不同生育期花生叶片蛋白质含量及氮代谢相关酶活性分析

以5个珍珠豆型花生(Arachis hypogaea Linn.)品种(系)‘汕E’(‘Shan E’)、‘汕G’(‘Shan G’)、‘TH’、‘TJ’和‘泉花7号’(‘Quanhua No.7’)为研究对象,分析了花针期、结荚期和饱果期花生叶片中可溶性蛋白质含量及硝酸还原酶(NR)、谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)活性的变化趋势,并比较了5个品种(系)荚果和秆产量的差异。结果表明:在3个生育期内,5个花生品种(系)叶片可溶性蛋白质含量和GDH活性的变化趋势基本一致,而NR和GS活性的变化趋势则有差异。其中,可溶性蛋白质含量均呈"低—高—低"的变化趋势,在结荚期最高;GDH活性均逐渐升高,至饱果期达最高;‘泉花7号’叶片NR活性呈"高—低—高"的变化趋势,而其他4个品种(系)叶片NR活性均逐渐降低;‘汕E’、‘TJ’和‘泉花7号’叶片GS活性呈逐渐降低趋势,而‘汕G’和‘TH’叶片GS活性呈"低—高—低"的变化趋势。总体上看,5个品种(系)中,‘汕G’和‘泉花7号’叶片的可溶性蛋白质含量及NR和GDH活性、‘汕E’叶片的NR和GS活性以及‘TH’叶片的GDH活性均较高。5个品种(系)的2个产量指标(单株荚果鲜质量和单株秆鲜质量)均有明显差异,总体上看,‘汕G’、‘泉花7号’和‘TH’的2个产量指标均较高,而‘汕E’和‘TJ’的2个产量指标均较低。综合分析结果显示:‘汕G’和‘泉花7号’叶片可溶性蛋白质含量及NR和GDH活性均相对较高,其荚果和秆产量也均较高,表明花生荚果和秆产量与不同生育期叶片氮代谢水平有一定关系。     

水稻种子萌发期间谷氨酰胺合成酶和NADH-谷氨酸合酶的变化

测定了水稻种子不同萌发时期胚乳、胚芽鞘和幼根的谷氨酰胺合成酶（GS）和依赖于NADH的谷氨酸合酶（NADH－GOGAT）活性变化。胚乳和胚芽鞘的GS活性在萌发过程中升高,幼根的GS活性则有所降低。NADH－GOGAT的活性变化趋势与GS相同。Native-PAGE活性染色表明,在萌发阶段的水稻种子胚乳和幼根里,始终只观察到一种GS活性带。但是,在水稻种子萌发3d后,在胚芽鞘中除继续检测到GS1的活性外,还可以观察到GS2的活性。蛋白质印迹显示,水稻种子胚乳中的GS（GSe)和GS1和GSra一样是一种胞质型GS。实验结果提示,这些不同组织中的GS与NADH－GOGAT构成的循环途径也许是水稻种子萌发时氨同化的主要途径。     

Ni胁迫对不同基因型谷子幼苗生长及氮素代谢的影响

采用盆栽土培法,研究了不同浓度Ni2+(0、25、50、100、150、200 mg/kg)对4种基因型谷子(13-36、B-7、晋谷51号、晋谷52号)幼苗生长,Ni2+富集与转运能力,叶片中硝态氮、氨态氮、可溶性蛋白质、脯氨酸含量及氮代谢相关酶硝酸还原酶(NR)、谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、谷氨酸脱氢酶(GDH)活性的影响。结果表明:Ni2+胁迫下,4种基因型谷子幼苗的根长、苗长、生物量随Ni2+浓度增加逐渐降低,体内Ni2+含量逐渐增加,与对照组差异显著(P0.05)。在所试浓度范围内,4种基因型谷子幼苗叶片中的硝态氮含量、NR、GS、GOGAT活性表现为低浓度(25—50mg/kg)增高和高浓度(50—200 mg/kg)降低,而GDH活性在Ni2+浓度为100mg/kg以上时下降,氨态氮含量在50—150 mg/kg处理组中为对照的1.14—3.02倍。不同浓度Ni2+处理后,4种基因型谷子幼苗叶片中的脯氨酸含量均有不同程度的提高,而可溶性蛋白质含量呈明显下降趋势。实验结果证明,Ni2+胁迫抑制了谷子幼苗对硝态氮的吸收,降低了叶片中NR、GS、GOGAT活性,影响了氨的同化作用,使谷子幼苗的氮素代谢发生紊乱,不同基因型谷子对Ni2+胁迫的毒性效应存在差异。4种基因型谷子对Ni2+的耐性顺序为13-36B-7晋谷51晋谷52。     

