Kev ntsuas Gibberellin Biosensor qhia txog lub luag haujlwm ntawm Gibberellins hauv Internode Specification hauv Shoot Apical Meristem

Kev loj hlob ntawm cov noob caj noob ces (SAM) yog qhov tseem ceeb rau kev tsim cov qia. Cov tshuaj hormones ntawm cov nroj tsuaggibberellins(GAs) ua lub luag haujlwm tseem ceeb hauv kev sib koom tes ntawm cov nroj tsuag loj hlob, tab sis lawv lub luag haujlwm hauv SAM tseem tsis tau nkag siab zoo. Ntawm no, peb tau tsim ib lub biosensor ratiometric ntawm GA signaling los ntawm kev tsim cov protein DELLA kom tswj nws txoj haujlwm tseem ceeb hauv GA transcriptional teb thaum khaws nws qhov kev puas tsuaj thaum GA lees paub. Peb qhia tau tias lub biosensor raws li kev puas tsuaj no sau cov kev hloov pauv hauv GA theem thiab kev hnov ​​​​​​ntawm lub hlwb thaum lub sijhawm txhim kho. Peb siv lub biosensor no los kos duab GA signaling kev ua ub no hauv SAM. Peb qhia tau tias cov teeb liab GA siab muaj nyob rau hauv cov hlwb nyob nruab nrab ntawm lub cev primordia, uas yog cov precursors rau internode hlwb. Siv cov kev nce-thiab-kev poob-ntawm-kev ua haujlwm, peb qhia ntxiv tias GA tswj hwm qhov kev taw qhia ntawm lub dav hlau faib cell, tsim kom muaj lub koom haum cellular canonical ntawm internodes, yog li txhawb nqa internode specification hauv SAM.
Lub shoot apical meristem (SAM), nyob ntawm lub apex ntawm tua, muaj ib lub niche ntawm cov stem cell uas nws cov haujlwm tsim cov lateral organ thiab stem nodes hauv ib qho modular thiab iterative txoj kev thoob plaws lub neej ntawm cov nroj tsuag. Txhua lub repeating units, lossis cov nroj tsuag nodes, suav nrog internodes thiab lateral organ ntawm cov nodes, thiab axillary meristems hauv cov nplooj axils1. Kev loj hlob thiab kev teeb tsa ntawm cov nroj tsuag nodes hloov pauv thaum lub sijhawm loj hlob. Hauv Arabidopsis, kev loj hlob internodal raug suppressed thaum lub sijhawm vegetative, thiab axillary meristems tseem dormant hauv axils ntawm nplooj rosette. Thaum lub sijhawm hloov mus rau theem paj, SAM dhau los ua inflorescence meristem, tsim cov internodes ntev thiab axillary buds, branchlets hauv axils ntawm nplooj cauline, thiab tom qab ntawd, leafless paj2. Txawm hais tias peb tau ua tiav ntau yam hauv kev nkag siab txog cov txheej txheem uas tswj kev pib ntawm nplooj, paj, thiab ceg, me ntsis paub txog yuav ua li cas internodes tshwm sim.
Kev nkag siab txog qhov kev faib tawm ntawm spatiotemporal ntawm GAs yuav pab kom nkag siab zoo dua txog cov haujlwm ntawm cov tshuaj hormones no hauv cov nqaij sib txawv thiab ntawm cov theem kev loj hlob sib txawv. Kev pom ntawm qhov kev puas tsuaj ntawm RGA-GFP fusion uas qhia nyob rau hauv qhov kev ua ntawm nws tus kheej promoter muab cov ntaub ntawv tseem ceeb ntawm kev tswj hwm tag nrho cov theem GA hauv cov hauv paus hniav15,16. Txawm li cas los xij, RGA kev qhia tawm sib txawv thoob plaws cov nqaij17 thiab raug tswj hwm los ntawm GA18. Yog li, kev qhia tawm sib txawv ntawm RGA promoter yuav ua rau muaj cov qauv fluorescence pom nrog RGA-GFP thiab yog li txoj kev no tsis yog qhov ntau. Tsis ntev los no, bioactive fluorescein (Fl)-labeled GA19,20 tau qhia txog kev sib sau ua ke ntawm GA hauv cov hauv paus hniav endocortex thiab kev tswj hwm ntawm nws cov theem cellular los ntawm GA thauj. Tsis ntev los no, GA FRET sensor nlsGPS1 tau qhia tias GA theem sib raug zoo nrog kev nthuav dav ntawm tes hauv cov hauv paus hniav, filaments, thiab cov hypocotyls tsaus nti21. Txawm li cas los xij, raws li peb tau pom, GA concentration tsis yog tib qho parameter tswj hwm GA signaling kev ua ub no, vim nws nyob ntawm cov txheej txheem kev xav nyuaj. Ntawm no, los ntawm peb txoj kev nkag siab txog DELLA thiab GA signaling pathways, peb tshaj tawm txog kev tsim thiab kev piav qhia ntawm lub degradation-based ratiometric biosensor rau GA signaling. Txhawm rau tsim cov quantitative biosensor no, peb tau siv mutant GA-sensitive RGA uas tau sib xyaw nrog cov protein fluorescent thiab ubiquitously expressed hauv cov ntaub so ntswg, nrog rau GA-insensitive fluorescent protein. Peb qhia tau tias cov mutant RGA protein fusions tsis cuam tshuam nrog endogenous GA signaling thaum ubiquitously expressed, thiab tias biosensor no tuaj yeem ntsuas qhov kev ua haujlwm signaling los ntawm ob qho tib si GA input thiab GA signal processing los ntawm cov khoom siv sensing nrog qhov kev daws teeb meem spatiotemporal siab. Peb siv cov biosensor no los kos duab qhov kev faib tawm spatiotemporal ntawm GA signaling kev ua haujlwm thiab ntsuas seb GA tswj hwm tus cwj pwm ntawm tes hauv SAM epidermis li cas. Peb qhia tau tias GA tswj hwm qhov kev taw qhia ntawm lub dav hlau faib ntawm SAM hlwb nyob nruab nrab ntawm lub cev primordia, yog li txhais tau tias lub koom haum cellular canonical ntawm internode.
