Shoot apical meristem (SAM) kukura kwakakosha kune stem architecture. Mahomoni emitigibberellins(GAs) vanoita basa rakakosha mukubatanidza kukura kwezvirimwa, asi basa ravo muSAM rinoramba risinganzwisisike. Pano, takagadzira ratiometric biosensor yeGA kusaina neinjiniya iyo DELLA puroteni kudzvanya basa rayo rakakosha rekutonga mumhinduro yeGA yekunyorwa uku ichichengetedza kuparara kwayo pakuzivikanwa kweGA. Isu tinoratidza kuti iyi degradation-based biosensor inorekodha shanduko mumazinga eGA uye maseru sensing panguva yekusimudzira. Isu takashandisa iyi biosensor kumepu GA kusaina chiitiko muSAM. Isu tinoratidza kuti yakakwira GA masiginecha aripo zvakanyanya mumaseru ari pakati peorgan primordia, ayo anotungamira kune internode maseru. Tichishandisa nzira dzekuwana- uye kurasikirwa-kwe-kushanda, isu tinowedzera kuratidza kuti GA inodzora kutaridzika kwendege yekuparadzanisa masero, kumisikidza canonical cellular sangano re internodes, nekudaro ichisimudzira internode kutsanangurwa muSAM.
Iyo pfuti apical meristem (SAM), iri panzvimbo yepfuti, ine niche yemaseru maseru ane basa rinogadzira nhengo dzeparutivi uye node dzemadzinde mune modular uye iterative maitiro ehupenyu hwechirimwa. Imwe neimwe yeaya mayuniti anodzokorora, kana mapfundo ezvirimwa, anosanganisira internodes uye lateral nhengo dziri panhongonya, uye axillary meristems mushizha axils1. Kukura uye kurongeka kwemanodhi ezvirimwa zvinoshanduka panguva yekukura. MuArabidopsis, kukura kwemukati kunodzvanywa panguva yezvinomera, uye axillary meristems inoramba yakarara muaxils yemashizha erosette. Munguva yekuchinja kuenda kuchikamu chemaruva, iyo SAM inova inflorescence meristem, ichigadzira elongated internodes uye axillary buds, matavi mumaaxils emashizha ecauline, uye gare gare, maruva asina mashizha2. Kunyange zvazvo takaita kufambira mberi kukuru mukunzwisisa nzira dzinodzora kutangwa kwemashizha, maruva, uye mapazi, zvishoma zvinozivikanwa pamusoro pekuti internodes inomuka sei.
Kunzwisisa kugoverwa kwespatiotemporal kweGAs kuchabatsira kunzwisisa zviri nani mashandiro emahomoni aya mumatishu akasiyana uye pamatanho ekukura akasiyana. Kuona kwekuparara kweRGA-GFP fusion inoratidzwa pasi pechiito chekusimudzira kwayo inopa ruzivo rwakakosha pamusoro pekugadzirisa kwehuwandu hweGA mazinga mumidzi15,16. Nekudaro, kutaura kweRGA kunosiyana mumatishu17 uye inodzorwa neGA18. Nokudaro, kutaura kwakasiyana kweRGA kukurudzira kunogona kukonzera fluorescence pattern yakaonekwa neRGA-GFP uye naizvozvo nzira iyi haisi yehuwandu. Munguva pfupi yapfuura, bioactive fluorescein (Fl) -yakanyorwa GA19,20 yakaratidza kuunganidzwa kweGA mumudziyo endocortex uye kudzorwa kwemazinga ayo emasero neGA kutakura. Munguva pfupi yapfuura, iyo GA FRET sensor nlsGPS1 yakaratidza kuti GA mazinga anowirirana nesero elongation mumidzi, filaments, uye yakasviba-yakakura hypocotyls21. Nekudaro, sezvataona, GA yekumisikidza haisiriyo yega parameter inodzora GA kusaina chiitiko, nekuti zvinoenderana neakaomesesa ekunzwa maitiro. Pano, tichivaka pamusoro pekunzwisisa kwedu kweDELLA neGA nzira dzekusaina, tinoshuma kuvandudzwa uye hunhu hwekudzikisira-based ratiometric biosensor yeGA kusaina. Kugadzira iyi yakawanda biosensor, takashandisa mutant GA-sensitive RGA iyo yakasanganiswa neprotein yefluorescent uye inoratidzirwa pese mumatishu, pamwe neGA-insensitive fluorescent protein. Isu tinoratidza kuti mutant RGA protein fusions haikanganise endogenous GA siginecha kana ichiratidzwa pese pese, uye kuti iyi biosensor inogona kuverengera kusaina chiitiko chinobva kune ese ari maviri GA kupinza uye GA chiratidzo chekugadzirisa nemidziyo yekunzwa ine yakakwirira spatiotemporal resolution. Isu takashandisa iyi biosensor kumepu kugoverwa kwespatiotemporal kweGA kusaina chiitiko uye kuona kuti GA inogadzirisa sei cellular maitiro muSAM epidermis. Isu tinoratidza kuti GA inodzora kutaridzika kweiyo division ndege yeSAM masero ari pakati peorgan primordia, nekudaro ichitsanangura iyo canonical cellular sangano reiyo internode.
Chekupedzisira, isu takabvunza kana qmRGA yaigona kuzivisa shanduko mune endogenous GA mazinga uchishandisa kukura hypocotyls. Isu takamboratidza kuti nitrate inokurudzira kukura nekuwedzera GA synthesis uye, zvakare, DELLA34 kudzikira. Saizvozvowo, takaona kuti urefu hwe hypocotyl mu pUBQ10 :: qmRGA mbesa dzakakura pasi pehuwandu hwe nitrate (10 mM NO3-) yakanga yakareba kudarika iyo mumichero yakakura pasi pemamiriro asina kukwana nitrate (Supplementary Fig. 6a). Zvichienderana nemhinduro yekukura, zviratidzo zveGA zvaive zvakakwirira mu hypocotyls yemadzinde akakura pasi pe 10 mM NO3 - mamiriro ezvinhu pane mbesa dzakakura pasina nitrate (Supplementary Fig. 6b, c). Nekudaro, qmRGA inogonesawo kutarisa kwekuchinja kweGA kusaina kunokonzerwa nekuchinja kwekupedzisira muG.
