Transient occurrence of an ebulin-related D-galactose-lectin in shoots of Sambucus ebulus L

Young shoots of Sambucus ebulus L. contain a monomeric d-galactose binding lectin (SELlm), which disappears upon shoot development, and was previously undetected since it co-purifies with the non-toxic type 2 ribosome-inactivating protein ebulin l and the dimeric lectin SELld. Molecular cloning of cDNA coding for SELlm and mass spectrometry analysis revealed a protein with a molecular mass of 34,239 Da, which displays 80%, 77% and 45% of amino acid sequence identity with the ebulin l-B chain, SELld and ricin-B chain, respectively. Furthermore, the cloned precursor, with respect to the ebulin l precursor is truncated and contains the signal peptide, a piece of the A chain, a piece of the connecting peptide and the B chain. Further processing yields the lectin protein, which contains only the B chain. Despite the fact that SELlm displays the same d-galactose-binding sites than ricin, it was found that the lectin has different binding properties to D-galactose-containing matrix than ricin. Notably, and unlike ricin, the binding of SELlm and other Sambucus lectins to such matrix was maximum in range of 0-10 degrees C and abolished at 20 degrees C.     

Presence of polymerized and free forms of the non-toxic type 2 ribosome-inactivating protein ebulin and a structurally related new homodimeric lectin in fruits of Sambucus ebulus L.

Mature leaves of dwarf elder (Sambucus ebulus L.) contain the non-toxic type 2 ribosome-inactivating protein ebulin 1 (Girbés et al., 1993b, J. Biol. Chem. 268: 18195–18199). We have now found that the green fruits of dwarf elder contain both free and polymerized forms of ebulin (ebulin f) and a new homo-dimeric D-galactose-binding lectin (SELfd). Polymerized material containing ebulin and lectin is composed of aggregates of variable relative molecular mass, some of them being close to 250 000. These aggregate forms are maintained in part by reducible disulphide bridges and reconstitute from reductant-free dialyzed material previously reduced with 2-mercaptoethanol. Direct incubation of free ebulin f with the free SELfd did not lead to polymerization, thus indicating that polymerization triggers some kind of substantial and perhaps catalyzed change in the structure of these proteins. Ebulin-containing polymerized material reacts with anti-ebulin f antibodies. Our results indicate that ebulin f is a fruit-form of ebulin 1. In contrast to green fruits, mature fruits lack both polymerized material and ebulin f, thus indicating some kind of reserve role for them in green fruits. Polymerization of ebulin and the dimeric lectin may represent a novel means of storing the non-toxic type 2 ribosome-inactivating proteins and lectins found in highly metabolic tissues, such as green fruits. Received: 4 April 1997 / Accepted: 17 June 1997     

2.8-A crystal structure of a nontoxic type-II ribosome-inactivating protein, ebulin l

Ebulin l is a type-II ribosome-inactivating protein (RIP) isolated from the leaves of Sambucus ebulus L. As with other type-II RIP, ebulin is a disulfide-linked heterodimer composed of a toxic A chain and a galactoside-specific lectin B chain. A normal level of ribosome-inactivating N-glycosidase activity, characteristic of the A chain of type-II RIP, has been demonstrated for ebulin l. However, ebulin is considered a nontoxic type-II RIP due to a reduced cytotoxicity on whole cells and animals as compared with other toxic type-II RIP like ricin. The molecular cloning, amino acid sequence, and the crystal structure of ebulin l are presented and compared with ricin. Ebulin l is shown to bind an A-chain substrate analogue, pteroic acid, in the same manner as ricin. The galactoside-binding ability of ebulin l is demonstrated crystallographically with a complex of the B chain with galactose and with lactose. The negligible cytotoxicity of ebulin l is apparently due to a reduced affinity for galactosides. An altered mode of galactoside binding in the 2gamma subdomain of the lectin B chain primarily causes the reduced affinity.     

Non-toxic type 2 ribosome-inactivating proteins (RIPs) from Sambucus: occurrence, cellular and molecular activities and potential uses.

