New
Phytologist
Research
Molecular features of secondary vascular tissue
regeneration after bark girdling in Populus
Jing Zhang1, Ge Gao1, Jia-Jia Chen1, Gail Taylor2, Ke-Ming Cui1 and Xin-Qiang He1
1
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China; 2School of Biological
Sciences, University of Southampton SO16 7PX, UK
Summary
Author for correspondence:
Xin-Qiang He
Tel: +86 10 62757016
Email: [email protected]
Received: 17 May 2011
Accepted: 19 July 2011
New Phytologist (2011) 192: 869–884
doi: 10.1111/j.1469-8137.2011.03855.x
Key words: bark girdling, Populus
tomentosa, regeneration, secondary vascular
tissue, transcriptome profiling.
• Regeneration is a common strategy for plants to repair damage to their tissue
after attacks from other organisms or physical assaults. However, how differentiating cells acquire regenerative competence and rebuild the pattern of new tissues
remains largely unknown.
• Using anatomical observation and microarray analysis, we investigated the
morphological process and molecular features of secondary vascular tissue
regeneration after bark girdling in trees.
• After bark girdling, new phloem and cambium regenerate from differentiating
xylem cells and rebuild secondary vascular tissue pattern within 1 month.
Differentiating xylem cells acquire regenerative competence through epigenetic
regulation and cell cycle re-entry. The xylem developmental program was blocked,
whereas the phloem or cambium program was activated, resulting in the secondary vascular tissue pattern re-establishment. Phytohormones play important roles
in vascular tissue regeneration.
• We propose a model describing the molecular features of secondary vascular tissue regeneration after bark girdling in trees. It provides information for
understanding mechanisms of tissue regeneration and pattern formation of the
secondary vascular tissues in plants.
Introduction
Plants and animals are subject to injuries and a variety of
assaults during their life span, and regeneration is a common way for them to repair the damaged body parts. In
animals, most cells, except for stem cells, lose their regenerative capacity during their differentiation into specific cell
types. Recent evidence also shows that the introduction of
‘pluripotency-inducing factors’ (PIFs) may cause a cell to
change its ‘fate’ (Yamanaka, 2009; Passier & Mummery,
2010; Vierbuchen et al., 2010). In plants, cells can regenerate new organs and tissues through different pathways; for
example, organ or tissue restoration via dedifferentiation
and redifferentiation or transdifferentiation, organogenesis
via meristem formation in callus, and embryogenic-like
ontology via somatic embryogenesis (Cui, 1997; Birnbaum
& Alvarado, 2008).
Two basic steps are common in the plant regeneration
process: acquisition of competence to regenerate via dedifferentiation, and repatterning of regenerated tissues
2011 The Authors
New Phytologist 2011 New Phytologist Trust
(Birnbaum & Alvarado, 2008). During the first step, chromatin remodeling and cell cycle re-entry have been
suggested to regulate cellular plasticity which is necessary
for regeneration (Sustar & Schubiger, 2005; Costa & Shaw,
2007; Grafi et al., 2007; Sena et al., 2009). It is also
believed that the specification and regulation mechanisms
operating during regeneration are similar to those during
normal development because multicellular organisms would
not adopt different genetic networks to produce the same
structures (Birnbaum & Alvarado, 2008). Furthermore,
studies on shoot and root regeneration in Arabidopsis have
suggested that the redistribution of phytohormone gradients may induce organ regeneration. Auxin transport is
necessary for root regeneration; establishing opposing cytokinin and auxin domains is an important step in shoot
meristem regeneration from callus (Xu et al., 2006; Gordon
et al., 2007; Grieneisen et al., 2007; Sena et al., 2009).
Thanks to the utilization of cell-specific markers in model
organisms, significant progress has been made in understanding regeneration mechanisms in plants (Xu et al.,
New Phytologist (2011) 192: 869–884 869
www.newphytologist.com
New
Phytologist
870 Research
2006; Sena et al., 2009; Sugimoto et al., 2010). However,
how differentiating cells acquire regenerative competence
and rebuild the pattern of new tissues is still largely
unknown, and further investigation in other species and different regeneration systems is necessary.
It is well known that plants can regenerate new bark or
vasculature upon wounding (Brown & Sax, 1962;
Thompson, 1967; Noel, 1970; Stobbe et al., 2002). When
a strip of bark is removed from trees, newly formed periderm and wound cambium develop from the callus on the
surface of the secondary xylem, and new phloem is subsequently derived from the wound cambium (Stobbe et al.,
2002). However, the response is different in the case of
large-scale (as much as 1–2 m) bark girdling in trees. The
optimal conditions for secondary vascular tissue (SVT)
regeneration after bark girdling on a large scale were first
established in Eucommia ulmoides, initially to allow repeated
harvesting of bark used in Chinese traditional medicine (Li
et al., 1981). The process was subsequently observed in
several other trees, including Broussonetia papyrifera, Betula
pubescens and Populus tomentosa (Lu et al., 1987; Li & Cui,
1988; Cui et al., 1995; Du et al., 2006; Wang et al., 2009).
It is interesting to note that after girdling on a large scale,
the newly formed sieve elements and wound cambium are
derived from differentiating xylem cells rather than from
callus (Li et al., 1981; Pang et al., 2008). Proteomic analysis
showed that the changes in gene expression pattern corresponded to the progression of SVT regeneration (Du et al.,
2006). However, owing to the limitations of sampling and
analyzing methodologies, the molecular mechanisms that
enable the secondary xylem cells to switch their fates into
multipotent cells and rebuild the SVT pattern are largely
unknown.
We show here the morphological and molecular features
of the process of cell fate switching in differentiating xylem
cells during SVT regeneration after bark girdling in
Populus. Because this regeneration system is different from
plant shoot and root meristem generation and embryogenic
processes, our study provides new information for understanding the mechanisms of tissue regeneration in plants; it
is also significant for unveiling the regulation of the SVT
pattern formation.
Materials and Methods
Girdling and sampling procedure
Four-year-old trees from a single clonal plantation of
healthy Populus tomentosa Carriere located in Renqiu,
Hebei province, in northern China (3850¢N, 11610¢E)
were girdled in early June 2006. A 1.2-m strip of bark was
peeled from trees, starting 0.5 m from the base, and the
exposed tissue was wrapped with a transparent plastic sheet.
Samples were collected at 0, 4, 6, 9 and 12 d after girdling
New Phytologist (2011) 192: 869–884
www.newphytologist.com
(DAG). Rectangular blocks (2–2.5 · 4 cm2) consisting of
regenerated tissues plus mature xylem were smoothly
removed from the stem as described by Uggla & Sundberg
(2002). Blocks were then cut into halves. One half of the
block was immediately frozen in liquid nitrogen after briefly
trimming and then stored at )70C for tangential cryosectioning, while the other half was divided into small
blocks (1.5 · 2 mm2) and fixed in 4% paraformaldehyde
in 0.01 M phosphate-buffered saline (PBS; pH 7.2) for
microscopic observation. Small strips (3 · 20 mm2) were
fixed in 70% ethanol for free hand sectioning.
Histological studies
To detect sieve elements, hand-cut sections were stained
with 0.005% aniline blue in 0.15 M K3PO4 (pH 8.2) and
then observed under UV light by fluorescence microscopy
(Zeiss Axioskop2 plus). Sections of 5 lm thickness were cut
from small blocks embedded in LR White resin (Sigma) on
a microtome (Leitz 1512, Ernst Leitz GmbH, Wetzlar,
Germany), and stained with toluidine blue O and ⁄ or aniline blue.
Tangential cryo-sectioning
A series of 20-lm-thick tangential sections was taken for
each section sample as described by Uggla & Sundberg
(2002) with modification. Regenerated tissues at different
stages were isolated by tangential cryosectioning at )24C
with a Leica CM1850 Cryostat (Leica Microsystems
Nussloch GmbH, Nussloch, Germany). Transverse sections
taken from both ends of the specimen and stained with aniline blue were used to locate the position of tangential
sections. Cryosections of regenerated tissues from the same
tree at certain stages were collected in a 1.5 ml Eppendorf
tube and frozen in liquid nitrogen immediately and then
stored at )70C.
Microarray processing and data analysis
Genomic DNA was isolated from cryosections of differentiating xylem from two trees using a modified CTAB method
(Doyle & Doyle, 1990). Total RNA was extracted from
cryosections representing five tissues, differentiating xylem,
dedifferentiating xylem, regenerated phloem, differentiating
regenerated cambium and regenerated cambium, and used
for microarray hybridization and following quantitative
reverse transcription polymerase chain reaction (qRT-PCR)
experiments (see Supporting Information, Methods S1 and
Table S5 for details). Three biological replicates were initially used for each tissue; however, failures as a result of
low RNA quantities or poor quality led to only two replicates being used for the microarray experiment. The 61K
Affymetrix GeneChip Poplar Genome Array (Affymetrix
2011 The Authors
New Phytologist 2011 New Phytologist Trust
New
Phytologist
Inc., Santa Clara, CA, USA) was employed in this study. A
genomic DNA-based probe-selection (Xspecies) strategy
was adopted to re-annotate probe-sets as described by
Hammond et al. (2005, 2006). Sample preparation and
array processing were carried out in Nottingham
Arabidopsis Stock Centre (NASC, UK) following a onecycle eukaryotic protocol. RNA signal intensity (.CEL) files
were generated using the Microarray Analysis Suite (MAS
version 5.0; Affymetrix). Data were further normalized
using the Invariant Set Normalization method with dChip
(Li & Wong, 2001). Nonlogged mean signal intensity
(MSI) values for selected probe-sets across the five samples
are presented in Tables S1–S4.
