Absolute stereochemistry of leucinopine,a crown gall opine

Crown gall tumors incited by Agrobacterium tumefaciens strain Bo542 have been reported to synthesize a tumor-specific substance identified as N-(1,3-dicarboxypropyl)-leucine (leucinopine), a compound with two centers of asymmetry. We report here evidence that leucinopine is indeed a crown gall opine, in that it is specifically catabolized by A. tumefaciens strains carrying the tumor-inducing plasmid pTi Bo542, as well as strains carrying closely related plasmids pTi AT1 and pTi AT4. We further report catabolism of leucinopine by the succinamopine-type strains A518, A519 and A532, carrying pTi EU6, pTi AT181 and pTi T10/73, respectively. Strains lacking any virulence plasmid, as well as those carrying octopine or nopaline type Ti plasmids or mannopine type Ri plasmids, did not catabolize leucinopine. On the basis of specificity of catabolism by bacteria carrying pTi Bo542, we conclude that the stereochemistry of natural leucinopine is l-threo, i.e. l^glu,l^leu. Such stereochemistry is novel in the opines known thus far: octopine, nopaline and succinamopine have d,l-stereochemistry: d^ala,l^arg (octopine), d^glu,l^arg (nopaline) and d^glu,l^asn (succinamopine).     

Detection of opines by colorimetric assay

A colorimetric procedure for confirming the presence of arginine-derived opines (nopaline and octopine) in plant tissue extracts is described. Those materials are widely used as markers of plant cell transformation and tumorigenesis mediated by the tumor-inducing plasmids of Agrobacterium tumefaciens. Nopaline and octopine are generally detected, following resolution by paper electrophoresis, by observation of the uv-fluorescent products formed upon reaction with phenanthrenequinone. We found that a further heat treatment step, compatible with paper electrophoresis, results in rapid production of a red-purple pigment. Our colorimetric assay is sensitive to 1.25-micrograms quantities of opine and eliminates problems of background fluorescence encountered with crude plant extract in the usual assay.     

植物冠瘿生长调控及相关基因研究进展

阐述了植物冠瘿产生的影响因素及冠瘿的生长和调控,并对冠瘿发生有关的基因进行了综述。     

Development of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil

AIMS: To develop a specific, sensitive and rapid PCR-based method for detection of tumorigenic Agrobacterium in soil. METHODS AND RESULTS: Three newly designed primers complementary to tms2 gene amplified DNA of only the tumor-inducing agrobacteria of 113 strains tested, resulting in 617 bp and 458 bp products in the first and second rounds of semi-nested PCR respectively. As optimized method of pre-incubation of soil suspensions on selective medium, DNA isolation and two-round semi-nested PCR enabled detection of 1-2 bacterial cells in 1 g of soil. Using this method tumour-inducing Agrobacterium was detected in 67 of 69 samples of naturally infested soil originating from the field, where plants with crown gall symptoms occurred. The pathogen was detected only in two samples of 15 tested, collected from a nursery where crown gall symptoms were not observed. CONCLUSIONS: The semi-nested PCR-based method allowed for sensitive and rapid detection of tumorigenic agrobacteria in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is proposed for testing of soil in fields intended for nursery production of fruit trees, roses or other plants susceptible to crown gall, as well as a tool for ecological studies.     

Thiophene production by crown galls and callus tissues of tagetes patula

Thiophene concentrations were measured in crown gall tissues produced by Agrobacterium tumefaciens infections (strains A208, A277) of Tagetes patula plants and compared with those of normal and transformed callus tissues. The results showed that it is not possible to predict the amounts of secondary motabolites produced as a result of transfers of genetic material from infected plants to crown galls and then to transformed callus tissues. There appears to be an accumulation of intermediates in some of the biosynthetic routes.     

Cytokinin biosynthesis in crown gall tissue of Vinca rosea: Metabolism of isopentenyladenine

When isopentenyl[8-¹⁴C]adenine was incubated with crown gall tumour tissue of Vinca rosea, it was stereospecifically hydroxylated to trans-zeatin and its derivatives, which are the endogenous free cytokinins in this tissue. Adenine, adenosine and adenine nucleotides were the major degradation products.     

Characterization of the tRNA and aminoacyl-tRNA synthetases of healthy and tumorous callus tissues from Nicotiana tabacum

Aminoacyl-tRNA synthetases extracted from healthy and crown gall tumor tissues (induced by Agrobacterium tumefaciens strain B6) from Nicotiana tabacum (strain Wisconsin 38) grown in vitro, showed the same ability to charge Phaseolus vulgaristRNA, for all the 15 amino acids tested. For each amino acid, optimal charging conditions (enzyme concentration, Mg²⁺/ATP ratios, K⁺ ion effects) have been determined with Phaseolus vulgaristRNA and were found to be the same whether aminoacyl-tRNA synthetases from healthy or tumor tissues were used. In each case, valyl- and glutamyl-tRNA synthetases were very sensitive to an excess of Mg²⁺ and K⁺ ions. Although tRNA's extracted from healthy and tumor tissues gave the same electrophoretic patterns, charging levels obtained with turner tRNAs were generally 45% higher than those obtained with tRNA's from healthy tissues.     

