Isoflavonoid-specific prenyltransferase gene family in soybean: GmPT01, a pterocarpan 2-dimethylallyltransferase involved in glyceollin biosynthesis.

Phytoalexin glyceollins are soybean-specific antimicrobial compounds that are derived from the isoflavonoid pathway. They are synthesized by soybean in response to extrinsic stress such as pathogen attack or injury, thereby conferring partial resistance if synthesized rapidly at the site of infection and at the required concentration. Soybean produces multiple forms of glyceollins that result from the differential prenylation reaction catalyzed by prenyltransferases (PTs) on either the C-2 or C-4 carbon of a pterocarpan glycinol. The soybean genome contains 77 PT-encoding genes (GmPTs) where at least 11 are (iso)flavonoid-specific. Transcript accumulation of five candidates GmPTs was increased in response to Phytophthora sojae infection, suggesting their role in phytoalexin synthesis. The induced GmPTs localize to plastids and display tissue-specific expression. We have in this study identified two additional GmPTs: an isoflavone dimethylallyltransferase 3 (IDT3); and a glycinol 2-dimethylallyl transferase GmPT01. GmPT01 prenylates (-)-glycinol at the C-2 position, localizes in the plastid, and exhibits root-specific gene expression. Furthermore, its expression is induced rapidly in response to stress, and is associated with a quantitative trait loci linked with resistance to P. sojae. Based on these results, we conclude that GmPT01 are possibly one of the loci involved in conferring partial resistance against stem and root rot disease in soybean.

Slow darkening of pinto bean seed coat is associated with significant metabolite and transcript differences related to proanthocyanidin biosynthesis.

BACKGROUND: Postharvest seed coat darkening in pinto bean is an undesirable trait resulting in a loss in the economic value of the crop. The extent of darkening varies between the bean cultivars and their storage conditions. RESULTS: Metabolite analysis revealed that the majority of flavonoids including proanthocyanidin monomer catechin accumulated at higher level in a regular darkening (RD) pinto line CDC Pintium than in a slow darkening (SD) line 1533-15. A transcriptome analysis was conducted to compare gene expression between CDC Pintium and 1533-15 and identify the gene (s) that may play a role in slow darkening processes in 1533-15 pinto. RNAseq against total RNA from RD and SD cultivars found several phenylpropanoid genes, metabolite transporter genes and genes involved in gene regulation or modification to be differentially expressed between CDC Pintium and 1533-15. CONCLUSION: RNAseq analysis and metabolite data of seed coat tissue from CDC Pintium and 1533-15 revealed that the whole proanthocyanidin biosynthetic pathway was downregulated in 1533-15. Additionally, genes that encode for putative transporter proteins were also downregulated in 1533-15 suggesting both synthesis and accumulation of proanthocyanidin is reduced in SD pintos.

Soybean CCA1-like MYB transcription factor GmMYB133 modulates isoflavonoid biosynthesis.

MYB transcription factors play important roles in the regulation of phenylpropanoid biosynthesis. However, the knowledge regarding the roles of CCA1-like MYBs in phenylpropanoid pathway is limited in plants. Previously, we identified 54 CCA1-like proteins in soybean. In the study, a CCA1-like MYB (GmMYB133) was functionally characterized as a positive regulator in isoflavonoid synthesis. GmMYB133 encodes a 330 aa protein featured with one CCA1 conserved motif. Further analysis indicated that the expression pattern of GmMYB133 was near-perfectly correlated with isoflavonoid accumulation as soybean embryos develop. GmMYB133 over-expression promoted the expression of two key isoflavonoid biosynthetic genes (GmCHS8 and GmIFS2) and increased total isoflavonoid content in hairy roots. Protein-protein interaction assays indicated that GmMYB133 might form hetero- and homodimers with an isoflavonoid regulator GmMYB176 and itself, respectively, while the subcellular localization of GmMYB133 can be altered by its interaction with 14-3-3 protein. These findings provided new insights into the functional roles of CCA1-like MYB proteins in the regulation of phenylpropanoid pathway, and will contribute to the future genetic engineering in the improvement of soybean seed quality.

Genome-wide identification and localization of chalcone synthase family in soybean (Glycine max [L]Merr).

