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Comprehensive analysis of the laccase gene family in tea plant highlights its roles in development and stress responses

Abstract

Background

Laccase (LAC) is the pivotal enzyme responsible for the polymerization of monolignols and stress responses in plants. However, the roles of LAC genes in plant development and tolerance to diverse stresses are still largely unknown, especially in tea plant (Camellia sinensis), one of the most economically important crops worldwide.

Results

In total, 51 CsLAC genes were identified, they were unevenly distributed on different chromosomes and classified into six groups based on phylogenetic analysis. The CsLAC gene family had diverse intron–exon patterns and a highly conserved motif distribution. Cis-acting elements in the promoter demonstrated that promoter regions of CsLACs encode various elements associated with light, phytohormones, development and stresses. Collinearity analysis identified some orthologous gene pairs in C. sinensis and many paralogous gene pairs among C. sinensis, Arabidopsis and Populus. Tissue-specific expression profiles revealed that the majority of CsLACs had high expression in roots and stems and some members had specific expression patterns in other tissues, and the expression patterns of six genes by qRT‒PCR were highly consistent with the transcriptome data. Most CsLACs showed significant variation in their expression level under abiotic (cold and drought) and biotic (insect and fungus) stresses via transcriptome data. Among them, CsLAC3 was localized in the plasma membrane and its expression level increased significantly at 13 d under gray blight treatment. We found that 12 CsLACs were predicted to be targets of cs-miR397a, and most CsLACs showed opposite expression patterns compared to cs-miR397a under gray blight infection. Additionally, 18 highly polymorphic SSR markers were developed, these markers can be widely used for diverse genetic studies of tea plants.

Conclusions

This study provides a comprehensive understanding of the classification, evolution, structure, tissue-specific profiles, and (a)biotic stress responses of CsLAC genes. It also provides valuable genetic resources for functional characterization towards enhancing tea plant tolerance to multiple (a)biotic stresses.

Introduction

The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most important woody cash crops, which tender buds and leaves are the raw material for the most widely consumed non-alcoholic beverages worldwide [1, 2]. The stems and leaves of tea plants have excellent physical and mechanical properties. The structure of the cell wall, which consists of cellulose, hemicellulose, pectin, protein and lignin, is one of the most pivotal contributing factors to these properties [3]. With the published genome of the tea plant [4, 5], genome-wide analysis of genes encoding enzymes participate in the lignin biosynthesis can be implemented. Studies have revealed that PAL, C4H, C3H, 4CL, HCT, CCR, CCoAOMT, CAD, F5H and COMT participate in lignin biosynthesis in plants [6,7,8,9]. However, how laccase is involved in lignin biosynthesis in tea plants remains unclear.

Lignin is the second most abundant biopolymer, and primarily consists of three canonical monomers, namely coniferyl (G), sinapyl (S) and p-coumaryl (H) alcohols [10]. Lignin monomers are synthesized in the cytosol and then exported to the apoplastic region, followed by oxidation and polymerization into lignin through a random coupling process [11]. During the polymerization process, laccase (p-diphenol:dioxygen oxidoreductase, EC 1.10.3.2) is the critical enzyme implementing single electron oxidation of phenolic compounds generating resonance structures [12]. Laccase is the largest subfamily of multicopper oxidases (MCOs), which have three conserved catalytic sites (Cu-oxidase, Cu-oxidase_2, and Cu-oxidase_3) that combine with four copper (Cu) ions and have a wide range of substrates. Laccases are widely present in bacteria, fungi, insects and plants, and many studies have shown the role of laccases in lignin biosynthesis and stress responses in plants.

Earlier studies have verified that many laccases can catalyze the oxidative polymerization of lignin precursors [13, 14]. Subsequent studies have further confirmed that laccase genes play crucial roles in the biosynthesis of lignin in some model and economically important plants, such as Arabidopsis thaliana [15], Brachypodium distachyon [16], Oryza sativa [17], Cleome hassleriana [18], Pyrus bretschneideri [19], and Phyllostachys edulis [11]. Among the 17 AtLACs in Arabidopsis thaliana, both AtLAC4 and AtLAC17 contribute to the constitutive lignification of stems, and lignin contents were slightly decreased in the double mutants Atlac4 lac11 and Atlac4 lac17; AtLAC11 was also found to participate in lignin polymerization, and the lignin content was tremendously decreased in the triple mutant Atlac4 lac11 lac17 [20]. Liu et al. (2017a) found that OsLAC10 was not only involved in lignin biosynthesis but also participated in the copper stress response in Oryza sativa. In the seed coats of Cleome hassleriana, ChLAC8 was essential for catechyl lignin polymerization and determined the lignin composition when caffeyl alcohol was available. In Gossypium hirsutum, overexpression of GhLac1 enhanced broad-spectrum biotic defense responses to both pathogens and pests by increasing lignin deposition [21]. The identified PeLAC10 in Phyllostachys edulis was overexpressed in Arabidopsis, demonstrating that the lignin content was increased and the adaptability to phenolic acid and drought stresses were improved in transgenic Arabidopsis [11]. Overall, these studies have shown that laccases play a pivotal role in plant development and responses to stresses by mediating lignin biosynthesis.

To clarify the role of the laccase genes in lignin biosynthesis in tea plant, we comprehensively analyzed the CsLAC gene family. Our analyses included determining chromosomal locations, evolutionary relationships, collinearity, gene structures and conserved motifs, cis-acting elements, protein interaction networks, target gene prediction of miR397, tissue-specific expression patterns, and expression profiles in response to biotic and abiotic stresses, as well as the development of polymorphic SSR markers. This study provides an important basis for further investigation of the role of CsLACs in the regulation of lignin biosynthesis and stress tolerance in tea plants.

