Suberins of Malus pumila stem and root corks

The suberin contents of the isolated superficial cork layers of Malus pumila stems and root ranged from 15 to 35% of the dry weight. The qualitative composition of the aliphatic monomers obtained after alkaline depolymerization of the extractive-free corks was similar but some quantitative differences were found according to cultivar and age of the cork layer. 1-Alkanols (mainly 22:0, 24:0 and 26:0), alkanoic acids (mainly 22:0 and 24:0), α, ω-alkanedioic acids (mainly 16:0, 18:1 (9) and 18:0) and ω-hydroxyalkanoic acids (mainly 18:1 (9) and 22:0) were major constituents of all the samples examined and together they comprised 40–50% of the total monomeric mixture. The remainder was composed mainly of 9,10-epoxy-18-hydroxy-and 9,10,18-trihydroxyoctadecanoic acids. The corresponding dibasic acids, 9,10-epoxy- and 9,10-dihydroxyoctadecane-1,18-dioic, were minor components as were C₁₆ and C₁₈ dihydroxyalkanoic acids (mainly 10,16-dihydroxyhexadecanoic and 10,18-dihydroxyoctadecanoic acids, respectively). The root suberin dittered from that of the stem in containing larger amounts of 9,10-epoxy- 18-hydroxyoctadecanoic and 18-hydroxyoctadec-9-enoic acids.     

Metabolism of added gibberellic acid in Malus pumilla in relation to cool storage breakdown

Gibberellic acid (GA₃) that was injected into the core of apples to reduce internal breakdown, was found to accumulate in the cortical tissue during cool storage only to a limited extent and never exceeded 0.5 % of the added dose. Limitations on the commercial use of GA₃ would appear to be associated with the low level of incorporation into susceptible tissue.     

Soluble and wall-bound glycoproteins of apple fruit tissue

Apple fruit tissue contains small amounts of readily soluble glycoproteins, rich in hydroxyproline; polymethylgalacturonide is not covalently bound to the soluble glycoproteins. Barium hydroxide hydrolysis of apple fruit cell walls liberated glycopeptides containing 4 arabinosyl residues per hydroxyprolyl residue, which were attacked very slowly by α-l-arabinofuranosidase. Hydrazinolysis liberated similar glycopeptides, which were difficult to separate from a polysaccharide containing galactose residues. Protease treatment of walls also released glycopeptides containing hydroxyproline, and a small proportion of these were associated with polyuronide. Polygalacturonase pretreatment of walls led to increased release of hydroxyprolyl residues by protease. Susceptibility of the hydroxyproline containing glycoprotein in the cell wall to attack by protease and arabinosidase did not change during fruit ripening. The amount of an unknown hexosamine associated with the wall was less in ripethan in unripe fruit.     

Properties of polygalacturonate and cell cohesion in apple fruit cortical tissue

Examination of the hydrodynamic properties of polygalacturonate fractions from unripe and ripe apple tissue suggested that the wall bound fraction was degraded during ripening but that the soluble fraction was not. Esterification of cell wall preparations with CH₂N₂ caused solubilisation of polygalacturonate. Acid MeOH caused more extensive solubilization, but this reagent hydrolysed arabinofuranosyl linkages. Both reagents reduced the cohesion of EtOH extracted apple tissue. This effect could also be achieved by treatment with sodium polyphosphate at pH 4 but not by EDTA or chaotropic agents. Free carboxyl groups on polygalacturonate probably maintain cell cohesion through co-operative binding of Ca²⁺ ions. The integrity of primary wall structure is thought to depend upon non-covalent bonding between cellulose, protein and polygalacturonate.     

