Kukura kwemuti we apical meristem (SAM) kwakakosha pakuvaka hunde.gibberellins(GAs) dzinoita basa guru mukubatanidza kukura kwezvirimwa, asi basa radzo muSAM harisati ranzwisiswa zvakanaka. Pano, takagadzira ratiometric biosensor yeGA signaling nekugadzira protein yeDELLA kuti idzvinyirire basa rayo rekutonga muGA transcriptional response uku tichichengetedza kuora kwayo pakuzivikanwa kweGA. Tinoratidza kuti iyi biosensor inonyora zvakarurama shanduko mumazinga eGA uye kunzwa kwemasero panguva yekugadzirwa. Takashandisa iyi biosensor kugadzira chirongwa cheGA signaling muSAM. Tinoratidza kuti masaini eGA akakwira aripo zvakanyanya mumasero ari pakati penhengo dzemuviri, ayo ari ekutanga kumasero einternode. Tichishandisa nzira dzekuwana nekudzikira kwebasa, tinoratidza zvakare kuti GA inotonga kurongeka kwesero rekupatsanura, ichigadzira sangano remaseru ecanonical ema internodes, nokudaro ichisimudzira kutsanangurwa kwema internode muSAM.
Muti we apical meristem (SAM), uri pamusoro pemuti, une niche yemasero emuti ane basa rinogadzira nhengo dzeparutivi uye ma stem nodes nenzira ye modular uye iterative muhupenyu hwese hwemuti. Chimwe nechimwe chezvikamu izvi zvinodzokorora, kana kuti ma plant nodes, chinosanganisira internodes uye lateral organs pama nodes, uye axillary meristems mu axils yemashizha1. Kukura nekurongeka kwema plant nodes kunochinja panguva yekukura. MuArabidopsis, kukura kwe internodal kunodziviswa panguva yekukura, uye axillary meristems inoramba yakarara mu axils yemashizha e rosette. Munguva yekuchinja kuenda kuchikamu chemaruva, SAM inova inflorescence meristem, ichigadzira internodes refu uye axillary buds, matavi mu axils emashizha e cauline, uye gare gare, maruva asina mashizha2. Kunyangwe tafambira mberi zvakanyanya mukunzwisisa mashandiro anodzora kutanga kwemashizha, maruva, uye matavi, zvishoma zvinozivikanwa nezvekuti internodes dzinomuka sei.
Kunzwisisa kugoverwa kweGA munzvimbo nenguva kunobatsira kunzwisisa zviri nani mashandiro emahormone aya mumatishu akasiyana uye pamatanho akasiyana ekukura. Kuona kuora kwekubatanidzwa kweRGA-GFP kunoratidzwa pasi pechiito chepromoter wayo kunopa ruzivo rwakakosha pamusoro pekutonga kwemazinga ese eGA mumidzi15,16. Zvisinei, kutaura kweRGA kunosiyana mumatishu17 uye kunodzorwa neGA18. Saka, kusiyana kwekutaura kwepromoter yeRGA kunogona kukonzera patani yefluorescence inoonekwa neRGA-GFP uye nekudaro nzira iyi haisi yehuwandu. Munguva pfupi yapfuura, GA19,20 ine bioactive fluorescein (Fl) yakaratidza kuunganidzwa kweGA mumidzi endocortex uye kudzora mazinga ayo emasero neGA transport. Munguva pfupi yapfuura, GA FRET sensor nlsGPS1 yakaratidza kuti mazinga eGA ane hukama nekureba kwemasero mumidzi, filaments, uye hypocotyls dzakakura rima21. Zvisinei, sezvataona, kuwanda kweGA hakusi iko kwega parameter inodzora GA signaling activity, sezvo zvichienderana nemaitiro akaomarara ekuona. Pano, tichivakira pakunzwisisa kwedu nzira dzeDELLA neGA signaling, tinoshuma nezvekuvandudzwa uye hunhu hwe biosensor inocherwa nekuora kwe ratiometric yeGA signaling. Kuti tigadzire iyi quantitative biosensor, takashandisa mutant GA-sensitive RGA yakabatanidzwa ku fluorescent protein uye inoratidzwa munzvimbo dzese, pamwe neGA-insensitive fluorescent protein. Tinoratidza kuti mutant RGA protein fusions hadzipindire endogenous GA signaling kana ikaratidzwa munzvimbo dzese, uye kuti iyi biosensor inogona kuyera masaini anoitwa kubva kuGA input uye GA signal processing ne sensor apparatus ine high spatiotemporal resolution. Takashandisa iyi biosensor kuti tiongorore kugoverwa kwe spatiotemporal kweGA signaling activity uye kuyera kuti GA inotonga sei maitiro emasero muSAM epidermis. Tinoratidza kuti GA inotonga kurongeka kwe division plane yeSAM cells iri pakati pe organ primordia, nokudaro ichitsanangura canonical cellular organization ye internode.
Pakupedzisira, takabvunza kana qmRGA yaigona kutaura shanduko mumazinga eGA emukati memuviri tichishandisa ma hypocotyls ari kukura. Takamboratidza kuti nitrate inokurudzira kukura nekuwedzera kugadzirwa kweGA uye, zvakare, kuora kweDELLA34. Saka, takaona kuti hurefu hwehypocotyl mumbeu dzepUBQ10::qmRGA dzakarimwa pasi pehuwandu hwakawanda hwenitrate (10 mM NO3−) hwakareba zvakanyanya kupfuura huri mumbeu dzakarimwa pasi pemamiriro ekushomeka kwenitrate (Supplementary Fig. 6a). Zvichienderana nemhinduro yekukura, zviratidzo zveGA zvaive zvakakwirira muhypocotyls dzembeu dzakarimwa pasi pemamiriro e10 mM NO3− pane mumbeu dzakarimwa pasina nitrate (Supplementary Fig. 6b, c). Saka, qmRGA inobvumirawo kutarisa shanduko muGA signaling inokonzerwa nekuchinja kwega kwega muhuwandu hweGA.
