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H.P.V. Rupasinghe, D.P. Murr and G. Paliyath

`McIntosh' apples were treated at 20 °C with 0.0, 0.01, 0.1, 1.0, 10, and 100 ppm 1-methylcyclopropene (1-MCP; EthylBloc™) a day after harvest for 18 h and stored at 0 °C in air. Apples were also continuously exposed to 0.0 and 25 ppm 1-MCP under controlled atmosphere (CA; 0 °C in 4.5 kPa CO2 and 3 kPa O2) by re-establishing the initial concentration at week 9 and 17. The threshold concentration of 1-MCP at 20 °C to inhibit de novo ethylene production in apple fruit was determined to be 1.0 ppm. Interestingly, the ethylene antagonist completely inhibited (99.67%) ethylene production in apples, which were removed from 0 °C in air and CA after 9 weeks and held at 20 °C up to 6 days. Overall, ethylene production was 10- to 100-fold less in apples treated with 1 ppm and above 1-MCP than in untreated apples. 1-MCP-treated apples showed less softening; fruit firmness was 2-4 Lb higher compared to untreated apples. Total soluble solids of apples was not affected by 1-MCP treatment. Total hydrophobic volatiles, including the sesquiterpene hydrocarbon α-farnesene, from apples measured by SPME/GC showed an inverse relation to 1-MCP concentration. Contents of α-farnesene and its putative superficial scald-causing catabolite, conjugated triene alcohol, in the skin were reduced 60% to 90% by 1-MCP. However, 1-MCP did not suppress the incidence of scald or other disorders, e.g., stem cavity, browning and brown core, in `McIntosh' apples.

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M.D. Whiting, G. Paliyath and D.P. Murr

Apple fruits (Malus domestica Borkh. cv. `Red Delicious') stored for 6 months at 2°C in air were analyzed for headspace volatiles by SPME-GC and for surface components by HPLC of hexane extracts. Analysis of headspace volatiles evolved from whole fruit showed five major volatiles that were identified previously as: acetic acid, hexyl ester; hexanoic acid, butyl ester; octanoic acid, propyl ester; hexanoic acid, hexyl ester; and the sesquiterpene, α-farnesene. No significant differences existed in these volatiles between scald-developing and non-scald developing apples. To explore potential differences in volatile evolution, fruit developing scald were cut (axial plane) into scalding and non-scalding halves for analysis. In all cases, volatile emission was much higher from the non-scalding side of the fruit, and the ratio of volatile levels from non-scalding to scalding averaged greater that 2. Various regions of tissue from the same fruit were extracted in hexane for estimation of levels of α-farnesene and its potential catabolites by HPLC. The levels and proportions of the components were nearly identical to those observed during headspace volatile analysis of half fruit. The results suggest that there are potential differences in α-farnesene metabolism an/or permeability of apple cuticle to volatiles between scald-developing and non-scald developing regions of apple fruit.

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H.P.V. Rupasinghe, G. Paliyath and D.P. Murr

α-Farnesene is an acyclic sesquiterpene hydrocarbon that is a constituent of the surface wax of apples (Malus domestica Borkh.). Although, oxidation products of α-farnesene have been implicated in the development of the physiological disorder superficial scald in apple, the mechanism of α-farnesene biosynthesis has not been studied in detail. We are currently investigating α-farnesene biosynthesis in relation to superficial scald development in apples. Radiolabelled feeding experiments using isolated tissue segments indicated that α-farnesene is derived from trans,trans-farnesyl pyrophosphate (FPP), mainly in the skin rather than cortex. Among the other labeled products detected, farnesol level was over a hundred-fold higher compared to α-farnesene. However, [1-3H] trans,trans-Farnesol was not incorporated into α-farnesene. Feeding radiolabelled FPP to skin tissue segments of scald-developing and normal apples showed differential incorporation of radiolabel into various products. Though the incorporation into α-farnesene was nearly the same, there was higher levels of incorporation into farnesyl esters in normal apples. As well, the levels of radiolabelled in the farnesol fraction was three times higher in scald-developing regions. These results indicate that there are potential difference in the biosynthesis and metabolism of farnesyl components between scald-developing and normal apples. In studies using cell-free extracts, farnesol formation was observed from labeled FPP and was two-fold higher in crude membrane extract compared to crude cytosol. Our results indicate that α-farnesene formation in apple fruit tissue is through FPP and is possibly catalyzed by a single sesquiterpene synthase enzyme. Purification and characterization of this enzyme are in progress.