Materials and methods

Plant material

Eight different tissues, including the apical bud, first leaf, second leaf, third leaf, young stem, young root, budding flower in autumn, and young fruit in summer, were collected from the 6-year-old cloned tea cultivar ‘Shuchazao’, which was planted in the Tea Plant Cultivar and Germplasm Resource Nursery (Hefei, Anhui, China) with good field management [2].

Two-year-old cloned tea plants (Camellia sinensis cv. ‘Shuchazao’) were cultured in plastic pots (30 cm diameter and 35 cm height) and grown under controlled conditions (23 ± 3 °C with 65 ± 5% humidity and a 16/8 h day/light photoperiod) at Anhui Agricultural University (Hefei, China). Plants with uniform growth (25–30 cm height) and without signs of disease and insects were used for our experiments. For the insect feeding treatment, tea geometrids (Ectropis obliqua) at the 3rd larval stage were starved for 8 h and distributed evenly on the tea plant leaves (leaves were at the same position on each plant, with 20 insects per plant), and then insects were removed after one-third of the leaves were consumed [22]. Leaves from the nontreated tea plants were used as controls. All treated and control leaves were collected at 3, 6, 12, and 24 h. Three biological replicates were harvested for each group of samples. All collected samples were immediately frozen in liquid nitrogen and subsequently stored at − 80 °C for further use.

Identification of the CsLAC gene family

A total of 17 Arabidopsis laccase members containing Cu-oxidase (PF00394), Cu-oxidase_2 (PF07731), and Cu-oxidase_3 (PF07732) domains were obtained [23]. To identify the CsLAC gene family in the Camellia sinensis ‘Shuchazao’ genome [5], BLASTp was performed using AtLAC protein sequences as queries, and sequences with an E-value < 10–10 were retained. The obtained candidate sequences with no conserved laccase domain were deleted and gene family identification was performed using the SMART (http://smart.embl-heidelberg.de/) and Pfam (http://pfam.xfam.org/) databases. A total of 51 unique CsLAC genes were identified, which were named from CsLAC1 to CsLAC51 based on their chromosomal location. The CDs and protein sequences of the 51 CsLAC genes are listed in Additional file 1: Table S1. To further explore the characteristics of their domain-containing proteins, the ExPasy program (http://web.expasy.org/protparam/) was used to calculate the molecular weight (MW) and isoelectric point (pI), and the online software Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) was used to predict their subcellular localization.

Chromosomal location, phylogenetic analysis and collinearity analysis

To identify their physical locations, the starting position of each CsLAC gene on each chromosome was determined by BlastN searches against the database of the complete tea plant genome [5]. The chromosomal locations of all CsLAC genes were confirmed by TBtools software (http://www.tbtools.com/). The amino acid sequences were used to construct phylogenetic relationships with MEGA6.0 using the neighbour-joining method (1000 bootstrap replications) [24]. The collinearity analysis of LAC genes within tea plants and among different plant species (Arabidopsis thaliana, Camellia sinensis and Populus trichocarpa) was performed by TBtools using MCScanX software.

Gene structures, conserved motifs and cis-elements

The exon‒intron structures were determined using the Gene Structure Display Server (http://gsds.gao-lab.org/). The conserved motifs of CsLAC protein sequences were analyzed by the MEME (http://meme-suite.org/tools/meme) program with previously described parameter settings and finally viewed by TBtools [25]. To determine the cis-elements, we obtained the 2000-bp sequence upstream from each CsLAC initiation codon and predicted their cis-elements using the online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) as described previously [26, 27].

Protein interaction network analysis

STRING (https://string-db.org/cgi/input.pl?sessionId) [28] was used for protein interaction network analysis and Cytoscape version 3.4.0 was used for construction of corresponding protein‒protein interaction networks.

Expression patterns of CsLAC genes

The transcriptome data generated from eight tissues, including the bud, the first leaf, the second leaf, the third leaf, stem, root, flower and fruit, were obtained from our previously published RNA-seq data [29]. The transcriptome data of CsLACs in response to drought and cold stresses were obtained from previous studies [30, 31]. The RNA-seq data of CsLACs in response to fungus and insect stresses were obtained from our previously published data [32] and unpublished data (NCBI SRA: PRJNA901518), respectively.

After obtaining the raw transcriptome data from our previous studies or the SRA database from NCBI, we converted the sra files to fastq format by the SRA Toolkit with fastq-dump and –split-3 parameters. Trimmomatic software was used to filter all raw reads based on standard criteria. The details are as follows: remove technical sequences, set a 5 bp sliding window from the 5’ end of the read and then remove the windows with an average quality below 20, cut off bases with a threshold lower than 3 at the beginning and end of reads, and retain the filtered reads with lengths greater than 40 bp. The obtained reads were used for comparison and assembly by Hisat2 and StringTie. The TPM (transcripts per million reads) values of CsLACs were calculated by StringTie and collated as a reference to evaluate the transcript abundance [33]. Heatmaps were drawn by TBtools software to show the different expression profiles.

