## 3 Results  
### Bioinformatic Analysis of AttPR10 Function  
The _Arabidopsis_ genome encodes more than 21 AtTPR proteins. These proteins are classified on the basis of their molecular structure and the presence or absence of the TPR domain and/or other motifs (Figure 1). Among the proteins, _AtTPR10_ (At3g04710) showed a peculiar structure with both TPR and ANK domains in a single polypeptide that is possibly involved in protein-protein interactions, protein assembly, and signal transduction [1; 30; 31; 32; 33]. The AtTPR10 protein is a 456 amino acid (aa) protein with seven N-terminal ANK domains (54-280 aa) and three C-terminal TPR domains (328-429 aa) (Figure 2). Figure 1: Schematic representation of the tetratricopeptide (TPR) repeat-containing proteins encoded by the _Arabidopsis_ genome. These proteins contain several copies of the TPR domain (orange boxes), along with other functional domains (blue boxes). Among them, TPR10 domains selected in this study are indicated as blue characters. The scale at the bottom indicates the number of amino acid residues in these proteins. To determine the function of AtTPR10 in plants, we investigated its mRNA expression under diverse abiotic stresses using microarray data (_Arabidopsis_ eFP Browser v2.0; [[http://bar.u-toronto.ca/efp2/Arabidopsis/Arabidopsis_eFPbrowser2.html](http://bar.u-toronto.ca/efp2/Arabidopsis/Arabidopsis_eFPbrowser2.html)]([http://bar.u-toronto.ca/efp2/Arabidopsis/Arabidopsis_eFPbrowser2.html](http://bar.u-toronto.ca/efp2/Arabidopsis/Arabidopsis_eFPbrowser2.html))). Results showed that the transcript level of _AtTPR10_ was specifically upregulated by heat stress (Figure 3a) but not by the other abiotic stresses, such as cold, salt, and drought. This prediction was confirmed by real-time polymerase chain reaction (RT-PCR), which revealed the upregulation of _AtTPR10_ transcripts by heat stress treatment at 37 \({}^{\circ}\)C and 45 \({}^{\circ}\)C (Figure 3b). Additionally, _AtTPR10_ expression was gradually induced by heat stress (37 \({}^{\circ}\)C) within 1 h after treatment (Figure 3c), suggesting that the functional role of the protein might be involved in heat resistance in plants. By contrast, _AtTPR10_ was non-responsive to other abiotic stresses, including cold, salt, and drought (Figure 3d). In addition to _AtTPR10_ expression analysis under heat shock treatment, we also examined the heat stability of the AtTPR10 protein. Recombinant AtTPR10 purified from _E. coli_ was treated with high temperatures (50 \({}^{\circ}\)C, and 60 \({}^{\circ}\)C) for 15 min and then centrifuged at 13,000\(\times\)_g_ for 20 min. The supernatant and pellet fractions were analyzed by SDS-PAGE, followed by Coomassieie brilliant blue staining. Most of the AtTPR10 protein showed high stability at all temperatures tested, unlike the malate dehydrogenase MDH used as a control (Figure 4). At 60 \({}^{\circ}\)C, more than 95% of the AtTPR10 protein was present in the supernatant fraction; by contrast, less than 2% of the MDH remained in the supernatant, while most of the MDH was precipitated by heat-induced aggregation (Figure 4a,b). These results strongly suggest that AtTPR10 is highly thermo-stable and its function possibly includes heat shock resistance in plant cells. Figure 2: Typical features of _AtTPR10_. (**a**) Schematic representation of AtTPR10 structure containing seven N-terminal ankyrin (ANK) repeats and three C-terminal TPR repeats. (**b**) Nucleotide and amino acid sequences of AtTPR10. The ANK repeats are indicated in blue, while the TPR domains are indicated in orange. Asterisk (*) indicates the stop codon. Figure 4: Stability of AtTPR10 under heat stress. (**a**) Heat stability analysis of AtTPR10 and malate dehydrogenase (MDH; control). Approximately 1 \(\upmu\)g each of AtTPR10 and MDH was incubated at 23 \({}^{\circ}\)C, 50 \({}^{\circ}\)C, and 60 \({}^{\circ}\)C for 15 min and then centrifuged at 13,000\(\times\)\(g\) for 20 min. The supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE, followed by Coomassie brilliant blue staining.