Shoot apical meristem (SAM) uto dị oke mkpa maka nhazi ụlọ. Hormone osisigibberellins(GA) na-arụ ọrụ dị mkpa n'ịkwado uto osisi, mana ọrụ ha na SAM ka aghọtachaghị nke ọma. N'ebe a, anyị mepụtara biosensor biometric nke mgbaàmà GA site na injinia protein DELLA iji kwụsị ọrụ nhazi ya dị mkpa na nzaghachi ntụgharị GA ka ọ na-echekwa mbibi ya na nnabata GA. Anyị na-egosipụta na biosensor dabere nbibi a na-edekọ nke ọma mgbanwe na ọkwa GA yana nghọta ekwentị n'oge mmepe. Anyị jiri biosensor a mee ka eserese GA ọrụ mgbama na SAM. Anyị na-egosi na mgbama GA dị elu dị na sel ndị dị n'etiti primordia organ, bụ ndị na-ebute sel internode. N'iji ụzọ uru na nkwụsị ọrụ na-abịaru nso, anyị na-egosipụtakwa na GA na-achịkwa nhazi nke ụgbọelu nkewa cell, na-eguzobe nzukọ cellular canonical nke internodes, si otú ahụ na-akwalite nkọwa internode na SAM.
The shoot apical meristem (SAM), nke dị na elu oge ịse, nwere niche nke mkpụrụ ndụ stem nke ọrụ ya na-ewepụta akụkụ akụkụ ya na ọnụ ụzọ ya n'ụdị modular na ntughari n'oge ndụ nke osisi ahụ. Nke ọ bụla n'ime akụkụ ndị a na-emegharị ugboro ugboro, ma ọ bụ ọnụ osisi, gụnyere internodes na akụkụ akụkụ na ọnụ ọnụ, na axillary meristems na akwukwo axils1. Uto na nhazi nke ọnụ osisi na-agbanwe n'oge mmepe. Na Arabidopsis, a na-ebelata uto internodal n'oge ogbo ahịhịa, na axillary meristems na-anọkwa n'akụkụ axils nke akwụkwọ rosette. N'oge mgbanwe na oge ifuru, SAM na-aghọ inflorescence meristem, na-emepụta elongated internodes na axillary buds, alaka ụlọ ọrụ na axils nke cauline epupụta, na mgbe e mesịrị, okooko osisi na-enweghị akwụkwọ2. Ọ bụ ezie na anyị enweela ọganihu dị ukwuu n'ịghọta usoro ndị na-achịkwa mmalite nke akwụkwọ, okooko osisi, na alaka, ọ dị ntakịrị ihe a maara banyere otú internodes si ebili.
Ịghọta nkesa spatiotemporal nke GA ga-enyere aka ịghọta nke ọma ọrụ nke homonụ ndị a na anụ ahụ dị iche iche na n'oge mmepe dị iche iche. Nleba anya nke nbibi nke RGA-GFP fusion gosipụtara n'okpuru omume nke onye na-akwalite ya na-enye ozi dị mkpa na nhazi nke mkpokọta GA na mgbọrọgwụ15,16. Agbanyeghị, okwu RGA dịgasị iche n'ofe anụ ahụ17 ma GA18 na-ahazi ya. Ya mere, okwu dị iche iche nke onye na-akwalite RGA nwere ike ime ka usoro fluorescence hụrụ na RGA-GFP ma ya mere usoro a abụghị ọnụọgụ. N'oge na-adịbeghị anya, bioactive fluorescein (Fl) nke akpọrọ GA19,20 kpughere mkpokọta GA na mgbọrọgwụ endocortex na nhazi ọkwa cellular ya site na njem GA. N'oge na-adịbeghị anya, ihe mmetụta GA FRET nlsGPS1 gosiri na ọkwa GA kwekọrọ na elongation cell na mgbọrọgwụ, filaments, na hypocotyls21 toro ọchịchịrị. Agbanyeghị, dị ka anyị hụworo, itinye uche GA abụghị naanị oke na-achịkwa ọrụ mgbaàmà GA, n'ihi na ọ dabere na usoro nghọta dị mgbagwoju anya. N'ebe a, na-ewulite na nghọta anyị maka ụzọ mgbaàmà DELLA na GA, anyị na-akọ mmepe na njirimara nke biosensor nke dabeere na mmebi nke ihe nrịbama GA. Iji zụlite biosensor nke ọnụ ọgụgụ a, anyị jiri mutant GA-sensitive RGA nke etinyere na protein fluorescent ma gosipụta ya n'ebe niile n'anụ ahụ, yana protein fluorescent nke GA-enweghị mmetụta. Anyị na-egosi na mutant RGA protein fusions adịghị egbochi endogenous GA mgbaàmà mgbe a na-egosipụta n'ebe nile, na nke a biosensor nwere ike ịkọwapụta ọrụ mgbaàmà sitere na ma ntinye GA na GA nhazi mgbaàmà site na-ahụ ihe ngwa na elu spatiotemporal mkpebi. Anyị jiri biosensor a mee ka eserese spatiotemporal nkesa ọrụ mgbaàmà GA wee kọwapụta etu GA si ahazi omume ekwentị na SAM epidermis. Anyị na-egosi na GA na-achịkwa nghazi nke ụgbọ elu nkewa nke mkpụrụ ndụ SAM dị n'etiti primordia organ, si otú ahụ na-akọwa nhazi cellular canonical nke internode.
N'ikpeazụ, anyị jụrụ ma qmRGA nwere ike ịkọ mgbanwe na ọkwa GA endogenous site na iji hypocotyls na-eto eto. Anyị gosipụtara na mbụ na nitrate na-akpali uto site n'ịba ụba njikọ GA na, n'aka nke ya, mmebi DELLA34. N'ihi ya, anyị chọpụtara na ogologo hypocotyl na pUBQ10 ::qmRGA osisi toro n'okpuru ụbara nitrate ọkọnọ (10 mM NO3-) bụ nke ukwuu ogologo karịa nke seedlings toro n'okpuru nitrate-erughị eru ọnọdụ (Ngbakwunye Fig. 6a). N'ikwekọ na nzaghachi uto, mgbaàmà GA dị elu na hypocotyls nke mkpụrụ osisi toro n'okpuru ọnọdụ 10 mM NO3- karịa na mkpụrụ osisi toro na enweghị nitrate (Mgbakwunye Fig. 6b, c). Ya mere, qmRGA na-enyekwa aka nleba anya mgbanwe mgbanwe na mgbama GA kpatara site na mgbanwe endogenous na ntinye GA.
