Ukukhula kwe-apical meristem (SAM) yehlumela kubalulekile ekwakhiweni kwesiqu.ama-gibberellins(ama-GA) adlala indima ebalulekile ekuhlanganiseni ukukhula kwezitshalo, kodwa indima yawo ku-SAM ayikaqondakali kahle. Lapha, sakha i-ratiometric biosensor ye-GA signaling ngobunjiniyela beprotheyini ye-DELLA ukuze sicindezele umsebenzi wayo obalulekile wokulawula empendulweni yokubhala ye-GA ngenkathi silondoloza ukuwohloka kwayo lapho kuqashelwa i-GA. Sibonisa ukuthi le biosensor esekelwe ekuwohlokeni iqopha ngokunembile izinguquko emazingeni e-GA kanye nokuzwa kwamaseli ngesikhathi sokuthuthukiswa. Sisebenzise le biosensor ukudweba umsebenzi wokubonisa we-GA ku-SAM. Sibonisa ukuthi izimpawu ze-GA eziphezulu zikhona kakhulu kumaseli atholakala phakathi kwe-organ primordia, okuyizinto ezandulela amaseli angaphakathi. Sisebenzisa izindlela zokuzuza nokulahlekelwa umsebenzi, sibonisa ukuthi i-GA ilawula ukuqondiswa kwendiza yokuhlukaniswa kwamaseli, isungula inhlangano yamaseli angaphakathi, ngaleyo ndlela ikhuthaze ukucaciswa kwama-internode ku-SAM.
I-meristem ye-apical yehlumela (i-SAM), etholakala esicongweni sehlumela, iqukethe indawo yamaseli esiqu anomsebenzi wawo okhiqiza izitho eziseceleni kanye nama-stem node ngendlela eguquguqukayo nephindaphindayo kuyo yonke impilo yesitshalo. Ngayinye yalezi zingxenye eziphindaphindayo, noma ama-node esitshalo, ifaka ama-internode kanye nezitho eziseceleni kuma-node, kanye nama-meristem e-axillary kuma-axils eqabunga1. Ukukhula nokuhleleka kwama-node esitshalo kuyashintsha ngesikhathi sokukhula. Ku-Arabidopsis, ukukhula kwama-internodal kuyacindezelwa ngesikhathi sesigaba sokukhula, kanti ama-meristem e-axillary ahlala elele kuma-axils amaqabunga e-rosette. Ngesikhathi sokushintshela esigabeni sezimbali, i-SAM iba yi-meristem ye-inflorescence, ikhiqiza ama-internode amade kanye nama-axillary buds, amagatsha ema-axils amaqabunga e-cauline, kanye nezimbali ezingenamaqabunga2 kamuva. Nakuba senze intuthuko enkulu ekuqondeni izindlela ezilawula ukuqala kwamaqabunga, izimbali, namagatsha, kuncane okwaziwayo mayelana nokuthi ama-internode avela kanjani.
Ukuqonda ukusatshalaliswa kwe-GAs kwendawo kuzosiza ukuqonda kangcono imisebenzi yala ma-hormone ezicutshini ezahlukene kanye nasezigabeni ezahlukene zokukhula. Ukubona ukuwohloka kokuhlanganiswa kwe-RGA-GFP okuvezwe ngaphansi kwesenzo se-promoter yayo kunikeza ulwazi olubalulekile mayelana nokulawulwa kwamazinga e-GA aphelele ezimpandeni15,16. Kodwa-ke, ukubonakaliswa kwe-RGA kuyahlukahluka kuzo zonke izicubu17 futhi kulawulwa yi-GA18. Ngakho-ke, ukubonakaliswa okuhlukile kwe-promoter ye-RGA kungabangela iphethini ye-fluorescence ebonwa yi-RGA-GFP ngakho-ke le ndlela ayilingani. Muva nje, i-GA19,20 ene-bioactive fluorescein (Fl) iveze ukuqongelela kwe-GA ku-endocortex yempande kanye nokulawulwa kwamazinga ayo eselula ngokuthuthwa kwe-GA. Muva nje, i-GA FRET sensor nlsGPS1 ibonise ukuthi amazinga e-GA ahlobene nokwandiswa kweseli ezimpandeni, kuma-filaments, kanye nama-hypocotyls akhule emnyama21. Kodwa-ke, njengoba sesibonile, ukuhlushwa kwe-GA akuyona yodwa ipharamitha elawula umsebenzi wokubonisa we-GA, njengoba kuncike ezinqubweni eziyinkimbinkimbi zokuzwa. Lapha, sakha ukuqonda kwethu izindlela zokubonisa izimpawu ze-DELLA kanye ne-GA, sibika ukuthuthukiswa kanye nokuchazwa kwe-biosensor esekelwe ekuwohlokeni kwe-GA signaling. Ukuze sithuthukise le biosensor yobuningi, sisebenzise i-RGA ezwela i-GA eguquguqukayo ehlanganiswe neprotheni ekhanyayo futhi evezwa kabanzi ezicutshini, kanye neprotheni ezwela i-GA engabonakali. Sibonisa ukuthi ukuhlanganiswa kwamaprotheni e-RGA eguquguqukayo akuphazamisi ukusayinwa kwe-GA okungokwemvelo lapho kuvezwa kabanzi, nokuthi le biosensor ingalinganisa umsebenzi wokusayina ovela kokubili ekufakweni kwe-GA kanye nokucutshungulwa kwesignali ye-GA yidivayisi yokuzwa enesisombululo esiphezulu se-spatiotemporal. Sisebenzise le biosensor ukumaka ukusatshalaliswa kwe-spatiotemporal komsebenzi wokusayina we-GA futhi silinganise ukuthi i-GA ilawula kanjani ukuziphatha kweseli ku-SAM epidermis. Sibonisa ukuthi i-GA ilawula ukuqondiswa kwendiza yokuhlukanisa yamaseli e-SAM atholakala phakathi kwe-organ primordia, ngaleyo ndlela ichaza inhlangano yamaseli e-canonical ye-internode.
Ekugcineni, sibuze ukuthi i-qmRGA ingabika yini izinguquko emazingeni e-GA angaphandle sisebenzisa ama-hypocotyls akhulayo. Ngaphambilini sibonise ukuthi i-nitrate ivuselela ukukhula ngokwandisa ukwenziwa kwe-GA, futhi, ukuwohloka kwe-DELLA34. Ngakho-ke, sibonile ukuthi ubude be-hypocotyl ezitshalweni ze-pUBQ10::qmRGA ezikhuliswe ngaphansi kokuhlinzekwa kwe-nitrate eningi (10 mM NO3−) bude kakhulu kunasezitshalweni ezikhuliswe ngaphansi kwezimo zokuntuleka kwe-nitrate (Isifanekiso Esingeziwe 6a). Ngokuhambisana nempendulo yokukhula, izimpawu ze-GA zaziphezulu kuma-hypocotyls ezitshalo ezikhuliswe ngaphansi kwezimo ze-10 mM NO3− kunasezitshalweni ezikhuliswe ngaphandle kwe-nitrate (Isifanekiso Esingeziwe 6b, c). Ngakho-ke, i-qmRGA iphinde ivumele ukuqapha izinguquko ekusayinweni kwe-GA okubangelwa izinguquko ezingokwemvelo ekugxilweni kwe-GA.
