Isoflavone Synthase Genes Research Articles

Transgenic Rice Expressing Isoflavone Synthase Gene from Soybean Shows Resistance Against Blast Fungus (Magnaporthe oryzae).

The isoflavones are a group of plant secondary metabolites primarily synthesized in legumes and are known for their role in improving human health and plant disease resistance. The isoflavones, especially genistein, act as precursors for the production of phytoalexins, which may induce broad-spectrum disease resistance in plants. In this study, we screened transgenic rice lines expressing the isoflavone synthase (GmIFS1) gene from soybean for rice blast (Magnaporthe oryzae) resistance. Two homozygous transgenic lines (I2 and I10), based on single copy gene integration, were identified. The expression of GmIFS1 in transgenic lines was confirmed by quantitative real-time PCR. Genistein was detected in the transgenic lines using liquid chromatography with tandem mass spectrometry. Subsequently, the transgenic lines were evaluated against the rice blast pathogen, isolate YJ54 (race IB-54). The results indicated that >60% of the plants in both the lines (I2 and I10) showed resistance against the blast pathogen. The progenies of one of the resistant transgenic lines (I10) also showed >65% resistance against rice blast. The resistance of these transgenic lines against rice blast may be attributed to the synthesis of isoflavone (genistein) in rice.

Isoflavone levels, nodulation and gene expression profiles of a CRISPR/Cas9 deletion mutant in the isoflavone synthase gene of red clover.

Isoflavones are not involved in rhizobial signaling in red clover, but likely play a role in defense in the rhizosphere. Red clover (Trifolium pratense) is a high-quality forage legume, well suited for grazing and hay production in the temperate regions of the world. Like many legumes, red clover produces a number of phenylpropanoid compounds including anthocyanidins, flavan-3-ols, flavanols, flavanones, flavones, and isoflavones. The study of isoflavone biosynthesis and accumulation in legumes has come into the forefront of biomedical and agricultural research due to potential for medicinal, antimicrobial, and environmental implications. CRISPR/Cas9 was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase (IFS1). A hemizygous plant carrying a 9-bp deletion in the IFS1 gene was recovered and was intercrossed to obtain homozygous mutant plants. Levels of the isoflavones formononetin, biochanin A and genistein were significantly reduced in the mutant plants. Wild-type and mutant plants were inoculated with rhizobia to test the effect of the mutation on nodulation, but no significant differences were observed, suggesting that these isoflavones do not play important roles in nodulation. Gene expression profiling revealed an increase in expression of the upstream genes producing the precursors for IFS1, namely, phenylalanine ammonium lyase and chalcone synthase, but there were no significant differences in IFS1 gene expression or in the downstream genes in the production of specific isoflavones. Higher expression in genes involved in ethylene response was observed in the mutant plants. This response is normally associated with biotic stress, suggesting that the plants may have been responding to cues in the surrounding rhizosphere due to lower levels of isoflavones.

Comparative Genomics of Spatholobus suberectus and Insight Into Flavonoid Biosynthesis.

Spatholobus suberectus Dunn (S. suberectus), has been widely used in traditional medicines plant source of the Leguminosae family. Its vine stem of which plays an important role in the prevention and treatment of various diseases because it contains various flavonoids. Comparative genome analysis suggested well-conserved genomic components and genetic collinearity between the genome of S. suberectus and other genera of Leguminosae such as Glycine max. We discovered two whole genome duplications (WGD) events in S. suberectus and G. max lineage underwent a WGD after speciation from S. suberectus. The determination of expansion and contractions of orthologous gene families revealed 1,001 expanded gene families and 3,649 contracted gene families in the S. suberectus lineage. Comparing to the model plants, many novel flavonoid biosynthesis-related genes were predicted in the genome of S. suberectus, and the expression patterns of these genes in the roots are similar to those in the stems [such as the isoflavone synthase (IFS) genes]. The expansion of IFS from a single copy in the Leguminosae ancestor to four copies in S. suberectus, will accelerate the biosynthesis of flavonoids. MYB genes are widely involved in plant flavonoid biosynthesis and the most abundant member of the TF family in S. suberectus. Activated retrotransponson positive regulates the accumulation of flavonoid in S. suberectus by introducing the cis-elements of tissue-specific expressed MYBs. Our study not only provides significant insight into the evolution of specific flavonoid biosynthetic pathways in S. suberectus, but also would facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding for alleviating resource shortage of S. suberectus.

