Tua apical meristem (SAM) kev loj hlob yog qhov tseem ceeb rau qia architecture. Cov tshuaj hormones coggibberellins(GAs) ua lub luag haujlwm tseem ceeb hauv kev sib koom tes cog qoob loo, tab sis lawv lub luag haujlwm hauv SAM tseem tsis to taub. Ntawm no, peb tau tsim ib qho piv txwv biosensor ntawm GA signaling los ntawm engineering DELLA protein los txwv nws txoj haujlwm tseem ceeb hauv GA transcriptional teb thaum khaws cia nws degradation raws li GA lees paub. Peb pom tau hais tias qhov degradation-based biosensor no tau sau cov kev hloov pauv hauv GA qib thiab kev paub ntawm tes thaum lub sijhawm txhim kho. Peb siv biosensor no los qhia GA signaling kev ua hauv SAM. Peb qhia tau hais tias cov teeb liab GA siab yog tam sim no feem ntau nyob rau hauv cov hlwb nyob nruab nrab ntawm lub cev primordia, uas yog precursors rau internode hlwb. Siv cov kev nce- thiab poob-ntawm-kev ua haujlwm, peb qhia ntxiv tias GA tswj kev taw qhia ntawm lub xov tooj ntawm tes faib dav hlau, tsim kom muaj lub koom haum canonical cellular ntawm internodes, yog li txhawb kev sib koom ua ke hauv SAM.
Qhov tua apical meristem (SAM), nyob rau ntawm qhov tua apex, muaj cov niche ntawm cov qia hlwb uas nws cov haujlwm ua rau cov kab mob sab nraud thiab cov kab mob hauv cov qauv thiab rov ua dua thoob plaws hauv lub neej ntawm cov nroj tsuag. Txhua qhov kev rov ua dua tshiab no, lossis cov nroj tsuag nodes, suav nrog internodes thiab lateral organs ntawm cov nodes, thiab axillary meristems nyob rau hauv nplooj axils1. Kev loj hlob thiab kev koom tes ntawm cov nroj tsuag nodes hloov thaum lub sij hawm kev loj hlob. Hauv Arabidopsis, kev loj hlob ntawm internodal yog suppressed thaum lub sij hawm vegetative theem, thiab axillary meristems nyob twj ywm nyob rau hauv lub axils ntawm rosette nplooj. Thaum lub sij hawm hloov mus rau floral theem, lub SAM ua lub inflorescence meristem, tsim elongated internodes thiab axillary buds, branchlets nyob rau hauv lub axils ntawm cauline nplooj, thiab tom qab ntawd, leafless paj2. Txawm hais tias peb tau ua tiav qhov tseem ceeb hauv kev nkag siab txog cov txheej txheem uas tswj kev pib ntawm nplooj, paj, thiab ceg ntoo, kuj tsis tshua paub txog yuav ua li cas internodes tshwm sim.
Kev nkag siab txog spatiotemporal faib ntawm GAs yuav pab kom nkag siab zoo dua cov haujlwm ntawm cov tshuaj hormones hauv cov ntaub so ntswg sib txawv thiab ntawm cov theem sib txawv. Kev pom ntawm qhov degradation ntawm RGA-GFP fusion qhia nyob rau hauv qhov kev txiav txim ntawm nws tus kheej txhawb nqa muab cov ntaub ntawv tseem ceeb ntawm kev tswj hwm tag nrho GA qib hauv cov hauv paus hniav15,16. Txawm li cas los xij, RGA qhia txawv ntawm cov ntaub so ntswg17 thiab raug tswj los ntawm GA18. Yog li, qhov sib txawv ntawm RGA tus txhawb nqa tuaj yeem ua rau cov qauv fluorescence pom nrog RGA-GFP thiab yog li cov qauv no tsis yog qhov ntau. Tsis ntev los no, bioactive fluorescein (Fl)-labeled GA19,20 tau qhia txog kev sib sau ntawm GA hauv cov hauv paus hniav endocortex thiab kev tswj hwm ntawm nws cov cellular theem los ntawm GA thauj. Tsis ntev los no, GA FRET sensor nlsGPS1 tau pom tias GA qib sib cuam tshuam nrog cell elongation hauv cov hauv paus hniav, filaments, thiab tsaus-loj hlob hypocotyls21. Txawm li cas los xij, raws li peb tau pom, GA concentration tsis yog tib qho kev tswj hwm GA signaling kev ua ub no, vim nws nyob ntawm cov txheej txheem sensing nyuaj. Ntawm no, tsim los ntawm peb txoj kev nkag siab ntawm DELLA thiab GA kev taw qhia txoj hauv kev, peb tshaj tawm txoj kev txhim kho thiab kev ua tus cwj pwm ntawm degradation-based ratiometric biosensor rau GA signaling. Txhawm rau tsim kom muaj nuj nqis biosensor no, peb siv cov mutant GA-sensitive RGA uas tau fused rau cov fluorescent protein thiab nthuav tawm nyob rau hauv cov ntaub so ntswg, nrog rau GA-insensitive fluorescent protein. Peb qhia tau hais tias cov mutant RGA protein fusions tsis cuam tshuam nrog endogenous GA signaling thaum ubiquitously qhia, thiab hais tias lub biosensor no muaj peev xwm quantify signaling kev ua los ntawm ob qho tib si GA input thiab GA teeb liab ua los ntawm lub sensing apparatus nrog siab spatiotemporal daws teeb meem. Peb siv biosensor no los qhia txog kev faib tawm ntawm GA signaling kev ua haujlwm thiab ntsuas seb GA tswj tus cwj pwm ntawm tes hauv SAM epidermis. Peb pom tau hais tias GA tswj kev taw qhia ntawm kev faib dav hlau ntawm SAM hlwb nyob nruab nrab ntawm lub cev primordia, yog li txhais lub koom haum canonical cellular ntawm lub internode.
Thaum kawg, peb nug seb qmRGA puas tuaj yeem tshaj tawm cov kev hloov pauv hauv endogenous GA qib uas siv cov hypocotyls loj hlob. Peb yav dhau los tau pom tias nitrate txhawb kev loj hlob los ntawm kev nce GA synthesis thiab, dhau los, DELLA34 degradation. Raws li, peb tau pom tias qhov ntev hypocotyl nyob rau hauv pUBQ10::qmRGA cov qoob loo loj hlob nyob rau hauv ntau cov khoom siv nitrate (10 mM NO3−) tau ntev dua li cov yub loj hlob nyob rau hauv cov xwm txheej tsis muaj nitrate (Ntawv qhia txog daim duab 6a). Raws li kev loj hlob teb, GA teeb liab tau siab dua nyob rau hauv hypocotyls ntawm seedlings loj hlob nyob rau hauv 10 mM NO3- tej yam kev mob tshaj li nyob rau hauv lub seedlings loj hlob nyob rau hauv tsis muaj nitrate (Ntxiv daim duab 6b, c). Yog li, qmRGA tseem tuaj yeem saib xyuas cov kev hloov pauv hauv GA teeb liab tshwm sim los ntawm kev hloov pauv endogenous hauv GA concentration.
