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. 2016 Sep 6;172(3):1612–1624. doi: 10.1104/pp.16.01230

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements1,[OPEN]

Misato Ohtani 1,2,3,4,5,6, Keiko Morisaki 1,2,3,4,5,6, Yuji Sawada 1,2,3,4,5,6, Ryosuke Sano 1,2,3,4,5,6, Abigail Loren Tung Uy 1,2,3,4,5,6, Atsushi Yamamoto 1,2,3,4,5,6, Tetsuya Kurata 1,2,3,4,5,6, Yoshimi Nakano 1,2,3,4,5,6, Shiro Suzuki 1,2,3,4,5,6, Mami Matsuda 1,2,3,4,5,6, Tomohisa Hasunuma 1,2,3,4,5,6, Masami Yokota Hirai 1,2,3,4,5,6, Taku Demura 1,2,3,4,5,6,*
PMCID: PMC5100780  PMID: 27600813

Primary metabolism is actively regulated for the biosynthesis of secondary wall polymers during the differentiation of protoxylem vessel elements.

Abstract

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell’s metabolic activity for the biosynthesis of secondary wall polymers.

Xylem vessels conduct water and nutrients in vascular plants (Myburg and Sederoff, 2001; Turner et al., 2007). Xylem vessel cells have a characteristic thick and patterned secondary cell wall (SCW) and undergo programmed cell death (PCD). SCW biopolymers such as cellulose, hemicellulose, and lignin are major constituents of terrestrial lignocellulosic biomass. As lignocellulose is recalcitrant to enzymatic hydrolysis, it has limited utility as a renewable resource. Improving our understanding of the molecular mechanisms underlying xylem vessel cell differentiation will facilitate the development of strategies to overcome this limitation (Abramson et al., 2010).

Xylem vessel cell differentiation, which has been studied extensively using an in vitro induction system (Fukuda and Komamine, 1980; Demura et al., 2002; Kubo et al., 2005; Pesquet et al., 2010), initiates with the transcriptional up-regulation of SCW- and PCD-related genes by the VASCULAR-RELATED NAC-DOMAIN (VND) master regulatory transcription factors (Kubo et al., 2005; Yamaguchi et al., 2008; Ohashi-Ito et al., 2010; Zhong et al., 2010; Yamaguchi et al., 2011; Endo et al., 2015; Nakano et al., 2015). The first detectable cytological sign of differentiation is the alignment of cortical microtubules, which determine patterned SCW thickening by guiding the cellulose synthase complex (Bashline et al., 2014; McFarlane et al., 2014). Next, cellulose and hemicellulose accumulate, followed by lignin deposition (Faik, 2010; Vanholme et al., 2010). Vacuole disruption triggers PCD, which results in proteases and nucleases being released into the cytosol (Bollhöner et al., 2012). During the final stages of differentiation, the end walls are perforated and the cell contents exit the cells, completing the differentiation of the hollow tube structure (Turner et al., 2007).

Transcriptome analysis of xylem tissues (for loblolly pine [Pinus taeda], Allona et al., 1998; for poplar [Populus spp.], Sterky et al., 2004; for spruce [Picea spp.], Ralph et al., 2008; and for eucalyptus [Eucalyptus spp.], Rengel et al., 2009) and studies in the in vitro system (Demura et al., 2002; Ohashi-Ito et al., 2010; Zhong et al., 2010; Yamaguchi et al., 2011) have identified many genes involved in xylem cell differentiation. These include VND family genes and SCW-related genes encoding MYB transcription factors (McCarthy et al., 2010; Ko et al., 2012, 2014; Zhong and Ye, 2012; Hussey et al., 2013); based on this information, NAC-MYB-based transcriptional networks have been proposed for xylem vessel cell differentiation (Nakano et al., 2015). Moreover, proteome data have been reported for xylem tissues of hybrid aspen (Populus spp.; Kalluri et al., 2009) and Hydrangea paniculata (Pagter et al., 2014). A recent study also reported a quantitative proteome analysis targeting microtubule-interacting proteins involved in xylem vessel cell differentiation (Derbyshire et al., 2015).

While these transcriptome and proteome studies have provided insight into xylem cell differentiation, little is known about the primary metabolic changes that occur during xylem vessel cell differentiation. Regulation of the biosynthesis of SCW-related metabolites, such as cellulose and its precursor UDP-Glc, the hemicellulosic polysaccharide xylan, and lignin monomers, has attracted substantial attention (Faik, 2010; Vanholme et al., 2010; Bashline et al., 2014; McFarlane et al., 2014). However, we lack information about the regulation of primary metabolism during xylem vessel cell differentiation.

In this study, we performed a wide-target metabolome analysis of protoxylem vessel element differentiation using a tobacco (Nicotiana tabacum) BY-2 in vitro system (VND7-VP16-GR; Yamaguchi et al., 2010; Goué et al., 2013) that allows the effective induction of protoxylem-type vessel element differentiation. Induced BY-2 cells were subjected to liquid chromatography-mass spectrometry (LC-MS)-based wide-target metabolome analysis as established by Sawada et al. (2009), which can automatically detect approximately 700 metabolites that are the products of metabolic regulatory pathways (Sawada et al., 2009). This method can provide data on general metabolites during protoxylem vessel element formation with high reproducibility. Indeed, we successfully obtained time-course metabolome data during protoxylem vessel element differentiation. In addition, we performed RNA sequencing (RNA-seq) analysis on the same differentiating BY-2 cells and examined the trends in transcript levels of genes encoding enzymes involved in the catalytic pathways of the metabolites detected by the metabolome analysis. Subsequent target quantification analysis showed active changes in Fru-6-P, phosphoenolpyruvate (PEP), and UDP-Glc during cell differentiation. These data suggested that the active regulation of glycolysis and amino acid biosynthesis at the transcriptional level alters cell activity, shifting metabolism toward the biosynthesis of specific kinds of metabolites, such as Fru-6-P, a precursor of nucleoside diphosphate (NDP)-sugar, and PEP, a precursor of Phe for lignin monomers, which are important building blocks of the SCW of protoxylem vessel element.

RESULTS

Widely Targeted Metabolome Analysis of Protoxylem Vessel Element Differentiation in a Tobacco BY-2 in Vitro System

For the widely targeted metabolome analysis during protoxylem vessel element differentiation, we used the established tobacco BY-2 in vitro system (VND7-VP16-GR) for protoxylem vessel element differentiation (Yamaguchi et al., 2010; Goué et al., 2013). In this system, the VND7-VP16-GR fusion protein is expressed continuously under the control of the 35S promoter, and its transcription factor activity can be posttranslationally activated by treatment with dexamethasone (DEX). Following DEX treatment, the VND7-VP16-GR BY-2 cells effectively transdifferentiate into protoxylem vessel elements (Yamaguchi et al., 2010; Goué et al., 2013). Figure 1A shows the transitional differentiation rates of VND7-VP16-GR BY-2 cells after DEX treatment. The first visible sign of cell differentiation was the weak deposition of helically patterned SCWs after 24 h of DEX treatment (Fig. 1C). At this stage, the cells with patterned SCWs had visible nuclei, indicating that these cells were living and undergoing differentiation as protoxylem vessel elements (Fig. 1C). Then, after 36 h of DEX treatment, we observed cells with thick helical SCWs but without cell contents (i.e. differentiated protoxylem vessel elements; Fig. 1D). Ultimately, approximately 70% of VND7-VP16-GR BY-2 cells differentiated into protoxylem vessel elements (Fig. 1A). Previously, we observed that the up-regulation of SCW biosynthetic genes could be detected after 6 h of DEX treatment (Goué et al., 2013), suggesting that protoxylem vessel element differentiation initiated at least at 6 h of incubation with DEX, even though no morphological changes were visible in the cells. Based on these observations, we sampled VND7-VP16-GR BY-2 cells, as well as nontransgenic BY-2 cells, after 0, 6, 12, 24, 36, and 48 h of DEX or mock treatment for further metabolome analysis.

