Kukula kwa mphukira za apical meristem (SAM) ndikofunikira kwambiri pa kapangidwe ka tsinde.gibberellins(GAs) amachita mbali yofunika kwambiri pakugwirizanitsa kukula kwa zomera, koma ntchito yawo mu SAM sikumveka bwino. Pano, tapanga ratiometric biosensor ya GA signaling mwa kupanga puloteni ya DELLA kuti ichepetse ntchito yake yofunikira yolamulira mu GA transcriptional response pamene ikusunga kuwonongeka kwake pamene GA yazindikirika. Tikuwonetsa kuti biosensor iyi yochokera ku kuwonongeka imalemba molondola kusintha kwa milingo ya GA ndi kuzindikira kwa maselo panthawi yopangidwa. Tagwiritsa ntchito biosensor iyi polemba zochitika za GA signaling mu SAM. Tikuwonetsa kuti zizindikiro za GA zapamwamba zimapezeka makamaka m'maselo omwe ali pakati pa ziwalo zamkati, zomwe ndi zoyambira maselo a internode. Pogwiritsa ntchito njira zopezera ndi kutaya ntchito, tikuwonetsanso kuti GA imawongolera momwe maselo amagawidwira, ndikukhazikitsa dongosolo la maselo ovomerezeka a internode, potero tikulimbikitsa ma internode specification mu SAM.
Mphukira ya apical meristem (SAM), yomwe ili pamwamba pa mphukira, ili ndi malo a maselo oyambira omwe ntchito yake imapanga ziwalo za m'mbali ndi ma tsinde m'njira yofanana komanso yobwerezabwereza nthawi yonse ya chomera. Chigawo chilichonse chobwerezabwerezachi, kapena ma node a chomera, chimaphatikizapo ma internode ndi ziwalo za m'mbali pa ma node, ndi ma axillary meristems mu axils ya tsamba1. Kukula ndi kakonzedwe ka ma node a chomera kumasintha panthawi yakukula. Mu Arabidopsis, kukula kwa ma node kumachepetsedwa panthawi ya kukula kwa zomera, ndipo ma axillary meristems amakhalabe opanda maluwa m'ma axils a masamba a rosette. Panthawi yosinthira ku gawo la maluwa, SAM imakhala inflorescence meristem, ndikupanga ma internode ataliatali ndi ma axillary buds, nthambi zazing'ono m'ma axils a masamba a cauline, ndipo pambuyo pake, maluwa opanda masamba2. Ngakhale tapita patsogolo kwambiri pakumvetsetsa njira zomwe zimalamulira kuyambika kwa masamba, maluwa, ndi nthambi, zochepa zomwe zimadziwika za momwe ma internode amaonekera.
Kumvetsetsa kufalikira kwa ma GA m'malo osiyanasiyana kudzathandiza kumvetsetsa bwino ntchito za mahomoni awa m'maselo osiyanasiyana komanso pazigawo zosiyanasiyana za chitukuko. Kuwona kuwonongeka kwa kusakanikirana kwa RGA-GFP komwe kumawonetsedwa ndi ntchito ya promoter yake kumapereka chidziwitso chofunikira pakuwongolera kuchuluka kwa GA mu mizu15,16. Komabe, kufotokozera kwa RGA kumasiyana m'maselo17 ndipo kumayendetsedwa ndi GA18. Chifukwa chake, kufotokozera kosiyana kwa promoter wa RGA kungayambitse mawonekedwe a fluorescence omwe amawonedwa ndi RGA-GFP ndipo motero njira iyi si yochuluka. Posachedwapa, GA19,20 yolembedwa ndi bioactive fluorescein (Fl) idavumbulutsa kusonkhana kwa GA mu endocortex ya mizu ndi kuwongolera kuchuluka kwa maselo ake ndi GA transport. Posachedwapa, GA FRET sensor nlsGPS1 idawonetsa kuti kuchuluka kwa GA kumagwirizana ndi kutalikitsa kwa maselo mu mizu, ulusi, ndi ma hypocotyls amdima21. Komabe, monga taonera, kuchuluka kwa GA sikokhako komwe kumalamulira ntchito yolumikizirana ya GA, chifukwa kumadalira njira zovuta zowunikira. Apa, poganizira za kumvetsetsa kwathu njira zolumikizirana za DELLA ndi GA, tikupereka lipoti la chitukuko ndi mawonekedwe a biosensor yochokera ku kuwonongeka kwa ratiometric ya GA signaling. Kuti tipange biosensor iyi yochuluka, tidagwiritsa ntchito RGA yosinthika ya GA yomwe idalumikizidwa ku puloteni yowala ndipo imawonetsedwa paliponse m'maselo, komanso puloteni yowala ya GA-insensitive. Tikuwonetsa kuti kusakanikirana kwa mapuloteni a RGA osinthika sikusokoneza chizindikiro cha GA chokhazikika pamene chikuwonetsedwa paliponse, ndipo kuti biosensor iyi imatha kuyeza ntchito yolumikizirana yomwe imachokera ku GA input ndi GA signal processing ndi chipangizo chowunikira chokhala ndi resolution yapamwamba ya spatiotemporal. Tidagwiritsa ntchito biosensor iyi kuti tilembe kufalikira kwa spatiotemporal kwa ntchito yolumikizirana ya GA ndikuyesa momwe GA imayendetsera machitidwe a maselo mu epidermis ya SAM. Tikuwonetsa kuti GA imawongolera momwe maselo a SAM amagawidwira pakati pa organ primordia, potero timafotokoza dongosolo la maselo ovomerezeka a internode.
Pomaliza, tinafunsa ngati qmRGA inganene kusintha kwa milingo ya GA yochokera ku endogenous pogwiritsa ntchito ma hypocotyls omwe akukula. Poyamba tidawonetsa kuti nitrate imalimbikitsa kukula mwa kuwonjezera kapangidwe ka GA ndipo, kenako, kuwonongeka kwa DELLA34. Chifukwa chake, tidawona kuti kutalika kwa hypocotyl mu mbande za pUBQ10::qmRGA zomwe zimamera pansi pa nitrate yochuluka (10 mM NO3−) kunali kotalika kwambiri kuposa mbande zomwe zimamera pansi pa mikhalidwe yosowa nitrate (Chithunzi Chowonjezera 6a). Mogwirizana ndi momwe kukula kumayankhira, zizindikiro za GA zinali zapamwamba mu ma hypocotyls a mbande zomwe zimamera pansi pa mikhalidwe ya 10 mM NO3− kuposa mbande zomwe zimamera popanda nitrate (Chithunzi Chowonjezera 6b, c). Chifukwa chake, qmRGA imathandizanso kuwunika kusintha kwa chizindikiro cha GA komwe kumachitika chifukwa cha kusintha kwa endogenous mu kuchuluka kwa GA.
