Characterization of Ferredoxin-Dependent Glutamine-Oxoglutarate Amidotransferase (Fd-GOGAT) Genes and Their Relationship with Grain Protein Content QTL in Wheat

Background

In higher plants, inorganic nitrogen is assimilated via the glutamate synthase cycle or GS-GOGAT pathway. GOGAT enzyme occurs in two distinct forms that use NADH (NADH-GOGAT) or Fd (Fd-GOGAT) as electron carriers. The goal of the present study was to characterize wheat Fd-GOGAT genes and to assess the linkage with grain protein content (GPC), an important quantitative trait controlled by multiple genes.

Results

We report the complete genomic sequences of the three homoeologous A, B and D Fd-GOGAT genes from hexaploid wheat (Triticum aestivum) and their localization and characterization. The gene is comprised of 33 exons and 32 introns for all the three homoeologues genes. The three genes show the same exon/intron number and size, with the only exception of a series of indels in intronic regions. The partial sequence of the Fd-GOGAT gene located on A genome was determined in two durum wheat (Triticum turgidum ssp. durum) cvs Ciccio and Svevo, characterized by different grain protein content. Genomic differences allowed the gene mapping in the centromeric region of chromosome 2A. QTL analysis was conducted in the Svevo×Ciccio RIL mapping population, previously evaluated in 5 different environments. The study co-localized the Fd-GOGAT-A gene with the marker GWM-339, identifying a significant major QTL for GPC.

Conclusions

The wheat Fd-GOGAT genes are highly conserved; both among the three homoeologous hexaploid wheat genes and in comparison with other plants. In durum wheat, an association was shown between the Fd-GOGAT allele of cv Svevo with increasing GPC - potentially useful in breeding programs.     

Mn<Superscript>2+</Superscript> enhances theanine-forming activity of recombinant glutamine synthetase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4 mM IPTG in LB medium, and maximal theanine-forming activity of the recombinant GS induced in LB is 6.4 U/mg at a series concentration (0–100 mM) of Mn²⁺ at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9-A (details are described in “Materials and methods”) and 0.1% (w/v) lactose were selected as culture medium and inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the specific activity of the recombinant GS is 6.2 U/mg which is approximate to that (6.4 U/mg) induced in LB in the presence of 10 mM Mn²⁺ at optimal pH 7.5. The activity is markedly higher activated by Mn²⁺ than that by other nine bivalent cations. Furthermore, M9-B (5 μM Mn²⁺ was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced in M9-B was improved 20% (up to 7.6 U/mg). Finally, theanine production experiment coupled with yeast fermentation system was carried out in a 1.0 ml reaction system with 0.1 mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and 13.1 g/L by paper chromatography and HPLC, respectively.     

Relationships between grain protein content and grain yield components through quantitative trait locus analyses in a recombinant inbred line population derived from two elite durum wheat cultivars

Grain protein content (GPC) in durum wheat (Triticum turgidum var. durum) is negatively correlated with grain yield. To evaluate possible genetic interrelationships between GPC and grain yield per spike, thousand-kernel weight and kernel number per spike, quantitative trait loci (QTL) for GPC were mapped using GPC-adjusted data in a covariance analysis on yield components. Phenotypic data were evaluated in a segregating population of 120 recombinant inbred lines derived from crossing the elite cultivars Svevo and Ciccio. The material was tested at five environments in southern Italy. QTL were determined by composite interval mapping based on the Svevo?×?Ciccio linkage map described in Gadaleta et al. (2009) and integrated with DArT markers. The close relationship between GPC and yield components was reflected in the negative correlation between the traits and in the reduction of variance when GPC values were adjusted to yield components. Ten independent genomic regions involved in the expression of GPC were detected, six of which were associated with QTL for one or more grain yield components. QTL alleles with increased GPC effects were associated with QTL alleles with decreased effects on one or more yield component traits, or vice versa (i.e. the allelic effects were in opposite direction). Four QTL for GPC showed always significant effects, and these QTL should represent genes that influence GPC independently from variation in the yield components. Such genes are of special interest in wheat breeding since they would allow an increase in GPC without a concomitant decrease in grain yield.     