Kuti tinzwisise kana iyo GA yekusaina chiitiko yakaonekwa neqmRGA inoenderana neGA kusungwa uye GA maonero, sezvaitarisirwa zvichibva pane sensor dhizaini, takaongorora kutaura kweatatu GID1 receptors mune zvinomera uye zvekubereka tishu. Mumichero, GID1-GUS mutori wenhau mutsara wakaratidza kuti GID1a uye c zvakaratidzwa zvakanyanya mu cotyledons (Fig. 3a-c). Mukuwedzera, zvose zvitatu zvakagamuchirwa zvakaratidzwa mumashizha, lateral root primordia, midzi mazano (kunze kwemudzi weGID1b), uye vascular system (Fig. 3a-c). Mune inflorescence SAM, takaona zviratidzo zveGUS chete zveGID1b uye 1c (Supplementary Fig. 7a–c). In situ hybridization yakasimbisa aya maitiro ekutaura uye yakaratidza zvakare kuti GID1c yakaratidzwa zvakafanana pamazinga akaderera muSAM, nepo GID1b yakaratidza kutaura kwepamusoro padivi peSAM (Supplementary Fig. 7d-l). Iyo pGID1b :: 2xmTQ2-GID1b yekushandura fusion yakaratidzawo giredhi renji yeGID1b kutaura, kubva pasi kana pasina kutaura pakati peSAM kusvika kumusoro kutaura kumiganhu yenhengo (Supplementary Fig. 7m). Saka, GID1 receptors haina kugovaniswa zvakafanana mukati uye mukati mematishu. Muzviedzo zvakazotevera, takaonawo kuti kuwedzeredza kweGID1 (pUBQ10 :: GID1a-mCherry) kwakawedzera kunzwisisika kweqmRGA mu hypocotyls kune kunze kweGA application (Fig. 3d, e). Kusiyana neizvi, fluorescence yakayerwa neqd17mRGA mu hypocotyl yakanga isinganzwisisi GA3 kurapwa (Fig. 3f, g). Kune ese maviri assay, mbesa dzakabatwa nehupamhi hwepamusoro hweGA (100 μM GA3) kuti iongorore kukurumidza maitiro eiyo sensor, uko kugona kusunga kune GID1 receptor kwakakwidziridzwa kana kurasika. Pamwe chete, mhedzisiro iyi inosimbisa kuti iyo qmRGA biosensor inoshanda yakasanganiswa basa seGA uye GA sensor, uye inokurudzira kuti mutsauko wekutaura weGID1 receptor unogona zvakanyanya kuenzanisa kubuda kweiyo sensor.
Kusvika parizvino, kugoverwa kwezviratidzo zveGA muSAM kunoramba kusingazivikanwe. Nokudaro, takashandisa qmRGA-inoratidza zvirimwa uye pCLV3 :: mCherry-NLS stem cell reporter35 kuverenga mamepu epamusoro-resolution quantitative mapeji ebasa rekuita reGA, achitarisa paL1 layer (epidermis; Fig. 4a, b, ona Nzira uye Supplementary Methods), sezvo L3 inobata basa rinokosha reSAM. Pano, pCLV3 ::mCherry-NLS kutaura kwakapa yakagadziriswa geometric referensi poindi yekuongorora spatiotemporal kugovera kweGA kusaina chiitiko37. Kunyange zvazvo GA ichionekwa seyakakosha kuti lateral organ development4, takaona kuti zviratidzo zveGA zvaive zvakaderera mumaruva ekutanga (P) kutanga kubva paP3 nhanho (Fig. 4a, b), asi P1 uye P2 primordiums vechidiki vaiva nemaitiro ane mwero akafanana neaya ari munharaunda yepakati (Fig. 4a, b). Higher GA chiratidzo chebasa chakaonekwa pamiganhu ye organ primordium, kutanga paP1 / P2 (pamativi emuganhu) uye ichikwira paP4, pamwe chete nemasero ose enharaunda yenharaunda iri pakati pekutanga (Fig. 4a, b uye Supplementary Fig. 8a, b). Iyi yepamusoro GA yekuratidzira basa yakaonekwa kwete mu epidermis chete asiwo muL2 uye kumusoro L3 zvidimbu (Supplementary Fig. 8b). Maitiro ezviratidzo zveGA akaonekwa muSAM achishandisa qmRGA zvakare yakaramba isingashanduki nekufamba kwenguva (Supplementary Fig. 8c-f, k). Kunyange zvazvo qd17mRGA yakagadzirwa yakanyatsogadziriswa muSAM yemiti yeT3 kubva kumitsara mishanu yakasununguka yatakaratidza zvakadzama, takakwanisa kuongorora maitiro efluorescence akawanikwa ne pRPS5a :: VENUS-2A-TagBFP kuvaka (Supplementary Fig. 8g-j, l). Mumutsara uyu wekutonga, shanduko shoma chete muyero yefluorescence yakaonekwa muSAM, asi muSAM centre takaona kuderera kwakajeka uye kusingatarisirwi muVENUS yakabatana neTagBFP. Izvi zvinosimbisa kuti chiratidzo chechiratidzo chakacherechedzwa neqmRGA chinoratidza GA-inotsamira kuderedzwa kwemRGA-VENUS, asi zvakare inoratidza kuti qmRGA inogona kuwedzeredza GA kusaina chiitiko munzvimbo yemeristem. Muchidimbu, mhedzisiro yedu inoratidza GA siginecha pateni iyo inonyanya kuratidza kugoverwa kweprimordia. Uku kugoverwa kwenharaunda ye-inter-primordial (IPR) kunokonzerwa nekugadzwa zvishoma nezvishoma kwepamusoro GA kusaina basa pakati pekutanga primordium nenharaunda yepakati, nepo panguva imwe chete GA kusaina basa mu primordium inoderera (Fig. 4c, d).
