PlLAC15 Facilitates Syringyl Lignin Deposition to Enhance Stem Strength in Herbaceous Peony

Abstract

Stems are prone to bending or lodging due to inadequate stem strength, which seriously reduces the cut-flower ornamental quality of herbaceous peony (Paeonia lactiflora Pall.). Plant LACCASE (LAC), a copper-containing polyphenol oxidase, has been shown to participate in the polymerization process of monolignols; however, the role of LAC in regulating the stem strength of P. lactiflora remains unclear. Here, the full-length cDNA of PlLAC15, which demonstrated a positive association with stem strength, was isolated. It consisted of 1790 nucleotides, encoding 565 amino acids that had four typical laccase copper ion-binding domains. Moreover, PlLAC15 was highly expressed in the stem, and its expression level gradually significantly increased during stem development. Furthermore, PlLAC15 was found to be localized specifically to the cell wall, and its recombinant protein exhibited laccase activity. Additionally, the role of PlLAC15 in regulating the stem strength of P. lactiflora was confirmed by transgenic studies. When PlLAC15 was overexpressed in tobacco, stem strength increased by more than 50%, S-lignin was significantly deposited, and the lignification degree of stem xylem fiber cells increased. These results suggested that PlLAC15 facilitated S-lignin deposition to enhance stem strength in P. lactiflora, which would provide precious information that benefits future exploration of stem bending or lodging resistance in plants.

1. Introduction

Stem development is essential for plant production. Due to inadequate strength, the stems are prone to bending or lodging. For crops, this is crucial for their yield and quality, as the stems of crops need to support the weight of the aboveground parts and resist external forces, such as wind and rain, to ensure normal growth and ultimately achieve high yields [1]. Nevertheless, for ornamental plants, the verticality and curvature of the stem can affect the appearance of ornamental plants, thereby affecting their ornamental value [2]. Therefore, enhancing stem strength is an immediate challenge that needs to be addressed in plant production. The stem strength of plants is tightly associated with the secondary wall components of mechanical tissues. Among these components, cellulose comprises the primary substance of the cell wall skeleton, and its deposition is essential to stabilize the cell wall structure [3]. In barley [4], soybean [5], maize [6], and cassava [7], the cellulose content was an important factor influencing stem strength. Moreover, lignin presented in the secondary cell wall can provide plants with essential structural support. In wheat [8], chrysanthemum [9], and alfalfa [10], there was a marked association noted between lignin content and stem strength. Some studies have demonstrated a stronger impact of lignin content on stem strength compared to cellulose [11,12].

Lignin is classified as a structurally complex polyphenolic heteropolymer that is found widely in plants and includes p-hydroxyphenyl lignin (H-lignin), guaiacyl lignin (G-lignin), and syringyl lignin (S-lignin), which result from the polymerization of monolignols, namely, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, respectively [13]. The biosynthesis of lignin is a very complex process that can be divided into two stages: monolignol biosynthesis and monolignol polymerization. Among them, monolignol biosynthesis is carried out in the cytoplasm and includes two biochemical reaction processes, the phenylpropanoid pathway and the lignin-specific pathway, and finally leads to the generation of the three monolignols [14]. A large number of enzymes that participate in these processes, such as phenylalanine ammonia-lyase (PAL), caffeic acid O-methyltransferase (COMT), hydroxycinnamoyl CoA (HCT), etc., are extensively isolated and studied. As far as monolignol polymerization is concerned, it is widely believed that the monolignols, after being synthesized in the cytoplasm, were relocated to the cell wall where they further catalyzed polymerization to form lignin. Studies on the model plants Arabidopsis thaliana and poplar have demonstrated that laccase (LAC) and peroxidase (PER) could successively catalyze monolignol polymerization [14,15,16]. Among them, LAC plays an important role in increasing lignin content. For instance, Cesarino et al. [17] discovered that overexpression of sugarcane SofLAC had the ability to restore lignin content in the A. thaliana lac17 mutant. Overexpression of pear PbLAC1 caused a substantial rise in lignin contents, along with thicker cell walls in both interfascicular fibers and xylem cells of A. thaliana [18].

