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First published online June 24, 2005; 10.1105/tpc.105.031542 © 2005 American Society of Plant Biologists Identification of Novel Genes in Arabidopsis Involved in Secondary Cell Wall Formation Using Expression Profiling and Reverse Genetics
a Faculty of Life Science, University of Manchester, Manchester M13 9PT United Kingdom 1 To whom correspondence should be addressed. E-mail simon.turner{at}manchester.ac.uk; fax 44-0161-2753938.
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.
The plant cell wall has many functions: it regulates cell expansion, contributes to cell adhesion, acts as a barrier to potential pests and pathogens, and determines the physical properties of the plant (Braam, 1999  ; Jones and Takemoto, 2004; Scheible and Pauly, 2004; Vorwerk et al., 2004) The differing functions of the cell wall are reflected in the large variation in cell wall composition between different cell types and during cell differentiation. One estimate suggests that as many as 15% of the genes in the genome may be concerned with cell wall synthesis, remodeling, or turnover (Carpita et al., 2001). The Arabidopsis thaliana genome contains >800 identifiable carbohydrate active enzymes. This figure represents a large proportion of the genome compared with nonplant organisms, and it is suggested that the overrepresentation of carbohydrate active enzymes is a requirement for synthesis, remodeling, and degradation of the plant cell wall (Coutinho et al., 2003). A large number of other genes are also required for synthesis of cell wall polymers, such as lignin, phenylpropanoids, structural proteins, and other cell wall components. Identifying and determining the function of genes involved in cell wall synthesis and modification remains a major challenge.
The deposition of a thick lignified secondary cell wall only occurs once cells have attained their final shape and size. As the major constituent of wood and plant fibers, understanding the synthesis of the secondary cell wall has important biological and economic implications. During inflorescence stem development in Arabidopsis, the xylem and interfascicular cells form a thick secondary cell wall that constitutes a large proportion of the dry weight of the stem (Turner and Somerville, 1997
Defects in the secondary cell wall are characterized by a collapse of xylem vessels that are unable to withstand the negative pressure generated during water transport through the xylem. This phenotype, described as irregular xylem (irx) has been used to isolate Arabidopsis mutants defective in the biosynthesis of both cellulose (Turner and Somerville, 1997
The irx1, irx3, and irx5 mutants are all caused by defects in members of the CesA gene family. The AtCesA4 (IRX5), AtCesA7 (IRX3), and AtCesA8 (IRX1) proteins all function in a nonredundant manner as part of a complex that is required to synthesize cellulose in the secondary cell wall (Gardiner et al., 2003
Recent annotation of the Arabidopsis genome suggests that of the 27,139 identified functional genes, 7592 (28%) are predicted as protein of either hypothetical or unknown function (http://arabidopsis.org/). Many of the remaining genes are annotated solely on the basis of a conserved domain or assigned to a gene family based on sequence homology that groups these genes into broad categories. Estimates suggest that only In this study, microarray data generated from developing stems and hypocotyls have been used to identify those genes coregulated with IRX1, IRX3, and IRX5. By cross-referencing publicly available data, several genes were selected for reverse genetic analysis. The results of this analysis suggest that several, if not all, of the genes identified are essential for secondary cell wall formation. Only one mutant, however, exhibited a severe cellulose-deficient phenotype, suggesting that the other genes are involved in other, potentially novel, pathways required for secondary cell wall formation.
Expression of IRX1 and IRX3 At the onset of this study, few publicly available data sets exhibited high levels of expression of the secondary cell wall–specific genes IRX1 and IRX3. This presumably reflects the fact that few studies use tissues such as mature stems that contain a high proportion of cells undergoing secondary cell wall formation. To examine the expression of these genes in more detail, real-time RT-PCR was performed on RNA isolated from four parts of the stem, mature hypocotyls, and leaves. This material was selected on the basis that secondary cell wall formation had previously been shown to increase dramatically from the top to the base of the stem and to be high in hypocotyls and low in leaves (Turner and Somerville, 1997 ). The expression levels of IRX3 and IRX1 both mirrored this pattern. Their expression increased dramatically from the tip to the base of the stem and was high in hypocotyls and low in leaves (Figure 1). Although IRX3 was expressed consistently at a higher level than IRX1, they both exhibited a very similar pattern of expression that varied dramatically with >60-fold higher levels of expression in the hypocotyls compared with leaves.