Yeast extract elicited isoflavonoid accumulation and biosynthetic gene expression in Pueraria candollei var. mirifica cell cultures

Yeast extract (YE) has emerged as a potent biotic elicitor that can induce plant defense responses, leading to enhanced phytoalexin accumulation. Increased production of two isoflavones—daidzein and genistein—that are widely used in pharmaceutical industries was elicited using YE in suspension cell cultures of Pueraria candollei var. mirifica. Compared with the controls, cells treated with 2 mg/L YE for 21 days produced 11-fold increased amounts of daidzein and genistein in the suspension cultures (5.12 and 0.34 mg/g dry weight (DW), respectively). Furthermore, YE treatment significantly upregulated isoflavonoid biosynthesis, as revealed via gene expression studies. In particular, among genes involved in daidzein and genistein biosynthesis, the isoflavone synthase and isoflavone reductase genes were significantly upregulated and the chalcone isomerase and 2,7,4′-trihydroxyisoflavanone dehydratase genes were significantly downregulated; moreover, these changes were associated with the accumulation of these two isoflavones in suspension cell cultures. Overall, the results obtained in this study both emphasize the utility of YE for enhancing the in vitro production of the two bioactive isoflavones examined for pharmaceutical and nutraceutical utilization and advance our understanding of their biosynthesis in response to YE elicitation. Yeast extract modulated the expression of genes involved in isoflavonoid biosynthesis in suspension cultures of P. candollei var. mirifica cells.

Identification of novel MYB transcription factors involved in the isoflavone biosynthetic pathway by using the combination screening system with agroinfiltration and hairy root transformation.

Soybean isoflavones are functionally important secondary metabolites that are mainly accumulated in seeds. Their biosynthetic processes are regulated coordinately at the transcriptional level; however, screening systems for key transcription factors (TFs) are limited. Here we developed a combination screening system comprising a simple agroinfiltration assay and a robust hairy root transformation assay. First, we screened for candidate MYB TFs that could activate the promoters of the chalcone synthase (CHS) gene GmCHS8 and the isoflavone synthase (IFS) genes GmIFS1 and GmIFS2 in the isoflavone biosynthetic pathway. In the agroinfiltration assay, we co-transformed a LjUbi (Lotus japonicus polyubiquitin gene) promoter-fused MYB gene with target promoter-fused GUS (β-glucuronidase) gene constructs, and identified three genes (GmMYB102, GmMYB280, and GmMYB502) as candidate regulators of isoflavone biosynthesis. We then evaluated the functional regulatory role of identified three MYB genes in isoflavone biosynthesis using hairy roots transformation assay in soybean for the accumulation of isoflavones. Three candidate MYB genes showed an increased accumulation of total isoflavones in hairy root transgenic lines. Accumulation of total isoflavones in the three MYB-overexpressing lines was approximately 2-to 4-folds more than that in the vector control, confirming their possible role to regulate isoflavone biosynthesis. However, the significant accumulation of authentic GmCHS8, GmIFS1, and GmIFS2 transcripts could not be observed except for the GmMYB502-overexpressing line. Therefore, the analysis of isoflavone accumulation in transgenic hairy root was effective for evaluation of transactivation activity of MYB TFs for isoflavone biosynthetic genes. Our results demonstrate a simple and robust system that can potentially identify the function of orphan TFs in diverse plant metabolic pathways.

