Ukukhula kwe-apical meristem (SAM) yehlumela kubaluleke kakhulu kwisakhiwo sesiqu.ii-gibberellins(ii-GA) zidlala indima ebalulekileyo ekuququzeleleni ukukhula kwezityalo, kodwa indima yazo kwi-SAM ayikaqondwa kakuhle. Apha, siphuhlise i-ratiometric biosensor ye-GA signaling ngobunjineli beprotheyini ye-DELLA ukuze icinezele umsebenzi wayo obalulekileyo wokulawula kwimpendulo ye-GA transcriptional ngelixa igcina ukuwohloka kwayo xa iqondwa i-GA. Sibonisa ukuba le biosensor esekelwe ekuwohlokeni irekhoda ngokuchanekileyo utshintsho kumanqanaba e-GA kunye nokuva kweeseli ngexesha lophuhliso. Sisebenzise le biosensor ukumakisha umsebenzi we-GA signaling kwi-SAM. Sibonisa ukuba imiqondiso ephezulu ye-GA ikhona ikakhulu kwiiseli ezikwi-organ primordia, ezizii-precursors zeeseli ze-internode. Sisebenzisa iindlela zokufumana kunye nokulahlekelwa ngumsebenzi, sibonisa ngakumbi ukuba i-GA ilawula ukujongwa kweplani yokwahlulahlula iiseli, iseka umbutho we-canonical cellular wee-internode, ngaloo ndlela ikhuthaza iinkcukacha ze-internode kwi-SAM.
I-shoot apical meristem (SAM), ekwincopho yehlumelo, iqulethe indawo yeeseli zesiqu ezisebenza ngayo ukuvelisa amalungu asecaleni kunye namaqhuqhuva esiqu ngendlela yemodyuli kunye nephindaphindayo ubomi bonke besityalo. Nganye kwezi yunithi ziphinda-phindayo, okanye amaqhuqhuva esityalo, iquka amaqhuqhuva kunye namaqhuqhuva asecaleni kumaqhuqhuva, kunye namaqhuqhuva e-axillary kwi-axils yamagqabi1. Ukukhula kunye nokuhlelwa kwamaqhuqhuva esityalo kuyatshintsha ngexesha lokukhula. Kwi-Arabidopsis, ukukhula kwamaqhuqhuva kuyacinezelwa ngexesha lesigaba sokukhula, kwaye amaqhuqhuva e-axillary ahlala elele kumaqhuqhuva amagqabi e-rosette. Ngexesha lokutshintshela kwisigaba seentyatyambo, i-SAM iba yi-inflorescence meristem, ivelisa amaqhuqhuva amade kunye namaqhuqhuva e-axillary, amasebe kumaqhuqhuva amagqabi e-cauline, kwaye kamva, iintyatyambo ezingenamagqabi2. Nangona senze inkqubela phambili ebalulekileyo ekuqondeni iindlela ezilawula ukuqaliswa kwamagqabi, iintyatyambo, kunye namasebe, kuncinci okwaziwayo malunga nendlela amaqhuqhuva avela ngayo.
Ukuqonda ukusasazwa kwe-GAs ngokwendawo kuya kunceda ukuqonda ngcono imisebenzi yala ma-hormone kwizicubu ezahlukeneyo nakwizigaba ezahlukeneyo zophuhliso. Ukubona ukuwohloka kwe-RGA-GFP fusion ebonakaliswa phantsi kwesenzo se-promoter yayo kubonelela ngolwazi olubalulekileyo malunga nokulawulwa kwamanqanaba e-GA iyonke kwiingcambu15,16. Nangona kunjalo, ukubonakaliswa kwe-RGA kuyahluka kwiizicubu17 kwaye kulawulwa yi-GA18. Ke ngoko, ukubonakaliswa okwahlukileyo kwe-promoter ye-RGA kunokubangela ipateni ye-fluorescence ebonwa yi-RGA-GFP kwaye ngaloo ndlela le ndlela ayilingani. Kutshanje, i-GA19,20 ene-bioactive fluorescein (Fl) ityhile ukuqokelelwa kwe-GA kwi-root endocortex kunye nokulawulwa kwamanqanaba ayo eseli ngothutho lwe-GA. Kutshanje, i-GA FRET sensor nlsGPS1 ibonise ukuba amanqanaba e-GA ahambelana nokwandiswa kweseli kwiingcambu, ii-filaments, kunye ne-hypocotyls ezikhule emnyama21. Nangona kunjalo, njengoko sibonile, ukugxila kwe-GA akusiyo kuphela iparameter elawula umsebenzi we-GA signaling, njengoko kuxhomekeke kwiinkqubo ezintsonkothileyo zokuva. Apha, sakha phezu kokuqonda kwethu iindlela zokubonisa i-DELLA kunye ne-GA, sixela uphuhliso kunye nokuchazwa kwe-biosensor esekelwe kwi-degradation-based ratiometric ye-GA signaling. Ukuphuhlisa le biosensor yobungakanani, sisebenzise i-RGA enovakalelo lwe-GA eguqulweyo eyadibaniswa neproteni ye-fluorescent kwaye yabonakaliswa ngokubanzi kwizicubu, kunye neproteni ye-fluorescent engenavakalelo lwe-GA. Sibonisa ukuba ukuhlanganiswa kweproteni ye-RGA eguqulweyo akuphazamisi i-GA signaling endogenous xa ibonakaliswa ngokubanzi, kwaye le biosensor inokulinganisa umsebenzi wokubonisa ovela kwi-GA input kunye ne-GA signal processing yisixhobo sokuva esinesisombululo esiphezulu se-spatiotemporal. Sisebenzise le biosensor ukumakisha ukusasazwa kwe-spatiotemporal yomsebenzi wokubonisa i-GA kunye nokulinganisa indlela i-GA elawula ngayo ukuziphatha kweseli kwi-SAM epidermis. Sibonisa ukuba i-GA ilawula ukujongwa kwendawo yokwahlulahlula iiseli ze-SAM ezikwi-organ primordia, ngaloo ndlela ichaza umbutho we-canonical cellular we-internode.
Ekugqibeleni, sibuze ukuba i-qmRGA ingaxela utshintsho kumanqanaba e-GA endalo kusetyenziswa ii-hypocotyls ezikhulayo. Ngaphambili sibonise ukuba i-nitrate ikhuthaza ukukhula ngokwandisa ukwenziwa kwe-GA kwaye, ngokuqhubekayo, ukuwohloka kwe-DELLA34. Ngokuhambelana noko, siqaphele ukuba ubude be-hypocotyl kwizithole ze-pUBQ10::qmRGA ezikhuliswe phantsi kobonelelo oluninzi lwe-nitrate (10 mM NO3−) bude kakhulu kunobo kwizithole ezikhuliswe phantsi kweemeko zokunqongophala kwe-nitrate (Umzobo oNcedisayo 6a). Ngokuhambelana nempendulo yokukhula, imiqondiso ye-GA yayiphezulu kwi-hypocotyls yezithole ezikhuliswe phantsi kweemeko ze-10 mM NO3− kunakwizithole ezikhuliswe ngaphandle kwe-nitrate (Umzobo oNcedisayo 6b, c). Ke ngoko, i-qmRGA ikwavumela ukujonga utshintsho kwi-GA signaling olubangelwa lutshintsho lwe-endogenous kwi-GA concentration.