Txhawm rau nkag siab seb GA signaling kev ua haujlwm tau pom los ntawm qmRGA nyob ntawm GA concentration thiab GA kev nkag siab, raws li xav tau raws li tus qauv tsim, peb tau txheeb xyuas qhov kev qhia ntawm peb GID1 receptors hauv cov nqaij mos thiab cov menyuam yaus. Hauv kev cog qoob loo, GID1-GUS tus neeg sau xov xwm kab tau pom tias GID1a thiab c tau nthuav tawm heev hauv cotyledons (Fig. 3a–c). Tsis tas li ntawd, tag nrho peb cov receptors tau qhia nyob rau hauv nplooj, lateral root primordia, cov lus qhia hauv paus (tshwj tsis yog lub hauv paus cap ntawm GID1b), thiab vascular system (Fig. 3a–c). Hauv inflorescence SAM, peb tau kuaj pom GUS cov cim rau GID1b thiab 1c (Ntxiv Fig. 7a–c). Nyob rau hauv situ hybridization tau lees paub cov qauv qhia no thiab qhia ntxiv tias GID1c tau qhia tsis sib xws ntawm qib qis hauv SAM, qhov GID1b tau qhia ntau dua ntawm qhov chaw ntawm SAM (Sab Ntxiv Fig. 7d–l). Lub pGID1b::2xmTQ2-GID1b kev txhais lus fusion kuj tau nthuav tawm ntau yam ntawm GID1b qhia, los ntawm qis lossis tsis muaj kev qhia nyob rau hauv nruab nrab ntawm SAM mus rau siab qhia ntawm lub cev ciam teb (Ntxiv Fig. 7m). Yog li, GID1 receptors tsis sib npaug sib faib thoob plaws hauv cov ntaub so ntswg. Hauv cov kev sim tom ntej, peb kuj tau pom tias overexpression ntawm GID1 (pUBQ10::GID1a-mCherry) nce rhiab heev ntawm qmRGA hauv hypocotyls rau sab nraud GA daim ntawv thov (Fig. 3d, e). Nyob rau hauv sib piv, fluorescence ntsuas los ntawm qd17mRGA nyob rau hauv lub hypocotyl yog insensitive rau GA3 kev kho mob (Fig. 3f, g). Rau ob qho tib si kev soj ntsuam, cov noob tau raug kho nrog qhov siab ntawm GA (100 μM GA3) txhawm rau ntsuas tus cwj pwm nrawm ntawm lub sensor, qhov twg lub peev xwm los khi rau GID1 receptor tau txhim kho lossis ploj. Ua ke, cov txiaj ntsig no tau lees paub tias qmRGA biosensor ua haujlwm ua ke raws li GA thiab GA sensor, thiab qhia tias qhov sib txawv ntawm GID1 receptor tuaj yeem hloov kho qhov emissivity ntawm lub sensor.
Txog niaj hnub no, kev faib tawm ntawm GA cov cim hauv SAM tseem tsis meej. Yog li ntawd, peb siv qmRGA-expressing nroj tsuag thiab pCLV3::mCherry-NLS qia cell reporter35 los xam high-resolution quantitative maps ntawm GA signaling kev ua, tsom rau lub L1 txheej (epidermis; Fig. 4a, b, saib Methods thiab Supplementary Methods a), txij li thaum L13 txoj kev loj hlob. Ntawm no, pCLV3::mCherry-NLS qhia tau muab cov ntsiab lus geometric ruaj khov rau kev tshuaj xyuas qhov kev faib tawm ntawm GA signaling kev ua 37. Txawm hais tias GA suav hais tias yog qhov tseem ceeb rau kev loj hlob ntawm lub cev 4, peb pom tias GA cov cim qhia tau qis hauv cov paj primordium (P) pib los ntawm theem P3 (Fig. 4a, b), thaum cov tub ntxhais hluas P1 thiab P2 primordiums muaj kev ua haujlwm nruab nrab zoo ib yam li hauv cheeb tsam nruab nrab (Fig. 4a, b). Kev ua haujlwm siab dua GA tau kuaj pom ntawm thaj tsam ntawm lub cev primordium, pib ntawm P1 / P2 (ntawm ob sab ntawm ciam teb) thiab peaking ntawm P4, nrog rau hauv txhua lub hlwb ntawm thaj chaw peripheral nyob nruab nrab ntawm lub primordia (Fig. 4a, b thiab Supplementary Fig. 8a, b). Qhov siab dua GA teeb liab kev ua haujlwm no tau pom tsis yog nyob rau hauv cov epidermis nkaus xwb tab sis kuj nyob rau hauv L2 thiab sab sauv L3 khaubncaws sab nraud povtseg (Ntxiv daim duab 8b). Tus qauv ntawm GA teeb liab pom nyob rau hauv SAM siv qmRGA kuj tseem tsis tau hloov lub sij hawm (Supplementary Fig. 8c–f, k). Txawm hais tias qd17mRGA kev tsim kho tau raug txo qis hauv SAM ntawm T3 cov nroj tsuag los ntawm tsib kab kev ywj pheej uas peb tau hais meej meej, peb tuaj yeem txheeb xyuas cov qauv fluorescence tau nrog pRPS5a::VENUS-2A-TagBFP kev tsim kho (Ntxiv Fig. 8g–j, l). Hauv kab tswj hwm no, tsuas yog kev hloov pauv me me hauv qhov sib piv fluorescence tau kuaj pom hauv SAM, tab sis hauv SAM qhov chaw peb tau pom qhov pom tseeb thiab poob qis hauv VENUS cuam tshuam nrog TagBFP. Qhov no lees paub tias cov qauv qhia pom los ntawm qmRGA qhia txog GA-dependent degradation ntawm mRGA-VENUS, tab sis kuj qhia tau tias qmRGA yuav overestimate GA signaling kev ua si nyob rau hauv meristem center. Hauv cov ntsiab lus, peb cov txiaj ntsig tau nthuav tawm GA teeb liab qauv uas feem ntau cuam tshuam txog kev faib tawm ntawm primordia. Qhov kev faib tawm ntawm thaj tsam nruab nrab ntawm thaj tsam thawj zaug (IPR) yog vim qhov kev tsim kho me ntsis ntawm kev ua haujlwm siab GA signaling ntawm kev loj hlob primordium thiab hauv nruab nrab cheeb tsam, thaum tib lub sij hawm GA signaling kev ua si nyob rau hauv lub primordium txo (Fig. 4c, d).
