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A High-Resolution Transcript Profile across the
Wood-Forming Meristem of Poplar Identifies Potential
Regulators of Cambial Stem Cell Identity
W
Jarmo Schrader,a,1 Jeanette Nilsson,a Ewa Mellerowicz,a Anders Berglund,b Peter Nilsson,c
Magnus Hertzberg,a and Göran Sandberga,2
a Umeå
Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of
Agricultural Sciences, 90183 Umeå, Sweden
b Department of Chemistry, Umeå University, 90187 Umeå, Sweden
c Department of Biotechnology, Royal Institute of Technology, 10044 Stockholm, Sweden
Plant growth is the result of cell proliferation in meristems, which requires a careful balance between the formation of new
tissue and the maintenance of a set of undifferentiated stem cells. Recent studies have provided important information on
several genetic networks responsible for stem cell maintenance and regulation of cell differentiation in the apical meristems
of shoots and roots. Nothing, however, is known about the regulatory networks in secondary meristems like the vascular
cambium of trees. We have made use of the large size and highly regular layered organization of the cambial meristem to
create a high-resolution transcriptional map covering 220 mm of the cambial region of aspen (Populus tremula). Clusters of
differentially expressed genes revealed substantial differences in the transcriptomes of the six anatomically homogenous
cell layers in the meristem zone. Based on transcriptional and anatomical data, we present a model for the position of the
stem cells and the proliferating mother cells in the cambial zone. We also provide sets of marker genes for different stages
of xylem and phloem differentiation and identify potential regulators of cambial meristem activity. Interestingly, analysis of
known regulators of apical meristem development indicates substantial similarity in regulatory networks between primary
and secondary meristems.
INTRODUCTION
All postembryonic production of plant tissue is mediated by
specialized structures known as meristems. Meristems provide
a reservoir of undifferentiated stem cells as well as a population
of proliferating cells that will produce the various tissues of
a plant. Recent years have seen a tremendous increase in
knowledge about the cellular organization and molecular mechanisms underlying a functional meristem (Nakajima and Benfey,
2002; Traas and Vernoux, 2002; Carles and Fletcher, 2003). Most
of the research has focused on the two primary meristems at the
shoot and root apices because these can be easily studied in
annual species like Arabidopsis thaliana. A large part of the
biomass on this planet, however, is produced by secondary
meristems, namely the vascular cambium of forest trees. The
vascular cambium forms a continuous cylinder of meristematic
cells in the stem, producing secondary phloem tissue on the
1 Current
address: Zeutrum für Molekularbiologie der Pflanze, Entwicklungsgenetik, Universität Tübingen, Auf der Morgenstelle 3, 72076
Tübingen, Germany.
2 To whom correspondence should be addressed. E-mail goran.
[email protected]; fax 46-90-786-5901.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Göran Sandberg
([email protected]).
W
Online version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.104.024190.
outside and secondary xylem or wood on the inside (Figure 1A).
The term cambium sensu stricto refers to one or several layers of
initials, analogous to the stem cells proposed for other meristems
(Larson, 1994; Laux, 2003). Periclinal (tangential) divisions of
these initials then produce phloem or xylem mother cells, which
in turn can undergo several rounds of cell division before
differentiating (Larson, 1994; Figure 1B). Cambial initials and
mother cells are collectively referred to as the cambial zone. The
subdivision of the cambial zone into initials and mother cells
is mainly a conceptual one, however, because microscopic
and ultrastructural analysis provides only little information that
can distinguish both cell types from each other (Larson, 1994;
Lachaud et al., 1999). Molecular markers that are able to
differentiate between initials and mother cells would therefore
be of great value for future characterizations of the cambial zone.
It is generally believed that the identity of stem cells and the
developmental fate of their derivatives are determined primarily
by positional cues, rather than cell lineage (Laux, 2003). The
molecular nature of these positional cues has been the subject of
intense research, and many regulatory components and interactions have been identified (Doerner, 2003). In the Arabidopsis
shoot apical meristem (SAM), a stable stem cell pool is maintained by a feedback loop involving CLAVATA (CLV) and
WUSCHEL (WUS) genes, and differentiation of the stem cells is
prevented by SHOOTMERISTEMLESS (STM) and related homeobox genes (for a review, see Gross-Hardt and Laux, 2003). In
the root apical meristem, on the other hand, the SCARECROW
(SCR) gene has been connected to the maintenance of stem cells
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The Plant Cell
are extremely difficult to perform in trees because of their size,
relatively slow growth, and, above all, long generation times.
Reverse genetics offers a viable alternative, if a limited set of
target genes can be selected based on prior information.
Transcript profiling using microarrays has made it possible to
test the potential involvement of thousands of genes in a
biological process, thus providing valuable information for
the selection of target genes. This technology has already generated important data on gene expression profiles during the
transdifferentiation of mesophyll cells into xylem cells in
Zinnia elegans (Demura et al., 2002) and identified candidate
genes involved in xylem formation in hybrid aspen (Populus
tremula 3 tremuloides) (Hertzberg et al., 2001b).
Here, we report on a high-resolution transcript profile across
the cambial zone of aspen (Populus tremula) for more than
13,000 genes. The data reveal that the anatomically nearly
indistinguishable cells of the cambial zone differ markedly in
their transcriptomes, providing molecular support for a subdivision of the cambial zone into a stem cell region and proliferating
mother cells. A comparison between apical and cambial meristems indicates that several regulators of apical meristems show
highly specific expression in the cambial zone, suggesting that
primary and secondary meristems share common regulatory
mechanisms.
RESULTS
Sampling Strategy
Figure 1. Sampling Scheme.
(A) Schematic overview of the cambial region. Horizontal bars and letters
indicate the positions of samples taken by Hertzberg et al. (2001b), and
the box marks the region sampled for this study.
(B) Cross section through the cambial region, with white and gray bars
marking the position of samples for the two different cutting series CSA
and CSB. In most graphs the sections are identified by labels X1 to X11
corresponding to the bar at the bottom of this figure. The figure was
drawn based on an actual cross section from series CSA.