Expression Analysis of Secondary Cell Wall Formation mRNA from the six stages described above were used for near genome-wide expression profiling using the Affymetrix ATH1 microarray chip (Santa Clara, CA). Experiments were performed using three biological replicates from each developmental stage. Principal component analysis (PCA) was used to analyze the variance within the data set. All the biological replicates for a particular developmental stage clustered closely together in this unsupervised multivariate analysis of all the genes on the microarrays. Similarly, the hypocotyls, leaf, and stem samples were all well separated from one another (Figure 2A). More importantly, the different stem samples separated on principal component 1 on the basis of their stage of development (i.e., from tip to base) (Figure 2A). This clear clustering of the microarray data in the PCA indicates highly reproducible and discrete mRNA expression patterns in the different stages of stem development that were collected.
A comparison of changes in gene expression between the top and the bottom of the stem is shown in Figure 2B. This analysis identifies several hundred genes that apparently show an increase in expression at the base of the stem compared with the tip. This was considered as too many genes to examine efficiently. Consequently, to perform a detailed study, this set of genes was further refined (see below). For reference, the genes eventually selected are indicated in Figure 2B by crosses. This clearly shows that there is no clear relationship between fold change and those genes eventually selected.
Selection of Candidate Secondary Cell Wall Genes for Further Analysis
The identification of two further genes that closely match the expression of IRX3 further increased the confidence in this method of analysis. At5g15630 (position 5) encodes a member of the COBRA family (COBL4) (Schindelman et al., 2001 ; Roudier et al., 2002). The ortholog of this gene in rice (Oryza sativa) is essential for cellulose deposition in the secondary cell wall (Li et al., 2003). Similarly, At3g16920 (position 8) encodes for a member of a family that exhibits homology to chitinase genes. A member of this family has been shown to be required for cellulose synthesis in the primary cell wall (Mouille et al., 2003). By contrast, expression of the endoglucanase KORRIGAN (KOR) (Nicol et al., 1998) does not closely match the pattern of IRX3 expression. Similarly, many genes required for lignin biosynthesis, such as IRX4 that encodes a cinnamoyl CoA reductase (Jones et al., 2001), do not exhibit a good match with IRX3 expression.
Many of the genes in Table 1 that exhibit a similar expression pattern to IRX3 are likely to be involved in carbohydrate metabolisms. Twelve of the 25 genes shown in Table 1 have been identified as belonging to various families of glycosyltransferase (GT) or glycosylhydrolase enzymes, including members of both the GT8 and GT47 families (http://afmb.cnrs-mrs.fr/CAZY/). Mutations in members of both of these gene families have been shown to result in cell wall defects in Arabidopsis (Bouton et al., 2002
Cross-Referencing Publicly Accessible Data
To further verify the coregulation of these genes with IRX3, the top 200 genes were used in a pairwise comparison using the two-gene scatterplot at the Nottingham Arabidopsis Stock Centre (NASC; http://nasc.nott.ac.uk/) and the Gene Correlator at the Genevestigator Web site (https://www.genevestigator.ethz.ch/) (Zimmermann et al., 2004 ). Both of these programs plot the expression of two selected genes from a large number of publicly available data sets. In addition, Gene Correlator generates a linear correlation coefficient (Table 1).
In general, any genes that did not exhibit a correlation coefficient of >0.6 were not selected for further study. However, there were exceptions, for example, a putative GT47 member (At2g28110) was retained despite a poor correlation (r2 = 0.01) because a member of the same family (At1g27440) did exhibit a good match with IRX3. Similarly, Xylem Insertion Mutant 12 (XIM12), an insertion in At5g16600, was selected for further analysis because it is a member of the MYB gene family that has been demonstrated to be involved in the regulation of secondary cell wall polymers (Tamagnone et al., 1998 To independently confirm the microarray data independently, 12 genes of the gene studied were analyzed using real-time RT-PCR. Consistent with the microarray data, all genes selected exhibited the highest level of expression in either the hypocotyls or at the base of the stem, low levels of expression in leaves, and a decline in expression levels going from the base to the tip of the stem (Figure 4).
