Five-month-old plants of C. arabica cv. Catuaí Vermelho IAC 144 and Obatã IAC 1669-20 (4–5 leaf pairs) were used in this study. Plants were selected based on size uniformity and were transferred to pots containing 3 L of aerated nutrient solution (ANS), adapted from Clark (1975) by de Carvalho et al. (2013). The hexanoic acid experiment was carried out as described in Silva et al. (2020), under controlled temperature (23 ± 2°C) and light/dark cycle (12/12 h, photosynthetically active photon flux density of ~400 μmol.m⁻².s⁻¹). The following treatments were assessed: (a) ANS (control); (b) ANS + hexanoic acid (Merck, final concentration 0.55 mM) for 48 h. Plants were grown in 3 to 6 plastic pots in which three pots received each treatment. The experiments were repeated 3 times to obtain biological replicates. The potted plants were grouped in “pools” (made of 9–18 plants), which were considered a biological replicate. Three biological replicates were used. The mature leaves of the middle third of the plants were collected within the 3rd hour of the light period and stored at −80°C to further analyses.

Total RNA was extracted from leaves pools using the RNeasy Plant kit (Qiagen, Hilden, North Rhine-Westphalia, Germany). Total RNA samples were purified using the RNeasy Minielute Cleanup kit (Qiagen, Hilden, North Rhine-Westphalia, Germany). The purity of RNA was determined using a NanoDrop ND-100 spectrophotometer (Thermo Scientific, San Jose, CA, USA) and concentrations were obtained using Qubit fluorimeter (Thermo Fisher Scientific, Wilmington, DE, USA). Poly(A) RNA sequencing library was prepared following Illumina's TruSeq-stranded-mRNA sample preparation protocol (Illumina Technologies, SanDiego, CA). Paired-ended sequencing (2 X 150 bp) was performed on Illumina's NovaSeq 6000 sequencing system at LC Sciences (Houston, TX, USA). Data were deposited into the European Nucleotide Archive (ENA), submission ERA6282544 and in Zenodo (doi: 10.5281/zenodo.5517785).

Adaptor contamination, low quality bases and undetermined bases were removed by using Cutadapt (Martin, 2011) and in house PERL scripts. Sequence quality was verified using FastQC (Andrews, 2010). HISAT2 (Kim et al., 2015) was used to map reads to the C. arabica genome (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/003/713/225/GCF_003713225.1_Cara_1.0/.Mapped) reads were assembled using StringTie (Pertea et al., 2015). Table 1 shows the statistics of the transcriptome analysis. StringTie was also used to detect expression level for mRNAs by calculating FPKM. The differentially expressed genes (DEGs) were selected with log2 (fold change) >1 or log2 (fold change) < -1 and with statistical significance (p value < 0.05) by R package edgeR (Robinson et al., 2010). A second analysis was done on the differentially expressed mRNAs and only the ones with FPKM (ratio) ≥ 2 or FPKM (ratio) ≤ −2; coefficient of variation ≤ 30% and average FPKM ≥ 5. These genes are reported in Supplementary Table 1.

Sequence annotation and functional analysis were done for the DEGs by using Blast2GO (Conesa et al., 2005), at the OmicsBox (Götz et al., 2008) platform. Sequences were annotated by blasting nucleotide sequences against the NCBI NR database (BLASTX, evalue ≤ 1.10⁻⁵). A total of 57 and 63 DEGs were found in Catuai Vermelho and Obatã, respectively. Most DEGs correspond to upregulated genes in response to Hx, in both cultivars (86% Catuai Vermelho and 73% Obatã). Eight DEGs were commonly found in the Catuai Vermelho and Obatã cultivars (AAA-ATPase, auxin-induced protein (two transcripts), ferredoxin-NADP reductase, premnaspirodiene oxygenase-like, glutathionyl-hydroquinone reductase, flavonol 3-O-glucosyltransferase and phenylalanine ammonia-lyase). DEGs were analyzed by Gene Ontology and were functionally assigned to the relevant terms. Biological process and molecular function categorizations revealed that DEGs related to cellular, oxidation-reduction, organic substance, and primary metabolic process, as well as transferase and ion binding activity might play a dominant role in the leaf response to priming. Most of the DEGs have a role in plant defense, corroborating to our hypothesis. These data contribute to identify key genes differentially expressed in response to Hx as well as indicate pathways modulated by this eliciting agent.