Study On The Salt Uptake Mechanisms Of Roots In Halophyte Halogeton Glomeratus

Halogeton glomeratus(H.glomeratus)has strong salt resistance,the research of salt tolerance mechanism is of great significance for revealing the salt tolerance mechanism of plants,excavating salt tolerant genes and cultivating new salt tolerant varieties.Some genes that are related to salt enrichment are identified via the combination of transcriptome and iTRAQ-based proteomic approaches in leaves,but little is known about the Na~+uptake mechanisms of roots under salinity stress in H.glomeratus.In this study,we systematically researched the Na~+uptake mechanisms of H.glomeratus by the physiology,transcriptome and proteomics methods,and successfully obtained the regeneration system of H.glomeratus,which will lay the foundation for the functional identification of candidate genes.The main findings are as follows:1.The concentrations of NaCl and the incubation time had a certain effect on the germination rate of the seeds in H.glomeratus,the germination rates of the seeds were lower when the concentrations of NaCl were higher than 300 mM.It showed optimal growth under the 100-150 mM NaCl condition.The growth of H.glomeratus roots was influenced by the higher and lower salt stress.2.The aseptic seedlings of H.glomeratus were cultivated on agar with different concentrations(0,50,100,150,200,250 and 300 mM)of NaCl for 2 months.It was found that the content of Na~+increased significantly in the roots of H.glomeratus with the increasing of salt concentration,and which tended to be stable when the concentration of NaCl was higher than 150 mM,but the content of Na~+invariably increased in the surrounding agar.The Na~+content in the surrounding agar was higher than that of the roots in H.glomeratus under the condition of each treatment;the Na weight percentages of the root cross-sections first increased,and then tended to be stable,and it showed increased in the outside agar solid culture with the increasing of salt concentration,the Na weight percentage on the surface of agar section was higher than that of H.glomeratus root.CoroNa was used to stain the root tips which were treated with different NaCl concentrations,it was found that the fluorescence intensity of root tips was very weak at the different concentrations of NaCl and there was no obvious differences.Therefore,we confirmed that the process of Na~+uptake for root tissues of H.glomeratus presents a limited absorption characteristic.3.Transcriptome sequencing was performed in five sequencing libraries that were prepared from root samples treated with 200 mM NaCl for 2,6,24 and 72 h and a control sample.We have identified 550,590,1411 and 2063 DEIs that are mainly involved in metabolic process,cellular process,single-organism process,biological regulation,response to stimulus,catalytic activity and transporter activity,and a total of 45 up-regulated,13 down-regulated salt induced DEIs were common to be observed at four stages of salt stress,most of these DEIs were related to signal transduction and transporters.Whether salt-induced DEIs are responsible for exploring salt-tolerance mechanisms in the roots of H.glomeratus requires further study.4.The iTRAQ proteomic approach was conducted to investigate the molecular mechanisms underlying the salt response of roots in halophyte H.glomeratus under the concentration of 200 mM NaCl for 2,24 and 72 h.In total,there were 157,478and 1,002 differentially expressed proteins at the different stages of salt stress.The differentially expressed proteins significantly enriched in biological processes which were mainly the energy metabolism,stress and defense signal transduction under the treatment of 200 mM NaCl for 72 h.we also obtained 9 up-regulated proteins and 21down-regulation proteins by Veeny chart.Cluster analysis was used to divide salt induced proteins,and the up-regulated proteins were divided into 2 groups and the down-regulated proteins were divided into 3 groups.Meanwhile,the proteins related to Na~+transporter were mianly located in the plasma membrane of H.glomeratus roots,and there was no significant difference for the expression of plasma membrane Na~+/H~+antiporter and tonoplast Na~+/H~+antiporter after salt stress.5.We have developed a rapid and efficient system for plantlet regeneration from sterile apical meristems of leaves in H.glomeratus.Callus mass was the highest and an induction frequency of 100%could be obtained on MS medium supplemented with2 mg/L 2,4-D and 10 mg/L L-glutamic acid.Callus subculture with 1 mg/L 2,4-D and10 mg/L L-glutamic acid was optimal for callus status improvement and embryogenic callus formation.A frequency of shoot regeneration of approximately 100%and optimal shoot health was obtained using MS medium supplemented with 0.5 mg/L6-BA,2 mg/L Kn,0.2 mg/L NAA,10 mg/L L-glutamic acid and 2 g/L casamino acids.Roots were easily produced on MS medium alone and the regenerated plants were obtained eventually.As detailed in our findings above,the mechanisms of salt limited absorption in the roots of H.glomeratus have been preliminarily revealed,and the high quality regenerated system of H.glomeratus has been constructed.But we were still unable to clearly address the question of how the roots of H.glomeratus restrictively uptake Na~+under the salt stress so far.It is necessary to further study for the sodium transporters research in the roots of H.glomeratus.