Insights into the Molecular Regulation of Lignin Content in Triploid Poplar Leaves

After polyploidization, plants usually undergo some morphological and physiological changes, including the lignin content of polyploids usually becoming lower than that of diploids. However, the regulatory mechanism of the variation of lignin content in polyploid plants remains unclear. Therefore, in this research, we used full-sib poplar triploids and diploids to explore the molecular regulatory basis of lignin content in poplar triploid leaves through the determination of lignin content, the observation of xylem cells, and transcriptome sequencing. The results showed that the lignin content of triploid leaves was significantly lower than that of diploid leaves. The xylem cells of triploid leaves were significantly larger than those of diploids. Transcriptome sequencing data show that most lignin biosynthesis genes were significantly downregulated, and genes related to cell growth were mostly upregulated in triploid leaves compared with diploid leaves. In addition, co-expression network analysis showed that several transcription factors might be involved in the regulation of lignin biosynthesis. Consequently, the altered expression of genes related to lignin might lead to the reduced lignin content in triploids. These results provide a theoretical basis for further exploring the molecular mechanism of the variation of polyploid lignin content and the utilization of polyploid lignocellulosic resources.


Introduction
Lignin is a major component of the secondary cell wall that is deposited when cell differentiation is completed and the secondary cell wall starts thickening [1]. Lignin is also the second most abundant plant lignocellulosic material in nature [2]. Lignin plays an important role in mechanical support, water transportation, and stress defense during plant growth and development [3]. However, it interweaves with cellulose and hemicellulose in the secondary cell wall, which seriously affects the depolymerization and utilization of plant lignocellulosic resources [4]. The high lignin content in plants is the main restrictive factor for lignocellulosic bioenergy production, pulp and paper making, and forage digestion [4][5][6]. Therefore, plants with properly low lignin content can better meet the needs for human social production practices. Plant polyploidy, accompanied by an increase in chromosome numbers, usually brings about some morphological and physiological changes through affecting the various physiological activities and metabolic processes of plants [7,8]. For example, plant polyploidy usually improves the growth rate, wood quality, and stress resistance [4,[9][10][11]. Studies have shown that the cell-wall components of polyploid plants tend to be altered. In Populus tomentosa, compared with diploids, triploids were characterized by longer and wider fiber cells, lower lignin content, which decreased by 17.9%, and higher cellulose content; therefore, they were easier to cook and soften in pulp and paper industrial processes, and energy consumption was reduced [12]. In triploid shrub willows, the lignin content significantly decreased, and the lignin monomer composition also changed [13]. Compared with Arabidopsis diploids, the lignin contents of tetraploids, hexaploids, and octaploids were decreased by 20%, 50%, and 55%, respectively [14]. A similar phenomenon was found for polyploid crops such as rice and potatoes; the lignin content of polyploids was significantly lower than that of diploids [15,16].
With the development of high-throughput sequencing technology, an increasing number of studies have led to in-depth discussions about the gene expression and regulatory mechanisms of polyploid plants. For example, in poplar triploids, the expression of genes related to vegetative growth has a dose effect, which enhances the capacities for photosynthesis, carbon fixation, and sucrose and starch metabolism, accompanied by a higher chlorophyll content and lower chlorophyll degradation, delaying leaf senescence [17]. Due to the gene dosage effect of polyploidy, some plant physiological and biochemical processes are strengthened along with more vigorous metabolism; consequently, the contents of biochemical components are also increased. However, in contrast to the higher photosynthesis, carbon fixation, and sucrose and starch metabolism, lignin content was lower in poplar triploids than in diploids. Why does the lignin content decrease in triploids? The mechanism behind the variation remains unclear. The analysis of the regulation in leaf lignin biosynthesis is beneficial to the development and utilization of polyploid-based leaf bioenergy. The existing studies on stems could not completely characterize the mechanism of lignin biosynthesis in leaves. The development and utilization of leaf-based biomass energy or important secondary products in polyploids still requires a more accurate regulatory mechanism model. Therefore, in this study, we used full-sib poplar triploids and diploids as study materials to explore the molecular mechanism of the regulation of lignin content in poplar triploids by determining the lignin content, observing xylem cells and performing transcription sequencing analysis. These results provide a theoretical basis for further exploring the molecular mechanism of lignin trait variation in other polyploid plants or tissues, e.g., woody tissues, and the utilization of polyploid lignocellulosic resources. Moreover, it can also enrich the theories on the molecular mechanisms behind the formation of advantageous traits of poplar triploids.