Figure 1.

Figure 1.

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Transitional differentiation rates of protoxylem vessel elements in VND7-VP16-GR BY-2 cells. A, Ratios of undifferentiated (light gray bars), differentiating (hatched bars), and differentiated (dark gray bars) cells at each time point of the incubation with DEX. Data show means ± sd (n = 3). B, Undifferentiated BY-2 cell with a clear nucleus structure (arrow). C, Differentiating BY-2 cell with a thin helical SCW (arrowheads) and a nucleus (arrow). D, Differentiated cell with thick SCWs (arrowheads) lacking nuclear and cytosolic structures. Shrunken cell contents (arrow) were observed in cells that had completed differentiation. Bar = 20 µm for B to D.

Using the LC-MS-based wide-target metabolome analysis established by Sawada et al. (2009), we identified 490 metabolites from the control and VND7-VP16-GR BY-2 cells and obtained relative amount data for 128 metabolites based on a cutoff signal-to-noise ratio of greater than 3 (Supplemental Tables S1 and S2). Principal component analysis (PCA) of these 128 metabolites successfully identified changes in the metabolome associated with the progression of cell differentiation; DEX-treated VND7-VP16-GR samples, mock-treated VND7-VP16-GR samples, and nontransgenic samples were separated in the PCA plot (Fig. 2). The first two principal components (PC1 and PC2) explained 36% of the total variance, and as the cell differentiation progressed, PC2 became larger in the DEX-treated VND7-VP16-GR samples (Fig. 2). PC1 possibly reflected the difference between VND7-VP16-GR samples and nontransgenic samples, since the wild-type samples were distributed in the region with negative values of PC1, whereas VND7-VP16-GR samples were found in the region with positive values of PC1 (Fig. 2). The PCA model in Figure 2 showed that amino acids and their derivatives tended to be highly correlated to PC1 and PC2 (P < 0.05; Tables I and II; Supplemental Tables S1 and S2). The 15 metabolites with the highest and lowest PC1 correlation scores (P < 0.05) included 11 amino acids (Asp, Glu, Thr, His, Pro, Lys, Gln, allo-Thr, α-methyl-His, l-threo-3-methyl-Asp, and 1-aminocyclopentane carboxylate) and five amino acid-related metabolites (Ala, Phe, 4-aminobutanoate, S-methyl-Met, and d-alanyl-d-Ala; Table I). The 15 metabolites with the highest and lowest PC2 correlation scores (P < 0.05) included five amino acids [Leu, Ile, N6-(l-1,3-dicarboxypropyl)-l-Lys, nor-Leu, and carnosine] and four amino acid-related metabolites (Ala, Ser, O-acetyl-l-Ser, and N-acetyl-dl-Ser; Table II). These results suggested that amino acids and their derivatives were the main metabolites contributing to the dispersion of the samples on PC1 and PC2.

Figure 2.

Figure 2.

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Metabolic changes associated with protoxylem vessel element differentiation. PCA plots are shown for mock-treated (i.e. ethanol-treated) and DEX-treated VND7-VP16-GR BY-2 and nontransgenic wild-type (WT) cells. Treatment times (in hours) are indicated. The x and y axes indicate the first component (PC1) and the second component (PC2), respectively. Independent triplicate samples were collected and examined for each time point.

Table I. Top 15 metabolites with high and low correlation scores for PC1 within the PCA analysis of time-sequential wide-target metabolome data for protoxylem vessel element differentiation.

Metabolitea Type of Metabolism Class of Metabolites Correlation P
Top 15 metabolites with high correlation scores for PC1
 Asp Primary Amino acid 0.920638002 7.76E-28
 Glu Primary Amino acid 0.915312495 5.70E-27
 Thr Primary Amino acid 0.897000219 2.25E-24
 His Primary Amino acid 0.896197896 2.84E-24
 l-Allo-Thr Primary Amino acid 0.890719092 1.35E-23
 Pro Primary Amino acid 0.877349881 4.39E-22
 Malate Primary Other 0.860575996 2.03E-20
 Lys Primary Amino acid 0.853053294 9.67E-20
 Methylguanidine Primary Other 0.840276945 1.13E-18
 2-Aminoethylphosphonate Primary Other 0.800972489 6.78E-16
 α-Methyl-His Primary Amino acid 0.787859709 4.20E-15
 l-threo-3-Methyl-Asp Primary Amino acid 0.774852194 2.27E-14
 1-Aminocyclopentane carboxylate Primary Amino acid 0.767925228 5.32E-14
 Gln Primary Amino acid 0.740290592 1.22E-12
 1,3-Diaminopropane Primary Other 0.72246723 7.51E-12
Top 15 metabolites with low correlation scores for PC1
 Threonate Primary Other −0.684909553 2.26E-10
 2,3-Diaminopropanoate Primary Other −0.656676739 2.14E-09
 Guanine Primary Other −0.606504405 6.76E-08
 Ala Primary Amino acid −0.598142463 1.14E-07
 Psicose Primary Other −0.554363533 1.37E-06
 4-Aminobutanoate Primary Amino acid −0.5504808 1.68E-06
 S-Methyl-Met Primary Amino acid −0.544953166 2.24E-06
 Glycerate Primary Other −0.510460119 1.19E-05
 Deoxyinosine Primary Other −0.506239536 1.45E-05
 Adenosine Primary Other −0.471957628 6.32E-05
 Uridine Primary Other −0.444140615 0.000187238
 Glc-1-P Primary Other −0.437642617 0.000238154
 Inosine Primary Other −0.427176328 0.000347355
 d-Alanyl-d-Ala Primary Amino acid −0.376960084 0.001809199
 Phe Primary Amino acid −0.366290228 0.002488166
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a

Amino acid-related metabolites are shown in boldface.

Table II. Top 15 metabolites with high and low correlation scores for PC2 within the PCA analysis of time-sequential wide-target metabolome data for protoxylem vessel element differentiation.

Metabolitea Type of Metabolism Class of Metabolites Correlation P
Top 15 metabolites with high correlation scores for PC2
 Cytidine Primary Other 0.851704009 1.27E-19
 Guanosine Primary Other 0.849274728 2.05E-19
 Adenine Primary Other 0.819066281 4.33E-17
 N6-(l-1,3-Dicarboxypropyl)-l-Lys Primary Amino acid 0.796352172 1.31E-15
 Tyramine Primary Other 0.788972897 3.62E-15
 Nor-Leu Primary Amino acid 0.755687444 2.24E-13
 Pipecolate Primary Other 0.714945999 1.55E-11
 Carnosine Primary Amino acid 0.699673617 6.31E-11
 Uridine Primary Other 0.671463343 6.80E-10
 l-Leu Primary Amino acid 0.663135214 1.31E-09
 Deoxyadenosine Primary Other 0.661653026 1.46E-09
 3′-CMP Primary Other 0.637400317 8.67E-09
 Deoxyguanosine Primary Other 0.63691194 8.98E-09
 l-Ile Primary Amino acid 0.61833074 3.16E-08
 Adenosine Primary Other 0.611578222 4.90E-08
Top 15 metabolites with low correlation scores for PC2
 Carnitine Primary Other −0.723203992 6.98E-12
 d-Arabitol Primary Other −0.679416094 3.57E-10
 Pelargonidin 3-O-Glc Secondary Flavonoid −0.652569752 2.90E-09
 Hypotaurine Primary Other −0.626529626 1.83E-08
 Pyridoxamine Primary Other −0.594416155 1.42E-07
 O-Acetyl-l-Ser Primary Amino acid −0.541259749 2.70E-06
 Man-6-P Primary Other −0.525065728 6.01E-06
 N-Acetyl-dl-Ser Primary Amino acid −0.514816671 9.76E-06
 Galactosamine Primary Other −0.510753618 1.18E-05
 Ala Primary Amino acid −0.474843701 5.62E-05
 N-Acetylneuraminate Primary Other −0.473072912 6.04E-05
 Thiamine Primary Other −0.469162515 7.08E-05
 Ser Primary Amino acid −0.447716665 0.000163683
 Man-1-P Primary Other −0.417155822 0.000492976
 Ethanolamine phosphate Primary Other −0.407927601 0.000674143
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a

Amino acid-related metabolites are shown in boldface.

