Shoot apical meristem (SAM) girma yana da mahimmanci ga tsarin gine-gine. Shuka hormonesgibberellins(GAs) suna taka muhimmiyar rawa wajen daidaita haɓakar shuka, amma ba a fahimci rawar da suke takawa a cikin SAM ba. Anan, mun haɓaka siginar biometric na GA ta hanyar injiniyan furotin DELLA don murkushe mahimman aikin sa na tsari a cikin amsawar rubutun GA yayin da muke kiyaye ɓarna akan ƙimar GA. Mun nuna cewa wannan na tushen biosensor na lalata daidai yana rikodin canje-canje a matakan GA da fahimtar salon salula yayin haɓakawa. Mun yi amfani da wannan biosensor don taswirar ayyukan siginar GA a cikin SAM. Mun nuna cewa manyan sigina na GA suna kasancewa galibi a cikin sel waɗanda ke tsakanin primordia na gabobin jiki, waɗanda ke gabatowar ƙwayoyin internode. Yin amfani da hanyoyin samun riba da asarar aiki, muna ƙara nuna cewa GA yana daidaita daidaitawar jirgin sama mai rarraba tantanin halitta, yana kafa ƙungiyar tantanin halitta ta internodes, ta haka yana haɓaka ƙayyadaddun ƙayyadaddun internode a cikin SAM.
Shooting apical meristem (SAM), wanda yake a koli mai harbi, yana ƙunshe da ɗimbin ɗimbin ƙwayoyin sel waɗanda ayyukansu ke haifar da gaɓoɓin gaɓoɓin gefe da kuɗaɗɗen kara a cikin tsari da juzu'i a tsawon rayuwar shukar. Kowane ɗayan waɗannan raka'o'in maimaitawa, ko nodes na shuka, sun haɗa da internodes da gabobin gefe a cikin nodes, da axillary meristems a cikin axils leaf1. Girma da tsarin nodes na shuka suna canzawa yayin haɓakawa. A cikin Arabidopsis, ana danne ci gaban internodal a lokacin matakin ciyayi, kuma axillary meristems sun kasance suna barci a cikin axils na ganyen rosette. A lokacin canzawa zuwa lokacin fure, SAM ya zama meristem inflorescence, yana haifar da internodes masu tsayi da buds na axillary, rassan rassan a cikin axils na ganyen cauline, kuma daga baya, furanni maras leaf2. Ko da yake mun sami ci gaba sosai wajen fahimtar hanyoyin da ke sarrafa farawar ganye, furanni, da rassa, amma kaɗan ba a san yadda internodes ke tasowa ba.
Fahimtar rarraba sararin samaniya na GAs zai taimaka wajen fahimtar ayyukan wadannan kwayoyin halitta a cikin kyallen takarda da kuma a matakai daban-daban na ci gaba. Ganin lalacewar haɗin RGA-GFP da aka bayyana a ƙarƙashin aikin mai tallata kansa yana ba da mahimman bayanai game da ƙa'idar jimlar matakan GA a cikin tushen15,16. Koyaya, maganganun RGA ya bambanta a cikin kyallen takarda17 kuma GA18 ke tsara shi. Don haka, bambance-bambancen mai talla na RGA na iya haifar da ƙirar haske da aka gani tare da RGA-GFP don haka wannan hanyar ba ta ƙididdigewa ba. Kwanan nan, bioactive fluorescein (Fl) -labeled GA19,20 ya bayyana tarin GA a cikin tushen endocortex da tsarin matakan salula ta hanyar jigilar GA. Kwanan nan, firikwensin GA FRET nlsGPS1 ya nuna cewa matakan GA sun daidaita tare da haɓakar tantanin halitta a cikin tushen, filaments, da hypocotyls21 masu girma. Koyaya, kamar yadda muka gani, tattarawar GA ba shine kawai ma'auni mai sarrafa ayyukan siginar GA ba, saboda ya dogara da tsarin fahimtar sarkar. Anan, ginawa akan fahimtarmu akan hanyoyin sigina na DELLA da GA, muna ba da rahoton haɓakawa da haɓakar haɓakar tushen abubuwan da ke haifar da ƙima don siginar GA. Don haɓaka wannan ƙididdiga na biosensor, mun yi amfani da mutant GA-sensitive RGA wanda aka haɗe zuwa furotin mai kyalli da kuma bayyana a ko'ina cikin kyallen takarda, da kuma furotin mai kyalli na GA. Mun nuna cewa mutant RGA furotin fusions ba sa tsoma baki tare da endogenous GA sigina lokacin da aka bayyana a ko'ina, kuma wannan biosensor na iya ƙididdige ayyukan sigina sakamakon duka shigarwar GA da sarrafa siginar GA ta hanyar na'urar ganowa tare da ƙudurin sararin samaniya. Mun yi amfani da wannan biosensor don taswirar rarraba lokaci-lokaci na ayyukan siginar GA da ƙididdige yadda GA ke daidaita halayen salula a cikin SAM epidermis. Mun nuna cewa GA yana daidaita daidaitawar jirgin sama na sel SAM da ke tsakanin gabobin jiki, ta haka ne ke bayyana ƙungiyar salula ta internode.
A ƙarshe, mun tambayi ko qmRGA zai iya ba da rahoton canje-canje a cikin matakan GA na ƙarshe ta amfani da hypocotyls masu girma. Mun nuna a baya cewa nitrate yana ƙarfafa haɓaka ta hanyar haɓaka haɗin GA kuma, bi da bi, lalata DELLA34. Saboda haka, mun lura cewa tsayin hypocotyl a pUBQ10 ::qmRGA da aka girma a ƙarƙashin wadataccen nitrate wadata (10 mM NO3-) ya fi tsayi fiye da haka a cikin tsire-tsire da aka girma a ƙarƙashin yanayin rashin nitrate (Ƙarin Hoto 6a). Daidai da amsawar girma, alamun GA sun kasance mafi girma a cikin hypocotyls na tsire-tsire da aka girma a ƙarƙashin yanayin 10 mM NO3- fiye da a cikin tsire-tsire da aka girma a cikin rashin nitrate (Ƙarin Hoton 6b, c). Don haka, qmRGA kuma yana ba da damar sa ido kan canje-canje a cikin siginar GA wanda aka haifar da canje-canje na ƙarshe a cikin tattarawar GA.
