Nickel-induced depression of nitrogen assimilation in wheat roots

The influence of 50 and 100 μM Ni on the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) was studied in the wheat roots. Root fresh weight, tissue Ni, nitrate, ammonium, glutamate and protein concentrations were also determined. Exposure to Ni resulted in a marked reduction in fresh weight of the roots accompanied by a rapid accumulation of Ni in these organs. Both nitrate and ammonium contents in the root tissue were considerably enhanced by Ni stress. While protein content was not significantly influenced by Ni application, glutamate concentration was slightly reduced on the first day after treatment with the higher Ni dose. Treatment of the wheat seedlings with 100 μM Ni led to a decrease in NR activity; however, it did not alter the activation state of this enzyme. Decline in NiR activity observed after application of 100 μM Ni was more pronounced than that in NR. The activities of GS and NADH-GOGAT also showed substantial decreases in response to Ni stress with the latter being more susceptible to this metal. Starting from the fourth day, both aminating and deaminating GDH activities in the roots of the seedlings supplemented with Ni were lower in comparison to the control. While the activity of AspAT remained unaltered after Ni application that of AlaAT showed a considerable enhancement. The results indicate that exposure of the wheat seedlings to Ni resulted in a general depression of nitrogen assimilation in the roots. Increase in the glutamate-producing activity of AlaAT may suggest its involvement in supplying the wheat roots with this amino acid under Ni stress.     

Hypoxically inducible barley alanine aminotransferase: cDNA cloning and expression analysis

A 1.75 kb cDNA containing the entire coding sequence of the hypoxically inducible alanine aminotransferase (AlaAT) from barley roots was isolated and sequenced. This clone has an open reading frame of 1446 bp, and a deduced amino acid sequence of 482 residues, giving an estimated protein molecular mass of 52 885 Da. RNA blot analysis of barley root tissue showed a 4-fold increase of a single AlaAT-2 mRNA band after 12–24 hours of hypoxic stress, followed by a decrease in message levels after 48 h of hypoxic conditions. AlaAT-2 protein concentration increased in a similar pattern to AlaAT activity in root tissue, to almost 6-fold the aerobic level after 96 h of hypoxic stress. AlaAT-2 activity increased more than 2-fold in roots of Panicum miliaceum exposed to hypoxia, and is the same isoform as the light inducible AlaAT in P. miliaceum leaves. The unique expression patterns of AlaAT-2 in root and leaf tissue upon exposure to different environmental stimuli is also discussed.     

根际低氧胁迫对黄瓜幼苗根系呼吸代谢的影响

采用营养液栽培方法,研究了低氧胁迫对两个耐低氧能力不同的黄瓜品种根系呼吸代谢的影响.结果表明：低氧胁迫下,两个黄瓜品种根系三羧酸循环显著受阻,无氧呼吸代谢被促进.与耐低氧能力较弱的中农8号相比,耐低氧能力较强的绿霸春4号根系琥珀酸脱氢酶和异柠檬酸脱氢酶活性的降低幅度较小,乳酸脱氢酶活性、乳酸和丙酮酸含量的增加幅度较小,而丙酮酸脱羧酶、乙醇脱氢酶活性及乙醇、丙氨酸含量的增加幅度较大;低氧胁迫8 d时,与相应对照相比,绿霸春4号根系乙醇脱氢酶活性及乙醇和丙氨酸含量分别增加了409.30%、112.13%和30.64%,中农8号根系分别增加了110.42%、31.84%和4.78%,这是两个黄瓜品种耐低氧能力差异的主要生理原因.两品种幼苗根系丙氨酸氨基转移酶活性和乙醛含量没有显著差异.表明低氧胁迫下黄瓜根系乙醇发酵代谢途径的增强和丙氨酸的积累有利于防御低氧伤害.     

ACandida maltosa mutant defective in alanine aminotransferase: isolation andl-alanine assimilation

Candida maltosa JCM1504 can grow well onl-alanine as a sole carbon and nitrogen source. We found that the activities of alanine aminotransferase (AlaAT) and NAD-dependent glutamate dehydrogenase were remarkably induced when glucose-grown cells were transferred to medium containingl-alanine. This suggested thatC. maltosa has an induciblel-alanine degradation system including the above two enzymes. To assess whether AlaAT is essential for the first step ofl-alanine degradation, we isolated mutant N-07, which was unable to usel-alanine as a nitrogen source, from the wild strain. Mutant N-07 was very similar to the wild strain in terms of growth on pyruvate and on various amino acids other thanl-alanine, suggesting that N-07 lacked onlyl-alanine-assimilating ability. The AlaAT activity in the cell extract of N-07 was very low and was not induced byl-alanine, whereas the NAD-dependent glutamate dehydrogenase activity was the same as that of the wild strain and was inducible. Western blots with antibody raised against purified AlaAT fromC. maltosa indicated that no AlaAT protein was expressed in the mutant N-07. The low level of AlaAT activity described above was possibly due to the pyruvate-forming activity of other enzymes under the assay conditions. From these results, we concluded that AlaAT is an indispensable key enzyme forl-alanine assimilation inC. maltosa.     

