He mea koʻikoʻi ka ulu ʻana o ka pana apical meristem (SAM) no ka hoʻolālā kumu. Nā hormones kanugiberellin(GAs) pāʻani koʻikoʻi i ka hoʻonohonoho ʻana i ka ulu ʻana o nā mea kanu, akā ʻaʻole maopopo ʻole kā lākou kuleana i ka SAM. Ma ʻaneʻi, ua hoʻomohala mākou i kahi biosensor ratiometric o ka hōʻailona GA ma o 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 hoʻohaʻahaʻa ʻana i ka ʻike ʻana o GA. Hōʻike mākou e hoʻopaʻa pololei kēia biosensor i hoʻohaʻahaʻa ʻia i nā loli i nā pae GA a me ka ʻike kelepona i ka wā o ka hoʻomohala ʻana. Ua hoʻohana mākou i kēia biosensor e palapala i ka hana hōʻailona GA ma ka SAM. Hōʻike mākou aia nā hōʻailona GA kiʻekiʻe i loko o nā cell i loaʻa ma waena o ka organ primordia, nā mea mua i nā cell internode. Ke hoʻohana nei i ka loaʻa a me ka nalowale o ka hana, hōʻike hou mākou i ka hoʻoponopono ʻana o GA i ka hoʻonohonoho ʻana o ka mokulele ʻāpana cell, hoʻokumu i ka hui canonical cellular o internodes, a laila e hoʻolaha ana i ka kikoʻī internode i ka SAM.
ʻO ka pana apical meristem (SAM), aia ma ka piko o ka pana, aia kahi niche o nā pūnaewele puʻupuʻu nona ka hana e hoʻohua ai i nā ʻāpana ʻaoʻao a me nā ʻōpuʻu kumu ma ke ʻano modular a me ke ʻano o ke ola o ka mea kanu. Loaʻa i kēlā me kēia o kēia mau ʻāpana hou, a i ʻole nā nodes mea kanu, nā internodes a me nā ʻāpana ʻaoʻao ma nā nodes, a me nā meristem axillary i nā axils lau. Hoʻololi ka ulu a me ka hoʻonohonoho ʻana o nā nodes mea kanu i ka wā o ka ulu ʻana. I loko o Arabidopsis, hoʻopau ʻia ka ulu ʻana o ka internodal i ka wā kanu, a noho mau nā axillary meristem 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 hana ana i nā internodes elongated a me nā ʻōpuʻu axillary, nā lālā i nā axils o nā lau cauline, a ma hope, nā pua lau ʻole2. ʻOiai ua holomua nui mākou i ka hoʻomaopopo ʻana i nā hana e hoʻomalu ai i ka hoʻomaka ʻana o nā lau, nā pua, a me nā lālā, ʻaʻole liʻiliʻi ka ʻike e pili ana i ke ala ʻana o nā internodes.
ʻO ka hoʻomaopopo ʻana i ka puʻunaue spatiotemporal o GA e kōkua i ka hoʻomaopopo maikaʻi ʻana i nā hana o kēia mau hormones i loko o nā ʻāpana like ʻole a ma nā pae hoʻomohala like ʻole. ʻO kaʻikeʻana i ka hoʻohaʻahaʻaʻana o ka hui RGA-GFP i hōʻikeʻia ma lalo o ka hana a kāna mea hoʻolaha pono'ī e hāʻawi i kaʻike koʻikoʻi e pili ana i ka hoʻoponoponoʻana i nā pae GA a pau i roots15,16. Eia naʻe, ʻokoʻa ka ʻōlelo RGA ma waena o nā tissues17 a ua hoʻoponopono ʻia e GA18. No laila, hiki i ka wehewehe ʻokoʻa o ka mea hoʻolaha RGA ke hopena i ke ʻano fluorescence i ʻike ʻia me RGA-GFP a no laila ʻaʻole i helu ʻia kēia ʻano. ʻO ka mea hou loa, bioactive fluorescein (Fl) -labeled GA19,20 hōʻike i ka hōʻiliʻili ʻana o GA i loko o ka endocortex aʻa a me ka hoʻoponopono ʻana o kāna mau pae kelepona e ka lawe GA. I kēia mau lā, ua hōʻike ka GA FRET sensor nlsGPS1 e pili ana nā pae GA me ka elongation cell i nā aʻa, nā filament, a me nā hypocotyls21 ulu ʻeleʻele. Eia nō naʻe, e like me kā mākou i ʻike ai, ʻaʻole ka manaʻo o GA ka mea hoʻohālikelike e hoʻomalu i ka hana hōʻailona GA, no ka mea, pili ia i nā kaʻina hana ʻike paʻakikī. Ma ʻaneʻi, e kūkulu ana ma luna o ko mākou ʻike i nā ala hōʻailona DELLA a me GA, hōʻike mākou i ka hoʻomohala ʻana a me ke ʻano o kahi biosensor ratiometric e pili ana 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 ʻia i loko o nā kiko, a me kahi protein fluorescent GA-insensitive. Hōʻike mākou ʻaʻole keʻakeʻa ka mutant RGA protein fusions i ka endogenous GA hōʻailona ke hōʻike ʻia i nā wahi āpau, a hiki i kēia biosensor ke helu i ka hana hōʻailona ma muli o ka hoʻokomo ʻana o GA a me ka hōʻailona hōʻailona GA e ka mea ʻike me ka hoʻonā spatiotemporal kiʻekiʻe. Ua hoʻohana mākou i kēia biosensor no ka palapala ʻana i ka māhele spatiotemporal o ka hana hōʻailona GA a helu i ke ʻano o ka hoʻoponopono ʻana o GA i ke ʻano kelepona i ka epidermis SAM. Hōʻike mākou e hoʻoponopono ʻo GA i ka hoʻonohonoho ʻana o ka mokulele mahele o nā cell SAM aia ma waena o ka organ primordia, a laila e wehewehe ana i ka hui cellular canonical o ka internode.
I ka hopena, ua nīnau mākou inā hiki i ka qmRGA ke hōʻike i nā loli i nā pae GA endogenous e hoʻohana ana i nā hypocotyls ulu. Ua hōʻike mua mākou i ka hoʻoulu ʻana o ka nitrate i ka ulu ʻana ma o ka hoʻonui ʻana i ka synthesis GA a, ʻo ia hoʻi, ʻo DELLA34 degradation. No laila, ua ʻike mākou i ka lōʻihi o ka hypocotyl ma pUBQ10::qmRGA nā mea kanu i ulu ma lalo o ka nui o ka nitrate (10 mM NO3−) i ʻoi aku ka lōʻihi ma mua o nā mea kanu i ulu ʻia ma lalo o nā kūlana nitrate-deficient (Fig 6a). E like me ka pane ulu, ua kiʻekiʻe nā hōʻailona GA i nā hypocotyls o nā mea kanu i ulu ma lalo o 10 mM NO3− kūlana ma mua o nā mea kanu i ulu i ka nele o ka nitrate (Supplementary Fig. 6b, c). No laila, hiki i ka qmRGA ke nānā i nā hoʻololi i ka hōʻailona GA i hoʻoulu ʻia e nā loli endogenous i ka manaʻo o GA.
