Genome-wide identification, classification, and expression profiling of LAC gene family in sesame

Abstract

Background

Laccases (LACs) are vital plant growth and development enzymes, participating in lignin biopolymerization and responding to stress. However, the role of LAC genes in plant development as well as stress tolerance, is still not well understood, particularly in sesame (Sesamum indicum L.), an important oilseed crop.

Results

In this study, 51 sesame LAC genes (SiLACs) were identified, which were unevenly distributed across different chromosomes. The phylogeny of Arabidopsis LAC (AtLACs) subdivided the SiLAC proteins into seven subgroups (Groups I-VII), of which Group VII contained only sesame LACs. Within the same subgroup, SiLACs exhibit comparable structures and conserved motifs. The promoter region of SiLACs harbors various cis-acting elements that are related to plant growth, phytohormones, and stress responses. Most SiLACs were expressed in the roots and stems, whereas some were expressed specifically in flowers or seeds. RNA-seq analysis revealed that 19 SiLACs exhibited down-regulation and three showed up-regulation in response to drought stress, while 15 SiLACs were down-regulated and four up-regulated under salt stress. Additionally, qRT-PCR analysis showcased that certain SiLAC expression was significantly upregulated as a result of osmotic and salt stress. SiLAC5 and SiLAC17 exhibited the most significant changes in expression under osmotic and salt stresses, indicating that they may serve as potential targets for improving sesame resistance to various stresses.

Conclusions

Our study offers a thorough comprehension of LAC gene structure, classification, evolution, and abiotic stress response in sesame plants. Furthermore, we provide indispensable genetic resources for sesame functional characterization to enhance its tolerance to various abiotic stresses.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-024-05982-w.

Background

Laccases (LACs; EC 1.10.3.2, benzenediol: oxygen oxidoreductases) catalyze various substrate oxidation. This enzymatic process involves the transfer of four electrons, thereby reducing molecular oxygen into water [1]. Typically, LACs belong to the multicopper oxidase family, which comprises ceruloplasmin, ascorbate oxidase, bilirubin oxidases, and ferroxidases [2]. Structurally, LACs possess three conserved copper-oxidase sites: Type 1 (T1), Type 2 (T2), and binuclear T3 copper sites. These copper sites are stabilized by three disulfide bridges found between domains I and II, I and III, and within domain III. In the maize LAC (ZmLac3) structure, the T1 copper ion is coordinated in a trigonal manner with two nitrogen atoms from histidine residues His451/519 and a sulfur atom from cysteine residue Cys514. At the trinuclear copper cluster (TNC) site, the coordination of two T3 copper ions is conducted by six nitrogen atoms from histidine residues and that of T2 copper ion by two nitrogen atoms from histidine residues. The ten histidine residues and one cysteine residue responsible for coordinating the four copper ions are highly conserved across LACs, indicating that these amino acids are crucial for the catalytic activity and structural stability of LAC [3]. During the enzymatic reaction, when a substrate binds to and is oxidized at the T1 site, an electron is transferred to the T2/T3 TNC. This electron transfer facilitated the combination of free hydrogen atoms with molecular oxygen (O₂), forming water molecules (H₂O) [4].

