Molecular cloning and characterization of lipoxygenase from ripening tomato fruit

Kurt David Kausch, Purdue University

Abstract

Climacteric fruit ripening and senescence are characterized by physiological and biochemical changes associated with altered gene expression. A 94 kD protein that predominately accumulates during tomato fruit ripening was purified and antibodies raised against this purified protein were used to isolate its clones from a red-ripe fruit cDNA library. Sequence analysis of these cDNAs identified the 94 kD protein as a member of the lipoxygenase (LOX, E.C. 1.13.11.12) family. Cross-reactivity of 94 kD antibodies to soybean LOX L-1, L-2 and L-3 and anti-soybean LOX L-1 to the 94 kD protein further established the 94 kD protein as a LOX. Maximum levels of 94 kD LOX mRNA and protein were obtained from the breaker to ripe and red-ripe stages, respectively, but were absent in root and leaf tissues. Immunocytolocalization using tissue printing revealed the highest accumulation of LOX protein in the locular jelly followed by the columella, radial walls and pericarp of ripening fruits. However, characterization of 94 kD LOX gene expression in different tissues from unripe and ripe fruits revealed maximum expression in radial walls of ripe fruit. In the ripening-impaired mutants Never-ripe and nonripening, 94 kD LOX mRNA was absent in aging nonripening fruits, while its accumulation pattern in Never-ripe fruits was similar to that of wild-type fruits. However, the 94 kD LOX protein was undectecable in Never-ripe and nonripening fruits of any age. LOX incorporates oxygen into unsaturated fatty acids producing hydroperoxides and is associated with numerous physiological processes in plants and animals. Tomato fruits with altered levels of LOX were engineered to elucidate the role(s) of LOX during fruit ripening and senescence. Fruits from transgenic plants expressing a sense chimeric 94 kD LOX gene exhibited a cosuppression effect with reduced levels of LOX mRNA and protein. Both greenhouse and field-grown transgenic fruit displayed a significant increase in firmness and a decreased rate of electrolyte leakage seven days after reaching breaker stage compared to control fruits. The cosuppression effect was stably inherited and the increase in fruit firmness was attributed to decreased LOX expression. I posit the direct involvement of LOX in textural-associated changes in ripening tomato fruits.

Degree

Ph.D.

Advisors

Handa, Purdue University.

Subject Area

Botany|Molecular biology

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