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First published online November 18, 2005; 10.1104/pp.105.070805 Plant Physiology 139:1649-1665 (2005) © 2005 American Society of Plant Biologists Cuticular Lipid Composition, Surface Structure, and Gene Expression in Arabidopsis Stem Epidermis1,[W]Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824 (M.P., J.O., F.B.); Department of Plant Biotechnology and Agricultural Plant Stress Research Center, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500–757, Korea (M.C.S.); and Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.L.S., R.J., L.K.)
All vascular plants are protected from the environment by a cuticle, a lipophilic layer synthesized by epidermal cells and composed of a cutin polymer matrix and waxes. The mechanism by which epidermal cells accumulate and assemble cuticle components in rapidly expanding organs is largely unknown. We have begun to address this question by analyzing the lipid compositional variance, the surface micromorphology, and the transcriptome of epidermal cells in elongating Arabidopsis (Arabidopsis thaliana) stems. The rate of cell elongation is maximal near the apical meristem and decreases steeply toward the middle of the stem, where it is 10 times slower. During and after this elongation, the cuticular wax load and composition remain remarkably constant (32 µg/cm2), indicating that the biosynthetic flux into waxes is closely matched to surface area expansion. By contrast, the load of polyester monomers per unit surface area decreases more than 2-fold from the upper (8 µg/cm2) to the lower (3 µg/cm2) portion of the stem, although the compositional variance is minor. To aid identification of proteins involved in the biosynthesis of waxes and cutin, we have isolated epidermal peels from Arabidopsis stems and determined transcript profiles in both rapidly expanding and nonexpanding cells. This transcriptome analysis was validated by the correct classification of known epidermis-specific genes. The 15% transcripts preferentially expressed in the epidermis were enriched in genes encoding proteins predicted to be membrane associated and involved in lipid metabolism. An analysis of the lipid-related subset is presented.
The plant cuticle is a continuous lipophilic layer covering the surface of all epidermal cell types (Esau, 1977  ) and is one of their distinctive characteristics (Holloway, 1982; Jeffree, 1996; Kunst et al., 2005). It forms a vital hydrophobic barrier over the aerial surfaces of land plants during primary stages of development, limiting nonstomatal water loss and gaseous exchanges, controlling the absorption of lipophilic compounds, providing mechanical strength and viscoelastic properties (Baker et al., 1982; Hoffmann-Benning and Kende, 1994; Riederer and Schreiber, 2001), preventing organ fusion during development (Lolle et al., 1998; Sieber et al., 2000), as well as protecting the plant from nonbiotic and biotic stressors from the environment (Schweizer et al., 1996). The cuticle is composed of cutin (Kolattukudy, 2001), a polymer of fatty acid derivatives, which abuts the cell wall and is embedded in and covered with a mixture of ubiquitous aliphatic compounds (mainly C24-C34 alkanes, alcohols, and ketones) called cuticular waxes (Kunst and Samuels, 2003), as well as minor, extremely diverse, compounds (Jetter, 2000; Jetter et al., 2002; Vermeer et al., 2003). Due to their close physical association, it is difficult to distinguish between the relative contribution of the cutin matrix and cuticular waxes to the physical properties and the biological functions of the cuticle. However, experimentally, the cutin and cuticular waxes are usually considered and analyzed separately, as the waxes are easily extracted in organic solvents while the cutin polymer remains insoluble.
