Effect of Narrow Bandwidth Infrared Radiation on Arabidopsis thaliana Growth and Gene Expression

本研究的目的，是設計一種紅外線發射器，利用其所發出的窄頻寬的紅外線去照射阿拉伯芥72小時，找出某特定波段的紅外光對阿拉伯芥發芽及生長影響。本研究使用的紅外光發射器，是依據表面電漿子原理製作，此紅外光源結構是在矽基板上鍍上鉬金屬，再鍍上銀/二氧化矽/銀三層薄膜，而最上層的銀作週期排列的孔洞，藉由鉬金屬通電流加熱，此結構便會發出窄頻寬高功率的紅外光；設計出發射波長為3, 3.5, 4, 4.5, 5μm，半高寬最細可達0.5μm，加熱到160oC時發射功率可達80mW/cm2之紅外光發射器。將阿拉伯芥經過不同波長之紅外光72小時的照射後，測量其下胚軸的長度，並使用熱休克蛋白來驗證此現象非為熱效應造成，再使用北方墨點分析法分析GASA4、CHS和RbcS這三個基因之表現，便可發現不同波段的紅外光對基因表現的影響。5.0及4.0μm的紅外光照射，對阿拉伯芥的下胚軸長度分別有9.0及13.1%的抑制，3.0、3.5及4.5μm的紅外光不影響阿拉伯芥下胚軸長度，CHS和RbcS的表現量在此實驗中出現明顯差異，顯示此二基因不只受可見光誘導也會受紅外光照射所影響。The purpose of this study is to design an infrared emitter, and utilize its narrow bandwidth infrared radiation to irradiate Arabidopsis for 72 hours, in order to identify the specific waveband of infrared radiation which may affect the seed germination and seedling development of Arabidopsis thaliana. Infrared radiation emitters used in this study are fabricated based on the principle of surface plasmon. The heat is generated by sending electric current to the molybdenum film on silicon substrate. The infrared source can be achieved by heating the triple layer structure which consists of a SiO2 layer between two Ag films on the molybdenum film. The top Ag layer is perforated by periodic holes, and the emission wavelength can be altered by changing the lattice constant and diameter of the hole arrays. In this study, plasmonic thermal emitters with different emission peak wavelengths, i.e., 3, 3.5, 4.0, 4.5, and 5.0 μm, were designed and fabricated. The smallest full width at half maximum (FWHM) could be shrunk down to 0.5μm. The highest emitting power can reach 80mW/cm2 at a temperature of 160oC. After 72 hours exposure to infrared radiation with different wavelength, the hypocotyl lengths of Arabidopsis thaliana seedlings were measured. HSP101 (heat shock protein) was used to verify that this phenomenon is not caused by thermal effects. Furthermore, Northern blot analysis is also applied to investigate the gene expression pattern of chosen genes GASA4, CHS and RbcS. Thus, different wavebands of infrared radiation affecting gene expression could be found. The infrared of wavelengths 5.0 and 4.0 μm inhibit the elongation of hypocotyl lengths by 9.0 and 13.1%, respectively. The 3.0, 3.5 and 4μm infrared irradiation do not affect the hypocotyl length of Arabidopsis thaliana. The gene expression of CHS and RbcS has significant change in this experiment, showing that the gene expressions of these two genes are not only induced by visible light but also influenced by infrared exposure.口試委員會審定書（中文）…………………………………………………..i試委員會審定書（英文）………………………………………………….ii謝…………………………………………………………………………………iiibstract (Chinese)…………………………………………………………...…ivbstract (English)……………………………………………………………….vhapter 1 Introduction…………………………………………………..1.1 Plant photo-physiology…………………………………………1.2 Purpose of this research…………………………………………3.3 Selected genes of Arabidopsis thaliana……………………5.4 Framework of the thesis…………………………………………7hapter 2 Chapter 2 The Fundamentals of Plasmonic Thermal Emitters……………………………………………………………..9.1 The fundamentals of surface plasmons………………….....9.1.1 Surface plasmons on smooth surfaces……………………..9.1.2 Surface plasmons on the surface with hole arrays…….15.1.3 Dispersion relation of surface plasmon in hexagonally ordered hole arrays…………………………………...19.2 Process flow………………………………………………………..22.2.1 Fabrication processes of metal hole arrays……………..22.2.1 Fabrication processes of plasmonic thermal emitter….24.3 Measuring Systems……………………………………………...27.3.1 Introduction of FTIR………………………………………..27.3.2 Thermal emitter chamber…………………………………..29hapter 3 Experimental Materials and Methods…………...31.1 Plant material and experimental setup……………………31.1.1 Plant material………………………………………………...31.1.2 Experimental setup………………………………………….32.2 Experiment flow………………………………………………….38.3 Student’s T-test……………………………………………………40.4 Gene expression pattern analysis………………………...…42.4.1 RNA isolation……………………………………..............…42.4.2 Northern blotting…………………………………………....43.5 The effect of temperature……………………...……………...46hapter 4 Results and discussion………….....................................47.1 Phenotype results…………...........................................................47.1.1 Infrared exposure experiment #1 (λp= 5 μm)...................47.1.2 Infrared exposure experiment #1 (λp= 4.5 μm)...............50.1.3 Infrared exposure experiment #1 (λp= 4 μm)...................53.1.4 Infrared exposure experiment #1 (λp= 3.5 μm)...............56.1.5 Infrared exposure experiment #1 (λp= 3 μm)...................59.2 Genotype results…………….........................................................63.2.1 Heat shock protein (HSP101)..............................................63.2.2 GASA4 expression..................................................................64.2.3 CHS and RbcS expression…………….