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The wp allele generates a pink flowered phenotype in soybean [Glycine max (L.) Merr.]. Genotypes with W1_wp wp are associated with larger seed size and higher seed protein content. The influence of a single locus on the anthocyanin pathway and seed protein accumulation has not been documented in other species. It is of interest to clone wp by initially identifying linked molecular markers. The objective of this research was to use restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) molecular markers to position the wp locus on the soybean public genetic map. Of the 74 markers tested, two RFLP and 14 SSR markers were identified as linked to wp. The two RFLP markers linked to wp in this study were previously reported to be associated with quantitative trait loci (QTL) controlling seed protein in a G. soja Siebold & Zucc. x G. max population. It is proposed that wp is located between RFLP locus K011 and SSR locus Satt600. The molecular markers linked to wp were located on linkage group D1b+W of the public soybean genetic map. The knowledge of linked molecular markers will assist in cloning wp, potentially leading to further characterization of how wp influences the anthocyanin pathway and seed protein accumulation., PINK FLOWERED SOYBEAN plants were discovered by Stephens and Nickell (1991). Genetic analysis revealed the unique color was due to homozygous recessive wp alleles in the presence of WI (Stephens [...]

Transposable element systems have been proposed to explain instability in floral pigmentation of several plant species, including soybean [Glycine max (L.) Merr.]. Soybean lines with chimeric (purple and pink sectored) flowers are hypothesized to contain wp-m, an active transposable element that is able to excise from the wp locus during morphological development. The objectives of this research were (i) to determine the inheritance of the chimeric flower phenotype when crossed to stable pink or purple flowered revertant lines and (ii) to determine the effect of wp on agronomic traits in stable flowered lines derived from wp-m. Chimeric flowers crossed to pink flowered revertant (wp*) lines produced four F2 populations with unusual segregation ratios of 52 pink, three purple, and two chimeric flowered plants. Crossing chimeric flowers to revertant purple flowered (Wp*) lines resulted in [F.sub.2] populations that did not have the chimeric flower phenotype evident. In the agronomic evaluations, stable wp* lines were later in maturity and averaged 4 g [kg.sup.-1] higher in protein content and 3 g [kg.sup.-1] lower in oil content than Wp* lines. The data suggest wp acts in a pleiotropic manner to influence protein synthesis, as purple flowered revertant lines from a pink flower source had lower protein content than sister lines with wp*. Pink flowered lines derived from a purple flower source had higher levels of protein than sister lines with Wp*. The influence of wp on the anthocyanin pathway, plant morphology, and protein accumulation is a unique phenomenon that has not been reported in other plant species., TRANSPOSABLE ELEMENT SYSTEMS have been well characterized in maize (Zea mays L.) since their initial discovery by Barbara McClintock in 1944 (Peterson, 1987; Fedoroff, 1988; Vodkin, 1989). A transposable element [...]

The article discusses the identification and phylogenetic analysis of sequences of bean pod mottle virus (BPMV), soybean mosaic virus and cowpea chlorotic mottle virus in expressed sequence tag data from soybean. The soybean sequence was derived from the North Central and Northern Great Plains regions, where an outbreak of BPMV has been reported. The strains of BPMV may be a mixture of different BPMV RNA-1 and RNA-2 genotypes.

Abstract Background Small non-coding RNAs (smRNAs) are known to have major roles in gene regulation in eukaryotes. In plants, knowledge of the biogenesis and mechanisms of action of smRNA classes including microRNAs (miRNAs), short interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs) has been gained mostly through studies with Arabidopsis. In recent years, high throughput sequencing of smRNA populations has enabled extension of knowledge from model systems to plants with larger, more complex genomes. Soybean (Glycine max) now has many genomics resources available including a complete genome sequence and predicted gene models. Relatively little is known, however, about the full complement of its endogenous smRNAs populations and the silenced genes. Results Using Illumina sequencing and computational analysis, we characterized eight smRNA populations from multiple tissues and organs of soybean including developing seed and vegetative tissues. A total of 41 million raw sequence reads collapsed into 135,055 unique reads were mapped to the soybean genome and its predicted cDNA gene models. Bioinformatic analyses were used to distinguish miRNAs and siRNAs and to determine their genomic origins and potential target genes. In addition, we identified two soybean TAS3 gene homologs, the miRNAs that putatively guide cleavage of their transcripts, and the derived tasiRNAs that could target soybean genes annotated as auxin response factors. Tissue-differential expression based on the flux of normalized miRNA and siRNA abundances in the eight smRNA libraries was evident, some of which was confirmed by smRNA blotting. Our global view of these smRNA populations also revealed that the size classes of smRNAs varied amongst different tissues, with the developing seed and seed coat having greater numbers of unique smRNAs of the 24-nt class compared to the vegetative tissues of germinating seedlings. The 24-nt class is known to be derived from repetitive elements including transposons. Detailed analysis of the size classes associated with ribosomal RNAs and transposable element families showed greater diversity of smRNAs in the 22- and 24-nt size classes. Conclusions The flux of endogenous smRNAs within multiple stages and tissues of seed development was contrasted with vegetative tissues of soybean, one of the dominant sources of protein and oil in world markets. The smRNAs varied in size class, complexity of origins, and possible targets. Sequencing revealed tissue-preferential expression for certain smRNAs and expression differences among closely related miRNA family members.