Iji ghọta ma ọrụ nrịbama GA nke qmRGA achọpụtara dabere na ntinye GA na nghọta GA, dịka a tụrụ anya dabere na ihe mmetụta, anyị nyochara okwu nke ndị nnabata GID1 atọ na anụ ahịhịa na ịmụ nwa. Na mkpụrụ osisi, ahịrị onye nta akụkọ GID1-GUS gosipụtara na GID1a na c gosipụtara nke ukwuu na cotyledons (Fig 3a-c). Tụkwasị na nke ahụ, e gosipụtara ndị nabatara atọ ahụ na akwụkwọ, mgbọrọgwụ mgbọrọgwụ primordia, ndụmọdụ mgbọrọgwụ (ma e wezụga maka mgbọrọgwụ mgbọrọgwụ nke GID1b), na usoro vaskụla (Fig. 3a-c). N'ime inflorescence SAM, anyị chọpụtara akara GUS naanị maka GID1b na 1c (Fig agbakwunyere. 7a-c). N'ebe a na-emekọ ihe ọnụ kwadoro usoro okwu ndị a ma gosikwa na GID1c ka egosipụtara n'otu n'otu na ọkwa dị ala na SAM, ebe GID1b gosipụtara okwu dị elu na mpụta nke SAM (Mgbakwunye 7d-l). Njikọ ntụgharị pGID1b :: 2xmTQ2-GID1b kpughekwara ọkwa dị iche iche nke GID1b okwu, site na okwu dị ala ma ọ bụ enweghị okwu n'etiti SAM ruo n'ókè dị elu na oke akụkụ (Mgbakwunye 7m). Ya mere, ndị na-anabata GID1 adịghị ekesa n'otu n'otu gafee na n'ime anụ ahụ. N'ime nnwale ndị sochirinụ, anyị hụkwara na oke ngosipụta nke GID1 (pUBQ10 :: GID1a-mCherry) mụbara mmetụta nke qmRGA na hypocotyls na ngwa GA mpụga (Fig 3d, e). N'ụzọ dị iche, fluorescence tụrụ site na qd17mRGA na hypocotyl enweghị mmetụta na ọgwụgwọ GA3 (Fig 3f, g). Maka nyocha abụọ ahụ, a na-emeso mkpụrụ osisi na nnukwu ọkwa nke GA (100 μM GA3) iji chọpụta ngwa ngwa ngwa ngwa nke ihe mmetụta, ebe ikike ijikọ na onye na-anabata GID1 na-abawanye ma ọ bụ furu efu. Ọnụ, nsonaazụ ndị a na-akwado na qmRGA biosensor na-arụ ọrụ jikọtara ọnụ dị ka ihe mmetụta GA na GA, ma na-atụ aro na ngosipụta dị iche iche nke onye na-anabata GID1 nwere ike gbanwee nrịanrịa nke ihe mmetụta.
Ruo ugbu a, nkesa nke mgbaàmà GA na SAM ka edoghị anya. Ya mere, anyị na-eji qmRGA-ekwupụta osisi na pCLV3 :: mCherry-NLS stem cell reporter35 iji gbakọọ elu-mkpebi quantitative maps nke GA signaling ọrụ, na-elekwasị anya na L1 oyi akwa (epidermis; fig. 4a, b, lee Ụzọ na usoro mgbakwunye), ebe L1 na-arụ ọrụ dị mkpa n'ịchịkwa SAM. N'ebe a, pCLV3 :: mCherry-NLS okwu nyere nrụtụ aka geometric edoziri maka nyocha nkesa nkesa nke GA nrịbama37. Ọ bụ ezie na a na-ewere GA dị ka ihe dị mkpa maka mmepe akụkụ akụkụ akụkụ4, anyị chọpụtara na mgbaàmà GA dị ala na floral primordium (P) malite na ọkwa P3 (Fig 4a, b), ebe ndị na-eto eto P1 na P2 primordiums nwere ọrụ na-agafeghị oke yiri nke dị na mpaghara etiti (Fig 4a, b). Achọpụtara ọrụ nrịbama GA dị elu na akụkụ primordium organ, malite na P1 / P2 (n'akụkụ nke ókèala) na peaking na P4, yana na sel niile nke mpaghara mpaghara dị n'etiti primordia (Fig. 4a, b na Fig. 8a, b). A na-ahụta ọrụ mgbaàmà GA dị elu ọ bụghị nanị na epidermis kamakwa na L2 na elu L3 layers (Mgbakwunye 8b). Ụkpụrụ nke mgbaàmà GA achọpụtara na SAM na-eji qmRGA anọgideghịkwa na-agbanwe agbanwe ka oge na-aga (Mgbakwunye 8c-f, k). Ọ bụ ezie na qd17mRGA gbadara n'usoro n'usoro na SAM nke osisi T3 site na ahịrị ise nwere onwe anyị nke anyị ji mara nke ọma, anyị nwere ike nyochaa usoro fluorescence enwetara na pRPS5a:: VENUS-2A-TagBFP (Mgbakwunye Fig. 8g-j, l). N'ahịrị njikwa a, ọ bụ naanị obere mgbanwe dị na nha fluorescence ka achọpụtara na SAM, mana na etiti SAM anyị hụrụ mbelata doro anya na nke a na-atụghị anya ya na VENUS jikọtara ya na TagBFP. Nke a na-akwado na usoro nrịbama nke qmRGA hụrụ na-egosipụta nbibi nke GA-dabere na mRGA-VENUS, mana gosikwara na qmRGA nwere ike ibuli ọrụ nrịbama GA na etiti meristem. Na nchịkọta, nsonaazụ anyị na-ekpughe ụkpụrụ nrịbama GA nke na-egosipụta isi nkesa nke primordia. Nkesa a nke mpaghara inter-primordial (IPR) bụ n'ihi nguzobe nke ntanye nke ọrụ mgbaàmà GA dị elu n'etiti primordium na-emepe emepe na mpaghara etiti, ebe n'otu oge ahụ GA-arụ ọrụ na-egosi na primordium na-ebelata (Fig 4c, d).
