Phototropins是藍光接受器，在葉綠體移動、氣孔打開及植物的生長及發育扮演著重要的角色。由於植物不能移動，在高光照環境下透過葉綠體躲避可減少光抑制之傷害。然而，在低光照環境下為了增加光合作用效率，植物會累積較多的葉綠體於細胞表面。在台灣蝴蝶蘭是一種很重要的經濟花卉作物。然而屆至目前為止，尚無對蝴蝶蘭葉綠體移動的研究。故本論文由三個方向做有系統的探勘: 1)選殖蝴蝶蘭兩個 Phototropins (簡稱: PaPhot1 及 PaPhot2)全長序列; 2) 探討Phototropins 對於光線的反應及葉綠體移動進行生理研究;及3) 確認蝴蝶蘭 Phototropins 的基因功能。首先我們利用 5’RACE 以及3’RACE (rapid amplification of cDNA end, cDNA末端快速擴增) 技術選殖 PaPhot1 及 PaPhot2 的全長 cDNA 序列，獲得基因全長分別為 3448 bp 以及 4954 bp。蝴蝶蘭 Phototropins 胺基酸序列與阿拉伯芥相似，具有兩個 LOV 重要區域及一個 Ser/Thr kinase 區域。親緣演化分析發現PaPhot1 及 PaPhot2 與單子葉植物親緣關係較相近。蝴蝶蘭葉片經由不同光源及不同藍光強度照射處理後分析PaPhot1 及 PaPhot2 在不同光源下皆有表現，但在藍光下表現特別高。在持續的黑暗環境下，蝴蝶蘭 PaPhots 在第四天有較多的表現，但在一週後其表現量則開始降低。經由不同藍光強度檢測顯示，在藍光20 μmole m-2.s-1 下蝴蝶蘭的葉綠體不移動，然而藍光低於15 μmole m-2.s-1 時葉綠體會累積至平週細胞壁。藍光高於25 μmole m-2.s-1 葉綠體會躲避至背斜細胞壁，特別是藍光高於100 μmole m-2.s-1 時葉綠體有明顯地躲避作用。PaPhots 在低藍光下表現量皆有上升，然而在高藍光下只有 PaPhot2 有較高的表現。自然界中蝴蝶蘭為附生的習性，在蝴蝶蘭根部發現 PaPhot1 及 PaPhot2 是有表現。此外相較於較年輕的根部部位，成熟的根部含有較多的葉綠素以及表現較多的 Phototropins。原位雜交技術顯示在根冠、葉片表皮及維管束有表達 PaPhot1 及 PaPhot2。為了確認Phototropins 參與葉綠體移動的生物功能，本研究利用病毒誘導基因靜默 (virus induced gene silencing , VIGS) 技術指出，蝴蝶蘭VIGS的葉子PaPhots 基因表現量減少且葉綠體移動的能力也跟著減低。此外，我們也利用互補實驗過度表達外源 PaPhot1 及 PaPhot2 於阿拉伯芥突變株中， RT-PCR 確認AtPhot1 及 AtPhot2 基因在突變株中已被剔除。結果顯示PaPhots基因過度表達於阿拉伯芥轉殖植株中有表現且恢復了葉綠體移動的能力。綜合以上結果，本研究成功地選殖蝴蝶蘭中的 Phototropins 基因，對該基因的特性有更多的瞭解，且證實他們的生物功能為藍光接受器。

Phototropins are blue light receptors and they play important roles in chloroplast movement, stomatal opening, and affect plant growth and development. Phalaenopsis orchid is one of the most important ornamental crops in Taiwan. However, study on chloroplast movement in Phalaenopsis orchid is limited. We cloned full-length cDNAs of PaPhot1 and PaPhot2. Phylogenetic analysis showed that PaPhot1 and PaPhot2 are clustered in the same group of monocots. PaPhot1 and PaPhot2 increased their expression level under different light sources, but significance up-regulated in BL. Slit assay results showed that chloroplast didn’t moved under 20 μmole m-2.s-1, however, low BL (< 15 μmole m-2.s-1), orchid accumulated more chloroplast on the periclinal cell walls. Under high BL (> 25 μmole m-2.s-1), chloroplast starts moving to the anticlinal cell walls and significant chloroplast avoidance at BL higher than 100μmole m-2.s-1. Interestingly, PaPhot1 and PaPhot2 expressed in orchid root might associate with the nature of epiphytic habitat. In situ hybridization showed that PaPhot1 and PaPhot2 expression in root cap, leaf epidermis and vascular. To verify biological function of Phototropins in chloroplast movement, virus induced gene silencing (VIGS) treated leaves showed decreasing gene expression of PaPhots and less chloroplast movement phenomena. In addition, we used heterologous overexpression PaPhot1 and PaPhot2 in Arabidopsis knockout mutants. RT-PCR confirmed Arabidopsis mutant lines knockout AtPhot1 and AtPhot2. The overexpressed PaPhots Arabidopsis transgenic lines expressed PaPhots and can complement Arabidopsis lines and recovered their chloroplast movement phenotype.