The cutin matrix is composed mainly of C16 and C18 hydroxy and epoxy fatty acid monomers. Minor amounts of aromatic compounds are also present (Kolattukudy, 1980
Knowledge on the biosynthesis of cutin is based largely on a few early studies, mostly done with broad bean (Vicia faba) leaves. These studies indicated that cutin biosynthesis involves
Cuticular waxes of the Arabidopsis shoot are found both above the cutin matrix (epicuticular) as well as embedded in the cutin (intracuticular). The chemical compositions of the Arabidopsis stem and leaf waxes have been investigated in both wild-type and eceriferum (glossy) plant lines (Hannoufa et al., 1993
The cuticle is known to be synthesized at very early stages of embryo and organ development (Szczuka and Szczuka, 2003
The Length of Epidermal Cells Increases More Than 80-Fold during Stem Elongation The bolting inflorescence stem of Arabidopsis provides an opportunity to study the biosynthesis of cuticular lipids by the epidermis during cell expansion. When growth was measured over a 24-h period for 9- to 11-cm-long bolting stems, the total length of the stems increased by 3.7 cm ± 0.3 (mean ± SE, n = 6). The elongation of apical, but not basal, 0.5-cm segments was observed (Fig. 1). Most of the increase in total length (about 85%) occurred in the top 3-cm segment of the stem, 15% in the middle part, while no increase was detected below 7 cm from the top. The elongation rate was greater nearer to the apex and maximal in the first 0.5-cm portion. Given typical mean diameters of the stem segments, this elongation corresponds to an increase in epidermis surface area of 60 mm2/24 h in the top 3 cm of the stem and 8 mm2/24 h in the middle 3-cm segment.
Growth of epidermal cells was followed with cryo-scanning electron microscopy (SEM) and confocal scanning laser microscopy with propidium iodide staining of the cell wall. Near the shoot apex (Fig. 1A), the cells of the epidermis were nearly isodiametric, but they soon underwent anisotropic growth to elongate greatly in the axial dimension (Fig. 1B). At the base of a 10-cm stem, the cells were elongated and trichomes were seen (Figs. 1C and 2C). No trichomes were present in the middle and top segments (Fig. 2, A and B). When the cell dimensions were quantified using cryo-SEM, the length of the cells increased from 6 µm ± 0.3 (mean ± SE, n = 24) to 532 ± 44 µm (mean ± SE, n = 14), an increase of 87-fold. In contrast, cell widths did not increase significantly.
The Load and Composition of Waxes Remain Constant along the Stem To test whether the biosynthesis of cuticular waxes is coordinated with the rapid expansion of the stem epidermal cells, cryo-SEM and gas chromatography (GC) with mass spectroscopy (MS) and flame ionization detection (FID) were used. Cryo-SEM provided information about the epicuticular wax crystals, while the GC-FID and GC-MS provided quantitative wax load and composition information. Epicuticular wax crystals were found over the entire stem surface, both vertical rods, tubes, longitudinal bundles of rodlets, and horizontal, reticulate plates (Fig. 2, D–F). In the region of most rapid elongation, the cuticle showed striations and crystals distorted along the axis of elongation. The cuticle around stomata did not have epicuticular crystals, only a smooth epicuticular film (Fig. 2, E and F).
Qualitative and quantitative chemical analyses were performed to compare the cuticular wax in the top, middle, and basal 3-cm segments of the bolting inflorescence stem. The wax load, expressed on a per-unit-area basis, was constant between these segments of the stem (Fig. 3). The composition along the stem did not vary significantly (Fig. 4a), with C29 alkane, ketone, and secondary alcohols predominating, and the overall composition is similar to that reported previously (Hannoufa et al., 1993
Polyester Composition Is Constant along the Stem, But the Load Decreases at the Base
To quantify the polyester monomers in the stem, the method bypassing cuticle isolation we have reported earlier (Bonaventure et al., 2004
We also determined the fatty acid content of the intracellular acyl lipids (membrane and storage lipids) of epidermal peels. When compared to the amounts of fatty acid derivatives measured in waxes and polyesters, the intracellular lipids represented less than one-half of the total cell lipids (Fig. 3). The fatty acid composition of the stem epidermis was found to be similar to that of the whole stem (Supplemental Fig. 1). In all stem segments, the ratio of surface lipids to epidermal intracellular acyl lipids was about 3:2 in mass (Fig. 3). This means that the epidermal cells of the elongating stem top need to have machinery capable of exporting onto the surface more than one-half of the approximately 75 µg of fatty acids produced per square centimeter of epidermis. Given an average rate of expansion of 0.6 cm2/24 h for the stem top, the accumulation of total fatty acids in the corresponding epidermis can be estimated to be around 1.9 µg cm–2 h–1 and the net rate of export 1.1 µg cm–2 h–1 (assuming that fatty acid accumulation commences when cell elongation starts). This represents a considerable flux of hydrophobic compounds, possibly partially polymerized, that must go through the plasma membrane and the aqueous cell wall. Since this specialized machinery of synthesis and export of surface lipids is specific to the epidermis, it is reasonable to assume that most of the genes encoding the enzymes, transporters, and other proteins that are part of the machinery will be transcriptionally up-regulated in the epidermis compared to the other tissues of the stem. Expression of these genes could be stronger in the elongating top epidermis or restricted to it. The above rationale provided the basis for the microarray analysis described below.