Ribosome-inactivating proteins (RIPs) are a family of enzymes that trigger the catalytic inactivation of ribosomes. The most known member of the family is the highly poisonous two-chain ricin isolated from Ricinus communis L. Sambucus species contain a number of two-chain RIPs structurally and enzymatically related to ricin which have the noteworthy feature that, having an enzymatic activity on ribosomes, leading to the inhibition of protein synthesis, higher than ricin, they are lacking of the tremendous unspecific toxicity of ricin. Therefore, they have been called non-toxic type 2 RIPs. The most representative and studied members are nigrin b present in the bark of the common (black) elder Sambucus nigra L. and ebulin 1 present in the leaves of the dwarf elder Sambucus ebulus L. The molecular basis for the low unspecific activities of nigrin b and ebulin 1 as compared with ricin seems to be related with single changes of amino acids in the high affinity sugar binding sites of the B chains. These changes determine the intracellular traffic of these proteins and thus the cellular toxicity. Conjugation ofnigrin b or ebulin 1 to either transferrin or monoclonal antibodies provided highly active conjugates targeting cancer. Thus these non-toxic type 2 RIPs are promising tools for cancer therapy.     

Ebulin-RP,a novel member of the Ebulin gene family with low cytotoxicity as a result of deficient sugar binding domains

Background

Sambucus ebulus is a rich source of ribosome-inactivating proteins (RIPs) and RIP-related lectins generated from multiple genes. These proteins differ in their structure, enzymatic activity and sugar binding specificity.

Ebulitins: A new family of type 1 ribosome-inactivating proteins (rRNA N-glycosidases) from leaves of Sambucus ebulus L. that coexist with the type 2 ribosome-inactivating protein ebulin 1

A new family of single chain (type 1) ribosome-inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non-toxic two chain (type 2) RIP named ebulin 1 in its leaves. Ebulitins are basic proteins of M_r 32,000, 29,000 and 29,000 for ebulitins , β and γ, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.     

Sialic acid-binding dwarf elder four-chain lectin displays nucleic acid N-glycosidase activity

Sialic acid-binding dwarf elder agglutinin (SEA) present only in rhizomes of the medicinal plant Sambucus ebulus L., was found to be a tetrameric glycoprotein consisting of two covalently-associated dimers of an enzymic A chain with rRNA N-glycosidase activity (EC 3.2.2.22) linked to a B chain with agglutinin properties. The lectin inhibited protein synthesis by a cell-free system and depurinated ribosomes. Cloning of the corresponding gene and molecular modeling of the deduced amino acid sequence demonstrated that SEA has a three-dimensional structure which resembles that reported for other two tetrameric type 2 RIPs from Sambucus (SNAI and SSA). The lectin agglutinated red blood cells and displayed sugar affinity for sialic acid residues apart from d-galactose, binding to the mucin-producing gut goblet cells. Since sialic acid is present in animal cells, especially in epithelial lining gut cells, but not in plants, SEA could play a role in the defense against insect attack.     

水稻矮缩病毒昆明分离物抗血清制备及免疫捕捉PCR检测

由水稻矮缩病毒(Rice dwarf virus,RDV)引起的水稻矮缩病害,最早由日本报道,随后在东南亚等国以及我国的福建、云南等南方稻区普遍发生,云南主要发生于中部及南部地区[1]。水稻在苗期至分蘖期感病后,植株矮缩,分蘖增多,叶片浓绿,僵直,出现白斑,生长后期病稻不能抽穗结实,在暴发流行年份可以引起水稻的严重减产。RDV在分类上属于呼肠孤病毒科(Reoviridae)植物呼肠孤病毒属(Phytoreovirus)成员,病毒粒子为正十二面体球形结构,直径约为69.3nm,无刺突,无脂蛋白外膜包被,具有双层的蛋白衣壳[2]。病毒基因组由12条双链RNA片段组成,其中S8编…     

The genetically unstable dwarf locus in azuki bean (Vigna angularis (Willd.) Ohwi & Ohashi)

