https://orcid.org/0000-0003-4268-8774
Figures
Abstract
Abiotic stressors, such as drought, salinity, and heavy metals, induce physiological changes, nutritional imbalances, molecular alterations, and oxidative stress in plants, which significantly reduce productivity. However, the secondary transporters, multidrug and toxic compound extrusion (MATE) proteins, transport substrates and metabolites. Accordingly, in response to abiotic stressors, these proteins strengthen plants’ immune systems, detoxify toxins, and enhance growth and development. Although the roles of MATE proteins responding to abiotic stresses have been investigated in several plants, their functions in sunflower have not yet been discovered. Therefore, this study identified 74 MATE proteins in sunflower (HanMATE) based on phylogenetic analysis, which were distributed into four subgroups. Their MATE-like properties were then validated using the domain, motif, gene structure, gene duplication, and physicochemical analysis. The HanMATE proteins in various cell organelles play a crucial role in abiotic stress tolerance, scavenging reactive oxygen species (ROS), and regulating transcription. Subsequently, Most HanMATE genes are enriched with biological processes and molecular functions that transport micro- and macro-molecules, drugs, negatively charged ions, organic anions, and citrate. The important Cis-regulatory elements (CREs), abscisic acid-, light-, and MeJA-responsive elements in HanMATE genes regulate plants’ growth and development in stress conditions. The synteny analysis indicated that 41 HanMATE proteins exhibit over 75% sequence similarity with 40 established stress-responsive (SR) MATE proteins from various plant species, suggesting their potential SR characteristics. Furthermore, this study identified 136 microRNAs linked to 58 HanMATE proteins, including 19 major hub microRNAs and 31 hub HanMATE proteins, which may enhance sunflower agronomic traits and abiotic stress resistance. The HanMATE proteins are conserved in other species that contribute to detoxification and have stable binding affinity with flavonoids and citric acid, validated from 3D structural modeling, molecular docking (MD), dynamic simulation, and functional prediction. These findings demonstrate that HanMATE genes are essential for sunflower abiotic stress tolerance (AST), and genetic engineering can be applied to develop more robust sunflower.
Citation: Hasan MN, Islam MR, Shahrier R, Hossen MB (2026) Genome-wide bioinformatics analysis of the MATE gene family for abiotic stress tolerance in sunflower (Helianthus annuus L.). PLoS One 21(4): e0346769. https://doi.org/10.1371/journal.pone.0346769
Editor: Shailender Kumar Verma, University of Delhi, INDIA
Received: April 3, 2025; Accepted: March 24, 2026; Published: April 13, 2026
Copyright: © 2026 Hasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The financial and technical support for this study was obtained from the Ministry of Science and Technology, Bangladesh, and the Department of Agricultural and Applied Statistics, Gazipur Agricultural University, Bangladesh. The Ministry of Science and Technology covered the costs for the study design, data collection, and analysis. The additional cost for further data analysis, technical assistance, and manuscript preparation was sourced from the Department of Agricultural and Applied Statistics, Gazipur Agricultural University, Bangladesh.
Competing interests: There are no conflicting interests that the authors need to disclose.
1. Introduction
Climate change brings about various environmental changes, which result in abiotic stresses in plants, including drought, salinity, heavy metal stress, and extreme temperatures [1,2]. Plants’ stationary nature significantly intensifies their vulnerability, affecting growth, reproduction, and productivity [3–5]. Among the abiotic stresses, drought occurs more frequently and with greater severity. It exposes plants to closing their stomata, reducing transpiration, limiting photosynthesis, causing nutrient imbalance, and enhancing ROS production in different cellular compartments, ultimately bringing the plant under salt stress [6–9]. High salinity, resulting from excessive concentrations of Na+ and Cl- in soil, causes high salinity and creates hyperosmotic and hyperionic conditions. These circumstances impede plant water and nutrient absorption, causing ionic and osmotic stresses, stomatal closure, limiting CO₂ uptake and photosynthesis, and oxidative stress [10–13]. Heavy metals, such as copper (Cu), cadmium (Cd), lead (Pb), iron (Fe), molybdenum (Mo), zinc (Zn), nickel (Ni), manganese (Mn), and cobalt (Co), are essential micronutrients for plant growth and development [14]. However, their excessive uptake can cause toxic effects, including inducing water-passive stomatal closure, impairing nutrient uptake channels, and excessive production of ROS in plants [15–22]. Furthermore, aluminum (AL) toxicity affects plants by inhibiting root growth and branching, binding to phosphorus, competing with essential cations, causing oxidative stress, and altering gene expression. It also affects signal transduction pathways, impacting plant responses to stress [23,24]. In conclusion, plants react to these stressors in comparable physiological and molecular ways, such as osmotic imbalance, cell dehydration, altered gene expression, and ROS generation [16,21–23,25,26].
The MATE proteins are secondary transporters primarily responsible for transporting many substrates, including chemical molecules, secondary metabolites, and phytohormones. Thus, MATE proteins detoxify internal and external toxins while simultaneously promoting the growth and development of plants. The MATE members are present in both prokaryotes and eukaryotes, and they are essential for multiple biological activities [27,28]. The MATE proteins comprise 400–700 amino acids and have a 40% sequence similarity among twelve transmembrane helices [27–29]. In mammals, MATE proteins were first identified in humans and mice. The SLC47A1 and SLC47A2 genes encode the human MATE1 and MATE2 proteins, which are mostly expressed in the kidney and liver [30–32]. Mammalian MATE proteins act as multispecific, electron-neutral organic cation transporters, facilitating the discharge of various organic cations and cationic therapeutics [33,34]. However, the MATE family is more diverse in plants than bacteria and animals [35]. In A. thaliana, the MATE gene AtDTX1 plays a crucial role in transporting and detoxifying alkaloids, antibiotics, and heavy metals such as Cd [36]. A mutation in AtDTX50 causes more abscisic acid (ABA) accumulation, upregulates many ABA marker genes, and is critical for ABA-mediated growth inhibition and drought stress responses [37]. In diploid cotton, GrMATE18/34/41/51 were considerably upregulated in response to stress from drought, salt, and Cd; these genes may be suitable candidates for breeders looking to produce more AST genotypes [38]. Khan et al. [39] discovered that HuMATE7/11/12/28 genes may aid in plant toxin detoxification from heavy metals and high soil salinity, paving the way for AST dragon fruit development. The cis-elements and expression pattern analysis identified GmMATE75 as a candidate gene for AL tolerance in soybean [40]. OsMATE4 and OsMATE9 are likely involved in the physiological process of transporting and detoxifying metal ions [41]. In potato, StMATE18/60/40/33/5 and StMATE33 were significantly upregulated by Cu2+ and Cd2+ stress, respectively, while StMATE59 was induced considerably by Ni2+ stress [42]. Therefore, MATE transporters play a crucial role in how plants respond to various abiotic stresses, including drought, salt, and heavy metal toxicity. Additionally, stress-resilient plants might be developed utilizing biotechnological approaches to MATE genes.
