Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.     

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.     

Purine Alkaloid Production and Accumulation in Cocoa Callus and Suspension Cultures

Cocoa callus and suspension cultures were found to produce caffeine,theobromine, and theophylline which are typical of the purinealkaloids found in cocoa beans. Production of these purine alkaloidswas monitored in callus cultures for over 2 years and shownto stabilize at concentrations of about 10% those found in vivo.Caffeine and theobromine were produced concomitant with logphase growth of the cultures whilst theophylline productionreached a maximum during stationary phase, reflecting the possiblerole of the latter as a catabolite of caffeine. The effectsof choice of cytokinin, explant tissue, cocoa type, light conditionsand time in culture on purine alkaloid production by callushave been examined. Purine alkaloid production by cocoa suspensioncultures has also been examined and these cultures were shownto be less productive and more variable than callus cultures.The results demonstrate that cocoa tissue cultures can be usefulfor studying secondary metabolism in vitro. Key words: Theobroma cacao, caffeine, theobromine, tissue culture, secondary metabolism     

Ethylene Accumulation in Flooded Plants

Ethylene concentration in sunflower (Helianthus annuus L.) cuttings increased 5-fold within 6 h after submersion in distilled water and then declined. When only the basal half of the cutting was steeped in water, ethylene concentration was slightly over half the concentration of the completely submerged cutting. Ethylene concentration also increased when cuttings were wrapped with moist paper tissue. When wrapped with Saran transparent plastic film, ethylene concentration increased continuously for 12 h. When part of the stem of an intact plant was wrapped with Saran, ethylene also increased in that part of the stem. When wrapping was removed or submersion was discontinued, accumulated ethylene in the cuttings decreased, much faster from unwrapped cuttings than from previously submerged ones. During 3 h submersion, ethylene production rate in submerged cuttings was approximately 10% of that for the controls and over 97% ethylene escaped out of the control cuttings while only 22-52% escaped from the submerged cuttings. Water content increased during submersion and decreased when submersion was discontinued. Water content did not change significantly during wrapping, but decreased when the cuttings were unwrapped. High water content in the submerged cuttings was apparently not related to the high ethylene concentration in the cuttings. Causes of ethylene increase in flooded plants were discussed and it was concluded that one of the first and major causes is the accumulation of ethylene in flooded portions of the plants due to the blockade of ethylene escape by water.     

Membranotropic action of cardiotonic agents and ubiquinone

Interaction of cardiotonic drugs (strophantidine acetate, suphan, para-oxybenzoic acid) and ubiquinone with phospholipid bilayers has been studied. Exothermic effect of the reaction followed by an increase in microviscosity and hydrophobicity of the bilayer from cardiolipin, but by a decrease of the microviscosity of the bilayer from lecithin has been estimated. A correlation is observed between changes in the lecithin bilayer fluidity and the heat effect of the interaction at the initial period of time after mixing of reagents.     

Bacterial Accumulation of Metallic Compounds

Xanthomonas maltophilia accumulated metals or metallic compounds within the cell in the form of one or a few particles, in a peculiar condition similar to that of an electrolyte solution in an electric cell. The particles consisted of many little granules, most of which were assumed to be composed mainly of a compound of zinc and phosphorus in an amorphous state.     

植物对Si的吸收、运输和沉积

本文综述了植物中的S i及其吸收、运输和沉积的研究进展:(1)植物含S i量因植物种、品种甚至无性系与器官的不同而有较大差异,S i在细胞中呈现区隔化分配;(2)植物中沉积的S i主要是非晶态的S iO2.nH2O(又称硅胶、植物蛋白石、植硅石等),是有机物质矿质化的产物,不同植物所形成的S i化结构及其形态不同,可用于植物分类、鉴定及古气候与环境变化的研究;(3)硅藻和高等植物主要吸收S i(OH)4,高等植物中存在S i主动吸收机制;(4)有机大分子物质(包括多胺、碳水化合物、纤维素)作为有机衬质参与了植物的S i沉积过程;(5)突变体的鉴定和应用,对S i营养研究具有重要作用.     

