RESUMO
Enzymes with fructan exohydrolase (FEH) activity are present not only in fructan-synthesizing species but also in non-fructan plants. This has led to speculation about their functions in non-fructan species. Here, a cell wall invertase-related Zm-6&1-FEH2 with no "classical" invertase motif was identified in maize. Following heterologous expression in Pichia pastoris and in Nicotiana benthamiana leaves, the enzyme activity of recombinant Zm-6&1-FEH2 displays substrate specificity with respect to inulin and levan. Subcellular localization showed Zm-6&1-FEH2 exclusively localized in the apoplast, and its expression profile was strongly dependent on plant development and in response to drought and abscisic acid. Furthermore, formation of 1-kestotriose, an oligofructan, was detected in vivo and in vitro and could be hydrolyzed by Zm-6&1-FEH2. In summary, these results support that Zm-6&1-FEH2 enzyme from maize can degrade both inulin-type and levan-type fructans, and the implications of the co-existence of Zm-6&1-FEH2 and 1-kestotriose are discussed.
Assuntos
Frutanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Inulina/metabolismo , Trissacarídeos/metabolismo , Zea mays/metabolismo , Glicosídeo Hidrolases/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Zea mays/crescimento & desenvolvimentoRESUMO
Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as a renewable resource for energy and bio-based products. Transcriptional regulators of the lignin biosynthetic pathway represent promising targets for tailoring lignin content and composition in plant secondary cell walls. However, knowledge about the transcriptional regulation of lignin biosynthesis in lignocellulosic feedstocks, such as Miscanthus, is limited. In Miscanthus leaves, MsSCM1 and MsMYB103 are expressed at growth stages associated with lignification. The ectopic expression of MsSCM1 and MsMYB103 in N. benthamiana leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin compositions. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 and MsMYB103 overexpression showed an extensive overlap with the response to the NAC master transcription factor MsSND1, but were distinct from each other, underscoring the inherent complexity of secondary cell wall formation. Furthermore, conserved and previously described promoter elements as well as novel and specific motifs could be identified from the target genes of the three transcription factors. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of lignin content and composition in Miscanthus towards a tailored biomass.
Assuntos
Lignina/biossíntese , Proteínas de Plantas/metabolismo , Poaceae/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Biomassa , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Poaceae/genética , Poaceae/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , RNA-Seq/métodos , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
Plant γ-glutamylcysteine ligase (GCL), catalyzing the first and tightly regulated step of glutathione (GSH) biosynthesis, is redox-activated via formation of an intramolecular disulfide bond. In vitro, redox-activation of recombinant GCL protein causes formation of homo-dimers. Here, we have investigated whether dimerization occurs in vivo and if so whether it contributes to redox-activation. FPLC analysis indicated that recombinant redox-activated WT (wild type) AtGCL dissociates into monomers at concentrations below 10-6â M, i.e. below the endogenous AtGCL concentration in plastids, which was estimated to be in the micromolar range. Thus, dimerization of redox-activated GCL is expected to occur in vivo To determine the possible impact of dimerization on redox-activation, AtGCL mutants were generated in which salt bridges or hydrophobic interactions at the dimer interface were interrupted. WT AtGCL and mutant proteins were analyzed by non-reducing SDS-PAGE to address their redox state and probed by FPLC for dimerization status. Furthermore, their substrate kinetics (KM, Vmax) were compared. The results indicate that dimer formation is not required for redox-mediated enzyme activation. Also, crystal structure analysis confirmed that dimer formation does not affect binding of GSH as competitive inhibitor. Whether dimerization affects other enzyme properties, e.g. GCL stability in vivo, remains to be investigated.
