Evolution and expression analysis of the caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) gene family in jute (Corchorus L.)

Abstract

Background

Jute is considered one of the most important crops for fiber production and multipurpose usages. Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a crucial enzyme involved in lignin biosynthesis in plants. The potential functions of CCoAOMT in lignin biosynthesis of jute have been reported in several studies. However, little is known about the evolution of the CCoAOMT gene family, and either their expression level at different developing stages in different jute cultivars, as well as under abiotic stresses including salt and drought stress.

Results

In the present study, 66 CCoAOMT genes from 12 species including 12 and eight CCoAOMTs in Corchorus olitorius and C. capsularis were identified. Phylogenetic analysis revealed that CCoAOMTs could be divided into six groups, and gene expansion was observed in C. olitorius. Furthermore, gene expression analysis of developing jute fibers was conducted at different developmental stages (15, 30, 45, 60, and 90 days after sowing [DAS]) in six varieties (Jute-179 [J179], Lubinyuanguo [LB], and Qiongyueqing [QY] for C. capsularis; Funong No.5 [F5], Kuanyechangguo [KY], and Cvlv [CL] for C. olitorius). The results showed that CCoAOMT1 and CCoAOMT2 were the dominant genes in the CCoAOMT family. Of these two dominant CCoAOMTs, CCoAOMT2 showed a constitutive expression level during the entire growth stages, while CCoAOMT1 exhibited differential expression patterns. These two genes showed higher expression levels in C. olitorius than in C. capsularis. The correlation between lignin content and CCoAOMT gene expression levels indicated that this gene family influences the lignin content of jute. Using real-time quantitative reverse transcription PCR (qRT-PCR), a substantial up-regulation of CCoAOMTs was detected in stem tissues of jute 24 h after drought treatment, with an up to 17-fold increase in expression compared to that of untreated plants.

Conclusions

This study provides a basis for comprehensive genomic studies of the entire CCoAOMT gene family in C. capsularis and C. olitorius. Comparative genomics analysis among the CCoAOMT gene families of 12 species revealed the close evolutionary relationship among Corchorus, Theobroma cacao and Gossypium raimondii. This study also shows that CCoAOMTs are not only involved in lignin biosynthesis, but also are associated with the abiotic stress response in jute, and suggests the potential use of these lignin-related genes to genetically improve the fiber quality of jute.

Background

Jute, one of the most important sources of natural fibers, provides approximately 80% of the world’s bast fiber output. In recent years, jute is considered as an important cash crop with multipurpose usages like paper and textile industries, herbal medicine, leafy vegetable, and renewable biofuel source, etc. [1, 2]. Although there are more than 100 Corchorus species in the Malvaceae family, only C. capsularis and C. olitorius are commercially cultivated [3].

Lignin is a heterogeneous polymer without a defined primary structure and has monolignol monomers as precursors [4]. Recent studies have indicated that lignin plays a crucial role in determining stem strength, cell wall structural integrity, pathogen resistance, and water transfer efficiency [5,6,7]. Lignin provides mechanical support for the growth of vertical land plants. Nonetheless, high lignin content has a negative effect on jute fiber separation as well as fiber fineness [2, 8]. In fact, the lignin content of jute fibers is comparatively high (~ 15%), which distinguishes them with other non-wood bast fibers such as ramie, hemp and flax fibers (< 5%). Islam et al. reported that C. olitorius fibers contain more lignin and less cellulose than those of C. capsularis [3]. Therefore, it is essential to identify the regulatory factors involved in plant lignin biosynthesis to improve our ability to grow plants with low lignin levels. Lignification in plants is influenced by a number of plant-related factors. Compared to flax, jute exhibits duplications in the lignin biosynthetic genes cinnamoyl-CoA reductase (CCR), 4-coumarate: CoA ligase (4CL), caffeic acid O-methyltransferase (COMT), and Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT). Many studies have reported that transcriptionally co-expressed genes are functionally associated and might interact with each other at the molecular and physiological levels. Monolignols are the precursors of lignin, which are synthesized as a branch of the phenylpropanoid pathway in the cytoplasm near the ER membrane and then transported to the cell wall to get polymerized into lignin [2, 9]. There are more than 10 gene families associated with monolignol biosynthesis, among which, CCoAOMT is one of the most important genes for monolignol biosynthesis [4, 10]. Chakraborty et al. discovered a total of 43 isoforms of all ten genes involved in monolignol biosynthesis of jute, including five isoforms for CCoAOMT [2]. The enzyme CCoAOMT is a S-adenosyl-L-methionine methyltransferase (SAM) central to the lignin biosynthetic pathway [5]. This protein is involved in the biosynthesis of lignin in plants as it catalyzes the methylation of caffeoyl CoA, an important metabolite for the biogenesis of guaiacyl lignin [11, 12]. The role of CCoAOMT in lignification was also supported by the fact that overexpression of jute CCoAOMT in A. thaliana resulted in the effect of increasing lignin content [13], and inhibition of CCoAOMT activity in transgenic tobacco significantly reduced lignin content in that species [14, 15]. Therefore, it is crucial to determine the expression of CCoAOMT genes in plants. Chakraborty et al. found CcCCoAOMT2 was down-regulated in mutant bast tissues at an early growth stage of jute, while it was over-expressed at later growth stage [2]. Liu et al. investigated changes in the expression of CCoAOMT in loquat fruits stored at low temperatures, and found that the expression of this gene in green loquat fruits was lower than that in mature fruits [16]. Low temperature storage stimulated its transcription at the beginning of the storage process; however, its expression decreased throughout the rest of the storage period [16]. Zhang et al. reported that CCoAOMT is expressed in leaf tissues, roots, stem bark, and stems of jute, with an obviously greater transcription in the stem [13]. CCoAOMT expression was found to be particularly associated to all lignifying tissues, including phloem fibers, xylem fibers, and tracheary elements (TEs) in the stems of forsythia [17], tobacco [18], alfalfa [19,20,21], tomato [22], and soybean [23]. The existence of an intimate connection between lignification and levels of CCoAOMT transcripts has been reported in dicot crops [17], supporting the hypothesis that CCoAOMT-mediated methylation is commonly implied in the biosynthesis of lignin during normal growth and development of dicot plants.

Water deficiency occurs in plants whenever water provision is inadequate for development, transpiration, and photosynthesis [24], and ultimately leads to yield reduction [25]. Alvarez et al. reported that polyethylene glycol (PEG) treatment inhibited lignin biosynthesis in maize [25]. In the current context of global climatic change, water deficit stress is a major problem limiting production and productivity of jute. It causes 20% ~ 30% loss of fiber yield and also deteriorates the quality [26]. Meanwhile, salt stress negatively affects jute growth and physiological parameters, which subsequently reduces yield and quality [27]. Liu et al. studied the levels of CCoAOMT transcripts under abiotic stress in switchgrass and observed substantial up-regulation of this gene in stem tissues, as its expression increased by 33-fold in comparison to that of untreated plants [28]. The study of CCoAOMT expression under abiotic stress in switchgrass and other plants showed that this gene can be greatly impacted by drought stress [29]. However, the effects of NaCl and PEG stresses on the expression of jute CCoAOMT genes have not been thoroughly examined.

