A high-resolution transcript profile across the wood-forming meristem of poplar identifies potential regulators of cambial stem cell identity

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 microm 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.

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 CS_A and CS_B. 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 CS_A. (C) Samples for meristem comparison experiment. Micrographs of poplar shoot and root apices with boxes showing the approximate location of tissue samples. Bars = 100 μm.

Figure 2.

Principal Component Analysis. Raw expression data from all samples of CS_A and CS_B 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 CS_A and circles CS_B.

Figure 3.

Cluster Analysis. 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 CS_A and CS_B. (B) Cluster averages plotted into the same graph, showing overlap of expression between CS_A (dotted lines) and CS_B (solid lines). Shaded areas mark the approximate location of the cambial zone.

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.

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.

Figure 6.

Expression of Selected Cell Cycle–Related Genes. Dashed lines, expression in CS_A; solid lines, expression in CS_B. 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).

Figure 7.

Expression of Meristem Identity and Other Marker Genes. Dashed lines, expression in CS_A; solid lines, expression in CS_B. 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.

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.

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.