氮素水平对冬小麦籽粒灌浆过程中淀粉合成关键酶活性的影响

小麦籽粒灌浆过程中,淀粉合成关键酶腺苷二磷酸葡萄糖焦磷酸化酶(ADPG-PPase)、可溶性淀粉合成酶(SSS)、淀粉分支酶(SBE)和束缚态淀粉合成酶(GBSS)均随着灌浆进程呈单峰曲线变化,峰值出现在花后25d;不同氮肥施用量对灌浆前期酶活性的影响较小,而在花后20d之后影响较大;随着氮肥施用量的增加,4种酶活性均呈增加趋势,但氮肥过量时酶活性下降,表明适当增加施氮量有利于淀粉合成关键酶活性的提高。     

The level of enzymes involved in the allantoin metabolism of Bacillus fastidiosus grown under different conditions

Bacillus fastidiosus was cultivated in batch and continuous culture on various carbon and nitrogen sources. The enzymes involved in allantoin degradation (allantoinase, urease, carboligase) of B. fastidiosus were hardly affected by either carbon or nitrogen source. In contrast, the enzymes involved in glycerol utilization (glycerol kinase, glycerol 3-phosphate dehydrogenase) were induced during growth on glycerol, but were not affected by the amount of allantoin present.     

Regulation of nitrogen catabolic enzymes in Bacillus spp.

The levels of the inducible nitrogen catabolic enzymes arginase (L-arginine amidinohydrolase, EC 3.5.3.1) and alanine dehydrogenase (L-alanine:NAD+ oxidoreductase [deaminating], EC 1.4.1.1) from Bacillus licheniformis and histidase (L-histidine ammonia-lyase, EC 4.3.1.3) from Bacillus subtilis and the ammonia assimilatory enzymes from B. licheniformis were determined in cultures grown in the presence of different nitrogen sources. Although the levels of these enzymes were dependent upon the nitrogen source present, induction of the catabolic enzymes in response to the addition of inducer occurred even in the presence of preferred nitrogen sources. Intracellular pool sizes of ammonia, glutamate, glutamine, and alpha-ketoglutarate were measured in continuous cultures of b. licheniformis growing in the presence of different nitrogen sources. A comparison of the pool sizes of these metabolites with the ammonia assimilatory enzyme levels showed that the pools of the metabolites did not change in a manner consistent with their use as regulators of the synthesis of any of these enzymes.     

Regulation of extracellular chitinases and proteases in the entomopathogen and acaricide Metarhizium anisopliae

Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS-PAGE.     

Responses of Ribulose-1,5-Bisphosphate Carboxylase,Cytochrome f,and Sucrose Synthesis Enzymes in Rice Leaves to Leaf Nitrogen and Their Relationships to Photosynthesis

The photosynthetic gas-exchange rates and various biochemical components of photosynthesis, including ribulose-1,5-bisphosphate carboxylase (Rubisco) content, cytochrome (Cyt) f content, and the activities of two sucrose synthesis enzymes, were examined in young, fully expanded leaves of rice (Oryza sativa L.) grown hydroponically in different nitrogen concentrations. The light-saturated rate of photosynthesis at an intercellular CO2 pressure of 20 Pa (CO2-limited photosynthesis) was linearly dependent on leaf nitrogen content, but curvilinearly correlated with Rubisco content. This difference was due to a greater than proportional increase in Rubisco content relative to leaf nitrogen content and the presence of a CO2 transfer resistance between the intercellular air spaces and the carboxylation sites. CO2-limited photosynthesis was proportional to Cyt f content, one of the key components of electron transport, but was not proportional to the activities of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, the two regulatory enzymes of sucrose synthesis. Light-saturated photosynthesis above an intercellular CO2 pressure of 60 Pa (CO2-saturated photosynthesis) was curvilinearly dependent on leaf nitrogen content. This CO2-saturated photosynthesis was proportional to Cyt f content in the low- and normal-nitrogen leaves, and correlated better with the activities of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase in the high-nitrogen leaves. The increase in the activities of these two enzymes with increasing leaf nitrogen was not as great as the increase in Cyt f content. Thus, as leaf nitrogen increased, the limitation caused by the activities of sucrose synthesis enzymes came into play, which resulted in the curvilinear relationship. However, this limitation by sucrose synthesis enzymes did not affect photosynthesis under normal ambient air.     