Kev faib tawm ntawm GID1b thiab GID1c receptors (saib saum toj no) qhia tias qhov sib txawv ntawm GA receptors pab tsim cov qauv ntawm GA signaling kev ua haujlwm hauv SAM. Peb xav paub seb qhov sib txawv ntawm GA puas tuaj yeem koom nrog. Txhawm rau tshawb xyuas qhov ua tau no, peb siv nlsGPS1 GA FRET sensor21. Kev ua kom muaj zog ntau ntxiv tau pom nyob rau hauv SAM ntawm nlsGPS1 kho nrog 10 μM GA4 + 7 rau 100 min (Cov duab ntxiv. 9a–e), qhia tias nlsGPS1 teb rau kev hloov pauv hauv GA concentration hauv SAM, raws li nws ua hauv cov hauv paus hniav21. Spatial faib ntawm nlsGPS1 kev ua kom nquag nquag tau qhia txog qib GA tsawg hauv cov txheej txheem sab nrauv ntawm SAM, tab sis pom tias lawv tau nce siab hauv nruab nrab thiab ntawm ciam teb ntawm SAM (Fig. 4e thiab Ntxiv Fig. 9a,c). Qhov no qhia tau hais tias GA kuj tau muab faib rau hauv SAM nrog tus qauv spatial piv rau qhov uas qhia los ntawm qmRGA. Raws li kev sib txuas ntxiv, peb kuj tau kho SAM nrog fluorescent GA (GA3-, GA4-, GA7-Fl) lossis Fl ib leeg raws li kev tswj tsis zoo. Lub teeb liab Fl tau muab faib thoob plaws hauv SAM, suav nrog thaj av nruab nrab thiab thawj zaug, txawm hais tias ntawm qhov siv qis dua (Fig. 4j thiab Ntxiv Fig. 10d). Nyob rau hauv sib piv, tag nrho peb GA-Fl accumulated tshwj xeeb nyob rau hauv lub primordium ciam teb thiab mus txawv degrees nyob rau hauv tas li ntawd ntawm lub IPR, nrog GA7-Fl accumulating nyob rau hauv lub loj tshaj plaws sau nyob rau hauv lub IPR (Fig. 4k thiab Supplementary Fig. 10a, b). Kev ntsuas ntawm fluorescence siv tau qhia tias IPR rau qhov tsis yog-IPR siv qhov sib piv yog siab dua hauv GA-Fl-kho SAM piv rau Fl-kho SAM (Fig. 4l thiab Supplementary Fig. 10c). Ua ke, cov txiaj ntsig no qhia tias GA muaj nyob rau hauv ntau dua hauv IPR cov hlwb uas nyob ze rau ntawm lub cev ciam teb. Qhov no qhia tau hais tias tus qauv ntawm SAM GA qhov taw qhia kev ua haujlwm tshwm sim los ntawm ob qho tib si sib txawv ntawm GA receptors thiab sib txawv ntawm GA hauv IPR hlwb nyob ze ntawm lub cev ciam teb. Yog li, peb qhov kev soj ntsuam tau nthuav tawm qhov kev npaj txhij txog spatiotemporal qauv ntawm GA signaling, nrog kev ua haujlwm qis dua hauv nruab nrab thiab primordium ntawm SAM thiab kev ua haujlwm siab dua hauv IPR hauv cheeb tsam peripheral.
Txhawm rau nkag siab txog lub luag haujlwm ntawm qhov sib txawv GA signaling kev ua haujlwm hauv SAM, peb tau txheeb xyuas qhov kev sib raug zoo ntawm GA signaling kev ua, kev nthuav dav ntawm tes, thiab kev faib ntawm tes siv sijhawm-lapse duab ntawm SAM qmRGA pCLV3::mCherry-NLS. Muab lub luag haujlwm ntawm GA hauv kev tswj hwm kev loj hlob, yuav tsum muaj kev sib raug zoo nrog kev nthuav dav ntawm tes. Yog li ntawd, peb thawj zaug piv GA cov phiaj xwm kev ua ub no nrog cov duab qhia ntawm cov xov tooj ntawm tes qhov loj hlob ntawm tus nqi (raws li tus neeg sawv cev rau lub zog ntawm kev nthuav dav ntawm tes rau ib lub xovtooj ntawm tes thiab rau cov menyuam ntxhais ntawm kev faib tawm) thiab nrog daim duab qhia kev loj hlob anisotropy, uas ntsuas qhov taw qhia ntawm kev nthuav dav ntawm tes (tseem siv ntawm no rau ib lub xovtooj ntawm tes thiab rau cov menyuam cell ntawm kev faib khoom; Fig. 5a,b, saib Methods and Supplement). Peb daim duab qhia chaw ntawm SAM cell nto qhov kev loj hlob zoo ib yam nrog cov kev soj ntsuam yav dhau los38,39, nrog rau kev loj hlob tsawg kawg nkaus ntawm ciam teb thiab qhov kev loj hlob siab tshaj plaws hauv kev tsim paj (Fig. 5a). Tus Thawj Saib Xyuas Kev Tshawb Fawb (PCA) tau pom tias GA qhov taw qhia kev ua haujlwm tsis zoo cuam tshuam nrog kev loj hlob ntawm tes ntawm tes (Daim duab 5c). Peb kuj tau pom tias cov axes tseem ceeb ntawm kev hloov pauv, suav nrog GA teeb liab tawm tswv yim thiab kev loj hlob siv, yog orthogonal rau cov kev taw qhia uas tau txiav txim siab los ntawm siab CLV3 qhia, lees paub qhov kev cais tawm ntawm cov hlwb los ntawm SAM centre nyob rau hauv cov kev soj ntsuam ntxiv. Spearman correlation tsom xam tau lees paub cov txiaj ntsig ntawm PCA (Daim duab 5d), qhia tias siab dua GA cov cim hauv IPR tsis ua rau muaj kev nthuav dav ntawm tes ntau dua. Txawm li cas los xij, kev txheeb xyuas kev sib raug zoo tau nthuav tawm me ntsis kev sib raug zoo ntawm GA signaling kev ua thiab kev loj hlob anisotropy (Daim duab 5c, d), qhia tias siab dua GA signaling hauv IPR cuam tshuam cov kev taw qhia ntawm kev loj hlob ntawm tes thiab tej zaum txoj hauj lwm ntawm lub cell division dav hlau.
