J. Japan. Soc. Hort. Sci. 81 (1): 101–108. 2012. Available online at www.jstage.jst.go.jp/browse/jjshs1 JSHS © 2012

Anatomical Characterization of -bud Blasting and Suppression Following Hormone Application in Eustoma grandiflorum (Raf.) Shinn.

Kyoko Kawakatsu, Ayuko Ushio and Naoko Fukuta*

NARO Institute of Floricultural Sciences, Fujimoto, Tsukuba 305-8519, Japan

Flower-bud blasting is a constraint for producing Eustoma grandiflorum so a preventative strategy is needed. Flower-bud blasting occurs under low light intensity and high fertilizer input. To gain insight into the mechanisms of flower-bud blasting, we conducted a detailed characterization of flower development under normal and blast- inducing conditions. We found that floral buds under low light intensity ceased to grow at the and differentiation stages, although and were initiated normally. Aborted rarely had normal ovules. Moreover, an expanded apical was observed. These results show that the differentiation and development of reproductive organs are critically suppressed by blast-inducing conditions; however, combined application of 300 ppm benzylaminopurine and 200 ppm gibberellic acid-3 to floral buds resulted in about five-fold greater frequency of flower opening compared to controls. Blasting inhibition also resulted from excising the inflorescent branch, suggesting the decrease in assimilates in flower buds would be attributed to flower-bud blasting. Moreover, hormone application combined with excision had an additive effect for preventing flower-bud blasting, suggesting that these treatments independently inhibit flower-bud blasting. These results suggest that flower-bud blasting in Eustoma is a break in floral development around the stamen and gynoecium initiation stages and is integrally induced by the factors related to hormone biosynthesis and the decrease in assimilates.

Key Words: assimilate, cytokinin, Eustoma, flower-bud blasting, gibberellin.

Flower-bud blasting is observed in many popular Introduction flowering plants, such as lily, Iris, Zinnia elegansm, and Eustoma grandiflorum (Raf.) Shinn (lisianthus) origi- Chrysanthemum. Although experiments have been nated from North America and is widely grown as an conducted to identify the cause of blasting, the ornamental flower in temperate areas (Shimizu-Yumoto mechanism remains poorly understood. Many studies and Ichimura, 2010). When the external environmental have focused on two possible factors that may be conditions are inappropriate, damage such as rosette forma- involved in regulating flower-bud blasting, hormones tion, tip burn, and flower-bud blasting is inevitable, and photoassimilation. Ethylene promotes flower-bud resulting in unsuccessful flower opening. Flower-bud blasting in the tulip and lily (Demunk, 1973; Mason and blasting is a type of abortion in flowers and commonly Miller, 1991). Hormonal changes may also be associated refers to the arrest of perfect flower development. The with flower-bud blasting, such as smaller amounts of mechanism of flower-bud blasting remains uncertain, cytokinin and larger amount of abscisic acid in Iris (Vonk although low light intensity and the application of high and Ribot, 1982; Vonk et al., 1986). Exogenous concentrations of nitrogen promote blasting in Eustoma gibberellin and cytokinin treatment results in the (Ushio and Fukuta, 2010). decreased occurrence of flower-bud blasting in tulip and Cymbidium (Demunk and Gijzenberg, 1977; Ohno, 1991), suggesting that blasting is induced by the factors Received; August 4, 2011. Accepted; September 23, 2011. related to hormone biosynthesis in tulip and Cymbidium. This work was supported by grants from The research and develop- Kernel abortion in maize, however, is apparently not ment projects for application in promoting new policy of agriculture, forestry and fisheries: No. 2007 and by Grant-in-Aid for Research initiated by a hormone signal, as a reduction in hormones Activity Start-up 22880042. is observed only after abortion occurs (Reed and * Corresponding author (E-mail: [email protected]). Singletary, 1989). This suggests that a careful examina-

