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Studies of cuticle drugs from natural sources. II. Inhibitory effects of Prunus plants on melanin biosynthesis.

The inhibitory effects of 50% ethanolic extracts from dried leaves of 38 plants collected in the herbal garden of Kinki University were investigated in vitro on melanin biosynthesis which is closely related to hyperpigmentation. Of the 38 extracts, Prunus yedoensis, P. zippeliana, P. amygdalus, P. persica, P. armeniaca, Thea sinensis and Chaenomeles sinensis showed a potent inhibition of tyrosinase, the enzyme which converts 3-(3,4-dihydroxyphenyl)alanine (dopa) to dopachrome in the biosynthetic process. Furthermore, the extracts from the leaves of P. yedoensis and P. zippeliana among the Prunus plants used in this experiment inhibited the production of melanin from dopachrome by autoxidation. These inhibitory effects of P. zippeliana on melanin biosynthesis were observed in cultured B-16 mouse melanoma cells. These results suggest that the leaves of P. zippeliana inhibit melanin biosynthesis which is involved in hyperpigmentation and could be used as a whitening agent for the skin.

Animals↗

Phylogenetic relationships in the stone fruit group of Prunus as revealed by restriction fragment analysis of chloroplast DNA.

In order to clarify the genetic relationships among stone fruits, a restriction fragment analysis of chloroplast DNAs (cpDNAs) was applied to cultivated Prunus species, whose genetic diagnoses are difficult because of their heterogeneity and long life span. Chloroplast DNAs (cpDNAs) were extracted from leaves of nine stone fruit accessions covering six species of Prunus. A restriction fragment analysis was conducted by gel electrophoresis after digestion with these endonucleases. The genome sizes of the cpDNAs were about 135-139 kbp, and the fruits were classified into seven chloroplast genome types according to their restriction fragment patterns. Two peach cultivars and nectarine were found to harbor identical plastomes, differing from those of two wild peaches and the European plum. This suggests that two cultivated peaches (P. persica) did not receive the cytoplasm from the wild peaches, P. mira and P. davidiana. Phylogenetic relationships among these types were then estimated based on the shared common fragments among the species.

Chloroplasts↗

Population Genomics of Almond (Prunus dulcis) Reveals Region-Specific Selection and a Complex History of Domestication.

The domestication of perennial crops in the Mediterranean Basin remains unclear, particularly regarding the genomic consequences of human-mediated demographic shifts and selection. We analysed 8.1 million single nucleotide polymorphisms from 96 cultivated almond (Prunus dulcis) accessions from Europe, North America, Central Asia, and New Zealand, alongside four wild relatives. Population structure analyses revealed four geographically differentiated cultivated groups (Central Asian, North American, and two European) and three wild populations (P. spinosissima, P. orientalis, and P. fenzliana). Cultivated almonds retained high genetic diversity, consistent with weak domestication bottlenecks typical of outcrossing perennials. Elevated diversity and private allele counts in Central Asian cultivars, together with limited evidence of crop-wild gene flow, support Central Asia as an important reservoir of ancestral cultivated diversity that may have played a major role during the early stages of almond domestication. In contrast, allele sharing consistent with historical wild-to-crop introgression-especially involving P. orientalis-has contributed to the genomic composition of European and North American almonds. Genome-wide scans for selective sweeps showed most genes overlapping candidate sweep regions were population-specific, though often associated with similar biological functions, including stress responses and agronomic traits. This suggests repeated targeting of comparable pathways during and post-domestication, despite distinct selection histories. Notably, a subset of candidate genes detected in cultivated populations also occurs in wild relatives, particularly P. orientalis. This overlap is consistent with shared ancestral variation, introgression/gene flow between wild and cultivated lineages, and/or parallel adaptation. Altogether, our results support a complex domestication and diversification history for almonds, shaped by geographic expansion, gene flow with wild relatives, and recurrent selection acting in different regions. This study highlights wild relatives as important reservoirs of genetic diversity and emphasises the need for broader geographic sampling to clarify their contributions to almond domestication and adaptation.

Prunus dulcis↗

Development of a process model to describe the synthesis of (R)-mandelonitrile by Prunus amygdalus hydroxynitrile lyase in an aqueous-organic biphasic reactor.

