华东师范大学：《植物生理学》课程教学资源（实验）阅读文献——Necessity of high temperature for the dormancy release of Narcissus tazetta var.chinensis

Journal of Plant Physiology 169(2012)1340-1347 Contents lists available at SciVerse Science Direct Journal of plant physiology ELSEVIER journalhomepagewww.elsevier.deliplph Necessity of high temperature for the dormancy release of Narcissus tazetta var chinensis Kiao-Fang Lia,, Xing-Hua Shao, Xin-Jie Deng, Yang Wang, Xue-Ping Zhang, Lin-Yan Jia, ing Xua Dong-Mei Zhang, Yue Sun, Ling Xu School of life Sciences, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, PR China b Shanghai Institute of Landscape Architecture, 899 Longwu Road, Shanghai 200232, PR China ARTICLE INFO A BSTRACT Winter dormancy has nsively studied in many plants, while much less information is available eceived 22 November 2011 eceived in revised form 24 April 2012 for summer dormancy tazetta var chinensis is characterized by a prolonged period of summer Accepted 8 May 2012 cle. In the present study, we characterized the nature of dormancy in the controlled growth release Cessation of growth and senescence of aerial tissues occurred even under conditions favorable arcissus tazetta var Chinensis for growth, suggesting an endo-dormancy process. Bulbs failed to sprout when they were exposed to lot storage temperatures, while high temperature treatment preceding low storage temperatures, or heating interruption during low storage temperatures, could make bulbs sprouting. These results suggest that high temperatures are necessary for endo-dormancy release Ethylene application during a later storage stage showed an obvious accelerative effect on bulb sprouting, whereas ethylene application during the middle stage had no effect on sprouting. The biological progression of dormancy was further studied hrough cytological and physiological analyses Under natural conditions, the ethylene level was reduced o trace amounts with the transition and progression of dormancy. a transient peak in ethylene release vas found when the plugged plasmodesmata(PD) began to re-open and flower initiation began. The nost common PD possessed electron-dense deposits in endo-dormancy. These data indicate that endo- dormancy ends when flower initiation begins and ethylene peak occurs. Ethylene application had ne effect on dormancy release, while it hastened sprouting only after the release from endo-dormancy by o 2012 Elsevier GmbH. All rights reserved. Introduction future growth and reproduction( rinne et al., 2001; Phillips, 2010 van der Schoot and rinne, 2011). Dormancy is an adaptive response that evolves from the envi- he definition of eco-dormancy as conditional dormancy or fac ronment of origin of various species, enabling their survival during ultative growth suspension dormancy has been controversial(rees, hreatening seasons(Lang et al, 1987). While this behavior has 1981; van der Schoot et al, 1995 ) However, the definition of endo- een extensively studied in plant species that experience severe dormancy as the most stable trapped state of the meristem even winter conditions, perennial plant species that survive summer under conditions conducive to growth has gained common accep- drought have not been given much attention. Plant species exhibit- tance(van der Schoot, 1996: Rinne et al, 2001: Horvath et al g summer dormancy usually inhabit semi-arid regions with a 2003: Volaire and Norton, 2006: Rohde and bhalerao, 2007). The Mediterranean type of climate. Such plants are characterized by strategy of eco-dormancy is favored where summer conditions a period of intensive growth and flowering during mild weather, are unpredictable, whereas endo-dormancy is advantageous rainy winter, and spring, followed by a prolonged rest period dur- habitats where season conditions are predictable(vaughton and ing the hot and dry summer(ofir and Kigel, 2006). This resting stage Ramsey, 2001). The morphogenetic activity of the shoot meristem increases the probability of plant survival during summer, allowing (SM)that changes during winter dormancy cycling in wood species is reflected by changes in cell-cell networking, including symplas- mic pathways created by plasmodesmata(PD)(Rinne et al., 2001 2011: van der Schoot and rinne, 2011). During endo-dormancy, the SM assumes a state of self-arrest by sealing off all PD at its orifices ponding author tel :+862 2;fax:+862162233754. with callose-containing dormancy sphincter complexes(DSCs)and address: xili@bioecnu.edu mpregnating cell walls with as yet unidentified substances that 0176-1617/S-see front matter o 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j-jplph.2012.05.017