Lignin, a primary plant cell wall component, has well-studied biosynthetic pathways in plants. In the cytoplasm, phenylalanine is exposed to a sequence of enzymatic reactions: deamination, hydroxylation, methylation, and reduction, thereby producing lignin monomers (G-, H-, and S-). Afterward, these monomers are transported to the extracellular matrix, where they are polymerized into lignin by peroxidase or LAC enzymes within the cell wall [5]. Firstly, LACs were discovered in Rhus vernicifera [6] and are broadly distributed across plants, bacteria, fungi, and insects [7–9]. Their various biological functions are influenced by the origin and developmental stage of the organism that produces them [10]. Numerous studies have shown that LACs catalyze the oxidative polymerization of plant lignin precursors and play a role in various biological processes. For example, both AtLAC4/17 play key roles in lignin biosynthesis in Arabidopsis. Knockdown of AtLAC4/17/11 leads to significant growth inhibition, reduced root diameter, indehiscent anthers, and impaired vascular development due to deficient lignification. Moreover, deleting different combinations of LACs leads to specific changes in lignin chemistry [11–14]. In addition, LACs are involved in the oxidative polymerization of flavonoids [15, 16]. For instance, suppressing GhLAC1 expression leads to constitutive accumulation of jasmonic acid (JA) and flavonoids in ovules and fiber cells [17]. Inhibition of the LAC gene decreases the oxidative polymerization of flavonoids, thereby reducing fruit browning [18–20]. Lignification in plants is frequently correlated with enhanced resistance to environmental and biological stressors [21, 22]. Upregulation of the LAC gene results in the lignification of xylem cell walls, creating a physical barrier that prevents ionic flow [23, 24]. Furthermore, increased lignification positively affects plant disease resistance. Elevating the levels of GhLAC1/15 promotes greater lignification and higher lignin accumulation in plant cell walls, which improves resistance to biotic threats like cotton bollworms, aphids, and fungal pathogens [25, 26]. MicroRNAs (miRNAs) represent tiny, non-coding RNA molecules with a length of 20–24 nucleotides, which modulate gene expression by directing the degradation or inhibition of mRNAs. Studies have demonstrated that LAC genes are influenced by miRNAs, including miRNA397 [27], miRNA408 [28], and miRNA528 [29], which control plant growth, development, and responses to exogenous stresses by modulating the expression of LAC genes [30, 31].

Recent studies have consistently identified and characterized LAC genes on a genome-wide scale across various plant species, including A. thaliana (17) [32], Oryza sativa (30) [23], Phyllostachys edulis (23) [33], Salvia miltiorrhiza (29) [34], Camellia sinensis (51) [35], Sorghum bicolor (27) [36], Populus trichocarpa (49) [37], and soybean (93) [38]. Sesame LAC gene family members have been identified based on the sesame genome v1.0 [39]; however, their sequence characterization, gene structure, and expression patterns have not been thoroughly elucidated. Accordingly, we conducted a comprehensive bioinformatics analysis of the LAC gene family in sesame, utilizing our newly released high-quality chromosome-level reference genome. Analysis revealed these genes’ structure, chromosomal distribution, repeat events, phylogeny, and conserved motifs. Additionally, sesame LAC gene expression has been studied under different stress conditions, such as drought, salinity, and osmotic pressure, to understand their diverse functions. Our results offer significant insights into the LAC gene family in sesame, providing critical foundations for its functional characterization.

Materials and methods

LAC gene identification, physicochemical attributes, subcellular localization in sesame

Herein, we employed two approaches for LAC gene identification in our assembled sesame genome utilizing third-generation sequencing data [40]. Method 1: LAC protein sequences from A. thaliana were used as queries to search for sesame LAC proteins using BLASTP with default parameters. Method 2: Hidden Markov Model (HMM) profiles of the Cu-oxidase_1 (PF07732), Cu-oxidase_2 (PF00394), and Cu-oxidase_3 (PF07731) domains [41] were acquired by accessing the InterPro Database (https://www.ebi.ac.uk/interpro/) and employed to search for LAC proteins (E-value ≤ 1e-5) in sesame genome-wide protein sequences using HMMER3.0 [42] software. The results from Methods 1 and 2 were combined to determine the final total number of SiLACs. Thereafter, SiLAC physicochemical properties and subcellular localization were analyzed via the online tools ExPASY [43] (https://web.expasy.org/protparam) and Plant-mPLoc [44] (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi), respectively.

Chromosomal localization, gene duplication analyses, and prediction of Si-miRNA target LAC genes

Chromosomal localization of all SiLAC genes was achieved by mapping them to sesame chromosomes based on positional data using the TBtools software [45]. Using the One-Step MCScanX function in TBtools, we conducted gene duplication analyses, visualizing the results with the Circle Gene View function [45]. Collinear gene pairs between sesame and three other plant species (Arabidopsis, tomato, and grape) were identified, calculating non-synonymous (Ka) and synonymous (Ks) substitution values by McScanX and TBTools. The selection pressure assessment was carried out using the Ka/Ks substitution ratio for each collinear LAC gene pair. A Ka/Ks ratio > 1, < 1, and = 1 positive, purifying, and neutral selection [46], respectively. To estimate divergence time for each collinear gene pair, we utilized the formula: T = Ks / (2 × 1.5 × 10⁻⁸) × 10⁻⁶ million years ago (MYA), where Ks denotes synonymous substitution number between two genes or sequences [47]. Fifty-one SiLAC transcript sequences were uploaded to the psRNATarget server (http://plantgrn.noble.org/psRNATarget/?function=3), and sesame miRNA397 and miRNA408 sequences were obtained from Zhang et al. [48]. Sequences having a cut-off score ≤ 3 indicated putative targets.

