E.H. Rühl

Activity-based profiling of a physiologic aglycone library reveals sugar acceptor promiscuity of family 1 UDP-glucosyltransferases from grape.

A method based on an aglycone library was developed for the targeted analysis of acceptor molecules of UDP-glycosyltransferases. Monoterpenols serve various biological functions and accumulate in grape (Vitis vinifera), where a major fraction occurs as nonvolatile glycosides. We have screened the grape genome for sequences with similarity to terpene URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASES (UGTs) from Arabidopsis (Arabidopsis thaliana). A ripening-related expression pattern was shown for three candidates by spatial and temporal expression analyses in five grape cultivars. Transcript accumulation correlated with the production of monoterpenyl β-d-glucosides in grape exocarp during ripening and was low in vegetative tissue. Targeted functional screening of the recombinant UGTs for their biological substrates was performed by activity-based metabolite profiling (ABMP) employing a physiologic library of aglycones built from glycosides isolated from grape. This approach led to the identification of two UDP-glucose:monoterpenol β-d-glucosyltransferases. Whereas VvGT14a glucosylated geraniol, R,S-citronellol, and nerol with similar efficiency, the three allelic forms VvGT15a, VvGT15b, and VvGT15c preferred geraniol over nerol. Kinetic resolution of R,S-citronellol and R,S-linalool was shown for VvGT15a and VvGT14a, respectively. ABMP revealed geraniol as the major biological substrate but also disclosed that these UGTs may add to the production of further glycoconjugates in planta. ABMP of aglycone libraries provides a versatile tool to uncover novel biologically relevant substrates of small-molecule glycosyltransferases that often show broad sugar acceptor promiscuity.

A UDP-Glucose:Monoterpenol Glucosyltransferase Adds to the Chemical Diversity of the Grapevine Metabolome

A glucosyltransferase forms geranyl and neryl glucosides during grape ripening. Terpenoids represent one of the major classes of natural products and serve different biological functions. In grape (Vitis vinifera), a large fraction of these compounds is present as nonvolatile terpene glycosides. We have extracted putative glycosyltransferase (GT) sequences from the grape genome database that show similarity to Arabidopsis (Arabidopsis thaliana) GTs whose encoded proteins glucosylate a diversity of terpenes. Spatial and temporal expression levels of the potential VvGT genes were determined in five different grapevine varieties. Heterologous expression and biochemical assays of candidate genes led to the identification of a UDP-glucose:monoterpenol β-d-glucosyltransferase (VvGT7). The VvGT7 gene was expressed in various tissues in accordance with monoterpenyl glucoside accumulation in grape cultivars. Twelve allelic VvGT7 genes were isolated from five cultivars, and their encoded proteins were biochemically analyzed. They varied in substrate preference and catalytic activity. Three amino acids, which corresponded to none of the determinants previously identified for other plant GTs, were found to be important for enzymatic catalysis. Site-specific mutagenesis along with the analysis of allelic proteins also revealed amino acids that impact catalytic activity and substrate tolerance. These results demonstrate that VvGT7 may contribute to the production of geranyl and neryl glucoside during grape ripening.

Dynamic grapevine clones—an AFLP-marker study of the Vitis vinifera cultivar Riesling comprising 86 clones

Grapevine cultivars are planted in worldwide viticulture and are asexually propagated. Horticultural clones are asexually derived from a single individual, and clonal variation can only occur through mutations. Molecular markers are an important tool for the differentiation and identification of clones and mutations. For breeding purposes and clonal selection, knowledge upon the variability of a given clone is essential. The aim of this study was to assess amplified fragment length polymorphism (AFLP) markers for classifying mutations in 86 Riesling clones of Vitis vinifera and to enhance our understanding on the dynamic of grapevine clones analysed by AFLP fingerprints. AFLP markers detected 135 polymorphic bands of a total amount of 305 bands. AFLP markers detected two different types of mutations: single-event mutations, only detected once in one clone, displaying the variation of the grape genome and specific loci mutations where the mutation could be found frequently in the set of clones and therefore stand for the stability of grapevine genome. A general grouping of clones according to age, sub-clonal lineage or origin could not be determined by the set of AFLP markers employed.

