https://orcid.org/0009-0003-6140-2716
Figures
Abstract
Fatty acid desaturases (FADs) play a pivotal role in the accumulation of oils in plant seeds. To elucidate the role of FADs in oil accumulation in the seeds of Xanthoceras sorbifolium, this study employed bioinformatics analysis methods to identify and analyze the FAD gene family. A total of 25 XsFAD genes were unevenly distributed across 11 chromosomes. Systematic phylogenetic analysis revealed that the XsFAD gene family is divided into three branches, with each branch exhibiting similar gene structures. The regulatory elements within the XsFAD gene promoter indicate that XsFAD genes are regulated by multiple factors. Quantitative real-time polymerase chain reaction (qRT-PCR) validation revealed a positive correlation between the expression levels of XsFAD genes and the oil content of X. sorbifolium. By conducting phenotypic measurements of the X. sorbifolium fruit and seeds, as well as quantitative analysis of the XsFAD gene expression, it has been preliminarily identified that the Liudong-5 variety may poseess the potential to be developed into a high-yield oil-producing variety, which may be related to the habitat of the X. sorbifolium germplasm and requires further investagation. In summary, this study provides a foundational understanding of the FAD gene family of X. sorbifolium, and the research outcomes will contribute to the theoretical basis for the selection of high-yielding oil varieties of X. sorbifolium in the Xinjiang region, as well as further genetic breeding and cultivation system studies.
Citation: Xing Q, He H, Wang Z, Li Y, Didar X, Zhou T (2025) Genome-wide characterization of FAD gene family in Xanthoceras sorbifolium Bunge and germplasm assessment. PLoS ONE 20(3): e0318900. https://doi.org/10.1371/journal.pone.0318900
Editor: Muhammad Qasim Shahid, South China Agricultural University, CHINA
Received: September 8, 2024; Accepted: January 23, 2025; Published: March 27, 2025
Copyright: © 2025 Xing 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: This research is supported by Xinjiang Uygur Autonomous Region Natural Science Foundation Youth Science Fund Project in the form of a grant [2024D01B27 to TZ] and Xinjiang Uygur Autonomous Region Public Welfare Research Institute Basic Research Service Fee Special Fund Project in the form of a grant [KY2024033 to TZ].
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
1.1. FAD gene functions
Fatty Acid Desaturase (FAD) is a key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs) in plants, catalyzing the dehydrogenation of C-C at specific positions on fatty acid chains to form C = C [1], thereby generating PUFAs with varying double bond positions and numbers. These PUFAs are essential for numerous biological processes, including the formation and stability of plant cell membranes, signal transduction, and resistance to environmental stresses. The FAD gene family is widely distributed and abundant in plants, typically featuring multiple exons and introns with complex and highly conserved structures.
The expression of FAD genes is tissue-specific and developmentally regulated, often displaying differential expression patterns in different tissues or developmental stages [2,3]. Environmental factors such as light, temperature, and water stress can significantly influence the expression levels of FAD genes, thereby regulating the synthesis and accumulation of PUFAs in plants. Recent studies have concentrated on the specific functions and mechanisms of FAD genes in plants through cloning, expression analysis, and functional validation [4]. In response to environmental stresses such as drought, salinity, and high and low temperatures, plants frequently adjust the expression of FAD genes. For instance, in Cicer arietinum, drought priming mitigates membrane damage and chlorophyll degradation by increasing membrane unsaturated fatty acids (UFAs) alongside the upregulation of various fatty acid desaturase (CaFAD) genes and antioxidative machinery during flowering, which improves seed yield in PBG5 [5]. In tobacco, overexpression of CbFAD3 results in a sustained increase in linolenic acid (LA) in leaves and roots, enhancing membrane fluidity and stability and improving plant tolerance to cold, drought, and salt stress [6]. FAD genes may also participate in the response of plants to pathogen infection [7,8], heavy metal pollution, and other stresses. Given the significant role of FAD genes in plant fatty acid synthesis and stress response, they have extensive applications in crop breeding. By genetically engineering FAD genes for targeted modification and expression regulation, it is possible to develop crop varieties with superior fatty acid composition and resistance to adverse conditions. For instance, overexpression of FAD genes can increase the content and proportion of PUFAs in crop seeds, improving the nutritional value and health benefits of edible oils [9]. Conversely, knocking out or suppressing the expression of FAD genes can reduce the sensitivity of crops to unfavorable environmental conditions, enhancing their resistance and yield [10].
