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Effects of a Bacterial ACC Deaminase on Plant Growth

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Effects of a Bacterial ACC Deaminase on Plant Growth Effects of a bacterial ACC deaminase on plant growth-promotion by Jennifer Claire Czarny A thesis presented to the University of Waterloo in fulfilment of the thesis requirement for the degree of Doctor of Philosophy in Biology Waterloo Ontario, Canada, 2008 c Jennifer Claire Czarny 2008 Author's declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract Plants often live in association with growth-promoting bacteria, which provide them with several benefits. One such benefit is the lowering of plant ethylene levels through the action of the bacterial enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase that cleaves the immediate biosynthetic precursor of ethylene, ACC. The plant hormone ethylene is responsible for many aspects of plant growth and development but under stressful conditions ethylene exacerbates stress symptoms. The ACC deaminase-containing bacterium Pseudomonas putida UW4, isolated from the rhizosphere of reeds, is a potent plant growth- promoting strain and as such was used, along with an ACC deaminase minus mutant of this strain, to study the role of ACC deaminase in plant growth-promotion. Also, transgenic plants expressing a bacterial ACC deaminase gene were used to study the role of this enzyme in plant growth and stress tolerance in the presence and absence of nickel. Transcriptional changes occurring within plant tissues were investigated with the use of an Arabidopsis oligonucleotide microarray. The results showed that transcription of genes involved in hormone regulation, secondary metabolism and the stress response changed in all treatments. In particular, the presence of ACC deaminase caused genes for auxin response factors to be up-regulated in plant tis- sues suggesting a de-repression of auxin signaling in the absence of high levels of ethylene. Also, transgenic plants had longer roots and grew faster than the non-transformed plants and genes involved in the stress response and secondary metabolism were up-regulated. Plants inoculated with bacteria had lower levels of secondary metabolism gene expression and slightly higher stress response gene expression than uninoculated plants. Yet, inoc- ulation with the ACC deaminase-expressing bacterium caused less up-regulation of plant genes involved in stress and defense responses and the down-regulation of genes involved in nitrogen metabolism in comparison to plants inoculated with the ACC deaminase minus mutant. Nickel stress caused the down-regulation of genes involved in photosynthesis and carbon iii fixation and the up-regulation of genes involved in stress responses, and amino acid and lipid breakdown suggesting energy starvation. When transgenic plants expressing ACC deaminase in the roots were exposed to nickel stress, plant stress symptoms were significantly lower and biomass was significantly higher suggesting that lowering the level of ethylene relieved many of the stress symptoms. In fact, genes involved in photosynthesis, secondary metabolism and nitrate assimilation were up-regulated in transgenic plants compared with non-transformed plants in the presence of nickel, suggesting that ACC deaminase is effective at reducing the severe effects of this metal stress. iv Acknowledgments I am very grateful to Dr. Bernard R. Glick for giving me an opportunity to work in his lab and for always treating me like a scientist and encouraging me to do likewise. I am also grateful to him for his guidance and compassion, both of which have helped me to thrive during my graduate studies. It takes a village to raise a child and I feel like the Department of Biology has been my village for the past several years. I'd like to say thank you to everyone who kindly provided equipment, technical support and/or their time to help me with my experiments, there are far too many of you to name here, but I am truly grateful. In particular, thank you to Dr. Marianne Hopkins who mischievously encouraged me to use microarrays to study canola gene expression and whose invaluable technical support helped to make it happen. Also, Owen Woody who performed all of the stats on the vast amount of raw microarray data, then patiently explained to me what he did. Thank you to Dr. Saleh Shah for making the transgenic plants used in this study and for his keen interest in my progress throughout the years. Thanks to Dr. Margie Gruber at Agriculture and Agri-Food Canada (Saskatoon), for providing me with Brassica EST sequences, which saved me months of work. I would like to sincerely thank my friends and family for their devotion and support, which are treasures that I'm lucky to have. Finally, I'm very grateful to my husband Pawel, whom I admire and love, with whom I share all my joy and sorrow and without whose support I wouldn't have made it. v Contents 1 Introduction 1 1.1 Ethylene . .1 1.1.1 Ethylene biosynthesis . .2 1.1.2 Ethylene perception . .5 1.1.3 Interaction between ethylene and auxin . 