耕作方式与施氮量对小麦-玉米复种系统玉米季土壤氮素转化及产量的影响

在黄淮砂姜黑土区冬小麦-夏玉米复种两熟种植体系中,研究了小麦季3种耕作方式(常规翻耕、旋耕和深松)结合夏玉米播前3个施氮量(120、225和330 kg·hm^-2)对玉米季主要生育时期根际土壤氮素转化微生物作用强度及酶活性、无机氮含量和产量的影响.结果表明: 旋耕方式下氨化作用强度最高,且随着施氮量的增加,土壤氮素转化微生物作用强度及酶活性增强.深松方式下根际土壤硝化、反硝化作用强度与脲酶活性明显高于常规与旋耕方式.增施氮肥可加强深松方式对土壤氮素转化的促进作用,而过量施氮虽然提高了土壤无机氮含量及玉米产量,但会对土壤氮素转化微生物作用强度及酶活性产生抑制.深松方式结合225 kg·hm^-2施氮量更有利于砂姜黑土区夏玉米土壤氮素转化,而深松方式结合330 kg·hm^-2施氮处理下产量最高.     

Cuticle-Degrading Proteases Produced by <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Their Induction in Different Media

Protease production by fourteen M. anisopliae isolates differing in geographical origin and host insect were investigated. Highest protease activity was observed during 4–8 days of culture incubation. Pr1 and Pr2 activity was investigated in various media containing different carbon and nitrogen source to evaluate the induction mechanism of these enzymes. Basal levels of Pr1 and Pr2 activity were observed in minimal medium suggesting constitutive production. Casein (1%) as an exogenous protein supplement was not able to induce significant release of Pr1 and Pr2 enzymes, whereas high levels of activity were observed in the medium containing colloidal chitin (2%) as sole carbon and nitrogen source. The pH, ammonia and oxalic acid production in in vitro conditions was also investigated and the alteration in pH for protease production was not significant in the different media used except for the medium containing casein (1%) as a supplement.     

Implication of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in the production of pectinolytic enzymes during cocoa fermentation

The role of bacilli in cocoa fermentation is not well known. Their potential of production of pectinolytic enzymes during this process was evaluated. Bacillus growth was monitored and pectinolytic strains were screened for their use of pectin as sole carbon source. Effects of cocoa fermentation parameters susceptible to influence on enzyme production were analysed. Among 98 strains isolated, 90 were positive for pectin degradation and 80% of them presented detectable pectinolytic activities in submerged fermentation. Forty-eight strains produced polygalacturonase (PG), 47 yielded pectin lyase (PL) and 23 strains produced both enzymes. Bacilli growth was not significantly affected during fermentation. PL production was favoured by galactose, lactose, glucose as sugars, and arginine, glutamine, cysteine and ammonium sulphate as nitrogen compounds. Pectin at low concentration (0.05%) and iron stimulated PL production. It was strongly repressed by galacturonic acid (1%), and negatively affected by nitrogen starvation, zinc and temperatures above 45°C. PL yield was very weak below pH 4.0 and in anaerobic conditions. PG production was weakened by sucrose and cation depletion. It was increased slightly by cysteine, ammonium nitrate and nitrogen starvation and significantly above 40°C. PG synthesis was not affected by acidic pH (3.0–6.0) or oxygen availability. As fermentation products, lactate and acetate lowered the production of both enzymes while ethanol had no effect. The high proportion of pectinolytic producers among the strains studied and analysis of factors influencing pectinolytic enzymes production, suggest that Bacillus sp. is liable to produce at least one enzyme during cocoa fermentation.     