…………..……….66.3 The absorption spectra of Arabidopsis………………..….67.4 Discussion…………….....................................................................71hapter 5 Conclusions.............................................................................75ppendix I – Arabidopsis seed sterilization protocol.....77ppendix II – Arabidopsis seed planting protocol.....................78ppendix III – RNA isolation protocol...........................................80ppendix IV – Northern blot analysis protocol............................82ppendix V – T-test results of experiment #1............................85ppendix VI – T-test results of experiment #2............................86ppendix VII – T-test results of experiment #3............................87ppendix VIII – T-test results of experiment #4…........................88ppendix IX – T-test results of experiment #5............................89eferences................................................................................................90igure Captionsig. 1.1…Three photosystems and corresponding absorption region of light...............2ig. 1.2…The (a) transmission spectra of filtered heat lamp and (b) emission spectra of a plasmonic thermal emitter at different temperatures…………………..5ig. 2.1… (a) SPs at the interface between a metal and a dielectric material. (b) The dispersion relation of the SP (solid line). (c) Electric filed in the direction perpendicular to the surface.........................................................................10ig. 2.2… (a) Missing momentum provided by the periodic hole arrays. (b) Top view of the periodic hole arrays in square lattice. (c) Definition of the direction of incident light.................................................................................................17ig. 2.3…The momentum conservation law of SPs on a reciprocal lattice of a square hole arrays…………………………………………………………………18ig. 2.4 ...(a) Dispersion relation of a plasmon on a metal surface with periodic hole array. (b) Charge distribution of +and － modes in a two-dimensional periodic hole array.(c) For a hexagonal-ordered hole array (a=3.3μm, d=1.8μm)……………………………………………………………………21ig. 2.5…Schematic plasmonic infrared emitter structure, (a) side view and (b) top view..............................................................................................................25ig. 2.6…The fabrication processes of a plasmonic infrared emitter..........................26ig. 2.7…The principle of Michelson interferometer………………………………..28ig. 2.8…Thermal emitter chamber, (a) inside view and (b) top view........................30ig. 3.1…Plant growth chamber..................................................................................33ig. 3.2…The transmission of three top cover materials…………….…...………….34ig. 3.3…(a) The measurement method of the infrared spatial intensity. (b) The measurement results of spatial intensity of infrared irradiation at the distance of 10 cm…………….....................................................................37ig. 3.4…Experimental flow chart...............................................................................39ig. 3.5…Three cases of data spread...........................................................................41ig. 3.6…The procedures of Northern blot analysis…………………………………45ig. 4.1…The emission spectra of the plasmonic thermal emitter at different temperatures used in experiment#1. The peak wavelength is at 5.03 μm, and FWHM is 0.52 μm…...................................................................................48ig. 4.2…The photographs showing the hypocotyls phenotype of 3-day-old Arabidopsis seedlings under (a) control (CK) and (b) 5.03μm infrared light illumination (IR)...........................................................................................49ig. 4.3…The hypocotyl length measurement results of experiment#1. The IR treated group is shorter than the control group by 9.0%...........................................50ig. 4.4…The emission spectra of the plasmonic thermal emitter at different temperatures used in experiment#2. The peak wavelength is at 4.51 μm, the FWHM is 0.25 μm………………………………………………...51ig. 4.5…The photographs showing the hypocotyls phenotype of 3-day-old Arabidopsis seedlings under (a) control (CK) and (b)4.51μm infrared light illumination (IR)….......................................................................................52ig. 4.6…The hypocotyl length measurement results of experiment#2. The IR treated group is longer than