The sunflower is moderately abiotic stress-tolerant due to the special structure of its vital organs: the root, stem, leaves, and head [43,44]. Thereby, the abiotic stress tolerance of sunflower has been studied in many approaches, such as controlling specialized metabolites [44], counterbalancing oxidative stress and detoxifying enzymes [45,46], non-targeted metabolomics and proteomics have been used to profile a set of inbred lines and hybrid genotypes [47,48], biomarkers identification for drought tolerance [49], identification of hub transcription factors involved in drought stress response in sunflower [50], time-dependent transcriptome analysis to identify drought response mechanisms in sunflower [51], drought response genomic biomarker identification in leaves and roots [52,53], genomic, phenotypic, and physicochemical connections to understand the drought response mechanism in sunflower [54], and phenotypic and transcriptomic responses to cultivated sunflower in multiple abiotic stresses [55,56]. However, the abiotic stress tolerance properties of MATE genes in sunflower have not yet been studied, even though they have been discovered in many plants and shown to be SR. For example, there are 66 MATE genes in apple [57], 85 in pear [58], 56 in Arabidopsis, 46 in rice [36,59], 69 in citrus fruit [60], 138 in tobacco [61], 71 in Populus [62], 49 in maize [63], 67 in tomato [64], 35 in dragon fruit [39], 39 in melon [65], 128, 70, and 72 genes in Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii, respectively [66], and 64 in potato [42]. Therefore, in this study, we attempted to identify the MATE gene family and explore its role in AST in sunflower.
2. Materials and methods
This section was broadly classified into three categories: sequence and physicochemical data collection, identification of the HanMATE genes, and exploration of their roles in abiotic stress tolerance. A conceptual and working flowchart for identifying HanMATE genes and their predicted roles in AST in sunflower is visualized in Fig 1. Ethical approval is not applicable for this study.
The flowchart section (A) outlines the consequences of abiotic stresses on plants. The healthy and stressed plants of this section were obtained from Rani et al. 2021 [67]. Section (B) of the flowchart describes the identification techniques of HanMATE genes. In section (C), the identified HanMATE genes’ AST properties were characterized. In the final section (D), the future stress-tolerant sunflower was depicted; the image was obtained from https://www.dreamstime.com/illustration/sunflower-plant-growth-stages.html.
2.1. Sequence and physicochemical data collection of HanMATE genes
2.1.1. Sequence data collection.
To investigate the HanMATE genes, we used the sunflower (Helianthus annuus r1.2) Genome’s [68] gene, protein coding sequence (CDS), and proteome sequences, which were collected from the Phytozome Database [69] with Phytozome genome ID: 494, NCBI taxonomy ID: 4232, and website: https://phytozome-next.jgi.doe.gov/info/Hannuus_r1_2. The 46 AtMATE protein sequences that were downloaded from the Arabidopsis Information Resource (TAIR) database [70] (https://www.arabidopsis.org/) used as query sequences to explore the HanMATE gene, protein, and CDS sequences using the BLASTP [71] search. We mainly consider HanMATE genes that have an identity score > 30%, a bit score > 50, and an E-value < 10e − 10, as these provide a more trustworthy homology [72,73]. However, the sequence duplication was prevented considering the primary sequence exclusively.
2.1.2. Data collection on basic physicochemical properties.
We leveraged the Phytozome database to obtain the HanMATE genes’ genomic length, protein ID, CDS length, and encoded protein length. The ExPASy database was utilized to ascertain the significant protein sequences’ grand average of hydropathicity (GRAVY), isoelectric point (pI), protein length (aa), and molecular weight (MW) [74].
2.2. Identification of the HanMATE genes
2.2.1. Phylogenetic analysis.
This study used the integrated tools MEGA11 [75] and iTOL [76] to construct the phylogenetic tree. The ClustalW program aligned protein sequences for 74 HanMATE and 46 AtMATE members. The phylogenetic tree was then built using the neighbor-joining algorithm, with the bootstrap replicate value set at 1000.
2.2.2. Domain, motif, and gene structure analysis.
The conserved domain structure of the 74 HanMATE protein sequences was constructed using Pfam [77] and TBtools [78]. Motifs within the HanMATE proteins were identified using the MEME Suite [79] database (https://meme-suite.org/meme/tools/meme) and Tbtools, with the default settings of motif width between 6 and 50 (inclusive) and the maximum number of motifs being 10. Gene structure analysis was performed using the Gene Structure Display Server (GSDS) [80] (https://gsds.gao-lab.org/) and Tbtools.
2.3. Role of HanMATE genes in AST
2.3.1. Sub-cellular localization analysis.
The subcellular location of certain proteins regulates the biological functions of plant cells. In conjunction, a web-based application, WoLF PSORT [81] (https://wolfpsort.hgc.jp/), was employed to predict the likelihood of HanMATE proteins within cells’ membrane-bound compartments/organelles. However, the co-cluster analysis simultaneously clusters the row (HanMATE proteins) and column (organelles) entities of a data matrix. Consequently, HanMATE proteins and organelles cluster together based on the strength of their association [82]. Finally, the R package “rhcoclust” [83] was utilized to analyze the relationship between HanMATE proteins and cell organelles based on the WoLF PSORT data. The rhcoclust is a robust approach to hierarchical co-clustering [84] obtained from the logistic transformation of data, which converts the original data into a range from 0.00 to +1.00. However, this study customized this range into WoLF PSORT-generated data.
2.3.2. The gene ontology enrichment (GOE) analysis.
The GOE analysis of the 74 HanMATE genes to the biological processes, molecular functions, and cellular components was analyzed using the online platform agriGO v2.0 [85] (https://systemsbiology.cau.edu.cn/agriGOv2/).
2.3.3. CREs in the promoter regions of HanMATE genes.
The 1.8 kbp upstream sequence of the translation initiation codon of the HanMATE and abiotic SR (ASR) MATE genes in other plants (potato, soybean, rice, cotton, Arabidopsis, dragon fruit, wheat, and maize) was curated from the Phytozome database. The CREs in the 1.8 kbp upstream region were then predicted using the web tool PlantCARE [86] (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The frequency of presence of CREs in the 1.8 kbp upstream region of HanMATE and ASR MATE genes was visualized using the hierarchical co-clustering algorithm [82,83].