Chromatographic separation of synthetic nopaline and isonopaline and their absolute configuration

Synthetic nopaline and isonopaline were separated preparatively by anion exchange chromatography and their configurations were ascertained by an enzymi     

Auxin-independent growth of maize tissue culture cells

Wall-regenerating protoplasts and suspension culture cells of Zea mays L. ‘Black Mexican Sweet’ were co-cultivated] with Ti plasmid-containing Agrobacterium tumefaciens strain ACH5. After elimination of the bacteria, putative transformants were selected for their ability to grow in the absence of added auxin. Althoug some of the treated cells grew, untreated controls and cells treated with the Ti plsmidless strain ACH5C3 also proliferated. The frequency of auxin-independent growth was similar in all treatments. Because none of 75 candidate transforms contained detectable T-DNA, the hormone-independent phenotype appears to be a consequence of habituation. Although some of the habituated colonies grew as undifferentiated callus, others produced rootlike structures. Miaze cells subcultured in liquid media containing progressively reduced concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) also became habituated.     

Agrobacterium radiobacter strains K84, K1026 and K84 Agr‐ produce an antibiotic‐like substance,active in vitro against A. tumefaciens and phytopathogenic Erwinia and Pseudomonas spp.

Agrobacterium radiobacter strains K84, K1026 and K84 Agr^‐ produced in vitro an antibiotic‐like substance (ALS 84), different from agrocin 84 and observed in mannitol‐glutamate medium. Twenty five out of 39 A. tumefaciens strains of biovars 1, 2 and 3 were sensitive to ALS 84 regardless of their sensitivity to agrocin 84. Sensitivity in A. tumefaciens strain C58 was not encoded by the Ti‐plasmid. Most isolates tested of Erwinia carotovora subsp. carotovora E. carotovora subsp. atroseptica, Pseudomonas corrugata P. cichorii and unidentified isolates from galls were also sensitive to this substance. ALS 84 was not affected by the proteases studied, nor by treatment at 62°C for 30 min and had a bacteriostatic effect. The production of ALS 84 might play a role in the complex mechanism of biological control of crown gall, especially in strains resistant to agrocin 84 and sensitive to ALS 84, and by the creation of an ecological niche favourable to A. radiobacter strains K84, K1026 or K84 Agr^‐.     

桃根癌病菌拮抗放线菌抑菌物质的分离纯化与结构鉴定

[目的]为明确根癌拮抗放线菌G-19的抑菌活性物质.[方法]采用SephadexLH-20柱层析、高效液相色谱及中压制备色谱等技术,对桃根癌拮抗放线菌G-19发酵液中的抑菌活性成分进行分离纯化,并通过LC-MS、NMR对其结构进行鉴定.[结果]分离得到化合物G-19-Ⅰ的结构为邻苯二甲酸二丁酯(DBP),化合物G-19-Ⅱ的结构为2-(4-羟基苯基)-3,4-二氢-2H-苯并吡喃-3,4,5,7-四醇,俗称无色天竺葵素.[结论]阐明了放线菌G-19抑菌活性成分的物质基础.     

Control of in vivo protein synthesis and peroxidase formation by DNA-containing extracts of normal and crown gall tissues

Nucleic acids extracted from normal bean hypocotyl tissue (NE) and crown gall tumors (TE) affect amino acid incorporation into protein and the development of peroxidase activity when vacuum infiltrated into normal receptor tissues. TE enhances and NE inhibits both processes; NE from successively older tissues produces progressively greater inhibitions per unit of infiltrated nucleic acid. The active material has an absorption maximum at 257 nm with an A_260:280 ratio of more than 2·0. On acrylamide gel electrophoresis it shows a small DNA peak, four typical r-RNA peaks and a small low molecular weight RNA peak. Activity in such extracts is completely destroyed by hydrolysis with 0·3 N KOH or DNAase; RNAase is only slightly effective and pronase ineffective. It is deduced that the effective material contains DNA that may be complexed with RNA or other materials in the extract. Pretreatment of donor tissues with actinomycin d or 5-fluorouracil diminishes or annuls the activity of the extract. Pretreatment of receptor tissue with actinomycin d inhibits the action of TE but not of NE; pretreatment with cycloheximide prevents the action of both NE and TE.     

