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In soilborne diseases, the plant-pathogen interaction begins as soon as the seed germinates and develops into a seedling. Aphanomyces euteiches , an oomycete, stays dormant in soil and is activated by sensing the host through chemical signals present in the root exudates. The composition of plant exudates may, thus, play an important role during the early phase of infection. To better understand the role of root exudates in plant resistance, we investigated the interaction between partially resistant lines (PI660736 and PI557500) and susceptible pea cultivars (CDC Meadow and AAC Chrome) against A. euteiches during the pre-invasion phase. The root exudates of the two sets of cultivars clearly differed from each other in inducing oospore germination. PI557500 root exudate not only had diminished induction but also inhibited the oospore germination. The contrast between the root exudates of resistant and susceptible cultivars was reflected in their metabolic profiles. Data from fractionation and oospore germination inhibitory experiments identified a group of saponins that accumulated differentially in susceptible and resistant cultivars. We detected 56 saponins and quantified 44 of them in pea root and 30 from root exudate; the majority of them, especially soyasaponin I and dehydrosoyasaponin I with potent in vitro inhibitory activities, were present in significantly higher amounts in both roots and root exudates of PI660736 and PI557500 compared with Meadow and Chrome. Our results provide evidence for saponins as deterrents against A. euteiches , which might have contributed to the resistance against root rot in the studied pea cultivars. [Formula: see text] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada and the National Research Council of Canada. This is an open access article distributed under the CC BY 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Suberin biosynthesis involves the coordinated regulation of both phenolic and aliphatic metabolisms. HXXXD/BAHD acyltransferases occupy a unique place in suberization, as they function to crosslink phenolic and aliphatic monomers during suberin assembly. To date, only one suberin-associated HXXXD/BAHD acyltransferase, StFHT, has been described in potatoes, whereas, in Arabidopsis , at least two are implicated in suberin biosynthesis. RNAseq data from wound-induced potato tubers undergoing suberization indicate that transcripts for 28 HXXXD/BAHD acyltransferase genes accumulate in response to wounding. In the present study, we generated RNAi knockdown lines for StFHT and another highly wound-induced HXXXD/BAHD acyltransferase, designated StHCT , and characterized their wound-induced suberin phenotype. StFHT-RNAi and StHCT-RNAi knockdown lines share the same aliphatic suberin phenotype of reduced esterified ferulic acid and ferulates, which is similar to the previously described StFHT-RNAi knockdown suberin phenotype. However, the phenolic suberin phenotype differed between the two knockdown genotypes, with StHCT-RNAi knockdown lines having proportionately more p -hydroxyphenyl-derived moieties than either StFHT-RNAi knockdown or empty vector control lines. Analysis of soluble polar metabolites revealed that StHCT catalyzes a step upstream from StFHT. Overall, our data support the involvement of more than one HXXXD/BAHD acyltransferase in potato suberin biosynthesis.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Susanne E. Kohalmi and His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada for the contribution of Emily J. Clayton, Nishat S. Islam, Kelsey Pannunzio, Kuflom Kuflu, Ramtin Sirjani and Sangeeta Dhaubhadel.)

