Ukukhula kwe-apical meristem (SAM) kubalulekile kuyilo lwesikhondo. Iihomoni zezityaloiigibberellins(ii-GAs) zidlala indima ephambili ekulungelelaniseni ukukhula kwezityalo, kodwa indima yazo kwi-SAM ihlala ingaqondwa kakuhle. Apha, siphuhlise i-ratiometric biosensor ye-GA yokubonisa ngobunjineli iprotein ye-DELLA ukucinezela umsebenzi wayo obalulekileyo wolawulo kwimpendulo ye-GA yokubhala ngelixa igcina ukuthotywa kwayo ekuqatshelweni kwe-GA. Sibonisa ukuba le biosensor esekwe kwi-degradation irekhoda ngokuchanekileyo utshintsho kumanqanaba e-GA kunye nokubona kweselula ngexesha lophuhliso. Sisebenzise le biosensor ukwenza imephu yomsebenzi wokubonisa i-GA kwi-SAM. Sibonisa ukuba imiqondiso ye-GA ephezulu ikhona ikakhulu kwiiseli ezibekwe phakathi kwe-organ primordia, ezingazandulela kwiiseli ze-internode. Ukusebenzisa iindlela zokuzuza kunye nokulahlekelwa komsebenzi, sibonisa ngakumbi ukuba i-GA ilawula ukuqhelaniswa kwendiza yolwahlulo lweeseli, ukuseka umbutho weselula we-canonical we-internodes, ngaloo ndlela ukhuthaza ukucaciswa kwe-internode kwi-SAM.
I-shoot apical meristem (SAM), ekwindawo yokudubula, iqulethe i-niche ye-stem cells umsebenzi wayo uvelisa amalungu asecaleni kunye ne-stem nodes ngendlela eyimodyuli kunye nephindaphindayo kubo bonke ubomi besityalo. Nganye kwezi yunithi ziphinda-phindayo, okanye iindawo zezityalo, zibandakanya ii-internodes kunye namalungu asemacaleni kwiindawo, kunye ne-axillary meristems kwi-axils yamagqabi1. Ukukhula kunye nokuhlelwa kweendawo zezityalo zitshintsha ngexesha lophuhliso. Kwi-Arabidopsis, ukukhula kwe-internodal kunyanzeliswa ngexesha lezityalo, kwaye i-axillary meristems ihlala ilala kwi-axils yamagqabi e-rosette. Ngethuba lokutshintshela kwisigaba seentyatyambo, i-SAM iba yi-inflorescence meristem, ivelisa i-internodes emide kunye ne-axillary buds, i-branchlets kwi-axils yamagqabi e-cauline, kwaye kamva, iintyatyambo ezingenamagqabi2. Nangona siye senza inkqubela ebonakalayo ekuqondeni iindlela ezilawula ukuqaliswa kwamagqabi, iintyatyambo, kunye namasebe, kuncinane okwaziwayo ngendlela ezikhula ngayo ii-internodes.
Ukuqonda ukuhanjiswa kwe-spatiotemporal ye-GAs kuya kunceda ukuqonda kakuhle imisebenzi yala mahomoni kwizicubu ezahlukeneyo kunye nezigaba ezahlukeneyo zophuhliso. Ukubonakala kokuthotywa kwe-RGA-GFP fusion echazwe phantsi kwesenzo somgqugquzeli wayo inikezela ngolwazi olubalulekileyo ekulawuleni amanqanaba e-GA apheleleyo kwiingcambu15,16. Nangona kunjalo, ukubonakaliswa kwe-RGA kuyahluka kwi-tissue17 kwaye ilawulwa yi-GA18. Ngaloo ndlela, ukubonakaliswa okwahlukileyo komgqugquzeli we-RGA kunokubangela ukuba ipateni ye-fluorescence ibonwe nge-RGA-GFP kwaye ngoko le ndlela ayinayo ubuninzi. Kutshanje, i-bioactive fluorescein (Fl) -ebhalwe i-GA19,20 ibonakalise ukuqokelela kwe-GA kwi-endocortex yeengcambu kunye nokulawulwa kwamanqanaba eselula ngothutho lwe-GA. Kutshanje, i-GA FRET sensor nlsGPS1 ibonise ukuba amanqanaba e-GA ahambelana nokwandiswa kweeseli kwiingcambu, iifilaments, kunye ne-hypocotyls21 emnyama. Nangona kunjalo, njengoko sesibonile, ugxininiso lwe-GA ayisiyiyo yodwa ipharamitha elawula umsebenzi wokubonakaliswa kwe-GA, njengoko kuxhomekeke kwiinkqubo zokuva ezintsonkothileyo. Apha, ukwakha ekuqondeni kwethu iindlela zokubonisa i-DELLA kunye ne-GA, sichaza ukuphuhliswa kunye nokubonakaliswa kwe-degradation-based ratiometric biosensor yokubonakalisa i-GA. Ukuphuhlisa le biosensor yobungakanani, sasebenzisa i-GA-sensitive RGA eguqukileyo eyadityaniswa kwiprotheyini ye-fluorescent kwaye yabonakaliswa kuyo yonke indawo kwiithishu, kunye neprotein ye-GA-insensitive fluorescent. Sibonisa ukuba ukuguqulwa kweprotheyini ye-RGA eguquguqukayo ayiphazamisi ukubonakaliswa kwe-GA engapheliyo xa ibonakaliswe kwindawo yonke, kwaye le biosensor inokulinganisa umsebenzi womqondiso ophuma kuzo zombini igalelo le-GA kunye nokusetyenzwa komqondiso we-GA ngesixhobo sokuvalela ngesisombululo esiphezulu se-spatiotemporal. Sisebenzise le biosensor ukwenza imephu yokusasazwa kwe-spatiotemporal yomsebenzi wokubonisa i-GA kunye nokulinganisa indlela i-GA elawula ngayo ukuziphatha kweselula kwi-SAM epidermis. Sibonisa ukuba i-GA ilawula ukuqhelaniswa kwendiza yolwahlulo lweeseli ze-SAM ezibekwe phakathi kwe-organ primordia, ngaloo ndlela ichaza umbutho weselula we-canonical we-internode.
Okokugqibela, sibuze ukuba ingaba i-qmRGA ingaxela na utshintsho kumanqanaba e-GA engapheliyo kusetyenziswa i-hypocotyls ekhulayo. Ngaphambili sabonisa ukuba i-nitrate ikhuthaza ukukhula ngokwandisa i-GA synthesis kwaye, emva koko, ukuthotywa kwe-DELLA34. Ngokufanelekileyo, siye sabona ukuba ubude be-hypocotyl kwi-pUBQ10 :: izithole ze-qmRGA ezikhule phantsi konikezelo lwe-nitrate eninzi (10 mM NO3-) yayinde kakhulu kunezithombo ezikhule phantsi kweemeko ezinqongopheleyo ze-nitrate (Fig. 6a). Ngokuhambelana nempendulo yokukhula, iimpawu ze-GA zaziphezulu kwi-hypocotyls yezithole ezikhuliswe phantsi kweemeko ze-10 mM NO3- kunezithole ezikhule ngokungabikho kwe-nitrate (I-Supplementary Fig. 6b, c). Ke, i-qmRGA ikwavumela ukuba kubekwe iliso kutshintsho kuphawu lwe-GA olubangelwa lutshintsho olungapheliyo kugxininiso lwe-GA.