(C) Samples for meristem comparison experiment. Micrographs of
poplar shoot and root apices with boxes showing the approximate
location of tissue samples. Bars ¼ 100 mm.
surrounding the quiescent center (Di Laurenzio et al., 1996;
Sabatini et al., 2003). Given our increasing understanding of
meristem regulation in primary meristems, can the models that
have been developed there be used as basis for the identification
of regulatory mechanisms in secondary meristems like the vascular cambium? Recent findings indicate that a CLV-mediated
patterning system might be active in both the root and shoot
meristems (Casamitjana-Martinez et al., 2003; Hobe et al., 2003),
suggesting that both primary meristems are regulated by similar
mechanisms. It is thus conceivable that even the vascular
cambium shares common regulatory elements with the apical
meristems. If this was the case, known meristem regulators or
their homologs should show similar, specific expression patterns
in the cambial zone as they do in the apical meristems.
One of the most powerful tools for meristem biology has been
the analysis of mutants. Unfortunately, classical genetic screens
The vascular cambium has been thoroughly described in numerous anatomical studies, but information about the molecular
identity of different cell types and the regulatory systems acting
on them is scarce. As a first step toward a more detailed picture
of cambial meristem regulation, we used cDNA microarrays to
create a transcriptional map of the cambial zone and screen for
potential regulators of cell identity and differentiation in the
cambial meristem of P. tremula. A series of 20-mm-thin tangential
sections was taken across the cambial region, starting with the
youngest mature phloem and ending in the early stages of xylem
cell expansion. One section corresponds to approximately three
cell layers of the cambial zone, thus providing near cell-specific
resolution for the obtained profiles. Cross sections taken after
every third tangential section were used to locate the position of
each section in the cambial region as shown in Figure 1B. The
samples were taken on July 7th in the middle of the growing
season, when the cambial zone is wide and the majority of newly
produced cells are found on the xylem side. To increase the
general validity of the experiment, two replicate series were
sampled from two individual trees denoted CSA and CSB. Section
6 in CSA and sections 3 and 5 in CSB failed to produce sufficient
amounts of cDNA and were excluded from the analysis.
mRNA was isolated from each section, reverse transcribed,
and PCR amplified using a method that has previously been
demonstrated to yield highly reproducible results in microarray
experiments (Hertzberg et al., 2001a) . The POP1 array used in
these experiments contained 13,824 spots representing 13,526
aspen EST clones as well as 298 controls (Bhalerao, 2003).
Amplified material from each section was hybridized against
Cambial Meristem Transcript Profile
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a pool of all sections from the same series, and all expression
values reported are log2 ratios of sample versus pool.
Both CSA and CSB only covered the immediate vicinity of the
cambial zone. To obtain a picture of a gene’s expression profile
over the entire wood-forming region, we used data from
Hertzberg et al. (2001b). In this experiment, later referred to as
CSG, samples were taken from the cambial zone (A and B),
expansion zone (C), and the zone of secondary wall formation (D)
and late cell maturation (E) as depicted in Figure 1A. Because the
original data of Hertzberg and coworkers only provided information on 2995 clones, their samples were rehybridized on the
POP1 array to obtain data for the full set of 13,526 clones.
Comparison of Biological Replicates
Replication is an important aspect of microarray experiments,
and replicates were therefore included both at the level of
technical replicates (at least three per section) and, most
importantly, biological replicates in form of the two series CSA
and CSB taken from individual trees. The experimental system
used presents a challenge in assessing whether a gene has the
same expression pattern in the biological replicates. There are
considerable differences in the thickness of the cambial region
between individual trees, and the exact location of a series of
sections can only be determined after sampling. It is thus nearly
impossible to obtain two series that overlap perfectly so that
corresponding sections contain exactly the same cell type. The
result of this variation in sampling and biology is that for a gene
with the same tissue-specific expression pattern one will obtain
graphs with similar shapes but which might be scaled or shifted
in both x (tissue position) and y (expression level) directions.
In this experiment, comparison of cross sections showed that
the CSA and CSB series are shifted relative to each other by
;2.5 sections.
We used principal component analysis (PCA) as an unbiased
method of comparing the results of CSA and CSB. A score plot of
the first two principal components in Figure 2 shows that the
samples were clearly ordered along the horizontal axis corresponding to their sampling position in the tree stem (Figure 1B).
This type of plot will cluster samples with similar expression
patterns for all the genes closely together. Two things can be
observed. First, the individual technical replicates clustered
closely together and were clearly separated from those of the
next sample, demonstrating that the results were reproducible
and the resolution was sufficient to distinguish the individual
sections from each other. More importantly, the analysis shows
that the individual samples of CSA and CSB both followed a very
similar curve, essentially demonstrating that both series describe
the same process.
Analysis of variance (ANOVA) produced a list of 2280 genes
that were differentially expressed in either CSA or CSB. We further
identified a set of stably expressed genes, these being the genes
with 5% least differential expression across both series as
measured by the SD of the log2 ratio across the samples. The
set of differentially expressed genes was submitted to cluster
analysis using self-organizing maps giving a collection of 16
expression sets, including the 5% least SD set (Figure 3A).
Figure 2. Principal Component Analysis.
Raw expression data from all samples of CSA and CSB were subjected to
PCA, and scores for the first two principal components for each
individual hybridization are plotted. These components have no direct
biological meaning but rather summarize a part of the total variation
in gene expression found in the different samples. Replicate hybridizations coming from the same section are grouped together by ellipses.
Triangles represent CSA and circles CSB.
The PCA analysis suggested good reproducibility between
CSA and CSB. This was further explored by calculating the
averages for all genes in the 16 expression sets and plotting them
in the same graph. Based on anatomical data, the CSA series was
shifted by 2.5 sections toward the xylem (Figure 3B). The overlap
of the averages for CSA and CSB was good for most of the sets;
marked differences were only observed in sets 10, 12, and 15.
Apart from variation arising from the microarray analysis itself,
these differences could have arisen from several factors. The two
trees likely differed in some aspect of their physiology; nutrient
and stress status as well as growth rate were probably the most
variable factors. Any genes involved in the response to these
factors were likely to also differ in their expression patterns. A
second explanation is related to the way the samples were taken.
Although very regular in composition, the vascular cambium is
not absolutely straight. The individual sections therefore represented highly enriched but not pure samples of cells from
a certain developmental stage. In particular, genes with specific
expression in mature phloem could show differences between
CSA and CSB because CSA did not extend into the mature
phloem. For reasons of simplicity, most graphs show results from
both CSA and CSB plotted against a common axis with sections
labeled X1 to X11, whereby B1 corresponds to X1 and A1
corresponds to X3.5 (Figure 1B). The region of anatomically
homogenous cambial zone cells from X5 to X7 is marked by
a shaded area in most graphs.