Cs-miR397a targeted gene prediction and their expression profiles

miRNA-targeted gene prediction was performed by the online toolbox psRobot (http://omicslab.genetics.ac.cn/psRobot/). Based on small RNAome and transcriptome data, we analysed the expression patterns of miR397/CsLACs in response to gray blight treatment [32]. The expression abundance of miR397a was normalized to one million against the total clean reads in each library with the following formula: TPM = actual count of miRNA/total count of clean reads × 1,000,000 [34]. The differential expression of miR397a was analyzed using Student’s t-test, and the threshold for individual time points was set as P ≤ 0.05 and log2 (fold change) > 1 [32].

RNA extraction and qRT‒PCR analysis

Total RNA was isolated by the RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The concentration and integrity of the total RNA were examined using an Agilent 2100 Bioanalyzer. The specific primers were designed by Primer 5.0, and GAPDH was used as an internal reference gene based on our previous studies [2, 35]. The relative expression levels of CsLACs were determined by qRT‒PCR using SYBR Green Mix (Takara, Dalian, China) on a CFX96 real time detection system (Bio-Rad, USA). The detailed reaction system and procedures were performed according to our previous studies [22, 35]. The fluorescence was detected during the extension step and the specificity of the amplicon for each primer pair was confirmed by melting curve analysis.

All reactions were implemented in three biological replicates, and each replicate was measured in triplicate. The relative gene expression levels were calculated by the 2−ΔΔCt method [36]. The primer pairs of the six CsLACs used for qRT‒PCR analysis are listed in Additional file 2: Table S2.

Subcellular localization of the CsLAC3 protein

The open reading frame (ORF) of CsLAC3 was amplified by RT‒PCR and cloned into the pCAMBIA1305 vector to construct the fusion protein. The empty vector and constructed plasmids were introduced into EHA105 competent cells for transient expression in Nicotiana benthamiana leaves. The tobacco leaves were held at 25 °C in the dark and collected for fluorescence examination at 48 h after infection [35]. GFP signals in the transiently infected leaves were observed using an Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan). The relevant primers are listed in Additional file 2: Table S2.

SSR identification and primer design

Simple sequence repeats (SSRs) are generally defined as repeats consisting of 2–6 bp motifs. Thus, SSRs with these basic motifs were identified from the CsLAC gene family. The minimum repeat unit was defined as 6, 5, 4, 4, and 4 for dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides, respectively [37]. Subsequently, oligonucleotide primers were designed for the sequences flanking the SSRs by Primer 5.0 software. Amplicons needed to be 100–400 bp in length, and primers were designed with the following parameters: primer length 20–22 bp, with 20 bp as the optimum; GC content 40–60%, with the optimum value of 50%; and Tm 50–60 °C, with 56 °C as the optimum value.

SSR genotyping and data analysis

A total of 36 SSR loci from 30 CsLAC genes were selected for designing primers. To validate the primers, 45 tea cultivars or varieties were used for PCR amplification and subsequent resolution by electrophoresis. The reaction mixtures, thermocycling conditions and protocols for PCR product separation were performed based on a previous study [38]. The amplified fragments were separated on a 96-capillary automated DNA fragment analyzer (Fragment Analyzer™ 96, Advanced Analytical Technologies, Inc., Ames, IA). The separated DNA bands were visually scored using PROSize™ 2.0 software, which was included in the advanced Fragment Analyzer™ 96 system. Only one or two fragments were collected for each individual [37].

The number of alleles (Na), Shannon’s information index (I), observed heterozygosity (Ho), expected heterozygosity (He), genetic diversity (GD) and polymorphism information content (PIC) values were calculated with PowerMarker version 3.25 (http://statgen.ncsu.edu/powermarker/downloads.htm) (Liu and Muse 2005).

Results

Identification of the CsLAC gene family in tea plant

To identify the CsLAC genes, we used the tea plant reference genome [5] and searched the genome with HAMMER 3.0 software for three conserved cupredoxin domains (Cu-oxidase, Cu-oxidase_2, and Cu-oxidase_3). A total of 51 CsLAC genes were identified after elimination of redundant genes with only one or two cupredoxin domains or without integral ORFs. The identified CsLAC genes were named CsLAC1 to CsLAC51 and were analyzed for their basic characteristics, including the amino acid (aa) length, protein molecular weight (MW), isoelectric point (pI), and subcellular localizations (Table 1). The amino acid length of the 51 CsLAC proteins ranged from 454 (CsLAC4) to 608 aa (CsLAC19), while the MW ranged from 50.18 (CsLAC4) to 67.87 kDa (CsLAC19), and the pI ranged from 5.15 (CsLAC35) to 9.71 (CsLAC40). The prediction of subcellular localization showed that all CsLAC genes were located in the cell membrane.

Table 1 Characteristics of the 51 identified CsLAC genes from tea plant genome

Chromosomal distribution and phylogenetic analysis

The 49 identified CsLAC genes were unequally mapped onto 14 out of 15 chromosomes, while the chromosomal locations of the remaining 2 CsLAC genes were on unassigned contigs (Fig. 1). Among these chromosomes, Chr4 had the highest number of CsLAC genes, with a total of 15 members (CsLAC9 to CsLAC23). However, Chr6, Chr8, Chr12 and Chr15 each contained only one CsLAC member, and there were no CsLAC genes on Chr13. Moreover, some members of the CsLAC family on Chr4, Chr7, Chr9 and Chr10 exist in the form of gene clusters.