抗桃根癌病菌的耐氰放线菌筛选及其抑菌作用研究

为了筛选耐氰并抗桃根癌病的微生物菌株,以不同浓度的KCN培养基作为选择培养基,以根癌农杆菌为靶标菌,通过平板对峙培养的方法,筛选出了耐氰放线菌G-19,其发酵液代谢产物对根癌农杆菌有明显的抑制作用,抑菌圈直径为21.66 mm。其抑菌作用主要表现在3个方面:使得病原菌的生长对数期缩短,菌体浓度处于低水平状态;使病原菌的细胞壁弯曲、皱缩,继之结构发生破裂,细胞质向边缘凝聚,中心变得稀薄,甚至形成空腔;使细胞膜的通透性增加,导致病原菌的细胞质液外泄;但未见到病原菌DNA的损伤。因此,认为G-19菌株抑菌的作用机理是使根癌病原菌的细胞壁结构破坏和膜透性增加。     

Quorum-sensing signal production by Agrobacterium vitis strains and their tumor-inducing and tartrate-catabolic plasmids

Agrobacterium vitis strains, their tumor-inducing (pTi) and tartrate utilization (pTr) plasmid transconjugants and grapevine tumors were analyzed for the presence of N -acyl-homoserine lactones (AHLs). All wild-type A. vitis strains produced long-chain signals. PCR analysis of the A. vitis long-chain AHL synthase gene, avsI , showed the predicted amplicon. Agrobacterium tumefaciens UBAPF2 harboring various A. vitis pTi plasmids produced N -(3-oxo-octanoyl)- l -homoserine lactone encoded also by pTis of A. tumefaciens . UBAPF2 transconjugants carrying pTrs except for pTrTm4 and pTrAB3, also produced an AHL. UBAPF2 transconjugants carrying pTrAT6, pTrAB4 and pTrRr4 or pTiNi1 produced two additional AHLs not observed in the corresponding wild-type strains. We also provide evidence for in situ production of AHLs in grapevine crown gall tumors of greenhouse and field origin.     

丹参的冠瘿组织培养和丹参酮的产生

用根癌农杆菌感染丹参无菌苗获得冠瘿组织,除菌后的冠瘿组织在无激素的Ms培养基上生长良好。经高压纸电泳检查,冠瘿组织中含有冠痿碱,证实根癌农杆菌的Ti质粒转化成功。冠瘿组织的生长和丹参酮的积累与基本培养基有关,B5和Ms培养基有利于生长．月增殖倍数分别达到102倍和90倍,而67-V和WP培养基则有利于丹参酮的合成,在培养过程中丹参酮能分泌到培养液中。研究表明用冠瘿组织作为培养系统,生产药用植物有效成分具有良好的开发前景。     

Expression of nopaline synthesis in tumorous and regenerant tobacco crown galls

Summary Tissues formed in liquid cultures of tobacco (Nicotiana tabacum cv. Wisconsin 38) crown galls incited byAgrobacterium tumefaciens C58 were of three types: unorganized callus, organized teratoma, and organized normal appearing. These tissues contained 400±12, 410±17, and 614±53 μg nopaline/g fresh weight, respectively. Using [¹⁴C]arginine, methods were developed for measuring in vivo nopaline biosynthetic rates. Tissues were incubated in a low concentration (i.e., 3 μM) of [¹⁴C]arginine to minimize disruption of the internal pool (approximately 140 μM free arginine). Radioactivity in the tissue was assayed and the specific radioactivity of free arginine, the precursor of nopaline, was determined. The linear rate of incorporation of radioactivity into nopaline was used to calculate the following biosynthetic rates (expressed as microgram nopaline per gram fresh weight per 24 h): callus, 14; teratoma, 21; normal appearing, 24. These results show conclusively that normal appearing tissues obtained from crown gall tumors can synthesize nopaline. Abnormal growth and opine biosynthesis, therfore, can be expressed independently.     

樱桃砧木实生苗对根癌菌侵染的抗性及生理响应

以贵州樱桃的6个砧木实生苗为材料,采用人工接种根癌菌的方法,探讨它们对根癌菌侵染的抗性及生理响应特征,评价其抗病性能力。结果显示:(1)接种根癌菌120d后,青岩樱桃实生苗发病率较低(6.67%),冠瘿瘤直径较小(1.76mm),感病系数也较低(6.67),属高抗砧木;沿河樱桃实生苗发病率高达50.0%,冠瘿瘤直径达6.43mm,感病系数达33.76,属高感砧木。(2)感染根癌菌后,各樱桃砧木幼苗的生理特性受到一定影响,其叶片游离脯氨酸含量增加,根系过氧化物酶(POD)和超氧化物歧化酶(SOD)活性增强,而过氧化氢酶(CAT)活性降低,其中,青岩樱桃的游离脯氨酸含量升高幅度最大,沿河樱桃的保护酶活性变化幅度明显较大。(3)樱桃砧木幼苗的发病率与感病系数呈显著正相关(0.98*);发病率与SOD活性、感病系数与POD活性和SOD活性均呈显著正相关,相关系数分别为0.86、0.82、0.83;其余指标间相关性不显著。研究表明,6个樱桃砧木实生苗对根癌菌的抗性有明显差异,并以青岩樱桃最强,且其抗性强弱与其感病后叶片脯氨酸含量和根系的POD、SOD活性增加幅度有密切关系。