I-Quantitative Gibberellin Biosensor Yembula Iqhaza Le-Gibberellins Ekucacisweni Kwe-Internode ku-Shoot Apical Meristem

Ukukhula kwe-shoot apical meristem (SAM) kubalulekile ekwakhiweni kwesiqu. Amahomoni ezitshaloama-gibberellins(Ama-GA) adlala indima ebalulekile ekuxhumaniseni ukukhula kwezitshalo, kodwa indima yawo ku-SAM isalokhu iqondwa kabi. Lapha, sithuthukise i-ratiometric biosensor yokusayina kwe-GA ngokunjiniyela iphrotheni ye-DELLA ukuze icindezele umsebenzi wayo wokulawula obalulekile ekuphenduleni kokubhaliwe kwe-GA kuyilapho ilondoloza ukuwohloka kwayo ekuqashelweni kwe-GA. Sibonisa ukuthi le biosensor esekwe ekucekeleni phansi irekhoda ngokunembile izinguquko kumazinga e-GA kanye nokuzwa kwamaselula ngesikhathi sokuthuthukiswa. Sisebenzise le biosensor ukwenza kumephu umsebenzi wokusayina we-GA ku-SAM. Sibonisa ukuthi amasignali e-GA aphezulu akhona ikakhulukazi kumaseli atholakala phakathi kwe-organ primordia, angazandulela kumaseli e-internode. Sisebenzisa izindlela zokuzuza nokulahlekelwa-ukusebenza, siqhubeka sibonisa ukuthi i-GA ilawula umumo wendiza yokuhlukaniswa kwamaseli, isungula inhlangano yamaselula e-canonical yama-internode, ngaleyo ndlela ikhuthaze ukucaciswa kwe-internode ku-SAM.
I-shoot apical meristem (SAM), etholakala ku-shoot apex, iqukethe i-stem cells umsebenzi wayo okhiqiza izitho ezisemaceleni kanye nama-stem node ngendlela ephindaphindayo nephindaphindayo kuyo yonke impilo yesitshalo. Ngayinye yalezi zingxenye eziphindaphindayo, noma iziqhumane zezitshalo, ihlanganisa ama-internode nezitho ezisemaceleni ezindaweni, kanye nama-axillary meristem kuma-axils amaqabunga1. Ukukhula nokuhleleka kwamanodi ezitshalo kuyashintsha ngesikhathi sokuthuthukiswa. E-Arabidopsis, ukukhula kwe-internodal kuyacindezelwa ngesikhathi sezitshalo, futhi ama-axillary meristems ahlala elele kuma-axils amaqabunga e-rosette. Ngesikhathi sokushintshela esigabeni sezimbali, i-SAM iba i-inflorescence meristem, ekhiqiza ama-internode amade nama-axillary buds, amagatsha kuma-axils amaqabunga e-cauline, futhi kamuva, izimbali ezingenamaqabunga2. Nakuba senze inqubekelaphambili ebalulekile ekuqondeni izindlela ezilawula ukuqaliswa kwamaqabunga, izimbali, namagatsha, kuncane kakhulu okwaziwayo ngendlela ama-internode avela ngayo.
Ukuqonda ukusatshalaliswa kwe-spatiotemporal kwama-GA kuzosiza ukuqonda kangcono imisebenzi yala mahomoni ezicutshini ezihlukene nasezigabeni zokuthuthuka ezihlukene. Ukubona ngeso lengqondo ukucekelwa phansi kwe-RGA-GFP fusion evezwe ngaphansi kwesenzo somgqugquzeli wayo kunikeza ulwazi olubalulekile mayelana nokulawulwa kwamazinga e-GA aphelele ezimpandeni15,16. Kodwa-ke, isisho se-RGA siyahlukahluka kuzo zonke izicubu17 futhi silawulwa yi-GA18. Ngakho-ke, ukubonakaliswa okuhlukile komgqugquzeli we-RGA kungase kubangele iphethini ye-fluorescence ebonwe nge-RGA-GFP futhi ngaleyo ndlela le ndlela ayilona inani. Muva nje, i-bioactive fluorescein (Fl)-ebhalwe ukuthi GA19,20 iveze ukuqoqwa kwe-GA ku-endocortex yempande kanye nokulawulwa kwamazinga ayo eselula ngokuthuthwa kwe-GA. Muva nje, inzwa ye-GA FRET nlsGPS1 ibonise ukuthi amazinga e-GA ahambisana nokwelulwa kweseli ezimpandeni, imicu, kanye nama-hypocotyls amnyama21. Kodwa-ke, njengoba sesibonile, ukugxila kwe-GA akuyona ukuphela kwepharamitha elawula umsebenzi wokusayina we-GA, njengoba kuncike ezinqubweni zokuzwa eziyinkimbinkimbi. Lapha, sakhela ekuqondeni kwethu izindlela zokusayina ze-DELLA kanye ne-GA, sibika ngokuthuthuka kanye nokubonakaliswa kwe-biosensor ye-ratiometric esekelwe ekonakaleni yokusayina kwe-GA. Ukuze sithuthukise le-biosensor yobuningi, sisebenzise i-RGA ezwelayo ye-GA eguquguqukayo eyahlanganiswa nephrotheni ekhanyayo futhi yavezwa yonke indawo kumathishu, kanye nephrotheni yefluorescent engezwani ne-GA. Sibonisa ukuthi ukuhlanganiswa kwamaprotheni e-RGA aguquguqukayo akuphazamisi ukusayinda kwe-GA kwe-endo native lapho kuvezwa yonke indawo, nokuthi le biosensor ingakwazi ukulinganisa umsebenzi wokusayinda obangelwa kokubili okokufaka kwe-GA kanye nokucubungula isignali ye-GA ngezisetshenziswa zezinzwa ezinokulungiswa okuphezulu kwe-spatiotemporal. Sisebenzise le biosensor ukwenza imephu yokusatshalaliswa kwe-spatiotemporal komsebenzi wokusayina we-GA futhi silinganise ukuthi i-GA ilawula kanjani ukuziphatha kwamaselula ku-SAM epidermis. Sibonisa ukuthi i-GA ilawula umumo wendiza ehlukanisayo yamaseli e-SAM atholakala phakathi kwe-organ primordia, ngaleyo ndlela ichaza inhlangano yeselula ye-canonical ye-internode.
