Identification of ABF/AREB gene family in tomato (Solanum lycopersicum L.) and functional analysis of ABF/AREB in response to ABA and abiotic stresses

Identification of the ABF/AREB family members in tomato

Firstly, the nine Arabidopsis thaliana ABF/AREB (AtABF1, AtABF2/AREB1, AtABF3, AtABF4/AREB2, AtDPBF1/ABI5, AtDPBF2, AtAREB3/DPBF3, AtDPBF4/EEL, and AtbZIP15) amino acid protein conserved sequences, downloaded from TAIR (https://www.arabidopsis.org/), were used to query the ABF/AREB subfamily members of tomato (Li et al., 2020). The members of ABF/AREB gene family were screened as candidate genes by homology comparison in the database of tomato gene testing (https://solgenomics.net/organism/Solanum_lycopersicum/genome). Secondly, an E-value of 1e-20 was used to reduce the false positive, and the PFAM database (http://pfam.xfam.org/) and SMART database (http://smart.embl-heidelberg.de/) were used to further verify the ABF/AREB protein domain of tomato. Candidate genes that did not contain a specific domain of the ABF/AREB gene (PF00170) were manually eliminated. The ABF/AREB gene family was represented by the remaining genes (Liu et al., 2019).

Tomato genome sequence and annotation information were downloaded by Ensemble plants-tomato genome database (http://plants.ensembl.org/index.html). The whole genome information (GFF3, FASTA, PEP, CDS) of tomato was sorted out by using TBtools (toolbox for ecological battle) v1.0985 software (Qu et al., 2021), and finally the whole genome information file of tomato ABF/AREB was screened out for mapping according to the gene ID of the identified members of tomato ABF/AREB gene family.

Characterization of ABF/AREB transcription factor in tomato

The chromosome position, amino acid length, molecular weight, isoelectric point, molecular formula, and other physical and chemical characteristics data were used to examine the tomato ABF/AREB protein sequence (https://web.expasy.org/protparam/). Online analysis of the tomato ABF/AREB transcription factor subcellular location prediction was done using WoLFPSORT (https://wolfpsort.hgc.jp/). The secondary structure of tomato ABF/AREB family proteins were examined using the online website prabi (http://www.prabi.fr/) and the associated data was exported and imaged (Ataee et al., 2022).

Conserved motifs and protein conserved domain analysis

MEME (http://meme-suite.org/tools/meme) was used to input all protein sequences of tomato ABF/AREB online program to analyze tomato ABF/AREB transcription factor family conserved motifs (Chen et al., 2018). The number of predicted motifs was set to 10, while the other parameters were set as default. The multiple sequence alignment of tomato family was done through ClustalX and GeneDox software.

Phylogenetic tree and cis-acting elements analysis

ABF/AREB protein sequences of Arabidopsis thaliana, Solanum tuberosum, and Populus orientalis were obtained from TAIR (https://www.arabidopsis.org/). Plant Gene Database (https://phytozome.jgi.doe.gov) (Liu et al., 2019) and article (Yong et al., 2021) (File S1). Phylogenetic trees were constructed using Mega 7.0 software. A phylogenetic tree of 33 ABF/AREB protein sequences was constructed by neighbor-joining method (Bootstrap parameter was set to 1,000) (Rusinko & McPartlon, 2017). In addition, the evolutionary tree was beautified using EvolView (https://evolgenius.info//evolview-v2/#login) website. A DNA sequence of 2,000 bp upstream of the tomato ABF/AREB gene was obtained from the genome-wide information of tomato ABF/AREB and submitted to PlantCare online database (http://bioinformatics.psb.ugent.be/) for analysis. The cis-acting element analysis results of PlantCARE website were deleted and integrated, and TBtools software was used for analysis (Zhao et al., 2023).

Tissue expression analysis of ABF/AREB gene in tomato

The IDs of the SlABF genes were searched in the eFP (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) database. Then, the data were sorted out and the expression patterns of SlABF in different tissues were drawn by TBtools (Kim et al., 2013).

Gene location, Ka (nonsynonymous)/Ks (synonymous) analysis and gene structure analysis

The GFF3 file in the whole genome information of tomato ABF/AREB was visualized and analyzed by TBtools software. ABF/AREB gene members were renamed according to their chromosomal distribution (Duan et al., 2022). The CDS sequences of tomato ABF/AREB genes were further used to calculate the relationship between Ka (non-synonymous substitution rate) and Ks (synonymous substitution rate) among family members by calculating Ka and Ks values by Simple Ka/Ks calculator (NG) of TBtools software (Liu et al., 2022b). The exon-intron structure distribution of ABF/AREB gene in tomato was analyzed by using the GFF3 file of ABF/AREB genome-wide information with TBtools software (Chen et al., 2020).

