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First published online November 10, 2006; 10.1104/pp.106.086306 Plant Physiology 143:326-338 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Transcriptome Analysis Reveals a Critical Role of CHS7 and CHS8 Genes for Isoflavonoid Synthesis in Soybean Seeds1,[W],[OA]Southern Crop Protection and Food Research Center, Agriculture and Agri-Food Canada, London, Ontario, Canada N5V 4T3
We have used cDNA microarray analysis to examine changes in gene expression during embryo development in soybean (Glycine max) and to compare gene expression profiles of two soybean cultivars that differ in seed isoflavonoid content. The analysis identified 5,910 genes that were differentially expressed in both soybean cultivars grown at two different locations for two consecutive years in one of the five different stages of embryo development. An ANOVA analysis with P value < 0.05 and < 0.01 indicated that gene expression changes due to environmental factors are greater than those due to cultivar differences. Most changes in gene expression occurred at the stages when the embryos were at 30 or 70 d after pollination. A significantly larger fraction of genes (48.5%) was expressed throughout the development and showed little or no change in expression. Transcript accumulation for genes related to the biosynthesis of storage components in soybean embryos showed several unique temporal expressions. Expression patterns of several genes involved in isoflavonoid biosynthesis, such as Phenylalanine Ammonia-Lyase, Chalcone Synthase (CHS) 7, CHS8, and Isoflavone Synthase2, were higher at 70 d after pollination in both the cultivars. Thus, expression of these genes coincides with the onset of accumulation of isoflavonoids in the embryos. A comparative analysis of genes involved in isoflavonoid biosynthesis in RCAT Angora (high seed isoflavonoid cultivar) and Harovinton (low seed isoflavonoid cultivar) revealed that CHS7 and CHS8 were expressed at significantly greater level in RCAT Angora than in Harovinton. Our study provides a detailed transcriptome profiling of soybean embryos during development and indicates that differences in the level of seed isoflavonoids between these two cultivars could be as a result of differential expression of CHS7 and CHS8 during late stages of seed development.
Soybean (Glycine max) L. Merr. is the world's most widely grown grain legume. It combines in one crop both the major supply of vegetable oil and protein, with a variety of uses in human food and animal feeds. Soybean also contains eight essential amino acids that are crucial for human nutrition and are not made naturally in the body (Carpenter et al., 2002  ).
Soybean seed is virtually devoid of endosperm and comprises a well-developed embryo and a surrounding seed coat. During embryo development, the fertilized egg cell differentiates into a mature embryo containing cells with different roles. The entire embryogenesis can be divided into five stages: globular, heart, cotyledon, maturation, and dormancy (Walbot, 1978
Soybean seed has a unique chemical composition that makes it a valuable industrial and agricultural commodity. It contains 40% of the seed dry weight as proteins. Soybean seeds are a rich source of isoflavonoids that are associated with many health benefits (Dixon and Ferreira, 2002 In this study, we have examined the temporal changes in gene expression during embryo development in soybean when seeds undergo major changes in metabolism. Soybean cDNA microarray chips consisting of over 18,000 cDNAs were used to define patterns of gene expression during five different embryo developmental stages in two soybean cultivars. We describe here the global gene expression profile during embryo development in soybean and discuss the transcriptional networks that synchronize the response to developmental programs, leading to the production of various components that accumulate in seeds. We also portray a detailed comparison of gene expression between soybean cultivars that differ in the seed isoflavonoid content and discuss the potential regulation points for isoflavonoid synthesis in soybean seeds.
The 18 K-A microarray slides were obtained from Dr. Lila Vodkin, University of Illinois (Vodkin et al., 2004 ). The arrays were comprised of 18,462 single-spotted PCR products from cDNAs of the low redundancy Gm-c1021, Gm-c1083, and Gm-c1070 unigene cDNAs and 64 choice clones spotted multiple times on the slide. The unigene set represented on the array provides highly representative mRNAs expressed in roots of seedlings and adult plants, flower buds, flowers, pods, and various stages of immature embryos and seed coats. These microarray slides have platform GPL3015 in the Gene Expression Omnibus (GEO). We focused our study on five different developmental stages of embryos from two different soybean cultivars (RCAT Angora and Harovinton) to examine the steady-state transcript abundance. These stages included the early maturation stage as represented by 30, 40, and 50 d after pollination (DAP) to late maturation stage (60 and 70 DAP), when seeds had attained their full size but the process of desiccation was not complete (Fig. 1 ). A decrease in chlorophyll content of the embryos was noticed at 50 DAP. Embryos were collected from plants grown at two different locations (London and Delhi, Ontario, Canada) for two consecutive years (2003 and 2004). A dye swap and four biological replications were conducted per gene for each developmental stage to minimize the technical and biological variations, respectively. A total of 32 analyses per choice clone were performed, because they were represented four times in each array. The data analysis was done using computer software (GeneSpring v7.3, Agilent Technologies), and normalization was performed by per spot, per chip, intensity-dependent LOWESS. Only those genes that showed intensity >10 were combined together and selected for further analysis. This method allowed increased reliability in expression analysis.
