Changes in the localization and levels of starch and lipids in cambium and phloem during cambial reactivation by artificial heating of main stems of Cryptomeria japonica trees

Background and Aims

Cambial reactivation in trees occurs from late winter to early spring when photosynthesis is minimal or almost non-existent. Reserve materials might be important for wood formation in trees. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules in cambium and phloem were examined from cambial dormancy to the start of xylem differentiation in locally heated stems of Cryptomeria japonica trees in winter.

Methods

Electric heating tape was wrapped on one side of the stem of Cryptomeria japonica trees at breast height in winter. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules were determined by image analysis of optical digital images obtained by confocal laser scanning microscopy.

Key Results

Localized heating induced earlier cambial reactivation and xylem differentiation in stems of Cryptomeria japonica, as compared with non-heated stems. There were clear changes in the respective localizations and levels of starch and lipids (as droplets) determined in terms of relative areas on images, from cambial dormancy to the start of xylem differentiation in heated stems. In heated stems, the levels and number of starch granules fell from cambial reactivation to the start of xylem differentiation. There was a significant decrease in the relative area occupied by lipid droplets in the cambium from cambial reactivation to the start of xylem differentiation in heated stems.

Conclusions

The results showed clearly that the levels and number of storage starch granules in cambium and phloem cells and levels of lipids (as droplets) in the cambium decreased from cambial reactivation to the start of xylem differentiation in heated stems during the winter. The observations suggest that starch and lipid droplets might be needed as sources of energy for the initiation of cambial cell division and the differentiation of xylem in Cryptomeria japonica.     

Patterns of protein synthesis in the cambial region of Scots pine shoots during reactivation

Changes in protein synthesis in cambial region cells were monitored in 1-year-old cuttings of Scots pine ( Pinus sylvestris L.) collected in November, when the cambium was dormant, and subjected to environmental conditions that promoted or inhibited cambial growth. The proteins were labelled in vivo with L-[³⁵S]-methionine and separated using 2-dimensional polyacrylamide gel electrophoresis. In budded cuttings cultured under environmental conditions favoring cambial reactivation, there was a reproducible quantitative change in 55 proteins (33 induced and 22 repressed), a less certain increase or decrease in 40 proteins, and no apparent change in about 150 proteins. Under the same conditions, 8 proteins were induced and 6 others were repressed in debudded cuttings treated apically with 1 mg indole-3-acetic acid (IAA) in 1 g lanolin, in which cambial reactivation occurred, compared with debudded cuttings treated with plain lanolin in which the cambium did not reactivate. Three of the proteins induced in the IAA-reated cuttings only appeared after cambial cell division and derivative differentiation actually began, and the same proteins were found in budded cuttings after their cambium had become reactivated. In contrast, protein expression in cuttings exposed to environmental conditions that prevented cambial reactivation was similar at the beginning and end of the experimental period. These results indicate that the cambium was in the quiescence stage of dormancy at the start of the experiment, that quiescent cambial region cells can synthesize proteins as soon as exposed to environmental conditions favoring reactivation, and that only 3 of the approximately 250 proteins detected were specifically involved in cambial growth     

Relationships between cambial activity,cell differentiation and the localization of starch in storage tissues around the cambium in locally heated stems of<Emphasis Type="Italic"> Abies sachalinensis</Emphasis> (Schmidt) Masters

A study was made, in a cool-temperate zone, of the extent of cell division in the cambium, the extent of differentiation of cambial derivatives, and the localization of storage starch around the cambium in locally heated (22–26°C) stems of the evergreen conifer Abies sachalinensis (Schmidt) Masters during cambial dormancy and immediately after natural reactivation of the cambium. In locally heated regions of stems during cambial dormancy, heating induced localized reactivation of the cambium. However, the cells in the heated and reactivated cambium stopped dividing soon after only a few cells had been generated. In addition, no differentiation of the xylem and the disappearance of starch from storage tissues around the cambium were observed. In regions of stem that had been locally heated after natural reactivation of the cambium, cell division continued in the cambium and earlywood tracheids with a large radial diameter and secondary walls were formed, with abundant starch in the storage tissues around the cambium. Our results suggest that the extent of both cell division in the cambium and cell differentiation depends on the amount of starch in storage tissues around the cambium in the locally heated stems of an evergreen conifer growing in a cool-temperate zone.     

Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassica oleracea L. var. botrytis) stems

Pectic substances are a major component of cell walls in vegetable plants and have an important influence on plant food texture. Cauliflower (Brassica oleracea L. var. botrytis) stem sections at different regions of the mature plant stem have been monitored for tissue-related changes in the native pectic polysaccharides. Chemical analysis detected appreciable differences in the degree of methyl-esterification (ME) of pectic polysaccharides. About 65% of galacturonic acid (GalpA) residues were methyl-esterified in floret tissues. Relative ME showed a basipetal decrease, from 94% in the upper stem to 51% in the lower-stem vascular tissues. The decrease was not related to a basipetal increase in glucuronic acid (GlcpA) residues. The monoclonal antibodies, JIM 5 and JIM 7, produced distinct labelling patterns for the relatively low-methyl-esterified and high-methyl-esterified pectin epitopes, respectively. Labelling was related to cell type and tissue location in the stem. Floret cell walls contained epitopes for both JIM 5 and JIM 7 throughout the wall. Stem vascular tissues labelled more strongly with JIM 5. Whereas pith parenchyma in the upper stem labelled more strongly with JIM 7, in the lower-stem pith parenchyma, JIM 5 labelling predominated. Localization of pectic polysaccharide epitopes in cell walls provides an insight into how structural modifications might relate to the textural and nutritional properties of cell walls. Received: 16 August 1997 / Accepted: 20 December 1997     

Induction of cambial reactivation by localized heating in a deciduous hardwood hybrid poplar (Populus sieboldii x P. grandidentata)

BACKGROUND AND AIMS: The timing of cambial reactivation plays an important role in the control of both the quantity and the quality of wood. The effect of localized heating on cambial reactivation in the main stem of a deciduous hardwood hybrid poplar (Populus sieboldii x P. grandidentata) was investigated. METHODS: Electric heating tape (20-22 degrees C) was wrapped at one side of the main stem of cloned hybrid poplar trees at breast height in winter. Small blocks were collected from both heated and non-heated control portions of the stem for sequential observations of cambial activity and for studies of the localization of storage starch around the cambium from dormancy to reactivation by light microscopy. KEY RESULTS: Cell division in phloem began earlier than cambial reactivation in locally heated portions of stems. Moreover, the cambial reactivation induced by localized heating occurred earlier than natural cambial reactivation. In heated stems, well-developed secondary xylem was produced that had almost the same structure as the natural xylem. When cambial reactivation was induced by heating, the buds of trees had not yet burst, indicating that there was no close temporal relationship between bud burst and cambial reactivation. In heated stems, the amount of storage starch decreased near the cambium upon reactivation of the cambium. After cambial reactivation, storage starch disappeared completely. Storage starch appeared again, near the cambium, during xylem differentiation in heated stems. CONCLUSIONS: The results suggest that, in deciduous diffuse-porous hardwood poplar growing in a temperate zone, the temperature in the stem is a limiting factor for reactivation of phloem and cambium. An increase in temperature might induce the conversion of storage starch to sucrose for the activation of cambial cell division and secondary xylem. Localized heating in poplar stems provides a useful experimental system for studies of cambial biology.     

活性氮、活性氧及植物激素在种子休眠解除中的作用及相互关系研究进展

休眠是植物种子对环境变化的适应机制,其机理至今未完全清楚阐明。前期对种子休眠机制的研究主要集中在激素调节上,近期的研究结果表明,一氧化氮（nitric oxide,NO）参与打破种子的休眠,并与其所引起的种子中活性氧的变化有关。本文简要综述活性氮（reactive nitrogen species,RNS）、活性氧（reactive oxygen species,R0s）和植物激素在种子休眠解除中的作用及相互关系研究进展。     