Ukuze siqonde ukuthi umsebenzi wokubonisana we-GA otholwe yi-qmRGA uncike ekugxilweni kwe-GA kanye nokubona kwe-GA, njengoba bekulindelekile ngokusekelwe ekwakhiweni kwenzwa, sihlaziye ukuvezwa kwama-receptor amathathu e-GID1 ezicutshini zezitshalo nezokuzala. Ezitshalweni, umugqa wentatheli we-GID1-GUS ukhombisile ukuthi i-GID1a kanye ne-c zazivezwa kakhulu kuma-cotyledons (Isithombe 3a–c). Ngaphezu kwalokho, wonke ama-receptor amathathu ayevezwa emaqabunga, izimpande ze-lateral root primordia, amathiphu ezimpande (ngaphandle kwe-root cap ye-GID1b), kanye nesistimu yemithambo yegazi (Isithombe 3a–c). Ku-inflorescence SAM, sithole izimpawu ze-GUS kuphela ze-GID1b kanye ne-1c (Isithombe Esingeziwe 7a–c). I-In situ hybridization iqinisekisile lawa maphethini okubonakaliswa futhi yabonisa ukuthi i-GID1c yayivezwa ngokufanayo emazingeni aphansi ku-SAM, kanti i-GID1b ibonise ukuvezwa okuphezulu emngceleni we-SAM (Isithombe Esingeziwe 7d–l). Ukuhlanganiswa kokuhumusha kwe-pGID1b::2xmTQ2-GID1b kuphinde kwembula ububanzi obulinganisiwe bokubonakaliswa kwe-GID1b, kusukela ekubonakalisweni okuphansi noma okungekho ndawo phakathi kwe-SAM kuya ekubonakalisweni okuphezulu emingceleni yezitho (Isithombe Esingeziwe 7m). Ngakho-ke, ama-receptor e-GID1 awasatshalaliswa ngokulinganayo kuzo zonke izicubu nangaphakathi kwazo. Ekuhlolweni okulandelayo, siphinde sabona ukuthi ukubonakaliswa ngokweqile kwe-GID1 (pUBQ10::GID1a-mCherry) kwandisa ukuzwela kwe-qmRGA kuma-hypocotyls kuya ekusetshenzisweni kwe-GA yangaphandle (Isithombe 3d, e). Ngokuphambene nalokho, i-fluorescence elinganiswe yi-qd17mRGA ku-hypocotyl yayingazweli ekwelashweni kwe-GA3 (Isithombe 3f, g). Kuzo zombili izivivinyo, izithombo zaphathwa ngamanani aphezulu e-GA (100 μM GA3) ukuhlola ukuziphatha okusheshayo kwenzwa, lapho ikhono lokubopha ku-receptor ye-GID1 lakhuliswa noma lalahleka. Ndawonye, le miphumela iqinisekisa ukuthi i-qmRGA biosensor isebenza umsebenzi ohlangene njenge-GA kanye ne-GA sensor, futhi iphakamisa ukuthi ukuvezwa okuhlukile kwe-GID1 receptor kungashintsha kakhulu ukukhishwa kwenzwa.
Kuze kube manje, ukusatshalaliswa kwezimpawu ze-GA ku-SAM akukacaci. Ngakho-ke, sisebenzise izitshalo eziveza i-qmRGA kanye ne-pCLV3::mCherry-NLS stem cell reporter35 ukuze sibale amamephu ezibalo aphezulu omsebenzi wokubonisa i-GA, sigxile kungqimba lwe-L1 (i-epidermis; Umfanekiso 4a, b, bheka Izindlela Nezindlela Zokwengeza), njengoba i-L1 idlala indima ebalulekile ekulawuleni ukukhula kwe-SAM36. Lapha, ukubonakaliswa kwe-pCLV3::mCherry-NLS kunikeze iphuzu lokubhekisela eliqinile le-geometric lokuhlaziya ukusatshalaliswa kwendawo komsebenzi wokubonisa i-GA37. Nakuba i-GA ibhekwa njengebalulekile ekuthuthukisweni kwezitho eziseceleni4, sibonile ukuthi izimpawu ze-GA zaziphansi ku-primordium yezimbali (P) kusukela esigabeni se-P3 (Umfanekiso 4a, b), kanti ama-primordium amancane e-P1 ne-P2 ayenomsebenzi olinganiselayo ofana nalowo osesifundeni esiphakathi (Umfanekiso 4a, b). Umsebenzi wokubonisa we-GA ophezulu utholakale emingceleni ye-primordium yesitho, uqala ku-P1/P2 (ezinhlangothini zomngcele) futhi ufinyelela esicongweni ku-P4, kanye nakuwo wonke amaseli esifunda esingaphandle esitholakala phakathi kwe-primordia (Isithombe 4a, b kanye nesithombe esengeziwe 8a, b). Lo msebenzi wokubonisa we-GA ophezulu ubonwe hhayi kuphela e-epidermis kodwa nakwezendlalelo ze-L2 kanye ne-L3 ephezulu (Isithombe Esengeziwe 8b). Iphethini yezibonakaliso ze-GA ezitholwe ku-SAM kusetshenziswa i-qmRGA nayo ayizange ishintshe ngokuhamba kwesikhathi (Isithombe Esengeziwe 8c–f, k). Nakuba ukwakheka kwe-qd17mRGA kwehliswe ngokuhlelekile ku-SAM yezitshalo ze-T3 kusuka emigqeni emihlanu ezimele esiyichaze ngokuningiliziwe, sikwazile ukuhlaziya amaphethini e-fluorescence atholwe ngokwakhiwa kwe-pRPS5a::VENUS-2A-TagBFP (Isithombe Esengeziwe 8g–j, l). Kulo mugqa wokulawula, kutholakale izinguquko ezincane kuphela ku-fluorescence ratio ku-SAM, kodwa esikhungweni se-SAM sibone ukwehla okucacile nokungalindelekile kwe-VENUS okuhlobene ne-TagBFP. Lokhu kuqinisekisa ukuthi iphethini yokubonisa ebonwe yi-qmRGA ibonisa ukwehla okuncike ku-GA kwe-mRGA-VENUS, kodwa futhi kukhombisa ukuthi i-qmRGA ingase ilinganisele ngokweqile umsebenzi wokubonisa we-GA esikhungweni se-meristem. Ngamafuphi, imiphumela yethu yembula iphethini yokubonisa ye-GA ekhombisa ngokuyinhloko ukusatshalaliswa kwe-primordia. Lokhu kusatshalaliswa kwesifunda esiphakathi kwe-primordial (IPR) kungenxa yokusungulwa kancane kancane komsebenzi wokubonisa we-GA ophezulu phakathi kwe-primordium ekhulayo kanye nesifunda esiphakathi, kuyilapho ngesikhathi esifanayo umsebenzi wokubonisa we-GA esikhungweni esiphakathi wehla (Umfanekiso 4c, d).