Journal of Plant Physiology 169 (2012) 1340–1347 Contents lists available at SciVerse ScienceDirect Journal of Plant Physiology journal h omepage: www.elsevier.de/jplph Necessity of high temperature for the dormancy release of Narcissus tazetta var. chinensis Xiao-Fang Li a,∗ , Xing-Hua Shaoa , Xin-Jie Denga , Yang Wanga , Xue-Ping Zhanga , Lin-Yan Jiaa , Jing Xua , Dong-Mei Zhang b, Yue Suna, Ling Xua a School of Life Sciences, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, PR China b Shanghai Institute of Landscape Architecture, 899 Longwu Road, Shanghai 200232, PR China a r t i c l e i n f o Article history: Received 22 November 2011 Received in revised form 24 April 2012 Accepted 8 May 2012 Keywords: Narcissus tazetta var. Chinensis Dormancy release Temperature Ethylene Plasmodesmata a b s t r a c t Winter dormancy has been extensively studied in many plants, while much less information is available for summer dormancy. Narcissus tazetta var. chinensis is characterized by a prolonged period of summer dormancy during the annual cycle. In the present study, we characterized the nature of dormancy in the controlled growth conditions and investigated the effects of temperature and ethylene on dormancy release. Cessation of growth and senescence of aerial tissues occurred even under conditions favorable for growth, suggesting an endo-dormancy process. Bulbs failed to sprout when they were exposed to low storage temperatures, while high temperature treatment preceding low storage temperatures, or heating interruption during low storage temperatures, could make bulbs sprouting. These results suggest that high temperatures are necessary for endo-dormancy release. Ethylene application during a later storage stage showed an obvious accelerative effect on bulb sprouting, whereas ethylene application during the middle stage had no effect on sprouting. The biological progression of dormancy was further studied through cytological and physiological analyses. Under natural conditions, the ethylene level was reduced to trace amounts with the transition and progression of dormancy. A transient peak in ethylene release was found when the plugged plasmodesmata (PD) began to re-open and flower initiation began. The most common PD possessed electron-dense deposits in endo-dormancy. These data indicate that endo￾dormancy ends when flower initiation begins and ethylene peak occurs. Ethylene application had no effect on dormancy release, while it hastened sprouting only after the release from endo-dormancy by high temperature. © 2012 Elsevier GmbH. All rights reserved. Introduction Dormancy is an adaptive response that evolves from the envi￾ronment of origin of various species, enabling their survival during threatening seasons (Lang et al., 1987). While this behavior has been extensively studied in plant species that experience severe winter conditions, perennial plant species that survive summer drought have not been given much attention. Plant species exhibit￾ing summer dormancy usually inhabit semi-arid regions with a Mediterranean type of climate. Such plants are characterized by a period of intensive growth and flowering during mild weather, rainy winter, and spring, followed by a prolonged rest period dur￾ing the hot and dry summer (Ofir andKigel, 2006). This resting stage increases the probability of plant survival during summer, allowing Abbreviations: SM, shoot meristem; PD, plasmodesmata. ∗ Corresponding author. Tel.: +86 21 62233582; fax: +86 21 62233754. E-mail address: xfli@bio.ecnu.edu.cn (X.-F. Li). future growth and reproduction (Rinne et al., 2001; Phillips, 2010; van der Schoot and Rinne, 2011). The definition of eco-dormancy as conditional dormancy or fac￾ultative growth suspension dormancy has been controversial(Rees, 1981; van der Schoot et al., 1995). However, the definition of endo￾dormancy as the most stable trapped state of the meristem even under conditions conducive to growth has gained common accep￾tance (van der Schoot, 1996; Rinne et al., 2001; Horvath et al., 2003; Volaire and Norton, 2006; Rohde and Bhalerao, 2007). The strategy of eco-dormancy is favored where summer conditions are unpredictable, whereas endo-dormancy is advantageous in habitats where season conditions are predictable (Vaughton and Ramsey, 2001). The morphogenetic activity of the shoot meristem (SM) that changes during winter dormancy cycling in wood species is reflected by changes in cell–cell networking, including symplas￾mic pathways created by plasmodesmata (PD) (Rinne et al., 2001, 2011; van der Schoot and Rinne, 2011). During endo-dormancy,the SM assumes a state of self-arrest by sealing off all PD at its orifices with callose-containing dormancy sphincter complexes (DSCs) and impregnating cell walls with as yet unidentified substances that 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.05.017