Nkesa nke ndị na-anabata GID1b na GID1c (lee n'elu) na-egosi na ngosipụta dị iche iche nke ndị na-anabata GA na-enyere aka ịkpụzi ụkpụrụ nke ọrụ mgbaàmà GA na SAM. Anyị na-eche ma mkpokọta GA dị iche iche nwere ike itinye aka. Iji nyochaa ohere a, anyị jiri nlsGPS1 GA FRET sensọ21. Achọpụtara ugboro ugboro ugboro ugboro na SAM nke nlsGPS1 na-emeso ya na 10 μM GA4 + 7 maka 100 min (Mgbakwunye 9a-e), na-egosi na nlsGPS1 na-anabata mgbanwe na ntinye GA na SAM, dịka ọ na-eme na mgbọrọgwụ21. Nkesa oghere nke nlsGPS1 ugboro ịgbalite gosipụtara ọkwa GA dị ntakịrị na elu elu nke SAM, mana gosipụtara na ebuliri ha n'etiti yana n'ókè nke SAM (Fig. 4e na Fig. 9a, c). Nke a na-egosi na a na-ekesakwa GA na SAM yana ụkpụrụ gbasara oghere yiri nke qmRGA kpughere. Dị ka usoro nkwado, anyị na-emeso SAM na fluorescent GA (GA3-, GA4-, GA7-Fl) ma ọ bụ Fl naanị dị ka njikwa na-adịghị mma. E kesara akara Fl na SAM dum, gụnyere mpaghara etiti na primordium, n'agbanyeghị na ike dị ala (Fig. 4j na Fig. 10d). N'ụzọ dị iche, atọ GA-Fl na-agbakọba kpọmkwem n'ime primordium ókèala yana na ọkwa dịgasị iche iche na ndị ọzọ nke IPR, na GA7-Fl na-agbakọta na ngalaba kachasị na IPR (Fig. 4k na Fig. 10a,b). Ọnụ ọgụgụ nke fluorescence siri ike gosipụtara na IPR na nke na-abụghị IPR dị elu na GA-Fl-emeso SAM ma e jiri ya tụnyere Fl-mere SAM (Fig. 4l na Fig. 10c). Ọnụ, nsonaazụ ndị a na-egosi na GA dị na ọkwa dị elu na sel IPR nke dị nso na oke akụkụ ahụ. Nke a na-egosi na ụkpụrụ nke ọrụ mgbaàmà SAM GA na-esite na ma okwu dị iche iche nke ndị na-anabata GA na nchịkọta dị iche iche nke GA na sel IPR dị nso n'akụkụ akụkụ ahụ. Ya mere, nyocha anyị gosipụtara ụkpụrụ spatiotemporal a na-atụghị anya ya nke mgbaàmà GA, na-arụ ọrụ dị ala na etiti na primordium nke SAM na ọrụ dị elu na IPR na mpaghara mpaghara.
Iji ghọta ọrụ dị iche iche GA mgbaàmà ọrụ na SAM, anyị nyochara mmekọrịta dị n'etiti GA signaling ọrụ, cell mgbasa ozi, na cell nkewa site na iji ezigbo oge-oge-oge imaging nke SAM qmRGA pCLV3 :: mCherry-NLS. N'inye ọrụ GA na nhazi uto, mmekọrịta dị mma na parampat mgbasawanye cell ka a tụrụ anya. Ya mere, anyị buru ụzọ jiri maapụ ọrụ mgbaàmà GA tụnyere maapụ nke mkpụrụ ndụ elu cell (dị ka onye nnọchiteanya maka ike nke mgbasawanye cell maka cell nyere na maka sel ụmụ nwanyị na nkewa) yana maapụ nke anisotropy nke uto, nke na-atụle ntụziaka nke mgbasawanye cell (nke a na-ejikwa ebe a maka cell nyere na sel nwa nwanyị na nkewa; Fig. 5a,b, lee Ụzọ na Usoro mgbakwunye). Maapụ anyị nke SAM cell elu ọnụego uto kwekọrọ na nleba anya gara aga38,39, na ọnụ ọgụgụ uto pere mpe na oke na oke uto na-eto eto na ifuru (Fig 5a). Ntụle akụrụngwa nke isi (PCA) gosiri na arụrụ ọrụ nrịbama GA na-emetụta oke uto elu cell (Njirimara 5c). Anyị gosikwara na isi axes nke mgbanwe dị iche iche, gụnyere ntinye akara GA na ike uto, bụ orthogonal na ntụziaka nke okwu CLV3 dị elu kpebisiri ike, na-akwado mwepu nke mkpụrụ ndụ site na etiti SAM na nyocha ndị ọzọ. Nyocha mmekọrịta Spearman kwadoro nsonaazụ PCA (Njirimara 5d), na-egosi na akara GA dị elu na IPR emeghị ka mgbasawanye cell dị elu. Otú ọ dị, nyocha mmekọrịta gosipụtara ntakịrị mmekọrịta dị mma n'etiti ọrụ mgbaàmà GA na anisotropy uto (Njirimara 5c, d), na-atụ aro na akara GA dị elu na IPR na-emetụta ntụziaka nke uto cell na ikekwe ọnọdụ nke ụgbọelu nkewa cell.
