He mea koʻikoʻi ka ulu ʻana o ka apical meristem (SAM) no ke kūkulu ʻana i ke kumu. Nā hormones o nā mea kanunā gibberellins(GAs) ke kuleana koʻikoʻi i ka hoʻonohonoho ʻana i ka ulu ʻana o nā mea kanu, akā ʻaʻole maopopo loa ko lākou kuleana ma SAM. Maanei, ua hoʻomohala mākou i kahi biosensor ratiometric o ka hōʻailona GA ma ka ʻenekinia ʻana i ka protein DELLA e kāohi i kāna hana hoʻoponopono koʻikoʻi i ka pane transcriptional GA ʻoiai e mālama ana i kona hōʻino ʻia ma ka ʻike ʻana o GA. Hōʻike mākou e hoʻopaʻa pololei ana kēia biosensor e pili ana i ka hōʻino ʻana i nā loli i nā pae GA a me ka ʻike ʻana o ke kelepona i ka wā o ka hoʻomohala ʻana. Ua hoʻohana mākou i kēia biosensor e palapala i ka hana hōʻailona GA ma SAM. Hōʻike mākou aia nā hōʻailona GA kiʻekiʻe ma nā pūnaewele i loaʻa ma waena o ka primordia organ, ʻo ia nā precursors i nā pūnaewele internode. Ma ka hoʻohana ʻana i nā ʻano hana loaʻa a me ka nalowale o ka hana, hōʻike hou mākou e hoʻoponopono ana ʻo GA i ke kuhikuhi ʻana o ka mokulele mahele cell, e hoʻokumu ana i ka hoʻonohonoho cellular canonical o nā internodes, no laila e hoʻolaha ana i ka kikoʻī internode ma SAM.
ʻO ka meristem apical shoot (SAM), aia ma ka piko o ka shoot, aia kahi niche o nā cell kumu nona ka hana e hoʻopuka i nā ʻōpū lateral a me nā node kumu ma ke ʻano modular a iterative i ke ola o ka mea kanu. ʻO kēlā me kēia o kēia mau ʻāpana hana hou, a i ʻole nā node mea kanu, e komo pū ana me nā internodes a me nā ʻōpū lateral ma nā nodes, a me nā meristems axillary i nā axils lau1. Hoʻololi ka ulu ʻana a me ka hoʻonohonoho ʻana o nā node mea kanu i ka wā o ka hoʻomohala ʻana. Ma Arabidopsis, ua kāohi ʻia ka ulu ʻana o ka internodal i ka wā vegetative, a noho mau nā meristems axillary i nā axils o nā lau rosette. I ka wā o ka hoʻololi ʻana i ka wā pua, lilo ka SAM i meristem inflorescence, e hoʻopuka ana i nā internodes elongated a me nā ʻōpuʻu axillary, nā lālā ma nā axils o nā lau cauline, a ma hope, nā pua lau ʻole2. ʻOiai ua holomua nui mākou i ka hoʻomaopopo ʻana i nā ʻano hana e kāohi ai i ka hoʻomaka ʻana o nā lau, nā pua, a me nā lālā, ʻaʻole maopopo iki e pili ana i ke ʻano o ka puka ʻana o nā internodes.
ʻO ka hoʻomaopopo ʻana i ka hoʻolaha spatiotemporal o nā GA e kōkua i ka hoʻomaopopo maikaʻi ʻana i nā hana o kēia mau hormones i nā ʻiʻo like ʻole a ma nā pae hoʻomohala like ʻole. ʻO ka ʻike ʻana i ka hōʻino ʻana o ka hui ʻana o RGA-GFP i hōʻike ʻia ma lalo o ka hana a kāna mea hoʻolaha ponoʻī e hāʻawi i ka ʻike koʻikoʻi e pili ana i ka hoʻoponopono ʻana o nā pae GA holoʻokoʻa i nā aʻa15,16. Eia nō naʻe, ʻokoʻa ka hōʻike ʻana o RGA ma waena o nā ʻiʻo17 a ua hoʻoponopono ʻia e GA18. No laila, ʻo ka hōʻike ʻokoʻa o ka mea hoʻolaha RGA e hopena i ke ʻano fluorescence i ʻike ʻia me RGA-GFP a no laila ʻaʻole quantitative kēia ʻano hana. ʻO ka mea hou loa, ua hōʻike ʻo bioactive fluorescein (Fl)-labeled GA19,20 i ka hōʻiliʻili ʻana o GA i loko o ke aʻa endocortex a me ka hoʻoponopono ʻana o kona mau pae cellular e ka lawe ʻana o GA. I kēia manawa, ua hōʻike ka sensor GA FRET nlsGPS1 e pili ana nā pae GA me ka elongation cell i nā aʻa, nā filaments, a me nā hypocotyls i ulu ʻeleʻele21. Eia naʻe, e like me kā mākou i ʻike ai, ʻaʻole ʻo ka nui o GA wale nō ke ʻano e hoʻomalu ana i ka hana hōʻailona GA, no ka mea, hilinaʻi ia i nā kaʻina hana ʻike paʻakikī. Maanei, e kūkulu ana ma luna o ko mākou ʻike o nā ala hōʻailona DELLA a me GA, hōʻike mākou i ka hoʻomohala ʻana a me ke ʻano o kahi biosensor ratiometric i hoʻokumu ʻia i ka degradation no ka hōʻailona GA. No ka hoʻomohala ʻana i kēia biosensor quantitative, ua hoʻohana mākou i kahi mutant GA-sensitive RGA i hoʻohui ʻia i kahi protein fluorescent a hōʻike nui ʻia i loko o nā ʻiʻo, a me kahi protein fluorescent GA-insensitive. Hōʻike mākou ʻaʻole e hoʻopilikia nā fusions protein RGA mutant i ka hōʻailona GA endogenous i ka wā e hōʻike nui ʻia ai, a hiki i kēia biosensor ke helu i ka hana hōʻailona i loaʻa mai ka hoʻokomo ʻana o GA a me ka hana hōʻailona GA e ka mea ʻike me ka hoʻonā spatiotemporal kiʻekiʻe. Ua hoʻohana mākou i kēia biosensor e palapala i ka hoʻolaha spatiotemporal o ka hana hōʻailona GA a helu pehea e hoʻoponopono ai ʻo GA i ke ʻano o ke kelepona ma ka epidermis SAM. Hōʻike mākou e hoʻoponopono ana ʻo GA i ke kuhikuhi ʻana o ka mokulele mahele o nā cell SAM i loaʻa ma waena o ka primordia organ, no laila e wehewehe ana i ka hoʻonohonoho kelepona canonical o ka internode.
ʻO ka mea hope loa, ua nīnau mākou inā hiki i ka qmRGA ke hōʻike i nā loli i nā pae GA endogenous me ka hoʻohana ʻana i nā hypocotyls e ulu ana. Ua hōʻike mua mākou e hoʻoulu ka nitrate i ka ulu ʻana ma ka hoʻonui ʻana i ka synthesis GA a, ma ka huli ʻana, ka hoʻohaʻahaʻa ʻana o DELLA34. No laila, ua ʻike mākou ʻo ka lōʻihi o ka hypocotyl i nā hua kanu pUBQ10::qmRGA i ulu ʻia ma lalo o ka lako nitrate nui (10 mM NO3−) ua ʻoi aku ka lōʻihi ma mua o nā hua kanu i ulu ʻia ma lalo o nā kūlana nitrate-deficient (Supplementary Fig. 6a). E like me ka pane ulu ʻana, ʻoi aku ke kiʻekiʻe o nā hōʻailona GA i nā hypocotyls o nā hua kanu i ulu ʻia ma lalo o nā kūlana 10 mM NO3− ma mua o nā hua kanu i ulu ʻia i ka loaʻa ʻole o ka nitrate (Supplementary Fig. 6b, c). No laila, hiki i ka qmRGA ke nānā i nā loli i ka hōʻailona GA i hoʻokumu ʻia e nā loli endogenous i ka nui o GA.