2.3.4. Syntenic relationship analysis of HanMATE genes with other plants’ ASR MATE genes.
The syntenic relationship of the ASR MATE genes from different plant species with the HanMATE gene was analyzed to predict the functional roles of HanMATE genes in AST. A Circos diagram between HanMATE and ASR MATE proteins was drawn with >75% homology, using the web tool Circoletto [87] (https://bat.infspire.org/circoletto/).
2.3.5. Regulatory Network with microRNAs (miRNAs).
In plants and animals, miRNAs are single-stranded noncoding RNA molecules of 19–24 nucleotides (nt). The miRNAs control plant growth, development, and stress response, regulating gene expression at the transcriptional and posttranscriptional levels [88–91]. In this section, we used the Plant miRNA Encyclopedia (PmiREN) [92] (https://www.pmiren.com/download) to analyze the association between miRNA and HanMATE genes using mature miRNA sequences and their expression in sunflower. The interaction between the miRNAs and HanMATE genes was then visualized using Cytoscape 3.7.1.
2.3.6. 3D structural modeling, functional prediction, molecular docking, and dynamic simulations.
To explore the structural and functional properties of HanMATE proteins, three-dimensional (3D) models were generated using the SWISS-MODEL web server [93]. Homology-based modeling offered insights into potential protein conformations and functional roles. Nonetheless, secondary metabolites play a crucial role in plants’ adaptation to abiotic stress through physiological and biochemical mechanisms. Among these metabolites, flavonoids regulate plant growth and development, hormone signaling, and the maintenance of ROS homeostasis [94,95]. We retrieve the molecular 3D structure of the important flavonoids from the online PubChem database [96]. The HanMATE proteins and flavonoids were pre-processed using AutoDock tools [97], and then MD analysis between HanMATE receptors and compounds was performed, and their binding affinity scores (kcal/mol) were computed using AutoDock Vina [98]. To explore the dynamic characteristics of the best protein-flavonoid complexes, we performed molecular dynamics simulations using the YASARA software [99].
3. Results
The findings were then separated into two primary sections: results related to identifying the HanMATE genes and their role in AST in sunflower.
3.1. Identification of HanMATE genes
3.1.1. HanMATE gene identification using phylogeny, domain, and motif analysis.
The best candidate HanMATE genes were identified by comparing the phylogenetic relationship between the candidate HanMATE and AtMATE proteins, considering 1000 bootstrap replicates. Accordingly, 74 HanMATE proteins and 46 AtMATE proteins were categorized into four clades (I–IV) (Fig 2). All HanMATE proteins have the conserved domain MATE-like superfamily except HanMATE 48 and 49, which have the MatE conserved domain. HanMATE 2, 3, 5, 19, 47, 59, 67, and 72 proteins have the conserved domain Polysacc_synt_C along with the MATE-like superfamily. In contrast, HanMATE 2, 3, 4, 5, 6, 19, 47, 52, and 60 have the conserved domain MurJ (Fig 3). Additionally, evolutionary relationships within HanMATE protein groups were investigated using 10 conserved motifs in each HanMATE protein. As we observed from Fig 3, these 10 conserved motifs were found in 45 HanMATE proteins. HanMATE 10, 3, 4, and 8 proteins consist of 9, 8, 4, and 3 motifs, respectively. HanMATE 59 and 72 have a single motif. Only 2 motifs were found in HanMATE74, while 6 motifs were found in HanMATE68. These analyses help to understand the evolutionary relationships of closely related proteins within the group. The protein sequences of the 74 HanMATE genes were provided in supplementary file in S1 File.
The colors in the figure represent different subgroups of HanMATE along with AtMATE proteins. Accordingly, we found four subgroups (I–IV) of 74 HanMATE and 46 AtMATE proteins in the figure.
The horizontal line at the bottom of each figure represents the protein’s length (0-600 aa).
3.1.2. Basic physicochemical properties of the HanMATE genes.
The basic physicochemical properties of all 74 identified HanMATE genes were studied, including the number and position of each gene on the chromosome, the starting and ending position of genes, the CDS length, molecular weight, the protein length, the protein pI, and GRAVY. Data from these properties were summarized in Table 1. The coding region of the HanMATE genes ranges from 528 bases (HanMATE20: HanXRQChr05g0160891) to 1791 bases (HanMATE53: HanXRQChr14g0459011). The protein ranged from 176 (HanMATE20: HanXRQChr05g0160891) to 597 (HanMATE52: HanXRQChr14g0459011) aa. The protein’s molecular weight ranges from 18707.89 Da (HanMATE20: HanXRQChr05g0160891) to 63887.8 Da (HanMATE52: HanXRQChr14g0459011). All HanMATE proteins are polar (positively charged), since the GRAVY value of these proteins is above 0, ranging from 0.328 (HanMATE63:HanXRQChr16g0521421) to 0.907 (HanMATE24:HanXRQChr06g0168521). The Ip values of HanMATE proteins vary from 4.94 (HanMATE20: HanXRQChr05g0160891) to 9.65 (HanMATE52: HanXRQChr14g0459011), with an average of 7.29.
3.1.3. Gene structure analysis.
The HanMATE gene’s structure was examined by arranging exons and introns, which were identified by comparing the CDS sequences with their corresponding genomic sequences. The HanMATE genes were found to contain 1–17 exons and 0–16 introns (Fig 4). Additionally, the evolutionary tree’s gene clusters exhibit almost similar exon and intron structures, based on the number of exons and introns (Fig 2 and Fig 4). For instance, Group I has between 4 and 11 exons and 3–10 introns; Group II has 6–11 exons and 5–10 introns; Group III has 1–5 exons and 0–4 introns; and Group IV has 12–17 exons and 11–16 introns. Nevertheless, 1–17 exons and 0–16 introns are present in the HanMATE genes, with the lowest, median, mode, 75th percentile, and maximum exon counts being 1, 9, 9, 10, and 17, in that order (Fig 4). The HanMATE gene and CDS sequences are provided in files S2 and S3 in S1 File.
The horizontal line at the bottom of the figure represents the gene’s length (0-55000 bp).