Polyamine catabolism in dormant embryos of the spindle tree (Euonymus europaeus L.) and in dormancy break obtained after treatment with gibberellic acid

Previously we showed that dormancy break of spindle tree embryos after gibberellic acid (GA₃) treatment was followed by an increase in arginine decarboxylase (ADC) activity (Béranger-Novat N. et al., Plant Sc. 102: 139–145, 1994). These results indicated that arginine decarboxylase pathway mediate hormone-induced growth responses in spindle tree embryos. In the present investigation we show that in GA₃-treated embryos diamine oxidase (DAO) increases immediately after putrescine content and the increase in DAO activity paralleles the accumulation of putrescine at the beginning of the culture (before the visible appearance of the radicle). In this system polyamine oxidase (PAO) increases immediately after DAO activity and follows closely the increase in spermidine content. These results demonstrate a direct correlation between the biosynthesis and oxidation of putrescine and spermidine. At every stage of development DAO and putrescine levels are lower than spermidine and PAO levels. Dormant embryos can be distinguished from GA₃-treated embryos by a complete lack of putrescine accumulation. In dormant embryos compared to GA₃-treated embryos DAO changed more or less in parallel and on the whole seemed to follow the same content and distribution, but the kinetics of the activation of DAOs were different in dormant embryos with a delay of 1.5 day for the first and 1 day for the second peak. During the first days of culture at least up to 4 days the distribution of spermidine and PAO in GA₃-treated embryos followed the same pattern observed in dormant embryos, but the levels of spermidine and PAO were greatly reduced in dormant embryos. On the other hand the kinetics of the activation of PAOs were different in dormant embryos with a delay of 1 day. The results suggest that dormant embryos are deficient in their ability to synthesize polyamines efficiently and support the view that spermidine catabolism (via PAO pool) is limiting in untreated embryos during the first days of culture.     

Meiotic cytokinetic apparatus in the formation of the linear spore tetrads of Conocephalum japonicum (Bryophyta)

Most bryophytes produce tetrahedral spore tetrads. However, linear spore tetrads have been reported to occur in Conocephalum japonicum (Thunb.) Grolle. In this study, the distribution of microtubules (MTs) during meiosis in C. japonicum was examined to determine the division pattern resulting in a linear tetrad. Spore mother cells in the pre-meiotic stage were cylindrical with randomly distributed cytoplasmic MTs. In the prophase-metaphase transition, spindle MTs replaced cytoplasmic MTs and a barrel-shaped spindle with two flattened poles developed. Cortical MT arrays were not detectable throughout meiosis. Although a phragmoplast appeared between sister nuclei in telophase-I, it disappeared without expanding to the parental cell wall. Metaphase-II spindles oriented parallel to the long axis of the cell and in tandem to each other resulted in a linear arrangement of telophase nuclei. Radial arrays of MTs developed from the nuclear surfaces and three phragmoplasts appeared among the four nuclei to produce four spores. Two phragmoplasts separating the paired sister nuclei appeared prior to the appearance of a phragmoplast between non-sister nuclei. The MT cycle is basically the same as that reported in meiosis of C. conicum, which produces non-linear tetrads. A morphometric study indicated that the difference in the division pattern between C. conicum and C. japonicum is due to a difference in the shape of spore mother cells. The cylindrical shape of sporocytes of C. japonicum restricts the orientation of spindles and phragmoplasts so that the four resultant spores are arranged linearly. Received: 22 April 1998 / Accepted: 15 May 1998     

Molecular cloning of the cDNA coding for the (R)-(+)-mandelonitrile lyase of Prunus amygdalus: temporal and spatial expression patterns in flowers and mature seeds

A gene highly expressed in the floral organs of almond (Prunus amygdalus Batsch), and coding for the cyanogenic enzyme (R)-(+)-mandelonitrile lyase (EC 4.1.2.10), has been identified and the full-length cDNA sequenced. The temporal expression pattern in maturing seeds and during floral development was analyzed by RNA blot, and the highest mRNA levels were detected in floral tissues. The spatial mRNA accumulation pattern in almond flower buds was also analyzed by in-situ hybridization. The mRNA levels were compared during seed maturation and floral development in fruit and floral samples from cultivars classified as homozygous or heterozygous for the sweet-almond trait or homozygous for the bitter trait. No correlation was found between these characteristics and levels of mandelonitrile lyase mRNA, suggesting that the presence of this protein is not the limiting factor in the production of hydrogen cyanide. Received: 3 December 1997 / Accepted: 18 April 1998     

