FT-Like NFT1 Gene May play a role in Flower Transition Induced by Heat Accumulation in Narcissus tazetta var chinensis Xiao-Fang Li, Lin-Yan Jia, Jing Xu, Xin-Jie Deng, Yang Wang, Wei Zhang, Xue-Ping Zhang Qi Fang, Dong-Mei Zhang Yue Sun'and Ling Xu' School of Life Science, East China Normal University, 500 Dongchuan Rd, Shanghai, PR China 200241 Shanghai Institute of Landscape Architecture, 899 Longwu Rd, Shanghai, PR China 200232 Correspondingauthor:E-mail,xfi@bioecnu.edu.cn;Fax,+86-21-62233754. ( Received July 23, 2012; Accepted December 18, 2012) The low-temperature Alowering-response pathway, used as The nucleotide sequence of NFTI reported in this paper has an inductive stimulus to induce flowering in plant species been submitted to NCBl under accession numbers JX316221 for from temperate regions in response to cold temperature, has cDNA and JX316222 for genomic DNA. been extensively studied. However, limited information is available on the flower transition of several bulbous species, Introduction which require high temperature for flower differentiation. Narcissus tazetta var chinensis(Chinese narcissus)exhibits a Timing of transition to flowering in higher plants is controlled 2 year juvenile phase, and flower initiation within its bulbs through environmental and endogenous cues. Genetic and mo- occurs during summer dormancy. The genetic factors that lecular studies in the model plant Arabidopsis thaliana character control flower initiation are mostly unknown in Chinese ize a complex network of genetic pathways that regulate narcissusIn the present study, we found that a high storage fowering(Amasino and Michaels 2010). Vemalization, ambient temperature is necessary for flower initiation. Flower initi- temperature and photoperiod pathways regulate fowering in ation was advanced in bulbs previously exposed to extended response to environmental cues, whereas autonomous, gibberel high temperature. The heat accumulation required for lin and development stage pathways regulate flowering in re- flower transition was also determined. High temperature sponse to endogenous signals (Samach and Wigge 2005, treatment rescued the low flower percentage resulting Kobayashi and Weigel 2007, Farrona et al. 2008 Turck et al. from short storage duration under natural conditions. In 2008, Kim et al. 2009, Mutasa-Gottgens and Hedden 2009, addition, extended high storage temperature was found to Wang et al 2009a, Amasino and Michaels 2010).Several key com increase the fowering percentage of 2-year-old plants, which ponent genes involved in these pathways have orthologs in a wide can be applied in breeding. Narcissus FLOWERING LOCUs variety of plants, induding other monocots and perennials T1(NFT1, a homolog of the Arabidopsis thaliana gene (Amasino 2010). Arabidopsis Flowering Locus T(FT) protein is FLOWERING LOCUS T, was isolated in this study NFT1 tran- now widely accepted as a mobile forigen( Corbesier et al. 2007, scripts were abundant during fower initiation in mature Giakountis and Coupland 2008). FT is activated in the leaf in bulbs and were up-regulated by high temperature The gen- response to an inductive photoperiod, and subsequently moves etic experiments, coupled with an expression profiling assay, to the shoot apex FT interacts with the product of Flowering suggest that NFT1 possibly takes part in flower transition Locus D(FD), a bZIP protein, at the vegetative shoot apex, and control in response to high temperature. then activates transcription of foral meristem genes to start the fowering process(Abe et al. 2005, Wigge et al. 2005). FT orthold Keywords: Flower initiation Narcissus tazetta var. have been discovered in several plant species(Bohlenius et al chinensis·NFT1· Temperature. 2006, Yan et al. 2006, Faure et al. 2007, Gyllenstrand et al. 2007, Abbreviations: CO,CONSTANS; FD, Flowering Locus D, Hayama et aL. 2007, Danilevskaya et al. 2008, Colasanti and Coneva FLC, FLOWERING LOCUS C: FT, FLOWERING LOCUS T: 2009, Hou and Yang 2009, Kikuchi et al. 2009, Komiya et al. 2009, qRT-PCR, quantitative real-time PCR; RACE, rapid ampli- Blackman et al. 2010, Kong et al. 2010) fication of cDNA ednds; SEM, scanning electron microscopy, Seasonal temperature changes elicit seasonal flowering re- SM, shoot meristem; SOC1, SUPPRESSOR OF OVEREX sponses that allow the synchronization of fowering with opti- PRESSION OF CONSTANST; TFL1, TERMINAL FLOWER 1; mal conditions(King and Heide 2009, Hemming and Trevaskis WT, wild type 2011). Several plant species from temperate regions use cold PlantCellPhysiol54(2):270-281(2013)doi:10.1093/pcp/pcs181,availableonlineatwww.pcp.oxfordjournalsorg C The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologis AllrightsreservedForpermissionspleaseemailjournalspermissions@oup.com 270 Plant Cell Physiol. 54(2): 270-281(2013)doi: 10.1093/pcp/pcs181 C The Author 2013
FT-Like NFT1 Gene May Play a Role in Flower Transition Induced by Heat Accumulation in Narcissus tazetta var. chinensis Xiao-Fang Li1,*, Lin-Yan Jia1 , Jing Xu1 , Xin-Jie Deng1 , Yang Wang1 , Wei Zhang1 , Xue-Ping Zhang1 , Qi Fang1 , Dong-Mei Zhang2 , Yue Sun1 and Ling Xu1 1 School of Life Science, East China Normal University, 500 Dongchuan Rd., Shanghai, PR China 200241 2 Shanghai Institute of Landscape Architecture, 899 Longwu Rd., Shanghai, PR China 200232 *Corresponding author: E-mail, xfli@bio.ecnu.edu.cn; Fax, +86-21-62233754. (Received July 23, 2012; Accepted December 18, 2012) The low-temperature flowering-response pathway, used as an inductive stimulus to induce flowering in plant species from temperate regions in response to cold temperature, has been extensively studied. However, limited information is available on the flower transition of several bulbous species, which require high temperature for flower differentiation. Narcissus tazetta var. chinensis (Chinese narcissus) exhibits a 2 year juvenile phase, and flower initiation within its bulbs occurs during summer dormancy. The genetic factors that control flower initiation are mostly unknown in Chinese narcissus. In the present study, we found that a high storage temperature is necessary for flower initiation. Flower initiation was advanced in bulbs previously exposed to extended high temperature. The heat accumulation required for flower transition was also determined. High temperature treatment rescued the low flower percentage resulting from short storage duration under natural conditions. In addition, extended high storage temperature was found to increase the flowering percentage of 2-year-old plants, which can be applied in breeding. Narcissus FLOWERING LOCUS T1 (NFT1), a homolog of the Arabidopsis thaliana gene FLOWERING LOCUS T, was isolated in this study. NFT1 transcripts were abundant during flower initiation in mature bulbs and were up-regulated by high temperature. The genetic experiments, coupled with an expression profiling assay, suggest that NFT1 possibly takes part in flower transition control in response to high temperature. Keywords: Flower initiation Narcissus tazetta var. chinensis NFT1 Temperature. Abbreviations: CO, CONSTANS; FD, Flowering Locus D, FLC, FLOWERING LOCUS C; FT, FLOWERING LOCUS T; qRT-PCR, quantitative real-time PCR; RACE, rapid ampli- fication of cDNA ednds; SEM, scanning electron microscopy; SM, shoot meristem; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1; TFL1, TERMINAL FLOWER 1; WT, wild type. The nucleotide sequence of NFT1 reported in this paper has been submitted to NCBI under accession numbers JX316221 for cDNA and JX316222 for genomic DNA. Introduction Timing of transition to flowering in higher plants is controlled through environmental and endogenous cues. Genetic and molecular studies in the model plant Arabidopsis thaliana characterize a complex network of genetic pathways that regulate flowering (Amasino and Michaels 2010). Vernalization, ambient temperature and photoperiod pathways regulate flowering in response to environmental cues, whereas autonomous, gibberellin and development stage pathways regulate flowering in response to endogenous signals (Samach and Wigge 2005, Kobayashi and Weigel 2007, Farrona et al. 2008, Turck et al. 2008, Kim et al. 2009, Mutasa-Gottgens and Hedden 2009, Wang et al. 2009a, Amasino and Michaels 2010). Several key component genes involved in these pathways have orthologs in a wide variety of plants, including other monocots and perennials (Amasino 2010). Arabidopsis Flowering Locus T (FT) protein is now widely accepted as a mobile florigen (Corbesier et al. 2007, Giakountis and Coupland 2008). FT is activated in the leaf in response to an inductive photoperiod, and subsequently moves to the shoot apex. FT interacts with the product of Flowering Locus D (FD), a bZIP protein, at the vegetative shoot apex, and then activates transcription of floral meristem genes to start the flowering process (Abe et al. 2005, Wigge et al. 2005). FT orthologs have been discovered in several plant species (Bohlenius et al. 2006, Yan et al. 2006, Faure et al. 2007, Gyllenstrand et al. 2007, Hayama et al. 2007, Danilevskaya et al. 2008, Colasanti and Coneva 2009, Hou and Yang 2009, Kikuchi et al. 2009, Komiya et al. 2009, Blackman et al. 2010, Kong et al. 2010). Seasonal temperature changes elicit seasonal flowering responses that allow the synchronization of flowering with optimal conditions (King and Heide 2009, Hemming and Trevaskis 2011). Several plant species from temperate regions use cold Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181, available online at www.pcp.oxfordjournals.org ! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com 270 Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. Regular Paper at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from
