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First published online July 8, 2005; 10.1104/pp.105.062539 Plant Physiology 138:2245-2259 (2005) © 2005 American Society of Plant Biologists Metabolic Engineering of Isoflavonoid Biosynthesis in Alfalfa1,[w]Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401
The potential health benefits of dietary isoflavones have generated considerable interest in engineering the synthesis of these phytoestrogens into plants. Genistein glucoside production (up to 50 nmol g–1 fresh weight) was engineered in alfalfa (Medicago sativa) leaves by constitutive expression of isoflavone synthase from Medicago truncatula (MtIFS1). Glucosides of biochanin A (4'-O-methylgenistein) and pratensein (3'-hydroxybiochanin A) also accumulated. Although MtIFS1 was highly expressed in all organs examined, genistein accumulation was limited to leaves. MtIFS1-expressing lines accumulated several additional isoflavones, including formononetin and daidzein, in response to UV-B or Phoma medicaginis, whereas the chalcone and flavanone precursors of these compounds accumulated in control lines. Enhanced accumulation of the phytoalexin medicarpin was observed in P. medicaginis-infected leaves of MtIFS1-expressing plants. Microarray profiling indicated that MtIFS1 expression does not significantly alter global gene expression in the leaves. Our results highlight some of the challenges associated with metabolic engineering of plant natural products, including tissue-specific accumulation, potential for further modification by endogenous enzyme activities (hydroxylation, methylation, and glycosylation), and the differential response of engineered plants to environmental factors.
Isoflavonoids are a predominantly legume-specific subclass of flavonoid secondary metabolites, with roles in plant defense and nodulation (Dixon, 1999  ). The protective effects of the soy (Glycine max) isoflavones genistein and daidzein against hormone-dependent cancers, cardiovascular disease, osteoporosis, and menopausal symptoms (for review, see Cornwell et al., 2004; Dixon, 2004) have generated considerable interest in engineering these isoflavones into more commonly consumed food crops (Dixon and Steele, 1999). Isoflavones are synthesized from common flavanone intermediates (either liquiritigenin or naringenin; Fig. 1) by aryl migration catalyzed by the enzyme 2-hydroxyisoflavanone synthase (also referred to as isoflavone synthase or IFS). Cloning of IFS has allowed isoflavone synthesis to be engineered into plants that normally do not make isoflavones, as demonstrated in Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and cell cultures of maize (Zea mays; Jung et al., 2000a, 2000b; Yu et al., 2000; Liu et al., 2002). These studies have also highlighted some of the factors impacting isoflavone accumulation in plants. For example, in IFS-expressing Arabidopsis, accumulation of genistein was limited by competition from the flavonol pathway (Liu et al., 2002), whereas in IFS-expressing tobacco, it was correlated with the activity of the phenylpropanoid pathway leading to the accumulation of anthocyanins (Yu et al., 2000). In all cases, levels of genistein accumulating in transgenic plants were several orders of magnitude lower than levels of genistein in soybean.
Engineering isoflavonoid biosynthesis in leguminous plants may provide enhanced levels of these compounds for plant-based dietary supplements and additional benefits related to plant defense and nodulation (Jung et al., 2003 ). There are only two examples of isoflavonoid engineering in legumes. A preliminary report of IFS-expressing soybean seeds showed that only two of 14 independent transgenic lines had significantly higher levels of seed isoflavones (maximal levels were 1.7-fold greater than controls), while in four lines isoflavone levels were reduced (Jung et al., 2003). In an alternative approach to increase isoflavone accumulation in soybean, the phenylpropanoid pathway-activating transcription factors C1 and R from maize were expressed in seed (Yu et al., 2003). The transgenic seed had a reduced genistein-to-daidzein ratio and only a modest (approximately 2-fold) increase in total isoflavones. Additional suppression of flavonol synthesis through down-regulation of flavanone 3-hydroxylase allowed for higher levels of isoflavone accumulation, as reported for IFS-expressing Arabidopsis (Liu et al., 2002). In this study, we have generated transgenic alfalfa (Medicago sativa) expressing IFS from the model legume Medicago truncatula and examined the effect of constitutive IFS expression on the flavonoid composition of alfalfa, the metabolic responses of the transgenic plants to abiotic and biotic stress, and the impact of IFS expression on global gene expression patterns. These studies reveal differential tissue- and environment-specific effects on flavonoid metabolism as a result of IFS transgene expression. We also discuss the potential for using metabolic engineering to generate novel plant material for dietary studies designed to address the effect of isoflavones on animal health.
