PdWND3A, a wood-associated NAC domain-containing protein, affects lignin biosynthesis and composition in Populus
- 301 Downloads
Plant secondary cell wall is a renewable feedstock for biofuels and biomaterials production. Arabidopsis VASCULAR-RELATED NAC DOMAIN (VND) has been demonstrated to be a key transcription factor regulating secondary cell wall biosynthesis. However, less is known about its role in the woody species.
Here we report the functional characterization of Populus deltoides WOOD-ASSOCIATED NAC DOMAIN protein 3 (PdWND3A), a sequence homolog of Arabidopsis VND4 and VND5 that are members of transcription factor networks regulating secondary cell wall biosynthesis. PdWND3A was expressed at higher level in the xylem than in other tissues. The stem tissues of transgenic P. deltoides overexpressing PdWND3A (OXPdWND3A) contained more vessel cells than that of wild-type plants. Furthermore, lignin content and lignin monomer syringyl and guaiacyl (S/G) ratio were higher in OXPdWND3A transgenic plants than in wild-type plants. Consistent with these observations, the expression of FERULATE 5-HYDROXYLASE1 (F5H1), encoding an enzyme involved in the biosynthesis of sinapyl alcohol (S unit monolignol), was elevated in OXPdWND3A transgenic plants. Saccharification analysis indicated that the rate of sugar release was reduced in the transgenic plants. In addition, OXPdWND3A transgenic plants produced lower amounts of biomass than wild-type plants.
PdWND3A affects lignin biosynthesis and composition and negatively impacts sugar release and biomass production.
KeywordsF5H Lignin Populus Saccharification Sinapyl alcohol S/G ratio VND
Cinnamoyl alcohol dehydrogenase
Cinnamoyl CoA reductase
V-myb myeloblastosis viral oncogene homolog
No Apical Meristem (NAM), Arabidopsis Transcriptional Activation Factor (ATAF1/2), Cup-shaped Cotyledon (CUC2)
NAC secondary wall thickening promoting factor1
Populus deltoides transgenic plants overexpressing PdWND3A
Populus deltoides wood-associated NAC domain protein 3A; Potri.015G127400
Populus deltoides wood-associated NAC domain protein 3B; Potri.012G126500
Secondary wall NAC binding element
Secondary wall-associated NAC domanin protein1
Vascular-related NAC domain
Plant cell walls define cellular space and protect internal cellular component against extracellular biotic and abiotic stimuli. In addition to the structural roles, plant cell walls have become an attractive target for conversion into biofuels and biomaterials due to their abundance, alternate chemical composition properties, and renewability. Plant cell walls are generally composed of two types of walls, i.e., the primary cell wall and secondary cell wall. The primary cell wall typically consists of cellulose, hemicellulose and pectin whereas the secondary cell wall contains a larger proportion of lignin [22, 56]. Of these secondary cell wall components, cellulose and hemicellulose are polysaccharides and are being considered as substrates for conversion into biofuels [4, 10, 26]. Lignin as a polyphenolic biopolymer contributes to cell rigidity and protection against pathogens . In addition, lignin facilitates hydrophilic transport by coating the interior of vessels which helps regulate water relations in the plant. However, from the perspective of biofuels production lignin is regarded as a major recalcitrance factor limiting access to cell wall polysaccharides. Therefore, genetic modification of lignin biosynthesis pathway has become an effective approach for reducing recalcitrance and improving biofuel conversion and production.
