Characterization of the Fatty Acyl-CoA Reductase (FAR) Gene Family and Its Response to Abiotic Stress in Rice (Oryza sativa L.)

Fatty acyl-CoA reductase (FAR) is an important NADPH-dependent enzyme that can produce primary alcohol from fatty acyl-CoA or fatty acyl-carrier proteins as substrates. It plays a pivotal role in plant growth, development, and stress resistance. Herein, we performed genome-wide identification and expression analysis of FAR members in rice using bioinformatics methods. A total of eight OsFAR genes were identified, and the OsFARs were comprehensively analyzed in terms of phylogenetic relationships, duplication events, protein motifs, etc. The cis-elements of the OsFARs were predicted to respond to growth and development, light, hormones, and abiotic stresses. Gene ontology annotation analysis revealed that OsFAR proteins participate in biological processes as fatty acyl-CoA reductase during lipid metabolism. Numerous microRNA target sites were present in OsFARs mRNAs. The expression analysis showed that OsFARs were expressed at different levels during different developmental periods and in various tissues. Furthermore, the expression levels of OsFARs were altered under abiotic stresses, suggesting that FARs may be involved in abiotic stress tolerance in rice. The findings presented here serve as a solid basis for further exploring the functions of OsFARs.

Previous studies have shown that different FARs usually exhibit different specificities for acyl chain lengths, resulting in the production of fatty alcohols with different chain lengths that not only affect the composition of plant epidermal wax but are also closely related to plant stress tolerance [7].The first gene encoding a FAR protein was cloned from Simmondsia chinensis [8,9].Since then, eight FAR genes have been identified in Arabidopsis thaliana based on the FAR sequence from S. chinensis [10,11].Among them, AtFAR3/CER4 is expressed in various tissues and is mainly responsible for the synthesis of cuticular wax in the epidermal cells in stems.Heterologous expression of AtFAR3/CER4 in yeast can generate C24:0 and C26:0, while the mutation in CER4 leads to a specific blockade of the alcohol formation pathway [12,13].Ectopic expressions of wheat (Triticum aestivum L.) TaFAR2, TaFAR3, and TaFAR4 in the Arabidopsis cer4 mutant increased the primary alcohol production of C22:0 to C30:0 and partially restored the function of AtCER4.In addition, when the transgenic plants were grown under drought conditions, all the TaFARs were upregulated, and more leaf cuticular wax was accumulated, which was an important strategy for limiting the dehydration of the plant cells and other organisms under drought conditions [14].Inhibiting the expressions of BnA1.CER4 and BnC1.CER4 in Brassica napus can decrease the content of branched primary alcohols and increase the content of other branched components that disrupt the wax crystal type, leading to increased cuticle permeability and enhanced resistance to fungal pathogens [15].Both poplar (Populus tomentosa Carr.)PtoMYB142 and Arabidopsis AtMYB94 can bind to the promoter regions of PtoCER4 and AtCER4, respectively, through an abscisic acid (ABA) signaling pathway, which, when induced by drought, results in upregulation of CER4, deposition of epidermal waxes, and enhancement of drought resistance [16,17].OsFAR1 in rice (Oryza sativa L.) responds positively to abiotic stress and ABA induction.Particularly, overexpressing OsFAR1 can increase the content of primary alcohols and total waxes in Arabidopsis and rice, reduce the accumulation of reactive oxygen species (ROS), and enhance drought tolerance in rice [18].
FAR also affects plant fertility by regulating lipid metabolism [19].For example, AtFAR2/MS2 can reduce palmitoyl-ACP in the cytoplasm of tapetum cells to C16:0 alcohols, which are essential primary alcohols in the synthesis of pollen walls.Thus, a loss-offunction mutation in AtFAR2/MS2 leads to abnormal development of the pollen wall [20].OsDPW/FAR2 from rice and ZmMS6021 from maize (Zea mays L.), highly homologous to AtMS2, also exhibit similar functions in the biosynthesis of anther keratin and the development of pollen outer walls [21,22].With the large-scale completion of plant genome sequencing and the wide application of bioinformatics technology, FAR genes have been identified in most species [20,[22][23][24][25].However, no systematic and detailed analysis of the FAR family members in rice has been carried out thus far.Therefore, the study of OsFARs' responses to abiotic stresses is necessary for enhancing crop tolerance.
In the study, eight members of the rice FAR family were identified and their physicochemical properties, motif patterns, gene structures, phylogenetic relationships, cis-acting elements, and gene duplication events were comprehensively analyzed.Furthermore, miRNA target prediction and gene ontology (GO) enrichment analysis were also performed.Finally, we investigated the expression patterns of OsFARs in different organs/tissues and in response to various abiotic stresses using gene microarray and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).This study provides a theoretical framework for understanding the role of FARs in the evolution, growth, development, and survival of rice under stresses, and also provides a reference for the subsequent functional verification.

