Plant Physiology Preview. Published on September 6, 2016, as DOI:10.1104/pp.16.01230
1
Short title: Metabolic control for protoxylem cell formation
2
3
Corresponding author:
4
Taku Demura
5
Professor
6
Graduate School of Biological Sciences
7
Nara Institute of Science and Technology
8
Ikoma, 630-0192, Japan
9
Tel: +81-743-72-5460
10
Fax: +81-743-72-5469
11
E-mail: [email protected]
12
1
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Copyright 2016 by the American Society of Plant Biologists
13
Primary metabolism during biosynthesis of secondary wall polymers of protoxylem vessel
14
elements
15
16
Misato Ohtani, Keiko Morisaki, Yuji Sawada, Ryosuke Sano, Abigail Loren Tung Uy, Atsushi
17
Yamamoto, Tetsuya Kurata, Yoshimi Nakano, Shiro Suzuki, Mami Matsuda, Tomohisa
18
Hasunuma, Masami Yokota Hirai, Taku Demura*
19
20
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara,
21
630-0192 Japan (M.O., K.M., R.S., A.L.T.U., A.Y., T.K., Y.N., T.D.); Graduate School of Life
22
Sciences, Tohoku University, Sendai, Miyagi, 980-8578 Japan (T.K.); Bioproduction Research
23
Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki,
24
305-8566, Japan (Y.N.); Research Institute for Sustainable Humanosphere, Kyoto University, Uji,
25
Kyoto 611-0011, Japan (S.S.); Graduate School of Science, Technology and Innovation, Kobe
26
University 1-1 Rokkodai, Nada, Kobe 657-8501, Japan (M.M., T.H.); and RIKEN Center for
27
Sustainable Resource Science, Yokohama, Kanagawa, 230-0045 Japan (M.O., Y.S., M.Y.H.,
28
T.D.)
29
30
One sentence summary
31
Primary metabolism is actively regulated for the biosynthesis of secondary wall polymers during
32
differentiation of protoxylem vessel elements.
33
2
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
34
1
35
Promotion of Science (KAKENHI Grant Number 25291062 to T.D.), and the Ministry of
36
Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific
37
Research on Innovative Areas “The Plant Cell Wall as Information Processing System” Grant
38
Number 25114520 and 15H01235 to M.O., 24114002 to T.D., and Grants-in-Aid from the
39
NC-CARP project to T.D.), and Japan Advanced Plant Science Network.
40
* Address correspondence to [email protected].
This work was supported in part by The Naito Foundation (to M.O.), Japan Society for the
41
The authors responsible for distribution of materials integral to the findings to the findings
42
presented in this article in accordance with the policy described in the Instructions for Authors
43
(www.plantphysiol.org) is: Taku Demura ([email protected]).
44
M.O. and T.D. designed the research; M.O., K.M., Y.S., Y.N., S.S., and M.M. performed the
45
experiments; M.O., K.M., Y.S., R.S., A.L.T.U., T.K., T.H., M.H., and T.D. analyzed the data;
46
M.O. and T.D. wrote the article.
3
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
47
Abstract
48
Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary
49
wall deposition and programmed cell death. These processes are regulated by the
50
VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes
51
in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco
52
BY-2 suspension culture cells carrying an inducible VND7 system to LC/MS-based wide-target
53
metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed
54
dynamic changes in metabolites related to amino acid biosynthesis. The concentration of
55
glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately
56
decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific
57
amino acids accumulated, including the shikimate-related amino acids and the translocatable
58
nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved
59
active upregulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis
60
from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and Phe
61
biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the
62
amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation.
63
Taken together, our results show that protoxylem vessel element differentiation is associated with
64
changes in primary metabolism, which could facilitate the production of polysaccharides and
65
lignin monomers, and thus promote formation of the secondary cell wall. Also, these metabolic
66
shifts correlate with active transcriptional regulation of specific enzyme genes. Therefore, our
67
observations indicate that primary metabolism is actively regulated during protoxylem vessel
4
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
68
element differentiation to alter the cell’s metabolic activity for the biosynthesis of secondary wall
69
polymers.
5
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
70
INTRODUCTION
71
72
Xylem vessels conduct water and nutrients in vascular plants (Myburg and Sederoff, 2001;
73
Turner et al., 2007). Xylem vessel cells have a characteristic thick and patterned secondary cell
74
wall (SCW) and undergo programmed cell death (PCD). SCW biopolymers such as cellulose,
75
hemicellulose, and lignin are major constituents of terrestrial lignocellulosic biomass. As
76
lignocellulose is recalcitrant to enzymatic hydrolysis, it has limited utility as a renewable
77
resource. Improving our understanding of the molecular mechanisms underlying xylem vessel
78
cell differentiation will facilitate the development of strategies to overcome this limitation
79
(Abramson et al., 2010).
80
Xylem vessel cell differentiation, which has been extensively studied using an in vitro
81
induction system (Fukuda and Komamine, 1980; Demura et al., 2002; Kubo et al., 2005; Pesquet
82
et al., 2010), initiates with the transcriptional upregulation of SCW- and PCD-related genes by
83
the VASCULAR-RELATED NAC-DOMAIN (VND) master regulatory transcription factors
84
(Kubo et al., 2005; Yamaguchi et al., 2008; Zhong et al., 2010; Ohashi-Ito et al., 2010;
85
Yamaguchi et al., 2011; Endo et al., 2015; Nakano et al., 2015). The first detectable cytological
86
sign of differentiation is the alignment of cortical microtubules, which determine patterned SCW
87
thickening by guiding the cellulose synthase complex (Bashline et al., 2014; McFarlane et al.,
88
2014). Next, cellulose and hemicellulose accumulate, followed by lignin deposition (Faik, 2010;
89
Vanholme et al., 2010). Vacuole disruption triggers PCD, which results in proteases and nucleases
90
being released into the cytosol (Bollhöner et al., 2012). During the final stages of differentiation,
6
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
91
the end walls are perforated and the cell contents exit the cells, completing the differentiation of
92
the hollow tube structure (Turner et al., 2007).
93
Transcriptome analysis of xylem tissues (for loblolly pine: Allona et al., 1998; for poplar:
94
Sterky et al., 1998; for spruce: Ralph et al., 2008; for eucalyptus: Rengel et al., 2009) and studies
95
in the in vitro system (Demura et al., 2002; Ohashi-Ito et al., 2010; Zhong et al., 2010;
96
Yamaguchi et al., 2011) have identified many genes involved in xylem cell differentiation. These
97
include VND family genes and SCW-related genes encoding MYB transcription factors
98
(McCarthy et al., 2009; Ko et al., 2012, 2014; Zhong and Ye, 2012; Hussey et al., 2013); based
99
on this information, NAC-MYB-based transcriptional networks have been proposed for xylem
100
vessel cell differentiation (Nakano et al., 2015). Moreover, proteome data have been reported for
101
xylem tissues of hybrid aspen (Kalluri et al., 2009) and Hydrangea paniculata (Pagter et al.,
102
2014).
103
microtubule-interacting proteins involved in xylem vessel cell differentiation (Derbyshire et al.,
104
2015).
A
recent
study
also
reported
a
quantitative
proteome
analysis
targeting
105
While these transcriptome and proteome studies have provided insight into xylem cell
106
differentiation, little is known about the primary metabolic changes that occur during xylem
107
vessel cell differentiation. Regulation of the biosynthesis of SCW-related metabolites, such as
108
cellulose and its precursor UDP-glucose, the hemicellulosic polysaccharide xylan, and lignin
109
monomers, has attracted substantial attention (Bashline et al., 2014; McFarlane et al., 2014; Faik,
110
2010; Vanholme et al., 2010). However, we lack information about the regulation of primary
111
metabolism during xylem vessel cell differentiation.