Phenylpropanoids, derived from phenylalanine, are the pivotal metabolic precursors to monolignol synthesize [16, 29, 45]. The general phenylpropanoid pathway includes three initial steps that are catalyzed by L-phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL) [15, 35, 37]. 4-coumaryl-CoA is the final product of general phenylpropanoid pathway and is the precursor chemical for synthesizing three different chemical families, i.e., flavonoids, monolignols, and phenolic acids. The lignin biosynthetic pathway has been well characterized and most biosynthetic enzymes have been identified [2, 29, 44]. Lignin is composed of three monomers known as hydroxyphenyl (H), guaiacyl (G) and syringyl (S) that are derived from p-coumaryl, coniferyl and sinapyl alcohols, respectively, and whose productions are regulated by caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamoyl CoA reductase (CCR), and cinnamoyl alcohol dehydrogenase (CAD) [12, 14, 25, 29, 31, 52]. The regulation and expression of lignin biosynthetic genes is associated with several transcription factors, including NAC (No Apical Meristem (NAM), Arabidopsis Transcriptional Activation Factor (ATAF1/2), Cup-shaped Cotyledon (CUC2)), and V-myb myeloblastosis viral oncogene homolog (MYB) [11, 16, 56]. Of these transcription factors, NAC family proteins function as the master switch regulator of secondary cell wall formation. Kubo et al.  suggested that the NAC transcription factors of VASCULAR-RELATED NAC-DOMAIN (VND) 1–7 subfamily act as master regulators of meta and proto xylem vessel formation in Arabidopsis root. NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and NST3/SECONDARY WALL-ASSOCIATED NAC DOMANIN PROTEIN1 (SND1) have also been shown to act as master transcriptional regulators of secondary cell wall formation and fiber cell differentiation [13, 20, 21, 23, 41, 47]. SND1 has been reported to bind directly to the promoter of MYB46 . SND1 also acts as a switch to regulate the expression of many downstream genes related to the secondary cell wall biosynthesis including cellulose and lignin biosynthesis. In Arabidopsis, the intricate network of transcriptional regulation of secondary cell wall biosynthesis has been summarized in several recent review articles [16, 22, 49, 55, 56].
As NAC family members, Arabidopsis VND 1–7 (AtVND1–7) were initially identified in the early stage of xylem vessel cell trans-differentiation using Arabidopsis suspension cultures . The transgenic Arabidopsis overexpressing AtVND1–7 resulted in ectopic formation of xylem vessel element [6, 13, 39, 58]. The comparative transcriptome analysis of inducible expression of AtVND6 and AtSND1 in transgenic Arabidopsis system showed that the upregulated genes by AtVND6 were overlapped with those genes by AtSND1 . However, there were also genes that were preferentially regulated by AtVND6 or AtSND1 . Furthermore, a total of 63 genes encoding a broad range of proteins, including both transcription factors and non-transcription factors involved in the programmed cell death were identified as target genes of AtVND7 in an overexpression study . Therefore, AtVNDs share with AtSND a common set of downstream target genes but also regulate the expression of target genes that are distinct from those regulated by AtSND. Electrophoretic mobility shift assay of AtVND1–7 and transactivation analysis of AtVND6 and AtVND7 showed that AtVNDs bind to the 19-bp consensus DNA sequence of secondary wall NAC binding element (SNBE) and the 11-bp tracheary-element-regulating cis-elements (TERE) in the promoter region of a group of genes involved in the secondary cell wall biosynthesis, cell wall modification, and programmed cell death [6, 23, 48]. Both TERE and SNBE were also found in the promoter sequences of some direct target genes of AtSND1 [23, 28, 48, 50, 51].
In the woody perennial species Populus trichocarpa, a total of eight genes among 16 Populus NAC domain protein genes were sub-grouped as Populus VND (PtrWND/PtVNS) [22, 24, 48]. Dominant repression of PtrWND2B/PtVNS10 and PtrWND6B/PtVNS08 using EAR-induced dominant repression approach in hybrid Populus (P. tremula × P. alba) resulted in reduction of wall thickness of xylary fibers , whereas ectopic secondary wall thickening phenotype was observed in transgenic Populus plants overexpressing all PtrWND/PtVNS genes driven by cauliflower mosaic virus 35S promoter . Moreover, ectopic deposition of lignin, cellulose, and hemicellulose was observed in transgenic Arabidopsis and Populus overexpressing PtrWND6B (an AtVND7 homolog) [48, 51]. Therefore, Populus VND-related proteins (PtVND) appeared to function similarly as AtVND in the regulation of vascular vessel formation and secondary cell wall biosynthesis [24, 48, 51]. This was further supported by the observation that heterologous expression of PtrWND3A/PtVNS05 and PtrWND3B/PtVNS06 (AtVND4 and 5 homologs) in Arabidopsis resulted in ectopic secondary wall deposition in leaf .