Kuti timvetse ngati ntchito yolumikizira ya GA yomwe yapezeka ndi qmRGA imadalira kuchuluka kwa GA ndi kuwona kwa GA, monga momwe timayembekezera kutengera kapangidwe ka sensa, tidasanthula momwe ma receptor atatu a GID1 amaonekera m'maselo a zomera ndi zoberekera. Mu mbande, mzere wa reporter wa GID1-GUS udawonetsa kuti GID1a ndi c zidawonekera kwambiri mu ma cotyledons (Chithunzi 3a–c). Kuphatikiza apo, ma receptor onse atatu adawonekera m'masamba, mizu ya lateral root primordia, nsonga za mizu (kupatula chivundikiro cha mizu ya GID1b), ndi dongosolo la mitsempha (Chithunzi 3a–c). Mu inflorescence SAM, tidapeza ma signal a GUS okha a GID1b ndi 1c (Chithunzi Chowonjezera 7a–c). In situ hybridization idatsimikizira mawonekedwe awa ndipo idawonetsanso kuti GID1c idawonekera mofanana pamlingo wotsika mu SAM, pomwe GID1b idawonetsa kufalikira kwakukulu m'mphepete mwa SAM (Chithunzi Chowonjezera 7d–l). Kusakanikirana kwa pGID1b::2xmTQ2-GID1b kunawonetsanso kuchuluka kwa ma significant a GID1b, kuyambira pa ma significant otsika kapena osapezeka pakati pa SAM mpaka ma significant apamwamba m'malire a ziwalo (Chithunzi Chowonjezera 7m). Chifukwa chake, ma receptor a GID1 sagawidwa mofanana m'thupi lonse komanso mkati mwa minofu. Mu zoyeserera zotsatira, tinawonanso kuti ma significant a GID1 (pUBQ10::GID1a-mCherry) adawonjezera mphamvu ya qmRGA mu ma hypocotyls kupita ku external GA application (Chithunzi 3d, e). Mosiyana ndi zimenezi, fluorescence yoyesedwa ndi qd17mRGA mu hypocotyl sinali yogwirizana ndi chithandizo cha GA3 (Chithunzi 3f, g). Pa mayeso onse awiriwa, mbande zinachiritsidwa ndi kuchuluka kwa GA (100 μM GA3) kuti ziwone momwe sensor imagwirira ntchito mwachangu, komwe kuthekera kolumikizana ndi cholandirira cha GID1 kunakulitsidwa kapena kutayika. Zonsezi pamodzi zimatsimikizira kuti qmRGA biosensor imagwira ntchito yogwirizana ngati sensa ya GA ndi GA, ndipo zikusonyeza kuti kusiyana kwa GID1 receptor kumatha kusintha kwambiri kutulutsa kwa sensa.
Mpaka pano, kufalikira kwa zizindikiro za GA mu SAM sikukudziwika bwino. Chifukwa chake, tagwiritsa ntchito zomera zotulutsa qmRGA ndi pCLV3::mCherry-NLS stem cell reporter35 kuti tiwerengere mamapu ochulukitsa a ntchito yowonetsa zizindikiro za GA, kuyang'ana kwambiri gawo la L1 (epidermis; Chithunzi 4a, b, onani Njira ndi Njira Zowonjezera), popeza L1 imagwira ntchito yofunika kwambiri pakulamulira kukula kwa SAM36. Pano, pCLV3::mCherry-NLS inapereka malo ofunikira owunikira kufalikira kwa mlengalenga kwa ntchito yowonetsa zizindikiro za GA37. Ngakhale GA imaonedwa kuti ndi yofunika kwambiri pakukula kwa ziwalo za m'mbali4, tawona kuti zizindikiro za GA zinali zochepa mu primordium ya maluwa (P) kuyambira pa siteji ya P3 (Chithunzi 4a, b), pomwe primordiums zazing'ono za P1 ndi P2 zinali ndi ntchito yocheperako yofanana ndi yomwe ili m'chigawo chapakati (Chithunzi 4a, b). Ntchito yayikulu yowonetsa GA idapezeka pamalire a ziwalo za primordium, kuyambira pa P1/P2 (m'mbali mwa malire) ndikukwera pa P4, komanso m'maselo onse a dera lozungulira lomwe lili pakati pa primordia (Chithunzi 4a, b ndi Chithunzi Chowonjezera 8a, b). Ntchito yayikulu yowonetsa GA iyi idawonedwa osati mu epidermis yokha komanso mu L2 ndi zigawo zapamwamba za L3 (Chithunzi Chowonjezera 8b). Kapangidwe ka zizindikiro za GA komwe kadapezeka mu SAM pogwiritsa ntchito qmRGA sikunasinthenso pakapita nthawi (Chithunzi Chowonjezera 8c–f, k). Ngakhale kapangidwe ka qd17mRGA kanachepetsedwa mwadongosolo mu SAM ya zomera za T3 kuchokera ku mizere isanu yodziyimira payokha yomwe tidafotokoza mwatsatanetsatane, tidatha kusanthula mawonekedwe a fluorescence omwe adapezeka ndi kapangidwe ka pRPS5a::VENUS-2A-TagBFP (Chithunzi Chowonjezera 8g–j, l). Mu mzere wowongolera uwu, kusintha kochepa kokha kwa chiŵerengero cha fluorescence kunapezeka mu SAM, koma mu SAM center tinawona kuchepa komveka bwino komanso kosayembekezereka kwa VENUS kogwirizana ndi TagBFP. Izi zikutsimikizira kuti mawonekedwe owonetsera omwe awonedwa ndi qmRGA akuwonetsa kuwonongeka kwa mRGA-VENUS komwe kumadalira GA, komanso kukuwonetsa kuti qmRGA ikhoza kuwerengera kwambiri ntchito ya GA signaling mu meristem center. Mwachidule, zotsatira zathu zikuwonetsa mawonekedwe a GA signaling omwe makamaka akuwonetsa kufalikira kwa primordia. Kufalikira kumeneku kwa chigawo chapakati (IPR) kumachitika chifukwa cha kukhazikitsidwa pang'onopang'ono kwa ntchito yayikulu ya GA signaling pakati pa primordium yomwe ikukula ndi chigawo chapakati, pomwe nthawi yomweyo ntchito ya GA signaling mu primordium imachepa (Chithunzi 4c, d).
