Anr Gene Research Articles

Citric acid treatment inhibits fading of sorghum (Sorghum bicolor) by modulating the accumulation of flavonoids

Sorghum seeds can discolor during storage. Treatment of seeds with citric acid improves sensory quality and antioxidant activity. This study compared the differences in phenotypic and antioxidant activity between citric acid-treated and water-treated sorghum seeds. The study used transcriptomics and metabolomics approaches to investigate the regulatory mechanisms. The ∆a, ∆b and ∆l values of citric acid-treated sorghum seeds significantly increased after 6 months of storage. The SOD, POD and CAT enzyme activities of the citric acid-treated group were 1.94, 1.91 and 2.45 times higher than those of the control, respectively. The joint transcriptome and metabolome analysis showed that the citric acid-induced changes were mainly focused on the flavonoid biosynthetic pathway. Citric acid treatment up-regulated CHS, ANR, MYB and bHLH genes and promoted flavonoid accumulation. In conclusion, citric acid treatment promotes flavonoid accumulation, delays sorghum seed discoloration, and enhances antioxidant activity and storage life.

Metabolic and molecular mechanisms of spine color formation in Chinese red chestnut.

The spines of Chinese red chestnut are red and the depth of their color gradually increases with maturity. To identify the anthocyanin types and synthesis pathways in red chestnut and to identify the key genes regulating the anthocyanin biosynthesis pathway, we obtained and analyzed the transcriptome and anthocyanin metabolism of red chestnut and its control variety with green spines at 3 different periods. GO and KEGG analyses revealed that photosynthesis was more highly enriched in green spines compared with red spines, while processes related to defense and metabolism regulation were more highly enriched in red spines. The analysis showed that the change in spine color promoted photoprotection in red chestnut, especially at the early growth stage, which resulted in the accumulation of differentially expressed genes involved in the defense metabolic pathway. The metabolome results revealed 6 anthocyanins in red spines. Moreover, red spines exhibited high levels of cyanidin, peonidin and pelargonidin and low levels of delphinidin, petunidin and malvidin. Compared with those in the control group, the levels of cyanidin, peonidin, pelargonidin and malvidin in red spines were significantly increased, indicating that the cyanidin and pelargonidin pathways were enriched in the synthesis of anthocyanins in red spines, whereas the delphinidin pathways were inhibited and mostly transformed into malvidin. During the process of flower pigment synthesis, the expression of the CHS, CHI, F3H, CYP75A, CYP75B1, DFR and ANS genes clearly increased, that of CYP73A decreased obviously, and that of PAL, 4CL and LAR both increased and decreased. Notably, the findings revealed that the synthesized anthocyanin can be converted into anthocyanidin or epicatechin. In red spines, the upregulation of BZ1 gene expression increases the corresponding anthocyanidin content, and the upregulation of the ANR gene also promotes the conversion of anthocyanin to epicatechin. The transcription factors involved in color formation included 4 WRKYs.

Identification of bacterial dissimilatory antimonate reductase AnrA: genes and proteins involved in antimonate respiration and resistance in Geobacter sp. strain SVR.

