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1.
New Phytol ; 225(1): 400-412, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411742

RESUMO

Effective defense of Arabidopsis against filamentous pathogens requires two mechanisms, both of which involve biosynthesis of tryptophan (Trp)-derived metabolites. Extracellular resistance involves products of PEN2-dependent metabolism of indole glucosinolates (IGs). Restriction of further fungal growth requires PAD3-dependent camalexin and other, as yet uncharacterized, indolics. This study focuses on the function of CYP71A12 monooxygenase in pathogen-triggered Trp metabolism, including the biosynthesis of indole-3-carboxylic acid (ICA). Moreover, to investigate the contribution of CYP71A12 and its products to Arabidopsis immunity, we analyzed infection phenotypes of multiple mutant lines combining pen2 with pad3, cyp71A12, cyp71A13 or cyp82C2. Metabolite profiling of cyp71A12 lines revealed a reduction in ICA accumulation. Additionally, analysis of mutant plants showed that low amounts of ICA can form during an immune response by CYP71B6/AAO1-dependent metabolism of indole acetonitrile, but not via IG hydrolysis. Infection assays with Plectosphaerella cucumerina and Colletotrichum tropicale, two pathogens with different lifestyles, revealed cyp71A12-, cyp71A13- and cyp82C2-associated defects associated with Arabidopsis immunity. Our results indicate that CYP71A12, but not CYP71A13, is the major enzyme responsible for the accumulation of ICA in Arabidopsis in response to pathogen ingression. We also show that both enzymes are key players in the resistance of Arabidopsis against selected filamentous pathogens after they invade.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Sistema Enzimático do Citocromo P-450/metabolismo , Imunidade Vegetal , Triptofano/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Ascomicetos/patogenicidade , Resistência à Doença/imunologia , Regulação da Expressão Gênica de Plantas , Glucosinolatos/metabolismo , Hidrólise , Indóis/metabolismo , Mutação/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Triptofano/biossíntese
2.
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
3.
Plant Cell ; 31(11): 2697-2710, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511315

RESUMO

Arabidopsis (Arabidopsis thaliana) efficiently synthesizes the antifungal phytoalexin camalexin without the apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting that the biosynthetic pathway of this compound is channeled by the formation of an enzyme complex. To identify such protein interactions, we used two independent untargeted coimmunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits and determined that the camalexin biosynthetic P450 enzymes copurified with these enzymes. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, the interaction of CYP71A13 and Arabidopsis P450 Reductase1 was observed. We detected increased substrate affinity of CYP79B2 in the presence of CYP71A13, indicating an allosteric interaction. Camalexin biosynthesis involves glutathionylation of the intermediary indole-3-cyanohydrin, which is synthesized by CYP71A12 and especially CYP71A13. FRET-FLIM and co-IP demonstrated that the glutathione transferase GSTU4, which is coexpressed with Trp- and camalexin-specific enzymes, is physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knockout and reduced in GSTU4-overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather plays a role in a competing mechanism.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Vias Biossintéticas/fisiologia , Indóis/metabolismo , Tiazóis/metabolismo , Arabidopsis/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Vias Biossintéticas/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Sesquiterpenos , Tabaco/genética , Tabaco/metabolismo
4.
Phytochemistry ; 161: 11-20, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30798200

RESUMO

Characteristic for cruciferous plants is the synthesis of a complex array of defence-related indolic compounds. In Arabidopsis, these include indol-3-ylmethyl glucosinolates (IMGs), as well as stress-inducible indole-3-carbaldehyde (ICHO)/indole-3-carboxylic acid (ICOOH) derivatives and camalexin. Key enzymes in the biosynthesis of the inducible metabolites are the cytochrome P450 enzymes CYP71A12, CYP71A13 and CYP71B6 and Arabidopsis Aldehyde Oxidase 1 (AAO1). Multiple mutants in the corresponding genes were generated and their metabolic phenotypes were comprehensively analysed in untreated, UV exposed and silver nitrate-treated leaves. Most strikingly, ICOOH and ICHO derivatives synthesized in response to UV exposure were not metabolically related. While ICHO concentrations correlated with IMGs, ICOOH derivatives were anti-correlated with IMGs and partially dependent on CYP71B6. The AAO1 genotype was shown to not only be important for ICHO metabolism but also for the accumulation of 4-pyridoxic acid, suggesting a dual role of AAO1 in vitamin B6 metabolism and IMG degradation in Arabidopsis.