Ukuqonda ukuba ingaba umsebenzi wokubonisa i-GA ofunyenwe yi-qmRGA ixhomekeke kwi-GA yoxinaniso kunye nombono we-GA, njengoko kulindelekile ngokusekelwe kuyilo lwenzwa, sihlalutye ukubonakaliswa kwee-receptors ezintathu ze-GID1 kwizicubu zezityalo kunye nokuzala. Kwizithole, umgca wentatheli we-GID1-GUS ubonise ukuba i-GID1a kunye ne-c zibonakaliswe kakhulu kwi-cotyledons (Umfanekiso 3a-c). Ukongezelela, zonke i-receptors ezintathu zibonakaliswe ngamagqabi, i-root lateral primordia, iingcebiso zeengcambu (ngaphandle kwe-root cap ye-GID1b), kunye nenkqubo ye-vascular (Fig. 3a-c). Kwi-inflorescence ye-SAM, sifumene iimpawu ze-GUS kuphela kwi-GID1b kunye ne-1c (i-Supplementary Fig. 7a-c). I-In situ hybridization iqinisekise ezi patheni zokubonisa kwaye yabonisa ngakumbi ukuba i-GID1c ibonakaliswe ngokufanayo kumanqanaba aphantsi kwi-SAM, ngelixa i-GID1b ibonise ukubonakaliswa okuphezulu kwi-periphery ye-SAM (i-Supplementary Fig. 7d-l). I-pGID1b:: i-2xmTQ2-GID1b yoguqulelo lwe-fusion iphinde yabonisa uluhlu oluhleliweyo lwentetho ye-GID1b, ukusuka kwintetho ephantsi okanye engekho embindini we-SAM ukuya kwinkcazo ephezulu kwimida ye-organ (Fig. 7m). Ke, ii-receptors ze-GID1 azihanjiswa ngokufanayo ngaphakathi nangaphakathi kwezicubu. Kwiimvavanyo ezilandelayo, siphinde sabona ukuba ukugqithiswa kwe-GID1 (pUBQ10:: GID1a-mCherry) kwandise uvakalelo lwe-qmRGA kwi-hypocotyls kwisicelo sangaphandle se-GA (Umfanekiso 3d, e). Ngokwahlukileyo, i-fluorescence elinganiswe yi-qd17mRGA kwi-hypocotyl yayingenamvakalelo kunyango lwe-GA3 (Umfanekiso 3f, g). Kuzo zombini iimvavanyo, izithole ziphathwe ngokugxininiswa okuphezulu kwe-GA (100 μM GA3) ukuvavanya ukuziphatha ngokukhawuleza kwenzwa, apho ukukwazi ukubopha kwi-receptor ye-GID1 kwandiswa okanye kulahleka. Ngokudibeneyo, ezi ziphumo ziqinisekisa ukuba i-biosensor ye-qmRGA isebenza umsebenzi odibeneyo njenge-GA kunye ne-GA sensor, kwaye iphakamisa ukuba ukubonakaliswa okwahlukileyo kwe-GID1 receptor kunokulungelelanisa kakhulu ukukhutshwa kwenzwa.
Ukuza kuthi ga ngoku, ukuhanjiswa kweempawu ze-GA kwi-SAM akukacaci. Ngoko ke, sasebenzisa izityalo ezibonisa i-qmRGA kunye ne-pCLV3 :: i-mCherry-NLS i-stem cell reporter35 ukubala i-high-resolution quantitative map ye-GA yokubonisa umsebenzi, ugxininise kwi-L1 layer (i-epidermis; Umzobo 4a, b, bona Iindlela kunye neeNdlela ezongezelelweyo), ekubeni i-L1 idlala indima ebalulekileyo ye-SAM. Apha, i-pCLV3 ::inkcazo ye-mCherry-NLS ibonelele ngesalathisi esisisigxina sejometri yokuhlalutya ukuhanjiswa kwe-spatiotemporal ye-GA umsebenzi wokubonisa37. Nangona i-GA ithathwa njengento ebalulekileyo ekuphuhliseni i-lateral organ4, siye sabona ukuba izibonakaliso ze-GA zaziphantsi kwi-primordium yentyatyambo (P) ukusuka kwinqanaba le-P3 (Umfanekiso 4a, b), kanti i-P1 kunye ne-P2 primordiums encinci yayinomsebenzi ophakathi kunye nommandla ophakathi (Umfanekiso 4a, b). Umsebenzi ophakamileyo wokubonakalisa i-GA ufunyenwe kwimida ye-primordium ye-organ, ukuqala kwi-P1 / P2 (emacaleni omda) kunye nokunyuka kwi-P4, kunye nazo zonke iiseli zengingqi ye-peripheral ephakathi kwe-primordia (Umfanekiso 4a, b kunye ne-Supplementary Fig. 8a, b). Lo msebenzi ophezulu wokubonakalisa i-GA awuzange ubonwe kuphela kwi-epidermis kodwa nakwi-L2 kunye ne-L3 ephezulu (i-Supplementary Fig. 8b). Ipatheni yeempawu ze-GA ezifunyenwe kwi-SAM usebenzisa i-qmRGA nazo zahlala zingatshintshi ngokuhamba kwexesha (i-Supplementary Fig. 8c-f, k). Nangona ulwakhiwo lwe-qd17mRGA lwathotywa ngokucwangcisiweyo kwi-SAM yezityalo ze-T3 ukusuka kwimigca emihlanu ezimeleyo esiye sabonakalisa ngokucacileyo, siye sakwazi ukuhlalutya iipateni ze-fluorescence ezifunyenwe nge-pRPS5a :: VENUS-2A-TagBFP ukwakhiwa (I-Supplementary Fig. 8g-j, l). Kulo mgca wokulawula, kuphela utshintsho oluncinci kwi-fluorescence ratio lufunyenwe kwi-SAM, kodwa kwiziko le-SAM sabona ukuhla okucacileyo kunye nokungalindelekanga kwi-VENUS ehambelana ne-TagBFP. Oku kuqinisekisa ukuba ipateni yomqondiso ebonwa yi-qmRGA ibonisa ukuthotywa kwe-GA exhomekeke kwi-mRGA-VENUS, kodwa ikwabonisa ukuba i-qmRGA inokugqithisa umsebenzi wokubonisa i-GA kwiziko le-meristem. Isishwankathelo, iziphumo zethu zityhila ipateni yokubonisa i-GA ebonisa ngokuyintloko ukuhanjiswa kwe-primordia. Oku kusasazwa kommandla we-inter-primordial (IPR) kubangelwa ukusekwa ngokuthe ngcembe kwe-GA ephezulu yokubonisa umsebenzi phakathi kwe-primordium ekhulayo kunye nommandla ophakathi, ngelixa ngexesha elifanayo umsebenzi wokubonakalisa i-GA kwi-primordium uyancipha (Umfanekiso 4c, d).