a, b Maapụ okpomọkụ nke uto elu elu (a) na anisotropy (b) na SAM gbakọrọ ihe karịrị osisi asaa nọọrọ onwe ya (eji dị ka proxies maka ike na ntụziaka nke mgbasawanye cell, n'otu n'otu). c PCA nyocha gụnyere mgbanwe ndị a: mgbama GA, ogo uto elu, anisotropy elu elu, na okwu CLV3. Akụkụ PCA 1 bụ nke emetụtara nke ọma n'ụzọ na-adịghị mma na uto elu elu yana nke ọma na mgbama GA. Akụkụ PCA 2 bụ nke ọma jikọtara ya na anisotropy uto elu yana ejikọtaghị ya na okwu CLV3. Pasent na-anọchite anya mgbanwe nke akụrụngwa ọ bụla kọwara. d Nyocha mmekọrịta Spearman n'etiti mgbaàmà GA, mmụba elu elu, na anisotropy elu elu na ọnụ ọgụgụ anụ ahụ ewezuga CZ. Nọmba dị n'aka nri bụ uru Spearman rho n'etiti mgbanwe abụọ. Asterisks na-egosi ọnọdụ ebe njikọ/mmekọrịta adịghị mma dị oke mkpa. e 3D anya nke mkpụrụ ndụ Col-0 SAM L1 site na microscopy confocal. Mgbidi cell ọhụrụ e guzobere na SAM (ma ọ bụghị primordium) na 10 h na-agba agba dịka ụkpụrụ akụkụ ha si dị. Egosiri mmanya agba na akuku aka nri ala. Ntinye na-egosi onyonyo 3D kwekọrọ na 0 h. Emegharịrị nnwale ahụ ugboro abụọ yana nsonaazụ yiri ya. f Igbe igbe na-egosipụta ọnụego nkewa cell na IPR na ndị na-abụghị IPR Col-0 SAM (n = 10 osisi kwụụrụ onwe ya). Ahịrị etiti na-egosi etiti, na oke igbe na-egosi 25th na 75th percentiles. Whiskers na-egosi opekempe na ụkpụrụ kachasị nke ejiri ngwa R ekpebie. Ejiri t-ule nwere ọdụ abụọ Welch nweta ụkpụrụ P. g, h Eserese eserese nke na-egosi (g) otu esi atụta akụkụ nke mgbidi cell ọhụrụ (magenta) gbasara ntụzịaka radial site na etiti SAM (ahịrị ntụpọ ọcha) (naanị nnukwu akụkụ ụkpụrụ, ya bụ, 0-90 Celsius, ka a na-atụle), na (h) ntụgharị okirikiri / mpụta na radial n'ime meristem. i Ihe onyonyo ugboro ugboro nke nhazi ụgbọ elu cell nkewa gafee SAM (acha anụnụ anụnụ), IPR (acha anụnụ anụnụ ọkara), na ndị na-abụghị IPR (acha anụnụ anụnụ), n'otu n'otu. E nwetara ụkpụrụ P site na ule Kolmogorov-Smirnov ọdụdụ abụọ. Emegharịrị nnwale ahụ ugboro abụọ yana nsonaazụ yiri ya. j Ihe ndekọ ugboro ugboro nke nhazi ụgbọ elu cell nkewa nke IPR gburugburu P3 (acha ndụ ndụ ndụ ndụ), P4 (ọkara akwụkwọ ndụ akwụkwọ ndụ), na P5 (acha ndụ ndụ gbara ọchịchịrị), n'otu n'otu. E nwetara ụkpụrụ P site na ule Kolmogorov-Smirnov ọdụdụ abụọ. Emegharịrị nnwale ahụ ugboro abụọ yana nsonaazụ yiri ya.
Ya mere, anyị nyochara njikọ dị n'etiti mgbaàmà GA na ọrụ nkewa cell site n'ịchọpụta mgbidi cell ọhụrụ e guzobere n'oge nyocha (Fig 5e). Ụzọ a nyere anyị ohere ịlele ugboro na ntụziaka nke nkewa cell. N'ụzọ dị ịtụnanya, anyị chọpụtara na ugboro ole nkewa nke cell na IPR na ndị ọzọ SAM (na-abụghị IPR, Fig. 5f) yiri nke ahụ, na-egosi na ọdịiche dị na GA na-egosi n'etiti IPR na sel ndị na-abụghị IPR adịghị emetụta nkewa cell. Nke a, na mmekọrịta dị mma n'etiti mgbaàmà GA na anisotropy uto, kpaliri anyị ịtụle ma ọrụ mgbaàmà GA nwere ike imetụta nhazi nke ụgbọelu nkewa cell. Anyị tụlere nghazi nke mgbidi cell ọhụrụ dị ka akụkụ dị ukwuu n'akụkụ radial axis na-ejikọta meristem center na etiti mgbidi cell ọhụrụ (Fig 5e-i) wee hụ ihe doro anya maka mkpụrụ ndụ na-ekewa n'akụkụ dị nso na 90 ° n'akụkụ radial axis, nke kachasị elu nke a na-ahụ na 70-820%) na 23 ° (23) (Fig 5e, i), kwekọrọ na nkewa sel na ntụgharị okirikiri / ntụgharị (Fig 5h). Iji nyochaa ntinye aka nke GA na-egosi na omume nkewa cell a, anyị nyochara usoro nkewa cell na IPR na ndị na-abụghị IPR iche (Fig 5i). Anyị chọpụtara na nkesa akụkụ nkewa na sel IPR dị iche na nke ahụ na sel ndị na-abụghị IPR ma ọ bụ na sel na SAM dum, na mkpụrụ ndụ IPR na-egosipụta akụkụ dị elu nke akụkụ akụkụ akụkụ / okirikiri, ya bụ, 70-80 ° na 80-90 ° (33.86% na 30.71%, n'otu n'otu, nha nha). Ya mere, ihe anyị chọpụtara gosiri njikọ dị n'etiti elu GA mgbaàmà na a cell nkewa ụgbọelu nghazi nso na okirikiri direction, yiri njikọ n'etiti GA mgbaàmà ọrụ na uto anisotropy (Fig. 5c, d). Iji nwetakwuo nchekwa nchekwa ohere nke mkpakọrịta a, anyị tụlere nhazi ụgbọ elu nkewa na sel IPR gbara gburugburu primordium malite na P3, ebe ọ bụ na achọpụtara ọrụ mgbaàmà GA kachasị elu na mpaghara a malite na P4 (Fig 4). Akụkụ nkewa nke IPR na gburugburu P3 na P4 egosighi ọdịiche dị iche iche dị ịrịba ama, ọ bụ ezie na a na-ahụta ugboro ugboro nke akụkụ sel n'akụkụ na IPR gburugburu P4 (Fig 5j). Otú ọ dị, n'ime sel IPR dị gburugburu P5, ọdịiche dị na nhazi nke ụgbọelu nkewa cell ghọrọ ihe dị ịrịba ama, na-enwe mmụba dị ukwuu na ugboro nke transverse cell division (Fig 5j). Ọnụ, nsonaazụ ndị a na-egosi na mgbaàmà GA nwere ike ijikwa nghazi nke nkewa sel na SAM, nke kwekọrọ na akụkọ gara aga40,41 na akara ngosi GA dị elu nwere ike ime ka ntụgharị ihu nke akụkụ cell na IPR.