Generation of Transgenic Alfalfa with Constitutive Expression of MtIFS1
A cDNA encoding M. truncatula IFS (MtIFS1) was initially isolated by screening an M. truncatula root cDNA library with the soybean IFS CYP93C1v2 (Steele et al., 1999 Alfalfa plants expressing MtIFS1 under the control of the cauliflower mosaic virus (CaMV) 35S promoter (Fig. 2A) were generated. Because alfalfa contains multiple copies of the IFS gene (data not shown), plants were screened by PCR using primers specific to the NPTII gene; a subset of these lines was additionally confirmed to harbor the NPTII gene by DNA gel-blot analysis (Fig. 2B).
Metabolite Analysis of MtIFS1-Expressing Alfalfa
Alfalfa shoots accumulate GlcUA conjugates of the 5-hydroxy flavones apigenin, luteolin, tricin, and chrysoeriol (Stochmal et al., 2001a
Relative to controls, leaf extracts of MtIFS1-expressing lines contained five new peaks with UV spectra similar to genistein and related isoflavones (Fig. 3B). These peaks were not shifted after treatment with  -glucuronidase (Fig. 3D), but were shifted after digestion with purified -glucosidase (Fig. 3E). The major peak present in -glucosidase-treated extracts of MtIFS1-expressing lines was identified as genistein by comparison of Rt (51.8 min) and UV spectrum with those of an authentic standard, and by liquid chromatography (LC)-mass spectrometry (MS; molecular ion at m/z 269.0 [M-H]–). The other two peaks were identified as pratensein (5,7,3'-trihydroxy-4'-methoxyisoflavone, Rt = 53.9 min, molecular ion at m/z 299.0 [M-H]–) and biochanin A (5,7-dihydroxy-4'-methoxyisoflavone, Rt = 66.1 min, molecular ion at m/z 283.5 [M-H]–). Based on the results of the enzyme digestion, the five peaks present in unhydrolyzed MtIFS1-expressing leaf extracts likely represent Glc conjugates of these three isoflavones. Peaks 1 (Rt = 36.2 min) and 4 (Rt = 46.9 min) were further identified as genistin (7-O-glucosyl-genistein) and sissotrin (7-O-glucosyl-biochanin A), respectively, by comparison of Rts and UV spectra with those of authentic standards, and by LC-MS analysis.
In preliminary experiments using plants grown under standard greenhouse conditions, the amount of genistein in
RNA gel-blot analysis demonstrated that MtIFS1 was highly expressed in leaf, stem, and root tissues of line C22, whereas in the vector control line VC11, IFS expression was only detected in the roots (Fig. 5). Surprisingly, no differences in flavonoid/isoflavonoid composition were observed in methanol extracts of stem, root, or flower tissues from lines C22 and B20 when compared to vector control lines (data not shown). Low levels of flavone conjugates were detected in stem extracts, similar to what was observed in leaf extracts. Root extracts of MtIFS1-expressing and vector control lines contained conjugates of the isoflavonoids formononetin, medicarpin, and coumestrol, but there was no significant difference in their levels in MtIFS1-expressing roots compared to controls. Flowers contained conjugates of the flavonols kaempferol and quercetin.
