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First published online May 13, 2009; 10.1104/pp.109.137497 Plant Physiology 150:1174-1191 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Functional Characterization of the Arabidopsis β-Ketoacyl-Coenzyme A Reductase Candidates of the Fatty Acid Elongase1,[W],[OA]Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts AL5 2JQ, United Kingdom (F.B., R.P.H., J.A.N.); University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (X.W., F.L., H.Z., L.K.); and Donald Danforth Plant Science Center, St. Louis, Missouri 63132 (J.E.M.)
In plants, very-long-chain fatty acids (VLCFAs; >18 carbon) are precursors of sphingolipids, triacylglycerols, cuticular waxes, and suberin. VLCFAs are synthesized by a multiprotein membrane-bound fatty acid elongation system that catalyzes four successive enzymatic reactions: condensation, reduction, dehydration, and a second reduction. A bioinformatics survey of the Arabidopsis (Arabidopsis thaliana) genome has revealed two sequences homologous to YBR159w encoding a Saccharomyces cerevisiae β-ketoacyl reductase (KCR), which catalyzes the first reduction during VLCFA elongation. Expression analyses showed that both AtKCR1 and AtKCR2 genes were transcribed in siliques, flowers, inflorescence stems, leaves, as well as developing embryos, but only AtKCR1 transcript was detected in roots. Fluorescent protein-tagged AtKCR1 and AtKCR2 were localized to the endoplasmic reticulum, the site of fatty acid elongation. Complementation of the yeast ybr159  mutant demonstrated that the two KCR proteins are divergent and that only AtKCR1 can restore heterologous elongase activity similar to the native yeast KCR gene. Analyses of insertional mutants in AtKCR1 and AtKCR2 revealed that loss of AtKCR1 function results in embryo lethality, which cannot be rescued by AtKCR2 expression using the AtKCR1 promoter. In contrast, a disruption of the AtKCR2 gene had no obvious phenotypic effect. Taken together, these results indicate that only AtKCR1 is a functional KCR isoform involved in microsomal fatty acid elongation. To investigate the roles of AtKCR1 in postembryonic development, transgenic lines expressing RNA interference and overexpression constructs targeted against AtKCR1 were generated. Morphological and biochemical characterization of these lines confirmed that suppressed KCR activity results in a reduction of cuticular wax load and affects VLCFA composition of sphingolipids, seed triacylglycerols, and root glycerolipids, demonstrating in planta that KCR is involved in elongation reactions supplying VLCFA for all these diverse classes of lipids.
Very-long-chain fatty acids (VLCFAs) with chain lengths between C20 and C34 are essential and ubiquitous constituents of eukaryotic cells. They are most commonly found as building blocks of sphingolipids, but they are also important components of glycerophospholipids, triacylglycerols, sterol esters, and wax esters. Depending on their cellular and tissue localization and chain length, VLCFAs perform a wide range of physiological and structural roles. For example, they are involved in the stabilization of highly curved nuclear pore membranes (Schneiter et al., 1996  , 2004) and the creation of membrane domains involved in lipid and protein trafficking (Dickson et al., 2006; Toulmay and Schneiter, 2007) and cell signaling (Leonard et al., 2002). In mammals, C20 VLCFAs also serve as precursors of biologically active eicosanoids implicated in inflammation responses (Karimi et al., 2007) and are critical components required for myelin production in the central nervous system (Poulos et al., 1992) as well as for normal functioning of photoreceptor cells in the retina (Zhang et al., 2001; McMahon et al., 2007). Furthermore, VLCFAs play an important structural role in skin barrier function (Wertz and Downing, 1983; Westerberg et al., 2004; Vasireddy et al., 2007). Similarly, in higher plants, VLCFAs give rise to cuticular waxes, lipid molecules deposited on the primary plant surfaces that serve as a barrier preventing excessive water loss and pathogen attack (Jenks et al., 1994), and mediate plant-insect interactions (Eigenbrode and Espelie, 1995) and pollen-stigma signaling required for fertilization (Preuss et al., 1993). Modified VLCFAs are also present in suberin, an extracellular plant polyester that controls water and solute fluxes in plant tissues (Bernards, 2002; Franke and Schreiber, 2007).