Promising strategy for improving calycosin-7-O-β-D-glucoside production in Astragalus membranaceus adventitious root cultures

Culture duration, elicitation, and precursor feeding can significantly affect secondary metabolite production in adventitious root cultures. Hydrogen peroxide (H2O2) can act as a diffusible signal to induce the production of secondary metabolites. L-phenylalanine (L-Phe) is the precursor in the biosynthesis of the bioactive compound calycosin-7-O-β-D-glucoside (CG). To improve the accumulation of CG in Astragalus membranaceus adventitious roots (AMARs), the present study investigated the effects of culture duration and H2O2 and L-Phe treatment on the accumulation of CG in AMARs. The CG content fluctuated during AMAR culture, with the highest productivity observed after 35 d of suspension culture. Furthermore, the H2O2 treatment efficiently enhanced CG content in a concentration- and time-dependent manner, and under 20 μM H2O2, the CG reached its maximum level after 12 h of treatment. However, the addition of L-Phe to H2O2 treatment remarkably improved the CG accumulation, with a maximum CG level observed after 120 h of treatment with 200 μM H2O2 and 300 μM L-Phe, which was 8.6-fold greater than the cultures treated with 200 μM H2O2 alone. The effectiveness of these parameters was confirmed by measuring the expression of four major CG-biosynthesis genes, and both the chalcone reductase and isoflavone synthase genes were identified as key regulatory nodes in the CG biosynthetic pathway. The improvement strategy was also applied using a large-scale bioreactor system. Both the CG content and antioxidant activity of the H2O2-elicited and L-Phe-fed bioreactor culture were greater than those of the untreated bioreactor culture and field-grown plants. Thus, the simultaneous application of H2O2 elicitation and L-Phe-feeding in 35-d-old AMAR cultures is a promising strategy to economically improve the industrial production of CG.

Cytosine Methylation of Isoflavone Synthase Gene in the Genic Region Positively Regulates Its Expression and Isoflavone Biosynthesis in Soybean Seeds.

Plants, being sessile organisms, have evolved several dynamic mechanisms of gene regulation. Epigenetic modification especially cytosine methylation and demethylation actively regulates the expression of genes. To understand the role of cytosine methylation during isoflavonoid biosynthesis and accumulation, we performed cytosine methylation analysis in the coding region of two isoforms IFS1 and IFS2 gene, in two contrasting soybean genotypes differing in total isoflavone content (NRC37: high isoflavone; and NRC7: low isoflavone). The results indicated increased 5-mC in both the isoforms in NRC37 (∼20.51% in IFS2 and ∼85% in IFS1) compared with NRC7 (∼7.8% in IFS2 and ∼2.5% in IFS1) genotype, which signifies the positive role of 5-mC in the coding region of the gene leading to enhanced expression. In addition, temporal expression profiling [35 days after flowering (DAF), 45, 55, and 65 DAF] of both the isoforms showed increasing trend of accumulation in both the genotypes with maximum in NRC37 at 65 DAF. To further establish a correlation between methylation and expression of transcripts, we quantified the different isoforms of isoflavone in both the genotypes across all the stages. Therefore, the finding of this study would certainly increase our understanding of epigenetic regulation of isoflavone biosynthetic pathway mediated by the cytosine methylation that would assist molecular breeders to get high-performing soybean genotypes with better isoflavone yield.

Legume isoflavone synthase genes have evolved by whole-genome and local duplications yielding transcriptionally active paralogs

Isoflavone synthase (IFS) is the key enzyme of isoflavonoid biosynthesis. IFS genes were identified in numerous species, although their evolutionary patterns have not yet been reconstructed. To address this issue, we performed structural and functional genomic analysis.Narrow leafed lupin, Lupinus angustifolius L., was used as a reference species for the genus, because it has the most developed molecular tools available. Nuclear genome BAC library clones carrying IFS homologs were localized by linkage mapping and fluorescence in situ hybridization in three chromosome pairs. Annotation of BAC, scaffold and transcriptome sequences confirmed the presence of three full-length IFS genes in the genome.Microsynteny analysis and Bayesian inference provided clear evidence that IFS genes in legumes have evolved by lineage-specific whole-genome and tandem duplications. Gene expression profiling and RNA-seq data mining showed that the vast majority of legume IFS copies have maintained their transcriptional activity. L. angustifolius IFS homologs exhibited organ-specific expression patterns similar to those observed in other Papilionoideae. Duplicated lupin IFS homologs retained non-negligible levels of substitutions in conserved motifs, putatively due to positive selection acting during early evolution of the genus, before the whole-genome duplication. Strong purifying selection preserved newly arisen IFS duplicates from further nonsynonymous changes.

