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PubMed1992

Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds.

In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and d-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two beta-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase.

Journal Article↗
PubMed1992

Cell Wall Changes in Nectarines (Prunus persica) : Solubilization and Depolymerization of Pectic and Neutral Polymers during Ripening and in Mealy Fruit.

Nectarine fruit (Prunus persica L. Batsch var nectarina [Ait] maxim) cultivar Fantasia were either ripened immediately after harvest at 20 degrees C or stored for 5 weeks at 2 degrees C prior to ripening. Fruit ripened after 5 weeks of storage did not soften to the same extent as normally ripened fruit, they lacked juice, and had a dry, mealy texture. Pectic and hemicellulosic polysaccharides were solubilized from the mesocarp of the fruit using phenol:acetic acid:water (PAW) treatment to yield PAW-soluble material and cell wall material (CWM). The carbohydrate composition and relative molecular weight (M(r)) of polysaccharide fractions released from the CWM by sequential treatment with cyclohexane-trans-1,2-diamine tetra-acetate, 0.05 m Na(2)CO(3), 6 m guanidinium thiocyanate, and 4 m KOH were determined. Normal ripening of nectarines resulted in solubilization of pectic polymers of high M(r) from CWM during the first 2 d at ripening temperatures. Concurrently, galactan side chains were removed from pectic polymers. Solubilized pectic polymers were depolymerized to lower M(r) species during the latter stages of ripening. Upon removal from cool storage, fruit had undergone some pectic polymer solubilization, and after ripening, pectins were not depolymerized and were of high M(r). Side chains did not appear to be removed from insoluble pectic polymers and branched pectins accumulated in the CWM. The molecular weight profiles obtained by gel filtration of the hemicellulosic fractions from normally ripening and mealy fruit were similar. The results suggest that mealiness results as a consequence of altered pectic polymer breakdown, including that associated with neutral side chains.

Journal Article↗
PubMed1994

Peach (Prunus persica) endopolygalacturonase cDNA isolation and mRNA analysis in melting and nonmelting peach cultivars.

Two distinct partial cDNAs, PRF1 and PRF3, similar in sequence to previously described polygalacturonases, were amplified from ripe peach (Prunus persica L. Batsch cv Flavorcrest) fruit cDNA by the polymerase chain reaction. PRF1-related RNA was present in fruit from early ripening at levels not detected by northern analysis. PRF3-related RNA was readily detectable in ripe fruit by northern analysis. PRF3 was used to isolate a cDNA with a complete open reading frame, PRF5, from a lambda ZAP II cDNA library prepared from poly(A)+ RNA of ripe peach fruit. PRF5 coded for a predicted protein of 393 amino acids with a molecular mass of 41,500 D. The derived amino acid sequence of PRF5 included a putative leader sequence of 23 amino acids, followed by a sequence that matched the N terminus of endopolygalacturonase protein purified from ripe peach fruit. By northern analysis, PRF3-related RNA was undetectable in firm, unripe Flavorcrest fruit. It appeared at low levels as a 1.7-kb transcript in fruit that had begun to ripen and soften and was very abundant in ripe fruit that had undergone the "melting" stage of softening. The marked increase in PRF3-related RNA levels took place over a period of less than 2 d at 20 degrees C and coincided with the climacteric peak in ethylene evolution. Levels of 1-aminocyclopropane-1-carboxylate oxidase-related RNA increased during ripening at a much earlier stage than levels of PRF3-related RNA. Lower levels of 1.7-kb RNA transcript were detected by PRF3 in ripe fruit of the melting cultivar Fragar, which are firmer than Flavorcrest fruit. In ripe fruit of the nonmelting cultivar Carolyn, PRF3 detected a 1.45-kb RNA transcript that was present at low levels. Transcripts of a peach polygalacturonase-related genomic sequence were not detected in ripening fruit.

Amino Acid Sequence↗
PubMed1994

Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch). II. A 60-kilodalton bark protein in cold-acclimated tissues of peach is heat stable and related to the dehydrin family of proteins.

