Plants modify harmful substances through an inducible detoxification system. In Arabidopsis (Arabidopsis thaliana), chemical induction of the cytochrome P450 gene CYP81D11 and other genes linked to ...the detoxification program depends on class II TGA transcription factors. CYP81D11 expression is also induced by the phytohormone jasmonic acid (JA) through the established pathway requiring the JA receptor CORONATINE INSENSITIVE1 (COI1) and the JA-regulated transcription factor MYC2. Here, we report that the xenobiotic-and the JA-dependent signal cascades have become interdependent at the CYP81D11 promoter. On the one hand, MYC2 can only activate the expression of CYP81D11 when both the MYC2-and the TGA binding sites are present in the promoter. On the other hand, the xenobiotic-regulated class II TGA transcription factors can only mediate maximal promoter activity if TGA and MYC2 binding motifs, MYC2, and the JA-isoleucine biosynthesis enzymes DDE2/AOS and JAR1 are functional. Since JA levels and degradation of JAZ1, a repressor of the JA response, are not affected by reactive chemicals, we hypothesize that basal JA signaling amplifies the response to chemical stress. Remarkably, stress-induced expression levels were 3-fold lower in coil than in the JA biosynthesis mutant dede2-2, revealing that COI1 can contribute to the activation of the promoter in the absence of JA. Moreover, we show that deletion of the MYC2 binding motifs abolishes the JA responsiveness of the promoter but not the responsiveness to COI1. These findings suggest that yet unknown cis-element(s) can mediate COI1-dependent transcriptional activation in the absence of JA.
Glutaredoxins (GRXs) are small proteins which bind glutathione to either reduce disulfide bonds or to coordinate iron sulfur clusters. Whereas these well-established functions are associated with ...ubiquitously occurring GRXs that encode variants of a CPYC or a CGFS motif in the active center, land plants also possess CCxC/S-type GRXs (named ROXYs) for which the biochemical functions are yet unknown. ROXYs physically and genetically interact with bZIP transcription factors of the TGA family. In Arabidopsis, ectopically expressed ROXY19 (originally named GRX480 or GRXC9) negatively regulates expression of jasmonic acid/ethylene-induced defense genes through an unknown mechanism that requires at least one of the redundant transcription factors TGA2, TGA5 or TGA6.
Ectopically expressed ROXY19 interferes with the activation of TGA-dependent detoxification genes. Similar to the tga2 tga5 tga6 mutant, 35S:ROXY19 plants are more susceptible to the harmful chemical TIBA (2,3,5-triiodobenzoic acid). The repressive function of ROXY19 depends on the integrity of the active site, which can be either CCMC or CPYC but not SSMS. Ectopic expression of the related GRX ROXY18/GRXS13 also led to increased susceptibility to TIBA, indicating potential functional redundancy of members of the ROXY gene family. This redundancy might explain why roxy19 knock-out plants did not show a phenotype with respect to the regulation of the TIBA-induced detoxification program. Complementation of the tga2 tga5 tga6 mutant with either TGA5 or TGA5C186S, in which the single potential target-site of ROXY19 had been eliminated, did not reveal any evidence for a critical redox modification that might be important for controlling the detoxification program.
ROXY19 and related proteins of the ROXY gene family can function as negative regulators of TGA-dependent promoters controlling detoxification genes.
For understanding the mechanism of transcriptional regulation, it is essential to know which transcription factor is bound in vivo to the promoter to be analysed. If transcription from a given ...promoter is regulated by developmental or environmental stimuli, the question of inducible versus constitutive binding has to be answered, particularly if the transcriptional regulator is expressed both under uninduced and induced conditions. Chromatin immunoprecipitation (ChIP) assays constitute the most adequate approach to address these issues as proteins are cross-linked to the DNA before disruption of the tissue. Thus, the DNA-protein interaction is stabilized during purification of the chromatin. The specific DNA-protein complex is immuno-enriched employing specific antibodies against the transcription factor to be analysed. After reversal of cross-links, the recovered DNA is amplified by PCR using specific primers that match sequences flanking the suspected binding site. The amount of PCR product is indicative of the relative abundance of the DNA-protein complex in vivo. A protocol for ChIP assays for Arabidopsis thaliana leaves is described.