101 102 K. Kawakatsu, A. Ushio and N. Fukuta tion considering the timing of sampling is needed. Histological analysis Another factor, photoassimilation, seems to be Flower buds were collected 5 times at 5-day intervals associated with flower-bud blasting among various from 3 days after transition to blast-inducing conditions. species (Chrysanthemun, Cymbidium, Iris, Lathyrus To obtain conclusive information, flower buds were odoratus; Fudano et al., 2009; Fukai et al., 1981; Ohno, collected 35 days after transition to blast-inducing 1991; Vonk and Ribot, 1982). In the pepper, flower-bud conditions. For paraffin sectioning, we collected floral blasting appears to be caused by the low accumulation bud samples and fixed them overnight in formalin : of assimilates in the floral bud (Aloni et al., 1997). glacial acetic acid : 70% ethanol; 1 : 1 : 18, followed by Information on the development of floral organs in dehydration in a graded ethanol series. Following Eustoma is essential to overcome flower-bud blasting; substitution with xylene, the samples were embedded in however, the developmental profile of the Eustoma Paraplast Plus (Oxford Labware, St. Louis, USA) and flower is not fully understood, even under normal sectioned to a thickness of 8 μm using a rotary micro- conditions, except for gametophytes (Yang et al., 2007). tome. Sections were stained with 0.05% toluidine blue Here, we conducted a detailed investigation into the and observed with a light microscope. initial stages of flower development. Additionally, we investigated the effect of the plant hormones cytokinin Branch removal and gibberellin on flower-bud blasting. We demonstrated branches located next to the first flower that gibberellin and cytokinin treatments inhibit flower- were removed when benzylaminopurine (BA), for- bud blasting caused by blast-inducing conditions in chlorfenuron (CPPU), and gibberellic acid-3 (GA3) and Eustoma. control liquid were applied to the first flower. To assess the effect of branch removal independent of hormone Materials and Methods application, we removed an inflorescence branch and Plant material and growth conditions applied the below-mentioned control liquid instead of Eustoma grandiflorum ‘Piccorosa Snow’ seeds were hormones to the first flower. sown in plug trays containing fertilized soil and maintained at 10°C in the dark for 5 weeks in a growth Hormone treatments cabinet. Trays were then transferred to a growth cham- We assessed the effects of exogenously applied BA, ° ber (Nihonika Co., Tokyo, Japan) maintained at 28 C CPPU, and GA3 on flower-bud blasting. Three weeks under a 12 h light phase with a fluorescent lamps after inducing flower-bud blasting, approximately 4 mm (50 μmol·m−2·s−1 PPFD) and 18°C under a 12 h dark lengths of flower bud were inoculated with 300 ppm BA phase until the two-pair stage. Each seedling was and 30 ppm CPPU in 20% (v/v) aqueous acetone transplanted to a 10.5 cm plastic pot containing fertilized containing 0.05% Tween20, respectively. Aqueous medium (Kureha Engeibaido, Kureha Chemical Industry acetone (20%, v/v) containing 0.05% Tween20 was Co., Ltd., Tokyo, Japan) and placed in a growth chamber applied to flower buds of control plants. Five pots were at 28°C/18°C under a 14 h photoperiod supplemented used to administer each compound. with fluorescent lamps (400 μmol·m−2·s−1 PPFD). Seventeen days after inducing flower-bud blasting, approximately 4 mm lengths of flower bud were Induction of flower-bud blasting and evaluation of inoculated with various concentrations of BA (0, 30, flower-bud blasting 100, 300, and 3000 ppm) in 20% (v/v) aqueous acetone At the transition stage from the vegetative to the containing 0.05% Tween20. Six pots were used for each reproductive phases (6 weeks after transplant), the plants concentration with and without branch removal. were transferred to a growth chamber at 25°C in a 12 h Two weeks after inducing flower-bud blasting, light phase with fluorescent lamps (130 μmol·m−2·s−1 approximately 2.5 mm lengths of flower bud were ° PPFD) and 15 C in a 12 h dark phase. The solution of inoculated with various concentrations of GA3 (0, 40, a compound fertilizer, OKF1 (N : P2O5 : K2O, 15 : 8 : 17, 200, and 400 ppm) in 20% aqueous ethanol solution Otsuka Chemical Co., Tokyo, Japan) including 75 ppm (v/v) containing 0.05% Tween20. Eight pots were used nitrogen was applied instead of water. Control plants for each concentration with and without branch removal. were grown under the same conditions as in the Two weeks after inducing flower-bud blasting, vegetative phase. The first flower of each plant was approximately 2.5 mm lengths of flower bud were evaluated for flower-bud blasting. inoculated with both 200 ppm GA3 and 300 ppm BA. Eight pots were used. Measurement of flower buds Results As sepals grew normally even if flower-bud blasting occurred, the length of flower buds was defined as the 1. Developmental course of flower differentiation and distance from the bottom end of the to the top of flower-bud blasting in E. grandiflorum the . Low light conditions and an increased level of nitrogen fertilizer significantly increased flower-bud blasting J. Japan. Soc. Hort. Sci. 81 (1): 101–108. 2012. 103

