Scopoletin 8-hydroxylase: a novel enzyme involved in coumarin biosynthesis and iron-deficiency responses in Arabidopsis.
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ABSTRACT:
SUBMITTER: Siwinska J
PROVIDER: S-EPMC5888981 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The fungus Phakopsora pachyrhizi (Pp) causes Asian soybean rust (SBR) disease which provokes tremendous losses in global soybean production. Pp is mainly controlled with synthetic fungicides to which the fungus swiftly develops fungicide resistance. To substitute or complement synthetic fungicides in Asian soybean rust control, we aimed to identify antifungal metabolites in Arabidopsis which is not a host for Pp. Comparative transcriptional and metabolic profiling of the Pp-inoculated Arabidopsis non-host and the soybean host revealed induction of phenylpropanoid metabolism-associated genes in both species but activation of scopoletin biosynthesis only in the resistant non-host. Scopoletin is a coumarin and an antioxidant. In vitro experiments disclosed fungistatic activity of scopoletin against Pp, associated with reduced accumulation of reactive oxygen species (ROS) in fungal pre-infection structures. Non-antioxidant and antioxidant molecules including coumarins with a similar structure to scopoletin were inactive or much less effective at inhibiting fungal accumulation of ROS and germination of Pp spores. When sprayed onto Arabidopsis leaves, scopoletin also suppressed the formation of Pp pre-infection structures and penetration of the plant. However, scopoletin neither directly activated defence nor did it prime Arabidopsis for enhanced defence, therefore emphasizing fungistatic activity as the exclusive mode of action of scopoletin against Pp. Because scopletin also protected soybean from Pp infection, the coumarin may serve as a natural fungicide or as a lead for the development of near-to-nature fungicides against Asian soybean rust.
| S-EPMC6852345 | biostudies-literature
Project description:Among the mineral nutrients that are required for plant metabolism, iron (Fe) and sulphur (S) play a central role as both elements are needed for the activity of several proteins involved in essential cellular processes. A combination of physiological, biochemical and molecular approaches was employed to investigate how S availability influences plant response to Fe deficiency, using the model plant Arabidopsis thaliana. We first observed that chlorosis symptom induced by Fe deficiency was less pronounced when S availability was scarce. We thus found that S deficiency inhibited the Fe deficiency induced expression of several genes associated with the maintenance of Fe homeostasis. This includes structural genes involved in Fe uptake (i.e. IRT1, FRO2, PDR9, NRAMP1) and transport (i.e. FRD3, NAS4) as well as a subset of their upstream regulators, namely BTS, PYE and the four clade Ib bHLH. Last, we found that the over accumulation of manganese (Mn) in response to Fe shortage was reduced under combined Fe and S deficiencies. These data suggest that S deficiency inhibits the Fe deficiency dependent induction of the Fe uptake machinery. This in turn limits the transport into the root and the plant body of potentially toxic divalent cations such as Mn and Zn, thus limiting the deleterious effect of Fe deprivation.
| S-EPMC7440645 | biostudies-literature
Project description:Plants face varying nutrient conditions, to which they have to adapt to. Adaptive responses are nutrient-specific and strategies to ensure supply and homeostasis for one nutrient might be opposite to another one, as shown for phosphate (Pi) and iron (Fe) deficiency responses, where many genes are regulated in an opposing manner. This was also observed on the metabolite levels. Whereas root and exudate levels of catechol-type coumarins, phenylpropanoid-derived 2-benzopyranones, which facilitate Fe acquisition, are elevated after Fe deficiency, they are decreased after Pi deficiency. Exposing plants to combined Pi and Fe deficiency showed that the generation of coumarin profiles in Arabidopsis thaliana roots by Pi deficiency considerably depends on the availability of Fe. Similarly, the effect of Fe deficiency on coumarin profiles is different at low compared to high Pi availability. These findings suggest a fine-tuning of coumarin profiles, which depends on Fe and Pi availability. T-DNA insertion lines exhibiting aberrant expression of genes involved in the regulation of Pi starvation responses (PHO1, PHR1, bHLH32, PHL1, SPX1) and Fe starvation responses (BRUTUS, PYE, bHLH104, FIT) were used to analyze the regulation of the generation of coumarin profiles in Arabidopsis thaliana roots by Pi, Fe, and combined Pi and Fe deficiency. The analysis revealed a role of several Fe-deficiency response regulators in the regulation of Fe and of Pi deficiency-induced coumarin profiles as well as for Pi deficiency response regulators in the regulation of Pi and of Fe deficiency-induced coumarin profiles. Additionally, the regulation of Fe deficiency-induced coumarin profiles by Fe deficiency response regulators is influenced by Pi availability. Conversely, regulation of Pi deficiency-induced coumarin profiles by Pi deficiency response regulators is modified by Fe availability.