Changes in Amino Acid-Related Metabolites during Protoxylem Vessel Element Differentiation

We further explored the changes in amino acid-related metabolites during protoxylem vessel element differentiation. In this work, we could not obtain data for Gly, Val, and Cys; however, we did detect the remaining 17 proteinogenic amino acids and additional related metabolites (Supplemental Tables S3 and S4). Parts of the LC-MS-based profiles for these metabolites are shown in Figures 3 and 4 and Supplemental Figures S1 and S2. Amino acids are derived from intermediates of the glycolysis pathway, and glyceraldehyde 3-phosphate (GAP), an intermediate of the glycolysis pathway, showed an immediate decrease after DEX treatment (Fig. 3). This early change was observed only in the DEX-treated VND7-VP16-GR BY-2 cells (Figs. 3 and 4; Supplemental Figs. S1 and S2; Supplemental Tables S3 and S4); therefore, the immediate decrease in GAP appears to be associated with the initiation of protoxylem vessel element differentiation.

Figure 3.

Figure 3.

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Changes in amino acid-related metabolites derived from intermediate compounds in the glycolysis pathway during protoxylem vessel element differentiation. Mock-treated (white squares) and DEX-treated (gray diamonds) VND7-VP16-GR BY-2 cells were collected at the indicated time points, and relative amounts of metabolites were obtained. Data are means ± sd (n = 3). Asterisks indicate statistically significant differences between mock-treated and DEX-treated samples at each time point (*, P < 0.05 and **, P < 0.01; Student’s t test).

Figure 4.

Figure 4.

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Changes in amino acid-related metabolites derived from oxaloacetate and 2-oxoglutarate during protoxylem vessel element differentiation. Mock-treated (white squares) and DEX-treated (gray diamonds) VND7-VP16-GR BY-2 cells were collected at the indicated time points, and relative amounts of metabolites were obtained. Data are means ± sd (n = 3). Asterisks indicate statistically significant differences between mock-treated and DEX-treated samples at each time point (*, P < 0.05 and **, P < 0.01; Student’s t test). GABA, γ-aminobutyrate.

The amino acids with high PC1 correlation scores (i.e. Asp, Glu, Thr, His, Pro, Lys, and Gln; Table I; Supplemental Table S1) all decreased in VND7-VP16-GR cells as cell differentiation progressed, whereas the amino acids with high PC2 correlation scores (i.e. Leu, Ile, Arg, and Trp; Table II; Supplemental Table S2) increased after 24 h of DEX treatment (Figs. 3 and 4; Supplemental Figs. S1 and S2; Supplemental Tables S3 and S4). These results indicated that the progression of protoxylem vessel element differentiation was associated with changes in amino acid metabolism. Among the increased amino acids, Trp, Phe, and Tyr are biosynthesized from d-erythrose-4-phosphate and PEP through the shikimate pathway (Maeda and Dudareva, 2012). The increase in Trp and tyramine produced from Tyr suggested that the shikimate pathway was activated after 24 h of DEX treatment (Fig. 3; Supplemental Fig. S1). The amounts of Tyr and Phe increased gradually during cell culture as well as in the wild-type cells (Fig. 3; Supplemental Fig. S1). However, the concentration of tyramine, a derivative of Tyr, increased greatly after 24 h of DEX treatment (Fig. 3), suggesting that most Tyr biosynthesized after DEX treatment is converted into tyramine. Similarly, although a transient increase in Phe was detected after 12 h of DEX treatment, the Phe level was the same as that after mock treatment (Fig. 3; Supplemental Table S3); thus, most of the Phe produced after DEX treatment would be converted into lignin monomer to generate SCWs (Vanholme et al., 2010). The other increased amino acids, Leu, Ile, and Arg, are located at the end of each respective metabolic pathway (Figs. 3 and 4). Other amino acids generated from the downstream metabolites of GAP decreased after DEX treatment (Figs. 3 and 4; Supplemental Figs. S1 and S2). The large increase of l-saccharopine and l-2-aminoadipate after 24 h of DEX treatment (Fig. 3) indicated that the Lys catabolic pathway was activated during protoxylem vessel element differentiation (Arruda et al., 2000).

Taken together, these data suggested that amino acid metabolism is changed to produce several specific kinds of amino acids, such as the branched-chain amino acids Leu and Ile, the aromatic amino acid Trp, and the translocatable nitrogen-rich amino acid Arg, and that such regulation could be initiated from the rapid use of the GAP supplied from the glycolysis pathway. In addition, the increased tyramine, l-saccharopine, and l-2-aminoadipate could imply that the catabolism of amino acids is activated, at least partly, during protoxylem vessel element differentiation. Since tyramine is known to be a precursor for hydroxycinnamic acid amides bound to cell walls (Facchini et al., 2000), it is possible that the synthesized tyramine is partly further catalyzed into such cell wall-bound amides and then incorporated into SCWs.

Transcriptome Analysis of VND7-VP16-GR BY-2 Cells

To obtain insight into the metabolic regulation of amino acids during protoxylem vessel element differentiation, we conducted RNA-seq analysis of VND7-VP16-GR BY-2 cells treated with DEX or mock for 0, 6, 12, 24, and 36 h. After the de novo assembly of sequence reads based on the tobacco reference genome (Sierro et al., 2014), we obtained 74,932 unique contigs from the mRNA-seq data for the VND7-VP16-GR BY-2 cells. Previous work reported the set of direct targets of Arabidopsis (Arabidopsis thaliana) VND6 and VND7 (Ohashi-Ito et al., 2010; Zhong et al., 2010; Yamaguchi et al., 2011); based on this, we first examined the expression patterns of genes homologous to the 63 VND7 direct target genes reported by Yamaguchi et al. (2011). Using BLAST searches, we identified 603 contigs with high sequence similarity to direct target genes of Arabidopsis VND7 (Supplemental Table S5). Two-thirds of these contigs were up-regulated after DEX treatment, and the remaining contigs decreased or did not change in response to DEX treatment in RNA-seq data (Supplemental Table S5; Supplemental Fig. S3). Most of up-regulation occurred after 6 h of DEX treatment (Supplemental Table S5; Supplemental Figs. S3 and S4); thus, the initiation of protoxylem vessel element differentiation occurred within 6 h of DEX treatment.