Phylogenetic analysis of sorghum demonstrated that one sorghum CCoAOMT-like enzyme is closely related to ancestral cyanobacterial CCoAOMT-like proteins. The remaining CCoAOMT-like enzymes, including those highly expressed in the leaves and stem, are closely related to the CCoAOMT gene family. Genes from these two groups have dissimilar or conserved gene structures [30]. Conversely, the identification and evolutionary analysis of CCoAOMT genes in jute have not yet been reported, and their transcriptional patterns were seldom studied. In order to determine the evolution of CCoAOMT gene family in jute, and to investigate the expression pattern of CCoAOMT genes in different situations of jute, we analyzed the evolution and expression patterns of members of the CCoAOMT gene family in the stem of distinct varieties of jute at different developmental stages, and revealed the levels of transcription of these genes under abiotic stress.

Results

Identification and cloning of CCoAOMT genes in jute

The availability of jute (C. capsularis and C. olitorius) genome sequences [1] made it possible to pinpoint the members of the CCoAOMT gene family in jute. Through a comparative genomics approach, 12 CCoAOMT genes were identified in C. olitorius, referred to as Co.CCoAOMT1–Co.CCoAOMT9, and eight orthologous genes were identified in C. capsularis, referred to as Cc.CCoAOMT1–Cc.CCoAOMT9 (Table 1). For consistency, C. capsularis and C. olitorius genes were named according to their phylogenetic relationships. These genes were used as references for primer design. DNA sequences from CCoAOMTs were cloned using reverse transcription PCR (Table 2, Additional file 1). These cloned CCoAOMT sequences were estimated to include complete open reading frames (ORFs). The size of the CCoAOMT proteins ranged from 99 to 1631 amino acid (aa) residues, with an average length of 365.5 aa. The corresponding molecular mass ranged from 11.36 to 181.62 kDa, with an average molecular weight of 41.03 kDa, and the computed theoretical isoelectric points ranged from 4.61 to 8.93 (Table 3). An analysis of the subtracted protein sequences of CCoAOMTs demonstrated that they are highly conserved. All Cc.CCoAOMT and Co.CCoAOMT proteins share a conserved domain which is “AdoMet_MTases” with the exception of Cc.CCoAOMT2 and Co.CCoAOMT2 proteins which share “PLN02589” as their conserved domain (Additional file 2). Six of the Cc.CCoAOMTs and eleven of the Co.CCoAOMTs shared a conserved domain (AdoMet_MTases) with A. thaliana and Oryza sativa (Additional file 2). In addition to seven â-strands, eight á-helices and a typical á/â Rossmann fold were discovered in both types of proteins (Fig. 1). Comparative analysis of protein sequences showed that CCoAOMTs exhibited protein sequence similarity, ranging from 20% to 100% (Table 4). Pairwise comparisons among the Co.CCoAOMTs showed that these genes shared protein sequence similarities that ranged from 19.30% to 94.17% with an average of 51.63%, while among the Cc.CCoAOMTs varied from 20.75% to 91.09% with an average of 46.09% (Additional file 3). Using the MEME online tool, a comparison of dissimilarity in protein structure was made to identify the conserved motifs among the jute CCoAOMT proteins. Altogether, 12 distinct motifs were discovered of CCoAOMTs in jute (Additional file 4).

Table 1 Basic information of the putative CCoAOMT gene family in jute

Table 2 Comparison of CCoAOMT genes between jute and PCR cloning sequences

Table 3 Comparison of the characterization of the CCoAOMT gene family in jute

Fig. 1

Protein sequence arrangement of the CCoAOMT genes in jute. Level of similarity of the CCoAOMT protein sequences were exhibited in dissimilar colors (cyan, cherry red). α-helices depicted as cylinders and β-strands as arrows indicate conserved secondary structural elements

Table 4 Pairwise comparisons of amino acid sequence among CCoAOMT genes in jute

Remarkably, two pairs of tandem duplicated genes (Co.CCoAOMT3a & Co.CCoAOMT3b, Co.CCoAOMT5a & Co.CCoAOMT5b) were discovered in the C. olitorius genome, and were likely originated from the C. olitorius as these two tandem duplicated genes of CCoAOMTs were not present in C. capsularis (Additional file 5). These two pairs of CCoAOMTs shared high (71.89% and 94.17%) sequence similarity, suggesting that they were originated from the very recent tandem duplication event (Additional file 5).

Analysis of the synonymous to non-synonymous interchange ratio (Ka/Ks) was performed to examine the evolutionary functional hindrance in these jute genes. The results showed there are no non-synonymous sites in gene pairs of Cc.CCoAOMT1-Co.CCoAOMT1 and Cc.CCoAOMT2-Co.CCoAOMT2. Meanwhile, the Ka/Ks ratios of the other six CCoAOMT gene pairs investigated were below 0.5, indicating that purifying selection was the principal driver of the evolution of CCoAOMT members (Fig. 2, Additional file 6).

Fig. 2

The synonymous (Ks) and non-synonymous (Ka) interchange percentages of 8 CCoAOMT members of jute. For statistical analysis of the ka/ks p-value, student's t-test was performed. ** refers p value < 0.01 and *** refers p value < 0.001 respectively

Phylogenetic analysis of the CCoAOMT family in 12 different plant species

To broadly assay the evolutionary relationships between the CCoAOMT gene family of jute and those of ten plants, we arranged 66 protein sequences from representatives of A. thaliana, Boehmeria nivea, Hibiscus cannabinus, Glycine max, Gossypium raimondii, Linum usitatissimum, Theobroma cacao, Synechocystis sp, Oryza sativa, and Streptomyces hygroscopicus as an outgroup, to construct an un-rooted phylogenetic tree using Mega 7.0 (Fig. 3, Additional files 7 and 8). Based on this phylogenetic analysis, CCoAOMTs were divided into two groups, referred to as group 1 (1a, 1b, 1c, 1d) and group 2, consistent with the distribution shown in Fig. 4. Group 1a was the largest, consisting of 28 CCoAOMTs, whereas groups 1b, 1c, 1d and 2 contained 23, 4, 9 and 2 CCoAOMTs, respectively. This difference in the number of CCoAOMT genes was mainly due to the occurrence of gene gain or loss in the two groups, independent of the level of similarity between the organisms.

Fig. 3

The phylogenetic analysis of CCoAOMTs in twelve plant species. Unrooted phylogenetic tree of CCoAOMT proteins formed using the Neighbor-joining approach with MEGA7 software. Note: Cc.CCoAOMT: C.capsularis, Co.CCoAOMT: C.olitorius, AtCCoAOMT: Arabidopsis Thaliana, OsCCoAOMT: Oryza sativa, BnCCoAOMT: Boehmeria Nivea, GmCCoAOMT: Glycine max, HcCCoAOMT: Hibiscus cannabinus, GrCCoAOMT: Gossypium Raimondii, LuCCoAOMT: Linum usitatissimum, TcCCoAOMT: Theobroma Cacao, SsCCoAOMT: Synechocystis sp, ShCCoAOMT: Streptomyces hygroscopicus, respectively

Fig. 4

The distribution of CCoAOMT gene family members in ten plant species. α, β, γ, σ and ρ represent whole genome duplication events

Among these two groups, subclade 1c had only Os.CCoAOMT proteins; in contrast, genes in subclades 1a, 1b, 1c, 1d and clade 2 included diverse plant species. The CCoAOMT genes could be classified into two groups in the phylogenetic tree, with Cc.CCoAOMT1/2/3/4/5/6/8 and Co.CCoAOMT1/2/3a/3b/4/5a/5b/6/7 s/7b in one group and the remaining genes in the other group, suggesting that CCoAOMTs originated from two ancestral genes. The earliest diverging bacteria (Streptomyces hygroscopicus) carried only one CCoAOMT, whereas the remaining included 65 CCoAOMTs (Figs. 3 and 4). Among the two clades, subclade 1a was found to include the largest number of CCoAOMTs (Figs. 3 and 4).

