Poplar Bark Storage Protein and a Related Wound-Induced Gene Are Differentially Induced by Nitrogen

Poplars (Populus deltoides Bartr. ex Marsh) accumulate a 32-kD bark storage protein (BSP) in phloem parenchyma and xylem ray cells during autumn and winter. Accumulation of poplar BSP is associated with short-day (SD) photoperiods. Poplar BSP shares sequence similarity with the product of the wound-inducible poplar gene win4. The influence of nitrogen availability and photoperiod on the levels of BSP, BSP mRNA, and win4 mRNA was investigated. In long-day (LD) plants BSP, BSP mRNA, and win4 mRNA levels were correlated with the amount of NH4NO3 provided to the plant. BSP mRNA and BSP were detected only in bark, whereas win4 mRNA was detected only in leaves. In LD plants treated with NH4NO3, BSP mRNA levels were significantly greater than those of win4. In nitrogen-deficient plants exposed to SD conditions, the accumulation of BSP mRNA and BSP was delayed for 2 weeks. This delay was eliminated by further SD exposure, and after 6 weeks of SD treatment similar levels of BSP and BSP mRNA were detected in the bark of SD plants regardless of the level of NH4NO3 treatment. win4 mRNA levels declined to undetectable levels in young leaves of SD plants but increased in mature leaves. These results indicate that BSP accumulation in both LD and SD plants is influenced by nitrogen availability. Although both BSP and win4 appear to be involved in nitrogen storage, our data suggest that BSP is probably the primary protein involved in both seasonal and short-term nitrogen storage in poplar. These results also suggest that nitrogen cycling and storage in poplar could involve a two-component system. In this system the win4 gene product may modulate accumulation and mobilization of leaf nitrogen, whereas BSP is involved in seasonal and short-term nitrogen storage during periods of excess nitrogen availability.     

Influence of N and Ni supply on nitrogen metabolism and urease activity in rice (Oryza sativa L.)

Nickel is considered to be an essential micronutrient in plants because of its role in the metalloenzyme urease. In order to characterize the metabolic consequences of Ni deprivation, the significance of Ni supply for growth and N metabolism of rice plants grown with either NH4NO3 or urea as sole N source was evaluated. Growth of plants receiving NH4NO3 was not affected by the Ni status, and neither were the activities of arginase and glutamine synthetase. However, urease activity was not detectable in leaves of low-Ni plants, which in conjunction with arginase action, led to the accumulation of urea in plants grown with NH4NO3. Amino acid contents and mineral nutrient status (except Ni) were not affected by the Ni treatment.Urea-grown Ni-deprived plants showed reduced growth and accumulated large amounts of urea owing to the lack of urease activity. These plants were further characterized by low amino acid contents indicating impaired usage of the N supplied. They also exhibited reduced levels of the urea precursor arginine, which is merely attributed to the overall N economy in these plant. When urea-grown plants were supplied with 0.5 mmol m^-3 Ni in the nutrient solution, the dry weight and the amino acid N contents were increased at the expense of the urea contents, indicating efficient use of urea N in Ni-supplemented plants.A critical Ni concentration in the shoot regarding dry matter production of NH4NO3-grown plants could not be deduced, while 25 g Ni kg^-1 DW is certainly inadequate for urea-grown plants. This suggests that the Ni requirement strongly depends on the N source employed.Keywords: Amino acids, ornithine cycle, Ni supply, rice, urea, urease activity.      

Growth and nitrogen metabolism of Catasetum fimbriatum (orchidaceae) grown with different nitrogen sources

Catasetum fimbriatum is an epiphytic orchid from South America that has been used for 15 years as a model plant for metabolic and developmental studies in our laboratory. In this work, C. fimbriatum plants were aseptically grown with 6 mol m(-3) of either glutamine or inorganic nitrogen forms (NO(3)(-):NH(4)(+) ratios). The highest biomass accumulation was found in plants supplied with glutamine; no significant difference was observed in plants incubated in the presence of inorganic nitrogen sources. Nitrogen assimilation was limited in the presence NO(3)(-) as a sole nitrogen source. C. fimbriatum did not accumulate NO(3)(-) and very low rates of in vivo nitrate reductase activity were observed. Most nitrate reductase activity (70%) was detected in the 2 cm apical roots. Nitrate-treated plants exhibited relatively lower amounts of free amino-N, chlorophyll and free NH(4)(+) contents and higher soluble sugar contents than the NH(4)(+)-treated plants. While shoot glutamine synthetase activity was only slightly affected by nitrogen sources, root glutamine synthetase activity was not modified by any nitrogen form. Glutamate dehydrogenase-NADH activity in shoot tissues was not influenced by any nitrogen source. However, the glutamate dehydrogenase-NADH activity in roots was enhanced when NH(4)(+) tissue contents was augmented by increasing NH(4)(+) in the medium and by the presence of glutamine. Our results strongly suggest that organic nitrogen and NH(4)(+) are probably the most important nitrogen sources to C. fimbriatum plants.     