Leaves of MtIFS1-expressing and control plants contained low levels of transcripts encoding the enzymes 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) and isoflavone 3'-hydroxylase (I3'H; Fig. 5), the activities of which are most likely responsible for the synthesis of biochanin A and pratensein in MtIFS1-expressing plants (Fig. 1). HI4'OMT has been shown to methylate the 2,7,4'-trihydroxyisoflavanone product of IFS on the 4' position to form 2,7-dihydroxy-4'-methoxyisoflavanone, an intermediate in the synthesis of 5-deoxyisoflavonoids in legumes (Akashi et al., 2003 ). This enzyme is also predicted to methylate 5-hydroxyisoflavanones leading to the formation of biochanin A. I3'H from M. truncatula has been previously shown to hydroxylate biochanin A on the 3' position in vitro to form pratensein (Liu et al., 2003). Expression levels of HI4'OMT and I3'H in the different tissues of MtIFS1-expressing line C22 and vector control line VC11 were similar, suggesting that MtIFS1 expression does not alter the normal expression level of these genes.
The accumulation of phenylpropanoid compounds in response to UV-B light is well known, and UV-B induces isoflavonoids in legumes (Hadwiger and Schwochau, 1971
UV-B treatment led to the appearance of several peaks on HPLC chromatograms in both MtIFS1-expressing and vector control lines, consistent with induction of isoflavonoid biosynthesis (Fig. 6). Most notably, UV-B treatment induced two compounds of Rt = 19.6 and 46.2 min, identified as the 5-deoxyisoflavones daidzein and formononetin, respectively, in MtIFS1-expressing plants (Fig. 6B, peaks D and F, and Fig. 1). Formononetin is a known alfalfa phytoalexin (Paiva et al., 1994
Although genistein levels were not significantly different after UV exposure, UV-B treatment more than doubled the level of biochanin A (Fig. 7A) and increased total isoflavone levels over 2-fold in MtIFS1-expressing lines C22 and B20 (from 22.8–60.8 nmol g–1 FW for C22 and from 31.9–78.1 nmol g–1 FW for B20), 40% to 50% of the increase in total isoflavones being attributable to the accumulation of formononetin. The plants used in this experiment were maintained in a growth chamber, and the lower levels of genistein and biochanin A in their leaves (when compared to levels in greenhouse-grown plants; Fig. 4) may be attributed to the different environmental conditions, in particular the much higher light intensity in the greenhouse (detailed in "Materials and Methods"). The amount of formononetin accumulated in the UV-B-treated MtIFS1-expressing lines was more than 15-fold higher than in the vector control lines (Fig. 7B, white bars). However, total levels of 5-deoxy compounds induced in response to UV-B (formononetin and daidzein in MtIFS1-expressing lines; isoliquiritigenin, liquiritigenin, and dihydroxyflavone in vector control lines) were similar (Fig. 7B, black bars).
Response of MtIFS1-Expressing Plants to Phoma medicaginis
Infection with Phoma medicaginis, a fungal pathogen responsible for spring black stem and leaf spot disease of alfalfa (Leath, 1990
There was no change in the flavonoid profile of vector control plants sprayed with a control solution (0.1% Tween 20) over the 72-h period (data not shown). However, MtIFS1-expressing plants sprayed with the same solution accumulated a small amount of formononetin at 24 to 72 h (Fig. 8A). Formononetin was not detected in any of the samples harvested at the start of the experiment (T = 0), and its synthesis may be in response to the cool, humid conditions used during the experiment.