We present an ensemble transfer learning method to predict suicide from Veterans Affairs (VA) electronic medical records (EMR). A diverse set of base models was trained to predict a binary outcome constructed from reported suicide, suicide attempt, and overdose diagnoses with varying choices of study design and prediction methodology. Each model used twenty cross-sectional and 190 longitudinal variables observed in eight time intervals covering 7.5 years prior to the time of prediction. Ensembles of seven base models were created and fine-tuned with ten variables expected to change with study design and outcome definition in order to predict suicide and combined outcome in a prospective cohort. The ensemble models achieved c-statistics of 0.73 on 2-year suicide risk and 0.83 on the combined outcome when predicting on a prospective cohort of [Formula: see text] 4.2 M veterans. The ensembles rely on nonlinear base models trained using a matched retrospective nested case-control (Rcc) study cohort and show good calibration across a diversity of subgroups, including risk strata, age, sex, race, and level of healthcare utilization. In addition, a linear Rcc base model provided a rich set of biological predictors, including indicators of suicide, substance use disorder, mental health diagnoses and treatments, hypoxia and vascular damage, and demographics., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Objective: To apply deep neural networks (DNNs) to longitudinal EHR data in order to predict suicide attempt risk among veterans. Local explainability techniques were used to provide explanations for each prediction with the goal of ultimately improving outreach and intervention efforts., Materials and Methods: The DNNs fused demographic information with diagnostic, prescription, and procedure codes. Models were trained and tested on EHR data of approximately 500 000 US veterans: all veterans with recorded suicide attempts from April 1, 2005, through January 1, 2016, each paired with 5 veterans of the same age who did not attempt suicide. Shapley Additive Explanation (SHAP) values were calculated to provide explanations of DNN predictions., Results: The DNNs outperformed logistic and linear regression models in predicting suicide attempts. After adjusting for the sampling technique, the convolutional neural network (CNN) model achieved a positive predictive value (PPV) of 0.54 for suicide attempts within 12 months by veterans in the top 0.1% risk tier. Explainability methods identified meaningful subgroups of high-risk veterans as well as key determinants of suicide attempt risk at both the group and individual level., Discussion and Conclusion: The deep learning methods employed in the present study have the potential to significantly enhance existing suicide risk models for veterans. These methods can also provide important clues to explore the relative value of long-term and short-term intervention strategies. Furthermore, the explainability methods utilized here could also be used to communicate to clinicians the key features which increase specific veterans' risk for attempting suicide., (Published by Oxford University Press on behalf of the American Medical Informatics Association 2023.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Khatri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 ( PvMATE s). 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada for the contribution of Nishat S. Islam, Kishor Duwadi, Ling Chen, Aga Pajak, Tim McDowell, Frédéric Marsolais and Sangeeta Dhaubhadel.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Istvan Rajcan, and Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada for the contribution of Praveen Khatri, Owen Wally, and Sangeeta Dhaubhadel.)

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.

In southern Ontario, Canada, the two-spotted spider mite (Tetranychus urticae) is an emerging pest of soybean (Glycine max) due to the increasing incidence of warmer, drier weather conditions. One key strategy to manage soybean pests is breeding resistant cultivars. Resistance to pathogens and herbivores in soybean has been associated with isoflavonoid phytoalexins, a group of specialized metabolites commonly associated with root, leaf and seed tissues. A survey of 18 Ontario soybean cultivars for spider mite resistance included evaluations of antibiosis and tolerance in relation to isoflavonoid and other metabolites detected in the leaves. Ten-day and 4-week trials beginning with early growth stage plants were used to compare survival, growth, fecundity as well as damage to leaves. Two-spotted spider mite (TSSM) counts were correlated with HPLC measurements of isoflavonoid concentration in the leaves and global metabolite profiling by high resolution LC-MS to identify other metabolites unique to the most resistant (R) and susceptible (S) cultivars. Within 10 days, no significant difference (P>0.05) in resistance to TSSM was determined between cultivars, but after 4 weeks, one cultivar, OAC Avatar, was revealed to have the lowest number of adult TSSMs and their eggs. Other cultivars showing partial resistance included OAC Wallace and OAC Lakeview, while Pagoda was the most tolerant to TSSM feeding. A low, positive correlation between isoflavonoid concentrations and TSSM counts and feeding damage indicated these compounds alone do not explain the range of resistance or tolerance observed. In contrast, other metabolite features were significantly different (P<0.05) in R versus S cultivars. In the presence of TSSM, the R cultivars had significantly greater (P<0.05) concentrations of the free amino acids Trp, Val, Thr, Glu, Asp and His relative to S cultivars. Furthermore, the R cultivar metabolites detected are viable targets for more in-depth analysis of their potential roles in TSSM defense., Competing Interests: The authors have declared that no competing interests exist.

