在蝴蝶蘭產業，一般商業上用來催花的方式就是低溫處理，雖然我們早已熟知低溫處理可以誘導蝴蝶蘭開花，但是詳細的分子機制仍未明。陳等人在2008年提出：涼爽夜溫會誘導蝴蝶蘭抽梗同時也會增加糖類的含量。本篇研究主要是架構在陳等人的結果進行兩個層面的探討，第一是為什麼涼爽的夜溫會使蔗糖含量上升？第二是為什麼涼爽夜溫會促使蝴蝶蘭抽梗。對於第一個問題，我們從三個蔗糖合成酶、三個蔗糖轉化酶及一個蔗糖磷酯合成酶著手進行即時定量RT-PCR分析。然而，除了PaSUS1, PaSUS6 及PaINV1，其它四個基因並沒有顯著關聯性。由於蔗糖在植物細胞內可以扮演營養分子也可同時扮演訊息傳導分子，我們將目標轉移至蔗糖的訊息傳導並假設蔗糖在SnRK1的訊息傳導上扮演一個重要的角色。很有趣的，我們的結果顯示涼爽夜溫雖然沒有影響PaSnRK1的RNA合成量但卻會會降低PaSnRK1的酵素活性，這個結果顯示高蔗糖會降低PaSnRK1活性以達到誘導蝴蝶蘭開花，顯然蔗糖可扮演一個訊息傳遞分子，透過PaSnRK1將訊息傳遞到下游開花基因。對於第二個問題，我們從十個開花基因的表現量著手分析，由即時定量RT-PCR結果發現在四條開花路徑中，自發性路徑基因(PaFCA, PaFPA)與光周期路徑基因(PaGI, PaFT)的表現量在涼爽夜溫處理時都會升高，而春化路徑(PaVIN3-1, PaVIN3-2, PaVRN)基因的表現變化較不顯著。這個結果顯示自發性路徑與光周期路徑是蝴蝶蘭主要在低夜溫誘導時的開花路徑。根據以上結果，我們提出一個假設，低夜溫處理會使蔗糖含量增加，進而抑制PaSnRK1的酵素活性，接著PaSnRK1將訊息傳遞至下游目標並影響蝴蝶蘭的開花時間。

The common way to induce spiking of Phalaenopsis is by low temperature treatment. Although we know it flowers under low temperature treatment, the detail mechanism is still unknown. In 2008, Chen et al. reported that cool-night temperature not only induces spike emergence but also increase free sugars in Phalaenopsis aphrodite. Based on this, we proposed to answer both why sucrose content elevates and why Phalaenopsis starts spiking under cool-night temperature. First, we analyzed the expression level of three sucrose synthase genes, three invertase genes and one sucrose phosphate synthase gene. By using quantitative real-time RT-PCR, our results showed that PaINV1, PaSUS1 and PaSUS6 showed decrease in their expression, while the rest four genes were not strongly correlated to the elevated sucrose content. In plant cell, sucrose is not only a nutrient but also a signaling molecule. We hypothesized that sucrose may play a key role via SnRK1 signaling. Interestingly, we found that the kinase activity of PaSnRK1 reduced during cool-night temperature although its mRNA expression remained unaffected. This implied that PaSnRK1 is involved in the flowering process of Phalaenopsis aphrodite. Secondly, we analyzed the expression levels of ten flowering genes from four flowering pathways by using quantitative real-time RT-PCR. We found that autonomous pathway genes (PaFCA and PaFPA) and photoperiod pathway genes (PaGI and PaFT) were upregulated under cool-night treatment, while vernalization pathway genes (PaVIN3-1, PaVIN3-2 and PaVRN) were not significantly affected. This indicated that autonomous and photoperiod pathways were two major pathways in the cool-night induced flowering process of Phalaenopsis. Finally, we proposed that cool-night temperature increases the level of sucrose that reduces the kinase activity of the PaSnRK1, and the down-regulated PaSnRK1 transfers its signal to unidentified downstream targets and then affects the flowering gene expression that eventually induces flowering of Phalaenopsis.

Abe, M., Kobayashi, Y., Yamamoto, S., Daimon, Y.,Yamaguchi, A., Ikeda, Y., Ichinoki, H., Notaguchi, M., Goto, K., and Araki, T. (2005). FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309, 1052-1056.
Bachmann, M., Shiraishi, N., Campbell, W.H., Yoo, B.C., Harmon, A.C., and Huber, S.C. (1996). Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. Plant Cell 8, 505-517.