乐果对菠菜叶片POD、SOD、CAT活性及MDA含量的影响

研究了喷施乐果后不同时间及不同施用量对菠菜(Spinacia oleracea)叶片POD、SOD和CAT活性及MDA含量的影响;同时检测了喷药后不同时间菠菜中乐果的残留量。结果表明,喷施乐果可促使菠菜叶片中POD和CAT活性增强以及MDA含量明显增加,而SOD活性在部分乐果处理中呈下降趋势;随着喷药后时间的延长,乐果在菠菜中的残留量亦随之减少。     

烤烟叶片衰老期氨气挥发特征及其生理调控研究

以烤烟品种K326为试验材料,利用氨气收集装置测定烟叶的氨气挥发量,并利用谷氨酰胺合成酶(GS)抑制剂(Glufosinate)处理叶片和质外体提取等方法,研究了叶片氨气挥发及其与氮代谢相关生理指标的关系。结果表明:(1)随着叶片的衰老,氨气挥发量在叶龄70d时最大(10.96μg.m-2.h-1),与衰老初期(叶龄40d)相比增加了2.15倍;质外体NH4+浓度和pH、氨气补偿点逐渐上升,GS和硝酸还原酶(NR)活性下降,谷氨酸脱氢酶(GDH)活性升高,可溶性蛋白和总氮降解,叶片NH4+浓度升高。(2)GS抑制剂处理后,叶片组织NH4+浓度和氨气补偿点升高,氨气挥发量增大,与对照相比差异显著。(3)氨气挥发量与质外体NH4+浓度、质外体pH和氨气补偿点呈极显著或显著正相关,与GS活性呈显著负相关,与GDH活性呈显著正相关,与叶片组织NH4+浓度等其他指标相关性不显著。研究认为,烤烟叶片衰老期间氨挥发量大幅上升,挥发量的大小受气孔氨气补偿点、GS和GDH活性的直接调控,以及其他氮素代谢相关指标的间接调控,其中GS起主导作用。     

植物抗逆性与光呼吸作用之间的关系

用Na_2SO_3溶液浸泡,可以影响菠菜叶片中超氧物歧化酶的活性。较低浓度(0.1、1、10ppm)的该溶液使酶活性增加,而100ppm的该溶液则降低此酶的活性。同时发现叶片中光呼吸关键酶,即乙醇酸氧化酶活性也随着发生正相关的变化。讨论植物受SO_3~(2-)胁迫后,乙醇酸氧化酶的活性变化与植物抗逆性的产生可能存在密切的关系。     

植物抗逆性与光呼吸作用之间的关系

用Na2SO3溶液浸泡,可以影响菠菜叶片中超氧物歧化酶的活性。较低浓度(0.1、1、10ppm)的该溶液使酶活性增加,而100ppm的该溶液则降低此酶的活性。同时发现叶片中光呼吸关键酶,即乙醇酸氧化酶活性也随着发生正相关的变化。讨论植物受SO32-胁迫后,乙醇酸氧化酶的活性变化与植物抗逆性的产生可能存在密切的关系。     

密度和种植方式对夏玉米酶活性和产量的影响

在豫北灌区的生产条件下,以郑单958和浚单20为试验材料,研究了不同密度和种植方式对夏玉米碳氮关键酶的影响。结果表明:生育后期夏玉米叶片硝酸还原酶(NR)、谷氨酰胺合成酶(GS)和籽粒蔗糖磷酸合成酶(SPS)、蔗糖合成酶(SS)活性均呈先升高后降低的变化趋势,叶片NR和GS活性峰值出现在吐丝期,籽粒SPS和SS活性峰值出现在灌浆后15d。基因型、密度和种植方式对夏玉米生育后期叶片NR、GS和籽粒SPS、SS活性均有显著影响,而3因素间总体上没有显著的互作效应。郑单958生育后期叶片NR、GS和籽粒SPS、SS活性均显著高于浚单20,分别提高5.02%、7.40%、6.25%和4.43%。在6.75—9.00万株hm2的范围内,随着密度的增大,夏玉米生育后期叶片NR、GS和籽粒SPS、SS活性显著降低。与60cm等行距种植方式相比,80cm-40cm宽窄行种植方式下夏玉米生育后期叶片NR、GS和籽粒SPS、SS活性均显著提高,分别提高6.23%、9.25%、6.87%和2.84%。在采用宽窄行种植、密度为8.25万株hm2时,产量最高。     