Abstract Background MicroRNAs (miRNAs) regulate the expression of target genes by mediating gene silencing in both plants and animals. The miRNA targets have been extensively investigated in Arabidopsis and rice using computational prediction, experimental validation by overexpression in transgenic plants, and by degradome or PARE (parallel analysis of RNA ends) sequencing. However, miRNA targets mostly remain unknown in soybean (Glycine max). More specifically miRNA mediated gene regulation at different seed developmental stages in soybean is largely unexplored. In order to dissect miRNA guided gene regulation in soybean developing seeds, we performed a transcriptome-wide experimental method using degradome sequencing to directly detect cleaved miRNA targets. Results In this study, degradome libraries were separately prepared from immature soybean cotyledons representing three stages of development and from seed coats of two stages. Sequencing and analysis of 10 to 40 million reads from each library resulted in identification of 183 different targets for 53 known soybean miRNAs. Among these, some were found only in the cotyledons representing cleavage by 25 miRNAs and others were found only in the seed coats reflecting cleavage by 12 miRNAs. A large number of targets for 16 miRNAs families were identified in both tissues irrespective of the stage. Interestingly, we identified more miRNA targets in the desiccating cotyledons of late seed maturation than in immature seed. We validated four different auxin response factor genes as targets for gma-miR160 via RNA ligase mediated 5’ rapid amplification of cDNA ends (RLM-5’RACE). Gene Ontology (GO) analysis indicated the involvement of miRNA target genes in various cellular processes during seed development. Conclusions The miRNA targets in both the cotyledons and seed coats of several stages of soybean seed development have been elucidated by experimental evidence from comprehensive, high throughput sequencing of the enriched fragments resulting from miRNA-guided cleavage of messenger RNAs. Nearly 50% of the miRNA targets were transcription factors in pathways that are likely important in setting or maintaining the developmental program leading to high quality soybean seeds that are one of the dominant sources of protein and oil in world markets.

Abstract Background Trichome hairs affect diverse agronomic characters such as seed weight and yield, prevent insect damage and reduce loss of water but their molecular control has not been extensively studied in soybean. Several detailed models for trichome development have been proposed for Arabidopsis thaliana, but their applicability to important crops such as cotton and soybean is not fully known. Results Two high throughput transcript sequencing methods, Digital Gene Expression (DGE) Tag Profiling and RNA-Seq, were used to compare the transcriptional profiles in wild-type (cv. Clark standard, CS) and a mutant (cv. Clark glabrous, i.e., trichomeless or hairless, CG) soybean isoline that carries the dominant P1 allele. DGE data and RNA-Seq data were mapped to the cDNAs (Glyma models) predicted from the reference soybean genome, Williams 82. Extending the model length by 250 bp at both ends resulted in significantly more matches of authentic DGE tags indicating that many of the predicted gene models are prematurely truncated at the 5' and 3' UTRs. The genome-wide comparative study of the transcript profiles of the wild-type versus mutant line revealed a number of differentially expressed genes. One highly-expressed gene, Glyma04g35130, in wild-type soybean was of interest as it has high homology to the cotton gene GhRDL1 gene that has been identified as being involved in cotton fiber initiation and is a member of the BURP protein family. Sequence comparison of Glyma04g35130 among Williams 82 with our sequences derived from CS and CG isolines revealed various SNPs and indels including addition of one nucleotide C in the CG and insertion of ~60 bp in the third exon of CS that causes a frameshift mutation and premature truncation of peptides in both lines as compared to Williams 82. Conclusion Although not a candidate for the P1 locus, a BURP family member (Glyma04g35130) from soybean has been shown to be abundantly expressed in the CS line and very weakly expressed in the glabrous CG line. RNA-Seq and DGE data are compared and provide experimental data on the expression of predicted soybean gene models as well as an overview of the genes expressed in young shoot tips of two closely related isolines.

Abstract Background To understand gene expression networks leading to functional properties of the soybean seed, we have undertaken a detailed examination of soybean seed development during the stages of major accumulation of oils, proteins, and starches, as well as the desiccating and mature stages, using microarrays consisting of up to 27,000 soybean cDNAs. A subset of these genes on a highly-repetitive 70-mer oligonucleotide microarray was also used to support the results. Results It was discovered that genes related to cell growth and maintenance processes, as well as energy processes like photosynthesis, decreased in expression levels as the cotyledons approached the mature, dry stage. Genes involved with some storage proteins had their highest expression levels at the stage of highest fresh weight. However, genes encoding many transcription factors and DNA binding proteins showed higher expression levels in the desiccating and dry seeds than in most of the green stages. Conclusions Data on 27,000 cDNAs have been obtained over five stages of soybean development, including the stages of major accumulation of agronomically-important products, using two different types of microarrays. Of particular interest are the genes found to peak in expression at the desiccating and dry seed stages, such as those annotated as transcription factors, which may indicate the preparation of pathways that will be needed later in the early stages of imbibition and germination.