Kugawa kwa ma receptor a GID1b ndi GID1c (onani pamwambapa) kukusonyeza kuti kufotokozera kosiyana kwa ma receptor a GA kumathandiza kupanga mawonekedwe a ntchito ya GA signaling mu SAM. Tinadzifunsa ngati kusonkhanitsa kosiyana kwa GA kungakhalepo. Kuti tifufuze kuthekera kumeneku, tinagwiritsa ntchito nlsGPS1 GA FRET sensor21. Kuwonjezeka kwa ma frequency activation kunapezeka mu SAM ya nlsGPS1 yothandizidwa ndi 10 μM GA4+7 kwa mphindi 100 (Chithunzi Chowonjezera 9a–e), kusonyeza kuti nlsGPS1 imayankha kusintha kwa kuchuluka kwa GA mu SAM, monga momwe zimachitikira mu mizu21. Kugawa kwa malo kwa ma frequency activation a nlsGPS1 kunawonetsa milingo yochepa ya GA m'magawo akunja a SAM, koma kunawonetsa kuti anali okwera pakati ndi m'malire a SAM (Chithunzi 4e ndi Chithunzi Chowonjezera 9a,c). Izi zikusonyeza kuti GA imagawidwanso mu SAM ndi mawonekedwe a malo ofanana ndi omwe adawululidwa ndi qmRGA. Monga njira yowonjezera, tinagwiritsanso ntchito SAM ndi GA ya fluorescent (GA3-, GA4-, GA7-Fl) kapena Fl yokha ngati njira yowongolera yoyipa. Chizindikiro cha Fl chinagawidwa mu SAM yonse, kuphatikiza dera lapakati ndi primordium, ngakhale kuti chinali ndi mphamvu yochepa (Chithunzi 4j ndi Chithunzi Chowonjezera 10d). Mosiyana ndi zimenezi, GA-Fl zonse zitatu zinasonkhana makamaka mkati mwa malire a primordium ndi madigiri osiyanasiyana m'malo ena onse a IPR, ndipo GA7-Fl inasonkhana m'dera lalikulu kwambiri mu IPR (Chithunzi 4k ndi Chithunzi Chowonjezera 10a,b). Kuyeza mphamvu ya fluorescence kunawonetsa kuti chiŵerengero cha mphamvu ya IPR ku non-IPR chinali chokwera mu SAM yothandizidwa ndi GA-Fl poyerekeza ndi SAM yothandizidwa ndi Fl (Chithunzi 4l ndi Chithunzi Chowonjezera 10c). Pamodzi, zotsatirazi zikusonyeza kuti GA ilipo pamlingo wapamwamba m'maselo a IPR omwe ali pafupi ndi malire a ziwalo. Izi zikusonyeza kuti kachitidwe ka SAM GA signaling activity kamachokera ku kufotokozera kosiyana kwa ma GA receptors komanso kusonkhanitsa kwa GA m'maselo a IPR pafupi ndi malire a ziwalo. Motero, kusanthula kwathu kunavumbula kachitidwe kosayembekezereka ka GA signaling, komwe ntchito yake inali yochepa pakati ndi pakati pa SAM ndi ntchito yake inali yapamwamba mu IPR m'chigawo chapakati.
Kuti timvetse ntchito ya ntchito yosiyanitsa ya GA mu SAM, tinasanthula mgwirizano pakati pa ntchito yosonyeza GA, kukula kwa maselo, ndi kugawa kwa maselo pogwiritsa ntchito chithunzi cha nthawi yeniyeni cha SAM qmRGA pCLV3::mCherry-NLS. Popeza ntchito ya GA mu kayendetsedwe ka kukula, mgwirizano wabwino ndi magawo okulitsa maselo unali kuyembekezera. Chifukwa chake, choyamba tinayerekeza mamapu a ntchito yosonyeza GA ndi mamapu a kuchuluka kwa kukula kwa maselo (monga choyimira mphamvu ya kukula kwa maselo a selo loperekedwa ndi maselo aakazi pa kugawa) komanso mamapu a kukula kwa anisotropy, omwe amayesa momwe kukula kwa maselo kumayendera (komwe kumagwiritsidwanso ntchito pano pa selo loperekedwa ndi maselo aakazi pa kugawa; Chithunzi 5a,b, onani Njira ndi Njira Zowonjezera). Mamapu athu a kuchuluka kwa kukula kwa maselo a SAM akugwirizana ndi zomwe adaziwona kale38,39, ndi kuchuluka kochepa kwa kukula pamalire ndi kuchuluka kwakukulu kwa kukula mu maluwa omwe akukula (Chithunzi 5a). Kusanthula kwa zigawo zazikulu (PCA) kunawonetsa kuti ntchito yosonyeza GA inali yogwirizana molakwika ndi kukula kwa kukula kwa maselo pamwamba (Chithunzi 5c). Tinawonetsanso kuti ma axes akuluakulu a kusiyanasiyana, kuphatikizapo kulowetsa kwa chizindikiro cha GA ndi kukula kwake, anali olunjika ku njira yomwe imatsimikiziridwa ndi kuwonetsa kwakukulu kwa CLV3, kutsimikizira kuchotsedwa kwa maselo kuchokera pakati pa SAM mu kusanthula kotsala. Kusanthula kwa mgwirizano wa Spearman kunatsimikizira zotsatira za PCA (Chithunzi 5d), kusonyeza kuti zizindikiro zapamwamba za GA mu IPR sizinapangitse kuti maselo achuluke. Komabe, kusanthula kwa mgwirizano kunawonetsa mgwirizano wabwino pang'ono pakati pa ntchito ya chizindikiro cha GA ndi kukula kwa anisotropy (Chithunzi 5c, d), zomwe zikusonyeza kuti chizindikiro cha GA chapamwamba mu IPR chimakhudza njira ya kukula kwa maselo komanso mwina malo a gawo la maselo.
