Genome-wide identification and evolution of the SAP gene family in sunflower (Helianthus annuus L.) and expression analysis under salt and drought stress

Identification of HaSAP gene family members and analysis of their physicochemical properties

The genomic data of A. officinalis, O. sativa, Z. mays, A. thaliana, B. rapa, C. lanatus, S. lycopersicum V. vinifera, and H. annuus were downloaded from Ensembl Plants (http://plants.ensembl.org/index.html). A. lappa, C. endivia, and C. intybus genomic and proteomic data were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/). C. morifolium genomic and proteomic data were downloaded from the Chrysanthemum Genome Database (http://www.amwayabrc.com/zh-cn/). C. tinctorius genomic and proteomic data were downloaded from the NGINX database (http://118.24.202.236:11010/filedown/). The hidden Markov model (HMM) analysis of the SAP gene domain (PF01754 and PF01428) in HMMER 3.2.1 software (http://HMMER.org/) was used to identify SAP protein sequences (Potter Simon, Aurélien & Eddy Sean, 2018). The CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) database was used to determine whether the candidate sequences had full A20 and/or AN1 functional domains, and the sequences were finalized for subsequent analysis and named according to their chromosomal order (Shennan, Jiyao & Farideh, 2020). The physicochemical properties of the HaSAP proteins were predicted using the Swiss Bioinformatics Resource Portal, ExPASy (https://web.ExPASy.org/protparam/; Julien, Davide & Alessandra, 2018).

Phylogenetic analysis of HaSAP

Using the ClustalW default settings in MEGA 7 software, A. officinalis, O. sativa, Z. mays, A. thaliana, B. rapa, C. lanatus, S. lycopersicum, V. vinifera, H. annuus, A. lappa, C. endivia, C. intybus, C. morifolium, L. sativa var. angustata, and C. tinctorius SAP protein sequences were aligned with multiple sequences. A phylogenetic tree was constructed using the neighbor-joining (NJ) method (the bootstrap method value was set to 1,000 and the remaining parameters were set to default values; Sudhir, Glen & Koichiro, 2016) and the EvolView website (https://www.evolgenius.info/evolview/).

Gene structure and motif analysis of the SAP gene family in Compositae

The motifs of the candidate genes were analyzed using the MEME website (https://meme-suite.org/meme/), with the number of functional domains set to 10. Gene structure information was extracted from the genome database GFF3 file. The xml file obtained from MEME, the nwk file of the evolutionary tree, and the GFF3 file of the gene structure were processed and visualized using TBtools software (Chen et al., 2023).

Analysis of HaSAP cis-acting elements

The 2000 bp sequence upstream of the HaSAP start codon was selected and submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict the cis-acting elements of the gene promoter region, which were then processed and visualized using TBtools software (Chen et al., 2023).

RNA-seq analysis

The transcriptome sequencing data of sunflower plants under salt stress were downloaded from the NCBI database (PRJNA866668). The raw data were filtered and quality controlled using fastp software (Chen et al., 2018). The filtered data were then aligned with the sunflower genome using hisat2 software, and expression quantification was performed using StringTie software (Pertea et al., 2016). Gene expression levels were quantified using the fragments per kilobase of transcript sequence per million mapped reads (FPKM) method. To visualize the expression patterns of the genes, an expression heatmap was generated using TBtools software (Chen et al., 2023).

Plant material

The plant material used in this study was the sunflower salt-tolerant inbred line 19S05, which was bred by the Bayannur Institute of Agricultural and Animal Sciences. This particular inbred line was chosen based on its demonstrated tolerance to high salt conditions. To prepare the plant material, the completed 19S05 seeds were washed with water and then disinfected with 3% hydrogen peroxide for 10 min. The plants were subsequently placed in plastic jars filled with disinfected soil. The incubation chamber maintained a temperature of 24 (±2) °C and a humidity level of 60–70%. The plants were exposed to 16 h of light with an intensity of 16,000 lx, followed by 8 h of darkness. An incandescent lamp was used for illumination. After germination, the first pair of true leaves was transplanted into the ordinary hydroponic solution once the leaves were fully expanded. The Hoagland nutrient solution was replaced every 2 days. When the plants reached the stage of having four true leaves, samples of the rhizome leaves were taken. For the salt stress treatment, the nutrient solution was replaced with a 200 mmol/L NaCl solution. Samples were taken at various time points after the treatment: 0, 1, 3, 6, 12, and 24 h. For drought stress, the nutrient solution was replaced with a 15% PEG6000 solution, and samples were taken at the same time points as the salt stress treatment. Each sample was biologically replicated in triplicate, and the samples were flash frozen with liquid nitrogen and stored at −80 °C to preserve the biological material for subsequent analysis.