A process model for the enzymatic synthesis of (R)-cyanohydrins in an aqueous-organic biphasic-stirred tank reactor was developed. The conversion of benzal-dehyde into (R)-mandelonitrile, catalyzed by Prunus amygdalus hydroxynitrile lyase at 5 degrees C and pH 5.5, was chosen as a model system with methyl tert-butyl ether as the organic phase. The process model consisted of a description of the reaction kinetics, mass transfer kinetics, and the mass balances for both the aqueous and the organic phase. Values for the enzyme kinetic parameters, according to ordered bi-uni kinetics, the lumped mass transfer coefficient for benzaldehyde, and the partition coefficients were determined separately. The process model is validated by using 11 experimental data sets of batch conversions in the aqueous-organic biphasic-stirred tank reactor. In these 11 experiments, different enzyme concentrations and phase volume ratios were used. The model was found to be valid with respect to both the conversion and the enantiomeric excess. To synthesize cyanohydrins with a high enantiomeric excess, the enzyme is required to work at mass transfer limited conditions. The developed process model will be used to investigate other process concepts and other substrates.

Acetonitriles↗

Variation in the composition of the heartwood flavonoids of Prunus avium by on-column capillary gas chromatography.

A fast and simple extraction procedure, coupled with on-column gas chromatography, has been developed in order to investigate the relative amounts of flavonoids in individual trees within several homogeneous populations of Prunus avium. Eight known flavonoid aglycones were isolated from the heartwood of P. avium and multivariate analysis was employed in order to characterise population groups.

Chromatography, Gel↗

Protein changes between dormant and dormancy-broken seeds of Prunus campanulata Maxim.

Seed dormancy is regulated by complex networks in order to optimize the timing of germination. However, the biochemical basis of the regulation of seed dormancy is still poorly understood. Many temperate timber species, which are of ecological and/or economic interest, are deeply dormant in seeds, such as Prunus campanulata. Freshly harvested seeds require warm plus cold stratification to break dormancy before they can begin to germinate. According to the results of germination, both warm and cold stratifications are the critical influences for breaking seed dormancy. Significant variations in seed proteins were observed by 2-DE before and after the breaking of seed dormancy. Among the 320, 455, and 491 reproducibly detected spots on the cotyledons, embryos, and testae, respectively, 71 dramatic changes in abundances were observed following warm and/or cold stratification. Among these protein spots, dehydrin, prunin 1 precursor, prunin 2 precursor, and prunin 2 were identified by MS and sequence comparison. The implications of protein changes in relation to the breaking of seed dormancy and germination are discussed. This is the first report of a proteomic analysis of dormancy breaking in woody plant seeds.

Amino Acid Sequence↗

Toxicity of chloroform extract of prunus africana stem bark in rats: gross and histological lesions.

Chloroform extract of Prunus africana (Hook f. (Rosaceae) did not cause clinical signs or pathology in rats at daily oral doses of up to 1,000 mg/kg for 8 weeks. The extract caused marked clinical signs, organ damage and a 50% mortality rate at a dose of 3.3 g/kg for 6 days. The main lesions observed at this dose were marked centrilobular hepatocellular degeneration and necrosis, diffuse nephrosis, myocardial degeneration, lymphocytic necrosis and neuronal degeneration. The morphological damage in these tissues caused a corresponding rise in blood biochemical parameters namely, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatine kinase and blood urea nitrogen. The target organs of toxicity of this extract are the liver, kidney and heart. Overt toxicity occurred only after the administration of multiple doses of 3.3 g/kg body weight. These findings confirm the suitability of this extract for therapeutic use, since the doses used in the therapy of prostate gland are much lower than those used in this study and would therefore not be expected to cause pathological changes.

Alanine Transaminase↗

The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus.

The complete nucleotide sequence of RNA 3 of the PE-5 peach isolate of Prunus necrotic ringspot ilarvirus (PNRSV) was obtained from cloned cDNA. The RNA sequence is 1941 nucleotides and contains two open reading frames (ORFs). ORF 1 consisted of 284 amino acids with a calculated molecular weight of 31,729 Da and ORF 2 contained 224 amino acids with a calculated molecular weight of 25,018 Da. ORF 2 corresponds to the coat protein gene. Expression of ORF 2 engineered into a pTrcHis vector in Escherichia coli results in a fusion polypeptide of approximately 28 kDa which cross-reacts with PNRSV polyclonal antiserum. Analysis of the coat protein amino acid sequence reveals a putative "zinc-finger" domain at the amino-terminal portion of the protein. Two tetranucleotide AUGC motifs occur in the 3'-UTR of the RNA and may function in coat protein binding and genome activation. ORF 1 homologies to other ilarviruses and alfalfa mosaic virus are confined to limited regions of conserved amino acids. The translated amino acid sequence of the coat protein gene shows 92% similarity to one isolate of apple mosaic virus, a closely related member of the ilarvirus group of plant viruses, but only 66% similarity to the amino acid sequence of the coat protein gene of a second isolate. These relationships are also reflected at the nucleotide sequence level. These results in one instance confirm the close similarities observed at the biophysical and serological levels between these two viruses, but on the other hand call into question the nomenclature used to describe these viruses.