Thaum kawg, peb nug seb qmRGA puas tuaj yeem tshaj tawm cov kev hloov pauv hauv cov theem endogenous GA siv cov hypocotyls loj hlob. Peb tau qhia ua ntej tias nitrate txhawb kev loj hlob los ntawm kev nce GA synthesis thiab, ua rau, DELLA34 degradation. Yog li ntawd, peb tau pom tias hypocotyl ntev hauv pUBQ10::qmRGA cov noob cog qoob loo uas cog rau hauv qab cov khoom noj nitrate ntau (10 mM NO3−) ntev dua li ntawm cov noob cog qoob loo uas cog rau hauv qab cov xwm txheej tsis muaj nitrate (Daim Duab Ntxiv 6a). Sib xws nrog kev teb rau kev loj hlob, cov cim GA siab dua hauv hypocotyls ntawm cov noob cog qoob loo uas cog rau hauv qab 10 mM NO3− xwm txheej dua li cov noob cog qoob loo uas cog rau hauv qab tsis muaj nitrate (Daim Duab Ntxiv 6b, c). Yog li, qmRGA kuj tseem ua rau muaj kev saib xyuas cov kev hloov pauv hauv GA signaling uas tshwm sim los ntawm kev hloov pauv endogenous hauv GA concentration.
Yuav kom nkag siab seb qhov kev ua haujlwm ntawm GA signaling uas qmRGA ntes tau puas yog nyob ntawm GA concentration thiab GA perception, raws li xav tau raws li kev tsim sensor, peb tau tshuaj xyuas qhov kev qhia tawm ntawm peb lub GID1 receptors hauv cov nqaij vegetative thiab reproductive. Hauv cov noob, GID1-GUS reporter line qhia tau tias GID1a thiab c tau qhia tawm ntau heev hauv cotyledons (Daim duab 3a-c). Tsis tas li ntawd, tag nrho peb lub receptors tau qhia tawm hauv nplooj, lateral root primordia, cov hauv paus hniav (tshwj tsis yog lub hauv paus ntawm GID1b), thiab cov hlab ntsha (Daim duab 3a-c). Hauv inflorescence SAM, peb tau pom GUS signals tsuas yog rau GID1b thiab 1c (Supplementary Fig. 7a-c). In situ hybridization lees paub cov qauv kev qhia tawm no thiab qhia ntxiv tias GID1c tau qhia tawm sib npaug ntawm cov qib qis hauv SAM, thaum GID1b tau qhia tawm ntau dua ntawm qhov chaw ntawm SAM (Supplementary Fig. 7d-l). Qhov pGID1b::2xmTQ2-GID1b kev sib txuas lus kuj tau qhia txog ntau yam kev qhia tawm ntawm GID1b, los ntawm qis lossis tsis muaj kev qhia tawm hauv nruab nrab ntawm SAM mus rau kev qhia tawm siab ntawm cov ciam teb ntawm lub cev (Daim Duab Ntxiv 7m). Yog li, GID1 receptors tsis tau faib sib npaug thoob plaws thiab hauv cov ntaub so ntswg. Hauv kev sim tom qab, peb kuj tau pom tias kev qhia tawm ntau dhau ntawm GID1 (pUBQ10::GID1a-mCherry) ua rau muaj kev nkag siab ntawm qmRGA hauv hypocotyls rau kev siv GA sab nraud (Daim Duab 3d, e). Qhov sib piv, fluorescence ntsuas los ntawm qd17mRGA hauv hypocotyl tsis muaj kev cuam tshuam rau kev kho GA3 (Daim Duab 3f, g). Rau ob qho kev sim, cov noob tau kho nrog cov concentration siab ntawm GA (100 μM GA3) los ntsuas tus cwj pwm sai ntawm lub sensor, qhov twg lub peev xwm los khi rau GID1 receptor tau txhim kho lossis ploj mus. Ua ke, cov txiaj ntsig no lees paub tias qmRGA biosensor ua haujlwm ua ke ua GA thiab GA sensor, thiab qhia tias qhov kev qhia sib txawv ntawm GID1 receptor tuaj yeem hloov kho qhov emissivity ntawm lub sensor.
Txog niaj hnub no, qhov kev faib tawm ntawm GA cov cim hauv SAM tseem tsis meej. Yog li ntawd, peb siv cov nroj tsuag qmRGA-expressing thiab pCLV3::mCherry-NLS stem cell reporter35 los xam cov ntawv qhia txog kev ua haujlwm GA signaling siab, tsom mus rau L1 txheej (epidermis; Daim duab 4a, b, saib Cov Txheej Txheem thiab Cov Txheej Txheem Ntxiv), txij li L1 ua lub luag haujlwm tseem ceeb hauv kev tswj hwm SAM kev loj hlob36. Ntawm no, pCLV3::mCherry-NLS kev qhia tau muab qhov chaw siv geometric ruaj khov rau kev tshuaj xyuas qhov kev faib tawm spatiotemporal ntawm GA signaling kev ua haujlwm37. Txawm hais tias GA raug suav hais tias tseem ceeb rau kev loj hlob ntawm lub cev sab nraud4, peb pom tias GA cov cim qis hauv paj primordium (P) pib los ntawm theem P3 (Daim duab 4a, b), thaum cov tub ntxhais hluas P1 thiab P2 primordiums muaj kev ua haujlwm nruab nrab zoo ib yam li hauv thaj chaw nruab nrab (Daim duab 4a, b). Kev ua haujlwm GA signaling siab dua tau pom ntawm cov ciam teb ntawm lub cev primordium, pib ntawm P1/P2 (ntawm ob sab ntawm ciam teb) thiab nce mus txog P4, nrog rau hauv txhua lub hlwb ntawm thaj chaw nyob nruab nrab ntawm primordia (Daim duab 4a, b thiab Daim duab ntxiv 8a, b). Kev ua haujlwm GA signaling siab dua no tau pom tsis yog hauv epidermis xwb tab sis kuj nyob rau hauv L2 thiab L3 txheej sab saud (Daim duab ntxiv 8b). Tus qauv ntawm cov teeb liab GA pom hauv SAM siv qmRGA kuj tseem tsis hloov pauv dhau sijhawm (Daim duab ntxiv 8c-f, k). Txawm hais tias qd17mRGA construct tau downregulated hauv SAM ntawm T3 cov nroj tsuag los ntawm tsib kab ywj pheej uas peb tau piav qhia meej, peb tau tuaj yeem tshuaj xyuas cov qauv fluorescence tau txais nrog pRPS5a::VENUS-2A-TagBFP construct (Daim duab ntxiv 8g-j, l). Hauv kab tswj no, tsuas yog muaj kev hloov me me ntawm qhov piv ntawm fluorescence xwb hauv SAM, tab sis hauv SAM center peb pom qhov txo qis meej thiab tsis tau xav txog hauv VENUS cuam tshuam nrog TagBFP. Qhov no lees paub tias tus qauv signaling uas qmRGA pom qhia txog kev puas tsuaj ntawm GA-dependent ntawm mRGA-VENUS, tab sis kuj qhia tau tias qmRGA yuav kwv yees qhov kev ua haujlwm GA signaling hauv meristem center ntau dhau. Hauv kev xaus, peb cov txiaj ntsig qhia txog tus qauv GA signaling uas feem ntau qhia txog kev faib tawm ntawm primordia. Qhov kev faib tawm ntawm thaj chaw inter-primordial (IPR) no yog vim muaj kev tsim maj mam ntawm kev ua haujlwm GA signaling siab ntawm thaj chaw tsim kho primordium thiab thaj chaw nruab nrab, thaum tib lub sijhawm GA signaling kev ua haujlwm hauv primordium txo qis (Daim duab 4c, d).
