Controlling ethylene responses in flowers at the receptor level.

For a vast number of ornamental species, blocking the plant's response to ethylene is an efficient strategy to enhance the longevity of the flowers. The most effective ways to conduct such interference will be reviewed in this paper. A large number of chemical compounds have been evaluated for their effects on ethylene production and perception. Among these are a range of strained olefines. This has resulted in the discovery that cyclopropenes, among them 1-methylcyclopropene (1-MCP) and a number of other substituted cyclopropenes effectively block ethylene responses at the receptor level. A lot of testing remains to be done to uncover the full potential of these compounds, but they do offer promising new ways to extend the postharvest life of ornamentals. Also genetic modification appears to be a very effective way in controlling of ethylene synthesis and perception. Attempts to use both a reduced endogenous ethylene production and a reduced sensitivity to ethylene will be reviewed. Among these the use of the mutant ethylene receptor gene, etr1-1, from Arabidopsis seems most promising, especially when it is expressed under the control of a flower specific promoter.

Ethylene perception by the ERS1 protein in Arabidopsis.

Ethylene perception in Arabidopsis is controlled by a family of five genes, including ETR1, ERS1 (ethylene response sensor 1), ERS2, ETR2, and EIN4. ERS1, the most highly conserved gene with ETR1, encodes a protein with 67% identity to ETR1. To clarify the role of ERS1 in ethylene sensing, we biochemically characterized the ERS1 protein by heterologous expression in yeast. ERS1, like ETR1, forms a membrane-associated, disulfide-linked dimer. In addition, yeast expressing the ERS1 protein contains ethylene-binding sites, indicating ERS1 is also an ethylene-binding protein. This finding supports previous genetic evidence that isoforms of ETR1 also function in plants as ethylene receptors. Further, we used the ethylene antagonist 1-methylcyclopropene (1-MCP) to characterize the ethylene-binding sites of ERS1 and ETR1. We found 1-MCP to be both a potent inhibitor of the ethylene-induced seedling triple response, as well as ethylene binding by yeast expressing ETR1 and ERS1. Yeast expressing ETR1 and ERS1 showed nearly identical sensitivity to 1-MCP, suggesting that the ethylene-binding sites of ETR1 and ERS1 have similar affinities for ethylene.

Partial Purification of an Ethylene-binding Component from Plant Tissue.

An ethylene binding component(s) has been partially purified from mung bean sprouts. Tissue was homogenized in 0.3 molar sucrose and 0.2 molar potassium phosphate buffer (pH 7.0). The homogenate was centrifuged, and resuspended fractions were assayed by incorporating them onto cellulose fibers (0.7 grams per milliliter). These were exposed to [(14)C]ethylene (3.7 x 10(-2) microliters per liter of 120 millicurie per millimole) in the presence or absence of 1000 microliters per liter unlabeled ethylene. The cellulose was transferred to separate containers and the [(14)C]ethylene was absorbed in mercury perchlorate and counted. Distribution of ethylene binding to various fractions was: 0 to 3,000g, 3%; 3,000 to 12,000g; 4%; 12,000 to 100,000g, 69%; cellular debris, 24%; 100,000g supernatant, 0%. Adjustment of the pH to 4.0 precipitates the ethylene-binding component. Neutralization, addition of Triton X-100, and readjustment of the pH to 4.0 "solubilized" most of the binding component. Further purification was obtained by chromatography on CM-Sephadex in 10 millimolar potassium acetate buffer, (pH 5.0) containing 1% Triton X-100. Elution was with 200 millimolar potassium phosphate (pH 6.0) containing 1% Triton X-100. Upon treatment of the Triton "solubilized" component with cold acetone, over 90% of the binding capacity was lost. Extraction of the acetone-precipitated residue with 2% Triton X-100 restored some of the binding capacity which was found in the soluble fraction. The pH optimum for binding is 6.0. Passing the Triton X-100 extract of the acetone powder through Sepharose 6B provides considerable purification. The binding component moved ahead of most of the protein.

Measurement of ethylene binding in plant tissue.

Tobacco leaves were exposed to (14)C-labeled ethylene (3.7 x 10(-2) microliters per liter) in the presence and absence of unlabeled ethylene and other compounds. Most of the [(14)C]ethylene appears to be bound to displaceable sites. Lineweaver-Burk plots for a one-half maximum response in a tobacco leaf respiration test gave a value of 0.3 microliter per liter for ethylene, 50 microliters per liter for propylene, and 266 microliters per liter for carbon monoxide. Scatchard plots for displacement of [(14)C]ethylene from the site gave 0.27 microliters per liter for ethylene, 42 microliters per liter for propylene, and 746 microliters per liter for carbon monoxide. At 2%, CO(2) displaces about 35% of the bound ethylene, but increasing the concentration to 10% does not displace the remaining [(14)C]ethylene. A value of 3.5 nanomolar was calculated for the concentration of ethylene-binding sites available to exogenous ethylene. This does not account for the sites occupied by endogenous ethylene, and the total number of binding sites is probably somewhat higher. Using tissue culture material, the system was shown to be stable to freezing and thawing; and the pi-acceptors, carbon monoxide, cyanide, n-butyl isocyanide, phosphorous trifluoride, and tetrafluoroethylene, were shown to compete with ethylene for binding.

Transport of bovine milk xanthine oxidase into mammary glands of the rat.

This research investigated transport of bovine milk xanthine oxidase into mammary glands of the lactating rat. Transport capability suggested an exogenous, nonmammary, source for the enzyme. Five lactating rats were injected intracardially with 100 microgram of purified iodine-125 labeled xanthine oxidase and five were injected with 100 microgram of the enzyme unpurified. Four hours later the rodents were hand-milked, and radiation was confirmed in all samples by liquid scintillation counting. Counts were recorded per volume of milk and the percentage radiation was computed. Autoradiographs of the rats indicated radiation almost exclusively associated with the mammary glands. Greatest concentration of radioactivity was in the micellar casein fraction of milk, and a compound of high molecular weight, presumably [iodine-125]xanthine oxidase, was confirmed by gel filtration of the casein. Results suggest that the compound was transported into the mammary glands. The degree of transport was dependent upon the stage of lactation.