(Fig. 1A, B) (Ushio and Fukuta, 2010). We compared Under low light intensity, all floral initiated sequential changes of flower development under blast- the development of normal sepal and petal primordia inducing conditions versus under control conditions. (Fig. 1B, C, J), whereas development of and Under control conditions, floral buds become larger with gynoecium was severely inhibited. Some plants initiated age (Fig. 1C). In contrast, floral buds maintained under stamen primordia 2 weeks after treatment (Fig. 1K) and low light intensity ceased to grow (Fig. 1C). several floral buds achieved stages 3 and 4 (Fig. 1L, M). To clarify the stage at which flower-bud blasting Low light intensity was associated with a low frequency occurred, we observed longitudinal sections of flower of normal ovule differentiation. In addition to the pistil, buds. Under control conditions, flower meristems petal development was also inhibited (Fig. 1B, N). We initiated sepal primordia and subsequently several layers confirmed that the petal size at stage 4 under control of petals protruded (Fig. 1D, stage 1). Floral meristems condition was 4.2 mm and the cell division of petals produced stamen primordia when the first petal continued from stage 4 onward (data not shown). These primordia were about 2 mm in length (Fig. 1E, stage 2). results suggested that blast-inducing conditions which Carpals and placenta were produced when the first petal caused smaller petals influenced cell proliferation as well was approximately 3–5 mm in length (Fig. 1F and G, as organ differentiation. stages 3 and 4, respectively). It took about 2 weeks from To obtain conclusive information regarding flower- stages 1 to 4. Subsequently, numerous ovules differ- bud blasting, we observed the floral buds 35 days after entiated (Fig. 1H, stage 5) and megasporocyte differen- the plants had been transferred to blast-inducing tiation occurred (Fig. 1I, stage 6). conditions. Under control conditions, all flower buds

Fig. 1. Developmental profiles of flower-bud blasting in Eustoma grandiflorum. A, Appearance of the plants under control (left) and blast- inducing conditions (right) 47 days after induction. All floral buds aborted under low light (arrows). Scale bar = 5 cm. B, Higher magnification of flower-bud blasting shown in A. Scale bar = 1 cm. C, Changing patterns in the length of floral buds during development. Vertical bars indicate SE (n = 7). D–M, Developmental profiles of flowers under normal conditions (D–I) and blast-inducing conditions (J–M). D, J, Petal initiation stage (stage 1). E, K, Stamen initiation stage (stage 2). F, L, Carpel initiation stage (stage 3). G, M, Placental differentiation stage (stage 4). H, Ovule differentiation stage (stage 5). I, Megasporogenesis stage (stage 6). Abbreviations: se, sepal; p, petal; p1, petal in the first layer; st, stamen; ca, carpel. Scale bars = 400 μm in D–M. N, Correlation of petal length with pistil length under normal and inducing conditions. 104 K. Kawakatsu, A. Ushio and N. Fukuta went through stage 6 (Fig. 2A). In contrast, up to 50% when flower buds next to them were approximately 2.5– of floral buds ceased to produce stamens and gynoecium 4 mm long. Branch removal resulted in the decreased under blast-inducing conditions (Fig. 2A), and none of frequency of flower-bud blasting (Figs. 3A, C, D and the flower buds enclosed ovules. Furthermore, aberra- 4A). The rate of flower-bud blasting appeared to be tions in floral meristems were occasionally found. For slightly but significantly (P < 0.05, t-test) lower than in example, just after initiating stamen primordial develop- ment, normal floral meristems were about 50 μm in width (Fig. 1E). In contrast, floral meristems in which flower-bud blasting occurred were up to 200 μm in width (Fig. 2B). Cells in the rib zone of blasting flowers expanded compared to those in normal flowers (Fig. 2B). These results demonstrated that blast-inducing condi- tions arrested two phenomena, organ differentiation and supply of undifferentiated cells, although they were not coincidental.