qRT–PCR analysis

The FastPure Plant Total RNA Isolation Kit (Novozan Biotechnology Co., Ltd., Nanjing, China) was used to isolate total RNA. After RNA isolation, cDNA synthesis was performed using PrimeScript Reverse Transcriptase (TaKaRa, Dalian, China). For quantitative real-time PCR, primers were designed using Primer Premier 5.0 software. The primer sequences can be found in Table S1. The sunflower Actin gene was used as an internal control for normalization purposes. The qRT‒PCR was carried out using the ChamQ Universal SYBR qPCR Master Mix (Novozan Biotechnology Co., Ltd., Nanjing, China). The qRT‒PCR protocol involved an initial denaturation step at 95 °C for 30 s, followed by denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 20 s. This cycle was repeated for a total of 40 cycles. All PCRs were performed in triplicate to ensure the reliability of the results. The quantitative data obtained from qRT‒PCR were analyzed using the 2^−∆∆CT method (O’Connell et al., 2017). Excel 2010 was also used for statistical analysis of the qRT-PCR data. The R language (version 4.2.3) ggplot2 software package was used for data visualization. Additionally, the ggpubr software package was used to perform a t-test on the data, specifically comparing the samples before 0 h (CK) and after stress, and the significance level was calculated to determine if there were statistically significant differences in gene expression between the control and stress conditions.

Identification of the SAP gene family in sunflowers

To study changes in the copy number of the SAP gene family during the evolution of Compositae plants, the SAP genes were comprehensively searched from the genomes of seven Compositae species (H. annuus, A. lappa, C. endivia, C. intybus, C. morifolium, L. sativa var. angustata, and C. tinctorius) via hmmsearch. The results of the search were verified in the NCBI-CDD database. A total of 27, 17, 19, 14, 18, 15, and 11 SAP sequences were identified, respectively. The 27 sunflower sequences on the chromosome were named HaSAP01–HaSAP27 (Table 1). The open reading frame (OFR) length of the HaSAP family gene ranged from 402 to 873 bp. The encoded protein contained between 133 and 290 amino acid residues, with a relative molecular weight ranging from 14.89 to 31.74 kDa. The theoretical isoelectric point of these proteins ranged from 7.85 to 9.71. In terms of genomic distribution, 27 HaSAP genes were found on 14 chromosomes of sunflower (chr1, chr3, chr4, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr16, and chr17). Among those chromosomes, chr11 contained the highest number of HaSAP genes, with six genes. Chr14 contained four HaSAP genes, while chr1, chr7, chr8, chr10, and chr13 each contained two HaSAP genes. The remaining chromosomes (chr3, chr4, chr6, chr9, chr12, chr16, and chr17) each contained one HaSAP gene.

Phytogenetic analysis of the SAP gene family in sunflower

To better understand the evolutionary relationship of sunflower SAP family genes, a phylogenetic tree was constructed based on 238 SAP protein sequences from seven Compositae species (H. annuus, A. lappa, C. endivia, C. intybus, C. morifolium, L. sativa var. Angustata, and C. tinctorius), as well as eight other plant species (A. officinalis, O. sativa, Z. mays, A. thaliana, B. rapa, C. lanatus, S. lycopersicum, V. vinifera). The evolutionary tree was divided into three distinct groups (Fig. 1A). Group 1 consisted of 72 SAP genes, with C. endivia and B. rapa having the highest number of SAP genes (8 and 7, respectively). A. thaliana, C. morifolium, and H. annuus each had six SAP genes, while Z. mays only had two SAP genes (Fig. 1B). Group 2 contained a total of 68 SAP genes, with B. rapa having the highest number (15), while A. officinalis did not have any SAP genes. A. thaliana, A. lappa, and H. annuus each had six SAP genes. Group 3 consisted of 98 SAP genes, with H. annuus having the highest number (15), and A. thalianas having only two SAP genes. O. sativa and C. morifolium each contained nine SAP genes. Notably, B. rapa, despite having the highest number of SAP genes overall (29), was mainly distributed in Group 2. The phylogenetic tree results indicated that the SAP genes of the seven Compositae plants were more closely related within each branch. Additionally, the number of SAP genes in the other seven Compositae plants showed similarity, except for H. annuus in Group 3, which suggests that the SAP genes of H. annuus in Group 3 had undergone amplification.

