The Mechanism Of CmHKT1;1 Increasing Salt Tolerance Of Grafted Cucumber Seedling And Identification Of Related MicroRNAs

The use of rootstock is a valid strategy in increasing cucumber abiotic stress tolerance.In our previous studys,we found that pumpkin rootstock improve cucumber salt tolerance by limiting Na~+ transport from root to the scion shoot,but the molecular mechanism is still unclear.HKT1 protein is a kind of Na~+ transporter.Its main function is to load Na~+ into the stele from xylem sap to reduce the Na~+ content in xylem.But the function of pumpkin HKT gene in cucumber grafted seedlings during salt stress is still unknown.microRNA is a kind of non-coding RNA widely involved in plant abiotic stresses,but the response and regulation mechanism in cucumber grafted seedlings during salt tolerance is not clear.Therefore,in this study we focus on the effects of rootstock on the Na~+ distribution in grafted seedlings,functional analysis of pumpkin CmHKT1;1 and microRNA response to salt stress and regulation network in grafted seedlings.The results were as follows:1.Salt tolerance-pumpkin rootstock is the key to limit Na~+ transport from root to salt sensitive-cucumber scion,and the scion also affects the Na~+ content in the rootstock.We constructed 4 pumpkin and cucumber reciprocal grafting combinations,75 mM NaCl for120 h.It was observed that the grafting combinations with pumpkin as the rootstock could effectively limit the accumulation of Na~+ in the scion shoot part and alleviate the degree of salt damage.Whereas,grafting combinations with cucumber as rootstock did not limit Na~+ accumulation in sicon,indicating that the pumpkin rootstock plays a key role in the salt tolerance of grafted cucumber.In addition,the sicon with different genotypes also affects the Na~+ content in the root of the rootstock.These results indicate that the rootstock and scion of the grafted seedlings both affect the accumulation of Na~+ in the salt-tolerant process,and there is an interaction between the scion and rootstocks.2.CmHKT1;1 is up-regulated by salt stress,and the expression concentrated in the stele xylem parenchymatous cell of pumpkin root and localized on the cell membrane.The HKT gene was cloned in a salt-tolerant pumkin rootstock and named as CmHKT1;1.RT-PCR and qRT-PCR showed that CmHKT1;1 was mainly up-regulated in pumpkin roots under salt stress.Subcellular localization indicated that CmHKT1;1 was localized on the cell membrane and was a membrane protein.In situ hybridization showed that CmHKT1;1expression was mainly concentrated at stele xylem parenchymatous cell of the pumpkin root.3.CmHKT1;1 is a Na~+ specific transporter.Using the yeast mutant to analyze the function of CmHKT1;1,in the salt-sensitive yeast strain G19,growth inhibition experiments showed that heterologous expression of CmHKT1;1 can increase the salt sensitivity of G19,indicating that CmHKT1;1 is a Na~+ transporter.In the functional complementation experiment,deficient endogenous K~+uptake mutant strain WΔ6heterologously expressed CmHKT1;1,unable to restore K~+uptake under sufficient K~+conditions,indicating that CmHKT1;1 has no transport ability to K~+.In ion depletion curve experiment,yeast transformated CmHKT1;1 caused a decrease in Na~+ content and a constant K~+content.In the medium containing Na~+ /K~+,K~+had no effect on the decrease of Na~+ content,indicating that CmHKT1;1 has no Na~+ /K~+co-transport function.4.CmHKT1;1 limit Na~+ unload to the xylem at the root of the rootstock,reduced the Na~+ transport to the upper part of the root,and improved the salt tolerance of the grafted cucumber seedlings.Agrobacterium rhizogenes-mediated CRISPR/Cas9 gene editing technology was used to transiently transform CmHKT1;1 in pumpkin roots,knocking out CmHKT1;1 in roots,the results showed that the gene was edited and reduced the salt tolerance of pumpkin,significantly increased the Na~+ content in xylem sap and the accumulation of Na~+ in the shoot.Overexpression of CmHKT1;1 in Arabidopsis HKT mutant hkt1;1,CmHKT1;1 can restore the salt tolerance of hkt1 to some extent,Na~+ specific dye stains the root Na~+ ,and found that CmHKT1;1 could reduce Na~+ unloading to the xylem.Overexpression of CmHKT1;1 in cucumber increased the salt tolerance of cucumber,increased chlorophyll content,increased dry weight of plants,and decreased Na~+ content in shoots.Using cucumber overexpressing CmHKT1;1 as rootstock and wild type cucumber as scion,we found that the salt tolerance of grafted seedlings could be improved,the chlorophyll content increased,the leaf ion leakage reduced,the dry weight of plants and the Na~+ content of shoot reduced.It is proved that CmHKT1;1 plays an important role in grafting salt tolerance.5.Aiming at the change of Na~+ content in grafted seedlings under salt stress,high-throughput sequencing was used to identify the miRNA expression of four grafted combinations seedlings under salt stress.The results showed that there were 323 miRNAs known in cucumber,119 novell miRNAs,374 known miRNAs and 91 novell miRNAs.The miRNAs of pumpkin and cucumber were highly homologous,and 236 miRNA sequences were identical.The miRNAs in response to salt stress were identified,and the miRNAs in the grafting combinations were mainly down-regulated.But in the roots of grafted cucumber seedlings,67 miRNAs were up-regulated,and 22 were down-regulated.The comparison of miRNA species in response to salt stress revealed that miRNA398 was significantly differentially expressed in all tissues,indicating that miR398 may be a salt-tolerant basal-responsive miRNA.The expression of differentially expressed miRNA was detected by qRT-PCR,and the reliability of sequencing data was verified.The target gene of miRNAs was predicted,and most of the target genes were found to be transcription factors such as MYB,NAC,HD-ZIP,etc.GO analysis and KEGG analysis of differentially expressed miRNA target genes in cucumber/pumpkin grafted seedlings and found that target genes are enriched in processes or pathways such as metabolic processes,catalytic activity,transcriptional activity,stimuli response and plant hormone signaling,etc.;The signal response pathways under salt stress were compared.It was found that miRNAs are involved in the regulation of salt tolerance by various hormonal signal transduction.For example,the scion cucumber part is involved in auxin signaling through the regulation of TIR1 by miR393,and the pumpkin rootstock is involved in Gibberellin signal transduction by miR159.In summary,in this study,we found that the expression of CmHKT1;1 was induced by salt stress and localized on the cell membrane of the parenchyma cells in the pumpkin root stele.As a Na~+ specific transporter,CmHKT1;1 reduced Na~+ accumulation in scion by limiting Na~+ unload to xylem,improved the salt tolerance of cucumber grafted seedlings.The response of salt-stressed miRNAs in grafted seedlings were identified and the physiological and biochemical processes and pathways involved in salt-responsive miRNAs were speculated and analyzed.