Qhov kev faib tawm ntawm GID1b thiab GID1c receptors (saib saum toj no) qhia tias qhov kev qhia sib txawv ntawm GA receptors pab tsim cov qauv ntawm GA signaling kev ua haujlwm hauv SAM. Peb xav paub seb puas yuav muaj kev sib sau ua ke ntawm GA. Txhawm rau tshawb nrhiav qhov ua tau no, peb tau siv nlsGPS1 GA FRET sensor21. Kev ua haujlwm ntau ntxiv tau pom hauv SAM ntawm nlsGPS1 kho nrog 10 μM GA4 + 7 rau 100 feeb (Daim Duab Ntxiv 9a-e), qhia tias nlsGPS1 teb rau kev hloov pauv hauv GA concentration hauv SAM, zoo li nws ua hauv cov hauv paus21. Kev faib tawm ntawm qhov chaw ntawm nlsGPS1 qhov zaus ua haujlwm tau qhia txog qib GA qis hauv cov txheej sab nraud ntawm SAM, tab sis qhia tau tias lawv tau nce siab hauv nruab nrab thiab ntawm ciam teb ntawm SAM (Daim Duab 4e thiab Daim Duab Ntxiv 9a, c). Qhov no qhia tau tias GA kuj tau faib tawm hauv SAM nrog tus qauv sib piv rau qhov uas tau qhia los ntawm qmRGA. Ua ib txoj hauv kev sib pab, peb kuj tau kho SAM nrog fluorescent GA (GA3-, GA4-, GA7-Fl) lossis Fl ib leeg ua ib qho kev tswj tsis zoo. Lub teeb liab Fl tau faib thoob plaws SAM, suav nrog thaj chaw nruab nrab thiab primordium, txawm tias muaj zog tsawg dua (Daim Duab 4j thiab Daim Duab Ntxiv 10d). Qhov sib txawv, tag nrho peb GA-Fl tau sib sau ua ke tshwj xeeb hauv ciam teb primordium thiab rau ntau qib hauv seem ntawm IPR, nrog GA7-Fl sib sau ua ke hauv thaj chaw loj tshaj plaws hauv IPR (Daim Duab 4k thiab Daim Duab Ntxiv 10a,b). Kev ntsuas ntawm qhov muaj zog fluorescence tau qhia tias qhov sib piv ntawm IPR rau qhov tsis yog IPR siab dua hauv GA-Fl-treated SAM piv rau Fl-treated SAM (Daim Duab 4l thiab Daim Duab Ntxiv 10c). Ua ke, cov txiaj ntsig no qhia tias GA muaj nyob rau ntawm qhov muaj zog ntau dua hauv IPR hlwb uas nyob ze tshaj plaws rau ciam teb ntawm lub cev. Qhov no qhia tau hais tias tus qauv ntawm SAM GA signaling kev ua ub no yog los ntawm ob qho kev qhia sib txawv ntawm GA receptors thiab kev sib sau ua ke ntawm GA hauv IPR hlwb ze ntawm ciam teb ntawm lub cev. Yog li, peb qhov kev tshuaj xyuas tau qhia txog tus qauv spatiotemporal tsis tau xav txog ntawm GA signaling, nrog rau kev ua haujlwm qis dua hauv nruab nrab thiab primordium ntawm SAM thiab kev ua haujlwm siab dua hauv IPR hauv thaj chaw peripheral.
Yuav kom nkag siab txog lub luag haujlwm ntawm kev ua haujlwm sib txawv ntawm GA signaling hauv SAM, peb tau tshuaj xyuas qhov sib raug zoo ntawm GA signaling kev ua haujlwm, kev nthuav dav ntawm tes, thiab kev faib tes siv lub sijhawm tiag tiag ntawm SAM qmRGA pCLV3::mCherry-NLS. Muab lub luag haujlwm ntawm GA hauv kev tswj kev loj hlob, kev sib raug zoo nrog cov kev ntsuas kev nthuav dav ntawm tes tau xav tias yuav muaj. Yog li ntawd, peb thawj zaug piv GA signaling kev ua haujlwm daim ntawv qhia nrog daim ntawv qhia ntawm qhov ceev ntawm qhov chaw ntawm tes (ua tus sawv cev rau lub zog ntawm kev nthuav dav ntawm tes rau ib lub tes thiab rau cov hlwb ntxhais ntawm kev faib) thiab nrog daim ntawv qhia ntawm kev loj hlob anisotropy, uas ntsuas qhov kev taw qhia ntawm kev nthuav dav ntawm tes (tseem siv ntawm no rau ib lub tes thiab rau cov hlwb ntxhais ntawm kev faib; Daim duab 5a, b, saib Cov Txheej Txheem thiab Cov Txheej Txheem Ntxiv). Peb daim ntawv qhia ntawm SAM cell qhov chaw loj hlob tus nqi yog sib xws nrog cov kev soj ntsuam yav dhau los 38,39, nrog rau qhov tsawg kawg nkaus ntawm kev loj hlob ntawm ciam teb thiab qhov siab tshaj plaws ntawm kev loj hlob hauv cov paj tsim (Daim duab 5a). Kev tshuaj xyuas cov khoom tseem ceeb (PCA) qhia tau hais tias GA signaling kev ua haujlwm tau sib raug zoo nrog qhov muaj zog ntawm qhov chaw ntawm tes (Daim duab 5c). Peb kuj tau qhia tias cov kab sib txawv tseem ceeb, suav nrog GA signaling input thiab kev loj hlob muaj zog, yog orthogonal rau qhov kev taw qhia txiav txim siab los ntawm CLV3 siab qhia, lees paub qhov kev tshem tawm ntawm cov hlwb los ntawm SAM chaw hauv cov kev tshuaj xyuas seem. Spearman correlation analysis tau lees paub cov txiaj ntsig PCA (Daim Duab 5d), qhia tias cov teeb liab GA siab dua hauv IPR tsis ua rau cov hlwb nthuav dav dua. Txawm li cas los xij, kev tshuaj xyuas correlation tau qhia txog kev sib raug zoo me ntsis ntawm GA signaling kev ua ub no thiab kev loj hlob anisotropy (Daim Duab 5c, d), qhia tias GA signaling siab dua hauv IPR cuam tshuam rau qhov kev taw qhia ntawm kev loj hlob ntawm cov hlwb thiab tej zaum qhov chaw ntawm lub dav hlau faib hlwb.