GmMYB176 is an R1 MYB transcription factor that regulates multiple genes in the isoflavonoid biosynthetic pathway, thereby affecting their levels in soybean roots. While GmMYB176 is important for isoflavonoid synthesis, it is not sufficient for the function and requires additional cofactor(s). The aim of this study was to identify the GmMYB176 interactome for the regulation of isoflavonoid biosynthesis in soybean. Here, we demonstrate that a bZIP transcription factor GmbZIP5 co-immunoprecipitates with GmMYB176 and shows protein-protein interaction in planta. RNAi silencing of GmbZIP5 reduced the isoflavonoid level in soybean hairy roots. Furthermore, co-overexpression of GmMYB176 and GmbZIP5 enhanced the level of multiple isoflavonoid phytoallexins including glyceollin, isowighteone and a unique O-methylhydroxy isoflavone in soybean hairy roots. These findings could be utilized to develop biotechnological strategies to manipulate the metabolite levels either to enhance plant defense mechanisms or for human health benefits in soybean or other economically important crops.

Type I Diacylglycerol acyltransferase (DGAT1) catalyzes the final step of the biosynthesis process of triacylglycerol (TAG), the major storage lipids in plant seeds, through the esterification of diacylglycerol (DAG). To characterize the function of DGAT1 genes on the accumulation of oil and other seed composition traits in soybean, transgenic lines were generated via trans-acting siRNA technology, in which three DGAT1 genes (Glyma.13G106100, Glyma.09G065300, and Glyma.17G053300) were downregulated. The simultaneous downregulation of the three isoforms in transgenic lines was found to be associated with the reduction of seed oil concentrations by up to 18 mg/g (8.3%), which was correlated with increases in seed protein concentration up to 42 mg/g (11%). Additionally, the downregulations also influenced the fatty acid compositions in the seeds of transgenic lines through increasing the level of oleic acid, up to 121 mg/g (47.3%). The results of this study illustrate the importance of DGAT1 genes in determining the seed compositions in soybean through the development of new potential technology for manipulating seed quality in soybean to meet the demands for its various food and industrial applications.

Pinto bean ( Phaseolus vulgaris ) is one of the leading market classes of dry beans that is most affected by postharvest seed coat darkening. The process of seed darkening poses a challenge for bean producers and vendors as they encounter significant losses in crop value due to decreased consumer preference for darker beans. Here, we identified a novel allele of the P gene, P sd , responsible for the slow darkening seed coat in pintos, and identified trait-specific sequence polymorphisms which are utilized for the development of new gene-specific molecular markers for breeding. These tools can be deployed to help tackle this economically important issue for bean producers., Summary: Postharvest seed coat darkening in pinto bean is an undesirable trait that reduces the market value of the stored crop. Regular darkening (RD) pintos darken faster after harvest and accumulate higher level of proanthocyanidins (PAs) compared to slow darkening (SD) cultivars. Although the markers cosegregating with the SD trait have been known for some time, the SLOW DARKENING ( Sd ) gene identity had not been proven.Here, we identified P sd as a candidate for controlling the trait. Genetic complementation, transcript abundance, metabolite analysis, and inheritance study confirmed that P sd is the Sd gene. P sd is another allele of the P ( Pigment ) gene, whose loss-of-function alleles result in a white seed coat. P sd encodes a bHLH transcription factor with two transcript variants but only one is involved in PA biosynthesis. An additional glutamate residue in the activation domain, and/or an arginine to histidine substitution in the bHLH domain of the P sd -1 transcript in the SD cultivar is likely responsible for the reduced activity of this allele compared to the allele in a RD cultivar, leading to reduced PA accumulation.Overall, we demonstrate that a novel allele of P , P sd , is responsible for the SD phenotype, and describe the development of new, gene-specific, markers that could be utilized in breeding to resolve an economically important issue for bean producers., (© 2020 Her Majesty the Queen in Right of Canada. Plants, People, Planet published by John Wiley & Sons Ltd on behalf of New Phytologist Trust. Reproduced with the permission of Minister of Agriculture and Agri-Food Canada.)

