Isoflavone Reductase Research Articles

Root plasticity and Pi recycling within plants contribute to low-P tolerance in Tibetan wild barley

BackgroundBarley is a low phosphorus (P) demand cereal crop. Tibetan wild barley, as a progenitor of cultivated barley, has revealed outstanding ability of tolerance to low-P stress. However, the underlying mechanisms of low-P adaption and the relevant genetic controlling are still unclear.ResultsWe identified low-P tolerant barley lines in a doubled-haploid (DH) population derived from an elite Tibetan wild barley accession and a high-yield cultivar. The tolerant lines revealed greater root plasticity in the terms of lateral root length, compared to low-P sensitive lines, in response to low-P stress. By integrating the QTLs associated with root length and root transcriptomic profiling, candidate genes encoding isoflavone reductase, nitrate reductase, nitrate transporter and transcriptional factor MYB were identified. The differentially expressed genes (DEGs) involved the growth of lateral root, Pi transport within cells as well as from roots to shoots contributed to the differences between low-P tolerant line L138 and low-P sensitive lines L73 in their ability of P acquisition and utilization.ConclusionsThe plasticity of root system is an important trait for barley to tolerate low-P stress. The low-P tolerance in the elite DH line derived from a cross of Tibetan wild barley and cultivated barley is characterized by enhanced growth of lateral root and Pi recycling within plants under low-P stress.

Genome-wide association study of feruloyl arabinoxylan content in common wheat grain

Arabinoxylan (AX) is an important component of dietary fiber, and exerts significant effects on the processing quality and nutritional value of flour. Feruloyl arabinoxylan (FAX) is a major factor influencing the functional properties of AX. Here, a genome-wide association study (GWAS) was conducted for FAX content using a high-density SNP platform. FAX content was evaluated in 256 wheat varieties (two populations) in four environments. Results showed that 83 and 33 marker-trait associations (MTAs) for FAX content were identified, explaining 6.7–9.2% and 10.2–21.2% phenotypic variation in the two populations. Two and three MTAs were repeatedly detected in at least two environments. Five MTAs were detected in both populations. Linear regression indicated a clear dependence of FAX content on the number of favorable alleles, indicating that QTL pyramiding would be an effective approach to modify FAX content. Associated with these significant MTAs, five candidate genes: isoflavone reductase, carnitine dehydrogenase, acylamino acid releasing enzyme, glucose isomerase, and GMP synthase were found to be involved in FAX biosynthesis. Lastly, our study contributes important information for genetic improvement of wheat quality and contributes to developing marker-assisted selection.

ITRAQ-based proteomic analysis reveals changes in response to sodium nitroprusside treatment in soybean sprouts

In recent years, nitric oxide (NO) has been considered a plant signaling compound involved in antioxidant systems and flavonoid production enhancement. Nevertheless, its mechanism of action, from the perspective of protein expression, remains largely unknown. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) was employed to investigate NO donor sodium nitroprusside treatment-induced proteomic changes in soybean sprouts. Among the 3033 proteins identified, compared with the control, sodium nitroprusside treatment up- and down-regulated 256 proteins. These proteins were involved in antioxidant system pathways, such as the thioredoxin, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione reductase (GR), glutathione S-transferase (GST), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR) and lipoxygenase (LOX) pathways, including allene oxide synthase and lipoxygenase. In addition, heat shock proteins (HSPs) and flavonoid biosynthetic proteins, such as cinnamate 4-hydroxylase, chalcone isomerase, chalcone synthase, isoflavone synthase and isoflavone reductase, were also modulated in response to sodium nitroprusside treatment.

ITRAQ-based analysis of proteins involved in secondary metabolism in response to ABA in soybean sprouts