Kugoverwa kweGID1b uye GID1c receptors (ona pamusoro) inoratidza kuti kusiyanisa kutaura kweGA receptors kunobatsira kuumba maitiro eGA kusaina chiitiko muSAM. Isu takashamisika kana kuunganidzwa kwakasiyana kweGA kungave kubatanidzwa. Kuti tiongorore mukana uyu, takashandisa iyo nlsGPS1 GA FRET sensor21. Kuwedzera activation frequency yakaonekwa muSAM yenlsGPS1 yakabatwa ne 10 μM GA4 + 7 ye 100 min (Supplementary Fig. 9a-e), zvichiratidza kuti nlsGPS1 inopindura kuchinja kweGA kusungwa muSAM, sezvainoita mumidzi21. Spatial distribution of nlsGPS1 activation frequency yakaratidza zvishoma zvishoma GA mazinga muzvikamu zvekunze zveSAM, asi zvakaratidza kuti zvakasimudzwa pakati uye pamiganhu yeSAM (Fig. 4e uye Supplementary Fig. 9a, c). Izvi zvinoratidza kuti GA inogoverwawo muSAM ine spatial pateni inofananidzwa neyakaburitswa neqmRGA. Senzira yekuwedzera, takabatawo iyo SAM nefluorescent GA (GA3-, GA4-, GA7-Fl) kana Fl chete sekutonga kwakashata. Chiratidzo cheFl chakagoverwa muSAM yose, kusanganisira nzvimbo yepakati uye primordium, kunyange zvazvo pakadzika pasi (Fig. 4j uye Supplementary Fig. 10d). Kusiyana neizvi, ese matatu GA-Fl akaunganidzwa zvakananga mukati memiganhu yekutanga uye kune madhigirii akasiyana mune yasara IPR, neGA7-Fl ichiunganidza munzvimbo huru muIPR (Fig. 4k uye Supplementary Fig. 10a, b). Quantification yefluorescence intensity yakaratidza kuti IPR kune isiri-IPR intensity ratio yakanga yakakwirira muGA-Fl-treated SAM kana ichienzaniswa neFl-treated SAM (Fig. 4l uye Supplementary Fig. 10c). Pamwe chete, mhedzisiro idzi dzinoratidza kuti GA iripo pamatunhu akakwirira muIPR maseru ari padyo nemuganhu wenhengo. Izvi zvinoratidza kuti iyo pateni yeSAM GA yekusaina chiitiko inobuda kubva kune ese ari maviri ekutaura kweGA receptors uye mutsauko kuunganidzwa kweGA mumaseru eIPR pedyo nemiganhu yenhengo. Nekudaro, ongororo yedu yakaratidza isingatarisirwe spatiotemporal pateni yeGA kusaina, ine yakaderera basa pakati uye primordium yeSAM uye yakakwirira chiitiko muIPR munharaunda yeperipheral.
Kuti tinzwisise basa rekusiyanisa GA kusaina chiitiko muSAM, takaongorora kuwirirana pakati peGA kusaina chiitiko, kuwedzera kwesero, uye kupatsanurwa kwesero tichishandisa chaiyo-nguva-yenguva-yekupedzisira imaging yeSAM qmRGA pCLV3 :: mCherry-NLS. Tichifunga nezve basa reGA mukukura mutemo, kuwirirana kwakanaka nemasero ekuwedzera paramita aitarisirwa. Naizvozvo, takatanga taenzanisa mamepu ebasa rekuita GA nemamepu ekukura kwesero pamusoro (semumiriri wesimba rekuwedzera kwesero kwesero rakapihwa uye neremwanasikana maseru pakupatsanurwa) uye nemamepu ekukura anisotropy, iyo inoyera kurongeka kwekuwedzera kwesero (inoshandiswawo pano sero rakapihwa uye kune masero emwanasikana pakupatsanurwa; Fig. 5a,b, Onawo Mamiriro Ekuwedzera uye Nzira). Mamepu edu eSAM cell surface growth rate inopindirana nekucherechedza kwekare38,39, ine zvishoma zvishoma zvekukura pamuganhu uye hukuru hwekukura mukukura kwemaruva (Fig. 5a). Principal component analysis (PCA) yakaratidza kuti GA kusaina basa raive rakabatana zvisina kunaka nesero rekukura kukura kwesimba (Mufananidzo 5c). Isu takaratidzawo kuti maaxes makuru ekusiyana, kusanganisira GA kusaina kupinza uye kukura kwakasimba, aive orthogonal kugwara rakatemwa nepamusoro CLV3 kutaura, zvichisimbisa kubviswa kwemaseru kubva kuSAM centre mune yakasara ongororo. Spearman correlation analysis yakasimbisa migumisiro yePCA (Mufananidzo 5d), zvichiratidza kuti zviratidzo zvepamusoro zveGA muIPR hazvina kukonzera kuwedzera kwesero. Nekudaro, ongororo yehuwirirano yakaratidza kuwirirana kwakanaka pakati peGA kusaina chiitiko uye kukura anisotropy (Mufananidzo 5c, d), zvichiratidza kuti yakakwira GA kusaina muIPR inopesvedzera kutungamira kwekukura kwesero uye pamwe chinzvimbo chendege yekuparadzanisa masero.
