCF fluorescence intensity by 0.30 times,and decreased the content of ABA by 0.79 times as compared with salt stress.Application of inhibitors of diamine oxidase（AG）,polyamine oxidase（1,8-DO）,nicotinamide adenine dinucleotide phosphate oxidase（DPI）,and cell wall peroxidase（SHAM）up-regulated the ABA synthesis gene（NCED,9-cis-cyclocarotenoid dioxygenase）,and down-regulated the reduced glutathione（GSH）synthesis gene（GSHS）.These inhibitors also decreased the stomatal diameter as well as H₂O₂and GSH contents,but increased ABA level.The influence ability is AG>1,8-DO>DPI>SHAM.However,Put induced down-regulation of ABA content and up-regulation of GSH content under salt stress effectively blocked by H₂O₂scavenger（DMTU）and GSH scavenger（CNDB）treatment.These results indicated that Put could degrade endogenous Put through diamine oxidase（DAO）,induced H₂O₂signal formation,increased GSH content,inhibited ABA accumulation in leaves,and promoted stomatal opening of cucumber under salt stress.3.In order to verify the function of CsADC gene and its regulatory mechanism in the stomatal opening under salt stress,tobacco was used as the material to construct overexpression lines of CsADC gene by genetic transformation.The results showed that there was no significant difference between the CsADC overexpression lines（OE-1 and OE-2）and the wild type（WT）under the control conditions,except for the higher expression of CsADC.After Na Cl treatment,comparing with WT,OE-1 and OE-2 showed strong salt tolerance,and plant height,stem diameter,shoot fresh weight and dry weight were increased by 0.78 times and 0.66 times,0.11 times and 0.23 times,0.5 times and 0.4times,0.66 times and 0.5 times,the total content of PAs increased by 0.91 times and 0.38times,the content of H₂O₂in leaves increased by 3.07 times and 1.27 times,the content of GSH in leaves increased by 1.23 times and 0.64 times,the fluorescence intensity of DCF and GSB increased significantly,the expression levels of NtADC,CsADC,NtDAO,NtPAO and GSHS increased by 14.13 times and 8.53 times,44.46 times and 38.07 times,0.53times and 0.28 times,0.91 times and 0.48 times,1.15 times and 0.42 times,the stomatal aperture increased significantly,the content of ABA decreased by 0.5 times and 0.42 times,the expression levels of NCED decreased by 0.64 times and 0.37 times.The salt tolerance of OE-1 was higher than that of OE-2.These results indicated that the accumulation of ABA in leaves were inhibited by H₂O₂signal formed by DAO degradation of endogenous Put,which could alleviate the stomatal closure induced by salt stress,and improve the salt tolerance of cucumber plants.