3.1.4. Chromosomal location and gene duplication analysis.
These chromosomal locations are shown in Fig 5A, where the 74 HanMATE genes are distributed across 17 chromosomes and one contig. Chromosomes 5 and 16 contain the maximum number of HanMATE genes, with nine each; specifically, HanMATE 14–22 is on chromosome 5, and HanMATE 60–68 is on chromosome 16. Chromosomes 1 and 13 each have 7 HanMATE genes, with HanMATE 1–7 on chromosome 1 and HanMATE 43–49 on chromosome 13. Chromosomes 2, 3, and 4, along with one contig, each contain two HanMATE genes. Chromosomes 6, 14, 15, and 17 have 3, 4, 6, and 4 HanMATE genes, respectively (Fig 5A). Tandem duplicated genes contribute significantly to plant evolution and adaptation to environmental changes. The duplicated genes within or between chromosomes were marked with red lines in Fig 5B. A total of 22 pairs of duplicated HanMATE genes were identified, with 11 pairs duplicated between chromosomes and 11 pairs duplicated within the same chromosome (Fig 5B). For example, HanMATE26 and HanMATE53 were duplicated on chromosomes 7 and 14, while HanMATE39 and HanMATE40 were duplicated within chromosome 10 (Fig 5B).
In the figure, plot A displays the chromosome number on the left side and the HanMATE name on the right side for each of the chromosomes. Plot B showed HanMATE gene duplication occurrences within the genome. Duplicated gene pairs in sunflower relate to lines, and inside the chromosome numbers are stated.
3.2. Role of HanMATE genes in AST in sunflower
3.2.1. Subcellular localization analysis.
Proteins’ subcellular location is connected to eukaryotic cells’ biological functions. The placement of proteins within cells helps to explain their functional activities at the cellular level [100,101]. The HanMATE proteins were in the nucleus, vacuole, chloroplast, endoplasmic reticulum (ER), plastid, and Golgi body (Fig 6 and Table 2). On the right side of the figure, a color scale bar ranging from white to black shows the likelihood of HanMATE proteins in relation to cellular organelles; white denotes the absence of HanMATE proteins, and the intensity of black signifies the likelihood. Subsequently, the vacuoles, plastids, ER, Golgi body, nucleus, cytoplasm, mitochondria, chloroplast, peroxisomes, and extracellular organelles contained 62, 69, 48, 42, 5, 10, 7, 11, 3, and 7 of the 74 HanMATE proteins (Table 2).
3.2.2. GOE analysis of HanMATE genes.
The GOE analysis of the HanMATE genes was performed to determine their relationship with various biological processes, molecular functions, and cellular components. Fig 7A and 7B represent the HanMATE proteins’ enrichment in the molecular functions and biological processes. According to Fig 7A, 71, 71, 70, 64, 70, 64, and 64 out of 74 HanMATE proteins significantly enriched the molecular functions of transporter activity (GO:0005215, p-value = 0.000), transmembrane transporter activity (GO:0022857, p-value = 0.000), drug transporter activity (GO:0090484), active transmembrane transporter activity (GO:0022804, p-value = 0.000), drug transmembrane transporter activity (GO:0015238, p-value = 0.000), secondary active transmembrane transporter activity (GO:0015291, p-value = 0.000), and antiporter activity (GO: 0015297, p-value = 0.000), respectively. Additionally, 5 HanMATE proteins were also significantly enriched in the molecular fluctuations of anion transmembrane transporter activity (GO:0008509, p-value = 0.001), organic anion transmembrane transporter activity (GO:0008514, p-value = 0.000), and citrate transmembrane transporter activity (GO:0015137, p-value = 0.000). On the other hand, 71, 71, 70, 71, 70, 70, 71, 70, 70, 71, 70, and 70 out of 74 HanMATE genes were significantly enriched in the biological processes such as localization (GO:0051179, p-value = 0.000), single-organism process (GO:0050896, p-value = 0.000), establishment of localization (GO:0051234, p-value = 0.000), single-organism localization (GO:1902578, p-value = 0.000), response to chemical (GO:0042221, p-value = 0.000), transport (GO:0006810, p-value = 0.000), response to drug (GO:0042493 p-value = 0.000), single-organism transport (GO:0044756), transmembrane transport (GO:0055085, p-value = 0.000), drug transport (GO:0015893, p-value = 0.000), and drug transmembrane transport (GO:0006855, p-value = 0.000), respectively. The biological processes like anion transport (GO:0006820, p-value = 0.023), organic anion transport (GO:0015711, p-value = 0.001), carboxylic acid transport (GO:0046942, p-value = 0.001), tricarboxylic acid transport (GO:0006842, p-value = 0.000), and citrate transport (GO:0015746, p-value = 0.000) were significantly enriched with 5 HanMATE genes. The detailed results of the GOE analysis were given in file S4 in S1 File.
Plot A in the figure illustrates the enrichment of HanMATE genes with molecular functions, while plot B shows the enrichment of HanMATE genes with biological processes. Each of the rectangles in the plot shows the GO ID with p-value, the GO term, and the number of HanMATE genes that were enriched for that GO term.
3.2.3. CREs in the promoter regions of HanMATE genes.
CREs are regions on a gene that can bind to transcription factors. CREs are crucial for gene regulation and control plant growth, development, differentiation, and stress response. In sunflower and other plants, we identified 25 CREs in the 1.8 kbp upstream of the HanMATE and ASR MATE genes, respectively, using the PlantCARE database and co-clustering technique (Figs. 8 and 9). The CREs LRE, MeJARE, ABRE, and AIE were found in the 1.8 kbp upstream region of most HanMATE and ASR MATE genes (Figs. 8 and 9). As a result, ASR MATE and HanMATE genes share 20 of these cis-regulatory elements. CREs for the HanMATE genes are provided in file S5 in S1 File.
In the figure ABRE = Abscisic Acid-Responsive Element, AIE = Anaerobic Induction Element, LRE = Light Responsive Element, MeJARE = MeJA Responsive Element, ARE = Auxin Responsive Element, DIE = Drought Inducibility, GRE = Gibberellin Responsive Element, LTRE = Low Temperature Responsive Element, MEE = Meristem Expression Element, MYBHv1_BS = MYBHv1 binding site, SARE = Salicylic Acid Responsive Element, ZME = Zein Metabolism Element, X60K_PBS = 60K protein binding site, AnIE = Anoxic Inducibility Element, ATBP.1_BS = ATBP-1 Binding Site, CCRE = Cell Cycle Regulation Element, CCE = Circadian Control Element, CRE = Cis Regulatory Element, CMA3 Element = CMA3 Element, EEE = Endosperm Expression Element, FBRE = Flavonoid Biosynthesis Regulation Element, HTLE = High Transcription Level, SSRE = Seed Specific Regulation Element, and SDRE = Stress and Defense Responsive.