X-F.Li et al.Journal of Plant Physiology 169(2012)1340-1347 1341 impede the movement of water,water-soluble ligands,and other that endo-dormancy ended in early August when flower initi- molecules(Rinne and van der Schoot,1998:Rinne et al.,2001).DSCs ation began.Heat treatment was necessary for the release of are composed of an extracellular callose ring and an intracellular endo-dormancy,whereas ethylene hastened sprouting rather than cytoplasmic plug built inside the PD entrance around the internal release endo-dormancy. macromolecular complex.They can be inspected as electron-dense deposits by transmission electron microscopy (Rinne et al.,2001). Materials and methods Formation of DSCs on PDs impairs intrinsic signaling networks that integrate cellular functions and sustain SM behavior,result- Plant materials and growth conditions ing in a dormant state no longer reversible by growth-promoting conditions(Rinne et al.,2001).When the SM is released from endo- Narcissus tazetta var.chinensis bulbs were commercially dormancy.PDs are restored by the breakdown of plasmodesmal obtained from Chongming.Shanghai,China.Healthy bulbs with DSCs(van der Schoot and Rinne,2011).Cellular changes thatappear similar sizes(one-year-old bulbs with5±1 cm circumference and in SM cells during the annual cycle of the plant exhibiting summer three-year old bulbs with15±1 cm circumference)were grouped dormancy are unknown. and stored in natural or controlled conditions.A dry and venti- Considerable variation is present within multiple species lated warehouse with ambient light and temperature was used as regarding the timing of the onset and release of bulb summer dor- the natural conditions.The average temperatures in Shanghai are mancy(Phillips et al.,2008.2010).Differences in the timing of the 20.8,25.0.29.2.28.5.and 25.1C in May.June.July,August,and onset of dormancy within species are habitat-correlated and likely September,respectively.One-year-old and three-year-old bulbs tied to differences in temperature.photoperiod,and/or soil mois- were used as materials for ethylene measurement and dormancy ture (Kamenetsky and Rabinoswitch,2006:Phillips et al.,2008. release assays,respectively,to distinguish changes during the dor- 2010).Based on the limited literature available on bulb dormancy mant state from those during flower differentiation. release,a period of low temperatures is required for breaking bulb dormancy in some species,such as Allium acuminatum,Allium bran- Determining the nature of dormancy degei,and Allium passeyi(Phillips,2010),whereas hot treatment stimulates dormancy release in Allium schoenoprasum (Folster and Chinese narcissus plants were grown in a controlled green- Krug.1977).Many other aspects of dormancy,such as common- house with favorable conditions,specifically.10 h/14 h light/dark alities or variations in summer dormancy induction and release, photoperiod at20Cand70%humidity,to determine whether require further study in many species. the nature of summer dormancy in the plant species is imposed Chinese narcissus(Narcissus tazettavar.chinensis)is a plant from (eco-dormancy)or physiological(endo-dormancy).Tests were con- family Amaryllidaceae that exhibits dormancy for approximately ducted over three consecutive years. five months from late May to the end of September.Plants grow actively during winter and early spring.Aboveground parts of the Treatment method to break dormancy plants senesce in late spring and early summer.Dormant bulbs are usually harvested and stored during the hot summer.Florogene- Bulbs were stored under natural conditions and planted on dif- sis is initiated within larger sized bulbs during summer dormancy. ferent dates,specifically,25 July.15 August,1 September,and 15 and high summer temperatures trigger transition of the bulb SM September(Nos.CK1,CK2,CK3 and CK4 in Fig.1A).and sprouting from the vegetative to the reproductive stage (Noy-Porata et al.. rates were recorded to analyze differences in the release of dor- 2009).Compared with the abundant information on flowering.less mancy between different bulbs.The experiment was repeated three knowledge about dormancy in narcissus is available (Kamenetsky, times in the year of 2006,2007 and 2008 respectively. 2009).Different temperatures,photoperiods,and/or soil moisture The effects of natural temperature,high storage temperature stimuli induce dormancy release in different species(Kamenetsky (30C).and low storage temperature (15C)(Nos.CK,1,and 2 in and Rabinoswitch,2006:Phillips,2010:van der Schoot and Rinne, Fig.2A)on bulb sprouting were analyzed to determine whether 2011).Hormonal control,which involves a gradual increase in the high or low temperatures favor the release of dormancy.To test ratio of sprouting promoters to inhibitors,may underlie the loss the effect of heating before or during low-temperature storage on of dormancy with time (van der Schoot and Rinne,2011).Numer- sprouting,the treatment of 30C for 20 d with storage at 15C for ous reports on the effect of ethylene on breaking the dormancy of 60 d(No.3 in Fig.2A).and the treatment of 15C for 60 d with geophytes are available (Masuda and Asahira,1980:Bufler,2009: heating at 30C for 20 d,and followed by 15C for another 30 d Suttle,2009).Notwithstanding the conflicting scientific reports on (No.4 in Fig.2A).were then conducted. its effects,the real function of ethylene on dormancy release is Results of storage at natural temperature with or without ethy- related to the application duration,conditions,or application tim- lene application just before planting(Nos.5 and CK in Fig.3A)were ing(Suttle,2009).In agricultural