Reverse Genetics For those genes identified for further analysis, insertion lines were selected using the SIGnaL database (http://signal.salk.edu/). Where multiple insertions were available in the same genes, lines were selected on the basis of the position of the insertion and the highest likelihood of it disrupting gene function. In most cases, insertions were obtained within the exon of a gene or within the 5' noncoding region (Table 1). Primers flanking the insertion site were used in conjunction with a primer from the left border of the T-DNA to identify lines homozygous for the T-DNA insertion (see Methods for details). Where problems with poor seed set or sterility occurred, the seeds from plants heterozygous for the insertion were collected. To verify the effect of the insertion on mRNA expression, RT-PCR was performed using gene-specific primers designed to be close to the 5' and 3' ends of the gene. Line XIM43 exhibited no difference in the expression of the target gene compared with the wild-type control. This line contains an insertion in the promoter region, and this presumably does not significantly alter the expression of the target gene. Similarly, after 50 cycles of PCR, XIM29 and XIM30 generated a band of the predicted size, although at a greatly reduced level. Interestingly, both of these lines generated a phenotype (see below). These lines contain an insertion in the 5' untranslated region and an intron of the target, respectively (Table 1). All of the other primer pairs generated a PCR fragment of the predicted size in the wild type, but that was undetectable in the corresponding insertion line (data not shown).
Phenotypic Analysis
In comparison with the wild type, irx3-4 plants are small, grow more slowly, are darker green, have narrower leaves, and were almost completely sterile (Figure 6). Similar phenotypes were obtained from insertions in the IRX1 and IRX5 genes (data not shown). This characteristic plant morphology was obtained for three other mutants: irx7, irx8, and irx9 (Figure 6). These lines correspond to the plants that exhibit a severe irx phenotype described above. Because of poor fertility, these lines were maintained by selecting heterozygotes and selecting homozygous insertion lines from their progeny. Removal of these plants from the growth chamber often resulted in severe wilting of the end of the inflorescence stems that was irreversible. Maintaining plants at very high humidity by permanently covering them with a plastic dome did help to generate plants that grew more vigorously, but all the phenotypes described above were still evident and mutants were very clearly distinguishable from the wild type.
For all the other lines examined, the plants appeared morphologically wild-type (Figure 6); however, two lines did exhibit other phenotypes. irx6 plants containing an insertion in the COBL4 gene exhibited a normal growth habit (Figure 6) but resulted in a plant with dramatically reduced stem strength that caused the inflorescence stem to be easily broken. To a lesser extent, this was also true of XIM9 containing an insertion in a GT8 family gene (At3g18660). A weak stem is a characteristic of known secondary cell wall mutants (Turner and Somerville, 1997 ; Jones et al., 2001).
Cell Wall Analysis
To overcome the potential problem with measuring cellulose content of plants with altered morphology, it is important to ensure that comparisons are made at the same developmental stage. Those lines that exhibited a reduction in cellulose content were examined at three stages of stem development. Wild-type plants exhibit an increase in cellulose content in the stem during development that reflects the increase in secondary cell wall deposition (Figure 8). Consistent with previous studies, plants with an insertion in IRX3 do not show this increase, and the cellulose content remains constant during stem development. Similarly, irx6 plants also exhibit no increase in cellulose during stem development (Figure 8). By contrast, irx7, irx8, and irx9 show a clear increase in cellulose content during development. The pattern of accumulation is similar to the wild type, but at each stage the cellulose content is less than that of corresponding wild-type plants (Figure 8).