Ecophysiological responses of Abies fabri seedlings to drought stress and nitrogen supply

Abies fabri (Mast.) Craib. (A. fabri) is an endemic and dominant species in typical subalpine dark coniferous forests distributed in mountainous regions of Western Sichuan, China. We investigated the ecophysiological responses of A. fabri seedlings to short‐term experimental drought, nitrogen supply and their interaction. Drought stress was created by excluding natural precipitation with automatically controlled plastic roof that covered the seedlings. Nitrogen fertilization was applied weekly by spraying over seedlings ammonium nitrate solution (50 kg N ha^?1 year^?1) during the growing season of 2009. The results showed that drought stress decreased leaf relative water content (RWC), whereas it caused marked increases in root mass ratio (RMR) and root/shoot mass ratio by 6.19 and 10.39%, respectively, as compared with the control. Drought stress increased malondialdehyde (MDA) content, electrolyte leakage, proline content, soluble sugars content and the activities of antioxidant enzymes, whereas nitrogen supply decreased MDA content, but enhanced activities of some antioxidant enzymes [especially peroxidase (POD)]. In the drought stressed plots, nitrogen supply increased RWC and decreased the content of MDA. The combination of drought stress and nitrogen supply also decreased the activities of antioxidant enzymes. These results indicated that the negative effects of drought stress on A. fabri seedlings might be alleviated by nitrogen supply.     

绿僵菌产海藻糖水解酶培养条件研究

丝状真菌绿僵菌能产生一系列二糖水解酶,其中包括海藻糖水解酶。这些酶在绿僵菌对昆虫的致病过程中起着重要的作用。本文研究了不同碳源、氮源对金龟子绿僵菌Metarhizium anisopliae var. acridum菌株CQMa102产生与分解昆虫血淋巴中海藻糖等二糖相关的海藻糖水解酶活性的影响。结果表明:分别以葡萄糖、麦芽糖、蔗糖、山梨醇和可溶性淀粉为碳源,金龟子绿僵菌均可产生海藻糖水解酶,但最佳碳源是可溶性淀粉,因为由其诱导产生的海藻糖水解酶具有最高的总活性和比活性以及更多的同工酶,山梨醇次之。硝态氮(NaNO₃)作为唯一氮源时,几乎检测不出海藻糖水解酶活性,而铵态氮((NH₄)₂SO₄)或NaNO₃和有机氮(蛋白胨和酵母浸膏)混合氮源作氮源时,海藻糖水解酶活性都很高。在绿僵菌菌丝提取液和滤液的海藻糖水解酶活性比较中发现:CQMa102在多数碳源的培养基中产生的海藻糖水解酶主要分泌到培养基中,仅有少数结合在细胞壁上。     

Influence of nitrogen and phosphorus fertilizers on ammonia-assimilating enzymes of Azolla

Summary A field experiment was conducted and studied the effect of nitrogen and phosphorus on ammonia assimilating enzymes of Azolla. Nitrogen and phosphorus at 30 and 60 kg/ha respectively were tested andAzolla pinnata was inoculated at 200 g/m². The Azolla samples were drawn on 24th hr, 7th day and 14th day and the ammonia assimilating enzymes glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamine dehydrogenase (GDH) were estimated. Nitrogen and phosphorus have markedly suppressed the GDH activity but fertilizer nitrogen has no significant influence in inhibiting the enzyme activity of GOGAT and GS. In general phosphorus application also has stimulated the GS activity significantly during the first sampling period of 24th hour.     

Activities of chitinolytic enzymes during primary and secondary colonization of wood by basidiomycetous fungi

The nitrogen (N) content of wood is usually suboptimal for fungal colonization. During decomposition of wood, an increasing fraction of the N becomes incorporated into fungal mycelium. Between 5 and 50% of the N in wood-degrading mycelium may be incorporated into chitin. Chitinolytic enzymes render this N available for re-utilization. Here, the activities of chitinolytic enzymes produced by wood-rotting fungi during degradation of spruce (Picea abies) wood were quantified in situ using fluorogenic 4-methylumbelliferyl substrates. A new method was developed that enables spatial quantification of enzyme activities on solid surfaces. All of the three tested fungi produced endochitinases, chitobiosidases and N-acetylhexosaminidases during colonization of wood. N-acetylhexosaminidase activity, and in some cases also chitobiosidase and endochitinase activities, were higher during secondary overgrowth of another fungus than during primary colonization of noncolonized wood. The results suggest that wood-degrading fungi degrade their own cell walls as well as the hyphae of earlier colonizers. Recycling of cell wall material within single mycelia and between fungal individuals during succession may lead to retention of N within woody debris.     