BACKGROUND: Soybean is a paleopolyploid that has undergone two whole genome duplication events. Gene duplication is a type of genomic change that can lead to novel functions of pre-existing genes. Chalcone synthase (CHS) is the plant-specific type III polyketide synthase that catalyzes the first committed step in (iso)flavonoid biosynthesis in plants. RESULTS: Here we performed a genome-wide search of CHS genes in soybean, and identified 21 GmCHS loci containing 14 unique GmCHS (GmCHS1-GmCHS14) that included 5 newly identified GmCHSs (GmCHS10-GmCHS14). Furthermore, 3 copies of GmCHS3 and 2 copies of GmCHS4 were found in soybean. Analysis of gene structure of GmCHSs revealed the presence of a single intron in protein-coding regions except for GmCHS12 that contained 3 introns. Even though GmCHS genes are located on 8 different chromosomes, a large number of these genes are present on chromosome 8 where they form 3 distinct clusters. Expression analysis of GmCHS genes revealed tissue-specific expression pattern, and that some GmCHS isoforms localize in the cytoplasm and the nucleus while other isoforms are restricted to cytoplasm only. CONCLUSION: Overall, we have identified 21 GmCHS loci with 14 unique GmCHS genes in the soybean genome. Their gene structures and genomic organization together with the spatio-temporal expression and protein localization suggest their importance in the production of downstream metabolites such as (iso)flavonoids and their derived phytoalexins.

Twin anchors of the soybean isoflavonoid metabolon: evidence for tethering of the complex to the endoplasmic reticulum by IFS and C4H.

Isoflavonoids are specialized plant metabolites, almost exclusive to legumes, and their biosynthesis forms a branch of the diverse phenylpropanoid pathway. Plant metabolism may be coordinated at many levels, including formation of protein complexes, or 'metabolons', which represent the molecular level of organization. Here, we have confirmed the existence of the long-postulated isoflavonoid metabolon by identifying elements of the complex, their subcellular localizations and their interactions. Isoflavone synthase (IFS) and cinnamate 4-hydroxylase (C4H) have been shown to be tandem P450 enzymes that are anchored in the ER, interacting with soluble enzymes of the phenylpropanoid and isoflavonoid pathways (chalcone synthase, chalcone reductase and chalcone isomerase). The soluble enzymes of these pathways, whether localized to the cytoplasm or nucleus, are tethered to the ER through interaction with these P450s. The complex is also held together by interactions between the soluble elements. We provide evidence for IFS interaction with upstream and non-consecutive enzymes. The existence of such a protein complex suggests a possible mechanism for flux of metabolites into the isoflavonoid pathway. Further, through interaction studies, we identified several candidates that are associated with GmIFS2, an isoform of IFS, in soybean hairy roots. This list provides additional candidates for various biosynthetic and structural elements that are involved in isoflavonoid production. Our interaction studies provide valuable information about isoform specificity among isoflavonoid enzymes, which may guide future engineering of the pathway in legumes or help overcome bottlenecks in heterologous expression.

Transcriptomic evidence for the control of soybean root isoflavonoid content by regulation of overlapping phenylpropanoid pathways.

BACKGROUND: Isoflavonoids are a class of specialized metabolites found predominantly in legumes. They play a role in signaling for symbiosis with nitrogen-fixing bacteria and inhibiting pathogen infection. RESULTS: A transcriptomic approach using soybean cultivars with high (Conrad and AC Colombe) and low (AC Glengarry and Pagoda) root isoflavonoid content was used to find elements that underlie this variation. Two genes, encoding the flavonoid-metabolizing enzymes, flavonoid 3'-hydroxylase (GmF3'H) and dihydroflavonol 4-reductase (GmDFR), had lower expression levels in high isoflavonoid cultivars. These enzymes compete with isoflavonoid biosynthetic enzymes for the important branch-point substrate naringenin and its derivatives. Differentially expressed genes, between the two sets of cultivars, encode transcription factors, transporters and enzymatic families of interest, such as oxidoreductases, hydrolases and transferases. In addition, genes annotated with stress and disease response were upregulated in high isoflavonoid cultivars. CONCLUSIONS: Coordinated regulation of genes involved in flavonoid metabolism could redirect flux into the isoflavonoid branch of the phenylpropanoid pathway, by reducing competition for the flavanone substrate. These candidate genes could help identify mechanisms to overcome the endogenous bottleneck to isoflavonoid production, facilitate biosynthesis in heterologous systems, and enhance crop resistance against pathogenic infections.

