Purification and properties of urease from the leaf of mulberry, Morus alba

Urease was purified from leaves of mulberry (Morus alba, L.) by ammonium sulfate fractionation, acetone fractionation and sequential column chromatography including Q-Sepharose HP, Phenyl-Sepharose HP, Superdex 200 HR and Mono Q. The enzyme was purified 5700-fold to apparent homogeneity with a recovery of 3.6%. The molecular mass of the enzyme was determined to be 90.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and 175 kDa by gel filtration, indicating that the enzyme was a homodimer. In the western blot analysis, 90.5 kDa subunit of the mulberry leaf urease cross-reacted with antiserum raised against jack bean seed urease. The N-terminal sequence of the first 20 residues of the enzyme revealed that it has a high similarity (80-90%) to ureases from other plant sources, suggesting that the mulberry leaf urease is closely related to other plant ureases. However, the mulberry leaf enzyme showed an optimum pH for activity of 9.0, while the optimum pH of most ureases isolated from plants and bacterial is neutral. In addition, the K(m) value for urea was 0.16 mM, which is lower than those of ureases from other sources. It is also proposed that urease activity ingested by browsing silkworm releases ammonia that is subsequently used in silkworm protein synthesis.     

Apical dominance in mulberry (Morus alba): Effects of position of lateral and accessory buds and leaves

Suzuki, T. 1990. Apical dominance in mulberry ( Morus alba ): Effects of position of lateral and accessory buds and leaves. – Physiol. Plant. 78: 468-474.
Removing apical portions of current growth coppice shoots from field-grown, low-pruned stumps of mulberry ( Morus alba L. cv. Shin-ichinose) caused sprouting of one or more upper main buds, almost concurrently with that of accessory buds. However, removal of the new sprouts, including those from accessory buds, slightly enhanced the sprouting of buds immediately below them, and did not affect buds lower down. In contrast, mature leaves inhibited the buds in their axils. Budless, leafy nodes on the upper part of pruned shoots tended to swell after treatment, perhaps due to the accumulation of substances translocated from the roots and possibly from the remaining leaves. Lateral buds at different positions along the shoot differed in their sprouting ability with buds lower on the shoot being more inhibited. This inhibition gradient dissappeared when all coppice shoots on one stump were pruned to the same bud position, suggesting inhibition from neighboring, actively growing shoots. These results demonstrate that acropetal influences are important in bud dominance relationships.     

Dormancy and spring development of lateral buds in mulberry (Morus alba)

Patterns of spring development of lateral buds of mulberry (Morus alba L. cv. Shin-ichinose) coppice shoots on 11-year-old low-pruned stumps varied in response to girdling, pruning and arching. The erect controls showed a weak acrotonic (apex-favoring) growth habit, in which the majority of the buds, including the basal ones, sprouted and elongated in mid- and late April, and hence there was a prolonged imposition of dominance on the upper laterals in mid- and late May. In contrast, early spring girdling or pruning enhanced the activity of the upper buds of the proximal (lower) halves of the girdled stems or of the pruned stems, resulting in considerable dominance of the laterals from such buds in late April. Arching markedly inhibited buds on the under side of the arched stems, leading to poor shoots. By late April, the buds on the adaxial (upper) side readily grew into new vertical shoots, which dominated over the lateral ones. When studied by a multiple-node-cutting test, increased length of segments of post-dormant mulberry stems was accompanied by decreased bud activity of the segments and by decreased breaking ability of the lower buds within the segments, suggesting the importance of roots in the weak acrotonic habit of the erect stem in spring. By contrast, the acropetal influences of the attached stems can in part affect dominance relationships, perhaps mediated through competition for factors translocated from the roots. Continuous basal applications of abscisic acid inhibited bud break and shoot growth of the postdormant stem segments, but these inhibitory effects could be reversed by applied gibberellic acid A₃ (GA₃). Two phases of lateral bud dormancy in erect mulberry coppice shoots were identified. The first was characterized by a smaller breaking capacity in the upper buds than in the lower ones and hence by a basitonic (base-favoring) gradient in bud growth potential. The second phase corresponded to a restoration of these capabilities in the upper buds and to a change towards a linear gradient in bud growth potential, with disappearance of the dormant condition, in February and March. This gradient change during dormancy release may represent the physiological basis for the weak acrotonic habit of erect mulberry stems in spring.     