Spore inoculation had a similar effect on the leaf flavonoid composition as did UV-B treatment, the major difference being the significant accumulation of medicarpin in P. medicaginis-infected tissues (Fig. 8, B and D). Only traces of medicarpin were detected in UV-B-treated leaves (data not shown), which suggests that its accumulation in response to P. medicaginis may be related to its antifungal activity (Blount et al., 1993 MtIFS1-expressing plants accumulated much higher concentrations of formononetin and medicarpin than the vector control lines 24 to 72 h after infection (Fig. 9). Levels of formononetin, which ranged from 173 to 286 nmol g–1 FW in infected leaves of C22 and B20, were 4- to 7-fold higher than levels accumulating in the vector control lines and more than 2-fold higher than those of genistein. In infected leaves of VC11 and VB2, medicarpin represented a minor peak (Fig. 8D), whereas much higher levels were found to accumulate in MtIFS1-expressing lines (Fig. 9). Maximal levels of medicarpin in leaves of MtIFS1-expressing lines were observed 24 h after infection and exceeded levels observed in the vector control lines by >25-fold (Fig. 9).
Microarray Analysis of MtIFS1-Expressing Plants
Genistein is a known signaling molecule in soybean-rhizobium interactions (Pan et al., 1998
Pairwise comparisons of gene expression between lines C22 and VC11 and lines B20 and VC11 showed that, out of 16,086 spots, only 10 (C22 versus VC11) and 13 (B20 versus VC11) spots showed a statistically significant 2-fold or greater change in expression (P
To address whether the experimental and/or biological variation was too great to identify differentially expressed genes, we analyzed a set of housekeeping genes that were spotted 17 times per array and found that the within-array coefficient of variation (range from 9%–29%) was similar, but often slightly higher than the between-array variation (range from 4%–31%) when identical spots were compared. This suggests that the analytical and biological variation, although quite high, would not limit the statistical resolution of the experiment. We predicted that genes whose expression was regulated in response to genistein accumulation would be similarly up- or down-regulated in both MtIFS1-expressing lines when compared to vector control line VC11. Using this criterion, only one tentative consensus (TC) sequence (TC46175, up-regulated 2.5- and 2.7-fold) was identified with a statistically significant change in gene expression in lines C22 and B20 relative to the vector control line VC11 (Table I; Supplemental Tables I and II). However, we could not detect expression of this TC in any of the leaf samples used in the microarray experiment when examined by reverse transcription (RT)-PCR or northern gel-blot analysis. Furthermore, TC46175 could not be amplified from M. truncatula genomic DNA or leaf cDNA. TC46175 shares homology with the 3'-untranslated region of a ubiquitin/ribosomal extension protein, and it is not clear how expression of this TC may be related to isoflavone biosynthesis. From these results, we conclude that constitutive MtIFS1 expression does not significantly alter global gene expression in the leaves of alfalfa.
Factors Impacting Isoflavone Engineering in Plants
The numerous health benefits ascribed to dietary intake of soy isoflavones has generated significant interest in engineering these compounds in more widely consumed plants, despite conflicting evidence of their benefits to consumers (Dixon, 2004
Although MtIFS1 was expressed in all tissues examined, changes in isoflavonoid/flavonoid composition were, surprisingly, only detected in the leaves. This was unexpected based on a previous report that tobacco plants expressing a soybean IFS accumulated a higher concentration of genistein in flowers than in leaves, presumably due to the anthocyanin pathway supplying naringenin in flowers (Yu et al., 2000
In all reports of metabolic engineering of isoflavones, genistein accumulated as glycosides (Yu et al., 2000