Ukusasazwa kwe-GID1b kunye ne-GID1c i-receptors (jonga ngasentla) iphakamisa ukuba ukubonakaliswa okungafaniyo kwee-receptors ze-GA kunceda ukubumba iphethini ye-GA yokubonisa umsebenzi kwi-SAM. Siye sazibuza ukuba ingaba ukuqokelelwa kokwahluka kwe-GA kunokubandakanyeka. Ukuphanda oku kunokwenzeka, sisebenzise i-nlsGPS1 GA FRET sensor21. Ukunyuka kwe-activation frequency yafunyanwa kwi-SAM ye-nlsGPS1 iphathwe nge-10 μM GA4 + 7 ye-100 min (i-Fig eyongezelelweyo 9a-e), ebonisa ukuba i-nlsGPS1 iphendula kwiinguqu kwi-GA yoxinaniso kwi-SAM, njengoko isenza kwiingcambu21. Ukusasazwa kwendawo ye-nlsGPS1 i-activation frequency ibonise amanqanaba aphantsi e-GA kwimigangatho yangaphandle ye-SAM, kodwa ibonise ukuba iphakanyiswe embindini nakwimida ye-SAM (Umfanekiso we-4e kunye ne-Supplementary Fig. 9a, c). Oku kuphakamisa ukuba i-GA ikwasasazwa kwi-SAM ngepateni yendawo ethelekiswa naleyo ityhilwe yi-qmRGA. Njengendlela yokuncedisana, siye saphatha i-SAM nge-fluorescent GA (GA3-, GA4-, GA7-Fl) okanye iFl yodwa njengolawulo olubi. Isignali ye-Fl yasasazwa kulo lonke i-SAM, kubandakanywa nommandla ophakathi kunye ne-primordium, nangona kuncinci kakhulu (Umfanekiso we-4j kunye ne-Supplementary Fig. 10d). Ngokwahlukileyo, zonke ezintathu ze-GA-Fl ziqokelelwe ngokukodwa ngaphakathi kwimida ye-primordium kunye namazinga ahlukeneyo kwi-IPR eseleyo, kunye ne-GA7-Fl eqokelela kwi-domain enkulu kwi-IPR (Umfanekiso we-4k kunye ne-Supplementary Fig. 10a, b). I-Quantification of fluorescence intensity ibonise ukuba i-IPR ukuya kwi-non-IPR intensity ratio yayiphezulu kwi-GA-Fl-treated SAM xa kuthelekiswa ne-Fl-treated SAM (Fig. 4l kunye ne-Supplementary Fig. 10c). Ngokudibeneyo, ezi ziphumo zibonisa ukuba i-GA ikhona kwiindawo eziphezulu kwiiseli ze-IPR ezibekwe kufutshane nomda womzimba. Oku kuphakamisa ukuba iphethini ye-SAM GA yokubonisa umsebenzi isiphumo esivela kuzo zombini ukubonakaliswa okwahlukileyo kwe-receptors ye-GA kunye nokuqokelela okwahlukileyo kwe-GA kwiiseli ze-IPR kufuphi nemida yamalungu. Ngaloo ndlela, uhlalutyo lwethu lubonakalise ipateni ye-spatiotemporal engalindelekanga yokubonakaliswa kwe-GA, kunye nomsebenzi ophantsi kwiziko kunye ne-primordium ye-SAM kunye nomsebenzi ophezulu kwi-IPR kummandla we-peripheral.
Ukuze siqonde indima ye-GA yokwahlukana komsebenzi wokubonakalisa uphawu kwi-SAM, sihlalutye ulungelelwaniso phakathi komsebenzi wokubonakaliswa kwe-GA, ukwandiswa kweeseli, kunye nokwahlula kweeseli kusetyenziswa i-real-time-lapse imaging ye-SAM qmRGA pCLV3 :: mCherry-NLS. Ukunikezelwa kwendima ye-GA kulawulo lokukhula, ulungelelwaniso oluhle kunye neeparitha zokwandisa iiseli kulindeleke. Ke ngoko, saqala sathelekisa iimephu ze-GA zokubonisa imephu kunye neemephu zenqanaba lokukhula komphezulu weseli (njengommeleli wokomelela kokwandiswa kweeseli kwiseli enikiweyo kunye neeseli zentombi kwisahlulo) kunye neemephu zokukhula kwe-anisotropy, elinganisa umkhombandlela wokwandiswa kweeseli (ekwasetyenziswa apha kwiseli enikiweyo kunye neeseli zentombi kulwahlulo; Umzobo 5a,b, jonga Iindlela ezongezelelweyo kunye neeNdlela). Iimephu zethu zezinga lokukhula kweeseli ze-SAM zihambelana nokuqwalaselwa kwangaphambili38,39, kunye namazinga amancinci okukhula kumda kunye namazinga aphezulu okukhula ekuphuhliseni iintyatyambo (umzobo 5a). Uhlalutyo lwecandelo eliphambili (PCA) lubonise ukuba umsebenzi wokubonakaliswa kwe-GA wawunxulunyaniswa kakubi kunye nokukhula komgangatho weseli (Umfanekiso 5c). Siphinde sabonisa ukuba ii-axes eziphambili zokwahluka, kubandakanywa igalelo lokubonisa i-GA kunye nokuqina kokukhula, yayiyi-orthogonal ukuya kwisalathiso esinqunywe yintetho ye-CLV3 ephezulu, eqinisekisa ukukhutshwa kweeseli kwiziko le-SAM kuhlalutyo oluseleyo. Uhlalutyo lokulungelelaniswa kwe-Spearman luqinisekisile iziphumo ze-PCA (Umfanekiso we-5d), ebonisa ukuba iimpawu eziphezulu ze-GA kwi-IPR azizange zibangele ukwanda kweeseli eziphezulu. Nangona kunjalo, uhlalutyo lolungelelwaniso luveze ulungelelwaniso oluncinci olulungileyo phakathi komsebenzi wokubonakaliswa kwe-GA kunye nokukhula kwe-anisotropy (Umfanekiso 5c, d), ecebisa ukuba umqondiso we-GA ophezulu kwi-IPR uphembelela isalathiso sokukhula kweeseli kwaye mhlawumbi indawo yendiza yolwahlulo lweeseli.