Don fahimtar ko aikin siginar GA da qmRGA ya gano ya dogara da tattara GA da kuma fahimtar GA, kamar yadda aka sa ran dangane da ƙirar firikwensin, mun bincika maganganun masu karɓar GID1 guda uku a cikin kyallen ciyayi da na haifuwa. A cikin tsire-tsire, layin mai ba da rahoto na GID1-GUS ya nuna cewa GID1a da c sun bayyana sosai a cikin cotyledons (Fig. 3a-c). Bugu da ƙari, dukkanin masu karɓa guda uku an bayyana su a cikin ganye, tushen primordia na gefe, tushen tukwici (sai dai tushen tushen GID1b), da tsarin jijiyoyin jini (Fig. 3a-c). A cikin inflorescence SAM, mun gano alamun GUS kawai don GID1b da 1c (Ƙarin Hoton 7a-c). A cikin yanayin haɓakawa ya tabbatar da waɗannan sifofin magana kuma ya ƙara nuna cewa GID1c an bayyana shi daidai a ƙananan matakan a cikin SAM, yayin da GID1b ya nuna mafi girma magana a gefen SAM (Ƙarin Hoton 7d-l). Haɗin fassarar pGID1b :: 2xmTQ2-GID1b kuma ya bayyana ƙimar ƙimar GID1b magana, daga ƙananan ko babu magana a tsakiyar SAM zuwa babban magana a kan iyakokin gabobin (Ƙarin Hoton 7m). Don haka, masu karɓar GID1 ba a rarraba su daidai gwargwado a ko'ina cikin kyallen takarda. A cikin gwaje-gwajen da suka biyo baya, mun kuma lura da cewa wuce gona da iri na GID1 (pUBQ10 :: GID1a-mCherry) ya karu da hankali na qmRGA a cikin hypocotyls zuwa aikace-aikacen GA na waje (Fig. 3d, e). Sabanin haka, hasken haske da aka auna ta qd17mRGA a cikin hypocotyl ba shi da hankali ga maganin GA3 (Fig. 3f, g). Don duka gwaje-gwajen, an bi da tsire-tsire tare da babban taro na GA (100 μM GA3) don tantance saurin halayen firikwensin, inda ikon ɗaure ga mai karɓar GID1 ya haɓaka ko ya ɓace. Tare, waɗannan sakamakon sun tabbatar da cewa qmRGA biosensor yana aiki da haɗin gwiwar aiki azaman firikwensin GA da GA, kuma yana ba da shawarar cewa bambance-bambancen mai karɓa na GID1 na iya daidaita haɓakar firikwensin.
Har zuwa yau, rarraba siginar GA a cikin SAM ya kasance ba a sani ba. Saboda haka, mun yi amfani da qmRGA-bayyana tsire-tsire da pCLV3 :: mCherry-NLS stem cell reporter35 don ƙididdige taswirar ƙididdige ƙididdiga na ayyukan siginar GA, mai da hankali kan Layer L1 (epidermis; Fig. 4a, b, duba Hanyoyi da Ƙarin Hanyoyin), tun da L1 yana taka muhimmiyar rawa wajen sarrafa SAM. Anan, pCLV3 :: mCherry-NLS magana ta ba da ƙayyadaddun ma'anar jumloli don nazarin rarraba sararin samaniya na ayyukan siginar GA37. Kodayake GA yana da mahimmanci ga ci gaban sassan sassan jiki na gefe4, mun lura cewa alamun GA sun kasance ƙananan a cikin primordium na fure (P) da suka fara daga mataki na P3 (Fig. 4a, b), yayin da matasan P1 da P2 primordiums suna da matsakaicin aiki irin na yankin tsakiya (Fig. 4a, b). An gano ayyukan siginar GA mafi girma a kan iyakokin primordium gabobin, farawa a P1 / P2 (a gefen iyakar) da kuma tsalle a P4, da kuma a cikin dukkanin kwayoyin halitta na yankin da ke tsakanin primordia (Fig. 4a, b da Ƙarin Hoto 8a, b). Wannan aikin siginar siginar GA mafi girma an lura da shi ba kawai a cikin epidermis ba har ma a cikin L2 da L3 na sama (Ƙarin Hoton 8b). Misalin siginar GA da aka gano a cikin SAM ta amfani da qmRGA shima ya kasance baya canzawa cikin lokaci (Ƙarin Hoton 8c–f, k). Ko da yake an rushe ginin qd17mRGA cikin tsari a cikin SAM na tsire-tsire na T3 daga layukan masu zaman kansu guda biyar waɗanda muka siffanta dalla-dalla, mun sami damar yin la'akari da ƙirar haske da aka samu tare da ginin pRPS5a:: VENUS-2A-TagBFP (Ƙarin Hoton 8g-j, l). A cikin wannan layin sarrafawa, kawai ƙananan canje-canje a cikin rabo mai haske an gano su a cikin SAM, amma a cikin cibiyar SAM mun lura da raguwa a fili da rashin tsammani a cikin VENUS da ke hade da TagBFP. Wannan yana tabbatar da cewa siginar siginar da qmRGA ya lura yana nuna lalacewar dogara ga GA na mRGA-VENUS, amma kuma yana nuna cewa qmRGA na iya ƙima aikin siginar GA a cikin cibiyar meristem. A taƙaice, sakamakon mu yana nuna alamar sigina ta GA wanda da farko ke nuna rarrabawar primordia. Wannan rarraba na yanki na tsakiya (IPR) shine saboda a hankali kafa babban aikin siginar GA tsakanin masu tasowa da kuma yankin tsakiya, yayin da a lokaci guda GA ayyukan siginar a cikin primordium ya ragu (Fig. 4c, d).
