Glutamine Synthetase Gene Research Articles

Gene Therapy for Treatment of Chronic Hyperammonemia in a Rat Model of Hepatic Encephalopathy

Introduction and aim. Hepatic encephalopathy (HE), caused by hyperammonemia resulting from liver disease, is a spectrum of neuropsychiatric and motor disorders that can lead to death. Existing therapies are deficient and alternative treatments are needed. We have shown that gene therapy with a baculovirus vector containing the glutamine synthetase (Bac-GS) gene is efficient for reducing ammonia levels in an acute hyperammonemia rat model. However, the most common condition resulting from liver disease is chronic hyperammonemia. In this work, Bac-GS was evaluated in bile-duct ligated rats, a chronic liver disease model with hyperammonemia and some characteristics of Type C HE.Material and methods. Bac-GS was tested for mediating GS over-expression in HeLa cells and H9C2 myotubes. For determining the utility of Bac-GS for the reduction of ammonia levels in a chronic hyperammonemia animal model, four groups of rats were treated: control, sham, ligated with Bac-GS and ligated with Bac-GFP. Baculoviruses were injected i.m. 18 days post-surgery. Blood was drawn 2, 3 and 4 weeks post-surgery and plasma ammonia concentrations were quantified.Results. In protein lysates of cells and myotubes transduced with Bac-GS, a 44 kDa band corresponding to GS was detected. Significant results were obtained in the hyperammonemic bile-duct ligated rat model, as plasma ammonia was reduced to normal levels 3 days after treatment with Bac-GS. Furthermore, a transitory effect of Bac-GS was observed.Conclusion. Our results show that gene therapy by delivering GS is a promising alternative for treatment of hyperammonemia in acute-on-chronic liver failure patients with HE.

Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis

In this study, a novel gene for Glutamine synthetase was cloned and characterized for its activities and stabilities from a marine bacterium Providencia vermicola (PveGS). A mutant S54A was generated by site directed mutagenesis, which showed significant increase in the activity and stabilities at a wide range of temperatures. The Km values of PveGS against hydroxylamine, ADP-Na2 and L-Glutamine were 15.7 ± 1.1, (25.2 ± 1.5) × 10−5 and 32.6 ± 1.7 mM, and the kcat were 17.0 ± 0.6, 9.14 ± 0.12 and 30.5 ± 1.0 s−1 respectively. In-silico-analysis revealed that the replacement of Ser at 54th position with Ala increased the catalytic activity of PveGS. Therefore, catalytic efficiency of mutant S54A had increased by 3.1, 0.89 and 2.9-folds towards hydroxylamine, ADP-Na2 and L-Glutamine respectively as compared to wild type. The structure prediction data indicated that the negatively charged pocket becomes enlarged and hydrogen bonding in Ser54 steadily promotes the product release. Interestingly, the residual activity of S54A mutant was increased by 10.7, 3.8 and 3.8 folds at 0, 10 and 50 °C as compared to WT. Structural analysis showed that S54A located on the loop near to the active site improved its flexibility due to the breaking of hydrogen bonds between product and enzyme. This also facilitated the enzyme to increase its cold adaptability as indicated by higher residual activity shown at 0 °C. Thus, replacement of Ala to Ser54 played a pivotal role to enhance the activities and stabilities at a wide range of temperatures.

The Unexpected Essentiality of glnA2 in Mycobacterium smegmatis Is Salvaged by Overexpression of the Global Nitrogen Regulator glnR, but Not by L-, D- or Iso-Glutamine.

