The jasmonoyl-isoleucine receptor CORONATINE INSENSITIVE1 suppresses defense gene expression in Arabidopsis roots independently of its ligand.

The F-box protein CORONANTINE INSENSITIVE1 (COI1) serves as the receptor for the plant hormone jasmonoyl-isoleucine (JA-Ile). COI1, its co-receptors of the JASMONATE ZIM-domain (JAZ) protein family, and JA-Ile form a functional unit that regulates growth or defense mechanisms in response to various stress cues. Strikingly, COI1, but not JA-Ile, is required for susceptibility of Arabidopsis thaliana towards the soil-borne vascular pathogen Verticillium longisporum. In order to obtain marker genes for further analysis of this JA-Ile-independent COI1 function, transcriptome analysis of roots of coi1 and allene oxide synthase (aos) plants (impaired in JA biosynthesis) was performed. Intriguingly, nearly all of the genes that are differentially expressed in coi1 versus aos and wild type are constitutively more highly expressed in coi1. To support our notion that COI1 acts independently of its known downstream signaling components, coi1 plants were complemented with a COI1 variant (COI1AA ) that is compromised in its interaction with JAZs. As expected, these plants showed only weak induction of the expression of the JA-Ile marker gene VEGETATIVE STORAGE PROTEIN2 after wounding and remained sterile. On the other hand, genes affected by COI1 but not by JA-Ile were still strongly repressed by COI1AA . We suggest that COI1 has a potential moonlighting function that serves to repress gene expression in a JA-Ile- and JAZ-independent manner.

N-hydroxypipecolic acid-induced transcription requires the salicylic acid signaling pathway at basal SA levels.

Systemic acquired resistance (SAR) is a plant immune response established in uninfected leaves after colonization of local leaves with biotrophic or hemibiotrophic pathogens. The amino acid-derived metabolite N-hydroxypipecolic acid (NHP) travels from infected to systemic leaves, where it activates salicylic acid (SA) biosynthesis through the isochorismate pathway. The resulting increased SA levels are essential for induction of a large set of SAR marker genes and full SAR establishment. In this study, we show that pharmacological treatment of Arabidopsis thaliana with NHP induces a subset of SAR-related genes even in the SA induction-deficient2 (sid2/isochorismate synthase1) mutant, which is devoid of NHP-induced SA. NHP-mediated induction is abolished in sid2-1 NahG plants, in which basal SA levels are degraded. The SA receptor NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and its interacting TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors are required for the NHP-mediated induction of SAR genes at resting SA levels. Isothermal titration analysis determined a KD of 7.9 ± 0.5 µM for the SA/NPR1 complex, suggesting that basal levels of SA would not bind to NPR1 unless yet unknown potentially NHP-induced processes increase the affinity. Moreover, the nucleocytoplasmic protein PHYTOALEXIN DEFICIENT4 is required for a slight NHP-mediated increase in NPR1 protein levels and NHP-induced expression of SAR-related genes. Our experiments have unraveled that NHP requires basal SA and components of the SA signaling pathway to induce SAR genes. Still, the mechanism of NHP perception remains enigmatic.

The mobile SAR signal N-hydroxypipecolic acid induces NPR1-dependent transcriptional reprogramming and immune priming.

N-hydroxypipecolic acid (NHP) accumulates in the plant foliage in response to a localized microbial attack and induces systemic acquired resistance (SAR) in distant leaf tissue. Previous studies indicated that pathogen inoculation of Arabidopsis (Arabidopsis thaliana) systemically activates SAR-related transcriptional reprogramming and a primed immune status in strict dependence of FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), which mediates the endogenous biosynthesis of NHP. Here, we show that elevations of NHP by exogenous treatment are sufficient to induce a SAR-reminiscent transcriptional response that mobilizes key components of immune surveillance and signal transduction. Exogenous NHP primes Arabidopsis wild-type and NHP-deficient fmo1 plants for a boosted induction of pathogen-triggered defenses, such as the biosynthesis of the stress hormone salicylic acid (SA), accumulation of the phytoalexin camalexin and branched-chain amino acids, as well as expression of defense-related genes. NHP also sensitizes the foliage systemically for enhanced SA-inducible gene expression. NHP-triggered SAR, transcriptional reprogramming, and defense priming are fortified by SA accumulation, and require the function of the transcriptional coregulator NON-EXPRESSOR OF PR GENES1 (NPR1). Our results suggest that NPR1 transduces NHP-activated immune signaling modes with predominantly SA-dependent and minor SA-independent features. They further support the notion that NHP functions as a mobile immune regulator capable of moving independently of active SA signaling between leaves to systemically activate immune responses.

