All plant materials were obtained from a provenance test plantation of L. chinense in Xiashu Forest Farm, Jurong City, Jiangsu Province (119°13′E, 32°7′N), China. The provenance of the sample tree was the Lushan Natural Reserve, Jiangxi Province (116°0′E, 29°32′N). From March to July, 2021, we collected leaves, shoots, roots, petals, and stems, which were quickly frozen in liquid nitrogen and stored in the refrigerator at –80°C before RNA extraction.

Nicotiana alata seedlings were sterilized with 10% NaClO for 15 min and then sown in 1/2 MS medium. After 2 days of vernalization (4°C dark), the seedlings were placed in an incubator (22°C with 16-h light and 8-h dark photoperiod) for a week. They were then transplanted into a medium for culturing.

We downloaded the Liriodendron Genome resources from the NCBI database (https://www.ncbi.nlm.nih.gov/; PRJNA418360; Chen et al., 2019). Thirteen AtCCRs that were identified to be involved in the biosynthesis of lignin precursors in A. thaliana were obtained from the TAIR databases (https://www.arabidopsis.org/). Thirteen AtCCR proteins ( Table S1 ) were used as alignment sequences to perform BLAT alignment with the protein database of L. chinense. The E-value was set to 0.001 to obtain the candidate CCR sequences of L. chinense. All candidate CCR sequences were assessed based on the presence of the conserved domain with InterPro (http://www.ebi.ac.uk/interpro/search/sequence/) and CDD search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) procedures. Finally, sequences with complete CCR domains were selected, and the sequences with more than 97% similarity between the different databases were deleted. The physical and chemical characteristics of LcCCR proteins identified from the L. chinense genome were predicted by ProtParam online tool (https://web.expasy.org/protparam/; Wilkins et al., 1999). The WoLF PSORT (https://wolfpsort.hgc.jp/) and TargetP-2.0 Server (http://www.cbs.dtu.dk/services/TargetP/) were used to predict the subcellular localization of the LcCCR proteins.

To group the CCR proteins in L. chinense, we analyzed the phylogenetic relationships between L. chinense and other plants. We constructed a phylogenetic tree using the CCR protein sequences from A. thaliana, P. tomentosa, L. perenne, Oryza sativa, Z. mays, and L. chinense ( Table S1 ). DNAMAN (version 6.0) software was used to perform multiple comparisons among these protein sequences, and a phylogenetic tree was constructed by the maximum likelihood method via MEGA (version 5.1) software. Substitution and site change rates were calculated using the Jones–Taylor–Thornton (JTT) model and the Gamma distributed with Invariant sites (G+I) model. Bootstrap analysis was performed with 1000 replicates to calculate the reliability of the phylogenetic tree. Finally, the network profile of the phylogenetic tree was visualized by Evoview (https://www.evolgenius.info/evolview/) (He et al., 2016). Last, we used DNAMAN (version 6.0) to compare the 13 candidate LcCCR genes with other bona fide CCR genes.

To analyze the exon-intron structure of CCR genes, the annotation profile was retrieved from the L. chinense genome (Chen et al., 2019). Information on the introns and exons of the LcCCR gene family was visualized by GSDS (version 2.0) (http://gsds.cbi.pku.edu.cn/) online tool (Hu et al., 2014). The conserved motifs in LcCCRs were visualized by MEME (version 5.3, https://meme-suite.org/meme/tools/meme) online tool (Bailey et al., 2006). The parameters were set as follows: an optimum motif could contain no less than 6 and no greater than 200 residues; the maximum number of motifs allowed was 10 (Chen et al., 2020b). The results of these motifs were then visualized via the TBtools software (Chen et al., 2020a).

To identify the cis-acting elements in the promoter sequences of the 13 CCRs in L. chinense, the 2000-bp upstream sequence of the start codon (ATG) in CCRs was analyzed. The types and numbers of cis-elements in LcCCRs were evaluated via the Plant CARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) online tool (Lescot et al., 2002).