Nitrogen metabolism plays a central role in the physiology of microorganisms, and Glutamine Synthetase (GS) genes are present in virtually all bacteria. In M. tuberculosis, four GS genes are present, but only glnA1 is essential, whereas glnA2 was shown to be non-essential for in-vitro as well as in-vivo growth and pathogenesis, and is postulated to be involved in D-glutamine and iso-glutamine synthesis. Whilst investigating the activity of an antimicrobial compound in M. smegmatis, we found a spontaneous temperature-sensitive mutant in glnA2 (I133F), and used it to investigate the role of glnA2 in M. smegmatis. We deleted the native glnA2 and replaced it with a mutated allele. This re-created the temperature sensitivity—as after 3–4 seemingly normal division cycles, glnA2 became essential for growth. This essentiality could not be salvaged by neither L, D- nor iso-glutamine, suggesting an additional role of glnA2 in M. smegmatis over its role in M. tuberculosis. We also found that overexpression of the global nitrogen regulator glnR enabled bypassing the essentiality of glnA2, allowing the creation of a complete deletion mutant. The discrepancy between the importance of glnA2 in Mtb and M. smegmatis stresses the caution in which results in one are extrapolated to the other.

First Report of Colletotrichum siamense Causing Anthracnose on Partridge tea (Mallotus oblongifolius) in China

Partridge tea (Mallotus oblongifolius [Miq.] Mull.Arg.) is an important medicinal plant in Hainan province of China used in tea making. During April 2017, circular necrotic lesions were observed on partridge tea plants with a disease incidence of 25% in an experimental station of Hainan University (110.19° E, 20.03° N) in Haikou, Hainan province, China. The lesions began as small circular spots, which later developed into larger spots (diameter of 1.2 to 2.9 mm) with a grayish white center and a dark brown banding pattern on the edges. To identify the pathogen, 10 symptomatic leaves from five plants were collected. Diseased tissue (2 × 2 mm) was cut from the margins of lesions, surface disinfected with 70% ethanol for 5 s, and rinsed four to five times with sterile distilled water. The excised sections were transferred to potato dextrose agar (PDA) plates and incubated at 28°C for 3 to 4 days in the dark. A total of six isolates of Colletotrichum sp. were obtained from diseased tissue, and three strains were randomly selected (Z1, Z4, and Z6) for further analysis. The colonies appeared white to gray on the upper surface of PDA medium at 28°C. Conidia were cylindrical to subcylindrical, rarely clavate, hyaline, and guttulate with 3.96 to 4.21 µm (width) and 12.30 to 18.90 µm (length). These morphological characteristics were compatible with those of species from the Colletotrichum gloeosporioides complex (Weir et al. 2012). For accurate identification, fungal DNA of the three isolates was extracted. Fragments of internal transcribed spacer (ITS) and genomic regions including chitin synthase (CHS-1), actin (ACT), glutamine synthetase (GS), the Apn2–Mat1–2 intergenic spacer, and the mating type (Mat1–2) gene (ApMAT) were amplified and sequenced. These nucleotide sequences were deposited in GenBank (A70814043, ITS; A7110043, CHS-1; A7110049, ACT; A7110052, GS; and A7110046, ApMAT). A BLAST search of the amplified sequences of ITS resulted in 99% similarity with those of C. siamense strain CCL8. ACT sequences resulted in 100% similarity with those of C. siamense strain ICK. CHS-1 sequences showed 99% similarity with those of C. siamense strain WC-OILPALM4. GS gene sequences showed 99% similarity with those of C. siamense strain LQ22, and the ApMAT sequences showed 100% similarity with those of C. siamense strain PAS. Phylogenetic analysis based on the ITS, ACT, CHS-1, GS, and ApMAT amplified sequences was undertaken using MEGA6 software. This analysis clustered the three isolates with the C. siamense strain CBS130417 with high bootstrap support (99%). Therefore, based on their morphological characteristics and sequence similarity data, these isolates were identified as C. siamense. Pathogenicity was confirmed by spraying 10 45-day-old partridge tea plants with a 1 × 10⁶ CFU/ml monosporic suspension. Three strains (Z1, Z4, and Z6) were used to pathogenicity test in a growth greenhouse. Three control plants were treated with sterile water only. After 6 days at 25°C and 85% relative humidity, sunken necrotic spots similar to those observed in the field developed on the inoculated plants. The control plants remained symptomless. C. siamense was successfully reisolated from lesions. C. siamense has been reported on many crops in China (Cheng et al. 2013; Liu et al. 2017; Niu et al. 2016). To our knowledge, this is the first report of anthracnose caused by C. siamense on partridge tea in China.