No ka hoʻomaopopo ʻana inā pili ka hana hōʻailona GA i ʻike ʻia e qmRGA i ka manaʻo o GA a me ka ʻike GA, e like me ka mea i manaʻo ʻia e pili ana i ka hoʻolālā sensor, ua loiloi mākou i ka ʻōlelo o nā mea loaʻa GID1 ʻekolu i nā mea kanu a me nā ʻiʻo hānau. I loko o nā mea kanu, ua hōʻike ʻia ka laina hōʻike GID1-GUS ua hōʻike nui ʻia ʻo GID1a a me c i nā cotyledons (Fig. 3a-c). Eia kekahi, ua hōʻike ʻia nā mea loaʻa ʻekolu a pau i nā lau, lateral root primordia, aʻa kumu (koe wale no ke kumu aʻa o GID1b), a me ka ʻōnaehana vascular (Fig. 3a-c). I ka inflorescence SAM, ua ʻike mākou i nā hōʻailona GUS wale nō no GID1b a me 1c (Supplementary Fig. 7a-c). Ua hōʻoia ʻo In situ hybridization i kēia mau hiʻohiʻona hōʻike a hōʻike hou aku ua hōʻike like ʻia ʻo GID1c ma nā haʻahaʻa haʻahaʻa i ka SAM, ʻoiai ua hōʻike ʻo GID1b i ka ʻōlelo kiʻekiʻe ma ka ʻaoʻao o ka SAM (Supplementary Fig. 7d-l). ʻO ka pGID1b:: 2xmTQ2-GID1b unuhi unuhi i hōʻike pū ʻia i kahi pae helu o ka hōʻike GID1b, mai ka haʻahaʻa a i ʻole ka ʻōlelo i waenakonu o ka SAM a i ka ʻōlelo kiʻekiʻe ma nā palena o ke kino (Supplementary Fig. 7m). No laila, ʻaʻole hoʻokaʻawale ʻia nā mea loaʻa GID1 ma waena a i loko o nā kiko. Ma nā hoʻokolohua ma hope, ua ʻike pū mākou i ka overexpression o GID1 (pUBQ10 :: GID1a-mCherry) i hoʻonui i ka naʻau o qmRGA i nā hypocotyls i waho o ka noi GA (Fig. 3d, e). ʻO ka hoʻohālikelike, ʻo ka fluorescence i ana ʻia e qd17mRGA i loko o ka hypocotyl he insensitive i ka mālama GA3 (Fig. 3f, g). No nā ho'āʻoʻelua, ua mālamaʻia nā mea kanu me nā kiʻekiʻe kiʻekiʻe o GA (100 μM GA3) e nānā i ka hana wikiwiki o ka meaʻike, kahi i hoʻonui ai a nalowale paha ka hiki ke hoʻopaʻa i ka GID1. ʻO kēia mau hopena e hōʻoia i ka lawelawe ʻana o ka biosensor qmRGA i kahi hana i hui pū ʻia e like me kahi sensor GA a me GA, a ke manaʻo nei e hiki ke hoʻololi i ka hōʻike ʻokoʻa o ka loaʻa GID1 i ka emissivity o ka sensor.
I kēia lā, ʻaʻole maopopo ka māhele ʻana o nā hōʻailona GA i ka SAM. No laila, ua hoʻohana mākou i nā mea kanu hōʻike qmRGA a me ka pCLV3::mCherry-NLS stem cell reporter35 no ka helu ʻana i nā palapala kiʻekiʻe kiʻekiʻe o ka hana hōʻailona GA, e kālele ana i ka papa L1 (epidermis; Fig. 4a, b, e ʻike i nā ʻano hana a me nā hana hoʻohui), no ka mea, he kuleana ko L1 i ka mālama ʻana i ka ulu ʻana o SAM36. Maʻaneʻi, hāʻawi ka pCLV3::mCherry-NLS hōʻike i kahi kikoʻī geometric paʻa no ka nānā ʻana i ka puʻunaue spatiotemporal o ka hana hōʻailona GA37. ʻOiai ua manaʻo ʻia ʻo GA he mea koʻikoʻi no ka hoʻomohala ʻana o ke kino lateral4, ua ʻike mākou he haʻahaʻa nā hōʻailona GA i ka primordium pua (P) e hoʻomaka ana mai ka pae P3 (Fig. 4a, b), ʻoiai ʻo P1 a me P2 primordiums he haʻahaʻa haʻahaʻa e like me ka mea ma ke kikowaena waena (Fig. 4a, b). Uaʻikeʻia ka hana hōʻailona GA kiʻekiʻe ma nā palena o ka organ primordium, e hoʻomaka ana ma ka P1 / P2 (ma nāʻaoʻao o ka palena) a me ka piʻiʻana ma ka P4, a me nā pūnaewele a pau o ka'āpana'āpana i waena o ka primordia (Fig. 4a, b a me Supplementary Fig. 8a, b). ʻAʻole ʻike ʻia kēia hana hōʻailona GA kiʻekiʻe ma ka epidermis akā ma ka L2 a me nā papa L3 luna (Supplementary Fig. 8b). ʻO ke ʻano o nā hōʻailona GA i ʻike ʻia ma ka SAM me ka hoʻohana ʻana i qmRGA ʻaʻole i hoʻololi ʻia i ka manawa (Supplementary Fig. 8c-f, k). ʻOiai ua hoʻohaʻahaʻa ʻia ka hana qd17mRGA i loko o ka SAM o nā mea kanu T3 mai nā laina kūʻokoʻa ʻelima a mākou i hōʻike ai i nā kikoʻī, ua hiki iā mākou ke ʻimi i nā ʻano fluorescence i loaʻa me ka pRPS5a :: VENUS-2A-TagBFP kūkulu (Supplementary Fig. 8g-j, l). Ma kēia laina mana, ʻike wale ʻia nā hoʻololi liʻiliʻi o ka fluorescence ratio i ka SAM, akā ma ke kikowaena SAM ʻike mākou i ka emi ʻana a me ka manaʻo ʻole o VENUS pili me TagBFP. Hōʻoia kēia i ka hōʻailona hōʻailona i ʻike ʻia e qmRGA e hōʻike ana i ka hoʻohaʻahaʻa ʻana o ka mRGA-VENUS, akā hōʻike pū kekahi e hiki i ka qmRGA ke hoʻonui i ka hana hōʻailona GA ma ke kikowaena meristem. I ka hōʻuluʻulu ʻana, hōʻike kā mākou mau hopena i kahi kumu hoʻohālike GA e hōʻike mua ana i ka puʻunaue o ka primordia. ʻO kēia māhele o ka māhele waena (IPR) ma muli o ka hoʻokumu mālieʻana o ka hana hōʻailona GA kiʻekiʻe ma waena o ka uluʻana o ka primordium a me ke kikowaena waena, i ka manawa like e emi iho ai ka hana hōʻailona GA i ka primordium (Fig. 4c, d).