a, b Mamapu a kutentha a kukula kwapakati pa pamwamba (a) ndi kukula kwa anisotropy (b) mu SAM anali ndi avareji ya zomera zisanu ndi ziwiri zodziyimira pawokha (zomwe zimagwiritsidwa ntchito ngati ma proxies a mphamvu ndi malangizo a kukula kwa maselo, motsatana). c Kusanthula kwa PCA kunaphatikizapo zosintha zotsatirazi: chizindikiro cha GA, kukula kwa pamwamba, kukula kwa anisotropy, ndi kufotokozera kwa CLV3. Gawo la PCA 1 linali logwirizana kwambiri ndi kukula kwa pamwamba ndipo linagwirizana bwino ndi chizindikiro cha GA. Gawo la PCA 2 linali logwirizana kwambiri ndi kukula kwa pamwamba ndipo linagwirizana molakwika ndi kufotokozera kwa CLV3. Maperesenti akuyimira kusiyana komwe kwafotokozedwa ndi gawo lililonse. d Kusanthula kwa mgwirizano wa Spearman pakati pa chizindikiro cha GA, kukula kwa pamwamba, ndi kukula kwa pamwamba pa anisotropy pamlingo wa minofu kupatula CZ. Nambala kumanja ndi mtengo wa Spearman rho pakati pa zosintha ziwiri. Asterisks amasonyeza milandu pomwe kulumikizana/kusagwirizana ndikofunikira kwambiri. e Kuwonetsa kwa 3D kwa maselo a Col-0 SAM L1 pogwiritsa ntchito microscopy ya confocal. Makoma atsopano a maselo opangidwa mu SAM (koma osati primordium) pa maola 10 amapakidwa utoto malinga ndi ma angle awo. Mzere wamitundu ukuwonetsedwa pakona yakumanja kumunsi. Chithunzi cha mkati chikuwonetsa chithunzi chofanana cha 3D pa 0 h. Kuyeseraku kunabwerezedwa kawiri ndi zotsatira zofanana. f Ma Box plots akuwonetsa kuchuluka kwa magawano a maselo mu IPR ndi non-IPR Col-0 SAM (n = 10 zomera zodziyimira pawokha). Mzere wapakati ukuwonetsa wapakati, ndipo malire a bokosi akuwonetsa ma percentiles a 25 ndi 75. Ndevu zimasonyeza ma values ocheperako komanso apamwamba kwambiri omwe atsimikiziridwa ndi pulogalamu ya R. Ma values a P adapezeka ndi Welch's two-tailed t-test. g, h Schematic diagram yowonetsa (g) momwe mungayezere ngodya ya khoma latsopano la selo (magenta) poyerekeza ndi njira yozungulira kuchokera pakati pa SAM (mzere woyera wokhala ndi madontho) (ma values acute angle okha, mwachitsanzo, 0–90°, amaganiziridwa), ndi (h) malangizo ozungulira/ambali ndi a radial mkati mwa meristem. i Ma histogram afupipafupi a njira yogawanitsira maselo kudutsa SAM (buluu wakuda), IPR (buluu wapakati), ndi non-IPR (buluu wopepuka), motsatana. Ma P values anapezedwa ndi mayeso a Kolmogorov-Smirnov okhala ndi michira iwiri. Kuyeseraku kunabwerezedwa kawiri ndi zotsatira zofanana. j Ma frequency histograms a mawonekedwe a cell division plane a IPR mozungulira P3 (wobiriwira wopepuka), P4 (wobiriwira wapakatikati), ndi P5 (wobiriwira wakuda), motsatana. Ma P values anapezedwa ndi mayeso a Kolmogorov-Smirnov okhala ndi michira iwiri. Kuyeseraku kunabwerezedwa kawiri ndi zotsatira zofanana.
Chifukwa chake, kenako tinafufuza za ubale pakati pa GA signaling ndi ntchito yogawa maselo pozindikira makoma atsopano a maselo panthawi yoyesa (Chithunzi 5e). Njira imeneyi inatithandiza kuyeza kuchuluka ndi komwe maselo amagawikana. Chodabwitsa n'chakuti, tinapeza kuti kuchuluka kwa magawikana a maselo mu IPR ndi SAM yonse (yosakhala IPR, Chithunzi 5f) kunali kofanana, zomwe zikusonyeza kuti kusiyana kwa GA signaling pakati pa maselo a IPR ndi omwe si a IPR sikukhudza kwambiri kugawa kwa maselo. Izi, komanso ubale wabwino pakati pa GA signaling ndi kukula kwa anisotropy, zinatipangitsa kuganizira ngati ntchito ya GA signaling ingakhudze momwe maselo amagawikana. Tinayesa momwe khoma latsopano la selo limayendera ngati ngodya yolunjika poyerekeza ndi mzere wozungulira womwe umalumikiza pakati pa meristem ndi pakati pa khoma latsopano la selo (Chithunzi 5e-i) ndipo tinaona kuti maselo amagawanika pa ngodya pafupi ndi 90° poyerekeza ndi mzere wozungulira, ndipo ma frequency apamwamba kwambiri amawonedwa pa 70–80° (23.28%) ndi 80–90° (22.62%) (Chithunzi 5e,i), mogwirizana ndi magawano a maselo mu circumferential/transverse direction (Chithunzi 5h). Kuti tiwone momwe GA signaling imathandizira pa khalidwe la magawano a maselo, tinasanthula magawo a magawano a maselo mu IPR ndi non-IPR padera (Chithunzi 5i). Tinaona kuti kugawa kwa ngodya ya magawano m'maselo a IPR kunali kosiyana ndi kwa m'maselo omwe si a IPR kapena m'maselo onse a SAM, pomwe maselo a IPR akuwonetsa kuchuluka kwa magawano a maselo a lateral/circular, mwachitsanzo, 70–80° ndi 80–90° (33.86% ndi 30.71%, motsatana, magawo ofanana) (Chithunzi 5i). Chifukwa chake, zomwe tawona zawonetsa mgwirizano pakati pa chizindikiro cha GA chapamwamba ndi mawonekedwe a ndege ya magawano a maselo pafupi ndi njira yozungulira, yofanana ndi mgwirizano pakati pa ntchito ya chizindikiro cha GA ndi kukula kwa anisotropy (Chithunzi 5c, d). Kuti titsimikizire kusungidwa kwa malo kwa mgwirizanowu, tinayesa mawonekedwe a ndege ya magawano m'maselo a IPR ozungulira primordium kuyambira pa P3, popeza ntchito yayikulu kwambiri ya chizindikiro cha GA idapezeka m'derali kuyambira pa P4 (Chithunzi 4). Ma ngodya a magawano a IPR ozungulira P3 ndi P4 sanawonetse kusiyana kwakukulu, ngakhale kuchuluka kwa magawano a maselo a lateral kunawonedwa mu IPR yozungulira P4 (Chithunzi 5j). Komabe, m'maselo a IPR ozungulira P5, kusiyana kwa malo ozungulira gawo logawa maselo kunakhala kofunikira kwambiri, ndi kuwonjezeka kwakukulu kwa kuchuluka kwa magawo opingasa a maselo (Chithunzi 5j). Pamodzi, zotsatirazi zikusonyeza kuti chizindikiro cha GA chingalamulire malo ozungulira magawano a maselo mu SAM, zomwe zikugwirizana ndi malipoti am'mbuyomu40,41 kuti chizindikiro cha GA chapamwamba chingayambitse malo ozungulira magawano a maselo mu IPR.