Co.CCoAOMT3a was more closely related to other plant CCoAOMTs than to its orthologous gene in C. capsularis. Finally, Co.CCoAOMT9 and Cc.CCoAOMT9 created their own distinct clade beyond the outgroup gene, indicating that they are evolutionarily ancient.

The distribution of CCoAOMTs revealed that the increase in the number of CCoAOMTs, many of which were species specific, could be attributed to an enormous duplication rate. Altogether, two clades, clade 1 (1a, 1b, 1c, 1d) and 2 included CCoAOMTs from both jute and other species, showing that these CCoAOMTs were greatly conserved across species during plant evolution. Intriguingly, the distribution of CCoAOMTs in the investigated plant species demonstrated that ancient whole-genome duplications (WGDs) did not lead to the expansion of the CCoAOMT family (Fig. 4); however, the number of CCoAOMTs slowly increased in every group. Phylogenetic analysis also revealed that Cc.CCoAOMTs and Co.CCoAOMTs have very close affinity relationship. However, these genes are more closely related to those of T. cacao and G. raimondii because they always clustered together with one of them in subclades (1a, 1b and 1d), indicating that TcCCoAOMTs and CrCCoAOMTs can be utilized as references for jute genes.

We estimated the divergence time among the two clades of CCoAOMTs based on the pairwise synonymous substitution rates (Ks) in jute. The median values of pairwise Ks varied from 1.882 to 4.098, corresponding to a divergence time varying from 154.2 to 335.9 Mya, revealing that the CCoAOMTs were ancient and divergent (Additional file 9). Furthermore, the divergence time among the paralogous Co.CCoAOMTs (5a, 7a, 7b) varied from 54.95 to 389.82 Mya with an average of 140.48 Mya (Additional file 10). These results show that the Co.CCoAOMTs are ancient gene family with recent gene duplication events in jute.

Structural analysis of the CCoAOMT genes of jute and other species

Based on gene structural analysis, the CCoAOMT genes of jute and other species could be divided into two clades (Fig. 5). To examine the structural features and evolution of CCoAOMTs in different species, we assayed the structural characteristics and patterns of CCoAOMTs. CCoAOMT genes showed a high variability in exon number and size. The exon number of CCoAOMTs ranged from 1 to 14 with an average number about 5, and of the 66 analyzed CCoAOMTs, 54 CCoAOMTs possess a number of exons varying from one to six (Additional files 11 and 12), and their introns are arranged following the GT-AG rule for splicing sites (Fig. 5). Therefore, we theorized that the number of exons of the CCoAOMT gene of the last common ancestor (LCA) of angiosperms was between one and six. CCoAOMT genes within the same subgroup exhibiting similar exon/intron patterns, particularly in paralogous gene pairs, and most of them possessed a conserved exon/intron pattern in terms of gene length or number of introns.

Fig. 5

Phylogeny and schematic diagram for exon–intron organization of CCoAOMT genes from12 plant species

Cc.CCoAOMT3, Cc.CCoAOMT5, and Cc.CCoAOMT6 have larger frontal introns than their corresponding orthologous genes in C. olitorius (Fig. 5). Co.CCoAOMT3a/b and Co.CCoAOMT7a/b have three exons, and Cc.CCoAOMT4 has only five exons, a small number compared to that of the other CCoAOMT genes of jute. In subclade 1a, the number of exons of CCoAOMTs ranges from three to twelve, while that of Co.CCoAOMTs/Cc.CCoAOMTs is conserved. Cc.CCoAOMT1 and Cc.CCCoAOMT2 showed great similarity in terms of exon/intron number and size with their orthologous genes in C. olitorius, consistent with their amino acid sequence similarity; however, they varied in terms of exon/intron patterns (Fig. 5 and Table 4). Cc.CCoAOMT1/2 have one more exon than monocot CCoAOMTs, which was likely originated by exonization. In subclade 1b, the number of exons of CCoAOMTs ranged from one to five. In subclade 1c, the number of exons of CCoAOMTs ranged from three to five, but the gene structure was conserved. CCoAOMTs of subclade 1d varied from 1 to 9 exons. In this subclade, Cc.CCoAOMT8 and Co.CCoAOMT8 were found to have dissimilar intron sizes; however, they shared a similar gene structure (Fig. 5). These results indicated that CCoAOMTs underwent gene restructuring under dissimilar evolutionary dynamics in jute. In clade 2, CCoAOMTs had only two exons.

Chromosomal distribution, gene duplication and collinearity and synteny analysis

We estimated gene duplication events in the C. capsularis and C. olitorius genomes by collinearity analysis. A total of 7 collinearity pairs of Co.CCoAOMT genes and 7 of Cc.CCoAOMT genes were identified by Blastp for all protein sequences and evaluated with MCScanX (Fig. 6). The results showed that 14 genes mapped to jute genome chromosomes, 7 to C. capsularis genome, 6 to C. olitorius genome and one to contig. The collinearity relationships revealed that over half of the Co.CCoAOMT and Cc.CCoAOMT collinearity genes were concentrated in Cc2, Cc4, Co2 and Co4 chromosomes, respectively (Fig. 6). Some of the chromosomes (Cc2, Cc4, Co2 and Co4) contained three or two CCoAOMT genes, while some others (Cc1, Cc7 and Co7) contained only one gene. There were no CCoAOMT genes identified on chromosomes Cc3, Cc5, Cc6 in C. capsularis and Co1, Co3, Co5, Co6 in C. olitorius (Fig. 6). The absence and uneven distribution of CCoAOMT genes on some C. capsularis and C. olitorius genome chromosomes indicate that gene loss may have occurred during evolution but an incomplete genome assembly could also be a factor.

Fig. 6

Collinear analysis of CCoAOMT gene pairs in jute genome. CCoAOMT gene pairs present on duplicated chromosomal segments are connected by different colored lines according to different classes. Col to Co7 (light blue blocks) represent C. olitorius chromosomes, while Ccl-Cc7 (pink color blocks) represent C. capsularis chromosomes

Additionally, the analysis of genetic evolution of the CCoAOMT members was carried out by the synteny analysis among C. capsularis and C. olitorius with the CCoAOMT genes of A. thaliana, and Oryza sativa (Additional file 13). Most of Cc.CCoAOMTs and Co.CCoAOMTs are paired with A. thaliana, producing a total of 5 collinear pairs. However, it was found that Cc.CCoAOMT3 was paired with two genes from A. thaliana, while Cc.CCoAOMT9 was paired with an orthologous gene from A. thaliana and Oryza sativa (Additional file 13). Therefore, it was observed that the strongest collinearity relationships are between dicot plants.