ʻO ka hāʻawi ʻana i nā mea loaʻa GID1b a me GID1c (e ʻike i luna) e hōʻike ana i ka hōʻike ʻokoʻa o nā mea loaʻa GA kōkua i ke ʻano o ka hana hōʻailona GA i ka SAM. Ua noʻonoʻo mākou inā pili paha ka hōʻiliʻili ʻokoʻa o GA. No ka noiʻi ʻana i kēia hiki, ua hoʻohana mākou i ka nlsGPS1 GA FRET sensor21. Uaʻikeʻia ka hoʻonui pinepineʻana i ka SAM o nlsGPS1 i mālamaʻia me 10 μM GA4 + 7 no 100 min (Supplementary Fig. 9a-e), e hōʻike ana i ka paneʻana o nlsGPS1 i nā hoʻololi o ka manaʻo GA i ka SAM, e like me ka roots21. Ua hōʻike ʻia ka hāʻawi ʻana o ka spatial activation frequency nlsGPS1 haʻahaʻa haʻahaʻa i nā pae o waho o ka SAM, akā ua hōʻike ʻia ua hoʻokiʻekiʻe ʻia lākou i ke kikowaena a me nā palena o ka SAM (Fig. 4e a me Supplementary Fig. 9a, c). Hōʻike kēia ua puʻunaue ʻia ʻo GA i ka SAM me kahi ʻano kikoʻī e like me ka mea i hōʻike ʻia e qmRGA. Ma ke ʻano he hoʻohui hoʻohui, mālama pū mākou i ka SAM me ka fluorescent GA (GA3-, GA4-, GA7-Fl) a i ʻole Fl wale nō ma ke ʻano he mana maikaʻi ʻole. Ua māhele ʻia ka hōʻailona Fl i loko o ka SAM, me ka ʻāpana waena a me ka primordium, ʻoiai ma kahi haʻahaʻa haʻahaʻa (Fig. 4j a me Supplementary Fig. 10d). I ka hoʻokaʻawale ʻana, ʻo nā GA-Fl ʻekolu i hōʻiliʻili kikoʻī i loko o nā palena primordium a me nā degere like ʻole i ke koena o ka IPR, me ka GA7-Fl e hōʻiliʻili ana i ka waihona nui loa i ka IPR (Fig. 4k a me Supplementary Fig. 10a,b). ʻO ka nui o ka nui o ka fluorescence i hōʻikeʻia uaʻoi aku ka kiʻekiʻe o ka IPR i ka lākiōʻole IPR ma GA-Fl-treated SAM i hoʻohālikelikeʻia me Fl-treated SAM (Fig. 4l and Supplementary Fig. 10c). ʻO kēia mau hopena e hōʻike ana aia ʻo GA ma nā kiʻekiʻe kiʻekiʻe o nā pūnaewele IPR e kū kokoke ana i ka palena o ke kino. Hōʻike kēia i ka hopena o ka hana hōʻailona SAM GA mai ka hōʻike ʻokoʻa ʻana o nā mea loaʻa GA a me ka hōʻiliʻili ʻokoʻa o GA ma nā pūnaewele IPR kokoke i nā palena o ke kino. No laila, ua hōʻike kā mākou loiloi i kahi hiʻohiʻona spatiotemporal i manaʻo ʻole ʻia o ka hōʻailona GA, me ka haʻahaʻa haʻahaʻa i 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 i ka SAM, ua ʻimi mākou i ka pilina ma waena o ka hana hōʻailona GA, ka hoʻonui ʻana o ka cell, a me ka mahele cell me ka hoʻohana ʻana i ke kiʻi manawa-manawa maoli o ka SAM qmRGA pCLV3::mCherry-NLS. Hāʻawi ʻia i ke kuleana o GA i ka hoʻoponopono ulu ʻana, ua manaʻo ʻia kahi pilina maikaʻi me nā ʻāpana hoʻonui cell. No laila, ua hoʻohālikelike mua mākou i nā palapala ʻāina hana hōʻailona GA me nā palapala ʻāina o ka ulu ʻana o ka cell surface (ma ke ʻano he koho no ka ikaika o ka hoʻonui ʻana o ke kelepona no kahi cell i hāʻawi ʻia a no nā cell kaikamahine ma ka mahele) a me nā palapala ʻāina o ka anisotropy ulu, e ana i ka directionality o ka hoʻonui cell (e hoʻohana pū ʻia ma ʻaneʻi no kahi cell i hāʻawi ʻia a no nā ʻāpana kaikamahine ma ka mahele; Fig. ʻO kā mākou mau palapala 'āina o ka SAM cell surface growth rate e kūlike me ka nānā muaʻana38,39, me ka liʻiliʻi o ka uluʻana ma ka palena a me ka nui o ka uluʻana i ka uluʻana i nā pua (Fig. 5a). Ua hōʻike ʻia ka loiloi ʻāpana kumu (PCA) ua pili maikaʻi ʻia ka hana hōʻailona GA me ka ikaika o ka ulu ʻana o ka ʻili o ke kelepona (Figure 5c). Ua hōʻike pū mākou i nā koʻi koʻikoʻi o ka hoʻololi ʻana, me ka hoʻokomo ʻana i ka hōʻailona GA a me ka ikaika o ka ulu ʻana, he orthogonal i ke kuhikuhi i hoʻoholo ʻia e ka ʻōlelo CLV3 kiʻekiʻe, e hōʻoia ana i ka wehe ʻana o nā cell mai ke kikowaena SAM i ke koena anana. Ua hōʻoia ʻo Spearman correlation analysis i nā hopena PCA (Figure 5d), e hōʻike ana i nā hōʻailona GA kiʻekiʻe ma ka IPR ʻaʻole i hopena i ka hoʻonui ʻana o ka cell cell. Eia naʻe, ua hōʻike ʻia ka loiloi correlation i kahi pilina maikaʻi ma waena o ka hana hōʻailona GA a me ka anisotropy ulu (Figure 5c, d), e hōʻike ana i ke kiʻekiʻe o ka hōʻailona GA i ka IPR e hoʻohuli i ke kuhikuhi o ka ulu ʻana o ka cell a me ke kūlana o ka mokulele mahele cell.
