Kukula kwa apical meristem (SAM) ndikofunikira kwambiri pamapangidwe a tsinde. Mahomoni a zomeragibberellins(GAs) amagwira ntchito yofunika kwambiri pakuwongolera kukula kwa mbewu, koma gawo lawo mu SAM silikudziwika bwino. Apa, tidapanga ratiometric biosensor ya GA siginecha popanga puloteni ya DELLA kuti ipondereze ntchito yake yofunikira pakuyankha kwa GA polemba ndikusunga kuwonongeka kwake pakuzindikirika kwa GA. Tikuwonetsa kuti biosensor yochokera pakuwonongekayi imalemba molondola kusintha kwa ma GA komanso kumva kwa ma cell pakukula. Tidagwiritsa ntchito biosensor iyi kupanga mapu akuwonetsa zochitika za GA mu SAM. Tikuwonetsa kuti ma siginecha apamwamba a GA amapezeka makamaka m'maselo omwe ali pakati pa organ primordia, omwe amatsogolera ma cell a internode. Pogwiritsa ntchito njira zopezera phindu komanso zotayika, tikuwonetsanso kuti GA imayang'anira momwe ndege yogawanitsa ma cell imayendera, ndikukhazikitsa gulu lovomerezeka la ma internodes, potero limalimbikitsa mafotokozedwe a internode mu SAM.
Mphukira ya apical meristem (SAM), yomwe ili pamtunda wa mphukira, imakhala ndi kagawo kakang'ono ka ma cell tsinde omwe zochita zake zimapanga ziwalo zoyandikana ndi tsinde mozungulira komanso mobwerezabwereza moyo wonse wa chomera. Iliyonse mwa mayunitsi obwerezabwerezawa, kapena mfundo za zomera, imaphatikizapo ma internodes ndi ziwalo za m'mphepete mwa mfundo, ndi ma axillary meristems mu axils yamasamba1. Kukula ndi kulinganiza kwa mfundo za zomera kumasintha panthawi ya chitukuko. Mu Arabidopsis, kukula kwa internodal kumaponderezedwa panthawi ya zomera, ndipo ma axillary meristems amakhalabe matalala mu ma axils a masamba a rosette. Pakusintha kupita ku gawo lamaluwa, ma SAM amakhala inflorescence meristem, kupanga ma internodes otalikirana ndi axillary masamba, nthambi mu axils a masamba a cauline, kenako, maluwa opanda masamba2. Ngakhale kuti tapita patsogolo kwambiri pomvetsetsa njira zomwe zimayendetsa kuyambika kwa masamba, maluwa, ndi nthambi, ndizochepa zomwe zimadziwika za momwe internodes zimayambira.
Kumvetsetsa kugawa kwa spatiotemporal kwa ma GA kumathandizira kumvetsetsa bwino ntchito za mahomoniwa m'magulu osiyanasiyana komanso pazigawo zosiyanasiyana zachitukuko. Kuwonetseratu kuwonongeka kwa kusakanikirana kwa RGA-GFP komwe kumasonyezedwa pansi pa zochita za woyambitsa wake kumapereka chidziwitso chofunikira pa kayendetsedwe ka chiwerengero cha GA mu mizu15,16. Komabe, mafotokozedwe a RGA amasiyana m'magulu 17 ndipo amayendetsedwa ndi GA18. Choncho, kufotokozera kusiyana kwa wolimbikitsa RGA kungapangitse chitsanzo cha fluorescence chomwe chimawonedwa ndi RGA-GFP ndipo motero njira iyi siili yochuluka. Posachedwapa, bioactive fluorescein (Fl) -yotchedwa GA19,20 idawulula kudzikundikira kwa GA muzu endocortex ndikuwongolera ma cell ake ndi zoyendera za GA. Posachedwapa, sensor ya GA FRET nlsGPS1 idawonetsa kuti milingo ya GA imagwirizana ndi kutalika kwa ma cell mumizu, filaments, ndi hypocotyls21 yakuda. Komabe, monga tawonera, GA ndende sizinthu zokhazo zomwe zimayang'anira ntchito zowonetsera GA, chifukwa zimadalira njira zovuta zomvera. Apa, tikukulitsa kumvetsetsa kwathu kwa njira zowonetsera za DELLA ndi GA, timapereka lipoti la chitukuko ndi mawonekedwe a ratiometric biosensor yochokera pakuwonongeka kwa chizindikiro cha GA. Kuti tipange biosensor yochulukirayi, tidagwiritsa ntchito GA-sensitive RGA yomwe idasakanikirana ndi puloteni ya fulorosenti ndipo imawonetsedwa paliponse m'matishu, komanso puloteni ya GA-yosamva fulorosenti. Tikuwonetsa kuti kuphatikizika kwa mapuloteni a RGA sikusokoneza ma siginecha amtundu wa GA akamawonetsedwa paliponse, komanso kuti biosensor iyi imatha kuwerengera zomwe zimachitika chifukwa cha kulowetsa kwa GA komanso kusinthidwa kwa ma siginecha a GA ndi zida zomverera zokhala ndi mawonekedwe apamwamba a spatiotemporal. Tidagwiritsa ntchito biosensor iyi kupanga mapu kugawa kwapadziko lapansi kwa ntchito zosayina za GA ndikuwerengera momwe GA imayendetsera ma cellular mu epidermis ya SAM. Tikuwonetsa kuti GA imayang'anira momwe magawano a ma cell a SAM ali pakati pa organ primordia, motero amatanthauzira gulu la ma cell a internode.
Pomaliza, tidafunsa ngati qmRGA inganene zosintha zamagawo amtundu wa GA pogwiritsa ntchito ma hypocotyl omwe akukula. Tidawonetsa kale kuti nitrate imathandizira kukula ndikuwonjezera kaphatikizidwe ka GA komanso kuwonongeka kwa DELLA34. Momwemo, tidawona kuti kutalika kwa hypocotyl mu pUBQ10 :: qmRGA mbande zomwe zidakula pansi pa kuchuluka kwa nitrate (10 mM NO3-) zinali zazitali kwambiri kuposa mbande zomwe zimakula pansi pakusowa kwa nitrate (Supplementary Fig. 6a). Mogwirizana ndi kuyankha kwakukula, zizindikiro za GA zinali zapamwamba mu hypocotyls za mbande zomwe zakula pansi pa 10 mm NO3- mikhalidwe kusiyana ndi mbande zomwe zimakula popanda nitrate (Supplementary Fig. 6b, c). Chifukwa chake, qmRGA imathandizanso kuyang'anira kusintha kwa kusanja kwa GA komwe kumabwera chifukwa cha kusintha kosasinthika kwa ndende ya GA.