Herbaceous peony (Paeonia lactiflora Pall.) enjoys a storied reputation as a famed flower in China but has also become an emerging high-end cut flower in recent years. However, stem bending due to inadequate stem strength seriously decreases the ornamental quality and commodity price of P. lactiflora cut-flowers. Until now, some advancements have been achieved in researching the stem strength of P. lactiflora. For instance, Zhao et al. [19] discovered that lignin was the key component that imparted mechanical strength to stems in P. lactiflora and obtained the candidate-related genes and transcription factors involved in lignin biosynthesis. Subsequently, three R2R3-MYBs, PlMYB43, PlMYB83, and PlMYB103, were identified in P. lactiflora to be involved in modulating stem strength by influencing lignin biosynthesis and secondary cell wall thickening [20]. PlWRKY41a could activate the expression of the XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 4 gene (PlXTH4) to stimulate secondary cell wall thickening for improved stem strength of P. lactiflora [21]. Additionally, applying calcium and silicon to P. lactiflora through foliar spraying would contribute to a substantial improvement in stem strength resulting from an increase in lignin content [22,23]. However, the genetic regulation of monolignol polymerization has yet to be investigated. Here, a differentially expressed PlLAC15 was screened based on our previous transcriptome sequencing data of P. lactiflora stems [19]. Subsequently, the full-length cDNA of PlLAC15 was isolated, and its expression pattern as well as subcellular localization were examined. Moreover, its role in regulating the stem strength of P. lactiflora by changing S-lignin deposition was systematically studied using overexpression assays. These results could broaden our understanding of the mechanism behind the regulation of P. lactiflora stem strength.

2. Materials and Methods

2.1. Gene Isolation and Bioinformatic Analysis

The full-length cDNA of PlLAC15 was isolated by polymerase chain reaction (PCR) technology based on previous transcriptome [19], and gene-specific primers were designed (forward primer: 5′-TTTGCTCTGTTTACTTGTGACG-3′, reverse primer: 5′-GCATCACTCTATTTCCACTACA-3′). The total RNA of stems from P. lactiflora cv. ‘Hongyan Zhenghui’ was extracted by the MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China), and the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China) was used for reverse transcription. After separation via agarose gel electrophoresis, the PCR product was then sent for sequencing.

The molecular weight and theoretical isoelectric point (pI) of PlLAC15 were calculated through the ExPASy server (https://web.expasy.org/protparam/, accessed on 1 July 2020). The LAC protein sequences from A. thaliana were acquired from the TAIR database (https://www.arabidopsis.org/, accessed on 31 July 2024). The domains of these LACs and PlLAC15 were aligned utilizing the CLUSTALW tool within MEGA11, and a phylogenetic tree was subsequently constructed employing the bootstrap neighbor-joining method with 1000 replicates to obtain support values for each branch.

2.2. Gene Expression Pattern Analysis

Different tissues and stems at four different development stages of P. lactiflora cv. ‘Hongyan Zhenghui’ [20] were used for extracting total RNA. Then, a HiScript^® III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China) was used to reverse RNA transcribed into cDNA. A qRT-PCR detection system (Bio-Rad CFX96^TM RT PCR Detection System, USA) was used to perform qRT-PCR analysis using a ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The gene-specific primers of PlLAC15 were designed (forward primer: 5′-GCTATTGTCATCTACCCC-3′, reverse primer: 5′-GACCATTGATAAGATACGCA-3′), and the γ-PlTUB used as the internal reference gene (forward primer: 5′-GAAATCGTGGGAGGACAG-3′, reverse primer: 5′-ATGATCGGGTGAAAAGGA-3′) [24]. The relative gene expression was calculated using the 2^−ΔΔCt method [25].