Assuntos
Aldeído Oxidase/genética , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Indóis/metabolismo , Aldeído Oxidase/metabolismo , Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Indóis/análise , Mutação
5.
Plant Physiol ; 178(1): 468-487, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30076223

RESUMO

Nitrogen dioxide (NO2) forms in plants under stress conditions, but little is known about its physiological functions. Here, we explored the physiological functions of NO2 in plant cells using short-term fumigation of Arabidopsis (Arabidopsis thaliana) for 1 h with 10 µL L-1 NO2. Although leaf symptoms were absent, the expression of genes related to pathogen resistance was induced. Fumigated plants developed basal disease resistance, or pattern-triggered immunity, against the necrotrophic fungus Botrytis cinerea and the hemibiotrophic bacterium Pseudomonas syringae Functional salicylic acid and jasmonic acid (JA) signaling pathways were both required for the full expression of NO2-induced resistance against B. cinerea An early peak of salicylic acid accumulation immediately after NO2 exposure was followed by a transient accumulation of oxophytodienoic acid. The simultaneous NO2-induced expression of genes involved in jasmonate biosynthesis and jasmonate catabolism resulted in the complete suppression of JA and JA-isoleucine (JA-Ile) accumulation, which was accompanied by a rise in the levels of their catabolic intermediates 12-OH-JA, 12-OH-JA-Ile, and 12-COOH-JA-Ile. NO2-treated plants emitted the volatile monoterpene α-pinene and the sesquiterpene longifolene (syn. junipene), which could function in signaling or direct defense against pathogens. NO2-triggered B. cinerea resistance was dependent on enhanced early callose deposition and CYTOCHROME P450 79B2 (CYP79B2), CYP79B3, and PHYTOALEXIN DEFICIENT3 gene functions but independent of camalexin, CYP81F2, and 4-OH-indol-3-ylmethylglucosinolate derivatives. In sum, exogenous NO2 triggers basal pathogen resistance, pointing to a possible role for endogenous NO2 in defense signaling. Additionally, this study revealed the involvement of jasmonate catabolism and volatiles in pathogen immunity.


Assuntos
Arabidopsis/genética , Resistência à Doença/efeitos dos fármacos , Resistência à Doença/genética , Dióxido de Nitrogênio/farmacologia , Doenças das Plantas/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Botrytis/fisiologia , Ciclopentanos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Oxidantes Fotoquímicos/farmacologia , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Pseudomonas syringae/fisiologia , Ácido Salicílico/metabolismo , Fatores de Tempo
6.
Plant J ; 89(1): 112-127, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27598402

RESUMO

Glucosinolates (GSL) of cruciferous plants comprise a major group of structurally diverse secondary compounds which act as deterrents against aphids and microbial pathogens and have large commercial and ecological impacts. While the transcriptional regulation governing the biosynthesis and modification of GSL is now relatively well understood, post-translational regulatory components that specifically determine the structural variation of indole glucosinolates have not been reported. We show that the cytoplasmic protein phosphatase 2A regulatory subunit B'γ (PP2A-B'γ) physically interacts with indole glucosinolate methyltransferases and controls the methoxylation of indole glucosinolates and the formation of 4-methoxy-indol-3-yl-methyl glucosinolate in Arabidopsis leaves. By taking advantage of proteomic approaches and metabolic analysis we further demonstrate that PP2A-B'γ is required to control the abundance of oligomeric protein complexes functionally linked with the activated methyl cycle and the trans-methylation capacity of leaf cells. These findings highlight the key regulatory role of PP2A-B'γ in methionine metabolism and provide a previously unrecognized perspective for metabolic engineering of glucosinolate metabolism in cruciferous plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Glucosinolatos/metabolismo , Folhas de Planta/metabolismo , Proteína Fosfatase 2/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Metionina/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Biológicos , Folhas de Planta/genética , Ligação Proteica , Proteína Fosfatase 2/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteômica/métodos , Homologia de Sequência de Aminoácidos
7.
Phytochemistry ; 122: 76-80, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26795461