Rarraba masu karɓa na GID1b da GID1c (duba sama) yana nuna cewa bambance-bambancen masu karɓa na GA suna taimakawa wajen tsara tsarin siginar GA a cikin SAM. Mun yi mamakin ko bambancin tarin GA na iya shiga ciki. Don bincika wannan yuwuwar, mun yi amfani da nlsGPS1 GA FRET sensor21. An gano ƙara yawan kunna kunnawa a cikin SAM na nlsGPS1 da aka bi da shi tare da 10 μM GA4 + 7 don 100 min (Ƙarin Hoton 9a-e), yana nuna cewa nlsGPS1 yana amsa canje-canje a cikin taro na GA a cikin SAM, kamar yadda yake a cikin tushen21. Rarraba sararin samaniya na nlsGPS1 mitar kunnawa ya bayyana ƙananan matakan GA kaɗan a cikin manyan yadudduka na SAM, amma ya nuna cewa an ɗaukaka su a tsakiya da kuma iyakokin SAM (Fig. 4e da Ƙarin Hoto 9a, c). Wannan yana nuna cewa GA kuma ana rarraba shi a cikin SAM tare da ƙirar sararin samaniya kwatankwacin wanda qmRGA ya bayyana. A matsayin madaidaicin hanya, mun kuma bi da SAM tare da fluorescent GA (GA3-, GA4-, GA7-Fl) ko Fl kadai a matsayin iko mara kyau. An rarraba siginar Fl a ko'ina cikin SAM, ciki har da yankin tsakiya da primordium, duk da haka a ƙananan ƙarfi (Fig. 4j da Ƙarin Hoto 10d). Sabanin haka, dukkanin GA-Fl guda uku sun tara musamman a cikin iyakokin farko da kuma digiri daban-daban a cikin sauran IPR, tare da GA7-Fl yana tarawa a cikin mafi girma a cikin IPR (Fig. 4k da Ƙarin Hoto 10a, b). Ƙididdigar ƙimar ƙarfin haske ya nuna cewa ƙimar IPR zuwa wanda ba IPR ba ya fi girma a cikin GA-Fl da aka yi wa SAM idan aka kwatanta da Fl-treated SAM (Fig. 4l da Ƙarin Fig. 10c). Tare, waɗannan sakamakon suna nuna cewa GA yana kasancewa a cikin mafi girma a cikin ƙwayoyin IPR waɗanda ke kusa da iyakar gabobin. Wannan yana nuna cewa tsarin aikin siginar SAM GA yana haifar da nau'ikan nau'ikan nau'ikan nau'ikan masu karɓa na GA da bambancin tarin GA a cikin ƙwayoyin IPR kusa da iyakokin gabobin. Don haka, bincikenmu ya nuna alamar yanayin sararin samaniya wanda ba zato ba tsammani na siginar GA, tare da ƙananan aiki a tsakiya da primordium na SAM da ayyuka mafi girma a cikin IPR a cikin yanki na yanki.
Don fahimtar rawar banbancin ayyukan siginar GA a cikin SAM, mun bincika alaƙar tsakanin ayyukan siginar GA, faɗaɗa tantanin halitta, da rarraba tantanin halitta ta amfani da hoto na lokaci-lokaci na SAM qmRGA pCLV3 :: mCherry-NLS. Ganin rawar GA a cikin ƙa'idodin girma, ana tsammanin kyakkyawar alaƙa tare da sigogin faɗaɗa tantanin halitta. Sabili da haka, mun fara kwatanta taswirar ayyukan siginar GA tare da taswira na ƙimar girma na sel (a matsayin wakili don ƙarfin haɓakar tantanin halitta don sel da aka ba da sel mata a rarraba) kuma tare da taswirorin anisotropy girma, wanda ke auna jagorar faɗaɗa tantanin halitta (kuma ana amfani da shi anan don tantanin halitta da aka ba da kuma ga sel ɗiya a rarraba; Hoto 5a,b, duba Hanyoyi da Ƙarin Hanyoyi). Taswirorin mu na ƙimar girman girman kwayar halitta ta SAM sun yi daidai da abubuwan lura na baya38,39, tare da ƙarancin girma a kan iyaka da ƙimar girma mafi girma a cikin furanni masu tasowa (Fig. 5a). Binciken babban ɓangaren (PCA) ya nuna cewa aikin siginar GA yana da alaƙa mara kyau tare da haɓakar haɓakar sel (Hoto 5c). Mun kuma nuna cewa manyan gatari na bambancin, ciki har da shigarwar siginar GA da ƙarfin girma, sun kasance daidaitattun zuwa jagorancin da aka ƙaddara ta babban CLV3, yana tabbatar da cire ƙwayoyin sel daga cibiyar SAM a cikin sauran nazarin. Binciken daidaitawar Spearman ya tabbatar da sakamakon PCA (Hoto na 5d), yana nuna cewa siginonin GA mafi girma a cikin IPR bai haifar da haɓakar tantanin halitta ba. Duk da haka, bincike na haɗin gwiwa ya nuna wani ɗan gajeren dangantaka mai kyau tsakanin ayyukan siginar GA da haɓakar anisotropy (Hoto 5c, d), yana nuna cewa mafi girma ga siginar GA a cikin IPR yana rinjayar jagorancin ci gaban tantanin halitta da yiwuwar matsayi na jirgin saman rarraba tantanin halitta.