Ekugcineni, sibuze ukuthi i-qmRGA ingakwazi yini ukubika izinguquko kumazinga e-GA engapheli isebenzisa ama-hypocotyl akhulayo. Ngaphambilini sibonise ukuthi i-nitrate ikhuthaza ukukhula ngokwandisa i-GA synthesis futhi, ngokulandelayo, ukuwohloka kwe-DELLA34. Ngokuvumelana nalokho, sabona ukuthi ubude be-hypocotyl ku-pUBQ10:: izithombo ze-qmRGA ezitshalwe ngaphansi kwe-nitrate eningi (10 mM NO3-) babubude kakhulu kunalokho ezitshalweni ezitshalwe ngaphansi kwezimo zokushoda kwe-nitrate (I-Supplementary Fig. 6a). Ngokuvumelana nokusabela kokukhula, izimpawu ze-GA zaziphakeme kuma-hypocotyl ezithombo ezitshalwe ngaphansi kwezimo ezingu-10 mM NO3− kunezithombo ezitshalwe ngaphandle kwe-nitrate (I-Supplementary Fig. 6b, c). Ngakho-ke, i-qmRGA iphinde inikeze amandla ukuqapha izinguquko ekusayineni kwe-GA okubangelwa izinguquko ezingapheli ekugxilweni kwe-GA.
Ukuze siqonde ukuthi ingabe umsebenzi wokusayinda we-GA otholwe yi-qmRGA uncike ekugxiliseni kwe-GA nasekuboneni kwe-GA, njengoba kulindelekile ngokusekelwe ekwakhiweni kwezinzwa, sihlaziye ukuvezwa kwama-receptor amathathu e-GID1 ezicutshini zezitshalo nezokuzala. Kuzithombo, ulayini wentatheli we-GID1-GUS ubonise ukuthi i-GID1a kanye ne-c zivezwe kakhulu kuma-cotyledons (Fig. 3a–c). Ngaphezu kwalokho, wonke ama-receptors amathathu abonakaliswe emaqabunga, i-lateral root primordia, amathiphu ezimpande (ngaphandle kwe-root cap ye-GID1b), kanye nesistimu ye-vascular (Fig. 3a-c). Ku-inflorescence ye-SAM, sithole amasignali e-GUS kuphela ku-GID1b no-1c (I-Supplementary Fig. 7a–c). I-In situ hybridization iqinisekise la maphethini enkulumo futhi yabonisa futhi ukuthi i-GID1c iboniswe ngendlela efanayo emazingeni aphansi ku-SAM, kuyilapho i-GID1b ibonise ukuvezwa okuphezulu kumaphethelo e-SAM (I-Supplementary Fig. 7d–l). Ukuhlanganisa kokuhumusha kwe-pGID1b::2xmTQ2-GID1b kuphinde kwembula uhla olulinganiselwe lwesisho se-GID1b, ukusuka ekuboniseni okuphansi noma okungekho phakathi nendawo ye-SAM ukuya ekukhulumeni okuphezulu emingceleni yesitho (I-Supplementary Fig. 7m). Ngakho-ke, ama-receptors e-GID1 awasakazwa ngokufanayo kuwo wonke nangaphakathi kwezicubu. Ekuhloleni okwalandela, siphinde sabona ukuthi ukuveza ngokweqile kwe-GID1 (pUBQ10:: GID1a-mCherry) kwandise ukuzwela kwe-qmRGA kuma-hypocotyls kuhlelo lokusebenza lwe-GA lwangaphandle (Fig. 3d, e). Ngokuphambene, i-fluorescence elinganiswa nge-qd17mRGA ku-hypocotyl yayinganaki ekwelashweni kwe-GA3 (Fig. 3f, g). Kuzo zombili izivivinyo, izithombo ziphathwe ngokugxila okuphezulu kwe-GA (100 μM GA3) ukuze kuhlolwe ukuziphatha okusheshayo kwenzwa, lapho ikhono lokubopha isamukeli se-GID1 lathuthukiswa noma lalahleka. Ndawonye, ​​le miphumela iqinisekisa ukuthi i-qmRGA biosensor isebenzisa umsebenzi ohlanganisiwe njengenzwa ye-GA ne-GA, futhi iphakamisa ukuthi ukuvezwa okuhlukile kwesamukeli se-GID1 kungamodela 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 ukubala amamephu wobuningi bokulungiswa okuphezulu komsebenzi wokusayina we-GA, sigxile kungqimba lwe-L1 (i-epidermis; i-Fig. 4a, b, bheka Izindlela Nezindlela Zokungezelela), njengoba i-L3 idlala indima ebalulekile ye-SAM. Lapha, i-pCLV3::inkulumo ye-mCherry-NLS inikeze indawo eyireferensi yejiyomethri engaguquki ukuze kuhlaziywe ukusatshalaliswa kwe-spatiotemporal komsebenzi wokusayina we-GA37. Nakuba i-GA ibhekwa njengebalulekile ekuthuthukisweni kwesitho se-lateral4, sibonile ukuthi izimpawu ze-GA zaziphansi ku-primordium yezimbali (P) kusukela esigabeni se-P3 (Fig. 4a, b), kanti i-P1 encane ne-P2 primordiums yayinomsebenzi olinganiselayo ofana nalowo esifundeni esimaphakathi (Fig. 4a, b). Umsebenzi wokubonisa i-GA ophakeme utholwe emingceleni ye-organ primordium, eqala ku-P1 / P2 (ezinhlangothini zomngcele) futhi ifinyelela phezulu ku-P4, kanye nakuwo wonke amaseli wesifunda se-peripheral ephakathi kwe-primordia (Fig. 4a, b kanye ne-Supplementary Fig. 8a, b). Lo msebenzi wokubonisa i-GA ophezulu wawungabonwa kuphela ku-epidermis kodwa futhi nasezindabeni ze-L2 nezingaphezulu ze-L3 (I-Supplementary Fig. 8b). Iphethini yamasiginali e-GA atholwe ku-SAM kusetshenziswa i-qmRGA nayo yahlala ingashintshile ngokuhamba kwesikhathi (I-Supplementary Fig. 8c–f, k). Nakuba ukwakhiwa kwe-qd17mRGA kwehliswa ngokuhlelekile ku-SAM yezitshalo ze-T3 kusukela emigqeni emihlanu ezimele esiyiphawule ngokuningiliziwe, sikwazile ukuhlaziya amaphethini e-fluorescence atholwe ngokwakhiwa kwe-pRPS5a::VENUS-2A-TagBFP (I-Supplementary Fig. 8g–j, l). Kulo mugqa wokulawula, kutholwe izinguquko ezincane kuphela kusilinganiso se-fluorescence ku-SAM, kodwa esikhungweni se-SAM sabona ukwehla okucacile nokungalindelekile ku-VENUS okuhlotshaniswa ne-TagBFP. Lokhu kuqinisekisa ukuthi iphethini yokusayina ebonwe yi-qmRGA ibonisa ukucekelwa phansi kwe-GA kwe-mRGA-VENUS, kodwa futhi kubonisa ukuthi i-qmRGA ingase ilinganisele ngokweqile umsebenzi wokusayina we-GA esikhungweni se-meristem. Kafushane, imiphumela yethu yembula iphethini yokusayina ye-GA ebonisa ngokuyinhloko ukusatshalaliswa kwe-primordia. Lokhu kusatshalaliswa kwesifunda se-inter-primordial (IPR) kubangelwa ukusungulwa kancane kancane komsebenzi wokubonisa i-GA ephezulu phakathi kwe-primordium ekhulayo kanye nesifunda esimaphakathi, kuyilapho ngesikhathi esifanayo umsebenzi wokusayinda i-GA ku-primordium uyancipha (Fig. 4c, d).