In several plant species, certain cold-regulated proteins share unique properties. These proteins are (a) heat stable and (b) hydrophilic and are related to the Group 2 late embryogenesis abundant or dehydrin family of proteins. Our previous work with sibling deciduous and evergreen peach genotypes demonstrated a correlation between the level of accumulation of certain bark proteins and cold-acclimation potential of these tissues. Here we identify a 60-kD bark protein in peach (Prunus persica [L.] Batsch), PCA60 ("peach cold acclimation"), that is accumulated during cold acclimation and is heat stable. Immunological studies indicated that this protein is related to the dehydrin family of proteins and accumulates at much higher levels in the bark tissues of the deciduous genotype than in the evergreen. Amino acid composition indicated that the 60-kD protein has a compositional bias for glycine (24%), glutamic acid/glutamine (11.4%), aspartic acid/asparagine (10%), and threonine (9.6%), contains relatively low levels of aromatic amino acids (phenylalanine and tyrosine), and is rich in hydrophilic amino acids. A novel characteristic of the 60-kD cold-acclimation protein is the presence of a repeating nine-amino acid sequence. A five-amino acid stretch, which is included within this repeating motif, shares striking homology with other cold-regulated proteins and dehydrins.

Acclimatization↗
PubMed1994

Utilization of Amygdalin during Seedling Development of Prunus serotina.

Cotyledons of mature black cherry (Prunus serotina Ehrh.) seeds contain the cyanogenic diglucoside (R)-amygdalin. The levels of amygdalin, its corresponding monoglucoside (R)-prunasin, and the enzymes that metabolize these cyanoglycosides were measured during the course of seedling development. During the first 3 weeks following imbibition, cotyledonary amygdalin levels declined by more than 80%, but free hydrogen cyanide was not released to the atmosphere. Concomitantly, prunasin, which was not present in mature, ungerminated seeds, accumulated in the seedling epicotyls, hypocotyls, and cotyledons to levels approaching 4 [mu]mol per seedling. Whether this prunasin resulted from amygdalin hydrolysis remains unclear, however, because these organs also possess UDPG:mandelonitrile glucosyltransferase, which catalyzes de novo prunasin biosynthesis. The reduction in amygdalin levels was paralleled by declines in the levels of amygdalin hydrolase (AH), prunasin hydrolase (PH), mandelonitrile lyase (MDL), and [beta]-cyanoalanine synthase. At all stages of seedling development, AH and PH were localized by immunocytochemistry within the vascular tissues. In contrast, MDL occurred mostly in the cotyledonary parenchyma cells but was also present in the vascular tissues. Soon after imbibition, AH, PH, and MDL were found within protein bodies but were later detected in vacuoles derived from these organelles.

Journal Article↗
PubMed1994

Immunocytochemical Localization of Prunasin Hydrolase and Mandelonitrile Lyase in Stems and Leaves of Prunus serotina.

In macerates of black cherry (Prunus serotina Ehrh.) leaves and stems, (R)-prunasin is catabolized to HCN, benzaldehyde, and D-glucose by the sequential action of prunasin hydrolase (EC 3.2.1.21) and (R)-(+)-mandelonitrile lyase (EC 4.1.2.10). Immuno-cytochemical techniques have shown that within these organs prunasin hydrolase occurs within the vacuoles of phloem parenchyma cells. In arborescent leaves, mandelonitrile lyase was also located in phloem parenchyma vacuoles, but comparison of serial sections revealed that these two degradative enzymes are usually localized within different cells.

Journal Article↗
PubMed1995

Analysis of Sweet cherry (Prunus avium L.) Leaves for Plant Signal Molecules That Activate the syrB Gene Required for Synthesis of the Phytotoxin, Syringomycin, by Pseudomonas syringae pv syringae.

An important aspect of the interaction of Pseudomonas syringae pv syringae with plant hosts is the perception of plant signal molecules that regulate expression of genes, such as syrB, required for synthesis of the phytotoxin, syringomycin. In this study, the leaves of sweet cherry (Prunus avium L.) were analyzed to determine the nature of the syrB-inducing activity associated with tissues of a susceptible host. Crude leaf extracts yielded high amounts of total signal activity of more than 12,000 units g-1 (fresh weight) based on activation of a syrB-lacZ fusion in strain B3AR132. The signal activity was fractionated by C18 reversed-phase high-performance liquid chromatography and found to be composed of phenolic glycosides, which were resolved in three regions of the high-performance liquid chromatography profile, and sugars, which eluted with the void volume. Two flavonol glycosides, quercetin 3-rutinosyl-4[prime]-glucoside and kaempferol 3-rutinosyl-4[prime]-glucoside, and a flavanone glucoside, dihydrowogonin 7-glucoside, were identified. The flavonoid glycosides displayed similar specific signal activities and were comparable in signal activity to arbutin, a phenyl [beta]-glucoside, giving rise to between 120 and 160 units of [beta]-galactosidase activity at 10 [mu]M. Although D-fructose exhibits intrinsic low level syrB-inducing signal activity, D-fructose enhanced by about 10-fold the signal activities of the flavonoid glycosides at low concentrations (e.g. 10 [mu]M). This demonstrates that flavonoid glycosides, which represent a new class of phenolic plant signals sensed by P. s. syringae, are in sufficient quantities in the leaves of P. avium to activate phytotoxin synthesis.