Overexpression of a cytosolic NADP+-isocitrate dehydrogenase causes alterations in the vascular development of hybrid poplars.

Cytosolic NADP+-isocitrate dehydrogenase (ICDH) is one of the major enzymes involved in the production of 2-oxoglutarate for amino acid biosynthesis in plants. In most plants studied, ICDH is encoded by either one gene or a small gene family, and the protein sequence has been highly conserved during evolution, suggesting it plays different and essential roles in metabolism and differentiation. To elucidate the role of ICDH in hybrid poplar (Populus tremula x P. alba), transgenic plants overexpressing the Pinus pinaster gene were generated. Overexpression of ICDH resulted in hybrid poplar (Populus tremula × P. alba) trees with higher expression levels of the endogenous ICDH gene and higher enzyme content than control untransformed plants. Transgenic poplars also showed an increased expression of glutamine synthetase (GS1.3), glutamate decarboxylase (GAD) and other genes associated with vascular differentiation. Furthermore, these plants exhibited increased growth in height, longer internodes and enhanced vascular development in young leaves and the apical region of stem. Modifications in amino acid and organic acid content were observed in young leaves of the transgenic lines, suggesting an increased biosynthesis of amino acids for building new structures and also for transport to other sink organs, as expanding leaves or young stems. Taken together, these results support an important role of ICDH in plant growth and vascular development.

Differential expression of multiple glutamine synthetase genes in air-breathing magur catfish, Clarias magur and their induction under hyper-ammonia stress

The present study demonstrates the unique presence of three different gs genes (cmgs01, cmgs02, and cmgs03) in air-breathing ureogenic magur catfish (Clarias magur), which is otherwise reported to be encoded by a single gene in higher vertebrates. Of these three genes, two (cmgs01and cmgs03) were identified as ‘liver’ form, predominantly expressed in liver cells, and the third one as ‘brain’ form (cmgs02), expressed chiefly in brain cells. Molecular characterization studies have revealed conservation of homologous active site residues in all the three gs genes. In silico analysis, accompanied by GS enzyme assay and Western blot analysis of different GS isoforms in different subcellular fractions indicated the mitochondrial localization of cmGS01 and cmGS03 in liver and kidney cells and cytosolic localization of cmGS02 in brain cells. Further, exposure of magur catfish to high external ammonia (HEA; 25 mM NH4Cl) led to a significant induction of multiple gs genes as evidenced by higher expression of different gs mRNAs at variable levels in different tissues. The cmgs01 and cmgs03 mRNA levels elevated significantly in liver, kidney, muscle, and gills, whereas the cmgs02 mRNA level increased considerably in the brain after 14 days of exposure to HEA. These increases in mRNA levels were associated with a significant rise in cmGS01 and cmGS03 proteins in liver, kidney, muscle, and gills, and the cmGS02 protein in the brain after 14 days of exposure to HEA. Therefore, it can be concluded that the unique differential expression of three gs genes and their induction under high ammonia level probably helps in detoxification of ammonia to glutamine and further to urea via the ornithine-urea cycle in ureogenic as well as non-ureogenic tissues of these magur catfish.

Effect of Glutamine Synthetase Gene Overexpression in Birch (Betula pubescens) Plants on Auxin Content and Rooting in vitro.