Kuti timvetsetse ngati ntchito yosayina ya GA yodziwika ndi qmRGA imadalira ndende ya GA ndi malingaliro a GA, monga momwe zimayembekezeredwa kutengera kapangidwe ka sensa, tidasanthula mafotokozedwe a ma GID1 atatu olandirira muzomera ndi zoberekera. Mu mbande, mzere wa mtolankhani wa GID1-GUS unasonyeza kuti GID1a ndi c zinawonetsedwa kwambiri mu cotyledons (Mkuyu 3a-c). Kuphatikiza apo, zolandilira zonse zitatu zidawonetsedwa m'masamba, lateral root primordia, nsonga za mizu (kupatula muzu wa GID1b), ndi dongosolo la mitsempha (mkuyu 3a-c). Mu inflorescence SAM, tinazindikira zizindikiro za GUS za GID1b ndi 1c (Zowonjezera mkuyu 7a-c). In situ hybridization inatsimikizira machitidwewa ndikuwonetsanso kuti GID1c idawonetsedwa mofanana pamagulu otsika mu SAM, pamene GID1b inasonyeza mawu apamwamba pamphepete mwa SAM (Supplementary Fig. 7d-l). Kuphatikizika komasulira kwa pGID1b::2xmTQ2-GID1b kunavumbulutsanso mitundu yosiyanasiyana ya mawu a GID1b, kuchokera ku mawu otsika kapena osamveka pakati pa SAM kupita ku mawu apamwamba pamalire a ziwalo (Supplementary Fig. 7m). Chifukwa chake, ma receptor a GID1 samagawidwa mofanana ndi mkati mwa minofu. M'mayesero otsatirawa, tinawonanso kuti kuwonjezereka kwa GID1 (pUBQ10 :: GID1a-mCherry) kumawonjezera chidwi cha qmRGA mu hypocotyls ku ntchito yakunja ya GA (Mkuyu 3d, e). Mosiyana ndi izi, fluorescence anayeza ndi qd17mRGA mu hypocotyl anali wosamva mankhwala GA3 (mkuyu 3f, g). Pazoyesa zonse ziwiri, mbande zimathandizidwa ndi kuchuluka kwa GA (100 μM GA3) kuti awone momwe sensor imayendera mwachangu, pomwe kuthekera komanga ku GID1 receptor kumakulitsidwa kapena kutayika. Pamodzi, zotsatirazi zikutsimikizira kuti qmRGA biosensor imagwira ntchito yophatikizika ngati GA ndi GA sensa, ndikuwonetsa kuti kusiyanitsa kwa GID1 receptor kumatha kusintha kwambiri kutulutsa kwa sensor.
Mpaka pano, kugawidwa kwa ma sign a GA mu SAM sikunadziwikebe. Choncho, tinagwiritsa ntchito zomera zowonetsera qmRGA ndi pCLV3 :: mCherry-NLS stem cell reporter35 kuti tiwerenge mapu okwera kwambiri a ntchito zowonetsera GA, poyang'ana pa L1 wosanjikiza (epidermis; Chithunzi 4a, b, onani Njira ndi Njira Zowonjezerapo), popeza L3 imagwira ntchito yofunika kwambiri ya SAM. Apa, pCLV3 ::mawu a mCherry-NLS adapereka malo okhazikika a geometric posanthula kugawa kwapakatikati kwa ntchito yowonetsa GA37. Ngakhale kuti GA imaonedwa kuti ndi yofunika kwambiri pa chitukuko cha lateral organist4, tinawona kuti zizindikiro za GA zinali zochepa mu primordium yamaluwa (P) kuyambira pa P3 siteji (Mkuyu 4a, b), pamene achinyamata a P1 ndi P2 primordiums anali ndi ntchito zolimbitsa thupi zofanana ndi zomwe zili m'chigawo chapakati (Mkuyu 4a, b). Ntchito yapamwamba ya GA yowonetserako inapezedwa pamalire a primordium organ, kuyambira pa P1 / P2 (m'mphepete mwa malire) ndikukwera pa P4, komanso m'maselo onse a chigawo chapakati chomwe chili pakati pa primordia (mkuyu 4a, b ndi Supplementary Fig. 8a, b). Ntchito yowonetsera GA yapamwambayi sinawonedwe kokha mu epidermis komanso mu L2 ndi pamwamba L3 zigawo (Supplementary Fig. 8b). Chitsanzo cha zizindikiro za GA zomwe zapezeka mu SAM pogwiritsa ntchito qmRGA zinakhalabe zosasinthika pakapita nthawi (Supplementary Fig. 8c-f, k). Ngakhale kuti mapangidwe a qd17mRGA adachepetsedwa mwadongosolo mu SAM ya zomera za T3 kuchokera ku mizere isanu yodziimira yomwe tidadziwika mwatsatanetsatane, tinatha kusanthula machitidwe a fluorescence omwe anapezeka ndi pRPS5a :: VENUS-2A-TagBFP kumanga (Supplementary Fig. 8g-j, l). Mu mzere wowongolera uwu, kusintha kwakung'ono kokha kwa chiŵerengero cha fluorescence kunapezeka mu SAM, koma pakati pa SAM tinawona kuchepa koonekeratu komanso kosayembekezereka kwa VENUS yogwirizana ndi TagBFP. Izi zikutsimikizira kuti mawonekedwe owonetsera omwe qmRGA amawonetsa kuwonongeka kodalira GA kwa mRGA-VENUS, komanso zikuwonetsa kuti qmRGA ikhoza kuwonetsa ntchito yowonetsa GA mu meristem center. Mwachidule, zotsatira zathu zimawulula mawonekedwe a siginecha a GA omwe amawonetsa kugawa kwa primordia. Kugawa kumeneku kwa dera lapakati-primordial (IPR) kumachitika chifukwa cha kukhazikitsidwa kwapang'onopang'ono kwa ntchito yowonetsera GA yapamwamba pakati pa primordium yomwe ikukula ndi dera lapakati, pamene nthawi yomweyo GA yowonetsera ntchito mu primordium imachepa (mkuyu 4c, d).