Amino Acid Sequence↗

A phenolic glucoside isolated from Prunus serrulata var. spontanea and its peroxynitrite scavenging activity.

A new phenolic glucoside (1), pursargentoside, was isolated from the leaves of Prunus serrulata var. spontanea, along with three other known compounds, orobol 7-omicron-glucoside (2), 1beta, 2alpha, 3alpha, 24-tetrahydroxy-urs-12-en-28-oic acid (3), and chlorogenic acid (4). The structure of pursargentoside (1) was identified by spectroscopic data analysis including 1D and 2D NMR spectroscopy, as 2-omicron-beta-(6'-benzoyl)-glucopyranosyl omicron-(Z)-coumaric acid. Compounds 1, 2, and 4 exhibited ONOO scavenging activity, whereas compound 3 was determined to be virtually inactive.

Free Radical Scavengers↗

Inhibitory activity of flavonoids from Prunus davidiana and other flavonoids on total ROS and hydroxyl radical generation.

Since reactive oxygen species (ROS) and hydroxyl radicals (*OH) play an important role in the pathogenesis of many human degenerative diseases, much attention has focused on the development of safe and effective antioxidants. Preliminary experiments have revealed that the methanol (MeOH) extract of the stem of Prunus davidiana exerts inhibitory/scavenging activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, total ROS and peroxynitrites (ONOO-). In the present study, the antioxidant activities of this MeOH extract and the organic solvent-soluble fractions, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH), and the water layer of P. davidiana stem were evaluated for the potential to inhibit *OH and total ROS generation in kidney homogenates using 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA), and for the potential to scavenge authentic ONOO-. We also evaluated the inhibitory activity of seven flavonoids isolated from P. davidiana stem, kaempferol, kaempferol 7-O-beta-D-glucoside, (+)-catechin, dihydrokaempferol, hesperetin 5-O-beta-D-glucoside, naringenin and its 7-O-beta-D-glucoside, on the total ROS, *OH and ONOO- systems. For the further elucidation of the structure-inhibitory activity relationship of flavonoids on total ROS and *OH generation, we measured the antioxidant activity of sixteen flavonoids available, including three active flavonoids isolated from P. davidiana, on the total ROS and *OH systems. We found that the inhibitory activity on total ROS generation increases in strength with more numerous hydroxyl groups on their structures. Also, the presence of an ortho-hydroxyl group, whether on the A-ring or B-ring, and a 3-hydroxyl group on the C-ring increased the inhibitory activity on both total ROS and *OH generation.

1-Butanol↗

Cloning and characterization of genomic DNA sequences of four self-incompatibility alleles in sweet cherry ( Prunus avium L.).

Gametophytic self-incompatibility (GSI) in sweet cherry is determined by a locus S with multiple alleles. In the style, the S-locus codifies for an allele-specific ribonuclease ( S-RNase) that is involved in the rejection of pollen that carries the same S allele. In this work we report the cloning and genomic DNA sequence analysis including the 5' flanking regions of four S-RNases of sweet cherry ( Prunus avium L., Rosaceae). DNA from the cultivars Ferrovia, Pico Colorado, Taleguera Brillante and Vittoria was amplified through PCR using primers designed in the conserved sequences of sweet cherry S-RNases. Two alleles were amplified for each cultivar and three of them correspond to three new S-alleles named S23, S24 and S25 present in 'Pico Colorado', 'Vittoria' and 'Taleguera Brillante' respectively. To confirm the identity of the amplified fragments, the genomic DNA of these three putative S-RNases and the allele S12 amplified in the cultivar Ferrovia were cloned and sequenced. The nucleotide and deduced amino-acid sequences obtained contained the structural features of rosaceous S-RNases. The isolation of the 5'-flanking sequences of these four S-RNases revealed a conserved putative TATA box and high similarity among them downstream from that sequence. However, similarity was low compared with the 5'-flanking regions of S-RNases from the Maloideae. S6- and S24-RNase sequences are highly similar, and most amino-acid substitutions among these two RNases occur outside the rosaceous hypervariable region (RHV), but within another highly variable region. The confirmation of the different specificity of these two S-RNases would help elucidate which regions of the S-RNase sequences play a role in S-pollen specific recognition.

5' Untranslated Regions↗

QTL analysis of quality traits in an advanced backcross between Prunus persica cultivars and the wild relative species P. davidiana.

