Fruit ripening in Vitis vinifera: spatiotemporal relationships among turgor, sugar accumulation, and anthocyanin biosynthesis

This study reports the first observations indicating the spatiotemporal relationships among genetic and physiological aspects of ripening in the berry of Vitis vinifera. At the onset of ripening in the red flesh variety Alicante Bouschet, colour development began in the flesh at the stylar end of the fruit and progressed toward the pedicel end flesh and into the skin. Tissue solute potential and cell turgor also decreased first in the flesh. The decrease in flesh solute potential was due to accumulation of sugars, glucose and fructose, an accumulation that is integral to ripening. Expression of the anthocyanin biosynthesis-related genes VvMybA and VvUFGT was linearly related to the decrease in solute potential. Expression of VvMybA, and to a lesser extent VvUFGT, was correspondingly low in green tissue, higher in the red, stylar end flesh of berries beginning to ripen, and greatest in red berries. In contrast, expression of the abscisic acid biosynthesis-related genes VvNCED1 and VvNCED2 was not correlated with the other spatiotemporal aspects of the onset of ripening. These results, together with earlier work showing that sugar accumulation and acid loss also begin in the stylar flesh in other varieties, indicate that ripening in the grape berry originates in the stylar end flesh.     

Restriction fragment length polymorphism and molecular taxonomy in Vitis vinifera L.

Forty-six accessions of grapevine (V. vinifera L.) were compared by restriction fragment length polmorphism (RFLP) analysis, and 111 informative or unique restriction fragments were found that revealed an important level of polymorphism. RFLP patterns were compared in two ways: by calculating electrophoretic similarity degree values further analyzed by principal component analysis and by studying the distribution of rare restriction fragments. Six taxonomic groups could be defined, which partially confirmed relationships derived from ampelographical data. Our data support the existence of ecogeographical groups.     

Enzymes of Krebs-Henseleit Cycle in Vitis vinifera L: II. Arginosuccinate Synthetase and Lyase

Arginosuccinate (ASA) synthetase and lyase activities were detected in extracts from Vitis vinifera L. cv. Chenin blanc mature leaves and seedlings. Optimum reaction conditions for ASA synthetase were 10 millimolar l-citrulline, 7.5 millimolar l-aspartate, 3 to 4 millimolar ATP, 12 millimolar Mg(2+) (pH 7.5 to 8.0), enzyme extract up to equivalent of about 200 milligrams of fresh tissue, and incubation temperature of 38 to 40 C. Optimum reaction conditions for ASA lyase were 4 millimolar ASA-K salt (pH 7.3 to 7.8), amount of extract up to equivalent of about 180 milligrams of fresh tissue, and incubation temperature of 38 to 40 C.     

Induction of somatic embryogenesis and plantlets in tendrils of Vitis vinifera L.

Somatic embryogenesis was observed in callus initiated from tendril explants of Vitis vinifera L. cvs. Thompson, Sonaka and Tas-e-Ganesh on Emershad and Ramming medium supplemented with 1 μm 6-benzylaminopurine. Low-frequency conversion to shoots was obtained in the third and fourth subculture on the same medium. Emerging shoots subsequently formed complete plantlets on liquid rooting medium containing 1 μm indole-3-acetic acid. The possible use of tendrils as a novel explant for somatic embryogenesis in grape is discussed. Received: 3 March 1997 / Revision received: 21 May 1997 / Accepted: 25 June 1997     

葡萄病程相关蛋白1基因的克隆和表达分析

以葡萄品种‘左优红’组培苗叶片为材料,利用同源克隆法获得其病程相关蛋白1基因VvPR1的cDNA全长序列。扩增片段大小为486bp,编码161个氨基酸,分子量17.5kDa,等电点PI=8.69,含有6个保守半胱氨酸,4个allergenV5/Tpx-1related保守结构域。VvPR1与多种植物PR1高度同源。实时定量PCR检测结果表明VvPR1在葡萄叶片中相对表达量最高;霜霉病菌、低温、盐和干旱胁迫均可显著诱导其表达;水杨酸、脱落酸、茉莉酸、一氧化氮、过氧化氢和硫化氢等亦可诱导其大量表达,据此推测,VvPR1参与了多种生物胁迫和非生物胁迫过程。     

Morphogenetic Effects of Roots and of Some Synthetic Cytokinins in Vitis vinifera L.

