Nitrogen metabolism and seed composition as influenced by glyphosate application in glyphosate-resistant soybean

Previous research has demonstrated that glyphosate can affect nitrogen fixation or nitrogen assimilation in soybean. This 2-year field study investigated the effects of glyphosate application of 1.12 and 3.36 kg of ae ha(-1) on nitrogen metabolism and seed composition in glyphosate-resistant (GR) soybean. There was no effect of glyphosate application on nitrogen fixation as measured by acetylene reduction assay, soybean yield, or seed nitrogen content. However, there were significant effects of glyphosate application on nitrogen assimilation, as measured by in vivo nitrate reductase activity (NRA) in leaves, roots, and nodules, especially at high rate. Transiently lower leaf nitrogen or (15)N natural abundance in high glyphosate application soybean supports the inhibition of NRA. With the higher glyphosate application level protein was significantly higher (10.3%) in treated soybean compared to untreated soybean. Inversely, total oil and linolenic acid were lowest at the high glyphosate application rate, but oleic acid was greatest (22%) in treated soybean. These results suggest that nitrate assimilation in GR soybean was more affected than nitrogen fixation by glyphosate application and that glyphosate application may alter nitrogen and carbon metabolism.     

Effect of combined nitrogen supply and nodulation on nitrate reductase activity and growth of pea plants

Nitrate reductase activity (NRA; EC 1.6.6.1) was measured in leaves, stems and roots of Pisum sativum cv. Lincoln supplied with different nitrate concentrations and inoculated with selected Rhizobium leguminosarum strains. As a control, noninoculated plants were grown in the same nutrient medium. NRA was determined by an in vivo‐nitrate assay. Although differences in tissue NRA were mainly related to nitrate concentration in the growing medium, nodulation much affected the NRA in the different plant organs, especially in root. An increased proportion of total plant NRA occurred in the leaves and stems as nitrate concentration was increased. Nitrate accumulation in leaves, stems and roots was correlated with the concentration of this ion in the nutrient solution. Nodulation also affects the nitrate accumulation in the different plant organs.     

Glyphosate-resistant and -susceptible soybean (Glycine max) and canola (Brassica napus) dose response and metabolism relationships with glyphosate

Experiments were conducted to determine (1) dose response of glyphosate-resistant (GR) and -susceptible (non-GR) soybean [Glycine max (L.) Merr.] and canola (Brassica napus L.) to glyphosate, (2) if differential metabolism of glyphosate to aminomethyl phosphonic acid (AMPA) is the underlying mechanism for differential resistance to glyphosate among GR soybean varieties, and (3) the extent of metabolism of glyphosate to AMPA in GR canola and to correlate metabolism to injury from AMPA. GR50 (glyphosate dose required to cause a 50% reduction in plant dry weight) values for GR (Asgrow 4603RR) and non-GR (HBKC 5025) soybean were 22.8 kg ae ha-1 and 0.47 kg ha-1, respectively, with GR soybean exhibiting a 49-fold level of resistance to glyphosate as compared to non-GR soybean. Differential reduction in chlorophyll by glyphosate was observed between GR soybean varieties, but there were no differences in shoot fresh weight reduction. No significant differences were found between GR varieties in metabolism of glyphosate to AMPA, and in shikimate levels. These results indicate that GR soybean varieties were able to outgrow the initial injury from glyphosate, which was previously caused at least in part by AMPA. GR50 values for GR (Hyola 514RR) and non-GR (Hyola 440) canola were 14.1 and 0.30 kg ha-1, respectively, with GR canola exhibiting a 47-fold level of resistance to glyphosate when compared to non-GR canola. Glyphosate did not cause reduction in chlorophyll content and shoot fresh weight in GR canola, unlike GR soybean. Less glyphosate (per unit leaf weight) was recovered in glyphosate-treated GR canola as compared to glyphosate-treated GR soybean. External application of AMPA caused similar injury in both GR and non-GR canola. The presence of a bacterial glyphosate oxidoreductase gene in GR canola contributes to breakdown of glyphosate to AMPA. However, the AMPA from glyphosate breakdown could have been metabolized to nonphytotoxic metabolites before causing injury to GR canola. Injury in GR and non-GR canola from exogenous application of AMPA was similar.     

