David R. Dilley

Extraction, partial purification and characterization of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruit

Abstract ACC oxidase (ethylene-forming enzyme — EFE) was extracted, partially purified and characterized from Golden Delicious apple fruit cortical tissue. The presence of 5% ( w v ) polyvinylpolypyrrolidone (PVPP) was necessary in the extraction medium to obtain an active enzyme from apple tissue. The specific activity was 40–100 nl ethylene h−1 mg−1 protein in the crude extract; polyethylene glycol fractionation following by calcium phosphate gel adsorption resulted in ca. 10-fold purification. Enzyme activity was retained at −20°C when the enzyme was stored with 30% glycerol. Ascorbate and Fe2+ were not essential in the extraction medium for apples. Apple ACC oxidase has a Km for ACC of 6.4 μM, Km for O2 of 0.4%, and a pH optimum of 7.2 in Tris-Mes buffer and 7.6 in phosphate buffer. The enzyme was stereospecific for substrate; it converted (±)allocoronamic acid to 1-butene while (±)coronamic acid was 10-fold less effective as a substrate. The characteristics of apple ACC oxidase are consistent with those reported for the enzyme from Cucumis melo [Ververidis and John, Phytochemistry 30 (1991), p. 727] and as demonstrated in several intact tissues [Yip et al., Plant Physiol. 88 (1988), p. 553] and in isolated pea leaf vacuoles [Gyy and Kende, Planta 160 (1984) p. 281] when consideration is made of substrate availability in vivo.

Induction of ethylene synthesizing competency in Granny Smith apples by exposure to low temperature in air

Abstract Preclimacteric Granny Smith apples ( Malus domestica , Borkh.) were held in air at 20°C or in air at 0°C for periods of 2, 4, 8, 16, or 32 days before transfer to air at 20°C and assessed periodically for rates of C 2 H 4 and CO 2 production and for 1-aminocyclopropane-1-carboxylic acid (ACC) content. Fruits kept in air continuously at 20°C entered the autocatalytic phase of C 2 H 4 production after 22 days while fruits chilled for 2, 4, 8, 16 or 32 days before ripening at 20°C began autocatalytic C 2 H 4 production after 16, 14, 2, 0 and 0 days, respectively. ACC levels in peel tissue from fruits kept at 0°C in air for 8 days or less remained at low levels when transferred to air at 20°C, then the ACC level increased greatly. Beyond 8 days in air at 0°C, fruits accumulated low ACC levels which were rapidly converted to C 2 H 4 as indicated by a high and sustained C 2 H 4 production rate.

Apple Ripening-Related cDNA Clone pAP4 Confers Ethylene-Forming Ability in Transformed Saccharomyces cerevisiae

The apple ripening-related cDNA insert of clone pAP4 (G.S. Ross, M.L. Kinghton, M. Lay-Yee [1992] Plant Mol Biol 19: 231–)238) has previously been shown to have considerable nucleic acid and predicted amino acid sequence similarity to the insert of a tomato ripening-related cDNA clone (pTOM13) that is known to encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase (A.J. Hamilton, G.W. Lycett, D. Grierson [1990] Nature 346: 284–287; A.J. Hamilton, M. Bouzayen, D. Grierson [1991] Proc Natl Acad Sci USA 88: 7434–7437). The cDNA insert from the clone pAP4 was fused between the galactose-inducible promoter and the terminator of the yeast expression vector pYES2. Transformation of Saccharomyces cerevisiae strain F808- with this DNA construct and incubation of the yeast in the presence of D[+]-galactose allowed these cells to convert ACC to ethylene. The transformed yeast converted 1-amino-2-ethylcyclopropane-1-carboxylate isomers to 1-butene with the same 1R,2S-stereoselectivity as achieved by the native ACC oxidase from apples. Both ascorbate and Fe2+ ions stimulated the rate of the production of ethylene from ACC by the transformed yeast, whereas Cu2+ and Co2+ were strongly inhibitory; these are features of ACC oxidase. Northern analysis of the total RNA from nontransformed and transformed yeast showed that the ability to convert the ACC to ethylene was correlated with the synthesis and accumulation of a novel 1.2-kb mRNA that hybridized to the cDNA clone pAP4. We conclude that the cDNA sequence of the clone pAP4 encodes ACC oxidase.

