High polyphenol oxidase activity and low titratable acidity in browning bamboo tissue culture

Summary Tissue browning that frequently results in the early death of bamboo shoots in vitro correlated directly with polyphenol oxidase (PPO, EC 1.10.3.1) activity and inversely with titratable acidity. It was unrelated to the level of endogenous phenols. During the course of culture, timing of PPO activity paralleled that of explant browning. Browning was highest among shoots cultured in a medium of pH 8, which was consistent with the pH optinum of the bamboo enzyme. The pH optimum was first determined with the crude enzyme, then verified with two purified isozymes. Stability of the bamboo PPO was also highest at pH 10. PPO activities of the severely browning Dendrocalamus latiflorus, the moderately browning Phyllostachys nigra, and the relatively non-browning Bambusa oldhamii were inhibited strongly by ascorbic acid, cysteine, sodium diethyldithiocarbamate, and sodium sulfite. But characterization of bamboo PPO according to enzyme inhibitors was not possible because enzyme extracts of the three species gave varied responses to the traditional substances. Nutrient medium addenda of some PPO inhibitors, namely ascorbic acid, cysteine, kojic acid, and thiourea, mainly enhanced browning. However, ferulic acid at 3 mM and lower concentrations reduced the number of brown shoots per culture, although not the percentage of cultures that browned. Polyvinylpyrrolidone failed completely to suppress browning. The two purified isozymes showed different temperature optima for PPO activity: 60°C and 65°C. The purified isozymes displayed a substrate preference for dopamine, or a cathecol oxidase characteristics.     

DISTRIBUTION OF COAGULATION OF YOUNG SHOOT HOMOGENATES IN THE AURANTIOIDEAE

Distribution of coagulation of young shoot homogenates was surveyed in 430 accessions from the orange subfamily Aurantioideae. Representatives of four genera available for study (Clausena, Glycosmis, Aegle, Feronia) were coagulating taxa. The genus Citrus included both coagulating and noncoagulating taxa. Nineteen genera (Murraya, Merillia, Pamburus, Paramignya, Triphasia, Aeglopsis, Afraegle, Balsamocitrus, Feroniella, Swinglea, Atalantia, Citropsis, Hesperethusa, Pleiospermium, Severinia, Clymenia, Eremocitrus, Poncirus, Fortunella) were noncoagulating. The status of eight genera (Micromelum, Wenzelia, Monanthocitrus, Oxanthera, Merope, Luvunga, Burkillanthus, Limnocitrus) is not known. Homogenized tissue from coagulating taxa turned into a paste while tissue from noncoagulating taxa was thin and juicy. Coagulation did not take place when the pH of the grinding medium was 11 or higher but it would occur if the pH was lowered. Homogenates of noncoagulating taxa become coagulated below pH 5.2–5.3, but remained noncoagulating above this. Noncoagulating taxa contained an unidentified substance which inhibited coagulation of tissue from coagulating taxa. The mechanism of coagulation and noncoagulaton is not presently known. The significance of coagulation and its absence as a taxonomic and genetic marker is discussed.     

Biodiversity patterns of macro-epifauna on different lamina parts of Laminaria digitata and Saccharina latissima collected during spring and summer 2004 in Kongsfjorden, Svalbard

The epifauna associated with two of the most common species of kelp in the Arctic, Laminaria digitata (Hudson) Lamouroux, 1813 and Saccharina latissima (Linnaeus) Lane, , Mayes, Druehl and Saunders 2006 [synonym: L. saccharina (Linnaeus) Lamouroux, 1813] were examined in Kongsfjorden, Svalbard. The aim of this study was to test whether species richness of epifauna was influenced by seasonality of recruitment supply (spring and summer) or by age of the substrate (lamina). From 20 algae specimens (ten—L. digitata, ten—S. latissima), a total of 27 epifaunal taxa were identified with bryozoans as the most species-rich group. Total species richness ranged from one to nine species per lamina. No significant difference in species richness was observed between the two kelp species. In both macroalgae, more epifauna taxa were registered on lamina in May (mean 6) than in August (mean 3). This indicates that the time chosen for sampling had a significant impact on the species richness pattern. The number of epifaunal species was significantly negatively correlated with increasing age of the lamina. There are indications that diversity of the epifauna on lamina is influenced to a large extent by substrate (lamina) surface area, environmental stress and the presence of meroplankton in the water column.     