2. Effect of excising the inflorescence branch on flower- bud blasting The inflorescence branch next to the flower bud continues to grow in Eustoma even if flower-bud blasting occurs (Fig. 1A). The most common symptom of flower- bud blasting is vigorous growth of the inflorescence branch. As flowers and inflorescence branches grow simultaneously, some competition may occur. To inves- tigate the effect of accumulation and partitioning on floral organization, we removed inflorescent branches

Fig. 3. Effects of exogenous cytokinin and branch removal on flower- bud blasting. A, Flower-bud length after applying BA and CPPU to plants with and without branch removal. Vertical bars indicate SE (n = 5). Numbers followed by the same letter are not significantly different (P < 0.05, Tukey’s HSD test). B, Representative appearance following cytokinin treatment and branch removal. C, Influence of BA concentration on flower Fig. 2. Internal structure of the flower bud. A, Comparison of the opening (%). D, Influence of BA concentration on the flower developmental stages between normal flowers and aborted bud length in plants with and without branch removal. Vertical flowers. Stage numbers correspond to those in Figure 1. B, Floral bars indicate SE (n = 5). The flower-bud length increased meristems of an aborted flower after the third-layer petal following application of a higher concentration of BA, although initiation stage. Scale bars = 400 μm. none was statistical significant (P < 0.05, Tukey’s HSD test). J. Japan. Soc. Hort. Sci. 81 (1): 101–108. 2012. 105 plants in which the inflorescent branches were not removed. These results indicate that the inflorescent branch might arrest the development of the neighboring flower.

3. Effect of cytokinin treatment on flower-bud blasting. Our anatomical study indicated that the blast-inducing condition likely influenced cell proliferation of floral organs in Eustoma. Cytokinins are plant hormones that regulate plant cell division (Riou-Khamlichi et al., 1999). To estimate the effect of cytokinins on flower- bud blasting, we applied the purine cytokinin N6- benzylaminopurine (BA) and the phenylurea cytokinin forchlorfenuron (CPPU), a synthetic compound with cytokinin-like activity. Both BA and CPPU resulted in increased length of flower 42 days after hormone inoculation (Fig. 3A). The appearances of flowers with cytokinin treatment and branch removal under blast- inducing condition were comparable to those under normal conditions (Figs. 1A and 3B). These results demonstrated that cytokinins act to suppress flower-bud blasting. Next, we applied various concentrations of BA to further investigate the effect of BA on the suppression of blasting. The applications of 30 ppm, 100 ppm, and 300 ppm BA increased the frequency of flower opening, respectively (Fig. 3C). Flower-bud blasting was signifi- cantly prevented by applying 30 ppm BA, regardless of whether the inflorescent branch was removed (Fig. 3D). To investigate the relationship between excising the Fig. 4. Effects of exogenous GA3 and BA on flower-bud blasting. inflorescent branch and BA treatment, we applied a A, Rate of flower opening in plants with and without branch combined treatment of excising the inflorescent branch removal. B, Time courses of floral bud length after applying and treating with BA and CPPU. Combined treatment GA3 and BA in plants without branch removal. C, Time courses of flower bud length after applying GA3 to plants with branch was more effective than the individual treatments removal. Vertical bars indicate SE (n = 8). Numbers followed by (Fig. 3C, D), suggesting that excising the inflorescent the same letter are not significantly different (P < 0.05, Tukey’s branch and treating with cytokinins acted independently HSD test). to affect flower-bud blasting. GA3 to floral buds resulted in about five-fold greater 4. Effect of gibberellic acid-3 (GA3) treatment on flower- frequency of flower