Gene Clusters Identify a Functional Subdivision
of the Cambial Zone
The cambial zone of trees is often described as consisting of
the cambial initials flanked by phloem and xylem mother cells.
Figure 3. Cluster Analysis.
Cambial Meristem Transcript Profile
Although all three cell types are able to undergo cell divisions,
the mother cells will eventually loose their ability to divide
and undergo terminal differentiation, whereas the initials retain
their undifferentiated state. It is, however, almost impossible to
distinguish the initials from mother cells with any certainty
because the cambial zone shows only minor variations in
anatomy or ultrastructure (Larson, 1994; Figure 1B). A functional
definition of the cambial initials states that they are the only cells
in each radial file that are able to produce both phloem and xylem
derivatives and to initiate new cell files by anticlinal (radial)
divisions. We made use of this functional difference in the
direction of cell division to identify the approximate location of
the cambial initials in our samples. Stem cross sections were
examined for examples where a new cell file had been initiated by
anticlinal divisions. Such divisions always appeared on the
phloem side of the cambial zone, and they were in general
separated by only one or two cells from the differentiating
phloem, whereas there were on average five cells on the xylem
side (Figure 4). It thus appears that during the period of maximum
growth in early summer, the cambial zone consists mainly of
xylem mother cells and the cambial initials are located on the
outermost side of the cambial zone adjacent to only one or two
phloem mother cells.
For a more detailed distinction of different cell types in the
cambial zone, it is important to obtain molecular markers. The
microarray data set was therefore searched for candidate marker
genes showing a peak of expression anywhere in the sampling
region. Candidate genes were first manually selected from
various different cluster sets and then subjected to statistical
filtering to remove genes, which did not show significant (P <
0.05) difference between maximum and the minima on both
sides. The resulting peak set contains 159 clones representing
117 different genes. Similarly, 29 clones with a minimum in the
series were identified; these represent 23 different genes.
Clustering methods were used to divide the peak genes into
three different groups, which showed approximate maxima
in sections X4, X5.5, and X6.5, respectively (Figure 5A; see
Supplemental Table 1 online). The genes in each peak set were
divided into functional classes, and a comparison of these
classes reveals fundamental differences between the three peak
sets, essentially identifying distinct zones within the cambial
region (Figure 5B).
The cells expressing genes in Peakset1 were located on the
phloem side of the cambium encompassing the phloem mother
cells and the youngest differentiating phloem. This layer was
distinct from mature phloem cells as suggested by several genes
associated with mature phloem functions like the sucrose transporter SUC2 or a phloem lectin that showed expression patterns
distinct from those in Peakset1 (Figure 6; Stadler et al., 1995;
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Figure 4. Anticlinal Divisions.
Observed number and location of anticlinal divisions within the cambial
zone. The location of anticlinal divisions within the cambium was determined by counting the number of cells starting from the first cambial
cell on the phloem side of the section.
Dinant et al., 2003). Peakset1 contained a large number of
metabolic genes with a subgroup of eight genes involved in
flavonoid biosynthesis. The second largest group was formed by
stress-related genes like thaumatin/osmotin (Kim et al., 2002),
dehydrin (Pelah et al., 1997a), and a disease resistance protein.
The presence of multiple stress-related genes in Peakset1
suggests that cells in this layer might play a role in protecting
the cambium under stress conditions. Interestingly, a gene with
homology to sucrose synthase was also found in this set. A
possible role for sucrose synthase could be the reconversion of
transported sugars from hexoses to sucrose for further transport
into the cambial zone. There could also be a connection with the
group of stress-related genes because some sucrose synthase
genes have been reported to be stress inducible in Arabidopsis
and aspen (Pelah et al., 1997b; Dejardin et al., 1999). For one
clone in Peakset1 (PU04112), an open reading frame of 798
nucleotides could be identified that nevertheless gave no significant hit against any sequence in the public databases. This
clone might therefore represent a gene unique to aspen or other
trees, with a potential specific role in phloem development.
Peakset2 covered the center of the cambial zone. It was the
smallest set and contained only six known genes, two involved in
metabolism and four with roles in signal transduction and transcriptional regulation. The xylem side of the cambial zone,
defined by Peakset3, was easily identified as a zone of high cell
division activity. Genes related to cell division and expansion like
histones and a xyloglucan endotransglycosylase formed the
major group. There were several other genes related to protein
turnover like E2 ubiquitin conjugating enzyme and a ubiquitin
Figure 3. (continued).
A total of 2280 clones showing statistically significant differential expression were divided into 15 clusters using self-organizing maps. The 16th cluster
contains the 5% clones with least differential expression.
(A) Expression profiles in log2 scale of clusters with separate graphs for CSA and CSB.
(B) Cluster averages plotted into the same graph, showing overlap of expression between CSA (dotted lines) and CSB (solid lines). Shaded areas mark
the approximate location of the cambial zone.
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extension protein as well as two ribosomal proteins, suggesting
elevated rates of protein turnover, as might be expected for
actively dividing cells. Finally, Peakset4 appeared to be the
inverse of Peakset3, containing genes with a minimum in the cell
proliferation zone. The largest functional groups were genes
involved in secondary metabolism as well as transport and
secretion.
Combined evidence from cell proliferation markers indicates
the highest rate of cell proliferation in the presumptive xylem
mother cells. Similarly, previous studies have found that in the
middle of the growing season, when samples for this experiment
were taken, aspen and many other species produce about 10
times more xylem than phloem tissue (Larson, 1994). Our
analysis of anticlinal divisions shows that the cambial stem cells
were located on the phloem side of the cambial zone during this
period (Figure 4). Based on this experiment and the expression
patterns of genes known to mark phloem and xylem initiation
(Figure 6), we can place the stemcell(s) on the phloem side of the
meristem. We can also conclude that the cambial stem cells
divide infrequently, whereas our transcript data indicate that the
majority of divisions took place in the mother cells. In this
respect, the cambium shows similarity to apical meristems
where the stem cells are found in regions of low cell division
activity.
Regulation of Cell Division
Figure 5. Peak Genes.
Clones with a local maximum or minimum in the region covered by the
experiment were manually selected from the data set.
(A) Expression profiles of 188 clones in log2 scale grouped into four peak
sets. Shaded areas mark the approximate location of the cambial zone.
(B) Functional classification of genes in the peak sets. The number of
clones in each functional category is indicated.