Redox-active cysteines in TGACG-BINDING FACTOR 1 (TGA1) do not play a role in salicylic acid or pathogen-induced expression of TGA1-regulated target genes in Arabidopsis thaliana.

Salicylic acid (SA) is an important signaling molecule of the plant immune system. In Arabidopsis thaliana, SA biosynthesis is indirectly modulated by the closely related transcription factors TGACG-BINDING FACTOR 1 and 4 (TGA1 and TGA4, respectively). They activate expression of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1, the gene product of which regulates the key SA biosynthesis gene ISOCHORISMATE SYNTHASE 1. Since TGA1 interacts with the SA receptor NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) in a redox-dependent manner and since the redox state of TGA1 is altered in SA-treated plants, TGA1 was assumed to play a role in the NPR1-dependent signaling cascade. Here, we identified 193 out of 2090 SA-induced genes that require TGA1/TGA4 for maximal expression after SA treatment. One robustly TGA1/TGA4-dependent gene encodes for the SA hydroxylase DOWNY MILDEW RESISTANT 6-LIKE OXYGENASE 1, suggesting an additional regulatory role of TGA1/TGA4 in SA catabolism. Expression of TGA1/TGA4-dependent genes in mock/SA-treated or Pseudomonas-infected plants was rescued in the tga1 tga4 double mutant after introduction of a mutant genomic TGA1 fragment encoding a TGA1 protein without any cysteines. Thus, the functional significance of the observed redox modification of TGA1 in SA-treated tissues remains enigmatic.

TGACG-BINDING FACTORs (TGAs) and TGA-interacting CC-type glutaredoxins modulate hyponastic growth in Arabidopsis thaliana.

TGACG-BINDING FACTORs (TGAs) control the developmental or defense-related processes. In Arabidopsis thaliana, the functions of at least TGA2 and PERIANTHIA (PAN) can be repressed by interacting with CC-type glutaredoxins, which have the potential to control the redox state of target proteins. As TGA1 can be redox modulated in planta, we analyzed whether some of the 21 CC-type glutaredoxins (ROXYs) encoded in the Arabidopsis genome can influence TGA1 activity in planta and whether the redox active cysteines of TGA1 are functionally important. We show that the tga1 tga4 mutant and plants ectopically expressing ROXY8 or ROXY9 are impaired in hyponastic growth. As expression of ROXY8 and ROXY9 is activated upon transfer of plants from hyponasty-inducing low light to normal light, they might interfere with the growth-promoting function of TGA1/TGA4 to facilitate reversal of hyponastic growth. The redox-sensitive cysteines of TGA1 are not required for induction or reversal of hyponastic growth. TGA1 and TGA4 interact with ROXYs 8, 9, 18, and 19/GRX480, but ectopically expressed ROXY18 and ROXY19/GRX480 do not interfere with hyponastic growth. Our results therefore demonstrate functional specificities of individual ROXYs for distinct TGAs despite promiscuous protein-protein interactions and point to different repression mechanisms, depending on the TGA/ROXY combination.

Ectopically expressed glutaredoxin ROXY19 negatively regulates the detoxification pathway in Arabidopsis thaliana.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: ROXY19 and related proteins of the ROXY gene family can function as negative regulators of TGA-dependent promoters controlling detoxification genes.

Plant-specific CC-type glutaredoxins: functions in developmental processes and stress responses.

Glutaredoxins (GRXs) are small oxidoreductases of the thioredoxin family proteins that can either regulate the thiol redox state of proteins or are linked to iron metabolism because of their ability to incorporate iron-sulfur [2Fe-2S] clusters. Here we review recent research on a land plant-specific class of GRX-like proteins, which are characterized by the conserved CC motif in the active centre. Loss-of-function mutants of CC-type GRXs in Arabidopsis (also named ROXYs), maize, and rice have unraveled a role in floral development, including regulation of organ primordia initiation, control of organ identity gene expression, and progression into meiosis in the male germ line. Other CC-type GRXs play a role in stress responses, most likely through their capacity to regulate nuclear gene expression. Consistently, CC-type GRXs, physically and genetically interact with individual members of the TGA transcription factor family. One of the challenges in the future is to unravel whether ROXYs control the redox state of TGA factors or other yet unknown target proteins or whether they regulate gene expression through other processes. Other intriguing questions concern the original function of the first CC-type GRXs in basal land plants and their potential contribution to the extremely successful radiation of angiosperms.