7
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
112
In the present study, we performed a wide-target metabolome analysis of protoxylem
113
vessel element differentiation using a tobacco BY-2 in vitro system (VND7-VP16-GR; Yamaguchi
114
et al., 2010; Goué et al., 2013) that allows the effective induction of protoxylem-type vessel
115
element differentiation. Induced BY-2 cells were subjected to LC/MS-based wide-target
116
metabolome analysis as established by Sawada et al. (2009), which can automatically detect
117
approximately 700 metabolites that are the product of metabolic regulatory pathways (Sawada et
118
al., 2012). This method can provide data on general metabolites during protoxylem vessel
119
element formation with high reproducibility. Indeed, we successfully obtained time-course
120
metabolome data during protoxylem vessel element differentiation. In addition, we performed
121
mRNA-seq analysis on the same differentiating BY-2 cells and examined the trends in transcript
122
levels of genes encoding enzymes involved in the catalytic pathways of the metabolites detected
123
by the metabolome analysis. Subsequent target quantification analysis showed active changes in
124
fructose 6-phosphate (Fru-6-P), phosphoenolpyruvate (PEP), and UDP-glucose during cell
125
differentiation. These data suggested that active regulation of glycolysis and amino acid
126
biosynthesis at the transcriptional level alters cell activity, shifting metabolism toward the
127
biosynthesis of specific kinds of metabolites, such as Fru-6-P, a precursor of NDP-sugar, and PEP,
128
a precursor of Phe for lignin monomers, which are important building blocks of the SCW of
129
protoxylem vessel element.
8
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
130
9
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
131
RESULTS
132
133
Widely targeted metabolome analysis of protoxylem vessel element differentiation in a
134
tobacco BY-2 in vitro system
135
For the widely targeted metabolome analysis during protoxylem vessel element
136
differentiation, we used the established tobacco BY-2 in vitro system for protoxylem vessel
137
element differentiation (VND7-VP16-GR; Yamaguchi et al., 2010; Goué et al., 2013). In this
138
system, the VND7-VP16-GR fusion protein is continuously expressed under the control of the
139
35S promoter, and its transcription factor activity can be post-translationally activated by
140
treatment with dexamethasone (DEX). Following DEX treatment, the VND7-VP16-GR BY-2
141
cells effectively trans-differentiate into protoxylem vessel elements (Yamaguchi et al., 2010;
142
Goué et al., 2013). Figure 1 shows the transitional differentiation rates of VND7-VP16-GR BY-2
143
cells after DEX treatment (Fig. 1A). The first visible sign of cell differentiation was the weak
144
deposition of helically patterned-SCW after 24 h of DEX treatment (Fig. 1C). At this stage, the
145
cells with patterned SCWs had visible nuclei, indicating that these cells were living and
146
undergoing differentiation as protoxylem vessel elements (Fig. 1C). Then, after 36 h of DEX
147
treatment, we observed cells with thick helical SCWs but without cell contents, i.e., differentiated
148
protoxylem vessel elements (Fig. 1D). Ultimately, ~70% of VND7-VP16-GR BY-2 cells
149
differentiated into protoxylem vessel elements (Fig. 1A). Previously we observed that the
150
upregulation of SCW biosynthetic genes could be detected after 6 h of DEX treatment (Goué et
151
al., 2013), suggesting that protoxylem vessel element differentiation initiated at least at 6 h of
10
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
152
incubation with DEX, even though no morphological changes were visible in the cells. Based on
153
these observations, we sampled VND7-VP16-GR BY-2 cells, as well as non-transgenic BY-2 cells,
154
after 0, 6, 12, 24, 36, and 48 h of DEX or mock treatment for further metabolome analysis.
155
Using the LC/MS-based wide-target metabolome analysis established by Sawada et al.
156
(2009), we identified 490 metabolites from the control and VND7-VP16-GR BY-2 cells, and
157
obtained relative amount data for 128 metabolites based on a cutoff signal-to-noise ratio of >3
158
(Table S1 and S2). Principal component analysis (PCA) of these 128 metabolites successfully
159
identified changes in the metabolome associated with progression of cell differentiation;
160
DEX-treated
161
non-transgenic samples were separated in the PCA plot (Fig. 2). The first two principal
162
components (PC1 and PC2) explained 36% of the total variance, and as the cell differentiation
163
progressed, PC2 became larger in the DEX-treated VND7-VP16-GR samples (Fig. 2). The PC1
164
component possibly reflected the difference between VND7-VP16-GR samples and
165
non-transgenic samples, since the wild-type samples were distributed in the region with negative
166
values of PC1, whereas VND7-VP16-GR samples were found in the region with positive values
VND7-VP16-GR
samples,
mock-treated
VND7-VP16-GR
11
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
samples,
and
167
of PC1 (Fig. 2). The PCA model in Fig. 2 showed that amino acids and their derivatives tended to
168
be highly correlated to PC1 and PC2 (p<0.05; Table 1, 2, S1 and S2). The 15 metabolites with the
169
highest and lowest PC1 correlation scores (p<0.05) included 11 amino acids (Asp, Glu, Thr, His,
170
Pro,
171
1-aminocyclopentanecarboxylate)
172
4-aminobutanoate, S-methylmethionine, and
173
with the highest and lowest PC2 correlation scores (p<0.05) included 5 amino acids (Leu, Ile,
174
N6-(L-1,3-dicarboxypropyl)-L-lysine, norleucine, and carnosine) and 4 amino acid-related
175
metabolites (Ala, Ser, O-acetyl-L-serine, N-acetyl-DL-serine) (Table 2). These results suggested
Lys,
Gln,
allothreonine,
α -methylhistidine,
and
5
amino
L-threo-3-methylaspartate,
acid-related
metabolites
D-alanyl-D-alanine)(Table
(Ala,
and
Phe,
1). The 15 metabolites
12
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
176
that amino acids and their derivatives were the main metabolites contributing to the dispersion of
177
the samples on PC1 and PC2.
178
179
Changes in amino acid-related metabolites during protoxylem vessel element differentiation
180
We further explored the changes in amino acid-related metabolites during protoxylem vessel
181
element differentiation. In the present work, we could not obtain data for Gly, Val, and Cys;
182
however, we did detect the remaining 17 proteinogenic amino acids and additional related
183
metabolites (Table S1 and S2). Parts of the LC/MS-based profiles for these metabolites are shown
184
in Fig. 3, Fig. 4, Fig. S1 and Fig. S2. Amino acids are derived from intermediates of the
185
glycolysis pathway and glyceraldehyde 3-phosphate (GAP), an intermediate of the glycolysis
186
pathway, showed an immediate decrease after DEX treatment (Fig. 3). This early change was
187
observed only in the DEX-treated VND7-VP16-GR BY-2 cells (Fig. 3, 4, S1, and S2, Table S3 and
188
S4); therefore, the immediate decrease in GAP appears to be associated with the initiation of
189
protoxylem vessel element differentiation.
190
The amino acids with high PC1 correlation scores, i.e., Asp, Glu, Thr, His, Pro, Lys, and Gln
191
(Table 1 and S1), all decreased in VND7-VP16-GR cells as cell differentiation progressed,
192
whereas the amino acids with high PC2 correlation scores, i.e., Leu, Ile, Arg, and Trp (Table 2
193
and S2), increased after 24 h of DEX treatment (Fig. 3, 4, S1 and S2, Table S3 and S4). These
194
results indicated that the progression of protoxylem vessel element differentiation was associated
195
with changes in amino acid metabolism. Among the increased amino acids, Trp, Phe, and Tyr are
196
biosynthesized from D-erythrose-4-phosphate and phosphoenolpyruvate through the shikimate
13
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
197
pathway (Maeda and Dudareva, 2012). The increase in Trp and tyramine produced from Tyr
198
suggested that the shikimate pathway was activated after 24 h of DEX treatment (Fig. 3 and S1).
