Elucidation of the role of glutamine synthetase seed isoform GLN1;5 in Arabidopsis thaliana (L.) with a reverse genetics approach
Keywords:germination, glutamine synthetase, grain filling, knockout mutant, phenotype
Abstract: Glutamine synthetase (E.C. 126.96.36.199) 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.
Received: March 15, 2019; Accepted: April 4, 2019; Published online: April 10, 2019
How to cite this article: Dragićević MB, Ćuković KB, Zdravković-Korać SR, Simonović AD, Bogdanović MD, Todorović SI. Elucidation of the role of glutamine synthetase seed isoform GLN1;5 in Arabidopsis thaliana (L.) with a reverse genetics approach. Arch Biol Sci. 2019;71(3):443-53.
Lothier J, Gaufichon L, Sormani R, Lemaître T, Azzopardi M, Morin H, Chardon F, Reisdorf-Cren M, Avice JC, Masclaux-Daubresse C. The cytosolic glutamine synthetase GLN1;2 plays a role in the control of plant growth and ammonium homeostasis in Arabidopsis rosettes when nitrate supply is not limiting. J Exp Bot. 2011;62(4):1375-90.
Ishiyama K, Inoue E, Watanabe-Takahashi A, Obara M, Yamaya T, Takahashi H. Kinetic Properties and Ammonium-dependent Regulation of Cytosolic Isoenzymes of Glutamine Synthetase in Arabidopsis. J Biol Chem. 2004;279(16):16598-605.
Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Schölkopf B, Weigel D, Lohmann JU. A gene expression map of Arabidopsis thaliana development. Nat Genet. 2005;37(5):501-6.
Guan M, Moller IS, Schjoerring JK. Two cytosolic glutamine synthetase isoforms play specific roles for seed germination and seed yield structure in Arabidopsis. J Exp Bot. 2015;66(1):203-12.
Konishi N, Ishiyama K, Beier MP, Inoue E, Kanno K, Yamaya T, Takahashi H, Kojima S. Contributions of two cytosolic glutamine synthetase isozymes to ammonium assimilation in Arabidopsis roots. J Exp Bot. 2017;68(3):613-25.
Oliveira IC, Coruzzi GM. Carbon and Amino Acids Reciprocally Modulate the Expression of Glutamine Synthetase in Arabidopsis. Plant Physiol. 1999;121(1):301-10.
Dragićević M, Simonović A, Bogdanović M, Subotić A, Ghalawenji N, Dragićević I, Todorović S. Differential regulation of GS-GOGAT gene expression by plant growth regulators in Arabidopsis seedlings. Arch Biol Sci. 2016;68(2):399-404.
Wang R, Okamoto M, Xing X, Crawford NM. Microarray Analysis of the Nitrate Response in Arabidopsis Roots and Shoots Reveals over 1,000 Rapidly Responding Genes and New Linkages to Glucose, Trehalose-6-Phosphate, Iron, and Sulfate Metabolism. Plant Physiol. 2003;132(2):556-67.
Coschigano KT, Melo-Oliveira R, Lim J, Coruzzi GM. Arabidopsis gls Mutants and Distinct Fd-GOGAT Genes: Implications for Photorespiration and Primary Nitrogen Assimilation. Plant Cell. 1998;10(5):741-52.
Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature. 2000;408(6814):796-815.
Guan M, de Bang TC, Pedersen C, Schjoerring JK. Cytosolic Glutamine Synthetase Gln1;2 Is the Main Isozyme Contributing to GS1 Activity and Can Be Up-Regulated to Relieve Ammonium Toxicity. Plant Physiol. 2016;171(3):1921-33.
Dragićević M, Todorović S, Bogdanović M, Filipović B, Misić D, Simonović A. Knockout mutants as a tool to identify the subunit composition of Arabidopsis glutamine synthetase isoforms. Plant Physiol Biochem. 2014;79:1-9.
