Ikura ry'umumero w'igiti (SAM) ni ingenzi cyane mu miterere y'igiti.gibberellins(GA) zigira uruhare runini mu guhuza imikurire y'ibimera, ariko uruhare rwabyo muri SAM ntirusobanuka neza. Aha, twakoze uburyo bwo gupima imiterere y'ibinyabutabire bya GA binyuze mu gukora poroteyine ya DELLA kugira ngo ihagarike imikorere yayo y'ingenzi mu kugenzura imikorere ya GA mu gihe cyo kuyihindura mu buryo, ariko ikarinda kwangirika kwayo igihe GA imenyekanye. Tugaragaza ko iyi biosensor ishingiye ku kwangirika yandika neza impinduka mu rugero rwa GA n'uburyo bwo kumva mu ngirabuzimafatizo mu gihe cyo kuyiteza imbere. Twakoresheje iyi biosensor kugira ngo dushyire ahagaragara ibikorwa bya GA mu gupima imikorere muri SAM. Tugaragaza ko ibimenyetso bya GA byinshi biboneka cyane cyane mu turemangingo turi hagati y'ingingo za mbere z'umubiri, ari zo zibanziriza uturemangingo twa internode. Dukoresheje uburyo bwo kwiyongera no gutakaza imikorere, tugaragaza ko GA igenzura icyerekezo cy'urwego rw'igabanywa ry'uturemangingo, ishyiraho imiterere y'uturemangingo twa internode, bityo igateza imbere uburyo internode zigenwa muri SAM.
Ishu ry’umuhondo (SAM), riherereye ku gasongero k’uruti, rigizwe n’urusobe rw’uturemangingo tw’uruti aho ibikorwa byatwo bitanga ingingo z’uruhande n’uturemangingo tw’uruti mu buryo busanzwe kandi busubiramo mu buzima bwose bw’ikimera. Buri gice cy’ibi bisubirwamo, cyangwa uturemangingo tw’ibimera, kirimo uturemangingo tw’uruti n’uturemangingo tw’uruti ku turemangingo, n’uturemangingo tw’uruti mu turemangingo tw’uruti mu turemangingo tw’uruti 1. Gukura no gutunganya uturemangingo tw’ibimera birahinduka mu gihe cyo gukura. Muri Arabidopsis, gukura kw’uturemangingo tw’uruti bigabanuka mu gihe cy’ibimera, kandi uturemangingo tw’uruti tuguma mu turemangingo tw’amababi ya rosette. Mu gihe cyo guhinduka kugera ku cyiciro cy’indabyo, SAM iba meristem y’uruti, ikabyara uturemangingo tw’uruti turerure n’uturemangingo tw’uruti, amashami mu turemangingo tw’amababi ya cauline, hanyuma, indabyo zitagira amababi 2. Nubwo twateye imbere cyane mu gusobanukirwa uburyo bugenzura gutangira kw’amababi, indabyo, n’amashami, ni bike cyane bizwi ku buryo uturemangingo tw’uruti tuvuka.
Gusobanukirwa uburyo GA zikwirakwira mu buryo bw’ikirere bizafasha gusobanukirwa neza imikorere y’iyi misemburo mu ngingo zitandukanye no mu byiciro bitandukanye by’iterambere. Kureba ukwangirika kwa RGA-GFP fusion igaragazwa n’igikorwa cya promoter yayo bwite bitanga amakuru y’ingenzi ku igenzura ry’urugero rwa GA mu mizi15,16. Ariko, igaragara rya RGA riratandukanye mu ngingo17 kandi rigenzurwa na GA18. Bityo, igaragara ry’itandukaniro rya promoter ya RGA rishobora gutuma habaho imiterere ya fluorescence igaragara kuri RGA-GFP bityo ubu buryo ntibufite ingano. Vuba aha, GA19,20 ifite ikirango cya bioactive fluorescein (Fl) yagaragaje ukwiyongera kwa GA mu gice cy’umuzi wa endocortex no kugenzurwa kw’urwego rwayo rw’uturemangingo binyuze mu gutwara GA. Vuba aha, sensor ya GA FRET nlsGPS1 yagaragaje ko urugero rwa GA rufitanye isano no kurekura k’uturemangingo mu mizi, mu turemangingo, no mu turemangingo tw’umwijima21. Ariko, nk’uko twabibonye, ingano ya GA si yo yonyine igenzura ibikorwa byo kwerekana GA, kuko biterwa n’uburyo bugoye bwo kumva. Aha, dushingiye ku gusobanukirwa kwacu inzira zo gutanga ibimenyetso za DELLA na GA, dutanga raporo ku iterambere n'imiterere ya biosensor ishingiye ku kwangirika kw'ibimenyetso bya GA. Kugira ngo duteze imbere iyi biosensor y'ingano, twakoresheje RGA ifite imiterere ya GA yahujwe na poroteyine y'urumuri kandi igaragara hose mu turemangingo, ndetse na poroteyine ifite imiterere ya GA. Tugaragaza ko imikoranire ya poroteyine ya RGA idahuza n'ibimenyetso bya GA iyo igaragara hose, kandi ko iyi biosensor ishobora gupima ibikorwa byo gutanga ibimenyetso bituruka ku kwinjira kwa GA no gutunganya ibimenyetso bya GA hakoreshejwe igikoresho cyo kumva gifite ubushobozi bwo hejuru bwo gusuzuma. Twakoresheje iyi biosensor kugira ngo dushushanye uburyo ibikorwa byo gutanga ibimenyetso bya GA bikwirakwira mu gihe runaka no gupima uburyo GA igenzura imyitwarire y'uturemangingo mu gice cya SAM. Tugaragaza ko GA igenzura icyerekezo cy'imiterere y'uturemangingo twa SAM turi hagati y'ingingo za mbere z'umubiri, bityo tugasobanura imiterere y'uturemangingo tw'internode.
Amaherezo, twabajije niba qmRGA ishobora gutanga raporo ku mpinduka mu rugero rwa GA rw’umwimerere hakoreshejwe hypocotyls zikura. Mbere twagaragaje ko nitrate itera imbaraga mu gukura binyuze mu kongera GA synthesis, hanyuma DELLA34 ikaranduka. Bityo, twabonye ko uburebure bwa hypocotyl mu mbuto za pUBQ10::qmRGA zatewe mu gihe cy’ubushyuhe bwinshi bwa nitrate (10 mM NO3−) bwari burebure cyane kurusha ubw’imbuto zatewe mu gihe cy’ubukene bwa nitrate (Ishusho y’inyongera ya 6a). Dukurikije uko ikura ryagenze, ibimenyetso bya GA byari byinshi mu mbuto zatewe mu gihe cy’ubushyuhe bwa mM NO3− 10 kurusha mu mbuto zatewe mu gihe nta nitrate irimo (Ishusho y’inyongera ya 6b, c). Bityo, qmRGA kandi ituma habaho gukurikirana impinduka mu bimenyetso bya GA biterwa n’impinduka mu buryo bwa GA.