Journal Article↗
PubMed2001

Xylem cavitation in the leaf of Prunus laurocerasus and its impact on leaf hydraulics.

This paper reports how water stress correlates with changes in hydraulic conductivity of stems, leaf midrib, and whole leaves of Prunus laurocerasus. Water stress caused cavitation-induced dysfunction in vessels of P. laurocerasus. Cavitation was detected acoustically by counts of ultrasonic acoustic emissions and by the loss of hydraulic conductivity measured by a vacuum chamber method. Stems and midribs were approximately equally vulnerable to cavitations. Although midribs suffered a 70% loss of hydraulic conductance at leaf water potentials of -1.5 MPa, there was less than a 10% loss of hydraulic conductance in whole leaves. Cutting and sealing the midrib 20 mm from the leaf base caused only a 30% loss of conduction of the whole leaf. A high-pressure flow meter was used to measure conductance of whole leaves and as the leaf was progressively cut back from tip to base. These data were fitted to a model of hydraulic conductance of leaves that explained the above results, i.e. redundancy in hydraulic pathways whereby water can flow around embolized regions in the leaf, makes whole leaves relatively insensitive to significant changes in conductance of the midrib. The onset of cavitation events in P. laurocerasus leaves correlated with the onset of stomatal closure as found recently in studies of other species in our laboratory.

Desiccation↗
PubMed2001

Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development.

The seasonal development of adaxial Prunus laurocerasus leaf surfaces was studied using newly developed methods for the mechanical removal of epicuticular waxes. During epidermal cell expansion, more than 50 microg leaf(-1) of alkyl acetates accumulated within 10 d, forming an epicuticular wax film approximately 30 nm thick. Then, alcohols dominated for 18 d of leaf development, before alkanes accumulated in an epicuticular wax film with steadily increasing thickness (approximately 60 nm after 60 d), accompanied by small amounts of fatty acids, aldehydes, and alkyl esters. In contrast, the intracuticular waxes stayed fairly constant during development, being dominated by triterpenoids that could not be detected in the epicuticular waxes. The accumulation rates of all cuticular components are indicative for spontaneous segregation of intra- and epicuticular fractions during diffusional transport within the cuticle. This is the first report quantifying the loss of individual compound classes (acetates and alcohols) from the epicuticular wax mixture. Experiments with isolated epicuticular films showed that neither chemical conversion within the epicuticular film nor erosion/evaporation of wax constituents could account for this effect. Instead, transport of epicuticular compounds back into the tissue seems likely. Possible ecological and physiological functions of the coordinate changes in the composition of the plant surface layers are discussed.

Biological Transport↗
PubMed1967

Metabolic Changes in After-Ripening Seed of Prunus cerasus.

Metabolic changes were studied in embryonic axes and cotyledons isolated from after-ripening seeds of Prunus cerasus. During the seventh week of after-ripening, a striking increase in the respiration rate at 25 degrees of embryonic axes occurred along with a sharp change from the dormant to non-dormant state of the seed. On the basis of C-6/C-1 ratio determinations this change may be related to an increased activity of the pentose phosphate cycle.

Journal Article↗
PubMed1972

The occurrence of nitrate reductase in leaves of prunus species.