Ukusatshalaliswa kwama-receptor e-GID1b kanye ne-GID1c (bheka ngenhla) kusikisela ukuthi ukuvezwa okuhlukile kwama-receptor e-GA kusiza ekwakheni iphethini yomsebenzi wokubonisana kwe-GA ku-SAM. Sizibuze ukuthi ukuqongelela okuhlukile kwe-GA kungenzeka yini. Ukuze sihlole lokhu kungenzeka, sisebenzise inzwa ye-nlsGPS1 GA FRET21. Imvamisa yokuqalisa eyandayo itholakale ku-SAM ye-nlsGPS1 ephathwe nge-10 μM GA4+7 imizuzu eyi-100 (Isithombe Esingeziwe 9a–e), okubonisa ukuthi i-nlsGPS1 iyasabela ezinguqukweni zokuhlushwa kwe-GA ku-SAM, njengoba kwenza ezimpandeni21. Ukusatshalaliswa kwendawo kwemvamisa yokuqalisa ukusebenza kwe-nlsGPS1 kwembule amazinga e-GA aphansi kakhulu ezingqimbeni zangaphandle ze-SAM, kodwa kwabonisa ukuthi ayephakeme phakathi nasemingceleni ye-SAM (Isithombe 4e kanye nesithombe Esingeziwe 9a,c). Lokhu kusikisela ukuthi i-GA nayo isatshalaliswa ku-SAM ngephethini yendawo efana naleyo eyembulwe yi-qmRGA. Njengendlela ehambisanayo, siphinde saphatha i-SAM nge-GA ekhanyayo (GA3-, GA4-, GA7-Fl) noma i-Fl yodwa njengokulawula okungekuhle. Isignali ye-Fl yasatshalaliswa kulo lonke i-SAM, kufaka phakathi isifunda esiphakathi kanye ne-primordium, yize yayinamandla aphansi (Isithombe 4j kanye ne-Supplementary Fig. 10d). Ngokuphambene nalokho, wonke ama-GA-Fl amathathu aqoqene ngqo ngaphakathi kwemingcele ye-primordium kanye namazinga ahlukahlukene kulo lonke i-IPR, kanti i-GA7-Fl iqoqene endaweni enkulu kunazo zonke ku-IPR (Isithombe 4k kanye ne-Supplementary Fig. 10a,b). Ukulinganiswa kokuqina kwe-fluorescence kwembule ukuthi isilinganiso se-IPR kuya kwe-non-IPR intensity sasiphezulu ku-SAM ephathwe yi-GA-Fl uma kuqhathaniswa ne-SAM ephathwe yi-Fl (Isithombe 4l kanye ne-Supplementary Fig. 10c). Ndawonye, le miphumela iphakamisa ukuthi i-GA ikhona ekugxilweni okuphezulu kumaseli e-IPR aseduze nomngcele we-organ. Lokhu kusikisela ukuthi iphethini yomsebenzi wokubonisana we-SAM GA ibangelwa kokubili ukubonakaliswa okuhlukile kwama-receptor e-GA kanye nokuqongelela okuhlukile kwe-GA kumaseli e-IPR eduze kwemingcele yezitho. Ngakho-ke, ukuhlaziywa kwethu kwembule iphethini engalindelekile yendawo yokubonisana kwe-GA, enomsebenzi ophansi phakathi nendawo kanye ne-primordium ye-SAM kanye nomsebenzi ophezulu ku-IPR esifundeni esingaphandle.
Ukuze siqonde indima yomsebenzi wokubonisana we-GA ohlukile ku-SAM, sihlaziye ubudlelwano phakathi komsebenzi wokubonisana we-GA, ukwandiswa kweseli, kanye nokwahlukaniswa kweseli sisebenzisa isithombe sesikhathi sangempela se-SAM qmRGA pCLV3::mCherry-NLS. Njengoba sinikezwe indima ye-GA ekulawulweni kokukhula, kwakulindelwe ubudlelwano obuhle namapharamitha okukhulisa iseli. Ngakho-ke, siqale saqhathanisa amamephu omsebenzi wokubonisana we-GA namamephu wesilinganiso sokukhula kobuso beseli (njengesivikelo samandla okukhulisa iseli leseli elinikeziwe kanye namaseli endodakazi ekuhlukanisweni) kanye namamephu we-anisotropy yokukhula, elinganisa ukuqondiswa kokwanda kweseli (okusetshenziswe lapha kuseli elinikeziwe kanye namaseli endodakazi ekuhlukanisweni; Umfanekiso 5a,b, bheka Izindlela Nezindlela Zokwengeza). Amamephu ethu esilinganiso sokukhula kobuso beseli le-SAM ahambisana nokubonwa kwangaphambilini38,39, ngamazinga okukhula amancane emngceleni kanye namazinga okukhula aphezulu ezimbalini ezikhulayo (Umfanekiso 5a). Ukuhlaziywa kwengxenye eyinhloko (i-PCA) kubonise ukuthi umsebenzi wokubonisana we-GA wawuhlobene kabi nokuqina kokukhula kobuso beseli (Umfanekiso 5c). Siphinde sabonisa ukuthi izingqimba eziyinhloko zokwehluka, okuhlanganisa nokufakwa kwesignali ye-GA kanye nokuqina kokukhula, zazihambisana nesiqondiso esinqunywe ukubonakaliswa okuphezulu kwe-CLV3, okuqinisekisa ukukhishwa kwamaseli esikhungweni se-SAM ekuhlaziyweni okusele. Ukuhlaziywa kokuxhumana kwe-Spearman kuqinisekisile imiphumela ye-PCA (Isithombe 5d), okubonisa ukuthi izimpawu ze-GA eziphezulu ku-IPR azizange ziholele ekukhuleni okuphezulu kwamaseli. Kodwa-ke, ukuhlaziywa kokuxhumana kwembula ukuhlangana okuncane okuhle phakathi komsebenzi wokubonisana we-GA kanye ne-anisotropy yokukhula (Isithombe 5c, d), okuphakamisa ukuthi ukubonakaliswa okuphezulu kwe-GA ku-IPR kuthonya isiqondiso sokukhula kwamaseli kanye mhlawumbe nendawo yendiza yokuhlukaniswa kwamaseli.