Phylogenetic, gene structure, conserved motifs, and cis-acting elements analyses

The predicted Arabidopsis and rice LAC protein sequences were obtained from the TAIR (https://www.arabidopsis.org/) and RAP-DB (http://rapdb.dna.affrc.go.jp/) databases, respectively. To conduct protein sequence alignment and phylogenetic analysis, MEGA11 software was deployed [49]. The phylogenetic tree generation was performed through the neighbor-joining method with a bootstrap value of 1000 while editing and identifying the final phylogenetic tree via TVBOT (https://www.chiplot.online/tvbot.html) [50]. Based on the gene structure annotation information in the GFF3 file, the SiLAC gene structure was analyzed using TBtools software [45]. Conserved motifs within the SiLACs were identified using Multiple Em for Motif Elicitation (MEME) v5.3.3 (http://meme-suite.org/tools/meme) [51], setting the predicted motif number to 10. For cis-acting element analysis in the promoter region, 1500-bp sequences upstream of the ATG start codon of SiLAC genes were extracted, followed by submission to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [52].

Tissue expression analysis and expression profiling of SiLACs under drought and salt

The extraction of SiLAC expression levels across different sesame tissues (root, stem, leaf, flower, capsule, and seed) and at various stages of seed development followed previously published RNA-seq data [53, 54]. Consistently, their expression levels under drought [55] and salt [56] stress conditions were derived from previously developed transcriptome data. Heatmaps were generated using TBtools software, applying Log2-transformed TPM values or fold changes [45].

Plant material and osmotic and salt stress treatments

Sesame cultivar Zhongzhi No. 13 [53] was employed to explore changes in SiLAC expression under osmosis and high salinity stress treatments. Initially, the seeds were sterilized with 3% sodium hypochlorite (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) and rinsed on three occasions using sterile water. Seedlings were exposed to two-week hydroponic cultivation in a container with half-strength Hoagland solution. Subsequently, the induction of osmotic and salt stresses was performed by transferring the seedlings into fresh nutrient solutions that contained 15% PEG6000 (Sigma-Aldrich, St. Louis, USA) and 150 mM NaCl (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China), respectively [57, 58]. Post-treatment, we collected leaf samples at 0, 3, 6, and 12 h for gene expression analysis using qRT-PCR. The samples were immediately frozen in liquid nitrogen and maintained at − 80 °C until further usage.

qRT-PCR analysis

Total RNA was isolated with the TransZol Up Plus RNA Kit (TransGen, Beijing, China). The first-strand cDNAs were synthesized per earlier outlined protocols [58]. Quantitative real-time PCR (qRT-PCR) was conducted using a Roche LightCycler 480 system equipped with the ChamQ SYBR qPCR Master Mix (Vazyme Biotech, China). Here, three biological replicates were performed. Relative expression levels were determined using the 2^–ΔΔCT method [59], conducting normalization against the reference gene Histone H3.3 (LOC105159325) [60]. Table S3 summarizes the used gene-specific primers.

Results

LAC family gene identification and characterization in sesame

Here, 51 putative LAC genes were identified and designated as SiLAC1 to SiLAC51, according to their chromosomal positions in sesame. Table S1 lists detailed information about each SiLAC, including gene name, gene ID, chromosome location, gene start and end positions, Cu-oxidase domain locations, protein length, and exon and intron number. The deduced protein sequences of the 51 SiLACs range from 535 amino acids (aa) in SiLAC42 to 597 aa in SiLAC25. The SiLAC molecular weights (MWs) range between 59.58 kDa (SiLAC11) and 66.68 kDa (SiLAC25), while their isoelectric points (pIs) range from 5.15 (SiLAC15) to 9.68 (SiLAC45). Subcellular localization prediction indicated that all SiLACs were located in the cell membrane. Table S2 provides detailed information regarding the physicochemical properties of each SiLAC protein.