VLCFAs are formed by elongation of C16 and C18 fatty acids by endoplasmic reticulum (ER) membrane-bound enzymes (Cinti et al., 1992
Biochemical studies of the VLCFA biosynthesis and analyses of mutants with defects in fatty acid elongation revealed that multiple FAEs with unique substrate chain length specificities are involved in generating the complete array of C20 to C34 acyl chains required by eukaryotic cells (Sprecher, 1974
In contrast to the KCSs that have strict substrate and tissue specificities, the other three enzymes of the fatty acyl elongase, the KCR, the HCD, and the ECR, are believed to be common to all VLCFA biosynthetic reactions, have (presumed) broad substrate specificities, and are expressed in all cells (Millar and Kunst, 1997
Two KCR orthologs have also been annotated in the Arabidopsis genome (Dietrich et al., 2005
Arabidopsis Has Two KCR Genes
The isolation of the yeast KCR gene YBR159w resulted in the identification of a single Arabidopsis ortholog, At1g67730 (Beaudoin et al., 2002
Isolation of Mutants Disrupted in the AtKCR Genes
To investigate the in planta function of the two predicted AtKCR enzymes, we initiated a reverse genetic approach by identifying insertional mutants disrupted in AtKCR1 and AtKCR2 genes. A single line, RATH12-5282-1-G, obtained from Plant Functional Genomics Research Group of RIKEN Genomic Sciences Center (Kuromori et al., 2004
Seeds from siliques developing on heterozygous plants were further analyzed by differential interference contrast (DIC) microscopy (Fig. 2 ) to compare the progression of embryogenesis in these two seed types. In young siliques, all the seeds were white and contained embryos at the globular stage. However, in older siliques, only wild-type-appearing green seeds continued to develop normally with embryos proceeding from globular stage (Fig. 2A) to maturation (Fig. 2F) over the course of 8 d after flowering. In contrast, embryos from white seeds arrested at the globular stage (Fig. 2, G–L).
We also obtained two T-DNA insertion lines in the AtKCR2 gene, SALK_096487 (Alonso et al., 2003 ) and SAIL _536_H04 (Sessions et al., 2002), with T-DNA insertions in the second and fourth exons, respectively (Fig. 1B). No visible developmental phenotypes were observed in homozygous atkcr2 progenies from either T-DNA mutant line.
To determine if the two apparent Arabidopsis homologs are both constitutively expressed and to delineate their expression domains, we performed RT-PCR analyses and β-glucuronidase (GUS) assays in transgenic plants transformed with AtKCR1promoter-GUS and AtKCR2promoter-GUS constructs. As shown in Figure 3 , both genes are expressed in green siliques, flowers, inflorescence stems, and leaves, with the highest expression levels detected in siliques and flowers. High AtKCR1 transcript levels were also present in roots, whereas AtKCR2 transcript was almost undetectable in root tissues. These expression results were confirmed by GUS activity assays (Fig. 4 ). In addition, AtKCR1promoter- and AtKCR2promoter-directed GUS activity was also detected in embryos of different stages (Fig. 4, A–F and L–R) and young developing seedlings (Fig. 4, G and S) but was absent from mature seeds (Fig. 4, K and W) and stem bases (data not shown).