The effects of transformation of downy birch (Betula pubescens Ehrh.) with the GS1 gene encoding the cytosolic form of glutamine synthetase on the rooting of plants in vitro was studied. The transgenic plants had an elevated content of glutamine as well as glutamic and aspartic acids and rooted more rapidly than the control plants. Rooting on a medium containing the glutamine synthetase inhibitor phosphinothricin prevented the accumulation of auxin in birch plants carrying the GS1 gene, indicating the involvement of this enzyme in raising the level of auxins in the transgenic plants. The correlation between the increase in the auxin levels in the transgenic plants carrying the glutamine synthetase gene and the increase in the rooting rate is shown for the first time.

Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality

Tea, one of the world's most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R2 ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 μM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.

Identification and characterization of glutamine synthetase isozymes in Gracilaria lemaneiformis

Glutamine synthetase (GS) isozymes are necessary during the conversion of inorganic to organic nitrogen (N) and they also play a crucial role in the growth of Gracilaria lemaneiformis. Six genes encoding GS-protein were found and identified from the transcriptome data of G. lemaneiformis genome. No intron was found in the six GS genes. Among these six GS genes, Unigene0016999 and Unigene0017002 genes encode two GSI subfamily proteins with 426 and 507 amino acid (AA), respectively. These two GSI proteins are localized to the mitochondria and contain only one conserved Gln-syn C domain. Three GSII subfamily proteins (Unigene0015608, Unigene0022741 and Unigene0002184 with 350 AA, 350 AA and 402 AA respectively) harbor a conserved Gln-synt_C and Gln-synt_N domains. While Unigene0002194 gene encodes a GSIII protein (735 AA) and is comprised of a conserved Gln-synt_C and a specific GSIII_N domain. Phylogenetic analyses provided strong support for the classification of three GS subfamilies in G. lemaneiformis and insights into the evolutionary relationship of GS subfamilies between different organisms.

Glutamine synthetase gene knockout-human embryonic kidney 293E cells for stable production of monoclonal antibodies.

Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX.

Functional Transcripts Indicate Phylogenetically Diverse Active Ammonia-Scavenging Microbiota in Sympatric Sponges.

Symbiotic ammonia scavengers contribute to effective removal of ammonia in sponges. However, the phylogenetic diversity and in situ activity of ammonia-scavenging microbiota between different sponge species are poorly addressed. Here, transcribed ammonia monooxygenase genes (amoA), hydrazine synthase genes (hzsA), and glutamine synthetase genes (glnA) were analyzed to reveal the active ammonia-scavenging microbiota in the sympatric sponges Theonella swinhoei, Plakortis simplex, and Phakellia fusca, and seawater. Archaeal amoA and bacterial glnA transcripts rather than bacterial amoA, hzsA, and archaeal glnA transcripts were detected in the investigated sponges and seawater. The transcribed amoA genes were ascribed to two Thaumarchaeota ecotypes, while the transcribed glnA genes were interspersed among the lineages of Cyanobacteria, Tectomicrobia, Poribacteria, Alpha-, Beta-, Gamma-, and Epsilonproteobacteria. In addition, transcribed abundances of archaeal amoA and bacterial glnA genes in these sponges have been quantified, showing significant variation among the investigated sponges and seawater. The transcriptome-based qualitative and quantitative analyses clarified the different phylogenetic diversity and transcription expression of functional genes related to microbially mediated ammonia scavenging in different sympatric sponges, contributing to the understanding of in situ active ecological functions of sponge microbial symbionts in holobiont nitrogen cycling.

Cytosolic Glutamine Synthetase Isozymes Play Redundant Roles in Ammonium Assimilation Under Low-Ammonium Conditions in Roots of Arabidopsis thaliana.