Abstract Background The molecular organization of very few genetically defined CACTA transposon systems have been characterized thoroughly as those of Spm/En in maize, Tam1 of Antirrhinum majus Candystripe1 (Cs1) from Sorghum bicolor and CAC1 from Arabidopsis thaliana, for example. To date, only defective deletion derivatives of CACTA elements have been described for soybean, an economically important plant species whose genome sequence will be completed in 2008. Results We identified a 20.5 kb insertion in a soybean flavonoid 3'-hydroxylase (F3'H) gene representing the t* allele (stable gray trichome color) whose origin traces to a single mutable chimeric plant displaying both tawny and gray trichomes. This 20.5 kb insertion has the molecular structure of a putative autonomous transposon of the CACTA family, designated Tgmt*. It encodes a large gene that was expressed in two sister isolines (T* and tm) of the stable gray line (t*) from which Tgmt* was isolated. RT-PCR derived cDNAs uncovered the structure of a large precursor mRNA as well as alternatively spliced transcripts reminiscent of the TNPA-mRNA generated by the En-1 element of maize but without sequence similarity to the maize TNPA. The larger mRNA encodes a transposase with a tnp2 and TNP1-transposase family domains. Because the two soybean lines expressing Tgmt* were derived from the same mutable chimeric plant that created the stable gray trichome t* allele line from which the element was isolated, Tgmt* has the potential to be an autonomous element that was rapidly inactivated in the stable gray trichome t* line. Comparison of Tgmt* to previously described Tgm elements demonstrated that two subtypes of CACTA transposon families exist in soybean based on divergence of their characteristic subterminal repeated motifs and their transposases. In addition, we report the sequence and annotation of a BAC clone containing the F3'H gene (T locus) which was interrupted by the novel Tgmt* element in the gray trichome allele t*. Conclusion The molecular characterization of a 20.5 kb insertion in the flavonoid 3'-hydroxylase (F3'H) gene of a soybean gray pubescence allele (t*) identified the structure of a CACTA transposon designated Tgmt*. Besides the terminal inverted repeats and subterminal repeated motifs,Tgmt* encoded a large gene with two putative functions that are required for excision and transposition of a CACTA element, a transposase and the DNA binding protein known to associate to the subterminal repeated motifs. The degree of dissimilarity between Tgmt* transposase and subterminal repeated motifs with those of previously characterized defective CACTA elements (Tgm1-7) were evidence of the existence of two subfamilies of CACTA transposons in soybean, an observation not previously reported in other plants. In addition, our analyses of a genetically active and potentially autonomous element sheds light on the complete structure of a soybean element that is useful for annotation of the repetitive fraction of the soybean genome sequence and may prove useful for transposon tagging or transposon display experiments in different genetic lines.

Abstract Background The soybean (Glycine max) cotyledon is a specialized tissue whose main function is to serve as a nutrient reserve that supplies the needs of the young plant throughout seedling development. During this process the cotyledons experience a functional transition to a mainly photosynthetic tissue. To identify at the genetic level the specific active elements that participate in the natural transition of the cotyledon from storage to photosynthetic activity, we studied the transcript abundance profile at different time points using a new soybean oligonucleotide chip containing 19,200 probes (70-mer long). Results After normalization and statistical analysis we determined that 3,594 genes presented a statistically significant altered expression in relation to the imbibed seed in at least one of the time points defined for the study. Detailed analysis of this data identified individual, specific elements of the glyoxylate pathway that play a fundamental role during the functional transition of the cotyledon from nutrient storage to photosynthesis. The dynamics between glyoxysomes and peroxisomes is evident during these series of events. We also identified several other genes whose products could participate co-ordinately throughout the functional transition and the associated mechanisms of control and regulation and we described multiple unknown genetic elements that by association have the potential to make a major contribution to this biological process. Conclusion We demonstrate that the global transcript profile of the soybean cotyledon during seedling development is extremely active, highly regulated and dynamic. We defined the expression profiles of individual gene family members, enzymatic isoforms and protein subunits and classified them accordingly to their involvement in different functional activities relevant to seedling development and the cotyledonary functional transition in soybean, especially the ones associated with the glyoxylate cycle. Our data suggests that in the soybean cotyledon a very complex and synchronized system of control and regulation of several metabolic pathways is essential to carry out the necessary functions during this developmental process.

Abstract Background Iron is one of fourteen mineral elements required for proper plant growth and development of soybean (Glycine max L. Merr.). Soybeans grown on calcareous soils, which are prevalent in the upper Midwest of the United States, often exhibit symptoms indicative of iron deficiency chlorosis (IDC). Yield loss has a positive linear correlation with increasing severity of chlorotic symptoms. As soybean is an important agronomic crop, it is essential to understand the genetics and physiology of traits affecting plant yield. Soybean cultivars vary greatly in their ability to respond successfully to iron deficiency stress. Microarray analyses permit the identification of genes and physiological processes involved in soybean's response to iron stress. Results RNA isolated from the roots of two near isogenic lines, which differ in iron efficiency, PI 548533 (Clark; iron efficient) and PI 547430 (IsoClark; iron inefficient), were compared on a spotted microarray slide containing 9,728 cDNAs from root specific EST libraries. A comparison of RNA transcripts isolated from plants grown under iron limiting hydroponic conditions for two weeks revealed 43 genes as differentially expressed. A single linkage clustering analysis of these 43 genes showed 57% of them possessed high sequence similarity to known stress induced genes. A control experiment comparing plants grown under adequate iron hydroponic conditions showed no differences in gene expression between the two near isogenic lines. Expression levels of a subset of the differentially expressed genes were also compared by real time reverse transcriptase PCR (RT-PCR). The RT-PCR experiments confirmed differential expression between the iron efficient and iron inefficient plants for 9 of 10 randomly chosen genes examined. To gain further insight into the iron physiological status of the plants, the root iron reductase activity was measured in both iron efficient and inefficient genotypes for plants grown under iron sufficient and iron limited conditions. Iron inefficient plants failed to respond to decreased iron availability with increased activity of Fe reductase. Conclusion These experiments have identified genes involved in the soybean iron deficiency chlorosis response under iron deficient conditions. Single linkage cluster analysis suggests iron limited soybeans mount a general stress response as well as a specialized iron deficiency stress response. Root membrane bound reductase capacity is often correlated with iron efficiency. Under iron-limited conditions, the iron efficient plant had high root bound membrane reductase capacity while the iron inefficient plants reductase levels remained low, further limiting iron uptake through the root. Many of the genes up-regulated in the iron inefficient NIL are involved in known stress induced pathways. The most striking response of the iron inefficient genotype to iron deficiency stress was the induction of a profusion of signaling and regulatory genes, presumably in an attempt to establish and maintain cellular homeostasis. Genes were up-regulated that point toward an increased transport of molecules through membranes. Genes associated with reactive oxidative species and an ROS-defensive enzyme were also induced. The up-regulation of genes involved in DNA repair and RNA stability reflect the inhospitable cellular environment resulting from iron deficiency stress. Other genes were induced that are involved in protein and lipid catabolism; perhaps as an effort to maintain carbon flow and scavenge energy. The under-expression of a key glycolitic gene may result in the iron-inefficient genotype being energetically challenged to maintain a stable cellular environment. These experiments have identified candidate genes and processes for further experimentation to increase our understanding of soybeans' response to iron deficiency stress.