Fig. 1
figure 1

Chromosomal distribution of CsLAC family genes in the tea plant genome. The chromosomal position of each CsLAC gene was mapped based on the tea plant genome. The ruler on the left represents the physical map distance (Mb). Chromosome 1–15 are arranged from left to right, and two contigs are located on the bottom right corner

To investigate the phylogenetic relationships of laccases between tea plant and Arabidopsis, we constructed a phylogenetic tree using the full-length protein sequences of 51 CsLACs and 17 AtLACs. Based on the classification standard of Arabidopsis laccases, 51 CsLACs were divided into six groups, and their distribution in each group was rather uneven (Fig. 2). In detail, six CsLACs were clustered with four AtLACs (AtLAC4, AtLAC10, AtLAC11 and AtLAC16) in Group 1, Group 2 contained eight CsLACs and four AtLACs (AtLAC1, AtLAC2, AtLAC6 and AtLAC17), twelve CsLACs were clustered with three AtLACs (AtLAC7, AtLAC8 and AtLAC9) in Group 3, Group 4 contained only CsLAC11 and four AtLACs (AtLAC3, AtLAC5, AtLAC12 and AtLAC13), seven CsLACs were clustered with AtLAC14 and AtLAC15 in Group 5, and seventeen CsLACs were clustered without AtLACs in Group 6. The results revealed that CsLAC genes underwent specific evolutionary events after the divergence of tea plant and Arabidopsis.

Fig. 2
figure 2

Phylogenetic analysis of LAC genes from Arabidopsis and Camellia sinensis. A phylogenetic tree was constructed with 17 Arabidopsis protein sequences and 51 Camellia sinensis protein sequences. A total of six subclades of the family are highlighted in distinct colours. Green pentacles and red circles represent the LAC genes from Arabidopsis and Camellia sinensis, respectively

Homology analysis of the LAC gene family

Gene duplication is considered to be one of the most important driving forces of genome evolution. Generally, gene duplication includes tandem repeats, segmental duplication and interspersed repeats, while segmental and tandem duplication are considered as the main factors of gene family expansion in plants. Studies have shown that the tea plant genome underwent two rounds of whole-genome duplication (WGD) events since they diverged from their common paleopolyploid ancestor [4, 5]. To investigate the gene duplication pattern of CsLACs, we performed collinear analysis. As a result, 30 out of 51 genes were tandem repeats, including 10 clusters of tandem repeat genes on eight chromosomes. Additionally, we found 16 CsLAC genes to be segmentally duplicated genes on seven chromosomes (Chr1, Chr2, Chr4, Chr10, Chr11, Chr12 and Chr14) (Fig. 3A).

Fig. 3
figure 3

Collinearity of LAC gene pairs. (A) Collinearity analysis of the CsLAC gene family. All CsLAC genes were located on the chromosomes, and the identified CsLAC gene pairs are marked in red and connected by red lines. (B) Collinearity analysis of LAC genes across Arabidopsis, Camellia sinensis and Populus trichocarpa. The chromosomes of each species are represented by distinct colours, and the collinear gene pairs are connected by red lines

To predict the function of CsLACs, we performed a homology analysis of CsLAC genes with LAC family genes from the model plant Arabidopsis and the woody model plant Populus (Fig. 3B). As a result, 25 homologous gene pairs were identified between C. sinensis and Arabidopsis, and 58 homologous gene pairs were obtained between C. sinensis and Populus.

Motif compositions and gene structures

We analyzed the 51 CsLAC proteins to reveal their conserved motifs using the MEME program and identified six types of motifs (Fig. 4B). As expected, all of the identified proteins contained three motifs (Cu-oxidase, Cu-oxidase_2, and Cu-oxidase_3). Many classes of CsLAC proteins had completely identical motif compositions, suggest that the possibility of functional redundancy among these genes. In addition, varying numbers or length differences of motifs across the CsLAC proteins may indicate functional divergence among some members.

Fig. 4
figure 4

Phylogenetic tree, conserved domains and gene structure of CsLACs. (A) Phylogenetic relationship of CsLACs. (B) Conserved motifs and their distribution. The conserved motifs are named in the top-right corner and presented in different colours. (C) Gene structure of CsLACs. The UTR, CDS, and introns are represented by yellow boxes, green boxes and gray lines, respectively

To gain more insights into gene evolution, the exon‒intron organization of CsLACs was investigated by aligning coding sequences against their corresponding genomic sequences. The results showed that the gene structure of CsLACs exhibited diverse intron–exon patterns (Fig. 4C). For instance, except for both CsLAC45 and CsLAC51, which had only one exon, the number of exons varied from 5 to 10 among the other CsLAC genes. Notably, CsLAC10 had seven exons and was the longest gene with 25,167 bp in total. The CsLAC members with high homology had highly similar intron‒exon structures (intron number and exon length).

Identification of cis-acting regulatory elements

The cis-acting regulatory elements are located in the promoter region of target genes and can bind to appropriate transcription factors to regulate target gene expression in plants. To obtain insight into the regulation of CsLAC gene expression, we analyzed the cis-acting elements in the 2000 bp upstream sequences of the 51 CsLAC genes. A total of 40 types of cis-acting elements were obtained in the promoter regions of CsLAC family genes; these elements were divided into four categories, including stress responsive elements, light responsive elements, hormone responsive elements, and plant growth and development responsive elements (Fig. 5). Six types of elements belong to the stress‒responsive elements groups, including ARE (anaerobic inductive elements), GC-motif (enhancer-like element involved in anoxic specific inducibility), LTR (low temperature-inducible elements), MBS (MYB binding site involved in drought-inducible elements), MRE (MYB binding site involved in light responsiveness), and TC-rich repeats. The light‒responsive element group contained eighteen types of cis-acting elements; Box 4, GT1-motif, G-box and GATA-motif were relatively abundant among them. There were eleven types of cis-acting elements in the hormone responsive element group, and several important responsive elements were abundant, such as ABRE (abscisic acid-responsive element), CGTCA-motif (MeJA-responsive element), TCA-element (salicylic acid-responsive elements), TGACG-motif (MeJA-responsive element), and TGA-box (auxin-responsive element). The plant growth and development responsive element group included five types of cis-acting elements, such as CAT-box, circadian, GCN4-motif, HD-Zip1 and O2-site elements.