Ammonium is a major nitrogen source for plants; it is assimilated into glutamine via a reaction catalyzed by glutamine synthetase (GLN). Arabidopsis expresses four cytosolic GLN genes, GLN1; 1, GLN1; 2, GLN1; 3 and GLN1; 4, in roots. However, the function and organization of these GLN1 isozymes in ammonium assimilation in roots remain unclear. In this study, we aimed to characterize the four GLN1 isozymes. The levels of growth of the wild type and gln1 single and multiple knockout lines were compared in a hydroponic culture at ammonium concentrations of 0.1 and 3 mM. Under the low-ammonium concentration, in single mutants for each GLN1 gene, there was little effect on growth, whereas the triple mutant for GLN1; 1, GLN1; 2 and GLN1; 3 grew slowly and accumulated ammonium. Under the high-ammonium concentration, the single mutant for GLN1; 2 showed 50% decreases in fresh weight and glutamine, whereas the other gln1 single mutants did not show notable changes in the phenotype. The double mutant for GLN1; 1 and GLN1; 2 showed less growth and a lower glutamine concentration than the single mutant for GLN1; 2. Promoter analysis indicated an overlapping expression of GLN1; 1 with GLN1; 2 in the surface layers of the roots. We thus concluded that: (i) at a low concentration, ammonium was assimilated by GLN1; 1, GLN1; 2 and GLN1; 3, and they were redundant; (ii) low-affinity GLN1; 2 could contribute to ammonium assimilation at concentrations ranging from 0.1 to 3 mM; and (iii) GLN1; 1 supported GLN1; 2 within the outer cell layers of the root.

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.

Assessment of Genetic Diversity of Freshwater Mud Eel (Monopterus cuchia) Using RAPD and RFLP Markers

Background: Monopterus cuchia, an economically important eel of Bangladesh, is confronting the challenge of population reduction and germplasm degeneration since the vast majority of fingerlings are collected from natural habitats and artificial cultivation of the fish is yet to be established.Materials and Methods: 30 individuals of M. cuchia from a natural population of Northern-East part of Bangladesh were analyzed using 8 decamer primers and glutamine synthetase gene was digested with 2 restriction enzymes.Results: A total of 735 bands with 228 polymorphic loci were detected among the selected 30 individuals by using RAPD assay while 100% polymorphism was revealed by all the primers. The genetic distance among the individuals was calculated by using the data from pair-wise similarity index where 36 groups of genetic diversities were measured while the lowest and highest genetic distance were found 0.58 and 0.97 respectively with an average genetic diversity of 0.81. The Nei’s genetic similarity values were found from 0.06 to 0.6 where average value was recorded 0.301746 which was found significant regarding genetic distance. Phylogenetic relationships using UPGMA clustering revealed linkage distance ranged from 3.6 to 6.24 and generated 6 clusters by 11 clades with the involvement of 22 individuals while the rest of the samples were connected to those clades with specific linkage distances. Genetic diversity of glutamine synthetase gene was analyzed with two restriction enzymes, e.g. +CfrI and +Hpy178III while both the enzymes digested the gene fragment at a length of 541 bp and polymorphism was detected in terms of wild type homogygotes, polymorphic homozygote and heterozygosity. The genetic diversity was observed by using the RFLP band analysis and four different groups of individuals were identified with the p-values of 0, 0.033, 0.05 and 1 respectively whereas different distances were found among the groups indicating the polymorphism among experimental individuals of glutamine synthetase gene.Conclusion: Herein, RAPD and RFLP analysis indicating the rich genetic diversity of this fish in the experimental ecological habitat and this would come helpful for the conservation of germplasm diversity and to support the sustainable breeding program of M. cuchia.