Species richness and composition patterns of clitellate (Annelida) assemblages in the treeless spring fens: the effect of water chemistry and substrate

Spring fens are isolated treeless wetlands of a high conservation value. Their environmental conditions are strongly related to their groundwater chemistry, which controls species distribution within various groups of organisms. Clitellates, a dominant group of non-insect aquatic fauna, however, have never been studied in these habitats. It is unclear from previous studies to what extent the distribution of aquatic non-insect taxa reflects water chemistry rather than the substrate structure. We studied 34 spring fens sampled in 17 isolated sites in the Western Carpathian Mountains to determine mainly the effects of water chemistry and substrate structure on variation in species richness and composition of clitellate assemblages as examples of the non-insect fauna. A total of 34 taxa were found, with 3–15 taxa collected per sample. Species richness was negatively correlated with water mineral concentration measured as water electric conductivity (r = −0.57, P < 0.001) and positively with TOC (r = 0.60, P < 0.001). Surprisingly, the lowest number of taxa was found in calcareous fens and richness increased towards Sphagnum-fens. There was a species turnover related to changes in mineral richness and substrate characters. The main change of species composition was promoted by changes in substrate structure. The second gradient of species composition was linked with the amount of nutrients, moisture, and dominance of sphagna, and was associated with an increase of eurytopic species in fens with high nutrient availability. It was difficult to separate the effects of water chemistry and substrate on clitellate species distributions owing to the fact that variation in tufa precipitation and vegetation was driven by water chemistry changes. This study presented the first quantitative data on fen clitellate assemblages, which appear to have an unusual pattern of species richness. In contrast to plants and molluscs, calcareous fens appeared to be a harsh environment for clitellate species. Only few specialized species, mainly Trichodrilus strandi, were able to establish viable populations. The significant effect of water chemistry on clitellate distribution patterns raises questions about the direct influence of water chemistry on non-insect aquatic taxa, which have previously been considered to be mostly determined by substrate characteristics.     

Characterisation of phenol oxidase and peroxidase from maize silk

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non‐browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o‐diphenolic substrates, was abundant only in browning silk, and low or absent in non‐browning silk. Pollination increased POD but not PPO activity. Isoelectric‐focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN₃, EDTA, KCN, and L‐cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination.     

RESTRICTION FRAGMENT ANALYSIS OF CHLOROPLAST DNA AND THE SYSTEMATICS OF VIGUIERA AND RELATED GENERA (ASTERACEAE: HELIANTHEAE)

Phylogenetic relationships among 31 species representing variously Helianthus, Helianthopsis, Heliomeris, Simsia, Viguiera, and Tithonia were assessed by chloroplast DNA restriction site mapping. A total of 147 mutations including 92 informative ones was detected using 16 restriction enzymes, with an estimated sequence divergence within the group of 1.4%. Parsimony analysis produced a single most parsimonious tree with a length of 161 steps and a consistency index of 0.91. Statistically significant clades, as assessed by the bootstrap method, correspond to a number of taxa recognized previously, including Helianthus (3 species), Helianthopsis (5 species), and several groups within Viguiera, including sect. Maculatae (4 species), the Baja California group (6 species), sect. Paradosa (2 species) and V. dentata (3 samples). However, species of Viguiera collectively form a highly paraphyletic group relative to species of other genera. Helianthus and Helianthopsis were separated into different clades, supporting their recent segregation. Placement of H. porteri in Helianthus rather than Heliomeris was confirmed; the single sample of the latter genus was most similar to the Baja California group of Viguiera. An expected relationship between Simsia (2 species) and one member of Viguiera ser. Grammatoglossae was confirmed (although not with two other putatively related members of Viguiera) and an unexpected relationship between Simsia and Tithonia was suggested. The presence of Mexican taxa as the more basal groups in the tree points toward a possible Mexican origin for Viguiera and related genera. A molecular clock hypothesis is rejected in many pairwise comparisons involving woody taxa with herbaceous ones, although it could not be rejected in most pairwise comparisons involving taxa of similar habit.     

Properties of Chlorogenic Acid Quinone: Relationship between Browning and the Formation of Hydrogen Peroxide from a Quinone Solution

Chlorogenic acid is the major polyphenol in foods derived from plants and is a good substrate for polyphenol oxidase. Chlorogenic acid quinone (CQA-Q), which is an oxidative product of chlorogenic acid by polyphenol oxidase, is an important intermediate compound in enzymatic browning. CQA-Q was prepared, and its properties and the relationship with browning were examined. The quinone solution was yellow or orange, and its molecular absorption coefficient was estimated to be 1.7×10³ for 325 nm and 9.7×10² for 400 nm in an acidic aqueous solution. Chlorogenic acid and H₂O₂ were spontaneously generated in the CQA-Q solution as the yellowish color of the solution gradually faded. A pale colored polymer was the major product in the reaction solution. Amino acids such as lysine and arginine added to CQA-Q solution did not repress the fading of the yellowish color of the solution. We concluded from these results that CQA-Q itself and a mixture of CQA-Q and amino acids did not form intensive brown pigments in the acidic aqueous solution. H₂O₂ spontaneously formed in the CQA-Q solution, and other polyphenols might have played an important role in the formation of the brown color by enzymatic browning.     