Whole-Genome Characterization of FARs in Rice
Through comparative analysis and domain identification, eight OsFAR genes were identified across the entire rice genome (Table 1).Analysis of physicochemical properties revealed that the lengths of CDS sequences range from 1242 to 1827 bp, and protein lengths range from 413 to 608 aa.The average MW is 56,261.35Da, ranging from 47,928.54 to 65,228.14Da, and the pI is between 7.03 and 8.98, with an average of 8.27.Except for OsFAR1 and OsFAR7, the GRAVY values of all other OsFARs are less than 0, indicating that they are hydrophilic proteins.Subcellular localization prediction reveals that OsFAR1 and OsFAR7 reside in the nucleus, OsFAR2 is situated within the chloroplast, and the other OsFARs are located in the cytoplasm.

FAR Protein Structure Analysis
The secondary structures of the OsFARs were analyzed (Supplementary Table S2).The results indicated that the OsFAR proteins are mainly composed of an alpha helix (42.43~49.31%),random coil (31.09~37.83%),extended strand (14.47~16.46%),and extended helix (4.01~5.3%).The alpha helix and the random coil play a major role in the secondary structure of OsFAR proteins.Differences in gene functions are inextricably linked to their structures [26].Tertiary structural homology modeling of the OsFAR proteins indicated that all OsFAR proteins, except OsFAR2, are similar (Figure 1), indicating a high level of conservation.

FAR Protein Structure Analysis
The secondary structures of the OsFARs were analyzed (Supplementary Table S2).The results indicated that the OsFAR proteins are mainly composed of an alpha helix (42.43~49.31%),random coil (31.09~37.83%),extended strand (14.47~16.46%),and extended helix (4.01~5.3%).The alpha helix and the random coil play a major role in the secondary structure of OsFAR proteins.Differences in gene functions are inextricably linked to their structures [26].Tertiary structural homology modeling of the OsFAR proteins indicated that all OsFAR proteins, except OsFAR2, are similar (Figure 1), indicating a high level of conservation.

Multiple Sequence Alignment, Phylogenetic Analysis, and Classification of OsFAR Proteins
To explore the evolutionary relationships between the OsFARs, a neighbor-joining (NJ) phylogenetic tree was constructed using protein sequences of FARs from rice, Arabidopsis, barley, wheat, maize, and soybean.The results showed that compared to Arabidopsis and soybean, the FAR proteins from rice, barley, wheat, and maize exhibited a closer genetic relationship across the entire evolutionary tree (Figure 2).Based on the phylogenetic analysis, the FARs were categorized into five groups (I~V), with each group, except Group V, containing 1~3 OsFARs.

Multiple Sequence Alignment, Phylogenetic Analysis, and Classification of OsFAR Proteins
To explore the evolutionary relationships between the OsFARs, a neighbor-joining (NJ) phylogenetic tree was constructed using protein sequences of FARs from rice, Arabidopsis, barley, wheat, maize, and soybean.The results showed that compared to Arabidopsis and soybean, the FAR proteins from rice, barley, wheat, and maize exhibited a closer genetic relationship across the entire evolutionary tree (Figure 2).Based on the phylogenetic analysis, the FARs were categorized into five groups (I~V), with each group, except Group V, containing 1~3 OsFARs.

Conserved Motifs and Gene Structures of OsFAR Gene Family Members
The analysis of gene structure and conserved motif composition of OsFARs showed that the members belonging to the same subfamily exhibit similar motif structures, especially in terms of the number, type, and arrangement of motifs, which display a significant degree of overlap.Except for OsFAR6, which lacks motifs 3, 4, and 10, all the other OsFARs contain 10 motifs.We also analyzed the protein domains of the OsFARs and found that each OsFAR contains the NAD_binding_4 and FAR_C domains (Figure 3C).Each OsFAR gene contains 6~10 exons and 5~9 introns (Figure 3D), and the 5′ UTR and 3′ UTR of OsFARs are usually present in the OsFAR genomic sequence, with the exception of OsFAR7, which lacks both the 5′ UTR and 3′ UTR (Figure 3D).Perhaps due to the specific evolutionary history, function, or genome organization, OsFAR7 does not have UTR regions, which may affect gene expression regulation, post-transcriptional processing, or mRNA stability [27].OsFARs in related subfamilies often exhibit similar exon/intron structures, indicating a high correlation between phylogenetic relationships among gene family members and gene structure.Different colors represent different subfamilies as follows: Group I (red), Group II (orange), Group III (green), Group IV (blue), and Group V (purple).

Conserved Motifs and Gene Structures of OsFAR Gene Family Members
The analysis of gene structure and conserved motif composition of OsFARs showed that the members belonging to the same subfamily exhibit similar motif structures, especially in terms of the number, type, and arrangement of motifs, which display a significant degree of overlap.Except for OsFAR6, which lacks motifs 3, 4, and 10, all the other Os-FARs contain 10 motifs.We also analyzed the protein domains of the OsFARs and found that each OsFAR contains the NAD_binding_4 and FAR_C domains (Figure 3C).Each OsFAR gene contains 6~10 exons and 5~9 introns (Figure 3D), and the 5 ′ UTR and 3 ′ UTR of OsFARs are usually present in the OsFAR genomic sequence, with the exception of OsFAR7, which lacks both the 5 ′ UTR and 3 ′ UTR (Figure 3D).Perhaps due to the specific evolutionary history, function, or genome organization, OsFAR7 does not have UTR regions, which may affect gene expression regulation, post-transcriptional processing, or mRNA stability [27].OsFARs in related subfamilies often exhibit similar exon/intron structures, indicating a high correlation between phylogenetic relationships among gene family members and gene structure.