1.2. Xanthoceras sorbifolium characteristics
Xanthoceras sorbifolium, a member of the Sapindaceae family, is a plant species that is widely distributed across northeastern, northwestern, and northern China. With a large adaptive range, it facilitates easy reproduction and exhibits tolerance to drought and nutrient-poor. It is well-suited for cultivation in Chinese farmland, which has rich mountainous resources. The oil content of X. sorbifolium seeds ranges from 50% to 67%, with high levels of unsaturated fatty acids, including oleic acid (52.8% to 53.3%), linoleic acid (37.8% to 39.4%), and other fatty acids such as eicosapentaenoic acid, palmitic acid, and linolenic acid. Its oil is rich in nutrients and is an ideal raw material for producing high-quality edible oils and biodiesel. X. sorbifolium oil also has significant medicinal value, demonstrating preventive and therapeutic effects on cardiovascular diseases [11]. Qiang Liang et al. have assembled a high-quality genome sequence of X. sorbifolium using advanced technologies such as Illumina sequencing, Pacific Biosciences (PacBio) single-molecule real-time sequencing, 10X Genomics linked reads, Bionano optical maps, and high-throughput chromosome conformation capture (Hi-C) [2]. The assembled genome size is 439.97 Mb, with 15 pseudo-chromosomes accounting for 95.42% (419.84 Mb) of the assembled genome, and 56.39% of the genome is repetitive. This genome contains 21,059 protein-coding genes, from which multiple genes with tissue-specific expression patterns have been identified. However, the FAD gene family in these oil plants has not been well studied, and little is known about the molecular systematics and differential expression characteristics of the FAD gene family across different growth regions. By studying the FAD gene family of X. sorbifolium, the FAD gene related to seed oil content can be identified, providing a basis for subsequent genetic breeding to improve the oil content of X. sorbifolium.
1.3. The study objectives
Therefore, this study aims to analyze the structure, physical properties, and expression patterns of FAD genes in X. sorbifolium, to further understand the molecular evolutionary characteristics of the XsFAD gene family. Moreover, as the main distribution area of X. sorbifolium in the western region of China, Xinjiang holds a wealth of local, wild, and introduced superior germplasm resources. The significant differences in environmental and climatic conditions have led to the accumulation of substantial genetic variation during the evolutionary process, obtaining the best varieties through the analysis of germplasm resources. This lays the foundation for breeding varieties with high economic value and specific traits and serves as a reference for genetic improvement research focusing on oil content, stress tolerance, and disease resistance in X. sorbifolium.
2. Materials and methods
2.1. Identification of XsFAD genes
To identify the FAD genes in X. sorbifolium, we downloaded the genome data, protein files, and gff3 annotation files (GCA_020796215.1) from the NCBI database (https://www.ncbi.nlm.nih.gov/). Utilizing the Hmmer-search 3.0 program to search protein sequences containing FAD domains (PF00487, PF03405, and PF10520) within the X. sorbifolium protein database, with a search threshold of E < 1e-10. Submitting the preliminarily selected X. sorbifolium FAD protein sequences to the SMART (http://smart.embl-heidelberg.de/), NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd/), and PFAM (http://pfam.xfam.org/) websites to ensure they possess complete FAD domains. After removing suspicious sequences, obtain the members of the XsFAD gene family. The XsFAD gene family members were named according to their physical location order on the 15 chromosomes of X. sorbifolium. Molecular weight (MW) and isoelectric point (PI) were predicted in Expasy (https://web.expasy.org). Using BUSCA (http://busca.biocomp.unibo.it/) to predict the subcellular localization of the XsFAD genes encoded proteins. Predicting the localization of XsFAD-encoded proteins helps us understand the specific biological processes they are involved in and can identify other proteins that they interact with.
2.2. Phylogenetic tree construction of XsFAD genes
The protein sequences of the XsFAD gene family members were aligned using ClustalW v2.1. The phylogenetic tree was constructed using MEGA 11 with the Neighbor-Joining method. The phylogenetic tree was edited and beautified using the iTOL 2.0 website (https://itol.embl.de/).
2.3. Analysis of cis-acting elements of XsFAD genes
Using TBtools, sequences of the 2000 bp region upstream of the ATG start codon of the XsFAD gene family genes were extracted based on the genomic annotation and sequence files of X. sorbifolium. These sequences were submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for analysis of promoter cis-acting elements. The analysis results were organized and imported into TBtools for visualization.
2.4. Chromosomal location analysis of XsFAD genes
With TBtools, the lengths of each chromosome, gene density, and chromosomal localization information of the XsFAD genes were extracted from the plant genome database and didn’t include genetic distance for the creation of a chromosomal localization map of the XsFAD genes.
2.5. Analysis of conserved motifs and gene structure of XsFAD genes
The MEME online tool (http://meme-suite.org/) was used to analyze the conserved motifs in the XsFAD proteins, with the number of motifs set to 10. The GSDS online tool (http://gsds.cbi.pku.edu.cn/) was used to analyze the gene structures of XsFAD genes, and the obtained files were imported into TBtools for visualization.
2.6. Gene duplication relationships and synteny analysis of XsFAD genes
To study duplication events of XsFAD genes, MCScanX was used for collinearity analysis. The extracted gene collinearity results were visualized using TBtools to generate an XsFAD gene family collinearity map.
2.7. Selection pressure analysis of XsFAD genes
ClustalX (http://www.clustal.org/) was used to align the XsFAD gene sequences, and KaKs_Calculator 3.0 (https://ngdc.cncb.ac.cn/biocode/tools/BT000001/releases/3.0) was utilized to analyze the selection pressure between gene pairs.