Ukusatshalaliswa kwama-GID1b kanye nama-GID1c receptors (bona ngenhla) kuphakamisa ukuthi ukuvezwa okuhlukile kwama-receptor e-GA kusiza ukulolonga iphethini yomsebenzi wokusayina we-GA ku-SAM. Sizibuze ukuthi ngabe ukuqoqwa okuhlukile kwe-GA kungenzeka yini kubandakanyeke. Ukuphenya lokhu okungenzeka, sisebenzise i-nlsGPS1 GA FRET sensor21. Ukunyuka kokuvama kokuvula kutholwe ku-SAM ye-nlsGPS1 ephathwa nge-10 μM GA4+7 imizuzu engu-100 (I-Supplementary Fig. 9a–e), okubonisa ukuthi i-nlsGPS1 iphendula kuzinguquko ekugxiliseni kwe-GA ku-SAM, njengoba yenza ku-root21. Ukusatshalaliswa kwendawo kwe-nlsGPS1 imvamisa yokuvula yembule amazinga e-GA aphansi ngokuqhathaniswa ezendlalelo zangaphandle ze-SAM, kodwa abonisa ukuthi anyuswe phakathi nendawo nasemingceleni ye-SAM (Fig. 4e kanye ne-Supplementary Fig. 9a,c). Lokhu kuphakamisa ukuthi i-GA nayo isatshalaliswa ku-SAM ngephethini yendawo eqhathaniswa naleyo evezwe yi-qmRGA. Njengendlela ehambisanayo, siphinde saphatha i-SAM nge-fluorescent GA (GA3-, GA4-, GA7-Fl) noma i-Fl iyodwa njengokulawula okungalungile. Isignali ye-Fl yasakazwa kuyo yonke i-SAM, kuhlanganise nesifunda esimaphakathi kanye ne-primordium, nakuba inamandla aphansi (I-Fig. 4j kanye ne-Supplementary Fig. 10d). Ngokuphambene, zontathu i-GA-Fl ziqoqwe ngokuqondile ngaphakathi kwemingcele yasekuqaleni kanye namazinga ahlukahlukene kuyo yonke i-IPR esele, i-GA7-Fl iqongelela esizindeni esikhulu kunazo zonke ku-IPR (Fig. 4k kanye ne-Supplementary Fig. 10a,b). I-Quantification of fluorescence intensity iveze ukuthi i-IPR kuya ku-non-IPR intensity ratio yayiphezulu ku-GA-Fl-treated SAM uma kuqhathaniswa ne-Fl-treated SAM (Fig. 4l kanye ne-Supplementary Fig. 10c). Ngokuhlangene, le miphumela iphakamisa ukuthi i-GA ikhona ekugxiliseni okuphezulu kumaseli e-IPR atholakala eduze kakhulu nomngcele wesitho. Lokhu kuphakamisa ukuthi iphethini yomsebenzi wokusayina we-SAM GA iphumela kukho kokubili ukuvezwa okuhlukile kwama-receptors e-GA kanye nokunqwabelana okuhlukile kwe-GA kumaseli e-IPR eduze kwemingcele yesitho. Ngakho-ke, ukuhlaziya kwethu kwembula iphethini ye-spatiotemporal engalindelekile yokusayina kwe-GA, nomsebenzi ophansi phakathi nendawo kanye ne-primordium ye-SAM kanye nomsebenzi ophezulu ku-IPR esifundeni esiseduze.
Ukuze siqonde indima yomsebenzi wokusayina we-GA ohlukile ku-SAM, sihlaziye ukuhlobana phakathi komsebenzi wokusayina we-GA, ukunwetshwa kweseli, nokuhlukaniswa kwamaseli sisebenzisa isithombe sesikhathi sangempela sokuphelelwa yisikhathi kwe-SAM qmRGA pCLV3::mCherry-NLS. Ngokunikezwa kwendima ye-GA ekulawuleni ukukhula, ukuhlobana okuhle namapharamitha wokunwetshwa kwamaseli bekulindeleke. Ngakho-ke, siqale saqhathanisa amamephu omsebenzi wokusayinda we-GA namamephu wezinga lokukhula kweseli (njengommeleli wamandla okunwetshwa kweseli kweseli elinikeziwe kanye namaseli endodakazi ekuhlukaniseni) kanye namamephu we-anisotropy yokukhula, ekala isiqondiso sokunwetshwa kwamangqamuzana (okubuye kusetshenziswe lapha kuseli elinikeziwe kanye namaseli endodakazi ekuhlukaniseni; Fig. 5a,b, bheka Izindlela Zokungezelela). Amamephu ethu wezinga lokukhula kwamaseli e-SAM ahambisana nokubhekwa kwangaphambilini38,39, namazinga amancane okukhula emngceleni kanye namazinga aphezulu okukhula ekukhuleni kwezimbali (Fig. 5a). Ukuhlaziywa kwengxenye eyinhloko (PCA) kubonise ukuthi umsebenzi wokusayinda i-GA wawuhlotshaniswa kabi nokukhula kwe-cell surface (Figure 5c). Siphinde sabonisa ukuthi ama-eksisi ayinhloko okuhlukahluka, okuhlanganisa okokufaka kokusayina kwe-GA kanye nokuqina kokukhula, aye-orthogonal esiqondisweni esinqunywe isisho esiphezulu se-CLV3, okuqinisekisa ukukhishwa kwamaseli esikhungweni se-SAM ekuhlaziyeni okusele. Ukuhlaziywa kokulungiswa kwe-Spearman kuqinisekisile imiphumela ye-PCA (Umfanekiso we-5d), okubonisa ukuthi amasignali aphezulu e-GA ku-IPR awazange abangele ukwanda kwamaseli. Kodwa-ke, ukuhlaziywa kokuhlobanisa kwembula ukuhlobana okuncane okuhle phakathi komsebenzi wokusayinda i-GA kanye nokukhula kwe-anisotropy (Umfanekiso 5c, d), okuphakamisa ukuthi ukusayinda okuphezulu kwe-GA ku-IPR kuthonya isiqondiso sokukhula kwamaseli futhi ngokunokwenzeka nendawo yendiza yokuhlukaniswa kwamaseli.