Isolation and Cloning of Isoflavone Synthase Gene from Soybean (Glycin max) Williams III Cultivar

Isolation and Cloning of Isoflavone Synthase Gene from Soybean (Glycin max) Williams III Cultivar

Tremendous enhancements of isoflavonoid biosynthesis, associated gene expression and antioxidant capacity in Astragalus membranaceus hairy root cultures elicited by methyl jasmonate

In this work, Astragalus membranaceus hairy root cultures (AMHRCs) were induced to boost isoflavonoid biosynthesis upon elicitations of methyl jasmonate (MJ), salicylic acid (SA) and acetylsalicylic acid (ASA). The optimal enhancement of isoflavonoid production was obtained in 34 day-old AMHRCs elicited by MJ under the concentration of 283μM and the exposure time of 33.75h. The resulting isoflavonoid yield was 2250.10±71.88μg/g dry weight (DW), which tremendously increased 9.71-fold relative to that of non-treated control (231.64±6.51μg/g DW). Moreover, transcriptions of eight isoflavonoid biosynthetic genes were significantly up-regulated in response to MJ elicitation, and chalcone isomerase and isoflavone synthase genes were found to be key regulatory nodes in isoflavonoid biosynthetic pathway. Additionally, an enormous increase was observed in antioxidant capacities of extracts from MJ-elicited AMHRCs (IC50 values: 0.51 and 0.72mg/mL) as against those of non-treated control (1.39 and 1.71mg/mL). Overall, this study offered a feasible strategy to promote isoflavonoid production in AMHRCs with higher antioxidant activity, and also paved the way for achieving isoflavonoid overproduction via metabolic manipulations in the future.

Cloning and transformation analysis of isoflavone synthase gene into Minshan Trifolium pratense.

The aim of this study was to clone the isoflavone synthase (IFS) gene and establish the recombinant Minshan Trifolium pratense. The IFS gene was cloned from the callus of Minshan T. pratense using reverse transcription-polymerase chain reaction. The plant expression vector pRI101-AN-IFS was constructed and introduced into Agrobacterium tumefaciens strain LBA4404, and then screened under cephalosporin. IFS expression was detected by reverse transcription-polymerase chain reaction. The IFS gene was cloned successfully. Sequence analysis indicated that IFS gene had high homology with similar genes from other plants. The IFS-overexpressing callus was obtained by introducing the LBA4404-harboring IFS-pRI101-AN-IFS vector into T. pratense calluses.

Genistein production in rice seed via transformation with soybean IFS genes

To produce genistein in rice, the isoflavone synthase (IFS) genes, SpdIFS1 and SpdIFS2 were cloned from the Korean soybean cultivar, Sinpaldalkong II as it has a higher genistein content than other soybean varieties. SpdIFS1 and SpdIFS2 show a 99.6% and 98.2% identity at the nucleotide level and 99.4% and 97.9% identity at the amino acid level, respectively, with IFS1 and IFS2 from soybean (GenBank accession Nos. AF195798 and AF195819). Plant expression vectors were constructed harboring SpdIFS1 or SpdIFS2 under the control of a rice globulin promoter that directs seed specific expression, and used to transform two rice varieties, Heugnam, a black rice, and Nakdong, a normal rice cultivar without anthocyanin pigment. Because naringenin, the substrate of SpdIFS1 and SpdIFS2, is on the anthocyanin biosynthesis pathway, the relative production rate of genistein was compared between SpdIFS-expressing transgenic Heugnam and Nakdong. Southern blot analysis of eight of the resulting transgenic rice plants revealed that the T0 plants had one to three copies of the SpdIFS1 or SpdIFS2 gene. The highest level of genistein content found in rice seeds was 103μg/g. These levels were about 30-fold higher in our transgenic rice lines than the genistein aglycon content of a non-leguminous IFS-expressing transgenic tobacco petal, equaling about 12% of total genistein content of Sinpaldalkong II. There were no significant differences found between the genistein content in Heugnam and Nakdong transgenic rice plants.