Ukuze siqonde ukuba umsebenzi we-GA signaling ofunyenwe yi-qmRGA uxhomekeke kuxinzelelo lwe-GA kunye nokuqonda kwe-GA, njengoko bekulindelwe ngokusekelwe kuyilo lwesensor, sihlalutye ukubonakaliswa kwee-receptors ezintathu ze-GID1 kwizicubu zezityalo nezokuzala. Kwizithombo, umgca wentatheli ye-GID1-GUS ubonise ukuba i-GID1a kunye ne-c zibonakaliswe kakhulu kwi-cotyledons (Umzobo 3a–c). Ukongeza, zonke ii-receptors ezintathu zibonakaliswe kumagqabi, i-lateral root primordia, iincam zeengcambu (ngaphandle kwentloko yengcambu ye-GID1b), kunye nenkqubo yemithambo yegazi (Umzobo 3a–c). Kwi-inflorescence SAM, sifumene imiqondiso ye-GUS kuphela kwi-GID1b kunye ne-1c (Umzobo oNcedisayo 7a–c). I-In situ hybridization iqinisekisile ezi patheni zokubonakaliswa kwaye yabonisa ngakumbi ukuba i-GID1c ibonakaliswe ngokufanayo kumanqanaba aphantsi kwi-SAM, ngelixa i-GID1b ibonise ukubonakaliswa okuphezulu kumda we-SAM (Umzobo oNcedisayo 7d–l). I-pGID1b::2xmTQ2-GID1b translational fusion ikwatyhile uluhlu olulinganisiweyo lwe-GID1b expression, ukusuka kwi-expression ephantsi okanye engekhoyo embindini we-SAM ukuya kwi-expression ephezulu kwimida yezitho (Umzobo oNcedisayo 7m). Ke ngoko, ii-receptors ze-GID1 azisasazwanga ngokulinganayo kuzo zonke izicwili nangaphakathi kwezicubu. Kuvavanyo olulandelayo, sikwabone ukuba i-GID1 expression engaphezulu (pUBQ10::GID1a-mCherry) yonyusa uvakalelo lwe-qmRGA kwi-hypocotyls kwi-external GA application (Umzobo 3d, e). Ngokwahlukileyo koko, i-fluorescence elinganiswe yi-qd17mRGA kwi-hypocotyl yayingenamvakalelo kunyango lwe-GA3 (Umzobo 3f, g). Kuzo zombini iimvavanyo, izithole zanyangwa ngoxinzelelo oluphezulu lwe-GA (100 μM GA3) ukuvavanya ukuziphatha okukhawulezayo kwe-sensor, apho amandla okubophelela kwi-GID1 receptor aphuculwe okanye alahlekile. Zonke ezi ziphumo ziqinisekisa ukuba i-qmRGA biosensor isebenza umsebenzi odibeneyo njenge-GA kunye ne-GA sensor, kwaye zibonisa ukuba ukubonakaliswa okwahlukileyo kwe-GID1 receptor kunokuguqula kakhulu ukukhupha kwe-sensor.
Ukuza kuthi ga ngoku, ukusasazwa kwemiqondiso ye-GA kwi-SAM akukacaci. Ke ngoko, sisebenzise izityalo ezibonisa i-qmRGA kunye ne-pCLV3::mCherry-NLS stem cell reporter35 ukubala iimaphu zobungakanani ezichanekileyo zomsebenzi we-GA signaling, sigxile kumaleko we-L1 (i-epidermis; Umzobo 4a, b, bona Iindlela kunye neendlela ezongezelelweyo), kuba i-L1 idlala indima ephambili ekulawuleni ukukhula kwe-SAM36. Apha, ukubonakaliswa kwe-pCLV3::mCherry-NLS kubonelele ngendawo yokubhekisa yejiyometri ezinzileyo yokuhlalutya ukusasazwa kwendawo yomsebenzi we-GA signaling37. Nangona i-GA ithathwa njengebalulekileyo kuphuhliso lwelungu elisecaleni4, siqaphele ukuba iimpawu ze-GA zaziphantsi kwi-primordium yeentyantyambo (P) ukusuka kwinqanaba le-P3 (Umzobo 4a, b), ngelixa ii-primordiums ezincinci ze-P1 kunye ne-P2 zazinomsebenzi ophakathi ofana nalowo ukwindawo ephakathi (Umzobo 4a, b). Umsebenzi ophezulu wokubonisa i-GA ufunyenwe kwimida ye-organ primordium, uqala kwi-P1/P2 (emacaleni omda) kwaye ufikelela kwincopho kwi-P4, kunye nazo zonke iiseli zommandla ophakathi kwe-primordia (Umzobo 4a, b kunye noMfanekiso oNcedisayo 8a, b). Lo msebenzi ophezulu wokubonisa i-GA ubonwe kungekuphela nje kwi-epidermis kodwa nakwi-L2 kunye neengqimba ze-L3 eziphezulu (Umzobo oNcedisayo 8b). Ipateni yeempawu ze-GA ezifunyenwe kwi-SAM kusetyenziswa i-qmRGA nayo ayizange itshintshe ngokuhamba kwexesha (Umzobo oNcedisayo 8c–f, k). Nangona ulwakhiwo lwe-qd17mRGA lwancitshiswa ngokucwangcisiweyo kwi-SAM yezityalo ze-T3 ukusuka kwimigca emihlanu ezimeleyo esiyichazile ngokweenkcukacha, sikwazile ukuhlalutya iipateni ze-fluorescence ezifunyenwe nge-pRPS5a::VENUS-2A-TagBFP (Umzobo oNcedisayo 8g–j, l). Kulo mgca wolawulo, kufunyenwe utshintsho oluncinci kuphela kumlinganiselo we-fluorescence kwi-SAM, kodwa kwiziko le-SAM sibone ukwehla okucacileyo nokungalindelekanga kwi-VENUS okunxulunyaniswa neTagBFP. Oku kuqinisekisa ukuba ipateni yokubonisa ebonwe yi-qmRGA ibonisa ukuwohloka okuxhomekeke kwi-GA kwe-mRGA-VENUS, kodwa ikwabonisa ukuba i-qmRGA inokulinganisela umsebenzi we-GA signaling kwiziko le-meristem. Ngamafutshane, iziphumo zethu zibonisa ipateni yokubonisa ye-GA ebonisa ngokuyintloko ukusasazwa kwe-primordia. Olu sasazo lwengingqi ephakathi kwe-primordial (IPR) lubangelwa kukusekwa kancinci komsebenzi we-GA signaling ophezulu phakathi kwe-primordium ephuhlisayo kunye nommandla ophakathi, ngelixa kwangaxeshanye umsebenzi we-GA signaling kwi-primordium uyancipha (Umzobo 4c, d).
