Genome-wide identification, classification and expression analysis of laccase family members in Solanum lycopersicum

Abstract

Laccase, a copper-containing polyphenol oxidase, is integral to plant growth and development, primarily due to its involvement in lignin and flavonoid biosynthesis, as well as its role in mediating plant responses to diverse biotic and abiotic stresses. Despite its importance, a comprehensive and systematic analysis of the laccase gene family in tomato has not been conducted until now. This study identifies eighteen laccase genes, unevenly distributed across seven chromosomes, within the tomato genome. Phylogenetic analysis categorizes these eighteen SlLACs into five distinct groups, corresponding to the established groups in Arabidopsis. Motif analysis indicates the presence of ten conserved motifs within the SlLACs. Gene duplication analysis reveals collinearity between the eighteen SlLACs and LAC genes in Arabidopsis, eggplant, and potato. Furthermore, cis-regulatory element analysis suggests that numerous SlLACs may be implicated in tomato morphogenesis, flavonoid biosynthesis, and various stress and growth processes. Expression analysis suggests that certain SlLAC genes may function in different tomato organs and potentially participate in diverse stress responses via hormone signaling pathways. This study contributes to elucidating the physiological roles of SlLAC family genes in tomatoes and may offer valuable genetic resources for tomato breeding programs.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08166-w.

Introduction

Laccase, an enzyme of significant importance in various biological processes, is a member of the copper blue oxidase family. It can efficiently catalyze the oxidation of polyphenols while reducing molecular oxygen to water. This enzyme is encoded by a distinct class of genes characterized by a Cu-oxidase_3/Cu-oxidase/Cu-oxidase_2 zinc finger domain [1–3]. Laccase was first isolated from the Japanese lacquer tree (Rhus vernicifera) in 1883, and was subsequently named for this tree [4]. It is released from damaged parts of lacquer trees, facilitating wound healing through the oxidation of sap alkyl catechols, known as urushiols [5]. Laccase is part of the ceruloplasmin oxidase protein family, which includes human plasma ceruloplasmin and plant ascorbic acid oxidase [6]. Research on laccases has advanced significantly across various fields, including food processing, biosensor design, microbial applications, medicine, healthcare, and nanotechnology [7–16]. With increasing research on laccases, research in plants has gradually increased.

In plants, laccases play essential roles in lignification and oxidation processes [17–19]. Specifically, they oxidize monolignols to produce lignin, a component crucial for plant growth and stress responses [20, 21]. In Arabidopsis thaliana, AtLAC4 and AtLAC17 are key mediators of lignin biosynthesis [22]. In cotton, suppression of GhLac1 elevates jasmonic acid (JA) and flavonoid accumulation in ovules and fiber cells [23], while its overexpression enhances lignification, which in turn improves resistance to the fungal pathogen Verticillium dahliae and insect pests including the cotton aphid (Aphis gosypii) and cotton bollworm (Helicoverpa armigera) [24]; this regulatory pathway is further modulated by the interaction of GhWRKY30 and GhWRKY41, which activates the GhLac1 promoter and reinforces cotton defense responses via lignin metabolism [25]. In poplar, coordinated expression of AtLAC2, AtLAC4, and peroxidase52 remodels lignin content and S/G ratios, leading to a marked increase in saccharification efficiency [26].

Laccases play crucial roles in plant responses to diverse abiotic and biotic stresses, underscoring their core functions in stress resistance and environmental adaptation. Plants are confronted with a multitude of environmental challenges (e.g., drought, extreme temperatures) during growth, which can disrupt normal development, induce severe damage, or even lead to plant death. Elucidating and harnessing the biological functions of laccases is therefore a key strategy to enhance plant stress resilience, thus advancing sustainable agriculture and environmental protection [27–29]. In response to stress, plants can activate a series of transcriptional regulatory genes, which help mitigate or eliminate the damage caused by that stress [30]. The stress response in plants involves complex signaling pathways, multiple genes, and the synergy of various gene products [31, 32]. For example, maize ZmLAC5, ZmLAC10, and ZmLAC17 are highly expressed under various stress conditions, highlighting their critical roles in stress responses [33]. Heterologous overexpression of rice OsLAC10 in Arabidopsis enhances copper tolerance by promoting root lignification and reducing excessive copper uptake [34]. Moreover, rice OsmiR397 modulates brassinosteroid signaling by repressing its target OsLAC, thereby improving rice yield [35].

The tomato (Solanum lycopersicum) is a vital fruit vegetable with significant global economic importance and serves as a key model for studying growth and ripening. However, its production is severely threatened by multiple abiotic stresses such as drought, high salinity, flooding, and extreme temperatures, all of which can cause drastic yield losses. Addressing these adverse environmental challenges is thus critical for safeguarding global food security. Laccase genes are crucial for plant growth, development, and stress response. Studying tomato laccase genes are expected to provide novel strategies to increase tomato quality and resistance to stress. Notably, laccase genes have been successfully isolated and characterized in various species, including Arabidopsis [36], maize [37], soybean [38], cotton [34], rice [39], common bean [40], Citrus sinensis [41], eggplant [42] and other species. Nevertheless, a comprehensive and systematic genome-wide analysis of the laccase gene family in tomato remains lacking.

This study aims to fill the gaps in the systematic analysis of the tomato laccase gene family by comprehensively identifying laccase genes from the tomato genome and performing multi-dimensional in-depth analyses. In this study, 18 laccase genes were identified from the tomato genome, and their physicochemical properties, phylogenetic evolution, chromosome localization, promoter cis-regulatory elements, conserved domains, gene structures, and collinearity were systematically analyzed. Furthermore, we explored the expression patterns of SlLACs in various vegetative organs across different developmental stages, revealing their significant responsiveness to abiotic stresses and diverse hormonal treatments. Through the in-depth study of tomato laccase gene family, it is expected to excavate gene resources related to important traits such as stress resistance and quality of tomato, and provide new gene targets and technical support for cultivating new tomato cultivars with enhanced stress resistance and superior quality, so as to improve the adaptability and yield of tomato under different environmental conditions, and promote the sustainable development of tomato industry.

