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First published online February 11, 2005; 10.1104/pp.104.055715 Plant Physiology 137:1067-1081 (2005) © 2005 American Society of Plant Biologists Differential Expression and Evolution of the Arabidopsis CYP86A Subfamily1,[w]Department of Cell and Structural Biology, University of Illinois, Urbana, Illinois 61801
Some members of the Arabidopsis (Arabidopsis thaliana) CYP86A and CYP94B cytochrome P450 monooxygenase subfamilies, which share some sequence homology with the animal and fungal fatty acid hydroxylases, have been functionally defined as fatty acid  -hydroxylases. With these activities, these and other fatty acid hydroxylases have potential roles in the synthesis of cutin, production of signaling molecules, and prevention of accumulation of toxic levels of free fatty acids. The constitutive and stress-inducible patterns of the five Arabidopsis CYP86A subfamily members have been defined in 7-d-old seedlings and 1-month-old plant tissues grown under normal conditions, and 7-d-old seedlings treated with different hormones (indole-3-acetic acid, abscisic acid, gibberellin, methyl jasmonic acid, brassinosteroid, salicylic acid), chemicals (clofibrate, 1-aminocyclopropane-1 carboxylic acid), or environmental stresses (cold, wounding, drought, mannitol, etiolation). Very distinct expression patterns exist for each of these fatty acid hydroxylases under normal growth conditions and in response to environmental and chemical stresses. Analysis of the promoter sequences for each of these genes with their expression patterns has highlighted a number of elements in current databases that potentially correlate with the responses of individual genes.
Multiple forms of cytochrome P450-dependent monooxygenases (P450s) catalyze in-chain hydroxylations, end-terminal hydroxylations, and epoxidations of medium- and long-chain fatty acids (Salaün and Helvig, 1995  ; Scheller et al., 1996; Simpson, 1997). Evidence exists that some of these enzymes have distinct substrate preferences and regiospecificities, with some modifying terminal positions on medium-chain fatty acids and others modifying internal positions on long-chain fatty acids. In plants, fatty acid hydroxylases are particularly important in the synthesis of plant cuticles and signaling molecules derived from fatty acids (Kolattukudy, 1980; Schweizer et al., 1996a; Fauth et al., 1998; Pinot et al., 1999). They also represent the primary enzymes responsible for preventing the accumulation of high, possibly toxic, concentrations of free fatty acids in the plant cells liberated by phospholipases in the early stress responses (Kahn and Durst, 2000). Because the fatty acid profiles of many crop plants are not ideal, there is much potential for the genetic manipulation of fatty acid hydroxylases aimed at improving commercial properties of crop plants.
In the broad set of activities catalyzed by fatty acid hydroxylases, those that catalyze the hydroxylation of the terminal methyl group on aliphatic fatty acid chains are designated as
Heterologous expressions of these fatty acid hydroxylases have indicated that each has distinct substrate specificities. For example, CYP94A1 Given the involvement of many P450 families in mediating fatty acid hydroxylations occurring in different plants and animals, it is clear that P450s metabolizing these types of endogenous compounds are among the earliest and most diversified of the P450s that exist in eukaryotes. But, because of the multiplicity of related P450 genes in various plant species with potentially overlapping functions and the dearth of information on their expression patterns, the physiological significance of many of these enzymes has still not been completely established.
