Girman harbe-harbe apical meristem (SAM) yana da mahimmanci ga tsarin tushen tushe.gibberellins(GAs) suna taka muhimmiyar rawa wajen daidaita ci gaban tsirrai, amma har yanzu ba a fahimci rawar da suke takawa a cikin SAM ba. A nan, mun ƙirƙiri na'urar auna siginar GA ta hanyar injiniyan furotin DELLA don danne muhimman ayyukansa na sarrafawa a cikin martanin kwafi na GA yayin da muke kiyaye lalacewarsa bayan an gane GA. Mun nuna cewa wannan na'urar auna siginar bio mai tushen lalacewa tana rubuta canje-canje a matakan GA da kuma fahimtar ƙwayoyin halitta yayin haɓakawa. Mun yi amfani da wannan na'urar auna siginar GA don taswirar ayyukan siginar GA a cikin SAM. Mun nuna cewa manyan siginar GA suna nan galibi a cikin ƙwayoyin da ke tsakanin organ primordia, waɗanda sune abubuwan da suka riga suka kai ga ƙwayoyin internode. Ta amfani da hanyoyin samun da asarar aiki, mun ƙara nuna cewa GA tana daidaita yanayin rarraba ƙwayoyin halitta, tana kafa tsarin ƙwayoyin halitta na canonical na internodes, don haka tana haɓaka ƙayyadaddun internode a cikin SAM.
Meristem na harbe-harbe (SAM), wanda ke kan kololuwar harbe-harbe, ya ƙunshi wani yanki na ƙwayoyin tushe waɗanda ayyukansu ke haifar da gabobin gefe da kuma ƙwayoyin tushe ta hanyar zamani da kuma ta maimaitawa a tsawon rayuwar shukar. Kowanne daga cikin waɗannan raka'o'in da ke maimaitawa, ko ƙwayoyin shuka, ya haɗa da internodes da gabobin gefe a cikin ƙwayoyin, da kuma axillary meristems a cikin ƙwayoyin ganye1. Girma da tsarin ƙwayoyin shuka suna canzawa yayin ci gaba. A cikin Arabidopsis, ana danne girman internodal a lokacin matakin ciyayi, kuma axillary meristems suna barci a cikin axils na ganyen rosette. A lokacin sauyawa zuwa lokacin fure, SAM ya zama meristem na inflorescence, yana samar da internodes masu tsayi da buds na axillary, rassan a cikin axils na ganyen cauline, kuma daga baya, furanni marasa ganye2. Kodayake mun sami ci gaba mai mahimmanci wajen fahimtar hanyoyin da ke sarrafa farawar ganye, furanni, da rassan, kaɗan ne aka sani game da yadda internodes ke tasowa.
Fahimtar rarrabawar GAs ta spatiotemporal zai taimaka wajen fahimtar ayyukan waɗannan hormones a cikin kyallen takarda daban-daban da kuma a matakai daban-daban na ci gaba. Ganin lalacewar haɗin RGA-GFP da aka bayyana a ƙarƙashin aikin mai haɓaka nasa yana ba da mahimman bayanai kan daidaita jimillar matakan GA a cikin tushen15,16. Duk da haka, bayyanar RGA ya bambanta a cikin kyallen takarda17 kuma GA18 ne ke tsara shi. Don haka, bambancin bayyanar mai haɓaka RGA na iya haifar da tsarin haske da aka lura da shi tare da RGA-GFP don haka wannan hanyar ba ta da adadi. Kwanan nan, GA19,20 mai suna bioactive fluorescein (Fl) ya bayyana tarin GA a cikin tushen endocortex da kuma daidaita matakan ƙwayoyin halittarsa ta hanyar jigilar GA. Kwanan nan, firikwensin GA FRET nlsGPS1 ya nuna cewa matakan GA suna da alaƙa da tsawaita ƙwayoyin halitta a cikin tushen, filaments, da hypocotyls21 masu duhu. Duk da haka, kamar yadda muka gani, yawan GA ba shine kawai siga da ke sarrafa ayyukan siginar GA ba, saboda ya dogara ne akan hanyoyin ji mai rikitarwa. A nan, bisa ga fahimtarmu game da hanyoyin siginar DELLA da GA, mun bayar da rahoton ci gaba da halayyar na'urar auna siginar ratiometric bisa lalata don siginar GA. Don haɓaka wannan na'urar auna siginar adadi, mun yi amfani da RGA mai auna siginar GA wanda aka haɗa shi da furotin mai haske kuma an bayyana shi a ko'ina a cikin kyallen takarda, da kuma furotin mai haske wanda ba shi da hankali ga GA. Mun nuna cewa haɗuwar furotin RGA mai canzawa ba sa tsoma baki ga siginar GA ta ciki lokacin da aka bayyana a ko'ina, kuma wannan na'urar auna siginar bio na iya ƙididdige ayyukan siginar da suka samo asali daga shigarwar GA da sarrafa siginar GA ta hanyar na'urar ji tare da babban ƙudurin spatiotemporal. Mun yi amfani da wannan na'urar auna siginar bio don zana taswirar rarraba ayyukan siginar GA na spatiotemporal da kuma ƙididdige yadda GA ke daidaita halayen ƙwayoyin halitta a cikin epidermis na SAM. Mun nuna cewa GA yana daidaita yanayin rarraba ƙwayoyin SAM da ke tsakanin organ primordia, ta haka ne ke bayyana tsarin ƙwayoyin halitta na internode.
A ƙarshe, mun tambayi ko qmRGA zai iya bayar da rahoton canje-canje a cikin matakan GA na ciki ta amfani da hypocotyls masu girma. Mun nuna a baya cewa nitrate yana ƙarfafa girma ta hanyar ƙara yawan haɗin GA kuma, bi da bi, lalacewar DELLA34. Saboda haka, mun lura cewa tsawon hypocotyl a cikin tsire-tsire na pUBQ10::qmRGA da aka girma a ƙarƙashin wadataccen nitrate (10 mM NO3−) ya fi tsayi fiye da na tsire-tsire da aka shuka a ƙarƙashin yanayin rashin nitrate (Hoto na Ƙarin 6a). Daidai da martanin girma, siginar GA ta fi girma a cikin hypocotyls na tsire-tsire da aka shuka a ƙarƙashin yanayin 10 mM NO3− fiye da na tsire-tsire da aka shuka a rashin nitrate (Hoto na Ƙarin 6b, c). Don haka, qmRGA kuma yana ba da damar sa ido kan canje-canje a cikin siginar GA da canje-canje na ciki a cikin yawan GA ke haifarwa.