NFT1 involved in flowering initiation induced by heating temperature signaling for reproduction (Wilkie et al. 2008, arid areas(e.g Cyclamen, Pancratium and Bellevalia) require Hemming and Trevaskis 2011), a phenomenon known as'ver- high summer temperatures for flower transition within nalization. The MADS-box gene FLOWERING LOCUS C(FLC)is the bulb. No cold induction is required for Aoral development a key regulator of vernalization-induced Lowering in A thaliana and stalk elongation (Kamenetsky and Fritsch 2002, nd related species(Sheldon et al. 2008, Sheldon et al. 2009, Kamenetsky and Rabinowitch 2002, Flaishman and Wang et al. 2009b). FLC inhibits fowering by repressing the Kamenetsky 2006). However, limited information on the mo- genes that promote fowering such as FT and the lecular mechanisms that regulate Rower initiation in response SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1) to temperature in bulbous geophytes is available to date ( Helliwell et al. 2006, Searle et al. 2006). Long durations of A high summer temperature signal is often used to advance cold temperatures suppress FLC transcription quantitatively, narcissus flowering. Chinese narcissus(Narcissus tazetta var. thereby allowing rapid flowering(Sheldon et al. 200 chinensis)is a plant from the Amaryllidaceae family that ex- A thaliana, FLC transcription is stably repressed by vernaliza. hibits summer dormancy. Its bulbs sprout in October to tion, and FLC expression remained low after the plants returned November(when soil temperature drops), grow throughout to warm conditions(Sheldon etal. 2000, Sheldonet al 2009). This the winter and flower in January to February. The above-ground effect provides a molecular 'memory of winter and allows rapid parts of the plants begin to senesce in late spring. Chinese fowering as temperature and daylength increase during spring. narcissus exhibits a 2 year juvenile phase. Florogenesis is The vernalization response in cereals is controlled by another initiated within large-sized bulbs during summer dormancy MADS-box gene, namely VERNALIZATION1(VRNI)(Trevaskis Timing of fower initiation in its dormant bulbs varies, depend et al. 2007). In contrast to FLC, VRNI promotes flowering, and its ing on where they were cultivated. Flower initiation occurs in transcript levels are low in plants that have not been vernalized. early June in Guangzhou, early July in Zhangzhou and late july in Exposure to cold temperatures increases VRNI transcript levels Shanghai(Zhong 1984, Li et al. 1987, Zhang and Yang 1987, Li quantitatively, thereby accelerating transition to reproductive et al. 2012). Noy-Porata et al. (2009) showed that foral initi- growth at the shoot apex, and also makes plants respond to ation and reproductive development in Galilee(N. tazetta) long days by de-repressing the long-day flowering-response path- cultivated in Israel is promoted by high temperature at an op- way in the leaves (Yan et al. 2003, von Zitzewitz et al. 2005, Sasani timum of 25C, whereas low temperatures(12C)inhibit for 号月已 et aL. 2009). In addition, the different roles of FT-like proteins in ogenesis completely. The foral transition in Chinese narcissus response to temperature, which regulates the reproductive tran- in response to environmental conditions and the molecular sition in some biennials and perennials, were revealed( Lifschitz mechanisms that regulate these responses remain unknown et al. 2006, Pin et al. 2010, Hsuet al. 2011). Two paralogs of the FT In this study, different temperature regimes were designed gene(BuFT1 and BvFT2)have evolved antagonistic functions in and different planting dates were employed for >3 years to biennial sugar beets(Beta vulgaris ssp. vulgaris). BvFT2 is func- determine the right inductive stimuli that will predict the re- tionally conserved with FT and is essential to fowering In con- productive development in the bulb of Chinese narcissus. The rast, BvFTI represses fowering, and its down-regulation is crucial reproductive organogenesis in 3-year-old bulbs and fowering for the vernalization response in beets. In the woody perennial percentage were assayed. Different storage temperature re- poplar(Populus spp ) the FLOWERING LOCUS T1(FT1) and gimes were also designed and performed on 2-year-old bulbs FLOWERING LOCUS T2( FT2) paralogs coordinate repeated to address the relationship between juvenile-adult phase ycles of vegetative and reproductive growth. Reproductive change and temperature. One Ft homolog, Narcissus onset is determined by FTi in response to winter temperatures, Flowering Locus T1(NFT1), was also isolated from Chinese nar- C29/=9E whereas vegetative growth and inhibition of bud set are pro- cissus. Its function was assayed to determine whether the genes moted by FT2 in response to warm temperatures and long days shown previously in A. thaliana also regulate flowering. This during the growing season(Hsu et aL. 2011). These advances sug. study showed that extended high temperature exposure not gest that the duplication or changes in FT genes contributed to only triggers the transition of the bulb shoot meristem(SM) the evolution of plant adaptation to environmental cues. Flower transition requires different temperature conditions, shortens the juvenile phase. In addition, NFTI was shown to depending on the ecological origin of the bulbous geophyte mediate flower transition in response to high temperature species(Halevy 1990, Flaishman and Kamenetsky 2006). es(eg. Lilium, Galtonia and Allium cepa)usually require low temperatures for fower differ Results entiation, such as vernalization in the winter annual model plant Arabidopsis Species with thermoperiodic cycles(such High storage temperature is essential to flower as Tulipa, Narcissus and Hyachinthus), from the Irano- initiation in Chinese narcissus ranian and Mediterranean regions, require relatively high The scanning electron microscopy(SEM)assay showed that temperatures for Lower differentiation inside the bulb, as well flower initiation occurred earlier in 3-year-old bulbs stored at as a period of low temperatures to allow foral stem elongation 30C compared with those under natural conditions( Fig. 1) and anthesis( Flaishman and Kamenetsky 2006). Species from Flower transition began in late july under natural conditions, Plant Cell Physiol. 54(2): 270-281(2013)doi: 10. 1093/pcp/pcs181 C The Author 2013. 271
temperature signaling for reproduction (Wilkie et al. 2008, Hemming and Trevaskis 2011), a phenomenon known as ‘vernalization’. The MADS-box gene FLOWERING LOCUS C (FLC) is a key regulator of vernalization-induced flowering in A. thaliana and related species (Sheldon et al. 2008, Sheldon et al. 2009, Wang et al. 2009b). FLC inhibits flowering by repressing the genes that promote flowering, such as FT and the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) (Helliwell et al. 2006, Searle et al. 2006). Long durations of cold temperatures suppress FLC transcription quantitatively, thereby allowing rapid flowering (Sheldon et al. 2009). In A. thaliana, FLC transcription is stably repressed by vernalization, and FLC expression remained low after the plants returned to warm conditions (Sheldonet al. 2000, Sheldonet al. 2009). This effect provides a molecular ‘memory’ of winter and allows rapid flowering as temperature and daylength increase during spring. The vernalization response in cereals is controlled by another MADS-box gene, namely VERNALIZATION1 (VRN1) (Trevaskis et al. 2007). In contrast to FLC, VRN1 promotes flowering, and its transcript levels are low in plants that have not been vernalized. Exposure to cold temperatures increases VRN1 transcript levels quantitatively, thereby accelerating transition to reproductive growth at the shoot apex, and also makes plants respond to long days by de-repressing the long-day flowering-response pathway in the leaves (Yan et al. 2003, von Zitzewitz et al. 2005, Sasani et al. 2009). In addition, the different roles of FT-like proteins in response to temperature, which regulates the reproductive transition in some biennials and perennials, were revealed (Lifschitz et al. 2006, Pin et al. 2010, Hsu et al. 2011). Two paralogs of the FT gene (BvFT1 and BvFT2) have evolved antagonistic functions in biennial sugar beets (Beta vulgaris ssp. vulgaris). BvFT2 is functionally conserved with FT and is essential to flowering. In contrast,BvFT1represses flowering, and itsdown-regulation is crucial for the vernalization response in beets. In the woody perennial poplar (Populus spp.), the FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2) paralogs coordinate repeated cycles of vegetative and reproductive growth. Reproductive onset is determined by FT1 in response to winter temperatures, whereas vegetative growth and inhibition of bud set are promoted by FT2 in response to warm temperatures and long days during the growing season (Hsu et al. 2011). These advances suggest that the duplication or changes in FT genes contributed to the evolution of plant adaptation to environmental cues. Flower transition requires different temperature conditions, depending on the ecological origin of the bulbous geophyte species (Halevy 1990, Flaishman and Kamenetsky 2006). Species from temperate zones (e.g. Lilium, Galtonia and Allium cepa) usually require low temperatures for flower differentiation, such as vernalization in the winter annual model plant Arabidopsis. Species with thermoperiodic cycles (such as Tulipa, Narcissus and Hyachinthus), from the IranoTuranian and Mediterranean regions, require relatively high temperatures for flower differentiation inside the bulb, as well as a period of low temperatures to allow floral stem elongation and anthesis (Flaishman and Kamenetsky 2006). Species from arid areas (e.g. Cyclamen, Pancratium and Bellevalia) require high summer temperatures for flower transition within the bulb. No cold induction is required for floral development and stalk elongation (Kamenetsky and Fritsch 2002, Kamenetsky and Rabinowitch 2002, Flaishman and Kamenetsky 2006). However, limited information on the molecular mechanisms that regulate flower initiation in response to temperature in bulbous geophytes is available to date. A high summer temperature signal is often used to advance narcissus flowering. Chinese narcissus (Narcissus