Differences in the flavonoid composition of MtIFS1-expressing and vector control plants were even greater when these plants were subjected to UV-B or infection with P. medicaginis. In both cases, vector control and MtIFS1-expressing lines responded by accumulating the phytoalexins formononetin and medicarpin (the latter of which accumulated primarily in response to P. medicaginis), with levels of these isoflavonoids in MtIFS1-expressing plants greatly exceeding those in vector control lines (Figs. 7 and 9). In addition, MtIFS1-expressing plants accumulated daidzein, an isoflavone that was not detected in treated leaves of vector control plants and has not been reported to accumulate in alfalfa. The presence of daidzein in treated leaves of MtIFS1-expressing plants suggests that the activity of the 4'-O-methyltransferase immediately downstream of IFS in the pathway to formononetin and medicarpin (Fig. 1) may, under some conditions, be limiting. The instability of the 2-hydroxyisoflavanone product of IFS coupled with potential activity of a recently cloned 2-hydroxyisoflavanone dehydratase (Akashi et al., 2005
The variable chemical composition of research material has been acknowledged as a major factor contributing to inconsistencies in the literature regarding the health effects of isoflavones (Messina et al., 2004
Microarray analysis suggests that constitutive MtIFS1 expression in alfalfa leaves does not alter the expression of genes leading to new or altered levels of metabolites that might pose food safety concerns or limit the use of transgenic plants in feeding studies. The lack of a global alteration of gene expression in MtIFS1-expressing plants is favorable from the perspective of genetic engineering since it may provide support for substantial equivalence (Kuiper et al., 2001
Plant Material and Greenhouse Conditions
Plants were potted in Metro Mix 350 and grown in a controlled environment greenhouse with a 16-h photoperiod. The temperature was maintained at 20°C to 23°C during the day and 18°C to 20°C at night. Humidity was set at 50% during the day and 75% at night. During the course of these experiments, measured light intensity in the greenhouse ranged from 266 to 417 µmol m–2 s–1 photosynthetically activated radiation (PAR). Plants were watered daily with Peters 20:10:20 fertilizer (100 ppm N; Scotts, Marysville, OH) by flood irrigation. Independent transgenic lines were vegetatively propagated by cuttings as described (Hipskind and Paiva, 2000
The MtIFS1 cDNA was subcloned into the EcoRI/KpnI sites of the pRTL2 vector (Restrepo et al., 1990
Genomic DNA was extracted from 100 mg of leaf tissue using plant DNAzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. PCR was performed with 250 ng of genomic DNA and NPT-II-specific primers (Trieu et al., 2000
RNA was extracted from plant tissues using TRI reagent (Molecular Research Center, Cincinnati) according to the manufacturer's instructions. Twenty micrograms of RNA were fractionated on a 1% (w/v) formaldehyde-agarose gel and transferred to a nylon membrane by standard protocols (Ausubel et al., 1994 For RT-PCR, 2 µg of total RNA were transcribed into cDNA using ready-to-go RT-PCR beads (Amersham) and oligo(dT) primer. Two microliters of cDNA were used in each PCR reaction (50 µL total) with Ex-Taq PCR reagents (TaKaRa Shuzo, Shiga, Japan) and the following primers: actin, 5'-CAATTTCTCGCTCTGCTGAGGTGG-3', 5'-GGCTGGATTTGCTGGAGATGATGC-3'; MtIFS1, 5'-CCCATGAAGCTACTTCC-3', 5'-AGTCGTTCATGATAAGC-3'; TC54732, 5'-GTTCGTGCTTTACGGGTTGT-3', 5'-AAAGCAGGGGGCAACATAGT-3'; TC76580, 5'-GTGGCGCTAAGAAGCGTAAG-3', 5'-TCAAAACGCACAAAATTGTATTG-3'; TC56026, 5'-CGGTGTCGTGCTTTACCTTT-3', 5'-ACCTACACAATGCCCCTCAA-3'; TC52595, 5'-GTTTTTGCACCAACTGCAAC-3', 5'-GCCTCCACCCTTCTTCTTTT-3'; TC46499, 5'-TGCTAGTAAATGGTCTGCCAAT-3', 5'-GCTAGGCAACGCTAGGACAT-3'; and TC44464, 5'-GACCGAGACGGAAATAGCAG-3', 5'-ATGGAGCCGTTTTGTTCTTG-3'. PCR conditions were 94°C, 5 min, 25 to 32 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, followed by 72°C for 10 min. PCR products were resolved on a 1% (w/v) Tris-acetate EDTA-agarose gel, visualized with ethidium bromide, and band intensities were quantified using LabWorks software version 4.0 (UVP, Upland, CA). Band intensities were normalized relative to actin.