To compare the subcellular localization of the AtKCR1 and AtKCR2, yellow fluorescent protein (YFP)-AtKCR1 and cyan fluorescent protein (CFP)-AtKCR2 fusion constructs were initially transiently expressed in tobacco (Nicotiana tabacum) under the control of the 35S promoter. Visualization of tobacco leaves revealed that both proteins were localized in the ER (data not shown). We also introduced both fluorescent protein fusion constructs into Arabidopsis. To ensure that the YFP-AtKCR1 is fully functional, we expressed it in AtKCR1/atkcr1 heterozygous plants. Sixteen out of 20 recovered independent transgenic lines had siliques with only green wild-type-looking seeds and no white seeds, indicating complementation (Fig. 5B ) and demonstrating that the mutation in the AtKCR1 gene is responsible for the embryo lethality. In all the complemented YFP-AtKCR1 transgenic lines examined by confocal microscopy, YFP fluorescence labeled a reticulate network typical of the ER (Fig. 5C). Because the disruption of AtKCR2 did not result in a detectable phenotype, we could not verify if the CFP-AtKCR2 fusion protein is active by complementation of a loss-of-function mutant, but we decided to proceed with its localization. For this purpose, we transformed the 35Spromoter-CFP-AtKCR2 fusion construct into wild-type Arabidopsis. This experiment revealed that the AtKCR2 protein also resides in the ER (Fig. 5G).
Only AtKCR1 Complements the Yeast ybr159 Mutant and Embryo Lethality of the Arabidopsis atkcr1 Mutant
AtKCR1 has been previously shown to restore heterologous elongase activity of the yeast ybr159
The fact that atkcr1 loss-of-function mutation is embryo lethal suggests that either AtKCR2 is not a functional KCR or that AtKCR2 is not expressed during embryogenesis. GUS activity assays in transgenic plants transformed with the AtKCR2promoter:GUS construct (Fig. 4, L–W) and global gene expression analyses during Arabidopsis embryogenesis (Schmid et al., 2005 ) indicate that AtKCR2 is transcribed at all times during embryo development, albeit at a much lower level than AtKCR1. To determine if the low expression level of AtKCR2 was the reason why AtKCR2 could not support embryogenesis in the absence of AtKCR1 or whether the AtKCR2 was indeed inactive, we expressed AtKCR2 using the AtKCR1 promoter in the AtKCR1/atkcr1 background. In contrast to AtKCR1, which complemented the atkcr1 mutant (Fig. 5B), AtKCR2 could not rescue the embryo lethality of atkcr1 even when expressed in the AtKCR1 manner behind the AtKCR1 promoter (data not shown), confirming that it is not a functional KCR isoform.
Embryo lethality prevented further phenotypic analysis on homozygous atkcr1 lines, and no developmental differences or changes in seed VLCFA composition or cuticular wax accumulation were detected in AtKCR1/atkcr1 heterozygotes in comparison with wild-type plants. We therefore generated transgenic plants in which AtKCR1 was down-regulated by RNAi to characterize their morphological and biochemical phenotypes and determine the role of AtKCR1 in the different VLCFA metabolic pathways. To test the specificity of our RNAi construct and ensure that the phenotypes that we describe are due to suppression of AtKCR1 alone, we performed RT-PCR analysis of the RNAi suppressed plants using mRNA prepared from flowers that showed high KCR1 and KCR2 expression in the wild type in previous assays (Fig. 3). This experiment confirmed that the RNAi construct specifically down-regulates the expression of only AtKCR1, but not AtKCR2 (Supplemental Fig. S2). Twenty T1 RNAi transformants were chosen for further evaluation. These plants displayed a variety of phenotypes, including growth retardation and fused rosette leaves (Fig. 7, A and D ). Individuals with the most severe morphological phenotype never formed rosettes, but instead produced incompletely developed leaf-like structures (Fig. 7A). They were also highly sensitive to dehydration and needed to be cultivated in a controlled environment with atmospheric humidity above 85% in wet soil at all times. Eleven T1 plants failed to grow to maturity, developed necrotic spots, and died. From the nine remaining plants, three (designated 4, 6, and 7) required mechanical organ separations to allow distorted leaves and inflorescence stems to develop (Fig. 7, C and D).