Grapevine ( Vitis ssp.): Example of Clonal Reproduction in Agricultural Important Plants

This review approaches the concept of clonality (asexual reproduction) and its implications for phenotypic and genetic stability. Grapevine cultivars (Vitis vinifera L.) are composed of clones showing homogeneous ampelographic characteristics with minor differences. The concept of clonal selection (through vegetative propagation) implies very low genetic variation within a “population of genotypes identical to the ancient progenitor except of mutations”. Yet, the accumulated genetic variation is higher than expected in cultivated grapevine clones. This variation is combined with numerous mechanisms of asexual strategies to enhance variation and to provide an open system for adaptation and selection processes. This chapter presents insights into the clonal selection of grapevine exemplifying the cultivar Pinot noir (V. vinifera L.) to elucidate the potential sources of its phenotypic and genotypic variation. The impact of clonal propagation of this agricultural important crop will also be discussed.

Cultivar Identity, Intravarietal Variation, and Health Status of Native Grapevine Varieties in Croatia and Montenegro

Croatia and Montenegro (located in southeast Europe) host numerous native (autochthonous) minor grapevine (Vitis vinifera L.) varieties that might irreversibly disappear in the near future because of limited conservation efforts and lack of virus-free propagation material. To prevent their disappearance and help sustain these native populations, a collaborative project was launched to gather and evaluate extremely rare and neglected native grapevine genotypes by establishing true-to-type and virus-tested stock material, ready to use for nurseries. During several field expeditions, 284 grapevine accessions collected in 22 different locations in Croatia and Montenegro were subjected to ampelographic in situ description, genetic identification by nine simple sequence repeat (SSR) markers, serological testing for the presence of common grape viruses, and intravarietal variability assessment by sequence-specific amplification polymorphism (S-SAP) markers. We observed 53 unique SSR profiles, of which 25 genotypes have not been previously reported and are thus worthy of further research. Comparison of these genetic profiles with those from some very rare and poorly described accessions within the European Vitis Database and other published studies revealed several previously unknown synonyms and homonyms, which helped properly name some of the profiles and provided novel information on the history of these cultivars and their distribution in the region. Virus testing revealed high frequencies of infection with Grapevine leafroll-associated viruses 1 and 3 and indicated the urgent need for sanitary selection. Assessment of intravarietal genetic variation indicated the presence of accumulated mutations, which strongly suggests a need for clonal selection.

Variability in vitis berlandieri

The invasion of phylloxera during the 19th century nearly led to the collapse of viticulture in Europe. This could only be prevented by the introduction of phylloxera tolerant American wild grapevine species, used henceforth as rootstocks for grapevine grafting. While phylloxera control is the major aim of rootstock breeding, a wide ranged suitability to different abiotic environmental stress factors, particularly with regards to soil adaptation, is of great importance as well. The majority of the European vineyards, typically characterized by higher lime contents, are not compatible with most of the American wild Vitis species. However, the American species Vitis berlandieri is very well adapted to limey soils, and thus makes it an interesting partner for rootstock breeding. In the beginnings of rootstock breeding only a few V. berlandieri individuals had been available. So most of all of the rootstock cultivars used today comprise only a very small fraction of V. berlandieri?s genetic information. To study the variability of Vitis berlandieri, seeds were collected in their natural habitat in Texas in the year 2005 (Schmid et al., 2009) and have been germinated and planted in an observation block at Geisenheim. This population of nearly 3500 individuals should be very well suited to represent a more complete picture of V. berlandieri?s gene pool. In our ongoing project, the phenotypic and genetic characteristics of these Vitis berlandieri individuals are being evaluated continuously. Currently, 2000 seedlings of 21 different mother plants are being monitored by using ampelographic and physiological characteristics and genetic markers. Results point out that the offspring of different accessions are showing various distinctive characteristic features, either within one accession, or between different accessions. This indicates a larger variability of V. berlandieri than primarily assumed, and it offers a more ample spectrum for future rootstock breeding programs.