Glyceollin is an Important Component of Soybean Plant Defense Against Phytophthora sojae and Macrophomina phaseolina

The response of soybean transgenic plants, with suppressed synthesis of isoflavones, and nontransgenic plants to two common soybean pathogens, Macrophomina phaseolina and Phytophthora sojae, was studied. Transgenic soybean plants of one line used in this study were previously generated via bombardment of embryogenic cultures with the phenylalanine ammonia lyase, chalcone synthase, and isoflavone synthase (IFS2) genes in sense orientation driven by the cotyledon-preferable lectin promoter (to turn genes on in cotyledons), while plants of another line were newly produced using the IFS2 gene in sense orientation driven by the Cassava vein mosaic virus constitutive promoter (to turn genes on in all plant parts). Nearly complete inhibition of isoflavone synthesis was found in the cotyledons of young seedlings of transgenic plants transformed with the IFS2 transgene driven by the cotyledon-preferable lectin promoter compared with the untransformed control during the 10-day observation period, with the precursors of isoflavone synthesis being accumulated in the cotyledons of transgenic plants. These results indicated that the lectin promoter could be active not only during seed development but also during seed germination. Downregulation of isoflavone synthesis only in the seed or in the whole soybean plant caused a strong inhibition of the pathogen-inducible glyceollin in cotyledons after inoculation with P. sojae, which resulted in increased susceptibility of the cotyledons of both transgenic lines to this pathogen compared with inoculated cotyledons of untransformed plants. When stems were inoculated with M. phaseolina, suppression of glyceollin synthesis was found only in stems of transgenic plants expressing the transgene driven by a constitutive promoter, which developed more severe infection. These results provide further evidence that rapid glyceollin accumulation during infection contributes to the innate soybean defense response.

Co‐expression of Arabidopsis transcription factor, AtMYB12, and soybean isoflavone synthase, GmIFS1, genes in tobacco leads to enhanced biosynthesis of isoflavones and flavonols resulting in osteoprotective activity

Isoflavones, a group of flavonoids, restricted almost exclusively to family Leguminosae are known to exhibit anticancerous and anti-osteoporotic activities in animal systems and have been a target for metabolic engineering in commonly consumed food crops. Earlier efforts based on the expression of legume isoflavone synthase (IFS) genes in nonlegume plant species led to the limited success in terms of isoflavone content in transgenic tissue due to the limitation of substrate for IFS enzyme. In this work to overcome this limitation, the activation of multiple genes of flavonoid pathway using Arabidopsis transcription factor AtMYB12 has been carried out. We developed transgenic tobacco lines constitutively co-expressing AtMYB12 and GmIFS1 (soybean IFS) genes or independently and carried out their phytochemical and molecular analyses. The leaves of co-expressing transgenic lines were found to have elevated flavonol content along with the accumulation of substantial amount of genistein glycoconjugates being at the highest levels that could be engineered in tobacco leaves till date. Oestrogen-deficient (ovariectomized, Ovx) mice fed with leaf extract from transgenic plant co-expressing AtMYB12 and GmIFS1 but not wild-type extract exhibited significant conservation of trabecular microarchitecture, reduced osteoclast number and expression of osteoclastogenic genes, higher total serum antioxidant levels and increased uterine oestrogenicity compared with Ovx mice treated with vehicle (control). The skeletal effect of the transgenic extract was comparable to oestrogen-treated Ovx mice. Together, our results establish an efficient strategy for successful pathway engineering of isoflavones and other flavonoids in crop plants and provide a direct evidence of improved osteoprotective effect of transgenic plant extract.

Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18

Isoflavone synthase (IFS; CYP93C) plays a key role in the biosynthesis of phenolic secondary metabolites, isoflavonoids. These compounds, which are well-known for their benefits to human health and plant defence, are produced mostly in legumes. However, more than 200 of them have been described in 59 other plant families without any knowledge of their respective IFS orthologue genes (with the sole exception of sugar beet). In this study, we selected IFS from Pisum sativum L. (CYP93C18) for functional expression. CYP93C18 was isolated, cloned, and introduced into Arabidopsis thaliana. The presence of the gene was shown by Southern blot analysis and its expression in the transgenic Arabidopsis was proven by RT-PCR and Western blots. The functional activity of the heterologous IFS was verified by HPLC-MS analysis of the metabolite levels: the isoflavone genistein and its derivatives tectorigenin and biochanin A were detected in the overexpressing lines. In addition, 35S::CYP93C18::GFP fused proteins were transiently expressed in the leaves of Nicotiana benthamiana and the localization of the GFP signal was observed on the endoplasmic reticulum using confocal microscopy which is consistent with the data from the literature and with our in silico predictions. The putative mode of attachment of IFS to the endoplasmic reticulum membrane is suggested. The undemanding methodology presented in this paper is applicable to the functional analysis of newly-identified isoflavone synthase genes from various species.

Diversifying Selection on Flavanone 3-Hydroxylase and Isoflavone Synthase Genes in Cultivated Soybean and Its Wild Progenitors

Soybean isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H) are two key enzymes catalyzing the biosynthesis of isoflavonoids and flavonoids, both of which play diverse roles in stress responses. However, little is known about the evolutionary pattern of these genes in cultivated soybean and its wild progenitors. Herein, we investigated the nucleotide polymorphisms in Isoflavone synthase (IFS1, IFS2) and Flavanone 3-hydroxylase (F3H2) genes from 33 soybean accessions, including 17 cultivars (Glycine max) and 16 their wild progenitors (Glycine soja). Our data showed that the target genes shared the levels of nucleotide polymorphism with three reference genes involved in plant-microbe interactions, but possessed a much higher nucleotide polymorphism than other reference genes. Moreover, no significant genetic differentiation was found between cultivated soybean and its wild relatives in three target genes, despite of considering bottleneck and founder effect during domestication. These results indicate that IFS and F3H genes could have experienced gene introgressions or diversifying selection events during domestication process. Especially, F3H2 gene appears to evolve under positive selection and enjoy a faster evolutionary rate than IFS1 and IFS2 genes.

Molecular Cloning and Characterization of Isoflavone Synthase Gene from Pueraria lobata

Isoflavones are the main active component of kudzu(Pueraria lobata).The first committed step in the production of isoflavones is performed by a cytochrome P450 enzyme,IFS.While IFS genes have been isolated from kudzu,no biological functions have been identified to date.We cloned an IFS gene via RT-PCR,named PlIFS,using kudzu collected from Langxi(Anhui,China)as plant material.The PlIFS contained an open reading frame(ORF)encoding 521 amino acid residues.The amplified PlIFS gene was cloned into pESC-TRP vector,with yeast GAL1 divergent promoters as the control.The constructed recombinant plasmid,named pESC-TRP-PlIFS,was transformed into Saccharomyces cerevisiae strain WAT11 according to the LiAc/ssDNA/PEG method.The PlIFS expressed in yeast was able to catalyze daidzein formation from liquiritigenin,indicating that it had IFS activity.Gene expression pattern analysis revealed that PlIFS was expressed in roots at the highest level,which was consistent with isoflavones accumulation in kudzu plantlets.