a, b Heat maps of mean ntog loj hlob (a) thiab kev loj hlob anisotropy (b) nyob rau hauv SAM nruab nrab ntau tshaj xya tus nroj tsuag ywj siab (siv raws li proxies rau lub zog thiab kev taw qhia ntawm cell expansion, ntsig txog). c PCA tsom xam suav nrog cov kev hloov pauv hauv qab no: GA teeb liab, qhov kev loj hlob ntawm qhov chaw, qhov loj hlob anisotropy, thiab CLV3 qhia. PCA tivthaiv 1 feem ntau tsis zoo cuam tshuam nrog kev loj hlob ntawm qhov chaw thiab muaj kev cuam tshuam zoo nrog GA teeb liab. PCA tivthaiv 2 feem ntau zoo sib raug zoo nrog kev loj hlob ntawm anisotropy thiab tsis zoo cuam tshuam nrog CLV3 qhia. Cov feem pua sawv cev qhov kev hloov pauv uas tau piav qhia los ntawm txhua qhov khoom. d Spearman correlation tsom xam ntawm GA teeb liab, nto kev loj hlob siv, thiab nto anisotropy loj hlob ntawm cov ntaub so ntswg scale tsis suav CZ. Tus lej ntawm sab xis yog Spearman rho tus nqi ntawm ob qhov sib txawv. Asterisks qhia txog cov xwm txheej uas qhov kev sib raug zoo / kev sib raug zoo tsis zoo yog qhov tseem ceeb heev. e 3D visualization ntawm Col-0 SAM L1 hlwb los ntawm confocal microscopy. Tshiab cell phab ntsa tsim nyob rau hauv lub SAM (tab sis tsis yog lub primordium) ntawm 10 h yog xim raws li lawv lub kaum sab xis qhov tseem ceeb. Cov xim bar yog qhia nyob rau hauv sab xis ces kaum. Lub inset qhia cov duab 3D sib raug ntawm 0 h. Qhov kev sim tau rov ua dua ob zaug nrog cov txiaj ntsig zoo sib xws. f Box plots qhia cell division tus nqi hauv IPR thiab non-IPR Col-0 SAM (n = 10 cov nroj tsuag ywj pheej). Cov kab nruab nrab qhia qhov nruab nrab, thiab lub thawv ciam teb qhia txog 25 thiab 75 feem pua. Whiskers qhia qhov tsawg kawg nkaus thiab siab tshaj qhov tseem ceeb txiav txim siab nrog R software. P qhov txiaj ntsig tau txais nrog Welch's ob-tailed t-test. g, h Schematic daim duab qhia (g) yuav ua li cas ntsuas lub kaum sab xis ntawm phab ntsa cell tshiab (magenta) hais txog lub radial kev taw qhia los ntawm qhov chaw ntawm lub SAM (dawb dotted kab) (tsuas yog mob lub kaum sab xis qhov tseem ceeb, piv txwv li, 0–90 °, raug txiav txim), thiab (h) lub circumferential / lateral thiab radial cov lus qhia nyob rau hauv lub meristem. i Frequency histograms ntawm cell division dav hlau orientation hla SAM (dub xiav), IPR ( nruab nrab xiav), thiab tsis-IPR (lub teeb xiav), feem. P tus nqi tau txais los ntawm ob-tailed Kolmogorov-Smirnov xeem. Qhov kev sim tau rov ua dua ob zaug nrog cov txiaj ntsig zoo sib xws. j Ntau zaus histograms ntawm cell division dav hlau orientation ntawm IPR nyob ib ncig ntawm P3 (lub teeb ntsuab), P4 (nruab nrab ntsuab), thiab P5 (ntsuab ntsuab), feem. P tus nqi tau txais los ntawm ob-tailed Kolmogorov-Smirnov xeem. Qhov kev sim tau rov ua dua ob zaug nrog cov txiaj ntsig zoo sib xws.
Yog li ntawd, peb tom ntej no tau tshawb xyuas qhov kev sib raug zoo ntawm GA signaling thiab cell division kev ua los ntawm kev txheeb xyuas cov phab ntsa tshiab ntawm tes thaum lub sij hawm ntsuam xyuas (Fig. 5e). Txoj hauv kev no tso cai rau peb ntsuas qhov zaus thiab kev taw qhia ntawm kev faib cell. Kuj ceeb tias, peb pom tias qhov zaus ntawm kev sib faib ntawm tes hauv IPR thiab qhov seem ntawm SAM (tsis yog-IPR, Fig. 5f) yog qhov zoo sib xws, qhia tias qhov sib txawv ntawm GA signaling ntawm IPR thiab non-IPR hlwb tsis cuam tshuam rau cell division. Qhov no, thiab qhov kev sib raug zoo ntawm GA signaling thiab kev loj hlob anisotropy, ua rau peb xav txog seb GA signaling kev ua ub no tuaj yeem cuam tshuam rau kev taw qhia ntawm lub cell division dav hlau. Peb ntsuas qhov taw qhia ntawm phab ntsa cell tshiab raws li lub kaum sab xis ntawm lub radial axis txuas lub meristem nruab nrab thiab qhov nruab nrab ntawm cov phab ntsa tshiab ntawm tes (Fig. 5e-i) thiab pom qhov pom tseeb ntawm cov hlwb sib faib ntawm kaum ze li ntawm 90 ° txheeb ze rau lub radial axis, nrog qhov siab tshaj plaws frequencies pom ntawm 703-80 ° (90 ° ~ 208%) (22.62%) (Fig. 5e, i), sib thooj rau cell divisions nyob rau hauv lub circumferential/transverse kev taw qhia (Fig. 5h). Txhawm rau tshuaj xyuas qhov kev koom tes ntawm GA qhov taw qhia rau qhov kev faib tawm ntawm tes no, peb tau txheeb xyuas cov cell division tsis nyob hauv IPR thiab tsis yog-IPR cais (Daim duab 5i). Peb pom tias kev faib lub kaum sab xis hauv IPR cov hlwb txawv ntawm qhov tsis yog IPR cov hlwb lossis hauv cov hlwb hauv tag nrho SAM, nrog cov IPR cov hlwb nthuav tawm ntau dua ntawm kev faib tawm sab nraud / voj voog ntawm tes, piv txwv li, 70–80° thiab 80–90° (33.86% thiab 30.71%, feem, 5). Yog li, peb cov kev soj ntsuam tau nthuav tawm ib qho kev sib koom ua ke ntawm siab GA signaling thiab lub cell division dav hlau orientation ze rau lub circumferential kev taw qhia, zoo ib yam li kev sib raug zoo ntawm GA signaling kev ua si thiab kev loj hlob anisotropy (Fig. 5c, d). Txhawm rau tsim kom muaj kev txuag chaw ntxiv ntawm lub koom haum no, peb tau ntsuas qhov kev faib dav dav ntawm kev taw qhia hauv IPR cov hlwb nyob ib puag ncig lub primordium pib los ntawm P3, txij li qhov siab tshaj plaws GA signaling ua haujlwm tau pom nyob rau hauv cheeb tsam no pib ntawm P4 (Fig. 4). Kev faib cov kaum sab xis ntawm IPR nyob ib ncig ntawm P3 thiab P4 tsis pom qhov sib txawv tseem ceeb, txawm hais tias muaj kev sib faib ntawm cov xov tooj ntawm sab nraud tau pom hauv IPR ib ncig ntawm P4 (Daim duab 5j). Txawm li cas los xij, hauv IPR cov hlwb nyob ib ncig ntawm P5, qhov sib txawv ntawm kev taw qhia ntawm lub xov tooj ntawm tes faib dav hlau tau dhau los ua qhov tseem ceeb, nrog kev nce ntxiv ntawm qhov zaus ntawm kev sib faib ntawm tes (Fig. 5j). Ua ke, cov txiaj ntsig no qhia tias GA signaling tuaj yeem tswj kev taw qhia ntawm kev faib tawm ntawm tes hauv SAM, uas yog raws li cov ntaub ntawv dhau los 40,41 tias GA signaling siab tuaj yeem ua rau muaj kev taw qhia sab nraud ntawm kev faib cell hauv IPR.