Abscisic acid (ABA), as a sesquiterpenoid hormone, could regulate lots of physiological processes, especially secondary metabolism in plants. Nevertheless, its mechanism of action, from the perspective of protein expression, remains largely unknown. In the study, isobaric tags for relative and absolute quantitation (iTRAQ) was employed to investigate ABA treatment-induced proteomic changes related to secondary metabolism in soybean sprouts. Among the 3033 proteins identified, compared with the control, ABA treatment up- and down-regulated 350 proteins. These proteins were involved in GABA biosynthesis, such as glutamate synthase, glutamate decarboxylase (GAD), methionine synthetase, 5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase 1, aminoaldehyde dehydrogenase (AMADH) and inositol phosphate metabolism pathways, including phosphoinositide phospholipase C (PI-PLC), purple acid phosphatase (PAP) and inositol polyphosphate 5-phosphatase. In addition, flavonoid biosynthetic proteins, such as cinnamate 4-hydroxylase, chalcone isomerase, chalcone synthase, isoflavone synthase and isoflavone reductase, were also modulated in response to ABA treatment. What's more, ABA treatment regulated proteins involved in ABA signal transduction, such as SNF1-related protein kinase (SnRK), protein phosphatase 2C (PP2C), guanine nucleotide-binding protein and calreticulin-3.

Harnessing genetic potential of wheat germplasm banks through impact-oriented-prebreeding for future food and nutritional security

The value of exotic wheat genetic resources for accelerating grain yield gains is largely unproven and unrealized. We used next-generation sequencing, together with multi-environment phenotyping, to study the contribution of exotic genomes to 984 three-way-cross-derived (exotic/elite1//elite2) pre-breeding lines (PBLs). Genomic characterization of these lines with haplotype map-based and SNP marker approaches revealed exotic specific imprints of 16.1 to 25.1%, which compares to theoretical expectation of 25%. A rare and favorable haplotype (GT) with 0.4% frequency in gene bank identified on chromosome 6D minimized grain yield (GY) loss under heat stress without GY penalty under irrigated conditions. More specifically, the ‘T’ allele of the haplotype GT originated in Aegilops tauschii and was absent in all elite lines used in study. In silico analysis of the SNP showed hits with a candidate gene coding for isoflavone reductase IRL-like protein in Ae. tauschii. Rare haplotypes were also identified on chromosomes 1A, 6A and 2B effective against abiotic/biotic stresses. Results demonstrate positive contributions of exotic germplasm to PBLs derived from crosses of exotics with CIMMYT’s best elite lines. This is a major impact-oriented pre-breeding effort at CIMMYT, resulting in large-scale development of PBLs for deployment in breeding programs addressing food security under climate change scenarios.

Transformation of oil palm calli using CRISPR/Cas9 System: toward genome editing of oil palm

Genome editing using CRISPR/Cas9 construct containing sgRNA is the newest technology used to assemble organisms with desired characters through knock out of the target gene. As part of the effort to develop oil palm planting material tolerant to Ganoderma, this research aimed to obtain a technique for introducing CRISPR/Cas9 modules containing sgRNA into oil palm tissue. Based on differential expression studies, two genes encoding isoflavone reductase (IFR) and methallothionine-like protein (MT) were selected as the target for edited genes. The CRISPR-IFR and CRISPR-MT modules each carrying a sequence of gene recognition (sgRNA) were constructed and transformed into oil palm calli using Agrobacterium tumefaciens. The transformed calli were cultured on a modified DF medium for callus propagation, and then sub-cultured into DF media for induction of somatic embryos. PCR analyses using specific primers flanking the DNA targeted region were carried out and the results showed that both constructs had been introduced into the cells. DNA sequence analysis indicated the presence of substitution of several bases in the target area of both genes. The transformed calli grew in a selection medium containing hygromycin. Development of somatic embryos was detected after 3-4 weeks culturing on modified DF media.

Gene expression analysis in Eucalyptus globulus exposed to drought stress in a controlled and a field environment indicates different strategies for short- and longer-term acclimation.

Previous knowledge suggested the involvement of specific pathways/proteins that could be identified as potential molecular indicators linked to enhanced drought tolerance in Eucalyptus globulus. Here, we looked for specific variations in key transcripts of two Eucalyptus globulus clones (AL-18 and AL-13) exposed to water deficit and rehydration with two main goals: (i) to check if and how transcripts potentially associated with stress response and protection are modulated in a controlled experiment; and (ii) to verify if the transcript response is robust in a field case study. Our results showed that the controlled experiment induced a severe acute stress that resulted in a strong realignment of gene expression resulting from an overwhelming of physiological adjustments to water limitation. A number of transcripts exhibited altered abundance after the acute water stress: reduction of RuBisCO activase and mitochondrial glycine cleavage system H protein, and increase of isoflavone reductase. Malate dehydrogenase, catalase, dehydration response element B1A and potassium channel GORK showed a different abundance pattern in each clone. The stress in the field was more moderate and chronic and the plants were able to deal with the stress primarily through physiological adjustments resulting in much smaller changes in gene expression. The transcripts of clone AL-18 showed few alterations between irrigated and non-irrigated plants throughout the experiment, while the transcript changes found in clone AL-13 highlighted the impact of early rewatering rather than growing under extended drought typical of a Mediterranean summer. Although a few concurrent responses were found, the results obtained in the field study draw a very distinct picture when compared with the controlled experiment.