Cell division is one of the key processes taking place in the
cambial zone. Although several global surveys of gene expression during the cell cycle in plants have provided lists of genes
with differential expression during the cell cycle (Breyne et al.,
2002; Menges et al., 2002), information about the spatial distribution of cell cycle–related genes in meristems has only been
available for individual genes (Hemerly et al., 1993; Ferreira et al.,
1994; Rohde et al., 1997; Mironov et al., 1999; Burssens et al.,
2000; Gaudin et al., 2000; Barroco et al., 2003). We identified
members of the core cell cycle machinery in aspen based on a list
of Arabidopsis genes published by Vandepoele et al. (2002). At
least 21 different genes from this list were present on the POP1
array. To obtain a more complete picture of the distribution of cell
cycle gene expression across the entire cambial region, we used
data from the CSG global wood formation survey of Hertzberg
et al. (2001b) in addition to the cambial zone data (see Figure 1 for
the location of samples). Approximately two-thirds of the cell
cycle–related genes showed highly similar profiles across the
cambial zone with a steep increase in expression toward the
xylem side that levels off at section X6 (Figure 6). These genes
include Cyclin A1 and Cyclin D3, cyclin-dependent kinase
CDKB2, CKS1, and a DP-E2F-like (DEL) gene. The same set of
genes also had a maximum of expression in sections A and B of
the Hertzberg et al. wood formation survey, which corresponds to
the region covered by the high-resolution data (insets in Figure 6).
Whereas the majority of cell cycle associated genes showed
similar expression profiles, there were considerable differences
between individual members of the different gene families. This is
exemplified in the comparison of Cylin A1 (PttCYCA1) and Cyclin
A2 (PttCYCA2). Whereas PttCYCA1 followed the general trend
of other cell cycle genes, PttCYCA2 showed only a very small
Cambial Meristem Transcript Profile
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Figure 6. Expression of Selected Cell Cycle–Related Genes.
Dashed lines, expression in CSA; solid lines, expression in CSB. Please refer to Figure 1B for location of sections X1 to X10. The shaded areas mark the
approximate location of the cambial zone. Insets show expression across the entire wood-forming region with samples A to E referring to sections as
indicated in Figure 1A. All expression data indicate relative expression in log2 scale with error bars showing SD. For the insets, distance between the tick
marks on the y axis is two log2 units. Data are taken from the following clones: cyclins CYCA1 (PU05347), CYCA2 (PU13262), CYCB2 (PU01835), CYCD3
(PU13230); cyclin dependent kinases CDKA (PU06293) and CDKB2 (PU00348); other regulators, DEL (PU04263), DPB (PU08368), Rb (PU01146);
histones H2A;1 (PU00913), H2A;2 (PU11928), H4 (PU02867); cell expansion marker aquaporin (PU06836); xyloglucan endotransglycosylase PttXET16C
(PU07373); expansin PttExp1 (PU02594); phloem markers, phloem lectin (PU04091); sucrose carrier SUC2 (PU03539).
increase toward the xylem. More interestingly, in the global
profile, PttCYCA2 expression remained high until well into
sample D, the zone of secondary wall formation (inset in Figure
6). It has been suggested that AtCYCA2 expression in Arabidopsis is associated with competence to divide (Burssens et al.,
2000). This would mean that xylem cells retain a certain competence to divide until very late in their development. PttCYCA1 on
the other hand, with its distinct peak in the cell division zone, is
most likely associated with the actual cell division events.
Similar to the A-type cyclins, PttCDKA1 and PttCDKB2 differed in their profiles. PttCDKA1 showed no differential expression across the wood-forming zone, which was consistent with
data indicating that Arabidopsis CDKA activity is regulated on
a posttranscriptional level (Mironov et al., 1999; Menges and
Murray, 2002). B-type CDKs, on the other hand, showed cell
cycle–dependent expression (Menges and Murray, 2002), and
correspondingly we found a strong peak of PttCDKB2 signal in
the proliferation zone.
Two genes for CDKs of the C- and D-type likewise showed no
differential expression (data not shown). These genes might have
roles unrelated to cell division as has recently been suggested for
some Arabidopsis C-type CDKs (Barroco et al., 2003). One
member of the DEL family of genes (Vandepoele et al., 2002) had
a distinct peak similar to the other cell cycle–related genes.
Members of the DEL family lack the dimerization domain found in
DP and E2F proteins, instead they possess a duplicate DNA
binding domain (Vandepoele et al., 2002). The function of these
genes is unknown at present, but the expression pattern of the
aspen DEL gene suggests a role during cell division.
Another well-known marker for cell division activity is the
histone H4 because its expression is associated with DNA
replication (Chaubet et al., 1996). Histone H4 expression was
maximal in X6-X8 and declined toward both xylem and phloem,
supporting the conclusions drawn from the cell cycle genes that
cell proliferation is highest around sample X7. Interestingly, two
genes coding for histone H2A showed contrasting expression
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profiles. Whereas H2A;1 was expressed constitutively across the
entire wood formation zone, H2A;2 showed a peak similar to that
of the other cell cycle–related genes. This could suggest a role for
H2A;2 in de novo synthesis of histones during DNA replication,
whereas H2A;1 would be responsible for maintenance and
replacement synthesis in nondividing cells.
Regulation of Cell Expansion
After cell division, cells in the cambial zone will expand in axial
and radial directions (Larson, 1994). The data set contains
several genes known to be involved in the cell expansion processes (for a review, see Darley et al., 2001; Mellerowicz et al.,
2001). Several genes encoding xyloglucan endotransglycosylase, pectin methylesterase, and expansin showed very similar
expression profiles, with an increase of expression at the
beginning of the cambial zone followed by constant levels across
the zones of cell proliferation and early expansion (Figure 6; data
not shown). Expanding cells increase their volumes mainly by
taking up water. Accordingly, there were several clones representing aquaporins with expansion zone–specific expression
patterns similar to those of xyloglucan endotransglycosylase or
pectin methylesterase. These expression profiles indicate that
cell expansion is initiated in parallel with cell division, which is in
agreement with anatomical observations of tip growth and radial
expansion, which are initiated in the dividing cells of the cambial
zone followed by massive radial expansion in both directions in
the expansion zone. For all the expansion-related genes mentioned above, there were also close homologs with constant
expression across the entire experiment (data not shown). Such
differences in expression pattern therefore provide initial evidence for distinguishing family members involved in specific
expansion processes from others with more general roles in wall
modification.