Nws tau kwv yees tias cov hlwb hauv IPR yuav tsis raug muab tso rau hauv primordia tab sis theej rau hauv internodes2,42,43. Kev hloov pauv ntawm kev faib tawm ntawm tes hauv IPR tuaj yeem ua rau lub koom haum raug cai ntawm cov kab sib txuas ntev ntawm cov kab mob epidermal hauv internodes. Peb cov kev soj ntsuam tau piav qhia saum toj no qhia tias GA teeb liab yuav ua lub luag haujlwm hauv cov txheej txheem no los ntawm kev tswj cov kev taw qhia ntawm cell division.
Kev poob haujlwm ntawm ntau DELLA noob ua rau muaj kev sib koom ua ke GA teb, thiab della mutants tuaj yeem siv los ntsuas qhov kev xav no44. Peb thawj zaug txheeb xyuas cov qauv qhia ntawm tsib DELLA noob hauv SAM. Transcriptional fusion ntawm GUS kab 45 tau qhia tias GAI, RGA, RGL1, thiab RGL2 (rau qhov tsawg dua) tau qhia hauv SAM (Cov Lus Ntxiv 11a–d). Nyob rau hauv situ hybridization ntxiv qhia tau hais tias GAI mRNA accumulates tshwj xeeb tshaj yog nyob rau hauv primordia thiab tsim paj (Ntxiv Fig. 11e). RGL1 thiab RGL3 mRNA raug kuaj pom thoob plaws hauv SAM canopy thiab hauv cov paj laus, qhov RGL2 mRNA muaj ntau dua nyob rau thaj tsam ciam teb (Cov duab ntxiv 11f–h). Confocal imaging ntawm pRGL3::RGL3-GFP SAM tau lees paub qhov kev qhia tau pom los ntawm hauv situ hybridization thiab pom tias RGL3 protein accumulates nyob rau hauv nruab nrab ntawm lub SAM (Sab Ntxiv Fig. 11i). Siv cov pRGA::GFP-RGA kab, peb kuj pom tias RGA protein ntau nyob rau hauv lub SAM, tab sis nws abundance txo nyob rau hauv ciam teb pib ntawm P4 (Ntxiv daim duab 11j). Qhov tseem ceeb, cov qauv qhia ntawm RGL3 thiab RGA yog ib qho kev ua haujlwm siab dua GA signaling hauv IPR, raws li pom los ntawm qmRGA (Fig. 4). Ntxiv mus, cov ntaub ntawv no qhia tau hais tias tag nrho DELLAs tau nthuav tawm hauv SAM thiab tias lawv cov lus qhia sib sau ua ke rau tag nrho SAM.
Peb tom ntej no soj ntsuam cov cell division tsis nyob rau hauv cov qus-hom SAM (Ler, tswj) thiab cov gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Fig. 6a, b). Interestingly, peb tau pom qhov kev hloov pauv tseem ceeb hauv kev faib tawm ntawm tes faib lub kaum ntse ntse hauv della ntiaj teb mutant SAM piv rau hom tsiaj qus (Fig. 6c). Qhov kev hloov pauv no hauv della ntiaj teb mutant yog vim qhov nce ntawm 80-90 ° kaum (34.71% vs. 24.55%) thiab, rau qhov tsawg dua, 70-80 ° kaum (23.78% vs. 20.18%), piv txwv li, sib piv rau transverseg.6 cell divisions. Qhov zaus ntawm kev sib faib tsis hloov pauv (0–60 °) kuj qis dua hauv della ntiaj teb mutant (Fig. 6c). Qhov zaus ntawm transverse cell divisions tau nce ntxiv hauv SAM ntawm della global mutant (Fig. 6b). Qhov zaus ntawm transverse cell divisions nyob rau hauv lub IPR kuj yog siab dua nyob rau hauv lub della ntiaj teb no mutant piv rau cov qus hom (Fig. 6d). Sab nraum IPR cheeb tsam, hom tsiaj qus muaj kev faib tawm ntawm tes faib cov ces kaum, qhov della ntiaj teb mutant nyiam tangential faib zoo li IPR (Fig. 6e). Peb kuj tau txheeb xyuas qhov kev taw qhia ntawm kev faib cell hauv SAM ntawm ga2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, thiab ga2ox6-2), GA-inactive mutant keeb kwm uas GA accumulates. Raws li kev nce qib hauv GA, SAM ntawm quintuple ga2ox mutant inflorescence loj dua li ntawm Col-0 (Supplementary Fig. 12a, b), thiab piv rau Col-0, quintuple ga2ox SAM pom qhov sib txawv ntawm qhov sib txawv ntawm cov cell division kaum sab xis, nrog rau lub kaum sab xis dua 50 ° frequency. tangential divisions (Ntxiv daim duab 12a–c). Yog li, peb qhia tau hais tias kev ua kom muaj txiaj ntsig ntawm GA signaling thiab GA tsub zuj zuj ua rau muaj kev sib faib ntawm tes hauv IPR thiab lwm yam ntawm SAM.