Abstract Background The recent discoveries of transposable elements carrying host gene fragments such as the Pack-MULEs (Mutator-like transposable elements) of maize (Zea mays), rice (Oryza sativa) and Arabidopsis thaliana, the Helitrons of maize and the Tgm-Express of soybeans, revealed a widespread genetic mechanism with the potential to rearrange genomes and create novel chimeric genes affecting genomic and proteomic diversity. Not much is known with regard to the mechanisms of gene fragment capture by those transposon elements or the expression of the captured host gene fragments. There is some evidence that chimeric transcripts can be assembled and exist in EST collections. Results We report results obtained from analysis of RT-PCR derived cDNAs of the Glycine max mutant flower color gene, wp, that contains a 5.7-kb transposon (Tgm-Express1) in Intron 2 of the flavanone 3-hydroxylase gene (F3H) and is composed of five unrelated host gene fragments. The collection of cDNAs derived from the wp allele represents a multiplicity of processed RNAs varying in length and sequence that includes some identical to the correctly processed mature F3H transcript with three properly spliced exons. Surprisingly, the five gene fragments carried by the Tgm-Express1 were processed through complex alternative splicing as additional exons of the wp transcript. Conclusion The gene fragments carried by the Tgm inverted repeat ends appear to be retained as functional exons/introns within the element. The spliceosomes then select indiscriminately the canonical intron splice sites from a pre-mRNA to assemble diverse chimeric transcripts from the exons contained in the wp allele. The multiplicity and randomness of these events provide some insights into the origin and mechanism of alternatively spliced genes.

Abstract Background Reports of plant molecular responses to pathogenic infections have pinpointed increases in activity of several genes of the phenylpropanoid pathway leading to the synthesis of lignin and flavonoids. The majority of those findings were derived from single gene studies and more recently from several global gene expression analyses. We undertook a global transcriptional analysis focused on the response of genes of the multiple branches of the phenylpropanoid pathway to infection by the Pseudomonas syringae pv. glycinea with or without the avirulence gene avrB to characterize more broadly the contribution of the multiple branches of the pathway to the resistance response in soybean. Transcript abundance in leaves was determined from analysis of soybean cDNA microarray data and hybridizations to RNA blots with specific gene probes. Results The majority of the genes surveyed presented patterns of increased transcript accumulation. Some increased rapidly, 2 and 4 hours after inoculation, while others started to accumulate slowly by 8 – 12 hours. In contrast, transcripts of a few genes decreased in abundance 2 hours post inoculation. Most interestingly was the opposite temporal fluctuation in transcript abundance between early responsive genes in defense (CHS and IFS1) and F3H, the gene encoding a pivotal enzyme in the synthesis of anthocyanins, proanthocyanidins and flavonols. F3H transcripts decreased rapidly 2 hours post inoculation and increased during periods when CHS and IFS transcripts decreased. It was also determined that all but one (CHS4) family member genes (CHS1, CHS2, CHS3, CHS5, CHS6 and CHS7/8) accumulated higher transcript levels during the defense response provoked by the avirulent pathogen challenge. Conclusion Based on the mRNA profiles, these results show the strong bias that soybean has towards increasing the synthesis of isoflavonoid phytoalexins concomitant with the down regulation of genes required for the synthesis of anthocyanins and proanthocyanins. Although proanthocyanins are known to be toxic compounds, the cells in the soybean leaves seem to be programmed to prioritize the synthesis and accumulation of isoflavonoid and pterocarpan phytoalexins during the resistance response. It was known that CHS transcripts accumulate in great abundance rapidly after inoculation of the soybean plants but our results have demonstrated that all but one (CHS4) member of the gene family member genes accumulated higher transcript levels during the defense response.

Abstract Background Microarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays. Results We report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants. The set was assembled from the 5' sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3' end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines. Conclusions Global expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype.