Kuti tinzwisise kana basa reGA signaling rinoonekwa neqmRGA richienderana nehuwandu hweGA uye maonero eGA, sezvaitarisirwa zvichibva pakugadzirwa kwesensor, takaongorora kuratidzwa kwemareceptor matatu eGID1 mumatishu ezvirimwa neanobereka. Mumbeu, GID1-GUS reporter line yakaratidza kuti GID1a na c zvairatidzwa zvakanyanya mu cotyledons (Mufananidzo 3a–c). Pamusoro pezvo, mareceptor ese matatu akaratidzwa mumashizha, lateral root primordia, midzi mirefu (kunze kwe root cap yeGID1b), uye vascular system (Mufananidzo 3a–c). Mu inflorescence SAM, takaona masaini eGUS chete eGID1b na1c (Mufananidzo Wekuwedzera 7a–c). In situ hybridization yakasimbisa matauriro aya uye yakaratidza zvakare kuti GID1c yakaratidzwa zvakaenzana pamazinga akaderera muSAM, nepo GID1b yakaratidza kuratidzwa kwepamusoro pamucheto weSAM (Mufananidzo Wekuwedzera 7d–l). Kusanganiswa kwepGID1b::2xmTQ2-GID1b kwakaratidzawo huwandu hwakatarwa hwekutaura kweGID1b, kubva pakuderera kana kusavapo pakati peSAM kusvika pakukwira kwekutaura pamiganhu yenhengo (Supplementary Fig. 7m). Saka, maGID1 receptors haana kupararira zvakaenzana mukati metishu. Mukuyedza kwakatevera, takaonawo kuti kuwedzera kweGID1 (pUBQ10::GID1a-mCherry) kwakawedzera kunzwa kweqmRGA muhypocotyls kune external GA application (Fig. 3d, e). Kusiyana neizvi, fluorescence yakayerwa neqd17mRGA muhypocotyl yakanga isinganzwe kurapwa kweGA3 (Fig. 3f, g). Pakuyedzwa kwese, mbesa dzakarapwa nehuwandu hwakawanda hweGA (100 μM GA3) kuti dziongorore maitiro ekukurumidza kwesensor, uko kugona kubatana neGID1 receptor kwakawedzerwa kana kurasikirwa. Pamwe chete, mhinduro idzi dzinosimbisa kuti qmRGA biosensor inoshanda pamwe chete seGA neGA sensor, uye dzinoratidza kuti differential expression yeGID1 receptor inogona kuchinja zvakanyanya emissivity yesensor.
Kusvika pari zvino, kugoverwa kwezviratidzo zveGA muSAM hakusati kwanyatsojeka. Saka, takashandisa qmRGA-expressing plants uye pCLV3::mCherry-NLS stem cell reporter35 kuverenga mamepu ehuwandu hwepamusoro-soro hwechiitiko cheGA signaling, tichitarisa pane L1 layer (epidermis; Fig. 4a, b, ona Nzira uye Supplementary Methods), sezvo L1 ichiita basa guru mukudzora kukura kweSAM36. Pano, pCLV3::mCherry-NLS expression yakapa nzvimbo yakatarwa yekuongorora kugoverwa kwenzvimbo kwechiitiko cheGA signaling37. Kunyangwe GA ichionekwa seyakakosha pakukura kwenhengo dzeparutivi4, takaona kuti zviratidzo zveGA zvaive zvishoma mu floral primordium (P) kutanga kubva padanho reP3 (Fig. 4a, b), nepo P1 neP2 primordiums diki dzaive nekushanda kwakaringana kwakafanana nekwemunzvimbo yepakati (Fig. 4a, b). Basa repamusoro reGA signaling rakaonekwa pamiganhu yenhengo, kutanga paP1/P2 (kumativi emuganhu) uye kusvika pakakwirira paP4, pamwe chete nemumasero ese enharaunda iri pakati pe primordia (Mufananidzo 4a, b uye Mufananidzo Wekuwedzera 8a, b). Basa iri repamusoro reGA signaling rakaonekwa kwete mu epidermis chete asiwo muL2 neL3 layers yepamusoro (Mufananidzo Wekuwedzera 8b). Maitiro ezviratidzo zveGA akaonekwa muSAM achishandisa qmRGA akarambawo asina kuchinja nekufamba kwenguva (Mufananidzo Wekuwedzera 8c–f, k). Kunyangwe kuvakwa kweqd17mRGA kwakaderedzwa zvishoma nezvishoma muSAM yezvirimwa zveT3 kubva pamitsetse mishanu yakazvimirira yatakatsanangura zvakadzama, takakwanisa kuongorora mapatani e fluorescence akawanikwa ne pRPS5a::VENUS-2A-TagBFP construct (Mufananidzo Wekuwedzera 8g–j, l). Mumutsetse uyu wekudzora, shanduko diki chete muhuwandu hwe fluorescence dzakaonekwa muSAM, asi muSAM centre takaona kudzikira kwakajeka uye kusingatarisirwi kweVENUS kwakabatana neTagBFP. Izvi zvinosimbisa kuti patani yezviratidzo yakaonekwa neqmRGA inoratidza kudzikira kweGA-dependent kwemRGA-VENUS, asi zvinoratidzawo kuti qmRGA inogona kuwedzeredza chiitiko cheGA signaling mu meristem centre. Mukupfupisa, zvatakawana zvinoratidza patani yezviratidzo yeGA inoratidza zvakanyanya kupararira kweprimordia. Kupararira uku kwenzvimbo yepakati peprimordial (IPR) kunokonzerwa nekugadzwa zvishoma nezvishoma kwechiitiko chezviratidzo cheGA chakakwira pakati pe primordium iri kukura nenharaunda yepakati, nepo panguva imwe chete chiitiko chezviratidzo cheGA mu primordium chichiderera (Mufananidzo 4c, d).
