Characterization of the unfolded protein response in arabidopsis thaliana

Abstract

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits an ER-to-nucleus signaling pathway known as the unfolded protein response (UPR) in eukaryotes. In yeast, Ire1p, a kinase/endoribonuclease in the ER membrane, plays as a key proximal sensor molecule in the UPR signaling. To elucidate the molecular mechanism involved in the unfolded protein response in plant, I isolated two cDNA homologs of IRE1 gene from Arabidopsis (AtIRE1a, AtIRE1b). The two IRE1 homologs were predicted to encode type I transmembrane proteins, which contain lumenal domain at N-terminal halves and a kinase/endoribonuclease domains at their C-terminal halves. The expressions of the two genes were detected in various organ tissues of the Arabidopsis plant in Northern blot analysis. Using GST-fused recombinant protein, it was demonstrated that the C-terminal half of the AtIre1a protein has in vitro autophosphorylation activity. However, I could not detect endoribonuclease activity of the AtIre1a protein when I used yeast HAC1 RNA as a substrate in vivo in transient expression using Arabidopsis protoplast. As an attempt to find the UPR-regulated genes in Arabidopsis by overall genome scale, I have performed the gene expression analysis using oligonucleotide genome array, which contains probes against more than 8,200 genes and 100 EST clusters for Arabidopsis thaliana. From the Northern analysis, optimized condition for the tunicamycin treatment to Arabidopsis seedlings were established, that is 5㎍/ml$ tunicamycin treatment for 4 hr. Through gene expression profiling experiments using 4 genechips for mock-treated control and 4 genechips for tunicamycin(Tm)-treated respectively, 32 genes, which occupies 0.4 % of the tested total genes, were revealed as tunicamycin-upregulated. Among them, ER chaperones such as BiPs, protein disulfide isomerases, calnexins, some of heat-shock proteins working in the ER lumen, calreticulins, and the genes involved in protein transport suc...