Proteomic insights into synthesis of isoflavonoids in soybean seeds.

Soybean seeds are the major human dietary source of isoflavonoids, a class of plant natural products almost entirely exclusive to legumes. Isoflavonoids reduce the risk of a number of chronic human illnesses. Biosynthesis and accumulation of this class of compounds is a multigenic and complex trait, with a great deal of variability among soybean cultivars and with respect to the environment. There is a wealth of genomic, transcriptomic, and metabolomics data regarding isoflavonoid biosynthesis, but the connection between multigene families and their cognate proteins is a missing link that could provide us with a great deal of functional information. The changing proteome of the developing seed can shed light on the correlative increase in isoflavonoids, while the maternal seed coat proteome can provide the link with inherited metabolic and signaling machinery. In this effort, 'seed-filling' proteomics has revealed key secondary metabolite enzymes that quantitatively vary throughout seed development. Seed coat proteomics has revealed the existence of metabolic apparatus specific to isoflavonoid biosynthesis (isoflavonoid reductase) that could potentially influence the chemical content of this organ. The future of proteomic analysis of isoflavonoid biosynthesis should be centered on the development of quantitative, tissue-specific proteomes that emphasize low-abundance metabolic proteins to extract the whole suite of factors involved.

Soybean chalcone isomerase: evolution of the fold, and the differential expression and localization of the gene family.

MAIN CONCLUSION: Soybean chalcone isomerase (CHI) family contains twelve members with unique evolutionary background, expression patterns and is compartmentalized to specific subcellular locations. The phenylpropanoid pathway produces a diverse array of plant natural products. A key branch-point enzyme, chalcone isomerase, catalyzes the reaction producing flavanones, the backbone for many downstream metabolites such as flavonoids and isoflavonoids. We have identified twelve soybean GmCHIs that fall into four subfamilies. The study of this family in soybean in the context of various CHIs and CHI-like proteins, across divisions in the plant kingdom and beyond, shows an evolutionary journey from fatty acid-binding proteins (FAPs) to sterically restricted folds that gave rise to the chalcone-to-flavanone isomerase. There are four GmCHIs with this functionality, three of which belong to a legume-specific clade known as 'type II' CHIs. Tissue-specific expression of eight core members of the soybean CHI family showed differential temporal and spatial expression, pointing to the potential function of GmCHI1A in seed isoflavonoid production. Promoter analysis of the GmCHIs described the minutiae of sub-organ expression patterns. Subcellular localization of the family was conducted to investigate the possibility of pathway-specific compartmentalization. Subfamilies 1, 2 and 4 localized to the nucleus and cytoplasm, with nuclear localization of CHIs raising questions about alternate function. GmCHI3 isoforms localized to the chloroplast, which, in conjunction with their position on the phylogenetic tree and expression patterns, closely associates them with the FAPs. This study provides the first comprehensive look at soybean CHIs, a family of unique evolutionary background and biochemical function, with the catalytically active members producing the backbone substrate in an important plant metabolic pathway.

Genome-wide analysis of Cyclophilin gene family in soybean (Glycine max).

BACKGROUND: Cyclophilins (CYPs) belong to the immunophilin superfamily, and have peptidyl-prolyl cis-trans isomerase (PPIase) activity. PPIase catalyzes cis- and trans-rotamer interconversion of the peptidyl-prolyl amide bond of peptides, a rate-limiting step in protein folding. Studies have demonstrated the importance of many PPIases in plant biology, but no genome-wide analysis of the CYP gene family has been conducted for a legume species. RESULTS: Here we performed a comprehensive database survey and identified a total of 62 CYP genes, located on 18 different chromosomes in the soybean genome (GmCYP1 to GmCYP62), of which 10 are multi- and 52 are single-domain proteins. Most of the predicted GmCYPs clustered together in pairs, reflecting the ancient genome duplication event. Analysis of gene structure revealed the presence of introns in protein-coding regions as well as in 5' and 3' untranslated regions, and that their size, abundance and distribution varied within the gene family. Expression analysis of GmCYP genes in soybean tissues displayed their differential tissue specific expression patterns. CONCLUSIONS: Overall, we have identified 62 CYP genes in the soybean genome, the largest CYP gene family known to date. This is the first genome-wide study of the CYP gene family of a legume species. The expansion of GmCYP genes in soybean, and their distribution pattern on the chromosomes strongly suggest genome-wide segmental and tandem duplications.