The phenolics and a hydrolysable tannin polyphenol oxidase of Medinilla magnifica

The production of viable meristem cultures of Medinilla magnifica has proved to be very difficult. This may be due, in part, to a pronounced ‘browning’ response of the tissues on cutting. For this reason the phenolic compounds and the hydrolysable-tannin polyphenol oxidase from Medinilla were studied. The distribution of the compounds was: simple phenols 19% , flavonoids 5% , hydrolysable tannins 69% , condensed tannins 7%. Amongst the simple phenols and phenolic acids, the following were identified: phloroglucinol, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, gallic acid (both in free and bound form the most abundant simple phenol), syringic acid, trans-p-coumaric acid, trans-ferulic acid and trans-caffeic acid. No kaempferol or quercetin or their derivatives were detected but condensed tannins are present. Methods for the extraction, fractionation and quantitative determination of phloroglucinol and the phenolic acids, as well as correction factors for losses during the extraction, alkali treatment and derivatization, are presented in a supplementary publication. With regard to the hydrolysable tannin polyphenol oxidase activity of Medinilla stems, the enzyme(s) is rather specific since at neither of its two pH optima (6 and 7) could a classical polyphenol oxidase activity be detected. The enzyme was strongly inhibited by 2-mercaptoethanol. Preliminary experiments have further shown that in addition to the hydrolysable tannins of the tissue, the ferrous ions of the medium, and oxygen together with the hydrolysable tannin polyphenol oxidase could play a role in the browning response. Ways to overcome this difficulty have been suggested.     

Glutamate oxaloacetate transaminase activity in developing Lycopersicon esculentum fruit

Glutamate oxaloacetate transaminase (GOT) occurs in both the mitochondrial and cytoplasmic fractions from tomato fruit tissue. Changes in activity of the enzyme from fruit at selected developmental stages have been followed. The combined activity fell from the immature green stage to the full red condition whilst the proportion in the mitochondria reached a peak in green-orange fruit. The activity of cytoplasmic, but not mitochondrial, GOT was stimulated by the addition of pyridoxal-5-phosphate. In the green areas of fruit showing blotchy ripening, the combined activity was equivalent to that in normal immature green fruit but with a much higher proportion of the activity in the mitochondria. Mitochondrial GOT could constitute a system in ripening tomato fruit whereby the accumulation of inhibitory concentrations of oxaloacetate affecting the oxidation of succinate and malate might be controlled.     

Metabolism of polymethylgalacturonate in apple fruit cortical tissue during ripening

Changes in the composition and metabolism of polymethylgalacturonate were followed in ripening apples. After the onset of ethylene production and fruit softening total polygalacturonate decreased and the water soluble fraction increased. No change was detected in the overall degree of esterification but the esterification of the water soluble fraction increased. Incorporation of radioactivity from methionine-[¹⁴C] into Me groups on polygalacturonate continued during ripening but incorporation from inositol-[³H] decreased sharply. Cell separation probably depends upon the removal of low ester polygalacturonate from the middle lamella by exopolygalacturonase; the continued incorporation from methionine-[¹⁴C] is probably due to synthesis of new polymethylgalacturonate.     

苹果多酚氧化酶的纯化与表征

运用丙酮浸漬干燥、磷酸盐缓冲液提取、低温离心、硫酸铵沉淀、DEAE-Sephadex(A-50)、Sephadex(G-75) 和DEAE-celluse(DE-52)层析等方法从苹果中分离获得一种新的含铜酶蛋白,该酶被命名为多酚氧化酶Ⅱ(polyphenol oxidase Ⅱ, PPOⅡ),纯化倍数是215,纯化收率是23%.PAGE、SDS-PAGE和MALDI-TOF 等技术用于测定所获的酶的纯度和分子量.在PAGE和SDS-PAGE 均显示一条带,表明PPOⅡ只由一个亚基组成,且已达到单一组分(MALDI-TOF的结果更证实了这一点).SDS-PAGE 和 MALDI-TOF 的结果都表明PPO的分子量为 38204 Da.pH值对酶活性和稳定性研究的结果显示,从pH值4.0～7.0随着pH值的增加,酶活性也不断增加;从pH值 7.0～11.0, 酶活性不断降低.PPOⅡ的最适pH值为6.6最适温度为30℃.     

Separate monophenolase and o-diphenolase enzymes in Triticum aestivum

Monophenolase and o-diphenolase activities of polyphenol oxidase are usually thought to be a part of the same enzyme complex. It has now been demonstrated that the two catalytic activities of the polyphenol oxidase of wheat grains are separable and reside in different enzymes. The electrophoretically separated monophenolase enzyme showed specificity only for monophenol (l-tyrosine) after its elution from acrylamide gels. Further, this enzyme is confined to the endosperm tissue and is undetectable in the embryonic region of the seedling.     

