Control of Ethylene Production in Flowers Progress Report — June 1997
Date 5/28/97
Title of Project: CONTROL OF ETHYLENE PRODUCTION IN FLOWERS
Institute where work is being conducted: Pennsylvania State University
Amount of Endowment Grant: $10,000
Covering Period: 9/11/96 - 8/31/97
Anticipated Date of Project Completion/Final Report: 8/31/98
Individual Conducting Project:
Kathleen Brown - Assoc Professor
Objective 1. Determine the activity of ACC synthase in flower tissues of pollinated and unpollinated flowers.
We have determined the activity of ACC synthase in two ways. In vivo activity. We began by measuring in vivo ACC synthase activity. Flowers were treated with COC12 for 2 hours to inhibit ACC oxidase activity, allowing the product of ACC synthase activity, ACC, to accumulate in the tissue rather than be converted to ethylene. ACC and its primary conjugated form, MACC, were extracted from pollinated and unpollinated flowers and assayed according to Lizada and Yang (Lizada and Yang 1979). ACC synthase activity was estimated from the accumulation of ACC and MACC. Conjugated ACC, presumably MACC (but other conjugates are possible), constituted the bulk of the ACC found. The activity of ACC synthase in geranium pistils was estimated at 1.8 nmoles/pistil/hr at 2 hours after pollination, which was 5-6x higher than unpollinated controls.
In vitro activity. The usual method of estimating enzyme activity is to make an extract of the tissue, partially purify the enzyme, and then measure its activity by supplying the extract with substrate (S-adenosyl methionine) and measuring the product of the reaction (ACC). ACC synthase is particularly difficult to extract and purify because half the activity is lost every 30 Minutes. Also our tissue, the pistil of a pollinated geranium flower, is very small and it is difficult to obtain a large quantity of pollinated pistils. The activity is lost if tissue is kept frozen for more than a few days. We have therefore spent a great deal of time working out procedures for this assay and testing them, first on tomato and then on geranium. We have, on occasion, found a small amount of activity in geranium pistils, and in some experiments found 4-1Ox as much activity in pollinated pistils as in controls, but other times we get no activity or very little difference. We are still working on extraction and tissue storage/handling techniques to improve our ability to get reliable results.
Objective 2. Determine the activity of ACC oxidase in flower tissues of pollinated and unpollinated flowers.
We have measured the activity of ACC oxidase by an in vivo assay. Pollinated and unpollinated pistils were divided into two parts: sterile overy+ovary and stigma+style, and 5 of each were placed in vials containing 0.1 mM ACC (the substrate). The amount of ethylene (the reaction product) accumulating in the vials was measured after 1.5 hours. As expected, geranium pistils had high ACC oxidase activity even before pollination. Activity increased after pollination by about 50% in the stigma+style tissue and by less than 20% in the ovary+sterile ovary. There was much more activity in the stigma+style tissue than in the ovary+ sterile ovary, but the latter tissue contains the abscission zone, where ethylene has its effect. Since there was little increase in ACC oxidase in the vicinity of the abscission zone, and since there was
already considerable activity before pollination, we concluded that changes in ACC oxidase activity are probably not very important for the enhancement of ethylene production by pollination.
Objective 3. Use inhibitors to determine whether mRNA and protein synthesis and/or protein phosphorylation are required for enzyme activity changes in response to pollination.
We tested the effect of cycloheximide, a protein synthesis inhibitor, on ethylene production in pollinated and unpollinated flowers. We had previously shown that cycloheximide can completely block petal abscission (Evensen et al. 1993). However, it had no effect whatsoever on ethylene production. This means that protein synthesis, i.e. the synthesis of new enzyme, is not required for ethylene production in geranium flowers.
Conclusions
Our results support our original hypothesis that ACC synthase activity is the most important determinant of ethylene production in pollinated pistils. Unpollinated flowers make only low amounts of ethylene, insufficient to cause petal abscission. These flowers express genes coding for both ACC synthase and ACC oxidase and have fairly high ACC oxidase activity even before pollination, so none of these can be limiting to ethylene production. ACC synthase activity, however, is low in unpollinated flowers and is strongly induced in pollinated flowers. Given the fact that ethylene production is not blocked by the protein synthesis inhibitor cycloheximide, the synthesis of new proteins (enzymes) must not be required for ethylene production in response to pollination. It seems likely that ACC synthase genes are expressed (or “on”) in the sense that mRNA is formed and the protein is synthesized even before pollination. However, in unpollinated flowers, there must be a factor that suppresses ACC synthase activity, keeping ethylene production too low to cause petal abscission. Pollination removes or inactivates this factor, so that ethylene production can increase and effect petal abscission.
Plans for the future
We would like to get accurate measurements of in vitro ACC synthase activity, if possible, both to verify the in vivo results and to use in tests of phosphorylation and dephosphorylation inhibitors (objective 3). If we can reliably get high enough activity to do so, we will check whether inhibitors of protein phosphatase and protein kinase affect activity. If we cannot get adequate in vitro activity, we will try feeding the inhibitors into living flowers and measuring in vivo activity. These results will give us clues about the suppression of ACC synthase activity in unpollinated flowers. The factor responsible for suppressing ACC synthase could be an important target for genetic and chemical methods to manipulate ethylene production.
References
Evensen, K. B., Page, A. M. & Stead, A. D. 1993. Anatomy of
ethylene-induced petal abscission in Pelargonium x hortorum. - Ann
Bot 71: 559-566.
Lizada, M. C. C. & Yang, S. F. 1979. A simple and sensitive assay
for I-aminocyclopropane-1- carboxylic acid. - Anal. Biochem. 100: 140-145.