a, b Kupisa mamepu ezvinoreva kukura kwepasi (a) uye kukura anisotropy (b) muSAM avhareji yezvinomera zvinomwe zvakazvimirira (zvinoshandiswa semapuroxisi esimba negwara rekuwedzera kwesero, zvichiteerana). c PCA kuongororwa kwaisanganisira zvinotevera: GA chiratidzo, kuwedzera kwekukura kwepamusoro, kukura kwepamusoro anisotropy, uye CLV3 kutaura. PCA chikamu 1 yainyanya kurongeka zvisina kunaka nekusimba kwekukura kwepamusoro uye yakabatana neiyo GA chiratidzo. PCA chikamu 2 chainyanya kuenderana nekukura kwepamusoro anisotropy uye yakashata yakabatana neCLV3 kutaura. Mazana anomiririra musiyano unotsanangurwa nechikamu chimwe nechimwe. d Spearman kuwirirana kwekuongorora pakati peGA chiratidzo, kukura kwepamusoro, uye kukura kwepamusoro anisotropy pachiyero chetishu kunze kweCZ. Nhamba iri kurudyi ndiyo Spearman rho kukosha pakati pezviviri zvakasiyana. Asterisks inoratidza zviitiko apo kuwirirana/negative kuwirirana kwakakosha zvakanyanya. e 3D kuona kweCol-0 SAM L1 masero ne confocal microscopy. Mitsva yesero madziro akaumbwa muSAM (asi kwete primordium) pa10 h ane mavara zvichienderana nemakona avo. Ruvara rwemavara runoratidzwa mukona yezasi yekurudyi. Iyo inset inoratidza inoenderana 3D mufananidzo pa0 h. Kuedza kwakadzokororwa kaviri nemigumisiro yakafanana. f Mabhokisi ezvirongwa anoratidza maseru ekupatsanurwa mazinga muIPR uye asiri IPR Col-0 SAM (n = 10 zvirimwa zvakazvimirira). Mutsetse wepakati unoratidza wepakati, uye miganhu yebhokisi inoratidza 25th uye 75th percentiles. Whiskers inoratidza hushoma uye hwakanyanya kukosha hwakatemerwa neR software. P maitiro akawanikwa neWelch's maviri-tailed t-bvunzo. g, h Schematic diagram inoratidza (g) nzira yekuyera kona yerutsva rutsva (magenta) neruremekedzo kune radial gwara kubva pakati peSAM (white dotted line) (chete acute angle values, kureva, 0–90 °, inoonekwa), uye (h) denderedzwa / lateral uye radial nzira mukati memeristem. i Frequency histograms ye cell division plane yakatarisa mhiri kweSAM (yakasviba bhuruu), IPR (yepakati bhuruu), uye isiri-IPR (yakajeka bhuruu), zvichiteerana. P maitiro akawanikwa neaviri-tailed Kolmogorov-Smirnov bvunzo. Kuedza kwakadzokororwa kaviri nemigumisiro yakafanana. j Frequency histograms of cell division plane orientation yeIPR yakatenderedza P3 (light green), P4 (yepakati girinhi), uye P5 (yakasviba girinhi), zvichiteerana. P maitiro akawanikwa neaviri-tailed Kolmogorov-Smirnov bvunzo. Kuedza kwakadzokororwa kaviri nemigumisiro yakafanana.
Naizvozvo, isu takazoongorora kuwirirana pakati peGA kusaina uye sero kupatsanura basa nekuzivisa achangobva kuumbwa maseru madziro panguva yekuedzwa (Fig. 5e). Iyi nzira yakatibvumira kuyera kuwanda uye mafambiro ekupatsanurwa kwesero. Zvinotoshamisa ndezvokuti takaona kuti kuwanda kwezvikamu zvemasero muIPR uye mamwe ose eSAM (kwete-IPR, Fig. 5f) akanga akafanana, zvichiratidza kuti kusiyana kweGA kuratidzwa pakati pe IPR uye kwete-IPR masero haaiti zvakanyanya kukanganisa kupatsanurwa kwesero. Izvi, uye kuwirirana kwakanaka pakati peG kusaina uye kukura anisotropy, zvakatikurudzira kuti tifunge kana basa rekusaina reGA rinogona kukanganisa mafambiro endege yekuparadzanisa masero. Takayera kutarisisa kwesero madziro matsva seacute angle inoenderana neradial axis inobatanidza meristem centre uye pakati pesero rusvingo rutsva (Fig. 5e-i) uye takacherechedza maitiro akajeka ekuti masero aparadzanise pamakona ari pedyo ne90 ° maererano neiyo radial axis, nepamusoro-soro mafrequency akaonekwa pa 70-80-60 ° 2% (23%) uye 70-80 ° 6 (2%). (Fig. 5e, i), inopindirana nemasero ekuparadzana munharaunda ye circumferential / transverse direction (Fig. 5h). Kuti tiongorore mupiro weGA kusaina kune iyi cell division maitiro, takaongorora masero ekupatsanura paramita mu IPR uye isiri-IPR zvakasiyana (Fig. 5i). Takacherechedza kuti kupatsanurwa kwemakona ekuparadzanisa mumasero eIPR kwakasiyana kubva kune asiri-IPR masero kana mumasero muSAM yose, neIPR masero anoratidza chikamu chepamusoro che lateral / circular cell divisions, kureva, 70-80 ° uye 80-90 ° (33.86% uye 30.71%, maererano, inoenderana nezvikamu 5). Nokudaro, kucherechedza kwedu kwakaratidza kushamwaridzana pakati pechiratidzo cheGA yakakwirira uye kutaridzika kwendege yekuparadzanisa kwesero pedyo nekutenderera kutenderera, kwakafanana nekubatana pakati peGA basa rekuita uye kukura anisotropy (Fig. 5c, d). Kuti tiwedzere kusimbisa kuchengetedza kwepakati kwesangano iri, takayera kutaridzika kwendege yekuparadzanisa mumasero eIPR akapoteredza primordium kutanga kubva kuP3, sezvo basa repamusoro reG GA rakaonekwa munzvimbo ino kubva kuP4 (Fig. 4). Makona ekuparadzanisa eIPR akapoteredza P3 neP4 haana kuratidza kusiyana kwakasiyana-siyana, kunyange zvazvo kuwedzera kwehuwandu hwemashure emagetsi emagetsi akaonekwa muIPR kumativi eP4 (Fig. 5j). Zvisinei, mumasero eIPR akapoteredza P5, kusiyana kwekutsvaga kwendege yekuparadzanisa kwesero kwakava nhamba inokosha, nekuwedzera kwakanyanya muhuwandu hwezvikamu zvemasero (Fig. 5j). Pamwe chete, mhedzisiro iyi inoratidza kuti kusaina kweGA kunogona kudzora kutaridzika kwesero kukamukana muSAM, iyo inopindirana nemishumo yapfuura40,41 iyo yakakwira GA siginecha inogona kukonzeresa kutarisisa kwesero kukamukana muIPR.