a, b Iimephu zobushushu zentsingiselo yokukhula komphezulu (a) kunye ne-anisotropy yokukhula (b) kwi-SAM i-avareji kwizityalo ezisixhenxe ezizimeleyo (ezisetyenziswa njenge proxies ukomelela kunye nesalathiso sokwandiswa kweeseli, ngokulandelelanayo). c Uhlalutyo lwe-PCA lubandakanya oku kuguquguqukayo okulandelayo: isignali ye-GA, ukuqina kokukhula komphezulu, i-anisotropy yokukhula komphezulu, kunye nokubonakaliswa kwe-CLV3. Icandelo le-PCA le-1 lalinxulunyaniswe ngokungalunganga kunye nokuqina kokukhula komphezulu kunye nokuhambelana kakuhle nomqondiso we-GA. I-PCA component 2 yayinxulunyaniswe ngokufanelekileyo kunye ne-anisotropy yokukhula komphezulu kwaye ihambelana kakubi ne-CLV3 yokubonisa. Iipesenti zibonisa ukwahluka okucaciswe licandelo ngalinye. d Uhlalutyo lokulungelelaniswa kwe-Spearman phakathi komqondiso we-GA, ukuqina kokukhula komphezulu, kunye ne-anisotropy yokukhula komphezulu kwisikali se-tissue ngaphandle kwe-CZ. Inani elisekunene yi Spearman rho ixabiso phakathi kwezinto ezimbini ezahlukeneyo. Iinkwenkwezi zibonisa iimeko apho unxulumano/unxulumano olubi lubaluleke kakhulu. e Ukubonwa kwe-3D yeeseli ze-Col-0 SAM L1 nge-confocal microscopy. Iindonga zeeseli ezintsha ezenziwe kwi-SAM (kodwa kungekhona i-primordium) kwi-10 h zinemibala ngokwemilinganiselo yazo. Ibha yombala iboniswe kwikona esezantsi ekunene. I-inset ibonisa umfanekiso we-3D ohambelanayo kwi-0 h. Uvavanyo luphindwe kabini ngeziphumo ezifanayo. f Iibhokisi zebhokisi zibonisa amazinga okwahlukana kweeseli kwi-IPR kunye ne-non-IPR Col-0 SAM (n = 10 izityalo ezizimeleyo). Umgca ophakathi ubonisa i-median, kwaye imida yebhokisi ibonisa ipesenti ze-25 kunye ne-75. I-Whiskers ibonisa ubuncinci kunye namaxabiso aphezulu agqitywe nge-software ye-R. Amaxabiso e-P afunyenwe ngovavanyo luka-Welch olunemisila emibini. g, h Umzobo weSchematic obonisa (g) indlela yokulinganisa i-engile yodonga lweseli entsha (magenta) ngokubhekiselele kulwalathiso lwe-radial ukusuka kumbindi we-SAM (umgca onamachaphaza amhlophe) (kuphela amaxabiso e-angle acute, okt, 0–90 °, ayaqwalaselwa), kunye (h) ne-circumferential/lateral and radial directions within themeristem. i I-histograms eziphindaphindiweyo ze-cell division plane orientation kwi-SAM (eluhlaza okwesibhakabhaka), i-IPR (i-blue blue), kunye ne-non-IPR (eluhlaza okwesibhakabhaka), ngokulandelanayo. Amaxabiso e-P afunyenwe ngovavanyo olunemisila emibini ye-Kolmogorov-Smirnov. Uvavanyo luphindwe kabini ngeziphumo ezifanayo. j Iihistograms zamaxesha okuqhelaniswa nenqwelomoya yolwahlulo lweseli ye-IPR malunga ne-P3 (uhlaza olukhanyayo), i-P4 (uhlaza oluphakathi), kunye ne-P5 (luhlaza mnyama), ngokulandelelanayo. Amaxabiso e-P afunyenwe ngovavanyo olunemisila emibini ye-Kolmogorov-Smirnov. Uvavanyo luphindwe kabini ngeziphumo ezifanayo.
Ngoko ke, ngokulandelayo siphande ulungelelwaniso phakathi kokubonakaliswa kwe-GA kunye nomsebenzi wokwahlula iiseli ngokuchonga iindonga zeseli ezisanda kwakhiwa ngexesha lokuvavanya (umzobo 5e). Le ndlela yasivumela ukuba silinganise ukuphindaphinda kunye nesalathiso sokwahlukana kweeseli. Okumangalisa kukuba, sifumene ukuba ukuphindaphinda kwezahlulo zeeseli kwi-IPR kunye nayo yonke i-SAM (engekho i-IPR, i-Fig. 5f) yayifana, ebonisa ukuba ukungafani kweempawu ze-GA phakathi kwe-IPR kunye ne-non-IPR iiseli azichaphazeli kakhulu ukuhlukana kweeseli. Oku, kunye nokulungelelaniswa okuhle phakathi kokubonakaliswa kwe-GA kunye nokukhula kwe-anisotropy, kwasishukumisela ukuba sicinge ukuba umsebenzi wokubonakalisa i-GA unokuchaphazela ukuqhelaniswa kwendiza yokwahlukana kweeseli. Silinganise ukuqhelaniswa kodonga olutsha lweseli njenge-angle ebukhali ehambelana ne-radial axis edibanisa i-meristem center kunye nombindi wodonga olutsha lweseli (umzobo 5e-i) kwaye saqaphela ukuthambekela okucacileyo kweeseli ukuba zihlulwe kwii-angles ezikufutshane ne-90 ° ngokumalunga ne-radial axis, kunye ne-frequencies ephezulu ebonwa kwi-70-80-80 ° 62% (2%) kunye ne-70. (Umfanekiso 5e, i), ehambelana nokwahlula kweeseli kwi-circumferential / transverse direction (Fig. 5h). Ukuphonononga igalelo le-GA ukubonakaliswa kolu kuziphatha lolwahlulo lweeseli, sihlalutye iiparamitha zokwahlula kweeseli kwi-IPR kunye ne-non-IPR ngokwahlukileyo (umzobo 5i). Siye saqaphela ukuba ukuhanjiswa kwe-angle yolwahlulo kwiiseli ze-IPR zahluke kwiiseli ezingezizo ze-IPR okanye kwiiseli kwi-SAM yonke, kunye neeseli ze-IPR ezibonisa umlinganiselo ophezulu we-lateral / circular cell divisions, oko kukuthi, i-70-80 ° kunye ne-80-90 ° (33.86% kunye ne-30.71%, ngokulandelanayo, imilinganiselo ehambelanayo). Ngaloo ndlela, ukujonga kwethu kubonakalise umbutho phakathi kwe-GA ephezulu yokubonisa kunye ne-cell division plane orientation kufuphi ne-circumferential direction, efana nokulungelelaniswa phakathi komsebenzi we-GA wokubonisa kunye nokukhula kwe-anisotropy (Fig. 5c, d). Ukuqhubela phambili ukuseka ukulondolozwa kwendawo yalo mbutho, silinganise ukuqhelaniswa kwendiza yokwahlula kwiiseli ze-IPR ezijikeleze i-primordium eqala kwi-P3, ekubeni umsebenzi ophezulu wokubonakalisa i-GA ufunyenwe kulo mmandla ukususela kwi-P4 (umzobo 4). I-angles yokwahlula kwe-IPR malunga ne-P3 kunye ne-P4 ibonise ukuba akukho ntlukwano ephawulekayo, nangona ukunyuka kwe-frequency ye-lateral cell division kubonwe kwi-IPR malunga ne-P4 (Umfanekiso 5j). Nangona kunjalo, kwiiseli ze-IPR ezijikeleze i-P5, umehluko kwindlela yokuqhelaniswa nendiza ye-cell division yaba yinto ephawulekayo, ngokunyuka okubukhali kwi-frequency of transverse cell divisions (Fig. 5j). Ngokudibeneyo, ezi ziphumo zibonisa ukuba ukubonakaliswa kwe-GA kunokulawula ukuqhelaniswa kwezahlulo zeeseli kwi-SAM, ehambelanayo neengxelo zangaphambili40,41 ukuba ukubonakalisa i-GA ephezulu kunokubangela ukuqhelaniswa kwecala lokuhlukana kweeseli kwi-IPR.