199
The amounts of Tyr and Phe gradually increased during cell culture, as well as in the wild-type
200
cells (Fig. 3 and S3). However, the concentration of tyramine, a derivative of Tyr, greatly
201
increased after 24 h of DEX treatment (Fig. 3), suggesting that most Tyr biosynthesized after
14
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
202
DEX treatment is converted into tyramine. Similarly, although transient increase in Phe was
203
detected after 12 h of DEX treatment, the Phe level was the same as that after mock treatment
204
(Fig. 3 and Table S3); thus, most of the Phe produced after DEX treatment would be converted
205
into lignin monomer to generate SCWs (Vanholme et al., 2010). The other increased amino acids,
206
Leu, Ile, and Arg, are located at the end of each respective metabolic pathway (Fig. 3 and 4).
207
Other amino acids generated from the downstream metabolites of GAP decreased after DEX
15
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
208
treatment (Fig. 3, 4, S3, and S4). The large increase of L-saccharopine and L-2-aminoadipate after
209
24 h of DEX treatment (Fig. 3) indicated that the Lys catabolic pathway was activated during
210
protoxylem vessel element differentiation (Arruda et al., 2000).
211
Taken together, these data suggested that amino acid metabolism is changed to produce
212
several specific kinds of amino acids, such as the branched-chain amino acids Leu and Ile,
213
aromatic amino acids Trp, and translocatable nitrogen-rich amino acid Arg, and that such
214
regulation could be initiated from the rapid use of the GAP supplied from the glycolysis pathway.
215
In addition, the increased tyramine, L-saccharopine, and L-2-aminoadipate could imply that the
216
catabolism of amino acids is, at least partly, activated during protoxylem vessel element
217
differentiation. Since tyramine is known to be a precursor for hydroxycinnamic acid amides bound
218
to cell walls (Facchini et al., 2000), it could be possible that the synthesized tyramine is partly
219
further catalyzed into such cell wall-bound amides and then incorporated into SCW.
220
221
Transcriptome analysis of VND7-VP16-GR BY-2 cells
222
To obtain insight into the metabolic regulation of amino acids during protoxylem vessel
223
element differentiation, we conducted mRNA-seq analysis of VND7-VP16-GR BY-2 cells treated
224
with DEX or mock for 0, 6, 12, 24, and 36 h. After de novo assembly of sequence reads based on
225
the Nicotiana tabacum reference genome (Sierro et al., 2014), we obtained 74,932 unique contigs
226
from the mRNA-seq data for the VND7-VP16-GR BY-2 cells. Previous work reported the set of
227
direct targets of Arabidopsis VND6 and VND7 (Ohashi-Ito et al., 2010; Zhong et al., 2010;
228
Yamaguchi et al., 2011); based on this, we first examined the expression patterns of genes
16
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
229
homologous to the 63 VND7 direct target genes reported by Yamaguchi et al. (2011). Using
230
BLAST searches, we identified 603 contigs with high sequence similarity to direct target genes of
231
Arabidopsis VND7 (Table S5). Two-thirds of these contigs were upregulated after DEX treatment,
232
and the remaining contigs decreased or did not change in response to DEX treatment in
233
mRNA-seq data (Table S5, Figure S3). Most of upregulation occurred after 6 h of DEX treatment
234
(Table S5, Figure S3); thus, the initiation of protoxylem vessel element differentiation occurred
235
within 6 h of DEX treatment.
236
237
Active transcriptional regulation of genes encoding glycolysis and shikimate pathway
238
enzymes during protoxylem vessel element differentiation
239
Next, we searched for changes in transcript levels of genes encoding enzymes involved in the
240
glycolysis pathway (Fig. 4 and S4, and Table S6). In accordance with the fact that the GAP
241
contents decreased after 6 h of DEX treatment (Fig. 3), we also detected changes after 6 h of DEX
242
treatment in the transcript levels of genes corresponding to fructose-bisphosphate aldolase
243
(EC4.1.2.13), which catalyzes the reversible conversion of fructose 1,6-bisphosphate (Fru-1,6-bP)
244
into dihydroxyacetone-phosphate (DHAP) and GAP (Fig. 4). We detected 19 contigs putatively
245
corresponding to fructose-bisphosphate aldolase (Table S6); two of these were statistically
246
significantly upregulated (FC>4, p<0.05; Student’s t-test) and one was statistically-significantly
247
downregulated within 6 h of DEX treatment (FC<0.5, p<0.05; Student’s t-test) (Fig. 5; Table S6),
248
whereas the mock treatment did not significantly affect their expression (Fig. S6 and Table S6).
249
All 5 contigs that tended to decrease at 6 h of DEX treatment (FC<1) were similar to Arabidopsis
17
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
250
genes encoding plastidic-type fructose-bisphosphate aldolase (Table S6); thus, it is possible that
251
the cytosolic types of fructose-bisphosphate aldolase are upregulated at the transcriptional level
18
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
252
during the initial stages of protoxylem vessel element differentiation. In addition, the expression
253
of fructose 1,6-bisphosphatase (EC.3.1.3.11), which converts Fru-1,6-bP to fructose 6-phosphate
254
(Fru-6-P), was upregulated after the DEX treatment (Fig. 5 and Table S6). Three contigs putatively
255
encoding fructose 1,6-bisphosphatase were greatly upregulated at 6 h of DEX treatment (FC>40,
256
p<0.05; Student’s t-test), while only 1 contig was significantly upregulated at 6 h of mock
257
treatment (FC=5.3, p<0.05; Student’s t-test) (Fig. S6 and Table S6). Conversely, the expression of
258
genes encoding phosphofructokinase (EC2.7.1.11), which phosphorylates Fru-6-P, decreased after
259
12 h of DEX treatment (Fig. 5 and S6, and Table S6). Considering that these enzymes catalyze
260
these reactions in only one direction, these observations indicate that the conversion of GAP to
261
Fru-6-P through Fru-1,6-bP increased during the early stages of cell differentiation.
262
Metabolome analysis data also showed increases in aromatic amino acids, such as Trp, Phe,
263
and Tyr during protoxylem vessel element differentiation (Fig. 3). Phe is an important metabolite
264
because it is the precursor of lignin monomer biosynthesis (Vanholme et al., 2010); therefore, we
265
further examined the expression of genes encoding enzymes involved in Phe biosynthesis,
266
including the shikimate pathway (Fig. 6 and S7). The data showed that all of the metabolic steps
267
except the one catalyzed by 3-dehydroquinate synthase (EC4.2.3.4) were actively regulated at the
268
transcriptional level, and most of them were upregulated during protoxylem vessel element
269
differentiation (Fig. 6 and S7).
270
Genes encoding phosphoenolpyruvate carboxykinase (EC4.1.1.49), which converts
271
oxaloacetate into phosphoenolpyruvate (PEP), were also upregulated during the early stages of
272
cell differentiation (Fig. 5 and S5). The decreased GAP (Fig. 3) and changes in the transcription
19
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
273
of genes involved in the glycolysis pathway (Fig. 5 and S5) suggested that PEP biosynthesis from
274
Fru-6-P was reduced after DEX treatment. Based on the upregulation of phosphoenolpyruvate
20
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
275
carboxykinase (EC4.1.1.49), we propose that the PEP required for Phe biosynthesis is derived
276
from oxaloacetate during protoxylem vessel element differentiation (Fig. 5).
277
278
Quantification of key metabolites for SCW polymer biosynthesis and lignin content during
279
protoxylem vessel element formation
280
Finally, we tried to quantify Fru-6-P and PEP, which were assumed to be key metabolites from
281
the glycolysis pathway for SCW polymer biosynthesis as shown above, and UDP-glucose, a
282
precursor of SCW oligosaccharides, by the CE/MS analysis established by Hasunuma et al. (2016).