Ammonium and nitrate uptake by soybean during recovery from nitrogen deprivation

Soybean [Glycine max (L.) Merrill] plants that had been subjected to 15 d of nitrogen deprivation were resupplied for 10 d with 1.0 mol m-3 nitrogen provided as NO3-, NH4+, or NH4(+) + NO3- in flowing hydroponic culture. Plants in a fourth hydroponic system received 1.0 mol m-3 NO3- during both stress and resupply periods. Concentrations of soluble carbohydrates and organic acids in roots increased 210 and 370%, respectively, during stress. For the first day of resupply, however, specific uptake rates of nitrogen, determined by ion chromatography as depletion from solution, were lower for stressed than for non-stressed plants by 43% for NO3- resupply, by 32% for NH4(+) + NO3- resupply, and 86% for NH4+ resupply. When specific uptake of nitrogen for stressed plants recovered to rates for non-stressed plants at 6 to 8 d after nitrogen resupply, carbohydrates and organic acids in their roots had declined to concentrations lower than those of non-stressed plants. Recovery of nitrogen uptake capacity of roots thus does not appear to be regulated simply by the content of soluble carbon compounds within roots. Solution concentrations of NH4+ and NO3- were monitored at 62.5 min intervals during the first 3 d of resupply. Intermittent 'hourly' intervals of net influx and net efflux occurred. Rates of uptake during influx intervals were greater for the NH4(+)-resupplied than for the NO3(-)-resupplied plants. For NH4(+)-resupplied plants, however, the hourly intervals of efflux were more numerous than for NO3(-)-resupplied plants. It thus is possible that, instead of repressing NH4+ influx, increased accumulation of amino acids and NH4+ in NH4(+)-resupplied plants inhibited net uptake by stimulation of efflux on NH4+ absorbed in excess of availability of carbon skeletons for assimilation. Entry of NH4+ into root cytoplasm appeared to be less restricted than translocation of amino acids from the cytoplasm into the xylem.     

UV-B辐射对香蕉光合作用和不同氮源利用的影响

生长在NO3^--N、NH4^--N和NH4NO3－N的香蕉叶片有相近似的最大光合速率,UV－B辐射引起生长在不同氮源的香蕉叶片光合速率、表现量子产率和光肥利用效率的降低。UV－B辐射使生长在不同氮源的植株叶面积干重和叶氮含是降低。生长在NH4^--N的植株Vcmax和Jmax均较生长在其它氮源的高。UV－B辐射引起生长在NH4^-N的植株Vcmax和Jmax降低较相同处理的NO3^-－N和NH4NO3－N植株明显,表明生长在NH4^ －N的香蕉对UV－B辐射更加敏感。UV－B辐射改变植株的叶片的碳氢比和碳氮比。经过UV－B辐射处理的NH4^ －N生长植株的碳氮生长在NO3^--N和NH4NO3－N的低。UV－B辐射可能改变植株对不同氮源的吸收利用,从而引起碳氮代谢和酸碱调节的变化。UV－B辐射降低叶氮在Rubisco和生物力能学组分的分配系数,可能使这些组分合成减少,使叶片光调节的变化。UV－B辐射降低叶氮在Rubisco和生物力能学组分的分配系数,可能使这些组分合成减少,使叶片光合速率下降。结果表明,生长在不同氮源的香蕉植树对UV－B辐射有不同响应,NH4^ -N有利于主要光合参数增高,但其对UV－B辐射亦最为敏感。氮供应受限制或植株生长在中性盐如NH4NO3－N则对UV－B辐射不甚敏感。     

Regulation of amino acid uptake in conifers by exogenous and endogenous nitrogen

Although an accumulating amount of research clearly indicates that plants are capable of taking up exogenous amino acids, the actual importance of such organic N sources for plant N nutrition is under debate. In this study, we show that amino acid uptake by Scots pine (Pinus sylvestris L.) is significantly decreased by elevated internal NH(4)(+) levels, while it increases following exposure to exogenous amino acids. Furthermore, amino acid uptake is larger in N-deficient plants than in plants grown with a large access of N. The regulatory pattern of amino acid uptake shows important similarities to the regulation of NO(3)(-) and NH(4)(+) transport as well as to the regulation of yeast amino acid transporters. In addition, our data suggest that uptake may be regulated by factors not originating from N metabolism. The up-regulation of uptake in response to N deficiency suggests that amino acid uptake may be a significant contributor to the N economy of P. sylvestris.     

