*4.2. Microarray Analysis*

Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and RNA integrity was tested with the 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Transcriptome analyses were carried out according to Minimum Information About a Microarray Experiment (MIAME) guidelines. We used the Agilent *Arabidopsis* (V4) Gene Expression 4 × 44K Microarray in a one-color experimental design. The microarray contained 43,803 probes (60-mer oligonucleotides). Four biological replicates were analyzed for each treatment (time points 0 and 24 h at 22 ◦C and 24 h at 37 ◦C).

Half a μg of RNA was amplified and labeled with the Agilent Low Input Quick Amp Labeling Kit. To assess the labeling and hybridization efficiencies we used an Agilent Spike-In Kit. Hybridization and slide washing were performed with the Gene Expression Hybridization Kit (Agilent) and Gene Expression Wash Buffers (Agilent), respectively. Then, slides were scanned at 5 μm resolution in an Agilent G2565AA microarray scanner, and image files were analyzed with the Feature Extraction software 9.5.1. We used the GeneSpring 12.1 software (Agilent) to perform the interarray analyses. To ensure a high-quality data set we removed control features, and selected only features for which the 'IsWellAboveBG' parameter was one in at least three out of four biological replicates (31,921 features from 43,803). Our microarray data sets have been submitted to the Gene Expression Omnibus database (accession number: GSE116592).

A new gene annotation of probes in the microarray was carried out using BLASTN searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi), using the sequences of each probe as query against the Arabidopsis genome annotation in The Arabidopsis Information Resource (TAIR; www.arabidopsis. org), version 10. BLAST results for each probe were filtered with a minimum *<sup>E</sup>*-value of 9.9 × <sup>10</sup>−6, a minimum sequence identity of 98% between probe and transcript, and a minimum overlap of the 75% of the probe sequence length. Probes matching multiple genes were not considered. Results for this gene annotation are quite similar to those obtained in similar analyses performed by TAIR (ftp://ftp.arabidopsis.org/Microarrays/Agilent/).

### *4.3. Gene Overexpression/Repression Analysis*

For each probe and experimental condition (three conditions: 0 h at 22 ◦C, 24 h at 22 ◦C, and 24 h at 37 ◦C), expression levels were averaged across the four biological replicates. For those genes that mapped to more than one probe, expression levels were averaged across all probes. As a result, a single expression level was obtained for each gene and experimental condition.

For each gene with available probes (*n* = 20,491), the response (*R*) of its expression to heat stress was computed as:

$$R = \log\_2 \frac{E\_{24,37}}{E\_{24,22}}\tag{1}$$

where *E*24,37 is expression level at 37 ◦C at 24 h, and *E*24,22 is expression level at 22 ◦C at 24 h. *R* takes positive values for genes that are overexpressed at 37 ◦C compared to 22 ◦C, and negative values for those that are repressed.

#### *4.4. Protein and Gene Sequence Analysis*

All *Arabidopsis* protein sequences were obtained from Ensembl Plants [38] (assembly: TAIR10). For each gene encoding multiple proteins (alternative splicing isoforms), the longest protein was selected for analysis. For each protein, the frequency of each amino acid was computed by dividing the number of occurrences of the amino acid by the length of the protein. GC content of each gene was retrieved from Ensembl Plants' Biomart [38,39]. For each protein, the most likely subcellular location was retrieved from the SUBA4 database [40]. The consensus location was used. Only proteins located to a single compartment were used in compartment-specific analyses.
