Isolation And Genetic Analysis Of Arabidopsis Salt-sensitive-mutants,the Proteasomeβ5 Subunit Relate To Plant Oxidative Stress Tolerance

Chapter 1: Isolation and Genetic Analysis of Arabidopsis Salt-Sensitive-MutantsSoil salinization is one of the major stress factors that limiting the productivity and qualityof crops. High sodium (Na⁺) concentration in soil is toxic to most higher plants, severely affectthe plants growth and development, accounting for large decrease in the yields of a wide varietyof crops all over the world. Thus, analyzing the mechanism of plants response to salt stress,exploring genes related to salt tolerance not only have important theoretical significance butalso have important practical significance for cultivation of salt-tolerant crops. The mechanismof plants adapt to salt stress is very complex, so improving the salt tolerance of crops still facedgreat challenges.For the past few years, many salt-sensitive mutants had been gained and had laid a goodresearch basis to further identify salt-sensitive mutants, example for the contribution of theseries of sos mutants. The modulating mechanism of the SOS signal pathway consisted ofSOS1-SOS5 in Arabidopsis has been illuminated, studies showed that the SOS signal pathwayplays an important role in ion homeostasis and salt tolerance of plants.In this dissertation work, T-DNA-mutagenized seeds of Arabidopsis thanlina inWassilewskaja genetic background was used to screen salt-sensitive mutants. By isolatingseedlings after treated with 75 mM NaCl conditions, we obtained the dds1 which hasdevelopmental defects and salt sensitive phenotype. Genetic analysis showed that the dds1carries a single, recessive nuclear mutation. We have obtained two sequence flanking T-DNA byplasmid rescue method, but PCR and kanamycin resistance assay showed that the mutationwere not co-segregated with the T-DNA insertion. Then the mutated DDS1 was mapped to thebottom of chromosome 2 and located in a region near M1836 and M1836 which are located in 18361670 bp and 18878730 bp of chromosome II.To determine the roles of DDS1 in plant growth and development, a few characteristic traitsrelated to growth and development were investigated physiologically in the dds1 mutant. TheDDS1 mutation causes a pleiotropic phenotype. Under normal conditions, the phenotype ofmutant exhibited reduced roots and rosette leaves elongation and reduced the number of lateralroots. Further analysis indicated that dds1 was more sensitive to 150 mM NaCl stress thanwild-type plant at seed germination and seedling stages. The dds1 mutant was also moresensitive to high ABA, Mannitol and MV stress. sos1 are more sensitive to low NaCl (50 mM)and LiCl (20 mM) stress, while dds1 showed no difference to 20 mM LiCl treatment andsensitive to high NaCl (150 mM) treatment. The results indicate that these two mutated genesmay be involved in different signaling pathways in plant response to salt stress.The leaf blades area and abaxial epidermal cells area was measured and compared betweendds1 and wild type. The area of dds1 leaves was smaller than wild type but abaxial epidermalcells area was wider than wild type. The increased of dds1 leaf expansion may associated withcell division defection. Finally the mutant responses to exogenous IAA and KT were alsoobserved. Incubation on low IAA containing MS medium has no effect on dds1 but reducedroot elongation in wild type, and low IAA has no effect on salt sensitive of dds1. But incubationon KT containing MS medium was no effect on dds1. Indicating that the IAA hormonesignaling pathway may changed in dds1 mutant.Taken together, these results suggest that DDS1 is a key component required for thenormal regulation of signal pathway during plant growth development.Chapter 2: The Proteasome 5 Subunit Relate to Plant Oxidative Stress ToleranceOxidative stress is one of the major threats to the plants. The oxidative stress is caused byan imbalance between the production of reactive oxygen and a biological system's ability toclean it. Disturbances in this normal redox state can finally affect metabolism by the damage ofthe proteins, lipids and DNA, severe oxidative stress can even cause cell death.The ubiquitin-26S proteasome pathway is considered to be an effective way of degrading unneeded or damaged proteins by proteolysis. The ubiquitin-26S proteasome pathway alsoplays an important role in hormone signaling, photomorphogenesis, flower development andPCD by degrading regulatory proteins.20S proteasome is the core particle (CP) of the 26S proteasome. All 20S particles consist offour stacked heptameric ring structures which composed of two different types of subunits: theouter two rings in the stack consist of sevenαsubunits and the inner two rings each consist ofseven subunits. The 1, 2, and 5 subunits have protease activities which have threedifferent substrate specificities.Stable overexpression of proteasome 5 subunit in WI38/T and HL60 cells increases theamount of assembled proteasome and confers ameliorated response to oxidative stress andhigher survival rates. So far, the overexpression of AtPBE in high plants has not been reported,so study the effect of overexpression AtPBE gene in Arabidopsis may give us muchinformations of improving the anti-oxidative ability of plant cells and the plant's resistance toabiotic stress by genetic engineering means.In order to study the function of AtPBE (the gene of proteasome 5 subunit in Arabidopsisthaliana), in this experiment, four vectors had been constructed: the overexpression vectors ofgene AtPBE, the GFP transient expression vectors of gene AtPBE. The AtPBE1 and AtPBE2genes were isolated from Arabidopsis thaliana by RT-PCR method and confirmed bysequencing. Than the AtPBE1 and AtPBE2 PCR products were inserted into binary plant vectorpCAMBIA3301. The resulting plasmids named pCAMBIA3301-AtPBE1 and pCAMBIA3301-AtPBE2 vectors, were introduced into Arabidopsis thaliana by Agrobaterium tumefaciens-mediated transformation with floral-dipping method. Transformants were selected for bastaresistance and gained T3homozygous lines. We also obtained mutant lines from ABRC andgained homozygous mutants pbe1 and pbe2. The homozygous lines were selected and used forfurther analysis molecularly and physiologically.The main results as following:1)The transgenic lines were detected by PCR, about 1 kb band was obtained while wild type has no band indicating that the AtPBE gene has been introduced into Arabidopsis genome.RT-PCR and Realtime-PCR analysis revealed the induced expression of AtPBE in T3transgeniclines than wild type Arabidopsis. RT-PCR analysis revealed no expression of AtPBE in pbe1and pbe2.2) Realtime-PCR analysis revealed that the transcript levels of several genes involved inPBA1, RPN10 and RPN12 subunits were constitutively up-regulated in the transgenic plants,but the transcript levels of PAG was reduced in transgenic plants and mutants.3) The analysis of the stress tolerance showed that the tolerance to high NaCl, MV andH₂O₂stress were the same between the transgenic plants and wild type in seeds germinationand seedling stages. The results implied that the overexpression of proteasome 5 subunit inArabidopsis were not results in increased oxidative stress resistance.4) The AtPBE::GFP transient expression were mostly located mainly in the nucleoplasm. Adispersed punctate pattern was also evident in the cytoplasm.The innovation of this thesis were shown as follows:1) We obtained the dds1, which has developmental defects and salt sensitive phenotype, afew characteristic traits related to growth and development were investigated physiologically inthe dds1 mutant.2) Abtained some markers suited for Arabidopsis thanlina mapping between Columbia-0and Wassilewskaja genetic background according to resequenceing.3) This work showed that overexpression of proteasome 5 subunit in Arabidopsis notresult in increased oxidative stress resistance.