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1.
Ann Bot ; 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34549262

RESUMO

BACKGROUND AND AIMS: ATP-dependent phosphofructokinases (PFKs) catalyse phosphorylation of the carbon-1 position of fructose-6-phosphate, to form fructose-1,6-bisphosphate. In the cytosol, this is considered a key step in channelling carbon into glycolysis. Arabidopsis thaliana has seven genes encoding PFK isoforms, two chloroplastic and five cytosolic. This study focusses on the four major cytosolic isoforms of PFK in vegetative tissues of A. thaliana. METHODS: We have isolated homozygous knock-out individual mutants (pfk1, pfk3, pfk6, pfk7) and two double mutants (pfk1/7 and pfk3/6) and characterized their growth and metabolic phenotypes. KEY RESULTS: In contrast to single mutants and the double mutant pfk3/6 for the hypoxia-responsive isoforms, the double mutant pfk1/7 had reduced PFK activity and shows a clear visual and metabolic phenotype with reduced shoot growth, early flowering, and elevated hexose levels. This mutant also has an altered ratio of short/long aliphatic glucosinolates and an altered root-shoot distribution. Surprisingly, this mutant does not show any major changes in short-term carbon flux and in levels of hexose-phosphates. CONCLUSIONS: We conclude that the two isoforms PFK1 and PFK7 are important for sugar homeostasis in leaf metabolism and apparently source/sink relations in Arabidopsis, while PFK3 and PFK6 only play a minor role under normal growth conditions.

2.
Int J Mol Sci ; 22(1)2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33466276

RESUMO

Intrinsically disordered proteins and regions with their associated short linear motifs play key roles in transcriptional regulation. The disordered MYC-interaction motif (MIM) mediates interactions between MYC and MYB transcription factors in Arabidopsis thaliana that are critical for constitutive and induced glucosinolate (GLS) biosynthesis. GLSs comprise a class of plant defense compounds that evolved in the ancestor of the Brassicales order. We used a diverse set of search strategies to discover additional occurrences of the MIM in other proteins and in other organisms and evaluate the findings by means of structural predictions, interaction assays, and biophysical experiments. Our search revealed numerous MIM instances spread throughout the angiosperm lineage. Experiments verify that several of the newly discovered MIM-containing proteins interact with MYC TFs. Only hits found within the same transcription factor family and having similar characteristics could be validated, indicating that structural predictions and sequence similarity are good indicators of whether the presence of a MIM mediates interaction. The experimentally validated MIMs are found in organisms outside the Brassicales order, showing that MIM function is broader than regulating GLS biosynthesis.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sequências Hélice-Alça-Hélice/genética , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Glucosinolatos/genética , Proteínas Intrinsicamente Desordenadas/genética , Fatores de Transcrição/genética
3.
New Phytol ; 229(2): 1021-1035, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32901916

RESUMO

Although ammonium (NH4 + ) is a key intermediate of plant nitrogen metabolism, high concentrations of NH4 + in the soil provoke physiological disorders that lead to the development of stress symptoms. Ammonium nutrition was shown to induce the accumulation of glucosinolates (GSLs) in leaves of different Brassicaceae species. To further understand the link between ammonium nutrition and GSLs, we analysed the ammonium stress response of Arabidopsis mutants impaired in GSL metabolic pathway. We showed that the MYB28 and MYB29 double mutant (myb28myb29), which is almost deprived of aliphatic GSLs, is highly hypersensitive to ammonium nutrition. Moreover, we evidenced that the stress symptoms developed were not a consequence of the lack of aliphatic GSLs. Transcriptomic analysis highlighted the induction of an iron (Fe) deficiency response in myb28myb29 under ammonium nutrition. Consistently, ammonium-grown myb28myb29 plants showed altered Fe accumulation and homeostasis. Interestingly, we showed overall that growing Arabidopsis with increased Fe availability relieved ammonium stress symptoms and that this was associated with MYB28 and MYB29 expression. Taken together, our data indicated that the control of Fe homeostasis was crucial for the Arabidopsis response to ammonium nutrition and evidenced that MYB28 and MYB29 play a role in this control.