TGA Transcription Factors Activate the Salicylic Acid-Suppressible Branch of the Ethylene-Induced Defense Program by Regulating ORA59 Expression.

Salicylic acid (SA), a hormone essential for defense against biotrophic pathogens, triggers increased susceptibility of plants against necrotrophic attackers by suppressing the jasmonic acid-ethylene (ET) defense response. Here, we show that this disease-promoting SA effect is abolished in plants lacking the three related TGACG sequence-specific binding proteins TGA2, TGA5, and TGA6 (class II TGAs). After treatment of plants with the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC), activation of all those genes that are suppressed by SA depended on class II TGAs. Rather than TGA binding sites, GCC-box motifs were significantly enriched in the corresponding promoters. GCC-box motifs are recognized by members of the superfamily of APETALA2/ETHYLENE RESPONSE FACTORs (ERFs). Of 11 activating ACC-induced APETALA2/ERFs, only ORA59 (for OCTADECANOID-RESPONSIVE ARABIDOPSIS APETALA2/ETHYLENE RESPONSE FACTOR domain protein59) and ERF96 were strongly suppressed by SA. ORA59 is the master regulator of the jasmonic acid-ET-induced defense program. ORA59 transcript levels do not reach maximal levels in the tga2 tga5 tga6 triple mutant, and this residual activity cannot be suppressed by SA. The ORA59 promoter contains an essential TGA binding site and is a direct target of class II TGAs as revealed by chromatin immunoprecipitation experiments. We suggest that class II TGAs at the ORA59 promoter constitute an important regulatory hub for the activation and SA suppression of ACC-induced genes.

Soluble phenylpropanoids are involved in the defense response of Arabidopsis against Verticillium longisporum.

Verticillium longisporum is a soil-borne vascular pathogen causing economic loss in rape. Using the model plant Arabidopsis this study analyzed metabolic changes upon fungal infection in order to identify possible defense strategies of Brassicaceae against this fungus. Metabolite fingerprinting identified infection-induced metabolites derived from the phenylpropanoid pathway. Targeted analysis confirmed the accumulation of sinapoyl glucosides, coniferin, syringin and lignans in leaves from early stages of infection on. At later stages, the amounts of amino acids increased. To test the contribution of the phenylpropanoid pathway, mutants in the pathway were analyzed. The sinapate-deficient mutant fah1-2 showed stronger infection symptoms than wild-type plants, which is most likely due to the lack of sinapoyl esters. Moreover, the coniferin accumulating transgenic plant UGT72E2-OE was less susceptible. Consistently, sinapoyl glucose, coniferyl alcohol and coniferin inhibited fungal growth and melanization in vitro, whereas sinapyl alcohol and syringin did not. The amount of lignin was not significantly altered supporting the notion that soluble derivatives of the phenylpropanoid pathway contribute to defense. These data show that soluble phenylpropanoids are important for the defense response of Arabidopsis against V. longisporum and that metabolite fingerprinting is a valuable tool to identify infection-relevant metabolic markers.

From pioneers to team players: TGA transcription factors provide a molecular link between different stress pathways.

The plant immune system encompasses an arsenal of defense genes that is activated upon recognition of a pathogen. Appropriate adjustment of gene expression is mediated by multiple interconnected signal transduction cascades that finally control the activity of transcription factors. These sequence-specific DNA-binding proteins act at the interface between the DNA and the regulatory protein network. In 1989, tobacco TGA1a was cloned as the first plant transcription factor. Since then, multiple studies have shown that members of the TGA family play important roles in defense responses against biotrophic and necrotrophic pathogens and against chemical stress. Here, we review 22 years of research on TGA factors which have yielded both consistent and conflicting results.

The vascular pathogen Verticillium longisporum requires a jasmonic acid-independent COI1 function in roots to elicit disease symptoms in Arabidopsis shoots.