RESUMO

In Arabidopsis thaliana phytoalexin biosynthesis is tightly regulated. The camalexin biosynthetic gene CYP71B15/PAD3 is highly expressed in response to pathogens and specific abiotic triggers, while constitutive expression is very low. Based on this property we expressed artificial antimicrobial peptides under control of the CYP71B15 promoter avoiding potential toxic effects to the plant related to constitutive expression. Significant and substantial growth inhibition of Pseudomonas syringae was observed, demonstrating that expression of these peptides under control of a phytoalexin promoter is an effective approach for enhancement of resistance against bacterial pathogens.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Indóis , Pseudomonas syringae/efeitos dos fármacos , Tiazóis , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas
8.
Mol Plant ; 9(5): 682-695, 2016 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-26802248

RESUMO

MYB34, MYB51, and MYB122 transcription factors are known as decisive regulators of indolic glucosinolate (IG) biosynthesis with a strong impact on expression of genes encoding CYP79B2 and CYP79B3 enzymes that redundantly convert tryptophan to indole-3-acetaldoxime (IAOx). This intermediate represents a branching point for IG biosynthesis, and pathways leading to camalexin and indole-carboxylic acids (ICA). Here we investigate how these MYBs affect the pathogen-triggered Trp metabolism. Our experiments indicated that these three MYBs affect not only IG production but also constitutive biosynthesis of other IAOx-derived metabolites. Strikingly, the PENETRATION 2 (PEN2)-dependent IG-metabolism products, which are absent in myb34/51/122 and pen2 mutants, were indispensable for full flg22-mediated induction of other IAOx-derived compounds. However, gene induction and accumulation of ICAs and camalexin upon pathogen infection was not compromised in myb34/51/122 plants, despite strongly reduced IG levels. Hence, in comparison with cyp79B2/B3, which lacks all IAOx-derived metabolites, we found myb34/51/122 an ideal tool to analyze IG contribution to resistance against the necrotrophic fungal pathogen Plectosphaerella cucumerina. The susceptibility of myb34/51/122 was similar to that of pen2, but much lower than susceptibility of cyp79B2/B3, indicating that MYB34/51/122 contribute to resistance toward P. cucumerina exclusively through IG biosynthesis, and that PEN2 is the main leaf myrosinase activating IGs in response to microbial pathogens.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Fatores de Transcrição/metabolismo , Triptofano/metabolismo , Ascomicetos/patogenicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Indóis/metabolismo , N-Glicosil Hidrolases/metabolismo , Oximas/metabolismo , Imunidade Vegetal/fisiologia
9.
Front Plant Sci ; 6: 654, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26379682

RESUMO

The phytoalexin camalexin and indolic glucosinolates share not only a common evolutionary origin and a tightly interconnected biosynthetic pathway, but regulatory proteins controlling the shared enzymatic steps are also modulated by the same R2R3-MYB transcription factors. The indolic phytoalexin camalexin is a crucial defense metabolite in the model plant Arabidopsis. Indolic phytoalexins and glucosinolates appear to have a common evolutionary origin and are interconnected on the biosynthetic level: a key intermediate in the biosynthesis of camalexin, indole-3-acetaldoxime (IAOx), is also required for the biosynthesis of indolic glucosinolates and is under tight control by the transcription factors MYB34, MYB51, and MYB122. The abundance of camalexin was strongly reduced in myb34/51 and myb51/122 double and in triple myb mutant, suggesting that these transcription factors are important in camalexin biosynthesis. Furthermore, expression of MYB51 and MYB122 was significantly increased by biotic and abiotic camalexin-inducing agents. Feeding of the triple myb34/51/122 mutant with IAOx or indole-3-acetonitrile largely restored camalexin biosynthesis. Conversely, tryptophan could not complement the low camalexin phenotype of this mutant, which supports a role for the three MYB factors in camalexin biosynthesis upstream of IAOx. Consistently expression of the camalexin biosynthesis genes CYP71B15/PAD3 and CYP71A13 was not negatively affected in the triple myb mutant and the MYBs could not activate pCYP71B15::uidA expression in trans-activation assays with cultured Arabidopsis cells. In conclusion, this study reveals the importance of MYB factors regulating the generation of IAOx as precursor of camalexin.