海水胁迫对菠菜叶绿素代谢的影响

以耐海水品种‘荷兰3号’和海水敏感品种‘圆叶菠菜’为试验材料,采用营养液栽培,研究海水胁迫对菠菜叶绿素代谢的影响。结果表明,海水胁迫下,2个菠菜品种叶片的叶绿素a(Chl a)、叶绿素b(Chl b)和总叶绿素含量以及叶绿素合成前体———原叶绿素酸(Pchl)、镁原卟啉IX(Mg-proto IX)、原卟啉IX(Proto IX)和尿卟啉原III(UroIII)含量均明显降低,而胆色素原(PBG)和δ-氨基酮戊酸(ALA)积累,‘圆叶菠菜’的变化幅度大于‘荷兰3号’。海水胁迫下,‘荷兰3号’叶片的叶绿素酶(Chlase)活性无显著变化,胆色素原脱氨酶(PBGD)和尿卟啉原III合酶(UROS)活性在胁迫第3天显著下降,而‘圆叶菠菜’Chlase活性显著上升,PBGD和UROS活性显著下降。研究发现,在海水胁迫条件下,菠菜叶片的叶绿素合成代谢受阻,受阻位点位于PBG→UroIII的转化过程,其中‘圆叶菠菜’的受阻程度大于‘荷兰3号’;耐海水品种‘荷兰3号’叶片叶绿素含量降低主要由叶绿素合成代谢受阻引起,而海水敏感品种‘圆叶菠菜’叶绿素含量的降低则是由叶绿素合成受阻和叶绿素降解共同作用的结果。     

光对水稻非光合组织谷氨酰胺合成酶同工酶表达的影响

以前的研究表明,高等植物叶绿体谷氨酰胺合成酶（GS2）受光调节,但叶片胞液GS（GS1）和非光合作用组织中的GS很少受光的影响,在本报道中,笔者运用GS活性染色和Western blotting研究了光对非光合作用组织水稻根GS同工酶表达的影响,在阳光的直接照射下以及在室内不同光照强度下,可以很清楚地观察到GSra和GS rb的活性带及其蛋白质带,但是,当用尼龙网档住阳光的直接照射下,GSrb的活性带和蛋白质带消失,当阳光被尼龙网遮挡住后,其光强度仍然比室内光照强度大得多,表明光照强度不是影响GSrb表达的主要因素,当分析生长在暗处以及生长在光／暗转换下的水稻幼苗根GS同工酶变化时,仍然可以观察到GSrb的在,在所有实验条件下,GSra都未发生明显变化,这些结果提示,光对GSrb表达的影响可能是由某些光谱相互作用所产生的未知因素造成的。     

Four isoforms of glutamine synthetase in light-grown soybeans

Glutamine synthetase (GS) activity recovered from linear sucrose gradients was associated with the cytosol of cells isolated from etiolated soybean hypocotyls whereas light-grown tissue contained increased GS activity localized in both the cytosol and chloroplasts. DEAE-cellulose chromatography indicated two GS isoforms in etiolated hypocotyls whereas light-grown hypocotyls and primary leaves contained four isoforms. Only one GS isoform was recovered from both etiolated and light-grown cotyledons.     

The role of cytosolic glutamine synthetase in wheat

The role of glutamine synthetase (GS; EC 6.3.1.2) was studied in wheat. GS isoforms were separated by HPLC and the two major leaf isoforms (cytosolic GS1 and chloroplastic GS2) were found to change in content and activity throughout plant development. GS2 dominated activity in green, rapidly photosynthesising leaves compared to GS1 which was a minor component. GS2 remained the main isoform in flag leaves at the early stages of grain filling but GS1 activity increased as the leaves aged. During senescence, there was a decrease in total GS activity which resulted largely from the loss of GS2 and thus GS 1 became a greater contributor to total GS activity. The changes in the activities of the GS isoforms were mirrored by the changes in GS proteins measured by western blotting. The changes in GS during plant development reflect major transitions in metabolism from a photosynthetic leaf (high GS2 activity) towards a senescencing leaf (relatively high GS1 activity). It is likely that, during leaf maturation and subsequently senescence, GS1 is central for the efficient reassimilation of ammonium released from catabolic reactions when photosynthesis has declined and remobilisation of nitrogen is occurring. Preliminary analysis of transgenic wheat lines with increased GS1 activity in leaves showed that they develop an enhanced capacity to accumulate nitrogen in the plant, mainly in the grain, and this is accompanied by increases in root and grain dry matter. The possibility that the manipulation of GS may provide a means of enhancing nitrogen use in wheat is discussed.     