Composition and structure of secondary fatty alcohols and ketones,isolated from Pyrus malus skin wax

A homologous series of long-chain secondary fatty alcohols from C₂₁ to C₂₉ (C₂₉ being Predominant) has been isolated from the skin wax of a Bulgarian apple variety, Bouhavitsa. The alcohols were found only in a free state. Long-chain fatty ketones, with C₂₉ again markedly prevalent, have been isolated for the first time from the skin wax of this, and another variety, Tetovka. The C₂₉-ketone from both samples was identified by means of MS as nonacosan-10-one. A complete similarity has been found in the relative amounts of C₂₉ in the mixtures of secondary alcohols, ketones and paraffins.     

A triterpenoid glycoside from Tetrapleura tetraptera fruit

The fruit pulp of Tetrapleura tetraptera has yielded aridanin, a novel 3-O-[β-D-glucopyranosyl-2′-acetamido-2′-deoxy]-oleanolic acid. Hentriacontane, phenylpropanoids and carbohydrate residues were also isolated and identified.     

Enzymic oxidation of linolenate by Ginkgo leaves: Fractionation and characterization of active fractions

An aqueous extract of defatted, macerated leaves of Ginkgo biloba L. catalysed the oxidation of linolenate. Extracts prepared from quickly frozen Ginkgo leaves had almost the same activity as the extract from fresh leaves but no trans-2-hexenal was formed. The activity was surprisingly stable at 100° and to acids. However, at pH 12 a marked loss of activity was observed, particularly when the soln was heated, and Pronase, also destroyed most of the activity.     

Flavonol tetraglycosides from the leaves of Prunus cerasus and P. Avium

Four new flavonol glycosides have been identified from fresh leaves and fruits of sweet and sour cherries (Prunus avium and P. cerasus) as minor flavonoids: quercetin 3-O-rutinosyl-7,3′-O-bisglucoside; two quercetin 3-O-rutinosyl-4′-di-O-glucosides; kaempferol 3-O-rutinosyl-4′-di-O-glucoside.     

Two new sterols from Physalis franchetii fruit

Two new steroids physanol A, 3β-benzoyloxy-6-oxo-stigmast-7,20-diene-11α-ol and physanol B, 3β-benzoyloxy-6-oxo-stigmast-7-ene-11α-ol have been isolated from the fruit of Physalis franchetti.     

Significance of flower and fruit thinning on fruit quality

The effects of mechanical or chemical flower and fruit thinning on fruitquality were primarily by altering crop load. However, there were alsodirect effects of thinning agents. Fruit size was directly related tothinning intensity. In addition to crop load, age of wood, flower budquality, competition within clusters and canopy were important factorsaffecting the response to thinning. Short- and long-term thinningstudies identified two groups of quality components: Group 1characteristics include size, colour, skin performance, firmness andsugar and acid content of the fruit. Group 2 characteristics wererepresented by inorganic components, especially calcium and potassiumwhich are implicated in the susceptibility of fruit to physiologicaldisorders. While group 1 characteristics were improved by increasingthinning intensity, storability of the fruit was better at high than atlow crop loads. Therefore, a compromise between all quality requirementsmust be found for a good economic return. Establishing the trends ofthinning on the different quality parameters can help to select athinning strategy for local or regional conditions typically beingdetermined by growing and market conditions.     

Properties of polyphenol oxidase from tubers of the yam Dioscorea bulbifera

The subunit MW of Dioscorea bulbifera polyphenol oxidase (MW 115 000 ± 2000) determined by SDS-PAGE is ca. 31 000 indicating that the enzyme is an oligomeric protein with four subunits. K_i values of various inhibitors and their modes of inhibition have been determined with catechol and pyrogallol as substrates. p-Nitrophenol, p-cresol, quinoline and resorcinol are competitive inhibitors of catechol binding while only orcinol and p-nitrophenol behave in the same way towards pyrogallol as substrate. From the effect of pH on V_max, groups with pK values ca. 4.7 and 6.8 have been identified to be involved in catalytic activity. The Arrhenius activation energy (E_a) at pH 4.0 is 8.9 kcal/mol between 40–65°. At pH 7.0, the value is 22.1 kcal/mol between 40 and 60°. The enthalpies (ΔH) at pH 4.0 and pH 7.0 are 2.3 kcal/mol and 32.4 kcal/mol respectively. The results are discussed considering the conformational changes of the enzyme during substrate binding.     