Ukusasazwa kwee-receptors ze-GID1b kunye ne-GID1c (jonga ngasentla) kubonisa ukuba ukubonakaliswa okwahlukileyo kwee-receptors ze-GA kunceda ekwakheni ipateni yomsebenzi we-GA signaling kwi-SAM. Sizibuze ukuba ingaba ukuqokelelwa okwahlukileyo kwe-GA kusenokuba negalelo na. Ukuphanda oku kunokwenzeka, sisebenzise i-nlsGPS1 GA FRET sensor21. Ukwanda kwe-activation frequency kufunyenwe kwi-SAM ye-nlsGPS1 ephathwe nge-10 μM GA4+7 kangangemizuzu eli-100 (Umzobo ongezelelweyo 9a–e), okubonisa ukuba i-nlsGPS1 iphendula kutshintsho kuxinzelelo lwe-GA kwi-SAM, njengoko isenza kwiingcambu21. Ukusasazwa kwendawo kwe-activation frequency ye-nlsGPS1 kubonise amanqanaba e-GA aphantsi kakhulu kumaleko angaphandle e-SAM, kodwa kubonise ukuba aphakanyisiwe embindini nakwimida ye-SAM (Umzobo 4e kunye nomzobo ongezelelweyo 9a,c). Oku kubonisa ukuba i-GA ikwasasazwa kwi-SAM ngepatheni yendawo efana naleyo ityhilelwe yi-qmRGA. Njengendlela encedisayo, sikwayiphathe i-SAM nge-GA ekhanyayo (GA3-, GA4-, GA7-Fl) okanye i-Fl yodwa njengolawulo olungalunganga. Isignali ye-Fl yasasazwa kulo lonke i-SAM, kubandakanya ummandla ophakathi kunye ne-primordium, nangona yayinamandla aphantsi (Umzobo 4j kunye nomzobo ongezelelweyo 10d). Ngokwahlukileyo koko, zonke ezi zintathu ze-GA-Fl zaqokelelana ngokukodwa ngaphakathi kwemida ye-primordium kunye namanqanaba ahlukeneyo kwezinye ii-IPR, kunye ne-GA7-Fl eqokelelana kwindawo enkulu kwi-IPR (Umzobo 4k kunye nomzobo ongezelelweyo 10a,b). Ukulinganiswa kobunzulu be-fluorescence kutyhile ukuba umlinganiselo we-IPR kwi-non-IPR intensity wawuphezulu kwi-SAM ephathwe yi-GA-Fl xa kuthelekiswa ne-SAM ephathwe yi-Fl (Umzobo 4l kunye nomzobo ongezelelweyo 10c). Zizonke, ezi ziphumo zibonisa ukuba i-GA ikhona kwiindawo eziphezulu kwiiseli ze-IPR ezikufutshane nomda we-organ. Oku kubonisa ukuba ipateni yomsebenzi wokubonisa i-SAM GA ivela kukubonakaliswa okwahlukileyo kwee-receptors ze-GA kunye nokuqokelelwa kwe-GA kwiiseli ze-IPR kufutshane nemida yezitho. Ngoko ke, uhlalutyo lwethu lutyhile ipateni engalindelekanga yendawo yokubonisa i-GA, kunye nomsebenzi ophantsi embindini nakwi-primordium ye-SAM kunye nomsebenzi ophezulu kwi-IPR kummandla ongaphandle.
Ukuze siqonde indima yomsebenzi we-GA signaling ohlukeneyo kwi-SAM, sihlalutye ulwalamano phakathi komsebenzi we-GA signaling, ukwandiswa kweseli, kunye nokwahlukana kweseli sisebenzisa umfanekiso we-time-lapse we-SAM qmRGA pCLV3::mCherry-NLS. Ngenxa yendima ye-GA kulawulo lokukhula, kulindeleke ulwalamano oluhle kunye neeparameter zokwandiswa kweseli. Ke ngoko, siqale sathelekisa iimaphu zomsebenzi we-GA signaling kunye neemaphu zesantya sokukhula komphezulu weseli (njenge-proxy yamandla okwandiswa kweseli kwiseli ethile kunye neeseli zentombi ekwahlulweni) kunye neemaphu ze-anisotropy yokukhula, elinganisa indlela yokwandiswa kweseli (ikwasetyenziswa apha kwiseli ethile kunye neeseli zentombi ekwahlulweni; Umzobo 5a,b, jonga Iindlela kunye neendlela ezongezelelweyo). Iimaphu zethu zesantya sokukhula komphezulu weseli ye-SAM ziyahambelana nokubonwa kwangaphambili38,39, kunye namazinga okukhula amancinci kumda kunye namazinga okukhula aphezulu kwiintyatyambo ezikhulayo (Umzobo 5a). Uhlalutyo lwecandelo eliphambili (PCA) lubonise ukuba umsebenzi we-GA signaling wawunxulumene kakubi nobunzulu bokukhula komphezulu weseli (Umfanekiso 5c). Sikwabonise ukuba ii-axis eziphambili zokwahluka, kubandakanya igalelo le-GA signaling kunye nokuqina kokukhula, zazihambelana necala elimiselwe kukubonakaliswa okuphezulu kwe-CLV3, okuqinisekisa ukukhutshwa kweeseli kwiziko le-SAM kuhlalutyo olusele. Uhlalutyo lolwalamano lweSpearman luqinisekisile iziphumo ze-PCA (Umfanekiso 5d), olubonisa ukuba imiqondiso ephezulu ye-GA kwi-IPR ayizange ibangele ukwanda okuphezulu kweeseli. Nangona kunjalo, uhlalutyo lolwalamano lubonakalise ulwalamano oluncinci oluhle phakathi komsebenzi womqondiso we-GA kunye ne-anisotropy yokukhula (Umfanekiso 5c, d), olubonisa ukuba umqondiso ophezulu we-GA kwi-IPR unefuthe kwicala lokukhula kweeseli kwaye mhlawumbi nendawo yendiza yokwahlulahlula iiseli.