a, b Amamephu okushisa okukhula kwendawo emaphakathi (a) kanye ne-anisotropy yokukhula (b) ku-SAM enesilinganiso esingaphezu kwezitshalo eziyisikhombisa ezizimele (asetshenziswa njengama-proxies wamandla nesiqondiso sokunwetshwa kwamaseli, ngokulandelana). c Ukuhlaziywa kwe-PCA kufaka phakathi okuguquguqukayo okulandelayo: isignali ye-GA, ukuqina kokukhula kwendawo, ukukhula kwe-surface anisotropy, kanye nenkulumo ye-CLV3. Ingxenye ye-PCA engu-1 yayihlotshaniswa kabi kakhulu nokuqina kokukhula kwendawo futhi ihlotshaniswa kahle nesiginali ye-GA. Ingxenye ye-PCA engu-2 yayihlotshaniswa kahle kakhulu ne-anisotropy yokukhula kwendawo futhi ihlotshaniswa kabi nenkulumo ye-CLV3. Amaphesenti amelela ukuhluka okuchazwe ingxenye ngayinye. d Ukuhlaziywa kokuhlobana kwe-Spearman phakathi kwesiginali ye-GA, ukuqina kokukhula kwendawo, kanye ne-anisotropy yokukhula kwendawo esikalini sethishu ngaphandle kwe-CZ. Inombolo engakwesokudla iyinani elithi Spearman rho phakathi kweziguquguqukayo ezimbili. Izinkanyezi zibonisa izimo lapho ukuhlobana/negative ukuhlobana kubaluleke kakhulu. e Ukuboniswa kwe-3D kwamaseli e-Col-0 SAM L1 nge-confocal microscopy. Izindonga zamaseli amasha ezakhiwe ku-SAM (kodwa hhayi i-primordium) ngo-10 h zinemibala ngokuvumelana nama-engeli azo amanani. Ibha yombala iboniswa ekhoneni elingezansi kwesokudla. Isingeniso sibonisa isithombe esihambisanayo se-3D ngo-0 h. Ukuhlolwa kuphindwe kabili ngemiphumela efanayo. f Iziqephu zebhokisi zibonisa izilinganiso zokuhlukaniswa kwamaseli ku-IPR kanye ne-non-IPR Col-0 SAM (n = izitshalo ezizimele eziyi-10). Umugqa omaphakathi ubonisa i-median, futhi imingcele yebhokisi ikhombisa amaphesenti angama-25 kanye nama-75. Ama-Whiskers akhombisa ubuncane kanye namanani aphezulu anqunywe nge-software ye-R. Amanani we-P atholwe ngokuhlolwa kuka-Welch okunemisila emibili. g, h Umdwebo weschematic obonisa (g) indlela yokulinganisa i-engeli yodonga lweseli entsha (imagenta) ngokuphathelene nesiqondiso se-radial ukusuka maphakathi ne-SAM (umugqa wamachashazi amhlophe) (amavelu e-engeli e-acute kuphela, okungukuthi, 0–90°, ayacatshangelwa), kanye (h) nezikhombisi-ndlela eziyindingilizi/ezingemuva kanye ne-radial ngaphakathi kwe-meristem. i-Frequency histograms of cell division plane orientation kuyo yonke i-SAM (blue blue), IPR (blue blue), kanye non-IPR (light blue), ngokulandelanayo. Amanani we-P atholwe ukuhlolwa kwe-Kolmogorov-Smirnov enemisila emibili. Ukuhlolwa kuphindwe kabili ngemiphumela efanayo. j Ama-histograms okuvama kokuma kwendiza yokuhlukaniswa kweseli ye-IPR eduze kwe-P3 (okuluhlaza okukhanyayo), i-P4 (okuluhlaza okumaphakathi), kanye ne-P5 (okuluhlaza okumnyama), ngokulandelanayo. Amanani we-P atholwe ukuhlolwa kwe-Kolmogorov-Smirnov enemisila emibili. Ukuhlolwa kuphindwe kabili ngemiphumela efanayo.
Ngakho-ke, ngokulandelayo siphenye ukuhlobana phakathi kokusayina kwe-GA nomsebenzi wokuhlukaniswa kwamaseli ngokuhlonza izindonga zamaseli ezisanda kwakhiwa ngesikhathi sokuhlolwa (Fig. 5e). Le ndlela yasivumela ukuba silinganise imvamisa nesiqondiso sokuhlukaniswa kwamaseli. Ngokumangalisayo, sithole ukuthi imvamisa yokuhlukaniswa kwamaseli ku-IPR kanye nayo yonke i-SAM (okungeyona i-IPR, i-Fig. 5f) yayifana, okubonisa ukuthi umehluko ekuboniseni i-GA phakathi kwamaseli we-IPR nama-non-IPR akuthinti kakhulu ukuhlukaniswa kwamaseli. Lokhu, kanye nokuhlobana okuhle phakathi kokusayina kwe-GA nokukhula kwe-anisotropy, kusishukumisele ukuba sicabangele ukuthi umsebenzi wokusayinda i-GA ungathonya ukuma kwendiza yokuhlukaniswa kwamaseli. Silinganise umumo wodonga lweseli entsha njenge-engeli ebukhali ehlobene ne-radial axis exhuma isikhungo se-meristem kanye nendawo emaphakathi yodonga lweseli entsha (Fig. 5e-i) futhi sabona ukuthambekela okucacile kokuthi amaseli ahlukane ngama-engeli asondele ku-90° ngokuhlobene ne-radial axis, namafrikhwensi aphakeme kakhulu abonwa ku-70-80–60° (2%). (I-Fig. 5e, i), ehambisana nokuhlukaniswa kwamaseli ku-circumferential/transverse direction (Fig. 5h). Ukuhlola umnikelo wokusayina kwe-GA kulokhu kuziphatha kokuhlukaniswa kweseli, sihlaziye amapharamitha wokuhlukaniswa kwamaseli ku-IPR kanye nokungeyona i-IPR ngokuhlukana (Fig. 5i). Siqaphele ukuthi ukusabalalisa kwe-division angle kumaseli e-IPR kuhluke kulokho kumaseli angewona e-IPR noma kumaseli kuyo yonke i-SAM, namaseli e-IPR abonisa ingxenye ephezulu yokuhlukaniswa kwamaseli angemuva/ayisiyingi, okungukuthi, 70–80° kanye no-80–90° (33.86% kanye no-30.71%, ngokulandelana, izilinganiso ezihambisanayo (i-Fig.5). Ngakho-ke, ukuqaphela kwethu kwembula ukuhlangana phakathi kokubonisa i-GA ephezulu kanye nendiza ye-cell division orientation eduze nesiqondiso se-circumferential, efana nokuhlobana phakathi komsebenzi wokubonisa i-GA kanye nokukhula kwe-anisotropy (Fig. 5c, d). Ukuze siqhubeke sisungula ukulondolozwa kwendawo yale nhlangano, silinganise umumo wendiza yokuhlukanisa kumaseli e-IPR azungeze i-primordium kusukela ku-P3, njengoba umsebenzi wokubonisa i-GA ophakeme kakhulu utholwe kulesi sifunda kusukela ku-P4 (Fig. 4). Ama-angles okuhlukaniswa kwe-IPR azungeze i-P3 ne-P4 abonisanga umehluko ophawulekayo wezibalo, nakuba imvamisa ekhulayo yokuhlukaniswa kwamaseli e-lateral yabonwa ku-IPR ezungeze i-P4 (Fig. 5j). Kodwa-ke, kumaseli e-IPR azungeze i-P5, umehluko ekuqondeni kwendiza yokuhlukaniswa kwamaseli waba ophawulekayo ngokwezibalo, ngokunyuka okubukhali kwemvamisa yokuhlukaniswa kwamaseli aguquguqukayo (Fig. 5j). Ngokuhlangene, le miphumela iphakamisa ukuthi ukusayinda kwe-GA kungalawula umumo wokuhlukaniswa kwamaseli ku-SAM, okuhambisana nemibiko yangaphambilini40,41 yokuthi ukusayinda okuphezulu kwe-GA kungabangela ukuma kwe-lateral kokuhlukana kwamaseli ku-IPR.