Kugawa kwa ma GID1b ndi GID1c receptors (onani pamwambapa) kukuwonetsa kuti kusiyanitsa kwa ma receptor a GA kumathandiza kupanga mawonekedwe a ntchito yowonetsa GA mu SAM. Tidadabwa ngati kudzikundikira kosiyana kwa GA kungakhudzidwe. Kuti tifufuze izi, tidagwiritsa ntchito nlsGPS1 GA FRET sensor21. Kuwonjezeka kwafupipafupi kutsegulira kunapezeka mu SAM ya nlsGPS1 yothandizidwa ndi 10 μM GA4 + 7 kwa mphindi 100 (Supplementary Fig. 9a-e), kusonyeza kuti nlsGPS1 imayankha kusintha kwa GA ku SAM, monga momwe imachitira mizu21. Kugawidwa kwa malo a nlsGPS1 kutsegulira kwafupipafupi kunavumbulutsa ma GA otsika kwambiri m'zigawo zakunja za SAM, koma adawonetsa kuti adakwezedwa pakati ndi kumalire a SAM (Mkuyu 4e ndi Supplementary Fig. 9a, c). Izi zikusonyeza kuti GA imagawidwanso mu SAM ndi mawonekedwe a malo ofanana ndi omwe adavumbulutsidwa ndi qmRGA. Monga njira yophatikizira, tidachitiranso SAM ndi fulorosenti GA (GA3-, GA4-, GA7-Fl) kapena Fl yokha ngati chowongolera choyipa. Chizindikiro cha Fl chinagawidwa mu SAM yonse, kuphatikizapo dera lapakati ndi primordium, ngakhale pang'onopang'ono (mkuyu 4j ndi Supplementary Fig. 10d). Mosiyana ndi zimenezi, GA-Fl yonse itatu inasonkhanitsidwa makamaka m'malire oyambirira komanso kumadera osiyanasiyana mu IPR yonse, ndi GA7-Fl ikuwonjezeka mu dera lalikulu kwambiri la IPR (Mkuyu 4k ndi Supplementary Fig. 10a, b). Kuwerengera kwa mphamvu ya fluorescence kunawonetsa kuti IPR kwa osakhala IPR intensity ratio inali yapamwamba mu GA-Fl-treated SAM poyerekeza ndi Fl-treated SAM (Fig. 4l ndi Supplementary Fig. 10c). Pamodzi, zotsatirazi zikuwonetsa kuti GA imapezeka m'maselo apamwamba a IPR omwe ali pafupi kwambiri ndi malire a ziwalo. Izi zikuwonetsa kuti mawonekedwe a SAM GA siginecha amachokera ku mafotokozedwe osiyanitsira a GA receptors komanso kudzikundikira kosiyana kwa GA m'maselo a IPR pafupi ndi malire a ziwalo. Chifukwa chake, kusanthula kwathu kunawonetsa mawonekedwe osayembekezeka a spatiotemporal a GA signing, ndi zochitika zotsika pakati ndi primordium ya SAM ndi zochitika zapamwamba mu IPR m'dera lozungulira.
Kuti timvetsetse gawo la ntchito yosiyanitsira ma GA mu SAM, tidasanthula kulumikizana pakati pa ntchito yosayina ya GA, kukulitsa ma cell, ndi kugawikana kwa ma cell pogwiritsa ntchito kujambula kwanthawi yeniyeni kwa SAM qmRGA pCLV3::mCherry-NLS. Poganizira gawo la GA pakuwongolera kukula, kulumikizana kwabwino ndi magawo akukulitsa ma cell kumayembekezeredwa. Choncho, poyamba tinkayerekezera mapu a ntchito zowonetsera GA ndi mapu a kukula kwa maselo (monga choyimira mphamvu ya kukula kwa selo kwa selo lopatsidwa ndi kwa ana aakazi pagawikana) ndi mapu a kukula kwa anisotropy, omwe amayesa njira ya kukula kwa selo (yomwe imagwiritsidwanso ntchito pano kwa selo lopatsidwa ndi kwa ana aakazi agawanika; Mkuyu 5a,b, onani Njira Zowonjezera ndi Njira). Mapu athu a kuchuluka kwa kukula kwa maselo a SAM amagwirizana ndi zomwe zachitika kale38,39, ndi kukula kochepa kumalire ndi kukula kwakukulu kwa maluwa omwe akukula (mkuyu 5a). Principal component analysis (PCA) inasonyeza kuti ntchito yowonetsera GA inali yogwirizana kwambiri ndi kukula kwa maselo (Chithunzi 5c). Tidawonetsanso kuti ma axx akuluakulu amitundu yosiyanasiyana, kuphatikiza kulowetsa kwa GA ndi kukula kwamphamvu, anali orthogonal kumayendedwe omwe amatsimikiziridwa ndi mafotokozedwe apamwamba a CLV3, kutsimikizira kuchotsedwa kwa ma cell kuchokera pakatikati pa SAM pakuwunika kotsalira. Kusanthula kogwirizana kwa Spearman kunatsimikizira zotsatira za PCA (Chithunzi 5d), kusonyeza kuti zizindikiro zapamwamba za GA mu IPR sizinapangitse kukula kwa maselo. Komabe, kusanthula kwamalumikizidwe kunawonetsa kulumikizana pang'ono kwabwino pakati pa ntchito yowonetsa GA ndi kukula kwa anisotropy (Chithunzi 5c, d), kutanthauza kuti kuwonetsa kwapamwamba kwa GA mu IPR kumakhudza momwe ma cell amakulira komanso mwina malo a ndege yogawa ma cell.