a, b Iimephu zobushushu zokukhula komphezulu ophakathi (a) kunye ne-anisotropy yokukhula (b) kwi-SAM ziphakathi kwezityalo ezisixhenxe ezizimeleyo (ezisetyenziswa njengeeproxies zamandla kunye nesikhokelo sokwanda kweseli, ngokulandelelana). c Uhlalutyo lwe-PCA luquke ezi zinto zilandelayo: isignali ye-GA, ubunzulu bokukhula komphezulu, i-anisotropy yokukhula komphezulu, kunye nokubonakaliswa kwe-CLV3. Icandelo le-PCA 1 lalinxulumene kakubi nobunzulu bokukhula komphezulu kwaye lahambelana kakuhle nesignali ye-GA. Icandelo le-PCA 2 lalinxulumene kakuhle nokukhula komphezulu kwaye ladibana kakubi nokubonakaliswa kwe-CLV3. Iipesenti zimele umahluko ochazwe licandelo ngalinye. d Uhlalutyo lolwalamano lwe-Spearman phakathi kwesignali ye-GA, ubunzulu bokukhula komphezulu, kunye ne-anisotropy yokukhula komphezulu kwisikali sezicubu ngaphandle kwe-CZ. Inani elingasekunene lixabiso le-Spearman rho phakathi kwezinto ezimbini eziguquguqukayo. Ii-Asterisks zibonisa iimeko apho ulwalamano/ulwalamano olungalunganga lubaluleke kakhulu. e Ukuboniswa kwe-3D kweeseli ze-Col-0 SAM L1 nge-confocal microscopy. Iindonga ezintsha zeeseli ezenziwe kwi-SAM (kodwa kungekhona i-primordium) kwiiyure ezili-10 zinombala ngokwexabiso lazo lee-angle. Ibha yombala iboniswe kwikona esezantsi ngasekunene. I-inset ibonisa umfanekiso we-3D ohambelanayo ngeyure eli-0. Olu vavanyo luphindwe kabini ngeziphumo ezifanayo. f Iiploti zebhokisi zibonisa amazinga okwahlulahlula iiseli kwi-IPR nakwi-non-IPR Col-0 SAM (n = izityalo ezizimeleyo ezili-10). Umgca osembindini ubonisa i-median, kwaye imida yebhokisi ibonisa iipesenti ezingama-25 kunye nama-75. Iiwhiskers zibonisa amaxabiso amancinci kunye nawona aphezulu amiselwe ngesoftware ye-R. Amaxabiso e-P afunyenwe ngovavanyo lwe-t olunemisila emibini lukaWelch. g, h Umzobo weSchematic obonisa (g) indlela yokulinganisa i-engile yodonga olutsha lweseli (i-magenta) ngokubhekiselele kwicala le-radial ukusuka embindini we-SAM (umgca omhlophe onamachaphaza) (amaxabiso e-angle abukhali kuphela, oko kukuthi, 0–90°, aqwalaselwayo), kunye (h) nezikhokelo ze-circumferential/lateral kunye ne-radial ngaphakathi kwe-meristem. i Ii-histograms ze-frequency ze-orientation yeplane yokwahlulahlula iiseli kwi-SAM (uluhlaza okwesibhakabhaka obumnyama), i-IPR (uluhlaza okwesibhakabhaka ophakathi), kunye ne-non-IPR (uluhlaza okwesibhakabhaka okhanyayo), ngokulandelelana. Amaxabiso e-P afunyenwe ngovavanyo lwe-Kolmogorov-Smirnov olunemisila emibini. Olu vavanyo luphindwe kabini ngeziphumo ezifanayo. j Ii-histograms zefrikhwensi yolwahlulo lweseli lwe-IPR ejikeleze i-P3 (luhlaza olukhanyayo), i-P4 (luhlaza oluphakathi), kunye ne-P5 (luhlaza olumnyama), ngokwahlukeneyo. Amaxabiso e-P afunyenwe ngovavanyo lwe-Kolmogorov-Smirnov olunemisila emibini. Olu vavanyo luphindwe kabini ngeziphumo ezifanayo.
Ngoko ke, emva koko sihlolisise ulwalamano phakathi kwe-GA signaling kunye nomsebenzi wokwahlulahlula iiseli ngokuchonga iindonga zeeseli ezisandula ukwenziwa ngexesha lovavanyo (Umzobo 5e). Le ndlela isivumele ukuba silinganise imvamisa kunye necala lokwahlulahlula iiseli. Okumangalisayo kukuba, sifumanise ukuba imvamisa yokwahlulahlula iiseli kwi-IPR nakwezinye ii-SAM (ezingeyiyo i-IPR, Umfanekiso 5f) yayifana, nto leyo ebonisa ukuba umahluko kwi-GA signaling phakathi kweeseli ze-IPR kunye nezingezizo i-IPR awuchaphazeli kakhulu ukwahlulwahlulwa kweeseli. Oku, kunye nolwalamano oluhle phakathi kwe-GA signaling kunye ne-growth anisotropy, kusishukumisele ukuba sicinge ukuba umsebenzi we-GA signaling unokuchaphazela na ukujongwa kweplani yokwahlulahlula iiseli. Silinganise indlela olujongwa ngayo udonga olutsha lweseli njenge-acute angle enxulumene ne-radial axis edibanisa iziko le-meristem kunye neziko lodonga olutsha lweseli (Umzobo 5e-i) saza sabona ukuthambekela okucacileyo kokuba iiseli zahlule kwii-engile ezikufutshane ne-90° xa kuthelekiswa ne-radial axis, apho amaza aphezulu abonwa kwi-70–80° (23.28%) kunye ne-80–90° (22.62%) (Umzobo 5e,i), ehambelana nokwahlukana kweeseli kwicala elijikelezayo/elinqamlezileyo (Umzobo 5h). Ukuze sihlolisise igalelo le-GA signaling kule ndlela yokuziphatha yokwahlukana kweeseli, sihlalutye iiparameter zokwahlukana kweeseli kwi-IPR nakwi-non-IPR ngokwahlukeneyo (Umzobo 5i). Siqaphele ukuba ukusasazwa kwe-engile yokwahlulahlula kwiiseli ze-IPR kwahlukile kuleyo kwiiseli ezingezizo ze-IPR okanye kwiiseli kwi-SAM iyonke, iiseli ze-IPR zibonisa inani eliphezulu lokwahlulahlula kweeseli ezisecaleni/ezijikelezayo, oko kukuthi, i-70–80° kunye ne-80–90° (33.86% kunye ne-30.71%, ngokulandelanayo, iipesenti ezihambelanayo) (Umzobo 5i). Ke ngoko, ukuqaphela kwethu kutyhile unxulumano phakathi kwe-GA signaling ephezulu kunye ne-cell division plane orientation kufutshane necala elijikelezileyo, efana nolwalamano phakathi komsebenzi we-GA signaling kunye ne-growth anisotropy (Umzobo 5c, d). Ukuze siqinisekise ngakumbi ukugcinwa kwendawo yale manyano, silinganise i-division plane orientation kwiiseli ze-IPR ezijikeleze i-primordium siqala kwi-P3, kuba umsebenzi we-GA signaling ophezulu ufunyenwe kule ndawo uqala kwi-P4 (Umzobo 4). Ii-division angles ze-IPR ezijikeleze i-P3 kunye ne-P4 azibonisanga mahluko abalulekileyo ngokwezibalo, nangona ukwanda kokwahlulwa kweeseli ezisecaleni kubonwe kwi-IPR ejikeleze i-P4 (Umzobo 5j). Nangona kunjalo, kwiiseli ze-IPR ezijikeleze i-P5, umahluko kwindlela ebekwe ngayo iplani yokwahlulahlula iiseli uye waba mkhulu ngokwezibalo, kunye nokwanda okukhulu kwimvamisa yokwahlulahlula kweeseli ezinqamlezileyo (Umzobo 5j). Zizonke, ezi ziphumo zibonisa ukuba i-GA signaling inokulawula indlela ebekwe ngayo ukwahlulwahlulwa kweeseli kwi-SAM, nto leyo ehambelana neengxelo zangaphambili40,41 zokuba i-GA signaling ephezulu inokubangela indlela ebekwe ngayo ecaleni yokwahlulahlula kweeseli kwi-IPR.