a, b Cov ntawv qhia cua sov ntawm qhov nruab nrab ntawm kev loj hlob ntawm qhov chaw (a) thiab kev loj hlob anisotropy (b) hauv SAM nruab nrab tshaj xya cov nroj tsuag ywj pheej (siv ua tus sawv cev rau lub zog thiab kev coj ntawm kev nthuav dav ntawm tes, feem). c PCA kev tshuaj xyuas suav nrog cov hloov pauv hauv qab no: GA teeb liab, qhov hnyav ntawm kev loj hlob ntawm qhov chaw, anisotropy ntawm kev loj hlob ntawm qhov chaw, thiab CLV3 kev qhia. PCA Cheebtsam 1 feem ntau yog qhov tsis zoo sib xws nrog qhov hnyav ntawm kev loj hlob ntawm qhov chaw thiab muaj feem cuam tshuam zoo nrog GA teeb liab. PCA Cheebtsam 2 feem ntau yog qhov zoo sib xws nrog qhov hnyav ntawm kev loj hlob ntawm qhov chaw thiab muaj feem cuam tshuam tsis zoo nrog CLV3 kev qhia. Feem pua ​​sawv cev rau qhov sib txawv piav qhia los ntawm txhua yam khoom. d Spearman kev tshuaj xyuas kev sib raug zoo ntawm GA teeb liab, qhov hnyav ntawm kev loj hlob ntawm qhov chaw, thiab qhov hnyav ntawm kev loj hlob ntawm qhov chaw ntawm cov ntaub so ntswg tsis suav nrog CZ. Tus lej ntawm sab xis yog tus nqi Spearman rho ntawm ob qho hloov pauv. Cov cim hnub qub qhia txog cov xwm txheej uas qhov sib raug zoo / kev sib raug zoo tsis zoo yog qhov tseem ceeb heev. e 3D pom ntawm Col-0 SAM L1 hlwb los ntawm confocal microscopy. Cov phab ntsa tshiab tsim hauv SAM (tab sis tsis yog primordium) ntawm 10 h yog xim raws li lawv cov nqi kaum sab xis. Lub kab xim tau pom nyob rau hauv kaum sab xis hauv qab. Daim duab inset qhia txog daim duab 3D sib xws ntawm 0 teev. Qhov kev sim tau rov ua dua ob zaug nrog cov txiaj ntsig zoo sib xws. f Cov phiaj xwm thawv qhia txog cov nqi faib cell hauv IPR thiab tsis yog IPR Col-0 SAM (n = 10 cov nroj tsuag ywj pheej). Kab nruab nrab qhia txog qhov nruab nrab, thiab cov ciam teb thawv qhia txog 25th thiab 75th feem pua. Whiskers qhia txog qhov tsawg kawg nkaus thiab siab tshaj plaws txiav txim siab nrog R software. P tus nqi tau txais nrog Welch's two-tailed t-test. g, h Daim duab kos qhia (g) yuav ua li cas ntsuas lub kaum sab xis ntawm phab ntsa cell tshiab (magenta) nrog rau kev taw qhia radial los ntawm qhov chaw ntawm SAM (kab dawb dotted) (tsuas yog cov nqi kaum sab xis, piv txwv li, 0–90 °, raug txiav txim siab), thiab (h) cov lus qhia circumferential / lateral thiab radial hauv meristem. i Cov histograms zaus ntawm kev faib cell dav hlau orientation hla SAM (xiav tsaus), IPR (xiav nruab nrab), thiab tsis yog IPR (xiav teeb), feem. Cov nqi P tau txais los ntawm kev sim Kolmogorov-Smirnov ob-tailed. Qhov kev sim tau rov ua ob zaug nrog cov txiaj ntsig zoo sib xws. j Cov histograms zaus ntawm kev faib cell dav hlau ntawm IPR nyob ib puag ncig P3 (ntsuab daj), P4 (ntsuab nruab nrab), thiab P5 (ntsuab tsaus), raws li. Cov nqi P tau txais los ntawm kev sim Kolmogorov-Smirnov ob-tailed. Qhov kev sim tau rov ua ob zaug nrog cov txiaj ntsig zoo sib xws.
Yog li ntawd, peb tom qab ntawd tshawb nrhiav qhov sib raug zoo ntawm GA signaling thiab cell division activity los ntawm kev txheeb xyuas cov phab ntsa cell tshiab thaum lub sijhawm assay (Daim Duab 5e). Txoj hauv kev no tso cai rau peb ntsuas qhov zaus thiab kev coj ntawm cell division. Qhov xav tsis thoob, peb pom tias qhov zaus ntawm cell divisions hauv IPR thiab lwm qhov ntawm SAM (non-IPR, Daim Duab 5f) zoo sib xws, qhia tias qhov sib txawv ntawm GA signaling ntawm IPR thiab non-IPR cell tsis cuam tshuam rau cell division. Qhov no, thiab qhov sib raug zoo ntawm GA signaling thiab kev loj hlob anisotropy, ua rau peb xav txog seb GA signaling activity puas tuaj yeem cuam tshuam rau qhov kev taw qhia ntawm cell division plane. Peb ntsuas qhov kev taw qhia ntawm phab ntsa cell tshiab ua lub kaum ntse ntse piv rau lub radial axis txuas lub meristem nruab nrab thiab lub hauv paus ntawm phab ntsa cell tshiab (Daim duab 5e-i) thiab pom tseeb tias cov cell faib ntawm cov ces kaum ze rau 90 ° piv rau lub radial axis, nrog rau cov zaus siab tshaj plaws pom ntawm 70–80 ° (23.28%) thiab 80–90 ° (22.62%) (Daim duab 5e, i), sib raug rau kev faib cell hauv qhov kev taw qhia circumferential / transverse (Daim duab 5h). Txhawm rau tshuaj xyuas qhov kev koom tes ntawm GA signaling rau tus cwj pwm faib cell no, peb tau tshuaj xyuas cov kev faib cell hauv IPR thiab tsis yog IPR sib cais (Daim duab 5i). Peb tau pom tias lub kaum sab faib hauv IPR hlwb txawv ntawm qhov hauv cov hlwb uas tsis yog IPR lossis hauv cov hlwb hauv tag nrho SAM, nrog rau IPR hlwb qhia txog qhov feem pua ​​​​​​ntawm kev faib hlwb sab nraud / ncig, piv txwv li, 70–80 ° thiab 80–90 ° (33.86% thiab 30.71%, feem, sib xws) (Daim duab 5i). Yog li, peb cov kev soj ntsuam tau qhia txog kev sib raug zoo ntawm kev tshaj tawm GA siab thiab kev taw qhia ntawm lub dav hlau faib hlwb ze rau qhov kev taw qhia ncig, zoo ib yam li kev sib raug zoo ntawm kev ua haujlwm GA thiab kev loj hlob anisotropy (Daim duab 5c, d). Txhawm rau kom paub ntxiv txog kev txuag chaw ntawm kev sib raug zoo no, peb tau ntsuas qhov kev taw qhia ntawm lub dav hlau faib hlwb hauv IPR hlwb nyob ib puag ncig primordium pib ntawm P3, txij li qhov kev ua haujlwm GA siab tshaj plaws tau pom hauv thaj chaw no pib ntawm P4 (Daim duab 4). Lub kaum sab faib ntawm IPR nyob ib puag ncig P3 thiab P4 tsis qhia txog qhov sib txawv tseem ceeb, txawm hais tias muaj qhov nce ntxiv ntawm kev faib hlwb sab nraud tau pom hauv IPR nyob ib puag ncig P4 (Daim duab 5j). Txawm li cas los xij, hauv cov hlwb IPR nyob ib puag ncig P5, qhov sib txawv ntawm kev taw qhia ntawm lub dav hlau faib hlwb tau dhau los ua qhov tseem ceeb, nrog rau kev nce ntxiv ntawm qhov zaus ntawm kev faib hlwb transverse (Daim duab 5j). Ua ke, cov txiaj ntsig no qhia tias GA signaling tuaj yeem tswj hwm kev taw qhia ntawm kev faib hlwb hauv SAM, uas yog sib xws nrog cov ntawv ceeb toom dhau los40,41 tias GA signaling siab tuaj yeem ua rau muaj kev taw qhia sab nraud ntawm kev faib hlwb hauv IPR.