Known Regulators of Cell Differentiation and
Meristem Identity
Our knowledge of the mechanisms conferring identity to the
different zones of both the shoot and the root apical meristems
has increased notably in recent years. Analysis of mutants with
defects in meristems has identified feedback loops and intercellular transport of proteins as important regulatory mechanisms and provided several paradigms for the regulation of
meristem identity and the differentiation of tissue types (reviewed
in Nakajima and Benfey, 2002). It can be speculated that similar
mechanisms govern the regulation of cell fate in the vascular
cambium. We therefore scrutinized the expression patterns of
several homologs of known regulators of meristem identity and
cell differentiation in the cambial meristem (Figure 7). The poplar
homologs of the Arabidopsis meristem regulators were identified
by building phylogenetic trees of the closely related Arabidopsis
and available poplar gene sequences and selecting the poplar
gene that clusters closest to a given Arabidopsis gene (see
Supplemental Figure 1 online). If necessary, information from an
ongoing poplar genome sequencing project (http://genome.jgipsf.org/poplar0/poplar0.info.html) was used to obtain additional
sequence information for phylogenetic tree construction.
The available data on meristem regulators suggest that the
shoot and root apical meristems might share at least some
regulatory mechanisms. To explore the relationship of the
cambial zone to other meristems, we compared global gene
expression between apical meristems and the vascular cambium
using the POP1 microarray. For the SAM, sample tissue was
pooled from 30 trees consisting of 100 mm 3 50 mm from the
center of the apical meristem (Figure 1C), whereas the root
sample covered the distal 0.5 mm of an actively growing root. A
30-mm section was taken from the cambial zone, and tissue from
a mature leaf was added as a nonproliferating control. Such
a comparison of tissues with different organization is, however,
not straightforward because apparent differences in gene expression might be dependent on differences in cell-type composition. Results from this comparison therefore have to be
interpreted with caution but can nevertheless give a good
indication whether a gene is likely to play a role in a certain
meristem.
In the Arabidopsis SAM, the CLV and WUS genes form
a feedback loop that ensures the maintenance of a stable
population of slowly dividing cells in the central zone (Brand
et al., 2000; Schoof et al., 2000). The POP1 array contains
a putative ortholog of CLV1 named PttCLV1 and two highly
similar receptor-like kinases, PttRLK2 and PttRLK3. PttCLV1 and
PttRLK3 were expressed in distinct domains on both sides of
section X5 (Figure 7), whereas PttRLK2 showed a slight peak of
expression at section X7 (data not shown). Potential ligands for
the PttCLV1 receptors are members of the CLAVATA3/ESRrelated (CLE) family (Fletcher et al., 1999; Brand et al., 2000;
Sharma et al., 2003). Putative orthologs of AtCLV3 and AtWUS
were identified in the PopulusDB EST database, but they were
not represented on the array. Their expression was therefore
measured using radiolabeled probes hybridized to a dot-blot
filter of cDNA from CSA, CSB, and the meristem comparison
experiment. Both PttCLV3 and PttWUS gave a clear signal for the
apical meristem sample but not for any of the other tissues
(Figure 8). Based on their high sequence similarity and specific
expression in the apical meristem, PttCLV3 and PttWUS are very
likely to be functional orthologs of their Arabidopsis counterparts.
These data further suggest that neither PttCLV3 nor PttWUS are
likely to play a regulatory role in the cambial meristem. Seven
other clones encoding CLE proteins were identified on the POP1
array. Two of them, PttCLE;1 and PttCLE;3, showed differential
expression with a fourfold decrease between X4 and X6 (Figure
7). On the other hand, PttHB2 and PttHB3, two members of the
WUSCHEL-related homeobox family of WUS-like genes,
showed a steep increase in expression toward the xylem side
of the cambial zone. Maintenance of the SAM in Arabidopsis
further requires the STM gene that is specifically expressed in the
central zone (Mayer et al., 1998). One role of STM is to suppress
expression of the ASYMMETRIC LEAVES1 (AS1) gene, which in
turn allows transcription of KNOX genes, such as KNAT1 and
KNAT6 (Byrne et al., 2000). The putative poplar ortholog of STM,
PttSTM, was not represented on the POP1 array, but filter
hybridization showed the strongest signal in the cambial sample
and a weaker expression in the apex (Figure 8). No signal was
detected for the closest poplar homolog of AS1 in the cambial
zone (data not shown). KNOX genes such as PttKNOX1,
Cambial Meristem Transcript Profile
9 of 15
Figure 7. Expression of Meristem Identity and Other Marker Genes.
Dashed lines, expression in CSA; solid lines, expression in CSB. Please refer to Figure 1B for location of sections X1 to X10. The shaded areas mark the
approximate location of the cambial zone. All expression data are presented in log2 scale with error bars indicating SD. Data are taken from the following
clones: PttCLV1, PU04960; PttRLK3, PU07633; PttCLE;1, PU04444; PttCLE;3, PU04153; PttHB2, PU05428; PttHB3, PU01627; PttKNOX1, PU00342;
PttKNOX2, PU01263; PttKNOX6, PU04548; PttANT;1, PU04170; PttPNH, PU02954; PttKAN1, PU01005; PttHB9, PU02134; PttHB15, PU07361;
PttHB8, PU00973; PttSHR1, PU04350; PttSHR2, PU05677; PttSCL6, PU08408.
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Figure 8. Expression of PttWUS, PttCLV3, and PttSTM in Different
Meristems.
mRNA isolated from apical meristems (A), the cambial zone (C), root tips
(R), and mature leaves (L) was PCR amplified, spotted onto a membrane,
and hybridized with radiolabeled probes.
PttKNOX2, and PttKNOX6, on the other hand, showed high
expression in both the shoot apex and cambial samples and very
low signal from roots and leaves (Figure 7). Expression of these
genes was slightly higher on the phloem side of the cambial zone.
These data are compatible with a model analogous to the apical
meristem where PttSTM expression in the cambial zone would
allow transcription of KNOX genes via repression of the poplar
AS1 homolog. The comparison of expression levels in the
different meristems further supports a role for KNOX genes in
shoot apical and cambial meristems but not in the root. No root
phenotypes have been reported for KNOX mutants either.
The Arabidopsis AINTEGUMENTA (ANT) gene has been connected to the control of cell proliferation. It is expressed in young
organ primordia and also in procambial cells (Elliott et al., 1996).