Gene expression and function of CCoAOMTs in jute

Detailed analysis of gene expression can provide an initial reference for assessing the potential function of genes in plants. In this study, all Cc.CCoAOMT and Co.CCoAOMT expression analysis was conducted using stem samples. The analysis of the result showed that Cc.CCoAOMT1/2 and Co.CCoAOMT1/2 possessed high transcript levels at all developmental stages (Fig. 7), suggesting that these two genes are the principal members of the CCoAOMT gene family. Conversely, Cc.CCoAOMT6 and Co.CCoAOMT3a/3b/4/6 showed low transcript levels at all developmental stages and in all examined stem tissues (Fig. 7A, B). Also, the expression of Cc.CCoAOMT4 was almost undetectable at all developmental stages and in all tissues investigated. Finally, Cc.CCoAOMT3/5/8/9 and Co.CCoAOMT5a/5b/7a/7b/8/9 exhibited differential gene transcript profiles in the two jute species at different developmental stages.

Fig. 7

Transcript levels of CCoAOMT genes under different growth stages and varieties of jute. A The transcript levels of CCoAOMT members in the stem of C. olitorius at five dissimilar developmental stages (15, 30, 45, 60 and 90 DAS) under F5 variety. B The expression patterns of CCoAOMT genes in C. capsularis under five different developmental stages (15, 30, 45, 60 and 90 DAS) in J179 variety. DAS refers days after sowing. C Comparison of expression patterns of CCoAOMT2 in two jute species (C. olitorius and C. capsularis) under different developmental stages and varieties. Note: SDs and mean values were acquired from three biological replicates. The letter S refers to stage. Turkey test was used for statistical analysis, and different letters expressed significant differences between developmental stages (p < 0.05)

CCoAOMT expression levels at different developmental stages

CCoAOMT genes could be categorized into three types according to their expression patterns: period-specific expression, constitutive expression, and low expression. Among the 12 assayed CCoAOMT genes from C. olitorius, four genes (Co.CCoAOMT3a, Co.CCoAOMT3b, Co.CCoAOMT4, and Co.CCoAOMT6) showed undetectable or very low transcript levels across the different growth stages. In contrast to other genes, Co.CCoAOMT1 showed differential gene expression levels in these five developmental stages and displayed the highest transcript levels at 90 DAS (Fig. 7A). Co.CCoAOMT2 was the most highly expressed among the members of C. olitorius CCoAOMT family at different developmental stages (F5 variety), and exhibited a constitutive transcript level (Fig. 7A). Co.CCoAOMT2 displayed higher expression levels at 60 and 90 DAS than at 15, 30, or 45 DAS (Fig. 7C), and higher expression in F5 than in the other two varieties (CL and KY) at 60 DAS (Additional file 14). Additionally, this gene exhibited the highest transcript levels in F5 and CL at 90 DAS (Additional file 14). Overall, during the early developmental stages from 15 to 45 DAS, the CCoAOMT2 expression levels were relatively low and had no significant difference among each stage except LB and KY (Fig. 7C). Furthermore, the CCoAOMT2 expression levels of most cultivars reached highest expression level during maturing stages at 60 DAS and 90 DAS except LB, which reached the highest at 30 DAS (Fig. 7C). At the seedling stage, Co.CCoAOMT7a showed low transcript levels; thus, Co.CCoAOMT7a probably plays a role in the premature development of jute. In contrast to that of Co.CCoAOMT7a, the expression of Co.CCoAOMT7b was abundant at all five developmental stages; however, this gene displayed greater transcript levels in mature stems than in premature ones (Fig. 7A). Both Co.CCoAOMT8 and Co.CCoAOMT9 were found to display greater transcript levels at 60 and 90 DAS than at 45 DAS. Finally, Co.CCoAOMT5a, Co.CCoAOMT5b, Co.CCoAOMT7b, Co.CCoAOMT8, and Co.CCoAOMT9 exhibited differential expression patterns across the five developmental stages (Fig. 7A).

The eight Cc.CCoAOMTs of C. capsularis generally followed a similar expression pattern to that of their orthologous genes in C. olitorius. Only a slight difference was observed in the expression of Cc.CCoAOMT8/9, which was lower at seedling stages (15 and 30 DAS) and greater at the mature stage (90 DAS) than that of their orthologs (Fig. 7B). In contrast to Co.CCoAOMT3a, Cc.CCoAOMT3 displayed high expression levels during the mature stage (Fig. 7B). Similarly, Cc.CCoAOMT1/2 exhibited higher expression levels than those of their orthologous genes in C. olitorius (Fig. 7B). The abundance of Co.CCoAOMTs and Cc.CCoAOMTs were compared for their relative transcript patterns at 15, 30, 45, 60, and 90 DAS by normalizing with the lowest expressed gene and presented as fold change in expression (Additional file 15).

To identify the expression levels of Co.CCoAOMTs and Cc.CCoAOMTs at different parts (top, middle and bottom) of the C. capsularis and C. olitorius stem tissues, a comparison of their transcript patterns was carried out. For Co.CCoAOMTs, the results exhibited that these genes were deferentially expressed at the top, middle and bottom tissues, with an average expression of these genes being higher in the middle tissues and mild in the top or the bottom, suggesting a gradient of CCoAOMT activity in the C. olitorius. Similarly, the expression levels of Cc.CCoAOMTs showed differential expression, with an average transcript being greater in the bottom tissues and moderate in the top or the middle (Additional file 16).

To comprehend the overall function of CCoAOMTs during different developmental stages of the jute stem, the integrated transcript levels of all CCoAOMTs were calculated. This analysis revealed a 0.5-fold decrease in CCoAOMT gene expression between 15 and 30 DAS, a 0.12-fold increase between 30 and 45 DAS, a 0.02-fold decrease between 45 and 60 DAS, and a 3.4-fold increase between 60 and 90 DAS (Additional file 17). Overall, the transcription of CCoAOMTs was enhanced throughout jute stem growth, consistent with their major role in lignin biosynthesis. In particular, CCoAOMT2 was the most abundantly expressed CCoAOMT across all developmental stages and species. Therefore, this gene was selected for further analysis, and its expression patterns were compared for six different varieties and phases (Fig. 7C). The results revealed that the expression of this gene was enhanced gradually in parallel with the growth of the plant and reached its peak at the mature stages (60 and 90 DAS) (Fig. 7C).

Analysis of lignin content in different varieties at various developmental stages

The measurement of lignin content using the acetyl bromide method revealed an overall increase in the lignin content of jute during fiber development (Fig. 8). The lignin content of J179, QY, LB, F5, and CL at the mature stage (90 DAS) increased by 20.7%, 28%, 36.3%, 27.5%, and 38.6%, respectively, compared with that of the premature stage (15 DAS) (Fig. 8A, B, C, E, F). This drastic increase in lignin content was observed during the transition from the vegetative to the reproductive growth phase, and was mainly ascribed to the lignification triggered by CCoAOMTs. The lignin content of J179, LB, and CL decreased at 30 DAS, but then increased steadily in parallel with the growth of the plant (Fig. 8B, C, F). The lignin content of QY increased steadily along plant development, peaking at stage 45 DAS (Fig. 8A), whereas that of F5 fluctuated during plant growth (Fig. 8C). The lignin content of KY did not show any substantial increment during seedling and vegetative phases (15, 30 and 45 DAS) but suddenly dropped at stage 60 and finally increased at maturing stage (90 DAS) (Fig. 8E). Furthermore, the correlation coefficient between lignin content and CCoAOMT2 gene expression level of each cultivar was inconsistent, both positive and negative correlations were found. Among which, CL variety showed the highest correlation coefficient (0.879), indicating a positive correlation between the lignin content and the CCoAOMT2 gene expression level in this cultivar (Table 5, Additional file 18). This result indicates that the down-regulation of CCoAOMTs may reduce the lignin content of jute (CL variety), thereby enhancing fiber quality.