Geobacter sp. strain SVR uses antimonate [Sb(V)] as a terminal electron acceptor for anaerobic respiration. Here, we visualized a possible key enzyme, periplasmic Sb(V) reductase (Anr), via active staining and non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that a novel dimethyl sulfoxide (DMSO) reductase family protein, WP_173201954.1, is involved in Anr. This protein was closely related with AnrA, a protein suggested to be the catalytic subunit of a respiratory Sb(V) reductase in Desulfuribacillus stibiiarsenatis. The anr genes of strain SVR (anrXSRBAD) formed an operon-like structure, and their transcription was upregulated under Sb(V)-respiring conditions. The expression of anrA gene was induced by more than 1 µM of antimonite [Sb(III)]; however, arsenite [As(III)] did not induce the expression of anrA gene. Tandem mass tag-based proteomic analysis revealed that, in addition to Anr proteins, proteins in the following categories were upregulated under Sb(V)-respiring conditions: (i) Sb(III) efflux systems such as Ant and Ars; (ii) antioxidizing proteins such as ferritin, rubredoxin, and thioredoxin; (iii) protein quality control systems such as HspA, HslO, and DnaK; and (iv) DNA repair proteins such as UspA and UvrB. These results suggest that strain SVR copes with antimony stress by modulating pleiotropic processes to resist and actively metabolize antimony. To the best of our knowledge, this is the first report to demonstrate the involvement of AnrA in Sb(V) respiration at the protein level. Furthermore, this is the first example to show high expression of the Ant system proteins in the Sb(V)-respiring bacterium.IMPORTANCEAntimony (Sb) exists mainly as antimonite [Sb(III)] or antimonate [Sb(V)] in the environment, and Sb(III) is more toxic than Sb(V). Recently, microbial involvement in Sb redox reactions has received attention. Although more than 90 Sb(III)-oxidizing bacteria have been reported, information on Sb(V)-reducing bacteria is limited. Especially, the enzyme involved in dissimilatory Sb(V) reduction, or Sb(V) respiration, is unclear, despite this pathway being very important for the circulation of Sb in nature. In this study, we demonstrated that the Sb(V) reductase (Anr) of an Sb(V)-respiring bacterium (Geobacter sp. SVR) is a novel member of the dimethyl sulfoxide (DMSO) reductase family. In addition, we found that strain SVR copes with Sb stress by modulating pleiotropic processes, including the Ant and Ars systems, and upregulating the antioxidant and quality control protein levels. Considering the abundance and diversity of putative anr genes in the environment, Anr may play a significant role in global Sb cycling in both marine and terrestrial environments.

Exploration of cross-cultivar group characteristics of a new cultivar of Prunus mume 'Zhizhang Guhong Chongcui'

'Zhizhang Guhong Chongcui' is a new cultivar of Prunus mume with cross-cultivar group characteristics. It has typical characteristics of cinnabar purple cultivar group and green calyx cultivar group. It has green calyx, white flower, and light purple xylem, but the mechanism remains unclear. In order to clarify the causes of its cross-cultivar group traits, the color phenotype, anthocyanin content and the expression levels of genes related to anthocyanin synthesis pathway of 'Zhizhang Guhong Chongcui', 'Yuxi Zhusha' and 'Yuxi Bian Lü'e' were determined. It was found that the red degree of petals, sepals and fresh xylem in branches was positively correlated with the total anthocyanin content. MYBɑ1, MYB1, and bHLH3 were the key transcription factor genes that affected the redness of the three cultivars of flowers and xylem. The transcription factors further promoted the high expression of structural genes F3'H, DFR, ANS and UFGT, thereby promoting the production of red traits. Combined with phenotype, anthocyanin content and qRT-PCR results, it was speculated that the white color of petals of 'Zhizhang Guhong Chongcui' were derived from the high expression of FLS, F3'5'H, LAR and ANR genes in other branches of cyanidin synthesis pathway, and the low expression of GST gene. The green color of sepals might be originated from the relatively low expression of F3'H, DFR and ANS genes. The red color of xylem might be derived from the high expression of ANS and UFGT genes. This study made a preliminary explanation for the characteristics of the cross-cultivar group of 'Zhizhang Guhong Chongcui', and provided a reference for molecular breeding of flower color and xylem color of Prunus mume.

Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp.

Main conclusionThe complexes involving MYBPA2, TT2b, and TT8 proteins are the critical regulators of ANR and LAR genes to promote the biosynthesis of proanthocyanidins in the leaves of Lotus spp.The environmental impact and health of ruminants fed with forage legumes depend on the herbage's concentration and structure of proanthocyanidins (PAs). Unfortunately, the primary forage legumes (alfalfa and clover) do not contain substantial levels of PAs. No significant progress has been made to induce PAs to agronomically valuable levels in their edible organs by biotechnological approaches thus far. Building this trait requires a profound knowledge of PA regulators and their interplay in species naturally committed to accumulating these metabolites in the target organs. Against this background, we compared the shoot transcriptomes of two inter-fertile Lotus species, namely Lotus tenuis and Lotus corniculatus, polymorphic for this trait, to search for differentially expressed MYB and bHLH genes. We then tested the expression of the above-reported regulators in L. tenuis x L. corniculatus interspecific hybrids, several Lotus spp., and different L. corniculatus organs with contrasting PA levels. We identified a novel MYB activator and MYB-bHLH-based complexes that, when expressed in Nicotiana benthamiana, trans-activated the promoters of L. corniculatus anthocyanidin reductase and leucoanthocyanidin reductase 1 genes. The last are the two critical structural genes for the biosynthesis of PAs in Lotus spp. Competition between MYB activators for the transactivation of these promoters also emerged. Overall, by employing Lotus as a model genus, we refined the transcriptional network underlying PA biosynthesis in the herbage of legumes. These findings are crucial to engineering this trait in pasture legumes.