Over-production of hydantoinase and N-carbamoylamino acid amidohydrolase enzymes by regulatory mutants of Agrobacterium tumefaciens

While the hydantoin-hydrolysing enzymes from Agrobacterium strains are used as biocatalysts in the commercial production of D-p-hydroxyphenylglycine, they are now mostly produced in heterologous hosts such as Escherichia coli. This is due to the fact that the activity of these enzymes in the native strains is tightly regulated by growth conditions. Hydantoinase and N-carbamoylamino acid amidohydrolase (NCAAH) activities are induced when cells are grown in the presence of hydantoin or an hydantoin analogue, and in complete medium, enzyme activity can be detected only in early stationary growth phase. In this study, the ability of Agrobacterium tumefaciens RU-OR cells to produce active enzymes was found to be dependent upon the choice of nitrogen source and the presence of inducer, 2-thiouracil, in the growth medium. Growth with (NH4)2SO4 as the nitrogen source repressed the production of both enzymes (nitrogen repression) and also resulted in a rapid, but reversible loss of hydantoinase activity in induced cells (ammonia shock). Mutant strains with inducer-independent production of the enzymes and/or altered response to nitrogen control were isolated. Of greatest importance for industrial application was strain RU-ORPN1F9, in which hydantoinase and NCAAH enzyme activity was inducer-independent and no longer sensitive to nitrogen repression or ammonia shock. Such mutants offer the potential for native enzyme production levels equivalent to those achieved by current heterologous expression systems.     

Regulation of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium.

The regulation of three Salmonella typhimurium phosphatases in reponse to different nutritional limitations has been studied. Two enzymes, an acid hexose phosphatase (EC 3.1.3.2) and a cyclic phosphodiesterase (EC 3.1.4.d), appear to be regulated by the cyclic adenosine 3' ,5'-monophosphate (AMP) catabolite repression system. Levels of these enzymes increased in cells grown on poor carbon sources but not in cells grown on poor nitrogen or phosphorus sources. Mutants lacking adenyl cyclase did not produce elevated levels of these enzymes in response to carbon limitation unless cyclic AMP was supplied. Mutants lacking the cyclic AMP receptor protein did not produce elevated levels of these enzymes in response to carbon limitation regardless of the presence of cyclic AMP. Since no specific induction of either enzyme could be demonstrated, these enzymes appear to be controlled solely by the cyclic AMP system. Nonspecific acid phsphatase activity (EC 3.1.3.2) increased in response to carbon, nitrogen, phosphorus, or sulfur limitation. The extent of the increase depended on growth rate, with slower growth rates favoring greater increases, and on the type of limitation. Limitation for either carbon or phosphorus resulted in maximum increases, whereas severe limitation of Mg2+ caused only a slight increase. The increase in nonspecific acid phosphatase during carbon limitation was apparently not mediated by the catabolite repression system since mutants lacking adenyl cyclase or the cyclic AMP receptor protein still produced elevated levels of this enzyme during carbon starvation. Nor did the increase during phosphorus limitation appear to be mediated by the alkaline phosphatase regulatory system. A strain of Salmonella bearing a chromosomal mutation, which caused constitutive production of alkaline phosphatase (introduced by an episome from Escherichia coli), did not have constitutive levels of nonspecific acid phosphatase.     

Amino-acid enzyme activities in liver and kidney of developing rats

The amino-acid enzymes (aspartate-, alanine- and tyrosine transaminases, serine dehydratase, glutamate dehydrogenase, glutamine synthetase, adenylate deaminase and arginase) activities in the liver and kidney of developing rats (days 19 and 21 after conception and 1, 5, 10, 20 and 30 after birth) compared with adults were determined in crude homogenates. Most enzymes attained the adult levels early after birth or at weaning, showing a marked trend towards amino-acid nitrogen conservation during late foetal and specially during the neonatal period, increasing their activity during lactation. It is postulated that these changes are closely related to availability of low grade protein in diet as well as to maturation of amino-acid homeostasis maintenance for growth.     

