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