a, b Taswirorin zafi na ma'anar girma (a) da girma anisotropy (b) a cikin SAM sun daidaita sama da tsire-tsire bakwai masu zaman kansu (an yi amfani da su azaman proxies don ƙarfi da jagorar faɗaɗa tantanin halitta, bi da bi). c Binciken PCA ya haɗa da masu canji masu zuwa: GA siginar, girman girman girma, anisotropy girma, da kuma CLV3 magana. Bangaren PCA na 1 ya kasance yana da alaƙa da rashin daidaituwa tare da ƙarfin girma na saman kuma yana da alaƙa da alaƙa da siginar GA. Bangaren PCA 2 ya kasance yana da alaƙa da inganci da haɓakar anisotropy na saman kuma yana da alaƙa mara kyau tare da magana ta CLV3. Kashi ɗari suna wakiltar bambancin da kowane bangare ya bayyana. d Spearman daidaitaccen bincike tsakanin siginar GA, girman girman girma, da anisotropy girma a saman sikelin nama ban da CZ. Lambar da ke hannun dama ita ce ƙimar Spearman rho tsakanin masu canji biyu. Asterisks suna nuna lokuta inda haɗin kai/mummuna ke da mahimmanci. e 3D hangen nesa na Col-0 SAM L1 Kwayoyin ta microscopy confocal. Sabbin ganuwar tantanin halitta da aka kafa a cikin SAM (amma ba primordium ba) a cikin sa'o'i 10 suna da launi bisa ga ƙimar kusurwarsu. Ana nuna sandar launi a ƙananan kusurwar dama. Saitin yana nuna hoton 3D mai dacewa a 0 h. An maimaita gwajin sau biyu tare da sakamako iri ɗaya. f Shirye-shiryen Akwatin suna nuna ƙimar rabon tantanin halitta a cikin IPR da waɗanda ba IPR Col-0 SAM ba (n = 10 tsire-tsire masu zaman kansu). Layin tsakiya yana nuna tsaka-tsaki, kuma iyakokin akwatin suna nuna kashi 25 da 75. Whiskers suna nuna mafi ƙanƙanta da matsakaicin ƙimar da aka ƙayyade tare da software R. An sami ƙimar P tare da t-gwajin Welch mai wutsiya biyu. g, h Tsarin tsari yana nuna (g) yadda ake auna kusurwar sabon bangon tantanin halitta (magenta) dangane da jagorar radial daga tsakiyar SAM (layin farar dige-dige) (ƙididdigar kusurwa kawai, watau, 0-90 °, ana la'akari), da (h) kewayawa / gefe da kwatance radial a cikin meristem. i Mitar histograms na tsarin rarraba jirgin sama a fadin SAM (duhu blue), IPR (matsakaici blue), da kuma wadanda ba IPR (mai haske blue), bi da bi. An samo ƙimar P ta hanyar gwajin Kolmogorov-Smirnov mai tsayi biyu. An maimaita gwajin sau biyu tare da sakamako iri ɗaya. j Mitar histogram na rarrabuwar jirgin sama na IPR a kusa da P3 (kore mai haske), P4 (matsakaicin kore), da P5 (koren duhu), bi da bi. An samo ƙimar P ta hanyar gwajin Kolmogorov-Smirnov mai tsayi biyu. An maimaita gwajin sau biyu tare da sakamako iri ɗaya.
Sabili da haka, mun binciko ma'amala tsakanin siginar GA da ayyukan rarraba tantanin halitta ta hanyar gano sabbin ganuwar tantanin halitta yayin gwajin (Fig. 5e). Wannan hanya ta ba mu damar auna mita da alkiblar rarraba tantanin halitta. Abin mamaki, mun gano cewa yawancin sassan sel a cikin IPR da sauran SAM (marasa IPR, Fig. 5f) sun kasance iri ɗaya, yana nuna cewa bambance-bambance a cikin siginar GA tsakanin IPR da sel marasa IPR ba su da tasiri sosai akan rarraba tantanin halitta. Wannan, da ingantacciyar alaƙa tsakanin siginar GA da haɓakar anisotropy, ya sa mu yi la'akari da ko ayyukan siginar GA na iya yin tasiri kan daidaitawar jirgin sama na sel. Mun auna ma'auni na sabon bangon tantanin halitta a matsayin kusurwa mai mahimmanci dangane da radial axis da ke haɗa cibiyar meristem da tsakiyar sabon bangon tantanin halitta (Fig. 5e-i) kuma mun lura da yanayin da ya dace don sel don rarraba a kusurwoyi kusa da 90 ° dangane da radial axis, tare da mafi girman mitoci da aka lura a 70.0-82%) da 23 ° 70-82% (23 °) (Fig. 5e, i), daidai da sassan sel a cikin kewayawa / juyawa (Fig. 5h). Don bincika gudummawar siginar GA zuwa wannan halayen rarraba tantanin halitta, mun bincika sigogin rarraba tantanin halitta a cikin IPR da waɗanda ba IPR daban (Fig. 5i). Mun lura cewa rarraba kusurwar rabe-rabe a cikin sel IPR ya bambanta da wannan a cikin sel marasa IPR ko a cikin sel a cikin dukkan SAM, tare da ƙwayoyin IPR suna nuna mafi girman rabo na sassan layi na gefe / madauwari, watau 70-80 ° da 80-90 ° (33.86% da 30.71%, bi da bi, madaidaicin rabo) (Fig.5). Don haka, abubuwan da muka lura sun nuna wata ƙungiya tsakanin babban siginar GA da madaidaicin rabewar jirgin tantanin halitta kusa da jagorar kewayawa, kama da alaƙa tsakanin ayyukan siginar GA da haɓakar anisotropy (Fig. 5c, d). Don ci gaba da tabbatar da kiyaye sararin samaniya na wannan ƙungiya, mun auna ma'auni na rarraba jirgin sama a cikin kwayoyin IPR da ke kewaye da primordium farawa daga P3, tun lokacin da aka gano aikin siginar GA mafi girma a wannan yanki daga P4 (Fig. 4). Matsakaicin rabe-raben IPR a kusa da P3 da P4 ba su nuna bambance-bambancen ƙididdiga ba, ko da yake an sami karuwar adadin sassan sassan layi a cikin IPR a kusa da P4 (Fig. 5j). Duk da haka, a cikin sel IPR da ke kusa da P5, bambanci a cikin daidaitawa na jirgin sama mai rarraba tantanin halitta ya zama mahimmancin ƙididdiga, tare da karuwa mai girma a cikin mitar sassan sassan jiki (Fig. 5j). Tare, waɗannan sakamakon sun ba da shawarar cewa siginar GA na iya sarrafa daidaitawar sassan sel a cikin SAM, wanda ya yi daidai da rahotannin da suka gabata40,41 cewa babban siginar GA na iya haifar da daidaitawar gefe na sassan tantanin halitta a cikin IPR.
