Transcriptomic Analysis Of Cucumber And Pumpkin Grafted Combinations In Reponse To Salt Stress And Functional Analysis Of CmoNAC1 Gene

Grafting onto pumpkin rootstock is an effective way to increase cucumber salt tolerance.Both pumpkin and cucumber belong to Cucurbitaceae,the genome sequencing has been completed.However,there is still a lack of molecular mechanism of the different salt tolerance in cucumber and pumpkin grafted combinations.In this study,four grafted combinations,including self-grafted cucumber and pumpkin,cucumber grafted onto pumpkin,and pumpkin grafted onto cucumber,were constructed.The transcriptome of root,rootstock stem,stem,leaf petiole,leaf,and stem apex was analyzed.Functional analysis of the key salt responsive gene Cmo NAC1 was conducted.The main results are as follows:1. There is a rootstock-scion interaction in K⁺content in the cucumber and pumpkin grafted combinations under salt stress. The root had significantly lower K⁺content under salt stress when pumpkin was used as rootstock,compared with cucmber as rootstock.On the contrary,pumpkin scion leaf had significantly higher K⁺content than cucumber scion leaf under salt stress.The scion leaf can feedback regulate the root K⁺content,compared with self-grafted pumpkin,the root of pumpkin grafted onto cucmber had significantly lower K⁺content under salt stress.Compared with cucumber leaf,pumpkin leaf had higher water retention ability and tissue salt tolerance.2. Transcriptome study showed that the differential gene expression?DEGs?in the root and leaf of cucmber and pumpkin grafted combinations in response to salinity was regulated by rootstock-scion interaction.Most of the DEGs in the pumpkin roots was up-regulated,while it was opposite in cucumber roots.Salt tolerance genes,including HAK5,POD,APX,RBOHD and RBOHH,were up-regulated in pumpkin root under salt stress,while the salt tolerance genes,inckuding HKT1,CHX18,POD and RBOHF,were down-regulated.The expression of POD,RBOHF,CHX and AVP were up-regulated in the leaf of cucumber grafted onto pumpkin.While in self-grafted cucumber,these salt tolerance genes were down-regulated.There was a feedback regulation on the expression of key potassium channel protein SKOR and PIP1 subfamily members from scion leaf to rootstock root.Genomic comparison showed that transcription factors had higher number and proportion in pumpkin,compared with cucumber.There are more than 2200 transcription factors in Cucurbita maxima and Cucurbita moschata,accounting for 7%.But there are about 1400transcription factors in cucumber,accounting for 5%.The proportion of NAC in cucumber and pumpkin is 6%,but the proportion of differentially expressed NAC in pumpkin is increased to 8%,while those in cucumbers decreased to 5%under salt stress.In addition,NAC,transporter and antioxidant gene were co-expressed in pumpkin roots.Therefore,NAC plays an important role in salt tolerance in pumpkin.3. The efficient hairy roots transformation and gene editing system of pumpkin was established,the function of NAC transcription factor Cmo NAC1 was identified. Firstly,130 NAC family genes were identified in pumpkin genome,among which 6 members of the same branch of ATAF gathered together in pumpkin,the number of ATAF members was3 in cucumber.Furthermore,4 members in roots and 6 members in leaves of pumpkin were up-regulated under salt stress,Cmo NAC1 was the most abundantly expressed in pumpkin.The members of cucumber ATAF were down-regulated.Subcellular localization results show that Cmo NAC1 is located in the nucleus,Full-length protein and C-terminal sequence have transcriptional activity,while N-terminal sequence does not have transcriptional activity,and mainly for DNA binding.Overexpression of Cmo NAC1 in Arabidopsis improved salt tolerance of Arabidopsis.In addition,Cmo NAC1 can promote H₂O₂ level,and knock out Cmo NAC1 by CRISPR/Cas9 reduces the salt tolerance of pumpkin,the production of H₂O₂ and the expression of RBOHD significantly under salt stress.The results showed that Cmo NAC1 could bind RBOHD promoter sequence contained NBS fragment by yeast one hybrid system,which indicated that pumpkin RBOHD was one of the potential downstream regulatory genes of Cmo NAC1,and then affected the ROS signal transmission under salt stress and salt tolerance of plants.In conclusion,there is a rootstock-scion interaction on the K⁺content,salt tolerance gene expression such as HAK5,SKOR,RBOHF and PIP1 in the cucumber and pumpkin grafted combinations.The potassium transporter and antioxidant system were mostly up-regulated when pumpkin is used as rootstock,the leaf had higher K⁺content,can maintain better water status,and higher salt tolerance.The transcription factors in pumpkin roots play an important role in the regulation of gene expression and salt tolerance.Cmo NAC1 is the key NAC of root in response to salt stress.Cmo NAC1 can regulate the expression of RBOHD,participate in ROS signal regulation,and affect the salt tolerance of pumpkin.