10.
BMC Plant Biol ; 15: 137, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-26063239

RESUMO

BACKGROUND: Cruciferous plants synthesize a large variety of tryptophan-derived phytoalexins in response to pathogen infection, UV irradiation, or high dosages of heavy metals. The major phytoalexins of Eutrema salsugineum (Thellungiella salsuginea), which has recently been established as an extremophile model plant, are probably derivatives of indole glucosinolates, in contrast to Arabidopsis, which synthesizes characteristic camalexin from the glucosinolate precursor indole-3-acetaldoxime. RESULTS: The transcriptional response of E. salsugineum to UV irradiation and AgNO3 was monitored by RNAseq and microarray analysis. Most transcripts (respectively 70% and 78%) were significantly differentially regulated and a large overlap between the two treatments was observed (54% of total). While core genes of the biosynthesis of aliphatic glucosinolates were repressed, tryptophan and indole glucosinolate biosynthetic genes, as well as defence-related WRKY transcription factors, were consistently upregulated. The putative Eutrema WRKY33 ortholog was functionally tested and shown to complement camalexin deficiency in Atwrky33 mutant. CONCLUSIONS: In E. salsugineum, UV irradiation or heavy metal application resulted in substantial transcriptional reprogramming. Consistently induced genes of indole glucosinolate biosynthesis and modification will serve as candidate genes for the biosynthesis of Eutrema-specific phytoalexins.


Assuntos
Brassicaceae/genética , Reprogramação Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Nitrato de Prata/farmacologia , Transcriptoma/genética , Raios Ultravioleta , Vias Biossintéticas/genética , Brassicaceae/efeitos dos fármacos , Brassicaceae/efeitos da radiação , Reprogramação Celular/efeitos da radiação , Técnicas de Inativação de Genes , Glucosinolatos/biossíntese , Indóis/metabolismo , Metais Pesados/toxicidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sesquiterpenos/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Estresse Fisiológico/efeitos da radiação , Tiazóis/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/efeitos da radiação , Transcriptoma/efeitos dos fármacos , Transcriptoma/efeitos da radiação , Triptofano/biossíntese
11.
New Phytol ; 208(3): 873-86, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26075497

RESUMO

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/metabolismo
12.
Plant Physiol ; 168(3): 849-58, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25953104

RESUMO

In Arabidopsis (Arabidopsis thaliana), a number of defense-related metabolites are synthesized via indole-3-acetonitrile (IAN), including camalexin and indole-3-carboxylic acid (ICOOH) derivatives. Cytochrome P450 71A13 (CYP71A13) is a key enzyme for camalexin biosynthesis and catalyzes the conversion of indole-3-acetaldoxime (IAOx) to IAN. The CYP71A13 gene is located in tandem with its close homolog CYP71A12, also encoding an IAOx dehydratase. However, for CYP71A12, indole-3-carbaldehyde and cyanide were identified as major reaction products. To clarify CYP71A12 function in vivo and to better understand IAN metabolism, we generated two cyp71a12 cyp71a13 double knockout mutant lines. CYP71A12-specific transcription activator-like effector nucleases were introduced into the cyp71a13 background, and very efficient somatic mutagenesis was achieved. We observed stable transmission of the cyp71a12 mutation to the following generations, which is a major challenge for targeted mutagenesis in Arabidopsis. In contrast to cyp71a13 plants, in which camalexin accumulation is partially reduced, double mutants synthesized only traces of camalexin, demonstrating that CYP71A12 contributes to camalexin biosynthesis in leaf tissue. A major role of CYP71A12 was identified for the inducible biosynthesis of ICOOH. Specifically, the ICOOH methyl ester was reduced to 12% of the wild-type level in AgNO3-challenged cyp71a12 leaves. In contrast, indole-3-carbaldehyde derivatives apparently are synthesized via alternative pathways, such as the degradation of indole glucosinolates. Based on these results, we present a model for this surprisingly complex metabolic network with multiple IAN sources and channeling of IAOx-derived IAN into camalexin biosynthesis. In conclusion, transcription activator-like effector nuclease-mediated mutation is a powerful tool for functional analysis of tandem genes in secondary metabolism.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/deficiência , Sistema Enzimático do Citocromo P-450/metabolismo , Desoxirribonucleases/metabolismo , Técnicas de Inativação de Genes , Indóis/metabolismo , Tiazóis/metabolismo , Transativadores/metabolismo , Arabidopsis/enzimologia , Sequência de Bases , Padrões de Herança/genética , Metabolômica , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese/genética , Mutação/genética , Oximas/metabolismo , Metabolismo Secundário
13.
Plant Physiol ; 165(2): 841-853, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24728709