Loss-of-function ant mutants have smaller organs because of
changes in cell number, whereas ANT overexpression leads to
enlarged organs and the formation of calli in mature organs
(Mizukami and Fischer, 2000). Similarly, the PINHEAD/ZWILLE
(PNH) gene is believed to play a role in regulating determinate
versus indeterminate growth based on insufficient SAM activity
in pnh mutants and the formation of ectopic meristems in plants
with ectopic PNH expression (Moussian et al., 1998; Newman
et al., 2002). In aspen, close homologs of ANT and PNH showed
a distinct peak of expression around X6 (Figure 7) in the zone
defined by Peakset2. Both aspen genes were expressed before
cells enter the zone of maximum cell proliferation; it is therefore
possible that these genes are involved in regulating the entry of
cambial zone cells into the proliferation zone.
As cells leave the proliferation zone, they need to acquire
characteristics of xylem or phloem cells. In the cambial zone, this
acquisition of cell fate is intimately connected with the radial
organization of the stem where phloem is always formed toward
the outside and xylem toward the inside of the stem. This
organization corresponds to abaxial/adaxial patterning in leaves
where the YABBY, KANADI, and HD-ZIP III gene families have
been implicated in the establishment of abaxial and adaxial
domains (reviewed in Bowman et al., 2002). Based on gain- and
loss-of-function mutants as well as expression studies, members of the YABBY and KANADI families are believed to promote
abaxial fate in leaves in a partly redundant manner. Three
different poplar YABBY genes were represented on the POP1
array and all showed the highest signal in mature leaf and lowest
in the cambial zone, and no differential expression could be
detected across the cambial region (data not shown). On the
other hand, a close homolog of AtKAN1, PttKAN1, showed
higher signal in the cambial sample compared with leaves and an
increase in expression toward the phloem side of the cambial
zone similar to the abaxial expression of AtKAN1 in leaves (Figure
7). Similarly, the cambial expression patterns of poplar HD-ZIP III
genes appear to match their presumed role of promoting adaxial
fate in leaves. Poplar homologs of PHAVOLUTA/PHABULOSA
(PttHB9) and ATHB-15 (PttHB15) exhibited a steep increase in
expression on the xylem side of the cambial zone (Figure 7).
These data suggest that both KANADI and HD-ZIP III genes
could be involved in radial patterning of the wood-forming
tissues, whereas the YABBY family might be more specific to
leaves and related organs like flowers. One of the earliest known
markers of vascular identity is the Arabidopsis HD-ZIP III gene
ATHB-8. It is expressed specifically in procambial strands and its
overexpression stimulates xylem formation (Baima et al., 1995,
2000, 2001). The closest aspen homolog of ATHB-8, PttHB8,
was expressed specifically on the xylem side of the cambial zone
and in the adjacent expanding xylem. These data are in agreement with an orthologous function of PttHB8 in promoting xylem
formation in the cambial zone and make PttHB8 a good marker
for early commitment to xylem production.
In the root apical meristem the SHORT-ROOT (SHR) and SCR
transcription factors are involved in specifying tissue identity (Di
Laurenzio et al., 1996; Helariutta et al., 2000). In the cambial
zone, two close homologs of AtSHR, PttSHR1 and PttSHR2,
appear to be expressed and showed an increased expression
toward the phloem (Figure 7). The closest poplar homolog of
AtSCR was not represented on the array, and among nine other
SCR-like clones, only one showed clear differential expression; it
increased steeply in the mature phloem starting at section X4, so
it is probably not involved in early differentiation events (data not
shown).
Conclusions
Molecular Markers Identify Zones within the
Cambial Region
Although anatomical investigations have provided a wealth of
information on the structure of the wood-forming tissues (Steves
and Sussex, 1989; Larson, 1994), details on the very early stages
of differentiation in the cambial zone have not been resolved. It
has been difficult to substantiate the conceptual division of the
cambial zone into xylem and phloem mother cells and a layer of
initials or stem cells. Although the existence of a cambial initial is
nevertheless generally acknowledged (Larson, 1994), it has been
a matter of debate whether the function of the initial is restricted
to one specific cell per radial file or whether there is a population
of stem cells, any of which can act as an initial. The main obstacle
in answering this question has been the lack of reliable markers
for cambial initial identity. On a transcriptional level, however, the
cambial zone is far from uniform with more than 100 genes
showing greater than fourfold change within 60 mm (X5-X7) of the
cambial zone. Based on the data in this study, we can provide
molecular support for a subdivision of the cambial zone and
identify several distinct layers in this meristem (Figure 9): first,
Cambial Meristem Transcript Profile
11 of 15
techniques or promoter studies. We have initiated this work
for a set of genes covering the entire region described in
this study.
Models for Stem Cell Regulation in the
Cambial Meristem
Figure 9. Summary of the Distribution of Different Cell Types in the
Cambium and the Average Expression Profiles of Different Sets of Genes
Peaking in This Meristem.
a layer of differentiating phloem and phloem mother cells
expressing genes in Peakset1, namely flavonoid biosynthetic
genes, stress-related genes and the putative poplar unique clone
PU04412. Second, a distinct layer on the phloem side of the
cambial zone marked by Peakset 2. Based on the identification
of anticlinal divisions, the cambial stem cells are located in this
layer. This putative stem cell layer is clearly distinct from the
proliferation layer identified by Peakset3, which is located on the
xylem side of the cambial zone and extends into the zone of radial
expansion.
The combination of anatomical studies with molecular markers
has proven a powerful technique for identifying different zones
and regulatory interactions in both the root and SAM (van den
Berg et al., 1995; Sabatini et al., 1999; Brand et al., 2000;
Nakajima and Benfey, 2002). A particularly successful approach
has been to experimentally disturb normal development using
mutants or chemicals and follow the resulting changes in tissue
patterns and cell fate using molecular markers. The application of
this technique to questions of cambial meristem identity has
been hampered by the limited number of marker genes for cell
identity in early stages of cambial differentiation. Although there
are markers for phloem identity (bark storage protein, phloem
lectin, and SUC2; Figure 6) and xylem fate (PttHB8, Figure 7) and
for the entire meristem region (Johansson et al., 2003), they do
not resolve the early stages of differentiation. Furthermore, most
of these genes have been described in different species, some of
them herbaceous, so their expression patterns in a tree system
like aspen need to be verified. In this study, we were able to
confirm the expected expression patterns for several marker
genes and identified several novel markers for early stages of
cambial cell differentiation. They complement and expand the
existing range of anatomical markers for cambial cell differentiation and will make it possible to track changes in cell fate after
experimental manipulations even in the absence of anatomical or
ultrastructural indicators. Because this study does not provide
single cell resolution, the expression patterns of selected markers will have to be verified at higher resolution using in situ
A major aim of this study was to investigate whether available
knowledge on regulatory systems in primary plant meristems can
be used to understand processes in the vascular cambium. The
data presented here, particularly the specific cambial expression
profiles of homologs of known apical meristem regulators like
PttCLV1, PttANT, and PttKNOX, suggest that similar regulatory
mechanisms are active in the cambium and apical meristems.