An annabta cewa sel a cikin IPR ba za a haɗa su cikin primordia ba amma a cikin internodes2,42,43. Madaidaicin juzu'i na sassan tantanin halitta a cikin IPR na iya haifar da tsari na yau da kullun na layuka masu tsayi na sel epidermal a cikin internodes. Abubuwan lura da mu da aka bayyana a sama suna nuna cewa alama ta GA mai yiwuwa tana taka rawa a cikin wannan tsari ta hanyar daidaita alkiblar rarraba tantanin halitta.
Asarar ayyuka na ƙwayoyin halittar DELLA da yawa yana haifar da amsawar GA mai ƙima, kuma ana iya amfani da mutantan della don gwada wannan hasashe44. Mun fara bincikar sifofin magana na kwayoyin halittar DELLA guda biyar a cikin SAM. Haɗin rubutun na GUS line45 ya bayyana cewa GAI, RGA, RGL1, da RGL2 (zuwa ƙarami) an bayyana su a cikin SAM (Ƙarin Hoton 11a-d). A cikin situ hybridization ya kara nuna cewa GAI mRNA ya taru musamman a cikin primordia da furanni masu tasowa (Ƙarin Hoto 11e). RGL1 da RGL3 mRNA an gano su a ko'ina cikin rufin SAM kuma a cikin tsofaffin furanni, yayin da RGL2 mRNA ya fi yawa a yankin iyaka (Ƙarin Hoton 11f–h). Hoton Confocal na pRGL3 :: RGL3-GFP SAM ya tabbatar da maganganun da aka lura da shi a cikin haɓakar yanayi kuma ya nuna cewa furotin RGL3 yana tarawa a tsakiyar ɓangaren SAM (Ƙarin Hoton 11i). Yin amfani da layin pRGA :: GFP-RGA, mun kuma gano cewa sunadaran RGA suna tarawa a cikin SAM, amma yawansa yana raguwa a kan iyakar da ya fara daga P4 (Ƙarin Hoton 11j). Musamman ma, sifofin magana na RGL3 da RGA sun yi daidai da ayyukan siginar GA mafi girma a cikin IPR, kamar yadda qmRGA ya gano (Fig. 4). Bugu da ƙari, waɗannan bayanan sun nuna cewa duk DELLA an bayyana su a cikin SAM kuma cewa maganganunsu tare ya haɗa da dukan SAM.
Mu na gaba mun bincika sigogin rarraba tantanin halitta a cikin nau'in daji na SAM (Ler, sarrafawa) da gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (duniya) mutants (Fig. 6a, b). Abin sha'awa, mun lura da wani canji mai mahimmanci a cikin rarraba mitoci na sassan sassan cell a cikin della mutant SAM na duniya idan aka kwatanta da nau'in daji (Fig. 6c). Wannan canji a cikin mutant na duniya na della ya kasance saboda karuwa a cikin mita 80-90 ° (34.71% vs. 24.55%) kuma, zuwa ƙarami, 70-80 ° kusurwa (23.78% vs. 20.18%), watau, daidai da sassan sassan cell (Fig. 6c). Yawan rabe-raben da ba a juyewa ba (0-60°) kuma ya kasance ƙasa da ƙasa a cikin mutant na duniya na della (Fig. 6c). Matsakaicin rabe-raben sel masu juyawa ya karu sosai a cikin SAM na della mutant na duniya (Fig. 6b). Yawan rabe-raben sel masu juyayi a cikin IPR kuma ya kasance mafi girma a cikin mutantan della na duniya idan aka kwatanta da nau'in daji (Fig. 6d). A waje da yankin IPR, nau'in daji yana da mafi daidaituwa na rarraba kusurwoyi na sel, yayin da della mutant na duniya ya fi son sassan tangential kamar IPR (Fig. 6e). Mun kuma ƙididdige daidaitawar sassan tantanin halitta a cikin SAM na ga2 oxidase (ga2ox) mutants quintuple (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, da ga2ox6-2), yanayin mutant na GA-mara aiki wanda GA ke tarawa. Daidai da haɓakar matakan GA, SAM na quintuple ga2ox mutant inflorescence ya fi girma fiye da na Col-0 (Ƙarin Hoton 12a, b), kuma idan aka kwatanta da Col-0, quintuple ga2ox SAM ya nuna bambanci daban-daban na rarraba kusurwoyi na cell, tare da mitar kusurwa yana karuwa daga 500 ° zuwa 9. Hoto na 12a-c). Don haka, muna nuna cewa ƙaddamar da siginar GA da tarin GA yana haifar da rarrabuwar sel ta gefe a cikin IPR da sauran SAM.
a, b 3D hangen nesa na L1 Layer na PI-stained Ler (a) da duniya della mutant (b) SAM ta amfani da microscopy confocal. Sabbin ganuwar tantanin halitta da aka kafa a cikin SAM (amma ba primordium ba) sama da tsawon awanni 10 ana nuna su kuma an yi musu launi gwargwadon ƙimar kusurwarsu. Saitin yana nuna SAM a 0 h. Ana nuna sandar launi a cikin ƙananan kusurwar dama. Kibiya a cikin (b) tana nuna misali na fayilolin salula masu daidaitawa a cikin mutantan della na duniya. An maimaita gwajin sau biyu tare da sakamako iri ɗaya. ce kwatankwacin mitar rarraba rabe-raben jirgin sama a cikin dukkan SAM (d), IPR (e), da wadanda ba IPR (f) tsakanin Ler da della na duniya ba. An samo ƙimar P ta amfani da gwajin Kolmogorov-Smirnov mai tsayi biyu. f, g 3D na gani na hotuna masu ɓoye na PI-stained SAM na Col-0 (i) da pCUC2 :: gai-1-VENUS (j) tsire-tsire masu canzawa. Panels (a, b) suna nuna sabon bangon tantanin halitta (amma ba primordia) da aka kafa a cikin SAM cikin sa'o'i 10 ba. An maimaita gwajin sau biyu tare da sakamako iri ɗaya. h–j Kwatanta mitar rarraba hanyoyin jirgin sama na rabon tantanin halitta dake cikin dukkan SAM (h), IPR (i) da wadanda ba IPR (j) tsakanin Col-0 da pCUC2 :: gai-1-VENUS shuke-shuke. An samo ƙimar P ta amfani da gwajin Kolmogorov-Smirnov mai wutsiya biyu.
