Gabaculine as a tool to investigate the polyamine biosynthesis pathway in habituated callus of Beta vulgaris (L.)

The polyamine content of a habituated callus of Beta vulgaris (L.) is strongly diminished after treatment with gabaculine, a potent inhibitor of ornithine aminotransferase. The inhibitory effect of gabaculine is reversed if ornithine is supplied. This result may indicate that proline catabolism provides ornithine for polyamine synthesis.     

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H₂O₂ detoxifying systems.Abbreviations 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - H fully habituated callus - HK hexokinase - HMP hexoses monophosphate - HPI hexose phosphate isomerase - N normal callus - PFK phosphofructokinase     

Polyamine metabolism and ethylene biosynthesis in normal and habituated sugar beet callus

The optimal assay conditions and the trend with time in culture (28 days) of arginine decarboxylase (ADE; EC 4.1.1.19), omithine decarboxylase (ODC; EC 4.1.1.17) and diamine oxidase (DAO; EC 1.4.3.6) activities in habituated (H) and normal (N) auxin- and cytokinin-requiring sugar beet callus were compared. Although the response to variations in buffer pH and EDTA and pyridoxal phosphate (PLP) concentrations varied for ADC and ODC activities between the two callus types, pH 8.3, 50 μ M PLP and 5 m M EDTA were generally optimal or near-optimal for both H and N callus. In most cases the addition of ornithine or arginine in the ADC and ODC assays, respectively, given to block the interconversion between the two substrates, resulted in lower ¹⁴CO₂ recovery. DAO activity was very differently affected in H and N callus by the presence of polyvinylpyrrolidone in the extration buffer. However, in both cases, this activity increased with time in culure. ADC activity was always predominant in both cell lines and always higher in N callus. In the latter, ADC activity rose sharply between days 14 and 21 and then leveled off while in H callus it incresed steadily from day 14 onwards. ODC activity was also higher in N callus and peaked sharply on day 21 while in H callus it was not detectable in the second half of the culture period. In both cell lines this activity was low or nil on day 28. 3,4-[¹⁴C]-methionine incorporation into ethylene and polyamines was also compared in N and H callus. In the latter, ethylene synthesis was lower and [¹⁴C]-spermidine formation higher than in N callus. This is in accord with the significantly higher spermidine titres found in H callus.     

Polyamine levels in relation to growth and NaCl concentration in normal and habituated sugarbeet callus cultures

Abstract. The concentrations of putrescine, spermidine and spermine, the only polyamines detectable in normal and habituated calli of Beta vulgaris L. ssp. altissima , were much higher in the habituated callus than the normal callus, irrespective of experimental conditions. These results suggest that, in normal (tolerant to NaCl) and habituated (sensitive to NaCl) calli, there exists a competition for the common precursor of ethylene and polyamine biosynthesis viz. S-adenosylmethionine. A disequilibrium favouring the synthesis of putrescine and spermidine in the habituated callus might be linked to structural deterioration of the cell membrane following extended culture or severe osmotic stress (68 mol m⁻¹ NaCl). The maintenance of membrane integrity by the normal callus coincides with ethylene production at the expense of polyamine synthesis. In contrast to the habituated callus, the salinity tolerance of the normal callus is accompanied by the accumulation of proline under hypersaline conditions (274mol m⁻³). The important osmoregulatory role played by quaternary ammonium compounds in the-aerial parts of Chenopodiaceae, especially the sugarbeet, is not observed in the calli, these compounds being found in very low concentrations in saline conditions.     

Abnormal growth of habituated sugarbeet callus and cell suspensions

Summary Habituated sugarbeet callus and cell suspension cultures derived therefrom, compared to hormone-dependent normal cultures, exhibit a shorter linear growth phase, although they divide actively. Microscopic observations indicate deficiencies in cell expansion and absence of cell differentiation. Cell expansion apparently is interrupted by a cell “budding” process. Some of the cells seem to be empty due to ballooning out of the protoplasm and the bursting of the cell membrane by defective cell wall development. A low amount of cellulose was confirmed by microspectrophotometric estimation. Such cultures exhibit all the characteristics of vitrified tissue.     

Does altered nitrogen metabolism and H2O2 accumulation explain the vitrified status of the fully habituated callus of Beta vulgaris (L.)?

