Zhenyong Wang

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.

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.

1-Aminocyclopropane-1-carboxylic acid oxidase reaction mechanism and putative post-translational activities of the ACCO protein

ACC oxidase (Malus. domestica ACCO1) catalyzes the final step in the biosynthesis of the plant hormone ethylene. ACCO converts 1-aminocyclopropane-1-carboxylic acid(ACC) to ethylene, cyanide, carbon dioxide and water in the presence of ferrous ion, oxygen, ascorbic acid and bicarbonate. Cyanide, a product of the reaction, activates ACCO. Site-directed mutagenesis investigations revealed binding sites for ACC, bicarbonate and ascorbic acid to include; Arg175, Arg244, Ser246, Lys158, Lys292, Arg299 and Phe300. ACCO may be involved in the ethylene signal transduction pathway not directly linked to the ACCO reaction through post-translational modifications. ACCO is subject to auto-phosphorylaton in vitro and promotes phosphorylation of some apple fruit proteins in a ripening-dependent manner.

Analysis of ACC Oxidase Activity by Site-Directed Mutagenesis of Conserved Amino Acid Residues

Site-directed mutagenesis of ACC oxidase (ACO) was used to determine the nature and role of conserved amino acid residues in the mechanism by which CO2 activates the enzyme. Mutants of ACO were expressed in E. coli as His-Tag fusion proteins. A consensus sequence search of 38 known or putative ACO revealed 8 completely conserved lysine residues; K72, K144, K158, K172, K199, K230 K292 and K296. All of the lysine mutant forms were typically activated by CO2 indicating that none of them is essential for CO2 activation by a carbamylation mechanism. H177, H234 and D179 are essential ligands for Fe. The H177, H234 and D179 ligands for Fe in ACO have equivalent residues in isopenicillin N synthase (IPNS) as H214, D216 and H270. ACO, a non-heme Fe2+/ascorbate requiring enzyme, belongs to the IPNS protein structure family. The Cterminal sequence from K292 through E301 is important for enzyme activity and CO2 activation; Arg299 may be involved in the mechanism of CO2 activation. We prepared R244K and S246A mutants to determine if these Arg and Ser residues may serve as ligands for the carboxyl group of ACC. The R244K and S246A mutants were 5.4% and 35% as active, respectively, as the native enzyme but were typically activated by CO2; the Km values for ACC for the R244K and S246A mutants were increased 2- to 3-fold compared to the native enxyme. This supports a putative role of Arg244 and Ser246 as ligands for the ACC carboxyl group.

Temperature and atmosphere regimens to control a CO2-linked disorder of ‘Empire’ apples

Abstract Preclimacteric ‘Empire’ apples are prone to develop a CO2-linked disorder (CLD); the symptoms resemble superficial scald. Effects of acclimatizing preclimacteric fruit at 3°C, either in air or at low O2 levels prior to elevation of the CO2 concentration during subsequent long-term controlled atmosphere (CA) storage, were investigated. The ‘Empire’ CLD was effectively controlled by conditioning fruit at 3°C (but not at 0°C) for 3–4 weeks at 1.5–3% O2 without CO2 prior to CA storage at 1.5% O2+3% CO2. Reduction of the disorder was also achieved by storage at 3°C in air, but excessive ripening and associated loss of flesh firmness occurred during subsequent CA storage.