Kuqikelelwa ukuba iiseli kwi-IPR aziyi kufakwa kwi-primordia kodwa endaweni ye-internodes2,42,43. I-orientation enqamlezileyo yolwahlulo lweeseli kwi-IPR inokubangela umbutho oqhelekileyo wemiqolo emide enxuseneyo yeeseli ze-epidermal kwii-internodes. Uqwalaselo lwethu oluchazwe ngasentla lucebisa ukuba umqondiso we-GA unokudlala indima kule nkqubo ngokulawula isalathiso sokwahlulwa kweeseli.
Ukulahlekelwa ngumsebenzi weemfuza ezininzi ze-DELLA kubangela impendulo ye-GA ekhoyo, kwaye i-della mutants ingasetyenziselwa ukuvavanya le ngcamango44. Siqale sahlalutya iipateni zokuvakalisa iijene ze-DELLA ezintlanu kwi-SAM. I-Transcriptional fusion ye-GUS line45 ibonise ukuba i-GAI, i-RGA, i-RGL1, kunye ne-RGL2 (incinci kakhulu) ibonakaliswe kwi-SAM (i-Supplementary Fig. 11a-d). I-In situ hybridization ibonise ngakumbi ukuba i-GAI mRNA iqokelelana ngokukodwa kwi-primordia kunye nokuphuhlisa iintyatyambo (Supplementary Fig. 11e). I-RGL1 kunye ne-RGL3 mRNA ziye zachongwa kuyo yonke ikhenopi ye-SAM nakwiintyatyambo ezindala, ngelixa i-RGL2 mRNA yayininzi kakhulu kummandla wemida (I-Supplementary Fig. 11f–h). I-confocal imaging ye-pRGL3 :: RGL3-GFP SAM iqinisekisile intetho ebonwe yi-situ hybridization kwaye yabonisa ukuba iprotheni ye-RGL3 iqokelela kwindawo ephakathi ye-SAM (i-Supplementary Fig. 11i). Ukusebenzisa i-pRGA :: Umgca we-GFP-RGA, siphinde safumanisa ukuba iprotheni ye-RGA iqokelela kwi-SAM, kodwa ubuninzi bayo buyancipha kumda oqala kwi-P4 (i-Supplementary Fig. 11j). Ngokucacileyo, iipatheni zokubonisa i-RGL3 kunye ne-RGA zihambelana nomsebenzi ophezulu wokubonisa i-GA kwi-IPR, njengoko ifunyenwe yi-qmRGA (umzobo 4). Ngaphezu koko, ezi datha zibonisa ukuba zonke ii-DELLA zichazwe kwi-SAM kwaye intetho yazo ngokudibeneyo ithatha yonke i-SAM.
Ngokulandelayo sihlalutye iiparamitha zokwahlula kweeseli kwi-SAM yasendle (Ler, ulawulo) kunye ne-gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Fig. 6a, b). Okubangel 'umdla kukuba, siye sabona utshintsho olubalulekileyo lwezibalo ekuhanjisweni kwee-engile ze-cell division frequencies kwi-della global mutant SAM xa kuthelekiswa nohlobo lwasendle (Fig. 6c). Olu tshintsho kwi-della global mutant ngenxa yokunyuka kwe-frequency ye-80-90 ° angles (34.71% vs. 24.55%) kwaye, kwinqanaba elingaphantsi, i-70-80 ° angles (23.78% vs. 20.18%), okt, ehambelana nokuhlukana kweeseli ezinqamlekileyo (Umfanekiso 6c). I-frequency of non-transverse divisions (0-60 °) nayo yayiphantsi kwi-della global mutant (Umfanekiso 6c). Ubuninzi bezahlulo zeeseli ezinqamlezayo zanda kakhulu kwi-SAM ye-della global mutant (Umfanekiso 6b). Ubuninzi bezahlulo zeeseli ezinqamlezayo kwi-IPR zaziphezulu kwi-della global mutant xa kuthelekiswa nohlobo lwasendle (Umfanekiso 6d). Ngaphandle kommandla we-IPR, uhlobo lwasendle lwalunokusasazwa okufanayo kwee-angles ze-cell division, kanti i-della global mutant ikhetha ulwahlulo lwe-tangential njenge-IPR (Fig. 6e). Siphinde silinganise ukuqhelaniswa kwezahlulo zeeseli kwi-SAM ye-ga2 oxidase (ga2ox) i-quintuple mutants (ga2ox1-1, i-ga2ox2-1, i-ga2ox3-1, i-ga2ox4-1, kunye ne-ga2ox6-2), i-GA-inactive mutant background apho i-GA iqokelela khona. Ngokuhambelana nokunyuka kwamanqanaba e-GA, i-SAM ye-quintuple ga2ox mutant inflorescence yayinkulu kune-Col-0 (I-Fig. Isazobe 12a–c). Ngaloo ndlela, sibonisa ukuba ukusetyenziswa kwe-GA kunye nokuqokelela kwe-GA kubangela ukuhlukana kweeseli ezisecaleni kwi-IPR kunye nayo yonke i-SAM.
a, b Ukuboniswa kwe-3D ye-L1 umaleko we-PI-stained Ler (a) kunye ne-global della mutant (b) i-SAM usebenzisa i-microscopy edibeneyo. Iindonga zeeseli ezintsha ezenziwe kwi-SAM (kodwa kungekhona i-primordium) kwixesha le-10-h ziboniswa kwaye zinemibala ngokwemilinganiselo yazo. I-inset ibonisa i-SAM ngo-0 h. Ibha yombala iboniswa kwikona esezantsi ekunene. Utolo oluku-(b) lukhomba kumzekelo weefayile zeseli ezilungelelanisiweyo kwi-global della mutant. Uvavanyo luphindwe kabini ngeziphumo ezifanayo. ce uthelekiso lokusasazwa okuphindaphindiweyo kokuqhelaniswa kwendiza yolwahlulo lweeseli kuyo yonke iSAM (d), IPR (e), kunye ne-non-IPR (f) phakathi kweLer kunye ne-global della. Amaxabiso e-P afunyenwe kusetyenziswa uvavanyo lwe-Kolmogorov-Smirnov olunemisila emibini. f, g Umboniso we-3D wemifanekiso ye-confocal ye-PI-stained SAM ye-Col-0 (i) kunye ne-pCUC2:: i-gai-1-VENUS (j) izityalo ze-transgenic. Iiphaneli (a, b) zibonisa iindonga zeeseli ezintsha (kodwa kungekhona i-primordia) ezenziwe kwi-SAM ngaphakathi kwe-10 h. Uvavanyo luphindwe kabini ngeziphumo ezifanayo. h–j Ukuthelekiswa kwe-frequency distribution of cell division plane orientations ebekwe kuyo yonke i-SAM (h), IPR (i) kunye ne-non-IPR (j) phakathi kwe-Col-0 kunye ne-pCUC2:: i-gai-1-VENUS izityalo. Amaxabiso e-P afunyenwe kusetyenziswa uvavanyo lwe-Kolmogorov-Smirnov olunemisila emibini.