Transcriptional analysis of ABF/AREB gene in tomato under different abiotic stresses and hormone treatments

RNA extraction and quantitative qRT-PCR

Total RNA was extracted from the samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) Take advantage of FastQuant First Strand cDNA Synthesis Kit (Tianen, Beijing, China) to synthesize cDNA. These reactions were executed under the following conditions: 37 °C for 15 min, 85 °C for 5 s, and finally ended at 4 °C. LightCycler 480 Real-Time PCR System (Roche Applied Science, Penzberg, Germany) and SYBR Green Premix Pro Taq HS Premix kit was used for qRT-PCR. The reaction system was 2×SYBR Green Pro Taq HS Premix 10 μL, primer F 0.4 μL, primer R 0.4 μL, cDNA 2 μL, ddH₂O 7.2 μL. The primers used in qRT-PCR were designed with Primer 5.0, and the internal reference was Actin (NC 015447.3) as shown in Table 1.

Data statistics and analysis

The data were analyzed using the 2^−∆∆Ct calculation method. GraphPad Prism software was used for statistical analysis (Schmittgen & Livak, 2008). ANOVA was used to detect the significant level of difference between different times or different treatments (Bertinetto, Engel & Jansen, 2020) (File S2).

Identifification of ABF/AREB genes in tomato

Ten tomatoes ABF/AREB genes were obtained by homologous alignment, which were named SlABF1-SlABF10 according to the location of the genes on the different chromosomes (Table 2). The tomato ABF/AREB transcription factor family is unevenly distributed on five chromosomes of tomato. Among them, SlABF1, SlABF2 and SlABF3 are located on Chr-01, SlABF4 and SlABF5 are located on Chr-04, SlABF6 is located on Chr-09. SlABF7, SlABF8 and SlABF9 are distributed on Chr-10 and SlABF10 is located on Chr-11 (Fig. 1). To investigate the selection pressure during the evolution of ABF/AREB genes, we calculated Ka/Ks values of tomato ABF/AREB (File S3). The results showed that three pairs of replication genes (SlABF7/SlABF9, SlABF7/SlABF10 and SlABF8/SlABF9) in the tomato ABF/AREB family had Ka/Ks ratios less than one (between 0.24 and 0.30), with two pairs of tandem repeats and one pair of fragment repeats. This indicates that the tomato ABF/AREB gene family underwent purifying selection after the replication event. A Ka/Ks value of less than one implies purifying selection, Ka/Ks = 1 represents neutral selection and Ka/Ks >1 indicates positive selection (Yong et al., 2021).

The amino acid size of tomato ABF/AREB transcription factor family is between 146 aa (SlABF1) and 447 aa (SlABF5). The molecular weight is between 16,689.92 and 47,977.73 kDa. The isoelectric point (pI) is between 6.41 (SlABF5) and 9.72 (SlABF4). In the ABF/AREB family, only SlABF4 and SlABF8 are acidic proteins (pI < 7), and the rest are alkalescent (pI > 7) (Table 2). The instability index is greater than 40 for all 10 tomato genes, showing that the ABF/AREB genes are unstable proteins. From the perspective of subcellular location analysis, SlABF1-S1ABF10 are all expressed in the nucleus, which speculate that the gene is related to the storage and replication of genetic material. SlABF2 is expressed in chloroplasts, suggesting that SlABF2 may be involved in photosynthesis.

Conserved domain and conserved motifs of tomato ABF/AREB family

ABF/AREB has a highly conserved protein structure including four conserved phosphorylation sites, three conserved domains of C1, C2 and C3 at the N-terminus, and a highly conserved domain of C4 at the C-terminus (Fujita et al., 2005). The tomato ABF/AREB protein has four conserved phosphorylation sites. The N-terminal is made up of C1, C2, and C3, whereas the C-terminal is made up of C4 and the bZIP region (basic region and leucine zipper). The C-terminus of tomato ABF/AREB proteins has the unique BRLZ domain of bZIP transcription factor, which has the function of recognizing and binding specific DNA sequence (Fig. 2).

In this study, 10 conserved motifs are found in the tomato ABF/AREB proteins (Fig. 3). Sequence information for the identified conserved motifs is presented in Table 3, and the amino acid sequences of different conserved motifs are shown by the stack of letters at each position (File S4). The length of each motif is between 10 and 50 amino acids. The results show that the 10 identified tomato ABF/AREB motifs are quite similar. Motif1 is the basal core of the bZIP domain. Motif5 and Motif4 constitute the C1 conserved phosphate site. Motif3, Motif2, and Motif6 constitute the C2, C3, and C4 conserved phosphate sites, respectively. Both Motif1 and Motif2 are presented in all tomato ABF/AREB proteins. Both Motif3 and Motif4 are presented in nine tomato ABF/AREB proteins except for SlABF1. Except for SlABF1 and SlABF10, the other eight tomato ABF/AREB proteins contain Motif6. Motif7 and Motif9 are in four tomato ABF/AREB proteins (SlABF3, SlABF5, SlABF7 and SlABF10). Motif10 occurs in SlABF3, SlABF5 and SlABF7. Motif8 occurs in SlABF8 and SlABF9. It can be inferred that the tomato ABF/AREB members are highly conservative and may have similar functions.