Such a conservation of basic regulatory concepts between
different meristems is further supported by recent findings indicating that a CLV-based system might also be active in the root
meristem (Casamitjana-Martinez et al., 2003). Many details,
however, will be different. Essential SAM regulators like CLV3
and WUS, for example, are not expressed in the root (Mayer et al.,
1998; Fletcher et al., 1999), and their putative orthologs PttCLV3
and PttWUS are likewise not detectable in the cambial meristem.
Because recent data indicate that WUS might have the ability to
confer shoot apex identity to cells (Gallois et al., 2004), lack of
PttWUS in the cambium appears reasonable. On the other hand,
cambial expression of PttCLV1 and the overlapping expression
of PttRLK3 and the WUS homolog PttHB3 on the xylem side
of the cambial zone suggests the existence of a cambial stem
cell regulating feedback loop similar to the one found in the
apical meristem. Particularly the opposing expression profiles
of PttCLV1 and PttRLK3 are interesting because they have the
potential to be part of two separate regulatory loops acting on
either side of the cambial initials. Throughout a growing season,
the cambium in trees can alter the relative rates of xylem and
phloem production (Larson, 1994); PttCLV1 and PttRLK3 could
therefore be part of a system that allows independent regulation
of xylem and phloem proliferation.
In addition to similarities on the level of stem cell maintenance,
there appears to be a certain degree of conservation in the radial
patterning system between the shoot apex and the cambial
meristem. The expression profiles of genes like PttKAN1 and
PttHB9 in the stem correspond well to the expression patterns
reported for their Arabidopsis orthologs in leaves. Because the
vasculature in leaves is continuous with that in the stem, they
might very well be the result of a common patterning mechanism.
These findings are also in agreement with data from Arabidopsis
where mutations in KANADI or HD-ZIP III genes alter the normal
organization of vascular bundles (Kerstetter et al., 2001).
The tentative identification of a cambial stem cell layer and the
possible involvement of PttCLV1, PttRLK3, PttHB3, PttANT, and
other genes in the regulation of cambial cell proliferation need
to be experimentally verified. We have therefore initiated the
creation of RNA interference lines and cell-specific studies of
expression patterns of the genes mentioned above. Further
insight into the organization of the cambial meristem could be
obtained by comparing expression profiles during xylem formation, as in this study with profiles taken early in the growing
season when mainly phloem is made. We would expect a shift of
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The Plant Cell
the proliferation-related expression clusters from the xylem side
to the phloem side of the stem cells. This would allow a separation of genes involved in proliferation in general from those
related to xylem production in particular and a further confirmation of the location of slowly dividing stem cells. Corresponding
experiments are currently in progress and will hopefully further
advance our understanding of cambial meristem organization.
METHODS
The POP1 Array
The POP1 array is based on a unigene set of 13,526 clones assembled
from a hybrid aspen EST collection containing 33,000 ESTs. The POP1
array is a spotted cDNA microarray containing 13,824 spots arrayed in
4 3 6 blocks of 576 spots. Various controls like empty spots and
heterologous DNA were included, amounting to 298 spots. The CSA
and CSB experiments were hybridized on the first generation of the POP1
chip with a feature size of 100 mm. The rehybridization of the CSG global
wood formation survey as well as the meristem comparison experiment
were performed on second generation POP1 arrays with a feature size of
50 mm and duplicate spots for each clone. A detailed description of the
array is given by Andersson et al. (2004). All spot annotations in this report
are based on an extended set of 100,000 ESTs that provided more
sequence information and, thus, more reliable annotation. The annotations of all genes discussed individually in the text were verified by manual
BLAST searches. Spots on the array are referred to by their unigene clone
ID of the form PU12345. Information on the sequences associated with
the clones can be found in PopulusDB (www.populus.db.umu.se).
Amplification and Hybridization
Extraction of mRNA, amplification of the transcript population, and
subsequent hybridization were performed as described previously
(Hertzberg et al., 2001a). Briefly, mRNA was isolated from crude extracts
using Dynabeads (Dynal, Norway, Sweden). Reverse transcription using
NotI-oligo(dT) primer (59-biotin-GAGGTCCCAACCGCGGCCGC(T)15-39)
was followed by second-strand cDNA synthesis and fragmentation of the
cDNA using a sonicator. Biotinylated 39 cDNA tags were isolated using
paramagnetic streptavidin coated beads (Dynal). An adaptor was ligated
to the fragments, and PCR with primers specific to the adaptor and the
NotI oligonucleotide were used to amplify the cDNA population. Labeling
was performed using PCR with the adaptor-specific primer only in the
presence of Cy3- or Cy5-dUTP. After 16 h hybridization under cover slips,
slides were washed in decreasing concentrations of SSC (0.13 SSC
final), dried, and scanned in a ScanArray4000 scanner (Perkin-Elmer Life
Sciences, Boston, MA) at a resolution of 10 mm (CSA and CSB) or 5 mm
(CSG, meristem comparison).
For CSA, CSB, and CSG, Cy3-labeled cDNA from individual sections
was hybridized against a pool of Cy5-labeled cDNA from all sections of
the same series. Three to five replicate hybridizations were used for each
sample. For the meristem comparison, all four samples were hybridized
against each other with four hybridizations for each comparison, two of
them with swapped dyes.
For dot-blot hybridizations, 250 ng of cDNA from each section of both
CSA and CSB as well as from different meristem samples were immobilized on nylon filters and hybridized with 32P-labeled probes for PttCLV3,
PttWUS, and PttSTM using standard techniques. Resulting signals were
detected on a phosphor imager system (Bio-Rad, Solna, Sweden).
Quality Control and Normalization
Experimental Design and Sampling
Two series of sequential tangential sections through the cambial region
were prepared from two individual trees designated CSA and CSB.
Amplified cDNA was prepared from each section individually. Separate
reference pools were created for each of the two series by pooling cDNA
from the individual sections. Section samples were labeled with Cy3 and
the pool with Cy5, and three to four replicate hybridizations were
performed for each section.