opening then in controls (Fig. 4A) bud blasting. and brought about flowers which were similar in size to Gibberellins (GAs) play an important role in the those under control condition (Figs. 1C and 4B). The development of flowers in Eustoma (Hisamatsu et al., exogenous, simultaneous application of GA3 and BA 1999, 2004); thus, we applied GA3 at various caused a higher frequency of flower opening than single concentrations to investigate the effect of GA3 on flower- applications (Figs. 3C and 4A), suggesting that GA3 and bud blasting. Treatment significantly inhibited flower- BA act to resolve flower-bud blasting independently. bud blasting (Fig. 4A, B). In particular, the flower Discussion opening rate was restored with the application of 200 ppm GA3 (Fig. 4A). Moreover, the effect was We conducted a detailed investigation of flower-bud enhanced by branch removal (Fig. 4A, C), suggesting blasting to develop an application to suppress flower- that GA3 and branch removal act independently to bud blasting. Our anatomical approach showed that prevent flower-bud blasting. Even low levels of GA3 flower-bud blasting is a spontaneous end to floral prevented flowers from blasting in case of branch development in the stamen and gynoecium initiation removal (Fig. 4A, C). The application of 200 ppm GA3 stages. An incomplete gynoecium was observed even in and excising the inflorescent branch reproducibly petals up to 10 mm long, demonstrating that floral-bud resulted in complete flower development (data not blasting represents a break in floral development rather shown). than necrosis of the flower bud. It is unlikely that low Combined application of 300 ppm BA and 200 ppm light intensity results in a shortage of energy sources 106 K. Kawakatsu, A. Ushio and N. Fukuta because the inflorescent branches next to aborted floral suggesting that cell division was suppressed. As buds grew normally (Fig. 1). Inadequate conditions did cytokinin stimulates cell division by regulating the not result in improper anabolism but rather in damage G1/S phase transition in Arabidopsis (Dewitte et al., associated with flower development. 2007; Riou-Khamlichi et al., 1999; Soni et al., 1995), To gain insight into this damage, we investigated the cytokinins in inoculation experiments would act as cell- effects of hormones by which floral organ development division activators in Eustoma. is almost certainly regulated. When we inoculated Not only cell division, but also organ differentiation hormones to flower buds under blast-inducing condition, was suppressed by blast-inducing conditions (Fig. 2A). the flower buds were 2.5–4 mm in length. Our anatomical It has been reported that cytokinins facilitate cell analysis demonstrated that the stages in which flower differentiation as well as cell division in Arabidopsis buds were about 2.5–4 mm in length were stage 2 to (Werner et al., 2001, 2003). Similarly, organ differenti- stage 3. GA3 and cytokinin applications resulted in ation is suppressed in cytokinin-deficient potato tubers increase of the frequency of flower opening. Thus, GA3 (Hartmann et al., 2011). Our results demonstrated that and cytokinins affected flower buds producing the the effects of blast-inducing conditions on organ stamen and gynoecium. differentiation and the supply of undifferentiated cells The highest frequency of blasting suppression by a were not coincidental. Hence, it is likely that exogenous single treatment was observed in experiments in which cytokinins promoted floral organ differentiation as well 200 ppm GA3 was applied (Fig. 4A). GA3 acts as a as cell division in Eustoma. As cytokinins are exported functional inhibitor of DELLA proteins, which are through the xylem and accumulate at high transpiration repressors of gibberellin signaling in Arabidopsis (Cheng sites (Aloni et al., 2005; Boonman et al., 2007), a et al., 2004; Yu et al., 2004). In Arabidopsis, GAs play reduction in active cytokinins in