Analysis of Cis-Regulatory Element in the Promoter Region of OsFARs
Cis-regulatory elements (CREs) are located in the promoter region and do not encode proteins but play a crucial role in the regulation of gene expression [28,29].This study identified a total of 33 major CREs (Figure 4A) that could be categorized into five functional groups: development-related elements, hormone-responsive elements, lightresponsive elements, stress-responsive elements, and MYB-related elements.OsFAR5 had the highest number of CREs (79), whereas OsFAR1 contained the lowest number (30).All OsFARs contained MYB-related elements and light-responsive elements, and most OsFARs contained methyl jasmonate (MeJA) elements (TGACG-motif and CGTCA-motif) and ABRE elements (ABA-related) (Figure 4).In addition, 26 stress-response elements (STRE) and 14 drought-response elements (MBS) were found in the promoter region of the OsFARs.The above analysis indicates that the OsFAR genes may be involved in the regulation of physiological processes such as plant growth and development, light regulation, plant hormone response, and stress.

Analysis of Cis-Regulatory Element in the Promoter Region of OsFARs
Cis-regulatory elements (CREs) are located in the promoter region and do not encode proteins but play a crucial role in the regulation of gene expression [28,29].This study identified a total of 33 major CREs (Figure 4A) that could be categorized into five functional groups: development-related elements, hormone-responsive elements, light-responsive elements, stress-responsive elements, and MYB-related elements.OsFAR5 had the highest number of CREs (79), whereas OsFAR1 contained the lowest number (30).All OsFARs contained MYB-related elements and light-responsive elements, and most OsFARs contained methyl jasmonate (MeJA) elements (TGACG-motif and CGTCA-motif) and ABRE elements (ABA-related) (Figure 4).In addition, 26 stress-response elements (STRE) and 14 drought-response elements (MBS) were found in the promoter region of the OsFARs.The above analysis indicates that the OsFAR genes may be involved in the regulation of physiological processes such as plant growth and development, light regulation, plant hormone response, and stress.

Analysis of Cis-Regulatory Element in the Promoter Region of OsFARs
Cis-regulatory elements (CREs) are located in the promoter region and do not encode proteins but play a crucial role in the regulation of gene expression [28,29].This study identified a total of 33 major CREs (Figure 4A) that could be categorized into five functional groups: development-related elements, hormone-responsive elements, lightresponsive elements, stress-responsive elements, and MYB-related elements.OsFAR5 had the highest number of CREs (79), whereas OsFAR1 contained the lowest number (30).All OsFARs contained MYB-related elements and light-responsive elements, and most OsFARs contained methyl jasmonate (MeJA) elements (TGACG-motif and CGTCA-motif) and ABRE elements (ABA-related) (Figure 4).In addition, 26 stress-response elements (STRE) and 14 drought-response elements (MBS) were found in the promoter region of the OsFARs.The above analysis indicates that the OsFAR genes may be involved in the regulation of physiological processes such as plant growth and development, light regulation, plant hormone response, and stress.

Analyses of Chromosomal Distribution and Gene Duplication of the OsFAR Genes
Based on the genomic data, we found that the OsFARs are unevenly distributed on the five chromosomes of rice (3, 4, 7, 8, 9) (Figure 5A), with one each on chromosomes 3 and 9, and two on Chromosomes 4, 7, and 8, respectively.

Analyses of Chromosomal Distribution and Gene Duplication of the OsFAR Genes
Based on the genomic data, we found that the OsFARs are unevenly distributed on the five chromosomes of rice (3, 4, 7, 8, 9) (Figure 5A), with one each on chromosomes 3 and 9, and two on Chromosomes 4, 7, and 8, respectively.Gene duplication is crucial for the generation of new genes and functions, and segmental duplication and tandem duplication are important driving forces for the expansion of gene families.Therefore, we analyzed gene duplication events in OsFAR genes.The results showed that the eight OsFARs are so far away from each other in the Gene duplication is crucial for the generation of new genes and functions, and segmental duplication and tandem duplication are important driving forces for the expansion of gene families.Therefore, we analyzed gene duplication events in OsFAR genes.The results showed that the eight OsFARs are so far away from each other in the genome that no tandem duplication occurs.However, we identified a pair of fragmentally duplicated FAR genes (OsFAR1 and OsFAR4) (Figure 5A).
Collinearity analysis can be used to study the evolution and affinity of species [30].Through collinearity analysis, it was found that there are one pair, four pairs, and six pairs of FAR collinear gene pairs between rice and tomato, barley and B. distachyon, respectively, but there was no FAR collinearity between rice and Arabidopsis (Figure 5B).Meanwhile, we found that OsFAR4 exhibited collinearity in several representative species, except Arabidopsis, suggesting that OsFAR4 is more conserved and plays an important role in the evolutionary process [31].
To further explore the selection of these fragment replication genes, the synonymous mutation frequency (Ks) and non-synonymous mutation frequency (Ka) values were calculated.The Ka/Ks ratio provides a powerful tool for elucidating the evolutionary process and selection pressure of OsFARs [32], with Ka/Ks = 1 suggesting neutral selection, values less than 1 indicating purifying selection, and a ratio greater than 1 pointing to positive selection.It was found that the Ka/Ks values of the segment repeat genes were consistently below 1 (0.445), suggesting that these replication genes were subjected to purifying selection during evolution and were involved in maintaining the conservative structure of the OsFARs.We evaluated the Ka/Ks of collinear gene pairs between rice and other species, resulting in a total of nine collinear gene pairs (Supplementary Table S3), with a range of Ka/Ks values between 0.178 and 0.306, which suggests that the cross-species FAR family was under purifying or stable selection during evolution.

