scholarly journals Elucidation of the role of glutamine synthetase seed isoform GLN1;5 in Arabidopsis thaliana (L.) with a reverse genetics approach

2019 ◽  
Vol 71 (3) ◽  
pp. 443-453
Author(s):  
Milan Dragicevic ◽  
Katarina Cukovic ◽  
Snezana Zdravkovic-Korac ◽  
Ana Simonovic ◽  
Milica Bogdanovic ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Glutamine Synthetase ◽  
Reverse Genetics ◽  
Technological Development ◽  
Expression Patterns ◽  
Dry Weight ◽  
Grain Filling ◽  
Kinetic Properties ◽  
Plant Nitrogen

Glutamine synthetase (E.C. 6.3.1.2) is a key enzyme of plant nitrogen metabolism that assimilates ammonia into glutamine. The Arabidopsis thaliana genome encodes one chloroplastic (GLN2) and five cytosolic (GLN1;1 ? GLN1;5) isoforms with different expression patterns, kinetic properties, regulation and functions. Physiological roles of different isoforms have been elucidated mainly by studying knockout mutants. However, the role of GLN1;5, which is expressed in dry seeds, remains unknown. To clarifty the function of GLN1;5, we studied a GLN1;5 knockout line (GLN1;5KO) homozygous for T-DNA insertion within the GLN1;5. GLN1;5 deficiency results in a phenotype with slightly delayed bolting and fewer siliques. The dry weight of GLN1;5KO seeds was 73.3% of wild-type (WT) seed weight, with seed length 90.9% of WT seeds. Finally, only 18.33% of the mutant seeds germinated in water within 10 days in comparison to 34.67% of WT seeds. KNO3 strongly stimulated germination of both GLN1;5KO and WT seeds, while germination in the presence of increasing NH4Cl concentrations potentiated the differences between the two genotypes. It can be concluded that GLN1;5 activity supports silique development and grain filling and that it has a role in ammonium reassimilation in the seed, as well as assimilation and/or detoxification of ammonium from the environment. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. ON173024 and Grant no. ON173015]

Expression of bacterial glutamine synthetase gene in Arabidopsis thaliana increases the plant biomass and level of nitrogen utilization

Biologia ◽  
2015 ◽  
Vol 70 (12) ◽  
Author(s):  
Chenguang Zhu ◽  
Guimin Zhang ◽  
Chunlei Shen ◽  
Shilin Chen ◽  
Yuanping Tang ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Glutamine Synthetase ◽  
Biochemical Characterization ◽  
Plant Biomass ◽  
Amino Acid Content ◽  
Dry Weight ◽  
Total Amino Acid ◽  
Bacterial Strains ◽  
Plant Nitrogen ◽  
Protein Levels

AbstractChanges in expression of glutamine synthetase (GS) have effect on plant nitrogen metabolism. In order to improve nitrogen use efficiency, several attempts at over-expressing GS II genes in plants have been previously undertaken, however few GSI and III genes are found such application. In this study, two GS I genes were cloned from bacterial strains and were transformed into Arabidopsis thaliana. Expression of the genes was confirmed under both mRNA and protein levels. Phenotypic studies revealed that all transgenic Arabidopsis lines showed enhanced fresh weight (12%) and dry weight (13%) compared with the wild-type plants at two concentrations of nitrate supplies. Further biochemical characterization confirmed that the transgenic lines had higher total nitrogen content (increased by 5-8%), soluble protein concentration (increased by 7-11%), total amino acid content (increased by 4-8%), leaf GS activity (enhanced by 8-14%) and free NO

scholarly journals Localization, Gene Expression, and Functions of Glutamine Synthetase Isozymes in Wheat Grain (Triticum aestivum L.)