Cloning and characterization of the ribosomal protein L3 (<Emphasis Type="Italic">RPL3</Emphasis>) gene family from <Emphasis Type="Italic">Triticum aestivum</Emphasis>

Plant pathogenic fungi of the genus Fusarium can cause severe diseases on small grain cereals and maize. The contamination of harvested grain with Fusarium mycotoxins is a threat to human and animal health. In wheat production of the toxin deoxynivalenol (DON), which inhibits eukaryotic protein biosynthesis, is a virulence factor of Fusarium, and resistance against DON is considered to be part of Fusarium resistance. Previously, single amino acid changes in RPL3 (ribosomal protein L3) conferring DON resistance have been described in yeast. The goal of this work was to characterize the RPL3 gene family from wheat and to investigate the potential role of naturally existing RPL3 alleles in DON resistance by comparing Fusarium-resistant and susceptible cultivars. The gene family consists of three homoeologous alleles of both RPL3A and RPL3B, which are located on chromosomes 4A (RPL3-B2), 4B (RPL3-B1), 4D (RPL3-B3), 5A (RPL3-A3), 5B (RPL3-A2) and 5D (RPL3-A1). Alternative splicing was detected in the TaRPL3-A2 gene. Sequence comparison revealed no amino acid differences between cultivars differing in Fusarium resistance. While using developed SNP markers we nevertheless found that one of the genes, namely, TaRPL3-A3 mapped close to a Fusarium resistance QTL (Qfhs.ifa-5A). The potential role of the RPL3 gene family in DON resistance of wheat is discussed.     

The two nitrogen mobilisation- and senescence-associated <Emphasis Type="Italic">GS1</Emphasis> and <Emphasis Type="Italic">GDH</Emphasis> genes are controlled by C and N metabolites

In tobacco, the two enzymes of nitrogen metabolism, cytosolic glutamine synthetase (GS1; E.C.6.3.1.2) and glutamate dehydrogenase (GDH; E.C.1.4.1.2), are induced during leaf senescence, whereas the chloroplastic glutamine synthetase (GS2; E.C.6.3.1.2) and nitrate reductase (NR; E.C.1.6.1.1) are repressed in the course of ageing. In this report, we showed in discs of fully expanded Nicotiana tabacum L. cv. Xanthi leaves that sucrose (Suc) and amino acids were involved in the regulation of the expression of GS1 and GDH genes. Suc induced the expression of GS1 and repressed that of GDH. Therefore, we concluded that in response to Suc, GS1 behaved as an early Senescence Associated Gene (SAG), whereas GDH behaved as a late SAG. Moreover, amino acids induced the expression of both genes. Among the amino acids tested as signal molecules, proline (Pro) and glutamate (Glu) were major inducers of GDH and GS1 expression, respectively. Interestingly, an opposite regulation of GS1 and GS2 by Pro and Glu was shown. The contrary effect of Suc on NIA (NR encoding gene) and GDH mRNA accumulation was also emphasized.     

Fitness analyses of Arabidopsis thaliana mutants depleted of FtsH metalloproteases and characterization of three FtsH6 deletion mutants exposed to high light stress, senescence and chilling

Glutamine synthetase (GS) plays a key role in the growth, nitrogen (N) use and yield potential of cereal crops. Investigating the haplotype variation of GS genes and its association with agronomic traits may provide useful information for improving wheat N-use efficiency and yield. We isolated the promoter and coding region sequences of the plastic glutamine synthetase isoform (GS2) genes located on chromosomes 2A, 2B and 2D in bread wheat. By analyzing nucleotide sequence variations of the coding region, two, six and two haplotypes were distinguished for TaGS2-A1 (a and b), TaGS2-B1 (a-f) and TaGS2-D1 (a and b), respectively. By analyzing the frequency data of different haplotypes and their association with N use and agronomic traits, four major and favorable TaGS2 haplotypes (A1b, B1a, B1b, D1a) were revealed. These favorable haplotypes may confer better seedling growth, better agronomic performance, and improved N uptake during vegetative growth or grain N concentration. Our data suggest that certain TaGS2 haplotypes may be valuable in breeding wheat varieties with improved agronomic performance and N-use efficiency.     

MicroRNAs in durum wheat seedlings under chronic and short-term nitrogen stress

Nitrogen is an essential macronutrient for plant growth and reproduction. In durum wheat, an appropriate nitrogen soil availability is essential for an optimal seed development. miRNAs contribute to the environmental change adaptation of plants through the regulation of important genes involved in stress processes. In this work, nitrogen stress response was evaluated in durum wheat seedlings of Ciccio and Svevo cultivars. Eight small RNA libraries from leaves and roots of chronically stressed plants were sequenced to detect conserved and novel miRNAs. A total of 294 miRNAs were identified, 7 of which were described here for the first time. The expression level of selected miRNAs and target genes was analyzed by qPCR in seedlings subjected to chronic (Ciccio and Svevo, leaves and roots) or short-term (Svevo roots) stress conditions. Some miRNAs showed an immediate stress response, and their level of expression was either maintained or returned to a basal level during a long-term stress. Other miRNAs showed a gradual up- or downregulation during the short-term stress. The newly identified miRNA ttu-novel-106 showed an immediate strongly downregulation after nitrogen stress, which was negatively correlated with the expression of MYB-A, its putative target gene. PHO2 gene was significantly upregulated after 24–48-h stress, corresponding to a downregulation of miR399b. Ttu-miR399b putative binding sites in the 5′ UTR region of the Svevo PHO2 gene were identified in the A and B genomes. Both MYB-A and PHO2 genes were validated for their cleavage site using 5′ RACE assay.     