2.3. Subcellular Localization of PlLAC15

The full-length cDNA (without its stop codon) of PlLAC15 was isolated (forward primer: 5′-cggggatcctctagagtcgacATGAAGTTTCCCATCGTCCAAT-3′, reverse primer: 5′-caccatggtactagtgtcgacACACGGTGGCATATCTGGTGG-3′) and inserted into the vector pCAMBIA2300 plasmid to generate p35S::PlLAC15-eGFP constructs. The recombinant plasmid and mCherry, labeled a cell wall localization protein (A. thaliana expansin A1, AtEXPA1, NM179538.1) plasmid, were transferred into GV3101, and then cotransformed into Nicotiana benthamiana leaves. After transformation for 36 h, a Leica TCS SP8 STED confocal laser scanning microscopy system was utilized to detect fluorescence signals, employing excitation wavelengths of 488 nm for eGFP and 561 nm for mCherry, respectively.

2.4. Expression and Purification of Recombinant PlLAC15 Protein and Its Enzyme Assays

The coding region of PlLAC15 was fused to His in the pCold TF vector (forward primer: 5′-gaaggtaggcatatggagctcATGAAGTTTCCCATCGTCCAAT-3′, reverse primer: 5′-gacaagcttgaattcggatccACACGGTGGCATATCTGGTGG-3′), and the fusion construct was then expressed in Escherichia coli (BL21). To enhance the expression of recombinant proteins, the cells were induced with a 0.5 mM isopropyl-1-thio-β-d-galactopyranoside (IPTG). Following the addition of IPTG, the cells were maintained at 16 °C, 150 rpm for 16 h for fusion His-tagged PlLAC15 protein expression. The recombinant proteins were purified by a His-tag Protein Purification Kit (Beyotime Biotechnology, Shanghai, China) in line with the recommendations provided in the manual. The recombinant protein samples combined with protein sample loading buffer were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis after incubating at 100 °C for 5 min. The mixture for the enzyme assays contained 50 mM acetate–sodium acetate buffer, pH 5.0, 1 mM ABTS, and 2 μg of purified protein with a final volume of 1 mL. The activity of the PlLAC15 protein was determined by oxidation of ABTS to produce stable cationic radicals at 25 °C and calculated by measuring the change in wavelength values at 420 nm for 0 min and 1 min.

2.5. Overexpressing PlLAC15 in Tobacco

Through the freeze–thaw method, the pCAMBIA1301-PlLAC15 plasmid (forward primer: 5′-caggtcgactctagaggatccATGAAGTTTCCCATCGTCCAAT-3′, reverse primer: 5′-ttcgagctcagatctggtaccACACGGTGGCATATCTGGTGG-3′) was transformed into the Agrobacterium tumefaciens strain GV3101. Then, it was introduced into tobacco (Nicotiana tabacum ‘k326’) through a leaf disc-based transformation. The identification of transgenic plants was achieved through PCR and qRT-PCR methods with tobacco NtActin (AB158612) (forward primer: 5′-TCCTCATGCAATTCTTCG-3′, reverse primer: 5′-ACCTGCCCATCTGGTAAC-3′) as the reference gene. After being cultured for 3 months, the phenotype of the wild-type (WT) and transgenic tobacco lines were observed, and stem diameter and strength were measured. Additionally, the stems were collected for deposition observation and content measurement of lignin.

2.6. Stem Strength, Lignin Content Determination, and Lignin Deposition Observation

To assess stem strength, a universal NK-2 digital force tester manufactured by Zhejiang Hui’er Instrument and Equipment Co. Ltd. (Zhejiang, China) was employed to quantify the force necessary for causing a kink in the stem when a perpendicular push was applied along its axis. Lignin monomers were quantified through the utilization of gas chromatography-mass spectrometry (GC-MS; Trace1310 ISQ, Thermo, Waltham, MA, USA) based on previous research [26] executed by Qingdao Sci-tech Innovation Inspection Co. Ltd. (Qingdao, China). Lignin deposition was observed through histochemical staining utilizing paraffin sections. After dehydration, transparency, waxing, and embedding, the stem segments fixed with FAA were sliced to a thickness of 5 μm with a rotary microtome (RM2245, Leica, Solms, Germany). Subsequently, some microsections were immersed in 0.05% toluidine blue solution for 2 min for observation of the degree of lignification of the stems. Some microsections were used to observe lignin monomer deposition using the Maüle method. The microsections were first immersed in 0.5% KMnO₄ for 10 min, followed by rinsing with water. Subsequently, they were exposed to 10% HCl for 5 min and rinsed again with water. Finally, the prepared microsections were mounted in concentrated NH₄OH. Finally, the micrographs were collected with an optical microscope (SZX7 and CX31RTSF, Olympus, Tokyo, Japan).