a, b Nā palapala ʻāina wela o ka ulu ʻana o ka ʻili (a) a me ka anisotropy ulu (b) ma SAM i ʻawelika ma luna o ʻehiku mau mea kanu kūʻokoʻa (hoʻohana ʻia ma ke ʻano he mau proxies no ka ikaika a me ke kuhikuhi o ka hoʻonui ʻana o ke kelepona, kēlā me kēia). ʻO ka nānā ʻana i ka PCA i komo i nā ʻano like ʻole: hōʻailona GA, ka ulu ʻana o ka ʻili, ka anisotropy ulu o ka ʻili, a me ka ʻōlelo CLV3. Hoʻopili maikaʻi ʻia ka ʻāpana PCA 1 me ka ulu ʻana o ka ʻili a hoʻopili maikaʻi ʻia me ka hōʻailona GA. Hoʻopili maikaʻi ʻia ka ʻāpana PCA 2 me ka anisotropy ulu o ka ʻili a hoʻopili maikaʻi ʻia me ka ʻōlelo CLV3. Hōʻike ka pākēneka i ka hoʻololi i wehewehe ʻia e kēlā me kēia ʻāpana. d Spearman correlation analysis between GA signal, surface growth intensity, and surface growth anisotropy at the tissue scale with CZ. ʻO ka helu ma ka ʻākau ka waiwai Spearman rho ma waena o ʻelua mau mea hoʻololi. Hōʻike nā Asterisk i nā hihia kahi i koʻikoʻi ka correlation/negative correlation. e ʻike 3D o Col-0 SAM L1 pūnaewele ma ka microscopy confocal. ʻO nā pā kelepona hou i hoʻokumu ʻia i ka SAM (akā ʻaʻole ka primordium) ma 10 mau hola e kala ʻia e like me ko lākou mau koina angle. Hōʻike ʻia ka pahu kala ma ka ʻaoʻao ʻākau lalo. Hōʻike ka inset i ke kiʻi 3D pili i 0 h. Ua hana hou ʻia ka hoʻokolohua me nā hopena like. f Hōʻike nā pahu pahu i ka nui o ka mahele pūnaewele ma IPR a me non-IPR Col-0 SAM (n = 10 mau mea kanu kūʻokoʻa). Hōʻike ka laina waena i ka waena, a hōʻike nā palena pahu i ka 25th a me 75th percentile. Hōʻike ʻo Whiskers i nā waiwai liʻiliʻi a me ka nui i hoʻoholo ʻia me ka polokalamu R. Ua loaʻa nā waiwai P me ka ho'āʻo t-huʻu ʻelua a Welch. g. i Nā histograms alapine o ka hoʻonohonoho ʻana o ka mokulele ma waena o ka SAM (uliuli ʻeleʻele), IPR (uliuli waena), a me ka IPR ʻole (uliuli māmā). Ua loaʻa nā waiwai P e kahi hōʻike Kolmogorov-Smirnov ʻelua huelo. Ua hana hou ʻia ka hoʻokolohua me nā hopena like. j Nā histograms alapine o ka hoʻonohonoho mokulele mahele pūnaewele o ka IPR a puni P3 ('ōmaʻomaʻo māmā), P4 ('ōmaʻomaʻo waena), a me P5 ('ōmaʻomaʻo ʻeleʻele). Ua loaʻa nā waiwai P e kahi hōʻike Kolmogorov-Smirnov ʻelua huelo. Ua hana hou ʻia ka hoʻokolohua me nā hopena like.
No laila, ua noiʻi hou mākou i ka pilina ma waena o ka hōʻailona GA a me ka hana hoʻokaʻawale cell ma o ka ʻike ʻana i nā paia cell hou i hana ʻia i ka wā o ka assay (Fig. 5e). Ua ʻae kēia ala iā mākou e ana i ke alapine a me ke kuhikuhi o ka māhele cell. ʻO ka mea kupanaha, uaʻike mākou heʻano like ke alapine o nā māhele pūnaewele i ka IPR a me ke koena o ka SAM (non-IPR, Fig. 5f), e hōʻike ana i nāʻokoʻa o ka hōʻailona GA ma waena o ka IPR a me nā pūnaewele non-IPR,ʻaʻole ia e pili nui i ka mahele pūnaewele. 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 ka orientation o ka mokulele mahele. Ua ana mākou i ka hoʻonohonoho ʻana o ka pā kelepona hou ma ke ʻano he kihi koʻikoʻi e pili ana i ka axis radial e hoʻopili ana i ke kikowaena meristem a me ke kikowaena o ka paia cell hou (Fig. 5e-i) a nānā i kahi ʻano maʻalahi o nā puʻupuʻu e puʻunaue i nā kihi kokoke i 90 ° pili i ke axis radial, me nā alapine kiʻekiʻe i ʻike ʻia ma 703-80° (203-80%). (22.62%) (Fig. 5e, i), e pili ana i nā māhele cell i ke kuhikuhi circumferential / transverse (Fig. 5h). No ka nānā ʻana i ka hāʻawi ʻana o ka hōʻailona GA i kēia ʻano hoʻokaʻawale cell, ua hoʻokaʻawale mākou i nā ʻāpana hoʻokaʻawale cell i ka IPR a me ka IPR ʻole (Fig. 5i). Ua ʻike mākou he ʻokoʻa ka māhele ʻāpana ʻāpana i nā cell IPR mai kēlā i loko o nā cell non-IPR a i ʻole nā pūnaewele i loko o ka SAM holoʻokoʻa, me nā cell IPR e hōʻike ana i kahi kiʻekiʻe o nā māhele cell lateral / circular, ʻo ia hoʻi, 70-80 ° a me 80-90 ° (33.86% a me 30.71%, i kēlā me kēia, pili pili 5i). No laila, ua hōʻike kā mākou mau ʻike i kahi hui ma waena o ka hōʻailona GA kiʻekiʻe a me kahi hoʻonohonoho mokulele hoʻokaʻawale cell kokoke i ke kuhikuhi circumferential, e like me ka pilina ma waena o ka hana hōʻailona GA a me ka anisotropy ulu (Fig. 5c, d). No ka hoʻokumu houʻana i ka mālama ākea o kēia hui, ua ana mākou i ka hoʻokaʻawaleʻana o ka mokulele i loko o nā pūnaewele IPR e hoʻopuni ana i ka primordium e hoʻomaka ana mai ka P3, no ka mea, uaʻikeʻia ka hana hōʻailona GA kiʻekiʻe loa ma kēia māhele e hoʻomaka ana mai P4 (Fig. 4). ʻAʻole i hōʻike ʻia nā ʻokoʻa koʻikoʻi o nā ʻāpana o ka IPR a puni ka P3 a me ka P4, ʻoiai ua ʻike ʻia ka piʻi ʻana o ke alapine o nā māhele cell lateral i ka IPR a puni ka P4 (Fig. 5j). Eia naʻe, i loko o nā pūnaewele IPR a puni ka P5, ua lilo ka ʻokoʻa i ka hoʻonohonoho ʻana o ka mokulele ʻāpana cell i ka helu helu, me ka piʻi nui ʻana o ke alapine o nā māhele cell transverse (Fig. 5j). ʻO ka hui pūʻana, hōʻike kēia mau hopena e hiki i ka hōʻailona GA ke hoʻomalu i ka hoʻonohonohoʻana o nā māhele pūnaewele i loko o ka SAM, i kūlike me nā hōʻike mua40,41 e hiki i ka hōʻailona GA kiʻekiʻe ke hoʻoulu i ka orientation lateral o nā māhele pūnaewele i ka IPR.
