Changes in the cellular and subcellular localization of glutamine synthetase and glutamate dehydrogenase during flag leaf senescence in wheat (Triticum aestivum L.)

In order to improve our understanding of the regulation of nitrogen assimilation and recycling in wheat (Triticum aestivum L.), we studied the localization of plastidic (GS2) and cytosolic (GS1) glutamine synthetase isoenzymes and of glutamate dehydrogenase (GDH) during natural senescence of the flag leaf and in the stem. In mature flag leaves, large amounts of GS1 were detected in the connections between the mestome sheath cells and the vascular cells, suggesting an active transfer of nitrogen organic molecules within the vascular system in the mature flag leaf. Parallel to leaf senescence, an increase of a GS1 polypeptide (GS1b) was detected in the mesophyll cytosol of senescing leaves, while the GS protein content represented by another polypetide (GS1a) in the phloem companion cells remained practically constant in both leaves and stems. Both GDH aminating activity and protein content were strongly induced in senescing flag leaves. The induction occurred both in the mitochondria and in the cytosol of phloem companion cells, suggesting that the shift in GDH cellular compartmentation is important during leaf nitrogen remobilization although the metabolic or sensing role of the enzyme remains to be elucidated. Taken together, our results suggest that in wheat, nitrogen assimilation and recycling are compartmentalized between the mesophyll and the vasculature, and are shifted in different cellular compartments within these two tissues during the transition of sink leaves to source leaves.     

Distribution of two isoforms of glutamine synthetase in bundle sheath and mesophyll cells of corn leaves

Localization of two isoforms of glutamine synthetase (GS; EC 6.3.1.2) was investigated in different cell types, mesophyll cells and bundle sheath cells, of corn ( Zea mays L. var. W64A × W182E) leaves by using ion exchange chrotnatography. In whole leaf extracts, relative activities of GS1 (cytosolic GS) and GS2 (chloroplastic GS) were almost equal. Purified mesophyll protoplasts and bundle sheath strands also showed similar proportions of GS1 and GS2. Methionine sulfoximine (1 mM ) enhanced the accumulation of ammonia when mesophyll protoplasts were incubated with nitrite or when bundle sheath strands were incubated with glycine. This clearly indicates a spatial separation of metabolism of NH⁺₄ derived from photorespiration and from reduction of NOJ.     

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.     

不同氮源对小麦幼苗谷氨酰胺合成酶的影响

利用ＤＥＡＥ－纤维素柱层析、酶活性测定、Ｎｏｒｔｈｅｒｎ 分子杂交等技术,研究了小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）幼苗的根、叶和离体叶在不同氮源培养条件下谷氨酰胺合成酶（ＧＳ）活性和同工酶变化, 以及不同氮源对ＧＳ基因转录－ＧＳ－ｍ ＲＮＡ 的影响． 同时与硝酸还原酶（ＮＲ）活性进行比较, 结果表明∶当以ＮＨ＋４ 作唯一氮源时,小麦幼苗根谷氨酰胺合成酶（ＧＳｒ）和叶细胞质谷氨酰胺合成酶（ＧＳ１）活性要比以ＮＯ－３ 作唯一氮源的高．当以ＮＯ－３ 为唯一氮源时, ＮＯ－３ 则促进完整叶片和离体叶片叶绿体谷氨酰胺合成酶（ＧＳ２）活性． 从转录水平上看,ＮＨ＋４ 促进根ＧＳ－ｍ ＲＮＡ 的合成,而ＮＯ－３ 促进叶ＧＳ－ｍ ＲＮＡ 的合成     

Changes in the activities of chloroplast and cytosolic isoenzymes of glutamine synthetase during normal leaf growth and plastid development in wheat

Soluble protein extracts and chloroplasts from a serial sequence of transverse sections of a 7-d-old wheat leaf (Triticum aestivum cv. Maris Huntsman) were used to study changes in the activity of glutamine synthetase (GS; EC 6.3.1.2) during cell and chloroplast development. Glutamine synthetase activity increased more than 50-fold per cell from the base to the tip of the wheat leaf. Two isoenzymes of GS were separated using fast protein liquid chromatography (FPLC). Glutamine synthetase localized in the cytoplasm (GS1) eluted at about 0.21 M NaCl, and the isoenzyme localized in the chloroplast (GS2) eluted at about 0.33 M NaCl. The increase in GS activity during leaf development was found to be caused primarily by an increase in the activity of the chloroplast GS2. The activity of the cytoplasmic GS1 remained constant as the cells were displaced from the base to the tip of the leaf, whereas GS2 activity increased within the chloroplast throughout development. At the base of the leaf, 26% of total GS activity was cytoplasmic; the remaining 74% was in the chloroplast. At 10 cm from the base, only 4% of the activity was cytoplasmic, and 96% was in the chloroplast. The results indicate that the chloroplast GS2 is probably responsible for most of the ammonia assimilation in the mature wheat leaf, whereas cytoplasmic GS1 may serve a role in immature developing leaf cells.Abbreviations FPLC fast protein liquid chromatography - GS glutamine synthetase - GS1 cytoplasmic glutamine synthetase - GS2 chloroplast glutamine synthetase     