Considering the relative shortage of replicates, we
performed model-based conservative estimation by introducing 90% lower confidence bounds (LCBs) of fold
change, which is the minimal fold change for a given comparison with at least 90% statistical confidence, to detect
the gene differential expression. Standard error (SE) is
employed to estimate LCBs and probe-sets with SE < 0.5
were selected for further analysis. A positive LCB indicates
that the higher signal was from the first member of the pairwise tissue comparison, while a negative LCB indicates that
the higher signal was from the second member of the comparison. A cutoff of LCB ‡ 1.5 in at least one out of the
four pairwise comparisons was chosen as the threshold.
Moreover, if a gene is expressed (or ‘present’) in one sample
group, but not expressed (or ‘absent’) in another group, it
was also indicated as ‘differentially expressed’ regardless of
the observed expression level. Furthermore, the coefficient
of variation (CV = SD ⁄ mean signal) was also calculated for
each probe-set and used to measure the fluctuation of
observed transcripts. Clustering analysis was performed
using a DNA-Chip Analyzer (dChip) (http://biosun1.
harvard.edu/complab/dchip) (Li & Wong, 2001). Microarray data and further details of the samples are
available through The National Center for Biotechnology
Information (NCBI) GEO (GEO submission GSE25309).
Results
Secondary vascular tissue regeneration after bark
girdling in Populus
The bark-girdled trees regenerated new SVT within 12 d
and formed new bark within 1 month in P. tomentosa
(Fig. 1). In the growing season, the vascular cambium produces secondary phloem outwards and secondary xylem
inwards. The surface of the exposed trunk was moist and
smooth when the bark of P. tomentosa was removed
(Fig. 1a). The cambium had been removed with the bark,
leaving only differentiating xylem cells on the surface of the
trunk (Fig. 2a,b). From 2 to 6 DAG, the girdled area of
trunk became protuberant with a soft surface (Fig. 1b).
2011 The Authors
New Phytologist 2011 New Phytologist Trust
Research
(a)
(b)
(c)
(d)
Fig. 1 Morphology of girdled trunk during bark regeneration in
Populus tomentosa. The surface of the exposed trunk was moist
and smooth when the bark was removed (a). The surface of the
girdled trunk became protuberant and soft at 4 d after girdling
(DAG), resulting from callus formation (b). It became green at
6 DAG (c), and then became dark green, dry and hard at
12 DAG (d).
Cross section revealed the formation of callus, resulting from
the division of ray cells of differentiating xylem (diX) at 2
DAG (Fig. 2c). Axial diX cells under callus underwent periclinal and transverse divisions, indicating xylem cell
dedifferentiation at 4 DAG (Fig. 2d,e,f). From 6 to 9 DAG,
the surface of the girdled area turned green (Fig. 1c). Sieve
elements were detected by aniline blue staining under fluorescence microscopy at 6 DAG (Fig. 2i), while cambium
was not observed at this time (Fig. 2g,h). The sieve elements
were present several cell layers under the isodiametric callus
cells, arranged between ray cells (Fig. S1a,d) and connected
longitudinally to each other (Fig. S1b), which is similar to
what was observed in E. ulmoides (Pang et al., 2008).
Moreover, typical phloem patterning was also observed tangentially (Fig. S1c). From 9 to 12 DAG, the girdled trunk
became greener, dry and hard (Fig. 1d). Continuous and flat
regenerated cambial cells were observed in several cell layers
under the regenerated phloem at 12 DAG (Fig. 2j–l).
Based upon our morphological observation of twiceperformed girdling experiments, we divided the regeneration
process into four stages: initially (Stage 0), differentiating
xylem cells are left on the surface of the trunk after bark
girdling (Figs 1a, 2a, b); Stage I, from 2 to 6 DAG, xylem cells
dedifferentiate (Figs 1b, 2c–f); stage II, from 6 to 9 DAG,
sieve element formation (Figs 1c, 2g–i); stage III, from 9 to
12 DAG, wound cambium formation (Figs 1d, 2j–l).
Transcriptome profiling indicates global changes of
gene expression during SVT regeneration
For transcriptome profiling analysis, a series of 20-lm-thick
tangential cryosections were collected at each stage of regeneration, as illustrated in Fig. 3(a). Five tissues, differentiating
xylem (diX, stage 0), dedifferentiating xylem (deX, stage I),
regenerated phloem (rPh, stage II), differentiating regenerated
New Phytologist (2011) 192: 869–884
www.newphytologist.com
871
New
Phytologist
872 Research
cambium (diC, stage II) and regenerated cambium (rC,
stage III) were sampled for Affymetrix poplar wholegenome array hybridization (Fig. 3a).
Four pairwise comparisons, (a)deX ⁄ diX, (b)rPh ⁄ deX,
(c)diC ⁄ deX and (d)rC ⁄ diC, were performed (Fig. 3b).
Transcripts with 90% LCBs ‡ 1.5 across at least one of the
four comparisons were chosen as differentially expressed
genes (DEGs). Extensive changes in gene expression were
observed across all stages of regeneration (Fig. 3c). SYBR
qRT-PCR of selected transcripts with different LCB values
suggested a good correlation between qRT-PCR and microarray results (Fig. 3d). The large numbers of up- and downregulated transcripts from diX to deX indicated dramatic
transcriptome changes during the process of xylem dediffer-
entiation. On the other hand, much smaller changes in gene
expression were observed from diC to rC, implying similar
molecular features of the two samples (Fig. 3c). DEGs were
then classified based on gene ontology (GO); the numbers
of up- or down-regulated genes in each GO category are
shown in Table 1 and Fig. S2. Genes associated with transcription, transport, stress response and response to
hormone stimulus were strongly regulated during regeneration (Fig. S2). Based on the GO annotation, it can be seen
that genes related to the chloroplast, plasma membrane,
nucleus and endomembrane system were enriched (Fig. S2).
Most of the GO biological process and cellular component
categories had similar numbers of genes that were up- or
down-regulated (Table 1 and Fig. S2). However, in early
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
Fig. 2 Anatomical observation of secondary vascular tissue (SVT) regeneration after bark girdling in Populus tomentosa. Cambium has been
removed with bark during girdling, leaving only differentiating xylem (diX) cells on the surface of the trunk, as shown in the cross section (a)
and radial section (b). At 2 d after girdling (DAG), ray cells of diX started to divide and form callus cells (Cal) as shown in the cross section (c).
At 4 DAG, more callus cells covered the wounded surface, and axial diX cells under callus underwent periclinal and transverse divisions;
dedifferentiating xylem cells (deX) were shown in the cross section (d) and radial section (e). No sieve elements can be detected in deX at this
time by aniline blue staining under fluorescence microscopy (f). At 6 DAG, sieve elements were detected by aniline blue staining (i), while
cambium was not observed at this time in both cross section (g) and radial section (h). Some of deX under regenerated phloem (rPh) will
differentiate into regenerated cambium (diC). At 12 DAG, continuous and flat regenerated cambium (rC) was observed in several cell layers in
both cross section (j) and radial section (k) under rPh; it was also shown in an aniline blue-stained radial section (l). Arrowheads show sieve
elements. Xy, xylem cells. Bars, 100 lm.
New Phytologist (2011) 192: 869–884
www.newphytologist.com
2011 The Authors
New Phytologist 2011 New Phytologist Trust
New
Phytologist
Research
(a)
(b)
(c)
(d)
Fig. 3 Illustration of sampling procedure and differentially expressed gene (DEG) distribution and quantitative reverse transcription
polymerase chain reaction (qRT-PCR) validation of microarray data. (a) Schematic illustration of sampling procedure for microarray and qRTPCR analysis. A series of 20-lm-thick tangential cryosections was taken for each sample: c. 60- to 80-lm-thick differentiating xylem cells (diX)
at stage 0, 40- to 60-lm-thick dedifferentiating xylem (deX) at stage I, regenerated phloem (rPh) and differentiating cambium (diC) at stage II
and regenerated cambium (rC) at stage III, as indicated by arrows. Bars with different patterning indicate various tissues (mx, mature xylem).