Phylogenetic analyses of the tomato ABF/AREB families

The 33 ABF/AREB (10 SlABFs, 9 AtABFs, 7 StABFs and 7 PdABFs) proteins are divided into two subfamilies (Group A and Group B) (Fig. 4). Among them, SlABF3, SlABF5, SlABF7 and SlABF10 belong to Group A. They have the highest homology with StAREB1, StAREB2, StAREB3 and StAREB4, respectively. SlABF1, SlABF2, SlABF4, SlABF6, SlABF8 and SlABF9 belong to Group B. SlABF6 is more closely related to StABI5. SlABF8 is more closely related to StABL2, and SlABF9 is more closely related to StABL1. SlABF2 and SlABF4 are closely related to AtDPBF2. It can be concluded from the entire evolutionary tree that SlABFs have the highest homology with StABFs, relatively low homology with AtABFs, and the lowest homology with PdABFs.

Analysis of the gene structure of tomato ABF/AREB family

By analyzing the phylogenetic tree and gene structure of the tomato ABF/AREB gene family, the overall number of introns and exons of the 10 tomato ABF/AREB genes had no significant difference, all ranging from 1 to 4. Tomato ABF/AREB genes are divided into two groups. SlABF3, SlABF5, SlABF6, SlABF7 and SlABF10 are divided into ClASS I, and SlABF1, SlABF2, SlABF4, SlABF8 and SlABF9 are divided into ClASS II (Fig. 5). We found that the number of exons in tomato ABF/AREB is between two and four. The number of introns is between one and four. Specifically, in ClASS I, SlABF5, SlABF6, and SlABF10 contain four introns and four exons, SlABF7 possesses three introns and three exons, SlABF3 contains one intron and two exons. Both SlABF2 and SlABF4 in ClASS II have three introns and four exons. There are three exons in SlABF1, SlABF8 and SlABF9, and two introns in SlABF1 and SlABF9. SlABF8 has four introns. In general, the genetic structures of different tomato ABF/AREB members are relatively similar. Interestingly, except for SlABF1 and SlABF3, the other eight genes have similar exon lengths. Thus, the function of the tomato ABF/AREB genes may also be relatively similar.

Analysis of protein secondary structure of tomato ABF/AREB family genes

The most abundant protein secondary structures within tomato ABF/AREB members are mainly alpha helix and random coil (Table 4). The 10 ABF/AREB encoded proteins have alpha helix (29.31–50.68%), extended strand (4.79–13.49%), beta turn (1.01–3.14%), and random coil (43.15–59.26%) as their secondary protein structures (File S5).

Table 4:

The secondary structure of ABF/AREB gene family protein sequence in tomato.

Blue indicates alpha helix; green indicates beta turn; red indicates extended strand; purple indicates random coil.

Protein	Alpha helix (%)	Extended strand (%)	Beta turn (%)	Random coil (%)	Distribution of secondary structure elements
SlABF1	50.68	4.79	1.37	43.15
SlABF2	40.07	7.07	1.01	51.85
SlABF3	31.40	11.59	3.14	53.86
SlABF4	44.48	4.87	1.62	49.03
SlABF5	29.31	10.07	1.57	59.06
SlABF6	34.51	6.81	1.41	57.28
SlABF7	32.54	13.49	2.91	51.06
SlABF8	33.95	5.25	1.54	59.26
SlABF9	34.29	7.14	1.43	57.14
SlABF10	32.88	9.04	1.92	56.16

DOI: 10.7717/peerj.15310/table-4

Analysis of cis-acting elements of tomato ABF/AREB family genes

The tomato ABF/AREB genes include a total of 18 homeopathic components (Fig. 6 and File S6). Among them, three elements (AE-box, GATA-motif, MRE) are related to light response, seven elements (ABRE, CGTCA-motif, GARE-motif, P-box, TGACG-motif, TCA-element, TATC-box) are related to hormone response, and four elements (ARE, LTR, MBS, TC-rich repeats) are related to stress response (Table 5). In order to further study cis-elements in the ABF/AREB promoter sequences, three main types of cis-acting elements are identified, including light, hormones, and stress response elements (Fig. 7). AE-box element is mainly distributed in SlABF9. ARE element is mainly distributed in SlABF8. ABRE is all tomato ABF/AREBgenes, with the exception of SlABF7 and SlABF8, and was prevalent in SlABF10. Both CGTCA-motif and TGACG-motif elements are mainly distributed in SlABF3 and SlABF7. Generally speaking, the cis-elements correlated to hormone is relatively more abundant, which manifesting that the tomato ABF/AREBs gene plays a vital role in regulating hormone response.