The genetics of nitrogen use in hexaploid wheat: N utilisation, development and yield

A genetic study is presented for traits relating to nitrogen use in wheat. Quantitative trait loci (QTLs) were established for 21 traits relating to growth, yield and leaf nitrogen (N) assimilation during grain fill in hexaploid wheat (Triticum aestivum L.) using a mapping population from the cross Chinese Spring × SQ1. Glutamine synthetase (GS) isozymes and estimated locations of 126 genes were placed on the genetic map. QTLs for flag leaf GS activity, soluble protein, extract colour and fresh weight were found in similar regions implying shared control of leaf metabolism and leaf size. Flag leaf traits were negatively associated with days to anthesis both phenotypically and genetically, demonstrating the complex interactions of metabolism with development. One QTL cluster for GS activity co-localised with a GS2 gene mapped on chromosome 2A, and another with the mapped GSr gene on 4A. QTLs for GS activity were invariably co-localised with those for grain N, with increased activity associated with higher grain N, but with no or negative correlations with grain yield components. Peduncle N was positively correlated, and QTLs co-localised, with grain N and flag leaf N assimilatory traits, suggesting that stem N can be indicative of grain N status in wheat. A major QTL for ear number per plant was identified on chromosome 6B which was negatively co-localised with leaf fresh weight, peduncle N, grain N and grain yield. This locus is involved in processes defining the control of tiller number and consequently assimilate partitioning and deserves further examination. Electronic Supplementary Material The online version of this article () contains supplementary material, which is available to authorized users.     

土壤水分对强筋小麦‘豫麦34’氮素同化酶活性和籽粒品质的影响

 以强筋型小麦(Triticum aestivum)品种‘豫麦34号’为材料,采用盆栽方法研究了土壤水分对氮素同化酶活性及籽粒品质的影响。结果表明：旗叶硝酸还原酶（NR）活性于花后呈下降趋势,且土壤含水量为田间持水量（FC）60%的处理活性最强,其次为40%FC,活性最低的是80%FC。旗叶和籽粒中谷氨酰胺合成酶（GS）活性于开花15 d前均呈下降趋势,15 d后均为上升趋势,各水分处理间酶活性大小关系是：80%FC＞60%FC＞40%FC。各水分处理间旗叶和籽粒谷氨酸合成酶（GOGAT）活性的大小关系同GS。60%FC籽粒产量及品质最优,80%FC产量次之,40%FC产量最低;40%FC品质次之,80%FC品质最低。不同水分处理下籽粒蛋白质含量与叶片NR、GS 和籽粒GOGAT活性均呈正相关,与旗叶GOGAT活性呈负相关。且40%FC和80%FC下籽粒蛋白质含量只与旗叶GS活性相关性达显著水平, 60%FC下蛋白质含量则与旗叶NR和籽粒GS活性均达显著相关,与旗叶GS活性达极显著相关。     

不同环境条件下小麦氮代谢关键酶活性及籽粒品质

研究了两种环境条件下3个不同蛋白含量小麦品种的氮代谢关键酶活性及籽粒品质的差异.结果表明,龙口试验点的小麦旗叶硝酸还原酶(NR)、谷胺酰氨合成酶(GS)和籽粒谷胺酰氨合成酶活性均显著高于泰安试验点,3个品种间的酶活性顺序均为：济麦20＞优麦3号＞PH971942.优质强筋小麦品种的籽粒综合品质性状在龙口试验点的表现优于泰安试验点. 灌浆期环境因素与小麦籽粒品质和酶活性存在显著的相关性,灌浆期间的较高气温、适当干旱和寡照环境有利于提高小麦籽粒品质.龙口试验点的中、强筋小麦品种和泰安试验点的中筋小麦品种蛋白质含量与旗叶NR和GS活性均达显著正相关.小麦品种用途不同,对环境条件的要求不同,适宜的环境条件提高了氮代谢关键酶的活性,利于改善小麦品质.     

Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production

The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.     

花后土壤水分状况对小麦籽粒淀粉和蛋白质积累关键调控酶活性的影响

在温室盆栽条件下,以2个不同蛋白质含量的冬小麦(Triticum aestivum L．)品种皖麦38和扬麦9为材料,研究了花后第4天开始的土壤干旱(SRWC=45％～50％)和渍水对籽粒蛋白质和淀粉积累关键调控酶活性的影响。小麦叶片和籽粒的测定结果均表明,小麦源库器官中籽粒蛋白质和淀粉积累的关键调控酶活性变化趋势在2个品种间基本一致。与对照(SRWC=75％～80％)相比,干旱和渍水均明显降低了花后旗叶中蔗糖含量和磷酸蔗糖合成酶(SPS)活性,而氨基酸含量和谷氨酰胺合成酶(GS)活性略有下降。干旱和渍水均降低了籽粒库蔗糖合成酶(SS)和结合态淀粉合成酶(GBSS)活性,可溶性淀粉合成酶(SSS)活性降低尤甚。其中干旱处理下SS的下降比渍水更为明显。与对照相比,渍水明显降低了籽粒谷丙转氨酶(GPT)和GS活性,而干旱的影响较小。相关性分析结果表明籽粒淀粉产量和含量与SPS,SSS和GBSS活性的关系比与SS活性的关系更为密切,籽粒蛋白质产量和含量与叶中GS和籽粒中GPT活性的关系比与籽粒中GS关系活性更为密切。这些结果表明小麦源库器官中调控籽粒蛋白质和淀粉积累的关键酶活性变化是花后不同水分状况影响籽粒淀粉和蛋白质特性的重要因素。     

