A new member of the Arabidopsis WRKY transcription factor family, AtWRKY6, is associated with both senescence- and defence-related processes.

WRKY proteins constitute a large family of plant-specific transcription factors whose precise functions have yet to be elucidated. Here we show that expression of one representative in Arabidopsis, AtWRKY6, is influenced by several external and internal signals often involved in triggering senescence processes and plant defence responses. Progressive 5' deletions of the AtWRKY6 promoter allowed separation of defined regions responsible for the expression in distinct organs or upon pathogen challenge. Nuclear localization of AtWRKY6 was demonstrated; protein truncations and gain-of-function studies enabled delineation of a region harbouring a novel type of functional nuclear localization signal (NLS).

The WRKY superfamily of plant transcription factors.

The WRKY proteins are a superfamily of transcription factors with up to 100 representatives in Arabidopsis. Family members appear to be involved in the regulation of various physio-logical programs that are unique to plants, including pathogen defense, senescence and trichome development. In spite of the strong conservation of their DNA-binding domain, the overall structures of WRKY proteins are highly divergent and can be categorized into distinct groups, which might reflect their different functions.

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley.

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.

A novel regulatory element involved in rapid activation of parsley ELI7 gene family members by fungal elicitor or pathogen infection.

Abstract In parsley (Petroselinum crispum), members of the ELI7 gene family were rapidly transcriptionally activated following treatment with an elicitor derived from the phytopathogen Phytophthora sojae. Several cDNA and genomic ELI7 clones were isolated. The deduced amino acid sequences revealed close similarity to fatty acid desaturases/hydroxylases, however, the precise functions are still unknown. Analysis of the promoters of two strongly elicitor-induced family members, ELI7.1 and ELI7.2, allowed us to functionally pinpoint a novel, independently acting regulatory region (S box), the only major sequence similarity between the two gene promoters. In situ RNA/RNA hybridization using an ELI7.1 gene-specific probe demonstrated that expression of this gene is rapidly and locally induced around infection sites in planta as well.

Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors.

Parsley WRKY proteins comprise a family of plant-specific zinc-finger-type factors implicated in the regulation of genes associated with pathogen defence. In vitro, these proteins bind specifically to functionally defined TGAC-containing W box promoter elements within the Pathogenesis-Related Class10 (PR-10) genes. Here we present in vivo data demonstrating that WRKY1 is a transcriptional activator mediating fungal elicitor-induced gene expression by binding to W box elements. In situ RNA hybridization revealed that the WRKY1 gene is rapidly and locally activated in parsley leaf tissue around fungal infection sites. Transient expression studies in parsley protoplasts showed that a specific arrangement of W box elements in the WRKY1 promoter itself is necessary and sufficient for early activation and that WRKY1 binds to such elements. Our results demonstrate that WRKY transcription factors play an important role in the regulation of early defence-response genes including regulation of WRKY1.

Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms.

The enzyme 4-coumarate:CoA ligase (4CL) plays a key role in channelling carbon flow into diverse branch pathways of phenylpropanoid metabolism which serve important functions in plant growth and adaptation to environmental perturbations. Here we report on the cloning of the 4CL gene family from Arabidopsis thaliana and demonstrate that its three members, At4CL1, At4CL2 and At4CL3, encode isozymes with distinct substrate preference and specificities. Expression studies revealed a differential behaviour of the three genes in various plant organs and upon external stimuli such as wounding and UV irradiation or upon challenge with the fungus, Peronospora parasitica. Phylogenetic comparisons indicate that, in angiosperms, 4CL can be classified into two major clusters, class I and class II, with the At4CL1 and At4CL2 isoforms belonging to class I and At4CL3 to class II. Based on their enzymatic properties, expression characteristics and evolutionary relationships, At4CL3 is likely to participate in the biosynthetic pathway leading to flavonoids whereas At4CL1 and At4CL2 are probably involved in lignin formation and in the production of additional phenolic compounds other than flavonoids.

Isolation of putative plant transcriptional coactivators using a modified two-hybrid system incorporating a GFP reporter gene.