Analysis of Lignin Content in Triploid and Diploid Leaves
The fifth leaf was in the growth stage, with its net photosynthetic rate and leaf area increasing. However, the net photosynthetic rate and leaf area peaked at the tenth leaf, meaning that the cell growth and leaf function tended to be stable [18]. In this study, juvenile and mature leaves represented the fifth and tenth leaves, respectively. In order to compare the lignin contents of different ploidy plants, the fifth leaves of triploids and diploids were used to determine the lignin content. The results show that the lignin content in triploid leaves was significantly lower than that in diploid leaves ( Figure 1A). Lignin is mainly deposited in specific vascular tissue cells. Thus, we further observed the xylem cell microstructure of the main veins by histochemical staining. The results show that the xylem vessels of triploid leaves were significantly larger than those of diploid leaves ( Figure 1B  Values are the means ± SE of three independent experiments. The asterisk indicates significant differences (* p < 0.05, ** p < 0.01). Bar = 50/20 µm.

Analysis of Transcriptome Sequencing Data
To analyze the gene expression differences between triploid and diploid poplar leaves, transcriptome sequencing was performed on juvenile and mature leaves of different ploidy plants. A total of 12 cDNA libraries were constructed, including three biological replicates. In total, 90.73%, 90.43%, 87.50%, and 89.27% of the average reads were matched to genomic locations, and uniquely mapped reads accounted for 84.90%, 84.13%, 82.13%, and 84.07% in the Dip_JL, Tri_JL, Dip_ML, and Tri_ML libraries, respectively (Table S2).
To assess the reliability of the tested samples, principal component analysis (PCA) was performed ( Figure 2A). The results show that there was a high degree of similarity among the biological replicates of each sample, and that different ploidy plants and different leaves had different gene expression patterns, indicating that the sequencing data were relatively reliable and suitable for further analysis. In this study, the screening threshold for differentially expressed genes (DEGs) was |FC| ≥ 2 and FDR < 0.05. Compared with diploids, there were 743 DEGs, including 320 upregulated and 423 downregulated genes in juvenile leaves ( Figure 2B). A total of 1886 DEGs were identified in mature leaves; 1012 were upregulated, and 874 were downregulated ( Figure 2B).

Functional Enrichment Analysis of DEGs
To characterize the biological roles of DEGs, Gene Ontology (GO) enrichment analysis was performed at three levels: biological process (BP), molecular function (MF), and cellular component (CC) (Figure 3A,B; Table S3). The major GO items are shown in Figure 3A,B (FDR < 0.05). In juvenile leaves, the DEGs were significantly enriched for 18 BP items, mainly including "metabolic process", "oxidation-reduction process", and "flavonoid biosynthesis process". The DEGs were significantly enriched for 45 MF items, the most significant being "transferase activity", "oxidation-reduction enzyme activity", and "catalytic activity". Four items were enriched for CC items, among which "extracellular region" and "cell wall" were the most significant. In mature leaves, the DEGs were significantly enriched for 53 BP items, primarily including "metabolic process", "defense stress response", "heat response", and "protein folding". A total of 15 MF items were enriched, the most significant being "transferase activity".  Table S3). All the significantly enriched KEGG pathways (p < 0.05) are shown in Figure 3C-F. In juvenile leaves, the upregulated genes were mainly enriched for "biosynthesis of secondary metabolites", "flavonoid biosynthesis", and "circadian rhythmplant" ( Figure 3C). The downregulated genes were mainly enriched for "the biosynthesis of secondary metabolites", "protein processing in endoplasmic reticulum", and "phenylpropanoid biosynthesis" ( Figure 3D). In mature leaves, the pathways of "protein processing in endoplasmic reticulum", "glutathione metabolism", and "plant-pathogen interaction" were mainly enriched in upregulated genes ( Figure 3E). The pathways of "biosynthesis of secondary metabolites", "sesquiterpenoid and triterpenoid biosynthesis", and "phenylpropanoid biosynthesis" were mainly enriched in differentially downregulated genes ( Figure 3F). Noteworthily, the downregulated genes were all significantly enriched in phenylpropanoid biosynthesis pathways in both leaves.
In conclusion, the enrichment results show that the DEGs in juvenile triploid leaves were mainly involved in the processes of cell growth, such as metabolite synthesis, physiological enzyme activities, and cell-wall activities. However, mature leaves gradually participated in the processes of various environmental interactions such as stimulus responses and defense stresses.