Among their many potential roles, fatty acid hydroxylases are important in the production of cutin in the aerial parts of plants and suberin in the roots of plants, which serve as structural components of the permeability barrier protecting plants against water loss and pathogen, insect, and mechanical damage. Derived from cellular lipids, cutin and suberin are polymeric networks of oxygenated C16 and C18 fatty acids cross-linked by ester bonds, such that the carboxyl group of one fatty acid is linked to a primary or secondary hydroxyl group of another (Kolattukudy, 2001
Once incorporated into elongated cutin, cutin polymers and monomers released from it play important roles in plant development and pathogen defense mechanisms. Cutin monomers released by fungal cutinase in the course of pathogenic infections are perceived as signals enhancing fungal cutinase expression and formation of fungal appressorial structures (Francis et al., 1996
Against this background of plant-fungal interactions, it is now evident that fatty acid hydroxylases play important roles in determining plant-bacterial interactions. Inactivation of the CYP86A2 protein in the att1 ethyl methanesulfonate mutant and the enhanced pathogenicity of Pseudomonas syringae associated with this mutation directly implicate cutin polymers derived from this P450 in establishing resistance to bacterial pathogens (Xiao et al., 2004
In addition to CYP86A1 and CYP86A8, whose fatty acid hydroxylase activities on various substrates have been functionally defined in heterologous expression systems, and CYP86A2, whose fatty acid hydroxylase activities have been inferred from mutational analysis, two additional CYP86A genes (CYP86A4 and CYP86A7) exist in the Arabidopsis genome (http://www.p450.kvl.dk/p450.shtml; http://Arabidopsis-P450.biotec.uiuc.edu). All five of these CYP86A sequences are typical non-A P450s with significant sequence homology to the fatty acid- and alkane-metabolizing CYP4 proteins and CYP52 proteins previously mentioned from mammals and fungi (Durst and Nelson, 1995
Comparison of the CYP86A Coding Sequences The five members of the CYP86A subfamily are scattered over four of the five Arabidopsis chromosomes. As indicated by their genomic locus numbers (At5g58860, At4g00360, At1g01600, At1g63710, At2g45970), CYP86A1 is located on the lower arm of chromosome 5, CYP86A2 is located on the very top of chromosome 2, CYP86A4 is located on the very top of chromosome 1, CYP86A7 is located on the lower arm of chromosome 1, and CYP86A8 is located very close to the bottom of chromosome 2 (http://www.p450.kvl.dk/p450.shtml). Supported by the existence of full-length cDNA clones for each of these five loci (http://Arabidopsis-P450.biotec.uiuc.edu), comparisons conducted at the nucleotide level indicate that these genes have very different organizations, with CYP86A1 having a single intron interrupting the coding sequence at position 1,140 nt (relative to ATG), CYP86A2 and CYP86A4 having a single intron interrupting the coding sequence at position 422 (relative to ATG), and CYP86A7 and CYP86A8 having no introns. The intron of CYP86A1 is relatively large (457 nt) compared to the smaller conserved introns of CYP86A2 (166 nt) and CYP86A4 (130 nt) that are 51.9% identical to one another. Except for the splice-site junctions, no significant matches exist between the CYP86A1 intron and the CYP86A2 and CYP86A4 introns. Pairwise comparisons between the five Arabidopsis CYP86A sequences indicate that, as shown in Table I, CYP86A2 and CYP86A4 have the highest degree of nucleotide identity within their coding sequence (82.3%). CYP86A2 and CYP86A4 have similarly high degrees of identity with CYP86A8 (73.6% and 73.3%, respectively). CYP86A7 shares comparably high identity with CYP86A8 (70.1%), CYP86A4 (69.8%), and CYP86A2 (68.4%). CYP86A1 has distinctly lower identity with the other four members of this P450 subfamily (62.3%–64.5%).
Pairwise comparisons conducted at the protein level indicate that these proteins are well conserved throughout most of their coding sequence and display the same derived relationships as seen at the nucleotide level (Table II). CYP86A2 and CYP86A4 have the highest degree of sequence identity (87%), with both equally close to CYP86A8 (75% and 76%, respectively) and, to a lesser degree, to CYP86A7 (70% and 68%, respectively). CYP86A1 has lower similarity with the other four members of the CYP86A subfamily (61%–62%). Quite unusually, these P450s differ in their amino acid lengths from 513 (CYP86A1), 524 (CYP86A7), 537 (CYP86A8), and 553 (CYP86A2), to 557 (CYP86A4). Alignments at the primary sequence level indicate that nearly all of these length differences exist at the C terminus, with CYP86A7 extending 11 residues, CYP86A8 extending 22 residues, CYP86A4 extending 36 residues, and CYP86A2 extending 37 residues beyond the termination point of CYP86A1 (Fig. 1). Analysis of the sequence composition in the extended tails of CYP86A2 and CYP86A4 shows a disproportionate number of nonpolar Val, Ala, and Gly residues (67% and 58%, respectively).