Effect of Environment and Cultivar Difference on Gene Expression One of the major objectives of this research was to delineate the temporal patterns of gene expression during embryo development in soybean and interpret the results in the context of our existing knowledge of seed development and storage reserve accumulation. We identified several developmentally regulated genes by hybridizing the microarrays with mRNA samples from five distinct stages of seed development. A total of 11,480 genes showed intensities >10 at any time point during the development. These genes were chosen for further analysis by examining their hybridization intensities using a single channel method. An ANOVA test with P value < 0.05 and < 0.01 with different parameters was performed to calculate the number of genes that show a significant difference in their expression. An estimation of the effect of environmental factors on gene expression is summarized in Table I and Figure 2 . A total of five representative genes (P < 0.01) were chosen from each parameter to demonstrate the common pattern of gene expression during the development. The results show that cultivar differences have very little effect compared to either the location or the growing season effects. The general patterns of gene expression were very similar in RCAT Angora and Harovinton for plants that were grown at the same location in different years, or in the same year at different locations, during different developmental stages (Fig. 2, A and B). A hierarchical clustering analysis using one-way ANOVA also grouped the cultivars together for particular developmental stages irrespective of location or the year (data not shown), confirming the greater effect of environmental factors on gene expression. A large difference in the temperature and precipitation was not observed between the locations during two growing seasons (Supplemental Table S1). However, the soil composition of the two locations was very different. Delhi soil had a significantly higher amount of sand compared to London (Table II ). When ANOVA analysis was performed on developmental stages, genes were selected that displayed consistent developmental changes in their expression profiles. Thus, for this set of genes, only small differences were observed between the different locations and years for both RCAT Angora and Harovinton. The representative genes in Figure 2, C and D for both the cultivars showed a similar pattern of expression during embryo development irrespective of environmental conditions. The ANOVA analysis on cultivar was the most restrictive filter and resulted in the selection of only five genes. These genes have the highest probability of differential expression between the cultivars at all five developmental time points that were sampled (Fig. 2, E and F). Other cultivar-specific gene expression profiles that are developmentally specific were identified by further analyses (below).
Cluster Analysis of 5,910 Differentially Expressed cDNAs in Developing Soybean Embryos
Our analysis grouped the total number of genes on the array into three categories based on the intensity and differential expression (Fig. 3A
). Using single channel intensities for each cultivar, data was normalized per chip to the 50th percentile and then per gene to the median of the measurement for that gene, with a cutoff value >0.5 and <2.0 in all developmental stages for both cultivars. Only the genes that passed the filtering criteria were considered as genes that changed in expression based upon the experimental conditions. A total of 5,910 genes changed in their expression in all the biological replicates in at least one embryo developmental stage used in the study. Only those genes with intensities >10 were included in this group. A group of 5,570 genes showed intensities >10; however, their expression did not change significantly during the development. Another group of 6,872 genes changed in their expression with time but possessed very low intensity. A complete list of genes belonging to each of three groups is deposited in GEO (see "Materials and Methods"). To acquire an overall picture of gene expression changes, we clustered 5,910 differentially expressed genes by a k-means analysis. This separated the differentially expressed genes into five sets according to their profiles irrespective of locations or growing seasons. The general hypothesis of k-means cluster analysis is that genes involved in a similar function or common metabolic pathway will have similar profile of expression and thus likely to be grouped into the same group. The analysis was carried out for RCAT Angora and Harovinton separately. These cultivars do not possess a close common lineage that is evident from their pedigree information (Buzzell et al., 1991
Genes in groups B and C showed similar expression profiles in that they contain genes that were up-regulated at the later stages of embryo development. However, a considerable increase in the level of gene expression was noticed for the genes included in group C at 70 DAP. Genes encoding minor allergen, homolog to zinc binding protein, oxalyl-CoA decarboxylase, pathogen inducible trypsin inhibitor-like protein, and calmodulin are included in group B, while group C included genes such as a lipid transfer protein precursor, late embryogenesis abundant proteins, desiccation protective protein, catalase 4, Pro-rich protein, plasma membrane Ca2+ ATPase, and many transcription factor genes such as TATA box binding protein, APETALA2 domain-containing protein, and WRKY family transcription factors. This group included many genes that are required for seed maturity or are reported to accumulate during embryo maturation. For example, maturity related protein, many Cyt P450, senescence related proteins, receptor kinases, and ethylene responsive proteins were all within this group. Most of the genes participating in isoflavonoid biosynthesis are also clustered in group C.