tazetta var. chinensis) is a plant from the Amaryllidaceae family that exhibits summer dormancy. Its bulbs sprout in October to November (when soil temperature drops), grow throughout the winter and flower in January to February. The above-ground parts of the plants begin to senesce in late spring. Chinese narcissus exhibits a 2 year juvenile phase. Florogenesis is initiated within large-sized bulbs during summer dormancy. Timing of flower initiation in its dormant bulbs varies, depending on where they were cultivated. Flower initiation occurs in early June in Guangzhou, early July in Zhangzhou and late July in Shanghai (Zhong 1984, Li et al. 1987, Zhang and Yang 1987, Li et al. 2012). Noy-Porata et al. (2009) showed that floral initiation and reproductive development in ‘Galilee’ (N. tazetta) cultivated in Israel is promoted by high temperature at an optimum of 25C, whereas low temperatures (12C) inhibit florogenesis completely. The floral transition in Chinese narcissus in response to environmental conditions and the molecular mechanisms that regulate these responses remain unknown. In this study, different temperature regimes were designed and different planting dates were employed for >3 years to determine the right inductive stimuli that will predict the reproductive development in the bulb of Chinese narcissus. The reproductive organogenesis in 3-year-old bulbs and flowering percentage were assayed. Different storage temperature regimes were also designed and performed on 2-year-old bulbs to address the relationship between juvenile–adult phase change and temperature. One FT homolog, Narcissus Flowering Locus T1 (NFT1), was also isolated from Chinese narcissus. Its function was assayed to determine whether the genes shown previously in A. thaliana also regulate flowering. This study showed that extended high temperature exposure not only triggers the transition of the bulb shoot meristem (SM) from the vegetative stage to the reproductive stage, but also shortens the juvenile phase. In addition, NFT1 was shown to mediate flower transition in response to high temperature. Results High storage temperature is essential to flower initiation in Chinese narcissus The scanning electron microscopy (SEM) assay showed that flower initiation occurred earlier in 3-year-old bulbs stored at 30C compared with those under natural conditions (Fig. 1). Flower transition began in late July under natural conditions, Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. 271 NFT1 involved in flowering initiation induced by heating at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from
PO X.F. Li et al LANT是 四1,1如1,何。砌 CK Storage under natural conditions Storage at3℃ Storage at I5℃ Tw lowest tene 35 口F0 8388=808号 6.16.106.207.1710120818.1082091 K No I No.2 CK No I No. CK No. 1 No. 2 CK Ne.I No. CK Ne. I No. CK NeI Ne2 treatments 75 storage days Fig. 1 High storage temperature promoted flower initiation in Chinese narcissus.(A)Illustration of different storage treatments of narcissus bulbs, with temperature regimes shown below the corresponding date.( B) Temperature of different treatments during storage. The average highest and lowest ofnaturalconditionsduring1951-2008inShanghaiwerebasedonhttp://php.weather.sina.com.cn/whd php?city.(C) Flower differentiation in Chinese narcissus is shown on scanning electron photomicrographs. The initiation process was classified into six stages, from FO to F5 FO is the vegetative meristem(VM), with only the differentiation of leaf primordium(LP). F1 is the meristem on the transition from VM to inflorescence meristem(IM) with the youngest LP F2 is the inflorescence meristem with the youngest LP removed. F3 is the spathe primordium(SP)beginning to initiate the periphery of the IM. f4 is the spathe enwrapping the IM. F5 shows that several floral meristems(FMs) begin to form in the spathe(the spathe was removed.( D) Percentage of meristems with a specific flower initiation stage on different days during storage. Five to 10 samples of each treatment at relevant time points were detected. Detailed temperature regimes in (D)are shown in(A)and( B) which were temperatures of between 19 and 33C in June, July High temperature can rescue the low flowering and August in Shanghai( Fig. 1B-D). Before mid-July, the SM in percentage due to short storage duration under the 3-year-old bulbs was sharp conical and only leaf primordia were differentiated around its edge(Fig. IC, FO). In late July (ie. natural conditions 50-60d post-harvest), some SMs began to fatten( F1 and F2 in The fowering percentage of 3-year-old bulbs became higher Fig 1C), spathe primordium began to initiate on the periphery as the planting date was delayed. Only 66.7% of the of the Rat inflorescence meristem(F3 in Fig. 1C), and the bulbs planted on July 25 flowered, which was significantly spathe enwrapped the inflorescence meristem(F4 in Fig. 10) lower than those planted on August 15 and September gradually. The flower meristems in the spathe began to initiate (87.7% and 94.1%, respectively). This preliminary test at about 7o d post-harvest(F5 in Fig. 1C, D). Afterwards, the showed that long natural storage duration improves fowering developed Rowers formed in one spathe inflorescence in late percentage, while short storage duration causes a low August. Early Aat inflorescence meristems(F1 in Fig. 1C)ap- the bulbs treated at 30oC for 20d, followed by short stor- peared at about the 40th day(Fig. 1D), and the whole Rower initiation process was advanced by about 10 d relative to that age at natural temperature(No. 1 in Fig. 2A), increased to under natural conditions, when the bulbs were stored at 300C 92.3%, which was not signifcantly different from those planted (Fig. 1D). The SM was kept at its sharp conical shape and at the on August 15( Fig. 2A, B). These data suggested that a FO stage when the 3-year-old bulbs were stored at 15 C No flat high percentage of fiowering required certain high temperature in florescence meristem was found throughout 1 year( Fig. 1D). accumulation during storage. 272 Plant Cell Physiol. 54(2): 270-281(2013)doi: 10.1093/pcp/pcs181 C The Author 2013
which were temperatures of between 19 and 33C in June, July and August in Shanghai (Fig. 1B–D). Before mid-July, the SM in the 3-year-old bulbs was sharp conical and only leaf primordia were differentiated around its edge (Fig. 1C, F0). In late July (i.e. 50–60 d post-harvest), some SMs began to flatten (F1 and F2 in Fig. 1C), spathe primordium began to initiate on the periphery of the flat inflorescence meristem (F3 in Fig. 1C), and the spathe enwrapped the inflorescence meristem (F4 in Fig. 1C) gradually. The flower meristems in the spathe began to initiate at about 70 d post-harvest (F5 in Fig. 1C, D). Afterwards, the floral organ primordia were soon initiated, and several fully developed flowers formed in one spathe inflorescence in late August. Early flat inflorescence meristems (F1 in Fig. 1C) appeared at about the 40th day (Fig. 1D), and the whole flower initiation process was advanced by about 10 d relative to that under natural conditions, when the bulbs were stored at 30C (Fig. 1D). The SM was kept at its sharp conical shape and at the F0 stage when the 3-year-old bulbs were stored at 15C. No flat inflorescence meristem was found throughout 1 year (Fig. 1D). High temperature can rescue the low flowering percentage due to short storage duration under natural conditions The flowering percentage of 3-year-old bulbs became higher as the planting date was delayed. Only 66.7% of the bulbs planted on July 25 flowered, which was significantly lower than those planted on August 15 and September 1 (87.7% and 94.1%, respectively). This preliminary test showed that long natural storage duration improves flowering percentage, while short storage duration causes a low flowering percentage. However, the flowering percentage of the bulbs treated at 30C for 20 d, followed by short storage at natural temperature (No. 1 in Fig. 2A), increased to 92.3%, which was not significantly different from those planted on August 15 (Fig. 2A, B). These data suggested that a high percentage of flowering required certain high temperature accumulation during storage. Fig. 1 High storage temperature promoted flower initiation in Chinese narcissus. (A) Illustration of different storage treatments of narcissus bulbs, with temperature regimes shown below the corresponding date. (B) Temperature of different treatments during storage. The average highest and lowest temperatures of natural conditions during 1951–2008 in Shanghai were based on http://php.weather.sina.com.cn/whd. php?city. (C) Flower differentiation in Chinese narcissus is shown on scanning electron photomicrographs. The initiation process was classified into six stages, from F0 to F5. F0 is the vegetative meristem (VM), with only the differentiation of leaf primordium (LP). F1 is the meristem on the transition from VM to inflorescence meristem (IM) with the youngest LP. F2 is the inflorescence meristem with the youngest LP removed. F3 is the spathe primordium (SP) beginning to initiate the periphery of the IM. F4 is the spathe enwrapping the IM. F5 shows that several floral meristems (FMs) begin to form in the spathe (the spathe was removed). (D) Percentage of meristems with a specific flower initiation stage on different days during storage. Five to 10 samples of each treatment at relevant time points were detected. Detailed temperature regimes in (D) are shown in (A) and (B). 272 Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. X.-F. Li et al. at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from