Fig. 8

Comparison of lignin content under dissimilar growth stages and varieties in jute. A QY variety under different stages. B J179 variety under different stages. C F5 variety under different stages. D LB variety under different stages. E KY variety under different stages. F CL variety under different stages. Note: SDs and mean values were acquired from three biological and five technical replicates. One-way ANOVA with Fisher’s test was used for statistical analysis, and different letters expressed significant differences between developmental stages (p < 0.01). The letter S refers to stage

Table 5 Correlation coefficient between lignin content and CCoAOMT2 expression level

Expression of CCoAOMTs under PEG or NaCl treatment

The expression of Co.CCoAOMTs and Cc.CCoAOMTs was examined under NaCl and PEG treatments at various time points (Fig. 9). At 24 h after PEG treatment, the expression of CCoAOMTs was highly up-regulated compared to that of control plants (Fig. 9A). In particular, Cc.CCoAOMT2 was induced by PEG stress, upon which it exhibited higher up-regulation greater than that of the control. The expression of Co.CCoAOMTs decreased during the second stage (48 h) of drought stress, whereas the transcript levels of Cc.CCoAOMTs were similar to those of control plants (Fig. 9B). The abundance of Co.CCoAOMT and Cc.CCoAOMT transcripts was substantially reduced at later stages (72 h) (Fig. 9C). This comparative analysis of CCoAOMT expression under PEG treatment showed that some C. olitorius and C. capsularis orthologous genes are either drought repressible or drought inducible in a similar way, indicating that these two species might share common transcriptional responses to drought stress. However, a small number of C. olitorius orthologs showed dissimilar responses to drought stress, suggesting the existence of species-specific drought responses.

Fig. 9

RT-qPCR expression patterns of CCoAOMT genes under drought and salt stress. The expression patterns were normalized to 24, 48 and 72 h (drought and salt stress treatments), respectively. A, B and C are expression patterns of CCoAOMT genes under PEG treatment; and D, E, F are expression levels of CCoAOMT genes under NaCl treatment. The asterisks indicate at most significantly difference (**p < 0.001, *p < 0.01)

Under salt stress, the expression of eight genes (Co.CCoAOMT1/2/5a/5b/7b and Cc.CCoAOMT1/2/3) was greatly induced (Fig. 9D). Nonetheless, more than half of all genes were repressed at 24 h (Fig. 9D). After 48 h from NaCl treatment, the expression levels of five Co.CCoAOMT genes (Co.CCoAOMT1/2/5a/5b/7b) were differentially expressed, whereas their orthologs (Cc.CCoAOMT1/2/3) were down-regulated (Fig. 9E). However, these five Co.CCoAOMT genes were down-regulated at 72 h (Fig. 9F). Co.CCoAOMT1/2/5a/5b/7b were differentially expressed, whereas their orthologues genes (Cc.CCoAOMT1/2/3) were down-regulated (Fig. 9E) in response to at least one treatment, and most of them could be induced by more than one stress treatment; this revealed that these genes are involved in the crosstalk between distinct signal transduction pathways in response to abiotic stresses. For example, Co.CCoAOMT2 was considerably down-regulated by PEG (48 h), while being up-regulated by NaCl treatment (48 h). Relatively greater Cc.CCoAOMT transcript levels were observed under drought and salt stress-tolerant cultivar J179, compared to those of the sensitive cultivar F5, confirming the essential role of these genes in the response to drought and salt stresses in jute (Fig. 9). Therefore, our study revealed that Co.CCoAOMTs from F5 and Cc.CCoAOMT from J179 are greatly induced by drought and salt stress and may play a crucial role in resistance to abiotic stress.

Discussion

Evolution of the CCoAOMT gene family in jute

Recent studies have investigated and examined the characteristics, evolution, expression, and function of some CCoAOMT genes in plants such as A. thaliana [31], citrus [32], sorghum [30], and maize [33, 34]. However, very little is known about CCoAOMT gene family in jute. In this study, 12 CCoAOMT genes from C. olitorius and eight CCoAOMTs from C. capsularis were identified, a higher number than in A. thaliana (seven CCoAOMTs), O. sativa (six CCoAOMTs), G. raimondii (six CCoAOMTs), and T. cacao (seven CCoAOMTs), suggesting the evolutionary expansion of CCoAOMT genes in jute.

Gene duplication events, mainly consisting of segmental and tandem duplications, have been suggested to be the principal driving force for the expansion and evolution of gene families [35]. The model plant A. thaliana is known to have undergone several whole genome duplication (WGD) events, resulting in the large-scale expansion of gene families in its genome [36]. The results of this study indicate that the mechanisms underlying CCoAOMT gene expansion may differ across species. WGDs are considered to be the typical driving force for the evolution of angiosperms, the latest evolved and most successful lineage of land plants [37,38,39]. Previous studies have discovered that the genome of pineapple has undergone a less ancient WGD (ñ) event than the other sequenced grass genomes. These currently accessible WGD data, along with the genomes of 10 plant species depicting major WGD events in angiosperms, facilitated the study of CCoAOMT gene evolution in angiosperms. New gene functions can be gained through four processes of gene evolution: chromosome insertion, duplication, transposition, and tandem duplication [40,41,42,43]. Since jute has only undergone a γ WGD, its gene duplication might be attributed to tandem duplication. It is worth noting that C. olitorius and C. capsularis have a different number of CCoAOMT genes (eight and four, respectively) belonging to group 1a, and this could depend on a recent gene duplication occurred after their speciation.

Based on the phylogenetic analysis of CCoAOMT genes from 12 plant species, the CCoAOMTs considered in this study could be divided into two clades. Clade 1 and 2 including CCoAOMT genes from both C. capsularis and C. olitorius. In particular, the Co.CCoAOMT7a/b genes (absent in C. capsularis) appeared to be the result of a recent duplication event occurred after the speciation of C. capsularis and C. olitorius. These two genes, detected only in C. olitorius, might have been lost by C. capsularis during evolution. Indeed, gene loss could occur due to the accumulation of harmful mutations, which is especially frequent soon after polyploidy events [44]. Phylogenetic analysis also indicated that the CCoAOMTs of jute have a closer affinity to T. cacao and G. raimondii CCoAOMTs than to those of the other investigated plant species, providing clear evidence that C. capsularis and C. olitorius might have a more recent common ancestor with T. cacao and G. raimondii than with other plant species. Consistent with the observed phylogenetic relationships, clade 2 CCoAOMTs belonged to the oldest branch of the CCoAOMT gene family. Therefore, CCoAOMTs in this group were hypothesized to have undergone more intron gain/loss events during their lengthy evolutionary process in line with the introns-early theory [45, 46]. Consequently, we presumed that the evolution of the gene structures of clade 2 CCoAOMTs was mainly due to exon splitting.

