臺灣博碩士論文加值系統

中文摘要

Phalaenopsis bellina 是具有香味的原生種蝴蝶蘭。P. bellina的主要香味成分是單萜類 (monoterpenes)中的 linalool 與 geraniol，其香味在開花後第五天達到最高。P. bellina的香味關鍵酵素是geranyl diphosphate synthase small subunit (PbGDPS­_SSU)，其表現量與香味釋放一致。先前研究中花香的生成於上午十點至下午四點最多，因此花朵香味的釋放極可能由溫度或光調控。我們分析linalool 在長日照 (16 h/8 h L/D): 連續白光與黑暗處理組的釋放形式，結果顯示原生種蝴蝶蘭花香味釋放主要是受光調控。 再者，P. bellina持續生長天數 (自開花當天至開花後第七天) 的轉錄體分析，結果顯示P. bellina其中13筆光受體 (photoreceptors) 轉錄體的表現模式和GDPS_SSU一致，包含光敏素 (PHY), 隱花素 (CRY), 向光素 (PHOT), 與紫外光受體 UVBR。 轉綠體與光受體基因進一步以定量real-time RT-PCR分析，兩個藍光受體PbCRY3 和 PbPHOT1被發現其表現模式與PbGDPS_SSU相似。 為深入研究藍光對蝴蝶蘭的香味釋放的調節，以兩種蝴蝶蘭商業品種進行研究多種光調節因子。香味成分 linalool 與 geraniol的釋放在長日照白光 (100 μmol m-2s-1, 16 h/8 h L/D) 下較遠紅光及黑暗處理組高。除此之外，高強度藍光 (270 μmol m-2s-1) 處理對兩原生種蝴蝶蘭linalool 與 geraniol的釋放呈現負調控，且對linalool的影響高於geraniol。 總結，這些結果顯示光對蘭花香味釋放為正調控，且光對linalool 與 geraniol的釋放調節可能有不同的機制。

ABSTRACT

Phalaenopsis bellina is a scented Phalaenopsis species. The main scent components of P. bellina are monoterpenes including linalool and geraniol with the maximal emission on day 5 post anthesis (D+5). The key enzyme gene for monoterpene biosynthesis in P. bellina is geranyl diphosphate synthase small subunit (PbGDPS_SSU), whose expression is concomitant with the maximal emission of floral scent. Previously, the floral scent production was detected mainly from 10 am to 4 pm for P. bellina. It is possible that the floral scent emission was either light or temperature regulated. We have analyzed whether the scent emission is circadian rhythm or light regulated by either under long-day (16 h/8 h light/dark) or continuous darkness conditions for 4 days since D+4. We found that the emission of orchid floral scent is light-regulated rather than circadian rhythm regulated. In addition, transcriptomic analysis of P. bellina flowers at consecutive stages (from the blooming day to D+7) showed that several photoreceptor genes displayed concurrent expression patterns as that of PbGDPS_SSU, including phytochrome (PHY), cryptochrome (CRY), phototropin (PHOT), and UVBR. Transcription of these unigenes was then verified by using quantitative real-time RT-PCR. Two blue light photoreceptors, PbCRY3 and PbPHOT1, showed similar expression patterns with PbGDPS_SSU. To investigate the role of blue light on floral scent emission in Phalaenopsis, the scent metabolic profiles of two native Phalaenopsis species and two commercial cultivars were analyzed by treating plants with various light factors. The emission levels of linalool and geraniol were higher in these plants under long day condition (white light 100 μmol m-2s-1, 16 h/8 h L/D) than those under far-red light or darkness. In addition, high intensity of blue light (270 μmol m-2s-1) showed a negative effect on the emission of linalool and geraniol in two native species, with more effect on linalool than geraniol. Taken together, these results indicate that light has played a positive effect on orchid floral scent emission, and the effect of light regulation on linalool and geraniol emission might be different.