To facilitate characterization and classification, mutant lines were examined using the metabolic fingerprinting method of Fourier transform infrared (FTIR) spectroscopy (Goodacre et al., 2004 ). A PCA scores plot derived from FTIR spectra data clearly separated the mutant lines into three distinct clusters on the basis of secondary cell wall defects (Figure 9A). irx1, irx3, and irx5 (represented as cluster 2) and irx7, irx8, and irx9 (represented as cluster 3) are separated from other mutant lines, including the wild type, in principal component 1 (PC1; Figure 9A). The loadings plot for PC1 in the polysaccharide fingerprint region (1250 cm–1 to 800 cm–1) (Figure 9B) showed characteristic IR bands that correlated well with purified cellulose, with peaks at 1161 cm–1, 1109 cm–1, 1059 cm–1, 1034 cm–1, and 1059 cm–1 (Liang and Marchessault, 1959; Sugiyama et al., 1991; Kacurakova et al., 2002). The spectrum from cotton (Gossypium hirsutum) lintels is shown for reference. The scores of clusters 2 and 3 are negative on PC1 relative to the mean (Figure 9A), suggesting a decrease in cellulose in secondary cell walls. Separation between clusters 2 and 3 suggests that discrimination on PC2 is based on noncellulosic differences between the groups. The PC2 loading plot (Figure 9C) is characterized by positive peaks that can be designated to xylan-type polysaccharides at 1240 cm–1, 1128 cm–1, 1082 cm–1, 1045 cm–1, and 978 cm–1 as described by Kacurakova and colleagues (Kacurakova et al., 1998, 1999, 2000). A spectrum of purified birch (Betula spp) xylan (Figure 9C) is shown for reference, showing that many these peaks match those found in the PC2 loading plot. This suggests that the cell walls of irx7, irx8, and irx9 are deficient in xylan. Further analysis of the FTIR spectra using discriminate function analysis (Manley, 1994) to reduce within group variance and maximize between group variance demonstrated that irx12, a putative laccase, also formed a separate cluster (data not shown). Because no other irx mutants grouped with irx12, it is likely that this is the only line with a defect in phenylpropanoid metabolism.
Figure 10 shows the noncellulosic carbohydrate composition of a crude cell wall fraction from stems. The proportion of sugars found in wild-type stems is in general agreement with previous studies (Turner and Somerville, 1997 ). The high xylose content, which is characteristic of secondary cell walls, reflects the typical composition of many angiosperms (Turner and Somerville, 1997). The cell wall sugar composition of cellulose-deficient mutants irx3 and irx6 plants were in approximate agreement with earlier studies (Turner and Somerville, 1997; Li et al., 2003).
The largest changes are evident in irx7, irx8, and irx9, which all exhibited a large reduction in xylose. The irx7 noncellulosic sugar fraction contained only 28% of wild-type xylose, a characteristic repeated to a lesser extent in both irx8 (35% of the wild type) and irx9 (45% of the wild type). In addition to a decrease in xylose, significant increases in the remaining cell wall sugars are observed in all three lines. This increase in proportion may be partly explained by the loss of xylose. irx10 stems had similar, but less dramatic, alterations in sugar composition with a decrease in xylose (80% of the wild type) and a small increase in the percentage of all other sugars.
The secondary cell wall of Arabidopsis is composed predominantly of cellulose, lignin, and xylan, making it an attractive model for the study of these pathways. However, several other components are likely to be important in the secondary cell wall. This is exemplified by work on a laccase gene in poplar (Populus spp). Downregulation of this gene results in fragmented secondary cell walls, distorted xylem vessels, and the accumulation of phenolic compounds that would presumably normally be cross-linked into the wall to give its proper structural properties (Ranocha et al., 2002 ). Before this study, the role of these compounds in wall formation had received little attention. It is likely given the current paucity of knowledge with respect to secondary metabolism in plants that other such pathways contribute essential components to the secondary cell but are as yet poorly characterized. Genetic screens, based upon the irx phenotype, have led to the isolation of several important genes required for secondary cell wall biosynthesis, including those involved in cellulose and lignin biosynthesis (Turner and Somerville, 1997; Jones et al., 2001; Taylor et al., 2003). To date, however, only five complementation groups have been identified.
