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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.

We demonstrate that phenylalanine ammonia‐lyase (PAL) in parsley (Petroselinum crispum) is encoded by a small family of at least four genes. The levels of mRNA from three identified PAL genes increase considerably upon treatment of cultured parsley cells with UV light or fungal elicitor and upon wounding of parsley leaves or roots. In cultured cells these changes were shown to involve transcriptional activation. We present the first primary structure of a plant PAL gene (parsley PAL‐1) and the deduced amino acid sequence of the enzyme. Inducible in vivo footprints in the PAL‐1 promoter define two nucleotide sequences, within the motifs CTCCAACAAACCCCTTC and ATTCTCACCTACCA, involved in the responses to both UV irradiation and elicitor application. These motifs are conserved at similar positions in several elicitor or light‐responsive genes from different species. In two cases they are found within short regions known to confer elicitor or UV‐light inducibility. The conserved motifs in the parsley 4‐coumarate:CoA ligase gene, which is coordinately regulated with PAL, also display UV‐light inducible in vivo footprints. Taken together, our findings suggest a general role of these putative cis‐acting elements in the responses of plants to such stresses.

Fungal elicitor stimulates a multicomponent defense response in cultured parsley cells (Petroselinum crispum). Early elements of this receptor-mediated response are ion f luxes across the plasma membrane and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Omission of Ca 2؉ from the culture medium or inhibition of elicitor-stimulated ion f luxes by ion channel blockers prevented the latter three reactions, all of which were triggered in the absence of elicitor by amphotericin B-induced ion f luxes. Inhibition of elicitor-stimulated ROS production using diphenylene iodonium blocked defense gene activation and phytoalexin accumulation. O 2 ؊ but not H 2 O 2 stimulated phytoalexin accumulation, without inducing proton f luxes. These results demonstrate a causal relationship between early and late reactions of parsley cells to the elicitor and indicate a sequence of signaling events from receptor-mediated activation of ion channels via ROS production and defense gene activation to phytoalexin synthesis. Within this sequence, O 2 ؊ rather than H 2 O 2 appears to trigger the subsequent reactions.

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Four cis‐acting elements, designated as Boxes I, II, III and IV, have previously been identified as functionally relevant components of the light‐responsive chalcone synthase (CHS) promoter in parsley (Petroselinum crispum). This paper describes the isolation of three cDNAs encoding proteins which bind specifically to Box II, one of two cis‐acting elements found within a 52 bp CHS promoter region shown here to be sufficient for light responsiveness in parsley. The deduced amino acid sequences of all three proteins reveal conserved basic and leucine zipper domains characteristic of transcription factors of the bZIP class. Nucleotide sequences recognized by these factors contain an ACGT motif common to many cis‐acting elements. Therefore, we have termed the proteins CPRF‐1, −2 and −3 (Common Plant Regulatory Factor). The characteristics of CPRF‐1 binding to Box II and the timing of transient CPRF‐1 mRNA accumulation during light exposure of previously dark‐grown parsley cells are consistent with the hypothesis that this factor participates in the light‐mediated activation of the CHS gene in parsley.

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