Assuntos
Proteínas de Arabidopsis/metabolismo , Glutamato-Cisteína Ligase/química , Glutamato-Cisteína Ligase/metabolismo , Glutationa/biossíntese , Sequência de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Domínio Catalítico , Ativação Enzimática , Glutamato-Cisteína Ligase/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Plantas Geneticamente Modificadas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: European grapevine cultivars (Vitis vinifera spp.) are highly susceptible to the downy mildew pathogen Plasmopara viticola. Breeding of resistant V. vinifera cultivars is a promising strategy to reduce the impact of disease management. Most cultivars that have been bred for resistance to downy mildew, rely on resistance mediated by the Rpv3 (Resistance to P. viticola) locus. However, despite the extensive use of this locus, little is known about the mechanism of Rpv3-mediated resistance. RESULTS: In this study, Rpv3-mediated defense responses were investigated in Rpv3+ and Rpv3- grapevine cultivars following inoculation with two distinct P. viticola isolates avrRpv3+ and avrRpv3-, with the latter being able to overcome Rpv3 resistance. Based on comparative microscopic, metabolomic and transcriptomic analyses, our results show that the Rpv3-1-mediated resistance is associated with a defense mechanism that triggers synthesis of fungi-toxic stilbenes and programmed cell death (PCD), resulting in reduced but not suppressed pathogen growth and development. Functional annotation of the encoded protein sequence of genes significantly upregulated during the Rpv3-1-mediated defense response revealed putative roles in pathogen recognition, signal transduction and defense responses. CONCLUSION: This study used histochemical, transcriptomic and metabolomic analyses of Rpv3+ and susceptible cultivars inoculated with avirulent and virulent P. viticola isolates to investigate mechanism underlying the Rpv3-1-mediated resistance response. We demonstrated a strong correlation between the expressions of stilbene biosynthesis related genes, the accumulation of fungi-toxic stilbenes, pathogen growth inhibition and PCD.
Assuntos
Resistência à Doença/genética , Genes de Plantas/fisiologia , Estilbenos/metabolismo , Vitis/genética , Regulação da Expressão Gênica de Plantas , Metaboloma , Oomicetos/patogenicidade , Doenças das Plantas/microbiologia , Transcrição Gênica , Transcriptoma , Vitis/imunologia , Vitis/microbiologiaRESUMO
BACKGROUND: Understanding lignin biosynthesis and composition is of central importance for sustainable bioenergy and biomaterials production. Species of the genus Miscanthus have emerged as promising bioenergy crop due to their rapid growth and modest nutrient requirements. However, lignin polymerization in Miscanthus is poorly understood. It was previously shown that plant laccases are phenol oxidases that have multiple functions in plant, one of which is the polymerization of monolignols. Herein, we link a newly discovered Miscanthus laccase, MsLAC1, to cell wall lignification. Characterization of recombinant MsLAC1 and Arabidopsis transgenic plants expressing MsLAC1 were carried out to understand the function of MsLAC1 both in vitro and in vivo. RESULTS: Using a comprehensive suite of molecular, biochemical and histochemical analyses, we show that MsLAC1 localizes to cell walls and identify Miscanthus transcription factors capable of regulating MsLAC1 expression. In addition, MsLAC1 complements the Arabidopsis lac4-2 lac17 mutant and recombinant MsLAC1 is able to oxidize monolignol in vitro. Transgenic Arabidopsis plants over-expressing MsLAC1 show higher G-lignin content, although recombinant MsLAC1 seemed to prefer sinapyl alcohol as substrate. CONCLUSIONS: In summary, our results suggest that MsLAC1 is regulated by secondary cell wall MYB transcription factors and is involved in lignification of xylem fibers. This report identifies MsLAC1 as a promising breeding target in Miscanthus for biofuel and biomaterial applications.
Assuntos
Lacase/genética , Lignina/química , Proteínas de Plantas/genética , Poaceae/fisiologia , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/fisiologia , Lacase/metabolismo , Lignina/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Poaceae/química , Poaceae/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Upon continuous stress exposure, plants display attenuated metabolic stress responses due to regulatory feedback loops. Here, we have tested the hypothesis that pulsed stress exposure with intervening recovery periods should affect these feedback loops, thereby causing increased accumulation of stress-induced metabolites. The response of Arabidopsis plantlets to continuous UV-B exposure (Cuv ) was compared with that of pulsed UV-B exposure (Puv ). The differential responses to Puv versus Cuv were monitored at the level of gene expression and metabolite accumulation, using wild type (WT) and different mutant lines. In comparison with Cuv , Puv increased sinapyl and flavonol (S + F) content, whereas adaptive growth attenuation was reduced. Furthermore, in a myb4 mutant (AtMYB4, repressor-type R2R3-MYB transcription factor), the S + F content was increased only for Cuv , but not beyond the level for Puv observed in WT. These observations and the ability of AtMYB4 to repress AtMYB12/AtMYB111-mediated activation of target gene promoters (pCHS and pFLS) indicate that the increase of S + F content after Puv observed in WT plants results from reduced feedback inhibition by AtMYB4. The results support the notion that stress-induced metabolic changes not necessarily cause a growth penalty. Furthermore, the observed Puv -induced increase in flavonol accumulation may stimulate reevaluation of commercial plant production practices.