GO Enrichment Analysis and miRNA Targeting Prediction
The PANNZER online website was used for the GO enrichment analysis of the OsFARs.The results (Supplementary Table S4) showed that eight OsFARs were significantly enriched for the lipid metabolic process (GO: 0006629).In addition to OsFAR6, seven other genes were involved in the suberin biosynthetic process (GO: 0010345).Furthermore, apart from OsFAR2 and OsFAR6, the others were involved in the acyl-CoA metabolic process (GO: 0006637).In terms of molecular function, all eight genes were predicted to be involved in alcohol-forming very long-chain fatty acyl-CoA reductase activity (GO: 0080019) and alcohol-forming long-chain fatty acyl-CoA reductase activity (GO: 0102965).In summary, OsFARs are mainly involved in the biological processes associated with lipid metabolism.
MicroRNA (miRNA) is an endogenous small-molecule non-coding RNA that mediates the post-transcriptional regulation of gene expression by recognizing and inhibiting target genes through sequence complementation and is also an important regulator of plant growth, reproduction, and stress responses [33].The CDS sequence of each OsFAR was used to predict miRNAs using the psRNATarget database, and a total of 65 mature miR-NAs (19-24 nt) were identified (Figure 6).Among these, eight targeted multiple OsFARs, while the remaining miRNAs exhibited specificity for each gene.The number of miRNAs targeting OsFAR8 was the highest (13), while the number of miRNAs targeting OsFAR3 and OsFAR5 was the lowest (1).Most predicted miRNAs targeting OsFAR had strong regulatory effects through cleavage, while only eight miRNAs regulated OsFAR expression through translation inhibition (Supplementary Table S5).It can be inferred that cleavage, serving as the primary function of miRNA, plays a crucial role in regulating the expressions of OsFARs.

Expression Patterns of FARs in Different Tissues
Analyzing the expression patterns of OsFARs in different tissues/stages of rice development can further reveal the potential functions of OsFARs.Expression data for rice FARs in different tissues of the IR64 were obtained.Expression data generated under drought, salt, and low-temperature conditions were also obtained from the Rice eFP Browser website.The results (Figure 7A) indicated that OsFAR1 and OsFAR4 exhibited similar expression patterns, with high expression at all stages of young root, SAM, and panicle development, while OsFAR8 was highly expressed at the P4, P5, and P6 stages of young root and panicle development.Given their unique tissue-specific features, these genes could be crucial for the morphogenesis of the panicle.For example, OsFAR5, OsFAR3, and OsFAR2 were highly expressed during a certain stage of panicle development.OsFAR6 and OsFAR7 were mainly expressed in young roots and leaves, and at different stages of seed development.However, there was almost no expression of OsFARs, except OsFAR7, in mature leaves.The results indicated that OsFARs exhibit differential expression patterns in various tissues and are potentially involved in panicle development.

Expression Patterns of FARs in Different Tissues
Analyzing the expression patterns of OsFARs in different tissues/stages of rice development can further reveal the potential functions of OsFARs.Expression data for rice FARs in different tissues of the IR64 were obtained.Expression data generated under drought, salt, and low-temperature conditions were also obtained from the Rice eFP Browser website.The results (Figure 7A) indicated that OsFAR1 and OsFAR4 exhibited similar expression patterns, with high expression at all stages of young root, SAM, and panicle development, while OsFAR8 was highly expressed at the P4, P5, and P6 stages of young root and panicle development.Given their unique tissue-specific features, these genes could be crucial for the morphogenesis of the panicle.For example, OsFAR5, OsFAR3, and OsFAR2 were highly expressed during a certain stage of panicle development.OsFAR6 and OsFAR7 were mainly expressed in young roots and leaves, and at different stages of seed development.However, there was almost no expression of OsFARs, except OsFAR7, in mature leaves.The results indicated that OsFARs exhibit differential expression patterns in various tissues and are potentially involved in panicle development.
To further explore the expression patterns of OsFARs, we analyzed microarray data of 7-day-old rice seedlings under three abiotic stresses.As shown in Figure 7B, except for OsFAR7 and OsFAR8, all the FAR genes were upregulated under cold stress, while OsFAR8 expression was upregulated under salt stress.Under drought stress, OsFAR5 was upregulated, while the other OsFAR genes were downregulated.The above results indicate that OsFARs are differentially sensitive to different abiotic stresses.

