Phosphorylation imayambitsa chowongolera kukula kwa DELLA, zomwe zimapangitsa kuti histone H2A igwirizane ndi chromatin mu Arabidopsis.

Mapuloteni a DELLA amasungidwaowongolera kukulazomwe zimagwira ntchito yofunika kwambiri pakukula kwa zomera poyankha zizindikiro zamkati ndi zakunja. Monga owongolera zolembera, ma DELLA amamangirira ku zinthu zolembera (TFs) ndi histone H2A kudzera m'magawo awo a GRAS ndipo amalembedwa kuti achitepo kanthu pa olimbikitsa. Kafukufuku waposachedwapa wasonyeza kuti kukhazikika kwa DELLA kumayendetsedwa pambuyo pa kumasulira ndi njira ziwiri: polyubiquitination yoyambitsidwa ndi homoni ya chomera.gibberellin, zomwe zimapangitsa kuti ziwonongeke mwachangu, komanso kuti zigwirizane ndi small ubiquitin-like modifier (SUMO), zomwe zimawonjezera kuchulukana kwawo. Komanso, ntchito ya DELLA imayendetsedwa mosiyanasiyana ndi njira ziwiri zosiyana za glycosylation: O-fucosylation imakulitsa kuyanjana kwa DELLA-TF, pomwe kusintha kwa O-linked N-acetylglucosamine (O-GlcNAc) kumaletsa kuyanjana kwa DELLA-TF. Komabe, ntchito ya DELLA phosphorylation siikudziwika bwino chifukwa maphunziro am'mbuyomu awonetsa zotsatira zotsutsana, pomwe ena akunena kuti phosphorylation imalimbikitsa kapena kuletsa kuwonongedwa kwa DELLA ndipo ena akunena kuti phosphorylation simakhudza kukhazikika kwawo. Pano, tikupeza malo a phosphorylation mu GA1-3 repressor (RGA), AtDELLA, yoyeretsedwa kuchokera ku Arabidopsis thaliana ndi mass spectrometry ndipo tikuwonetsa kuti phosphorylation ya ma peptide awiri a RGA m'madera a PolyS ndi PolyS/T imakulitsa ntchito ya RGA polimbikitsa kulumikizana kwa H2A ndi mgwirizano wa RGA ndi olimbikitsa omwe akufuna. Chodziwika bwino ndi chakuti, phosphorylation sinakhudze kuyanjana kwa RGA-TF kapena kukhazikika kwa RGA. Kafukufuku wathu akuwonetsa njira ya mamolekyu yomwe phosphorylation imayambitsa ntchito ya DELLA.
Kusanthula kwathu kwa mass spectrometric kunavumbulutsa kuti Pep1 ndi Pep2 zonse zinali ndi phosphorylation yambiri mu RGA kumbuyo kwa Ga1 yosowa GA. Kuwonjezera pa kafukufukuyu, maphunziro a phosphoproteomic avumbulutsanso phosphorylation ya Pep1 mu RGA, ngakhale kuti ntchito yake sinaphunziridwebe53,54,55. Mosiyana ndi zimenezi, phosphorylation ya Pep2 sinafotokozedwepo kale chifukwa peptide iyi inkapezeka pogwiritsa ntchito RGAGKG transgene. Ngakhale kuti kusintha kwa m1A, komwe kunathetsa phosphorylation ya Pep1, kunachepetsa pang'ono ntchito ya RGA mu zomera, kunali ndi zotsatira zowonjezera pamene kunaphatikizidwa ndi m2A pochepetsa ntchito ya RGA (Chithunzi Chowonjezera 6). Chofunika kwambiri, phosphorylation ya Pep1 inachepetsedwa kwambiri mu sly1 mutant yowonjezeredwa ndi GA poyerekeza ndi ga1, zomwe zikusonyeza kuti GA imalimbikitsa RGA dephosphorylation, kuchepetsa ntchito yake. Njira yomwe GA imaletsa RGA phosphorylation imafuna kufufuza kwina. Kutheka kwina ndikuti izi zimachitika kudzera mu kayendetsedwe ka protein kinase yosadziwika. Ngakhale kuti kafukufuku wasonyeza kuti kufotokozera kwa CK1 protein kinase EL1 kumachepetsedwa ndi GA mu rice41, zotsatira zathu zikusonyeza kuti kusintha kwapamwamba kwa Arabidopsis EL1 homologue (AEL1-4) sikuchepetsa phosphorylation ya RGA. Mogwirizana ndi zotsatira zathu, kafukufuku waposachedwa wa phosphoproteomic pogwiritsa ntchito mizere yowonjezereka ya Arabidopsis AEL ndi ael triple mutant sanazindikire mapuloteni aliwonse a DELLA ngati magawo a kinases awa56. Pamene tinakonza zolembazo, zinanenedwa kuti GSK3, jini yomwe imapanga GSK3/SHAGGY-like kinase mu tirigu (Triticum aestivum), imatha phosphorylate DELLA (Rht-B1b)57, ngakhale phosphorylation ya Rht-B1b ndi GSK3 sinatsimikizidwe mu planta. Machitidwe a enzymatic mu vitro pamaso pa GSK3 otsatiridwa ndi kusanthula kwa mass spectrometry adawonetsa malo atatu a phosphorylation omwe ali pakati pa madera a DELLA ndi GRAS a Rht-B1b (Chithunzi Chowonjezera 3). Kusintha kwa serine kupita ku alanine m'malo onse atatu a phosphorylation kudapangitsa kuti ntchito ya Rht-B1b ichepe mu tirigu wosinthika, mogwirizana ndi zomwe tapeza kuti kusintha kwa alanine mu Pep2 RGA kudachepetsa ntchito ya RGA. Komabe, mayeso a kuwonongeka kwa mapuloteni mu vitro adawonetsanso kuti phosphorylation imathanso kukhazikika Rht-B1b57. Izi zikusiyana ndi zotsatira zathu zomwe zikuwonetsa kuti kusintha kwa alanine mu Pep2 RGA sikusintha kukhazikika kwake mu zomera. GSK3 mu tirigu ndi mtundu wa mapuloteni osakhudzidwa ndi brassinosteroid 2 (BIN2) mu Arabidopsis 57. BIN2 ndi yolamulira yoyipa ya chizindikiro cha BR, ndipo BR imayatsa njira yake yolumikizirana poyambitsa kuwonongeka kwa BIN2 58. Tawonetsa kuti chithandizo cha BR sichichepetsa kukhazikika kwa RGA 59 kapena kuchuluka kwa phosphorylation mu Arabidopsis (Chithunzi Chowonjezera 2), zomwe zikusonyeza kuti RGA singathe kupangidwa ndi BIN2.
Deta yonse yowerengera idasanthulidwa pogwiritsa ntchito Excel, ndipo kusiyana kwakukulu kudapezeka pogwiritsa ntchito mayeso a t a Student. Palibe njira zowerengera zomwe zidagwiritsidwa ntchito podziwa kukula kwa zitsanzo. Palibe deta yomwe idachotsedwa mu kusanthula; kuyesaku sikunachitike mwachisawawa; ndipo ofufuzawo adadziwa za kugawa panthawi yoyesera ndi kuwunika zotsatira. Kukula kwa zitsanzo kumaperekedwa mu nthano za ziwerengero ndi m'mafayilo a data osaphika.