Domin fahimtar ko aikin siginar GA da qmRGA ta gano ya dogara ne akan yawan GA da fahimtar GA, kamar yadda aka zata bisa ga ƙirar firikwensin, mun yi nazarin bayyanar masu karɓar GID1 guda uku a cikin kyallen tsirrai da haihuwa. A cikin tsirrai, layin mai ba da rahoto na GID1-GUS ya nuna cewa GID1a da c sun bayyana sosai a cikin cotyledons (Hoto na 3a-c). Bugu da ƙari, duk masu karɓar guda uku an bayyana su a cikin ganye, tushen asali na gefe, ƙarshen tushe (banda murfin tushen GID1b), da tsarin jijiyoyin jini (Hoto na 3a-c). A cikin SAM mai fure, mun gano siginar GUS kawai don GID1b da 1c (Hoto na Ƙarin 7a-c). In situ hybridization ya tabbatar da waɗannan alamu na bayyanar kuma ya ƙara nuna cewa GID1c an bayyana shi daidai gwargwado a ƙananan matakan SAM, yayin da GID1b ya nuna mafi girma a gefen SAM (Hoto na Ƙarin 7d-l). Haɗakar fassarar pGID1b::2xmTQ2-GID1b ta kuma bayyana kewayon bayyanar GID1b mai daraja, daga ƙarancin ko babu magana a tsakiyar SAM zuwa babban magana a iyakokin gabobi (Hoto na Ƙarin 7m). Don haka, masu karɓar GID1 ba su rarraba daidai gwargwado a cikin kyallen takarda da kuma cikin kyallen takarda ba. A cikin gwaje-gwajen da suka biyo baya, mun kuma lura cewa yawan bayyanar GID1 (pUBQ10::GID1a-mCherry) ya ƙara yawan tasirin qmRGA a cikin hypocotyls zuwa aikace-aikacen GA na waje (Hoto na 3d, e). Sabanin haka, hasken da aka auna ta hanyar qd17mRGA a cikin hypocotyl bai damu da maganin GA3 ba (Hoto na 3f, g). Ga gwaje-gwajen guda biyu, an yi wa shuka magani da babban yawan GA (100 μM GA3) don tantance saurin halayen firikwensin, inda aka ƙara ko ɓace ikon ɗaurewa da mai karɓar GID1. Tare, waɗannan sakamakon sun tabbatar da cewa na'urar gano kwayoyin halitta ta qmRGA tana aiki tare a matsayin na'urar gano kwayoyin halitta ta GA da GA, kuma suna nuna cewa bambancin bayyanar mai karɓar GID1 na iya daidaita fitowar na'urar sosai.
Zuwa yanzu, rarrabawar siginar GA a cikin SAM har yanzu ba a fayyace ta ba. Saboda haka, mun yi amfani da tsire-tsire masu bayyana qmRGA da kuma pCLV3::mCherry-NLS stem cell reporter35 don ƙididdige taswirorin adadi masu girma na ayyukan siginar GA, suna mai da hankali kan layin L1 (epidermis; Hoto na 4a, b, duba Hanyoyi da Hanyoyin Ƙarin), tunda L1 tana taka muhimmiyar rawa wajen sarrafa ci gaban SAM36. A nan, bayyanar pCLV3::mCherry-NLS ta samar da wurin tunani mai tsayayye don nazarin rarrabawar spatiotemporal na ayyukan siginar GA37. Kodayake ana ɗaukar GA a matsayin mahimmanci ga ci gaban gabobi4, mun lura cewa siginar GA tana da ƙasa a cikin primordium na fure (P) tun daga matakin P3 (Hoto na 4a, b), yayin da ƙananan primordiums na P1 da P2 suna da matsakaicin aiki kamar na yankin tsakiya (Hoto na 4a, b). An gano mafi girman aikin siginar GA a iyakokin organ primordium, farawa daga P1/P2 (a gefen iyaka) da kuma kololuwa a P4, da kuma a cikin dukkan ƙwayoyin halitta na yankin da ke tsakanin primordia (Hoto na 4a, b da Hoto na Ƙarin 8a, b). An lura da wannan babban aikin siginar GA ba kawai a cikin epidermis ba har ma a cikin layukan L2 da na sama na L3 (Hoto na Ƙarin 8b). Tsarin siginar GA da aka gano a cikin SAM ta amfani da qmRGA shi ma ya kasance ba canzawa akan lokaci (Hoto na Ƙarin 8c–f, k). Kodayake an rage tsarin ginin qd17mRGA a cikin SAM na tsire-tsire na T3 daga layuka biyar masu zaman kansu waɗanda muka bayyana dalla-dalla, mun sami damar yin nazarin tsarin haske da aka samu tare da ginin pRPS5a::VENUS-2A-TagBFP (Hoto na Ƙarin 8g–j, l). A cikin wannan layin sarrafawa, ƙananan canje-canje ne kawai aka gano a cikin rabon hasken rana a cikin SAM, amma a cikin cibiyar SAM mun lura da raguwa bayyananne da ba zato ba tsammani a cikin VENUS da ke da alaƙa da TagBFP. Wannan ya tabbatar da cewa tsarin siginar da qmRGA ta lura yana nuna lalacewar mRGA-VENUS da ke dogara da GA, amma kuma yana nuna cewa qmRGA na iya ƙididdige ayyukan siginar GA a cikin cibiyar meristem. A taƙaice, sakamakonmu ya bayyana tsarin siginar GA wanda galibi yana nuna rarrabawar primordia. Wannan rarrabawar yankin tsakanin primordial (IPR) ya faru ne saboda kafa babban aikin siginar GA a hankali tsakanin primordium mai tasowa da yankin tsakiya, yayin da a lokaci guda aikin siginar GA a cikin primordium yana raguwa (Hoto na 4c, d).