Kuqikelelwa ukuba iiseli ezikwi-IPR aziyi kufakwa kwi-primordia kodwa ziya kufakwa kwi-internodes2,42,43. Ulwalathiso olunqamlezileyo lolwahlulo lweeseli kwi-IPR lunokubangela ulungelelwaniso oluqhelekileyo lwemigca emide ehambelanayo yeeseli ze-epidermal kwii-internodes. Izinto esizibonileyo apha ngasentla zibonisa ukuba i-GA signaling inokuba nendima kule nkqubo ngokulawula indlela yolwahlulo lweeseli.
Ukulahlekelwa ngumsebenzi we-DELLA genes ezininzi kubangela impendulo ye-GA, kwaye i-della mutants ingasetyenziselwa ukuvavanya le ngcamango44. Siqale sahlalutya iipatheni zokubonakaliswa kwe-genes ezintlanu ze-DELLA kwi-SAM. Ukuhlanganiswa kwe-transcriptional komgca we-GUS45 kutyhile ukuba i-GAI, i-RGA, i-RGL1, kunye ne-RGL2 (ngomlinganiselo omncinci kakhulu) zibonakaliswe kwi-SAM (Umzobo oNcedisayo 11a–d). I-In situ hybridization ibonise ngakumbi ukuba i-GAI mRNA iqokelelana ngokukodwa kwi-primordia nakwiintyatyambo ezikhulayo (Umzobo oNcedisayo 11e). I-RGL1 kunye ne-RGL3 mRNA zifunyenwe kuyo yonke i-SAM canopy nakwiintyatyambo ezindala, ngelixa i-RGL2 mRNA yayininzi kakhulu kummandla womda (Umzobo oNcedisayo 11f–h). I-Confocal imaging ye-pRGL3::RGL3-GFP SAM iqinisekisile intetho ebonwe yi-in situ hybridization kwaye ibonise ukuba i-RGL3 protein iqokelelana kwindawo ephakathi ye-SAM (Umzobo oNcedisayo 11i). Sisebenzisa umgca we-pRGA::GFP-RGA, sikwafumanise ukuba iprotheyini ye-RGA iqokelelana kwi-SAM, kodwa ubuninzi bayo buyehla kumda oqala kwi-P4 (Umzobo oNcedisayo 11j). Okuphawulekayo kukuba, iipateni zokubonakaliswa kwe-RGL3 kunye ne-RGA ziyahambelana nomsebenzi ophezulu wokubonisa i-GA kwi-IPR, njengoko kubonwe yi-qmRGA (Umzobo 4). Ngaphezu koko, ezi datha zibonisa ukuba zonke ii-DELLA zibonakaliswa kwi-SAM kwaye ukubonakaliswa kwazo ngokudibeneyo kugubungela i-SAM yonke.
Emva koko sihlalutye iiparameter zokwahlulwa kweseli kwi-wild-type SAM (Ler, control) kunye ne-gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Umzobo 6a, b). Okunomdla kukuba, sibonile utshintsho olubalulekileyo ngokwezibalo ekusasazweni kwee-frequency ze-angle zokwahlulwa kweseli kwi-della global mutant SAM xa kuthelekiswa nohlobo lwe-wild (Umzobo 6c). Olu tshintsho kwi-della global mutant lubangelwe kukwanda kwe-frequency yee-angles ezingama-80–90° (34.71% vs. 24.55%) kwaye, kancinci, ii-angles ezingama-70–80° (23.78% vs. 20.18%), oko kukuthi, ezihambelana nokwahlulwa kwe-transverse cell (Umzobo 6c). I-frequency yokwahlulwa kwe-non-transverse (0–60°) nayo yayiphantsi kwi-della global mutant (Umzobo 6c). Ubuninzi bokwahlulwa kweeseli ezinqamlezileyo bunyuswe kakhulu kwi-SAM ye-della global mutant (Umzobo 6b). Ubuninzi bokwahlulwa kweeseli ezinqamlezileyo kwi-IPR bukwaphezulu kwi-della global mutant xa kuthelekiswa nohlobo lwendalo (Umzobo 6d). Ngaphandle kommandla we-IPR, uhlobo lwendalo lwalunokusasazwa okufanayo kwee-angles zokwahlulwa kweeseli, ngelixa i-della global mutant ikhetha ukwahlulwa kwe-tangential njenge-IPR (Umzobo 6e). Sikwalinganise indlela yokwahlulwa kweeseli kwi-SAM ye-ga2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, kunye ne-ga2ox6-2), imvelaphi ye-GA-inactive mutant apho i-GA iqokelela khona. Ngokuhambelana nokwanda kwamanqanaba e-GA, i-SAM ye-quintuple ga2ox mutant inflorescence yayinkulu kune-Col-0 (Umzobo oNcedisayo 12a, b), kwaye xa ithelekiswa ne-Col-0, i-quintuple ga2ox SAM ibonise ukusasazwa okwahlukileyo kwee-angles zokwahlulahlula iiseli, kunye ne-angle frequency enyuka ukusuka kwi-50° ukuya kwi-90°, oko kukuthi kwakhona ikhetha ukwahlulwahlulwa kwe-tangential (Umzobo oNcedisayo 12a–c). Ke ngoko, sibonisa ukuba ukusebenza okubonakalayo kwe-GA signaling kunye nokuqokelelwa kwe-GA kubangela ukwahlulwahlulwa kweeseli ezisecaleni kwi-IPR nakwezinye ii-SAM.
a, b Umboniso we-3D womaleko we-L1 we-PI-stained Ler (a) kunye ne-global della mutant (b) SAM kusetyenziswa i-confocal microscopy. Iindonga ezintsha zeeseli ezenziwe kwi-SAM (kodwa kungekhona i-primordium) kwixesha leeyure ezili-10 ziboniswa kwaye zifakwe imibala ngokwexabiso lazo leengile. I-inset ibonisa i-SAM kwi-0 h. Ibha yombala iboniswa kwikona esezantsi ngasekunene. Utolo olukwi-(b) lubonisa umzekelo weefayile zeeseli ezihambelanayo kwi-global della mutant. Olu vavanyo luphindwe kabini kunye neziphumo ezifanayo. ce uthelekiso lokusasazwa rhoqo kweendlela zokwahlulwa kweseli kwi-SAM yonke (d), IPR (e), kunye ne-non-IPR (f) phakathi kweLer kunye ne-global della. Amaxabiso e-P afunyenwe kusetyenziswa uvavanyo lwe-Kolmogorov-Smirnov olunemisila emibini. f, g Umboniso we-3D wemifanekiso ye-confocal ye-PI-stained SAM ye-Col-0 (i) kunye ne-pCUC2::gai-1-VENUS (j) izityalo ze-transgenic. Iiphaneli (a, b) zibonisa iindonga ezintsha zeseli (kodwa kungekhona ii-primordia) ezenziwe kwi-SAM kwiiyure ezili-10. Olu vavanyo luphindwe kabini ngeziphumo ezifanayo. h–j Uthelekiso lokusasazwa rhoqo kweendawo zokwahlulwa kweseli ezikwi-SAM yonke (h), IPR (i) kunye ne-non-IPR (j) phakathi kwezityalo zeCol-0 kunye ne-pCUC2::gai-1-VENUS. Amaxabiso e-P afunyenwe kusetyenziswa uvavanyo lweKolmogorov–Smirnov olunemisila emibini.
