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.

Evidence for host-associated clones of grape phylloxera Daktulosphaira vitifoliae (Hemiptera: Phylloxeridae) in Australia.

Grape phylloxera, Daktulosphaira vitifoliae Fitch, is an important pest of grapevines (Vitis vinifera L.) (Vitaceae). Using microsatellite DNA markers it was demonstrated strong associations can exist between D. vitifoliae asexual lineages and vine host type within a vineyard. Also, in excised root bioassays, D. vitifoliae collected from three regions where different genotypic classes predominated showed host-specific differences in life table parameters of reproductive rate and intrinsic rate of increase. Lastly, comparisons of mitochondrial DNA (cytochrome oxidase I) sequences revealed that D. vitifoliae in Australia have paraphyletic origins and fall into two clades partially related to vine host usage. These findings indicate introduction of separate lineages of D. vitifoliae which have close host associations and as such, have important implications for management of this pest in Australia.

Clonal reproduction and population genetic structure of grape phylloxera, Daktulosphaira vitifoliae, in Australia.

The grape phylloxera, Daktulosphaira vitifoliae, is a viticultural pest that in the past has devastated vineyards worldwide, yet little is known about this insect's biology. The genetic structure of Australian populations of grape phylloxera and its mode of reproduction were studied following the development of four polymorphic microsatellite loci. Insects were collected from 28 vineyards, with a total of 361 insects included in the study. The majority of vineyards were infested by functionally parthenogenetic lineages of grape phylloxera that inhabit the root system and there was little support for the traditionally described holocyclic life cycle for this species. Clonal diversity was limited in all of the vineyard regions, with the exception of the Rutherglen region. A multiple founder scenario or occasional sex may contribute to diversity within the Rutherglen region. Leaf galling populations comprised classes distinct from the common genotypic classes identified on the roots, suggesting limited exchange between these groups. Implications for the management of D. vitifoliae are discussed.

Proline accumulation in developing grapevine fruit occurs independently of changes in the levels of delta1-pyrroline-5-carboxylate synthetase mRNA or protein.

Mature fruit of grapevine (Vitis vinifera) contains unusually high levels of free proline (Pro; up to 24 micromol or 2.8 mg/g fresh weight). Pro accumulation does not occur uniformly throughout berry development but only during the last 4 to 6 weeks of ripening when both berry growth and net protein accumulation have ceased. In contrast, the steady-state levels of both the mRNA encoding V. vinifera Delta1-pyrroline-5-carboxylate synthetase (VVP5CS), a key regulatory enzyme in Pro biosynthesis, and its protein product remain relatively uniform throughout fruit development. In addition, the steady-state protein levels of Pro dehydrogenase, the first enzyme in Pro degradation, increased throughout early fruit development but thereafter remained relatively constant. The developmental accumulation of free Pro late in grape berry ripening is thus clearly distinct from the osmotic stress-induced accumulation of Pro in plants. It is not associated with either sustained increases in steady-state levels of P5CS mRNA or protein or a decrease in steady-state levels of Pro dehydrogenase protein, suggesting that other physiological factors are important for its regulation.

Identification and characterization of a fruit-specific, thaumatin-like protein that accumulates at very high levels in conjunction with the onset of sugar accumulation and berry softening in grapes.

The protein composition of the grape (Vitis vinifera cv Muscat of Alexandria) berry was examined from flowering to ripeness by gel electrophoresis. A protein with an apparent molecular mass of 24 kD, which was one of the most abundant proteins in extracts of mature berries, was purified and identified by amino acid sequence to be a thaumatin-like protein. Combined cDNA sequence analysis and electrospray mass spectrometry revealed that this protein, VVTL1 (for V. vinifera thaumatin-like protein 1), is synthesized with a transient signal peptide as seen for apoplastic preproteins. Apart from the removal of the targeting signal and the formation of eight disulfide bonds, VVTL1 undergoes no other posttranslational modification. Southern, northern, and western analyses revealed that VVTL1 is found in the berry only and is encoded by a single gene that is expressed in conjunction with the onset of sugar accumulation and softening. The exact role of VVTL1 is unknown, but the timing of its accumulation correlates with the inability of the fungal pathogen powdery mildew (Uncinula necator) to initiate new infections of the berry. Western analysis revealed that the presence of thaumatin-like proteins in ripening fruit might be a widespread phenomenon.

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.

Rapid identification of Acremonium lolii and Acremonium coenophialum endophytes through arbitrarily primed PCR.

Using a random decamer 5'-CCGAGGTGAC-3' in an arbitrarily primed PCR, similar band patterns were observed between Acremonium lolii and A. coenophialum DNA, which were somewhat different from those formed by other fungal DNA. Despite sharing bands of around 0.7, 0.9 and 2.1 kb, A. lolii can be distinguished from A. coenophialum by the presence of an additional band at around 0.5 kb in the arbitrarily primed PCR.

Purification and characterization of ornithine acetyltransferase from Saccharomyces cerevisiae.