To investigate the mRNA levels of genes expressed in the epidermis of the apical and basal segments of the stems, we used the GeneChip Arabidopsis ATH1 Genome Array (Affymetrix) that represents 22,748 probe sets covering approximately 23,750 Arabidopsis genes (Redman et al., 2004
Transcripts from a total of about 13,000 genes were detected in the stem segments (Supplemental Table I), which is very close to the value of 60% of expressed genes in the stem reported recently (Ma et al., 2005 ). The Affymetrix Microarray Suite 5.0 (MAS 5.0) software identified more than 3,000 genes whose expression was increased in the epidermis compared to the whole stem (data not shown). Using some statistical parameters calculated by MAS 5.0 and a conservative cutoff value of 2.0 for the lower estimate of the epidermis-to-stem gene expression ratio (see "Materials and Methods"), we identified about 1,900 genes up-regulated in the stem epidermis. This list breaks down into three subsets of about 600 each (Supplemental Table I): up-regulated in the epidermis of stem segments (top and base), in top segments alone (elongating), and in basal segments alone (nonelongating). These three subsets of transcripts (see Supplemental Table III for the identity) are likely to be enriched in genes related to various aspects of epidermal biology, such as trichome formation for the basal-only subset or cell elongation for the apical-only subset. A list of the 40 most up-regulated, highly expressed transcripts in the apical-only subset is given in Table II as an example. Among these, several encode proteins belonging to expected categories, such as enzymes of cuticle biosynthesis or defense against pathogens. Interestingly, almost one-third of the proteins have putative regulatory functions, such as the seven putative protein kinases, a group highly represented in the epidermis (Fig. 6). This list is thus likely to yield good candidates for the cellular signaling pathways involved in the division, differentiation, or elongation of apical epidermal cells. Another important group representing about 40% of the total in Table II is proteins from families completely uncharacterized or whose function is so far unclear. The identification of these proteins as epidermis up-regulated should provide helpful clues in the determination of their function in the cell. Finally, the list contains a few proteins related to cell wall metabolism that could indicate the existence in the epidermis of a specific composition or structure of the external cell wall to which the cuticle may be anchored and through which surface lipids must be transported during cuticle formation.
The Epidermis Up-Regulated Set of Genes Is Enriched in Candidates Encoding Proteins Predicted to Be Membrane Associated, Extracellular, or Related to Stress/Stimulus or Lipid Metabolism
The subcellular location of the synthesis of waxes and cutin monomers and the mechanism of secretion are mostly unknown. However, due to the hydrophobicity of these molecules, it is likely to involve membrane-associated enzymes and (at least in the case of waxes) transporters like CER5 (Pighin et al., 2004
Among the 620 genes known or thought to be involved in acyl lipid metabolism in Arabidopsis (Beisson et al., 2003
For example, the new candidates for wax synthesis include specific members of multigenic families for which some members have already been previously shown to be involved in wax synthesis, e.g. the ketoacyl-CoA synthase (KCS) family. Figure 7 summarizes the level of gene expression and the epidermis-to-stem gene expression ratios found for the 20 members of the KCS family (out of 21) present on the ATH1 array. It can be clearly seen that there are five uncharacterized KCS genes showing an epidermal up-regulation higher than the CER60 gene, which is known to affect wax synthesis. These genes are thus good candidates for the putative specific elongases thought to be responsible for each of the multiple elongation steps of wax synthesis. In addition, and as expected, the KCS FAE1 isoform (At4g34520) that is responsible for the elongation of fatty acids for storage lipids in seeds is not among the epidermis up-regulated KCS genes.
