Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Modulations of visible light irradiance effects the photosynthetic phenotype in UV-B exposed Arabidopsis thaliana A thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology At Massey University, Palmerston North, New Zealand Briana C.W. Nelson 2015 i Abstract Photosynthesis is dependent upon energy provided by visible light from the electromagnetic spectrum. While such wavelengths of light are vital for resource assimilation to take place, we now also understand that other wavelengths of light may likely alter a plant’s photosynthetic capability, including the ultraviolet (UV) radiation spectrum. The ultraviolet spectrum includes UV-A (315nm-400nm) and UV-B radiation (280nm-315nm). UV-B light has been of particular interest in recent years as changes in the ozone has resulted in increased UV-B radiation levels reaching the Earth’s surface. Such scientific interest has resulted in many subsequent studies trying to understand how plants protect themselves against this powerful waveband. UV-B response in plants has been linked to both physiological and molecular changes in plants. That could be manipulated to protect plants against pathogens and increase crop yields. The quite recent discovery of the UV-B specific photoreceptor UVR8 showed how plants to respond to UV-B. A molecular pathway has begun to take shape for UVR8, with interactions with the transcription factors COP1 and HY5 necessary for activation. What is less understood are the subsequent interactions genes have with UVR8, to cause responses such as flavonoid accumulation and photosynthetic competency. After previous research showed an increase in photosynthetic rate in lettuce in response to UV-B radiation this study aimed to find the photosynthetic response of Arabidopsis thaliana and possibly re-create the increase. To do this the photosynthetic rate was studied under various PAR levels alongside UV-B exposure to characterise the photosynthetic response. The accumulation of photo-protective compounds was also studied to see if their accumulation affected photosynthetic responses. Three different lines were studied; Columbia-0, Landsberg erecta and uvr8-1. The uvr8-1 plants provided information on whether UVR8 is necessary for photosynthetic competency in Arabidopsis. qPCR studies of genes linked to the UVR8 pathway were also considered for their role in photosynthetic competency. The results in this thesis will show that manipulations of PAR, changes the UV-B photosynthetic response and that UVR8 is necessary for photosynthetic competency. ELIP1 and SIG5 are not mediated by UVR8 for photosynthetic competency. ELIP1 and SIG5 are possibly involved in UVR8 mediated accumulation of photo-protective compounds. ii iii Acknowledgements There are many people who have helped me to complete this thesis over the last couple of years. To my supervisor Dr Jason Wargent, thank-you for giving me that job during a summer in my undergrad degree, it started me on the path towards working with you on this thesis. I don’t think it would have happened without it. The conversations we have had helped me to understand how to go about my research, my data and the topic of UV greatly and also for the feedback as I wrote this thesis. I have learnt so much from you over the last few years, thank-you. I would also like to thank the PGU staff Steve, Lindsay and Lesley for all their help in growing my plants, as well as the technical help with things didn’t go quite right. Thanks also to Chris Rawlingson for helping when the IRGA broke down. To Dr Michael McManus for allowing me space in his lab to complete my qPCR work and to Srishti, Julia and Tina for teaching me how to do it and the rest of the C3.15 lab for their support. Thank-you Susanna Leung, who helped me to create the primers for my qPCR. I thank the JP Skipworth Scholarship (Plant Biology) for support during the second year of my thesis. I would also like to thank Rixta, you have been such an important sounding board for ideas and theories over the last year as I started to write up. Thank-you to my partner Nick, for supporting with me when I came home frustrated or happy about my work and for putting up with me when I stayed up to all hours working on different things. I want to also thank my parents, Julie and Warren, for their constant support as I have completed my study as well as my brother and sister and the rest of my family. Also thank-you to my sister, Olivia, for the grammatical checks even when you didn’t understand half the words I used. Thank-you all so much as without your help and support this would not have been a reality. iv v Table of Contents Abstract .............................................................................................................................. i Acknowledgements .......................................................................................................... iii Table of Contents .............................................................................................................. v List of Figures ..................................................................................................................viii List of Tables ..................................................................................................................... ix Abbreviations .................................................................................................................... x 1. Introduction .............................................................................................................. 1 1.1. Light in Plants ..................................................................................................... 1 1.2. Why look at UV responses in plants? ................................................................. 3 1.3. Classic Responses to UV-B .................................................................................. 6 1.3.1. Morphological responses ............................................................................ 6 1.3.2. Physiological responses............................................................................... 6 1.3.3. Known Molecular responses of genes of interest ...................................... 7 1.3.3.1. UVR8 .................................................................................................... 