2.8. Protein interaction network analysis of XsFAD genes
STRING (https://string-db.org/) was used to predict the protein interactions of the XsFAD-encoded proteins, establishing the corresponding protein relationship network and visualizing it.
2.9. Investigation of plant germplasm and oil content analysis
In this study, 10 germplasm resources of the plant were obtained from the Xinjiang Academy of Forestry Sciences. These germplasm were located in various regions of Xinjiang, China: Korla (41° 43′ 48″ N, 86° 1′ 12″ E, Germplasm ID: Liudong-1), Qitai County (44° 1′ 12″ N, 89° 30′ 36″ E, Germplasm ID: 131-75), Urumqi (43° 49′ 11.99″ N, 87° 36′ 36″ E, Germplasm ID: Liudong-5), Karamay (45° 36′ 0″ N, 84° 53′ 24″ E, Germplasm ID: 80 acres-1), Changji (44° 48′ 0″ N, 87° 12′ 0″ E, Germplasm ID: 80 acres-5), Kizilsu (44° 4′ 48″ N, 87° 16′ 12″ E, Germplasm ID: 80 acres-7), Aksu (41° 12′ 0″ N, 80° 12′ 0″ E, Germplasm ID: 49-4), Manas County (44° 12′ 0″ N, 86° 6′ 36″ E, Germplasm ID: 80-3), Ili (43° 54′ 36″ N, 81° 16′ 48″ E, Germplasm ID: 80-3-2), and Kuqa (41° 45′ 0″ N, 82° 56′ 24″ E, Germplasm ID: 81-6-1). The investigation included the shape, cracking mode, average length, and average diameter of the fruits under the same growth period. Furthermore, the oil content of the seeds was measured using the method referenced by Gu Yao et al. [12]. Seed oil is obtained by drying, grinding, leaching, filtering, and concentrating, and it is weighed to calculate the oil yield. Each variety was analyzed in triplicate.
2.10. Comprehensive assessment of germplasm from different regions
The original data of the 11 growth characteristics were listed in the original data table (Table S1). The maximum value for each indicator was identified as the standard value, normalizing it to a value of 1. Each value in that indicator column was divided by the standard value, and the resulting quotients were filled into the “Matrix Coordinate Table” (Table S2). The squared deviation sum of the values in each column of the matrix coordinate table relative to other standard values was calculated as , which was filled into the P2i comprehensive assessment table. The row sums provided the squared sums of the inter-point distances for each indicator:
. The evaluation order was determined according to the size of
[13].
2.11. Expression analysis of XsFAD genes in germplasm from different regions
Total RNA from the seeds of each variety was extracted using the RNAprep Pure Plant Total RNA Extraction Kit (DP432). The expression levels of relevant genes were detected using quantitative real-time polymerase chain reaction (qRT-PCR). The ABI 7500 fluorescence quantitative PCR detection system (Thermo Fisher Scientific Inc., Massachusetts, USA) was utilized according to the operational steps of Takara’s One Step TB Green® PrimeScript™ RT-PCR Kit II (Takara Bio Inc., Tokyo, Japan), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference gene. Each sample was biologically repeated three times, and the relative expression levels of each gene were calculated using the . The primers were designed using primer 5.0, the primer information is shown in Table 1.
3. Results
3.1. Identification and physicochemical property analysis of FAD genes in X. sorbifolium
Through the analysis of the whole-genome data of X. sorbifolium, 26 candidate genes containing FAD domains were identified, among which 25 members matched the characteristics of FAD family proteins. These 25 members were validated through the SMART, NCBI-CDD, and PFAM websites. The sequence was named XsFAD1-XsFAD25 based on their physical location on the chromosomes. The physicochemical property analysis of XsFAD-encoded proteins showed a sequence length range of 346-8,291 amino acids, a molecular weight distribution of 7,834.91-56,868.51 Da, and an isoelectric point range of 5.34-9.55. The predicted subcellular localization of XsFAD-encoded proteins includes chloroplasts, cytoplasm, and the endoplasmic reticulum (Table 2). These findings laid a foundation for further functional research and exploration of the role of these genes in biological processes.
3.2. Phylogenetic tree of XsFAD gene family
The 25 XsFAD protein sequences of X. sorbifolium were used to construct a phylogenetic tree based on their homology (Fig. 1). The results showed that the FAD genes of X. sorbifolium can be divided into three branches, with clade 1 containing 10 FAD genes at the base of the phylogenetic tree, clade 2 containing 8 FAD genes, and clade 3 containing 7 FAD genes. This clustering suggests potential functional similarities and evolutionary relationships among the FAD genes within the species.
3.3. Promoter cis-acting element analysis of XsFAD genes
To understand the role of XsFAD genes in stress response and signal transduction in X. sorbifolium, the cis-acting elements in the 2000 bp upstream of the ATG start codon in the promoter region of the 25 XsFAD genes were analyzed using the PlantCARE website. The results showed that the promoter region of XsFAD genes contains a series of important growth and abiotic stress response elements, such as ARE, MYB, and drought-inducibility (Fig. 2). Notably, the promoter regions of XsFAD1, XsFAD2, XsFAD4, XsFAD11, XsFAD20, and XsFAD21 contain response elements to antioxidant reactions. Studies suggested that regulatory elements in FAD promoters, such as WRI1, can influence oil content by modulating gene expression through interactions with enhancers and environmental conditions like darkness, ethylene, and temperature [14], indicating that XsFAD promoter may participate in environmental stress responses.