a, b Amamephu okushisa okukhula okumaphakathi kobuso (a) kanye ne-anisotropy yokukhula (b) ku-SAM alinganisela izitshalo eziyisikhombisa ezizimele (ezisetshenziswa njengezivikelo zamandla kanye nesiqondiso sokwanda kweseli, ngokulandelana). c Ukuhlaziywa kwe-PCA kufake phakathi izinto ezilandelayo eziguquguqukayo: isignali ye-GA, ukuqina kokukhula kobuso, i-anisotropy yokukhula kobuso, kanye nokubonakaliswa kwe-CLV3. Ingxenye ye-PCA 1 yayihlotshaniswa kakhulu nokuqina kokukhula kobuso futhi ihlotshaniswa kahle nesignali ye-GA. Ingxenye ye-PCA 2 yayihlotshaniswa kakhulu nokukhula kobuso futhi ihlotshaniswa kabi nokubonakaliswa kwe-CLV3. Amaphesenti amelela ukwehluka okuchazwe yingxenye ngayinye. d Ukuhlaziywa kokuxhumana kwe-Spearman phakathi kwesignali ye-GA, ukuqina kokukhula kobuso, kanye ne-anisotropy yokukhula kobuso esikalini sezicubu ngaphandle kwe-CZ. Inombolo ngakwesokudla yinani le-Spearman rho phakathi kwezinto ezimbili eziguquguqukayo. Ama-Asterisk akhombisa amacala lapho ukuhlangana/ukuhlangana okungekuhle kubaluleke kakhulu. e Ukuboniswa kwe-3D kwamaseli e-Col-0 SAM L1 nge-confocal microscopy. Izindonga ezintsha zamaseli ezakhiwe ku-SAM (kodwa hhayi i-primordium) emahoreni ayi-10 zinombala ngokuya ngamanani azo e-engeli. Ibha yombala iboniswe ekhoneni elingezansi kwesokudla. I-inset ikhombisa isithombe se-3D esihambisanayo ngo-0 h. Ukuhlolwa kuphindwe kabili ngemiphumela efanayo. f Ama-Box plots abonisa amazinga okuhlukaniswa kwamaseli ku-IPR kanye ne-non-IPR Col-0 SAM (n = izitshalo ezizimele eziyi-10). Umugqa ophakathi ukhombisa i-median, kanti imingcele yebhokisi ikhombisa ama-percentile angama-25 nama-75. Ama-whiskers akhombisa amanani amancane naphezulu anqunywe ngesofthiwe ye-R. Amanani e-P atholakale nge-t-test ka-Welch enemisila emibili. g, h Umdwebo we-Schematic obonisa (g) ukuthi ungalinganisa kanjani i-engeli yodonga olusha lweseli (i-magenta) maqondana nesiqondiso se-radial kusuka enkabeni ye-SAM (umugqa omhlophe onamachashazi) (kucatshangelwa amanani e-angle acute kuphela, okungukuthi, 0–90°), kanye (h) iziqondiso ze-circumferential/lateral kanye ne-radial ngaphakathi kwe-meristem. i Ama-histogram emvamisa yokuqondiswa kwendiza yokuhlukaniswa kwamaseli kulo lonke i-SAM (oluhlaza okwesibhakabhaka okumnyama), i-IPR (oluhlaza okwesibhakabhaka okuphakathi), kanye ne-non-IPR (oluhlaza okwesibhakabhaka okukhanyayo), ngokulandelana. Amanani e-P atholwe ngokuhlolwa kwe-Kolmogorov-Smirnov okunemisila emibili. Ukuhlolwa kuphindwe kabili ngemiphumela efanayo. j Ama-histogram emvamisa yokuqondiswa kwendiza yokuhlukaniswa kweseli kwe-IPR ezungeze i-P3 (oluhlaza okotshani okukhanyayo), i-P4 (oluhlaza okotshani okuphakathi), kanye ne-P5 (oluhlaza okotshani okumnyama), ngokulandelana. Amanani e-P atholwe ngokuhlolwa kwe-Kolmogorov-Smirnov okunemisila emibili. Ukuhlolwa kuphindwe kabili ngemiphumela efanayo.
Ngakho-ke, ngokulandelayo sihlole ubudlelwano phakathi kokubonisana kwe-GA nomsebenzi wokuhlukanisa amaseli ngokuhlonza izindonga zamaseli ezisanda kwakheka ngesikhathi sokuhlolwa (Umfanekiso 5e). Le ndlela yasivumela ukuthi silinganise imvamisa kanye nesiqondiso sokuhlukanisa amaseli. Ngokumangazayo, sithole ukuthi imvamisa yokuhlukanisa amaseli ku-IPR kanye nayo yonke i-SAM (engeyona i-IPR, Umfanekiso 5f) yayifana, okubonisa ukuthi umehluko ekuboniseni kwe-GA phakathi kwamaseli e-IPR kanye nalawo angewona i-IPR awuthinti kakhulu ukuhlukaniswa kwamaseli. Lokhu, kanye nobudlelwano obuhle phakathi kokubonisana kwe-GA kanye ne-anisotropy yokukhula, kwasenza sacabanga ukuthi umsebenzi wokubonisana we-GA ungathonya yini ukuqondiswa kwendiza yokuhlukanisa amaseli. Silinganise ukuqondiswa kodonga lweseli elisha njenge-acute angle ehlobene ne-radial axis exhumanisa isikhungo se-meristem kanye nesikhungo sodonga lweseli elisha (Isithombe 5e-i) futhi sabona ukuthambekela okucacile kwamaseli ukuhlukana ngama-engeli aseduze nama-90° uma kuqhathaniswa ne-radial axis, kanye nama-frequency aphezulu abonwe ku-70–80° (23.28%) kanye no-80–90° (22.62%) (Isithombe 5e,i), okuhambisana nokuhlukaniswa kwamaseli ohlangothini olujikelezayo/oluphambene (Isithombe 5h). Ukuze sihlole umnikelo we-GA signaling kulokhu kuziphatha kokuhlukaniswa kwamaseli, sihlaziye amapharamitha okuhlukaniswa kwamaseli ku-IPR kanye ne-non-IPR ngokwehlukana (Isithombe 5i). Sibonile ukuthi ukusatshalaliswa kwe-engeli yokuhlukanisa kumaseli e-IPR kuhlukile kulokho okusemaseli angewona ama-IPR noma kumaseli kuyo yonke i-SAM, lapho amaseli e-IPR ebonisa ingxenye ephezulu yokuhlukaniswa kwamaseli aseceleni/ayindilinga, okungukuthi, 70–80° kanye no-80–90° (33.86% kanye no-30.71%, ngokulandelana, izilinganiso ezihambisanayo) (Isithombe 5i). Ngakho-ke, okubonwe yithi kwembule ubudlelwano phakathi kokubonisana okuphezulu kwe-GA kanye nokuqondiswa kwendiza yokuhlukanisa iseli eduze kwesiqondiso esijikelezayo, okufana nobudlelwano phakathi komsebenzi wokubonisana we-GA kanye ne-anisotropy yokukhula (Isithombe 5c, d). Ukuze siqhubeke nokuqinisekisa ukulondolozwa kwendawo kwalokhu kuhlangana, silinganise ukuqondiswa kwendiza yokuhlukanisa kumaseli e-IPR azungeze i-primordium kusukela ku-P3, njengoba umsebenzi wokubonisana we-GA ophezulu kakhulu utholakale kulesi sifunda kusukela ku-P4 (Isithombe 4). Ama-engeli okuhlukanisa e-IPR azungeze i-P3 kanye ne-P4 awabonisanga umehluko obalulekile ngokwezibalo, yize imvamisa yokwanda kokuhlukaniswa kwamaseli aseceleni kubonwe ku-IPR azungeze i-P4 (Isithombe 5j). Kodwa-ke, kumaseli e-IPR azungeze i-P5, umehluko ekuqondisweni kwendiza yokuhlukaniswa kwamaseli waba mkhulu ngokwezibalo, ngokwanda okukhulu kokuvama kokuhlukaniswa kwamaseli okuphambene (Isithombe 5j). Ndawonye, le miphumela isikisela ukuthi ukusayina kwe-GA kungalawula ukuqondiswa kokuhlukaniswa kwamaseli ku-SAM, okuhambisana nemibiko yangaphambilini40,41 yokuthi ukusayina kwe-GA okuphezulu kungadala ukuqondiswa kwecala kokuhlukaniswa kwamaseli ku-IPR.