RESUMO

Indolic secondary metabolites play an important role in pathogen defense in cruciferous plants. In Arabidopsis (Arabidopsis thaliana), in addition to the characteristic phytoalexin camalexin, derivatives of indole-3-carbaldehyde (ICHO) and indole-3-carboxylic acid (ICOOH) are synthesized from tryptophan via the intermediates indole-3-acetaldoxime and indole-3-acetonitrile. Based on feeding experiments combined with nontargeted metabolite profiling, their composition in nontreated and silver nitrate (AgNO3)-treated leaf tissue was comprehensively analyzed. As major derivatives, glucose conjugates of 5-hydroxyindole-3-carbaldehyde, ICOOH, and 6-hydroxyindole-3-carboxylic acid were identified. Quantification of ICHO and ICOOH derivative pools after glucosidase treatment revealed that, in response to AgNO3 treatment, their total accumulation level was similar to that of camalexin. ARABIDOPSIS ALDEHYDE OXIDASE1 (AAO1), initially discussed to be involved in the biosynthesis of indole-3-acetic acid, and Cytochrome P450 (CYP) 71B6 were found to be transcriptionally coexpressed with camalexin biosynthetic genes. CYP71B6 was expressed in Saccharomyces cerevisiae and shown to efficiently convert indole-3-acetonitrile into ICHO and ICOOH, thereby releasing cyanide. To evaluate the role of both enzymes in the biosynthesis of ICHO and ICOOH derivatives, knockout and overexpression lines for CYP71B6 and AAO1 were established and analyzed for indolic metabolites. The observed metabolic phenotypes suggest that AAO1 functions in the oxidation of ICHO to ICOOH in both nontreated and AgNO3-treated leaves, whereas CYP71B6 is relevant for ICOOH derivative biosynthesis specifically after induction. In summary, a model for the biosynthesis of ICHO and ICOOH derivatives is presented.

14.
Mol Plant Pathol ; 14(8): 791-802, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23782494

RESUMO

The endoplasmic reticulum (ER)-resident BAX INHIBITOR-1 (BI-1) protein is one of a few cell death suppressors known to be conserved in animals and plants. The function of BI-1 proteins in response to various biotic and abiotic stress factors is well established. However, little is known about the underlying mechanisms. We conducted co-immunoprecipitation (co-IP) experiments to identify Arabidopsis thaliana BI-1-interacting proteins to obtain a potentially better understanding of how BI-1 functions during plant-pathogen interactions and as a suppressor of cell death. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 95 proteins co-immunoprecipitated with green fluorescing protein (GFP)-tagged BI-1. Five selected candidate proteins, a RIBOPHORIN II (RPN2) family protein, VACUOLAR ATP SYNTHASE SUBUNIT A (VHA-A), cytochrome P450 83A1 (CYP83A1), H(+) -ATPASE 1 (AHA1) and PROHIBITIN 2 (PHB2), were further investigated with regard to their role in BI-1-associated processes. To this end, we analysed a set of Arabidopsis mutants in the interaction with the adapted powdery mildew fungus Erysiphe cruciferarum and on cell death-inducing treatments. Two independent rpn2 knock-down mutants tended to better support powdery mildew, and a phb2 mutant showed altered responses to cell death-inducing Alternaria alternata f.sp. lycopersici (AAL) toxin treatment. Two independent cyp83a1 mutants showed a strong powdery mildew resistance phenotype and enhanced sensitivity to AAL toxin. Moreover, co-localization studies and fluorescence resonance energy transfer (FRET) experiments suggested a direct interaction of BI-1 with CYP83A1 at the ER.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/microbiologia , Ascomicetos/fisiologia , Interações Hospedeiro-Patógeno , Imunoprecipitação , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Morte Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem
15.
PLoS One ; 7(11): e48661, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23144921