To understand translational capacity on a genome-wide scale across three developmental stages of immature soybean seed cotyledons, ribosome profiling was performed in combination with RNA sequencing and cluster analysis. Transcripts representing 216 unique genes demonstrated a higher level of translational activity in at least one stage by exhibiting higher translational efficiencies (TEs) in which there were relatively more ribosome footprint sequence reads mapping to the transcript than were present in the control total RNA sample. The majority of these transcripts were more translationally active at the early stage of seed development and included 12 unique serine or cysteine proteases and 16 2S albumin and low molecular weight cysteine-rich proteins that may serve as substrates for turnover and mobilization early in seed development. It would appear that the serine proteases and 2S albumins play a vital role in the early stages. In contrast, our investigation of profiles of 19 genes encoding high abundance seed storage proteins, such as glycinins, beta-conglycinins, lectin, and Kunitz trypsin inhibitors, showed that they all had similar patterns in which the TE values started at low levels and increased approximately 2 to 6-fold during development. The highest levels of these seed protein transcripts were found at the mid-developmental stage, whereas the highest ribosome footprint levels of only up to 1.6 TE were found at the late developmental stage. These experimental findings suggest that the major seed storage protein coding genes are primarily regulated at the transcriptional level during normal soybean cotyledon development. Finally, our analyses also identified a total of 370 unique gene models that showed very low TE values including over 48 genes encoding ribosomal family proteins and 95 gene models that are related to energy and photosynthetic functions, many of which have homology to the chloroplast genome. Additionally, we showed that genes of the chloroplast were relatively translationally inactive during seed development.

Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factor proteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface. As a model system to test proof of concept of the silicon-substrate PC arrays to directly detect rare proteins in crude plant extracts, we selected representatives of four different transcription factor families (zinc finger GATA, basic helix-loop-helix, BTF3/NAC [for basic transcription factor of the NAC family], and YABBY) that have increasing transcript levels during the stages of seedling cotyledon development. Antibodies to synthetic peptides representing the transcription factors were printed on both glass slides and silicon-substrate PC slides along with antibodies to abundant cotyledon proteins, seed lectin, and Kunitz trypsin inhibitor. The silicon-substrate PC arrays proved more sensitive than those performed on glass slides, detecting rare proteins that were below background on the glass slides. The zinc finger transcription factor was detected on the PC arrays in crude extracts of all stages of the seedling cotyledons, whereas YABBY seemed to be at the lower limit of their sensitivity. Interestingly, the basic helix-loop-helix and NAC proteins showed developmental profiles consistent with their transcript patterns, indicating proof of concept for detecting these low-abundance proteins in crude extracts., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

During early seedling growth, the cotyledons transition from a storage tissue to become effectively the first leaf-like structures of the plant. In this programmed developmental process, they likely undergo a massive change in gene expression to redirect their metabolism and physiological processes. To define the developmental shifts in gene expression and begin to understand the gene regulatory networks that set this transition in motion, we carried out high-throughput RNA sequencing of cotyledons from seven developmental stages of soybean seedlings. We identified 154 gene models with high expression exclusively in the early seedling stages. A significant number (about 25 %) of those genes with known annotations were involved in carbohydrate metabolism. A detailed examination of glyoxylate cycle genes revealed the upregulation of their expression in the early stages of development. A total of approximately 50 % of the highly expressed genes whose expression peaked in the mid-developmental stages encoded ribosomal family proteins. Our analysis also identified 219 gene models with high expression at late developmental stages. The majority of these genes are involved in photosynthesis, including photosystem I- and II-associated genes. Additionally, the advantage of RNA-Seq to detect genes expressed at low levels revealed approximately 460 transcription factors with notable expression in at least one stage of the developing soybean seedling. Relatively over-represented transcription factor genes encode AP2, zinc finger, NAC, WRKY, and MYB families. These transcription factor genes may lead to the transcriptional reprogramming during the transition of seedling cotyledons from storage tissue to metabolically active organs that serve as the first functional leaves of the plant.

The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down-regulation of CHS7 and CHS8 target mRNAs during seed development leading to yellow seed coats lacking anthocyanin pigments. Here, we report small RNA sequencing of ten stages of seed development from a few days post fertilization through maturity, revealing the amplification from primary to secondary short interfering RNAs (siRNAs) occurring during development. The young seed populations had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs. More intriguingly, the very young seed had a higher proportion of 22-nt CHS siRNAs than did the mid-maturation seed. We infer that the primary CHS siRNAs increase during development to levels sufficient to trigger amplification of secondary CHS siRNAs from the CHS7/8 target mRNAs, enabling the total levels of 21-nt CHS siRNAs to rise dramatically. Further, we demonstrate that the soybean system exhibits tissue-specific CHS siRNA production because primary CHS siRNA levels are not sufficient to trigger secondary amplification in tissues other than the seed coat.

