THE REGULATORS AND BIOLOGICAL ROLES OF THE UNFOLDED PROTEIN RESPONSE IN ARABIDOPSIS THALIANA By YA-NI CHEN A DISSERTATION Submitted to Michigan State University in partial fulfillment of requirements for the degree of Plant Biology – Doctor of Philosophy 2013 ABSTRACT THE REGULATORS AND BIOLOGICAL ROLES OF THE UNFOLDED PROTEIN RESPONSE IN ARABIDOPSIS THALIANA By YA-NI CHEN The secretory pathway is fundamental for majority of cellular responses in eukaryotes. Most secretory proteins are folded and modified in the endoplasmic reticulum (ER). Thus, the ER is critical for operation of the secretory pathway. To adjust protein-folding capacity in the ER, eukaryotic cells activate intra-cellular signaling pathways termed the unfolded protein response (UPR). The UPR is triggered by stress sensors to respond the accumulation of unfolded proteins in the ER, a cellular condition referring to ER stress. To identify the plant UPR regulators, I performed mutant analyses of a conserved ER stress sensor IRE1 in Arabidopsis. I established that IRE1 is a functional ER stress sensor in plants. By showing that an ire1 mutant displays a short-root phenotype under normal growth conditions, I revealed a biological function of plant IRE1 in multicellular organisms. In addition, I found that a mutant of a component of G-protein complex, AGB1, enhances both the ER stress-sensitive and the short-root phenotype in ire1, suggesting that regulation of AGB1 on the UPR does not completely rely on IRE1. I further investigated regulatory relationship between the UPR and other cellular processes. Auxin is a major phytohormone essential for plant physiology. I found that ER stress down-regulates the transcription of auxin receptors and transporters, suggesting that ER stress represses the auxin response to coordinate stress adaption and growth regulation. By establishing that ER-localized auxin transporters and regulators are required for optimal UPR activation, I uncovered a previously unknown cellular function of ER-based auxin biology. The results also support the suggestion that regulation on auxin homeostasis is a plant-specific strategy to cope with ER stress. Moreover, I showed that ire1 displays a compromised rootinhibition phenotype under exogenous auxin treatment, indicating that IRE1 is required for the auxin response. The free auxin level is lower in ire1, supporting a role of IRE1 in regulation of the auxin homeostasis. I further examined the functional relationship between IRE1 and an ER-localized auxin transporter, PIN5. I found that pin5 enhances the defects of both the auxin response and the UPR activation in ire1. Together, my results have established the inter-regulation of the UPR and auxin signaling. In summary, my work has identified a conserved ER stress sensor IRE1 and previously unknown UPR mediators, ER-localized auxin regulators. By examination of the regulatory relationship between IRE1 and AGB1 or PIN5, my results contribute to important understanding of the plant UPR signaling network. I have also uncovered biological roles of plant IRE1 in primary root growth and auxin homeostasis. Thus, I have provided significant insights into regulators and physiological significance of the plant UPR. DEDICATION To my parents and my husband, Kyaw Aung, whose love and support sustained me through this journey. iv ACKNOWLEDGMENTS I would like to thank my mentor, Dr. Federica Brandizzi, for her tremendous support over the years. Federica provided me so many wonderful opportunities and trainings to become a better researcher. Right at the beginning, she gave me full trust and generous resources to explore my projects independently. Whenever I need her advices, she is always happy to help with full patience as well as passion. Because of her faith in me, I started to believe in myself. I am extremely grateful for such a valuable journey in her lab. I would also like to thank my committee, Dr. Gregg Howe, Dr. Rob Last, and Dr. ShinHan Shiu for their constructive advices and encouraging guidance. Whenever there is challenge in my research life, I always look up to them to retain my original intention to be a scientist. I especially like to thank Shin-Han and his wife, Melissa Lehti-Shiu, for their continual support and encouragement. I would like to acknowledge current and past members of the Brandizzi lab. I have learned incredibly by working with each of them. It is truly an influential and important experience for me. I especially like to thank Giovanni Stefano and Kathryn Walicki for their wonderful help in my research projects. v I am also very grateful for pursuit of my graduate study at MUS especially the Plant Research Laboratory. It is such a fantastic and cooperative research environment. I am truly honored to be a part of this community. I would like to thank all the friends and colleagues for their helpfulness and support. Most importantly, if I have conquered any challenge in this journey, it is all because I have my families and my husband, who always watch over me. I could never ever thank them enough for what they have given to me. vi TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ x   LIST OF FIGURES ......................................................................................................... xi   KEY TO ABBREVIATIONS ........................................................................................... xiii   CHAPTER 1 Literature review The unfolded protein response ....................................... 1   Introduction ................................................................................................................... 2   The ER quality control system ...................................................................................... 2   Activation of the UPR .................................................................................................... 3   ER stress sensors ......................................................................................................... 4   Biological roles of the UPR ........................................................................................... 5   Significance of the plant UPR ....................................................................................... 6   CHAPTER 2 Literature review IRE1: ER stress sensor and cell fate executor ................ 7   Introduction ................................................................................................................... 8   IRE1 signaling in cell fate determination ..................................................................... 10   Revised model of IRE1α signaling network in mammals......................................... 11   Is mammalian IRE1α the only major trigger of ER stress-induced apoptosis?........ 12   The substrate specificity of mammalian IRE1α ....................................................... 13   Plant IRE1 in ER stress response and cell fate determination ................................ 14   Shared components of the UPRosome and apoptosis ............................................... 16   IRE1 sensing mechanisms ......................................................................................... 17   Cell-type specific sensing mechanisms: the role of mammalian IRE1β .................. 18   Sensing mechanisms beyond protein-folding homeostasis ..................................... 19   Concluding remarks .................................................................................................... 20   Aims of the thesis research ......................................................................................... 21   Acknowledgements ..................................................................................................... 30   CHAPTER 3 IRE1 and AGB1 independently control two essential unfolded protein response pathways in Arabidopsis ................................................................................. 31   Abstract ....................................................................................................................... 32   Introduction ................................................................................................................. 33   Results ........................................................................................................................ 35   IRE1A and IRE1B are Essential for the Plant UPR ................................................. 35   Loss-of-Function of AGB1 Causes Sensitivity to ER Stress .................................... 37   agb1 enhances ER stress phenotype of ire1 ........................................................... 38   IRE1A and IRE1B Have a Role in Root Growth ...................................................... 40   The root growth phenotype of ire1 is associated with defects in cell elongation ..... 41   vii The expression of UPR target genes is lower in the root of ire1 agb1 .................... 42   Discussion ................................................................................................................... 43   IRE1A and IRE1B are Critical for the Plant UPR ..................................................... 43   Is bZIP60 the only AtIRE1 substrate in UPR Signaling? ......................................... 45   AGB1 has a Positive Role in Cell Survival upon ER Stress .................................... 46   Antagonistic Regulation of IRE1 and AGB1 on the Plant UPR ............................... 47   The Regulation of IRE1 and AGB1 on Root Growth ............................................... 47   Methods ...................................................................................................................... 71   Plant Materials and Growth Conditions ................................................................... 71   Tm Treatment .......................................................................................................... 71   Genotyping and Isolation of Multiple T-DNA Insertion Mutants ............................... 72   RNA Extraction and Quantitative RT–PCR (qRT–PCR) Analysis ........................... 72   Phenotypical Analyses ............................................................................................ 73   Arabidopsis Stable Transformation and Complementation ..................................... 74   Confocal Laser Scanning Microscopy ..................................................................... 74   Acknowledgements ................................................................................................. 75   CHAPTER 4 Inter-regulation of the unfolded protein response and auxin signaling ..... 76   Abstract ....................................................................................................................... 77   Introduction ................................................................................................................. 78   Results ........................................................................................................................ 80   ER Stress Alters the Expression of Auxin Regulators ............................................. 80   IRE1 is Required for the Auxin Responses and Homeostasis ................................ 83   ER-localized Auxin Regulators are Involved in UPR Activation .............................. 84   pin5 Enhances the ire1 Phenotype in Auxin Responses and UPR Activation ......... 86   Discussion ................................................................................................................... 87   Methods .................................................................................................................... 121   Plant material and growth conditions ..................................................................... 121   Tm treatment ......................................................................................................... 121   RNA extraction and quantitative RT-PCR analysis ............................................... 121   Phenotypic analysis ............................................................................................... 122   Immunoblotting and confocal microscopy analyses .............................................. 122   Free IAA analysis .................................................................................................. 123   Acknowledgements ................................................................................................... 123   CHAPTER 5 Conclusion and Future Perspectives ...................................................... 124   Plant IRE1 is a Functional ER Stress Sensor and Involved in Root Growth............. 125   Future directions .................................................................................................... 126   The Inter-regulation of UPR and Auxin Signaling ..................................................... 128   Future directions .................................................................................................... 128   The Significance of Plant UPR in Cellular Function .................................................. 130   Future directions .................................................................................................... 131   APPENDICES .............................................................................................................. 132   APPENDIX A Analysis of unfolded protein response in Arabidopsis ........................ 133   viii APPENDIX B Published manuscripts ....................................................................... 148   REFERENCES ............................................................................................................. 151   ix LIST OF TABLES Table 2.1. Interacting proteins of IRE1α ........................................................................ 28 Table 3.1. DNA primers used in this study .................................................................... 68 Table 4.1. DNA primers used in this study .................................................................. 117 Table A.1. DNA primers of UPR target genes ............................................................. 144 x LIST OF FIGURES Figure 2.1. Overview of UPR arms in eukaryotes. ........................................................ 22 Figure 2.2. IRE1α regulatory mechanisms during ER stress. ....................................... 24 Figure 2.3. Updated model of IRE1α and PERK signaling in cell fate determination during ER stress.. ........................................................................................................... 26 Figure 3.1. Genotyping of mutants of IRE1A, IRE1B, and AGB1. ................................. 49 Figure 3.2. Isolation of mutants of IRE1A, IRE1B, and AGB1. ...................................... 50 Figure 3.3. IRE1A and IRE1B are required for the plant UPR ...................................... 52 Figure 3.4. No significant differences in BiP3 induction in ire1a-4 and ire1b-2. ............ 54 Figure 3.5. Complementation of Tm sensitivity phenotype of ire1 by IRE1A or IRE1B. 55 Figure 3.6. Loss-of-function of AGB1 leads to oversensitivity of ER stress. ................. 57 Figure 3.7. agb1-3 enhances the Tm-sensitive phenotype in ire1. ............................... 59 Figure 3.8. agb1-3 enhances the short-root phenotype in ire1. .................................... 62 Figure 3.9. The elongation zone of ire1 and ire1 agb1 root is defective. ...................... 64 Figure 3.10. No significant differences in cell length in the root meristems of agb1-3, ire1, and ire1 agb1. ........................................................................................................ 66 Figure 3.11. Expression of UPR target genes is lower in the root of ire1 agb1. ............ 67 Figure 4.1. Tunicamycin induces activation of UPR target genes. ................................ 92 Figure 4.2. ER stress alters the expression of auxin regulators. ................................... 93 Figure 4.3. The transcripts of genes encoding ER-localized and nuclear proteins remain unchanged under Tm treatment. .................................................................................... 95 Figure 4.4. DTT transcriptionally activates UPR target genes and down-regulates auxin regulators. ...................................................................................................................... 96 xi Figure 4.5. IRE1 and TIR1/AFBs play fine-tuning roles in ER stress-induced downregulation of auxin regulators. ........................................................................................ 98 Figure 4.6. ire1 exhibits the compromised auxin responses. ..................................... 101 Figure 4.7. ire1 and ire1 pin5 display comparable sensitivity to JA, ACC, and ABA. .. 104 Figure 4.8. The UPR target genes were not altered under IAA or NPA treatment. ..... 106 Figure 4.9. The free auxin level is unchanged on ER stress. ...................................... 