Materials and methods

Identification of laccase genes in the tomato genome

To identify members of the tomato laccase gene family, the Sol Genomics Network (SGN, http://solgenomics.net/) database was used to download the tomato genome assembly and annotation files. Based on previously published research [18], Arabidopsis AtLAC protein sequences were retrieved from the TAIR database (https://www.arabidopsis.org/index.jsp). These sequences were used as queries to search for homologous predicted proteins in the tomato genome with an E-value cutoff of 1E-5. Candidate SlLAC proteins were identified by removing redundant and overlapping sequences. To further validate these candidates, their corresponding polypeptide sequences were subjected to BLASTP searches against the UniProtKB/Swiss-Prot (swissprot) database. All putative proteins were subsequently verified using the Pfam (http://pfam.xfam.org), NCBI Conserved Domain Database (Batch CD-Search, https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi), and SMART (http://smart.embl-heidelberg.de) databases. Finally, tomato SlLACs family members containing the conserved domains Cu-oxidase, Cu-oxidase_2, and Cu-oxidase_3 were identified. Additionally, the SGN (https://solgenomics.net/) website was used to obtain an annotation map of the tomato SlLAC genes on chromosomes. Based on the annotation data and gene density of the tomato genome, the identified SlLAC was mapped to the chromosome via TBtools. The physicochemical properties of the SlLACs, including their molecular weight (MW), theoretical isoelectric point (pI), instability index, aliphatic index, total average hydrophilicity were analyzed via the ExPASy server (https://www.ExPASy.org) [43].

Phylogenetic analysis of SlLACs

To further clarify the classification and evolutionary relationships of laccase family members between tomato and Arabidopsis thaliana, 18 SlLAC and 17 AtLAC proteins were subjected to multiple sequence alignment using MEGA 5.0. A phylogenetic tree of the SlLAC and AtLAC proteins was constructed via neighbor-joining (NJ) method with 1000 bootstrap replicates [42]. Finally, the phylogenetic tree was visualized using the Evolview online platform (http://www.evolgenius.info/evolview/#/).

Gene structure, conserved motif and conserved domain analysis of SlLACs

To further understand the functions and evolutionary relationships of SlLACs genes, the conserved domains, conserved motifs, and gene structures of tomato laccase family members were analyzed. Protein sequences of AtLACs and SlLACs were submitted to the online tool MEME (http://meme-suite.org/tools/meme) with default parameters, except that the number of motifs was set to 10. Conserved domains of SlLAC proteins were subsequently analyzed using the SMART database (http://smart.embl-heidelberg.de/). The annotation information for all the SlLAC was obtained from the Sol Genome Network (https://solgenomics.net/) website. Finally, the conserved domains, conserved motifs, and gene structures of tomato laccase family members were visualized using the Gene Structure Viewer tool in TBtools (v2.0) [42].

Signal peptide and glycosylation site analysis

The SignalP 6.0 server (https://services.healthtech.dtu.dk/service.php?SignalP-6.0) was used to predict the presence of putative N-terminal signal peptides, their lengths, and cleavage sites, while TargetP 2.0 (https://services.healthtech.dtu.dk/service.php?TargetP-2.0) was employed to further validate the existence of N-terminal signal peptides. Additionally, NetNGlyc 1.0 (https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0) was utilized to identify the number of putative N-glycosylation sites, and NetOGlyc 4.0 (https://services.healthtech.dtu.dk/service.php?NetOGlyc-4.0) was used to determine the number of putative O-glycosylation sites [18].

Gene replication and synteny analysis of SlLACs

The gene duplication of SlLAC was analyzed via TBtools (v2.0). According to the calculation method reported in a previous study [44], the nonsynonymous (Ka) and synonymous (Ks) substitution rates of duplicate genes were calculated. Collinearity analysis of LAC genes across tomato, potato, eggplant, Arabidopsis thaliana, and rice was performed via the MCScanX plugin embedded in TBtools (v2.0) [45]. A collinear graph was created via TBtools (v2.0). MCScanX was used to obtain the gene replication map in TBtools (v2.0) [46]. The proposed duplicate genes are indicated by the connecting line.

Cis-element analyses of SlLACs

To identify putative cis-regulatory elements, the 2,000 bp genomic sequence upstream of the start codon (ATG) was retrieved for each SlLAC gene from the tomato genome. Then we predicted these cis-elements using the PlantCARE server (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), and counted these elements with Excel 2019. Ultimately, TBtoolsII software was utilized for the subsequent visualization of the SlLAC promoter elements [47].

Plant materials and hormone and stress treatments

Wild-type (WT) tomato seeds (Solanum lycopersicum Ailsa Craig, AC⁺⁺) were cultivated in a greenhouse. The growth conditions included exposure to sodium light for 16 h at 28 °C and 8 h at 18 °C. The roots (Rt), stems (St), leaves (Le), and flowers (Fl) of the tomato plants were collected to analyze the expression patterns of SlLAC genes.

Tomato seedlings grown for 35 d were sprayed with 50 µM gibberellin (GA₃), distilled water, 50 µM abscisic acid (ABA), and 50 µM jasmonic acid (MeJA) solutions, and tomato leaves at the same position were collected to detect the hormone expression pattern of the SlLAC gene [48]. Three independent biological replicates (each consisting of tissues collected from separate plants).

The tomato seedlings cultured for 35 d were removed from the nutrient bowl, and the soil at the roots of the seedlings was washed away. The whole tomato seedlings were subsequently placed on dry filter paper for drying treatment. After treatment, tomato leaves from the same part were collected to detect the drought expression pattern of the SlLAC gene [49]. Three independent biological replicates (each consisting of tissues collected from separate plants).

Tomato seedlings cultured for 35 d were subjected to low-temperature treatment at 4 °C, with a light cycle of 16 h of daylight and 8 h of darkness. Leaves from the same area of the tomato plants were collected to analyze the expression pattern of the SlLAC gene under low-temperature conditions. Three independent biological replicates (each consisting of tissues collected from separate plants).