Ahmad, M., and Cashmore, A.R. (1993). Hy4 Gene of a-Thaliana Encodes a Protein with Characteristics of a Blue-Light Photoreceptor. Nature 366, 162-166.

Banas, A.K., Aggarwal, C., Labuz, J., Sztatelman, O., and Gabrys, H. (2012). Blue light signalling in chloroplast movements. J Exp Bot 63, 1559-1574.

Briggs, W.R., Beck, C.F., Cashmore, A.R., Christie, J.M., Hughes, J., Jarillo, J.A., Kagawa, T., Kanegae, H., Liscum, E., Nagatani, A., Okada, K., Salomon, M., Rudiger, W., Sakai, T., Takano, M., Wada, M., and Watson, J.C. (2001). The phototropin family of photoreceptors. Plant Cell 13, 993-997.

Cho, H.Y., Tseng, T.S., Kaiserli, E., Sullivan, S., Christie, J.M., and Briggs, W.R. (2007). Physiological roles of the light, oxygen, or voltage domains of phototropin 1 and phototropin 2 in Arabidopsis. Plant Physiol 143, 517-529.

Christie, J.M. (2007). Phototropin blue-light receptors. Annual review of plant biology 58, 21-45.

Christie, J.M., Salomon, M., Nozue, K., Wada, M., and Briggs, W.R. (1999). LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): Binding sites for the chromophore flavin mononucleotide. P Natl Acad Sci USA 96, 8779-8783.

Chung, J.P., Huang, C.Y., and Dai, T.E. (2010). Spectral effects on embryogenesis and plantlet growth of Oncidium 'Gower Ramsey'. Sci Hortic-Amsterdam 124, 511-516.

Clack, T., Mathews, S., and Sharrock, R.A. (1994). The Phytochrome Apoprotein Family in Arabidopsis Is Encoded by 5 Genes - the Sequences and Expression of Phyd and Phye. Plant Mol Biol 25, 413-427.

Demarsy, E., and Fankhauser, C. (2009). Higher plants use LOV to perceive blue light. Curr Opin Plant Biol 12, 69-74.

Folta, K.M., and Spalding, E.P. (2001). Unexpected roles for cryptochrome 2 and phototropin revealed by high-resolution analysis of blue light-mediated hypocotyl growth inhibition. Plant J 26, 471-478.

Folta, K.M., and Kaufman, L.S. (2003). Phototropin 1 is required for high-fluence blue-light-mediated mRNA destabilization. Plant Mol Biol 51, 609-618.

Harada, A., and Shimazaki, K.I. (2007). Phototropins and blue light-dependent calcium signaling in higher plants. Photochem Photobiol 83, 102-111.

Huala, E., Oeller, P.W., Liscum, E., Han, I.S., Larsen, E., and Briggs, W.R. (1997). Arabidopsis NPH1: A protein kinase with a putative redox-sensing domain. Science 278, 2120-2123.

Imaizumi, T., Tran, H.G., Swartz, T.E., Briggs, W.R., and Kay, S.A. (2003). FKF1 is essential for photoperiodic-specific light signalling in Arabidopsis. Nature 426, 302-306.

Inoue, S., Kinoshita, T., and Shimazaki, K. (2005). Possible involvement of phototropins in leaf movement of kidney bean in response to blue light. Plant Physiol 138, 1994-2004.

Jiao, Y.L., Lau, O.S., and Deng, X.W. (2007). Light-regulated transcriptional networks in higher plants. Nat Rev Genet 8, 217-230.

Kagawa, T., and Wada, M. (1999). Chloroplast-avoidance response induced by high-fluence blue light in prothallial cells of the fern Adiantum capillus-veneris as analyzed by microbeam irradiation. Plant Physiol 119, 917-923.

Kagawa, T., Sakai, T., Suetsugu, N., Oikawa, K., Ishiguro, S., Kato, T., Tabata, S., Okada, K., and Wada, M. (2001). Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response. Science 291, 2138-2141.

Kanegae, H., Tahir, M., Savazzini, F., Yamamoto, K., Yano, M., Sasaki, T., Kanegae, T., Wada, M., and Takano, M. (2000). Rice NPH1 homologues, OsNPH1a and OsNPH1b, are differently photoregulated. Plant & cell physiology 41, 415-423.

Kasahara, M., Kagawa, T., Sato, Y., Kiyosue, T., and Wada, M. (2004). Phototropins mediate blue and red light-induced chloroplast movements in Physcomitrella patens. Plant Physiol 135, 1388-1397.

Kasahara, M., Kagawa, T., Oikawa, K., Suetsugu, N., Miyao, M., and Wada, M. (2002). Chloroplast avoidance movement reduces photodamage in plants. Nature 420, 829-832.