283
The data indicated active changes in these metabolites in VND7-VP16-GR cells in a DEX
284
treatment-dependent manner (Fig. 7A-C). Although VND7-VP16-GR cells showed higher levels of
285
Fru-6-P and UDP-glucose than those in the wild-type cells even under mock conditions (Fig. 7A
286
and C), the induction of protoxylem vessel element differentiation by DEX treatment significantly
287
changed the trend of these metabolites in VND7-VP16-GR cells; both Fru-6-P and UDP-glucose
288
decreased at 6 h of DEX treatment, and then increased as cell differentiation progressed (Fig. 7A
289
and C). The increased level of Fru-6-P after 12 h of DEX treatment can be, at least partly, explained
290
by the early upregulation of fructose 1,6-bisphosphatase (EC.3.1.3.11) rather than the conversion
291
from Glu-6-P by glucose-6-phosphate isomerase (EC.5.3.1.9), because the expression levels of
292
glucose-6-phosphate isomerase were not changed at 6-12 h of DEX treatment (Fig. 5). We also
293
found that the PEP contents increased after 12 h of DEX treatment (Fig. 7B), in accordance with
294
the upregulation of phosphoenolpyruvate carboxykinase (EC4.1.1.49) shown in Figure 5.
21
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
295
In addition, to monitor lignification in our system, p-coumaric acid, a precursor of lignin
296
monomer generated from Phe, and the lignin contents were analyzed. The relative amount data of
22
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
297
p-coumaric acid by LC/MS analysis showed the transient increase of p-coumaric acid after 24 h of
298
DEX treatment in VND7-VP16-GR cells, and thioglycolic acid methods detected lignin deposition
299
after 36 h of DEX treatment only in DEX-treated VND7-VP16-GR cells (Fig. 7D). As shown in Fig.
300
3, Phe transiently increased after 12 h of DEX treatment in VND7-VP16-GR cells, suggesting the
301
activation of flow from Phe to lignin monomer through p-coumaric acid.
23
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
302
DISCUSSION
303
304
In the current work, we examined protoxylem vessel element differentiation using wide-target
305
metabolome and transcriptome analyses. The highly synchronous in vitro system for protoxylem
306
vessel element differentiation (VND7-VP16-GR; Yamaguchi et al., 2010) in tobacco BY-2 cells
307
provided time-sequential data for relative levels of 128 metabolites (Table S3). The data
308
described here revealed prominent changes in the contents of amino acids and the expression
309
patterns of genes encoding enzymes involved in the glycolysis and shikimate pathways (Fig. 2-6,
310
Table S1-S6). CE/MS analysis indicated that Fru-6-P and PEP, suggested to be key metabolites
311
linking the glycolysis pathway to SCW polymer biosynthesis based on transcriptome analysis, are
312
actively regulated (Fig. 7). These results suggest that active regulation of primary metabolism
313
could be the basis of protoxylem vessel element differentiation.
314
Based on the data described here, we can speculate the regulatory dynamics of primary
315
metabolism during protoxylem vessel element differentiation as below: at the initiation of xylem
316
vessel cell differentiation, the amounts of GAP decrease (Fig. 3), which is accompanied with the
317
upregulation of genes for enzymes that convert GAP to Fru-6-P (Fig. 5). Then, it seems that the
318
transcription-level of genes involved in the Fru-6-P-to-pyruvate flow in the glycolysis pathway is
319
downregulated, as the decreased expression of genes corresponding to several steps of the
320
glycolysis pathway became apparent after 12 h of DEX treatment (Fig. 5). These observations
321
suggest that GAP metabolism has important functions in the glycolysis pathway during protoxylem
322
vessel element differentiation, probably involving the concentration of carbon sources within the
24
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
323
glycolysis pathway to Fru-6-P by arresting glycolysis in the upstream steps of GAP. Fru-6-P is a
324
key metabolite converted into NDP-sugar and/or connecting glycolysis with the pentose phosphate
325
pathway (Bar-Peled and O’Neill, 2011; Roach et al., 2012). NDP-sugars are building units of cell
326
wall polysaccharides; therefore, activation of Fru-6-P biosynthesis is possibly connected to the
327
activation of SCW formation during protoxylem vessel element differentiation. Our data showed
328
similar patterns for Fru-6-P and UDP-glucose levels during cell differentiation (Fig. 7), supporting
329
the idea that Fru-6-P biosynthesis can influence NDP-sugar biosynthetic activity.
330
Previous studies suggested the importance of Fru-6-P biosynthesis by fructokinase activity for
331
xylem development and hydraulic conductance (Damari-Weissler et al., 2009; Roach et al., 2012;
332
Stein et al., 2016). Particularly the RNAi hybrid aspen plants for fructokinase isoforms, in which
333
the Fru-6-P producing activity by fructokinase was reduced, showed the decrease in both Fru-6-P
334
and UDP-glucose, resulting in the thinner fiber cell walls with a reduction in the proportion of
335
cellulose (Roach et al., 2012). Based on these results, the involvement of fructokinase in carbon
336
partitioning to cellulose during wood formation was revealed (Roach et al., 2012). Our
337
transcriptome data indicated that the expression levels of 3 of 17 contigs corresponding to
338
fructokinase genes were upregulated during protoxylem vessel element differentiation (Table S6).
339
Therefore, it is plausible that the Fru-6-P biosynthesis by multiple pathways might be activated for
340
SCW formation during protoxylem vessel element differentiation.
341
In differentiating protoxylem vessel elements, the transcriptional upregulation of enzyme
342
genes involved in Phe biosynthesis was prominent (Fig. 6). The expression of shikimate pathway
343
genes is decreased in Arabidopsis nst1 snd1/nst3 double mutants, which are defective in the
25
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
344
differentiation of fiber cells, SCW-containing supporting cells (Mitsuda et al., 2007). The NST1
345
and SND1/NST3 proteins are NAC transcription factors belonging to the sister group of VND
346
family genes; therefore, these SCW-related NAC proteins can commonly upregulate genes of the
347
shikimate pathway. Moreover, we found that genes for phosphoenolpyruvate carboxykinase
348
(EC4.1.1.49) were upregulated during the early stages of protoxylem vessel element differentiation
349
(Fig. 5), suggesting that the conversion of oxaloacetate into PEP, one of the starting materials of
350
Phe biosynthesis (Fig. 6), was activated by VND7 induction. Indeed, the PEP level increased as
351
cell differentiation progressed (Fig. 7B). These results imply that carbon sources within the TCA
352
cycle might be reallocated to Phe for lignin biosynthesis through PEP production.
353
Finally, we found that the contents of Leu, Ile, and Arg, which all represent the ends of
354
amino acid biosynthetic pathways, also increased after DEX treatment (Fig. 3 and 4, Table S3).
355
Long-distance transport of amino acids through the xylem is important for the proper distribution
356
of nitrogen in plants (Okumoto and Pilot, 2011). Arg is a translocatable amino acid that can be
357
used as a relatively effective nitrogen resource by plants (Furuhashi and Yatazawa, 1970). Thus,
358
these accumulated amino acids could be transferred to shoot regions for the recycling of nitrogen
359
within plant bodies, after the completion of PCD of developing protoxylem vessel elements.
360
Taken together, our findings point to a system that regulates plant cell metabolic activity to
361
biosynthesize SCW-specific polymers during protoxylem vessel element differentiation. In this
362
system, a shift from primary cell wall biosynthetic mode to SCW biosynthetic mode would be
363
achieved through active transcriptional regulation of genes encoding primary metabolic enzymes,
364
especially at key catalytic steps, such as GAP metabolism, including Fru-6-P production and PEP
26
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
365
metabolism. This work thus suggests a novel strategy for improving woody biomass production
366
that involves enhanced flow of carbon resources from primary metabolites to SCW polymers.