Assuntos
Compostos de Amônio , Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosinolatos , Histona Acetiltransferases/metabolismo , Homeostase , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
J Chem Ecol ; 47(1): 112-122, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33180275

RESUMO

Plants evolved in close contact with a myriad of microorganisms, some of which formed associations with their roots, benefitting from carbohydrates and other plant resources. In exchange, they evolved to influence important plant functions, e.g. defense against insect herbivores and other antagonists. Here, we test whether a fungus, Metarhizium brunneum, which is mostly known as an insect pathogen, can also associate with plant roots and contribute to above-ground plant defense. Cauliflower (Brassica oleracea var. botrytis) seeds were sown together with M. brunneum-inoculated rice grains, and the resulting plants subjected to leaf herbivory by the specialist Plutella xylostella. Activity of myrosinases, the enzymes activating glucosinolates, was measured before and after herbivory; larval consumption and plant weight at the end of experiments. Metarhizium brunneum clearly established in the plant roots, and after herbivory myrosinase activity was substantially higher in M. brunneum-treated plants than in controls; before herbivory, M. brunneum-treated and control plants did not differ. Leaf consumption was slightly lower in the M. brunneum-treated plants whereas total biomass and allocation to above- or below-ground parts was not affected by the Metarhizium treatment. Thus, M. brunneum associates with roots and primes the plant for a stronger or faster increase in myrosinase activity upon herbivory. Consistent with this, myrosinase function has been suggested to be rate-limiting for induction of the glucosinolate-myrosinase defense system. Our results show that M. brunneum, in addition to being an insect pathogen, can associate with plant roots and prime plant defense.


Assuntos
Brassica/enzimologia , Glicosídeo Hidrolases/metabolismo , Metarhizium/fisiologia , Mariposas/fisiologia , Defesa das Plantas contra Herbivoria , Raízes de Plantas/enzimologia , Animais , Brassica/crescimento & desenvolvimento , Brassica/microbiologia , Herbivoria , Larva/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia
5.
Plant Cell Environ ; 44(2): 548-558, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33131061

RESUMO

In Arabidopsis, two leaf-type ferredoxin-NADP+ oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP+ , while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using ß-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Ferredoxina-NADP Redutase/metabolismo , Oxirredutases/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Temperatura Baixa , Transporte de Elétrons , Ferredoxina-NADP Redutase/genética , Oxirredutases/metabolismo , Fotossíntese , Folhas de Planta/enzimologia , Folhas de Planta/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Plastídeos/enzimologia , Isoformas de Proteínas , Estresse Fisiológico
6.
Proc Natl Acad Sci U S A ; 117(42): 26197-26205, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33033229

RESUMO

MicroProteins are small, often single-domain proteins that are sequence-related to larger, often multidomain proteins. Here, we used a combination of comparative genomics and heterologous synthetic misexpression to isolate functional cereal microProtein regulators. Our approach identified LITTLE NINJA (LNJ), a microProtein that acts as a modulator of jasmonic acid (JA) signaling. Ectopic expression of LNJ in Arabidopsis resulted in stunted plants that resembled the decuple JAZ (jazD) mutant. In fact, comparing the transcriptomes of transgenic LNJ overexpressor plants and jazD revealed a large overlap of deregulated genes, suggesting that ectopic LNJ expression altered JA signaling. Transgenic Brachypodium plants with elevated LNJ expression levels showed deregulation of JA signaling as well and displayed reduced growth and enhanced production of side shoots (tiller). This tillering effect was transferable between grass species, and overexpression of LNJ in barley and rice caused similar traits. We used a clustered regularly interspaced short palindromic repeats (CRISPR) approach and created a LNJ-like protein in Arabidopsis by deleting parts of the coding sentence of the AFP2 gene that encodes a NINJA-domain protein. These afp2-crispr mutants were also stunted in size and resembled jazD Thus, similar genome-engineering approaches can be exploited as a future tool to create LNJ proteins and produce cereals with altered architectures.