Aminomethylphosphonic acid, a metabolite of glyphosate, causes injury in glyphosate-treated, glyphosate-resistant soybean

Glyphosate-resistant (GR) soybean [Glycine max (L.) Merr.] was developed by stable integration of a foreign gene that codes insensitive enzyme 5-enolpyruvylshikimate-3-phosphate synthase, an enzyme in the shikimate pathway, the target pathway of glyphosate. Application of glyphosate to GR soybean results in injury under certain conditions. It was hypothesized that if GR soybean is completely resistant to the glyphosate, injury could be caused by a metabolite of glyphosate, aminomethylphosphonic acid (AMPA), a known phytotoxin. Glyphosate and AMPA effects on one- to two-trifoliolate leaf stage (16-18-days old) GR and non-GR soybean were examined in the greenhouse. In GR soybean, a single application of glyphosate-isopropylammonium (1.12-13.44 kg/ha) with 0.5% Tween 20 did not significantly reduce the chlorophyll content of the second trifoliolate leaf at 7 days after treatment (DAT) or the shoot dry weight at 14 DAT compared with Tween 20 alone. A single application of AMPA (0.12-8.0 kg/ha) with 0.5% Tween 20 reduced the chlorophyll content of the second trifoliolate leaf by 0-52% at 4 DAT and reduced shoot fresh weight by 0-42% at 14 DAT in both GR and non-GR soybeans compared with Tween 20 alone. AMPA at 0.12 and 0.50 kg/ha produced injury in GR and non-GR soybean, respectively, similar to that caused by glyphosate-isopropylammonium at 13.44 kg/ha in GR soybean. AMPA levels found in AMPA-treated soybean of both types and in glyphosate-treated GR soybean correlated similarly with phytotoxicity. These results suggest that soybean injury to GR soybean from glyphosate is due to AMPA formed from glyphosate degradation.     

钼对甘蔗体内固氮菌的固氮酶活性的影响

以巴西固氮甘蔗品种B1和B8为材料,在温室桶栽砂培条件下,对甘蔗施以含不同钼水平的营养液,以了解钼对甘蔗体内固氮菌的固氮酶活性的效应。结果表明,在含氮条件下,较低浓度的钼处理能提高甘蔗根内固氮菌的固氮酶活性,高浓度钼处理则能提高B1茎、叶片和B8茎中固氮菌的固氮酶活性;在无氮条件下,钼处理提高了B1叶片和茎中固氮菌的固氮酶活性,而B1根及B8根、茎、叶中固氮菌的固氮酶活性有所降低。此外,甘蔗根、茎、叶中固氮菌的固氮酶活性之间关系较为密切,根中固氮菌的固氮酶活性与叶片和茎中的都呈负相关。上述结果说明:巴西甘蔗在本地也具有一定的固氮能力;在不缺氮条件下,钼处理较利于调节甘蔗体内固氮菌的固氮酶活性,促进其固氮;而在缺氮条件下,钼处理不利于调节甘蔗体内固氮菌的固氮酶活性,抑制其固氮作用。     

大豆植株苗期至结荚初期对肥料氮的吸收与分配

采用砂培与15N分阶段标记的方法,研究了苗期至结荚初期大豆植株对氮素的同化吸收,尤其是肥料氮在不同器官和节中的积累与分配。结果表明:(1)苗期至结荚初期大豆植株氮素积累量逐渐增加,由标记试验Ⅰ的64.08g/plant逐渐增加到标记试验Ⅴ的307.17mg/plant;(2)不同器官中肥料氮积累存在差异,叶是苗期至结荚初期肥料氮积累的主要器官,所占比例为55.2%,根系次之为25.9%,茎最小为18.9%;(3)苗期至结荚初期肥料氮积累量和所占比例逐渐减小,而根瘤固氮则逐渐增加,其中苗期至盛花期肥料氮是大豆植株氮素主要来源,盛花期以后以根瘤固氮为主;(4)不同成长程度叶对肥料氮的积累存在差异,表现为成长中叶>新生叶>成熟叶;(5)新生茎叶氮素构成随全株氮素构成的变化而变化,苗期至初花期以肥料氮为主,初花期至结荚初期根瘤固氮逐渐转为主体。     