Studies on the Chemical Regulation of Alkaloid Biosynthesis in Tobacco Plants

Effect of auxin treatment upon the alkaloid content of Nicotiana was examined using hydroponically grown plants. Nicotine content of Nicotiana tabacum was reduced by the addition of 3-indoleacetic acid or 2, 4-dichlorophenoxyacetic acid to nutrient solution, an the incorporation of nitrate-¹⁵N into nicotine was decreased by the addition of 2, 4-dichloro-phenoxyacetic acid. Contents of nicotine, nornicotine, anabasine and anatabine in Nicotiana glutinosa were decreased by 2, 4-dichlorophenoxyacetic acid added to the nutrient solution. Foliar applications of eleven kinds of synthetic auxins were effective in reducing nicotine content of Nicotiana tabacum and the effectiveness of the compounds seemed to be concerned with their auxin activities. Foliar application of auxin tended to increase the dry weight of stems whereas decreased that of roots, and, moreover, higher content of soluble sugars and lower content of starch in leaves were observed by the treatment. A possibility of a practical application of auxin to reduce the nicotine content of tobacco plants were drawn from this work.     

木荷属和柃木属植物的嘌呤代谢及嘌呤碱合成研究

以［8-¹⁴C］标记的腺嘌呤和黄嘌呤为底物,对两种可以合成少量咖啡碱和茶叶碱的木荷属和柃木属植物（Schima mertensiana,Eurya japonica）叶片的嘌呤代谢进行了检测研究。发现木荷属和柃木属植物中嘌呤代谢相似,¹⁴C标记的腺嘌呤可以整合到嘌呤核苷酸、RNA、酰脲（包括尿囊素和尿囊酸）、二氧化碳中。经过24 h培养,在叶片吸收的放射能中,仅有6%～7%用于甲基黄嘌呤类化合物的合成（3-甲基黄嘌呤、7-甲基黄嘌呤核苷、7-甲基黄嘌呤、茶叶碱）。和其他植物一样,绝大多数¹⁴C标记的黄嘌呤整合到嘌呤的分解代谢物中（二氧化碳和酰脲）,少量的放射能分布在3-甲基黄嘌呤及茶叶碱中。根据结果可以推断木荷属和柃木属植物具有N-甲基转移酶活性,可以用来合成咖啡碱和茶叶碱,相对于茶树而言,活性不高。综上,本文对木荷属和柃木属植物的嘌呤代谢以及嘌呤碱合成进行了研究。     

植物对土壤中铀的吸收与富集

核工业发展导致重金属铀排放和扩散,并造成了地表土壤的污染,对人类的生存环境产生了极其不利的影响。如何修复铀污染土壤成为亟待解决的问题。近年来发展起来的植物修复技术以其成本低廉、安全和环保的特点成为修复铀污染土壤的新选择。寻找理想的铀富集植物是这一技术的基础和关键。该文通过实验模拟铀污染的土壤（土壤中铀的浓度为100 mg.kg–1）,进行一次和二次铀污染土壤的植物修复后,从4个方面对植物修复铀污染土壤效果进行评估,即富集铀的浓度、生物提取量、生物富集系数（BFS）和转运系数（TFS）。实验结果表明：第1次修复时,四季香油麦菜（Lactuca dolichophylla）地上部富集铀的浓度为1.67×103 mg.kg–1,生物富集系数和转移系数均大于3;第2次修复时,麦冬（Ophiopogon japoni-cus）富集铀的浓度与第1次修复相比变化不大,而吊兰（Chlorophytum comosum）、四季豆（Phaseolus vulgaris）和艾蒿（Artemisia lavandulaefolia）富集铀的浓度与第1次修复相比均减少4–8倍;施加土壤改良剂鸡粪肥、海藻肥和柠檬酸后发现海藻肥和柠檬酸能够增强植物对铀污染土壤的修复;对两次修复土壤中铀的形态进行对比分析,发现二次修复时土壤中生物有效态铀的含量降低,造成第2次修复的难度增加。     

The Relationship between Growth Rate, Differentiation and Alkaloid Accumulation in Cell Cultures