3.2.4. Syntenic relationship between HanMATE and ASR MATE proteins.
Synteny analysis involves large blocks of conserved sequences across genomes, indicated by highly similar patterns between species [102–104]. This study compared 74 HanMATE genes in sunflower with 66 ASR MATE proteins from different plants, such as Arabidopsis, rice, wheat, maize, potato, soybean, and dragon fruit, based on their sequence similarity, as shown in Fig 10. Accordingly, 41 HanMATE protein sequences share > 75% sequence similarity with 40 ASR MATE proteins (Fig 10), which was summarized in Table 3. The table indicated that seven (StMATE5/18/33/40/59/60/61) proteins in potato were sensitive to heavy metal stress, which is associated with 21 HanMATE2/3/4/5/6/14/15/16/17/27/28/31/32/33/38/41/42/50/51/53/62 proteins. StMATE11/63 proteins synthesized flavonoid in potato under stress and showed >75% sequence similarity with HanMATE13/14/26/30/51/61/62 proteins. In soybean, GmMATE13/58/74/84 proteins are responsive to AL stress that aligned with HanMATE 2/3/4/5/6/19/26/42/47 proteins. Nevertheless, in rice, OsMATE4/9 are involved in metal ion detoxification and responsive to salt stress, having >75% sequence similarity with HanMATE 2/5/19/47 in sunflower. Similarly, six GaMATE18/34/51/53/54/58 (upregulated in multiple abiotic stresses) and 14 HanMATE9/14/26/31/32/33/37/42/43/46/57/58/61/62 proteins’ sequences are similar (>75%) in cotton and sunflower, respectively. The sequence of DTX18/19/45/47/50/51 proteins in Arabidopsis, involved in detoxification, transportation, and disease resistance, aligned with that of HanMATE10/26/37/43/52/55/59/67/72 proteins in sunflower. DTX48/50 maintain Fe homeostasis in the cell, aligned with HanMATE50, which shares >75% sequence similarity. Similarly, DTX26/28/30 transport Ca+ as well as lessen multiple abiotic stresses in Arabidopsis, having >75% sequence similarity with HanMATE50/51. In contrast, HuMATE7/11/12 proteins detoxify toxic compounds under heavy metal and salt stresses in dragon fruit, showing >75% sequence similarity with HanMATE14/38/43/46/55/56/57/58/62. Conversely, in wheat, TaMATE10/14 are upregulated under multiple abiotic stresses that share >75% sequence similarity with HanMATE17/18/61. However, in maize, ZmMATE1/16/23/32 proteins are responsive to AL stress and show sequence alignment (>75%) similar to HanMATE26/46/47/55/56/59/72.
3.2.5. Regulatory network analysis with miRNAs.
In our investigation, we identified 136 miRNAs associated with 58 HanMATE proteins (Fig 11 and File S6 in S1 File). The figure showed that there are 19 major hub miRNAs and 31 important hub HanMTE proteins. The interaction scores of these hub proteins and miRNAs are displayed in parentheses (Fig 11).
In the figure, the purple- and green-colored nodes represent the HanMATE proteins and the miRNAs, respectively. The highlighted HanMATE proteins and the miRNAs with interaction scores beside the respective HanMATE proteins and the miRNAs in parentheses are the hub proteins and miRNAs.
3.2.6. Protein 3D structural modeling, functional prediction, and molecular dynamic simulation.
The HanMATE proteins were 3D structurally modeled as monomers and exhibited high sequence identity to their respective templates, ranging from 70.92% to 100%, with near-full coverage (≥0.92). The aligned templates were primarily derived from Helianthus annuus, Artemisia annua, Mikania micrantha, Sesamum indicum, and other related plant species. Notably, several HanMATE proteins, including HanMATE15/24/25/43/52/61/70, showed 100% sequence identity and full coverage with Helianthus annuus templates, indicating exact or near-exact matches to known sunflower proteins. Functional prediction based on the matched template descriptions indicated that most HanMATE proteins are likely involved in protein detoxification or MATE efflux transport processes (File S7 in S1 File). Overall, homology-based modeling and functional annotation suggest that HanMATE proteins in sunflower are predominantly detoxification-related transporters with highly conserved structure.
MD was performed to assess the binding affinities of nine flavonoids (elatin, corylifolin, artocarpin, laurifolin, anthocyanins, quercetin, and catechin) and citric acid with the randomly selected thirteen HanMATE proteins (Table 4). Specific protein-flavonoid interactions highlighted variability among HanMATE members. For instance, HanMATE26 displayed the strongest binding with artocarpin (−9.9 kcal/mol), laurifolin (−9.6 kcal/mol), and Elatin (−9.4 kcal/mol); while HanMATE32 showed the highest binding affinity with Corylifolin (−9.8 kcal/mol). Other proteins, such as HanMATE57 and HanMATE4, showed preferential binding to Corylifolin (−9.5 kcal/mol) and Elatin (−9.2 kcal/mol), respectively. Overall, these results suggest that artocarpin, corylifolin, laurifolin, and elatin showed consistently high binding affinities for the selected HanMATE proteins. These binding affinities were then validated and further explored using molecular dynamics simulations and binding free energy calculations to assess the stability and dynamic behavior of these HanMATE-flavonoid complexes. In the molecular dynamic simulations of these complexes, RMSDLigMove (ligand movement RMSD) analysis provided insights into the stability of flavonoid binding within the HanMATE protein’s active site (Fig 12A). For HanMATE4_Elatin, the RMSD remained low and stable throughout the simulation compared to the other complexes, indicating that the ligand maintained its binding pose with minimal drift inside the pocket. Consistently, the complex exhibited favorable binding affinity with the lowest binding free energy, suggesting strong and stable interactions with the protein (Fig 12B).
(A) Root-mean-square deviation (RMSD) of flavonoid movement. (B) Binding free energy (kJ/mol).
4. Discussion
The abiotic stresses of drought, salinity, and heavy metal stress seriously impacted the plant’s growth and development, biodiversity, and productivity [3–5]. These stresses bring morphological, physiological, and molecular changes in plants. Consequently, closing stomata, reducing transpiration, limiting photosynthesis, ionic and osmotic stress, impairing nutrient uptake channels, and excessive production of ROS are the main indications of plants under abiotic stresses [6,7,11,13,15–22]. Nonetheless, the secondary transporter gene, MATE, transports substrates like chemical molecules, metabolites, and phytohormones. These transporter proteins promote plant growth and development, detoxify internal and external toxins, and defend prokaryotes and eukaryotes from stresses [27,28,36,102]. On the contrary, the special structure of the root, stem, leaves, and head makes the sunflower moderately AST [43,44]. Therefore, researchers worldwide studied the AST of sunflower in many ways [44–53]. However, researchers have not yet discovered the role of MATE genes in sunflower AST, though their functions have been studied in many plants. Therefore, in this study, we tried to find insights into the MATE gene family in sunflower for AST.