No ka hoʻomaopopo ʻana inā pili ka hana hōʻailona GA i ʻike ʻia e qmRGA i ka nui o GA a me ka ʻike ʻana o GA, e like me ka mea i manaʻo ʻia ma muli o ka hoʻolālā sensor, ua kālailai mākou i ka hōʻike ʻana o nā mea loaʻa ʻekolu GID1 i nā ʻiʻo vegetative a me reproductive. I nā mea kanu liʻiliʻi, ua hōʻike ka laina hōʻike GID1-GUS ua hōʻike nui ʻia ʻo GID1a a me c i nā cotyledons (Kiʻi 3a-c). Eia kekahi, ua hōʻike ʻia nā mea loaʻa ʻekolu i nā lau, ka primordia aʻa lateral, nā wēlau aʻa (koe wale nō ke poʻi aʻa o GID1b), a me ka ʻōnaehana vascular (Kiʻi 3a-c). I loko o ka inflorescence SAM, ua ʻike mākou i nā hōʻailona GUS no GID1b a me 1c wale nō (Kiʻi Hoʻohui 7a-c). Ua hōʻoia ka hybridization in situ i kēia mau ʻano hōʻike a ua hōʻike hou aku ua hōʻike like ʻia ʻo GID1c ma nā pae haʻahaʻa ma ka SAM, ʻoiai ʻo GID1b i hōʻike i ka hōʻike kiʻekiʻe ma ka periphery o SAM (Kiʻi Hoʻohui 7d-l). Ua hōʻike pū ka hui unuhi ʻana o pGID1b::2xmTQ2-GID1b i kahi pae i hoʻokaʻawale ʻia o ka hōʻike ʻana o GID1b, mai ka haʻahaʻa a ʻaʻohe hōʻike ma ke kikowaena o SAM a i ka hōʻike kiʻekiʻe ma nā palena o ke kino (Kiʻi Hoʻohui 7m). No laila, ʻaʻole i hoʻolaha like ʻia nā mea loaʻa GID1 ma waena a ma loko o nā ʻiʻo. Ma nā hoʻokolohua ma hope mai, ua ʻike pū mākou ua hoʻonui ka overexpression o GID1 (pUBQ10::GID1a-mCherry) i ka ʻike o qmRGA i nā hypocotyls i ka noi GA waho (Kiʻi 3d, e). I ka hoʻohālikelike ʻana, ʻo ka fluorescence i ana ʻia e qd17mRGA i loko o ka hypocotyl he mea ʻole ia i ka mālama ʻana iā GA3 (Kiʻi 3f, g). No nā hoʻāʻo ʻelua, ua mālama ʻia nā mea kanu me nā kiʻekiʻe kiʻekiʻe o GA (100 μM GA3) e loiloi i ka hana wikiwiki o ka sensor, kahi i hoʻonui ʻia ai a nalowale paha ka hiki ke hoʻopaʻa i ka mea loaʻa GID1. I ka hui pū ʻana, ua hōʻoia kēia mau hopena e lawelawe ana ka biosensor qmRGA i kahi hana i hui pū ʻia ma ke ʻano he sensor GA a me GA, a hōʻike e hiki i ka hōʻike ʻokoʻa o ka mea loaʻa GID1 ke hoʻololi nui i ka emissivity o ka sensor.
A hiki i kēia lā, ʻaʻole maopopo ka hoʻolaha ʻana o nā hōʻailona GA ma ka SAM. No laila, ua hoʻohana mākou i nā mea kanu qmRGA-expressing a me ka pCLV3::mCherry-NLS stem cell reporter35 e helu i nā palapala ʻāina quantitative kiʻekiʻe o ka hana hōʻailona GA, e kālele ana i ka papa L1 (epidermis; Fig. 4a, b, e ʻike i nā Methods and Supplementary Methods), ʻoiai he kuleana koʻikoʻi ko L1 i ka kaohi ʻana i ka ulu ʻana o SAM36. Maanei, ua hāʻawi ka hōʻike ʻana o pCLV3::mCherry-NLS i kahi kiko kuhikuhi geometric paʻa no ka nānā ʻana i ka hoʻolaha spatiotemporal o ka hana hōʻailona GA37. ʻOiai ua manaʻo ʻia ʻo GA he mea nui no ka hoʻomohala ʻana o nā ʻāpana lateral4, ua ʻike mākou he haʻahaʻa nā hōʻailona GA ma ka primordium floral (P) e hoʻomaka ana mai ke kahua P3 (Fig. 4a, b), ʻoiai ʻo nā primordiums ʻōpio P1 a me P2 he hana kaulike e like me ia ma ka ʻāpana waena (Fig. 4a, b). Ua ʻike ʻia ka hana hōʻailona GA kiʻekiʻe ma nā palena primordium o ke kino, e hoʻomaka ana ma P1/P2 (ma nā ʻaoʻao o ka palena) a piʻi i ka piko ma P4, a ma nā pūnaewele āpau o ka ʻāpana peripheral i loaʻa ma waena o ka primordia (Kiʻi 4a, b a me ke Kiʻi Hoʻohui 8a, b). ʻAʻole i ʻike ʻia kēia hana hōʻailona GA kiʻekiʻe ma ka epidermis wale nō akā ma nā papa L2 a me nā papa L3 kiʻekiʻe (Kiʻi Hoʻohui 8b). ʻAʻole i loli ke ʻano o nā hōʻailona GA i ʻike ʻia ma SAM me ka hoʻohana ʻana i ka qmRGA i ka hala ʻana o ka manawa (Kiʻi Hoʻohui 8c–f, k). ʻOiai ua hoʻohaʻahaʻa ʻia ke kūkulu qd17mRGA ma ka SAM o nā mea kanu T3 mai nā laina kūʻokoʻa ʻelima a mākou i wehewehe ai i nā kikoʻī, ua hiki iā mākou ke kālailai i nā ʻano fluorescence i loaʻa me ka kūkulu pRPS5a::VENUS-2A-TagBFP (Kiʻi Hoʻohui 8g–j, l). Ma kēia laina kaohi, ua ʻike ʻia nā loli liʻiliʻi wale nō i ka lakio fluorescence ma ka SAM, akā ma ke kikowaena SAM ua ʻike mākou i ka emi maopopo a me ka manaʻo ʻole ʻia ma VENUS e pili ana me TagBFP. Hōʻoia kēia e hōʻike ana ke ʻano hōʻailona i ʻike ʻia e qmRGA i ka hōʻino ʻana o mRGA-VENUS e pili ana iā GA, akā hōʻike pū kekahi e hiki i ka qmRGA ke hoʻonui i ka hana hōʻailona GA ma ke kikowaena meristem. I ka hōʻuluʻulu manaʻo, hōʻike kā mākou mau hopena i kahi ʻano hōʻailona GA e hōʻike nui ana i ka hoʻolaha ʻana o ka primordia. ʻO kēia hoʻolaha ʻana o ka ʻāpana inter-primordial (IPR) ma muli o ka hoʻokumu mālie ʻana o ka hana hōʻailona GA kiʻekiʻe ma waena o ka primordium e ulu ana a me ka ʻāpana waena, ʻoiai i ka manawa like e emi ana ka hana hōʻailona GA ma ka primordium (Kiʻi 4c, d).