Kugira ngo dusobanukirwe niba ibikorwa byo gutanga ibimenyetso bya GA byagaragaye na qmRGA biterwa n'ubwinshi bwa GA n'uburyo GA ibonwa, nk'uko byari byitezwe hashingiwe ku gishushanyo mbonera cy'imashini, twasesenguye uburyo receptor eshatu za GID1 zigaragara mu ngingo z'ibimera n'iz'imyororokere. Mu bimera, umurongo w'umunyamakuru wa GID1-GUS wagaragaje ko GID1a na c byagaragaye cyane muri cotyledons (Ishusho ya 3a-c). Byongeye kandi, receptor zose uko ari eshatu zagaragaye mu mababi, ku gice cy'imizi y'inyuma, ku mitwe y'imizi (usibye umutwe w'umuzi wa GID1b), no ku mitsi y'amaraso (Ishusho ya 3a-c). Muri SAM y'ururabo, twabonye ibimenyetso bya GUS kuri GID1b na 1c gusa (Ishusho y'inyongera ya 7a-c). Guhuza imiterere y'ishusho muri situ byemeza ubwo buryo bwo kugaragaza kandi berekana ko GID1c yagaragaye neza ku rugero rwo hasi muri SAM, mu gihe GID1b yagaragaje ubwinshi ku nkengero za SAM (Ishusho y'inyongera ya 7d-l). Uburyo bwo guhuza pGID1b::2xmTQ2-GID1b bwagaragaje kandi urwego rw’imiterere ya GID1b, kuva ku miterere mike cyangwa nta miterere iri hagati ya SAM kugeza ku mipaka y’ingingo (Ishusho y’inyongera ya 7m). Bityo, receptors za GID1 ntizikwirakwira mu buryo bumwe no mu ngingo. Mu bushakashatsi bwakurikiyeho, twabonye kandi ko imiterere myinshi ya GID1 (pUBQ10::GID1a-mCherry) yongereye ubushobozi bwo kumva qmRGA muri hypocotyls kugeza ku mikoreshereze ya GA yo hanze (Ishusho ya 3d, e). Mu buryo bunyuranye, fluorescence yapimwe na qd17mRGA muri hypocotyl ntiyashoboraga kumva GA3 (Ishusho ya 3f, g). Ku bipimo byombi, ingemwe zavuwe zifite GA nyinshi (100 μM GA3) kugira ngo hasuzumwe imikorere yihuse ya sensor, aho ubushobozi bwo gufatana na receptors za GID1 bwarushijeho kwiyongera cyangwa bukabura. Ibi bisubizo byemeza ko qmRGA biosensor ikora akazi gahuriweho nka sensor ya GA na GA, kandi bigaragaza ko itandukaniro ry’ishusho ya receptor ya GID1 rishobora guhindura cyane imikorere y’imashini.
Kugeza ubu, ikwirakwizwa ry'ibimenyetso bya GA muri SAM ntirirasobanuka neza. Kubwibyo, twakoresheje ibimera bitanga ibimenyetso bya qmRGA hamwe na pCLV3::mCherry-NLS stem cell reporter35 kugira ngo tubare amakarita y'ingano y'ibikorwa bya GA, twibanda ku gice cya L1 (epidermis; Ishusho ya 4a, b, reba Uburyo n'Uburyo bw'Inyongera), kubera ko L1 igira uruhare runini mu kugenzura ikura rya SAM36. Aha, pCLV3::mCherry-NLS yatangaga ahantu hahamye ho gusesengura ikwirakwizwa ry'ibikorwa bya GA37. Nubwo GA ifatwa nk'ingenzi mu iterambere ry'ingingo zo ku ruhande4, twabonye ko ibimenyetso bya GA byari bike mu gice cy'indabyo (P) uhereye ku cyiciro cya P3 (Ishusho ya 4a, b), mu gihe ibice bito bya P1 na P2 byari bifite ibikorwa biringaniye nk'ibyo mu gace ko hagati (Ishusho ya 4a, b). Igikorwa cyo gutanga ibimenyetso bya GA cyagaragaye ku mbibi z'ingingo, guhera kuri P1/P2 (ku mpande z'umupaka) no kuzamuka kuri P4, ndetse no mu turemangingo twose tw'akarere kari hagati y'inyongera (Ishusho ya 4a, b n'Ishusho y'inyongera 8a, b). Iki gikorwa cyo gutanga ibimenyetso bya GA cyagaragaye atari mu gice cy'imbere cy'umubiri gusa ahubwo no mu duce twa L2 na L3 two hejuru (Ishusho y'inyongera 8b). Imiterere y'ibimenyetso bya GA byabonetse muri SAM hakoreshejwe qmRGA nayo ntiyahindutse uko igihe cyagiye gihita (Ishusho y'inyongera 8c–f, k). Nubwo imiterere ya qd17mRGA yagabanijwe mu buryo burambuye muri SAM y'ibimera bya T3 uhereye ku mirongo itanu yigenga twagaragaje mu buryo burambuye, twashoboye gusesengura imiterere ya fluorescence yabonetse hamwe n'imiterere ya pRPS5a::VENUS-2A-TagBFP (Ishusho y'inyongera 8g–j, l). Muri uyu murongo w’igenzura, hagaragaye impinduka nto gusa mu gipimo cy’urumuri muri SAM, ariko muri SAM centre twabonye igabanuka rigaragara kandi ritunguranye rya VENUS rijyanye na TagBFP. Ibi byemeza ko imiterere y’ibimenyetso yagaragaye na qmRGA igaragaza kwangirika kwa mRGA-VENUS bitewe na GA, ariko kandi bigaragaza ko qmRGA ishobora kurenza urugero mu bikorwa by’ibimenyetso bya GA muri meristem centre. Muri make, ibyavuye mu bushakashatsi bwacu bigaragaza imiterere y’ibimenyetso bya GA igaragaza ahanini ikwirakwizwa rya primordia. Iri kwirakwizwa ry’akarere ka inter-primordial (IPR) riterwa no gushyiraho buhoro buhoro ibikorwa by’ibimenyetso bya GA byinshi hagati y’igice cy’ibanze kirimo gukura n’igice cyo hagati, mu gihe icyarimwe ibikorwa by’ibimenyetso bya GA muri primordium bigabanuka (Ishusho ya 4c, d).