In transgenic tobacco, pea Ferredoxin-1 (Fed-1) mRNA accumulates rapidly in response to photosynthesis even when the transgene is driven by a constitutive promoter. To investigate the role of ...photosynthesis on Fed-1 mRNA stability, we used the tetracycline repressible Top10 promoter system to specifically shut off transcription of the Fed-1 transgene. The Fed-1 mRNA has a half-life of approximately 2.4 hr in the light and a half-life of only 1.2 hr in the dark or in the presence of the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These data indicate that cessation of photosynthesis, either by darkness or DCMU results in a destabilization of the Fed-1 mRNA. Furthermore, the Fed-1 mRNA half-life is reduced immediately upon transfer to darkness, suggesting that Fed-1 mRNA destabilization is a primary response to photosynthesis rather than a secondary response to long-term dark adaptation. Finally, the two different methods for efficient tetracycline delivery reported here generally should be useful for half-life measurements of other mRNAs in whole plants
Summary
Transgenic potatoes (Solanum tuberosum) with either increased (sense transformants) or reduced (antisense transformants) phytochrome A (phyA) levels were used, in combination with specific ...light treatments, to investigate the involvement of phyA in the perception of signals that entrain the circadian clock. Far‐red or far‐red plus red light treatments given during the night reset the circadian rhythm of leaf movements in wild‐type plants and phyA over‐expressors, but had little effect in phyA under‐expressors. Far‐red light was also able to reset the rhythm of leaf movement in wild‐type Arabidopsis thaliana but was not effective in mutants without phyA. Blue light was necessary to reset the rhythm in phyA‐deficient potato plants. Resetting of the rhythm by far‐red plus red light was only slightly affected in transgenic plants with reduced levels of phytochrome B. The production of tubers was delayed by day extensions with far‐red plus red light, but this effect was reduced in transgenic lines deficient in phyA. We conclude that phyA is involved in resetting the circadian clock controlling leaf movements and in photoperiod sensing in light‐grown potato plants.
Volatile secondary metabolites emitted by plants contribute to plant-plant, plant-fungus, and plant-insect interactions. The C(16)-homoterpene TMTT (for 4,8,12-trimethyltrideca-1,3,7,11-tetraene) is ...emitted after herbivore attack by a wide variety of plant species, including Arabidopsis thaliana, and is assumed to play a role in attracting predators or parasitoids of herbivores. TMTT has been suggested to be formed as a degradation product of the diterpene alcohol (E,E)-geranyllinalool. Here, we report the identification of Terpene Synthase 04 (TPS04; At1g61120) as a geranyllinalool synthase (GES). Recombinant TPS04/GES protein expressed in Escherichia coli catalyzes the formation of (E,E)-geranyllinalool from the substrate geranylgeranyl diphosphate. Transgenic Arabidopsis lines carrying T-DNA insertions in the TPS04 locus are deficient in (E,E)-geranyllinalool and TMTT synthesis, a phenotype that can be complemented by expressing the GES gene under the control of a heterologous promoter. GES transcription is upregulated under conditions that induce (E,E)-geranyllinalool and TMTT synthesis, including infestation of plants with larvae of the moth Plutella xylostella and treatment with the fungal peptide alamethicin or the octadecanoid mimic coronalon. Induction requires jasmonic acid but is independent from salicylic acid or ethylene. This study paves the ground to address the contribution of TMTT in ecological interactions and to elucidate the signaling network that regulates TMTT synthesis.