Kugoverwa kweGID1b neGID1c receptors (ona pamusoro apa) kunoratidza kuti kusiyana kwekutaura kweGA receptors kunobatsira kugadzira patani yeGA signaling activity muSAM. Takashamisika kana kuunganidzwa kweGA kwakasiyana kungave kuripo. Kuti tiongorore mukana uyu, takashandisa nlsGPS1 GA FRET sensor21. Kuwedzera kwe activation frequency kwakaonekwa muSAM yenlsGPS1 yakarapwa ne10 μM GA4+7 kwemaminitsi zana (Supplementary Fig. 9a–e), zvichiratidza kuti nlsGPS1 inopindura kuchinja kwehuwandu hweGA muSAM, sezvainoita mumidzi21. Spatial distribution ye nlsGPS1 activation frequency yakaratidza GA levels yakaderera muzvikamu zvekunze zveSAM, asi yakaratidza kuti yakakwidziridzwa pakati nepamiganhu yeSAM (Fig. 4e neSupplementary Fig. 9a,c). Izvi zvinoratidza kuti GA yakagoverwawo muSAM ine spatial pattern yakafanana neyakaratidzwa neqmRGA. Senzira yekuwedzera, takabatawo SAM ne fluorescent GA (GA3-, GA4-, GA7-Fl) kana Fl chete senzira yekudzora isina kunaka. Chiratidzo cheFl chakaparadzirwa muSAM yese, kusanganisira nzvimbo yepakati neprimordium, kunyangwe paine simba rakaderera (Mufananidzo 4j neMuenzaniso wekuwedzera 10d). Kusiyana neizvi, ese matatu GA-Fl akaungana zvakananga mukati memiganhu yeprimordium uye kusvika kumadhigirii akasiyana mune imwe nzvimbo yeIPR, neGA7-Fl ichiungana munzvimbo yakakura muIPR (Mufananidzo 4k neMuenzaniso wekuwedzera 10a,b). Kuyera kwesimba refluorescence kwakaratidza kuti chiyero cheIPR kune non-IPR intensity chaive chakakwira muSAM yakarapwa neGA-Fl zvichienzaniswa neSAM yakarapwa neFl (Mufananidzo 4l neMuenzaniso wekuwedzera 10c). Pamwe chete, mhedzisiro iyi inoratidza kuti GA iripo pahuwandu hwakanyanya mumasero eIPR ari padyo nemuganhu wenhengo. Izvi zvinoratidza kuti maitiro eSAM GA signaling activity anobva mukusiyana kwezviratidzo zveGA receptors uye kuunganidzwa kweGA mumasero eIPR pedyo nemiganhu yenhengo. Saka, ongororo yedu yakaratidza maitiro asingatarisirwi eGA signaling, nemabasa mashoma pakati nepakati peSAM uye mabasa akakwira muIPR munzvimbo yekunze.
Kuti tinzwisise basa rebasa reGA signaling activity muSAM, takaongorora hukama huripo pakati peGA signaling activity, cell expansion, uye cell division tichishandisa real-time time-lapse imaging yeSAM qmRGA pCLV3::mCherry-NLS. Tichifunga nezvebasa reGA mukudzora kukura, hukama hwakanaka ne parameters dzekuwedzera masero hwaitarisirwa. Nokudaro, takatanga taenzanisa mamepu eGA signaling activity nemamepu ekukura kwemasero pamusoro pemasero (seproxy yesimba rekuwedzera masero kune imwe sero uye mwanasikana masero pakupatsanurwa) uye nemamepu ekukura anisotropy, ayo anoyera kutungamira kwekuwedzera kwemasero (anoshandiswawo pano kune imwe sero uye mwanasikana masero pakupatsanurwa; Mufananidzo 5a,b, ona Nzira neNzira Dzekuwedzera). Mamepu edu eSAM cell surface growth rate anoenderana nezvakaonekwa kare38,39, nekukura kushoma pamuganhu uye kukura kwakanyanya mumaruva ari kukura (Mufananidzo 5a). Principal component analysis (PCA) yakaratidza kuti basa reGA signaling raive rakabatana zvisina kunaka nekukura kwemasero pamusoro pemasero (Mufananidzo 5c). Takaratidzawo kuti ma axes makuru ekusiyana, kusanganisira GA signaling input uye kukura kwakasimba, aive akaenderana negwara rakatarwa ne high CLV3 expression, zvichisimbisa kubviswa kwemasero kubva pakati peSAM mukuongorora kwasara. Spearman correlation analysis yakasimbisa PCA results (Mufananidzo 5d), zvichiratidza kuti high GA signals muIPR haina kukonzera kuwedzera kwemasero. Zvisinei, correlation analysis yakaratidza hukama hwakanaka pakati peGA signaling activity uye growth anisotropy (Mufananidzo 5c, d), zvichiratidza kuti high GA signaling muIPR inokanganisa gwara rekukura kwemasero uye pamwe nzvimbo ye cell division plane.
