1.9.30
MANIPULATION OF SESQUITERPENOIDS IN LETTUCE FOR PEST AND DISEASE RESISTANCE

RA SESSA1, MH BENNETT1, M BEALE2 and J MANSFIELD1

1Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent TN25 5AH, UK; 2IACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol BS18 9AF, UK

Background and objectives
The main phytoalexin in lettuce is the induced sesquiterpene lactone lettucenin A, which possesses considerable antifungal activity and probably has an important role in the resistance of lettuce tissues to microbial colonization [1]. The principal constitutive sesquiterpenes in Lactuca sativa are lactucopicrin, lactucin and 8-deoxylactucin. The preformed compounds are predominantly found in the plant latex, stored in specialized laticiferous cells which form an interconnected network throughout the plant. The main role of these sesquiterpenes is thought to be as insect antifeedants, but they have also been found to have low levels of antimicrobial activity. The initial aim was the purification and characterization of the constitutive sesquiterpenoids. Studies were then made to investigate the relationship between changes in the concentrations of the preformed sesquiterpenes and the induced phytoalexin. The key enzymes in sesquiterpene biosynthesis is sesquiterpene cyclase. Preliminary experiments have been carried out to isolate this gene using PCR-based cloning.

Results and conclusions
Constitutive sesquiterpenes were extracted from 9-week-old bolted lettuces, while lettucenin A was extracted from CuSO4-elicited Iceberg lettuce leaves [1]. Compounds were separated and purified using several silica and Sep-pak columns. Reversed-phase HPLC, LCMS and proton and carbon13 NMR were then used for identification and characterization of the compounds.

Analysis of lettuce tissues (latex, hypocotyls and cotyledons) showed that in lettuce the main sesquiterpenes, contrary to the literature, are found mainly in the oxalate form rather than as glycosides [2]. Elicitation of 7-day-old cotyledons and hypocotyls with both abiotic (UV and CuSO4) and biotic (Bremia lactucae and Botrytis cinerea) treatments has revealed rapid accumulation of the phytoalexin, but little change to the constitutive pool of sesquiterpenoids.

Degenerate primers based on the sequence of previously identified clones of sesquiterpene cyclase were used for PCR amplification. Two sequences, amplified from genomic DNA, show homology to other sesquiterpene cyclase genes. The cloned genes will be used to modify levels of the cyclase, and to examine alterations in the pattern of sesquiterpenoids produced and any changes in the susceptibility of lettuce to fungal infection.

References
1. Bennett MH, Gallagher M, Bestwick CS et al., 1994. Physiological and Molecular Plant Pathology 44, 321-333.
2. Price KR, DuPont SM, Shepherd R et al., 1990. Journal of the Science of Food and Agriculture 53, 185-192.