Shoot-tip abscission and adventitious abscission of internodes in mulberry (Morus alba)

In mulberry ( Morus alba L. cv. Shin-ichinose), shoot-tip abscission following the cessation of apical growth could be induced in different internodes, depending on the vigour of the shoot and its apex and other internal and external factors. In the lateral, short shoots of 1-year-old stems of low-pruned trees, the apical growth cessation and shoot-tip abscission (May–June) resulted primarily from the dominance of the upper, long shoots and intense competition among laterals along the stem. Decapitation of the laterals, before abortion of their apices took place (early May), readily caused adventitious abscission of the distal internode. Similar decapitation-induced, adventitious abscission of the distal internode of the upper, long shoots of 1-year-old stems of pruned trees also occurred (May–September), demonstrating that the abscission itself is not directly associated with photoperiod. In May and June, decapitation induced abscission primarily in parallel with or after sprouting of lateral buds and shoot elongation, while in July, August and September, the abscission was induced by decapitation and independently of sprouting. Shoot (stem) orientation positively affected the abscission, which is related to gravimorphic effects on buds and shoots on the lower and lateral sides of the horizontally trained stem. These results suggest that the vigour of shoots and apices is an important determinant of growth and apex abscission in mulberry.     

Production of triploid plants of mulberry (Morus alba L) by endosperm culture

 A continuously growing callus was obtained from immature endosperm of Morus alba L Cv S-36 cultured on Murashige and Skoog medium containing 5 μm 2,4-dichlorophenoxyacetic acid. Shoot buds were produced when the callus was subcultured on a medium containing a cytokinin or a cytokinin and 1-naphthaleneacetic acid (NAA). The maximum number of shoots was formed on the medium containing thidiazuron (1 μM), or benzylaminopurine (5 μM) and NAA (1 μM). Shoots were multiplied by forced axillary branching and rooted in vitro. Endosperm-derived plants were established in soil. Each of the ten plants examined cytologically was triploid (3 n=42). Received:17 February 1999 / Revision received: 4 May 1999 / Accepted: 19 May 1999     

桑树肌动蛋白actin基因全长序列的克隆与分析

肌动蛋白在植物的各种生理活动中起着重要作用,是研究基因表达与调控模式的内标参考。通过染色体步移方法获得了桑树肌动蛋白actin基因1612bp的序列,该基因CDS长1312bp(GenBank登录号:HM623866),编码377个氨基酸残基,与水稻、葡萄等的同源基因的序列一致性在90%以上。基因内含子的数目及其在基因组上的位置也与水稻、葡萄等的相似。对来自不同物种的24个肌动蛋白基因进行聚类分析的结果显示,基因被分为ClassⅠ和ClassⅡ两个明显的亚群。     

Cloning and sequence analysis of full length of mulberry (Morus alba ) actin gene

肌动蛋白在植物的各种生理活动中起着重要作用,是研究基因表达与调控模式的内标参考.通过染色体步移方法获得了桑树肌动蛋白actin基因1 612 bp的序列,该基因CDS长1 312 bp(GenBank登录号:HM623866),编码377个氨基酸残基,与水稻、葡萄等的同源基因的序列一致性在90％以上.基因内含子的数目及其在基因组上的位置也与水稻、葡萄等的相似.对来自不同物种的24个肌动蛋白基因进行聚类分析的结果显示,基因被分为Class Ⅰ和ClassⅡ两个明显的亚群.     