Unlike the gene expression patterns for the genes belonging to group B and C, group D included genes that did not show major change in the level of expression from 30 to 60 DAP followed by a dramatic decrease in the level of transcript accumulation at 70 DAP. Examples include several chlorophyll a/b-binding proteins, Group E illustrates the expression patterns characteristic for a collection of genes that increased gradually in their transcript accumulation from 30 to 40 DAP followed by a slight increase or decrease in expression or reached a plateau. Examples include genes encoding lipooxygenase, WRKY family transcription factor, xyloglucan endotransglucosylase/hydrolase, seed calcium dependent protein kinase, and putative ATP-binding cassette transporters.
The major storage products of soybean seed are proteins and triacylglycerols. Significant amounts of isoflavonoids also accumulate in the seed during development (Dhaubhadel et al., 2003
The array used in this study allowed simultaneous analysis of 46 genes involved in fatty acid metabolism that showed intensity >10. These genes could be characterized by three main patterns of expression. The patterns of the first group followed a slow decline in expression from the early embryo development to late maturity stage (Fig. 4B, in red). Some of the representative genes in this category include: acyl carrier proteins (ACPs),  -3-fatty acid desaturase,  -ketoacyl-ACP synthetase, enoyl-ACP reductase, etc. The second group showed an increase in gene expression from 30 to 60 DAP followed by a sharp decrease in the expression. This group also includes many ACPs, two -6-desaturase FAD 2 to 1,  -12-fatty acid desaturase, and fatty acid elongase (Fig. 4B, in green). The third group showed a very different profile of gene expression. The expression of genes belonging to this group initially increased slowly and then more rapidly after 60 DAP.  -6-desaturase FAD 2 to 1 and FAD2-2, acetyl CoA carboxylase, and many putative AIM1 proteins are included in this group (Fig. 4B, in blue). We also studied the expression patterns of genes that are involved in shikimic acid and phenylpropanoid biosynthesis, because these routes lead to the isoflavonoid pathway. Out of 430 genes that are potentially involved in those pathways and were included on the array, only 168 (39%) showed the intensity >10. These genes were classified into three main groups according to their expression profiles (Fig. 4C). The first group started with high expression at 30 DAP, decreased at 40 DAP, and increased slightly at 50 DAP then decreased later on the development (Fig. 4C, in red). Some of the representative genes in this group are: Phe ammonia-lyase (PAL) 2, NAD(P)H-dependent 6-deoxychalcone synthase/reductase, and prephenate dehydrase. The second group was characterized by a set of genes with maximum expression at 60 DAP followed by a sharp decline in the expression level with time during the later stages of development (Fig. 4C, in green). For example, the expression of isoflavone reductase, 4-coumarate:CoA ligase isoenzyme 2, cinnamic acid 4-hydroxylase, and chalcone isomerase (CHI). The last group included genes with a distinctly different pattern, with maximum expression later in development (Fig. 4C, in blue). This group included PAL1, chalcone synthase (CHS) 7, CHS8, isoflavone synthase (IFS) 1, IFS2, and UDP-Glc:flavonoid glucosyltransferase. The complete list of genes for all three seed storage products belonging to each group is available in Supplemental Table S2.
We have previously shown that RCAT Angora accumulates a higher level of seed isoflavonoids compared to Harovinton (Dhaubhadel et al., 2003
For the comparison of genes involved in isoflavonoid biosynthesis between RCAT Angora and Harovinton, a total of 430 genes that are involved either in shikimic acid pathway or in phenylpropanoid pathway were chosen. Results from different years and locations but for identical developmental time points were pooled together, and the mean normalized ratio between RCAT Angora and Harovinton for each gene was calculated. Of 430 genes, only 168 genes had intensity >10, and 26 genes showed a 1.5-fold difference in expression between two cultivars at one of the five embryo developmental stages under study (Table III ). In fact, 19 genes were up-regulated, and seven genes were down-regulated in RCAT Angora compared to Harovinton. Only four genes revealed a 2-fold or greater change in expression, of which one was down-regulated. The expression of CHS7 and CHS8 genes was greater in RCAT Angora compared to Harovinton at 70 DAP (Fig. 5B; Table III). This difference was reproducible, statistically significant, and occurred at the stage when the embryo starts accumulating noticeably higher levels of isoflavonoids. The IFS2 (1.68-fold) and a putative dihydroflavonol reductase (2.19-fold) were also found to be expressed at higher levels in RCAT Angora versus Harovinton as were many upstream genes involved in the phenylpropanoid pathway, such as PAL, CHS, CHI, and certain Cyt P450 genes. The differences in isoflavonoid pathway gene expression between the two cultivars were even greater when separate analysis was performed for years and locations (data not shown).