Fig. 5
figure 5

Identification of cis-acting elements of CsLAC genes. The distinct colours and numbers in the grid represent the numbers of different promoter elements in CsLAC genes

Interaction network CsLAC proteins in tea plant

To investigate whether CsLAC proteins might function by forming homo- or hetero-protein complexes, we constructed a protein interaction network for CsLACs based on their orthology with AtLAC proteins (Fig. 6). A total of 197 interacting protein pairs were predicted in Arabidopsis and divided into eight subfamilies (Additional file 3: Table S3). The number and types of interacting proteins for each subfamily were obviously distinct. Nine proteins were predicted to interact with CsLAC1 and CsLAC23 proteins in the first subfamily; three members (CsLAC7, CsLAC13 and CsLAC39) had eight interacting proteins in the second subfamily; CsLAC8 had eight interacting proteins in the third subfamily; six members (CsLAC14, CsLAC15, CsLAC40, CsLAC41, CsLAC42 and CsLAC43) had ten interacting members in the fourth subfamily; CsLAC11 had nine interacting proteins in the fifth subfamily; twelve CsLAC proteins (CsLAC16, CsLAC17, CsLAC18, CsLAC19, CsLAC20, CsLAC21, CsLAC22, CsLAC24, CsLAC28, CsLAC29, CsLAC30 and CsLAC31) interacted with only four proteins in the sixth subfamily; four members (CsLAC9, CsLAC34, CsLAC35 and CsLAC50) in the seventh family had six interacting proteins; and only one member (CsLAC49) had six interacting proteins in the eighth subfamily.

Fig. 6
figure 6

Interaction network of CsLAC proteins. There are 197 pairs of interacting proteins for 8 CsLAC subfamilies. The pink rhombus represents the CsLAC proteins in each subfamily; the purple circle indicates the interaction proteins in each clade

Expression patterns in different tissues

To investigate the tissue-specific expression profiles of the CsLAC gene family, transcriptome data from eight distinct tissues were collected for further analyses. The tissue expression profiles were viewed in a heatmap, demonstrating that all CsLAC genes were detected in these tissues with diverse expression patterns (Fig. 7A). For instance, nine genes (CsLAC1, CsLAC9, CsLAC11, CsLAC12, CsLAC14, CsLAC23, CsLAC38, CsLAC40 and CsLAC44) had extremely high expression levels in stems, twenty-four genes showed the highest expression level in roots (47.1%), three genes (CsLAC35, CsLAC45 and CsLAC51) showed obviously higher expression levels in flowers than in the other tissues, and some genes had relatively higher expression levels in buds and leaves.

Fig. 7
figure 7

Expression profiles of CsLAC genes in eight different tissues. The eight tissues include the apical bud, the first leaf, the second leaf, the third leaf, budding flowers, young fruits, young roots and young stems. (A) Expression patterns of the 51 CsLAC genes in eight tissues based on mRNA-seq data. The colour scale on the right indicates log2 transformed TPM values, which represent high and low expression, respectively. (B) Expression levels of six genes in eight different tissues using qRT‒PCR. The expression values are the mean ± standard deviation of three independent biological replicates, and each biological replicate contained three technical replicates. Different letters above the bars denote significant differences at P < 0.05

Based on the transcriptome data, we randomly selected six genes (CsLAC3, CsLAC17, CsLAC21, CsLAC38, CsLAC43 and CsLAC51) for further validation of their expression patterns in eight different tissues by qRT‒PCR (Fig. 7B). As a result, the expression profiles of six genes based on qRT‒PCR were highly consistent with the results from transcriptome data. For instance, based on both qRT‒PCR and transcriptome data, four genes (CsLAC3, CsLAC17, CsLAC21 and CsLAC43) had the highest expression level in roots, CsLAC38 had a relatively higher expression level in leaves than in the other tissues, and CsLAC51 showed a significantly higher expression level in flowers than in the other tissues.

Expression patterns in response to drought and cold stresses

Many studies have reported that LAC family genes participate in the response to abiotic stress, such as drought and cold stresses. Based on RNA-seq data, we analyzed the expression patterns of CsLACs under drought treatment and identified a total of 39 CsLAC genes (Fig. 8A). Five genes were dominantly downregulated under recovery after drought treatment, ten genes were significantly downregulated under drought and recovery treatments compared with the control, seven genes were significantly upregulated under drought and then downregulated after recovery, and seventeen genes had the highest expression level under recovery compared with the control and drought treatments.

Fig. 8
figure 8

Expression profiles of CsLAC genes under drought and cold stresses. (A) A total of 39 CsLAC genes were distinctly expressed under drought stress compared to the control. (B) A total of 46 CsLAC genes were differently expressed under cold treatment compared to the control

We also analyzed the expression patterns of CsLACs under cold treatment based on transcriptome data. A total of 46 CsLAC genes were identified and showed diverse expression profiles (Fig. 8B). Four genes had significantly higher expression levels than the control after 48 h of treatment and at 24 h, six genes showed higher expression levels than the control at 24 and 48 h, five genes had the highest expression level at 24 h, and five genes showed the lowest expression level at 24 h.