Apical control of lateral bud development and shoot growth in mulberry (Morus alba)

Measurements of changes in the degree of dominance by upper laterals over lower ones in coppice shoots (1-year-old stems) of 12-year old low- pruned stumps of mulberry ( Morus alba L. cv. Shin-ichinose) were made by removal of upper stem sections (pruning) or of lateral buds (debudding.) before spring bud burst, as part of a study of the factors involved in dominance relationships between the developing buds and elongating shoots. Besides inhibition of lower laterals by the upper, leading shoots, there was evidence for mutual inhibition (competition) of neighboring laterals along the stem. Thus in stems in which every other bud, or 4 out of every 5 buds were removed, there was a delay in growth cessation of lower laterals and their greater elongation than in controls. Such competition was seen to exist even between the uppermost and sub-terminal laterals, since the former elongated more in the absence of the latter.
In contrast to high and middle pruned stems, the delay in sprouting of the buds in low-pruned stems resulted in limited elongation of the shoots from such buds. This inhibition was removed when all the stems on a stump were pruned to the same length, suggesting that it was associated with intact stems with actively growing laterals. Patterns of regrowth of the short shoots (lower laterals) after summer pruning (middle-pruned) depended on the extent of removal of other stems with vigorously growing, upper laterals. These results demonstrate that both acropetal and basipetal influences are important in bud and shoot dominance relationships.     

Genetic variations among mulberry genotypes (Morus alba) as revealed by Random Amplified Polymorphic DNA (RAPD) markers

Turkey is one of the most important mulberry fruit producers in the world. In particular, in Eastern and Inner part of Turkey, mulberry fruits are processed into several products such as `Mulberry Pekmez', `Mulberry Pestil', `Mulberry Kome' etc. Therefore, mulberry fruits give higher economic returns thus have greater employment potentials in Turkey. In order to improve the yield and fruit quality through breeding, the genetic variations and relationships among 15 selected white mulberry genotypes (Morus alba L.) were evaluated using Random Amplified Polymorphic DNA (RAPD). A total of 101 DNA markers were generated by 16 random primers, with an average of 6.3 easily detectable markers per primer. Several RAPD markers showed unique patterns of mean frequency that differed among the white mulberry genotypes. The distance matrix showed that the highest genetic distance (0.902) was between EMS13 and EMS8 and the least (0.413) was between EMS12 and EMS15. According to the results, RAPD analysis can be used for the characterization and grouping of mulberry genotypes. The genetically divergent genotypes identified from this study would be of much use in the future breeding program.     

Isolation and characterization of six novel microsatellite markers for mulberry (Morus indica)

Genetic characterization of germplasm resources is necessary for their effective management and efficient utilization, especially for species like mulberry in which the available germplasm exhibits rich phenotypic diversity with almost no information about its genetic base. Here we present the first report on the isolation of six novel microsatellite markers of mulberry, developed from an enriched genomic library of Morus indica. These markers revealed a high degree of polymorphism (14–26 alleles per locus; polymorphic information content, 0.85–0.90) and a broad cross‐species affinity when tested on a set of 43 elite genotypes including 13 related Morus species. The data thus demonstrate their utility as potentially efficient genetic markers for germplasm characterization, crop improvement and molecular systematics of mulberry.     

Ultrastructural and immunochemical features of the cell wall sac formed in mulberry (Morus alba) idioblasts

A peculiar inward growth, named a “cell wall sac”, formed in mulberry (Morus alba) idioblasts, is a subcellular site for production of calcium carbonate crystals. On the basis of ultrastructural observations, a fully expanded cell wall sac could be divided into two parts—an amorphous complex consisting of multi-layered compartments with multiple fibers originating from the innermost cell wall layer, and a peripheral plain matrix with fiber aggregates. Immunofluorescent localization showed that low and highly esterified pectin epitopes were detected at the early stages of development of the cell wall sac, followed by complete disappearance from the both parts of fully enlarged mature sac. In contrast, the xyloglucan epitope remained in the compartment complex; this was supported by the observation that the xyloglucan epitope labeled with immuno-gold particles is found on fibers in the complex part.     