To quantify the expression levels of LcCCR genes in the different tissues (leaf, shoot apex, and developing xylem at the stem (DXS)), we analyzed the expression profiles based on the transcriptome data (Unpublished data from our laboratory). The RPKM (reads per kilobase per million mapped reads) approach was used to represent the expression abundance of each LcCCRs. The TBtools (version 1.0) software was used to draw the gene expression heatmap (Chen et al., 2020a).

To identify genes involved in lignin biosynthesis, we conducted the RT-qPCR analysis of CCR genes in five tissues (leaves, shoots, roots, petals, and stems). The detailed protocols are as follows:

Total RNA was isolated from each sample using a total RNA isolation kit (TIANGEN, China) following the manufacturer’s protocol. RNA-free deoxyribonuclease (DNase I) was used to remove trace DNA from the extracted RNA. The integrity of the total RNA was detected via 1.0% agarose gel electrophoresis. The concentration and purity of the total RNA were analyzed by a NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Reverse transcription was performed using the Evo M-MLV RT Premix for qPCR (AG11706, ACCURATE BIOTECHNOLOGY, HUNAN, Co., Ltd). Quantitative primers for the CCR genes were designed using Oligo 7.0 software according to strict requirements ( Table S2 ). The RT-qPCR was run in a StepOnePlusTM System (Applied Biosystems) as a 10-μL reaction mixture containing 5 μL of 2× SYBR Premix Ex Taq, 0.2 μL of 50× ROX Reference Dye (SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, ACCURATE BIOTECHNOLOGY, HUNAN, Co., Ltd)), primers, and cDNA. The LcActin97 (Tu et al., 2019) and NtActin (AJ421411) primers were used for the amplification of the internal reference gene. To ensure the accuracy of the results, three biological replicates (where each biological replicate had three technical replicates) were conducted, and the data were examined using the 2^−ΔΔCT method. Significance was determined by the t-test using the SPSS statistical software (version 20, IBM, New York, NY, USA; *p < 0.05, **p < 0.01).

LcCCR13 was retrieved from the existing L. chinense genome data (Chen et al., 2019). We designed the intermediate fragment-specific PCR primers via the Oligo (version 7.0) software ( Table S2 ). Reverse-transcribed cDNA was used as a template for PCR to synthesize intermediate fragments. The PCR products were cloned into the pEASY-Blunt Zero Cloning Kit (Transgen Biotech, Beijing, China) and transformed into E. coli (DH5α). Finally, the amplicons were sequenced by Jie Li Biology (Shanghai, China).

The sequenced ORF of the LcCCR13 gene was inserted into the overexpression vector, which was digested with Xba I and BamH I QuickCut enzymes (Takara Biomedical Technology, Dalian, China). The construct was then transformed into Agrobacterium tumefaciens strain EHA105 by the freeze-thaw method (Holsters et al., 1978, Zhang et al., 2012). The target gene was transferred into WT tobacco by the leaf-disc conversion method. Six transgenic tobacco lines featuring independent recombination events were identified by molecular detection; they were all heterozygous with similar phenotypes. To detect the relative expression levels of LcCCR13 in the different lines, three transgenic lines together with the WT strain were subjected to a semi-quantitative PCR assay. The relative expression level of LcCCR13 was quantified from three biological replicates, where each replicate had three technical replicates.

Meanwhile, the histochemical staining of stem segments from transgenic tobacco was performed via the Phloroglucinol-HCL and safranin staining methods (Yang et al., 2022). The stained sections were observed under a Zeiss Axio microscope. The ImageJ software (version 2.3.0) was then used to calculate the size of the thickened and lignified cells (μm).

All the transgenic and WT plants were acclimated and grown in a greenhouse at 18–23°C with 60% humidity under a 16-h light and 8-h dark photoperiod at NJFU (Nanjing Forestry University, Nanjing, China).

The height and the diameter were measured in 2-month-old transgenic and WT plants. The measurements for each plant line included at least 5 replicates.

The CCR activity was determined following the method of Hamedan et al. (Hamedan et al., 2019).