Dual hybrid interacting screening in yeast led to the identification of two proteins from Arabidopsis both exhibiting sequence similarity to a family of transcriptional coactivators from a diverse range of organisms. Their discovery constitutes the first description of such plant proteins. A modified yeast two-hybrid approach utilising the green fluorescent protein (GFP) of Aequora victoria was developed and used to clone one of the putative plant transcriptional coactivators from an Arabidopsis cDNA library. The two proteins, designated KIWI and KELP, can associate both hetero- and homomerically and their genes were cloned and mapped on the Arabidopsis genome. Both proteins are believed to play a role in gene activation during pathogen defence and plant development. The involvement of these proteins in general plant transcription as well as the advantages of using GFP as a reporter gene for detecting protein-protein interactions are discussed.

Transcriptional control of plant genes responsive to pathogens.

Transcriptional activation of genes is a vital part of the plants defence system against pathogens. Cis-acting elements within the promoters of many of these genes have recently been defined and investigators have started to isolate their cognate trans-acting factors. Some of these factors have counterparts in animals, whereas others are present only in plants, reflecting the fact that plants have developed a unique defence system.

Developmental and auxin-induced expression of the Arabidopsis prha homeobox gene.

A single-copy Arabidopsis homeobox gene, prha, which encodes a homeodomain protein with a molecular weight of 90,500 has been characterized. The position of the gene was mapped to the distal part of chromosome 4. Expression of the gene differs in various vegetative and floral plant tissues and is positively influenced by the phytohormone auxin. In Arabidopsis plants, a complex pattern of prha promoter-driven GUS expression is observed, often associated with regions of developing vascular tissue. Exogenously applied auxin strongly increased endogenous prha transcript levels. In addition, the prha promoter is highly responsive to the synthetic auxin, naphthalene acetic acid, in transient assays using tobacco protoplasts. The PRHA protein has the capacity to bind to TAATTG core sequence elements but requires additional adjacent bases for high-affinity binding. These findings are discussed in relation to studies of other plant homeobox genes as well as possible in vivo target genes for PRHA.

Rapid and transient induction of a parsley microsomal delta 12 fatty acid desaturase mRNA by fungal elicitor.

Treatment of cultured parsley (Petroselinum crispum L.) cells with a structurally defined peptide elicitor (Pep25) of fungal origin has previously been shown to cause rapid and large changes in the levels of various desaturated fatty acids. We isolated two distinct parsley cDNAs sharing high sequence similarity with microsomal omega-6 fatty acid desaturases (FADs). One of them was functionally identified as a delta 12 FAD by expression in the yeast Saccharomyces cerevisiae. Two dienoic fatty acids, hexadecadienoic and linoleic, which were not detectable in control cells, together constituted up to 12% of the total fatty acids in the transformed yeast cells. delta 12 FAD mRNA accumulated rapidly and transiently in elicitor-treated parsley cells, protoplasts, and leaves. These and previous results indicate that fatty acid desaturation is an important early component of the complex defense response of parsley to attempted fungal infection.

A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

Rapid amplification of genomic ends (RAGE) as a simple method to clone flanking genomic DNA.

This report describes the amplification of upstream genomic sequences using the polymerase chain reaction (PCR) based solely on downstream DNA information from a cDNA clone. In this novel and rapid technique, genomic DNA (gDNA) is first incubated with a restriction enzyme that recognizes a site within the 5' end of a gene, followed by denaturation and polyadenylation of its free 3' ends with terminal transferase. The modified gDNA is then used as template for PCR using a gene-specific primer complementary to a sequence in the 3' end of its cDNA and an anchored deoxyoligothymidine primer. A second round of PCR is then performed with a second, nested gene-specific primer and the anchor sequence primer. The resulting PCR product is cloned and its sequence determined. Three independent plant genomic clones were isolated using this method that exhibited complete sequence identity to their cDNAs and to the primers used in the amplification.

Rapid, transient, and highly localized induction of plastidial omega-3 fatty acid desaturase mRNA at fungal infection sites in Petroselinum crispum.