Kwv yees tias cov hlwb hauv IPR yuav tsis raug koom ua ke rau hauv primordia tab sis mus rau hauv internodes2,42,43. Kev taw qhia transverse ntawm kev faib hlwb hauv IPR yuav ua rau muaj kev teeb tsa ib txwm ntawm cov kab sib luag ntawm cov hlwb epidermal hauv internodes. Peb cov kev soj ntsuam piav qhia saum toj no qhia tias GA signaling yuav ua lub luag haujlwm hauv cov txheej txheem no los ntawm kev tswj hwm kev taw qhia ntawm kev faib hlwb.
Kev poob ntawm kev ua haujlwm ntawm ntau lub noob caj noob ces DELLA ua rau muaj kev teb GA tas li, thiab cov noob caj noob ces della tuaj yeem siv los sim qhov kev xav no44. Peb thawj zaug tshuaj xyuas cov qauv kev qhia tawm ntawm tsib lub noob caj noob ces DELLA hauv SAM. Kev sib xyaw ua ke ntawm kab GUS45 tau qhia tias GAI, RGA, RGL1, thiab RGL2 (rau qhov tsawg dua) tau qhia tawm hauv SAM (Daim Duab Ntxiv 11a-d). Kev sib xyaw ua ke hauv qhov chaw tau qhia ntxiv tias GAI mRNA sib sau ua ke tshwj xeeb hauv primordia thiab cov paj uas tab tom loj hlob (Daim Duab Ntxiv 11e). RGL1 thiab RGL3 mRNA tau pom thoob plaws hauv SAM canopy thiab hauv cov paj laus dua, thaum RGL2 mRNA muaj ntau dua hauv thaj tsam ciam teb (Daim Duab Ntxiv 11f-h). Kev thaij duab confocal ntawm pRGL3::RGL3-GFP SAM tau lees paub qhov kev qhia tawm uas tau pom los ntawm kev sib xyaw ua ke hauv qhov chaw thiab qhia tias RGL3 protein sib sau ua ke hauv nruab nrab ntawm SAM (Daim Duab Ntxiv 11i). Siv cov kab pRGA::GFP-RGA, peb kuj pom tias RGA protein sib sau ua ke hauv SAM, tab sis nws qhov ntau tsawg zuj zus ntawm ciam teb pib ntawm P4 (Daim Duab Ntxiv 11j). Qhov tseem ceeb, cov qauv kev qhia tawm ntawm RGL3 thiab RGA yog sib xws nrog kev ua haujlwm GA signaling siab dua hauv IPR, raws li pom los ntawm qmRGA (Daim Duab 4). Ntxiv mus, cov ntaub ntawv no qhia tias txhua DELLAs tau qhia tawm hauv SAM thiab tias lawv qhov kev qhia tawm ua ke nthuav dav tag nrho SAM.
Tom ntej no peb tau tshuaj xyuas cov kev faib tawm ntawm tes hauv cov tsiaj qus SAM (Ler, tswj) thiab gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Daim duab 6a, b). Qhov nthuav yog, peb tau pom qhov kev hloov pauv tseem ceeb hauv kev faib tawm ntawm lub kaum sab xis ntawm tes hauv della global mutant SAM piv rau hom tsiaj qus (Daim duab 6c). Qhov kev hloov pauv no hauv della global mutant yog vim muaj kev nce ntxiv ntawm lub kaum sab xis 80–90° (34.71% vs. 24.55%) thiab, tsawg dua, lub kaum sab xis 70–80° (23.78% vs. 20.18%), piv txwv li, sib raug rau kev faib tawm ntawm tes (Daim duab 6c). Lub zaus ntawm kev faib tsis yog transverse (0–60°) kuj qis dua hauv della global mutant (Daim duab 6c). Qhov zaus ntawm kev faib cov cell transverse tau nce ntxiv ntau hauv SAM ntawm della global mutant (Daim Duab 6b). Qhov zaus ntawm kev faib cov cell transverse hauv IPR kuj siab dua hauv della global mutant piv rau hom qus (Daim Duab 6d). Sab nraum thaj tsam IPR, hom qus muaj kev faib cov ces kaum faib cell sib npaug ntau dua, thaum della global mutant nyiam kev faib tangential zoo li IPR (Daim Duab 6e). Peb kuj tau ntsuas 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), keeb kwm yav dhau los GA-inactive mutant uas GA sib sau ua ke. Raws li qhov nce ntxiv ntawm GA theem, SAM ntawm quintuple ga2ox mutant inflorescence loj dua li ntawm Col-0 (Supplementary Fig. 12a, b), thiab piv rau Col-0, quintuple ga2ox SAM tau qhia txog kev faib tawm ntawm cov ces kaum faib cell sib txawv, nrog rau qhov zaus ntawm lub kaum sab xis nce ntxiv ntawm 50° txog 90°, piv txwv li rov nyiam kev faib tangential (Supplementary Fig. 12a–c). Yog li, peb qhia tau tias kev ua haujlwm ntawm GA signaling thiab GA accumulation ua rau muaj kev faib cell sab hauv IPR thiab seem ntawm SAM.
