A dual positional specific lipoxygenase functions in the generation of flavor compounds during climacteric ripening of apple.
Lipoxygenase (LOX) is an important contributor to the formation of aroma-active C6 aldehydes in apple (Malus × domestica) fruit upon tissue disruption but little is known about its role in autonomously produced aroma volatiles from intact tissue. We explored the expression of 22 putative LOX genes in apple throughout ripening, but only six LOXs were expressed in a ripening-dependent manner. Recombinant LOX1:Md:1a, LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b proteins showed 13/9-LOX, 9-LOX, 13/9-LOX and 13-LOX activity with linoleic acid, respectively. While products of LOX1:Md:1c and LOX2:Md:2b were S-configured, LOX1:Md:1a and LOX2:Md:2a formed 13(R)-hydroperoxides as major products. Site-directed mutagenesis of Gly567 to an alanine converted the dual positional specific LOX1:Md:1a to an enzyme with a high specificity for 9(S)-hydroperoxide formation. The high expression level of the corresponding MdLOX1a gene in stored apple fruit, the genetic association with a quantitative trait locus for fruit ester and the remarkable agreement in regio- and stereoselectivity of the LOX1:Md:1a reaction with the overall LOX activity found in mature apple fruits, suggest a major physiological function of LOX1:Md:1a during climacteric ripening of apples. While LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b may contribute to aldehyde production in immature fruit upon cell disruption our results furnish additional evidence that LOX1:Md:1a probably regulates the availability of precursors for ester production in intact fruit tissue.
Biotechnology of Natural Products, Technische Universität München , Liesel-Beckmann-Str. 1, D-85354 Freising, Germany.
Doreen
D
Schiller
Michigan State University, Department of Horticulture , 1066 Bogue Street, East Lansing, MI 48824, USA.
Carolina
C
Contreras
Julius Kühn-Institut (JKI) - Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Fruit Crops, Pillnitzer Platz 3a , D-01326 Dresden, Germany.
Jörg
J
Vogt
Julius Kühn-Institut (JKI) - Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Horticultural Crops , Erwin-Baur-Str. 27, D-06484 Quedlinburg, Germany.
Frank
F
Dunemann
INIA La Platina, PO Box 439-9 , Santiago, Chile.
Bruno G
BG
Defilippi
Michigan State University, Department of Horticulture , 1066 Bogue Street, East Lansing, MI 48824, USA.
Randolph
R
Beaudry
Biotechnology of Natural Products, Technische Universität München , Liesel-Beckmann-Str. 1, D-85354 Freising, Germany.
Wilfried
W
Schwab