2021 ◽  
Vol 12 ◽  
Author(s):  
Yihao Wei ◽  
Shuping Xiong ◽  
Zhiyong Zhang ◽  
Xiaodan Meng ◽  
Lulu Wang ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Nitrogen Metabolism ◽  
Glutamate Synthase ◽  
Grain Filling ◽  
Maximum Activity ◽  
Transfer Cells ◽  
Aleurone Layer ◽  
Wheat Grain ◽  
Plant Nitrogen ◽  
Endosperm Transfer Cells

Glutamine synthetase (GS) plays a major role in plant nitrogen metabolism, but the roles of individual GS isoforms in grains are unknown. Here, the localization and expression of individual TaGS isozymes in wheat grain were probed with TaGS isoenzyme-specific antibodies, and the nitrogen metabolism of grain during the grain filling stage were investigated. Immunofluorescence revealed that TaGS1;1, TaGS1;3, and TaGS2 were expressed in different regions of the embryo. In grain transporting tissues, TaGS1;2 was localized in vascular bundle; TaGS1;2 and TaGS1;1 were in chalaza and placentochalaza; TaGS1;1 and TaGS1;3 were in endosperm transfer cells; and TaGS1;3 and TaGS2 were in aleurone layer. GS exhibited maximum activity and expression at 8 days after flowering (DAF) with peak glutamine content in grains; from then, NH4+ increased largely from NO3- reduction, glutamate dehydrogenase (GDH) aminating activity increased continuously, and the activities of GS and glutamate synthase (GOGAT) decreased, while only TaGS1;3 kept a stable expression in different TaGS isozymes. Hence, GS-GOGAT cycle and GDH play different roles in NH4+ assimilation of grain in different stages of grain development; TaGS1;3, located in aleurone layer and endosperm transfer cells, plays a key role in Gln into endosperm for gluten synthesis. At 30 DAF, grain amino acids are mainly transported from maternal phloem.

Molecular cloning, gene expression and functional expression of a phosphoenolpyruvate carboxylase Osppc1 in developing rice seeds: implication of involvement in nitrogen accumulation

2014 ◽  
Vol 24 (1) ◽  
pp. 23-36 ◽  
Author(s):  
Naoki Yamamoto ◽  
Tatsuya Kubota ◽  
Takehiro Masumura ◽  
Naomasa Shiraishi ◽  
Kunisuke Tanaka ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Grain Filling ◽  
Functional Expression ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Nitrogen Accumulation ◽  
Consecutive Reaction ◽  
Gramineous Plant ◽  
Rice Seeds ◽  
Regulatory Properties

AbstractWe isolated two cDNAs of phosphoenolpyruvate carboxylase (PEPC) from developing rice seeds, Osppc1 and Osppc3. The deduced amino acid sequences of both cDNAs share several conserved motifs with other non-photosynthetic PEPCases, and these common motifs are known to be functionally important to their regulatory properties. The deduced protein sequence of Osppc1 was clustered into a monocotyledonous plant-specific clade, and Osppc3 was clustered into a gramineous plant-specific clade in the phylogenetic tree of plant PEPCases. The mRNA accumulations of Osppc1 and Osppc3 were found in developing rice seeds throughout the grain-filling stages, although their expression patterns differed: Osppc1 was strongly expressed at 7 d after flowering, and Osppc3 was strongly expressed at 4 d after flowering. The kinetic properties of the Osppc1 recombinant protein were quite similar to those of maize root-type PEPCase, except that the sensitivity for malate at pH 7.3 was weaker. Mining rice microarray data, we observed that Osppc1 was co-expressed with aspartate aminotransferase and alanine aminotransferase, which are involved in seed nitrogen metabolism. Moreover, reannotation of the co-expressed genes revealed that Osppc1, the two aminotransferases and the enolase were mapped on to the consecutive reaction from 2-phosphoglycerate to glutamate and pyruvate in the cytosol. These results imply that Osppc1 functions cooperatively with the two aminotransferases in the synthesis of amino acids that are used for storage protein synthesis in developing rice seeds.