Widely Targeted Metabolomics Analyses Clarify the Biosynthetic Pathways Responsible for Flavonoids in Sweet Potato (Ipomoea batatas (L.) Lam.) Storage Roots

Sweet potatoes (Ipomoea batatas (L.) Lam.) are a widely cultivated member of the Convolvulaceae family. Despite intensive agricultural interest, the metabolic determinants of sweet potato quality remain poorly understood owing to a lack of reliable or systematic sweet potato metabolite analyses. This study aimed to reveal the mechanism of flavonoid biosynthesis using widely targeted metabolomics and qRT-PCR analysis of white (S19) and yellow (BS) sweet potatoes. We found that the PAL, C4H, 4CL, CHS, CHI, IFS, F3H, F3’H, DFR, ANS, and ANR genes were differentially expressed in BS. Upregulation of PAL, C4H, 4CL, and CHS led to the accumulation of large amounts of chalcone, which is highly expressed in yellow flesh, resulting in the yellow color of BS. In S19, the high expression of FLS and the low expression of DFR inhibited pigment accumulation, while the low expression of CHS also inhibited flavonoid synthesis, ultimately leading to the white color. In conclusion, this study identified the main differentially expressed genes and their metabolites in the flavonoid biosynthesis pathway, and preliminarily elucidated the mechanism underlying the different flesh colors in sweet potato, thus providing new insights into the composition and abundance of metabolites in sweet potatoes with different-colored flesh.

Functional Identification of ANR Gene in Apple (Malus halliana) Demonstrates It Reduces Saline-alkali Stress Tolerance.

As one of the major abiotic stresses restricting the development of global agriculture, saline-alkali stress can cause osmotic stress, ion poisoning, reactive oxygen species damage and high pH value damage, which seriously restricts the sustainable development of fruit industry. Therefore, it is of great significance to cultivate saline-alkali resistant apple rootstocks to improve the yield and quality of apple in China. Based on transcriptome data, MhANR (LOC114827797), which was significantly induced by saline-alkali stress, was cloned from Malus halliana. The physicochemical properties, evolutionary relationships and cis-acting elements were analyzed. Subsequently, the tolerance of MhANR overexpression in Arabidopsis thaliana, tobacco and apple calli to saline-alkali stress was verified by genetic transformation. Transgenic plants presented lower chla, chlb and proline contents, lower Superoxide dismutase (SOD), Peroxidase enzyme (POD) and Catalase enzyme (CAT) activities, and higher relative electrical conductivity (REC) compared to those in WT plants under saline-alkali stress. In addition, the expression levels of saline-alkali stress related genes in overexpressed apple calli also diaplayed lower values compared to WT calli, including antioxidant genes MhSOD and MhCAT, Na+ transporter genes MhCAX5, MhCAX5, MhSOS1, MhALT1, and H+ -ATPase genes MhAHA2 and MhAHA8, while the expression levels of K+ transporter genes MhSKOR and MhNHX4 displayed higher values. In conclusion, MhANR reduced the tolerance of Arabidopsis thaliana, tobacco and apple calli under saline-alkali stress by regulating osmoregulatory substances, Chlorophyll content, antioxidant enzyme activity and expression of saline-alkali stress-related genes, which provided a theoretical basis for cultivating apple rootstocks with effective saline-alkali stress tolerance.

Antioxidant processes involving epicatechin decreased symptoms of pine wilt disease.

