Robyn Van Heeswijck

Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests

The ornamental tobacco (Nicotiana alata) produces one 6-kDa chymotrypsin inhibitor and four 6-kDa trypsin inhibitors from a single 40.3-kDa precursor protein. Three different approaches have been used to assess the potential of these proteinase inhibitors (PIs) in insect control. The first was an in-vitro approach in which all five inhibitors, the single chymotrypsin inhibitor or three of the four trypsin inhibitors were tested for their ability to inhibit gut protease activity in insects from four orders. The second approach was to incorporate the N. alata PIs in the artificial diet of the native budworm (Helicoverpa punctigera) and the black field cricket (Teleogryllus commodus). H. punctigera larvae and T. commodus nymphs had a significant (P<0.01) reduction in growth after ingestion of the PI and were more lethargic than insects on the control diet. Several of the H. punctigera larvae also failed to complete moulting at the third or fourth instar. The third approach was to express the N. alata PIs in transgenic tobacco under the control of the 35S CaMV promoter. When H. punctigera larvae were fed tobacco leaves expressing the N. alata PIs at 0.2% soluble protein, significant (P<0.01) differences in mortality and/or growth rate were observed.

Ethanol triggers grape gene expression leading to anthocyanin accumulation during berry ripening

Recent studies have shown that low doses of ethanol stimulate the maturation of some fruits. The present work showed that spraying Cabernet Sauvignon grapes, with 5% ethanol at veraison enhances the anthocyanin accumulation. Veraison is the time when the berries turn from green to purple. HPLC analysis showed a marked increase in the total concentrations of the derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin from the fourth day after the ethanol treatment until harvest. This was not linked to a difference in berry weight in comparison to controls. Two distinct expression patterns were found for anthocyanin biosynthesis genes in the treated and untreated berries. For one group, consisting of chalcone synthase, flavanone-3-hydroxylase, dihydroxyflavonol-4-reductase and leucoanthocyanidin dioxygenase, the expression was inhibited or unchanged by the ethanol treatment, whereas for UDP glucose-flavonoid 3-O-glucosyltransferase (UFGT) there was a marked increase in expression from 1 to 20 days after ethanol treatment. These results suggest that the UFGT gene is a key factor in the observed anthocyanin accumulation following ethanol treatment.

Complementation of the Aspergillus nidulansarg B1 mutation by ornithine transcarbamylase cDNA from rat liver

An Aspergillus nidulans strain which is deficient in ornithine transcarbamylase due to the arg B1 mutation was transformed with a plasmid containing the ornithine transcarbamylase cDNA from rat liver under the control of the amd S promoter. Stable transformants were obtained by selection on arginine free medium indicating complementation of the arg B mutation. Proof of expression of the rat enzyme in transformants was obtained by immunoprecipitation of all ornithine transcarbamylase activity from cell extracts with antibodies specific for the rat enzyme. The presence of catalytically active rat ornithine transcarbamylase in the transformants indicated that it is capable of being imported into mitochondria in A. nidulans, proteolytically processed and assembled into its homotrimeric form. In vitro uptake experiments using isolated A. nidulans mitochondria demonstrate that processing of the precursor of rat ornithine transcarbamylase occurs in two temporally separated steps as it does in rat liver mitochondria suggesting evolutionary conservation of the processing machinery. Up to 560 ng of active rat enzyme was produced per gm wet weight mycelia. Use of beta-D-alanine, an inducer of amd S, as sole N-source resulted in increased levels of active rat ornithine transcarbamylase relative to uninduced cultures.

In vitro systems for studying the interaction of root-knot nematode with grapevine

A simple system for in vitro dual culture of grapevine (Vitis spp.) plantlets and root-knot nematode (Meloidogyne javanica (Treub) Chitwood) is described. Based on the presence or absence of mature females, or the total number of nematodes in the roots after 36-day co-culture, the system reliably discriminated resistant (cv. Ramsey) and susceptible (cv. Chardonnay) grapevines. The system was sensitive enough to differentiate between infestation levels of cvs Borner and Chardonnay, both susceptible in the in vitro conditions. A modification of the system to use plantlets from rooted petioles has reduced labour and space requirements and would suit mass screening of grapevine genotypes in traditional or genetic engineering-based breeding programmes. In both systems, nematodes in roots of cv. Ramsey tended to be associated with brown tissue and, compared with those in roots of cv. Chardonnay, were more likely to be confined to tips rather than be distributed along the root after co-culture for 11 or 36 days.