The nitrogen economy of winter wheat

1977 ◽  
Vol 88 (1) ◽  
pp. 159-167 ◽  
Author(s):  
R. B. Austin ◽  
M. A. Ford ◽  
J. A. Edrich ◽  
R. D. Blackwell
Keyword(s):  
Nitrogen Uptake ◽  
Nitrate Uptake ◽  
Nitrogen Concentration ◽  
Carbon Assimilation ◽  
Dry Weight ◽  
Grain Filling ◽  
Strong Positive Correlation ◽  
Plant Weight ◽  
Plant Nitrogen ◽  
High Yields

SUMMARYIn a field experiment with 47 wheat genotypes, plant samples were taken at anthesis and maturity and analysed for nitrogen. Taking means over all genotypes, the plants contained at anthesis 83 % of the total present at maturity, while at maturity 68 % of the plant nitrogen was present in the grain.There was significant genetic variation in most of the component attributes determining nitrogen uptake. At anthesis, the heaviest plants contained most nitrogen. Because the nitrogen concentration in the leaves (average 2·8%) was much greater than in the stems and ears (average O8 %) and the leaves comprised, on average, 37 % of the plant weight at anthesis, variation in leanness was the main cause of variation in the nitrogen content of plants of a given weight. During grain filling, plants which lost the most dry weight from their stems and leaves took up the least nitrogen.It is suggested that the strong positive correlation between the accumulation of dry matter and of nitrogen, both until anthesis and during grain filling, occurred because both carbon assimilation and nitrate reduction depend on energy made available from chloroplasts. In addition, assimilate is required to sustain the growth of roots which is necessary for continued nitrate uptake.The results show that it should be easier for breeders to select for high nitrogen uptake from among heavy than from among light genotypes. However, although dwarf genotypes tended to be light, none of the correlations found was strong enough to render it impossible to produce dwarf genotypes capable of giving high yields of high-protein grain.

N-terminal truncation affects the kinetics and structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana

2001 ◽  
Vol 359 (3) ◽  
pp. 591-597 ◽  
Author(s):  
Dorthe VILLADSEN ◽  
Tom H. NIELSEN
Keyword(s):  
Arabidopsis Thaliana ◽  
Gel Filtration ◽  
Full Length ◽  
Dihydroxyacetone Phosphate ◽  
Kinetic Properties ◽  
N Terminus ◽  
Fructose 1,6 Bisphosphate ◽  
Substrate Affinities ◽  
Allosteric Properties

The enzyme fructose-6-phosphate 2-kinase (F6P,2K; 6-phosphofructo-2-kinase)/fructose-2,6-bisphosphatase(F26BPase) catalyses the formation and degradation of the regulatory metabolite fructose 2,6-bisphosphate. A cDNA encoding the bifunctional plant enzyme isolated from Arabidopsis thaliana (AtF2KP) was expressed in yeast, and the substrate affinities and allosteric properties of the affinity-purified enzyme were characterized. In addition to the known regulators 3-phosphoglycerate, dihydroxyacetone phosphate, fructose 6-phosphate and Pi, several metabolites were identified as important new effectors. PPi, phosphoenolpyruvate and 2-phosphoglycerate strongly inhibited F6P,2K activity, whereas fructose 1,6-bisphosphate and 6-phosphogluconate inhibited F26BPase activity. Furthermore, pyruvate was an activator of F6P,2K and an inhibitor of F26BPase. Both kinase and phosphatase activities were rapidly inactivated by mild heat treatment (42°C, 10min), but the presence of phosphate protected both enzyme activities from inactivation. In addition to the catalytic regions, the Arabidopsis enzyme comprises a 345-amino-acid N-terminus of unknown function. The role of this region was examined by the expression of a series of N-terminally truncated enzymes. The full-length and truncated enzymes were analysed by gel-filtration chromatography. The full-length enzyme was eluted as a homotetramer, whereas the truncated enzymes were eluted as monomers. Deletion of the N-terminus decreased the kinase/phosphatase activity ratio by 4-fold, and decreased the affinity for the substrate fructose 6-phosphate. The data show that the N-terminus is important both for subunit assembly and for defining the kinetic properties of the enzyme.

scholarly journals Nitrogen Regulating the Expression and Localization of Four Glutamine Synthetase Isoforms in Wheat (Triticum aestivum L.)

