Genome-wide identification of CaARR-Bs transcription factor gene family in pepper and their expression patterns under salinity stress

Identification of members of the CaARR-Bs gene family in pepper

Using The Arabidopsis Information Resource (TAIR) database (https://www.arabidopsis.org/), we were able to retrieve Arabidopsis thaliana structural domains for ARR-Bs genes. The obtained sequences were used as probes for homology searches using BLASTp (Wang et al., 2020). BLASTP searches selected the sequences of CaARR-Bs genes to the online pepper genomics database (https://solgenomics.net/organism/Capsicum_annuum/genome), where the genome database of the C. annuum cultivar Znula was selected (Qin et al., 2014). The search yielded pepper ARR-Bs candidate genes. The physicochemical properties of the ARR-Bs family genes were estimated using Expasy (http://web.expasy.org/) (Gasteiger et al., 2005). Finally, the subcellular localization analysis of the pepper CaARR-Bs gene family was performed using Cell-Ploc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/). The CaARR-Bs gene family protein sequences were aligned with A. thaliana ARR-B genes by MAFFT aligner (Katoh & Standley, 2013) using the embedded algorithms in Geneious Prime. Subsequently, the rooted phylogenetic trees were generated using the maximum likelihood methods (ML). The ML tree was constructed using FastTree V2 (Kumar et al., 2018), embedded in Geneious Prime.

Chromosomal location, gene structure, domain annotation, and secondary structure analysis

The chromosomal location of CaARR-Bs genes throughout the pepper genome was investigated using the MG2C V2 online tool (Chao et al., 2021). GSDS was used to identify CaARR-Bs gene exon-intron structures by comparing the genomic sequences vs cDNA sequences (http://gsds.cbi.pku.edu.cn/). The CaARR-Bs and the Myb DNA binding domains (a domain that distinguishes the ARR-A from ARR-B genes) were detected using CDD (Marchler-Bauer et al., 2015), SMART, pfam and InterProScan databases (Wang et al., 2022). SPOMA was used to predict CaARR-Bs secondary structures (Sapay, Guermeur & Deléage, 2006).

Potential cis-element analysis in promoter regions

A BLASTn search was conducted on the pepper genome using the cultivar Znula as the query sequence to identify CaARR-Bs genes. For each gene, a 1,500 bp upstream of the initiation codon was retrieved and analyzed for cis-elements using PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Plant materials and salinity stress treatment

Seeds of the pepper cultivar Gedeon F1 (Capsicum annuum L.; https://www.syngenta.com/en) were sterilized with 1% sodium hypochlorite for 30 min, washed with sterile water, and then sown in perlite beds at 28 °C (Qin et al., 2014). Seedlings were grown under 16 h/light at 25 °C and 8 h/night at 18 °C with a relative humidity of 60% until they had six leaves. Plants were irrigated with Hoagland solution at half-strength pH 5.6. Leaves and roots were harvested from the seedlings with three biological replicates as control. For the salt stress experiment, five-week-old plants were irrigated with 200 mM NaCl (Wang et al., 2022). After 12 and 24 h of treatment, each sample (3–4 leaves) was collected in three biological replicates. Liquid nitrogen was used to rapidly freeze samples, followed by −80 °C storage until RNA extraction.

RNA extraction and qPCR analysis

Total RNA was extracted from pepper tissues (leaves and roots) subjected to different salt stress treatments using the EasyPure^® Plant RNA Kit (TransGen Biotech, Beijing, China) following the instructions provided by the kit manufacturer. The quality and quantity of the extracted RNA were evaluated using electrophoresis on 2% agarose gels and a Quantus^™ Fluorometer (Promega, Madison, WI, USA). The cDNA synthesis was performed using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and adjusted to a concentration of 100 ng µL⁻¹. During the PCR amplification, a range of 108 to 147 base pairs was targeted while avoiding the conserved region (Table 1).

