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© 2004 American Society of Plant Biologists Tissue-Specific Gene Silencing Mediated by a Naturally Occurring Chalcone Synthase Gene Cluster in Glycine max
a Program in Physiological and Molecular Plant Biology, University of Illinois, Urbana, Illinois 61801 3 To whom correspondence should be addressed. E-mail l-vodkin{at}uiuc.edu; fax 217-333-4582.
Chalcone synthase, a key regulatory enzyme in the flavonoid pathway, constitutes an eight-member gene family in Glycine max (soybean). Three of the chalcone synthase (CHS) gene family members are arranged as inverted repeats in a 10-kb region, corresponding to the I locus (inhibitor). Spontaneous mutations of a dominant allele (I or ii) to a recessive allele (i) have been shown to delete promoter sequences, paradoxically increasing total CHS transcript levels and resulting in black seed coats. However, it is not known which of the gene family members contribute toward pigmentation and how this locus affects CHS expression in other tissues. We investigated the unusual nature of the I locus using four pairs of isogenic lines differing with respect to alleles of the I locus. RNA gel blots using a generic open reading frame CHS probe detected similar CHS transcript levels in stems, roots, leaves, young pods, and cotyledons of the yellow and black isolines but not in the seed coats, which is consistent with the dominant I and ii alleles mediating CHS gene silencing in a tissue-specific manner. Using real-time RT-PCR, a variable pattern of expression of CHS genes in different tissues was demonstrated. However, increase in pigmentation in the black seed coats was associated with release of the silencing effect specifically on CHS7/CHS8, which occurred at all stages of seed coat development. These expression changes were linked to structural changes taking place at the I locus, shown to encompass a much wider region of at least 27 kb, comprising two identical 10.91-kb stretches of CHS gene duplications. The suppressive effect of this 27-kb I locus in a specific tissue of the G. max plant represents a unique endogenous gene silencing mechanism.
Homology-dependent gene silencing (HDGS) is a comprehensive term that refers to epigenetic silencing effects based on recognition of nucleic acid sequence homology at either the DNA (transcriptional) or RNA (posttranscriptional) level (Meyer and Saedler, 1996  ). First discovered in transgenic plants of Petunia, where it was termed cosuppression (Jorgensen, 1990), it is now recognized that HDGS is a commonly observed outcome of transgenesis in plants. Originally proposed and documented as the immune system of the genome (reviewed in Plasterk, 2002; Wassenegger, 2002), several emerging lines of evidence are implicating the various components of the HDGS machinery to be a part of a sophisticated network of normal regulation of endogenous genes (Jacobsen et al., 1999; Reinhart et al., 2002; Carrington and Ambros, 2003).
Posttranscriptional gene silencing (PTGS) is one form of HDGS that is characterized by sequence-specific degradation of cytoplasmic RNA (Meins, 2000
Extensively studied in several plant species, anthocyanins form a group of secondary metabolites, serving a variety of functions in flowers, fruits, and seeds. In G. max, three genetic loci (I, R, and T) are known to control pigmentation of the seed coat (Bernard and Weiss, 1973
Though all wild Glycine accessions have pigmented seed coats (recessive i allele), most cultivated G. max varieties have been selected for a yellow seed coat (homozygous I or ii). Several spontaneous independent mutations have frequently occurred in cultivars having either the I or ii alleles (yellow seed coats or yellow seed coats with pigmented hila) to give rise to the recessive i alleles (pigmented seed coats). Some of these mutations have been preserved as near isogenic lines differing only in respect to the alleles at the I locus (Wilcox, 1988
Analysis of these isogenic pairs has shown that the I locus corresponds to a region of chalcone synthase (CHS) gene duplications (Todd and Vodkin, 1996
In this article, we demonstrate that silencing of chalcone synthase expression in the dominant I allele takes place in a tissue-specific manner. Secondly, we also determine the relative expression profile of the CHS gene family members in seed coats and cotyledons of the isogenic pairs and attribute the increase in seed coat pigmentation in the recessive i allele to increased expression of CHS7/CHS8. Finally, we corroborate these changes in CHS expression profile and consequential seed coat pigmentation to structural changes taking place at the chalcone synthase inverted repeat of not three but six CHS genes comprising the I locus.