Ua wānana ʻia ʻaʻole e hoʻokomo ʻia nā cell i ka IPR i loko o primordia akā i loko o internodes2,42,43. ʻO ka hoʻohālikelike ʻana o nā ʻāpana cell i ka IPR hiki ke hopena i ka hoʻonohonoho maʻamau o nā lālani longitudinal parallel o nā cell epidermal i loko o nā internodes. ʻO kā mākou mau ʻike i hōʻike ʻia ma luna nei e hōʻike ana he kuleana ko ka hōʻailona GA i kēia kaʻina hana ma o ka hoʻoponopono ʻana i ke kuhikuhi o ka māhele cell.
ʻO ka nalowale o ka hana o kekahi mau genes DELLA ka hopena i ka pane GA, a hiki ke hoʻohana ʻia nā della mutants e hoʻāʻo i kēia hypothesis44. Ua nānā mua mākou i nā hiʻohiʻona o nā genes DELLA ʻelima i ka SAM. Ua hōʻike ʻia ka fusion transcriptional o ka GUS line45 i hōʻike ʻia ʻo GAI, RGA, RGL1, a me RGL2 (i kahi liʻiliʻi liʻiliʻi) i ka SAM (Supplementary Fig. 11a-d). Ua hōʻike hou aku ka hybridization in situ i ka hōʻiliʻili ʻana o GAI mRNA i ka primordia a me ka hoʻomohala ʻana i nā pua (Supplementary Fig. 11e). Ua ʻike ʻia ʻo RGL1 a me RGL3 mRNA i loko o ka canopy SAM a me nā pua kahiko, akā ʻoi aku ka nui o ka RGL2 mRNA ma ka ʻāina palena (Supplementary Fig. 11f-h). ʻO ke kiʻi hoʻohālikelike o pRGL3 :: Ua hōʻoia ʻo RGL3-GFP SAM i ka ʻōlelo i ʻike ʻia e in situ hybridization a hōʻike ʻia e hōʻuluʻulu ʻia ka protein RGL3 ma ka ʻāpana waena o ka SAM (Supplementary Fig. 11i). Ke hoʻohana nei i ka laina pRGA :: GFP-RGA, ua ʻike pū mākou ua hōʻuluʻulu ʻia ka protein RGA i ka SAM, akā e emi ana kona nui ma ka palena e hoʻomaka ana mai P4 (Supplementary Fig. 11j). ʻO ka mea nui, ua kūlike nā hiʻohiʻona hōʻike o RGL3 a me RGA me ka hana hōʻailona GA kiʻekiʻe ma ka IPR, e like me ka ʻike ʻia e qmRGA (Fig. 4). Eia kekahi, hōʻike kēia mau ʻikepili ua hōʻike ʻia nā DELLA a pau i ka SAM a ʻo kā lākou ʻōlelo i hui pū ʻia i ka SAM holoʻokoʻa.
Hoʻopili hou mākou i nā ʻāpana ʻāpana cell i loko o ka SAM ʻano hihiu (Ler, control) a me ka gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (global) mutants (Fig. 6a, b). ʻO ka mea mahalo, ua ʻike mākou i ka neʻe ʻana o ka helu helu i ka hoʻohele ʻana i nā alapine angle māhele cell i ka della global mutant SAM i hoʻohālikelike ʻia me ke ʻano hihiu (Fig. 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 ka liʻiliʻi liʻiliʻi, nā kihi 70-80 ° (23.78% vs. 20.18%), ʻo ia hoʻi, e pili ana i nā māhele cell transverse (Fig 6c). ʻO ke alapine o nā māhele non-transverse (0-60 °) i emi iho ma ka della global mutant (Fig. 6c). Ua hoʻonui nuiʻia ka pinepine o nā māhele pūnaewele transverse i ka SAM o ka della global mutant (Fig. 6b). ʻO ke alapine o nā māhele cell transverse i ka IPR ua ʻoi aku ka kiʻekiʻe ma ka della global mutant i hoʻohālikelike ʻia me ke ʻano hihiu (Fig. 6d). Ma waho aʻe o ka ʻāina IPR, ʻoi aku ka like o ka māhele ʻana o ka ʻano hihiu i nā kihi hoʻokaʻawale cell, akā makemake ka della global mutant i nā māhele tangential e like me ka IPR (Fig. 6e). Ua helu pū mākou i ka hoʻonohonoho ʻana o nā māhele cell i ka SAM o ga2 oxidase (ga2ox) quintuple mutants (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, a me ga2ox6-2), he ʻano mutant GA-inactive kahi e hōʻiliʻili ai ʻo GA. Kūlike me ka piʻi ʻana o nā pae GA, ua ʻoi aku ka nui o ka SAM o ka quintuple ga2ox mutant inflorescence ma mua o ko Col-0 (Supplementary Fig. 12a, b), a hoʻohālikelike ʻia me Col-0, ua hōʻike ka quintuple ga2ox SAM i kahi mahele ʻokoʻa o nā kihi o nā cell division, me ka piʻi ʻana o ke alapine angle mai 50°ing a hiki i 9000. 12a–c). No laila, hōʻike mākou i ka hoʻoulu ʻana o ka hōʻailona GA a me ka hōʻiliʻili ʻana o GA i ka hoʻokaʻawale ʻana i nā cell lateral i ka IPR a me ke koena o ka SAM.