Effect of ammonium nutrition on the activity of nitrate reductase in the roots of apple seedlings

Active extracts of nitrate reductase were prepared from theroots of apple seedlings c.v. Granny Smith which were grownin nutrient solution under controlled enviromental conditions.The nutrient solutions contained various ratios of nitrate andammonium ions but all the treatments contained a total of 112ppm nitrogen. Maximum nitrate reductase activity in the roots was obtainedwhen plants were supplied with nitrate as the sole source ofnitrogen. Roots grown in solution containing only ammonium nitrogenhad little or no activity. When plants were supplied with bothforms of nitrogen in the nutrient solution, the presence ofammonium ions markedly lowered the activity of nitrate reductasein the roots. Plants supplied with 98 ppm nitrate nitrogen plus14 ppm ammonium nitrogen had activities only half those of plantsgrown in nitrate alone. Plants supplied with equal amounts ofammonium and nitrate nitrogen had activities less than one sixththose of plants grown in nitrate alone. (Received June 3, 1972; )     

Purification and characterization of an anaerobically induced alanine aminotransferase from barley roots

Alanine aminotransferase (AlaAT, EC 2.6.1.2) is an enzyme that is induced under anaerobic conditions in cereal roots. In barley (Hordeum vulgare L.) roots, there are a number of isoforms of AlaAT. We have identified the anaerobically induced isoform and have purified it to homogeneity. The isolation procedure involved a two-step ammonium sulfate precipitation, gel filtration, ion-exchange chromatography, and chromatofocusing. The enzyme was purified approximately 350-fold to a specific activity of 2231 units/milligram protein. The apparent molecular masses of the native and sodium dodecyl sulfate-denatured AlaAT proteins are 97 and 50 kilodaltons, respectively, indicating that the native enzyme is probably a homodimer. AlaAT has a number of interesting characteristics when compared with other plant aminotransferases. AlaAT does not require the presence of pyridoxyl-5-phosphate to retain its activity, and it appears to be very specific in the reactions that it will catalyze.     

Utilization of 15NO3 − by nodulated soybean plants under conditions of root hypoxia

Waterlogging of soils is common in nature. The low availability of oxygen under these conditions leads to hypoxia of the root system impairing the development and productivity of the plant. The presence of nitrate under flooding conditions is regarded as being beneficial towards tolerance to this stress. However, it is not known how nodulated soybean plants, cultivated in the absence of nitrate and therefore not metabolically adapted to this compound, would respond to nitrate under root hypoxia in comparison with non-nodulated plants grown on nitrate. A study was conducted with ¹⁵N labelled nitrate supplied on waterlogging for a period of 48 h using both nodulated and non-nodulated plants of different physiological ages. Enrichment of N was found in roots and leaves with incorporation of the isotope in amino acids, although to a much smaller degree under hypoxia than normoxia. This demonstrates that nitrate is taken up under hypoxic conditions and assimilated into amino acids, although to a much lesser extent than for normoxia. The similar response obtained with nodulated and non-nodulated plants indicates the rapid metabolic adaptation of nodulated plants to the presence of nitrate under hypoxia. Enrichment of N in nodules was very much weaker with a distinct enrichment pattern of amino acids (especially asparagine) suggesting that labelling arose from a tissue source external to the nodule rather than through assimilation in the nodule itself.     

Induction of lactate dehydrogenase isozymes by oxygen deficit in barley root tissue

Lactate dehydrogenase (LDH) activity in attached roots of barley and other cereals increased up to 20-fold during several days of severe hypoxia, reaching a maximum of about 2 micromoles per minute per gram fresh weight. In barley, induction of LDH activity was significant at 2.6% O₂ and greatest at 0.06%, the lowest O₂ concentration tested. Upon return to aerobic conditions, induced LDH activity declined with an apparent half-life of 2 days. The isozyme profile of barley LDH comprised 5 bands, consistent with a tetrameric enzyme with subunits encoded by two different Ldh genes. Changes in staining intensity of the isozymes as a function of O₂ level suggested that one Ldh gene was preferentially expressed in severe hypoxia. When tracer [U-¹⁴C]glucose was supplied to induced roots under hypoxic conditions, lactate acquired label, but much less than either ethanol or alanine. Most of the [¹⁴C] lactate was secreted into the medium, whereas most other labeled anionic products were retained in the root. Neither hypoxic induction of LDH, nor lactate secretion by induced roots, is predicted from the Davies-Roberts hypothesis, which holds that lactate glycolysis ceases soon after the onset of hypoxia due to acidosis brought about by lactate accumulation in the cytoplasm. These results imply a functional significance for LDH beyond that assigned it in this hypothesis.     