The Dominant I Allele Inhibits CHS Expression Only in the Seed Coats Previous work had shown that the I locus, corresponding to a region of chalcone synthase gene duplications, in its dominant form prevents the accumulation of anthocyanins in the G. max seed coat (Wang et al., 1994 ; Todd and Vodkin, 1996). Evidently, chalcone synthase, a key regulatory enzyme in the flavonoid pathway, is not only involved in the production of pigment in the seed coat, but plays a significant role in the synthesis of fundamental secondary metabolites functioning as UV protectants, phytoalexins, insect protectants, and symbiosis initiators in various plant tissues. Therefore, it was pertinent to analyze chalcone synthase expression levels in other plant tissues. We examined the steady state CHS mRNA levels in stem, root, young leaf, old leaf, and young pod of the isogenic pair Richland (IRt, yellow seed coat) and T157 (iRt, imperfect black seed coat) by probing RNA gels with an open reading frame CHS probe. The results shown in Figure 1A revealed that in both isolines, roots displayed the highest levels of CHS expression, and reasonable amounts also were detected in young pods and old leaves. Much weaker hybridization to the 1.4-kb RNA was detected in the young leaf and stem. Significantly, no obvious differences of expression were found in CHS transcript levels between the different tissues of the two isolines, thereby suggesting that the suppressive effect of the dominant I allele on CHS expression is absent at least in the vegetative plant parts and the young pod.
To further elucidate the tissue-specific nature of the silencing effect of the I locus, we decided to analyze CHS expression levels in the developing cotyledons. It has been shown that steady state CHS mRNA levels are reduced in yellow seed coats relative to the pigmented seed coats for all developmental stages (Wang et al., 1994 ). We therefore compared levels of CHS expression in the seed coats and cotyledons at various stages of seed development for the two isolines, Richland (I) and T157 (i). Hybridization of RNA gel blots to the generic CHS probe revealed that the levels of CHS transcripts did not vary between the cotyledons of the two isolines at all stages of seed development (Figure 1B, panel 1). In both Richland and T157, CHS was expressed at the highest levels very early during seed development (10 to 75 mg) and gradually decreased as the seed matured (100 to 200 mg). However, in striking contrast with the cotyledons, there was a drastic reduction of the 1.4-kb CHS mRNA in seed coats of Richland (I) relative to T157 (i) throughout all developmental stages (Figure 1B, panel 2). CHS transcript levels were highest during very early stages of seed coat development (10 to 75 mg) and decreased as the seed started to reach maturity (75 to 200 mg) in the pigmented isoline T157 (Figure 1B, panel 2). These results suggest that the gene silencing effect of the dominant I allele is seed coat specific.
High Nucleotide Similarity in the CHS Open Reading Frames Necessitates Design of Gene-Specific Primers and Probes in the 5' and 3' Untranslated Regions of the Multigene Family
Nucleotide sequences of the eight CHS genes, entered into GenBank (Akada et al., 1990a
The extreme homology among the gene family members necessitated the use of a sensitive and reproducible method, TaqMan RT-PCR. Gene-specific primers and probes were designed for CHS1 to CHS6 either in the 5' upstream region or the 3' untranslated region (Table 2). Considering nearly 97% homology between the other two members, CHS7 and CHS8, a common primer-probe set (CHS7/CHS8) spanning a portion of the second exon was used to quantify both CHS7 and CHS8. Additionally, primers and probes also were designed for phosphoenolpyruvate carboxylase (PEPC16) to be used as the endogenous control. Phosphoenolpyruvate carboxylase (PEPC), a ubiquitous cytosolic enzyme in higher plants, catalyzes the irreversible carboxylation of phosphoenolpyruvate to yield oxaloacetate and Pi. It forms a highly similar four-member gene family in G. max (Hata et al., 1998