NFT1 involved in flowering initiation induced by heating 21,22,mxm to that of the FT-like genes in the FT-tFL family genes, with a identity of 70% and 77% to Arabidopsis FT and rice Hd3a, re- spectively, as well as 52% identity to TFL in Arabidopsis. Sequence comparison between NFT1 and the FT-TFL family proteins showed that NFT1 carries the functionally important K2 Storage under natural conditions FT signatures Tyr85(Y)(Tyr79 in NFT1)and Gln 140(Q)(GIn 134 in NFT1)(Fig. 4A)(Hanzawa et al. 2005). In addition, the gene Storage under natural conditions structure of NFT1 was similar to that of FT(Fig. 4B), and NFTI exhibited a region identical to most other FT genes within seg- 30C for 20 d ment B of the fourth exon(encoding an external loop of PEBP (Fig. 4A; Supplementary Fig. S1), which is important to FT vs. TFLI function in Arabidopsis(Ahn et al. 2006). Phylogeny re- constructions with other published FT-TFL genes clearly showed that NFT1 falls into the FT-like subfamily, rather thar the TFL- and MFT-like subfamily(Supplementary Fig. $2) Expression pattern of NFT1 in Chinese narcissus The spatial expression pattern of NFT1 was detected through in situ hybridization analysis. Mature leaf blades, shoot apice from 3-year-old growing plants and fower buds were used Treatments for in situ hybridization. The transcripts of Arabidopsis FT, Fig. 2 High temperature treatment can rescue the low flowering per- rice Hd3a and RFTi were mainly detected in the leaf phloem centage of 3-year-old bulbs caused by a short duration of storage (Tamaki et al. 2007, Komiya et al. 2009). Similarly, hybridization under natural conditions.(A)llustration of the different storage treat- with NFT1 RNA antisense probe revealed signals over vascular ments of narcissus bulbs, with temperature regimes shown below the bundles within transverse leaf sections(Fig 5A, B). The signal 号月已 corresponding date.(B)Effect of different planting dates on the fow- ering percentage of 3-year-old bulbs. Different lower case letters indi. was detected primarily in the phloem, xylem parenchyma and cate significant differences between different treatments(Pearson's muscle cells at high magnification(Fig 5B). NFT1 signal was too x, P<0.05, n=30-50). Detailed temperature regimes in(B low to be detected in the shoot apices of the 3-year-old bulbs shown in(A). during endo-dormancy(ie. from April to early July under nat- ural conditions)(data not shown). However, signal was de- tected in the apices from late July(Fig. 5C)and in early Sufficient high temperature treatment can Rower buds. Gene-specific quantitative real-time PCR improve the flowering rate of 2-year-old bulbs (qRT-PCR)analyses were performed on mRNA samples from Fewer than 25% of the 2-year-old bulbs flowered when stored Rowers, leaves, shoot apices and bulbs during active growth. natural temperature( Fig. 3B). Almost no plants flowered when Strong expression levels of NFT1 were detected in the leaves the 2-year-old bulbs were initially subjected to 30 C for 20d, and shoot apices, whereas low levels were detected in opening with storage at 22-25%C until planting. The maximum flower- flowers and bulbs. The bulbs have several layers of scales, and ing percentage of the bulbs treated at 30 C for 40 d, with stor- the expression levels in scales at different locations did not age at 22-25.C, was 38.6%; whereas the flowering percentage of show any significant difference(Fig. 5D) those subjected to treatment at 30oC for 80d was 86.1%.The esults for the 2-year-old bulbs stored at natural temperature Ectopic expression of NFT1 in A thaliana and those treated at 30C for 40 d, with storage at 22-25oC, advanced flowering were not signifcantly different(Fig. 3B). Obviously, longer Ectopic expression through the 35S promoter provided evi- treatment at high temperature improves the Owering rate of dence that NFT1 encodes a protein that acts as floral regulator 2-year-old bulbs in Arabidopsis. A total of nine lines with the 35S: NFTI con- struct in Col plants [ wild type(WT))and five lines with trans- Isolation of NFTl, a homolog of FT, from Chinese formation of the 355: NFTI construct directly into ft-3 mutant narcissus plants plants were selected for flowering time analysis. No develop Using degenerate primers and the RACE (rapid amplification of mental abnormality, except for flowering time, was caused in DNA ends) method, a full-length cDNA of the NFTI clone, these lines. The flowering time of most lines was significantly comprising the complete coding regions, was isolated. Analysis earlier than that of WT or ft-3 mutant plants under inductive of the sequences showed that NFT1 CDNA was 816 bp long and long-day conditions(Fig. 6A, B, E, F). The lines of the 35S: NFT1 encodes a protein with a predicted length of 174 amino acids. construct in Col flowered 7-14 d earlier and made 5-7 leave The amino acid sequence encoded by NFTI was highly similar fewer than those of Col controls( Fig. 6B). Although the lines of Plant Cell Physiol. 54(2): 270-281(2013)doi: 10. 1093/pcp/pcs181 C The Author 2013. 273
Sufficient high temperature treatment can improve the flowering rate of 2-year-old bulbs Fewer than 25% of the 2-year-old bulbs flowered when stored at natural temperature (Fig. 3B). Almost no plants flowered when the 2-year-old bulbs were initially subjected to 30C for 20 d, with storage at 22–25C until planting. The maximum flowering percentage of the bulbs treated at 30C for 40 d, with storage at 22–25C, was 38.6%; whereas the flowering percentage of those subjected to treatment at 30C for 80 d was 86.1%. The results for the 2-year-old bulbs stored at natural temperature and those treated at 30C for 40 d, with storage at 22–25C, were not significantly different (Fig. 3B). Obviously, longer treatment at high temperature improves the flowering rate of 2-year-old bulbs. Isolation of NFT1, a homolog of FT, from Chinese narcissus plants Using degenerate primers and the RACE (rapid amplification of cDNA ends) method, a full-length cDNA of the NFT1 clone, comprising the complete coding regions, was isolated. Analysis of the sequences showed that NFT1 cDNA was 816 bp long and encodes a protein with a predicted length of 174 amino acids. The amino acid sequence encoded by NFT1 was highly similar to that of the FT-like genes in the FT-TFL family genes, with an identity of 70% and 77% to Arabidopsis FT and rice Hd3a, respectively, as well as 52% identity to TFL in Arabidopsis. Sequence comparison between NFT1 and the FT-TFL family proteins showed that NFT1 carries the functionally important FT signatures Tyr85(Y) (Tyr79 in NFT1) and Gln140(Q) (Gln134 in NFT1) (Fig. 4A) (Hanzawa et al. 2005). In addition, the gene structure of NFT1 was similar to that of FT (Fig. 4B), and NFT1 exhibited a region identical to most other FT genes within segment B of the fourth exon (encoding an external loop of PEBP) (Fig. 4A; Supplementary Fig. S1), which is important to FT vs. TFL1 function in Arabidopsis (Ahn et al. 2006). Phylogeny reconstructions with other published FT-TFL genes clearly showed that NFT1 falls into the FT-like subfamily, rather than the TFL- and MFT-like subfamily (Supplementary Fig. S2). Expression pattern of NFT1 in Chinese narcissus The spatial expression pattern of NFT1 was detected through in situ hybridization analysis. Mature leaf blades, shoot apices from 3-year-old growing plants and flower buds were used for in situ hybridization. The transcripts of Arabidopsis FT, rice Hd3a and RFT1 were mainly detected in the leaf phloem (Tamaki et al. 2007, Komiya et al. 2009). Similarly, hybridization with NFT1 RNA antisense probe revealed signals over vascular bundles within transverse leaf sections (Fig. 5A, B). The signal was detected primarily in the phloem, xylem parenchyma and muscle cells at high magnification (Fig. 5B). NFT1 signal was too low to be detected in the shoot apices of the 3-year-old bulbs during endo-dormancy (i.e. from April to early July under natural conditions) (data not shown). However, signal was detected in the apices from late July (Fig. 5C) and in early flower buds. Gene-specific quantitative real-time PCR (qRT-PCR) analyses were performed on mRNA samples from flowers, leaves, shoot apices and bulbs during active growth. Strong expression levels of NFT1 were detected in the leaves and shoot apices, whereas low levels were detected in opening flowers and bulbs. The bulbs have several layers of scales, and the expression levels in scales at different locations did not show any significant difference (Fig. 5D). Ectopic expression of NFT1 in A. thaliana advanced flowering Ectopic expression through the 35S promoter provided evidence that NFT1 encodes a protein that acts as floral regulator in Arabidopsis. A total of nine lines with the 35S::NFT1 construct in Col plants [wild type (WT)] and five lines with transformation of the 35S::NFT1 construct directly into ft-3 mutant plants were selected for flowering time analysis. No developmental abnormality, except for flowering time, was caused in these lines. The flowering time of most lines was significantly earlier than that of WT or ft-3 mutant plants under inductive long-day conditions (Fig. 6A, B, E, F). The lines of the 35S::NFT1 construct in Col flowered 7–14 d earlier and made 5–7 leaves fewer than those of Col controls (Fig. 6B). Although the lines of Fig. 2 High temperature treatment can rescue the low flowering percentage of 3-year-old bulbs caused by a short duration of storage under natural conditions. (A) Illustration of the different storage treatments of narcissus bulbs, with temperature regimes shown below the corresponding date. (B) Effect of different planting dates on the flowering percentage of 3-year-old bulbs. Different lower case letters indicate significant differences between different treatments (Pearson’s 2 , P < 0.05, n = 30–50). Detailed temperature regimes in (B) are shown in (A). Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. 273 NFT1 involved in flowering initiation induced by heating at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from