Ngokulandelayo sivavanye umphumo wokuthintela ukubonakaliswa kwe-GA ngokukodwa kwi-IPR. Ukuza kuthi ga ngoku, sasebenzisa i-cotyledon cup 2 (CUC2) umgqugquzeli wokuqhuba ukubonakaliswa kweprotheyini ephezulu ye-gai-1 edibeneyo kwi-VENUS (kwi-pCUC2 :: i-gai-1-VENUS line). Kwi-SAM yasendle, umgqugquzeli we-CUC2 uqhuba ukubonakaliswa kwee-IPR ezininzi kwi-SAM, kubandakanywa neeseli zomda, ukusuka kwi-P4 ukuya phambili, kunye nenkcazo efanayo efanayo yabonwa kwi-pCUC2 :: izityalo ze-gai-1-VENUS (jonga ngezantsi). Ukusasazwa kwee-angles ze-cell division kwi-SAM okanye i-IPR ye-pCUC2 :: izityalo ze-gai-1-VENUS azizange zihluke kakhulu kuhlobo lwasendle, nangona ngokungalindelekanga sifumene ukuba iiseli ezingenayo i-IPR kulezi zityalo zihlulwe kwi-frequency ephezulu ye-80-90 ° (Umfanekiso 6f-j).
Kuye kwacetyiswa ukuba ulwalathiso lolwahlulo lweeseli luxhomekeke kwijometri ye-SAM, ngokukodwa ukunyanzeliswa koxinzelelo olwenziwa yi-tissue curvature46. Ngoko ke sibuze ukuba imo ye-SAM yatshintshwa kwi-della global mutant kunye ne-pCUC2:: izityalo ze-gai-1-VENUS. Njengoko bekuxeliwe ngaphambili12, ubungakanani be-della global mutant SAM yayinkulu kunohlobo lwasendle (Fig. 13a, b, d). I-In situ hybridization ye-CLV3 kunye ne-STM RNA iqinisekisile ukwandiswa kwe-meristem kwi-della mutants kwaye yabonisa ngakumbi ukwandiswa kwecala le-stem cell niche (i-Supplementary Fig. 13e, f, h, i). Nangona kunjalo, i-SAM curvature yayifana kuzo zombini i-genotypes (i-Supplementary Fig. 13k, m, n, p). Siye sabona ukunyuka okufanayo kobukhulu kwi-gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant ngaphandle kokutshintsha kwi-curvature xa kuthelekiswa nohlobo lwasendle (I-Supplementary Fig. 13c, d, g, j, l, o, p). I-frequency ye-cell division orientation orientation nayo yachaphazeleka kwi-della quadruple mutant, kodwa kwinqanaba elingaphantsi kune-della monolithic mutant (i-Supplementary Fig. 12d-f). Esi siphumo sedosi, kunye nokungabikho kwempembelelo kwi-curvature, iphakamisa ukuba umsebenzi oseleyo we-RGL3 kwi-Della quadruple mutant imida yokutshintsha utshintsho kwi-cell division orientation ebangelwa ukulahlekelwa ngumsebenzi we-DELLA kunye nokuba utshintsho kwi-lateral cell divisions lwenzeka ekuphenduleni utshintsho kwi-GA yokubonisa umsebenzi kunokuba utshintsho kwi-SAM geometry. Njengoko kuchazwe ngasentla, umgqugquzeli we-CUC2 uqhuba ukubonakaliswa kwe-IPR kwi-SAM eqala kwi-P4 (i-Supplementary Fig. 14a, b), kwaye ngokuchaseneyo, i-pCUC2 :: i-gai-1-VENUS SAM yayinobukhulu obuncitshisiweyo kodwa i-curvature ephezulu (i-Supplementary Fig. 14c-h). Olu tshintsho kwi-pCUC2 :: i-gai-1-VENUS i-morphology ye-SAM inokubangela ukusabalalisa okungafaniyo koxinzelelo lomatshini xa kuthelekiswa nohlobo lwasendle, apho uxinzelelo oluphezulu olujikelezayo luqala kumgama omfutshane ukusuka kwiziko le-SAM47. Ngenye indlela, utshintsho kwi-pCUC2 :: i-gai-1-VENUS i-SAM morphology inokubangelwa utshintsho kwiipropati zengingqi zomatshini ezibangelwa yi-transgene expression48. Kuzo zombini ezi meko, oku kunokuphelisa ngokuyinxenye iziphumo zotshintsho kuphawu lwe-GA ngokwandisa amathuba okuba iiseli zahlukane kumjikelo ojikelezayo/oguqukileyo, sicacisa imigqaliselo yethu.
Kuthatyathwe kunye, idatha yethu iqinisekisa ukuba umqondiso ophezulu we-GA udlala indima esebenzayo kwi-lateral orientation yendiza ye-cell division kwi-IPR. Bakwabonisa ukuba i-meristem curvature ikwaphembelela ukuqhelaniswa kwendiza yolwahlulo lweeseli kwi-IPR.
I-interverse orientation ye-division plane kwi-IPR, ngenxa ye-GA ephezulu yokubonisa umsebenzi, iphakamisa ukuba i-GA iququzelele kwangaphambili ifayile ye-radial cell kwi-epidermis ngaphakathi kwe-SAM ukucacisa umbutho weselula oya kufumaneka kamva kwi-epidermal internode. Enyanisweni, ezo fayile zeseli zazihlala zibonakala kwimifanekiso ye-SAM ye-della global mutants (Umfanekiso 6b). Ngaloo ndlela, ukuqhubela phambili ukuphonononga umsebenzi wophuhliso wepateni yendawo yokubonisa i-GA kwi-SAM, sasebenzisa i-imaging ye-time-lapse ukuhlalutya intlangano yendawo yeeseli kwi-IPR kwi-wild-type (Ler kunye ne-Col-0), i-della global mutants, kunye ne-pCUC2:: i-gai-1-VENUS izityalo ze-transgenic.