the control group by 1.1%...........................................53ig. 4.7…The emission spectra of the plasmonic thermal emitter at different temperature used in experiment#3. The peak wavelength is at 3.98μm, the FWHM is 0.57 μm………………………………………………………....54ig. 4.8…The photographs showing the hypocotyls phenotype of 3-day-old Arabidopsis seedlings under (a) control (CK) and (b)3.98μm infrared light illumination (IR)……....................................................................................55ig. 4.9…The hypocotyl length measurement results of experiment#3. The IR treated group is shorter than the control group by 13.1%.........................................56ig. 4.10...The emission spectra of the plasmonic thermal emitter at different temperatures used in experiment#4. The peak wavelength is at 3.52 μm, the FWHM is 0.65 μm........................................................................................57ig. 4.11…The photographs showing the hypocotyls phenotype of 3-day-old Arabidopsis seedlings under (a) control (CK) and (b)3.52μm infrared light illumination (IR)...........................................................................................58ig. 4.12…The hypocotyl length measurement results of experiment#4. The IR treated group is shorter than the control group by 4.0%.............................59ig. 4.13…The emission spectra of band pass filter used in experiment#5. The passed wavelength is from 2.7 μm to 3.5 μm...........................................................61ig. 4.14…The emission spectra of plasmonic thermal emitter at different temperatures used in experiment#5. The peak wavelength is at 3.22 μm, the FWHM is 0.35 μm…………………………………………………………61ig. 4.15…The photographs showing the hypocotyls phenotype of 3-day-old Arabidopsis seedlings under (a) control (CK) and (b)3.22μm infrared light illumination (IR)............................................................................................62ig. 4.16…The hypocotyl length measurement results of experiment#5. The IR treated group is almost the same as the control group..................................63ig. 4.17…RT-PCR analysis of the HSP101 expression in the control group (D), experimental group (IR) and heat shock treated group (HS). UBQ10 represents the loading control. (by Yen-Chang Chiou, Institute of Plant Biology, National Taiwan University)............................................................................64ig. 4.18…RT-PCR analysis of GASA4 expression in the seedlings transferred from the darkness to infrared treatment for various time periods. UBQ10 represents the loading control. (by Mu-Huan Wu, Institute of Plant Biology, National Taiwan University).............................................................................65ig. 4.19…Northern blot analysis of GASA4 expreesion in Arabidopsis seedlings irradiated with various wavelengths of infrared irradiation. The numbers below the blot indicate the normalized values of GASA4 expression based on the signal intensity in these six groups of samples with the darkness set as 1.....................................................................................................................65ig. 4.20…The gene expression of CHS by Northern blot. The rRNA is used as a loading control……………….......................................................................67ig. 4.21…The expression of CHS and RbcS genes. UBQ10 represents a loading control………………………………………………………………………67ig. 4.22…The transmission spectra of Arabidopsis...................................................69ig. 4.23…The reflection spectra of Arabidopsis........................................................69ig. 4.24…The absorption spectra of Arabidopsis......................................................70ig. 4.25…The hypocotyl length with different wavelength infrared treatment…….73ig. 4.26...The relative expression level of GASA4, CHS and RbcS expression under infrared exposure with different wavelength………………………………74ist of Tablesable 2.1 Conditions and purposes of the cleaning solvent.................................23able 2.2 The photolithography conditions........................................................23able 3.1 Emitted intensity of plasmonic thermal emitter with peak emission wavelength 4 μm at different temperature..........................................36 able 4.1 assignments of absorption peaks in FTIR spectra of Arabidopsis.........................................................................................70able 4.2 T-test results of hypocotyl length of Arabidopsis treated by different wavelength infrared............................................................................7