Quantitative real-time RT-PCR (qRT-PCR) was performed using TransStart^® Green qPCR SuperMix (TransGen Biotech, Beijing, China). The amplification reactions were performed following: 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 20 s, and 72 °C for 30 s. Melting curve analysis, performed by increasing the temperature from 55 to 95 °C (0.5 °C per 10 s), and a gel electrophoresis of the amplified fragments confirmed that the product contained single amplicons. In each experiment, the relative fold differences were calculated using the ^ΔΔ Ct. Normalization was conducted using GAPDH Ct values, amplified using 5′-ATGATGATGTGAAAGCAGCG-3′ and 5′-TTTCAACTGGTGGCTGCTAC-3′ as a reference gene (Arce-Rodríguez & Ochoa-Alejo, 2015). In this experiment, three biological replicates per sample were used. A statistical student’s t-test was used to compare gene expression levels.

Identification of the CaARR-Bs family genes members in pepper

The identification of the CaARR-Bs family genes in the C. annuum genome (cultivar Gedeon F1) based on the pepper genome sequences database was performed using BLASTp database search to query Arabidopsis thaliana ARR-Bs genes. In total, ten sequences were putative as pepper ARR-Bs genes (CaARRs). The chromosome location, exon number, and genomic and physiochemical characteristics of each gene are in Table 2. In the current study, the shortest putative open reading frame (ORF) was 1,374 pb, while the longest was 2,025 pb, with the amino acid length of CaARRs proteins ranging from 389 to 684 pb, a molecular weight (Mw) of 43.09 to 75.16 kDa, and a theoretical isoelectric point (PI) varying from 5.39 to 9.47. In addition, the presence of the ARR-like domain was verified using the Pfam database, and its position was unfixed among all copies, while all copies were localized at the subcellular level in the nucleus.

Alignment and phylogenetic analysis of CaARR-Bs

Phylogenetic analysis was conducted to confirm the identified CaARRs copies and determine the evolutionary affinity of the CaARR-Bs genes and ARR-Bs genes of Arabidopsis. With full-length amino acid sequences, the alignment of the ARR-BS domain was conducted and used to perform an unrooted phylogenetic tree with a bootstrap test. The analyzed ARR-Bs proteins were grouped into five distinct clusters. The CaARR-Bs proteins in pepper were clustered into four of the five subclusters with strong bootstrap support (Fig. 1). Functional divergence could have resulted from the presence or absence of species-specific CaARR-Bs.

Chromosomal location and duplication event of CaARR-Bs genes

As indicated by the starting and ending positions of CaARR-Bs genes on the chromosomes in Table 1, the genomic DNA sequences of each CaARR-Bs gene were mapped to the chromosomal location (Fig. 2). With few exceptions, CaARR-Bs genes were mainly found at the extremities of their respective chromosomes. The CaARR-Bs genes were asymmetrically distributed on chromosomes 1, 5, 6, 7, 9, 11, and 12, and none were found on chromosomes 2, 3, 4, 8, and 10. The CaARR1 and 2 genes were located on chromosome 1, CaARR3 and 4 were located on chromosome 5 with a separation of 10 Mb, and CaARR5 was on chromosome 6. In addition, CaARR6 and 7 located on chromosome 7 with a separation of <1 Mb, while chromosome 9 had CaARR8, chromosome 11 had CaARR9, and CaARR10 was in chromosome 12 (Fig. 2).

Predicted secondary structures of CaARR-Bs proteins

The prediction of the secondary structure of CaARR-Bs proteins was conducted. The primary constituent forms of the secondary structure of the pepper ARR-Bs protein, with α-helices, β-turns, and extended chains, were investigated and measured. According to structural predictions, pepper ARR-Bs gene family members contain α-helices, irregularly coiled, extended chains, and β-turns. The most average proportion for alpha-helices was around 34.1 ± 8%; the highest percentage was from CaARR6 (84.7%) and the lowest was from CaARR2 (26.3%). Most genes have averaged around 21.6 ± 4% β-turns; the highest percentage was from CaARR3 (26.3%) and the lowest was from CaARR5 (13.8%). The results show that the irregular coiling average record was 23.3 ± 6%, as the highest proportion was for CaARR7 (30.3%), while the lowest was for CaARR6 (13.7%; Table 3).