氮肥形态对马铃薯氮素积累与分配的影响

采用田间试验的方法,研究了不同氮肥形态对氮素在马铃薯不同器官中的吸收和运转分配及产量的影响。试验结果表明:铵态氮肥对马铃薯地上干物质积累量的增加作用最明显,施氮处理马铃薯块茎干物质积累量比对照增加50.37%～71.38%;马铃薯各器官中含氮量随生育期推进逐渐下降,其中,茎和叶下降幅度较大;马铃薯各器官中氮含量生育前期表现为叶片>地上茎>根,进入块茎形成期以后,则叶片>根>地上茎>块茎。施氮在马铃薯生育前期有利于茎对氮素的吸收和储存,后期又可以促进茎中的氮素向叶片和块茎转移。施氮各处理产量较对照增加19.28%～63.86%,NH4+-N处理对氮的吸收、积累与分配影响最大,且产量最高,达到39 410.2 kg.hm-2。     

氮肥对大豆结瘤固氮、籽粒产量和蛋白质含量的影响

【目的】合理施用氮肥不仅可提高大豆结瘤固氮能力,还可减少农业污染,实现大豆生产的高产优质高效。研究施氮时期和施氮量对大豆结瘤固氮、产量及蛋白质含量的影响,为大豆高产优质提供理论基础及科学依据。【方法】采用盆栽试验,供试大豆品种为‘东生35’,试验设2个氮肥施用时期(V2期和R1期)和3个氮肥施用量[N 0、5、100 mg/(kg,土)],表示为N0、N5和N100。在大豆R2期(盛花期)和R5期(鼓粒期)取样分析了地上部干物质积累量、根瘤数量、根瘤干重和固氮酶活性。在R8期(成熟期)调查了大豆籽粒产量和蛋白质含量。【结果】施氮时期和施氮量对大豆地上干物质积累、结瘤和固氮能力均有显著影响。不论是V2期还是R1期施氮,大豆地上部干物质积累量均随着施氮量的增加而增加,而根瘤干重、数量则呈降低的趋势。R1期施氮条件下,N100处理的大豆盛花期根瘤数量和根瘤干重比N0分别下降了42.3%和32.8%,而固氮酶活性则均以N5处理最高;V2期施氮条件下,N5处理的大豆固氮酶活性在R2期和R5期较N0处理分别增加15.3%和27.1%。大豆籽粒产量和蛋白质含量均以N5处理最高,籽粒蛋白质含量较N0处...     

Tolerance and accumulation of shikimic acid in response to glyphosate applications in glyphosate-resistant and nonglyphosate-resistant cotton (Gossypium hirsutum L.).

Measurement of shikimic acid accumulation in response to glyphosate inhibition of 5-enolpyruvylshikimate-3-phosphate synthase is a rapid and accurate assay to quantify glyphosate-induced damage in sensitive plants. Two methods of assaying shikimic acid, a spectrophotometric and a high-performance liquid chromatography (HPLC) method, were compared for their accuracy of recovering known amounts of shikimic acid spiked into plant samples. The HPLC method recovered essentially 100% of shikimic acid as compared with only 73% using the spectrophotometric method. Relative sensitivity to glyphosate was measured in glyphosate-resistant (GR) and non-GR cotton leaves, fruiting branches, and squares (floral buds) by assaying shikimic acid. Accumulation of shikimic acid was not observed in any tissue, either GR or non-GR, at rates of 5 mM glyphosate or less applied to leaves. All tissues of non-GR plants accumulated shikimic acid in response to glyphosate treatment; however, only fruiting branches and squares of GR plants accumulated a slight amount of shikimic acid. In non-GR cotton, fruiting branches and squares accumulated 18 and 11 times, respectively, more shikimic acid per micromolar of translocated glyphosate than leaf tissue, suggesting increased sensitivity to glyphosate of reproductive tissue over vegetative tissue. GR cotton leaves treated with 80 mM of glyphosate accumulated 57 times less shikimic acid per micromolar of translocated glyphosate than non-GR cotton but only 12.4- and 4-fold less in fruiting branches and squares, respectively. The increased sensitivity of reproductive structures to glyphosate inhibition may be due to a higher demand for shikimate pathway products and may provide an explanation for reports of fruit abortion from glyphosate-treated GR cotton.     