Using rapidly elongating Arabidopsis stems, we have performed measurements of the elongation of epidermal cells in conjunction with quantitative analyses of the cuticular lipids. In brief, the results indicate that polyester and waxes are deposited in the elongating apical part of the stem at a rate that allows the cuticle to keep a constant load and composition of surface lipids, despite the fast rate of elongation of the epidermal cells. A major metabolic function of these epidermal cells is extracellular lipid synthesis as illustrated by the fact that more lipid is transported out of the cell than remains in membrane glycerolipids (Fig. 3) and by the enriched expression of transcripts for many enzymes of lipid metabolism (Fig. 6). These isoforms preferentially expressed in the epidermis are thus strong candidates for roles in wax and cutin synthesis (Table III).
The wax analysis shows that the amounts of individual wax constituent do not vary significantly along the stem (Figs. 2–4 Because surface area expansion is greatest and unequal in the top segment (Fig. 1), the constant wax load and composition found both along the stem and within the top segment implies that the rate of net deposition of wax is strictly synchronized with epidermal cell expansion and largely limited to the top zone of the stem, i.e. early on during development. All wax constituents are therefore formed at similar rates and deposited at the same time in the expanding epidermal cells. Expression of all proteins involved in wax biosynthesis is likely synchronized and must be highly up-regulated during rapid epidermal cell expansion either transcriptionally or posttranscriptionally.
Constant wax loads on the lower segments of the stems might reflect a dynamic equilibrium between wax accumulation and wax loss by erosion or back transport (Jetter and Schaffer, 2001
The fact that the highest polyester loads were observed in the youngest part of the stem is consistent with previous observations that the major cutin synthesis occurs in young tissues (Kolattukudy, 1970
The wax load of the leaf is about 2 times lower than the polyester load: 0.75 µg/cm2 (data not shown) versus 1.5 µg/cm2 (this study). Since the average polyester load of the stem is 6 µg/cm2, one would expect about 3 µg/cm2 of waxes to be present on the stem surface (assuming the wax-to-polyester ratio of the leaf is the same). However, a much higher wax load was found in the stem: on average, 32 µg/cm2 (this study). The difference is thus likely to come from the abundant epicuticular waxes of the stem, as evidenced by the wax crystals seen in the ultrastructure of the stem surface (Fig. 2), but not in that of the leaf surface (data not shown). It can therefore be estimated that epicuticular wax crystals represent about 90% of the total wax load in the stem. Finally, it should be stressed that the actual ratio of wax to matrix in the cuticle could be different from the wax-to-polyester ratio because the amount of cuticle lipid that cannot be depolymerized is unknown.