Rarraba masu karɓar GID1b da GID1c (duba sama) yana nuna cewa bambancin bayyanar masu karɓar GA yana taimakawa wajen tsara tsarin aikin siginar GA a cikin SAM. Mun yi mamakin ko tarin bambancin GA na iya shiga. Don bincika wannan yuwuwar, mun yi amfani da na'urar firikwensin nlsGPS1 GA FRET21. An gano ƙaruwar mitar kunnawa a cikin SAM na nlsGPS1 da aka yi wa magani da 10 μM GA4+7 na tsawon minti 100 (Hoto na Ƙarin 9a–e), yana nuna cewa nlsGPS1 yana amsawa ga canje-canje a cikin yawan GA a cikin SAM, kamar yadda yake yi a cikin roots21. Rarraba sarari na mitar kunnawa na nlsGPS1 ya nuna ƙarancin matakan GA a cikin yadudduka na waje na SAM, amma ya nuna cewa an ɗaga su a tsakiya da kuma iyakokin SAM (Hoto na 4e da Ƙarin Hoto na 9a,c). Wannan yana nuna cewa GA kuma an rarraba shi a cikin SAM tare da tsarin sarari wanda yayi daidai da wanda qmRGA ya bayyana. A matsayin hanyar da ta dace, mun kuma yi amfani da SAM da fluorescent GA (GA3-, GA4-, GA7-Fl) ko Fl kaɗai a matsayin wani abu mai hana konewa. An rarraba siginar Fl a ko'ina cikin SAM, gami da yankin tsakiya da primordium, kodayake a ƙaramin ƙarfi (Hoto na 4j da Hoto na Ƙarin 10d). Sabanin haka, dukkan GA-Fl guda uku sun taru musamman a cikin iyakokin primordium da kuma zuwa matakai daban-daban a cikin sauran IPR, tare da GA7-Fl sun taru a cikin mafi girman yanki a cikin IPR (Hoto na 4k da Hoto na Ƙarin 10a,b). Ƙididdige ƙarfin haske ya nuna cewa rabon ƙarfin IPR zuwa wanda ba IPR ba ya fi girma a cikin SAM da aka yi wa GA-Fl idan aka kwatanta da SAM da aka yi wa Fl (Hoto na 4l da Hoto na Ƙarin 10c). Tare, waɗannan sakamakon suna nuna cewa GA yana nan a cikin mafi yawan taro a cikin ƙwayoyin IPR waɗanda ke kusa da iyakar gabobi. Wannan yana nuna cewa tsarin aikin siginar SAM GA ya samo asali ne daga bambancin bayyanar masu karɓar GA da kuma tarin GA a cikin ƙwayoyin IPR kusa da iyakokin gabobi. Don haka, bincikenmu ya nuna wani yanayi na spatiotemporal na siginar GA, tare da ƙarancin aiki a tsakiya da primordium na SAM da kuma babban aiki a cikin IPR a yankin da ke kewaye.
Domin fahimtar rawar da bambancin aikin siginar GA ke takawa a cikin SAM, mun yi nazarin alaƙar da ke tsakanin aikin siginar GA, faɗaɗa ƙwayoyin halitta, da kuma rabuwar ƙwayoyin halitta ta amfani da hoton lokaci-lokaci na SAM qmRGA pCLV3::mCherry-NLS. Ganin rawar da GA ke takawa a cikin daidaita girma, an yi tsammanin samun kyakkyawar alaƙa da sigogin faɗaɗa ƙwayoyin halitta. Saboda haka, da farko mun kwatanta taswirar ayyukan siginar GA tare da taswirar ƙimar girmar saman ƙwayoyin halitta (a matsayin wakili don ƙarfin faɗaɗa ƙwayoyin halitta ga wata ƙwayar halitta da kuma ga ƙwayoyin 'ya mace a rarrabawa) da kuma taswirar anisotropy na girma, wanda ke auna alkiblar faɗaɗa ƙwayoyin halitta (wanda kuma aka yi amfani da shi a nan don wata ƙwayar halitta da kuma ga ƙwayoyin 'ya mace a rarrabawa; Hoto na 5a,b, duba Hanyoyi da Hanyoyin Ƙarin). Taswirorinmu na ƙimar girmar saman ƙwayoyin SAM sun yi daidai da abubuwan da aka lura a baya38,39, tare da ƙarancin ƙimar girma a kan iyaka da mafi girman ƙimar girma a cikin furanni masu tasowa (Hoto na 5a). Babban nazarin sassan (PCA) ya nuna cewa aikin siginar GA yana da alaƙa mara kyau da ƙarfin girmar saman ƙwayoyin halitta (Hoto na 5c). Mun kuma nuna cewa manyan ginshiƙan bambancin, gami da shigar da siginar GA da ƙarfin girma, sun kasance daidai da alkiblar da aka ƙayyade ta hanyar babban bayyanar CLV3, wanda ya tabbatar da cire ƙwayoyin halitta daga cibiyar SAM a cikin sauran nazarin. Binciken haɗin gwiwar Spearman ya tabbatar da sakamakon PCA (Hoto na 5d), yana nuna cewa manyan siginar GA a cikin IPR ba su haifar da faɗaɗa ƙwayar halitta ba. Duk da haka, nazarin haɗin gwiwa ya nuna ɗan alaƙa mai kyau tsakanin aikin siginar GA da anisotropy na girma (Hoto na 5c, d), yana nuna cewa mafi girman siginar GA a cikin IPR yana tasiri ga alkiblar haɓakar ƙwayar halitta da kuma wataƙila matsayin jirgin rarraba ƙwayar halitta.