According to the method of Bhaskara et al. (1999), a 10 mg powdered sample was weighed and ground into a homogenate in a mortar with 95% ethanol. The homogenate was then transferred to a 50-mL centrifuge tube for centrifugal separation at 4000 rpm for 5 min. The pellet thus obtained was washed twice with 5 mL of 95% ethanol and 6 mL of ethanol: n-hexane (1:2 (v/v)). The pellet was dried naturally. Six milliliters of 25% acetyl bromide solution in glacial acetic acid were used to dissolve the precipitate, followed by being covered and sealed in a water bath at 70°C for 30 min. Subsequently, 0.9 mL of 2 mol·L⁻¹ NaOH, 5 mL of glacial acetic acid, 0.1 mL of 7.5 mol·L⁻¹ hydroxylamine hydrochloride, and 15 mL of glacial acetic acid were added to the centrifuge tube. Instead of the substrate, distilled water was used for the same reaction as a control. The absorbance of the supernatant was measured at 280 nm using the GeneQuant pro ultraviolet spectrophotometer (Biochrom Ltd, Cambridge, UK), and the absorbance per gram of the dry sample at 280 nm represented the lignin content per gram (OD g⁻¹ DW) (OD: Optical Density, DW: dry weight). The statistical significance of the data was estimated by analysis of variance (ANOVA); the Pearson coefficient was calculated using the SPSS statistical software (version 20, IBM, New York, NY, USA; *p < 0.05, **p < 0.01).

The statistical significance of the data using ANOVA was assessed using the SPSS statistical software (version 20, IBM, New York, NY, USA). Multiple comparative analyses were performed using the t-test (*p < 0.05, **p < 0.01). To identify differences among the samples or plant lines. One-way ANOVA followed by Duncan’s multiple comparisons test was used.

Thirteen LcCCRs were identified in this study containing the complete “X-W-Y-X-X” functional domain. Detailed characteristics of the 13 LcCCRs are presented in Table 1 . These CCR genes were named LcCCR1-LcCCR13. Briefly, the length of the coding DNA sequences (CDS) ranged from 786 to 1188 bp with 5–7 exons in each, and the amino acid length ranged from 261 (LcCCR4) to 368 (LcCCR48) amino acids, with the number of amino acids being greater than 300, except for in two proteins (LcCCR4/6). In addition, the molecular weight (MW) of the proteins ranged from 28933.98 (LcCCR4) to 43564.06 (LcCCR11) Da, where most protein’s MW was greater than 30.00 kDa. The isoelectric points (pI) varied from 5.34 (LcCCR4) to 7.52 (LcCCR3/5). The average amino acid number, MW, and pI of these protein sequences were 330, 36309.11 Da, and 6.33, respectively. We used WoLF POST and online analysis with the TargetP-2.0 Server to predict the subcellular localization of the LcCCR proteins. Both online tools indicated that LcCCR proteins are localized in the cytoplasm or the chloroplast ( Table 1 ).

The subcellular localization was predicted by online tools: WoLF PSORT (https://wolfpsort.hgc.jp/) and TargetP-2.0 Server (http://www.cbs.dtu.dk/services/TargetP/). The physical and chemical characteristics of LcCCR proteins identified in the L. chinense genome were predicted using the online tool, ProtParam (https://web.expasy.org/protparam/).

To understand the sequence characteristics, a multiple sequence alignment analysis of the 13 LcCCR proteins was performed by DNAMAN software with default parameters. Two AtCCR proteins (AtCCR5/9) and three PtoCCR proteins (PtoCCR1/4/7) were selected as representatives for further comparison. The conserved domain structures in LcCCRs are displayed in Figure 1 . It was predicted that the amino acid sequences in each member of the LcCCRs were significantly different from other CCR proteins, but the C-terminal and the middle sequences were highly conserved. Almost all of the above sequences contained the conserved domain, “X-W-Y-X-X,” from the cinnamyl-CoA reductase family of proteins and the recognition domain of NAD(P)-dependent short-chain reductase (SDR). The structure also included the typical “Rossmann fold” and the conserved catalytic center, the Ser-Tyr-Lyr triad (Kallberg et al., 2002).

