Role of ethanol in inhibition of climacteric senescence in carnation (Dianthus caryophyllus L.) cut flowers : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Increases in the vase life of carnation flowers have been reported with ethanol and with acetaldehyde. The increase with ethanol has been attributed either to inhibition of ethylene biosynthesis or sensitivity to ethylene or both, whereas acetaldehyde has been reported to inhibit ethylene biosynthesis only. Tomato cultivars responded differently in terms of delay in ripening and inhibition of ethylene production when treated with ethanol. This research was designed to determine whether carnation cultivars responded differently to ethanol or acetaldehyde, and if they did, to determine the underlying reason for the differential responses. Further aims of the research were to determine whether ethanol or acetaldehyde is the beneficial compound which delays senescence in carnation flowers, and to assess the specific role of the effective compound in the biosynthesis of ethylene. Ethanol at various concentrations (0, 2, 4, 6 or 8%) was applied to five carnation cultivars (Yellow Candy, White Candy, Iury, Sandrosa and Francesco) and vase life was assessed by visual assessment. Ethanol at 4 or 6% gave maximum increase in vase life of carnation cvs Yellow Candy (9 or 11 days), White Candy (3 or 7 days) or Iury (5 or 3 days) but did not increase vase life of cvs Sandrosa or Francesco. From these carnation cultivars, three cultivars contrasting in terms of response to ethanol were selected and were treated with 0 or 4% ethanol with or without short term (8h) ethylene exposure (0.1 µ1.1⁻¹). Vase life was assessed visually and ethylene production was measured by gas chromatography using a flame ionisation detector (FID). Ethanol at 4% significantly (P< 0.05) inhibited ethylene biosynthesis and sensitivity to ethylene in cvs Yellow Candy, White Candy and Sandrosa. Cultivar Yellow Candy was a short lasting (10 days), high ethylene producer (245 nl flower⁻¹ h⁻¹) with a 10 day extension of vase life when treated with ethanol. Cultivar White Candy was intermediate lasting (14 days), medium ethylene producer (190 nl flower⁻¹ h⁻¹) and with a 3.5 day extension of vase life whereas cv. Sandrosa was long lasting (19 days), low ethylene producer (<100 nl flower⁻¹ h⁻¹) and non-responsive to ethanol (0%). Acetaldehyde, a metabolite of ethanol, was applied at various concentrations (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 or 0.5%) to two cultivars of carnation (Yellow Candy and Sandrosa). Acetaldehyde at 0.05% significantly (P < 0.001) increased the vase life of carnation cvs Yellow Candy and Sandrosa by 3 days irrespective of their short or long lasting nature. Two cultivars of carnation (Yellow Candy or Sandrosa) were then treated with acetaldehyde (0 or 0.05%) with or without short term (8h) ethylene exposure (0.1 µ1.1⁻¹). Vase life was assessed and ethylene production measured by gas chromatography. Acetaldehyde at 0.05% increased the vase life of carnation cv. Sandrosa by 2 days after short term ethylene exposure. However, acetaldehyde failed to increase vase life of cv. Yellow Candy after short term ethylene exposure or when ethylene was measured. The increase in vase life of cv. Sandrosa was associated with a 5 day delay in the ethylene climacteric peak without short term ethylene exposure and with a 3 day delay after short term ethylene exposure. Ethanol and acetaldehyde can be interconverted by alcohol dehydrogenase (ADH). In order to determine which of these compounds (ethanol or acetaldehyde) was responsible for the increase of flower vase life and inhibition of ethylene production, 4-methyl pyrazole (4-MP), a specific inhibitor of ADH, was applied and ADH activity measured. Ethanol at 4% in the presence or absence of 4-MP significantly (P< 0.001) inhibited ethylene biosynthesis and sensitivity to ethylene in cv. Yellow Candy. The ADH activity of cv. Yellow Candy was half that of cv. Sandrosa suggesting that in cv. Yellow Candy applied ethanol remained as ethanol. In cv. Sandrosa ADH activity was stimulated with exogenous ethanol or acetaldehyde suggesting interconversion of these compounds. To determine how ethanol was interfering with ethylene production, the activity of various enzymes and the concentration of metabolites involved in the ethylene production were measured. Ethanol was found to be effective in inhibiting ethylene biosynthesis in carnation cv. Yellow Candy by significantly inhibiting (P< 0.01) conversion of S-adenosyl methionine (SAM) to ACC. How the inhibition of ACC production occurs was not detennined but there was a trend towards reduction of ACC synthase activity. In addition, ethanol inhibited ethylene sensitivity, thereby inhibiting autocatylatic ethylene production. However, ethanol did not interfere in the ethylene biosynthesis step from methionine to SAM or ACC to ethylene. From the results of this research it is suggested that both ethanol and acetaldehyde can increase the vase life of carnation flowers; efficacy depends upon cultivar. It has been found that the compound that inhibits ethylene biosynthesis is ethanol but not acetaldehyde and the inhibition of ethylene biosynthesis with ethanol is due to inhibition of the conversion of SAM to ACC