Soybean AROGENATE DEHYDRATASES (GmADTs): involvement in the cytosolic isoflavonoid metabolon or trans-organelle continuity?

Soybean (Glycine max) produces a class of phenylalanine (Phe) derived specialized metabolites, isoflavonoids. Isoflavonoids are unique to legumes and are involved in defense responses in planta, and they are also necessary for nodule formation with nitrogen-fixing bacteria. Since Phe is a precursor of isoflavonoids, it stands to reason that the synthesis of Phe is coordinated with isoflavonoid production. Two putative AROGENATE DEHYDRATASE (ADT) isoforms were previously co-purified with the soybean isoflavonoid metabolon anchor ISOFLAVONE SYNTHASE2 (GmIFS2), however the GmADT family had not been characterized. Here, we present the identification of the nine member GmADT family. We determined that the GmADTs share sequences required for enzymatic activity and allosteric regulation with other characterized plant ADTs. Furthermore, the GmADTs are differentially expressed, and multiple members have dual substrate specificity, also acting as PREPHENATE DEHYDRATASES. All GmADT isoforms were detected in the stromules of chloroplasts, and they all interact with GmIFS2 in the cytosol. In addition, GmADT12A interacts with multiple other isoflavonoid metabolon members. These data substantiate the involvement of GmADT isoforms in the isoflavonoid metabolon.

14-3-3 proteins regulate the intracellular localization of the transcriptional activator GmMYB176 and affect isoflavonoid synthesis in soybean.

Isoflavonoids are legume-specific natural plant compounds that play important functions in nitrogen fixation as well as biotic and abiotic stress responses. Many clinical studies have suggested a role for isoflavonoids in human health and nutrition. We have recently identified an R1 MYB transcription factor GmMYB176 that regulates CHS8 gene expression and isoflavonoid biosynthesis. Here we demonstrate that binding of 14-3-3 proteins to GmMYB176 modulates this function. GmMYB176 interacts with all 16 14-3-3 proteins (SGF14s) in soybean (Glycine max) with varying activity. The detailed analysis of 14-3-3-binding sites within GmMYB176 identified a critical motif (D2) where Ser29 is potentially phosphorylated. Deletion of the D2 motif from GmMYB176 or substitution of Ser29 with an alanine abolished binding with SGF14 proteins, which altered the subcellular localization of GmMYB176. Overexpression of SGF14l in soybean hairy roots did not affect the transcript level of GmMYB176 but it reduced the expression levels of key isoflavonoid genes and isoflavonoid accumulation in soybean hairy root. Our results suggest that SGF14-GmMYB176 interaction regulates the intracellular localization of GmMYB176, thereby affecting isoflavonoid biosynthesis in soybean.

Identification of the factors that control synthesis and accumulation of a therapeutic protein, human immune-regulatory interleukin-10, in Arabidopsis thaliana.

Plants are one of the most economical platforms for large-scale production of recombinant proteins for biopharmaceutical and industrial uses. A large number of human recombinant proteins of therapeutic value have been successfully produced in plant systems. One of the main technical challenges of producing recombinant proteins in plants is to obtain sufficient level of protein. This research aims to identify the factors that control synthesis and accumulation of recombinant proteins in stable transgenic plants. A stepwise dissection of human immune-regulatory interleukin-10 (IL-10) protein production was carried out using Arabidopsis thaliana as a model system. EMS-mutagenized transgenic Arabidopsis IL-10 lines, at2762 and at3262, produced significantly higher amount of IL-10 protein than the non-mutagenized IL-10 line (WT-IL-10). The fates of trans-gene in these sets of plants were compared in detail by measuring synthesis and accumulation of IL-10 transcript, transcript stability, protein synthesis and IL-10 protein accumulation. The IL-10 transcripts were more stable in at2762 and at3262 lines than WT-IL-10, which may contribute to higher protein synthesis in these lines. To evaluate whether translational regulation of IL-10 controls its synthesis in non-mutagenized WT-IL-10 and higher IL-10 accumulating mutant lines, we measured the efficiency of the translational machinery. Our results indicate that mutant lines with higher trans-gene expression contain more robust and efficient translational machinery compared with the control line.

