**1. Introduction**

Proteins of thermophilic prokaryotes (those adapted to high temperatures) exhibit several distinctive features that increase their thermostability. One of the most consistent observations in thermophile proteins is an enrichment in salt bridges [1,2]. Salt bridges consist of electrostatic interactions among amino acid residues with positive (Lys and Arg) and negative (Glu and Asp) charges, and their contribution to increasing the stability of thermophilic bacteria was first proposed by Perutz and Raidt [3]. In addition, compared with proteins of mesophiles (adapted to intermediate temperatures) and psychrophiles (adapted to low temperatures), thermophile proteins tend to exhibit

more disulfide bonds and non-covalent interactions, including hydrogen bonds, and hydrophobic interactions, features that also tend to increase protein stability by linking together distant parts of the amino acid sequence [4,5]. These structural trends have an impact on the amino acid composition of thermophilic proteomes: the proteins of thermophilic bacteria tend to be enriched in charged amino acids and depleted in polar ones such as Ser, Thr, Asn, and Gln [6–12].

A few studies in prokaryotes have also shown that thermophile proteins are depleted in intrinsically disordered regions (IDRs), i.e., regions that lack a defined three-dimensional structure [13–15]. This observation is consistent with the fact that high temperatures induce disorder, but in contrast with the fact that IDRs confer thermoresistance [16–18].

Much less is known about how eukaryotic proteomes adapt to high temperatures. Some studies have suggested that the same biases in amino acid composition observed in thermophilic prokaryotes can be observed in thermophilic fungi (compared to other fungi; ref. [19]) and endothermic vertebrates (compared to ectothermic vertebrates; ref. [20]). In agreement with this notion, comparison of the orthologous proteins of two closely related fish, *Pachycara brachycephalum* (from Antarctica) and *Zoarces viviparous* (from a temperate zone) revealed an excess of Ser and a reduction of Glu and Asn in the cold-adapted species [21]. To our knowledge, the relationship between temperature and intrinsic disorder has not been investigated in eukaryotic proteomes.

Protein adaptation to high temperatures is expected to be observed not only in the proteins of thermophilic organisms, but also in some of the proteins of any mesophilic organism. When an organism is exposed to high temperatures, a subset of its proteins is overexpressed, whereas others are repressed (heat-induced and heat-repressed proteins, respectively, e.g., ref. [22]). As heat-induced function at relatively high temperatures, we hypothesize that they should be similar to those of thermophilic organisms.

Plants represent particularly suitable models to test this hypothesis, as they are sessile organisms that cannot escape from their environment, and they lack the effective thermoregulation mechanisms exhibited by homeotherms. Therefore, plants are expected to have developed adaptations to cope with heat stress [23]. To test our hypothesis, we grew *Arabidopsis thaliana* plants under normal (22 ◦C) and heat stress conditions (37 ◦C), and measured gene expression levels. Proteins overexpressed under heat stress were enriched in electrostatically charged amino acids and depleted in polar and hydrophobic amino acids. However, in contrast with our expectations, these proteins were also enriched in IDRs. These results indicate that *Arabidopsis* heat-induced proteins exploit some, but not all the same mechanisms as thermophile proteins to cope with high temperatures.

### **2. Results**