Assuntos
Arabidopsis/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Relação Dose-Resposta à Radiação , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Regiões Promotoras Genéticas , RNA de Plantas/genética , RNA de Plantas/metabolismo , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Plântula/efeitos da radiação , Estresse Fisiológico/efeitos da radiaçãoRESUMO
About 15% of higher plants have acquired the ability to convert sucrose into fructans. Fructan degradation is catalyzed by fructan exohydrolases (FEHs), which are structurally related to cell wall invertases (CWI). However, the biological function(s) of FEH enzymes in non-fructan species have remained largely enigmatic. In the present study, one maize CWI-related enzyme named Zm-6&1-FEH1, displaying FEH activity, was explored with respect to its substrate specificities, its expression during plant development, and its possible interaction with CWI inhibitor protein. Following heterologous expression in Pichia pastoris and in N. benthamiana leaves, recombinant Zm-6&1-FEH1 revealed substrate specificities of levan and inulin, and also displayed partially invertase activity. Expression of Zm-6&1-FEH1 as monitored by qPCR was strongly dependent on plant development and was further modulated by abiotic stress. To explore whether maize FEH can interact with invertase inhibitor protein, Zm-6&1-FEH1 and maize invertase inhibitor Zm-INVINH1 were co-expressed in N. benthamiana leaves. Bimolecular fluorescence complementation (BiFC) analysis and in vitro enzyme inhibition assays indicated productive complex formation. In summary, the results provide support to the hypothesis that in non-fructan species FEH enzymes may modulate the regulation of CWIs.
Assuntos
Glicosídeo Hidrolases/genética , Folhas de Planta/enzimologia , Zea mays/enzimologia , beta-Frutofuranosidase/genética , Sequência de Aminoácidos , Metabolismo dos Carboidratos/genética , Frutanos/genética , Frutanos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Glicosídeo Hidrolases/química , Folhas de Planta/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Especificidade por Substrato , Nicotiana/enzimologia , Nicotiana/genética , Zea mays/genética , beta-Frutofuranosidase/antagonistas & inibidoresRESUMO
Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.
Assuntos
Secas , Zea mays/metabolismo , Ácido Abscísico/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , beta-Frutofuranosidase/metabolismoRESUMO
In Cichorium intybus, inulin metabolism is mediated by fructan-active enzymes (FAZYs): sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT), and fructan 1-exohydrolases 1, 2a and 2b (1-FEH1, -2a and -2b), respectively. While these enzymes have been rigorously characterized, the transcriptional network orchestrating their development- and stress-related expression has remained largely unknown. Here, the possible role of R2R3-MYB transcription factors in FAZY regulation was explored via bioinformatic identification of R2R3-MYBs (using an RNA sequencing (RNAseq) database), studies of co-expression of these factors with target genes, in vivo transient transactivation assays of FAZY target promoters (dual luciferase assay), and a yeast one-hybrid assay investigating the specificity of the binding of these factors to cis-elements. The chicory MYB transcription factor CiMYB17 specifically activated promoters of 1-SST and 1-FFT by binding to the consensus DNA-motif DTTHGGT. Unexpectedly, CiMYB17 also activated promoters of fructan exohydrolase genes. The stimulatory effect on promoter activities of sucrose transporter and cell wall invertase genes points to a general role in regulating the source-sink relationship. Co-induction of CiMYB17 with 1-SST and 1-FFT (and, less consistently, with 1-FEH1/2) in nitrogen-starved or abscisic acid (ABA)-treated chicory seedlings and in salt-stressed chicory hairy roots supports a role in stress-induced fructan metabolism, including de novo fructan synthesis and trimming of pre-existing fructans, whereas the reduced expression of CiMYB17 in developing taproots excludes a role in fructan accumulation under normal growth conditions.