Expression patterns in response to insect and fungal stresses

An analysis of cis-acting elements and the functional validation of LAC genes in different plant species demonstrated that LAC genes played an important role in response to biotic stresses, including fungal and insect pest disease. The expression patterns of CsLACs were analyzed under simulated Ectropis obliqua attack based on the RNA-seq data, and a total of 45 CsLACs were identified (Fig. 9A). Nine genes showed the highest expression level at 6 h after treatment, seven genes had higher expression levels at 24 h than at other time points, three genes (CsLAC11, CsLAC22 and CsLAC26) displayed extremely high expression levels at 3 h, and eighteen genes had the highest expression level at 12 h time point.

Fig. 9
figure 9

Expression profiles of CsLAC genes under Ectropis obliqua feeding and gray blight treatment and subcellular localization of CsLAC3. (A) A total of 45 CsLAC genes were identified with significantly different expression levels compared to the control. (B) A total of 48 CsLAC genes were significantly differentially expressed compared to the control. (C) Expression patterns of CsLAC3 under gray blight treatment. The asterisks indicate the significant level (*** P < 0.001) based on a Student’s t-test. (D) Subcellular localization of the CsLAC3 protein. pCAMBIA1305 (empty vector) and pCAMBIA1305-CsLAC3 were transiently expressed in Nicotiana benthamiana leaves, scale bar = 25 μm

A total of 48 genes were differentially expressed after gray blight treatment compared with the control (Fig. 9B). Only one gene (CsLAC35) had an extremely higher expression level at 4 d compared with the control and at other time points, eighteen genes showed significantly increased expression at 1 d after treatment, fifteen genes had the highest expression level at 13 d, seven genes showed the highest expression level at 7 d, and only three genes had the highest expression level at 10 d compared with the other four time points. Subsequently, we validated the expression pattern of CsLAC3 under gray blight treatment (Fig. 9C). As a result, the expression level of CsLAC3 decreased slightly at 1 d and increased significantly at 13 d, displaying a similar result as that found with the transcriptome data. To obtain insight into the molecular function of the CsLAC3 protein, we transferred the CsLAC3-GFP plasmid into Agrobacterium to infect tobacco leaves, and the results showed that the CsLAC3 protein was localized in the plasma membrane (Fig. 9D).

Identification of cs-miR397a targeting CsLAC genes and their expression analysis in response to gray blight infection

In plants, it was reported that LAC genes can be targeted and regulated by miR397 [39]. In tea plant, a total of four miR397 were identified based on previous studies [32, 40, 41], including cs-miR397a, cs-miR397b and cs-miR397c (Fig. 10A). To investigate the possible role of miR397 in regulating CsLAC genes, all 51 CsLACs were used to analyze the presence of potential target sites. As a result, 12 (CsLAC1, CsLAC7, CsLAC12, CsLAC13, CsLAC15, CsLAC21, CsLAC22, CsLAC23, CsLAC39, CsLAC41, CsLAC42 and CsLAC43) out of 51 CsLACs were predicted to be the targets of cs-miR397a (Fig. 10B), while no CsLAC genes were targeted by cs-miR397b and cs-miR397c.

Fig. 10
figure 10

Putative miR397 target sites in CsLAC genes and the expression profile of ‘cs-miR397/CsLACs’ under gray blight disease stress

Moreover, the expression patterns of cs-miR397a and eleven predicted targets (except for CsLAC22) were analyzed under gray blight infection (Fig. 10C). The expression level of cs-miR397a was significantly downregulated at 1 and 13 d but upregulated at 4, 7 and 10 d. In comparison, most target genes (CsLAC1, CsLAC7, CsLAC12, CsLAC15, CsLAC23, CsLAC39, CsLAC41, CsLAC42 and CsLAC43) showed opposite expression patterns. Furthermore, we cloned and sequenced pre-miR397a and obtained its double-stranded stem‒loop precursor structure (Fig. 10D). The results provide an important foundation for further investigating the role of ‘cs-miR397a/CsLACs’ in tea plants.

Development and polymorphism analysis of SSR markers

A total of 36 SSR loci from 30 genes were obtained for designing primers. To test the reliability and polymorphism of these SSR loci, eight tea samples were selected for screening the primers. Among them, the markers without polymorphism of amplification, as well as those with ambiguous bands, were not used. As a result, a total of 18 SSR markers from 15 genes that generated both unambiguous and polymorphic bands were successfully developed. Subsequently, we selected 45 varieties/cultivars belonging to section Thea of the genus Camellia in the family Theaceae to test the tea plant germplasm resource transferability of these markers. The primer pairs of the 18 SSR markers and 45 tea samples are listed in Additional file 4: Table S4.

The majority of SSR markers displayed high polymorphism among the 45 tea samples, and the genetic properties of all the SSR markers were calculated (Fig. 11 and Table 2). The Na per locus ranged from 3 (CsLAC1-2, CsLAC6 and CsLAC39) to 8 (CsLAC49) with an average of 5.222 alleles. The I ranged from 0.468 (CsLAC1-2) to 1.598 (CsLAC36), with an average of 1.099. The Ho varied from 0.111 (CsLAC28) to 0.911 (CsLAC36), with an average of 0.510; the He ranged from 0.240 (CsLAC1-2) to 0.793 (CsLAC36), with an average of 0.566. The GD value ranged from 0.238 (CsLAC1-2) to 0.784 (CsLAC36), with an average of 0.560, and the PIC value varied from 0.221 (CsLAC1-2) to 0.750 (CsLAC36), with an average of 0.515. The results showed that these newly developed SSR markers from the CsLAC gene family are stable and highly polymorphic, providing a valuable resource for genetic research in tea plant.