a, b 3D visualization ntawm L1 txheej ntawm PI-stained Ler (a) thiab ntiaj teb della mutant (b) SAM siv confocal microscopy. Tshiab cell phab ntsa tsim nyob rau hauv SAM (tab sis tsis yog lub primordium) nyob rau hauv 10-h lub sij hawm yog qhia thiab xim raws li lawv lub kaum sab xis qhov tseem ceeb. Lub inset qhia SAM ntawm 0 h. Cov xim bar yog tso tawm nyob rau hauv lub kaum sab xis sab xis. Cov xub hauv (b) taw qhia rau ib qho piv txwv ntawm cov ntaub ntawv xovtooj sib raug zoo hauv ntiaj teb della mutant. Qhov kev sim tau rov ua dua ob zaug nrog cov txiaj ntsig zoo sib xws. ce kev sib piv ntawm cov zaus faib ntawm cell division dav hlau orientations nyob rau hauv tag nrho SAM (d), IPR (e), thiab non-IPR (f) ntawm Ler thiab ntiaj teb no della. P qhov txiaj ntsig tau txais los ntawm kev xeem ob-tailed Kolmogorov-Smirnov. f, g 3D visualization ntawm confocal dluab ntawm PI-stained SAM ntawm Col-0 (i) thiab pCUC2::gai-1-VENUS (j) transgenic nroj tsuag. Panels (a, b) qhia cov phab ntsa tshiab ntawm tes (tab sis tsis yog primordia) tsim hauv SAM hauv 10 teev. Qhov kev sim tau rov ua dua ob zaug nrog cov txiaj ntsig zoo sib xws. h–j Kev sib piv ntawm qhov zaus ntawm kev faib cov xov tooj ntawm tes dav hlau taw qhia nyob rau hauv tag nrho SAM (h), IPR (i) thiab non-IPR (j) ntawm Col-0 thiab pCUC2::gai-1-VENUS cov nroj tsuag. P qhov txiaj ntsig tau txais los ntawm kev sim ob-tailed Kolmogorov-Smirnov.
Peb tom ntej no sim cov txiaj ntsig ntawm inhibiting GA signaling tshwj xeeb hauv IPR. Txog rau qhov kawg no, peb siv cov cotyledon khob 2 (CUC2) tus txhawb nqa los tsav kev qhia ntawm qhov tsis zoo gai-1 protein fused rau VENUS (hauv pCUC2:: gai-1-VENUS kab). Hauv cov tsiaj qus-hom SAM, CUC2 tus neeg txhawb nqa tsav kev qhia ntawm feem ntau IPRs hauv SAM, suav nrog ciam teb hlwb, los ntawm P4 mus ntxiv, thiab cov lus qhia tshwj xeeb zoo sib xws tau pom hauv pCUC2::gai-1-VENUS cov nroj tsuag (saib hauv qab). Kev faib tawm ntawm tes faib cov kaum sab xis hla SAM lossis IPR ntawm pCUC2::gai-1-VENUS cov nroj tsuag tsis txawv ntawm cov tsiaj qus, txawm tias poob nthav peb pom tias cov hlwb tsis muaj IPR hauv cov nroj tsuag no tau muab faib ua ntau zaus ntawm 80-90 ° (Fig. 6f-j).
Nws tau raug pom tias cov kev taw qhia ntawm cell division nyob ntawm qhov geometry ntawm SAM, tshwj xeeb tshaj yog cov tensile stress generated los ntawm cov ntaub so ntswg curvature46. Yog li ntawd peb thiaj nug seb tus qauv ntawm SAM puas tau hloov nyob rau hauv della ntiaj teb no mutant thiab pCUC2::gai-1-VENUS nroj tsuag. Raws li tau tshaj tawm yav dhau los12, qhov loj ntawm della ntiaj teb mutant SAM yog loj dua li cov tsiaj qus (Ntxiv Fig. 13a, b, d). Nyob rau hauv situ hybridization ntawm CLV3 thiab STM RNA tau lees paub qhov meristem expansion nyob rau hauv della mutants thiab ntxiv qhia lub lateral expansion ntawm qia cell niche (Ntxiv daim duab 13e, f, h, i). Txawm li cas los xij, SAM curvature tau zoo sib xws hauv ob qho tib si genotypes (Ntxiv daim duab 13k, m, n, p). Peb tau pom qhov zoo sib xws ntawm qhov loj me hauv gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant yam tsis muaj kev hloov pauv hauv curvature piv rau hom tsiaj qus (Ntxiv Fig. 13c, d, g, j, l, o, p). Qhov zaus ntawm kev faib ntawm tes taw qhia kuj raug cuam tshuam rau hauv della quadruple mutant, tab sis kom tsawg dua li ntawm della monolithic mutant (Ntxiv daim duab 12d–f). Qhov kev siv ntau npaum no, nrog rau qhov tsis muaj qhov cuam tshuam ntawm curvature, qhia tias qhov seem RGL3 kev ua haujlwm hauv Della quadruple mutant txwv kev hloov pauv hauv cell division orientation los ntawm kev poob ntawm DELLA kev ua ub no thiab cov kev hloov pauv hauv cov kev faib tawm ntawm tes tshwm sim hauv kev teb rau cov kev hloov hauv GA signaling kev ua es tsis yog kev hloov hauv SAM geometry. Raws li tau piav qhia saum toj no, CUC2 tus neeg txhawb nqa tsav IPR kev qhia hauv SAM pib ntawm P4 (Supplementary Fig. 14a, b), thiab qhov sib txawv, lub pCUC2::gai-1-VENUS SAM tau txo qhov loj me tab sis siab dua curvature (Ntxiv Fig. 14c–h). Qhov kev hloov pauv no hauv pCUC2::gai-1-VENUS SAM morphology yuav ua rau muaj kev sib txawv ntawm kev sib txawv ntawm cov neeg kho tshuab kev nyuab siab piv rau cov tsiaj qus, uas qhov kev ntxhov siab nyob rau hauv siab pib ntawm qhov luv luv ntawm SAM center47. Xwb, cov kev hloov pauv hauv pCUC2::gai-1-VENUS SAM morphology tuaj yeem tshwm sim los ntawm kev hloov pauv hauv cheeb tsam cov khoom siv hluav taws xob cuam tshuam los ntawm transgene expression48. Nyob rau hauv ob qho tib si, qhov no tuaj yeem cuam tshuam qhov cuam tshuam ntawm kev hloov pauv hauv GA signaling los ntawm kev ua kom cov hlwb yuav sib faib nyob rau hauv lub voj voog / kev hloov pauv, piav qhia peb cov kev soj ntsuam.
Ua ke, peb cov ntaub ntawv lees paub tias siab dua GA signaling plays lub luag haujlwm tseem ceeb hauv kev taw qhia sab nraud ntawm lub xov tooj ntawm tes faib dav hlau hauv IPR. Lawv kuj qhia tau hais tias meristem curvature kuj cuam tshuam rau kev taw qhia ntawm cell division dav hlau hauv IPR.
Lub transverse orientation ntawm lub dav hlau division nyob rau hauv lub IPR, vim yog siab GA signaling kev ua, qhia tias GA pre-organizes ib tug radial cell ntaub ntawv nyob rau hauv lub epidermis nyob rau hauv lub SAM los txhais lub cellular lub koom haum uas yuav tom qab pom nyob rau hauv lub epidermal internode. Tseeb tiag, cov ntaub ntawv xovtooj ntawm tes tau pom ntau zaus hauv SAM cov duab ntawm della global mutants (Fig. 6b). Yog li, txhawm rau tshawb nrhiav kev loj hlob ntxiv ntawm cov qauv spatial ntawm GA signaling hauv SAM, peb siv sijhawm-lapse duab los tshuaj xyuas lub koom haum spatial ntawm cov hlwb hauv IPR hauv hom tsiaj qus (Ler thiab Col-0), della ntiaj teb mutants, thiab pCUC2:: gai-1-VENUS transgenic nroj tsuag.