a, b Mapu otentha a kukula kwapamwamba (a) ndi kukula kwa anisotropy (b) mu SAM pafupifupi zomera zisanu ndi ziwiri zodziimira (zogwiritsidwa ntchito ngati ma proxies a mphamvu ndi njira ya kukula kwa maselo, motsatira). c PCA kusanthula kunaphatikizapo zotsatirazi: chizindikiro cha GA, kukula kwapamwamba, kukula kwa anisotropy, ndi mawu a CLV3. Gawo la PCA 1 linali logwirizana kwambiri ndi kukula kwapamwamba komanso kulumikizidwa bwino ndi chizindikiro cha GA. PCA chigawo cha 2 chinali chogwirizana kwambiri ndi kukula kwa anisotropy komanso kugwirizana molakwika ndi mawu a CLV3. Maperesenti amayimira kusiyanasiyana komwe kumafotokozedwa ndi gawo lililonse. d Kusanthula kwamalumikizidwe a Spearman pakati pa chizindikiro cha GA, kukula kwapamwamba, ndi kukula kwa anisotropy pamtunda wa minofu kupatula CZ. Nambala yomwe ili kumanja ndi mtengo wa Spearman rho pakati pa mitundu iwiri. Nyenyezi zimawonetsa zochitika zomwe kulumikizana / kusagwirizana kuli kofunikira kwambiri. e 3D mawonedwe a Col-0 SAM L1 maselo ndi confocal microscopy. Makoma atsopano omwe amapangidwa mu SAM (koma osati primordium) pa 10 h amapangidwa molingana ndi milingo yawo. Chojambula chamtundu chikuwonetsedwa kumunsi kumanja. Tsambali likuwonetsa chithunzi chofananira cha 3D pa 0 h. Kuyesera kunabwerezedwa kawiri ndi zotsatira zofanana. f Magawo a bokosi amawonetsa kuchuluka kwa magawo a cell mu IPR ndi omwe si a IPR Col-0 SAM (n = 10 zomera zodziyimira pawokha). Mzere wapakati umasonyeza wapakatikati, ndipo malire a bokosi amasonyeza 25th ndi 75th percentiles. Whiskers amawonetsa zochepa komanso zotsika mtengo zomwe zimatsimikiziridwa ndi pulogalamu ya R. Makhalidwe a P adapezedwa ndi mayeso a Welch okhala ndi michira iwiri. g, h Chithunzi chojambula chosonyeza (g) momwe mungayezere ngodya ya khoma la selo latsopano (magenta) potsata njira yozungulira kuchokera pakati pa SAM (mzere wa madontho oyera) (makona aang'ono okha, mwachitsanzo, 0-90 °, amaganiziridwa), ndi (h) mayendedwe ozungulira / ozungulira ndi ma radial mkati mwa meristem. i Ma frequency histograms a cell division plane orientation kudutsa SAM (dark blue), IPR (medium blue), and non-IPR (light blue), motsatana. Makhalidwe a P adapezedwa ndi mayeso amichira awiri a Kolmogorov-Smirnov. Kuyesera kunabwerezedwa kawiri ndi zotsatira zofanana. j Mafupipafupi histograms of cell division plane orientation of the IPR around P3 (light green), P4 (medium green), ndi P5 (dark green), motsatana. Makhalidwe a P adapezedwa ndi mayeso amichira awiri a Kolmogorov-Smirnov. Kuyesera kunabwerezedwa kawiri ndi zotsatira zofanana.
Choncho, tinafufuzanso mgwirizano pakati pa zizindikiro za GA ndi ntchito zogawanitsa ma cell pozindikira makoma a cell omwe angopangidwa kumene panthawi yoyesa (mkuyu 5e). Njirayi inatithandiza kuyeza pafupipafupi komanso momwe ma cell amagawanika. Chodabwitsa n'chakuti, tapeza kuti mafupipafupi a magawo a maselo mu IPR ndi ena onse a SAM (osati IPR, Fig. 5f) anali ofanana, kusonyeza kuti kusiyana kwa zizindikiro za GA pakati pa IPR ndi maselo omwe si a IPR sikumakhudza kwambiri magawano a maselo. Izi, komanso kulumikizana kwabwino pakati pa GA signing ndi kukula kwa anisotropy, zidatipangitsa kulingalira ngati ntchito yowonetsa ma GA ingakhudze momwe ndege yogawira ma cell ikuyendera. Tinayesa kutsata kwa khoma la selo latsopano ngati ngodya yowopsya yokhudzana ndi radial axis yomwe imagwirizanitsa pakati pa meristem ndi pakati pa khoma la selo latsopano (mkuyu 5e-i) ndipo tinawona chizolowezi chodziwikiratu chakuti maselo amagawanika pamakona pafupi ndi 90 ° pokhudzana ndi ma radial axis, ndi maulendo apamwamba kwambiri omwe amawonedwa pa 70-80 ° 66 ° 22% (70-80-62%) ndi 70-80 ° 62% (2%). (Mkuyu 5e, i), zogwirizana ndi magawano a selo mu circumferential / transverse direction (Fig. 5h). Kuti tiwone momwe ma GA amaperekera kugawikana kwa ma cell, tidasanthula magawo a magawo a cell mu IPR ndi omwe si a IPR padera (mkuyu 5i). Tinawona kuti kugawanika kwa magawo a magawo m'maselo a IPR kumasiyana ndi omwe si a IPR kapena m'maselo a SAM yonse, ndi maselo a IPR akuwonetsa gawo lapamwamba la magawo a lateral / ozungulira maselo, mwachitsanzo, 70-80 ° ndi 80-90 ° (33.86% ndi 30.71%, motsatira, chiwerengero cha 5). Choncho, zomwe taziwona zinawonetsa mgwirizano pakati pa zizindikiro za GA zapamwamba ndi kayendedwe ka ndege kagawidwe ka selo pafupi ndi njira yozungulira, yofanana ndi kugwirizana pakati pa ntchito yowonetsera GA ndi kukula kwa anisotropy (Mkuyu 5c, d). Kuti tipitirize kukhazikitsa malo osungiramo malo a mgwirizanowu, tinayesa kayendetsedwe ka ndege zogawanika m'maselo a IPR ozungulira primordium kuyambira ku P3, popeza ntchito yapamwamba kwambiri ya GA yodziwika bwino inapezeka m'dera lino kuyambira P4 (Mkuyu 4). Magawo a magawo a IPR ozungulira P3 ndi P4 sanawonetse kusiyana kwakukulu, ngakhale kuti kuwonjezeka kwafupipafupi kwa ma cell a lateral kunawonedwa mu IPR kuzungulira P4 (mkuyu 5j). Komabe, m'maselo a IPR ozungulira P5, kusiyana kwa kayendetsedwe ka ndege yogawanitsa maselo kunakhala kofunika kwambiri, ndi kuwonjezeka kwakukulu kwafupipafupi kugawanika kwa maselo (mkuyu 5j). Pamodzi, zotsatirazi zikuwonetsa kuti kuwonetsa kwa GA kumatha kuwongolera momwe ma cell agawika mu SAM akuyendera, zomwe zimagwirizana ndi malipoti am'mbuyomu40,41 omwe kuwonetsa kwapamwamba kwa GA kungayambitse kutsata kwa magawo a cell mu IPR.