Exon–intron structural differences between orthologous genes have been shown to be generated by three main mechanisms: exon/intron gain/loss, exonization/pseudoexonization, and insertion/deletion [47]. Comparative analyses of the gene structures of CCoAOMTs made it possible to evaluate the structural evolution of CCoAOMT genes in jute. The CCoAOMT genes of jute displayed great variability in the number of exons. Compared with the variable structures of the CCoAOMT genes from jute, the sequences of the corresponding proteins were more conserved. Thus, gene structural evolution after the divergence of these plant species did not cause significant differences in the coding region. Therefore, CCoAOMT structural variants mainly evolved through intron gain/loss and insertion/deletion, but not through exonization/pseudoexonization. According to their phylogenetic distribution, the evolution of CCoAOMT genes in jute travelled along significantly distinct paths over evolutionary history. In addition, the fact that the majority (54.55%) of CCoAOMTs have five exons led us to infer a basic gene model containing five exons and four introns for the ancestral CCoAOMT genes, from which all observed gene structures could develop by insertion and/or loss of introns. In comparison with the CCoAOMT gene structure and exon counts of Physcomitrella patens, Selaginella and Plagipchasma, the CCoAOMT gene structure and exon number from the two jute species and the other eight plant species analyzed in this study are more diverse in exon number, size and pattern [30, 48]. The exon number of CCoAOMT genes from Physcomitrella patens, Selaginella and Plagipchasma range from three to seven while the exon number of CCoAOMT genes from C. capsularis, C. olitorius, Oryza sativa, A. thaliana, Boehmeria nivea, Glycine max, Hibiscus cannabinus, Gossypium raimondii, Linum usitatissimum and Theobroma cacao varied from one to fourteen respectively.

The study of variations in CCoAOMT gene structures is key to unravel the genome dosage of the two jute species, and further investigation may provide the foundation for understanding the molecular basis of jute genetics.

The orthologous CCoAOMT gene pairs of C. capsularis and C. olitorius exhibited high identity (82% ~ 100%). The structural features of some Co.CCoAOMTs corresponded to those of the respective Cc.CCoAOMTs, demonstrating that the functions of these genes remained highly conserved during evolution. Moreover, Cc.CCoAOMT6/Co.CCoAOMT6 had the lowest protein sequence similarity (82%) among the orthologous pairs. The pI values of CCoAOMTs of jute varied from 4.61 (Cc.CCoAOMT6) to 8.93 (Cc.CCoAOMT8), with majority of the members (11) showing pI values higher than 5 (Table 3). The pI values of the remaining nine genes were lower than 5, indicating the independence between pI divergence and gene evolution.

The Ka/Ks assay showed that most of the homologous CCoAOMT genes from jute have experienced purifying selection, since most Co.CCoAOMTs exhibited very high similarity in gene sequences and highly conserved functions to their orthologs from C. capsularis. This assay also revealed that CCoAOMT4 had a lower selective constraint than the other CCoAOMTs. Furthermore, CCoAOMT4 was distinct from the other CCoAOMTs in terms of protein size and showed the lowest gene expression levels among all CCoAOMTs. Thus, the evolution of CCoAOMT4 could have depended on its functional specialization in jute, given the long-term divergence of CCoAOMT4 from the LCA with respect to the other CCoAOMTs. In summary, the study of the evolutionary relationships and gene structures of CCoAOMTs is a vital step towards the comprehension of their potential functions in jute.

Expression of CCoAOMT genes at different growth stages

Gene transcript profiles are highly correlated with gene function in plants [49]. Previous studies on CCoAOMT genes indicated that they may play a role in lignin biosynthesis, disease, and abiotic stress resistance in some plant species [40,41,42,43,44,45,46,47,48,49,50,51,52,53]. In the present study, the expression levels of CCoAOMTs were examined using stem tissues at five distinct stages and in different varieties of jute. Among group 1b genes, CCoAOMT1 showed greater expression at the mature stage (90 DAS) than at the premature stage (15 DAS) in C. olitorius than in C. capsularis. CCoAOMT2 was the most highly expressed gene among all CCoAOMTs, and its transcript levels peaked at the last stage (90 DAS) (F5, CL varieties), suggesting that this gene is the predominant gene involved in lignin biosynthesis in jute. The CCoAOMT2 showed different expression levels in the six cultivars at each developmental stage. Cc.CCoAOMT2 of J179 had relatively lower transcript level at 15 DAS and 30 DAS, while the one in KY and LB varieties had higher expression level at the same stages. At 45 DAS, Cc.CCoAOMT2 of LB variety expressed relatively low transcription level and showed significant difference with other cultivars. Cc.CCoAOMT2 expression level of J179 reached highest at 60 DAS, in comparison with other cultivars at the same stage. Moreover, at 90 DAS, QY, LB and KY showed lower transcript levels, in contrast to CL, F5 and J179 had higher transcript level at the same stage. Furthermore, the Cc.CCoAOMT2 expression levels of QY were relatively low at all stages in comparison with other cultivars. This gene showed higher expression levels in C. olitorius than in C. capsularis.

Among group 1a genes, Co.CCoAOMT3a and Co.CCoAOMT3b showed very low transcript levels at all growth stages, while their orthologous Cc.CCoAOMT3 exhibited greater transcript levels at all five developmental stages, which peaked at 90 DAS. This led us to hypothesize that CCoAOMT3 may be functionally divergent in the two species.

Co.CCoAOMT4 and Cc.CCoAOMT4 were undetectable throughout the developmental stages and in all varieties. CCoAOMT4 was presumed to have originated after the divergence of C. olitorius and C. capsularis, in accordince with the results of the phylogenetic analysis described before, supporting the hypothesis that the CCoAOMT4 lineage is functionally redundant in malvids. Additionally, the lack of an orthologous gene to Co.CCoAOMT7 in C. capsularis was considered to be due to a gene deletion event occurred after the divergence of C. olitorius and C. capsularis. Co.CCoAOMT5b was highly expressed at the maturing (45 DAS) and mature stages (60 DAS), and its ortholog Cc.CCoAOMT5 exhibited similar expression levels at the mature stage (60 DAS). CCoAOMT6 showed very low transcript at all developmental stages and in all investigated tissues, possibly as a result of gene functional redundancy due to the recent ρ WGD. Co.CCoAOMT7a/b are recently duplicated genes that showed different expression patterns in the examined tissues and developmental stages. The expression of both Co.CCoAOMT7a and Co.CCoAOMT7b peaked at 90 DAS. Furthermore, the expression of Co.CCoAOMT7s dramatically increased from the maturing to the mature phase, suggesting that these genes play a significant role in lignin biosynthesis.

Among group 1d genes, CCoAOMT8 showed increased expression at the maturation stage (60 DAS) and had higher transcript levels in C. olitorius than in C. capsularis at the same stage. This gene also exhibited a similar expression pattern in different tissues of the stem in both jute species. Among group 2, the expression of CCoAOMT9 peaked at the maturation phase and it was lower in C. olitorius than in C. capsularis, whereas it was undetectable in both species at the premature stage. Overall, gene expression analysis revealed that orthologous genes had similar expression patterns in both species.

In the present study, CCoAOMT1 and CCoAOMT2 were found to be constitutively expressed in the whole stem of both species and across all growth stages, indicating their essential role in lignin biosynthesis in jute. Six genes (Co.CCoAOMT3a, Co.CCoAOMT3b, Co.CCoAOMT4, Co.CCoAOMT6, Cc.CCoAOMT4, and Cc.CCoAOMT6) exhibited very low or undetectable transcript levels in all the investigated tissues of the two species and at all growth stages, indicating that these genes may play non-essential roles in lignin biosynthesis in jute.