Organ-specific changes in the activity and subunit composition of glutamine-synthetase isoforms of barley (Hordeum vulgare L.) after growth on different levels of NH+ 4

One cytosolic glutamine synthetase (GS, EC 6.3.1.2) isoform (GS 1a) was active in the germinating seeds of barley (Hordeum vulgare L.). A second cytosolic GS isoform (GS 1b) was separated from the leaves as well as the roots of 10-d-old seedlings. The chloroplastic isoform (GS 2) was present and active only in the leaves. The three GS isoforms were active in N-supplied (NH⁺ ₄ or NO ₃ ^– ) as well as in N-free-grown seedlings. This indicates (i) that a supply of nitrogen to the germinating seeds was not necessary for the induction of the GS isoforms and (ii) that no nitrogen-specific isoforms appeared during growth of seedlings with different nitrogen sources. The activity of GS, however, depended on the seedlings' nitrogen source: the specific activity was much higher in the leaves and much lower in the roots of NH⁺ ₄-grown barley than in the respective organs of NO ₃ ^– -fed or N free-grown plants. With increasing concentrations of NH⁺ ₄ (supplied hydroponically during growth), the specific activity of GS 1b increased in the leaves, but decreased in the roots. The activity of GS 2 (leaf) also increased with increasing NH⁺ ₄ supply, whereas GS 1a activity (leaf and root) was not affected. The changes in the activities of GS 1b and GS 2 were correlated with changes in the subunit compositions of the active holoenzymes: growth at increased levels of external NH⁺ ₄ resulted in an increased abundance of one of the four GS subunits, and of two of the five GS 1b subunits in the leaves. In the roots, however, the abundance of these two GS 1b subunits was decreased under the same growth conditions, indicating an organ-specific difference either in the expression of the genes coding for the respective GS 1b subunits or in the assembly of the GS 1b holoenzymes. Furthermore, growth at different levels of NH⁺ ₄ resulted in changes in the substrate affinities of the isoforms GS 1b (root and leaf) and GS 2 (leaf), presumably due to the changes in the subunit compositions of the active holoenzymes.Abbreviations FPLC fast protein liquid chromatography - GHA -glutamyl hydroxamate - GS glutamine synthetase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Activity and immunocytochemical localization of glutamine synthetase inLathraea clandestins L.

Summary The activity of glutamate synthetase (GS) was determined in the different organs ofLathraea clandeslina L., a holoparasitic Scrophulariaceae. It was very low throughout the plant but levels were slightly higher in the scale leaves. Immunoprecipitation reactions carried out with immune serums raised against the isoforms GS1 or GS2 of the enzyme showed that, in the scale leaves, isoenzyme GS1 was present, but the existence of small amounts of GS2 remained in question on account of possible cross reactions. On the other hand, the study of intracellular localization of GS in the scale leaves by indirect immunofluorescence, using the same antibodies anti-GS1 and anti-GS2, clearly demonstrated the occurrence of two GS forms: a GS1 isoenzyme located in the cytoplasm of glandular and parenchymatous cells and a GS2-type isoenzyme only detected in the stroma of the large amyloplasts present in the outer parenchyma. This amyloplastidial isoenzyme seems to be a peculiar GS form, distinct from both GS1 and GS2.Abbreviations GS glutamate synthetase - GS₁, GS₂, GSR glutamate synthetase isoforms - PBS phosphate buffered saline - PEG poly ethylene glycol - GP peltate glands - GB shield glands - P amyloplasts     

Change in Levels of Acyl Carrier Proteins in Greening Plants

The levels of acyl carrier proteins (ACP) in greening spinachcotyledons and greening oat leaves were examined by immunoblottingwith antiserum raised against spinach ACP I. Two isoforms ofACP, ACP I and ACP II, were found in spinach cotyledons, asthey were in the green leaves. The level of ACP II was higherthan that of ACP I in etiolated cotyledons. The level of ACPI increased markedly with greening. In the greened cotyledons,the major isoform was ACP I as was the case in green spinachleaves. In oat leaves, two isoforms were also identified, oatACPI (about 12kDa) and ACP II (about 17kDa), which cross-reactedwith the antiserum against spinach ACP I, but which were differentfrom spinach ACPs I and II. The levels of oat ACPs I and IIwere very low in etiolated leaves. The increase in levels ofboth ACPs corresponded to the change in the activity of fattyacid synthesis during illumination for 24 h. During furtherillumination for 24 h, the level of ACP II increased a littlein parallel with the change in the activity of fatty acid synthesis,whereas the level of ACP I increased somewhat more. The functionof oat ACPs I and II is discussed in connection with the formationof chloroplast. (Received March 27, 1989; Accepted September 18, 1989)     

Glutamine synthetase oligomers and isoforms in sugarbeet (Beta vulgaris L.)