Zimanenedweratu kuti maselo mu IPR sadzaphatikizidwa mu primordia koma m'malo mwa internodes2,42,43. Kuzungulira kwa magawo a maselo mu IPR kungapangitse kuti pakhale mizere yofananira ya ma cell a epidermal mu internodes. Zomwe taziwona pamwambapa zikuwonetsa kuti kusaina kwa GA kumakhala ndi gawo pakuchita izi pakuwongolera momwe ma cell amagawika.
Kutayika kwa ntchito kwa majini angapo a DELLA kumabweretsa kuyankha kwa GA, ndipo della mutants angagwiritsidwe ntchito kuyesa lingaliro ili44. Tidasanthula koyamba mawonekedwe amitundu isanu ya DELLA mu SAM. Kuphatikizika kwa transcriptal kwa GUS line45 kunavumbula kuti GAI, RGA, RGL1, ndi RGL2 (mpaka pang'ono) adawonetsedwa mu SAM (Supplementary Fig. 11a-d). Mu situ hybridization zinasonyeza kuti Gai mRNA amasonkhana makamaka primordia ndi kukula maluwa (Supplementary mkuyu. 11e). RGL1 ndi RGL3 mRNA zinadziwika pamtundu wonse wa SAM ndi maluwa akale, pamene RGL2 mRNA inali yochuluka kwambiri m'dera la malire (Supplementary Fig. 11f-h). Kujambula kwa confocal kwa pRGL3 :: RGL3-GFP SAM inatsimikizira mawu omwe amawonedwa ndi in situ hybridization ndipo amasonyeza kuti mapuloteni a RGL3 amasonkhana pakati pa SAM (Supplementary Fig. 11i). Pogwiritsa ntchito pRGA :: GFP-RGA mzere, tinapezanso kuti mapuloteni a RGA amasonkhana mu SAM, koma kuchuluka kwake kumachepa kumalire kuyambira P4 (Supplementary Fig. 11j). Mwachidziwitso, machitidwe a RGL3 ndi RGA akugwirizana ndi zochitika zapamwamba za GA zowonetsera mu IPR, monga zazindikirika ndi qmRGA (Mkuyu 4). Kuphatikiza apo, izi zikuwonetsa kuti ma DELLA onse amawonetsedwa mu SAM ndikuti mawu awo onse pamodzi amapitilira SAM yonse.
Kenako tidasanthula magawo ogawa ma cell mumitundu yakuthengo ya SAM (Ler, control) ndi gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Mkuyu 6a, b). Chochititsa chidwi n'chakuti, tinawona kusintha kwakukulu pakugawidwa kwa ma cell division angle frequency mu della global mutant SAM poyerekeza ndi mtundu wakutchire (mkuyu 6c). Kusintha kumeneku kwa della global mutant kunali chifukwa cha kuwonjezeka kwafupipafupi kwa 80-90 ° angles (34.71% vs. 24.55%) ndipo, pang'onopang'ono, 70-80 ° angles (23.78% vs. 20.18%), mwachitsanzo, mogwirizana ndi magawo a cell transverse (mkuyu 6c). Mafupipafupi a magawo osasinthika (0-60 °) analinso otsika mu della global mutant (Mkuyu 6c). Mafupipafupi a magawano a cell transverse anawonjezeka kwambiri mu SAM ya della global mutant (Mkuyu 6b). Mafupipafupi a magawo osinthika a cell mu IPR analinso apamwamba mu della global mutant poyerekeza ndi mtundu wakuthengo (mkuyu 6d). Kunja kwa dera la IPR, mtundu wakuthengo unali ndi gawo lofananirako la ma ngodya zogawikana ma cell, pomwe della global mutant ankakonda magawano tangential monga IPR (mkuyu 6e). Tidawerengeranso momwe magawo amagawidwira ma cell mu SAM ya ga2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, ndi ga2ox6-2), GA-inactive mutant maziko momwe GA imadziunjikira. Mogwirizana ndi kuwonjezeka kwa ma GA, SAM ya quintuple ga2ox mutant inflorescence inali yaikulu kuposa ya Col-0 (Supplementary Fig. 12a, b), ndipo poyerekeza ndi Col-0, quintuple ga2ox SAM inasonyeza kugawidwa kosiyana kwambiri kwa ma angles ogawa ma cell, ndi ma angles omwe akuwonjezeka kuchokera ku 050 °, kugawanitsa kwa 9 Chithunzi 12a-c). Chifukwa chake, tikuwonetsa kuti kutsegulira koyambitsa kwa GA siginecha ndi GA kudzikundikira kumapangitsa magawano a cell mu IPR ndi ma SAM ena onse.
a, b mawonekedwe a 3D a L1 wosanjikiza wa PI-stained Ler (a) ndi global della mutant (b) SAM pogwiritsa ntchito confocal microscopy. Makoma atsopano a cell opangidwa mu SAM (koma osati oyambira) pa nthawi ya 10-h amawonetsedwa ndikujambulidwa molingana ndi milingo yawo. Tsambali likuwonetsa SAM pa 0 h. Chojambula chamtundu chikuwonetsedwa pansi pakona yakumanja. Mivi yomwe ili mu (b) ikuloza ku chitsanzo cha mafayilo a cell omwe ali mu global della mutant. Kuyesera kunabwerezedwa kawiri ndi zotsatira zofanana. ce kufanizitsa kagawidwe kafupipafupi kagawo kagawo ka ndege mu SAM (d), IPR (e), ndi non-IPR (f) pakati pa Ler ndi global della. Makhalidwe a P adapezedwa pogwiritsa ntchito mayeso amichira awiri a Kolmogorov-Smirnov. f, g Kuwonera kwa 3D kwa zithunzi za confocal za PI-stained SAM ya Col-0 (i) ndi pCUC2:: gai-1-VENUS (j) zomera za transgenic. Mapanelo (a, b) amawonetsa makoma atsopano a cell (koma osati primordia) opangidwa mu SAM mkati mwa 10 h. Kuyesera kunabwerezedwa kawiri ndi zotsatira zofanana. h-j Kuyerekeza kugawidwa kwafupipafupi kwa maulendo a ndege ogawa ma cell omwe ali mu SAM (h), IPR (i) ndi non-IPR (j) pakati pa Col-0 ndi pCUC2 :: gai-1-VENUS zomera. Makhalidwe a P adapezedwa pogwiritsa ntchito mayeso amichira awiri a Kolmogorov-Smirnov.