a, b Taswirorin zafi na matsakaicin girman saman (a) da anisotropy na girma (b) a cikin SAM sun kai matsakaicin tsire-tsire bakwai masu zaman kansu (wanda aka yi amfani da su azaman wakili don ƙarfi da alkiblar faɗaɗa tantanin halitta, bi da bi). c Binciken PCA ya haɗa da masu canji masu zuwa: siginar GA, ƙarfin girman saman, anisotropy na girma saman, da bayyanar CLV3. An haɗa ɓangaren PCA na 1 da mummunan alaƙa da ƙarfin girman saman kuma an yi alaƙa mai kyau da siginar GA. ɓangaren PCA na 2 galibi yana da alaƙa mai kyau da anisotropy na girma saman kuma an yi alaƙa mara kyau da bayyanar CLV3. Kashi yana wakiltar bambancin da kowane sashi ya bayyana. d Binciken hulɗar Spearman tsakanin siginar GA, ƙarfin girman saman, da anisotropy na girma saman a sikelin nama ban da CZ. Lambar da ke dama ita ce ƙimar Spearman rho tsakanin masu canji biyu. Taurari suna nuna lokuta inda haɗin kai/mara kyau yana da matuƙar mahimmanci. e 3D gani na ƙwayoyin Col-0 SAM L1 ta hanyar na'urar microscopy ta confocal. Sabbin bangon tantanin halitta da aka samar a cikin SAM (amma ba primordium ba) a cikin awanni 10 an yi musu launi bisa ga ƙimar kusurwarsu. An nuna sandar launi a kusurwar dama ta ƙasa. Saitin yana nuna hoton 3D mai dacewa a awanni 0. An maimaita gwajin sau biyu tare da sakamako iri ɗaya. f Tsarin akwatin yana nuna ƙimar rarraba tantanin halitta a cikin IPR da wanda ba IPR Col-0 SAM ba (n = tsire-tsire masu zaman kansu 10). Layin tsakiya yana nuna matsakaici, kuma iyakokin akwatin suna nuna kashi 25 da 75. Wutsiya suna nuna mafi ƙarancin ƙima da matsakaicin ƙima da aka ƙayyade tare da software na R. An sami ƙimar P tare da gwajin t-jeri biyu na Welch. g, h Tsarin zane yana nuna (g) yadda ake auna kusurwar sabon bangon tantanin halitta (magenta) dangane da alkiblar radial daga tsakiyar SAM (layin fari mai dige-dige) (ana la'akari da ƙimar kusurwa mai tsanani kawai, watau, 0-90°,), da (h) alkiblar kewaye/gefen da radial a cikin meristem. i Histograms na mitar daidaitawar jirgin sama na rarraba tantanin halitta a fadin SAM (shuɗi mai duhu), IPR (shuɗi matsakaici), da kuma wanda ba IPR ba (shuɗi mai haske), bi da bi. An samo ƙimar P ta hanyar gwajin Kolmogorov-Smirnov mai wutsiya biyu. An maimaita gwajin sau biyu tare da sakamako iri ɗaya. j Mitar histograms na yanayin rarraba tantanin halitta na IPR a kusa da P3 (kore mai haske), P4 (kore mai matsakaici), da P5 (kore mai duhu), bi da bi. An sami ƙimar P ta hanyar gwajin Kolmogorov-Smirnov mai wutsiya biyu. An maimaita gwajin sau biyu tare da sakamako iri ɗaya.
Saboda haka, mun sake bincika alaƙar da ke tsakanin siginar GA da ayyukan rarraba tantanin halitta ta hanyar gano sabbin bangon tantanin halitta da aka samar yayin gwajin (Hoto na 5e). Wannan hanyar ta ba mu damar auna mita da alkiblar rarraba tantanin halitta. Abin mamaki, mun gano cewa mitar rarrabuwar tantanin halitta a cikin IPR da sauran SAM (wanda ba IPR ba ne, Hoto na 5f) iri ɗaya ne, yana nuna cewa bambance-bambancen siginar GA tsakanin ƙwayoyin IPR da waɗanda ba IPR ba ba sa yin tasiri sosai kan rarraba tantanin halitta. Wannan, da kuma kyakkyawar alaƙa tsakanin siginar GA da anisotropy na girma, ya sa muka yi la'akari da ko ayyukan siginar GA na iya yin tasiri ga yanayin rarraba tantanin halitta. Mun auna yanayin sabuwar bangon tantanin halitta a matsayin kusurwa mai tsanani dangane da axis na radial da ke haɗa tsakiyar meristem da tsakiyar sabon bangon tantanin halitta (Hoto na 5e-i) kuma mun lura da yanayin da ƙwayoyin halitta ke rabawa a kusurwoyi kusa da 90° dangane da axis na radial, tare da mafi girman mitoci da aka lura a 70–80° (23.28%) da 80–90° (22.62%) (Hoto na 5e,i), wanda ya dace da rarrabuwar tantanin halitta a cikin alkiblar da'ira/juyawa (Hoto na 5h). Don bincika gudummawar siginar GA ga wannan ɗabi'ar rarraba tantanin halitta, mun bincika sigogin rarraba tantanin halitta a cikin IPR da wanda ba IPR ba daban (Hoto na 5i). Mun lura cewa rarraba kusurwar rarrabuwa a cikin ƙwayoyin IPR ya bambanta da na ƙwayoyin da ba IPR ba ko kuma a cikin ƙwayoyin SAM gaba ɗaya, tare da ƙwayoyin IPR suna nuna babban rabo na rarrabuwar ƙwayoyin gefe/da'ira, watau, 70–80° da 80–90° (33.86% da 30.71%, bi da bi, rabon da ya dace) (Hoto na 5i). Don haka, abubuwan da muka lura sun nuna alaƙa tsakanin siginar GA mai girma da kuma yanayin rarraba ƙwayoyin halitta kusa da alkiblar da'ira, kama da alaƙar da ke tsakanin aikin siginar GA da anisotropy na girma (Hoto na 5c, d). Don ƙara tabbatar da kiyaye sararin samaniya na wannan haɗin, mun auna yanayin rarraba a cikin ƙwayoyin IPR da ke kewaye da primordium tun daga P3, tunda an gano mafi girman aikin siginar GA a wannan yanki tun daga P4 (Hoto na 4). Kusurwoyin rarrabuwa na IPR a kusa da P3 da P4 ba su nuna bambance-bambance masu mahimmanci na ƙididdiga ba, kodayake an lura da ƙaruwar yawan rarrabuwar ƙwayoyin gefe a cikin IPR a kusa da P4 (Hoto na 5j). Duk da haka, a cikin ƙwayoyin IPR da ke kewaye da P5, bambancin da ke cikin yanayin rarraba ƙwayoyin halitta ya zama mai mahimmanci a kididdiga, tare da ƙaruwa mai yawa a cikin yawan rarraba ƙwayoyin halitta (Hoto na 5j). Tare, waɗannan sakamakon sun nuna cewa siginar GA na iya sarrafa yanayin rarrabuwar ƙwayoyin halitta a cikin SAM, wanda ya yi daidai da rahotannin da suka gabata40,41 cewa babban siginar GA na iya haifar da yanayin rarrabuwar ƙwayoyin halitta a cikin IPR.