Wild grown Populus tremula with a height of ;6 m and a stem diameter
of ;40 mm were used for all experiments. Sampling was performed on
July 7, 2000 during a period of maximal growth. Ten-centimeter-long
stem segments were frozen at 808C and subsequently trimmed to
blocks of 1.5-cm length and ;2-mm width. Tangential sections, 20-mm
thick, were taken in a cryomicrotome and frozen in liquid nitrogen. After
every third section, a cross section was prepared to determine the
position of the tangential sections. The experiment comprises two series
CSA and CSB from independent trees, each encompassing eight sections
(A1 to A8 and B1 to B8, respectively) and covering a radial width of
160 mm. Sections A6, B3, and B5 failed to produce high-quality cDNA
and were excluded from the experiment.
For the meristem, comparison samples were obtained from greenhouse grown Populus tremula 3 tremuloides. For the apical meristem,
sample tissue sections of ;100 mm 3 50 mm were dissected from the
centers of the apical meristems of 30 trees. The cambium sample is
a 30-mm tangential section covering the cambial zone, the root sample
covers the first 0.5 mm of the root tip, and the leaf sample was taken
from the blade of a mature, fully expanded leaf.
The CSG data was obtained by rehybridizing the amplified cDNA from
Hertzberg et al. (2001b) on POP1 arrays as described below.
Spot intensities were quantified using either Quantarray (Perkin-Elmer
Life Sciences) with the adaptive circle option (CSA) or GenePixPro 4.1
(CSB, CSG, and meristem comparison; Axon Instruments, Union City, CA).
Spots with high background, uneven morphology, or dust speckles were
manually flagged as bad. Mean foreground intensity minus median
background values were used for the subsequent steps. A quality filter
was applied to all spots as follows: a spot was considered good if (1) it
was not manually flagged as bad, and (2) <50% of the pixels were
saturated in both channels and (3) the foreground intensity was higher
than two times background standard deviation in both channels, or (4) the
foreground intensity was higher than four times background standard
deviation in one channel and higher than one background standard
deviation in the other channel.
Several hybridizations showed systematic intensity- and spacedependent artifacts. These were removed by applying a global intensity
and space-dependent (lowess) normalization using the smooth function
in the R/maanova package (version 0.91, http://www.jax.org/staff/
churchill/labsite/software/anova/rmaanova/index.html; Wu et al., 2003)
to all samples. At the same time, this function essentially normalizes
each array to a median Cy3/Cy5 ratio of one. Data reported in the
article are log2 ratios of Cy3 (sample section)/Cy5 (reference).
The six different contrasts of the meristem comparison were summarized into relative expression values by fitting an ANOVA model (for an
overview on the use of ANOVA for microarray analysis, see Wu et al.,
2003). The following model was used: yijkg ¼ m þ Ai þ Dj þ ADij þ Gg þ
VGkg þ DGjg þ AGig þ eijkg, where yijkg denotes the signal measured for
gene g, on array i, with dye j and sample k. The terms A, D, and AD capture
the average effect of the array, dye, and array 3 dye interaction on the
expression level, respectively. G can be interpreted as the average
intensity of a given gene, and DG and AG correspond to interactions
between genes and dyes or genes and arrays, respectively. The term of
Cambial Meristem Transcript Profile
interest is the sample (variety) 3 gene interaction VG. This term can be
interpreted as the relative expression level of a gene in the different
samples. The R/maanova package was used for fitting the ANOVA model.
Cluster and Principal Component Analysis
A subset of genes for cluster analysis was selected based on the following
criteria: genes had to show significant differential expression in either CSA
or CSB as determined by the ANOVA F-test implemented in the R/
maanova package with P < 0.001 after Bonferroni multiple test correction.
They further had to have at least two valid replicates in at least four
samples of each series. This set comprises 2280 genes. Clusters were
generated using self-organizing maps as implemented in the GeneSpring
5 software (Silicon Genetics, Redwood City, CA). A separate set16 was
assembled from 5% clones with the smallest standard deviation of the
relative log2 expression across samples. For the peak sets in Figure 4,
candidate genes were first manually selected from various clusters
created from the 2280 clone set to capture all genes with a potential
maximum or minimum in the cambial zone. These genes were then
filtered by a t test (Bonferroni adjusted P < 0.01) for significantly different
maximum and minimum values in either CSA or CSB. The final set of 188
genes was clustered as above.
Raw expression data from all samples of CSA and CSB were subjected
to PCA (Joliffe, 1986; Jackson, 1991). PCA is a method for analyzing large
data tables where the number of variables (in this study, genes) is much
larger than the number of objects (experiments). This is done by
projecting down the objects in the multidimensional data space onto
a set of few principal components. These principal components are
derived so they describe the direction in the multidimensional space with
maximum variation. The data can then be described with a set of vectors
in the following form: X ¼ t1*p1 þ t2*p2 þ . . . tA*pA þ e, where ti is the score
vector, pi is the loading vector, e is the residual matrix, and A is the number
of principal components used. The scores, ti, describe how similar
different objects (experiments) are to each other. Plotting scores against
each other, in a so-called score plot (Figure 2), can reveal the presence of
groups, clusters, or other trends in the data. The PCA calculations were
done with the multivariate analysis module in Evince (www.umbio.com)
using log2 expression ratios, which were normalized by standardizing
each gene to mean zero.
Analysis of Anticlinal Divisions
Stem sections from actively growing Populus tremula 3 temuloides were
fixed immediately after harvesting with 3.7% paraformaldehyde in 13
PBS, pH 7.0 (10 mM Na phosphate and 130 mM NaCl, pH 7.2) for 3 h at
room temperature and frozen to 808C. Frozen tissue was used to obtain
50-mm-thick cross sections using a cryomicrotome. After staining for cell
walls using Safranin/Alcacian blue, sections were scored for anticlinal
divisions in the cambial zone under a light microscope (Zeiss Axiovision 2;
Jena, Germany).
ACKNOWLEDGMENTS
We thank I. Sandström and E. Valfridsson for technical assistance. This
work was supported by the Swedish Research Council, FORMAS, and
the Swedish Foundation for Strategic Research.
Received May 12, 2004; accepted June 30, 2004.
13 of 15
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A High-Resolution Transcript Profile across the Wood-Forming Meristem of Poplar Identifies
Potential Regulators of Cambial Stem Cell Identity
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Plant Cell; originally published online August 17, 2004;
DOI 10.1105/tpc.104.024190
This information is current as of June 16, 2017
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