production,ethylene is often used compared to determine the effect of ethylene on bud sprouting. to advance narcissus flowering.However,the specific role of ethy- Bulbs were initially subjected to high temperature(30C)for 40 d lene and other environment factors in the regulation of dormancy and then stored at room temperature until planting.Ethylene was and sprouting of narcissus bulbs remains unknown. applied either after high temperature treatment or prior to plant- In the present study,controlled growth conditions were adopted ing or not at all (Nos.6,7,and 8 in Fig.3A)to measure the effects over three years to determine whether dormancy is imposed of the timing of ethylene treatment on sprouting rate.Three-year- (eco-dormancy)or physiological (endo-dormancy)to address the old bulbs were used here and the experiment was conducted in cardinal question about the nature of summer dormancy in Chi- triplicate with independent materials. nese narcissus.Different temperature regimes were designed and Temperature-controlled incubators were used for different tem- combined with ethylene applications to address how dormancy perature treatments.For ethylene treatment,bulbs were incubated is released and ascertain which environmental conditions and for 8 h in 20 mg L-1 Ethrel daily,and then dried at room tempera- whether or not ethylene affects this process.Additionally,ethy- ture.The treatment lasted for 3 d.Unless otherwise stated,samples lene production during the annual cycle of Chinese narcissus in each treatment were comprised of at least 40 bulbs.The detailed was measured.The annual cycle was also analyzed at the cyto- methods are illustrated in the figures.After treatments,all bulbs logical and physiological levels.This study not only ascertained were planted in plastic pots(20 cm height,15 cm diameter)filled the endo-dormant nature of Chinese narcissus but also showed with similar quantities of substrates (75%vermiculite,10%perlite

X.-F. Li et al. / Journal of Plant Physiology 169 (2012) 1340–1347 1341 impede the movement of water, water-soluble ligands, and other molecules (Rinne and van der Schoot, 1998;Rinne et al., 2001). DSCs are composed of an extracellular callose ring and an intracellular cytoplasmic plug built inside the PD entrance around the internal macromolecular complex. They can be inspected as electron-dense deposits by transmission electron microscopy (Rinne et al., 2001). Formation of DSCs on PDs impairs intrinsic signaling networks that integrate cellular functions and sustain SM behavior, result￾ing in a dormant state no longer reversible by growth-promoting conditions (Rinne et al., 2001). When the SM is released from endo￾dormancy, PDs are restored by the breakdown of plasmodesmal DSCs (van der Schoot andRinne, 2011). Cellular changes that appear in SM cells during the annual cycle of the plant exhibiting summer dormancy are unknown. Considerable variation is present within multiple species regarding the timing of the onset and release of bulb summer dor￾mancy (Phillips et al., 2008, 2010). Differences in the timing of the onset of dormancy within species are habitat-correlated and likely tied to differences in temperature, photoperiod, and/or soil mois￾ture (Kamenetsky and Rabinoswitch, 2006; Phillips et al., 2008, 2010). Based on the limited literature available on bulb dormancy release, a period of low temperatures is required for breaking bulb dormancy in some species, such as Allium acuminatum, Allium bran￾degei, and Allium passeyi (Phillips, 2010), whereas hot treatment stimulates dormancy release in Allium schoenoprasum (Folster and Krug, 1977). Many other aspects of dormancy, such as common￾alities or variations in summer dormancy induction and release, require further study in many species. Chinese narcissus (Narcissus tazetta var. chinensis)is a plantfrom family Amaryllidaceae that exhibits dormancy for approximately five months from late May to the end of September. Plants grow actively during winter and early spring. Aboveground parts of the plants senesce in late spring and early summer. Dormant bulbs are usually harvested and stored during the hot summer. Florogene￾sis is initiated within larger sized bulbs during summer dormancy, and high summer temperatures trigger transition of the bulb SM from the vegetative to the reproductive stage (Noy-Porata et al., 2009). Compared with the abundant information on flowering, less knowledge about dormancy in narcissus is available (Kamenetsky, 2009). Different temperatures, photoperiods, and/or soil moisture stimuli induce dormancy release in different species (Kamenetsky and Rabinoswitch, 2006; Phillips, 2010; van der Schoot and Rinne, 2011). Hormonal control, which involves a gradual increase in the ratio of sprouting promoters to inhibitors, may underlie the loss of dormancy with time (van der Schoot and Rinne, 2011). Numer￾ous reports on the effect of ethylene on breaking the dormancy of geophytes are available (Masuda and Asahira, 1980; Bufler, 2009; Suttle, 2009). Notwithstanding the conflicting