Nitrate reductase was found in leaves of apricot Prunus armeniaca, sour cherry P. cerasus, sweet cherry P. avium, and plum P. domestica, but not in peach P. persica, from trees grown in sand culture receiving a nitrate containing nutrient solution. Nitrate was found in the leaves of all species. Nitrate and nitrate reductase were found in leaves of field-grown apricot, sour cherry, and plum trees. The enzyme-extracting medium contained insoluble polyvinylpyrrolidone, and including dithiothreitol or mercaptobenzothiazole did not improve enzyme recovery. Inclusion of cherry leaf extract diminished, and peach leaf extract abolished, recovery of nitrate reductase from oat tissue. Low molecular weight phenols liberated during extraction were probably responsible for inactivation of the enzyme. The enzyme from apricot was two to three times as active as from the other species. Both nicotine adenine diphosphopyridine nucleotide and flavin mononucleotide were effective electron donors. The enzyme was readily induced in apricot leaves by 10 mm nitrate supplied through the leaf petiole.

Journal Article↗
PubMed1982

Ethephon-Induced Gummosis in Sour Cherry (Prunus cerasus L.) : I. Effect on Xylem Function and Shoot Water Status.

Ethephon, (2-chloroethyl)phosphonic acid, was sprayed at concentrations up to 69.2 millimolar to enhance gum formation in 1-year-old shoots of mature Prunus cerasus L. cv Montmorency trees. Gum accumulation caused rupturing of the shoot periderm, followed by gum extrusion. Lower ethephon concentrations were required to induce gum formation in spring and early summer (1.7-3.5 millimolar) then in late summer and fall (13.8-69.2 millimolar). The number of functional vessels, shoot hydraulic conductance, and water potential of both leaf and internode tissue decreased as gum content of shoots increased. Nontreated control shoots also contained small quantities of gum. There was no difference in neutral sugar composition of gum exuded by the tree, obtained from aqueous shoot extracts, or flushed from the vessels of shoots, whether induced by ethephon or not. Severe decrease in shoot and leaf water potential was associated with shoot die-back. Recovery of xylem function may occur where gummosis is less severe. Discrepancy between measured and predicted hydraulic conductance increased as shoot gum content increased, suggesting that decrease in number of functional vessels alone was not sufficient to explain the effects of gum on loss of shoot hydraulic conductance. Increased gum content in those vessels remaining functional would increase vessel sap viscosity and further reduce hydraulic conductance. The viscosities necessary to account for discrepancy between measured and predicted hydraulic conductance were calculated. Gum concentration less than 1.0% (w/v) would produce these viscosities.

Journal Article↗
PubMed1982

Ethephon-Induced Gummosis in Sour Cherry (Prunus cerasus L.) : II. Flow Characteristics of Gum Solutions.

Flow of sour cherry (Prunus cerasus L. cv. Montmorency) gum solutions through a glass capillary was Newtonian for pressure gradients from 0 to 1.8 megapascals per meter, and hydraulic conductance was inversely proportional to solution viscosity in this range. However, flow became plastic at pressure gradients above 1.8 megapascals per meter, resulting in a decrease in solution viscosity. The magnitude of this effect diminished as gum concentration increased. Flow of water, a solution of the component sugar monomers of sour cherry gum, and sucrose solutions remained Newtonian over the entire pressure gradient range examined (0-4 megapascals per meter). Plastic flow of gum solutions in the vessels of intact sour cherry shoots is possible under pressure gradients induced by transpiration when high resistance to flow occurs over short distances.

Journal Article↗
PubMed1984

Xylem development in prunus flower buds and the relationship to deep supercooling.

Xylem development in eight Prunus species was examined and the relationship to deep supercooling assessed. Dormant buds of six species, P. armeniaca, P. avium, P. cerasus, P. persica, P. salicina, and P. sargentii deep supercooled. Xylem vessel elements were not observed within the dormant floral primordia of these species. Instead, discrete bundles containing procambial cells were observed. Vascular differentiation resumed and xylem continuity was established during the time that the capacity to deep supercool was lost. In P. serotina and P. virginiana, two species which do not supercool, xylem vessels ran the length of the inflorescence and presumably provided a conduit for the spread of ice into the bud. The results support the hypothesis that the lack of xylem continuity is an important feature of buds which deep supercool.

Journal Article↗
PubMed1985

Identification of Gibberellins A(1), A(5), A(29), and A(32) from Immature Seeds of Apricot (Prunus armeniaca L.).