ʻO ka hoʻolaha ʻana o nā mea loaʻa GID1b a me GID1c (e nānā i luna) e hōʻike ana he kōkua ka hōʻike ʻokoʻa o nā mea loaʻa GA i ke ʻano o ka hana hōʻailona GA ma ka SAM. Ua noʻonoʻo mākou inā paha e pili ana ka hōʻiliʻili ʻokoʻa o GA. No ka noiʻi ʻana i kēia hiki, ua hoʻohana mākou i ka sensor nlsGPS1 GA FRET21. Ua ʻike ʻia ka piʻi ʻana o ka alapine hoʻāla ma ka SAM o nlsGPS1 i mālama ʻia me 10 μM GA4+7 no 100 min (Kiʻi Hoʻohui 9a–e), e hōʻike ana e pane ana ʻo nlsGPS1 i nā loli i ka nui o GA ma ka SAM, e like me ia ma nā aʻa21. Ua hōʻike ka hoʻolaha ʻana o ka alapine hoʻāla nlsGPS1 i nā pae GA haʻahaʻa ma nā papa o waho o ka SAM, akā ua hōʻike ʻia ua kiʻekiʻe lākou ma ke kikowaena a ma nā palena o ka SAM (Kiʻi 4e a me ke Kiʻi Hoʻohui 9a,c). Hōʻike kēia ua hoʻolaha pū ʻia ʻo GA ma ka SAM me kahi ʻano spatial e like me ka mea i hōʻike ʻia e qmRGA. Ma ke ʻano he ala hoʻohui, ua mālama pū mākou i ka SAM me ka fluorescent GA (GA3-, GA4-, GA7-Fl) a i ʻole Fl wale nō ma ke ʻano he kaohi maikaʻi ʻole. Ua hoʻolaha ʻia ka hōʻailona Fl ma loko o ka SAM, me ka ʻāpana waena a me ka primordium, ʻoiai ma ka ikaika haʻahaʻa (Kiʻi 4j a me ke Kiʻi Hoʻohui 10d). I ka hoʻohālikelike ʻana, ua hōʻiliʻili nā GA-Fl ʻekolu i loko o nā palena primordium a i nā kekelē like ʻole ma ke koena o ka IPR, me GA7-Fl e hōʻiliʻili ana ma ka ʻāpana nui loa ma ka IPR (Kiʻi 4k a me ke Kiʻi Hoʻohui 10a,b). Ua hōʻike ka helu ʻana o ka ikaika fluorescence ua ʻoi aku ka kiʻekiʻe o ka lakio ikaika IPR i ka non-IPR ma ka SAM i mālama ʻia me GA-Fl i hoʻohālikelike ʻia me ka SAM i mālama ʻia me Fl (Kiʻi 4l a me ke Kiʻi Hoʻohui 10c). I ka hui pū ʻana, hōʻike kēia mau hopena aia ʻo GA ma nā ʻano kiʻekiʻe ma nā pūnaewele IPR e kokoke loa ana i ka palena o ke kino. Hōʻike kēia i ke ʻano o ka hana hōʻailona SAM GA mai ka hōʻike ʻokoʻa o nā mea loaʻa GA a me ka hōʻiliʻili ʻokoʻa o GA i nā pūnaewele IPR kokoke i nā palena o ke kino. No laila, ua hōʻike kā mākou loiloi i kahi ʻano spatiotemporal i manaʻo ʻole ʻia o ka hōʻailona GA, me ka hana haʻahaʻa ma ke kikowaena a me ka primordium o ka SAM a me ka hana kiʻekiʻe ma ka IPR ma ka ʻāpana peripheral.
No ka hoʻomaopopo ʻana i ke kuleana o ka hana hōʻailona GA ʻokoʻa ma ka SAM, ua kālailai mākou i ka pilina ma waena o ka hana hōʻailona GA, ka hoʻonui ʻana o ke kelepona, a me ka mahele ʻana o ke kelepona me ka hoʻohana ʻana i ke kiʻi manawa-lapse manawa maoli o ka SAM qmRGA pCLV3::mCherry-NLS. Ma muli o ke kuleana o GA i ka hoʻoponopono ʻana i ka ulu ʻana, ua manaʻo ʻia kahi pilina maikaʻi me nā palena hoʻonui kelepona. No laila, ua hoʻohālikelike mua mākou i nā palapala hana hōʻailona GA me nā palapala o ka ulu ʻana o ka ʻili o ke kelepona (ma ke ʻano he proxy no ka ikaika o ka hoʻonui ʻana o ke kelepona no kahi kelepona i hāʻawi ʻia a no nā kelepona kaikamahine ma ka mahele ʻana) a me nā palapala ʻāina o ka anisotropy ulu, e ana ana i ke kuhikuhi ʻana o ka hoʻonui ʻana o ke kelepona (hoʻohana ʻia ma ʻaneʻi no kahi kelepona i hāʻawi ʻia a no nā kelepona kaikamahine ma ka mahele ʻana; Kiʻi 5a,b, e ʻike i nā ʻAno Hana a me nā ʻAno Hana Hoʻohui). ʻO kā mākou mau palapala ʻāina o ka ulu ʻana o ka ʻili o ke kelepona SAM e kūlike me nā nānā mua38,39, me nā helu ulu liʻiliʻi ma ka palena a me nā helu ulu nui loa i nā pua e ulu ana (Kiʻi 5a). Ua hōʻike ka loiloi ʻāpana nui (PCA) ua pili maikaʻi ʻole ka hana hōʻailona GA me ka ikaika o ka ulu ʻana o ka ʻili o ke kelepona (Kiʻi 5c). Ua hōʻike pū mākou ua orthogonal nā koʻi nui o ka loli, me ka hoʻokomo ʻana o ka hōʻailona GA a me ka ikaika o ka ulu ʻana, i ke kuhikuhi i hoʻoholo ʻia e ka hōʻike CLV3 kiʻekiʻe, e hōʻoia ana i ka hoʻokaʻawale ʻana o nā cell mai ke kikowaena SAM i nā loiloi i koe. Ua hōʻoia ka loiloi correlation Spearman i nā hopena PCA (Kiʻi 5d), e hōʻike ana ʻaʻole i hopena nā hōʻailona GA kiʻekiʻe ma ka IPR i ka hoʻonui ʻana o nā cell. Eia nō naʻe, ua hōʻike ka loiloi correlation i kahi pilina maikaʻi iki ma waena o ka hana hōʻailona GA a me ka anisotropy ulu (Kiʻi 5c, d), e hōʻike ana he hoʻohuli ka hōʻailona GA kiʻekiʻe ma ka IPR i ke kuhikuhi o ka ulu ʻana o nā cell a me ke kūlana paha o ka mokulele mahele cell.