Profiling of Differentially Expressed Lignin Biosynthesis Genes
Lignin is synthesized through the phenylpropanoid biosynthesis pathway, with the participation of many enzymes and genes. The lignin biosynthesis genes are mainly expressed during secondary cell-wall biosynthesis and eventually lead to lignin deposition in the cell wall [1]. In our current study, cell growth was relatively stable in mature leaves, while cell growth, including cell-wall activities, was ongoing in juvenile leaves.
Compared with diploids, a total of eight and 11 DEGs related to lignin biosynthesis were observed in triploid juvenile and mature leaves, respectively ( Figure 4; Table S4). Among them, there were five common genes differentially expressed in both leaves. PAL (phenylalanine ammonia lyase) is the first rate-limiting enzyme in this pathway. Two PAL1 genes were upregulated in mature triploid leaves compared with diploid leaves. A key enzyme that catalyzes the formation of CoA esters is 4CL (4-coumaroyl: CoA ligase). Two 4CL2 genes were downregulated in both triploid leaves compared with diploid leaves. HCT (hydroxycinnamoyl-CoA shikimate) is also a key enzyme in lignin biosynthesis. In triploids, there was one HCT gene downregulated in juvenile leaves and two HCTs upregulated in mature leaves compared with diploids. F5H (ferulic acid 5-hydroxylase) is necessary for lignin monomer synthesis. FAH1 was downregulated in both triploid leaves compared with diploids. In addition, PODs (peroxidases) play a role in the oxidative polymerization of lignin monomers at the end of this pathway. Compared with diploids, there were four PODs differentially expressed, three of which were downregulated in juvenile triploid leaves. There were also four PODs differentially expressed in mature triploid leaves, two of which were downregulated. A shared POD (Potri.006G129900) was downregulated in both triploid leaves. These results indicate that most lignin biosynthesis genes were downregulated in triploids, especially in juvenile leaves, which might be related to the decrease in lignin content.

Profiling of DEGs Related to Cell Growth
In our research, we found that the xylem cells in triploid leaves were significantly larger than those in diploid leaves. Therefore, we further analyzed the expression pattern of genes related to cell growth, and some DEGs between triploid and diploid leaves were found ( Figure 5A; Table S4).
XTHs (xyloglucan endotransglucosylase/hydrolases) are key genes related to cell-wall remodeling. In this study, a total of five XTHs were significantly differentially expressed in triploids compared with diploids, most of which were upregulated. Two XTH9 genes were upregulated in juvenile leaves, whereas XTH15 and XTH30 were upregulated in mature leaves. Expansins are also important regulators involved in cell expansion. In this study, most genes encoding expansins were not differentially expressed, but EXPA15 was differentially downregulated in mature triploid leaves compared with diploid. Phytohormones are also important for cell expansion during plant growth and development. In this study, we found that some auxin-related genes were differentially expressed between triploids and diploids. Genes associated with auxin transportation were mostly upregulated in triploid leaves compared with diploid leaves, such as PISL, LAX2, and PDR9. Three auxin signal transduction inhibitors AUX2-11 were downregulated in both triploid leaves compared with diploid leaves. Genes related to auxin responses, such as ARF2, IAA11, and ILL6, also showed higher expression in mature triploid leaves than in diploid leaves. In addition, as the other main cell-wall components, cellulose-and hemicelluloserelated genes were also analyzed in this study. The results show that only SUS6 (sucrose synthase 6) was differentially downregulated in the cellulose biosynthesis of triploid leaves compared with diploids, while the CESAs (cellulose synthase), which were crucial for cellulose biosynthesis, were not differentially expressed. Only DUF57 and CSCL6 (cellulose synthase-like C6) were differentially downregulated in the hemicellulose biosynthesis of triploid mature leaves compared with diploid, while IRXs (irregular xylem), which are important for xylan synthesis, were not differentially expressed.
Consequently, XTHs and auxin-related genes might be the major regulators to promote the cell growth in triploid leaves compared with diploids.