107 Figure 4.10. Mutants impaired in intracellular auxin transport display a defective UPR phenotype. ................................................................................................................... 108 Figure 4.11. pin5 enhances the auxin and ER stress response phenotype in ire1. .... 111 Figure 4.12. ire1 and ire1 pin5 display normal root density and hypocotyl elongation. ..................................................................................................................................... 114 Figure 4.13. Working model. ....................................................................................... 115 Figure A.1. Plant phenotype under tunicamycin treatments.………………………...142 Figure A.2. Vertical growth of plant seedlings…………………………………………143 xii KEY TO ABBREVIATIONS 35S Cauliflower Mosaic Virus 35S promoter ABA Abscisic acid ABRC Arabidopsis Biological Resource Center ACC 1-aminocyclopropane-1-carboxylic acid AGB1 Guanine nucleotide-binding protein subunit beta At Arabidopsis thaliana ATF4 Activating transcription factor 4 ATF6 Activating transcription factor 6 BiP Immunoglobulin binding protein bZIP Basic leucine zipper CASP2 Caspase-2 C.elegans Caenorhabditis elegans CHOP CCAAT enhancer-binding protein homology protein Col-0 Columbia-0 CRT Calreticulin C-terminus Carboxyl-terminus DNA Deoxyribonucleic acid DR5 Auxin responsive element DTT Dithiothreitol eIF2α Eukaryotic initiation factor 2α ER Endoplasmic reticulum xiii GFP Green fluorescent protein G-protein GTP-binding protein GTP Guanosine-5’-triphosphate H Hours IAA Indole-3-acetic acid IRE1 Inositol-requiring enzyme 1 JA Jasmonic acid JNK C-Jun N-terminal kinase MEFs Mouse embryonic fibroblasts MiRs MicroRNAs mRNA Messenger RNA MUC2 Mucin2 N-linked Asparagine-linked N-terminus Amine-terminus PCR Polymerase chain reaction PERK Protein kinase R-like ER kinase qRT-PCR Quantitative reverse transcriptase PCR RIDD Regulated IRE1-Dependent Decay RNA Ribonucelic acid RNase Ribonuclease SDS Sodium dodecyl sulfate SDS-PAGE SDS-polyacrylamide gel electrophoresis TIR1 Transport inhibitor response 1 xiv TXNIP Thioredoxin interacting protein UBQ Ubiquitin UPR Unfolded protein response XBP-1 X-box binding protein 1 YFP Yellow fluorescent protein xv CHAPTER 1 Literature review The unfolded protein response 1 Introduction Approximately one-third of the eukaryotic proteome is synthesized in the secretory pathway. Most secretory proteins first enter the endoplasmic reticulum (ER) for folding and maturation. The ER can sense an imbalance between demand and capacity of secretory protein synthesis. To increase protein-folding capacity in the ER, cells invoke protective signaling pathways known as the unfolded protein response (UPR) [1, 2]. The UPR is triggered by stress sensors to respond the accumulation of unfolded proteins in the ER, a cellular condition referring to ER stress. The initial response of the UPR aims to rebalance the protein-folding homeostasis in the ER. If cells fail to recover from ER stress, the UPR triggers apoptosis on irremediable ER stress [3-8]. The ER quality control system The ER is a specialized sub-cellular compartment for protein folding and modification. Because the ER has many unique features, including an oxidizing environment and abundance of protein-folding enzymes, certain important protein modifications only occur at the ER. The fidelity of protein functions relies on the precise protein modifications and conformation. Unfolded or misfolded proteins are not only nonfunctional, but they could be harmful for cells. To ensure that only properly modified and folded proteins exit from the ER, eukaryotic cells operate a robust system termed the ER quality control to scrutinize processes of protein maturation and transportation [6, 2 7, 9-12]. One major component of the ER quality control system is molecular chaperones. Molecular chaperones not only contribute to protein folding; they are also important in recognition of proteins that fail to be folded or modified. Once proteins are recognized for degradation, they are relocated back to the cytosol, tagged with ubiquitin, and degraded by an ER-associated degradation system [6, 7, 9-12]. Activation of the UPR If the ER quality control system is sufficient to support the demand of protein folding in the ER, activation status of the UPR is relatively low. By contrast, certain physiological or environmental conditions can overwhelm the ER quality control system. To rebalance the protein-folding homeostasis, eukaryotic cells activate the UPR to increase the capacity of the ER quality control system. The UPR is triggered by an overload of unfolded proteins in the ER mediated by ER stress sensors [7, 9, 10, 13-15]. ER stress sensors are ER transmembrane proteins with a stress-sensing domain facing the ER lumen and enzymatic domains facing the cytosol. When levels of unfolded proteins are relatively low at the ER, the activity of ER stress sensors is restricted through physical association with BiP, the most abundant ER-resident chaperone. Dissociation with BiP or interaction with unfolded proteins is an important mechanism to activate ER stress sensors [16]. 3 ER stress sensors Inositol-requiring enzyme 1 (IRE1) is the most conserved ER stress sensor in eukaryotes [17, 18]. IRE1 is a type I membrane protein, consisting of a stress-sensing domain at the N-terminus as well as a kinase and an endoribonuclease domain at the C-terminus. Upon sensing an accumulation of unfolded proteins in the ER, the endoribonuclease domain of IRE1 is activated through trans-autophosphorylation and oligomerization. Active IRE1 unconventionally splices an intron of a transcription factor, HAC1 in yeast [19] and XBP-1 in animals [20]. To recover protein-folding capacity in the ER, the spliced transcription factors induce the expression of UPR target genes, which encode protein involved in assisting protein folding and degrading misfolded protein [19, 21]. To manage more complex UPR signaling, animal cells adopt the IRE1, the PKRlike ER kinase (PERK), and the activating transcription factor 6 (ATF6) regulatory pathways [22]. Like IRE1, PERK and ATF6 have a luminal stress-sensing domain and a cytosolic enzymatic domain that initiates downstream responses. PERK mediates the UPR by repression of protein synthesis through phosphorylation of eukaryotic initiation factor 2α (eIF2α) [23]. ATF6 is a membrane-tethered transcription factor. To activate the UPR, ATF6 is translocated to the Golgi and its transcriptional activation domain is released from the membrane by proteolytic cleavage. The released domain enters the nucleus to regulate UPR target genes [24]. It is undetermined that PERK homologs or inhibition of protein synthesis on ER stress exist in plants. By contrast, Arabidopsis genome encodes two sequence homologs of IRE1, IRE1A (At2g17520) and IRE1B (At5g24360) [25]. It was reported that the stress-sensing domain of Arabidopsis IRE1A or IRE1B functionally replaces that of yeast IRE1 to activate the UPR in yeast [25]. Also, 4 auto-phosphorylation activity of IRE1A was shown in vitro [26]. These observations hint that plant IRE1 also regulates UPR signaling. Furthermore, bZIP17 and bZIP28 are two functional homologs of AFT6 in plants. In addition of UPR, bZIP17 and bZIP28 regulate salt stress and heat stress [27]. Biological roles of the UPR The UPR is essential for stress adaption and physiological regulation. The mammalian UPR is required for many fundamental biological processes. One of the most intensively studied fields is contribution of the UPR in professional secretory cells. In mammalian cells, individual UPR arm plays specific roles in regulation of professional secretory cells [28]. The IRE1α-XBP-1 UPR arm supports the high demand of secretory protein production when B lymphocytes differentiated into plasma cells [29]. IRE1β regulates the expression of Mucin2 (MUC2), the most prominent protein secreted from goblet cells [30]. These observations indicate the significance of mammalian IRE1dependent UPR in professional secretory cells. In addition, PERK is important for proinsulin synthesis and insulin secretion [31]. The mammalian UPR is also essential for regulation of metabolism, cardiovascular development, immune responses, and sterol homeostasis By contrast, our knowledge of the plant UPR has relatively more restricted to stress adaption [27]. Biological roles of the plant UPR are still largely unexplored. The further characterization of regulatory relationships between the plant UPR and other cellular processes will not only uncover plant-specific features of the UPR, but also reveal the evolutional significance of the UPR in multicellular organisms. 5 Significance of the plant UPR Yeast, plants, and animals share conserved components of the UPR. Compared to the UPR studies in yeast and animals, our knowledge of the plant UPR is still in its infants. Nevertheless, Arabidopsis is one of idealist model systems to enable in vivo UPR studies using intact organisms. Also, functional relationships between UPR transducers are more easily examined under physiological conditions in plants. Thus, the plant UPR research is valuable for understanding of essential regulation on the secretory pathway in eukaryotes. Moreover, organism-specific features of secretory pathway also exist in plants. Plants have evolved unique molecular mechanisms to coordinate the UPR signaling network. Thus, to gain more comprehensive understanding of the plant UPR, it is necessary to explore the molecular mechanisms and biological roles of the UPR using plants as a model system. 6 CHAPTER 2 Literature review IRE1: ER stress sensor and cell fate executor This section has been previously published in Trends in Cell Biology YA-NI CHEN and FEDERICA BRANDIZZI (2013) Trends in Cell Biology 23:2547-555 7 Introduction Cells operate a signaling network termed the unfolded protein response (UPR) to monitor protein-folding capacity in the endoplasmic reticulum (ER). IRE1 is an ER transmembrane sensor that activates the UPR to maintain ER and cellular function. Inositol-requiring enzyme (IRE1) is the only identified ER stress sensor in yeast and essential for the UPR in animals and plants [37-40] (Figure 2.1). As an ER transmembrane protein, IRE1 monitors ER homeostasis through an ER luminal stresssensing domain and triggers the UPR through a cytoplasmic kinase domain and an RNase domain [37, 38]. On ER stress, IRE1 RNase is activated through conformational change, autophosphorylation, and higher order oligomerization [41-43]. Mammalian IRE1 initiates diverse downstream signaling of the UPR either through unconventional splicing of the transcription factor Xbp-1 or and through posttranscriptional modifications via Regulated IRE1-Dependent Decay (RIDD) of multiple substrates [37, 38, 44-47]. In addition, PERK and ATF6 function as two distinct mammalian ER stress sensors to cope with complex UPR scenarios [14, 48] (Figure 2.1). Similar to IRE1, PERK and ATF6 are ER transmembrane proteins that contain an ER luminal stress-sensing domain and a cytoplasmic enzymatic domain. To prevent a further increase in proteinfolding demand in the ER, PERK transiently inhibits general protein translation through phosphorylation of eukaryotic initiation factor 2 alpha (elF2α). Phosphorylated elF2α can also selectively activate translation of mRNAs including ATF4 transcription factor to regulate UPR target genes [49]. ER stress triggers relocation of ATF6 from the ER to the Golgi where it undergoes proteolytic cleavage. The cleaved transcription factor domain of ATF6 enters the nucleus for UPR regulation [36, 50, 51] (Figure 2.1). 8 The main molecular mechanisms underlying IRE1 unconventional splicing are conserved in eukaryotes. In budding yeast, mammals, and plants, there is only one transcription factor identified as a splicing target of IRE1 (Figure 2.1). The stem-loop structure and cleavage site of the IRE1 splicing substrate are conserved among species. By contrast, RIDD appears more divergent in eukaryotes. Among yeast, RIDD operates in fission yeast Schizosaccharomyces pombe, but not in budding yeast Saccharomyces cerevisiae [52]. Intriguingly, RIDD-mediated decrease in protein-folding demand is the only identified mechanism of UPR in fission yeast [52]. Although plant RIDD may potentially degrade a significant portion of mRNAs encoding secretory proteins [53], it is undetermined whether plant RIDD processes various substrates to direct UPR outputs like mammalian RIDD. Unlike the mammalian UPR, plant PERK orthologs remain to be identified; however, two functional homologs of ATF6, bZIP28 and bZIP17, exist in plants [27] (Figure 2.1). Moreover, a component of G-protein complex, AGB1, is essential for the plant UPR [39] and an alternative G-protein-coupled receptor is involved in non-canonical UPR in Caneorhabditis elegans [54]. Due to the large number of members of the mammalian G-protein complex, its roles in the classical UPR may be more challenging to reveal. While the IRE1 and ATF6 arms are partially conserved between plants and animals, it will be interesting to establish the degree of UPR diversification between the two kingdoms. This chapter presents the latest advances and viewpoints on IRE1-dependent UPR research. I focus on the recent groundbreaking discoveries that define IRE1 as a master regulator in cell fate determination on ER stress. IRE1 was long considered as a positive regulator of cell survival. Thus, the repression of IRE1 was believed to 9 potentiate apoptosis. The recent identification of novel IRE1 regulatory events reveals that IRE1 signaling is persistent during ER stress. Namely, IRE1 can no longer be considered simply as a driving force for cell survival, but rather as an administrator/executor of cell fate determination on ER stress. Through presentation of the recent evidence establishing that IRE1 triggers diverse signaling, we delineate current IRE1-signaling models. It has also become clear that IRE1 monitors cellular homeostasis beyond protein-folding status in the ER; therefore, the functional relevance of the UPR within physiological processes will be discussed. Finally, we compare convergent and divergent features of IRE1 between plants and mammals to provide an integrated view of IRE1 in multicellular eukaryotes. IRE1 signaling in cell fate determination Life-versus-death determination is constantly scrutinized and tightly controlled. The prevalence of malfunctioning cells due to irremediable ER stress contributes to significant diseases, including cancer and diabetes. Conversely, overcommitment to cell death may result in organ damage or cell-degenerative diseases [55-59]. To reach optimal fitness under ER stress, cell fates are determined through tight coordination of adaptive and apoptotic responses [57, 60, 61]. In mammals, PERK-eIF2α-ATF4 regulates the transcription factor CHOP to activate ER stress-triggered apoptosis. In parallel, IRE1 controls cell fate determination through mitogen protein kinase JNK on ER stress [7, 14, 62, 63] (Figure 2.1). By contrast, although ER stress plays a role in programmed cell death in plants [64], very little is known about ER stress-induced cell death in plants [39, 53, 65]. Furthermore, lack of sequence homologs of most 10 mammalian apoptosis regulators in plants hints that divergent mechanisms of ER stress-induced cell death exist among organisms. Revised model of IRE1α signaling network in mammals The mammalian genome encodes two isoforms of IRE1, IRE1α and IRE1β. IRE1α is expressed ubiquitously and IRE1α knockout mice exhibit embryonic lethality. By contrast, IRE1β expression is restricted and IRE1β knockout mice are viable [30, 66]; therefore, most mammalian UPR research conducts on IRE1α. IRE1α was identified as a positive regulator of cell survival. It was believed that IRE1α signaling was terminated during irremediable ER stress to enable apoptosis [6, 9, 14, 37, 67, 68]. Nevertheless, recent studies have challenged this concept by showing that IRE1α persistently adjusts protein-folding capacity, actively directs UPR signaling, and executes cell fate determination [69, 70] (Figure 2.2). IRE1α employs splicing and RIDD to direct cell fate throughout ER stress. Despite Xbp-1 being the only identified IRE1α splicing target, numerous types of RNA are proven to be RIDD substrates [44, 69, 70]. Although the significance of RIDD targets is not completely understood, some RIDD events are critical for IRE1α-dependent cell fate determination. During the adaptive response, IRE1α conducts RIDD on mRNAs encoding ER-translocating proteins to prevent further increases in protein-folding demand in the ER [70]. To augment proteinfolding capacity, IRE1α splices the transcription factor Xbp-1 mRNA to induce the transcription of ER quality control components. If attempts to restore ER homeostasis fail, IRE1α ceases to splice Xbp-1 mRNA. Alternatively, IRE1α represses adaptive responses and activates apoptosis through RIDD [69, 70]. During the transition phase, 11 