Accumulation of heavy metals in epidermal glands of the waterlily (Nymphaeaceae)

This study investigates the anatomical aspects of heavy-metal accumulation in the waterlily (Nymphaea `Aurora', Nymphaeaceae). Epidermal glands were identified by light microscopy on the abaxial side of the leaf laminae and on the epidermis of the rhizome; glandular trichomes were observed in the petiole epidermis. Glands were not observed in the roots. Accumulation of heavy metals in these glands was monitored using a scanning electron microscope equipped for energy-dispersive spectroscopy. Further experiments showed maximal cadmium and calcium accumulation in the mature leaf lamina in daylight, and this accumulation was inhibited by the herbicide 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. These results suggest that, in Nymphaea, heavy metals are accumulated primarily in association with glands found in plant organs that have direct contact with water or mud. Deposition and storage of heavy metals by these glands may represent a stage in the sequestration and detoxification of the metals. Our results raise the possibility of utilizing waterlilies for the removal of heavy metals from polluted environments. Received: 29 April 2000 / Accepted: 8 June 2000     

Evaluation of the diagnostic potential of recombinant leptospiral OMP A-like protein (Loa22) and transmembrane (OmpL37) protein in latex agglutination test for serodiagnosis of leptospirosis in animals

Leptospirosis is a re-emerging zoonotic disease of animals and humans caused by pathogenic Leptospira, which has major public health concerns. The study is aimed to express the recombinant outer membrane protein (OMP) A-like protein (rLoa22) and transmembrane (rOmpL37) protein of Leptospira interrogans serovar Hardjo in the Escherichia coli and their evaluation as a diagnostic antigen in the latex agglutination test (LAT) to detect anti-leptospiral antibodies in the sera of animals. The Loa22 and OmpL37 genes lacking signal peptide coding sequences were individually amplified (522 and 963 bp), by polymerase chain reaction, and directionally cloned into a pETite N-His Kan vector for expression. The expressed purified proteins were characterized by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblot, which confirmed leptospiral specific reactive protein with a molecular weight of ~19 and 36 kDa, respectively. The sensitized latex beads coated with these OM proteins separately were evaluated in LAT using cattle sera of microscopic agglutination test (MAT) confirmed positive (n = 53) and negative (n = 52) cases of leptospirosis. The rLoa22 LAT and rOmpL37 LAT revealed the relative diagnostic sensitivity of 94·34 and 96·23%, diagnostic specificity of 92·31 and 96·15% and accuracy of 93·33 and 96·19%, with the excellent agreement of Cohen's kappa value of 0·87 and 0·92, respectively. After extensive evaluation, this rapid recombinant protein-based field diagnostic test can be applied as a screening test for the detection of anti-leptospiral antibodies in the sera of animals in the field conditions.     

Use of Competitive PCR to Detect and Quantify Haplosporidium nelsoni Infection (MSX disease) in the Eastern Oyster (Crassostrea virginica)

This study was undertaken to develop a quantitative polymerase chain reaction assay that would improve the utility of PCR for detecting Haplosporidium nelsoni (MSX), a serious parasite of the eastern oyster Crassostrea virginica. A competitive PCR sequence was generated from the H. nelsoni small subunit ribosomal DNA fragment, originally described by Stokes and colleagues, that was amplified by the same PCR primers and had similar amplification performance. Assays performed using competitor dilutions ranging from 0.05 to 500 pg/μl DNA were used to test oyster samples designated using histological techniques as having ``light' or ``heavy' MSX infections. Visual diagnoses were confirmed equally well with three methods: densitometry of ethidium-bromide-stained agarose, densitometry of SYBRGreen-stained polyacrylamide gels, and analysis by GeneScan 3.0 of fluorescent products detected in ultrathin gels. Oysters diagnosed as negative for MSX tested as negative or light by PCR. Oysters with light MSX infections generally had less than 5 pg/μl infectious DNA. Oysters with heavy infections generally corresponded to 5 pg/μl or greater competitor dilutions. Received September 3, 1999; accepted March 3, 2000.     