CHS Gene Family Members Are Expressed in all Tissues but at Varying Levels Though multiple CHS genes have been shown to be transcribed in various tissues in some studies, it has been reported for other plants that only one or two members of the gene family account for most of the mRNA produced (Koes et al., 1986 ; Fukada-Tanaka et al., 1997). This is in agreement with the presence of single CHS genes in Arabidopsis (Arabidopsis thaliana) and Petroselinum crispum (parsley). To ascertain the tissue-specific expression profile, we investigated the relative expression levels of the CHS gene family members in different tissues of our standard G. max cultivar, Williams 43 (ii, yellow seed coats with pigmented hilum) using the gene-specific primers and probes designed for the TaqMan assay. Figure 2 shows the expression levels of all CHS genes in four tissues, namely roots, young leaves, cotyledons, and seed coats. Our results indicate that all members of the CHS family are transcribed, though their relative mRNA amounts might vary considerably from tissue to tissue. On the whole, significantly higher total CHS transcript levels were detected in roots than in any other tissue (Figure 2A). These results substantiate those from RNA gel blot analysis on another yellow seed coat line (Richland, I) presented above (Figure 1). CHS transcripts also have been found in roots of Pisum sativum (pea) and parsley (Koes et al., 1989; Wingender et al., 1989; Harker et al., 1990; Estabrook and Sengupta-Gopalan, 1991). The higher CHS transcript levels in roots reflects the obvious function of synthesis of flavonoid derivatives in the legume roots, which are secreted into the rhizosphere and induce the nod genes of Rhizobium. Moderate accumulation of CHS RNA was observed in seed coats and cotyledons, and very little was found in leaves (Figures 2B to 2D).
At the transcript level, maximal amount of CHS7/CHS8 expression was detected in roots, followed by seed coats (Figures 2A and 2B). In the cotyledons, on the other hand, CHS2 was nearly fourfold more abundant than CHS7/CHS8 (Figure 2D). The other green tissue, leaves, predominantly expressed CHS1 and CHS7/CHS8 (Figure 2C). Of the three CHS genes constituting the 10-kb inverted duplicated repeat (CHS1, CHS3, and CHS4) (Akada and Dube, 1995 ), CHS1 and CHS3 transcripts were low to moderate abundance in the four tissues. However, CHS4 transcripts were primarily low abundance in all the tissues.
Pigmentation in the Seed Coats Results from Release of the Silencing Effect Primarily on CHS7/CHS8 Expression
ii
I  i MutationsThe second set of spontaneous mutations, whereby mutation of the dominant I allele (yellow seed coats) to the recessive i allele (pigmented seed coats) has been correlated with loss of the duplicated CHS1 promoter (Todd and Vodkin, 1996 ), were analyzed using the two pairs Richland-T157 and UC31-UC33 (Figure 4A). As observed in the first set of spontaneous mutations, there was a significant increase in total CHS transcripts in the pigmented seed coats upon release of silencing (Figure 4A). The increased seed coat pigmentation in the i isolines T157 and UC33 relative to their nonpigmented counterparts Richland and UC31, respectively, was attributed mainly to the higher expression of CHS7/CHS8 and CHS2, the former contributing to as much as a 15-fold increase in T157 and 25-fold increase in UC33 (Figure 4A). The other CHS genes were expressed at fairly low levels, with the exception of CHS6, which was not detected both in UC31 and UC33 (Figure 4A). Also, CHS5 transcripts, absent in the yellow seed coats, were detected at a very low level in the black seed coats. Of the two pairs, CHS4 transcripts were not detected in cultivar UC33, which has been shown to maintain the restiction fragment length polymorphism (RFLP) pattern of its nonrecurrent parent, UC9, exhibiting the loss of the 2.3-kb CHS4 fragment (Todd and Vodkin, 1996).