2020 ◽  
Vol 21 (17) ◽  
pp. 6299
Author(s):  
Yihao Wei ◽  
Xiaochun Wang ◽  
Zhiyong Zhang ◽  
Shuping Xiong ◽  
Xiaodan Meng ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Nitrogen Assimilation ◽  
Vascular Tissue ◽  
Mrna Level ◽  
Triticum Aestivum L ◽  
Kinetic Properties ◽  
High Catalytic Activity ◽  
Root Tips ◽  
Plant Nitrogen ◽  
Key Enzyme

Glutamine synthetase (GS), the key enzyme in plant nitrogen assimilation, is strictly regulated at multiple levels, but the most relevant reports focus on the mRNA level. Using specific antibodies as probes, the effects of nitrogen on the expression and localization of individual wheat GS (TaGS) isoforms were studied. In addition to TaGS2, TaGS1;1 with high affinity to substrate and TaGS1;3 with high catalytic activity were also localized in mesophyll, and may participate in cytoplasmic assimilation of ammonium (NH4+) released from photorespiration or absorbed by roots; TaGS1;2 was localized in xylem of leaves. In roots, although there were hundreds of times more TaGS1;1 than TaGS1;2 transcripts, the amount of TaGS1;1 subunit was not higher than that of TaGS1;2; NH4+ inhibited TaGS1;1 expression but stimulated TaGS1;3 expression. In root tips, nitrate stimulated TaGS1;1, TaGS1;3, and TaGS2 expression in meristem, while NH4+ promoted tissue differentiation and TaGS1;2 expression in endodermis and vascular tissue. Only TaGS1;2 was located in vascular tissue of leaves and roots, and was activated by glutamine, suggesting a role in nitrogen transport. TaGS1;3 was induced by NH4+ in root endodermis and mesophyll, suggesting a function in relieving NH4+ toxicity. Thus, TaGS isoforms play distinct roles in nitrogen assimilation for their different kinetic properties, tissue locations, and response to nitrogen regimes.

scholarly journals A New Perspective on the Role of Glutamine Synthetase in Nitrogen Remobilization in Wheat (Triticum aestivum L.)

2021 ◽  
Vol 22 (20) ◽  
pp. 11083
Author(s):  
Yihao Wei ◽  
Lulu Wang ◽  
Butan Qin ◽  
Huiqiang Li ◽  
Xiaoran Wang ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Vascular System ◽  
Grain Filling ◽  
Triticum Aestivum L ◽  
Nitrogen Remobilization ◽  
Plant Nitrogen ◽  
Key Enzyme ◽  
N Remobilization ◽  
New Perspective ◽  
Ureide Degradation

Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is closely related to nitrogen remobilization. However, how GS isoforms participate in nitrogen remobilization remains unclear. Here, the spatiotemporal expression of the TaGS gene family after anthesis was investigated, and the results showed that TaGS1;1 was mainly encoded by TaGS1;1-6A, while the other isozymes were mainly encoded by TaGS localized on the A and D subgenomes. TaGS1;2-4A/4D had the highest expression level, especially in rachis and peduncle. Furthermore, immunofluorescence showed TaGS1;2 was located in the phloem of rachis and peduncle. GUS (β-glucuronidase) staining confirmed that ProTaGS1;2-4A/4D::GUS activity was mainly present in the vascular system of leaves, roots, and petal of Arabidopsis. Ureides, an important transport form of nitrogen, were mainly synthesized in flag leaves and transported to grains through the phloem of peduncle and rachis during grain filling. TaAAH, which encodes the enzyme that degrades ureides to release NH4+, had a higher expression in rachis and peduncle and was synchronized with the increase in NH4+ concentration in phloem, indicating that NH4+ in phloem is from ureide degradation. Taking the above into account, TaGS1;2, which is highly expressed in the phloem of peduncle and rachis, may participate in N remobilization by assimilating NH4+ released from ureide degradation.

scholarly journals Arabidopsis thaliana exudates induce growth and proteomic changes in Gluconacetobacter diazotrophicus

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9600
Author(s):  
Tamires Cruz dos Santos ◽  
Mariana Ramos Leandro ◽  
Clara Yohana Maia ◽  
Patrícia Rangel ◽  
Fabiano S. Soares ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Bacterial Growth ◽  
Proteomic Analysis ◽  
Reverse Genetics ◽  
Fatty Acid Biosynthesis ◽  
Gluconacetobacter Diazotrophicus ◽  
Comparative Proteomic Analysis ◽  
Plant Exudates ◽  
Insertion Mutants

