Evaluation Of Melon Cold Tolerance And The Participatory Mechanism Of Putrescine At Seedling Stage

Cucumis Melo is a cold sensitive plant,which originated from tropical regions such as Africa and India.According to our investigation,in the Northeast,North China and the middle and lower reaches of the Yangtze River,melon sawing and transplanting are concentrated in the middle and late December to the late April of the next year.This stage is in winter and early spring in China,with low temperature,frequent rain and snow weather,and frequent cold in late spring.Melon is vulnerable to low temperature in the seedling stage of early cultivation.Based on the extensive collection of melon germplasm resources,the evaluation system of low temperature tolerance in germination stage and the identification method in seedling stage were established by using artificial simulated low temperature.On this basis,CBF(C repeat binding factor),ABA and polyamine(PA)pathway were used to analyze the molecular mechanism of cold tolerance in melon seedling stage,so as to provide scientific basis for rapid screening of melon germplasm with cold tolerance,breeding cold-tolerant melon and solving the cold injury problem of melon early cultivation.The main results are as follows:1.There are fifty melon germplasms are collected for cold tolerance evaluation at the germination stage.Seeds germinated at constant 15℃and 28℃for eight days.The results show that under the influence of low temperature,the germination time of seeds is greatly delayed,the vigor of seeds decreased,the radicle elongated slowly.The principal component analysis is used to weight each index,and the membership function value of each index is integrated to determine the cold tolerance index of each germplasm.With the cluster analysis and multiple linear regression analysis,an evaluation model is established:Y=1.224 RGI+0.291 RRL+0.254 RGP-0.022(R~2=0.996,P<0.001 RGI,relative germination index;RRL,relative radicle length;RGP,relative germination potential).When the predicted value Y≤0,it can be directly determined that the tested materials are cold sensitive;Y>0,the predicted accuracy of only two materials is 80.8%and 87.0%;the average predicted accuracy of the other 46 germplasms is 97.4%.The results show that the selected indexes had significant effects on the chilling tolerance of melon seedlings,and the mathematical model can be used to evaluate the cold tolerance of melon seedlings at germination stage.2.To obtain a rapid and accurate method for evaluating cold tolerance of melon,ten genotypes were selected to investigate their cold tolerance at seedling stage.Chilling stress(15°C/6°C,D/N)increased leaf angles and caused leaves wilted.Nevertheless,the phenotypes of the ten genotypes were obviously different.Thus,a new predicted method for chilling injury index(CII)of melon was constructed based on the change of leaf angle and leaf state.Firstly,the reliability of this method was verified by the data of 10 materials.The results showed that CII is significantly correlated with the survival rate,the maximum photochemical quantum yield of PSII(Fv/Fm)and the SPAD values.In addition,the validity of the method is further verified by the seedling growth,photosynthesis,membrane permeability and metabolite accumulation of the four screened genotypes.3.We identified a total of 25 key genes,including 6 genes(2 Cm ICEs,4 Cm CBFs)in CBF(C-repeat binding factor)pathway,9 genes(4 Cm NCEDs,5 Cm ABFs)and 10 genes(1Cm ADC,1 Cm ODC,4 Cm SAMDCs,2 Cm SPDSs,2 Cm SPMSs)in ABA and polyamine(PA)pathway,respectively.IVF571(cold-tolerant)and IVF004(cold-sensitive)are selected to explore their tolerance mechanism in this study.Cm ICEs,Cm CBFs,Cm NCED3,Cm NCED3-2,Cm ABFs,Cm ADC,Cm SAMDCs,Cm SPDS2 and Cm SPMS1 were significantly induced by low temperature(15°C/6°C,D/N),and the corresponding products accumulation were consistent with gene expression.The transcriptional levels of Cm NCED3,Cm NCED3-2and Cm ADC in cold-tolerant genotype were significantly higher than those in cold sensitive genotype,and the accumulation levels of putrescine and ABA in cold tolerant genotype were significantly higher than those in cold sensitive genotype.Taken together,the cold-tolerant melon seedlings could trigger CBF,ABA and PA pathways to increase cold tolerance under low temperature.4.ICE is located in the upstream of CBF,and there is ICE binding site in Cm ADC promoter.The results show that the cold tolerance of melon seedlings decreased significantly after Cm ICE1 silencing,and the expression of Cm ADC and putrescine accalimation decreased sharply.The results showed that the chilling tolerance of melon seedlings decreased after Cm ICE1 silencing,which is related to the inhibition of putrescine.Exogenous ABA inhibitor(dimethoxystrobic acid)and Cm NCED3 silenced significantly decrease the cold tolerance of melon seedlings,also cause a reduction in putrescine level,which prove that the accumulation of ABA under low temperature can partly affect the level of putrescine accumulation and improve the cold tolerance of melon seedlings.5.It is proved that Cm ADC mediated putrescine biosynthesis played an important role in melon cold tolerance through putrescine inhibitor and virus induced gene silencing(VIGS)test.The sequence of Cm ADC promoter is analyzed and cloned.There are three ABRE motifs and three DRE motifs in the promoter.All the four cloned promoters have promoter activity.Subcellular localization shows that five Cm ABFs and four Cm CBFs are located in the nucleus.Transcriptional activation assay shows that Cm ABF1/3/4/5 and Cm CBF1/2/4 have transcriptional activation activity.Cm ABFs and Cm CBFs can couple ABRE and DRE motif within the Cm ADC promoter fragment in vitro,and Cm ABF1/3/4/5 and Cm CBF1/2/4 are screened as candidate TFs by yeast one-hybrid assay.GUS activity and luciferase reporter assays are further confirmed that this cotransformation activated the expression of Cm ADC regulations in tobacco leaves and melon cotyledons.Virus induced gene silencing(VIGS)system proved mono-gene silenced melon seedlings are more sensitive to low temperature,with Cm ADC expression and putrescine accumulation dramatically reduced.These results suggest Cm ABF1/3/4/5 and Cm CBF1/2/4 promoted putrescine biosynthesis by triggering the expression of Cm ADC which positively enhanced the cold tolerance of melon seedlings.Our work resulted in novel evidence that ABA pathway and CBF pathway intersect at Cm ADC that mediates cold tolerance of melon seedlings.