a, b 3D pom ntawm L1 txheej ntawm PI-stained Ler (a) thiab thoob ntiaj teb della mutant (b) SAM siv confocal microscopy. Cov phab ntsa cell tshiab tsim nyob rau hauv SAM (tab sis tsis yog primordium) dhau 10-teev lub sijhawm tau pom thiab xim raws li lawv cov nqi kaum sab xis. Daim duab qhia SAM ntawm 0 teev. Lub bar xim tau tso tawm hauv kaum sab xis qis. Tus xub hauv (b) taw rau ib qho piv txwv ntawm cov ntaub ntawv cell sib phim hauv thoob 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 qhov zaus faib ntawm cell division plane orientations hauv tag nrho SAM (d), IPR (e), thiab tsis yog IPR (f) ntawm Ler thiab thoob ntiaj teb della. P tus nqi tau txais los ntawm kev siv ob-tailed Kolmogorov-Smirnov xeem. f, g 3D pom ntawm cov duab confocal ntawm PI-stained SAM ntawm Col-0 (i) thiab pCUC2::gai-1-VENUS (j) transgenic nroj tsuag. Cov vaj huam sib luag (a, b) qhia cov phab ntsa cell tshiab (tab sis tsis yog primordia) tsim nyob rau 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 faib ntawm cov kev taw qhia ntawm lub dav hlau faib cell nyob hauv tag nrho SAM (h), IPR (i) thiab tsis yog IPR (j) ntawm Col-0 thiab pCUC2::gai-1-VENUS cov nroj tsuag. Cov nqi P tau txais los ntawm kev siv ob-tailed Kolmogorov-Smirnov xeem.
Tom ntej no peb tau sim qhov cuam tshuam ntawm kev txwv tsis pub GA signaling tshwj xeeb hauv IPR. Txog qhov kawg no, peb tau siv lub khob cotyledon 2 (CUC2) promoter los tsav kev qhia tawm ntawm cov protein gai-1 tsis zoo uas tau sib xyaw rau VENUS (hauv kab pCUC2::gai-1-VENUS). Hauv SAM hom qus, CUC2 promoter tsav kev qhia tawm ntawm feem ntau IPRs hauv SAM, suav nrog cov hlwb ciam teb, txij li P4 mus tom ntej, thiab kev qhia tshwj xeeb zoo sib xws tau pom hauv cov nroj tsuag pCUC2::gai-1-VENUS (saib hauv qab no). Kev faib tawm ntawm cov ces kaum faib cell hla SAM lossis IPR ntawm cov nroj tsuag pCUC2::gai-1-VENUS tsis txawv ntawm hom qus, txawm hais tias peb tsis tau xav tias peb pom tias cov hlwb tsis muaj IPR hauv cov nroj tsuag no faib ntawm qhov zaus siab dua ntawm 80–90 ° (Daim duab 6f–j).
Nws tau hais tias qhov kev taw qhia ntawm kev faib cell nyob ntawm qhov geometry ntawm SAM, tshwj xeeb tshaj yog qhov kev ntxhov siab tensile tsim los ntawm cov ntaub so ntswg curvature46. Yog li ntawd peb nug seb cov duab ntawm SAM puas tau hloov pauv hauv della global mutant thiab pCUC2::gai-1-VENUS nroj tsuag. Raws li tau tshaj tawm ua ntej12, qhov loj ntawm della global mutant SAM loj dua li ntawm hom qus (Supplementary Fig. 13a, b, d). In situ hybridization ntawm CLV3 thiab STM RNA tau lees paub qhov kev nthuav dav meristem hauv della mutants thiab ntxiv qhia txog kev nthuav dav ntawm cov qia cell niche (Supplementary Fig. 13e, f, h, i). Txawm li cas los xij, SAM curvature zoo sib xws hauv ob qho tib si genotypes (Supplementary Fig. 13k, m, n, p). Peb pom muaj qhov loj me zoo sib xws hauv gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant yam tsis muaj kev hloov pauv ntawm qhov nkhaus piv rau hom qus (Daim Duab Ntxiv 13c, d, g, j, l, o, p). Qhov zaus ntawm kev faib cell kuj tau cuam tshuam rau hauv della quadruple mutant, tab sis tsawg dua li hauv della monolithic mutant (Daim Duab Ntxiv 12d-f). Qhov txiaj ntsig ntawm kev noj tshuaj no, nrog rau qhov tsis muaj kev cuam tshuam rau qhov nkhaus, qhia tias qhov seem RGL3 kev ua haujlwm hauv Della quadruple mutant txwv tsis pub muaj kev hloov pauv hauv kev faib cell los ntawm kev poob ntawm DELLA kev ua haujlwm thiab tias kev hloov pauv hauv kev faib cell sab nraud tshwm sim los teb rau kev hloov pauv hauv GA signaling kev ua haujlwm es tsis yog kev hloov pauv hauv SAM geometry. Raws li tau piav qhia saum toj no, tus txhawb nqa CUC2 tsav IPR kev qhia tawm hauv SAM pib ntawm P4 (Daim Duab Ntxiv 14a, b), thiab qhov sib piv, pCUC2::gai-1-VENUS SAM muaj qhov loj me me tab sis nkhaus dua (Daim Duab Ntxiv 14c-h). Qhov kev hloov pauv no hauv pCUC2::gai-1-VENUS SAM morphology yuav ua rau muaj kev faib tawm sib txawv ntawm kev ntxhov siab piv rau hom qus, uas kev ntxhov siab siab pib ntawm qhov deb luv dua ntawm SAM center47. Xwb, kev hloov pauv hauv pCUC2::gai-1-VENUS SAM morphology yuav yog los ntawm kev hloov pauv hauv cov khoom siv hauv cheeb tsam uas tshwm sim los ntawm kev qhia transgene48. Hauv ob qho tib si, qhov no tuaj yeem them ib feem ntawm cov teebmeem ntawm kev hloov pauv hauv GA signaling los ntawm kev ua kom muaj feem ntau uas cov hlwb yuav faib hauv kev taw qhia circumferential / transverse, piav qhia peb cov kev soj ntsuam.
Thaum muab peb cov ntaub ntawv los sib sau ua ke, peb cov ntaub ntawv lees paub tias qhov kev taw qhia GA siab dua ua lub luag haujlwm tseem ceeb hauv kev taw qhia sab nraud ntawm lub dav hlau faib cell hauv IPR. Lawv kuj qhia tau tias qhov nkhaus ntawm meristem kuj cuam tshuam rau kev taw qhia ntawm lub dav hlau faib cell hauv IPR.
