The Mechanism of Resistance of EUROPEAN Plum to Plum pox virus Mediated by Hypersensitive Response Is Linked to VIRAL NIa and Its Protease Activity.

Plum pox virus (PPV) infects Prunus trees across the globe, causing the serious Sharka disease. Breeding programs in the past 20 years have been successful, generating plum varieties hypersensitive to PPV that show resistance in the field. Recently, a single tree displaying typical PPV symptoms was detected in an orchard of resistant plums. The tree was eradicated, and infected material was propagated under controlled conditions to study the new PPV isolate. Performing overlapping PCR analysis, the viral sequence was reconstructed, cloned and tested for infectivity in different 'Jojo'-based resistant plums. The results confirmed that the isolate, named PPV-D 'Herrenberg' (PPVD-H), was able to infect all these varieties. Analyses of chimeras between PPVD-H and a PPV-D standard isolate (PPVD) revealed that the NIa region of PPD-H, carrying three amino acid changes, was enough to break the resistance of these plums. Experiments with single and double mutants showed that all changes were essential to preserve the escaping phenotype. Additionally, one of the changes at the VPg-NIapro junction suggested the involvement of controlled endopeptidase cleavage in the viral response. Transient expression experiments in Nicotiana benthamiana confirmed that NIa cleavage in PPVD-H was reduced, compared to PPVD, linking the observed behavior to an NIa cleavage modulation.

Histochemical Analysis of Anthocyanins, Carotenoids, and Flavan-3-ols/Proanthocyanidins in Prunus domestica L. Fruits during Ripening.

As a result of the high variability of fruit properties in the European plum Prunus domestica, a histochemical analysis of fruits at different stages of development was performed to understand the ripening process in cv. 'Colora' (yellow-red skinned) and cv. 'Topfive' (purple skinned). Histological analysis showed that carotenoids in the fruit had two different origins. In the fruit flesh, they derived from chloroplasts that turned into chromoplasts, whereas carotenoids in the fruit skin derived probably from proplastids. Flavan-3-ols and proanthocyanidins showed differential localization during ripening. They were visible in the vacuole in different fruit tissues or organized in tannosomes in the fruit flesh. Tanninoplasts were observed only in hypodermal cells of 'Colora'. Toward maturity, anthocyanins were detected in the epidermis and later in the hypodermis of both cultivars. The study forms a basis for the analysis of the biosynthesis of secondary metabolites in European plums and their biological effects.

The rare orange-red colored Euphorbia pulcherrima cultivar 'Harvest Orange' shows a nonsense mutation in a flavonoid 3'-hydroxylase allele expressed in the bracts.

BACKGROUND: Commercially available poinsettia (Euphorbia pulcherrima) varieties prevalently accumulate cyanidin derivatives and show intense red coloration. Orange-red bract color is less common. We investigated four cultivars displaying four different red hues with respect to selected enzymes and genes of the anthocyanin pathway, putatively determining the color hue. RESULTS: Red hues correlated with anthocyanin composition and concentration and showed common dark red coloration in cultivars 'Christmas Beauty' and 'Christmas Feeling' where cyanidin derivatives were prevalent. In contrast, orange-red bract color is based on the prevalent presence of pelargonidin derivatives that comprised 85% of the total anthocyanin content in cv. 'Premium Red' and 96% in cv. 'Harvest Orange' (synonym: 'Orange Spice'). cDNA clones of flavonoid 3'-hydroxylase (F3'H) and dihydroflavonol 4-reductase (DFR) were isolated from the four varieties, and functional activity and substrate specificity of the corresponding recombinant enzymes were studied. Kinetic studies demonstrated that poinsettia DFRs prefer dihydromyricetin and dihydroquercetin over dihydrokaempferol, and thus, favor the formation of cyanidin over pelargonidin. Whereas the F3'H cDNA clones of cultivars 'Christmas Beauty', 'Christmas Feeling', and 'Premium Red' encoded functionally active enzymes, the F3'H cDNA clone of cv. 'Harvest Orange' contained an insertion of 28 bases, which is partly a duplication of 20 bases found close to the insertion site. This causes a frameshift mutation with a premature stop codon after nucleotide 132 and, therefore, a non-functional enzyme. Heterozygosity of the F3'H was demonstrated in this cultivar, but only the mutated allele was expressed in the bracts. No correlation between F3'H-expression and the color hue could be observed in the four species. CONCLUSIONS: Rare orange-red poinsettia hues caused by pelargonidin based anthocyanins can be achieved by different mechanisms. F3'H is a critical step in the establishment of orange red poinsettia color. Although poinsettia DFR shows a low substrate specificity for dihydrokaempferol, sufficient precursor for pelargonidin formation is available in planta, in the absence of F3'H activity.

Constitutive Polyphenols in Blades and Veins of Grapevine ( Vitis vinifera L.) Healthy Leaves.

Despite the economic importance and the diffusion of grapevine cultivation worldwide, little is known about leaf chemical composition. We characterized the phenolic composition of Nebbiolo, Barbera, Pinot noir, Cabernet Sauvignon, Grenache, and Shiraz ( Vitis vinifera L.) healthy leaves (separating blades and veins) during the season. Quantitative and qualitative differences were found between leaf sectors and among genotypes. In healthy grapevine leaves, anthocyanins, dihydromyricetin-rhamnoside, hexosides of dihydroquercetin, and dihydrokaempferol exclusively accumulated in veins. Astilbin was the only flavanonol detected in blades and the prevalent flavanonol in veins. Barbera distinguished for the lowest proanthocyanidin and the highest hydroxycinnamate content, and Pinot noir for the absence of acylated-anthocyanins. Nebbiolo, and Cabernet Sauvignon displayed a high concentration of epigallocatechin gallate in veins. Nebbiolo leaves showed the highest concentrations of flavanonols and the widest profile differentiation. Knowledge derived from the present work is a contribution to find out leaf polyphenol potential as a part of grapevine defense mechanisms and to dissect genotype-related susceptibility to pathogens; moreover, it represents a starting point for future deepening about grapevine and vineyard byproducts as a source of bioactive phenolic compounds.