Soil-grown T2 progeny of these AtKCR-RNAi primary transformants exhibited two predominant phenotypes: glossy inflorescence stems (Fig. 7G) or twisted and curled fiddlehead (fdh)-like stems that were not obviously glossy (Fig. 7H). Both glossy and nonglossy plants were present in the progeny of the 4, 6, and 7 lines. These three lines, together with a fourth line (line 9, showing a less extreme phenotype), were selected for a detailed morphological and biochemical characterization. Examination of the flowers from glossy plants revealed an unusual bushy stigma morphology with longer papillae and stamen with shorter filaments unable to position anthers at the height of the stigma, similar to that reported for cer10 (Zheng et al., 2005 ; Fig. 7E). These plants also produced short and crooked siliques, containing mostly aborted seeds, suggesting a problem with pollen viability/fertility (Fig. 7F). Cross-pollination of glossy plants with wild-type pollen resulted in longer siliques and a considerably increased number of seeds produced (data not shown). Similar short and crooked siliques were also found on the nonglossy plants. Interestingly, mature dried seeds collected from both glossy and nonglossy AtKCR-RNAi plants were considerably larger than seeds collected from wild-type plants grown under identical conditions (Table II
).
An aspect of the AtKCR-RNAi phenotype that has not previously been reported in other mutants impaired in VLCFA biosynthesis is the abnormal root morphology of the glossy plants (Fig. 7J). When grown horizontally on plates, the roots of these plants ran on the surface of the gel matrix, lacked lateral branches, and had dramatically reduced root hairs (Fig. 7J, inset). Closer observation revealed that lateral root primordia were formed but did not elongate. In contrast, roots of nonglossy plants with curly inflorescences appeared wild-type. To determine if the phenotypes observed were due to AtKCR1 RNAi suppression, AtKCR1 transcript levels in transgenic lines were determined by RT-PCR in 15-d-old-seedlings and in a number of tissues from 8-week-old T2 plants. As shown in Figure 8 , in the developing seedlings and in all of the tissues analyzed, the levels of AtKCR1 transcripts were lower than in the wild type. The reduction in KCR transcripts was more pronounced in the glossy plants compared with the more limited reduction observed in the nonglossy lines. Within each line and for each plant phenotype (glossy or nonglossy), levels of AtKCR1 transcripts were similar in all tissues. These results show a clear correlation between reduced levels of AtKCR1 transcripts and increased severity of the phenotype in the T2 generation.
We also overexpressed the AtKCR1 cDNA under the control of 35S promoter. About 50% of transgenic lines displayed abnormal phenotypes, similar to those of the AtKCR1-RNAi lines, including dwarfism, curly cauline leaves, fused flower buds, and bright green glossy stems (Supplemental Fig. S3, A–C). To investigate if these phenotypes were caused by AtKCR1 cosuppression, AtKCR1 transcript levels in a variety of tissues of transgenic plants were determined by RT-PCR. As shown in Supplemental Figure S3D, in cauline leaves, stems, and floral buds, the organs exhibiting the most pronounced phenotypes, AtKCR1 transcript levels were considerably lower than in the wild type.
As previously reported for cer10, fdh, wbc11, and wax2/pel6, both glossy and nonglossy AtKCR-RNAi plants display clearly altered trichome morphology (Fig. 9, A–F ). As shown in Figure 9B, trichomes are sparsely distributed, have fused branches in glossy plants, and have shorter or bent branches in nonglossy plants (Fig. 9C). Scanning electron microscopy examination showed that the basal cells of these trichomes are swollen and bulging out of the epidermal surface (Fig. 9, D–F). A further inspection of the surface of young leaves revealed impaired integrity of the epidermis in glossy plants. Groups of cells appeared swollen to the point were they did not adhere tightly to each other, resulting in deep intercellular grooves (Fig. 9, G–I) and probably contributing to the extreme sensitivity of glossy AtKCR1-RNAi plants to dehydration. This type of abnormal epidermal structure could not be detected in nonglossy plants.