occurring between the adaptive and apoptotic response, RIDD increases ER stress intensity through degradation of selective UPR target genes including ER protein chaperone BiP [70]. Once ER stress intensity reaches its threshold, RIDD initiates apoptosis through repression of antiapoptotic pre-miRNAs [69]. Caspase-2 (CASP2) is a proapoptotic protease essential for the execution of apoptosis [71]. Upregulation of CASP2 is an indicator of apoptotic initiation. Through decay of anti-Casp2 pre-miRNAs, IRE1α activates apoptosis through upregulation of Casp2 (Figure 2.2) [69]. A close association of IRE1α activity and cell fate determination has been proposed for years [6, 9, 14, 37, 67, 68]. These findings provide direct evidence that IRE1α is a molecular switch and apoptosis executioner during ER stress [69]. It was previously proposed that the attenuation of IRE1 activity allows cells to initiate apoptosis [6, 9, 14, 37, 67, 68]. The identification of the IRE1α-Casp2 pathway elaborates an intriguing IRE1α signaling model: IRE1α-Xbp-1 is active in the adaptive phase and attenuated in the apoptotic phase. In parallel, activation of IRE1α-Casp2 event initiates cell death in the apoptotic phase (Figure 2.3). Is mammalian IRE1α the only major trigger of ER stress-induced apoptosis? IRE1α is necessary and sufficient to trigger apoptosis, whereas PERK and ATF6 are dispensable in the apoptosis activation [69]. Nonetheless, it cannot be excluded that distinct ER stress sensors may serve as major executioners of cell death in a contextspecific manner. Using chemical genetic tools, the regulatory roles of the phosphortransfer and RNase activity of IRE1α in the UPR can be examined separately. The phosphor-transfer function is dispensable for Xbp-1 mRNA splicing and upregulation of 12 CASP2 expression; however, it is required for the subsequent CASP2 cleavage and apoptosis activation, indicating that IRE1α phosphor-transfer function is essential for cell fate switch during ER stress [69, 70]. Notably, the phosphor-transfer function is mostly studied through an in vitro conditional IRE1α induction that mimics ER stress. Although this experimental system is valuable to distinguish phosphor-transfer and RNase function of IRE1α, it is important to note that ATF6 and PERK are not activated through ER stress. A potentially compromised crosstalk among the UPR arms raises a possibility that the IRE1α induction system may not completely resemble a genuinely biological scenario of ER stress. Hence, careful data interpretation from the conditional induction system and integration of in vivo analyses are necessary to determine whether IRE1α is the master trigger in ER stress-induced apoptosis. The substrate specificity of mammalian IRE1α Although the four identified IRE1α-cleaved miRNAs, miR-17, miR-34a, miR-96, and miR-125b repress the common substrate Casp2, TXNIP is another target of miR-17 [72]. TXNIP is involved in β-cell death and was selected to potentially regulate ER stress-induced apoptosis based on its rapidly elevated expression under severe ER stress. Similar to the IRE1α mutation, TXNIP mutation leads to compromised apoptosis activation, indicating that TXNIP is essential for ER stress-induced apoptosis [72, 73]. Wheras PERK-eIF2α activates TXNIP transcription. IRE1α increases TXNIP expression by degradation of miR-17. Accordingly, it is conceivable that each of four IRE1α-cleaved miRNAs may have specific substrates such as TXNIP. Based on this scenario, IRE1α may differentially degrade its individual target miRNA for fine-tuning of the UPR. 13 Another interesting feature of mammalian RIDD is that distinct substrates have a degree of sequence similarity within the cleavage site, whereas the flanking sequences of the cleavage sites are relatively divergent [69, 74]. This suggests that the cleavage mechanisms are likely to be conserved, whereas the flanking sequence determines the specificity of substrate recognition. This scenario would support the hypothesis that IRE1α adjust its RNase substrate specificity to activate diverse UPRs. The flexibility of IRE1α to target different substrates may rely on combinations of phosphorylation status, conformational changes, and physical associations with IRE1α regulators. Because alterations of IRE1α substrate specificity lead to opposite cell fates [70], further understanding of IRE1α substrate preferences will reveal how IRE1α coordinates cellular homeostasis to determine cell fate under ER stress. Currently, target switching of RIDD has been reported only in animals. Therefore, to gain a deeper understanding of evolution of the UPR in eukaryotes, further studies are needed to determine whether similar mechanisms exist in yeast and plants. Plant IRE1 in ER stress response and cell fate determination Despite the conservation of IRE1 among eukaryotes, divergent IRE1-dependent regulatory events have also been observed between plants and mammals. These evolutionarily divergent mechanisms are likely the reason for different ER stress and cell fate phenotypes observed between plants and mammals. Unlike mammalian IRE1 isoforms, the two Arabidopsis IRE1 isoforms are expressed ubiquitously with a limited tissue-specific expression pattern [26, 75]. There is no significant defect of the UPR in single mutants of Arabidopsis IRE1A or IRE1B while Arabidopsis ire1 double mutants 14 display compromised ER stress tolerance and a UPR activation phenotype [39, 40]. These observations indicate that the two Arabidopsis IRE1 homologues share partially overlapping function during the UPR. Evidence for established, dominant or specific roles of individual Arabidopsis IRE1 isoforms during the UPR and cell fate regulation need to be further elucidated. Notably, it is experimentally undetermined whether viable Arabidopsis ire1b are knockouts or partial loss-of-function mutants. Failure to recover a homozygous plant of putative IRE1B knockout hints that Arabidopsis IRE1B may be an essential gene similar to mammalian IRE1α [76]. Interestingly, although mammalian IRE1α is essential for the UPR in goblet cells, in other cell types, there is no detectable defect in UPR target gene induction in a mammalian ire1 double mutant likely due to partially overlapping function with ATF6 and PERK [30, 77]. By contrast, although two functional homologs of ATF6, bZIP28 and bZIP17, exist in Arabidopsis [27] (Figure 2.1), Arabidopsis ire1 double mutants exhibit dramatic reduction of UPR target gene activation [39, 40]. These data indicate that the UPR is partially diversified between mammals and plants. Nonetheless, similar IRE1 features are also observed between plants and mammals. For instance, ire1 and xbp1-1 mutants display differential phenotypes despite both being essential genes. Likewise, the mutant of Arabidopsis IRE1 splicing target bZIP60 shows comparable ER stress tolerance with wild type plants as opposed to Arabidopsis ire1 double mutants [39], supporting the hypothesis that the function of Arabidopsis IRE1 is not restricted to unconventional splicing like mammalian IRE1. Interestingly, mutations of IRE1 in plants and mammals lead to opposite effects in ER stress-induced cell death phenotypes [39, 40, 53]. Ire1α−/− mouse embryonic 15 fibroblasts (MEFs) exhibit a greater survival rate than Ire1α+/+ MEFs on ER stress, supporting the suggestion that mammalian IRE1 is an apoptosis executioner. By contrast, Arabidopsis ire1 double mutants display compromised ER stress tolerance, instead of a greater survival rate [39, 40]. Consistently, DNA fragmentation and ion leakage are enhanced in the Arabidopsis ire1 double mutant on ER stress [53], suggesting that plant IRE1 may not function as an apoptosis executioner like its mammalian counterpart. Nevertheless, it cannot be excluded that the differences are related to dissimilar experimental settings; mammalian UPR research is mostly conducted in cell culture, whereas intact organisms are used in plant UPR studies. Moreover, except potential roles in protein-loading reduction under ER stress [53], biological significance of the plant RIDD in cell fate determination is unknown. Further experimental validation will reveal whether plant RIDD could process multiple substrates to control cell fate decisions, similar to that seen in mammals. Shared components of the UPRosome and apoptosis IRE1α activation is tightly controlled by its interacting protein complex, termed the UPRosome [37]. Most UPRosome components are involved in apoptosis, supporting the suggestion that intense crosstalk exists between IRE1α activity and apoptosis activation (Table 2.1). Specifically, although the UPRosome comprises multiple components, loss-of-function mutation of a single component, such as PARP16, Bi-1, Aip-1, PTP-1B, NMHCIIB, Jab1, Nck1, and Ask1, is sufficient to alter IRE1α splicing activity or apoptosis activation [78-85] (Table 2.1). Moreover, IRE1α-interactor mutants displaying either elevated or declined IRE1α splicing activity can show enhanced 16 apoptosis, indicating that a precise level of activation of IRE1α splicing is important for cell survival [78-85]. This further suggests IRE1α activation is controlled by a signaling network that maintains a delicate equilibrium of adaptive and apoptotic responses. A subtle imbalance of the equilibrium could disturb cellular homeostasis and thus alter cell fate determination [78-95]. Furthermore, the observation that a single mutation of the UPRosome leads to significant defects in IRE1α signaling hints that IRE1α is differentially regulated in a context-specific manner (Table 2.1). Because UPRosome analyses are conducted under various conditions, systematic and comparable analyses of UPRosome members will connect each hub and thus give a clearer view of IRE1α signaling network. IRE1 sensing mechanisms ER stress-sensing mechanisms are intensively studied in yeast and animals [16]. The ER stress sensors are relatively more inactive through physical association with BiP, the most abundant ER-resident chaperone. Dissociation with BiP or interaction with unfolded proteins is the major trigger of IRE1 activation. Yeast IRE1 is activated through association with unfolded proteins rather than disassociation with BiP [96]; however, the physical interaction of BiP is a fine-tuning mechanism to ensure that yeast IRE1 is appropriately activated [97]. Unlike yeast IRE1, the activation mechanisms of mammalian IRE1α rely on its dissociation with BiP as opposed to a direct interaction with unfolded proteins [98]. The differences in activation mechanisms between yeast and mammalian IRE1α can be partially explained by the dissimilarity in protein structure within the sensor domain [37]. Surprisingly, a recent study revealed that mammalian 17 IRE1β tends to interact with unfolded proteins like yeast IRE1 and it is unable to associate with BiP [99]. Accordingly, it is possible that, like yeast IRE1, binding of unfolded proteins is the primary trigger of mammalian IRE1β activation. Despite intense studies in mammals and yeast, the plant IRE1 sensing mechanisms are completely undefined. Further structural and functional analyses of plant IRE1 will be instrumental in revealing ER stress-sensing mechanisms in plants. Cell-type specific sensing mechanisms: the role of mammalian IRE1β How the cellular homeostasis is maintained in a cell-type specific manner is a fundamental question of cell biology. It has been recently shown that IRE1β is essential for the UPR specifically in goblet cells [30]. In goblet cells, IRE1β is dispensable for Xbp-1 splicing and BiP induction. Instead, IRE1β mutation leads to enhance ER stress intensity evidenced by higher level of Xbp-1 splicing and BiP induction. Moreover, IRE1β−/− mice display a distended ER phenotype, potentially due to overaccumulation of Mucin2 (MUC2), the most prominent protein secreted from goblet cells. This suggests that IRE1β controls MUC2 expression in goblet cells. Thus, IRE1β mutation leads to MUC2 overload in the ER and in turns trigger ER stress [30]. RIDD was proposed to be the mechanism underlying IRE1β regulation on MUC2 levels in goblet cells [30]. The cell type-specific target of IRE1β provides a molecular explanation of how the UPR maintains a dynamic and specific secretory ability in multicellular organisms. Consistent with the notion that unfolded proteins trigger IRE1β activation, IRE1β may interact physically with certain types of unfolded protein. In the case of goblet cells, IRE1β may specifically monitor the MUC2 level in the ER and adjusts its loading into the ER 18 through RIDD. Based on this scenario, the mammalian ER stress sensors may distinguish the type of unfolded proteins accumulated in the ER and trigger differential UPR signaling. More specifically, if the unfolded proteins are dispensable for cell survival, ER stress sensors could repress the expression of unfolded proteins through RNA decay or translational repression. Conversely, if unfolded proteins are essential for cellular function, the UPR may preferentially augment the expression of chaperones to recover the production of unfolded proteins. While ER stress duration and intensity are considered major factors in the apoptosis threshold, the type of misfolded protein may be also critical for determination of UPR signaling outputs. Sensing mechanisms beyond protein-folding homeostasis Emerging evidence shows that IRE1 monitors cellular homeostasis beyond sensing unfolded protein accumulation. For instance, CRY1/CRY2-mediated circadian rhythm regulates IRE1α activity in the liver [100], suggesting that IRE1α coordinates ER function to cope with circadian-related physiological processes. These observations provide a link between the IRE1α-dependent UPR, circadian regulation, and liver metabolic processes. More importantly, because circadian rhythm has a substantial influence on UPR activation, time-course studies of the UPR will require diligent experimental design and appropriate controls to avoid biases. Recently, lipid homeostasis is proven to impact UPR activation through an unconventional sensing mechanism, because the unfolded-protein-sensing domain of IRE1α and PERK is dispensable for lipid-dependent UPR activation [101]. Together, these observations support the suggestion that the UPR perceives physiological and cellular signaling 19 beyond ER protein folding homeostasis. Although it is unclear whether plant IRE1 senses signaling beyond protein-folding capacity, an Arabidopsis ire1 double mutant displaying a root-specific phenotype under unstressed conditions hints that plant IRE1 also integrates physiological signals to maintain specific secretory activity [39]; however, this hypothesis awaits experimental validation. Concluding remarks Significant progress in defining IRE1 mechanisms has been achieved. We now know that IRE1 activities are coordinated at a systemic level to cope with dynamic secretion activity. Although in vitro experimental systems and conditional IRE1 induction approaches have made groundbreaking discoveries in basic UPR knowledge [69, 70, 102-105], we remain far from a comprehensive understanding of the UPR in intact organisms. The lethality of the mammalian IRE1α mutant represents a challenge to gaining insights into the IRE1 function in vivo. By contrast, the viability of plant IRE1 mutants enables in vivo analyses to reveal its roles in organ growth, pathogen defense, and abiotic stress responses [27, 39, 106, 107]. Moreover, with the ease of building high-order plant mutants, in vivo phenotypic analyses show that a conserved component of the G protein complex enhances Arabidopsis ire1 phenotype in both plant UPR activation and growth regulation. The study underscores the building of UPR networks in intact organisms using plants as a model system [39]. With more systematic and quantitative studies of the UPR in vivo, there are significant findings ahead that will decipher the dynamic UPR maps close to a genuinely physiological scenario. 20 Aims of the thesis research This thesis is aimed at understanding the plant UPR by characterizing plant ER stress regulators and identifying biological roles of the plant UPR. In Chapter 3, I established that Arabidopsis IREIA and IRE1B are functional ER stress sensors. I found that an ire1a ire1b (ire1) double mutant displays the ER stress-sensitive and the compromised UPR activation phenotype. To reveal regulatory relationship between IRE1 and other plant ER stress regulators, I showed that a mutant of G-protein complex component, AGB1, enhances both the ER stress sensitivity and the UPR activation phenotype in ire1. Moreover, I uncovered a role of the plant UPR in primary root growth by showing that the short-root phenotype of ire1 under normal growth condition. In Chapter 4, I further explored the regulatory interaction between the plant UPR and the auxin response. I found that ER stress regulates the transcription of auxin receptors and transporters. Also, mutants of ER-localized transporters show the compromised UPR activation phenotype, indicating that ER-based auxin transport may play a role in the plant UPR. Moreover, I provided evidence that IRE1 is required for the auxin homeostasis and signaling. Together, the results reveal the inter-regulation of the plant UPR and auxin signaling. In appendices, I provided protocols of ER stress phenotypic assays and quantification of UPR activation. 