This study found 74 HanMATE genes in sunflower and grouped them into four clades (I-IV). Groups I, II, III, and IV comprise 27, 19, 16, and 12 of the 74 HanMATE proteins. The gene, domain, and motif structures of the HanMATE genes are nearly identical, corresponding to the respective phylogenetic groups. For instance, the domain MATE-like superfamily is included in Groups I and II. In addition to the domain MATE-like superfamily, HanMATE proteins in Group III also contain the domain MATE (HanMATE 48 and 49). In Group IV, the HanMATE proteins contain the domains MurJ, MATE-like superfamily, and Polyasacc_synt_C superfamily. Likewise, the HanMATE genes and proteins organization displayed nearly the same pattern across the various phylogenetic groups. Additionally, the motifs’ distribution over the HanMATE proteins shows almost similar patterns in the respective phylogenetic groups. Similarly, in the case of the HanMATE gene structure, genes in the same group often have similar structures according to the distribution of exons and introns (Figs 2 and 4). As a result, the genes or proteins in the corresponding evolutionary Groups I, II, III, and IV share nearly identical structures. Conversely, in other plants like melon, soybean, and cotton, similar results were also found [38,40,65].
The domain MATE and MATE-like superfamily transport secondary metabolites and toxic chemicals across the cell membrane and maintain homeostasis in cells in response to abiotic and biotic stresses [113]. Though the roles of the domains Polyasacc_synt_C and the Polyasacc_synt_C superfamily have not yet been identified, MurJ is crucial for flipping Lipid II across the cytoplasmic membrane in bacteria for biosynthesizing the peptidoglycan [114]. The peptidoglycan precursor flippers MurJ’s homologous genes MltB and VanY in the moss Physcomitrella patens play an important role in chloroplast division [115]. Although chloroplasts are known to carry out photosynthesis, they also enable the assimilation of nitrogen and sulfur as well as the production of amino acids, fatty acids, nucleotides, and hormones in plants [116]. Nevertheless, protein motifs are crucial structural elements, serving as signatures of protein families and aiding in protein function prediction [117]. The 1–10 predicted motifs in HanMATE proteins are MATE and MATE-like superfamily, which are linked with predicted domains responsible for exporting secondary metabolites and toxic chemicals across the cell membrane and maintaining homeostasis in cells in response to abiotic and biotic stresses [113].
Proteins are linear assemblies of amino acids, with their functional properties determined by their physical and chemical characteristics. Usually, most MATE proteins consist of 450–550 amino acid residues, while some members with 9–12 transmembrane helices may exceed 700 amino acids. The predicted length of the HanMATE protein ranges from 112 to 597 amino acids, with a weight range of 18.70 to 63.88 KD and an Isoelectric Point (Ip) between 4.94 and 7.29. In contrast, rice MATE proteins typically range from 370 to 598 amino acids, weigh between 39.41 and 61.65 KD, and have an Ip from 5.01 to 11.98, correlating with responses to drought and salt stresses. Similarly, in maize, MATE proteins vary from 125 to 692 amino acids, with a weight range of 13.8 to 73.10 KD, and an Ip from 4.34 to 10.0, responding to aluminum stress. The patterns of MATE proteins in dragon fruit are comparable to those in rice, maize, and sunflower, with sizes from 124 to 686 amino acids, weights between 13.19 and 74.37 KD, and Ip values from 5.46 to 8.99, responding to abiotic stresses. Additionally, hydrophilic proteins tend to accumulate in plants under various stress conditions, indicating their protective role. Similarly, dragon fruit MATE proteins respond to abiotic stresses with a GRAVY ranging from 0.361 to 0.856, whereas HanMATE proteins vary from 0.328 to 0.890. These observations regarding the physicochemical properties of MATE proteins sunflower and other plants suggest that these HanMATE proteins may play a significant role in AST.
The identified 74 HanMATE genes are distributed across seventeen chromosomes and one contig (Fig 5A). These genes are duplicated between or within chromosomes, resulting in the numerous MATE genes found in sunflower. The synteny analysis revealed 22 pairs of duplicated HanMATE genes, with 11 pairs duplicated between chromosomes and 11 pairs duplicated within chromosomes (Fig 5B). Subsequently, duplicated genes are important in plant evolution and adaptation to environmental changes, including biotic and abiotic stresses [118–121]. Therefore, it could be concluded that the HanMATE gene duplication might increase the AST in sunflower.
Abiotic stress significantly impacts plant growth and development by altering subcellular organelles and affecting intracellular compartments. However, subcellular proteins, including transporters, ROS scavengers, and signaling and transcriptional regulators, play a crucial role in stress tolerance [122,123]. These proteins have developed intricate detoxification mechanisms to handle a wide range of potentially toxic compounds, including exogenous xenobiotics and endogenous metabolites, particularly secondary metabolites. Membrane transporters play a vital role in maintaining ionic balance and facilitating the movement of substances across organellar membranes during abiotic stress in plants. They assist in reducing ROS production by regulating ions and metabolites, activating antioxidant enzymes, and improving ROS scavenging [124–126]. After enzymatic modification or synthesis, such compounds are transported and stored in the central vacuoles in plant cells [126]. Nonetheless, nearly 50% of the mitochondrial proteome is dedicated to energy production. Consequently, it generates ATP and ROS through oxidative phosphorylation and the electron transport chain, contributing to plant stress responses, which serve as environmental sensors [127–129]. The inner mitochondrial membrane transporters manage energy and calcium exchange, promoting metabolic stability and enhancing plant resilience to environmental stress [130]. Nevertheless, 74 HanMATE proteins were observed in the nucleus, vacuole, chloroplast, endoplasmic reticulum, plastid, and Golgi bodies (Fig 6), which may participate in stress tolerance in sunflower. Almost all HanMATE genes were present in the plastids with higher likelihood. Plastids, essential plant cell organelles present in all plant cells and green algae, include chloroplasts, amyloplasts, chromoplasts, and leucoplasts. The components of plastids are responsible for important biological functions. For example, chloroplasts convert CO₂ to carbohydrates, chromoplasts regulate the colors of flowers and fruits, and amyloplasts and leucoplasts store starch and lipids in the cell [131–133]. Plant cells’ vacuoles, which house the majority of the HanMATE proteins, are another crucial organelle. It is essential for controlling development and growth, maintaining the equilibrium of the cell’s acidity and pressure, controlling the storage and flow of materials, regulating the mobility and positioning of critical proteins within the cell, and responding to both living and non-living stressors are the primary functions of vacuoles [134,135]. Conversely, the ER is a cell’s organelle that stocks two-thirds of the HanMATE proteins. These proteins are involved in signaling and the organism’s or cells’ reaction to stressors and the environment [45,136]. In addition, oxidation, hydroxylation, and deamination of xenobiotics or biological components occur in the ER. Stress-induced H2O₂ generation interacts outside of the ER, which triggers antioxidant defense components to balance the cell’s redox state [137–140]. The Golgi apparatus plays important roles in cell wall formation, protein sorting, and glycosylation, which are essential for plant cell development and division [141]. On the other hand, the antioxidants superoxide dismutase, glutathione peroxidases, and catalases are localized in the cytosol, mitochondria, and chloroplasts and scavenge ROS. In Arabidopsis, these five ascorbate peroxidases, involved in scavenging ROS generated during photosynthesis [142–144]. Therefore, it could be concluded that HanMATE proteins in the subcellular compartment may take part in transporting toxic elements, ROS scavenging, signaling, and transcriptional regulation.