Here we report the functional characterization of PdWND3A, an AtVND4/5 sequence homolog, using Populus transgenics. The transgenic Populus overexpressing PdWND3A displayed increased vessel formation in the stem. Both lignin content and lignin S/G ratio were increased in the transgenic plants. Interestingly, RT-PCR analysis indicated that among tested secondary cell wall biosynthesis-related genes, the expression of F5H1 was predominantly up-regulated in the transgenic plants, suggesting that PdWND3A may affect lignin biosynthesis and composition by regulating F5H1 expression.
Phylogenetic analysis of Populus NAC domain-containing proteins
In a previous studies, Zhong and Ye  used AtSND1 (AT1G32770) as a template to search for Populus homologs and defined their nomenclatures. In another study, Ohtani et al. identified 16 NAC domain protein genes in the Populus genome based on the protein homology analysis with Arabidopsis VND/NST/SND protein . With the availability of the latest P. trichocarpa genome annotation (v3.1), we used AtSND1 as a template to search of all possible AtSND1 sequence homologs in Phytozome (https://phytozome.jgi.doe.gov)  and identified a total of 21 Populus loci with a cutoff of amino acid sequence identity > 30% (Additional file 2). Among these proteins, a group of eight Populus proteins showing high amino acid sequence identity with respective AtVND proteins were selected for further study (Additional file 3). Two clades, including four Populus proteins (Potri.012G126500, Potri.015G127400, Potri.001G120000 and Potri.003G113000), shared a cluster with AtVND4 and AtVND5. On the basis of these results, we selected Potri.015G127400, which was previously designated as PtrWND3A  for further characterization. PtrWND3B (Potri.012G126500), in the same clade with PtrWND3A, shared 95.3% similarity with PtrWND3A at the amino acid level (Additional file 3). A DNA fragment of 24 bp is absent in the middle of coding sequence of PtrWND3A; therefore, we were able to use gene-specific primer for this region to distinguish PtrWND3A from PtrWND3B (Additional file 4). Similar to Arabidopsis VND proteins, NAC domain at the N-terminus of PtrWND3A is the only predictable domain (Additional file 3).
Expression pattern of PdWND3A
Transgenic Populus plants overexpressing PdWND3A
AtVND family proteins are viewed as master switch transcription factors regulating vessel formation in xylem tissue . To examine whether such a function is conserved in Populus, we examined vessel formation in OXPdWND3A transgenic plants. Cytological analysis with cross-section specimen of mature stem revealed a dense vessel formation in the stem of OXPdWND3A (Fig. 2c), with the number of xylem vessel significantly higher in OXPdWND3A transgenic plants compared to wild-type WV94 (Fig. 2d). These results support the view that the regulation of vessel formation is a common function of VND proteins in both Arabidopsis and Populus.
Chemical composition analysis of secondary cell wall components in OXPdWND3A transgenic plants
Lignin physicochemical characterization
Analysis of lignin monolignols and interunit linkage in OXPdWND3A transgenic plants. Structural information of lignin was obtained by 2D 1H-13C HSQC NMR analysis. The contents of monolignols (S and G), PB (p-hydroxybenzoate), and lignin interunit linkage [β-aryl ether (β-O-4), phenylcoumaran (β-5), and resinol (β-β)] were calculated as a fraction of total lignin subunits (S + G). Two biological replicates of stem tissues were used for the analysis. The number in parenthesis displayed standard deviation
Lignin interunit linkages
Gene expression analysis
To determine whether PdWND3A impacts expression of genes involved in lignin biosynthesis or monomer composition, we performed quantification analysis of the expression of lignin biosynthetic genes. We measured the relative transcript abundance of eight representative lignin biosynthetic genes, including PAL1 and PAL4, CAD1 and 4CL for general phenylprophenoid pathway, and CCoAOMT1, F5H1, COMT3 and COMT4 for monolignol biosynthesis. Among these eight tested genes, F5H1, a gene involved in S unit lignin monomer biosynthesis, was upregulated in both leaf and young stem tissues in the transgenic lines compared to the wild-type control (Fig. 4b). Although COMT and CAD had been reported to regulate S unit lignin monomer biosynthesis , no significant difference in their transcript level was observed between OXPdWND3A transgenic plants and the wild-type WV94. The transcript level of CCoAOMT1, another key enzyme involved in the monolignol biosynthesis of G- and S-type lignin [18, 19], in leaf tissue was also higher in the transgenic plants than WV94. Other tested genes were not altered in either tissues between the transgenic lines and WV94 (Fig. 4b). Collectively, these results support that PdWND3A has a role in regulating the expression of genes involved in lignin biosynthesis and lignin monomer composition.