3.4. Chromosome localization analysis of XsFAD genes
As shown in Fig. 3, the 25 XsFAD genes are unevenly distributed on 11 chromosomes of X. sorbifolium, with the densest distribution on chromosomes 3 and 6, each containing 5 and 6 genes, respectively. The number of XsFAD genes on chromosomes 1, 4, 7, 9, 10, 12, 13, 14, and 15 is 1, 3, 2, 1, 1, 2, 1, 1, 2, respectively, while no XsFAD genes were found on chromosomes 2, 5, 8, and 11. The uneven distribution of XsFAD genes across chromosomes suggests potential genomic hotspots for FAD gene evolution and regulation in X. sorbifolium.
3.5. Analysis of conserved motifs, domain and gene structure of XsFAD genes
Based on evolutionary analysis, the sequence characteristics of the 25 XsFAD genes in X. sorbifolium were analyzed for their conserved motifs and gene structure features. The analysis of conserved motifs showed that the proteins in the same branch of XsFAD genes have similar motif compositions (Fig. 4), with motif 5 in all 9 proteins of clade 1, and motif 1, motif 2, motif 3, motif 4, motif 5, motif 8, and motif 9 in all 9 proteins, motif 6 and motif 7 in all 8 proteins of clade 2, motif 6 being present in all 7 proteins of clade 3. It can be seen that clade 1 members have more motif types and more regular motif compositions. The analysis of gene structure showed that XsFAD genes can contain up to 2 exons, with 8 genes lacking exons. Among these, proteins in clade 3 do not have exons. The absence of exons may be attributed to a combination of genetic mutations, evolutionary processes, alternative splicing, and gene regulation mechanisms.
By predicting the conserved domain of FAD genes, we found that FAD genes contain several conserved domains. Ye et al. studied the FAD gene family of Camellia oleifera and found that membrane-bound FADs can be further classified into five subfamilies, including FAB2, FAD4, FAD2/FAD6 (Omega-6 desaturases, ω6), FAD3/FAD7/FAD8 (Omega-3 desaturases, ω3), and ADS/SLD/DES, and identified conserved domains, including FA_desaturase, Lipid_DES, cyt-b5l, and TMEM189_B [15]. By analyzing the conserved domains of the XsFAD gene, clade 1 can be identified as FAB2, which contained one lengthy FA_desaturase_2 domain; clade 2 can be identified as FAD2/FAD6, including Delta12-FADS domain; the clade 3 can be identified as ADS/SLD/DES, which includes the FA_desaturase domain. This comprehensive analysis provides insights into the evolutionary relationships, motif conservation, gene structure variation, and conserved domain composition of XsFAD genes in X. sorbifolium.
3.6. Analysis of gene duplication and colinearity of XsFAD genes
To further analyze the evolutionary relationship of FAD genes in X. sorbifolium, the gene duplication events of the 25 XsFAD genes were analyzed. The results showed that there are three pairs of colinear genes among XsFAD genes: XsFAD20/XsFAD22, XsFAD20/XsFAD23, and XsFAD22/XsFAD23, indicating that the XsFAD gene family has undergone gene duplication events (Fig. 5A). By analyzing the gene collinearity of X. sorbifolium, Zea mays and Glycine max, we found that there are three genes collinear with XsFAD genes in maize and 21 genes collinear with XsFAD genes in soybean, which may have similar functions to XsFAD genes (Fig. 5B). The identification of colinear gene pairs highlights potential mechanisms contributing to the expansion and diversification of the FAD gene family within the X. sorbifolium genome.
(A) Collinearity analysis of genes within species. (B) Collinearity analysis of genes between species.
3.7. Analysis of selection pressure on XsFAD genes
To study the pressure on XsFAD genes during evolution, a selection pressure analysis was conducted, revealing that there are eight pairs of genes under selection pressure, with their Ka/Ks values all being less than 1. This indicates that these gene sequences have fewer non-synonymous mutations than synonymous mutations, thus experiencing purifying selection. Purifying selection is important for maintaining the functionality of genes, as it helps to eliminate deleterious mutations that could disrupt the protein’s structure or function (Table 3)[16]. The prevalence of purifying selection in XsFAD genes underscores the evolutionary conservation and functional significance of these genes in X. sorbifolium.
3.8. Protein interaction network analysis of XsFAD genes
To analyze the possible protein interactions of XsFAD-encoded proteins in X. sorbifolium, the homologous genes were used to construct a protein interaction network using the STRING software. The results showed that there may be various regulatory relationships among XsFAD-encoded proteins, with XsFAD2, XsFAD7, XsFAD8, XsFAD11, XsFAD18, XsFAD20 interacting with multiple XsFAD-encoded proteins, indicating potential functional collaboration among XsFAD proteins and their coordinating role in biological pathways and processes (Fig. 6).