An yi hasashen cewa ƙwayoyin da ke cikin IPR ba za a haɗa su cikin primordia ba, sai dai a cikin internodes2,42,43. Tsarin rarraba ƙwayoyin halitta a cikin IPR na iya haifar da tsarin da aka saba da shi na layukan tsayi masu layi ɗaya na ƙwayoyin epidermal a cikin internodes. Abubuwan da muka lura da su a sama sun nuna cewa siginar GA wataƙila tana taka rawa a cikin wannan tsari ta hanyar daidaita alkiblar rarraba ƙwayoyin halitta.
Rashin aiki na kwayoyin halittar DELLA da yawa yana haifar da amsawar GA mai tsari, kuma ana iya amfani da della mutants don gwada wannan hasashe44. Da farko mun yi nazari kan yanayin bayyanar kwayoyin halittar DELLA guda biyar a cikin SAM. Haɗakar da aka yi a layin GUS45 ta nuna cewa GAI, RGA, RGL1, da RGL2 (zuwa ƙasa da haka) an bayyana su a cikin SAM (Hoto na Ƙarin 11a–d). Haɗakar da ke cikin situ ta ƙara nuna cewa mRNA na GAI yana taruwa musamman a cikin furanni na primordia da masu tasowa (Hoto na Ƙarin 11e). An gano mRNA na RGL1 da RGL3 a cikin rufin SAM da kuma cikin tsofaffin furanni, yayin da mRNA na RGL2 ya fi yawa a yankin iyaka (Hoto na Ƙarin 11f–h). Hoto na Confocal na pRGL3::RGL3-GFP SAM ya tabbatar da bayyanar da aka lura da hybridization na in situ kuma ya nuna cewa furotin na RGL3 yana taruwa a tsakiyar ɓangaren SAM (Hoto na Ƙarin 11i). Ta amfani da layin pRGA::GFP-RGA, mun kuma gano cewa furotin RGA yana taruwa a cikin SAM, amma yawansa yana raguwa a kan iyaka tun daga P4 (Hoto na Ƙarin 11j). Abin lura shi ne, tsarin bayyanar RGL3 da RGA sun yi daidai da babban aikin siginar GA a cikin IPR, kamar yadda qmRGA ta gano (Hoto na 4). Bugu da ƙari, waɗannan bayanai sun nuna cewa duk DELLAs an bayyana su a cikin SAM kuma cewa bayyanar su ta haɗu ta mamaye dukkan SAM.
Mun sake yin nazarin sigogin rarraba tantanin halitta a cikin nau'in SAM na daji (Ler, control) da kuma gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (duniya) masu maye gurbi (Hoto na 6a, b). Abin sha'awa, mun lura da wani gagarumin sauyi a kididdiga a cikin rarrabawar mitoci na kusurwar rarraba tantanin halitta a cikin della global mutant SAM idan aka kwatanta da nau'in daji (Hoto na 6c). Wannan canji a cikin della global mutant ya faru ne saboda karuwar mitar kusurwoyi 80-90° (34.71% vs. 24.55%) kuma, zuwa ƙasa da haka, kusurwoyi 70-80° (23.78% vs. 20.18%), watau, daidai da rarrabuwar tantanin halitta masu juyawa (Hoto na 6c). Mitawar rarrabuwar da ba ta juyawa ba (0-60°) shi ma ya yi ƙasa a cikin della global mutant (Hoto na 6c). Yawan rarrabuwar ƙwayoyin halitta ya ƙaru sosai a cikin SAM na della global mutant (Hoto na 6b). Yawan rarrabuwar ƙwayoyin halitta a cikin IPR ya fi girma a cikin della global mutant idan aka kwatanta da nau'in daji (Hoto na 6d). A wajen yankin IPR, nau'in daji yana da rarrabawar kusurwoyin rarraba ƙwayoyin halitta iri ɗaya, yayin da della global mutant ya fi son rarrabuwar tangential kamar IPR (Hoto na 6e). Mun kuma ƙididdige yanayin rarrabuwar ƙwayoyin halitta a cikin SAM na ga2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, da ga2ox6-2), asalin GA-mara aiki wanda GA ke taruwa a ciki. Daidai da ƙaruwar matakan GA, SAM na furen ga2ox mai canzawa na quintuple ya fi na Col-0 girma (Hoto na Ƙarin 12a, b), kuma idan aka kwatanta da Col-0, quintuple ga2ox SAM ya nuna rarrabawar kusurwoyin rarraba tantanin halitta daban-daban, tare da mitar kusurwoyin yana ƙaruwa daga 50° zuwa 90°, watau kuma yana fifita rarrabuwar tangential (Hoto na Ƙarin 12a–c). Don haka, mun nuna cewa kunna siginar GA da tarin GA yana haifar da rarrabuwar tantanin halitta a cikin IPR da sauran SAM.