To clarify the phylogenetic relationship between the LcCCRs and other CCR proteins, an unrooted phylogenetic tree was constructed with 59 CCR proteins (13 LcCCR, 13 AtCCR, 11 PtoCCR, 10 LpCCR, 10 OsCCR, and 2 ZmCCR proteins) based on the maximum likelihood method. In the phylogenetic tree, all 59 CCRs mentioned above could be roughly divided into four groups (Groups I to IV; Figure 2 ). Our results showed that Group I was divided into three subgroups: subgroups Ia, Ib, and Ic. Subgroup Ia contained 14 CCR members (3 LcCCRs, 2 AtCCRs, 3 LpCCRs, 3 PtCCRs, 2 OsCCRs, and 1 ZmCCRs), which were related to lignin biosynthesis [AtCCR5/9 (Goujon et al., 2003), ZmCCR1 (Pichon et al., 1998), PtCCR1/4/7 (Chao et al., 2017)]. The entire Group I comprised CCRs from dicotyledons and monocotyledons. Subgroup Ib contained 8 CCR members (4 LpCCRs, 3 OsCCRs, and 1 ZmCCR). Subgroup Ic contained 8 CCR members (3 LpCCRs and 5 OsCCRs). Subgroups Ib and Ic consisted only of CCR proteins from the monocotyledons. Group II, shown in blue color, contained 5 CCR members (3 LcCCRs and 2 PtoCCRs). Group III in green contained 8 CCR members (3 LcCCRs, 3 PtoCCRs, and 2 AtCCRs). Group IV in pink contained 16 CCR members (4 LcCCRs, 4 PtoCCRs, and 8 AtCCRs) ( Figure 2 ). Notably, most CCRs were evenly distributed in four groups (Barakat et al., 2011). Moreover, it was found that the LcCCRs had close phylogenetic relations with their ancestors’ species in each group. The distribution of CCR proteins in the phylogenetic tree suggested that CCR proteins were duplicated multiple times before becoming specific to the monocotyledon and dicotyledon species in which they were observed.

The exon-intron configurations in the LcCCR genes were examined to further explore the probable structural evolution of this family of genes ( Figure 3A ). Similar structures were usually observed in the same group. e.g., the members of Group II contained 5 exons, and the members of Group III/IV contained 6 exons. The length and number of introns were generally similar in each group, which was consistent with the clusters of LcCCRs. However, there were some exceptions. For example, the members of Group I did not have a similar exon-intron pattern.

Furthermore, we investigated the full-length protein sequences of the 13 LcCCRs to identify their conserved motifs. 10 conserved motifs were predicted to be present in 13 LcCCRs. The number of amino acids in the 10 motifs ranged from 11 to 50 ( Table S3 ). The patterns of the conserved motifs are shown in Figure 3B . As motif 1 constituted the main structure of the CCR domain (“NWYCY”), it was identified in all LcCCR proteins. LcCCR proteins in the same group tended to have similar motif compositions, suggesting that protein structures were conserved in a specific subfamily (Song and Peng, 2019). Furthermore, only the members of Group I of LcCCR proteins and LcCCR7 did not contain Motif 8. We speculate that LcCCR7 may have a deletion, leading to the lack of this discrepancy. Overall, members within each group shared the same genetic structure, which was consistent with their phylogenetic relationships. The stability of group classifications was maintained as per our findings from surveys on the conserved motif compositions, gene structures, and phylogenetic relationships.

To explore gene function and regulation patterns, we surveyed the cis-elements in a region 2000 bp upstream of the initiation codon in each LcCCR gene. The cis-acting elements of the LcCCR genes were then predicted by searching the promoter sequences from the PlantCARE database (Song and Peng, 2019). As shown in Figure 4 , almost all LcCCR gene promoters contained an AC element (Raes et al., 2003), along with the methyl jasmonate (MejA), salicylic acid (SA), and abscisic acid (ABA) elements. Some promoters contained light response seed-specific regulation (Chen et al., 2020b) or low-temperature response elements. The number of cis-acting elements for each of the 13 LcCCR genes ranged from 4 to 22. Promoters of different genes in the same group contained similar elements with only minor differences.

