STUDY ON THE DOWNSTREAM TARGETS OF DELLA PROTEINS IN ARABIDOPSIS THALIANA FANG LEI NATIONAL UNIVERSITY OF SINGAPORE 2010 I STUDY ON THE DOWNSTREAM TARGETS OF DELLA PROTEINS IN ARABIDOPSIS THALIANA FANG LEI (M.Sc, NJU) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2010 II ACKNOWLEGEMENTS First of all, I would like to express my sincere and profound gratitude to my supervisor, Associate Professor Yu Hao, for his excellent guidance and constant support during the whole course of my PhD. I have benefited greatly working under Prof Yu from his extensive knowledge in research and innovative ideas. Besides academic research, Prof Yu‟s immense talent, work rate and easy-going personality are also areas for me to learn from. In addition to the great help rendered to my project, Prof Yu is also concerned about my day-to-day life and is thoughtful about my future. His constant advice will be highly treasured as I leave the lab. Secondly, I would like to express my special thanks to my lab mates Xing Liang and Candy, for their kindness and patience. I thank Xing Liang for his ingenious ideas in guiding my experiments and Candy for teaching me some crucial experimental techniques and for the critical reading of my thesis. Without their help, my project would not be able to proceed as smoothly. Thirdly, I would like to give my heartful thanks to my other lab mates, Li-sha, Wanyan, Liu Chang, Liu Lu, Wang Yue, Xiao-jing, Li-hua, Zhong-hui, Tao Zhen, Wei Yan, Zhang Lei, Chow-lih, Cai-ping, Wan-ying, and all the other members in our group. Thank you all for your kind help and warm friendship! I also thank our lab manager, Chye Fong for her great efficiency in providing support to many of my experiments and Mr Ou Yang for his help in TEM and SEM. i Last but not least, I would like to thank my family, for their love, support, and confidence in me. This thesis is dedicated to them. ii TABLE OF CONTENTS Page Acknowledgements i Table of contents iii List of abbreviations vi List of tables viii List of figures ix Summary xii Chapter 1. Introduction 1.1 DELLA proteins 1.2 The SnRK1 protein complex 1.3 The ERF/AP2 transcription factor family 12 1.4 microRNAs 21 1.5 Objectives of this study 26 Chapter 2. Materials and methods 27 2.1 Plant materials and growth conditions 28 2.2 Crossing of Arabidopsis 29 2.3 Hormone and stress treatments 30 2.4 Seed germination assay 31 2.5 Measurement of flowering time 32 2.6 Transverse sections and light microscopy 32 2.7 Scanning electron microscopy (SEM) 33 2.8 Transmission electron microscopy (TEM) 33 2.9 Aniline blue staining of pollen tubes 34 iii 2.10 Genomic DNA extraction and genotyping PCR 35 2.11 RNA isolation, reverse transcription, and real-time PCR 38 2.12 Plasmid construction 38 2.13 Escherichia coli and Agrobacterium tumefaciens transformation 40 2.14 Arabidopsis transformation 41 2.15 Particle bombardment 43 2.16 Non radioactive in situ hybridization 44 2.17 Bioinformatics tools 48 2.18 Graphics software 48 Chapter 3. AtPV42a and AtPV42b redundantly regulate reproductive 49 development in Arabidopsis thaliana 3.1 Abstract 50 3.2 Results 3.21 Regulation of the expression of AtPV42a and AtPV42b 51 3.2.2 AtPV42a and AtPV42b are putative γ-type subunits of 57 the plant SnRK1 complexes 3.2.3 Expression of AtPV42a and AtPV42b in Arabidopsis 59 3.2.4 Artificial microRNA-mediated silencing of AtPV42a 60 and AtPV42b 3.2.5 amiR-atpv42b is defective in late stamen development 67 3.2.6 Female gametophytes of amiR-atpv42b are defective in 72 pollen tube reception 3.3 Discussion 79 iv Chapter 4. RAP2.6 is involved in plant responses to ABA and abiotic stresses 86 4.1 Abstract 87 4.2 Results 4.2.1 Regulation of RAP2.6 expression 88 4.2.2 The RAP2.6 protein belongs to the ERF/AP2 93 transcription factor family 4.2.3 Expression of the RAP2.6 gene in Arabidopsis 95 4.2.4 The RAP2.6 protein is localized in the nucleus 97 4.2.5 RAP2.6 is induced by ABA and abiotic stress treatments 99 4.2.6 Artificial microRNA-mediated silencing of RAP2.6 and 100 35S promoter-driven overexpression of RAP2.6 4.2.7 amiR-rap2.6 confers resistance to abiotic stresses 105 and ABA 4.2.8 amiR-rap2.6 confers PAC resistance during 111 seed germination 4.2.9 Silencing and overexpressing RAP2.6 affect the 115 expression of stress-responsive marker genes 4.2.10 amiR-rap2.6 exhibits a late flowering phenotype 123 at low temperature 4.3 Discussion 127 Chapter 5. Conclusion 134 References 136 List of publications 163 v LIST OF ABBREVIATIONS ABA abscisic acid amiRNA artificial microRNA AMPK AMP-activated protein kinase AP2 APETALA2 BSA bovine serum albumin CaMV Cauliflower mosaic virus CBF C-repeat-binding factor CBS cystathionine-β-synthase Cyc cycloheximide DAPI 4', 6-diamidino-2-phenylindole DELLAs DELLA proteins Dex dexamethasone DRE dehydration-responsive element DREB DRE-binding protein EREBPs ethylene-responsive element binding proteins ET ethylene ERF ethylene response factor GA gibberellin GFP green fluorescent protein GR glucocorticoid receptor GRAS GAI, RGA, and SCR JA jasmonic acid miRNA microRNA vi MS Murashige and Skoog PAC paclobutrazol PIFs phytochrome interacting factors piRNA piwi-interacting RNA SA salicylic acid SAIL Syngenta Arabidopsis Insertion Library SEM scanning electron microscopy siRNA short interfering RNA SNF1 Sucrose non-fermenting SnRK1 SNF1-related kinase sRNA small RNA TEM transmission electron microscopy TF transcription factor Tm melting temperature vii LIST OF TABLES Page Table 1.1 Classfication of the Arabidopsis ERF/AP2 transcription factors Table 2.1 List of primers 16 36, 37 viii responsive mitogen-activated protein kinase cascade induces the biosynthesis of ethylene in plants. 