Verticillium longisporum is a soil-borne vascular pathogen that causes reduced shoot growth and early senescence in Arabidopsis (Arabidopsis thaliana). Here, we report that these disease symptoms are less pronounced in plants that lack the receptor of the plant defense hormone jasmonic acid (JA), CORONATINE INSENSITIVE1 (COI1). Initial colonization of the roots was comparable in wild-type and coi1 plants, and fungal DNA accumulated to almost similar levels in petioles of wild-type and coi1 plants at 10 d post infection. Completion of the fungal life cycle was impaired in coi1, as indicated by the reduced number of plants with microsclerotia, which are detected on dead plant material at late stages of the disease. Contrary to the expectation that the hormone receptor mutant coi1 should display the same phenotype as the corresponding hormone biosynthesis mutant delayed dehiscence2 (dde2), dde2 plants developed wild-type-like disease symptoms. Marker genes of the JA and the JA/ethylene defense pathway were induced in petioles of wild-type plants but not in petioles of dde2 plants, indicating that fungal compounds that would activate the known COI1-dependent signal transduction chain were absent. Grafting experiments revealed that the susceptibility-enhancing COI1 function acts in the roots. Moreover, we show that the coi1-mediated tolerance is not due to the hyperactivation of the salicylic acid pathway. Together, our results have unraveled a novel COI1 function in the roots that acts independently from JA-isoleucine or any JA-isoleucine mimic. This COI1 activity is required for a yet unknown root-to-shoot signaling process that enables V. longisporum to elicit disease symptoms in Arabidopsis.

Xenobiotic- and jasmonic acid-inducible signal transduction pathways have become interdependent at the Arabidopsis CYP81D11 promoter.

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 coi1 than in the JA biosynthesis mutant dde2-2, [corrected] 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.

Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis.

Terpene volatiles play important roles in plant-organism interactions as attractants of pollinators or as defense compounds against herbivores. Among the most common plant volatiles are homoterpenes, which are often emitted from night-scented flowers and from aerial tissues upon herbivore attack. Homoterpene volatiles released from herbivore-damaged tissue are thought to contribute to indirect plant defense by attracting natural enemies of pests. Moreover, homoterpenes have been demonstrated to induce defensive responses in plant-plant interaction. Although early steps in the biosynthesis of homoterpenes have been elucidated, the identity of the enzyme responsible for the direct formation of these volatiles has remained unknown. Here, we demonstrate that CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the Arabidopsis CYP82 family, is responsible for the breakdown of the C(20)-precursor (E,E)-geranyllinalool to the insect-induced C(16)-homoterpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Recombinant CYP82G1 shows narrow substrate specificity for (E,E)-geranyllinalool and its C(15)-analog (E)-nerolidol, which is converted to the respective C(11)-homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Homology-based modeling and substrate docking support an oxidative bond cleavage of the alcohol substrate via syn-elimination of the polar head, together with an allylic C-5 hydrogen atom. CYP82G1 is constitutively expressed in Arabidopsis stems and inflorescences and shows highly coordinated herbivore-induced expression with geranyllinalool synthase in leaves depending on the F-box protein COI-1. CYP82G1 represents a unique characterized enzyme in the plant CYP82 family with a function as a DMNT/TMTT homoterpene synthase.

Jasmonic acid perception by COI1 involves inositol polyphosphates in Arabidopsis thaliana.