Kubikezelwa ukuthi amaseli aku-IPR ngeke afakwe kuma-primordia kodwa afakwe kuma-internode2,42,43. Ukuqondiswa okuphambene kokuhlukaniswa kwamaseli ku-IPR kungabangela ukuhlelwa okuvamile kwemigqa emide efanayo yamaseli e-epidermal kuma-internode. Okubonwe ngenhla kusikisela ukuthi i-GA signaling cishe idlala indima kule nqubo ngokulawula isiqondiso sokuhlukaniswa kwamaseli.
Ukulahlekelwa umsebenzi wezakhi zofuzo eziningana ze-DELLA kuholela empendulweni ye-GA ehlanganisiwe, futhi i-della mutants ingasetshenziswa ukuhlola lo mbono44. Siqale sahlaziya amaphethini okubonakaliswa kwezakhi zofuzo ezinhlanu ze-DELLA ku-SAM. Ukuhlanganiswa kombhalo komugqa we-GUS45 kwembule ukuthi i-GAI, i-RGA, i-RGL1, kanye ne-RGL2 (ngezinga elincane kakhulu) kwavezwa ku-SAM (Isithombe Esingeziwe 11a–d). I-In situ hybridization yabonisa futhi ukuthi i-GAI mRNA iqoqana ikakhulukazi ezimbalini zokuqala kanye nasezitshalweni ezisathuthuka (Isithombe Esingeziwe 11e). I-RGL1 kanye ne-RGL3 mRNA zatholakala kulo lonke i-SAM canopy kanye nasezimbalini ezindala, kanti i-RGL2 mRNA yayiningi kakhulu esifundeni somngcele (Isithombe Esingeziwe 11f–h). Ukuthwebula izithombe ze-pRGL3::RGL3-GFP SAM kuqinisekisile ukubonakaliswa okubonwe yi-in situ hybridization futhi kwabonisa ukuthi iphrotheni ye-RGL3 iqoqana engxenyeni ephakathi ye-SAM (Isithombe Esingeziwe 11i). Sisebenzisa umugqa we-pRGA::GFP-RGA, sithole nokuthi iphrotheni ye-RGA iqoqana ku-SAM, kodwa ubuningi bayo buyancipha emngceleni kusukela ku-P4 (Isithombe Esingeziwe 11j). Okuphawulekayo ukuthi amaphethini okubonakaliswa kwe-RGL3 kanye ne-RGA ahambisana nomsebenzi wokubonisa we-GA ophezulu ku-IPR, njengoba kutholwe yi-qmRGA (Isithombe 4). Ngaphezu kwalokho, le datha ikhombisa ukuthi wonke ama-DELLA avezwa ku-SAM nokuthi ukubonakaliswa kwawo ndawonye kuhlanganisa yonke i-SAM.
Ngokulandelayo sihlaziye amapharamitha okuhlukaniswa kwamaseli ku-SAM yohlobo lwe-wild-type (Ler, control) kanye ne-gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Fig. 6a, b). Ngokuthakazelisayo, sibone ushintsho olubalulekile ngokwezibalo ekusabalalisweni kwama-angle okuhlukaniswa kwamaseli ku-della global mutant SAM uma kuqhathaniswa nohlobo lwe-wild (Fig. 6c). Lolu shintsho ku-della global mutant lubangelwe ukwanda kwemvamisa yama-angles angu-80-90° (34.71% vs. 24.55%) kanye, ngezinga elincane, ama-angles angu-70-80° (23.78% vs. 20.18%), okungukuthi, okuhambisana nokuhlukaniswa kwamaseli okuguquguqukayo (Fig. 6c). Imvamisa yokuhlukaniswa okungeyona i-transverse (0-60°) nayo yayiphansi ku-della global mutant (Fig. 6c). Imvamisa yokuhlukaniswa kwamaseli ajikelezayo yanda kakhulu ku-SAM ye-della global mutant (Isithombe 6b). Imvamisa yokuhlukaniswa kwamaseli ajikelezayo ku-IPR nayo yayiphezulu ku-della global mutant uma kuqhathaniswa nohlobo lwasendle (Isithombe 6d). Ngaphandle kwesifunda se-IPR, uhlobo lwasendle lwalunokusatshalaliswa okufanayo kwama-engeli okuhlukaniswa kwamaseli, kuyilapho i-della global mutant ikhetha ukuhlukaniswa kwe-tangential njenge-IPR (Isithombe 6e). Siphinde salinganisa ukuqondiswa kokuhlukaniswa kwamaseli ku-SAM kwama-ga2 oxidase (ga2ox) ama-quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, kanye ne-ga2ox6-2), isizinda se-GA-inactive mutant lapho i-GA iqoqa khona. Ngokuhambisana nokwanda kwamazinga e-GA, i-SAM ye-quintuple ga2ox mutant inflorescence yayinkulu kuneye-Col-0 (Isithombe Esingeziwe 12a, b), futhi uma iqhathaniswa ne-Col-0, i-quintuple ga2ox SAM ibonise ukusatshalaliswa okuhlukile kakhulu kwama-engeli okuhlukaniswa kwamaseli, kanye nemvamisa ye-engeli ekhuphuka kusukela ku-50° kuya ku-90°, okungukuthi futhi ithanda ukuhlukaniswa kwe-tangential (Isithombe Esingeziwe 12a–c). Ngakho-ke, sibonisa ukuthi ukusebenza okuhlanganisiwe kwe-GA signaling kanye nokuqongelela kwe-GA kubangela ukuhlukaniswa kwamaseli aseceleni ku-IPR kanye nayo yonke i-SAM.