a, b Nā palapala wela o ka ulu ʻana o ka ʻili maʻamau (a) a me ka anisotropy ulu (b) ma SAM i ʻawelike ʻia ma luna o ʻehiku mau mea kanu kūʻokoʻa (i hoʻohana ʻia ma ke ʻano he proxies no ka ikaika a me ke kuhikuhi o ka hoʻonui ʻana o ke kelepona, kēlā me kēia). c Ua komo pū ka loiloi PCA i nā loli aʻe: ka hōʻailona GA, ka ikaika o ka ulu ʻana o ka ʻili, ka anisotropy ulu ʻana o ka ʻili, a me ka hōʻike ʻana o CLV3. Ua pili maikaʻi ʻole ka ʻāpana PCA 1 me ka ikaika o ka ulu ʻana o ka ʻili a ua pili maikaʻi me ka hōʻailona GA. Ua pili maikaʻi ʻia ka ʻāpana PCA 2 me ka anisotropy ulu ʻana o ka ʻili a ua pili maikaʻi ʻole me ka hōʻike ʻana o CLV3. Hōʻike nā pakeneka i ka ʻokoʻa i wehewehe ʻia e kēlā me kēia ʻāpana. d Ka loiloi pilina Spearman ma waena o ka hōʻailona GA, ka ikaika o ka ulu ʻana o ka ʻili, a me ka anisotropy ulu ʻana o ka ʻili ma ka pae ʻiʻo koe naʻe ʻo CZ. ʻO ka helu ma ka ʻākau ʻo ia ka waiwai Spearman rho ma waena o ʻelua mau loli. Hōʻike nā Asterisks i nā hihia kahi i koʻikoʻi loa ai ka pilina/pili maikaʻi ʻole. e ʻIke 3D o nā kelepona Col-0 SAM L1 e ka microscopy confocal. Ua kala ʻia nā paia kelepona hou i hana ʻia ma SAM (akā ʻaʻole ka primordium) ma 10 h e like me ko lākou mau waiwai kihi. Hōʻike ʻia ka pā kala ma ke kihi ʻākau haʻahaʻa. Hōʻike ka inset i ke kiʻi 3D e pili ana i ka hola 0. Ua hana hou ʻia ka hoʻokolohua i ʻelua manawa me nā hopena like. f Hōʻike nā kiʻi pahu i nā helu mahele cell ma IPR a me nā mea kanu kūʻokoʻa ʻole IPR Col-0 SAM (n = 10 mau mea kanu kūʻokoʻa). Hōʻike ka laina waena i ka median, a hōʻike nā palena pahu i ka 25th a me 75th percentiles. Hōʻike nā Whiskers i nā waiwai liʻiliʻi a me ka nui loa i hoʻoholo ʻia me ka polokalamu R. Ua loaʻa nā waiwai P me ka hoʻāʻo t ʻelua huelo a Welch. g, h Kiʻikuhi schematic e hōʻike ana (g) pehea e ana ai i ke kihi o ka paia cell hou (magenta) e pili ana i ke kuhikuhi radial mai ke kikowaena o SAM (laina kiko keʻokeʻo) (ʻo nā waiwai kihi ʻākea wale nō, ʻo ia hoʻi, 0-90°, i manaʻo ʻia), a me (h) nā kuhikuhi circumferential/lateral a me radial i loko o ka meristem. i Nā histograms alapine (frequency) o ke kuhikuhi mokulele mahele cell ma waena o ka SAM (uliuli ʻeleʻele), IPR (uliuli waena), a me ka non-IPR (uliuli māmā). Ua loaʻa nā waiwai P ma o kahi hoʻāʻo Kolmogorov-Smirnov ʻelua-huelo. Ua hana hou ʻia ka hoʻokolohua i ʻelua manawa me nā hopena like. j Nā histograms alapine (frequency) o ke kuhikuhi ʻana o ka mokulele mahele cell o ka IPR a puni ʻo P3 (ʻōmaʻomaʻo māmā), P4 (ʻōmaʻomaʻo waena), a me P5 (ʻōmaʻomaʻo ʻeleʻele), kēlā me kēia. Ua loaʻa nā waiwai P ma o kahi hoʻāʻo Kolmogorov-Smirnov ʻelua-huelo. Ua hana hou ʻia ka hoʻokolohua i ʻelua manawa me nā hopena like.
No laila, ua noiʻi hou mākou i ka pilina ma waena o ka hōʻailona GA a me ka hana mahele cell ma ka ʻike ʻana i nā paia cell i hana hou ʻia i ka wā o ka hoʻāʻo ʻana (Kiʻi 5e). Ua ʻae kēia ʻano hana iā mākou e ana i ke alapine (frequency) a me ke kuhikuhi o ka mahele cell. ʻO ka mea kupanaha, ua ʻike mākou ua like ke alapine (frequency) o nā mahele cell ma ka IPR a me ke koena o ka SAM (non-IPR, Fig. 5f), e hōʻike ana ʻaʻole i hoʻopilikia nui nā ʻokoʻa o ka hōʻailona GA ma waena o nā cell IPR a me nā cell non-IPR i ka mahele cell. ʻO kēia, a me ka pilina maikaʻi ma waena o ka hōʻailona GA a me ka anisotropy ulu, ua hoʻoikaika iā mākou e noʻonoʻo inā hiki i ka hana hōʻailona GA ke hoʻololi i ke kuhikuhi o ka mokulele mahele cell. Ua ana mākou i ke kuhikuhi o ka paia cell hou ma ke ʻano he kihi ʻoi e pili ana i ke axis radial e hoʻopili ana i ke kikowaena meristem a me ke kikowaena o ka paia cell hou (Kiʻi 5e-i) a ua ʻike ʻia kahi ʻano maopopo no nā cell e māhele ma nā kihi kokoke i 90° e pili ana i ke axis radial, me nā alapine kiʻekiʻe loa i ʻike ʻia ma 70-80° (23.28%) a me 80-90° (22.62%) (Kiʻi 5e,i), e pili ana i nā mahele cell ma ke kuhikuhi circumferential/transverse (Kiʻi 5h). No ka nānā ʻana i ka hāʻawi ʻana o ka hōʻailona GA i kēia ʻano mahele cell, ua kālailai mākou i nā palena mahele cell ma ka IPR a me ka non-IPR ma ke kaʻawale (Kiʻi 5i). Ua ʻike mākou ua ʻokoʻa ka hoʻokaʻawale ʻana o ke kihi mahele i loko o nā cell IPR mai nā cell non-IPR a i ʻole i loko o nā cell i ka SAM holoʻokoʻa, me nā cell IPR e hōʻike ana i kahi hapa kiʻekiʻe o nā mahele cell lateral/circular, ʻo ia hoʻi, 70-80° a me 80-90° (33.86% a me 30.71%, kēlā me kēia, nā hapa like) (Kiʻi 5i). No laila, ua hōʻike kā mākou mau nānā ʻana i kahi pilina ma waena o ka hōʻailona GA kiʻekiʻe a me kahi kuhikuhi mokulele mahele cell kokoke i ke kuhikuhi circumferential, e like me ka pilina ma waena o ka hana hōʻailona GA a me ka anisotropy ulu (Kiʻi 5c, d). No ka hoʻokumu hou ʻana i ka mālama spatial o kēia hui, ua ana mākou i ke kuhikuhi mokulele mahele i loko o nā cell IPR e hoʻopuni ana i ka primordium e hoʻomaka ana mai P3, ʻoiai ua ʻike ʻia ka hana hōʻailona GA kiʻekiʻe loa ma kēia ʻāpana e hoʻomaka ana mai P4 (Kiʻi 4). ʻAʻole i hōʻike nā kihi mahele o ka IPR a puni P3 a me P4 i nā ʻokoʻa koʻikoʻi, ʻoiai ua ʻike ʻia ka piʻi ʻana o ka pinepine o nā mahele cell lateral ma ka IPR a puni P4 (Kiʻi 5j). Eia nō naʻe, i loko o nā pūnaewele IPR a puni ʻo P5, ua lilo ka ʻokoʻa o ke kuhikuhi ʻana o ka mokulele mahele kelepona i mea koʻikoʻi ma ka helu helu, me ka piʻi nui ʻana o ke alapine o nā mahele kelepona transverse (Kiʻi 5j). I ka hui pū ʻana, hōʻike kēia mau hopena e hiki i ka hōʻailona GA ke kāohi i ke kuhikuhi ʻana o nā mahele kelepona ma SAM, kahi e kūlike me nā hōʻike mua40,41 hiki i ka hōʻailona GA kiʻekiʻe ke hoʻoulu i ka kuhikuhi ʻaoʻao o nā mahele kelepona ma ka IPR.