高浓度CO2对冬小麦旗叶和穗部氮吸收的影响

利用农田开放式CO2浓度升高(Free Air Carbon dioxide Enrichment,FACE)试验平台,以强筋小麦中麦175为供试材料,研究高浓度CO2和不同施氮量对冬小麦旗叶和穗部含N量、氮吸收量和旗叶硝酸还原酶活性(NRA)的影响。CO2浓度处理设对照(Ambient,CO2415±16μL·L-1)和高浓度(FACE,CO2550±17μL.L-1)两个水平;施N处理设常规施氮(NN,底肥含N 118kg·hm-2+追肥含N 70kg·hm-2)和低氮(LN,底肥含N 66kg·hm-2+追肥含N 17kg·hm-2)两个水平。结果表明,在乳熟期,不考虑氮肥的影响,CO2浓度升高使穗部N含量和氮吸收量分别增加7.59%和16.3%,说明CO2浓度升高加大了穗部对氮素的需求;不考虑各生育期的差异,CO2浓度升高导致冬小麦旗叶N含量下降10.98%;同时使旗叶硝酸还原酶活性(NRA)降低22.24%。说明CO2浓度升高抑制了NO3-的还原,导致旗叶含N量降低,高浓度CO2可能影响以NO3-为主要氮源的植物生长。与低氮处理相比,常规施氮处理没有显著增加小麦吸氮量和硝酸还原酶活性。因此,为了满足高CO2条件下,穗部对氮素的更多需求,不能单一增加施氮量,而需要合理配施NH4+肥,以环境友好的形式提高小麦产量。     

Effects of long‐term nitrogen dioxide fumigation and nitrate supply on nitrite and Leghemoglobin concentrations,pH, and nitrogenase activity of soybean root nodules

Exposing 12‐day‐old soybean plants to 0.2 ppm nitrogen dioxide (NO₂) for four weeks increased the nitrite concentration and acidity, and decreased the Leghemoglobin (LHb) concentration and the nitrogenase activity of root nodules. The supply of 1 mol.m^‐3 nitrate to the roots intensified the nitrite accumulation, decreased the acidity of the nodules, and alleviated the inhibition of nitrogenase activity by NO₂ fumigation. These results suggested that the inhibition of nitrogen (N₂) fixation by N fertilizer supply might relate to the acid‐alkali balance in nodules.     

淹涝胁迫和氮形态对苗期玉米糖、氮代谢底物量的影响

采用砂培培养方法,比较研究淹水和不同氮形态(铵态氮、硝态氮以及铵态氮︰硝态氮为1︰1)对苗期玉米根、茎鞘和叶的糖、氮代谢底物——可溶性糖、还原糖、硝态氮和游离氨基酸等物质含量的影响。结果表明,当淹涝胁迫持续7 d时,在非淹涝胁迫条件下,铵态氮处理的根、茎鞘和叶的可溶性糖和游离氨基酸含量均显著高于硝态氮处理(P<0.05);在淹涝胁迫条件下,硝态氮处理的根、茎鞘和叶的生物量干重显著低于铵态氮处理(P<0.05),其根和叶的生物量干重也显著低于铵态氮、硝态氮混合处理(P<0.05)。与非淹涝条件相比,在淹涝胁迫条件下,硝态氮处理的根系和叶的硝态氮含量显著降低(P<0.05),降低幅度分别高达62.6%和30.0%;此外,与非淹涝条件相比,在淹涝胁迫条件下,铵态氮处理的根的可溶性糖、还原糖以及游离氨基酸含量,茎鞘的可溶性糖和还原糖含量以及叶的可溶性糖和游离氨基酸含量均显著升高(P<0.05),而硝态氮处理仅根、茎鞘和叶的还原糖含量以及叶的游离氨基酸含量显著升高(P<0.05)。因此,在本试验条件下,由于糖、氮代谢底物含量充足,铵态氮处理的苗期玉米具有相对较强的耐淹涝胁迫能力。     