The promoter sequences of most genes (70%) had AC elements, such as LcCCR1/2/3/6/7/8/8/10/11/12 ( Figure 4A ), suggesting that these genes may be involved in the regulation of phenylpropanol metabolism and other unknown functions. In addition, these cis-elements are evenly distributed in the promoter region of LcCCRs ( Figure 4B ).

Cis-acting elements, such as ABRE, TCA-element, and TGACG-motif, are related to the signaling pathways of ABA, SA, and MejA, respectively. All of these metabolites are related to stress resistance in plants. Almost all promoters contain these three cis-acting elements, therefore, it can be reasonably inferred that some LcCCRs might be involved in plants’ responses to biotic and abiotic stresses.

Other promoters contained cis-elements for seed-specific regulation, light response, and low-temperature response, indicating that they may have a certain influence on the seed. The low-temperature response was related to the light response. Detailed information is shown in Table S4 .

We investigated the expression patterns of LcCCRs in different tissues (leaf, shoot apex, and DXS) with Illumina RNA-seq data (Unpublished data from our laboratory). The results showed that the 13 LcCCR genes were expressed in all three tissues but some genes exhibited tissue-specific expression. For instance, LcCCR1/3/4/6/9 were highly expressed in the leaf. While in the shoot apex, the expression levels of LcCCR2/11 were the highest. Meanwhile, LcCCR8 displayed high expression in the leaf and DXS. Furthermore, it was found that LcCCR5/7/10/12/13 had the highest expression in the DXS ( Figure 5 ). Collectively, the different expression patterns of LcCCRs suggest that they may play different roles in the various developmental stages/tissues in L. chinense.

To obtain insights into the potential roles of LcCCR5/7/10/12/13, which were predicted to be highly expressed in DXS ( Figure 5 ), the expression level of LcCCR genes was further determined by RT-qPCR in five tissues (root, stem, leaf, shoot, and petal). The results showed that the LcCCR5/7/10/12/13 were expressed in all the tested tissues, but their expression levels were different ( Figure 6 ). The relative expressions of LcCCR5/10/12 were similar in all tissues with the highest expression level in the root. Moreover, the relative expression level of LcCCR7 in the petal was the highest. Furthermore, LcCCR13 had the highest expression level in the stem, too, which was extremely significantly different from the expression in the root, leaf, petal, and shoot. Although the expression of the LcCCR13 gene was not xylem-specific, it was mainly expressed in lignin-forming tissues, such as stem, suggesting that LcCCR13 may play an important role in lignin biosynthesis in L. chinense.

To further investigate the function of the LcCCR13 gene in lignification, a transgenic assay was used. Tobacco leaves were transformed with the 35s::LcCCR13 vector to induce high expression of the gene ( Figures 7A, B ); six LcCCR13 positive lines were thus obtained ( Figure 7C ). To investigate the influence of the LcCCR13 gene on plant growth, the WT controls and the three transgenic tobacco lines were monitored. Significant differences were observed in the growth phenotypes of the transgenic lines and the WT plants ( Figures 8A, B ). For example, there was a significant height difference between the L4 lines and the WT plants (0.7 fold; Figure 8C ). We found that the transgenic lines had a significantly greater stem diameter than the WT (1.6 fold; Figure 8D ). All in all, transgenic lines exhibited altered growth characteristics as compared to the WT plants.

A significant increase was noted in the lignin content of the transgenic lines (1.3 fold; Figure 8E ), and the L4 lines were selected for histochemical staining due to the highest LcCCR13 gene relative expression in them ( Figures 8F, G ). The difference in the CCR activity of transgenic lines was measured. We found that the relative activity of CCR in transgenic plants was significantly higher than that in the WT plants, and the L4 lines had the highest CCR activity compared to other transgenic lines ( Figure 8H ).

When observed under higher magnification, the tissue sections of the L4 lines showed a more highly developed xylem with wider and deeper brown color than in the WT plants ( Figures 9A–F ). Furthermore, the size of the thickened and lignified cells in transgenic tobacco was 306.20 μm, and they were ~1.75× (p < 0.01) as thick as the WT cells (174.52 μm; Figure 9G ). Therefore, higher lignin deposition occurred in the L4 lines than in the WT plants. These results suggest that the overexpression of LcCCR13 might positively regulate lignin synthesis.