(b) Illustration of the regeneration of phloem and cambium and four pairwise sample comparisons. (c) Numbers of DEGs identified to be
up-regulated (striped bars) or down-regulated (filled bars) across distinct regeneration stages. Transcripts with lower confidence bounds
(LCBs) ‡ 1.5 in at least one of the four comparisons were flagged as differentially expressed. (d) Comparison between the 90% LCB of fold
change reported by microarray and fold change (FC) obtained by qRT-PCR. Genes with LCB > 2, close to 2, 1.5, 1 were chosen for validation.
Data were obtained from 28 probe-sets across four comparisons (as shown in Fig. 1b). Log2FC for each comparison is plotted against log2LCB.
The intercept of the linear regression line was set to pass through the origin.
regeneration (dedifferentiation, deX ⁄ diX), genes related to
cell cycle, DNA methylation and chromatin regulation were
dominantly up-regulated while genes related to cytoskeleton
and cell wall biogenesis and Golgi apparatus were strongly
down-regulated (Fig. S2). During phloem and cambium
regeneration (rPh ⁄ deX and diC ⁄ deX), there was a preponderance of up-regulated genes related to transcription
(Table 1 and Fig. S2). In addition, genes involved in trans-
2011 The Authors
New Phytologist 2011 New Phytologist Trust
port, stress and hormone response were dramatically altered
during phloem reformation (Fig. S2).
Xylem cells may acquire regenerative competence
through epigenetic regulation and cell cycle re-entry
Investigation of the expression patterns of the related genes
indicated that epigenetic regulation and cell cycle re-entry
New Phytologist (2011) 192: 869–884
www.newphytologist.com
873
New
Phytologist
874 Research
Table 1 Gene ontology (GO) classification of differentially expressed genes during second vascular tissue regeneration
(a) deX ⁄ diX
(b) rPh ⁄ deX
(c) diC ⁄ deX
(d) rC ⁄ diC
Up
Up
Up
Up
GO category
Function category
Biological process
Transcription
Transport
Response to stress
Response to hormone stimulus
Proteolysis
Signal transduction
Cell cycle
DNA methylation and chromatin
assembly or disassembly
Amino acid metabolic process
Programmed cell death and apoptosis
Cytoskeleton
Ion homeostasis
Cell wall biogenesis
Others
Unannotated
235
257
160
110
65
41
49
37
268
353
84
102
52
77
9
6
186
139
109
67
49
27
3
6
91
110
51
44
26
15
37
22
146
119
74
45
37
31
3
7
52
88
42
31
16
11
14
1
17
22
9
10
5
3
0
0
17
10
3
2
4
2
3
1
27
15
8
6
3
3168
799
2
18
20
14
34
2658
968
9
33
5
11
5
1275
536
7
3
14
1
4
1103
315
8
21
4
11
2
1117
455
5
1
6
2
0
604
189
2
1
0
0
1
128
80
0
2
0
0
0
104
47
Chloroplast
Plasma membrane
Nucleus
Endomembrane system
Cytosol
Cell wall
Ribosome
Plastid
Other intracellular components
Mitochondria
Endoplasmic reticulum
Golgi apparatus
Extracellular
Other cellular components
1162
551
469
350
291
218
174
97
81
59
58
21
16
1283
410
761
365
487
122
146
19
17
74
20
81
131
17
1884
297
298
208
281
54
73
16
13
26
3
37
12
10
1022
272
236
185
154
92
90
44
34
21
45
30
20
8
551
182
234
175
313
43
55
8
12
32
4
32
6
10
892
124
154
91
136
28
49
8
10
6
16
24
3
7
375
15
22
20
39
4
11
0
0
1
0
6
0
5
142
23
26
19
25
7
3
2
1
1
0
5
0
1
77
Total
4830
4534
2350
1782
1998
1031
265
190
Cellular component
Down
Down
Down
Down
deX, dedifferentiating xylem; diX, differentiating xylem; diC, differentiating regenerated cambium; rC, regenerated cambium; rPh, regenerated phloem.
The number of up- or down-regulated genes under each GO category for each sample pairwise comparison is shown.
were involved in early SVT regeneration. Three DNA
methyltransferases, METHYLTRANSFERASE 1 (MET1),
DOMAINS REARRANGED METHYLASE 1 and 2
(DRM1 ⁄ 2), and CHROMOMETHYLASE3 (CMT3)
(Finnegan & Kovac, 2000), and DEMETER (DME), a
DNA glycosylase with DNA demethylation activity
(Gehring et al., 2006; Morales-Ruiz et al., 2006), are
involved in DNA methylation regulation in plants. Our
data showed that one putative CMT3 and two MET transcripts were up-regulated, whereas two candidate DME
genes were down-regulated in early regeneration in deX
(Fig. 4a). The opposite expression patterns of putative
DNA methyltransferases and DME suggested that
DNA methylation regulation is involved during early SVT
regeneration.
In addition to DNA methylation regulation, we found
that the expression of histone modification enzymes was
changed in SVT regeneration. Two histone demethylase
New Phytologist (2011) 192: 869–884
www.newphytologist.com
families, amine oxidase (AOD) and Jumonji C (JmjC)
domain-containing histone demethylase families, have been
identified in the Populus genome (Zhou & Ma, 2008).
Among these putative histone demethylases, six AOD
domain-containing transcripts (PtAOD1B, PtAOD2B,
PtAOD7A, PtAOD8B, and another two AOD family genes)
and five JmjC domain-containing transcripts (PtKDM3D,
PtPKDM11, PtPKDM8A, PtPKDM8B and PtPDKM7E)
showed diverse expression patterns during regeneration
(Fig. 4a and Table S1).
Two putative histone methyltransferases, HMT1 and
HMT3, were also found to have expression peaks at diX
and rC, respectively (Fig. 4a). In addition, probe-sets representing putative Populus histone acetyltransferase (HAT)
and histone deacetylase (HDAC) genes (Pandey et al.,
2002) were identified in our dataset. Four HAT (HAG2,
HAG3, HAF2 and HAC4) and five HDAC (HDA6, 8, 14,
19 and one plant-specific HDAC, HDT1) transcripts were
2011 The Authors
New Phytologist 2011 New Phytologist Trust
New
Phytologist
Research
(a)
(b)
Fig. 4 Transcript expression patterns of
epigenetic regulation and cell cycle-related
genes during Populus tomentosa secondary
vascular tissue (SVT) regeneration.
(a) Candidate genes involved in DNA
methylation, histone methylation, histone
acetylation, chromatin remodeling and
polycomb group (PcG) proteins were selected
and clustered. (b) Cyclins and cyclindependent kinase (CDK) genes with
differential expression levels across all
samples were identified and clustered.
Expression values for each probe-set across
all samples were standardized (linearly
scaled) to have mean 0 and standard
deviation 1 as indicated by red ⁄ greencolored squares. The coefficient of variation
(CV) for each transcript across samples was
used to indicate fluctuation in expression.
Complete information for each probe-set in
the gene lists can be found in the Supporting
Information, Table S1.
2011 The Authors
New Phytologist 2011 New Phytologist Trust
New Phytologist (2011) 192: 869–884
www.newphytologist.com
875
876 Research
differentially expressed. However, they exhibited different
expression patterns at early regeneration stages (Fig. 4a).
Among the homologous genes of chromatin remodelingrelated proteins (Boyko & Kovalchuk, 2008) identified in
our dataset, some of them showed significant changes in
transcriptional level. In early regeneration, one group of
genes consisting of DDM1 and LHP1 were up-regulated
while another group of genes, including putative MBD2
and MBD8, MOM1 and one ISW2-like gene, were downregulated (Fig. 4a).
Polycomb group (PcG) proteins and the Polycomb
repressive complexes PRC1 and PRC2 play important roles
during cell fate switch and regeneration (Costa & Shaw,
2007; Birnbaum & Alvarado, 2008). All putative homologs
of Arabidopsis PcG genes (Pien & Grossniklaus, 2007) were
identified in our dataset, including several PRC2 subunits,
such as SWINGER (SWN), FIE and MSI1 (Fig. 4a). SWN
was down-regulated in deX and then increased at a later
stage in rPh and rC. Conversely, MSI1, MSI2, ICU2 and
FIE were up-regulated in deX and diC (Fig. 4a). In our earlier proteomic study, a protein homolog of SWN was found
at 6 DAG and then disappeared at 10 DAG (Du et al.,
2006). Our present transcript data together with previous
proteomic data suggest a role for PcG in xylem cell dedifferentiation during SVT regeneration.
During SVT regeneration, almost all differentially
expressed type-A cyclins (CYCAs) and type-B cyclins
(CYCBs) exhibited highly similar profiles with a significant
peak in expression at early regeneration in deX (Fig. 4b).