Assuntos
Cichorium intybus/genética , Frutanos/biossíntese , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/fisiologia , Fatores de Transcrição/fisiologia , Cichorium intybus/metabolismo , Frutanos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In the biennial Cichorium intybus, inulin-type fructans accumulate in the taproot during the first year. Upon cold or drought exposure, fructans are degraded by fructan exohydrolases, affecting inulin yield and degree of polymerization. While stress-induced expression of 1-FEH genes has been thoroughly explored, the transcriptional network mediating these responses has remained unknown. In this study, several R2R3-MYB transcriptional regulators were analysed for their possible involvement in 1-FEH regulation via transient transactivation of 1-FEH target promoters and for in vivo co-expression with target genes under different stress and hormone treatments. CiMYB3 and CiMYB5 selectively enhanced promoter activities of 1-FEH1, 1-FEH2a, and 1-FEH2b genes, without affecting promoter activities of fructosyltransferase genes. Both factors recognized the MYB-core motifs (C/TNGTTA/G) that are abundantly present in 1-FEH promoters. In chicory hairy root cultures, CiMYB5 displayed co-expression with its target genes in response to different abiotic stress and phytohormone treatments, whereas correlations with CiMYB3 expression were less consistent. Oligofructan levels indicated that the metabolic response, while depending on the balance of the relative expression levels of fructan exohydrolases and fructosyltransferases, could be also affected by differential subcellular localization of different FEH isoforms. The results indicate that in chicory hairy root cultures CiMYB5 and CiMYB3 act as positive regulators of the fructan degradation pathway.
Assuntos
Cichorium intybus/genética , Frutanos/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosídeo Hidrolases/metabolismo , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Cichorium intybus/metabolismo , Redes e Vias Metabólicas , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Cell wall invertase (CWI) and vacuolar invertase (VI) play multiple functions in plant growth. As well as depending on transcriptional and post-transcriptional regulation, there is growing evidence that CWI and VI are also subject to post-translational control by small inhibitory proteins. Despite the significance of this, genes encoding inhibitors, their molecular and biochemical properties, and their potential roles in regulating seed production have not been well documented in soybean (Glycine max). In this study, two invertase inhibitor isoforms, GmCIF1 and GmC/VIF2, were characterized to possess inhibitory activities in vitro via heterologous expression. Transcript analyses showed that they were predominantly expressed in developing seeds and in response to ABA. In accordance with this, surveys of primary targets showed subcellular localizations to the apoplast in tobacco epidermis after expressing YFP-fusion constructs. Investigations using RNAi transgenic plants demonstrated marked elevations of CWI activities and improvements in seed weight in conjunction with higher accumulations of hexoses, starch, and protein in mature seeds. Further co-expression analyses of GmCIF1 with several putative CWI genes corroborated the notion that GmCIF1 modulation of CWI that affects seed weight is mainly contingent on post-translational mechanisms. Overall, the results suggest that post-translational elevation of CWI by silencing of GmCIF1 expression orchestrates the process of seed maturation through fine-tuning sucrose metabolism and sink strength.
Assuntos
Regulação da Expressão Gênica de Plantas , Glycine max/fisiologia , Proteínas de Plantas/genética , Sementes/fisiologia , beta-Frutofuranosidase/genética , Sequência de Aminoácidos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Glycine max/genética , beta-Frutofuranosidase/química , beta-Frutofuranosidase/metabolismoRESUMO
The brassinosteroid (BR) signaling module is a central regulator of plant morphogenesis, as indicated by the large number of BR-responsive cell wall-related genes and the severe growth defects of BR mutants. Despite a detailed knowledge of the signaling components, the logic of this auto-/paracrine signaling module in growth control remains poorly understood. Recently, extensive cross-talk with other signaling pathways has been shown, suggesting that the outputs of BR signaling, such as gene-expression changes, are subject to complex control mechanisms. We previously provided evidence for a role of BR signaling in a feedback loop controlling the integrity of the cell wall. Here, we identify the first dedicated component of this feedback loop: a receptor-like protein (RLP44), which is essential for the compensatory triggering of BR signaling upon inhibition of pectin de-methylesterification in the cell wall. RLP44 is required for normal growth and stress responses and connects with the BR signaling pathway, presumably through a direct interaction with the regulatory receptor-like kinase BAK1. These findings corroborate a role for BR in controlling the sensitivity of a feedback signaling module involved in maintaining the physico-chemical homeostasis of the cell wall during cell expansion.
