Plant Molecular Biology Reporter pages 192-194 Volume 9(3) 1991

Commentary

H o w Plant Molecular Biologists Revealed a Surprising Relationship Between Two Enzymes,

Which Took an Enzyme Out of a Membrane Where It was not Located, and Put it into the

Soluble Phase Where It Could be Studied

Philip John

Department of Agricultural Botany, Plant Science Laboratories, University of Reading, Reading RG6 2AS, UK

T he final step in the biosynthesis of the plant growth regulator, ethylene, is catalysed by the ethylene-forming enzyme (EFE), which is readily assayed in vivo by supplying tissues with its

substrate, 1-aminocyclopropane-l-carboxylic acid (ACC), but until re- cently the enzyme had never been studied in vitro because it was invariably found that activity disappears when tissue is homogenised (Yang and Hoffman, 1984). Some EFE activity is retained by vacuoles isolated from leaf rnesophyll (Guy and Kende, 1984) and by membrane vesicles in the juice squeezed from kiwifruits (Mitchell et al., 1988), but these systems retain only about 1% (Porter et al., 1986) or less (Mitchell et al., 1988) of the in-vivo activity, and their activity disappears com- pletely when membrane integrity is lost (Porter et al, 1986; Mayne and Kende, 1986).

The earlier work had given rise to a firm belief in everyone's mind that enzyme activity depended on membrane integrity (Yang and Hoffman, 1984). The present author even went so far as to propose that EFE activity was associated with proton translocation across the plasma membrane, and that activity depended on the maintenance of a membrane potential (John, 1983). However this Mitchellian EFE did not readily find experi- mental support, even from our own laboratory (John et al., 1986)! False

Abbreviations: ACC, 1-aminocyclopropane-l-carboxylic acid; EFE, ethylene- forming enzyme.

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Commentary 193

trails were also started up by the discovery of cell-free systems (always membrane systems) which were capable of converting ACC to ethylene, but in these preparations ethylene was shown to be generated by nonen- zymatic reactions between ACC and free radicals which resulted from lipoxygenase activity CYang and Hoffman, 1984). All in all, little progress was being made in characterising EFE. Until, that is, the molecular biologists entered the scene.

In 1990 Hamilton et al. reported that they had identified a gene (pTOM13') in tomato which encoded for the EFE, or at least a polypep- tide component of the EFE. The amino acid sequence of this polypeptide resembled that of flavanone-3-hydroxylase, a soluble enzyme respon- sible for catalysing the hydroxylation of 2S flavanones to form 2R, 3R dihydrof lavonols in the biosynthetic pa thway to flavonols and anthocyanidins. There is no obvious relationship enzymatically be- tween flavanone-3-hydroxylase and the EFE. No biochemist had previ- ously proposed an affinity between the two enzymes. Yet the structural relationship implied by the sequence homology provided the vital clue which enabled us to break the impasse reached with the EFE. Stabilisation of the flavanone-3-hydroxylase activity required inter alia anoxic condi- tions, and the presence of Fe 2. and ascorbate (Britsch and Grisebach, 1986). When these conditions were used to extract the EFE from melon fruits, there was a complete recovery of the authentic EFE activity as a soluble enzyme (Ververidis and John, 1991). It is now clear that EFE is a member of a group of soluble oxygenases that require Fe 2. and ascorbate for full activity, and it is now as amenable to biochemical studies as any other enzyme (Ververidis et al., 1991).

What then is the moral of this tale? Quite simply that plant molecular biologists need not wait for their protein to be characterised biochemi- cally; molecular biology can be a very useful prelude to the biochemistry, pointing the biochemists in the right direction.

References

Britsch, L. and H. Grisebach. 1986. Purification and characterization of 2S flavanone-3- hydroxylase from Petunia hybrida. Eur. J. Biochem. 156:569-577.

Guy, M. and H. Kende. 1984. Conversion of 1-amino-cyclopropane-l-carboxylic acid to ethylene in plant tissues. Planta 160:281-287.

Editor's Note. The symbol "pTOM13" is not an accepted form for the name of a gene. It was originally employed by Slater et al. (Plant Mol. Biol, 5:137-147, 1985) to designate a plasmid containing a specific cDNA, but the term was extended in the article cited (Hamilton et al., 1990) to designate the gene encoded by the cDNA.

194 John

Hamilton, A.J., G.W. Lycett, and D. Grierson. 1990. Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346: 284-287.

John, P. 1983. The coupling of ethylene biosynthesis to a transmembrane, electrogenic proton flux. FEBS Lett 152:141-143.

John, P., A.J.R. Porter, and A.J. Miller. 1985. Activity of the ethylene-forming enzyme measured in vim at different cell potentials. J. Plant Physiol. 121:397406.

Mayne, R.G. and H. Kende. 1986. Ethylene biosynthesis in isolated vacuoles of Vicia faba L. requirement for membrane integrity. Planta 167:159-165.

Mitchell, T., A.J.R. Porter, and P. John. 1988. Authentic activity of the ethylene-forming enzyme observed in membranes obtained from kiwifruit (Actinidia deliciosa) New Phytol. 109:313-319.

Porter, A.J.R., J.T. Borlakoglu, and P. John. 1986. Activity of the ethylene-forming enzyme in relation to plant cell structure and organization. J. Plant Physiol.125:207-216.

Ververidis, P. and P. John. 1991. Complete recovery in vitro of ethylene-forming enzyme activity. Phytochemistry 30:725-727.

Ververidis, P., J.J. Smith, and P. John. 1991. Characterization of the ACC oxygenase responsible for ethylene production by plants. Plant Physiol. (suppl.). 96:153.

Yang, S.F. and N.E. Hoffman. 1984. Ethylene biosynthesis and its regulation in higher plants. Ann. Rev. Plant Physiol. 35:155-189.