21 Figure 2.1. Overview of UPR arms in eukaryotes. 22 Figure 2.1 (cont’d) The IRE1 arm is conserved in eukaryotes. IRE1 unconventionally splices the bZIP transcription factors Xbp-1 and bZIP60 and Hac1 mRNA in mammals, plants, and yeast respectively. The spliced bZIP transcription factor enters the nucleus to regulate UPR target genes. In addition, two distinct arms mediated by PERK and ATF6 regulate the mammalian UPR. ATF6 is an ER transmembrane transcription factor. ER stress triggers the relocation of ATF6 from the ER to the Golgi apparatus, where it undergoes proteolytic cleavage. Subsequently, the transcription factor domain of ATF6 enters the nucleus to modulate transcription of UPR target genes. Two functional homologues of ATF6, bZIP17 and bZIP28, exist in plants. PERK, an ER transmembrane protein kinase is identified only in animals. On ER stress, PERK phosphorylates eukaryotic initiation factor 2 alpha (elF2α), which leads to transient inhibition of general protein translation and selective translation of the transcription factor ATF4. Under irremediable ER stress, PERK-elF2α-ATF4-CHOP and IRE1-JNK initiate apoptosis in mammals. Moreover, the beta subunit of the heterotrimeric G protein complex, AGB1, is essential for the plant UPR. Although the G protein complex is conserved in eukaryotes, its significance in the UPR is unclear in other eukaryotic organisms. Color code: blue, eukaryotes; black, mammals; green, plants; red, yeast. For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation. 23 Cell death ! Figure 2.2. IRE1α regulatory mechanisms during ER stress. 24 ! Figure 2.2 (cont’d) Mammalian IRE1α is repressed through a physical interaction with BiP when demand and capacity of protein folding is balanced in the ER. Dissociation of IRE1α from BiP due to an elevated level of unfolded protein in the ER leads to activation of IRE1α. The IRE1α-activating processes include its auto-phosphorylation, conformational change, and higher-order assembly. IRE1α directs cell fate decisions through unconventional splicing and Regulated IRE1-Dependent Decay (RIDD). To prevent increasing demand of ER protein folding, IRE1α conducts RIDD to degrade the transcripts of ERtranslocating proteins. In parallel, IRE1α unconventionally splices the transcript of Xbp-1 transcription factor. The spliced XBP-1 enters the nucleus to transcriptionally reprogram UPR target genes, including ER chaperones. Under irremediable ER stress, IRE1α ceases to splice Xbp-1 mRNA. Instead, IRE1α operates RIDD on selective UPR target genes including BiP to enhance the intensity of the ER stress. Once the ER stress intensity reaches its threshold, IRE1α represses anti-Casp2 microRNA (miR-17, miR34a, miR-96, and miR-125b) through RIDD. IRE1α-mediated degradation of anti-Casp2 miRNAs leads to activation of apoptotic initiator Casp2 and subsequently triggers mitochondrion-dependent apoptosis. 25 Transition Apoptotic response PERK Activation level of signaling Adaptive response IRE1α-Casp2 ER stress intensity IRE1α-Xbp-1 Duration of ER stress Figure 2.3. Updated model of IRE1α and PERK signaling in cell fate determination during ER stress. 26 Figure 2.3 (cont’d) UPR signaling aimed for cell survival is considered an adaptive response during ER stress. Under irremediable ER stress, UPR represses the adaptive response and triggers an apoptotic response. IRE1α and PERK are two ER stress sensors that decrease ER protein-folding demand through mRNA decay and translational inhibition, respectively. Both PERK and IRE1α signaling appear to persist throughout ER stress. IRE1α differentially triggers diverse UPRs according to need. In the adaptive phase, to increase protein-folding capacity, IRE1α-mediated Xbp-1 mRNA splicing is activated for transcriptional regulation of UPR target genes. In a transition phase between the adaptive and apoptotic responses, the signaling mediated by IRE1α-Xbp-1 is attenuated. In parallel, IRE1α increases the intensity of the ER stress through mRNA decay of selective UPR target genes, including ER chaperones. During the apoptotic phase, IRE1α-Casp2 signaling is activated to initiate cell death. 27 Table 2.1. Interacting proteins of IRE1α IRE1α Observed phenotype of loss-of-function interators mutations PARP16 Bi-1 Decreased Xbp-1 splicing / increased cell death Increased Xbp-1 splicing/ increased cell death AiP-1 Decreased Xbp-1 splicing / decreased cell death Ptp-1b Decreased Xbp-1 splicing / decreased cell death Decreased Xbp-1 splicing/ compromised IRE1α foci NMHCIIB formation Decreased Xbp-1 splicing/ impaired IRE1α Bax/Bak oligomerization Bim/Puma Decreased Xbp-1 splicing and UPR genes activation Jab1 Decreased Xbp-1 splicing and UPR genes activation Nck1 Decreased cell death Ask1 Decreased cell death/altered JNK activation 28 Table 2.1 (cont’d) IRE1α interators Observed phenotype of induction or overexpression Traf2 Altered activation of JNK pathway Jik Altered activation of JNK pathway Hsp90 Decreased IRE1α protein stability Usp14 Altered activation of ERAD SYVN1 Increased IRE1 ubiquitination and degradation Hsp72 Increased Xbp-1 splicing/ decreased cell death Rack1 Decreased phosphorylation of PP2A 29 Acknowledgements We thank Dr Danielle Loughlin for helpful comments and suggestions. They apologize to those authors whose work could not be cited owing to space constraints. This work was supported by grants from the National Institutes of Health (R01 GM101038-01), Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. DOE (DE-FG02-91ER20021), NASA (NNX12AN71G) and the National Science MCB1243792). 30 Foundation (MCB0948584 and CHAPTER 3 IRE1 and AGB1 independently control two essential unfolded protein response pathways in Arabidopsis This section has been previously published in The Plant Journal YA-NI CHEN and FEDERICA BRANDIZZI (2012) The plant journal 69:266-277 31 Abstract The endoplasmic reticulum (ER) has the ability to maintain the balance between demand for and synthesis of secretory proteins. To ensure protein-folding homeostasis in the ER, cells invoke signaling pathways known as the unfolded protein response (UPR). To initiate the UPR, yeasts largely rely on a conserved sensor, IRE1. In plants, some UPR transducers have been identified, but no functional relationship among them has yet been examined. The Arabidopsis genome encodes two IRE1 sequence homologues, IRE1A and IRE1B. Here we provide evidence that IRE1A and IRE1B have overlapping functions that essential for the plant UPR. A double mutant of IRE1A and IRE1B, ire1a ire1b (ire1), showed reduced ER stress tolerance and a compromised UPR activation phenotype. By demonstrating that ire1 has a root growth phenotype, we attribute a role to UPR transducers in organ growth regulation. We have also established that a mutant of Arabidopsis AGB1, a subunit of the ubiquitous heterotrimeric GTP-binding protein family, enhances both the ER stress and the shortroot phenotype in ire1. 32 Introduction Environmental or physiological factors that cause an accumulation of unfolded proteins in the ER lead to ER stress. To restore ER protein-folding homeostasis, eukaryotic cells invoke protective signaling pathways known as the unfolded protein response (UPR) [1, 2]. The regulatory mechanisms of the UPR have been extensively explored in yeast and metazoans. Inositol-requiring enzyme 1 (IRE1) is a highly conserved protein in eukaryotes and the major ER stress sensor in yeast [17, 18]. IRE1 is a type I membrane protein, which consists of an ER stress sensor domain at the Nterminus as well as a kinase and an endoribonuclease domains at the C-terminus. Upon sensing the accumulation of unfolded proteins in the ER, the endoribonuclease domain of IRE1 is activated through oligomerization and trans-autophosphorylation. Activated IRE1 splices the transcript of a specific transcription factor- HAC1 in yeasts [19] and XBP-1 in animals [20]. The spliced transcription factors control the expression of UPR target genes, which are involved in assisting protein folding, degrading misfolded protein, and regulating programmed cell death [19, 21]. To manage the UPR induced by various physiological or environmental conditions, animal cells use IRE1 pathway and two additional UPR regulatory pathways: the PKR-like ER Kinase (PERK) pathway and the Activating Transcription Factor 6 (ATF6) pathway [22]. Similar to IRE1, both PERK and ATF6 have a stress-sensing domain that protrudes into the ER lumen and a cytosolic region that initiates downstream responses. PERK mediates the UPR by repression of protein synthesis through phosphorylation of eukaryotic initiation factor 2α [23]. ATF6 is a membrane-tethered transcription factor that is activated by ER stress. The transcriptional activation domain of ATF6 is released from the ER membrane by 33 protease cleavage and translocated into the nucleus to regulate UPR target genes on ER stress [24]. A few conserved UPR transducers have been identified in plants. In Arabidopsis, bZIP28 is known to be a functional homolog of the mammalian ATF6. To activate the UPR, bZIP28 undergoes proteolytic release of its transcriptional activation domains to regulate expression of ER chaperones [108, 109]. The Arabidopsis genome also encodes two sequence homologues of IRE1—IRE1A (At2g17520) and IRE1B (At5g24360) [25]—that are ubiquitously expressed in plant tissues [110]. Analyses of three individual T-DNA insertion lines of IRE1A show that the induction of UPR target genes was similar between the ire1a mutants and wild-type Col-0 on ER stress [111]. Recently, it was demonstrated that the UPR target gene induction is reduced in a TDNA insertion line of IRE1B (ire1b-2) [112]. In addition, the activation of bZIP60 upon ER stress relies on its splicing by IRE1B, but not by IRE1A, suggesting that IRE1B is the only functional IRE1 homolog in plants [112]. However, it cannot be excluded that IRE1A also plays a role in the plant UPR, as the functional redundancy between IRE1A and IRE1B has not yet been test. In addition to the conserved counterparts of mammalian UPR regulators, unexpected UPR mediators have also been identified in plants. It has been shown that GTP-binding protein beta 1 (AGB1), an ER-localized heterotrimeric GTP-binding protein (G protein), is involved in the UPR in Arabidopsis [113]. G proteins are ubiquitous signaling molecules in eukaryotes [114]. In plants, AGB1 is known to be involved in vegetative and reproductive development as well as in light and oxidative stress responses [115-118]. 34 Here, we have performed functional characterization of an ire1 double mutant, providing genetic and molecular evidence showing that IRE1A and IRE1B are essential plant UPR regulators. We also show that an agb1 loss-of-function mutant enhanced the ire1 phenotype with respect to UPR activation. The functional relationship between IRE1 and AGB1 was also examined in term of the short-root phenotype of ire1. Hence, this study also sheds light on regulation of organ-specific growth by UPR transducers in plants. Results IRE1A and IRE1B are Essential for the Plant UPR To determine whether IRE1A and IRE1B control the plant UPR, we performed functional analyses by isolating loss-of-function mutants of IRE1A and IRE1B. The ire1a-4 (WISCDSLOX420D09) and ire1b-2 (SAIL_238_F07) alleles were obtained from the Arabidopsis Biological Resource Center (ABRC). Homozygous lines of ire1a-4 and ire1b-3 were confirmed by genomic polymerase chain reaction (PCR) (Figure 3.1). To determine whether ire1a-4 and ire1b-2 represent RNA null alleles, 4 specific IRE1A and IRE1B primer sets—IRE1A-N, IRE1A-C, IRE1B-N, and IRE1B-C, annealing upstream or downstream of the T-DNA insertion sites, were used in RT-PCR analyses (Figure 3.2a, b). We detected no IRE1A transcript in ire1a-4 using either primer sets, suggesting that ire1a-4 is a knock-out mutant. By contrast, the IRE1B amplicon was found to be present in ire1b-2 using the upstream primers set (IRE1B-N) while there was no IRE1B amplicon was detectable in ire1b-2 using the downstream primer set 35 (IRE1B-C). The RT-PCR results indicate that ire1b-2 is not an RNA null mutant, but it does not express the full-length IRE1B transcript. To test ER stress tolerance, ire1a-4, ire1b-2, and wild-type Col-0 were germinated on medium containing DMSO (mock control) or 25 or 50 ng/ml tunicamycin (Tm), which is a typical UPR inducer that blocks protein N-glycosylation. Because ire1a4, ire1b-2, and wild-type Col-0 displayed similar responses with respect to both ER stress tolerance and the UPR target gene induction (Figure 3.3a and 3.4), we hypothesized that the two IRE1 isoforms could compensate for each other in the single mutants. To test this possibility, we generated an ire1a ire1b (ire1) double mutant by crossing ire1a-4 and ire1b-2 (Figure 3.1 and 3.2). ire1 was over-sensitive to Tm compared with wild-type Col-0 and the single mutants (Figure 3.3a). RT-PCR analyses showed that, after 6 h Tm treatment, induction of a known UPR activation indicator, BiP3 [119], was drastically reduced in ire1 compared with wild-type Col-0 (Figure 3.3b). Quantitative real time RT-PCR (qRT-PCR) confirmed that the expression level of BiP3 in ire1 was three to four times lower than that of wild-type Col-0 over 3-day time course of Tm treatment (Figure 3.3c). Consistently, the induction of two other UPR target genes, AtERdj3A and AtERdj3B [120], was lower in ire1 than wild-type Col-0 on ER stress (Figure 3.3d). Thus, we concluded that the lower ER stress tolerance phenotype in ire1 arose from defects in UPR target gene induction. To prove that Tm-sensitive phenotype was due to loss-of-function of IRE1A and IRE1B, we complemented ire1 using the IRE1A clone under control of the native promoter (pIRE1A-IRE1A) as well as a dexamethasone (Dex)-inducible clone of either IRE1A or IRE1B. We found that IRE1A or IRE1B alone at least partially rescued the ER 36 stress-sensitive phenotype of ire1 (Figure 3.5). These data show that the Tm-sensitive phenotype of ire1 is caused by the loss-of-function mutations in IRE1A and IRE1B. Therefore, the IRE1 signaling is essential for the plant UPR, and IRE1A and IRE1B share partially overlapping functions in the plant UPR activation. Loss-of-Function of AGB1 Causes Sensitivity to ER Stress Our data support that IRE1 is essential for the plant UPR. We next aimed to get insight into the plant UPR signaling networks by examination of functional relationship between IRE1 and other plant UPR regulators. Therefore, we first compared the ER stress tolerance between mutants of known UPR components, including bZIP28, bZIP60, BiP2, and AGB1 [109, 113, 119, 121]. Among bzip28-1, bzip60-1, bip2, and agb1-3, only agb1-3 showed a Tm-sensitive phenotype. We found that the agb1-3 mutant was more sensitive to ER stress compared with wild-type Col-0 (Figure 3.6a). However, another T-DNA allele of AGB1, the agb1-2 mutant, was previously shown to be more resistant to ER stress [113]. To clarify whether different agb1 allelic mutants have similar ER stress sensitivity, we compared the ER stress tolerance between agb13 and two other null allele of AGB1, agb1-1 and agb1-2 (Figure 3.6b) [113, 115]. Similar to the agb1-3 mutant, the agb1-1 and agb1-2 mutants displayed over-sensitivity to ER stress compared with wild-type Col-0 (Figure 3.6b). Hence, we conclude that there is no substantial difference in ER stress tolerance among the agb1-1, agb1-2, and agb1-3 mutants. To clarify that the opposite ER stress tolerance phenotype of the agb1 mutants reported in here and Wang et al. (2007) was not due to use of different phenotypic assays, the higher ER stress sensitivity of agb1-3 compared with wild-type Col-0 was 37 further confirmed using the same ER stress phenotypic assay as used by Wang et al. 2007. In detail, agb 1-3 and wild-type seeds were germinated on high concentrations of Tm (300 ng/ml) for 6 days, and then transferred to normal growth medium without Tm for 10 days. Wild-type Col-0 seeds recovered from the ER stress as indicated by germination, but the agb1-3 seeds failed to survive under the same treatment (Figure 3.6c). In addition, complementation of agb1-3 by AGB1 showed that the ER stress phenotype of agb1-3 is due to the AGB1 loss-of-function (Figure 3.6d). Therefore, these data provide evidence that an AGB1 loss-of-function causes over-sensitivity to ER stress. agb1 enhances ER stress phenotype of ire1 To our knowledge, functional relationship between plant UPR transducers has not been reported yet. We have shown that both IRE1 and AGB1 are essential for the plant UPR. We further investigated regulatory relationship between IRE1 and AGB1 by generating an ire1 agb1 triple mutant. Interestingly, compared with the ire1 double mutant and the agb1-3 