Mu na gaba mun gwada tasirin hana siginar GA musamman a cikin IPR. Don wannan karshen, mun yi amfani da cotyledon Cup 2 (CUC2) mai tallata don fitar da furci na babban furotin gai-1 da aka haɗa zuwa VENUS (a cikin pCUC2 :: gai-1-VENUS line). A cikin nau'in SAM na daji, mai tallata CUC2 yana fitar da maganganun mafi yawan IPRs a cikin SAM, gami da sel kan iyaka, daga P4 gaba, kuma an lura da takamaiman takamaiman magana a cikin pCUC2 :: gai-1-VENUS shuke-shuke (duba ƙasa). Rarraba kusurwoyi na sel a fadin SAM ko IPR na pCUC2 :: gai-1-VENUS tsire-tsire ba su bambanta da na nau'in daji ba, ko da yake ba zato ba tsammani mun gano cewa kwayoyin halitta ba tare da IPR ba a cikin waɗannan tsire-tsire sun raba a mafi girma na 80-90 ° (Fig. 6f-j).
An ba da shawarar cewa alkiblar rarraba tantanin halitta ya dogara ne akan juzu'i na SAM, musamman damuwa mai ƙarfi da ke haifar da curvature nama46. Saboda haka mun tambayi ko an canza siffar SAM a cikin della global mutant da pCUC2 :: gai-1-VENUS shuke-shuke. Kamar yadda aka ruwaito a baya12, girman della mutant SAM ya fi girma fiye da na nau'in daji (Ƙarin Hoton 13a, b, d). A cikin yanayin haɓakar CLV3 da STM RNA sun tabbatar da haɓakar meristem a cikin mutantan della kuma ya ƙara nuna haɓakar haɓakar ƙwayar ƙwayar ƙwayar ƙwayar cuta (Ƙarin Hoto 13e, f, h, i). Koyaya, curvature SAM ya kasance iri ɗaya a cikin nau'ikan genotypes (Ƙarin Hoton 13k, m, n, p). Mun lura da irin wannan karuwa a cikin gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant ba tare da canji a cikin curvature ba idan aka kwatanta da nau'in daji (Ƙarin Hoton 13c, d, g, j, l, o, p). An kuma shafi mitar daidaitawar rarraba tantanin halitta a cikin mutantan della quadruple, amma zuwa ƙarami fiye da na della monolithic mutant (Ƙarin Hoton 12d-f). Wannan sakamako na sashi, tare da rashin tasiri akan curvature, yana nuna cewa ragowar ayyukan RGL3 a cikin Della quadruple mutant mutant yana iyakance canje-canje a cikin daidaitawar rarraba tantanin halitta wanda ya haifar da asarar ayyukan DELLA da kuma cewa canje-canje a cikin sassan sel na gefe suna faruwa a cikin martani ga canje-canje a cikin ayyukan siginar GA maimakon canje-canje a cikin lissafin SAM. Kamar yadda aka bayyana a sama, mai tallata CUC2 yana fitar da maganganun IPR a cikin SAM farawa a P4 (Ƙarin Hoton 14a, b), kuma da bambanci, pCUC2 :: gai-1-VENUS SAM yana da raguwar girman girman amma mafi girma (Ƙarin Hotuna 14c-h). Wannan canji a cikin pCUC2 :: gai-1-VENUS SAM ilimin halittar jiki na iya haifar da rarraba daban-daban na damuwa na inji idan aka kwatanta da nau'in daji, wanda babban damuwa na kewaye ya fara a ɗan gajeren nesa daga cibiyar SAM47. A madadin, canje-canje a cikin pCUC2 :: gai-1-VENUS SAM ilimin halittar jiki na iya haifar da canje-canje a cikin kayan aikin injiniya na yanki wanda transgene expression48 ya haifar. A cikin duka biyun, wannan na iya ɗan daidaita tasirin canje-canje a cikin siginar GA ta hanyar ƙara yuwuwar cewa sel za su rarraba a cikin madaidaicin kewayawa / juzu'i, yana bayyana abubuwan da muka lura.
Haɗe tare, bayananmu sun tabbatar da cewa siginar GA mafi girma yana taka rawa sosai a cikin karkatar da keɓaɓɓiyar jirgin tantanin halitta a cikin IPR. Har ila yau, sun nuna cewa curvature na meristem shima yana tasiri akan yanayin jirgin rabon tantanin halitta a cikin IPR.
Matsakaicin juzu'i na rarraba jirgin sama a cikin IPR, saboda babban aikin siginar GA, yana nuna cewa GA ya riga ya tsara fayil ɗin radial cell a cikin epidermis a cikin SAM don ayyana ƙungiyar salula wanda daga baya za a samu a cikin internode na epidermal. Lallai, ana yawan ganin irin waɗannan fayilolin tantanin halitta a cikin hotunan SAM na della mutants na duniya (Fig. 6b). Don haka, don kara nazarin aikin ci gaba na tsarin sararin samaniya na GA siginar a cikin SAM, mun yi amfani da hotunan lokaci-lokaci don nazarin tsarin sararin samaniya na sel a cikin IPR a cikin nau'in daji (Ler da Col-0), della mutants na duniya, da pCUC2 :: gai-1-VENUS transgenic shuke-shuke.