砧木对梨叶芽休眠的影响及其与多胺代谢的关系

以分别嫁接在杜梨和豆梨上的砂梨品种‘丰水’为试材,研究了2008和2009年11～12月气温变化和不同砧木对‘丰水’梨叶芽休眠进程的影响,分析叶芽中游离态和束缚态内源多胺种类和含量的变化,结果表明：嫁接在豆梨上的‘丰水’叶芽自然休眠结束的时间要比嫁接在杜梨上的‘丰水’叶芽早10d左右,且游离态和束缚态腐胺（Put）、戊二胺（Cad）、己二胺（Hex）、亚精胺（Spd）和精胺（Spm）5种内源多胺含量开始升高的时间与供试材料叶芽自然休眠结束的时间一致,表明梨叶芽的休眠进程与砧木种类和多胺代谢有密切关系,尤其是与束缚态多胺含量变化的关系更为密切。     

中国大鲵消化系统13种器官的蛋白水解酶种类和活性分析

蛋白水解对生命活动是必不可少的(Vassali et al., 1994),蛋白质的酶解修饰(Xu et al.,1999)、细胞的迁移、组织再生与修复、消化系统对食物中蛋白质的消化等均与蛋白水解酶有关(Baimbridge et al.,1992),许多病理过程也与蛋白水解酶功能失调有关(Teichert et al., 1989; Monard, 1988).因此开展大鲵消化系统各器官的蛋白水解酶种类和性质的研究,对了解大鲵消化系统各器官的功能、演化及大鲵的营养需求、食性、消化生理等是必要的.本文对大鲵消化系统各器官的蛋白水解酶特征进行了初步分析,现将结果报道如下.     

Simulation of the maximum nitrate inflow (Imax) of lettuce (Lactuca sativa L.) grown under fluctuating climatic conditions in the greenhouse

Lettuce was grown in nutrient solution under fluctuating climatic conditions in the greenhouse. The maximum nitrate inflow (Imax) was measured twice a week to validate a model for calculating Imax, that was developed for constant conditions in a growth chamber.Growth and Imax were very similar between greenhouse and growth chamber plants, so that the model was able to predict Imax very precisely. The daily maximum nitrate inflow was calculated and its dependency on fluctuating temperature could be shown.     

Subcellular distribution of glucose transporter (GLUT-1) during development of the blood-brain barrier in rats

Electron microscopy was used to quantify the subcellular distribution of the GLUT-1 isoform of the glucose transporter in developing microvessels of the brain of embryonic rats from E (embryonic stage) 13 to E19 and in adult rats. Gold-conjugated secondary antibodies were used to localize, on ultrathin sections of brain, a rabbit polyclonal antiserum (anti-GLUT-1) raised against a synthetic peptide encoding 13 amino acids of the C-terminus of the human glucose transporter. Staining was weak at E13 but increased in density during development into adulthood. The increase represented an increase in the absolute amount of transporter per vessel profile, with a concomitant decrease in vessel size with the narrowing of the wall. At early stages, the percentages of total particles per profile of lumenal membrane, ablumenal membrane, and cytoplasm were approximately equivalent. The ratio of lumenal to ablumenal particle density then shifted from below 1 at E13 to above 2 at E19 and to 4 in the adult. In contrast, vessels of the choroid plexus were devoid of labeling, but the choroid plexus epithelium stained as early as E15. In the brain, no astrocytes, neurons, or pericytes were stained at any stage examined. Developmental upregulation of the GLUT-1 glucose transporter therefore seems to occur at the blood-brain barrier, and the modulation of the subcellular distribution of the transporter can be correlated with other observed changes in the microvessels as they develop the blood-brain barrier phenotype. Received: 18 November 1995 / Accepted: 12 January 1996     