The Effect of Activated Charcoal Supplemented Media to Browning of In Vitro Cultures of Piper species

With the aim to effectively minimise the browning of tissue cultures of different Piper species (P. longum, P. attenuatum, P. betle, P. nigrum) explants from stem (nodal and internodal segments), petiole, and leaf were planted on Murashige and Skoog's basal medium supplemented with activated charcoal (AC). AC in the concentration of 200 mg dm^-3 proved to be the best. Among the taxa studied, P. longum showed the least browning whereas, wild P. nigrum showed maximum browning.     

Role of Chlorogenic Acid Quinone and Interaction of Chlorogenic Acid Quinone and Catechins in the Enzymatic Browning of Apple

Chlorogenic acid (CQA) is one of the major polyphenols in apple and a good substrate for the polyphenol oxidase (PPO) in apple. Apple contains catechins as well as CQA, and the role of CQA quinone and its interaction with catechins in the enzymatic browning of apple were examined. Browning was repressed and 2-cysteinyl-CQA was formed when cysteine was added to apple juice. CQA quinone was essential for browning to occur. Although catechins and CQA were oxidized by PPO, some catechins seemed to be non-enzymatically oxidized by CQA quinone.     

Role of chlorogenic acid quinone and interaction of chlorogenic acid quinone and catechins in the enzymatic browning of apple

Chlorogenic acid (CQA) is one of the major polyphenols in apple and a good substrate for the polyphenol oxidase (PPO) in apple. Apple contains catechins as well as CQA, and the role of CQA quinone and its interaction with catechins in the enzymatic browning of apple were examined. Browning was repressed and 2-cysteinyl-CQA was formed when cysteine was added to apple juice. CQA quinone was essential for browning to occur. Although catechins and CQA were oxidized by PPO, some catechins seemed to be non-enzymatically oxidized by CQA quinone.     

Properties of chlorogenic acid quinone: relationship between browning and the formation of hydrogen peroxide from a quinone solution

Chlorogenic acid is the major polyphenol in foods derived from plants and is a good substrate for polyphenol oxidase. Chlorogenic acid quinone (CQA-Q), which is an oxidative product of chlorogenic acid by polyphenol oxidase, is an important intermediate compound in enzymatic browning. CQA-Q was prepared, and its properties and the relationship with browning were examined. The quinone solution was yellow or orange, and its molecular absorption coefficient was estimated to be 1.7 x 10(3) for 325 nm and 9.7 x 10(2) for 400 nm in an acidic aqueous solution. Chlorogenic acid and H2O2 were spontaneously generated in the CQA-Q solution as the yellowish color of the solution gradually faded. A pale colored polymer was the major product in the reaction solution. Amino acids such as lysine and arginine added to CQA-Q solution did not repress the fading of the yellowish color of the solution. We concluded from these results that CQA-Q itself and a mixture of CQA-Q and amino acids did not form intensive brown pigments in the acidic aqueous solution. H2O2 spontaneously formed in the CQA-Q solution, and other polyphenols might have played an important role in the formation of the brown color by enzymatic browning.     

THE EFFECTS OF SOLUBILIZATION PROCEDURES ON THE RELEASE AND MOLECULAR STATE OF ACETYLCHOLINESTERASE FROM ELECTRIC ORGAN TISSUE