Plant responses to wounding are part of their defense responses against insects, and are tightly regulated. The isoleucin conjugate of jasmonic acid (JA-Ile) is a major regulatory molecule. We have previously shown that inositol polyphosphate signals are required for defense responses in Arabidopsis; however, the way in which inositol polyphosphates contribute to plant responses to wounding has so far remained unclear. Arabidopsis F-box proteins involved in the perception of JA-Ile (COI1) and auxin (TIR1) are structurally similar. Because TIR1 has recently been shown to contain inositol hexakisphosphate (InsP6) as a co-factor of unknown function, here we explored the possibility that InsP6 or another inositol polyphosphate is required for COI1 function. In support of this hypothesis, COI1 variants with changes in putative inositol polyphosphate coordinating residues exhibited a reduced interaction with the COI1 target, JAZ9, in yeast two-hybrid tests. The equivalent COI1 variants displayed a reduced capability to rescue jasmonate-mediated root growth inhibition or silique development in Arabidopsis coi1 mutants. Yeast two-hybrid tests using wild-type COI1 in an ipk1Δ yeast strain exhibiting increased levels of inositol pentakisphosphate (InsP5) and reduced levels of InsP6 indicate an enhanced COI1/JAZ9 interaction. Consistent with these findings, Arabidopsis ipk1-1 mutants, also with increased InsP5 and reduced InsP6 levels, showed increased defensive capabilities via COI1-mediated processes, including wound-induced gene expression, defense against caterpillars or root growth inhibition by jasmonate. The combined data from experiments using mutated COI1 variants, as well as yeast and Arabidopsis backgrounds altered in inositol polyphosphate metabolism, indicate that an inositol polyphosphate, and probably InsP5, contributes to COI1 function.

The glutaredoxin ATGRXS13 is required to facilitate Botrytis cinerea infection of Arabidopsis thaliana plants.

Botrytis cinerea is a major pre- and post-harvest necrotrophic pathogen with a broad host range that causes substantial crop losses. The plant hormone jasmonic acid (JA) is involved in the basal resistance against this fungus. Despite basal resistance, virulent strains of B. cinerea can cause disease on Arabidopsis thaliana and virulent pathogens can interfere with the metabolism of the host in a way to facilitate infection of the plant. However, plant genes that are required by the pathogen for infection remain poorly described. To find such genes, we have compared the changes in gene expression induced in A. thaliana by JA with those induced after B. cinerea using genome-wide microarrays. We have identified genes that are repressed by JA but that are induced by B. cinerea. In this study, we describe one candidate gene, ATGRXS13, that encodes for a putative glutaredoxin and that exhibits such a crossed expression. In plants that are infected by this necrotrophic fungus, ATGRXS13 expression was negatively controlled by JA and TGA transcription factors but also through a JA-salicylic acid (SA) cross-talk mechanism as B. cinerea induced SA production that positively controlled ATGRXS13 expression. Furthermore, plants impaired in ATGRXS13 exhibited resistance to B. cinerea. Finally, we present a model whereby B. cinerea takes advantage of defence signalling pathways of the plant to help the colonization of its host.

Arabidopsis thaliana class-II TGA transcription factors are essential activators of jasmonic acid/ethylene-induced defense responses.

The three closely related Arabidopsis basic leucine zipper (bZIP) transcription factors TGA2, TGA5 and TGA6 are required for the establishment of the salicylic acid (SA)-dependent plant defense response systemic acquired resistance, which is effective against biotrophic pathogens. Here we show that the same transcription factors are essential for the activation of jasmonic acid (JA)- and ethylene (ET)-dependent defense mechanisms that counteract necrotrophic pathogens: the tga256 triple mutant is impaired in JA/ET-induced PDF1.2 and b-CHI expression, which correlates with a higher susceptibility against the necrotroph Botrytis cinerea. JA/ET induction of the trans-activators ERF1 and ORA59, which act upstream of PDF1.2, was slightly increased (ERF1) or unaffected (ORA59). PDF1.2 expression can be restored in the tga256 mutant by increased expression of ORA59, as observed in the tga256 jin1 quadruple mutant, which lacks the transcription factor JIN1/AtMYC2 that functions as a negative regulator of the JA/ET-dependent anti-fungal defense program. Whereas JA/ET-induced PDF1.2 expression is strongly suppressed by SA in wild-type plants, no negative effect of SA on PDF1.2 expression was observed in the tga256 jin1 quadruple mutant. These results imply that the antagonistic effects of TGA factors and JIN1/AtMYC2 on the JA/ET pathway are necessary to evoke the SA-mediated suppression of JA/ET-induced defense responses.

The Arabidopsis PR-1 promoter contains multiple integration sites for the coactivator NPR1 and the repressor SNI1.