Assuntos
Brassinosteroides/química , Pectinas/química , Proteínas de Plantas/fisiologia , Proteínas de Arabidopsis/fisiologia , Parede Celular/metabolismo , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Homeostase , Ligantes , Microscopia Confocal , Mutação , Fenótipo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de SinaisRESUMO
In higher plants, cell wall invertase (CWI) and vacuolar invertase (VI) are recognized as essential players in sugar metabolism and sugar signaling, thereby affecting source-sink interactions, plant development and responses to environmental cues. CWI and VI expression levels are transcriptionally controlled; however, both enzymes are also subject to posttranslational control by invertase inhibitor proteins. The physiological significances of inhibitor proteins during seed germination and early seedling development are not yet fully understood. Here, we demonstrate that the inhibitor isoform AtCIF1 impacted on seed germination and early seedling growth in Arabidopsis. The primary target of AtCIF1 was shown to be localized to the apoplast after expressing an AtCIF1 YFP-fusion construct in tobacco epidermis and transgenic Arabidopsis root. The analysis of expression patterns showed that AtCWI1 was co-expressed spatiotemporally with AtCIF1 within the early germinating seeds. Seed germination was observed to be accelerated independently of exogenous abscisic acid (ABA) in the AtCIF1 loss-of-function mutant cif1-1. This effect coincided with a drastic increase of CWI activity in cif1-1 mutant seeds by 24 h after the onset of germination, both in vitro and in planta. Accordingly, quantification of sugar content showed that hexose levels were significantly boosted in germinating seeds of the cif1-1 mutant. Further investigation of AtCIF1 overexpressors in Arabidopsis revealed a markedly suppressed CWI activity as well as delayed seed germination. Thus, we conclude that the posttranslational modulation of CWI activity by AtCIF1 helps to orchestrate seed germination and early seedling growth via fine-tuning sucrose hydrolysis and, possibly, sugar signaling.
Assuntos
Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Transdução de Sinais , beta-Frutofuranosidase/antagonistas & inibidores , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Parede Celular/enzimologia , Expressão Gênica , Genes Reporter , Germinação , Mutação , Filogenia , Epiderme Vegetal/enzimologia , Epiderme Vegetal/genética , Epiderme Vegetal/crescimento & desenvolvimento , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Nicotiana/enzimologia , Nicotiana/genética , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismoRESUMO
Plant stilbenes are phytoalexins that accumulate in a small number of plant species, including grapevine (Vitis vinifera), in response to biotic and abiotic stresses and have been implicated in many beneficial effects on human health. In particular, resveratrol, the basic unit of all other complex stilbenes, has received widespread attention because of its cardio-protective, anticarcinogenic, and antioxidant properties. Although stilbene synthases (STSs), the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying stilbene biosynthesis is unknown. Here, we report the identification and functional characterization of two R2R3-MYB-type transcription factors (TFs) from grapevine, which regulate the stilbene biosynthetic pathway. These TFs, designated MYB14 and MYB15, strongly coexpress with STS genes, both in leaf tissues under biotic and abiotic stress and in the skin and seed of healthy developing berries during maturation. In transient gene reporter assays, MYB14 and MYB15 were demonstrated to specifically activate the promoters of STS genes, and the ectopic expression of MYB15 in grapevine hairy roots resulted in increased STS expression and in the accumulation of glycosylated stilbenes in planta. These results demonstrate the involvement of MYB14 and MYB15 in the transcriptional regulation of stilbene biosynthesis in grapevine.