367
27
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
368
MATERIALS AND METHODS
369
370
Plant material and DEX treatment
371
Nicotiana tabacum L. cv. BY-2 suspension culture cells with the VND7-VP16-GR system
372
(VND7-VP16-GR BY-2 cells) were used (Yamaguchi et al., 2010; Goue et al., 2013). Batches of
373
100 mL BY-2 cell suspension cultures were grown in 300-mL conical flasks. Liquid suspension
374
cultures were diluted 95-fold at weekly intervals with medium containing Murashige & Skoog
375
salt mixture (Wako), 0.2 mg/ml KH2PO4, 0.1 mg/mL myo-inositol, 1 mg/L thiamine-HCl, 0.2
376
mg/l 2,4-dichlorophenoxyacetic acid, and 30 g/L sucrose (pH 5.8), and then transferred to a
377
rotary shaker at 130 rpm at 27°C in the dark. The medium was supplemented with 100 mg/L
378
kanamycin. For time-series sampling in the metabolome and transcriptome analyses, the culture
379
scale was increased to 300 mL BY-2 cell cultures in 1-L flasks.
380
To induce differentiation of protoxylem vessel elements, dexamethasone (DEX) was added
381
to the 4-d-old cell cultures to a final concentration of 1 µM. Samples were cultured with agitation
382
in the dark at 27°C for 0, 6, 12, 24, 36, and 48 h prior to harvesting. At each sampling, 5 mL cell
383
culture was harvested and centrifuged (240 g, 2 min, at room temperature) to collect the cells.
384
The centrifugation was repeated 10 times with distilled water for washing, and then the cells were
385
subjected to metabolome and transcriptome analyses. The DEX treatment and cell harvesting
386
were performed three times for triplicate experiments.
387
388
Wide-target metabolome analysis
28
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
389
After harvesting, the cells were freeze-dried in a vacuum freeze dryer (FZ-2.5, Labconco)
390
and then subjected to the wide-target metabolome analysis by the methods described in Sawada
391
et al. (2009) with minor modifications. The freeze-dried BY-2 cell samples were crushed with a
392
bead shaker (1,000 rpm, 2 min; ShakeMaster Neo, BMS), and 4±0.2 mg sample was extracted
393
with 80% MeOH by crushing with 5 mm zirconia beads (1,000 rpm, 2 min). The extracts were
394
diluted 10-fold with 80% MeOH, 25 µL of each extract was transferred to a 96-well plate, and
395
the solutions were dried under N2 gas using a 96-well format spray instrument (40°C, 25 min and
396
30°C, 20 min). To prepare the samples for LC-MS/MS analysis, the dried samples were dissolved
397
in 250 μL water and then filtered twice through a 384-well filter plate (Whatman). The prepared
398
samples were analyzed in a UPLC-TQC machine (Waters), with solvents A, 0.1% formic acid in
399
water (Thermo), and B, 0.1% formic acid in acetonitrile (Wako). The gradient program and
400
TQMS conditions were described in Sawada et al. (2009).
401
After missing values were set to 20, the signal intensities of 3 samples were averaged. The
402
metabolites whose signal-to-noise ratios were less than 3 in all 18 experimental groups were
403
removed. Metabolites whose relative standard deviation was greater than 30% in all 18
404
experimental groups were also removed, leaving 128 metabolites for further analyses (criterion
405
A). Subsequent data analysis was performed according to Sawada et al. (2009). Finally, the
406
metabolome data for three replicate experiments of DEX treatment were obtained.
407
408
409
RNA-seq analysis
The VND7-VP16-GR BY-2 cells were sampled after 0, 6, 12, 24, and 36 h of DEX treatment
29
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
410
as described above, and subjected to total RNA extraction with the RNeasy Mini Kit (Qiagen).
411
The quality of each total RNA sample was examined using an Agilent RNA 6000 Pico LabChip
412
Kit in Agilent 2100 bioanalyzer. The mRNA fractions were isolated from 800 ng total RNAs with
413
the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). The cDNA
414
libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (New
415
England Biolabs), with NEBNext Multiplex Oligos for Illumina (New England Biolabs). All
416
procedures were performed according to the manufacturer's instructions. The quality and quantity
417
of each library were analyzed using a High Sensitivity DNA Kit (Agilent) and by subsequent
418
quantitative PCR (KAPA), respectively. Sequencing was carried out with a Genome Analyzer IIx
419
(Illumina).
420
The obtained short reads (ca. 530 million reads of 32-bp-length) were assembled using
421
Trinity (ver. 2.0.6, Grabherr et al., 2011), via the 'genome_guided_bam' option with the
422
Nicotiana tabacum "TN90" genome sequence (Sierro, et al. 2014). A total of 74,932 unique
423
contigs were constructed. The longest contig was 5,437 bp, the shortest contig was 150 bp, the
424
average length of contigs was 707.310 bp, and the N50 length was 1,006 bp. The sequence tags
425
were mapped to the contigs using TopHat2 (ver. 2.0.13, Kim et al., 2013), to calculate the Reads
426
Per Kilobase per Million Mapped reads (RPKM) for each contig by Cuffdiff2 (ver. 2.2.1,
427
Trapnell et al., 2013). For the expression analysis of putative homologous genes of Arabidopsis
428
VND7-target genes, BLAST searches were performed using the Arabidopsis genes listed as
429
target genes of VND7 as queries (Yamaguchi et al., 2011; Xu et al., 2014) (TBLASTN search;
430
E-value < 0.00001). For the enzyme genes involved in glycolysis and amino acid biosynthesis,
30
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
431
BLAST searches were performed (TBLASTN search; E-value<0.00001) using Arabidopsis and
432
tomato gene sequences that were annotated in the Plant Metabolic Network website
433
(http://www.plantcyc.org/, Dreher, 2014) as queries. For clustering representation of expression
434
(RPKM) patterns of the BY-2 contigs putatively homologous to Arabidopsis VND7 direct target
435
genes, the soft clustering algorithm was utilized with the R software package Mfuzz (Kumar &
436
Futschik, 2007).
437
438
Quantitative RT-PCR analysis
439
To check the reliability of the mRNA-seq analysis, quantitative RT-PCR was performed with
440
the selected putative homologous genes of Arabidopsis VND7-target genes (Figure S2). Total
441
RNAs extracted as described above were subjected to quantitative RT-PCR. Five micrograms of
442
total RNAs was reverse-transcribed with oligo(dT)15 primer (Roche) and the Transcriptor 1st
443
Strand cDNA Synthesis Kit (Roche) to synthesize template cDNA. After 10-fold dilution, 1 µL
444
template cDNA was subjected to absolute quantification analysis using LightCycler 480 SYBR
445
Green I Master (Roche) and the LightCycler 480 System (Roche). NtEF1α was used as the
446
internal control. The results of the quantitative RT-PCR analysis showed good agreement with
447
RNA-seq data. The primer sequences are shown in Table S7.
448
449
Sampling procedures for CE/MS-based metabolic profiling
450
Lyophilized cells (equivalent to 2 to 3 mg dry weight) were suspended in the mixture of 1
451
mL prechilled (-30°C) methanol containing 30.9 μM piperazine-1,4-bis(2-ethanesulfonic acid) as
31
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
452
an internal standard, 300 μL chloroform, and 100 μL prechilled (4°C) water. After 1 h of
453
bead-beater cell-breaking and centrifugation at 14,000 g for 5 min at 4°C, 800 μL of supernatant
454
was transferred to a new tube. The cell extract was mixed with 325 μL prechilled (4°C) water by
455
vortexing. After centrifugation at 14,000 g for 5 min at 4°C, 600 μL of the aqueous layer was
456
transferred onto a Millipore 3-kDa cut-off filter for the removal of solubilized proteins. After the
457
filtration, 400 μL of the aqueous layer extract was evaporated under vacuum using a CVE-3100
458
freeze dry system (Tokyo Rikakikai Co. Ltd., Tokyo, Japan). Dried extracts were stored at -80°C
459
until analysis by CE/MS. CE/MS analysis followed the methods described in Hasunuma et al.