a, b 3D hiʻohiʻona o ka papa L1 o PI-stained Ler (a) a me global della mutant (b) SAM me ka hoʻohana ʻana i ka microscopy confocal. Hōʻike ʻia nā pā kelepona hou i hoʻokumu ʻia i ka SAM (akā ʻaʻole ka primordium) ma kahi manawa 10-h a ʻulaʻula e like me ko lākou ʻano koina. Hōʻike ka inset i ka SAM ma 0 h. Hōʻike ʻia ka pahu kala ma ka ʻaoʻao ʻākau lalo. Hōʻike ka pua ma (b) i kahi laʻana o nā faila kelepona i hoʻopaʻa ʻia ma ka honua della mutant. Ua hana hou ʻia ka hoʻokolohua me nā hopena like. ka hoʻohālikelike o ka puʻunaue alapine o ka hoʻokaʻawale ʻana o ka mokulele ma ka SAM holoʻokoʻa (d), IPR (e), a me ka IPR ʻole (f) ma waena o Ler a me global della. Ua loaʻa nā waiwai P me ka hoʻohana ʻana i kahi hōʻike Kolmogorov-Smirnov ʻelua-huelo. f, g 3D hiʻona o nā kiʻi confocal o PI-stained SAM o Col-0 (i) a me pCUC2 :: gai-1-VENUS (j) mea kanu transgenic. Hōʻike nā panela (a, b) i nā paia kelepona hou (akā ʻaʻole primordia) i hoʻokumu ʻia i ka SAM i loko o 10 mau hola. Ua hana hou ʻia ka hoʻokolohua me nā hopena like. h–j Ka hoohalike ana o ka puunaue alapine (frequency distribution) o na hookuonoono ana o ka mokulele mahele cell i loko o ka SAM (h), IPR (i) a me IPR ole (j) ma waena o Col-0 a me pCUC2 ::gai-1-VENUS mea kanu. Ua loaʻa nā waiwai P me ka hoʻohana ʻana i kahi hōʻike 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 hoʻokau i ka hōʻike ʻana o kahi protein gai-1 maikaʻi ʻole i hoʻohui ʻia iā VENUS (ma ka laina pCUC2::gai-1-VENUS). I loko o ka SAM ʻano hihiu, hoʻolaha ka mea hoʻolaha CUC2 i ka hōʻike ʻana o ka hapa nui o nā IPR i ka SAM, e komo pū ana me nā ʻāpana palena, mai P4 a hiki i luna, a ua ʻike ʻia ka ʻōlelo kikoʻī like i nā mea kanu pCUC2:: gai-1-VENUS (e ʻike i lalo). ʻAʻole i ʻokoʻa loa ka māhele ʻana o nā kihi ʻāpana cell ma waena o ka SAM a i ʻole IPR o pCUC2 :: gai-1-VENUS mea kanu mai ka ʻano hihiu, ʻoiai ʻaʻole i manaʻo ʻia ua ʻike mākou ua māhele ʻia nā cell me ka IPR i kēia mau mea kanu i ke alapine kiʻekiʻe o 80-90 ° (Fig. 6f-j).
Ua manaʻo ʻia ʻo ke kuhikuhi o ka mahele cell e pili ana i ka geometry o ka SAM, ʻo ia hoʻi ke koʻikoʻi tensile i hana ʻia e ka curvature kiko46. No laila ua nīnau mākou inā ua hoʻololi ʻia ke ʻano o ka SAM i ka della global mutant a me pCUC2 :: gai-1-VENUS mea kanu. E like me ka mea i hōʻike mua ʻia 12, ʻoi aku ka nui o ka della global mutant SAM ma mua o ka ʻano hihiu (Supplementary Fig. 13a, b, d). ʻO ka hybridization in situ o CLV3 a me STM RNA i hōʻoia i ka hoʻonui meristem i nā della mutants a hōʻike hou i ka hoʻonui ʻana i ka ʻaoʻao o ka niche stem cell (Supplementary Fig. 13e, f, h, i). Eia naʻe, ua like ka curvature SAM i nā genotypes ʻelua (Supplementary Fig. 13k, m, n, p). Ua ʻike mākou i ka hoʻonui like ʻana o ka nui ma ka gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant me ka ʻole o ka hoʻololi ʻana i ka curvature i hoʻohālikelike ʻia me ke ʻano hihiu (Supplementary Fig. 13c, d, g, j, l, o, p). Ua hoʻopili pū ʻia ke alapine o ka hoʻokaʻawale ʻana o ka cell i ka della quadruple mutant, akā ma kahi liʻiliʻi ma mua o ka della monolithic mutant (Supplementary Fig. 12d-f). ʻO kēia hopena dosage, a me ka nele o ka hopena ma ka curvature, e hōʻike ana i ke koena o ka hana RGL3 i ka Della quadruple mutant e kaupalena i ka hoʻololi ʻana i ka hoʻokaʻawale ʻana o ka cell division ma muli o ka nalo ʻana o ka hana DELLA a hiki i nā loli i nā ʻāpana cell lateral ke pane i nā loli i ka hana hōʻailona GA ma mua o nā loli i ka geometry SAM. E like me ka mea i hōʻike ʻia ma luna nei, hoʻolale ka CUC2 mea hoʻolaha i ka hōʻike IPR i ka SAM e hoʻomaka ana ma P4 (Supplementary Fig. 14a, b), a i ka hoʻohālikelike ʻana, ua emi ka nui o ka pCUC2 :: gai-1-VENUS SAM akā kiʻekiʻe curvature (Supplementary Fig. 14c–h). ʻO kēia hoʻololi i ka pCUC2::gai-1-VENUS SAM morphology hiki ke hopena i ka mahele ʻokoʻa o nā koʻikoʻi mechanical i hoʻohālikelike ʻia me ke ʻano hihiu, kahi e hoʻomaka ai nā koʻikoʻi pōʻai kiʻekiʻe ma kahi mamao pōkole mai ke kikowaena SAM47. ʻO kahi ʻē aʻe, hiki ke hoʻololi i ka pCUC2:: gai-1-VENUS SAM morphology ma muli o nā loli i nā waiwai mechanical regional i hoʻokomo ʻia e ka transgene expression48. Ma nā hihia ʻelua, hiki i kēia ke hoʻopau i nā hopena o ka hoʻololi ʻana i ka hōʻailona GA ma o ka hoʻonui ʻana i ka likelihood e māhele ʻia nā cell i ka circumferential / transverse orientation, e wehewehe ana i kā mākou nānā.
Hoʻohui pū ʻia, hōʻoia kā mākou ʻikepili i ka hoʻokō ʻana o ka hōʻailona GA kiʻekiʻe i ka ʻaoʻao ʻaoʻao o ka mokulele ʻāpana cell i ka IPR. Hōʻike pū lākou e pili ana ka curvature meristem i ka orientation o ka mokulele ʻāpana cell i ka IPR.
ʻO ka hoʻonohonoho transverse o ka mokulele mahele i ka IPR, ma muli o ka hana hōʻailona GA kiʻekiʻe, e hōʻike ana e hoʻonohonoho mua ʻo GA i kahi faila radial cell i loko o ka epidermis i loko o ka SAM e wehewehe i ka hui kelepona e ʻike ʻia ma ka epidermal internode. ʻOiaʻiʻo, ʻike pinepine ʻia nā faila kelepona i nā kiʻi SAM o della global mutants (Fig. 6b). No laila, e ʻimi hou aku i ka hana hoʻomohala o ke ʻano spatial o ka hōʻailona GA i ka SAM, ua hoʻohana mākou i ke kiʻi manawa manawa e kālailai i ka hui spatial o nā cell i ka IPR ma nā ʻano hihiu (Ler a me Col-0), della global mutants, a me pCUC2 :: gai-1-VENUS transgenic mea kanu.
