In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries.

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 (< or = 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.

LE-ACS4, a fruit ripening and wound-induced 1-aminocyclopropane-1-carboxylate synthase gene of tomato (Lycopersicon esculentum). Expression in Escherichia coli, structural characterization, expression characteristics, and phylogenetic analysis.

ACC (1-aminocyclopropane-1-carboxylic acid) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene and is encoded by a highly divergent multigene family in tomato (Rottmann, W. H., Peter, G. F., Oeller, P. W., Keller, J. A., Shen, N. F., Nagy, B. P., Taylor, L. P., Campbell, A. D., and Theologis, A. (1991) J. Mol. Biol. 222, 937-961). Two members of the family, LE-ACS2 and LE-ACS4, are induced during fruit ripening and upon treatment of mature green fruits with exogenous ethylene (C2H4) in a dose-dependent manner. Both genes are superinduced by wounding of pericarp tissue during various stages of ripening. The wound-induced accumulation of LE-ACS2 mRNA is more rapid and greater than that of LE-ACS4. Both mRNAs accumulate in the absence of protein synthesis, suggesting that their induction is a primary response to the inducer. The LE-ACS4 gene was isolated and structurally characterized. The function of the LE-ACS4 protein (53,509 Da, pI 5.4) was verified by expression experiments in Escherichia coli. The promoters of LE-ACS2 and LE-ACS4 contain potential cis-acting regulatory elements responsible for induction by ethylene, wounding, and anaerobiosis. In addition, elements for binding the transcriptional factors EmBP1, GBF-1, and OCSBF-1 are also present. Phylogenetic analysis of 20 ACC synthases from dicots and monocots indicate that the LE-ACS2 and LE-ACS4 proteins belong to an unique sublineage that includes an additional member of the tobacco family, NT-ACS1. The divergence of this sublineage is a relatively recent event in the evolution of ACC synthase protein.

Transcriptional Analysis of Polygalacturonase and Other Ripening Associated Genes in Rutgers, rin, nor, and Nr Tomato Fruit.

We have studied the transcription of polygalacturonase (PG) and several other riponing-associated genes in wild-type tomato (Lycopersicon esculentum) fruit and three ripening-impaired mutants, rin, nor, and Nr. In wild-type fruit, the PG gene becomes transcriptionally active early in ripening and remains transcriptionally active during the ripening process. Fruit of the three ripening-impaired mutants, which have reduced levels of PG mRNA, have correspondingly reduced PG transcription rates. Other ripening-associated genes showed diverse patterns of expression in the ripening-impaired mutant backgrounds. These results indicate that transcriptional activation of the PG gene is an important control point regulating the expression of PG during ripening in wild-type fruit and that PG expression in rin, nor, and Nr fruit is blocked at the level of transcription. A comparison of PG transcription rates and mRNA levels with those of other ripening-associated genes suggests that posttranscriptional processes may also contribute to the large accumulation of PG mRNA during ripening.

Regulation of Gene Expression by Ethylene in Wild-Type and rin Tomato (Lycopersicon esculentum) Fruit.

Levels of ethylene biosynthesis and ethylene-inducible gene expression in wild-type tomato (Lycopersicon esculentum) fruit and in nonripening fruit from the tomato mutant rin (ripening inhibitor) were compared in order to investigate the mechanism of ethylene action. Whereas wild-type tomato fruit dramatically increase the rate of ethylene biosynthesis at the onset of ripening, rin fruit constitutively produce ethylene at a low basal level. We have compared the mRNA levels and transcription rates of four cloned ethylene-inducible genes (JE Lincoln, S Cordes, E Read, RL Fischer 1987 Proc Natl Acad Sci USA 84: 2793-2797) during wild-type and rin fruit development. In wild-type fruit, both mRNA levels and transcription rates of these genes increase. The effect of the rin mutation on gene expression is different for each ethylene-inducible gene. In one case expression is completely suppressed, while in other instances it is either partially inhibited or relatively unaffected by the mutation. The mRNA levels of each of these genes in response to exogenous ethylene in rin fruit was also measured. The mRNAs for all four genes accumulate to similar levels in both ethylene treated rin and ethylene treated wild-type fruit. These results are discussed with regard to the response of plants to ethylene hormone at the level of gene expression.

Diverse mechanisms for the regulation of ethylene-inducible gene expression.

We have investigated the mechanism of action of the plant hormone ethylene by analyzing the expression of ethylene-inducible genes isolated from tomato (Lycopersicon esculentum). We have found that the expression of each cloned gene is regulated by ethylene in a unique manner. That is, for certain genes ethylene affects transcriptional processes, while for another gene it affects both transcriptional and post-transcriptional processes. Furthermore, induction of gene transcription by ethylene is organ specific for one gene, while for others it is not. In addition, we have measured gene expression as a function of ethylene concentration and have found that each gene displays a unique ethylene dose-response curve. Our results suggest that ethylene modulates gene expression by a variety of mechanisms.

Regulation of gene expression by ethylene during Lycopersicon esculentum (tomato) fruit development.

We have investigated the regulation of gene expression by the plant hormone ethylene by cloning mRNAs that accumulate in unripe tomato fruit (Lycopersicon esculentum) exposed to exogenous ethylene. The response to exogenous ethylene is rapid; within 30-120 min we detect an increase in the cloned mRNA concentrations. DNA sequence analysis indicates that one of the ethylene-inducible genes is related to a gene encoding wound-inducible proteinase inhibitor I. We have measured ethylene production during fruit development and detect low basal levels in unripe fruit and much higher levels in ripening fruit. Blot hybridization experiments show that expression of the cloned genes is developmentally regulated by ethylene during fruit ripening: the mRNAs produced by these genes are more abundant in ripe fruit than in unripe fruit, and this mRNA accumulation is repressed by a competitive inhibitor of ethylene action, norbornadiene. However, during fruit development some of the cloned mRNAs begin to accumulate when ethylene production is at a basal level, whereas other mRNAs begin to accumulate later when the endogenous ethylene concentration increases, suggesting that gene expression during fruit development can be activated by ethylene in two ways. In some cases gene expression is primarily activated by an increase in sensitivity to basal ethylene levels, whereas in other cases it may be regulated by an increase in ethylene concentration.