scientific reports on its effects, the real function of ethylene on dormancy release is related to the application duration, conditions, or application tim￾ing (Suttle, 2009). In agricultural production, ethylene is often used to advance narcissus flowering. However, the specific role of ethy￾lene and other environment factors in the regulation of dormancy and sprouting of narcissus bulbs remains unknown. Inthepresent study, controlled growthconditions were adopted over three years to determine whether dormancy is imposed (eco-dormancy) or physiological (endo-dormancy) to address the cardinal question about the nature of summer dormancy in Chi￾nese narcissus. Different temperature regimes were designed and combined with ethylene applications to address how dormancy is released and ascertain which environmental conditions and whether or not ethylene affects this process. Additionally, ethy￾lene production during the annual cycle of Chinese narcissus was measured. The annual cycle was also analyzed at the cyto￾logical and physiological levels. This study not only ascertained the endo-dormant nature of Chinese narcissus but also showed that endo-dormancy ended in early August when flower initi￾ation began. Heat treatment was necessary for the release of endo-dormancy, whereas ethylene hastened sprouting rather than release endo-dormancy. Materials and methods Plant materials and growth conditions Narcissus tazetta var. chinensis bulbs were commercially obtained from Chongming, Shanghai, China. Healthy bulbs with similar sizes (one-year-old bulbs with 5 ± 1 cm circumference and three-year old bulbs with 15 ± 1 cm circumference) were grouped and stored in natural or controlled conditions. A dry and venti￾lated warehouse with ambient light and temperature was used as the natural conditions. The average temperatures in Shanghai are 20.8, 25.0, 29.2, 28.5, and 25.1 ◦C in May, June, July, August, and September, respectively. One-year-old and three-year-old bulbs were used as materials for ethylene measurement and dormancy release assays, respectively, to distinguish changes during the dor￾mant state from those during flower differentiation. Determining the nature of dormancy Chinese narcissus plants were grown in a controlled green￾house with favorable conditions, specifically, 10 h/14 h light/dark photoperiod at 20 ◦C and 70% humidity, to determine whether the nature of summer dormancy in the plant species is imposed (eco-dormancy) orphysiological(endo-dormancy). Tests were con￾ducted over three consecutive years. Treatment method to break dormancy Bulbs were stored under natural conditions and planted on dif￾ferent dates, specifically, 25 July, 15 August, 1 September, and 15 September (Nos. CK1, CK2, CK3 and CK4 in Fig. 1A), and sprouting rates were recorded to analyze differences in the release of dor￾mancy betweendifferent bulbs. The experiment was repeatedthree times in the year of 2006, 2007 and 2008 respectively. The effects of natural temperature, high storage temperature (30 ◦C), and low storage temperature (15 ◦C) (Nos. CK, 1, and 2 in Fig. 2A) on bulb sprouting were analyzed to determine whether high or low temperatures favor the release of dormancy. To test the effect of heating before or during low-temperature storage on sprouting, the treatment of 30 ◦C for 20 d with storage at 15 ◦C for 60 d (No. 3 in Fig. 2A), and the treatment of 15 ◦C for 60 d with heating at 30 ◦C for 20 d, and followed by 15 ◦C for another 30 d (No. 4 in Fig. 2A), were then conducted. Results of storage at natural temperature with or without ethy￾lene application just before planting (Nos. 5 and CK in Fig. 3A) were compared to determine the effect of ethylene on bud sprouting. Bulbs were initially subjected to high temperature (30 ◦C) for 40 d and then stored at room temperature until planting. Ethylene was applied either after high temperature treatment or prior to plant￾ing or not at all (Nos. 6, 7, and 8 in Fig. 3A) to measure the effects of the timing of ethylene treatment on sprouting rate. Three-year￾old bulbs were used here and the experiment was conducted in triplicate with independent materials. Temperature-controlledincubators wereusedfordifferenttem￾perature treatments. For ethylene treatment, bulbs were incubated for 8 h in 20 mg L−1 Ethrel daily, and then dried at room tempera￾ture. The treatment lasted for 3 d. Unless otherwise stated, samples in each treatment were comprised of at least 40 bulbs. The detailed methods are illustrated in the figures. After treatments, all bulbs were planted in plastic pots (20 cm height, 15 cm diameter) filled with similar quantities of substrates (75% vermiculite, 10% perlite