Nitrate assimilation in pea leaves in the presence of cadmium

Supply of 0.01 to 1.0 mM Cd acetates either to the intact seedlings or to the excised leaves of 15 dPisum sativum L. Cv. Bonvilla seedlings inhibitedin vivo nitrate reductase activity (NRA), total soluble protein, chlorophyll, and carotenoids. The inhibition was independent of metal concentrations. In excised leaf tissues, higher concentrations (0.5 to 1.0 mM) of Cd had no conspicuous effect on nitrate assimilation. When NRA was assayed byin vitro method, an increase of 12 to 45% was obtained with Cd in intact and excised leaves. It appears that NRA is more sensitive to Cd concentration than any other parameter examined.     

New insights on glyphosate mode of action in nodular metabolism: Role of shikimate accumulation

The short-term effects of the herbicide glyphosate (1.25-10 mM) on the growth, nitrogen fixation, carbohydrate metabolism, and shikimate pathway were investigated in leaves and nodules of nodulated lupine plants. All glyphosate treatments decreased nitrogenase activity rapidly (24 h) after application, even at the lowest and sublethal dose used (1.25 mM). This early effect on nitrogenase could not be related to either damage to nitrogenase components (I and II) or limitation of carbohydrates supplied by the host plant. In fact, further exposure to increasing glyphosate concentrations (5 mM) and greater time after exposure (5 days) decreased nodule starch content and sucrose synthase (SS; EC 2.4.1.13) activity but increased sucrose content within the nodule. These effects were accompanied by a great inhibition of the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31). There were remarkable and rapid effects on the increase of shikimic and protocatechuic (PCA) acids in nodules and leaves after herbicide application. On the basis of the role of shikimic acid and PCA in the regulation of PEPC, as potent competitive inhibitors, this additional effect provoked by glyphosate on 5-enolpyruvylshikimic-3-phosphate synthase enzyme (EPSPS; EC 2.5.1.19) inhibition would divert most PEP into the shikimate pathway, depriving energy substrates to bacteroids to maintain nitrogen fixation. These findings provide a new explanation for the effectiveness of glyphosate as a herbicide in other plant tissues, for the observed differences in tolerance among species or cultivars, and for the transitory effects on glyphosate-resistant transgenic crops under several environmental conditions.     

不同品种油菜氮效率差异及其成因分析

采用盆栽试验，以氮胁迫与正常供氮条件下籽粒产量的比值作为氮效率系数，探讨了不同品种油菜氮效率差异及其生理基础。结果表明，所测定的8个油菜品种氮效率系数的变化范围是0．37～0．69；氮胁迫与正常供氮条件下不同油菜品种植株氮素累积吸收量、生长后期茎叶氮素转运率及氮素生理效率的比值不同，氮效率高的品种，其比值高，反之亦然;在氮素供应水平低的情况下，氮效率高的品种具有较长的根长、较多的侧根、较高的茎叶硝态氮再利用量和叶片硝酸还原酶活性。     

Nitrogen assimilation studies using 15N in soybean plants treated with imazethapyr, an inhibitor of branched-chain amino acid biosynthesis

The pattern of nitrogen assimilation in soybean plants treated with a herbicide that inhibits branched-chain amino acid biosynthesis was evaluated by (15)N isotopic analysis. The herbicide imazethapyr caused a strong decrease in nitrate uptake by roots, partly due to a reduced stomatal conductance. The inhibition of (15)N uptake was accompanied by a decrease in the (15)N content in the plant and, concomitantly, an inhibition of translocation to the shoot. Imazethapyr inhibited nitrate reductase activity in leaves and roots. Among all parameters studied, "de novo" synthesis of proteins was the first parameter of the N assimilation metabolism affected by the herbicide. These results show that this class of herbicides totally damages N metabolism and indicates a regulatory effect on N uptake and translocation that would be mediated by the increase in free amino acid pool provoked by the inhibition of branched-chain amino acid biosynthesis.     