a, b Mepu dzekupisa dzekukura kwepakati pepamusoro (a) uye kukura kwemasero (b) muSAM dzakaita avhareji yemiti minomwe yakazvimirira (inoshandiswa senzira dzekusimbisa simba nekutungamira kwekukura kwemasero, zvichiteerana). c Kuongororwa kwePCA kwakabatanidza zvinotevera: chiratidzo cheGA, kukura kwemasero, kukura kwemasero, uye CLV3 expression. Chikamu chePCA 1 chakanyanya kuwirirana nekusimba kwekukura kwemasero uye chakabatana zvakanaka nechiratidzo cheGA. Chikamu chePCA 2 chakanyanya kuwirirana nekusimba kwekukura kwemasero uye chakabatana zvisina kunaka nekutaura kweCLV3. Maperesenti anomiririra mutsauko unotsanangurwa nechikamu chimwe nechimwe. d Kuongororwa kwehukama hweSpearman pakati pechiratidzo cheGA, kukura kwemasero, uye kukura kwemasero pachiyero chetishu kusasanganisa CZ. Nhamba iri kurudyi ndiyo Spearman rho value pakati pezvinhu zviviri. Asterisks inoratidza zviitiko apo hukama/kusawirirana kwakakosha zvikuru. e 3D visualization yeCol-0 SAM L1 cells ne confocal microscopy. Madziro matsva emasero akaumbwa muSAM (asi kwete primordium) pamaawa gumi anopendwa zvichienderana ne angle values dzawo. Ruvara rwebhawa rwunoratidzwa mukona yepazasi kurudyi. Chinyorwa chinoratidza mufananidzo we3D unoenderana nenguva ye0 awa. Kuedza kwakadzokororwa kaviri nemigumisiro yakafanana. f Mabhokisi ebhokisi anoratidza mwero wekupatsanurwa kwemasero muIPR uye asiri eIPR Col-0 SAM (n = 10 zvirimwa zvakazvimiririra). Mutsetse wepakati unoratidza midhiya, uye miganhu yebhokisi inoratidza 25th ne75th percentiles. Whiskers dzinoratidza hunhu hushoma uye hwepamusoro hwakatarwa neR software. P hunhu hwakawanikwa neWelch's two-tailed t-test. g, h Schematic diagram inoratidza (g) maitiro ekuyera kona yemadziro matsva esero (magenta) maererano negwara reradial kubva pakati peSAM (mutsetse muchena une madota) (hunhu hweacute angle chete, kureva, 0–90°, ndihwo hunofungwa), uye (h) circumferential/lateral uye radial directions mukati me meristem. i Frequency histograms ye cell division plane orientation kuburikidza neSAM (rima bhuruu), IPR (pakati bhuruu), uye kwete-IPR (light blue), zvichiteerana. MaP values akawanikwa kuburikidza nebvunzo yeKolmogorov-Smirnov ine miswe miviri. Kuedza uku kwakadzokororwa kaviri nemhedzisiro yakafanana. j Ma frequency histograms ekutarisa kwenzvimbo yekupatsanurwa kwemasero eIPR yakatenderedza P3 (girini rakapfava), P4 (girini repakati), uye P5 (girini rakasviba), zvichiteerana. MaP values akawanikwa kuburikidza nebvunzo yeKolmogorov-Smirnov ine miswe miviri. Kuedza uku kwakadzokororwa kaviri nemhedzisiro yakafanana.
Saka, takazoongorora hukama huripo pakati peGA signaling uye masero ekupatsanurwa nekuona madziro matsva emasero panguva yekuongorora (Mufananidzo 5e). Maitiro aya akatibvumira kuyera frequency uye gwara rekupatsanurwa kwemasero. Zvinoshamisa kuti takaona kuti frequency yekupatsanurwa kwemasero muIPR nedzimwe SAM (kwete IPR, Mufananidzo 5f) yakanga yakafanana, zvichiratidza kuti musiyano muGA signaling pakati peIPR nemasero asiri eIPR haukanganisi zvakanyanya kupatsanurwa kwemasero. Izvi, pamwe nehukama hwakanaka pakati peGA signaling uye kukura kweanisotropy, zvakatikurudzira kufunga kana GA signaling activity inogona kukanganisa kutungamira kwe cell division plane. Takayera kurongeka kwemadziro matsva esero sekona yakapinza kana tichienzanisa ne radial axis inobatanidza meristem centre nepakati pemadziro matsva esero (Mufananidzo 5e-i) uye takaona kuti masero anopatsanurana pamakona ari pedyo ne90° kana tichienzanisa ne radial axis, nema frequency akanyanya kuonekwa pa 70–80° (23.28%) uye 80–90° (22.62%) (Mufananidzo 5e,i), zvichienderana nekupatsanurwa kwemasero munzira ye circumferential/transverse (Mufananidzo 5h). Kuti tiongorore mupiro weGA signaling kune maitiro aya ekupatsanurwa kwemasero, takaongorora ma parameter ekupatsanurwa kwemasero mu IPR ne non-IPR zvakasiyana (Mufananidzo 5i). Takaona kuti kugoverwa kwekona yekuparadzanisa mumasero eIPR kwakasiyana nekwemumasero asiri eIPR kana mumasero ari muSAM yese, nemasero eIPR achiratidza chikamu chikuru chekupatsanurwa kwemasero edivi/denderedzwa, kureva, 70–80° uye 80–90° (33.86% uye 30.71%, zvichiteerana, zviyero zvinoenderana) (Mufananidzo 5i). Saka, zvatakaona zvakaratidza hukama pakati pechiratidzo cheGA chakakwira uye kurongeka kwendege yekupatsanurwa kwemasero pedyo negwara redenderedzwa, zvakafanana nehukama huripo pakati pechiitiko cheGA chekusaina uye kukura kweanisotropy (Mufananidzo 5c, d). Kuti tiwedzere kusimbisa kuchengetedzwa kwenzvimbo kwehukama uhwu, takayera kurongeka kwendege yekuparadzanisa mumasero eIPR akakomberedza primordium tichitanga kubva paP3, sezvo chiitiko chepamusoro cheGA chekusaina chakaonekwa munzvimbo iyi chichitanga kubva paP4 (Mufananidzo 4). Makona ekuparadzanisa eIPR akakomberedza P3 neP4 haana kuratidza mutsauko wakakosha, kunyangwe kuwanda kwekupatsanurwa kwemasero edivi kwakaonekwa muIPR akakomberedza P4 (Mufananidzo 5j). Zvisinei, mumasero eIPR akakomberedza P5, musiyano uripo pakurongwa kwechikamu chemasero wakava wakakosha, nekuwedzera kwakanyanya kwekugoverwa kwemasero anochinjika (Mufananidzo 5j). Pamwe chete, mhedzisiro iyi inoratidza kuti GA signaling inogona kudzora kurongwa kwekugoverwa kwemasero muSAM, izvo zvinoenderana nemishumo yapfuura40,41 yekuti GA signaling yakakwira inogona kukonzera kurongeka kwechikamu chemasero muIPR.