Relationship between lignin content and CCoAOMT gene expression

Despite being one of the most important fiber sources, jute has relatively high lignin content [1]. Nevertheless, little information is available on lignin biosynthesis in jute. In this study, the expression levels of the lignin-related CCoAOMT genes and lignin accumulation patterns in jute were thoroughly examined for the first time. The results showed an overall increase in lignin content during the development (from 30 DAS) of three jute varieties (CL, J179, and LB), whereas the lignin content of the other two varieties (F5, and KY) fluctuated across the five developmental phases. The lignin content of QY enhanced steadily during plant development, peaking at stage 45. A significant increase in lignin content was observed at 90 DAS compared to that at 45 DAS (J179, LB, F5 and CL varieties). Additionally, the analysis of CCoAOMT activity revealed greater expression levels at 90 DAS, perfectly correlating with the rapid increase in lignin content occurring at the same stage. The integrated transcript level of all CCoAOMT genes was greatest at 90 DAS, further supporting the correlation between CCoAOMT gene expression and lignin accumulation. Furthermore, the correlation of lignin content and CCoAOMT2 expression level was inconsistent among the six jute varieties investigated. A positive correlation between lignin content and CCoAOMT expression was observed under CL varieties (Table 5). The production of transgenic jute plants with diverse lignin structures and the least damage from other cell wall constituents could be a useful strategy for examining the impact of lignin properties on fiber quality in the future.

Analysis of the expression of CCoAOMTs under abiotic stresses

Profiling gene expression under many abiotic stresses is vital for a mechanistic understanding of stress resistance in plants. Lignin biosynthesis via the phenylpropanoid pathway is associated to a complex regulatory network, in which secondary metabolites synthesized by various interacting enzymes perform essential functions in stress responses [54]. CCoAOMT was first reported to be instantly up-regulated by fungal elicitors in carrot and parsley cell cultures [55]. Subsequent studies indicated that the corresponding enzyme may play a crucial role in tolerance to stresses such as drought, cold, wounding, salt, and phytohormones [56,57,58,59]. Even though there is little information on the mechanisms by which drought affects lignin biosynthesis, several studies have demonstrated that CCoAOMTs can be considerably or slightly up-regulated by drought at the protein and mRNA levels in the roots of A. thaliana [60] and in the leaves of wild watermelon [61], soybean [62], peanut [63], and H. cannabinus [57].

Understanding how stressors impact the transcription of Co.CCoAOMTs and Cc.CCoAOMTs may prompt the development of novel methods for genetic improvement of jute for its cultivation in marginal areas. Our study showed that Co.CCoAOMTs and Cc.CCoAOMTs were substantially up-regulated at 24 h after exposure to drought stress. This result was consistent with those of Liu et al. [28], who discovered the up-regulation of CCoAOMTs in both stems and leaves of switchgrass during later time points (24 ~ 48 h) of drought stress. The expression of Co.CCoAOMT genes was down-regulated in dehydrated stem tissues, whereas the transcript levels of Cc.CCoAOMTs were unchanged with respect to those in untreated controls at 48 h. In contrast, the transcript levels of both Co.CCoAOMTs and Cc.CCoAOMTs decreased at 72 h after drought stress.

The lignin content and gene transcript levels might depend on the plant tissue. For instance, the slower growth at the bottom of maize roots in comparison to the top was associated with greater up-regulation of cinnamoyl-CoA reductases (CAD) 1 and 2, and increased deposition of lignin [24]. Interestingly, we discovered that the expression of CCoAOMTs in the stems of jute after PEG treatment was substantially greater at 24 h (approximately 17-fold that of the control) than at 48 or 72 h. This significant increase in jute CCoAOMT expression indicates its potential functional value in drought resistance. Hence, it is essential to thoroughly investigate the function of jute CCoAOMTs in lignin deposition under drought stress, particularly in stem tissues.

We observed that salt stress (24 h) induced CCoAOMT expression in both jute species but at different levels, suggesting that increased monolignol biosynthesis is one of the plant’s strategies to avoid salt damage. The expression of CCoAOMTs showed a substantial increase in C. olitorius, followed by a noticeable decline along the saline stress treatment (48 h) in C. capsularis. Additionally, the transcript levels of CCoAOMTs were initially, slightly reduced in C. olitorius and subsequently returned to the control levels at 72 h after salt stress. In this study, five of 12 Co.CCoAOMTs were found to be up-regulated or down-regulated under both drought and salt stresses, while the remaining genes showed similar transcript levels as those of control plants. Similarly, three of the eight Cc.CCoAOMTs were up-regulated and down-regulated under the same stresses. Furthermore, some genes (CCoAOMT1/2) up-regulated in drought-stressed stems were also induced in salt-stressed stems (24 h). In contrast, the majority (78% of Cc.CCoAOMTs and 83% of Co.CCoAOMTs) of the CCoAOMTs of the two jute species exhibited different transcript levels under various stress conditions, suggesting that these genes may be involved in the communication between distinct signal transduction pathways. Taken together, these results suggest that jute CCoAOMTs may participate in the stress response to drought and salt treatment. These results also provide a molecular basis for functional characterization of CCoAOMT genes by overexpression or silencing. Furthermore, additional proteomic assays of CCoAOMTs under abiotic stress may also help to reveal their physiological functions.

Conclusion

We identified 66 CCoAOMT genes from 12 plant genomes including 20 CCoAOMTs in Corchorus olitorius and C. capsularis. The study indicated that CCoAOMTs underwent gene restructuring under dissimilar evolutionary dynamics in jute and angiosperms. Phylogenetic analysis revealed that gene structures and amino acid sequences of CCoAOMTs are conserved between C. capsularis and C. olitorius, and CCoAOMT genes of Corchorus have the closest evolutionary relationship with Theobroma cacao and Gossypium raimondii. Furthermore, the expression level of CCoAOMT2 indicated that this gene plays a leading role in lignin biosynthesis of jute. Meanwhile, we found out that CCoAOMTs had positive correlation with lignin content (CL variety), and CCoAOMTs were also involved in the regulation of salt and drought resistance on jute. These findings revealed evolutionary, expression and function aspects of the CCoAOMT genes in jute and identify genes that may be useful for genetic manipulation on improving fiber quality of jute.

Methods

Plant materials and culture conditions

Six varieties were used in this study, namely three for C. capsularis (J179, LB, and QY) and three for C. olitorius (F5, KY, and CL). These were provided by the Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops (Fuzhou, China), and Institute of Bast Fiber Crops, Chinese Academy of Agriculture Sciences (Changsha, China). The seeds were sterilized with 5% NaOCl for 10 min and rinsed three times with sterilized water before planting in pots containing nutrient soil, and the diameter and height of the pots are both 20 cm. The plants were grown for up to 90 days in a greenhouse with temperatures of 30/26 °C (day/night) in a 16/8 h light/dark cycle. Samples were taken from the stems of jute plants at five stages (15, 30, 45, 60, and 90 "DAS"). Additionally, the whole jute stem of 60 DAS was separated into three parts (top, middle, bottom) according to the plant height, and the middle stem of each part was taken as the samples to identify the expression levels of CCoAOMT genes. For abiotic stress treatment, two jute cultivars, the drought-sensitive C. olitorius F5 and the drought-tolerant C. capsularis J179 were used. The seedlings were cultivated in 1/4 Hoagland solution in the same greenhouse. The seedlings were subjected to abiotic stresses at 30 DAS. The plants were exposed to 10% (w/v) PEG6000 and 200 mM sodium chloride, and collected at three time points (24, 48, and 72 h) [64]. Subsequently, the samples were quickly placed in liquid nitrogen and stored at -80 °C for RNA extraction.

