Studies on Nitrogen Metabolism in Tobacco Plants

Δ¹-Pyrroline-5-carboxylate reductase has been considerably purified from tobacco leaves. This enzyme uses NADPH or NADH for the formation of proline, although the former is better used. This enzyme was found in washed chloroplast extract as well as in cytoplasmic fluid and utilized NADPH, formed by the photosynthetic NADP reduction, for the sythesis of proline in the light.     

Studies on the Nitrogen Metabolism in Tobacco Plants. A.

The nitrogenous compounds of tobacco saps have been studied both qualitatively and quantitatively and the following results were obtained.

(1) Nitrate nitrogen accounts for 40 to 70% of the total nitrogen and the rest is composed mostly of amino and alkaloid nitrogen.

(2) Amides and basic amino acids compose a large part of the amino and amide nitrogen. Among the amino acids and amides of the tobacco saps glutamine is the highest in the content and asparagine, lysine, leucine and serine follow glutamine.

(3) Topping procedure increased remarkably the alkaloid contents in the sap but decreased the amino acid nitrogen as compared with those of the untopped plant sap.     

Studies on Nitrogen Metabolism in Tobacco Plants,B

Nitrate reductase from the leaves of Burley 21 tobacco (Nicotiana tabacum L.) was partially purified by ammonium sulfate fractionation, protamine sulfate treatment and calcium-phosphate gel adsorption.

The enzyme has optimum pH at 7.4 and is specific for reduced diphosphopyridine nucleotide (DPNH) as the electron donor. The nitrite formed increased in proportion to the rate at which DPNH disappeared in the reaction mixtures. Addition of flavin adenine dinucleo-tide (FAD) to the assay system enhanced the activity. FAD content in the “highly purified” enzyme was also determined. The enzyme was sensitive to heavy metals and SH-group inhibitors.

Discussions are presented on the metal and the properties of the enzyme in comparison to those published on other higher plants.     

Studies on Protein Metabolism in Higher Plants

A leaf protease of tobacco whose activity was enhanced during curing was purified about 60 times with ammonium sulfate fractionation, ethanol precipitation, calcium phosphate gel treatment and Sephadex G-200 column chromatography, and some properties of the protease were examined. The purified enzyme showed the optimum pH at 5.5 and the optimum temperature at 60°C. The protease activity was stable between pH 4.5 and 5.5 at 50°G or at pH 5.5 below 40°C for 1 hr, but completely destroyed at 70°C during 1 hr. The protease activity was greatly activated by reducing agents such as cysteine, glutathione or mercaptoethanol and inhibited by p-chloromercuribenzoate, phenyl- mercuric acetate or silver ions. Metal ions except for silver ion and ethylenediamine tetraacetic acid did not affect the protease activity so far examined.     

Studies on Protein Metabolism in Higher Plants

Changes in ribulose diphosphate (RuDP) carboxylase activity and the content of fraction 1 protein, which had been elucidated to be identical with protein of RuDP carboxylase, in tobacco leaves were examined with age, comparing with change in total protein content. Fraction 1 protein was determined by an immunological precipitin method developed in this experiment. Fraction 1 protein decreased with age and the rate was similar or a little larger than those of total protein and total chlorophyll. The carboxylase activity decreased more rapidly than fraction 1 protein during the senescent process. In a plant, upper leaves showed higher values of the carboxylase activity and fraction 1 protein content than lower leaves. The specific activity, RuDP carboxylase activity per unit fraction 1 protein, in upper leaves was higher than that in lower leaves.     

Studies on the Nitrogen Metabolism in Ectomycorrhizae

Concentrations of free and bound amino acids were determined in 1) the mycorrhizal fungus Boletus variegatus Fr. 2) nonmycorrhizal root systems of aseptically grown Pinus sylvestris L. seedlings, and 3) mycorrhizal root systems of seedlings developed aseptically using the two symbionts. Arginine (total) was the major amino acid constituent in the mycelium of B. variegatus (18%–22%) during the exponential phase of growth. While 59%–86% of the available arginine was bound during the acceleration phase of growth, in the logarithmic phase 63%–75% was in the free pool. There were differences in the proportions between the individual amino acids in the bound fraction at different stages of growth suggesting production of diverse proteins. Twenty per cent of the amino acid content of uninfected P. sylvestris root systems was arginine. Infection of the root systems by the fungal symbiont did not result in an increase but a slight decrease in the free arginine content of the composite structure. Almost all other amino acids in the free pool were found in higher concentrations in the mycorrhizal root system. It is suggested that arginine synthesis in B. variegatus is repressed by the arginine available in the host. The mycorrhizal fungus possibly metabolizes the host arginine pool ultimately resulting in more efficient protein synthesis in both the partners.     