籼稻桂华占、八桂香花后营养器官干物质流转与籽粒灌浆关系

以桂华占和八桂香2个籼稻品种为材料,研究籼稻花后不同部位器官物质积累、运转与籽粒生长的动态特征及相互关系。结果表明:(1)叶、叶鞘、节间干物质流转存在一定差异,倒2叶鞘对籽粒的贡献超过倒3叶鞘和倒1叶鞘,倒3节间对籽粒的贡献超过倒2节间和倒1节间;(2)不同部位籽粒的灌浆速率和拐点粒重呈现UPG(上部籽粒)MPG(中部籽粒)BPG(下部籽粒)变化趋势,拐点时间和活跃灌浆时间及持续灌浆时间均呈现BPGMPGUPG变化规律,UPG启动早,灌浆速率大,BPG的灌浆速率小,灌浆时间滞后,籽粒粒重呈现UPGMPGBPG;(3)叶片、叶鞘及节间干物质运转速度和运转率都与籽粒起始灌浆势呈正相关,其中器官间与起始灌浆势的相关系数大小表现为节间叶鞘叶片,不同叶位间与起始灌浆势的相关系数大小表现为倒2叶倒3叶倒1叶,其中节间干物质运转对籽粒生长的作用大于叶鞘,叶片干物质运转与籽粒生长的相关性最小。倒1节鞘物质输出与BPG生长时间上同步,倒2节鞘与MPG生长同步,倒3节鞘与UPG生长同步。     

Lead-induced oxidative stress and role of antioxidant defense in wheat ( Triticum aestivum L.)

The aim of this study was to investigate soil lead pollution on biochemical properties and gene expression pattern of antioxidant enzymes in three wheat cultivars (Morvarid, Gonbad and Tirgan) at flag leaf sheath swollen stage. Lead (Pb(NO3)2) was used at four different concentrations (0, 15, 30 and 45 mg/kg of soil). The leaf and roots samples were taken at late-booting stage (Zadoks code, GS: 45). The results showed that lead heavy metal toxicity increased the expression of some genes and the activity of key enzymes of the antioxidant defense system in wheat. Moreover, the cell oxidation levels (MDA, LOX) enhanced under lead stress conditions. The relative gene expression and activity of antioxidant enzymes (CAT, SOD, GPX and APX) increased significantly in the both leaves and root tissues under lead stress conditions. The level of gene expression and enzymatic activity were higher in the root than the leaf tissue. There was no significant difference among cultivars in each of lead concentrations but Morvarid and Tirgan cultivars had more tolerance to toxic concentrations of lead when compared to Gonbad cultivar.     

Cytosolic localization in tomato mesophyll cells of a novel glutamine synthetase induced in response to bacterial infection or phosphinothricin treatment

In tomato (Lycopersicon esculentum Mill.) leaves, the predominant glutamine synthetase (GS; EC 6.3.1.2) is chloroplastic (GS2; 45 kDa) whereas the cytosolic isoform (GS1; 39 kDa) is represented as a minor enzyme. Following either infection by Pseudomonas syringae pv. tomato (Pst) or treatment with phosphinothricin (PPT), a GS inhibitor, GS1 accumulated in the leaves. In contrast to healthy control leaves, where GS1 was restricted to the veins, in infected and PPT-treated leaves the GS1 polypeptide was also detected in the leaf blade; moreover, it was more abundant than GS2. Different immunological approaches were therefore used to investigate whether or not the GS1 polypeptide expressed in Pst-infected and PPT-treated tomato leaves was distributed among different tissues and subcellular compartments in the same way as the constitutive GS1 expressed in healthy leaves. By tissue-printing analysis, a similar GS immunostaining was observed in epidermis, mesophyll and phloem of leaflet midrib cross-sections of control, infected and PPT-treated leaves. Immunocytochemical localization revealed that GS protein was present in the chloroplast of mesophyll cells and the cytoplasm of phloem cells in healthy leaves; however, in Pst-infected or PPT-treated leaves, a strong labelling was observed in the cytoplasm of mesophyll cells. Two-dimensional analysis of GS polypeptides showed that, in addition to the constitutive GS1, a GS1 polypeptide different in charge was present in tomato leaflets after microbial infection or herbicide treatment. All these results indicate that a novel cytosolic GS is induced in mesophyll cells of Pst-infected or PPT-treated leaves. A possible role for this new cytosolic GS in the remobilization of leaf nitrogen during infection is proposed. Received: 16 January 1998 / Accepted 21 April 1998