TaqMan Expression Profiling of the CHS Gene Family Members in Cotyledons Confirms the Seed Coat–Specific Suppressive Effect of the I Locus Having attributed pigmentation to increased expression of CHS7/CHS8 in the seed coats, we investigated relative transcript levels of members of this multigene family in a nonsilencing representative tissue, cotyledons (50 to 75 mg seed fresh weight), using TaqMan RT-PCR (Figures 3B and 4B). In cotyledons of both sets of spontaneous mutations, though no significant differences in the total CHS transcript levels were observed among the pigmented and nonpigmented isolines using RNA gel blots (Figure 1B), differences at the individual transcript level were detected in all four pairs using TaqMan RT-PCR (Figures 3B and 4B). Thus, a relative increase in a particular transcript level in the pigmented genotype was being compensated for by a decrease in expression of another member. Unlike the seed coats, CHS2 expression levels were predominantly higher than any of the other genes in all the genotypes, whereas CHS7/CHS8 and CHS4 were moderately expressed (Figures 3B and 4B). The relative levels of CHS2 decreased in the pigmented isoline of one pair (Williams 44) but increased in the other pigmented isoline (Williams 55), though not significantly. A similar kind of variation in expression was observed for many other genes (CHS1, CHS5, and CHS7/CHS8). However, no specific pattern of increase or decrease of transcripts could be identified among the four pairs. This analysis further substantiated the results from RNA gel blots presented above (Figure 1), thereby reinforcing that the suppressive effect of the I locus is seed coat specific.
Silencing Effect in the Seed Coats Is Mediated at All Stages of Seed Development
The striking feature, however, was a significant difference in CHS7/CHS8 transcript levels between the pigmented and the hilum pigmented seed coats at all stages of development (Figure 5A). This difference in expression rose from as much as fourfold in the earliest stage of seed coat development (25 to 50 mg) to almost 24-fold in the mature seed. Contrary to significantly higher (twofold to threefold) transcript levels of CHS2 in the pigmented seed coats than the hilum pigmented ones, CHS3 expression differences were not considerable (Figure 5B). These results clearly illustrate that a release of the silencing effect of the ii allele is manifested via an increased expression of most CHS genes at all stages of seed coat development, with CHS7/CHS8 being the key player.
Structural Changes Associated with the 27-kb Duplication Region in Different Alleles of the I Locus The presence of an exact duplication of the 10.91-kb CHS inverted repeat in the G. max line carrying the ii allele raised important questions about the presence of this structure in the other I locus alleles and how it might affect seed coat pigmentation. To look for the presence/absence of this 27-kb inverted repeat structure, PCR amplifications of genomic DNA from the four pairs of isogenic lines were performed.
Of the two isogenic pairs analyzed for the first set of spontaneous mutations (ii to i mutation), Williams 55 (i) in comparison to Williams 54 (ii, which was found to contain an intact duplicated repeat) showed complete absence of the second cluster along with the spacer region (Figures 6 and 7). With the tested primers, the deletion extended into the first cluster to as close as 36 bases of the start codon for CHS4, thereby abolishing most of the promoter, including the HindIII site on its way. This is in agreement with the absence of the 2.3-kb HindIII fragment in the CHS RFLP patterns observed by Todd and Vodkin (1996)
On the other hand, in the other isogenic pair, Williams 43-Williams 44, both the genotypes seemed to show the presence of the two clusters along with the spacer region. Though we could not amplify a 3.8-kb region extending from the promoter of CHS4 in the first cluster into the spacer region (fragment 4) as one intact band in Williams 44, we could successfully amplify this large stretch of DNA as three contiguous subfragments in both Williams 43 and Williams 44 (Figure 7).