Kenako tidayesa zotsatira zoletsa kusaina kwa GA makamaka mu IPR. Kuti izi zitheke, tidagwiritsa ntchito wotsatsa wa cotyledon cup 2 (CUC2) kuwonetsa puloteni yoyipa ya gai-1 yolumikizidwa ku VENUS (mu pCUC2 :: gai-1-VENUS mzere). Mu SAM yamtundu wakutchire, wolimbikitsa CUC2 amayendetsa kufotokoza kwa IPRs ambiri mu SAM, kuphatikizapo maselo a malire, kuchokera ku P4 kupita mtsogolo, ndipo kufotokozera kofananako kunawonedwa mu pCUC2 :: gai-1-VENUS zomera (onani pansipa). Kugawidwa kwa ma angles ogawa ma cell kudutsa SAM kapena IPR ya pCUC2 :: zomera za gai-1-VENUS sizinali zosiyana kwambiri ndi zamtundu wa nyama zakutchire, ngakhale mosayembekezereka tinapeza kuti maselo opanda IPR mu zomera izi amagawidwa pamtunda wapamwamba wa 80-90 ° (Mkuyu 6f-j).
Zanenedwa kuti mayendedwe agawidwe a cell amadalira geometry ya SAM, makamaka kupsinjika kwamphamvu komwe kumapangidwa ndi minyewa yopindika46. Choncho tinafunsa ngati mawonekedwe a SAM adasinthidwa mu della global mutant ndi pCUC2:: gai-1-VENUS zomera. Monga tanena kale12, kukula kwa della global mutant SAM kunali kokulirapo kuposa mtundu wakutchire (Supplementary Fig. 13a, b, d). In situ hybridization ya CLV3 ndi STM RNA inatsimikizira kukula kwa meristem mu della mutants ndikuwonetsanso kufalikira kwapambuyo kwa tsinde cell niche (Supplementary Fig. 13e, f, h, i). Komabe, kupindika kwa SAM kunali kofanana ndi ma genotypes (Supplementary Fig. 13k, m, n, p). Tinawona kuwonjezeka kofanana kwa kukula kwa gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant popanda kusintha kwa kupindika poyerekeza ndi mtundu wakutchire (Supplementary Fig. 13c, d, g, j, l, o, p). Mafupipafupi a magawano a maselo adakhudzidwanso mu della quadruple mutant, koma pang'onopang'ono kusiyana ndi della monolithic mutant (Supplementary Fig. 12d-f). Zotsatira za mlingowu, pamodzi ndi kusowa kwa zotsatira pa kupindika, zimasonyeza kuti ntchito yotsalira ya RGL3 mu Della quadruple mutant imalepheretsa kusintha kwa magawano a selo chifukwa cha kutayika kwa ntchito ya DELLA komanso kuti kusintha kwa maselo amtundu wa lateral kumachitika chifukwa cha kusintha kwa ntchito yowonetsera GA kusiyana ndi kusintha kwa SAM geometry. Monga tafotokozera pamwambapa, wolimbikitsa CUC2 amayendetsa mawu a IPR mu SAM kuyambira pa P4 (Supplementary Fig. 14a, b), ndipo mosiyana, pCUC2 :: gai-1-VENUS SAM inali ndi kukula kochepa koma kupindika kwapamwamba (Supplementary Fig. 14c-h). Kusintha kumeneku kwa pCUC2 :: gai-1-VENUS SAM morphology kungapangitse kugawidwa kosiyana kwa zovuta zamakina poyerekeza ndi mtundu wakutchire, momwe kupanikizika kwakukulu kumayambira pamtunda waufupi kuchokera ku SAM center47. Kapenanso, kusintha kwa pCUC2 :: gai-1-VENUS SAM morphology kungabwere chifukwa cha kusintha kwazinthu zamakina am'deralo zomwe zimayambitsidwa ndi transgene expression48. Pazochitika zonsezi, izi zitha kuchepetsa pang'ono zotsatira za kusintha kwa kusaina kwa GA powonjezera mwayi woti ma cell agawikane mozungulira / mozungulira, kufotokozera zomwe taziwona.
Kuphatikizidwa pamodzi, deta yathu imatsimikizira kuti kusaina kwapamwamba kwa GA kumagwira ntchito yoyang'ana kutsogolo kwa ndege yogawa ma cell mu IPR. Amawonetsanso kuti kupindika kwa meristem kumathandizanso momwe ma cell amagawika ndege amayendera mu IPR.
Kuzungulira kwa ndege yogawanitsa mu IPR, chifukwa cha ntchito yayikulu yowonetsera GA, ikuwonetsa kuti GA imakonzekereratu fayilo ya ma radial cell mu epidermis mkati mwa SAM kuti ifotokozere gulu la ma cell omwe pambuyo pake adzapezeka mu epidermal internode. Zoonadi, mafayilo amtundu woterewa ankawoneka kawirikawiri muzithunzi za SAM za della global mutants (Mkuyu 6b). Choncho, kuti tipitirize kufufuza ntchito yachitukuko cha mawonekedwe a malo a chizindikiro cha GA mu SAM, tinagwiritsa ntchito kujambula kwa nthawi kuti tifufuze gulu la malo a maselo mu IPR mumtundu wamtundu (Ler ndi Col-0), della global mutants, ndi pCUC2 :: gai-1-VENUS zomera zowonongeka.