Peb pom tias qmRGA tau pom tias GA qhov taw qhia kev ua haujlwm hauv IPR tau nce los ntawm P1 / P2 thiab nce siab ntawm P4, thiab tus qauv no tseem nyob tas li nyob rau lub sijhawm (Fig. 4a–f thiab Ntxiv Fig. 8c–f, k). Txhawm rau txheeb xyuas lub koom haum spatial ntawm cov hlwb hauv IPR nrog nce GA teeb liab, peb sau npe Ler IPR hlwb saum toj no thiab rau sab ntawm P4 raws li lawv txoj hmoo kev loj hlob tau soj ntsuam 34 h tom qab kev soj ntsuam thawj zaug, piv txwv li, ntau tshaj ob lub sij hawm plastid, cia peb ua raws li IPR hlwb thaum lub sij hawm primordium txoj kev loj hlob ntawm P1 / P2 rau P4. Peb siv peb cov xim sib txawv: daj rau cov hlwb uas tau koom ua ke rau hauv cov primordium ze P4, ntsuab rau cov uas nyob hauv IPR, thiab ntshav rau cov uas koom nrog ob txoj kev (Fig. 7a–c). Ntawm t0 (0 h), 1-2 txheej ntawm IPR hlwb tau pom nyob rau pem hauv ntej ntawm P4 (Fig. 7a). Raws li qhov xav tau, thaum cov hlwb sib faib, lawv tau ua ntau dhau ntawm lub dav hlau transverse division (Fig. 7a–c). Cov txiaj ntsig zoo sib xws tau txais los ntawm kev siv Col-0 SAM (tsom rau P3, uas nws ciam teb zoo ib yam li P4 hauv Ler), txawm hais tias nyob rau hauv cov genotype no cov quav tsim ntawm cov paj ciam teb zais cov IPR hlwb ntau dua (Fig. 7g–i). Yog li, cov qauv faib ntawm IPR hlwb ua ntej npaj cov hlwb rau hauv kab radial, xws li hauv internodes. Lub koom haum ntawm cov kab radial thiab thaj chaw ntawm IPR cov hlwb ntawm cov kab mob ua tau zoo qhia tias cov hlwb no yog cov kab mob internodal.
Ntawm no, peb tau tsim qhov ratiometric GA signaling biosensor, qmRGA, uas tso cai rau kom muaj nuj nqis ntawm GA signaling kev ua los ntawm kev sib xyaw GA thiab GA receptor concentrations thaum txo kev cuam tshuam nrog endogenous signaling pathways, yog li muab cov ntaub ntawv ntawm GA muaj nuj nqi ntawm cov cellular. Txog rau qhov kawg no, peb tau tsim kho DELLA protein, mRGA, uas tau poob lub peev xwm los khi DELLA cov koom tes sib koom tes tab sis tseem muaj kev cuam tshuam rau GA-induced proteolysis. qmRGA teb rau ob qho tib si exogenous thiab endogenous hloov nyob rau hauv GA qib, thiab nws dynamic sensing zog pab kev ntsuam xyuas ntawm spatiotemporal hloov nyob rau hauv GA signaling kev ua si thaum lub sij hawm txoj kev loj hlob. qmRGA kuj yog ib qho cuab yeej hloov tau yooj yim heev vim nws tuaj yeem hloov kho rau cov ntaub so ntswg sib txawv los ntawm kev hloov cov neeg txhawb nqa siv rau nws cov lus qhia (yog tias tsim nyog), thiab muab qhov kev txuag ntawm GA teeb liab txoj hauv kev thiab PFYRE motif hla angiosperms, nws yog qhov yuav hloov mus rau lwm hom 22. Raws li qhov no, qhov sib npaug ntawm kev hloov pauv hauv cov nplej SLR1 DELLA protein (HYY497AAA) kuj tau pom tias muaj kev cuam tshuam kev loj hlob ntawm kev tawm tsam ntawm SLR1 thaum tsuas yog txo qis nws cov GA-mediated degradation, zoo ib yam li mRGA23. Qhov tseem ceeb, cov kev tshawb fawb tsis ntev los no hauv Arabidopsis tau pom tias ib qho kev hloov pauv ntawm cov amino acid nyob rau hauv PFYRE domain (S474L) hloov pauv cov kev ua haujlwm ntawm RGA yam tsis muaj kev cuam tshuam rau nws lub peev xwm los cuam tshuam nrog cov neeg koom tes sib koom ua ke50. Txawm hais tias qhov kev hloov pauv no nyob ze rau ntawm 3 amino acid hloov pauv tam sim no hauv mRGA, peb cov kev tshawb fawb qhia tias ob qhov kev hloov pauv no hloov cov yam ntxwv ntawm DELLA. Txawm hais tias feem ntau cov neeg koom tes sib sau ua ke khi rau LHR1 thiab SAW domains ntawm DELLA26,51, qee cov amino acids khaws cia hauv PFYRE domain tuaj yeem pab tswj cov kev sib cuam tshuam no.