460
(2016).
461
462
Thioglycolic acid lignin determination
463
Thioglycolic acid lignin was determined as described by Suzuki et al. (2009) with some
464
modifications after the starch removal treatment. Briefly, about 3 mg of freeze-dried cells was
465
treated with α-amylase (SIGMA, St. Louis, MO) solution (1 mL) for 16 h at 37°C. After the
466
treatment, the mixture was centrifuged at 20,000 g for 10 min, and the supernatant was discarded.
467
After washing of the pellet with water and methanol (each 2 mL), the pellet was dried and
468
weighed. The residue (about 1.5 mg) was treated with 0.5 mL 3M HCl and 0.1 mL thioglycolic
469
acid (Nakalaitesque, Kyoto, Japan) for 3 h at 80°C. The reaction mixture was cooled on ice for 5
470
min and centrifuged at 20,000 g for 10 min. The pellet was vortexed in water (2 mL) and
471
centrifuged at 20,000 g for 10 min. This washing and centrifugation was repeated once more. The
472
resulting pellet was dissolved in 1 mL 1 M NaOH for 16 h at room temperature and centrifuged at
32
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
473
20,000 g for 10 min. The supernatant was acidified with 0.1 mL concentrated HCl and incubated
474
at 4 °C overnight. The resulting precipitates were collected by centrifugation at 20,000 g for 10
475
min, washed with 2 mL water twice, and dried. The dried pellet was dissolved in 0.25 mL 1 M
476
NaOH. A portion of the solution (0.2 mL) was used for measurement of absorbance at 280 nm on
477
a Greiner UV Star 96-well plate (Greiner bio-one, Frickenhausen, Germany) in a microplate
478
reader (SH-1000 Lab, Corona Electric, Hitachinaka, Japan). The calibration curve used was as
479
described by Suzuki et al. (2009).
480
481
Large datasets
482
The mRNA-seq data presented in this study were submitted to DDBJ Sequence Read
483
Archive (http://trace.ddbj.nig.ac.jp/dra/index_e.html) and can be retrieved via accession number
484
PRJDB5152.
485
486
Supplemental Materials
487
This paper contains the following supplemental materials:
488
Figure S1. Changes in amino acid-related metabolites derived from intermediate compounds in
489
the glycolysis pathway in the wild-type BY-2 cells.
490
Figure S2. Changes in amino acid-related metabolites derived from oxaloacetate and
491
2-oxoglutarate in the wild-type BY-2 cells.
492
Figure S3. Clustering analysis of BY-2 contigs putatively homologous to Arabidopsis VND7
493
direct target genes.
33
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
494
Figure S4. Quantitative RT-PCR analysis of selected BY-2 contigs putatively homologous to
495
Arabidopsis VND7 direct target genes during protoxylem vessel element differentiation.
496
Figure S5. Changes in the expression of genes putatively involved in glycolysis in mock-treated
497
VND7-VP16-GR BY-2 cells.
498
Figure S6. Changes in expression of genes putatively involved Phe biosynthesis in mock-treated
499
VND7-VP16-GR BY-2 cells.
500
501
Table S1. Correlated metabolites of PC1 within PCA analysis of time-sequential wide-target
502
metabolome data for protoxylem vessel element differentiation.
503
Table S2. Correlated metabolites of PC2 within PCA analysis of time-sequential wide-target
504
metabolome data for protoxylem vessel element differentiation.
505
Table S3. Relative amount data for 128 metabolites in VND7-VP16-GR BY-2 cells during
506
protoxylem vessel element differentiation.
507
Table S4. Relative amount data for 128 metabolites in wild-type BY-2 cells after DEX treatment.
508
Table S5. Expression profiles of BY-2 contigs putatively homologous to Arabidopsis VND7
509
direct target genes.
510
Table S6. Expression profiles of BY-2 contigs putatively involved in primary metabolic
511
regulation.
512
Table S7. Primer sequences used in this study.
513
34
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
514
Acknowledgments
515
516
We thank Mr. Muneo Sato, Mr. Yutaka Yamada, and Ms. Akane Sakata (RIKEN) for their
517
excellent technical assistance in LC/MS analysis, Ms. Megumi Ozaki and Ms. Yumiko Oku
518
(Kyoto University) for their excellent technical assistance in lignin determination, and Dr.
519
Minoru Kubo, Dr. Arata Yoneda, Dr, Ko Kato, Dr. Hitoshi Endo, and Dr. Bo Xu (NAIST) for
520
fruitful discussions. Lignin analysis was carried out by the FBAS collaborative program of the
521
Research Institute for Sustainable Humanosphere, Kyoto University.
522
35
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
523
Table 1. Top 15 metabolites with high and low correlation scores for PC1 within the PCA
524
analysis of time-sequential wide-target metabolome data for protoxylem vessel element
525
differentiation.
Metabolitea
Type of metabolism
Class of
Correlation
p value
metabolites
Top 15 metabolites with high correlation scores for PC1
Aspartic acid
Primary
Amino acid
0.920638002
7.76E-28
Glutamic acid
Primary
Amino acid
0.915312495
5.70E-27
Threonine
Primary
Amino acid
0.897000219
2.25E-24
Histidine
Primary
Amino acid
0.896197896
2.84E-24
L-Allothreonine
Primary
Amino acid
0.890719092
1.35E-23
Proline
Primary
Amino acid
0.877349881
4.39E-22
Malate
Primary
Other
0.860575996
2.03E-20
Lysine
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
alpha-Methyl-histidine
Primary
Amino acid
0.787859709
4.20E-15
L-threo-3-Methylaspartate
Primary
Amino acid
0.774852194
2.27E-14
1-Aminocyclopentanecarboxyl
Primary
Amino acid
0.767925228
5.32E-14
Primary
Amino acid
0.740290592
1.22E-12
ate
Glutamine
36
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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
Alanine
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-Methylmethionine
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
Glucose 1-phosphate
Primary
Other
-0.437642617
0.000238154
Inosine
Primary
Other
-0.427176328
0.000347355
D-Alanyl-D-alanine
Primary
Amino acid
-0.376960084
0.001809199
Phenylalanine
Primary
Amino acid
-0.366290228
0.002488166
Deoxyadenosine
Primary
Other
-0.365496858
0.002546748
526
527
a
Amino acid-related metabolites are shown in bold.
528
37
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
529
Table 2. Top 15 metabolites with high and low correlation scores for PC2 within the PCA
530
analysis of time-sequential wide-target metabolome data for protoxylem vessel element
531
differentiation.
Metabolitea
Type of
Class of
metabolism
metabolites
Correlation
p value
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-lysine
Primary
Amino acid
0.796352172
1.31E-15
Tyramine
Primary
Other
0.788972897
3.62E-15
Norleucine
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-Leucine
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-Isoleucine
Primary
Amino acid
0.61833074
3.16E-08
Adenosine
Primary
Other
0.611578222
4.90E-08
38
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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-serine
Primary
Amino acid
-0.541259749
2.70E-06
Mannose 6-phosphate
Primary
Other
-0.525065728
6.01E-06
N-acetyl-DL-serine
Primary
Amino acid
-0.514816671
9.76E-06
Galactosamine
Primary
Other
-0.510753618
1.18E-05
Alanine
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
Serine
Primary
Amino acid
-0.447716665
0.000163683
Mannose 1-phosphate
Primary
Other
-0.417155822
0.000492976
Ethanolamine phosphate
Primary
Other
-0.407927601
0.000674143
Noradrenaline
Primary
Other
-0.399776603
0.00088232
532
533
a
Amino acid-related metabolites are shown in bold.