The habituated callus is a vitrified tissue which has two main biochemical characteristics both leading to production of toxic forms of oxygen: first the blockage of the porphyrin pathway and a lack of H₂O₂ detoxifying enzymes (catalase and peroxidases); secondly a deviation of the nitrogen metabolism induced by NH₃ accumulation. Ammonia detoxification is ensured by increased glutamate dehydrogenase activity and accumulation of both proline and polyamines. A putative linkage between proline synthesis and the HMP pathway, as proposed for animal proliferating tissues (Phang 1985), might explain a high purine biosynthesis and cytokinin autonomy.Abbreviations FFA free fatty acids - 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - GLU glutamate - GDH glutamate dehydrogenase - GR glutathion reductase - H habituated callus - HMP hexoses-monophosphate - IAA indolyl-acetic acid - LOX lipoxygenase - MDA malondialdehyde - N normal callus - OAT ornithine aminotransferase - ORN ornithine - PAs polyamines - P₅C pyrroline-5-carboxylate - P₅CR pyrroline-5-carboxylate reductase - PP-ribose-P phosphoribosyl pyrophosphate - SOD superoxide dismutase     

Differential growth dependency of normal and habituated sugarbeet cell lines upon endogenous ethylene production and exogenous ethylene application

A fully habituated (auxin‐ and cytokinin‐independent) nonorganogenic (HNO) sugarbeet ( Beta vulgaris ) callus produces very little ethylene as compared with a normal (N) hormone‐requiring callus of the same strain. Both callus types react by growth changes to application of inhibitors of ethylene biosynthesis and ethylene action, of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) as the immediate precursor of ethylene, to transfer from light to darkness, and also to application of exogenous ethylene or an ethylene trapper. This indicates their growth dependency upon their endogenously biosynthesized ethylene and also their sensitivity to exogenous gas. However, the sensitivity was generally higher for the HNO callus producing naturally less ethylene. The weaker reaction of the HNO callus to the exogenous ethylene was attributed to its hyperhydric status (a water layer surrounding the cells). Because low ethylene production appears as a general characteristic of habituated cell lines, the causal and/or consequential relationships of this low ethylene production with other characteristics of habituated tissues (absence of exogenous hormones in the culture media, deficiency of cell differentiation, accumulation of polyamines in neoplastic tissues) are discussed.     

Effect of Azotobacter strains on sugar beet callus proliferation and nitrogen metabolism enzymes

The effect of five Azotobacter chroococcum strains and nitrogen content in nutrient media on callus growth of two Beta vulgaris L. cultivars were investigated, as well as the activity of nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in inoculated callus tissue. On medium with full nitrogen content (1 N) the inoculation with A. chroococcum strain A2 resulted in the highest calli mass, while strains A8 and A14 maximally increased NR activity. On media with 1/8 N the highest effect on calli growth, GS and GDH activity had the strain A8. The strain A2/1 significantly increased callus proliferation on medium without N. Asymbiotic association between sugar beet calli and Azotobacter depended on genotype/strain interaction and was realised in presence of different nitrogen levels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Water stress-induced alterations in the proline metabolism of drought-susceptible and -tolerant cassava (Manihot esculenta) cultivars

The free proline levels and activities of ornithine aminotransferase (EC 2.6.1.13) and proline oxidase (EC 1.5.2.2), two of the enzymes involved in proline metabolism were studied during the induction of water stress in a drought susceptible (M-4) and a drought tolerant (S-1315) cultivar of cassava ( Manihot esculenta Crantz). Water stress induced by polyethylene glycol (MW 6000, osmotic potential — 1.65 MPa) caused a ca 25-fold increase in proline in young excised leaves of the susceptible cultivar (M-4) while the increase was about 9-fold in the tolerant cultivar (S-1315). The activity of ornithine aminotransferase (OAT), a key enzyme involved in the biosynthesis of proline, was found to increase 3-fold in water stressed leaves of M-4 and about 2-fold in those of S-1315. The activity of proline oxidase, which is involved in the degradation of proline to pyrroline-5-carboxylate, was reduced by 50% in M-4 and nearly 25% in S-1315 on water stress. Comparison of the kinetic properties of OAT showed that the enzyme from water-stressed leaves is more stable to heat inactivation compared to that of control. These results indicate that during water stress there are alterations in the metabolism of proline in cassava, and the extent of alteration varies between drought-susceptible and -tolerant cultivars.     

Interactions of polyamines and nitrogen nutrition in plants

Biogenic amines occupy an important position among the many nitrogenous plant compounds. Polyamines are part of the overall metabolism of nitrogenous compounds, yet they do not seem to function in the 'normal' nitrogen nutrition. Rather, these widespread polycations (e. g. putrescine, spermidine and spermine) are involved in the regulation of growth and stress, probably by binding to negatively charged macromolecules. In addition, some diamines and polyamines are metabolized to yield 'secondary 'metabolites such as nicotine and other alkaloids. Previous studies have indicated that the ratio of nitrate to ammonium nutrition affects polyamine biosynthesis and content in intact plants. Thus, an increase in putrescine accumulation was found under conditions of excess ammonium ions, relative to nitrate. Modifications of nitrogen sources in the culture medium of tobacco cell suspensions (depletion of ammonium nitrate, or potassium nitrate, or both) resulted in marked changes in the content of cellular free polyamines. Considerable changes in the content of specific polyamines were also found with exposure to specific inhibitors of polyamine biosynthesis (difluoromethyl ornithine, difluoromethyl arginine, cyclohexylamine, methylglyoxal-bis-guanylhydrazone). However, a combination of nitrogen depletion of the medium and some inhibitors resulted in a very marked over-production of spermidine and spermine. The significance of these findings is discussed in relation to the assumption that polyamines act as a metabolic buffer, and maintain cellular pH under conditions where ammonium assimilation produces an excess of protons.     

