Rapid increase of NO release in plant cell cultures induced by cytokinin.
4,5-Diaminofluorescein, a fluorescence indicator for NO, was applied to detect the release of NO from plant cells. NO production was increased within 3 min when plant cell cultures (Arabidopsis, parsley, and tobacco) were treated by cytokinin and was dose-dependent and signal-specific in that other plant hormones and inactive cytokinin analog were not effective in stimulating of NO release. The response was quenched by addition of 2-(aminoethyl)-2-thiopseudourea, an inhibitor of the animal NO synthase, and by addition of an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide. These results imply that NO may act in cytokinin signal transduction. (+info
Crosstalk among stress responses in plants: pathogen defense overrides UV protection through an inversely regulated ACE/ACE type of light-responsive gene promoter unit.
Plants often have to cope with two or more environmental hazards simultaneously. Such coincidences require instantaneous decisions on relative severity and consequential crosstalk between the respective signaling cascades. Among the frequently encountered threats are pathogen infections and UV irradiation, both of which trigger specifically targeted defense responses by means of changes in gene transcription rates. In Petroselinum crispum, pathogen defense has been shown to be associated with extensive metabolic reprogramming, including strong repression of the UV-protective flavonoid biosynthetic pathway. Here we show that one of the involved genes, encoding acyl-CoA oxidase, responds positively to UV light and negatively to a pathogen-derived elicitor through an inversely regulated promoter unit consisting of two almost identical ACGT-containing elements (ACEs). This unit, when either introduced into an unrelated promoter or generated by mutation of a differently composed unit, confers the same type of response pattern on the recipient genes, confirming its general functionality at a convergence site of two largely distinct signaling pathways. Similarly large, rapid, and partly inverse effects of UV light and elicitor were observed for several mRNAs encoding common plant regulatory factors (CPRFs) that exhibit distinct dimerization and DNA-binding properties. This striking coincidence suggests a major role of common plant regulatory factors in mediating the apparent switch in the function of ACGT-containing elements from positive UV light to negative elicitor or pathogen responsiveness. (+info
Leucine zipper-containing WRKY proteins widen the spectrum of immediate early elicitor-induced WRKY transcription factors in parsley.
Two new WRKY transcription factors from parsley (Petroselinum crispum), WRKY4 and WRKY5, were isolated using the yeast one-hybrid system. In yeast, both proteins interacted sequence-specifically with W boxes (TTGACC) and activated transcription. They appear to contain functional leucine zippers, which increase their affinities for W boxes. Co-transfection experiments in parsley protoplasts confirmed their in vivo-binding specificity for W boxes. Elicitor-mediated expression of the WRKY5 gene, the first parsley member of the group III family of WRKY proteins, is extremely transient, with high mRNA levels occurring within a time window of less than 1 h. WRKY4 and -5, as well as the previously identified parsley transcription factors WRKY1 and -3, are encoded by immediate early elicitor-activated genes that differ in their sensitivity to cycloheximide (CHX) and their activation kinetics. We propose that a number of the pathways activated during the plant defense response require the induction of several distinct WRKY transcription factors with different DNA binding-site preferences to fine-tune the activation of a wide spectrum of target genes. (+info
Pretreatment with salicylic acid primes parsley cells for enhanced ion transport following elicitation.
Pretreatment with salicylic acid (SA), an inducer of plant disease resistance, enhanced the capacity of parsley cells for the induction of a rapid K(+)/pH response and the subsequent coumarin (phytoalexin) secretion. In SA-primed cells, a low elicitor dose induced these two responses to a similar extent as did a high elicitor dose in non-primed cells. These observations suggest that the SA-mediated augmentation of the early K(+)/pH response may contribute to the enhancement of subsequent coumarin secretion. As the amphotericin B-induced K(+)/pH response was not enhanced in SA-primed cells, it is concluded that signaling components that are improved by priming are located between elicitor signal perception and the plasma membrane transporters mediating the K(+)/pH response. (+info
An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum.
The plant enzyme phenylalanine ammonia-lyase (PAL, EC 220.127.116.11) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138. Determination of the kinetic constants of the overexpressed and purified enzymes revealed that mutagenesis led in each case to diminished activity. Mutants S203A, R354A and Y351F showed a decrease in kcat by factors of 435, 130 and 235, respectively. Mutants F400A, Q488A and L138H showed a 345-, 615- and 14-fold lower kcat, respectively. The greatest loss of activity occurred in the PAL mutants N260A, Q348A and Y110F, which were 2700, 2370 and 75 000 times less active than wild-type PAL. To elucidate the possible function of the mutated amino-acid residues in PAL we built a homology model of PAL based on structural data of HAL and mutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowed us to propose possible roles for the corresponding residues in PAL catalysis. (+info
Mitogen-activated protein kinases play an essential role in oxidative burst-independent expression of pathogenesis-related genes in parsley.