a, b Ukuboniswa kwe-3D kwesendlalelo se-L1 se-PI-stained Ler (a) kanye ne-global della mutant (b) SAM kusetshenziswa i-confocal microscopy. Izindonga ezintsha zamaseli ezakhiwe ku-SAM (kodwa hhayi i-primordium) esikhathini samahora ayi-10 ziyaboniswa futhi zifakwe imibala ngokwezinga lazo le-engeli. Isethi engezansi ikhombisa i-SAM ku-0 h. Ibha yombala iboniswa ekhoneni elingezansi kwesokudla. Umcibisholo ku-(b) ukhomba isibonelo samafayela eseli aqondanisiwe ku-global della mutant. Ukuhlolwa kuphindwe kabili ngemiphumela efanayo. ce ukuqhathaniswa kokusatshalaliswa kwemvamisa kokuqondiswa kwendiza yokuhlukaniswa kwamaseli kuyo yonke i-SAM (d), i-IPR (e), kanye ne-non-IPR (f) phakathi kwe-Ler kanye ne-global della. Amanani e-P atholwe kusetshenziswa ukuhlolwa kwe-Kolmogorov-Smirnov okunemisila emibili. f, g Ukuboniswa kwe-3D kwezithombe ze-confocal ze-PI-stained SAM ye-Col-0 (i) kanye ne-pCUC2::gai-1-VENUS (j) izitshalo ze-transgenic. Amaphaneli (a, b) abonisa izindonga ezintsha zamaseli (kodwa hhayi ama-primordia) ezakhiwe ku-SAM zingakapheli amahora ayi-10. Ukuhlolwa kuphindwe kabili ngemiphumela efanayo. h–j Ukuqhathaniswa kokusatshalaliswa kwemvamisa kokuqondiswa kwendiza yokuhlukaniswa kwamaseli okutholakala kuyo yonke i-SAM (h), i-IPR (i) kanye ne-non-IPR (j) phakathi kwezitshalo ze-Col-0 kanye ne-pCUC2::gai-1-VENUS. Amanani e-P atholwe kusetshenziswa ukuhlolwa kwe-Kolmogorov–Smirnov okunemisila emibili.
Ngokulandelayo sihlole umphumela wokuvimbela ukusayinwa kwe-GA ikakhulukazi ku-IPR. Ngenxa yalokhu, sisebenzise i-cotyledon cup 2 (CUC2) promoter ukuze siqhubekisele phambili ukubonakaliswa kweprotheyini ye-gai-1 engemihle ehlanganisiwe ku-VENUS (kumugqa we-pCUC2::gai-1-VENUS). Ku-SAM yohlobo lwe-wild, i-CUC2 promoter iqhubekisele phambili ukubonakaliswa kwama-IPR amaningi ku-SAM, kufaka phakathi amaseli omngcele, kusukela ku-P4 kuya phambili, futhi ukubonakaliswa okufanayo okuqondile kwabonwa ezitshalweni ze-pCUC2::gai-1-VENUS (bheka ngezansi). Ukusatshalaliswa kwama-engeli okuhlukaniswa kwamaseli ku-SAM noma i-IPR yezitshalo ze-pCUC2::gai-1-VENUS kwakungafani kakhulu nokwezinhlobo zasendle, yize ngokungalindelekile sithole ukuthi amaseli angenayo i-IPR kulezi zitshalo ahlukaniswe ngesivinini esiphezulu sama-80–90° (Isithombe 6f–j).
Kuye kwaphakanyiswa ukuthi isiqondiso sokuhlukaniswa kwamaseli sincike ku-geometry ye-SAM, ikakhulukazi ukucindezeleka kokudonsa okubangelwa ukugoba kwezicubu46. Ngakho-ke sibuze ukuthi isimo se-SAM sishintshiwe yini ezitshalweni ze-della global mutant kanye ne-pCUC2::gai-1-VENUS. Njengoba kubikiwe ngaphambilini12, usayizi we-della global mutant SAM wawumkhulu kunowohlobo lwasendle (Isifanekiso Esingeziwe 13a, b, d). I-In situ hybridization ye-CLV3 kanye ne-STM RNA iqinisekisile ukwanda kwe-meristem kuma-della mutants futhi yabonisa nokwanda kwe-lateral kwe-stem cell niche (Isifanekiso Esingeziwe 13e, f, h, i). Kodwa-ke, ukugoba kwe-SAM kwakufana kuzo zombili izinhlobo ze-genotype (Isifanekiso Esingeziwe 13k, m, n, p). Sibone ukwanda okufanayo kosayizi ku-gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant ngaphandle kokushintsha kokugoba uma kuqhathaniswa nohlobo lwasendle (Isithombe Esingeziwe 13c, d, g, j, l, o, p). Ukuvama kokuqondiswa kokuhlukaniswa kwamaseli nakho kuthintekile ku-della quadruple mutant, kodwa ngezinga elincane kunaku-della monolithic mutant (Isithombe Esingeziwe 12d–f). Lo mphumela womthamo, kanye nokuntuleka komphumela ekugobeni, kusikisela ukuthi umsebenzi osele we-RGL3 ku-Della quadruple mutant ukhawulela izinguquko ekuqondisweni kokuhlukaniswa kwamaseli okubangelwa ukulahlekelwa umsebenzi we-DELLA nokuthi izinguquko ekuhlukanisweni kwamaseli aseceleni zenzeka ngenxa yezinguquko emsebenzini wokubonisana we-GA kunezinguquko ku-geometry ye-SAM. Njengoba kuchaziwe ngenhla, umgqugquzeli we-CUC2 uqhuba ukuvezwa kwe-IPR ku-SAM kusukela ku-P4 (Isithombe Esingeziwe 14a, b), futhi ngokuphambene nalokho, i-pCUC2::gai-1-VENUS SAM yayinosayizi oncishisiwe kodwa ijika eliphakeme (Isithombe Esingeziwe 14c–h). Lolu shintsho ku-pCUC2::gai-1-VENUS SAM lungaholela ekusabalaleni okuhlukile kokucindezeleka komshini uma kuqhathaniswa nohlobo lwasendle, lapho ukucindezeleka okuphezulu kokujikeleza kuqala ibanga elifushane ukusuka esikhungweni se-SAM47. Ngaphandle kwalokho, izinguquko ku-pCUC2::gai-1-VENUS SAM ziqukethwe izinguquko kuzakhiwo zesifunda zomshini ezibangelwa ukubonakaliswa kwe-transgene48. Kuzo zombili izimo, lokhu kunganciphisa kancane imiphumela yezinguquko kusignali ye-GA ngokwandisa amathuba okuthi amaseli azohlukana ekuqondisweni kwe-circumferential/transverse, kuchaza okubonwe yithi.
Uma sibheka ndawonye, idatha yethu iqinisekisa ukuthi ukusayina kwe-GA okuphezulu kudlala indima ebalulekile ekuqondisweni kwecala kwendiza yokuhlukaniswa kwamaseli ku-IPR. Zibonisa nokuthi ukugoba kwe-meristem nakho kuthonya ukuqondiswa kwendiza yokuhlukaniswa kwamaseli ku-IPR.
Ukuqondiswa okuphambene kwendiza yokuhlukanisa ku-IPR, ngenxa yomsebenzi ophezulu wokubonisana kwe-GA, kusikisela ukuthi i-GA ihlela kusengaphambili ifayela leseli eliyi-radial ku-epidermis ngaphakathi kwe-SAM ukuze ichaze inhlangano yeselula ezotholakala kamuva ku-epidermal internode. Ngempela, amafayela anjalo eseli ayebonakala kaningi ezithombeni ze-SAM ze-della global mutants (Umfanekiso 6b). Ngakho-ke, ukuze sihlole kabanzi umsebenzi wokuthuthukiswa kwephethini yendawo ye-GA signaling ku-SAM, sisebenzise i-time-lapse imaging ukuhlaziya inhlangano yendawo yamaseli ku-IPR ku-wild-type (Ler and Col-0), i-della global mutants, kanye ne-pCUC2::gai-1-VENUS transgenic plants.