Isoflavonoids are a group of plant natural compounds synthesized almost exclusively by legumes, and are abundant in soybean seeds and roots. They play important roles in plant-microbial interactions and the induction of nod gene expression in Rhizobia that form nitrogen-fixing nodules on soybean roots. Isoflavonoids also contribute to the positive health effects associated with soybean consumption by humans and animals. An R1 MYB transcription factor GmMYB176 regulates isoflavonoid biosynthesis by activating chalcone synthase ( CHS ) 8 gene expression in soybean. Using a combination of transcriptomic and metabolomic analyses of GmMYB176-RNAi silenced (GmMYB176-Si), GmMYB176-overexpressed (GmMYB176-OE), and control soybean hairy roots, we identified a total of 33 differentially expressed genes (DEGs) and 995 differentially produced metabolite features (DPMF) in GmMYB176-Si hairy roots, and 5727 DEGs and 149 DPMFs in GmMYB176-OE hairy roots. By a targeted approach, 25 isoflavonoid biosynthetic genes and 6 metabolites were identified as differentially regulated in GmMYB176-OE and GmMYB176-Si soybean hairy roots. Taken together, our results demonstrate the complexity of isoflavonoid biosynthesis in soybean roots and suggest that a coordinated expression of pathway genes, substrate flux and product threshold level may contribute to the dynamic of the pathway regulation.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

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., (© 2018 Her Majesty the Queen in Right of Canada. The Plant Journal © 2018 John Wiley & Sons Ltd and Society for Experimental Biology. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.)

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 ( Glycine max [L.] Merr) is one of the main grain legumes worldwide. Soybean farmers lose billions of dollars' worth of yield annually due to root and stem rot disease caused by the oomycete Phytophthora sojae . Many strategies have been developed to combat the disease, however, these methods have proven ineffective in the long term. A more cost effective and durable approach is to select a trait naturally found in soybean that can increase resistance. One such trait is the increased production of phytoalexin glyceollins in soybean. Glyceollins are isoflavonoids, synthesized via the legume-specific branch of general phenylpropanoid pathway. The first key enzyme exclusively involved in glyceollin synthesis is chalcone reductase (CHR) which coacts with chalcone synthase for the production of isoliquiritigenin, the precursor for glyceollin biosynthesis. Here we report the identification of 14 putative CHR genes in soybean where 11 of them are predicted to be functional. Our results show that GmCHR s display tissue-specific gene expression, and that only root-specific GmCHR s are induced upon P. sojae infection. Among 4 root-specific GmCHR s, GmCHR2A is located near a QTL that is linked to P. sojae resistance suggesting GmCHR2A as a novel locus for partial resistance that can be utilized for resistance breeding.

A subset of WD40 proteins with DWD motif has been proposed to serve as substrate receptor of DDB-CUL4-ROC1 complex, thereby getting involved in protein degradation via ubiquitination pathway. Here, we identified a total of 161 potential DWD proteins in soybean (Glycine max) by searching DWD motif against the genome-wide WD40 repeats, and classified them into 20 groups on the basis of their functional domains and annotations. These putative DWD genes in soybean displayed tissue-specific expression patterns, and their genome localization and analysis of evolutionary relationship identified 48 duplicated gene pairs within 161 GmDWDs. Among the 161 soybean DWD proteins, Gm08DWD was previously found to interact with an isoflavonoid regulator, GmMYB176. Therefore, Gm08DWD and its homologue Gm05DWD were further investigated. Expression profile of both genes in different soybean tissues revealed that Gm08DWD was expressed higher in embryo, while Gm05DWD exhibited maximum transcript accumulation in leaf. Our protein-protein interaction studies demonstrated that Gm08DWD interacts with GmMYB176. Although Gm08DWD was localized both in nucleus and cytoplasm, the resulting complex of Gm08DWD and GmMYB176 was mainly observed in the nucleus. This finding is consistent with the functional localization of CUL4-E3 ligase complex. In conclusion, the survey on soybean potential DWD protein is useful reference for the further functional investigation of their DDB1-binding ability. Based on the functional investigation of Gm08DWD, we speculate that protein-protein interaction between Gm08DWD and GmMYB176 may lead to the degradation of GmMYB176 through CUL4-DDB1complex.