Fig. 11
figure 11

Gel electrophoresis image of 18 SSR markers among 45 tea varieties/cultivars

Table 2 Characteristics of 18 developed SSR markers

Discussion

Laccase enzymes are multicopper oxidases that play critical roles in the biosynthesis of lignin, which is involved in plant development and various stress responses. Systematic analyses have been conducted to identify laccase gene families in many model, crop and woody plants. Tea plant is one of the most important woody cash crops worldwide; however, there is little information about CsLAC genes. Here, a total of 51 CsLACs were identified based on the tea plant genome, and comprehensive analysis of the CsLAC gene family was performed. The number of CsLAC genes in tea plants is higher than that in most other plants studied, including Arabidopsis thaliana (17) [42, 43], Brachypodium distachyon (29) [16], Oryza sativa (30) [17], Phyllostachys edulis (23) [11], Citrus sinensis (24) [44], Pyrus bretschneideri (41) [19], and Populus trichocarpa (49) [45], while it is less than that in soybean (93) [23] and Eucalyptus grandis (54) [46]. For gene family number, tandem and segmental duplication events are the major reasons for gene expansion [47]. In tea plant, 49 CsLACs are unevenly distributed on 14 chromosomes and 2 CsLACs are on unassigned contigs, including 10 clusters of tandem repeat genes on eight chromosomes and 16 segmentally duplicated genes on seven chromosomes (Fig. 3A).

All 51 identified CsLACs had conserved copper-binding domains, while most of them had distinct gene structures, implying that they had similar genetic origins but had divergent biological functions. Notably, some transcription factors (TFs) may be involved in regulating the expression of CsLAC genes by the recognition of their cis-acting elements, such as G-box elements are generally exist in the promoters of light-responsive genes and can be bound by bZIP and bHLH TFs [48, 49], ABRE elements are often discovered in the promoters of ABA hormone‒responsive genes [50]. To understand the potential regulation of CsLAC expression, we analyzed cis-acting elements in the 51 CsLAC promoter regions. Four classes of cis-acting elements, including plant growth and development elements, stress responsive elements, light responsive elements and hormone responsive elements, were obtained. The putative cis-acting elements suggested that CsLACs function in various physiological processes, such as development, morphogenesis, and response to stresses. Additionally, tissue-specific expression profiles of the 51 CsLACs were analyzed, demonstrating that they had diverse expression patterns and preferred to a particular organ. The majority of CsLACs had the highest expression level in roots (47.1%), and nine CsLACs were preferentially expressed in stems (Fig. 7A). Similarly, most laccase genes are mostly expressed in roots and stems in several other plant species, such as Arabidopsis [42, 51], Oryza sativa [17], and Eucalyptus grandis [46]. Since both roots and stems contain a predominant amount of lignified tissues, these CsLAC genes might play important roles in lignin biosynthesis. It was shown that some CsLACs had predominant expression levels in buds and leaves, suggesting that they are involved in the growth and development of buds and leaves. Interestingly, CsLAC35, CsLAC45 and CsLAC51 had extremely high expression levels in flowers but lower to no-expression levels in other tissues; some genes were also found to be mainly expressed in flowers in other plant species including Oryza sativa [17], Solanum melongena [52], and Phyllostachys edulis [11]. The results indicate that these three CsLAC genes may play a major role in flower development.

Based on multiple sequence alignments, a phylogenetic tree containing 17 AtLACs and 51 CsLACs was constructed, and six groups were identified based on phylogenetic analysis (Fig. 2). AtLAC4 and AtLAC11 in Group 1 and AtLAC2 and AtLAC17 in Group 2 have been verified to be related to lignin biosynthesis [15, 20, 53], implying that CsLACs in the two groups are probably involved in lignin biosynthesis. In Group 4, twelve CsLACs were clustered with Arabidopsis laccases AtLAC7, AtLAC8 and AtLAC9, which respond to environmental cues [42]. In Group 5, seven CsLACs were clustered with AtLAC14 and AtLAC15, which have been reported to be involved in the polymerization of phenolic compounds [43, 54]. In upland cotton, GhLAC1 and GhLAC15 were phylogenetically related to AtLAC14 and AtLAC15, which were participate in positively regulating defense-induced lignification to enhance the broad-spectrum biotic stress response [21, 55]. Based on the heatmap, all the CsLACs were involved in the response to herbivory feeding except CsLAC36, and the expression of seven CsLACs was positively regulated by fungal stress treatment (Fig. 9). Therefore, the CsLACs in Group 5 are probably involved in lignin biosynthesis and defense responses to biotic stresses. A total of seventeen CsLACs, but no AtLACs, were classified into Group 6, implying that these CsLAC genes may have distinct roles during tea plant evolution. CsLAC3 in Group 6 was selected for further validation, displaying that CsLAC3 protein was localized in plasma membrane, had high expression in roots and leaves (Fig. 7) and was involved in the response to gray blight treatment (Fig. 9), while CsLAC3 functions should be further validated.