We characterized a spontaneous dwarf mutant showing extremely short internodes and dark green leaves originating from azuki bean (Vigna angularis (Willd.) Ohwi & Ohashi) cultivar "Erimo-shouzu." F(1) plants of 3 cross combinations between the dwarf mutant and several representative wild-type plants, Erimo-shouzu, V. angularis accession Acc2265 and wild relative V. riukiuensis accession Acc2482, supported the dwarf genotype being recessive. In a total of 3328 F(2) progeny of these 3 crosses, 65 dwarfs (2.0%) and 5 chimeric dwarfs (0.2%) segregated and the remainder were wild-type plants (97.8%). In F(3) progeny derived from self-pollinated dwarf F(2) plants, we observed wild type (54.3%), dwarf (39.1%), and chimeric dwarf (6.5%) plants. Two types of chimeric plants were observed: dwarf branches on the axils of wild-type plant stems and wild-type branches on the axils of dwarf stems. In 21 dwarf F(2) plants, the dwarf trait cosegregated with simple sequence repeat marker CEDG154 on chromosome 4. Conversely, homozygote F(2) plants at this chromosomal segment from the dwarf mutant frequently (>90%) expressed the wild-type phenotype. We concluded that the dwarf phenotype is mitotically and meiotically inheritable and controlled by a single genetically unstable locus, designated Azuki Dwarf1 (AD1), which converts between 2 phenotypic states bidirectionally.     

夏谷群体呼吸特性及其与群体光合的关系

中秆大穗型和矮秆紧凑型2种不同类型的夏谷群体呼吸速率在整个生育时期的变化呈单峰曲线,且随密度的增大而提高,抽穗开花期达最大值,紧凑型品种后期呼吸速率高于中秆大穗型品种,后期呼吸消耗大是低产的重要原因,群体呼吸日变化为一随温度而变化的单峰曲线,群体呼吸速率在冠层中的分布,依此是上3叶＞茎鞘＞中3叶＞穗＞下部叶,群体呼吸／光合的比值,两品种相近,在47％左右。     

Comparison of growth and gibberellin concentrations in shoots from orchard-grown standard and thermosensitive dwarf apple trees

Apple (Malus domestica Borkh.) trees were propagated by budding from selected fully grown hybrids that ranged in height from 1.5 to 8 m. The growth and development of the selected budded trees after 7 years in the orchard was similar to that of the parent trees. Additional grafting studies showed that the dwarfism was not associated with the roots. Differences in photosynthetic activity and associated processes were not related to the size difference between tissue culture-propagated orchard-grown standard cv. Golden Delicious and dwarf hybrid trees. Applications of GA₃ did not stimulate elongation of shoots of dwarf trees. Shoots of both standard and dwarf trees started to develop in mid-April when they contained nearly the same amounts of GA₁, GA₃ and GA₈, but standard shoots contained higher concentrations of GA₁₉, GA₂₀ and GA₂₉. On 2 June standard shoots were almost three times the length of dwarf shoots, but the number of leaves and area per leaf were nearly the same. The relative amounts of GAs on 12 May and 2 June for both plant types were similar to those on 20 April, except that GA₁₉, GA₂₀, GA₁ and GA₂₉ levels had declined. Gibberellin levels in standard shoots declined further between 2 and 22 June, after which there was no further shoot elongation or production of new leaves. Between 2 June and the end of the growing season, when summer temperatures were high, dwarf shoots continued to elongate slowly and to develop new leaves, which expanded little. During this time, the GA₁₉ content of dwarf shoots nearly doubled, whereas the amounts of GA₂₀, GA₁, GA₂₉ and GA₈ declined. By the end of the season, standard shoots were 40 cm in length with 20 leaves and dwarf shoots were 28 cm in length, but with 36 leaves. High summer temperatures appear to induce loss of GA-responsiveness in orchard-grown dwarf trees and to cause a reduced rate of conversion of GA₁₉ to GA₂₀ in these genotypes.     