Internode txoj kev loj hlob yog ib qho tseem ceeb hauv cov nroj tsuag architecture thiab yield txhim kho. qmRGA nthuav tawm ntau dua GA signaling kev ua hauv IPR internode progenitor hlwb. Los ntawm kev sib txuas cov duab ntau thiab cov noob caj noob ces, peb tau pom tias GA cov cim qhia cov qauv superimpose voj voog / transverse cell division dav hlau nyob rau hauv SAM epidermis, shaping lub cell division lub koom haum xav tau rau internode txoj kev loj hlob. Ob peb tus tswj hwm ntawm cell division dav hlau taw qhia tau raug txheeb xyuas thaum lub sij hawm kev loj hlob52,53. Peb txoj haujlwm muab ib qho piv txwv meej txog yuav ua li cas GA signaling kev ua ub no tswj cov cellular parameter. DELLA tuaj yeem cuam tshuam nrog prefolding protein complexes41, yog li GA signaling tuaj yeem tswj hwm cell division plane orientation los ntawm kev cuam tshuam ncaj qha rau cortical microtubule orientation40,41,54,55. Peb tau npaj txhij txog pom tias hauv SAM, qhov sib cuam tshuam ntawm GA teeb liab siab dua tsis yog cell elongation lossis faib, tab sis tsuas yog kev loj hlob anisotropy, uas yog ib qho kev cuam tshuam ncaj qha ntawm GA ntawm kev taw qhia ntawm cell division hauv IPR. Txawm li cas los xij, peb tsis tuaj yeem cais tawm tias qhov kev cuam tshuam no tuaj yeem ua tsis ncaj, piv txwv li kev kho los ntawm GA-induced cell phab ntsa softening56. Cov kev hloov pauv hauv cov phab ntsa ntawm tes ua rau muaj kev ntxhov siab rau cov neeg kho tshuab57,58, uas tuaj yeem cuam tshuam rau kev taw qhia ntawm lub xov tooj ntawm tes faib dav hlau los ntawm kev cuam tshuam rau kev taw qhia ntawm cortical microtubules39,46,59. Kev sib xyaw ua ke ntawm GA-vim kev ntxhov siab thiab kev tswj hwm ncaj qha ntawm microtubule kev taw qhia los ntawm GA tuaj yeem koom nrog hauv kev tsim cov qauv tshwj xeeb ntawm cell division orientation hauv IPR los txhais cov internodes, thiab cov kev tshawb fawb ntxiv yog xav tau los ntsuas lub tswv yim no. Ib yam li ntawd, cov kev tshawb fawb yav dhau los tau hais txog qhov tseem ceeb ntawm DELLA-interacting proteins TCP14 thiab 15 hauv kev tswj hwm ntawm internode formation60,61 thiab cov yam ntxwv no tuaj yeem kho qhov kev txiav txim ntawm GA ua ke nrog BREVIPEDICELLUS (BP) thiab PENNYWISE (PNY), uas tswj kev txhim kho internode thiab tau pom tias muaj kev cuam tshuam, 2 GA teeb liab. Muab hais tias DELLAs cuam tshuam nrog brassinosteroid, ethylene, jasmonic acid, thiab abscisic acid (ABA) qhia txoj hauv kev 63,64 thiab cov tshuaj hormones no tuaj yeem cuam tshuam rau microtubule orientation65, cov teebmeem ntawm GA ntawm cell division orientation kuj tuaj yeem kho los ntawm lwm cov tshuaj hormones.
Cov kev tshawb fawb cytological thaum ntxov tau pom tias ob qho tib si sab hauv thiab sab nrauv ntawm Arabidopsis SAM yog xav tau rau kev txhim kho internode2,42. Qhov tseeb tias GA nquag tswj kev faib tawm ntawm tes hauv cov ntaub so ntswg 12 txhawb kev ua haujlwm ntawm GA hauv kev tswj meristem thiab internode loj hauv SAM. Tus qauv ntawm kev faib cov xov tooj ntawm tes kuj tseem tswj nruj rau hauv cov ntaub so ntswg SAM, thiab cov kev cai no yog qhov tseem ceeb rau qia kev loj hlob52. Nws yuav txaus siab los tshuaj xyuas seb GA kuj tseem ua lub luag haujlwm hauv kev taw qhia lub dav hlau faib ntawm tes hauv lub koom haum SAM sab hauv, yog li synchronizing cov lus qhia thiab kev txhim kho ntawm internodes hauv SAM.
Cov nroj tsuag tau cog rau hauv vitro hauv av lossis 1x Murashige-Skoog (MS) nruab nrab (Duchefa) ntxiv nrog 1% sucrose thiab 1% agar (Sigma) raws li cov txheej txheem (16 h teeb, 22 ° C), tshwj tsis yog rau kev sim hypocotyl thiab hauv paus kev loj hlob uas cov noob tau cog rau ntawm daim phiaj ntsug hauv qab lub teeb thiab 22 ° C. Rau kev sim nitrate, cov nroj tsuag tau cog rau ntawm kev hloov kho MS nruab nrab (bioWORLD cog nruab nrab) ntxiv nrog cov nitrate txaus (0 lossis 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose thiab 1% A-agar (Sigma) nyob rau hauv cov hnub ntev.
GID1a cDNA tso rau hauv pDONR221 yog recombined nrog pDONR P4-P1R-pUBQ10 thiab pDONR P2R-P3-mCherry rau hauv pB7m34GW los tsim pUBQ10::GID1a-mCherry. IDD2 DNA tso rau hauv pDONR221 tau rov ua dua rau hauv pB7RWG266 los tsim p35S:IDD2-RFP. Txhawm rau tsim pGID1b::2xmTQ2-GID1b, ib qho 3.9 kb fragment upstream ntawm GID1b coding cheeb tsam thiab 4.7 kb fragment uas muaj GID1b cDNA (1.3 kb) thiab terminator (3.4 kb) tau ua ntej amplified siv cov primers NR muab tso rau hauv Supplement. P4-P1R (Thermo Fisher Scientific) thiab pDONR P2R-P3 (Thermo Fisher Scientific), feem, thiab thaum kawg recombined nrog pDONR221 2xmTQ268 rau hauv pGreen 012567 lub hom phiaj vector siv Gateway cloning. Txhawm rau tsim pCUC2::LSSmOrange, CUC2 txhawb nqa ib ntus (3229 bp nce toj ntawm ATG) ua raws li cov txheej txheem coding ntawm Stokes-hloov mOrange (LSSmOrange)69 nrog N7 nuclear localization teeb liab thiab NOS transcriptional terminator tau sib sau ua ke rau hauv pGreen fractioning 3 vector. recombination system (Invitrogen). Cov nroj tsuag binary vector tau nkag mus rau hauv Agrobacterium tumefaciens strain GV3101 thiab nkag rau hauv Nicotiana benthamiana nplooj los ntawm Agrobacterium infiltration method thiab mus rau Arabidopsis thaliana Col-0 los ntawm floral dip method, feem. pUBQ10::qmRGA pUBQ10::GID1a-mCherry thiab pCLV3::mCherry-NLS qmRGA raug cais tawm ntawm F3 thiab F1 progenies ntawm tus ntoo khaub lig, raws li.
RNA nyob rau hauv situ hybridization tau ua ntawm kwv yees li 1 cm ntev tua cov lus qhia 72, uas tau sau thiab kho tam sim ntawd hauv FAA tov (3.7% formaldehyde, 5% acetic acid, 50% ethanol) ua ntej txias rau 4 ° C. Tom qab 2 × 15 feeb kev kho lub tshuab nqus tsev, cov khoom kho tau hloov pauv thiab cov qauv raug tsim tawm thaum hmo ntuj. GID1a, GID1b, GID1c, GAI, RGL1, RGL2, thiab RGL3 cDNAs thiab antisense probes rau lawv cov 3′-UTRs tau tsim los siv cov primers uas qhia nyob rau hauv Supplementary Table 3 raws li piav los ntawm Rosier li al.73. Digoxigenin-labeled probes tau txhaj tshuaj tiv thaiv kab mob uas siv cov tshuaj digoxigenin antibodies (3000-fold dilution; Roche, tus lej catalog: 11 093 274 910), thiab ntu tau stained nrog 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 2-foldlue-trosphate) (NBT, 200-fold dilution) daws.
Lub sij hawm xa tuaj: Feb-10-2025