Ua wānana ʻia ʻaʻole e hoʻokomo ʻia nā pūnaewele i loko o ka IPR i loko o ka primordia akā i loko o nā internodes2,42,43. ʻO ke kuhikuhi transverse o nā mahele pūnaewele i loko o ka IPR e hopena paha i ka hoʻonohonoho maʻamau o nā lālani longitudinal like o nā pūnaewele epidermal i loko o nā internodes. Hōʻike kā mākou mau nānā ʻana i hōʻike ʻia ma luna nei he kuleana ko ka hōʻailona GA i kēia kaʻina hana ma o ka hoʻoponopono ʻana i ke kuhikuhi o ka mahele pūnaewele.
ʻO ka nalowale ʻana o ka hana o kekahi mau genes DELLA e hopena i kahi pane GA mau, a hiki ke hoʻohana ʻia nā mutants della e hoʻāʻo i kēia kuhiakau44. Ua kālailai mua mākou i nā ʻano hōʻike o ʻelima mau genes DELLA i loko o ka SAM. Ua hōʻike ka hui transcriptional o ka laina GUS45 ua hōʻike ʻia ʻo GAI, RGA, RGL1, a me RGL2 (i kahi liʻiliʻi loa) i loko o ka SAM (Kiʻi Hoʻohui 11a–d). Ua hōʻike hou aku ka hybridization in situ e hōʻiliʻili ana ʻo GAI mRNA i loko o ka primordia a me nā pua e ulu ana (Kiʻi Hoʻohui 11e). Ua ʻike ʻia ʻo RGL1 a me RGL3 mRNA ma loko o ka canopy SAM a ma nā pua kahiko, ʻoiai ʻoi aku ka nui o RGL2 mRNA ma ka ʻāpana palena (Kiʻi Hoʻohui 11f–h). Ua hōʻoia ka kiʻi confocal o pRGL3::RGL3-GFP SAM i ka hōʻike i ʻike ʻia e ka hybridization in situ a ua hōʻike ʻia e hōʻiliʻili ana ka protein RGL3 ma ka ʻāpana waena o ka SAM (Kiʻi Hoʻohui 11i). Ma ka hoʻohana ʻana i ka laina pRGA::GFP-RGA, ua ʻike pū mākou ua hōʻiliʻili ka protein RGA i loko o ka SAM, akā ua emi kona nui ma ka palena e hoʻomaka ana mai P4 (Kiʻi Hoʻohui 11j). ʻIke ʻia, kūlike nā ʻano hōʻike o RGL3 a me RGA me ka hana hōʻailona GA kiʻekiʻe ma ka IPR, e like me ka ʻike ʻia e qmRGA (Kiʻi 4). Eia kekahi, hōʻike kēia ʻikepili ua hōʻike ʻia nā DELLA āpau i loko o ka SAM a ʻo kā lākou hōʻike ʻana e uhi pū ana i ka SAM holoʻokoʻa.
Ua kālailai mākou i nā palena mahele cell ma ka SAM ʻano hihiu (Ler, kaohi) a me nā mutants gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) (Kiʻi 6a, b). ʻO ka mea hoihoi, ua ʻike mākou i kahi neʻe koʻikoʻi i ka hoʻolaha ʻana o nā alapine kihi mahele cell ma ka della global mutant SAM i hoʻohālikelike ʻia me ke ʻano hihiu (Kiʻi 6c). ʻO kēia hoʻololi i ka della global mutant ma muli o ka piʻi ʻana o ke alapine o nā kihi 80-90° (34.71% vs. 24.55%) a, i kahi liʻiliʻi, 70-80° kihi (23.78% vs. 20.18%), ʻo ia hoʻi, e pili ana i nā mahele cell transverse (Kiʻi 6c). ʻOi aku ka haʻahaʻa o ke alapine o nā mahele non-transverse (0-60°) ma ka della global mutant (Kiʻi 6c). Ua hoʻonui nui ʻia ke alapine o nā mahele cell transverse ma ka SAM o ka della global mutant (Kiʻi 6b). ʻOi aku ke kiʻekiʻe o ka alapine (frequency) o nā mahele cell transverse ma ka IPR i ka della global mutant i hoʻohālikelike ʻia me ke ʻano hihiu (Fig. 6d). Ma waho o ka ʻāpana IPR, ʻoi aku ka like o ka hoʻolaha ʻana o nā kihi mahele cell ma ke ʻano hihiu, ʻoiai makemake ka della global mutant i nā mahele tangential e like me ka IPR (Fig. 6e). Ua helu pū mākou i ke kuhikuhi o nā mahele cell ma ka SAM o nā ga2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, a me ga2ox6-2), kahi hope mutant GA-inactive kahi e hōʻiliʻili ai ʻo GA. E kūlike ana me ka piʻi ʻana o nā pae GA, ʻoi aku ka nui o ka SAM o ka inflorescence mutant quintuple ga2ox ma mua o ko Col-0 (Kiʻi Hoʻohui 12a, b), a i hoʻohālikelike ʻia me Col-0, ua hōʻike ka quintuple ga2ox SAM i kahi hoʻolaha ʻokoʻa o nā kihi mahele cell, me ka piʻi ʻana o ke alapine kihi mai 50° a 90°, ʻo ia hoʻi ke makemake hou nei i nā mahele tangential (Kiʻi Hoʻohui 12a–c). No laila, ke hōʻike nei mākou ʻo ka hoʻoulu mau ʻana o ka hōʻailona GA a me ka hōʻiliʻili GA e hoʻoulu ai i nā mahele cell lateral i ka IPR a me ke koena o ka SAM.