Ikwirakwizwa rya za receptors za GID1b na GID1c (reba hejuru) rigaragaza ko itandukaniro ry’imvugo za GA receptors rifasha mu gushyiraho imiterere y’ibikorwa bya GA byo gutanga ibimenyetso muri SAM. Twibajije niba ubwiyongere bw’itandukaniro bwa GA bushobora kuba burimo. Kugira ngo dukore ubushakashatsi kuri ibi bishoboka, twakoresheje sensor ya nlsGPS1 GA FRET21. Inshuro nyinshi zo gukoresha zabonetse muri SAM ya nlsGPS1 yavuwe na 10 μM GA4+7 mu gihe cy’iminota 100 (Ishusho y’inyongera ya 9a–e), bigaragaza ko nlsGPS1 isubiza impinduka mu gipimo cya GA muri SAM, nkuko bigenda mu mizi21. Ikwirakwizwa ry’inshuro zo gukoresha za nlsGPS1 ryagaragaje urugero rwa GA ruri hasi mu bice byo hanze bya SAM, ariko ryagaragaje ko zari hejuru hagati no ku mbibi za SAM (Ishusho ya 4e n’Ishusho y’inyongera ya 9a,c). Ibi bigaragaza ko GA nayo ikwirakwizwa muri SAM ifite imiterere y’ahantu igereranywa n’iyahishuwe na qmRGA. Nk'uburyo bwuzuzanya, twakoresheje SAM fluorescent GA (GA3-, GA4-, GA7-Fl) cyangwa Fl gusa nk'uburyo bwo kugenzura nabi. Ikimenyetso cya Fl cyakwirakwijwe muri SAM yose, harimo n'akarere ko hagati n'inyongera, nubwo byari bifite imbaraga nke (Ishusho ya 4j n'Ishusho y'inyongera ya 10d). Mu buryo bunyuranye, GA-Fl zose uko ari eshatu zakusanyije by'umwihariko mu mbibi za primordium no ku rugero rutandukanye mu bindi bice bya IPR, aho GA7-Fl yikusanyiriza mu gice kinini muri IPR (Ishusho ya 4k n'Ishusho y'inyongera ya 10a,b). Ingano y'imbaraga za fluorescence yagaragaje ko igipimo cya IPR ku mbaraga zitari iza IPR cyari kinini muri SAM yavuwe na GA-Fl ugereranije na SAM yavuwe na Fl (Ishusho ya 4l n'Ishusho y'inyongera ya 10c). Ibi bisubizo byose hamwe bigaragaza ko GA iboneka ku rugero rwo hejuru mu turemangingo twa IPR turi hafi y'umupaka w'ingingo. Ibi bigaragaza ko imiterere y'ibikorwa byo gutanga ibimenyetso bya SAM GA ituruka ku miterere itandukanye ya za receptor za GA ndetse no ku kwegeranya GA mu turemangingo twa IPR hafi y'imipaka y'ingingo. Bityo, isesengura ryacu ryagaragaje imiterere itunguranye y'ibikorwa byo gutanga ibimenyetso bya GA, aho ibikorwa bike biri hagati no mu gice cya mbere cya SAM n'ibikorwa byinshi muri IPR mu gace k'inyuma.
Kugira ngo dusobanukirwe uruhare rw'ibikorwa by'ibimenyetso bya GA bitandukanye muri SAM, twasesenguye isano iri hagati y'ibikorwa by'ibimenyetso bya GA, kwaguka kw'uturemangingo, no kugabana kw'uturemangingo dukoresheje ishusho ya SAM qmRGA pCLV3::mCherry-NLS mu gihe nyacyo. Bitewe n'uruhare rwa GA mu kugenzura imikurire, byari byitezwe ko habaho isano nziza n'ibipimo byo kwaguka kw'uturemangingo. Kubwibyo, twabanje kugereranya amakarita y'ibikorwa by'ibimenyetso bya GA hamwe n'amakarita y'igipimo cyo gukura kw'ubuso bw'uturemangingo (nk'ikimenyetso cy'imbaraga zo kwaguka kw'uturemangingo ku turemangingo runaka no ku turemangingo tw'ingore mu kugabana) hamwe n'amakarita y'ikura ry'uturemangingo, ripima icyerekezo cyo kwaguka kw'uturemangingo (nanone yakoreshejwe hano ku turemangingo runaka no ku turemangingo tw'ingore mu kugabana; Ishusho ya 5a, b, reba Uburyo n'Uburyo bw'Inyongera). Amakarita yacu y'igipimo cyo gukura kw'ubuso bw'uturemangingo twa SAM ahuye n'ibyo twabonye mbere38,39, hamwe n'igipimo gito cyo gukura ku mupaka n'igipimo kinini cyo gukura mu ndabyo zikura (Ishusho ya 5a). Isesengura ry'ibice by'ingenzi (PCA) ryagaragaje ko ibikorwa by'ibimenyetso bya GA bifitanye isano mbi n'ubukana bw'imikurire y'ubuso bw'uturemangingo (Ishusho ya 5c). Twagaragaje kandi ko imiterere y’ingenzi y’impinduka, harimo n’ubwiyongere bw’ibimenyetso bya GA n’ubukana bw’ikura, byari bikurikije icyerekezo cyagenwe n’izamuka ry’ishusho rya CLV3, byemeza ko utunyangingo twakuwe mu gice cya SAM mu isesengura risigaye. Isesengura ry’isano rya Spearman ryemeje ibyavuye muri PCA (Ishusho ya 5d), bigaragaza ko ibimenyetso bya GA byinshi muri IPR bitatumye utunyangingo twiyongera. Ariko, isesengura ry’isano ryagaragaje isano nto hagati y’ibikorwa bya GA byo kwerekana no gukura kwa anisotropi (Ishusho ya 5c, d), bigaragaza ko ibimenyetso bya GA byinshi muri IPR bigira ingaruka ku cyerekezo cy’ikura ry’utunyangingo ndetse wenda n’aho urwego rw’igabanywa ry’utunyangingo ruherereye.