Since the pine wood nematode (PWN, Bursaphelenchus xylophilus) invasion of Northeast China, both symptomatic and asymptomatic PWN carriers have been found. Asymptomatic PWN carriers, which are more dangerous than symptomatic carriers, constitute a source of infection in the following spring. The simultaneous presence of symptomatic and asymptomatic PWN carriers indicates that Pinus koraiensis has different tolerance levels to PWN. In this study, validity of susceptibility testing discovered differential types of P. koraiensis including Latent Reservoirs, Low Susceptibles, High Susceptibles and Bell Ringers. Among those types, the Low Susceptibles and Latent Reservoirs were asymptomatic PWN carriers, and Latent Reservoirs were the most dangerous. Transcriptome and metabolomic data showed that 5 genes (3 ans and 2 anr gene) involved in the epicatechin (EC) synthesis pathway were significantly upregulated, which increased the content of EC antioxidants in Latent Reservoirs. Hydrogen peroxide (H2O2) staining and content determination showed that the hypersensitive response (HR) and H2O2, which functions as a signaling molecule in systemic acquired resistance, decreased in Latent Reservoirs. However, low contents of EC and high contents of H2O2 were found in the High Susceptibles of P. koraiensis. RT-PCR results showed that the expression of ans and anr was upregulated together only in Latent Reservoirs. These results show that the susceptibility of P. koraiensis to PWN differed among different individuals, although no resistant individuals were found. Latent Reservoirs, in which more PWNs resided without visible symptoms via prolonged incubation period, inhibited the symptoms caused by H2O2 because of increased contents of the EC antioxidants.

Novel Insights into Anthocyanin Synthesis in the Calyx of Roselle Using Integrated Transcriptomic and Metabolomic Analyses.

Roselle (Hibiscus sabdariffa L.) is an annual herbaceous plant of the genus Hibiscus in family Malvaceae. Roselle calyxes are rich in anthocyanins, which play important roles in human health. However, limited information is available on anthocyanin biosynthesis in the roselle calyx. In this study, transcriptomic and metabolomic analyses were performed to identify the key genes involved in anthocyanin biosynthesis in the roselle calyx. Three roselle cultivars with different calyx colors, including FZ-72 (red calyx, R), Baitao K (green calyx, G), and MG5 (stripped calyx, S), were used for metabolomic analyses with UPLC-Q-TOF/MS and RNA-seq. Forty-one compounds were quantified, including six flavonoids and 35 anthocyanins. The calyx of FZ-72 (red calyx) had the highest contents of anthocyanin derivatives such as delphinidin-3-O-sambubioside (955.11 μg/g) and cyanidin-3-O-sambubioside (531.37 μg/g), which were responsible for calyx color, followed by those in MG5 (stripped calyx) (851.97 and 330.06 μg/g, respectively). Baitao K (green calyx) had the lowest levels of these compounds. Furthermore, RNA-seq analysis revealed 114,415 differentially expressed genes (DEGs) in the calyxes at 30 days after flowering (DAF) for the corresponding cultivars FZ-72 (R), Baitao K (G), and MG5(S). The gene expression levels in the calyxes of the three cultivars were compared at different flowering stages, revealing 11,555, 11,949, and 7177 DEGs in R vs. G, R vs. S, and G vs. S, respectively. Phenylpropanoid and flavonoid biosynthesis pathways were found to be enriched. In the flavonoid pathway, 29, 28, and 27 genes were identified in G vs. R, G vs. S, and S vs. R, respectively. In the anthocyanin synthesis pathway, two, two, and one differential genes were identified in the three combinations; these differential genes belonged to the UFGT gene family. After joint analysis of the anthocyanin content in roselle calyxes, nine key genes belonging to the CHS, CHI, UFGT, FLS, ANR, DFR, CCoAOMT, SAT, and HST gene families were identified as strongly related to anthocyanin synthesis. These nine genes were verified using qRT-PCR, and the results were consistent with the transcriptome data. Overall, this study presents the first report on anthocyanin biosynthesis in roselle, laying a foundation for breeding roselle cultivars with high anthocyanin content.

Transcriptomic profiling analysis to identify genes associated with PA biosynthesis and insolubilization in the late stage of fruit development in C-PCNA persimmon