Tissue-specific expression pattern of tomato ABF/AREB genes

In order to investigate the expression of the ABF/AREB gene in various tomato tissues during various growth stages. The expression of ABF/AREB genes in 14 tomato tissues is analyzed, including unopened flower bud, fully opened flower, leaf, root, 1 cm fruit, 2 cm fruit, 3 cm fruit, mature green fruit, breaker fruit, breaker fruit + 10, pimpinellifolium immature, green fruit, pimpinellifolium breaker fruit, pimpinellifolium breaker + 5 fruit and pimpinellifolium leaf (Fig. 8). Some SlABFs, including SlABF2, SlABF3, and SlABF10, are highly expressed in all tissues. In contrast, SlABF6 and SlABF7 are expressed at low levels in all tissues. The expression level of SlABF5 in roots is much higher than that in other tissues. The expression of SlABF9 is higher in pimpinellifolium leaf, but lower in other tissues. In addition, SlABF1, SlABF4 and SlABF8 also show similar expression patterns.

Expression profles analysis ABF/AREB genes in tomato under ABA and FLD treatment

The relative expression of SlABF1, SlABF2, SlABF3, SlABF4, SlABF5, SlABF8, SlABF9 and SlABF10 is significantly up-regulated under ABA and FLD treatments (Fig. 9). SlABF6 expression decreases after 12 h of ABA treatment and then increases gradually. There is a downward trend under the treatment of FLD. SlABF7 is up-regulated by ABA treatment, but increases first and then decreases under FLD treatment. Seven genes (SlABF1, SlABF2, SlABF5, SlABF6, SlABF7, SlABF9, and SlABF10) have higher relative expression levels under ABA than under FLD treatment. When compared to FLD treatment, the relative expression of SlABF3 and SlABF8 under ABA treatment at 12 h is marginally greater, whereas at 24 h, it was marginally lower. At 12 h, the relative expression of SlABF4 is higher in FLD treatment than in ABA treatment, while, at 24 h, it is lower in FLD treatment than in ABA treatment.

Expression profles analysis of ABF/AREB genes in tomato under NaCl, UV, cold and PEG treatments

In order to clarify the role of ABF/AREB in tomato under abiotic stress, the expression levels of 10 ABF/AREB genes in tomato under NaCl, UV, cold and PEG treatments were studied. As shown in Fig. 10A, the relative expression levels of 10 ABF/AREB genes in tomato are different under NaCl treatment. SlABF6 and SlABF7 expression levels is decreased by NaCl and cold treatments (Figs. 10A and 10C). In contrast, the expression of SlABF3, SlABF5 and SlABF10 is upregulated by NaCl and cold treatments. Moreover, SlABF8 is also significantly upregulated by cold stress. Under NaCl treatment, four genes (SlABF1, SlABF2, SlABF3, and SlABF4) reach the highest levels at 12 h, with SlABF3 showing the greatest change and increasing approximately 9.06-fold compared to 0 h. The expression levels of SlABF6, SlABF7 and SlABF9 decrease gradually with the increase of treatment time. The expression levels of the remaining three genes (SlABF5, SlABF8, and SlABF10) are highest at 24 h with NaCl treatment. Under cold treatment, the expression of the four genes (SlABF1, SlABF3, SlABF4 and SlABF5) gradually increases and reaches the highest level at 24 h. Compared to 0 h, it is increased by 2.26, 16.80, 3.59 and 10.56-folds, respectively.

The relative expression levels of SlABF6, SlABF7 and SlABF9 are significantly inhibited by UV treatment (Fig. 10B). After 12 h, the relative expression levels of SlABF2 and SlABF8 remain essentially unaltered. As the amount of time spent receiving UV treatment is extended, the relative expression levels of SlABF3, SlABF4 and SlABF5 steadily increase. SlABF3 has the largest change trend, increasing by over 38.50 times at 24 h compared to 0 h. After 12 h, SlABF1 and SlABF10 expression levels dramatically increase, and at 24 h, they marginally reduce.

Under PEG treatment, the relative expression levels of 10 tomato ABF/AREB genes have a similar trend: the expression levels all reach the highest at 24 h (Fig. 10D). The biggest changes are seen in the expression levels of SlABF1, SlABF3, and SlABF5, which are increased by (14.98, 110.16 and 11.13 times compared with 0 h, respectively). The expression levels of SlABF1 and SlABF4 decrease at first and then increase with the extension of treatment time.