Zvinofungidzirwa kuti masero ari muIPR haazobatanidzwi mu primordia asi kuti asanganiswe mu internodes2,42,43. Kurongeka kwezvikamu zvemasero muIPR kunogona kukonzera kurongeka kwakajairika kwemitsara yakareba yakafanana yemasero e epidermal mu internodes. Zvatakaona pamusoro apa zvinoratidza kuti GA signaling inogona kunge iine basa mukuita uku nekudzora kutungamira kwekupatsanurwa kwemasero.
Kurasikirwa nebasa kwemajini akati wandei eDELLA kunoguma nemhinduro yeGA, uye della mutants inogona kushandiswa kuyedza iyi fungidziro44. Takatanga taongorora maitiro ekutaura kwemajini mashanu eDELLA muSAM. Kubatanidzwa kweGUS line45 kwakaratidza kuti GAI, RGA, RGL1, uye RGL2 (zvishoma) zvakaratidzwa muSAM (Supplementary Fig. 11a–d). In situ hybridization yakaratidza zvakare kuti GAI mRNA inoungana zvakanyanya mumaruva ekutanga neari kukura (Supplementary Fig. 11e). RGL1 neRGL3 mRNA zvakaonekwa mukati meSAM canopy nemumaruva ekare, nepo RGL2 mRNA yaive yakawanda munharaunda yemuganhu (Supplementary Fig. 11f–h). Confocal imaging ye pRGL3::RGL3-GFP SAM yakasimbisa kutaura kwakaonekwa ne in situ hybridization uye yakaratidza kuti RGL3 protein inoungana pakati peSAM (Supplementary Fig. 11i). Tichishandisa mutsetse we pRGA::GFP-RGA, takaonawo kuti mapuroteni eRGA anoungana muSAM, asi kuwanda kwawo kunoderera pamuganhu kutanga kubva paP4 (Mufananidzo Wekuwedzera 11j). Zvinonyanya kukosha kuti maitiro ekutaura eRGL3 neRGA anoenderana nebasa repamusoro reGA signaling muIPR, sezvakaonekwa neqmRGA (Mufananidzo 4). Uyezve, data iri rinoratidza kuti maDELLA ese anoratidzwa muSAM uye kuti kutaura kwawo pamwe chete kunobatanidza SAM yese.
Takazoongorora maparamita ekupatsanurana kwemasero muSAM yemhando yeWild-type (Ler, control) uye gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Mufananidzo 6a, b). Zvinofadza kuti, takaona shanduko yakakosha mukupararira kwemafrequencies eangle ekupatsanurana kwemasero muSAM yeDella global mutant zvichienzaniswa neWild type (Mufananidzo 6c). Kuchinja uku muDella global mutant kwakakonzerwa nekuwedzera kwefrequency ye80–90° angles (34.71% vs. 24.55%) uye, kusvika padanho diki, 70–80° angles (23.78% vs. 20.18%), kureva kuti, zvinoenderana nekupatsanurana kwemasero (Mufananidzo 6c). Kuwanda kwekupatsanurana kusiri kwetransverse (0–60°) kwaive kwakadererawo muDella global mutant (Mufananidzo 6c). Kuwanda kwekupatsanurwa kwemasero e transverse kwakawedzera zvakanyanya muSAM ye della global mutant (Mufananidzo 6b). Kuwanda kwekupatsanurwa kwemasero e transverse muIPR kwaive kwakanyanyawo mu della global mutant zvichienzaniswa ne wild type (Mufananidzo 6d). Kunze kwenzvimbo yeIPR, wild type yaive nekupararira kwakafanana kwema angle ekupatsanurwa kwemasero, nepo della global mutant yaifarira kupatsanurwa kwe tangential seIPR (Mufananidzo 6e). Isu takayerawo kurongeka kwekupatsanurwa kwemasero muSAM ye ga2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, uye ga2ox6-2), iyo GA-inactive mutant background umo GA inoungana. Zvichienderana nekuwedzera kwemazinga eGA, SAM ye quintuple ga2ox mutant inflorescence yaive yakakura kupfuura yeCol-0 (Supplementary Fig. 12a, b), uye zvichienzaniswa neCol-0, quintuple ga2ox SAM yakaratidza kugoverwa kwakasiyana kwemakona ekuparadzanisa maseru, ne frequency ye angle ichiwedzera kubva pa50° kusvika 90°, kureva zvakare ichifarira kupatsanurwa kwe tangential (Supplementary Fig. 12a–c). Saka, tinoratidza kuti constitutive activation ye GA signaling uye GA accumulation inokonzera kupatsanurwa kwemasero e lateral muIPR uye mune zvimwe zvese zveSAM.
