Analiza subjediničnog sastava i regulacije izoformi glutamin-sintetaze kod biljaka Arabidopsis thalliana (L.) Heynh. i Lotus corniculatus L.

Abstract

Glutamine synthetase (GS) is the key enzyme involved in the assimilation ofammonia derived from nitrate reduction, photorespiration, amino acid catabolism, N2fixation in legumes, and other metabolic processes in plants. A small gene familyencodes for different cytosolic (GS1) isoforms in higher plants, while a single geneencodes for the plastidic isoform (GS2). GS operates with glutamate synthаse(GOGAT) in a two enzyme cycle. The net outcome of this cycle is the production ofglutamate, which, through the action of aminotransferases, is used to synthesize otheramino acids. Gaining further knowledge about GS is essential for understandingnitrogen assimilation in higher plants, which can lead to better nitrogen use efficiencyand lowering of fertilizer input.The structure and regulation of GS isoforms from two model plants, Arabidopsisthaliana (L.) Heynh and Lotus corniculatus L. were investigated in the presented study.Since the A. thaliana genome encodes five GS1 subunits (GLN1;1-1,5), one of theobjectives was to investigate whether different GS1 proteins from this plant are able toform heteromeric enzymes. In order to identify the subunit composition of A. thalianaGS1 isoforms, electrophoretic profiles of GS isoforms from GS1 SALK and SAILknockout mutants were compared. It was concluded that GLN1;1 and GLN1;3, as wellas GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 are able to form functionalheterodecamers in all stoichiometric proportions. Furthermore, in the knockout mutantslacking GLN1;2 and GLN1;3, higher GLN1;1 transcript levels were found implicatingthat GLN1;1, at least in part, compensates for the missing isoforms. The secondobjective of this study was to investigate the regulation of expression of A. thaliana GSand GOGAT genes by plant growth regulators: kinetin, abscisic acid, giberelic acid and2,4-dichlorophenoxyacetic acid. The expression of GS and GOGAT genes isdifferentially regulated by growth regulators in the leaf and root. The observed patternsof expression provide insights into the hormonal regulation of these genes duringdevelopment and as response to environmental cues. The third objective was to investigate phosphinothricin (PPT) induced hormesis in L. corniculatus. PPT is a potentGS inhibitor used as a non selective post emergence herbicide. GS inhibition in plantscauses ammonia accumulation, glutamine depletion and eventually death. However, thegrowth response of L. corniculatus plants immersed in solutions with a broad range ofPPT concentrations is biphasic, with pronounced stimulating effect on biomassproduction at concentrations ≤ 50 μM and growth inhibition at higher concentrations.The growth stimulation at low PPT concentrations is a result of activation of GS2, whilethe growth suppression is caused by inhibition of both GS1 and GS2 at higher PPTconcentrations. A detailed molecular mechanism of concentration-dependent interactionof PPT with the GS holoenzymes from L. corniculatus is proposed. The mechanism isin concurrence with all experimental and literature data.

Glutamin-sintetaza (GS) katališe asimilaciju amonijum-jona poreklomod redukcije nitrata, fotorespiracije, katabolizma aminokiselina, fiksacijeN2 kod leguminoza i drugih metaboličkih procesa kod biljka. Kod viših biljaka,mala familija gena kodira različite citosolne (GS1) izoforme, dok jedan genkodira izoformu lokalizovanu u plastidima (GS2). Zajedno sa glutamat-sintazom(GOGAT), GS je deo dvoenzimskog ciklusa odgovornog za biosintezu glutamata,koji je donor amino-grupe aminokiselinama u reakcijama katalisanimrazličitim transaminazama. Sticanje dodatnih saznanja o GS je od presudnogznačaja za razumevanje procesa asimilacije azota kod viših biljaka, što možedovesti do bolje efikasnosti u korišćenju dostupnog azota i smanjenoj upotrebiveštačkih đubriva.U prezentovanom istraživanju ispitivana je struktura i regulacija GSizoformi iz dve model-biljke, Arabidopsis thaliana (L.) Heynh i Lotus corniculatusL. Jedan od ciljeva bio je ispitivanje mogućnosti različitih GS1 proteina A.thaliana, čiji genom kodira pet GS1 subjedinica (GLN1;1-1,5), da formirajuheteromere. Kako bi se ispitao subjedinični sastav GS1 izoformi A. thaliana,upoređeni su elektroforetski profili GS izoformi različitih GS1 SALK iSAIL knockout mutanata. Zaključeno je da GLN1;1 i GLN1;3, kao i GLN1;2 iGLN1;3, a moguće i GLN1;1 i GLN1;2 subjedinice mogu da se kombinuju u svimstehiometrijskim odnosima formirajući funkcionalne dekamerne enzime.Pored toga, kod knockout mutanata u GLN1;2 i GLN1;3 detektovana je povišenakoličina GLN1;1 transkripata što upućuje da GLN1;1 delom kompenzujenedostajuće izoforme. Drugi cilj ovog istraživanja bio je ispitivanjeregulacije ekspresije GS i GOGAT gena A. thaliana biljnim regulatorimarastenja: kinetinom, abscisinskom kiselinom, giberelinskom kiselinom i2,4-dihlorfenoksiacetatom. Ekspresija GS i GOGAT gena je diferencijalnoregulisana regulatorima rastenja u listu i korenu, a dobijeni obrasci ekspresije upućuju na mogući fiziološki kontekst hormonalne regulacije ovihgena tokom razvića biljke i kao odgovoru na uslove sredine. Treći cilj je bioispitivanje hormeze indukovane fosfinotricinom (PPT) kod zvezdana (L.corniculatus). PPT je inhibitor GS koji je pronašao primenu kao neselektivniherbicid. Inhibicija GS u biljci dovodi do nagomilavanja amonijaka,nedostatka glutamina i konačno, do smrti. Utvrđeno je da biljke L. corniculatustretirane širim opsegom koncentracija PPT-a, pokazuju dvofazni odgovor kojiobuhvata stimulaciju produkcije biomase pri koncentracijama nižim od 50 μM,i inhibiciju rasta i smrt biljke pri višim koncentracijama. Stimulacija rastaniskim koncentracijama PPT-a je posledica aktivacije GS2 izoforme, dok jeinhibicija rasta i smrt biljke pri visokim koncentracijama herbicidaprouzrokovana inhibicijom GS1 i GS2 izoformi. Predložen je detaljanmolekulski mehanizam koncentraciono-zavisne interakcije GS holoenzima saPPT-om, koji je u skladu sa eksperimentalnim i literaturnim podacima.