Expression Profiles of OsFARs under Abiotic Stresses
In order to study the response patterns of the OsFARs under different stress conditions, two-week-old rice seedlings were treated with salt, drought, high temperature, low temperature, and ABA, and samples were taken at 0, 6, and 24 h.The To further explore the expression patterns of OsFARs, we analyzed microarray data of 7-day-old rice seedlings under three abiotic stresses.As shown in Figure 7B, except for OsFAR7 and OsFAR8, all the FAR genes were upregulated under cold stress, while OsFAR8 expression was upregulated under salt stress.Under drought stress, OsFAR5 was upregulated, while the other OsFAR genes were downregulated.The above results indicate that OsFARs are differentially sensitive to different abiotic stresses.

Expression Profiles of OsFARs under Abiotic Stresses
In order to study the response patterns of the OsFARs under different stress conditions, two-week-old rice seedlings were treated with salt, drought, high temperature, low temperature, and ABA, and samples were taken at 0, 6, and 24 h.The relative expression levels of OsFARs in the rice leaves under different stresses were detected using RT-qPCR.The results showed that, except for OsFAR7, which was not detected due to its low expression in the leaves, the other OsFARs exhibited different responses and regulatory mechanisms under various abiotic stresses (Figure 8).

Discussion
Many FARs have been identified in plants such as Arabidopsis [11][12][13]20,34], wheat [14,24,35], rice [18,21], B. distachyon [23,36], and Gossypium hirsutum [25].FARs mediating plant growth, development, and stress response have also been reported.However, it is still important to comprehensively evaluate the genetic characteristics of the FAR family in rice and their role under abiotic stress.Hence, it is crucial to comprehensively Under salt, drought, ABA, heat, and cold stress treatments, most OsFARs were downregulated compared to the control at most time points.However, excluding heat stress, OsFAR2 was significantly upregulated by all other stresses and reached a peak at 24 h.The expression of OsFAR1 was induced by drought, salt stress, and ABA, the same as in previous studies [18].In addition, OsFAR6 actively responded to heat stress, while OsFAR8 actively responded to drought stress and ABA induction.Collectively, these results suggested that OsFARs may play a role in responding to environmental stresses.However, the trend of OsFARs expression under various abiotic stresses may be inconsistent with the microarray data due to disparities in rice varieties, seedling age, and stress conditions.