Genetic control of the different attributes involved in peach quality has been investigated in an advanced backcross population derived from a cross between Prunus davidiana clone P1908, a wild parent with poor agronomic performance, and a commercial variety, Summergrand. A total of 24 physical and biochemical traits were investigated. Quantitative trait loci (QTLs) were detected for all the traits studied. We identified alleles from P. davidiana with agronomically favorable effects regarding fruit and stone sizes, sugar and acid concentrations and red flesh coloration, in clear contrast to its phenotype. We identified three main regions of the genome where alleles from P. davidiana had negative effects on multiple traits. In other regions, co-locations of QTLs with opposite effects on quality traits were also detected. We discuss the nature of these co-locations in the light of the probable physiological mechanisms involved. Strategies to cope with negative correlations between favorable traits and co-locations of P. davidiana alleles with negative effects on quality traits and positive effects regarding resistance to powdery mildew are discussed from a breeding point of view.

Agriculture↗

SSR allelic variation in almond (Prunus dulcis Mill.).

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach.

Alleles↗

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.).

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.

Amino Acid Sequence↗

Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plants (Prunus domestica L.).

We describe here an improved system for routinely developing transgenic plum plants (Prunus domestica L.) through the use of Agrobacterium tumefaciens. The production of non-transformed "escapes" has been virtually eliminated, and rates of plant establishment in the greenhouse have been dramatically improved. The system is based on the regeneration of shoots from hypocotyls extracted from mature seed. The shoot regeneration medium is Murashige and Skoog (MS) salts and vitamins supplemented with 7.5 microM thidiazuron and 0.25 microM indole-butyric acid. Transferring the explants after co-cultivation to shoot regeneration medium containing 80 mg l(-1) of kanamycin and 300 mg l(-1) of Timentin reduced the total number of regenerated shoots without affecting the transformation rate. Transformation rates using the described system averaged 1.2% of the hypocotyl slices producing transgenic plants, with a range of 0-4.2%. The transgenic shoots rooted at a rate of 90% on half-strength MS salts and vitamins supplemented with 5 microM alpha-naphthaleneacetic acid and 0.01 microM kinetin. Plantlets were transferred to a greenhouse directly from culture tubes with a 90% average survival.

Acclimatization↗

Factors affecting somatic embryogenesis in Prunus incisa cv. February Pink.

Factors affecting somatic embryogenesis from root explants of Prunus incisa Thunb. cv. February Pink were investigated. Using a medium containing Murashige and Skoog salts and vitamins supplemented with 10 microM 2,4-dichlorophenoxyacetic (2,4-D), we evaluated the effects of light, growth regulators, amino acids, carbohydrate source, and root induction medium. Explants cultured under light or dark conditions both resulted in the formation of embryos. Embryogenesis was inhibited by the addition of 6-benzyladenine, thidiazuron, or gibberellic acid to the medium. Amino acids were not effective in promoting embryogenesis, with high levels of amino acids actually inhibiting it. Sucrose and glucose effectively induced embryogenesis, while sorbitol and mannitol completely inhibited it. Sucrose and glucose also promoted secondary embryogenesis. Embryos that formed in medium containing 4% or 5% sucrose were abnormally shaped and did not fully develop, while those that formed in medium with sucrose concentrations of 2% or 3% were much more vigorous. Root explants that were induced on medium containing 1.0 micro M indole-3-butyric acid (IBA) produced more somatic embryos than explants induced on medium without IBA. Approximately 50% of the roots induced on medium containing 1.0 microM IBA produced somatic embryos on medium containing 10 microM 2,4-D and 3% sucrose.

Amino Acids↗

Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus x P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens.

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro.

Agrobacterium tumefaciens↗

In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.).

Regeneration of adventitious shoots from leaves and, for the first time, from internode sections were compared and optimized for five economically important sweet cherry cultivars, i.e. "Schneiders", "Sweetheart", "Starking Hardy Giant", "Kordia" and "Regina" (Prunus avium L.). The influence of basal media, carbon source, combination and dosage of phytohormones, ethylene inhibitor such as silver thiosulfate and a 16 h:8 h light:dark photoperiod versus complete darkness were evaluated. Both, DKW/WPM (1:1) and Quoirin/Lepoivre (QL) basal media stimulated organogenesis more than QL/WPM (1:1), Chee and Pool (CP), Murashige Skoog (MS), Driver and Kuniyuki (DKW) or woody plant (WPM) media did. An induction phase in darkness resulted in lower or zero regeneration rates. The best regeneration efficiencies were generally obtained with thidiazuron in combination with indole-3-butyric-acid. The addition of silver thiosulfate resulted in a similar or reduced regeneration efficiency. Significant genotypic variability in adventitious bud formation was evident for both explant sources, leaf and internode section. Adventitious shoots were obtained from 11% of leaf explants and 50% of internode sections indicating that shoot regeneration from internodes was significantly more efficient than from leaves.

Plant Leaves↗