Lignin synthesis starts from the common phenylpropanoid pathway and is regulated via a variety of enzymes, but the cinnamyl-CoA reductase encoded by the CCR is one of the key enzymes in lignin biosynthesis (Lewis and Yamamoto, 1990; Raes et al., 2003). As for most forest tree species and associated metabolic pathways, genome-wide analysis is a crucial method to elucidate the biological functions of the CCR family in plants. Here, we report the phylogeny and genome structure of the CCR genes in basal angiosperms for the first time. After further characterization, 13 LcCCR genes were identified in L. chinense.

The expansion of the CCR family members is important to plant evolution. After years of continuous research, studies have identified and cloned the full-length or partial coding sequence (CDS) of CCR genes from the xylem or other tissues in many plant species. For example, seven CCR genes involved in lignin biosynthesis have been identified in P. tomentosa (Chao et al., 2017), 11 in Populus tremuloides (Li et al., 2006), 4 in Boehmeria nivea (Tang et al., 2019), and 11 in A. thaliana (Raes et al., 2003). In general, there are more CCR genes in woody species. We hypothesize that the number of CCR family members in different species may be affected by genomic differences, the redundancy of gene functions, or species differences.

In this study, 13 LcCCR genes were screened by proteomic mining of the complete sequence genome of L. chinense. The molecular weight of these LcCCR proteins was greater than 35 kDa each, which is the same as most CCR proteins reported in plants (Prasad et al., 2010). Their isoelectric points were between 5.5 and 7.5, which is consistent with those for the BnCCR proteins (Tang et al., 2019). Lauvergeat et al. examined the protein structures of the bona fide Arabidopsis CCR proteins, AtCCR5 (AT1G15950) and AtCCR9 (AT1G80820) (Lauvergeat et al., 2001), to reveal that the region of the N-terminal portion of AtCCR5/9 involved in the NADP(H) cofactor binding site was conserved. A very well-conserved motif, KNWYCY, which is thought to be involved in the catalytic site of this enzyme, also exhibits the signature of CCRs (Lacombe et al., 1997). Previous studies have shown that the second and third amino acids (“W” and “Y”) are crucial for the binding of the enzyme to the substrate, and thus, they are rarely replaced (Barakat et al., 2011). The conserved domain, “X-W-Y-X-X”, in the cinnamyl-CoA reductase family of proteins was found in all 13 LcCCR protein sequences here. A similar phenomenon was observed in Pyrus bretschneideri (Chen et al., 2020b) indicating that LcCCRs remained highly conserved during the evolution of L. chinense.

Based on the results of MEME analysis with default parameters, the same group of LcCCR proteins shared common motifs in the phylogenetic tree, suggesting that these LcCCRs are highly conserved, strongly supporting the reliability of the group classifications (Bailey et al., 2006). In addition, gene structure and motif analysis methods were employed to discover the potential features of genes (Barakat et al., 2011). Phylogenetic analysis shows that LcCCR genes could be divided into four groups. The structures of exon-intron regions in the genes from the same group were similar. The change in the number of CCR genes and the diversification of characteristic motifs provides new insights to understand the evolution and gene function of the LcCCR gene family.

The phenylpropanoid pathway, the main pathway in lignin biosynthesis that is regulated by a variety of enzymes, is well understood. Cinnamoyl-CoA reductase (CCR) is considered to be the first committed enzyme in the lignin-specific branch because it can catalyze the conversion of cinnamoyl-CoA to cinnamaldehyde in the monolignol biosynthetic pathway, which can further be transferred into three lignin monomers (G, S, and H) (Gayoso et al., 2010). Besides, cinnamoyl-CoA can be synthesized into phenolic substances (such as anthocyanins and flavonoids) when the function of CCR has been lost. For this reason, the key role of the CCR gene in lignin synthesis has been confirmed in many plant species, and changing its expression levels might significantly affect the lignin content and growth of plants (Liu et al., 2021). In our study, we measured the plant height and the lignin content in transgenic lines and observed that the overexpression of the LcCCR13 gene significantly increased lignin accumulation in the stems and decreased plant height. These results corroborate that the LcCCR13 is a key gene for lignin synthesis in the stems of L. chinense.