Molecular models have been developed for these five proteins using molecular operating environment (MOE, Chemical Computing Group, Montreal) programs as detailed by Rupasinghe et al. (2004) for other Arabidopsis P450s. Based on these structural predictions, assignments of the substrate recognition site (SRS; Gotoh, 1992) regions have indicated that all five P450s are nearly identical in SRS1, more variable in SRS4 and SRS5, and most variable in SRS2, SRS3, and SRS6 (Fig. 1; Supplemental Fig. 1). The identities in these regions, which are thought to contribute to defining catalytic site specificity, are striking given the level of overall sequence divergence in these proteins. Even more striking is the degree to which the backbones predicted for these proteins overlay one another (data not shown), with all displaying a longer J-K loop or  -pleated sheet structure compared with the crystal structure determined for bacterial CYP102 (Ravichandran et al., 1993; Li and Poulos, 1994, 1997), and all except CYP86A2 displaying a longer F-G loop (SRS2). Among the few more divergent regions that do exist, CYP86A2 and CYP86A8 have longer G-H loops, potentially affecting interactions with P450 reductase; CYP86A2 and CYP86A4 have longer B'-C loops containing SRS1; and CYP86A2, CYP86A4, and CYP86A8 have longer exterior surface loops between -sheets 3-2 and 4-2. While this relatively small number of backbone differences between these proteins suggests that their catalytic sites have similar configurations, amino acid variations in SRS4, SRS5, and SRS6 suggest that these P450s have the potential to metabolize different ranges of fatty acids.
An unrooted phylogenetic tree of potentially orthologous fatty acid
From the unrooted tree (Fig. 2A), it is apparent that, except for the less well-characterized CYP78A1, CYP92B1, and CYP703A1 proteins, all fatty acid -hydroxylases exist in one phylogenetic group, regardless of the fact that they are present in different species and even different kingdoms. The fact that CYP703A1, CYP92B1, and CYP78A1 show up in the plant-specific A-type group, represented by the CYP73A5 and three others, suggests that conserved activities, such as fatty acid hydroxylation normally mediated by non-A-type P450s found in a variety of organisms, can be mediated at some marginal level by A-type P450s.
As noted previously, CYP86A1 and CYP86A8 have been functionally defined as
Tissue Profiling of CYP86A Transcripts
To elucidate the physiological functions of different members within this subfamily, we next defined the constitutive expression levels of the CYP86A transcripts in different tissues of Arabidopsis seedlings and mature plants. For this, RNA samples from 7-d-old shoots and roots, 3-week-old rosettes, and 1-month-old mature leaves, roots, siliques, and flowers were reverse transcription (RT)-PCR amplified using 5' gene-specific primers and a 3' primer complementary to the poly(A) tract present on mature mRNAs, and varying PCR cycle numbers determined to quantitatively amplify each transcript. RT-PCR products were subsequently gel blotted, hybridized with probes corresponding to the gene-specific microarray elements, and quantified by phosphor imager analysis. Normalization of each total RNA sample against its level of constitutive elongation factor EF-l
Stress Profiling of CYP86A Transcripts
To further elucidate the inducibilities of transcripts in this P450 subfamily, the inducible transcript levels were defined by RT-PCR gel-blot analysis, with RNAs isolated from 7-d-old seedlings treated with different hormones such as indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellin (GA), methyl jasmonic acid (MeJA), brassinosteroid (BR), and salicylic acid (SA) or chemicals such as clofibrate and 1-aminocyclopropane-1-carboxylic acid (ACC), for varying times, or stressed with cold, wounding, drought, mannitol, or etiolation. Normalization of each total RNA sample was done against its level of constitutive EF-l
Promoter Analysis Searches for identifiable promoter elements in sequences 2 kb upstream of the translation start site were performed using two databases, Arabidopsis Gene Regulatory Information Server (AGRIS; http://Arabidopsis.med.ohio-state.edu) and PlantCARE (http://intra.psb.ugent.be:8080/PlantCARE). The first one contains cis-elements characterized only in Arabidopsis studies, and the second one contains cis-elements characterized in a variety of different plant species. Elements from the AGRIS database found in the CYP86A promoters and 5' untranslated region are listed in Table III (under Elements Found in AGRIS) and a partial list of elements from the PlantCARE database is included in Table III (under Elements Found in PlantCARE). The chemical/stress treatments inducing transcript accumulation at least 1.5-fold in our RT-PCR analyses are listed below each column, with an asterisk indicating the existence of a known cis-element for a particular chemical/environmental inducer. Among the elements identified in the first Arabidopsis-specific database, there is good agreement between the cis-element search and RT-PCR analysis of responses to ABA for all five CYP86A genes, as well as response to IAA for CYP86A4. Likewise, the CYP86A1 and CYP86A2 responses to cold/mannitol and drought/mannitol treatments correlate with the presence of ABA response element (ABRE)-like elements (dehydration/low temperature) and/or AtMYC2 elements (drought/ABA) in their promoters. However, stress-specific elements correlating with the CYP86A4 response to cold at 3 and 27 h, the CYP86A7 response to MeJA at 3 and 27 h, the CYP86A2 response to wounding at 3 h, and the CYP86A1/CYP86A4 responses to ethylene do not exist either due to the existence of novel elements in these promoters or to the high stringency of search functions in this database. In the second plant-specific database, multiple cis-elements not highlighted in the first search correlate with the responses of individual genes. Additional elements identified in this search include some correlating with the CYP86A1/CYP86A4 responses to ACC, the CYP86A2 response to wounding, the CYP86A4 response to cold, and the CYP86A7 response to MeJA. But, in three of these cases (ACC, wounding, MeJA), the additional elements identified in this search exist in multiple copies in promoters not responding to these stresses. In addition, emphasizing the differences between the array of consensus sequences present in these two promoter search sites, the second broader search identified no auxin response elements putatively mediating the CYP86A2 and CYP86A4 response to IAA. Based on these comparisons, the Arabidopsis-specific promoter search process more accurately highlights elements that may be involved in the responses of individual promoters, although, because of the stringencies applied, there is potential to miss response elements.