| S-EPMC6378295 | biostudies-literature
Project description:Iron (Fe) is an essential micronutrient that participates in various biological processes important for plant growth. Ethylene production induced by Fe deficiency plays important roles in plant tolerance to stress induced by Fe deficiency. However, the activation and regulatory mechanisms of 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) genes in this response are not clear. In this study, we demonstrated that Fe deficiency increased the abundance of ACS2, ACS6, ACS7, and ACS11 transcripts in both leaves and roots as well as the abundance of ACS8 transcripts in leaves and ACS9 transcripts in roots. Furthermore, we investigated the role of mitogen-activated protein kinase 3 and 6 (MPK3/MPK6)-regulated ACS2/6 activation in Fe deficiency-induced ethylene production. Our results showed that MPK3/MPK6 transcript abundance and MPK3/MPK6 phosphorylation are elevated under conditions of Fe deficiency. Furthermore, mpk3 and mpk6 mutants show a lesser induction of ethylene production under Fe deficiency and a greater sensitivity to Fe deficiency. Finally, in mpk3, mpk6, and acs2 mutants under conditions of Fe deficiency, induction of transcript expression of the Fe-deficiency response genes FRO2, IRT1, and FIT is partially compromised. Taken together, our results suggest that the MPK3/MPK6 and ACS2 are part of the Fe starvation-induced ethylene production signaling pathway.
| S-EPMC4630569 | biostudies-literature
Project description:The lipophilic biopolyester suberin forms important boundaries to protect the plant from its surrounding environment or to separate different tissues within the plant. In roots, suberin can be found in the cell walls of the endodermis and the hypodermis or periderm. Apoplastic barriers composed of suberin accomplish the challenge to restrict water and nutrient loss and prevent the invasion of pathogens. Despite the physiological importance of suberin and the knowledge of the suberin composition of many plants, very little is known about its biosynthesis and the genes involved. Here, a detailed analysis of the Arabidopsis aliphatic suberin in roots at different developmental stages is presented. This study demonstrates some variability in suberin amount and composition along the root axis and indicates the importance of omega-hydroxylation for suberin biosynthesis. Using reverse genetics, the cytochrome P450 fatty acid omega-hydroxylase CYP86A1 (At5g58860) has been identified as a key enzyme for aliphatic root suberin biosynthesis in Arabidopsis. The corresponding horst mutants show a substantial reduction in omega-hydroxyacids with a chain length <C(20), demonstrating that CYP86A1 functions as a hydroxylase of root suberized tissue. Detailed expression studies revealed a strong root specificity and a localized expression in the root endodermis. Transgenic expression of CYP86A1 fused to GFP distributed CYP86A1 to the endoplasmic reticulum, indicating that suberin monomer biosynthesis takes place in this sub-cellular compartment before intermediates are exported in the apoplast.
| S-EPMC2423664 | biostudies-literature
Project description:A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (-)-pinoresinol to (-)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)- and (-)-pinoresinols to (+)- and (-)-lariciresinols efficiently with comparative k(cat)/K(m) values. Analysis of lignans and spatiotemporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol.
| S-EPMC3259658 | biostudies-literature
Project description:Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24-72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava's marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6'-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6'-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6'-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.