Abstract— A study was made of the effect of various solubilization procedures on the release of AChE from electric organ tissue of the electric eel and on the molecular state of the enzyme. The procedures employed included homogenization in different ionic media or in the presence of detergents, etuymic treatment and chemical modification. Studies were performed on intact electroplax, tissue homogenates and membrane fractions. The apparent AChE activity of intact cells, homogenates and membrane fractions was shown to be governed by diffusion-controlled substrate and hydrogen ion gradients, generated by AChE-catalyscd hydrolysis, leading to a lower substrate concentration and a lower pH in the vicinity of the particulate enzyme.
Treatment of homogenates with NaCl solutions or with NaCl solutions containing the nonionic detergent Triton X-100 causes release of the native'molecular forms of the enzyme (primarily the 18 S species) which aggregate at low ionic strength. For optimal extraction both high ionic strength (e.g. 1 M-NaCl) and the detergent are needed AChE is also solubilized by treatment of tissue homogenates with trypsin, bacterial protease or collagenase. The first two enzymes caused its release as an 11 S non-aggregating form, while collagenase also produces a minor non-aggregating - 16 S component. Treatment of tissue homogenates with maleic anhydride causes release of AChE as a non-aggregating 18 S species. On the basis of the solubilization experiments it is concluded that the interaction of AChE with the excitable membrane is primarily electrostatic. The possible orientation of the enzyme within the synaptic gap is discussed.     

PRO-LONG涂膜对采后贮藏荔枝果实色泽和酶活性变化的影响

本文以荔枝品种“槐枝”为材料,以1．5％和2．5％Pro-long涂膜处理果实,研究Pro-long涂膜对果后贮于4℃的荔枝果皮色泽变化和有关酶活性的变化。对照和处理的HunterL值和b值均随贮藏时间的延长而降低,处理的比对照的下降慢。采后贮藏开始21d内,对照和处理的HuntCra值变化很小,保持相对稳定,之后的贮藏时间内有较显著的下降,说明贮藏初期果实的红色特性变化较少,之后变化很明显,这与果皮花色素苷、类黄酮、总酚的变化是相对应的。在贮藏后期,处理的HuntCra值下降明显较对照慢。Hunter值的变化与在贮藏过程中果实外观逐渐变成暗红和揭变以及在果实的来后根变中起着重要作用的多酚氧化酶和部分地涉及褐变过程的过氧化物酶的变化相对应。处理的过氧化物酶活性增加较对照慢,而且,与对照比较,多酚氧化酶的峰值稍稍延迟了。未发现l‘5％和2．5％处理间有明显差别。因此,Pro-long涂胶对荔枝的应用可以部分地降低多酚氧化酶和过氧化物酶,影响果皮花色素苷的降解、类黄酮和总酚的变化,延迟了褐变过程的发生。     

SOME PROPERTIES OF GLUTAMATE DECARBOXYLASE AND THE CONTENT OF PYRIDOXAL PHOSPHATE IN BRAINS OF THREE VERTEBRATE SPECIES

—Some properties of glutamate decarboxylase (GAD) were studied in the brain of the carp (Carassius auratus), the pigeon (Columbia livia) and the mouse (Mus musculus). The optimum pH for GAD in the three species was 6·3-6·5. In the three species studied, GAD activity of brain homogenates in water was higher than that of homogenates in buffer. The supernatant from homogenates in Triton-X-100 gave an enzyme preparation which showed greater activation by pyridoxal phosphate than those obtained from complete water or buffer homogenates or from the supernatant of Water homogenates. In the absence of pyridoxal phosphate, the activity of carp GAD was considerably lower than that of mouse or pigeon GAD. The addition of pyridoxal phosphate resulted in a much greater activation of carp GAD than that of pigeon or mouse GAD. Pyridoxal phosphate content was also measured in brains of the species studied. The difference between coenzyme levels in carp and mouse was very small in comparison to the difference in GAD activity in the absence of exogenous coenzyme. The pyridoxal phosphate content of pigeon brain was higher than that of the other two species.     

A CHEMOSYSTEMATIC STUDY OF SELECTED TAXA OF CAPSICUM

Six taxa of Capsicum were chosen for a comparative chemosystematic study. A “key” individual from each taxon was selected for intensive chemical investigation. Thirteen flavonoids were isolated from leaf material and characterized by paper chromatography and absorption spectroscopy. The C-glycosylflavones vitexin and isovitexin, and orientin and iso-orientin, as well as O-glycosides of the flavones apigenin, luteolin, and chrysoeriol, were isolated from the key individuals. Chromatographic analysis of collections from various regions of South America, Central America, and Mexico showed in general that flavonoid variability is more common in cultivated taxa than in wild. Three groups of Capsicum were recognized and the main systematic conclusions were: (1) The white-flowered taxa in Group I, C. baccatum var. baccatum and C. baccatum var. pendulum, have identical flavonoids, corroborating previous conclusions that they are one species. The absence of chrysoeriol in this group separates it from Groups II and III. (2) The purple-to-white-flowered C. eximium var. eximium and C. eximium var. lomenlosum, Group II, have a complex flavonoid chemistry which appears to link Groups I and III. (3) Two purple-flowered species, C. cardenasii and C. pubescens, Group III, are chemically distinct from the other taxa examined.     