The identified HanMATE genes in sunflower were enriched in GO terms relating to molecular function (GO:0003674), biological process (GO:0008150), and cellular component (GO:0005575). Almost all the HanMATE genes enriched in the molecular function (GO:0003674) (Fig 7A) participate in the process of directing the movement of substances, including macromolecules, small molecules, and ions, into, out of, within, or between the cells. They also contribute to transferring substances from one side of a membrane to another and the movement of drugs, which can affect an organism’s structure or functioning. They also actively transport a solute across a membrane. The HanMATE genes also transfer negatively charged ions, organic anions, and citrate. These genes also play a role in the localization of cells, substances, or cellular entities, such as protein complexes or organelles, through selective degradation [145–147]. Nonetheless, around all 74 HanMATE genes enriched to different biological processes significantly (Fig 7B), which are involved in transporting xenobiotics, foreign compounds, into, out of, or within a cell or between cells, is a process that involves agents like transporters or pores. This process can be tethered to or maintained in a specific location and can result in changes in cell or organism activity. Localization of substances or cellular components can also occur through movement, tethering, or selective degradation [146,147].
CREs play a crucial role in gene expression patterning and development, providing the proper spatiotemporal patterning for environmental reactions [148]. On the other hand, phytohormones serve as vital regulators for several physiological and biochemical processes that govern plant growth, development, and productivity in both favorable and stressful conditions [149,150]. The ABRE, AIE, LRE, and MeJARE are important CREs clustered together that are present in the 1.8 kbp upstream region in almost every HanMATE and ASR MATE genes. ABARE is a crucial phytohormone for plant growth and development, regulating stress responses. It is constantly adjusted in response to physiological and environmental changes. Transcription factors like DREB2A/2B, AREB1, RD22 BP1, and MYC/MYB regulate ABA-responsive gene expression through interactions with CRE such as ABRE and MYCRS/MYBRS [151–153]. In plants, AIEs are DNA sequences that stimulate gene expression in the presence of low oxygen. They serve as binding sites for transcription factors, activating genes to assist plants in adjusting to stress when oxygen levels fall [154–156]. On the other hand, LREs are located within the promoter regions of light-responsive genes, which act as binding sites for proteins that mediate the effects of light on gene expression. In Arabidopsis, LREs are involved in the light regulation of the nuclear genes GapA and GapB that encode the A and B subunits of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) [157,158]. G3PDH, a conserved glycolytic enzyme, functions as a moonlighting protein, regulating gene expression, cell signaling, and hormone signaling pathways, thereby influencing plant growth and development [159]. However, the phytohormones, particularly MeJA, salicylic acid, Gibberellin, Auxin, and abscisic acid, can help mitigate the effects of abiotic stresses. MeJA inhibits the uptake of harmful ions and lessens the negative consequences of osmotic stress by controlling either organic or inorganic penetrating ions [160]. According to the current study’s findings, TGACG and CGTCA motifs regulate plant defense against abiotic stressors such as heat, salinity, drought, and cold, and they also have a role in MeJA response [161]. Salicylic acid plays a crucial role in enhancing plant tolerance to abiotic stress by regulating key metabolic processes. Recent research has highlighted the pivotal role of phytohormones in mediating plant responses to environmental changes, encompassing growth, development, and responses to environmental stress [162–164]. Another phytohormone, Gibberellin, regulates various plant functions, including blooming, fruit patterning, root and shoot elongation, and seed germination. Its signaling pathway influences plant growth under stress conditions, and its increased signal transduction promotes tolerance to abiotic stressors [165–168]. Auxin, or indole-3-acetic acid, is another phytohormone that regulates plant growth and development and responds to abiotic stressors. It influences gene expression through auxin response factors, which act as an effector of auxin response and translate chemical signals into gene regulation [169,170]. Consequently, MeJARE, SARE, GRE, ARE, and ABRE, CREs were predicted in the HanMATE proteins as well as ASR MATE proteins in other plants (Fig 8 and Fig 9) that signify HanMATE proteins might work for stress tolerance in sunflower. Among the other CREs in HanMATE proteins and stress-responsive MATE proteins, circadian control or the circadian clock, responsible for synchronizing environmental signals with biological processes, plays a crucial role in plants’ ability to tolerate and adapt to environmental stress. Stress-related extremes like extreme temperatures, drought, and salinity can lead to crop losses and agricultural limitations. Recent evidence suggests the CCE controls gene expression and stress response hormone signaling [171–173]. However, the evolution of biology is linked to atmospheric dioxygen formation, which reprograms gene expression through regulatory interactions between transcription factors and anaerobic-responsive elements, responding to abiotic stresses like anaerobic, drought, and salt by the conciliation of ABA and ethylene hormones [154]. Sunflower’s AIE could contribute to its ability to endure stress. Plants’ abiotic stress responses and tolerance are greatly influenced by DIE, which controls the expression of several SR genes. Abiotic stressors like heat, cold, salt, and drought are known to be regulated by members of the DIE binding gene family [174–177]. The DIEs are closely connected with the dehydration-responsive element binding regulators [178] and make sunflower AST. FBRE, low-molecular-weight polyphenolic compounds found in plants, have various physiological functions in stress response [179]. The WRKY transcription factors regulate flavonoid biosynthesis during biotic and abiotic stress [180], and since HanMATE proteins contain flavonoid biosynthesis regulation CREs in sunflower, they may have stress tolerance properties.
