Xylem Cell Wall Formation in Pioneer Roots and Stems of Populus trichocarpa (Torr. & Gray)

Abstract

Regulation of gene expression, as determined by the genetics of the tree species, is a major factor in determining wood quality. Therefore, the identification of genes that play a role in xylogenesis is extremely important for understanding the mechanisms shaping the plant phenotype. Efforts to develop new varieties characterized by higher yield and better wood quality will greatly benefit from recognizing and understanding the complex transcriptional network underlying wood development. The present study provides a detailed comparative description of the changes that occur in genes transcription and the biosynthesis of cell-wall-related compounds during xylogenesis in Populus trichocarpa pioneer roots and stems. Even though results of microarray analysis indicated that only approximately 10% of the differentially expressed genes were common to both organs, many fundamental mechanisms were similar; e.g. the pattern of expression of genes involved in the biosynthesis of cell wall proteins, polysaccharides, and lignins. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) shows that the composition of monosaccharides was also very similar, with an increasing amount of xylose building secondary cell wall hemicellulose and pectins, especially in the stems. While hemicellulose degradation was typical for stems, possibly due to the intensive level of cell wall lignification. Notably, the main component of lignins in roots were guiacyl units, while syringyl units were dominant in stems, where fibers are especially needed for support. Our study is the first comprehensive analysis, at the structural and molecular level, of xylogenesis in under- and aboveground tree parts, and clearly reveals the great complexity of molecular mechanisms underlying cell wall formation and modification during xylogenesis in different plant organs.

Keywords: Populus trichocarpa; cell wall biogenesis; microarrays; wood; xylogenesis.

Figure 1

Global aspects of the black cottonwood transcriptome during three stages of xylem development in pioneer roots. (A) Expression pattern of the 1,914 statistically significant transcript sets at three time points during pioneer roots xylogenesis in black cottonwood and the overlaps among them are shown. Venn diagram comparing the differentially expressed transcripts. (B) Heatmap of 1914 Populus trichocarpa genes that showed, at last, two-fold differences with a p-value ≤ 0.001 in pioneer roots. Hierarchical clustering shows the gene expression clusters. (C) Functional classification of the genes up-regulated in the stage PR2 and PR3, respectively as compared to PR1 in pioneer roots of black cottonwood using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The x-axis represents the Gene Ontology (GO) annotation categories and the y-axis represents the number of total matched genes from a specific category. (D) Functional classification of the genes down-regulated in the stage PR2 and PR3, respectively as compared to PR1 in pioneer roots of P. trichocarpa using the DAVID. The x-axis represents the GO annotation categories and the y-axis represents the number of total matched genes from a specific category.

Figure 2

Global aspects of the black cottonwood transcriptome during three stages of xylem development in stem. (A) Expression pattern of the 1171 statistically significant transcript sets at three time points during pioneer roots xylogenesis in black cottonwood and the overlaps among them are shown. Venn diagram comparing the differentially expressed transcripts. (B) Expression pattern of statistically significant transcript sets in pioneer roots and stem of black cottonwood during xylogenesis process and the overlaps among them are shown. Venn diagram comparing the differentially expressed transcripts. (C) Heatmap of 1,171 Populus trichocarpa genes that showed, at last, two-fold differences with a p-value ≤ 0.001 in stem. Hierarchical clustering shows the gene expression clusters. (D) Functional classification of the genes up-regulated in the stage PR2 and PR3, respectively as compared to PR1 in pioneer roots of black cottonwood using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The x-axis represents the GO annotation categories and the y-axis represents the number of total matched genes from a specific category. (E) Functional classification of the genes down-regulated in the stage PR2 and PR3, respectively as compared to PR1 in pioneer roots of P. trichocarpa using the DAVID. The x-axis represents the GO annotation categories and the y-axis represents the number of total matched genes from a specific category.

Figure 3

(A) Expression profile of genes related to cellulose metabolism in pioneer roots and (B) stem of black cottonwood. CesA, cellulose synthase; TED7, tracheary element differentiation-related 7 membrane protein; SuS, sucrose synthase. (C) Cell wall monosaccharide composition in pioneer roots and stem of Populus trichocarpa. Cell wall monosaccharide composition was measured from alcohol-insoluble residues extracted from root and stem tissues. Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Man, mannose; Gal, galactose; Glu, glucose. (D) Cellulose content in cell walls in pioneer roots and stem of P. trichocarpa. Crystalline cellulose content was measured in alcohol-insoluble residue (AIR) of cell walls with Updegraff method. Means designated by different letters indicate changes statistically significant according to Tukey´s post hoc test, (P < 0.05). Error bars indicate ± SE (n = 4).

Figure 4

(A) Autofluorescence of lignins in pioneer roots and (B) stem of Populus trichocarpa. (C) Expression profile of genes related to lignin metabolism in pioneer roots and (D) stem of black cottonwood. SAMT, S-adenosyl-L-methionine:carboxyl methyltransferase; COMT, caffeic acid O-methyltransferase; CCR, cinnamoyl-CoA reductase; LAC, laccase; CAD, cinnamyl alcohol dehydrogenase; SAM, S-adenosylmethionine synthetase; PAL, phenylalanine ammonia-lyase; 4CL, 4-coumarate:CoA ligase. (E) Cell wall lignin composition in pioneer roots and stem of P. trichocarpa. Cell wall lignin composition was measured from alcohol-insoluble residues extracted from stem and root tissues. H (p-hydroxyphenyl), G (guaiacyl), S (syringyl) lignin monolignols.

Figure 5

Immunolocalization of extensins (A–D), xyloglucan (E–H), homogalacturonan (I–L), and their co-localization with lignins in stems and pioneer roots during different developmental stages. [x, primary xylem; ph, phloem; sx, secondary xylem; pf, phloem fibers; c, cambium; ct, cork tissue (phellem); pc, parenchyma cortex]. Bars 50 µm.

Figure 6

Immunolocalization of arabinogalactan proteins (A–D), arabinan (E–H), galactan (I–L)and their co-localization with ligninsin in pioneer roots and stem during different developmental stages. [x, primary xylem; ph, phloem; sx, secondary xylem; pf, phloem fibers; c, cambium; ct, cork tissue (phellem)]. Bars 50 µm.

Figure 7

A simplified model to describe the major changes in developing pioneer roots and stems of Populus trichocarpa. The overlapping region corresponds to common processes in pioneer roots and stems. The upward green arrows show up-regulated features and the downward red arrows show down-regulated features. CW, cell wall; PCW, primary cell wall; SCW, secondary cell wall; PX, primary xylem; PXG, primary xylem formation; SX, secondary xylem; SXG, secondary xylem formation; G, guiacyl units in lignins; S, syringyl units in lignins; AGPs, arabinogalactan-proteins; FLAs, fasciclin-like arabinogalactan-proteins.