Verification of microarray results with reverse transcription (RT)-PCR analysis using gene-specific primers confirmed that IFS1 transcripts were present at similar levels in the developing embryos from early embryo development until maturity, while IFS2 transcript levels increased in both the cultivars during embryo development. Both CHS7 and CHS8 genes were expressed at a higher level in RCAT Angora compared to Harovinton at 70 DAP (Fig. 5C).
To identify other differentially expressed genes between RCAT Angora and Harovinton developing embryos, we prepared a list of genes that showed 2-fold or greater differences in expression between the cultivars at any particular stage of development (Table IV ). To supplement the fold-change analysis, we performed a t test with P value of 0.01 by comparing a specific developmental stage between the two cultivars. The relationship between the P value from the t test and fold difference are represented by volcano plot for different stages of embryo development (Fig. 6 ).
The genes selected by this analysis show a significant difference between the two cultivars that are independent of location or year. Most of the differences observed between the two cultivars were at 30 DAP or near maturity. Three genes that were up-regulated in RCAT Angora at most of the developmental stages under study were a NADPH-protochlorophyllide oxidoreductase (NPR), a Bowman-Birk type protease isoinhibitor C (BBI), and a homolog to hypothetical protein from Arabidopsis. The maximum difference in normalized intensity of BBI between RCAT Angora and Harovinton was 12.8-fold at 30 DAP. The gene encoding NPR was expressed 6.1-fold higher in RCAT Angora at 30 DAP, and the minimum fold difference was 2.9 at 50 and 70 DAP. The transcripts for an auxin response factor (ARF) 17 and an unknown protein were accumulated to a significantly higher level in Harovinton compared to RCAT Angora. The normalized intensity of ARF17 was 6.3-fold higher at 60 DAP and 3-fold higher at other stages of development in Harovinton than in RCAT Angora.
A Finally, to determine whether the differential gene expression observed between Harovinton and RCAT Angora was due to the difference in their genome structure and copy number, we performed microarray hybridization of an 18 K-A chip with the probes derived from the genomic DNA from each of the cultivars. The result indicated that the differences in transcript accumulation for the genes described above are not due to major differences in gene copy number with the genomes of each of the cultivars.
One of the major challenges of plant developmental biology is to identify the genes involved in seed development and determine their functions. During seed development, various amino acids and metabolites are transported into the developing embryo and distributed to different biosynthetic pathways for the synthesis of major seed storage compounds. Transcriptome analysis is an important step toward gaining an understanding of the complexity and coordination of the various pathways. Here, we present a comprehensive analysis of the soybean transcriptome at five stages of embryo development. Our analysis identified 11,480 genes that are expressed in developing embryos of soybean. To select constitutively expressed genes that do not change, we filtered for genes that possessed the normalized intensity >0.5 and <2.0 in both the cultivars at both the locations and years. A total of 5,570 genes showed consistent expression level throughout development in our study. Thus, almost 50% of the genes that were detected as expressed genes did not change in their expression level during seed development. Using solution hybridization, Goldberg et al. (1981) have shown that many mRNAs present in the maturation stage in soybean embryos are also present in the cotyledon stage embryo, suggesting that many of these messages are present in the embryos throughout development. We identified 5,910 genes that changed in their expression at least once during the development. Grouping of these genes into different categories according to their expression profiles led to clusters of genes that change in a similar fashion and possibly share certain functional characteristics (Fig. 3). Of 5,910 genes that showed differential expression, 760 genes (12.8%) were annotated as unknown. Some of the unknown genes were highly expressed at certain stages of development and may possibly play a major role in seed development and metabolism. Therefore, this new information on expression profiling can guide potential work in functional genomics and may offer the foundation for reverse genetic methods to identify the function of these highly expressed genes during embryo development in soybean. The expression profiles of the vast majority of the genes follow the same pattern in RCAT Angora and Harovinton. The similarity in expression suggests that there are minor cultivar-specific differences in soybean, and thus the results may be broadly applicable. Two genes that displayed greater hybridization signals in RCAT Angora were BBI and NPR (Fig. 2, E and F). BBI is a sulfur-rich protein that inhibits trypsin and chymotrypsin proteases (Wilson, 1997). NPR catalyzes the first light-dependent reaction in chlorophyll biosynthesis, and its expression is related to chlorophyll synthesis in green tissues (Kuroda et al., 1995). Because both the cultivars were grown under the same conditions and possess a similar maturity profile, it is not clear why RCAT Angora embryos accumulate higher levels of these transcripts compared to Harovinton.