X-F. Li et aL/ Joumal of plant Physiology 169(2012)1340-1347 p, Month - CKI-k-CK2 +CK3+CK4 0203010day Natural temperature Natural temperature CK4 Natural temperature 24283236 routing (A) Schematic illustration of different storage phases with the corresponding date on top. CK1. CK2, CK3 and CK4 are the ture conditions with ent durations. (B) The effect of different planting dates on the sprouting percent at different days from he three-year tests are shown; vertical bars represent standard deviation. Asterisks indicate significant differences between different 0.05, n=30-50). Detailed storage durations in(B)are shown in(A). 231=2 ep. Month a10 CK-1 3110203010dny80 8323640444852 3[30℃20d15℃60d 15℃60d30℃20d15℃30d 40 Days post planting below the o is necessary for bulb sprouting.(A) Schematic illustration of the different storage treatments of narcissus bulbs, in which temperature regimes are rresponding date on top. (B and c) The effect of storage ure on the sprouting percentage at different days from the date of planting Mean shown; vertical bars represent standard deviation. Different lowercase letters indicate significant differences between different treatments at the given time point Chi-square, x2, P<0.05, n=30-50). Detailed temperature regimes in(B)and(c)are shown in(A). and 15% clay)in a controlled greenhouse with a short photoperiod time course of dormancy. In the present study, data were pre- (10h/14 h light/dark)at 18-20oC and 70% humidity The tion of the macro-element in 1 5 Murashige and Skoog was applied in the soil bi-monthly after planting. The og medium sented as the mean of at least three samples of three-year-old plants proportion was recorded every 4 d after planting. Light and transmission electron microscopy Shoot meristems(SMs)of three-year-old bulbs were collected The ethylene level in the whole plant was measured at differ- weekly since their harvest Apices were dissected under an anatom ent developmental stages by sealing four plants or bulbs(during ical lens as rapidly as possible, fixed in formaldehyde-acetic acid transition into dormancy )in airtight jars for 12 h at 22-23C, after (solution composed of 63% ethanol, 5% formaldehyde, and 6% acetic which a 1 mL sample of the headspace was obtained and injected acid)at 4 C overnight, and then dehydrated in an ethanol series. into a Hewlett-Packard 5890 series ll gas chromatograph equipped Median longitudinal paraffin sections (7 um thick)were stained with a flame ionization detector GC9800 (Guangzhou, China). Ethy- with 1% toluidine blue and investigated microscopically(rinne lene levels in bulbs were measured twice every month during the et al, 2001)

1342 X.-F. Li et al. / Journal of Plant Physiology 169 (2012) 1340–1347 Fig. 1. Effects of planting date on bulb sprouting. (A) Schematic illustration of different storage phases with the corresponding date on top. CK1, CK2, CK3 and CK4 are the treatment numbers of natural temperature conditions with different durations. (B) The effect of different planting dates on the sprouting percent at different days from the date of planting. Mean values of the three-year tests are shown; vertical bars represent standard deviation. Asterisks indicate significant differences between different treatments at the given time point (P < 0.05, n = 30–50). Detailed storage durations in (B) are shown in (A). Fig. 2. High temperature is necessary for bulb sprouting. (A) Schematic illustration of the different storage treatments of narcissus bulbs, in which temperature regimes are shown just below the corresponding date on top. (B and C) The effect of storage temperature on the sprouting percentage at different days from the date of planting. Mean values are shown; vertical bars represent standard deviation. Different lowercase letters indicate significant differences between different treatments at the given time point (Pearson’s Chi-square, 2, P < 0.05, n = 30–50). Detailed temperature regimes in (B) and (C) are shown in (A). and 15% clay) in a controlled greenhouse with a short photoperiod (10 h/14 h light/dark) at 18–20 ◦C and 70% humidity. The salt solu￾tion of the macro-element in 1/5 Murashige and Skoog medium was applied in the soil bi-monthly after planting. The sprouting proportion was recorded every 4 d after planting. Ethylene measurements The ethylene level in the whole plant was measured at differ￾ent developmental stages by sealing four plants or bulbs (during transition into dormancy) in airtight jars for 12 h at 22–23 ◦C, after which a 1 mL sample of the headspace was obtained and injected into a Hewlett-Packard 5890 series II gas chromatograph equipped with a flame ionization detector GC9800 (Guangzhou, China). Ethy￾lene levels in bulbs were measured twice every month during the time course of dormancy. In the present study, data were pre￾sentedas themeanof atleastthree samples ofthree-year-oldplants (L h−1 g−1 fresh weight). Light and transmission electron microscopy Shoot meristems (SMs) of three-year-old bulbs were collected weekly since their harvest.Apices were dissected under an anatom￾ical lens as rapidly as possible, fixed in formaldehyde–acetic acid (solution composed of 63% ethanol, 5% formaldehyde, and 6% acetic acid) at 4 ◦C overnight, and then dehydrated in an ethanol series. Median longitudinal paraffin sections (7 m thick) were stained with 1% toluidine blue and investigated microscopically (Rinne et al., 2001).