3.9. Germplasm survey and oil content analysis of X. sorbifolium
After collecting sample information, it was found that the 10 X. sorbifolium germplasm fruit shapes are mainly spherical and peach-shaped, with Liudong-5 and 80-3 being cylindrical; the fruit opening method is generally 3-lobed; the largest fruit length and width are 73.03 mm and 74.17 mm for Liudong-1, respectively, and the smallest are 44.11 mm and 44.11 mm for 80 acres-1; the largest single fruit weight is 51.90 g for Liudong-1, and the smallest is 22.39 g for 80 acres-7; the largest seed diameter is 25.20 mm for 80 acres-7, and the smallest is 12.78 mm for 131–75; the highest thousand-seed weight is 1230.8 seeds for Liudong-5, and the lowest is 716 seeds for 81-6-1; the highest number of fruits is 303 for 131–75, and the lowest is 58 for 80 acres-7. In addition, the oil content of the seeds of 131-75 is the highest at 72.45%, and the lowest is 56.10% for 49-4 (Fig. 7A, Table S1). This data provides a valuable foundation for selecting high-oil content germplasm and breeding improved X. sorbifolium varieties tailored to specific traits and productivity goals.
(A)Oil Content of 10 X. sorbifolium Germplasm Seeds and FAD gene expression. (B) Correlation heatmap between oil content between FAD gene expression.
3.10. Comprehensive evaluation of different varieties of X. sorbifolium
To objectively evaluate the different varieties of X. sorbifolium germplasm, the diameter, number, weight, and oil content of the seeds were comprehensively evaluated using the multidimensional space (Euclidean) Multi-vector theoretical comprehensive evaluation mathematical model, “coordinate comprehensive evaluation method”. According to the
, the X. sorbifolium germplasm was ranked, with a higher
indicating poorer germplasm quality and a lower
indicating superior X. sorbifolium germplasm quality (Table S2, Table S3 ). Among the ten varieties, Liudong-5 had the highest evaluation, followed by Liudong-1 and 131-75. Liudong-5 has large fruit and seeds, with a high thousand-seed weight; although the oil content is low, the number of fruits and single plant seed weight values are high. Liudong-1 has a high number of single fruit seeds, and 131-75 has a high number of fruits, single plant seed weight, and oil content, indicating that these varieties have advantages. These three varieties of X. sorbifolium show promise for breeding superior cultivars through hybridization and biotechnological methods.
3.11. Correlation analysis of FAD gene family and oil content in X. sorbifolium seeds
To study the correlation between the FAD gene family and the oil content in X. sorbifolium seeds, the expression levels of three genes from each clade were detected in the seeds of different varieties of X. sorbifolium. The expression levels of the three FAD genes were significantly correlated with the oil content of X. sorbifolium seeds. In Liudong-5, with lower oil content, the gene expression level is also relatively low, while in 131-75, with higher oil content, the FAD gene expression level is relatively high. According to the correlation heatmap, we can find that seed oil content is highly correlated with the FAD18 gene, indicating that the FAD2 clade might play an important role in oil content accumulation (Fig. 7B). These findings underscore the potential regulatory role of the FAD gene family in determining oil content variations among different X. sorbifolium varieties, offering insights into the genetic mechanisms underlying oil accumulation in X. sorbifolium seeds.
4. Discussion
Fatty acid desaturases (FADs) play a critical role in plant lipid metabolism by introducing double bonds into hydrocarbon chains, thus influencing the composition of fatty acids in oils. FAD enzymes are essential in plant growth, development, and defense mechanisms, enabling resistance against various abiotic stresses, including temperature extremes. With the advancement of genomic sequencing and molecular biology, the FAD gene family has been elucidated in multiple plant species, including soybean, tomato, banana, and others [1,3,7,17]. The oil of X. sorbifolium holds significant medicinal value, and exploring the XsFAD gene family can facilitate the utilization and development of this oil.
4.1. XsFAD genes phylogenetics and evolution
XsFAD genes encode proteins that are classified into three branches in phylogenetic analysis, suggesting that proteins within the same branch may share similar biological functions. The gene structure, conserved motifs, and domains provide evidence for the phylogenetic tree, with a cluster of genes sharing structural similarity. The sizes of XsFAD genes vary widely, with differences in the number of exons, indicating evolutionary divergence within the XsFAD gene family. Plant FADs can be divided into soluble desaturases and membrane-bound desaturases according to their solubility. Membrane-bound FADs can be further classified into five subfamilies according to their distinct functions. Fatty acid desaturase-2 (FAD2) encodes a ω-6 desaturase that functions in the endoplasmic reticulum, whereas fatty acid desaturase-6 (FAD6) encodes a ω-6 desaturase that functions in the plastids [18]. In Arabidopsis thaliana, three gene products of FAD3, FAD7, and FAD8 are responsible for the production of trienoic fatty acids via unsaturation at the ω-3 position [19,20]. Moreover, the FAD gene family exhibits tissue-specific expression, with CtFAD2-1 and CtFAD2-10 in Carthamus tinctorius being specifically expressed in developing seeds and flower heads, respectively, while CtFAD2-2 shows relatively low oleic acid desaturase activity across the plant. CtFAD2-5 and CtFAD2-8 are specifically expressed in the root tissue of red flower seedlings, whereas CtFAD2-3 is mainly expressed in cotyledons and hypocotyls [21]. Tomato SlFAD is involved in the expression of root, stem, and leaf tissues; SlFAD8 is predominantly expressed in leaves [3]. XsFAD genes may also exhibit tissue specificity, with distinct functions in different tissues.