Active Transcriptional Regulation of Genes Encoding Glycolysis and Shikimate Pathway Enzymes during Protoxylem Vessel Element Differentiation

Next, we searched for changes in transcript levels of genes encoding enzymes involved in the glycolysis pathway (Fig. 5; Supplemental Fig. S5; Supplemental Table S6). In accordance with the fact that the GAP contents decreased after 6 h of DEX treatment (Fig. 3), we also detected changes after 6 h of DEX treatment in the transcript levels of genes corresponding to Fru-bisphosphate aldolase (EC 4.1.2.13), which catalyzes the reversible conversion of fructose 1,6-bisphosphate (Fru-1,6-bP) into dihydroxyacetone phosphate and GAP (Fig. 5). We detected 19 contigs putatively corresponding to Fru-bisphosphate aldolase (Supplemental Table S6); two of these were statistically significantly up-regulated (fold change [FC] > 4, P < 0.05; Student’s t test) and one was statistically significantly down-regulated within 6 h of DEX treatment (FC < 0.5, P < 0.05; Student’s t test; Fig. 5; Supplemental Table S6), whereas the mock treatment did not significantly affect their expression (Supplemental Fig. S5; Supplemental Table S6). All five contigs that tended to decrease at 6 h of DEX treatment (FC < 1) were similar to Arabidopsis genes encoding plastid-type Fru-bisphosphate aldolase (Supplemental Table S6); thus, it is possible that the cytosolic types of Fru-bisphosphate aldolase are up-regulated at the transcriptional level during the initial stages of protoxylem vessel element differentiation. In addition, the expression of Fru 1,6-bisphosphatase (EC 3.1.3.11), which converts Fru-1,6-bP to Fru-6-P, was up-regulated after the DEX treatment (Fig. 5; Supplemental Table S6). Three contigs putatively encoding Fru 1,6-bisphosphatase were greatly up-regulated at 6 h of DEX treatment (FC > 40, P < 0.05; Student’s t test), while only one contig was significantly up-regulated at 6 h of mock treatment (FC = 5.3, P < 0.05; Student’s t test; Supplemental Fig. S5; Supplemental Table S6). Conversely, the expression of genes encoding phosphofructokinase (EC 2.7.1.11), which phosphorylates Fru-6-P, decreased after 12 h of DEX treatment (Fig. 5; Supplemental Fig. S5; Supplemental Table S6). Considering that these enzymes catalyze these reactions in only one direction, these observations indicate that the conversion of GAP to Fru-6-P through Fru-1,6-bP increased during the early stages of cell differentiation.

Figure 5.

Figure 5.

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Changes in the expression of genes putatively involved in glycolysis during protoxylem vessel element differentiation in DEX-treated VND7-VP16-GR BY-2 cells. Expression data are shown for genes encoding the indicated enzymes. Each circle indicates the proportion of contigs increased (red [FC > 4] and orange [2 < FC < 4]), unchanged (yellow [0.5 < FC < 2]), and decreased (sky blue [0.25 < FC < 0.5] and deep blue [FC < 0.25]) compared with the expression level at 0 h of DEX treatment at the indicated time points. 1,3-bPG, 1,3-Bisphospho-d-glycerate; DHAP, dihydroxy acetone phosphate; Glu-6-P, d-Glc-6-P; 2-PG, 2-phospho-d-glycerate; 3-PG, 3-phospho-d-glycerate; Pyr, pyruvate; TCA, tricarboxylic acid.

Metabolome analysis data also showed increases in aromatic amino acids, such as Trp, Phe, and Tyr, during protoxylem vessel element differentiation (Fig. 3). Phe is an important metabolite because it is the precursor of lignin monomer biosynthesis (Vanholme et al., 2010); therefore, we further examined the expression of genes encoding enzymes involved in Phe biosynthesis, including the shikimate pathway (Fig. 6; Supplemental Fig. S6). The data showed that all of the metabolic steps except the one catalyzed by 3-dehydroquinate synthase (EC 4.2.3.4) were actively regulated at the transcriptional level, and most of them were up-regulated during protoxylem vessel element differentiation (Fig. 6; Supplemental Fig. S6).

Figure 6.

Figure 6.

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Changes in the expression of genes putatively involved in Phe biosynthesis during protoxylem vessel element differentiation in DEX-treated VND7-VP16-GR BY-2 cells. Expression data are shown for genes encoding the indicated enzymes. Each circle indicates the proportion of contigs increased (red [FC > 4] and orange [2 < FC < 4]), unchanged (yellow [0.5 < FC < 2]), and decreased (sky blue [0.25 < FC < 0.5] and deep blue [FC < 0.25]) compared with the expression level at 0 h of DEX treatment at the indicated time points. DAHP, 3-Deoxy-d-arabino-heptulosonate-7-phosphate; E4P, erythrose-4-phosphate; EPSP, 5-enolpyruvyl-shikimate 3-phosphate.

Genes encoding PEP carboxykinase (EC 4.1.1.49), which converts oxaloacetate into PEP, also were up-regulated during the early stages of cell differentiation (Fig. 5; Supplemental Fig. S5). The decreased GAP (Fig. 3) and changes in the transcription of genes involved in the glycolysis pathway (Fig. 5; Supplemental Fig. S5) suggested that PEP biosynthesis from Fru-6-P was reduced after DEX treatment. Based on the up-regulation of PEP carboxykinase, we propose that the PEP required for Phe biosynthesis is derived from oxaloacetate during protoxylem vessel element differentiation (Fig. 5).

Quantification of Key Metabolites for SCW Polymer Biosynthesis and Lignin Content during Protoxylem Vessel Element Formation

Finally, we tried to quantify Fru-6-P and PEP, which were assumed to be key metabolites from the glycolysis pathway for SCW polymer biosynthesis as shown above, and UDP-Glc, a precursor of SCW oligosaccharides, by the capillary electrophoresis-mass spectrometry (CE-MS) analysis established by Hasunuma et al. (2016). The data indicated active changes in these metabolites in VND7-VP16-GR cells in a DEX treatment-dependent manner (Fig. 7, A–C). Although VND7-VP16-GR cells showed higher levels of Fru-6-P and UDP-Glc than the wild-type cells even under mock conditions (Fig. 7, A and C), the induction of protoxylem vessel element differentiation by DEX treatment significantly changed the trend of these metabolites in VND7-VP16-GR cells: both Fru-6-P and UDP-Glc decreased at 6 h of DEX treatment and then increased as cell differentiation progressed (Fig. 7, A and C). The increased level of Fru-6-P after 12 h of DEX treatment can be explained, at least partly, by the early up-regulation of Fru 1,6-bisphosphatase (EC 3.1.3.11) rather than the conversion from Glu-6-P by Glc-6-P isomerase (EC 5.3.1.9), because the expression levels of Glc-6-P isomerase were not changed at 6 to 12 h of DEX treatment (Fig. 5). We also found that the PEP contents increased after 12 h of DEX treatment (Fig. 7B), in accordance with the up-regulation of PEP carboxykinase shown in Figure 5.

Figure 7.

Figure 7.

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Changes in Fru-6-P, PEP, UDP-Glc, p-coumaric acid, and lignin contents during protoxylem vessel element differentiation. A to C, Mock-treated (white squares) and DEX-treated (gray diamonds) wild-type (WT; left) and VND7-VP16-GR (right) BY-2 cells were collected at the indicated time points, and Fru-6-P (A), PEP (B), and UDP-Glc (C) were quantified by CE-MS analysis. DW, Dry weight. D, Relative amounts of p-coumaric acid detected in LC-MS analysis (left) and lignin contents measured by thioglycolic acid methods (right). Data are means ± sd (n = 3) for mock-treated wild-type (white bars), DEX-treated wild-type (light gray bars), mock-treated VND7-VP16-GR (dark gray bars), and DEX-treated VND7-VP16-GR (black bars) plants. Asterisks indicate statistically significant difference between mock-treated and DEX-treated samples at each time point (*, P < 0.1; **, P < 0.05; and ***, P < 0.01; Student’s t test).

