Abstract 0 Investigations into senescence and oxidative metabolism in gentian and petunia flowers A thesis Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Plant Biotechnology at the University of Canterbury by Shugai Zhang 2008 Abstract I Abstract Using gentian and petunia as the experimental systems, potential alternative post-harvest treatments for cut flowers were explored in this project. Pulsing with GA3 (1 to 100 µM) or sucrose (3%, w/v) solutions delayed the rate of senescence of flowers on cut gentian stems. The retardation of flower senescence by GA3 in both single flower and half petal systems was accompanied by a delay in petal discoloration. The delay in ion leakage increase or fresh weight loss was observed following treatment with 5 or 10 µM GA3 of the flowers at the unopen bud stage. Ultrastructural analysis showed that in the cells of the lower part of a petal around the vein region, appearance of senescence-associated features such as degradation of cell membranes, cytoplasm and organelles was faster in water control than in GA3 treatment. In particular, degeneration of chloroplasts including thylakoids and chloroplast envelope was retarded in response to GA3 treatment. In the cells of the top part of a petal, more carotenoids-containing chromoplasts were found after GA3 application than in water control. In petunia, treatment with 6% of ethanol or 0.3 mM of STS during the flower opening stage was effective to delay senescence of detached flowers. The longevity of isolated petunia petals treated with 6% ethanol was nearly twice as long as when they were held in water. Senescence-associated petal membrane damage, weight decline, ovary growth and decrease in protein and total RNA levels were counteracted in ethanol-treated petals. The accumulation of ROS, particularly superoxide and hydrogen peroxide, was also inhibited or delayed by ethanol application. Anti-senescence mechanisms, particularly the changes of oxidative / antioxidant metabolism involved in petal senescence, were investigated. In gentian, activities of AP and SOD but not POD in the GA3-treated petals were significantly higher than those of the control. In isolated petunia petals, the decreased trends of antioxidative SOD and AP Abstract II activities during senescence were apparently prevented in response to ethanol treatment although the levels of ascorbate and photo-protective carotenoids were not affected. Furthermore, by optimizing a range of critical PCR parameters such as primer combinations, cDNA concentrations and annealing temperatures, a reliable protocol has been established for quantifying the expression level of Cu-Zn SOD gene in petunia petals using SYBR Green I based real-time RT-PCR. A 228 bp gene fragment of Cu-Zn SOD was isolated from petunia (var. ‘hurrah’) using RT-PCR. It was found that the mRNA level (relative to 18S rRNA level) of Cu-Zn SOD decreased significantly after 6 days in water. However, there was about a 55-fold increase in Cu-Zn mRNA level after 6 days of ethanol treatment when compared to water-treated petals. Similarly, down- regulation of the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed during senescence of petunia petals. Increased vase life of petunia petals by ethanol treatment was correlated with promotion of GAPDH expression by a factor of about 16 on day 6. Taking together, the anti-senescence effects of GA3 and ethanol are at least partially associated with an increased efficiency of petal system utilizing ROS since the selected antioxidants were significantly maintained when compared to the corresponding values for the control. Keywords: Petunia; Gentian; Senescence; Flower; Ethanol; Gibberellic acid; Reactive oxygen species; Superoxide; Hydrogen peroxide; Antioxidant; Superoxide dismutase; Ascorbate peroxidase. Acknowledgements III Acknowledgements I would like to sincerely thank all those people who have helped out in some way with my project. To my supervisor, Dr David Leung, thank you so much for introducing me to enter the beautiful world of cut flowers and to start the interesting study of cut flower senescence. Your invaluable advice, encouragement, support, help, criticism when required and your frequently loud and happy laugh during this study will be unforgettable for the rest of my life. Thanks you too for reading all the drafts of the thesis. Many thanks to the staff of School of Biological Sciences who gave me help in various ways over these years, particularly Dave Conder for helpful and assistance with my field and glass house plants, Manfred Ingerfeld for his various patient help on all the processes of TEM work, Nicole Lauren, for technical advice, Matt Walters for photography, Graham Young and John Scoh for assistance with the computer. I never forget Max and Miriann from a gentian farm in Christchurch who kindly and freely supported on the beautiful cut gentian stems whenever needed during the course of this thesis. I would also like to acknowledge the considerable input of Jason Song from School of Biological Sciences who gave huge advice, assistance and support to the molecular part of this project particularly on real time PCR whenever required. Many thanks to lovely sandy from Biochemistry and my colleagues Seetha, Aruni, Maggrete, Ali, especially, my sisters Lily and Ying, in the Biotechnology laboratory for your friendship, helpfulness and various entertaining discussions. In particular, special thanks to my husband Junjie Xue and my lovely daughter Yiru Xue for their help, unease love and inspiration throughout this study. Without their Acknowledgements IV understanding, support and assistance both professionally and privately, this thesis would never have been finished. I can not forget the priceless love from my big family (Dad, Mum, brothers and sisters) for all their support during my time studying. Also, thanks for the financial support of doctoral scholarship from University of Canterbury during my study. And thanks to the Creator of all and His Son, thank you for letting me work with you among those beautiful flowers and the truth of flower senescence. Table of contents V Table of contents ABSTRACT........................................................................................................................I ACKNOWLEDGEMENTS ...........................................................................................III TABLE OF CONTENTS ................................................................................................ V LIST OF PLATES .......................................................................................................XIII LIST OF FIGURES .....................................................................................................XVI LIST OF TABLES.........................................................................................................XX ABBREVIATIONS......................................................................................................XXI CHAPTER ONE ............................................................................................................- 1 - INTRODUCTION........................................................................................................- 1 - 1.1 PLANTS USED IN THIS PROJECT ............................................................................... - 1 - 1.1.1 Gentian...........................................................................................................- 1 - 1.1.2 Petunia ...........................................................................................................- 2 - 1.2 CUT FLOWER INDUSTRY IN NEW ZEALAND ............................................................ - 4 - 1.3 SENESCENCE AND PROGRAMMED CELL DEATH....................................................... - 5 - 1.4 SENESCENCE: STRUCTURAL AND FUNCTIONAL CHANGES AT THE CELLULAR AND BIOCHEMICAL LEVEL.................................................................................................... - 6 - 1.4.1 DNA fragmentation and nuclear degradation ...............................................- 6 - 1.4.2 Protein degradation.......................................................................................- 7 - 1.4.3 Lipid degradation...........................................................................................- 8 - 1.4.4 Pigment and chloroplasy degradation...........................................................- 8 - 1.4.5 Membrane degradation..................................................................................- 9 - 1.4.6 Mitochondria degradation...........................................................................- 10 - 1.5 SENESCENCE: CHANGES IN OXIDATIVE METABOLISM ........................................... - 11 - 1.5.1 Generation of reactive oxygen species in plants..........................................- 11 - 1.5.2 Antioxidant defence in plants.......................................................................- 13 - Table of contents VI 1.5.3 Senescence and oxidative stress...................................................................- 14 - 1.5.4 Senescence and antioxidants........................................................................- 16 - 1.6 GENE EXPRESSION AND REGULATION ASSOCIATED WITH SENESCENCE ................ - 20 - 1.6.1 Senescence-associated genes.......................................................................- 20 - 1.6.2 The technical approach for the study of gene expression used