Gene duplication is a crucial mechanism for the expansion and evolution of plant gene families. Fragment and tandem duplication are considered the primary modes of gene family expansion. In this study, only three pairs of tandemly repeated genes were identified in the 14 chromosome arrays, indicating that fragment duplication played a significant role in the amplification of the XsFAD gene family. Similar amplification patterns have been observed in other plant gene families, such as the expansion of sweet potato (Ipomoea batatas) CYP450 genes through tandem and fragment duplication, and the large-scale fragment repetition in the tea tree (Camellia sinensis) ABC gene family, with tandem duplication serving a supplementary role [22,23]. By analyzing the gene collinearity of X. sorbifolium, Zea mays and Glycine max, we found interesting gene conservation patterns and potential functional similarities. The identification of collinear genes in these species means that some genetic factors in FAD-related pathways are conserved. The common collinearity pattern implies the potential functional conservation or convergence of these genes in fatty acid metabolism, oil biosynthesis, or other biological processes related to FAD genes.
4.2. Functional insights
Transcription factors bind to cis-acting elements in the promoter regions of XsFAD genes, regulating gene expression. Identified transcription factors in the promoter regions of XsFAD genes are associated with drought resistance, antioxidant activity, light response, gibberellin response, and flavonoid synthesis, indicating that FAD genes can enhance the resistance of the plant to both biotic and abiotic stresses. FADs maintain cell membrane fluidity and integrity by altering the synthesis and ratio of unsaturated fatty acids, thus influencing plant stress tolerance [24]. For example, rice mutants lacking the ω-3 fatty acid desaturase (FADs) are unable to adapt to cold temperatures [25]. Light also affects fatty acid desaturation in plants, with dark treatment reducing the expression levels of FAD3 and FAD8 in soybean cell cultures and FAD2 in olive fruits, indicating light-dependent transcriptional regulation of FAD genes [22,26,27]. Additionally, tomato SlFAB5 can regulate various stages of fruit development under the influence of exogenous hormones [3]. The stress response of XsFAD genes to the environment will be further verified by experiments.
Fatty acid desaturase 2 (FAD2) enzymes facilitate the conversion of oleic acid (OA) to linoleic acid (LA) in plants. This crucial polyunsaturated fatty acid is a significant component of plant oils; the catalytic activity of FAD2 is closely related to the oil content in plant seeds [28]. Artemisia sphaerocephala, a desert shrub, is rich in linoleic acid and it possesses a large FAD2 gene family. The proteins encoded by the FAD2 gene family exhibit high sequence similarity and are relatively conserved during evolution. The heterologous expression in Saccharomyces cerevisiae demonstrates that AsFAD2-1, AsFAD2-10, and AsFAD2-3 can convert oleic acid to linoleic acid. The AsFAD2-1 gene is strongly expressed in developing seeds, which may be closely related to the high accumulation capacity of LA in seeds [29]. Mutations in the FAD2-1 gene in soybeans increase the content of oleic acid in seeds [17]. By detecting the oil content in different varieties of X. sorbifolium and quantifying the FAD genes, it can be shown that the expression level of FAD genes is significantly correlated with the oil content of X. sorbifolium seeds. Among them, 131-75 has the highest oil content, and the relative expression levels of XsFAD18 in this variety are relatively high. This indicates that FAD2 genes can promote an increase in seed oil content.
4.3. Environmental adaptation
The results showed that the comprehensive evaluation of the characteristics of different varieties of X. sorbifolium could lay a foundation for selecting high-oil-content germplasm and cultivating improved varieties. Among them, Liudong-5 was rated as the best variety, followed by Liudong-1 and 131-75. Liudong-5 has large fruits and seeds with a high 1000-grain weight. Liudong-1 has a high number of seeds per fruit, while 131-75 is excellent in fruit number, seed weight per plant, and oil content. These varieties show the potential to cultivate excellent varieties through hybridization and biotechnology. 131-75 has a significantly higher oil content than other varieties, this may be related to environmental differences in the Xinjiang region of China, as this variety was collected from a poor area in the southern Xinjiang region, where the average annual precipitation is extremely low, and the soil pH is around 8.8-9. Studies show that the FAD gene family plays an important role in abiotic stress and drought often increases oil content [30, 31]. This finding suggests that drought and salt tolerance signaling pathways in X. sorbifolium may regulate lipid synthesis at the molecular level, this requires further systematic experimental validation.