Profiling of DEGs Related to Plant Stress Resistance
Plant polyploidy usually results in stronger resistance to various stresses compared to diploidy. Lignin accumulation is an effective pathway for plant stress resistance. However, in our study, triploids had a lower lignin content than diploids; thus, we speculated that there may be some other pathways for plant stress resistance in triploids.
We found that a few antioxidant enzyme genes associated with stress resistance were differentially expressed ( Figure 5B; Table S4). APX2 is a gene encoding ascorbate peroxidase, which was upregulated in triploids compared with diploids. There were eight GSTs related to glutathione metabolism that were differentially expressed in juvenile triploid leaves, and half of them were upregulated. In mature triploid leaves, 20 GSTs were differentially expressed, and 18 GSTs were upregulated, indicating that mature leaves were more active in response to stress defense than juvenile leaves.
In addition, we also found that some DEGs involved in flavonoid biosynthesis shared the phenylpropanoid metabolic pathway with lignin biosynthesis, and most of them had higher expression in triploids than in diploids (Figure 4; Table S4). Flavonoids play an important role in defending against stresses and promoting growth and development in plants. CHS (chalcone synthase) is a key enzyme involved in the biosynthesis of flavonoids. In this study, there were three CHSs and one CHS gene that were upregulated in juvenile and mature leaves, respectively. F3H encodes flavanone 3-hydroxylase and regulates flavonoid biosynthesis. There were three F3Hs gene differentially expressed in triploids compared with diploids, one of which was upregulated in juvenile leaves and two of which were upregulated in mature leaves. F3 H encodes flavonoid 3 hydroxylase, related to flavonoid biosynthesis. Compared with diploids, seven F3 H and two F3 H genes were differentially expressed in juvenile and mature triploid leaves, respectively, and most of the F3 Hs were downregulated in juvenile triploid leaves. FLS1 encodes a flavonol synthase involved in the synthesis of flavonols, which was differentially upregulated in both triploid leaves. LDOX encodes a leucoanthocyanidin dioxygenase involved in proanthocyanin biosynthesis, which was upregulated in both triploid leaves. DFR encodes dihydroflavonol reductase, involved in the biosynthesis of anthocyanins. There were three DFRs differentially expressed in mature triploid leaves, two of which were upregulated.
These results show that some other DEGs related to stress resistance were mostly upregulated in triploids compared with diploids, indicating that triploids may gain stronger resistance through other pathways.