In woody cuttings of the grape vine, inflorescences of fertilebuds usually fail to develop and atrophy soon after bud burst.Results presented here indicate that this effect is relatedto absence of roots at planting. Inflorescences were retainedin pre-rooted cuttings which were propagated by holding thetemperature of the medium at 25° C and the air temperatureat 4° C. Similar effects on inflorescence growth were obtainedby applying synthetic cytokinins to the bases of unrooted cuttingsin solution cultures, and by applications to emergent inflorescences.Promotion of inflorescence growth was found with 6-benzyl-aminopurine(BAP) and 6-(benzylamino)-9-(2-tetrahydropyranyl)-9H purine(‘SD 8₃₃₉’). Stimulation of inflorescence growthby BAP was accompanied by reduction in vegetative growth, andby development of red pigments in inflorescences and leaves.Results of cincturing experiments indicate that BAP is transportedacropetally in the xylem of vines. Effects of roots, and effectsof synthetic cytokinins, are discussed in relation to recentdiscoveries of endogenous cytokinins in the ascending sap ofvines.     

Regeneration of transgenic shape Vitis vinifera L. Sultana plants: genotypic and phenotypic analysis

Different approaches to producing transgenic grapevines based on regeneration via embryogenesis were investigated. Embryogenic callus was initiated from anther tissue of Vitis vinifera cv. Sultana and three embryogenic culture types (embryogenic callus, tissue type I; proliferating embryos, tissue type II; and a suspension) were established. The three culture types were incolucaled with Agrobacterium tumefaciens harbouring a binary vector which contained a uidA reporter gene and either a hpt or nptII selectable marker gene or the cultures were bombarded with microprojectiles carrying a uidA/nptII binary vector. Transgenic plants were produced only from Agrobacterium transformation experiments. Transformed embryos were selected with kanamycin or hygromycin antibiotics and recovered with the highest efficiency from inoculated type I cultures. Southern analysis of genomic DNA extracted from ten transgenic plants showed that the number of T-DNA insertions in the genome ranged from 1 to at least 4. Evidence for methylation of the T-DNA at cytosine and adenine residues in transgenic plants was found by Southern analysis of DNA digested with two isoschizomer pairs of restriction endonucleases. No evidence for genotype alterations or somatic meiosis was found when DNA from 80 somatic embryos and seven plants regenerated from embryogenic culture were analysed at six sequence-tagged sites which are heterozygous in cv. Sultana. Expression of the uidA gene in in vitro grown leaves of transgenic plants was most often high and uniform but GUS staining was occasionally observed to be low and/or patchy. Transgenic plants and all plants regenerated from embryogenic culture produced red veined, lobed leaves which are uncharacteristic of the accepted ampelographic phenotype of Sultana. It is suggested that this phenotype may represent a juvenile growth stage.     

Enzymes of Krebs-Henseleit Cycle in Vitis vinifera L: I. Ornithine Carbamoyltransferase: Isolation and Some Properties

Ornithine carbamoyltransferase (OCT) activity was detected in extracts from mature leaves, fruit, germinating seeds, and seedlings of Vitis vinifera L. Michaelis-Menten constants for OCT were 3.5 millimolar for carbamyl phosphate and 5.5 millimolar for l-ornithine. Concentrations of l-ornithine greater than 10 millimolar slightly inhibited the enzyme, whereas carbamyl phosphate at concentrations greater than the optimal (about 10 millimolar) did not affect OCT activity. l-Citrulline formation was linear with incubation period for the first 25 minutes and with increasing amounts of enzyme up to an equivalent of about 200 milligrams of fresh tissue. The optimum pH for in vitro OCT activity was between 8.4 and 8.8, and the optimum incubation temperature was 38 C.     

Investigations of 5S rDNA of Vitis vinifera L.: sequence analysis and physical mapping.