Baena-Gonzalez, E., Rolland, F., Thevelein, J.M., and Sheen, J. (2007). A central integrator of transcription networks in plant stress and energy signalling. Nature 448, 938-942.
Barratt, D.H., Barber, L., Kruger, N.J., Smith, A.M., Wang, T.L., and Martin, C. (2001). Multiple, distinct isoforms of sucrose synthase in pea. Plant Physiol 127, 655-664.
Bernier, G., and Perilleux, C. (2005). A physiological overview of the genetics of flowering time control. Plant Biotechnol J 3, 3-16.
Blanchard, M.G., and Runkle, E.S. (2008). Benzyladenine promotes flowering in Doritaenopsis and Phalaenopsis orchids. J Plant Growth Regul 27, 141-150.
Blazquez, M. (2000). Flower development pathways. J Cell Sci 113 ( Pt 20), 3547-3548.
Bouly, J.-P., Gissot, L., Lessard, P., Kreis, M., and Thomas, M. (1999). Arabidopsis thaliana proteins related to the yeast SIP and SNF4 interact with AKINα1, an SNF1-like protein kinase The Plant Journal 18, 541-550.
Chen, W.H., Tseng, Y.C., Liu, Y.C., Chuo, C.M., Chen, P.T., Tseng, K.M., Yeh, Y.C., Ger, M.J., and Wang, H.L. (2008). Cool-night temperature induces spike emergence and affects photosynthetic efficiency and metabolizable carbohydrate and organic acid pools in Phalaenopsis aphrodite. Plant Cell Rep 27, 1667-1675.
Chen, W.S., Chang, H.W., Chen, W.H., and Lin, Y.S. (1997). Gibberellic acid and cytokinin affect Phalaenopsis flower morphology at high temperature. Hortscience 32, 1069-1073.
Chourey, P.S., Taliercio, E.W., Carlson, S.J., and Ruan, Y.L. (1998). Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis. Mol Gen Genet 259, 88-96.
Corbesier, L., Lejeune, P., and Bernier, G. (1998). The role of carbohydrates in the induction of flowering in Arabidopsis thaliana: comparison between the wild type and a starchless mutant. Planta 206, 131-137.
Daie, J. (1993). Cytosolic fructose-1,6-bisphosphatase - a key enzyme in the sucrose biosynthetic-pathway. Photosynth Res 38, 5-14.
Dale, S., Arro, M., Becerra, B., Morrice, N.G., Boronat, A., Hardie, D.G., and Ferrer, A. (1995). Bacterial expression of the catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase (isoform HMGR1) from Arabidopsis thaliana, and its inactivation by phosphorylation at Ser577 by Brassica oleracea 3-hydroxy-3-methylglutaryl-CoA reductase kinase. Eur J Biochem 233, 506-513.
Dickinson, J.R., and Schweizer, M. (1999). Carbon metabolism. (London & Philadelphia: Taylor & Francis).
Dielen, V., Lecouvet, V., Dupont, S., and Kinet, J.M. (2001). in vitro control of floral transition in tomato (Lycopersicon esculentum Mill.), the model for autonomously flowering plants, using the late flowering uniflora mutant. J Exp Bot 52, 715-723.
Douglas, P., Morrice, N., and MacKintosh, C. (1995). Identification of a regulatory phosphorylation site in the hinge 1 region of nitrate reductase from spinach (Spinacea oleracea) leaves. FEBS Lett 377, 113-117.
Essmann, J., Schmitz-Thom, I., Schon, H., Sonnewald, S., Weis, E., and Scharte, J. (2008). RNA interference-mediated repression of cell wall invertase impairs defense in source leaves of tobacco. Plant Physiol 147, 1288-1299.
Gissot, L., Polge, C., Bouly, J.P., Lemaitre, T., Kreis, M., and Thomas, M. (2004). AKINbeta3, a plant specific SnRK1 protein, is lacking domains present in yeast and mammals non-catalytic beta-subunits. Plant Mol Biol 56, 747-759.
Gissot, L., Polge, C., Jossier, M., Girin, T., Bouly, J.P., Kreis, M., and Thomas, M. (2006). AKINbetagamma contributes to SnRK1 heterotrimeric complexes and interacts with two proteins implicated in plant pathogen resistance through its KIS/GBD sequence. Plant Physiol 142, 931-944.