a, b Kuonekwa kwe3D kweL1 layer yePI-stained Ler (a) uye global della mutant (b) SAM uchishandisa confocal microscopy. Madziro matsva emasero akaumbwa muSAM (asi kwete primordium) kwenguva ye10-awa anoratidzwa uye anopendwa zvichienderana ne angle values dzawo. Chinyorwa chinoratidza SAM pa 0 h. Ruvara runoratidzwa mukona yezasi yekurudyi. Museve uri mu (b) unonongedzera kumuenzaniso wemafaira emasero akarongwa mu global della mutant. Kuedza kwakadzokororwa kaviri nemhedzisiro yakafanana. ce kuenzanisa kwekugoverwa kwefrequency kwe cell division plane orientations muSAM yese (d), IPR (e), uye isiri-IPR (f) pakati peLer ne global della. P values dzakawanikwa uchishandisa bvunzo yeKolmogorov-Smirnov ine miswe miviri. f, g Kuonekwa kwe3D kwemifananidzo ye confocal yePI-stained SAM yeCol-0 (i) uye pCUC2::gai-1-VENUS (j) transgenic plants. Mapaneru (a, b) anoratidza madziro matsva emasero (asi kwete primordia) akaumbwa muSAM mukati memaawa gumi. Kuedza uku kwakadzokororwa kaviri nemigumisiro yakafanana. h–j Kuenzanisa kwekugoverwa kwefrequency kwenzvimbo dzekupatsanurwa kwemasero dziri muSAM yese (h), IPR (i) uye isiri-IPR (j) pakati peCol-0 ne pCUC2::gai-1-VENUS plants. P values dzakawanikwa uchishandisa bvunzo yeKolmogorov–Smirnov ine miswe miviri.
Takazoedzazve mhedzisiro yekudzivisa GA signaling kunyanya muIPR. Kuti tiite izvi, takashandisa cotyledon cup 2 (CUC2) promoter kukurudzira kuratidzwa kweproteni ine gai-1 ine simba guru yakabatana neVENUS (mumutsara we pCUC2::gai-1-VENUS). Mu wild-type SAM, CUC2 promoter inosimudzira kuratidzwa kweIPR dzakawanda muSAM, kusanganisira border cells, kubva paP4 zvichienda mberi, uye kutaura kwakafanana kwakaonekwa mu pCUC2::gai-1-VENUS plants (ona pazasi). Kugoverwa kwemakona ekupatsanurwa kwemasero muSAM kana IPR ye pCUC2::gai-1-VENUS plants kwakanga kusina kusiyana zvakanyanya nekwe wild type, kunyangwe zvisingatarisirwi takawana kuti masero asina IPR mumiti iyi akapatsanurwa pa frequency yakakwira ye80–90° (Mufananidzo 6f–j).
Zvakataurwa kuti kutungamira kwekupatsanurwa kwemasero kunoenderana nejometri yeSAM, kunyanya kushushikana kwekumanikidzwa kunokonzerwa nekukombama kwetishu46. Saka takabvunza kana chimiro cheSAM chakachinjwa mumiti yedella global mutant uye pCUC2::gai-1-VENUS. Sezvakambotaurwa kare12, saizi yedella global mutant SAM yaive yakakura kupfuura yerudzi rwemusango (Supplementary Fig. 13a, b, d). In situ hybridization yeCLV3 neSTM RNA yakasimbisa kuwedzera kwemeristem mu della mutants uye yakaratidza kuwedzera kwelateral kwe stem cell niche (Supplementary Fig. 13e, f, h, i). Zvisinei, SAM curvature yaive yakafanana mu genotypes dzese dziri mbiri (Supplementary Fig. 13k, m, n, p). Takaona kuwedzera kwakafanana muhukuru mu gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant pasina shanduko mukukombama zvichienzaniswa ne wild type (Supplementary Fig. 13c, d, g, j, l, o, p). Kuwanda kwekupatsanurwa kwemasero kwakakanganiswawo mu della quadruple mutant, asi zvishoma pane mu della monolithic mutant (Supplementary Fig. 12d–f). Iyi dosage effect, pamwe chete nekushaikwa kwemhedzisiro pakukombama, inoratidza kuti residual RGL3 activity mu Della quadruple mutant inoderedza shanduko mukupatsanurwa kwemasero kunokonzerwa nekurasikirwa nebasa reDELLA uye kuti shanduko mukupatsanurwa kwemasero eparutivi dzinoitika nekuda kwekuchinja kwebasa reGA signaling pane shanduko mu SAM geometry. Sezvakatsanangurwa pamusoro apa, CUC2 promoter inofambisa IPR expression muSAM kutanga paP4 (Supplementary Fig. 14a, b), uye zvakasiyana, pCUC2::gai-1-VENUS SAM yaive nehukuru hwakaderera asi hwakakwira (Supplementary Fig. 14c–h). Kuchinja uku mu pCUC2::gai-1-VENUS SAM morphology kunogona kukonzera kugoverwa kwakasiyana kwe mechanical stress zvichienzaniswa ne wild type, umo high circumferential stress dzinotanga padaro pfupi kubva pakati peSAM47. Neimwe nzira, shanduko mu pCUC2::gai-1-VENUS SAM morphology inogona kukonzerwa nekuchinja mu regional mechanical properties inokonzerwa ne transgene expression48. Muzviitiko zvese izvi, izvi zvinogona kuderedza zvishoma mhedzisiro yekuchinja mu GA signaling nekuwedzera mukana wekuti masero achapatsanurana mu circumferential/transverse orientation, tichitsanangura zvatakaona.
Kana zvabatanidzwa, data redu rinosimbisa kuti GA signaling yakakwira ine basa guru mukutungamira kwechikamu chechikamu chemasero muIPR. Zvinoratidzawo kuti meristem curvature inokanganisawo kuenderera kwechikamu chechikamu chemasero muIPR.
Kutarisa kwechikamu chedivision plane muIPR, nekuda kwekushanda kweGA signaling kwakawanda, kunoratidza kuti GA inoronga faira re radial cell mu epidermis mukati meSAM kuti itsanangure sangano remasero richazowanikwa gare gare mu epidermal internode. Zvechokwadi, mafaira emasero akadaro aiwanzoonekwa mumifananidzo yeSAM ye della global mutants (Mufananidzo 6b). Saka, kuti tiongorore zvakanyanya basa rekukura kwe spatial pattern yeGA signaling muSAM, takashandisa time-lapse imaging kuti tiongorore spatial organization yemasero muIPR mu wild-type (Ler naCol-0), della global mutants, uye pCUC2::gai-1-VENUS transgenic plants.
