Topic 22.4

ACC Synthase Gene Expression and Biotechnology

As we have seen, ACC synthase is a cytosolic enzyme that catalyzes the first committed step in ethylene biosynthesis in higher plants. It is a key regulatory enzyme that catalyzes the production of the ethylene precursor ACC from AdoMet (S-adenosylmethionine). However, this enzyme is difficult to purify because it is labile and present in low abundance in plant tissues. The molecular cloning and functional expression of ACC synthase in E. coli and yeasts have facilitated biochemical and structural studies of this enzyme.

ACC synthase cDNAs and genomic sequences have been cloned from numerous plant species. The emerging picture from the study of these genes is that ACC synthase is encoded by members of a divergent multigene family in which each gene is differentially regulated by various environmental and developmental factors during the growth of a plant. Arabidopsis, for example, has twelve and tomato has at least nine gene members, which are differentially regulated by inducers such as auxin, fruit ripening, and wounding (Olson et al. 1991; Rottmann et al. 1991; Abel et al. 1995).

The protein synthesis inhibitor cycloheximide is a potent inducer of many ACC synthase genes. Cycloheximide inducibility is a characteristic of primary response genes such as the early auxin-inducible genes (see textbook Chapter 19). Two mechanisms have been proposed to explain this inducibility.

First, the transcription of these genes may be under the control of a short-lived repressor protein whose synthesis is prevented by the addition of cycloheximide;

Second, the mRNA transcripts of these genes may be short-lived and are stabilized when cycloheximide prevents the synthesis of a nuclease.

The induction of at least one of the genes for ACC synthase (ACS2) in Arabidopsis by cycloheximide appears to be due to transcriptional activation (Liang et al. 1996), and thus is likely regulated by a short-lived transcriptional repressor.

A clue to the signal transduction mechanism involved in the regulation of ACC synthase gene expression has come from experiments with lithium (Li⁺). Li⁺ is known to interfere with the phosphoinositide signaling pathway (see Chapter 14 on this web site) by inhibiting the activity of inositolphosphate phosphatase. Li⁺ is also one of the strongest inducers of ACC synthase activity in plants (Liang et al. 1996). This finding suggests that the regulation of at least some ACC synthase genes may involve Ca²⁺ mobilization mediated by inositol trisphosphate (IP₃) (see Chapter 14 on this web site).

Because ethylene plays such an important role in fruit ripening, the cloning of the genes for ACC synthase and ACC oxidase has enabled scientists to use biotechnology to reduce ethylene production during fruit ripening, and thus to prolong the storage life of fruit.

In the case of tomato, two different strategies have been employed to control ethylene production.

The first strategy was to use the Ti plasmid of Agrobacterium tumefaciens to transform tomato tissue with antisense DNA for ACC synthase or ACC oxidase. When the genes for the two enzymes are expressed in the reverse direction in the cell, the resulting "antisense transcript" binds to and inactivates the "sense transcript" produced by the wild-type gene, effectively shutting down expression of the wild-type gene (Hamilton et al. 1990; Oeller et al. 1991; Picton et al. 1993). After transformation, intact plants can be regenerated in tissue culture.

In the second strategy for reducing ethylene production, tomato tissue was transformed (using A. tumefaciens) with genes encoding enzymes that metabolize ACC or AdoMet (Klee et al. 1991; Good et al. 1993). In this case, the ACC that is produced is rapidly degraded, and no ethylene can be synthesized.

Regardless of the strategy used, all fruits from these genetically engineered plants show significant delays in ripening. Tomato fruits from plants transformed with the ACC synthase antisense DNA fail to ripen unless treated with ethylene (Web Figure 22.3.A).

Web Figure 22.3.A Inhibition of tomato fruit ripening in detached tomato fruits by antisense ACC synthase RNA. Antisense tomato fruits (center) remain firm and green unless treated by ethylene. Ethylene-treated antisense fruits (left) are indistinguishable from naturally ripened fruits (right) in terms of color, flavor, aroma, and texture. (Reprinted with permission from Oeller et al. 1991.) (Click image to enlarge.)

Similar approaches are now being carried out in other agronomically important crops, such as melon and banana. In addition, these approaches are being used to retard floral senescence in carnations, thereby reducing postharvest losses. The research on ACC synthase and other ethylene biosynthesis genes thus provides an excellent example of how basic research can lead to improvements in agricultural and horticultural crops, the full benefits of which we are only beginning to realize.

References

Abel, S., Nguyen, M., Chow, W., and Theologis, A. (1995) ACS4, a primary indole-acetic acid-responsive gene encoding 1-aminocyclopropane-1-carboxylate synthase in Arabidopsis thaliana. J. Biol. Chem. 270: 19093–19099.

Olson, D. C., White, J. A., Edelman, L., Harkins, R. N., and Kende, H. (1991) Differential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruits. Proc. Natl. Acad. Sci. USA 88: 5340–5344.

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) 1-aminocyclopropane-1-carboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence. J. Mol. Biol. 222: 937–961.