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Plant Cell. Sep 2004; 16(9): 2278–2292.
PMCID: PMC520933

A High-Resolution Transcript Profile across the Wood-Forming Meristem of Poplar Identifies Potential Regulators of Cambial Stem Cell IdentityW in Box

Abstract

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 μm 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 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.

Figure 1.
Sampling Scheme.

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 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 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 × 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

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-μm-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 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.

Figure 2.
Principal Component Analysis.

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 3.
Cluster Analysis.

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. 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.

Figure 4.
Anticlinal Divisions.

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).

Figure 5.
Peak Genes.

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; 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.

Figure 6.
Expression of Selected Cell Cycle–Related Genes.

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 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

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 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 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.jgi-psf.org/poplar0/poplar0.info.html) was used to obtain additional sequence information for phylogenetic tree construction.

Figure 7.
Expression of Meristem Identity and Other Marker Genes.

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 μm × 50 μm 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-μm 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/ESR-related (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, 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.

Figure 8.
Expression of PttWUS, PttCLV3, and PttSTM in Different Meristems.

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 μm (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, 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.

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.

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 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

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 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 × 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 μm. 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 μm 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).

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 −80°C and subsequently trimmed to blocks of 1.5-cm length and ~2-mm width. Tangential sections, 20-μm 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 μm. 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 × tremuloides. For the apical meristem, sample tissue sections of ~100 μm × 50 μm were dissected from the centers of the apical meristems of 30 trees. The cambium sample is a 30-μm 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.

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 (5′-biotin-GAGGTCCCAACCGCGGCCGC(T)15-3′) was followed by second-strand cDNA synthesis and fragmentation of the cDNA using a sonicator. Biotinylated 3′ 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 (0.1× SSC final), dried, and scanned in a ScanArray4000 scanner (Perkin-Elmer Life Sciences, Boston, MA) at a resolution of 10 μm (CSA and CSB) or 5 μm (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

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 space-dependent 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 = μ + Ai + Dj + ADij + Gg + VGkg + DGjg + AGig + εijkg, 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 × 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 interest is the sample (variety) × 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 = t1*p1 + t2*p2 + … tA*pA + ε, where ti is the score vector, pi is the loading vector, ε 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 × temuloides were fixed immediately after harvesting with 3.7% paraformaldehyde in 1× PBS, pH 7.0 (10 mM Na phosphate and 130 mM NaCl, pH 7.2) for 3 h at room temperature and frozen to −80°C. Frozen tissue was used to obtain 50-μm-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).

Supplementary Material

[Supplemental Data]

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.

Notes

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 (es.uls.syfneg@grebdnas.narog).

W in BoxOnline 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.

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