Tinapeza kuti qmRGA inasonyeza kuti ntchito yowonetsera GA mu IPR inakula kuchokera ku P1 / P2 ndipo inafika pa P4, ndipo chitsanzochi chinakhalabe chokhazikika pakapita nthawi (Mkuyu 4a-f ndi Supplementary Fig. 8c-f, k). Kuti tifufuze gulu la malo a maselo mu IPR ndi kuwonjezeka kwa chizindikiro cha GA, tinalemba ma cell a Ler IPR pamwamba ndi kumbali za P4 molingana ndi chitukuko chawo chomwe chinafufuzidwa 34 h pambuyo poyang'ana koyamba, mwachitsanzo, nthawi zoposa ziwiri za plastid, zomwe zimatilola kuti tizitsatira maselo a IPR panthawi ya chitukuko choyambirira kuchokera ku P1 / P2 mpaka P4. Tinagwiritsa ntchito mitundu itatu yosiyana: yachikasu kwa maselo omwe anaphatikizidwa mu primordium pafupi ndi P4, obiriwira kwa omwe anali mu IPR, ndi ofiirira kwa omwe adagwira nawo ntchito zonse ziwiri (mkuyu 7a-c). Pa t0 (0 h), zigawo za 1-2 za maselo a IPR zinkawoneka kutsogolo kwa P4 (mkuyu 7a). Monga momwe zimayembekezeredwa, pamene maselowa adagawanika, adachita izi makamaka kudzera mu ndege yogawanitsa (Mkuyu 7a-c). Zotsatira zofananazo zinapezedwa pogwiritsa ntchito Col-0 SAM (yoyang'ana pa P3, yomwe malire ake amapindika mofanana ndi P4 ku Ler), ngakhale mu genotype iyi khola lopangidwa pamalire amaluwa linabisala maselo a IPR mofulumira (Mkuyu 7g-i). Chifukwa chake, magawano a ma cell a IPR amakonzekeratu ma cell kukhala mizere yozungulira, monga mu internodes. Kapangidwe ka mizere yozungulira komanso kuyika kwa ma cell a IPR pakati pa ziwalo zotsatizana zikuwonetsa kuti ma cellwa ndi oyambira apakati.
Apa, tidapanga ratiometric GA signing biosensor, qmRGA, yomwe imalola kuwerengera kuchuluka kwa ntchito zosayina za GA zochokera kuphatikizika kwa GA ndi GA receptor ndikuchepetsa kusokonezedwa ndi njira zama siginecha zamkati, potero zimapereka chidziwitso pa ntchito ya GA pama cell. Kuti izi zitheke, tidapanga puloteni yosinthidwa ya DELLA, mRGA, yomwe yasiya kutha kumangiriza ma DELLA omwe amalumikizana nawo koma amakhalabe okhudzidwa ndi GA-induced proteinolysis. qmRGA imayankha pazosintha zonse zakunja komanso kwanthawi yayitali m'magawo a GA, ndipo mawonekedwe ake owoneka bwino amathandizira kuwunika kwakusintha kwapadziko lonse muzochita zosayina za GA panthawi yachitukuko. qmRGA ndi chida chosinthika kwambiri chifukwa chimatha kusinthidwa kukhala minofu yosiyanasiyana posintha wolimbikitsa omwe amagwiritsidwa ntchito pofotokozera (ngati kuli kofunikira), ndikupatsidwa mawonekedwe otetezedwa a GA signing pathway ndi PFYRE motif kudutsa angiosperms, zitha kusamutsidwa ku mitundu ina22. Mogwirizana ndi izi, kusinthika kofananako kwa puloteni ya mpunga SLR1 DELLA (HYY497AAA) kunawonetsedwanso kuti kulepheretsa kukula kwa SLR1 pomwe kumachepetsa pang'ono kuwonongeka kwake kwa GA, kofanana ndi mRGA23. Makamaka, kafukufuku waposachedwa ku Arabidopsis adawonetsa kuti kusintha kumodzi kwa amino acid mu PFYRE domain (S474L) kudasinthiratu zolemba za RGA popanda kusokoneza kuthekera kwake kolumikizana ndi omwe amagwirizana nawo50. Ngakhale kusinthaku kuli pafupi kwambiri ndi ma 3 amino acid omwe amapezeka mu mRGA, kafukufuku wathu akuwonetsa kuti masinthidwe awiriwa amasintha mawonekedwe a DELLA. Ngakhale ambiri omwe amalumikizana nawo amalumikizana ndi madera a LHR1 ndi SAW a DELLA26,51, ma amino acid ena osungidwa mu domain la PFYRE atha kuthandiza kukhazikika kuyanjana uku.
Kukula kwa Internode ndichinthu chofunikira kwambiri pakumanga mbewu ndikuwongolera zokolola. qmRGA idawulula zochitika zapamwamba za GA mu ma cell a progenitor a IPR internode. Pophatikiza kuyerekezera kwachulukidwe ndi ma genetics, tidawonetsa kuti ma signing a GA amawongolera ma circular / transverse cell division plan mu SAM epidermis, kupanga gulu logawa ma cell lomwe limafunikira kuti chitukuko cha internode chikule. Owongolera angapo amagawo amtundu wa ma cell adadziwika panthawi ya chitukuko52,53. Ntchito yathu imapereka chitsanzo chodziwikiratu cha momwe ntchito yosayinira ya GA imawongolera ma parameter awa. DELLA imatha kuyanjana ndi ma protein complexes41, kotero kuwonetsa kwa GA kumatha kuwongolera kayendetsedwe ka ndege kagawidwe ka cell pokhudza mwachindunji ma cortical microtubule orientation40,41,54,55. Tinawonetsa mosayembekezereka kuti mu SAM, kugwirizanitsa kwa ntchito yowonetsera GA yapamwamba sikunali kutalika kwa selo kapena kugawanika, koma kukula kwa anisotropy, komwe kumagwirizana ndi zotsatira zachindunji za GA pa kayendetsedwe ka magawo a maselo mu IPR. Komabe, sitingasankhe kuti izi zitha kukhala zosalunjika, mwachitsanzo, kulumikizidwa ndi GA-induced cell wall softening56. Kusintha kwa makoma a cell kumapangitsa kuti makina azipanikizika57,58, zomwe zingakhudzenso kayendedwe ka ndege yogawanitsa ma cell pokhudza kayendedwe ka cortical microtubules39,46,59. Zotsatira zophatikizana za kupsinjika kwa makina opangidwa ndi GA komanso kuwongolera kwachindunji kwa microtubule orientation ndi GA zitha kuphatikizidwa pakupanga mawonekedwe apadera a magawo a cell mu IPR kuti afotokozere internodes, ndipo maphunziro ena amafunikira kuyesa lingaliro ili. Mofananamo, kafukufuku wam'mbuyomu adawonetsa kufunikira kwa mapuloteni ogwirizana ndi DELLA TCP14 ndi 15 poyang'anira internode formation60,61 ndipo zinthuzi zikhoza kugwirizanitsa zochita za GA pamodzi ndi BREVIPEDICELLUS (BP) ndi PENNYWISE (PNY), zomwe zimayang'anira chitukuko cha internode ndipo zasonyezedwa kuti zimakhudza 2 GA2. Popeza kuti DELLAs amalumikizana ndi brassinosteroid, ethylene, jasmonic acid, ndi abscisic acid (ABA) njira zowonetsera63,64 komanso kuti mahomoniwa amatha kukhudza microtubule orientation65, zotsatira za GA pamagulu a magawano a maselo amathanso kuthandizidwa ndi mahomoni ena.