In addition, to monitor lignification in our system, p-coumaric acid, a precursor of a lignin monomer generated from Phe, and the lignin contents were analyzed. The relative amount data for p-coumaric acid by LC-MS analysis showed a transient increase of p-coumaric acid after 24 h of DEX treatment in VND7-VP16-GR cells, and thioglycolic acid methods detected lignin deposition after 36 h of DEX treatment only in DEX-treated VND7-VP16-GR cells (Fig. 7D). As shown in Figure 3, Phe increased transiently after 12 h of DEX treatment in VND7-VP16-GR cells, suggesting the activation of flow from Phe to lignin monomer through p-coumaric acid.

DISCUSSION

In this work, we examined protoxylem vessel element differentiation using wide-target metabolome and transcriptome analyses. The highly synchronous in vitro system for protoxylem vessel element differentiation (VND7-VP16-GR; Yamaguchi et al., 2010) in tobacco BY-2 cells provided time-sequential data for relative levels of 128 metabolites (Supplemental Table S3). The data described here revealed prominent changes in the contents of amino acids and the expression patterns of genes encoding enzymes involved in the glycolysis and shikimate pathways (Figs. 2–6; Supplemental Tables S1–S6). CE-MS analysis indicated that Fru-6-P and PEP, suggested to be key metabolites linking the glycolysis pathway to SCW polymer biosynthesis based on transcriptome analysis, are actively regulated (Fig. 7). These results suggest that active regulation of primary metabolism could be the basis of protoxylem vessel element differentiation.

Based on the data described here, we can speculate on the regulatory dynamics of primary metabolism during protoxylem vessel element differentiation as follows. At the initiation of xylem vessel cell differentiation, the amounts of GAP decrease (Fig. 3), which is accompanied by the up-regulation of genes for enzymes that convert GAP to Fru-6-P (Fig. 5). Then, it seems that the transcription levels of genes involved in the Fru-6-P-to-pyruvate flow in the glycolysis pathway are down-regulated, as the decreased expression of genes corresponding to several steps of the glycolysis pathway became apparent after 12 h of DEX treatment (Fig. 5). These observations suggest that GAP metabolism has important functions in the glycolysis pathway during protoxylem vessel element differentiation, probably involving the concentration of carbon sources within the glycolysis pathway to Fru-6-P by arresting glycolysis in the upstream steps of GAP. Fru-6-P is a key metabolite converted into NDP-sugar and/or connecting glycolysis with the pentose phosphate pathway (Bar-Peled and O’Neill, 2011; Roach et al., 2012). NDP-sugars are building units of cell wall polysaccharides; therefore, the activation of Fru-6-P biosynthesis is possibly connected to the activation of SCW formation during protoxylem vessel element differentiation. Our data showed similar patterns for Fru-6-P and UDP-Glc levels during cell differentiation (Fig. 7), supporting the idea that Fru-6-P biosynthesis can influence NDP-sugar biosynthetic activity.

Previous studies suggested the importance of Fru-6-P biosynthesis by fructokinase activity for xylem development and hydraulic conductance (Damari-Weissler et al., 2009; Roach et al., 2012; Stein et al., 2016). Particularly, the RNA interference hybrid aspen plants for fructokinase isoforms, in which the Fru-6-P-producing activity by fructokinase was reduced, showed the decrease in both Fru-6-P and UDP-Glc, resulting in thinner fiber cell walls with a reduction in the proportion of cellulose (Roach et al., 2012). Based on these results, the involvement of fructokinase in carbon partitioning to cellulose during wood formation was revealed (Roach et al., 2012). Our transcriptome data indicated that the expression levels of three of 17 contigs corresponding to fructokinase genes were up-regulated during protoxylem vessel element differentiation (Supplemental Table S6). Therefore, it is plausible that the Fru-6-P biosynthesis by multiple pathways might be activated for SCW formation during protoxylem vessel element differentiation.

In differentiating protoxylem vessel elements, the transcriptional up-regulation of enzyme genes involved in Phe biosynthesis was prominent (Fig. 6). The expression of shikimate pathway genes is decreased in Arabidopsis nac secondary wall thickening promoting factor1 (nst1) secondary wall-associated nac domain protein 1 (snd1)/nst3 double mutants, which are defective in the differentiation of fiber cells, SCW-containing supporting cells (Mitsuda et al., 2007). The NST1 and SND1/NST3 proteins are NAC transcription factors belonging to the sister group of VND family genes; therefore, these SCW-related NAC proteins can commonly up-regulate genes of the shikimate pathway. Moreover, we found that genes for PEP carboxykinase were up-regulated during the early stages of protoxylem vessel element differentiation (Fig. 5), suggesting that the conversion of oxaloacetate into PEP, one of the starting materials of Phe biosynthesis (Fig. 6), was activated by VND7 induction. Indeed, the PEP level increased as cell differentiation progressed (Fig. 7B). These results imply that carbon sources within the tricarboxylic acid cycle might be reallocated to Phe for lignin biosynthesis through PEP production.

Finally, we found that the contents of Leu, Ile, and Arg, all of which represent the ends of amino acid biosynthetic pathways, also increased after DEX treatment (Figs. 3 and 4; Supplemental Table S3). Long-distance transport of amino acids through the xylem is important for the proper distribution of nitrogen in plants (Okumoto and Pilot, 2011). Arg is a translocatable amino acid that can be used as a relatively effective nitrogen resource by plants (Furuhashi and Yatazawa, 1970). Thus, these accumulated amino acids could be transferred to shoot regions for the recycling of nitrogen within plant bodies after the completion of PCD of developing protoxylem vessel elements.

Taken together, our findings point to a system that regulates plant cell metabolic activity to biosynthesize SCW-specific polymers during protoxylem vessel element differentiation. In this system, a shift from primary cell wall biosynthetic mode to SCW biosynthetic mode would be achieved through the active transcriptional regulation of genes encoding primary metabolic enzymes, especially at key catalytic steps, such as GAP metabolism, including Fru-6-P production and PEP metabolism. This work thus suggests a novel strategy for improving woody biomass production that involves the enhanced flow of carbon resources from primary metabolites to SCW polymers.

MATERIALS AND METHODS

Plant Material and DEX Treatment

Tobacco (Nicotiana tabacum) BY-2 suspension culture cells with the VND7-VP16-GR system (VND7-VP16-GR BY-2 cells) were used (Yamaguchi et al., 2010; Goué et al., 2013). Batches of 100 mL of BY-2 cell suspension cultures were grown in 300-mL conical flasks. Liquid suspension cultures were diluted 95-fold at weekly intervals with medium containing Murashige and Skoog salt mixture (Wako), 0.2 mg mL−1 KH2PO4, 0.1 mg mL−1 myoinositol, 1 mg L−1 thiamine-HCl, 0.2 mg L−1 2,4-dichlorophenoxyacetic acid, and 30 g L−1 Suc (pH 5.8) and then transferred to a rotary shaker at 130 rpm at 27°C in the dark. The medium was supplemented with 100 mg L−1 kanamycin. For time-series sampling in the metabolome and transcriptome analyses, the culture scale was increased to 300 mL of BY-2 cell cultures in 1-L flasks.

To induce the differentiation of protoxylem vessel elements, DEX was added to the 4-d-old cell cultures to a final concentration of 1 µm. Samples were cultured with agitation in the dark at 27°C for 0, 6, 12, 24, 36, and 48 h prior to harvesting. At each sampling, 5 mL of cell culture was harvested and centrifuged (240g, 2 min, at room temperature) to collect the cells. The centrifugation was repeated 10 times with distilled water for washing, and then the cells were subjected to metabolome and transcriptome analyses. The DEX treatment and cell harvesting were performed three times for triplicate experiments.