Saccharification efficiency of OXPdWND3A
OXPdWND3A biomass production
Woody biomass is mainly composed of cellulose, hemicellulose and lignin. The development of applicable method to convert biomasses to biofuel has been regarded as a pivotal research for cost-effective biofuel production. In the last decade, molecular and genetic studies of woody plants suggested that transcription factors are critical for regulating secondary cell wall biosynthesis. Of these transcription factors, NAC family proteins are viewed as master switches [22, 56]. We provide evidence here that PdWND3A, a member of NAC domain-containing protein family, is involved in the regulation of lignin biosynthesis and composition.
PdWND3A and lignin biosynthesis and composition
The physicochemical analysis of OXPdWND3A transgenic lines suggest that overexpression of PdWND3A affects lignin biosynthesis (Fig. 4). Consistent with the physicochemical analysis, the histochemical image showed more xylem vessel formation in OXPdWND3A than WV94 (Fig. 2c and d). In earlier reports, overexpression of PtrWND6B, a homolog of AtVND7 protein, induced ectopic deposition of lignin in leaf epidermal and mesophyll cells [48, 51]. In addition, inducible expression of AtVND6 or AtVND7 in Populus resulted in ectopic lignin deposition . More specifically, overexpression of PtrWND3A was shown to induce ectopic secondary cell wall deposition in poplar leaves . Although a microscopic examination of ectopic secondary cell wall deposition in the leaves of transgenic plants overexpressing PdWND3A was not conducted in the present study, we provided physicochemical analysis which confirms that the lignin is accumulated in both the leaf and the stem of OXPdWND3A transgenic plants (Fig. 4a). In addition, monolignol composition between S and G unit was altered by overexpression of PdWND3A (Table 1). The structural properties in lignin were also altered in OXPdWND3A. We observed significant increase of resinol (β-β) linkages (Table 1). It was reported that β-β linages are primarily associated with S unit whereas phenylcoumaran is associated with G unit . Therefore, the observed increased lignin resinol abundance is consistent with the observed increased lignin S/G ratio in OXPdWND3A transgenic lines. Collectively, these results suggest that PdWND3A is involved in the regulation of both lignin biosynthesis and lignin monomer composition. It remains unclear whether PdWND3A preferentially regulates lignin biosynthesis or composition and how PdWND3A achieves it. Because PdWND3A functions as a transcription factor, it may do so through the regulation of specific lignin pathway genes.
PdWND3A and F5H1 expression
Gene expression analysis of lignin biosynthetic genes in OXPdWND3A transgenic plants indicated that among all tested genes, PdWND3A overexpression primarily affected the expression of F5H1 in both stem and leaf. This was in contrast to previous studies in Arabidopsis in which overexpression of every AtVND gene (AtVND1 to AtVND7) was shown to induce the expression of PAL1, CCoAOMT1 and 4CL of lignin biosynthetic genes but not F5H1 [23, 40, 58]. Zhou et al.,  demonstated that the promoters of CCoAOMT1 and 4CL are directly activated by AtVND proteins (AtVND1 to AtVND5). In another study, Populus transgenic plants expressing AtVND7 showed increased expression of genes encoding cationic peroxidase, laccase, CCR, and phenylcoumaran benzylic ether reductase related to lignin biosynthesis . Arabidopsis transgenic plants expressing PtrWND6B, a Populus homolog of AtVND6, also showed increased expression of 4CL1 and CCoAMT1 . The transactivation assay using PtrWND6B as the effector construct identified laccase, CCoAMT1 and COMT1 as direct target genes of PtrWND6B [24, 48]. Therefore, the regulation of gene expression of lignin biosynthetic genes by VND homologs appeared to be conserved between Arabidopsis and Populus. However, no report had shown the relationship between F5H1 expression and VND in previous studies. Our study showed that the expression of F5H1 is upregulated by PdWND3A, implying that there may be regulatory specificity among members of VND/WND transcription factor family regarding their downstream direct or indirect target genes.