Samples were taken after 0, 2, 4, 8, 12, 24 h of hormone, drought and low-temperature stress treatment. All the collected samples were labeled, frozen in liquid nitrogen, and stored in a refrigerator at -80 °C.

Total RNA extraction and qRT-PCR analysis

Total RNA was extracted from the collected samples via RNAiso Plus reagent according to the manufacturer’s instructions (Takara, China). Oligo (dT) primers were used to synthesize cDNA from one microgram of total RNA through reverse transcription-polymerase chain reaction (M-MLV reverse transcriptase, Takara) [50].

The CFX96™ Real-Time System (Bio-Rad, USA) was used for fluorescence quantitative PCR (qRT-PCR) experiments. The reaction system included 1 µL of cDNA (20 ng/µL), 0.5 µL of primer pair (10 mM), 5 µL of 2×GoTaq^® qRT-PCR Master Mix (Promega), and 3.5 µL of nuclease-free water. The SlCAC (Solyc08g006960) gene was chosen as the internal reference gene to normalize gene expression in tomato [51]. Relative gene expression levels were calculated using the 2^−ΔΔCT method [52].

Each biological replicate was subjected to three technical replicates in the qPCR assay. The sequences of primers used in this study are detailed in Table S1.

Statistical analysis

SPSS 26.0 was used for analysis of variance. The significance of the difference (p < 0.05) between treatments was tested by Tukey’s test, and different letters were used to represent the significance of the difference. All the data are presented as the means of at least three independent biological replicates.

Results

Genome-wide identification of the SlLAC gene family in tomato

Tomato SlLAC family members were identified based on the amino acid sequences of Arabidopsis AtLAC proteins reported in previous studies [18]. BLASTP searches were performed against the tomato genome sequence using the NCBI and Sol Genomics Network (SGN) databases with an E-value cutoff of 1E-5 to identify putative laccase proteins. After removing redundant sequences, a total of 18 SlLAC members were identified and numbered SlLAC1 to SlLAC18 according to their chromosomal locations. Notably, these 18 SlLAC genes are unevenly distributed across only 7 of the tomato chromosomes.

Specifically, chromosome 6 harbors five SlLAC genes, chromosome 9 contains four, and chromosome 2 has three. Additionally, chromosomes 5 and 10 each carry two SlLAC members, while chromosomes 4 and 7 each contain one (Fig. 1). This uneven distribution may be associated with gene evolution, gene duplication, and chromosomal structural variation.

Fig. 1.

Chromosomal distributions and tandem replication analysis of SlLACs in tomato. The scale on the left represents the chromosome length (Mb). Columns with different colors represent the gene density on each chromosome. The red sections symbolize the distribution of various SlLAC genes on each chromosome, whereas the red arcs indicate the presence of tandemly duplicated genes among the laccase members in tomato

The 18 tomato laccase family genes were renamed, and Table S2 lists the amino acid length, theoretical isoelectric points (pIs), and relative molecular weights (MWs) of the laccase family genes. The protein length ranged from 476 (SlLAC10) amino acids to 700 (SlLAC6) amino acids, and the corresponding molecular weight ranged from 52815.71 (SlLAC10) to 78037.83 Da (SlLAC6). The predicted pI values ranged from 6.05 (SlLAC4) to 9.70 (SlLAC15), the instability index ranged from 25.19 (SlLAC5) to 35.76 (SlLAC8), the aliphatic index varied from 76.03 (SlLAC6) to 90.46 (SlLAC9), and the hydrophilic values ranged from − 0.35 (SlLAC6) to -0.001 (SlLAC18), indicate that all SlLAC proteins are alkaline and hydrophilic. These differences in physical properties may be related to the function and evolution of genes, which provides basic data for further study of the function of laccase genes. The subcellular localization of the SlLAC protein was predicted. The results revealed that seven SlLAC proteins were located in the extracellular space, five SlLAC proteins were located in both the extracellular space and the lysosome, four SlLAC proteins were located in both the extracellular space and the plasma membrane, one SlLAC protein was located in the lysosome, and one SlLAC protein was located in the extracellular space and nucleus (Table S2).

Analysis of the phylogeny of the tomato laccase family

To further analyze the evolutionary relationships between laccase family proteins from tomato and Arabidopsis, a phylogenetic tree was constructed using their amino acid sequences (Fig. S1). Seventeen Arabidopsis laccase family members were classified into six distinct groups, while eighteen tomato SlLACs were classified into five groups. Specifically, the number of laccase members in each group was not uniform. Specifically, there were seven laccase genes in Group II, four laccase genes in Group III, three laccase genes in Group I, and two laccase genes in Groups V and VI (Fig. 2). Notably, tomato SlLAC members in Group IV were subdivided into two Subgroups: Subgroup IVa (SlLAC6) and Subgroup IVb (SlLAC4). Group IV was divided into two subgroups, probably due to the unique selection pressure they may have experienced during the evolution process, resulting in changes in gene structure or expression regulation to adapt to the unique growth environment and physiological needs of tomato.

Fig. 2.

Phylogenetic analysis of laccase family members in tomato and Arabidopsis. A total of 18 SlLACs (tomato laccase proteins) and 17 AtLACs (Arabidopsis laccase proteins) were analyzed, all of which exhibit typical characteristics found in Arabidopsis. Using the full-length peptide sequence of the laccase, a phylogenetic tree with 1,000 bootstrap repeats was constructed via the maximum likelihood (ML) method. The tomato LAC proteins were divided into five categories. Different color branches represent different evolutionary branches

Analysis of conserved domains, motif compositions and gene structure of tomato laccase family members

To further analyze the structural diversity of SlLAC proteins, conserved motifs and domains were analyzed in conjunction with the constructed phylogenetic tree (Fig. 3). Conserved motifs of the 18 SlLAC proteins were identified using the MEME program, and seven conserved motifs (Motif 1, Motif 3, Motif 4, Motif 6, Motif 8, Motif 9, and Motif 10) were present in all putative SlLAC members. Additionally, motif composition and arrangement were similar among members of the same group. Within each group, all SlLACs shared identical motif compositions except for SlLAC13 (Group I), SlLAC1 (Group II), SlLAC10 (Group III), and SlLAC4 (Group IV), suggesting potential functional redundancy among these genes. Furthermore, SMART database analysis confirmed that all SlLAC proteins contain the conserved Cu-oxidase, Cu-oxidase_2, and Cu-oxidase_3 domains (Fig. 3A)—a hallmark of laccase proteins and essential for their catalytic activity. Specifically, the Cu-oxidase domain harbors conserved cysteine (Cys) residues, while the Cu-oxidase_2 and Cu-oxidase_3 domains contain conserved histidine (His) residues. These residues coordinate with copper ions to form a stable active center, which is critical for laccase-mediated redox reactions.