39
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
534
Figure legends
535
536
Figure 1. Transitional differentiation rates of protoxylem vessel elements in VND7-VP16-GR
537
BY-2 cells. A, Ratios of undifferentiated (light gray), differentiating (hatched), and differentiated
538
(dark gray) cells at each time point of the incubation with DEX. Data show the means ± SD (n =
539
3). B, Undifferentiated BY-2 cell with a clear nucleus structure (arrow). C, Differentiating BY-2
540
cells with a thin helical SCW (arrowheads) and a nucleus (arrow). D, Differentiated cell with
541
thick SCWs (arrowheads) lacking nuclear and cytosolic structures. Shrunken cell contents
542
(asterisk) were observed in cells that had completed differentiation. Scale bar = 20 µm (B-D).
543
544
Figure 2. Metabolic changes associated with protoxylem vessel element differentiation. Principle
545
component analysis (PCA) plots of mock-treated (i.e. ethanol-treated) and DEX-treated
546
VND7-VP16-GR BY-2 and non-transgenic wild-type (WT) cells. Treatment times (in hours) are
547
indicated. The x- and y-axes indicate the first component (PC1) and second component (PC2),
548
respectively. The independent triplicate samples were collected and examined for each time point.
549
550
Figure 3. Changes in amino acid-related metabolites derived from intermediate compounds in the
551
glycolysis pathway during protoxylem vessel element differentiation. Mock-treated (white
552
squares) and DEX-treated (gray diamonds) VND7-VP16-GR BY-2 cells were collected at the
553
indicated time points and relative amounts of metabolites were obtained. Data are the means ±
554
S.D. (n = 3). Asterisks indicate statistically significant differences between mock-treated and
40
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
555
DEX-treated samples at each time point (*, p < 0.05; **, p < 0.01; Student’s t-test).
556
557
Figure 4. Changes in amino acid-related metabolites derived from oxaloacetate and
558
2-oxoglutarate during protoxylem vessel element differentiation. Mock-treated (white squares)
559
and DEX-treated (gray diamonds) VND7-VP16-GR BY-2 cells were collected at the indicated
560
time points and relative amounts of metabolites were obtained. Data are the means ± S.D. (n = 3).
561
Asterisks indicate statistically significant differences between mock-treated and DEX-treated
562
samples at each time point (*, p < 0.05; **, p < 0.01; Student’s t-test).
563
564
Figure 5. Changes in the expression of genes putatively involved in glycolysis during
565
protoxylem vessel element differentiation in DEX-treated VND7-VP16-GR BY-2 cells.
566
Expression data are shown for genes encoding the indicated enzymes. Each circle indicates the
567
proportion of contigs increased (red, FC>4 and orange, 2<FC<4), unchanged (yellow, 0.5<FC<2),
568
and decreased (sky blue, 0.25<FC<0.5 and deep blue, FC<0.25) compared with the expression
569
level at 0 h of DEX treatment, at the indicated time point. Glu-6-P, D-glucose-6-P; Fru-6-P,
570
fructose-6-phosphate;
571
dihydroxy-acetone-phosphate;
572
1,3-bisphospho-D-glycerate; 3-PG, 3-phospho-D-glycerate; 2-PG, 2-phospho-D-glycerate; PEP,
573
phosphoenolpyruvate; and Pyr, pyruvate. FC, fold change.
Fru-1,6-bP,
GAP,
fructose-1,6-bisphosphate;
glyceraldehyde
3-phosphate;
DHAP,
1,3-bPG,
574
575
Figure 6. Changes in expression of genes putatively involved Phe biosynthesis during
41
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
576
protoxylem vessel element differentiation in DEX-treated VND7-VP16-GR BY-2 cells.
577
Expression data are shown for genes encoding the indicated enzymes. Each circle indicates the
578
proportion of contigs increased (red, FC>4 and orange, 2<FC<4), unchanged (yellow, 0.5<FC<2),
579
and decreased (sky blue, 0.25<FC<0.5 and deep blue, FC<0.25) compared with the expression
580
level at 0 h of DEX treatment, at the indicated time point. E4P, erythrose-4-phosphate; PEP,
581
phosphoenolpyruvate;
582
5-enolpyruvyl-shikimate 3-phosphate. FC, fold change.
DAHP,
3-deoxy-D-arabino-heptulosonate-7-phosphate;
and
EPSP,
583
584
Figure 7. Changes in fructose-6-phosphate, phosphoenolpyruvate, UDP-glucose, p-coumaric
585
acid and lignin contents during protoxylem vessel element differentiation. A-C, Mock-treated
586
(white squares) and DEX-treated (gray diamonds) wild-type (WT, left panels) and
587
VND7-VP16-GR (right panels) BY-2 cells were collected at the indicated time points, and
588
fructose-6-phosphate (A), phosphoenolpyruvate (B), and UDP-glucose (C) were quantified by
589
CE/MS analysis. D, Relative amounts of p-coumaric acid detected in LC/MS analysis (left) and
590
lignin contents measured by thioglycolic acid methods (right). Data are the means ± S.D. (n = 3)
591
for mock-treated wild-type (WT, white bars), DEX-treated wild-type (WT, light gray bars),
592
mock-treated VND7-VP16-GR (dark gray bars), and DEX-treated VND7-VP16-GR (black bars).
593
Asterisks indicate statistically significant difference between mock-treated and DEX-treated
594
samples at each time point (*, p < 0.1; **, p < 0.05; ***, p < 0.01; Student’s t-test).
595
596
Figure S1. Changes in amino acid-related metabolites derived from intermediate compounds in
42
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
597
the glycolysis pathway in the wild-type BY-2 cells. Mock-treated (white squares) and
598
DEX-treated (gray diamonds) wild-type BY-2 cells were collected at the indicated time points
599
and relative amounts of metabolites were obtained. Data are the means ± S.D. (n = 3). Asterisks
600
indicate statistically significant difference between mock-treated and DEX-treated samples at
601
each time point (*p < 0.05, **p < 0.01; Student’s t-test).
602
603
Figure S2. Changes in amino acid-related metabolites derived from oxaloacetate and
604
2-oxoglutarate in the wild-type BY-2 cells. Mock-treated (white squares) and DEX-treated (gray
605
diamonds) wild-type BY-2 cells were collected at the indicated time points and relative amounts
606
of metabolites were obtained. Data are the means ± S.D. (n = 3). Asterisks indicate statistically
607
significant difference between mock-treated and DEX-treated samples at each time point (*, p <
608
0.05; **, p < 0.01; Student’s t-test).
609
610
Figure S3. Clustering analysis of tobacco contigs putatively homologous to Arabidopsis VND7
611
direct target genes. The 603 contigs with high sequence similarities to VND7 direct target genes
612
of Arabidopsis were subjected to clustering analysis. All clusters other than Clusters 1, 3, and 5
613
showed upregulation of expression under DEX treatment. i, DEX-treated samples to induce
614
VND7 activity; m, mock-treated samples.
615
616
Figure S4. Quantitative RT-PCR analysis of selected BY-2 genes putatively homologous to
617
Arabidopsis VND7 direct target genes during protoxylem vessel element differentiation. Left
43
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
618
y-axis shows the relative expression levels of IRX7, IRX8, IRX9, IRX10, IRX12, IRX14, and
619
MAP70-5, and right y-axis shows the relative expression levels of CesA and XCP. Results are
620
means ± S.D. (n = 3).
621
622
Figure S5. Changes in the expression of genes putatively involved in glycolysis in mock-treated
623
VND7-VP16-GR BY-2 cells. Expression data are shown for genes encoding the indicated
624
enzymes. Each circle indicates the proportion of contigs increased (red, FC>4 and orange,
625
2<FC<4), unchanged (yellow, 0.5<FC<2), and decreased (sky blue, 0.25<FC<0.5 and deep blue,
626
FC<0.25) compared with the expression level at 0 h of DEX treatment, at the indicated time point.