F5H mediates the chemical conversion from coniferaldehyde to 5-OH coniferaldehyde in the S monolignol biosynthesis pathway . Overexpression of F5H from Liquidambar styraciflua in Pinus radiate produced more sinapyl alcohol in the lignin polymer . Accumulative evidence suggested that the regulation of F5H1 gene expression may be distinct from the common regulation of other lignin biosynthetic genes. For example, overexpression of AtMYB58 and AtMYB63 activated lignin biosynthetic genes except F5H1 [45, 57]. This result is consistent with the observation that the AC cis-acting element, a binding site for AtMYB58, is absent in the promoter of F5H1 [46, 57]. In Arabidopsis, AtSND1 has been reported to regulate F5H1 gene expression . In the present study, we showed that the expression of F5H1 is up regulated by PdWND3A overexpression (Fig. 4b). Because AtVNDs (AtVND1 to AtVND7) have been shown to bind to the consensus DNA sequence of secondary wall NAC binding element (SNBE) in the promoter region of a group of genes associated with cell wall biosynthesis [6, 23, 48] and the SNBE consensus is present in the F5H1 promoter , it is plausible that F5H1 may serve as a direct target PdWND3A. The biochemical determination of F5H1 as a potential PdWND3A target gene (i.e., via protein-DNA binding assays) deserves further investigation.
PdWND3A and sugar release
Previous studies using P. trichocarpa natural variants showed that both lignin content and S/G ratio affect saccharification efficiency [34, 43]. Glucose release was significantly correlated with both lignin content and S/G ratio [34, 43]. However, the glucose release depended on lignin content but not on S/G ratio when sugar release was measured without pretreatment . In the present study, OXPdWND3A transgenic lines showed both higher lignin content and higher S/G ratio (Fig. 3, Fig. 4, Table 1) with lower saccharification efficiency measured without pretreatment (Fig. 5), which is consistent with the observation in P. trichocarpa natural variants . Therefore, lignin content seems to play a more dominant role than S/G ratio in the process of saccharification without pretreatment. Collectively, PdWND3A, when overexpressed, negatively impacts saccharification efficiency. As a future study, creating and characterizing Populus PdWND3A knockdown or knockout transgenic plants may complement and potentially strengthen the conclusion on the role of PdWND3A in lignin biosynthesis and sugar release drawn from overexpression study.
Our results indicate that PdWND3A, a member of NAC domain-containing protein family, impacts both lignin biosynthesis and lignin monomer composition. Specifically, PdWND3A regulates the expression of F5H gene. Overexpression of PdWND3A negatively impacts saccharification efficiency and biomass production.
The full-length open reading frame of PdWND3A was amplified from Populus deltoides genotype WV94 and cloned into the pAGW560 binary vector for transformation into WV94. We followed the same procedure for growing and maintaining transgenic plants in the greenhouses as reported in a previous publication . The growth conditions were set with constant 25 °C with 16 h/8 h photoperiod.
Amino acid sequence alignment and phylogenetic analysis
AtSND1 (AT1G32770) was subjected to Phytozome v12.0 (https://phytozome.jgi.doe.gov)  and BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)  to identify NAC domain-containing proteins in the Populus (P. trichocarpa) and Arabidopsis (A. thaliana) genomes. The full-length amino acid sequence homologs of AtSND1 from each species were subsequently used to perform reciprocal sequence homolog search with > 30% amino acid similarity cutoff (e-value< 0.01). The collected proteins were used as subjects in the Pfam database to predict putative protein domains and functional motifs . The phylogenetic tree was constructed by PhyML (a phylogeny software based on the maximum-likelihood principle) using Jones-Taylor-Thornton (JTT) model matrix of amino acid substitution with 1000 bootstrap replication . Nearest-Neighbor-Interchange (NNI) algorithm was used to perform tree topology search.