meristems may be a role in upregulating the expression of floral homeotic induced by low light intensity. genes that control stamen and carpel identity (Yu et al., In contrast to cytokinins, it has not been reported that 2004). They also promote the expression of the flower low light intensity decreased endogenous GA in flower meristem identity gene LEAFY, which directly activates buds; therefore, GAs may act as negative blasting genes including stamen and carpel identity genes regulators by decreasing sensitivity to other factor(s) that (Blazquez and Weigel, 2000; Busch et al., 1999; Parcy are induced by blast-inducing conditions and that et al., 1998). In addition, exogenous GA modulates promote blasting. flower development in various plants (Demunk and It has been reported that removing branches changes Gijzenberg, 1977; Ohno, 1991; Zhang et al., 2008; assimilate partitioning (Fudano et al., 2001) and the Zieslin et al., 1977). In light of these findings and our correlation between low tolerance to light stress and analysis, GA-mediated promotion of flower develop- levels of assimilate in flower buds (Aloni et al., 1996; ment appears to be conserved in Eustoma. Turner and Wien, 1994). Our results demonstrate that In flower meristems of Arabidopsis, the gibberellin the effect of branch removal was independent of hormone 3-oxidase (GA3ox) gene, which catalyzes the final step treatments; therefore, it is likely that branch removal in the synthesis of bioactive GAs, is only expressed in leads to the suppression of blasting by increasing the stamens and the gynoecium (Hu et al., 2008), and the assimilate in flower buds. Branch removal, however, flower homeotic gene AGAMOUS, which facilitates unexpectedly did not lead to extreme inhibition of organ identity of stamens and the gynoecium, binds to blasting. This result indicated that the increased levels the GA3ox promoter (Gomez-Mena et al., 2005). Thus, of assimilation in floral buds might not be sufficient for once floral organ differentiation proceeds through perfective flower development. Decapitation together stamen and gynoecium stages, flower buds themselves with hormone treatment led to marked inhibition of would become capable of supplying sufficient GA. In blasting compared to each single hormone treatment and Eustoma, almost all aborted-flowers were less than single branch removal. These additive effects suggest 10 mm long. Floral meristems under control conditions, that low amounts of assimilates in the floral bud were in which flower buds are about 10 mm long, differ- rate-limiting for overcoming blasting in the single entiated ovule primordia. This suggested that flower- hormone application experiment and vice versa. bud blasting tend not to occur once the stamen and Flower-bud blasting studies in diverse plant species gynoecium initiation stages had passed; therefore, have predicted that flower-bud blasting is caused by low GA3ox induction at stamen and gynoecium primordia levels of assimilate as an energy source (Aloni et al., might be conserved in Eustoma. 1997; Fudano et al., 2009; Fukai et al., 1981; Ohno, In addition to GA, applying BA and CPPU reversed 1991; Vonk and Ribot, 1982). A possible explanation blasting in Eustoma. Cytokinins regulate a variety of for the slight effect of branch removal in this study is developmental processes, including the development of that not only absolute amounts of assimilates in flower floral organs (Bartrina et al., 2011; Nishijima and Shima, buds but also those in the whole plants which have not 2006). Blast induction arrested the development of been exploited in vegetative organs may act as a signal floral organs which had been differentiated (Fig. 1C), that controls flower development. This is consistent with J. Japan. Soc. Hort. Sci. 81 (1): 101–108. 2012. 107 the previous result that the shortage of acquired Fukai, M., K. Yamada and K. Funakoshi. 