Parsley (Petroselinum crispum) plants and suspension-cultured cells have been used extensively for studies of non-host-resistance mechanisms in plant/pathogen interactions. We now show that treatment of cultured parsley cells with a defined peptide elicitor of fungal origin causes rapid and large changes in the levels of various unsaturated fatty acids. While linoleic acid decreased and linolenic acid increased steadily for several hours, comparatively sharp increases in oleic acid followed a biphasic time course. In contrast, the overall level of stearic acid remained unaffected. Using a PCR-based approach, a parsley cDNA was isolated sharing high sequence similarity with omega-3 fatty acid desaturases. Subsequent isolation and characterization of a full-length cDNA enabled its functional identification as a plastid-localized omega-3 fatty acid desaturase by complementation of the Arabidopsis thaliana fad7/8 double mutant which is low in trienoic fatty acids. omega-3 Fatty acid desaturase mRNA accumulated rapidly and transiently in elicitor-treated cultured parsley cells, protoplasts, and leaves, as well as highly localized around fungal infection sites in parsley leaf buds. These results indicate that unsaturated fatty acid metabolism is yet another component of the highly complex, transcriptionally regulated pathogen defense response in plants.

Arabidopsis thaliana defense-related protein ELI3 is an aromatic alcohol:NADP+ oxidoreductase.

We expressed a cDNA encoding the Arabidopsis thaliana defense-related protein ELI3-2 in Escherichia coli to determine its biochemical function. Based on a protein database search, this protein was recently predicted to be a mannitol dehydrogenase [Williamson, J. D., Stoop, J. M. H., Massel, M. O., Conkling, M. A. & Pharr, D. M. (1995) Proc. Natl. Acad. Sci. USA 92, 7148-7152]. Studies on the substrate specificity now revealed that ELI3-2 is an aromatic alcohol: NADP+ oxidoreductase (benzyl alcohol dehydrogenase). The enzyme showed a strong preference for various aromatic aldehydes as opposed to the corresponding alcohols. Highest substrate affinities were observed for 2-methoxybenzaldehyde, 3-methoxybenzaldehyde, salicylaldehyde, and benzaldehyde, in this order, whereas mannitol dehydrogenase activity could not be detected. These and previous results support the notion that ELI3-2 has an important role in resistance-related aromatic acid-derived metabolism.

Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes.

PR1 is a pathogenesis-related protein encoded in the parsley genome by a family of three genes (PR1-1, PR1-2 and PR1-3). Loss- and gain-of-function experiments in a transient expression system demonstrated the presence of two fungal elicitor responsive elements in each of the PR1-1 and PR1-2 promoters. These elements, W1, W2 and W3, contain the sequence (T)TGAC(C) and mutations that disrupt this sequence abolish function. Gel shift experiments demonstrated that W1, W2 and W3 are bound specifically by similar nuclear proteins. Three cDNA clones encoding sequence-specific DNA-binding proteins were isolated by South-Western screening and these proteins, designated WRKY1, 2 and 3, also bind specifically to W1, W2 and W3. WRKY1, 2 and 3 are members of the family of sequence-specific DNA-binding proteins, which we call the WRKY family. Treatment of parsley cells with the specific oligopeptide elicitor Pep25 induced a transient and extremely rapid increase in mRNA levels of WRKY1 and 3. WRKY2 mRNA levels in contrast showed a concomitant transient decrease. These rapid changes in WRKY mRNA levels in response to a defined signal molecule suggest that WRKY1, 2 and 3 play a key role in a signal transduction pathway that leads from elicitor perception to PR1 gene activation.

Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle-related genes.

The effects of UV light or fungal elicitors on plant cells have so far been studied mostly with respect to defense-related gene activation. Here, an inverse correlation of these stimulatory effects with the activities of several cell cycle-related genes is demonstrated. Concomitant with the induction of flavonoid biosynthetic enzymes in UV-irradiated cell suspension cultures of parsley (Petroselinum crispum), total histone synthesis declined to about half the initial rate. A subclass of the histone H3 gene family was selected to demonstrate the close correlation of its expression with cell division, both in intact plants and cultured cells. Using RNA-blot and run-on transcription assays, it was shown that one arbitrarily selected subclass of each of the histone H2A, H2B, H3 and H4 gene families and of the genes encoding a p34cdc2 protein kinase and a mitotic cyclin were transcriptionally repressed in UV-irradiated as well as fungal elicitor-treated parsley cells. The timing and extent of repression differed between the two stimuli; the response to light was more transient and smaller in magnitude. These differential responses to light and elicitor were inversely correlated with the induction of phenylalanine ammonia-lyase, a key enzyme of phenylpropanoid metabolism. Essentially the same result was obtained with a defined oligopeptide elicitor, indicating that the same signaling pathway is responsible for defense-related gene activation and cell cycle-related gene repression. A temporary (UV light) or long-lasting (fungal elicitor) cessation of cell culture growth is most likely due to an arrest of cell division which may be a prerequisite for full commitment of the cells to transcriptional activation of full commitment of the cells to transcriptional activation of pathways involved in UV protection or pathogen defense. This conclusion is corroborated by the observation that the histone H3 mRNA level greatly declined around fungal infection sites in young parsley leaves.