a, b ʻIke 3D o ka papa L1 o Ler (a) i hoʻoluʻu ʻia i ka PI a me ka global della mutant (b) SAM me ka hoʻohana ʻana i ka microscopy confocal. Hōʻike ʻia a hoʻoluʻu ʻia nā paia cell hou i hana ʻia ma ka SAM (akā ʻaʻole ka primordium) ma luna o kahi manawa 10-h e like me kā lākou mau waiwai kihi. Hōʻike ka inset i ka SAM ma 0 h. Hōʻike ʻia ka pā kala ma ke kihi ʻākau haʻahaʻa. Kuhikuhi ka pua ma (b) i kahi laʻana o nā faila cell i hoʻonohonoho ʻia ma ka global della mutant. Ua hana hou ʻia ka hoʻokolohua i ʻelua manawa me nā hopena like. hoʻohālikelike ce o ka hoʻolaha pinepine ʻana o nā kuhikuhi mokulele mahele cell ma ka SAM holoʻokoʻa (d), IPR (e), a me ka non-IPR (f) ma waena o Ler a me ka global della. Ua loaʻa nā waiwai P me ka hoʻohana ʻana i kahi hoʻāʻo Kolmogorov-Smirnov ʻelua-tailed. f, g ʻIke 3D o nā kiʻi confocal o SAM i hoʻoluʻu ʻia i ka PI o Col-0 (i) a me pCUC2::gai-1-VENUS (j) nā mea kanu transgenic. Hōʻike nā panela (a, b) i nā paia cell hou (akā ʻaʻole ka primordia) i hoʻokumu ʻia i loko o ka SAM i loko o 10 h. Ua hana hou ʻia ka hoʻokolohua ʻelua me nā hopena like. h–j Hoʻohālikelike o ka hoʻolaha pinepine ʻana o nā kuhikuhi mokulele mahele cell i loaʻa i loko o ka SAM holoʻokoʻa (h), IPR (i) a me ka non-IPR (j) ma waena o nā mea kanu Col-0 a me pCUC2::gai-1-VENUS. Ua loaʻa nā waiwai P me ka hoʻohana ʻana i kahi hoʻāʻo Kolmogorov-Smirnov ʻelua-huelo.
Ua hoʻāʻo hou mākou i ka hopena o ke kāohi ʻana i ka hōʻailona GA ma ka IPR. No kēia hopena, ua hoʻohana mākou i ka mea hoʻolaha cotyledon cup 2 (CUC2) e hoʻokele i ka hōʻike ʻana o kahi protein gai-1 maikaʻi ʻole i hoʻohui ʻia iā VENUS (ma ka laina pCUC2::gai-1-VENUS). Ma ka SAM ʻano hihiu, hoʻokele ka mea hoʻolaha CUC2 i ka hōʻike ʻana o ka hapa nui o nā IPR i loko o ka SAM, me nā cell palena, mai P4 a i mua, a ua ʻike ʻia ka hōʻike kikoʻī like ma nā mea kanu pCUC2::gai-1-VENUS (e nānā ma lalo). ʻAʻole ʻokoʻa loa ka hoʻolaha ʻana o nā kihi mahele cell ma waena o ka SAM a i ʻole IPR o nā mea kanu pCUC2::gai-1-VENUS mai ko ke ʻano hihiu, ʻoiai ua ʻike koke mākou ua māhele ʻia nā cell me ka ʻole o ka IPR i loko o kēia mau mea kanu ma kahi alapine kiʻekiʻe o 80-90° (Kiʻi 6f-j).
Ua manaʻo ʻia e pili ana ke kuhikuhi o ka mahele ʻana o ke kelepona i ke ʻano o ka SAM, ʻoiai ke koʻikoʻi tensile i hana ʻia e ka curvature o ke kino46. No laila ua nīnau mākou inā ua loli ke ʻano o ka SAM i loko o ka della global mutant a me nā mea kanu pCUC2::gai-1-VENUS. E like me ka mea i hōʻike mua ʻia12, ʻoi aku ka nui o ka della global mutant SAM ma mua o ke ʻano hihiu (Kiʻi Hoʻohui 13a, b, d). Ua hōʻoia ka hybridization in situ o CLV3 a me STM RNA i ka hoʻonui ʻana o ka meristem i loko o nā della mutants a hōʻike hou aku i ka hoʻonui ʻana o ka ʻaoʻao o ka niche cell stem (Kiʻi Hoʻohui 13e, f, h, i). Eia naʻe, ua like ka curvature SAM ma nā genotypes ʻelua (Kiʻi Hoʻohui 13k, m, n, p). Ua ʻike mākou i kahi hoʻonui like o ka nui i loko o ka gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant me ka ʻole o kahi loli i ka curvature i hoʻohālikelike ʻia me ke ʻano hihiu (Kiʻi Hoʻohui 13c, d, g, j, l, o, p). Ua hoʻopilikia pū ʻia ke alapine o ke kuhikuhi ʻana o ka mahele cell i loko o ka della quadruple mutant, akā i kahi liʻiliʻi ma mua o ka della monolithic mutant (Kiʻi Hoʻohui 12d–f). ʻO kēia hopena dosage, me ka nele o ka hopena ma ka curvature, e hōʻike ana i ka hana RGL3 koena i loko o ka Della quadruple mutant e kaupalena i nā loli i ke kuhikuhi ʻana o ka mahele cell i hoʻokumu ʻia e ka nalowale o ka hana DELLA a ʻo nā loli i nā mahele cell lateral e hana ʻia ma ke ʻano he pane i nā loli i ka hana hōʻailona GA ma mua o nā loli i ka geometry SAM. E like me ka mea i wehewehe ʻia ma luna, hoʻokele ka mea hoʻolaha CUC2 i ka hōʻike IPR i loko o ka SAM e hoʻomaka ana ma P4 (Kiʻi Hoʻohui 14a, b), a ma ke ʻano ʻē aʻe, ua emi ka nui o ka pCUC2::gai-1-VENUS SAM akā ʻoi aku ka kiʻekiʻe o ka curvature (Kiʻi Hoʻohui 14c–h). ʻO kēia hoʻololi i ke ʻano pCUC2::gai-1-VENUS SAM hiki ke hopena i kahi hoʻolaha ʻokoʻa o nā pilikia mechanical i hoʻohālikelike ʻia me ke ʻano hihiu, kahi e hoʻomaka ai nā pilikia circumferential kiʻekiʻe ma kahi mamao pōkole mai ke kikowaena SAM47. ʻOkoʻa, ʻo nā loli i ke ʻano o ka pCUC2::gai-1-VENUS SAM e hopena paha mai nā loli i nā waiwai mechanical ʻāpana i hoʻokumu ʻia e ka transgene expression48. Ma nā hihia ʻelua, hiki i kēia ke hoʻopau hapa i nā hopena o nā loli i ka hōʻailona GA ma ka hoʻonui ʻana i ka likelika e māhelehele nā pūnaewele i ke kuhikuhi circumferential/transverse, e wehewehe ana i kā mākou mau nānā ʻana.
I ka hui pū ʻia ʻana, hōʻoia kā mākou ʻikepili e pāʻani ana ka hōʻailona GA kiʻekiʻe i kahi hana ikaika i ke kuhikuhi ʻaoʻao o ka mokulele mahele cell ma ka IPR. Hōʻike pū lākou e hoʻopilikia pū ka curvature meristem i ke kuhikuhi ʻana o ka mokulele mahele cell ma ka IPR.
ʻO ke kuhikuhi transverse o ka mokulele mahele ma ka IPR, ma muli o ka hana hōʻailona GA kiʻekiʻe, e hōʻike ana ua hoʻonohonoho mua ʻo GA i kahi faila cell radial i loko o ka epidermis i loko o ka SAM e wehewehe i ka hoʻonohonoho cellular e loaʻa ma hope ma ka internode epidermal. ʻOiaʻiʻo, ua ʻike pinepine ʻia kēlā mau faila cell i nā kiʻi SAM o nā mutants della global (Kiʻi 6b). No laila, no ka ʻimi hou ʻana i ka hana hoʻomohala o ke ʻano spatial o ka hōʻailona GA ma ka SAM, ua hoʻohana mākou i ke kiʻi time-lapse e kālailai i ka hoʻonohonoho spatial o nā cell ma ka IPR ma ke ʻano hihiu (Ler a me Col-0), nā mutants della global, a me nā mea kanu transgenic pCUC2::gai-1-VENUS.