PO LANT是 呵°,,_L^叫Pp:,Mom 203110203010203110203010Day 1匚 Storage un der natural condition 2 30C for 20 h 30℃for40d Storage at30℃for80d 1-2+3-4 B 8890号月 61 73798593 Days post plantin Fig. 3 High storage temperature increases the fowering percentage of 2-year-old bulbs.(A)Illustration of different storage treatments of narcissus bulbs, with temperature regimes shown below the corresponding date, and continuous lines representing periods between 22 an 25C.(B)Effect of storage temperature on the percentage of bolting plants at different days from the planting date. Different lower case letters indicate signifcant differences between different treatments at a given time point(Pearsons x, P<0.05, n= 30-0). Detailed temperature regimes in(b)are shown in(A). the 35S: NFT1 construct in ft-3 Lowered later than the WT Ler Expression of NFT1 in Chinese narcissus induced plants, these lines flowered about 15 d earlier and made 3-10 by foral inductive treatment leaves fewer than the ft-3 controls(Fig. 6F). Several transgenic The foregoing data showed that flowers were differentiated in lines were selected for the semi-quantitative or qRT-PCR ana- the bulbs during dormancy, when the species has no leaf, and lysis of the expression levels in mature leaves. All transgenic that flower initiation in Chinese narcissus was induced and lines showed NFT1 expression(Fig. 6C, G; Supplementary advanced by high temperature. The change in transcription Fig. $3).SOC1 is one of the targets of FT (Lee and Lee 2010), abundance in the bulb apices was assayed using RT-PCR and and the SOCI transcript was up-regulated by the ectopic ex- RT-PCR during the entire storage time to determine whether pression of NFTI(Fig. 6D, H; Supplementary Fig. $3). SoC1 flower transition is related to NFT1 expression NFT1 gene tran- expression levels in the transgenic lines of ft-3 were higher scription increased gradually at 1 month before the early Alower than those of the parent line of ft-3. However, they were not transition (indicated by the triangle in Fig. 7A)under natural consistently than that of WT Ler plants(Fig. 6H; conditions, and reached its peak at the moment of flower tran- Supplementa S3B). The expression of endogenous sition( Fig. 7A, B). NFTI gene transcription also increased grad AtFT in the transgenic lines was also not consistently higher ually in the apices of the bulbs stored at high temperature than in the parent lines(Fig. 6D, H; Supplementary Fig S3). (300C). Moreover, NFTI gene transcription in the bulb apices 274 Plant Cell Physiol. 54(2): 270-281(2013)doi: 10.1093/pcp/pcs181 C The Author 2013
the 35S::NFT1 construct in ft-3 flowered later than the WT Ler plants, these lines flowered about 15 d earlier and made 3–10 leaves fewer than the ft-3 controls (Fig. 6F). Several transgenic lines were selected for the semi-quantitative or qRT-PCR analysis of the expression levels in mature leaves. All transgenic lines showed NFT1 expression (Fig. 6C, G; Supplementary Fig. S3). SOC1 is one of the targets of FT (Lee and Lee 2010), and the SOC1 transcript was up-regulated by the ectopic expression of NFT1(Fig. 6D, H; Supplementary Fig. S3). SOC1 expression levels in the transgenic lines of ft-3 were higher than those of the parent line of ft-3. However, they were not consistently higher than that of WT Ler plants (Fig. 6H; Supplementary Fig. S3B). The expression of endogenous AtFT in the transgenic lines was also not consistently higher than in the parent lines (Fig. 6D, H; Supplementary Fig. S3). Expression of NFT1 in Chinese narcissus induced by floral inductive treatment The foregoing data showed that flowers were differentiated in the bulbs during dormancy, when the species has no leaf, and that flower initiation in Chinese narcissus was induced and advanced by high temperature. The change in transcription abundance in the bulb apices was assayed using RT-PCR and qRT-PCR during the entire storage time to determine whether flower transition is related to NFT1 expression. NFT1 gene transcription increased gradually at 1 month before the early flower transition (indicated by the triangle in Fig. 7A) under natural conditions, and reached its peak at the moment of flower transition (Fig. 7A, B). NFT1 gene transcription also increased gradually in the apices of the bulbs stored at high temperature (30C). Moreover, NFT1 gene transcription in the bulb apices Fig. 3 High storage temperature increases the flowering percentage of 2-year-old bulbs. (A) Illustration of different storage treatments of narcissus bulbs, with temperature regimes shown below the corresponding date, and continuous lines representing periods between 22 and 25C. (B) Effect of storage temperature on the percentage of bolting plants at different days from the planting date. Different lower case letters indicate significant differences between different treatments at a given time point (Pearson’s 2 , P < 0.05, n = 30–0). Detailed temperature regimes in (B) are shown in (A). 274 Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. X.-F. Li et al. at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from
NFT1 involved in flowering initiation induced by heating NFTI 194bp 23b862bp 94bp235bp 201bp 815bp 118 bp FT 9即224bp245bp Fig. 4 Chinese narcissus NFT1 belongs to the FT-like gene family. (A)Partial amino acid sequence alignment of FT-TFL family members. Black stars on the upper row indicate the Tyr85(Y)/His88(H)and GIn 140(Q)/Asp144(D)residues distinguishing all FT-like and TFL1/CEN-like members The homeotic subfamily gene groups are labeled at the right margin based on phylogeny. B)Gene structures of NFT1 and FT. Boxes represent exons,whereas thin lines represent introns. The 5-and 3-untranslated regions are indicated by a shaded box. as strongly up-regulated, and its peak appeared earlier than temperature(30C)compared with those stored under natura that under natural conditions( Fig. 7A), which was consistent conditions(Fig. 1). In addition, the flowering percentage of with the early flower transition in the bulbs stored at high 3-year-old bulbs was reduced when the storage duration under natural conditions was short(i.e. the planting date was earlier than the time of normal fower initiation) and growth temperatures were lower than 25C, which is not suitable for Discussio Rower initiation(Fig. 2B). This preliminary result suggests that Heat accumulation is necessary for flower heat accumulation was not enough for flowering initiation, re- initiation in Chinese narcissus sulting in a reduced Flowering percentage. Consistent with this hypothesis, treatment at 300C for 20 d, combined with a short Flowering initiation of Chinese narcissus was induced gradually storage duration, accumulated enough heat for flowering by summer heating. Flowering is initiated in the dormant bulbs initiation, which complemented the low fowering percentage during summer, wherein the reported critical growth tempera-( Fig. 2A, B). These data indicate that extended heat is necessary ture was 25C, above which growth stops(Lin 2002). In for fower transition in Chinese narcissus, which is in contrast to Shanghai, summer usually starts in early June, with a mean the requirement for extended cold temperature(i.e. ver mperature of 250-292C; Flowering initiation in Chinese nar- tion)in some temperate plant species. This phenomenon may cissus occurs between late June and early August( Fig. 1). No be the adaptive strategy of certain plant species to detect when Lowering initiation was found in bulbs stored at 13-150C, even the hot and dry summer is past. Less heat accumulation due to after 1 year, whereas it was advanced in bulbs stored at high early planting or low storage temperature will reduce the Plant Cell Physiol. 54(2): 270-281(2013)do: 10. 1093/pcp/pcs181 The Author 2013. 275
was strongly up-regulated, and its peak appeared earlier than that under natural conditions (Fig. 7A), which was consistent with the early flower transition in the bulbs stored at high temperature. Discussion Heat accumulation is necessary for flower initiation in Chinese narcissus Flowering initiation of Chinese narcissus was induced gradually by summer heating. Flowering is initiated in the dormant bulbs during summer, wherein the reported critical growth temperature was 25C, above which growth stops (Lin 2002). In Shanghai, summer usually starts in early June, with a mean temperature of 25.0–29.2C; flowering initiation in Chinese narcissus occurs between late June and early August (Fig. 1). No flowering initiation was found in bulbs stored at 13–15C, even after 1 year, whereas it was advanced in bulbs stored at high temperature (30C) compared with those stored under natural conditions (Fig. 1). In addition, the flowering percentage of 3-year-old bulbs was reduced when the storage duration under natural conditions was short (i.e. the planting date was earlier than the time of normal flower initiation) and growth temperatures were lower than 25C, which is not suitable for flower initiation (Fig. 2B). This preliminary result suggests that heat accumulation was not enough for flowering initiation, resulting in a reduced flowering percentage. Consistent with this hypothesis, treatment at 30C for 20 d, combined with a short storage duration, accumulated enough heat for flowering initiation, which complemented the low flowering percentage (Fig. 2A, B). These data indicate that extended heat is necessary for flower transition in Chinese narcissus, which is in contrast to the requirement for extended cold temperature (i.e. vernalization) in some temperate plant species. This phenomenon may be the adaptive strategy of certain plant species to detect when the hot and dry summer is past. Less heat accumulation due to early planting or low storage temperature will reduce the Fig. 4 Chinese narcissus NFT1 belongs to the FT-like gene family. (A) Partial amino acid sequence alignment of FT-TFL family members. Black stars on the upper row indicate the Tyr85(Y)/His88(H) and Gln140(Q)/Asp144(D) residues distinguishing all FT-like and TFL1/CEN-like members. The homeotic subfamily gene groups are labeled at the right margin based on phylogeny. (B) Gene structures of NFT1 and FT. Boxes represent exons, whereas thin lines represent introns. The 50 - and 30 -untranslated regions are indicated by a shaded box. Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. 275 NFT1 involved in flowering initiation induced by heating at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from