Mun gano cewa qmRGA ya nuna cewa aikin siginar GA a cikin IPR ya karu daga P1/P2 kuma ya yi girma a P4, kuma wannan tsarin ya kasance akai-akai a tsawon lokaci (Fig. 4a-f da Ƙarin Hoto 8c-f, k). Don nazarin tsarin sararin samaniya na sel a cikin IPR tare da ƙarar siginar GA, mun lakafta Ler IPR Kwayoyin a sama da kuma zuwa ga bangarorin P4 bisa ga ci gaban da suka yi nazari 34 h bayan lura na farko, watau, fiye da sau biyu plastid sau, ƙyale mu mu bi IPR Kwayoyin a lokacin primordium ci gaban daga P1/P2 zuwa P4. Mun yi amfani da launuka daban-daban guda uku: rawaya ga waɗancan sel waɗanda aka haɗa a cikin primordium kusa da P4, kore ga waɗanda ke cikin IPR, da shunayya ga waɗanda suka shiga cikin matakai biyu (Fig. 7a-c). A t0 (0 h), 1-2 yadudduka na kwayoyin IPR sun kasance a gaban P4 (Fig. 7a). Kamar yadda aka zata, lokacin da waɗannan sel suka rarraba, sun yi haka ne ta hanyar jirgin sama mai jujjuyawa (Figs. 7a-c). An samu irin wannan sakamakon ta amfani da Col-0 SAM (mai da hankali kan P3, wanda iyakarsa ta ninka daidai da P4 a cikin Ler), kodayake a cikin wannan genotype ɗin da aka kafa a kan iyakar furen ya ɓoye ƙwayoyin IPR da sauri (Fig. 7g-i). Don haka, tsarin rarrabuwa na sel IPR sun riga sun tsara sel zuwa layuka na radial, kamar a cikin internodes. Ƙungiya na layuka na radial da ƙaddamar da ƙwayoyin IPR tsakanin gabobin da suka biyo baya sun nuna cewa waɗannan kwayoyin halitta ne na cikin mahaifa.
Anan, mun haɓaka siginar siginar rabometric GA biosensor, qmRGA, wanda ke ba da damar yin taswirar ƙididdige ayyukan siginar GA wanda ya haifar da haɗakar GA da ƙididdigar masu karɓar GA yayin da rage tsangwama tare da hanyoyin sigina na ƙarshe, don haka samar da bayanai kan aikin GA a matakin salula. Don wannan ƙarshen, mun gina ingantaccen furotin DELLA, mRGA, wanda ya rasa ikon ɗaure abokan hulɗar DELLA amma ya kasance mai kula da sinadari mai haifar da GA. qmRGA yana ba da amsa ga canje-canje na ban mamaki da na ƙarshe a cikin matakan GA, kuma ƙayyadaddun abubuwan ganowa suna ba da damar kimanta canje-canjen yanayi a cikin ayyukan siginar GA yayin haɓakawa. qmRGA kuma kayan aiki ne mai sauƙi kamar yadda za'a iya daidaita shi zuwa kyallen takarda daban-daban ta hanyar canza mai gabatarwa da aka yi amfani da shi don magana (idan ya cancanta), kuma an ba da yanayin kiyaye yanayin hanyar siginar GA da kuma PFYRE motif a fadin angiosperms, yana yiwuwa a iya canjawa wuri zuwa wasu nau'in22. Daidai da wannan, an kuma nuna madaidaicin maye gurbi a cikin furotin SLR1 DELLA shinkafa (HYY497AAA) don murkushe ayyukan danne haɓakar haɓakar SLR1 yayin da ɗan rage raguwar ɓarna na tsaka-tsakin GA, kama da mRGA23. Musamman, binciken da aka yi kwanan nan a Arabidopsis ya nuna cewa maye gurbin amino acid guda ɗaya a cikin yankin PFYRE (S474L) ya canza aikin rubutun RGA ba tare da ya shafi ikonsa na yin hulɗa tare da abokan haɗin gwiwa ba50. Kodayake wannan maye gurbi yana kusa da sauye-sauyen amino acid guda 3 da ke cikin mRGA, bincikenmu ya nuna cewa waɗannan maye gurbi guda biyu suna canza halaye na musamman na DELLA. Ko da yake yawancin abokan aikin rubutawa suna ɗaure ga yankunan LHR1 da SAW na DELLA26,51, wasu amino acid da aka adana a cikin yankin PFYRE na iya taimakawa wajen daidaita waɗannan hulɗar.
Ci gaban Internode shine mabuɗin sifa a cikin gine-ginen tsire-tsire da haɓaka yawan amfanin ƙasa. qmRGA ya bayyana mafi girman ayyukan siginar GA a cikin IPR internode progenitor sel. Ta hanyar haɗa ƙididdiga masu ƙididdigewa da kwayoyin halitta, mun nuna cewa alamun sigina na GA sun fi ƙarfin jiragen madauwari/mai juyawa a cikin epidermis na SAM, suna tsara ƙungiyar rarraba tantanin halitta da ake buƙata don ci gaban internode. An gano da yawa masu kula da daidaitawar jirgin sama na rabon tantanin halitta yayin haɓaka52,53. Ayyukanmu yana ba da cikakken misali na yadda ayyukan siginar GA ke sarrafa wannan siginar salula. DELLA na iya yin mu'amala tare da rukunin furotin na farko41, don haka siginar GA na iya daidaita yanayin rarrabuwar jirgin tantanin halitta ta hanyar yin tasiri kai tsaye na cortical microtubule orientation40,41,54,55. Mun nuna ba zato ba tsammani a cikin SAM, daidaitaccen aikin siginar GA mafi girma ba shine haɓakar cell ko rarraba ba, amma anisotropy girma ne kawai, wanda ya dace da tasirin GA kai tsaye a kan jagorancin rarraba tantanin halitta a cikin IPR. Duk da haka, ba za mu iya ware cewa wannan tasirin zai iya zama kaikaice ba, misali mai shiga tsakani ta GA-induced cell bango softening56. Canje-canje a cikin kaddarorin bangon tantanin halitta suna haifar da damuwa na inji57,58, wanda kuma zai iya yin tasiri kan daidaitawar jirgin sama na rarraba tantanin halitta ta hanyar tasiri na cortical microtubules39,46,59. Haɗuwa da abubuwan da ke haifar da damuwa na inji da GA da ke haifar da kai tsaye na daidaitawar microtubule ta GA na iya shiga cikin samar da ƙayyadaddun tsari na daidaitawar rarraba tantanin halitta a cikin IPR don ayyana internodes, kuma ana buƙatar ƙarin karatu don gwada wannan ra'ayin. Hakazalika, binciken da aka yi a baya ya nuna muhimmancin DELLA-interacting proteins TCP14 da 15 a cikin kula da tsarin haɗin gwiwar internode60,61 kuma waɗannan abubuwan zasu iya daidaita aikin GA tare da BREVIPEDICELLUS (BP) da PENNYWISE (PNY), wanda ke tsara ci gaban internode kuma an nuna su da tasiri ga siginar GA2,62. Ganin cewa DELLAs suna hulɗa tare da brassinosteroid, ethylene, jasmonic acid, da abscisic acid (ABA) hanyoyin siginar sigina63,64 da kuma cewa waɗannan hormones na iya rinjayar microtubule orientation65, tasirin GA akan daidaitawar rarraba tantanin halitta na iya zama tsaka-tsaki ta wasu hormones.
