Arabidopsis thaliana plants treated with exogenous cytokinins accumulate anthocyanin pigments. We have characterized this response because it is potentially useful as a genetic marker for cytokinin responsiveness. Levels of mRNAs for four genes of the anthocyanin biosynthesis pathway, phenylalanine ammonia lyase 1 (PAL1 ), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) were shown to increase coordinately in response to benzyladenine (BA). However, nuclear run-on transcription experiments suggested that although CHS and DFR are controlled by BA at the transcriptional level, PALl and CHI are controlled by BA posttranscriptionally. CHS mRNA levels increased within 2 h of BA spray application, and peaked by 3 h. Levels of PALl mRNA did not increase within 6 h of BA spray. We also showed that PAL1, CHS, CHI, and DFR mRNA levels fluctuate during a 24-h period and appear to be controlled by a circadian clock. The relation between cytokinin regulation and light regulation of CHS gene transcription is discussed.Cytokinins are important regulators of many aspects of plant development, including cell division, nutrient mobilization, senescence, chloroplast development, and apical dominance (Binns, 1994). Despite the widely acknowledged importance of cytokinin in plant development, very little is known about its mechanism of action at the molecular level.Cytokinins have been shown to affect the expression of specific genes by both increasing and decreasing the abundance of particular proteins or "As.A partia1 list of genes that are up-regulated by cytokinin includes nitrate reductase, rbcS, cab, hydroxypyruvate reductase in excised pumpkin cotyledons, a "multiple stimulus response" gene in Nicotiana plumbaginifolia cells, the early nodulin gene SrEnod2 from Sesbania rostrata, and a number of unidentified cDNAs from cultured soybean cells (Flores and Tobin, 1986;Chen, 1989;Crowell et al., 1990;Stabel et al., 1990;Dehio and de Bruijn, 1992;Dominov et al., 1992). Expression of phytochrome and a cucumber gene encoding a cDNA called CR9 are repressed by cytokinin (Cotton et al., 1990;Teramoto et al., 1994).

Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes,prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of l-tryptophan to form 7-chloro-l-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-l-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.

Pyrrolnitrin is a secondary metabolite of Pseudomonas and Burkholderia sp. strains with strong antifungal activity. Production of pyrrolnitrin has been correlated with the ability of some bacteria to control plant diseases caused by fungal pathogens, including the damping-off pathogen Rhizoctonia solani. Pseudomonas fluorescens BL915 has been reported to produce pyrrolnitrin and to be an effective biocontrol agent for this pathogen. We have isolated a 32-kb genomic DNA fragment from this strain that contains genes involved in the biosynthesis of pyrrolnitrin. Marker-exchange mutagenesis of this DNA with Tn5 revealed the presence of a 6.2-kb region that contains genes required for the synthesis of pyrrolnitrin. The nucleotide sequence of the 6.2-kb region was determined and found to contain a cluster of four genes that are required for the production of pyrrolnitrin. Deletion mutations in any of the four genes resulted in a pyrrolnitrin-nonproducing phenotype. The putative coding sequences of the four individual genes were cloned by PCR and fused to the tac promoter from Escherichia coli. In each case, the appropriate tac promoter-pyrrolnitrin gene fusion was shown to complement the pyrrolnitrin-negative phenotype of the corresponding deletion mutant. Transfer of the four gene cluster to E. coli resulted in the production of pyrrolnitrin by this organism, thereby demonstrating that the four genes are sufficient for the production of this metabolite and represent all of the genes required to encode the pathway for pyrrolnitrin biosynthesis.

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