RESUMO

Little is known about how drought stress influences plant secondary metabolite accumulation and how this affects plant defense against different aphids. We therefore cultivated Arabidopsis thaliana (L.) plants under well-watered, drought, and water-logged conditions. Two aphid species were selected for this study: the generalist Myzus persicae (Sulzer) and the crucifer specialist Brevicoryne brassicae (L.). Metabolite concentrations in the phloem sap, which influence aphid growth, changed particularly under drought stress. Levels of sucrose and several amino acids, such as glutamic acid, proline, isoleucine, and lysine increased, while concentrations of 4-methoxyindol-3-ylmethyl glucosinolate decreased. M. persicae population growth was highest on plants under drought stress conditions. However, B. brassicae did not profit from improved phloem sap quality under drought stress and performed equally in all water treatments. Water stress and aphids generally had an opposite effect on the accumulation of secondary metabolites in the plant rosettes. Drought stress and water-logging led to increased aliphatic glucosinolate and flavonoid levels. Conversely, aphid feeding, especially of M. persicae, reduced levels of flavonoids and glucosinolates in the plants. Correspondingly, transcript levels of aliphatic biosynthetic genes decreased after feeding of both aphid species. Contrary to M. persicae, drought stress did not promote population growth of B. brassicae on these plants. The specialist aphid induced expression of CYP79B2, CYP79B3, and PAD3 with corresponding accumulation of indolyl glucosinolates and camalexin. This was distinct from M. persicae, which did not elicit similarly strong camalexin accumulation, which led to the hypothesis of a specific defense adaptations against the specialist aphid.


Assuntos
Afídeos/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Herbivoria , Estresse Fisiológico , Água/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosinolatos/metabolismo , Indóis/metabolismo , Floema/metabolismo , Tiazóis/metabolismo
16.
Mol Plant Microbe Interact ; 25(9): 1186-97, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22852809

RESUMO

The growth-promoting and root-colonizing endophyte Piriformospora indica induces camalexin and the expression of CYP79B2, CYP79B3, CYP71A13, PAD3, and WRKY33 required for the synthesis of indole-3-acetaldoxime (IAOx)-derived compounds in the roots of Arabidopsis seedlings. Upregulation of the mRNA levels by P. indica requires cytoplasmic calcium elevation and mitogen-activated protein kinase 3 but not root-hair-deficient 2, radical oxygen production, or the 3-phosphoinositide-dependent kinase 1/oxidative signal-inducible 1 pathway. Because P. indica-mediated growth promotion is impaired in cyp79B2 cyp79B3 seedlings, while pad3 seedlings-which do not accumulate camalexin-still respond to the fungus, IAOx-derived compounds other than camalexin (e.g., indole glucosinolates) are required during early phases of the beneficial interaction. The roots of cyp79B2 cyp79B3 seedlings are more colonized than wild-type roots, and upregulation of the defense genes pathogenesis-related (PR)-1, PR-3, PDF1.2, phenylalanine ammonia lyase, and germin indicates that the mutant responds to the lack of IAOx-derived compounds by activating other defense processes. After 6 weeks on soil, defense genes are no longer upregulated in wild-type, cyp79B2 cyp79B3, and pad3 roots. This results in uncontrolled fungal growth in the mutant roots and reduced performance of the mutants. We propose that a long-term harmony between the two symbionts requires restriction of root colonization by IAOx-derived compounds.


Assuntos
Arabidopsis/efeitos dos fármacos , Arabidopsis/microbiologia , Basidiomycota/fisiologia , Indóis/química , Indóis/farmacologia , Oximas/química , Oximas/farmacologia , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/microbiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Microbiologia do Solo , Fatores de Tempo
17.
Nat Prod Rep ; 28(8): 1381-405, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21681321

RESUMO

Phytoalexins are antimicrobial secondary metabolites produced de novo by plants in response to stress, including microbial attack. In general, phytoalexins are important components of plant defenses against fungal and bacterial pathogens. The phytoalexins of crucifers are indole alkaloids derived from (S)-tryptophan, most of which contain a sulfur atom derived from cysteine. Beside their antimicrobial activity against different plant pathogenic species, cruciferous phytoalexins have shown anticarcinogenic effects on various human cell lines. This review focuses on the phytoalexins produced by cruciferous plants reported to date, with particular emphasis on their chemical synthesis, biosynthesis, metabolism by plant fungal pathogens and biological activities. A summary table containing all phytoalexins, their cultivated and wild cruciferous sources, their synthetic starting materials, biotransformation products and biological activities is provided.