Here we report the first results of a study of 5S rDNA of Vitis vinifera. 5S rDNA sequences from seven genotypes were amplified by PCR, cloned, and sequenced. Three types of repeats were found. Two variants, denominated long repeat and short repeat, appeared to be the main components of the 5S rDNA of this species, since they were found in all genotypes analyzed. They differed markedly from each other in both the length and the nucleotide composition of the spacers. The third variant, classified as DEL short repeat, differs from the short repeat owing to a large deletion in the spacer region. It appears to be the most recent repeat type, since it was identified in only one genotype. The organization of the 5S rDNA repeat unit variants was investigated by amplifying the genomic DNA with primers designed on the sequence of the long and short spacers. The PCR-amplified fragments showed that the long repeat is associated with the other two repeats, indicating that in V. vinifera different repeat units coexist within the same tandem array. FISH analysis demonstrated that 5S rRNA genes are localized at a single locus. The variability of 5S rDNA repeats is discussed in relation to the putative allopolyploid origin of V. vinifera.     

Cloning and characterization of Vine-1, a LTR-retrotransposon-like element in Vitis vinifera L., and other Vitis species.

We report the organization of a grapevine chimeric gene Adhr-Vine-1, composed by an Adhr gene, into which a retroelement, Vine-1, was inserted. Sequence analysis revealed that Adhr is a member of the Adh multigene family, but does not correspond to any other grapevine Adh described to date. Vine-1, albeit defective, is the most complete LTR (long terminal repeat)-retrotransposon-like element described in Vitis vinifera L. It is 2392 bp long, with two almost identical LTRs (287 bp) in the same orientation, and flanked by direct repeats of a 5 bp host DNA. This element presents other features, characteristic of retroviruses and retrotransposons including inverted repeats, a primer binding site, and a polypurine tract. It has a single open reading frame (ORF) of 581 amino acids, potentially encoding for a gag protein and parts of the protease and integrase proteins. Vine-1 is most likely related to the copia-like type family, but with no significant similarity to any previously described plant retrotransposon or inserted element, nor to any eukaryotic element described to date. Vine-1 element has been found in Adhr at the same location in different V. vinifera cultivars, but not in some other analyzed Vitis species. These data suggest that Vine-1 insertion in Adhr is specific to V. vinifera, and has occurred after the Adh isogene separation, but prior to cultivar development. Sequences related to Vine-1 were revealed in multiple copies in the V. vinifera genome and, to a lesser extent, in other analyzed Vitis species. The polymorphism observed prompts us to question the role played by transposition in the evolution of the Vitis genus.     

Haemolymph amino acid and sugar levels in locusts fed nutritionally unbalanced diets

Aspects of pre- and post-ingestive compensation were investigated in locusts (Locusta migratoria) fed nutritionally unbalanced artificial diets containing 7% protein and 21% digestible carbohydrate (7:21) or 21% protein and 7% digestible carbohydrate (21:7). Feeding behaviour and haemolymph levels of amino acids and sugars were measured in locusts fed ad libitum on these diets. Locusts fed the high-protein diet had chronically elevated haemolymph levels of 15 out of 19 amino acids measured compared to locusts fed the low protein diet. However, haemolymph levels of lysine, alanine, aspartic acid and glutamic acid did not differ between diets, suggesting some specific regulatory mechanism for these amino acids. Haemolymph glucose and trehalose reflected levels of carbohydrate in the diets, being high in insects fed diet 7:21 relative to those given diet 21:7. These data are discussed in relation to the physiological and behavioural bases of nutritional homeostasis.Abbreviations AA amino acid(s) - PRO protein - CHO carbohydrate - PBS phosphate-buffered saline - MW molecular weight     

Restructuring of endophytic bacterial communities in grapevine yellows-diseased and recovered Vitis vinifera L. plants

Length heterogeneity-PCR assays, combined with statistical analyses, highlighted that the endophytic bacterial community associated with healthy grapevines was characterized by a greater diversity than that present in diseased and recovered plants. The findings suggest that phytoplasmas can restructure the bacterial community by selecting endophytic strains that could elicit a plant defense response.     

Small intestinal sugar and amino acid transport in semistarvation.

The effect of semistarvation on small intestinal transport of D-glucose, L-valine, and NaCl was studied in an in vitro system of isolated rat brush border membrane vesicles. Whereas semistarvation enhanced the transport rate for L-valine by 19-29%, there was no change in D-glucose transport. When energy in the form of a NaSCN gradient was supplied to the membrane vesicles prepared from semistarved animals, L-valine was concentrated to a greater extent than those from well-fed animals. Strain differences were observed in the manner semistarvation affected NaCl transport across the brush border membrane. Semistarvation increased the NaCl transport rate by a factor of 3.5 in one rat strain and not at all in another. These results provide a partial explanation for the cellular basis of elevated neutral amino acid absorption by the small intestine in semistarvation.     