The -amino-N compounds that accumulate in the thickening storage root of sugarbeet (Beta vulgaris L.) were synthesized in the leaves (NO ₃ ^– nutrition) and also in the lateral roots (NH ₄ ⁺ nutrition). Ammonium stimulated glutamine synthetase (GS, EC 6.3.1.2) activity, especially in the lateral roots. With non-denaturing polyacrylamide-gel isoelectric focussing, simultaneously active charge-isomers of GS were separated in both leaves and roots. The leaf isoforms were active in an octameric and also in a tetrameric form. In the root only octameric isoforms were found. The tetramer was more active than the octamer in the leaf blade and vice versa in the leaf stem. Only the tetramer needed -mercaptoethanol for activity stabilization in vitro. A reactivation, however, of an inactive tetramer by the addition of thiol/thioredoxin was not possible. The same isoforms of GS were separated in different organs of sugarbeet but with different patterns of relative activity. The activity pattern depended also on the N-source of the plant. With increasing age of the plant the number of active GS isoforms declined in both leaves and roots although the in-vitro activity remained unchanged (NO ₃ ^– -fed plants) or even increased (NH ₄ ⁺ -fed plants).Abbreviations GS glutamine synthetase (E.C. 6.3.1.2.) - IEF isoelectric focussing - PAGE polyacrylamide gel electrophoresis This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.     

Glutamine synthetase from mesophyll and bundle sheath maize cells: isoenzyme complements and different sensitivities to phosphinothricin

 Anion-exchange FPLC has been used to resolve the isoforms of glutamine synthetase (GS, EC 6.3.1.2) from Zea mays mesophyll (MC) and bundle sheath cells (BSC). Two different isoforms were detected in both types of photosynthetic cells. The predominantly active isoform was GS1 (61%) in MC and GS2 (67%) in BSC. The relative contribution of GS1 and GS2 to the overall GS activity in BSC in maize here reported resembles the proportion described for most C3 plants. Differences among these isoforms in terms of their susceptibility to phosphinothricin (PPT), an analogue of glutamate and known inhibitor of GS, were found. The GS1 isoenzyme from MC was the most sensitive form, being inhibited by 50% at approximately 2.0 μM DL-PPT, whereas the GS2 from BSC presented the highest tolerance to the inhibitor (I₅₀=30 μM). The transferase-to-semibiosynthetic activity ratio for the MC isoforms, which was higher than the ratio for the BSC isoforms, and the differences shown by the isoforms in susceptibility to PPT predict important differences in the biochemical properties and regulation of GS isoenzymes. In this regard, the cytoplasmic isoenzymes, and especially the one in MC, due to its relatively high contribution to mesophyll cell GS activity, could play a vital role in nitrogen metabolism in maize. Received: 1 December 1999 / Revised: 7 February 2000 / Accepted: 23 February 2000     

Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants

Over‐expression of glutamine synthetase (GS, EC 6.3.1.2), a key enzyme in nitrogen assimilation, may be a reasonable approach to enhance plant nitrogen use efficiency. In this work phenotypic and biochemical characterizations of young transgenic poplars showing ectopic expression of a pine cytosolic GS transgene in photosynthetic tissue (Gallardo et al., Planta 210, 19–26, 1999) are presented. Analysis of 22 independent transgenic lines in a 6 month greenhouse study indicated that expression of the pine GS transgene affects early vegetative growth and leaf morphology. In comparison with non‐transgenic controls, transgenic trees exhibited significantly greater numbers of nodes and leaves (12%), and higher average leaf length and width resulting in an increase in leaf area (25%). Leaf shape was not altered. Transgenic poplars also exhibited increased GS activity (66%), chlorophyll content (33%) and protein content (21%). Plant height was correlated with GS content in young leaves, suggesting that GS can be considered a marker for vegetative growth. Molecular and kinetic characterization of GS isoforms in leaves indicated that poplar GS isoforms are similar to their counterparts in herbaceous plants. A new GS isoenzyme that displayed molecular and kinetic characteristics corresponding to the octomeric pine cytosolic GS1 was identified in the photosynthetic tissues of transgenic poplar leaves. These results indicate that enhanced growth and alterations in biochemistry during early growth are the consequence of transgene expression and assembly of pine GS1 subunits into a new functional holoenzyme in the cytosol of photosynthetic cells.