However, exceptions, such as CYCA2 and type-D cyclins
(CYCDs), were observed. Transcripts for CYCA2;2 and
CYCA2;4 showed the highest expression in diX and downregulation during regeneration (Fig. 4b). In Populus,
PttCYCA2 expression remains high until very late in xylem
development (Schrader et al., 2004). Expression of
AtCYCA2 in Arabidopsis is considered to be associated with
competence for cell division (Burssens et al., 2000). These
data suggest that diX cells retained their developmental
plasticity and have the capacity to change into other cell
types in certain conditions, such as wounding. Several
probe-sets representing Cyclin D3:1 exhibited high expression in diC or rC (Fig. 4b). Cyclin-dependent kinases
(CDKs) and CDK-like (CKL) displayed different expression profiles; of these, two plant-specific CDKB genes
(CDKB1;2 and CDKB2;1) followed the general expression
trend of most CYCA and CYCB genes (Fig. 4b). Similarly,
most histone transcripts also exhibited an expression peak
in deX (Table S1). In cell cycle re-entry experiments conducted in Arabidopsis, most CYCAs and CYCBs exhibit a
distinct peak in early mitosis and are both considered as
mitotic cyclins, while CDKB genes exhibit their expression
peak at either the G2 phase or the G2 ⁄ M transition
(Menges et al., 2005). Comparing the observed specific
expression pattern of mitotic cyclins and CDKB genes dur-
New Phytologist (2011) 192: 869–884
www.newphytologist.com
New
Phytologist
ing SVT regeneration with the regulatory mechanism found
in cell re-entry experiments suggests the activation of cell
cycle re-entry of differentiating xylem cells in their early cell
fate switching process. This is also consistent with our anatomical findings during early SVT regeneration in both
P. tomentosa (Fig. 2) and Eucommia, in which different cell
division phases of immature xylem cells have been observed
(Pang et al., 2008).
Down-regulation of the xylem-specific program and
activation of phloem and cambium developmental
programs during SVT regeneration
To address the identity change of the diX cells, we examined reported xylem marker genes, such as cellulose
synthase (CesA) genes, xyloglucan endotransglycosylase
genes PttXET16C ⁄ PtXTH35, programmed cell death
(PCD)-associated XYLEM CYSTEINE PEPTIDASE (XCP),
lignin biosynthesis-related genes and xylem specific transcription factors (TFs) (Schrader et al., 2004; Zhao et al.,
2005; Geisler-Lee et al., 2006; Suzuki et al., 2006). Eight
probe-sets representing xylem-specific Populus CesA and
CesA-like (Csl) genes (PtCesA18 ⁄ 3-2, PtrCesA3, PtCesA3,
PtCesA7, PtCesA4 ⁄ 5, PtCslA1 ⁄ 2) were identified from our
dataset and all eight CesA transcripts exhibited high expression in diX but were absent or had very low expression in
regenerated phloem and cambium tissues (Fig. 5a and
Table S2). The transcript of PtXTH35 and homolog of
Arabidopsis XCP2 also show preferential expression in diX
(Fig. 5a and Table S2). Populus lignin biosynthesis-related
enzymes (Hamberger et al., 2007; Shi et al., 2010), including
PAL, C4H, C3H, 4CL (Poptr4CL3L), HCT, CCoAOMT
(PttCCoAOMT1), COMT (PttCCoAOMT1 and 2) and
CAD (PoptrCAD1 and PoptrCAD4 ⁄ PtrCAD2 ⁄ PtCAD)
genes, exhibited a similar expression profile with enrichment
in diX and a dramatic decline in regenerating tissues, implying the loss of xylem characteristics in cells during
regeneration (Fig. 5a and Table S2).
HD-ZIP III genes have been demonstrated to promote
xylem specification (Emery et al., 2003; Carlsbecker &
Helariutta, 2005). In Populus, homologs of ATHB-8
(PttHB8), ATHB-9 ⁄ PHV (PttHB9) and ATHB-15
(PttHB15) exhibit a steep increase in expression on the xylem
side of the cambial zone (Schrader et al., 2004). In our study,
PttHB8, PttHB9 and PttHB15 were specifically expressed in
diX and distinctly down-regulated in regenerating tissues
(Fig. 5b and Table S2). In addition, examination of xylembiased NAC domain TFs and MYB TFs (Wilkins et al.,
2009; Zhong & Ye, 2009; Yamaguchi & Demura, 2010)
showed six NAC TFs and a group of Populus R2R3-MYB
TFs were diX-enriched with dramatic down-regulation
during regeneration, as shown in Fig. 5b and Table S2.
Taken together, the enrichment of these genes in diX validated the enrichment of these genes in secondary xylem
2011 The Authors
New Phytologist 2011 New Phytologist Trust
New
Phytologist
Research
(a)
(b)
Fig. 5 Down-regulation of xylem
development-related genes during Populus
tomentosa secondary vascular tissue (SVT)
regeneration. (a) Xylem-specific marker
genes and lignification-related enzyme
genes. (b) Xylem specification-related
transcription factors. Complete information
for each probe-set can be found in
Supporting Information, Table S2.
cells; furthermore, the significant decrease in expression of
these genes in regenerating tissues indicates the shuttingoff of the xylem specification program and thus the loss
of xylem cell identity during regeneration.
On the other hand, our data suggested that phloem and
cambium gene expression programs were activated during
SVT regeneration. Transcription factors ALTERED
PHLOEM DEVELOPMENT (APL) and KANADI (KAN)
are required for phloem specification and differentiation
(Kerstetter et al., 2001; Bonke et al., 2003; Emery et al.,
2003). During SVT regeneration, transcripts of Populus
APL and PttKAN1, PttKAN2 were accumulated in the
phloem formation stage (stage II) and their expression level
remained high until the appearance of cambium at stage III
(Fig. 6a). Besides, several other groups of TFs were also
found highly expressed in rPh, including G2-like MYB,
Dof and NAM (NO APICAL MERISTEM) (Fig. 6a and
2011 The Authors
New Phytologist 2011 New Phytologist Trust
Table S3). MYB-RELATED PROTEIN 1 (MYR1) and Dof
genes have been found to be phloem-abundant in various
species (Zhao et al., 2005; Le Hir et al., 2008). A recent
report also shows that Dof5.6 ⁄ HCA2 is expressed in phloem
and cambium of Arabidopsis inflorescence stems (Guo
et al., 2009). For all Dof genes identified in the Populus
genome (Yang et al., 2006), six transcripts showed clear
enrichment in rPh or ⁄ and rC (Fig. 6a). Among them,
PtrDOF03 ⁄ 33 was the closest homolog of Arabidopsis
Dof5.6. Collectively, our results provided molecular evidence that groups of TFs such as MYB, Dof and NAM
contributed to phloem and cambium regeneration.
Furthermore, to assess the structure and function of the
newly formed sieve elements as transport phloem cells,
potential phloem structure and function-associated genes
were examined. Genes encoding sieve elements ⁄ companion
cells specific jacalin-related lectins, sieve element specifically
New Phytologist (2011) 192: 869–884
www.newphytologist.com
877
878 Research
New
Phytologist
(a)
(b)
Fig. 6 Up-regulation of transcription factors
involved in phloem specification (a) and
cambium regulation (b) during Populus
tomentosa secondary vascular tissue (SVT)
regeneration. Complete information for each
probe-set in the gene lists can be found in
Supporting Information, Table S3.
synthesized serpins, sucrose synthase and phloem-specific
metabolic enzymes could highlight common features of
phloem functions in various plant species (Le Hir et al.,
2008; Lin et al., 2009). Clustering analysis showed that
these related genes were abundant in rPh (Table S3). As
shown in Figs 3, S1, callose, the marker for sieve elements,
was detected in stages II and III by aniline blue.
Accordingly, transcripts of callose synthase were up-regulated in rPh (Table S3). Transcripts of hormone
biosynthesis-related genes, such as GA4 ⁄ gibberellin 3 betahydroxylase and ACC synthase, and stress response genes
were also present in rPh, (Table S3). In our earlier in vitro
experiments, isotope tracing of twigs with [14C]sucrose
showed that newly formed sieve elements are capable of
transporting sugar during the bark regeneration process in
Eucommia (Pang et al., 2008). Genes encoding sugar transporters, amino acid carriers, H+-transporting ATPase
(AHA10), potassium channels as well as RNA-binding pro-
New Phytologist (2011) 192: 869–884
www.newphytologist.com
teins showed an increase in expression in rPh (Table S3)
and thus provided molecular evidence of transport function
recovery of phloem. Together, our data indicate the activation of the phloem program during SVT regeneration.