Zikunenedwa kuti maselo omwe ali mu IPR sadzaphatikizidwa mu primordia koma m'malo mwake mu internodes2,42,43. Kuyang'ana kopingasa kwa magawano a maselo mu IPR kungayambitse kukonzedwa kwa mizere yofanana ya maselo a epidermal mu internodes. Zomwe tawona pamwambapa zikusonyeza kuti GA signaling mwina imagwira ntchito pa njirayi poyang'anira njira yogawanitsira maselo.
Kutayika kwa ntchito kwa majini angapo a DELLA kumabweretsa yankho la GA, ndipo ma della mutants angagwiritsidwe ntchito kuyesa lingaliro ili44. Choyamba tinasanthula machitidwe a ma gene asanu a DELLA mu SAM. Kuphatikizika kwa mzere wa GUS45 kunavumbula kuti GAI, RGA, RGL1, ndi RGL2 (kochepa kwambiri) zinafotokozedwa mu SAM (Chithunzi Chowonjezera 11a–d). Kusakaniza kwa malo ozungulira kunawonetsanso kuti GAI mRNA imasonkhana makamaka mu maluwa oyamba ndi omwe akukula (Chithunzi Chowonjezera 11e). RGL1 ndi RGL3 mRNA zinapezeka m'mphepete mwa SAM komanso m'maluwa akale, pomwe RGL2 mRNA inali yochuluka kwambiri m'chigawo cha malire (Chithunzi Chowonjezera 11f–h). Chithunzi chachinsinsi cha pRGL3::RGL3-GFP SAM chinatsimikizira mawu omwe adawonedwa ndi in situ hybridization ndipo adawonetsa kuti mapuloteni a RGL3 amasonkhana pakati pa SAM (Chithunzi Chowonjezera 11i). Pogwiritsa ntchito mzere wa pRGA::GFP-RGA, tinapezanso kuti mapuloteni a RGA amasonkhana mu SAM, koma kuchuluka kwake kumachepa pamalire kuyambira pa P4 (Chithunzi Chowonjezera 11j). Chodziwika bwino ndi chakuti, mawonekedwe a RGL3 ndi RGA akugwirizana ndi ntchito yayikulu ya GA signaling mu IPR, monga momwe zapezekera ndi qmRGA (Chithunzi 4). Komanso, deta iyi ikuwonetsa kuti ma DELLA onse amafotokozedwa mu SAM ndipo kuti mawonekedwe awo onse amakhudza SAM yonse.
Kenako tinasanthula magawo a kugawa kwa maselo mu mtundu wa wild-type SAM (Ler, control) ndi gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Chithunzi 6a, b). Chosangalatsa n'chakuti, tinaona kusintha kwakukulu kwa ziwerengero pakugawa kwa ma frequency a ngodya ya kugawa kwa maselo mu della global mutant SAM poyerekeza ndi mtundu wa wild (Chithunzi 6c). Kusintha kumeneku mu della global mutant kunabwera chifukwa cha kuwonjezeka kwa ma frequency a 80–90° angles (34.71% vs. 24.55%) ndipo, pang'ono, ma angle a 70–80° (23.78% vs. 20.18%), mwachitsanzo, mogwirizana ndi magawano a maselo opingasa (Chithunzi 6c). Ma frequency a magawano osapingasa (0–60°) anali otsikanso mu della global mutant (Chithunzi 6c). Kuchuluka kwa magawano a maselo opingasa kunawonjezeka kwambiri mu SAM ya della global mutant (Chithunzi 6b). Kuchuluka kwa magawano a maselo opingasa mu IPR kunalinso kwakukulu mu della global mutant poyerekeza ndi wild type (Chithunzi 6d). Kunja kwa dera la IPR, wild type inali ndi kufalikira kofanana kwa ma angles a magawano a maselo, pomwe della global mutant idakonda magawano a tangential monga IPR (Chithunzi 6e). Tinayesanso momwe magawano a maselo amayendera mu SAM ya ga2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, ndi ga2ox6-2), maziko a GA-inactive mutant omwe GA imasonkhanamo. Mogwirizana ndi kuwonjezeka kwa milingo ya GA, SAM ya quintuple ga2ox mutant inflorescence inali yayikulu kuposa ya Col-0 (Chithunzi Chowonjezera 12a, b), ndipo poyerekeza ndi Col-0, quintuple ga2ox SAM inawonetsa kugawa kosiyana kwa ma angles ogawa maselo, ndi ma frequency a ngodya akukwera kuchokera pa 50° mpaka 90°, mwachitsanzo, kukondanso magawano a tangential (Chithunzi Chowonjezera 12a–c). Chifukwa chake, tikuwonetsa kuti kuyambitsa kwa GA signaling ndi GA accumulation kumayambitsa magawano a cell lateral mu IPR ndi SAM yonse.