Chromosomal localization and gene duplication analysis of SiLAC genes

The distribution of the 51 SiLACs among the 13 chromosomes of sesame is uneven, except for Chr10, where SiLACs are absent (Fig. 1). The Chr9 region exhibited the highest number of SiLAC genes, with a total of nine. In contrast, Chr12/13 contained only one SiLAC. Some SiLACs form clusters on Chr2/3/6/9/11.

Fig. 1.

Chromosomal location of SiLAC genes in sesame genome

Investigation gene duplication is crucial in exploring LAC gene family evolution and expansion in sesame. Collinearity analysis showed that 15 SiLAC gene pairs participated in segmental duplication involving 24 SiLACs (SiLAC1/3/4/6/7/8/11/12/13/17/18/26/27/28/29/30/32/33/34/35/42/44/49) located on ten chromosomes (Chr1/2/3/4/5/6/7/8/9/11) (Fig. S1). Additionally, 14 SiLACs (SiLAC5/9/20/21/22/36/37/38/39/40/43/45/46/47) originated from tandem duplications (Table S1). The results manifested that both segmental and tandem duplications contributed to SiLAC gene family growth. The evolutionary pressure analysis results revealed that all Ka/Ks ratios for gene pairs were < 1 (Table S3), suggesting that the SiLAC genes have likely undergone substantial purifying selection throughout their evolutionary history, which has played a significant role in preserving the function of the SiLAC gene family.

Phylogenetic analysis and mapping of orthologous genes

To understand the phylogenetic relationships among the LAC gene families, we constructed an evolutionary tree by aligning 28 OsLACs, 17 AtLACs, and 51 SiLACs (Fig. 2). Seventeen AtLACs were categorized into six clusters [32]. Based on the classification standard used for Arabidopsis, sesame LAC genes were assigned to seven groups (Groups I–VII) that contained six, six, five, seven, one, two, and 24 SiLACs, respectively. Notably, only sesame LACs were present in group VII. Additionally, we found that 10 members of Group VII were involved in segmental duplication events, while six underwent tandem duplication events.

Fig. 2.

Phylogenetic tree of LACs from sesame, rice and Arabidopsis. The different-colored arcs indicate subgroups of the LAC proteins. The unrooted Neighbor-Joining phylogenetic tree was constructed using MEGA11 with full-length amino acid sequences and the bootstrap test replicate was set as 1000 times

To further analyze the timing of replication and evolutionary origin of the SiLAC genes, we conducted a genome-wide collinearity analysis between sesame and three representative plants: Arabidopsis, tomato, and grape. We identified 45, 56, and 40 collinear gene pairs in sesame compared to those in Arabidopsis, tomato, and grape, respectively (Fig. S2). To better understand the selection pressures, evolutionary dynamics, and divergence times, the Ka and Ks values, along with the Ka/Ks ratios, were computed for these orthologous gene pairs. This study identified 106 orthologous gene pairs with Ka/Ks ratios < 1, and no pairs showing ratios of one or above. This suggests that purifying selection was the predominant force driving SiLAC gene evolution (Table S4). The divergence times for these orthologous gene pairs ranged between 34.95 and 204.48 million years. The maximum Ka/Ks ratio recorded was 0.18 (SiLAC25 and Solyc11T000289.2), while the minimum was 0.025 (SiLAC17 and Solyc10T002792.1). In addition, 16 SiLAC genes (SiLAC2/6/11/12/13/16/17/18/26/27/28/32/44/49/50/51) show a collinear correlation with the other three species. This suggests that these SiLAC genes likely predated the divergence of these species and may have been crucial in the evolutionary development of these plants.

Gene structure, conserved motif, and miRNA Target Site Prediction of SiLACs

To study the structural diversity of SiLAC genes, we used TBtools software to visualize the gene structure of SiLAC family members and compare their exon-intron arrangements. The results showed that Group VII exhibited the largest variation in exon number, ranging from one to nine, while the remaining subgroups had between five and seven exons, with most (41.17%) SiLACs having six exons (Fig. 3A). Overall, we identified 10 conserved motifs (motifs 1–10) using the MEME web server (Fig. 3B). These motifs ranged in size from 22 to 50 aa (Fig. S3). Conserved motifs were generally similar among members of the same group. All SiLAC members share motifs 1/2/3/5/6/7, suggesting a high level of conservation of the LAC family genes in sesame. In contrast, motifs 8/9 were exclusive to Group VII, which did not include motifs 4 or 10.