Cuticular Wax Load Is Reduced in AtKCR1-RNAi Lines
Organ fusions and stem glossiness observed in the AtKCR1-RNAi plants are typically associated with defects in plant cuticles. Cuticle abnormalities can be detected by a toluidine blue (TB) test, which results in staining of organs with permeable cuticles (Tanaka et al., 2004
Because VLCFAs are precursors of all the aliphatic components of cuticular wax, we suspected that cuticle permeability of AtKCR1-RNAi lines may at least in part be due to reduced wax accumulation. We therefore examined the glossy inflorescence stem surfaces of the AtKCR1-RNAi plants by scanning electron microscopy. As shown in Figure 10B, wild-type stems are densely covered with epicuticular wax crystals. In contrast, glossy stem surfaces of AtKCR1-RNAi plants are virtually devoid of wax crystals. Wax analyses confirmed major reductions in wax loads in AtKCR1-RNAi plants. The total wax coverage on wild-type Columbia-0 (Col-0) inflorescence stems under the conditions used here was 1086 µg per g fresh weight. In contrast, wax loads measured in glossy AtKCR1-RNAi plants reached 124.2 µg per g fresh weight (11.4% of the wild type) in line 7 (7 GL) and 42.8 µg per g fresh weight (3.9% of the wild type) in line 6 (6 GL; Fig. 11 ). Quantification of wax components demonstrated that reduced wax loads were due to decreased accumulation of all compound classes (Fig. 11). In nonglossy AtKCR1-RNAi plants, the decrease in wax load was far less pronounced, and it was proportional to the levels of AtKCR1 transcripts detected in the stem of those plants (Fig. 8). Wax loads measured for nonglossy plants from lines 7 and 9 were 811 and 869 µg per g fresh weight (75% and 80% of the wild type), respectively (Fig. 11, inset). However, these data indicate that while lines 7 and 9 were phenotypically classified as nonglossy, they do have a reduction in wax load, as might be expected for plants with an impaired capacity to synthesize VLCFAs.
VLCFA Content Is Decreased in Root Lipids and Seed Triacylglycerols of AtKCR1-RNAi Lines
Since Arabidopsis leaves contain very low levels of VLCFAs, we analyzed the fatty acid composition in the roots of wild-type and AtKCR-RNAi plants that exhibit abnormal root morphology (Fig. 7J). A clear reduction in the content of C20, C22, and C24 fatty acids was detected in glossy lines 4 and 6 (Fig. 12, A and B
). VLCFAs accounted for 5.6% of the total root fatty acid extracted from wild-type Col-0 plants grown in this study, predominantly reported in phospholipids, such as phosphatidylethanolamine and phosphatidylserine (Devaiah et al., 2006
Surprisingly, in the root fatty acid methyl ester (FAME) extracts prepared from glossy plants, we detected three novel peaks (Fig. 12, A and B, peaks 1–3) that were either absent or low in wild-type extracts. These peaks were tentatively identified by mass spectrometry as C16 and C18  , -dicarboxylic acids and an -hydroxy fatty acid, compounds that are abundant in the cutin and suberin in Arabidopsis (Bonaventure et al., 2004, Franke et al., 2005, Beisson et al., 2007). The presence of these usually highly polymerized constituents in the AtKCR1-RNAi FAME samples after mild methanolic acid extraction may reflect the altered composition and/or structure of the suberin when the synthesis of longer C20, C22, and C24 compounds is reduced. The nonglossy plant extracts did not contain the three additional compounds founds in the roots of glossy plants. Because Arabidopsis seed oil also contains VLCFAs, we examined the fatty acid composition and content of mature dry seeds. Compositional analyses of oil produced by the AtKCR1-RNAi seeds revealed that only the oil from glossy plants was depleted in C20 and C22 saturated and monounsaturated fatty acids. The total VLCFAs content in these seeds was about 30% lower compared to the wild type (Table III ; Fig. 13 ). The larger seeds collected from both glossy and nonglossy AtKCR1-RNAi plants had a higher fatty acid content than wild-type seeds (Tables II and III). Overall, the increase in total fatty acid content was largely proportional to the increase in seed size and did not result from a higher proportion of oil as a percentage of the seed weight (data not shown).