Uaʻike mākou ua hōʻike ka qmRGA i ka hoʻonuiʻana o ka hana hōʻailona GA i ka IPR mai ka P1 / P2 a piʻi i ka P4, a ua mau kēiaʻano maʻamau i ka manawa (Fig. 4a-f a me ka Fig. 8c-f, k). No ke kālailai ʻana i ka hoʻonohonoho spatial o nā cell i loko o ka IPR me ka hoʻonui ʻana i ka hōʻailona GA, ua kapa mākou i nā pūnaewele Ler IPR ma luna a ma nā ʻaoʻao o P4 e like me kā lākou hopena hoʻomohala i kālai ʻia 34 mau hola ma hope o ka nānā mua ʻana, ʻo ia hoʻi, ʻoi aku ma mua o ʻelua mau plastid, e ʻae iā mākou e hahai i nā cell IPR i ka wā o ka ulu 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 i P4, ʻōmaʻomaʻo no nā mea i loko o ka IPR, a me ka poni no ka poʻe i komo i nā kaʻina ʻelua (Fig. 7a-c). Ma ka t0 (0 h), uaʻikeʻia nā papa 1-2 o nā pūnaewele IPR i mua o P4 (Fig. 7a). E like me ka mea i manaʻo ʻia, i ka wā i māhele ʻia ai kēia mau pūnaewele, ua hana nui lākou ma o ka mokulele mahele transverse (Fig. 7a–c). Ua loaʻa nā hopena like me ka hoʻohana ʻana iā Col-0 SAM (ka nānā ʻana i ka P3, nona ka palena e like me ka P4 ma Ler), ʻoiai ma kēia genotype ua hūnā ʻia ka paʻi i hana ʻia ma ka palena pua i ka wikiwiki o nā cell IPR (Fig. 7g–i). No laila, ʻo ke ʻano mahele o nā pūnaewele IPR e hoʻonohonoho mua i nā cell i nā lālani radial, e like me nā internodes. ʻO ka hoʻonohonoho ʻana o nā lālani radial a me ka hoʻokaʻawale ʻana o nā cell IPR ma waena o nā ʻāpana e hōʻike ana he progenitors internodal kēia mau cell.
Maʻaneʻi, ua hoʻomohala mākou i kahi biosensor hōʻailona GA ratiometric, qmRGA, e ʻae ai i ka palapala ʻana i ka nui o ka hana hōʻailona GA ma muli o ka hui pū ʻana o GA a me GA hoʻokipa ʻana i ka wā e hoʻemi ana i ka hoʻopilikia ʻana i nā ala hōʻailona endogenous, e hāʻawi i ka ʻike e pili ana i ka hana GA ma ka pae kelepona. I kēia hopena, ua kūkulu mākou i kahi pūmua DELLA i hoʻololi ʻia, mRGA, i nalowale ka hiki ke hoʻopaʻa i nā hoa pili DELLA akā ua paʻa mau i ka proteolysis i hoʻokomo ʻia e GA. Ua pane ʻo qmRGA i nā hoʻololi exogenous a endogenous i nā pae GA, a ʻo kāna mau ʻano ʻike ikaika e hiki ai ke loiloi i nā loli spatiotemporal i ka hana hōʻailona GA i ka wā o ka hoʻomohala ʻana. He mea maʻalahi loa ka qmRGA no ka mea hiki ke hoʻololi i ka mea hoʻolaha i hoʻohana ʻia no kāna hōʻike (inā pono), a hāʻawi ʻia i ke ʻano mālama ʻia o ke ala hōʻailona GA a me ke kumu PFYRE ma waena o nā angiosperms, hiki ke hoʻololi ʻia i nā ʻano ʻē aʻe22. E like me kēia, ua hōʻike pū ʻia kahi mutation like i ka laiki SLR1 DELLA protein (HYY497AAA) e kāohi i ka hana repressor ulu o SLR1 ʻoiai e hoʻemi iki ana i kāna degradation GA-mediated, e like me mRGA23. ʻO ka mea nui, ua hōʻike ʻia nā haʻawina hou ma Arabidopsis i hoʻololi ʻia kahi mutation amino acid hoʻokahi i ka domain PFYRE (S474L) i ka hana transcriptional o RGA me ka ʻole o ka hoʻopili ʻana i kona hiki ke launa pū me nā hoa transcription factor50. ʻOiai ua kokoke loa kēia hoʻololi ʻana i ka 3 amino acid substitutions i loko o ka mRGA, hōʻike kā mākou mau haʻawina e hoʻololi kēia mau hoʻololi ʻelua i nā ʻano ʻokoʻa o DELLA. ʻOiai ʻo ka hapa nui o nā hoa transcription factor e hoʻopaʻa i nā kāʻei LHR1 a me SAW o DELLA26,51, hiki i kekahi mau amino acid i mālama ʻia ma ka domain PFYRE ke kōkua i ka hoʻopaʻa ʻana i kēia mau pilina.
ʻO ka ulu 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 IPR internode progenitor cell. Ma ka hoʻohui ʻana i nā kiʻi kiʻi nui a me nā genetics, ua hōʻike mākou i nā kumu hōʻailona GA e hoʻokau i nā mokulele circular / transverse cell division i loko o ka epidermis SAM, e hana ana i ka hui ʻāpana cell i koi ʻia no ka ulu ʻana o internode. Ua ʻike ʻia kekahi mau mea hoʻoponopono o ka hoʻokaʻawale mokulele mokulele i ka wā hoʻomohala52,53. Hāʻawi kā mākou hana i kahi hiʻohiʻona maopopo o ka hoʻoponopono ʻana o ka hana hōʻailona GA i kēia ʻāpana kelepona. Hiki iā DELLA ke launa pū me ka prefolding protein complexes41, no laila hiki i ka hōʻailona GA ke hoʻoponopono i ka hoʻonohonoho ʻana o ka mokulele ma o ka hoʻohuli pololei ʻana i ka cortical microtubule orientation40,41,54,55. Ua hōʻike ʻole mākou i ka SAM, ʻo ka correlate o ka hana hōʻailona GA kiʻekiʻe ʻaʻole ia ka elongation cell a i ʻole ka mahele, akā ʻo ka anisotropy ulu wale nō, e kūlike me ka hopena pololei o GA ma ke kuhikuhi o ka māhele cell i ka IPR. Eia nō naʻe, ʻaʻole hiki iā mākou ke hoʻokaʻawale i kēia hopena hiki ke hoʻokaʻawale ʻole, no ka laʻana i hoʻopili ʻia e GA-induced cell wall softening56. ʻO nā hoʻololi i nā waiwai o ka pā hale e hoʻoulu ai i ka stress mechanical57,58, hiki ke hoʻololi i ka orientation o ka mokulele mahele cell ma o ka hoʻopili ʻana i ka orientation o cortical microtubules39,46,59. ʻO nā hopena i hui pū ʻia o GA-induced mechanical stress a me ka hoʻoponopono pololei ʻana o ka microtubule orientation e GA hiki ke komo i ka hana ʻana i kahi ʻano kikoʻī o ka hoʻokaʻawale ʻana i nā cell i ka IPR e wehewehe i nā internodes, a pono nā noiʻi hou e hoʻāʻo i kēia manaʻo. Pēlā nō, ua hōʻike nā haʻawina mua i ke koʻikoʻi o ka DELLA-interacting proteins TCP14 a me 15 i ka mana o ka internode formation60,61 a hiki i kēia mau mea ke hoʻopili i ka hana a GA me BREVIPEDICELLUS (BP) a me PENNYWISE (PNY), nāna e hoʻoponopono i ka ulu ʻana o ka internode a ua hōʻike ʻia e hoʻoikaika i ka signaling GA2,62. Ma muli o ka launa pū ʻana o DELLA me ka brassinosteroid, ethylene, jasmonic acid, a me abscisic acid (ABA) nā ala hōʻailona63,64 a hiki i kēia mau hormones ke hoʻoikaika i ka microtubule orientation65, hiki ke hoʻopili ʻia nā hopena o GA ma ka hoʻokaʻawale ʻana i nā cell e nā hormones ʻē aʻe.