10 1.1.4 Stress ethylene . 10 1.2 Plant stress . 12 1.2.1 Biotic stress . 14 1.2.2 Abiotic stress . 15 1.3 Plant growth-promoting bacteria . 17 1.3.1 ACC deaminase . 18 1.3.2 Relief of plant stress by PGPR . 19 1.4 Conclusion . 21 2 Materials and Methods 22 2.1 Plant varieties . 22 2.2 Plant growth conditions . 23 2.2.1 Plate method . 23 2.2.2 Growth pouch method . 23 2.2.3 Soil method . 24 vi 2.2.4 Vermiculite method . 24 2.3 Bacterial strains . 25 2.4 Biomass of plant tissues . 25 2.5 Chlorophyll measurements . 25 2.6 Nickel accumulation . 26 2.7 RNA isolation . 26 2.8 Microarray experiments . 28 2.8.1 Experimental design . 28 2.8.2 Labeling, hybridization and scanning . 28 2.8.3 Data analysis . 30 2.9 Validation of microarray experiments . 33 2.9.1 Primer design . 35 2.9.2 Real-time RT-PCR . 38 3 Results 39 3.1 The role of ACC deaminase in PGPR-induced plant growth . 39 3.1.1 Functional categorization of expression data . 41 3.1.2 Metabolic pathways . 47 3.1.3 Gene lists . 52 3.1.4 Clustering . 52 3.1.5 Validation of microarray expression data . 56 3.2 Transgenic canola expressing bacterial ACC deaminase . 65 3.2.1 Physiological effects of the transgene . 65 3.2.2 Transcriptional changes occurring within trAcdS-rolD plants . 72 3.2.3 Functional categorization of expression data . 72 3.2.4 Metabolic pathways . 77 3.2.5 Gene lists . 78 3.3 Transgenic and non-transformed plants under nickel stress . 79 vii 3.3.1 Physiological effects of excess nickel . 79 3.3.2 Transcriptional changes occurring due to the presence of nickel . 88 3.3.3 Functional categorization of expression data . 88 3.3.4 Metabolic pathways . 94 3.3.5 Gene lists . 95 4 Discussion 97 4.1 Phytohormones . 98 4.1.1 Ethylene biosynthesis and response genes . 98 4.1.2 Auxin biosynthesis and response genes . 100 4.1.3 Jasmonic acid biosynthesis and response genes . 103 4.2 Effect of Pseudomonas putida UW4 on plant gene expression . 106 4.2.1 Role of ACC deaminase in plant growth-promotion . 107 4.2.2 Plants with a bacterial ACC deaminase transgene . 107 4.3 Effects of nickel stress . 108 4.3.1 Stress tolerance conferred by bacterial ACC deaminse . 109 4.4 Conclusions . 111 A Expression data organized by metabolic pathway 113 B MvA plots 120 C Lists of genes with significant expression changes 129 Bibliography 208 viii List of Tables 2.1 Genes chosen for validation of microarray data . 37 2.2 Reat-time RT-PCR primer sequences . 37 3.1 Summary of expression changes in shoots of plants treated with bacteria . 50 3.2 Summary of expression changes in roots of plants treated with bacteria . 51 3.3 Blastn (within Brassicaceae) results for real-time RT-PCR products . 59 A.1 Expression data organized by metabolic pathway . 113 C.1 Significant expression changes in shoots of 6 day-old seedlings treated with bacteria . 129 C.2 Significant expression changes in roots of 6 day-old seedlings treated with bacteria . 151 C.3 Significant expression changes in shoots of 6 day-old trAcdS-rolD seedlings . 156 C.4 Significant expression changes in roots of 6 day-old trAcdS-rolD seedlings . 163 C.5 Significant expression changes in shoots of 3 week-old trAcdS-rolD plants . 170 C.6 Significant expression changes in roots of 3 week-old trAcdS-rolD plants . 182 C.7 Significant expression changes in shoots of 3 week-old plants grown in nickel- spiked soil . 190 C.8 Significant expression changes in shoots of 3 week-old trAcdS-rolD plants grown in nickel-spiked soil . 203 ix C.9 Significant expression changes in roots of 3 week-old trAcdS-rolD plants grown in nickel-spiked soil . 205 x List of Figures 1.1 Methionine cycle and ethylene biosynthesis pathway . .4 1.2 Post-translational regulation of ACC synthase . .6 1.3 Ethylene perception and signal cascade . .8 1.4 Stress ethylene . 13 1.5 Model of bacterial ACC deaminase mode of action . 20 2.1 Microarray experimental design . 29 2.2 Fluorescence image of microarray slide . 31 2.3 Validation of microarray expression data using real-time RT-PCR . 34 2.4 Alignments between probes from Arabidopsis microarray and Brassica ESTs 36 3.1 Photo of inoculated and uninoculated seedlings . 40 3.2 Venn diagram of expression changes within tissues of plants treated with bacteria 43 3.3 Functional categorization of genes up-regulated in shoot tissue of plants treated with bacteria .
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