Synthesis of glutamine and glutamate by intact bundle-sheath cells of maize (Zea mays L.)

Intact bundle-sheath cells with functional plasmodesmata were isolated from leaves of Zea mays L. cv. Mutin, and the capacity of these cells to synthesize glutamine and glutamate was determined by simulating physiological substrate concentrations in the bathing medium. The results show that glutamine synthetase can operate at full rate in the presence of added 8 mM ATP. At lower concentrations of ATP a higher rate of glutamine synthesis was found in the light than in darkness. Glutamate-synthase activity, on the other hand, was strictly light dependent. It appears that in bundle-sheath cells of maize the nitrate-assimilatory capacities of glutamine synthetase (located mainly in the cytosol) and of glutamate synthase (located in the stroma) are high enough to meet the demands of whole maize leaves.Abbreviations Gln glutamine - Glu glutamate - GOGAT glutamate synthase - GS glutamine synthetase - 2-OG 2-oxoglutarate This work was supported by the Bundesminister für Forschung und Technologie (0319296A). We thank Mr. Bernd Raufeisen for the art work of Fig. 1.     

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     

Cloning,characterization and expression analysis of tonoplast intrinsic proteins and glutamine synthetase in ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Perennial ryegrass (Lolium perenne L.) is the most important turf and forage grass species of the temperate regions. It requires substantial input of nitrogen fertilizer for optimum yield. Improved nitrogen use efficiency (NUE) is therefore one of the main breeding targets. However, limited knowledge is currently available on the genes controlling NUE in perennial ryegrass. The aim of the present study was to isolate genes involved in ammonium transport and assimilation. In silico screening of a Lolium EST-library using known sequences of tonoplast intrinsic proteins (TIPs) and cytosolic glutamine synthetase (GS1) revealed a number of homologous sequences. Using these sequences, primers were designed to obtain the full-length sequences by RACE-PCR. Three TIP genes (LpTIP1;1, LpTIP1;2 and LpTIP2;1) and two GS genes (LpGS1a and LpGS1b) were isolated. Characterization in S. cerevisiae confirmed a function in ammonium transport for LpTIP1;1 and LpTIP2;1 and in synthesis of glutamine for LpGS1a and LpGS1b. Cytoimmunochemical studies showed that GS protein was present in the chloroplasts and cytosol of leaf cells, while TIP1 proteins localized to the tonoplast. At the expression level, Lolium GS1 genes responded to N starvation and re-supply in a manner consistent with functions in primary N assimilation and N remobilization. Similarly, the expression of LpTIPs complied with a role in vacuolar ammonium storage. Together, the reported results provide new understanding of the genetic basis for N assimilation and storage in ryegrass.     

Inhibition of F plasmid replication in htpR mutants of Escherichia coli deficient in sigma 32 protein

Summary In Drosophila melanogaster there are two glutamine synthetase (GS) (EC 6.3.1.2) isozymes. They are called GSI and GSII. The two enzymes have different subunits and different genetic determination. A DNA fragment that comprises 80% of the coding region of the glutamine synthetase gene of Chinese hamster ovary (CHO) cells allowed the identification and cloning of an homologous DNA fragment of Drosophila. This sequence is located at the 10B8-11 region on the X chromosome. Dose variation of a chromosomal segment from 9F3 to 10C1-2, which encompasses the 10B region, leads to proportional variations of GSII without apparently influencing the amount of GSI.     

The asthma candidate gene <Emphasis Type="Italic">NPSR1</Emphasis> mediates isoform specific downstream signalling

Background

Neuropeptide S Receptor 1 (NPSR1, GPRA, GPR154) was first identified as an asthma candidate gene through positional cloning and has since been replicated as an asthma and allergy susceptibility gene in several independent association studies. In humans, NPSR1 encodes two G protein-coupled receptor variants, NPSR1-A and NPSR1-B, with unique intracellular C-termini. Both isoforms show distinct expression pattern in asthmatic airways. Although NPSR1-A has been extensively studied, functional differences and properties of NPSR1-B have not yet been clearly examined. Our objective was to investigate downstream signalling properties of NPSR1-B and functional differences between NPSR1-A and NPSR1-B.

Methods

HEK-293 cells transiently overexpressing NPSR1-A or NPSR1-B were stimulated with the ligand neuropeptide S (NPS) and downstream signalling effects were monitored by genome-scale affymetrix expression-arrays. The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²⁺ assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays.

Results

NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells.

Conclusions

We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B. Our findings suggest an isoform-specific link to pathogenetic processes in asthma and allergy.