Factors affecting the in vivo nitrate reductase assay for mm 106 apple trees

Abstract

Factors affecting the in vivo nitrate reductase (NR) assay were investigated in order to optimize the assay conditions for various tissues of MM 106 apple trees. The addition of nitrate and phosphate to the assay medium significantly increased nitrate reductase activity (NRA), but high concentrations of nitrate and phosphate inhibited the NRA. The optimum concentrations of nitrate and phosphate for in vivo NRA ranged from SO to 100 mM and were tissue‐specific. The optimum pH of the assay medium was 7.5. The addition of 2% (v/v) n‐propanol to the assay medium stimulated NRA, but concentrations of n‐propanol greater than 2% significantly decreased the NRA. Vacuum infiltration was effective in stimulating NRA. The in vivo NR assays for leaves and stems were linear for at least 60 minutes following an initial 30 minute lag, whereas there was no lag phase in root tissues.     

氮、磷肥对杉木幼苗生物量及养分分配的影响

采用盆栽试验,研究了不同氮、 磷肥对杉木（Cunninghamia lanceolata）幼苗生物量及养分分配的影响。结果表明,供磷可促进杉木幼苗植株和各器官生物量的增加,并影响叶、 茎、 根生物量的分配比例,氮、 磷处理幼苗叶生物量占全株生物量的45% 以上, 施氮反而降低杉木叶、 茎、 根的生物量; 施氮显著增加根和叶的氮含量,而显著降低根和叶的磷含量,对茎的氮、 磷含量没有明显影响; 施磷显著降低叶、 茎、 根的氮含量,叶、 茎、 根的磷含量随供磷水平的增加而逐渐增加。氮磷配施显著影响叶、 茎、 根的氮、 磷含量和氮、 磷累积量。叶片是主要的氮、 磷养分存储器官。氮（或磷）水平的增加可降低杉木幼苗的磷（或氮）利用效率,提高氮（或磷）的利用效率; 氮、 磷肥显著影响杉木幼苗叶、 茎、 根的N/P比。研究结果说明,氮、 磷肥增加了杉木幼苗各器官生物量和氮、 磷含量,影响了幼苗的养分分配和营养平衡。     

IMPACT OF LOW RATES OF NITROGEN APPLIED AT PLANTING ON SOYBEAN NITROGEN FIXATION

Environmental conditions in the northern Great Plains can delay emergence, nitrogen (N) fixation and growth of soybean due to cool and wet soil conditions at planting. The objective was to evaluate the impact of low rates of N applied at planting on soybean N fixation and crop growth. A field experiment was established within corn soybean rotation using a split-plot design with four replications. Whole plots were no-tillage and conventional tillage and split plots were starter fertilizer. Nitrogen sources were ammonium nitrate or urea applied at four rates. The amount of plant N fixation increased with growth stage reaching a maximum fixation at the R5 growth stages. Plant ureide content decrease with increase N applied for all growth stages except R7. The increase in plant biomass contributed to an overall increase in yield indicating that in unfavorable environments application of N at planting can have a positive impact on soybean growth.     

Changes in ureide synthesis,transport and assimilation following ammonium nitrate fertilization of nodulated Soybeans 1

The effect of ammonium nitrate fertilization on ureide synthesis, xylem transport and assimilation was examined in four week old nodulated soybean plants. In nodules the activity of 5‐phosphoribosylpyrophosphate amidotransferase, a key enzyme of ureide biosynthesis, declined 75%, while enzymes of purine oxidation (xanthine dehydrogenase and uricase) showed no response to nitrogen treatment. Xylem sap concentrations of allantoin and allantoate, as compared to untreated controls, were reduced by about 85% and 65%, respectively. Despite the substantial decline in ureide synthesis and transport, allantoate concentration in leaves of ammonium nitrate treated plants increased by about three‐fold. No ureide accumulation was observed in leaf tissue following a suppression of ureide formation in nodules by allopurinol, an inhibitor of xanthine dehydrogenase. In the pathway of ureide assimilation in leaves, application of inorganic nitrogen had no effect on activity of allantoinase, but caused a 50% drop in activity of allantoate amidohydrolase. Therefore, the observed accumulation of allantoate in soybean leaves in response to nitrogen fertilization was due to new ureide synthesis in plant tissues other than nodules, or to retardation of ureide degradation in leaves caused by the alternative source of nitrogen.