Because both databases lack elements known to mediate responses to some of these chemicals, these promoters were searched for novel sequence identities using the PromoterWise program. The initial results from this pairwise comparison were manually checked to eliminate direct and inverted matches having less than seven contiguous nucleotides within aligned regions and less than two guanosines or cytosines. The final compilation of conserved elements, shown in Table IV, indicates that the CYP86A2/CYP86A8 and CYP86A4/CYP86A8 promoter pairs contain the greatest number of extended sequence elements (nine total), with many in the CYP86A4/CYP86A8 alignment occurring in the same orientation (designated D in Table IV). The CYP86A1/CYP86A2 and CYP86A1/CYP86A4 promoter pairs contain slightly fewer shared sequences with seven and eight extended sequence elements, respectively. The CYP86A2/CYP86A4 promoter pair has the fewest extended sequence elements (three total), but two of these are the longest ones (14 and 25 nt) identified in this search process. Interestingly, pairwise alignments between the CYP86A2 and CYP86A4 promoters indicate that they have high degrees of identity, with 52.1% identity in 1.5 kb upstream from the translation start site decreasing only slightly to 50.5% identity in 2.0 kb upstream from the translation start site. Notably, the region from –442 to –373 (Fig. 6) has more than 88% (61/69) identity. Pairwise alignments of the other CYP86A promoters indicated no other significant identities.
Current data indicate that plants have different types of enzymes responsible for fatty acid hydroxylations (Blee and Schuber, 1993 ; van De Loo et al., 1993; Cabello-Hurtado et al., 1998), but among these, only P450-dependent enzymes are responsible for hydroxylations at the -position (Wellesen et al., 2001). Multiple P450 families appear to mediate this hydroxylation with the plant CYP86 and CYP94 families representing two major groups of fatty acid -hydroxylases (Benveniste et al., 1998; Tijet et al., 1998; Pinot et al., 1999; Kahn et al., 2001; LeBouquin et al., 2001; Wellesen et al., 2001; Petkova-Andonova et al., 2002). In the CYP86A subfamily of Arabidopsis, which contains five different members that function as fatty acid -hydroxylases, CYP86A1 is the oldest member and CYP86A2 and CYP86A4 are the most recently duplicated members. Compared to other plant P450 subfamilies, these five CYP86A proteins share high degrees of primary sequence identity (between 62% and 82%) despite very different lengths (between 513 and 557 amino acids). Nearly all of the length differences are attributable to C-terminal extensions on these proteins, with CYP86A2 and CYP86A4 having 14 to 15 more C-terminal amino acids than CYP86A8, 25 to 27 more amino acids than CYP86A7, and 36 to 37 more amino acids than CYP86A1. Because these additional amino acids lie outside presumed SRSs, it is not known whether they affect the biochemical activities and/or physiological functions of the CYP86A proteins. Molecular modeling of these C-terminal extensions, which contain an unusually high number of nonpolar Val, Ala, and Gly residues, predicts that they form random coil structures extending from the external surface of these proteins.