Kubikezelwa ukuthi amaseli ku-IPR ngeke afakwe ku-primordia kodwa kunalokho kuma-internodes2,42,43. Ukuma okuphambene kokuhlukaniswa kwamaseli ku-IPR kungase kubangele inhlangano evamile yemigqa yobude obufanayo yamaseli e-epidermal kuma-internode. Ukuqaphela kwethu okuchazwe ngenhla kuphakamisa ukuthi ukusayinda kwe-GA kungenzeka kubambe iqhaza kule nqubo ngokulawula isiqondiso sokuhlukaniswa kwamaseli.
Ukulahleka kokusebenza kwezakhi zofuzo ezimbalwa ze-DELLA kuphumela ekuphenduleni kwe-GA, futhi i-della mutants ingasetshenziswa ukuhlola le mbono44. Siqale sahlaziya amaphethini enkulumo yezakhi zofuzo ezinhlanu ze-DELLA ku-SAM. Ukuhlanganisa okubhaliwe komugqa we-GUS45 kwembula ukuthi i-GAI, i-RGA, i-RGL1, ne-RGL2 (ngezinga eliphansi kakhulu) zavezwa ku-SAM (I-Supplementary Fig. 11a–d). I-In situ hybridization iphinde yabonisa ukuthi i-GAI mRNA iqongelela ikakhulukazi kuma-primordia kanye nezimbali ezikhulayo (I-Supplementary Fig. 11e). I-RGL1 ne-RGL3 mRNA zitholwe kuyo yonke i-SAM canopy nasezimbalini ezindala, kuyilapho i-RGL2 mRNA yayichichima kakhulu esifundeni somngcele (I-Supplementary Fig. 11f–h). I-confocal imaging ye-pRGL3::RGL3-GFP SAM iqinisekise inkulumo ebonwe yi-in situ hybridization futhi yabonisa ukuthi amaprotheni e-RGL3 aqoqana engxenyeni emaphakathi ye-SAM (I-Supplementary Fig. 11i). Sisebenzisa ulayini we-pRGA::GFP-RGA, siphinde sathola ukuthi amaprotheni e-RGA anqwabelana ku-SAM, kodwa ukuchichima kwawo kuyehla emngceleni kusukela ku-P4 (I-Supplementary Fig. 11j). Ngokuphawulekayo, amaphethini enkulumo we-RGL3 ne-RGA ahambisana nomsebenzi wokusayina we-GA ophezulu ku-IPR, njengoba kutholwe i-qmRGA (Fig. 4). Ngaphezu kwalokho, le datha ibonisa ukuthi wonke ama-DELLA avezwa ku-SAM nokuthi inkulumo yawo ihlangene ihlanganisa yonke i-SAM.
Ngokulandelayo sihlaziye amapharamitha wokuhlukaniswa kwamaseli ku-SAM yohlobo lwasendle (Ler, control) kanye ne-gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) eziguquguqukayo (Fig. 6a, b). Kuyathakazelisa ukuthi sibone uguquko olubalulekile ngokwezibalo ekusabalaliseni ama-engeli e-cell division ku-della global mutant SAM uma kuqhathaniswa nohlobo lwasendle (Fig. 6c). Lolu shintsho ku-della global mutant lwabangelwa ukwanda kwemvamisa yama-angles angu-80-90 ° (34.71% vs. 24.55%) futhi, kancane kancane, ama-angles angu-70-80 ° (23.78% vs. 20.18%), okungukuthi, ahambisana nokuhlukaniswa kwamaseli aphambene (Fig. 6c). Imvamisa yokuhlukaniswa okungaguquki (0-60 °) nayo yayiphansi ku-della global mutant (Fig. 6c). Imvamisa yokuhlukaniswa kwamaseli aguquguqukayo yanda kakhulu ku-SAM ye-della global mutant (Fig. 6b). Imvamisa yokuhlukaniswa kwamaseli aguquguqukayo ku-IPR nayo yayiphezulu ku-della global mutant uma kuqhathaniswa nohlobo lwasendle (Fig. 6d). Ngaphandle kwesifunda se-IPR, uhlobo lwasendle lwalunokusabalalisa okufanayo kwama-engeli wokuhlukaniswa kwamaseli, kuyilapho i-della global mutant ikhetha ukuhlukaniswa okune-tangential njenge-IPR (Fig. 6e). Siphinde salinganisa umumo wokuhlukaniswa kwamaseli ku-SAM ye-ga2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, ne-ga2ox6-2), ingemuva eliguquguqukayo le-GA elingasebenzi lapho i-GA inqwabelana khona. Ngokuvumelana nokwenyuka kwamazinga e-GA, i-SAM ye-quintuple ga2ox mutant inflorescence yayinkulu kunaleyo ye-Col-0 (I-Supplementary Fig. 12a, b), futhi uma iqhathaniswa ne-Col-0, i-quintuple ga2ox SAM ibonise ukusatshalaliswa okuhluke ngokuphawulekayo kwama-engeli e-cell division, ne-engeli ekhuphuka imvamisa isuka ku-50 °, i-engeli yokuhlukanisa iphinde ibe ngu-9 Umfanekiso 12a–c). Ngakho, sibonisa ukuthi ukwenziwa kusebenze okuyisisekelo kokusayina kwe-GA kanye nokunqwabelana kwe-GA kudala ukuhlukana kwamaseli asemaceleni ku-IPR nakuyo yonke i-SAM.
