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.

Genetic transformation of major wine grape cultivars of Vitis vinifera L.

We have developed an Agrobacterium-mediated transformation system for a number of important grapevine cultivars used in wine production. Transgenic plants were obtained for the seven cultivars: Cabernet Sauvignon, Shiraz, Chardonnay, Riesling, Sauvignon Blanc, Chenin Blanc and Muscat Gordo Blanco. Embryogenic callus was initiated from anther filaments and genotypic differences were observed for initiation and subsequent proliferation with Chardonnay responding most favourably to culture conditions. The transformation system allowed the recovery of germinating transgenic embryos 10-12 weeks after Agrobacterium inoculation and plants within 18 weeks. Examination of the expression patterns of the green fluorescent protein gene under the control of the CAMV35S promoter in leaf tissue of transgenic plants showed that for up to 35% of plants the pattern was not uniform. The successful transformation of a genetically diverse group of wine grape cultivars indicates that the transformation system may have general application to an even wider range of Vitis vinifera cultivars.

cDNA cloning and expression of a new form of human aryl sulfotransferase.

To date, four human cytosolic sulfotransferases have been cloned and characterised. The aim of the present study was to identify new forms of these enzymes using molecular cloning techniques. Two full length human aryl sulfotransferase (HAST) cDNAs were cloned from a lambda gt10 liver cDNA library. The COS cell expression system was used to express the cDNAs and to determine the ability of the encoded proteins to metabolise the model substrates p-nitrophenol and dopamine. The two cDNAs were 1036 bp (HAST4) and 1060 bp (HAST4v) in length, and encoded proteins that differed by two amino acids (Thr-7 to Ile and Thr-235 to Asn). The coding domains of HAST4 and HAST4v were 97 and 94% homologous to previously reported phenol (HAST1) and monoamine (HAST3) sulfonating forms of sulfotransferase, respectively. On expression of these cDNAs in COS cells the encoded proteins were capable of sulfonating p-nitrophenol with markedly different affinities: the K(m)s for HAST4 and HAST4v being 73.7 and 7.75 microM, respectively. For the same reaction HAST1 and HAST3 have K(m)s of 0.7 and 2200 microM, respectively. Unlike HAST1 and HAST3, the expressed HAST4/4v proteins could not sulfonate dopamine. In addition to having markedly different K(m)s for p-nitrophenol as a substrate, the expressed HAST4/4 proteins also differed significantly in their affinity for the cofactor 3'-phosphoadenosine-5'-phosphosulfate. This report on the functional dissimilarity between two allelic variants of HAST4 highlights that substitution at two residues, Thr-7 and -235, markedly alters their substrate specificities and provides insight into the domains that determine these characteristics.