The sunflower SAP gene was used as the core gene to explore collinear relationships of SAP genes between H. annuus and A. officinalis, O. sativa, Z. mays, A. thaliana, B. rapa, C. lanatus, S. lycopersicum, and V. vinifera (Fig. 2). H. annuus produced three pairs of collinearity genes with two SAP genes from A. officinalis, two pairs of collinearity genes with two SAP genes from A. thaliana, eight pairs of collinearity genes with six SAP genes from B. rapa, one pair of collinearity genes with one SAP gene from C. lanatus, seven pairs of collinearity genes with four SAP genes from O. sativa, six pairs of collinearity genes with two SAP genes from S. lycopersicum, seven pairs of collinearity genes with three SAP genes from V. vinifera, and 12 pairs of collinearity genes with two SAP genes from Z. mays. Although the most collinearity gene pairs were found between H. annuus SAP and Z. mays, only two ZmSAP genes (ZmSAP1 and ZmSAP4) were formed.

The collinear relationships of SAP genes were also analyzed for seven Compositae plants (H. annuus, A. lappa, C. endivia, C. intybus, C. morifolium, L. sativa var. Angustata, and C. tinctorius; Fig. 3). The H. annuus genome contains 16 collinearity SAP gene pairs. H. annuus produced 10 collinearity pairs with eight SAP genes from A. lappa, nine collinearity pairs with six SAP genes from C. endivia, 10 collinearity pairs with six SAP genes from C. intybus, 12 collinearity pairs with seven SAP genes from C. morifolium, six collinearity pairs with three SAP genes from C. tinctorius, and eight collinearity pairs with five SAP genes from L. sativa. H. annuus contained the most collinearity gene pairs with C. morifolium, and H. annuus and C. morifolium had the closest distance in the phylogenetic tree. The collinearity of SAP gene pairs between sunflower and other Compositae plants was not much different, and the number of branches in the phylogenetic tree was relatively similar, indicating that the SAP gene family was conserved in the evolution of Compositae plants.

Evolution, gene structure, and motif analysis of the Compositae species SAP gene family

A phylogenetic tree was created and gene structure and motif analyses were performed based on the full-length, CDS, and protein sequences of SAP genes in seven Compositae plants to further understand the composition and structure of sunflower SAP genes (Fig. 4). The phylogenetic tree revealed that the SAP genes from the seven Compositae plants could be divided into three subgroups. This subdivision in subgroups is consistent with similar research conducted on other plant species, indicating a certain level of evolutionary conservation within the SAP gene family. A motif is a structural component of a protein molecule with a specific spatial conformation and a specific function and is a subunit of the domain. Members of the same subgroup exhibited similar motifs. Groups 2 and 3 displayed the simplest gene structure and the least number of motifs. Except for CmSAP3, the SAP genes in Group 1 contained at least two exons and four motifs (motif 4, motif 5, motif 6, and motif 7). In Group 2, with a few exceptions (CiSAP1, CeSAP1, LsSAP1, HaSAP3, HaSAP6, HaSAP9, and HaSAP24), the SAP genes contained one exon and four motifs (motif 1, motif 3, motif 4, and motif 9). In Group 3, excluding a few genes that contained two exons (HaSAP7, HaSAP11, HaSAP17, CmSAP5, CmSAP11, CmSAP15, CmSAP18, CeSAP6, CeSAP17, CiSAP7, LsSAP8, and AlSAP15), the remaining SAP genes had one exon and seven motifs (motif 1, motif 2, motif 3, motif 5, motif 8, motif 9, and motif 10). These findings suggest that the three subgroups of SAP genes may have undergone genetic structure changes during the evolutionary process, potentially resulting in different biological functions.

Analysis of promoter cis-acting elements of the SAP gene family in sunflower

The analysis of the 2000 bp upstream promoter region of the HaSAP gene provided insights into its potential functions. The cis-acting element analysis revealed an uneven distribution of cis-acting elements associated with stress and hormonal responses. Four cis-acting elements related to stress responses were identified; among them, the most abundant element was the anaerobic response element (ARE), which plays a crucial role in anaerobic induction. The majority of HaSAP genes (25 out of 27) contained at least one ARE. Additionally, HaSAP18 had eight low-temperature responsiveness (LTR) elements, suggesting its potential involvement in low-temperature tolerance. The promoter region of the HaSAP gene contained nine cis-acting elements associated with five hormones: auxin (TGA-element and AuxRR-core), abscisic acid (ABRE), MeJA (TGACG-motif and CGTCA-motif), gibberellin (P-box, GARE-motif and TATC-box), and salicylic acid (TCA-element; Fig. 5). These cis-acting elements suggest the HaSAP gene is involved in various hormone signaling pathways. Notably, the largest number of cis-acting elements was associated with abscisic acid responses, with a total of 64 elements identified, suggesting that the HaSAP gene family may primarily function through synergistic effects on ABA biosynthesis or signal transduction genes.