Oxidative stress and antioxidant responses of mulberry (Morus alba) plants subjected to deficiency and excess of manganese

The aim of the study was to relate the effects of deficiency and excess of Mn with the generation of reactive oxygen species (ROS) and altered cellular redox environment in mulberry (Morus alba L.) cv. Kanva-2 plants. Mn deficiency symptom appeared as mild interveinal chlorosis in middle leaves. Mn-excess did not produce any specific symptom. Leaf water potential (Ψ) was increased in Mn-deficient and Mn-excess mulberry plants. Mn-deficient leaves contained less Mn, less chloroplastic pigments and high tissue Fe, Zn and Cu concentrations. Starch content was increased with increasing Mn supply. While reducing sugar content increased in Mn-deficient and Mn-excess plants as well, non-reducing sugars remained unaffected in Mn-deficient plants and decreased in Mn-excess plants. Moreover, study of antioxidative responses, oxidative stress (H₂O₂ and lipid peroxidation) and cellular redox environment [dehydroascorbate (DHA)/ascorbic acid (AsA) ratio] in Mn-stressed mulberry plants was also undertaken. Both hydrogen peroxide and lipid peroxidation were enhanced in the leaves of Mn-deficient plants. Increased H₂O₂ concentration in Mn-excess leaves did not induce oxidative damage as indicated by no change in lipid peroxidation. The ratio of the redox couple (DHA/AsA) was increased both in Mn-deficient or Mn-excess plants. The activities of superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) increased in Mn-deficient plants. The activity of ascorbate peroxidase (EC 1.11.1.11) increased with increasing Mn supply. The results suggest that deficiency or excess of Mn induces oxidative stress through enhanced ROS generation and disturbed redox couple in mulberry plants.     

Chemical characterization of cytotoxic indole acetic acid derivative from mulberry fruit (Morus alba L.) against human cervical cancer

The fruit of the white mulberry tree (Morus alba L.) is a multiple fruit with a sweet flavor commonly consumed around the world. Chemical investigation of the fruits led to the isolation of two indole acetic acid derivatives (1 −2) including a new compound, which turned out to be an isolation artifact, 3S-(β-D-glucopyranosyloxy)-2,3-dihydro-2-oxo-1H-indole-3-acetic acid butyl ester (1), along with five known compounds (3 −7). Compounds 2 and 7 were newly identified from mulberry fruit. The new isolation artifact (1) exhibited cytotoxic effect on human cervical cancer Hela cells in a dose-dependent manner. Compound 1 activated caspase-8, caspase-9, and caspase-3, followed by cleavage of PARP, a substrate of caspase-3, in a dose-dependent manner. Simultaneous alterations in protein expression of mitochondrial factors Bax, BID and Bcl-2 were also observed. A comparison between compounds 1 and 2 led to a structure-activity relationship analysis of the cytotoxic effect. These results suggest that compound 1 could be beneficial in human cervical cancer treatment, and provide a theoretical basis for further application of compound 1.     

桑树(Morus alba)朱砂叶螨（Tetranychus cinnabarinus）实验种群的温度效应

采用生命表分析、生存分析、"王-兰-丁"模型及线性模型等分析方法,对15～28℃温区的5个恒温处理朱砂叶螨(Tetranychus cinnabarinus)实验种群进行了系统研究.结果表明,此温区内,种群在生殖、发育和生存3方面,有明显的温度效应:内禀增长力、周限增长力、净增殖力和平均日产卵量、世代平均周期、及种群倍增时间的倒数呈线性增长;而平均寿命、最大死亡年龄,随温度升高而递减;性比和实际产卵天数对温度不敏感.种群在生殖、发育和生存三者之间,采取了较为"折衷"的策略:8℃为发育临界点;13℃左右为生殖和种群增长临界温度;22℃左右为生殖和种群增长最适温度;30℃左右为发育最适温度.     