Table of content

中文摘要 Ⅰ
ABSTRACT ⅠⅠ
誌謝..............................................................................................................................ⅠⅠⅠ
List of table.................................................................................................................Ⅵ
List of figure………………………………………………………….……………...Ⅶ
List of supplemental………………………………………………………………...Ⅷ
1. INTRODUCTION 1
1.1Phalaenopsis scent species 1
1.2 The economic value of orchids in Taiwan 1
1.3 Plant volatile compound and function: attract pollinator and defense enemy 1
1.3.1 Fragrance compound in plants 2
1.3.2 Fragrance compound in Phalaenopsis bellina 2
1.3.3 Biosynthesis of terpenoids 2
1.4 Environmental factors affect plant growth condition 3
1.4.1 Light impact on plants 3
1.4.2 Different kinds of photoreceptors and functions in plants 4
1.5 PURPOSE 5
2. MATERIAL and METHODS 6
2.1 Plant materials 6
2.2 Light experiment condition 6
2.3 Head-space extraction and GC-MS analysis 6
2.4 RNA extraction 7
2.5 cDNA transcription 7
2.6 Primer design 7
2.7 Real time quantitative RT-PCR 8
3. RESULT 9
3.1 Collection and analysis of Phalaenopsis scent flower 9
3.2 Effects of light on P. violacea scent emission 9
3.3 Effect of different light wavelength on monoterpene emission 10
3.3.1 Native species: P. bellina and P. violacea 10
3.3.2 Cultivars: P. Meidarland Bellina Age ‘LM128’ and P. I-Hsin Venus ‘KHM2212’ 10
3.3.3 Relationship between scent and photoreceptors expression 11
4. DISCUSSION 12
4.1 Effect of light and circadian rhythm on the emission of monoterpene in petunia, snapdragon and agarwood 12
4.2 P. I-Hsin Venus volatile and GDPS_SSU 12
4.3 The expression of Photoreceptors correlated with the floral scent emission 12
4.4 The effect of blue light on emitted linalool and geraniol 13
5. CONCLUSION 14
REFERENCES...........................................................................................................15
Table 18
Figure 19
Supplemental 30































List of table

Table 1. primer used in this study 18

































List of Figure

Figure 1. Emission of linalool and geraniol from P. violacea flowers exposed to different Light/Dark conditions 19

Figure 2. Emission of linalool and geraniol from P. violacea flowers exposed to different Light/Dark conditions 20

Figure 3. Linalool and geraniol emission in P. violacea after different light treatment……………………………………………………………………………...21

Figure 4. Linalool and geraniol emission in different light wavelength 22

Figure 5. Monoterpene emitting of native scent Phalaenopsis, P. violacea (a,b) and P. bellina (c,d) 23

Figure 6. Monoterpene emission in hybrid P. Meidarland Bellina Age ‘LM128’ 24

Figure 7. Phylogeny tree of P.bellina photoreceptors related genes 25

Figure 8. RNA sequencing pattern of putative P.bellina photoreceptors. (from Yu-Chen Chuang) 26

Figure 9. Expression pattern of Photoreceptors in P. bellina. 27

Figure 10. Expression pattern of scent enzyme GDPS_SSU, photoreceptor CRY3, PHOT_1 and UVR8 28

Figure 11. Linalool emission in different light wavelength 29








List of supplemental

Supplemental 1. Putative metabolic pathway of P. bellina (Hsiao et al., 2006) 30

Supplemetal 2. Transcripition level and scent emission of P. bellina (Hsiao et al., 2008)………………………………………………………………………………….31

Supplemental 3. Plant material of Phalaenopsis scent flowers 32

Supplemental 4. P. I-Hsin Venus ‘KHM2212’ pedigree chart 33

Supplemental 5. P. Meidarland Bellina Age ‘LM128’ pedigree chart 34

Supplemetal 6. Range of various light wavelength (nm) and their photoreceptors 35

Supplemental 7. Emission of linalool and geraniol in P. Meidarland Bellina Age ‘LM128’ after flowering 36

Supplemental 8. Emission of linalool P. I-HsinVenus in continuous flowering (from Yu-Chen Chuang) 37

Supplemental 9. Wavelength of different LED light treated (from Hipoint company) 38

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