Analysis of Differentially Expressed Transcription Factors
Transcription factors (TFs) play crucial roles in regulating lignin biosynthesis. In this study, 38 differentially expressed TFs were identified in juvenile leaves, composed of 17 TF families. A total of 122 differentially expressed TFs involving 28 TF families were identified in mature leaves. The overall distribution patterns of the TFs are shown in Figure 6, most of which belonged to TF families such as MYB, ERF, NAC, and bHLH ( Figure 6A; Table S5).
To identify TFs significantly associated with lignin synthesis, we constructed a coexpression network using DEGs related to lignin including 15 lignin biosynthesis structural genes, nine genes related to cell growth, 11 genes related to stress resistance, and all the differentially expressed TFs ( Figure 6B; Table S5). The screening threshold for high correlation was r > 0.85 and p < 0.05. The larger nodes have stronger connectivity degrees, indicating that the genes may be more important. In this co-expression network, we identified 162 pairs correlated between 14 lignin structural genes and 53 TFs. Among the lignin structural genes, HCT (Potri.001G042900), 4CL2 (Potri.003G188500), FAH1 (Potri.007G016400), and POD (Potri.006G129900) directly connected with the greatest numbers of TFs, all downregulated in triploids compared with diploids. We further analyzed TFs directly linked to lignin structural genes in the network, and the results show that 50 TFs came from 20 families, among which the MYB family had the largest number, with 14 TFs. In addition, the network showed that TFs with high degrees of connectivity were mainly involved in the processes of phenylpropanoid biosynthesis, cell growth, leaf development, and stress response, and they were mainly composed of MYB and bHLH family members (Table S5). Among them, MYB63 is a transcriptional activator of lignin biosynthesis. MYB63 connected with the largest numbers of lignin structural genes, and its expression was lower in mature triploid leaves than in diploid leaves. We also found that MYB115, associated with proanthocyanidin biosynthesis, was upregulated in both juvenile and mature triploid leaves compared with diploid leaves, which may play an important role in the regulation of flavonoids synthesis. MYC4, a bHLH transcription factor, could participate in the regulation of secondary cell-wall synthesis induced by blue light [19]. It was differentially upregulated in triploids with low expression and may work under the induction of blue light. MYB4, a repressor involved in the phenylpropanoid pathway, was differentially downregulated in mature triploid leaves compared with diploids. Other TFs have not yet been reported to be involved in lignin synthesis; whether they can directly or indirectly participate in lignin synthesis remains to be studied in the future.

Expression Analysis by qRT-PCR
To verify the reliability of the transcriptome sequencing data, 10 DEGs related to lignin were chosen for real-time fluorescence quantification (Figure 7 and Table S1). These selected genes included structural genes and TFs. The results of the qRT-PCR show that the expression patterns of these genes were similar to the transcriptome sequencing data, indicating that the transcriptome sequencing results were reliable.