Fig. 3.

Phylogenetic relationships, conserved protein motifs, and gene structure of SiLACs. The phylogenetic tree was constructed based on sesame LAC proteins using MEGA 11 software. (A) Exon-intron structure of SiLAC genes. Green boxes indicate exons; black lines indicate introns. (B) The motif composition of SiLAC proteins. The motifs, numbers 1–10, are displayed in different colored boxes. The sequence information for each motif is provided in Supplementary Figure S3

miRNAs regulate gene expression through mechanisms such as cleavage of target mRNAs or repression of their translation. The involvement of miRNAs in regulating LAC gene expression has been reported. In our previous small RNA sequencing analysis, we identified two types of conserved miRNAs in sesame, miR397/408 (Table S5). The specific sequence information detailed in Table S4 predicted that 16 SiLAC genes were targeted by miR397, while miR408 targeted two SiLAC genes. Notably, SiLAC13 was identified as a target gene for both miR397/408, while SiLAC27 was specifically targeted by miR408 (Table S6).

Cis- acting elements analyses of SiLAC genes

To detect potential cis-acting regulatory elements within SiLAC promoter regions, we analyzed the sequences located 1500-bp upstream of the start codons (ATG) for each gene using the PLACE database [61]. The identified cis-elements are assigned into three main groups: abiotic and biotic stress, phytohormone-responsive, and plant growth and development [62]. Within the first category, we observed elements related to various stress responses, including oxidation, defense, drought, wounding, heat, and low temperature stress. Analyzing the SiLAC genes revealed that the majority of these stress-related elements were associated with two key motifs: the MYB (CCAAT box) and MYC (CACATG box) binding sites, accounting for 24% and 25% of the total stress-responsive elements, respectively. Regarding the second group of phytohormone-responsive elements, ABRE (contributed to ABA responsiveness), the CGTCA motif (contributed to MeJA responsiveness), as-1 elements (contributed to SA and auxin responsiveness), and ERE elements (contributed to ethylene responsiveness) accounted for 77% of the elements linked to phytohormone responses. In the last group, 10 cis-elements associated with plant growth and development were recognized, including nine light-responsive elements and one meristemic-specific expression element (CAT-box). Box-4 (ATTAAT) was the most prevalent light-responsive element in the SiLAC promoter region, accounting for 41% of the total (Fig. 4).

Fig. 4.

Predicted cis-elements in the promoter of SiLACgenes. The left is the number of each cis-element in the promoter of SiLAC genes. Based on the functional annotation, the cis-elements were classified into three major categories: abiotic and biotic stresses, phytohormone responsive, and plant growth and development. The right is the sum of the cis-elements in each gene and category

Expression analysis of SiLACs in various sesame tissues

The SiLAC gene expression patterns were investigated across six tissues (stem, flower, leaf, root, capsule, and seed) in sesame with our previously released transcriptome data [53]. Most SiLAC genes exhibited tissue-specific expression patterns (Fig. 5). For example, SiLAC14/33/35/36/37/38/39/40 were highly expressed in the flowers. However, SiLAC6/11/18/24/29 were overexpressed in all tissues, suggesting their involvement in multiple biological regulatory processes.

Fig. 5.

Expression profiles of SiLAC genes in various tissues of sesame. Log2-transformed (TPM + 1) values of each gene were used to construct heatmap. The expression level was shown in color as the scale. The outer color blocks represent different subgroup classifications

Analysis of LAC gene expression in black and white sesame seeds at different developmental stages revealed distinct patterns among SiLAC family members [54] (Fig. 6). Most members of Group VII (SiLAC24/51/18/12/26/29/43/32/6/11) showed high expression at the early developmental stage, followed by a trend of decreasing expression. Most SiLAC genes in groups I, II, V, and IV exhibited downregulation during the developmental period. Both SiLAC13/21 in Group III maintained overexpression during the developmental period. SiLAC47 exhibited very high expression in black seeds at the early developmental stage but was not expressed in white seeds. SiLAC28 in Group II showed a similar expression pattern to SiLAC47 in Group I, suggesting that they may play similar roles. Specific expression patterns of LAC family genes at various developmental stages coordinately regulate the development of sesame seeds.