Assuntos
Arabidopsis/metabolismo , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas , Hordeum/metabolismo , Oryza/metabolismo , Oxilipinas/farmacologia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Perfilação da Expressão Gênica , Hordeum/efeitos dos fármacos , Hordeum/genética , Oryza/efeitos dos fármacos , Oryza/genética , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Isoformas de Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais
7.
Plant Physiol ; 183(3): 1376-1390, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32321840

RESUMO

Glucosinolates (GSLs) are sulfur-containing defense metabolites produced in the Brassicales, including the model plant Arabidopsis (Arabidopsis thaliana). Previous work suggests that specific GSLs may function as signals to provide direct feedback regulation within the plant to calibrate defense and growth. These GSLs include allyl-GSL, a defense metabolite that is one of the most widespread GSLs in Brassicaceae and has also been associated with growth inhibition. Here we show that at least three separate potential catabolic products of allyl-GSL or closely related compounds affect growth and development by altering different mechanisms influencing plant development. Two of the catabolites, raphanusamic acid and 3-butenoic acid, differentially affect processes downstream of the auxin signaling cascade. Another catabolite, acrylic acid, affects meristem development by influencing the progression of the cell cycle. These independent signaling events propagated by the different catabolites enable the plant to execute a specific response that is optimal to any given environment.


Assuntos
Glucosinolatos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Acrilatos/farmacologia , Glucosinolatos/química , Glucosinolatos/farmacologia , Ácidos Indolacéticos/farmacologia , Meristema/efeitos dos fármacos , Meristema/crescimento & desenvolvimento , Modelos Biológicos , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Tiazóis/análise , Tionas/análise
8.
FEMS Microbiol Ecol ; 96(5)2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32239208

RESUMO

Little is known about the influence of host genotype and phytohormones on the composition of fungal endophytic communities. We investigated the influence of host genotype and phytohormones on the structure of the fungal endophytic communities of tomato roots by amplicon sequencing of the ITS1 region and combined this approach with isolation and functional characterization of the isolates. A significant effect of the host genotype on the dominant fungal species was found by comparing the cultivars Castlemart and UC82B and, surprisingly, root pathogens were among the most abundant taxa. In contrast, smaller changes in the relative abundance of the dominant species were found in mutants impaired in jasmonic acid biosynthesis (def1) and ethylene biosynthesis (8338) compared to the respective wild types. However, def1 showed significantly higher species richness compared to the wild type. Analysis of the phytohormone profiles of these genotypes indicates that changes in the phytohormone balance may contribute to this difference in species richness. Assessing the lifestyle of isolated fungi on tomato seedlings revealed the presence of both beneficial endophytes and latent pathogens in roots of asymptomatic plants, suggesting that the interactions between members of the microbiome maintain the equilibrium in the community preventing pathogens from causing disease.


Assuntos
Endófitos , Lycopersicon esculentum , Endófitos/genética , Fungos , Estilo de Vida , Reguladores de Crescimento de Plantas , Raízes de Plantas
9.
Metab Eng ; 57: 129-139, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31722246

RESUMO

In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, these organisms do not directly compete with agricultural resources. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. Amino acids are traditionally produced by extraction from protein hydrolysates, chemical synthesis or fermentation pathways using heterotrophic microorganisms. In this work we demonstrate for the first time the efficient overproduction of phenylalanine and tyrosine from CO2 in a Synechocystis sp. PCC 6803 strain heterologously expressing the feedback-inhibition-resistant AroG and TyrA enzymes from E. coli. Production titers reached 904 ±â€¯53 mg/gDW (580 ±â€¯34 mg/L) of phenylalanine and 64 ±â€¯3.7 mg/gDW (41 ±â€¯2.3 mg/L) of tyrosine after 10 days of photoautotrophic growth. We estimate that the production of the two amino acids corresponds to 56% of the total fixed carbon. Phenylalanine and tyrosine are the precursors for phenylpropanoids, thus, we tested the functionality of several phenylpropanoid biosynthetic enzymes in the generated cyanobacterium strains and successfully achieved the production of 470 ±â€¯70 mg/gDW (207 mg/L) of p-coumaric acid, 267 ±â€¯31 mg/gDW (114 mg/L) of cinnamic acid and 47.4 ±â€¯13.9 mg/gDW (12.6 mg/L) of caffeic acid after 6 days of photoautotrophic growth. All compounds were secreted to the growth medium. Our work enlarges the repertoire and yield of heterologous chemicals produced by Synechocystis and contributes to extend the molecular knowledge about this cyanobacterium.