Ornithine acetyltransferase has been purified 4000-fold to homogeneity from Saccharomyces cerevisiae. The enzyme catalyses the freely reversible interchange of an acetyl group between N-acetylornithine and glutamate and has a specific activity of 22 mumol.min-1.mg-1 at 37 degrees C and pH 7.5. The Km values were determined for the substrates of the forward and reverse directions to be 1.0 mM for N-acetylornithine, 7.2 mM for glutamate, 1.5 mM for ornithine and 17.1 mM for N-acetylglutamate. The enzyme was localised to the mitochondrial matrix and was found to be a 57-kDa heterodimer consisting of subunits of 31 kDa and 26 kDa. Antibodies raised against the small subunit immunoprecipitated a single in vitro translation product of approximately 57 kDa suggesting that the subunits are processed from a single precursor protein. This is supported by N-terminal sequence analysis which shows that the 26-kDa subunit exhibits 40% sequence identity (8 out of 20) with the N-terminus of ornithine acetyltransferase from Neisseria gonorrhoeae whereas the N-terminus of the 31-kDa subunit exhibits 45% identity (9 out of 20) with a sequence located in the middle of the 60-kDa N. gonorrhoeae enzyme. The N-terminal sequence of the small subunit has the potential to form an amphiphilic helix, further suggesting that the precursor protein with the small subunit at its N-terminus could be targeted to mitochondria and processed into two subunits.

The amdR product and a CCAAT-binding factor bind to adjacent, possibly overlapping DNA sequences in the promoter region of the Aspergillus nidulans amdS gene.

The amdS gene of Aspergillus nidulans is regulated by a number of positively acting regulatory genes which act additively and independently. Using gel mobility shift assays with crude nuclear extracts we show here that the product of one of these regulatory genes, the amdR gene, binds to DNA fragments containing part of the promoter region of the amdS gene. This confirms the earlier prediction from DNA sequence data that amdR encodes a DNA-binding protein containing a cysteine-rich 'zinc finger' motif. In addition we detected the binding of another previously unidentified protein to an adjacent, possibly overlapping region of the amdS 5' sequence at the site of a consensus 'CCAAT-box' sequence. Replacement of the CCAAT sequence with CCTTT abolished the binding of this protein which we have designated as an A. nidulans 'CCAAT-box' binding factor (AnCF). The 'CCAAT-box' sequence appears to be involved in determining the basal level of transcription of amdS (T.G. Littlejohn and M.J.H., unpublished data). This suggests that AnCF is a transcription factor, and that the 'CCAAT-box' sequences found in the promoters of some filamentous fungal genes function as binding sites for these factors, as in other eucaryotes.

Site-directed mutagenesis of Arg60 and Cys271 in ornithine transcarbamylase from rat liver.

We have investigated the putative carbamylphosphate- and ornithine-binding domains in ornithine transcarbamylase from rat liver using site-directed mutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-X and therefore implicated in the binding of the phosphate moiety of carbamylphosphate has been replaced with a leucine. This results in a dramatic reduction of catalytic activity, although the enzyme is synthesized in cells stably transfected with the mutant clone and imported, correctly processed and assembled into a homotrimer in mitochondria. The sole cysteine residue (Cys271) has been implicated in ornithine binding by the chemical modification studies of Marshall and Cohen in 1972 and 1980 (J. Biol. Chem., 247, 1654-1668, 1669-1682; 255, 7291-7295, 7296-7300). Replacement of this residue with serine did not eliminate enzyme activity but affected the Michaelis constant for ornithine (Kb), increasing it 5-fold from 0.71 to 3.7 mM and reduced the kcat at pH 8.5 by 20-fold. These changes represent a loss in apparent binding energy for the enzyme--ornithine complex of 2.9 kcal/mol, suggesting that Cys271 is normally involved in hydrogen bonding to the substrate, ornithine. The cysteine to serine substitution also caused the dissociation constant (Kii) for the competitive inhibitor, L-norvaline to be increased 10-fold, from 12 to 120 microM. The small loss in binding energy and relatively high residual catalytic activity of the mutant strongly suggests that a number of other residues are involved in the binding of ornithine.(ABSTRACT TRUNCATED AT 250 WORDS)

Complementation of the Aspergillus nidulans arg 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.

Characterization of a leuA gene and an ARS element from Mucor circinelloides.

A 4.4-kb PstI restriction endonuclease fragment of Mucor circinelloides DNA has previously been shown to both complement a leuA- mutation, and to enable the autonomous replication of plasmids within this organism. The complete nucleotide (nt) sequence of this fragment has been determined and an open reading frame of 1935 bp with no introns has been identified, which exhibits significant similarity (75% at the nt level) with 114 bp of the 5' coding region of the Saccharomyces cerevisiae LEU1 gene. Based on this and on the fact that the fragment weakly complements a leu1 auxotroph of S. cerevisiae, we concluded that the Mucor leu gene encodes alpha-isopropylmalate (alpha-IPM) isomerase and designated it leuA+ accordingly. Primer extension analysis of leuA mRNA and Northern-blot hybridization, indicated the leuA transcript to be approx. 2.3 kb in size, with 5'- and 3'-untranslated regions of 16-20 nt and approx. 450 nt, respectively. Specific Mucor ARS sequence(s) were not identified, although the general location of ARS was indicated by subcloning experiments. Nucleotide sequences are present within this region, which show some similarity with the core consensus of the S. cerevisiae ARS; however, any functional homology is doubtful, since insertion of the 4.4-kb PstI fragment into YIp5 did not increase the transformation frequency of S. cerevisiae with such a vector.