Functional characterization of Cinnamate 4-hydroxylase gene family in soybean (Glycine max).

Cinnamate 4-hydroxylase (C4H) is the first key cytochrome P450 monooxygenase (P450) enzyme in the phenylpropanoid pathway. It belongs to the CYP73 family of P450 superfamily, and catalyzes the conversion of trans-cinnamic acid to p-coumaric acid. Since p-coumaric acid serves as the precursor for the synthesis of a wide variety of metabolites involved in plant development and stress resistance, alteration in the expression of soybean C4H genes is expected to affect the downstream metabolite levels, and its ability to respond to stress. In this study, we identified four C4H genes in the soybean genome that are distributed into both class I and class II CYP73 family. GmC4H2, GmC4H14 and GmC4H20 displayed tissue- and developmental stage-specific gene expression patterns with their transcript accumulation at the highest level in root tissues. GmC4H10 appears to be a pseudogene as its transcript was not detected in any soybean tissues. Furthermore, protein homology modelling revealed substrate docking only for GmC4H2, GmC4H14 and GmC4H20. To demonstrate the function of GmC4Hs, we modified a cloning vector for the heterologous expression of P450s in yeast, and used it for microsomal protein production and enzyme assay. Our results confirmed that GmC4H2, GmC4H14 and GmC4H20 contain the ability to hydroxylate trans-cinnamic acid with varying efficiencies.

Transcripts of sulphur metabolic genes are co-ordinately regulated in developing seeds of common bean lacking phaseolin and major lectins.

The lack of phaseolin and phytohaemagglutinin in common bean (dry bean, Phaseolus vulgaris) is associated with an increase in total cysteine and methionine concentrations by 70% and 10%, respectively, mainly at the expense of an abundant non-protein amino acid, S-methyl-cysteine. Transcripts were profiled between two genetically related lines differing for this trait at four stages of seed development using a high density microarray designed for common bean. Transcripts of multiple sulphur-rich proteins were elevated, several previously identified by proteomics, including legumin, basic 7S globulin, albumin-2, defensin, albumin-1, the Bowman-Birk type proteinase inhibitor, the double-headed trypsin inhibitor, and the Kunitz trypsin inhibitor. A co-ordinated regulation of transcripts coding for sulphate transporters, sulphate assimilatory enzymes, serine acetyltransferases, cystathionine ß-lyase, homocysteine S-methyltransferase and methionine gamma-lyase was associated with changes in cysteine and methionine concentrations. Differential gene expression of sulphur-rich proteins preceded that of sulphur metabolic enzymes, suggesting a regulation by demand from the protein sink. Up-regulation of SERAT1;1 and -1;2 expression revealed an activation of cytosolic O-acetylserine biosynthesis. Down-regulation of SERAT2;1 suggested that cysteine and S-methyl-cysteine biosynthesis may be spatially separated in different subcellular compartments. Analysis of free amino acid profiles indicated that enhanced cysteine biosynthesis was correlated with a depletion of O-acetylserine. These results contribute to our understanding of the regulation of sulphur metabolism in developing seed in response to a change in the composition of endogenous proteins.

Relationship between asparagine metabolism and protein concentration in soybean seed.