Identification of QTLs for nitrate and ammonium use efficiency under direct seeded condition and differential expression of genes involved in their absorption and metabolic pathway

Considering water and labor crises, area under direct seeded rice cultivation has increased. With the direct seeding during the early seedling stage the rice crop is very sensitive to water stress and nutrient deficiencies. The availability of nutrients is highly influenced by water status. Over the years, we have critically observed that under direct seeded condition at seedling stage if drought occurs the plants suffer more because of nutrient deficiency rather than water deficiency. The continued plant growth and development under changed rhizosphere depends heavily on availability of nitrogen and other nutrients (P, Fe and S) and the ability of the root system to absorb these nutrients efficiently and use in metabolism under their changed forms. Rice has evolved and adopted for efficient utilization of ammonical form of nitrogen, while nitrate from is less efficiently used at least during seedling and early vegetative phases. Most of the genotypes (except a few) we evaluated exhibited extremely low level of Nitrate transporter and Nitrate Reductase activities which are key factor and enzyme in absorption and utilization of nitrate form of nitrogen. Therefore, the identification of lines with greater nitrate use efficiency, mainly because of more efficient High Affinity Transporters, increased expression of nitrate reductase and nitrite reductase along with ammonium transporters and GS and GOGAT will certainly have key role in N efficiency of rice genotypes particularly under water stress conditions. In study 1, one hundred twenty two and selected thirty two recombinant inbred lines (RILs) of two indica genotypes, Danteshwari × Dagaddeshi were evaluated under three nitrogen forms and three environments (Irrigated, Rainfed and Terminal stage drought) along with root studies under soil filled glass rhizotrons at IGKV, Raipur, for generation of phenotypic data. The genotypic data of 162 SSR and HvSSR based markers were developed and used for identifying QTLs for agronomically complex trait i.e. NUE. The phenotypic and genotypic data was statistically analyzed for QTLs identification for yield & NUE traits using QTL Cartalographer. QTLs for nitrate and ammonical use efficiency varied significantly. A presence of QTL clusters between HvSSR 1–87(38.9cM) to HvSSR 1–89 (40.6cM) on chromosome 6, RM 449 (81.9cM) to RM5 (9.4cM) on chromosome 5, HvSSR 9–25 (14.6cM) to HvSSR 9–27 (15.5cM) on chromosome 9 and RM 434 (57.7cM) to RM 410 (64.4cM) on chromosome 5 signifies important genomic regions associated with evaluated traits under different conditions and will be useful in marker assisted breeding for NUE in rice. In study 2, we investigated the different members AMT (Ammonium transporters), NRT (Nitrate transporters), GS (Glutamine Synthetase) & GOGAT (Glutamate Synthase) genes, involved in NUE and analyzed the expression pattern of each gene using gene-specific primer in young rice seedlings by quantitative real time PCR, revealing a distinct expression pattern of these genes. Collectively, OsGln1;1, OsGln1;2, OsGln1;3, OsGln2, OsGlt1 and OsGlt2 manifested different and reciprocal responses to nitrate and ammonium supply. The relationship of these genes/enzymes and its interaction with water stress will be presented during the conference.

First Report of Colletotrichum aenigma Causing Anthracnose on Sedum kamtschaticum in Korea

