Functional Analysis Of Peach PpSnRK1 Kinase α Subunit Under Salt Stress

At present,soil salt stress is a global problem,seriously threatening the growth of horticultural plants and reducing yields.In the world,the planting area of peach in China ranks first.Therefore,studying the resistance mechanism of peach trees to salt stress is of great significance for improving the resistance of peach trees and the yield and quality of peach.Previous studies have shown that SNF1-related protein kinase 1(SnRK1)is a key component of cell signal transduction.SnRK1 can respond to a variety of stresses,but its specific mechanism in salt stress response and tolerance is still unknown.To this end,this study took the peach SnRK1α subunit(PpSnRK1α)as the entry point,and studied the resistance of PpSnRK1α to salt stress through the transgenic PpSnRK1α tomato lines,and further studied the role of PpSnRK1α in the salt tolerance mechanism of peach trees through genetic transformation.The main findings are as follows:1.PpSnRK1α participates in salt stress and improves salt toleranceFirst of all,it was found that the increased tolerance of PpSnRK1α-overexpressing tomatoes to salt stress is related to the increase in the expression level of PpSnRK1α and SnRK1 kinase activity.Compared with the wild type(WT),the PpSnRK1α overexpression lines exhibited a lower level of leaf damage,as well as increased proline content and lower malondialdehyde(MDA)in the leaves under salt stress.Also,overexpression of PpSnRK1α can enhance the metabolism of reactive oxygen species(ROS)by increasing the expression level of antioxidant enzyme genes and antioxidant enzyme activities.After that,transcriptome sequencing was used to analyze WT and three PpSnRK1α overexpression lines and identified about 1000 PpSnRK1α-regulated genes,including many antioxidant enzymes,and these genes were significantly enriched in the MAPK signaling pathway(plant),plant-pathogen interactions,and plant hormone signal transduction and can respond to stimuli,metabolic processes,and biological regulation.Furthermore,it was found that the transcriptional levels of several salt stress-responsive genes and abscisic acid(ABA)signal transduction pathway genes,Sl PP2C37,Sl PYL4,Sl PYL8,Sl NAC022,Sl NAC042,and Sl SnRK2 family were altered significantly by PpSnRK1α under salt stress,signifying that SnRK1α may be involved in the ABA signaling pathway to improve tomato salt tolerance.2.PpSnRK1α regulates the sensitivity of plants to ABATo further explore the molecular mechanism of PpSnRK1α improving plant salt tolerance,and to verify whether PpSnRK1α can participate in the ABA signal transduction pathway to improve plant salt tolerance,firstly,the wild-type peach seedlings were treated with exogenous ABA,and it was found that the expression level of PpSnRK1α could significantly respond the treatment of exogenous ABA.Secondly,by detecting the germination rate of PpSnRK1α overexpressing tomato seeds under ABA treatment,it was found that PpSnRK1αoverexpressing plants could respond to ABA treatment and thus reduce the germination rate of seeds.Then,Agrobacterium rhizogenes-mediated hairy root transformation protocol was used to produce composite plants with wild-type shoot and transgenic roots.Therefore,the transgenic composite peach seedlings with PpSnRK1α overexpression(PpSnRK1α-1302)and PpSnRK1α inhibited expression(PpSnRK1α-TRV)were obtained.Under normal growth conditions,the expression of SnRK2 family genes PpSRK2 I,PpSRK2E-1,PpSRK2E-2,and downstream transcription factors PpABI3 and PpABI5 in the root of PpSnRK1α-overexpressing composite peach seedlings increased.And the expression of SnRK2 family genes PpSRK2E-1,PpSRK2E-1,and PpSnRK2E-1 in PpSnRK1α-TRV composite peach were inhibited,but the expression of downstream transcription factors PpABI3 and PpABI5 remained unchanged.The above results indicate that PpSnRK1α is sensitive to ABA and may regulate the expression of PpABI3,PpABI5,and SnRK2 family genes.3.Interaction between PpSnRK1α and ABA signal transduction pathway transcription factors To further study the molecular mechanism of PpSnRK1α in resisting salt stress,firstly,PpSnRK1α was tested for its self-activation and toxicity.Secondly,a transcription factor PpABI3,which can interact with PpSnRK1α protein and participate in the ABA signal transduction pathway,was screened by yeast two-hybrid assay.The interaction between PpSnRK1α protein and PpABI3 protein was further verified by Bimolecular Fluorescent Complimentary(Bi FC).The response mechanism of PpSnRK1α and PpABI3 to exogenous ABA needs to be further explored.