RNA isolation and first strand cDNA synthesis

Total RNA was isolated from nearly 100 mg of fresh stem tissue using the OMEGA isolation kit (R6827-01, USA) and the Huayueyang Biotechnology isolation kit (Beijing, China), according to the manufacturers’ instructions. Genomic DNA contamination was removed by RNase-free DNase I (TaKaRa, Japan) treatment. The quality of RNA was assessed using a NanoDrop 2000 spectrophotometer (NanoDrop, Thermo Fisher Scientific). The integrity of RNA was evaluated by 2.0% agarose gel electrophoresis. Lastly, RNA specimens with an A260/A230 ratio of more than 1.8 and an A260/A280 ratio between 1.8 and 2.2 were used for further analysis. Subsequently, using the PrimeScript RT reagent kit (TaKaRa, Japan), first-strand cDNA was synthetized from 1 μg of complete RNA in a 20μL reaction mixture, according to the manufacturer’s guidelines. The final cDNA was diluted in water for "qRT-PCR".

Identification of the CCoAOMT gene family in jute

The genome sequence of jute was downloaded from the NCBI database [1]. To identify the CCoAOMT gene family in jute, the sequences of A. thaliana and rice CCoAOMT proteins [61, 65] were used as queries to search against the C. capsularis and C. olitorius genomes using tBLASTn and BLASTp on the NCBI database [66] with default parameters. All hits were confirmed using the InterProScan program [67] to verify the existence of the protein domain.

Cloning of jute CCoAOMT genes

To clone the full gene and open reading frame (ORF) sequences of the jute CCoAOMT genes, pairs of primers (Additional file 19) were designed according to the coding sequences (CDS) of CCoAOMTs reported in GenBank resources for jute. Forward and reverse primers were designed to align to the opposite endings of the ORF. DNA was extracted from leaf samples of young jute plants using the TIANcombi DNA Lyse & Det PCR Kit (Tiangen, China), according to the manufacturer’s guidelines. PCR products derived from genomic DNA amplification were cloned into the pMD19-T cloning vector (TaKaRa Biotechnology, Dalian, China) and subsequently sequenced by BGI Tech Solutions Co., Ltd. (BGI-Tech). The obtained sequences were BLAST-searched against the NCBI database for validation.

BLAST searches of CCoAOMT gene family members in eight representative dicot and monocot plant species

The sequences of seven known CCoAOMT genes from A. thaliana) [59] were utilized as queries to identify the complete sequences of CCoAOMT genes in rice (Oryza sativa), ramie (Boehmeria nivea), soybean (Glycine max), cotton (Gossypium raimondii), kenaf (Hibiscus cannabinus), flax (Linum usitatissimum), cacao (Theobroma cacao), and Amborella (Amborella trichopoda). Candidate sequences that fulfilled the criteria of a probability score < 10⁻⁴ and a similarity score > 50.0 were selected. These candidate CCoAOMT sequences were further confirmed by searching against the annotated databases of Phytozome [68] and NCBI using BLASTX and BLAST. Moreover, candidate CCoAOMT proteins were assessed by investigating their conserved domains using the CD-Search and InterPro tools [69, 70]. All Cc.CCoAOMT and Co.CCoAOMT proteins share a conserved domain which is “AdoMet_MTases” with the exception of Cc.CCoAOMT2 and Co.CCoAOMT2 proteins which share “PLN02589” as their conserved domain. Using the ExPASy Translate tool [71], the confirmed DNA sequences were translated into proteins. Additionally, the theoretical isoelectric point (pI) and molecular weight (Mw) of the proteins were calculated using the ExPASy Compute pI/Mw tool [72]. Prediction of subcellular localization was conducted using the Prediction Servers of Signal P5.0 Server [73], PlantmPLoc [74], MitoProt [75], and ChloroP [76]. The CCoAOMT protein motifs were assayed using the online tool MEME Suite [77] with the frameworks of minimum motif width being 6, maximum motif number being 15, maximum motif width being 50 and dissemination of motif occurrences being one per sequence or Zero. The gene schematic structures were drawn, using the Gene Structure Display Server [78, 79].

Phylogenetic analysis

The evolutionary history of CCoAOMTs in 12 plant species was deduced using the neighbor-joining method [80]. The ratio of replicate trees was displayed next to the branches [81]. Evolutionary distances were calculated using the Poisson correction method [82] and were expressed as the number of amino acid substitutions per site. Sixty-six amino acid sequences were used in the analysis [83]. By applying the Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances, the initial trees for the heuristic search were obtained automatically. Subsequently, the topology showing a superior log-likelihood value was chosen. Using the Easy_KaKs calculation program [84], the non-synonymous (Ka) and synonymous (Ks) substitution ratios of eight pairs of orthologous CCoAOMT genes were computed. Finally, Fisher’s exact test was used to confirm the validity and compute the p-value of the Ka and Ks ratios [85] and the divergence time (T) was computed by using this formula: $\mathrm{T}=\mathrm{Ks}/(2\times 6.1\times {10}^{^-9})\times {10}^{^-6}\mathrm{Mya}$ [86]. Utilizing MCScanX analysis [87], collinearity relationships of CCoAOMT genes and classifier program were used to sort gene duplication types. The identified collinear gene pairs were mapped to their respective locus in the jute genome in a circular diagram using Circos 0.69 [88].

Analysis of gene expression by qRT-PCR

One microgram of total RNA from each of the samples collected from plants at various developmental stages subjected to different treatments was reverse-transcribed to cDNA using the Reverse Transcriptase Kit (TaKaRa) in a 20uL reaction mixture, according to the manufacturer’s guidelines. The resulting cDNA was diluted for qRT-PCR analysis. Based on the annotated sequences of CCoAOMTs, qPCR primers (Additional file 20) were designed using the PrimerQuest tool at Integrated DNA Technologies [89]. The expression patterns of 12 Co.CCoAOMT genes and eight orthologous Cc.CCoAOMT genes were compared across the different developmental stages. qRT-PCR was performed using the Multicolor Real-Time PCR Detection System (Bio-Rad). The qRT-PCR reactions were incubated at 94 °C for 2 min, and then subjected to 39 cycles of 94 °C for 5 s, 65 °C for 5 s, and 72 °C for 5 s. Melting curve analysis was conducted to verify qPCR specificity. For the developmental stage group, ubiquitin extension protein (UBI) was selected as a reference gene. For the salt stress group, actin 7 (ACT7) was selected as an internal control; finally, for drought stress, ubiquitin-conjugating enzyme-like protein (UBC) was used as a reference gene (Additional file 20) [90]. The 2^{−ΔΔCT −}method [91] was utilized to calculate relative expression levels for each CCoAOMT gene.

Lignin content and composition of developing jute fibers

The measurement of lignin content was conducted on jute stem samples (J179, QY, LB, F5, KY, and CL) collected at 15, 30, 45, 60, and 90 DAS. Three biological replicates were used for each sample. The determination of lignin constituents in jute threads was carried out through the acetyl bromide method [92, 93]. The results were expressed as mg lignin g⁻¹ cell wall. SPSS20.0 was used to perform statistical analysis of lignin content data for 6 genotypes and 5 developmental stages, as well as the correlation between lignin content and CCoAMOT2 expression level.