7 1.3.3.2. CHS ....................................................................................................... 9 1.3.3.3. ELIPs ..................................................................................................... 9 1.3.3.4. SIG5 ...................................................................................................... 9 1.3.3.5. CP12 ................................................................................................... 10 1.4. Effects of PAR on UV-B responses .................................................................... 10 1.5. Project and Aims ............................................................................................... 10 2. Materials and Methods ........................................................................................... 13 2.1. Plant Material ....................................................................................................... 13 2.2. Growing Environments ........................................................................................ 13 2.2.1. Nursery Set-up .............................................................................................. 13 2.2.2. Growth Chamber Set-up ............................................................................... 13 vi 2.3. Experimental Conditions .................................................................................. 15 2.4. Planting Method ............................................................................................... 17 2.5. Physiological Measurements ............................................................................ 17 2.5.1. Photosynthetic Measurements ................................................................. 17 2.5.2. Leaf metabolism and Secondary metabolite measurements ................... 18 2.5.3. Destructive Harvesting .............................................................................. 18 2.6. Molecular Measurements ................................................................................ 19 2.6.1. Plant Material Preparation ....................................................................... 19 2.6.2. Primer Design ............................................................................................ 19 2.6.3. RNA Isolation ............................................................................................. 20 2.6.3.1. RNA Quantification ............................................................................ 20 2.6.3.2. DNAse Treatment .............................................................................. 20 2.6.3.3. cDNA synthesis .................................................................................. 20 2.6.4. Polymerase Chain Reaction (PCR) ............................................................. 21 2.6.5. Gel Electrophoresis ................................................................................... 21 2.6.6. Quantitative Polymerase Chain Reaction (qPCR) ..................................... 22 2.7. Statistical Analysis ............................................................................................ 22 2.7.1. Physiological Measurements .................................................................... 22 2.7.2. qPCR statistics ........................................................................................... 23 3. Results ......................................................................................................................... 25 3.1. Question 1: Does manipulation of PAR levels before or during UV-B exposure affect photosynthetic response? ................................................................................ 25 3.2. Question 2: Does plant age affect the UV-B photosynthesis phenotype or the level of photo-protective pigmentation? ............................................................. 28 3.3. Question 3: How does pre-treatment with higher PAR affect UV-B photosynthetic response? .......................................................................................... 34 vii 3.4. Question 4: What is the role of the UVR8 photoreceptor in UV-B photosynthetic response?........................................................................................... 42 3.4.1. Physiological Measurements .................................................................... 42 3.4.2. Quantitative-PCR Results .......................................................................... 49 4. Discussion ................................................................................................................ 55 4.1. Manipulation of PAR levels does change the UV-B photosynthetic response .... 55 4.2. Age had an effect on photosynthetic response but not photo-protective pigmentation. .............................................................................................................. 56 4.3. Pre-treatment at a higher PAR lowers the initial photosynthetic rate ................ 58 4.4. UVR8 has a role in maintaining photosynthetic competency ............................. 60 4.5. ELIP1 and SIG5 do not mediate UVR8 photosynthetic response ......................... 61 4.6. Future Directions and Conclusion ........................................................................ 62 Appendices ...................................................................................................................... 65 Appendix 1 Age responses physical plant data ........................................................... 65 Appendix 2 Physical data for plants pre-treated 21DAS ............................................. 67 Appendix 3 Physical data for plants pre-treated 23DAS ............................................. 68 Appendix 4 Physical Data for Ler and uvr8-1 .............................................................. 70 4.1 Ler ...................................................................................................................... 70 4.2 uvr8-1 ................................................................................................................ 71 Appendix 5 Relative transcript levels of UVR8 and CP12-2. ....................................... 72 References ....................................................................................................................... 