a, b Kuwonetsa kwa 3D kwa gawo la L1 la PI-stained Ler (a) ndi global della mutant (b) SAM pogwiritsa ntchito microscopy ya confocal. Makoma atsopano a maselo opangidwa mu SAM (koma osati primordium) kwa nthawi ya maola 10 akuwonetsedwa ndikupakidwa utoto malinga ndi ngodya zawo. Chithunzicho chikuwonetsa SAM pa 0 h. Mtundu wa bala ukuwonetsedwa pakona yakumanja yakumunsi. Muvi mu (b) ukuwonetsa chitsanzo cha mafayilo a maselo ogwirizana mu global della mutant. Kuyeseraku kunabwerezedwa kawiri ndi zotsatira zofanana. kuyerekeza kwa kufalikira kwa pafupipafupi kwa ma cell division plane directions mu SAM yonse (d), IPR (e), ndi non-IPR (f) pakati pa Ler ndi global della. Ma P values adapezeka pogwiritsa ntchito mayeso a Kolmogorov-Smirnov okhala ndi michira iwiri. f, g Kuwonetsa kwa 3D kwa zithunzi za confocal za PI-stained SAM ya zomera za Col-0 (i) ndi pCUC2::gai-1-VENUS (j). Mapanelo (a, b) akuwonetsa makoma atsopano a maselo (koma osati primordia) opangidwa mu SAM mkati mwa maola 10. Kuyeseraku kunabwerezedwa kawiri ndi zotsatira zofanana. h–j Kuyerekeza kufalikira kwa pafupipafupi kwa malo ogawa maselo omwe ali mu SAM yonse (h), IPR (i) ndi osakhala IPR (j) pakati pa zomera za Col-0 ndi pCUC2::gai-1-VENUS. Ma P values anapezeka pogwiritsa ntchito mayeso a Kolmogorov–Smirnov okhala ndi michira iwiri.
Kenako tinayesa zotsatira za kuletsa chizindikiro cha GA makamaka mu IPR. Pachifukwa ichi, tinagwiritsa ntchito cotyledon cup 2 (CUC2) promoter kuti tiyendetse kuwonetsa kwa puloteni ya gai-1 yoyipa kwambiri yomwe idalumikizidwa ku VENUS (mu mzere wa pCUC2::gai-1-VENUS). Mu mtundu wa SAM wachilengedwe, CUC2 promoter imayendetsa kuwonetsa kwa ma IPR ambiri mu SAM, kuphatikiza maselo am'malire, kuyambira P4 kupita mtsogolo, ndipo kuwonetsa kofananako kunawonedwa mu zomera za pCUC2::gai-1-VENUS (onani pansipa). Kugawa kwa ma angles ogawa maselo kudutsa SAM kapena IPR ya zomera za pCUC2::gai-1-VENUS sikunali kosiyana kwambiri ndi kwa mtundu wachilengedwe, ngakhale mosayembekezereka tinapeza kuti maselo opanda IPR m'zomera izi amagawikana pafupipafupi ya 80–90° (Chithunzi 6f–j).
Zanenedwa kuti njira yogawa maselo imadalira mawonekedwe a SAM, makamaka kupsinjika kwa tensile komwe kumapangidwa ndi kupindika kwa minofu46. Chifukwa chake tidafunsa ngati mawonekedwe a SAM adasinthidwa mu zomera za della global mutant ndi pCUC2::gai-1-VENUS. Monga momwe tafotokozera kale12, kukula kwa della global mutant SAM kunali kwakukulu kuposa kwa mtundu wakuthengo (Chithunzi Chowonjezera 13a, b, d). Kusakanikirana kwa CLV3 ndi STM RNA mu situ kunatsimikizira kukula kwa meristem mu ma mutants a della ndipo kunawonetsanso kukula kwa mbali ya niche ya stem cell (Chithunzi Chowonjezera 13e, f, h, i). Komabe, kupindika kwa SAM kunali kofanana mu ma genotype onse awiri (Chithunzi Chowonjezera 13k, m, n, p). Tinaona kukula kofananako mu gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant popanda kusintha kwa kupindika poyerekeza ndi mtundu wachilengedwe (Chithunzi Chowonjezera 13c, d, g, j, l, o, p). Kuchuluka kwa momwe maselo amagawidwira kunakhudzidwiranso mu della quadruple mutant, koma pang'ono poyerekeza ndi momwe maselo amagawidwira (Chithunzi Chowonjezera 12d–f). Kuchuluka kumeneku, pamodzi ndi kusowa kwa mphamvu pa kupindika, kukusonyeza kuti ntchito yotsalira ya RGL3 mu Della quadruple mutant imaletsa kusintha kwa momwe maselo amagawidwira chifukwa cha kutayika kwa ntchito ya DELLA ndipo kusintha kwa magawo a maselo a lateral kumachitika poyankha kusintha kwa ntchito ya GA signaling m'malo mwa kusintha kwa SAM geometry. Monga tafotokozera pamwambapa, chothandizira cha CUC2 chimayambitsa kufotokozedwa kwa IPR mu SAM kuyambira pa P4 (Chithunzi Chowonjezera 14a, b), ndipo mosiyana, pCUC2::gai-1-VENUS SAM inali ndi kukula kocheperako koma kupindika kwakukulu (Chithunzi Chowonjezera 14c–h). Kusintha kumeneku kwa mawonekedwe a pCUC2::gai-1-VENUS SAM kungayambitse kufalikira kosiyana kwa kupsinjika kwa makina poyerekeza ndi mtundu wachilengedwe, momwe kupsinjika kwakukulu kozungulira kumayambira patali pang'ono kuchokera pakati pa SAM47. Kapenanso, kusintha kwa mawonekedwe a pCUC2::gai-1-VENUS SAM kungachitike chifukwa cha kusintha kwa mawonekedwe a makina am'deralo omwe amachitika chifukwa cha kufotokozedwa kwa transgene48. M'zochitika zonsezi, izi zitha kuchepetsa pang'ono zotsatira za kusintha kwa chizindikiro cha GA powonjezera mwayi woti maselo agawikane mu circumferential/transverse, kufotokozera zomwe tawona.
Poganizira zonse, deta yathu ikutsimikizira kuti chizindikiro cha GA chapamwamba chimagwira ntchito yofunikira pakuyang'ana mbali ya gawo logawanitsa maselo mu IPR. Zikuwonetsanso kuti kupindika kwa meristem kumakhudzanso momwe gawo logawanitsa maselo limayendera mu IPR.
Kuyang'ana kopingasa kwa gawo logawa mu IPR, chifukwa cha ntchito yayikulu yowonetsa ma GA, kukuwonetsa kuti GA imakonza kale fayilo ya ma radial cell mu epidermis mkati mwa SAM kuti ifotokoze bungwe la ma cell lomwe lidzapezeka pambuyo pake mu epidermal internode. Zoonadi, ma cell oterewa ankawoneka kawirikawiri mu zithunzi za SAM za della global mutants (Chithunzi 6b). Chifukwa chake, kuti tifufuze bwino ntchito ya chitukuko cha mawonekedwe a GA signaling mu SAM, tidagwiritsa ntchito time-lapse imaging kuti tiwunikenso dongosolo la ma cell mu IPR mu wild-type (Ler ndi Col-0), della global mutants, ndi pCUC2::gai-1-VENUS transgenic plants.