Zvinofanotaurwa kuti maseru ari muIPR haazopinzwa muprimordia asi pane internodes2,42,43. Iyo yakachinjika yekumisikidzwa kwemaseru kukamukana muIPR inogona kuguma neyakajairwa kurongeka kweparallel longitudinal mitsara yemaseru epidermal muinternodes. Zvatinoona zvakatsanangurwa pamusoro apa zvinoratidza kuti kusaina kweGA kunogona kuita basa mukuita uku nekugadzirisa mafambiro ekukamura maseru.
Kurasikirwa kwekushanda kwemajini akawanda eDELLA kunoguma nemhinduro yeGA, uye della mutants inogona kushandiswa kuedza iyi hypothesis44. Takatanga kuongorora maitiro ekutaura emajini mashanu eDELLA muSAM. Transcriptional fusion yeGUS line45 yakaratidza kuti GAI, RGA, RGL1, uye RGL2 (kusvika zvishoma zvishoma) yakaratidzwa muSAM (Supplementary Fig. 11a-d). In situ hybridization yakaratidza zvakare kuti GAI mRNA inounganidza zvakananga muprimordia uye kukura maruva (Supplementary Fig. 11e). RGL1 uye RGL3 mRNA zvakaonekwa mukati meSAM canopy uye mumaruva akare, asi RGL2 mRNA yakanga yakawanda munzvimbo yemuganhu (Supplementary Fig. 11f-h). Confocal imaging ye pRGL3 :: RGL3-GFP SAM yakasimbisa kutaura kwakaonekwa ne in situ hybridization uye yakaratidza kuti RGL3 protein inounganidza muchikamu chepakati cheSAM (Supplementary Fig. 11i). Kushandisa pRGA :: GFP-RGA mutsara, takaonawo kuti RGA protein inounganidza muSAM, asi kuwanda kwayo kunoderera pamuganhu kubva kuP4 (Supplementary Fig. 11j). Zvinonyanya kukosha, maitiro ekutaura eRGL3 neRGA anopindirana nepamusoro GA chiratidzo chekuita muIPR, sezvakaonekwa neqmRGA (Fig. 4). Zvakare, idzi data dzinoratidza kuti ese maDELLA anoratidzwa muSAM uye kuti kutaura kwavo pamwe chete kunotambanudzira SAM yese.
Isu takazoongorora iyo sero division parameters mune yemusango-mhando SAM (Ler, control) uye gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Fig. 6a, b). Sezvineiwo, takaona kushanduka kwakakosha mukugoverwa kwesero division angle frequencies mu della global mutant SAM zvichienzaniswa nemhando yemusango (Fig. 6c). Kuchinja uku mu della global mutant kwakakonzerwa nekuwedzera kwehuwandu hwe80-90 ° angles (34.71% vs. 24.55%) uye, kusvika zvishoma, 70-80 ° angles (23.78% vs. 20.18%), kureva, inopindirana ne transverse cell divisions (Fig. 6c). Kuwanda kwezvikamu zvisingashanduki (0-60 °) zvakare zvakaderera mu della global mutant (Fig. 6c). Iyo yakawanda ye transverse cell division yakawedzera zvakanyanya muSAM ye della global mutant (Fig. 6b). Iyo yakawanda ye transverse cell division muIPR yaivewo yakakwirira mu della global mutant ichienzaniswa nemhando yemusango (Fig. 6d). Kunze kwenharaunda yeIPR, rudzi rwemusango rwakanga rwakanyanya kufanana kugoverwa kwemakona ekuparadzanisa masero, nepo della global mutant yaifarira kupatsanurwa kwetangential se IPR (Fig. 6e). Isu takaverengerawo kutariswa kwesero division muSAM yega2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, uye ga2ox6-2), GA-inactive mutant kumashure uko GA inounganidza. Zvichienderana nekuwedzera kweGA mazinga, iyo SAM ye quintuple ga2ox mutant inflorescence yaive yakakura kupfuura yeCol-0 (Supplementary Fig. 12a, b), uye ichienzaniswa neCol-0, iyo quintuple ga2ox SAM yakaratidza kupararira kwakasiyana kwakasiyana kwesero division angles, neiyo angle frequency ichiwedzera kubva ku050 ° tashing division zvakare ku9, Mufananidzo 12a–c). Nekudaro, isu tinoratidza kuti kuitisa activation yeGA kusaina uye GA kuunganidza kunoita kuti lateral cell division muIPR uye yese yeSAM.
a, b 3D visualization yeL1 layer yePI-stained Ler (a) uye global della mutant (b) SAM uchishandisa confocal microscopy. Mitsva yesero madziro akaumbwa muSAM (asi kwete primordium) pamusoro penguva ye10-h anoratidzwa uye ane mavara zvichienderana nemakona avo. Iyo inset inoratidza iyo SAM pa0 h. Ruvara rwemavara runoratidzwa mukona yezasi yekurudyi. Museve muna (b) unonongedza kumuenzaniso wemasero akaiswa mafaira muglobal della mutant. Kuedza kwakadzokororwa kaviri nemigumisiro yakafanana. ce kuenzanisa kweiyo frequency kugoverwa kwesero division ndege yekumisikidza mune yese SAM (d), IPR (e), uye isiri-IPR (f) pakati peLer nepasi rose della. P maitiro akawanikwa uchishandisa maviri-tailed Kolmogorov-Smirnov bvunzo. f, g 3D kuona kwemifananidzo yakasanganiswa yePI-yakasvibiswa SAM yeCol-0 (i) uye pCUC2 :: gai-1-VENUS (j) miti ye transgenic. Panel (a, b) inoratidza madziro matsva emasero (asi kwete primordia) akaumbwa muSAM mukati me10 h. Kuedza kwakadzokororwa kaviri nemigumisiro yakafanana. h-j Kufananidza kugoverwa kwehuwandu hwemasero ekuparadzanisa ndege inotenderera iri muSAM yose (h), IPR (i) uye isiri IPR (j) pakati peCol-0 uye pCUC2 :: gai-1-VENUS zvirimwa. P maitiro akawanikwa uchishandisa maviri-tailed Kolmogorov-Smirnov bvunzo.