a, b Imbonerahamwe y'ubushyuhe bw'imikurire y'ubuso (a) na anisotropi y'imikurire (b) muri SAM byapimwe ku bimera birindwi byigenga (byakoreshejwe nk'ibihamya by'imbaraga n'icyerekezo cyo kwaguka kw'uturemangingo, uko bikurikirana). c Isesengura rya PCA ryari rikubiyemo ibi bikurikira: ikimenyetso cya GA, ubukana bw'imikurire y'ubuso, anisotropi y'imikurire y'ubuso, n'igaragazwa rya CLV3. Igice cya PCA cya 1 cyari gifitanye isano mbi n'ubukana bw'imikurire y'ubuso kandi gifitanye isano nziza n'ikimenyetso cya GA. Igice cya PCA cya 2 cyari gifitanye isano nziza n'igaragazwa rya anisotropi y'imikurire y'ubuso kandi gifitanye isano mbi n'igaragazwa rya CLV3. Ijanisha rigaragaza itandukaniro ryasobanuwe na buri gice. d Isesengura ry'isano rya Spearman hagati y'ikimenyetso cya GA, ubukana bw'imikurire y'ubuso, na anisotropi y'imikurire y'ubuso ku gipimo cy'uturemangingo hatarimo CZ. Umubare uri iburyo ni agaciro ka Spearman rho hagati y'ibipimo bibiri. Inyenyeri zigaragaza aho isano/isano mbi ari ingenzi cyane. e Ishusho ya 3D y'uturemangingo twa Col-0 SAM L1 hakoreshejwe mikorosikopu ya confocal. Inkuta nshya z'uturemangingo zakozwe muri SAM (ariko ntabwo ari primordium) mu masaha 10 zibara ibara hakurikijwe agaciro kazo k'inguni. Akarongo k'amabara kagaragara mu mfuruka yo hasi iburyo. Ishusho y'imbere yerekana ishusho ya 3D ijyanye nayo mu isaha 0. Igerageza ryasubiwemo kabiri hamwe n'ibisubizo bisa. f Imiterere y'agasanduku yerekana igipimo cyo kugabana kw'uturemangingo muri IPR na non-IPR Col-0 SAM (n = 10 plants zigenga). Umurongo wo hagati ugaragaza ikigereranyo, naho imbibi z'agasanduku zigaragaza percentile ya 25 na 75. Udupira tw'inyonjo tugaragaza agaciro gato n'akatari gake kagenwe na porogaramu ya R. Agaciro ka P kabonetse hakoreshejwe ikizamini cya t-test cya Welch gifite imirizo ibiri. g, h Ishusho ya Schematic igaragaza (g) uburyo bwo gupima inguni y'urukuta rushya rw'uturemangingo (magenta) ugereranije n'icyerekezo cya radial uhereye hagati ya SAM (umurongo w'umweru ufite utudomo) (hatekerezwa gusa inguni ikaze, ni ukuvuga, 0–90°,), na (h) icyerekezo cy'uruziga/uruhande n'uruhande rwa radial muri meristem. i Histograms z'impinduka z'agasanduku k'itsinda ry'uturemangingo muri SAM (ubururu bwijimye), IPR (ubururu bwo hagati), na non-IPR (ubururu bworoshye), uko bikurikirana. Agaciro ka P kabonetse hakoreshejwe ikizamini cya Kolmogorov-Smirnov gifite imirizo ibiri. Ikizamini cyasubiwemo kabiri gifite ibisubizo bisa. j Histogramu z'impinduka z'icyerekezo cy'uturemangingo cya IPR hafi ya P3 (icyatsi kibisi cyoroshye), P4 (icyatsi kibisi cyo hagati), na P5 (icyatsi kibisi cyijimye), uko bikurikirana. Agaciro ka P kabonetse hakoreshejwe ikizamini cya Kolmogorov-Smirnov gifite imirizo ibiri. Ikizamini cyasubiwemo kabiri gifite ibisubizo bisa.
Bityo rero, twasuzumye isano iri hagati y’ibimenyetso bya GA n’ibikorwa byo kugabana kw’uturemangingo dushya mu gihe cyo gusuzuma (Ishusho ya 5e). Ubu buryo bwatumye dushobora gupima inshuro n’icyerekezo cyo kugabana kw’uturemangingo. Igitangaje ni uko twasanze inshuro zo kugabana kw’uturemangingo muri IPR no mu zindi SAM (zitari IPR, Ishusho ya 5f) zisa, bigaragaza ko itandukaniro mu bimenyetso bya GA hagati y’uturemangingo twa IPR n’ututari IPR ritagira ingaruka zikomeye ku kugabana kw’uturemangingo. Ibi, hamwe n’isano nziza iri hagati y’ibimenyetso bya GA n’ibyiyongera bya anisotropi, byatumye dutekereza niba ibikorwa bya GA bishobora kugira ingaruka ku cyerekezo cy’urwego rw’igabana ry’uturemangingo. Twapimye icyerekezo cy'urukuta rushya rw'ingirabuzimafatizo nk'inguni igaragara ugereranije n'umurongo wa radial uhuza hagati ya meristem n'igice cyo hagati cy'urukuta rushya rw'ingirabuzimafatizo (Ishusho ya 5e-i) kandi tubona ko ingirabuzimafatizo zigabanya ku nguni hafi ya 90° ugereranije n'umurongo wa radial, aho imirongo yo hejuru yagaragaye kuri 70–80° (23.28%) na 80–90° (22.62%) (Ishusho ya 5e, i), ijyanye n'igabanywa ry'ingirabuzimafatizo mu cyerekezo cyo kuzenguruka/kunyura (Ishusho ya 5h). Kugira ngo dusuzume uruhare rw'ibimenyetso bya GA muri iyi myitwarire yo gutandukanya ingirabuzimafatizo, twasesenguye ibipimo byo gutandukanya ingirabuzimafatizo muri IPR no muri IPR ukwabyo (Ishusho ya 5i). Twabonye ko ikwirakwizwa ry'inguni y'igabana mu turemangingo twa IPR ritandukanye n'iryo mu turemangingo tutari utw'iPR cyangwa mu turemangingo two muri SAM yose, aho utw'iPR tugaragaza igipimo kinini cy'igabana ry'uturemangingo two ku ruhande/ku ruziga, ni ukuvuga 70-80° na 80-90° (33.86% na 30.71%, uko bikurikirana, ibipimo bihuye) (Ishusho ya 5i). Bityo, ibyo twabonye byagaragaje isano iri hagati y'ibimenyetso bya GA byinshi n'icyerekezo cy'igabana ry'uturemangingo hafi y'icyerekezo cyo kuzenguruka, bisa n'isano iri hagati y'ibikorwa bya GA byo kwerekana no gukura kwa anisotropi (Ishusho ya 5c, d). Kugira ngo turusheho kwemeza uburyo iyi sano ikomeza, twapimye icyerekezo cy'igabana mu turemangingo twa IPR dukikije primordium duhereye kuri P3, kubera ko ibikorwa bya GA byo kwerekana byinshi byabonetse muri aka gace bihereye kuri P4 (Ishusho ya 4). Inguni z'igabana za IPR zikikije P3 na P4 nta tandukaniro rikomeye ryagaragaye mu mibare, nubwo inshuro nyinshi z'igabana ry'uturemangingo two ku ruhande byagaragaye muri IPR hafi ya P4 (Ishusho ya 5j). Ariko, mu turemangingo twa IPR hafi ya P5, itandukaniro mu cyerekezo cy’urwego rw’igabanywa ry’uturemangingo ryabaye ingenzi mu mibare, hamwe n’ubwiyongere bukabije bw’igabanywa ry’uturemangingo (Ishusho ya 5j). Hamwe na hamwe, ibi byavuye mu bushakashatsi bigaragaza ko ikimenyetso cya GA gishobora kugenzura icyerekezo cy’igabanywa ry’uturemangingo muri SAM, ibi bikaba bihuye na raporo zabanje40,41 zerekana ko ikimenyetso cya GA kinini gishobora gutera icyerekezo cy’igabanywa ry’uturemangingo muri IPR.