Metabolic activities of the sugarbeet pathogens Fusarium solani (Mart.) Sacc. and Sclerotium rolfsii Sacc. under pyramin stress

Effects of different concentrations of active ingredient of the herbicide pyramin on metabolic activities of Fusarium solani and Sclerotium rolfsii were examined. High concentrations of this herbicide (1000 and 2000 g mL^-1 for F. solani and 100 and 200 g mL^-1 for S. rolfsii) had inhibitory effects on the metabolic activities of both fungi. These were demonstrated by significant decreases in growth, and increases in rates of CO₂ evolved, O₂ consumed and keto acids produced. These were accompanied by increased rates of sugar, nitrate and inorganic phosphorus absorption as well as lowered rates of synthesis of carbohydrates and insoluble nitrogenous (including protein) and phosphorus (including RNA-P and DNA-P) compounds. In addition, rates of excretion of both nitrogen and phosphorus fractions by the mycelial mats were increased.A concentration of 25 g mL^-1 exerted little or no effect on the metabolic activities of these fungi, although S. rolfsii was somewhat sensitive to this concentration.     

PII,the key regulator of nitrogen metabolism in the cyanobacteria

PII proteins are a protein family important to signal transduction in bacteria and plants. PII plays a critical role in regulation of carbon and nitrogen metabolism in cyanobacteria. Through conformation change and covalent modification, which are regulated by 2-oxoglutarate, PII interacts with different target proteins in response to changes of cellular energy status and carbon and nitrogen sources in cyanobacteria and regulates cellular metabolism. This article reports recent progress in PII research in cyanobacteria and discusses the mechanism of PII regulation of cellular metabolism.     

PII,the key regulator of nitrogen metabolism in the cyanobacteria

PII proteins are a protein family important to signal transduction in bacteria and plants. PII plays a critical role in regulation of carbon and nitrogen metabolism in cyanobacteria. Through conformation change and covalent modification, which are regulated by 2-oxoglutarate, PII interacts with different target proteins in response to changes of cellular energy status and carbon and nitrogen sources in cyanobacteria and regulates cellular metabolism. This article reports recent progress in PII research in cyanobacteria and discusses the mechanism of PII regulation of cellular metabolism .     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( BvcycII ).     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( Bvcycll ).     

Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment

In rape leaf discs the response to osmotic stress has been found to be associated with increases in putrescine and 1,3-diaminopropane (an oxidation product of spermidine and/or spermine) and decreases in spermidine titers. In contrast, agmatine and spermine titers showed small changes while cadaverine accumulated massively. Similar results were observed in whole rape seedlings subjected to drought conditions. -DL-difluoromethylarginine (DFMA), a specific irreversible inhibitor of arginine decarboxylase, strongly inhibited polyamine accumulation in unstressed rape leaf discs, which suggested that the arginine decarboxylase pathway is constitutively involved in putrescine biosynthesis. In leaf discs treated under high osmotic stress conditions, both DFMA and DFMO (-DL-difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase) inhibited the accumulation of polyamines. Although the stressed discs treated with DFMA had a lower concentration of putrescine than those treated with DFMO, we propose that under osmotic stress the synthesis of putrescine might involve both enzymes. DFMA, but not DFMO, was also found to inhibit cadaverine formation strongly in stressed explants. The effects on polyamine biosynthesis and catabolism of cyclohexylamine, the spermidine synthase inhibitor, aminoguanidine, the diamine-oxidase inhibitor and -aminobutyric acid, a product of putrescine oxidation via diamine oxidase or spermidine oxidation via polyamine oxidase were found to depend on environmental osmotic challenges. Thus, it appears that high osmotic stress did not block spermidine biosynthesis, but induced a stimulation of spermidine oxidation. We have also demonstrated that in stressed leaf discs, exogenous ethylene, applied in the form of (2-chloroethyl) phosphonic acid or ethephon, behaves as an inhibitor of polyamine synthesis with the exception of agmatine and diaminopropane. In addition, in stressed tissues, when ethylene synthesis was inhibited by aminooxyacetic acid or aminoethoxyvinylglycine, S-adenosylmethionine utilization in polyamine synthesis was not promoted. The relationships between polyamine and ethylene biosynthesis in unstressed and stressed tissues are discussed.     