Fig. 6.

Expression profile of SiLACs during the seed development of black and white sesame. Log2-transformed (TPM + 1) values of each gene were used to construct heatmap. DPA, Days post-anthesis

SiLAC gene expression profiles in response to abiotic stresses

To explore how SiLAC genes respond to drought and salt stresses, their expression was analyzed at various time points under these abiotic conditions using RNA-seq data from earlier research [56, 60]. Unfortunately, RNA-seq data were missing for 14 genes (SiLAC2/3/4/10/15/19/20/21/22/25/27/30/37/42) under progressive drought stress and for 16 genes (SiLAC2/3/4/10/15/21/22/25/30/35/36/38/39/40/42/45) under salt stress. SiLAC genes displayed significant changes in response to drought stress (Fig. 7A). Specifically, SiLAC5 exhibited a strong increase in expression at every time point during drought stress treatment. Eight genes (SiLAC6/7/11/18/32/33/40/50) were significantly downregulated at 5, 7, and 10 d following drought stress. Seven gene expressions (SiLAC9/16/31/34/41/47/49) gradually decreased with increasing drought stress. Most SiLAC genes were differentially down-regulated by salt stress at different time points (Fig. 7B), specifically, four genes (SiLAC6/18/26/33) showed decreased expression at all four periods, and 10 genes (SiLAC16/17/28/31/34/41/43/47/49/50) were increased with increasing salt stress duration and the down-regulation gradually increased. In contrast, four SiLAC genes (SiLAC8/9/13/32) were up-regulated by salt stress at two or three time points.

Fig. 7.

Transcriptome-based expression profiles of SiLAC under drought (A) and salt stress (B). Heatmap displaying expression changes of SiLAC genes in sesame plants stressed for 3 d, 5 d, 7 d, and 10 h of drought or 2 h, 6 h, 12 h, and 24 h of salt compared with the control. The colored scale for the different fold change is shown

The qRT-PCR results indicated that six genes (SiLAC5/12/17/18/21/26) were significantly upregulated at least once during salt stress, with SiLAC17 exhibiting the highest fold-change. All six genes displayed a peaked expression at 12 h of treatment (Fig. 8). Eleven SiLACs (SiLAC5/12/17/18/20/21/22/26/27/32/38) were significantly upregulated under osmotic stress, and SiLAC17 showed significantly higher expression under osmotic stress at all three-time points (Fig. 9). SiLAC18/20/22/27/38 showed significantly higher expression levels during the early stage of stress, followed by a decrease. SiLAC17/21/32 showed significantly higher expression levels during the early stage of stress, and their expression gradually increased with prolonged stress duration. In summary, the expression levels of SiLAC5/12/17/18/21/26 were significantly upregulated in response to both osmotic and salt stresses, suggesting their potential role in enhancing resistance to multiple abiotic stresses.

Fig. 8.

Expression profiles of 15 SiLAC genes in response to salt stress. Relative expression levels of SiLAC genes at 0 h, 3 h, 6 h and 12 h after high salinity treatment (150 mM NaCl) were analyzed by qRT-PCR. Sesame SiH3.3 gene was used as the internal control. Error bars indicate standard error based on three replicates. * P < 0.05; ** P < 0.01; *** P < 0.001, t test

Fig. 9.

Expression profiles of 15 SiLAC genes in response to osmotic stress. Relative expression levels of SiLAC genes at 0 h, 3 h, 6 h and 12 h after osmotic stress treatment (15% PEG 6000) were analyzed by qRT-PCR. Sesame SiH3.3 gene was used as the internal control. Error bars indicate standard error based on three replicates. * P < 0.05; ** P < 0.01; *** P < 0.001, t test

