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Genetic Analysis Of Salt Acclimation Mechanisms In Arabidopsis Thaliana

Posted on:2015-05-01Degree:DoctorType:Dissertation
Country:ChinaCandidate:X Y ShenFull Text:PDF
GTID:1220330467456438Subject:Botany
Abstract/Summary:PDF Full Text Request
Soil salinization is one of the important factors which affect plant growth, foodproduction and the area of arable land in the worldwide. Salt stress affects almost allaspects of plant development, including seeds germination, vegetative andreproductive growth. Although plants have formed a series of salt responsivemechanism to protect themselves in the long-term evolution, the capacity of plantswhich cope with high salt stress is limited. Soil salinization is much severer year byyear. So to elaborate mechanism of salt tolerance and to explore the strategies ofincreasing salt tolerence has been of important theoretical and practical significance.Plants can successfully improve their salt resistance to previously lethal salinitystress by a short exposure to low levels of salt stress, a process known as saltacclimation. In the natural, salt shock rarely occur and most salt stresses aregradual.The gradual salt application is the same as the process of salt acclimation. Inspite of its fundamental significance in theoretical study and agricultural practice, themechanisms underlying plant acclimation remain elusive. In recent years, the study ofacclimation has been some progress, such as pathogen priming, cold accliamtion, heatacclimation, salt acclimation and so on, but the study of molecuar mechanism are rare.In this study, the effective salt acclimation scheme was designed. The salttreatment samples and the control were prepared for RNA-seq which was performedto analyze the genome-wide transcriptional response under salt acclimation conditions.We observe the phenotype of mutants in salt acclimation process to deduce the geneparticipating salt acclimation process or not. This study provides some clues to theimportant role of salt acclimation in the mechanism of salt tolerence. The results wereas follows:1. Scheme of salt acclimation treatments used in this study: seven-day-oldArabidopsis seedlings were pretreated on MS plates containing50mM NaCl for48 h before being moved to MS plates containing200mM NaCl. Conversely,non-acclimated salt shock was applied by transferring seedlings first to MS platesfor48h and then to MS plates containing200mM NaCl. The Arabidopsis youngseedlings which have undergone salt acclimation showed an albino phenotype muchlater and could survive at least13days longer on200mM NaCl than seedlingswithout salt acclimation.They showed an increased salt tolerance against a normallylethal level of salt stress.2. To further study on the molecular mechanism of salt accliamtion, we sampledArabidopsis young seedlings at different treatments of salt accliamtion for RNA-seq,including the control,50mM NaCl treatment for48h(salt acclimation, SA),200mMNaCl for12h after acclimation(salt acclimated salt stress, SASS), salt shock on200mM NaCl for12h without acclimation(non-acclimated salt stress, NASS). Based onthe data of RNA-seq,518differentially expressed genes(DEGs)were detected undersalt acclimation compared to the control. Among them,366up-regulated genes wereenriched for cell wall biosynthesis, osmoregulation, oxidative stress, or transcriptionfactors and152gene showed down-regulated. Seven DEGs participated in thesynthesis of lignin and24DEGs encoded plant cell wall proteins, suggesting theimportance of cell wall remodeling under salt acclimation. Furthermore, incomparison to non-acclimated salt stress,245DEGs were found in acclimated saltstress. Interestingly, only17genes were up-regulated in acclimated salt stress and8genes encode plant cell wall proteins.228genes were repressed by acclimated saltstress. Of them,95were up-regulated in both SASS and NASS samples comparedwith SA samples, but to a much lesser extent under SASS conditions,including manygenes related to ethylene biosynthesis,such as ACS2, ACS6, ACS7, and ACS8.Therefore, our datas suggest that, after salt acclimation, the ethylene biosynthesis wasrepressed during subsequent salt stress, which may slow down plant senescence andimprove resistance against high salinity.3. MAPK6, a major component of the ethylene signaling pathway, mediatesethylene biosynthesis by phosphorylating ACSs. We performed salt acclimation tomapk6and mapk3.In contrast to the wild