Assuntos
Engenharia Metabólica , Fenilalanina , Fenilpropionatos/metabolismo , Synechocystis , Tirosina , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Fenilalanina/biossíntese , Fenilalanina/genética , Synechocystis/genética , Synechocystis/crescimento & desenvolvimento , Tirosina/biossíntese , Tirosina/genética
10.
Phytochemistry ; 169: 112100, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31771793

RESUMO

The glucosinolates (GSLs) is a well-defined group of plant metabolites characterized by having an S-ß-d-glucopyrano unit anomerically connected to an O-sulfated (Z)-thiohydroximate function. After enzymatic hydrolysis, the sulfated aglucone can undergo rearrangement to an isothiocyanate, or form a nitrile or other products. The number of GSLs known from plants, satisfactorily characterized by modern spectroscopic methods (NMR and MS) by mid-2018, is 88. In addition, a group of partially characterized structures with highly variable evidence counts for approximately a further 49. This means that the total number of characterized GSLs from plants is somewhere between 88 and 137. The diversity of GSLs in plants is critically reviewed here, resulting in significant discrepancies with previous reviews. In general, the well-characterized GSLs show resemblance to C-skeletons of the amino acids Ala, Val, Leu, Trp, Ile, Phe/Tyr and Met, or to homologs of Ile, Phe/Tyr or Met. Insufficiently characterized, still hypothetic GSLs include straight-chain alkyl GSLs and chain-elongated GSLs derived from Leu. Additional reports (since 2011) of insufficiently characterized GSLs are reviewed. Usually the crucial missing information is correctly interpreted NMR, which is the most effective tool for GSL identification. Hence, modern use of NMR for GSL identification is also reviewed and exemplified. Apart from isolation, GSLs may be obtained by organic synthesis, allowing isotopically labeled GSLs and any kind of side chain. Enzymatic turnover of GSLs in plants depends on a considerable number of enzymes and other protein factors and furthermore depends on GSL structure. Identification of GSLs must be presented transparently and live up to standard requirements in natural product chemistry. Unfortunately, many recent reports fail in these respects, including reports based on chromatography hyphenated to MS. In particular, the possibility of isomers and isobaric structures is frequently ignored. Recent reports are re-evaluated and interpreted as evidence of the existence of "isoGSLs", i.e. non-GSL isomers of GSLs in plants. For GSL analysis, also with MS-detection, we stress the importance of using authentic standards.


Assuntos
Glucosinolatos , Plantas/metabolismo , Glucosinolatos/síntese química , Glucosinolatos/química , Glucosinolatos/metabolismo , Estrutura Molecular , Plantas/química
11.
Protein Sci ; 29(1): 169-183, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31642121