Studies have shown that some LAC genes are targets of miR397, which is conserved across most plant species [56]. The plant miR397 family mainly targets LAC genes functioning in lignin biosynthesis and is involved in plant development and stress responses, such as floral organ and seed development, fruit development, drought and cold stresses, heavy metal stress, and pathogen stress [39]. We identified 12 CsLAC genes as potential targets of Cs-miR397a, which had only one base difference from At-miR397a (Fig. 10). Based on small RNA sequencing data, we analyzed the expression patterns of Cs-miR397 under cold [57], drought [58], insect herbivory [22], and fungal disease stresses [32]. However, no differentially expressed miR397 was identified under cold, drought and insect herbivory stresses, whereas Cs-miR397a was identified under gray blight infection. Under gray blight infection, the expression level of Cs-miR397a was downregulated at 1 and 13 d but upregulated significantly at 4, 7 and 10 d, while most of the potential CsLAC targets had the opposite expression patterns (Fig. 10C). In Malus hupehensis, it was reported that Mh-miR397b negatively regulates resistance to Botryosphaeria dothidea disease by modulating MhLAC7, which is involved in lignin biosynthesis. Therefore, we predicted that Cs-miR397a may be involved in fungal disease resistance by targeting CsLACs in tea plant, but further research is needed.

SSR molecular markers have gained considerable importance in plant genetic research due to their multiple-allelic nature, codominant inheritance, stability, and high abundance in the genome [37, 59]. After screening 36 SSR markers, we obtained 18 SSR markers that showed stable and unambiguous amplification bands in 45 tea samples (Fig. 11). The majority of SSR markers displayed high polymorphism with an average PIC value of 0.515, while CsLAC1-2 and CsLAC28 had low polymorphism with PIC values of 0.221 and 0.248, respectively. The polymorphism of SSR markers can be influenced by several factors, including the location of SSR loci in the genome, the number of markers, the sampling scheme, the accuracy of genotyping data, and the type of SSR motif repeats [37, 38]. In tea plant, several previous studies of genomic SSR marker development showed that the average PIC values for 13, 30 and 36 markers were 0.860, 0.704 and 0.862, respectively [38, 59, 60], while two studies showed that the average PIC values of SSR markers were similar to the average PIC value in our study [61, 62]. Overall, the newly developed SSR markers can be used for various genetic studies in tea plant, such as genetic variation, evolutionary origin, fingerprinting, QTL mapping, and marker-assisted selection breeding.

Conclusions

In this study, we performed a genome-wide analysis of the CsLAC gene family, generated a wide range of expression data, including tissue-specific expression patterns and expression profiles of CsLACs responding to abiotic and biotic stresses, and developed some highly polymorphic SSR markers. This study provides target genes for regulating lignin biosynthesis in tea plant and lays the foundation for understanding the function of CsLAC genes.

Availability of data and materials

The data generated and analyzed in this study are included in this article and its Supplementary materials. RNA-Seq data of Ectropis obliqua feeding treatment are available at the NCBI SRA database (https://www.ncbi.nlm.nih.gov/) under project accession number PRJNA901518.

Abbreviations

PAL:

Phenylalanine ammonia-lyase

4CL:

4-(Hydroxy) cinnamoyl CoA ligase

C3H:

P-coumarate 3-hydroxylase

C4H:

Cinnamate 4-hydroxylase

CAD:

Cinnamyl alcohol dehydrogenase

CCoAOMT:

Caffeoyl CoA O-methyltransferase

HCT:

Hydroxycinna-moyl-CoA:shikimate (SA)/quinate (QA) hydroxycinnamoyl transferase (HCT); CCR: Cinnamoyl CoA reductase

COMT:

Caffeic acid/5-hydroxyferulic acid O-methyltransferase

F5H:

Ferulate 5-hydroxylase

LAC:

Laccase; TPM: Transcripts Per Million

SSR:

Simple sequence repeat

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Acknowledgements

We appreciate the editor and anonymous reviewers for critically evaluating the manuscript and providing constructive comments for its improvement. We thank the Tea Plant Cultivar and Germplasm Resource Garden (Guohe town, Lu Jiang County, Anhui Province, China) for providing tea plant samples.

Funding

This work was supported by the National Natural Science Foundation of China (32272770 and 31800585), the Project of Science and Technology of Yunnan Province (202102AE090038), and the Natural Science Foundation of Anhui Province (1808085QC92).

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JXZ performed data analysis and real-time PCR. HXZ and KLH conducted real-time PCR, subcellular localization, data analysis, and SSR marker development. RG, HX, JYZ, JJZ, HLG, HRC, and GQL are involved in data analysis, sample collection, and DNA extraction. CLW revised the manuscript. SRL designed the research and wrote the manuscript. All authors have read and approved the manuscript.

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Correspondence to Shengrui Liu.

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Supplementary Information

Additional file 1:

Table S1. CsLAC gene family CDs and protein sequences. 

Additional file 2:

Table S2. Primers developed for six CsLAC genes for qRT‒PCR and CsLAC3 for subcellular localization.

Additional file 3:

Table S3. Proteins interacting with LAC proteins in Arabidopsis and C. sinensis. 

Additional file 4:

Table S4. Primer pairs for 18 SSR markers and 45 tea plant samples used for SSR marker development.

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Zhu, J., Zhang, H., Huang, K. et al. Comprehensive analysis of the laccase gene family in tea plant highlights its roles in development and stress responses. BMC Plant Biol 23, 129 (2023). https://doi.org/10.1186/s12870-023-04134-w

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Keywords

  • Laccase
  • Tea plant
  • Development
  • (A)biotic stress
  • Expression patterns
  • SSR markers