Assuntos
Proteínas de Plantas/metabolismo , Estilbenos/metabolismo , Fatores de Transcrição/metabolismo , Vitis/metabolismo , Aciltransferases/metabolismo , Clonagem Molecular , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Vitis/genéticaRESUMO
Root colonization by the beneficial fungus Piriformospora indica is controlled by plant innate immunity, but factors that channel this interaction into a mutualistic relationship are not known. We have explored the impact of abscisic acid (ABA) and osmotic stress on the P. indica interaction with Arabidopsis thaliana. The activation of plant innate immunity in roots was determined by measuring the concentration of the phytoalexin camalexin and expression of transcription factors regulating the biosynthesis of tryptophan-related defence metabolites. Furthermore, the impact of the fungus on the content of ABA, salicylic acid, jasmonic acid (JA) and JA-related metabolites was examined. We demonstrated that treatment with exogenous ABA or the ABA analogue pyrabactin increased fungal colonization efficiency without impairment of plant fitness. Concomitantly, ABA-deficient mutants of A. thaliana (aba1-6 and aba2-1) were less colonized, while plants exposed to moderate stress were more colonized than corresponding controls. Sustained exposure to ABA attenuated expression of transcription factors MYB51, MYB122 and WRKY33 in roots upon P. indica challenge or chitin treatment, and prevented an increase in camalexin content. The results indicate that ABA can strengthen the interaction with P. indica as a consequence of its impact on plant innate immunity. Consequently, ABA will be relevant for the establishment and outcome of the symbiosis under stress conditions.
Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/microbiologia , Basidiomycota/fisiologia , Raízes de Plantas/microbiologia , Arabidopsis/imunologia , Arabidopsis/metabolismo , Etilenos , Regulação da Expressão Gênica de Plantas , Imunidade Inata , Indóis/metabolismo , Naftalenos , Pressão Osmótica , Raízes de Plantas/imunologia , Raízes de Plantas/metabolismo , Estresse Fisiológico , Sulfonamidas , Simbiose , Tiazóis/metabolismo , Triptofano/metabolismoRESUMO
The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed.
Assuntos
Glutationa/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nicotiana/enzimologia , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Ácido Salicílico/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Reporter , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Oxirredução , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pseudomonas syringae/fisiologia , Nicotiana/genética , Nicotiana/imunologiaRESUMO
A complex of R2R3-MYB and bHLH transcription factors, stabilized by WD40 repeat proteins, regulates gene transcription for plant cell pigmentation and epidermal cell morphology. It is the MYB component of this complex which specifies promoter target activation. The Arabidopsis MYB TT2 regulates proanthocyanidin (PA) biosynthesis by activating the expression of ANR (anthocyanidin reductase), the gene product of which catalyzes the first committed step of this pathway. Conversely the closely related MYB PAP4 (AtMYB114) regulates the anthocyanin pathway and specifically activates UFGT (UDP-glucose:flavonoid-3-O-glucosyltransferase), encoding the first enzyme of the anthocyanin pathway. Both at the level of structural and regulatory genes, evolution of PA biosynthesis proceeded anthocyanin biosynthesis and we have identified key residues in these MYB transcription factors for the evolution of target promoter specificity. Using chimeric and point mutated variants of TT2 and PAP4 we found that exchange of a single amino acid, Gly/Arg(39) in the R2 domain combined with an exchange of a four amino acid motif in the R3 domain, could swap the pathway selection of TT2 and PAP4, thereby converting in planta specificity of the PA towards the anthocyanin pathway and vice versa. The general importance of these amino acids for target specificity was also shown for the grapevine transcription factors VvMYBPA2 and VvMYBA2 which regulate PAs and anthocyanins, respectively. These results provide an insight into the evolution of the different flavonoid regulators from a common ancestral gene.