Sifumene ukuba i-qmRGA ibonise ukuba umsebenzi wokubonisa i-GA kwi-IPR yanda ukusuka kwi-P1 / P2 kwaye yanyuka kwi-P4, kwaye le pateni yahlala ihleli ixesha elide (umzobo 4a-f kunye ne-Supplementary Fig. 8c-f, k). Ukuhlalutya umbutho wendawo yeeseli kwi-IPR ngokunyuka kwesibonakaliso se-GA, sibhale iiseli ze-Ler IPR ngasentla kunye namacala e-P4 ngokwexesha labo lophuhliso elihlalutyelwe kwi-34 h emva kokuqwalaselwa kokuqala, oko kukuthi, ngaphezu kwamaxesha amabini e-plastid, okusivumela ukuba silandele iiseli ze-IPR ngexesha lophuhliso lwe-primordium ukusuka kwi-P1 / P2 ukuya kwi-P4. Sasebenzisa imibala emithathu eyahlukeneyo: tyheli kwezo seli zidityaniswe kwi-primordium kufuphi ne-P4, eluhlaza kwezo zazikwi-IPR, kunye nemfusa kwabo bathatha inxaxheba kuzo zombini iinkqubo (Umfanekiso 7a-c). Kwi-t0 (0 h), i-1-2 iileyile zeeseli ze-IPR zazibonakala phambi kwe-P4 (umzobo 7a). Njengoko bekulindelekile, xa ezi seli zahlulahlula, zenza oko ikakhulu nge-plane yokwahlula okunqamlezayo (Imifanekiso 7a-c). Iziphumo ezifanayo zifunyenwe kusetyenziswa i-Col-0 SAM (igxininise kwi-P3, i-folders yayo yomda ngokufana ne-P4 e-Ler), nangona kule genotype i-fold fold yakhiwe kumda weentyatyambo ifihle iiseli ze-IPR ngokukhawuleza (Fig. 7g-i). Ke, ipateni yolwahlulo lweeseli ze-IPR zicwangcisa kwangaphambili iiseli zibe yimiqolo yeradial, njengakwi-internodes. Ukulungelelaniswa kwemiqolo yeradial kunye nokubekwa kwendawo kweeseli ze-IPR phakathi kwamalungu alandelelanayo kubonisa ukuba ezi seli zi-internodal progenitors.
Apha, siphuhlise i-ratiometric GA yokubonisa i-biosensor, i-qmRGA, evumela ukuba imephu yobungakanani bomsebenzi womqondiso we-GA obangelwa kugxininiso lwe-GA kunye ne-GA ye-receptor ngelixa unciphisa ukuphazamiseka kweendlela ze-endogenous signaling, ngaloo ndlela zibonelela ngolwazi malunga nomsebenzi we-GA kwinqanaba leselula. Ukuza kuthi ga ngoku, sakhe iprotein ye-DELLA elungisiweyo, i-mRGA, ephulukene namandla okubopha amaqabane entsebenziswano ye-DELLA kodwa ihlala inovelwano kwi-GA-induced proteolysis. I-qmRGA iphendula kuzo zombini iinguqu zangaphandle kunye nezangaphandle kumanqanaba e-GA, kunye neempawu zayo eziguquguqukayo zezivamvo zenza uvavanyo lotshintsho lwespatiotemporal kumsebenzi wophawu lwe-GA ngexesha lophuhliso. I-qmRGA ikwasisixhobo esibhetyebhetye kakhulu njengoko sinokulungelelaniswa kwizicubu ezahlukeneyo ngokuguqula umgqugquzeli osetyenziselwe ukubonakaliswa kwayo (ukuba kukho imfuneko), kwaye inikwe ubume obugciniweyo bendlela yokubonisa i-GA kunye ne-PFYRE motif kwii-angiosperms, kunokwenzeka ukuba idluliselwe kwezinye iintlobo ze-22. Ngokungqinelana noku, uguqulo olulinganayo kwiprotein ye-SLR1 DELLA yerayisi (HYY497AAA) nayo yaboniswa ukucinezela umsebenzi wokucinezela ukukhula kwe-SLR1 ngelixa inciphisa kancinci ukuthotywa kwayo kwe-GA, efana ne-mRGA23. Ngokucacileyo, izifundo zamva nje kwi-Arabidopsis zibonise ukuba ukuguqulwa kwe-amino acid enye kwindawo ye-PFYRE (S474L) iguqule umsebenzi wokukhutshelwa kwe-RGA ngaphandle kokuchaphazela amandla ayo okunxibelelana namaqabane ezinto ezishicilelweyo50. Nangona olu tshintsho lusondele kakhulu kwi-3 amino acid substitution ekhoyo kwi-mRGA, izifundo zethu zibonisa ukuba ezi zinguqu zimbini ziguqula iimpawu ezahlukileyo ze-DELLA. Nangona uninzi lwamaqabane oshicilelo lubophelela kwi-LHR1 kunye nemimandla ye-SAW ye-DELLA26,51, ezinye ii-amino acids ezigciniweyo kwi-domain ye-PFYRE zinokunceda ukuzinzisa olu nxibelelwano.
Uphuhliso lwe-Internode luphawu oluphambili kuyilo lwezityalo kunye nokuphuculwa kwesivuno. I-qmRGA ibonakalise umsebenzi ophezulu wokubonisa i-GA kwiiseli ze-IPR internode progenitor. Ngokudibanisa i-imaging yobuninzi kunye ne-genetics, sibonise ukuba iipatheni zokubonisa i-GA zenza iiplani zesetyhula / ezinqamlezayo zokwahlukana kweeseli kwi-epidermis ye-SAM, ukubumba umbutho wokwahlula iiseli ezifunekayo kuphuhliso lwe-internode. Izilawuli ezininzi zokuqhelaniswa kwendiza yolwahlulo lweeseli ziye zachongwa ngexesha lophuhliso52,53. Umsebenzi wethu ubonelela ngomzekelo ocacileyo wendlela umsebenzi wokubonakaliswa kwe-GA ulawula le parameter yeselula. I-DELLA inokusebenzisana ne-protein yangaphambili ye-protein complexes41, ngoko ukubonakaliswa kwe-GA kunokulawula ukuqhelaniswa kwendiza ye-cell division ngokuchaphazela ngokuthe ngqo i-cortical microtubule orientation40,41,54,55. Sibonise ngokungalindelekanga ukuba kwi-SAM, i-correlate ye-GA ephezulu yokubonisa umsebenzi yayingekho i-cell elongation okanye i-division, kodwa i-anisotropy yokukhula kuphela, ehambelana nefuthe elithe ngqo le-GA kwisalathiso sokuhlukana kweeseli kwi-IPR. Nangona kunjalo, asinakukhuphela ngaphandle ukuba esi siphumo sinokungathanga ngqo, umzekelo ukulamlwa yi-GA-induced cell wall softening56. Utshintsho kwiipropathi zodonga lweseli lubangela uxinzelelo lomatshini57,58, olunokuthi luchaphazele ukuqhelaniswa kwendiza yolwahlulo lweeseli ngokuchaphazela ukuqhelaniswa kwe-cortical microtubules39,46,59. Iziphumo ezidibeneyo ze-GA-induced mechanical stress kunye nokulawulwa ngokuthe ngqo kwe-microtubule orientation yi-GA inokubandakanyeka ekuveliseni iphethini ethile ye-cell division orientation kwi-IPR ukuchaza i-internodes, kunye nezifundo ezongezelelweyo zifunekayo ukuvavanya le ngcamango. Ngokufanayo, izifundo zangaphambili zibonise ukubaluleka kwe-DELLA-interacting proteins TCP14 kunye ne-15 ekulawuleni i-internode formation60,61 kwaye ezi zinto zinokuthi zidibanise isenzo se-GA kunye ne-BREVIPEDICELLUS (BP) kunye ne-PENNYWISE (PNY), elawula ukuphuhliswa kwe-internode kwaye ibonakaliswe ukuba ichaphazele i-2GA22. Ngenxa yokuba i-DELLAs isebenzisana ne-brassinosteroid, i-ethylene, i-jasmonic acid, kunye ne-abscisic acid (ABA) indlela yokubonisa indlela63,64 kunye nokuba ezi hormone zinokuphembelela i-microtubule orientation65, iziphumo ze-GA kwi-cell division orientation inokuthi ixutywe ngamanye amahomoni.