Discussion
Lignin biosynthesis is a very complex process, which begins with phenylalanine entering the phenylpropanoid metabolic pathway, followed by the lignin-specific synthesis pathway. Finally, three lignin monomers are oxidatively polymerized to three types of lignin by peroxidases in the cell wall [2,20]. In this study, the cell growth of mature leaves was relatively stable, while the cells of juvenile leaves were growing and constructing a cell wall. Thus, it is more important to analyze the expression of lignin biosynthesis genes in juvenile leaves.
The phenylpropanoid metabolic pathway mainly involves the PAL, C4H, and 4CL genes. PAL is the first rate-limiting enzyme-encoding gene located at the beginning of this pathway and determines the metabolic flow of the entire pathway. As the last key gene, 4CL regulates the formation of coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA, which determines the synthesis of lignin precursors. According to the results of this study, two 4CL2 genes were significantly downregulated in both triploid leaves compared with diploid leaves. Two PAL1 genes showed no significant difference in juvenile triploid leaves, although they were significantly upregulated in mature triploid leaves compared with diploid leaves. We know that PAL is upstream of 4CL, and that its expression also responds to various outside stresses and plant hormone regulation [21]. Studies have shown that, when genes regulating lignin synthesis change, some other chemical components can also change correspondingly, aiming to ensure the normal physiological metabolism, growth, and development of plants [22,23]. In this study, we found that some flavonoid synthesis genes such as CHS, F3H, and FLS were significantly upregulated in mature triploid leaves. Therefore, we speculated that, during leaf growth and development, PAL1, an upstream pathway gene, may lead to more precursors flowing to the branch of flavonoid synthesis, compensating for the reduced lignin accumulation, which might enhance the stress resistance and growth of polyploids. As the final regulatory gene, 4CL2 is widely involved in the synthesis of lignin precursors. Its expression was more in line with the actual lignin synthesis in triploid leaves. Thus, the results indicate that the downregulated expression of key genes in the phenylpropanoid metabolic pathway was one of the reasons leading to the decrease in the lignin content in triploid leaves.
Genes such as C3H, HCT, F5H, COMT, and POD are the main regulators of the lignin-specific pathway [24][25][26][27]. Among them, HCT is key for the synthesis of G and S monomers. F5H is necessary for S monomer synthesis. POD determines the final oxidative polymerization of monolignols. According to the results of this study, DEGs involved in the lignin-specific pathway were most significantly downregulated in triploids compared with diploids. Among them, HCT was downregulated in juvenile triploid leaves. FAH1 was downregulated in both triploid leaves. A total of four PODs were differentially expressed in juvenile triploid leaves, three of which were significantly downregulated. Consequently, these results also indicate that the downregulation of the expression of key genes in this pathway might result in the reduction in lignin content in triploid leaves during leaf growth and development.
Lignin synthesis not only requires the cooperative regulation of structural genes, but also requires the regulation of TFs. Previous studies have shown that a multilevel regulatory network mainly composed of NAC and MYB TFs can regulate lignin biosynthesis [28]. NACs (VND1-7 and NST1-3) are primary switches that regulate secondary cell-wall synthesis. MYB46 and MYB83 are secondary switches that can regulate downstream TFs, regulating the expression of secondary cell-wall synthesis genes [28,29]. Among the downstream TFs, MYB58, MYB63, and MYB85 can specifically bind to and activate the expression of lignin structural genes [30], whereas MYB4, MYB7, and MYB32 are transcriptional inhibitors of lignin synthesis [3]. In this study, the co-expression network between related structural genes and TFs showed that TFs with high degrees of connectivity were mainly MYBs. As an activator of lignin synthesis, MYB63 was differentially downregulated in triploid leaves compared with diploids, which may lead to decrease lignin accumulation. MYB4, a repressor of phenylpropanoid biosynthesis, has been suggested to repress not only the synthesis of monolignols, but also the synthesis of flavonoids [3,31]. In our study, MYB4 was downregulated in the mature leaves, which may decrease the inhibition of phenylpropanoid pathway and possibly participate in flavonoid synthesis. However, during leaf growth and development, most MYBs have no significant differences, MYB115 was significantly upregulated in juvenile triploid leaves compared with diploid leaves. MYB115 shows high homology in amino acid sequence to Arabidopsis MYB5, promoting proanthocyanin biosynthesis [32]. Previous studies have shown that certain MYB transcription factors can regulate both flavonoid metabolism and secondary cell-wall formation, such as MYB5-like transcription factors [33][34][35]. VvMYB5a and VvMYB5b from grapevine positively regulate proanthocyanin and anthocyanin biosynthesis, but negatively affect lignin metabolism [34]. In Populus tomentosa, MYB6, the homolog of MYB5-like transcription factors, can promote proanthocyanin and anthocyanin biosynthesis and inhibit secondary cell-wall biosynthesis by interacting with KNAT7, regulating multiple branches of the phenylpropanoid pathway [35]. In our study, we found that most lignin biosynthesis genes were negatively correlated with flavonoid biosynthesis genes. MYB115, as a positive activator of proanthocyanin synthesis, might also participate in the regulation of lignin synthesis. However, further studies are needed to verify whether MYB115 has a regulatory effect on lignin synthesis.
In this study, our results showed that most genes involved in the regulation of lignin biosynthesis were downregulated during leaf growth and development, leading to a decrease in lignin content in triploid leaves. However, what causes the downregulation of the expression of these genes in triploids? It is known that plant polyploidy can result in cellular giganticity, and poplar triploids are no exception [8,17,36,37]. Zhang [38] showed that phytohormones that including auxin can participate in cell growth, modulating the expression of genes related to cell expansion, and promoting the formation of huge cells. In the study, we analyzed genes related to cell growth, such as XTHs, expansins, and plant hormone-related genes, and the results showed that XTHs and auxin-related genes may play a major regulatory role in triploid leaves. Their expressions were mostly upregulated, which may lead to the cell growth in triploid leaves. In addition, from the results of this study, most cellulose and hemicellulose biosynthesis genes had no significant difference between triploid leaves and diploids, unlike lignin biosynthesis genes. Only several related genes were differentially downregulated in triploid leaves, indicating that there may be not significant changes or downregulation of chemical component contents between triploid and diploid leaves. However, this needs to be further studied.
From the results of our study, we suppose that polyploids have larger cells, usually accompanied by decreased cell-surface areas per unit volume, which possibly decrease the demand for lignin accumulation. Lignin content is usually reduced in multiple polyploid plants, such as poplar in this study, shrub willow, Arabidopsis, rice, and potato [12][13][14][15][16]. In contrast, the cellulose and hemicellulose contents of polyploids showed different trends in different species. For example, the cellulose content increased in triploid shrub willow [13], while it decreased in polyploid Arabidopsis, tetraploid rice, and tetraploid potato [14][15][16]. These results indicated that triploids may have a certain mechanism of regulating lignin biosynthesis to adapt their own demands, which are usually achieved by decreasing the expression of lignin structural genes and changing the expression of transcription factors, together regulating lignin biosynthesis. With an increase in chromosome numbers, how do triploids regulate the expression of lignin synthesis genes to adapt to the altered lignin demand due to cell enlargement? In the future, more in-depth and comprehensive studies need to be conducted to explore the molecular regulation mechanism of lignin trait variation in polyploids, such as focusing on woody tissues and studying at the post-transcriptional level. This research provides an important theoretical basis for future studies.