Current evidence indicates that a WUSCHEL-CLAVATAlike mediated regulatory model is active in cambial meristems, as in the apical meristems (Elo et al., 2009; Zhang
et al., 2011). In Arabidopsis, the PXY-CLE41 ⁄ CLE44 loop
maintains the undifferentiated pluripotent status in the
cambium (Etchells & Turner, 2010). WOX4, a
WUSCHEL-related HOMEOBOX gene, may function in
the TDIF ⁄ CLE41 ⁄ CLE44-TDR/PXY signaling pathway to
promote procambial ⁄ cambial cell proliferation (Hirakawa
et al., 2010). In Populus, Schrader et al. (2004) have also
proposed the existence of a similar feedback loop in the
cambium. PttCLV1, a putative ortholog of CLAVATA1,
showed very low expression in diX and deX and displayed
significant up-regulation in regenerated cambial and
2011 The Authors
New Phytologist 2011 New Phytologist Trust
New
Phytologist
Research
(a)
Fig. 7 Transcript expression patterns of
auxin carriers and auxin receptors genes
during secondary vascular tissue (SVT)
regeneration. Auxin carrier AUX and PIN
genes, auxin receptor TIR and auxin-binding
protein1 (ABP1) genes were identified and
clustered. The complete information for each
probe-set in the gene lists can be found in
Supporting Information, Table S4. diX,
differentiating xylem; deX, dedifferentiating
xylem; rPh, regenerated phloem; diC,
differentiating regenerated cambium; rC,
regenerated cambium; PAT, polar auxin
transport; CV, coefficient of variation.
(b)
phloem cells (Fig. 6b). Transcripts of CLV2 and several
other leucine-rich repeat kinases also exhibited similar
expression patterns to PttCLV1 (Table S3). Conversely, the
receptor-like kinase PttRLK3 and a WUSCHEL-related
homeobox family gene PttHB2 showed preferential expression in diX and down-regulated across regeneration. This
was similar to what has been found in the normal cambial
region, where PttHB2 shows a steep increase in expression
toward the xylem side (Schrader et al., 2004).
PttANT and PttPNH, Populus homologs of
AINTEGUMENTA and PINHEAD ⁄ ZWILLE, show a distinct expression peak in the cambium zone, which implies
their involvement in regulation of cambial cell proliferation
(Schrader et al., 2004). Probe-sets for PttANT;1 and
PttPNH were identified and they all exhibited an expression
peak in regenerated cambium (Fig. 6b, Table S3).
Interestingly, the accumulation of PttANT transcripts was
also detected as early as in deX. The up-regulation of one
PINHEAD gene during cambium regeneration was
reported and predicted to function in cambium initiation
rather than maintenance (Wang et al., 2009). However, our
data revealed that the expression of PttPNH remained high
until cambium formation, indicating that PINHEAD might
be involved in cambium maintenance as well (Fig. 6b).
Populus orthologs of Arabidopsis Class I KNOX genes were
highly expressed in the cambial zone and functional analysis
suggested that they are likely involved in the cambial cell
identity maintenance (Schrader et al., 2004; Groover et al.,
2006; Du et al., 2009). During regeneration, KNOX1 and
KNOX6 showed an increasing trend in regenerated phloem
and cambium (Fig. 6b). In addition, a class II KNOX gene,
KNAT3 ⁄ KNOX3, showed high expression in rPh and diC
but lower signals in rC (Fig. 6b). The GRAS family TFs
SHORTROOT (SHR) and SCARECROW (SCR) contribute
2011 The Authors
New Phytologist 2011 New Phytologist Trust
to vascular cell patterning in Arabidopsis roots (Helariutta
et al., 2000; Nakajima & Benfey, 2002). During cambium
regeneration, two Populus homologs of SHR (PttSHR1 and
PttSHR2) exhibited maximum expression in rC, while four
putative SCR-like 6 (SCL6) revealed different expression
patterns toward rC (Fig. 6b and Table S3). The expression
profiling of these TFs indicates their potential contribution
in cambium regeneration.
Further expression profile analysis revealed that members
of additional TF families, such as R2R3-MYB, WRKY,
bHLH, bZIP, Whirly and CONSTANS, were enriched in
regenerated phloem and cambium (Table S3).
Phytohormone regulation during SVT regeneration
Phytohormones play crucial roles in regulating tissue or
organ regeneration in plants (Xu et al., 2006; Grieneisen
et al., 2007; Birnbaum & Alvarado, 2008; Sena et al.,
2009). An auxin gradient provides a positional signal in pattern specification during SVT development (Uggla et al.,
1996, 1998; Mwange et al., 2005). Redistribution of auxin
gradients may induce root regeneration (Xu et al., 2006)
and roots failed to regenerate when auxin transport was
blocked (Sena et al., 2009). To look into the molecular evidence for auxin redistribution during SVT regeneration, we
checked the expression patterns of polar auxin transport
(PAT) genes including auxin influx carriers (PttLAX,
AUX1-like) and efflux carriers (PttPIN, PIN1-lke) identified in Populus (Schrader et al., 2003). The results showed
that most AUX1-like genes were highly expressed in diX;
however, PttLAX2 and another AUX1-like genes were also
enriched in rC (Fig. 7a). PttLAX3, on the other hand,
showed a similar expression pattern to PttPIN1 and a
PIN1-like gene with high expression in rC. A very different
New Phytologist (2011) 192: 869–884
www.newphytologist.com
879
New
Phytologist
880 Research
result was found for PttPIN3, which was enriched in deX
(Fig. 7a). In the wood-forming region, PttLAX1 and
PttLAX2 are both expressed in secondary cell wall-forming
xylem but PttLAX2 is also expressed in the cambial zone.
PttLAX3, PttPIN1 and PttPIN2 are expressed in the cambium and dividing xylem mother cells, while PttPIN3
shows highest expression in the cortex layer (Schrader et al.,
2003). The correlation between the dynamic expression
patterns of PAT genes across SVT regeneration and their
spatial expression patterns in the normal wood-forming
region implied that the expression changes of PAT genes
might contribute to the resetting up of auxin gradients
during phloem and cambium recovery. By using high-performance liquid chromatography and immunolocalization
techniques, we have previously shown the significant change
of auxin concentration and distribution within the girdled
areas during bark reconstitution (Mwange et al., 2003).
Alongside polar auxin transport, auxin signaling also plays
an essential role in tissue regeneration. A list of auxin-responsive genes is involved in Arabidopsis root-tip regeneration
(Sena et al., 2009) and one IAA-induced TF is up-regulated
during poplar secondary vascular system regeneration
(Wang et al., 2009). Homologs of auxin receptors (TIR1)
and genes involved in auxin response, including Auxin ⁄
Indole-3-Acetic Acid (Aux ⁄ IAA) and Auxin Response Factor
(ARF), were identified (Table S4). Analysis of TIR1 and
auxin-binding protein1 (ABP1) genes demonstrated that
putative TIR1 transcripts were up-regulated during SVT
regeneration, whereas ABP1 transcript was down-regulated
(Fig. 7b). Recent reports indicate that ABP1 is required for
the regulation of early auxin-regulated genes and PAT
(Robert et al., 2010; Effendi et al., 2011). Our observation
might imply different modes of action for the two potential
auxin receptors in SVT regeneration. A total of 35 Aux ⁄ IAA
genes denoted as PoptrIAA and 39 ARF denoted as
PoptrARF were discovered in the Populus genome (Kalluri
et al., 2007). Twenty-five probe-sets representing 15
PoptrIAA and 18 probe-sets representing 14 PoptrARF were
identified from our dataset and they showed differential
expression profiles and tissue preference during regeneration
(Table S4). The presence of PoptrARF8 in rPh (Table S4)
confirmed previous detection of this gene transcript in
phloem (Zhao et al., 2005; Le Hir et al., 2008). Several
auxin-responsive genes (examined by Sena et al. (2009)) and
enzymes putatively involved in Aux ⁄ IAA proteolysis were
also up-regulated in rPh and rC (Table S4). The regulatory
role of auxin during SVT regeneration has also been supported in our experiments carried out in Eucommia, in
which application of exogenous IAA on the wound surface
accelerates sieve element differentiation (Pang et al., 2008).
In addition to auxin, genes involved in other phytohormone signalling pathways, such as those of cytokinin, GA
and ethylene, were also differentially expressed during SVT
regeneration (Table S4). Recent studies demonstrate that
New Phytologist (2011) 192: 869–884
www.newphytologist.com
cytokinins are central hormonal regulators of cambium
development (Matsumoto-Kitano et al., 2008; Nieminen
et al., 2008; Dettmer et al., 2009). Cytokinins can accelerate
sieve tube regeneration and promote callose production
around the wound of Coleus internodes (Aloni et al., 1990).
Analysis of the expression patterns of cytokinin receptors,
type-A and type-B response regulators (RRs) identified in
Populus (Ramirez-Carvajal et al., 2008) revealed that transcripts of putative Populus CRE1B and histidine kinase-like
(PtHK3a and PtHK3b), four type-A RRs (PtRR1, 2, 7 and
10) and three type-B RRs (PtRR19, 21 and 22) were upregulated in rPh and ⁄ or rC (Table S4). The GA signal transduction protein (spindly), signal response protein
GIBBERELLIC ACID INSENSITIVE (GAI) and GAregulated protein GASA were differentially expressed during
regeneration. However, multiple expression patterns for
these genes were observed (Table S4). Homologs of ethylene
receptor members EIN4, ERT2 and ERS were differentially
expressed during regeneration. A number of Populus ERFs
(ethylene response factors) and DREBs (dehydrationresponsive element binding proteins) were enriched in
regenerating tissues (Table S4).