RESUMO

Protein domains constitute regions of distinct structural properties and molecular functions that are retained when removed from the rest of the protein. However, due to the lack of tertiary structure, the identification of domains has been largely neglected for long (>50 residues) intrinsically disordered regions. Here we present a sequence-based approach to assess and visualize domain organization in long intrinsically disordered regions based on compositional sequence biases. An online tool to find putative intrinsically disordered domains (IDDomainSpotter) in any protein sequence or sequence alignment using any particular sequence trait is available at http://www.bio.ku.dk/sbinlab/IDDomainSpotter. Using this tool, we have identified a putative domain enriched in hydrophilic and disorder-promoting residues (Pro, Ser, and Thr) and depleted in positive charges (Arg and Lys) bordering the folded DNA-binding domains of several transcription factors (p53, GCR, NAC46, MYB28, and MYB29). This domain, from two different MYB transcription factors, was characterized biophysically to determine its properties. Our analyses show the domain to be extended, dynamic and highly disordered. It connects the DNA-binding domain to other disordered domains and is present and conserved in several transcription factors from different families and domains of life. This example illustrates the potential of IDDomainSpotter to predict, from sequence alone, putative domains of functional interest in otherwise uncharacterized disordered proteins.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/química , Arabidopsis/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Viés , Sítios de Ligação , Histona Acetiltransferases , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Desdobramento de Proteína , Espalhamento a Baixo Ângulo , Fatores de Transcrição/metabolismo , Difração de Raios X
12.
J Plant Physiol ; 244: 153088, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31812906

RESUMO

Zinc (Zn) is one of the important elements of plant growth, however, at elevated level it is toxic. Exposure of Chinese cabbage to elevated Zn2+ concentrations (5 and 10 µM ZnCl2) resulted in enhancement of total sulfur and organic sulfur concentration. Transcript level of APS reductase (APR) as a key enzyme in biosynthesis of primary sulfur compounds (cysteine and thiols), was up-regulated in both shoot and root upon exposure to elevated Zn2+, which was accompanied by an increase in the concentration of cysteine in both tissues. In contrast, the concentration of thiols increased only in the root by 5.5 and 15-fold at 5 and 10 µM Zn2+, respectively, which was in accompanied by an upregulation of ATP sulfurylase, an enzyme responsible for activation of sulfate. An elevated content of glucosinolates, mostly indolic glucosinolates, only in the shoot of plants exposed to excess level of Zn2+ coincided with an increase in gene expression of key biosynthetic enzymes and regulators (CYP79B3, CYP83B1, MYB34). Thus distinct acuumulation patterns of sulfur containing compounds in root and shoot of Chinese cabbage may be a strategy for Chinese cabbage to combat with exposure to excess Zn.


Assuntos
Brassica/metabolismo , Glucosinolatos/metabolismo , Proteínas de Plantas/metabolismo , Compostos de Sulfidrila/metabolismo , Zinco/administração & dosagem , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Regulação para Cima
13.
Plant Physiol ; 182(2): 1161-1181, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31659127

RESUMO

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'γ is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'γ regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'γ to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'γ is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'γ depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'γ age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Botrytis/imunologia , Senescência Celular/genética , Resistência à Doença/genética , Doenças das Plantas/imunologia , Folhas de Planta/metabolismo , Proteína Fosfatase 2/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Senescência Celular/fisiologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Resistência à Doença/imunologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/genética , Genótipo , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Mutação , Fenótipo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/genética , Ácido Salicílico/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genética
14.
Front Plant Sci ; 10: 1560, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31867028

RESUMO

Dynamically changing environmental conditions promote a complex regulation of plant metabolism and balanced resource investments to development and defense. Plants of the Brassicales order constitutively allocate carbon, nitrogen, and sulfur to synthesize glucosinolates as their primary defense metabolites. Previous findings support a model in which steady-state levels of glucosinolates in intact tissues are determined by biosynthesis and turnover through a yet uncharacterized turnover pathway. To investigate glucosinolate turnover in the absence of tissue damage, we quantified exogenously applied allyl glucosinolate and endogenous glucosinolates under different nutrient conditions. Our data shows that, in seedlings of Arabidopsis thaliana accession Columbia-0, glucosinolate biosynthesis and turnover are coordinated according to nutrient availability. Whereas exogenous carbon sources had general quantitative effects on glucosinolate accumulation, sulfur or nitrogen limitation resulted in distinct changes in glucosinolate profiles, indicating that these macronutrients provide different regulatory inputs. Raphanusamic acid, a breakdown product that can potentially be formed from all glucosinolate structures appears not to reflect in planta turnover rates, but instead correlates with increased accumulation of endogenous glucosinolates. Thus, raphanusamic acid could represent a metabolic checkpoint that allows glucosinolate-producing plants to measure the flux through the biosynthetic and/or turnover pathways and thereby to dynamically adjust glucosinolate accumulation in response to internal and external signals.

