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 Nitrogen Metabolism in Tobacco Plants A

The effect of age on non-protein constituents of tobacco leaves (N. tabacum L., var. Bright Yellow) has been studied. For this purpose leaves in three different stalk positions, upper, middle and lower, which represent young, mature and over-mature leaves, respectively, were harvested in the day-time and at night.

The total amino nitrogen content both in the day-time and at night decreases from the upper leaf position downwards. As for the individual groups, the content of both upper and middle leaves increases in the day-time and that of the lower ones increases at night.

In general, the content of the individual amino acids is high in the upper leaves and low in the lower ones. Proline and γ-aminobutyric acid, as a ratio of the total amino acid content, show a marked difference with position, in other words with age of the leaves.

The levels of proline decrease very sharply from the upper leaf position downwards and that of γ-aminobutyric acid exhibits an opposite trend in both samples at night and in the day-time. These trends are very prominent in the case of the midribs.

The contents of other amino acids, regardless of position, show similar trends with time to those reported in the previous paper₁₎, and the aspartic acid content increases at night.     

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 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.     

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.     

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.     

Effects of Irradiance-Sulphur Interactions on Enzymes of Carbon,Nitrogen, and Sulphur Metabolism in Maize Plants

The effect of sulphur deprivation and irradiance (180 and 750 µmol m^–2 s^–1) on plant growth and enzyme activities of carbon, nitrogen, and sulphur metabolism were studied in maize (Zea mays L. Pioneer cv. Latina) plants over a 15-d-period of growth. Increase in irradiance resulted in an enhancement of several enzyme activities and generally accelerated the development of S deficiency. ATP sulphurylase (ATPs; EC 2.7.7.4) and o-acetylserine sulphydrylase (OASs; EC 4.2.99.8) showed a particular and different pattern as both enzymes exhibited maximum activity after 10 d from the beginning of deprivation period. Hence in maize leaves the enzymes of C, N, and S metabolism were differently regulated during the leaf development by irradiance and sulphur starvation.     

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。     

Studies on Vitamin B6 Metabolism in Microorganisms

Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

An enzyme, pyridoxamine 5′-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5′-phosphate transaminase did not contain pyridoxal 5′-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5′-phosphate.

The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio and Flavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.     

Studies on Vitamin B6 Metabolism in Microorganisms

Oxidation of pyridoxine-P and pyridoxamine-P to pyridoxal-P, inhibition and reactivation of the oxidases were investigated, using the Alcaligenes faecalis oxidase and the Azotobacter agilis oxidase catalyzing. Zone electrophoretic experiments indicated that the oxidases obtained from Alcaligenes faecalis and Azotobacter agilis moved to cathode and anode, respectively, under the same conditions. The oxidation-reduction potential of the both oxidase was found to be about ?50 mV. The oxidation of both pyridoxine-P and pyridoxamine-P was strongly inhibited by pyridoxal-P, pyridoxal, pyridine-4-aldehyde and 4-pyridoxic acid phosphate. This inhibition was markedly decreased by Tris-HCl buffer, and other amino compounds that form Schiff’s base of pyridoxal-P.

An enzyme “pyridoxamine-P transaminase” which catalyzed the transamination between pyridoxamine-P and α-ketoglutaric acid was found in certain anaerobic bacteria, such as Clostridium acetobutylicum, Cl. kainantoi, Cl. kaneboi and Cl. butyricum. The pyridoxamine-P transaminase in the cell-free extract of Cl. kainantoi was purified and some properties were investigated. α-Ketoglutaric acid appeared to be the dominant amino acceptor. Pyridoxamine-P was found to be active as amino donor, but other amino compounds were inert. Since the results were inconclusive, the possibility of vitamin B₆-enzyme of pyridoxamine-P transaminase was not shown by the inhibitor studies. Physiological role of the pyridoxamine-P transaminase was discussed in the relation to vitamin B₆ metabolism in anaerobic bacteria.     

Studies on Vitamin B6 Metabolism in Microorganisms

A large number of bacteria were searched for the activity of the synthesis of pyridoxine 5′-phosphate by the transphosphorylation between pyridoxine and p-nitrophenyl phosphate. Several properties of the transphosphorylation by the partially purified enzyme prepared from one of the isolated bacteria, Escherichia freundii K–1, were investigated accompanying with phosphatase activity. The behavior of the phosphotransferase and phosphatase activities in various reaction conditions were almost parallel. It was pointed out that the transphosphorylation might be catalyzed by the function of acid phosphatase. The phosphoryl donor specificity for the enzyme system was found to be broad.

The enzyme which catalyzed the transphosphorylation of pyridoxine accompanying with the hydrolyzation of phosphoryl donor substrates was purified and crystallized from the cell free extract of Escherichia freundii K–1. The purification procedures involved heat treatment, ammonium sulfate fractionation and DEAE-cellulose, hydroxylapatite, and CM-sephadex column chromatographies. The crystalline enzyme showed the sedimentation coefficient of 7.5 S and the diffusion coefficient of 6.15 × 10^?7 cm²/sec. The molecular weight was calculated to be 120,000. Several properties of the purified enzyme were also investigated. It was recognized that the transphosphorylation of pyridoxine might be catalyzed by the action of acid phosphatase.     

Studies on Vitamin B6 Metabolism in Microorganisms

Pyridoxine-P and pyridoxamine-P oxidase in the extract of Alcaligenes faecalis was purified and some properties of the enzyme were investigated. Several lines of evidence indicated that both pyridoxine-P oxidation and pyridoxamine-P oxidative deamination were catalyzed with a single enzyme. The enzyme is a flavoprotein, and the treatment of the enzyme with acid ammonium sulfate resolved the enzyme into apo- and coenzyme. Flavin mononucleotide reactivated the apoenzyme for the oxidation of both substrates. Physiological role of the pyridoxine-P and pyridoxamine-P oxidase was suggested in relation to the transformation of vitamin B₆ in microorganisms.     

Studies on Vitamin B6 Metabolism in Microorganisms

A new phosphotransferring reaction which phosphorylated pyridoxine through the phosphoryl group transfer from p-nitrophenylphosphate was found, and the distribution of the reaction in several microorganisms was investigated. The transferring activity was widely distributed in various kinds of microorganisms, especially in fungi belonging to genus such as Aspergillus. The phosphorylated product was isolated from the reaction mixture with the dried cells of Aspergillus flavus and identified as pyridoxine 5′-phosphate.