Halford, N.G., and Paul, M.J. (2003). Carbon metabolite sensing and signalling. Plant Biotechnol J 1, 381-398.
Halford, N.G., Hey, S., Jhurreea, D., Laurie, S., McKibbin, R.S., Paul, M., and Zhang, Y. (2003). Metabolic signalling and carbon partitioning: role of Snf1-related (SnRK1) protein kinase. J Exp Bot 54, 467-475.
He, Y., Michaels, S.D., and Amasino, R.M. (2003). Regulation of flowering time by histone acetylation in Arabidopsis. Science 302, 1751-1754.
Helliwell, C.A., Wood, C.C., Robertson, M., James Peacock, W., and Dennis, E.S. (2006). The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex. Plant J 46, 183-192.
Henderson, I.R., and Dean, C. (2004). Control of Arabidopsis flowering: the chill before the bloom. Development 131, 3829-3838.
Heyer, A.G., Raap, M., Schroeer, B., Marty, B., and Willmitzer, L. (2004). Cell wall invertase expression at the apical meristem alters floral, architectural, and reproductive traits in Arabidopsis thaliana. Plant J 39, 161-169.
Hruz, T., Laule, O., Szabo, G., Wessendorp, F., Bleuler, S., Oertle, L., Widmayer, P., Gruissem, W., and Zimmermann, P. (2008). Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes. Adv Bioinformatics 2008, 420747.
Hsiao, Y.Y., Jeng, M.F., Tsai, W.C., Chuang, Y.C., Li, C.Y., Wu, T.S., Kuoh, C.S., Chen, W.H., and Chen, H.H. (2008). A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2_4D motif. Plant J 55, 719-733.
Jang, S., Marchal, V., Panigrahi, K.C., Wenkel, S., Soppe, W., Deng, X.W., Valverde, F., and Coupland, G. (2008). Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photoperiodic flowering response. EMBO J 27, 1277-1288.
Jiang, R., and Carlson, M. (1997). The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex. Mol Cell Biol 17, 2099-2106.
Kardailsky, I., Shukla, V.K., Ahn, J.H., Dagenais, N., Christensen, S.K., Nguyen, J.T., Chory, J., Harrison, M.J., and Weigel, D. (1999). Activation tagging of the floral inducer FT. Science 286, 1962-1965.
Kataoka, K., Sumitomo, K., Fudano , T., and Kawase, K. (2004). Changes in sugar content of Phalaenopsis leaves before floral transition Scientia Horticulturae 102, 121-132
Kim, J.Y., Mahe, A., Brangeon, J., and Prioul, J.L. (2000). A maize vacuolar invertase, IVR2, is induced by water stress. Organ/tissue specificity and diurnal modulation of expression. Plant Physiol 124, 71-84.
Koornneef, M., Hanhart, C.J., and van der Veen, J.H. (1991). A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana. Mol Gen Genet 229, 57-66.
Lee, N., and Lin, G. (1984). Effect of temperature on growth and flowering of Phalaenopsis white hybrid. J Chin Soc Hortic Sci 30, 223-231.
Lee, N., Lee C. H. (1996). Changes in carbohydrate in Phalaenopsis during floral induction and inflorescence development. J. Chinese Soc. Hort. Sci 42, 262-275.
Lu, C.A., Lin, C.C., Lee, K.W., Chen, J.L., Huang, L.F., Ho, S.L., Liu, H.J., Hsing, Y.I., and Yu, S.M. (2007). The SnRK1A protein kinase plays a key role in sugar signaling during germination and seedling growth of rice. Plant Cell 19, 2484-2499.
Macmillan, C.P., Blundell, C.A., and King, R.W. (2005). Flowering of the grass Lolium perenne: effects of vernalization and long days on gibberellin biosynthesis and signaling. Plant Physiol 138, 1794-1806.
Michaels, S.D., and Amasino, R.M. (1999). FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11, 949-956.
Michaels, S.D., and Amasino, R.M. (2001). Loss of FLOWERING LOCUS C activity eliminates the late-flowering phenotype of FRIGIDA and autonomous pathway mutations but not responsiveness to vernalization. Plant Cell 13, 935-941.
Mutasa-Gottgens, E., and Hedden, P. (2009). Gibberellin as a factor in floral regulatory networks. J Exp Bot 60, 1979-1989.
Onouchi, H., Igeno, M.I., Perilleux, C., Graves, K., and Coupland, G. (2000). Mutagenesis of plants overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes. Plant Cell 12, 885-900.
