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.

Keywords: (A)biotic stress; Development; Expression patterns; Laccase; SSR markers; Tea plant.

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 10

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

Fig. 11

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