Background Plants interact with a variety of microorganisms during their life cycle, among which beneficial bacteria deserve special attention. Gluconacetobacter diazotrophicus is a beneficial bacterium able to fix nitrogen and promote plant growth. Despite its biotechnological potential, the mechanisms regulating the interaction between G. diazotrophicus and host plants remain unclear. Methods We analyzed the response of G. diazotrophicus to cocultivation with Arabidopsis thaliana seedlings. Bacterial growth in response to cocultivation and plant exudates was analyzed. Through comparative proteomic analysis, G. diazotrophicus proteins regulated during cocultivation were investigated. Finally, the role of some up-accumulated proteins in the response G. diazotrophicus to cocultivation was analyzed by reverse genetics, using insertion mutants. Results Our results revealed the induction of bacterial growth in response to cocultivation. Comparative proteomic analysis identified 450 bacterial proteins, with 39 up-accumulated, and 12 down-accumulated in response to cocultivation. Among the up-accumulated pathways, the metabolism of pentoses and protein synthesis were highlighted. Proteins potentially relevant to bacterial growth response such as ABC-F-Etta, ClpX, Zwf, MetE, AcnA, IlvC, and AccC were also increased. Reverse genetics analysis, using insertion mutants, revealed that the lack of ABC-F-Etta and AccC proteins severely affects G. diazotrophicus response to cocultivation. Our data demonstrated that specific mechanisms are activated in the bacterial response to plant exudates, indicating the essential role of “ribosomal activity” and “fatty acid biosynthesis” in such a process. This is the first study to demonstrate the participation of EttA and AccC proteins in plant-bacteria interactions, and open new perspectives for understanding the initial steps of such associations.

Accumulation of microelements by different invertebrate trophic groups on wasted lands

2017 ◽  
Vol 7 (4) ◽  
pp. 30-34 ◽  
Author(s):  
O. A. Didur ◽  
Yu. L. Kulbachko ◽  
V. Y. Gasso
Keyword(s):  
Active Agent ◽  
Dry Weight ◽  
Nature Management ◽  
Habitat Transformation ◽  
Natural Landscapes ◽  
Invertebrate Animals ◽  
Risk Of Disease ◽  
Ore Dressing ◽  
Processing Enterprise

<p>The problem of transformation of natural landscapes resulted from the negative technogenic impact is highlighted. It is shown that mining enterprises are powerful anthropo-technical sources of organic and inorganic toxicants entering the environment. Their wastes pollute all components of the ecosystems and negatively influence human health by increasing a risk of disease. The nature of the accumulation of trace elements (Fe, Cu, Zn, Ni, Cd, and Pb) by invertebrate animals of various functional groups under conditions of anthropo-technogenic pressure was studied. The sample plots were located on self-overgrowing sites with ruderal vegetation located in the immediate vicinity of the Mangan ore-dressing and processing enterprise (Dnipropetrovsk region). It is quite naturally that among the studied biogenic microelements (Fe, Cu, Zn and Ni), the phyto-, zoo-, and saprophages in the investigated zone of technogenic pollution most actively accumulate Fe:<em> </em>22758, 17516 and 18884 mg/kg dry weight on average, respectively. There are significant differences (p ≤ 0.05) in the content of studied microelements between saprophages and phytophages. The saprophages accumulate such trace metals as Mn, Cu, Zn and Cd in high quantities, but Ni and Pb – in smaller ones. The saprophagous functional group of invertebrates is an active agent of detritogenesis, in the conditions of modern nature management it acts as a powerful element of ecosystem engineering (habitat transformation), the main ecological role of which is to modify the habitat of other soil biota. In addition, the saprophages fulfil their concentrating geochemical function. They actively participate in the most important soil biochemical process: the formation of humus, the migration of microelements along trophic chains, the biological cycle in general, and provide such supporting ecosystem services as increasing soil fertility and nutrient cycling.</p>

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