Background: Two plant-specific transcription factors, NAC and YABBY, are involved in important plant developmental processes. However their molecular mechanisms, especially DNA binding sites and co-regulated genes, are largely unknown during soybean seedling development., Results: In order to identify genome-wide binding sites of specific members of the NAC and YABBY transcription factors and co-regulated genes, we performed Chromatin Immunoprecipitation Sequencing (ChIP-Seq) and RNA Sequencing (RNA-Seq) using cotyledons from soybean seedling developmental stages. Our RNA-Seq data revealed that these particular NAC and YABBY transcription factors showed a clear pattern in their expression during soybean seedling development. The highest level of their expression was found in seedling developmental stage 4 when cotyledons undergo a physiological transition from non-photosynthetic storage tissue to a metabolically active photosynthetic tissue. Our ChIP-Seq data identified 72 genes potentially regulated by the NAC and 96 genes by the YABBY transcription factors examined. Our RNA-Seq data revealed highly differentially expressed candidate genes regulated by the NAC transcription factor include lipoxygense, pectin methyl esterase inhibitor, DEAD/DEAH box helicase and homeobox associated proteins. YABBY-regulated genes include AP2 transcription factor, fatty acid desaturase and WRKY transcription factor. Additionally, we have identified DNA binding motifs for the NAC and YABBY transcription factors., Conclusions: Genome-wide determination of binding sites for NAC and YABBY transcription factors and identification of candidate genes regulated by these transcription factors will advance the understanding of complex gene regulatory networks during soybean seedling development. Our data imply that there is transcriptional reprogramming during the functional transition of cotyledons from non-photosynthetic storage tissue to metabolically active photosynthetic tissue.

Background: Trichome hairs affect diverse agronomic characters such as seed weight and yield, prevent insect damage and reduce loss of water but their molecular control has not been extensively studied in soybean. Several detailed models for trichome development have been proposed for Arabidopsis thaliana, but their applicability to important crops such as cotton and soybean is not fully known., Results: Two high throughput transcript sequencing methods, Digital Gene Expression (DGE) Tag Profiling and RNA-Seq, were used to compare the transcriptional profiles in wild-type (cv. Clark standard, CS) and a mutant (cv. Clark glabrous, i.e., trichomeless or hairless, CG) soybean isoline that carries the dominant P1 allele. DGE data and RNA-Seq data were mapped to the cDNAs (Glyma models) predicted from the reference soybean genome, Williams 82. Extending the model length by 250 bp at both ends resulted in significantly more matches of authentic DGE tags indicating that many of the predicted gene models are prematurely truncated at the 5' and 3' UTRs. The genome-wide comparative study of the transcript profiles of the wild-type versus mutant line revealed a number of differentially expressed genes. One highly-expressed gene, Glyma04g35130, in wild-type soybean was of interest as it has high homology to the cotton gene GhRDL1 gene that has been identified as being involved in cotton fiber initiation and is a member of the BURP protein family. Sequence comparison of Glyma04g35130 among Williams 82 with our sequences derived from CS and CG isolines revealed various SNPs and indels including addition of one nucleotide C in the CG and insertion of ~60 bp in the third exon of CS that causes a frameshift mutation and premature truncation of peptides in both lines as compared to Williams 82., Conclusion: Although not a candidate for the P1 locus, a BURP family member (Glyma04g35130) from soybean has been shown to be abundantly expressed in the CS line and very weakly expressed in the glabrous CG line. RNA-Seq and DGE data are compared and provide experimental data on the expression of predicted soybean gene models as well as an overview of the genes expressed in young shoot tips of two closely related isolines.

Background: The molecular organization of very few genetically defined CACTA transposon systems have been characterized thoroughly as those of Spm/En in maize, Tam1 of Antirrhinum majus Candystripe1 (Cs1) from Sorghum bicolor and CAC1 from Arabidopsis thaliana, for example. To date, only defective deletion derivatives of CACTA elements have been described for soybean, an economically important plant species whose genome sequence will be completed in 2008., Results: We identified a 20.5 kb insertion in a soybean flavonoid 3'-hydroxylase (F3'H) gene representing the t* allele (stable gray trichome color) whose origin traces to a single mutable chimeric plant displaying both tawny and gray trichomes. This 20.5 kb insertion has the molecular structure of a putative autonomous transposon of the CACTA family, designated Tgmt*. It encodes a large gene that was expressed in two sister isolines (T* and tm) of the stable gray line (t*) from which Tgmt* was isolated. RT-PCR derived cDNAs uncovered the structure of a large precursor mRNA as well as alternatively spliced transcripts reminiscent of the TNPA-mRNA generated by the En-1 element of maize but without sequence similarity to the maize TNPA. The larger mRNA encodes a transposase with a tnp2 and TNP1-transposase family domains. Because the two soybean lines expressing Tgmt* were derived from the same mutable chimeric plant that created the stable gray trichome t* allele line from which the element was isolated, Tgmt* has the potential to be an autonomous element that was rapidly inactivated in the stable gray trichome t* line. Comparison of Tgmt* to previously described Tgm elements demonstrated that two subtypes of CACTA transposon families exist in soybean based on divergence of their characteristic subterminal repeated motifs and their transposases. In addition, we report the sequence and annotation of a BAC clone containing the F3'H gene (T locus) which was interrupted by the novel Tgmt* element in the gray trichome allele t*., Conclusion: The molecular characterization of a 20.5 kb insertion in the flavonoid 3'-hydroxylase (F3'H) gene of a soybean gray pubescence allele (t*) identified the structure of a CACTA transposon designated Tgmt*. Besides the terminal inverted repeats and subterminal repeated motifs,Tgmt* encoded a large gene with two putative functions that are required for excision and transposition of a CACTA element, a transposase and the DNA binding protein known to associate to the subterminal repeated motifs. The degree of dissimilarity between Tgmt* transposase and subterminal repeated motifs with those of previously characterized defective CACTA elements (Tgm1-7) were evidence of the existence of two subfamilies of CACTA transposons in soybean, an observation not previously reported in other plants. In addition, our analyses of a genetically active and potentially autonomous element sheds light on the complete structure of a soybean element that is useful for annotation of the repetitive fraction of the soybean genome sequence and may prove useful for transposon tagging or transposon display experiments in different genetic lines.