Takaona kuti qmRGA yakaratidza kuti basa reGA signaling muIPR rakawedzera kubva paP1/P2 uye rakasvika paP4, uye maitiro aya akaramba aripo nekufamba kwenguva (Mufananidzo 4a–f uye Supplementary Fig. 8c–f, k). Kuti tiongorore kurongeka kwemasero muIPR nekuwedzera kwechiratidzo cheGA, takaisa mazita kumasero eLer IPR pamusoro uye kumativi eP4 zvichienderana nekukura kwawo kwakaongororwa maawa makumi matatu nemana mushure mekutarisa kwekutanga, kureva, kanopfuura kaviri plastid, zvichitibvumira kutevera masero eIPR panguva yekukura kweprimordium kubva paP1/P2 kusvika paP4. Takashandisa mavara matatu akasiyana: yero kune masero akabatanidzwa muprimordium pedyo neP4, girini kune avo vaiva muIPR, uye pepuru kune avo vakapinda mumaitiro ese ari maviri (Mufananidzo 7a–c). Pa t0 (0 h), 1–2 layers yemasero eIPR aionekwa pamberi peP4 (Mufananidzo 7a). Sezvaitarisirwa, apo masero aya akapatsanurwa, akaita izvi zvakanyanya kuburikidza ne transverse division plane (Mifananidzo 7a–c). Zvakafanana zvakawanikwa uchishandisa Col-0 SAM (yakatarisana neP3, iyo muganhu wayo unopetwa zvakafanana neP4 muLer), kunyangwe mu genotype iyi danga rakaumbwa pamuganhu wemaruva rakavanza masero eIPR nekukurumidza (Mifananidzo 7g–i). Saka, maitiro ekupatsanurwa kwemasero eIPR anoronga masero kuita mitsara ye radial, sezvakaita muma internodes. Kurongeka kwemitsara ye radial uye nzvimbo yemasero eIPR pakati penhengo dzinotevedzana zvinoratidza kuti masero aya ndiwo madzitateguru e internodal.
Pano, takagadzira ratiometric GA signaling biosensor, qmRGA, inobvumira quantitative mapping yeGA signaling activity inobva mu combined GA neGA receptor concentrations uku tichideredza kukanganiswa ne endogenous signaling pathways, nokudaro tichipa ruzivo pamusoro peGA function pa cellular level. Kuti tiite izvi, takagadzira modified DELLA protein, mRGA, iyo yakarasikirwa nekugona kusunga DELLA interaction partners asi inoramba ichinzwa GA-induced proteolysis. qmRGA inopindura kune exogenous uye endogenous changes muGA levels, uye dynamic sensing properties yayo inobvumira kuongororwa kwe spatiotemporal changes muGA signaling activity panguva yekukura. qmRGA chishandiso chinochinjika zvikuru sezvo chinogona kugadziriswa kune akasiyana matishu nekushandura promoter inoshandiswa pakutaura kwayo (kana zvichidikanwa), uye tichifunga nezvekuchengetedzwa kweGA signaling pathway uye PFYRE motif kuburikidza ne angiosperms, inogona kutamisirwa kune mamwe marudzi22. Zvichienderana neizvi, shanduko yakafanana mupuroteni yeSLR1 DELLA yemupunga (HYY497AAA) yakaratidzwawo kuti inodzvinyirira basa reSLR1 rekuderedza kukura uku ichideredza zvishoma kuora kwayo kweGA, kwakafanana nemRGA23. Zvinoshamisa kuti zvidzidzo zvechangobva kuitwa muArabidopsis zvakaratidza kuti shanduko imwe chete yeamino acid muPFYRE domain (S474L) yakachinja basa rekunyora reRGA pasina kukanganisa kugona kwayo kutaurirana nevanobatana netranscription factor50. Kunyangwe shanduko iyi iri pedyo zvikuru ne3 amino acid substitutions dziri mumRGA, zvidzidzo zvedu zvinoratidza kuti shanduko mbiri idzi dzinochinja hunhu hwakasiyana hweDELLA. Kunyangwe vazhinji vanobatana netranscription factor vachibatana neLHR1 neSAW domains dzeDELLA26,51, mamwe maamino acids akachengetedzwa muPFYRE domain anogona kubatsira kudzikamisa kusangana uku.
Kukura kwepakati pemanode chinhu chakakosha mukuvaka kwemiti uye kuvandudza goho. qmRGA yakaratidza basa repamusoro reGA signaling muIPR internode progenitor cells. Nekubatanidza quantitative imaging uye genetics, takaratidza kuti GA signaling patterns dzinoisa denderedzwa/transverse cell division planes muSAM epidermis, zvichigadzira sangano re cell division rinodiwa pakukura kwe internode. Vatongi vakati wandei ve cell division plane orientation vakaonekwa panguva yekukura52,53. Basa redu rinopa muenzaniso wakajeka wekuti GA signaling activity inotonga sei iyi cellular parameter. DELLA inogona kushanda ne prefolding protein complexes41, saka GA signaling inogona kudzora cell division plane orientation nekuchinja zvakananga cortical microtubule orientation40,41,54,55. Takaratidza tisingatarisiri kuti muSAM, correlate ye higher GA signaling activity yakanga isiri cell elongation kana division, asi kungori growth anisotropy, iyo inoenderana nemhedzisiro yakananga yeGA panzira yekuparadzaniswa kwemasero muIPR. Zvisinei, hatigone kubvisa kuti mhedzisiro iyi inogonawo kunge isina kunanga, semuenzaniso inodzorwa neGA-induced cell wall softening56. Kuchinja kwehunhu hwemadziro esero kunokonzera kushushikana kwemuchina57,58, izvo zvinogonawo kukanganisa kurongeka kwenzvimbo yekupatsanurana kwesero nekuchinja kurongeka kwe cortical microtubules39,46,59. Migumisiro yakabatana yekushushikana kwemuchina kunokonzerwa neGA uye kutonga kwakananga kweiyo microtubule orientation neGA zvinogona kunge zvichibatanidzwa mukugadzira pateni chaiyo yeiyo cell division orientation muIPR kuti itsanangure internodes, uye zvimwe zvidzidzo zvinodiwa kuti kuyedze pfungwa iyi. Saizvozvowo, zvidzidzo zvekare zvakaratidza kukosha kwemapuroteni anoshanda eDELLA TCP14 na15 mukudzora kuumbwa kwe internode60,61 uye zvinhu izvi zvinogona kubatsira pakuita kweGA pamwe chete neBREVIPEDICELUS (BP) nePENNYWISE (PNY), izvo zvinodzora kukura kwe internode uye zvakaratidzwa kuti zvinokanganisa GA signaling2,62. Zvichienderana nekuti maDELLA anoshanda pamwe chete nebrassinosteroid, ethylene, jasmonic acid, uye abscisic acid (ABA) signaling pathways63,64 uye kuti mahormone aya anogona kukanganisa microtubule orientation65, mhedzisiro yeGA pa orientation ye cell division inogonawo kutungamirirwa nemamwe mahormone.