PA-enhanced content causes astringency in persimmon fruit. PCNA persimmons can lose their astringency naturally and they become edible when still on the tree, which allows for conserves of physical and financial resources. C-PCNA persimmon originates in China. Its deastringency trait primarily depends on decreased PA biosynthesis and PA insolubilization at the late stage of fruit development. Although some genes and transcription factors that may be involved in the deastringency of C-PCNA persimmon have been reported, the expression patterns of these genes during the key deastringency stage are reported less. To investigate the variation in PA contents and the expression patterns of deastringency-related genes during typical C-PCNA persimmon ‘Xiaoguo-tianshi’ fruit development and ripening, PA content and transcriptional profiling were carried out at five late stages from 70 to 160 DAF. The combinational analysis phenotype, PA content, and DEG enrichment revealed that 120–140 DAF and 140–160 DAF were the critical phases for PA biosynthesis reduction and PA insolubilization, respectively. The expression of PA biosynthesis-associated genes indicated that the downregulation of the ANR gene at 140–160 DAF may be associated with PA biosynthesis and is decreased by inhibiting its precursor cis-flavan-3-ols. We also found that a decrease in acetaldehyde metabolism-associated ALDH genes and an increase in ADH and PDC genes might result in C-PCNA persimmon PA insolubilization. In addition, a few MYB-bHLH-WD40 (MBW) homologous transcription factors in persimmon might play important roles in persimmon PA accumulation. Furthermore, combined coexpression network analysis and phylogenetic analysis of MBW suggested that three putative transcription factors WD40 (evm.TU.contig1.155), MYB (evm.TU.contig8910.486) and bHLH (evm.TU.contig1398.203), might connect and co-regulate both PA biosynthesis and its insolubilization in C-PCNA persimmon. The present study elucidated transcriptional insights into PA biosynthesis and insolubilization during the late development stages based on the C-PCNA D. kaki genome (unpublished). Thus, we focused on PA content variation and the expression patterns of genes involved in PA biosynthesis and insolubilization. Our work has provided additional evidence on previous knowledge and a basis for further exploration of the natural deastringency of C-PCNA persimmon.

Expression of Structural Flavonoid Biosynthesis Genes in Dark-Blue and White Myrtle Berries (Myrtus communis L.)

Within the myrtle (Myrtus communis L.) species, different genotypes may produce dark-blue berries or white berries depending on the peel color upon ripening. One dark-blue cultivar and one white myrtle cultivar were used to study the molecular mechanisms underlying flavonoid biosynthesis. The relative expression levels of common (PAL, CHS, CHI, DFR and LDOX) and specific (FLS, ANR, LAR and UFGT) flavonoid genes were analyzed during fruit development by means of quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, the anthocyanin content was determined, and it showed an increase with the ripening of the berries of the dark-blue cultivar. The results showed an increased transcript abundance of PAL, CHI, DFR, LDOX and UFGT gene expression in the dark-blue cultivar compared to the white one, as well as a strong positive correlation between the changes in gene expression and anthocyanin accumulation. The transcript levels of UFGT showed sharp increases at 150 and 180 days after full blooming (DAF) in the dark-blue cultivar, which corresponded with anthocyanin accumulation. However, ripening seemed to modulate the expression of genes implicated in flavonols (i.e., FLS) and flavan-3-ols (i.e., LAR and ANR) in different manners. However, whereas FLS transcript accumulation increased at the end of the ripening period in the dark-blue cultivar, LAR and ANR gene expression decreased in both cultivars.

Comprehensive ESI-Q TRAP-MS/MS based characterization of metabolome of two mango (Mangifera indica L) cultivars from China

Polyphenols based bioactive compounds from vegetables and fruits are known for impressive antioxidant activity. Ingestion of these antioxidants may promote human health against cardiovascular diseases and cancer. Mango is a popular tropical fruit with special taste, high nutritional value and health-enhancing metabolites. The aim was to investigate the diversity of phytochemicals between two mango cultivars of china at three stages of fruit maturity. We used ESI-QTRAP-MS/MS approach to characterize comprehensively the metabolome of two mango cultivars named Hongguifei (HGF) and Tainong (TN). HPLC was used to quantify selected catechin based phenolic compounds. Moreover, real-time qPCR was used to study the expression profiles of two key genes (ANR and LAR) involved in proanthocyanidin biosynthesis from catechins and derivatives. A total of 651 metabolites were identified, which include at least 257 phenolic compounds. Higher number of metabolites were differentially modulated in peel as compared to pulp. Overall, the relative quantities of amino acids, carbohydrates, organic acids, and other metabolites were increased in the pulp of TN cultivar. While the contents of phenolic compounds were relatively higher in HGF cultivar. Moreover, HPLC based quantification of catechin and derivatives exhibited cultivar specific variations. The ANR and LAR genes exhibited an opposite expression profile in both cultivars. Current study is the first report of numerous metabolites including catechin-based derivatives in mango fruit. These findings open novel possibilities for the use of mango as a source of bioactive compounds.

Stacking triple genes increased proanthocyanidins level in Arabidopsis thaliana.