Estimates based upon the frequency of known mutations and the numbers of unique genes suggest that perhaps mutations in only one in 10 genes would give a clear phenotype (Meinke et al., 2003 The advantage of targeting specific processes with expression data followed by reverse genetic analysis of the genes is highlighted in this study. This is illustrated by insertions in the IRX3 gene and several other lines described in this study. These plants have greatly altered morphology that is presumably caused by the collapse of the xylem (Figure 5). Because most mutant analysis does not include cross sections of the xylem, the irx phenotype would not be obvious, and the altered morphology may be attributed to some other factor. Similarly, some mutants, such as irx6, appear morphologically normal (Figure 6) but exhibit a clear collapsed xylem phenotype that would be impossible to detect without making cross sections of the xylem. Why a collapse xylem causes alteration in plant morphology is unclear, but there is a correlation between the severity of the irx phenotype and the severity of the whole-plant phenotype. Those plants classified as very severe irx exhibit the most severe whole-plant morphology, whereas those plants with a less severe irx phenotype appear similar to the wild type (Figures 5 and 6).
Of the genes that do not give a phenotype, their correlation with the expression patterns of IRX1 and IRX3 would support a role for them in secondary cell wall formation. Of the nine lines that exhibited no phenotype, RT-PCR analysis suggests that one line contained normal levels of mRNA for the target gene; however, for the other eight lines, the corresponding mRNA was undetectable. It is not possible to preclude that these plants still make a truncated or altered mRNA that would lead to the production of protein that retains at least some activity. A more likely explanation of why some lines identified do not exhibit a clear phenotype is probably a combination of gene redundancy and a screening method that is specific for only one cell wall function. The former point can be illustrated by XIM54; this line is annotated as a gene of unknown function but contains a specific motif recognized by the program pfam (http://www.sanger.ac.uk/Software/Pfam/) known as DUF579. The Arabidopsis genome possesses 10 genes that contain this domain. Four of these genes, however, can be found within the list of 60 genes whose expression patterns most closely match that of IRX3. The extent to which redundancy contributes to the absence of an irx phenotype can only be assessed once lines containing two or more gene knockouts have been generated by crossing. The alternative explanation is the limitation of screening using the irx phenotype. This phenotype selects for plants whose secondary cell wall lacks the strength to withstand the forces generated within the xylem (Turner and Somerville, 1997
It is striking that only mutations in COBL4 gave a severe cellulose-deficient phenotype (Figure 7) in a manner similar to its rice homolog (Li et al., 2003
irx7, irx8, and irx9 exhibit much smaller but significant changes in cellulose content of the stem. These plants, however, exhibit gross alteration in plant growth and morphology (Figure 6), and it is likely that this contributes to an overall reduction in secondary cell wall deposition in the stem. This would be analogous to the irx4 mutant caused by a defect in the gene encoding cinnamoyl CoA reductase. irx4 plants have large reduction in cell wall phenolics but also exhibit a decrease in cellulose that is presumably a result of alteration in plant morphology (Jones et al., 2001
Apart from COBL4, this study suggests that most genes that are specific for cellulose synthesis in secondary cell wall synthesis have been identified by forward genetics. It is clear, however, that not all genes involved in cellulose synthesis during secondary cell wall formation will be identified in our analysis. For example, KOR plays a role in cellulose synthesis during both primary and secondary cell wall formation (Lane et al., 2001