Discussion
Root and shoot regeneration in plants has been widely studied but with limited attention paid to vascular tissue
regeneration. In this study, we report an in situ plant regeneration system, the SVT regeneration after bark girdling in
Populus. Unlike most other de novo regeneration system in
plants (Sena & Birnbaum, 2010), SVT regeneration does not
lead to entirely new individuals, but only the regeneration of
missing tissues in the vasculature (Fig. 2). Furthermore,
compared with other in situ regeneration systems, such as
Arabidopsis root tip cut regeneration, our system includes cell
dedifferentiation, transdifferentiation and new tissue pattern
rebuilding, which happens over a longer time span, and this
provides us with a good opportunity for higher temporal
resolution analysis of plant regeneration.
In SVT regeneration after bark girdling, the wound cambium and sieve elements are not derived from callus but
from differentiating xylem cells. After girdling, differentiating xylem cells at different positions transdifferentiate to
form phloem cells or dedifferentiate to form the wound
cambium (Pang et al., 2008). Furthermore, microarray
analysis with different regenerated tissues at distinct stages
revealed the molecular features of the regeneration process.
It is commonly accepted that cells dedifferentiate to form
pluripotent cells (as callus) during regeneration. However,
recent findings in plants have suggested that organ regeneration does not need complete dedifferentiation to a
pluripotent state (Pang et al., 2008; Sena et al., 2009). On
the other hand, although callus has long been regarded as
dedifferentiated and pluripotent cell mass, recent studies
2011 The Authors
New Phytologist 2011 New Phytologist Trust
New
Phytologist
demonstrated that callus possesses root meristem characteristics even when it is derived from shoot tissues (Atta et al.,
2009; Sugimoto et al., 2010). Our experiments showed that
phloem or cambium regeneration does not need cell dedifferentiation into a callus state during SVT regeneration in
Populus.
To address whether the molecular program of early SVT
regeneration is similar to organogenesis via callus, we compared our profiling data in early SVT regeneration
(deX ⁄ diX) with published profiling data of early callus
induction (CIM3 ⁄ CIM0) in Populus (Bao et al., 2009).
During early SVT regeneration, 13% of up-regulated genes
(649) and 6% of down-regulated genes (294) were common
with these callus-expressed genes, suggesting only modest
overlap of molecular processes by these two systems. The
enriched GO classes of genes that were common and upregulated include those related to chloroplast, transcription,
transport and response to stress, while the largest common
down-regulated class was genes related to the plasma membrane (Table S1, Fig. S2). However, different expression
patterns of specific groups of genes were observed when the
two processes were compared. Among the 223 up-regulated
and 353 down-regulated transcription factors during early
callus induction in Populus (Bao et al., 2009), only 47 were
up-regulated and 80 down-regulated during early SVT
regeneration. The regulation of cell cycle genes was different.
CYCA2 genes are up-regulated in CIM3 during early callogenesis (Bao et al., 2009) while down-regulated in deX
(Fig. 4b). Moreover, most CYCAs and CYCBs were
up-regulated in deX, which is not observed in early callus
induction (Bao et al., 2009). Auxin and cytokinin signalingrelated genes were also differently regulated during
early SVT regeneration (Table S4) and early callus induction
(Bao et al., 2009). These data provide molecular evidence
that SVT regeneration is dissimilar from in vitro callogenesis.
Chromatin remodeling and cell cycle re-entry are necessary for organ regeneration (Costa & Shaw, 2007;
Birnbaum & Alvarado, 2008). Histone methylation controls telomere lengthening in Arabidopsis mesophyll cells
Research
undergoing dedifferentiation and is necessary for maintaining the dedifferentiating state of cells and cell cycle re-entry
(Grafi et al., 2007). During Drosophila leg to wing transdifferentiation, PcG proteins involved in PRC1 are downregulated in transdifferentiating cells, and the frequency of
transdifferentiation is enhanced in PcG mutant flies (Lee
et al., 2005). In Arabidopsis, double mutation of two PcG
genes CURLY LEAF (CLF) and SWN induces the formation
of ectopic callus and somatic embryos (Chanvivattana et al.,
2004), which implies that down-regulation of PcG promotes dedifferentiation from differentiated cells. Our
results indicate that epigenetic modulation is involved in
the early stages of SVT regeneration and differentiating
xylem cells may acquire regenerative competence through
cell cycle re-entry during SVT regeneration.
Phytohormones are important in the regulation of new
tissue reconstruction. During phloem regeneration, both
exogenous auxin and cytokinin accelerate sieve element
differentiation in different vascular regeneration systems
(Aloni et al., 1990; Pang et al., 2008). On the other hand,
auxin transport is necessary for root regeneration, and
setting up opposing cytokinin and auxin domains is an
important step in shoot meristem regeneration from callus
(Xu et al., 2006; Gordon et al., 2007; Grieneisen et al.,
2007; Sena et al., 2009). Dramatic expression changes of
auxin signaling genes as well as the dynamic expression
patterns of PAT genes (Fig. 7 and Table S4) indicate that
auxin signaling and redistribution play important roles
during SVT regeneration. The expression changes of PAT
genes might contribute to re-establishing the auxin gradients and thus provide positional signaling for the
specification of phloem and cambium. Based on the gene
expression patterns of related genes (Table S4), we conclude
that cytokinin and ethylene signaling are also activated during SVT regeneration.
Based upon our morphological observation and transcriptome profiling, we proposed a model for SVT
regeneration in Populus (Fig. 8). After bark girdling, differentiating xylem cells which remain on the trunk acquire the
Fig. 8 A proposed model for differentiating xylem cells switch fate into phloem and cambium during secondary vascular tissue (SVT)
regeneration after girdling. After bark girdling, differentiating xylem cells remained on the trunk. Chromatin remodeling and cell cycle re-entry
can help to reset the genomic state in early regeneration. While the xylem developmental program is blocked, the phloem or cambium
program is activated, resulting in their progressive reestablishment. Phytohormones also play an important role in the regulation of SVT
regeneration. NAC:NAM, ATAF and CUC; Dof, DNA binding with one finger; CLV, CLAVATA; ANT1, AINTEGUMENTA1. APL, ALTERED
PHLOEM DEVELOPMENT; SCR, SCARECROW; SHR, SHORTROOT; TF, transcription factor;
2011 The Authors
New Phytologist 2011 New Phytologist Trust
New Phytologist (2011) 192: 869–884
www.newphytologist.com
881
882 Research
competence to switch their fate through epigenetic regulation and cell cycle re-entry. Then, the xylem differentiation
program is blocked and the cell fates of the remaining differentiating xylem cells at different locations change at
different time points after girdling. This is decided by ‘positional information’ which is coordinated by specific signal
molecules. When xylem cells change into a competent state,
phloem specification-related genes such as APL, KANADI
and Dof are activated and hormone distribution is repatterned, resulting in the re-establishment of an expression
program for phloem development. In a similar way, genes
involved in cambium determination and maintenance are
up-regulated and the hormone gradient is repositioned to
promote the reformation of cambium. Concurrently, a cell
signaling network consisting of transcription factors and
hormone signaling transduction pathways is re-established
in regenerated tissues. Thus, the lost phloem and cambium
tissues are reconstituted from differentiating xylem cells.
Our hypothetical model describes the molecular features of
SVT regeneration after bark girdling in trees. It also provides information for revealing the mechanisms of tissue
regeneration in plants and for understanding pattern formation of the SVTs.
Acknowledgements
We thank Ykä Helariutta (University of Helsink, Finland),
Hiroo Fukuda (The University of Tokyo, Japan) and
Jing-Chu Luo (Peking University, China) for the valuable
suggestions on this project and critical comments on the
manuscript. We thank Sedeer El-Showk (University of
Helsink, Finland) for critical reading and editing of the
manuscript. This work was supported by the National
Natural Science Foundation of China (31070156), the
China Ministry of Agriculture Transgenic Breeding Projects
(no. 2009ZX08009-095B) and the China Ministry of
Science and Technology 863 Program (no. 2006AA02Z334;
2007AA02Z165).
References
Aloni R, Baum SF, Peterson CA. 1990. The role of cytokinin in sieve tube
regeneration and callose production in wounded Coleus internodes.
Plant Physiology 93: 982–989.
Atta R, Laurens L, Boucheron-Dubuisson E, Guivarc’h A, Carnero E,
Giraudat-Pautot V, Rech P, Chriqui D. 2009. Pluripotency of
Arabidopsis xylem pericycle underlies shoot regeneration from root and
hypocotyl explants grown in vitro. Plant Journal 57: 626–644.
Bao Y, Dharmawardhana P, Mockler TC, Strauss SH. 2009. Genome
scale transcriptome analysis of shoot organogenesis in Populus. BMC
Plant Biology 9: 132.
Birnbaum KD, Alvarado AS. 2008. Slicing across kingdoms: regeneration
in plants and animals. Cell 132: 697–710.