Studies on the Nitrogen Metabolism in Ectomycorrhizae

Free and bound amino acids in the mycorrhizal and nonmycorrhizal root systems of Pinus nigra Arnold and Corylus avellna L. grown under semi-natural conditions were analyzed through automatic amino acid analyzers. Tuber brumale Vitt. and T. melanosporum Vitt. were the respective fungal symbionts. Arginine and citrulline were found to accumulate in large quantities in the free pool of the uninoculated P. nigra and C. avellana root systems respectively. In the mycorrhizal root systems these substances decreased in their levels with a parallel increase in the concentrations of glutamine and asparagine. Implications of these changes are discussed with reference to ectomycorrhizae. In general the majority of the identified free amino acids were found in larger concentrations in both the types of mycorrhizal root systems. Results obtained suggested that this may not be due to proteolysis but due to increased biosynthesis. Possible interrelationships in ectomycorrhizal root systems with reference to nitrogen metabolism are presented in the form of a scheme.     

外来入侵植物的氮代谢及其土壤氮特征

研究了4种外来入侵植物(五爪金龙、南美蟛蜞菊、金腰箭和马缨丹)和1种本地植物鸡矢藤(对照)的氮代谢及其土壤氮特征.结果表明:外来人侵植物的组织硝酸还原酶活性、根际土壤NH4-N、NO3-N含量、蛋白酶活性和脲酶活性均较高,分别为鸡矢藤的1.65～4.34、1.56～2.15、1.72～3.11、1.43～3.23和1.41～3.33倍,而植物组织硝态氮含量则较低,仅为鸡矢藤的17.5%～50.6%.相关分析表明:植物组织硝酸还原酶活性与根际土壤总氮、NH4-N、NO3-N含量呈显著正相关(P<0.05),与蛋白酶活性和脲酶活性呈极显著正相关(P<0.01).这说明,外来植物入侵使土壤氮代谢加快,氮的生物有效性增强,氮同化能力提高,并且较好地将植物体氮素代谢与土壤氮素代谢协调起来.因此,较强的氮素同化能力与加速土壤氮素的转化可能是植物成功入侵的重要机制之一.     

Chemical Studies on Brown-spot Disease of Tobacco Plants

Tenuazonic acid has been isolated as a halo-inducing toxin from the culture filtrate of Alternaria longipes, a fungus causative of the tobacco brown-spot disease. Further, its occurrence in leaves of diseased plants was confirmed by a spectrometric method. The role of the acid in the host-parasite interaction was elucidated from the phytopathological standpoint.     

Effect of Light Quality on Nitrogen Metabolism of Radish Plants

The long-term action of blue or red light on nitrogen metabolism was studied in radish (Raphanus sativus L.) plants. The potential activity of nitrate reductase (NR) in vivo and its maximum activity in vitro, the content of soluble protein and free amino acids were determined in the course of the growth of a third leaf of radish plants. The effect of light quality on NR activity was found to depend significantly on the stage of leaf development. Blue light (BL) stimulated NR activity in leaves, when their areas were about 11–13% of the fully developed leaves. The efficiency of red light (RL) was significantly lower, because the maximum NR activity was observed in the leaves developed to the stage, when their areas were 38–40% of the final one. The comparative analysis of the pool of free amino acids in expanding leaves of BL- or RL-grown plants revealed significant changes in the contents of individual amino acids. Despite a higher accumulation of two amino acids in the leaves of BL-grown plants, namely, Asp (27% as compared to 13–16% in the RL-grown leaves) and Gly (5% against 2.5% in RL-grown leaves), the BL-grown leaves also demonstrated a significant decrease in Ala (10% as compared to 23% in the RL-grown leaves) and some decrease in the amounts of Ser and Gly. The content of soluble protein in a juvenile BL-grown leaf was observed to decrease gradually during leaf development. However, the protein content in the BL-grown leaf was always higher than in the RL-grown leaf of the same age. We concluded that the photoregulatory action of BL on NR activity determined the different rates of nitrogen assimilation in BL- and RL-grown plants.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 349–356.Original Russian Text Copyright © 2005 by Maevskaya, Bukhov.     