Analysis of the expression patterns of the SAP gene family in sunflower plants under salt stress

The expression patterns of HaSAP genes were investigated by analyzing RNA-seq data under salt stress conditions (Fig. 6). Out of all the HaSAP genes analyzed, a subset of 10 genes (HaSAP1, HaSAP3, HaSAP8, HaSAP10, HaSAP15, HaSAP16, HaSAP21, HaSAP22, HaSAP23, and HaSAP26) exhibited significant changes in expression in response to salt stress. These 10 genes showed specific induction upon exposure to high salt levels, while the remaining HaSAP genes either displayed minor changes in expression or remained relatively stable under salt stress conditions. This suggests that these 10 genes may be specifically involved in the plant’s response to salt stress. The expression levels of the five genes with the most significant changes (HaSAP3, HaSAP10, HaSAP15, HaSAP16, and HaSAP26) reached their minimums at 3 h. However, since RNA-seq data were only measured for a duration of 12 h, it is possible that the expression levels of these genes continued to change beyond the 12-h time point. These results showed that approximately one-third of the HaSAP family genes demonstrated significant changes in expression levels under salt stress conditions, indicating that the HaSAP family genes may play a role in the plant’s response to salt stress.

qRT-PCR analysis of candidate gene expression under drought and salt stress and tissue-specific expression

The qRT‒PCR analysis of the selected genes (HaSAP1, HaSAP3, HaSAP8, HaSAP10, HaSAP15, HaSAP16, HaSAP21, HaSAP22, HaSAP23, and HaSAP26) under salt stress conditions validated the findings of the expression analysis. The transcript levels of the selected genes were measured at different time points after stress treatment. Compared to the expression levels at 0 h under salt stress, significant changes in transcript levels were observed for all ten genes (Fig. 7). Four genes (HaSAP1, HaSAP3, HaSAP16, and HaSAP26) showed a significant decrease in expression after salt stress, reaching their minimum levels at 6 or 12 h, while five genes (HaSAP8, HaSAP10, HaSAP21, HaSAP22, and HaSAP23) exhibited a significant increase in expression after salt stress, reaching their maximum levels at 1, 6, or 12 h. The expression of HaSAP15 under salt stress was particularly notable as it showed a significant increase after 1 h of salt stress, followed by a significant decrease after 3 h, reaching its lowest expression level at 6 h.

The expression patterns of the 10 genes (HaSAP1, HaSAP3, HaSAP8, HaSAP10, HaSAP15, HaSAP16, HaSAP21, HaSAP22, HaSAP23, and HaSAP26) were further investigated under drought stress conditions using qRT‒PCR analysis (Fig. 8). Under drought stress, eight genes (HaSAP1, HaSAP3, HaSAP8, HaSAP15, HaSAP16, HaSAP21, HaSAP23, and HaSAP26) exhibited significant increases in transcript levels compared to their expression levels at 0 h, indicating that these eight genes were strongly induced at the transcriptional level in response to drought stress. Under salt stress, the expression levels of two genes (HaSAP1 and HaSAP8) significantly decreased, reaching their lowest levels at 6 h, while the expression levels of six genes (HaSAP3, HaSAP15, HaSAP16, HaSAP21, HaSAP23, and HaSAP26) increased significantly after salt stress. HaSAP1, HaSAP21, and HaSAP23 all displayed consistent expression patterns under both salt and drought stress conditions. These findings suggest that these three genes may play crucial roles in the mechanisms of salt tolerance and drought resistance in sunflowers.

Tissue-specific expression patterns of the 10 genes (HaSAP1, HaSAP3, HaSAP8, HaSAP10, HaSAP15, HaSAP16, HaSAP21, HaSAP22, HaSAP23, and HaSAP26) were also analyzed in the roots, stems, and leaves (Fig. 9). Six genes (HaSAP1, HaSAP3, HaSAP15, HaSAP16, HaSAP22, and HaSAP26) displayed significantly higher expression levels in the roots compared to levels in the stems and leaves, indicating that these genes are likely involved in root-specific functions, including some functions related to the plant’s response to salt stress. Conversely, the HaSAP8, HaSAP10, and HaSAP23 genes exhibited significantly higher expression levels in the leaves compared to levels in the roots and stems, indicating that these genes may play specific roles in leaf-related processes, including some processes associated with the plant’s response to drought stress. Notably, the HaSAP21 gene displayed the lowest expression in stems, and had similar expression levels in the roots and leaves. This suggests that HaSAP21 may have a more uniform expression pattern across different tissues than the other genes. The significant changes observed in the expression of HaSAP genes in plant leaves and roots after stress treatment likely contribute to improved drought and salt tolerance in sunflower plants. These findings highlight the importance of the HaSAP gene family in the stress tolerance traits of sunflower plants. These findings also provide a foundation for validating the functional roles of these genes and investigating the underlying molecular mechanisms involved in plant stress resistance.