Characterization of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase multigene family of Malus domestica Borkh

Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA isolated from leaf tissue of apple (Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an ORF of 990 bp) and MD-ACO3 (966 bp) have been compared with a previously cloned gene of apple, MD-ACO1 (with an ORF of 942 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9% in the ORF but diverge in the 3′ untranslated regions (3′-UTR) (69.5%). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% (MD-ACO1) and 71% (MD-ACO2) in the 3′-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a distinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression analysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli confirmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further, the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Analysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli revealed apparent Michaelis constants (K_m) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) for the substrate ACC, catalytic constants (K_cat) of 6.6 × 10⁻² (MD-ACO1), 3.44 × 10⁻² (Md-ACO2) and 9.14 × 10⁻² (MD-ACO3) and K_cat/K_m (μM s⁻¹) values of 7.38 × 10⁻⁴ μM s⁻¹ (MD-ACO1), 0.86 × 10⁻⁴ M s⁻¹ (MD-ACO2) and 3.8 × 10⁻⁴ μM s⁻¹ (MD-ACO3). These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression pattern that is supported by some variation in kinetic properties.     

Carotenoids of Eriobotrya japonica

The carotenoids of the loquat fruit Eriobotrya japonica Golden Nugget variety, were investigated. They were identified according to their chromatographic, spectrophotometric and chemical properties and compared with standard pigments. For some of the carotenoids, MS were determined. Pulp and peels were investigated separately. The main pattern of the pulp carotenoids was β-carotene (33%), γ-carotene (6%), cryptoxanthin (22%), lutein, violaxanthin and neoxanthin, each about 3–4%. The peel, with a carotenoid content 5 times as high, had a similar pattern, but the ratio between the main pigments differed: β-carotene (50%); γ-carotene (5%); cryptoxanthin (5%); lutein (13%); violaxanthin, neoxanthin, 3–4%. The carotenoids of the loquat (subfamily Maloideae) were very similar to those of the apricot (Prunus armeniaca-subfamily Prunoideae) both of the family of Rosaceae. The intergeneric differences are more pronounced, which is of possible taxonomic significance. The lower concentration of cryptoxanthin and the high concentration of lutein in the peels is noteworthy and of biosynthetic interest.     

Melanin biosynthesis during differentiation of Physarum polycephalum

Melanin synthesis in the myxomycete Physarum polycephalum occurs during sporulation but not during spherule formation. Melanin-like pigment was extracted from spores. An almost identical substance of polyphenols was extracted from spherules and characterized by its ultraviolet and infrared absorbance spectra. Polyphenol oxidase activity in spherules was very low and showed only one weak isoenzyme band in isoelectric focusing polyacrylamide gels. A much higher activity, and an increasing number of isoenzymes, were detected in sporulating cultures after illumination during the differentiation process. The addition of melanin precursors resulted in the synthesis of brownish-yellow spherules, probably containing dopachrome, whereas the addition of polyphenol oxidase inhibitors resulted in yellow sporangia. The results indicate that melanin synthesis is probably only a stage in maturation but not an essential part of the morphogenetic process itself.     

Electrophoretic investigation of enzymes from developing Lycopersicon esculentum fruit

Acetone powders were prepared from tomato fruit tissue sampled during development and the proteins were separated by polyacrylamide disc gel electrophoresis. The gels were stained to show up general proteins, lipoproteins, glycoproteins and certain enzymes. Minor changes in protein and glycoprotein patterns accompanied development. Most enzymes exhibited more than one active band, with maximum diversity and specific activities usually appearing in extracts from mature green tissue and least with over-ripe tissue. The results support the view that enzyme synthesis accompanies the climacteric respiration rise at the expense of non-metabolic protein.