Plant tissue was routinely harvested in the greenhouse, snap frozen in liquid N2, and stored at –80°C. Tissues were ground in liquid N2 and 0.5 to 1.0 g extracted two times in 10 volumes of 80% (v/v) MeOH. Extracts were combined and dried under a stream of N2. For analysis of isoflavone conjugates, residues were resuspended by vortexing in 80% or 100% MeOH, briefly sonicated, and clarified by centrifugation before loading on the HPLC. For digestion with
Cuttings rooted in 96-well flats were grown in a growth chamber under fluorescent lighting (approximately 150–255 µmol m–2 s–1 PAR). The chamber was set to a 16-h photoperiod, with a day/night temperature of 23°C/19°C, and 50% humidity. Plants (140- to 260-cm tall over the course of three independent experiments) were exposed to UV-B light (XX-15MR bench lamp, MR 302 nm, 115 V; UVP) for 6 h. The intensity of the light as measured with a 310-nm UV meter was 0.370 to 0.734 mW cm–2 from the top of the soil to the average maximal plant height. Prior to the start of the experiment, the fluorescent lighting in the chamber was lowered to half-intensity (approximately 80–130 µmol m–2 s–1 PAR). Control plants (non-UV treated) were kept in a separate growth chamber under identical conditions. In each experiment, leaves from three replicate plants were pooled and frozen in liquid N2 and the experiment was repeated three times. Flavonoid extraction and hydrolysis were performed as described above. Hydrolyzed isoflavones were further purified using C18 cartridges (Waters Sep-Pak C18 cartridges; Waters, Milford, MA) as follows. Extracts, dissolved in a small volume of MeOH, were diluted 1,000-fold with water and loaded on a C18 cartridge that had been preconditioned by washing with 10 volumes of MeOH followed by water. After washing with water, flavonoids were eluted with 6 volumes of MeOH. Eluates were dried under N2 and residues resuspended in 100 µL MeOH. Samples were loaded on the HPLC and eluted in 1% (v/v) phosphoric acid with an increasing gradient of acetonitrile (0 to 5 min, 25%; 5 to 80 min, 25% to 45%; 80 to 81 min, 45% to 100%) at a flow rate of 0.8 mL min–1.
Phoma medicaginis Malbranche et Roumeguere was from the American Type Culture Collection (Manassas, VA). Conidial suspensions of P. medicaginis were plated on potato dextrose agar and incubated in the dark at 21°C until sporangia were visible (approximately 4–6 weeks). Spores were collected by flooding the surface of the plate with a sterile 0.1% (v/v) Tween 20 solution followed by gentle scraping with a spatula. Chunks of agar and mycelia were removed by filtration. Spore concentrations, as determined by hemacytometer counting, were routinely 1 to 2 x 108 spores mL–1. Plants were sprayed with the spore suspension or a control solution (0.1% Tween 20) in a growth chamber until dripping wet and bagged for 24 h. The chamber was set to a 12-h photoperiod with a day/night temperature of 21°C/16°C and 95% humidity, conditions reported to be optimal for growth chamber infection of Medicago sativa with P. medicaginis (Barbetti, 1987
The Medicago Genome Oligo Set version 1.0 (Qiagen Operon, Alameda, CA) includes 70-mer oligonucleotides representing 16,086 M. truncatula TC sequences from TIGR Gene Index Database (www.tigr.org/tdb/tgi/mtgi) MtGI Release 5.0, as well as both positive and negative controls. Details of this oligo set are available at http://oligos.qiagen.com/arrays/omad.php. The oligos were spotted in 3x SSC onto Superamine (Telechem, Sunnyvale, CA) glass slides by the Galbraith lab at the University of Arizona (Tucson, AZ). A single array element was printed per oligo. After printing, slides were baked at 80°C for 1 h and stored at room temperature. Prior to use, slides were UV-cross-linked at 200 mJ and prehybridized in 5x SSC, 0.1% (w/v) SDS, and 1% (w/v) bovine serum albumin for 45 min at 42°C. Slides were then rinsed five times in distilled, deionized water, spun dry, and used immediately. Approximately 90% of spots on this array showed significant signal when hybridized with labeled alfalfa leaf cDNA. PolyA RNA was isolated