Peng Y, Zhang X, Takano T, Liu S, Bu Y. Functional Comparative Study of Arabidopsis thaliana Glutamine Synthetase gene GLN1;1 and GLN1;5 in Response to Salt Stress during Germination Molecular Soil Biology. 2014;5(4):23-30.
Debouba M, Dguimi HM, Ghorbel M, Gouia H, Suzuki A. Expression pattern of genes encoding nitrate and ammonium assimilating enzymes in Arabidopsis thaliana exposed to short term NaCl stress. J Plant Physiol. 2013;170(2):155-60.
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 2003;301(5633):653-7.
Meng L, Feldman L. A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds. Biotechnology Journal. 2010;5(2):183-6.
Haymes K. Mini-prep method suitable for a plant breeding program. Plant Mol Biol Report. 1996;14(3):280-4.
McNair JN, Sunkara A, Frobish D. How to analyse seed germination data using statistical time-to-event analysis: non-parametric and semi-parametric methods. Seed Sci Res. 2012;22(02):77-95.
Therneau TM, Grambsch PM. Modeling Survival Data: Extending the Cox Model. New York: Springer; 2000.
Grambsch PM, Therneau TM. Proportional Hazards Tests and Diagnostics Based on Weighted Residuals. Biometrika. 1994;81(3):515-26.
Harrington DP, Fleming TR. A class of rank test procedures for censored survival data. Biometrika. 1982;69(3):553-66.
Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Görlach J. Growth Stage-Based Phenotypic Analysis of Arabidopsis: A Model for High Throughput Functional Genomics in Plants. Plant Cell. 2001;13(7):1499-510.
Martin A, Lee J, Kichey T, Gerentes D, Zivy M, Tatout C, Dubois F, Balliau T, Valot B, Davanture M, Tercé-Laforgue T, Quilleré I, Coque M, Gallais A, Gonzalez-Moro MB, Bethencourt L, Habash DZ, Lea PJ, Charcosset A, Perez P, Murigneux A, Sakakibara H, Edwards KJ, Hirel B. Two Cytosolic Glutamine Synthetase Isoforms of Maize Are Specifically Involved in the Control of Grain Production. Plant Cell. 2006;18(11):3252-74.
Muhitch MJ, Liang H, Rastogi R, Sollenberger KG. Isolation of a promoter sequence from the glutamine synthetase1–2 gene capable of conferring tissue-specific gene expression in transgenic maize. Plant Sci. 2002;163(4):865-72.
Tabuchi M, Sugiyama K, Ishiyama K, Inoue E, Sato T, Takahashi H, Yamaya T. Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase1;1. Plant J. 2005;42(5):641-51.
Baud S, Boutin J-P, Miquel M, Lepiniec L, Rochat C. An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiol Biochem. 2002;40(2):151-60
Limami AM, Rouillon C, Glevarec G, Gallais A, Hirel B. Genetic and physiological analysis of germination efficiency in maize in relation to nitrogen metabolism reveals the importance of cytosolic glutamine synthetase. Plant Physiol. 2002;130(4):1860-70.
Miflin BJ, Habash DZ. The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. J Exp Bot. 2002;53(370):979-87.
Alboresi A, Gestin C, Leydecker MT, Bedu M, Meyer C, Truong HN. Nitrate, a signal relieving seed dormancy in Arabidopsis. Plant Cell Environ. 2005;28(4):500-12.
Bethke PC, Libourel IG, Reinohl V, Jones RL. Sodium nitroprusside, cyanide, nitrite, and nitrate break Arabidopsis seed dormancy in a nitric oxide-dependent manner. Planta. 2006;223(4):805-12.
Batak I, Dević M, Gibal Z, Grubišić D, Poff KL, Konjević R. The effects of potassium nitrate and NO-donors on phytochrome A- and phytochrome B-specific induced germination of Arabidopsis thaliana seeds. Seed Sci Res. 2002;12(4):253-9.