Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics

Abstract

Forward genetic screens have led to the isolation of several genes involved in secondary cell wall formation. A variety of evidence, however, suggests that the list of genes identified is not exhaustive. To address this problem, microarray data have been generated from tissue undergoing secondary cell wall formation and used to identify genes that exhibit a similar expression pattern to the secondary cell wall-specific cellulose synthase genes IRREGULAR XYLEM1 (IRX1) and IRX3. Cross-referencing this analysis with publicly available microarray data resulted in the selection of 16 genes for reverse genetic analysis. Lines containing an insertion in seven of these genes exhibited a clear irx phenotype characteristic of a secondary cell wall defect. Only one line, containing an insertion in a member of the COBRA gene family, exhibited a large decrease in cellulose content. Five of the genes identified as being essential for secondary cell wall biosynthesis have not been previously characterized. These genes are likely to define entirely novel processes in secondary cell wall formation and illustrate the success of combining expression data with reverse genetics to address gene function.

Figure 1.

Analysis of IRX1 and IRX3 Expression in the Stem, Leaf, and Hypocotyl. Expression of IRX1 (open bars) and IRX3 (closed bars) are represented as a percentage of actin expression. Standard error bars are shown (n = 3).

Figure 2.

Interpretation and Analysis of Microarray Data. (A) PCA of microarray data from leaves, hypocotyls, and four stages of stem development. The data include three biological replicates for each stage. The first two principal components are shown (component 1 accounts for 45% of the variance and component 2 accounts for 23%). Circles are drawn as a guide and have no statistical significance. (B) Comparison of the gene expression between the base and tip of the stem. Changes in expression levels have been plotted against P values derived from a t test. Horizontal lines indicate a change in gene expression of twofold or more. The vertical line corresponds to a P value of 0.01. Shaded area shows genes that have a greater than twofold increase in gene expression between the tip and the base of the stem and a P value of <0.01 (n = 3). Crosses represent genes selected for further analysis. (C) Slope profile analysis of microarray data showing the expression profile of the top 25 genes that most closely match that of IRX3 (thick line).

Figure 3.

Graphical Representation of Root Microarray Data (Birnbaum et al., 2003). Stage 1 closest to the root tip; stage 3 furthest from the root tip. Cell layers in the root indicated are lateral root cap (lrc), epidermis (epi), endodermis and cortex (endo and cor), endodermis (end), and stele. (A) At4g18780 (IRX1); (B) At5g17420 (IRX3); (C) At3g18660; (D) At3g12955.

Figure 4.

Real-Time RT-PCR Analysis of Selected Genes in Different Tissues. The expression of genes was measured as the percentage of actin expression. For convenience, the results have been expressed as a proportion of the maximum expression level for that gene and are an average of three biological replicates.

Figure 5.

Cross Sections of Stem Vascular Bundles in Wild-Type and irx Mutant Plants. Transverse stem sections stained with toluidine blue. A single representative vascular bundle is shown from each mutant. The phloem (ph) and xylem vessels (arrows) are indicated. (A) Columbia wild type; (B) irx3-4; (C) irx6; (D) irx7; (E) irx8; (F) irx9; (G) irx10; (H) irx11; (I) irx12. Bars = 50 μm.

Figure 6.

Whole-Plant Morphology of Wild-Type and irx Mutants. All plants shown are 5 weeks old.

Figure 7.

Cellulose Content of Stems from Wild-Type and Insertion Mutant Lines. Cellulose content is expressed as a proportion of the ethanol insoluble cell wall material collected at the late stage of stem development. Numbers correspond to insertion line number (see Table 1). Closed bars indicate measurement from irx1-5 and irx3-4 plants. Hatched bars highlight novel irx mutants. Shaded bars highlight Ws background. Asterisks show a significant reduction in cellulose. Standard error bars are shown (n = 4).

Figure 8.

Cellulose Content of Developing Stems from Wild-Type and irx Mutant Plants. Cellulose content is expressed as a proportion of the ethanol insoluble cell wall material. Standard error bars are shown (n = 4).

Figure 9.

Cell Wall Analysis of Insertion Mutant Lines. (A) PCA of FTIR spectra from wild-type and mutant stem material. PC1 accounted for 62.5% of the total explained variance, whereas PC2 accounted for 20.2%. Cluster 2 contains irx1, irx3, and irx5. Cluster 3 contains irx7, irx8, and irx9. Cluster 1 contains wild-type and all other insertion lines. Numbers refer to XIM line numbers. Circles are drawn as a guide and have no statistical significance. (B) Loading plot of PC1 in the polysaccharide fingerprint region. A spectrum derived from cotton linters cellulose is also shown for reference. Peaks characteristic of cellulose (1161 cm⁻¹, 1109 cm⁻¹, 1059 cm⁻¹, 1034 cm⁻¹, and 1059 cm⁻¹) are indicated. (C) Loading plot of principal component 2 in the polysaccharide fingerprint region. A spectrum derived from birchwood xylan is also shown for reference. Peaks characteristic of xylan (1240 cm⁻¹, 1082 cm⁻¹, 1045 cm⁻¹, and 978 cm⁻¹) are indicated.

Figure 10.

The Noncellulosic Carbohydrate Composition of Cell Wall Material from Stems of Wild-Type and Insertion Mutant Lines. Individual sugars are expressed as a percentage of the total cell wall sugar. Material used was from the late stage of development. Standard error bars are shown (n = 5).