Daidzein reductase of Eggerthella sp. YY7918, its octameric subunit structure containing FMN/FAD/4Fe-4S, and its enantioselective production of R-dihydroisoflavones

S-Equol is a metabolite of daidzein, a type of soy isoflavone, and three reductases are involved in the conversion of daidzein by specific intestinal bacteria. S-Equol is thought to prevent hormone-dependent diseases. We previously identified the equol producing gene cluster (eqlABC) of Eggerthella sp. YY7918. Daidzein reductase (DZNR), encoded by eqlA, catalyzes the reduction of daidzein to dihydrodaidzein (the first step of equol synthesis), which was confirmed using a recombinant enzyme produced in Escherichia coli. Here, we purified recombinant DZNR to homogeneity and analyzed its enzymological properties. DZNR contained FMN, FAD, and one 4Fe-4S cluster per 70-kDa subunit as enzymatic cofactors. DZNR reduced the CC bond between the C-2 and C-3 positions of daidzein, genistein, glycitein, and formononetin in the presence of NADPH. R-Dihydrodaidzein and R-dihydrogenistein were highly stereo-selectively produced from daidzein and genistein. The Km and kcat for daidzein were 11.9μM and 6.7s-1, and these values for genistein were 74.1μM and 28.3s-1, respectively. This enzyme showed similar kinetic parameters and wide substrate specificity for isoflavone molecules. Thus, this enzyme appears to be an isoflavone reductase. Gel filtration chromatography and chemical cross-linking analysis of the active form of DZNR suggested that the enzyme consists of an octameric subunit structure. We confirmed this by small-angle X-ray scattering and transmission electron microscopy at a magnification of×200,000. DZNR formed a globular four-petal cloverleaf structure with a central vertical hole. The maximum particle size was 173Å.

Differential accumulation of proteins in oil palms affected by fatal yellowing disease.

There is still no consensus on the true origin of fatal yellowing, one of the most important diseases affecting oil palm (Elaeis guineensis Jacq.) plantations. This study involved two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-UPLC-MSE) analyses to identify changes in protein profiles of oil palms affected by FY disease. Oil palm roots were sampled from two growing areas. Differential accumulation of proteins was assessed by comparing plants with and without symptoms and between plants at different stages of FY development. Most of the proteins identified with differential accumulation were those related to stress response and energy metabolism. The latter proteins include the enzymes alcohol dehydrogenase and aldehyde dehydrogenase, related to alcohol fermentation, which were identified in plants with and without symptoms. The presence of these enzymes suggests an anaerobic condition before or during FY. Transketolase, isoflavone reductase, cinnamyl alcohol dehydrogenase, caffeic acid 3-O-methyltransferase, S-adenosylmethionine synthase, aldehyde dehydrogenase and ferritin, among others, were identified as potential marker proteins and could be used to guide selection of FY-tolerant oil palm genotypes or to understand the source of this anomaly. When comparing different stages of FY, we observed high accumulation of alcohol dehydrogenase and other abiotic stress related-proteins at all disease stages. On the other hand, biological stress-related proteins were more accumulated at later stages of the disease. These results suggest that changes in abiotic factors can trigger FY development, creating conditions for the establishment of opportunistic pathogens.

Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii.