Isu takazoedza mhedzisiro yekudzivisa GA kusaina chaizvo muIPR. Kuti izvi zviitike, takashandisa iyo cotyledon mukombe 2 (CUC2) mutsigiri kutyaira kutaura kweakanyanya kunaka gai-1 protein yakasanganiswa kuVENUS (mu pCUC2 :: gai-1-VENUS mutsara). Mumusango-mhando yeSAM, mutsigiri weCUC2 anotyaira kutaura kwevakawanda IPRs muSAM, kusanganisira masero emuganhu, kubva kuP4 zvichienda mberi, uye kutaura kwakafanana kwakaonekwa mu pCUC2 :: gai-1-VENUS miti (ona pazasi). Kugoverwa kwemakona ekuparadzanisa masero kumativi ose eSAM kana IPR ye pCUC2 :: gai-1-VENUS zvirimwa zvakanga zvisina kunyanya kusiyana nemhando yemusango, kunyange zvisingatarisirwi takaona kuti masero asina IPR mumiti iyi yakakamurwa pane imwe nguva yakawanda ye80-90 ° (Fig. 6f-j).
Izvo zvave zvataurwa kuti kutungamira kwesero kupatsanurwa kunoenderana neiyo geometry yeSAM, kunyanya kushushikana kwekushushikana kunogadzirwa netishu curvature46. Saka takabvunza kana chimiro cheSAM chakashandurwa mu della global mutant uye pCUC2 :: gai-1-VENUS zvirimwa. Sezvambotaurwa kare12, ukuru hwe della global mutant SAM hwakanga hwakakura kupfuura hwemhando yemusango (Supplementary Fig. 13a, b, d). In situ hybridization yeCLV3 uye STM RNA yakasimbisa kuwedzera kwemeristem mu della mutants uye yakaratidzazve kuwedzera kwe lateral ye stem cell niche (Supplementary Fig. 13e, f, h, i). Zvisinei, SAM curvature yakanga yakafanana mune zvose genotypes (Supplementary Fig. 13k, m, n, p). Takaona kuwedzera kwakafanana muhukuru mu gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant pasina kuchinja kwe curvature kana ichienzaniswa nemhando yemusango (Supplementary Fig. 13c, d, g, j, l, o, p). Kuwanda kwemaitiro ekuparadzanisa kwesero kwakakanganiswawo mu della quadruple mutant, asi kusvika zvishoma kudarika mu della monolithic mutant (Supplementary Fig. 12d-f). Iyi dhizaini mhedzisiro, pamwe nekushaikwa kwemhedzisiro pa curvature, inoratidza kuti yakasara RGL3 chiitiko muDella quadruple mutant miganhu shanduko mu cell division orientation inokonzerwa nekurasikirwa kwekuita kweDELLA uye kuti shanduko mu lateral cell divisions inoitika mukupindura kune shanduko muGA kusaina chiitiko pane shanduko muSAM geometry. Sezvakatsanangurwa pamusoro apa, mutsigiri weCUC2 anotyaira IPR kutaura muSAM kutanga paP4 (Supplementary Fig. 14a, b), uye zvakasiyana, pCUC2 :: gai-1-VENUS SAM yakanga ine saizi yakaderedzwa asi yakakwirira curvature (Supplementary Fig. 14c-h). Kuchinja uku mu pCUC2 :: gai-1-VENUS SAM morphology inogona kukonzera kugoverwa kwakasiyana kwemaitiro emagetsi kuenzaniswa nemhando yemusango, umo kushushikana kwepamusoro kunotangira pane imwe nzvimbo shoma kubva kuSAM centre47. Neimwe nzira, shanduko mu pCUC2 :: gai-1-VENUS SAM morphology inogona kuguma nekushandurwa kwenzvimbo dzemakina zvivakwa zvinokonzerwa ne transgene expression48. Muzviitiko zvese izvi, izvi zvinogona kudzikamisa zvishoma mhedzisiro yekuchinja kweGA kusaina nekuwedzera mukana wekuti maseru apatsanure mukutenderera / kuchinjika, tichitsanangura zvatinoona.
Takatorwa pamwechete, data redu rinosimbisa kuti yakakwira GA siginecha inoita basa rinoshanda mukutarisisa kumberi kwendege yekuparadzanisa masero muIPR. Ivo zvakare vanoratidza kuti meristem curvature zvakare inopesvedzera kutaridzika kweiyo cell division ndege muIPR.
Iyo yakachinjika kutenderera kweiyo ndege yekuparadzanisa muIPR, nekuda kweiyo yakakwira GA kusaina chiitiko, inoratidza kuti GA inofanoronga radial cell faira mu epidermis mukati meSAM kutsanangura iyo cellular sangano rinozowanikwa mune epidermal internode. Zvechokwadi, mafaira akadaro aigara achionekwa muSAM mifananidzo ye della global mutants (Fig. 6b). Nokudaro, kuti tiwedzere kuongorora basa rekusimudzira kwechimiro chechimiro cheG kusaina muSAM, takashandisa kufungidzira kwekufamba kwenguva kuti tiongorore sangano renzvimbo yemasero mu IPR mumhando yemhuka (Ler naCol-0), della global mutants, uye pCUC2 :: gai-1-VENUS transgenic plant.