PCR amplification studies of the second class of I locus mutations, for example, from a yellow seed coat (I) to a fully pigmented seed coat (i), were performed using the isogenic pair Richland-T157 and UC31-33. In the Richland-T157 pair, presence of the duplicated CHS clusters was verified by the amplification of all fragments except for fragment 8. The 2.59-kb fragment 8, extending further into the 3' end of the second cluster, could not be amplified in both Richland and T157 (Figure 7). However, some subfragments within this 2.59-kb region amplified in Richland. The absence of the 2.59-kb fragment could potentially indicate some structural divergence downstream of the 3' end of the second cluster, which may or may not play a role in relieving gene silencing. This hypothesis is supported from RFLP data on the dominant I allele (in Richland), demonstrating the presence of an extra 12.1-kb HindIII fragment that hybridizes to both the CHS1 promoter and the coding region probe, thereby indicating the existence of an extra CHS1 very close to the I locus. A deletion in the promoter of this duplicated CHS1 (referred to as dCHS1 or ICHS1) leading to a shift from 12.1 kb to either a 10.6- or 9.4-kb HindIII fragment, restored a higher level of CHS mRNA in the seed coats (Todd and Vodkin, 1996 DNA from the second pair in this set of spontaneous mutations, UC31-UC33, also was amplified using the nine primer sets to verify the existence of the 27-kb inverted repeat structure (Figure 7). As mentioned in Table 1, UC31 and UC33 are near isogenic lines. Both these lines have been created from repeated backcrossing of the I and i alleles from the lines T201 (I) and UC9 (i) into Clark ii as the recurrent parent. UC31 and UC33 therefore showed the pattern of their respective nonrecurrent parents on PCR amplification of genomic DNA with the nine primer pairs. UC31 showed the absence of fragment 8 as seen in Richland, whereas UC33, harboring the recessive i allele, showed the complete absence of the 10.91-kb CHS cluster B and the spacer region, as seen in Williams 55.
Expression of Closely Related CHS Gene Family Members in Different G. max Tissues as Determined by TaqMan RT-PCR Of considerable study in plants, chalcone synthase, a member of the plant polyketide superfamily, catalyzes the first committed step of flavonoid biosynthesis. In most dicots, particularly the legumes, chalcone synthase forms a 6- to 12-member multigene family, though it appears to consist of only two copies in the very limited number of grass species studied (Wienand et al., 1986 ; Rohde et al., 1991; Christensen et al., 1998).
Sequence analysis of CHS genes cloned from several plant species has shown them to share a high degree of nucleotide sequence homology. As part of this study, at least 80% nucleotide identity was observed among all the eight members of the G. max chalcone synthase gene family. Furthermore, sequence analysis classified the gene family into two highly homologous subgroups based on nucleotide similarity in the open reading frame. Previous phylogenetic analysis of
Although total CHS expression has been widely documented to be developmentally and environmentally regulated in many plant species (van der Meer et al., 1993
Evolution of multigene families has been explained as a gene duplication process that involves the acquisition of new enzymatic activities. Our results with tissue-specific expression profiling show that all CHS genes are transcribed and have diversified expression profiles, although the predicted polypeptides encoded by this gene family are fairly constant. Presumably, this diversification is more closely associated with specialization of tissue-specific expression than to variation in enzymatic functions. Additionally, CHS7/CHS8 are the most abundant transcripts in roots. Confirmatory evidence for this is provided by the analysis of root EST libraries from the G. max EST project (Shoemaker et al., 2002
Existence of Tandem CHS Clusters in the Different I Locus Isolines
Considering the dynamics of this chalcone synthase gene–rich region and its effect on G. max seed coat pigmentation, we sequenced and assembled a 103-kb BAC clone, BAC104J7, harboring the ii allele from cultivar Williams. This BAC clone was found to represent a very gene-rich region of the G. max genome, with ORFs separated on average by
A recent study has presented evidence that recruitment of new duplicate CHS genes is occurring at a remarkably rapid rate in grass genomes, and these novel genes have been indicated to be functional (Oberholzer et al., 2000
Furthermore, though the two maize CHS genes, c2 and whp are believed to be the result of an allotetraploid event that occurred some 15 million years ago (Gaut and Doebley, 1997