The protein superfamily of short-chain dehydrogenases/reductases (SDR), including members of the atypical type (aSDR), covers a huge range of catalyzed reactions and in vivo substrates. This superfamily also comprises isoflavone reductase-like (IRL) proteins, which are aSDRs highly homologous to isoflavone reductases from leguminous plants. The molecular function of IRLs in non-leguminous plants and green microalgae has not been identified as yet, but several lines of evidence point at their implication in reactive oxygen species homeostasis. The Chlamydomonas reinhardtii IRL protein IFR1 was identified in a previous study, analyzing the transcriptomic changes occurring during the acclimation to sulfur deprivation and anaerobiosis, a condition that triggers photobiological hydrogen production in this microalgae. Accumulation of the cytosolic IFR1 protein is induced by sulfur limitation as well as by the exposure of C. reinhardtii cells to reactive electrophile species (RES) such as reactive carbonyls. The latter has not been described for IRL proteins before. Over-accumulation of IFR1 in the singlet oxygen response 1 (sor1) mutant together with the presence of an electrophile response element, known to be required for SOR1-dependent gene activation as a response to RES, in the promoter of IFR1, indicate that IFR1 expression is controlled by the SOR1-dependent pathway. An implication of IFR1 into RES homeostasis, is further implied by a knock-down of IFR1, which results in a diminished tolerance toward RES. Intriguingly, IFR1 knock-down has a positive effect on photosystem II (PSII) stability under sulfur-deprived conditions used to trigger photobiological hydrogen production, by reducing PSII-dependent oxygen evolution, in C. reinhardtii. Reduced PSII photoinhibition in IFR1 knock-down strains prolongs the hydrogen production phase resulting in an almost doubled final hydrogen yield compared to the parental strain. Finally, IFR1 knock-down could be successfully used to further increase hydrogen yields of the high hydrogen-producing mutant stm6, demonstrating that IFR1 is a promising target for genetic engineering approaches aiming at an increased hydrogen production capacity of C. reinhardtii cells.

Charting novel allergens from date palm pollen (Phoenix sylvestris) using homology driven proteomics

Pollen grains from Phoenix sylvestris (date palm), a commonly cultivated tree in India has been found to cause severe allergic diseases in an increasing percentage of hypersensitive individuals. To unearth its allergenic components, pollen protein were profiled by two-dimensional gel electrophoresis followed by immunoblotting with date palm pollen sensitive patient sera. Allergens were identified by MALDI-TOF/TOF employing a layered proteomic approach combining conventional database dependent search and manual de novo sequencing followed by homology-based search as Phoenix sylvestris is unsequenced. Derivatization of tryptic peptides by acetylation has been demonstrated to differentiate the ‘b’ from the ‘y’ ions facilitating efficient de novo sequencing. Ten allergenic proteins were identified, out of which six showed homology with known allergens while others were reported for the first time. Amongst these, isoflavone reductase, beta-conglycinin, S-adenosyl methionine synthase, 1, 4 glucan synthase and beta-galactosidase were commonly reported as allergens from coconut pollen and presumably responsible for cross-reactivity. One of the allergens had IgE binding epitope recognized by its glycan moiety. The allergenic potency of date palm pollen has been demonstrated using in vitro tests. The identified allergens can be used to develop vaccines for immunotherapy against date palm pollen allergy. The significance of the studyIdentification of allergenic proteins from sources harboring them is essential in developing therapeutic interventions. This is the first comprehensive study on the identification of allergens from Phoenix sylvestris (date palm) pollen, one of the major aeroallergens in India using a proteomic approach. Proteomic methods are being increasingly used to identify allergens. However, since many of these proteins arise from species which are un-sequenced, it becomes difficult to interpret those using conventional proteomics. Date palm being an unsequenced species, the IgE-reactive proteins have been identified using a stratified proteomic workflow incorporating manual de novo sequencing and homology-based proteomics. This study also gives an insight into the presence of glycan nature of the IgE binding epitopes. Five proteins have been found to be common with coconut pollen allergens and presumably responsible for cross-reactivity. These can be used in diagnostics to differentiate patient cohorts allergic to both coconut and date palm pollen from true date palm pollen allergic subjects. This would also determine better specific immunotherapy regimes between the two cohorts. The allergens identified herein have potential towards vaccine development in date palm pollen allergy as well as in enriching the existing catalogue of allergenic proteins.

Transcriptomic and histological responses of African rice (Oryza glaberrima) to Meloidogyne graminicola provide new insights into root-knot nematode resistance in monocots.