75 viii List of Figures Figure 1.1. Representation of light spectrum and associated photoreceptors ................ 2 Figure 1.2. Possible interactions of UV with plant morphology and crop quality outcomes ............................................................................................................... 5 Figure 1.3. Model of UVR8 mediated signalling ............................................................... 8 Figure 2.1. Set-up of Nursery room and Growth Chamber and Spectral Irradiances of environments .................................................................................................. 14 Figure 3.1. Pilot study reveals increased net photosynthetic rate with or without UV-B treatment over time................................................................................... 26 Figure 3.2. UV-B treated Arabidopsis increase in net photosynthetic rate slower than non UV-B treated plants ............................................................................. 27 Figure 3.3. UV-B treated Arabidopsis have a lower photosynthetic rate than untreated plants .................................................................................................. 27 Figure 3.4. 35 day old Arabidopsis have higher photosynthetic rate than 28 day old plants, with no differences between UV treatments ......................................... 29 Figure 3.5. Age and UV treatment does not affect chlorophyll accumulation ............... 30 Figure 3.6. Accumulation of flavonoids in Arabidopsis is more rapid in UV treated plants and in older plants after 3 days of higher PAR ......................................... 31 Figure 3.7. 35DAS UV-B treated plants had a significant increase from 35DAS no UV-B plants in anthocyanin content on Day 2. The 28DAS plants didn’t have a significant increase due to UV-B until day 6 .................................................... 32 Figure 3.8. UV treatment causes sharper decrease in NBI ............................................. 33 Figure 3.9. No difference seen in net photosynthetic rate of Arabidopsis that had a pre-treatment or UV-B exposure ........................................................................ 35 Figure 3.10. Exposure of Arabidopsis plants to higher PAR before UV-B exposure decreases net photosynthetic rate initially ......................................................... 37 Figure 3.11. Exposure of plants to higher PAR before UV-B treatment does not change chlorophyll accumulation ....................................................................... 38 Figure 3.12. UV-B exposure increased the flavonoid accumulation in the first repeat only with plants that had no pre-treatment exhibiting a greater increase than those pre-treated ......................................................................... 39 ix Figure 3.13. UV-B exposure leads to greater decrease in NBI regardless pre- exposure to high PAR .......................................................................................... 41 Figure 3.14. Early exposure of Arabidopsis Ler plants to higher PAR lowers net photosynthetic rate and no difference is seen due to UV-B treatment unlike those plants that were not pre-treated .............................................................. 43 Figure 3.15. Net photosynthetic rate of uvr8-1 Arabidopsis decreases in presence of higher PAR and UV .......................................................................................... 44 Figure 3.16. Treatments do not affect the amount of chlorophyll in wild-type Ler and uvr8-1 mutant............................................................................................... 45 Figure 3.17. Flavonoid content increases during experiment in both Ler and uvr8- ..... 47 Figure 3.18. NBI decreases in both Ler and uvr8-1 plants .............................................. 48 Figure 3.19. UV exposure causes significant increase in CHS transcripts ....................... 50 Figure 3.20. UV exposure leads to increased ELIP1 transcription in Ler and a decrease in uvr8-1. .............................................................................................. 51 Figure 3.21. UV exposure causes an increased SIG5 transcript level in Ler and decreased transcription in uvr8-1 ....................................................................... 52 List of Tables Table 2.1. Growing conditions of experiments in which data was collected ................. 16 Table 2.2. Primer sequences used for qPCR ................................................................... 19 Table 2.3. Photosynthetic measurements replicate numbers ........................................ 24 Table 2.4. Leaf metabolism and secondary metabolite replicate numbers ................... 24 x Abbreviations Amax net photosynthetic rate BLRP blue light responsive promoter bp base pairs CFCs chlorofluorocarbons CHI CHALCONE ISOMERASE CHS CHALCONE SYNTHASE Col-0 Columbia-0 COP1 CONSTITUTIVELY PHOTOMORPHOGENIC 1 CP12 CHLOROPLAST PROTEIN 12 CP12-1 CHLOROPLAST PROTEIN 12-1 CP12-2 CHLOROPLAST PROTEIN 12-2 CP12-3 CHLOROPLAST PROTEIN 12-3 CRY Cryptochrome cry1 cryptochrome1 cry2 cryptochrome2 DAS Days after sowing ELIP1 EARLY LIGHT-INDUCIBLE PROTEIN 1 ELIP2 EARLY LIGHT-INDUCIBLE PROTEIN 2 FAD Flavin Adenine Dinucleotide FLS FLAVONOL SYNTHASE FMN Flavin Mononucleotide FR far-red light GADPH glyceraldehyde-3-phosphate HY5 ELONGATED HYPOCOTYL 5 HYH HY5 HOMOLOG IRGA Infra-red gas analyser Ler Landsberg erecta LOV light, oxygen, voltage MAPK mitogen-activated protein kinases MT Metal halides MTHF methenyltetrahydrofolate MYB111 MYB DOMAIN PROTEIN 111 MYB12 MYB DOMAIN PROTEIN 12 NBI nitrogen balance index nm Nanometers PAR Photosynthetically active radiation PCR polymerase chain reaction PHR1 PHOTOLYASE 1 PHY Phytochrome PHYA phytochrome A PHYB phytochrome B PHYE phytochrome E xi PKR Phosphoribulokinase PSII photosystem II qPCR quantitative PCR R red light RFR red: far-red light ROS reactive oxygen species RUP1 REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 RUP2 REPRESSOR OF UV-B PHOTOMORPHOGENESIS 2 SE Standard error SIG5 SIGMA FACTOR 5 TRP Tryptophan UV Ultraviolet UV-A Ultraviolet-A UV-B Ultraviolet-B UVBE Biologically effective UV UV-C Ultraviolet-C UVR3 UV REPAIR DEFECTIVE 3 UVR8 UV RESISTANCE LOCUS 8 VOC volatile organic compounds WFT White fluorescent tubes WL White light WUE water use efficiency ZTL zeitlupes xii