Nazarin cytological na farko ya nuna cewa duka yankuna na ciki da na waje na Arabidopsis SAM ana buƙatar ci gaban internode2,42. Gaskiyar cewa GA yana daidaita rarraba tantanin halitta a cikin tsokoki na ciki12 yana goyan bayan aikin dual na GA wajen daidaita girman meristem da internode a cikin SAM. Hakanan ana daidaita tsarin rarraba tantanin halitta a cikin nama na SAM na ciki, kuma wannan ƙa'idar tana da mahimmanci don haɓakar kara52. Zai zama mai ban sha'awa don bincika ko GA kuma yana taka rawa wajen daidaita jirgin rarrabuwar tantanin halitta a cikin ƙungiyar SAM ta ciki, ta haka yana daidaita ƙayyadaddun ƙayyadaddun bayanai da haɓaka internodes a cikin SAM.
An shuka tsire-tsire a cikin ƙasa ko 1x Murashge-Skoog (MS) matsakaici (Duchefa) tare da 1% sucrose da 1% agar (Sigma) a ƙarƙashin yanayin daidaitaccen yanayi (haske 16 h, 22 ° C), ban da gwaje-gwajen haɓakar hypocotyl da tushen tushen wanda aka girma a kan faranti a tsaye a ƙarƙashin haske akai-akai da 22 ° C. Don gwaje-gwajen nitrate, an shuka tsire-tsire akan gyare-gyaren matsakaicin MS (matsakaicin shuka bioWORLD) wanda aka haɓaka tare da isassun nitrate (0 ko 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose da 1% A-agar (Sigma) a ƙarƙashin yanayin tsawon rana.
An sake haɗa GID1a cDNA da aka saka cikin pDONR221 tare da pDONR P4-P1R-pUBQ10 da pDONR P2R-P3-mCherry cikin pB7m34GW don samar da pUBQ10::GID1a-mCherry. IDD2 DNA da aka saka cikin pDONR221 an sake haɗa shi cikin pB7RWG266 don samar da p35S: IDD2-RFP. Don samar da pGID1b :: 2xmTQ2-GID1b, guntun 3.9kb a sama na yankin coding na GID1b da guntuwar 4.7kb mai ɗauke da GID1b cDNA (1.3 kb) da mai ƙarewa (3.4 kb) an fara haɓaka ta amfani da firam a cikin ƙarin P1PN da aka saka a cikin P1M R. Fisher Scientific) da pDONR P2R-P3 (Thermo Fisher Scientific), bi da bi, kuma a ƙarshe an haɗa su tare da pDONR221 2xmTQ268 a cikin pGreen 012567 manufa vector ta amfani da cloning Gateway. Don samar da pCUC2 :: LSSmOrange, jerin masu tallata CUC2 (3229 bp na sama na ATG) wanda ya biyo bayan jerin lambobin manyan Stokes-shifted mOrange (LSSmOrange) 69 tare da siginar Nukiliya N7 da NOS transcriptional m. tsarin sake hadewa (Invitrogen). An gabatar da vector binaryar shuka a cikin Agrobacterium tumefaciens iri GV3101 kuma an gabatar da shi a cikin ganyen Nicotiana benthamiana ta hanyar shigar Agrobacterium da cikin Arabidopsis thaliana Col-0 ta hanyar tsoma fure, bi da bi. pUBQ10::qmRGA pUBQ10::GID1a-mCherry da pCLV3::mCherry-NLS qmRGA an ware su daga zuriyar F3 da F1 na giciye daban-daban, bi da bi.
RNA in situ hybridization da aka yi a kan kusan 1 cm tsayi tips72, wanda aka tattara kuma nan da nan gyarawa a cikin FAA bayani (3.7% formaldehyde, 5% acetic acid, 50% ethanol) pre-sanyi zuwa 4 °C. Bayan 2 × 15 min jiyya mara amfani, an canza gyara kuma an sanya samfuran cikin dare. GID1a, GID1b, GID1c, GAI, RGL1, RGL2, da RGL3 cDNAs da antisense probes zuwa 3'-UTRs an haɗa su ta amfani da firam ɗin da aka nuna a Ƙarin Table 3 kamar yadda Rosier et al.73 ya bayyana. Digoxigenin-labeled probes an immunodetected ta yin amfani da digoxigenin antibodies (3000-dilution dilution; Roche, catalog number: 11 093 274 910), da kuma sassan da aka tabo da 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 250-ninka diluphosphate) 200-ninka dilution) bayani.
Lokacin aikawa: Fabrairu-10-2025