The relationship between asparagine metabolism and protein concentration was investigated in soybean seed. Phenotyping of a population of recombinant inbred lines adapted to Illinois confirmed a positive correlation between free asparagine levels in developing seeds and protein concentration at maturity. Analysis of a second population of recombinant inbred lines adapted to Ontario associated the elevated free asparagine trait with two of four quantitative trait loci determining population variation for protein concentration, including a major one on chromosome 20 (linkage group I) which has been reported in multiple populations. In the seed coat, levels of asparagine synthetase were high at 50 mg and progressively declined until 150 mg seed weight, suggesting that nitrogenous assimilates are pre-conditioned at early developmental stages to enable a high concentration of asparagine in the embryo. The levels of asparaginase B1 showed an opposite pattern, being low at 50 mg and progressively increased until 150 mg, coinciding with an active phase of storage reserve accumulation. In a pair of genetically related cultivars, ∼2-fold higher levels of asparaginase B1 protein and activity in seed coat, were associated with high protein concentration, reflecting enhanced flux of nitrogen. Transcript expression analyses attributed this difference to a specific asparaginase gene, ASPGB1a. These results contribute to our understanding of the processes determining protein concentration in soybean seed.

Comprehensive Analysis of Cytochrome P450 Monooxygenases Reveals Insight Into Their Role in Partial Resistance Against Phytophthora sojae in Soybean.

Cytochrome P450 monooxygenases (P450) participate in the catalytic conversion of biological compounds in a plethora of metabolic pathways, such as the biosynthesis of alkaloids, terpenoids, phenylpropanoids, and hormones in plants. Plants utilize these metabolites for growth and defense against biotic and abiotic stress. In this study, we identified 346 P450 (GmP450) enzymes encoded by 317 genes in soybean where 26 GmP450 genes produced splice variants. The genome-wide comparison of both A-type and non-A-type GmP450s for their motifs composition, gene structure, tissue-specific expression, and their chromosomal distribution were determined. Even though conserved P450 signature motifs were found in all GmP450 families, larger variation within a specific motif was observed in the non-A-type GmP450s as compared with the A-type. Here, we report that the length of variable region between two conserved motifs is exact in the members of the same family in majority of the A-type GmP450. Analyses of the transcriptomic datasets from soybean-Phytophthora sojae interaction studies, quantitative trait loci (QTL) associated with P. sojae resistance, and co-expression analysis identified some GmP450s that may be, in part, play an important role in partial resistance against P. sojae. The findings of our CYPome study provides novel insights into the functions of GmP450s and their involvements in metabolic pathways in soybean. Further experiments will elucidate their roles in general and legume-specific function.

Investigation of Metabolic Resistance to Soybean Aphid (Aphis glycines Matsumura) Feeding in Soybean Cultivars.

Soybean aphid (Aphis glycines) is a major soybean (Glycine max) herbivore pest in many soybean growing regions. High numbers of aphids on soybean can cause severe reductions in yield. The management of soybean aphids includes monitoring, insecticide applications when required, and the use of resistant cultivars. Soybean aphid-resistant soybean varieties are associated with genes that confer one or more categories of resistance to soybean aphids, including antibiosis (affects survival, growth, and fecundity), antixenosis (affects behaviour such as feeding), and tolerance (plant can withstand greater damage without economic loss). The genetic resistance of soybean to several herbivores has been associated with isoflavonoid phytoalexins; however, this correlation has not been observed in soybean varieties commonly grown in southern Ontario, Canada. Isoflavonoids in the leaves of 18 cultivars in the early growth stage were analyzed by HPLC and the concentration by fresh weight was used to rate the potential resistance to aphids. Greenhouse and growth cabinet trials determined that the cultivars with greater resistance to aphids were Harosoy 63 and OAC Avatar. The most susceptible cultivar was Maple Arrow, whereas Pagoda and Conrad were more tolerant to aphid feeding damage. Overall, there was a low correlation between the number of aphids per leaf, feeding damage, and leaf isoflavonoid levels. Metabolite profiling by high-resolution LC-MS determined that the most resistant cultivar had on average lower levels of certain free amino acids (Met, Tyr, and His) relative to the most susceptible cultivar. This suggests that within the tested cultivars, nutritional quality stimulates aphid feeding more than isoflavonoids negatively affect aphid feeding or growth. These findings provide a better understanding of soybean host plant resistance and suggest ways to improve soybean resistance to aphid feeding through the breeding or metabolic engineering of leaf metabolites.