Emva koko sivavanye isiphumo sokuthintela i-GA signaling ngokukodwa kwi-IPR. Ngenxa yesi sizathu, sisebenzise i-cotyledon cup 2 (CUC2) promoter ukuqhuba ukubonakaliswa kweprotheyini ye-gai-1 engalunganga edityaniswe kwi-VENUS (kumgca we-pCUC2::gai-1-VENUS). Kwi-SAM yohlobo lwe-wild, i-CUC2 promoter iqhuba ukubonakaliswa kwe-IPR ezininzi kwi-SAM, kuquka iiseli zomda, ukusuka kwi-P4 ukuya phambili, kwaye ukubonakaliswa okufanayo kwabonwa kwizityalo ze-pCUC2::gai-1-VENUS (jonga ngezantsi). Ukusasazwa kwee-engile zokwahlulwa kweeseli kwi-SAM okanye i-IPR yezityalo ze-pCUC2::gai-1-VENUS kwakungahlukanga kakhulu kohlobo lwe-wild, nangona ngokungalindelekanga sifumanise ukuba iiseli ezingenayo i-IPR kwezi zityalo zahlulwe kwi-frequency ephezulu ye-80–90° (Umzobo 6f–j).
Kuye kwacetyiswa ukuba indlela yokwahlulahlula iiseli ixhomekeke kwijiyometri ye-SAM, ngakumbi uxinzelelo lokuxinana oluveliswa kukugoba kwezicubu46. Ngoko ke sibuze ukuba imo ye-SAM itshintshiwe na kwizityalo ze-della global mutant kunye ne-pCUC2::gai-1-VENUS. Njengoko bekuxeliwe ngaphambili12, ubungakanani be-della global mutant SAM babubukhulu kunobo be-wild type (Umzobo oNcedisayo 13a, b, d). I-In situ hybridization ye-CLV3 kunye ne-STM RNA iqinisekisile ukwanda kwe-meristem kwi-della mutants kwaye yabonisa ukwanda okusecaleni kwe-stem cell niche (Umzobo oNcedisayo 13e, f, h, i). Nangona kunjalo, ukugoba kwe-SAM kwakufana kuzo zombini ii-genotype (Umzobo oNcedisayo 13k, m, n, p). Sibone ukwanda okufanayo kobukhulu kwi-gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant ngaphandle kotshintsho kwi-curvature xa kuthelekiswa nohlobo lwe-wild (Umzobo oNcedisayo 13c, d, g, j, l, o, p). Ubuninzi bokuqhelaniswa kokwahlulwa kweseli buchaphazelekile nakwi-della quadruple mutant, kodwa kancinci kunakwi-della monolithic mutant (Umzobo oNcedisayo 12d–f). Esi siphumo somthamo, kunye nokungabikho kwesiphumo kwi-curvature, sibonisa ukuba umsebenzi oseleyo we-RGL3 kwi-Della quadruple mutant uthintela utshintsho kwi-orientation ye-cell division ebangelwa kukulahlekelwa ngumsebenzi we-DELLA kwaye utshintsho kwi-lateral cell divisions lwenzeka ngenxa yotshintsho kumsebenzi we-GA signaling endaweni yotshintsho kwi-SAM geometry. Njengoko kuchaziwe apha ngasentla, umgqugquzeli we-CUC2 uqhuba ukubonakaliswa kwe-IPR kwi-SAM ukuqala kwi-P4 (Umzobo oNcedisayo 14a, b), kwaye ngokwahlukileyo, i-pCUC2::gai-1-VENUS SAM yayinobukhulu obuncitshisiweyo kodwa ijikajike kakhulu (Umzobo oNcedisayo 14c–h). Olu tshintsho kwimo ye-pCUC2::gai-1-VENUS SAM lunokubangela ukusasazwa okwahlukileyo koxinzelelo loomatshini xa kuthelekiswa nohlobo lwasendle, apho uxinzelelo oluphezulu olujikelezayo luqala kumgama omfutshane ukusuka kwiziko le-SAM47. Kungenjalo, utshintsho kwimo ye-pCUC2::gai-1-VENUS SAM lunokubangela utshintsho kwiipropati zoomatshini zengingqi ezibangelwa kukubonakaliswa kwe-transgene48. Kuzo zombini iimeko, oku kunokunciphisa imiphumo yotshintsho kwi-GA signaling ngokwandisa amathuba okuba iiseli ziya kwahlulwa kwi-circumferential/transverse orientation, echaza izinto esizibonileyo.
Xa zizonke, idatha yethu iqinisekisa ukuba i-GA signaling ephezulu idlala indima ebalulekileyo kwindlela ebekwe ngayo ecaleni kwendlela yokwahlulahlula iseli kwi-IPR. Zikwabonisa ukuba ukugoba kwe-meristem kukwachaphazela nendlela ebekwe ngayo indlela yokwahlulahlula iseli kwi-IPR.
Indlela enqamlezileyo yokwahlulahlula i-division plane kwi-IPR, ngenxa yomsebenzi ophezulu we-GA signaling, ibonisa ukuba i-GA ilungiselela kwangaphambili ifayile yeseli ye-radial kwi-epidermis ngaphakathi kwe-SAM ukuchaza umbutho weseli oza kufunyanwa kamva kwi-epidermal internode. Enyanisweni, iifayile zeseli ezinjalo zazibonakala rhoqo kwimifanekiso ye-SAM ye-della global mutants (Umzobo 6b). Ke ngoko, ukuze sihlolisise ngakumbi umsebenzi wophuhliso lwepatheni yendawo ye-GA signaling kwi-SAM, sisebenzise i-time-lapse imaging ukuhlalutya umbutho wendawo weeseli kwi-IPR kwi-wild-type (Ler kunye ne-Col-0), i-della global mutants, kunye ne-pCUC2::gai-1-VENUS transgenic plants.