A ‘scald-like’ controlled atmosphere storage disorder of Empire apples — a chilling injury induced by CO2

Abstract Empire apples ( Malus domestica Borkh.) were harvested prior to the onset of the ethylene climacteric and stored for six weeks in controlled atmospheres (CA) ranging from 1.3 to 7.3% CO 2 and 1.6 to 5.1% O 2 and in air at 1 and 3 °C with or without treatment with 1000 mg l −1 diphenylamine (DPA) scald inhibitor. Fruits were examined for incidence of ‘scald-like’ disorder upon removal from storage and after two weeks at 20 °C. A physiological disorder resembling superficial scald was evident on fruits not treated with DPA upon removal from CA and the severity of symptoms increased only slightly after holding the fruits in air at 20 °C. The disorder only occurred on fruit held in CA and was much more prevalent at 1 °C as compared to 3 °C storage. The disorder predominantly affected the non-red portions of the fruit surface and involved only the hypodermal cells. The fruit surface affected was tan or brown and became wrinkled and sunken as the subtending cells collapsed and dehydrated. The disorder incidence was markedly associated with high CO 2 concentration during CA at 1 °C and this was exacerbated at 2.0% CO 2 with 1.5% O 2 . We propose that the mechanism responsible for the Empire fruit ‘scald-like’ disorder is a free radical-catalyzed oxidation of susceptible amino acid residues in proteins essential for cell function and perhaps other macromolecules, such as membrane lipids containing oxidizable reactive groups.

Initial low oxygen stress controls superficial scald of apples

Abstract The effectiveness of initial low oxygen stress (ILOS) to control apple scald without postharvest application of scald inhibitors and fungicides was investigated. Scald susceptible (‘Granny Smith’, ‘Law Rome’, ‘Red Delicious’) and not susceptible (‘Idared’) fruits were harvested preclimacterically and treated at 1°C with (1) ILOS pretreatment or (2) no pretreatment as control. Fruit were then stored at 0.5–1°C in 3% O 2 with 0% CO 2 , 1.5% O 2 with 3% CO 2 , or 0.7–0.8% O 2 with 3% CO 2 in flow-through controlled atmosphere (CA) or in air. ILOS of 0.5 and 0.25% O 2 for 2 weeks resulted in excellent control of scald when fruits were subsequently held in CA (3% O 2 , 0% CO 2 ). Initial treatment with 0.25% O 2 for 2 weeks, and when this treatment was supplemented with an additional 2 weeks of low O 2 stress after 2 months of storage, were more effective treatment regimens for scald control than all other treatments. ILOS followed by CA storage at 1.5% O 2 gave complete control of scald. A commercial test of initial low O 2 stress confirmed its efficacy for controlling scald in several cultivars of apples. The production of α-farnesene and its volatile oxidation product 6-methyl-5-hepten-2-one (MHO) was inhibited by initial low O 2 stress treatments and 1.5% O 2 CA. The 0.25% O 2 initial stress treatment caused stronger inhibition of α-farnesene and MHO production than 0.5% O 2 initial stress treatment. The accumulation of MHO was related to scald development of apples.

Molecular cloning of a novel heat induced/chilling tolerance related cDNA1 in tomato fruit by use of mRNA differential display2

Chilling injury was circumvented by heat-treating mature green tomatoes (Lycopersicon esculentum, cv. Mountain Springs) at 42 °C for two days prior to storing them at 2 °C for one or two weeks, whereas fruits stored at 2 °C without preheating developed typical chilling injury symptoms and failed to ripen at 20 °C. Using mRNA differential display and screening of the cDNA libraries, we have cloned from tomato fruit a full-length HCT1 cDNA (heat induced/chilling tolerance related). The protein (17.6 kDa) predicted from coding region of HCT1 cDNA has high identity with class II cytosolic small HSPs. The gene corresponding to HCT1 cDNA was termed as LeHSP 17.6. Southern-blot hybridization indicates that LeHSP 17.6 belongs to a two-member gene family. Northern blot analysis indicates the heat-induced transcript of the LeHSP 17.6 remains up-regulated during subsequent exposure of the fruit to chilling temperatures for at least one week and upon transfer to ripening temperatures for one day. Fruits which were only chilled show a low level of expression of the LeHSP 17.6 transcript. We hypothesize that LeHSP 17.6 may be involved in protecting the cell from metabolic dysfunctions leading to ripening failure caused by chilling injury. This is the first report of a class II cytosolic smHSPs encoding gene in tomato.

Hypobaric storage removes scald-related volatiles during the low temperature induction of superficial scald of apples