Two pathogen-responsive genes in parsley encode a tyrosine-rich hydroxyproline-rich glycoprotein (hrgp) and an anionic peroxidase.

Two recently isolated cDNAs representing genes that are transcriptionally activated in fungus-infected parsley leaves or elicitor-treated, cultured parsley cells are shown to encode a hydroxyproline-rich glycoprotein (HRGP) and an anionic peroxidase. The deduced HRGP protein is rich in tyrosine residues, a feature also found in other pathogen- and wound-induced plant HRGPs. Expression of the peroxidase gene(s) is induced rapidly upon elicitation and precedes that of the HRGP gene. In situ hybridization experiments demonstrate the presence of HRGP and peroxidase mRNAs in parsley tissue around fungal infection sites. Peroxidase mRNA accumulation is particularly sharply restricted to plant cells directly adjacent to fungal hyphae. These results provide further evidence for an important role of specific cell wall modifications in plant defense.

The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana.

Phenylpropanoid derivatives are a complex class of secondary metabolites that have many important roles in plants during normal growth and in responses to environmental stress. Phenylalanine ammonialyase (PAL) catalyzes the first step in the biosynthesis of phenylpropanoids, and is usually encoded by a multi-gene family. Genomic clones for three Arabidopsis thaliana PAL genes containing the entire protein-coding region and upstream and downstream sequences have been obtained and completely sequenced. Two A. thaliana PAL genes (PAL1 and PAL2) are structurally similar to PAL genes that have been cloned from other plant species, with a single intron at a conserved position, and a long highly conserved second exon. Previously identified promoter motifs plus several additional sequence motifs were found in the promoter regions of PAL1 and PAL2. Expression of PAL1 and PAL2 is both qualitatively and quantitatively similar in different plant organs and under various inductive conditions. A third A. thaliana PAL gene, PAL3, differs significantly from PAL1 and PAL2 and other sequenced plant PAL genes. PAL3 contains an additional intron, and its deduced amino acid sequence is less homologous to other PAL proteins. The PAL3 promoter region lacks several sequence motifs conserved between A. thaliana PAL1 and PAL2, as well as motifs described in other genes involved in phenylpropanoid metabolism. A. thaliana PAL3 was expressed at very low levels under the conditions examined.

Plant homeodomain protein involved in transcriptional regulation of a pathogen defense-related gene.

Transcription of the parsley pr2 gene, encoding pathogenesis-related protein 2 (PR2), is rapidly stimulated by fungal or bacterial elicitors. Previous work has revealed a 125-bp region within the pr2 promoter; this region encompasses all important cis-regulatory elements required for fungal elicitor-mediated expression. We now report the identification of a functionally relevant 11-bp DNA motif (CTAATTGTTTA) contained within this region; it specifically binds to factors present in both parsley and Arabidopsis nuclear protein extracts. From both plant species, full-length cDNA clones were isolated that encode proteins with high affinity fo this DNA motif. The proteins from both species contain stretches of 61 amino acids that are characteristic of homeodomain (HD) proteins. Binding studies and use of a polyclonal antiserum raised against a fusion polypeptide of glutathione S-transferase with the HD portion of the parsley protein indicated that the 11-bp DNA motif is a potential in vivo target site and that the HD protein is contained within the observed complex formed between the DNA motif and nuclear protein extracts. Transient expression studies using the authentic and a mutated target site suggested a functional role of the HD-DNA interaction in the regulation of the pr2 gene expression.