Wide-Target Metabolome Analysis

After harvesting, the cells were freeze-dried in a vacuum freeze dryer (FZ-2.5; Labconco) and then subjected to wide-target metabolome analysis by the methods described by Sawada et al. (2009) with minor modifications. The freeze-dried BY-2 cell samples were crushed with a bead shaker (1,000 rpm, 2 min; ShakeMaster Neo; BMS), and 4 ± 0.2 mg of sample was extracted with 80% (v/v) methanol by crushing with 5-mm zirconia beads (1,000 rpm for 2 min). The extracts were diluted 10-fold with 80% (v/v) methanol, 25 µL of each extract was transferred to a 96-well plate, and the solutions were dried under N2 gas using a 96-well format spray instrument (40°C for 25 min and 30°C for 20 min). To prepare the samples for liquid chromatography-tandem mass spectrometry analysis, the dried samples were dissolved in 250 μL of water and then filtered twice through a 384-well filter plate (Whatman). The prepared samples were analyzed in a UPLC-TQC machine (Waters) with solvents A, 0.1% (v/v) formic acid in water (Thermo), and B, 0.1% (v/v) formic acid in acetonitrile (Wako). The gradient program and tandem quadrupole-mass spectrometry conditions were described by Sawada et al. (2009).

After missing values were set to 20, the signal intensities of three samples were averaged. The metabolites whose signal-to-noise ratios were less than 3 in all 18 experimental groups were removed. Metabolites whose relative sd was greater than 30% in all 18 experimental groups also were removed, leaving 128 metabolites for further analyses (criterion A). Subsequent data analysis was performed according to Sawada et al. (2009). Finally, the metabolome data for three replicate experiments of DEX treatment were obtained.

RNA-seq Analysis

The VND7-VP16-GR BY-2 cells were sampled after 0, 6, 12, 24, and 36 h of DEX treatment as described above and subjected to total RNA extraction with the RNeasy Mini Kit (Qiagen). The quality of each total RNA sample was examined using the Agilent RNA 6000 Pico LabChip Kit in the Agilent 2100 bioanalyzer. The mRNA fractions were isolated from 800 ng of total RNAs with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). The cDNA libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs), with NEBNext Multiplex Oligos for Illumina (New England Biolabs). All procedures were performed according to the manufacturer’s instructions. The quality and quantity of each library were analyzed using the High Sensitivity DNA Kit (Agilent) and by subsequent quantitative PCR (KAPA), respectively. Sequencing was carried out with the Genome Analyzer IIx (Illumina).

The obtained short reads (approximately 530 million reads of 32-bp length) were assembled using Trinity (version 2.0.6; Grabherr et al., 2011) via the genome_guided_bam option with the tobacco “TN90” genome sequence (Sierro et al., 2014). A total of 74,932 unique contigs were constructed. The longest contig was 5,437 bp, the shortest contig was 150 bp, the average length of contigs was 707.31 bp, and the N50 length was 1,006 bp. The sequence tags were mapped to the contigs using TopHat2 (version 2.0.13; Kim et al., 2013) to calculate the reads per kilobase per million mapped reads for each contig by Cuffdiff2 (version 2.2.1; Trapnell et al., 2013). For the expression analysis of putative homologous genes of Arabidopsis (Arabidopsis thaliana) VND7-target genes, BLAST searches were performed using the Arabidopsis genes listed as target genes of VND7 as queries (Yamaguchi et al., 2011; Xu et al., 2014; TBLASTN search; E < 0.00001). For the enzyme genes involved in glycolysis and amino acid biosynthesis, BLAST searches were performed (TBLASTN search; E < 0.00001) using Arabidopsis and tomato (Solanum lycopersicum) gene sequences that were annotated in the Plant Metabolic Network Web site (http://www.plantcyc.org/; Zhang et al., 2010) as queries. For clustering representation of expression (reads per kilobase per million mapped reads) patterns of the BY-2 contigs putatively homologous to Arabidopsis VND7 direct target genes, the soft clustering algorithm was utilized with the R software package Mfuzz (Kumar and Futschik, 2007).

Quantitative Reverse Transcription-PCR Analysis

To check the reliability of the mRNA-seq analysis, quantitative reverse transcription (RT)-PCR was performed with the selected putative homologous genes of Arabidopsis VND7-target genes (Supplemental Fig. S2). Total RNAs extracted as described above were subjected to quantitative RT-PCR. Five micrograms of total RNAs was reverse transcribed with oligo(dT)15 primer (Roche) and the Transcriptor First Strand cDNA Synthesis Kit (Roche) to synthesize template cDNA. After 10-fold dilution, 1 µL of template cDNA was subjected to absolute quantification analysis using the LightCycler 480 SYBR Green I Master (Roche) and the LightCycler 480 System (Roche). Nicotiana tabacum elongation factor 1-alpha gene (NtEF1α) was used as the internal control. The results of the quantitative RT-PCR analysis showed good agreement with RNA-seq data. The primer sequences are shown in Supplemental Table S7.

Sampling Procedures for CE-MS-Based Metabolic Profiling

Lyophilized cells (equivalent to 2–3 mg dry weight) were suspended in the mixture of 1 mL of prechilled (−30°C) methanol containing 30.9 μm piperazine-1,4-bis(2-ethanesulfonic acid) as an internal standard, 300 μL of chloroform, and 100 μL of prechilled (4°C) water. After 1 h of bead-beater cell breaking and centrifugation at 14,000g for 5 min at 4°C, 800 μL of supernatant was transferred to a new tube. The cell extract was mixed with 325 μL of prechilled (4°C) water by vortexing. After centrifugation at 14,000g for 5 min at 4°C, 600 μL of the aqueous layer was transferred onto a Millipore 3-kD cutoff filter for the removal of solubilized proteins. After the filtration, 400 μL of the aqueous layer extract was evaporated under vacuum using a CVE-3100 freeze-dry system (Tokyo Rikakikai). Dried extracts were stored at −80°C until analysis by CE-MS. CE-MS analysis followed the methods described by Hasunuma et al. (2016).

Thioglycolic Acid Lignin Determination

Thioglycolic acid lignin was determined as described by Suzuki et al. (2009) with some modifications after the starch-removal treatment. Briefly, about 3 mg of freeze-dried cells was treated with α-amylase (Sigma) solution (1 mL) for 16 h at 37°C. After the treatment, the mixture was centrifuged at 20,000g for 10 min, and the supernatant was discarded. After washing of the pellet with water and methanol (each 2 mL), the pellet was dried and weighed. The residue (about 1.5 mg) was treated with 0.5 mL of 3 m HCl and 0.1 mL of thioglycolic acid (Nakalaitesque) for 3 h at 80°C. The reaction mixture was cooled on ice for 5 min and centrifuged at 20,000g for 10 min. The pellet was vortexed in water (2 mL) and centrifuged at 20,000g for 10 min. This washing and centrifugation was repeated once more. The resulting pellet was dissolved in 1 mL of 1 m NaOH for 16 h at room temperature and centrifuged at 20,000g for 10 min. The supernatant was acidified with 0.1 mL of concentrated HCl and incubated at 4°C overnight. The resulting precipitates were collected by centrifugation at 20,000g for 10 min, washed with 2 mL of water twice, and dried. The dried pellet was dissolved in 0.25 mL of 1 m NaOH. A portion of the solution (0.2 mL) was used for the measurement of A280 on a Greiner UV Star 96-well plate (Greiner bio-one) in a microplate reader (SH-1000 Lab; Corona Electric). The calibration curve used was as described by Suzuki et al. (2009).

Large Data Sets

The mRNA-seq data presented in this study were submitted to the DNA Data Bank of Japan Sequence Read Archive (http://trace.ddbj.nig.ac.jp/dra/index_e.html) and can be retrieved via accession number PRJDB5152.

Supplemental Data

The following supplemental materials are available.