More recent work has included analysis of a large number of publicly available data sets along with our own data. Using several clustering methods, as well as the slope profile, it has been shown that eight genes exhibit very similar expression patterns and cluster together very closely. This group includes IRX1, IRX3, and IRX5 together with COBL4, a chitinase-like gene, a laccase, a GT8 family member, and a GT43 family member. It is unlikely, however, that all these genes are involved in cellulose biosynthesis. An insertion in the laccase gene results in a weak irx phenotype (Figure 5). There is, however, no associated reduction in cellulose content (Figure 7). This gene is the homolog of a poplar gene, lac3, which has been demonstrated to be essential for secondary cell wall integrity (Ranocha et al., 2002 In addition to the laccase, five other genes have an irx phenotype but have small or no alteration in cellulose content. These genes represent entirely novel aspects of secondary cell wall formation. One of these genes, At1g62990, exhibits similarity to the HD1 class of transcription factors and presumably regulates the expression of other genes that are likely to be required during the later stages of cell wall formation. At least four novel genes with homology to GTs have been identified as being essential for secondary cell wall formation. This includes two members of the GT47 family, one member of the GT43 family, and a member of the GT8 gene family. The development of a method for using metabolic fingerprinting data, generated by FTIR, to study the secondary cell wall has greatly facilitated the study of these mutants. Using FTIR to study secondary cell walls creates particular problems with generating homogenous material that is thin enough to allow the infrared light to pass though during sampling. The validity of the method developed is clearly shown by the loadings plot of PC1. All cellulose-deficient mutants are separated from the wild type by PC1, and the loading plot exhibits a remarkably good match to that of purified cellulose (Figure 9B). The FTIR data (Figure 9A) also suggest that irx7, irx8, and irx9 group together and may form part of the same metabolic pathway. These mutants are separated from the known cellulose-deficient mutant by PC2. The loading plot of PC2 exhibits many of the characteristics of purified xylan (Figure 9C). This idea is confirmed by analysis of the cell wall sugar compositions (Figure 10); all three mutants exhibit a decrease in xylose. These mutants also exhibit complex changes in other cell wall sugars, in particular irx7 and irx9 exhibit an increase in the proportion of all other cell wall sugars. The alterations in morphology of these mutants mean that this data should be interpreted with caution because the results will also reflect changes in plant morphology. It is important to note, however, that the cellulose-deficient mutant irx3-4 exhibits a similar alteration in morphology (Figure 6) but does not exhibit any decrease in xylose (Figure 10). Exactly when and where the genes identified in this study function during secondary cell wall formation await further analysis. The combination of expression analysis and reverse genetics has led to the identification of many genes that play a role in secondary cell wall synthesis. This targeted approach has led to the identification of seven novel secondary cell wall mutants. At least five of the genes identified define novel steps in secondary cell wall formation. The fact that these genes have not been identified by either biochemical studies or via forward genetic analysis emphasizes the efficiency of the approach taken in this study.
Since completing this work, it has come to our attention that Chris Somerville and colleagues have analyzed publicly available data to identify genes that are coexpressed with secondary cell wall CesA genes (Persson et al., 2005
Plant Material All plants were germinated and grown on plates containing 0.8 to 1.0% agar (w/v) and MS media with B5 vitamins for 1 to 2 weeks before transferring nine plants to a 4-inch pot containing compost with vermiculite and perlite (10:1:1). They were then grown in continuous light at 22°C in controlled environment cabinets (Percival, Perry, IA) at a light intensity of 120 to 150 µE m–2. Material for the study of stem development was grown until the inflorescence stem contained two to three expanded siliques. The siliques and flowers were removed before the stems being divided into four sections of equal length. Hypocotyls and leaves were harvested at the same developmental stage as stems. All material used was from the Columbia background other than FLAG_428B03, which was in the Ws background.
Screening of Homozygote Plants with T-DNA Insertions
Plant DNA was extracted from one to two young leaves using a previously described miniprep procedure (Guidet et al., 1991 In the case of the insertion from the FLAG collection, only a single left border primer was used FLAG-LB1 (5'-CGGCTATTGGTAATAGGACACTGG-3'). The genotype of selected plants was verified in the next generation using a single reaction containing the flanking primers and one of the left border primers. The amplification conditions used for all screening were as follows: 95°C for 5 min; 35 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 1 min; 4°C hold.