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AtPV42a and AtPV42b redundantly regulate reproductive development in Arabidopsis thaliana. (submitted manuscript) 2. Lei Fang, Hao Yu. ERFM, a downstream target of DELLAs, is involved in ABA and stress-induced responses. Joint 5th Structural Biology and Functional Genomics and 1st Biophysics International Conference, 2008, Singapore. (poster presentation) 163 [...]... Likewise, DELLA proteins also function as a converging point of the GA and ABA signalling pathways in regulating cotyledon expansion and controlling seed dormancy (Penfield et al., 2006) ABA blocks seed germination by inducing ABI5 (ABA-insensitive5), a basic domain/leucine zipper (bZIP) transcription factor (Finkelstein, 1994) RGL2 inhibits Arabidopsis seed germination by increasing ABA synthesis and... complexes in Arabidopsis and is possibly involved in plant-pathogen interactions (Gissot et al., 2006), the biological function of AKINγ- and PV42-type proteins remains unclear The conservation among γ-type subunits in fungi, mammals and plants partly lies in the four cystathionine-β-synthase (CBS) domains found in these proteins (Bateman, 1997; Polge & Thomas, 2007) The CBS domain was first discovered in the. .. members, all of which contain two copies of the AP2 domain The RAV subfamily members all contain an AP2 domain as well as the B3 domain of VP1/ABI3 transcription factors (Kagaya et al., 1999) Members belonging to the other three subfamilies are all single-AP2-domain-containing transcription factors and the majority of them (approximately 125 genes) fall into the DREB and the ERF subfamilies The DREB subfamily... subfamily (or Group A) consists of proteins with DNA binding domains most similar to the dehydration-responsive element-binding proteins (DREB) , while proteins with binding domains most similar to AtERF1 are grouped into the ERF subfamily (or Group B) (Sakuma et al., 2002; Gutterson & Reuber, 2004) On the basis of their conserved regions, the DREB and ERF subfamilies are further divided into 6 subgroups... CBS domain-containing proteins in plants So far, 48 Arabidopsis proteins have been designated as CBS domaincontaining proteins (Ignoul & Eggermont, 2005), which include γ-type subunits of the SnRK1 kinase complex 1.3 The ERF/AP2 transcription factor family ERF/AP2 proteins, which harbor one or two AP2 domains, comprise a large family of transcription factors in plants, with around 150 in Arabidopsis, ... sorting of proteins, channel gating, and ligand binding (Ignoul & Eggermont, 2005) In AMPK, the CBS motifs function as dimers to form two domains which constitute the site of fixation of the regulatory AMP or ATP molecules, suggesting that CBS domains might act as sensors of intracellular metabolites (Kemp, 2004; Scott et al., 2004) In contrast, very few information is available for the CBS domain-containing... domain (Peng et al., 1999) and a LXXLL consensus motif, which mediates the binding of steroid receptor co-activator complexes to nuclear receptors in mammals (Peng et al., 1997; Silverstone et al., 1998) The DELLA domain and C terminus of the DELLA proteins are essential for their degradation in response to GA (Silverstone et al., 2001) Deletion or specific amino acid substitutions within the conserved... downstream targets of DELLAs, AtPV42a and RAP2.6, which were involved in reproductive processes and plant adaptive growth in response to environmental stresses, respectively My findings would help to elucidate the underlying mechanisms of DELLAs function and enhance our knowledge of the GA signalling pathway xiv CHAPTER 1 Introduction 1 1.1 DELLA proteins The phytohormone gibberellins (GAs) function... are involved in plant responses to diverse abiotic and biotic stresses, it is likely that DELLAconferred plant growth restraint is an integrating response to different phytohormones and signals of adverse environment (Achard et al., 2006) The involvement of DELLAs in multiple pathways and its importance in plant development make the elucidation of the downstream mechanisms of DELLA proteins a topic of. .. degradation through interaction with SCFGID2 (Gomi et al., 2004) In barley, a tyrosine kinase inhibitor blocked the GA-induced degradation of SLN1 (Fu et al., 2002) However, more recent observations suggest that phosphorylation of DELLA proteins is not directly involved in GA-induced degradation (Itoh et al., 2005) DELLAs belong to a subfamily of the plant-specific GRAS gene superfamily of regulatory proteins, . STUDY ON THE DOWNSTREAM TARGETS OF DELLA PROTEINS IN ARABIDOPSIS THALIANA FANG LEI NATIONAL UNIVERSITY OF SINGAPORE 2010 II STUDY ON THE DOWNSTREAM TARGETS OF DELLA. development, ranging from seed germination to floral induction and pollen maturation. The nuclear-localized DELLA proteins (DELLAs), which confer plant growth restraint, are a key component in the GA. environmental signals. The involvement of DELLAs in multiple pathways and their importance in plant development, make the elucidation of their downstream mechanisms a topic of great interest. In