1981. Studies on the assimilates in the whole plant is not necessarily for blindness in Chrysanthemum morifolium Ramat. cv. Tenryu- blasting (Ushio and Fukuta, 2010). Sugars play pivotal no-asa. J. Japan. Soc. Hort. Sci. 50: 332–341 (In Japanese with English abstract). roles regulating the expression of genes controlling cell Gibson, S. 2005. Control of plant development and gene expression division, cell proliferation, and death (Gibson, 2005; by sugar signaling. Curr. Opin. Plant Biol. 8: 93–102. Leon and Sheen, 2003). To assess whether a decrease Gomez-Mena, C., S. de Folter, M. Costa, G. Angenent and R. in assimilates acts as a signal or not, further studies are Sablowski. 2005. Transcriptional program controlled by the needed. floral homeotic gene AGAMOUS during early organogenesis. Development 132: 429–438. Acknowledgements Hartmann, A., M. Senning, P. Hedden, U. Sonnewald and S. Sonnewald. 2011. Reactivation of meristem activity and We thank Drs. T. Nishijima and T. Hisamatsu for their sprout growth in potato tubers require both cytokinin and valuable suggestions and discussion, and Ms. K. gibberellin. Plant Physiol. 155: 776–796. Koshizuka and A. Mori for their technical assistance. Hisamatsu, T., M. Koshioka and L. Mander. 2004. Regulation of gibberellin biosynthesis and stem elongation by low Literature Cited temperature in Eustoma grandiflorum. J. Hort. Sci. Biotech. Aloni, B., L. Karni, Z. Zaidman and A. Schaffer. 1996. Changes 79: 354–359. of carbohydrates in pepper (Capsicum annuum L) flowers in Hisamatsu, T., M. Koshioka, N. Oyama and L. Mander. 1999. The relation to their abscission under different shading regimes. relationship between endogenous gibberellins and resetting Ann. Bot. 78: 163–168. in Eustoma grandiflorum. J. Japan. Soc. Hort. Sci. 68: 527– Aloni, B., L. Karni, Z. Zaidman and A. Schaffer. 1997. The 533. relationship between sucrose supply, sucrose-cleaving Hu, J., M. Mitchum, N. Barnaby, B. Ayele, M. Ogawa, E. Nam, enzymes and flower abortion in pepper. Ann. Bot. 79: 601– W. Lai, A. Hanada, J. Alonso, J. Ecker, S. Swain, S. 605. Yamaguchi, Y. Kamiya and T. Sun. 2008. Potential sites of Aloni, R., M. Langhans, E. Aloni, E. Dreieicher and C. Ullrich. bioactive gibberellin production during reproductive growth 2005. Root-synthesized cytokinin in Arabidopsis is distrib- in Arabidopsis. Plant Cell 20: 320–336. uted in the shoot by the transpiration stream. J. Exp. Bot. 56: Leon, P. and J. Sheen. 2003. Sugar and hormone connections. 1535–1544. Trends Plant Sci. 8: 110–116. Bartrina, I., E. Otto, M. Strnad, T. Werner and T. Schmulling. Mason, M. and W. Miller. 1991. Flower bud blast in Easter lily 2011. Cytokinin regulates the activity of reproductive is induced by ethephon and inhibited by silver thiosulfate. meristems, flower organ size, ovule formation, and thus seed HortScience 26: 1165–1167. yield in Arabidopsis thaliana. Plant Cell 23: 69–80. Nishijima, T. and K. Shima. 2006. Change in flower morphology Blazquez, M. A. and D. Weigel. 2000. Integration of floral of Torenia fournieri Lind. induced by forchlorfenuron inductive signals in Arabidopsis. Nature 404: 889–892. application. Sci. Hortic. 109: 254–261. Boonman, A., E. Prinsen, F. Gilmer, U. Schurr, A. Peeters, L. Ohno, H. 1991. Microsporogenesis and flower bud blasting Voesenek and T. Pons. 2007. Cytokinin import rate as a signal as affected by high-temperature and in Cymbidium for photosynthetic acclimation to canopy light gradients. Plant (Orchidaceae). J. Japan. Soc. Hort. Sci. 60: 149–157. Physiol. 143: 1841–1852. Parcy, F., O. Nilsson, M. A. Busch, I. Lee and D. Weigel. 1998. Busch, M. A., K. Bomblies and D. Weigel. 1999. Activation of a A genetic framework for floral patterning. Nature 395: 561– floral homeotic gene in Arabidopsis. Science 285; 585–587. 566. Cheng, H., L. Qin, S. Lee, X. Fu, D. Richards, D. Cao, D. Luo, Reed, A. and G. Singletary. 