-F Li et al. /Joumal of Plant Physiology 169(2012)1340-1347 1343 一CK+5 Natural temperature 6 16202428323640444852 30℃40d C 0000 121620243832364044485256 E小y3y°八少 Fig 3. Effects of ethylene combined with different te ture regimes during storage on bulb dormancy release. (A)Schematicillustration of the different storage f narcissus bulbs, in which temperature regimes combined with ethylene application are shown just below the corresponding date on top. Continuous lines represe of 22-25C. (B and C) Effects of ethylene application at different times on sprouting rate. Detailed treatment regimes in(b)and ( c)are letters indicate significant differences between different treatments at the given time point(Pearsons Chi-square, x2, P<0.05. n=30-50)(D)Ethylene release in bulbs at different times Mean values are shown; vertical bars represent standard deviations and different lower-case letters indicate significant differences between values(P<0.05)- bulbs were collected every weeacroscopy. SMs in one-year-old series(Rinne et al. 2001). Samples were embedded in Spur,'s resin For transmission electro ks during their annual cycle and (Sigma). Ultra-thin sections (70 nm) were obtained from median fixed in dual fixation solution. Samples were fixed for 4h at 4C longitudinal positions with an ultramicrotome (Leica, Nestzlar in 2.5%(v/v) glutaraldehyde in 200 mM phosphate buffer(pH 7. 4). Germany), stained with 2% aqueous uranyl acetate and Reynolds The fixed tissue was washed in buffer and post-fixed overnight at lead citrate, and then examined with a JEOL 1200 EXll electron 4C in 1%(w/v)OsOA and then dehydrated in a graded ethanol microscope at 80 kV (Tokyo, Japan). B D Apr 1 May1 Jun1 JuL3 Aug11 g. 4. The metabolism in narcissus namic changes. Transmission electron t nages of shoot apical cells during(A)active growth. (B)dormancy. ormancy progression. Mean values are shown; vertical bars represent standard deviations and different lower-case letters indicate significant differences between vang nd(c)dormancy release are shown. Amyloplasts are indicated by arrows, and n represents the nucleus. bars=l um. (D)The soluble sugar conte

X.-F. Li et al. / Journal of Plant Physiology 169 (2012) 1340–1347 1343 Fig. 3. Effects of ethylene combined with differenttemperature regimes during storage on bulb dormancy release.(A) Schematic illustration ofthe different storage treatments of narcissus bulbs, in which temperature regimes combined with ethylene application are shown just below the corresponding date on top. Continuous lines represent periods of 22–25 ◦C. (B and C) Effects of ethylene application at different times on sprouting rate. Detailed treatment regimes in (B) and (C) are shown in (A). Different lowercase letters indicate significant differences between different treatments at the given time point (Pearson’s Chi-square, 2, P < 0.05, n = 30–50). (D) Ethylene release in bulbs at different times. Mean values are shown; vertical bars represent standard deviations and different lower-case letters indicate significant differences between values (P < 0.05). For transmission electron microscopy, SMs in one-year-old bulbs were collected every 2 weeks during their annual cycle and fixed in dual fixation solution. Samples were fixed for 4 h at 4 ◦C in 2.5% (v/v) glutaraldehyde in 200 mM phosphate buffer (pH 7.4). The fixed tissue was washed in buffer and post-fixed overnight at 4 ◦C in 1% (w/v) OsO4 and then dehydrated in a graded ethanol series (Rinne et al., 2001). Samples were embedded in Spurr’s resin (Sigma). Ultra-thin sections (70 nm) were obtained from median longitudinal positions with an ultramicrotome (Leica, Nestzlar, Germany), stained with 2% aqueous uranyl acetate and Reynolds’ lead citrate, and then examined with a JEOL 1200 EXII electron microscope at 80 kV (Tokyo, Japan). Fig. 4. The metabolism in narcissus bulbs showing dynamic changes. Transmission electron microscopy images of shoot apical cells during (A) active growth, (B) dormancy, and (C) dormancy release are shown. Amyloplasts are indicated by arrows, and N represents the nucleus. Scale bars = 1 m. (D) The soluble sugar content in bulbs during dormancy progression. Mean values are shown; vertical bars represent standard deviations and different lower-case letters indicate significant differences between values (P < 0.05).