PO X-F. Li et al LANT是 Sense Antisense 之E9 令 8388=808号 Antisens Fig. 5 Expression pattern of NFT1 in Chinese narcissus (A-C)In situ localization of NFT1 transcripts in leaves and shoot meristems of Chinese narcissus during growth. Sections were hybridized with a digoxigenin-labeled sense 3-specific RNa probe (A)or antisense 3-specihc RNa probe (B and C).(D)Analysis of NFT1 expression levels in different organs using qRT-PCR. Relative expression levels were normalized to ACTIN and relative to those of middle scales. Data points represent the average of three biological replicates, with three technical replicates. Error bars represent the SD: LP, leaf primordium; MC, leaf muscle cell; XP, xylem parenchyma; and P, phloem Scale bars: 100 um in(A)and( B), and 400 um in(C) fowering percentage Based on our data, 40 d of constant high (Noy-Porata et al. 2009). We also found that treatment at 26 temperatures(30C)without a day and night temperature dif- and 30C caused a similar fowering rate ference, or 50-60 d of natural temperature(average tempera- ture >25.C, Fig. 1B)were enough for flower transition. NFT1 integrates endogenous and temperature Consistently, Lowering initiation in Chinese place in June in Guangzhou(Zhong 1984, Zhang and Yang FT1, an FT ortholog, might integrate endogenous and tempera- 1987),which was earlier than that in Shanghai, because the ture signals for fowering in Chinese narcissus. Chinese narcissus average temperature above 25 C begins in mid-April is not transformable, and the function of NFT1 in inducing early In agreement with our observations, high temperatures pro- Owering is conserved in transgenic Arabidopsis( Fig. 6) motes floral initiation in'Galilee, N tazetta cultivated in Israel,( Kardailsky et al. 1999). Although high signals of NFT1 transcrip- while lower temperatures(12 C)inhibits florogenesis com- tion were detected in leaves( Fig. 5), foral initiation takes place pletely, which was reported previously(Noy-Porata et al. in the bulbs when there is no leaf. Shoot tips from 3-year 2009). However, Noy et al showed that 30C treatments bulbs of Chinese narcissus were collected to test whether the Rower initiation in N. tazetta, which is inconsistent wit NFT1 expression level is an important determinant of flowering results in this study showing that heat accumulatio time. NFTI gene transcription increased gradually at 1 month 30C promotes fowering in Chinese narcissus. One explanation before the early flowering transition under natural conditions; might be that the temperature range for the iower initiation in and it achieved its peak at the moment of fower transition Galilee'and Chinese narcissus is different. In fact, in the re-(Fig. 7A, B), suggesting that a critical level of NFT1 expression port of Noy-Porata et al. (2009), the percentage of flower tran- was required to initiate flowering. Consistent with early flower sition in bulbs stored at 30 and 25 C was as not significantly ing initiation under high temperature, NFTI gene transcription different, although the former is lower than the latter in bulb apices was strongly up-regulated and the higher level 276 Plant Cell Physiol. 54(2): 270-281(2013)doi: 10.1093/pcp/pcs181 C The Author 2013
flowering percentage. Based on our data, 40 d of constant high temperatures (30C) without a day and night temperature difference, or 50–60 d of natural temperature (average temperature >25C, Fig. 1B) were enough for flower transition. Consistently, flowering initiation in Chinese narcissus takes place in June in Guangzhou (Zhong 1984, Zhang and Yang 1987), which was earlier than that in Shanghai, because the average temperature above 25C begins in mid-April. In agreement with our observations, high temperatures promotes floral initiation in ‘Galilee’, N. tazetta cultivated in Israel, while lower temperatures (12C) inhibits florogenesis completely, which was reported previously (Noy-Porata et al. 2009). However, Noy et al showed that 30C treatments delay flower initiation in N. tazetta, which is inconsistent with our results in this study showing that heat accumulation at 30C promotes flowering in Chinese narcissus. One explanation might be that the temperature range for the flower initiation in ‘Galilee’ and Chinese narcissus is different. In fact, in the report of Noy-Porata et al. (2009), the percentage of flower transition in bulbs stored at 30 and 25C was as not significantly different, although the former is lower than the latter (Noy-Porata et al. 2009). We also found that treatment at 26 and 30C caused a similar flowering rate. NFT1 integrates endogenous and temperature signals for flowering FT1, an FT ortholog, might integrate endogenous and temperature signals for flowering in Chinese narcissus. Chinese narcissus is not transformable, and the function of NFT1 in inducing early flowering is conserved in transgenic Arabidopsis (Fig. 6) (Kardailsky et al. 1999). Although high signals of NFT1 transcription were detected in leaves (Fig. 5), floral initiation takes place in the bulbs when there is no leaf. Shoot tips from 3-year-old bulbs of Chinese narcissus were collected to test whether the NFT1 expression level is an important determinant of flowering time. NFT1 gene transcription increased gradually at 1 month before the early flowering transition under natural conditions; and it achieved its peak at the moment of flower transition (Fig. 7A, B), suggesting that a critical level of NFT1 expression was required to initiate flowering. Consistent with early flowering initiation under high temperature, NFT1 gene transcription in bulb apices was strongly up-regulated and the higher level Fig. 5 Expression pattern of NFT1 in Chinese narcissus. (A–C) In situ localization of NFT1 transcripts in leaves and shoot meristems of Chinese narcissus during growth. Sections were hybridized with a digoxigenin-labeled sense 30 -specific RNA probe (A) or antisense 30 -specific RNA probe (B and C). (D) Analysis of NFT1 expression levels in different organs using qRT-PCR. Relative expression levels were normalized to ACTIN and relative to those of middle scales. Data points represent the average of three biological replicates, with three technical replicates. Error bars represent the SD; LP, leaf primordium; MC, leaf muscle cell; XP, xylem parenchyma; and P, phloem. Scale bars: 100 mm in (A) and (B), and 400 mm in (C). 276 Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. X.-F. Li et al. at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from
NFT1 involved in flowering initiation induced by heating 口35S: NFTI in Col Col 355::NFTI in Col 0000 678910l11213141516 leaf number 530000 25000 20000 15000 0.5 5000 col46486-39-910-7 Co4648639910.7136 35S: NFTI in Col 35S: NFTl in Col 口n3■35s: NFTl in f3口WTer 355: NFTin fi-335 er fi-3 5 56789101121314151617181920 G 口socI口AFT *8 Lineg LineR 35S:: NFTI in f-3 Fig 6 Ectopic expression of NFT1 promoted flowering in transgenic Arabidopsis (A)Growth of 355: NFT1 in Col, and Col plants under long-day (LD)conditions after 28d. (B)Effect of ectopic NFT1 expression in Col on the percentage of plants with a different number of rosette leaves when bolting under LD conditions. n= 62 and 20 for 35S NFT1 in Col and Col, respectively. (C )and (D)NFTl, Soc1 and AtFT expression was assayed via qRT-PCR in the selected transgenic lines of 35S: NFTI in Col.(E)Growth of 35S: NFT1 in ft-3 and ft-3(in the Ler background) plants under LD onditions after 32d. (F)Effect of ectopic NFT1 expression in ft-3 on the percentage of plants with a different number of rosette leaves when bolting under LD conditions. n=20, 17 and 15 for 355: NFTI in ft-3 transgenic plants, ft-3 and Ler plants, respectively.(G)and(H)Ectopic expression of NFTI increased SOC1 transcription in transgenic plants. Expression of NFTl, SOCI and the Arabidopsis FT gene(At-FT)was assayed via qRT-PCR in the selected transgenic lines. Plant Cell Physiol. 54(2): 270-281(2013)doi: 10. 1093/pcp/pcs181 C The Author 2013. 277
Fig. 6 Ectopic expression of NFT1 promoted flowering in transgenic Arabidopsis. (A) Growth of 35S::NFT1 in Col, and Col plants under long-day (LD) conditions after 28 d. (B) Effect of ectopic NFT1 expression in Col on the percentage of plants with a different number of rosette leaves when bolting under LD conditions. n = 62 and 20 for 35S::NFT1 in Col and Col, respectively. (C) and (D) NFT1, SOC1 and AtFT expression was assayed via qRT-PCR in the selected transgenic lines of 35S::NFT1 in Col. (E) Growth of 35S::NFT1 in ft-3 and ft-3 (in the Ler background) plants under LD conditions after 32 d. (F) Effect of ectopic NFT1 expression in ft-3 on the percentage of plants with a different number of rosette leaves when bolting under LD conditions. n = 20, 17 and 15 for 35S::NFT1 in ft-3 transgenic plants, ft-3 and Ler plants, respectively. (G) and (H) Ectopic expression of NFT1 increased SOC1 transcription in transgenic plants. Expression of NFT1, SOC1 and the Arabidopsis FT gene (At-FT) was assayed via qRT-PCR in the selected transgenic lines. Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. 277 NFT1 involved in flowering initiation induced by heating at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from
