The Involvement Of Arabidopsis AtFes1A In Thermotolerance

The HSP70 chaperone completes their functions by the ATPase cycles. There are numerous proteins and co-chaperones involved in the HSP70 chaperone system. In the HSP70 chaperone complex, the hydrolysis of ATP is stimulated by the'J'domain containing proteins and substrates, and the nucleotide is stripped by the nucleotide exchange factors (NEFs). Hence, NEFs are important co-factors in HSP70 complex. To days, most reports about NEFs come from the studies of GrpE in procaryotic cells, Fes1p and Sse1p in yeast cells as well as Bag-1 and HspBP1 in animal cells. NEFs in plants have rarely been reported, up to present. So, we performed a comprehensive survey to find Fes1p homologues in the Arabidopsis thaliana genome.The homologues of Fes1p in Arabidopsis have three members, all of which belong to the ARM (armadillo repeat) super family. The three Arabidopsis Fes1p homologues were named AtFes1A (At3g09350), AtFes1B (At3g53800) and AtFes1C (At5g02150). Among the three members, AtFes1A is the most abundant heat-response gene found by Real-Time PCR analysis. So we preferred to investigate the AtFes1A gene. We researched the expression character, intracellular location of AtFes1A。We found that AtFes1A was a heat induced protein, the location of AtFes1A was in the cytosolic.By ordering T-DNA insertion mutants salk-021784 and salk-012416 of AtFes1A from ABRC, we investigated the biologic function of AtFes1A. Finally, we pursued the firefly luciferase (Photinus pyralis), which was extremely sensitive to high temperature, for the primitive cause of heat sensitive in atfes1a mutants.The main research results were shown as follows:1. There were three homologus of Fes1 in Arabidopsis: AtFes1A, AtFes1B and AtFes1C. AtFes1A was the most abundant one of the three members. AtFes1A gene fused with the pYX242 vector was transferred into fes1△yeast, and the thermotolerance defect in fes1△yeast was complemented. This result implied that AtFes1A has a similar function to Fes1 in thermotolerance.To study the expression character of AtFes1A, we checked the AtFes1A protein level under heat shock, cold, drought, and NaCl treatment by western-blotting,the result represented that AtFes1A was strongly induced by heat shock, and was slightly induced by cold and drought. NaCl could not moudulate the expression of AtFes1A. By Agrobacterium tumefaciens-mediated method, a construct encoding the transit sequence of AtFes1A and GFP fusion protein was transfered into Arabidopsis, the expression assay of GFP fluorescence shows that GFP locates in cytosolic of Arabidopsis guarde cell and mesophyll protoplast from a transgenic Arabidopsis, indicating that the location of AtFes1A was cytoplasmic.We checked the effect of AtFes1A on the hydrolysis of HSP70 ATPase in steady-state, in order to verdict that whether AtFes1A could accelerate the strippring of ADP, by a Malachit Green and molybdic acid spectrophotometry and found that AtFes1A in high or low concentration had no influence on the steady-state activity of HSP70 ATPase.2. We identified two individual T-DNA insertion mutants (salk-021784 and salk-012416) of AtFes1A. To locate the T-DNA inserts at the AtFes1A genomic locus, we performed PCR analysis and sequenced the amplified fragments of PCR. These investigations revealed that salk-021784 and salk-012416 each had a T-DNA insertion in the sixth exon and fifth intron of At3g09350. No AtFes1A transcript and protein were detectable in the two homozygous mutants. Both of the two mutants showed visible heat sensitive phenotype in the acquired-thermotolerance assay during the hypocotyl enlongation and seedling period, as well as the seed germination. The complement assay of salk-021784 showed that the expression of AtFes1A could almostly repaire the heat sensitivity of salk-021784 mutant. These result sufficiently confirmed the fact that the acquired-thermotolerance defect in atfes1a is indeed the result of AtFes1A knock-out.3. In order to find out a possible reason for the defective acquired thermotolerance of atfes1a mutants, we measured the transcript levels of thermotolerance-related HSPs including HSP70, HSP101, sHSP class I and sHSP classII, and Hsfs including HsfA2 and HsfB1.. The Northern bolt showed that transcript amount of tested hsps and hsfs genes were higher in atfes1a mutant (salk-021784) than in the wild type. For the confirmation of Northern blot, we utilized the Real-time PCR to measure the transcripts in atfes1a mutants (salk-021784 and salk-012416). The results from Real-time PCR accorded with those from Northern blot. These investigations showed that the deletion of AtFes1A caused the increase of transcript levels in the heat tolerance related genes, hence, AtFes1A was a negative regulator of the HSPs gene signal transduction. The expression of HSPs was repressed by HSP70. But in the salk-021784 mutant, the HSP70 and other HSPs genes were up-regulated. The high expression of HSP70 did not act as a repressor. Hence, we evaluated the protein level of cytosolic HSP70 which was related to the thermotolerance in mutant salk-021784 and wild type Arabidopsis. The results showed that the HSP70 protein levels in a salk-021784 mutant did not correspond to the large accumulations of its transcripts, but were at levels lower than those seen for the wild type. This was the first report that AtFes1A were involved in the stability of HSP70.4. We checked the total protein ubiquitination status of salk-021784 mutant and wild type Arabidopsis after heat shock by western-blotting. The result indicated that more total protein were tagged with ubiqutin in atfes1a mutant (salk-021784) than in the wild type Arabidopsis, that was to say, there were more denatured substrates would be digested by protease.For deeper research of the possible mechanism about the heat sensitivity in atfes1a, we constructed a plant expression vector contained the firefly luciferase (Photinus pyralis) gene under the control of a heat shock promoter of LeHSP 23.8, and transferred the vector to the salk-021784 mutant and wild type Arabidopsis by the Agrobacterium tumefaciens-mediated method. The transformation plants were heated at 38℃for 2h to trigger the expression of luciferase, then heated at 45℃for 2h to denature the luciferase and then were shifted to room temperature for the recovery. During the recovery period, the activity of luciferase in afes1a mutant was slower than that in the wild type. Then, the level of soluble luciferase protein was measured using an anti-luciferase antibody. We found that less of luciferase protein in the atfes1a mutant existed in soluble, indicating that the lower activity of luciferase in atfes1a mutant was caused by the lower level of soluble luciferase. In addition, we evaluated the recovery of denatured luciferase in wheat germ extract with or without AtFes1A protein. The results presented that AtFes1A did not influenced the recovery of luciferase, indicating that AtFes1A had no effect in chaperone machine in vitro.In brief, AtFes1A is a novel gene involved in plant thermotolerance, by the way of keeping the stability of cytosolic HSP70 as well as modulating the denatured substrate. The innovations of this thesis were shown as follows:It was the first research to illustrate that the Arabidopsis AtFes1A was involved in plant thermotolerance. The location of AtFes1A, the effect of AtFes1A on the stability of HSP70, the modulation of AtFes1A on heat shock protein signal transduction as well as the modulation of AtFes1A on the renaturation of denatured substrates were firstly investigated in this dissertation.