Kawai, H., Kanegae, T., Christensen, S., Kiyosue, T., Sato, Y., Imaizumi, T., Kadota, A., and Wada, M. (2003). Responses of ferns to red light are mediated by an unconventional photoreceptor. Nature 421, 287-290.

Kinoshita, T., Doi, M., Suetsugu, N., Kagawa, T., Wada, M., and Shimazaki, K. (2001). phot1 and phot2 mediate blue light regulation of stomatal opening. Nature 414, 656-660.

Kong, S.G., Suetsugu, N., Kikuchi, S., Nakai, M., Nagatani, A., and Wada, M. (2013). Both Phototropin 1 and 2 Localize on the Chloroplast Outer Membrane with Distinct Localization Activity. Plant and Cell Physiology 54, 80-92.

Kwokki, H., Hockhin, Y., and Choysin, H. (1983). The Presence of Photosynthetic Machinery in Aerial Roots of Leafy Orchids. Plant and Cell Physiology 24, 1317-1321.

Lin, C.T., Yang, H.Y., Guo, H.W., Mockler, T., Chen, J., and Cashmore, A.R. (1998). Enhancement of blue-light sensitivity of Arabidopsis seedlings by a blue light receptor cryptochrome 2. P Natl Acad Sci USA 95, 2686-2690.

Lin, H.Y., Chen, J.C., Wei, M.J., Lien, Y.C., Li, H.H., Ko, S.S., Liu, Z.H., and Fang, S.C. (2014). Genome-wide annotation, expression profiling, and protein interaction studies of the core cell-cycle genes in Phalaenopsis aphrodite. Plant Mol Biol 84, 203-226.

Lin, M.J., and Hsu, B.D. (2004). Photosynthetic plasticity of Phalaenopsis in response to different light environments. Journal of plant physiology 161, 1259-1268.

Long, S.P., Humphries, S., and Falkowski, P.G. (1994). Photoinhibition of Photosynthesis in Nature. Annu Rev Plant Phys 45, 633-662.

Luesse, D.R., DeBlasio, S.L., and Hangarter, R.P. (2006). Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis. Plant Physiol 141, 1328-1337.

Ohgishi, M., Saji, K., Okada, K., and Sakai, T. (2004). Functional analysis of each blue light receptor, cry1, cry2, phot1, and phot2, by using combinatorial multiple mutants in Arabidopsis (vol 101, pg 2223, 2004). P Natl Acad Sci USA 101, 5696-5696.

Reymond, P., Short, T.W., and Briggs, W.R. (1992). Blue light activates a specific protein kinase in higher plants. Plant Physiol 100, 655-661.

Rizzini, L., Favory, J.J., Cloix, C., Faggionato, D., O'Hara, A., Kaiserli, E., Baumeister, R., Schafer, E., Nagy, F., Jenkins, G.I., and Ulm, R. (2011). Perception of UV-B by the Arabidopsis UVR8 Protein. Science 332, 103-106.

Sakai, T., Kagawa, T., Kasahara, M., Swartz, T.E., Christie, J.M., Briggs, W.R., Wada, M., and Okada, K. (2001). Arabidopsis nph1 and npl1: Blue light receptors that mediate both phototropism and chloroplast relocation. P Natl Acad Sci USA 98, 6969-6974.

Sakamoto, K., and Briggs, W.R. (2002). Cellular and subcellular localization of phototropin 1. Plant Cell 14, 1723-1735.

Schultz, T.F., Kiyosue, T., Yanovsky, M., Wada, M., and Kay, S.A. (2001). A role for LKP2 in the circadian clock of Arabidopsis. Plant Cell 13, 2659-2670.

Sharrock, R.A., and Quail, P.H. (1989). Novel phytochrome sequences in Arabidopsis thaliana: structure, evolution, and differential expression of a plant regulatory photoreceptor family. Genes & development 3, 1745-1757.

Somers, D.E., Schultz, T.F., Milnamow, M., and Kay, S.A. (2000). ZEITLUPE encodes a novel clock-associated PAS protein from Arabidopsis. Cell 101, 319-329.

Su, C.L., Chao, Y.T., Yen, S.H., Chen, C.Y., Chen, W.C., Chang, Y.C.A., and Shih, M.C. (2013). Orchidstra: An Integrated Orchid Functional Genomics Database. Plant and Cell Physiology 54, E11-+.

Sullivan, J.A., and Deng, X.W. (2003). From seed to seed: the role of photoreceptors in Arabidopsis development. Dev Biol 260, 289-297.

Takahashi, S., and Murata, N. (2008). How do environmental stresses accelerate photoinhibition? Trends Plant Sci 13, 178-182.

Tokutomi, S., Matsuoka, D., and Ziklhara, K. (2008). Molecular structure and regulation of phototropin kinase by blue light. Bba-Proteins Proteom 1784, 133-142.

Wada, M. (2013). Chloroplast movement. Plant science : an international journal of experimental plant biology 210, 177-182.