A novel low molecular weight alanine aminotransferase from fasted rat liver

Alanine is the most effective precursor for gluconeogenesis among amino acids, and the initial reaction is catalyzed by alanine aminotransferase (AlaAT). Although the enzyme activity increases during fasting, this effect has not been studied extensively. The present study describes the purification and characterization of an isoform of AlaAT from rat liver under fasting. The molecular mass of the enzyme is 17.7 kD with an isoelectric point of 4.2; glutamine is the N-terminal residue. The enzyme showed narrow substrate specificity for L-alanine with Km values for alanine of 0.51 mM and for 2-oxoglutarate of 0.12 mM. The enzyme is a glycoprotein. Spectroscopic and inhibition studies showed that pyridoxal phosphate (PLP) and free -SH groups are involved in the enzymatic catalysis. PLP activated the enzyme with a Km of 0.057 mM.     

蔗糖对不同氮源培养下水稻根部氨同化相关酶活性的影响

糖、有机酸以及氨基酸影响碳-氮代谢过程中的相关酶的基因表达和活性。将蔗糖分别加入到含有相同氮素浓度的（NH4）2SO4（NH4^＋）或丙氨酸（Ala）作为氮源的营养液中培养水稻,测定幼苗根的谷氨酰胺合成酶（GS）、依赖于NADH的谷氨酸合酶（NADH-GOGAT）、磷酸烯醇式丙酮酸羧化酶（PEPC）和依赖于NADP的异柠檬酸脱氢酶（NADP-ICDH）的活性。结果显示,蔗糖诱导NH4^＋氮源中幼苗根的GS、NADH-GOGAT活性,抑制Ala氮源中幼苗根的这两种酶活性,蔗糖对PEPC和NADP-ICDH活性的影响也不同;未加蔗糖时,以Ala作为氮源的幼苗根的GS、NADH-GOGAT、PEPC和NADP-ICDH的活性明显高于以NH4^＋为氮源时的活性;生物量和蛋白质水平的变化与上述参数的变化基本一致。基于Ala碳骨架的存在,这些结果表明,碳／氮平衡是影响这些酶活性差别表达的主要原因。     

Stimulation of alanine amino transferase (AlaAT) gene expression and alanine accumulation in embryo axis of the model legume Medicago truncatula contribute to anoxia stress tolerance

The efficiency of ethanolic fermentation in anoxia tolerance under sugar-limiting conditions, as in the field is still matter of debate. Due to higher rates of glycolysis and ethanol fermentation, faster depletion of sugar stores leads to decreased survival. In the present work the hypothesis that alanine amino transferase ( AlaAT ) fermentation be involved in anoxia tolerance was explored in Medicago truncatula during germination and seedling establishment. Expression of AlaAT and two low oxygen-responsive genes, alcohol dehydrogenase ( ADH ) and lactate dehydrogenase ( LDH ) were determined by real time quantitative RT-PCR and AlaAT activity was determined by ¹⁵N-Glutamate labelling coupled to amino acids analysis by gas chromatography–mass spectrometry and HPLC. Under anoxia not only ADH and LDH levels of expression increased but also AlaAT expression increased substantially. In parallel in vivo AlaAT activity increased and resulted in an increase in alanine synthesis that accumulated as the major amino acid instead of asparigine. These findings support the hypothesis that AlaAT expression and alanine accumulation contribute efficiently to anoxia tolerance. By competing with ethanolic fermentation for pyruvate, under sugar-limiting conditions alanine synthesis saves C3 skeletons avoiding a shortage in carbon availability and limits accumulation of acetaldehyde, a toxic compound. On another hand, increase in alanine was accompanied by an increase in γ-amino butyric acid, both amino acids may intervene in cytosolic pH regulation. Finally the role of alanine in anoxia tolerance was strengthened by the fact that when alanine synthesis was impaired germination and seedling development failed under anoxia.     