5. Conclusion
In conclusion, 25 XsFAD genes were identified. By analyzing gene structure, XsFAD genes were divided into three subfamilies, FAB2, FAD2/FAD6, and ADS/SLD/DES, and three pairs of colinear genes were found. Through gene expression analysis, it was found that the expression of XsFAD18 in FAD2 was high and significantly related to seed oil content. In future research, we will construct the genetic transformation system of X. sorbifolium and further verify the function of XsFAD genes. The molecular regulatory mechanisms in lipid synthesis will be revealed by joint analysis of the transcriptome and metabolomics. Then screen the key transcription factors by yeast hybridization and other experiments, and then analyze the promoter site of the transcription factor binding by ChIP and EMSA experiments. Meanwhile, we will design related stress experiments, such as drought stress and cold stress, to reveal the efficiency mechanism of environmental factors on oil production. This study provides a theoretical research basis and reference for the research of variety breeding, cultivation system, and genetic breeding of China Xinjiang X. sorbifolium Fruit.
Supporting information
S1 Table. Basic Information of 10 X. sorbifolium Germplasm.
https://doi.org/10.1371/journal.pone.0318900.s001
(PDF)
S2 Table. Coordinate Table of Matrix for Comprehensive Evaluation of 10 X. sorbifolium Germplasm.
https://doi.org/10.1371/journal.pone.0318900.s002
(PDF)
S3 Table.
Comprehensive Evaluation of
of 10 X. sorbifolium Provenances.
https://doi.org/10.1371/journal.pone.0318900.s003
(PDF)
References
- 1. Liu K, Zhao S, Wang S, Wang H, Zhang Z. Identification and analysis of the FAD gene family in walnuts (Juglans regia L.) based on transcriptome data. BMC Genomics. 2020;21(1):299. pmid:32293267
- 2. Liang Q, Li H, Li S, Yuan F, Sun J, Duan Q, et al. The genome assembly and annotation of yellowhorn (Xanthoceras sorbifolium Bunge). Gigascience. 2019;8(6):giz071. pmid:31241155
- 3. Xi R, Liu H, Chen Y, Zhuang H, Han H, Wang H, et al. Genome-Wide Characterization of Tomato FAD Gene Family and Expression Analysis under Abiotic Stresses. Plants (Basel). 2023;12(22):3818. pmid:38005715
- 4. Wang ML, Chen CY, Tonnis B, Pinnow D, Davis J, An Y-QC, et al. Changes of Seed Weight, Fatty Acid Composition, and Oil and Protein Contents from Different Peanut FAD2 Genotypes at Different Seed Developmental and Maturation Stages. J Agric Food Chem. 2018;66(14):3658–65. pmid:29558122
- 5. Saini R, Adhikary A, Juneja S, Kumar R, Singh I, Nayyar H, et al. Drought priming triggers diverse metabolic adjustments and induces chilling tolerance in chickpea (Cicer arietinum L.). Plant Physiol Biochem. 2023;194418–39. pmid:36493590
- 6. Shi Y, Yue X, An L. Integrated regulation triggered by a cryophyte ω-3 desaturase gene confers multiple-stress tolerance in tobacco. J Exp Bot. 2018;69(8):2131–48. pmid:29432580
- 7. Cheng C, Liu F, Sun X, Wang B, Liu J, Ni X, et al. Genome-wide identification of FAD gene family and their contributions to the temperature stresses and mutualistic and parasitic fungi colonization responses in banana. Int J Biol Macromol. 2022;204:661–76. pmid:35181326
- 8. Fang X, Jia Z, Yu T, Rui P, Zheng H, Lu Y, et al. FATTY ACID DESATURASE4 enhances plant RNA virus replication and undergoes host vacuolar ATPase-mediated degradation. Plant Physiol. 2024;196(2):1502–17. pmid:38935533
- 9. Xue Y-F, Fu C, Chai C-Y, Liao F-F, Chen B-J, Wei S-Z, et al. Engineering the Staple Oil Crop Brassica napus Enriched with α-Linolenic Acid Using the Perilla FAD2-FAD3 Fusion Gene. J Agric Food Chem. 2023;71(19):7324–33. pmid:37130169
- 10. Yara A, Yaeno T, Hasegawa M, Seto H, Montillet J-L, Kusumi K, et al. Disease resistance against Magnaporthe grisea is enhanced in transgenic rice with suppression of omega-3 fatty acid desaturases. Plant Cell Physiol. 2007;48(9):1263–74. pmid:17716996
- 11. Zang E, Qiu B, Chen N, Li C, Liu Q, Zhang M, et al. Xanthoceras sorbifolium Bunge: A Review on Botany, Phytochemistry, Pharmacology, and Applications. Front Pharmacol. 2021;12:708549. pmid:34526898
- 12. Gu Y, Zeng Y, Chen S, Zhou L, Yang L. Oil content and fatty acid composition of walnut produced from different areas in guangxi. Modern Agricultural Science and Technology. 2020;23(23):208–10.