Fig. 3.

Phylogenetic relationships, motifs, and gene structures of laccase members from tomato. A The phylogenetic tree was constructed on the basis of the amino acid sequence of tomato laccases, and 1000 replications were constructed via the maximum likelihood (ML) method. The laccase genes of tomato were divided into five groups, and the laccase genes of Arabidopsis were divided into six groups. The six major groups are indicated and differentiated with diverse colored backgrounds. Schematic of the conserved motifs in the LAC proteins from tomato elucidated by TBtools. The relative length of the protein is represented by black lines. The digital color box represents the amino acid motif in the SlLAC protein, and the conserved motif of the protein was identified via MEME. The conserved domains of the tomato LAC protein were analyzed via TBtools software. The conserved domains of Cu-oxidase, Cu-oxidase_2 and Cu-oxidase_3 are represented by pink, green and yellow boxes, respectively. The conserved domains were identified via SMART. B Gene structure of tomato laccase members. The UTRs, CDSs, and introns are represented by yellow frames, green frames, and black lines, respectively

To further investigate the characteristics of tomato SlLAC family genes, a phylogenetic tree of tomato laccase family members was constructed and visualized (Fig. 3B) according to the amino acid sequence of all SlLACs. The results revealed that most tomato SlLAC genes presented highly analogous gene structures in the phylogenetic tree. Specifically, approximately 72% of tomato laccase genes contain six exons, and the number of exons ranges from four to eight (Fig. 3B). Notably, in the phylogenetic tree, the SlLAC genes in the same evolutionary branch presented different intron/exon numbers. For instance, SlLAC7 has seven exons, and its neighboring paralogous gene SlLAC8, which has a 100% bootstrap value, has six exons. Similarly, SlLAC13 has eight exons, and its neighboring paralogous gene, SlLAC14, generally has six exons, despite also sharing a 100% bootstrap value in their evolutionary relationships. Different combinations of exons or alternative splicing of introns may produce different laccase isomers, which are different in catalytic activity, substrate specificity or subcellular localization to fulfill the physiological demands of tomato at different growth and development stages and adapt to diverse environmental conditions.

Analyze the N-terminal signal peptide and glycosylation of tomato laccase family genes

The core function of the N-terminal signal peptide in plant genes is to guide nascent proteins to accurately localize to specific sites within the cell, ensuring that proteins perform their functions at the correct locations [18]. Among the 17 laccase proteins in Arabidopsis thaliana, only AtLAC6 lacks an N-terminal signal peptide. The prediction results of the N-terminal signal peptides in tomato laccase family proteins indicated that most laccase proteins contain N-terminal signal peptides (Table 1 and Fig. 4). SlLAC2, SlLAC3, SlLAC4, SlLAC8, SlLAC9, SlLAC11, SlLAC12, SlLAC14, SlLAC17 and SlLAC18 proteins contain an N-terminal signal peptide of 21–26 amino acids, which may guide these proteins to localize correctly in cells (Fig. 4). In contrast, other tomato laccase proteins do not contain an N-terminal signal peptide, and their normal localization may not depend on the signal peptide-mediated endoplasmic reticulum-Golgi (ER-Golgi) secretion system (Fig. 4). Some proteins may pass through their own hydrophobic domains or bind to transporters/channels on the cell membrane, directly passing through the plasma membrane and releasing outside the cell.

Table 1.

Prediction of N-terminal signal peptides and glycosylation sites of tomato

Gene	Signal peptide length	Cleavage site	Probability	Predicted target site	Number of potential glycosylation sites
					N-Glyc	O-Glyc
SlLAC1	None	None	None	None	9	None
SlLAC2	25	VEA-AL	0.979934	Secretory	12	3
SlLAC3	25	AEA-AL	0.983453	Secretory	12	5
SlLAC4	26	AHA-FV	0.978615	Secretory	10	2
SlLAC5	None	None	None	None	8	5
SlLAC6	None	None	None	None	12	16
SlLAC7	None	None	None	None	6	11
SlLAC8	22	AKA-KV	0.981959	Secretory	7	11
SlLAC9	25	VLC-KG	0.979386	Secretory	11	12
SlLAC10	None	None	None	None	7	4
SlLAC11	22	ANA-EF	0.98472	Secretory	6	2
SlLAC12	26	VSA-EV	0.87481	Secretory	8	6
SlLAC13	None	None	None	None	14	15
SlLAC14	23	VSA-EI	0.981899	Secretory	15	7
SlLAC15	None	None	None	None	12	9
SlLAC16	None	None	None	None	10	16
SlLAC17	21	VEC-RI	0.984151	Secretory	8	12
SlLAC18	21	VFG-IT	0.983242	Secretory	9	11

Fig. 4.

Prediction of N-terminal signal peptide of tomato laccase protein. The yellow and red solid lines indicate that the laccase protein contains an N-terminal signal peptide

The prediction of N-glycosylation sites of tomato laccase proteins showed that all proteins contained 6–15 N-glycosylation sites (Table 1), which were involved in various biological processes such as protein folding, localization, stability maintenance and cell recognition. Among all SlLAC proteins, only the SlLAC1 lacks O-glycosylation sites; the remaining members contain putative O-glycosylation sites, though the number of potential sites varies substantially (Table 1). It indicated that different laccase proteins may require different numbers of O-glycosylation sites to maintain the stability of protein structure.