Anthocyanins and proanthocyanidins are two important plant secondary metabolites, and they contribute to plant survival and human health. In particular, proanthocyanidins could also prevent ruminants from the damage of pasture bloat. However, the improvement of proanthocyanidins content remain unsatisfied. In this study, we attempted to improve proanthocyanidins level by gene stacking in Arabidopsis thaliana as prove-of-concept. Two proanthocyanidin pathway genes from tea plant, CsF3’5’H and CsANR2, were co-expressed in the wild type and PAP1 over-expression Arabidopsis. Over-expression of CsF3’5’H slightly affected anthocyanins level in leaves and proanthocyanidins in mature seed when expressed alone in the pap1-D line. Over-expression of CsANR2 led to an obvious decrease in anthocyanins in leaves of both wild type and pap1-D lines, but increase in proanthocyanidin level in mature seeds. Over-expression of CsANR2 in pap1-D lines lead to production of DMACA-reactive soluble proanthocyanidins in leaves, but not in wild type or pap1-D lines. Anthocyanins level was decreased in the leaves of CsF3’5’H, CsANR2 and pap1-D co-expression lines, but proanthocyanidins were increased remarkably in both leaves and mature seeds in the co-expression line. It is concluded that co-expression of CsANR2 and PAP1 in Arabidopsis produce soluble proanthocyanidins in leaves, and co-expression of CsF3’5’H, CsANR2 and PAP1 lead to a significant increase in proanthocyanidins in mature seeds. The transcript levels of endogenous CHS, DFR, ANS and ANR genes in Arabidopsis were up-regulated in the triple genes co-expression line. Based on these studies, it is possible to develop new plant germplasm with improved proanthocyanidins by co-expressing of multiple genes.

Combined metabolomic and transcriptomic analysis reveals key candidate genes involved in the regulation of flavonoid accumulation in Anoectochilus roxburghii

Anoectochilus roxburghii, a species used in Chinese herbal medicine, has unique characteristics. This plant has important medicinal and ornamental value and is distributed primarily in China, including Fujian, Zhejiang, Jiangxi, and Guizhou provinces, and in Taiwan. Flavonoids are involved in leaf pigment formation and are major pharmacodynamic substances. However, the molecular mechanisms that regulate accumulation of flavonoids remain unclear, which has significantly limited their application. To elucidate the molecular mechanisms underlying the regulation of flavonoid accumulation in A. roxburghii, root, stem and leaf samples were collected for constructing transcriptomic and metabolomic datasets using RNA sequencing and liquid chromatography-mass spectrometry (LC–MS) techniques. Sequencing of the transcriptomes of the different organs generated 60.2 Gb of data, which were assembled into 186,865 unigenes. Metabolomic analysis resulted in the identification of 10,690 metabolites. Based on the ESI+ mode, 4,010, 4,008 and 4,013 metabolites were annotated in roots, stems, and leaves, respectively; based on the ESI– mode, 1,530, 1,530, and 1,531 metabolites were annotated, respectively. Differential analyses of the transcriptome and flavonoid metabolism of the different organs revealed the greatest significant differences between roots and leaves, followed by differences between stems and leaves; differences between roots and stems were the smallest. According to analysis of differentially expressed genes (DEGs), the secondary metabolism-related DEGs between roots and stems, between roots and leaves, and between leaves and stems were classified into 16, 21, and 19 secondary metabolic Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. Among these pathways, flavonoid biosynthesis, flavone and flavonol biosynthesis, and isoflavonoid biosynthesis significantly differed in terms of both gene expression and secondary metabolism. Moreover, the key genes involved in flavonoid accumulation were comprehensively analyzed using metabolic and transcriptomic data, and ten transcription factor-encoding genes and fourteen flavonoid biosynthesis genes were identified. Specifically, four of the ten transcription factor-encoding genes appear to activate CHS, CHI, CYP75A and ANR gene expression. The combined approach used in this study provides an in-depth understanding of the molecular regulatory mechanisms underlying flavonoid accumulation in A. roxburghii and is a powerful tool that can uncover valuable information for plant breeders.

Oxygen Limitation Enhances the Antimicrobial Activity of Fosfomycin in Pseudomonas aeruginosa Following Overexpression of glpT Which Encodes Glycerol-3-Phosphate/Fosfomycin Symporter.