The root-knot nematode Meloidogyne graminicola is responsible for production losses in rice ( Oryza sativa ) in Asia and Latin America. The accession TOG5681 of African rice, O. glaberrima , presents improved resistance to several biotic and abiotic factors, including nematodes. The aim of this study was to assess the cytological and molecular mechanisms underlying nematode resistance in this accession. Penetration and development in M. graminicola in TOG5681 and the susceptible O. sativa genotype 'Nipponbare' were compared by microscopic observation of infected roots and histological analysis of galls. In parallel, host molecular responses to M. graminicola were assessed by root transcriptome profiling at 2, 4 and 8d post-infection (dpi). Specific treatments with hormone inhibitors were conducted in TOG5681 to assess the impact of the jasmonic acid and salicylic acid pathways on nematode penetration and reproduction. Penetration and development of M. graminicola juveniles were reduced in the resistant TOG5681 in comparison with the susceptible accession, with degeneration of giant cells observed in the resistant genotype from 15 dpi onwards. Transcriptome changes were observed as early as 2 dpi, with genes predicted to be involved in defence responses, phenylpropanoid and hormone pathways strongly induced in TOG5681, in contrast to 'Nipponbare'. No specific hormonal pathway could be identified as the major determinant of resistance in the rice-nematode incompatible interaction. Candidate genes proposed as involved in resistance to M. graminicola in TOG5681 were identified based on their expression pattern and quantitative trait locus (QTL) position, including chalcone synthase, isoflavone reductase, phenylalanine ammonia lyase, WRKY62 transcription factor, thionin, stripe rust resistance protein, thaumatins and ATPase3. This study provides a novel set of candidate genes for O. glaberrima resistance to nematodes and highlights the rice- M. graminicola pathosystem as a model to study plant-nematode incompatible interactions.

Immunodominance in allergic T-cell reactivity to Japanese cedar in different geographic cohorts

BackgroundJapanese cedar (JC) pollen is a common trigger for allergic rhinitis in Japan. Pollen proteins targeted by IgE, including Cry j 1 and Cry j 2, and isoflavone reductase (IFR) have been identified. ObjectiveTo compare antigen-specific IgE titers and T-cell responses to JC pollen–derived extract and peptides in cohorts with high and low pollen exposure. MethodsPeripheral blood mononuclear cells from JC pollen allergic or nonallergic patients who have lived in Japan for at least 1 year and JC pollen allergic patients who have never been to Japan were tested for T-cell responses against JC pollen extract and peptide pools derived from Cry j 1, Cry j 2, or IFR. T-cell reactivity was assessed by interleukin 5 and interferon γ production by ELISPOT. ResultsJC pollen–specific T-cell reactivity and IgE titers were significantly higher in the allergic compared with the nonallergic Japanese cohort, which was also associated with different patterns of polysensitization. Interestingly, a significant overlap was observed in the hierarchy of the T-cell epitopes in the allergic Japanese cohort compared with the allergic non-Japanese cohort. In all 3 cohorts, T-cell reactivity was dominantly directed against peptides from the major allergens Cry j 1 and 2, with few T-cell responses detected against IFR. ConclusionOur studies identify common denominators of T-cell reactivity in patient populations with different sensitization patterns, suggesting that generally applicable immunotherapeutic approaches might be developed irrespective of exposure modality.

Transcriptomic and proteomic analyses of leaves from Clematis terniflora DC. under high level of ultraviolet-B irradiation followed by dark treatment

High level of UV-B irradiation followed by dark treatment (HUV-B+D) causes accumulation of secondary metabolites in Clematis terniflora DC. To investigate the response mechanism under HUV-B+D, transcriptomic and proteomic analyses were performed in leaves of C. terniflora. The number of genes related to tetrapyrrole synthesis, amino acid metabolism, tricarboxylic acid cycle, and mitochondrial electron transport chains was hierarchically changed in leaves of C. terniflora under HUV-B+D. Data from RNA-sequencing transcriptomics and gel-free/label-free proteomics were integrated. The genes related to biosynthesis of lignins and flavonoids/isoflavonoids were significantly upregulated. Luteolin 7-O-β-D-glucosiduronic acid, rutin, and kaempferol 3-O-rutinose were accumulated. The number of differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) related to protein metabolism were largely changed in posttranslational modification, ubiquitin proteasome, and ribosomal protein. The expression of NADP-dependent malic enzyme and the abundance of NADP-malate dehydrogenase were upregulated and increased, respectively. The activities of these two enzymes were also enhanced. These results suggest that the secondary metabolism pathway and tricarboxylic acid cycle might be activated in leaves of C. terniflora in response to HUV-B+D. Biological significanceThis study reported response mechanism in leaves of Clematis terniflora DC. under high level of UV-B irradiation followed by dark treatment using transcriptomic and proteomic techniques. Transcriptomic analysis revealed that the number of genes related to secondary metabolism was increased by >2 times. Phenylalanine ammonia-lyase, 4-Coumarate: CoA ligase, chalcone synthase, isoflavone reductase homolog, and cinnamoyl-CoA reductase were significantly upregulated, which suggests that the secondary metabolism pathway related to lignins and flavonoids/isoflavonoids might be activated. Genes and proteins related to protein metabolism were largely changed in postranslational modification, ubiquitin proteasome, and ribosomal protein. NADP-dependent malic enzyme and NADP-malate dehydrogenase were activated in tricarboxylic acid cycle, which suggests that tricarboxylic acid cycle might be enhanced in leaf of C. terniflora in response to HUV-B+D.