Bonke M, Thitamadee S, Mahonen AP, Hauser MT, Helariutta Y. 2003.
APL regulates vascular tissue identity in Arabidopsis. Nature 426:
181–186.
New Phytologist (2011) 192: 869–884
www.newphytologist.com
New
Phytologist
Boyko A, Kovalchuk I. 2008. Epigenetic control of plant stress response.
Environmental and Molecular Mutagenesis 49: 61–72.
Brown CL, Sax K. 1962. The influence of pressure on the differentiation
of secondary tissues. American Journal of Botany 49: 683–691.
Burssens S, de Almeida Engler J, Beeckman T, Richard C, Shaul O,
Ferreira P, Van Montagu M, Inzé D. 2000. Developmental expression
of the Arabidopsis thaliana CycA2; 1 gene. Planta 211: 623–631.
Carlsbecker A, Helariutta Y. 2005. Phloem and xylem specification: pieces
of the puzzle emerge. Current Opinion in Plant Biology 8: 512–517.
Chanvivattana Y, Bishopp A, Schubert D, Stock C, Moon YH, Sung ZR,
Goodrich J. 2004. Interaction of Polycomb-group proteins controlling
flowering in Arabidopsis. Development 131: 5263–5276.
Costa S, Shaw P. 2007. ‘Open minded’ cells: how cells can change fate.
Trends in Cell Biology 17: 101–106.
Cui KM. 1997. Switch and stages of plant cell differentiation. Life Science
9: 49–54.
Cui KM, Wu SQ, Wei LB, Little CHA. 1995. Effect of exogenous IAA
on the regeneration of vascular tissues and periderm in girdled Betula
pubescens stems. Chinese Journal of Botany 7: 17–23.
Dettmer J, Elo A, Helariutta Y. 2009. Hormone interactions during
vascular development. Plant Molecular Biology 69: 347–360.
Doyle JJ, Doyle JL. 1990. Isolation of plant DNA from fresh tissue. Focus
12: 13–15.
Du J, Mansfield SD, Groover AT. 2009. The Populus homeobox gene
ARBORKNOX2 regulates cell differentiation during secondary growth.
Plant Journal 60: 1000–1014.
Du J, Xie HL, Zhang DQ, He XQ, Wang MJ, Li YZ, Cui KM, Lu MZ.
2006. Regeneration of the secondary vascular system in poplar as a novel
system to investigate gene expression by a proteomic approach.
Proteomics 6: 881–895.
Effendi Y, Rietz S, Fischer U, Scherer GF. 2011. The heterozygous
abp1 ⁄ ABP1 insertional mutant has defects in functions requiring polar
auxin transport and in regulation of early auxin-regulated genes. Plant
Journal 65: 282–294.
Elo A, Immanen J, Nieminen K, Helariutta Y. 2009. Stem cell function
during plant vascular development. Seminars in Cell & Developmental
Biology 20: 1097–1106.
Emery JF, Floyd SK, Alvarez J, Eshed Y, Hawker NP, Izhaki A, Baum SF,
Bowman JL. 2003. Radial patterning of Arabidopsis shoots by class III
HD-ZIP and KANADI genes. Current Biology 13: 1768–1774.
Etchells JP, Turner SR. 2010. The PXY-CLE41 receptor ligand pair
defines a multifunctional pathway that controls the rate and orientation
of vascular cell division. Development 137: 767–774.
Finnegan EJ, Kovac KA. 2000. Plant DNA methyltransferases. Plant
Molecular Biology 43: 189–201.
Gehring M, Huh JH, Hsieh TF, Penterman J, Choi Y, Harada JJ,
Goldberg RB, Fischer RL. 2006. DEMETER DNA glycosylase
establishes MEDEA polycomb gene self-Imprinting by allele-specific
demethylation. Cell 124: 495–506.
Geisler-Lee J, Geisler M, Coutinho PM, Segerman B, Nishikubo N,
Takahashi J, Aspeborg H, Djerbi S, Master E, Andersson-Gunneras S
et al. 2006. Poplar carbohydrate-active enzymes. Gene identification
and expression analyses. Plant Physiology 140: 946–962.
Gordon SP, Heisler MG, Reddy GV, Ohno C, Das P, Meyerowitz EM.
2007. Pattern formation during de novo assembly of the Arabidopsis
shoot meristem. Development 134: 3539–3548.
Grafi G, Ben-Meir H, Avivi Y, Moshe M, Dahan Y, Zemach A. 2007.
Histone methylation controls telomerase-independent telomere
lengthening in cells undergoing dedifferentiation. Developmental Biology
306: 838–846.
Grieneisen VA, Xu J, Maree AFM, Hogeweg P, Scheres B. 2007. Auxin
transport is sufficient to generate a maximum and gradient guiding root
growth. Nature 449: 1008–1013.
2011 The Authors
New Phytologist 2011 New Phytologist Trust
New
Phytologist
Groover A, Mansfield S, DiFazio S, Dupper G, Fontana J, Millar R,
Wang Y. 2006. The Populus homeobox gene ARBORKNOX1 reveals
overlapping mechanisms regulating the shoot apical meristem and the
vascular cambium. Plant Molecular Biology 61: 917–932.
Guo Y, Qin GJ, Gu HY, Qu LJ. 2009. Dof5.6 ⁄ HCA2, a Dof
transcription factor gene, regulates interfascicular cambium formation
and vascular tissue development in Arabidopsis. Plant Cell 21:
3518–3534.
Hamberger B, Ellis M, Friedmann M, Souza CDA, Barbazuk B, Douglas
CJ. 2007. Genome-wide analyses of phenylpropanoid-related genes in
Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa: the Populus
lignin toolbox and conservation and diversification of angiosperm gene
families. Canadian Journal of Botany 85: 1182–1201.
Hammond JP, Bowen HC, White PJ, Mills V, Pyke KA, Baker AJM,
Whiting SN, May ST, Broadley MR. 2006. A comparison of the
Thlaspi caerulescens and Thlaspi arvense shoot transcriptomes. New
Phytologist 170: 239–260.
Hammond JP, Broadley MR, Craigon DJ, Higgins J, Emmerson ZF,
Townsend HJ, White PJ, May ST. 2005. Using genomic DNA-based
probe-selection to improve the sensitivity of high-density
oligonucleotide arrays when applied to heterologous species. Plant
Methods 1: 1746–4811.
Helariutta Y, Fukaki H, Wysocka-Diller J, Nakajima K, Jung J, Sena G,
Hauser MT, Benfey PN. 2000. The SHORT-ROOT gene controls
radial patterning of the Arabidopsis root through radial signaling. Cell
101: 555–567.
Hirakawa Y, Kondo Y, Fukuda H. 2010. TDIF peptide signaling regulates
vascular stem cell proliferation via the WOX4 homeobox gene in
Arabidopsis. Plant Cell 22: 2618–2629.
Kalluri U, DiFazio S, Brunner A, Tuskan G. 2007. Genome-wide analysis
of Aux ⁄ IAA and ARF gene families in Populus trichocarpa. BMC Plant
Biology 7: 59.
Kerstetter RA, Bollman K, Taylor RA, Bomblies K, Poethig RS. 2001.
KANADI regulates organ polarity in Arabidopsis. Nature 411: 706–709.
Le Hir R, Beneteau J, Bellini C, Vilaine F, Dinant S. 2008. Gene
expression profiling: keys for investigating phloem functions. Trends in
Plant Science 13: 273–280.
Lee N, Maurange C, Ringrose L, Paro R. 2005. Suppression of Polycomb
group proteins by JNK signalling induces transdetermination in
Drosophila imaginal discs. Nature 438: 234–237.
Li ZL, Cui KM. 1988. Differentiation of secondary xylem after girdling.
IAWA Bulletin 9: 375–383.
Li ZL, Cui KM, Yu CS, Chang XL. 1981. Anatomical studies of
regeneration after ringing of Eucomma ulmoides. Acta Botanica Sinica 23:
6–13.
Li C, Wong WH. 2001. Model-based analysis of oligonucleotide arrays:
expression index computation and outlier detection. Proceedings of the
National Academy of Sciences, USA 98: 31–36.
Lin MK, Lee YJ, Lough TJ, Phinney BS, Lucas WJ. 2009. Analysis of the
pumpkin phloem proteome provides insights into angiosperm sieve tube
function. Molecular & Cellular Proteomics 8: 343–356.
Lu PZ, Cui KM, Li ZL. 1987. Preliminary investigations of the
regeneration of 14 seed plants after girdling. Acta Botanica Sinica 29:
111–113.
Matsumoto-Kitano M, Kusumoto T, Tarkowski P, Kinoshita-Tsujimura
K, Vaclavikova K, Miyawaki K, Kakimoto T. 2008. Cytokinins are
central regulators of cambial activity. Proceedings of the National
Academy of Sciences, USA 105: 20027–20031.