欧李叶片过氧化物酶同工酶与叶果矿质元素含量变化的关系

以同一生境条件下113份燕山山脉野生欧李为试材,采用聚丙烯酰胺凝胶电泳方法,研究欧李叶片过氧化物酶(POD)同工酶与叶果中6种矿质元素的含量变化及其相关关系,为在酶水平上揭示欧李矿质营养代谢遗传差异提供理论参考。结果表明,欧李叶片过氧化物酶同工酶划分为7种酶谱类型,其中Rf 0.126和Rf 0.716酶带为共有的特征带,不同酶谱类型中单株个体间的相同酶带酶量大小以及叶果中的矿质元素含量存在明显差异,变异程度较大;相关分析结果显示,不同酶谱类型中相关酶带酶量大小与叶果中矿质元素含量变化存在显著或极显著的相关关系,表明欧李叶片过氧化物酶同工酶酶量大小变化在一定程度上反映矿质营养代谢的遗传差异和多样性。     

利用双向电泳技术分离大豆矮秆突变体相关蛋白

矮秆是大豆育种的重要目标性状之一。本实验以大豆野生型东农42和矮秆突变体东泽11为材料,利用近年来发展起来的双向电泳技术,在蛋白质水平对两个材料的差异蛋白质进行筛选,目的是鉴定与矮秆突变体相关的蛋白,为基因克隆提供依据。通过对酚(Phenol)法与TCA／丙酮沉淀法二种提取方法、100μg和200μg两种加样量、考马斯亮蓝染色和银染两种染色方法的比较,发现用丙酮沉淀法提取叶片可溶性总蛋白、加样量为200μg进行电泳,用考马斯亮蓝染色的效果较好,从而建立了大豆叶片总蛋白双向电泳技术优化体系。用该体系对野生型与突变体叶片全蛋白的差异分析,鉴定出９个蛋白差异点,其中６个上调表达,３个下调表达。     

The Role of Roots in Control of Bean Shoot Growth

Restriction of root growth by growing bean plants (Phaseolusvulgaris L.) in very small pots led to the development of dwarfplants. The leaves of those plants were smaller and their internodesshorter than those of control plants which were grown in largerpots and had developed a more extensive root system. A largequantity of starch—much more than in control plants —accumulated in the leaves and shoots of the dwarf plants. Increasingthe amount of minerals which was supplied to the roots, enhancedleaf growth of the control plants but failed to affect the dwarfones, in spite of the fact that in both cases the treatmentincreased the content of N, P and K in all the plant organs.The leaf water content was similar in both treatments, but theleaf water potential was higher in the dwarf plants. Exogenousapplication of gibberellic acid (GA₃) to the dwarf plants overcamethe reduction of stem growth completely, and that of the leavespartially. Application of the cytokinin, benzyladenine (BA)did not affect stem growth, but increased that of the primaryleaves. A combined supply of GA₂ + BA restored completely thegrowth of the stem and the primary leaves, and partially thatof the trifoliate leaves. It is concluded that a limited rootsystem restricts shoot growth through an hormonal system inwhich at least gibberellins and cytokinins are involved, andthat the dwarfing is not a consequence of mineral or assimilatedeficiency, or due to water stress. Phaseolus vulgaris L., leaf growth, stem growth, root restriction, gibberellic acid, benzyladenine, cytokinin     

Setting confidence limits for the detection of prune dwarf virus in Prunus avium with a monoclonal antibody-based triple antibody-sandwich ELISA†

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) with a monoclonal antibody was developed and evaluated for the detection of prune dwarf virus (PDV) in sweet cherry trees (Prunus avium). An independent reverse transcribed polymerase chain reaction test was also developed to establish, in conjunction with a bioassay, the incidence of PDV in 40 sweet cherry trees and to confirm the absence of virus in 15 control trees. Trees with two-thirds of their leaves positive for PDV would be identified with 99% probability by testing four leaves per tree with TAS-ELISA. The monoclonal antibody did not cross-react with Prunus necrotic ringspot virus in the TAS-ELISA.     