紫貂秋季生境选择的初步研究

采用无线电跟踪技术和全球定位系统（ＧＰＳ）对大兴安岭地区紫貂（Ｍａｒｔｅｓ ｚｉｂｅｌｌｉｎａ）秋季的生境选择进行研究。秋季紫貂对优势树种（以落叶松为主,７２％）、中下坡位（９０％）、小树密度（Ｐ＝０．０００４）、大树密度（Ｐ＝０００１）、大树胸径（Ｐ＝０．０００１）、倒木长度（Ｐ＝０．０００６）及密度（Ｐ＝０．０００１）都有一定的选择性,对坡向,灌丛盖度的选择性不明显,对中等盖度的林地有一定的选     

Ontogeny of the Major Xenobiotic‐Metabolizing Enzymes Expression and the Dietary Lipids Modulatory Effect in the Rat Dimethylbenz(a)anthracene‐Induced Breast Cancer Model

Breast cancer is the most common malignancy in women worldwide. Environmental factors such as xenobiotic exposure and lifestyle and nutrition play a key role in its etiology. This study was designed to evaluate the age‐related changes in the expression of major xenobiotic‐metabolizing enzymes (XMEs) in the rat liver and the mammary gland in the dimethylbenz(a)anthracene‐induced breast cancer model. The influence of dietary lipids on the ontogeny of XMEs was also evaluated. mRNA and protein levels of phase I (CYP1A1, CYP1A2, and CYP1B1) and phase II (NAD(P)H:quinone acceptor oxidoreductase 1 and GSTP1) enzymes were analyzed, as well as their regulation by AhR and Nrf2, respectively. Results showed differences in the phase I enzymes expression, whereas little changes were obtained in phase II. High corn oil and olive oil diets differentially influenced the expression of age‐related changes, suggesting that the different susceptibility to xenobiotic exposure depending upon the age may be modulated by dietary factors.     

淡水富营养型湖泊沉积物亚硝酸还原酶基因(nirS)的多样性和系统发育

【目的】以亚硝酸盐还原酶基因(nirS)为分子标记,探讨富营养化湖泊武汉东湖沉积物中NirS类反硝化细菌群落的多样性及系统发育,并分析环境因子对群落分布的影响。【方法】在武汉东湖4个典型子湖郭郑湖、汤菱湖、团湖和庙湖采集沉积物样品,测定环境参数;提取沉积物中微生物群落基因组DNA,分别构建4个子湖的反硝化微生物的nirS基因文库,利用限制性片段长度的多态性分析(Restriction Fragment LengthPolymorphism,RFLP)技术初步分群,确定各群的代表菌株并测定其nirS基因序列;利用DOTUR软件计算各群落多样性和丰富度指数,以Neighbor-Joining法构建供试菌与参比菌的系统发育树。【结果】环境参数测定结果表明东湖4个子湖中庙湖沉积物总氮(TN)和氨态氮(NH 4+-N)含量最高,团湖最低,郭郑湖沉积物中NO 3-浓度最高。基于NirS序列的生物多样性和丰富度分析表明团湖生物多样性和丰富度指数最高而庙湖各项指数均较低。各子湖供试序列及其代表序列综合RFLP聚类分析表明,武汉东湖沉积物中NirS类反硝化微生物种群具有丰富的多样性。NJ系统发育分析表明东湖沉积物NirS类反硝化菌群可分成3个较大群体(群I-III)。群I占总群体的67.7%,广泛分布于不同的生态环境;来自郭郑湖代表菌的81%分布于群I,而庙湖的代表菌中65%分布于群II。比较分析发现来自于东湖和人工湿地两种生境的NirS群落间具有较高的相似性。【结论】武汉东湖淡水富营养型湖泊沉积物中亚硝酸还原酶基因(nirS)具有丰富的多样性。东湖沉积物中TN、NH 4+和NO 3-的浓度可能是影响NirS类反硝化微生物多样性和空间分布的重要因素之一。