Background: The recent discoveries of transposable elements carrying host gene fragments such as the Pack-MULEs (Mutator-like transposable elements) of maize (Zea mays), rice (Oryza sativa) and Arabidopsis thaliana, the Helitrons of maize and the Tgm-Express of soybeans, revealed a widespread genetic mechanism with the potential to rearrange genomes and create novel chimeric genes affecting genomic and proteomic diversity. Not much is known with regard to the mechanisms of gene fragment capture by those transposon elements or the expression of the captured host gene fragments. There is some evidence that chimeric transcripts can be assembled and exist in EST collections., Results: We report results obtained from analysis of RT-PCR derived cDNAs of the Glycine max mutant flower color gene, wp, that contains a 5.7-kb transposon (Tgm-Express1) in Intron 2 of the flavanone 3-hydroxylase gene (F3H) and is composed of five unrelated host gene fragments. The collection of cDNAs derived from the wp allele represents a multiplicity of processed RNAs varying in length and sequence that includes some identical to the correctly processed mature F3H transcript with three properly spliced exons. Surprisingly, the five gene fragments carried by the Tgm-Express1 were processed through complex alternative splicing as additional exons of the wp transcript., Conclusion: The gene fragments carried by the Tgm inverted repeat ends appear to be retained as functional exons/introns within the element. The spliceosomes then select indiscriminately the canonical intron splice sites from a pre-mRNA to assemble diverse chimeric transcripts from the exons contained in the wp allele. The multiplicity and randomness of these events provide some insights into the origin and mechanism of alternatively spliced genes.

Iron deficiency chlorosis (IDC) in soybeans has proven to be a perennial problem in the calcareous soils of the U.S. upper Midwest. A historically difficult trait to study in fields, the use of hydroponics in a controlled greenhouse environment has provided a mechanism to study genetic variation while limiting environmental complications. IDC susceptible plants growing in calcareous soils and in iron-controlled hydroponic experiments often exhibit a characteristic chlorotic phenotype early in the growing season but are able to re-green later in the season. To examine the changes in gene expression of these plants, near-isogenic lines, iron efficient PI548553 (Clark) and iron inefficient PI547430 (IsoClark), developed for their response to iron deficiency stress [USDA, ARS, National Genetic Resources Program, Germplasm Resources Information Network - GRIN. (Online Database) National Germplasm Resources Laboratory, Beltsville, MD, 2004. Available: http://www.ars.grin.gov/cgi-bin/npgs/html/acc_search.pl?accid=PI+547430. [22] were grown in iron-deficient hydroponic conditions for one week, then transferred to iron sufficient conditions for another week. This induced a phenotypic response mimicking the growth of the plants in the field; initial chlorosis followed by re-greening. RNA was isolated from root tissue and transcript profiles were examined between the two near-isogenic lines using publicly available cDNA microarrays. By alleviating the iron deficiency stress our expectation was that plants would return to baseline expression levels. However, the microarray comparison identified four cDNAs that were under-expressed by a two-fold or greater difference in the iron inefficient plant compared to the iron efficient plant. This differential expression was re-examined and confirmed by real time PCR experimentation. Control experiments showed that these genes are not differentially expressed in plants grown continually under iron rich hydroponic conditions. The expression differences suggest potential residual effects of iron deficiency on plant health.

Three loci (I, R, and T) control pigmentation of the seed coats in Glycine max and are genetically distinct from those controlling flower color. The T locus also controls color of the trichome hairs. We report the identification and isolation of a flavonoid 3' hydroxylase gene from G. max (GmF3'H) and the linkage of this gene to the T locus. This GmF3'H gene was highly expressed in early stages of seed coat development and was expressed at very low levels or not at all in other tissues. Evidence that the GmF3'H gene is linked to the T locus came from the occurrence of multiple RFLPs in lines with varying alleles of the T locus, as well as in a population of plants segregating at that locus. GmF3'H genomic and cDNA sequence analysis of color mutant lines with varying t alleles revealed a frameshift mutation in one of the alleles. In another line derived from a mutable genetic stock, the abundance of the mRNAs for GmF3'H was dramatically reduced. Isolation of the GmF3'H gene and its identification as the T locus will enable investigation of the pleiotropic effects of the T locus on cell wall integrity and its involvement in the regulation of the multiple branches of the flavonoid pathway in soybean.

Expressed sequence tags (ESTs) exhibiting homology to a BURP domain containing gene family were identified from the Glycine max (L.) Merr. EST database. These ESTs were assembled into 16 contigs of variable sizes and lengths. Consistent with the structure of known BURP domain containing proteins, the translation products exhibit a modular structure consisting of a C-terminal BURP domain, an N-terminal signal sequence, and a variable internal region. The soybean family members exhibit 35-98% similarity in a -100-amino-acid C-terminal region, and a phylogenetic tree constructed using this region shows that some soybean family members group together in closely related pairs, triplets, and quartets, whereas others remain as singletons. The structure of these groups suggests that multiple gene duplication events occurred during the evolutionary history of this family. The depth and diversity of G. max EST libraries allowed tissue-specific expression patterns of the putative soybean BURPs to be examined. Consistent with known BURP proteins, the newly identified soybean BURPs have diverse expression patterns. Furthermore, putative paralogs can have both spatially and quantitatively distinct expression patterns. We discuss the functional and evolutionary implications of these findings, as well as the utility of EST-based analyses for identifying and characterizing gene families.