HomePlant DiseaseVol. 101, No. 12First Report of Colletotrichum aenigma Causing Anthracnose on Sedum kamtschaticum in Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Colletotrichum aenigma Causing Anthracnose on Sedum kamtschaticum in KoreaH.-W. Choi, Y. K. Lee, and S. K. HongH.-W. ChoiSearch for more papers by this author, Y. K. LeeSearch for more papers by this author, and S. K. Hong†Corresponding author: S. K. Hong; E-mail: E-mail Address: [email protected]Search for more papers by this authorAffiliationsAuthors and Affiliations H.-W. Choi Y. K. Lee S. K. Hong † , National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Republic of Korea. Published Online:10 Oct 2017https://doi.org/10.1094/PDIS-07-17-0938-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Orange stonecrop (Sedum kamtschaticum Fisch. and C.A. Mey.) is a perennial herbaceous plant belonging to the family Crassulaceae. It has been used as an ornamental vegetable and for folk medicine for treating inflammatory disorders in Korea. In October 2010, anthracnose occurred on leaves of orange stonecrop grown in a field in Bonghwa, Korea (36.92°N, 128.77°E). Diseased plants were about 5 to 10% of 1,000 total orange stonecrops in the field. Symptoms appeared as circular to irregular lesions with blackish brown discoloration on margins of leaves. Fragments of diseased leaves were surface sterilized with 1% NaOCl for 30 s, then rinsed in sterile water. Tissue pieces were placed on water agar (WA) and incubated at 25°C for 5 days. Three isolates were cultured on potato dextrose agar (PDA) at 25°C in darkness for 7 days. Colonies were at first cottony, floccose, grayish white, becoming gray with age, dark brown at the center in reverse, with mycelial growth of 58.3 mm after 7 days at 25°C. Conidiomata on PDA were slimy, dark brown, forming setae in abundance. Setae were brown, 1 to 3 septate, with tapering acute apices, and 41.5 to 103.2 × 4.6 to 5.9 μm (mean 78.1 × 5.2). There were some differences between shape and size of conidia from natural lesions and those from cultures. The former was cylindrical, tapering toward one or both ends, rarely fusiform, sometimes slightly curved, and 14.1 to 19.1 × 6.7 to 8.8 μm (mean 16.6 × 7.5, l/b 2.2) in size, while the latter was usually cylindrical, straight, with both ends rounded, rarely tapering toward one end, and 16.5 to 22.7 × 6.0 to 7.0 μm (mean 18.7 × 6.4, l/b 2.9) in size. Appressoria were brown to dark brown, usually clavate to ovate, crenate or irregular, and 8.7 to 15.2 × 6.1 to 10.2 μm (mean 11.1 × 8.6) in size. Based on morphological and cultural characteristics, the three isolates were identified as Colletotrichum gloeosporioides species complex (Phoulivong et al. 2010; Weir et al. 2012). To clarify taxonomic placement of the present isolate (IY1059) within the C. gloeosporioides complex, chitin synthase 1 (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), β-tubulin (TUB2), actin (ACT), rDNA internal transcribed spacers regions (ITS), and glutamine synthetase (GS) from IY1059 were amplified by PCR as previously described (Prihastuti et al. 2009). The PCR products were sequenced and deposited in GenBank with accession numbers KY820888–93, and MF401417, respectively. Six sequences of IY1059, except the GS gene, were 99 or 100% identical with the reference strains, C. aenigma C1253.4 or ICMP 18608. The GS sequence of IY1059 showed 98% identity with the reference strain. Based on morphological and molecular characteristics, the isolate was identified as C. aenigma. A pathogenicity test of the three isolates was made on wounded and unwounded leaves of orange stonecrop by dropping 30 µl of conidial suspensions (5 × 105 conidia ml−1). The same quantity of sterile distilled water placed on unwounded or wounded leaves was used as control. Both inoculated and control leaves were placed in plastic boxes with 100% relative humidity at 25°C. After 3 days, symptoms started to develop only on inoculated and wounded leaves. No symptoms developed on the unwounded and control leaves for 7 days after inoculation. The fungus was consistently reisolated from the only inoculated leaves, thus fulfilling Koch’s postulates. To our knowledge, this is the first report that C. aenigma causes anthracnose on leaves of orange stonecrop. Further studies are required to better define the host range of C. aenigma in Korea.

Allelic Variants of Glutamine Synthetase and Glutamate Synthase Genes in a Collection of Durum Wheat and Association with Grain Protein Content

Wheat is one of the most important crops grown worldwide. Despite the fact that it accounts for only 5% of the global wheat production, durum wheat (Triticum turgidum L. subsp. durum) is a commercially important tetraploid wheat species, which originated and diversified in the Mediterranean basin. In this work, the candidate gene approach has been applied in a collection of durum wheat genotypes; allelic variants of genes glutamine synthetase (GS2) and glutamate synthase (GOGAT) were screened and correlated with grain protein content (GPC). Natural populations and collections of germplasms are quite suitable for this approach, as molecular polymorphisms close to a locus with evident phenotypic effects may be closely associated with their character, providing a better physical resolution than genetic mapping using ad hoc constituted populations. A number of allelic variants were detected both for GS2 and GOGAT genes, and regression analysis demonstrated that some variations are positively and significantly related to the GPC effect. Additionally, these genes map into homoeologous chromosome groups 2 and 3, where several authors have localized important quantitative trait loci (QTLs) for GPC. The information outlined in this work could be useful in breeding and marker-assisted selection programs.