1989. Roles of carbohydrate supply N. Harberd and J. Peng. 2004. Gibberellin regulates and phytohormones in maize kernel abortion. Plant Physiol. Arabidopsis floral development via suppression of DELLA 91: 986–992. protein function. Development 131: 1055–1064. Riou-Khamlichi, C., R. Huntley, A. Jacqmard and J. Murray. 1999. Demunk, W. 1973. Flower-bud blasting of tulips caused by Cytokinin activation of Arabidopsis cell division through a ethylene. Neth. J. Plant Pathol. 79: 41–53. D-type cyclin. Science 283: 1541–1544. Demunk, W. and J. Gijzenberg. 1977. Flower-bud blasting in tulip Simizu-Yumoto, H. and K. Ichimura. 2010. Postharvest physiology plants mediated by hormonal status of plant. Sci. Hortic. 7: and technology of cut Eustoma flowers. J. Japan. Soc. Hort. 255–268. Sci. 79: 227–238. Dewitte, W., S. Scofield, A. Alcasabas, S. Maughan, M. Menges, Soni, R., J. Carmichael, Z. Shah and J. Murray. 1995. A family N. Braun, C. Collins, J. Nieuwland, E. Prinsen, V. Sundaresan of cyclin D-homologs from plants differentially controlled and J. Murray. 2007. Arabidopsis CYCD3 D-type cyclins by growth-regulators and containing the conserved retino- link cell proliferation and endocycles and are rate-limiting blastoma protein-interaction motif. Plant Cell 7: 85–103. for cytokinin responses. Proc. Natl. Acad. Sci. USA 36: Turner, A. and H. Wien. 1994. Photosynthesis, dark respiration 14537–14542. and bud sugar concentrations in pepper cultivars differing in Fudano, T., T. Hayashi and S. Yazawa. 2001. Partitioning of C- susceptibility to stress-induced bud abscission. Ann. Bot. 73: 13-photosynthate in sweet pea (Lathyrus odoratus L.) plants 623–628. during the flowering period. J. Japan. Soc. Hort. Sci. 70: 102– Ushio, A. and N. Fukuta. 2010. Effects of nitrogen fertilization 107 (In Japanese with English abstract). level in nutrient solution applied before/after flower budding Fudano, T., T. Hayashi and S. Yazawa. 2009. Dynamic model of on blasting in winter flowering of Eustoma grandiflorum dry matter distribution and stabilization in the number of buds (Raf.) Shinn. Hort. Res (Japan) 9: 191–196 (In Japanese with per inflorescence by overnight supplemental lighting in sweet English abstract). pea (Lathyrus odoratus L.). J. Japan. Soc. Hort. Sci. 78: 344– Vonk, C. and S. Ribot. 1982. Assimilate distribution and the role 349. of abscisic acid and zeatin in relation to flower-bud blasting, 108 K. Kawakatsu, A. Ushio and N. Fukuta

induced by lack of light, in Iris cv. Ideal. Plant Growth Regul. gametophytes in Eustoma grandiflorum. J. Japan. Soc. Hort. 1: 93–105. Sci. 76: 244–249. Vonk, C., E. Davelaar and S. Ribot. 1986. The role of cytokinins Yu, H., T. Ito, Y. Zhao, J. Peng, P. Kumar and E. Meyerowitz. in relation to flower bud blasting in Iris cv. Ideal: Cytokinin 2004. Floral homeotic genes are targets of gibberellin determination by an improved enzyme-linked immunosor- signaling in flower development. Proc. Natl. Acad. Sci. USA bent-assay. Plant Growth Regul. 4: 65–74. 101: 7827–7832. Werner, T., V. Motyka, R. Laucou, H. Smets, H. Van Onckelen Zhang, M., D. Ye, L. Wang, J. Pang, Y. Zhang, K. Zheng, H. Bian, and T. Schmulling. 2003. Cytokinin-deficient transgenic N. Han, J. Pan, J. Wang and M. Zhu. 2008. Overexpression Arabidopsis plants show multiple developmental alterations of the cucumber LEAFY homolog CFL and hormone indicating opposite functions of cytokinins in the regulation treatments alter flower development in gloxinia (Sinningia of shoot and root meristem activity. Plant Cell 15: 2532–2550. speciosa). Plant Mol. Biol. 67: 419–427. Werner, T., V. Motyka, M. Strnad and T. Schmulling. 2001. Zieslin, N., Y. Leshem, H. Spiegelstein and A. Halevy. 1977. Regulation of plant growth by cytokinin. Proc. Natl. Acad. Possible membrane-associated effects in gibberellic-acid and Sci. USA 98: 10487–10492. phenylalanine-induced rose coloration enhancement. Acta Yang, X., Q. Wang and Y. Li. 2007. Microsporogenesis, Botanica Neerlandica 26: 183–186. megasporogenesis, and the development of male and female