Phylogenetic analysis of all functionally defined plant, mammalian, and fungal fatty acid
Phylogenetic comparisons at the gene level indicate that the Arabidopsis CYP86A sequences, which contain introns at different positions, arose either from the divergence of a common ancestral gene by a complicated series of intron deletions and reinsertions or from the convergence of several ancestral genes for a common function. Alignments of the intron-containing CYP86A1, CYP86A2, and CYP86A4 genes with one another indicate that introns occur at different positions in the CYP86A1 and CYP86A2/CYP86A4 genes, suggesting that the single intron in the CYP86A1 gene was lost during the evolution of these genes and that the reinserted intron in the CYP86A2/CYP86A4 genes is not related to original CYP86A1 intron. The alignment of the intron in the CYP86A1 gene with single introns in the CYP86B1 and CYP86B2 genes in Arabidopsis supports this hypothesis of intron loss from the ancestral CYP86A gene existing in dicots. Phylogenetic comparisons of the Arabidopsis CYP86A genes with the recently identified three CYP86A genes and six CYP86A pseudogenes in the Oryza sativa genome (Nelson et al., 2004
The significant conservation that these five CYP86A proteins share at the primary sequence level becomes even more significant when one examines the very limited set of amino acid variations that occur in the six putative SRSs. Based on this higher level of primary sequence conservation and molecular modeling of these proteins, it appears that they might mediate some redundant substrate specificities. Our data support this notion in showing that all five of these proteins metabolize various lengths of saturated and unsaturated fatty acids and evolve it in showing that these proteins have different substrate preferences for long-chain (C18) fatty acids (H. Duan and M.A. Schuler, unpublished data). Importantly, these redundant in vitro catalytic activities do not necessarily translate into redundant in vivo functions since transcript analysis shows that each CYP86A gene is regulated in its own tissue-specific manner. The nonredundant nature of these genes is obvious in the case of CYP86A8 and CYP86A2, where cyp86a8 mutant plants show severe pleiotropic phenotypes associated with developmental cuticular deficiencies under normal growth conditions (Wellesen et al., 2001
Comparison of transcript profiles in various tissues by both microarray and RT-PCR analysis indicates that members of the CYP86A subfamily are expressed at quite different constitutive levels. CYP86A1 transcripts are expressed significantly only in root tissue, CYP86A4 and CYP86A7 transcripts are expressed at their highest levels in mature stems and flowers, and CYP86A2 and CYP86A8 transcripts are expressed at moderate levels in most tissues analyzed. The very high levels of CYP86A4 and CYP86A7 transcripts in reproductive tissues suggest that they may play roles in the recognition of the stigma by pollen and/or in the process of pollen tube growth, both processes that have substantial requirements for hydroxylated fatty acids (Koiwai and Matsuzaki, 1988
Just as these CYP86A genes show different tissue profiles, they also display different responses to chemical and environmental stresses. This is not unexpected, given the putative roles of CYP86A2, CYP86A8, and CYP94A1 in epidermal cutin and suberin synthesis (Pinot et al., 1999 The array of elements in each of these promoters picked up with available promoter search programs suggests that the Arabidopsis-specific promoter search identifies sets of putative regulatory elements that for ABA, IAA, and cold correlate more closely with the chemical and environmental inducers of individual genes. Correlations on the regulatory elements for clofibrate, BR, and wounding cannot be drawn since consensus elements for these stresses do not exist in this database. The failure to detect elements for the ethylene responses of CYP86A1/CYP86A4, the MeJA response of CYP86A7, and the root-specific expression of CYP86A1 suggests that these responses are mediated by novel elements or indirectly by other signaling cascades and/or by more divergent elements. Deletion and mutational analysis of some of the novel elements identified in our promoter comparisons will determine the extent to which any one of these is involved in stress signaling.
Chemicals Plant hormone and growth regulators, including IAA, ABA, MeJA, SA, ACC, BR, and clofibrate, were obtained from Sigma (St. Louis). NADPH, Glc-6-P, and Glc-6-P dehydrogenase were also obtained from Sigma, and [1-14C]lauric acid was obtained from Amersham Biosciences (Piscataway, NJ).