Sphingolipid Analyses
To evaluate the effect of reduced AtKCR1 expression on VLCFA incorporation into sphingolipids, we analyzed the complex sphingolipid composition in the stems of glossy and nonglossy AtKCR1-RNAi plants that all exhibit abnormal shoot morphology. All the examined lines accumulated free long-chain bases (LCBs), which are characteristic of impaired sphingolipid metabolism as reported for the yeast FAE mutants (Kohlwein et al., 2001
This study describes the functional characterization of the Arabidopsis orthologs of the yeast KCR YBR159w and confirms the central role of this enzyme activity in the synthesis of VLCFAs in plants, as well as the essential nature of these fatty acids. Like maize, Arabidopsis contains two KCR-like sequences (KCR1 and KCR2), but they do not share the same high degree of identity exhibited by the monocot genes. Our data indicate that only KCR1 is a bona fide KCR of the FAE in Arabidopsis, based on several independent criteria. First, KCR1 but not KCR2 is capable of rescuing fatty acid elongation in the yeast ybr159w mutant. Second, the expression of the KCR2 ORF under the control of the KCR1 promoter is unable to suppress the embryo lethality of the kcr1 insertional mutant. Finally, while insertional mutations of KCR1 are embryo lethal, similar mutations of KCR2 have no obvious phenotypic effect and do not affect the composition of lipids that are known to accumulate or are derived from VLCFAs, such as seed storage lipids, cuticular waxes, and sphingolipids. It is possible that this apparently normal phenotype of the kcr2 mutant is a result of compensation by the KCR1 protein, but we have not detected increased KCR1 transcript levels in the kcr2 mutant to support this idea. The precise function of KCR2 remains to be determined, although it is still likely to be as a member of the short-chain dehydrogenases/reductase superfamily. Thus, Arabidopsis differs from maize, in which two closely related KCRs (GL8A and GL8B) both contribute to the activity of the microsomal elongase.
Since KCR1 represents the sole microsomal elongase KCR, it is not surprising that disruption of the KCR1 gene is embryo lethal. This also indicates that the capacity to synthesize VLCFAs is crucial at early stages of embryo development and is in agreement with the recent data of Bach et al. (2008)
To evaluate the requirement for VLCFAs during the growth and development of Arabidopsis after embryogenesis, we generated a number of transgenic lines with reduced KCR1 transcript levels via RNAi-mediated silencing. Such plants displayed a number of developmental and biochemical phenotypes that correlated with the relative reduction in transcript levels for KCR1. In particular, lines with more pronounced reduction in KCR1 transcripts showed an almost complete loss of cuticular waxes and a strong fiddlehead-like phenotype, whereas less severe reduction in KCR1 transcripts still generated an fdh phenotype, but only a small reduction in waxes. Given that the KCSs required for wax synthesis (predominantly CER6) is expressed at significantly higher levels than the FDH KCS (Joubès et al., 2008
Our data confirm the essential nature of VLCFAs in Arabidopsis development, though given the evidence that these fatty acids accumulate in virtually all classes of lipids (sphingolipids, waxes, triacylglycerols, suberin, and some phospholipids), the precise reason(s) for the observed embryo lethality in insertion mutants and gross phenotypic perturbations in the KCR1-RNAi knockdowns requires further investigation. For example, there is clear evidence for an absolute requirement for VLCFAs in Arabidopsis for the synthesis of sphingolipids (Chen et al., 2006 The results obtained in our study do provide some new insights into the role of VLCFAs in Arabidopsis. First, it is clear that the ability to make waxes is intimately linked to the capacity to synthesize VLCFAs, with some of the KCR1-RNAi knockdown lines showing an almost complete absence of waxes. As previously reported, such wax-deficient mutants are still viable, but they have to be grown at high humidity. Thus, the essential nature of VLCFAs is unlikely to relate to defects in wax biosynthesis. Our study also shows that the more severe KCR1-RNAi knockdown lines are not able to form lateral roots and root hairs, which correlates with a strong reduction in C20+ saturated fatty acids. In Arabidopsis roots, saturated VLCFAs are present in phospholipids, predominantly phosphatidylethanolamine, and are also components of suberin. Strikingly, in lines 4 and 6, which show the strongest defect in lateral root formation, we detected the presence of several suberin-derived dicarboxyclic acids in simple fatty acid methyl-ester extractions. Dicarboxyclic acids are not normally extracted under such mild conditions and may indicate a perturbation to the VLCFA-containing suberin polymer in the root. Further work is needed to determine if impaired root development is linked to phospholipid- or suberin-related VLCFA deficiency.