Maphunziro oyambirira a cytological amasonyeza kuti madera onse amkati ndi akunja a Arabidopsis SAM amafunikira pa chitukuko cha internode2,42. Mfundo yakuti GA imayang'anira mwachangu kugawanika kwa maselo mu minofu yamkati12 imathandizira ntchito ziwiri za GA poyang'anira kukula kwa meristem ndi internode mu SAM. Mchitidwe wa kugawanika kwa maselo otsogolera kumayendetsedwanso mwamphamvu mkati mwa minofu ya SAM, ndipo lamulo ili ndilofunika kuti tsinde ikule52. Zingakhale zosangalatsa kuwona ngati GA imagwiranso ntchito pakuwongolera ndege yogawa ma cell mu bungwe la SAM lamkati, potero kugwirizanitsa mafotokozedwe ndi chitukuko cha ma internodes mkati mwa SAM.
Zomera zidabzalidwa m'nthaka kapena 1x Murashige-Skoog (MS) sing'anga (Duchefa) yowonjezeredwa ndi 1% sucrose ndi 1% agar (Sigma) pansi pamikhalidwe yokhazikika (16 h kuwala, 22 °C), kupatula kuyesa kwa hypocotyl ndi kukula kwa mizu komwe mbande zidakulitsidwa pa mbale zoyima pansi pa kuwala kosalekeza 22 ° C ndi 22 ° C. Pazoyesa za nitrate, mbewu zidabzalidwa pakatikati pa MS medium (bioWORLD plant medium) wophatikizidwa ndi nitrate yokwanira (0 kapena 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose ndi 1% A-agar (Sigma) pansi pamikhalidwe yamasiku atali.
GID1a cDNA yolowetsedwa mu pDONR221 idaphatikizidwanso ndi pDONR P4-P1R-pUBQ10 ndi pDONR P2R-P3-mCherry mu pB7m34GW kupanga pUBQ10::GID1a-mCherry. IDD2 DNA yolowetsedwa mu pDONR221 inaphatikizidwanso mu pB7RWG266 kuti ipange p35S:IDD2-RFP. Kuti apange pGID1b::2xmTQ2-GID1b, chidutswa cha 3.9 kb kumtunda kwa dera la GID1b ndi 4.7 kb fragment yomwe ili ndi GID1b cDNA (1.3 kb) ndi terminator (3.4 kb) idakulitsidwa koyamba pogwiritsa ntchito zoyambira mu Supplementary ppl-PdR4 (Thermo Fisher Scientific) ndi pDONR P2R-P3 (Thermo Fisher Scientific), motsatana, ndipo potsirizira pake anaphatikizananso ndi pDONR221 2xmTQ268 mu pGreen 012567 chandamale vekitala pogwiritsa ntchito Gateway cloning. Kupanga pCUC2 ::LSSmOrange, kutsatizana kwa CUC2 (3229 bp kumtunda kwa ATG) kutsatiridwa ndi kutsatizana kotsatizana kwa Stokes-shifted mOrange (LSSmOrange)69 yokhala ndi chizindikiro cha nyukiliya cha N7 ndi terminator ya NOS transcriptional zidasonkhanitsidwa munjira yolowera panjira yoyang'anira 3. (Invitrogen). Chomera cha binary vekitala chinayambitsidwa mu Agrobacterium tumefaciens strain GV3101 ndikulowetsedwa mu masamba a Nicotiana benthamiana ndi njira ya Agrobacterium infiltration ndi mu Arabidopsis thaliana Col-0 ndi njira ya dip yamaluwa, motsatana. pUBQ10 :: qmRGA pUBQ10 :: GID1a-mCherry ndi pCLV3 :: mCherry-NLS qmRGA anasiyanitsidwa ndi F3 ndi F1 ana a mitanda motsatira.
RNA in situ hybridization inkachitika pafupifupi 1 cm kutalika kwa nsonga72, yomwe idasonkhanitsidwa ndikukhazikika mu FAA solution (3.7% formaldehyde, 5% acetic acid, 50% ethanol) yokhazikika mpaka 4 °C. Pambuyo pa 2 × 15 min vacuum mankhwala, chokhazikikacho chinasinthidwa ndipo zitsanzo zinakulungidwa usiku wonse. GID1a, GID1b, GID1c, GAI, RGL1, RGL2, ndi RGL3 cDNAs ndi zofufuza za antisense ku 3'-UTRs zawo zinapangidwa pogwiritsa ntchito zoyambira zomwe zikuwonetsedwa mu Supplementary Table 3 monga momwe Rosier et al.73 anafotokozera. Ma probe olembedwa ndi Digoxigenin anali osatetezedwa pogwiritsa ntchito ma antibodies a digoxigenin (3000-fold dilution; Roche, nambala ya catalog: 11 093 274 910), ndipo magawo anali odetsedwa ndi 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 250-litrolution) /nitrolution 200-fold dilution) yankho.
Nthawi yotumiza: Feb-10-2025