Overexpression of a cytoplasm-localized allene oxide synthase promotes the wound-induced accumulation of jasmonic acid in transgenic tobacco.

Jasmonic acid (JA) is involved in regulating the expression of certain plant defense genes and response to various 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 overexpression of 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-induced JA which in turn directly or indirectly enhances the expression of plant defense genes.

Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens strain Chry5.

This report describes the disarming of Agrobacterium tumefaciens Chry5, a strain highly tumorigenic on soybean. Disarming was achieved by removing an approximately 16.5-kb segment of the 285-kb Ti plasmid pTiChry5, including approximately 4 kb of the oncogenic T-DNA and an extended region right of the T-DNA, and replacing it with a gene for carbenicillin resistance, through homologous recombination. The deletion was confirmed with Southern analysis, and the loss of tumorigenicity was verified in tobacco and tomato plant stem inoculation assays. The deletion mutant, named KYRT1, successfully transferred the ß-glucuronidase (GUS) gene into tobacco leaf tissue, producing GUS-expressing callus which could be regenerated into viable plants. In a comparative study, the transformation efficiency of A. tumefaciens KYRT1, GV3850, and EHA105 was assayed by inoculating cotyledonary node explants. The results of this study revealed that, in a binary vector system, KYRT1 is equally or more effective than EHA105 or GV3850 at delivering DNA into soybean.

Effect of volatile methyl jasmonate on the oxylipin pathway in tobacco, cucumber, and arabidopsis.

The effect of atmospheric methyl jasmonate on the oxylipin pathway was investigated in leaves of tobacco (Nicotiana tabacum L.), cucumber (Cucumis sativa L.), and Arabidopsis thaliana (L.). Differential sensitivities of test plants to methyl jasmonate were observed. Thus, different concentrations of methyl jasmonate were required for induction of changes in the oxylipin pathway. Arabidopsis was the least and cucumber the most sensitive to methyl jasmonate. Methyl jasmonate induced the accumulation of lipoxygenase protein and a corresponding increase in extractable lipoxygenase activity. Atmospheric methyl jasmonate additionally induced hydroperoxide lyase activity and the enhanced production of several volatile six-carbon products. It is interesting that lipid hydroperoxidase activity, which is a measure of hydroperoxide lyase plus allene oxide synthase plus possibly other lipid hydroperoxide-metabolizing activities, was not changed by methyl jasmonate treatment. Methyl jasmonate selectively altered the activity of certain enzymes of the oxylipin pathway (lipoxygenase and hydroperoxide lyase) and increased the potential of leaves for greatly enhanced six-carbon-volatile production.

Lipid-derived signals that discriminate wound- and pathogen-responsive isoprenoid pathways in plants: methyl jasmonate and the fungal elicitor arachidonic acid induce different 3-hydroxy-3-methylglutaryl-coenzyme A reductase genes and antimicrobial isoprenoids in Solanum tuberosum L.

Induction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR; EC 1.1.1.34) is essential for the synthesis of steroid derivatives and sesquiterpenoid phytoalexins in solanaceous plants following mechanical injury or pathogen infection. Gene-specific probes corresponding to different HMGR genes (hmg1 and hmg2) were used to study HMGR expression in potato tissue following treatment with methyl jasmonate, a lipoxygenase product of linolenic acid, or arachidonic acid, an elicitor present in the lipids of the potato late blight fungus Phytophthora infestans. Treatment of potato discs (2.2 cm in diameter) with low concentrations (0.45-45 nmol per disc surface) of methyl jasmonate nearly doubled the wound-induced accumulation of hmg1 transcripts and steroid-glycoalkaloid (SGA) accumulation, reduced the abundance of hmg2 transcripts, and did not induce phytoalexins. High concentrations of methyl jasmonate (2-4.5 mol per disc surface) suppressed hmg1 mRNA and SGA accumulation but did not affect hmg2 mRNA abundance or induce phytoalexins. In contrast, arachidonate treatment strongly suppressed hmg1 and strongly induced hmg2 mRNA in a concentration-dependent manner. There was a corresponding suppression of SGA accumulation and an induction of sesquiterpene phytoalexin accumulation by this elicitor. Lipoxygenase inhibitors reduced the wound-induced accumulation of hmg1 transcripts and suppressed SGA levels, effects that were overcome by exogenous methyl jasmonate (45 nmol per disc surface). The results (i) suggest that methyl jasmonate can function as a signal for hmg1 expression and SGA induction following wounding and (ii) indicate that the arachidonate- and jasmonate-response pathways are distinct in relation to HMGR gene expression and isoprenoid product accumulation. The results also are consistent with placement of the HMGR activities encoded by hmg1 and hmg2 within discrete steroid and sesquiterpenoid biosynthetic channels.