Biteganyijwe ko uturemangingo muri IPR tutazashyirwa muri primordia ahubwo tuzashyirwa muri internodes2,42,43. Icyerekezo cy’igabanywa ry’uturemangingo muri IPR gishobora gutuma habaho itegurwa risanzwe ry’imirongo miremire y’uturemangingo tw’inyuma mu turemangingo. Ibyo twabonye haruguru bigaragaza ko ibimenyetso bya GA bishobora kugira uruhare muri iki gikorwa mu kugenzura icyerekezo cy’igabanywa ry’uturemangingo.
Kubura imikorere ya genes nyinshi za DELLA bivamo igisubizo cya GA, kandi della mutants zishobora gukoreshwa mu gupima iki gitekerezo44. Twabanje gusesengura imiterere y’imvugo ya genes eshanu za DELLA muri SAM. Guhuza umurongo wa GUS45 byagaragaje ko GAI, RGA, RGL1, na RGL2 (ku rugero ruto cyane) byagaragajwe muri SAM (Ishusho y'inyongera 11a–d). Guhuza ahantu hatandukanye byagaragaje ko GAI mRNA yikusanyiriza cyane cyane mu ndabyo za mbere n’izikura (Ishusho y'inyongera 11e). RGL1 na RGL3 mRNA byabonetse mu mfuruka ya SAM no mu ndabyo zishaje, mu gihe RGL2 mRNA yari nyinshi mu karere k’umupaka (Ishusho y'inyongera 11f–h). Ishusho ya confocal ya pRGL3::RGL3-GFP SAM yemeje imvugo yabonwe na in situ hybridization kandi yerekana ko poroteyine ya RGL3 yikusanyiriza mu gice cyo hagati cya SAM (Ishusho y'inyongera 11i). Dukoresheje umurongo wa pRGA::GFP-RGA, twasanze kandi poroteyine ya RGA yikusanyiriza muri SAM, ariko ubwinshi bwayo bugabanuka ku mupaka uhereye kuri P4 (Ishusho y'inyongera ya 11j). Ikigaragara ni uko imiterere y'imvugo ya RGL3 na RGA ihuye n'imikorere y'ibimenyetso bya GA byinshi muri IPR, nk'uko byagaragajwe na qmRGA (Ishusho ya 4). Byongeye kandi, aya makuru agaragaza ko DELLA zose zigaragara muri SAM kandi ko isura yazo ihuriweho na SAM yose.
Hanyuma twasesenguye ibipimo byo kugabanya ingirangingo muri SAM yo mu bwoko bwa wild-type (Ler, control) na gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Ishusho ya 6a, b). Igishimishije ni uko twabonye impinduka zikomeye mu mibare mu gukwirakwiza inguni zo kugabanya ingirangingo muri della global mutant SAM ugereranije n'ubwoko bwo mu bwoko bwa wild (Ishusho ya 6c). Iri hinduka muri della global mutant ryatewe n'ubwiyongere bw'inguni za 80–90° (34.71% ugereranyije na 24.55%), kandi, ku rugero ruto, inguni za 70–80° (23.78% ugereranyije na 20.18%), ni ukuvuga, bihuye n'igabanywa ry'ingirangingo zinyuranamo (Ishusho ya 6c). Inshuro zo kugabanya ingirangingo zitanyuranamo (0–60°) nazo zari nke muri della global mutant (Ishusho ya 6c). Inshuro yo kugabana kw'uturemangingo tw’uruhererekane rw’uturemangingo tw’uruhererekane rwiyongereye cyane muri SAM ya della global mutant (Ishusho ya 6b). Inshuro yo kugabana kw'uturemangingo tw’uruhererekane rw’uturemangingo muri IPR nayo yari hejuru muri della global mutant ugereranije n'ubwoko bw’inyamaswa zo mu gasozi (Ishusho ya 6d). Hanze y’akarere ka IPR, ubwoko bw’inyamaswa bwo mu gasozi bwari bufite uburyo bumwe bwo gukwirakwiza inguni z’uturemangingo, mu gihe della global mutant yakundaga kugabana kw’uturemangingo nk’uko IPR ibibona (Ishusho ya 6e). Twanapimye icyerekezo cy’igabana ry’uturemangingo muri SAM ya ga2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, na ga2ox6-2), inyuma ya GA-inactive mutant aho GA yikusanyiriza. Bijyanye n’ubwiyongere bw’urugero rwa GA, SAM y’uruvange rwa ga2ox rwa quintuple rwari runini kurusha urwa Col-0 (Ishusho y’inyongera ya 12a, b), kandi ugereranije na Col-0, ga2ox SAM ya quintuple yagaragaje uburyo butandukanye bwo gutandukanya inguni z’uturemangingo, aho inguni ziyongera kuva kuri 50° kugeza kuri 90°, ni ukuvuga nanone gukundisha ibice bya tangential (Ishusho y’inyongera ya 12a–c). Bityo, tugaragaza ko gukora kwa GA signaling na GA accumulation bitera gutandukanya uturemangingo two ku ruhande muri IPR no mu bindi bice bya SAM.