氮肥形态对冬小麦根际土壤氮素生理群活性及无机氮含量的影响

采用大田盆栽方法研究了硝态氮肥、铵态氮肥、酰胺态氮肥3种氮肥形态对冬小麦品种豫麦50生育中后期(拔节期、开花期、花后14 d、花后28 d)根际土壤氮转化相关微生物活性、酶活性和根际土壤NH+4离子、NO-3离子含量的影响。结果表明:随着生育期的推进,除脲酶外,氨化细菌、硝化细菌、亚硝化细菌、反硝化细菌和蛋白酶活性变化的均为"倒V"型变化特征,以花后14 d活性最强;而脲酶活性在拔节期最强,并且其活性远大于其它微生物及酶。氮肥形态对根际土壤氮素生理群及无机氮的影响不同。酰胺态氮肥促进了根际氨化细菌、反硝化细菌、脲酶、蛋白酶的活性,而硝化细菌、亚硝化细菌在硝态氮肥条件下活性较强。除拔节期外,土壤中NH+4离子在铵态氮肥处理下含量较高,NO-3离子在酰氨态氮肥处理下含量较高。因此,酰胺态氮能够促进小麦根际土壤有机氮的分解,硝态氮肥可以促进土壤中氨的转化,以利于小麦根系的吸收与利用。氮肥形态主要是通过影响土壤中氮素生理类群及酶的活性,从而影响土壤中无机氮的含量。     

Regulation of Extracellular Chitinases and Proteases in the Entomopathogen and Acaricide <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS–PAGE. Received: 8 May 2002 / Accepted: 8 June 2002     

Induction and repression of arginase and ornithine transaminase in baker's yeast

The arginase and the ornithine transaminase of baker's yeast are induced byl-arginine. Both enzymes have been shown to be repressed by nitrogen compounds. This is evidenced primarily by the decrease in specific enzyme activities caused by the addition of readily assimilable nitrogen compounds to a yeast culture with arginine, secondly by the derepression of both enzymes during nitrogen starvation of the yeast grown in various arginine-free media. This derepression equals both in rate and in amount the enzyme synthesis during the adaptation of the yeast to a medium withl-arginine as the sole nitrogen source. It is inhibited by various assimilable and non-assimilable amino acids. The derepression is the result of the nitrogen deficiency itself, since during the starvation of the yeast for sulphate, phosphate or magnesium, neither of the two enzymes is derepressed, and since it is independent of the nature of the carbon source in the nitrogen starvation medium, provided the latter is immediately assimilable.The enzymes are not subject to catabolite repression by glucose metabolites.It is concluded that the synthesis of arginase and ornithine transaminase in yeast is regulated by induction and repression. Arginine induces the enzymes; they are repressed by nitrogen compounds, probably in cooperation with one or more vitamins.Thanks are due to Professor E. G. Mulder for his frequent encouragement, to the Heineken's Brouwerij, Rotterdam and to the Landbouwhogeschoolfonds for research grants, and to Miss H. P. M. Klinkers, to Mr. P. J. Buysman and to Mr. G. J. K. Pesch for their skilful technical assistance.     

植物氮代谢及其环境调节研究进展

氮代谢是植物的基本生理过程之一,也是参与地球化学循环的重要组成部分,植物氮素同化的主要途径是经过硝酸盐还原为铵后直接参与氨基酸的合成与转化,期间硝酸还原酶(NR)、谷氨酰胺合成酶(GS)、谷氨酰胺合酶(GOGAT)、天冬酰胺转氨酶(AspAT)等关键酶参与了催化和调节,以氨基酸为主要底物在细胞中合成蛋白质,再经过对蛋白质的修饰、分类、转运及储存等,成为植物有机体的组成部分,同时与植物的碳代谢等协调统一,共同成为植物生命活动的基本过程,文中概述了植物氮素同化的途径、几种关键酶的特性和调控机制,简述了氮素代谢的信号传导、植物细胞蛋白质的形成、转运、储存和降解过程,基于水分胁迫等关键生态因子对氮代谢的影响及其调节机制的评述,强调了未来需加强研究的7个方面。