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Project description:Many non-graminaceous species release various coumarins in response to iron (Fe) deficiency. However, the physiological relevance of these coumarins remains poorly understood. Here, we show that the three enzymes leading to sideretin biosynthesis co-exist in Arabidopsis (Arabidopsis thaliana) epidermal and cortical cells and that the shift to fraxetin at alkaline pH depends on MYB72-mediated repression of CYTOCHROME P450, FAMILY 82, SUBFAMILY C, POLYPEPTIDE 4 (CYP82C4). In vitro, only fraxetin and sideretin can reduce part of the Fe(III) that they mobilize. We demonstrate that coumarin-mediated Fe(III) reduction is critical under acidic conditions, as fraxetin and sideretin can complement the Fe(III)-chelate reductase mutant ferric reduction oxidase 2 (fro2), and disruption of coumarin biosynthesis in fro2 plants impairs Fe acquisition similar to in the Fe(II) uptake-deficient mutant iron-regulated transporter 1 (irt1). Disruption of sideretin biosynthesis in a fro2 cyp82C4-1 double mutant revealed that sideretin is the dominant chemical reductant that functions with FRO2 to mediate Fe(II) formation for root uptake. At alkaline pH, Fe(III) reduction by coumarins becomes almost negligible but fraxetin still sustains high Fe(III) mobilization, suggesting that its main function is to provide chelated Fe(III) for FRO2. Our study indicates that strategy-I plants link sideretin and fraxetin biosynthesis and secretion to external pH to recruit distinct coumarin chemical activities to maximize Fe acquisition according to prevailing soil pH conditions.
| S-EPMC10896297 | biostudies-literature
Project description:Although cells express hundreds of metalloenzymes, the mechanisms by which apoenzymes receive their metal cofactors are largely unknown. Poly(rC)-binding proteins PCBP1 and PCBP2 are multifunctional adaptor proteins that bind iron and deliver it to ferritin for storage or to prolyl and asparagyl hydroxylases to metallate the mononuclear iron center. Here, we show that PCBP1 and PCBP2 also deliver iron to deoxyhypusine hydroxylase (DOHH), the dinuclear iron enzyme required for hypusine modification of the translation factor eukaryotic initiation factor 5A. Cells depleted of PCBP1 or PCBP2 exhibited loss of DOHH activity and loss of the holo form of the enzyme in cells, particularly when cells were made mildly iron-deficient. Lysates containing PCBP1 and PCBP2 converted apo-DOHH to holo-DOHH in vitro with greater efficiency than lysates lacking PCBP1 or PCBP2. PCBP1 bound to DOHH in iron-treated cells but not in control or iron-deficient cells. Depletion of PCBP1 or PCBP2 had no effect on the cytosolic Fe-S cluster enzyme xanthine oxidase but led to loss of cytosolic aconitase activity. Loss of aconitase activity was not accompanied by gain of RNA-binding activity, a pattern suggesting the incomplete disassembly of the [4Fe-4S] cluster. PCBP depletions had minimal effects on total cellular iron, mitochondrial iron levels, and heme synthesis. Thus, PCBP1 and PCBP2 may serve as iron chaperones to multiple classes of cytosolic nonheme iron enzymes and may have a particular role in restoring metal cofactors that are spontaneously lost in iron deficient cells.
| S-EPMC4050543 | biostudies-literature
Project description:A family of dinuclear iron cluster-containing oxygenases that catalyze ?-hydroxylation tailoring reactions in natural product biosynthesis by nonribosomal peptide synthetase (NRPS) systems was recently described [Makris, T. M., Chakrabarti, M., Münck, E., and Lipscomb, J. D. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 15391-15396]. Here, the 2.17 Å X-ray crystal structure of the archetypal enzyme from the family, CmlA, is reported. CmlA catalyzes ?-hydroxylation of l-p-aminophenylalanine during chloramphenicol biosynthesis. The fold of the N-terminal domain of CmlA is unlike any previously reported, but the C-terminal domain has the ???? fold of the metallo-?-lactamase (MBL) superfamily. The diiron cluster bound in the C-terminal domain is coordinated by an acetate, three His residues, two Asp residues, one Glu residue, and a bridging oxo moiety. One of the Asp ligands forms an unusual monodentate bridge. No other oxygen-activating diiron enzyme utilizes this ligation or the MBL protein fold. The N-terminal domain facilitates dimerization, but using computational docking and a sequence-based structural comparison to homologues, we hypothesize that it likely serves additional roles in NRPS recognition and the regulation of O2 activation.
| S-EPMC3826434 | biostudies-literature