RNA Extraction and Expression Analysis Real-time quantitative PCR was performed using an ABI Prism 7000 machine (Applied Biosystems, Warrington, UK). Primers and probes, for both Taqman and SYBR green assays, were designed using Primer Express (version 1.0) (Applied Biosystems). Taqman probes were labeled with 6-carboxyfluorescein at the 5' end and tetramethylrhodamine at the 3' end. Total RNA was treated with DNAase I (Invitrogen, Paisley, UK). First strand synthesis was performed in a volume of 20 µL, containing 1 µg of total RNA with 500 ng of poly-(dT) primer and 100 units of reverse transcriptase (Promega, Southampton, UK) at 42°C for 60 min. PCR conditions for both assays were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 60 s. For SYBR green assays only, a melting curve was produced at the end of every experiment to ensure that only single products were formed. The reliability of SYBR green primers was also examined by running the products on an agarose gel to ensure that only a single band was present. PCR reactions were performed in a volume of 25 µL, containing 12.5 µL of 2x quantitative PCR Mastermix (Eurogentec, Seraing, Belgium), 25 pmol each primer and 5 pmol probe (Taqman assays only). Data analysis was performed using the Sequence Detector (version 1.7) program (Applied Biosystems). Actin2 (At3g18780) expression was used to normalize the transcript level in each sample.
Array Measurements The volcano plot shown in Figure 2B was generated using the fold change that for each probe set was calculated as (mean reading at base of stem/mean reading at the tip of the stem). This was plotted against the P value generated from the standard Student's t test for each probe set. To identify genes with expression patterns similar to the IRX3 gene (At5g17420), profile filtering with the slope mathematical algorithm (MAXDVIEW) was performed using IRX3 as the target profile. To calculate the slope metric, the profile of the mean expression level (log2) for each plant was used. This slope metric calculates the sum of the differences of the first derivatives of the expression profiles (i.e., the slopes of lines joining expression values). The profile with the smallest sum is regarded as the most similar to the target profile (IRX3).
For a gene (X), the slope profile was calculated using the following general formula:
Mutant Analysis
Metabolite Analyses For FTIR spectroscopy and gas chromatography analysis, a pool of five to seven stems was freeze-dried for 2 d and milled by rapid shaking with a ball bearing for 30 min using a tissue lyser (Qiagen) before analysis. For FTIR analysis, sample density was optimized such that good spectra with high signal-to-noise ratio were obtained. It was found that most optimal spectra were obtained at a dry weight of 50 mg/mL. Samples (5-µL aliquots) were evenly applied onto a silicon microplate containing 96 wells and oven dried at 50°C for 30 min (or until visibly dry). All samples were analyzed in quadruplicate.
A Bruker Equinox 55 FTIR spectrometer (Coventry, UK), fitted with a HTS-XT high-throughput microplate sampling accessory (Harrigan et al., 2004
To reduce problems arising from baseline shifts, Matlab (The Math Works, Natick, MA) was used to correct for CO2 vibrations by removing the peaks at 2403 to 2272 cm–1 and filling with a trend. To account for any differences in sample thickness, normalization to the total area of spectra was performed. To reduce the dimensionality of the FTIR data, PCA was performed according to the NIPALS algorithm (Wold, 1996
Analysis of monosaccharides in the noncellulose fraction of the cell wall fraction was performed using gas chromatography of alditol acetates as previously described (Reiter et al., 1993 Microarray data from this article have been deposited with the ArrayExpress data library under accession number E-MXP-265.
We are grateful to Andy Hayes and Leanne Wardleworth at the University of Manchester Microarray Facility for performing the microarray analysis. We would also like to thank Neil Taylor, Raymond Wightman, and Jon Pittman for their comments on the manuscript and Raymond Wightman for his assistance in analyzing the root microarray data (Birnbaum et al., 2003 ). Work performed by Leo Zeef and Leanne Wardleworth was funded by the Welcome Trust. David Brown was supported by a studentship from the Biotechnology and Biological Science Research Council. R.G. and J.E. are also very grateful to the Biotechnology and Biological Science Research Council for financial support.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Simon R. Turner (simon.turner{at}manchester.ac.uk). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.031542. Received February 7, 2005; Revision received May 6, 2005. accepted May 23, 2005.
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ABSTRACT
INTRODUCTION
10% of genes had any experimental data to support their function (Arabidopsis Genome Initiative, 2000