Ammonium uptake capacity and response of cytosolic glutamine synthetase 1;2 to ammonium supply are key factors for the adaptation of ammonium nutrition in Arabidopsis thaliana

ABSTRACTPlant requires nitrogen for the growth, and it use nitrate and ammonium from the environment. Plant suffers from the toxicity when excess ammonium is supplied as a sole nitrogen, although it could be a good nitrogen source for plant growth. We hypothesized that the different responses of ecotypes to ammonium nutrient could partly account for the adaptation of Arabidopsis to an ammonium environment. The purpose of this study is to understand the different responses of ecotypes in ammonium environment. The growth of Arabidopsis thaliana ecotypes, Columbia was compared to those of Arabidopsis thaliana ecotypes, Landsberg erecta in ammonium nutrient. The ratio of shoot dry weight to root dry weight was compared to evaluate the adaptation of two ecotypes. The shoot:root ratio of Landsberg was significantly higher than that of Columbia. T-DNA insertion in cytosolic glutamine synthetase 1;2, one of the essential ammonium assimilatory enzymes, led a decrease of shoot:root ratio. We also measured the isotope-labeled ammonium uptake and the expression levels of ammonium transporter genes, and also the expression of ammonium assimilatory genes, glutamine synthetase genes and glutamate synthase genes, in roots after ammonium re-supply using real-time polymerase chain reaction analysis. We found that (1) ammonium uptake of Landsberg erecta was higher than that of Columbia, when ammonium was supplied at higher concentration, and (2) cytosolic glutamine synthetase 1;2 was highly increased by ammonium supply in the root of Landsberg erecta. The present study suggested the importance of these two factors for adaptation of Arabidopsis to an ammonium-rich environment.

FIRST REPORT OF ANTHRACNOSE ON CHERIMOYA CAUSED BY COLLETOTRICHUM TROPICALE IN CUBA

Cherimoya (Annona cherimola Mill.) fruits with preharvest anthracnose symptoms were collected in Province Artemisa, Cuba, in April 2015. Necrotic, sunken lesions with orange spore masses were observed on 71% of the fruits. Single spore isolations on potato dextrose agar (PDA) were initially white and became light gray with scant aerial mycelium and abundant orange conidiomata in concentric rings. Conidia were 12.5-17.5 × 5-6.5 µm, subcylindrical with rounded ends, aseptate and hyaline, morphological characters consistent with Colletotrichum tropicale (Rojas et al., 2010). To confirm pathogen identity, the ITS region, the Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) gene (ApMat) and a partial sequence of the glutamine synthetase gene (GS) were amplified and sequenced (European Nucleotide Archive Accession Nos. LT853592, LT853593 and LT853594, respectively). A phylogenetic analysis using Bayesian inference with published ApMat, GS, and ITS data for Colletotrichum species in the C. gloeosporioides complex (Jayawardena et al., 2016) placed our isolate INIFAT 4144 in the C. tropicale clade. For assessing pathogenicity, five detached cherimoya fruits were inoculated with a suspension of 105 spores/ml by depositing 30 µl droplets of the suspension on the surfaces of nonwounded and wounded fruits with a sterile needle. Control fruits received sterile water. Typical anthracnose symptoms were observed in all inoculated fruits after 3 to 5 days. Control fruits presented no symptoms. The pathogen was consistently re-isolated from the infected fruits. To our knowledge, this is the first report of C. tropicale in Cuba and the first report on A. cherimola.