The Highly Structured 27-kb I Locus Directs Silencing of CHS7/CHS8 Specifically in the Seed Coats
Previously, Todd and Vodkin had associated spontaneous mutations of the dominant I and ii to the nonsilencing i locus to deletions in the promoters of CHS4 and ICHS1, respectively (Todd and Vodkin, 1996
Toward determining the precise nature of the I locus–mediated silencing mechanism operating in the G. max seed coat, in this article, our investigations on the relative expression profile of CHS gene family members show that the three CHS genes comprising the I locus are transcribed at comparable although low levels in both the pigmented and the nonpigmented isolines. It has been shown in IR-induced PTGS in transgenic P. hybrida that a high level of transcription of CHS genes is not essential (Stam et al., 1997
Accordingly, it could be speculated that the transcriptional orientation of the two genes comprising the inverted repeat, CHS3 and CHS4, converges, which makes it possible to synthesize dsRNA by read-through transcription. Furthermore, it is not hard to envision the origin of dsRNA from this 27-kb stretch of DNA, comprising six genes in multiple tandem or inverted orientations. Consistent with this hypothesis of dsRNA generation from the inverted CHS repeats, we observed deletion of the second 10.91-kb CHS cluster, extending into the spacer region and further into as close as 36 bp of the start codon of CHS4 in the first cluster, thereby eliminating most of the promoter. This deletion event from the ii
In the case of I
A widely accepted feature of dsRNA-induced gene silencing is that the dsRNA is cleaved into small RNAs of 21 to 25 nucleotides, known as siRNAs (Hamilton and Baulcombe, 1999 Multiple alignments of the CHS gene family members reveal quite a few stretches of 21 to 25 bp with 100% identity. Furthermore, CHS7 and CHS8 are nearly 80% identical in the transcribed region to the other gene family members. Thus, it can be speculated that the siRNAs triggering sequence-specific degradation of CHS7/CHS8 transcripts originate from the CHS dsRNA from the CHS1/CHS3/CHS4 cluster regions. However, we do not observe complete absence of the CHS7/CHS8 or CHS2 transcripts in the yellow seed coats. This could possibly be explained by the ability of the cells to sense and maintain a critical threshold of transcription of CHS because it is a key metabolic enzyme in the phenylpropanoid pathway leading to the production of other functional proteins involved in plant defense.
Significantly, as part of this study, using two different techniques for measuring CHS transcript levels in the various plant tissues of both the black and yellow isolines, we have provided compelling evidence for the I locus–mediated silencing effect to be seed coat specific, showing IR-mediated tissue-specific silencing in an endogenous plant system. In many transgenic plants, variable and spatial patterns of PTGS have been observed, for example, the simple and complex vein patterns of flower pigmentation of CHS PTGS in P. hybrida (Que et al., 1997
Many studies have documented the ability of certain viral proteins in overcoming the plant antiviral defense mechanism and thereby suppressing RNA silencing (Anandalakshmi et al., 1998 To summarize, we have described an endogenous duplicated IR repeat system in G. max that drives silencing of CHS genes in a novel tissue-specific manner, thereby inhibiting pigmentation of the seed coats. Further study of this system should provide insight into the mechanism of tissue-specific gene silencing, which could potentially be of practical use to target silencing to a restricted tissue/cell type.
Plant Materials and Genetic Nomenclature All isolines of G. max used for this study are described in Table 1. They were obtained from the USDA Soybean Germplasm Collections (Department of Crop Sciences, USDA Agricultural Research Service, University of Illinois), which totals >18,000 plant introductions. All lines are homozygous for the loci indicated, and for brevity, only one allele is indicated in the text and figures. Plants were grown in the greenhouse, and tissue was harvested from at least four plants of each isoline. Shoot tips, leaves, and roots were harvested from 4-week-old plants and frozen in liquid nitrogen. For seed coats and cotyledons, pods of mature plants were first harvested and seeds extracted from the pods and divided into the following categories by fresh weight of the entire seed: 10 to 25 mg, 25 to 50 mg, 50 to 75 mg, 75 to 100 mg, and 100 to 200 mg. Seed coats and cotyledons were then dissected from the seeds and frozen in liquid nitrogen. The frozen tissues were then freeze dried (Multi-Dry Lyophilizer; FTS Systems, Stone Ridge, NY) and stored at –20°C.