Zvidzidzo zvekutanga zve cytological zvakaratidza kuti nzvimbo dzemukati nedzekunze dzeArabidopsis SAM dzinodiwa pakukura kwema internode2,42. Chokwadi chekuti GA inodzora kupatsanurwa kwema cell mumatishu emukati12 chinotsigira basa reGA mukugadzirisa meristem nehukuru hwe internode muSAM. Maitiro ekupatsanurwa kwema cell anotungamirirwawo zvakasimba mumatishu emukati meSAM, uye mutemo uyu wakakosha pakukura kwema stem52. Zvichave zvinonakidza kuongorora kana GA iinewo basa mukutungamira plane yekupatsanurwa kwema cell muSAM yemukati, nokudaro ichibatanidza specification nekukura kwema internodes mukati meSAM.
Zvirimwa zvakarimwa muvhu kana kamwe chete Murashige-Skoog (MS) medium (Duchefa) yakawedzerwa 1% sucrose uye 1% agar (Sigma) pasi pemamiriro ezvinhu akajairwa (16 h chiedza, 22 °C), kunze kwekuyedza kwehypocotyl nekukura kwemidzi umo mbesa dzakarimwa pamaplate akamira pasi pechiedza chisingachinji uye 22 °C. Pakuyedza nitrate, zvirimwa zvakarimwa pane modified MS medium (bioWORLD plant medium) yakawedzerwa nitrate yakakwana (0 kana 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose uye 1% A-agar (Sigma) pasi pemamiriro ezuva refu.
GID1a cDNA yakaiswa mu pDONR221 yakasanganiswazve ne pDONR P4-P1R-pUBQ10 uye pDONR P2R-P3-mCherry mu pB7m34GW kuti igadzire pBQ10::GID1a-mCherry. IDD2 DNA yakaiswa mu pDONR221 yakasanganiswazve mu pB7RWG266 kuti igadzire p35S:IDD2-RFP. Kuti vagadzire pGID1b::2xmTQ2-GID1b, chidimbu che3.9 kb chiri kumusoro kwenzvimbo yeGID1b coding uye chidimbu che4.7 kb chine GID1b cDNA (1.3 kb) uye terminator (3.4 kb) zvakatanga kuwedzeredzwa uchishandisa ma primers ari muSupplementary Table 3 ndokuzoiswa mu pDONR P4-P1R (Thermo Fisher Scientific) uye pDONR P2R-P3 (Thermo Fisher Scientific), zvichiteerana, uye pakupedzisira zvakasanganiswa zvakare ne pDONR221 2xmTQ268 mu pGreen 012567 target vector uchishandisa Gateway cloning. Kuti vagadzire pCUC2::LSSmOrange, CUC2 promoter sequence (3229 bp kumusoro kweATG) ichiteverwa ne coding sequence yeStokes-shifted mOrange (LSSmOrange)69 yakakura ine N7 nuclear localization signal uye NOS transcriptional terminator zvakaunganidzwa mu pGreen kanamycin targeting vector uchishandisa Gateway 3-fragment recombination system (Invitrogen). Plant binary vector yakaunzwa muAgrobacterium tumefaciens strain GV3101 uye yakaunzwa muNicotiana benthamiana leaves neAgrobacterium infiltration method uye muArabidopsis thaliana Col-0 ne floral dip method, zvichiteerana. pBQ10::qmRGA pBQ10::GID1a-mCherry uye pCLV3::mCherry-NLS qmRGA zvakaparadzaniswa kubva kuF3 neF1 progenies dzemichinjiko yacho, zvichiteerana.
Kusanganiswa kweRNA mu situ kwakaitwa pamipendero yemashizha inenge 1 cm yakareba72, iyo yakaunganidzwa ndokugadziriswa pakarepo muFAA solution (3.7% formaldehyde, 5% acetic acid, 50% ethanol) yakatonhodzwa kusvika 4 °C. Mushure mekurapwa kwevacuum kwemaminitsi 2 × 15, fixative yakachinjwa uye samples dzakaiswa mu incubation husiku hwese. GID1a, GID1b, GID1c, GAI, RGL1, RGL2, uye RGL3 cDNAs uye antisense probes kune 3′-UTRs dzavo zvakagadzirwa uchishandisa ma primers anoratidzwa muSupplementary Table 3 sezvakatsanangurwa naRosier et al.73. Mapurobhu ane label yeDigoxigenin akaonekwa nemasoja ekudzivirira chirwere achishandisa ma antibodies e digoxigenin (3000-fold dilution; Roche, catalog number: 11 093 274 910), uye zvikamu zvakaiswa 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 250-fold dilution)/nitroblue tetrazolium (NBT, 200-fold dilution).
Nguva yekutumira: Kukadzi-10-2025