Accession-Level Differentiation of Urushiol Levels, and Identification of Cardanols in Nascent Emerged Poison Ivy Seedlings.

Poison ivy (Toxicodendron radicans (L.) Kuntze) shows accession-level differentiation in a variety of morphometric traits, suggesting local adaptation. To investigate whether the presumed defense compound urushiol also demonstrates accession-level accumulation differences, in vitro nascent germinated poison ivy seedlings from geographically isolated populations were germinated in vitro and then assayed for known urushiol congener accumulation levels. Significant accession-level differences in the accumulation levels of total C15- and C17-, total C15-, total C17-, specific C15 congeners, and specific C17 congeners of urushiol were identified. In addition, hereto novel C15- and C17-urushiol isomers were identified as well. Cardanols are assumed to be the penultimate metabolites giving rise to urushiols, but this assumption was not previously empirically validated. C15-cardanol congeners and isomers corresponding to expected substrates needed to produce the observed C15-urushiol congeners and isomers were identified in the same poison ivy seedling extracts. Total C15-cardanol and C15-cardanol congeners also showed significant accession-level differences. Based on the observed C15-cardanol congeners in poison ivy, the penultimate step in urushiol biosynthesis was proposed to be a cardanol-specific hydroxylase activity.

Retrotransposon-based markers from potato monoploids used in somatic hybridization.

In an attempt to remove lethal and deleterious genes and enhance the heterozygosity of the potato genome, we developed several diverse somatic hybrids through the electrofusion of selected monoploids. Somatic hybrids and somaclones resulting from fused and unfused protoplasts, respectively, were verified with microsatellites. Molecular markers anchored in the Tst1 retrotransposon were used to examine polymorphisms in the regenerated plants and to reveal any somaclonal variation. Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon display (sequence-specific amplified polymorphism (S-SAP), anchored in a retransposon) were examined on an ALFexpress DNA sequencer. Because of inconsistencies in the number and quality of bands revealed by the combination of either class of marker in combination with the ALFexpress, we cloned and sequenced 11 S-SAP bands to use as restriction fragment length polymorphism (RFLP) probes in Southern blot analyses of genetic relationships in our potato populations and among related Solanaceae. Readily scorable bands (n = 27) that separated somatic hybrids from parental monoploids and somaclones and grouped monoploids according to known genetic relationships were produced. Some of the probes could be used to differentiate tomato and Datura from potato. Sequence analysis of 5 cloned IRAP and 11 cloned S-SAP markers confirmed that they were anchored in the Tst1 retrotransposon. BLAST searches within GenBank produced 10 highly significant hits (5 nucleotide, 4 expressed sequence tag (EST), and 1 protein) within closely related Solanaceae, suggesting that Tst1 represents an old retroelement that was inserted before the diversion of genera within Solanaceae; however, most sequences were undescribed.

Association of diamine oxidase and S-adenosylhomocysteine hydrolase in Nicotiana tabacum extracts.

The oxidative deamination of methylated putrescine by a diamine oxidase activity (DAO) is an important step in the biosynthesis of nicotine in tobacco and tropane alkaloids in several Solanaceous plants. A polyclonal rabbit antiserum was previously developed to a purported purified DAO enzyme from Nicotiana tabacum. The antiserum bound to a single 53 kDa protein and immunoprecipitated 80% of DAO activity from tobacco root extracts. In an effort to obtain DAO cDNAs, this antiserum was used to screen a tobacco cDNA expression library and three distinct immunoreactive cDNA clones were isolated. These cDNAs encoded predicted proteins that were either identical or nearly identical to predicted S-adenosylhomocysteine hydrolase (SAHH) from two Nicotiana species. Thus, the rabbit antiserum was not specific to DAO, even though it immunodepleted the majority of DAO activity from root extracts. Alternative hypotheses to explain the DAO immunodepletion results (such as poisoning of DAO activity or that SAHH is a bifunctional enzyme) were tested and ruled out. Therefore, we hypothesize that SAHH associates with DAO as part of a larger multienzyme complex that may function in planta as a nicotine metabolic channel.