Phytostilbenoid production in white mulberry (Morus alba L.) cell culture using bioreactors and simple deglycosylation by endogenous enzymatic hydrolysis

Phytostilbenes are responsible for several biological activities of mulberry (Morus sp.), which has been widely used as a raw material in health products. This study aimed to investigate the capability of Morus alba L. cell in bioreactors to produce the major bioactive stilbenes. The cell obtained from air-driven bioreactors such as round bottom, flat bottom, and air-lift vessel shape bioreactors was collected and analyzed for the levels of mulberroside A and oxyresveratrol. The results showed that the cell culture in round bottom and air-lift vessel bioreactors had higher growth rate, as compared with the cell culture in shake flasks (1.38- and 1.41-fold, respectively). The optimized culture condition to produce mulberroside A was obtained from round bottom bioreactor culture (55.56 ± 11.41 μmol/L). Additionally, endogenous stilbenoid hydrolysis of cell from the bioreactor culture was examined. Under optimized hydrolytic conditions, mulberroside A in the cell was readily deglycosylated to give oxyresveratrol within 1 h. These results indicated that the glycoside mulberroside A in the cell is sensitive to the endogenous enzymatic hydrolysis. Interaction of the stilbenoid components with the endogenous hydrolytic enzyme triggered by cell disruption in M. alba samples was suggested to be the major cause of the alteration of the stilbenoid levels. These findings have provided a new approach to producing glycosidic compounds and corresponding aglycones in cell culture.

     

Hypolipidemic and antioxidant effects of Morus alba L. (Egyptian mulberry) root bark fractions supplementation in cholesterol-fed rats

The 70% alcohol extract of the Egyptian Morus alba L. root bark was fractionated over cellulose CC eluted with water, 50% methanol and finally with 100% methanol to yield 3 fractions (MRBF-1, MRBF-2 and MRBF-3), respectively. In continuation of chromatographic purification of 70% alcohol extract fractions of the Egyptian M. alba L. root bark, 4 compounds namely: mulberroside A, 5,7,2'-trihydroxyflavanone-4'-O-beta-D-glucoside and albanols A and B were isolated from MRBF-2 for the first time from the Egyptian plant. Experimentally induced atherosclerosis was produced by feeding rats a diet enriched in coconut oil (25% by weight) and cholesterol (2% by weight) for 21 days. Then, hypercholesterolemic rats were orally administered (MRBF-1, MRBF-2 and MRBF-3 fractions) in a dose of 500 mg kg(-1) day(-1) for 15 successive days, in order to evaluate their expected hypocholesterolemic activity. Lipid profile parameters such as plasma total cholesterol, LDL-C, VLDL-C, LDL:HDL ratio and triglycerides, as well as plasma and liver lipid peroxides and glutathione-S-transferase enzyme levels, serum paraoxonase enzyme level, LDL oxidation, LDL aggregation and LDL retention, were measured. Plasma and liver glutathione-S-transferase enzyme levels were unaffected in all studied groups. The results revealed that the administration of (MRBF-2 and/or MRBF-3) fractions resulted in alleviation of atherosclerotic state. Administration of MRBF-3 significantly retained plasma and liver peroxides towards their normal levels, and also, produced significant increase in resistance towards major atherogenic modifications; namely LDL oxidation, LDL aggregation and LDL retention by 44%, 30%, and 33%, respectively. Thus, it can be concluded that the consumption of MRBF-2 and (MRBF-3, in some extent) fractions of M. alba L. root bark 70% alcohol extract may act as a potent hypocholesterolemic nutrient and powerful antioxidant via the inhibition of LDL atherogenic modifications and lipid peroxides formation in hypercholesterolemic rats.     

Regulation of phenol oxidase activity with sulfur dioxide as the base for production of high-quality instant green tea

The possibility to regulate phenoloxidase activity with sulfur dioxide was studied. It was found that this compound is a potent inhibitor of phenoloxidase of the reversible and mixed type. The inhibitory effect of sulfur dioxide on phenoloxidase provided grounds for a new biotechnological approach to the production of instant green tea. This approach allows increasing the yield of the extractive and the proportion of phenolics in the extractive, thereby improving the organoleptic quality of the product.