a, b Ishusho ya 3D y'urwego rwa L1 rwa Ler (a) ya PI-stained Ler (a) na global della mutant (b) SAM hakoreshejwe mikorosikopi ya confocal. Inkuta nshya z'uturemangingo zakozwe muri SAM (ariko ntabwo ari primordium) mu gihe cy'amasaha 10 zirerekanwa kandi zigasigwa ibara hakurikijwe agaciro kazo. Ishusho yerekana SAM ku isaha ya 0. Akarongo k'amabara kagaragara mu mfuruka yo hepfo iburyo. Akambi muri (b) kerekana urugero rw'amadosiye y'uturemangingo ahujwe muri della mutant yo ku isi. Igerageza ryasubiwemo kabiri hamwe n'ibisubizo bisa. ce kugereranya ikwirakwizwa ry'inshuro z'icyerekezo cy'igabanywa ry'uturemangingo muri SAM yose (d), IPR (e), na non-IPR (f) hagati ya Ler na global della. Agaciro ka P kabonetse hakoreshejwe ikizamini cya Kolmogorov-Smirnov gifite imirizo ibiri. f, g Ishusho ya 3D y'amashusho ya confocal ya PI-stained SAM ya Col-0 (i) na pCUC2::gai-1-VENUS (j) transgenic plants. Uduce (a, b) tugaragaza inkuta nshya z'uturemangingo (ariko ntabwo ari primordia) twakozwe muri SAM mu masaha 10. Igerageza ryasubiwemo kabiri, ritanga ibisubizo bisa. h–j Igereranya ry'ikwirakwizwa ry'ibyiciro by'uturemangingo biri muri SAM yose (h), IPR (i) n'ibitari IPR (j) hagati ya Col-0 na pCUC2::gai-1-VENUS. Agaciro ka P kabonetse hakoreshejwe ikizamini cya Kolmogorov–Smirnov gifite imirizo ibiri.
Hanyuma twagerageje ingaruka zo kubuza ibimenyetso bya GA by’umwihariko muri IPR. Muri iyi ntego, twakoresheje promoteri ya cotyledon cup 2 (CUC2) kugira ngo dutere imbere poroteyine ya gai-1 igaragara nabi cyane yahujwe na VENUS (mu murongo wa pCUC2::gai-1-VENUS). Muri SAM yo mu bwoko bwa wild-type, promoteri ya CUC2 ituma habaho imikorere ya IPR nyinshi muri SAM, harimo n’uturemangingo tw’imipaka, kuva kuri P4 kugeza kuri, kandi imikorere isa nayo yagaragaye mu bimera bya pCUC2::gai-1-VENUS (reba hepfo). Ikwirakwizwa ry’inguni zo mu bice by’uturemangingo kuri SAM cyangwa IPR y’ibimera bya pCUC2::gai-1-VENUS ntiryari ritandukanye cyane n’iry’ubwoko bwa wild, nubwo mu buryo butunguranye twasanze uturemangingo tudafite IPR muri ibi bimera twigabanyijemo ku rugero rwo hejuru rwa 80–90° (Ishusho ya 6f–j).
Byavuzwe ko icyerekezo cy’igabanywa ry’uturemangingo giterwa n’imiterere ya SAM, cyane cyane imbaraga zo gukurura ziterwa no kugorama kw’uturemangingo46. Twabajije rero niba imiterere ya SAM yarahindutse mu bimera bya della global mutant na pCUC2::gai-1-VENUS. Nkuko byatangajwe mbere12, ingano ya della global mutant SAM yari nini kuruta iyo mu bwoko bw’inyamaswa (Ishusho y’inyongera 13a, b, d). Guhuza CLV3 na STM RNA muri situ byemeje kwaguka kwa meristem mu bitanda bya della ndetse berekana no kwaguka k’uruhande rw’uturemangingo tw’ingirabuzimafatizo (Ishusho y’inyongera 13e, f, h, i). Ariko, kugorama kwa SAM kwari gusa muri genotype zombi (Ishusho y’inyongera 13k, m, n, p). Twabonye ukwiyongera gukomeye nk'uku muri gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant nta mpinduka mu kuzunguruka ugereranije n'ubwoko bw'ishyamba (Ishusho y'inyongera 13c, d, g, j, l, o, p). Inshuro zo kwerekeza ku rugabano rw'uturemangingo nazo zagize ingaruka muri della quadruple mutant, ariko ku rugero ruto ugereranyije na della monolithic mutant (Ishusho y'inyongera 12d–f). Iyi ngaruka y'ingano, hamwe no kutagira ingaruka ku kuzunguruka, bigaragaza ko ibikorwa bya RGL3 bisigaye muri Della quadruple mutant bigabanya impinduka mu kwerekeza ku rugabano rw'uturemangingo biterwa no kubura kwa DELLA activity kandi ko impinduka mu kuzunguruka kw'uturemangingo ku ruhande bibaho bitewe n'impinduka mu bikorwa byo kwerekana ibimenyetso bya GA aho kuba impinduka mu miterere ya SAM. Nkuko byasobanuwe haruguru, promoter ya CUC2 itera IPR expression muri SAM guhera kuri P4 (Ishusho y'inyongera 14a, b), kandi mu buryo bunyuranye, pCUC2::gai-1-VENUS SAM yari ifite ingano nto ariko ifite curvature iri hejuru (Ishusho y'inyongera 14c–h). Iri hinduka mu miterere ya pCUC2::gai-1-VENUS SAM rishobora gutuma habaho ikwirakwizwa ry’imitsi ya mekanike itandukanye ugereranije n’ubwoko bw’ishyamba, aho imitsi myinshi izenguruka itangirira ku ntera ngufi uvuye hagati ya SAM47. Ubundi buryo, impinduka mu miterere ya pCUC2::gai-1-VENUS SAM zishobora guterwa n’impinduka mu miterere ya mekanike yo mu karere iterwa no guhinduka kwa gene48. Muri ibyo bihe byombi, ibi bishobora gukuraho ingaruka z’impinduka mu bimenyetso bya GA byongereye amahirwe y’uko uturemangingo tuzagabana mu cyerekezo cy’uruziga/uruhererekane, dusobanura ibyo twabonye.
Amakuru yacu yemeza ko ikimenyetso cya GA cyo hejuru gikora uruhare runini mu cyerekezo cy’uruhande rw’igice cy’uturemangingo muri IPR. Agaragaza kandi ko curvature ya meristem igira ingaruka ku cyerekezo cy’igice cy’uturemangingo muri IPR.
Uburyo imiterere y’aho imiterere y’uturemangingo iherereye muri IPR, bitewe n’imikorere myinshi y’ibimenyetso bya GA, bigaragaza ko GA itegura dosiye y’uturemangingo twa radial mu gice cy’imbere cya SAM kugira ngo igaragaze imiterere y’uturemangingo izaboneka nyuma muri epidermal internode. Mu by’ukuri, dosiye nk’izo zakunze kugaragara mu mashusho ya SAM ya della global mutants (Ishusho ya 6b). Bityo, kugira ngo dusuzume imikorere y’imiterere y’ahantu ya GA signaling muri SAM, twakoresheje amashusho ya time-lapse kugira ngo dusesengure imiterere y’uturemangingo muri IPR mu bwoko bwa wild-type (Ler na Col-0), della global mutants, na pCUC2::gai-1-VENUS transgenic plants.
