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Pseudomonas sp. strain ACP is capable of growth on l-aminocyclopropane-l-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to a-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The Pseudomonas sp. strain ACP and the yeast Hansenula saturnus are capable of utilizing the cyclopropanoid amino acid 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase (EC 4.1.99.4) in these organisms (8). Pseudomonas ACC deaminase has an estimated molecular mass of 110 kDa and is composed of three identical subunits, each with a molecular mass estimated to be 36.5 kDa (7,8). ACC deaminase utilizes pyridoxal 5'-phosphate as a cofactor in catalyzing the cleavage of ACC to a-ketobutyrate and ammonia (7,22).ACC was originally identified as a natural product in the juices of several fruits (2) and is now regarded as a key intermediate in the biosynthesis of the plant hormone ethylene (23). Ethylene is biosynthesized in higher plants from methionine via S-adenosylmethionine and ACC (1, 24). The rate-limiting step in ethylene biosynthesis is the production of ACC by a ring-closing displacement of methylthioadenosine from S-adenosylmethionine in a reaction catalyzed by ACC synthase (1). The reverse of this reaction can be viewed as analogous to the reaction catalyzed by ACC deaminase (22).Ethylene influences many aspects of plant development, including leaf and flower senescence and fruit ripening (23). Techniques for the integration and expression of bacterial genes in plants utilizing Agrobacterium tumefaciens are available (14), and the expression of an ACC deaminase gene in plants may provide a means to perturb ACC levels and ethylene biosynthesis, thus leading to a better understanding of the role of this plant hormone. As an initial step in this process, the ACC deaminase gene from Pseudomonas sp. strain ACP was isolated and characterized. The amino acid * Corresponding author. sequence of purified ACC deaminase was determined and used to design oligonucleotide primers. The primers were used in polymerase chain reactions (PCR) to isolate a portion of the gene, which was used to screen a Pseudomonas genomic library. The DNA sequence of the ACC deaminase gene is presented, and its authenticity is demonstrated by expression of the gene in Escherichia coli. MATERIALS AND METHODSBacterial strains and growth media. Lambda Zap II and E. coli XL1-Blue and SURE were obtained from Stratagene (La Jolla, Calif.). Bluescript plasmids pBCSK+, pBSK-, and pBCKS+ (Stratagene) in E. coli SURE or DH5a (5) were used for DNA sequencing. pUC19 (25) was used for subcloning in E. coli SURE. E. coli XL1-Blue and SURE strains were maintained on Luria-Bertani (LB) agar plates supplemented with 20 jig of tetracycline per ml and routinely grown at 37°C in LB medium or Terrific broth (T-broth) (13). Pseudomonas sp. strain ACP was grown and maintained in T-broth or 2% glucose4.5% Bacto-Peptone (Difco)-0.3% yeast e...

SummaryThis paper describes the analysis of tomato plants transformed with a chimeric gene consisting of the promoter region of a fruit specifically expressed tomato gene linked to the iptgene coding sequences from the Ti plasmid of Agrobacterium tumefaciens. The pattern of expression of this chimeric gene was found to be consistent with the expression of the endogenous fruit-specific gene and consequently, plants expressing the chimeric gene were phenotypically normal until fruit maturation and ripening. A dramatically altered fruit phenotype, islands of green pericarp tissue remaining on otherwise deep red ripe fruit, was then evident in many of the transformed plants. Cytokinin levels in transformed plant fruit tissues were 10 to lO0-fold higher than in control fruit. In the leaves of a fruitbearing transformant, despite a lack of detectable ipt mRNA accumulation, approximately fourfold higher than control leaf levels of cytokinin were detected. It is suggested that cytokinin produced in fruit is being transported to the leaves since accumulation in leaves of PR-1 and chitinase mRNAs, which encode defenserelated proteins known to be induced by cytokinin, occurred only when the transformant was reproductively active. Effects of elevated cytokinin levels on tomato fruit gene expression and cellular differentiation processes are also described.

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