Factors contributing to dwarfing in the mangrove Avicennia marina

BACKGROUND AND AIMS: In Richards Bay, South Africa, Avicennia marina frequently exhibits a distinct productivity gradient, with tree height decreasing markedly from 6-10 m in the fringe zone to <1.5 m in the dwarf zone which is 120 m inland at a slightly higher elevation. In this investigation, soil physico-chemical conditions between fringe and dwarf A. marina were compared and the constraints imposed by any differences on mangrove ecophysiology and productivity determined. METHODS: Soil and plant samples were analysed for inorganic ions using spectrophotometry. Gas exchange measurements were taken with an infrared gas analyser and chlorophyll fluorescence with a fluorometer. Xylem psi was determined with a pressure chamber and chlorophyll content with a chlorophyll absorbance meter. RESULTS: In the dwarf site, soil salinity, total cations, electrical conductivity and soil concentrations of Na(+), K(+), Ca(2+), Mg(2+), Zn(2+), Mn(2+) and Cu(2+) were significantly higher than those in the fringe zone. Soil water potential and the concentration of soil P, however, were significantly lower in the dwarf site. In the leaves, Na(+) was the predominant ion and its concentration was 24 % higher in dwarf than fringe mangroves. Leaf concentrations of K(+), Ca(2+), Mg(2+), Mn(2+) and P, however, were significantly lower in dwarf mangroves. Photosynthetic performance, measured by gas exchange and chlorophyll fluorescence, was significantly reduced in the dwarf plants. CONCLUSIONS: The results suggest that hydro-edaphic factors contribute to high soil salinities, low water potentials, water stress and ion imbalance within tissues including P deficiency, which in interaction, contribute to dwarfing in Avicennia marina.     

Growth Activity of the Gibberellins of Dwarf French Bean, Potato, and Lettuce

Zones with similar Rf to gibberellic acid (GA) on chromatogramsof ethylacetate extracts of dwarf French bean and potato leavesand stems and lettuce heads all promoted growth of disks ofetiolated dwarf French bean leaves in darkness. The presence of a gibberellin was confirmed by tests with stemsof dwarf pea, but that the substance was not GA was shown bythe lack of effect in the tests with lettuce hypocotyls. Thetests with dwarf maize, effects on d-3 and d-5, but not d-1,identified it as gibberelliin A₅ (bean factor II). Materialthat promoted the growth of lettuce hypocotyls occurred in zonesclose to the starting-line of chromatograms of extracts of beanand lettuce leaves. Calibration of the growth tests with GA gave an estimate ofthe gibberellin content of the tissues extracted. In the dwarf-peatest the apparent gibberellin contents were similar to the valuesin the leaf-disk test, but very low values occurred in the maizetests.     

玉米ZmNPR1基因的克隆及表达分析

该研究从玉米高抗自交系D863F中克隆了ZmNPR1基因(GenBank登录号为MH619241)的gDNA和cDNA序列,开放阅读框长1 866 bp,编码621个氨基酸,相对分子质量67.61 kD,等电点为5.46。系统进化树比对表明,玉米ZmNPR1蛋白和高粱SbNPR1蛋白的亲缘关系较近,相似性高达97%。实时荧光定量PCR结果表明,ZmNPR1在叶片中能够被水稻黑条矮缩病毒诱导并显著上调表达。同时ZmNPR1在玉米叶片、茎、根、雄穗、雌穗以及花丝中均有表达,在雌穗和叶片中的表达量较高。研究表明,ZmNPR1可能在玉米粗缩病抗病过程中起着重要作用。     

Localization of production and emission of pollinator attractant on whole leaves of Chamaerops humilis (Arecaceae)

Volatile compounds, which frequently play important roles in plant-insect interaction, can be produced either by flowers to attract pollinators or by leaves to deter herbivores. The specialized structures associated with odor production differ in these two organs. The European dwarf palm Chamaerops humilis represents a unique intermediate between these two. In previous work, its leaves were shown to produce volatile organic compounds (VOCs) that attract pollinators only during flowering. Because the leaf sinuses look like a gland, the sinus was examined histologically and with environmental scanning electron microscopy (ESEM) for evidence that the sinus emits VOCs. Volatile compounds emitted by the different parts of the leaf were extracted by washes and headspace then analyzed by gas chromatograph-mass spectrometer (GC-MS). The sinus does not have the expected gland-like structure; the VOCs are actually produced by the whole leaf, even if the composition of the VOCs emitted by the sinus slightly differs. Thus, attraction of pollinators does not result from specialized secreting cells in leaves of flowering European dwarf palms. The results are discussed in the context of a convergent evolution of leaves toward petals.