627
Glu-6-P, D-glucose-6-P; Fru-6-P, fructose-6-phosphate; Fru-1,6-bP, fructose-1,6-bisphosphate;
628
DHAP,
629
1,3-bisphospho-D-glycerate; 3-PG, 3-phospho-D-glycerate; 2-PG, 2-phospho-D-glycerate; PEP,
630
phosphoenolpyruvate; and Pyr, pyruvate. FC, fold change.
dihydroxy-acetone-phosphate;
GAP,
glyceraldehyde
3-phosphate;
1,3-bPG,
631
632
Figure S6. Changes in expression of genes putatively involved Phe biosynthesis in mock-treated
633
VND7-VP16-GR BY-2 cells. Expression data are shown for genes encoding the indicated
634
enzymes. Each circle indicates the proportion of contigs increased (red, FC>4 and orange,
635
2<FC<4), unchanged (yellow, 0.5<FC<2), and decreased (sky blue, 0.25<FC<0.5 and deep blue,
636
FC<0.25) compared with the expression level at 0 h of DEX treatment, at the indicated time point.
637
E4P,
638
3-deoxy-D-arabino-heptulosonate-7-phosphate; and EPSP, 5-enolpyruvyl-shikimate 3-phosphate.
erythrose-4-phosphate;
PEP,
phosphoenolpyruvate;
44
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
DAHP,
639
FC, fold change.
640
45
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
641
46
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Parsed Citations
Abramson M, Shoseyov O, Shani Z (2010) Plant cell wall reconstruction toward improved lignocellulosic production and
processability. Plant Sci 178: 61-72
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Allona I, Quinn M, Shoop E, Swope K, St Cyr S, Carlis J. Riedl J, Retzel E, Campbell MM, Sederoff R, Whetten RW (1998) Analysis of
xylem formation in pine by cDNA sequencing. Proc Natl Acad Sci USA 95: 9693-9698
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Arruda P, Kemper EL, Papes F, Leite A (2000) Regulation of lysine catabolism in higher plants. Trend Plant Sci 5: 324-330
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bar-Peled M, O'Neill MA (2011) Plant nucleotide sugar formation, interconversion, and salvage by sugar recycling. Annu Rev Plant
Biol 62: 127-155
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bashline L, Li S, Gu Y (2014) The trafficking of the cellulose synthase complex in higher plants. Ann Bot (Lond) 114: 1059-1067
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bollhöner B, Prestele J, Tuominen H (2012) Xylem cell death: emerging understanding of regulation and function. J Exp Bot 63:
1081-1094
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Damari-Weissler H, Rachamilevitch S, Aloni R, German MA, Cohen S, Zwieniecki MA, Michele Holbrook N, Granot D (2009) LeFRK2
is required for phloem and xylem differentiation and the transport of both sugar and water. Planta 230: 795-805
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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 (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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Endo H, Yamaguchi M, Tamura T, Nakano Y, Nishikubo N, Yoneda A, Kato K, Kubo M, Kajita S, Katayama Y, Ohtani M, Demura T
(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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Facchini PJ1, Huber-Allanach KL, Tari LW. (2000) Plant aromatic L-amino acid decarboxylases: evolution, biochemistry, regulation,
and metabolic engineering applications.Phytochem 54, 121-138
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Faik, A. (2010) Xylan biosynthesis: news from the grass. Plant Physiol 153: 396-402
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded
on June
18, 2017
by of
www.plantphysiol.org
Furuhashi K, Yatazawa M (1970) Amino
acids asfrom
nitrogen
sources
for- Published
the growth
rice callus tissue. Plant Cell Physiol 11: 559-567
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E,
Hacohen N, Gnirke A, Rhind N, di P, Federica, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length
transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29: 644-652
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hussey SG, Mizrachi E, Creux NM, Myburg AA (2013) Navigating the transcriptional roadmap regulating plant secondary cell wall
deposition. Front Plant Sci 4: 325
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kalluri UC, Hurst GB, Lankford PK, Ranjan P, Pelletier DA (2009) Shotgun proteome profile of Populus developing xylem.
Proteomics 9: 4871-4880
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg S (2013) TopHat2: accurate alignment of transcriptomes in the
presence of insertions, deletions and gene fusions. Genome Biol 14: R36
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ko JH, Kim WC, Kim JY, Ahn SJ, Han KH (2012) MYB46-mediated transcriptional regulation of secondary cell wall biosynthesis. Mol
Plant 5: 961-963
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kumar L, Futschik M (2007) Mfuzz: A software package for soft clustering of microarray data. Bioinformation 2: 5-7
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Maeda H, Dudareva N (2012) The shikimate pathway and aromatic amino Acid biosynthesis in plants. Annu Rev Plant Biol. 63: 73105
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
McCarthy RL, Zhong R, Fowler S, Lyskowski D, Piyasena H, Carleton K, et al. (2010) The poplar MYB transcription factors, PtrMYB3
and PtrMYB20, are involved in the regulation of secondary wall biosynthesis. Plant Cell Physiol 51: 1084-1090
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
McFarlane HE, Döring A, Persson S (2014) The cell biology of cellulose synthesis. Annu Rev Plant Biol 65: 69-94
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Myburg AA, Sederoff RR (2001) Xylem Structure and Function. In eLS. Nature Publishing Group
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Okumoto S, Pilot G (2011) Amino acid export in plants: a missing link in nitrogen cycling. Mol Plant 4: 453-463
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ralph SG, Chun HJ, Kolosova N, Cooper D, Oddy C, Ritland CE, Kirkpatrick R, Moore R, Barber S, Holt RA, Jones SJ, Marra MA,
Douglas CJ, Ritland K, Bohlmann J (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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Rengel D, Clemente HS, Servant F, Ladouce N, Paux E, Wincker P, et al. (2009). A new genomic resource dedicated to wood
formation in Eucalyptus. BMC Plant Biol. 9: 36
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Roach M, Gerber L, Sandquist D, Gorzsás A, Hedenström M, Kumar M, Steinhauser MC, Feil R, Daniel G, Stitt M, Sundberg B,
Niittylä T (2012) Fructokinase is required for carbon partitioning to cellulose in aspen wood. Plant J 70: 967-977
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Stein O, Damari-Weissler H, Secchi F, Rachamilevitch S, German MA, Yeselson Y, Amir R, Schaffer A, Holbrook NM, Aloni R,
Zwieniecki MA, Granot D (2016) The tomato plastidic fructokinase SlFRK3 plays a role in xylem development. New Phytol 209: 148495
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sterky F, Bhalerao RR, Unneberg P, Segerman B, Nilsson P, Brunner AM, et al. (2004) A populus EST resources for plant
functional genomics. Proc Natl Acad Sci USA 101: 13951-13956
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Turner S, Gallois P, Brown D (2007) Tracheary element differentiation. Annu Rev Plant Biol 58: 407-433
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W (2010). Lignin biosynthesis and structure. Plant Physiol 153: 895-905
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xu B, Ohtani M, Yamaguchi M, Toyooka K, Wakazaki M, Sato M, Kubo M, Hiwatashi Y, Murata T, Kurata T, Yoneda A, Kato K,
Hasebe M, Demura T (2014) Contribution of NAC transcription factors of plant adaptation to land. Science 343: 1505-1508
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yamaguchi M, Goué N, Igarashi H, Ohtani M, Nakano Y, Mortimer JC, 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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yamaguchi M, Mitsuda N, Ohtani M, Ohme-Takagi M, Demura T (2011) VASCULAR-RELATED NAC-DOMAIN 7 directly regulates the
expression of broad range of genes for xylem vessel formation. Plant J 66: 579-590
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhong R, Ye ZH (2012) MYB46 and MYB83 bind to the SMRE sites and directly activate a suit of transcription factors and
secondary wall biosynthetic genes. Plant Cell Physiol 53: 368-380
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.