15.
Nucleic Acids Res ; 47(18): 9592-9608, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400117

RESUMO

Physical interactions between members of the MYB and bHLH transcription factor (TF) families regulate many important biological processes in plants. Not all reported MYB-bHLH interactions can be explained by the known binding sites in the R3 repeat of the MYB DNA-binding domain. Noteworthy, most of the sequence diversity of MYB TFs lies in their non-MYB regions, which contain orphan small subgroup-defining motifs not yet linked to molecular functions. Here, we identified the motif mediating interaction between MYB TFs from subgroup 12 and their bHLH partners. Unlike other known MYB-bHLH interactions, the motif locates to the centre of the predicted disordered non-MYB region. We characterised the core motif, which enabled accurate prediction of previously unknown bHLH-interacting MYB TFs in Arabidopsis thaliana, and we confirmed its functional importance in planta. Our results indicate a correlation between the MYB-bHLH interaction affinity and the phenotypic output controlled by the TF complex. The identification of an interaction motif outside R3 indicates that MYB-bHLH interactions must have arisen multiple times, independently and suggests many more motifs of functional relevance to be harvested from subgroup-specific studies.


Assuntos
Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Filogenia , Fatores de Transcrição/genética , Sequência de Aminoácidos/genética , Arabidopsis/genética , Proteínas de Arabidopsis/classificação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/classificação , Regulação da Expressão Gênica de Plantas/genética , Plantas Geneticamente Modificadas/genética , Fatores de Transcrição/classificação
16.
Trends Plant Sci ; 24(10): 934-946, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31358471

RESUMO

Several transcription factor (TF) families, including the MYB family, regulate a wide array of biological processes. TFs contain DNA-binding domains (DBDs) and regulatory regions; although information on protein structure is scarce for plant MYB TFs, various in silico methods suggest that the non-MYB regions contain extensive intrinsically disordered regions (IDRs). Although IDRs do not fold into stable globular structures, they comprise functional regions including interaction motifs, and recent research has shown that IDRs perform crucial biological roles. We map here domain organization, disorder predictions, and functional regions across the entire Arabidopsis thaliana R2R3 MYB TF family, and highlight where an increased research focus will be necessary to shape a new understanding of structure-function relationships in plant TFs.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Sequência de Aminoácidos , DNA , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas , Fatores de Transcrição
17.
Metab Eng ; 55: 33-43, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31091467

RESUMO

Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products.


Assuntos
Cloroplastos , Sistema Enzimático do Citocromo P-450 , Engenharia Metabólica , Fotossíntese/genética , Proteínas de Plantas , Tabaco , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cloroplastos/enzimologia , Cloroplastos/genética , Cianobactérias/genética , Cianobactérias/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Transporte de Elétrons/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tabaco/enzimologia , Tabaco/genética
18.
Plant Physiol ; 179(4): 1298-1314, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30765478

RESUMO

Plant immunity depends on fast and specific transcriptional reprogramming triggered by the perception of biotic stresses. Numerous studies have been conducted to better understand the response of plants to the generalist herbivore two-spotted spider mite (Tetranychus urticae). However, how plants perceive mites and how this perception is translated into changes in gene expression are largely unknown. In this work, we identified a gene induced in Arabidopsis (Arabidopsis thaliana) upon spider mite attack that encodes a two-domain protein containing predicted lectin and Toll/Interleukin-1 receptor domains. The gene, previously named PP2-A5, belongs to the Phloem Protein2 family. Biotic assays showed that PP2-A5 confers tolerance to T. urticae Overexpression or knockout of PP2-A5 leads to transcriptional reprogramming that alters the balance of hormone accumulation and corresponding signaling pathways. The nucleocytoplasmic location of this protein supports a direct interaction with regulators of gene transcription, suggesting that the combination of two putative signaling domains in a single protein may provide a novel mechanism for regulating gene expression. Together, our results suggest that PP2-A5 improves the ability to defend against T. urticae by participating in the tight regulation of hormonal cross talk upon mite feeding. Further research is needed to determine the mechanism by which this two-domain protein functions and to clarify its molecular role in signaling following a spider mite attack.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Tetranychidae/fisiologia , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Feminino , Glucosinolatos/metabolismo , Herbivoria , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo
19.
Front Plant Sci ; 9: 550, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29755493