The relationship between alkaloid content, growth rate and differentiationwas investigated in seven solanaceous species in culture. Evidencewas obtained for an inverse correlation between the growth phaseon the one hand and alkaloid content and cell organization (aggregation)on the other. Linear phase and stationary phase cells of Daturainnoxia Mill, incorporated DL-(1_–14C)ornithine-HCl differentiallyinto protein and alkaloids with proportionately more label foundin alkaloids in the stationary phase cells. On transferringcallus of D. innoxia from high auxin medium (containing 10–6M 2, 4–D) to low auxin medium (containing 10–6 M,2, 4–D), the alkaloid content dropped concomitant witha decrease in chlorophyll content and an increase in growthrate and friability. The nature of the interrelationships betweengrowth, differentiation and accumulation are considered. Key words: Tropane alkaloids, Accumulation, Growth, Differentiation     

Studies on the Chemical Regulation of Alkaloid Biosynthesis in Tobacco Plants

Effects of helminthosporol and helminthosporic acid (H-acid) on nicotine biosynthesis in Nicotiana were examined. By the addition of these compounds into nutrient solution, nicotine formation in decapitated and intact plants of Nicotiana tabacum was decreased. In sterile culture of excised roots of Nicotiana rustica, H-acid reduced the yield of nicotine exerting no effect on growth rate. Incorporation of l-glutamic acid-U-¹⁴C and dl-ornithine-2-¹⁴C into nicotine in the roots was decreased by the addition of H-acid, and that of nicotinic acid-³H(G) was not influenced. Since H-acid did not enhance destruction of nicotine-³H exogenously added, it is probable that the decrease of nicotine yield by the acid in the root culture is due to reduction in the production rate of the alkaloid.     

Effect of Light on Alkaloid Accumulation in Cell Cultures of Nicotiana Species

The effect of light on alkaloid accumulation in a range of cellcultures of tobacco was determined. Cell suspension culturesof Nicoriana rabacwn L. cv. Wisconsin-38 with differing degreesof photosynthetic activity, callus cultures of N. glauca Graham,root cultures of N. rustica L. and shoot cultures of N. tabacumwere used. The alkaloid content of green illuminated cultureswas greatly reduced compared with non-green cultures grown inthe dark, but decreased accumulation did not correlate withincreasing photosynthetic activity. The accumulation of allof the major alkaloids was affected, regardless of the speciesof tobacco used. Transfer of N. glauca callus from the darkinto the light caused a decrease in alkaloid accumulation, whilemoving cultures from the light into the dark resulted in anincrease in alkaloid content. In root cultures light causeda reduction in growth, which affected alkaloid synthesis. Inshoot cultures there were only traces of alkaloid detectable,regardless of whether or not cultures were illuminated. Lightappeared to cause a non-photosynthetic suppression of alkaloidaccumulation in visibly undifferentiated cultures, and thiseffect was modified in visibly differentiated cultures. Key words: Nicoriana spp, tobacco, alkaloid accumulation, cell culture     

Silicon Compounds in Xylem Exudates of Plants

An account is given of a study of silicon compounds in the xylemexudates of Equisetum hyemale and Zea mays. When fractionatedby gel filtration the compounds behaved as silicic acid andnot as an organo-silicon complex.     

Membranotropic and pro-oxidative effects of alpha-tocopherol short-chain derivatives

The development of hemolysis and methemoglobin formation under influence of vitamin E derivatives was investigated on the in vitro model of rat red blood cells. It is established, that the shortening of lateral chains of alpha-tocopherol (T), alpha-tocopheryl acetate (TAc) and alpha-tocopheryl quinone (TQ) to 6 atoms of carbon (accordingly C6-T, C6-TAc, C6-TQ) results in occurrence of hemolysis and prooxidative properties, which are not characteristic of derivatives with native length of isoprenoid side chains. Modifications of chroman ring of short-chain derivatives, in turn, bring the contribution to mechanisms of their action. So, the effect of C6-TAc is caused, basically, physical destabilization of a plasmatic membrane, in C6-TQ effect prevails prooxidative mechanism, while C6-T equally shows membrane-destabilizing and prooxidative properties. Thus the derivatives T with the modified structure chroman nucleus and variability of isoprenoid side chains length show absolutely various properties and in each concrete case should be considered as independent biologically active substances irrespective of T properties.