Sithole ukuthi i-qmRGA ikhombisile ukuthi umsebenzi wokubonisa we-GA ku-IPR ukhuphuke kusuka ku-P1/P2 futhi wafinyelela esicongweni ku-P4, futhi leli phethini lahlala lingaguquki ngokuhamba kwesikhathi (Isithombe 4a–f kanye nesithombe esengeziwe 8c–f, k). Ukuze sihlaziye ukuhlelwa kwendawo kwamaseli ku-IPR ngesignali ye-GA ekhulayo, sibhale amaseli e-Ler IPR ngaphezulu nasezinhlangothini ze-P4 ngokwesiphetho sawo sokuthuthukiswa esihlaziywe amahora angu-34 ngemuva kokubuka kokuqala, okungukuthi, izikhathi ezingaphezu kwezimbili ze-plastid, okusivumela ukuthi silandele amaseli e-IPR ngesikhathi sokuthuthukiswa kwe-primordium kusuka ku-P1/P2 kuya ku-P4. Sisebenzise imibala emithathu ehlukene: ophuzi kulawo maseli ahlanganiswe ku-primordium eduze kwe-P4, oluhlaza kulawo ayese-IPR, kanye nophuzi kulabo abahlanganyele kuzo zombili izinqubo (Isithombe 7a–c). Ku-t0 (0 h), izendlalelo ezingu-1–2 zamaseli e-IPR zazibonakala phambi kwe-P4 (Isithombe 7a). Njengoba bekulindelekile, lapho la maseli ehlukana, akwenza lokhu ikakhulukazi ngendiza yokuhlukanisa eguquguqukayo (Izithombe 7a–c). Imiphumela efanayo yatholakala kusetshenziswa i-Col-0 SAM (egxile ku-P3, umngcele wayo ogoqeka ngendlela efanayo ne-P4 ku-Ler), yize kulolu hlobo lwe-genotype ukugoqeka okwakhiwe emngceleni wezimbali kwafihla amaseli e-IPR ngokushesha (Isithombe 7g–i). Ngakho-ke, iphethini yokuhlukanisa yamaseli e-IPR ihlela amaseli abe yimigqa ye-radial, njengakuma-internode. Ukuhlelwa kwemigqa ye-radial kanye nokwenziwa kwamaseli e-IPR phakathi kwezitho ezilandelanayo kusikisela ukuthi la maseli angama-internodal progenitors.
Lapha, sakha i-biosensor ye-GA signaling ye-ratiometric, i-qmRGA, evumela ukumepha okulinganiselwe komsebenzi we-GA signaling ovela ekugxilweni kwe-GA kanye ne-GA receptor okuhlangene ngenkathi kunciphisa ukuphazamiseka kwezindlela ze-GA signaling ezingeni leselula. Ngenxa yalokhu, sakhe iphrotheni ye-DELLA eguquliwe, i-mRGA, elahlekelwe yikhono lokubopha abalingani bokuxhumana be-DELLA kodwa ehlala ibucayi ku-proteolysis ebangelwa yi-GA. I-qmRGA isabela kokubili ezinguqukweni zangaphandle kanye nezangaphandle emazingeni e-GA, kanye nezakhiwo zayo zokuzwa ezinamandla zivumela ukuhlolwa kwezinguquko ze-spatiotemporal emsebenzini we-GA signaling ngesikhathi sokuthuthukiswa. I-qmRGA futhi iyithuluzi eliguquguqukayo kakhulu njengoba ingashintshwa ezicutshini ezahlukene ngokushintsha i-promoter esetshenziselwa ukuvezwa kwayo (uma kudingeka), futhi ngenxa yemvelo egciniwe yendlela ye-GA signaling kanye ne-PFYRE motif kuwo wonke ama-angiosperms, kungenzeka ukuthi ingadluliselwa kwezinye izinhlobo22. Ngokuvumelana nalokhu, ukuguquka okufanayo kuprotheni ye-SLR1 DELLA yerayisi (HYY497AAA) kuboniswe futhi ukuthi kuvimbela umsebenzi wokucindezela ukukhula kwe-SLR1 ngenkathi kunciphisa kancane ukuwohloka kwayo okubangelwa yi-GA, okufana ne-mRGA23. Okuphawulekayo ukuthi izifundo zakamuva ku-Arabidopsis zibonise ukuthi ukuguquka okukodwa kwe-amino acid kusizinda se-PFYRE (S474L) kushintshe umsebenzi wokubhala we-RGA ngaphandle kokuthinta ikhono layo lokuxhumana nabalingani be-transcription factor50. Nakuba lokhu kuguquka kusondele kakhulu ekushintsheni okungu-3 kwe-amino acid okukhona ku-mRGA, izifundo zethu zibonisa ukuthi lokhu kuguquka okubili kushintsha izici ezihlukile ze-DELLA. Nakuba abalingani abaningi be-transcription factor bebopha ezizindeni ze-LHR1 ne-SAW ze-DELLA26,51, amanye ama-amino acid agciniwe kusizinda se-PFYRE angasiza ekuqiniseni lokhu kuxhumana.
Ukuthuthukiswa kwama-internode kuyisici esibalulekile ekwakhiweni kwezitshalo nasekuthuthukisweni kwesivuno. I-qmRGA yembule umsebenzi wokubonisa we-GA ophezulu kumaseli e-IPR internode progenitor. Ngokuhlanganisa izithombe zokulinganisa kanye nezakhi zofuzo, sibonise ukuthi amaphethini okubonisa we-GA afaka izindiza zokuhlukaniswa kwamaseli eziyindilinga/ezinqamula ku-epidermis ye-SAM, akha inhlangano yokuhlukaniswa kwamaseli edingekayo ekuthuthukisweni kwama-internode. Abalawuli abaningana bokuqondisa kwendiza yokuhlukaniswa kwamaseli batholwe ngesikhathi sokuthuthukiswa52,53. Umsebenzi wethu unikeza isibonelo esicacile sendlela umsebenzi wokubonisa we-GA olawula ngayo le pharamitha yeselula. I-DELLA ingasebenzisana nama-complexes amaprotheni angaphambi kokugoqwa41, ngakho-ke ukubonisa kwe-GA kungalawula ukuqondiswa kwendiza yokuhlukaniswa kwamaseli ngokuthonya ngqo ukuqondiswa kwe-cortical microtubule40,41,54,55. Sibonise ngokungalindelekile ukuthi ku-SAM, ukuhambisana komsebenzi wokubonisa we-GA ophezulu kwakungekona ukunwebeka kwamaseli noma ukuhlukaniswa, kodwa i-anisotropy yokukhula kuphela, ehambisana nomthelela oqondile we-GA ekuqondisweni kokuhlukaniswa kwamaseli ku-IPR. Kodwa-ke, asikwazi ukukhipha ukuthi lo mphumela ungase ube ongaqondile, isibonelo oqondiswa ukuthamba kodonga lwamaseli olubangelwa yi-GA56. Izinguquko ezakhiweni zodonga lweseli zidala ukucindezeleka komshini57,58, okungathonya futhi ukuqondiswa kwendiza yokuhlukaniswa kweseli ngokuthinta ukuqondiswa kwama-microtubules e-cortical39,46,59. Imiphumela ehlanganisiwe yokucindezeleka komshini okubangelwa yi-GA kanye nokulawulwa okuqondile kokuqondiswa kwe-microtubule yi-GA kungase kuhileleke ekudaleni iphethini ethile yokuqondiswa kokuhlukaniswa kweseli ku-IPR ukuchaza ama-internode, futhi kudingeka izifundo ezengeziwe ukuhlola lo mbono. Ngokufanayo, izifundo zangaphambilini zigcizelele ukubaluleka kwamaprotheni asebenzisana ne-DELLA i-TCP14 kanye no-15 ekulawuleni ukwakheka kwama-internode60,61 futhi lezi zici zingase ziqondise isenzo se-GA kanye ne-BREVIPEDICELLUS (BP) kanye ne-PENNYWISE (PNY), ezilawula ukuthuthukiswa kwama-internode futhi ziboniswe ukuthi zithonya i-GA signaling2,62. Njengoba ama-DELLA esebenzisana ne-brassinosteroid, i-ethylene, i-jasmonic acid, kanye ne-abscisic acid (ABA) signaling pathways63,64 nokuthi la ma-hormone angathonya ukuqondiswa kwe-microtubule65, imiphumela ye-GA ekuqondisweni kokuhlukaniswa kwamaseli ingase futhi iqondiswe ngamanye ama-hormone.