Analysis of gene duplication and collinearity in the tomato laccase family

In the process of genome evolution, gene duplication, including fragment duplication, tandem duplication, and interspersed duplication, is the main driving force. In the tomato SlLAC family, there are four pairs of tandem repeats. Notably, the Ka/Ks values for these pairs were all less than 1, indicating that these genes have evolved under strong purifying selection. The repeat events were estimated to have occurred between 22.635 Mya (Ks = 0.118) and 266.239 Mya (Ks = 1.384) (Table 2).

Table 2.

Tandem duplications and segment duplication of the SlLACs gene pairs in tomato and inference of duplication time

Duplication gene pair	Duplicaiton type	Ka	Ks	Ka/Ks	Duplication date (Mya)
SlLAC2/SlLAC3	tandem	0.022866691	0.129650034	0.176372428	24.9326988
SlLAC10/SlLAC11	tandem	0.023906406	0.117703794	0.203106507	22.63534492
SlLAC14/SlLAC15	tandem	0.185945648	1.384445131	0.134310594	266.2394482
SlLAC1/SlLAC2	segment	0.064541848	0.628462856	0.102697951	120.8582415

Ka and Ks indicate nonsynonymous and synonymous substition rates, respectively. Ka/Ks is used to determine the selective pressure or strength after duplication. Ka/Ks values that = 1, > 1 or < 1 indicate neutral, positive, and purifying selection, respectively. The duplication date (Mya) was calculated using the formula: T = Ks/2λ × 10^− 6, where λ = 2.6 × 10^− 9

To investigate the homology of tomato SlLAC genes, collinearity analysis was performed between tomato and four other species (potato, eggplant, rice, and Arabidopsis) using the dual synteny plot function of MCScanX in TBtools. The collinearity analysis indicated that seven SlLAC genes exhibited collinearity with the laccase genes from Arabidopsis, potato, and eggplant. Additionally, only one tomato LAC gene was collinear with the LAC genes of eggplant, Arabidopsis and potato. In contrast, no collinearity was detected between the tomato laccase gene and the rice laccase gene. This lack of collinearity may be attributed to the fact that tomato is a dicotyledonous plant, while rice is a monocotyledonous plant, with far genetic relationship and low homology among laccase family members (Fig. 5).

Fig. 5.

Collinearity analysis of LAC genes in tomato, potato, eggplant, Arabidopsis, and rice. The collinear relationship is represented by a blue line

Analysis of the cis-regulatory elements predicted in the promoters of SlLACs

To investigate the potential function of tomato laccase family genes, a 2000 kb sequence upstream of the initiation codon of all tomato laccase family genes was extracted from TBtools. Plant CARE was used to detect cis-regulatory elements in the upstream promoter of the tomato SlLAC gene (Table S3). Notably, multiple light-responsive elements were detected in all tomato SlLAC upstream promoters, indicating that laccase genes play essential roles in tomato morphogenesis. Additionally, in the upstream promoter of the tomato LAC gene, multiple laccase genes were detected to contain multiple cis-acting elements related to the hormone response (Fig. 6). For example, among the 18 laccases, 13 laccases contained jasmonic acid response elements, 11 laccases contained ABA response cis-regulatory elements, 10 laccases contained gibberellin (GA) response elements, 7 laccases contained auxin response elements, and 5 laccases contained salicylic acid (SA) response elements. For example, when plants are subjected to stress such as drought and low temperature, the content of abscisic acid in the body increases, and the ABRE element can bind to abscisic acid-responsive transcription factors to initiate the expression of laccase genes, thereby participating in the response and adaptation of plants to stress.

Fig. 6.

Predicted cis-regulatory elements in the SlLACs promoter. A The number represents the quantity of cis-acting elements, and red indicates a greater number. B Positions of the cis-acting elements on the promoter of the SlLACs gene

Similarly, many stress response cis-acting elements related to the MYB binding site involved in drought induibility, defense and stress responsiveness, circadian control, low temperature, and anaerobic induction were also detected in the upstream promoter of the tomato SlLACs gene. For example, among the 18 SlLACs whose promoters were analyzed, the cis-regulatory elements related to anaerobic induction were the most prevalent, followed by those associated with low-temperature responses and those related to defense and stress responsiveness. Moreover, many specific cis-regulatory elements, such as the meristem, the endosperm, seed-specific regulation, zein metabolism regulation, and palisade mesophyll cell differentiation, were detected in the upstream promoters of multiple tomato LAC genes. In addition, multiple cis-regulatory elements related to zein metabolism were found in the upstream promoters of seven tomato laccase genes, and only cis-regulatory elements related to seed-specific regulation were found in the upstream promoter of the SlLAC18 gene. Furthermore, MYB binding sites (involved in regulating flavonoid biosynthesis) were found in the upstream promoter regions of SlLAC8 and SlLAC13, indicating that these two laccase genes may be involved in tomato flavonoid biosynthesis. Further visualization of the locations of these cis-acting elements on the SlLAC gene upstream promoter is shown in Fig. 6B. The diversity of promoter cis-regulatory elements indicates that the expression of tomato laccase genes is complexly regulated by a variety of environmental factors and hormone signals. These elements work synergistically under different physiological processes and environmental conditions to finely regulate the expression level of laccase genes to meet the needs of tomato growth and development and response to external environmental changes.

Tissue-specific expression profiles of the SlLAC gene family

To further analyze the potential function of SlLAC gene, different organs of tomato plants were collected, and the expression pattern of the SlLAC gene in tomato was detected via qRT-PCR (quantitative real-time polymerase chain reaction). Through promoter analysis of SlLAC family genes and functional studies of Arabidopsis laccase family genes, this family of genes was shown to be involved mainly in the growth and development of plants, and its role in fruits was less significant.