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H.P.V. Rupasinghe, G. Paliyath and D.P. Murr

α-Farnesene is an acyclic sesquiterpene hydrocarbon that is a constituent of the volatile components and the surface wax of apples (Malus ×domestica Borkh.). Although oxidation products of α-farnesene have been implicated in the development of superficial scald in apples, the relation between α-farnesene biosynthesis and scald development is not well understood. In vivo labeling studies using isolated tissue segments showed that α-farnesene is derived from trans,trans-[1,2-14Cor 1-3H]-farnesyl pyrophosphate (FPP) mostly in the skin rather than cortex tissue. Among other labeled products, farnesol was >100-fold higher compared to α-farnesene. However, HPLC analysis of hexane-extractable components from apple skin revealed farnesol is not a predominant natural constituent of apple skin tissue. In addition, trans,trans-[1-3H]-farnesol was not converted to α-farnesene by apple skin tissue. Our results indicate that biosynthesis of α-farnesene in apple tissue occurs through the isoprenoid pathway, and the conversion of FPP to α-farnesene is catalyzed by a single sesquiterpene synthase enzyme, trans,trans-α-farnesene synthase, rather than via farnesol as an intermediate. A comparison of α-farnesene biosynthesis between scald-developing and scald-free regions of the same apple showed that incorporation of radiolabel into α-farnesene from trans,trans-[1-3H]-FPP was nearly 3-fold lower in scald-developing skin tissue than in scald-free skin tissue.

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D.P. Murr, K. Hustwit, R. Tschanz, M.V. Rao and G. Paliyath

Heat treatment of apples (Malus domestica Borkh cvs. Red Delicious, Starkrimson) and its effect on scald development have been investigated. Several parameters indicative of scald, such as ethanol and acetaldehyde content, UV-absorbing components from skin, and fruit quality parameters, such as fruit firmness and soluble solids content, were monitored after exposing apples to heat therapy at 40C for 24 h, followed by storing them at room temperature in polyethylene bags. In general, heat-treated apples possessed higher ethanol and acetaldehyde levels. As well, heat-exposed apples appeared to possess a lower degree of scald. The content of soluble solids did not appear to be affected by heat treatment. The degree of firmness, however, was maintained in heat-treated apples. Effect of heat treatment on several other physiological and biochemical parameters will be presented.

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H.P.V. Rupasinghe, K.C. Almquist, G. Paliyath and D.P. Murr

We tested the hypothesis that conversion of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG CoA) to mevalonate (MVA) catalyzed by HMG CoA reductase (HMGR) is the rate limiting step for α-farnesene biosynthesis of apples. In higher plants, isopentenyl pyrophosphate (IPP) is derived via two pathways: 1) the classical mevalonate pathway, and 2) the novel glyceraldehyde-3-phosphate (GAP)/pyruvate pathway independent of HMGR action. When apple skin discs were incubated with MVA, or GAP and pyruvate, MVA increased α-farnesene levels in the skin but not GAP and pyruvate. Treating apple fruits with Lovastatin (1000 ppm), a competitive inhibitor of HMGR, inhibited α-farnesene accumulation in the skin by 20% to 50% during storage. Content of α-farnesene in the skin increased during the first 2 to 4 months in storage, and then decreased. In contrast, HMGR activity, as determined by the conversion of [4-3H]HMG CoA to MVA in the total membrane and soluble fraction, was the highest at the time of harvest and gradually decreased during 5 months of storage in air at 0 °C. The potent ethylene action inhibitor 1-MCP inhibited ethylene production and α-farnesene evolution by 99% and 97%, respectively. The effect of 1-MCP on in vitro activity of HMGR was marginal (≈30% inhibition). 1-MCP inhibited respiratory CO2 evolution by 50%, which suggests also that inhibition by 1-MCP of α-farnesene synthesis in apple could be regulated by the acetyl CoA pool. In plants, HMGR is encoded by a small gene family and differentially expressed. As the first step of studying the molecular mechanism of HMGR regulation, we have isolated a 444-bp fragment of apple hmgr gene using apple skin mRNA and degenerate oligonucleotides designed against conserved regions of plant hmgr genes.