About 1,900 genes (15% of the genes detected in the stem) were identified as preferentially expressed in the epidermis of top and/or basal segments of stems. The majority of these genes (70%) had a transcript epidermis-to-stem ratio that fell in the 2.0 to 4.0 range, while 30% had a ratio greater than 4.0 (Supplemental Table II). This is consistent with what has been observed in maize for transcripts accumulating to at least 2-fold higher levels in epidermal cells than in vascular tissues (Nakazono et al., 2003
The epidermal peel approach used here is based on the harvest of all epidermal cell types and thus transcripts specifically expressed in less abundant cell types, like guard cells, will be diluted by more abundant cell types. The candidate genes obtained here are therefore more likely to be genes expressed in all epidermal cells of a particular stem region. However, the example of the guard cell-specific MYB transcription factor At1g08810 (Cominelli et al., 2005 To identify gene candidates for wax and cutin synthesis, we have focused mostly on the epidermis-to-stem gene expression ratio for the top stem. Even if there is a turnover of surface lipids in the epidermal cells of the nonelongating stem base, the rapid elongation and the high wax-polyester content observed in the stem top indicate that this machinery must be more active in the epidermis of the top than in the epidermis of the base. In the case of transcriptional regulation, the transcripts of the corresponding genes are thus expected to be more abundant in the epidermis of the top segment than in the epidermis of the basal segment of the stem. However, it is striking that many genes involved in wax synthesis, such as the major elongase-condensing enzyme (CER6) that supplies alkane and alkane-derived (secondary alcohols and ketones) waxes, are still highly expressed or even up-regulated in the lower stem epidermis (Table III), where we would expect very low net flux into these components. It is possible that there is a turnover of the wax and/or cutin components in the nonelongating parts of the stems, requiring continued synthesis to maintain constant loads. An alternative hypothesis is that many of the genes involved in surface lipid metabolism could be expressed in the epidermis of all stem segments, but strongly controlled at the posttranscriptional level, depending on the elongation rate of the cell. This control might, for example, include a system that senses the wax-cutin loads to provide feedback to biosynthesis.
Lipid-related genes of Table III may not all be involved in surface lipid metabolism. Some are clearly related to cellular signaling, possibly playing a role in epidermal cell differentiation, elongation, and/or interaction with the environment. It is not likely that the signaling genes up-regulated in the epidermis are induced by wounding during the harvest of epidermal peels because peels were frozen in liquid nitrogen immediately and also several essential and well characterized genes of wound response (Delessert et al., 2004
Many reactions in the pathways for the synthesis of wax components remain obscure and some proteins identified by mapping of wax mutants cannot be assigned a clear molecular function (Kunst and Samuels, 2003
In addition to the KCS family (Fig. 7), another example of the utility of Table III for large gene families is provided by analysis of the expression of the subfamilies of putative lipid transfer proteins (LTPs) that have been suggested to be involved in wax-polyester monomer transport through the cell wall. In Arabidopsis, there are 72 putative LTPs that were classified into eight types based on the conserved Cys pattern (Beisson et al., 2003
Our list of candidates for wax synthesis (Table III) includes uncharacterized members of the KCS family, such as At5g16280 or At2g43760, that have been recently identified as highly expressed in the aerial parts of 15-d-old expanding seedlings (Costaglioli et al., 2005
Concerning cutin biosynthesis, putative fatty acid hydroxylases and acyltransferases are proteins of special interest. The protein affected in the Arabidopsis att1 cutin mutant (Xiao et al., 2004
The polyester synthases responsible for the assembly of cutin chains remain completely unknown and the acyltransferases listed in Table III are therefore potential candidates for this function. It is not likely that the epidermis up-regulated acyltransferases listed in Table III (1-acylglycerol-3-P acyltransferase [LPAT5] and glycerol-3-P acyltransferase [GPAT4], etc.) are involved in housekeeping membrane biogenesis because the cells of the tissues underlying the epidermis are also elongating and need to synthesize membrane lipids at about the same rate as epidermal cells. Thus, it is more likely that the acyltransferases of Table III are somehow involved in surface lipid synthesis. Indeed, LPAT activity was not detected for the recombinant LPAT5 (Kim et al., 2005
The accumulation of cuticular lipids by the plant epidermis has been studied by a combined quantitative and qualitative analysis of the surface lipids and of the transcripts present in expanding epidermal cells during the elongation of Arabidopsis stems. The high loads and constant composition of polyesters and waxes found in the elongating top and middle zone of stems indicated that the synthesis and the secretion of these two components of the cuticle are highly coregulated to keep up with the pace of rapid epidermal cell expansion. Our microarray approach identified a subset of the genome (about 15% of the genes detected in the stem) that is preferentially expressed in the epidermis and provided a list of potential gene candidates for the machinery of assembly, secretion, and synthesis of surface lipids that can be tested by reverse-genetics approaches. This dataset should accelerate progress toward elucidation of the wax biosynthesis pathways and aid in unraveling the biosynthesis of cutin.