A major change in gene regulation was observed at 30 and 70 DAP as compared to other stages of development. This was particularly true for the three main storage products of the soybean seeds (Fig. 4). The storage proteins of soybean seeds are comprised of two multimeric globulins, namely 7S and 11S globulins. The 7S globulin consists of
Regulation of fatty acid composition is one of the challenging areas in any oilseed breeding program, and this has gained a lot of attention in soybean breeding. Oils low in polyunsaturated fatty acids and high in 18:1 have increased stability and possibly nutritional benefits (Liu and White, 1992
One of the major emphases of this study was to analyze expression patterns of genes involved in isoflavonoid synthesis and to correlate the patterns with seed isoflavonoid accumulation. A rate-limiting enzyme for isoflavonoid synthesis is IFS. This Cyt P450 enzyme converts naringenin and liquiritigenin to their corresponding isoflavones and defines a branch point in the synthesis of these natural products (Steele et al., 1999
Past studies that have explored the control of isoflavonoid accumulation in plant tissues may help us to interpret our data. For example, expression of a chimeric R and C1 transcription factor, which increases anthocyanin levels in maize tissues (Bruce et al., 2000
For isoflavonoid biosynthesis, chalcone is a critical metabolite that is produced by CHS/CHR and is either used in isoflavonoid synthesis or diverted to the other branch of the pathway. It is possible that the increase in CHS expression enhances production of chalcone that may be diverted toward isoflavonoid synthesis in RCAT Angora and not affect the rest of the phenylpropanoid pathway. In contrast, no significant increase in isoflavone level was observed when CHS was expressed in soybean seed under the control of seed-specific promoter (Yu and McGonigle, 2005
The hypothesis that CHS7 and CHS8 expression may influence seed isoflavonoid content is supported by independent, quantitative trait loci (QTL) analyses that were performed to identify markers associated with isoflavone levels. These past studies have identified several QTL that lie in the same linkage group as CHS genes (Kassem et al., 2004 In conclusion, our results illustrate that transcriptional control during soybean embryo development is a highly coordinated process. We found much evidence demonstrating that the synthesis and transport of storage proteins, fatty acids, and isoflavonoids are transcriptionally regulated from the early developmental stage to maturity in soybean embryo. Our results show that environmental effects on the transcriptome of the developing seed are large and exceed cultivar-specific effects. The information obtained from this study provides a powerful tool for studying and understanding gene functions for many unidentified genes that may have crucial roles in regulating and coordinating the expression of nutrient partitioning genes during embryo development. This study also provides a detailed comparison of gene expression between two cultivars that differ in isoflavonoid content and points to CHS7 and CHS8 as genes that influence isoflavonoid biosynthetic flux in soybean seeds.
Plant Materials and Tissue Preparation Soybean (Glycine max) L. Merr. cv RCAT Angora (3150 CHU) and cv Harovinton (3100 CHU) were obtained from Dr. Istvan Rajcan (Department of Plant Agriculture, University of Guelph, Ontario) and Agriculture and Agri-Food Canada, Harrow, respectively. Both the cultivars belong to late maturity group I to early maturity group II. Soybean seeds were planted at two Agriculture and Agri-Food Canada experimental stations in southern Ontario, London and Delhi, in 2003 and 2004. Regular agronomic practices and planting dates were followed. The pods were tagged on the first day of pollination and harvested at 30, 40, 50, 60, and 70 DAP. The pods were collected randomly from five to seven plants, and embryos were excised from seeds, frozen in liquid nitrogen, and stored at –80°C.