single mutant, the triple mutant displayed an even more sensitive phenotype to ER stress using two phenotypic assays: Tm infiltration and germination on Tm-containing medium (Figure 3.7a, b). Leaf senescence and damage appeared more severe in the ire1 agb1 triple mutant than in the ire1 double mutant at 24 days after infiltration with 15 µg/ml Tm (Figure 3.7a). When germinated on Tmcontaining medium, the ire1 agb1 triple mutant was smaller than the ire1 double mutant (Figure 3.7b). The enhanced growth defects of the ire1 agb1 triple mutant compared 38 with t the ire1 double mutant on ER stress were further visualized by induced hypocotyl elongation under dark growth conditions (Figure 3.7b). We further investigated whether the lower ER stress tolerance in the ire1 agb1 triple mutant compared with the ire1 double mutant was the result of more extensive aberrant UPR target gene induction. We compared the expression of UPR target genes, BiP3, AtERdj3A, and AtERdj3B in the agb1-3 single mutant, the ire1 double mutant, and the ire1 agb1 triple mutant over the time course Tm treatment. The qRT-PCR results showed that the three UPR target genes were induced to a lower level in the ire1 double mutant compared with wild-type Col-0, but the induction was higher in the agb1-3 single mutant compared with wild-type Col-0 (Figure 3.7c, d). These data suggest that IRE1 and AGB1 play an antagonistic role in UPR target gene induction. Furthermore, although the fold changes of UPR target gene induction were altered in the ire1 double mutant and the agb1-3 single mutant, the expression patterns of UPR target genes in the ire1 double mutant and the agb1-3 single mutant were similar to those of wild-type Col-0 over the time course of Tm treatment (Figure 3.7d). By contrast, both the expression levels and patterns of UPR target genes were severely affected in the ire1 agb1 triple mutant (Figure 3.7d). Together with the evidence that the ER stress tolerance of the ire1 agb1 triple mutant was lower than that of the ire1 double mutant and the agb1-3 single mutant (Figure 3.7a, b), these results enable us to conclude that agb1 enhances ER stress phenotype in ire1. In metazoans, regulatory relationships have been established between components belonged to independent UPR pathways. For example, although ATF6 and IRE1 are known as two distinct UPR sensors in metazoans, ATF6 activates the transcription of XBP-1, whose product is the splicing 39 substrate of IRE1 and a key regulator of the IRE1-dependent signaling arm [122]. Because the transcription of AGB1 is down-regulated upon UPR activation [113], we investigated whether the AGB1 transcript is regulated by IRE1. Hence, we compared the AGB1 expression level between wild-type Col-0 and the ire1 double mutant over a time course of Tm treatment. Consistent with earlier findings [113], the AGB1 RNA level decreased on ER stress in wild-type Col-0 (Figure 3.7e). In ire1, however, the AGB1 transcript remained unchanged over the time course of Tm treatment (Figure 3.7e). These results imply that down-regulation of the AGB1 transcript on ER stress relies on the IRE1. IRE1A and IRE1B Have a Role in Root Growth In multicellular organisms, the demand of secretory protein varies during cell differentiation and proliferation, and the UPR is required for maintenance of the ER protein-folding capability in specialized cell types or at specific developmental stages in metazoans. For example, deletion of mammalian IRE1α causes embryo lethality due to placental defects [29, 66]. To investigate whether IRE1-mediated UPR is involved in growth and development in plants, we compared plant morphology between wild-type Col-0 and ire1 through developmental stages. We found that the primary root of the ire1 double mutant was significantly shorter than wild-type Col-0 (T-test, p=3.92449E-18) (Figure 3.8a, c), but there was no visible morphological phenotype in the aerial parts (Figure 3.8b, d). These results indicate that IRE1 is involved specifically in optimal root growth in plants. In addition, consistent with the previous findings [123, 124], the agb1-3 single mutant had longer roots compared with wild-type Col-0 40 (T-test, p=0.0064) (Figure 3.8a, c); however, the primary root of the ire1 agb1 triple mutant was significantly shorter than that of the ire1 double mutant (T-test, p=0.0024) (Figure 3.8a, c). The fact that the agb1-3 mutation enhanced both the ER stress sensitivity and the root growth defects phenotype of ire1 hints the possibility that IRE1 and AGB1 independently regulate two parallel UPR pathways, and that both these two plant UPR pathways contribute to root growth. The root growth phenotype of ire1 is associated with defects in cell elongation To further explore the short-root phenotype, we visualized the root tissue anatomy by counterstaining cell walls with propidium iodide (Figure 3.9a). Four wellcharacterized growth zones are defined in the Arabidopsis root apex: meristematic zone, transition zone, elongation zone, and growth-terminating zone [125]. The length of each zone in Arabidopsis thaliana ecotype Col-0 has been determined based on their unique cellular activities: meristem, 200 µm from root cap junction (RCJ); transition zone, 200-520 µm from RCJ; elongation zone, 520-850 µm from RCJ; growthterminating zone, 850-1500 µm from RCJ [125]. In addition, the onset of fast elongation in the elongation zone is indicated by root hair initiation (RHI) in epidermal cells. The results of propidium iodide staining show that the cell number and cell size within 400 µm from the RCJ were similar between wild-type Col-0, the agb1-3 single mutant, the ire1 double mutant, and ire1 agb1 triple mutant (Figures 3.10), suggesting an absence of significant defects in distal root patterning. By contrast, in the region above 400 µm, the cell length was abnormal in the ire1 double mutant and ire1 agb1 triple mutant. In wild-type Col-0 and the agb1-3 single mutant, the length of cells gradually increased 41 toward the growth-terminating zone (Figure 3.9); however, the pattern of cell elongation was different in the ire1 double mutant and ire1 agb1 triple mutant (Figure 3.9). Cells between 400–600 µm from the RCJ were significantly longer in the ire1 double mutant and ire1 agb1 triple mutant compared with that of wild-type Col-0 (Figure 3.9b). In addition, in the elongation zone, the mean cell length of cells showing root hair initiation was only 50% in the ire1 double mutant and 40% in the ire1 agb1 triple mutant compared with that of wild-type Col-0 (Figure 3.9c). These data indicate that the shortroot phenotype of the ire1 double mutant and the ire1 agb1 triple mutant is due to a disorder in cell elongation in transition zone/elongation rather than to defects in the meristem. The data further imply that the maintenance of optimal root cell elongation relies on the IRE1- and -dependent signaling pathways. The expression of UPR target genes is lower in the root of ire1 agb1 Our results from PI staining show that the ire1 double mutant and the ire1 agb1 triple mutant displayed defects in root cell elongation specifically (Figures 3.9); such elongation is characterized by rapid cell-wall synthesis [126]. The biosynthesis and assembly of plant cell wall relies on the secretory pathway. Hence, it is possible that the IRE1- and AGB1-dependent UPR pathways may be involved in the control of secretory pathway activities to achieve optimal root cell elongation. To test this hypothesis, we compared the transcription level of UPR target genes in the root tissue between wildtype Col-0, the agb1-3 mutant, the ire1 double mutant, and the ire1 agb1 triple mutant using qRT-PCR. We found that, while only two out of ten tested UPR target genes showed lower expression in the root of ire1 double mutant compared with wild-type Col- 42 0, the transcription of seven UPR target genes was significantly reduced in the ire1 agb1 triple mutant compared with wild-type Col-0 (Figure 3.11). These data suggest that the enhanced short-root phenotype in the ire1 agb1 triple mutant compared with the ire1 double mutant is associated with a lower abundance of UPR target gene transcripts in the root of ire1 agb1 triple mutant. Discussion We have established that IRE1A and IRE1B are critical for the plant UPR by showing that the loss of function of IRE1A and IRE1B leads to oversensitivity to ER stress and alteration of UPR target gene induction. By demonstrating that agb1 enhances the ER stress phenotype in Ire1, we have also revealed a functional relationship between IRE1 and a G protein-signaling component in UPR. In addition, we have shown that IRE1 and AGB1 contribute to root growth, which is considered an elegant model system for organogenesis studies. Hence, we have also associated the UPR transducers with a new biological role in a multicellular context. IRE1A and IRE1B are Critical for the Plant UPR IRE1 is essential for growth and development in mammals: inactivation of the IRE1α gene, encoding one of the two mammalian IRE1 isoforms, leads to lethality in mouse due to severe placental dysfunction [29, 66]. In yeast, however, the knockout mutant of the single-copy IRE1 gene is viable [17, 20]. Although ire1b-2 is not lethal, it is still not clear whether IRE1B is dispensable for normal growth and development in plants. A homozygous line of a putative IRE1B T-DNA insertion mutant (SALK_018150, 43 ire1b-1) could not be isolated after selfing of a heterozygous line [111]. If the embryonic or reproductive lethality in ire1b-1 is not caused by an IRE1B locus-linked mutation, then IRE1B is an essential gene. This possibility implies that the ire1b-2 mutant is not a null allele. This hypothesis is supported by our data showing that the ire1b-2 mutant is not an RNA null mutant (Figure 3.2b). In addition, mammalian IRE1α is an essential gene [29, 66] that mediates a diverse biological responses by physical interaction through its linker and kinase domains [37]. Since the T-DNA insertion site of the ire1b-2 mutant is located at the end of the kinase domain, we propose that stress-sensor and kinase function may be preserved but RNase activity is probably abolished in ire1b-2. Hence, the truncated IRE1B protein in ire1b-2 may still be able to sense ER stress and transduce the signals through protein-protein interaction or other unknown mechanisms. In particular, the partial loss of function of IRE1B in the ire1b-2 may be sufficient to maintain the signaling responses required for growth and development. Thus, the leaky ire1b-2 mutant is viable. By contrast, the T-DNA insertion site in the ire1b-1 mutant is located close to the transmembrane domain, implying that either the membrane insertion of IRE1B or the kinase and RNase activity are affected severely enough to result in complete loss of function in the ire1b-1 mutant. As IRE1B may be essential for embryonic or reproductive development, the null allele, ire1b-1, is lethal. Although it is undetermined whether IRE1B is an essential gene, our data clearly show that UPR activation is compromised in ire1 (Figure 3.3), and both IRE1A and IRE1B are critical UPR transducers in plants. While this paper was under review, Nagashima et al. reported a similar ER stress tolerance phenotype of ire1 using another viable T-DNA insertion line of IRE1B (ire1b-3) 44 [34]. The T-DNA insertion in ire1b-3 is at the N-terminus of transmembrane, suggesting that the transmembrane and kinase domain are probably disrupted in ire1b-3. The fact that ire1b-3 is viable implies that IRE1B may not be an essential gene; however, whether ire1b-3 is an RNA null mutant is still uncertain because the transcript analysis of IRE1B in ire1b-3 has only been examined by the primer set cross the T-DNA insertion. A complementation test of ire1b-1 with IRE1B will clarify that whether IRE1B is an essential gene in plants. Is bZIP60 the only AtIRE1 substrate in UPR Signaling? It has been recently demonstrated that the activation of bZIP60 on ER stress relies on mRNA splicing by IRE1B, but not by IRE1A [112]. Unlike the ire1 double mutant, neither the ire1b-2 nor bzip60-1 mutant showed reduced ER stress tolerance phenotype compared with wild-type Col-0 (Figure 3.3a and 3.6a). This indicates that IRE1A is sufficient to compensate the absence of IRE1B-bZIP60 regulation on the plant UPR. Hence, the splicing of bZIP60 mRNA may not be the only regulatory mechanism in IRE1-dependent UPR pathway. Furthermore, because there were no detectable phenotypic changes in ER stress tolerance in the bzip60-1 mutant (Figure 3.6a), we propose that the ER stress-sensitive phenotype of ire1 is not simply caused by the defects in bZIP60 mRNA splicing. Instead, other target(s) may exist that may be recognized and activated by either IRE1A alone or by both IRE1A and IRE1B. In animals and yeast, only one splicing substrate of IRE1 has been identified and confirmed as a UPR regulator; however, the existence of multiple splicing targets of IRE1 in plants may allow them to respond to a variety of stimuli that they encounter as 45 sessile organisms. Also, we cannot exclude the possibility that IRE1 may regulate the plant UPR through protein-protein interaction or some unidentified regulatory mechanisms. Again, while this paper was under review, Nagashima et al. reported that the defect of bZIP60 mRNA splicing was only detected in the ire1 double mutant, but not the ire1a or ire1b single mutants. Although there is discrepancy in the results regarding bZIP60 mRNA splicing in single mutants of IRE1B [16, 34], the fact that bzip60-1 did not show an ER stress-sensitive phenotype comparable to ire1 supports the hypothesis that IRE1 control the plant UPR not only through the splicing of bZIP60 mRNA. AGB1 has a Positive Role in Cell Survival upon ER Stress Unlike IRE1, a well-characterized ER stress sensor in yeasts and metazoans, the molecular mechanisms of AGB1 regulation on the UPR are yet unclear. In particular, it is unknown that whether AGB1 controls the UPR through a classical G-protein signaling function, such as maintaining ion homeostasis [127]. Sustained ER stress may lead to the apoptotic pathway. Because induction of UPR target genes is higher in the agb1-3 mutant compared with that of wild-type Col-0 (Figure 3.7c, d), we propose that AGB1 monitors the induction level of UPR target genes to ensure the UPR is not overactivated, which may lead to cell apoptotic pathway. G-protein components are involved in certain fundamental cellular function, including ion homeostasis and cell proliferation, in both plants and animals [36]; however, to our knowledge, G-protein signaling pathways have not been reported to regulate the UPR in metazoans. It is possible that in AGB1 has evolved unique functions in the UPR in plants, but we cannot exclude the 46 possibility that the G-protein signaling pathways are also involved in the metazoan UPR. Uncovering roles of-G protein signaling in the metazoan UPR may be challenging due to the functional redundancy resulted from the presence of multiple copies of G-protein components in metazoans. Antagonistic Regulation of IRE1 and AGB1 on the Plant UPR Studies of genetic interactions between UPR regulators in Caenorhabditis elegans have uncovered complex functional relationships of UPR regulators [128]. Our results suggest that there are two parallel signaling pathways mediated by IRE1 or AGB1, supporting the hypothesis that multiplicity of UPR arms is advantageous for cells in response to diverse stimuli in multicellular organisms. ER protein-folding homeostasis is known to be exquisitely dynamic and to require timely and fine-tuned regulation [129]. The evidence that induction of UPR target genes is lower in the ire1 double mutant but higher in the agb1-3 single mutant compared with that of wild-type Col-0 suggests that IRE1 and AGB1 act antagonistically to maintain a proper UPR (Figure 3.7c, d). The hypothesis is further supported by the fact that the down-regulation of AGB1 transcript on ER stress seen in wild-type Col-0 is disrupted in ire1. The Regulation of IRE1 and AGB1 on Root Growth The UPR is essential for growth and development in mammals. Although we cannot exclude the possibility that IRE1A and IRE1B regulate root growth via a mechanism unrelated to the UPR, the most likely scenario is that the IRE1- and AGB1dependent UPR pathways coordinately contribute to primary root growth. The fact that 47 the ire1 agb1 triple mutant displayed enhanced phenotypes compared with the ire1 double mutant in terms of both ER stress sensitivity and root growth defects. Also, the lower transcription level of UPR target genes in the root of the ire1 agb1 triple mutant suggests that the shorter root length is partially due to compromised secretory capacity (Figure 3.11). Identification and characterization of differentially expressed genes in root tissue between wild-type Col-0 and the ire1 agb1 triple mutant will further help to determine whether the UPR contribute to root growth, and possibly define novel regulatory pathways in root growth and development. 