Discussion
Many FARs have been identified in plants such as Arabidopsis [11][12][13]20,34], wheat [14,24,35], rice [18,21], B. distachyon [23,36], and Gossypium hirsutum [25].FARs mediating plant growth, development, and stress response have also been reported.However, it is still important to comprehensively evaluate the genetic characteristics of the FAR family in rice and their role under abiotic stress.Hence, it is crucial to comprehensively characterize the OsFAR gene family throughout the entire genome.This is essential for understanding the functional and evolutionary relationships of this gene family in major crop species, thereby enhancing our knowledge of its role in agricultural science.
In the study, we identified eight FARs in rice (Table 1) and classified them into five subfamilies (Figure 1).The gene structure and motif distribution of the OsFARs were similar within the same subfamily, indicating that OsFARs in the same subfamily originated from a common ancestor.Genes from a shared ancestor evolved independently at a consistent rate with minimal change.In terms of structural features, all OsFARs contain an NAD_binding_4 domain at the N-terminus, which is involved in their binding to the NAD(P)H cofactor, and a FAR_C domain at the C-terminus that has not yet been attributed a clear role [10].NAD_binding_4 contains the conserved GXXGXX(G/A) motif and the YXXXK active site motif [14], where tyrosine (Y) and lysine (K) residues are predicted to play a direct role in catalysis [37].Site-specific mutations of Y and K residues in the YXXXK motif of AtFAR5 prevent yeast from producing primary alcohols [38].Studies have found that transferring complementary vectors lacking GXXGXX (G/A) or YXXXK motifs into Arabidopsis ms2 could not restore the defects of the pollen wall, suggesting the essential role of these two conserved motifs in the NAD_binding_4 of MS2 [20].Subcellular localization prediction showed that OsFAR2 is located in the chloroplast, mainly due to the presence of a transport peptide in its N-terminal region [21] that is observed in other OsFAR2 homologs such as Arabidopsis MS2 [20].OsFAR6 lacks Motifs 3 (related to GXXGXXG motif) and 4 (related to YXXXK motif) (Supplementary Table S6, Supplementary Figure S1).Therefore, it is speculated that OsFAR6 cannot participate in the normal synthesis of primary alcohols in the fatty acid synthesis pathway, and its detailed biological function needs further exploration.
Gene duplication, mutation, and natural selection are the main sources for the generation of new genes with new functions, providing the basis for biodiversity [39].Gene duplication significantly contributes to the process of gene amplification.During plant development and growth, gene duplication can help plants adapt to various conditions [40].Exploring the amplification patterns of gene families can help elucidate their evolutionary processes and functions.Through gene duplication and collinearity analysis, we identified a pair of segmental duplications (OsFAR1 and OsFAR4).OsFAR1 was more closely related to OsFAR4, with 63.48% homology.In addition, the Ka/Ks ratio of this gene pair was <1, leading us to infer that the OsFAR gene family underwent purifying selection during evolution, but the expansion was gradual and highly conserved throughout the evolutionary process.Interspecies collinearity analysis showed that the collinearity between rice and monocotyledonous plants is stronger than that between rice and dicotyledonous plants, which aligns with their kinship distance.
CREs are essential for regulating the spatiotemporal expression of genes, which influences plant growth and development, and coordinates adaptation to the environment.In this study, a total of 23 major CREs were identified, including numerous CREs related to growth and development, hormone response, light response, stress response, and MYB.Among these, most were light-responsive elements (74), followed by jasmonic acid (MeJA)responsive elements, and ABA-responsive elements.The universal plant hormones MeJA and ABA are widely involved in abiotic stress responses [18,[41][42][43].During wound healing, the exogenous application of ABA could upregulate the expression of AchnFAR in kiwifruit and increase primary alcohol accumulation [44].So far, it has been found that some MYB transcription factors regulate the expression levels of wax-related FARs under abiotic stress.For example, BdMYB41 can directly interact with the promoter region of BdFAR4, activating its expression [45].The synergistic effect of wheat TaTDRL and TaMYB103 can bind to the promoter of TaTAA1a and regulate its expression, increasing transcription activity [35].Many FARs have been identified in different species related to plant drought resistance.A total of seven OsFARs contain 1~3 drought-responsive CREs, indicating that these genes may be involved in the regulation of drought stress.
The response of plants to external stimuli is regulated by miRNA-mediated gene regulation [46,47].Previous studies have shown that miRNAs regulate plant growth, development, and stress responses [48][49][50].For example, the FAR5 gene in wheat is regulated by miRNA (unconservative_chr5B_part2-30016), which may affect the seedsetting rate in plants [51].A total of 65 miRNAs were discovered as target genes in the OsFAR family, and eight miRNAs specifically target multiple genes.Among these, osa-miR5075 targets three different genes, suggesting that osa-miR5075 may respond to abiotic stresses by regulating the OsFARs.
To further understand the potential biological functions of OsFAR family members, we analyzed their expression profiles in various tissues/organs of rice and under abiotic stresses using gene microarray data.The results indicated differential expression of OsFARs across tissues, suggesting their involvement in panicle development.The accumulation of wax in higher plants is often influenced by various environmental factors, including low temperature, high temperature, and drought.Previous studies have shown that FARs can alleviate abiotic stress in rice.Silencing GhFAR3.1 reduced wax accumulation in upland cotton leaves and weakened drought resistance [25].The expression of OsFAR1 was affected by drought stress and it is considered a positive regulator of drought tolerance [18].Mutations in BnA1.CER4 and BnC1.CER4 resulted in a decrease in wax crystal types and a decrease in the content of branched primary alcohols in Brassica napus, thereby reducing the water retention capacity [15].To discover the roles of OsFARs in the response to abiotic stress, data from the gene microarray database and stress treatments were analyzed.The data from the database indicated that OsFARs exhibit differential expression under different abiotic stress conditions, with most actively responding to cold stress.The data from the stress treatments also showed that most OsFARs were affected by salt, drought, low temperature, high temperature, and ABA.Among these, OsFAR2 was significantly induced under low temperature, salt, drought, and ABA treatments.Previous research revealed that CREs in the promoter region can be bound by regulatory factors, leading to the induction of their expression under abiotic stress [52].Our analysis found CREs such as ABREs, MBS, and STREs in the OsFAR2 gene promoter, indicating its ability to induce OsFAR2 expression under abiotic stress.Secondly, some miRNAs related to drought responses, such as osa-miR5795 [53], target OsFAR2 and mediate abiotic stress.Under high-temperature treatment, only OsFAR1 and OsFAR6 were upregulated within 24 h.The participation of OsFAR6 in heat stress response may be related to the regulation of miRNAs such as osa-miR1865-3P [54] and osa-miR1862d [55], which may be involved in heat stress response.Additionally, OsFAR8 was upregulated by ABA treatment.These results collectively provide a valuable reference for the functional validation of OsFARs.

Identification of FARs in Rice
Rice genome, proteome, and related annotation files were obtained from the Ensembl Plants database (http://plants.ensembl.org/index.thml,accessed on 10 March 2023).We downloaded the hidden Markov models (HMM) of the NAD_binding_4 (PF07993) and FAR_C (PF03015) domains from the InterPro website (https://www.ebi.ac.uk/interpro/, accessed on 10 March 2023), and TBtools v2.069 software was used to screen protein sequences containing these domains in rice, with an E-value ≤ 10 −5 [56,57].In addition, utilizing the FAR protein sequence of Arabidopsis in the search queries, a BLASTP search was executed against the rice database in the Ensembl Plants database to screen potential OsFARs, with the criteria: E-value ≤ 10 −5 and Identity ≥ 50% [58].These candidate sequences were then validated for domain composition using both the SMART website (http://smart.embl-heidelberg.de/,accessed on 27 March 2023) and the InterPro website.Candidate genes were named according to previous research and chromosomal locations.

Multiple Alignments and Phylogenetic Analysis
To characterize the sequence characteristics of eight OsFARs, multiple sequence alignments were performed using DNAMAN v9 software.To explore the evolutionary relationships between FARs in rice, Arabidopsis, barley (Hordeum vulgare L.), wheat, soybean (Glycine max L.), and maize (Zea mays L.), a multiple sequence alignment of FARs from these six species was conducted using the MEGA v7.026 software.Subsequently, a phylogenetic tree of FARs was constructed using the neighbor-joining (NJ) method with the Poisson model.The bootstrap value was set to 1000, with all other parameters set to their default values.Lastly, Evolview (http://www.evolgenius.info/evolview,accessed on 27 April 2023) was employed to visualize the evolutionary tree.