Enzymes of Krebs-Henseleit Cycle in Vitis vinifera L: III. In Vivo and In Vitro Studies of Arginase

Ornithine carbamoyltransferase (OCT) activity was detected in extracts from mature leaves, fruit, germinating seeds, and seedlings of Vitis vinifera L. Michaelis-Menten constants for OCT were 3.5 millimolar for carbamyl phosphate and 5.5 millimolar for l-ornithine. Concentrations of l-ornithine greater than 10 millimolar slightly inhibited the enzyme, whereas carbamyl phosphate at concentrations greater than the optimal (about 10 millimolar) did not affect OCT activity. l-Citrulline formation was linear with incubation period for the first 25 minutes and with increasing amounts of enzyme up to an equivalent of about 200 milligrams of fresh tissue. The optimum pH for in vitro OCT activity was between 8.4 and 8.8, and the optimum incubation temperature was 38 C.     

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Axenic shoot cultures of virus-free Vitis vinifera L. cv. Soultanina were a highly efficient source for isolation of viable protoplasts. Optimum results were obtained with leaves of 50–100 mg fresh weight, leaf discs of 0.7 cm in diameter, 100 and 15 U ml^-1 Cellulase R-10 and Macerozyme R-10, respectively, and 18 h reaction time in either light or in darkness. Protoplast yield was approx. 25×10⁶ viable protoplasts per g fresh weight and their size ranged from 12 to 44 m. During a 20-day culture period, the maximum survival rate obtained was approx. 40%. A plating density of 10×10⁵ protoplasts per ml resulted in increased survival rates. Various growth regulators and glutamine did not significantly improve survival rates of protoplasts, whereas extract from coconut added to the culture medium caused an increase in the survival rates of protoplasts. Cell elongation at a significant rate and divisions were observed. [¹⁴C]glucose uptake was studied as an index of cell membrane integrity and functioning. Uptake rate of glucose by protoplasts was linear for up to 60 min, fully inhibited by NaN₃, with an optimum pH of 4.8. Protoplasts 24 h old exhibited significantly lower rates of glucose uptake.     

Manipulating anthocyanin composition in Vitis vinifera suspension cultures by elicitation with jasmonic acid and light irradiation

Jasmonic acid altered the accumulation of major anthocyanins in Vitis vinifera cell culture. Peonidin 3-glucoside content at day three was increased from 0.3 to 1.7 mg g^–1 dry cell wt while other major anthocyanins were increased by smaller increments. By day 14, the content of methylated and acylated anthocyanins (peonidin 3-p-coumaroylglucoside and malvidin 3-p-coumaroylglucoside) was 6.3 mg g^–1 DCW, in response to treatment with jasmonic acid, and comprising 45% (w/w) of total anthocyanins. In comparison, the untreated control culture contained 1.2 mg g^–1 DCW which made up 32% (w/w) of total anthocyanins. Light further enhanced anthocyanin accumulation induced by jasmonic acid elicitation. The content of peonidin 3-glucoside at day 3 was 6.6 mg g^–1 DCW, 22-fold higher than control cultures while the content in response to light irradiation alone was 0.6 mg g^–1 DCW. When a highly pigmented cell line was elicited with jasmonic acid total anthocyanins increased from 9.2 to 20.7 mg g^–1 DCW, but there was no change in the anthocyanin composition.     

Sequencing and assembly of highly heterozygous genome of Vitis vinifera L. cv Pinot Noir: problems and solutions

A new approach to sequencing and assembling a highly heterozygous genome, that of grape, species Vitis vinifera cv Pinot Noir, is described. The combining of genome shotgun of paired reads produced by Sanger sequencing and sequencing by synthesis of unpaired reads was shown to be an efficient procedure for decoding a complex genome. About 2 million SNPs and more than a million heterozygous gaps have been identified in the 500Mb genome of grape. More than 91% of the sequence assembled into 58,611 contigs is now anchored to the 19 linkage groups of V. vinifera.