Fosfomycin is resurfacing as a “last resort drug” to treat infections caused by multidrug resistant pathogens. This drug has a remarkable benefit in that its activity increases under oxygen-limited conditions unlike other commonly used antimicrobials such as β-lactams, fluoroquinolones and aminoglycosides. Especially, utility of fosfomycin has being evaluated with particular interest to treat chronic biofilm infections caused by Pseudomonas aeruginosa because it often encounters anaerobic situations. Here, we showed that P. aeruginosa PAO1, commonly used in many laboratories, becomes more susceptible to fosfomycin when grown anaerobically, and studied on how fosfomycin increases its activity under anaerobic conditions. Results of transport assay and gene expression study indicated that PAO1 cells grown anaerobically exhibit a higher expression of glpT encoding a glycerol-3-phosphate transporter which is responsible for fosfomycin uptake, then lead to increased intracellular accumulation of the drug. Elevated expression of glpT in anaerobic cultures depended on ANR, a transcriptional regulator that is activated under anaerobic conditions. Purified ANR protein bound to the DNA fragment from glpT region upstream, suggesting it is an activator of glpT gene expression. We found that increased susceptibility to fosfomycin was also observed in a clinical isolate which has a promoted biofilm phenotype and its glpT and anr genes are highly conserved with those of PAO1. We conclude that increased antibacterial activity of fosfomycin to P. aeruginosa under anaerobic conditions is attributed to elevated expression of GlpT following activation of ANR, then leads to increased uptake of the drug.

Metabolic engineering of proanthocyanidin production by repressing the isoflavone pathways and redirecting anthocyanidin precursor flux in legume.

Summary MtPAR is a proanthocyanidin (PA) biosynthesis regulator; the mechanism underlying its promotion of PA biosynthesis is not fully understood. Here, we showed that MtPAR promotes PA production by a direct repression of biosynthesis of isoflavones, the major flavonoids in legume, and by redirecting immediate precursors, such as anthocyanidins, flux into PA pathway. Ectopic expression of MtPAR repressed isoflavonoid production by directly binding and suppressing isoflavone biosynthetic genes such as isoflavone synthase (IFS). Meanwhile, MtPAR up‐regulated PA‐specific genes and decreased the anthocyanin levels without altering the expression of anthocyanin biosynthetic genes. MtPAR may shift the anthocyanidin precursor flux from anthocyanin pathway to PA biosynthesis. MtPAR complemented PA‐deficient phenotype of Arabidopsis tt2 mutant seeds, demonstrating their similar action on PA production. We showed the direct interactions between MtPAR, MtTT8 and MtWD40‐1 proteins from Medicago truncatula and Glycine max, to form a ternary complex to trans‐activate PA‐specific ANR gene. Finally, MtPAR expression in alfalfa (Medicago sativa) hairy roots and whole plants only promoted the production of small amount of PAs, which was significantly enhanced by co‐expression of MtPAR and MtLAP1. Transcriptomic and metabolite profiling showed an additive effect between MtPAR and MtLAP1 on the production of PAs, supporting that efficient PA production requires more anthocyanidin precursors. This study provides new insights into the role and mechanism of MtPAR in partitioning precursors from isoflavone and anthocyanin pathways into PA pathways for a specific promotion of PA production. Based on this, a strategy by combining MtPAR and MtLAP1 co‐expression to effectively improve metabolic engineering performance of PA production in legume forage was developed.

Identification of Two Anthocyanidin Reductase Genes and Three Red-Brown Soybean Accessions with Reduced Anthocyanidin Reductase 1 mRNA, Activity, and Seed Coat Proanthocyanidin Amounts

Anthocyanidin reductase (ANR; EC 1.3.1.77) catalyzes a key step in the biosynthesis of proanthocyanidins (PAs; also known as condensed tannins), flavonoid metabolites responsible for the brown pigmentation of seeds. Here, two ANR genes (ANR1 and ANR2) from the seed coat of brown soybean (Glycine max (L.) Merr.) have been isolated and their enzymatic function confirmed for the reduction of cyanidin to (-)-epicatechin in vitro. Biochemical and genetic comparisons of soybean lines differing in seed coat color revealed three red-brown lines to exhibit major reductions in the amounts of soluble PAs in the seed coat compared to brown soybean lines. Two spontaneous mutants with red-brown grain color had reduced ANR1 gene expression in the seed coat, and an EMS-mutagenized red-brown mutant had nonsynonymous substitutions that resulted in slightly reduced ANR1 activity in vitro. These results suggest that defects in the ANR1 gene can be associated with red-brown soybean grain color. These results suggest that suppressing ANR1 gene expression or activity may be a rational approach toward engineering seed coat color to enable the visual identification of genetically modified soybean grains.

Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine (Vitis vinifera L.)

Anthocyanins and tannins are two of the most abundant flavonoids found in grapevine, and their synthesis is derived from the phenylpropanoid pathway. As described for model species such as Arabidopsis thaliana, maize and petunia, the end-point branches of this pathway are tightly regulated by the combinatorial interaction of three families of regulatory factors; MYB, bHLH (also known as MYC) and WDR proteins. Among these, only MYB genes have been previously identified in grapes. Here, we report the isolation of the first members from the WDR and bHLH families found in Vitis vinifera, named WDR1, WDR2 and MYCA1. WDR1 contributed positively to the accumulation of anthocyanins when it was overexpressed in A. thaliana, although it was not possible to determine the function of WDR2 by ectopic expression. The sub-cellular localizations of WDR1 and MYCA1 were observed by means of GFP-fusion proteins, indicating both cytoplasm and nuclear localization, in contrast to the localization of a MYB factor exclusively in the nucleus. The expression patterns of these genes were quantified in coloured reproductive organs throughout development, and correlated with anthocyanin accumulation and the expression profiles of the flavonoid-related MYBA1-2, UFGT, and ANR genes. In vitro grapevine plantlets grown under high salt concentrations showed a cultivar-dependent response for anthocyanin accumulation, which correlated with the expression of MYBA1-2, MYCA1 and WDR1 genes. These results suggest that MYCA1 may regulate ANR and UFGT and that this last control is easier to distinguish whenever MYBA genes are absent or in low abundance. Future studies should address the specific interactions of these proteins and their quantitative contribution to flavonoid synthesis in grape berries.

The coordinate regulation of multiple terminal oxidases by the Pseudomonas putida ANR global regulator

Pseudomonas putida KT2440 contains a branched aerobic respiratory chain with multiple terminal oxidases. Their relative proportion varies according to environmental conditions. The role of the oxygen-responsive ANR global regulator on expression of these terminal oxidases was analysed. During exponential growth in a highly aerated complete medium, ANR activated expression of the Cbb3-1 terminal oxidase (equivalent to Pseudomonas aeruginosa Cbb3-2), but had little role on expression of other terminal oxidases. In early stationary phase, or under oxygen limitation, inactivation of the anr gene led to increased expression of the bo(3)-type cytochrome (Cyo) and cyanide-insensitive (CIO) terminal oxidases, and to a much lower expression of Cbb3-1. DNase I footprints identified ANR binding sites at the promoters for these oxidases. Their location suggests that ANR is a transcriptional activator of Cbb3-1 genes and a repressor of CIO genes, consistent with expression data. ANR binding sites at the promoter for Cyo genes suggests a complex regulation in combination with other factors. Therefore, ANR coordinates expression of Cyo, CIO and Cbb3-1, but does not influence cytochrome aa3 and Cbb3-2 terminal oxidases under the conditions analysed. Functional assays showed that Cyo has a leading role during aerobic exponential growth, while Cbb3-1 becomes very important in stationary phase.

In Schizosaccharomyces pombe the 14–3–3 protein Rad24p is involved in negative control of pho1 gene expression

Expression of Schizosaccharomyces pombe pho1-encoded acid phosphatase is transcriptionally regulated by adenine and phosphate. Four genes, anr1-3 and anr5, encode negative regulators of pho1 expression. Apart from being designated as loci, the anr genes have not been further characterized. In this study we provide evidence that a strain carrying the deletion of rad24, a 14-3-3 protein-encoding gene, exhibits an anr mutant like the phenotype (higher phosphatase activity, higher transcript levels of pho1, lower sensitivity to adenine of pho1 expression) and that rad24 is closely linked, probably allelic, to anr5. By sequencing the two exons of the rad24 gene in a strain carrying the mutant allele anr5-13, we found a T/A-to-C/G transition in the 225th codon of its ORF, causing a leucine-to-serine substitution in a highly conserved region of all proteins of the 14-3-3 family. anr2 and anr3 are not allelic to rad24. The mutant alleles of anr2 and anr3 are recessive to their wild-type alleles and do not belong to the same epistasis group as rad24.