Total RNA was isolated from developing embryos following the procedure of Wang and Vodkin (1994)
Soybean cDNA microarray slides consisting of 18,432 cDNAs spotted onto amine-coated glass slides (18 K-A) were obtained from Dr. Lila Vodkin (University of Illinois, Urbana). A total of 42 microarray slides were hybridized, 40 from four separate biological samples using independent samples of mRNA for each experiment with dye swaps to minimize technical variation. Each experiment included mRNA samples from soybean cv RCAT Angora and cv Harovinton at five different embryo developmental stages (30, 40, 50, 60, and 70 DAP). An additional two slides were hybridized with labeled genomic DNA to compare the two cultivars at the genomic level. Probe labeling was performed by using CyScribe First-Strand cDNA Labeling kit (Amersham BioSciences) according to the manufacturer's instruction. Samples of 1.5 µg mRNA were used in labeling reaction with CyDye-labeled dCTP. Purification of labeled cDNA and removal of unincorporated nucleotides were performed by using CyScribe GFX Purification kit (Amersham BioSciences) according to the manufacturer's instruction, except that the labeled cDNAs were eluted in two steps to a total elution volume of 80 µL. The incorporation of cyanine-3- and cyanine-5-labeled nucleotides into cDNA was determined spectrophotometrically by measuring the absorption at 550 nm and 650 nm, respectively.
Microarray slides were exposed to an additional cross-linking at 50 mJ cm–2 and then prehybridized for 45 min at 42°C in prehybridization buffer containing 5x SSC, 0.1% SDS, and 1% bovine serum albumin, followed by two washes in 0.1x SSC at room temperature. The slides were rinsed with sterile water and dried by centrifugation. Equal amounts of purified CyDye-labeled probes were combined together, dried under vacuum at 45°C (Speed Vac, Savant Instrument), and resuspended in a hybridization solution (40 µL total volume) containing 1.25 ng/µL poly(A) DNA, 50% (v/v) formamide, and 25% (v/v) hybridization buffer (Amersham BioSciences). The hybridization mix was denatured at 100°C for 2 min, cooled to room temperature, and applied to the prehybridized slide. The slide was covered with a 24- x 60-mm coverslip and placed in a hybridization chamber containing 10 µL water. The hybridization was carried out at 42°C for 20 h, the coverslip removed in 2x SSC, 0.1% SDS, followed by one posthybridization wash in 2x SSC, 0.1% SDS at 42°C for 5 min, two washes in 0.1x SSC, 0.1% SDS at 42°C for 2 min, and two room temperature washes in 0.1x SSC for 1 min. Slides were rinsed with sterile water and dried under nitrogen gas prior to laser scanning (Bio-Rad ChipReader with VersArray ChipReader v3.0, Bio-Rad). Spot intensities were quantified individually for background signals (Array Vision v6.0 software). Background subtracted intensities were imported into GeneSpring v7.3 (Agilent Technologies) and normalized by per spot, per chip, intensity-dependent LOWESS. A dye swap was performed in each experiment, and final normalized ratios were averaged from each location and year for a particular cultivar at a given time point. The differential gene expression between RCAT Angora and Harovinton at a particular stage of embryo development was monitored by taking the ratio of RCAT Angora and Harovinton from the two-color hybridization of each slide. The relative gene expression for embryo development as a function of time was calculated as an alternate approach to the data using a single channel input for each cultivar imported into GeneSpringGX, normalized by per chip to 50th percentile, and per gene to the median of the measurement of that gene. To further validate this approach, two-color hybridizations were performed on two time points of the same cultivar, RCAT Angora 30 DAP and 70 DAP. A comparative genomic DNA hybridization using genomic DNA from RCAT Angora and Harovinton was conducted as described in Gijzen et al. (2006)
To estimate technical variation, control experiments in which the same RNA was labeled with cyanine-3 and cyanine-5 were performed. The degree of biological and environmental variability in the embryos was assessed by growing them for two different years at two different locations.
Embryo samples of identical developmental stages that were used in the microarray experiment were ground to a fine powder in liquid nitrogen and extracted with 50% acetonitrile in water followed by sonication for 30 min in an ice water bath. The samples were centrifuged for 25 min at 3,000g and the supernatant was collected. The extraction process was repeated two times with the pellet and the supernatant fractions were pooled together and filtered (Acrodisc, nylon, 0.45 µm). The malonyl- and acetyl-isoflavonoids were converted to their corresponding glucosides by hydrolyzing the filtered samples with 1.3% KOH at room temperature for 4 h followed by neutralization of the sample with 3% KH2PO4. The solvent was evaporated and samples were redissolved in 40% dimethyl sulfoxide prior to HPLC analysis. Isoflavonoids were separated by injecting 20 µL of the samples on a C18 column (Symmetry Column, Waters Corporation, 5 µm). A guard column of the identical packing material was connected before the analytical column. The samples were run at room temperature applying a mobile-phase gradient of 10% to 35% acetonitrile in 0.1% acetic acid over 45 min at a flow rate of 1 mL/min (Waters Limited). The total separation time was 63 min, which included a 4-min wash and 14-min equilibration. Isoflavonoid peaks were compared with the retention time and UV spectra of the aglycone and glucoside standards (LC Laboratories) and quantified using the Millenium32 Software (Waters Limited).