48 e (i r 1) ag b1 e (i r 1 a ) b1 g 1b 1b i re i re l-0 e1 o-0 re1 ol-0 e1a ol-0 e1a primer i ir ir C C Co ir C 4 a- 3 b- RP+LP RP+T-DNA IRE1A primer IRE1B primer IRE1A primer IRE1B primer Figure 3.1. Genotyping of mutants of IRE1A, IRE1B, and AGB1. PCR of genomic DNA from wild-type Col-0, ire1a-4, ire1b-2, and ire1 showing the presence of azygous (wild-type Col-0) and homozygous insertion alleles of IRE1A and IRE1B. Gene-specific primers (LP and RP) for IRE1A and IRE1B were used to amplify the upper band. The lower band indicates the presence of a T-DNA insertion; it was amplified with a RP annealing in IRE1 and a T-DNA primer. Both upper and lower bands were sequenced. 49 (a) ire1a-4 IRE1A IRE1A-N IRE1A-C ire1b-2 ire1b-1 IRE1B ire1b-3 IRE1B-N agb1-1 (G->A) IRE1B-C agb1-2 agb1-3 100 bp AGB1 AGB1 (b) e (i r l Co -4 -0 e1a e1 ir ir 3 b- 1) 1b 1b ire -3 ire 1a b1 e1a re ag i ir ag b1 e (i r IRE1A-N 40 cycles IRE1A-C 40 cycles IRE1B-N 40 cycles IRE1B-C 40 cycles AGB1 35 cycles UBQ10 25 cycles Figure 3.2. Isolation of mutants of IRE1A, IRE1B, and AGB1. 50 1 a ) b1 g Figure 3.2. (cont’d) (a) Genomic structure of IRE1A, IRE1B, and AGB1. The coding regions and the UTRs are shown in black and gray rectangles, respectively. The T-DNA insertions in ire1a-4, ire1b-1, ire1b-2, ire1b-3, agb1-2 and agb1-3 are shown as open triangles; the point mutation in agb1-1 is shown with an arrow. (b) RT-PCR analyses of IRE1A, IRE1B, AGB1, and UBQ10 transcript in wild-type Col-0, ire1a-4, ire1b-2, ire1a ire1b (ire1), agb1-3, and ire1a ire1b agb1 (ire1 agb1). The primer location is shown in (a) and primer sequences are indicated in Table 3.1. 51 (a) DMSO 25 ng/ml Tm 50 ng/ml Tm a d a d a d b c b c b c (d1) Col-0 0 3 ire1 6 0 3 6 (h) BiP3 UBQ10 AtERdj3A Relative expression (b) 60 40 20 0 1 (c) 2 3 (day) (d2) AtERdj3B Col-0 2 Relative expression BiP3 Relative expression ire1 Col-0 ire1 1 0 1 2 3 (day) 4 4 2 2 00 1 Figure 3.3. IRE1A and IRE1B are required for the plant UPR. 52 ire1 Col-0 2 3 (day) Figure 3.3 (cont’d) (a) a, Wild-type Col-0; b, ire1a-4; c, ire1b-2; d, ire1 were germinated on LS medium containing DMSO, 25 ng/ml or 50 ng/ml Tm for 2 weeks. (b) RT-PCR of BiP3 transcript in 4-week-old wild-type Col-0 and ire1 seedlings after treatment with 10 µg/ml Tm for 0, 3, or 6 h in a hydroponic system. (c) qRT-PCR of BiP3 transcript in 2-week-old wild-type Col-0 and ire1 seedlings after treatment with 50 µg/ml Tm for 1, 2, or 3 days in a plate system. (d) qRT-PCR of AtERdj3A and AtERdj3B transcripts under the same condition as (c). 53 Relative expression BiP3 2 1.5 1 0.5 0 Col-0 ire1a-4 ire1b-3 ire1 Figure 3.4. No significant differences in BiP3 induction in ire1a-4 and ire1b-2. qRT-PCR of BiP3 transcript in 2-week-old wild-type Col-0, ire1a-4, ire1b-2, and ire1 after treatment with 50 µg/ml Tm for 4 h in a plate system. 54 (a) a DMSO b d c (b) a DMSO a c b a Tm b b d Tm b b d d c d d a b b a Tm + DEX b b c d c DEX (c) d c DMSO d d Tm a b c d DEX a b c a d b d b c d d Tm + DEX b a b c d Figure 3.5. Complementation of Tm sensitivity phenotype of ire1 by IRE1A or IRE1B. 55 Figure 3.5 (cont’d) (a) Complementation of ire1 by IRE1A; b, ire1 expressing pIRE1A-IRE1A#1-7; d, ire1 expressing pIRE1A-IRE1A#2-11; c, ire1; a, wild-type seeds germinated on ½ LS containing DMSO or 50 ng/ml Tm. (ire1 pIRE1A-IRE1A#1-7 and ire1 pIRE1A-IRE1A#2-11 refer to two independent homozygous lines). (b) Complementation of ire1 by Dex-inducible-IRE1A. a, ire1 expressing pDex-IRE1A#3; b, ire1 expressing pDex-IRE1A#6; c, ire1; d, wild-type seeds germinated on ½ LS containing DMSO, 30 µM Dex, 50ng/ml Tm, or 30 µM Dex+50ng g/ml Tm(ire1 pDexIRE1A#3 and ire1 pDex-IRE1A#6 refer to two independent T2-segregating lines). (c) Complementation of ire1 by Dex-inducible-IRE1B. a, ire1 expressing pDex-IRE1B#3; b, ire1 expressing pDex-IRE1B#6; c, ire1; d, wild-type seeds germinated on ½ LS containing DMSO, 30 µM Dex, 50ng/ml Tm, or 30 µM Dex+50ng /ml Tm (ire1 pDexIRE1B#3 and ire1 pDex-IRE1B#6 refer to two independent T2 segregating lines). 56 (a) DMSO (b) Tm (ng/ml ) 25 DMSO Tm (ng/ml ) 12.5 25 50 Col-0 Col-0 bzip28-1 agb1-1 bzip60-1 bip2 agb1-2 b ire1 agb1-3 agb1-3 ire1 (c) Mock Col-0 ire1 Recover from 300 (ng/ml ) Tm Col-0 ire1 agb1-3 ire1 agb1 (d) agb1-3 ire1 agb1 DMSO Col-0 agb1-3 25 ng/ ml Tm agb1-3/ 35S-YFPAGB1#3 Col-0 agb1-3/ 35S-YFP-AGB1#8 agb1-3 agb1-3/ 35S-YFPAGB1#3 agb1-3/ 35S-YFP-AGB1#8 Figure 3.6. Loss-of-function of AGB1 leads to oversensitivity of ER stress. 57 Figure 3.6 (cont’d) (a) Wild-type Col-0, bzip28-1, bzip60-1, bip2, agb1-3, and ire1 were germinated on LS medium containing DMSO, 25 ng/ml or 50 ng/ml Tm for 2 weeks. (b) Wild-type Col-0, agb1-1, agb1-2, agb1-3, and ire1 were germinated on LS medium containing DMSO, 12.5 ng/ml or 25 ng/ml Tm for 2 weeks. (c) Wild-type Col-0, agb1-3, ire1, and ire1 agb1 were germinated on ½ LS containing 300 ng/ml Tm for 6 days, and then transferred to ½ LS without Tm. The plants were photographed after 10-day recovery recovered on ½ LS without Tm. Mock control: The plants were germinated on ½ LS with DMSO for 6 days, and then transferred to ½ LS without DMSO or Tm. (d) Complementation of agb1-3 by 35S-YFP-AGB1. Wild-type Col-0, agb1-3, agb1-3 expressing 35S-YFP-AGB1 #3, and agb1-3 expressing 35S-YFP-AGB1 #8 seeds were germinated on ½ LS containing DMSO, or 50ng/ml Tm for 2 weeks (agb1-3 expressing 35S-YFP-AGB1 #3 and agb1-3 expressing 35S-YFP-AGB1 #8 refer to two independent T2-segregating lines). 58 (a) (b) c 25 ng/ml Tm (Dark) Tm 4 day b a b d b c a c a d b d b 25 ng/ml Tm Tm 2 day Tm 3 day b d a c d agb1-3 ire1 ire1 agb1 DMSO a d Col-0 a c DMSO c Figure 3.7. agb1-3 enhances the Tm-sensitive phenotype in ire1. 59 Figure 3.7 (cont’d) (c) (d1) Col-0 ire1 agb1-3 ire1 agb1 10 5 0 (e) Relative expression Relative expression BiP3 8 24 48 Col-0 0.5 1 2 3 30 20 10 4 0 4 8 24 48 72 (h) (d2) ire1 1 0 ire1 agb1 AtERdj3A 40 72 (h) AGB1 1.5 ire1 agb1-3 Relative expression Relative expression 15 4 Col-0 AtERdj3B 5 4 3 2 1 0 5 (day) 60 4 8 24 48 72 (h) Figure 3.7 (cont’d) (a) Leaves of 5-week-old wild-type Col-0, agb1-3, ire1, and ire1 agb1 were infiltrated with DMSO or 15 µg/ml Tm. (b) a, wild-type Col-0; b, agb1-3; c, ire1; and d, ire1 agb1 were germinated on LS medium containing DMSO or 25 ng/ml Tm under either normal growth or dark conditions for 10 days. (c) qRT-PCR of BiP3 transcript in 2-week-old wild-type Col-0, agb1-3, ire1 and ire1 agb1 after treatment with 50 µg/ml Tm for 4, 8, 24, 48, 72 h Tm in a plate system. (d) qRT-PCR of AtERdj3A (d1) and AtERdj3B (d2) transcripts in 2-week-old wild-type Col-0, agb1-3, ire1, and ire1 agb1 after treatment with 50 µg/ml Tm for 4, 8, 24, 48, 72 h Tm treatment in a plate system. (e) qRT-PCR of AGB1 transcript in 2-week-old wild-type Col-0 and ire1 after treatment with 50 µg/ml Tm for 1, 2, 3, 4, 5 day treatment in a plate system. 61 (a) Col-0 agb1-3 ire1 ire1 agb1 Col-0 agb1-3 ire1 ire1 agb1 (b) (c) (d) Primary root length 4 * 8 * 6 FW (mg) / plant Root length (cm) 10 * * 4 2 0 Co l -0 a 1 gb -3 Leaf fresh weight ire 1 ire g 1a b1 3 2 1 0 Co l-0 a 1 gb Figure 3.8. agb1-3 enhances the short-root phenotype in ire1. 62 -3 i re 1 i re 1 ag b1 Figure 3.8 (cont’d) (a) Wild-type Col-0, agb1-3, ire1, and ire1 agb1 were grown on LS medium for 2 weeks. (b) Macromorphology of wild-type Col-0, agb1-3, ire1, and ire1 agb1. (c) Measurement of primary root length (cm) of wild-type Col-0, agb1-3, ire1, and ire1 agb1. An asterisk indicates significant differences between agb1-3 and wild-type Col-0 or between ire1 and wild-type Col-0. The double asterisk indicates a significant difference between ire1 and ire1 agb1. (d) Fresh weight (mg) of leaves from rosettes of 2-week-old plants. 63 (a) Col-0 ire1 ire1 agb1 agb1-3 Meristem 1200- 1500 (µm) from RCJ Col-0 agb1-3 600µm ire1 400µm C ire1 agb1 0µm (RCJ) (c) Cell length between 400-600 µm * 40 30 Cell length (µm) Cell length (µm) (b) * 20 10 0 Col-0 agb1-3 ire1 ire1 agb1 Average cell length of RHI cells 60 40 * 20 0 Col-0 agb1-3 ire1 Figure 3.9. The elongation zone of ire1 and ire1 agb1 root is defective. 64 * ire1 agb1 Figure 3.9 (cont’d) (a) Confocal microscopy images of roots (longitudinal axis) of wild-type Col-0, ire1, ire1 agb1, and agb1-3 labeled with PI. '0 µm' is the position of the root cap junction (RCJ). (b) Mean cell length for wild-type Col-0 and mutants in the regions between the 400-600 µm from the RCJ. (c) Mean cell length for wild-type Col-0 and mutants in the regions showing root hair initiation (RHI). Asterisks indicate significant differences between the mutants and wild-type Col-0. 65 Cell length between 0-400 µm from RCJ Col-0 agb1-3 ire1 ire1 agb1 200-300um 300-400um 18 Cell length (µm) 12 6 0 0-100um 100-200um Figure 3.10. No significant differences in cell length in the root meristems of agb1-3, ire1, and ire1 agb1. Mean cell number for wild-type Col-0 and mutants in the regions within 400 µm from the root cap junction. 66 ire1 agb1 ire1 agb1-3 Col-0 1.4 1.2 Relative expression 1.0 * * 0.8 * * * ** * * 0.6 0.4 0.2 0.0 1 iP B /2 1 RT C 1 X CN Rd tE A j3A Rd tE A j3B Rd tE A j2A j2 Rd E At 1 B P5 PK 8I PD I6 PD I9 Figure 3.11. Expression of UPR target genes is lower in the root of ire1 agb1. qRT-PCR of BiP1/2, CRT1, CNX1, P58IPK1, PDI6, PDI9, AtERdj3A, AtERdj3B, AtERdj2A, and AtERdj2B transcripts in the root tissue of 2-week-old wild-type Col-0, agb1-3, ire1, and ire1 agb1. Asterisk indicate significant differences between ire1 and wild-type Col-0 or between ire1 agb1 and wild-type Col-0. 67 Table 3.1. DNA primers used in this study primers Sequence (5'–3') WiscDsLox420D09 RP tatctccgatccatcgttgac WiscDsLox420D09 LP caaaatcttcagtgctagcgg WiscDsLox LP aacgtccgcaat gtgttattaagttg SAIL_238_F07RP gaaggaaaacggacatccttc SAIL_238_F07LP cctctcgaacccttcaggtac SAIL-LB2 gcttcctattatatcttcccaaattaccaataca SALK_061896RP tgtgaatcctgctgtaatccc SALK_061896LP tcattagattggacaccggag LBa1 tggttcacgtagtgggccatcg AtIRE1A-N For agaccctgatttacgtcctagc AtIRE1A-N Rev ccgacaagttcctgaatttccg AtIRE1B-N For caaatttgagaccgagagcac AtIRE1B-N Rev ctagaatacagtggtcttag AtIRE1A-C For agaccctgatttacgtcctagc AtIRE1A-C Rev ccgacaagttcctgaatttccg AtIRE1B-C For caaatttgagaccgagagcac AtIRE1B-C Rev ctagaatacagtggtcttag 68 Table 3.1 (cont’d) AGB1 For gacacaccggaaaggtttattcattag AGB1 Rev caaacgcccatatctttagatttgaatc BiP3 For gtttggtttttctgactgtgcttgattttttaatg BiP3 Rev catcattgaaatacgctggaaccgtgatc UBQ10 For tcaattctctctaccgtgatcaagatgca UBQ10 Rev ggtgtcagaactctccacctcaagagta BiP3-qP For aaccgcgagcttggaaaat BiP3-qP Rev tcccctgggtgcaggaa AtERdj3A-qP For tcaagtggtggtggtttcaact AtEdj3A-qP Rev cccaccgcccatattttg AtERdj3B-qP For gaggaggcggcatgaatatg AtERdj3B-qP Rev ccatcgaacctccaccaaaa AGB1-qP For gcggcgcaaggacgta AGB1-qP Rev acaacaaaccagatccgttgct BiP1/2-qP For ccaccggccccaagag BiP1/2-qP Rev ggcgtccacttcgaatgtg PDI6-qP For cgaagtggctttgtcattcca PDI6-qP Rev gcggttgcgtccaatttt PDI9-qP For ggccctgttgaagtgactgaa PDI9-qP Rev cagcagaaccacacttcttttcc 69 Table 3.1 (cont’d) CNX1-qP For gtgtcctcgtcgccattgt CNX1-qP Rev ttgccaccaaagataagcttga CRT1-qP For gatcaagaaggaggtcccatgt CRT1-qP Rev gacggaggacgaaggtgtaca AtERdj2A-qP For tgggcttgtaggcgctctt AtERdj2A-qP Rev aacccaatagttttcctccttgtg AtERdj2B-qP For tgaaacgtcccaatggactca AtERdj2B-qP Rev cctctttgtggaaaggaaagtaagg AtP58IPK-qP For gcgttatagtgatgccctcgat AtP58IPK-qP Rev gaaagcgcagggtctgctt IPP2-qP For atttgcccatcgtcctctgt IPP2-qP Rev gagaaagcacgaaaattcggtaa attB1-AGB1+1 ggggacaagtttgtacaaaaaagcaggcttcatgtctgtctccgagctc attB2-AGB1+1132 ggggaccactttgtacaagaaagctgggtccaaatcactctcctgtgtc attB1-IRE1A-1503 ggggacaagtttgtacaaaaaagcaggcttctgactactaaaattttcaattc attB2-IRE1A+2526 ggggaccactttgtacaagaaagctgggtcttagatgatgtcgcatttgaag XhoI-IRE1B+1 ctgggtctcgagatgtggttattggccatctc SpeI-IRE1B+2646 ctgggtactagtctagaatacagtggtcttagag XhoI-IRE1A+1/F ctgggtctcgagatgccgccgagatgtcctttc XhoI-IRE1A+2526/ ctgggtctcgagttagatgatgtcgcatttgaag 70 Methods Plant Materials and Growth Conditions Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild-type control. Arabidopsis transfer-DNA insertion (T-DNA) mutants, ire1a-4 (WISCDSLOX420D09), ire1b-2 (SAIL_238_F07), agb1-3 (SALK_061896), agb1-2 (CS6535), bzip28-1 (SALK_132285), bzip60-1 (SALK_050203), bip2 (SALK_047956) and the mutant with point mutation agb1-1 (CS3976) were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus OH [109, 113, 119, 121]. The primers used for genotyping are listed in the Table S1. Surface-sterilized seeds were plated directly onto square Petri dishes containing ½ Linsmaier and Skoog's (LS) medium, 1.5% (w/v) sucrose, and 0.4% Phytagel. For the normal growth condition, plants were grown at 21°C under 16-h-light/8-h-dark conditions. Tm Treatment In a plate system, the Tm (Sigma, T7765; dissolved in DMSO) was directly added in the half-strength LS medium containing 1.5% (w/v) sucrose and 0.4% Phytagel, at the concentrations indicated in the text. Seeds were directly germinated in Tm-containing medium for observation of ER stress tolerance. To harvest tissue for UPR gene expression analysis, the seeds were germinated in half-strength LS medium without Tm for 2 weeks, and then transferred to Tm-containing medium. In a hydroponic system (Araponics), seedlings were grown in liquid medium (FloraSeries by GHE) without Tm for 4 weeks; then 10 mg/ml Tm dissolved in DMSO was added in the liquid medium. For the infiltration method, needleless syringe was used to infiltrate 15 µg/ml Tm into the 71 abaxial side of leaves. As mock control of the Tm treatment, a volume of DMSO equivalent to that of used to dissolve Tm in each method was used in the same experimental procedure. Genotyping and Isolation of Multiple T-DNA Insertion Mutants Genotyping of the T-DNA insertion mutants was accomplished by genomic DNA extraction followed by DNA amplification with T-DNA and gene-specific primers. The primers used for genotyping and phenotyping are listed in the Table S1. PCR experiments were performed in standard conditions and were carried out using 0.2 mM dNTPs, 0.2 µM primer, and 1 unit of Taq polymerase (Promega). Homozygous lines for T-DNA insertion of transgenic plants were isolated through segregation analyses on media containing the selective marker encoded in the T-DNA. Isolation of multi-allelic lines was performed through reciprocal crosses followed by genotyping of the F2 generation. RNA Extraction and Quantitative RT–PCR (qRT–PCR) Analysis Total RNA was extracted from whole seedlings using the RNeasy Plant Mini Kit (Qiagen) and treated with DNaseI (Qiagen). All samples within an experiment were reverse transcribed at the same time using High Capacity RNA-to-cDNA Master Mix Kit (ABI 4390777). A ‘‘No-RT’’ reaction, in which RNA was subjected to the same conditions of cDNA synthesis except without reverse transcriptase, was included as a negative control in all qRT–PCR assays to ensure the purity of RNA samples. Real-time qRT-PCR with SYBR green detection was performed in triplicate using the Applied 72 Biosystems 7500 Fast Real-Time PCR System. Data were analyzed by the DDCT method. Transcript level was normalized to ISOPENTENYL PYROPHOSPHATE (IPP2) for each sample. For AtERdj3A, AtERdj3B, and AGB1, the relative transcript level is expressed as the fold change (mean ± SEM) in each genotype under Tm treatment relative to the mock control (set to a value of 1). For BiP3, as the transcript level is extremely low in the mock control, the relative transcript level is expressed as the fold change (mean ± SEM) in ire1 under each time point of Tm treatment relative to the wildtype Col-0 under the same treatment condition (set to a value of 1). For all the UPRresponse genes examined in the root or shoot tissue, the relative transcript level is expressed as the fold change (mean ± SEM) in each genotype relative to the wild-type Col-0 under the normal growth condition (set to a value of 1). We performed three independent experiments in triplicates. Values are presented in the figures are averages of three samples from one representative experiment. A similar pattern was observed from three independent biological replicates. Phenotypical Analyses Root length measurements were averaged from 30 plants for each genotype; aerial tissue from 10 plants was pooled to estimate the fresh weight. Values were averaged from 3 individual samples for each genotype. Cell length calculations were performed on 10 roots for each genotype. Statistical analyses included the Student’s two-tailed ttest, assuming equal variance; data with p<0.05 were considered significant. 