a, b 3D na gani na Layer L1 na Ler (a) mai launi PI da kuma mutant della na duniya (b) SAM ta amfani da na'urar hangen nesa ta confocal. Sabbin bangon tantanin halitta da aka samar a cikin SAM (amma ba primordium ba) a cikin tsawon awanni 10 ana nuna su kuma ana canza su bisa ga ƙimar kusurwar su. Saitin yana nuna SAM a 0 h. An nuna sandar launi a kusurwar dama ta ƙasa. Kibiyar da ke cikin (b) tana nuna misalin fayilolin tantanin halitta masu daidaitawa a cikin mutant della na duniya. An maimaita gwajin sau biyu tare da sakamako iri ɗaya. kwatanta rarraba mita na yanayin rarraba tantanin halitta a cikin dukkan SAM (d), IPR (e), da kuma IPR (f) mara IPR (f) tsakanin Ler da della na duniya. An sami ƙimar P ta amfani da gwajin Kolmogorov-Smirnov mai wutsiya biyu. f, g 3D na gani na hotunan confocal na SAM mai launi PI na Col-0 (i) da pCUC2::gai-1-VENUS (j) tsire-tsire masu canzawa. Faifan (a, b) suna nuna sabbin bangon tantanin halitta (amma ba primordia) da aka samar a cikin SAM cikin awanni 10. An maimaita gwajin sau biyu tare da sakamako iri ɗaya. h–j Kwatanta rarrabawar mitar yanayin rarraba tantanin halitta da ke cikin dukkan SAM (h), IPR (i) da wanda ba IPR ba (j) tsakanin tsire-tsire Col-0 da pCUC2::gai-1-VENUS. An sami ƙimar P ta amfani da gwajin Kolmogorov-Smirnov mai wutsiya biyu.
Mun sake gwada tasirin hana siginar GA musamman a cikin IPR. Don haka, mun yi amfani da mai haɓaka cotyledon cup 2 (CUC2) don haɓaka bayyanar furotin gai-1 mara kyau wanda aka haɗa zuwa VENUS (a cikin layin pCUC2::gai-1-VENUS). A cikin SAM na nau'in daji, mai haɓaka CUC2 yana haifar da bayyanar yawancin IPRs a cikin SAM, gami da ƙwayoyin iyaka, daga P4 zuwa gaba, kuma an lura da irin wannan takamaiman bayyanar a cikin tsire-tsire na pCUC2::gai-1-VENUS (duba ƙasa). Rarraba kusurwoyin rarraba tantanin halitta a fadin SAM ko IPR na tsire-tsire na pCUC2::gai-1-VENUS bai bambanta sosai da na nau'in daji ba, kodayake ba zato ba tsammani mun gano cewa ƙwayoyin da ba su da IPR a cikin waɗannan tsire-tsire sun rabu a mafi yawan mita na 80-90° (Hoto na 6f-j).
An yi nuni da cewa alkiblar rabuwar tantanin halitta ta dogara ne da yanayin SAM, musamman ma matsin lamba da aka samu daga lanƙwasa nama46. Saboda haka muka tambaya 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 global mutant SAM ya fi na nau'in daji girma (Ƙarin Hoto na 13a, b, d). In situ hybridization na CLV3 da STM RNA sun tabbatar da faɗaɗawar meristem a cikin della mutants kuma sun ƙara nuna faɗaɗa ta gefe na niche na tushen tantanin halitta (Ƙarin Hoto na 13e, f, h, i). Duk da haka, lanƙwasa SAM yayi kama da juna a cikin duka nau'ikan halittu (Ƙarin Hoto na 13k, m, n, p). Mun lura da irin wannan ƙaruwa a girma a cikin gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant ba tare da canji a lanƙwasa ba idan aka kwatanta da nau'in daji (Hoto na Ƙarin 13c, d, g, j, l, o, p). Hakanan mitar yanayin rarraba tantanin halitta ya shafi a cikin della quadruple mutant, amma zuwa ƙasa da na della monolithic mutant (Hoto na Ƙarin 12d–f). Wannan tasirin allurar, tare da rashin tasiri akan lanƙwasa, yana nuna cewa ragowar aikin RGL3 a cikin Della quadruple mutant yana iyakance canje-canje a yanayin rarraba tantanin halitta wanda asarar aikin DELLA ya haifar kuma canje-canje a cikin rarrabuwar tantanin halitta na gefe suna faruwa ne sakamakon canje-canje a cikin aikin siginar GA maimakon canje-canje a cikin yanayin SAM. Kamar yadda aka bayyana a sama, mai haɓaka CUC2 yana haifar da bayyanar IPR a cikin SAM wanda ya fara daga P4 (Ƙarin Hoto na 14a, b), kuma akasin haka, pCUC2::gai-1-VENUS SAM yana da ƙaramin girma amma mafi girman lanƙwasa (Ƙarin Hoto na 14c-h). Wannan canji a cikin siffar pCUC2::gai-1-VENUS SAM na iya haifar da rarrabawar damuwa ta injiniya daban-daban idan aka kwatanta da nau'in daji, inda matsin lamba mai yawa na kewaye ya fara a ɗan gajeren nisa daga cibiyar SAM47. A madadin haka, canje-canje a cikin siffar pCUC2::gai-1-VENUS SAM na iya faruwa ne sakamakon canje-canje a cikin halayen injiniya na yanki wanda bayyanar transgene48 ta haifar. A cikin duka halayen biyu, wannan na iya rage tasirin canje-canje a cikin siginar GA ta hanyar ƙara yiwuwar ƙwayoyin za su rabu a cikin yanayin kewaye/juyawar, yana bayyana abubuwan da muka lura.
Idan aka haɗa bayanai, bayananmu sun tabbatar da cewa siginar GA mafi girma tana taka rawa a cikin yanayin gefen ɓangaren ɓangaren ƙwayoyin halitta a cikin IPR. Hakanan suna nuna cewa lanƙwasawar meristem kuma tana tasiri ga yanayin ɓangaren ɓangaren ƙwayoyin halitta a cikin IPR.
Juyawar yanayin rarrabawa a cikin IPR, saboda yawan ayyukan siginar GA, yana nuna cewa GA tana shirya fayil ɗin tantanin halitta mai radiyo a cikin epidermis a cikin SAM don ayyana tsarin tantanin halitta wanda daga baya za a same shi a cikin epidermal internode. Hakika, irin waɗannan fayilolin tantanin halitta suna bayyane akai-akai a cikin hotunan SAM na della global mutants (Hoto na 6b). Don haka, don ƙarin bincika aikin ci gaban tsarin sararin samaniya na siginar GA a cikin SAM, mun yi amfani da hoton lokaci-lokaci don nazarin tsarin sararin samaniya na ƙwayoyin halitta a cikin IPR a cikin nau'in daji (Ler da Col-0), della global mutants, da pCUC2::gai-1-VENUS transgenic shuke-shuke.