Takaona kuti qmRGA yakaratidza kuti GA chiratidzo chekuita muIPR yakawedzera kubva kuP1 / P2 uye yakakwira paP4, uye iyi muenzaniso yakaramba isingagumi nekufamba kwenguva (Fig. 4a-f uye Supplementary Fig. 8c-f, k). Kuti tiongorore kurongeka kwenzvimbo yemasero muIPR nekuwedzera kweGA chiratidzo, takanyora Ler IPR masero kumusoro uye kumativi eP4 maererano nehupenyu hwavo hwekuvandudza hwakaongororwa 34 h mushure mekutanga kutarisa, kureva, nguva dzinopfuura mbiri dzeplastid, dzichitibvumira kutevera IPR masero panguva yekukura kwekutanga kubva kuP1 / P2 kusvika kuP4. Takashandisa mavara matatu akasiyana: yero kune aya maseru akabatanidzwa mune yekutanga pedyo neP4, yegirini kune avo vaive muIPR, uye yepepuru kune avo vakatora chikamu mune zvese maitiro (Fig. 7a-c). Pa t0 (0 h), 1-2 zvikamu zvePIR masero zvakaonekwa pamberi peP4 (Fig. 7a). Sezvaitarisirwa, apo masero aya akapatsanurwa, aizviita kunyanya kuburikidza nendege yekuparadzanisa (Fig. 7a-c). Migumisiro yakafanana yakawanikwa uchishandisa Col-0 SAM (inotarisa paP3, iyo muganhu wayo unopeta zvakafanana neP4 muLer), kunyange mune iyi genotype kupeta kwakaumbwa pamuganhu wemaruva akavanza IPR masero nokukurumidza (Fig. 7g-i). Saka, kupatsanurwa kwemaseru eIPR anofanoronga maseru kuita mitsara yeradial, semu internodes. Kurongeka kwemitsara yeradial uye kuiswa kweIPR masero pakati penhengo dzinotevedzana zvinoratidza kuti masero aya ma internodal progenitors.
Pano, takagadzira ratiometric GA siginecha biosensor, qmRGA, iyo inobvumira kuwanda kwemepu yeGA kusaina chiitiko chinobva mukusanganiswa kweGA uye GA receptor kutariswa uku ichideredza kukanganiswa ne endogenous kusaina nzira, nekudaro ichipa ruzivo nezve GA basa padanho rema cellular. Kuti izvi zviitike, takagadzira puroteni yeDELLA yakagadziridzwa, mRGA, iyo yakarasikirwa nekukwanisa kusunga DELLA kudyidzana vadyidzani asi inoramba iine hanya neGA-induced proteolysis. qmRGA inopindura kune ese ari maviri exogenous uye endo native shanduko muGA mazinga, uye ayo ane simba ekunzwa zvimiro anogonesa kuongororwa kwe spatiotemporal shanduko muGA kusaina chiitiko panguva yekusimudzira. qmRGA zvakare chishandiso chinoshanduka-shanduka sezvo chinogona kuchinjika kune akasiyana matishu nekushandura anosimudzira anoshandiswa pakutaura kwawo (kana zvichidikanwa), uye nekupihwa kuchengetedzwa kwenzira yeG kusaina nzira uye PFYRE motif mhiri kweangiosperms, inogona kuchinjika kune mamwe marudzi22. Zvinoenderana neizvi, shanduko yakaenzana mumupunga SLR1 DELLA protein (HYY497AAA) yakaratidzwawo kudzvanyirira kukura kwekudzvanya chiitiko cheSLR1 ichingoderedza zvishoma kuderedzwa kwayo kweGA-mediated, yakafanana nemRGA23. Zvikuru, ongororo dzichangoburwa muArabidopsis dzakaratidza kuti imwe amino acid mutation muPFYRE domain (S474L) yakashandura iyo transcriptional chiitiko cheRGA isingakanganise kugona kwayo kuyanana nevatambi vezvinyorwa zvechinyorwa50. Kunyangwe shanduko iyi iri padhuze neiyo 3 amino acid inotsiva iripo muRGA, zvidzidzo zvedu zvinoratidza kuti idzi shanduko mbiri dzinoshandura hunhu hwakasiyana hweDELLA. Kunyangwe vazhinji vanonyorerana chinhu vanobatana vachisunga kune LHR1 uye SAW madomasi eDELLA26,51, mamwe akachengetedzwa amino acids muPFYRE domain anogona kubatsira kudzikamisa kudyidzana uku.
Kuvandudzwa kweInternode chinhu chakakosha mukuvaka kwezvirimwa uye kuvandudza goho. qmRGA yakaratidza yakakwira GA kusaina chiitiko muIPR internode progenitor masero. Nekubatanidza kuwanda kwekufungidzira uye genetics, takaratidza kuti GA masiginecha mapatani anopfuura denderedzwa / transverse cell division ndege muSAM epidermis, ichiumba sero division sangano rinodiwa pakuvandudza internode. Vanoverengeka vanodzora ve cell division ndege yekutarisa yakaonekwa panguva yekusimudzira52,53. Basa redu rinopa muenzaniso wakajeka wekuti GA kusaina basa rinogadzirisa sei iyi cellular parameter. DELLA inogona kupindirana neprefolding protein complexes41, saka GA siginecha inogona kudzora sero division ndege kutaridzika nekupesvedzera zvakananga cortical microtubule orientation40,41,54,55. Isu tisingatarisiri takaratidza kuti muSAM, iyo correlate yepamusoro GA kusaina chiitiko chaive chisiri kurebesa kwesero kana kupatsanurwa, asi kukura chete anisotropy, iyo inopindirana nemhedzisiro yakananga yeGA pakutungamira kwesero kupatsanurwa muIPR. Nekudaro, isu hatigone kusarura kuti mhedzisiro iyi inogona zvakare kuve isina kunanga, semuenzaniso inopindirana neGA-induced cell wall softening56. Kuchinja kwemasero emadziro emadziro kunokonzera mechanical stress57,58, iyo inogonawo kukanganisa kutaridzika kwendege yekuparadzanisa masero nekukanganisa kutaridzika kwe cortical microtubules39,46,59. Izvo zvakasanganiswa zvinokonzerwa neGA-induced mechanical stress uye zvakananga kurongeka kwe microtubule orientation ne GA inogona kubatanidzwa mukugadzira imwe nzira yemasero ekuparadzanisa maitiro mu IPR kutsanangura internodes, uye zvimwe zvidzidzo zvinodiwa kuedza pfungwa iyi. Saizvozvowo, zvidzidzo zvekare zvakaratidza kukosha kweDELLA-interacting proteins TCP14 uye 15 mukutonga kwe internode formation60,61 uye zvinhu izvi zvinogona kupindirana nekuita kweGA pamwe chete neBREVIPEDICELLUS (BP) uye PENNYWISE (PNY), iyo inodzora kukura kwe internode uye yakaratidzwa, 2 GA chiratidzo. Zvichipa kuti DELLAs inopindirana nebrassinosteroid, ethylene, jasmonic acid, uye abscisic acid (ABA) inoratidza nzira63,64 uye kuti mahomoni aya anogona kukanganisa microtubule orientation65, migumisiro yeGA pamusangano wekuparadzanisa masero inogonawo kupindirana nemamwe mahomoni.