Uptake, metabolism and distribution of organic and inorganic nitrogen sources by Pinus sylvestris

Although an increasing number of studies show that many plant species have the capacity to take up amino acids from exogenous sources, the importance of such uptake for plant nitrogen nutrition is largely unknown. Moreover, little is known regarding metabolism and distribution of amino acid-N following uptake or of the regulation of these processes in response to plant nitrogen status. Here results are presented from a study following uptake, metabolism, and distribution of nitrogen from NO(3)(-) NH(4)(+), Glu, or Ala in Scots pine (Pinus sylvestris L). In a parallel experiment, Ala uptake, processing, and shoot allocation were also monitored following a range of pretreatments intended to alter plant C- and N-status. Uptake data, metabolite profiles, N fluxes through metabolite pools and tissues, as well as alanine aminotransferase activity are presented. The results show that uptake of the organic N sources was equal to or larger than NH(4)(+) uptake, while NO(3)(-) uptake was comparatively low. Down-regulation of Ala uptake in response to pretreatments with NH(4)NO(3) or methionine sulphoximine (MSX) indicates similarities between amino acid and inorganic N uptake regulation. N derived from amino acid uptake exhibited a rapid flux through the amino acid pool following uptake. Relative shoot allocation of amino acid-N was equal to that of NH(4)(+) but smaller than for NO(3)(-) Increased N status as well as MSX treatment significantly increased relative shoot allocation of Ala-N suggesting that NH(4)(+) may have a role in the regulation of shoot allocation of amino acid-N.     

空气NH3增高情况下不同形式氮源对荫香光合作用和氮利用的影响

 生长在供给NO-3 N、NH+4 N和NH4NO3 N氮源下的荫香（Cinnamomum burmanni）幼树暴露在增高空气NH3浓度下30 d。利用气体交换测定和氮分析研究了植株的光合作用、氮利用和氮在光合过程一些组分中的分配,根据Farquhar-von Caemmerer模式得出相关光合参数。结果表明在增高空气NH3下生长于NO-3 N的植株Rubisco最大羧化速率（Vcmax）和最大光合电子传递速率（Jmax）较正常空气下的高,但生长于NH+4 N和NH4NO3 N的植株则较正常空气下的低。无论生长于何种形式氮下的植株,在空气NH3增高下以单位叶面积为基准的叶氮含量（Na）显著增高（p<0.05）。在增高空气NH3下,生长于NO-3 N下的植株,其类囊体氮量（NT）、Rubisco氮（NR）和结合于光合电子传递链的氮（NE）的含量较正常空气下的增高（p<0.05）;而生长于NH+4 N和NH4NO3 N下的植株则较正常空气下的低。表明在空气NH3增高下生长于NO－3 N的植株能有效地利用氮合成光合过程必要的组份,而生长于NH+4 N和NH4NO－3 N的植株氮在NT、NR和NE的分配受到部分限制。在空气NH3增高下生长于NO－3 N和NH4NO3 N的植株,其以单位干重为基准的有机氮量较正常空气下的高,但生长于NH+4 N的植株则较正常空气下的低,此外在空气NH3增高下生长于NO－3 N的植株的可溶性蛋白氮较正常空气下增高,而生长在NH+4 N的植株亦见降低。结果表明空气NH3增高可能有利于NO－3 N下生长的荫香植株利用空气中的氮,促进叶片光合速率提高,而空气NH3增高能抑制NH+4 N或NH4NO3 N下生长的荫香植株光合作用和氮的利用和再分配。     

Loblolly pine arginase responds to arginine in vitro

Following germination of loblolly pine (Pinus taeda L.) seeds, storage proteins in the embryo and megagametophyte are broken down to provide nitrogen, in the form of amino acids, to the developing seedling. A substantial portion of the free amino acids released in this process is arginine. Arginine is hydrolyzed in the cotyledons of the seedling by the enzyme arginase (EC 3.5.3.1), which is under developmental control. It has been shown previously that the seedling is able to initiate arginase gene expression in vitro in the absence of the megagametophyte, however, presence of the megagametophyte causes a greater accumulation of arginase protein and mRNA. Using an in vitro culture system we show that arginine itself may be responsible for up-regulating arginase activity. Application of exogenous arginine to cotyledons of seedlings germinated in the absence of the megagametophyte caused an increase in total shoot pole arginase activity as well as arginase specific activity. Arginine was also able to induce arginase mRNA accumulation in the same tissue.     