A na-ebu amụma na sel ndị dị na IPR agaghị etinye n'ime primordia kama na internodes2,42,43. Ntugharị ntụgharị nke nkewa sel na IPR nwere ike bute ụdị nhazi nke ahịrị ogologo ogologo nke sel epidermal na internodes. Nleba anya anyị kọwara n'elu na-egosi na mgbaàmà GA nwere ike na-ekere òkè na usoro a site n'ịhazi ntụziaka nke nkewa sel.
Ọnwụ nke ọtụtụ mkpụrụ ndụ ihe nketa DELLA na-ebute nzaghachi GA mejupụtara, na della mutant nwere ike iji nwalee hypothesis44 a. Anyị buru ụzọ nyochaa usoro okwu nke mkpụrụ ndụ ihe nketa DELLA ise na SAM. Ngwakọta ederede nke GUS line45 gosipụtara na GAI, RGA, RGL1, na RGL2 (ruo n'ókè dị ntakịrị) ka egosipụtara na SAM (Mgbakwunye Fig. 11a-d). N'ime ọnọdụ ngwakọ, gosipụtara n'ihu na GAI mRNA na-agbakọta kpọmkwem na primordia na okooko osisi na-eto eto (Fig 11e mgbakwunye). Achọpụtara RGL1 na RGL3 mRNA n'okirikiri SAM na okooko osisi ndị ochie, ebe RGL2 mRNA karịrị akarị na mpaghara oke (Foto mgbakwunye. 11f–h). Confocal imaging nke pRGL3 :: RGL3-GFP SAM kwadoro okwu a na-ahụ na ọnọdụ hybridization ma gosi na protein RGL3 na-agbakọba n'akụkụ etiti nke SAM (Mgbakwunye 11i). N'iji pRGA :: GFP-RGA ahịrị, anyị chọpụtakwara na protein RGA na-agbakọta na SAM, mana ụbara ya na-ebelata na oke na-amalite site na P4 (Mgbakwunye 11j). N'ụzọ doro anya, usoro okwu nke RGL3 na RGA kwekọrọ na ọrụ mgbaàmà GA dị elu na IPR, dịka qmRGA achọpụtara (Fig 4). Ọzọkwa, data ndị a na-egosi na egosipụtara DELLA niile na SAM yana na nkwupụta ha jikọtara ọnụ gbasara SAM dum.
Anyị na-enyocha usoro nkewa cell na ụdị anụ ọhịa SAM (Ler, control) na gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Fig. 6a, b). N'ụzọ na-akpali mmasị, anyị hụrụ mgbanwe dị ịrịba ama na nkesa nkesa akụkụ akụkụ cell na della mutant SAM ma e jiri ya tụnyere ụdị anụ ọhịa (Fig 6c). Mgbanwe a na della mutant zuru ụwa ọnụ bụ n'ihi mmụba nke ugboro nke 80-90 ° (34.71% vs. 24.55%) na, n'ókè dị nta, 70-80 ° akụkụ (23.78% vs. 20.18%), ya bụ, kwekọrọ na nkewa sel transverse (Fig 6c). Ugboro nke nkewa na-abụghị transverse (0-60°) dịkwa ala na della mutant zuru ụwa ọnụ (Fig 6c). Ugboro nke nkewa sel transverse abawanyela nke ukwuu na SAM nke della mutant zuru ụwa ọnụ (Fig 6b). Ugboro nke transverse cell nkewa na IPR dịkwa elu na della mutant zuru ụwa ọnụ ma e jiri ya tụnyere ụdị anụ ọhịa (Fig 6d). N'èzí mpaghara IPR, ụdị anụ ọhịa ahụ nwere nkesa otu akụkụ nke akụkụ cell, ebe della mutant zuru ụwa ọnụ na-ahọrọ nkewa tangential dị ka IPR (Fig 6e). Anyị kọwakwara nghazi nke nkewa sel na SAM nke ga2 oxidase (ga2ox) mutants quintuple (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, na ga2ox6-2), ihe ndabere GA-adịghị arụ ọrụ nke GA na-akwakọba. N'ikwekọ na mmụba na ọkwa GA, SAM nke quintuple ga2ox mutant inflorescence dị ukwuu karịa nke Col-0 (Foto mgbakwunye. 12a, b), ma e jiri ya tụnyere Col-0, quintuple ga2ox SAM gosipụtara nkesa dị iche iche nke akụkụ akụkụ cell nkewa, na nkuku na-abawanye site na 50 ° gential ọzọ 9. Ọgụgụ 12a–c). Ya mere, anyị na-egosi na ntinye aka nke GA-egosi na mkpokọta GA na-ebute nkewa sel n'akụkụ na IPR na ndị ọzọ nke SAM.
a, b 3D nleba anya nke oyi akwa L1 nke PI-stained Ler (a) na mutant della zuru ụwa ọnụ (b) SAM na-eji microscopy confocal. A na-egosi mgbidi ọhụrụ cell na SAM (ma ọ bụghị primordium) n'ime oge 10-h na agba dị ka ụkpụrụ akụkụ ha si dị. Ntinye na-egosi SAM na 0h. A na-egosipụta mmanya agba na akuku aka nri ala. Ụka dị na (b) na-atụ aka na atụ nke faịlụ sel agbakọtara na mutant della zuru ụwa ọnụ. Emegharịrị nnwale ahụ ugboro abụọ yana nsonaazụ yiri ya. ntụnyere nkesa ugboro ugboro nke sel nkewa ụgbọ elu na SAM (d), IPR (e), na ndị na-abụghị IPR (f) n'etiti Ler na Global della. A na-enweta ụkpụrụ P site na iji ule Kolmogorov-Smirnov ọdụdụ abụọ. f, g 3D nleba anya nke onyonyo confocal nke PI-stained SAM nke Col-0 (i) na pCUC2 :: gai-1-VENUS (j) osisi transgenic. Ogwe (a, b) na-egosi mgbidi sel ọhụrụ (ma ọ bụghị primordia) etolite na SAM n'ime awa 10. Emegharịrị nnwale ahụ ugboro abụọ yana nsonaazụ yiri ya. h –j Ntụle nkesa ugboro ugboro nke cell nkewa ụgbọelu orientations dị na dum SAM (h), IPR (i) na ndị na-abụghị IPR (j) n'etiti Col-0 na pCUC2 ::gai-1-VENUS osisi. A nwetara ụkpụrụ P site na iji ule Kolmogorov-Smirnov ọdụdụ abụọ.