Plant Materials
A synthetic poplar allotriploid (2n = 3x = 57) and diploid (2n = 2x = 38) were used as plant materials in this study, which were full-sib progeny induced by Populus pseudosimonii × P. nigra 'Zheyin3#' and P. × beijingensis hybridization [39]. All the materials were planted under 16 h/8 h (day/night) conditions and a relative humidity of 45-70% in the greenhouse of Beijing Forestry University (Beijing, China). The fifth and tenth leaves from the top were collected at the same time when they were 3 months old. Three clones for each ploidy were randomly selected as three biological replicates. After sampling, the materials were immediately frozen in liquid nitrogen and then stored at −80 • C for transcriptome sequencing and other experiments.

Determination of Lignin Content
The lignin content of leaves was determined by ultraviolet spectrophotometry, according to the instructions of a lignin content determination kit (COMINBIO, Suzhou, China). Samples were collected and dried at 105 • C. The dried samples were ground to a fine powder and used to prepare alcohol insoluble residues (AIRs) according to Foster [40]. The pretreated AIRs were used to determine lignin content according to the instructions. Firstly, 5 mg of AIRs were weighed into test tubes, leaving one tube empty for a blank. Then, 1 mL of acetyl bromide solution (25% v/v acetyl bromide in glacial acetic acid) and 40 µL of perchloric acid were gently added, fully mixed, and heated at 80 • C for 40 min with vortexing every 10 min. After cooling to room temperature, 1 mL of a solution of sodium hydroxide and glacial acetic acid was added to terminate the reaction. Finally, after centrifuging, 40 µL of supernatant was added to 1.96 mL of glacial acetic acid for dilution, and 1 mL of liquid was added to a quartz cuvette to determine absorbance at 280 nm using an ultraviolet spectrophotometer. The lignin content was calculated on the basis of the absorbance at 280 nm, and three biological replicates and three technical replicates were performed.