  • Supplemental Figure S1. Changes in amino acid-related metabolites derived from intermediate compounds in the glycolysis pathway in the wild-type BY-2 cells.

  • Supplemental Figure S2. Changes in amino acid-related metabolites derived from oxaloacetate and 2-oxoglutarate in the wild-type BY-2 cells.

  • Supplemental Figure S3. Clustering analysis of BY-2 contigs putatively homologous to Arabidopsis VND7 direct target genes.

  • Supplemental Figure S4. Quantitative RT-PCR analysis of selected BY-2 contigs putatively homologous to Arabidopsis VND7 direct target genes during protoxylem vessel element differentiation.

  • Supplemental Figure S5. Changes in the expression of genes putatively involved in glycolysis in mock-treated VND7-VP16-GR BY-2 cells.

  • Supplemental Figure S6. Changes in the expression of genes putatively involved in Phe biosynthesis in mock-treated VND7-VP16-GR BY-2 cells.

  • Supplemental Table S1. Correlated metabolites of PC1 within PCA analysis of time-sequential wide-target metabolome data for protoxylem vessel element differentiation.

  • Supplemental Table S2. Correlated metabolites of PC2 within PCA analysis of time-sequential wide-target metabolome data for protoxylem vessel element differentiation.

  • Supplemental Table S3. Relative amount data for 128 metabolites in VND7-VP16-GR BY-2 cells during protoxylem vessel element differentiation.

  • Supplemental Table S4. Relative amount data for 128 metabolites in wild-type BY-2 cells after DEX treatment.

  • Supplemental Table S5. Expression profiles of BY-2 contigs putatively homologous to Arabidopsis VND7 direct target genes.

  • Supplemental Table S6. Expression profiles of BY-2 contigs putatively involved in primary metabolic regulation.

  • Supplemental Table S7. Primer sequences used in this study.

Supplementary Material

Supplemental Data
supp_172_3_1612__index.html (1.2KB, html)

Acknowledgments

We thank Muneo Sato, Yutaka Yamada, and Akane Sakata (RIKEN) for excellent technical assistance in LC-MS analysis; Megumi Ozaki and Yumiko Oku (Kyoto University) for excellent technical assistance in lignin determination; and Dr. Minoru Kubo, Dr. Arata Yoneda, Dr. Ko Kato, Dr. Hitoshi Endo, and Dr. Bo Xu (NAIST) for fruitful discussions. Lignin analysis was carried out by the FBAS collaborative program of the Research Institute for Sustainable Humanosphere, Kyoto University.

Glossary

SCW

secondary cell wall

PCD

programmed cell death

LC-MS

liquid chromatography-mass spectrometry

mRNA-seq

mRNA sequencing

PEP

phosphoenolpyruvate

DEX

dexamethasone

PCA

principal component analysis

GAP

glyceraldehyde 3-phosphate

Fru-1,6-bP

fructose 1,6-bisphosphate

FC

fold change

CE-MS

capillary electrophoresis-mass spectrometry

RT

reverse transcription

Footnotes

1

This work was supported by the Naito Foundation (to M.O.), the Japan Society for the Promotion of Science (KAKENHI grant no. 25291062 to T.D.), the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant nos. 25114520 and 15H01235 to M.O., grant no. 24114002 to T.D., and Grants-in-Aid from the NC-CARP project to T.D.), and the Japan Advanced Plant Science Network.