RNA Extraction and Gel Blot Analysis
For RNA gel blot analysis, 10 µg of total RNA containing 400 ng of ethidium bromide was electrophoresed through 1.2% agarose/3% formaldehyde gels (Sambrook et al., 1989
The probe for CHS was a 2.0-kb HindIII fragment prepared from a genomic CHS subclone, pC2H2.0, which was isolated with a P. vulgaris gene and identified by sequence data to have 90% sequence similarity to the P. vulgaris gene (Frank and Vodkin, 1988
Real-Time Quantitative RT-PCR A 1-µg aliquot of purified RNA was then reverse transcribed in quadruplicate in a 20-µL reaction volume using the Superscript II first strand cDNA synthesis system for RT-PCR according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Parallel reactions for each RNA sample were run in the absence of Superscript II (no RT control) to assess any genomic DNA contamination. The reaction was terminated by heat inactivation at 70°C for 15 min. Subsequently, the cDNA product was treated with 2 units of RNase H for 20 min at 37°C. The cDNA samples for each tissue were pooled and stored at –20°C.
Design of Gene-Specific TaqMan Primers and Probes To design gene-specific primers and probes, a detailed analysis of the nucleotide sequences of the eight CHS genes was performed using Sequencher version 4 (Gene Codes, Ann Arbor, MI). Keeping in mind the extensive homology among the CHS gene family members, gene-specific regions (5' upstream sequences or 3' untranslated regions) were identified. Primer Express (Applied Biosystems, Foster City, CA) was then used to design gene-specific primer-probe sets for genes CHS1 to CHS6, whereas the seventh primer-probe set was used to quantify both CHS7 and CHS8 transcript levels. Additonally, primers and probes specific to the endogenous reference PEPC16 also were designed. The amplified products from these primer-probe sets ranged in size from 81 to 114 bp. The 5' and 3' ends of these probes were labeled with fluorescent dyes FAM (6-carboxyfluorescein) and TAMRA (6-carboxy-tetramethyl-rhodamine), respectively. Also, BLASTN searches against dbEST and nr (nonredundant set of GenBank, EMBL, and DDJB database sequences) were conducted to confirm the total gene specificity of the nucleotide sequences chosen for the primers and probes. The fluorescent probes were ordered from Applied Biosystems, and the forward and reverse primers were synthesized at the Keck Center (University of Illinois Biotechnology Center). Details of these are presented in Table 2.
PCR Amplification
Data Analysis
Confirmation of Primer/Probe Specificity
DNA Isolation and PCR Amplifications PCR amplifications were performed in a 50-µL reaction volume using 500 ng of genomic DNA in the presence of 1x PCR buffer, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 2.5 units of Taq polymerase (Invitrogen), and 0.4 µM forward and reverse primers. PCR reactions were conducted in a PTC-100 programmable thermocycler (MJ Research, Watertown, MA) via an initial denaturation step at 96°C for 2 min followed by 40 cycles of denaturing at 96°C for 20 s, annealing at 55°C for 1 min, and polymerization at 72°C for 2 min, to end with a 7-min extension at 72°C. The amplified reactions were separated on a 1% agarose gel and bands visualized via ethidium bromide staining.
We are grateful for the Physiological and Molecular Plant Biology Fellowship Award from the Interdisciplinary Physiological and Molecular Plant Biology Program, University of Illinois, to J.H.T. We thank Mark Band for advice on TaqMan RT-PCR. This work was supported by grants from the Illinois Soybean Program Operating Board, University of Illinois Research Board, and National Science Foundation (DBI9872565) to L.O.V.
1 Current address: USDA Agricultural Research Service, University of Illinois, Urbana, IL 61801. 
2 Current address: Department of Medicine, Washington University, St. Louis, MO 63130.
Online version contains Web-only data. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Lila Vodkin (l-vodkin{at}uiuc.edu). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.021352. Received January 28, 2004; accepted February 10, 2004.
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ABSTRACT
INTRODUCTION
90 cDNA sequences encoding CHS from various plant species also classified the G. max genes CHS1, CHS2, CHS3, CHS4, CHS5, and CHS6 as a closely related cluster from the more distantly related CHS7 (Fukada-Tanaka et al., 1997
library screen of the G. max cultivar Miyagi shirome (I) (Senda et al., 2002a
-32P]dATP by random primer reaction method (Feinberg and Vogelstein, 1983