Mun gano cewa qmRGA ya nuna cewa aikin siginar GA a cikin IPR ya ƙaru daga P1/P2 kuma ya kai kololuwa a P4, kuma wannan tsari ya kasance daidai akan lokaci (Hoto na 4a–f da Ƙarin Hoto na 8c–f, k). Don yin nazarin tsarin sararin samaniya na ƙwayoyin halitta a cikin IPR tare da ƙaruwar siginar GA, mun yiwa ƙwayoyin Ler IPR alama a sama da gefen P4 bisa ga ƙaddarar ci gaban su an yi nazari a cikin awanni 34 bayan lura ta farko, watau, fiye da lokutan plastid biyu, wanda ya ba mu damar bin ƙwayoyin IPR yayin ci gaban primordium daga P1/P2 zuwa P4. Mun yi amfani da launuka uku daban-daban: rawaya ga waɗancan ƙwayoyin da aka haɗa cikin primordium kusa da P4, kore ga waɗanda ke cikin IPR, da shunayya ga waɗanda suka shiga cikin duka hanyoyin (Hoto na 7a–c). A t0 (0 h), ana iya ganin layuka 1–2 na ƙwayoyin IPR a gaban P4 (Hoto na 7a). Kamar yadda aka zata, lokacin da waɗannan ƙwayoyin suka rabu, sun yi hakan galibi ta hanyar jirgin ƙasa mai rarrafe (Hoto na 7a–c). An samu irin wannan sakamako ta amfani da Col-0 SAM (wanda aka mayar da hankali kan P3, wanda iyakokinsa suka ninka kamar P4 a Ler), kodayake a cikin wannan nau'in halittar, ninki da aka samar a kan iyakar fure ya ɓoye ƙwayoyin IPR da sauri (Hoto na 7g–i). Don haka, tsarin rarraba ƙwayoyin IPR yana tsara ƙwayoyin zuwa layukan radial, kamar yadda yake a cikin internodes. Tsarin layukan radial da kuma gano ƙwayoyin IPR tsakanin gabobin da ke biye yana nuna cewa waɗannan ƙwayoyin sune magabatan internodal.
A nan, mun ƙirƙiri wani na'urar auna siginar GA mai auna rabo, qmRGA, wanda ke ba da damar yin taswirar adadi na ayyukan siginar GA wanda ya samo asali daga haɗakar yawan masu karɓar GA da GA yayin da ake rage tsangwama ga hanyoyin siginar endogenous, ta haka ne ke samar da bayanai kan aikin GA a matakin ƙwayoyin halitta. Don wannan dalili, mun gina wani ingantaccen furotin DELLA, mRGA, wanda ya rasa ikon ɗaure abokan hulɗar hulɗar DELLA amma yana ci gaba da kasancewa mai saurin kamuwa da proteolysis da GA ke haifarwa. qmRGA yana amsawa ga canje-canje na waje da na ciki a matakan GA, kuma halayensa na ji da motsin rai yana ba da damar kimanta canje-canje na spatiotemporal a cikin aikin siginar GA yayin haɓakawa. qmRGA kuma kayan aiki ne mai sassauƙa saboda ana iya daidaita shi zuwa kyallen takarda daban-daban ta hanyar canza mai haɓaka da ake amfani da shi don bayyana shi (idan ya cancanta), kuma idan aka yi la'akari da yanayin kiyaye hanyar siginar GA da kuma tsarin PFYRE a cikin angiosperms, yana yiwuwa a iya canja shi zuwa wasu nau'ikan22. Daidai da wannan, an kuma nuna cewa wani canji mai kama da na shinkafa SLR1 DELLA protein (HYY497AAA) yana danne aikin mai hana ci gaban SLR1 yayin da yake rage raguwar GA kaɗan, kamar mRGA23. Abin lura shi ne, 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 shafar ikonsa na hulɗa da abokan hulɗar rubutun bayanai50 ba. Kodayake wannan maye gurbin yana kusa da maye gurbin amino acid guda 3 da ke cikin mRGA, bincikenmu ya nuna cewa waɗannan maye gurbi guda biyu suna canza halaye daban-daban na DELLA. Kodayake yawancin abokan hulɗar rubutun bayanai suna ɗaure ga yankunan LHR1 da SAW na DELLA26,51, wasu amino acid da aka kiyaye a cikin yankin PFYRE na iya taimakawa wajen daidaita waɗannan hulɗar.
Ci gaban internode muhimmin abu ne a cikin tsarin tsirrai da haɓaka yawan amfanin ƙasa. qmRGA ya bayyana ƙarin aikin siginar GA a cikin ƙwayoyin halittar internode na IPR. Ta hanyar haɗa hoton adadi da kwayoyin halitta, mun nuna cewa tsarin siginar GA yana mamaye jiragen rarraba sel masu zagaye/juyawa a cikin fatalwar SAM, yana tsara ƙungiyar rarraba sel da ake buƙata don ci gaban internode. An gano masu kula da tsarin rarraba sel da yawa yayin ci gaba52,53. Aikinmu yana ba da misali bayyananne na yadda ayyukan siginar GA ke sarrafa wannan sigar sel. DELLA na iya hulɗa da hadaddun furotin da ke naɗewa41, don haka siginar GA na iya tsara yanayin rarraba sel ta hanyar tasiri kai tsaye kan yanayin microtubule na cortical40,41,54,55. Mun nuna ba zato ba tsammani cewa a cikin SAM, alaƙar aikin siginar GA mafi girma ba tsawaita ko rabuwar sel ba ne, amma kawai anisotropy na girma, wanda ya yi daidai da tasirin kai tsaye na GA akan alkiblar rarraba sel a cikin IPR. Duk da haka, ba za mu iya ware cewa wannan tasirin na iya zama kai tsaye ba, misali ta hanyar laushin bangon sel da GA ya haifar56. Canje-canje a cikin halayen bangon tantanin halitta suna haifar da damuwa ta injiniya57,58, wanda kuma zai iya yin tasiri ga yanayin rarraba tantanin halitta ta hanyar shafar yanayin microtubules na cortical39,46,59. Haɗakar tasirin damuwa ta injiniya da GA ke haifarwa da kuma daidaita kai tsaye na yanayin microtubule ta GA na iya shiga cikin samar da takamaiman tsari na yanayin rarraba tantanin halitta a cikin IPR don ayyana internodes, kuma ana buƙatar ƙarin bincike don gwada wannan ra'ayin. Hakazalika, binciken da aka yi a baya ya nuna mahimmancin sunadaran DELLA TCP14 da 15 a cikin sarrafa samuwar internode60,61 kuma waɗannan abubuwan na iya shiga tsakani na aikin GA tare da BREVIPEDICELLUS (BP) da PENNYWISE (PNY), waɗanda ke tsara ci gaban internode kuma an nuna suna yin tasiri ga siginar GA2,62. Ganin cewa DELLAs suna hulɗa da hanyoyin siginar brassinosteroid, ethylene, jasmonic acid, da abscisic acid (ABA)63,64 kuma cewa waɗannan hormones na iya yin tasiri ga yanayin microtubule65, tasirin GA akan yanayin rarraba tantanin halitta kuma ana iya yin sulhu da shi ta hanyar wasu hormones.