Reduction of soy isoflavones by use of Escherichia coli whole-cell biocatalyst expressing isoflavone reductase under aerobic conditions

Soy isoflavone metabolites are currently receiving much attention due to the stronger and wider bioactivities than that of isoflavones. Therefore, biosynthesis of isoflavone metabolites by isolated isoflavone biotransforming bacteria is important. However, the biosynthesis process must be under obligate anaerobic conditions due to the reduction reactions catalysed by isoflavone biotransforming bacteria. In this study, we cloned the daidzein and genistein reductase gene (dgr) from Slackia sp. AUH-JLC159. The recombinant Escherichia coli (E.coli) whole-cell was used for the first time as the biocatalyst for aerobic biosynthesis of dihydrodaidzein (DHD) and dihydrogenistein (DHG) from soy isoflavones daidzein and genistein. Our results indicated that the recombinant E.coli whole-cell was able to reduce daidzein and genistein to DHD and DHG under aerobic conditions, while the maximal concentration of the substrate daidzein or genistein that the E.coli whole-cell was able to convert efficiently was only 0·4mmoll(-1) . Under the optimized conditions, the maximal concentration of daidzein or genistein that the E.coli whole-cell was able to convert efficiently was increased to 1·4mmoll(-1) . Our results demonstrated that E.coli whole-cell is an efficient biocatalyst for biosynthesis of isoflavone metabolites under aerobic conditions. Soy isoflavone metabolites, which are more biologically active than their precursor isoflavones, are currently receiving much more attention. However, the non-natural isoflavone metabolites are synthesized or biosynthesized under obligate anaerobic conditions. Here, we describe a new approach to the reduction of soy isoflavones daidzein and genistein under aerobic conditions by use of the recombinant Escherichia coli whole-cell expressing isoflavone reductase. Our study provides the first evidence that isoflavone metabolites, such as dihydrodaidzein and dihydrogenistein, are able to be produced efficiently under aerobic conditions.

English

Among antibiotics, tetracyclines are the most commonly used and detected in the environment. In this study, the amount of tetracycline taken up from soil by pea seedlings was analyzed, identified its main site of accumulation in plants and determined also changes in the protein profile of pea. The study demonstrates that pea seedlings take up tetracycline from soil and transport the drug via roots to over-ground parts and then accumulate it in the youngest parts, such as upper stem and leaves. After the taken up of drug, the activity of guaiacol peroxidase is modified and changes in the profile of proteins, as determined by two-dimensional gel electrophoresis occur. The majority of proteins (∼40%) visualized possessed molecular weight between 25 and 37 kDa. Only 8% of the proteins had molecular weight lower than 20 kDa, and 2% greater than 75 kDa. The number of spots in the control samples was 194, which is less by 49 than at the concentration of 150 mg kg of soil. Isoflavone reductase was present only in seedlings growing with tetracycline. Tetracycline uptake from soil modify mainly the changes in biochemical processes connected with protein. © 2016 Friends Science Publishers

Proteome analysis of Pueraria mirifica tubers collected in different seasons.

Pueraria mirifica-derived tuberous powder has been long-term consumed in Thailand as female hormone-replacement traditional remedies. The protein profiles of tubers collected in different seasons were evaluated. Phenol extraction, 2D-PAGE, and mass spectrometry were employed for tuberous proteome analysis. Out of the 322 proteins detected, over 59% were functionally classified as being involved in metabolism. The rest proteins were involved in defense, protein synthesis, cell structure, transportation, stress, storage, and also unidentified function. The proteins were found to be differentially expressed with respect to harvest season. Importantly, chalcone isomerase, isoflavone synthase, cytochrome p450, UDP-glycosyltransferase, and isoflavone reductase, which are all involved in the biosynthesis pathway of bioactive isoflavonoids, were most abundantly expressed in the summer-collected tubers. This is the first report on the proteomic patterns in P. mirifica tubers in relevant with seasonal variation. The study enlights the understanding of variance isoflavonoid production in P. mirifica tubers.