Ua hōʻike ʻia nā haʻawina cytological mua e koi ʻia nā ʻāpana o loko a me waho o ka Arabidopsis SAM no ka ulu ʻana o internode2,42. ʻO ka ʻoiaʻiʻo e hoʻoponopono ikaika ʻo GA i ka mahele cell i loko o nā ʻiʻo i loko12 e kākoʻo i kahi hana pālua o GA i ka hoʻoponopono ʻana i ka meristem a me ka nui internode i ka SAM. Hoʻoponopono paʻa ʻia ke ʻano o ka mahele cell direction i loko o ka ʻiʻo SAM i loko, a he mea nui kēia hoʻoponopono no ka ulu ʻana o ke kumu52. He mea hoihoi ia ke nānā aku inā he kuleana ko GA i ka hoʻonohonoho ʻana i ka mokulele ʻāpana cell i loko o ka hui SAM i loko, a laila hoʻonohonoho i ka kikoʻī a me ka hoʻomohala ʻana o nā internodes i loko o ka SAM.
Ua ulu nā mea kanu i loko o ka 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 kukui, 22 ° C), koe wale no nā hoʻokolohua hypocotyl a me ke aʻa ulu kahi i ulu ai nā mea kanu ma nā papa kūpaʻa ma lalo o ka mālamalama mau a me 22 °C. No nā hoʻokolohua nitrate, ua ulu ʻia nā mea kanu ma luna o MS i hoʻololi ʻia (bioWORLD mea kanu 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 lā lōʻihi.
ʻO GID1a cDNA i hoʻokomo ʻia i loko o pDONR221 ua hui hou ʻia me pDONR P4-P1R-pUBQ10 a me pDONR P2R-P3-mCherry i loko o pB7m34GW e hoʻohua i ka pUBQ10:: GID1a-mCherry. Ua hui hou ʻia ka IDD2 DNA i hoʻokomo ʻia i loko o pDONR221 i pB7RWG266 e hoʻohua i ka p35S: IDD2-RFP. No ka hana ʻana i pGID1b:: 2xmTQ2-GID1b, ua hoʻonui mua ʻia kahi ʻāpana 3.9 kb i luna o ka ʻāpana coding GID1b a me kahi ʻāpana 4.7 kb i loaʻa ka GID1b cDNA (1.3 kb) a me ka terminator (3.4 kb) i hoʻonui ʻia me ka hoʻohana ʻana i nā primers i loko o ka PRmo hoʻohui PNR 3 a laila hoʻokomo ʻia i loko o ka PNR 3 a laila. ʻO Fisher Scientific) a me pDONR P2R-P3 (Thermo Fisher Scientific), i kēlā me kēia, a hoʻohui hou ʻia me pDONR221 2xmTQ268 i loko o ka pGreen 012567 pahu hopu e hoʻohana ana i ka cloning Gateway. No ka hana ʻana i ka pCUC2:: LSSmOrange, ʻo ka CUC2 promoter sequence (3229 bp upstream of ATG) a ukali ʻia e ka coding sequence o Stokes-shifted mOrange (LSSmOrange) 69 me ka N7 nuclear localization signal a me ka NOS transcriptional terminator i hōʻuluʻulu ʻia i loko o ka pGreen-fragment vector i hoʻohana ʻia i ka gateway recombin target. (Invitrogen). Ua hoʻokomo ʻia ka vector binary mea kanu i loko o ka Agrobacterium tumefaciens strain GV3101 a hoʻokomo ʻia i loko o nā lau Nicotiana benthamiana ma ke ʻano infiltration Agrobacterium a i loko o Arabidopsis thaliana Col-0 ma ke ʻano hoʻoheheʻe pua. pUBQ10::qmRGA pUBQ10::GID1a-mCherry a me pCLV3::mCherry-NLS qmRGA ua hoʻokaʻawale ʻia mai nā ʻohana F3 a me F1 o kēlā me kēia keʻa.
Hana ʻia ka RNA in situ hybridization ma kahi o 1 knm ka lōʻihi o ka pana ʻana i nā tips72, i hōʻiliʻili ʻia a hoʻopaʻa koke ʻia i loko o ka solution FAA (3.7% formaldehyde, 5% acetic acid, 50% ethanol) i hoʻomaha ʻia i 4 °C. Ma hope o 2 × 15 min hoʻomaʻamaʻa maʻemaʻe, ua hoʻololi ʻia ka fixative a ua incubated nā laʻana i ka pō. ʻO GID1a, GID1b, GID1c, GAI, RGL1, RGL2, a me RGL3 cDNAs a me nā antisense probes i kā lākou 3′-UTRs ua synthesized me ka hoʻohana ʻana i nā primers i hōʻike ʻia ma ka Pākuʻi Pākuʻi 3 e like me ka wehewehe ʻana e Rosier et al.73. Ua hoʻopaʻa ʻia ʻo Digoxigenin-labeled probes i immunodetected me ka hoʻohana ʻana i nā antibodies digoxigenin (3000-fold dilution; Roche, helu helu: 11 093 274 910), a ua hoʻopaʻa ʻia nā ʻāpana me ka 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 250-foldlue tetrafoldN0, 250-foldlyl phosphate) dilution) solution.
Ka manawa hoʻouna: Feb-10-2025