Relationship between the enzymatic browning and phenylalanine ammonia-lyase activity of cut lettuce, and the prevention of browning by inhibitors of polyphenol biosynthesis.

Cut lettuce stored at 4 degrees C gradually turned brown on the cut section after several days of storage. Three factors for enzymatic browning, the polyphenol content, polyphenol oxidase activity, and phenylalanine ammonia-lyase (PAL) activity, were examined during the cold storage of cut lettuce. A relationship between the browning and PAL activity was apparent. We tried to prevent this browning by using the two enzyme inhibitors, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the phenylpropanoid pathway, and glyphosate, an inhibitor of the shikimate pathway. AIP and glyphosate significantly inhibited the browning of cut lettuce. The polyphenol content and PAL activity were both reduced by the treatment with AIP. These results show that regulating the biosynthesis of polyphenols is essential to prevent the browning of cut lettuce.     

荔枝果皮多酚氧化酶酶促褐变的研究

从荔枝果皮分别提取多酚氧化酶及其天然底物,两者相作用,形成褐色产物,酶促褐变是荔枝果皮变褐的原因。 从荔枝果皮提取的酚类物质中分离出多酚氧化酶的天然底物。此底物的紫外吸收光谱分别在215和280 nm有一强的和一弱的吸收峰,它的红外吸收光谱在3190、1600、1500 cm~(-1)有很强的吸收峰。     

ORIGIN AND RECENT ENDEMIC DIVERGENCE OF A CASPIAN MYSIS SPECIES FLOCK WITH AFFINITIES TO THE “GLACIAL RELICT” CRUSTACEANS IN BOREAL LAKES

Aspects of the evolution of intralacustrine species flocks and of the origin of the Arctic or “glacial-relict” zoogeographical element in Eurasian inland waters were elucidated in an allozyme study of the crustacean genus Mysis. This element, of supposedly northern marine ancestry, is represented by vicarious taxa in the deeper parts of the Caspian Sea (an enclosed ancient basin) and in young boreal lakes. The three endemic Caspian Mysis species studied are very close genetically (Nei's D = 0.06), which suggests a recent intrabasin radiation and rapid morphological divergence. This is in contrast to the pattern in postglacial Holarctic boreal lakes, where the Mysis relicta group is represented by a set of morphologically uniform but probably much older sibling species (D = 0.3–0.6). The results provide a parallel to those on the recent diversification of some fish species flocks in ancient freshwater lakes. The situation is, however, unusual in that the Caspian sympatric Mysis flock is pelagic, and conditions promoting speciation through allopatric isolation or spatial segregation by trophic substrate specialization seem implausible. The monophyletic Caspian Mysis clade shows a relatively strong divergence from both the northern lacustrine and the Arctic marine congeners (D = 0.6–1.0); the phylogenetic branching order of these three zoogeographical groups is not conclusively resolved. The results contradict the prevailing hypothesis of a recent Pleistocene origin of the Caspian Arctic element by invasion from Eastern European continental proglacial lakes that drained south to the Caspian basin during the glacial maxima and served as refugia for the boreal lacustrine taxa.     

甘蔗茎尖离体培养褐变影响因素及细胞区室结构分析

该研究通过综合分析对甘蔗(ROC 22)茎尖离体培养褐变不同条件因素的影响以及褐变细胞区室结构的变化,探讨了甘蔗茎尖离体培养褐变的机理机制。结果表明:不同芽位茎尖诱导成活率具有明显差异,随着芽位的增加,诱导成活率不断降低;不同季节取芽对外植体茎尖总酚类物质含量无明显影响;但不同芽位及不同催芽天数,外植体芽的总多酚含量明显不同,随着催芽天数的增加,不同芽位的多酚含量呈现由低升高的趋势;蔗芽在培养4周时多酚含量较低,适宜进行采芽接种培养;从褐变甘蔗茎尖的解剖结构变化分析,褐变甘蔗茎尖细胞离体培养初期细胞核结构出现变形,线粒体有肿胀拉长,部分液泡膜开始分解;中后期质壁分离更为严重,胞质中出现大量溶酶体,线粒体等细胞器全分解,细胞膜、液泡膜、核膜、线粒体膜的双层膜结构出现破损和缺口;而正常发育的茎尖细胞,能基本保持细胞核的形态结构,只有少量的溶酶体出现。因此,可以推测细胞核和线粒体结构变形以及膜系统的大量破损是甘蔗茎尖培养褐变死亡的原因。