Tapeza kuti qmRGA yawonetsa kuti ntchito yowonetsa GA mu IPR idakwera kuchokera pa P1/P2 ndipo idafika pachimake pa P4, ndipo mawonekedwe awa adakhalabe osasintha pakapita nthawi (Chithunzi 4a–f ndi Chithunzi Chowonjezera 8c–f, k). Kuti tiwunike kapangidwe ka malo a maselo mu IPR ndi chizindikiro chowonjezeka cha GA, tidalemba ma cell a Ler IPR pamwamba ndi m'mbali mwa P4 malinga ndi tsogolo lawo la chitukuko lomwe lidawunikidwa maola 34 pambuyo poyang'ana koyamba, mwachitsanzo, nthawi zoposa ziwiri za pulasitiki, zomwe zidatilola kutsatira ma cell a IPR panthawi yopanga primordium kuyambira P1/P2 mpaka P4. Tinagwiritsa ntchito mitundu itatu yosiyana: yachikasu ya ma cell omwe adalumikizidwa mu primordium pafupi ndi P4, yobiriwira ya omwe anali mu IPR, ndi yofiirira ya omwe adachita nawo machitidwe onse awiri (Chithunzi 7a–c). Pa t0 (0 h), zigawo 1–2 za ma cell a IPR zidawoneka patsogolo pa P4 (Chithunzi 7a). Monga momwe zinalili, pamene maselowa anagawikana, anachita zimenezi makamaka kudzera mu ndege yogawa magawo (Zithunzi 7a–c). Zotsatira zofanana zinapezeka pogwiritsa ntchito Col-0 SAM (yoyang'ana kwambiri P3, yomwe malire ake amapinda mofanana ndi P4 mu Ler), ngakhale kuti mu genotype iyi, fold yomwe inapangidwa pamalire a maluwa inabisa maselo a IPR mwachangu (Chithunzi 7g–i). Motero, kapangidwe ka magawano a maselo a IPR kamakonzeratu maselowo kukhala mizere yozungulira, monga momwe zilili m'ma internodes. Kukonza mizere yozungulira ndi malo a maselo a IPR pakati pa ziwalo zotsatizana kumasonyeza kuti maselowa ndi oyambitsa ma node.
Apa, tinapanga ratiometric GA signaling biosensor, qmRGA, yomwe imalola kupanga mapu ochulukirapo a ntchito ya GA signaling yomwe imachokera ku kuchuluka kwa GA ndi GA receptor pamodzi pomwe ikuchepetsa kusokoneza njira zolumikizirana za endogenous, motero kupereka chidziwitso pa ntchito ya GA pamlingo wa maselo. Pachifukwa ichi, tinapanga puloteni yosinthidwa ya DELLA, mRGA, yomwe yataya mphamvu yomangirira ogwirizana a DELLA koma imakhalabe yokhudzidwa ndi proteolysis yomwe imayambitsa GA. qmRGA imayankha kusintha kwakunja komanso kwa endogenous mu milingo ya GA, ndipo mphamvu zake zomvera zimathandiza kuwunika kusintha kwa malo mu ntchito ya GA signaling panthawi yopanga. qmRGA ndi chida chosinthika kwambiri chifukwa chingasinthidwe ku minofu yosiyanasiyana posintha cholimbikitsira chomwe chimagwiritsidwa ntchito pofotokozera (ngati pakufunika), komanso poganizira momwe njira ya GA signaling ndi PFYRE zimasungidwira komanso momwe zimakhalira ndi ma angiosperms, zitha kusamutsidwira ku mitundu ina22. Mogwirizana ndi izi, kusintha kofanana mu puloteni ya mpunga ya SLR1 DELLA (HYY497AAA) kunawonetsedwanso kuti kumaletsa ntchito yoletsa kukula kwa SLR1 pomwe kumachepetsa pang'ono kuwonongeka kwake komwe kumayendetsedwa ndi GA, kofanana ndi mRGA23. Chodziwika bwino, kafukufuku waposachedwa mu Arabidopsis adawonetsa kuti kusintha kwa amino acid imodzi mu PFYRE domain (S474L) kunasintha ntchito yolembera ya RGA popanda kukhudza kuthekera kwake kolumikizana ndi ogwirizana ndi transcription factor50. Ngakhale kusinthaku kuli pafupi kwambiri ndi ma amino acid atatu omwe amapezeka mu mRGA, maphunziro athu akuwonetsa kuti kusinthaku kumasintha makhalidwe osiyana a DELLA. Ngakhale ogwirizana ambiri a transcription factor amamangirira ku LHR1 ndi SAW domains za DELLA26,51, ma amino acid ena osungidwa mu PFYRE domain angathandize kukhazikika kwa kuyanjana kumeneku.
Kukula kwa ma node pakati pa maselo ndi khalidwe lofunika kwambiri pa kapangidwe ka zomera ndi kukweza zokolola. qmRGA inavumbula ntchito yayikulu ya GA signaling mu maselo a IPR internode progenitor. Mwa kuphatikiza quantitative imaging ndi genetics, tinawonetsa kuti ma pattern a GA signaling amaika ma cell division planes ozungulira/opingasa mu SAM epidermis, ndikupanga bungwe logawa maselo lofunikira pakukula kwa ma internode. Oyang'anira angapo a cell division plane orientation apezeka panthawi ya chitukuko52,53. Ntchito yathu imapereka chitsanzo chomveka bwino cha momwe GA signaling activity imayendetsera parameter iyi ya selo. DELLA imatha kuyanjana ndi ma protein complexes a prefolding41, kotero GA signaling ikhoza kuwongolera cell division plane orientation mwa kukhudza mwachindunji cortical microtubule orientation40,41,54,55. Tinawonetsa mosayembekezereka kuti mu SAM, correlate ya high GA signaling activity sinali kutalikitsa maselo kapena kugawa, koma kukula kwa anisotropy kokha, komwe kumagwirizana ndi zotsatira za GA mwachindunji pa njira ya cell division mu IPR. Komabe, sitingachotse kuti zotsatirazi zitha kukhala zosalunjika, mwachitsanzo zomwe zimayendetsedwa ndi GA-induced cell wall softening56. Kusintha kwa mawonekedwe a khoma la selo kumayambitsa kupsinjika kwa makina57,58, komwe kungakhudzenso momwe maselo amagawidwira pokhudza momwe maselo amagawidwira ndi ma microtubules a cortical39,46,59. Zotsatira zophatikizana za kupsinjika kwa makina komwe kumachitika chifukwa cha GA komanso kuwongolera mwachindunji momwe maselo amagawidwira ndi GA zitha kukhala ndi gawo pakupanga mawonekedwe enieni a momwe maselo amagawidwira mu IPR kuti afotokoze ma internode, ndipo maphunziro ena amafunika kuti ayesere lingaliro ili. Mofananamo, maphunziro am'mbuyomu awonetsa kufunika kwa mapuloteni ogwirizana a DELLA TCP14 ndi 15 pakulamulira mapangidwe a internode60,61 ndipo zinthu izi zitha kuyambitsa ntchito ya GA pamodzi ndi BREVIPEDICELUS (BP) ndi PENNYWISE (PNY), zomwe zimawongolera chitukuko cha internode ndipo zawonetsedwa kuti zimakhudza GA signaling2,62. Popeza kuti ma DELLA amalumikizana ndi brassinosteroid, ethylene, jasmonic acid, ndi abscisic acid (ABA) signaling pathways63,64 ndipo mahomoni amenewa amatha kukhudza microtubule orientation65, zotsatira za GA pa cell division orientation zithanso kuthandizidwa ndi mahomoni ena.
