RESUMO
Following a previous microbial inoculation, plants can induce broad-spectrum immunity to pathogen infection, a phenomenon known as systemic acquired resistance (SAR). SAR establishment in Arabidopsis thaliana is regulated by the Lys catabolite pipecolic acid (Pip) and flavin-dependent-monooxygenase1 (FMO1). Here, we show that elevated Pip is sufficient to induce an FMO1-dependent transcriptional reprogramming of leaves that is reminiscent of SAR. In planta and in vitro analyses demonstrate that FMO1 functions as a pipecolate N-hydroxylase, catalyzing the biochemical conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates in plants after microbial attack. When exogenously applied, it overrides the defect of NHP-deficient fmo1 in acquired resistance and acts as a potent inducer of plant immunity to bacterial and oomycete infection. Our work has identified a pathogen-inducible L-Lys catabolic pathway in plants that generates the N-hydroxylated amino acid NHP as a critical regulator of systemic acquired resistance to pathogen infection.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Oxigenases/metabolismo , Ácidos Pipecólicos/metabolismo , Imunidade Vegetal/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Cromatografia Gasosa-Espectrometria de Massas , Lisina/metabolismo , Oomicetos/patogenicidade , Oxigenases/genética , Ácidos Pipecólicos/análise , Ácidos Pipecólicos/farmacologia , Folhas de Planta/enzimologia , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Pseudomonas syringae/patogenicidade , Transaminases/genética , Transaminases/metabolismoRESUMO
Isochorismate-derived metabolism enables biosynthesis of the plant defence hormone salicylic acid (SA) and its derivatives. In Arabidopsis thaliana, the stress-induced accumulation of SA depends on ISOCHORISMATE SYNTHASE1 (ICS1), and also requires the presumed isochorismate transporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) and the GH3 enzyme avrPphB SUSCEPTIBLE3 (PBS3). By comparative metabolite and structural analyses, we identified several hitherto unreported ICS1- and EDS5-dependent, biotic stress-inducible Arabidopsis metabolites. These involve meta-substituted SA derivatives (5-formyl-SA, 5-carboxy-SA, 5-carboxymethyl-SA), their benzoic acid (BA) analogues (3-formyl-BA, 3-carboxy-BA, 3-carboxymethyl-BA) and, besides the previously detected salicyloyl-aspartate (SA-Asp), the ester conjugate salicyloyl-malate (SA-Mal). SA functions as a biosynthetic precursor for SA-Mal and SA-Asp, but not for the meta-substituted SA- and BA-derivatives, which accumulate to moderate levels at later stages of bacterial infection. Interestingly, Arabidopsis leaves possess oxidising activity to effectively convert meta-formyl- into meta-carboxy-SA/BAs. In contrast to SA, exogenously applied meta-substituted SA/BA-derivatives and SA-Mal exert moderate impact on plant immunity and defence-related gene expression. While the isochorismate-derived metabolites are negatively regulated by the SA receptor NON-EXPRESSOR OF PR GENES1, SA conjugates (SA-Mal, SA-Asp, SA-glucose conjugates) and meta-substituted SA/BA-derivatives are oppositely affected by PBS3. Notably, our data indicate a PBS3-independent path to isochorismate-derived SA at later stages of bacterial infection, which does not considerably impact immune-related characteristics. Moreover, our results argue against a previously proposed role of EDS5 in the biosynthesis of the immune signal N-hydroxypipecolic acid and associated transport processes. We propose a significantly extended biochemical scheme of plant isochorismate metabolism that involves an alternative generation mode for benzoate- and salicylate-derivatives.
RESUMO
Tocopherols are lipid-soluble antioxidants synthesized in plastids of plants and other photosynthetic organisms. The four known tocopherols, α-, ß-, γ-, and δ-tocopherol, differ in number and position of methyl groups on their chromanol head group. In unstressed Arabidopsis (Arabidopsis thaliana) leaves, α-tocopherol constitutes the main tocopherol form, whereas seeds predominantly contain γ-tocopherol. Here, we show that inoculation of Arabidopsis leaves with the bacterial pathogen Pseudomonas syringae induces the expression of genes involved in early steps of tocopherol biosynthesis and triggers strong accumulation of γ-tocopherol, moderate production of δ-tocopherol, and generation of the benzoquinol precursors of tocopherols. The pathogen-inducible biosynthesis of tocopherols is promoted by the immune regulators ENHANCED DISEASE SUSCEPTIBILITY1 and PHYTOALEXIN-DEFICIENT4. In addition, tocopherols accumulate in response to bacterial flagellin and reactive oxygen species. By quantifying tocopherol forms in inoculated wild-type plants and biosynthetic pathway mutants, we provide biochemical insights into the pathogen-inducible tocopherol pathway. Notably, vitamin E deficient2 (vte2) mutant plants, which are compromised in both tocopherol and benzoquinol precursor accumulation, exhibit increased susceptibility toward compatible P. syringae and possess heightened levels of markers of lipid peroxidation after bacterial infection. The deficiency of triunsaturated fatty acids in vte2-1 fatty acid desaturase3-2 (fad3-2) fad7-2 fad8 quadruple mutants prevents increased lipid peroxidation in the vte2 background and restores pathogen resistance to wild-type levels. Therefore, the tocopherol biosynthetic pathway positively influences salicylic acid accumulation and guarantees effective basal resistance of Arabidopsis against compatible P. syringae, possibly by protecting leaves from the pathogen-induced oxidation of trienoic fatty acid-containing lipids.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Tocoferóis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Pseudomonas syringae/patogenicidadeRESUMO
The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/µ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction.