Kurasa apical meristem (SAM) gukura ningirakamaro muburyo bwububiko. Gutera imisemburogibberellins(GAs) bigira uruhare runini muguhuza imikurire yibihingwa, ariko uruhare rwabo muri SAM ntirwumvikana neza. Hano, twateje imbere biosensor ya ratiometric ya GA yerekana mugukora proteine ya DELLA kugirango ihagarike imikorere yingenzi yo kugenzura igisubizo cya GA mugihe twakomeje kwangirika kwa GA. Twerekana ko uku kwangirika gushingiye kuri biosensor yanditse neza neza impinduka murwego rwa GA hamwe na sensing selile mugihe cyiterambere. Twifashishije biosensor gushushanya ikarita ya GA ibikorwa byerekana muri SAM. Twerekana ko ibimenyetso bya GA bihari cyane cyane muri selile ziri hagati yingingo ya primordia, ibanziriza selile interode. Dukoresheje inyungu- no gutakaza-imikorere-yimikorere, turerekana kandi ko GA igenga icyerekezo cyindege igabanya selile, ishyiraho urutonde rwa selile ya selile ya internode, bityo igateza imbere interode muri SAM.
Kurasa apical meristem (SAM), iherereye hejuru yikirasa, irimo niche ya selile stem ibikorwa byayo bibyara ingingo zomuruhande hamwe nuduce twibiti muburyo bwa modular kandi butera mubuzima bwikimera. Buri kimwe muri ibyo bice bisubiramo, cyangwa ibimera, birimo interode ningingo zinyuranye kuri node, hamwe na meristem ya axillary mumababi yibabi1. Gukura no gutondekanya ibimera bihinduka mugihe cyiterambere. Muri Arabidopsis, imikurire yimbere ihagarikwa mugihe cyibimera, kandi meristem ya axillary ikomeza gusinzira mumirongo yamababi ya rosette. Mugihe cyo kwimuka mugice cyindabyo, SAM ihinduka meristem ya inflorescence, ikabyara interode ndende hamwe nuduti twa axillary, amashami mumirongo yamababi ya cauline, hanyuma, indabyo zitagira amababi2. Nubwo twateye intambwe igaragara mugusobanukirwa uburyo bugenzura itangizwa ryamababi, indabyo, n amashami, ugereranije ni bike bizwi kubyerekeranye na internode.
Gusobanukirwa ikwirakwizwa rya spatiotemporal GAs bizafasha kumva neza imikorere yiyi misemburo mubice bitandukanye no mubyiciro bitandukanye byiterambere. Kwerekana iyangirika rya RGA-GFP fusion yagaragajwe nigikorwa cya porotokoro yayo itanga amakuru yingenzi ku kugena urwego rwa GA mu mizi15,16. Ariko, imvugo ya RGA iratandukanye mubice 17 kandi igengwa na GA18. Rero, imvugo itandukanye ya porotokoro ya RGA irashobora kuvamo uburyo bwa fluorescence bwagaragaye hamwe na RGA-GFP bityo ubu buryo ntabwo ari bwinshi. Vuba aha, bioactive fluorescein (Fl) -yanditseho GA19,20 yerekanye kwirundanya kwa GA mumuzi ya endocortex no kugenzura urwego rwa selile hamwe nubwikorezi bwa GA. Vuba aha, sensor ya GA FRET nlsGPS1 yerekanye ko urwego rwa GA rufitanye isano no kurambura ingirabuzimafatizo mumizi, filaments, hamwe na hypocotyls ikuze yijimye21. Ariko, nkuko twabibonye, kwibanda kwa GA ntabwo aribyo byonyine bigenzura ibikorwa byerekana ibimenyetso bya GA, kuko biterwa nuburyo bukomeye bwo kumva. Hano, twubakiye kumyumvire yacu ya DELLA na GA inzira zerekana ibimenyetso, turatanga raporo yiterambere hamwe nibiranga kwangirika gushingiye kuri ratiometric biosensor ya signal ya GA. Kugira ngo duteze imbere iyi biosensor yuzuye, twakoresheje RGA ihindagurika ya GA ihujwe na poroteyine ya fluorescent kandi igaragara hose mu ngingo, ndetse na poroteyine ya GA-itumva. Twerekana ko intungamubiri za poroteyine za RGA zitabangamira amarenga ya GA ya endogenous GA igihe yerekanwe hose, kandi ko iyi biosensor ishobora kugereranya ibikorwa byerekana ibimenyetso biturutse ku kwinjiza GA hamwe no gutangiza ibimenyetso bya GA hamwe n’ibikoresho byifashishwa bikemurwa na spatiotemporal. Twifashishije iyi biosensor mugushushanya ikwirakwizwa rya spatiotemporal yibikorwa bya GA no kugereranya uburyo GA igenga imyitwarire ya selile muri epidermis ya SAM. Twerekana ko GA igenga icyerekezo cyindege igabanya selile ya SAM iri hagati yingingo ya primordia, bityo igasobanura ishyirahamwe ryemewe rya selile ya internode.
Hanyuma, twabajije niba qmRGA ishobora kwerekana impinduka murwego rwa endogenous GA ikoresheje hypocotyls ikura. Twerekanye mbere ko nitrate itera gukura mu kongera synthesis ya GA, nayo, kwangirika kwa DELLA34. Kubera iyo mpamvu, twabonye ko uburebure bwa hypocotyl muri pUBQ10 :: ingemwe za qmRGA zihingwa munsi ya nitrate nyinshi (10 mM NO3−) yari ndende cyane ugereranije n’ingemwe zahinzwe mu bihe bya nitrate (Ishusho yinyongera 6a). Bihuye nigisubizo cyo gukura, ibimenyetso bya GA byari hejuru muri hypocotyls yingemwe zahinzwe munsi ya mM 10 NO3− ugereranije ningemwe zahinzwe mugihe habuze nitrate (Ishusho yinyongera. 6b, c). Rero, qmRGA nayo ituma hakurikiranwa impinduka mubimenyetso bya GA biterwa nimpinduka za endogenous mumitekerereze ya GA.
Kugira ngo twumve niba ibikorwa byerekana ibimenyetso bya GA byagaragaye na qmRGA biterwa nubushakashatsi bwa GA hamwe n’imyumvire ya GA, nkuko byari byitezwe hashingiwe ku gishushanyo mbonera cya sensor, twasesenguye imvugo ya reseptor eshatu za GID1 mu ngingo z’ibimera n’imyororokere. Mu ngemwe, umurongo wabanyamakuru GID1-GUS werekanye ko GID1a na c byagaragaye cyane muri cotyledon (Ishusho 3a - c). Byongeye kandi, ibyakiriwe byose uko ari bitatu byagaragaye mumababi, imizi ya primordia kuruhande, inama zumuzi (usibye umuzi wumuzi wa GID1b), hamwe na sisitemu yimitsi (Ishusho 3a - c). Muri inflorescence SAM, twabonye ibimenyetso bya GUS kuri GID1b na 1c gusa (Ishusho yinyongera. 7a - c). Muri Hybridisation yemeje iyi mvugo kandi ikomeza kwerekana ko GID1c yerekanwe kimwe murwego rwo hasi muri SAM, mugihe GID1b yerekanaga imvugo ihanitse kuruhande rwa SAM (Ishusho yinyongera 7d - l). PGID1b :: 2xmTQ2-GID1b yo guhuza ibisobanuro byanagaragaje urwego rwerekana imvugo ya GID1b, kuva hasi cyangwa nta mvugo iri hagati ya SAM kugeza imvugo ndende kumupaka wurugingo (Ishusho yinyongera 7m). Rero, GID1 yakira ntabwo ikwirakwizwa kimwe no mubice. Mu bushakashatsi bwakurikiyeho, twabonye kandi ko gukabya gukabije kwa GID1 (pUBQ10 :: GID1a-mCherry) byongereye ubukana bwa qmRGA muri hypocotyls kuri GA yo hanze (Ishusho 3d, e). Ibinyuranye, fluorescence yapimwe na qd17mRGA muri hypocotyl ntiyumvaga kuvura GA3 (Ishusho 3f, g). Kuri ubwo bushakashatsi bwombi, ingemwe zavuwe hamwe na GA (100 μM GA3) kugira ngo zisuzume imyitwarire yihuse ya sensor, aho ubushobozi bwo guhuza reseptor ya GID1 bwongerewe cyangwa bwatakaye. Hamwe na hamwe, ibisubizo byemeza ko qmRGA biosensor ikora umurimo uhuriweho nka sensor ya GA na GA, ikanerekana ko imvugo itandukanye ya reseptor ya GID1 ishobora guhindura cyane imitekerereze ya sensor.
Kugeza ubu, gukwirakwiza ibimenyetso bya GA muri SAM ntibisobanutse neza. Kubwibyo, twakoresheje ibimera byerekana qmRGA hamwe na pCLV3 :: mCherry-NLS umunyamakuru w’akagari35 kugira ngo tubare amakarita arenze urugero yerekana umubare w’ibikorwa bya GA byerekana ibimenyetso, twibanze ku gipimo cya L1 (epidermis; Ishusho 4a, b, reba Uburyo n’uburyo bwiyongera), kubera ko L1 igira uruhare runini mu kugenzura iterambere rya SAM36. Hano, pCLV3 :: mCherry-NLS imvugo yatanze geometrike ihamye yo gusesengura ikwirakwizwa rya spatiotemporal ikwirakwizwa ryibikorwa bya GA37. Nubwo GA ifatwa nkibyingenzi mugutezimbere kwingingo4, twabonye ko ibimenyetso bya GA byari bike muri primordium yindabyo (P) guhera kuri P3 (Igishusho 4a, b), mugihe abasore ba P1 na P2 bari bafite ibikorwa bitarenze nkibyo mukarere ko hagati (Ishusho 4a, b). Igikorwa cyo hejuru cyerekana ibimenyetso bya GA cyagaragaye kumupaka wa primordium, guhera kuri P1 / P2 (kumpande zumupaka) no kugera kuri P4, ndetse no mu tugari twose two mukarere ka peripheri kari hagati ya primordia (Ishusho 4a, b hamwe ninyongera ya 8a, b). Iki gikorwa cyo hejuru cyerekana ibimenyetso bya GA nticyagaragaye gusa muri epidermis ahubwo no muri L2 no hejuru ya L3 (Ishusho yinyongera. 8b). Imiterere yibimenyetso bya GA byagaragaye muri SAM ukoresheje qmRGA nayo ntiyigeze ihinduka mugihe (Ishusho yinyongera. 8c - f, k). Nubwo kubaka qd17mRGA byagabanijwe kuri gahunda muri SAM y ibihingwa bya T3 duhereye kumirongo itanu yigenga twaranze muburyo burambuye, twashoboye gusesengura uburyo bwa fluorescence twabonye hamwe na pRPS5a :: VENUS-2A-TagBFP yubatswe (Ishusho yinyongera 8g - j, l). Muri uyu murongo wo kugenzura, hagaragaye impinduka zoroheje mu kigereranyo cya fluorescence zagaragaye muri SAM, ariko mu kigo cya SAM twabonye igabanuka rigaragara kandi ritunguranye muri VENUS ifitanye isano na TagBFP. Ibi biremeza ko uburyo bwo kwerekana ibimenyetso bwerekanwe na qmRGA bugaragaza kwangirika kwa GA guterwa na mRGA-VENUS, ariko kandi byerekana ko qmRGA ishobora gusuzugura ibikorwa byerekana ibimenyetso bya GA muri centre ya meristem. Muncamake, ibisubizo byacu byerekana GA ibimenyetso byerekana cyane cyane ikwirakwizwa rya primordia. Iri gabana ry’akarere k’ibanze (IPR) riterwa no gushiraho buhoro buhoro ibikorwa byinshi byerekana ibimenyetso bya GA hagati ya primordium igenda itera imbere n’akarere ko hagati, mu gihe kimwe ibikorwa bya GA byerekana ibimenyetso muri primordium bigabanuka (Ishusho 4c, d).
Ikwirakwizwa rya GID1b na GID1c (reba hejuru) byerekana ko imvugo itandukanye yerekana reseptor ya GA ifasha gushushanya uburyo bwibikorwa bya GA byerekana muri SAM. Twibajije niba kwirundanya gutandukanye kwa GA bishobora kubigiramo uruhare. Kugira ngo dukore iperereza kuri ibi bishoboka, twakoresheje sensor ya nlsGPS1 GA FRET21. Kwiyongera kwinshuro zagaragaye byagaragaye muri SAM ya nlsGPS1 yavuwe hamwe na 10 μM GA4 + 7 kuminota 100 (Ishusho yinyongera. Ikwirakwizwa ryumwanya wa nlsGPS1 inshuro nyinshi ryagaragaje urwego ruto rwa GA murwego rwo hanze rwa SAM, ariko rwerekana ko rwazamuwe hagati no kumupaka wa SAM (Igicapo 4e hamwe ninyongera Ishusho 9a, c). Ibi birerekana ko GA nayo ikwirakwizwa muri SAM hamwe nuburyo butandukanye ugereranije nibyavuzwe na qmRGA. Nuburyo bwo kuzuzanya, twafashe kandi SAM hamwe na fluorescent GA (GA3-, GA4-, GA7-Fl) cyangwa Fl yonyine nkigenzura ribi. Ikimenyetso cya Fl cyakwirakwijwe muri SAM, harimo akarere ko hagati na primordium, nubwo bifite ubukana buke (Ishusho 4j hamwe n’inyongera 10d). Ibinyuranye, GA-Fl zose uko ari eshatu zegeranije byumwihariko mumipaka ya primordium no kurwego rutandukanye mugice gisigaye cya IPR, hamwe na GA7-Fl yegeranije murwego runini muri IPR (Igishusho 4k hamwe ninyongera 10a, b). Umubare w'uburemere bwa fluorescence wagaragaje ko IPR ku gipimo cy’uburemere butari IPR yari hejuru muri SAM ivurwa na GA-Fl ugereranije na SAM ivurwa na Fl (Ishusho 4l hamwe n’inyongera 10c). Hamwe na hamwe, ibisubizo byerekana ko GA ihari murwego rwo hejuru muri selile IPR iherereye hafi yumupaka wingingo. Ibi birerekana ko uburyo bwibikorwa bya SAM GA byerekana ibisubizo bivuye muburyo butandukanye bwo kwerekana imiterere ya GA no kwegeranya kwa GA muri selile IPR hafi yumupaka wurugingo. Rero, isesengura ryacu ryerekanye uburyo butunguranye bwa spatiotemporal yerekana ibimenyetso bya GA, hamwe nibikorwa byo hasi muri centre na primordium ya SAM nibikorwa byinshi muri IPR mukarere ka peripheri.
Kugira ngo dusobanukirwe uruhare rwibikorwa bitandukanye byerekana ibimenyetso bya GA muri SAM, twasesenguye isano iri hagati yibikorwa byerekana ibimenyetso bya GA, kwaguka kwakagari, no kugabana kwakagari dukoresheje amashusho-nyayo-yerekana amashusho ya SAM qmRGA pCLV3 :: mCherry-NLS. Urebye uruhare rwa GA mugutunganya imikurire, byari biteganijwe ko habaho isano nziza hamwe no kwagura selile. Kubwibyo, twabanje kugereranya ikarita yerekana ibikorwa bya GA hamwe namakarita yikigereranyo cyikura ryimiterere ya selile (nka proksi yimbaraga zo kwaguka kwakagari kuri selile runaka no kubakobwa bagabanijwe) hamwe namakarita yo gukura anisotropy, igapima icyerekezo cyo kwaguka kwakagari (nayo ikoreshwa hano kumasoko yatanzwe hamwe na selile yumukobwa kugabana; Ishusho 5a, b, reba Uburyo nuburyo bwiyongera). Ikarita yacu yikigereranyo cyubwiyongere bwa selile ya SAM ihuye nubushakashatsi bwibanze 38,39, hamwe nubwiyongere bukabije bwikura kumupaka hamwe nubwiyongere bukabije bwiterambere ryindabyo (Ishusho 5a). Isesengura ryibanze ryibanze (PCA) ryerekanye ko ibikorwa byerekana ibimenyetso bya GA byari bifitanye isano mibi nimbaraga zo gukura kwingirabuzimafatizo (Ishusho 5c). Twerekanye kandi ko amashoka nyamukuru yo gutandukana, harimo GA yerekana kwinjiza no gukomera kwiterambere, byari hagati yicyerekezo cyagenwe n’imvugo ndende ya CLV3, yemeza ko ingirabuzimafatizo ziva mu kigo cya SAM mu isesengura risigaye. Isesengura rifitanye isano nicumu ryemeje ibisubizo bya PCA (Igicapo 5d), byerekana ko ibimenyetso bya GA biri hejuru muri IPR bitatumye kwaguka kwinshi kwakagari. Nyamara, isesengura rifitanye isano ryagaragaje isano iri hagati yibikorwa bya GA byerekana ibimenyetso na anisotropy yo gukura (Igicapo 5c, d), byerekana ko ibimenyetso bya GA biri hejuru muri IPR bigira ingaruka ku cyerekezo cyo gukura kwakagari ndetse bikaba bishoboka ko indege igabana.
A. c Isesengura rya PCA ryarimo impinduka zikurikira: ikimenyetso cya GA, ubukana bwikura ryikigereranyo, imikurire yimiterere ya anisotropy, hamwe nimvugo ya CLV3. Igice cya PCA 1 cyari gifitanye isano cyane nimbaraga zo gukura hejuru kandi bifitanye isano neza nikimenyetso cya GA. Igice cya PCA 2 cyari gifitanye isano cyane no gukura hejuru ya anisotropy kandi bifitanye isano nabi n'imvugo ya CLV3. Ijanisha ryerekana itandukaniro ryasobanuwe na buri kintu. d Isesengura ryicumu hagati yikimenyetso cya GA, ubukana bwikura ryikigereranyo, hamwe no gukura hejuru ya anisotropy kurwego rwa tissue ukuyemo CZ. Umubare uri iburyo ni Icumu rho agaciro hagati yimpinduka ebyiri. Asterisks yerekana ibihe aho ihuriro / ribi rifitanye isano cyane. e 3D iyerekwa rya Col-0 SAM L1 selile ikoresheje microscopi ya conocal. Urukuta rushya rwakozwe muri SAM (ariko ntabwo ari primordium) saa 10 h rufite amabara ukurikije agaciro kabo. Ibara ryibara ryerekanwe mugice cyo hepfo yiburyo. Inet yerekana ishusho ya 3D ihuye kuri 0 h. Ubushakashatsi bwasubiwemo kabiri hamwe nibisubizo bisa. f Isanduku yububiko yerekana igipimo cyo kugabana muri IPR hamwe na IPR Col-0 SAM (n = ibimera 10 byigenga). Umurongo wo hagati werekana median, naho imbago zerekana agasanduku zerekana 25 na 75%. Whiskers yerekana agaciro ntarengwa nagaciro kagenwe na software ya R. P indangagaciro zabonetse hamwe na Welch umurizo wa t-test ebyiri. g, h Igishushanyo mbonera cyerekana (g) uburyo bwo gupima inguni y'urukuta rushya rw'akagari (magenta) kubijyanye n'icyerekezo cya radiyo kuva hagati ya SAM (umurongo utudomo wera) (gusa indangagaciro zikomeye, ni ukuvuga 0-90 °, zifatwa), na (h) icyerekezo kizengurutse / cyegeranye na radiyo muri meristem. i Frequency histogrammes ya selile igabana indege yerekeza kuri SAM (ubururu bwijimye), IPR (ubururu buciriritse), na IPR (ubururu bwerurutse). P indangagaciro zabonetse kubizamini bibiri bya Kolmogorov-Smirnov. Ubushakashatsi bwasubiwemo kabiri hamwe nibisubizo bisa. j Frequency histogrammes ya selile igabana indege icyerekezo cya IPR hafi ya P3 (icyatsi kibisi), P4 (icyatsi kibisi), na P5 (icyatsi kibisi). P indangagaciro zabonetse kubizamini bibiri bya Kolmogorov-Smirnov. Ubushakashatsi bwasubiwemo kabiri hamwe nibisubizo bisa.
Noneho, ubutaha twakurikiranye iperereza riri hagati yikimenyetso cya GA nigikorwa cyo kugabana selile tumenye inkuta za selile nshya zakozwe mugihe cyo gusuzuma (Ishusho 5e). Ubu buryo bwatwemereye gupima inshuro nicyerekezo cyo kugabana selile. Igitangaje, twasanze inshuro zo kugabana selile muri IPR hamwe na SAM isigaye (itari IPR, Igishusho 5f) byari bisa, byerekana ko itandukaniro ryerekana ibimenyetso bya GA hagati ya IPR na selile zitari IPR ridafite ingaruka zikomeye kubigabana. Ibi, hamwe nisano nziza iri hagati yikimenyetso cya GA no gukura kwa anisotropy, byaduteye gusuzuma niba ibikorwa byerekana ibimenyetso bya GA bishobora guhindura icyerekezo cyindege igabana. Twapimye icyerekezo cy'urukuta rushya nk'inguni ikaze ugereranije n'umurongo wa radiyo uhuza ikigo cya meristem no hagati y'urukuta rushya (Ishusho 5e-i) maze tureba ko bigaragara ko ingirabuzimafatizo zigabanywa ku mpande zigera kuri 90 ° ugereranije n'umurongo wa radiyo, hamwe n'umuvuduko mwinshi wagaragaye kuri 70-80 ° (23.28%) na 80-90 ° (22.2%). 5h). Kugirango dusuzume uruhare rwa GA rwerekana iyi myitwarire yo kugabana, twasesenguye ibipimo byo kugabana muri IPR na IPR bitandukanye (Ishusho 5i). Twabonye ko gukwirakwiza inguni mu ngirabuzimafatizo za IPR bitandukanye cyane no mu ngirabuzimafatizo zitari IPR cyangwa mu tugari muri SAM yose, hamwe na selile IPR igaragaza umubare munini w'uduce duto duto duto, ni ukuvuga 70-80 ° na 80-90 ° (33.86% na 30.71%, uko bikurikirana) (Ishusho 5i). Rero, ibyo twabonye byagaragaje isano iri hagati yikimenyetso kinini cya GA nicyerekezo cyindege igabanya icyerekezo cyegereye icyerekezo cyizengurutse, bisa nisano iri hagati yibikorwa bya GA byerekana ibimenyetso na anisotropy yo gukura (Ishusho 5c, d). Kugirango turusheho gushimangira kubungabunga iri shyirahamwe, twapimye icyerekezo cyindege igabanywa muri selile IPR ikikije primordium duhereye kuri P3, kubera ko ibikorwa byinshi byerekana ibimenyetso bya GA byagaragaye muri kano karere guhera kuri P4 (Ishusho 4). Inguni zo kugabana IPR hafi ya P3 na P4 zerekanye ko nta tandukanyirizo rishingiye ku mibare rihari, nubwo ubwiyongere bw’amacakubiri yagabanijwe bwagaragaye muri IPR hafi ya P4 (Ishusho 5j). Nyamara, muri selile IPR ikikije P5, itandukaniro mubyerekezo byindege igabanije ingirabuzimafatizo ryabaye ingirakamaro mu mibare, hamwe no kwiyongera gukabije kwinshuro zigabanywa (selile 5j). Hamwe na hamwe, ibisubizo byerekana ko ibimenyetso bya GA bishobora kugenzura icyerekezo cy’amacakubiri muri SAM, ibyo bikaba bihuye na raporo zabanjirije 40,41 ko ikimenyetso kinini cya GA gishobora gutera icyerekezo cy’amacakubiri muri IPR.
Byahanuwe ko selile zo muri IPR zitazinjizwa muri primordia ahubwo zizinjira muri internode2,42,43. Icyerekezo cyo guhinduranya ibice by'utugari muri IPR bishobora kuvamo ishyirahamwe risanzwe rijyanye na parallel longitudinal imirongo ya epidermal selile muri internode. Ibyo twabonye byasobanuwe haruguru byerekana ko ibimenyetso bya GA bishobora kugira uruhare muriki gikorwa muguhuza icyerekezo cyo kugabana.
Gutakaza imikorere ya genes nyinshi za DELLA bivamo igisubizo cya GA, kandi mutant ya della irashobora gukoreshwa mugupima iyi hypothesis44. Twabanje gusesengura imiterere ya genes eshanu za DELLA muri SAM. Guhuza inyandiko-mvugo y'umurongo wa GUS45 byagaragaje ko GAI, RGA, RGL1, na RGL2 (ku rugero ruto cyane) byagaragaye muri SAM (Ishusho y'inyongera 11a - d). Muri Hybridisation yerekana kandi ko GAI mRNA yegeranya cyane muri primordia no guteza imbere indabyo (Ishusho yinyongera 11e). RGL1 na RGL3 mRNA byagaragaye mu kibanza cya SAM no mu ndabyo zishaje, mu gihe RGL2 mRNA yari myinshi mu karere gahana imbibi (Ishusho yinyongera 11f - h). Kwerekana amashusho ya pRGL3 :: RGL3-GFP SAM yemeje imvugo yagaragaye mugihe cyo guhuza imiterere kandi yerekana ko poroteyine ya RGL3 yegeranya mugice cyo hagati cya SAM (Ishusho yinyongera 11i). Twifashishije umurongo wa pRGA :: GFP-RGA, twasanze kandi proteine ya RGA yegeranya muri SAM, ariko ubwinshi bwayo bugabanuka kumupaka guhera kuri P4 (Ishusho yinyongera 11j). Ikigaragara ni uko imvugo ya RGL3 na RGA ihuye nigikorwa cyo hejuru cyerekana ibimenyetso muri GA muri IPR, nkuko byagaragaye na qmRGA (Ishusho 4). Byongeye kandi, aya makuru yerekana ko DELLAs zose zigaragarira muri SAM kandi ko imvugo yabo hamwe ikwirakwiza SAM yose.
Ubukurikira twasesenguye ibipimo by'utugingo ngengabuzima mu bwoko bwa SAM (Ler, kugenzura) na gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (isi) ihinduka (Ishusho 6a, b). Igishimishije, twabonye ihinduka rikomeye mu mibare mu gukwirakwiza ingirabuzimafatizo zigabanywa muri selile ya della ku isi yose ugereranije n'ubwoko bw'ishyamba (Ishusho 6c). Ihinduka muri della ku isi yose ryatewe no kwiyongera kwinshuro zingana na 80-90 ° (34,71% na 24.55%) kandi, ku rugero ruto, inguni 70-80 ° (23,78% na 20.18%), ni ukuvuga, bihuye n’amacakubiri ya selile (Ishusho 6c). Inshuro z’amacakubiri adahinduka (0-60 °) nayo yari hasi muri della yisi yose (Ishusho 6c). Inshuro zo gutandukanya ingirabuzimafatizo ziyongereye cyane muri SAM ya mutant ya della ku isi (Ishusho 6b). Inshuro zo kugabana utugingo ngengabuzima muri IPR nazo zari hejuru muri della ku isi yose ugereranije n'ubwoko bw'ishyamba (Ishusho 6d). Hanze y'akarere ka IPR, ubwoko bw'ishyamba bwari bufite uburyo bwo gukwirakwiza ingirabuzimafatizo zigabanywa, mu gihe della ku isi ya mutant yahisemo kugabana ibice nka IPR (Ishusho 6e). Twagereranije kandi icyerekezo cyo kugabana selile muri SAM ya ga2 oxydease (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, na ga2ox6-2), GA-idakora ihinduka ryimiterere ya GA. Bihuye n'ubwiyongere bw'urwego rwa GA, SAM ya quintuple ga2ox mutant inflorescence yari nini kuruta iya Col-0 (Igishushanyo cy'inyongera. Rero, twerekana ko ibikorwa bigize GA byerekana ibimenyetso hamwe no gukusanya GA bitera amacakubiri ya selile muri IPR hamwe na SAM isigaye.
a, b 3D iyerekwa rya L1 urwego rwa PI-Ler (a) hamwe na della mutant (b) SAM kwisi yose ukoresheje microscopi conocal. Urukuta rushya rwakorewe muri SAM (ariko ntabwo ari primordium) mugihe cya 10-h rwerekanwe kandi rufite amabara ukurikije agaciro kabo. Inet yerekana SAM kuri 0 h. Ibara ryibara ryerekanwa mugice cyiburyo cyiburyo. Umwambi muri (b) werekana urugero rwimikorere ya dosiye ya selile muri global della mutant. Ubushakashatsi bwasubiwemo kabiri hamwe nibisubizo bisa. ce kugereranya inshuro zo gukwirakwiza indege igabana indege yerekanwe muri SAM yose (d), IPR (e), na IPR (f) hagati ya Ler na della yisi. P indangagaciro zabonetse ukoresheje ikizamini cyumurizo wa Kolmogorov-Smirnov. f, g 3D amashusho yerekana amashusho asebanya ya PI-SAM ya Col-0 (i) na pCUC2 :: gai-1-VENUS (j) ibimera bya transgenji. Ikibaho (a, b) cyerekana urukuta rushya (ariko ntabwo ari primordia) rwakozwe muri SAM bitarenze 10 h. Ubushakashatsi bwasubiwemo kabiri hamwe nibisubizo bisa. h - j Kugereranya inshuro zo gukwirakwiza indege igabanije indege igana muri SAM yose (h), IPR (i) na IPR (j) hagati ya Col-0 na pCUC2 :: gai-1-VENUS. P indangagaciro zabonetse ukoresheje ikizamini cyumurizo wa Kolmogorov - Smirnov.
Ubukurikira twagerageje ingaruka zo kubuza ibimenyetso bya GA byumwihariko muri IPR. Kugira ngo ibyo bishoboke, twakoresheje poroteri ya cotyledon 2 (CUC2) kugirango dutware imvugo ya proteine mbi ya gai-1 yiganje kuri VENUS (mumurongo wa pCUC2 :: gai-1-VENUS). Mu bwoko bwa SAM bwo mu gasozi, porotokoro ya CUC2 itwara imvugo ya IPR nyinshi muri SAM, harimo selile zumupaka, kuva P4 gukomeza, kandi imvugo nkiyi yagaragaye muri pCUC2 :: gai-1-VENUS (reba hepfo). Ikwirakwizwa ryingirabuzimafatizo zigabanywa kuri SAM cyangwa IPR ya pCUC2 :: gai-1-VENUS ntago yari itandukanye cyane nubwoko bw’ishyamba, nubwo mu buryo butunguranye twasanze ingirabuzimafatizo zidafite IPR muri ibyo bimera zigabanijwe ku ntera ndende ya 80-90 ° (Ishusho 6f - j).
Hasabwe ko icyerekezo cyo kugabana selile giterwa na geometrie ya SAM, cyane cyane imihangayiko ikabije iterwa na tissue curvature46. Twabajije rero niba imiterere ya SAM yahinduwe muri della yisi ya mutant na pCUC2 :: gai-1-VENUS. Nkuko byavuzwe mbere12, ubunini bwa della ku isi yose mutant SAM yari nini kuruta iy'ishyamba (Ishusho yinyongera 13a, b, d). Muri Hybridisation ya CLV3 na STM RNA yemeje ko meristem yagutse muri mutant ya della kandi ikomeza kwerekana kwaguka kuruhande rwa selile niche (Umugereka wongeyeho 13e, f, h, i). Nyamara, kugabanuka kwa SAM kwarasa muri genotypes zombi (Ishusho yinyongera. 13k, m, n, p). Twabonye ubwiyongere busa nubunini muri gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant nta gihindutse ku kugabanuka ugereranije nubwoko bwishyamba (Ishusho yinyongera 13c, d, g, j, l, o, p). Inshuro yo kugabana ingirabuzimafatizo yerekanwe no muri mutant ya della ya kane, ariko ku rugero ruto ugereranije no muri della monolithic mutant (Ishusho yinyongera. 12d - f). Ingaruka ya dosiye, hamwe no kutagira ingaruka kuri curvature, yerekana ko ibikorwa bya RGL3 bisigaye muri Della quadruple mutant bigabanya impinduka mubyerekezo byo kugabana kwagateganyo biterwa no gutakaza ibikorwa bya DELLA kandi ko impinduka mumacakubiri y'utugari zibaho zisubiza impinduka mubikorwa bya GA byerekana ibimenyetso aho guhinduka muri geometrike ya SAM. Nkuko byasobanuwe haruguru, umushoramari wa CUC2 atwara imvugo ya IPR muri SAM guhera kuri P4 (Ishusho yinyongera. Ihinduka muri pCUC2 :: gai-1-VENUS SAM morphologie irashobora kuvamo gukwirakwiza ukundi guhangayikishwa nubukanishi ugereranije nubwoko bwishyamba, aho imihangayiko yo kuzenguruka itangirira ku ntera ngufi na santere ya SAM47. Ubundi, impinduka muri pCUC2 :: gai-1-VENUS SAM morphologie irashobora guturuka kumihindagurikire yimiterere yubukanishi bwakarere iterwa na transgene imvugo48. Muri ibyo bihe byombi, ibi birashobora gukuraho igice cyingaruka zimpinduka mubimenyetso bya GA byongera amahirwe yuko selile zigabanywa muburyo bwo kuzenguruka / guhinduranya, gusobanura ibyo twabonye.
Dufatiye hamwe, amakuru yacu yemeza ko ibimenyetso bya GA byo hejuru bigira uruhare runini mubyerekezo byindege igabana indege muri IPR. Berekana kandi ko meristem curvature nayo igira uruhare mubyerekezo byindege igabana muri IPR.
Icyerekezo cyo guhinduranya indege igabana muri IPR, kubera ibikorwa byinshi byerekana ibimenyetso bya GA, byerekana ko GA ibanziriza gutegura dosiye ya selile ya selile muri epidermis muri SAM kugirango isobanure ishyirahamwe ryimikorere rizaboneka nyuma muri internode epidermal. Mubyukuri, amadosiye nkaya yagaragaye kenshi mumashusho ya SAM ya della yisi yose (Ishusho 6b). Rero, kugirango turusheho gucukumbura imikorere yiterambere ryimiterere ya GA yerekana ibimenyetso muri SAM, twakoresheje amashusho yigihe kugirango dusesengure imitunganyirize yimiterere yingirabuzimafatizo muri IPR mubwoko bwishyamba (Ler na Col-0), della ihinduka ryimiterere yisi yose, na pCUC2 :: gai-1-VENUS ibimera.