One final role of VLCFAs that needs to be considered relates to their potential involvement in the establishment of cellular polarity, most likely through the action of VLCFA-containing sphingolipids. Sphingolipids, in conjunction with sterols, have been shown to be enriched in Arabidopsis detergent-insoluble membrane fractions (Borner et al., 2005
Plant Material and Growth Conditions
The Ds transposon insertional line (RATH12-5282-1-G) was obtained from Plant Functional Genomics Research Group of RIKEN Genomic Sciences Center (Kuromori et al., 2004 After transformation with the KCR1-RNAi construct, T1 seeds were surface sterilized, spread evenly onto agar plates containing kanamycin, stratified for 3 d in a cold room, and germinated at 22°C under continuous light (150 µE m–2 s–1). After 2 weeks, kanamycin-resistant plant were transferred to soil and grown in a controlled environment cabinet with a photoperiod of 16 h light (150 µE m–2 s–1) at 22°C and 8 h dark at 18°C, keeping the soil damp and atmospheric humidity above 80%.
Cuticular wax was extracted and analyzed as described by Rowland et al. (2006)
Total fatty acids from leaves and root tissues and from yeast cultures were extracted and methylated as described before (Sayanova et al., 1997
Total RNAs from aerial tissues of 2- to 6-week-old Arabidopsis (Arabidopsis thaliana) plants were extracted using TRIZOL reagent (Invitrogen Life Technologies) following the manufacturer's instructions. Total RNA from root tissue was isolated from 14-d-old seedlings grown on AT-agar plates placed vertically. For RT-PCR analysis of AtKCR1 mRNA levels in seeds from immature siliques, total RNA (2.5 µg) from each sample, treated with RNase-free DNase (Promega), was used for reverse transcriptase reactions. First-strand cDNA was synthesized with random hexamers using a SuperScript first-strand synthesis system according to the manufacturer's manual (Invitrogen Life Technologies), and the constitutive expression gene glyceraldehyde-3-P dehydrogenase C subunit was used as a control for RT-PCR experiments. One microliter of reverse transcription reaction mixture was used as a template in a 20-µL PCR. The primer sequences used were as follows: AtKCR1 forward primer 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGAGATCTGCACTTACTTCAAAT-3' (bold sequences = directional attB sites) and AtKCR1 reverse primer 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTCTTTCTTCATGGAGTCTTTTTGG-3'. The amplification conditions were 94°C (30 s), 56°C (30 s), and 72°C (30 s), and the number of cycles varied from 28 to 32 for different genes. Each RT-PCR was repeated twice. For RT-PCR analysis of AtKCR1 and AtKCR2 mRNA levels in 2- to 6-week-old plant tissues, total RNA (2.5 µg) from each sample, treated with Turbo DNA-free DNase (Ambion), was used for reverse transcriptase reactions. First-strand cDNA was synthesized using oligo(dT)12-18 primer and SuperScript II reverse transcriptase according to the manufacturer's instructions (Invitrogen). The constitutive expression gene tubulin (
The full-length cDNAs of AtKCR1 and AtKCR2 were obtained by RT-PCR with primers containing Gateway (Invitrogen) recombination sites (bold sequences), AtKCR1 forward primer 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGAGATCTGCACTTACTTCAAAT-3' and reverse primer 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTCTTTCTTCATGGAGTCTTTTTGG-3'; AtKCR2 forward primer 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCAGGGAGCATGCATCTCCGAGA-3' and reverse primer 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGATAAACTTCTTCTGCGAAGTCCG-3', and subcloned into Gateway donor vector (pDONR207; Curtis and Grossniklaus, 2003