Assuntos
Antocianinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proantocianidinas/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Selenium is an essential trace element required for seleno-protein synthesis in many eukaryotic cells excluding higher plants. However, a substantial fraction of organically bound selenide in human nutrition is directly or indirectly derived from plants, which assimilate inorganic selenium into organic seleno-compounds. In humans, selenium deficiency is associated with several health disorders Despite its importance for human health, selenium assimilation and metabolism is barely understood in plants. Here, we analyzed the impact of the two dominant forms of soil-available selenium, selenite and selenate, on plant development and selenium partitioning in plants. We found that the reference plant Arabidopsis thaliana discriminated between selenate and selenite application. In contrast to selenite, selenate was predominantly deposited in leaves. This explicit deposition of selenate caused chlorosis and impaired plant morphology, which was not observed upon selenite application. However, only selenate triggered the accumulation of the macronutrient sulfur, the sister element of selenium in the oxygen group. To understand the oxidation state-specific toxicity mechanisms for selenium in plants, we quantified the impact of selenate and selenite on the redox environment in the plastids and the cytosol in a time-resolved manner. Surprisingly, we found that selenite first caused the oxidation of the plastid-localized glutathione pool and had a marginal impact on the redox state of the cytosolic glutathione pool, specifically in roots. In contrast, selenate application caused more vigorous oxidation of the cytosolic glutathione pool but also impaired the plastidic redox environment. In agreement with the predominant deposition in leaves, the selenate-induced oxidation of both glutathione pools was more pronounced in leaves than in roots. Our results demonstrate that Se-species dependent differences in Se partitioning substantially contribute to whole plant Se toxicity and that these Se species have subcellular compartment-specific impacts on the glutathione redox buffer that correlate with toxicity symptoms.
RESUMO
The claim of methane (CH4) formation in plants has caused much controversy and debate within the scientific community over the past 4 years. Here, using both stable isotope and concentration measurements, we demonstrate that CH4 formation occurs in plant cell cultures that were grown in the dark under sterile conditions. Under non-stress conditions the plant cell cultures produced trace amounts [0.3-0.6 ng g⻹ dry weight (DW) h⻹] of CH4 but these could be increased by one to two orders of magnitude (up to 12 ng g⻹ DW h⻹) when sodium azide, a compound known to disrupt electron transport flow at the cytochrome c oxidase (complex IV) in plant mitochondria, was added to the cell cultures. The addition of other electron transport chain (ETC) inhibitors did not result in significant CH4 formation indicating that a site-specific disturbance of the ETC at complex IV causes CH4 formation in plant cells. Our study is an important first step in providing more information on non-microbial CH4 formation from living plants particularly under abiotic stress conditions that might affect the electron transport flow at the cytochrome c oxidase in plant mitochondria.
Assuntos
Beta vulgaris/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metano/biossíntese , Nicotiana/metabolismo , Vitis/metabolismo , Isótopos de Carbono/análise , Técnicas de Cultura de Células , Canamicina/farmacologia , Mitocôndrias/metabolismo , Rotenona/farmacologia , Salicilamidas/farmacologia , Azida Sódica/farmacologiaRESUMO
Selenium (Se)-fortified broccoli (Brassica oleracea var. italica) has been proposed as a functional food for cancer prevention, based on its high glucosinolate (GSL) content and capacity for Se accumulation. However, as selenate and sulphate share the initial assimilation route, Se fertilization could interfere with sulphur metabolism and plant growth. Consequently, GSL accumulation could be compromised. To evaluate these potentially adverse effects of Se fertilization, we performed a comprehensive study on sand-grown young broccoli plants (weekly selenate applications of 0.8 µmol plant(-1) via the root) and field-grown adult broccoli plants during head formation (single foliar selenate application: 25.3 or 253 µmol plant(-1) ). The results show that under these conditions, Se application does not affect plant growth, contents of cysteine, glutathione, total GSL, glucoraphanin (major aliphatic GSL) or the expression of BoMYB28 (encoding a functionally confirmed master regulator for aliphatic GSL biosynthesis). Conversely, due to the changed expression of sulphate transporters (BoSULTR1;1, 1;2, 2;1, and 2;2), sulphate and total S contents increased in the shoot of young plants while decreasing in the root. We conclude that broccoli can be fertilized with Se without reduction in GSL content, even with Se accumulation exceeding the level recommended for human consumption.