Twabonye ko qmRGA yerekanaga ko ibikorwa bya GA byerekana muri IPR byiyongereye kuva kuri P1 / P2 bikagera kuri P4, kandi ubu buryo bwakomeje guhoraho mugihe (Ishusho 4a - f hamwe nishusho ya 8c - f, k). Kugirango dusesengure imiterere yimikorere ya selile muri IPR hamwe no kongera ibimenyetso bya GA, twanditseho selile ya Ler IPR hejuru no kumpande za P4 dukurikije uko iterambere ryabo ryasesenguye 34 h nyuma yo kwitegereza bwa mbere, ni ukuvuga inshuro zirenze ebyiri za plastide, bituma dushobora gukurikira selile IPR mugihe cya primordium kuva P1 / P2 kugeza P4. Twakoresheje amabara atatu atandukanye: umuhondo kuri utwo tugari twinjijwe muri primordium hafi ya P4, icyatsi kubari muri IPR, n'umuhengeri ku bagize uruhare muri ibyo bikorwa byombi (Ishusho 7a - c). Kuri t0 (0 h), ibice 1-2 bya selile ya IPR byagaragaye imbere ya P4 (Ishusho 7a). Nkuko byari byitezwe, mugihe utugingo ngengabuzima twagabanijwe, babikoze cyane cyane binyuze mu ndege igabana (Isanamu 7a - c). Ibisubizo nkibi byabonetse ukoresheje Col-0 SAM (yibanda kuri P3, imipaka yayo isa na P4 muri Ler), nubwo muri iyi genotype ububiko bwakorewe kumupaka windabyo bwahishe selile IPR vuba (Ishusho 7g - i). Rero, igabana ryimikorere ya selile IPR ibanza gutegura selile mumirongo ya radiyo, nkuko biri muri internode. Gutondekanya imirongo ya radiyo hamwe no gutandukanya ingirabuzimafatizo za IPR hagati yingingo zikurikirana byerekana ko izo selile ari progenitor.
Hano, twateje imbere ratiometric GA yerekana biosensor, qmRGA, ituma ikarita yerekana umubare wibikorwa byerekana ibimenyetso bya GA biturutse ku guhuriza hamwe kwa GA hamwe na GA mugihe twagabanije kwivanga ninzira zerekana ibimenyetso, bityo tugatanga amakuru kumikorere ya GA kurwego rwa selire. Kugira ngo ibyo bishoboke, twubatsemo poroteyine ya DELLA yahinduwe, mRGA, yatakaje ubushobozi bwo guhuza abafatanyabikorwa ba DELLA ariko ikomeza kumva proteolysis iterwa na GA. qmRGA isubiza impinduka zidasanzwe ndetse na endogenous murwego rwa GA, kandi imbaraga zayo zo kwiyumvisha ibintu zituma hasuzumwa impinduka za spatiotemporal mubikorwa bya GA byerekana ibikorwa mugihe cyiterambere. qmRGA nayo nigikoresho cyoroshye cyane kuko gishobora guhuzwa nuduce dutandukanye muguhindura porotokoro ikoreshwa mukugaragaza (nibiba ngombwa), kandi ukurikije imiterere yabitswe yinzira ya GA yerekana inzira na motif ya PFYRE hakurya ya angiosperms, birashoboka ko ishobora kwimurirwa mubindi binyabuzima22. Bihuye nibi, ihinduka rihwanye numuceri SLR1 DELLA proteine (HYY497AAA) naryo ryerekanwe guhagarika ibikorwa byo gukumira imikurire ya SLR1 mugihe byagabanije gato kwangirika kwabo kwa GA, bisa na mRGA23. Ikigaragara ni uko ubushakashatsi buherutse gukorwa muri Arabidopsis bwerekanye ko ihindagurika rimwe rya aside amine muri domaine ya PFYRE (S474L) ryahinduye ibikorwa byo kwandikirana kwa RGA bitagize ingaruka ku bushobozi bwo gukorana nabafatanyabikorwa ba transcription50. Nubwo iyi mutation yegereye cyane insimburangingo 3 ya aminide acide igaragara muri mRGA, ubushakashatsi bwacu bwerekana ko ihinduka ryimiterere yombi rihindura ibintu bitandukanye biranga DELLA. Nubwo abafatanyabikorwa benshi bahinduranya bahuza LHR1 na SAW ya DELLA26,51, aminide acide yabitswe muri domeni ya PFYRE irashobora gufasha guhuza imikoranire.
Iterambere rya internode nimwe mumico yingenzi mububiko bwibimera no kuzamura umusaruro. qmRGA yerekanye ibikorwa byinshi bya GA byerekana muri IPR internode progenitor selile. Muguhuza amashusho yumubare hamwe na genetika, twerekanye ko GA yerekana ibimenyetso byerekana indege zigabanya uruziga / transvers selile igabana muri epidermis ya SAM, ishyiraho ishyirahamwe ryigabana ryakagari risabwa kugirango iterambere ryimbere. Abagenzuzi benshi bayobora indege igabana indege bamenyekanye mugihe cyiterambere 52,53. Akazi kacu gatanga urugero rusobanutse rwuburyo ibikorwa bya GA byerekana ibimenyetso bya selile. DELLA irashobora gukorana na poroteyine ibanziriza 41, bityo ibimenyetso bya GA birashobora kugenga icyerekezo cyindege igabanya icyerekezo cya microtubule ya cortical 40,41,54,55. Twerekanye mu buryo butunguranye ko muri SAM, ihuriro ryibikorwa byo hejuru bya GA bitari ukurambura selile cyangwa kugabana, ahubwo ni anisotropy yo gukura gusa, ibyo bikaba bihuye ningaruka itaziguye ya GA ku cyerekezo cyo kugabana selile muri IPR. Ariko, ntidushobora gukuraho ko izi ngaruka zishobora no kuba indirect, kurugero rwahujwe na GA iterwa nurukuta rwa selile koroshya 56. Imihindagurikire yimiterere yurukuta rwa selile itera ubukanishi 57,58, ishobora kandi guhindura icyerekezo cyindege igabana ingirabuzimafatizo bigira ingaruka kuri microtubules ya cortical 39,46,59. Ingaruka zifatika ziterwa na GA ziterwa no gukanika no kugenzura mu buryo butaziguye icyerekezo cya microtubule na GA zishobora kugira uruhare mugutanga uburyo bwihariye bwo kugabana selile muri IPR kugirango dusobanure interode, kandi birakenewe ubundi bushakashatsi kugirango dusuzume iki gitekerezo. Mu buryo nk'ubwo, ubushakashatsi bwibanze bwerekanye akamaro ka poroteyine DELLA ikorana na TCP14 na 15 mu kugenzura imiterere ya internode 60,61 kandi ibyo bintu bishobora guhuza ibikorwa bya GA hamwe na BREVIPEDICELLUS (BP) na PENNYWISE (PNY), bigenga iterambere rya internode kandi byagaragaye ko bigira ingaruka kuri GA signal2,62. Urebye ko DELLAs ikorana na brassinosteroid, Ethylene, acide jasmonic, na aside abcisic (ABA) yerekana inzira 63,64 kandi ko iyi misemburo ishobora kugira ingaruka kuri microtubule65, ingaruka za GA mubyerekezo byo kugabana selile zishobora no guhuzwa nindi misemburo.
