Consequences of transferring three sorghum genes for secondary metabolite (cyanogenic glucoside) biosynthesis to grapevine hairy roots.

A multigenic trait (biosynthesis of the secondary metabolite, dhurrin cyanogenic glucoside) was engineered de novo in grapevine (Vitis vinifera L.). This follows a recent report of transfer of the same trait to Arabidopsis (Arabidopsis thaliana) using three genetic sequences from sorghum (Sorghum bicolor): two cytochrome P450-encoding cDNAs (CYP79A1 and CYP71E1) and a UDPG-glucosyltransferase-encoding cDNA (sbHMNGT). Here we describe the two-step process involving whole plant transformation followed by hairy root transformation, which was used to transfer the same three sorghum sequences to grapevine. Transgenic grapevine hairy root lines that accumulated transcript from none, one (sbHMNGT), two (CYP79A1 and CYP71E1) or all three transgenes were recovered and characterisation of these lines provided information about the requirements for dhurrin biosynthesis in grapevine. Only lines that accumulated transcripts from all three transgenes had significantly elevated cyanide potential (up to the equivalent of about 100 mg HCN kg(-1) fresh weight), and levels were highly variable. One dhurrin-positive line was tested and found to release cyanide upon maceration and can therefore be considered 'cyanogenic'. In in vitro dual co-culture of this cyanogenic hairy root line or an acyanogenic line with the specialist root-sucking, gall-forming, aphid-like insect, grapevine phylloxera (Daktulosphaira vitifoliae, Fitch), there was no evidence for protection of the cyanogenic plant tissue from infestation by the insect. Consistently high levels of dhurrin accumulation may be required for this to occur. The possibility that endogenous grapevine gene expression is modulated in response to engineered dhurrin biosynthesis was investigated using microarray analysis of 1225 grapevine ESTs, but differences in patterns of gene expression associated with dhurrin-positive and dhurrin-negative phenotypes were not identified.

Cyanogenic glucosides in grapevine: polymorphism, identification and developmental patterns.

Twelve grapevine (Vitis vinifera L.) cultivars were surveyed for 'cyanide potential' (i.e. the total cyanide measured in beta-glucosidase-treated crude, boiled tissue extract) in mature leaves. Two related cultivars (Carignan and Ruby Cabernet) had mean cyanide potential (equivalent to 110 mgHCNkg-1fr.wt) ca. 25-fold greater than that of the other 10 cultivars, and so the trait is polymorphic in the species. In boiled leaf extracts of Carignan and Ruby Cabernet, free cyanide constituted a negligible fraction of the total cyanide potential because beta-glucosidase treatment was required to liberate the major cyanide fraction - which is therefore bound in glucosylated cyanogenic compound(s) (or cyanogenic glucosides). In addition, cyanide was liberated from ground leaf tissue of Ruby Cabernet but not Sultana (a cultivar with low cyanide potential). Hence, the high cyanide potential in Ruby Cabernet leaves is coupled with endogenous beta-glucosidase(s) activity and this cultivar may be considered 'cyanogenic'. A method was developed to detect and identify cyanogenic glucosides using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). Two putative cyanogenic glucosides were found in extracts from leaves of Carignan and Ruby Cabernet and were identified as the epimers prunasin and sambunigrin. Cyanide potential measured at three times over the growing season in young and mature leaves, petioles, tendrils, flowers, berries, seeds and roots of Ruby Cabernet was substantially higher in the leaves compared with all other tissues. This characterisation of cyanogenic glucoside accumulation in grapevine provides a basis for gauging the involvement of the trait in interactions of the species with its pests and pathogens.