Zvidzidzo zvepakutanga zve cytological zvakaratidza kuti zvose zviri mukati nekunze nharaunda dzeArabidopsis SAM dzinodiwa pakuvandudza internode2,42. Icho chokwadi chekuti GA inoshingairira kutonga kupatsanurwa kwesero mukati memukati matishu12 inotsigira maviri maviri basa reGA mukugadzirisa meristem uye saizi yeinternode muSAM. Iyo nzira yekutungamira sero kupatsanurwa inodzorwawo zvakasimba mukati memukati SAM tishu, uye mutemo uyu wakakosha pakukura kwe stem52. Zvichave zvinonakidza kuongorora kana GA ichiitawo basa mukutungamira ndege yekukamura maseru musangano remukati reSAM, nekudaro kuwiriranisa kutsanangurwa nekuvandudzwa kwemainternodes mukati meSAM.
Zvirimwa zvakarimwa mu vitro muvhu kana 1x Murashige-Skoog (MS) yepakati (Duchefa) yakawedzerwa ne1% sucrose uye 1% agar (Sigma) pasi pemamiriro akajairwa (16 h mwenje, 22 °C), kunze kweiyo hypocotyl uye midzi yekukura miedzo umo mbesa dzakarimwa pamahwendefa akatwasuka pasi pechiedza che22 ° C nguva dzose. Pakuedza nitrate, zvidyarwa zvakarimwa pane yakagadziridzwa MS medium (bioWORLD chirimwa chepakati) yakawedzerwa nenitrate yakakwana (0 kana 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose uye 1% A-agar (Sigma) pasi pemamiriro emazuva mazhinji.
GID1a cDNA yakaiswa mupDONR221 yakasanganiswa nepDONR P4-P1R-pUBQ10 uye pDONR P2R-P3-mCherry mupB7m34GW kugadzira pUBQ10::GID1a-mCherry. IDD2 DNA yakaiswa mupDONR221 yakasanganiswa zvakare mupB7RWG266 kugadzira p35S:IDD2-RFP. Kugadzira pGID1b::2xmTQ2-GID1b, chidimbu che3.9 kb kumusoro kwenzvimbo yeGID1b yekukodha uye chidimbu che 4.7 kb chine GID1b cDNA (1.3 kb) uye terminator (3.4 kb) zvakatanga kukwidziridzwa uchishandisa maprimers muSupplementary pDO1R 4. (Thermo Fisher Scientific) uye pDONR P2R-P3 (Thermo Fisher Scientific), zvichiteerana, uye pakupedzisira yakasanganiswa ne pDONR221 2xmTQ268 kupinda pGreen 012567 target vector uchishandisa Gateway cloning. Kugadzira pCUC2 ::LSSmOrange, iyo CUC2 inosimudzira sequence (3229 bp kumusoro kweATG) ichiteverwa nekodhesi kutevedzana kweStokes-shifted mOrange (LSSmOrange)69 ine N7 nyukireya chiratidzo chenzvimbo uye NOS transcriptal terminator yakaunganidzwa munzira yekunongedza yeGreen 3. (Invitrogen). Chirimwa chebinary vector chakaunzwa muAgrobacterium tumefaciens strain GV3101 uye chakaunzwa muNicotiana benthamiana mashizha neAgrobacterium infiltration method uye muArabidopsis thaliana Col-0 nenzira yemaruva dip, zvichiteerana. pUBQ10 :: qmRGA pUBQ10 :: GID1a-mCherry uye pCLV3 :: mCherry-NLS qmRGA akaparadzaniswa kubva kuF3 uye F1 zvizvarwa zvemichinjikwa yakatevedzana, zvichiteerana.
RNA in situ hybridization yakaitwa paanenge 1 cm kureba pfuti tips72, iyo yakaunganidzwa uye pakarepo yakagadziriswa mu FAA solution (3.7% formaldehyde, 5% acetic acid, 50% ethanol) isati yatonhodzwa kusvika 4 °C. Mushure me2 × 15 min vacuum kurapwa, iyo inogadziriswa yakashandurwa uye masampuli akaiswa mukati mehusiku humwe. GID1a, GID1b, GID1c, GAI, RGL1, RGL2, uye RGL3 cDNAs uye antisense probes kune yavo 3'-UTRs yakagadziriswa uchishandisa zvinyorwa zvakaratidzwa muSupplementary Table 3 sezvinotsanangurwa naRosier et al.73. Digoxigenin-yakanyorwa probes yakavharwa nekushandisa digoxigenin masoja ekudzivirira chirwere (3000-fold dilution; Roche, catalogue nhamba: 11 093 274 910), uye zvikamu zvakasvibiswa ne5-bromo-4-chloro-3-indolyl phosphate (BCIP, 250-BTlution, 250-diblueN). 200-fold dilution) mhinduro.
Nguva yekutumira: Feb-10-2025