Plant Molecular Biology 40: 783–793, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Overexpression of a cytoplasm-localized allene oxide synthase promotes
the wound-induced accumulation of jasmonic acid in transgenic tobacco
Cunxi Wang, Sergei Avdiushko and David F. Hildebrand
Department of Agronomy, University of Kentucky, Lexington, KY 40546, USA (
author for correspondence)
Received 10 December 1999; accepted in revised form 8 April 1999
Key words: allene oxide synthase, C
volatiles, jasmonic acid, Nicotina tabacum, pathogenesis-related gene,
Jasmonic acid (JA) is involved in regulating the expression of certain plant defense genes and response to var-
ious stresses. JA biosynthesis is hypothesized to occur both in chloroplasts and the cytoplasm. In order to test
whether or not a cytosol-localized allene oxide synthase (AOS) can promote JA biosynthesis, transgenic tobacco
plants containing a ﬂax AOS cDNA without a chloroplast transit sequence under the control of a tetracycline-
inducible promoter were generated. Induction of the ﬂax AOS gene in transgenic plants with chlor-tetracycline
(Tc) led to the expression of the ﬂax AOS mRNA and protein, which resulted in high level of metabolism of
13(S)-hydroperoxyoctadecatrienoic acid (13(S)-HPOT) and formation of 12-oxo-phytodienoic acid (12-O-PDA).
Subcellular fractionation demonstrated that the ﬂax AOS protein and activity were associated with the cytosol.
Overexpression of the ﬂax AOS in induced transgenic plants did not increase JA levels in healthy, undamaged leaf
tissues. However, in wounded tissues overexpressing a ﬂax AOS, levels of JA and the transcript of a pathogenesis-
related gene (PR-1) dramatically increased when compared to those not expressing the ﬂax AOS. Analysis of
the release of wound-induced C6 volatiles showed that the level of (Z)-3-hexen-1-ol decreased about 30% due to
overexpressionof the cytoplasm-localized AOS, while (Z)-3-hexenal and (Z)-3-hexenyl acetate appeared not to be
signiﬁcantly altered. The data indicate that cytoplasmic AOS responds to wounding by increasing the levels of the
wound-inducedJA which in turn directly or indirectly enhances the expression of plant defense genes.
Abbreviations: AOS, allene oxide synthase; HL, hydroperoxidelyase; 13(S)-HPOT, 13(S)-hydroperoxyoctadeca-
trienoic acid; JA, jasmonic acid; LOX, lipoxygenase; MS, Murashige and Skoog; 12-O-PDA, 12-oxo-phytodienoic
acid; pin2, proteinase inhibitor II; PR-1, pathogenesis-related genes; Tc, chlor-tetracycline.
Lipoxygenase (LOX) catalyzes the dioxygenation of
C18 polyunsaturatedfattyacids such as linoleic (18:2)
and linolenic (18:3) acids, leading to the forma-
tion of their corresponding hydroperoxy derivatives.
13(S)-HPOT can be further metabolized either into JA
throughthe AOS pathwayor into hexenals through the
hydroperoxide lyase (HL) activity, whereas hexanal
is derived from the 13(S)-hydroperoxy derivative of
18:2 (Croft et al., 1993; Vick, 1993; Gardner et al.,
1996). Hexanal and hexenals, along with the corre-
sponding alcohols, are the major volatile compounds
responsible for the ‘green odor’ of leaves (Hatanaka
et al., 1987; Gardner, 1989). These volatiles have
been shown to have antifungal, antimicrobial and anti-
insect activities (Croft et al., 1993; Gardner, 1995).
JA and its derivatives (often referred to as jasmonates)
are known to function as signaling molecules to stim-
ulate the expression of many wound-activated and
defense-related genes (Bergey et al., 1996; Creelman
and Mullet, 1997; Seo et al., 1997). Studies with
jasmonate-deﬁcient mutants of tomato (Howe et al.,
1996) and Arabidopsis (McConn et al., 1997; Vijayan
et al., 1998) have established an essential role of jas-
monates in insect and pathogen defenses. Jasmonates