Qhov kev taw qhia transverse ntawm lub dav hlau faib hauv IPR, vim muaj kev ua haujlwm siab ntawm GA signaling, qhia tias GA ua ntej npaj cov ntaub ntawv radial cell hauv epidermis hauv SAM los txhais cov cellular organization uas tom qab ntawd yuav pom nyob rau hauv epidermal internode. Tseeb tiag, cov ntaub ntawv cell zoo li no feem ntau pom hauv SAM cov duab ntawm della global mutants (Daim Duab 6b). Yog li, txhawm rau tshawb nrhiav ntxiv txog kev ua haujlwm ntawm tus qauv spatial ntawm GA signaling hauv SAM, peb siv time-lapse imaging los tshuaj xyuas qhov spatial organization ntawm cell hauv IPR hauv wild-type (Ler thiab Col-0), della global mutants, thiab pCUC2::gai-1-VENUS transgenic plants.
Peb pom tias qmRGA qhia tau tias GA signaling ua haujlwm hauv IPR tau nce ntxiv los ntawm P1/P2 thiab nce mus txog P4, thiab tus qauv no tseem nyob ruaj khov dhau sijhawm (Daim duab 4a-f thiab Daim duab ntxiv 8c-f, k). Txhawm rau tshuaj xyuas qhov chaw ntawm cov hlwb hauv IPR nrog kev nce ntxiv ntawm GA teeb liab, peb tau sau npe rau Ler IPR hlwb saum toj no thiab rau sab ntawm P4 raws li lawv txoj kev loj hlob uas tau tshuaj xyuas 34 teev tom qab thawj zaug soj ntsuam, piv txwv li, ntau dua ob lub sijhawm plastid, uas tso cai rau peb ua raws li IPR hlwb thaum lub sijhawm primordium los ntawm P1/P2 mus rau P4. Peb siv peb xim sib txawv: daj rau cov hlwb uas tau koom ua ke rau hauv primordium ze P4, ntsuab rau cov uas nyob hauv IPR, thiab ntshav rau cov uas koom nrog ob qho txheej txheem (Daim duab 7a-c). Ntawm t0 (0 h), 1-2 txheej ntawm IPR hlwb tau pom nyob rau pem hauv ntej ntawm P4 (Daim duab 7a). Raws li xav tau, thaum cov hlwb no faib, lawv tau ua li ntawd feem ntau ntawm lub dav hlau faib transverse (Figs. 7a-c). Cov txiaj ntsig zoo sib xws tau txais los ntawm kev siv Col-0 SAM (tsom mus rau P3, uas nws cov ciam teb zoo ib yam li P4 hauv Ler), txawm hais tias hauv genotype no qhov quav tsim ntawm ciam teb paj zais cov hlwb IPR sai dua (Fig. 7g-i). Yog li, tus qauv faib ntawm cov hlwb IPR ua ntej npaj cov hlwb rau hauv cov kab radial, zoo li hauv internodes. Kev teeb tsa ntawm cov kab radial thiab qhov chaw ntawm cov hlwb IPR ntawm cov kab ke sib law liag qhia tias cov hlwb no yog cov thawj coj internodal.
Ntawm no, peb tau tsim ib lub ratiometric GA signaling biosensor, qmRGA, uas tso cai rau kev ntsuas qhov ntau ntawm GA signaling kev ua ub no los ntawm kev sib xyaw ua ke GA thiab GA receptor concentration thaum txo qis kev cuam tshuam nrog endogenous signaling pathways, yog li muab cov ntaub ntawv ntawm GA kev ua haujlwm ntawm theem cellular. Txhawm rau qhov kawg no, peb tau tsim ib qho hloov kho DELLA protein, mRGA, uas tau poob lub peev xwm los khi DELLA cov neeg koom tes sib cuam tshuam tab sis tseem rhiab rau GA-induced proteolysis. qmRGA teb rau ob qho kev hloov pauv sab nraud thiab endogenous hauv GA theem, thiab nws cov khoom siv dynamic sensing ua rau muaj kev ntsuam xyuas ntawm spatiotemporal kev hloov pauv hauv GA signaling kev ua ub no thaum lub sijhawm kev loj hlob. qmRGA kuj yog ib lub cuab yeej hloov pauv tau yooj yim vim nws tuaj yeem hloov kho rau cov ntaub so ntswg sib txawv los ntawm kev hloov tus promoter siv rau nws qhov kev qhia tawm (yog tias tsim nyog), thiab muab qhov xwm txheej ntawm GA signaling pathway thiab PFYRE motif hla angiosperms, nws yuav hloov mus rau lwm hom tsiaj22. Raws li qhov no, qhov kev hloov pauv sib npaug hauv cov protein SLR1 DELLA (HYY497AAA) kuj tau pom tias yuav tiv thaiv kev loj hlob ntawm SLR1 thaum tsuas yog txo qis nws qhov kev puas tsuaj GA-mediated, zoo ib yam li mRGA23. Qhov tseem ceeb, cov kev tshawb fawb tsis ntev los no hauv Arabidopsis tau qhia tias ib qho kev hloov pauv amino acid hauv PFYRE domain (S474L) hloov pauv kev ua haujlwm ntawm RGA yam tsis cuam tshuam rau nws lub peev xwm los cuam tshuam nrog cov neeg koom tes transcription factor50. Txawm hais tias qhov kev hloov pauv no ze heev rau 3 qhov kev hloov pauv amino acid uas muaj nyob hauv mRGA, peb cov kev tshawb fawb qhia tau tias ob qho kev hloov pauv no hloov pauv cov yam ntxwv sib txawv ntawm DELLA. Txawm hais tias feem ntau cov neeg koom tes transcription factor khi rau LHR1 thiab SAW domains ntawm DELLA26,51, qee cov amino acids khaws cia hauv PFYRE domain yuav pab tswj cov kev sib cuam tshuam no.