Therefore, qRT-PCR was used to detect the expression levels of multiple tomato laccase genes in tomato roots (Rt), stems (St), leaves (Le), and flowers (Fl). The tissue expression patterns of the SlLAC gene family exhibit distinct differentiation characteristics. Stem/leaf-preferential high expression: SlLAC1 (Fig. 7A) shows relatively high expression levels in stems and leaves; SlLAC2 (Fig. 7B) and SlLAC14 (Fig. 7N) display a gradient expression pattern of stems, roots, flowers and leaves. Root-preferential high expression: SlLAC3 (Fig. 7C), SlLAC5 (Fig. 7E), SlLAC10 (Fig. 7J), SlLAC12 (Fig. 7L), SlLAC16 (Fig. 7P), and SlLAC17 (Fig. 7Q) exhibit significantly higher expression levels in roots than in stems, leaves, and flowers, with expression fold differences ranging from 2.5 to 600 relative to other tissues. Single-tissue specific expression: SlLAC4 (Fig. 7D) is a flower-specific high-expression gene; SlLAC6 (Fig. 7F), SlLAC7 (Fig. 7G), SlLAC9 (Fig. 7I), SlLAC11 (Fig. 7K), and SlLAC13 (Fig. 7M) show leaf-specific high-expression characteristics. Broad multi-tissue expression: SlLAC8 (Fig. 7H) is expressed in roots, leaves, and flowers; SlLAC15 (Fig. 7O) is expressed across roots, stems, and leaves; SlLAC18 (Fig. 7R) accumulates mainly in roots and leaves.

Fig. 7.

Expression levels of the SlLAC genes in different organs of tomato. A-R Relative expression patterns of SlLAC1- SlLAC18 in the WT. Rt represents the root, St represents the stem, Le represents the leaf, and Fl represents the flower. SPSS 26.0 software was used to analyze the variance of the data. Different letters indicate significant differences (p < 0.05) according to Tukey’s test. The data are presented as the means ± standard errors of three replicates

Notably, some SlLAC genes (e.g., SlLAC7, Fig. 7G; SlLAC12, Fig. 7L; SlLAC17, Fig. 7Q) exhibit extreme expression differences (e.g., the expression level of SlLAC7 in stems is approximately 2800-fold that in roots), which further reflects the strong tissue-specific transcriptional regulation of this gene family.

Analysis of SlLACs genes under abiotic stress treatment

Multiple SlLAC genes may respond to a variety of abiotic stresses via cis-regulatory elements in the upstream promoter of the SlLAC gene. To further explore the function of the SlLAC gene, the expression patterns of SlLACs under different stress conditions were analyzed. Therefore, on the basis of the cis-acting elements of the promoter, the SlLAC4, SlLAC9, SlLAC11, and SlLAC18 genes were selected, and their expression levels under various stress treatments were detected via qRT-PCR. As shown in Fig. 8A-C, during cold stress, the expression level of the SlLAC4 gene was significantly upregulated within 8 h before it decreased. The SlLAC11 gene showed a considerable decrease in expression starting at 2 h, whereas the expression level of the SlLAC18 gene was notably inhibited after 12 h.

Fig. 8.

qRT-PCR analysis of the expression patterns of SlLACs in response to low temperature and drought. A-C Relative expression patterns of SlLAC4, SlLAC11, and SlLAC18 under low-temperature treatment. D-G Relative expression patterns of SlLAC4, SlLAC9, SlLAC11, and SlLAC18 under drought treatment. ANOVA was performed via SPSS 26.0. Significant differences (p < 0.05) between treatments, as determined by Tukey’s tests, are indicated with different letters. The data are expressed as the means ± standard errors of three replicates

After 2 h of drought treatment, the SlLAC4 gene presented a relatively high expression level (Fig. 8D). Since the SlLAC4 gene can be induced to express under low-temperature and drought conditions, and the homologous CsLAC4 gene in tea plants is associated with lignin biosynthesis [53], it is speculated that the SlLAC4 gene may also be involved in lignin biosynthesis, thereby helping tomatoes resist the negative effects of low-temperature and drought stresses. The SlLAC9 and SlLAC11 genes have the same expression pattern; that is, their expression levels start at 2 h and are significantly inhibited until they are upregulated after 12 h (Fig. 8E-F). The short-term suppression followed by a subsequent increase in the expression levels of SlLAC9 and SlLAC11 may be due to the fact that tomatoes prioritize the activation of core emergency response pathways to cope with stress in the early stages of drought treatment. Once these emergency pathways are established, there is a need to develop long-term defense mechanisms. It is speculated that SlLAC9 and SlLAC11 genes are involved in the synthesis of lignin or flavonoids. These two types of substances can strengthen cell wall structures or enhance antioxidant capacity, so the increased expression of these genes in the later stage helps tomatoes establish a more stable stress resistance system. Moreover, the SlLAC18 gene presented a relatively high expression level after 12 h of drought treatment (Fig. 8G). The SlLAC18 gene is induced to express, and it is speculated that it may promote the synthesis of lignin and flavonoids to help tomatoes cope with stress responses. With the extension of drought treatment time, the expression of the SlLAC18 gene may be inhibited due to excessive defense which could suppress plant growth. These results indicated that the expression level of the SlLACs gene changes under low temperature and drought stress in response to stress.

Analysis of SlLAC genes under hormone treatment

On the basis of the cis-regulatory elements in the upstream promoter of the SlLACs gene, the SlLACs gene has the potential to respond to multiple hormones. To further investigate the potential function of the SlLAC gene, the expression level of the SlLAC gene under different hormone treatments was detected via qRT-PCR.