Plant Material and Growth Conditions The wild-type ecotype Col-0 of Arabidopsis (Arabidopsis thaliana) was used for all experiments. Seeds were stratified for 3 to 4 d at 4°C and plants were grown on a mixture of soil:vermiculite:perlite (1:1:1) under white fluorescent light (80–100 µE m–2 s–1) in an 18-h-light/6-h-dark photoperiod. The temperature was set at 20°C to 22°C and the relative humidity at 50% to 70%.
Each harvested 10- to 11-cm-long primary stem was cut into 3-cm-long segments (3 cm from the base of the stem, 3 cm from the base of the inflorescence, and 3 cm in the middle of the stem). The cauline leaves or siliques were cut off and the stem segments were kept in liquid nitrogen until use. Diameters were measured using transverse hand sections of stems and light microscopy and, in addition, diameters of intact stems were measured with cryo-SEM. Typical diameters were around 0.6 mm for the basal and middle segments of the stems and around 0.4 mm for the apical segment. The surface areas of the segments were calculated using an average diameter for each segment and assuming cylinder geometry. In order to detect any significant departure from these standard diameters in the samples used for routine lipid analysis, the stem segments used were photocopied prior to chemical analysis and diameters were estimated from magnified copies.
Segments from the apical 1 cm of stem were mounted onto cryo-SEM stubs with 25% dextran and plunged into liquid nitrogen. Frozen samples were transferred into an Emitech K1250 cryo-system, where water was sublimed for 30 min at –77°C and subsequently sputter coated with gold for 2.5 min at 35 mA. The coated samples were viewed with a Hitachi S4700 field emission SEM using an accelerating voltage of 2 kV and a working distance of 12 mm.
Stem segments of Arabidopsis plants were immersed in a solution of propidium iodide (100 µg/mL; Sigma) and examined with a Radiance 2000 confocal laser-scanning microscope (Bio-Rad). The excitation wavelength was 568 nm with the emission filter set at 580 to 600 nm. All confocal images obtained were processed with ImageJ (http://rsb.info.nih.gov/ij) and Photoshop 5.0 (Adobe Systems) software.
Thirty to 100 3-cm-long segments from 10- to 11-cm-long primary stems were used for each replicate. The analysis of the polyesters was conducted according to Bonaventure et al. (2004)
The cuticular waxes were extracted by immersing whole inflorescence stems or 3-cm-long segments of stems two times for 30 s into 5 mL of chloroform (CHCl3) at room temperature. Both CHCl3 solutions were combined and n-tetracosane was added as internal standard. The solvent was removed under a gentle stream of nitrogen gas, and the remaining wax mixture was redissolved in 1 mL of CHCl3 and stored at 4°C until used. The extracted area was determined by measuring the height and diameter of the stems assuming cylinder geometry. Prior to GC analysis, chloroform was evaporated from the samples under a gentle stream of nitrogen gas while heating to 50°C. Then the wax mixtures were treated with bis-N,N-(trimethylsilyl) trifluoroacetamide (BSTFA; Sigma) in pyridine (30 min at 70°C) to transform all hydroxyl-containing compounds into the corresponding trimethylsilyl derivatives. The qualitative composition was studied with capillary GC (5890 N; column 30 m Hewlett-Packard-1, 0.32-mm i.d., film thickness = 0.1 µm; Agilent) with He carrier gas inlet pressure regulated for constant flow of 1.4 mL min–1 and a MS detector (5973 N; Agilent). GC was carried out with temperature-programmed injection in a 50°C oven, 2 min at 50°C, raised by 40°C min–1 to 200°C, held for 2 min at 200°C, raised by 3°C min–1 to 320°C, and held for 30 min at 320°C. The quantitative composition of the mixtures was studied using capillary GC with FID under the same GC conditions as above, but H2 carrier gas inlet pressure regulated for constant flow of 2 mL min–1. Single compounds were quantified against the internal standard by automatically integrating peak areas.