from 400 µg of total leaf RNA using the Poly(A) Purist MAG kit from Ambion (Austin, TX). Two micrograms of polyA were transcribed into cDNA and fluorescently labeled with Cy3 and Cy5 dyes using the SuperScript indirect cDNA labeling system (Invitrogen) according to the manufacturer's instructions. Labeled cDNA for experimental (Cy5) and reference (Cy3) samples were combined, ethanol precipitated, and resuspended in 10 µL of water. The cDNA was heated at 100°C for 3 min before the addition of 70 µL of SlideHyb glass array hybridization buffer number 1 (Ambion). The hybridization solution was pipetted onto slides covered with a gapped coverslip (LifterSlips; Erie Scientific Company, Portsmouth, NH). Slides were incubated overnight at 42°C in Corning hybridization chambers, then washed in 1x SSC, 0.1% (w/v) SDS for 5 min at 42°C, followed by 5-min washes in 0.1x SSC, 0.1% SDS (w/v), and 0.05x SSC at room temperature. Slides were scanned using the ScanArray 4000 microarray analysis system. Based on an initial low-resolution (50 µm) scan, laser power settings were manually adjusted to balance the intensities between the two channels and slides were rescanned at 10-µm resolution. Typical laser settings were Cy3 laser power = 100, photomultiplier tube = 80; Cy5 laser power = 85, photomultiplier tube = 75. GPR files were generated using GenePix Pro 4.1 software and the mean pixel intensities for each spot were used in subsequent data analysis. Data files were imported into GeneTraffic Duo version 2.6, and data for each hybridization were normalized using the Lowess SubGrid method. Spots flagged by the program under the default settings (spots with a raw pixel intensity <100 or lower than the average background and spots with an intensity/background intensity ratio <1) were removed from further analysis. Significance analysis was done in GeneTraffic by performing two class, unpaired t tests with the variance stabilization and Benjamini-Hochberg P-value correction options. Analysis was first performed on a dataset that excluded spots lacking one or more value over the three replicates. A second analysis was performed on a dataset that included spots missing one value and in which case the missing value was imputed by determining its k-nearest neighbor. This second analysis increased the number of spots analyzed by approximately 2,000 and increased the number of significant spots by 1 to 5, depending on the comparison. Four TCs (TC54732, TC56026, TC57633, TC58892) that were initially identified with a statistically significant change in gene expression in either one or both MtIFS1-expressing lines were seen to be spotted in close proximity to the spot corresponding to IFS (TC45135) on the arrays used in this experiment. Expression of these spots could not be verified by RT-PCR or RNA gel-blot analysis, and the TCs were not identified as differentially expressed in a second experiment using a different array batch. Because of the possibility that the potential up-regulation of these TCs was artifactual, they were eliminated from our analysis. Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requester. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY939826 (MtIFS1). Gene expression data have been deposited with the GEO repository under accession number GSE2546.
We thank Jack Blount for help with HPLC analysis, Dr. Naveed Aziz for help with microarrays, David Huhman and Dr. Lloyd Sumner for assistance with LC-MS analysis, Yuanji Zhang for BLAST analysis, Corey Broeckling and members of the Dixon lab for helpful discussions, and Drs. Li Tian and Rujin Chen for critical review of the manuscript. Received March 8, 2005; returned for revision April 19, 2005; accepted May 6, 2005.
1 This work was supported by a grant from the Oklahoma Center for the Advancement of Science and Technology (project no. HR02–040R to R.A.D.) and by the Samuel Roberts Noble Foundation. 
[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.105.062539. * Corresponding author; e-mail radixon{at}noble.org; fax 580–224–6692.
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