The synthesis of lignin is tissue specific. In general, lignin synthesis occurs in tissues with higher lignification, and the CCR genes are strongly expressed in such tissues. For example, AtCCR5 is mainly concentrated in the tissues that are being lignified and participate in the process of tissue lignification (Lauvergeat et al., 2001; Goujon et al., 2003); the activity of PtoCCR1/4/7 affects the accumulation of lignin (Chao et al., 2017). The relative expression levels of the EgCCR gene are also the highest in the stems, followed by their expression in the roots and other tissues (Lacombe et al., 1997). TaCCR2 is mainly expressed in the roots but also participates in the lignin synthesis in stems, indicating that it plays an important role in lignin synthesis in T. aestivum (Lin et al., 2001). The expression analysis of 10 PoptrCCRs showed that all of them were expressed in the bark, leaves, and xylem, but only the bona fide PoptrCCR12/14 had the highest expression levels in the xylem, where they showed a significant difference from the expression levels in the leaf/bark (Barakat et al., 2011). Our transcriptome data indicated that only LcCCR5/7/10/12/13 genes were predominantly expressed in the DXS (stem developmental xylem). RT-qPCR assays showed that the LcCCR13 had the highest expression level in the stem, which was significantly different from the expression level in other tissues. These observations were largely consistent with previous studies; For instance, the bona fide CCR genes are highly expressed in the tissues/organs with high lignification (Zhang et al., 2015). In this study, only LcCCR13 was highly expressed in the stem, indicating that it might play potential roles in stem development. Therefore, we overexpressed LcCCR13 in tobacco to determine whether it was associated with lignin synthesis.

Transgenic technologies have been widely used to control the lignin content and composition in various plants. Up-regulation or down-regulation of AtCCR expression in A. thaliana significantly affected the lignin content and other characteristics related to plant growth and development (Goujon et al., 2003). As compared to the WT plants, the transgenic lines of B. napus over-expressing BnC.CCR2b had significantly higher lignin content in the stems (Prasad et al., 2010). When BnCCR1 activity was increased in B. platyphylla, the lignin content also increased, and the height of the transgenic plants was reduced (Zhang et al., 2015). The lignin content increased and the plant height decreased in the antisense transgenic tobacco plants, and their plant phenotypes (such as plant height, seed quality, and length of the leaves) was significantly altered (Chabannes et al., 2001). These findings indicate that the manipulation of CCR gene expression affects lignin content, and the changes in lignin content in the transgenic plants is incompatible with normal phenotypes.

In this study, we found that the over-expression of the LcCCR13 gene affects the growth and development of transgenic tobacco. As compared to other transgenic lines, transgenic plants with the highest CCR activity showed the highest lignin content, indicating that CCR might be the key gene involved in lignin biosynthesis. Hamedan et al. also reported that increased lignin content coincides with CCR activities in Gerbera jamesonii (Hamedan et al., 2019). Moreover, according to the measurement of lignin content and its deposition sites in transgenic plants, we conclude that the LcCCR13 gene has a significant effect on lignin synthesis in the stem. Based on the fact that increasing the expression of LcCCR13 reduced the height growth and increased the lignin content in the stem, we assume that LcCCR13 is involved in the thickening of the cell wall by increasing the levels of all three types of subunits. It further regulates the ligin content in plants. Tu et al. found that the downregulation of CCR1 expression in transgenic L. perenne plants reduced the lignin content by lowering the levels of all three lignin monomers (Tu et al., 2010). Interestingly, Zhang et al. found that BpCCR1 could control the height growth and lignin content by lignifying the cell wall (Zhang et al., 2015), which was consistent with our results. All in all, these findings might be significant for understanding the roles of the LcCCR13 gene in lignin biosynthesis in the stems of L. chinense.