Sifumanise ukuba i-qmRGA ibonise ukuba umsebenzi we-GA signaling kwi-IPR unyuke ukusuka kwi-P1/P2 waza wafikelela kwincopho kwi-P4, kwaye le patheni yahlala ihleli ngokuhamba kwexesha (Umzobo 4a–f kunye noMfanekiso oNcedisayo. 8c–f, k). Ukuhlalutya ulungelelwaniso lwendawo lweeseli kwi-IPR ngomqondiso we-GA okhulayo, sibhale iiseli ze-Ler IPR ngaphezulu namacala e-P4 ngokwekamva lazo lophuhliso ezihlalutyiweyo iiyure ezingama-34 emva kokuqwalaselwa kokuqala, oko kukuthi, amaxesha angaphezu kwambini e-plastid, okusivumela ukuba silandele iiseli ze-IPR ngexesha lophuhliso lwe-primordium ukusuka kwi-P1/P2 ukuya kwi-P4. Sisebenzise imibala emithathu eyahlukileyo: umthubi kwezo seli zihlanganiswe kwi-primordium kufutshane ne-P4, uluhlaza kwezo zazikwi-IPR, kunye nomsobo kubo bathathe inxaxheba kuzo zombini iinkqubo (Umzobo 7a–c). Kwi-t0 (0 h), iileya ezi-1–2 zeeseli ze-IPR zazibonakala phambi kwe-P4 (Umzobo 7a). Njengoko bekulindelekile, xa ezi seli zahlukana, zenze njalo ngokuyintloko ngeplani yokwahlulahlula enqamlezileyo (Iifig. 7a–c). Iziphumo ezifanayo zifunyenwe kusetyenziswa i-Col-0 SAM (igxile kwi-P3, enomda wayo ugoba ngendlela efanayo ne-P4 kwi-Ler), nangona kule genotype umqukumbelo owenziwe kumda weentyatyambo ufihle iiseli ze-IPR ngokukhawuleza (Umzobo 7g–i). Ngoko ke, ipateni yokwahlulahlula yeeseli ze-IPR ihlela iiseli zibe yimigca ye-radial, njengakwi-internodes. Ukuhlelwa kwemigca ye-radial kunye nokwenziwa kweeseli ze-IPR phakathi kwamalungu alandelelanayo kubonisa ukuba ezi seli zizizalana eziphakathi.
Apha, siphuhlise i-ratiometric GA signaling biosensor, i-qmRGA, evumela ukubalwa kwemephu yomsebenzi we-GA signaling ovela kwi-GA kunye ne-GA receptor conjunction contending concentrations ngelixa kunciphisa ukuphazamiseka kweendlela ze-GA signaling ezingaphakathi, ngaloo ndlela sinikezela ngolwazi malunga nomsebenzi we-GA kwinqanaba leselula. Ngenxa yesi sizathu, sakhe iprotheyini ye-DELLA eguquliweyo, i-mRGA, elahlekelwe yikhono lokubopha amaqabane e-DELLA interaction kodwa ihlala ivakalelwa yi-GA-induced proteolysis. I-qmRGA iphendula kutshintsho lwangaphandle nolwe-endogenic kumanqanaba e-GA, kwaye iipropati zayo zokuva amandla zivumela uvavanyo lotshintsho lwe-spatiotemporal kumsebenzi we-GA signaling ngexesha lophuhliso. I-qmRGA ikwasisixhobo esiguquguqukayo kakhulu njengoko sinokuhlengahlengiswa kwiithishu ezahlukeneyo ngokutshintsha i-promoter esetyenziselwa ukubonakaliswa kwayo (ukuba kuyimfuneko), kwaye ngenxa yendalo egciniweyo yendlela ye-GA signaling kunye ne-PFYRE motif kwi-angiosperms, kusenokwenzeka ukuba idluliselwe kwezinye iintlobo22. Ngokuhambelana noku, utshintsho olufanayo kwiproteni yerayisi ye-SLR1 DELLA (HYY497AAA) lukwabonakalisiwe ukuba lucinezela umsebenzi wokunciphisa ukukhula kwe-SLR1 ngelixa lunciphisa kancinci ukubola kwayo okubangelwa yi-GA, okufana ne-mRGA23. Okuphawulekayo kukuba, izifundo zakutshanje kwi-Arabidopsis zibonise ukuba utshintsho olunye lwe-amino acid kwi-PFYRE domain (S474L) lutshintshe umsebenzi wokubhala we-RGA ngaphandle kokuchaphazela amandla ayo okusebenzisana namaqabane e-transcription factor50. Nangona olu tshintsho lusondele kakhulu kwii-amino acid substitutions ezi-3 ezikhoyo kwi-mRGA, izifundo zethu zibonisa ukuba olu tshintsho lumbini lutshintsha iimpawu ezahlukeneyo ze-DELLA. Nangona uninzi lwamaqabane e-transcription factor ebopha kwiindawo ze-LHR1 kunye ne-SAW ze-DELLA26,51, ezinye ii-amino acids ezigciniweyo kwi-PFYRE domain zinokunceda ukuzinzisa olu nxibelelwano.
Uphuhliso lwangaphakathi kwe-node luphawu oluphambili kuyilo lwezityalo kunye nokuphuculwa kwesivuno. I-qmRGA ityhile umsebenzi ophezulu we-GA signaling kwiiseli ze-IPR internode progenitor. Ngokudibanisa i-quantitative imaging kunye ne-genetics, sibonise ukuba iipatheni ze-GA signaling zibeka ngaphezulu kwe-circular/transverse cell division planes kwi-SAM epidermis, zibumba ulungiselelo lwe-cell division olufunekayo kuphuhliso lwe-internode. Abalawuli abaninzi be-cell division plane orientation bachongiwe ngexesha lophuhliso52,53. Umsebenzi wethu ubonelela ngomzekelo ocacileyo wendlela i-GA signaling activity elawula ngayo le parameter yeselula. I-DELLA inokusebenzisana ne-prefolding protein complexes41, ngoko ke i-GA signaling inokulawula i-cell division plane orientation ngokuchaphazela ngokuthe ngqo i-cortical microtubule orientation40,41,54,55. Sibonise ngokungalindelekanga ukuba kwi-SAM, i-correlate yomsebenzi we-GA signaling ophezulu yayingekuko ukunwebeka kweseli okanye ukwahlukana, kodwa yi-growth anisotropy kuphela, ehambelana nesiphumo esithe ngqo se-GA kwicala lokwahlukana kweseli kwi-IPR. Nangona kunjalo, asinakuyikhupha ngaphandle into yokuba esi siphumo sinokuba singathanga ngqo, umzekelo silawulwa yi-GA-induced cell wall softening56. Utshintsho kwiipropati zodonga lweseli lubangela uxinzelelo loomatshini57,58, olunokuthi luchaphazele nolwalathiso lweplani yokwahlulahlula iseli ngokuchaphazela ulwalathiso lwee-microtubules ze-cortical39,46,59. Iziphumo ezidibeneyo zoxinzelelo loomatshini olubangelwa yi-GA kunye nolawulo oluthe ngqo lolwalathiso lwe-microtubule yi-GA zinokubandakanyeka ekuveliseni ipateni ethile yolwalathiso lokwahlulahlula iseli kwi-IPR ukuchaza ii-internodes, kwaye kufuneka izifundo ezongezelelweyo ukuvavanya le ngcamango. Ngokufanayo, izifundo zangaphambili zigxininise ukubaluleka kweeproteni ezisebenzisana ne-DELLA i-TCP14 kunye ne-15 kulawulo lokwakheka kwee-internode60,61 kwaye ezi zinto zinokulawula isenzo se-GA kunye ne-BREVIPEDICELLUS (BP) kunye ne-PENNYWISE (PNY), ezilawula uphuhliso lwee-internode kwaye zibonakaliswe ukuba zinefuthe kwi-GA signaling2,62. Njengoko i-DELLAs isebenzisana ne-brassinosteroid, i-ethylene, i-jasmonic acid, kunye neendlela zokubonisa i-abscisic acid (ABA)63,64 kwaye ezi hormone zinokuchaphazela ulwalathiso lwe-microtubule65, iziphumo ze-GA kwi-orientation yokwahlulahlula iseli zinokulawulwa zezinye iihomoni.