Whole-genome sequencing is fundamental to understanding the genetic composition of an organism. Given the size and complexity of the soybean genome, an alternative approach is targeted random-gene sequencing, which provides an immediate and productive method of gene discovery. In this study, more than 120000 soybean expressed sequence tags (ESTs) generated from more than 50 cDNA libraries were evaluated. These ESTs coalesced into 16928 contigs and 17336 singletons. On average, each contig was composed of 6 ESTs and spanned 788 bases. The average sequence length submitted to dbEST was 414 bases. Using only those libraries generating more than 800 ESTs each and only those contigs with 10 or more ESTs each, correlated patterns of gene expression among libraries and genes were discerned. Two-dimensional qualitative representations of contig and library similarities were generated based on expression profiles. Genes with similar expression patterns and, potentially, similar functions were identified. These studies provide a rich source of publicly available gene sequences as well as valuable insight into the structure, function, and evolution of a model crop legume genome.

We report the isolation of a novel soybean gene, Msg, which is highly expressed in developing soybean pods. The gene shows significant homology to a family of fruit- and flower-specific genes, designated the major latex protein (MLP) homologues, so far reported in only a few species and whose functions are unknown. The MLPs are more distantly related to a group of pathogenesis-related proteins (IPR or PR-10) whose functions are likewise unknown. This is the first report of a MLP homologue in a plant for which there is already an IPR-protein reported. We performed an analysis of the Msg promoter with 14 different promoter fragments ranging from 0.65 kb to 2.26 kb, fused to the uidA (GUS) gene. High transient expression was obtained with all the constructs upon particle bombardment in soybean and green bean pods. Stable Arabidopsis transformants were obtained with the Agrobacterium vacuum infiltration method. The promoter is fully active in Arabidopsis only in plants transformed with the 2.26 kb fragment promoter, expressing GUS in nectaries, nodes, short style and in guard cells of the silique, pedicel and stem but not in mature leaves. Surprisingly, the proximal 650 bp TATA-containing region cannot function on its own in Arabidopsis and can be deleted without a change in expression pattern in both Arabidopsis and soybean. Thus, tissue-specific regions of the complex Msg promoter reside in the distal 5' regions upstream of a dispensable TATA box in contrast to many examples of tissue-specific elements that reside much closer to the TATA box.

The pigmented seed coats of several soybean (Glycine max (L.) Merr.) plant introductions and isolines have unusual defects that result in cracking of the mature seed coat exposing the endosperm and cotyledons. It has previously been shown that the T (tawny) locus that controls the color of trichomes on stems and leaves also has an effect on both the structure and pigmentation of the seed coat. Distribution of pigmentation on the seed coat is controlled by alleles of the I (inhibitor) locus. It was also found that total seed coat proteins were difficult to extract from pigmented seed coats with i T genotypes because they have procyanidins that exhibit tannin properties. We report that the inclusion of poly-L-proline in the extraction buffer out-competes proteins for binding to procyanidins. Once this problem was solved, we examined expression of the proline-rich cell wall proteins PRP1 and PRP2 in pigmented genotypes with the dominant T allele. We found that both homozygous i T and i t genotypes have reduced soluble PRP1 levels. The epistatic interaction of the double recessive genotype at both loci is necessary to produce the pigmented, defective seed coat phenotype characteristic of seed coats with the double recessive i and t alleles. This implies a novel effect of an enzyme in the flavonoid pathway on seed coat structure in addition to its effect on flavonoids, anthocyanidins, and proanthocyanidins. No soluble PRP1 polypeptides were detectable in pigmented seed coats (i T genotypes) of isolines that also display a net-like pattern of seed coat cracking, known as the Net defect. PRP2 was also absent in one of the these lines. However, both PRP1 and PRP2 cytoplasmic mRNAs were found in the Net-defective seed coats. Together with in vitro translation studies, these results suggest that the absence of soluble PRP polypeptides in the defective Net lines is post-translational and could be due to a more rapid or premature insolubilization of PRP polypeptides within the cell wall matrix.

Two genes, Prp1 and Prp2, encode proline-rich proteins that are found in different stages of developing seed coats, hypocotyls, and roots of soybeans (Glycine max (L.) Merr.). PRP1 is found in young seed coats and PRP2 is found later during seed desiccation. In some soybean varieties, both proteins are smaller as determined by immunoblotting seed coat proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was found that Prp1 and Prp2 genes are linked to each other by testing seed coat protein extracts from an F2 population of a cross between the cultivar Richland, which exhibits the larger PRP proteins, and Blackhawk, which has the smaller PRP proteins. The Prp1 and Prp2 genes were separated by approximately 13% recombination. Simultaneous expression of soluble PRP2 polypeptides in the maternal seed coat and underlying aleurone layer of the embryo was found. The molecular basis for the size difference between the two varieties was examined using polymerase chain reaction to isolate the Prp genes from Blackhawk, the variety that exhibited the smaller proteins. Both of the genes from Blackhawk contained length polymorphisms that result in omission of some of the repeat units (pro pro val tyr lys) from the proteins. In Prp1, there were two separate deletions in different parts of the gene, each being two tandem repeats in length. In Prp2, there was only one deletion of two tandem repeats. These deletions occur within the coding regions in a manner that conserves the reading frame. The results are the first description of genetic variation in cell wall proteins and its molecular basis.(ABSTRACT TRUNCATED AT 250 WORDS)