To obtain the image of xylem vessel formation from OXPdWND3A transgenic plants and WV94 wild-type plants, stem tissues were collected at a position 15 cm above the stem base of 6-month-old plants. Cross-section specimen were sliced at 100 μm thickness without any fixation by using Leica RM2255 microtome (Leica biosystems, IL). Each slice was directly stained in 2% Phloroglucinol (Sigma-Aldrich, St. Louis, MO) dissolved in 95% ethanol for 5 min in dark. The red color was developed by adding 2–3 drops of concentrated Hydrochloride (HCl). Images were captured using SteREO Discovery V8 dissecting microscope (ZEISS, Thornwood, NY). The total count of vessel in each image was determined by ImageJ1 open source program .
RNA extraction and RT-PCR
To measure relative transcript abundance of PdWND3A and secondary cell wall biosynthesis-related genes, total RNA was extracted from young stem tissue (1–3 internode) and mature leaf (4-6th from apex) of six-month-old Populus plants with Plant Spectrum RNA extraction kit with treatment of in-column DNase following manufacture’s manual (Sigma-Aldrich). We performed quantitative reverse transcription polymerase chain reaction (sq- or qRT-PCR) to determine relative transcript abundance of selected genes. The single strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA by 1 h incubation with RevertAid reverse transcriptase (Thermo Fisher Scientific, Hudson, NH) at 42 °C. One μl of two-times diluted cDNA was used for real time PCR reaction. PCR reaction was performed with Maxima SYBR Green/ROX qPCR master mix including uracyl DNA glycosylase (UDG) (Thermo Fisher Scientific). Gene-specific primers used for PCR reactions were listed in the Additional file 1. PCR reaction was started with UDG activation at 50 °C for 2 min, a pre-denaturation of 95 °C for 10 min, followed by 40 cycles of combined two steps of 95 °C for 15 s and 60 °C for 30 s. The relative gene expression was calculated by 2–ΔΔCt equation . Populus UBIQUITIN C (PdUBCc, Potri.006G205700) was used as an internal control for all relative quantification analyses.
Chemical composition analysis
Carbohydrate contents were analyzed using a Dionex ICS-3000 ion chromatography system with external standards.
Lignin S/G ratio analysis
Nuclear magnetic resonance (NMR) analysis was used to measure the lignin S/G ratio. Stem samples were extracted as described above. Cellulolytic enzyme lignin was isolated from the extractives-free biomass as described in a previous study . The isolated lignin (~ 30 mg) was dissolved with DMSO-d6 in 5 mm NMR tube. A Bruker Avance III 400 MHz spectroscopy equipped with a 5 mm Broadband Observe probe and Bruker standard pulse sequence (‘hsqcetgpsi2’) was used for two-dimensional (2D) 1H-13C heteronuclear single quantum coherence (HSQC) NMR analysis at 300 K. The spectral widths of 11 ppm (1H, 2048 data points) and 190 ppm in F1 (13C, 256 data points) were employed for the 1H and 13C-dimensions, respectively. The number of transients was 64 and the coupling constant (1JCH) used was 145 Hz. Bruker Topspin software (v3.5) was used for data processing.
Saccharification efficiency assay
Stem tissues collected at a position 15 cm above the stem base of 6-month-old plants were dried and Wiley-milled to 40-mesh for sugar release measurement. Approximately 250 mg of sample was placed in 50 mM citrate buffer solution (pH 4.8) with 70 mg/g-biomass of Novozymes CTec2 (Novozymes, Franklinton, NC) loading. The enzymatic hydrolysis was conducted at 50 °C with 200 rpm in an incubator shaker for 48 h. Enzymes in the hydrolysate were deactivated in the boiling water for 5 min prior to the analysis of released sugars by using the Dionex ICS-3000 ion chromatography system. Each analysis was conducted in duplicates from single plant of each transgenic line.
T-test (against WV94) was performed at p < 0.01 by t-test function integrated in the Excel software (Microsoft, Redmond, WA) for all statistical analysis. Asterisk in each figure indicates significant difference from WV94 or control samples (p < 0.01).
Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. Department of Energy under Contract Number DE-AC05-00OR22725.
YY performed bioinformatics, RT-PCR and qRT-PCR analyses, and microscopic imaging. CGY, YP and AJR performed chemical composition analysis and measured sugar release. WR, KAW and CMC generated Populus transgenic lines. LEG and SSJ measured biomass production. XY designed the gene construct for Populus transformation. GAT and JGC conceived the study, coordinated research and contributed to experimental design and data interpretation. All authors read and approved the final manuscript.