Assuntos
Brassicaceae/química , Alcaloides Indólicos/química , Sesquiterpenos , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/metabolismo , Alcaloides Indólicos/farmacologia , Estrutura Molecular
18.
Phytochemistry ; 71(14-15): 1667-72, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20701934

RESUMO

Tryptophan synthase beta-subunits (TSBs) catalyze the last step in tryptophan biosynthesis, i.e. the condensation of indole and serine yielding tryptophan. In microorganisms two subfamilies of TSBs (here designated as type 1 and type 2) are known, which are only distantly related. Surprisingly, in all genomes of multicellular plants analyzed genes encoding both types are present. While type 1 enzymes are well established as components of tryptophan synthase complexes, type 2 enzymes in plants have not yet been characterized. Tissue specific expression of the TSB genes from Arabidopsis thaliana was analyzed. While AtTSB1 is the predominantly expressed isoform in vegetative tissues, AtTSB1 and AtTSBtype2 reach similar transcript levels in seeds. AtTSBtype2 protein was expressed in Escherichia coli and purified. It converted indole and serine to tryptophan with a strikingly low K(m)-value for indole of ca. 74 nM. Attsbtype2 T-DNA insertion mutants showed no obvious deviation from the wild type phenotype, indicating that AtTSBtype2 function is not essential under standard growth conditions. As example for a monocot enzyme, maize TSBtype 2 was analyzed and found to be transcribed in various tissues. ZmTSBtype2 was also catalytically active and here a K(m)-value for indole of ca. 7 microM was determined. These data indicate that TSB type 2 enzymes generally are functionally expressed in plants. Their potential biological role is discussed.


Assuntos
Arabidopsis/enzimologia , Plantas/enzimologia , Triptofano Sintase/metabolismo , Zea mays/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Indóis/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Subunidades Proteicas/metabolismo , RNA/análise , Serina/metabolismo , Triptofano/metabolismo , Triptofano Sintase/classificação , Triptofano Sintase/genética , Zea mays/genética , Zea mays/metabolismo
19.
Plant Cell ; 21(6): 1830-45, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19567706

RESUMO

Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, gamma-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate-treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys(IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Cisteína/metabolismo , Sistema Enzimático do Citocromo P-450/fisiologia , Indóis/metabolismo , Tiazóis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cianetos/química , Cianetos/metabolismo , Cisteína/química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Indóis/química , Espectrometria de Massas , Metabolômica , NADP/metabolismo , Tiazóis/química
20.
Phytochemistry ; 70(15-16): 1638-44, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19523656

RESUMO

Structurally related secondary products are rather rarely shared by organisms from different kingdoms. Consequently, the evolution of biosynthetic pathways of defence metabolites between distantly related organisms has not been broadly investigated. Thiazolylindoles are found in Arabidopsis thaliana, as the phytoalexin camalexin, and in a Streptomyces strain, which synthesizes a tumour-inhibitory derivative, designated BE-10988. Camalexin originates from cysteine and tryptophan, which is converted to indole-3-acetaldoxime and subsequently dehydrated to indole-3-acetonitrile. The metabolic origin of BE-10988 was determined by retrobiosynthetic NMR analysis and incorporation studies with direct precursors. Like camalexin, it is derived from tryptophan and cysteine. However, as BE-10988 is synthesized via indole-3-pyruvic acid, not via indole-3-acetaldoxime, independent mechanisms of tryptophan modification have evolved.


Assuntos
Arabidopsis/química , Evolução Molecular , Indóis/isolamento & purificação , Streptomyces/química , Tiazóis/isolamento & purificação , Triptofano/química , Arabidopsis/genética , Cisteína/metabolismo , Indóis/química , Indóis/metabolismo , Indóis/farmacologia , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oximas/metabolismo , Streptomyces/metabolismo , Tiazóis/química , Tiazóis/farmacologia , Triptofano/genética , Triptofano/metabolismo
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