The Arabidopsis thaliana NONEXPRESSER OF PR GENES1 (NPR1, also known as NIM1) protein is an essential positive regulator of salicylic acid (SA)-induced PATHOGENESIS-RELATED (PR) gene expression and ...systemic acquired resistance (SAR). PR gene activity is regulated at the level of redox-dependent nuclear transport of NPR1. NPR1 interacts with members of the TGA family of transcription factors that are known to bind to SA-responsive elements in the PR-1 promoter. In an attempt to identify proteins involved in SA-mediated signal transduction, we previously described the isolation of three novel genes encoding distinct albeit structurally related proteins designated NIMIN1 (for NIM1-INTERACTING1), NIMIN2, and NIMIN3 that interact with NPR1 in the yeast two-hybrid system. Here, we show that NIMIN1 and NPR1 can be copurified from plant extracts, providing biochemical evidence for their interaction. We provide functional evidence for this interaction by describing transgenic plants constitutively expressing high amounts of NIMIN1. These plants show reduced SA-mediated PR gene induction and a compromised SAR, thus mimicking the described phenotype conferred by npr1. Moreover, they showed reduced RESISTANCE gene-mediated protection. These effects were dependent on the ability of NIMIN1 to interact with NPR1. Mutant plants with a T-DNA insertion in NIMIN1 as well as transgenic plants with reduced NIMIN1 mRNA levels showed hyperactivation of PR-1 gene expression after SA treatment but no effect on the disease resistance phenotype. Our results strongly suggest that NIMIN1 negatively regulates distinct functions of NPR1, providing a mechanism to modulate specific features of SAR.
Cross-talk between salicylic acid (SA) and jasmonic acid (JA) defense signaling pathways allows a plant to finely tune its response to the attacker encountered. In Arabidopsis, pharmacological ...experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway. We investigated the putative role of histone modifications in the regulation of SA-mediated suppression of PDF1.2 transcription. Chromatin immunoprecipitation analysis using an antibody directed against acetylated histone H3 revealed that SA does not affect the association of this histone modification at the PDF1.2 promoter, suggesting that chromatin remodeling does not play a major role in SA/JA cross-talk.
Verticillium longisporum is a soil-borne vascular pathogen found primarily on oilseed rape in Northern Europe. Infection of the model plant Arabidopsis thaliana can be achieved under laboratory ...conditions. In the article related to this addendum, we have shown that Arabidopsis dde2-2 mutants that are compromised in their ability to synthesize the defense hormone jasmonoyl-isoleucine (JA-Ile) are slightly more susceptible than wild-type. Contrary to the expectation that hormone biosynthesis mutants and their respective receptor mutants should have the same phenotype, we found that plants that lack the JA-Ile receptor CORONATINE INSENSITIVE1 (COI1) are more tolerant to the disease. This addendum addressed the question whether the increased JA-Ile levels found in coi1 are responsible for its tolerance phenotype. Based on the evidence that the JA-Ile-deficient dde2-2 coi1-t double mutant is as tolerant as coi1-t, we conclude that increased JA-Ile levels do not protect Arabidopsis against the fungus in the absence of COI1.
Salicylic acid (SA) is a plant signalling molecule needed for the induction of defence responses upon attack by a variety of pathogens. Truncation of the Cauliflower Mosaic Virus (CaMV) 35S promoter ...down to 90 bp has identified activation sequence-1 (as-1) as an autonomous SA-responsive cis element. The as-1-like elements are found in a number of SA-inducible promoters like e.g. the tobacco PR-1a promoter. They are recognized by basic/leucine zipper (bZIP) transcription factors of the TGA family. In tobacco leaves, TGA2.2 is the most abundant TGA factor. TGA2.2 is required for the expression of as-1-containing promoters. Here we unravel clear differences between the "truncated" CaMV 35S and the PR-1a promoter with respect to in vivo TGA binding and histone acetylation. Chromatin immunoprecipitation (ChIP) analysis revealed SA-inducible recruitment of tobacco TGA2.2 as well as SA-inducible histone acetylation at the PR-1a promoter. In contrast, no influence of SA on TGA2.2 binding and histone acetylation was detectable at the "truncated" CaMV 35S promoter. The finding of SA-independent TGA factor binding in the absence of additional flanking regulatory sequences suggests that transcriptional activation is not necessarily mediated by inducible DNA binding of TGA factors. Plants with severely reduced TGA2.2 protein levels also showed SA-induced histone acetylation at the PR-1a promoter indicating that regulatory events independent from TGA2.2 function are initiated at the PR-1a promoter.
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