1344 X-F. Li et aL/ Joumal of plant Physiology 169(2012)1340-1347 ∴ Fig. 5. Changes in shoot SM during transition into flower differentiation. (A)The vegetative meristem(VM)with only leaf primordial (LP)differentiation (B)The meristem aring transition from VM to inflorescence meristem(IM)(C)IM. (D)Floral meristems begin to initiate around the Im. (e and F)The meristem during the formation of floral rgan primordia. The tepal primordium(TP). paracorolla primordium(PCP), stamen primordium(StaP), and carpel primordium(CarP)are shown by arrows. (G)Inflorescence without spathe. (H)Stamens and the corona were removed. ()Transverse section of an ovary Scale bars: 500um in(A)-(F) and 4 mm in(GH(I). and 48 d post planting were significantly different from those lanted on September 15th. For bulbs planted before September >> Statistical analyses were performed with SPSS Statistics version 1st, 25.5+2.5 d were required to reach 10% sprouting percentage, .O software Pearsons x2 test, ANOVA, and Students t-test were 36.3+2.9 d were needed for half-number sprouting, and 53.3+2.1 used to detect significant differences. d were necessary to obtain the maximum sprouting percentage. Results Significance of high-temperature treatment for dormancy release The dormancy nature and variation in the release of bulb The effects of natural temperature, low temperature, and high- storage temperature on the release of dormancy were analyzed In the three-year study, cessation of growth and senescence of Similar final percentages of bulbs sprouting were obtained under aerial tissues occurred even under conditions favorable for growth, 30C or natural storage temperature, while the low-storage tem- iggesting an endo-dormancy process. The later the planting date. perature resulted in no bulb sprouting two months after planting the higher the sprouting percentage was during the same period(Nos CK, 1, and 2 in Fig 2B). However, bulbs could sprout when tarting from the time of planting( Fig. lA and b). Up to 50% of the heating at 30 for 20 d when treated before or during the storage ulbs planted on September 15th sprouted within 25-30 d, and at 15C(Fig. 2C). The sprouting rate of the treatment of 30 for all narcissuses sprouted within 40 d. Statistical analysis showed 20 d preceding storage at 15C for 60 d was advanced markedly no significant differences between bulbs planted on August 15th Most bulbs with the treatment of heating at 30.C for 20 d preceded and September 1st. However, the sprouting percentages of bulbs by 15C for 60 d and followed by 15C for another 30 d began planted on August 15th and September 1st at 28, 32, 36, 40, 44, to sprout 40 d after planting(Fig. 2C), later than those stored at

1344 X.-F. Li et al. / Journal of Plant Physiology 169 (2012) 1340–1347 Fig. 5. Changes in shoot SM during transition into flower differentiation. (A) The vegetative meristem (VM) with only leaf primordial (LP) differentiation. (B) The meristem during transition from VM to inflorescence meristem (IM). (C) IM. (D) Floral meristems begin to initiate around the IM. (E and F) The meristem during the formation of floral organ primordia. The tepal primordium (TP), paracorolla primordium (PCP), stamen primordium (StaP), and carpel primordium (CarP) are shown by arrows. (G) Inflorescence without spathe. (H) Stamens and the corona were removed. (I) Transverse section of an ovary. Scale bars: 500 m in (A)–(F) and 4 mm in (G)–(I). Statistical analysis Statistical analyses were performed with SPSS Statistics version 17.0 software. Pearson’s 2 test, ANOVA, and Student’s t-test were used to detect significant differences. Results The dormancy nature and variation in the release of bulb dormancy In the three-year study, cessation of growth and senescence of aerial tissues occurred even under conditions favorable for growth, suggesting an endo-dormancy process. The later the planting date, the higher the sprouting percentage was during the same period starting from the time of planting (Fig. 1A and B). Up to 50% of the bulbs planted on September 15th sprouted within 25–30 d, and all narcissuses sprouted within 40 d. Statistical analysis showed no significant differences between bulbs planted on August 15th and September 1st. However, the sprouting percentages of bulbs planted on August 15th and September 1st at 28, 32, 36, 40, 44, and 48 d post planting were significantly different from those planted on September 15th. For bulbs planted before September 1st, 25.5 ± 2.5 d were required to reach 10% sprouting percentage, 36.3 ± 2.9 d were needed for half-number sprouting, and 53.3 ± 2.1 d were necessary to obtain the maximum sprouting percentage. Significance of high-temperature treatment for dormancy release The effects of natural temperature, low temperature, and high￾storage temperature on the release of dormancy were analyzed. Similar final percentages of bulbs sprouting were obtained under 30 ◦C or natural storage temperature, while the low-storage tem￾perature resulted in no bulb sprouting two months after planting (Nos. CK, 1, and 2 in Fig. 2B). However, bulbs could sprout when heating at 30 ◦C for 20 d when treated before or during the storage at 15 ◦C (Fig. 2C). The sprouting rate of the treatment of 30 ◦C for 20 d preceding storage at 15 ◦C for 60 d was advanced markedly. Most bulbs with the treatment of heating at 30 ◦C for 20 d preceded by 15 ◦C for 60 d and followed by 15 ◦C for another 30 d began to sprout 40 d after planting (Fig. 2C), later than those stored at