Abstract ‘Law Rome’ and ‘Granny Smith’ apples were stored hypobarically in air at 5 kPa total pressure and also in air or controlled atmosphere (CA) at 1.5 or 3% O2 with 0 or 3% CO2, for 8 months at 1°C. Fruit were placed under hypobaric storage immediately after harvest or after 0.5, 1, 2, 3, 4, 5 or 6 months storage in air at 1°C to determine the effects of delaying imposition of hypobaric storage on ripening and scald development and on the production of α-farnesene and its oxidation product 6-methyl-5-hepten-2-one (MHO). If fruit were placed under hypobaric conditions within 1 month after harvest, scald did not develop. After a 3-month delay, scald development was similar to that for fruit stored continuously in air. Both cvs. produced MHO which accumulated in their epicuticular wax when fruit were placed under hypobaric storage after a 1-month or more delay in air. MHO which had partitioned in the epicuticular wax of fruit stored hypobarically after 2 or more months delay was released upon transfer of fruit to atmospheric pressure of 20°C; MHO accumulated and/or was produced in direct proportion to the delay prior to hypobaric storage. In another experiment with five apple cvs., the production rates of α-farnesene and MHO were low during hypobaric storage, but upon removal of fruit from storage after 7 months, the rates increased over a 7 day period in air at 20°C and then sharply decreased afterward. After storage, α-farnesene and MHO production rates were similar and high for ‘Law Rome’, ‘Mutsu’, ‘Red Delicious’ and ‘Golden Delicious’ apples and were the lowest for ‘Granny Smith’. Scald did not develop on any hypobarically stored fruit whereas it did on all cvs. except ‘Golden Delicious’ stored in air. It was proposed that hypobaric ventilation removes a scald-related volatile substance that otherwise accumulates and partitions into the epicuticular wax of fruit stored in air at atmospheric pressure.

Carbon dioxide activation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase in ethylene biosynthesis

Abstract Carbon dioxide was found to be required for, and to markedly stimulate, the activity of ACC oxidase enzyme extracted from apple fruit. Without supplemental CO 2 enzyme activity was low and variable. The half-maximal concentration CO 2 is 0.68% (v/v). Undissociated CO 2 rather than CHO − 3 serves as the activator of ACC oxidase. CO 2 and dithiothreitol act synergistically to activate the enzyme but CO 2 activation is independent of dithiothreitol. CO 2 increases the K m value for substrates ACC and ascorbic acid several fold while it increases the V max about 10-fold. CO 2 activation of ACC oxidase resembles that of rubisco, the only other plant enzyme known to be activated by CO 2 . Whereas CO 2 is known to be an inhibitor of ethylene action, it is required to make ACC oxidase catalytically competent to produce ethylene.

Purification and Characterization of ACC Oxidase and Its Expression during Ripening in Apple Fruit

1-aminocyclopropane-1-carboxylic acid (ACC) oxidase was purified to apparent homogeneity from ripened apple fruits (Malus domestica Borkh. cv. Golden Delicious). Ascorbate is required as a cosubstrate (Km = 0.4 mM); L-ascorbic acid-6-palmitate inhibited ACC oxidase competitively (K1 = 20μM) with respect to L-ascorbate. The enzyme is markedly activated by CO2 (KA = 0.65%). Fe2+ is required (KA = 3μM); Co2+ is inhibitory (Ki = 3μM) competitively with respect to Fee+. ACC oxidase activity was positively correlated with the development of the ethylene climacteric in apples; in vivo or in vitro activity was not detectable in preclimacteric apples. Immunoblots of proteins and of in vitro translation products of poly(A)+ RNA from apples at progressive stages of the ethylene climacteric indicated that ACC oxidase and the mRNA encoding ACC oxidase was nil in unripe fruits but accumulated markedly during the ethylene climacteric.

Development of ACC oxidase activity during maturation and ripening of ‘Paulared’, ‘Empire’ and ‘Law Rome’ apples

Abstract The development of ACC oxidase activity was investigated in three cultivars of apple ( Malus domestica Borkh.), ‘Paulared’, ‘Empire’ and ‘Law Rome’, as typifying early, mid-season and late cultivars, respectively, during maturation and ripening. Fruits were selected at progressive stages of development of the autogenous ethylene climacteric during maturation and ripening on the tree. The level of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene was used as a relative measure of the activity of ACC synthase and the ability of fruit tissue to convert ACC to ethylene was used as a measure of the activity of ACC oxidase, the ethylene forming enzyme. ‘Paulared’ fruits accumulated high levels of ACC as the autogenous ethylene climacteric began and this was associated with slow development of ACC oxidase activity. ‘Law Rome’ apples showed ACC oxidase activity early in the development of the ethylene climacteric but this was associated with accumulation of low levels of ACC in fruits. ‘Empire’ fruits were found to be intermediate between the early and late cultivars. ACC oxidase activity of fruits of the three cultivars was found to increase concurrently with or subsequent to the accumulation of ethylene to ca. 0.1 μl 1 −1 in the internal atmosphere of the fruit. Thus, the ACC content of the fruit was found to be a function of the development of ACC synthase activity as well as the relative activity of ACC oxidase. The levels of these activities were cultivar dependent. Differential sequestering of ACC into malonyl ACC by malonyl Co A-ACC transferase among the three cultivars may also account for the wide disparity in changes in ACC content with maturation but this was not investigated.