Discussion

Typically, LAC enzymes, a multicopper oxidase, are crucial for lignin biosynthesis and are vital for plant development as well as responses to multiple stresses. In sesame, 51 SiLACs were identified, all possessing conserved copper-binding domains yet displaying variations in gene structure. This indicates that while they may share common genetic origins, they have evolved distinct biological functions. Gene duplication plays a crucial role in forming gene families, with tandem and segmental duplications acting as evolutionary driving forces contributing to gene family expansion [63, 64]. Expansion of the LAC family via tandem and segmental duplications has been extensively reported in plants, including Populus trichocarpa [65], Pyrus bretschneideri [66], and switchgrass [67]. Conversely, only tandem-duplicated LAC genes have been observed in Solanum melongena [68] and Setaria viridis [69]. In the sesame LAC gene family, 14 genes are involved in tandem duplication and 24 in segmental duplication, highlighting the significant role that these events have played in expanding the SiLAC gene family. Notably, some transcription factors (TFs) regulate gene expression by recognizing cis-acting elements, whereas miRNAs govern gene expression by targeting mRNAs for cleavage or repressing translational. The regulatory network involving MYB family members in plant secondary cell wall formation is well documented [70]. Interestingly, the SiLACs identified in this study featured numerous MYB-binding sites (Fig. 4). Presumably, MYB proteins act as transcription factors in this process. The involvement of MYB transcription factors in the regulation of LAC genes during xylem fiber lignification has been reported [71–73]. Previous reports have indicated that LAC genes can be recognized by various miRNAs, such as miR397/408/528/857. Among these, only miR397 and miR408 have been identified in sesame [48]. These two miRNAs were used for miRNA target prediction across the 51 SiLAC genes, revealing that 16 SiLACs are targets of miR397 and two SiLACs are targets of miR408. Notably, SiLAC13 is a target of both miR397/408, whereas SiLAC27 is a target of miR408.

The varied temporal and spatial expression patterns of the SiLACs imply that these genes may have tissue-specific physiological and biochemical functions in sesame growth and development. Most SiLACs were expressed in the roots (64.7%), and 11 were predominantly expressed in the stems [53] (Fig. 5). Similarly, in other plant species, including Arabidopsis [10, 32], Oryza sativa [23], Populus trichocarpa [65], Turnip and Chinese Cabbage [74], most LAC genes show high expression levels in roots and stems. Given that both roots and stems have substantial lignified tissues, it is likely that these SiLAC genes are crucial for lignin biosynthesis. Interestingly, eight genes (SiLAC14/33/35/36/37/38/39/40) exhibited very high expression levels, specifically in flowers, with significantly lower or no expression in other tissues. Similar patterns have been observed in other plant species, including Oryza sativa [23], Camellia sinensis [35], and Phyllostachys edulis [33]. This implies that these eight SiLAC genes may be crucial for flower development. LAC gene is also related to seed coat and fruit Browning, and the expression of exogenous tobacco proves that litchi LcLAC7 has a catalytic effect on epicatechin [75]. MdLAC7 was expressed instantaneously in apple fruit and callus, and anthocyanin, tannic acid and catechin acid were identified as its catalytic substrates [18]. Transparent testa10 is involved in oxidative polymerization of flavonoids [20]. In sesame seeds it has also been reported that black sesame seeds contain more flavonoids than white sesame seeds [76]. We examined the expression of SiLACs during the development of black and white sesame seeds [54]. Six genes (SiLAC7/13/21/43/50/51) were differentially expressed during the period of seed coat color change in sesame, suggesting that these SiLACs may be involved in the change in seed coat color (Fig. S4).

Herein, we generated a phylogenetic tree incorporating 28 OsLACs, 17 AtLACs, and 51 SiLACs through multiple sequence alignments (Fig. 2). The tree was divided into seven groups, adhering to the Arabidopsis classification standard. Notably, the seventh group comprised solely SiLACs, indicating that these 24 SiLACs might be unique to the species. Previous research has demonstrated that AtLAC4/11 in Group I and AtLAC2/17 in Group II play roles in lignin biosynthesis [12, 13, 77]. The LACs from both groups in other plant species, such as SofLAC [78], PbLAC1 [66], and MsLAC1 [71], have also been linked to this process. This suggests that SiLACs within these groups may similarly contribute to lignin biosynthesis. Tissue expression patterns lend support to this hypothesis, as SiLACs in these two groups exhibit higher expression in roots, stems, and capsules. Group III contains four AtLACs with distinct functions: AtLAC3 influences Casparian strip formation [79], AtLAC5 contributes to neolignan biosynthesis through seed-specific expression [14], AtLAC12 regulates iron transport from roots to shoots and enhances growth under iron-limited conditions [80], and AtLAC13 is linked to biomass production and seed yield [81]. Notably, SiLAC21 in Group III displayed seed-specific expression, suggesting a potential function akin to AtLAC5. In Group IV, seven SiLACs clustered with AtLAC14 and AtLAC15, which are known to be involved in phenolic compound polymerization [20, 82, 83].