Ua ʻike mākou ua hōʻike ʻo qmRGA ua piʻi ka hana hōʻailona GA ma ka IPR mai P1/P2 a piʻi i luna ma P4, a ua mau kēia ʻano ma ka hala ʻana o ka manawa (Kiʻi 4a–f a me ke Kiʻi Hoʻohui 8c–f, k). No ka nānā ʻana i ka hoʻonohonoho spatial o nā cell ma ka IPR me ka hoʻonui ʻana i ka hōʻailona GA, ua kapa mākou i nā cell Ler IPR ma luna a i nā ʻaoʻao o P4 e like me ko lākou hopena hoʻomohala i kālailai ʻia he 34 hola ma hope o ka nānā mua ʻana, ʻo ia hoʻi, ʻoi aku ma mua o ʻelua mau manawa plastid, e ʻae ana iā mākou e hahai i nā cell IPR i ka wā o ka hoʻomohala ʻana o ka primordium mai P1/P2 a i P4. Ua hoʻohana mākou i ʻekolu mau kala like ʻole: melemele no kēlā mau cell i hoʻohui ʻia i loko o ka primordium kokoke iā P4, ʻōmaʻomaʻo no ka poʻe i loko o ka IPR, a me ka poni no ka poʻe i komo i nā kaʻina hana ʻelua (Kiʻi 7a–c). Ma t0 (0 h), ʻike ʻia nā papa 1-2 o nā cell IPR i mua o P4 (Kiʻi 7a). E like me ka mea i manaʻo ʻia, i ka wā i māhele ʻia ai kēia mau cell, ua hana nui lākou ma o ka mokulele mahele transverse (Kiʻi 7a–c). Ua loaʻa nā hopena like me ka hoʻohana ʻana iā Col-0 SAM (e kālele ana ma P3, nona ka palena e pelu like me P4 ma Ler), ʻoiai ma kēia genotype ua hūnā koke ka pelu i hana ʻia ma ka palena pua i nā pūnaewele IPR (Kiʻi 7g–i). No laila, ʻo ke ʻano mahele o nā pūnaewele IPR e hoʻonohonoho mua i nā pūnaewele i nā lālani radial, e like me nā internodes. ʻO ka hoʻonohonoho ʻana o nā lālani radial a me ka localization o nā pūnaewele IPR ma waena o nā ʻōpū e pili ana e hōʻike ana he mau kūpuna internodal kēia mau pūnaewele.
Maanei, ua hoʻomohala mākou i kahi biosensor hōʻailona GA ratiometric, qmRGA, e ʻae ana i ka palapala ʻāina quantitative o ka hana hōʻailona GA i loaʻa mai ka hui pū ʻana o GA a me GA receptor concentrations me ka hoʻēmi ʻana i ka hoʻopilikia ʻana me nā ala hōʻailona endogenous, no laila e hāʻawi ana i ka ʻike ma ka hana GA ma ka pae cellular. No kēia hopena, ua kūkulu mākou i kahi protein DELLA i hoʻololi ʻia, mRGA, i nalowale ka hiki ke hoʻopaʻa i nā hoa launa pū ʻo DELLA akā ke noho nei i ka naʻau i ka proteolysis i hoʻokomo ʻia e GA. Pane ʻo qmRGA i nā loli exogenous a me endogenous i nā pae GA, a ʻo kāna mau waiwai ʻike dynamic e hiki ai ke loiloi i nā loli spatiotemporal i ka hana hōʻailona GA i ka wā o ka hoʻomohala ʻana. He mea hana maʻalahi loa ʻo qmRGA no ka mea hiki ke hoʻololi ʻia i nā ʻiʻo like ʻole ma ka hoʻololi ʻana i ka mea hoʻolaha i hoʻohana ʻia no kona hōʻike ʻana (inā pono), a hāʻawi ʻia i ke ʻano i mālama ʻia o ke ala hōʻailona GA a me ka motif PFYRE ma waena o nā angiosperms, hiki ke hoʻoili ʻia i nā ʻano ʻē aʻe22. E kūlike me kēia, ua hōʻike ʻia hoʻi kahi mutation like i loko o ka protein laiki SLR1 DELLA (HYY497AAA) e kāohi i ka hana hoʻopau ulu o SLR1 ʻoiai e hōʻemi iki ana i kāna hoʻohaʻahaʻa GA-mediated, e like me mRGA23. ʻIke ʻia, ua hōʻike nā haʻawina hou ma Arabidopsis ua hoʻololi kahi mutation amino acid hoʻokahi i loko o ka PFYRE domain (S474L) i ka hana transcriptional o RGA me ka ʻole o ka hoʻopilikia ʻana i kona hiki ke launa pū me nā hoa transcription factor50. ʻOiai ua kokoke loa kēia mutation i nā pani amino acid 3 i loaʻa i loko o mRGA, hōʻike kā mākou mau haʻawina e hoʻololi kēia mau mutations ʻelua i nā ʻano like ʻole o DELLA. ʻOiai ʻo ka hapa nui o nā hoa transcription factor e hoʻopaʻa i nā kikowaena LHR1 a me SAW o DELLA26,51, hiki i kekahi mau waikawa amino i mālama ʻia i loko o ka PFYRE domain ke kōkua i ka hoʻopaʻa ʻana i kēia mau pilina.
ʻO ka hoʻomohala ʻana o Internode kahi ʻano koʻikoʻi i ka hoʻolālā mea kanu a me ka hoʻomaikaʻi ʻana i ka hua. Ua hōʻike ʻo qmRGA i ka hana hōʻailona GA kiʻekiʻe ma nā cell progenitor IPR internode. Ma ka hoʻohui ʻana i ke kiʻi quantitative a me ka genetics, ua hōʻike mākou e hoʻopili nā ʻano hōʻailona GA i nā mokulele mahele cell circular/transverse i loko o ka epidermis SAM, e hoʻopili ana i ka hoʻonohonoho mahele cell e pono ai no ka hoʻomohala internode. Ua ʻike ʻia kekahi mau mea hoʻoponopono o ka hoʻonohonoho mokulele mahele cell i ka wā o ka hoʻomohala ʻana52,53. Hāʻawi kā mākou hana i kahi laʻana maopopo o ke ʻano o ka hoʻoponopono ʻana o ka hana hōʻailona GA i kēia parameter cellular. Hiki iā DELLA ke launa pū me nā complexes protein prefolding41, no laila hiki i ka hōʻailona GA ke hoʻoponopono i ka hoʻonohonoho mokulele mahele cell ma o ka hoʻohuli pololei ʻana i ka cortical microtubule orientation40,41,54,55. Ua hōʻike koke mākou ma SAM, ʻo ka correlate o ka hana hōʻailona GA kiʻekiʻe ʻaʻole ia he elongation cell a mahele paha, akā ʻo ka anisotropy ulu wale nō, kahi e kūlike me ka hopena pololei o GA ma ke kuhikuhi o ka mahele cell ma ka IPR. Eia naʻe, ʻaʻole hiki iā mākou ke kāpae i kēia hopena hiki ke pololei ʻole, no ka laʻana i hoʻopili ʻia e ka palupalu o ka paia cell i hoʻokomo ʻia e GA56. ʻO nā loli i nā waiwai o ka paia cell e hoʻoulu ai i ke kaumaha mechanical57,58, hiki ke hoʻohuli i ke kuhikuhi o ka mokulele mahele cell ma o ka hoʻopili ʻana i ke kuhikuhi o nā microtubules cortical39,46,59. ʻO nā hopena i hui pū ʻia o ke kaumaha mechanical i hoʻokomo ʻia e GA a me ka hoʻoponopono pololei ʻana o ke kuhikuhi microtubule e GA e pili ana paha i ka hoʻoulu ʻana i kahi ʻano kikoʻī o ke kuhikuhi mahele cell ma ka IPR e wehewehe i nā internodes, a pono nā haʻawina hou aʻe e hoʻāʻo i kēia manaʻo. Pēlā nō, ua hōʻike nā haʻawina mua i ke koʻikoʻi o nā protein DELLA-interacting TCP14 a me 15 i ka kaohi ʻana i ka hoʻokumu ʻana o ka internode60,61 a hiki i kēia mau mea ke hoʻoponopono i ka hana a GA me BREVIPEDICELLUS (BP) a me PENNYWISE (PNY), ka mea e hoʻoponopono ai i ka ulu ʻana o ka internode a ua hōʻike ʻia e hoʻohuli i ka hōʻailona GA2,62. No ka mea, launa pū nā DELLA me nā ala hōʻailona brassinosteroid, ethylene, jasmonic acid, a me ka abscisic acid (ABA)63,64 a hiki i kēia mau hormones ke hoʻohuli i ka microtubule orientation65, hiki ke hoʻopili ʻia nā hopena o GA ma ka cell division orientation e nā hormones ʻē aʻe.
