| Screening high temperature sensitive mutants is an effective way to explor the molecular mechanisms of plant thermotolerance. An increasing number of mutants have extended our understanding that plant heat shock response is a complex network, involving heat shock proteins, heat shock transcription factors, antioxidant system, hormones and calcium.Arabidopsis AtFes1 consists of three gene family: At3g09350 (AtFes1A), At3g53800 (AtFes1B) and At5g02150 (AtFes1C). Bioinformatics data indicate that At3g09350 gene encodes an armadillo / beta-catenin repeat protein which is induced by high-temperature, but its biological function is unknown yet.We obtained mutant lines Salk-021784 and Salk-072075 from the ABRC. Homozygous mutants were confirmed by using PCR and Western-blotting. Backcross experiment result indicates that Salk-021784 has only one locus of T-DNA insertion .We found that the mutants present a defection of acquired-thermotolerance in the 5d seedling. The phenotypic defection of Salk-021784 mutant were recovered by complementary experiment. These results indicate that absence of AtFes1A will lead to the defection of thermotolerance in Arabidopsis thaliana. To find the possible cause for defective acquired thermotolerance in AtFes1A, we determined the transcript levels and the expression profiles of some Hsps and Hsfs at different temperatures by Northern and Western. We found that the expression of Hsp70 in atfes1a mutants was more stronger, but its protein level was significantly weaker than the wild type. This indicate that Hsp70 was degradated under high temperature in the mutans, which may lead to the defection of thermotolerance in Arabidopsis.The interraction beteen AtFes1A and Hsp70 had been demonstrated. In this experiment we used a yeast two-hybrid system to further analysis the interraction between AtFes1A and Hsp70 . We found that the expression of Hsp70 in yeast was normal, but AtFes1A expressed with diversity immuno-hybridied bands, and the genetically modified yeast can not grow in the four defects medium.In addition, we constructed AtFes1A overexpression vector and transformed it into the wild type. We find that overexpression of AtFes1A have no effects on the accumulation of Hsp70 and Hsp101 transcripts and their corresponding proteins in Arabidopsis. |