Izifundo zangaphambili ze-cytological zibonise ukuba zombini imimandla yangaphakathi nangaphandle ye-Arabidopsis SAM iyadingeka kuphuhliso lwe-internode2,42. Inyaniso yokuba i-GA ilawula ngokusebenzayo ukuhlukana kweeseli kwizicubu zangaphakathi12 isekela umsebenzi obini we-GA ekulawuleni i-meristem kunye nobukhulu be-internode kwi-SAM. Ipateni yokwahlula kweeseli eziqondisayo iphinde ilawuleke ngokuqinileyo kwizicubu ze-SAM zangaphakathi, kwaye lo mmiselo uyimfuneko ekukhuleni kwesiqu52. Kuya kuba nomdla ukujonga ukuba ngaba i-GA nayo idlala indima ekuqhelaniseni indiza yolwahlulo lweeseli kumbutho wangaphakathi we-SAM, ngaloo ndlela ingqamanisa ukucaciswa kunye nophuhliso lwe-internodes ngaphakathi kwe-SAM.
Izityalo zakhuliswa kwi-vitro emhlabeni okanye i-1x Murashige-Skoog (MS) medium (Duchefa) yongezwa nge-1% sucrose kunye ne-1% ye-agar (Sigma) phantsi kweemeko eziqhelekileyo (ukukhanya kwe-16 h, i-22 °C), ngaphandle kwe-hypocotyl kunye nemifuniselo yokukhula kweengcambu apho izithole zakhuliswa kwiipleyiti ezithe nkqo phantsi kokukhanya okungaguqukiyo kwe-22 ° C. Kwiimvavanyo ze-nitrate, izityalo zakhuliswa kwi-MS medium elungisiweyo (i-bioWORLD plant medium) yongezwa nge-nitrate eyaneleyo (0 okanye 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose kunye ne-1% A-agar (Sigma) phantsi kweemeko zeentsuku ezinde.
I-GID1a cDNA efakwe kwi-pDONR221 yaphinda yadityaniswa ne-pDONR P4-P1R-pUBQ10 kunye ne-pDONR P2R-P3-mCherry kwi-pB7m34GW ukuvelisa i-pUBQ10 :: GID1a-mCherry. I-IDD2 DNA efakwe kwi-pDONR221 yaphinda yadityaniswa kwi-pB7RWG266 ukuvelisa i-p35S:IDD2-RFP. Ukuvelisa i-pGID1b::2xmTQ2-GID1b, iqhekeza le-3.9 kb phezulu kommandla wekhowudi ye-GID1b kunye neqhekeza le-4.7 kb eliqulethe i-GID1b cDNA (1.3 kb) kunye nesiterminator (3.4 kb) zaqala zandiswa kusetyenziswa iiprimers kwi-Supplementary pDOP4 (I-Thermo Fisher Scientific) kunye ne-pDONR P2R-P3 (i-Thermo Fisher Scientific), ngokulandelelana, kwaye ekugqibeleni yadityaniswa ne-pDONR221 2xmTQ268 kwi-pGreen 012567 ye-vector ekujoliswe kuyo usebenzisa i-Gateway cloning. Ukuvelisa i-pCUC2::LSSmOrange, ulandelelwano lomgqugquzeli weCUC2 (3229 bp phezulu komsinga we-ATG) lulandelwa lulandelelwano lwekhowudi ye-Stokes-shifted mOrange enkulu (LSSmOrange)69 kunye nomqondiso we-N7 we-nuclear yasekhaya kunye ne-NOS transcriptional terminator ziye zadityaniswa kwi-tagement ye-tagement ye-3 okanye i-Green com. (I-Invitrogen). Isityalo se-binary vector saziswa kwi-Agrobacterium tumefaciens strain GV3101 kwaye yaziswa kumagqabi e-Nicotiana benthamiana nge-Agrobacterium infiltration method kunye ne-Arabidopsis thaliana Col-0 ngendlela yediphu yeentyatyambo, ngokulandelelanayo. I-pUBQ10 :: qmRGA pUBQ10 :: GID1a-mCherry kunye ne-pCLV3 :: mCherry-NLS qmRGA zaye zahlukaniswa kwi-F3 kunye ne-F1 inzala yee-cross ezihlukeneyo, ngokulandelanayo.
I-RNA in situ hybridization yenziwa malunga ne-1 cm ubude bokudubula tips72, eziye zaqokelelwa zaza zalungiswa ngokukhawuleza kwisisombululo se-FAA (3.7% ye-formaldehyde, i-5% ye-acetic acid, i-50% ye-ethanol) epholileyo kwi-4 ° C. Emva kwe-2 × i-15 min yonyango lwe-vacuum, ukulungiswa kwatshintshwa kwaye iisampuli zafakwa ngobusuku. I-GID1a, i-GID1b, i-GID1c, i-GAI, i-RGL1, i-RGL2, kunye ne-RGL3 cDNAs kunye ne-antisense probes kwi-3'-UTRs yazo zenziwe zisebenzisa ii-primers eziboniswe kwi-Supplementary Table 3 njengoko kuchazwe nguRosier et al.73. Iiprobes ezibhalwe i-Digoxigenin zifakwe kwi-immunodetected usebenzisa i-digoxigenin antibodies (i-3000-fold dilution; Roche, inombolo yekhathalogu: 11 093 274 910), kwaye amacandelo ahlanjululwe nge-5-bromo-4-chloro-3-indolyl phosphate (BCIP, 250-BTlution, 250-dibluetrolution) Isisombululo se-200-fold dilution.
Ixesha lokuposa: Feb-10-2025