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References

  1. Abramson M, Shoseyov O, Shani Z (2010) Plant cell wall reconstruction toward improved lignocellulosic production and processability. Plant Sci 178: 61–72 [Google Scholar]
  2. Allona I, Quinn M, Shoop E, Swope K, St Cyr S, Carlis J, Riedl J, Retzel E, Campbell MM, Sederoff R, et al. (1998) Analysis of xylem formation in pine by cDNA sequencing. Proc Natl Acad Sci USA 95: 9693–9698 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arruda P, Kemper EL, Papes F, Leite A (2000) Regulation of lysine catabolism in higher plants. Trends Plant Sci 5: 324–330 [DOI] [PubMed] [Google Scholar]
  4. Bar-Peled M, O’Neill MA (2011) Plant nucleotide sugar formation, interconversion, and salvage by sugar recycling. Annu Rev Plant Biol 62: 127–155 [DOI] [PubMed] [Google Scholar]
  5. Bashline L, Li S, Gu Y (2014) The trafficking of the cellulose synthase complex in higher plants. Ann Bot (Lond) 114: 1059–1067 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bollhöner B, Prestele J, Tuominen H (2012) Xylem cell death: emerging understanding of regulation and function. J Exp Bot 63: 1081–1094 [DOI] [PubMed] [Google Scholar]
  7. Damari-Weissler H, Rachamilevitch S, Aloni R, German MA, Cohen S, Zwieniecki MA, Holbrook NM, Granot D (2009) LeFRK2 is required for phloem and xylem differentiation and the transport of both sugar and water. Planta 230: 795–805 [DOI] [PubMed] [Google Scholar]
  8. Demura T, Tashiro G, Horiguchi G, Kishimoto N, Kubo M, Matsuoka N, Minami A, Nagata-Hiwatashi M, Nakamura K, Okamura Y, et al. (2002) Visualization by comprehensive microarray analysis of gene expression programs during transdifferentiation of mesophyll cells into xylem cells. Proc Natl Acad Sci USA 99: 15794–15799 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Derbyshire P, Ménard D, Green P, Saalbach G, Buschmann H, Lloyd CW, Pesquet E (2015) Proteomic analysis of microtubule interacting proteins over the course of xylem tracheary element formation in Arabidopsis. Plant Cell 27: 2709–2726 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Endo H, Yamaguchi M, Tamura T, Nakano Y, Nishikubo N, Yoneda A, Kato K, Kubo M, Kajita S, Katayama Y, et al. (2015) Multiple classes of transcription factors regulate the expression of VASCULAR-RELATED NAC-DOMAIN7, a master switch of xylem vessel differentiation. Plant Cell Physiol 56: 242–254 [DOI] [PubMed] [Google Scholar]
  11. Facchini PJ, Huber-Allanach KL, Tari LW (2000) Plant aromatic L-amino acid decarboxylases: evolution, biochemistry, regulation, and metabolic engineering applications. Phytochemistry 54: 121–138 [DOI] [PubMed] [Google Scholar]
  12. Faik A. (2010) Xylan biosynthesis: news from the grass. Plant Physiol 153: 396–402 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fukuda H, Komamine A (1980) Establishment of an experimental system for the study of tracheary element differentiation from single cells isolated from the mesophyll of Zinnia elegans. Plant Physiol 65: 57–60 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Furuhashi K, Yatazawa M (1970) Amino acids as nitrogen sources for the growth of rice callus tissue. Plant Cell Physiol 11: 559–567 [Google Scholar]
  15. Goué N, Mortimer JC, Nakano Y, Zhang Z, Josserand M, Ohtani M, Paul D, Kakegawa K, Demura T (2013) Secondary cell wall characterization in a BY-2 inductive system. Plant Cell Tiss Org 115: 223–232 [Google Scholar]
  16. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, et al. (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29: 644–652 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hasunuma T, Matsuda M, Kondo A (2016) Improved sugar-free succinate production by Synechocystis sp. PCC 6803 following identification of the limiting steps in glycogen catabolism. Metab Eng Commun 3: 130–141 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hussey SG, Mizrachi E, Creux NM, Myburg AA (2013) Navigating the transcriptional roadmap regulating plant secondary cell wall deposition. Front Plant Sci 4: 325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kalluri UC, Hurst GB, Lankford PK, Ranjan P, Pelletier DA (2009) Shotgun proteome profile of Populus developing xylem. Proteomics 9: 4871–4880 [DOI] [PubMed] [Google Scholar]
  20. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14: R36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ko JH, Jeon HW, Kim WC, Kim JY, Han KH (2014) The MYB46/MYB83-mediated transcriptional regulatory programme is a gatekeeper of secondary wall biosynthesis. Ann Bot 114: 1099–1107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ko JH, Kim WC, Kim JY, Ahn SJ, Han KH (2012) MYB46-mediated transcriptional regulation of secondary wall biosynthesis. Mol Plant 5: 961–963 [DOI] [PubMed] [Google Scholar]
  23. Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T (2005) Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev 19: 1855–1860 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kumar L, Futschik ME (2007) Mfuzz: a software package for soft clustering of microarray data. Bioinformation 2: 5–7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maeda H, Dudareva N (2012) The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu Rev Plant Biol 63: 73–105 [DOI] [PubMed] [Google Scholar]
  26. McCarthy RL, Zhong R, Fowler S, Lyskowski D, Piyasena H, Carleton K, Spicer C, Ye ZH (2010) The poplar MYB transcription factors, PtrMYB3 and PtrMYB20, are involved in the regulation of secondary wall biosynthesis. Plant Cell Physiol 51: 1084–1090 [DOI] [PubMed] [Google Scholar]
  27. McFarlane HE, Döring A, Persson S (2014) The cell biology of cellulose synthesis. Annu Rev Plant Biol 65: 69–94 [DOI] [PubMed] [Google Scholar]
  28. Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, Ohme-Takagi M (2007) NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19: 270–280 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Myburg AA, Sederoff RR (2001) Xylem structure and function. In eLS. John Wiley & Sons, Chichester, UK: doi/10.1038/npg.els.0001302 [Google Scholar]
  30. Nakano Y, Yamaguchi M, Endo H, Rejab NA, Ohtani M (2015) NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants. Front Plant Sci 6: 288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ohashi-Ito K, Oda Y, Fukuda H (2010) Arabidopsis VASCULAR-RELATED NAC-DOMAIN6 directly regulates the genes that govern programmed cell death and secondary wall formation during xylem differentiation. Plant Cell 22: 3461–3473 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Okumoto S, Pilot G (2011) Amino acid export in plants: a missing link in nitrogen cycling. Mol Plant 4: 453–463 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pagter M, Sergeant K, Møller SM, Bertram HCS, Reneaut J (2014) Changes in the proteome and water state in bark and xylem of Hydrangea paniculata during loss of freezing tolerance. Environ Exp Bot 106: 99–111 [Google Scholar]
  34. Pesquet E, Korolev AV, Calder G, Lloyd CW (2010) The microtubule-associated protein AtMAP70-5 regulates secondary wall patterning in Arabidopsis wood cells. Curr Biol 20: 744–749 [DOI] [PubMed] [Google Scholar]
  35. Ralph SG, Chun HJ, Kolosova N, Cooper D, Oddy C, Ritland CE, Kirkpatrick R, Moore R, Barber S, Holt RA, et al. (2008) A conifer genomics resource of 200,000 spruce (Picea spp.) ESTs and 6,464 high-quality, sequence-finished full-length cDNAs for Sitka spruce (Picea sitchensis). BMC Genomics 9: 484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rengel D, San Clemente H, Servant F, Ladouce N, Paux E, Wincker P, Couloux A, Sivadon P, Grima-Pettenati J (2009) A new genomic resource dedicated to wood formation in Eucalyptus. BMC Plant Biol 9: 36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Roach M, Gerber L, Sandquist D, Gorzsás A, Hedenström M, Kumar M, Steinhauser MC, Feil R, Daniel G, Stitt M, et al. (2012) Fructokinase is required for carbon partitioning to cellulose in aspen wood. Plant J 70: 967–977 [DOI] [PubMed] [Google Scholar]
  38. Sawada Y, Akiyama K, Sakata A, Kuwahara A, Otsuki H, Sakurai T, Saito K, Hirai MY (2009) Widely targeted metabolomics based on large-scale MS/MS data for elucidating metabolite accumulation patterns in plants. Plant Cell Physiol 50: 37–47 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sierro N, Battey JND, Ouadi S, Bakaher N, Bovet L, Willig A, Goepfert S, Peitsch MC, Ivanov NV (2014) The tobacco genome sequence and its comparison with those of tomato and potato. Nat Commun 5: 3833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stein O, Damari-Weissler H, Secchi F, Rachamilevitch S, German MA, Yeselson Y, Amir R, Schaffer A, Holbrook NM, Aloni R, et al. (2016) The tomato plastidic fructokinase SlFRK3 plays a role in xylem development. New Phytol 209: 1484–1495 [DOI] [PubMed] [Google Scholar]
  41. Sterky F, Bhalerao RR, Unneberg P, Segerman B, Nilsson P, Brunner AM, Charbonnel-Campaa L, Lindvall JJ, Tandre K, Strauss SH, et al. (2004) A Populus EST resource for plant functional genomics. Proc Natl Acad Sci USA 101: 13951–13956 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Suzuki S, Suzuki Y, Yamamoto N, Hattori T, Sakamoto M, Umezawa T (2009) High-throughput determination of thioglycolic acid lignin from rice. Plant Biotechnol 26: 337–340 [Google Scholar]
  43. Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L (2013) Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol 31: 46–53 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Turner S, Gallois P, Brown D (2007) Tracheary element differentiation. Annu Rev Plant Biol 58: 407–433 [DOI] [PubMed] [Google Scholar]
  45. Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W (2010) Lignin biosynthesis and structure. Plant Physiol 153: 895–905 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Xu B, Ohtani M, Yamaguchi M, Toyooka K, Wakazaki M, Sato M, Kubo M, Nakano Y, Sano R, Hiwatashi Y, et al. (2014) Contribution of NAC transcription factors to plant adaptation to land. Science 343: 1505–1508 [DOI] [PubMed] [Google Scholar]
  47. Yamaguchi M, Goué N, Igarashi H, Ohtani M, Nakano Y, Mortimer JC, Nishikubo N, Kubo M, Katayama Y, Kakegawa K, et al. (2010) VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system. Plant Physiol 153: 906–914 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yamaguchi M, Kubo M, Fukuda H, Demura T (2008) Vascular-related NAC-DOMAIN7 is involved in the differentiation of all types of xylem vessels in Arabidopsis roots and shoots. Plant J 55: 652–664 [DOI] [PubMed] [Google Scholar]
  49. Yamaguchi M, Mitsuda N, Ohtani M, Ohme-Takagi M, Kato K, Demura T (2011) VASCULAR-RELATED NAC-DOMAIN7 directly regulates the expression of a broad range of genes for xylem vessel formation. Plant J 66: 579–590 [DOI] [PubMed] [Google Scholar]
  50. Zhang P, Dreher K, Karthikeyan A, et al. (2010) Creation of a Genome-Wide Metabolic Pathway Database for Populus trichocarpa Using a New Approach for Reconstruction and Curation of Metabolic Pathways for Plants. Plant Physiology 153: 1479–1491 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhong R, Lee C, Ye ZH (2010) Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis. Mol Plant 3: 1087–1103 [DOI] [PubMed] [Google Scholar]
  52. Zhong R, Ye ZH (2012) MYB46 and MYB83 bind to the SMRE sites and directly activate a suite of transcription factors and secondary wall biosynthetic genes. Plant Cell Physiol 53: 368–380 [DOI] [PubMed] [Google Scholar]

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supp_172_3_1612__index.html (1.2KB, html)
supp_pp.16.01230_PP2016-01230DR1_Supplemental_Figures.pdf (1.6MB, pdf)
supp_pp.16.01230_PP2016-01230DR1_Supplemental_Table.xlsx (448.9KB, xlsx)

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