Amaprotheni e-DELLA ayalondolozwaizilawuli zokukhulaezidlala indima ebalulekile ekuthuthukisweni kwezitshalo ekuphenduleni izimpawu zangaphakathi nezangaphandle. Njengabalawuli bokuloba, ama-DELLA abophezela ezintweni ezilotshiweyo (ama-TFs) kanye ne-histone H2A esebenzisa izizinda zawo ze-GRAS futhi abuthelwa ukusebenza kubathuthukisi. Ucwaningo lwakamuva lubonise ukuthi ukuzinza kwe-DELLA kulawulwa ngemva kokuhumusha ngezindlela ezimbili: i-polyubiquitination eyenziwa yi-hormone yesitshalo.i-gibberellin, okuholela ekonakaleni kwawo ngokushesha, kanye nokuhlangana ne-ubiquitin-like modifier (SUMO), okwandisa ukuqoqwa kwawo. Ngaphezu kwalokho, umsebenzi we-DELLA ulawulwa ngamandla yizindlela ezimbili ezihlukene ze-glycosylation: I-O-fucosylation ithuthukisa ukusebenzisana kwe-DELLA-TF, kuyilapho ukuguqulwa kwe-O-linked N-acetylglucosamine (O-GlcNAc) kuvimbela ukusebenzisana kwe-DELLA-TF. Kodwa-ke, indima ye-DELLA phosphorylation ayicacile njengoba izifundo zangaphambilini zibonise imiphumela ephikisanayo, abanye bephakamisa ukuthi i-phosphorylation ikhuthaza noma icindezela ukuwohloka kwe-DELLA kanti abanye baphakamisa ukuthi i-phosphorylation ayithinti ukuzinza kwabo. Lapha, sihlonza amasayithi e-phosphorylation ku-GA1-3 repressor (RGA), AtDELLA, ehlanjululwe ku-Arabidopsis thaliana nge-mass spectrometry futhi sibonisa ukuthi i-phosphorylation yama-peptide amabili e-RGA ezifundeni ze-PolyS kanye ne-PolyS/T ithuthukisa umsebenzi we-RGA ngokuphromotha ukubophezela kwe-H2A kanye nobudlelwane be-RGA nabagqugquzeli abaqondiwe. Ngokuphawulekayo, i-phosphorylation ayizange ithinte ukusebenzisana kwe-RGA-TF noma ukuzinza kwe-RGA. Ucwaningo lwethu lwembula indlela yamangqamuzana lapho i-phosphorylation idala umsebenzi we-DELLA.
Ukuhlaziywa kwethu kwe-mass spectrometric kwembula ukuthi zombili i-Pep1 ne-Pep2 zine-phosphorylated kakhulu ku-RGA ngemuva kwe-GA-deficient Ga1. Ngaphezu kwalolu cwaningo, izifundo ze-phosphoproteomic nazo zembule i-Pep1 phosphorylation ku-RGA, nakuba indima yayo ingakafundwa53,54,55. Ngokuphambene, i-Pep2 phosphorylation ayizange ichazwe ngaphambilini njengoba le peptide ingatholwa kuphela kusetshenziswa i-RGAGKG transgene. Nakuba ukuguqulwa kwe-m1A, okuqede i-phosphorylation ye-Pep1, kunciphise kancane kuphela umsebenzi we-RGA ku-planta, kube nomthelela ongeziwe lapho kuhlanganiswa ne-m2A ekwehliseni umsebenzi we-RGA (Umfanekiso Owengeziwe 6). Okubalulekile, i-Pep1 phosphorylation yehliswe kakhulu ku-GA-enhanced sly1 mutant uma kuqhathaniswa ne-ga1, iphakamisa ukuthi i-GA ikhuthaza i-RGA dephosphorylation, inciphisa umsebenzi wayo. Indlela i-GA ecindezela ngayo i-RGA phosphorylation idinga uphenyo olwengeziwe. Okunye okungenzeka ukuthi lokhu kufinyelelwa ngokulawulwa kwe-protein kinase engaziwa. Nakuba ucwaningo lubonise ukuthi ukubonakaliswa kwe-CK1 protein kinase EL1 yehliswa yi-GA ku-rice41, imiphumela yethu ibonisa ukuthi ukuguqulwa kwe-oda eliphezulu kwe-Arabidopsis EL1 homologue (AEL1-4) akunciphisi i-RGA phosphorylation. Ngokuvumelana nemiphumela yethu, ucwaningo lwakamuva lwe-phosphoproteomic olusebenzisa imigqa ye-Arabidopsis AEL overexpressing kanye ne-ael triple mutant ayizange ibone noma yimaphi amaprotheni e-DELLA njengama-substrates alezi kinases56. Lapho silungisa umbhalo wesandla, kwabikwa ukuthi i-GSK3, isakhi sofuzo esifaka i-GSK3/SHAGGY-like kinase kukolweni (Triticum aestivum), ingaba ne-phosphorylate DELLA (Rht-B1b)57, nakuba i-phosphorylation ye-Rht-B1b nge-GSK3 ayizange iqinisekiswe ku-planta. Ukusabela kwe-in vitro enzymatic phambi kwe-GSK3 kulandelwa ukuhlaziywa kwe-mass spectrometry kwembula amasayithi amathathu e-phosphorylation atholakala phakathi kwezizinda ze-DELLA ne-GRAS ze-Rht-B1b (I-Supplementary Fig. 3). Ukushintshwa kwe-Serine kuya ku-alanine kuzo zonke izingosi ezintathu ze-phosphorylation kuholele ekwehleni komsebenzi we-Rht-B1b kukolweni we-transgenic, ngokuhambisana nokuthola kwethu ukuthi ukushintshwa kwe-alanine ku-Pep2 RGA kwehlisa umsebenzi we-RGA. Kodwa-ke, ukuhlolwa kokuwohloka kwamaprotheni e-in vitro kuphinde kwabonisa ukuthi i-phosphorylation ingase futhi izinze i-Rht-B1b57. Lokhu kuphambene nemiphumela yethu ebonisa ukuthi ukushintshwa kwe-alanine ku-Pep2 RGA akuguquli ukuzinza kwayo ku-planta. I-GSK3 kukolweni i-ortholog ye-brassinosteroid-insensitive protein 2 (BIN2) ku-Arabidopsis 57. I-BIN2 isilawuli esingalungile sokusayina kwe-BR, futhi i-BR yenza kusebenze indlela yayo yokubonisa ngokubangela ukuwohloka kwe-BIN2 58. Sibonise ukuthi ukwelashwa kwe-BR akunciphisi ukuzinza kwe-RGA ku-59 noma i-phosphory ye-phosphory ye-phosphory iphakamisa ukuthi i-RGA mancane amathuba okuthi ibe yi-phosphorylated yi-BIN2.
Yonke idatha yobuningi yahlaziywa ngokwezibalo kusetshenziswa i-Excel, futhi umehluko omkhulu wanqunywa kusetshenziswa i-t-test Yomfundi. Azikho izindlela zezibalo ezisetshenzisiwe ukuze kunqunywe kusengaphambili usayizi wesampula. Ayikho idatha ekhishiwe ekuhlaziyweni; ukuhlolwa akuzange kwenziwe ngokungahleliwe; futhi abacwaningi bebazi ngesabelo ngesikhathi sokuhlolwa nokuhlolwa kwemiphumela. Osayizi besampula banikezwa kuzinganekwane zezibalo nakumafayela edatha eluhlaza.
Isikhathi sokuthumela: Apr-15-2025