Global analysis of common bean multidrug and toxic compound extrusion transporters (PvMATEs): PvMATE8 and pinto bean seed coat darkening.

In common bean (Phaseolus vulgaris L.), postharvest seed coat darkening is an undesirable trait that affects crop value. The increased accumulation of proanthocyanidins (PAs) in the seed coat results in darker seeds in many market classes of colored beans after harvest. The precursors of PAs are synthesized in the cytoplasm, and subsequently get glycosylated and then transported to the vacuoles where polymerization occurs. Thus, vacuolar transporters play an important role in the accumulation of PAs. Here, we report that common bean genome contains 59 multidrug and toxic compound extrusion genes (PvMATEs). Phylogenetic analysis of putative PvMATEs with functionally characterized MATEs from other plant species categorized them into substrate-specific clades. Our data demonstrate that a vacuolar transporter PvMATE8 is expressed at a higher level in the pinto bean cultivar CDC Pintium (regular darkening) compared to 1533-15 (slow darkening). PvMATE8 localizes in the vacuolar membrane and rescues the PA deficient (tt12) mutant phenotype in Arabidopsis thaliana. Analysis of PA monomers in transgenic seeds together with wild-type and mutants suggests a possible feedback regulation of PA biosynthesis and accumulation. Identification of PvMATE8 will help better understand the mechanism of PA accumulation in common bean.

A single-repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean.

Here we demonstrate that GmMYB176 regulates CHS8 expression and affects isoflavonoid synthesis in soybean. We previously established that CHS8 expression determines the isoflavonoid level in soybean seeds by comparing the transcript profiles of cultivars with different isoflavonoid contents. In the present study, a functional genomic approach was used to identify the factor that regulates CHS8 expression and isoflavonoid synthesis. Candidate genes were cloned, and co-transfection assays were performed in Arabidopsis leaf protoplasts. The results showed that GmMYB176 can trans-activate the CHS8 promoter with maximum activity. Transient expression of GmMYB176 in soybean embryo protoplasts increased endogenous CHS8 transcript levels up to 169-fold after 48 h. GmMYB176 encodes an R1 MYB protein, and is expressed in soybean seed during maturation. Furthermore, GmMYB176 recognizes a 23 bp motif containing a TAGT(T/A)(A/T) sequence within the CHS8 promoter. A subcellular localization study confirmed nuclear localization of GmMYB176. A predicted pST binding site for 14-3-3 protein is required for subcellular localization of GmMYB176. RNAi silencing of GmMYB176 in hairy roots resulted in reduced levels of isoflavonoids, showing that GmMYB176 is necessary for isoflavonoid biosynthesis. However, over-expression of GmMYB176 was not sufficient to increase CHS8 transcript and isoflavonoid levels in hairy roots. We conclude that an R1 MYB transcription factor, GmMYB176, regulates CHS8 expression and isoflavonoid synthesis in soybean.

Soybean 14-3-3 gene family: identification and molecular characterization.

The 14-3-3s are a group of proteins that are ubiquitously found in eukaryotes. Plant 14-3-3 proteins are encoded by a large multigene family and are involved in signaling pathways to regulate plant development and protection from stress. Recent studies in Arabidopsis and rice have demonstrated the isoform specificity in 14-3-3s and their client protein interactions. However, detailed characterization of 14-3-3 gene family in legumes has not been reported. In this study, soybean 14-3-3 proteins were identified and their molecular characterization performed. Data mining of soybean genome and expressed sequence tag databases identified 18 14-3-3 genes, of them 16 are transcribed. All 16 SGF14s have higher expression in embryo tissues suggesting their potential role in seed development. Subcellular localization of all transcribed SGF14s demonstrated that 14-3-3 proteins in soybean have isoform specificity, however, some overlaps were also observed between closely related isoforms. A comparative analysis of SGF14s with Arabidopsis and rice 14-3-3s indicated that SGF14s also group into epsilon and non-epsilon classes. However, unlike Arabidopsis and rice 14-3-3s, SGF14s contained only one kind of gene structure belonging to each class. Overall, soybean consists of the largest family of 14-3-3 proteins characterized to date. Our results provide a solid framework for further investigations into the role of SGF14s and their involvement in legume-specific functions.