2.7. Statistical Analysis

All data presented the mean values derived from three replicates, along with their respective standard deviations. The data were analyzed for normal distribution and homogeneity of variance before undergoing an analysis of variance (one-way ANOVA). If data were not normally distributed and the variances were heteroscedastic, the Kruskal–Wallis non-parametric test was used [27]. Tukey’s HSD test or Student’s t-test was used for comparisons among treatments (p < 0.05). SPSS29.0 statistical analysis software was used during data processing and analyses (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Isolation and Sequence Analysis of PlLAC15

Based on our previous transcriptome sequencing data of P. lactiflora stems [19], 10 LACs were differentially expressed between the stems of upright ‘Hong Feng’ and bending ‘Xixia Yingxue’ across three flower developmental stages (Figure 1a). Among them, four nonredundant LACs were identified, and one LAC (Gene ID: i2_HQ_PL_c36823) caught our attention. Its expression level changed most dramatically, which was notably higher in the stems of ‘Hong Feng’ than those of ‘Xixia Yingxue’ at each stage, and the FPKM value exhibited a positive correlation with stem strength (Figure 1b).

Moreover, its full-length cDNA was isolated using PCR technology, and PlLAC15 contained an open reading frame (ORF) of 1698 bp, accompanied by a 5′ untranslated region (UTR) of 22 bp, and a 3′ UTR of 70 bp, respectively. The ORF was translated into a putative protein consisting of 565 amino acid residues, which was anticipated to possess a molecular weight of 62,974.72 Da and a theoretical pI value of 7.69. A phylogenetic tree was constructed between PlLAC15 and 17 LACs from A. thaliana, which revealed it was evolutionarily closest to AtLAC15 and designated to PlLAC15 (Figure 1c). Alignment of protein sequences revealed that PlLAC15 had four typical laccase copper ion-binding domains, suggesting that this gene might be functionally conserved (Figure 1d).

3.2. Expression Pattern Analysis of PlLAC15

In order to confirm the role of PlLAC15 in regulating P. lactiflora stem strength, we further quantified its expression level by qRT-PCR. In different tissues of P. lactiflora, PlLAC15 expression was higher in the stem and root, while expression was significantly lower in the flower and leaf (Figure 2a). Furthermore, PlLAC15 expression gradually increased significantly during stem development and expression was nine times higher in P4 than in P1 (Figure 2b).

3.3. Subcellular Localization of PlLAC15

In order to confirm the subcellular localization of PlLAC15, the fluorescence of PlLAC15-eGFP was checked with the cell wall marker protein (A. thaliana expansin A1, AtEXPA1, NM179538.1). PlLAC15-eGFP had a strong fluorescence signal associated with the cell wall, which suggested that PlLAC15 localized specifically to the cell wall (Figure 3).

3.4. Enzyme Assays of the Recombinant PlLAC15

To access the enzymatic activity of PlLAC15, its recombinant protein was produced. Subsequently, the recombinant PlLAC15 was purified (Figure 4a). Compared to the control, the laccase activity of recombinant PlLAC15 was significantly higher after being induced by IPTG (Figure 4b). This result showed that PlLAC15 represented laccase activity.