Selecting appropriate forms of nitrogen fertilizer to enhance soil arsenic removal by Pteris vittata: a new approach in phytoremediation

Certain plant species have been shown to vigorously accumulate some metals from soil, and thus represent promising and effective remediation alternatives. In order to select the optimum forms of nitrogen (N) fertilizers for the arsenic (As) hyperaccumulator, Pteris vittata L., to maximize As extraction, five forms of N were added individually to different treatments to study the effect of N forms on As uptake of the plants under soil culture in a greenhouse. Although shoot As concentration tended to decrease and As translocation from root to shoot was inhibited, overall As accumulation was greater due to higher biomass when N fertilizer was added. Arsenic accumulation in plants with N fertilization was 100-300% more than in the plants without N fertilization. There were obvious differences in plant biomass and As accumulation among the N forms, i.e., NH4HCO3, (NH4)2S04, Ca(NO3)2, KNO3, urea. The total As accumulation in the plants grown in As-supplied soil, under different forms of N fertilizer, decreased as NH4HCO3>(NH4)2S04 > urea > Ca(NO3)2 >KNO3>CK. The plants treated with N and As accumulated up to 5.3-7.97 mg As/pot and removed 3.7-5.5% As from the soils, compared to approximately 2.3% of As removal in the control. NH4+ -N was apparently more effective than other N fertilizers in stimulating As removal when soil was supplied with As at initiation. No significant differences in available As were found among different forms of N fertilizer after phytoremediation. It is concluded that NH4+ -N was the preferable fertilizer for P. vittata to maximize As removal.     

Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana.

Here, we characterize a cDNA encoding a glutamine-dependent asparagine synthetase (ASN1) from Arabidopsis thaliana and assess the effects of metabolic regulation on ASN1 mRNA levels. Sequence analysis shows that the predicted ASN1 peptide contains a purF-type glutamine-binding domain. Southern blot experiments and cDNA clone analysis suggest that ASN1 is the only gene encoding glutamine-dependent asparagine synthetase in A. thaliana. The ASN1 gene is expressed predominantly in shoot tissues, where light has a negative effect on its mRNA accumulation. This negative effect of light on ASN1 mRNA levels was shown to be mediated, at least in part, via the photoreceptor phytochrome. We also investigated whether light-induced changes in nitrogen to carbon ratios might exert a metabolic regulation of the ASN1 mRNA accumulation. These experiments demonstrated that the accumulation of ASN1 mRNA in dark-grown plants is strongly repressed by the presence of exogenous sucrose. Moreover, this sucrose repression of ASN1 expression can be partially rescued by supplementation with exogenous amino acids such as asparagine, glutamine, and glutamate. These findings suggest that the expression of the ASN1 gene is under the metabolic control of the nitrogen to carbon ratio in cells. This is consistent with the fact that asparagine, synthesized by the ASN1 gene product, is a favored compound for nitrogen storage and nitrogen transport in dark-grown plants. We have put forth a working model suggesting that when nitrogen to carbon ratios are high, the gene product of ASN1 functions to re-direct the flow of nitrogen into asparagine, which acts as a shunt for storage and/or long-distance transport of nitrogen.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.     

Effects of Nitrate and Ammonium on Gene Expression of Phosphoenolpyruvate Carboxylase and Nitrogen Metabolism in Maize Leaf Tissue during Recovery from Nitrogen Stress