Isoflavone synthase (IFS) gene phylogeny in Trifolium species associated with plant isoflavone contents

This study presents the results of the identification and quantification of 12 isoflavones (prunetin, irilone, pseudobaptigenin, glycitein, daidzin, genistin, daidzein, pratensein, puerarin, biochanin A, formononetin and genistein) in 23 species of Trifolium (T. arvense, T. pratense, T. ligusticum, T. striatum, T. lappaceum, T. angustifolium, T. hirtum, T. subterraneum, T. isthmocarpum, T. stellatum, T. mutabile, T. strictum, T. fragiferum, T. alexandrinum, T. tomentosum. T. nigrescens subsp. petrisavii, T. nigrescens, T. glomeratum, T. subterraneum subsp. brachycalycinum, T. cherleri, T. resupinatum, T. campestre and T. repens). Isoflavones were extracted by an MSPD method and analyzed with HPLC coupled with a diode-array detector. The evaluation of molecular phylogeny of the IFS gene and the relation with isoflavone content was also performed. Five species (T. subterraneum subsp. brachycalycinum, T. alexandrinum, T. pratense, T. subterraneum and T. lappaceum) were identified with high levels of biochanin A (431–83 mg/kg), formononetin (72–365 mg/kg) and genistein (9–509 mg/kg), which could be utilized as alternative sources for the nutraceutical industry. Genetic phylogeny for the IFS gene was found in the species studied, with 20 out of 23 species having been divided into two clades, while the remaining three were genetically distant. Based on our results, we confirm the direct correlation between IFS gene polymorphism and isoflavones content in species of Trifolium particularly noted for formononetin. Therefore, the IFS gene can be utilized for screening Trifolium genotypes for formononetin. The relation of the three isoflavones' contents and the molecular phylogeny of plants determined by the IFS sequences, as a screening marker for plants with high isoflavone contents in Trifolium species, are to the best of our knowledge described for the first time.

Molecular characterization of isoflavone synthase gene from Pueraria candollei var. mirifica

Cloning, sequencing, and characterization of the isoflavone synthase gene (2-hydroxyisoflavone synthase;IFS) from Pueraria candollei var. mirifica were carried out in this study. The full-length open reading frame (ORF) of the P. candollei var. mirifica IFS gene or PcmIFS was obtained by reverse transcription polymerase chain reaction (RT-PCR). Nucleotide sequence of PcmIFS was 1,566 bp long, encoded for 521 amino acid residues with the relative molecular mass of 58.9 kDa. The pI value of the PcmIFS gene product was 8.9 and all of the main conserved motifs that are essential for IFS activities were found. The deduced amino acid sequence of the PcmIFS-encoded protein showed high degree of identity to those of IFS proteins fromPueraria montana var. lobata, Glycine max, Glycine soja, and Pisum sativum. Southern blot analysis indicated that the PcmIFS gene belonged to a multigene family. The expression of PcmIFS was detected in leaf, stem, and root of the plant and its expression levels was induced by both low and high temperature stresses as well as by UV-B and wounding treatments. Key words: Isoflavone synthase, isoflavones, Pueraria candollei var. mirifica, molecular cloning.

Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

BackgroundCytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship.ResultsWe have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an isoflavone synthase gene) is co-expressed with several genes encoding isoflavonoid-related metabolic enzymes. We then focused on nodulation-induced P450s and found that CYP728H1 was co-expressed with the genes involved in phenylpropanoid metabolism. Similarly, CYP736A34 was highly co-expressed with lipoxygenase, lectin and CYP83D1, all of which are involved in root and nodule development.ConclusionsThe genome scale analysis of P450s in soybean reveals many unique features of these important enzymes in this crop although the functions of most of them are largely unknown. Gene co-expression analysis proves to be a useful tool to infer the function of uncharacterized genes. Our work presented here could provide important leads toward functional genomics studies of soybean P450s and their regulatory network through the integration of reverse genetics, biochemistry, and metabolic profiling tools. The identification of nodule-specific P450s and their further exploitation may help us to better understand the intriguing process of soybean and rhizobium interaction.