3.5. Overexpressing PlLAC15 in Tobacco Enhances Stem Strength by Promoting S-Lignin Deposition

To confirm the function of PlLAC15 in regulating stem strength, it was heterologously overexpressed in tobacco (Figure 5a), and the identification of transgenic plants was achieved through PCR and qRT-PCR (Figure 5b,c). Further observation of the related traits of transgenic plants showed that overexpression of PlLAC15 promoted stem straightening and increased stem diameter and stem strength; the increase in stem strength was especially high in transgenic plants, exceeding 50% in comparison with the WT plants (Figure 5d). Subsequently, lignin deposition was observed through histochemical staining utilizing paraffin sections. It was found that the lignification degree of stem xylem fiber cells increased by toluidine blue staining when PlLAC15 was overexpressed (Figure 6a). Further, the change in G-lignin and S-lignin deposition in stem xylem fiber cells was observed by Maüle staining. It was found that overexpression of PlLAC15 resulted in the abundant deposition of S-lignin in stem xylem fiber cells (Figure 6b). In addition, GC-MS was used to detect the contents of S-lignin and G-lignin in stems. The total content of lignin in transgenic stems was significantly higher in comparison with the WT plants. The S-lignin content was significantly increased by 15.39% in transgenic stems, while the G-lignin content had little change (3.8%), which led to an extremely significant rise in the S/G ratio (Figure 7). These results revealed that overexpressing PlLAC15 in tobacco enhanced stem strength by promoting S-lignin deposition.

4. Discussion

LAC is a copper-containing polyphenol oxidase that is present in animals, plants, bacteria, and fungi. Plant LACs belong to a multi-gene family of proteins, and the number of LAC family members in different plants varies greatly. For instance, 17 AtLACs have been characterized in A. thaliana [28]. Similarly, 30 OsLACs have been identified in rice [29], 45 LuLACs in linen [30], 27 SbLACs in sweet sorghum [31], 29 BdLACs in Brachypodium distachyon [32], and 12 SpLACs in sugarcane [17], respectively. LACs serve as the terminal enzymes in lignin biosynthesis, facilitating the polymerization of monolignols into lignin. Research has revealed the positive role of LACs in lignin deposition. For example, overexpression of moso bamboo (Phyllostachys edulis) PeLAC10 or pear PbLAC1 was found to be beneficial in enhancing the lignin content in the stems of transgenic Arabidopsis [18,33]. Conversely, overexpression of sugarcane SofLAC in the Arabidopsis lac17 mutant led to the restoration of lignin content [17]. Although LACs were identified and classified in many species, the role of LACs in lignin deposition has primarily been reported in the model plants and crops [17,18,33]. So far, the only report suggesting a role in enhancing lignin deposition in an ornamental plant has been by Tang et al. [20], who found that PlLAC4 was capable of increasing lignin deposition in P. lactiflora stems. In this study, 10 LACs were differentially expressed between the stems of upright ‘Hong Feng’ and bending ‘Xixia Yingxue’ across three flower developmental stages in P. lactiflora, and four nonredundant LACs were then identified. Among them, PlLAC15 caught our attention as its FPKM value exhibited a positive correlation with stem strength. Xue et al. [34] identified a total of 36 ZjLACs from the Chinese jujube genome, encoding 439–621 amino acids; the molecular weight was 49.09–69.86 kD, the pI was 4.97–9.77, and most of them were hydrophilic proteins. Similarly, PlLAC15 isolated from P. lactiflora encoded a putative protein consisting of 565 amino acid residues, which was anticipated to possess a molecular weight of 62,974.72 Da and a theoretical pI value of 7.69. This result was consistent with ZjLACs from Chinese jujube, and PlLAC15 had four typical laccase copper ion-binding domains, suggesting the PlLAC15 sequence was right and might be functionally conserved.