Gibberellins (GAs) A(1), A(5), and A(29) were identified, and also GA(32) was confirmed, as endogenous GAs of immature seeds (3-4 weeks after anthesis, 0.25-0.5 gram fresh weight) of apricot (Prunus armeniaca L.) based on capillary gas chromatography (GC), retention time (Rt), and selected ion monitoring (SIM), in comparison with authentic standards. Fractions subjected to GC-SIM were purified and separated using sequential solvent partitioning --> paper chromatography --> reverse phase C(18) high performance liquid chromatography (HPLC) --> bioassay on dwarf rice cv Tan-ginbozu. Two other peaks of free GA-like bioactivity (microdrop and immersion dwarf rice assays) were eluted from C(18) HPLC at Rts where GA(4/7) and GA(8) (or other GAs with similar structures) would elute. Also, three unidentified GA glucoside-like compounds (based on bioactivity on the immersion assay, and no bioactivity on the microdrop assay) were noted. There were very high amounts of GA(32) (112 ng of GA(3) equivalents per gram fresh weight), and minor amounts (0.5 ng of GA(3) equivalents) for each of GA(1) and GA(5), respectively, based on the microdrop assay.

Journal Article↗
PubMed1987

Metabolism of [H]Gibberellin A(5) by Immature Seeds of Apricot (Prunus armeniaca L.).

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A(5) (GA(5)) as 1- and 1,2-[(3)H]GA(5) (5.3 Curies per millimole to 16 milliCuries per millimole) at doses (42 nanograms to 10.6 micrograms per seed) 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free [(3)H]GA-like metabolites were separated from the highly H(2)O-soluble [(3)H]metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted --> isocratic eluted reversed-phase C(18) high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 x expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of [(3)H] GA(5) were identified as GA(1), GA(3), and GA(6) by GC-SIM. The major highly water soluble metabolite of [(3)H]GA(5) at all levels of substrate GA(5) had chromatographic characteristics similar to authentic GA(1)-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [(3)H]GA(5) feeds (2 x expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA(1), GA(3), GA(5) methyl ester, GA(6), GA(22), GA(29) (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA(1), GA(3), GA(5), and GA(8) (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA(6) and GA(29) would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GA(5). Increasing substrate GA(5) levels increased the proportion of metabolites with HPLC Rts similar to GA(1), GA(6), and especially GA(1) glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA(3), GA(3)-glucosyl ester, and a postulated conjugate of GA(6). There was evidence that high doses of substrate GA(5) induced new metabolites which often, but not always, differed from GA(1), GA(3), and GA(6) in HPLC Rt. These same metabolites, when analyzed by GC-SIM yielded m/e ions the same as the M(+) and other characteristic m/e ions of the above GAs, albeit at differing GC Rt and relative intensities.

Journal Article↗
PubMed1990

Dormancy in Peach (Prunus persica L.) Flower Buds : I. Floral Morphogenesis and Endogenous Gibberellins at the End of the Dormancy Period.

Flower buds of peach (Prunus persica L.) trees, cv Novedad de Cordoba (Argentina), were collected near the end of the dormant period and immediately before anthesis. After removal of scale leaves, morphological observations of representative buds, made on transverse and longitudinal microtome sections, showed that all verticils making up the flower are present in an undifferentiated form during the dormant period (June). Flower buds collected at the end of dormant period (August) showed additional growth and differentiation, at which time formation of two ovules was beginning in the unicarpelar gynoecium. Dehiscence of anthers had not yet occurred 10 days before full bloom, and the ovules were still developing. Free endogenous gibberellin (GA)-like substances were quantified by bioassay (Tan-ginbozu dwarf rice microdrop) after SiO(2) partition column chromatography, reversed phase C18-high performance liquid chromatography, and finally Nucleosil [N(CH(3))(2)]high performance liquid chromatography. Bioactive fractions were then subjected to capillary gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM). Gibberellins A(1), A(3), and A(8) were tentatively identified in peach flower buds using GC-SIM and Kovat's retention indices, and relative amounts approximated by GC-SIM (2:8:6 for GA(1), GA(3), and GA(8), respectively). The highest concentration (330 nanograms per gram dry weight) of free GA(1)/GA(3) was found in dormant buds (June) and diminished thereafter. The concentration free of GA(1)/GA(3) did not increase immediately prior to bud break. However, high GA(1)/GA(3) concentrations occurred during stages where rate of growth and cellular differentiation of (mainly fertile) verticils can be influenced.

Journal Article↗