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.

Cyclophilins (CYPs) belong to the immunophilin superfamily with peptidyl-prolyl cis-trans isomerase (PPIase) activity. They catalyze the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptides. A yeast-two-hybrid screening using the isoflavonoid regulator GmMYB176 as bait identified GmCYP1 as one of the interacting proteins in soybean embryos. GmCYP1 localizes both in the nucleus and cytoplasm, and interacts in planta with GmMYB176, in the nucleus, and with SGF14l (a soybean 14-3-3 protein) in the nucleus and the cytoplasm. GmCYP1 contains a single cyclophilin-like domain and displays a high sequence identity with other plant CYPs that are known to have stress-specific function. Tissue-specific expression of GmCYP1 revealed higher expression in developing seeds compared to other vegetative tissues, suggesting their seed-specific role. Furthermore, GmCYP1 transcript level was reduced in response to stress. Since isoflavonoids are involved in plant stress resistance against biotic and abiotic factors, the interaction of GmCYP1 with the isoflavonoid regulators GmMYB176 and 14-3-3 protein suggests its role in defense in soybean.

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., (© Her Majesty the Queen in Right of Canada The Plant Journal © 2016 John Wiley & Sons Ltd.)

14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play important roles in a variety of cellular processes including response to diverse stresses. Although much has been learned about 14-3-3s in several plant species, it remains unknown in common bean. In this study, 9 common bean 14-3-3s (PvGF14s) were identified by exhaustive data mining against the publicly available common bean genomic database. A phylogenetic analysis revealed that each predicted PvGF14 was clustered with two GmSGF14 paralogs from soybean. Both epsilon-like and non-epsilon classes of PvGF14s were found in common bean, and the PvGF14s belonging to each class exhibited similar gene structure. Among 9 PvGF14s, only 8 are transcribed in common bean. Expression patterns of PvGF14s varied depending on tissue type, developmental stage and exposure of plants to stress. A protein-protein interaction study revealed that PvGF14a forms dimer with itself and with other PvGF14 isoforms. This study provides a first comprehensive look at common bean 14-3-3 proteins, a family of proteins with diverse functions in many cellular processes, especially in response to stresses.

Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL)-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (CysP) in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10) were selected for further characterization. The effect of CysP gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER), suggesting that ER may be the site of IL-10 degradation. Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

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., (© 2015 Her Majesty the Queen in Right of Canada, PROTEOMICS © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

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., (© Her Majesty the Queen in Right of Canada 2013 Reproduced with the permission of the Minister of Agricultural and Agri-Food Canada.)

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 β-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.

Isoflavonoids are plant natural compounds predominantly found in leguminous plant. They play important functions in both nitrogen fixation and stress resistance. Many clinical studies have linked dietary intake of isoflavonoids to human health benefits. Binding of 14-3-3 proteins to GmMYB176, an isoflavonoid regulator, modulates expression of key isoflavonoids gene expression and its biosynthesis. We have recently demonstrated that the interaction of 14-3-3 proteins with GmMYB176 regulates nuclear-cytoplasmic localization of GmMYB176 thereby affecting target gene expression. Here, we report GmMYB62 as a new R1 MYB client protein of soybean 14-3-3s that may function together with GmMYB176 for gene regulation 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., (© 2012 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri Food Canada The Plant Journal © 2012 Blackwell Publishing Ltd.)