Anyị ọzọ nwalere mmetụta nke igbochi GA-egosi kpọmkwem na IPR. Iji mezuo nke a, anyị na-eji cotyledon cup 2 (CUC2) onye na-akwalite ngosipụta nke protein gai-1 na-adịghị mma jikọtara na VENUS (na pCUC2 :: gai-1-VENUS line). N'ime ụdị SAM nke anụ ọhịa, onye na-akwalite CUC2 na-akwalite nkwupụta nke ọtụtụ IPRs na SAM, gụnyere mkpụrụ ndụ ókèala, site na P4 gaa n'ihu, a na-ahụkwa okwu yiri nke ahụ na pCUC2 :: gai-1-VENUS osisi (lee n'okpuru). Nkesa nkesa nkewa nke cell n'ofe SAM ma ọ bụ IPR nke pCUC2 :: gai-1-VENUS osisi adịghị iche na nke anụ ọhịa, ọ bụ ezie na anyị na-atụghị anya ya chọpụtara na sel ndị na-enweghị IPR na osisi ndị a kewara na ugboro dị elu nke 80-90 ° (Fig 6f-j).
A na-atụ aro na ntụzịaka nke nkewa sel dabere na geometry nke SAM, ọkachasị nrụrụ aka nke anụ ahụ curvature46 na-ebute. Ya mere, anyị jụrụ ma ọdịdị nke SAM agbanweela na della global mutant na pCUC2 :: gai-1-VENUS osisi. Dị ka a kọrọ na mbụ12, nha della mutant SAM zuru ụwa ọnụ buru ibu karịa nke ụdị anụ ọhịa (Fig. 13a, b, d). N'ime ọnọdụ hybridization nke CLV3 na STM RNA kwadoro mgbasawanye meristem na della mutants wee gosikwa n'akụkụ mpụta nke niche cell stem (Mgbakwunye 13e, f, h, i). Otú ọ dị, curvature SAM yiri nke ahụ na genotypes abụọ (Foto mgbakwunye 13k, m, n, p). Anyị hụrụ mmụba yiri nke ahụ na gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant na-enweghị mgbanwe na curvature ma e jiri ya tụnyere ụdị anụ ọhịa (Fig. 13c, d, g, j, l, o, p). A na-emetụtakwa usoro nhazi nkewa nke cell na della quadruple mutant, mana ọ dị ntakịrị karịa na della monolithic mutant (Foto mgbakwunye. 12d-f). Mmetụta ọgwụ a, yana enweghị mmetụta na curvature, na-egosi na ọrụ RGL3 fọdụrụnụ na Della quadruple mutant na-egbochi mgbanwe na nhazi nkewa nke cell kpatara site na ọnwụ nke ọrụ DELLA yana mgbanwe na nkewa sel n'akụkụ na-eme na nzaghachi mgbanwe na ọrụ mgbaàmà GA kama mgbanwe na geometry SAM. Dị ka akọwara n'elu, onye na-akwalite CUC2 na-eme ka okwu IPR dị na SAM malite na P4 (Mgbakwunye 14a, b), na n'ụzọ dị iche, pCUC2 :: gai-1-VENUS SAM nwere mbelata nha ma ọ dị elu curvature (Mgbakwunye 14c-h). Mgbanwe a na pCUC2 :: gai-1-VENUS SAM morphology nwere ike ịkpata nkesa nrụgide n'ibu dị iche iche ma e jiri ya tụnyere ụdị anụ ọhịa, nke nrụgide dị elu na-amalite n'ebe dị anya site na SAM center47. N'aka nke ọzọ, mgbanwe ndị dị na pCUC2 :: gai-1-VENUS SAM morphology nwere ike ịpụta site na mgbanwe na akụrụngwa mpaghara nke transgene expression48 kpatara. N'okwu abụọ a, nke a nwere ike imebi mmetụta nke mgbanwe na mgbaàmà GA site n'ịbawanye ohere nke mkpụrụ ndụ ga-ekewa na ntụgharị okirikiri / ntụgharị, na-akọwa ihe anyị chọpụtara.
Ejikọtara ọnụ, data anyị na-akwado na mgbama GA dị elu na-arụ ọrụ na-arụ ọrụ n'akụkụ akụkụ nke ụgbọelu nkewa cell na IPR. Ha gosikwara na curvature meristem na-emetụtakwa nghazi ụgbọ elu nkewa cell na IPR.
Ntugharị ntụgharị nke ụgbọ elu nkewa na IPR, n'ihi ọrụ mgbaàmà GA dị elu, na-atụ aro na GA na-ahazi faịlụ radial cell na epidermis n'ime SAM iji kọwaa nzukọ cellular nke ga-emesị hụ na epidermal internode. N'ezie, faịlụ cell ndị dị otú ahụ na-ahụkarị na foto SAM nke della mutants zuru ụwa ọnụ (Fig 6b). Ya mere, iji nyochaa ọrụ mmepe nke oghere oghere nke GA na-egosi na SAM, anyị na-eji ihe ngosi oge na-agafe iji nyochaa nhazi nhazi nke sel na IPR na ụdị anụ ọhịa (Ler na Col-0), della mutants zuru ụwa ọnụ, na pCUC2 :: gai-1-VENUS transgenic osisi.