缺氮及氮素形态对烟草幼苗糖代谢的影响机理初探

为了探明缺氮及不同氮素形态对烟草幼苗糖代谢的影响机理。该试验以‘中烟100’为试材,设置正常供氮为对照(CK,NO3-∶NH4+=1∶1)、缺氮(T1)、单一铵态氮(T2)和单一硝态氮(T3)4个处理的沙培试验,检测烟株功能叶片的全氮、还原糖、总糖、蔗糖淀粉的含量及糖代谢相关酶的活性,并分析根、茎、叶等3个部位糖代谢关键基因(SUT1、INV、GBSSI、AGPase)的表达差异。结果显示:(1)烟株功能叶片的全氮含量表现为T3CKT2T1。(2)与CK前期相比,T1处理显著增加了烟株叶片的还原糖、总糖、蔗糖及淀粉含量,降低了淀粉酶的活性,但对转化酶活性影响不显著;其中烟株叶片淀粉积累表现为T1T2CKT3。(3)缺氮及氮素形态对糖转运蛋白基因(SUT1)、焦磷酸化酶基因(AGPase)和颗粒淀粉合成酶基因(GBSSI)等关键基因的表达影响显著。研究认为,缺氮及不同氮素形态下烟草幼苗氮素利用率与淀粉积累成反比,其中蔗糖转运蛋白基因(SUT1)在氮素形态影响烟叶糖代谢过程中起关键作用。     

Effect of Salinity and Potassium on the Uptake of Nitrogen and on Nitrogen Metabolism in Young Barley Plants

In a solution culture experiment with 4-week-old barley plants (variety Villa) the influence of NaCl salinization and of KCl additions on the uptake and turnover of labelled N (¹⁵NH₄¹⁵NO₃) was studied. Labelled N was applied for 24 h at the end of the growth period. Salinization impaired growth and uptake of labelled N. The incorporation of labelled N into the protein fraction, however, was improved by NaCl salinization. Additions of KCl to the nutrient solution diminished the negative effect of NaCl salinization on growth. At both NaCl salinization levels (60 and 120 mM) K additions favoured the uptake of labelled N and particularly its incorporation into the protein fraction. It is suggested that the negative influence of the NaCl stress is not primarily due to an impaired protein synthesis, but is possibly caused by a deterimental effect of Na on other metabolic processes.     

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.     

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.     

Improving the Mineral Nutrition in Grafted Watermelon Plants: Nitrogen Metabolism

Watermelon (Citrullus lanatus [Trumb.] Mansfeld cv. Early Star), was used as scion grafted onto three cultivars of pumpkin (Cucurbita pepo L. cvs. Brava, Shintoza and Kamel) used as rootstocks and ungrafted Early Star plants were used as control. The rootstocks showed a high capacity for N uptake and transport to the scion where N reduction and assimilation improved growth of the scion in grafted plants with respect to the control.     

The Effects of Drought on the Water Balance and Nitrogen Metabolism in Sand-fixing Plants

The changes in water balance and nitrogen metabolism of sandfixing plants under soil drought condition in desert area were studied. During sustained drought, the free water content was decreased and the bound water content was increased in all of the experimental plants. Nitrogen metabolism in the plants was converted by drought. The protein synthesis was inhibited, in the meantime the non-protein nitrogen was accumulated. It was found that a notable difference exists in various plants as far as accumulated non-protein nitrogen is concerned, amide nitrogen was accumulated mainly by xerophyte Hedysarium scoparium and nitrate nitrogen by mesophyte Robinia psudoacacia. The water balance of plants and the role of bound water in drought resistance are also discussed.