Molecular and cellular mechanisms of diversity within grapevine varieties.

A grapevine variety consists of an array of clones descended by vegetative propagation from a single selected vine grown from a single seedling. A majority of the clones within a variety are identical, but some can show divergent genotypes and, to some extent, divergent phenotypes. Polymorphism results mainly from somatic mutations that occur naturally during plant growth. Various types of mutations were shown to be responsible for genetic diversity among clones: point mutations, large deletions, illegitimate recombination or variable number of repeats in microsatellite sequences. In most cases, somatic mutations do not affect the whole plant; rather, they affect only one cell layer, leading to periclinal chimeras. Such structures do not threaten the plant's fitness and are stable through vegetative propagation. Occasionally, cellular rearrangements in the chimera lead to homogenization of the genotype of the whole plant. Through these molecular and cellular mechanisms, the genotypes of clones drift over time and grapevine varieties evolve.

Genetic and biochemical analysis of intragenic complementation events among nitrate reductase apoenzyme-deficient mutants of Nicotiana plumbaginifolia.

Intragenic complementation has been observed between apoenzyme nitrate reductase-deficient mutants (nia) of Nicotiana plumbaginifolia. In vivo as in vitro, the NADH-nitrate reductase (NR) activity in plants heterozygous for two different nia alleles was lower than in the wild type plant, but the plants were able to grow on nitrate as a sole nitrogen source. NR activity, absent in extracts of homozygous nia mutants was restored by mixing extracts from two complementing nia mutants. These observations suggest that NR intragenic complementation results from either the formation of heteromeric NR or from the interaction between two modified enzymes. Complementation was only observed between mutants retaining different partial catalytic activities of the enzyme. Results are in agreement with molecular data suggesting the presence of three catalytic domains in the subunit of the enzyme.

Complete sequence of Tvv1, a family of Ty 1 copia-like retrotransposons of Vitis vinifera L., reconstituted by chromosome walking.

A chromosome-walking strategy was used to sequence and characterize retrotransposons in the grapevine genome. The reconstitution of a family of retroelements, named Tvv1, was achieved by six successive steps. These elements share a single, highly conserved open reading frame 4,153 nucleotides-long, putatively encoding the gag, pro, int, rt and rh proteins. Comparison of the Tvv1 open reading frame coding potential with those of drosophila copia and tobacco Tnt1, revealed that Tvv1 is closely related to Ty 1 copia-like retrotransposons. A highly variable untranslated leader region, upstream of the open reading frame, allowed us to differentiate Tvv1 variants, which represent a family of at least 28 copies, in varying sizes. This internal region is flanked by two long terminal repeats in direct orientation, sized between 149 and 157 bp. Among elements theoretically sized from 4,970 to 5,550 bp, we describe the full-length sequence of a reference element Tvv1-1, 5,343 nucleotides-long. The full-length sequence of Tvv1-1 compared to pea PDR1 shows a 53.3% identity. In addition, both elements contain long terminal repeats of nearly the same size in which the U5 region could be entirely absent. Therefore, we assume that Tvv1 and PDR1 could constitute a particular class of short LTRs retroelements.

Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms.

The domestication of the Eurasian grape (Vitis vinifera ssp. sativa) from its wild ancestor (Vitis vinifera ssp. sylvestris) has long been claimed to have occurred in Transcaucasia where its greatest genetic diversity is found and where very early archaeological evidence, including grape pips and artefacts of a 'wine culture', have been excavated. Whether from Transcaucasia or the nearby Taurus or Zagros Mountains, it is hypothesized that this wine culture spread southwards and eventually westwards around the Mediterranean basin, together with the transplantation of cultivated grape cuttings. However, the existence of morphological differentiation between cultivars from eastern and western ends of the modern distribution of the Eurasian grape suggests the existence of different genetic contribution from local sylvestris populations or multilocal selection and domestication of sylvestris genotypes. To tackle this issue, we analysed chlorotype variation and distribution in 1201 samples of sylvestris and sativa genotypes from the whole area of the species' distribution and studied their genetic relationships. The results suggest the existence of at least two important origins for the cultivated germplasm, one in the Near East and another in the western Mediterranean region, the latter of which gave rise to many of the current Western European cultivars. Indeed, over 70% of the Iberian Peninsula cultivars display chlorotypes that are only compatible with their having derived from western sylvestris populations.

Effect of ochre nonsense mutations on yeast URA1 mRNA stability.

The genetic map of 27 mutants of the URA1 yeast gene has been established by meiotic recombination and 16 nonsense mutations characterized. The half life of URA1 mRNA was determined by two independent methods in the wild-type and in two ochre mutants localized at each extremity of the genetic map. A halflife of 15 min was found for the wild-type and for one of the ochre mutants. This half-life was radically reduced in the other ochre mutant whereas the instantaneous rate of its mRNA synthesis remained constant. After subcloning various endonucleolytic fragments the coding sequence of the URA1 gene was restricted to a 1.65 kb fragment within a 5.7 kb yeast DNA segment. Direct visualization of the URA1 mRNA by Northern hybridization of denatured RNA with a URA1 specific DNA probe revealed a length of 1.5 kb.

Diversification within grapevine cultivars goes through chimeric states.

Vitis vinifera 'Pinot' clones were analysed at 50 microsatellite loci to assess intravarietal genetic diversity. When analysing leaf tissue DNAs, polymorphism mainly resulted from the appearance of a third allele when two were expected for heterozygous loci in a diploid species. The sequencing of the three microsatellite alleles at two loci has confirmed their simultaneous presence in the leaf tissues. A hypothesis explaining the triallelic profiles at a locus is the presence of a periclinal chimera meristem structure, in which genetically different cell layers coexist. The periclinal chimeric state of two Vitis vinifera 'Pinot gris' clones was confirmed by splitting and analysing the genotypes resulting from L1 and L2 cell layers in progeny derived from self-fertilization, in root tissues, and in plants regenerated from somatic embryogenesis. Prevalence of chimerism in polymorphic clones observed in a collection of 145 accessions belonging to 'Pinot gris', 'Pinot noir', Pinot blanc', 'Pinot meunier', and 'Pinot moure' cultivars was demonstrated. The accumulation of somatic mutations and cell layer rearrangements allowed us to deduce the relationships between the various genotypes and to open a way for understanding the diversification process and the phylogeny in the 'Pinot' group.

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia.

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.