Nazarin farko na cytology ya nuna cewa yankuna na ciki da na waje na Arabidopsis SAM ana buƙatar su don haɓaka internode2,42. Gaskiyar cewa GA tana sarrafa rarrabuwar ƙwayoyin halitta a cikin kyallen ciki12 tana goyan bayan aiki biyu na GA wajen daidaita girman meristem da internode a cikin SAM. Tsarin rarrabawar ƙwayoyin alkibla kuma ana tsara shi sosai a cikin kyallen SAM na ciki, kuma wannan ƙa'ida yana da mahimmanci don haɓakar tushe52. Zai zama abin sha'awa a bincika ko GA kuma tana taka rawa wajen daidaita yanayin rarraba ƙwayoyin halitta a cikin ƙungiyar SAM ta ciki, ta haka yana daidaita ƙayyadaddun bayanai da haɓaka internodes a cikin SAM.
An shuka shuke-shuke a cikin ƙasa ko kuma an ƙara musu 1x Murashige-Skoog (MS) matsakaici (Duchefa) tare da 1% sucrose da 1% agar (Sigma) a ƙarƙashin yanayi na yau da kullun (haske awanni 16, 22 °C), sai dai gwaje-gwajen girma na hypocotyl da tushen shuka inda aka shuka shuke-shuke a kan faranti a tsaye a ƙarƙashin haske mai ɗorewa da 22 °C. Don gwaje-gwajen nitrate, an shuka shuke-shuke a kan matsakaicin MS (bioWORLD) wanda aka ƙara masa isasshen nitrate (0 ko 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose da 1% A-agar (Sigma) a ƙarƙashin yanayi na dogon lokaci.
An sake haɗa GID1a cDNA da aka saka a cikin pDONR221 da pDONR P4-P1R-pUBQ10 da pDONR P2R-P3-mCherry cikin pB7m34GW don samar da pUBQ10::GID1a-mCherry. An sake haɗa DNA na IDD2 da aka saka a cikin pDONR221 cikin pB7RWG266 don samar da p35S:IDD2-RFP. Domin samar da pGID1b::2xmTQ2-GID1b, an fara ƙara girman guntu na 3.9 kb daga yankin lambar GID1b da guntu na 4.7 kb wanda ke ɗauke da GID1b cDNA (1.3 kb) da terminator (3.4 kb) ta amfani da firam ɗin a cikin Ƙarin Tebur na 3 sannan aka saka su cikin pDONR P4-P1R (Thermo Fisher Scientific) da pDONR P2R-P3 (Thermo Fisher Scientific), bi da bi, kuma a ƙarshe an sake haɗa su da pDONR221 2xmTQ268 cikin vector mai manufa na pGreen 012567 ta amfani da cloning na Gateway. Domin samar da pCUC2::LSSmOrange, an haɗa jerin masu haɓaka CUC2 (3229 bp sama da ATG) sannan aka biyo bayan jerin lambobi na manyan mOrange masu canzawa na Stokes (LSSmOrange)69 tare da siginar gano wuri na nukiliya na N7 da kuma mai ƙarewar NOS a cikin vector mai niyya na pGreen kanamycin ta amfani da tsarin sake haɗa sassan Gateway 3 (Invitrogen). An shigar da vector na binaryar shuka cikin nau'in Agrobacterium tumefaciens GV3101 kuma an shigar da shi cikin ganyen Nicotiana benthamiana ta hanyar hanyar shigar Agrobacterium da kuma cikin Arabidopsis thaliana Col-0 ta hanyar tsoma furanni, bi da bi. An ware pUBQ10::qmRGA pUBQ10::GID1a-mCherry da pCLV3::mCherry-NLS qmRGA daga zuriyar F3 da F1 na giciye daban-daban, bi da bi.
An yi amfani da sinadarin RNA a wurin da aka haɗa shi a kan tip ɗin harbi mai tsawon kusan santimita 172, waɗanda aka tattara kuma nan da nan aka gyara su a cikin maganin FAA (3.7% formaldehyde, 5% acetic acid, 50% ethanol) kafin a sanyaya su zuwa digiri 4 na Celsius. Bayan jiyya na minti 2 × 15, an canza abin da aka gyara kuma an saka samfuran a cikin kwantena na dare ɗaya. An haɗa GID1a, GID1b, GID1c, GAI, RGL1, RGL2, da RGL3 cDNAs da kuma binciken antisense ga 3′-UTRs ɗinsu ta amfani da firam ɗin da aka nuna a cikin Tebur na Ƙarin Bayani na 3 kamar yadda Rosier et al.73 suka bayyana. An gano ƙwayoyin cuta masu laƙabi da Digoxigenin ta amfani da ƙwayoyin cuta masu ɗauke da digoxigenin (narkewar ƙwayoyin cuta sau 3000; Roche, lambar kasida: 11 093 274 910), kuma an yi wa sassan fenti da maganin 5-bromo-4-chloro-3-indolyl phosphate (BCIP, narkewar ƙwayoyin cuta sau 250)/nitroblue tetrazolium (NBT, narkewar ƙwayoyin cuta sau 200).
Lokacin Saƙo: Fabrairu-10-2025