type seedlings, both mapk6mutant lines could not survive high salinity stress and showed albino even after salt acclimation,while the phenotype of mapk3show the same as wild type. Overexpression ofMAPK6in mapk6mutant lines resulted in restoration of the ability to survive highsalinity stress after salt acclimation. So we concluded MAPK6plays a crucial role insalt acclimation. According to the data of RNA-seq, the expression level of MAPK6was not changed during salt acclimation, MAPK6may be mainly regulated at thepost-translational level in salt acclimation.4. According to the data gained from the salt acclimation assay using350different ecotypes of Arabidopsis, Katori et al.(2010) concluded that salt acclimationimproved salt tolerance which was controlled by a single gene located in about18.5Mbp interval between SSLP markers At5g-102and nga129of chromosome5.According to this, we chose two genes which encode arginine methyltransferase andDNA methyltransferase, respectively, and ordered two genes T-DNA mutants(prmt4a-1,prmt4a-2,met1-3and met1-7) for salt acclimation. We only identifiedhomozygous T-DNA insertion mutants of arginine methyltransferase (prmt4a-1andprmt4a-2). The phenotype of the two arginine methyltransferase mutants showed thesame as wild type, which implied that the gene encoding arginine methyltransferasedidn’t participate in salt acclimation.Salt acclimation is regulated by a complex network including many genes andsignaling pathways. Our transcriptomic analysis provided critical perception on theroles of cell wall remodeling and the ethylene biosynthesis and signaling pathwaysduring salt acclimation in Arabidopsis. MAPK cascade pathways were also includedin salt acclimation. These provided some information to study the molecularmechanisms of salt acclimation and salt tolerance.Another part work of the study was to clone a low-K+sensitive and etiolatedmutant by map-based cloning which was screened from T-DNA insertion mutants.The results were as follows:1. The preliminary work of the mutant was to study the phenotype oflow-potassium sensitive. TAIL-PCR was performed. The results showed T-DNA inserted intergenic region and three genes encoding polyamine oxidase were aroundthe insertion site. Through analysing the mutant phenotype of the three genes, theydidn’t show low-potassium sensitive. So the T-DNA insertion was not the reason ofthe low-potassium sensitive.2. Subsequently the mutant was hybridized with Ler and the resultant F2population was used to map based cloning. Due to difficulty to distinguish the mutantin F2segregating populations and poor reproducibility of low-potassium condition,the potassium-sensitive phenotype can not be positioned. While the etiolatedphenotype in segregating populations is obvious and genetic analysis showed that theetiolated mutant was a single and recessive mutation. Then the etiolated mutant wasmapped to the chromosome2and located in a region between7,163,253bp and7,283,651bp. The region included31genes. We chose several genes associated withthe chloroplast to amplify and sequence their genomic DNA and analyzed ifdeletion or insertion of a fragment of T-DNA existed. The resluts showed that the fullsequence of AT2G16630and the majority of AT2g16640were deleted.3. AT2g16640encoded the outer envelope membrane protein of chloroplasts,TOC132. The phenotype of toc132in Col background was the same as the etiolatedmutant, which had been reported. The etiolated mutants in segregated population weretreated with different concentration of sucrose and H2O2. The results showed that theroots were significantly inhibited by high sucrose and H2O2.The innovations of this thesis were shown as follows:1. Identifing the50mM NaCl as an effective concentration of salt accclimation,which can effectively improve salt tolerence of Arabidopsis on high salt stress. Theexperimental system of salt acclimation is established for the first time.2. We studied the salt-tolerant mechanism of salt acclimation and enriched thecontent of the mechanism.3. Our transcriptomic analysis found many genes associated with salt acclimationand provided critical perception on the roles of cell wall remodeling and the ethylenebiosynthesis and signaling pathways during salt acclimation in Arabidopsis.
Keywords/Search Tags:Arabidopsis thaliana, differentially expressed genes, RNA-seq, saltacclimation, transcriptome analysis, map based cloning, etiolated mutant
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