Systemic acquired resistance is a broad-spectrum plant immune response involving massive transcriptional reprogramming. The Arabidopsis (Arabidopsis thaliana) PATHOGENESIS-RELATED-1 (PR-1) gene has been used in numerous studies to elucidate transcriptional control mechanisms regulating systemic acquired resistance. WRKY transcription factors and basic leucine zipper proteins of the TGA family regulate the PR-1 promoter by binding to specific cis-elements. In addition, the promoter is under the control of two proteins that do not directly contact the DNA: the positive regulator NONEXPRESSOR OF PR GENES1 (NPR1), which physically interacts with TGA factors, and the repressor SUPPRESSOR OF NPR1, INDUCIBLE1 (SNI1). In this study, we analyzed the importance of the TGA-binding sites LS5 and LS7 and the WKRY box LS4 for regulation by NPR1 and SNI1. In the absence of LS5 and LS7, NPR1 activates the PR-1 promoter through a mechanism that requires LS4. Since transcriptional activation of WRKY genes is under the control of NPR1 and since LS4 is not sufficient for the activation of a truncated PR-1 promoter by the effector protein NPR1-VP16 in transient assays, it is concluded that the LS4-dependent activation of the PR-1 promoter is indirect. In the case of NPR1 acting directly through TGA factors at its target promoters, two TGA-binding sites are necessary but not sufficient for NPR1 function in transgenic plants and in the NPR-VP16-based trans-activation assay in protoplasts. SNI1 exerts its negative effect in the noninduced state by targeting unknown proteins associated with sequences between bp -816 and -573. Under induced conditions, SNI1 negatively regulates the function of WRKY transcription factors binding to WKRY boxes between bp -550 and -510.

Induction of Jasmonoyl-Isoleucine (JA-Ile)-Dependent JASMONATE ZIM-DOMAIN (JAZ) Genes in NaCl-Treated Arabidopsis thaliana Roots Can Occur at Very Low JA-Ile Levels and in the Absence of the JA/JA-Ile Transporter JAT1/AtABCG16.

The plant hormone jasmonoyl-isoleucine (JA-Ile) is an important regulator of plant growth and defense in response to various biotic and abiotic stress cues. Under our experimental conditions, JA-Ile levels increased approximately seven-fold in NaCl-treated Arabidopsis thaliana roots. Although these levels were around 1000-fold lower than in wounded leaves, genes of the JA-Ile signaling pathway were induced by a factor of 100 or more. Induction was severely compromised in plants lacking the JA-Ile receptor CORONATINE INSENSITIVE 1 or enzymes required for JA-Ile biosynthesis. To explain efficient gene expression at very low JA-Ile levels, we hypothesized that salt-induced expression of the JA/JA-Ile transporter JAT1/AtABCG16 would lead to increased nuclear levels of JA-Ile. However, mutant plants with different jat1 alleles were similar to wild-type ones with respect to salt-induced gene expression. The mechanism that allows COI1-dependent gene expression at very low JA-Ile levels remains to be elucidated.

Chromatin immunoprecipitation experiments to investigate in vivo binding of Arabidopsis transcription factors to target sequences.

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.

Reconstitution of the Jasmonate Signaling Pathway in Plant Protoplasts.

The phytohormone jasmonic acid (JA) plays an important role in various plant developmental processes and environmental adaptations. The JA signaling pathway has been well-elucidated in the reference plant Arabidopsis thaliana. It starts with the perception of the active JA derivative, jasmonoyl-isoleucine (JA-Ile), by the F-box protein COI1 which is part of the E3-ligase SCFCOI1. Binding of JA-Ile enables the interaction between COI1 and JAZ repressor proteins. Subsequent degradation of JAZ proteins leads to the activation of transcription factors like e.g., MYC2. Here we demonstrate that the pathway can be reconstituted in transiently transformed protoplasts. Analysis of the stability of a JAZ1-fLuc fusion protein as a function of COI1 transiently expressed in coi1 protoplasts allows structure function analysis of both JAZs and COI1. Using this system, we found that conserved cysteines in COI1 influence steady state COI1 protein levels. Using a luciferase reporter gene under the control of the JAZ1 promoter enable to address those features of JAZ1 that are required for MYC2 repression. Interestingly, the conserved TIFY-motif previously described to interact with NINJA to recruit the corepressor TOPLESS is not necessary for repression. This result is in favor of the alternative repression mode that proposes a direct competition between repressive JAZs and promotive MEDIATOR25 at MYC2. Finally, using protoplasts from the aoscoi1 double mutant, which is deficient in JA synthesis and perception, we provide a system that has the potential to study the activity of different COI1 variants in the presence of different ligands.