A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (<0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.

We have exploited the positional gradient of cellular differentiation in Zea mays leaves to study the accumulation of mRNAs encoding subunits ofthe two COrfixing enzymes and the major chlorophyll-binding protein. These multi-step scheme of CO2 fixation called C4 photosynthesis. PEPCase3, the enzyme responsible for initially fixing the atmospheric CO2 in maize, is found only in mesophyll cells (22). The four-carbon acid resulting from this reaction is shuttled to bundle sheath cells where it is decarboxylated, supplying CO2 to carry out the reductive pentose phosphate cycle, the initial product of which is a three-carbon compound, as in C3 plants (4). RuBPCase is found only in the chloroplasts of bundle sheath cells (12). This compartmentalization of the carboxylases confers photosynthetic advantage to C4 plants under conditions of high temperature and light intensity (11). In addition to these enzymic differences between the mesophyll and bundle sheath cells of C4 plants, there are differences in the chloroplast membrane proteins of the two cell types. Membrane proteins from maize mesophyll cell chloroplasts are essentially identical to those of several C3 plants, containing polypeptides responsible for the light-harvesting and energy-transduction reactions of photosynthesis (2). The bundle sheath cell chloroplasts, however, contain little if any of the PSII protein complex and its associated LHCP (1; Schuster G, I Ohad, B Martineau, WC Taylor, unpublished). We have outlined the patterns of mRNA accumulation for four of the compartmentalized polypeptides related to C4 photosynthesis by using hybridization with DNA probes to measure accumulation of RuBPCase (LSu and SSu), PEPCase, and LHCP mRNAs in cells of increasing age and stage of differentiation. We compare our patterns of mRNA accumulation for these proteins to the patterns of their polypeptide accumulation established by Mayfield and Taylor (17) in a similar study. We have monitored leaf morphological development by using a cytological marker to indicate fully differentiated vascular bundles, again taking advantage of the gradient of cellular development in the young leaves we have chosen to study.MATERIALS AND METHODS Plant Material. Seeds from an inbred line of Zea mays (B73, a gift of Pioneer Hi-Bred International, Johnston, IA) were planted in a 2:1 mixture of soil and sand, covered with 2 cm of vermiculite, and germinated and grown in a growth chamber. Periods of 16 h light (4 x 104 lux) and 8 h dark, at 25°C, were used. Plants were harvested 10 to 14 d after germination when the third leaf to emerge from the coleoptile was 12 to 16 cm long. A third leaf of this age has not yet developed a ligule, the structure which delineates the leaf blade from its sheath. The coleoptile and first and second leaves were removed from each plant. The third leaf was cut at points 1, 2, 4, 6, and 8 cm from the leaf base. The six resulting leaf segments, representing populations of cells of the same relative age and stage of differentiation, were separated and...

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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.