73 Arabidopsis Stable Transformation and Complementation For cloning in the Dex-inducible vector (pTA7002), standard molecular cloning techniques were used. Complementation of agb1-3 was achieved by a 35S-AGB1-YFP fusion. pAtIRE1A-AtIRE1A and 35S-YFP-AGB1 were generated using binary vectors pGWB1 and pEarleygate104, respectively [130, 131]. The genomic or coding regions of genes were amplified with the Gateway-compatible primers from the cDNA synthesized from total RNA of wild-type (Col-0) seedlings using Phusion High-Fidelity DNA polymerase (New England Biolabs). The PCR fragments were cloned to the donor vector (pDonorTM207) and destination vectors pGWB1 and pEarleygate104. Primer sequences used in this work are listed in Table 3.1. Arabidopsis plants were transformed by the floral dip method [132] and transformants were selected on halfstrength LS media supplemented with hygromycin (final concentration, 20 µg/ml) and 0.8% (w/v) agar. Induction of Dex-inducible clone was achieved with DEX (30 µM; Sigma). Confocal Laser Scanning Microscopy An inverted laser scanning confocal microscope (LSM510 META, Carl Zeiss) was used for imaging analyses. Imaging of propidium iodide-labeled roots (10 µg/ml) was carried out using a 543-nm excitation of a He/Ne laser and a LP 560 emission filter. Postacquisition analyses were performed with Zeiss AIM software. Adobe Illustrator was used for further image handling. 74 Acknowledgements We acknowledge support by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (award number DE-FG02-91ER20021) and National Aeronautics and Space Agency (NNH08ZTT003N NRA – 08-FSB_Prop-0052) 75 CHAPTER 4 Inter-regulation of the unfolded protein response and auxin signaling This section has been accepted in the Plant Journal DOI: 10.1111/tpj.12373 76 Abstract The unfolded protein response (UPR) is a signaling network triggered by overload of protein-folding demand in the endoplasmic reticulum (ER), a condition termed ER stress. The UPR is critical for growth and development; nonetheless, connections between the UPR and other cellular regulatory processes remain largely unknown. Here, we identify a novel link between the UPR and the phytohormone auxin, a master regulator of plant physiology. We show that ER stress triggers down-regulation of auxin receptors and transporters in Arabidopsis thaliana. We also demonstrate that an Arabidopsis mutant of a conserved ER stress sensor IRE1 exhibits defects in the auxin response and levels. These data not only support that the plant IRE1 is required for auxin homeostasis, they also reveal a species-specific feature of IRE1 in multicellular eukaryotes. Furthermore, by establishing that UPR activation is reduced in mutants of ER-localized auxin transporters, including PIN5, we define a long-neglected biological significance of ER-based auxin regulation. We further reveal the functional relationship of IRE1 and PIN5 by showing that an ire1 pin5 triple mutant enhances defects of UPR activation and auxin homeostasis in ire1 or pin5. Our results imply that the plant UPR has evolved a hormone-dependent strategy for coordinating ER function with physiological processes. 77 Introduction The UPR adjusts the ER protein folding capacity to cope with the dynamic secretory protein demands in cells [1, 2]. When the ER protein folding machinery is competent, stress sensors are restrained in the ER by ER-resident chaperones [133, 134]. Accumulation of unfolded proteins in the ER activates ER stress sensors either by causing them to dissociate from protein chaperones or to associate with unfolded proteins [96, 133-135]. Activated ER stress sensors transmit signals to the nucleus for transcriptional regulation of UPR target genes [1, 2]. If ER stress is not resolved, the UPR triggers the activation of cell death [67]. IRE1, the only identified ER stress sensor in yeast, is conserved in multicellular eukaryotes [17, 136]. Two IRE1 homologues, IRE1A and IRE1B, have been proven to be functional ER stress sensors in Arabidopsis [39, 40]. The activation of IRE1 relies on auto-phosphorylation, conformational modification, and oligomerization. Activated IRE1 splices an intron from the mRNA of a UPR-specific bZIP transcription factor [19]. The spliced transcription factor enters the nucleus to control UPR target genes [19]. The UPR is critical for numerous fundamental cellular processes [28]. IRE1 alpha knockout mice exhibit embryonic lethality [66]. Dysregulation of the UPR contributes to the pathology of several significant diseases, including diabetes, neurodegeneration, and cancer [137]. In Arabidopsis, mutations of IRE1 lead to a short primary root phenotype [39]. Despite the high significance of the UPR in growth and development in multicellular eukaryotes, the regulatory connections between the UPR and other cellular responses remain unclear. 78 Because the hormone auxin has profound roles in most plant developmental processes, nucleus-based auxin signaling and plasma membrane (PM)-based intercellular auxin transport have been intensively studied. Three major classes of auxin signaling regulators exist in the nucleus: TIR1/AFB auxin co-receptors [138-140], AUX/IAA transcriptional repressors [141], and ARF transcription factors [142]. To initiate the auxin response in the nucleus, TIR1/AFBs and auxin coordinately promote degradation of AUX/IAA transcriptional repressors. Consequently, ARFs are released from repression and activate the transcription of auxin responsive genes [138-140]. Directional (polar) transport between cells is another crucial regulatory aspect of the auxin response. The auxin efflux carriers of the PIN family are the principal components of the polar auxin transport machinery [143, 144]. Based on protein topology and subcellular localization, PINs can be classified into PM- or ER-localized types [145-147]. While PM-based intercellular auxin transport has been considered the most critical point of regulation in the auxin response, it has recently been revealed that ER-based auxin regulation is also important. A putative auxin receptor, ABP1, and several auxin transporters (PIN5, PIN6, PIN8, and PILSs) have been shown to localize to the ER. The requirement of the ER-localized regulators in the auxin response underscores the existence of ER-based auxin biology [145-148]. Despite accumulating evidence that the ER is crucial for auxin regulation [149], the physiological impact of ER-based auxin signaling is largely unknown. As the UPR is critical for growth and development, we sought to identify the regulatory connection between the UPR and other cellular regulatory processes. Given the central roles of auxin in numerous aspects of plant physiology, we hypothesized that 79 the UPR regulates auxin signaling for coordinating secretory activities and physiological responses. Through biochemical, molecular biology, and genetic analyses, we demonstrate a connection between the UPR and auxin biology. Specifically, we show that ER stress negatively influences auxin signaling and that the ER-based auxin homoeostasis is important for UPR activation, supporting that the plant UPR alters auxin signaling to cope with ER stress. On the contrary, by establishing that IRE1 is required for the auxin responses, our work reveals that IRE1 has a specific role in hormonal signaling. The regulatory connections between the UPR and auxin biology revealed here hint that plants have evolved an organism-specific strategy to maintain balance between stress adaption and growth regulation. Results ER Stress Alters the Expression of Auxin Regulators To examine whether ER stress modulates the transcription of auxin regulators, we monitored the expression of four auxin co-receptors: TIR1, AFB1, AFB2, and AFB3 (TIR1/AFBs), under ER stress. The UPR was activated by inhibiting protein Nglycosylation using a classical ER stress inducer termed tunicamycin (Tm). Arabidopsis seedlings were subjected to Tm for various periods of time, as adopted in established protocols [26, 53, 75]. The transcriptional induction of UPR target genes is a molecular indication of UPR activation. To quantify the UPR activation levels, we monitored the transcription of classical UPR activation indicators, BiP1/2 and PDI6 over a 4-hr time course of Tm treatment using quantitative reverse transcription–polymerase chain 80 reaction (qRT-PCR) analyses [150]. BiP1/2 is an ER chaperone essential for the UPR and a primary UPR target gene. PDI6 encodes protein disulfide isomerase. Similar to BiP proteins, upregulation of PDI6 under ER stress contributes to increasing protein folding capacity in the ER. qRT-PCR showed that both BiP1/2 and PDI6 were induced more than 2-fold at 0.5 h of Tm treatment and their levels increased over the time course of treatment (Figure 4.1). Interestingly, we found that there was a 20 to 55% percent reduction in the level of TIR1/AFB transcripts 4 h after Tm treatment in seedlings (Figure 4.2a). These results imply that ER stress negatively influences auxin signaling by repressing TIR1/AFB transcripts. As TIR1/AFBs activate the auxin response by promoting degradation of the AUX/IAA transcriptional repressors, we sought to determine whether the ER stress-induced repression of TIR1/AFB transcripts resulted in the stabilization of AUX/IAA proteins. To do so, we conducted western blot analyses using transgenic Arabidopsis plants expressing DII-VENUS, a fluorescently tagged auxin-interaction domain (DII) of AUX/IAAs that contains the canonical degron responsible for auxin- and TIR1/AFB-mediated protein degradation [151]. As the stabilization of AUX/IAAs is a downstream response of TIR1/AFB reduction, we examined DII-VENUS protein levels 6 h after Tm treatment. Indeed, immunoblot analyses showed that the protein levels of DII-VENUS increased in wild-type Col-0 roots under ER stress treatment (Figure 4.2b). Consistent with the western blot analysis results, a confocal microscopy approach revealed that DII-VENUS fluorescence levels, and therefore AUX/IAA protein levels, were consistently greater in roots challenged by the ER stress inducer than in mock-treated ones (Figure 4.2c). Together these observations support that ER stress leads to an increase in AUX/IAA levels, which is 81 most likely a consequence of protein stabilization resulting from the down-regulation of TIR1/AFBs (Figure 4.2a). Next, we investigated whether ER stress could control the transcription of auxin transporters. Using qRT-PCR analyses, we detected a 30-80% decrease in the mRNA levels of PIN1, PIN2, PIN3, PIN4, PIN5, PIN6, and PIN7 in wild-type Col-0 seedlings during ER stress treatment (Figure 4.2d). In contrast, the transcriptional levels of an ERassociated ethylene receptor (ETR1), two ER-localized cytokinin receptors (AHK2 and AHK3), three nuclear protein (RAN2, ABH1, and FIB1), and two secretory proteins (VSR1 and SCAMP3) [152-158] remained unchanged in ER stress conditions (Figure 4.3). Thus, we conclude that the Tm-induced decrease in the abundance of TIR1/AFB and PIN transcripts is a specific cellular response. When ER stress was triggered by reduction of disulfide bond formation using dithiotreitol (DTT) treatment, similar downregulation of TIR1/AFB and PIN transcripts was observed (Figure 4.4). Overall, these results show that ER stress specifically modulates the auxin response by repressing the transcription of auxin co-receptors and transporters. Next, we examined whether either IRE1 or TIR1/AFBs is essential for ER stressinduced down-regulation of auxin regulators. To do so, we performed the same ER stress treatment coupled with qRT-PCR analyses in an ire1a ire1b (ire1) double mutant and a tir1 afb1 afb2 afb3 (tir1 afbs) mutant [39, 138]. In ire1, both TIR1/AFB and PIN transcripts were still reduced under ER stress conditions (Figure 4.5a-c), similar to the decreased TIR1/AFB and PIN transcripts pattern seen in wild-type Col-0 (Figure 4.2a). The PIN transcripts also decreased under ER stress conditions in tir1 afbs mutant backgrounds (Figure 4.5c). However, with the exception of PIN7, in ire1, the PIN and 82 TIR1/AFB transcription levels were further slightly reduced compared with wild-type Col0 (Figure 4.5b, d). In contrast, the reduction of PIN1, PIN2, and PIN4 transcript levels was larger in the tir1 afbs mutant compared with wild-type Col-0 (Figure 4.5d). These data indicate that down-regulation of PIN transcripts upon ER stress is partially and slightly affected by mutations of either IRE1 or TIR1/AFBs. These results further suggest that the IRE1 and TIR1/AFBs play unessential but fine-tuning roles in ER stress-mediated repression of auxin regulators. IRE1 is Required for the Auxin Responses and Homeostasis While the UPR is necessary for growth and development, the manner by which the UPR influences other cellular regulatory processes is largely unknown. Mammalian IRE1 controls multiple physiological responses under normal growth conditions. Our findings that auxin signaling is altered under ER stress hint that the UPR participates in the auxin response in plants. Although IRE1 is not essential for ER stress-triggered down-regulation of auxin regulators, we aimed to determine whether IRE1-dependent UPR is required for the auxin response without chemical induction of ER stress. Thus, we performed root-inhibition assays to test the sensitivity of exogenous auxin application in ire1. To do so, ire1 and wild-type Col-0 were germinated on medium that contain a synthetic auxin analog, 1-naphthaleneacetic-acid (NAA), a naturally occurring auxin, indole-3-acetic acid (IAA), or an auxin transport inhibitor, 1-N- naphthylphthalamic-acid (NPA). Interestingly, we found that ire1was significantly less sensitive to exogenously applied NAA, IAA, or NPA than wild-type Col-0 (Figure 4.6a-c). The findings that ire1 and wild-type Col-0 displayed comparable root-inhibition 83 responses to three other plant hormone, jasmonic acid (JA), abscisic acid (ABA) and ethylene, indicate that the plant IRE1 has a role specifically in the auxin response, as opposed to general hormones responses or growth regulation (Figure 4.7). To further confirm that IRE1 is involved in the auxin response, we investigated the transcriptional activation of auxin-responsive genes in ire1 upon external application of auxin. qRTPCR analyses showed that the transcriptional induction of five auxin-responsive genes, IAA3, IAA5, IAA19, IAA20, and GH3.6, was compromised after 2 and 4 h of NAA treatment in ire1 relative to wild-type Col-0 (Figure 4.6d). The lower induction of auxinresponsive genes was repeatedly observed after both 1 and 2 h of IAA treatment (Figure 4.6e). These data further indicate that ire1 exhibits an impaired response to exogenously applied auxin. The transcription of BiP1/2 or PDI6 was not significantly altered under IAA or NPA treatment in wild-type Col-0, suggesting that IAA or NPA treatment does not trigger ER stress like Tm treatment (Figure 4.8). Furthermore, we found that there was a 30% reduction in the free auxin level in roots of 10-day-old ire1plants compared with wild-type Col-0 (Figure 4.6f). While IRE1 is required to maintain the free auxin level without ER stress treatment, the free auxin level remained unaffected within 4 h after Tm treatment (Figure 4.9). All together, these data support that plant IRE1 is required for the auxin response and homeostasis. ER-localized Auxin Regulators are Involved in UPR Activation Our observations that auxin signaling is regulated by ER stress led us to test whether auxin homeostasis influences UPR activation. To this end, we examined whether mutations in auxin signaling, polar transport, or biosynthesis affected the 84 induction of UPR target genes. Intriguingly, we found that a mutation in PIN5 or PIN6, two ER-localized auxin transporters, compromised UPR activation under ER stress. Compared with wild type, pin5-5 [145] and pin6-4 [146] exhibited a 30-40% reduction in the level of BiP1/2 and PDI6 transcripts during ER stress treatment (Figure 4.10a). These data imply that ER-based auxin homeostasis contributes to UPR regulation in plants. This hypothesis was supported by the observation that mutants of other types of ER-localized auxin transporters, pils2-2, pils5-2, and pils2-2 pils5-2 [148] exhibited similar defects in UPR activation (Figure 4.10a). We found that the expression levels of BiP1/2 and PDI6 were comparable among wild-type Col-0 and the auxin mutants without ER stress treatment (Figure 4.10b), supporting that the ER-localized auxin transporters are involved in ER stress-triggered UPR activation. In contrast, we found no significant differences in the induction of UPR target genes in mutants defective in either PM-localized auxin exporters, pin1-1 (pin1), eir1-1 (pin2), pin3-4 (pin3), pin4-3 (pin4), pin7-2 (pin7), pin3-5 pin4-3 pin7-1 (pin3 pin4 pin7), an overexpressor of PIN1 (OxPIN1), or the auxin importer aux1-22 (aux1) compared with wild-type Col-0 under the same ER stress treatment conditions (Figure 4.10c). These results show that ERbased intracellular auxin transport, but not intercellular auxin transport, is required for the optimal UPR activation in plants. We next investigated whether the ER-localized putative auxin receptor ABP1 and the auxin biosynthesis enzyme YUC were also involved in the UPR activation. Similar to the auxin mutants defective in ER-based transport, the abp1-5 [160] and YUC [161] mutants also exhibited reduced levels in the activation of UPR target genes under ER stress (Figure 4.10a). Conversely, the transcription levels of UPR target genes were 85 higher in the tir1 afbs auxin co-receptor mutant than in wild-type Col-0 (Figure 4. 