Histochemical Staining
The leaves, including main veins, were fixed in FAA fixative solution for more than 24 h and used for paraffin sectioning. Firstly, the samples were dehydrated through a gradient of ethanol solutions (70%, 80%, and 100%; 1 h each), followed by ethanoldimethylbenzene (1:1, v/v, 30 min) and dimethylbenzene solutions (30 min). Secondly, each sample was transferred to mixtures of paraffin, dimethylbenzene (1:1, v/v, 60 • C for 6 h), and pure paraffin three times (60 • C for 2 h each time), after which each sample was embedded in a paper cup. Thirdly, the paraffin block was trimmed to a suitable size and sectioned (8 µm) using a paraffin-slicing machine. Lastly, after dewaxing, the paraffin section was stained using 1% safranin O and 0.1% fast green. All the materials were observed and photographed under an Olympus BX51 microscope. The cell areas were measured using ImageJ, and data statistical analysis was conducted using SPSS 20.0.

Transcriptome Sequencing and Mapping
Total RNA was extracted from the samples using TRIzol Reagent Kits (Invitrogen, Carlsbad, CA, USA), followed by using a NanoDrop 2000 bioanalyzer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) to determine the quality. The RNA integrity was verified using 1.5% agarose gels and then was used to construct cDNA libraries using an Ion Total RNA-Seq kit v2. Transcriptome sequencing was performed on the Ion Proton platform (Life Technologies, Carlsbad, CA, USA) by Shanghai Novelbio Biological Technology Co. Ltd., Shanghai, China. The adapter sequences were removed from the raw data. Lowquality reads and reads shorter than 50 bp were filtered out to obtain clean reads using Fast QC [41]. Then, the clean reads were mapped to the genome of Populus trichocarpa using MapSplice [42].

Identification of Differentially Expressed Genes and Functional Analysis
The transcript abundance was calculated in reads per kilobase per million mapped reads (RPKM) [43]. Differential expression analysis was performed using DESeq [44]. Genes were deemed to be differentially expressed when they had a fold change (FC) ≥2 and false discovery rate (FDR) <0.05. Differentially expressed genes (DEGs) were annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The GO enrichment analysis was performed using agriGO (http://bioinfo.cau.edu. cn/agriGO/) (accessed on 10 April 2022) [45]. GO terms with FDR <0.05 were considered significantly enriched terms. DEGs were mapped to the KEGG database (http://www. kegg.jp/) (accessed on 10 April 2022) to identify signaling and metabolic pathways [46]. Pathways were considered to be significantly enriched when p < 0.05.

Co-Expression Network Analysis
The RPKM of the genes was used for co-expression analysis. The gene correlation was calculated in terms of the Spearman correlation coefficient using the R package. The screening threshold was |r| ≥ 0.85 and p < 0.05. A positive value indicated a positive correlation, and a negative value indicated a negative correlation. The co-expression network diagram was visualized using the software Cytoscape 3.9.0, and the connectivity degree of genes was calculated using the same software. The node size was positively correlated with the degree of the connectivity of the genes.

Quantitative Real-Time PCR
Firstly, the total RNA was extracted using RNeasy Plant Mini Kits (Qiagen China, Shanghai, China). Then, cDNA was synthesized using FastQuant RT Kit (with gDNase) (Tiangen Biotech CO., LTD, Beijing, China). The qRT-PCR was accomplished with a SuperReal PreMix Plus (SYBR Green) kit (Tiangen Biotech) according to the manufacturer's recommendations using an Applied Biosystems 7500 Fast Instrument (AB Ltd., Lincoln, NE, USA). Three technical replicates and three biological replicates were used for each gene. The relative expression of the selected genes was calculated using the 2 −∆∆CT method. The sequences of the primers were designed using Primer3Plus (http://www.primer3plus. com/) (accessed on 10 April 2022). All the primer sequences used in this study are listed in Table S1. Actin (accession number: EF145577) was chosen as the reference gene.

Conclusions
In summary, our results show that the lignin content in triploid poplar leaves was significantly lower than that in diploid leaves. In the process of triploid lignin biosynthesis, most DEGs were significantly downregulated, which may lead to a decrease in lignin content. At the same time, transcription factors may play an important regulatory role, together resulting in the reduced lignin content. The change in the expression pattern of lignin-related genes in triploids may be a type of adjustment and adaptation to the decreased demand for lignin accumulation.