X.F. Li et al LANT是 A凹,r",,L个叫上p, Momth Similar to Arabidopsis F,BFT2 function is needed for fower- 20 31 10 20 30 10 20 31 10 20 30 10 ay ing. The vernalization response suppresses BvFTI expression and releases its inhibition against BvFT2 to induce fowering Storage under natural conditions FLC, other FT orthologs and more of the relevant genes in 30℃for40d Chinese narcissus need to be characterized in order to elucidate the regulation of NFT1 expression in the SM. 120 Heat treatment is beneficial to the adjustment of the flowering time of Chinese narcissus Heat accumulation was not only necessary for flowering initi- ation in the 3-year-old bulbs, but also promoted fowering in the 2-year-old bulbs Chinese narcissus is characterized by 2 year juvenile phases before the first fower is formed. During farming 3-year-old bulbs were selected as flowering plants according to their sizes. Our data also showed that the fowering percentage 6.9 6.23 7.7 7.21 8.4 8.20 9.2 Date in the 2-year-old bulbs increased with longer high-temperature treatment. In addition, treatment at 30oC for 80 d significantly No. 1 increased fowering percentage(86. 1% vS. 25% under natural temperature)(Fig. 3B). Setting the heat accumulation of one summer equal to the treatment at 30oC for 40 d was necessary FTI to induce flowering initiation in the 3-year-old bulbs. the Row- ACTIN ering capability in most 2-year-old bulbs treated at 30C for 80 Fig. 7 NFT1 expression in the bulb apices was induced by high tem- d was due to the heat accumulation, equivalent to two summer perature. (A)Effect of temperature on the NFT1 expression profile in seasons. In addition to heat treatment combined with a short RT-PCR. The upper part illustrates the storage duration to improve the fiowering percentage, these different storage treatments of narcissus bulbs, with temperature re- results may have interesting applications in farming to adjust gimes shown below the corresponding date, and continuous lines fowering time. presenting periods between 22 and 25C. The two time points indicated by arrows are the beginning of flower transition in bulbs with relevant treatment. Relative expression levels indicate the data relative to those obtained on June 9 under natural temperatures. B) Materials and Method Effect of temperature on the change of NFT1 transcripts in bulb apices during storage, shown via RT-PCR Detailed temperature regimes are Plant materials and growth conditions shown in(A) Narcissus tazetta var. chinensis bulbs were commercially ob tained from Shanghai, China. Healthy bulbs of similar sizes (2-year-old bulbs, each with a 10+ 1 cm circumference, and and peak appeared earlier(Fig. 7A, B). These results there- 3-year-old bulbs, each with a 15+ 1 cm circumference)were fore suggest that the NFT1 transcription level in the SM may grouped and stored in natural or controlled conditions. a dry be a regulator of heat accumulation-induced flowering and ventilated warehouse with ambient light and temperature Reproductive onset in perennial poplar was also determined was used as the natural environment. by FT1 signaling, in response to winter temperature( Bohlenius Arabidopsis thaliana, ecotype Col, Ler and ft-3(SALK line et al. 2006, Hsu et al. 2011) cs185 in the Ler background) plants were grown in a green The nature of the upstream mechanism that controls NFT1 house under constant illumination(approximately 80 umol- expression in the SM remains an open question. FT expression $)at 22+2C. Seeds were surface sterilized, stratifed at 4C in Arabidopsis leaves is directly activated by the Co gene, which for 3d, plated on half-strength Murashige and Skoog acts as a sensor of photoperiod signals through the circadian (Murashige and Skoog 1962)medium containing 50 ug clock(Turck et al. 2008). The NFT1 gene in Chinese narcissus kanamycin, and grown under continuous fuorescent light at was also expressed in the leaves( Fig. 5), suggesting a possible 22 C to screen the transgenic plants role in flowering control during the photoperiod. However, the NFTI transcription level in the SM is an important regulator of Treatment method to affect flowering heat accumulation-induced fowering. The mechanism of ver- The flower differentiation during storage of the 3-year-old bulbs nalization in Arabidopsis is the release from FT inhibition by the at natural temperature, high temperature(300C)or low tem action of FLC FT duplication products(BvFT1 and BvFT2)have perature(150C Nos. CK, 1 and 2 in Fig. 1A) was analyzed via divergent functions in biennial sugar beet( Beta spp )(Pin et al. SEM to determine whether high or low temperatures favor 2010). BvFT1 represses reproductive onset and bolting fowering initiation. The average natural temperatures in 278 Plant Cell Physiol. 54(2): 270-281(2013)doi: 10.1093/pcp/pcs181 C The Author 2013
and peak appeared earlier (Fig. 7A, B). These results therefore suggest that the NFT1 transcription level in the SM may be a regulator of heat accumulation-induced flowering. Reproductive onset in perennial poplar was also determined by FT1 signaling, in response to winter temperature (Bohlenius et al. 2006, Hsu et al. 2011). The nature of the upstream mechanism that controls NFT1 expression in the SM remains an open question. FT expression in Arabidopsis leaves is directly activated by the CO gene, which acts as a sensor of photoperiod signals through the circadian clock (Turck et al. 2008). The NFT1 gene in Chinese narcissus was also expressed in the leaves (Fig. 5), suggesting a possible role in flowering control during the photoperiod. However, the NFT1 transcription level in the SM is an important regulator of heat accumulation-induced flowering. The mechanism of vernalization in Arabidopsis is the release from FT inhibition by the action of FLC. FT duplication products (BvFT1 and BvFT2) have divergent functions in biennial sugar beet (Beta spp.) (Pin et al. 2010). BvFT1 represses reproductive onset and bolting. Similar to Arabidopsis FT, BvFT2 function is needed for flowering. The vernalization response suppresses BvFT1 expression and releases its inhibition against BvFT2 to induce flowering. FLC, other FT orthologs and more of the relevant genes in Chinese narcissus need to be characterized in order to elucidate the regulation of NFT1 expression in the SM. Heat treatment is beneficial to the adjustment of the flowering time of Chinese narcissus Heat accumulation was not only necessary for flowering initiation in the 3-year-old bulbs, but also promoted flowering in the 2-year-old bulbs. Chinese narcissus is characterized by 2 year juvenile phases before the first flower is formed. During farming, 3-year-old bulbs were selected as flowering plants according to their sizes. Our data also showed that the flowering percentage in the 2-year-old bulbs increased with longer high-temperature treatment. In addition, treatment at 30C for 80 d significantly increased flowering percentage (86.1% vs. 25% under natural temperature) (Fig. 3B). Setting the heat accumulation of one summer equal to the treatment at 30C for 40 d was necessary to induce flowering initiation in the 3-year-old bulbs. The flowering capability in most 2-year-old bulbs treated at 30C for 80 d was due to the heat accumulation, equivalent to two summer seasons. In addition to heat treatment combined with a short storage duration to improve the flowering percentage, these results may have interesting applications in farming to adjust flowering time. Materials and Methods Plant materials and growth conditions Narcissus tazetta var. chinensis bulbs were commercially obtained from Shanghai, China. Healthy bulbs of similar sizes (2-year-old bulbs, each with a 10 ± 1 cm circumference, and 3-year-old bulbs, each with a 15 ± 1 cm circumference) were grouped and stored in natural or controlled conditions. A dry and ventilated warehouse with ambient light and temperature was used as the natural environment. Arabidopsis thaliana, ecotype Col, Ler and ft-3 (SALK line cs185 in the Ler background) plants were grown in a greenhouse under constant illumination (approximately 80 mmol–2 s –1) at 22 ± 2C. Seeds were surface sterilized, stratified at 4C for 3 d, plated on half-strength Murashige and Skoog (Murashige and Skoog 1962) medium containing 50 mg ml–1 kanamycin, and grown under continuous fluorescent light at 22C to screen the transgenic plants. Treatment method to affect flowering The flower differentiation during storage of the 3-year-old bulbs at natural temperature, high temperature (30C) or low temperature (15C; Nos. CK, 1 and 2 in Fig. 1A) was analyzed via SEM to determine whether high or low temperatures favor flowering initiation. The average natural temperatures in Fig. 7 NFT1 expression in the bulb apices was induced by high temperature. (A) Effect of temperature on the NFT1 expression profile in the bulb apices shown via qRT-PCR. The upper part illustrates the different storage treatments of narcissus bulbs, with temperature regimes shown below the corresponding date, and continuous lines representing periods between 22 and 25C. The two time points indicated by arrows are the beginning of flower transition in bulbs with relevant treatment. Relative expression levels indicate the data relative to those obtained on June 9 under natural temperatures. (B) Effect of temperature on the change of NFT1 transcripts in bulb apices during storage, shown via RT-PCR. Detailed temperature regimes are shown in (A). 278 Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. X.-F. Li et al. at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from