     

Cytoplasmic glutamine synthetase gene expression regulates larval development in Bactrocera dorsalis (Hendel)

In insects, glutamine synthetase (GS), a key enzyme in the synthesis of glutamine, has been reported to be associated with embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on postembryonic development. In this study, we demonstrate that blocking the activity of GS in the oriental fruit fly (Bactrocera dorsalis) with use of a GS‐specific inhibitor (L‐methionine S‐sulfoximine), led to a significant delay in larval development, pupal weight loss, and inhibition of pupation. We further identify cloned and characterized two GS genes (BdGS‐c and BdGS‐m) from B. dorsalis. The two GS genes identified in B. dorsalis were predicted to be located in the cytosol (BdGS‐c) and mitochondria (BdGS‐m), and homology analysis indicated that both genes were similar to homologs from other Dipterans, such as Drosophila melanogaster and Aedes aegypti. BdGS‐c was highly expressed in the larval stages, suggesting that cytosolic GS plays a predominant role in larval development. Furthermore, RNA interference experiments against BdGS‐c, to specifically decrease the expression of cytosolic GS, resulted in delay in larval development as well as pupal weight loss. This study presents the prominent role played by BdGS‐c in regulating larval development and suggests that the observed effect could have been modulated through ecdysteroid synthesis, agreeing with the reduced expression of the halloween gene spook. Also, the direct effects of BdGS‐c silencing on B. dorsalis, such as larval lethality, delayed pupation, and late emergence, can be further exploited as novel insecticide target in the context of pest management.     

Hairy roots of Brassica napus: I. Applied glutamine overcomes the effect of phosphinothricin treatment

Summary Hairy roots of Brassica napus (rape cv. Giant) were produced by cocultivating leaf and cotyledon explants with Agrobacterium rhizogenes strain A4T. The hairy roots grew prolifically on solid and in liquid media. Incorporation of ammonium sulphate or phosphinothricin (PPT) into the media reduced growth. PPT treatment reduced glutamine synthetase (GS) activity and increased the ammonia content of the hairy roots. We have found that PPT treatment also induces a loss of glutamine from the roots and this may influence root growth. To test this we grew hairy roots in a liquid medium containing 10 mM glutamine. This glutamine treatment overcame the PPT induced suppression of growth but also significantly increased GS activity, reduced ammonia accumulation and increased the levels of glutamate and asparagine.     

Genetic,molecular and developmental analysis of the glutamine synthetase isozymes ofDrosophila melanogaster

The glutamine synthetase isozymes ofDrosophila melanogaster offer an attractive model for the study of the molecular genetics and evolution of a small gene family encoding enzymatic isoforms that evolved to assume a variety of specific and sometimes essential biological functions. InDrosophila melanogaster two GS. isozymes have been described which exhibit different cellular localisation and are coded by a two-member gene family. The mitochondrial GS structural gene resides at the 21B region of the second chromosome, the structural gene for the cytosolic isoform at the 10B region of the X chromosome. cDNA clones corresponding to the two genes have been isolated and sequenced. Evolutionary analysis data are in accord with the hypothesis that the twoDrosophila glutamine synthetase genes are derived from a duplication event that occurred near the time of divergence between Insecta and Vertebrata. Both isoforms catalyse all reactions catalysed by other glutamine synthetases, but the different kinetic parameters and the different cellular compartmentalisation suggest strong functional specialisation. In fact, mutations of the mitochondrial GS gene produce embryo-lethal female sterility, defining a function of the gene product essential for the early stages of embryonic development. Preliminary results show strikingly distinct spatial and temporal patterns of expression of the two isoforms at later stages of development.     

Relationships between nitrogenase,glutamine synthetase,glutamine, and energy charge in Azotobacter vinelandii

When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.Abbreviations GS glutamine synthetase - GS-S(Mg) Mg²⁺ dependent synthetic activity of GS - GS-T(Mn) Mn²⁺ dependent transferase activity of GS     

Isolation of Two Streptothricin-like Antibiotics,Nos. 6241-A and B,as Inhibitors of de novo Starch Synthesis and Their Herbicidal Activity

Two streptothricin-like antibiotics, Nos. 6241-A and B, were obtained as inhibitors of de novo starch synthesis in excised leaf segments of barnyard millet (Panicum crus-galli). No. 6241-B was identified as SF-701, and No. 6241-A was a new antibiotic, in which N,N-dimethylglycine was substituted for the N-methylglycine of No. 6241-B (SF-701). Both antibiotics also inhibited plant growth. The inhibitory activity of No. 6241-B (SF-701) was approximately ten times that of No. 6241-A. By foliar treatment, No. 6241-B (SF-701) showed remarkable herbicidal activity against barnyard millet at a concentration of more than 500 ppm with little phytotoxicity for the rice plant (Oryza sativa L. cv. Nihonbare).