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.

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.

We recently identified a novel R1 MYB transcription factor, GmMYB176, which regulates the CHS8 gene expression and influences isoflavonoid biosynthesis in soybeans. GmMYB176 recognizes a unique sequence motif [TAGT(T/A)(A/T)] in CHS8 promoter and binds with it. The in planta role of GmMYB176 was established by RNAi silencing of GmMYB176 in soybean hairy roots. Silencing of GmMYB176 reduced the expression of CHS8 gene expression and isoflavonoid accumulation in hairy roots. However, the overexpression of GmMYB176 did not lead to increase in both CHS8 expression and isoflavonoid level in hairy roots suggesting that GmMYB176 is essential but not sufficient for CHS8 gene activation.

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.

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network., (Crown Copyright (c) 2010. Published by Elsevier Inc. All rights reserved.)

Chalcone synthase (CHS) catalyzes the first reaction specific for flavonoid and isoflavonoid biosynthesis. The soybean genome consists of nine copies of CHS genes (CHS1-CHS9) and a duplicate copy of CHS1. Even though the soybean CHS gene family members share a high degree of sequence similarity, they play different roles during plant development or in response to environmental stimuli. Our previous work on the comparison of a global gene expression in two soybean cultivars that differ in the level of total isoflavonoid accumulation has denoted the involvement of CHS7 and CHS8 genes in isoflavonoid synthesis. We have extended our effort to understand expression patterns of these two genes in soybean and in transgenic Arabidopsis. Promoter regions of CHS7 and CHS8 genes were isolated and in silico analysis performed to investigate potential transcription factor binding sites (TFBSs). The TFBSs were verified by DNase I footprint analysis. Some unique and several common TFBSs were identified in CHS7 and CHS8 promoters. We cloned beta-glucuronidase (GUS) under CHS7 and CHS8 promoters and monitored the tissue-specific GUS expression in transformed Arabidopsis. Differential GUS activity was observed in young leaves, roots, and mature pod walls of transgenic CHS7 promoter-GUS and CHS8 promoter-GUS plants. The tissue-specific expression patterns of CHS7 and CHS8 genes were determined in soybean by quantitative RT-PCR. Both CHS7 and CHS8 genes were expressed at higher levels in roots; however, overall expression pattern of these genes varied in different tissues. The results suggest that the structural diversity within CHS7 and CHS8 promoters may lead into differential activation of these genes by different inducers as well as developmental stage- and tissue-specific differences in gene expression.

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.

The seed coat serves as a multifunctional organ with a role in protection and for the supply of nutrients to the embryo sac during development. The composition of the legume seed coat differs from other seed tissues in many ways including its protein composition. An abundant 24 kDa protein (SC24) has been purified and identified from soybean (Glycine max [L.] Merr) seed hulls. The corresponding cDNA and genomic DNA clones for SC24 were isolated and characterized, and expression patterns were determined. The deduced protein sequence of 219 amino acids included an N-terminal signal peptide. Transcripts encoding SC24 were present in the seed coat from 30 days after pollination (DAP) until maturity, but the protein was not detected until the final stages of seed maturation. In mature seeds, most of the SC24 protein was localized to the parenchyma and aleurone layers of the seed coat. The expression of SC24 was also induced in vegetative tissues by pathogen infection and by wounding. The SC24 protein bound to an affinity column containing an isophthalic acid ligand, and was eluted with 7 mM citrate. Polyclonal antibodies raised against recombinant SC24 cross-reacted with the seed coat peroxidase enzyme, suggesting that these two proteins may share an antigenic determinant. Overall, the results indicate that SC24 belongs to a novel class of plant defence proteins with carboxylate-binding activity.