This research was supported by the U.S. Department of Energy BioEnergy Science Center and the Center for Bioenergy Innovation. The BioEnergy Science Center and the Center for Bioenergy Innovation are U.S. Department of Energy Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the U.S. Department of Energy Office of Science. The funding body has no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
- 5.Demura T, Tashiro G, Horiguchi G, Kishimoto N, Kubo M, Matsuoka N, Minami A, Nagata-Hiwatashi M, Nakamura K, Okamura Y, Sassa N, Suzuki S, Yazaki J, Kikuchi S, Fukuda H. Visualization by comprehensive microarray analysis of gene expression programs during transdifferentiation of mesophyll cells into xylem cells. Proc Natl Acad Sci U S A. 2002;99:15794–9.PubMedPubMedCentralCrossRefGoogle Scholar
- 6.Endo H, Yamaguchi M, Tamura T, Nakano Y, Nishikubo N, Yoneda A, Kato K, Kubo M, Kajita S, Katayama Y, Ohtani M, Demura T. Multiple classes of transcription factors regulate the expression of vascular-related nac-domain7, a master switch of xylem vessel differentiation. Plant Cell Physiol. 2015;56:242–54.PubMedCrossRefGoogle Scholar
- 14.Lacombe E, Hawkins S, Van Doorsselaere J, Piquemal J, Goffner D, Poeydomenge O, Boudet AM, Grima-Pettenati J. Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant J. 1997;11:429–41.PubMedCrossRefGoogle Scholar
- 18.Marita JM, Ralph J, Harfield RD, Guo D, Chen F, Dixon RA. Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme a 3-O-methyltransferase. Phytochemistry. 2003;62:53–65.PubMedCrossRefPubMedCentralGoogle Scholar
- 19.Meyermans H, Morreel K, Lapierre C, Pollet B, De Bruyn A, Herdewijn B, Van Beeumen J, Marita JM, Ralph J, Chen C, Burggraeve B, Van Montagu M, Messens E, Boerjan W. Modifications in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme a O-methyltranseferase, an enzyme involved in lignin biosynthesis. J Biol Chem. 2000;275:36899–909.PubMedCrossRefGoogle Scholar
- 29.Ragauskas AJ, Beckham GT, Biddy MJ, Chandra R, Chen F, Davis MF, Davison BH, Dixon RA, Gilna P, Keller M, Langan P, Naskar AK, Saddler JN, Tschaplinski TJ, Tuskan GA, Wyman CE. Lignin valorization: improving lignin processing in the biorefinery. Science. 2014;344:1246843.PubMedCrossRefGoogle Scholar
- 32.Sluiter A, Hames B, Fuiz R, Scarlata C, Sluiter J, Templeton D, Crocker D. Determination of structural carbohydrates and lignin in biomass in: laboratory analytical procedure National Renewable Energy Laboratory; 2012. p. 3–13.Google Scholar
- 38.Yang Y, Yoo C, Guo H, Rottmann W, Winkeler K, Collins C, Gunter L, Jawdy S, Yang X, Guo H, Pu Y, Ragauskas A, Tuskan G, Chen J. Overexpression of a domain of unknown function 266-containing protein results in high cellulose content, reduced recalcitrance, and enhanced plant growth in the bioenergy corp Populus. Biotech Biofuels. 2017;10:74.CrossRefGoogle Scholar
- 39.Yamaguchi M, Goue N, Igarashi H, Ohtani M, Nakano Y, Mortimer JC, Nishikubo N, Kubo M, Katayama Y, Kakegawa K, Dupree P, Demura T. Vascular-related nac-domain6 and vascular-related nac-domain7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system. Plant Physiol. 2010;153:906–14.PubMedPubMedCentralCrossRefGoogle Scholar
- 43.Yoo CG, Yang Y, Pu Y, Meng X, Muchero W, Yee K, Thompson O, Rodriguez M, Bali G, Engle N, Lindquist E, Singan V, Schmutz J, DiFazio S, Tschaplinski T, Tuskan G, Chen J, Davison B, Ragauskas A. Insights of biomass recalcitrance in natural Populus trichocarpa variants for biomass conversion. Green Chem. 2017;19:5467–78.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.