Paltiel, J., Amin, R., Gover, A., Ori, N., and Samach, A. (2006). Novel roles for GIGANTEA revealed under environmental conditions that modify its expression in Arabidopsis and Medicago truncatula. Planta 224, 1255-1268.
Polge, C., and Thomas, M. (2007). SNF1/AMPK/SnRK1 kinases, global regulators at the heart of energy control? Trends Plant Sci 12, 20-28.
Purcell, P.C., Smith, A.M., and Halford, N.G. (1998). Antisense expression of a sucrose nonfermenting-1-related protein kinase sequence in potato results in decreased expression of sucrose synthase in tubers and loss of sucrose-inducibility of sucrose synthase transcripts in leaves. Plant J. 14, 195–202.
Roldan, M., Gomez-Mena, C., Ruiz-Garcia, L., Salinas, J., and Martinez-Zapater, J.M. (1999). Sucrose availability on the aerial part of the plant promotes morphogenesis and flowering of Arabidopsis in the dark. Plant J 20, 581-590.
Rolland, F., Baena-Gonzalez, E., and Sheen, J. (2006). Sugar sensing and signaling in plants: conserved and novel mechanisms. Annu Rev Plant Biol 57, 675-709.
Sanda, S.L., and Amasino, R.M. (1996). Interaction of FLC and late-flowering mutations in Arabidopsis thaliana. Mol Gen Genet 251, 69-74.
Sheldon, C.C., Rouse, D.T., Finnegan, E.J., Peacock, W.J., and Dennis, E.S. (2000). The molecular basis of vernalization: the central role of FLOWERING LOCUS C (FLC). Proc Natl Acad Sci U S A 97, 3753-3758.
Sheldon, C.C., Burn, J.E., Perez, P.P., Metzger, J., Edwards, J.A., Peacock, W.J., and Dennis, E.S. (1999). The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11, 445-458.
Shen, C.H., and Yeh, K.W. (2010). Hydrogen peroxide mediates the expression of ascorbate-related genes in response to methanol stimulation in Oncidium. Journal of Plant Physiology 167, 400-407.
Shen, C.H., Krishnamurthy, R., and Yeh, K.W. (2009a). Decreased l-ascorbate content mediating bolting is mainly regulated by the galacturonate pathway in Oncidium. Plant Cell Physiol 50, 935-946.
Shen, W., Reyes, M.I., and Hanley-Bowdoin, L. (2009b). Arabidopsis protein kinases GRIK1 and GRIK2 specifically activate SnRK1 by phosphorylating its activation loop. Plant Physiol 150, 996-1005.
Simpson, G.G., and Dean, C. (2002). Arabidopsis, the Rosetta stone of flowering time? Science 296, 285-289.
Suarez-Lopez, P., Wheatley, K., Robson, F., Onouchi, H., Valverde, F., and Coupland, G. (2001). CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis. Nature 410, 1116-1120.
Sugden, C., Donaghy, P.G., Halford, N.G., and Hardie, D.G. (1999). Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl- coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro. Plant Physiol 120, 257-274.
Valverde, F., Mouradov, A., Soppe, W., Ravenscroft, D., Samach, A., and Coupland, G. (2004). Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303, 1003-1006.
Vincent, O., and Carlson, M. (1999). Gal83 mediates the interaction of the Snf1 kinase complex with the transcription activator Sip4. EMBO J 18, 6672-6681.
Vincent, O., Townley, R., Kuchin, S., and Carlson, M. (2001). Subcellular localization of the Snf1 kinase is regulated by specific beta subunits and a novel glucose signaling mechanism. Genes Dev 15, 1104-1114.
Wang, C.Y., Chiou, C.Y., Wang, H.L., Krishnamurthy, R., Venkatagiri, S., Tan, J., and Yeh, K.W. (2008). Carbohydrate mobilization and gene regulatory profile in the pseudobulb of Oncidium orchid during the flowering process. Planta 227, 1063-1077.
Weckwerth, W., Loureiro, M.E., Wenzel, K., and Fiehn, O. (2004). Differential metabolic networks unravel the effects of silent plant phenotypes. Proc Natl Acad Sci U S A 101, 7809-7814.
Zhang, Y., Shewry, P.R., Jones, H., Barcelo, P., Lazzeri, P.A., and Halford, N.G. (2001). Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley. Plant J 28, 431-441.