Comparative genomics is a field of biology that predicts homologous genes or proteins based on their functional similarities. In this context, synteny analysis serves as a valuable tool to identify the conservation of homologous genes across species. It helps reveal genome structure and find shared markers in different genomes, offering insights into evolutionary relationships [102,181–185]. In this study, synteny analysis showed that over 75% of the sequences of 41 HanMATE proteins and 40 ASR proteins in Arabidopsis, rice, maize, potato, cotton, melon, and dragon fruit are similar, suggesting that HanMATE proteins may be ASR proteins that play a role in sunflowers’ stress tolerance.
miRNAs are small, non-coding RNAs that regulate gene expression in eukaryotes. They can potentially improve agronomic properties and enhance resistance to abiotic stress in plants. Because changes in miRNA expression in abiotic stress conditions suggest that miRNAs are potential targets for genetic manipulation to engineer AST [186–188]. This study found 19 major hub miRNAs linked to 31 important HanMATE genes (Fig 11). Among them, Han-miR169q has been linked with seven HanMATE genes. The miR169 regulates abiotic stress responses and plays critical roles in hormone accumulation, antioxidant activity, ion-channeling membrane components, and potential interactions with stress-regulating transcription factors such as AsHsfA and AsWRKYs in Agrostis stolonifera L. [189]. ZmmiR169q, a protein in maize, responds to stress-induced ROS signals, reducing their accumulation. Depleting it increases salt tolerance, while overexpressing it decreases. ZmmiR169q repressed the transcript abundance of its target nuclear factor YA8 (ZmNF-YA8), while overexpression of ZmNF-YA8 in maize improved salt tolerance [190]. Although the functions of miR5686, miR5701, miR5701, and miR5757 in abiotic stress responses have not been identified, miRNAs regulate gene expression in eukaryotes and play important roles in AST. However, in Arabidopsis, miRNA165/166 regulates targets, including HD-ZIPIII transcription factors, which in turn influence plant development and stress tolerance through ABA signaling [191,192]. The expression of gma-miR156a and gma-miR172a in chickpea significantly impacts plant morphology and physiology, particularly during reproductive development. These genes regulate multiple traits, potentially enhancing crop yield under changing climatic conditions and responding to heavy metal stress in chickpeas [193,194]. Consequently, 41 of the HanMATE proteins matched (>75%) with the 40 ASR HanMATE proteins, which are considered for stress tolerance in sunflower (Fig 11 and Table 2).
3D structural and functional prediction suggests that almost all HanMATE proteins are detoxification-related transporters in sunflower. However, molecular docking was performed to assess the binding affinities of nine flavonoids (elatin, corylifolin, artocarpin, laurifolin, anthocyanins, quercetin, and catechin) and citric acid with 13 randomly selected HanMATE proteins. The protein-flavonoid interactions have strong binding affinity that was validated by molecular dynamic simulation analysis, RMSD, and binding free energy analysis.
5. Conclusions
Numerous studies in the literature have examined sunflower abiotic stress tolerance using various methods, including metabolite regulation, oxidative stress defense, enzyme detoxification, and drought response mechanisms. These studies also identified key transcription factors, genomic markers, and phenotypic responses to abiotic stresses. Nevertheless, MATE proteins detoxify endogenous and external poisons, strengthening the plant’s immune system and fostering growth and development. These proteins also play an important role in adjusting the ROS inside cells and maintaining cellular homeostasis. However, although the functions of MATE proteins have been identified in some other plants, this has not been done yet in sunflower AST. Therefore, this study identified 74 HanMATE proteins/genes in sunflower compared with 46 AtMATE proteins in Arabidopsis. These genes were grouped into four clades in a phylogenetic tree. Comparing the results of domain, motif, gene structure, gene duplication, and physicochemical analysis with other plants, we confirmed that the identified 74 HanMATE genes have MATE-like properties. The ASR and detoxifying features of HanMATE genes were analyzed using sub-cellular localization, microRNA, gene ontology, CREs, syntenic relationship, and protein 3D structure and function, molecular docking, and dynamic simulation analysis. Most of the HanMATE proteins are present in the plastid, nucleus, and vacuole with a higher likelihood. The GOE analysis predicted that out of 74 HanMATE genes, 71 enriched the molecular functions: transporter activity and drug transporter activity, etc. On the other hand, 70 HanMATE genes enriched the biological processes: response to stimulus, response to drugs, and response to chemicals etc. This study also found that ABRE, AIE LRE, and MeJARE are the most important CREs in the 1.8 kbp upstream region of the HanMATE genes that play a crucial role in the plant’s immune boosting as well as growth and development. Furthermore, the syntenic relationship analysis revealed that 41 HanMATE proteins share more than 75% sequence similarity with 40 ASR MATE proteins in other plants, indicating that the HanMATE proteins may be ASR and contribute to sunflower’s stress tolerance. On the other hand, miRNAs regulate gene expression, may enhance plant agronomic traits and resistance to abiotic stress, indicating the potential for genetic manipulation to improve AST. This study identified 136 miRNAs associated with 58 HanMATE proteins, including 19 major hub miRNAs and 31 key hub HanMATE proteins. Additionally, these findings were also validated using protein 3D structure and function prediction based on their homologs in common ancestors, which found that the key HanMATE genes may participate in detoxification. Additionally, molecular docking and dynamic simulation validated that HanMATE genes have strong binding affinity with flavonoids and citric acid. Besides these in silico validations of HanMATE genes’ ASR properties, an experimental study could be conducted in the future for wet lab agreement. Consequently, the discovered HanMATE genes may be crucial for sunflowers’ ability to withstand abiotic stress and may also be applied to the development of robust, more resilient crops.
Supporting information
S1 File. S1. Protein sequences of HanMATE genes.
S2. HanMATE gene sequences. S3. CDS sequences of HanMATE genes. S4. HanMATE genes’ GOE analysis results. S5. HanMATE genes’ CRE analysis results. S6. HanMATE gene and miRNA analysis results. S7. HanMATE proteins’ 3D structure and functional prediction results.
https://doi.org/10.1371/journal.pone.0346769.s001
(ZIP)
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