Izifundo zokuqala ze-cytological zibonise ukuba zombini iindawo zangaphakathi nezangaphandle ze-Arabidopsis SAM ziyafuneka ukuze kuphuhliswe ii-internode2,42. Inyani yokuba i-GA ilawula ngokusebenzayo ukwahlulwahlulwa kweeseli kwizicubu zangaphakathi12 ixhasa umsebenzi ombaxa we-GA ekulawuleni ubungakanani be-meristem kunye ne-internode kwi-SAM. Ipatheni yokwahlulwa kweeseli ezibhekiselele kwicala ikwalawulwa ngokuqinileyo kwizicubu zangaphakathi ze-SAM, kwaye lo mgaqo ubalulekile ekukhuleni kwesiqu52. Kuya kuba nomdla ukuphonononga ukuba i-GA ikwadlala indima ekujoliseni iplani yokwahlulwa kweeseli kwintlangano yangaphakathi ye-SAM, ngaloo ndlela kuhambelana iinkcukacha kunye nophuhliso lwee-internode ngaphakathi kwe-SAM.
Izityalo zikhuliswe kwi-vitro emhlabeni okanye kwi-1x Murashige-Skoog (MS) medium (Duchefa) eyongezwe nge-1% ye-sucrose kunye ne-1% ye-agar (Sigma) phantsi kweemeko eziqhelekileyo (ukukhanya kweeyure ezili-16, ama-22 °C), ngaphandle kovavanyo lwe-hypocotyl kunye nokukhula kweengcambu apho izithole zikhuliswe kwiipleyiti ezithe nkqo phantsi kokukhanya okungaguqukiyo kunye nama-22 °C. Kwizilingo ze-nitrate, izityalo zikhuliswe kwi-modified MS medium (i-bioWORLD plant medium) eyongezwe nge-nitrate eyaneleyo (0 okanye 10 mM KNO3), i-0.5 mM NH4-succinate, i-1% ye-sucrose kunye ne-1% ye-A-agar (Sigma) phantsi kweemeko zemini ende.
I-GID1a cDNA efakwe kwi-pDONR221 yaphinda yadityaniswa ne-pDONR P4-P1R-pUBQ10 kunye ne-pDONR P2R-P3-mCherry kwi-pB7m34GW ukuvelisa i-pBQ10::GID1a-mCherry. I-IDD2 DNA efakwe kwi-pDONR221 yaphinda yadityaniswa kwi-pB7RWG266 ukuvelisa i-p35S:IDD2-RFP. Ukuvelisa i-pGID1b::2xmTQ2-GID1b, iqhekeza le-3.9 kb elingaphezulu kwengingqi yekhowudi ye-GID1b kunye neqhekeza le-4.7 kb eliqulethe i-GID1b cDNA (1.3 kb) kunye ne-terminator (3.4 kb) zaqala zandiswa kusetyenziswa ii-primers kwiTheyibhile eyoNgezelelweyo 3 zaza zafakwa kwi-pDONR P4-P1R (Thermo Fisher Scientific) kunye ne-pDONR P2R-P3 (Thermo Fisher Scientific), ngokwahlukeneyo, kwaye ekugqibeleni zadityaniswa kwakhona ne-pDONR221 2xmTQ268 kwi-pGreen 012567 target vector kusetyenziswa i-Gateway cloning. Ukuvelisa i-pCUC2::LSSmOrange, ulandelelwano lwe-CUC2 promoter (3229 bp phezulu kwe-ATG) olulandelwa lulandelelwano lwekhowudi ye-Stokes-shifted mOrange enkulu (LSSmOrange)69 enesignali ye-N7 nuclear localization kunye ne-NOS transcriptional terminator zahlanganiswa kwi-pGreen kanamycin targeting vector kusetyenziswa inkqubo ye-Gateway 3-fragment recombination (Invitrogen). I-plant binary vector yaziswa kwi-Agrobacterium tumefaciens strain GV3101 yaza yaziswa kumagqabi eNicotiana benthamiana ngendlela ye-Agrobacterium infiltration kunye nakwi-Arabidopsis thaliana Col-0 ngendlela ye-floral dip, ngokulandelelana. i-pBQ10::qmRGA pBQ10::GID1a-mCherry kunye ne-pCLV3::mCherry-NLS qmRGA zahlulwe kwi-F3 kunye ne-F1 progenies ze-crosses ezahlukeneyo, ngokulandelelana.
I-RNA in situ hybridization yenziwe kwiincam zokudubula ezimalunga ne-1 cm ubude72, ezaqokelelwa zaza zafakwa kwisisombululo se-FAA (3.7% formaldehyde, 5% acetic acid, 50% ethanol) ngaphambi kokuba zipholiswe ukuya kwi-4 °C. Emva konyango lwe-vacuum lwemizuzu emi-2 × 15, i-fixative yatshintshwa kwaye iisampulu zafakwa kwi-incubation ubusuku bonke. I-GID1a, GID1b, GID1c, GAI, RGL1, RGL2, kunye ne-RGL3 cDNAs kunye nee-antisense probes kwi-3′-UTRs zazo zenziwe kusetyenziswa ii-primers eziboniswe kwiTheyibhile eyoNgezelelweyo 3 njengoko kuchaziwe nguRosier et al.73. Iiprobe ezineelebheli zeDigoxigenin zifunyenwe kusetyenziswa ii-antibodies ze-digoxigenin (i-dilution ephindwe ka-3000; iRoche, inombolo yekhathalogu: 11 093 274 910), kwaye iindawo zadityaniswa nge-5-bromo-4-chloro-3-indolyl phosphate (BCIP, i-dilution ephindwe ka-250)/nitroblue tetrazolium (NBT, i-dilution ephindwe ka-200).
Ixesha leposi: Februwari-10-2025