Isoflavonoids are biologically active natural products that accumulate in soybean seeds during development. The amount of isoflavonoids present in soybean seed is variable, depending on genetic and environmental factors that are not fully understood. Experiments were conducted to determine whether isoflavonoids are synthesized within seed tissues during development, or made in other plant organs and transported to the seeds where they accumulate. An analysis of isoflavonoids by HPLC detected the compounds in all organs of soybean plant, but the amount of isoflavonoids present varied depending on the tissue and developmental stage. The greatest concentrations were found in mature seeds and leaves. The 2-hydroxyisoflavanone synthase genes IFS1 and IFS2 were studied to determine their pattern of expression in different tissues and developmental stages. The highest level of expression of IFS1 was observed in the root and seed coat, while IFS2 was most highly expressed in embryos and pods, and in elicitor-treated or pathogen-challenged tissues. Incorporation of radiolabel into isoflavonoids was observed when developing embryos and other plant organs were fed with [(14)C]phenylalanine. Embryos excised from developing soybean seeds also accumulated isoflavonoids from a synthetic medium. A maternal effect on seed isoflavonoid content was noted in reciprocal crosses between soybean cultivars that differ in seed isoflavonoids. From these results, we propose that developing soybean embryos have an ability to synthesize isoflavonoids de novo, but that transport from maternal tissues may in part contribute to the accumulation of these natural products in the seed.

In addition to their essential role in plant development, brassinosteroids have the ability to protect plants from various environmental stresses. Currently it is not understood how brassinosteroids control plant stress responses at the molecular level. We have begun an investigation into the molecular mechanisms underlying 24-epibrassinolide (EBR)-mediated stress resistance. Earlier we found that treatment of Brassica napus seedlings with EBR leads to a significant increase in their basic thermotolerance, and results in higher accumulation of four major classes of heat-shock proteins (hsps) as compared to untreated seedlings. Surprisingly, previous studies have shown that while hsp levels were significantly higher in treated seedlings during the recovery period, transcripts corresponding to these hsps were present at higher levels in untreated seedlings. To understand mechanisms controlling hsp synthesis in EBR-treated and untreated seedlings, we studied protein synthesis in vivo as well as in vitro, and assessed the levels of components of the translational machinery in these seedlings. We report here that increased accumulation of hsps in EBR-treated seedlings results from higher hsp synthesis, even when the mRNA levels are lower than in untreated seedlings, and that several translation initiation and elongation factors are present at significantly higher levels in EBR-treated seedlings as compared to untreated seedlings. These results suggest that EBR treatment limits the loss of some of the components of the translational apparatus during prolonged heat stress, and increases the level of expression of some of the components of the translational machinery during recovery, which correlates with a more rapid resumption of cellular protein synthesis following heat stress and a higher survival rate.

Brassinosteroids are plant growth-promoting compounds that exhibit structural similarities to animal steroid hormones. Recent studies have indicated that brassinosteroids are essential for proper plant development. In addition to a role in development, several lines of evidence suggest that brassinosteroids exert anti-stress effects on plants. However, the mechanism by which they modulate plant stress responses is not understood. We show here that Brassica napus and tomato seedlings grown in the presence of 24-epibrassinolide (EBR) are significantly more tolerant to a lethal heat treatment than are control seedlings grown in the absence of the compound. Since a preconditioning treatment of seedlings was not required to observe this effect, we conclude that EBR treatment increases the basic thermotolerance of seedlings. An analysis of heat shock proteins (HSPs) in B. napus seedlings by western blot analysis indicated that the HSPs did not preferentially accumulate in EBR-treated seedlings at the control temperature. However, after heat stress, HSP accumulation was higher in EBR-treated than in untreated seedlings. The results of the present study provide the first direct evidence for EBR-induced expression of HSPs. The higher accumulation of HSPs in EBR-treated seedlings raises the possibility that HSPs contribute, at least in part, to thermotolerance in EBR-treated seedlings. A search for factors other than HSPs, which may directly or indirectly contribute to brassinosteroid-mediated increase in thermotolerance, is underway.