We previously showed that the selective accumulation of phosphoenolpyruvate carboxylase (PEPC) in photosynthetically maturing maize (Zea mays L.) leaf cells induced by nitrate supply to nitrogen-starved plants was primarily a consequence of the level of its mRNA (B Sugiharto, K Miyata, H Nakamoto, H Sasakawa, T Sugiyama [1990] Plant Physiol 92: 963-969). To determine the specificity of inorganic nitrogen sources for the regulation of PEPC gene expression, nitrate (16 millimolar) or ammonium (6 millimolar) was supplied to plants grown previously in low nitrate (0.8 millimolar), and changes in the level of PEPC and its mRNA were measured in the basal region of the youngest, fully developed leaves of plants during recovery from nitrogen stress. The exogenous supply of nitrogen selectively increased the levels of protein and mRNA for PEPC. This increase was more pronounced in plants supplemented with ammonium than with nitrate. The accumulation of PEPC during nitrogen recovery increased in parallel with the increase in the activity of glutamine synthetase and/or ferredoxin-dependent glutamate synthase. Among the major amino acids, glutamine was the most influenced during recovery, and its level increased in parallel with the steady-state level of PEPC mRNA for 7 hours after nitrogen supply. The administration of glutamine (12 millimolar) to nitrogen-starved plants increased the steady-state level of PEPC mRNA 7 hours after administration, whereas 12 millimolar glutamate decreased the level of PEPC mRNA. The results indicate that glutamine and/or its metabolite(s) can be a positive control on the nitrogen-dependent regulation of PEPC gene expression in maize leaf cells.     

空气 NH3增高和不同氮源供应下大叶相思叶片光合参数的变化

生长在空气 NH3增高下 45 d的 NOˉ3- N大叶相思植株 ,其光饱和光合速率较对照的植株高 ;而生长在空气 NH3增高下的 NH 4- N和 NH4 NO3- N的大叶相思 ,当光强在 70 0 μmol·m- 2 ·s- 1左右时 Pn 达到最大值 ,较对照植株的要高。而当光强 >70 0 μmol·m- 2·s- 1时 ,Pn 降低 ,且较生长在对照条件下的低。表明在空气 NH3增高下生长的 NH 4- N和 NH4 NO3- N植株 ,其净光合速率 Pn会受到强光抑制。空气 NH3增高并不明显改变光呼吸 ( Rd)和无光呼吸下的 CO2 补充点 (Γ* )。无论生长在何种氮源下的大叶相思 ,其最大Ru BP饱和羧化速率 ( Vcmax)和最大电子传递速率 ( Jmax)均较生长在对照植株的高 ( P<0 .0 5 ) ,其叶氮含量亦较高 ( P<0 .0 5 ) ,其碳氮比较对照的低。在空气 NH3增高下 ,无论何种氮源生长的大叶相思 ,其 PR和 PB明显高于对照的植株 ,表明大叶相思能从空气 NH3中摄取和同化氮 ,增加氮积累和有利于 Rubisco和电子传递组分的合成 ,增高光合速率。空气 NH3增高可能有利于 Rubisco和电子传递组分的合成 ,在较低光强下能增高光合速率。空气 NH3增高可能有利于退化生态系统的生态恢复过程中的氮积累和先锋植物的早期生长。     

Regulation of glutamine synthetase 1 and amino acids transport in the phloem of young wheat plants

The possible regulation of amino acid remobilization via the phloem in wheat (Triticum aestivum L.) by the primary enzyme in nitrogen (N) assimilation and re-assimilation, glutamine synthetase (GS, E.C. 6.3.1.2) was studied using two conditions known to alter N phloem transport, N deficiency and cytokinins. The plants were grown for 15 days in controlled conditions with optimum N supply and then N was depleted from and/or 6-benzylaminopurine was added to the nutrient solution. Both treatments generated an induction of GS1, monitored at the level of gene expression, protein accumulation and enzyme activity, and a decrease in the exudation of amino acids to the phloem, obtained with EDTA technique, which correlated negatively. GS inhibition by metionine sulfoximide (MSX) produced an increase of amino acids exudation and the inhibitor successfully reversed the effect of N deficiency and cytokinin addition over phloem exudation. Our results point to an important physiological role for GS1 in the modulation of amino acids export levels in wheat plants.     

Diurnal variations in growth rate and growth substrate levels of spinach (Spinacia oleracea L.) under nitrogen-limiting conditions

Spinach plants were grown in hydroponic culture provided with variable limiting amounts of N. During a complete diurnal cycle, growth of the root and shoot parts, as well as levels of soluble and insoluble sugars and of free amino acids, were monitored. No clear relationship could be detected between the level of N feeding and the levels of free sugars and amino acids. Analysis of variance revealed that the variances in the relative growth rates of plant root and shoot could be correlated with the levels of sugars and amino acids. Root amino acid concentration could be correlated with shoot amino acid concentration and root sugar concentration. No relationship was found between the variances in root and shoot free sugar concentrations.