Genome-wide transcriptome analysis of the transition from primary to secondary stem development in Populus trichocarpa

Abstract

Background: With its genome sequence and other experimental attributes, Populus trichocarpa has become the model species for genomic studies of wood development. Wood is derived from secondary growth of tree stems, and begins with the development of a ring of vascular cambium in the young developing stem. The terminal region of the developing shoot provides a steep developmental gradient from primary to secondary growth that facilitates identification of genes that play specialized functions during each of these phases of growth.

Results: Using a genomic microarray representing the majority of the transcriptome, we profiled gene expression in stem segments that spanned primary to secondary growth. We found 3,016 genes that were differentially expressed during stem development (Q-value </= 0.05; >2-fold expression variation), and 15% of these genes encode proteins with no significant identities to known genes. We identified all gene family members putatively involved in secondary growth for carbohydrate active enzymes, tubulins, actins, actin depolymerizing factors, fasciclin-like AGPs, and vascular development-associated transcription factors. Almost 70% of expressed transcription factors were upregulated during the transition to secondary growth. The primary shoot elongation region of the stem contained specific carbohydrate active enzyme and expansin family members that are likely to function in primary cell wall synthesis and modification. Genes involved in plant defense and protective functions were also dominant in the primary growth region.

Conclusion: Our results describe the global patterns of gene expression that occur during the transition from primary to secondary stem growth. We were able to identify three major patterns of gene expression and over-represented gene ontology categories during stem development. The new regulatory factors and cell wall biogenesis genes that we identified provide candidate genes for further functional characterization, as well as new tools for molecular breeding and biotechnology aimed at improvement of tree growth rate, crown form, and wood quality.

Figure 1

Anatomy of stem internodes from developing Populus trichocarpa Nisqually-1 shoot apex. IN2, IN3, IN4, IN5 and IN9 are Toluidine Blue-O stained cross sections from stem internodes 2 to 9 (plastochron index) of an actively growing young tree under field conditions in Corvallis, Oregon. The IN3 stem section shows well developed primary vascular tissue. IN9 shows well developed secondary growth with well lignified xylem tissue. IIP, IIX - secondary phloem and xylem respectively; VC - vascular cambium; IP, IX - primary phloem and primary xylem respectively. Scale bar for IN2-IN9 whole stem cross sections is 0.5 mm; that for IN3 and IN9 stained partial cross sections is 50 um.

Figure 2

Apical shoot internode growth of Populus trichocarpa Nisqually 1. (A) elongation zone (red arrow) and (B) change in internode lengths along the developing stem of the two trees sampled for RNA.

Figure 3

Comparision of Populus trichocarpa genes on our array that is homologous to xylem-predominant genes from other studies. Normalized intensity values for IN3 (internode 3) plotted against IN9 (internode 9). Upper and low diagonal lines show two-fold change ratio intervals. (A) Comparison to postulated Arabidopsis core-xylem genes [23]. (B) Comparison to xylem-predominant transcription factor genes [24].

Figure 4

Hierarchical clustering of all differentially expressed genes and transcription factor genes in stem internode tissue (IN2 - IN9). Mean centered expression levels are represented with green denoting down regulation, and red indicating upregulation. (A) All 3,016 differentially expressed genes in stem internode tissue (IN2 - IN9). The 3 major clusters of genes with similar expression patterns are marked with vertical lines and numbered 1 to 3. The expression values, gene models, and closest hit for each of these genes are listed in Additional file 2. (B) 439 transcription factor genes regulated during stem development. The two major clusters of genes with similar expression patterns are marked with vertical lines. Gene identities are illustrated in Additional file 5.

Figure 5

Over-represented gene ontology categories during stem development. Statistically over-represented GO categories based on each of the three main gene expression pattern clusters depicted in figure 4A. (A) Cluster 1 genes, (B) Cluster 2 genes, and (C) Cluster 3 genes. The circles are shaded based on significance level (yellow = FDR below 0.05), and the radius of each circle denotes the number of genes in each category.

Figure 6

Clustering of transcription factor classes implicated in secondary development (i.e., dominant expression in IN9 and IN5). Cluster diagram of all expressed family members and gene identities are in additional file 7.

Figure 7

Clustering and identities of CesA and Csl gene family based on gene expression. Hierarchical clustering of expressed cellulose synthase (CesA) family and Ces like hemicelluloses-related genes (Csl) in stem internode tissue. Gene names/closest Arabidopsis hit information in Additional file 9.

Figure 8

Clustering and identities of expansin (A) and extensin (B) gene families based on gene expression. Hierarchical clustering of expressed (A) expansin gene family (EXPA, EXPB, EXLA, EXPLB) and (B) extensin gene family. Gene names/closest Arabidopsis hit information in Additional file 9.

Figure 9

Clustering and identities of cytoskeleton-related gene families based on gene expression. Hierarchical clustering of expressed (A) actin (B) tubulin (C) actin depolymerizing factor (ADFs) gene families. Gene names/closest Arabidopsis hit information in Additional file 9.

Figure 10

Expression of fasciclin-like AGP gene family. (A) Hierarchical clustering based on expression in stem internode tissue (B) Expression pattern in different Populus trichocarpa tissue types. Gene names/closest Arabidopsis hit information in Additional file 9.

Figure 11

Clustering and identities of phenylpropanoid genes based on gene expression. Gene names/closest Arabidopsis hit information in Additional file 9.