LAC has a crucial role in various plant development and growth processes [35]. Previous studies have shown that LAC expression and subcellular localization were closely related to its function. For example, in Chinese jujube, ZjLAC14 and ZjLAC15 were localized in the cytoplasm and extracellular, and their expression levels decreased with fruit development, which suggested that they might play a role in flavonoid metabolism [34]. Wang et al. [32] found that BdLAC5 and BdLAC6 were localized in the apoplasm, were mainly expressed in lignified tissues, and were required for the lignification of the B. distachyon stem. Additionally, investigations into expression pattern analysis further verified that a few SmLACs in eggplant might play a role in vegetative and reproductive organs during diverse growth stages and that they may also respond to the influence of either a single stress factor or multiple stress factors [36]. Here, we further quantified the expression level of PlLAC15 by qRT-PCR, to confirm its function in regulating the strength of P. lactiflora stems, and found that it was expressed highly in lignified tissues and demonstrated a marked rise along with stem development, which suggests it might be also be involved in P. lactiflora stem lignification. Lignification is a complex and dynamic process of lignin deposition in the cell wall. Afterward, the subcellular localization of PlLAC15 was observed and was discovered to be localized in the cell wall. This result further confirmed our speculation that PlLAC15 regulated the lignification of P. lactiflora stems. Based on this, the laccase activity of the recombinant PlLAC15 was verified, which laid a foundation for us to study its function.

In plants, LAC has been reported to participate in several biological processes. For example, Zhao et al. [37] found that Eucommia ulmoides EuLAC1 could significantly enhance resistance to gray mold by increasing lignin content. Overexpression of Populus euphratica PeuLAC2 improved drought tolerance by improving water transport capacity [38]. Overexpression of SmLAC25 could decrease salvianolic acid content in Salvia miltiorrhiza [39]. Additionally, Soni et al. [40] found that the protective role of wheat TaLAC4 in NIL-R was attributed to the pathogen-triggered hardening of the secondary cell walls within the rachis, which involved lignification. Moreover, LAC is involved in the formation of secondary cell walls by catalytic monolignol polymerization, but it is selective in this process [41]. For example, overexpression of poplar PtoLAC14 could preferentially catalyze the polymerization of G-lignin monomers [42], and BdLAC5 was also beneficial for the polymerization of G-lignin monomers in B. distachyon [32]. On the contrary, in vitro experiments showed that maize ZmLAC3 and Miscanthus MsLAC1 proteins were more inclined to choose S-lignin monomers as a substrate during polymerization [43,44]; PtrLAC16 also favored the polymerization of S-lignin monomer in Populus [45]. Among three types of lignin monomers, S-lignin significantly contributed to the improvement of stem strength in P. lactiflora [19,22], as further confirmed by other studies. For example, a reduction in the content of wheat S-lignin resulted in a decrease in stem strength under high planting density and nitrogen fertilizer consumption [46]. Overexpression of wheat TaCOMT-3D improved stem strength by promoting S-lignin accumulation in the transgenic lines [47]. These above studies suggested that finding the key LAC members regulating the polymerization of S-lignin monomers was crucial for improving stem strength in plants. Here, PlLAC15 was overexpressed in tobacco, which resulted in a more than 50% increase in stem strength, and a 15.39% increase in S-lignin content. These results suggest that PlLAC15 facilitated S-lignin deposition to enhance stem strength in P. lactiflora, which would lay a foundation for improving the stem straightness in P. lactiflora.

In the future, there are two main avenues of research that could significantly impact the improvement of P. lactiflora stems. On the one hand, conducting excellent haplotype identification of PlLAC15 would enable early screening of superior seedlings, thereby shortening the breeding period of P. lactiflora. On the other hand, exploring other genes involved in lignin biosynthesis within P. lactiflora could provide valuable genetic reserves and a theoretical basis for enhancing the stem strength of this plant.

5. Conclusions

In this study, a differentially expressed PlLAC15 was screened based on our previous transcriptome sequencing data of P. lactiflora stems. The results demonstrated that PlLAC15 expressed highest in the stems compared to other tissues, and its expression trend was positively correlated with stem strength. PlLAC15 was localized specifically to the cell wall and showed laccase activity. Overexpression of PlLAC15 promoted stem straightening and increased stem diameter and stem strength, which accounted for the increased lignification degree of stem xylem fiber cells, especially the increase in S-lignin content. Collectively, the findings suggested a positive role of PlLAC15 in S-lignin deposition and stem strength in P. lactiflora, a finding which would broaden our understanding of regulation in S-lignin deposition in plants and lay the foundation to improve stem strength in the future.