Research on Root Responses to Pb and Zn Combined Stress of Carex putuoshan.

Pb hyper-accumulated Carex putuoshan was taken as experimental material and subjected to combined stress of Pb and Zn. The differential expression of proteins in their roots were analyzed by Proteomic Approach. The protein that was directly involved in the cellular defense under the Pb and Zn combined stress was separated, and expression of those genes was analyzed with Carex Evergold as control. The results were obtained by MALDI-TOF/MS analysis. After applying Pb and Zn combined stress, the expression of 9 protein spots (including 7 different proteins, 2 identical proteins, 1 unknown protein) in Carex putuoshan root was found to be significantly up-regulated. Five proteins were obtained from the 9 proteins related to carbohydrate metabolism, including malate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, frutose-1,6-bisphosphate aldolase, enolase, and 6- phosphogluconate dehydrogenase. Two proteins were related to protein biosynthesis, including isoflavone reductase and phytochelatin synthase (PCS). From these proteins, the most important protein is PCS, which is a key enzyme in the synthesis of phytochelatins (PCs) and plays an important role in chelation. It is directly involved in cellular defense under Pb and Zn stress. After Pb and Zn combined stress, the CpPCS in Carex putuoshan was cloned. The full length of cDNA is 1461 bps, and it encodes 486 amino acids with molecular weight of 53.86 kD and pI value of 6.12. Two typical phytochelatin synthase subfamily domains constitute CpPCS protein, which includes three adjacent Cys-Cys elements in the C-terminal region. Phylogenetic analysis of PCS proteins from different species showed that it had the closest relationship with the Oryza sativa and Triticum aestivum. Real-time quantitative PCR analysis indicated that CpPCS and CePCS (Carex Evergold) genes were expressed in the root. The CpPCS and CePCS genes were up-regulated by Pb and Zn treatments. The expression of CpPCS was higher than that of CePCS under the same condition. The study found that CpPCS expression was increased by Pb and Zn stress in the Carex putuoshan enrichment process of Pb, which lead to high expression of PCS protein. CpPCS improved the accumulation ability and resistance of Carex putuoshan to heavy metals with the expression level of glucose metabolism related proteins increasing after Pb and Zn stress.

Identification of differentially expressed genes from Fusarium oxysporum f. sp cubense and Trichoderma asperellum (prr2) interaction in the susceptible banana cultivar Grand Naine

Identification of defense-related genes in the host is one of the most essential steps in understanding disease resistance mechanisms in plants. In this study, a suppression subtractive hybridization (SSH) library was constructed to study differential gene expression in banana plants mediated through a Fusarium wilt pathogen (Fusarium oxysporum f.sp cubense - Foc) and its interaction with the Foc effective biocontrol agent Trichoderma asperellum (prr2). Here cDNAs from the roots of banana cv. Grand Naine infected by Foc were used as the driver and cDNAs from Foc + T. asperellum inoculated banana plants as the tester population. After hybridization and cloning, an EST library of 300 nonredundant clones was obtained. Based on sequence analysis and a homology search in the NCBI database, the clones were assigned to different functional categories. The expression patterns of six selected defense-related genes, namely endochitinase, polyubiquitin, calmodulin binding protein, pleotropic drug resistant gene, isoflavone reductase, and mannose binding lectin, were analyzed through quantitative real-time PCR in Foc alone inoculated and Foc + T. asperellum inoculated banana plants. It was observed that the expression of these genes during initial progression of disease was higher in Foc + T. asperellum inoculated plants as compared to Foc alone inoculated plants. Our results constitute a step toward a better understanding of the role of mycoparasitic T. asperellum in plant defense during its interaction with Foc in the susceptible banana cultivar Grand Naine. Keywords: Foc-biocontrol interaction, Fusarium oxysporum f. sp. cubense, Musa defense genes, suppression subtractive hybridization Full Text: PDF

Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean.

Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.