As shown in Fig. 9A, D, G, J, under jasmonic acid (MeJA) treatment, the expression levels of the SlLAC4, SlLAC6, SlLAC9, and SlLAC13 genes all tended to increase, but their expression levels tended to decrease first and then increase, and their expression levels peaked at 24 h. The expression of SlLAC4, SlLAC6, SlLAC9, and SlLAC13 gene decreased in the early stage of MeJA treatment, which may be due to the fact that plants first start the upstream genes of the signal transduction pathway. After the signal transduction is complete, the downstream functional genes of SlLAC4, SlLAC6, SlLAC9, and SlLAC13 were activated, which may affect the synthesis of lignin and flavonoids, so that tomato responds to abiotic stress. Conversely, under ABA treatment, SlLAC6, SlLAC9, SlLAC13, and SlLAC11 were induced, but the expression levels of the SlLAC6, SlLAC9, and SlLAC13 genes gradually decreased with increasing treatment time, and their expression levels peaked at 2 h (Fig. 9B, E, H). In contrast, after ABA treatment, the expression of the SlLAC11 gene first increased but then decreased (Fig. 9K). In the early stage of ABA treatment, SlLAC6, SlLAC9 and SlLAC13 genes were highly expressed, suggesting that they may be involved in the rapid initiation of plant defense mechanisms. The expression pattern of SlLAC11 showed that there was a buffer period of signal accumulation or pathway activation in the response of SlLAC11 to ABA. After gibberellin treatment, the expression levels of the SlLAC6, SlLAC9, and SlLAC13 genes tended to increase first but then decreased, and their expression levels peaked at 2–4 h (Fig. 9C, F, I). In the early stage of gibberellin treatment, the signal is rapidly transmitted to the cell, activating the relevant transcription factors, and then starting the expression of SlLAC6, SlLAC9, and SlLAC13, indicating that these three genes may be directly or indirectly positively regulated by the gibberellin signaling pathway.

Fig. 9.

qRT-PCR analysis of the expression patterns of SlLACs in response to hormone treatment. A-I The relative expression patterns of SlLAC6, SlLAC9, and SlLAC13 under MeJA, ABA, and GA₃ treatments. J The relative expression patterns of SlLAC4 under MeJA treatments. K Relative expression patterns of SlLAC11 under ABA treatment. ANOVA was performed via SPSS 26.0. Significant differences (p < 0.05) between treatments, as determined by Tukey’s tests, are indicated with different letters. The data are expressed as the means ± standard errors of three replicates

Discussion

Laccases, as copper-containing polyphenol oxidases of the copper blue oxidase family, are conserved in mediating polyphenol oxidation and oxygen reduction across plants, and play pivotal roles in growth and development, lignin/flavonoid biosynthesis, and stress response [22, 54–59]. While laccase gene families have been systematically identified in multiple plant species (45 in Brassica napus [60], 84 in cotton [61], 44 in G. arboreum [61], 46 in Gossypium raimondii [61], 30 in rice [34], and 48 in eggplant [42]), systematic characterization and functional exploration of the tomato (Solanum lycopersicum) laccase family remain unreported. This study fills this gap by identifying 18 SlLAC genes in tomato, all harboring conserved Cu-oxidase_3, Cu-oxidase, or Cu-oxidase_2 domains—with Cu-oxidase_3 composed of motif 1 and 7, Cu-oxidase of motif 5, and Cu-oxidase_2 of motif 2 and 4. This domain-motif correspondence not only confirms the evolutionary conservation of the tomato laccase family but also provides direct evidence for their potential involvement in lignin synthesis and flavonoid metabolism via copper ion-dependent catalytic activity, laying the foundation for deciphering species-specific functional divergence of laccases in tomato.

Chromosomal localization analysis revealed uneven distribution of 18 SlLAC genes across seven tomato chromosomes, a pattern that may be linked to the family’s evolutionary expansion and functional differentiation. Physicochemical property prediction showed that most SlLAC proteins (16 out of 18) had an isoelectric point (pI) above 7.6, with only SlLAC2 (pI 6.05) and SlLAC8 (pI 6.8) being slightly acidic, suggesting that most tomato laccases preferentially function in an alkaline environment (Table S2)—a characteristic consistent with laccases from soybean [38] and eggplant [42], reflecting the conserved adaptability of laccases to intracellular microenvironments across Solanaceae and legumes. Notably, multiple SlLAC proteins were predicted to be secretory proteins with N-terminal signal peptides, which aligns with findings in soybean [38] and eggplant [42] and implies that tomato laccases may exert catalytic functions in the extracellular space (e.g., cell wall) to participate in cell wall remodeling or extracellular polyphenol metabolism. Further analysis of gene structure and protein motifs demonstrated that nearly all SlLAC proteins shared similar exon/intron architectures and motif compositions, strongly suggesting functional redundancy among SlLAC members with close genetic relationships. A striking tomato-specific evolutionary feature was identified in collinearity analysis: unlike eggplant, where one SmLAC gene exhibits collinearity with two rice OsLAC genes [42], no collinearity was detected between SlLAC and OsLAC genes. This indicates that during tomato evolution, the SlLAC family may have undergone unique gene duplication and loss events, leading to the disruption of orthologous relationships with rice laccases and potentially driving the evolution of tomato-specific functions.

Phylogenetic analysis integrating tomato and Arabidopsis laccase members (17 AtLAC genes categorized into six groups [18]) clustered the 18 SlLAC genes into five groups, with Groups II and III containing the most members—consistent with the distribution pattern in Arabidopsis [18], reflecting the evolutionary conservation of laccase family classification. More importantly, phylogenetic clustering combined with motif conservation and promoter cis-element analysis enabled the proposal of functional hypotheses for specific SlLAC genes. For example, SlLAC14 clustered with AtLAC17, which is involved in lignin polymerization under continuous light [22] ; given that SlLAC14 also harbors the conserved Cu-oxidase domains (motifs 1, 5, 7) essential for lignin synthesis, it is hypothesized that SlLAC14 participates in tomato lignin biosynthesis. SlLAC15 clustered with AtLAC2, which is induced by PEG and mediates abiotic stress response and root growth regulation [62]; notably, the SlLAC15 promoter contains multiple hormone- and stress-responsive cis-elements, and qRT-PCR results showed high expression of SlLAC15 in roots, stems, and leaves. This multi-dimensional evidence collectively suggests that SlLAC15 may be involved in tomato root development and abiotic stress response via hormone signal transduction—mirroring the miR397b-LAC2 module’s role in regulating root lignification and copper homeostasis under cadmium stress in Arabidopsis [63]. Additionally, SlLAC4 clustered with AtLAC15 (linked to seed color regulation [62]) and exhibited high expression in flowers (even higher than in stems), implying a potential role in tomato flower development or seed coat formation. SlLAC6 clustered with AtLAC6 (inhibited by cold stress [18]), and its expression was induced by MeJA, ABA, and GA₃, suggesting that SlLAC6 may mediate tomato cold stress response through hormone crosstalk. Members of SlLAC Group II clustered with AtLAC4 (a key lignin biosynthesis gene [22]), and Group III members clustered with AtLAC5 (associated with seed coat protective neolignans [64]); combined with their conserved lignin synthesis-related motifs, these results strongly support the involvement of multiple SlLAC genes in tomato lignin biosynthesis. In contrast, SlLAC Group V members clustered with AtLAC8 (linked to flowering time [62]), indicating a potential role in tomato flower opening—a process crucial for tomato reproduction and yield formation. Collectively, these findings suggest that SlLAC genes have evolved both conserved (e.g., lignin biosynthesis) and divergent (e.g., species-specific stress adaptation) functions to meet the physiological needs of tomato growth and environmental adaptation.