Arabidopsis (Arabidopsis thaliana ecotype Columbia) seeds were surface sterilized and grown on half-strength Murashige and Skoog-agar plates in a 22°C to 24°C growth room, with a 16-h-light/8-h-dark cycle. For IAA, ABA, MeJA, SA, and BR treatments, chemical compounds were dissolved in final 0.1% ethanol at the following concentrations: 100 µM (IAA, MeJA), 50 µM (ABA), 1 mM (SA), and 1 µM (BR). For ACC, clofibrate, and mannitol treatments, chemicals were dissolved in sterile water at the following concentrations: 1 µM (ACC), 1 mM (clofibrate), and 500 mM (mannitol). At the beginning of each treatment, 50 mL of each solution were poured onto the plates to cover the 7-d-old seedlings, and the plates were grown horizontally for the remainder of the treatment period. At the end of each treatment, whole seedlings were harvested directly from the Murashige and Skoog-agar plates, washed thoroughly with sterile water, and frozen in liquid nitrogen. For wounding treatment, 7-d-old seedlings were chopped with a sharp razor blade and left under a dim light for the indicated time period. For drought treatment, 7-d-old seedlings were placed on a piece of Whatman 3MM filter paper (Clifton, NJ) inside a chemical hood under dim light for the indicated time period. For etiolation, seeds were either germinated and grown in the dark for a total of 10 d (etiolated) or germinated under an 8-h-light/16-h-dark cycle for 3 d and then placed in the dark for an additional 4 d (dark adapted). Total RNA was isolated from each of the treatment and matched control samples (0.1% ethanol for IAA, ABA, JA, SA, BR; sterile water for ACC, mannitol, clofibrate) using TRIzol reagent (Invitrogen, Carlsbad, CA). For these, 1 g of tissue was ground in a mortar and pestle with 12 mL of TRIzol, transferred to a 15-mL Falcon tube, extracted with 3 mL of chloroform, incubated for 5 min at room temperature, and centrifuged at 5,000g at 4°C for 15 min. The supernatant was transferred to a fresh tube, and nucleic acids were precipitated by adding one-half volume of isopropanol, gently inverting the mixture, incubating at room temperature for 10 min, and centrifuging at 10,000g at 4°C for 10 min. Nucleic acids were redissolved in 500 µL of RNase-free water (Invitrogen), digested with 5 units of RQ DNase at 37°C for 1 h, reextracted with the same amount of phenol:chloroform (1:1) and then chloroform, reprecipitated with one-tenth volume of 3 M sodium acetate, pH 5.0, and two volumes of ethanol for 20 min at –20°C, centrifuged at 13,000g for 10 min at 4°C, dried, resuspended in 300 µL of sterile RNase-free water, and stored at –80°C.
Quantitative RT-PCR gel-blot analysis of individual P450 transcripts was carried out by amplifying approximately 0.1 µg of total RNA of each sample in one-step RT-PCR reactions containing 50 mM KCl, 10 mM Tris-HCl, pH 8.4, 200 µM each dNTP, 200 µg/mL gelatin, 40 pmol of a 5' gene-specific primer, 40 pmol of a 3' oligo(dT)17-EcoRI primer, 4 units of AMV reverse transcriptase (Promega, Madison, WI), 20 units of RNasin, and 2.5 units of Taq polymerase (Gibco-BRL, Cleveland). First-strand cDNAs were synthesized for 40 min at 42°C and subsequently PCR amplified for 18 to 22 cycles, with each cycle consisting of denaturation at 95°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 2.5 min, followed by a final extension step of 72°C for 10 min. The number of PCR cycles used for each transcript (20 for CYP86A1, CYP86A8, CYP86A2, and CYP86A4; 22 for CYP86A7; 18 for EF-l
Recombinant viruses containing each of the CYP86A subfamily members were constructed by RT-PCR strategies and expressed in Sf9 insect cells as described previously (Duan et al., 2004
Amino acid and nucleotide sequence alignments were performed using Vector NTI software version 8.0 (InforMax, Bethesda, MD) and ClustalW version 1.6 (Thompson et al., 1994
We gratefully acknowledge Dr. Shahjahan Ali, Dr. Yurdagul Ferhatoglu, Dr. Jyothi Thimmapuram, Dr. Mark Band, Dr. Daniele Werck-Reichhart, and Mr. Albert Bari for their assistance in construction and for analysis of the microarrays associated with this study. We also acknowledge Ms. Wen Gu for help with cloning CYP86A2 and CYP86A4, Mr. Sangeewa Rupasinghe for modeling the SRS domains within these proteins, and Mr. Saranyan Palaniswamy and Dr. Erich Grotewold for help in promoter database searches. Received October 28, 2004; returned for revision December 3, 2004; accepted December 9, 2004.
1 This work was supported by the National Science Foundation (NSF 2010 Project grant no. MCB 0115068 to M.A.S.). 
[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.104.055715. * Corresponding author; e-mail maryschu{at}uiuc.edu; fax 217–244–1336.
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ABSTRACT
RESULTS
transcript and subsequently against the level of P450 transcript present in seedling shoots was performed to allow for comparisons between tissues and with microarray data described elsewhere (http://Arabidopsis-P450.biotec.uiuc.edu). As shown in 