NFT1 involved in flowering initiation induced by heating Shanghai are 20.8, 25.0, 29. 2, 28.5 and 251@C in May, June, July, NFT-A28(5-CTACACCCTGGTGATGGTRGAYCCNGA-3. A August and September, respectively. The highest and lowest partial cDNA clone was isolated from Chinese narcissus emperatures are shown in Fig. 1B. BLAST searches in public databases, with the conceptual Three-year-old bulbs were stored under natural conditions amino acid sequence of the clone as query sequences, and planted on different dates, specifically, July 25, August 15 showed that the clone shared high sequence identity with and September 1(Nos CK1, CK2 and CK3 in Fig. 2A). The FT-like genes from various plants, and thus was named NFTI percentages of the flowering plants were recorded to analyze(Narcissus FT-1). The 3 and 5 regions corresponding to the e effects of different planting dates on flowering. The per- DNAs of NFTI were isolated using the RACE method(Roche centages of the flowering plants treated at 30C for 20 d stored Diagnostics) cDNA and dna clones with a complete open under natural conditions and planted on July 25(No. 1 in reading frame were isolated via PCR, with primers located in Fig 1A)were compared with those stored under natural con- the 5 and 3 regions ditions. The experiment was repeated three times: in 2006, 2007 d2008 Plant transformation and transgenic plant analysis Two-year-old bulbs were initially subjected to high tempera- An EcoRI fragment containing the full-length cDNA for the ture(30 C)for 20, 40 and 80 d, and then stored between 22 and NFT1 gene was introduced into the binary T-DNA vector 25 C until planting to determine the effects of different high pMon530(Monsanto), driven by the 35S promoter. Genet temperature durations on fowering. The results for the transformation lines were obtained, as previously reported(Li 2-year-old bulbs stored at natural temperature and with treat- et al. 2009). The phenotypic effects of NFT1 in transgenic Col ment at 30 C for 40d, with storage from 22 to 25C(Nos 1-4 plants were analyzed in nine independent transgenic lines. Five in Fig 3A), were also compared to test the effect of different independent lines were created by directly transforming the temperatures on Lowering. The experiment was conducted in 35S: NFT1 construct into ft-3 mutant plants, and were selected triplicate with independent materials and was repeated twice, for flowering time analy in2010and2011 Temperature-controlled incubators were used for different Sequence alignments 88号月 temperature treatments. The samples in each treatment con- Predicted amino acid sequences were used for phylogenetic sisted of at least 40 bulbs, unless otherwise stated. The detailed analysis. Protein sequences were aligned using Clustal w were planted in a controlled greenhouse with a short photo- (http://www.clustalorg/),thenmanuallyadjusted period (10 h/14 h light/dark) at 18-20oC. The growing condi- RT-PCR tions and planting order were previously described (Li et al 2012) Total RNA from different tissues was extracted from frozen tissue using TRIZOL reagent(Invitrogen) and purifed on Scanning electron microscopy RNeasy columns(QIAGEN). The first CDNA strand was gener ated according to the instructions for the Superscript RT SMs of 3-year-old bulbs were collected weekly after their har- (Toyobo ). PCR amplification was performed with gene-specific vest. Apices were dissected under an anatomical lens as rapidly primers,NFT (5-TAGCCCAAGTGACCCAAACT-3 )and as possible, and fixed in formaldehyde-acetic acid(cor NFT R2(5-GGACTCCACCCCCAAATTAT-3) ACTIN ex of 63% ethanol, 5% formaldehyde and 6% acetic acid) at 4"c sion levels were monitored to serve as a quantifying control, overnight. Afterwards, the samples were dehydrated in an etha- and the experiments were repeated at least three times ol series, critical-point dried in liquid COz, sputter-coated with qRT-PCR was performed with 3-6 independent biological gold-palladium,analyzed, and photographed with a Philips XL replicates and three technical replicates for each sampleData 30 FEG SEM. Five to 10 samples of each treatment were de- were analyzed via the tected at each time point, and the number of samples at dif- Schmittgen and Livak( 2008). Statistical significance between ferent development stages was analyzed any pair of relative expression means was determined by a t-test. The experiment was repeated with samples from another Cloning of NFT1 cDNAs year with a similar result. Partial cDNAs were isolated from younger inflorescences of Chinese narcissus using degenerate primers according to the In situ hybridization consensus-degenerate hybrid oligonucleotide primer method Samples were fixed in 4%(v/v) paraformaldehyde and 0.5%(v/v) (Rose et al. 1998, Rose et al. 2003, Staheli et al. 2011). The glutaraldehyde for 24 h at 4oC. Then samples were dehydrated first PCR was conducted using a pair of degenerated primers, through an ethanol series, embedded in Histoplast and sectioned NFT-A64(5-GCCGGGGGCGTANACNGTYTG-3)and NFT- at 7 um using a rotary microtome. a probe for NFTI was ampli- A20(5-GCACTGGCTGGTGACNGATATHCC-3) Nested PCr fied with primers 5-gaattcATGAGTAGGGATCCTTTGGTT-3 as performed using another pair of degenerate primers, and 5-gaattcTCATCATTAGGGGTACATCCTC-3. Digoxigenin- NFT-A65 (5-CCAGCCGGGGGCRTANACNGTYT-3) and labeled sense and antisense rna probes were synthesize Plant Cell Physiol. 54(2) 270-281(2013)doi: 10. 1093/pcp/pcs181 C The Author 2013. 279
Shanghai are 20.8, 25.0, 29.2, 28.5 and 25.1C in May, June, July, August and September, respectively. The highest and lowest temperatures are shown in Fig. 1B. Three-year-old bulbs were stored under natural conditions and planted on different dates, specifically, July 25, August 15 and September 1 (Nos. CK1, CK2 and CK3 in Fig. 2A). The percentages of the flowering plants were recorded to analyze the effects of different planting dates on flowering. The percentages of the flowering plants treated at 30C for 20 d stored under natural conditions and planted on July 25 (No. 1 in Fig. 1A) were compared with those stored under natural conditions. The experiment was repeated three times: in 2006, 2007 and 2008. Two-year-old bulbs were initially subjected to high temperature (30C) for 20, 40 and 80 d, and then stored between 22 and 25C until planting to determine the effects of different high temperature durations on flowering. The results for the 2-year-old bulbs stored at natural temperature and with treatment at 30C for 40 d, with storage from 22 to 25C (Nos. 1–4 in Fig. 3A), were also compared to test the effect of different temperatures on flowering. The experiment was conducted in triplicate with independent materials and was repeated twice, in 2010 and 2011. Temperature-controlled incubators were used for different temperature treatments. The samples in each treatment consisted of at least 40 bulbs, unless otherwise stated. The detailed methods are illustrated in Figs. 1–3. After treatment, all bulbs were planted in a controlled greenhouse with a short photoperiod (10 h/14 h light/dark) at 18–20C. The growing conditions and planting order were previously described (Li et al. 2012). Scanning electron microscopy SMs of 3-year-old bulbs were collected weekly after their harvest. Apices were dissected under an anatomical lens as rapidly as possible, and fixed in formaldehyde–acetic acid (composed of 63% ethanol, 5% formaldehyde and 6% acetic acid) at 4C overnight. Afterwards, the samples were dehydrated in an ethanol series, critical-point dried in liquid CO2, sputter-coated with gold–palladium, analyzed, and photographed with a Philips XL 30 FEG SEM. Five to 10 samples of each treatment were detected at each time point, and the number of samples at different development stages was analyzed. Cloning of NFT1 cDNAs Partial cDNAs were isolated from younger inflorescences of Chinese narcissus using degenerate primers according to the consensus-degenerate hybrid oligonucleotide primer method (Rose et al. 1998, Rose et al. 2003, Staheli et al. 2011). The first PCR was conducted using a pair of degenerated primers, NFT-A64 (50 -GCCGGGGGCGTANACNGTYTG-30 ) and NFTA20 (50 -GCACTGGCTGGTGACNGATATHCC-30 ). Nested PCR was performed using another pair of degenerate primers, NFT-A65 (50 -CCAGCCGGGGGCRTANACNGTYT-30 ) and NFT-A28 (50 -CTACACCCTGGTGATGGTRGAYCCNGA-30 ). A partial cDNA clone was isolated from Chinese narcissus. BLAST searches in public databases, with the conceptual amino acid sequence of the clone as query sequences, showed that the clone shared high sequence identity with FT-like genes from various plants, and thus was named NFT1 (Narcissus FT-1). The 30 and 50 regions corresponding to the cDNAs of NFT1 were isolated using the RACE method (Roche Diagnostics). cDNA and DNA clones with a complete open reading frame were isolated via PCR, with primers located in the 50 and 30 regions. Plant transformation and transgenic plant analysis An EcoRI fragment containing the full-length cDNA for the NFT1 gene was introduced into the binary T-DNA vector pMon530 (Monsanto), driven by the 35S promoter. Genetic transformation lines were obtained, as previously reported (Li et al. 2009). The phenotypic effects of NFT1 in transgenic Col plants were analyzed in nine independent transgenic lines. Five independent lines were created by directly transforming the 35S::NFT1 construct into ft-3 mutant plants, and were selected for flowering time analysis. Sequence alignments Predicted amino acid sequences were used for phylogenetic analysis. Protein sequences were aligned using Clustal W (http://www.clustal.org/), then manually adjusted. RT-PCR Total RNA from different tissues was extracted from frozen tissue using TRIZOL reagent (Invitrogen) and purified on RNeasy columns (QIAGEN). The first cDNA strand was generated according to the instructions for the Superscript RT (Toyobo). PCR amplification was performed with gene-specific primers, NFT F2 (50 -TAGCCCAAGTGACCCAAACT-30 ) and NFT R2 (50 -GGACTCCACCCCCAAATTAT-30 ). ACTIN expression levels were monitored to serve as a quantifying control, and the experiments were repeated at least three times. qRT-PCR was performed with 3–6 independent biological replicates and three technical replicates for each sample. Data were analyzed via the 2–Ct method, as described by Schmittgen and Livak (2008). Statistical significance between any pair of relative expression means was determined by a t-test. The experiment was repeated with samples from another year with a similar result. In situ hybridization Samples were fixed in 4% (v/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde for 24 h at 4C. Then samples were dehydrated through an ethanol series, embedded in Histoplast and sectioned at 7 mm using a rotary microtome. A probe for NFT1 was ampli- fied with primers 50 -gaattcATGAGTAGGGATCCTTTGGTT-30 and 50 -gaattcTCATCATTAGGGGTACATCCTC-30 . Digoxigeninlabeled sense and antisense RNA probes were synthesized Plant Cell Physiol. 54(2): 270–281 (2013) doi:10.1093/pcp/pcs181 ! The Author 2013. 279 NFT1 involved in flowering initiation induced by heating at East China Normal University on June 3, 2013 http://pcp.oxfordjournals.org/ Downloaded from