a, b ukubona ngeso lengqondo kwe-3D kwesendlalelo se-L1 se-PI-stained Ler (a) kanye ne-global della mutant (b) SAM kusetshenziswa imakroskopu e-confocal. Izindonga zamaseli amasha ezakhiwe ku-SAM (kodwa hhayi i-primordium) esikhathini esingamahora angu-10 ziyaboniswa futhi zinemibala ngokuya ngamavelu e-engeli yazo. Isingeniso sibonisa i-SAM ngo-0 h. Ibha yombala iboniswa ekhoneni elingezansi kwesokudla. Umcibisholo kokuthi (b) ukhomba isibonelo samafayela eseli aqondanisiwe ku-global della mutant. Ukuhlolwa kuphindwe kabili ngemiphumela efanayo. ce ukuqhathaniswa kokusatshalaliswa kwemvamisa kokuma kwendiza yokuhlukaniswa kweseli kuyo yonke i-SAM (d), i-IPR (e), kanye ne-non-IPR (f) phakathi kwe-Ler ne-della yomhlaba wonke. Amanani we-P atholwe kusetshenziswa ukuhlolwa kwe-Kolmogorov-Smirnov enemisila emibili. f, g Ukubukwa kwe-3D kwezithombe ezihlanganisiwe ze-PI-stained SAM ye-Col-0 (i) kanye ne-pCUC2::gai-1-VENUS (j) izitshalo ze-transgenic. Amaphaneli (a, b) abonisa izindonga zamaseli amasha (kodwa hhayi i-primordia) ezakhiwe ku-SAM phakathi kwamahora angu-10. Ukuhlolwa kuphindwe kabili ngemiphumela efanayo. h–j Ukuqhathaniswa kokusatshalaliswa kwemvamisa kokuma kwendiza yokuhlukaniswa kwamaseli okutholakala kuyo yonke i-SAM (h), IPR (i) kanye ne-non-IPR (j) phakathi kwe-Col-0 ne-pCUC2:: i-gai-1-VENUS izitshalo. Amanani we-P atholwe kusetshenziswa ukuhlolwa kwe-Kolmogorov-Smirnov enemisila emibili.
Ngokulandelayo sihlole umphumela wokuvimbela ukusayina kwe-GA ngokukhethekile ku-IPR. Kuze kube manje, sisebenzise umgqugquzeli wenkomishi ye-cotyledon 2 (CUC2) ukuze siqhubekisele phambili ukuvezwa kwephrotheni ebusayo eyi-gai-1 ehlanganiswe ne-VENUS (kulayini we-pCUC2::gai-1-VENUS). Ku-SAM yohlobo lwasendle, umgqugquzeli we-CUC2 ushayela ukuvezwa kwama-IPR amaningi ku-SAM, okuhlanganisa namaseli omngcele, kusukela ku-P4 kuya phambili, futhi inkulumo ethize efanayo yabonwa ku-pCUC2 ::gai-1-VENUS izitshalo (bona ngezansi). Ukusatshalaliswa kwama-angles okuhlukaniswa kwamaseli kuwo wonke ama-SAM noma i-IPR ye-pCUC2 :: izitshalo ze-gai-1-VENUS azizange zihluke kakhulu kuhlobo lwasendle, nakuba ngokungalindelekile sathola ukuthi amaseli angenayo i-IPR kulezi zitshalo ahlukaniswe imvamisa ephakeme ye-80-90 ° (Fig. 6f-j).
Kuye kwaphakanyiswa ukuthi ukuqondiswa kokuhlukaniswa kwamaseli kuncike kujometri ye-SAM, ikakhulukazi ukucindezeleka okuqinile okukhiqizwa i-tissu curvature46. Ngakho-ke sibuze ukuthi ngabe ukwakheka kwe-SAM kwashintshwa yini ku-della global mutant kanye ne-pCUC2::gai-1-VENUS izitshalo. Njengoba kubikwe ngaphambilini12, usayizi we-della global mutant SAM wawumkhulu kunohlobo lwasendle (I-Supplementary Fig. 13a, b, d). I-In situ hybridization ye-CLV3 kanye ne-STM RNA iqinisekise ukunwetshwa kwe-meristem kuma-della mutants futhi yabonisa ngokuqhubekayo ukunwetshwa kwe-lateral kwe-stem cell niche (I-Supplementary Fig. 13e, f, h, i). Kodwa-ke, i-curvature ye-SAM yayifana kuzo zombili izinhlobo ze-genotype (I-Supplementary Fig. 13k, m, n, p). Sibone ukwanda okufanayo kosayizi ku-gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant ngaphandle koshintsho ekujikeni uma kuqhathaniswa nohlobo lwasendle (I-Supplementary Fig. 13c, d, g, j, l, o, p). Imvamisa ye-orientation ye-cell division nayo yathinteka ku-della quadruple mutant, kodwa ngezinga elincane kune-della monolithic mutant (I-Supplementary Fig. 12d-f). Lo mphumela wokulinganisa, kanye nokuntuleka komthelela ekugobeni, uphakamisa ukuthi umsebenzi osele we-RGL3 ku-Della quadruple mutant ukhawula izinguquko ekuqondeni kokuhlukaniswa kwamaseli okubangelwa ukulahlekelwa umsebenzi we-DELLA nokuthi izinguquko ekuhlukaniseni kwamaseli angemuva zenzeka ngenxa yezinguquko kumsebenzi wokusayinda i-GA kunezinguquko ku-SAM geometry. Njengoba kuchazwe ngenhla, umgqugquzeli we-CUC2 ushayela inkulumo ye-IPR ku-SAM eqala ku-P4 (Fig. Lolu shintsho ku-pCUC2 :: i-gai-1-VENUS ye-SAM morphology ingase ibangele ukusatshalaliswa okuhlukile kokucindezeleka kwemishini uma kuqhathaniswa nohlobo lwasendle, lapho ukucindezeleka okuphezulu kwe-circumferential kuqala ebangeni elifushane ukusuka kusikhungo se-SAM47. Kungenjalo, izinguquko ku-pCUC2 ::gai-1-VENUS SAM morphology zingase zibe umphumela wezinguquko zezakhiwo zemishini zesifunda ezibangelwa i-transgene expression48. Kuzo zombili izimo, lokhu kungase kususe kancane imiphumela yezinguquko ekusayineni kwe-GA ngokwandisa amathuba okuthi amaseli azohlukana ngomumo oyisiyingi/oguquguqukayo, echaza esikubonile.
Sekuhlangene, idatha yethu iqinisekisa ukuthi ukusayinda okuphezulu kwe-GA kudlala indima ebalulekile kuma-lateral endiza yokuhlukaniswa kwamaseli ku-IPR. Ziphinde zibonise ukuthi i-meristem curvature iphinda ibe nomthelela ekumisweni kwendiza yokuhlukaniswa kwamaseli ku-IPR.
Ukuma okuphambene kwendiza ehlukanisayo ku-IPR, ngenxa yomsebenzi ophezulu wokusayinda i-GA, kuphakamisa ukuthi i-GA ihlela kusengaphambili ifayela le-radial cell ku-epidermis ngaphakathi kwe-SAM ukuze ichaze inhlangano yeselula ezotholakala kamuva ku-epidermal internode. Ngempela, amafayela weseli anjalo ayevame ukubonakala ezithombeni ze-SAM ze-della global mutants (Fig. 6b). Ngakho-ke, ukuze siqhubeke sihlola umsebenzi wokuthuthuka wephethini yendawo yokubonisa i-GA ku-SAM, sasebenzisa ukucabanga kokudlula isikhathi ukuze sihlaziye inhlangano yendawo yamaseli ku-IPR ngohlobo lwasendle (Ler noCol-0), i-della global mutants, kanye ne-pCUC2 ::gai-1-VENUS izitshalo ze-transgenic.