Analysis of Conserved Motifs and Gene Structures
To analyze conserved motifs within the OsFAR proteins, the MEME website (http://meme-suite.org/,accessed on 19 April 2023) was utilized.The default parameters were kept unchanged, except for the number of motifs, which was set to 10 [60].Functional domain information for the rice FAR family was obtained from NCBI Batch CD-Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi,accessed on 19 April 2023).The GFF3 (Generic Feature Format version 3 Data) annotation files of OsFARs were downloaded from the Ensembl Plants database and visualized using TBtools software.

Cis-Regulatory Element Analysis of OsFARs
In order to explore the CREs in the OsFARs' promoter region, the TBtools software was used to extract a 2 kb upstream sequence adjacent to each OsFARs start codon.Subsequently, these sequences were analyzed using the Plant Care website (http://bioinformatics. psb.ugent.be/webtools/plantcare/html/,accessed on 7 May 2023) and visualized using TBtools [61].

Chromosome Distribution, Gene Duplication, and Selective Pressure Analysis
The genome data for rice, Arabidopsis, tomato (Solanum lycopersicum L.), barley, and Brachypodium distachyon were downloaded from the Ensembl Plants and Phytozome v13 (https://phytozome-next.jgi.doe.gov/,accessed on 16 May 2023) databases [62].Based on the physical locations of the OsFARs in the rice database, the Circos and Dual Synteny Plot tools from TBtools software were used to examine and illustrate the chromosomal distribution, gene duplication events, and gene-level synteny across the various plant genomes.Moreover, the Ka/Ks ratio was calculated for duplicated gene pairs to assess the selective pressure on genes [63].

GO Functional Annotation and miRNA Targeting Prediction
The PANNZER2 (Protein ANNotation with Z-scoRE, http://ekhidna2.biocenter.helsinki.fi/sanspanz/, accessed on 22 May 2023) tool was used for GO enrichment analysis.The results were visualized using Excel 2010 software [64].
Potential miRNAs targeting OsFAR were predicted using the psRNATarget database (https://www.zhaolab.org/psRNATarget/,accessed on 22 May 2023).The CDS sequences of OsFARs were used as inputs files and searched against the reference miRNAs in rice.All parameters were kept at default values [65].The interaction network between the predicted miRNAs and their corresponding target genes was constructed using Cytoscape v3.9.1 software.

Gene Expression Profile Analysis
Expression data for OsFARs in 15 different organs/tissues and under 3 different abiotic stress conditions were obtained from the Rice eFP Browser website (https://bar.utoronto.ca/, accessed on 25 May 2023).In addition, the TBtools software was used to visualize the data and generate heatmaps.

Stress Treatment, RNA Extraction, and RT-qPCR Analysis
Seeds from Oryza sativa cv.Nipponbare were disinfected with 2.5% NaClO and incubated at 30 • C in darkness.Seedings with consistent growth were selected, transferred to a 96-well PCR plate, and cultivated in a greenhouse under a 14 h day (28 • C) light/10 h (25 • C) night cycle.After two weeks, the seedings were exposed to various stress conditions, including salt (150 mM NaCl), drought (20% (m/V), polyethylene glycol (PEG) 6000), low temperature (4 • C), high temperature (42 • C), and the exogenous hormone ABA (50 µM).The aboveground parts of whole plants were grown under various stress conditions and then collected 0, 6, and 24 h after treatment.Rice leaves were then randomly collected, immediately frozen in liquid nitrogen, and stored at -80 • C.
Total RNA was extracted using an Eastern ® Super Total RNA Extraction Kit (Promega, Beijing, China), and cDNA synthesis was performed using the Hifair ® III 1st Strand cDNA Synthesis SuperMix kit for qPCR (gDNA digester plus) (YEASEN, Shanghai, China) according to the manufacturer's instructions.RNA was amplified using RT-qPCR using the Hieff ® qPCR SYBR Green Master Mix (YEASEN, Shanghai, China).All the primers (Supplementary Table S1) were designed using Primer Premier 5 software, and OsUBQ5 (LOC_Os01g22490) was used as an internal reference gene.Three biological replicates of each sample were tested, and relative expression levels were calculated using the 2 −∆∆Ct method [66].The expression of OsFARs genes was visualized using GraphPad Prism.9.5 software.

Conclusions
The rice FAR gene family consists of eight members that are randomly distributed across five chromosomes.Based on evolutionary analysis, OsFARs can be divided into five subfamilies and include a pair of segmental duplicated genes that may be the way to expand and evolve the OsFAR gene family.Members of the same subfamily have similar conserved motifs, gene structures, and protein structures.Inter-species collinearity analysis showed that compared with dicotyledonous plants, OsFARs in rice share greater homology

Figure 1 .
Figure 1.Tertiary structures of OsFAR proteins.Random coils are shown in red.

Figure 1 .
Figure 1.Tertiary structures of OsFAR proteins.Random coils are shown in red.