Nitrate reductase activity of two leafy vegetables as affected by nickel and different nitrogen forms

The author studied the effect of different nickel concentrations (0, 0.4, 40 and 80 μM Ni) on the nitrate reductase (NR) activity of New Zealand spinach (Tetragonia expansa Murr.) and lettuce (Lactuca sativa L. cv. Justyna) plants supplied with different nitrogen forms (NO₃ ⁻–N, NH₄ ⁺–N, NH₄NO₃). A low concentration of Ni (0.4 μM) did not cause statistically significant changes of the nitrate reductase activity in lettuce plants supplied with nitrate nitrogen (NO₃ ⁻–N) or mixed (NH₄NO₃) nitrogen form, but in New Zealand spinach leaves the enzyme activity decreased and increased, respectively. The introduction of 0.4 μM Ni in the medium containing ammonium ions as a sole source of nitrogen resulted in significantly increased NR activity in lettuce roots, and did not cause statistically significant changes of the enzyme activity in New Zealand spinach plants. At a high nickel level (Ni 40 or 80 μM), a significant decrease in the NR activity was observed in New Zealand spinach plants treated with nitrate or mixed nitrogen form, but it was much more marked in leaves than in roots. An exception was lack of significant changes of the enzyme activity in spinach leaves when plants were treated with 40 μM Ni and supplied with mixed nitrogen form, which resulted in the stronger reduction of the enzyme activity in roots than in leaves. The statistically significant drop in the NR activity was recorded in the aboveground parts of nickel-stressed lettuce plants supplied with NO₃ ⁻–N or NH₄NO₃. At the same time, there were no statistically significant changes recorded in lettuce roots, except for the drop of the enzyme activity in the roots of NO₃ ⁻-fed plants grown in the nutrient solution containing 80 μM Ni. An addition of high nickel doses to the nutrient solution contained ammonium nitrogen (NH₄ ⁺–N) did not affect the NR activity in New Zealand spinach plants and caused a high increase of this enzyme in lettuce organs, especially in roots. It should be stressed that, independently of nickel dose in New Zealand spinach plants supplied with ammonium form, NR activity in roots was dramatically higher than that in leaves. Moreover, in New Zealand spinach plants treated with NH₄ ⁺–N the enzyme activity in roots was even higher than in those supplied with NO₃ ⁻–N.     

Involvement of nitrite in the nitrate-mediated modulation of fermentative metabolism and nitric oxide production of soybean roots during hypoxia

It is widely accepted that nitrate but not ammonium improves tolerance of plants to hypoxic stress, although the mechanisms related to this beneficial effect are not well understood. Recently, nitrite derived from nitrate reduction has emerged as the major substrate for the synthesis of nitric oxide (NO), an important signaling molecule in plants. Here, we analyzed the effect of different nitrogen sources (nitrate, nitrite and ammonium) on the metabolic response and NO production of soybean roots under hypoxia. Organic acid analysis showed that root segments isolated from nitrate-cultivated plants presented a lower accumulation of lactate and succinate in response to oxygen deficiency in relation to those from ammonium-cultivated plants. The more pronounced lactate accumulation by root segments of ammonium-grown plants was followed by a higher ethanol release in the medium, evidencing a more intense fermentation under oxygen deficiency than those from nitrate-grown plants. As expected, root segments from nitrate-cultivated plants produced higher amounts of nitrite and NO during hypoxia compared to ammonium cultivation. Exogenous nitrite supplied during hypoxia reduced both ethanol and lactate production and stimulated cyanide-sensitive NO emission by root segments from ammonium-cultivated plants, independent of nitrate. On the other hand, treatments with a NO donor or a NO scavenger did not affect the intensity of fermentation of soybean roots. Overall, these results indicate that nitrite participates in the nitrate-mediated modulation of the fermentative metabolism of soybean roots during oxygen deficiency. The involvement of mitochondrial reduction of nitrite to NO in this mechanism is discussed.     

Waterlogging affects nitrogen transport in the xylem of soybean

Transfer of nodulated and non-nodulated plants grown in vermiculite to hydroponic culture without soil was used to study waterlogging and nitrogen transport in the xylem of soybean. Non-aeration, aeration or aeration with nitrogen gas were used to obtain different levels of oxygen in the culture solutions. Ureides, the principal form of nitrogen transport in nodulated plants, were considerably reduced in waterlogged plants or after transfer to water-culture, especially when not aerated or aerated with nitrogen gas. Aeration of the water-culture following a period of non-aeration allowed some recovery of ureides, as did the return of plants to drained vermiculite. Although smaller changes in the total amino acid fraction were observed for the different treatments, marked changes occurred in the composition depending on the treatment imposed. A high proportion of asparagine and low glutamine characterised non-nodulated plants grown on nitrate, or nodulated plants subsequently fed nitrate. A higher level of glutamine and lower level of asparagine characterised nodulated plants dependent on nitrogen fixation. High levels of aspartic acid characterised plants transferred to water-culture with aeration, especially in N-deficient solution, while alanine and serine were very prominent in non-aerated or hypoxic water-culture. These changes also occurred in non-nodulated plants and plants kept in vermiculite in a flooded condition. Some of the changes in transport were accompanied by similar changes in the free amino acid fraction of the roots. It is suggested that an alteration in asparagine metabolism may underlie the changes in amino acid transport in the xylem associated with waterlogging.