Epidermal peels were manually dissected as a thin transparent film using thin forceps under a dissecting microscope (Fig. 5). For each freshly cut 3-cm-long stem segment of 10- to 11-cm-long primary stems, peels were collected, immediately frozen in liquid nitrogen, and stored at –80°C for RNA isolation or fatty acid analysis.
Epidermal peels from about two to three stem segments were heated at 85°C for 1.5 h in 1 mL of methanol containing 5% H2SO4 (v/v) with triheptadecanoylglycerol and
Total RNAs were isolated from epidermal peels or whole-stem segments using the RNeasy kit (Qiagen). Double-stranded cDNA was synthesized from approximately 15 µg of total RNA by using a SuperScript double-stranded cDNA synthesis kit (Invitrogen) with oligo d(T) primer containing a T7 RNA polymerase promoter sequence at its 5' end (GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGC-GG-(dT)24-3'; Genset). After synthesis of the second-strand cDNA, the cDNA was extracted with phenol-chloroform-isoamylalcohol, precipitated with ethanol, and resuspended in ribonuclease-free water. Labeled cRNA was generated from cDNA by in vitro transcription using a bioarray high-yield RNA transcript-labeling kit (Enzo Diagnostics) following the manufacturer's instructions and incorporating biotinylated CTP and UTP. After purification of biotin-labeled cRNA using an RNeasy column, 15 µg of the labeled cRNA were fragmented to a size of 35 to 200 bases by incubating at 94°C for 35 min in fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate).
The fragmented cRNA was used for hybridization of Arabidopsis ATH1 gene chips (Affymetrix), which was performed in the Research Technology Support Facility at Michigan State University. Hybridization, washing, and detection of labeled cRNA were done as recommended by Affymetrix. Image acquisition and global data scaling were performed with Affymetrix MAS 5.0 software. The signal intensity values were scaled to a mean of 500 for each chip. The signal intensities indicated in the "Results" section are the mean from two biological replicates. The ratios of gene expression are the mean of four ratios of epidermis versus whole-stem segments calculated using multiple pairwise comparisons of epidermis versus stem. For each gene, a 90% confidence interval was calculated for the epidermis-to-stem ratio using a T-score and a degree of freedom of 3. Genes were considered as up-regulated in the epidermis if they were called present by the MAS 5.0 software in both biological replicates, and if the lower bound of the 90% confidence interval for the epidermis-to-stem gene expression ratio was
We would like to thank Sherry Wu, Dale Chen, Swati Pardhi, and Khedidja Beldjilali for excellent technical help, as well as Dr. Kathy Schmid, Dr. Gustavo Bonaventure, and Dr. Yonghua Li for helpful discussions. Received August 31, 2005; returned for revision September 22, 2005; accepted September 23, 2005.
1 This work was supported by the Dow Chemical Company, Dow AgroSciences, a U.S. Department of Agriculture National Research Initiative grant (grant no. 05–35318–15419 to M.P. and F.B.), the Canadian National Science and Engineering Research Council of Canada Special Research Opportunity Grant (grant no. 305360–04 to A.L.S., R.J., and L.K.), and the Plant Signaling Network Research Center and the Agricultural Plant Stress Research Center Grant of the Korea Science and Engineering Foundation (grant no. R11–2001–0920301–0 to M.C.S.).  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.plantphysiol.org) is: Fred Beisson (beisson{at}msu.edu).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.070805. * Corresponding author; e-mail beisson{at}msu.edu; fax 517–353–1926.
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ABSTRACT
RESULTS
-hydroxy fatty acids, but dicarboxylic acids (Bonaventure et al., 2004
-ketoacyl-CoA synthase; LACERATA, fatty acid 
2.0 in stem tops and stem bases (90% confidence level). The complete charts showing all functional categories for each type of annotation are available from Supplemental Figure 2.