Gene structure of CaARR-Bs proteins

For each gene in the CaARR-Bs gene family, exon-intron distribution and conserved motifs were analyzed. For each gene, several isoforms were detected with the exception for CaARR2 and CaARRs 5–10 (unique isoform). The CaARR1 recorded two isoforms where one was complete and a shorter isoform (x₂: 1,431 bp and 477 aa). The CaARR3 recorded three isoforms: one complete and two shorter isoforms (x₂: 1,992 bp and 664 aa; x₃: 1,956 bp and 652 aa). Finally, the CaARR4 recorded five isoforms where one was complete, and five shorter isoforms (x₂: 1,785 bp and 595 aa; x₃: 1,896 bp and 632 aa; x₄: 1,899 bp and 633 aa; x₅: 1,908 bp and 636 aa).

There were differences in exonic and intronic regions between the 10 CaARR-Bs. Exons ranged from 5 to 11 while introns ranged from 4 to 10. The CaARR-Bs genes were clustered according to their sequence homology into three groups, showing similar gene structures within each group. Regardless of their chromosomal location, one cluster included CaARR6 and CaARR8, another included CaARR7, CaARR1, and CaARR5, and the other included CaARR2, CaARR4, and CaARR9 (Fig. 3).

Potential cis-element analysis in promoter regions of CaARR-Bs genes

To further characterize the potential regulatory mechanisms of CaARR-Bs, 1,500 bp upstream sequences from the translation start sites were analyzed to ensure the inclusion of potential enhancers (they can be located up to 1 Mbp away from the gene, but typically, they are found within a shorter distance, around 50–1,500 bp, upstream or downstream from the start site; Pennacchio et al., 2013). Based on their biological significance, cis-elements in CaARR-Bs were identified and categorized. The promoter sequences of 10 CaARR-Bs genes contained 146 possible cis-elements. Three main groups of cis-acting elements were identified: hormones, environmental stress, and photoresponses. The CaARR-Bs promoters included transcription factors binding sites related to abscisic acid responsiveness, auxin responsiveness, defense and stress responsiveness, endosperm expression, enhancer-like involved in anoxic specific inducibility, light responsiveness, low temperature responsiveness, meristem expression, zein metabolism regulation, MeJA responsiveness, Gibberellin responsiveness, ATBP 1 transcription factor, MYB binding site involved in drought inducibility, MYB binding site involved in flavonoid biosynthetic genes regulation, MYB binding site involved in light responsiveness, MYBHv1 binding site, and Salicylic acid responsiveness (Fig. 4). According to the cis-component, CaARR-Bs genes can respond to different abiotic stresses.

CaARR-Bs gene expression profiles in response to salinity stress

The expression level of the CaARR-Bs was measured with qRT-PCR for seedlings exposed to salt stress. The expression patterns of CaARR-Bs genes varied among stress levels and showed considerable variation in expression patterns of the CaARR-Bs genes over time in the roots of plants more than in leaves. A heat map was generated to show the expression profiles while the expression differences were color-indicated (Fig. 5). In response to salinity stress, the expression of patterns of CaARR2 and CaARR9 in the roots were up-regulated after 12 and 24 h; additionally, CaARR5 and CaARR6 were up-regulated in roots after 24 and 12 h, respectively. Meanwhile, the CaARR3, CaARR4, CaARR7, and CaARR8 were highly expressed in control samples and were down-regulated after 12 and 24 h of treatments. In the leaves, the expression level of CaARR1 was the only expressed CaARR gene, and only in control samples, followed by CaARR5, which showed slight expression after 24 h of salinity treatment. The expression of CaARR2, CaARR6, and CaARR9 increased slightly after 24 h of salinity treatment. The expression of CaARR3, CaARR4, CaARR7, and CaARR8 was down-regulated in 24 h. The expression level on the leaves of CaARR6, CaARR5, and CaARR67 was decreased, while CaARR3 was induced after 12 and 24 h of stress. The CaARR10 was the only copy that showed no amplification curves during the qPCR.