Sithole ukuthi i-qmRGA ibonise ukuthi umsebenzi wokusayinda i-GA ku-IPR unyuke usuka ku-P1/P2 futhi wafinyelela phezulu ku-P4, futhi le phethini yahlala injalo ngokuhamba kwesikhathi (Fig. 4a–f kanye ne-Supplementary Fig. 8c–f, k). Ukuhlaziya inhlangano yendawo yamaseli ku-IPR ngokukhula kwesignali ye-GA, sibhale amaseli e-Ler IPR ngenhla nasezinhlangothini ze-P4 ngokusho kwesiphetho sabo sokuthuthuka esihlaziywe amahora angu-34 ngemva kokubhekwa 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 ne-P4, oluhlaza kulawo abeku-IPR, kanye nonsomi kulawo abambe iqhaza kuzo zombili izinqubo (Fig. 7a–c). Ku-t0 (0 h), izingqimba ze-1-2 zamaseli e-IPR zazibonakala phambi kwe-P4 (Fig. 7a). Njengoba kulindelekile, lapho la maseli ehlukana, akwenza ikakhulukazi ngendiza ehlukanisayo enqamulayo (Amakhiwane 7a-c). Imiphumela efanayo yatholwa kusetshenziswa i-Col-0 SAM (egxile ku-P3, omngcele wayo ogoqa ngokufanayo ne-P4 ku-Ler), nakuba kule genotype ukugoqa okwakhiwa emngceleni wezimbali kufihle amaseli e-IPR ngokushesha (Fig. 7g–i). Ngakho-ke, iphethini yokuhlukanisa yamaseli e-IPR ihlela kusengaphambili amaseli abe yimigqa yeradial, njengakuma-internode. Ukuhlelwa kwemigqa ye-radial kanye nokwenza kwasendaweni kwamaseli e-IPR phakathi kwezitho ezilandelanayo kuphakamisa ukuthi lawa maseli angama-internodal progenitors.
Lapha, sithuthukise i-ratiometric GA signing biosensor, qmRGA, evumela ukwakheka kwemephu kobuningi bomsebenzi wokusayinda we-GA okubangelwa ukugxila okuhlanganisiwe kwe-GA kanye ne-GA ngenkathi kuncishiswa ukuphazamiseka kwezindlela zokusayina ze-endo native, ngaleyo ndlela inikeze ulwazi ngomsebenzi we-GA ezingeni lamaselula. Kuze kube manje, sakhe iphrotheni ye-DELLA eshintshiwe, i-mRGA, elahlekelwe amandla okuhlanganisa ozakwethu bokusebenzelana be-DELLA kodwa ihlala izwela ku-GA-induced proteinolysis. I-qmRGA iphendula kukho kokubili izinguquko zangaphandle nezingapheli kumazinga e-GA, futhi izici zayo zokuzwa ezinamandla zinika amandla ukuhlolwa kwezinguquko ze-spatiotemporal emsebenzini wokusayinda we-GA ngesikhathi sokuthuthukiswa. I-qmRGA futhi iyithuluzi elivumelana nezimo kakhulu njengoba lingakwazi ukujwayelana nezicubu ezihlukene ngokushintsha umgqugquzeli osetshenziselwa ukuvezwa kwayo (uma kunesidingo), futhi kunikezwe imvelo elondoloziwe yendlela yokusayina ye-GA kanye ne-PFYRE motif kuwo wonke ama-angiosperms, kungenzeka idluliselwe kwezinye izinhlobo zezilwane22. Ngokuvumelana nalokhu, ukuguqulwa okufanayo kwerayisi SLR1 DELLA protein (HYY497AAA) kuphinde kwaboniswa ukucindezela umsebenzi wokucindezela ukukhula kwe-SLR1 kuyilapho kunciphisa kancane ukuwohloka kwayo okune-GA, okufana ne-mRGA23. Ngokuphawulekayo, ucwaningo lwakamuva ku-Arabidopsis lubonise ukuthi ukuguqulwa kwe-amino acid eyodwa esizindeni se-PFYRE (S474L) kuguqule umsebenzi wokuloba we-RGA ngaphandle kokuphazamisa amandla ayo okusebenzelana nabalingani besici sokuloba50. Nakuba lokhu kuguqulwa kusondele kakhulu ekufakweni kwe-amino acid emi-3 okukhona ku-mRGA, ucwaningo lwethu lubonisa ukuthi lezi zinguquko ezimbili zishintsha izici ezihlukile ze-DELLA. Yize iningi lozakwethu besici sokuloba bebophezela kusizinda se-LHR1 ne-SAW se-DELLA26,51, amanye ama-amino acid alondoloziwe esizindeni se-PFYRE angasiza ukuzinzisa lokhu kusebenzisana.
Ukuthuthukiswa kwe-Internode kuyisici esibalulekile ekwakhiweni kwezitshalo nasekuthuthukisweni kwesivuno. I-qmRGA iveze umsebenzi ophezulu wokusayina we-GA kumaseli e-IPR internode progenitor. Ngokuhlanganisa i-imaging ye-quantitative kanye nezakhi zofuzo, sibonise ukuthi amaphethini wokusayina we-GA adlula izindiza eziyindilinga/eziguquguqukayo zokuhlukaniswa kwamaseli ku-SAM epidermis, ebumba inhlangano yokuhlukaniswa kwamaseli edingekayo ekuthuthukisweni kwe-internode. Izilawuli eziningana zokuma kwendiza yokuhlukaniswa kwamaseli zikhonjwe ngesikhathi sokuthuthukiswa52,53. Umsebenzi wethu unikeza isibonelo esicacile sokuthi umsebenzi wokusayina we-GA ulawula kanjani le pharamitha yeselula. I-DELLA ingasebenzisana ne-prefolding protein complexes41, ngakho-ke ukusayina kwe-GA kungase kulawule ukuma kwendiza yokuhlukaniswa kwamaseli ngokuthonya ngokuqondile i-cortical microtubule orientation40,41,54,55. Sibonise ngokungalindelekile ukuthi ku-SAM, ukuhlobana komsebenzi wokusayinda kwe-GA okuphezulu bekungekona ukwelulwa kweseli noma ukuhlukaniswa, kodwa kuphela i-anisotropy yokukhula, ehambisana nomthelela oqondile we-GA ekuqondeni kokuhlukaniswa kwamaseli ku-IPR. Kodwa-ke, asikwazi ukushiya ngaphandle ukuthi lo mphumela ungase ungaqondile, isibonelo ulamula ukuthambisa udonga lweseli lwe-GA-induced56. Izinguquko ezakhiweni zodonga lwamangqamuzana zibangela ukucindezeleka komshini57,58, okungase futhi kuthonye ukuqondiswa kwendiza yokuhlukaniswa kwamaseli ngokuthinta ukuqondiswa kwama-microtubules e-cortical39,46,59. Imiphumela ehlanganisiwe ye-GA-induced mechanical stress kanye nokulawulwa okuqondile kwe-microtubule orientation yi-GA kungase kuhileleke ekukhiqizeni iphethini ethile ye-cell division orientation ku-IPR ukuze kuchazwe ama-internode, futhi izifundo ezengeziwe ziyadingeka ukuze kuhlolwe lo mbono. Ngokufanayo, izifundo zangaphambilini zibonise ukubaluleka kwe-DELLA-interacting proteins TCP14 kanye ne-15 ekulawuleni ukwakheka kwe-internode60,61 futhi lezi zici zingase zixazulule isenzo se-GA kanye ne-BREVIPEDICELLUS (BP) kanye ne-PENNYWISE (PNY), elawula ukuthuthukiswa kwe-internode futhi iboniswe ukuthi ithonya i-2GA2 signaling. Njengoba kunikezwe ukuthi ama-DELLA asebenzisana ne-brassinosteroid, i-ethylene, i-jasmonic acid, kanye ne-abscisic acid (ABA) yokubonisa izindlela63,64 nokuthi lawa mahomoni angathonya i-microtubule orientation65, imiphumela ye-GA ekuqondeni kokuhlukaniswa kwamaseli ingase futhi ixoxwe ngamanye amahomoni.