The following materials are available in the online version of this article.
We thank Istvan Rajcan (University of Guelph) and Vaino Poysa (Agriculture and Agri-Food Canada, Harrow) for soybean seeds, Dr. Lila Vodkin (University of Illinois) for 18 K-A cDNA microarray slides, and Alex Molnar and Aldona Gaidauskas-Scott (Agriculture and Agri-Food Canada, London) for technical assistance. Received July 7, 2006; accepted October 31, 2006; published November 10, 2006.
1 This work was supported by Agriculture and Agri-Food Canada's Canadian Crop Genomics Initiative (grant to S.D.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Sangeeta Dhaubhadel (dhaubhadels{at}agr.gc.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.106.086306 * Corresponding author; e-mail dhaubhadels{at}agr.gc.ca; fax 519–457–3997.
Alba R, Payton P, Fei Z, McQuinn R, Debbie P, Martin GB, Tanksley SD, Giovannoni JJ (2005) Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development. Plant Cell 17: 2954–2965 Bruce W, Folkerts O, Garnaat C, Crasta O, Roth B, Bowen B (2000) Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factor CRC and P. Plant Cell 12: 65–79 Buzzell RI, Anderson TR, Hamill AS, Welacky TW (1991) Harovinton soybean. Can J Plant Sci 71: 525–526 Carpenter J, Felsot A, Goode T, Hamming M, Onstad D, Sankula S (2002) Comparative Environmental Impacts of Biotechnology Derived and Traditional Soybean, Corn and Cotton Crops. Council for Agricultural Science and Technology, Ames, IA, pp 15–50 Dhaubhadel S, McGarvey BD, Williams R, Gijzen M (2003) Isoflavonoid biosynthesis and accumulation in developing soybean seeds. Plant Mol Biol 53: 733–743[CrossRef][ISI][Medline] Dixon RA, Ferreira D (2002) Genestein. Phytochemistry 60: 205–211[CrossRef][ISI][Medline] Duan M, Sun SSM (2005) Profiling the expression of genes controlling rice grain quality. Plant Mol Biol 59: 165–178[CrossRef][ISI][Medline] Gijzen M, Kuflu K, Moy P (2006) Gene amplification of the Hps locus in Glycine max. BMC Plant Biol 6: 6[CrossRef][Medline] Goldberg RB, Hoschek G, Tam SA, Ditta GS, Breidenbach RW (1981) Abundance, diversity, and regulation of mRNA sequence sets in soybean embryogenesis. Dev Biol 83: 201–217[CrossRef][ISI][Medline] Hajduch M, Ganapathy A, Stein JW, Thelen JJ (2005) A systematic proteomic study of seed filling in soybean: establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteomic database. Plant Physiol 137: 1397–1419 Hennig L, Gruissem W, Grossniklaus U, Köhler C (2004) Transcriptional programs of early reproductive stages in Arabidopsis. Plant Physiol 135: 1765–1775 Heppard EP, Kinney AJ, Stecca KL, Miao G-H (1996) Developmental and growth temperature regulation of two different microsomal omega-6 desaturase genes in soybeans. Plant Physiol 110: 311–319[Abstract] Hill JE, Breidenbach RW (1974) Proteins of soybean seeds. II. Accumulation of the major protein components during seed development and maturation. Plant Physiol 53: 747–751 Jung W, Yu O, Lau SM, O'Keefe DP, Odell J, Fader G, McGonigle B (2000) Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes. Nat Biotechnol 18: 208–212[CrossRef][ISI][Medline] Kassem MA, Meksem K, Iqbal MJ, Njiti VN, Banj WJ, Winters TA, Wood A, Lightfoot DA (2004) Definition of soybean genomic regions that control seed phytoestrogen amounts. J Biomed Biotechnol 1: 52–60 Kuroda H, Masuda T, Ohta H, Shioi Y, Takamiya K (1995) Light enhanced gene expression of NADPH-protochlorophyllide oxidoreductase in cucumber. Biochem Biophys Res Commun 210: 310–316[CrossRef][ISI][Medline] Lee J-M, Williams ME, Tingey SV, Rafalski JA (2002) DNA array profiling of gene expression changes during maize embryo development. Funct Integr Genomic 2: 13–27[CrossRef] Liu C-J, Blount JW, Steele CL, Dixon RA (2002) Bottlenecks for metabolic engineering of isoflavone glycoconjugates in Arabidopsis. Proc Natl Acad Sci USA 99: 14578–14583 Liu HR, White PJ (1992) Oxidative stability of soybean oils with altered fatty acid composition. J Am Oil Chem Soc 69: 528–532[CrossRef][ISI] Matsumura H, Watanabe S, Harada K, Senda M, Akada S, Kawasaki S, Dubouzet EG, Minaka N, Takahashi R (2005) Molecular linkage mapping and phylogeny of the chalcone synthase multigene family in soybean. Theor Appl Genet 110: 1203–1209[CrossRef][ISI][Medline] Orthoefer FT (1978) Processing and utilization. In AG Norman, ed, Soybean Physiology, Agronomy, and Utilization. Academic Press, New York, pp 219–246 Primomo VS, Poysa V, Ablett GR, Jackson C-J, Gijzen M, Rajcan I (2005) Mapping QTL for individual and total isoflavone content in soybean seeds. Crop Sci 45: 2454–2464 Ruuska SA, Girke T, Benning C, Ohlrogge JB (2002) Contrapuntal networks of gene expression during Arabidopsis seed filling. Plant Cell 14: 1191–1206 Schena M, Shalon D, Davis RW, Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270: 467–470 Sreevidya VS, Srinivasa Rao C, Sullia SB, Ladha JK, Reddy PM (2006) Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia. J Exp Bot 57: 1957–1969 Steele CL, Gijzen M, Qutob D, Dixon RA (1999) Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean. Arch Biochem Biophys 367: 146–150[CrossRef][ISI][Medline] Thanh VH, Shibasaki K (1976) Heterogeneity of beta-conglycinin. Biochim Biophys Acta 181: 404–409 Tuteja JH, Clough SJ, Chan WC, Vodkin LO (2004) Tissue-specific gene silencing mediated by a naturally occurring chalcone synthase gene cluster in Glycine max. Plant Cell 16: 819–835 Vodkin LO, Khanna A, Shealy R, Clough SJ, Gonzalez DO, Philip R, Zabala G, Thibaud-Nissen F, Sidarous M, Stromvik MV, et al (2004) Microarray analysis for global expression constructed with a low redundancy set of 27,500 sequenced cDNAs representing an array of developmental stages and physiological conditions of the soybean plant. BMC Genomics 5: 73[CrossRef][Medline] Walbot V (1978) Control mechanisms for plant embryogeny. In M Clutter, ed, Dormancy and Developmental Arrest. Academic Press, New York, pp 113–166 Wang CS, Vodkin LO (1994) Extraction of RNA from tissues containing high levels of procyanidins that bind RNA. Plant Mol Biol 12: 132–145 Wilson KA (1997) The protease inhibitors of seeds. In BA Larkins, IK Vasil, eds, Cellular and Molecular Biology of Plant Seed Development. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 331–374 Wobus U, Weber H (1999) Seed maturation: genetic programmes and control signals. Curr Opin Plant Biol 2: 33–38[Medline] Yadav NS (1996) Genetic modification of soybean oil quality. In DPS Verma, RC Shoemaker, eds, Soybean Biotechnology. CAB International, Wallingford, UK, pp 165–188 Yu O, McGonigle B (2005) Metabolic engineering of isoflavone biosynthesis. Adv Agron 86: 147–190 Yu O, Shi J, Hession AO, Maxwell CA, McGonigle B, Odell CA (2003) Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochemistry 63: 753–763[CrossRef][ISI][Medline] Zhu T, Budworth P, Chen W, Nicholas P, Chang H-S, Guimil S, Su W, Estes B, Zou G, Wang X (2003) Transcriptional control of nutrient partitioning during rice grain filling. Plant Biotechnol J 1: 59–70[CrossRef][Medline] This article has been cited by other articles:
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TOP
ABSTRACT
RESULTS
(Gm-r1083-2907), pectinesterase (Gm-r1083-3987), hypothetical protein (Gm-r1070-9004), oligouridylate binding protein (Gm-r1021-2348), Asp aminotransferase (Gm-r1021-2151); B, hypothetical protein (Gm-r1070-8066, Gm-r1070-2773, Gm-r1021-3499), fiber protein Fb15 (Gm-r1021-3282), and ribosomal protein L28 (Gm-r1021-313); C and D, imbibition protein (Gm-r1083-2642), dehydrogenase (Gm-r1083-2142), splicing factor PRP8 homolog (Gm-r1070-7746), polyprotein (Gm-r1070-7423), annexin (Gm-r1070-7252); E and F, NPR (Gm-r1070-495), BBI (Gm-r1070-399), ARF 17 (Gm-r1070-8247), and hypothetical protein (Gm-r1070-591, Gm-r1070-1795).
-subunit of 