Kafukufuku woyambirira wa cytological adawonetsa kuti madera amkati ndi akunja a Arabidopsis SAM amafunikira pakukula kwa ma internode2,42. Mfundo yakuti GA imayang'anira kugawikana kwa maselo m'kati mwa minofu12 imathandizira ntchito ziwiri za GA pakulamulira kukula kwa meristem ndi internode mu SAM. Kapangidwe ka kugawikana kwa maselo kolunjika kamayendetsedwanso mwamphamvu m'kati mwa minofu ya SAM, ndipo lamuloli ndi lofunikira pakukula kwa tsinde52. Zidzakhala zosangalatsa kufufuza ngati GA imagwiranso ntchito poyang'anira gawo la kugawikana kwa maselo m'gulu lamkati la SAM, potero kulumikiza kufotokozera ndi chitukuko cha ma internode mkati mwa SAM.
Zomera zinalimidwa mu dothi kapena 1x Murashige-Skoog (MS) medium (Duchefa) yowonjezera 1% sucrose ndi 1% agar (Sigma) pansi pa mikhalidwe yokhazikika (maola 16 kuwala, 22 °C), kupatula kuyesa kwa hypocotyl ndi kukula kwa mizu komwe mbande zinalimidwa pa mbale zoyima pansi pa kuwala kosalekeza ndi 22 °C. Pa kuyesa kwa nitrate, zomera zinalimidwa pa modified MS medium (bioWORLD plant medium) yowonjezera nitrate yokwanira (0 kapena 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose ndi 1% A-agar (Sigma) pansi pa mikhalidwe ya tsiku lonse.
GID1a cDNA yomwe inalowetsedwa mu pDONR221 inaphatikizidwanso ndi pDONR P4-P1R-pUBQ10 ndi pDONR P2R-P3-mCherry mu pB7m34GW kuti ipange pBQ10::GID1a-mCherry. IDD2 DNA yomwe inalowetsedwa mu pDONR221 inaphatikizidwanso mu pB7RWG266 kuti ipange p35S:IDD2-RFP. Kuti apange pGID1b::2xmTQ2-GID1b, chidutswa cha 3.9 kb pamwamba pa dera lolemba ma GID1b ndi chidutswa cha 4.7 kb chokhala ndi GID1b cDNA (1.3 kb) ndi terminator (3.4 kb) chinakulitsidwa koyamba pogwiritsa ntchito ma primers mu Supplementary Table 3 kenako nkuyikidwa mu pDONR P4-P1R (Thermo Fisher Scientific) ndi pDONR P2R-P3 (Thermo Fisher Scientific), motsatana, ndipo potsiriza chinaphatikizidwanso ndi pDONR221 2xmTQ268 mu pGreen 012567 target vector pogwiritsa ntchito Gateway cloning. Kuti apange pCUC2::LSSmOrange, CUC2 promoter sequence (3229 bp pamwamba pa ATG) yotsatiridwa ndi coding sequence ya large Stokes-shifted mOrange (LSSmOrange)69 yokhala ndi N7 nuclear localization signal ndi NOS transcriptional terminator zinasonkhanitsidwa mu pGreen kanamycin targeting vector pogwiritsa ntchito Gateway 3-fragment recombination system (Invitrogen). Chomera cha binary vector chinalowetsedwa mu Agrobacterium tumefaciens strain GV3101 ndipo chinalowetsedwa mu masamba a Nicotiana benthamiana pogwiritsa ntchito Agrobacterium infiltration method ndi mu Arabidopsis thaliana Col-0 pogwiritsa ntchito floral dip method, motsatana. pBQ10::qmRGA pBQ10::GID1a-mCherry ndi pCLV3::mCherry-NLS qmRGA zinalekanitsidwa kuchokera ku ana a F3 ndi F1 a crosses omwewo, motsatana.
Kusakaniza kwa RNA mu situ kunachitika pa nsonga zazitali pafupifupi 1 cm72, zomwe zinasonkhanitsidwa ndikukhazikika nthawi yomweyo mu yankho la FAA (3.7% formaldehyde, 5% acetic acid, 50% ethanol) loziziritsidwa kale mpaka 4 °C. Pambuyo pa chithandizo cha vacuum cha mphindi 2 × 15, chosinthiracho chinasinthidwa ndipo zitsanzo zinayikidwa mu incubation usiku wonse. GID1a, GID1b, GID1c, GAI, RGL1, RGL2, ndi RGL3 cDNAs ndi ma antisense probes ku ma 3′-UTR awo adapangidwa pogwiritsa ntchito ma primers omwe akuwonetsedwa mu Supplementary Table 3 monga momwe Rosier et al.73 adafotokozera. Ma probe okhala ndi chizindikiro cha Digoxigenin adapezeka ndi chitetezo chamthupi pogwiritsa ntchito ma antibodies a digoxigenin (kusungunuka kwa nthawi 3000; Roche, nambala ya katalogi: 11 093 274 910), ndipo magawo adapakidwa utoto wa 5-bromo-4-chloro-3-indolyl phosphate (BCIP, kusungunuka kwa nthawi 250)/nitroblue tetrazolium (NBT, kusungunuka kwa nthawi 200).
Nthawi yotumizira: Feb-10-2025