Kev loj hlob ntawm cov noob yog ib qho tseem ceeb hauv kev tsim cov nroj tsuag thiab kev txhim kho cov qoob loo. qmRGA tau qhia txog kev ua haujlwm siab dua ntawm GA signaling hauv IPR internode progenitor cells. Los ntawm kev sib koom ua ke ntawm kev ntsuas thiab kev tshuaj ntsuam xyuas noob caj noob ces, peb tau qhia tias cov qauv GA signaling superimpose circular/transverse cell division planes hauv SAM epidermis, tsim cov cell division orientation uas xav tau rau kev loj hlob ntawm cov noob. Muaj ntau tus neeg tswj hwm ntawm cell division plane orientation tau txheeb xyuas thaum lub sijhawm loj hlob52,53. Peb txoj haujlwm muab ib qho piv txwv meej txog li cas GA signaling activity tswj hwm cov cellular parameter no. 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 tsis tau xav tias yuav pom tias hauv SAM, qhov sib raug zoo ntawm GA signaling activity tsis yog cell elongation lossis division, tab sis tsuas yog kev loj hlob anisotropy, uas yog sib xws nrog kev cuam tshuam ncaj qha ntawm GA ntawm kev coj ntawm cell division hauv IPR. Txawm li cas los xij, peb tsis tuaj yeem tsis suav tias qhov cuam tshuam no kuj tuaj yeem yog indirect, piv txwv li mediated los ntawm GA-induced cell wall softening56. Kev hloov pauv ntawm cov khoom ntawm phab ntsa cell ua rau muaj kev ntxhov siab mechanical57,58, uas kuj tseem tuaj yeem cuam tshuam rau qhov kev taw qhia ntawm lub dav hlau faib cell los ntawm kev cuam tshuam rau qhov kev taw qhia ntawm cortical microtubules39,46,59. Cov teebmeem ua ke ntawm GA-induced mechanical stress thiab kev tswj hwm ncaj qha ntawm microtubule orientation los ntawm GA tej zaum yuav koom nrog kev tsim cov qauv tshwj xeeb ntawm kev faib cell hauv IPR los txhais cov internodes, thiab kev tshawb fawb ntxiv yog qhov xav tau los sim 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 tsim60,61 thiab cov yam no yuav cuam tshuam rau kev ua haujlwm ntawm GA ua ke nrog BREVIPEDICELLUS (BP) thiab PENNYWISE (PNY), uas tswj hwm kev loj hlob internode thiab tau pom tias cuam tshuam rau GA signaling2,62. Muab hais tias DELLAs cuam tshuam nrog brassinosteroid, ethylene, jasmonic acid, thiab abscisic acid (ABA) signaling pathways63,64 thiab tias cov tshuaj hormones no tuaj yeem cuam tshuam rau microtubule orientation65, cov teebmeem ntawm GA ntawm cell division orientation kuj tseem tuaj yeem raug cuam tshuam los ntawm lwm cov tshuaj hormones.
Cov kev tshawb fawb cytological thaum ntxov qhia tau hais tias ob qho tib si sab hauv thiab sab nraud ntawm Arabidopsis SAM yog qhov yuav tsum tau rau kev loj hlob internode2,42. Qhov tseeb tias GA tswj hwm kev faib cell hauv cov ntaub so ntswg sab hauv12 txhawb nqa ob lub luag haujlwm ntawm GA hauv kev tswj hwm meristem thiab qhov loj me ntawm internode hauv SAM. Tus qauv ntawm kev faib cell directional kuj raug tswj hwm nruj nreem hauv cov ntaub so ntswg SAM sab hauv, thiab qhov kev tswj hwm no yog qhov tseem ceeb rau kev loj hlob ntawm qia52. Nws yuav nthuav kom tshuaj xyuas seb GA puas tseem ua lub luag haujlwm hauv kev taw qhia lub dav hlau faib cell hauv lub koom haum SAM sab hauv, yog li ua kom sib xws ntawm cov lus qhia thiab kev loj hlob 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) nyob rau hauv cov xwm txheej txheem (16 teev lub teeb, 22 ° C), tshwj tsis yog hypocotyl thiab kev loj hlob hauv paus kev sim uas cov noob tau cog rau ntawm cov phaj ntsug nyob rau hauv lub teeb tas li thiab 22 ° C. Rau kev sim nitrate, cov nroj tsuag tau cog rau ntawm cov khoom hloov kho MS (bioWORLD cog nruab nrab) ntxiv nrog nitrate txaus (0 lossis 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose thiab 1% A-agar (Sigma) nyob rau hauv cov xwm txheej ntev.
GID1a cDNA ntxig rau hauv pDONR221 tau rov ua ke nrog pDONR P4-P1R-pUBQ10 thiab pDONR P2R-P3-mCherry rau hauv pB7m34GW los tsim pUBQ10::GID1a-mCherry. IDD2 DNA ntxig rau hauv pDONR221 tau rov ua ke rau hauv pB7RWG266 los tsim p35S:IDD2-RFP. Txhawm rau tsim pGID1b::2xmTQ2-GID1b, ib daim 3.9 kb fragment upstream ntawm GID1b coding cheeb tsam thiab ib daim 4.7 kb fragment uas muaj GID1b cDNA (1.3 kb) thiab terminator (3.4 kb) tau raug amplified thawj zaug siv cov primers hauv Supplementary Table 3 thiab tom qab ntawd ntxig rau hauv pDONR P4-P1R (Thermo Fisher Scientific) thiab pDONR P2R-P3 (Thermo Fisher Scientific), raws li, thiab thaum kawg rov ua ke nrog pDONR221 2xmTQ268 rau hauv pGreen 012567 lub hom phiaj vector siv Gateway cloning. Txhawm rau tsim pCUC2::LSSmOrange, cov kab ke CUC2 promoter (3229 bp upstream ntawm ATG) ua raws li cov kab ke coding ntawm Stokes-shifted mOrange loj (LSSmOrange)69 nrog N7 nuclear localization signal thiab NOS transcriptional terminator tau sib sau ua ke rau hauv pGreen kanamycin targeting vector siv Gateway 3-fragment recombination system (Invitrogen). Lub plant binary vector tau qhia rau hauv Agrobacterium tumefaciens strain GV3101 thiab qhia rau hauv Nicotiana benthamiana nplooj los ntawm Agrobacterium infiltration method thiab rau hauv Arabidopsis thaliana Col-0 los ntawm floral dip method, raws li. pUBQ10::qmRGA pUBQ10::GID1a-mCherry thiab pCLV3::mCherry-NLS qmRGA tau cais tawm ntawm F3 thiab F1 progenies ntawm cov ntoo khaub lig, raws li.
RNA nyob rau hauv situ hybridization tau ua tiav rau ntawm kwv yees li 1 cm ntev tua tips72, uas tau sau thiab tam sim ntawd kho hauv FAA kua (3.7% formaldehyde, 5% acetic acid, 50% ethanol) ua ntej txias rau 4 ° C. Tom qab 2 × 15 feeb kev kho mob nqus tsev, cov fixative tau hloov pauv thiab cov qauv tau incubated ib hmos. GID1a, GID1b, GID1c, GAI, RGL1, RGL2, thiab RGL3 cDNAs thiab antisense probes rau lawv 3′-UTRs tau synthesized siv cov primers qhia nyob rau hauv Supplementary Table 3 raws li piav qhia los ntawm Rosier et al.73. Cov tshuaj probes uas muaj daim ntawv lo Digoxigenin tau kuaj pom los ntawm kev siv cov tshuaj tiv thaiv digoxigenin (3000-fold dilution; Roche, tus lej catalog: 11 093 274 910), thiab cov seem tau pleev xim nrog 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 250-fold dilution)/nitroblue tetrazolium (NBT, 200-fold dilution) kua.