Anyị chọpụtara na qmRGA gosipụtara na ọrụ mgbaàmà GA na IPR mụbara site na P1/P2 wee gbakọọ na P4, ụkpụrụ a na-adịgidekwa ogologo oge (Fig. 4a-f na Fig. 8c-f, k). Iji nyochaa nhazi ohere nke mkpụrụ ndụ na IPR na mgbaàmà GA na-arịwanye elu, anyị na-akpọ mkpụrụ ndụ Ler IPR n'elu na n'akụkụ nke P4 dị ka mmepe mmepe ha nyochachara 34 h mgbe nchọpụta mbụ, ya bụ, ihe karịrị ugboro abụọ plastid, na-enye anyị ohere ịgbaso sel IPR n'oge mmepe primordium site na P1/P2 ruo P4. Anyị na-eji agba atọ dị iche iche: odo maka sel ndị ahụ jikọtara na primordium dị nso P4, akwụkwọ ndụ akwụkwọ ndụ maka ndị nọ na IPR, na-acha odo odo maka ndị na-ekere òkè na usoro abụọ ahụ (Fig 7a-c). Na t0 (0 h), 1-2 oyi akwa nke mkpụrụ ndụ IPR na-ahụ n'ihu P4 (Fig 7a). Dị ka a tụrụ anya, mgbe mkpụrụ ndụ ndị a kewara, ha mere ya tumadi site na ụgbọ elu nkewa transverse (Figs. 7a-c). Enwetara nsonaazụ yiri nke ahụ site na iji Col-0 SAM (na-elekwasị anya na P3, onye ókèala ya na-agbakọ n'otu aka ahụ na P4 na Ler), ọ bụ ezie na na genotype a, nke a na-emepụta n'ogige nke ifuru na-ezobe sel IPR ngwa ngwa (Fig 7g-i). Ya mere, usoro nkewa nke mkpụrụ ndụ IPR na-ahazi sel ndị ahụ n'ime ahịrị radial, dịka na internodes. Nhazi nke ahịrị radial na nhazi nke mkpụrụ ndụ IPR n'etiti akụkụ ahụ ndị na-esote na-egosi na mkpụrụ ndụ ndị a bụ progenitors internodal.
N'ebe a, anyị mepụtara biosensor ratiometric GA, qmRGA, nke na-enye ohere ịdepụta ọnụọgụ nke ọrụ mgbaàmà GA sitere na mkpokọta GA na GA ndị na-anabata mkpokọta ebe na-ebelata nnyonye anya na ụzọ mgbama endogenous, si otú ahụ na-enye ozi gbasara ọrụ GA na ọkwa cellular. Iji mezuo nke a, anyị rụrụ protein DELLA gbanwetụrụ, mRGA, nke tụfuru ikike ijikọ ndị mmekọ DELLA mana ọ ka na-enwe mmetụta maka proteolysis nke GA-ebutere. qmRGA na-anabata ma mgbanwe exogenous na endogenous na ọkwa GA, yana ihe nhụta ya dị ike na-enyere aka nleba anya mgbanwe mgbanwe oge na ọrụ mgbaàmà GA n'oge mmepe. qmRGA bụkwa ngwá ọrụ na-agbanwe agbanwe dị ka enwere ike ịmegharị ya na anụ ahụ dị iche iche site n'ịgbanwe onye nkwalite eji eme ihe maka okwu ya (ọ bụrụ na ọ dị mkpa), ma nyekwa ọdịdị echekwara nke ụzọ mgbaàmà GA na PFYRE motif gafee angiosperms, ọ ga-abụ na a ga-ebufe ya na ụdị ndị ọzọ22. N'ikwekọ na nke a, egosiri ngbanwe nhata na protein osikapa SLR1 DELLA (HYY497AAA) iji kwụsị ọrụ mmegide uto nke SLR1 ebe ọ bụ naanị ntakịrị ibelata mmebi mgbasa ozi GA, yiri mRGA23. N'ụzọ doro anya, nnyocha e mere n'oge na-adịbeghị anya na Arabidopsis gosiri na otu amino acid mutation na ngalaba PFYRE (S474L) gbanwere ọrụ ederede nke RGA na-emetụtaghị ikike ya na ndị mmekọ ihe odide ederede50. Ọ bụ ezie na ngbanwe a dị ezigbo nso na nnọchi amino acid 3 dị na mRGA, ọmụmụ anyị na-egosi na ngbanwe abụọ a na-agbanwe àgwà dị iche iche nke DELLA. Agbanyeghị na ọtụtụ ndị mmekọ ihe ederede na-ejikọta na ngalaba LHR1 na SAW nke DELLA26,51, ụfọdụ amino acid echekwara na ngalaba PFYRE nwere ike inye aka mee ka mmekọrịta ndị a kwụsie ike.
Mmepe internode bụ isi ihe dị n'ime ihe owuwu ihe ọkụkụ na nkwalite mkpụrụ. qmRGA kpughere ọrụ nrịbama GA dị elu na IPR internode progenitor sel. Site na ijikọ ọnụọgụ ọnụọgụ na mkpụrụ ndụ ihe nketa, anyị gosipụtara na ụkpụrụ mgbaàmà GA na-ebuli ụgbọ elu nkewa okirikiri/transverse cell na SAM epidermis, na-akpụzi nzukọ nkewa sel achọrọ maka mmepe internode. Achọpụtala ọtụtụ ndị na-ahụ maka nhazi ụgbọ elu nkewa cell n'oge mmepe52,53. Ọrụ anyị na-enye ihe atụ doro anya ka ọrụ mgbaàmà GA si ahazi oke ekwentị a. DELLA nwere ike imekọrịta ihe na ogige protein na-ebu ụzọ 41, yabụ akara GA nwere ike ịhazi nhazi nkewa ụgbọ elu cell site na imetụta cortical microtubule orientation ozugbo40,41,54,55. Anyị na-atụghị anya na-egosi na na SAM, njikọ nke ọrụ mgbaàmà GA dị elu abụghị sel elongation ma ọ bụ nkewa, kama ọ bụ naanị anisotropy na-eto eto, bụ nke na-adabere na mmetụta GA na ntụziaka nke cell nkewa na IPR. Agbanyeghị, anyị enweghị ike iwepu na mmetụta a nwekwara ike ịbụ nke na-apụtaghị ìhè, dịka ọmụmaatụ nke GA-ebutere mgbidi cell softening56. Mgbanwe na ihe ndị dị na mgbidi cell na-ebute nrụgide n'ibu57,58, nke nwekwara ike imetụta nhazi nke ụgbọelu nkewa cell site na imetụta nhazi nke cortical microtubules39,46,59. Mmetụta dị iche iche nke GA-akpata nrụgide n'ibu na nhazi kpọmkwem nke microtubule orientation site GA nwere ike itinye aka n'ịmepụta usoro nhazi nkewa nke cell na IPR iji kọwaa internodes, na ọmụmụ ihe ọzọ dị mkpa iji nwalee echiche a. N'otu aka ahụ, ọmụmụ ndị gara aga egosila mkpa DELLA-protein na-emekọrịta ihe na TCP14 na 15 na njikwa nke internode formation60,61 na ihe ndị a nwere ike ịmekọrịta ihe omume GA na BREVIPEDICELLUS (BP) na PENNYWISE (PNY), nke na-achịkwa mmepe nke internode ma gosipụta na ọ ga-emetụta GA signaling2,62. Nyere na DELLAs na-emekọ ihe na brassinosteroid, ethylene, jasmonic acid, na abscisic acid (ABA) ụzọ akara ụzọ63,64 na hormones ndị a nwere ike imetụta microtubule orientation65, mmetụta nke GA na nhazi nhazi nke cell nwekwara ike bụrụ onye na-edozi ya site na hormones ndị ọzọ.