Menges M, de Jager SM, Gruissem W, Murray JAH. 2005. Global
analysis of the core cell cycle regulators of Arabidopsis identifies novel
genes, reveals multiple and highly specific profiles of expression and
provides a coherent model for plant cell cycle control. Plant Journal 41:
546–566.
2011 The Authors
New Phytologist 2011 New Phytologist Trust
Research
Morales-Ruiz T, Ortega-Galisteo AP, Ponferrada-Marı́n MI, Martı́nezMacı́as MI, Ariza RR, Roldán-Arjona T. 2006. Demeter and repressor
of silencing 1 encode 5-methylcytosine DNA glycosylases. Proceedings of
the National Academy of Sciences, USA 103: 6853–6858.
Mwange KNK, Hou HW, Cui KM. 2003. Relationship between
endogenous indole-3-acetic acid and abscisic acid changes and bark
recovery in Eucommia ulmoides Oliv. after girdling. Journal of
Experimental Botany 54: 1899–1907.
Mwange KNK, Hou HW, Wang YQ, He XQ, Cui KM. 2005. Opposite
patterns in the annual distribution and time-course of endogenous
abscisic acid and indole-3-acetic acid in relation to the periodicity of
cambial activity in Eucommia ulmoides Oliv. Journal of Experimental
Botany 56: 1017–1028.
Nakajima K, Benfey PN. 2002. Signaling in and out: control of cell
division and differentiation in the shoot and root. Plant Cell 14:
S265–S276.
Nieminen K, Immanen J, Laxell M, Kauppinen L, Tarkowski P, Dolezal
K, Thtiharju S, Elo A, Decourteix M, Ljung K et al. 2008. Cytokinin
signaling regulates cambial development in poplar. Proceedings of the
National Academy of Sciences, USA 105: 20032–20037.
Noel A. 1970. The girdled tree. The Botanical Review 36: 162–195.
Pandey R, Muller A, Napoli CA, Selinger DA, Pikaard CS, Richards EJ,
Bender J, Mount DW, Jorgensen RA. 2002. Analysis of
histone acetyltransferase and histone deacetylase families of Arabidopsis
thaliana suggests functional diversification of chromatin modification
among multicellular eukaryotes. Nucleic Acids Research 30: 5036–5055.
Pang Y, Zhang J, Cao J, Yin SY, He XQ, Cui KM. 2008. Phloem
transdifferentiation from immature xylem cells during bark regeneration
after girdling in Eucommia ulmoides Oliv. Journal of Experimental Botany
59: 1341–1351.
Passier R, Mummery C. 2010. Getting to the heart of the matter: direct
reprogramming to cardiomyocytes. Cell Stem Cell 7: 139–141.
Pien S, Grossniklaus U. 2007. Polycomb group and trithorax group
proteins in Arabidopsis. Biochimica et Biophysica Acta 1769: 375–382.
Ramirez-Carvajal GA, Morse AM, Davis JM. 2008. Transcript profiles of
the cytokinin response regulator gene family in Populus imply diverse
roles in plant development. New Phytologist 177: 77–89.
Robert S, Kleine-Vehn J, Barbez E, Sauer M, Paciorek T, Baster P,
Vanneste S, Zhang J, Simon S, Čovanová M et al. 2010. ABP1
mediates auxin inhibition of clathrin-dependent endocytosis in
Arabidopsis. Cell 143: 111–121.
Schrader J, Baba K, May ST, Palme K, Bennett M, Bhalerao RP,
Sandberg G. 2003. Polar auxin transport in the wood-forming tissues of
hybrid aspen is under simultaneous control of developmental and
environmental signals. Proceedings of the National Academy of Sciences,
USA 100: 10096–10101.
Schrader J, Nilsson J, Mellerowicz E, Berglund A, Nilsson P, Hertzberg
M, Sandberg G. 2004. A high-resolution transcript profile across the
wood-forming meristem of poplar identifies potential regulators of
cambial stem cell identity. Plant Cell 16: 2278–2292.
Sena G, Birnbaum KD. 2010. Built to rebuild: in search of organizing
principles in plant regeneration. Current Opinion in Genetics and
Development 20: 460–465.
Sena G, Wang X, Liu HY, Hofhuis H, Birnbaum KD. 2009. Organ
regeneration does not require a functional stem cell niche in plants.
Nature 457: 1150–1153.
Shi R, Sun YH, Li Q, Heber S, Sederoff R, Chiang VL. 2010. Towards a
systems approach for lignin biosynthesis in Populus trichocarpa:
transcript abundance and specificity of the monolignol biosynthetic
genes. Plant Cell Physiology 51: 144–163.
Stobbe H, Schmitt U, Eckstein D, Dujesiefken D. 2002. Developmental
stages and fine structure of surface callus formed after debarking of
living lime trees (Tilia sp.). Annals of Botany 89: 773–782.
New Phytologist (2011) 192: 869–884
www.newphytologist.com
883
New
Phytologist
884 Research
Sugimoto K, Jiao YL, Meyerowitz EM. 2010. Arabidopsis regeneration
from multiple tissues occurs via a root development pathway.
Developmental Cell 18: 463–471.
Sustar A, Schubiger G. 2005. A transient cell cycle shift in Drosophila
imaginal disc cells precedes multipotency. Cell 120: 383–393.
Suzuki S, Li L, Sun YH, Chiang VL. 2006. The cellulose synthase gene
superfamily and biochemical functions of xylem-specific cellulose
synthase-like genes in Populus trichocarpa. Plant Physiology 142:
1233–1245.
Thompson NP. 1967. The time course of sieve tube and xylem cell
regeneration and their anatomical orientation in Coleus stems. American
Journal of Botany 54: 588–595.
Uggla C, Mellerowicz EJ, Sundberg B. 1998. Indole-3-acetic acid controls
cambial growth in Scots Pine by positional signaling. Plant Physiology
117: 113–121.
Uggla C, Moritz T, Sandberg G, Sundberg B. 1996. Auxin as a positional
signal in pattern formation in plants. Proceedings of the National
Academy of Sciences, USA 93: 9282–9286.
Uggla C, Sundberg B. 2002. Sampling of cambial region tissues for high
resolution analysis. In: Chaffey N, ed. Wood formation in trees: cell and
molecular biology techniques. New York, NY, USA: Taylor and Francis,
215–228.
Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Südhof TC, Wernig
M. 2010. Direct conversion of fibroblasts to functional neurons by
defined factors. Nature 463: 1035–1042.
Wang M, Qi X, Zhao S, Zhang S, Lu MZ. 2009. Dynamic changes in
transcripts during regeneration of the secondary vascular system in
Populus tomentosa Carr. revealed by cDNA microarrays. BMC Genomics
10: 215.
Wilkins O, Nahal H, Foong J, Provart NJ, Campbell MM. 2009.
Expansion and diversification of the Populus R2R3-MYB family of
transcription factors. Plant Physiology 149: 981–993.
Xu J, Hofhuis H, Heidstra R, Sauer M, Friml J, Scheres B. 2006. A
molecular framework for plant regeneration. Science 311: 385–388.
Yamaguchi M, Demura M. 2010. Transcriptional regulation of secondary
wall formation controlled by NAC domain proteins. Plant Biotechnology
27: 237–242.
Yamanaka S. 2009. Elite and stochastic models for induced pluripotent
stem cell generation. Nature 460: 49–52.
Yang X, Tuskan GA, Cheng MZ. 2006. Divergence of the Dof gene
families in poplar, Arabidopsis, and rice suggests multiple modes of gene
evolution after duplication. Plant Physiology 142: 820–830.
Zhang J, Elo A, Helariutta Y. 2011. Arabidopsis as a model for wood
formation. Current Opinion in Biotechnology 22: 293–299.
Zhao C, Craig JC, Petzold HE, Dickerman AW, Beers EP. 2005. The
xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl. Plant Physiology 138: 803–818.
New Phytologist (2011) 192: 869–884
www.newphytologist.com
Zhong R, Ye ZH. 2009. Transcriptional regulation of lignin biosynthesis.
Plant Signaling and Behavior 4: 1028–1034.
Zhou X, Ma H. 2008. Evolutionary history of histone demethylase
families: distinct evolutionary patterns suggest functional divergence.
BMC Evolutionary Biology 8: 294.
Supporting Information
Additional supporting information may be found in the
online version of this article.
Fig. S1 The regenerated sieve elements at stage II during
secondary vascular tissue regeneration.
Fig. S2 Enriched GO categories during SVT regeneration.
Table S1 Expression data of genes involved in epigenetic
regulation and cell cycle
Table S2 Expression data of genes involved in xylem
development
Table S3 Expression data of genes involved in phloem and
cambium specification
Table S4 Expression data of genes involved in phytohormones
Table S5 Primers for qRT-PCR
Methods S1 Additional information for experimental
procedure.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting information
supplied by the authors. Any queries (other than missing
material) should be directed to the New Phytologist Central
Office.
2011 The Authors
New Phytologist 2011 New Phytologist Trust