Twasanze qmRGA yagaragaje ko ibikorwa byo gutanga ibimenyetso bya GA muri IPR byiyongereye kuva kuri P1/P2 bigera ku rwego rwa P4, kandi iki gishushanyo cyakomeje kuba kimwe uko igihe cyagiye gihita (Ishusho ya 4a–f n'Ishusho y'inyongera ya 8c–f, k). Kugira ngo dusesengure imiterere y'uturemangingo muri IPR hamwe n'ikimenyetso cya GA cyiyongera, twashyize amazina ku turemangingo twa Ler IPR hejuru no ku mpande za P4 dukurikije uko iterambere ryazo ryagenze ryasesenguwe amasaha 34 nyuma yo kwitegereza bwa mbere, ni ukuvuga inshuro zirenga ebyiri za plastid, bidufasha gukurikira uturemangingo twa IPR mu gihe cy'iterambere rya primordium kuva kuri P1/P2 kugeza kuri P4. Twakoresheje amabara atatu atandukanye: umuhondo kuri utwo turemangingo twashyizwe muri primordium hafi ya P4, icyatsi kibisi kuri utwo twari muri IPR, n'umuhengeri ku bagize uruhare muri izo nzira zombi (Ishusho ya 7a–c). Kuri t0 (0 h), urwego rwa 1–2 rw'uturemangingo twa IPR rwagaragaye imbere ya P4 (Ishusho ya 7a). Nkuko byari byitezwe, ubwo izi selile zagabanyaga, zabikoze cyane cyane binyuze mu gice cy’itsinda cya transverse division (Ishusho ya 7a–c). Ibisubizo bisa byabonetse hakoreshejwe Col-0 SAM (yibanda kuri P3, aho umupaka wayo ugenda uhinduka nk’uwa P4 muri Ler), nubwo muri iyi genotype, umukufi wakorewe ku mupaka w’indabyo wahishe selile za IPR vuba cyane (Ishusho ya 7g–i). Bityo, imiterere y’itsinda ry’uturemangingo twa IPR ibanza gushyira utwo turemangingo mu mirongo ya radial, nk’uko bimeze muri internodes. Imiterere y’imirongo ya radial n’aho selile za IPR ziherereye hagati y’ingingo zikurikiranye bigaragaza ko izi selile ari abakurambere b’internodal.
Aha, twakoze uburyo bwo gupima GA signaling biosensor, qmRGA, bwemerera gukora ikarita y'ibikorwa bya GA signaling bikomoka ku gipimo cya GA na GA receptor bihujwe mu gihe bigabanya uburyo bwo kubangamira inzira z'ibimenyetso bya endogenous, bityo tugatanga amakuru ku mikorere ya GA ku rwego rw'uturemangingo. Muri urwo rwego, twubatse poroteyine ya DELLA yahinduwe, mRGA, yatakaje ubushobozi bwo guhuza DELLA ikorana nayo ariko igakomeza kwiyumvamo poroteyine iterwa na GA. qmRGA isubiza impinduka zo hanze n'izo mu ngano za GA, kandi imiterere yayo yo kumva ituma habaho gusuzuma impinduka zo mu mwanya mu bikorwa bya GA signaling mu gihe cyo gutera imbere. qmRGA nayo ni igikoresho cyoroshye cyane kuko gishobora guhinduka ku ngingo zitandukanye binyuze mu guhindura promoter ikoreshwa mu kwerekana (niba bikenewe), kandi bitewe n'uburyo inzira ya GA signaling na PFYRE ibungabunzwe, birashoboka ko ishobora kwimurirwa mu zindi nkomoko22. Bijyanye n'ibi, impinduka zingana muri poroteyine ya SLR1 DELLA y'umuceri (HYY497AAA) nayo yagaragaye ko igabanya imikorere ya SLR1 mu gukura kwayo mu gihe igabanya gato gusa kwangirika kwayo kwa GA, kimwe na mRGA23. Icyitonderwa, ubushakashatsi buherutse gukorwa muri Arabidopsis bwagaragaje ko impinduka imwe ya aside amine mu gice cya PFYRE (S474L) yahinduye imikorere ya RGA mu kwandika nta ngaruka igira ku bushobozi bwayo bwo gukorana n'abafatanyabikorwa ba transcription factor50. Nubwo iyi mpinduka yegereye cyane aside amine 3 zisimbura ziboneka muri mRGA, ubushakashatsi bwacu bwerekana ko izi mpinduka ebyiri zihindura imiterere yihariye ya DELLA. Nubwo abafatanyabikorwa benshi ba transcription factor bifatanya n'urwego rwa LHR1 na SAW rwa DELLA26,51, aside amine zimwe na zimwe zibitswe mu gice cya PFYRE zishobora gufasha mu gutuza iyi mikoranire.
Iterambere ry’uturemangingo ni ikintu cy’ingenzi mu miterere y’ibimera no kunoza umusaruro. qmRGA yagaragaje imikorere yo hejuru y’ibimenyetso bya GA mu turemangingo twa IPR internode progenitor. Mu guhuza amashusho y’ingano n’imiterere y’uturemangingo, twerekanye ko imiterere y’ibimenyetso bya GA ishyira hejuru y’imiterere y’uturemangingo dutandukanya uruziga/transverse mu gice cya SAM, bigahindura imiterere y’uturemangingo ikenewe kugira ngo iterambere ry’uturemangingo rigerweho. Hari abagenzura icyerekezo cy’uturemangingo bagaragaye mu gihe cy’iterambere52,53. Akazi kacu gatanga urugero rugaragara rw’uburyo imikorere ya GA igenga iyi parametre y’uturemangingo. DELLA ishobora gukorana n’ibice bya poroteyine bibanza gupfunyika41, bityo GA irashobora kugenzura icyerekezo cy’uturemangingo dutandukanya imiterere y’uturemangingo binyuze mu kugira ingaruka zitaziguye ku cyerekezo cya microtubule ya cortical40,41,54,55. Twagaragaje mu buryo butunguranye ko muri SAM, isano y’imikorere yo hejuru y’ibimenyetso bya GA atari ukwiyongera cyangwa kwigabanya kw’uturemangingo, ahubwo ari ukwiyongera gusa, bihuye n’ingaruka za GA ku cyerekezo cy’igabanywa ry’uturemangingo muri IPR. Ariko, ntitwakwirengagiza ko iyi ngaruka ishobora no kuba itaziguye, urugero nk’iterwa no koroshya inkuta z’uturemangingo ziterwa na GA56. Impinduka mu miterere y'urukuta rw'uturemangingo zitera stress ya mekanike57,58, ishobora no kugira ingaruka ku cyerekezo cy'umuyoboro w'uturemangingo ugabanyijemo ibice binyuze mu kugira ingaruka ku cyerekezo cya microtubules za cortical39,46,59. Ingaruka zihuriweho z'stress ya mekanike iterwa na GA hamwe no kugenzura neza icyerekezo cya microtubule na GA bishobora kugira uruhare mu kurema imiterere yihariye y'icyerekezo cy'umuyoboro w'uturemangingo muri IPR kugira ngo hamenyekane internodes, kandi hakenewe izindi nyigo kugira ngo hagenzurwe iki gitekerezo. Mu buryo nk'ubwo, ubushakashatsi bwabanje bwagaragaje akamaro ka poroteyine zikorana na DELLA TCP14 na 15 mu kugenzura ikorwa rya internodes60,61 kandi ibi bintu bishobora guhuza ibikorwa bya GA hamwe na BREVIPEDICELLUS (BP) na PENNYWISE (PNY), bigenga iterambere rya internodes kandi byagaragaye ko bigira ingaruka ku bushobozi bwa GA2,62. Bitewe n'uko DELLA zikorana na brassinosteroid, ethylene, aside jasmonic, na aside abscisic (ABA) signaling pathways63,64 kandi ko iyi misemburo ishobora kugira ingaruka ku miterere ya microtubule65, ingaruka za GA ku miterere y'uturemangingo zishobora no guterwa n'indi misemburo.
