Plant body weight-induced secondary growth in Arabidopsis and its transcription phenotype revealed by whole-transcriptome profiling

Abstract

Wood is an important raw material and environmentally cost-effective renewable source of energy. However, the molecular biology of wood formation (i.e. secondary growth) is surprisingly understudied. A novel experimental system was employed to study the molecular regulation of secondary xylem formation in Arabidopsis. First, we demonstrate that the weight carried by the stem is a primary signal for the induction of cambium differentiation and the plant hormone, auxin, is a downstream carrier of the signal for this process. We used Arabidopsis whole-transcriptome (23 K) GeneChip analysis to examine gene expression profile changes in the inflorescent stems treated for wood formation by cultural manipulation or artificial weight application. Many of the genes up-regulated in wood-forming stems had auxin responsive cis-acting elements in their promoter region, indicating auxin-mediated regulation of secondary growth. We identified 700 genes that were differentially expressed during the transition from primary growth to secondary growth. More than 40% of the genes that were up-regulated (>5x) were associated with signal transduction and transcriptional regulation. Biological significance of these regulatory genes is discussed in light of the induction and development of secondary xylem.

Figure 1.

Secondary xylem tissue development of Arabidopsis stem. A, Secondary xylem development is related to the plant stem height. All plants are the same age (8 weeks old) and have similar stem thickness, but differ in the height growth of inflorescence stems. The heights of the stems are indicated in upper panels. Basal level of the stem was cross-sectioned and stained with 2% phloroglucinol-HCl to visualize secondary xylem as red color in the lower section. Scale bars represent 1 cm in upper sections and 0.5 cm in lower sections. B, Secondary xylem developed from vascular cambium of the mature Arabidopsis stem. Detailed structure of immature and mature stem cross-section was obtained from confocal laser microscopy (see “Materials and Methods”). EP, epidermis; IFR, interfascicular region; PC, procambium; PH, phloem; PX, primary xylem; VC, vascular cambium; PF, phloem fiber; SX, secondary xylem. Bar indicates 0.2 mm of length.

Figure 2.

Artificially-applied weight promotes secondary xylem development. All of the plants used were approximately 5 cm tall (immature) and treated for 3 d. A, Basal level cross-section of intact plant (11 cm tall). B, Basal level cross-section of decapitated immature plants. C, Basal level cross-section of the stem that received 2.5 g-weight treatment and (D) its magnified view. E and F, Weights (2.5 g) were applied to the top part of the decapitated immature stem without (E) or with (F) 5 μm NAA. Plants cut at the basal level are shown and the arrows indicate the basal level. G and H, Basal level cross-sections of E and F, respectively. Red asterisks indicate the auxiliary branch developed during the treatment. All staining was performed using 2% phloroglucinol-HCl except (D), which was stained with 0.05% toluidine blue O. 1, 2, 3, and 4, Detailed confocal laser microscopy images of the insets in G and H. 5, Magnified view of inset in 4 showing the vascular cambium cells. Scale bars represent 0.5 mm (A, B, C, G, and H); 100 μm (D, 1, 2, 3, and 4); 1 cm (E and F); and 20 μm (5).

Figure 3.

Artificially-applied weight increases basipetal auxin transport. A, Diagram of the experimental design. Weight (2.5 g) was applied on top of the stem placed upside down in the 15-mL conical tube containing [¹⁴C]IAA (see “Materials and Methods”). B, Basipetal [¹⁴C]IAA movement was facilitated by weight treatment. Error bar means se (n = 3).

Figure 4.

Confirmation of signal intensity of GeneChip data with conventional northern-blot analysis. A, Northern-blot analysis. RNA gel-blot analysis was performed with 15 μg of total RNA extracted from each sample indicated above and hybridized with the randomly labeled cDNA probes indicated on the left side. All of the RNA samples are duplicates of the RNAs used in the GeneChip analysis. Exposed to Kodak x-ray film for 1 d (Rochester, NY). 25S rRNA serves as a RNA loading control. B, Signal intensity of the GeneChip data.

Figure 5.

Comparison of gene expression patterns with weight treatment and intermediate stem. A, Gene finding using hierarchical clustering; 700 genes have similar expression pattern (with minimum correlation of 0.95). Normalization and K-Mean clustering was done by GeneSpring 4.2.1 software (Silicon Genetics, Redwood City, CA). The expression patterns of the selected 700 genes are selectively shown. B, Magnifying view of each cluster in (A). Supplemental Table provides the list of genes. Ct, control; Wt, 2.5-g weight treatment; Im, immature; Int, intermediate stem. C, Functional classifications. Classification was based on the Munich Information Center for Protein Sequences (http://mips.gsf.de/proj/thal/db/tables/tables_func_frame.html).

Figure 6.

Expression profiles of transcription factor candidates might be involved in the transition from primary to secondary growth. Gene expression (y axis) designates the mean value of signal intensity of each gene. Note the different scope. Ct, control; Wt, 2.5-g weight treatment; Im, immature; Int, intermediate stem; Mat, mature stem.

Figure 7.

Expression profiles of auxin-modulating genes during the development of wood-forming stems. Gene expression (y axis) designates the mean value of signal intensity of each gene. Note the different scope. Imm, immature; Int, intermediate; Mat, mature stem.