Figure 3 .
Figure 3.The phylogenetic relationships, conserved motifs, domains, and gene structures of OsFARs.(A) Phylogenetic tree of OsFAR proteins.(B) Distribution of conserved motifs in OsFARs.The scale bar represents 100 aa.(C) Distribution of the NAD_binding_4 and FAR_C domains of OsFARs.(D) The structures of the OsFARs.Black lines, yellow rectangles, and green rectangles indicate introns, exons, and UTRs, respectively.The scale bar represents 1000 bp.

Figure 4 .
Figure 4. Analysis of CREs in the rice FAR promoter region.(A) The CREs in the promoters of each OsFAR gene.(B) The distribution of various CREs in the promoter.Vertical bars with different colors indicate CREs.

Figure 3 .
Figure 3.The phylogenetic relationships, conserved motifs, domains, and gene structures of OsFARs.(A) Phylogenetic tree of OsFAR proteins.(B) Distribution of conserved motifs in OsFARs.The scale bar represents 100 aa.(C) Distribution of the NAD_binding_4 and FAR_C domains of OsFARs.(D) The structures of the OsFARs.Black lines, yellow rectangles, and green rectangles indicate introns, exons, and UTRs, respectively.The scale bar represents 1000 bp.

Figure 3 .
Figure 3.The phylogenetic relationships, conserved motifs, domains, and gene structures of OsFARs.(A) Phylogenetic tree of OsFAR proteins.(B) Distribution of conserved motifs in OsFARs.The scale bar represents 100 aa.(C) Distribution of the NAD_binding_4 and FAR_C domains of OsFARs.(D) The structures of the OsFARs.Black lines, yellow rectangles, and green rectangles indicate introns, exons, and UTRs, respectively.The scale bar represents 1000 bp.

Figure 4 .
Figure 4. Analysis of CREs in the rice FAR promoter region.(A) The CREs in the promoters of each OsFAR gene.(B) The distribution of various CREs in the promoter.Vertical bars with different colors indicate CREs.

Figure 4 .
Figure 4. Analysis of CREs in the rice FAR promoter region.(A) The CREs in the promoters of each OsFAR gene.(B) The distribution of various CREs in the promoter.Vertical bars with different colors indicate CREs.

Figure 5 .
Figure 5. Chromosomal location, collinearity, and evolutionary analysis of OsFARs.(A) Distribution and collinearity of OsFARs in the rice genome.The collinearity of gene pairs is represented by gray and red lines.Scale bars marked on the chromosomes indicate chromosome lengths (Mb).(B) Interspecific collinearity analysis of FARs in rice, Arabidopsis thaliana, tomato, barley, and Brachypodium distachyon.The purple curves show the homology between FAR genes in rice and other species.The red triangles show gene locations.

Figure 5 .
Figure 5. Chromosomal location, collinearity, and evolutionary analysis of OsFARs.(A) Distribution and collinearity of OsFARs in the rice genome.The collinearity of gene pairs is represented by gray and red lines.Scale bars marked on the chromosomes indicate chromosome lengths (Mb).(B) Interspecific collinearity analysis of FARs in rice, Arabidopsis thaliana, tomato, barley, and Brachypodium distachyon.The purple curves show the homology between FAR genes in rice and other species.The red triangles show gene locations.

Figure 7 .
Figure 7. Expressions of OsFARs in different tissues and under different abiotic stresses.(A) The expression patterns of OsFARs in different tissues and developmental stages.The samples were obtained from 7-day-old seedling roots, mature leaves, young leaves, shoot apical meristems, panicles at six stages (P1-P6) divided based on panicle length and days after pollination, and seeds at five distinct stages (S1-S5) classified based on the development stages.(B) Expression patterns of OsFARs in the shoots of rice seedlings grown under three different abiotic stress conditions.Red indicates a higher expression level while green indicates a lower expression level.

Figure 7 .
Figure 7. Expressions of OsFARs in different tissues and under different abiotic stresses.(A) The expression patterns of OsFARs in different tissues and developmental stages.The samples were obtained from 7-day-old seedling roots, mature leaves, young leaves, shoot apical meristems, panicles at six stages (P1-P6) divided based on panicle length and days after pollination, and seeds at five distinct stages (S1-S5) classified based on the development stages.(B) Expression patterns

18 Figure 8 .
Figure 8. Expression levels of OsFARs under different abiotic stresses.(A) Treatment at 4 °C.(B) Treatment at 42 °C.(C) Treatment with 150 mM NaCl.(D) Treatment with 20% PEG6000.(E) Treatment with 50 µM ABA.All experiments were performed independently at least three times.Error bars represent the standard deviation of replicates.The asterisks (* p < 0.05, ** p < 0.01, Student's t-test) represent significant differences between the controls and treatments.

Figure 8 .
Figure 8. Expression levels of OsFARs under different abiotic stresses.(A) Treatment at 4 • C. (B) Treatment at 42 • C. (C) Treatment with 150 mM NaCl.(D) Treatment with 20% PEG6000.(E) Treatment with 50 µM ABA.All experiments were performed independently at least three times.Error bars represent the standard deviation of replicates.The asterisks (* p < 0.05, ** p < 0.01, Student's t-test) represent significant differences between the controls and treatments.

Table 1 .
General information on and sequence characterization of eight OsFAR genes.

Table 1 .
General information on and sequence characterization of eight OsFAR genes.