However, the SlLAC gene family remains uncharacterized in tomato, and functional prediction of these genes provides a basis for further functional investigations. We thus examined SlLAC expression in various tomato organs via qRT-PCR, and found that the family displayed highly divergent tissue-specific expression profiles, classified into four distinct patterns: stem/leaf-biased high expression, root-dominant expression (2.5–600-fold higher than other tissues), flower/leaf-specific expression, and multi-tissue broad expression. These profiles indicate that SlLAC genes may participate in stem morphogenesis, elongation and lignification, as well as root elongation, differentiation, structural construction and nutrient absorption and transport during root development. Extreme expression disparities in specific members (e.g., SlLAC7) further emphasize strong spatiotemporal transcriptional regulation of the SlLAC family, reflecting functional divergence of its members in modulating tissue-specific biological processes.

Furthermore, an analysis of expression patterns under low-temperature and drought conditions revealed that the expression of the SlLAC4 gene could be induced by these stress factors. Under low temperature conditions, the high expression of SlLAC4 gene may help tomato improve the stability of cell membrane and enhance cold resistance. Under drought conditions, the high expression of SlLAC4 gene may be involved in the synthesis of cell wall lignin, thereby enhancing the mechanical strength of the cell wall and reducing the evaporation of water. In contrast, the expression levels of SlLAC9, SlLAC11, and SlLAC18 were inhibited under low-temperature and drought conditions. Variation in expression patterns was observed among members of the laccase family. The variation in expression could be due to mutations and changes in the upstream regulatory regions of the genes that have occurred during evolution [65, 66].

The analysis of tomato hormone expression patterns revealed that methyl jasmonate (MeJA) treatment could induce the expression of the SlLAC4 gene. Additionally, MeJA, abscisic acid (ABA), and gibberellin (GA₃) treatments could induce the expression of the SlLAC6, SlLAC9, and SlLAC13 genes. Furthermore, ABA specifically induced the expression of the SlLAC11 gene. Under gibberellin treatment, changes in gene expression may affect plant internode elongation and seed germination. Under abscisic acid treatment, the change of gene expression is closely related to the response of plants to stress and the regulation of dormancy. These results provide an important basis for further understanding the mechanism of tomato laccase genes in abiotic stress and hormone signal transduction pathways, and also provide potential targets for crop improvement using these genes.

In conclusion, this study systematically characterizes the tomato SlLAC gene family and proposes biologically meaningful functional hypotheses by integrating motif, promoter, phylogeny, and expression data. It can be speculated that the expression level of the SlLAC4 gene can be induced by drought, low temperature, and hormones treatments, and that this gene may play a role in the biological processes of flowers and leaves. On the other hand, the expression of the SlLAC9 gene is inducible by hormonal treatments, while being repressed by drought and low-temperature stress, and this gene may play a role in the biological processes of leaves. However, the specific biological functions of the SlLAC4 and SlLAC9 genes need to be further revealed. Future research will focus on investigating these two genes to better understand their roles in the response of tomato to hormones and improve disease resistance in tomatoes. In subsequent studies, gene editing techniques (such as CRISPR/Cas9) can be used to knock out or overexpress key laccase genes, and the function of genes can be further studied by observing the phenotypic changes of transgenic plants.

Conclusions

This study represents the first comprehensive and systematic genome-wide analysis of the laccase gene family in tomato, which successfully identified 18 SlLAC genes from the tomato genome and conducted multi-dimensional characterizations of these genes. Through bioinformatics analysis, the physical properties, phylogenetic relationship, chromosome localization, promoter cis-regulatory elements, conserved domains and gene structure of these genes were clarified. It was found that tomato laccase genes had diversity and complexity in the evolution process, and were unevenly distributed on chromosomes. Fragment repeat events play an important role in the evolution and expansion of gene families. Promoter cis-regulatory element analysis showed that the expression of laccase gene was complexly regulated by light signal, hormone signal and various environmental stresses.

In terms of gene expression research, the specific expression patterns of tomato laccase genes in different vegetative organs at different developmental stages were revealed, as well as the significant responses to abiotic stresses such as drought, low temperature and hormone treatments such as gibberellin, methyl jasmonate and abscisic acid. The potential function and mechanism of laccase genes in tomato growth and development and stress response were preliminarily clarified.

Collectively, our results substantially advance the current understanding of the tomato laccase gene family and unravel its complex biological and physiological roles in mediating tomato growth, development, and responses to environmental perturbations. This study not only provides a valuable reference for further functional investigations of laccase genes in plants, but also holds important theoretical implications for research in the field of plant gene function.

Authors’ contributions

F.W. and Y.C. designed and managed the research. X.L., X.W., Y.D., B.Q., and Y.J. provided the experimental methods and carried out the experiments. Y. C and J. G analyzed the data. Y.C. wrote the manuscript and revised it.

Funding

This work was supported by the Natural Science Foundation Project of Colleges and Universities in Jiangsu Province (Z413B24317) and the Scientific Research Start-up project of the Huaiyin Institute of Technology (Z301B23519).

Data availability

All data on the results of this study can be found in the paper and its supplemental materials published online.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors agreed to publish research results on academic journals, conference proceedings and related academic platforms.

Competing interests

The authors declare no competing interests.