Production of somatic embryos and shoots from sugarbeet callus: Effects of abscisic acid, other growth regulators, nitrogen source, sucrose concentration and genotype

Summary Two sugarbeet (Beta vulgaris L.) genotypes, REL-1 and REL-2, were used to measure the level of somatic embryo and shoot production from hormone-autonomous callus plated under varied nutrient medium combinations of abscisic acid with the growth regulators 6-benzyladenine, 1-naphthaleneacetic acid, or 2,4-dichlorophenoxyacetic acid, with eight sole nitrogen sources, or with different sucrose concentrations. Clone REL-2 produced embryos up to 35-fold more frequently than clone REL-1. Inclusion of abscisic acid at some concentrations consistently improved embryo production in all experiments and was observed to stimulate shoot production. At some concentrations, 1-naphthaleneacetic acid as well as urea and glutamine stimulated greater embryo production over the control, but only for REL-1, for which there was greater room for improvement. Three and five percent sucrose were superior to 1, 7, and 9%. Higher initial 6-benzyladenine concentration [in the range 0, 0.1−1.0 mg/L (0.44−4.44 μM)] was associated with lower embryo production but greater shoot regeneration for both clones. REL-2 was significantly better than REL-1 in shoot regeneration. The range of embryo production was more than 35-fold between genotypes, whereas the range of physiological effects was no greater than 10-fold. REL-2 has been released to sugarbeet researchers because of its superior embryogenic and shoot regeneration abilities for application in biotechnology.     

施氮时期对高产夏玉米氮代谢关键酶活性及抗氧化特性的影响

选用玉米品种登海661和郑单958为材料,研究了高产条件下施氮时期对夏玉米产量、氮素利用率、氮代谢相关酶及抗氧化酶活性的影响.结果表明:拔节期一次性施氮不利于夏玉米产量提高和氮素积累,分次施氮且增施花粒肥显著提高了植株和籽粒的吸氮量,并提高了籽粒产量.拔节期、10叶期、花后10d按2∶4∶4施氮,登海661产量最高可达14123.0kg· hm-2;基肥、拔节期、10叶期、花后10 d按1∶2∶5∶2施氮,郑单958产量最高可达14517.1 kg· hm-2,这2种施氮方式较拔节期一次性施氮分别增产14.5％和17.5％.花前分次施氮可以显著提高开花期硝酸还原酶活性;登海661和郑单958在花后0～42 d中,施氮处理的谷氨酰胺合成酶、谷氨酸合成酶、谷氨酸脱氢酶活性分别平均提高了32.6％、47.1％、50.4％和14.5％、61.8％、25.6％,减缓了其下降趋势;超氧化物歧化酶、过氧化氢酶活性提高了22.0％、36.6％和13.4％、62.0％,丙二醛含量显著降低.在高产条件下,分次施氮且适当增加花粒肥施入比例可以提高氮代谢相关酶活性,延缓植株衰老,促进氮素吸收利用,进而提高籽粒产量.     

Fertilizer nitrogen budgets of 15N-labelled sugarbeet (Beta vulgaris) tops and Na 15NO3 dressings split-applied to winter wheat (Triticum aestivum) in microplots on a loam soil

The fate of N from sugarbeet (Beta vulgaris L.) tops returned to the soil (50 T ha^-1) in autumn 1986 before sowing winter wheat (Triticum aestivum L.), and from NaNO₃ split-applied in 3 equal dressings (at tillering, stem elongation and flag leaf stages) was studied using isotopically labelled ¹⁵N in open stainless-steel cylinders pressed into the soil.At harvest, the percentage utilization (PU) of N from sugarbeet was very low (6.66%) and negatively influenced by fertilizer N (5.59%), while that of fertilizer N was rather high (69.64%) and unchanged by addition of tops. Residual N in soil represented 25.9% of the amount applied in tops and ranged from 33% for the tillering application to 21% for the flag leaf application. N losses (mainly denitrification) from sugar beet tops amounted to 67% and were very low for mineral fertilizer (less than 5%).     

好氧反硝化细菌碳氮代谢特点及途径的研究进展

好氧反硝化作用的发现打破了反硝化只能在严格厌氧条件下进行的传统认知,为生物脱氮提供了一条新的途径,已成为近些年的研究热点。碳源可为好氧反硝化过程提供能量和电子供体,其代谢难易程度直接影响着好氧反硝化细菌的脱氮效率,因此有必要明确碳源在好氧反硝化脱氮过程中的代谢机理。基于此,本文阐述了好氧反硝化细菌的种类及其对硝态氮与亚硝态氮的代谢途径;系统分析了不同好氧反硝化细菌对碳氮源代谢的差异与代谢机理;综合分析了碳代谢差异对好氧反硝化脱氮过程的影响,并对未来的研究方向进行了展望,旨在深入理解好氧反硝化细菌同时去除碳氮的机理,为提高废水生物脱氮除碳效率提供理论依据。