Wild-type and ybr159w
A length of 780 bp of the 5' promoter region of AtKCR1 was amplified from genomic DNA by PCR using forward primer 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCCTTTGGACTTACCAACG-3' and reverse primer 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGGTTGAGACTTTGGAGATGAGA-3'. The amplified product was introduced into Gateway entry vector by Gateway BP Clonase enzyme mix (Invitrogen). The confirmed entry clones were transferred into destination vector pMDC163 (Curtis and Grossniklaus, 2003 For GUS activity assays, 1- or 2-week-old seedlings, leaves, flowers, and siliques from 4-week-old plants, and embryos of different developmental stages were incubated in GUS assay buffer (50 mM sodium phosphate, pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% [v/v] Triton X-100, and 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronide) at 37°C overnight. Then, samples were cleared in 70% ethanol and visualized by light microscopy.
For rapid visualization of leaf cuticle defects, we used the TB test described by Tanaka et al. (2004)
For the YFP or CFP fusion protein localization analysis, leaf samples of transgenic plants were examined using confocal laser scanning microscope as described by Zheng et al. (2005)
Siliques of different developmental stages from heterozygous plants were dissected. Ovules from individual siliques were mounted on slides in Hoyer's clearing solution (chloral hydrate, water, glycerol, 8:2:1 v/v) and cleared overnight at 4°C (Liu and Meinke, 1998 For electron microscopy, sections of stem and leaves were mounted on Al cryo stubs using Optimal Cutting Temperature compound (Ted Pella) and plunge frozen in slushed liquid nitrogen. The samples were then transferred to the cryo chamber stage under vacuum and etched for 2 min at –95°C. The stage temperature was returned to –175°C, and the samples were coated for 90 s with Au Pd. The samples were then loaded on a JEOL 6700 FEG scanning electron microscope chamber stage maintained at –160°C for imaging using the on board software.
For routine sequence comparison, BLAST was used (Altschul et al., 1990 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers NY143811 (AtKCR1), NM102292 (AtKCR2), AY557868 (Ybr159), AF302098 (GL8a), and AF527771 (GL8b).
The following materials are available in the online version of this article.
We thank the Genomic Analysis Laboratory of the Salk Institute and RIKEN Genomic Sciences Center for providing Arabidopsis T-DNA insertion mutants, the Bioimaging Facility at the University of British Columbia for providing microscopy and technical support, and Jean Devonshire and the Centre for Bioimaging at Rothamsted who helped with the scanning electron microscopy work. We are also grateful to Louise Michaelson and Teresa Dunn for helpful discussions. Received February 20, 2009; accepted April 28, 2009; published May 13, 2009.
1 This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (UK) to Rothamsted Research and a grant from the Natural Sciences and Engineering Research Council of Canada to L.K. 
2 These authors contributed equally to the article.
3 Present address: Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1. The authors 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) are: Johnathan A. Napier (johnathan.napier{at}bbsrc.ac.uk) and Ljerka Kunst (kunst{at}interchange.ubc.ca).
[W] The online version of this article contains Web-only data.
[OA] Open access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.137497 * Corresponding author; e-mail kunst{at}interchange.ubc.ca.
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