Nnyocha cytological nke mbụ gosiri na ma mpaghara ime na mpụta nke Arabidopsis SAM chọrọ maka mmepe internode2,42. Eziokwu ahụ bụ na GA na-arụsi ọrụ ike na-achịkwa nkewa cell na anụ ahụ dị n'ime12 na-akwado ọrụ abụọ nke GA na-achịkwa meristem na internode size na SAM. A na-ahazikwa usoro nkewa mkpụrụ ndụ ntụzịaka nke ọma na anụ ahụ SAM dị n'ime, na ụkpụrụ a dị mkpa maka uto stem52. Ọ ga-adọrọ mmasị inyocha ma GA na-ekerekwa òkè n'ịhazi ụgbọ elu nkewa cell na nzukọ SAM dị n'ime, si otú a na-emekọrịta nkọwapụta na mmepe nke internodes n'ime SAM.
A na-akụ osisi na vitro na ala ma ọ bụ 1x Murashge-Skoog (MS) ọkara (Duchefa) gbakwunyere na 1% sucrose na 1% agar (Sigma) n'okpuru ọnọdụ ọkọlọtọ (16 h ìhè, 22 Celsius C), ma e wezụga n'ihi hypocotyl na mgbọrọgwụ ibu nnwale nke seedlings toro na vetikal efere n'okpuru ìhè mgbe niile na 22 Celsius C. Maka nnwale nitrate, a na-akụ osisi na ọkara MS gbanwere (ihe ọkụkụ bioWORLD) agbakwunyere na nitrate zuru oke (0 ma ọ bụ 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose na 1% A-agar (Sigma) n'okpuru ọnọdụ ogologo ụbọchị.
GID1a cDNA etinyere na pDONR221 ka ejikọtara ya na pDONR P4-P1R-pUBQ10 na pDONR P2R-P3-mCherry n'ime pB7m34GW iji mepụta pUBQ10::GID1a-mCherry. IDD2 DNA etinyere na pDONR221 ka eweghachiri n'ime pB7RWG266 iji mepụta p35S: IDD2-RFP. Iji wepụta pGID1b :: 2xmTQ2-GID1b, mpempe 3.9 kb dị elu nke mpaghara koodu GID1b yana mpempe 4.7 kb nwere GID1b cDNA (1.3 kb) na njedebe (3.4 kb) bu ụzọ gbasaa site na iji primers na mgbakwunye PNP4 na p Fisher Scientific) na pDONR P2R-P3 (Thermo Fisher Scientific), n'otu n'otu, na n'ikpeazụ ejikọta ya na pDONR221 2xmTQ268 n'ime pGreen 012567 vector lekwasịrị anya site na iji cloning Gateway. Iji mepụta pCUC2 :: LSSmOrange, usoro mgbasa ozi CUC2 (3229 bp elu nke ATG) sochiri usoro nzuzo nke nnukwu Stokes-shifted mOrange (LSSmOrange) 69 na akara ngosi N7 nke nuklia na NOS transcriptional terminator na-agbakọta n'ime pGreen vetegreen-3. usoro nchikota (Invitrogen). Ewebatara vector ọnụọgụ abụọ nke osisi n'ime Agrobacterium tumefaciens strain GV3101 wee weba ya n'ime akwụkwọ Nicotiana benthamiana site na usoro ntinye nke Agrobacterium yana banye Arabidopsis thaliana Col-0 site na usoro itinye ifuru, n'otu n'otu. pUBQ10::qmRGA pUBQ10::GID1a-mCherry na pCLV3::mCherry-NLS qmRGA kewapụrụ na ụmụ F3 na F1 nke obe dị iche iche, n'otu n'otu.
Emere ngwakọ RNA na ọnọdụ n'ihe dị ka 1 cm ogologo oge ịse tips72, nke anakọtara wee dozi ya ozugbo na ngwọta FAA (3.7% formaldehyde, 5% acetic acid, 50% ethanol) tupu oyi ruo 4 Celsius. Mgbe 2 × 15 min gwọchara oghere, a gbanwere ihe ndozi ahụ wee tinye ihe nlele n'otu abalị. GID1a, GID1b, GID1c, GAI, RGL1, RGL2, na RGL3 cDNAs na antisense probes na 3′-UTR ha ka ejikọtara site na iji primers egosiri na tebụl mgbakwunye 3 dị ka Rosier et al.73 kọwara. A na-enyocha nyocha nke akara Digoxigenin site na iji ọgwụ mgbochi digoxigenin (3000-fold dilution; Roche, nọmba katalọgụ: 11 093 274 910), na akụkụ nke nwere ntụpọ 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 250etrobt) diluphosphate (BCIP, 250-fold diluphosphate). 200-fold dilution) ngwọta.
Oge nzipu: Feb-10-2025