Ubushakashatsi bwa mbere bwagaragaje ko uturere tw’imbere n’inyuma twa Arabidopsis SAM dukenewe kugira ngo habeho iterambere ry’uturemangingo tw’imbere2,42. Kuba GA igenzura neza igabanywa ry’uturemangingo mu ngingo z’imbere12 bishyigikira imikorere ibiri ya GA mu kugenzura ingano ya meristem n’ingano y’uturemangingo tw’imbere muri SAM. Imiterere y’igabanywa ry’uturemangingo tw’icyerekezo nayo igenzurwa cyane mu ngingo z’imbere za SAM, kandi iri tegeko ni ingenzi mu mikurire y’uturemangingo52. Bizaba bishimishije gusuzuma niba GA ifite uruhare mu kwerekeza urwego rw’igabanywa ry’uturemangingo mu miterere ya SAM y’imbere, bityo bigahuza imiterere n’iterambere ry’uturemangingo tw’imbere muri SAM.
Ibimera byahinzwe mu butaka cyangwa mu butaka bwa Murashige-Skoog (MS) bwa 1x (Duchefa) bwongewemo 1% ya sucrose na 1% ya agar (Sigma) mu bihe bisanzwe (urumuri rw'amasaha 16, 22 °C), usibye igerageza rya hypocotyl n'iry'imizi aho ingemwe zahinzwe ku masahani ahagaze munsi y'urumuri ruhoraho na 22 °C. Ku igerageza rya nitrate, ibimera byahinzwe ku buryo bwa MS bwahinduwe (bioWORLD plant medium) bwongewemo nitrate ihagije (0 cyangwa 10 mM KNO3), 0.5 mM NH4-succinate, 1% ya sucrose na 1% A-agar (Sigma) mu bihe by'umunsi munini.
GID1a cDNA yinjijwe muri pDONR221 yongeye guhuzwa na pDONR P4-P1R-pUBQ10 na pDONR P2R-P3-mCherry muri pB7m34GW kugira ngo hakorwe pBQ10::GID1a-mCherry. ADN ya IDD2 yinjijwe muri pDONR221 yongeye guhuzwa muri pB7RWG266 kugira ngo hakorwe p35S:IDD2-RFP. Kugira ngo hakorwe pGID1b::2xmTQ2-GID1b, igice cya 3.9 kb kiri hejuru y’agace ka GID1b gashinzwe kwandika hamwe n’igice cya 4.7 kb kirimo GID1b cDNA (1.3 kb) na terminator (3.4 kb) byabanje kongerwamo imbaraga hakoreshejwe primers ziri mu Imbonerahamwe y’inyongera ya 3 hanyuma bishyirwa muri pDONR P4-P1R (Thermo Fisher Scientific) na pDONR P2R-P3 (Thermo Fisher Scientific), uko bikurikirana, hanyuma bivangwa na pDONR221 2xmTQ268 muri pGreen 012567 target vector hakoreshejwe Gateway cloning. Kugira ngo hakorwe pCUC2::LSSmOrange, urukurikirane rwa CUC2 promoter (3229 bp hejuru ya ATG) rukurikirwa n'urukurikirane rwa kode rwa mOrange nini ya Stokes-shifted (LSSmOrange)69 hamwe n'ikimenyetso cya N7 nuclear localization hamwe na NOS transcriptional terminator byakusanyijwe muri pGreen kanamycin targeting vector hakoreshejwe sisitemu ya Gateway 3-fragment recombination (Invitrogen). Vector y'ibimera yashyizwe mu bwoko bwa Agrobacterium tumefaciens GV3101 hanyuma yinjizwa mu mababi ya Nicotiana benthamiana hakoreshejwe uburyo bwa Agrobacterium infiltration na muri Arabidopsis thaliana Col-0 hakoreshejwe uburyo bwa floral dip, uko bikurikirana. pUBQ10::qmRGA pUBQ10::GID1a-mCherry na pCLV3::mCherry-NLS qmRGA byatandukanijwe n'ibisekuru bya F3 na F1 by'imiyoboro itandukanye, uko bikurikirana.
RNA in situ hybridization yakozwe ku mitwe y’amasasu ifite uburebure bwa cm 172, yakusanyijwe ihita ishyirwa mu gisubizo cya FAA (3.7% formaldehyde, 5% aside asetiki, 50% ethanol) mbere yo gukonjeshwa kugeza kuri 4 °C. Nyuma yo kuvurwa mu buryo bwa vacuum iminota 2 × 15, fixative yarahinduwe maze ingero zishyirwa mu icuba ijoro ryose. GID1a, GID1b, GID1c, GAI, RGL1, RGL2, na RGL3 cDNAs hamwe na antisense probes kuri 3′-UTRs byakozwe hakoreshejwe primers zigaragara mu mbonerahamwe y’inyongera ya 3 nk’uko byasobanuwe na Rosier et al.73. Imashini zipima Digoxigenin zafashwe hakoreshejwe antibodies za digoxigenin (ku nshuro 3000; Roche, nimero ya kataloge: 11 093 274 910), kandi ibice byasizwe irangi rya 5-bromo-4-chloro-3-indolyl phosphate (BCIP, ku nshuro 250) / nitroblue tetrazolium (NBT, ku nshuro 200).
Igihe cyo kohereza: Gashyantare-10-2025