Plants are continuously exposed to attack by potential phytopathogens. Disease prevention requires pathogen recognition and the induction of a multifaceted defense response. We are studying the non-host disease resistance response of parsley to the oomycete, Phytophthora sojae using a cell culture-based system. Receptor-mediated recognition of P. sojae may be achieved through a thirteen amino acid peptide sequence (Pep-13) present within an abundant cell wall transglutaminase. Following recognition of this elicitor molecule, parsley cells mount a defense response, which includes the generation of reactive oxygen species (ROS) and transcriptional activation of genes encoding pathogenesis-related (PR) proteins or enzymes involved in the synthesis of antimicrobial phytoalexins. Treatment of parsley cells with the NADPH oxidase inhibitor, diphenylene iodonium (DPI), blocked both Pep-13-induced phytoalexin production and the accumulation of transcripts encoding enzymes involved in their synthesis. In contrast, DPI treatment had no effect upon Pep-13-induced PR gene expression, suggesting the existence of an oxidative burst-independent mechanism for the transcriptional activation of PR genes. The use of specific antibodies enabled the identification of three parsley mitogen-activated protein kinases (MAPKs) that are activated within the signal transduction pathway(s) triggered following recognition of Pep-13. Other environmental challenges failed to activate these kinases in parsley cells, suggesting that their activation plays a key role in defense signal transduction. Moreover, by making use of a protoplast co-transfection system overexpressing wild-type and loss-of-function MAPK mutants, we show an essential role for post-translational phosphorylation and activation of MAPKs for oxidative burst-independent PR promoter activation. (+info
Non-self recognition, transcriptional reprogramming, and secondary metabolite accumulation during plant/pathogen interactions.
Disease resistance of plants involves two distinct forms of chemical communication with the pathogen: recognition and defense. Both are essential components of a highly complex, multifaceted defense response, which begins with non-self recognition through the perception of pathogen-derived signal molecules and results in the production, inter alia, of antibiotically active compounds (phytoalexins) and cell wall-reinforcing material around the infection site. To elucidate the molecular details and the genomic basis of the underlying chains of events, we used two different experimental systems: suspension-cultured cells of Petroselinum crispum (parsley) and wild-type as well as mutant plants of Arabidopsis thaliana. Particular emphasis was placed on the structural and functional identification of signal and defense molecules, and on the mechanisms of signal perception, intracellular signal transduction and transcriptional reprogramming, including the structural and functional characterization of the responsible cis-acting gene promoter elements and transacting regulatory proteins. Comparing P. crispum and A. thaliana allows us to distinguish species-specific defense mechanisms from more universal responses, and furthermore provides general insights into the nature of the interactions. Despite the complexity of the pathogen defense response, it is experimentally tractable, and knowledge gained so far has opened up a new realm of gene technology-assisted strategies for resistance breeding of crop plants. (+info
Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley.
Flavone synthases (FNSs) catalyze the oxidation of flavanones to flavones, i.e. the formation of apigenin from (2S)-naringenin. While many plants express a microsomal-type FNS II, the soluble FNS I appears to be confined to a few species of the Apiaceae and was cloned recently from parsley plants. FNS I belongs to the Fe(II)/2-oxoglutarate-dependent dioxygenases characterized by short conserved sequence elements for cofactor binding, and its evolutionary context and mode of action are under investigation. Using a homology-based reverse transcription polymerase chain reaction approach, two additional flavonoid-specific dioxygenases were cloned from immature parsley leaflets, which were identified as flavanone 3beta-hydroxylase (FHT) and flavonol synthase (FLS) after expression in yeast cells. Sequence alignments revealed marginal differences among the parsley FNS I and FHT polypeptides of only 6%, while much less identity (about 29%) was observed with the parsley FLS. Analogous to FNS I, FLS oxidizes the flavonoid gamma-pyrone by introducing a C2, C3 double bond, and (2R,3S)-dihydrokaempferol (cis-dihydrokaempferol) was proposed recently as the most likely intermediate in both FNS I and FLS catalysis. Incubation of either FNS I or FLS with cis-dihydrokaempferol exclusively produced kaempferol and confirmed the assumption that flavonol formation occurs via hydroxylation at C3 followed by dehydratation. However, the lack of apigenin in these incubations ruled out cis-dihydrokaempferol as a free intermediate in FNS I catalysis. Furthermore, neither (+)-trans-dihydrokaempferol nor unnatural (-)-trans-dihydrokaempferol and 2-hydroxynaringenin served as a substrate for FNS I. Overall, the data suggest that FNS I has evolved uniquely in some Apiaceae as a paraphyletic gene from FHT, irrespective of the fact that FNS I and FLS catalyze equivalent desaturation reactions. (+info