Izifundo zokuqala ze-cytological zibonise ukuthi zombili izifunda zangaphakathi nezangaphandle ze-Arabidopsis SAM ziyadingeka ekuthuthukisweni kwama-internode2,42. Iqiniso lokuthi i-GA ilawula ngokusebenzayo ukuhlukaniswa kwamaseli ezicutshini zangaphakathi12 lisekela umsebenzi ophindwe kabili we-GA ekulawuleni i-meristem kanye nosayizi wama-internode ku-SAM. Iphethini yokuhlukaniswa kwamaseli aqondisayo nayo ilawulwa ngokuqinile ezicutshini zangaphakathi ze-SAM, futhi lo mthetho ubalulekile ekukhuleni kwesiqu52. Kuzoba mnandi ukuhlola ukuthi i-GA nayo idlala indima ekuqondiseni indiza yokuhlukaniswa kwamaseli enhlanganweni yangaphakathi ye-SAM, ngaleyo ndlela ivumelanise imininingwane kanye nokuthuthukiswa kwama-internode ngaphakathi kwe-SAM.
Izitshalo zakhuliswa ngaphandle kwe-vitro enhlabathini noma i-Murashige-Skoog (MS) medium (Duchefa) engezwe nge-1% sucrose kanye ne-1% agar (Sigma) ngaphansi kwezimo ezijwayelekile (ukukhanya kwamahora angu-16, 22 °C), ngaphandle kokuhlolwa kwe-hypocotyl kanye nokukhula kwezimpande lapho izithombo zakhuliswa khona emapuletini aqondile ngaphansi kokukhanya okungaguquki kanye no-22 °C. Kokuhlolwa kwe-nitrate, izitshalo zakhuliswa ku-modified MS medium (bioWORLD plant medium) engezwe nge-nitrate eyanele (0 noma 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose kanye ne-1% A-agar (Sigma) ngaphansi kwezimo zosuku olude.
I-GID1a cDNA efakwe ku-pDONR221 yahlanganiswa kabusha ne-pDONR P4-P1R-pUBQ10 kanye ne-pDONR P2R-P3-mCherry ku-pB7m34GW ukuze kukhiqizwe i-pBQ10::GID1a-mCherry. I-IDD2 DNA efakwe ku-pDONR221 yahlanganiswa kabusha ku-pB7RWG266 ukuze kukhiqizwe i-p35S:IDD2-RFP. Ukuze kukhiqizwe i-pGID1b::2xmTQ2-GID1b, ingxenye engu-3.9 kb engenhla kwesifunda sekhodi se-GID1b kanye nengxenye engu-4.7 kb equkethe i-GID1b cDNA (1.3 kb) kanye ne-terminator (3.4 kb) yaqala yandiswa kusetshenziswa ama-primer kuThebula Elingeziwe 3 yabe isifakwa ku-pDONR P4-P1R (Thermo Fisher Scientific) kanye ne-pDONR P2R-P3 (Thermo Fisher Scientific), ngokulandelana, futhi ekugcineni yahlanganiswa kabusha ne-pDONR221 2xmTQ268 ku-vector ye-pGreen 012567 kusetshenziswa i-Gateway cloning. Ukuze kukhiqizwe i-pCUC2::LSSmOrange, i-CUC2 promoter sequence (3229 bp upstream ye-ATG) elandelwa i-code sequence ye-Stokes-shifted mOrange (LSSmOrange)69 enkulu enesignali ye-N7 nuclear localization kanye ne-NOS transcriptional terminator kwahlanganiswa kwi-pGreen kanamycin targeting vector kusetshenziswa i-Gateway 3-fragment recombination system (Invitrogen). I-plant binary vector yethulwa ku-Agrobacterium tumefaciens strain GV3101 futhi yethulwa kumaqabunga e-Nicotiana benthamiana ngendlela ye-Agrobacterium infiltration kanye ne-Arabidopsis thaliana Col-0 ngendlela ye-floral dip, ngokulandelana. i-pBQ10::qmRGA pBQ10::GID1a-mCherry kanye ne-pCLV3::mCherry-NLS qmRGA zahlukaniswa ku-F3 kanye ne-F1 progenies yeziphambano ezifanele, ngokulandelana.
Ukuhlanganiswa kwe-RNA in situ kwenziwa eziqongweni zokudubula ezingaba ngu-1 cm ubude72, ezaqoqwa futhi zafakwa ngokushesha kusisombululo se-FAA (3.7% formaldehyde, 5% acetic acid, 50% ethanol) ezipholiswe kusengaphambili ku-4 °C. Ngemva kokwelashwa nge-vacuum imizuzu engu-2 × 15, i-fixative yashintshwa futhi amasampula afakwa e-incubation ubusuku bonke. I-GID1a, GID1b, GID1c, GAI, RGL1, RGL2, kanye ne-RGL3 cDNAs kanye nama-antisense probes kuma-3′-UTR awo ahlanganiswa kusetshenziswa ama-primer aboniswe kuThebula Elingeziwe 3 njengoba kuchazwe nguRosier et al.73. Ama-probe anelebula le-Digoxigenin atholakale ngokuzivikela kusetshenziswa ama-antibodies e-digoxigenin (ukuxuba okuphindwe ka-3000; i-Roche, inombolo yekhathalogi: 11 093 274 910), kanti izingxenye zafakwa i-5-bromo-4-chloro-3-indolyl phosphate (BCIP, ukuxuba okuphindwe ka-250)/nitroblue tetrazolium (NBT, ukuxuba okuphindwe ka-200).
Isikhathi sokuthunyelwe: Feb-10-2025