Ua hōʻike ʻia nā haʻawina cytological mua e pono nā ʻāpana o loko a me waho o ka Arabidopsis SAM no ka hoʻomohala ʻana o ka internode2,42. ʻO ka ʻoiaʻiʻo e hoʻoponopono ikaika ana ʻo GA i ka mahele ʻana o ke kelepona i loko o nā ʻiʻo o loko12 e kākoʻo ana i kahi hana pālua o GA i ka hoʻoponopono ʻana i ka meristem a me ka nui o ka internode i loko o ka SAM. Hoʻoponopono paʻa ʻia ke ʻano o ka mahele ʻana o ke kelepona kuhikuhi i loko o ka ʻiʻo SAM o loko, a he mea nui kēia hoʻoponopono no ka ulu ʻana o ke kumu52. He mea hoihoi ia e nānā inā he kuleana ko GA i ke kuhikuhi ʻana i ka mokulele mahele kelepona i loko o ka hui SAM o loko, no laila e hoʻohālikelike ana i ka kikoʻī a me ka hoʻomohala ʻana o nā internodes i loko o ka SAM.
Ua ulu ʻia nā mea kanu ma ke ʻano in vitro i loko o ka lepo a i ʻole 1x Murashige-Skoog (MS) medium (Duchefa) i hoʻohui ʻia me 1% sucrose a me 1% agar (Sigma) ma lalo o nā kūlana maʻamau (16 h mālamalama, 22 °C), koe wale nō nā hoʻokolohua hypocotyl a me ke aʻa ulu kahi i ulu ai nā mea kanu ma nā papa kū pololei ma lalo o ka mālamalama mau a me 22 °C. No nā hoʻokolohua nitrate, ua ulu ʻia nā mea kanu ma ka MS medium i hoʻololi ʻia (bioWORLD plant medium) i hoʻohui ʻia me ka nitrate kūpono (0 a i ʻole 10 mM KNO3), 0.5 mM NH4-succinate, 1% sucrose a me 1% A-agar (Sigma) ma lalo o nā kūlana lā lōʻihi.
Ua hoʻohui hou ʻia ka GID1a cDNA i hoʻokomo ʻia i loko o pDONR221 me pDONR P4-P1R-pUBQ10 a me pDONR P2R-P3-mCherry i loko o pB7m34GW e hana i ka pUBQ10::GID1a-mCherry. Ua hoʻohui hou ʻia ka IDD2 DNA i hoʻokomo ʻia i loko o pDONR221 i loko o pB7RWG266 e hana i ka p35S:IDD2-RFP. No ka hana ʻana i ka pGID1b::2xmTQ2-GID1b, ua hoʻonui mua ʻia kahi ʻāpana 3.9 kb ma luna o ka ʻāpana coding GID1b a me kahi ʻāpana 4.7 kb e loaʻa ana ka GID1b cDNA (1.3 kb) a me ka terminator (3.4 kb) me ka hoʻohana ʻana i nā primers ma ka Papa Hoʻohui 3 a laila hoʻokomo ʻia i loko o pDONR P4-P1R (Thermo Fisher Scientific) a me pDONR P2R-P3 (Thermo Fisher Scientific), a ma hope ua hoʻohui hou ʻia me pDONR221 2xmTQ268 i loko o ka vector target pGreen 012567 me ka hoʻohana ʻana i ka Gateway cloning. No ka hana ʻana iā pCUC2::LSSmOrange, ua hoʻākoakoa ʻia ka moʻo hoʻolaha CUC2 (3229 bp ma luna o ATG) i ukali ʻia e ka moʻo coding o ka Stokes-shifted mOrange nui (LSSmOrange)69 me ka hōʻailona localization nuklea N7 a me ka terminator transcriptional NOS i loko o ka vector targeting pGreen kanamycin me ka hoʻohana ʻana i ka ʻōnaehana recombination Gateway 3-fragment (Invitrogen). Ua hoʻokomo ʻia ka vector binary mea kanu i loko o ke ʻano Agrobacterium tumefaciens GV3101 a ua hoʻokomo ʻia i loko o nā lau Nicotiana benthamiana e ke ʻano infiltration Agrobacterium a i loko o Arabidopsis thaliana Col-0 e ke ʻano floral dip, kēlā me kēia. Ua hoʻokaʻawale ʻia ʻo pUBQ10::qmRGA pUBQ10::GID1a-mCherry a me pCLV3::mCherry-NLS qmRGA mai nā hua F3 a me F1 o nā keʻa like ʻole.
Ua hana ʻia ka RNA in situ hybridization ma kahi o 1 cm ka lōʻihi o nā ʻōpuʻu72, i hōʻiliʻili ʻia a hoʻopaʻa koke ʻia i loko o ka hopena FAA (3.7% formaldehyde, 5% acetic acid, 50% ethanol) i hoʻomaʻalili mua ʻia i 4 °C. Ma hope o 2 × 15 min vacuum treatments, ua hoʻololi ʻia ka fixative a ua hoʻomoʻa ʻia nā laʻana i ka pō. Ua synthesized ʻia nā cDNA GID1a, GID1b, GID1c, GAI, RGL1, RGL2, a me RGL3 a me nā probes antisense i kā lākou 3′-UTR me ka hoʻohana ʻana i nā primers i hōʻike ʻia ma ka Pākuʻi Hoʻohui 3 e like me ka mea i wehewehe ʻia e Rosier et al.73. Ua ʻike ʻia nā probes i hōʻailona ʻia me ka Digoxigenin me ka hoʻohana ʻana i nā antibodies digoxigenin (3000-fold dilution; Roche, helu papa inoa: 11 093 274 910), a ua hoʻoluʻu ʻia nā ʻāpana me ka 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 250-fold dilution) / nitroblue tetrazolium (NBT, 200-fold dilution) solution.
Ka manawa hoʻouna: Pepeluali-10-2025