- 13. Zhang L, Zheng Y, Wu J, Sun P, Yang T. NaCl stress of new aigeiros clones seedlings from water cultures. Journal of Zhejiang Forestry College. 2000;17(2):121-125.
- 14. Zhang Q, Sun R, Zheng Y, Yuan Y, Li D. Isolation and characterization of the EgWRI1 promoter from oil palm (Elaeis guineensis Jacq.) and its response to environmental stress and ethylene. PLoS One. 2019;14(12):e0225115. pmid:31800586
- 15. Ye Z, Mao D, Wang Y, Deng H, Liu X, Zhang T, et al. Comparative genome-wide identification of the fatty acid desaturase gene family in tea and oil tea. Plants (Basel). 2024;13(11):1444. pmid:38891253
- 16. Gomathinayagam S, Kanagalingam S, Chandrasekaran S, Krishnan T, Kodiveri Muthukaliannan G. Millennial-scale microbiome analysis reveals ancient antimicrobial resistance conserved despite modern selection pressures. Environ Microbiome. 2024;19(1):110. pmid:39695840
- 17. Lakhssassi N, Zhou Z, Liu S, Colantonio V, AbuGhazaleh A, Meksem K. Characterization of the FAD2 gene family in soybean reveals the limitations of gel-based TILLING in genes with high copy number. Front Plant Sci. 2017;8:324. pmid:28348573
- 18. Zhang J-T, Zhu J-Q, Zhu Q, Liu H, Gao X-S, Zhang H-X. Fatty acid desaturase-6 (Fad6) is required for salt tolerance in Arabidopsis thaliana. Biochem Biophys Res Commun. 2009;390(3):469–74. pmid:19799856
- 19. Kodama H, Hamada T, Horiguchi G, Nishimura M, Iba K. Genetic enhancement of cold tolerance by expression of a gene for chloroplast [omega]-3 fatty acid desaturase in transgenic tobacco. Plant Physiol. 1994;105(2):601–5. pmid:12232227
- 20. Park M-E, Lee HJ, Kim I, Kim HU. Characterization of fab2 T-DNA insertion mutants in terms of fatty acid composition and plant phenotype. Plant Signal Behav. 2023;18(1):2213937. pmid:37204036
- 21. Cao S, Zhou X-R, Wood CC, Green AG, Singh SP, Liu L, et al. A large and functionally diverse family of Fad2 genes in safflower (Carthamus tinctorius L.). BMC Plant Biol. 2013;135. pmid:23289946
- 22. Saini R, Kumar S. Genome-wide identification, characterization and in-silico profiling of genes encoding FAD (fatty acid desaturase) proteins in chickpea (Cicer arietinum L.). Plant Gene. 2019;18:100180.
- 23. Tovuu A, Zulfugarov IS, Wu G, Kang IS, Kim C, Moon BY, et al. Rice mutants deficient in ω-3 fatty acid desaturase (FAD8) fail to acclimate to cold temperatures. Plant Physiol Biochem. 2016;109:525–35. pmid:27835850
- 24. Collados R, Andreu V, Picorel R, Alfonso M. A light-sensitive mechanism differently regulates transcription and transcript stability of omega3 fatty-acid desaturases (FAD3, FAD7 and FAD8) in soybean photosynthetic cell suspensions. FEBS Lett. 2006;580(20):4934–40. pmid:16930600
- 25. Hernández ML, Padilla MN, Sicardo MD, Mancha M, Martínez-Rivas JM. Effect of different environmental stresses on the expression of oleate desaturase genes and fatty acid composition in olive fruit. Phytochemistry 2011;72(2–3):178–87. pmid:21194717
- 26. Lin X, Tang B, Li Z, Shi L, Zhu H. Genome-wide identification and expression analyses of CYP450 genes in sweet potato (Ipomoea batatas L.). BMC Genomics 2024;25(1):58. pmid:38218763
- 27. Shen C, Li X. Genome-wide identification and expression pattern profiling of the ATP-binding cassette gene family in tea plant (Camelliasinensis). Plant Physiol Biochem. 2023;202:107930. pmid:37552927
- 28. Zhao M, Wang W, Wei L, Chen P, Peng L, Qin Z, et al. The evolution and biocatalysis of FAD2 indicate its correlation to the content of seed oil in plants. Int J Mol Sci. 2019;20(4):849. pmid:30781405
- 29. Miao X, Zhang L, Hu X, Nan S, Chen X, Fu H. Cloning and functional analysis of the FAD2 gene family from desert shrub Artemisia sphaerocephala. BMC Plant Biol. 2019;19(1):481. pmid:31703625
- 30. Xu B, Gao M, Hu W, Zhao W, Wang S, Zhou Z. Individual and interactive influences of elevated air temperature and soil drought at the flowering and boll-forming stage on cottonseed yield and nutritional quality. The Crop Journal. 2022;10(1):128–39.
- 31. Luisa Hernández M, Dolores Sicardo M, Arjona PM, Martínez-Rivas JM. Specialized functions of olive FAD2 gene family members related to fruit development and the abiotic stress response. Plant Cell Physiol. 2020;61(2):427–41. pmid:31730170