RESUMO

Plants release chemicals to deter attackers. Arabidopsis thaliana relies on multiple defense compounds, including indol-3-ylmethyl glucosinolate (I3G), which upon hydrolysis initiated by myrosinase enzymes releases a multitude of bioactive compounds, among others, indole-3-acetonitrile and indole-3-acetoisothiocyanate. The highly unstable isothiocyanate rapidly reacts with other molecules. One of the products, indole-3-carbinol, was reported to inhibit auxin signaling through binding to the TIR1 auxin receptor. On the contrary, the nitrile product of I3G hydrolysis can be converted by nitrilase enzymes to form the primary auxin molecule, indole-3-acetic acid, which activates TIR1. This suggests that auxin signaling is subject to both antagonistic and protagonistic effects of I3G hydrolysis upon attack. We hypothesize that I3G hydrolysis and auxin signaling form an incoherent feedforward loop and we build a mathematical model to examine the regulatory network dynamics. We use molecular docking to investigate the possible antagonistic properties of different I3G hydrolysis products by competitive binding to the TIR1 receptor. Our simulations reveal an uncoupling of auxin concentration and signaling, and we determine that enzyme activity and antagonist binding affinity are key parameters for this uncoupling. The molecular docking predicts that several I3G hydrolysis products strongly antagonize auxin signaling. By comparing a tissue disrupting attack - e.g., by chewing insects or necrotrophic pathogens that causes rapid release of I3G hydrolysis products - to sustained cell-autonomous I3G hydrolysis, e.g., upon infection by biotrophic pathogens, we find that each scenario gives rise to distinct auxin signaling dynamics. This suggests that plants have different defense versus growth strategies depending on the nature of the attack.

20.
G3 (Bethesda) ; 8(5): 1733-1746, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29563187

RESUMO

The theoretical ability of epigenetic variation to influence the heritable variation of complex traits is gaining traction in the study of adaptation. This theory posits that epigenetic marks can control adaptive phenotypes but the relative potential of epigenetic variation in comparison to genetic variation in these traits is not presently understood. To compare the potential of epigenetic and genetic variation in adaptive traits, we analyzed the influence of DNA methylation variation on the accumulation of chemical defense compounds glucosinolates from the order Brassicales. Several decades of work on glucosinolates has generated extensive knowledge about their synthesis, regulation, genetic variation and contribution to fitness establishing this pathway as a model pathway for complex adaptive traits. Using high-throughput phenotyping with a randomized block design of ddm1 derived Arabidopsis thaliana epigenetic Recombinant Inbred Lines, we measured the correlation between DNA methylation variation and mean glucosinolate variation and within line stochastic variation. Using this information, we identified epigenetic Quantitative Trait Loci that contained specific Differentially Methylated Regions associated with glucosinolate traits. This showed that variation in DNA methylation correlates both with levels and variance of glucosinolates and flowering time with trait-specific loci. By conducting a meta-analysis comparing the results to different genetically variable populations, we conclude that the influence of DNA methylation variation on these adaptive traits is much lower than the corresponding impact of standing genetic variation. As such, selective pressure on these traits should mainly affect standing genetic variation to lead to adaptation.


Assuntos
Arabidopsis/genética , Epigênese Genética , Variação Genética , Característica Quantitativa Herdável , Mapeamento Cromossômico , Metilação de DNA/genética , Flores/fisiologia , Genes de Plantas , Genética Populacional , Glucosinolatos/metabolismo , Endogamia , Padrões de Herança/genética , Fenótipo , Locos de Características Quantitativas/genética , Fatores de Tempo
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