Ubushakashatsi bwambere bwa cytologiya bwerekanye ko uturere twimbere ninyuma ya Arabidopsis SAM isabwa kugirango iterambere ryimbere2,42. Kuba GA igenga cyane igabana ryimikorere mubice byimbere12 ishyigikira imikorere ibiri ya GA muguhuza meristem nubunini bwa internode muri SAM. Uburyo bwo kugabana ingirabuzimafatizo kandi bugengwa cyane mubice by'imbere bya SAM, kandi aya mabwiriza ni ngombwa kugirango imikurire ikure 52. Bizaba bishimishije gusuzuma niba GA nayo igira uruhare mukuyobora indege igabana selile mumuryango wa SAM imbere, bityo igahuza ibisobanuro niterambere rya internode muri SAM.
Ibimera byahinzwe muri vitro mubutaka cyangwa 1x Murashige-Skoog (MS) iciriritse (Duchefa) hiyongeraho 1% sucrose na 1% agar (Sigma) mubihe bisanzwe (16 h urumuri, 22 ° C), usibye hypocotyl nubushakashatsi bwakuze bwumuzi aho ingemwe zahinzwe kumasahani ahagaritse munsi yumucyo uhoraho na 22 ° C. Kubushakashatsi bwa nitrate, ibihingwa byahinzwe kuri MS yahinduwe (bioWORLD igihingwa giciriritse) hiyongeraho nitrate ihagije (0 cyangwa 10 mM KNO3), 0.5 mM NH4-succinc, 1% sucrose na 1% A-agar (Sigma) mubihe byumunsi.
GID1a cDNA yinjijwe muri pDONR221 yahujwe na pDONR P4-P1R-pUBQ10 na pDONR P2R-P3-mCherry muri pB7m34GW kubyara pUBQ10 :: GID1a-mCherry. ADN IDD2 yinjijwe muri pDONR221 yongeye guhuzwa muri pB7RWG266 kugirango itange p35S: IDD2-RFP. Kubyara pGID1b :: 2xmTQ2-GID1b, agace ka 3.9 kb hejuru yubutaka bwakarere ka code ya GID1b hamwe nigice cya 4.7 kb kirimo GID1b cDNA (1.3 kb) na terminator (3.4 kb) babanje kongererwa imbaraga bakoresheje primers mumeza yinyongera 3 hanyuma binjizwa muri pDONR P4R P2R-P3 (Thermo Fisher Scientific), hanyuma, hanyuma yongera guhuzwa na pDONR221 2xmTQ268 muri pGreen 012567 yerekana icyerekezo ukoresheje clone ya Gateway. Kubyara pCUC2 :: LSSmOrange, urutonde rwa porotokoro ya CUC2 (3229 bp hejuru ya ATG) ikurikirwa na code ikurikirana ya mOrange nini ya Stoke yimuwe (LSSmOrange) 69 hamwe nikimenyetso cya N7 cyerekana ibimenyetso bya kirimbuzi hamwe na terminator ya NOS yakusanyirijwe muri pGreen kanamycin yibasiye vector ikoresheje sisitemu ya Gateway. Ikimera cya binary vector cyinjijwe muri Agrobacterium tumefaciens strain GV3101 hanyuma cyinjizwa mumababi ya Nicotiana benthamiana hakoreshejwe uburyo bwa Agrobacterium no kwinjira muri Arabidopsis thaliana Col-0 hakoreshejwe uburyo bwo kwibiza indabyo. pUBQ10 :: qmRGA pUBQ10 :: GID1a-mCherry na pCLV3 :: mCherry-NLS qmRGA bitandukanije na F3 na F1 kubyara imisaraba bijyanye.
RNA muburyo bwa Hybridisation yakorewe kuri cm 1 z'uburebure bwo kurasa 72, zegeranijwe zihita zishyirwa mubisubizo bya FAA (3,7% formaldehyde, 5% acide acike, 50% etanol) mbere yo gukonja kugeza kuri 4 ° C. Nyuma yo kuvura 2 × 15 min vacuum, gukosora byarahinduwe kandi ingero zaraye zakozwe ijoro ryose. GID1a, GID1b, GID1c, GAI, RGL1, RGL2, na RGL3 cDNAs hamwe na antisense probe kuri 3′-UTR zabo zahujwe hakoreshejwe primers zerekanwa mumbonerahamwe 3 nkuko byasobanuwe na Rosier et al.73. Ubushakashatsi bwa Digoxigenin bwarakingiwe hakoreshejwe antibodiyite za digoxigenin (inshuro 3000;
Igihe cyo kohereza: Gashyantare-10-2025