Throughout their growth and development, plants frequently encounter adverse abiotic environmental conditions, such as extreme temperatures, nutrient scarcity, and high levels of salt or toxic metals in the soil [84]. In response to these varied environmental challenges, plants have developed an intricate regulatory system [85]. Numerous studies suggest that LACs likely play a role in lignin production, which aids in tissue and organ development, offers mechanical defense or resistance to dieback, and facilitates responses to various biotic and abiotic stressors [86–89]. Numerous studies have shown that LAC gene expression is reduced under various abiotic stresses. For instance, in haseolus vulgaris, 28 LAC genes exhibited reduced expression under salt stress [90]. In rice, LAC expression decreased under both salt and drought stress [23], while more than half of the LAC genes in tea plants were downregulated under drought stress [35]. Conversely, LAC expression increased in response to cold stress [35, 91]. In this study, we examine the expression patterns of SiLAC genes under drought and salt stress. Consistent with previous research, the expression of 10 SiLACs (SiLAC 6/16/18/33/31/34/41/47/49/50) genes was downregulated under both stresses. Quantitative real-time PCR analysis demonstrated that both SiLAC5/17 were upregulated at multiple time points under salt and osmotic stress conditions, suggesting their potential involvement in various stress response mechanisms. Furthermore, tissue-specific expression profiles indicated that SiLAC5/17 were expressed at higher levels in tissues that accumulate lignin, indicating that these genes may participate in responding to salt and osmotic stresses. Plant roots play a crucial role in water and nutrient uptake and are highly lignified. Numerous studies have shown that lignification is linked to stress tolerance, such as enhanced tolerance to salt [92], drought [93], and heavy metals [94, 95], as well as changes in lignin composition that affect responses to biotic stress [96]. Additionally, research has demonstrated that laccase is involved in lignin accumulation [11–14]. Therefore, modulating root lignification through laccase regulation presents a promising strategy to enhance plant stress tolerance.

Conclusion

This research conducted a comprehensive analysis of the LAC gene family across the sesame genome, examining chromosomal positioning, structural characteristics, phylogenetic and syntenic associations, as well as expression patterns specific to tissues and as a result of abiotic stress. The investigation identified 51 members of the SiLAC gene family, which were unevenly distributed across 12 out of 13 chromosomes. Notably, 24 SiLACs are involved in segmental duplications, while 14 are involved in tandem duplications. Evolutionary analysis revealed that SiLAC genes have undergone significant purifying selection. Based on their evolutionary relationships, the SiLAC genes were classified into seven subgroups. Expression analysis revealed that some SiLAC genes showed tissue-specific expression pattern. Two SiLAC genes, SiLAC5 and SiLAC17, were notably upregulated under various abiotic stress conditions. These findings provide a groundwork for future studies to explore the functions of SiLACs and elucidate their involvement in enhancing sesame’s resilience to abiotic stress.

Electronic supplementary material

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Author contributions

JY and LW conceived and designed the experiments; JZ and FH performed the experiments; MB, RZ, DL, HL, LY, TZ, and YZ participated in data collection and analysis; JZ, JY, and MB wrote the paper. All authors reviewed the manuscript.

Funding

This work was funded by the National Key Research and Development Program of China (2024YFD1600100), the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2021-OCRI), the China Agriculture Research System (CARS-14), the Hubei International Science and Technology Cooperation Project (2022EHB034, 2024EHA055), the Science and Technology Innovation Project of Hubei province (2024-620-000-001-031), the High-end Foreign Expert Project of Hubei province (2024DJC011), The Central Public-interest Scientific Institution Basal Research Fund (1610172023003), the National Center for Crops Germplasm Resources (NCCGR-2024-016), and the General Projects of Hubei province (2024AFB988).

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

Not applicable. The research conducted in this study required neither approval from an ethics committee, nor involved any human or animal subjects.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.