Ucwaningo lokuqala lwe-cytological lubonise ukuthi kokubili izifunda zangaphakathi nezingaphandle ze-Arabidopsis SAM ziyadingeka ekuthuthukisweni kwe-internode2,42. Iqiniso lokuthi i-GA ilawula ngenkuthalo ukuhlukaniswa kwamaseli ezicutshini zangaphakathi12 isekela umsebenzi ombaxambili we-GA ekulawuleni i-meristem kanye nosayizi we-internode ku-SAM. Iphethini yokuhlukaniswa kwamaseli okuqondisayo nayo ilawulwa ngokuqinile kuzicubu ze-SAM zangaphakathi, futhi lo mthethonqubo ubalulekile ekukhuleni kwesiqu52. Kuzokujabulisa ukuhlola ukuthi ingabe i-GA nayo idlala indima ekuqondiseni indiza yokuhlukaniswa kwamaseli enhlanganweni ye-SAM yangaphakathi, ngaleyo ndlela ivumelanisa ukucaciswa nokuthuthukiswa kwama-internode ngaphakathi kwe-SAM.
Izitshalo zazitshalwa ku-vitro enhlabathini noma i-1x Murashige-Skoog (MS) medium (Duchefa) engezwe ngo-1% sucrose kanye no-1% we-agar (Sigma) ngaphansi kwezimo ezijwayelekile (ukukhanya kwamahora angu-16, 22 °C), ngaphandle kokuhlolwa kwe-hypocotyl nokukhulisa izimpande lapho izithombo zazitshalwa khona kumapuleti aqondile ngaphansi kokukhanya okungaguquki okungu-22 ° C. Ocwaningweni lwe-nitrate, izitshalo zatshalwa ku-MS medium elungisiwe (i-bioWORLD plant medium) engezwe nge-nitrate eyanele (0 noma 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose kanye no-1% A-agar (Sigma) ngaphansi kwezimo zosuku olude.
I-GID1a cDNA efakwe ku-pDONR221 iphinde yahlanganiswa ne-pDONR P4-P1R-pUBQ10 kanye ne-pDONR P2R-P3-mCherry ku-pB7m34GW ukuze kukhiqizwe i-pUBQ10::GID1a-mCherry. I-IDD2 DNA efakwe ku-pDONR221 iphinde yahlanganiswa yaba yi-pB7RWG266 ukuze kukhiqizwe i-p35S:IDD2-RFP. Ukuze ukhiqize i-pGID1b::2xmTQ2-GID1b, isinqamu esingu-3.9 kb esikhuphuka nomfula wesifunda sekhodi se-GID1b kanye nesiqephu esingu-4.7 kb esiqukethe i-GID1b cDNA (1.3 kb) nesinqamuli (3.4 kb) kwaqala kwakhuliswa kusetshenziswa ama-primers kokuthi Supplementary p-1R4 (Thermo Fisher Scientific) kanye ne-pDONR P2R-P3 (Thermo Fisher Scientific), ngokulandelana, futhi ekugcineni kwahlanganiswa kabusha ne-pDONR221 2xmTQ268 ku-pGreen 012567 ivector eqondiwe kusetshenziswa i-Gateway cloning. Ukuze ukhiqize i-pCUC2::LSSmOrange, ukulandelana komgqugquzeli we-CUC2 (3229 bp phezulu komfula we-ATG) kulandelwa ukulandelana kwekhodi kwe-Stokes-shifted mOrange enkulu (LSSmOrange)69 ngesignali yendawo ye-nuclear ye-N7 kanye nesinqamuli sokuloba se-NOS kwahlanganiswa ohlelweni oluqondisayo lwe-3 or Green. (I-Invitrogen). I-vector kanambambili yesitshalo yethulwa kuhlobo lwe-Agrobacterium tumefaciens GV3101 futhi yethulwa emaqabunga e-Nicotiana benthamiana ngendlela ye-Agrobacterium yokungena kanye naku-Arabidopsis thaliana Col-0 ngendlela ye-floral dip, ngokulandelana. I-pUBQ10::qmRGA pUBQ10::GID1a-mCherry kanye ne-pCLV3::mCherry-NLS qmRGA ahlukanisiwe kunzalo ye-F3 ne-F1 yeziphambano ngokulandelana kwazo.
I-RNA in situ hybridization yenziwa cishe ku-1 cm ubude bokudubula amathiphu72, aqoqwe futhi alungiswa ngokushesha kusixazululo se-FAA (3.7% formaldehyde, 5% acetic acid, 50% ethanol) epholiswe ngaphambili ku-4 °C. Ngemuva kokwelashwa kwe-vacuum eyi-2 × 15 min, ukulungiswa kwashintshwa futhi amasampuli afakwa ngobusuku bonke. I-GID1a, i-GID1b, i-GID1c, i-GAI, i-RGL1, i-RGL2, ne-RGL3 cDNAs kanye nama-antisense probes kuma-3′-UTR awo ahlanganiswa kusetshenziswa ama-primers aboniswe kuThebula 3 Lokwengeza njengoba kuchazwe ngu-Rosier et al.73. Ama-probe anelebuli ye-Digoxigenin ayengagonywanga kusetshenziswa amasosha omzimba e-digoxigenin (i-dilution ephindwe izikhathi ezingu-3000; i-Roche, inombolo yekhathalogi: 11 093 274 910), futhi izigaba zazingcoliswe nge-5-bromo-4-chloro-3-indolyl phosphate (BCIP, 250-litrolution, 250-litrolution) Isixazululo esingu-200-fold dilution).