10a), suggesting that TIR1/AFBs play a negative role in UPR target gene induction. Altogether, these data highlight that the ER-based regulation of auxin homeostasis may operate as a molecular link between the UPR and other cellular processes. pin5 Enhances the ire1 Phenotype in Auxin Responses and UPR Activation To investigate a functional relationship of IRE1 and PIN5 in the UPR and auxin response, we generated an ire1 pin5 triple mutant and performed phenotypic analyses. Consistent with previous reports, ire1 and pin5-5 displayed a short primary root phenotype [39, 145]. We found that the roots of the ire1 pin5 triple mutant were significantly shorter than those of ire1 or pin5-5 (Figure 4.11a). However, ire1, pin5-5, and ire1 pin5 showed comparable lateral root density and hypocotyl length to wild-type Col-0 (Figure 4.12), suggesting that the IRE1 and PIN5 have a role specifically in regulation of primary root elongation. In addition, ire1 pin5 also displayed lower free auxin levels compared with ire1 or pin5-5 (Fig 4.11b). Specifically, compared with wildtype Col-0, the roots of pin5-5 and ire1 exhibited a 15 and 30% reduction in free auxin levels, respectively. Nonetheless, the roots of ire1 pin5 displayed a 45% reduction in free auxin level (Figure 4.11b). Finally, in agreement with previous findings [145], rootinhibition assays showed that pin5-5 was less sensitive than wild-type Col-0 to low concentrations of IAA but displayed a normal response to NAA, NPA, or high concentrations of IAA [145]. Intriguingly, the ire1 pin5 mutant was significantly less sensitive than ire1 to all three treatments (Figure 4.11c-e). A comparable root-inhibition 86 response to JA, ABA, and ethylene in ire1 pin5 indicated that the genetic interaction between IRE1 and PIN5 is specific to the auxin response (Figure 4.7). We next tested the functional relationship of IRE1 and PIN5 under ER stress. qRT-PCR revealed that the induction of BiP1/2 and PDI6 was also reduced in ire1 pin5 compared with ire1 or pin5-5 (Figure 4.11f), supporting that PIN5 participates in the UPR activation in a manner not entirely dependent on IRE1. Altogether, our results imply that regulation of ER-based auxin homeostasis is part of ER stress adaptive mechanisms that plants have evolved to parallel the classical UPR signaling pathways. Discussion Our findings uncover an unpredicted but critical regulatory relationship between two fundamental signaling pathways in plants, the UPR and auxin response. Studies of the mammalian UPR indicate that distinct UPR signaling pathways mediate specific physiological processes [28]. While the IRE1-dependent mRNA splicing event is the most evolutionarily conserved UPR pathway in eukaryotes, IRE1 has also evolved specific functions in multicellular organisms to associate the UPR with more complex physiological processes [86, 87]. Nevertheless, our understanding of the connection between the UPR and other cellular processes is still in its infancy. Here, we have defined a plant-specific regulatory role for IRE1 in the auxin response. The auxin polar transport is one of the most crucial regulatory mechanisms in the auxin biology. As most regulatory components of the auxin polar transport system are secretory proteins, we speculate that the IRE1-dependent UPR maintains a robust and efficient membrane trafficking system for the supply of functional auxin regulators. The identification of auxin 87 regulators directly controlled by IRE1 will elucidate how the UPR modulates specific aspects of auxin biology to coordinate the secretory pathway with physiological responses. Together with the involvement of UPR-specific membrane tethered transcription factors in brassinosteroid signaling [162], our results support the significance of the plant UPR in hormone signaling. IRE1 regulates the UPR through various mechanisms including unconventional splicing, RNA decay, and protein-protein interaction. It has recently been reported that similar to its mammalian counterpart, plant IRE1 controls gene expression through RNA decay in addition of splicing bZIP60 transcription factor [53]. Mammalian IRE1 operates RNA decay to trigger diverse UPR signaling pathways. It would be interesting to test whether plant IRE1 also relies on its RNA decay function for the auxin response. If IRE1-dependent RNA decay contributed at least partially to the regulation of auxin signaling under ER stress, it would represent a specific regulatory event of the UPR as opposed to random RNA decay under stress. Notably, we have established that IRE1 is required for the optimal auxin response under exogenously applied auxin (Figure 4.6), but plays only partial role in ER stress-induced down-regulation of auxin regulators (Figure 4.5). These findings support that distinct mechanisms regulate auxin signaling under various conditions to achieve context-specific auxin responses. We have established that only ER-localized auxin transporters, but not PMlocalized auxin exporters or importers, are required for the optimal UPR activation (Figure 4.10). Studies of ER-localized auxin regulators suggest that a distinct auxin signaling pathway exists in the ER [149]. Accordingly, we propose that ER-based auxin signaling actively transports free auxin through the ER membranes to modulate the 88 signaling response in the nucleus. More specifically, plant cells can transmit signals between sub-cellular compartments by adjusting the free auxin level in the ER, cytosol, and nucleus. We thus propose a novel cellular function for auxin as a signaling molecule that connects subcellular compartments and maintains cellular homeostasis in plants. It has long been believed that intercellular polar auxin transport is the key regulatory component of the auxin response; however, the biological significance of intracellular auxin transport has been overlooked. Our findings support a specific cellular function of ER-based intracellular auxin distribution in the UPR activation, and thus emphasize the importance of ER auxin biology in plant physiology. We have shown that ER-localized auxin transporters have a role in the UPR activation. A plausible hypothesis to explain this is that the auxin levels in the ER lumen contribute to UPR activation under ER stress. Namely, ER-localized auxin transporters or their associated proteins may sense ER stress and rapidly adjust auxin levels in the ER lumen. The consequent fluctuation of auxin levels in the ER could in turn affect the magnitude of UPR activation. Nevertheless, because a mutation of PIN5 enhances the ire1 mutant phenotype in the UPR activation (Figure 4.11f), PIN5-dependent regulation of auxin levels under ER stress does not completely rely on IRE1. Whether ER-localized auxin transporters can directly monitor ER stress or indirectly sense ER stress-related cellular homeostasis is yet to be established; however, the findings presented here support that ER-localized transporters play a role in the UPR activation. Once a reliable system to monitor auxin levels in the ER lumen is developed, it will be interesting to experimentally confirm that ER-localized transporters mediate auxin transport between ER lumen and cytosol during ER stress. 89 PIN5 has been proposed to play a unique role in the auxin response since its transcriptional regulation and regulatory mechanisms appear to be different from PMlocalized PINs. It was reported that the transcription of PIN5 is decreased under exogenous application of auxin although PIN5 is required for the auxin response [145]. Likewise, our study also showed that ER stress induces a decrease in the transcription of PIN5 while PIN5 is required for the optimal induction of UPR activation. As the PIN5 protein levels have not been monitored under auxin or ER stress treatment, one possibility is that the down-regulation of PIN5 transcript represents a feedback regulatory mechanism. Namely, the cellular availability or the activity of PIN5 may be increased in response to ER stress (e.g. by protein stabilization or post-translational modifications). This in turn may cause reduction of PIN5 transcriptional levels to safeguard cellular auxin homeostasis. Another possibility is that ER stress represses general auxin responses including inter- and intra-cellular auxin transport to optimize cellular responses to cope with stress. Thus, both PM- and ER-localized transporter are down-regulated under ER stress; however, a basal level of ER-localized transporters may be still required for optimal induction of UPR target gene as they may be involved in stress signal transmission through transport the auxin between subcellular compartments. Thus, mutants of ER-localized auxin regulators would display a compromised UPR activation. Further experimental evidence are needed to verify the possibilities. Nonetheless, our data support that regulation of PIN5 transcripts is a mechanism to maintain PIN5-related cellular homeostasis. In contrast with animals, plants, as sessile organisms, have an extraordinary plasticity in post-embryonic development, responding to both internal and external cues. 90 Nonetheless, our understanding of how plants integrate developmental and environmental signals to balance growth and adaptive regulation is limited. The interregulation between the UPR and auxin response demonstrated in this study provides a new paradigm in plant physiology. Given the essential roles of the UPR in multiple stresses adaptation, the integrated action of the UPR and auxin response highlights a plant-specific strategy that evolved to maintain the crucial balance between stress response and growth regulation for ultimate fitness. 91 20 15 BiP1/2 PDI6 10 5 0 Figure 4.1. Tunicamycin induces activation of UPR target genes. qRT-PCR analyses of BiP1/2 and PDI6 transcripts in 10-day-old Col-0 Arabidopsis seedlings after treatment with 5 µg/ml Tm for 0.5, 1, 4, or 6 h. Error bars represent standard error of the mean (SEM) from three independent biological replicates. 92 Relative expression level (a) 1.2 TIR1 AFB1 AFB2 AFB3 1.0 0.8 0.6 0.4 0.2 0.0 0 0.5 (b) 1 4 h (Tm) (c) DII-VENUS DMSO DMSO Tm 55 36 130 95 72 55 Tm 36 Relative expression level (d) 1.2 ETR1 PIN1 PIN2 PIN7 PIN3 PIN4 PIN5 PIN6 1.0 0.8 0.6 0.4 0.2 0.0 0 0.5 1 4 h (Tm) Figure 4.2. ER stress alters the expression of auxin regulators. 93 Figure 4.2 (cont’d) (a) qRT-PCR analyses of TIR1, AFB1, AFB2, and AFB3 expression in 10-day-old Col-0 Arabidopsis seedlings after treatment with 5 µg/ml Tm for 0.5, 1, or 4 h. Error bars represent standard error of the mean (SEM) from three independent biological replicates. P-values were calculated by Student’s two-tailed t test against expression levels at 4 h relative to 0 h: TIR1 (P = 0.00036), AFB1 (P = 0.00041), AFB2 (P = 0.00048), AFB3 (P = 0.00032). (b) The levels of DII-VENUS fusion proteins increase upon ER stress. 10-day-old DIIVENUS transgenic plants were treated with 5 µg/ml Tm or dimethyl sulfoxide (DMSO) for 6 h. Proteins were extracted from root tissues and the fusion proteins were detected by immunoblot analysis using anti-GFP serum (upper panel). Comassie blue staining gel used as loading control (lower panel). (c) 10-day-old transgenic plants expressing DII-VENUS were treated with 5 µg/ml Tm or DMSO for 6 h. Primary root tips were subjected to confocal microscopy analyses. Scale bars = 50 µm. (d) PIN mRNA levels decrease upon exposure to ER stress. qRT-PCR analyses of PIN family transcripts in 10-day-old wild-type Col-0 seedlings during treatment with 5 µg/ml Tm for 0.5, 1, or 4 h. Error bars represent standard error of the mean (SEM) from three independent biological replicates. P-values were calculated against expression levels at 4 h relative to 0 h: PIN1 (P = 0.00221), PIN2 (P = 0.00316), PIN3 (P = 5.4E-05), PIN4 (P = 4.9E-05), PIN5 (P = 6.4E-05), PIN6 (P = 0.00012), PIN7 (P = 0.00353). The transcriptional level of ETR1, an ER-associated ethylene receptor, was unchanged after treatment with Tm for 0.5, 1, or 4 h. 94 Relative expression level 1.5 VSR1 SCAMP3 RAN2 1.0 ABH1 FIB1 0.5 AHK2 AHK3 0.0 0 1 4 h (Tm) Figure 4.3. The transcripts of genes encoding ER-localized and nuclear proteins remain unchanged under Tm treatment. qRT-PCR analyses of VSR1, SCAMP3, RAN2, ABH1, FIB1, AHK2, and AHK3 transcripts in 10-day-old Col-0 Arabidopsis seedlings after treatment with 5 µg/ml Tm for 0, 1, or 4 h. Error bars represent standard error of the mean (SEM) from three independent biological replicates. No statistical differences were observed between the expression levels of individual genes in the time course. 95 Relative expression level Relative expression level Relative expression level 14 12 PDI6 10 BiP1/2 8 6 4 2 0 0 1 2 h (DTT) 1.2 TIR1 1.0 AFB1 0.8 AFB2 0.6 AFB3 0.4 0.2 0.0 0 1 2 h (DTT) 1.5 PIN1 PIN2 PIN7 PIN3 PIN4 PIN5 PIN6 ETR1 1.0 0.5 0.0 0 1 2 h (DTT) Figure 4.4. DTT transcriptionally activates UPR target genes and down-regulates auxin regulators. 96 Figure 4.4 (cont’d) qRT-PCR analyses of BiP1/2, PDI6, TIR1, AFB1, AFB2, AFB3, and PIN family transcripts in 10-day-old Col-0 Arabidopsis seedlings after treatment with 2mM DTT for 0, 1, or 2 h. Error bars represent standard error of the mean (SEM) from three independent biological replicates. 97 Relative expression level (a) ire1 1.2 1.0 0.8 0.6 TIR1 AFB1 AFB2 AFB3 0.4 0.2 0.0 0 0.5 1 4 h (Tm) (b) Relative expression level 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Tm - + - + - + - + - + - + - + Col-0 ire1 Col-0 ire1 Col-0 ire1 Col-0 TIR1 AFB1 AFB2 - + ire1 AFB3 Figure 4.5. IRE1 and TIR1/AFBs play fine-tuning roles in ER stress-induced downregulation of auxin regulators. 98 Figure 4.5 (cont’d) ire1 tir1 afbs 1.2 PIN1 PIN2 PIN7 PIN3 PIN4 PIN5 PIN6 1.0 0.8 0.6 0.4 0.2 0.0 0 0.5 1 Relative expression level Relative expression level (c) 4 h (Tm) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 0.5 1 4 h (Tm) (d) Relative expression level (%) Mock Col-0 Tm ire1 Tm tir1 afbs Tm 1.2 1.0 0.8 0.6 0.4 0.2 0.0 PIN1 PIN2 PIN3 PIN4 99 PIN5 PIN6 PIN7 Figure 4.5 (cont’d) (a) qRT-PCR analyses of TIR1, AFB1, AFB2, and AFB3 expression in 10-day-old ire1 Arabidopsis seedlings after treatment with 5 µg/ml Tm for 0.5, 1, or 4 h. (b) qRT-PCR analyses of TIR1, AFB1, AFB2, and AFB3 expression in 10-day-old wild type Col-0 and ire1 Arabidopsis seedlings after treatment with 5 µg/ml Tm for 4 h. (c) PIN mRNA levels decrease upon exposure to ER stress in 10-day-old ire1 or tir1 afb. qRT-PCR analyses of PIN family transcripts in 10-day-old ire1 or tir1 afb seedlings after treatment with 5 µg/ml Tm for 0.5, 1, or 4 h. (d) Transcriptional repression of PINs after treatment with 5 µg/ml Tm for 4 h in 10-dayold Col-0, ire1, or tir1 afb Arabidopsis seedlings. Error bars represent standard error of the mean (SEM) from three independent biological replicates. 100 ire1 Col-0 Relative root length (%) (a) 0 50 100 200 100 80 60 40 Col-0 20 ire1 0 0 50 100 NAA (nM) NAA (nM) (c) 100 75 50 25 0 Col-0! ire1! 0 1 2 3 4 IAA (µM) 5 Relative root length (%) Relative root length (%) (b) 200 100 75 50 25 0 Col-0 0 2.5 NPA (µM) Figure 4.6. ire1 exhibits the compromised auxin responses. 101 ire1 5 5 4 3 2 1 0 2h 250 200 150 100 50 4 h (NAA) 0 ire1 10 5 0 Relative expression level (e) 250 2h 4 h (NAA) IAA5 Col-0 ire1 200 150 100 50 0 1h Relative expression level Col-0 2 h (IAA) Relative expression level Relative expression level IAA20 15 IAA5 Col-0 ire1 20 2h 4 h (NAA) IAA19 Col-0 ire1 60 40 20 0 2h 4 h (NAA) GH3.6 Col-0 ire1 15 10 5 0 2h 4 h (NAA) IAA19 Col-0 ire1 40 30 20 10 0 1h 102 2 h (IAA) (f) Free IAA (pmol per g FW) 6 IAA3 Col-0 ire1 Relative expression level Relative expression level (d) Relative expression level Figure 4.6 (cont'd) Free IAA 350 280 210 140 70 0 Col-0 ire1 Figure 4.6 (cont’d) (a-c) In ire1, root growth is largely resistant to treatments with auxin (NAA and IAA) or an auxin transport inhibitor (NPA). Relative primary root length of 10-day-old Col-0 and ire1 Arabidopsis seedlings grown in the presence 50, 100, 200 nM NAA (a), or 1, 2, 3, 4, 5 µM IAA (b), or 2.5, 5 µM NPA (c) compared with those grown in the absence of the chemicals. Error bars represent standard error of the mean (SEM), n > 30. Scale bars = 1 cm. P-values are relative to Col-0: 100, 200 nM NAA (P < 0.00078), 1, 2, 3, 4, 5 µM IAA (P < 0.00050), 2.5, 5 µM NPA (P < 0.00344). (d) qRT-PCR analyses of IAA3, IAA5, IAA19, IAA20, and GH3.6 expression in 10-dayold Col-0 and ire1 Arabidopsis seedlings after a 2- or 4-h treatment with 10 µM NAA. Error bars represent SEM from three independent biological replicates. P-values are relative to Col-0: IAA3 (P < 0.00098), IAA5 (P < 0.00016), IAA19 (P < 0.00479), IAA20 (P < 0.00036), and GH3.6 (P < 0.00391). (e) qRT-PCR analyses of IAA5 and IAA19 in 10-day-old Col-0 and ire1 Arabidopsis seedlings after a 1- or 2-h treatment with 10 µM IAA. P-values are relative to Col-0: IAA5 (P < 0.00086) and IAA19 (P < 0.00112). (f) Free IAA concentration in 10-day-old Col-0 and ire1 Arabidopsis roots. Error bars represent standard error of the mean (SEM) from three independent biological replicates. P-value is relative to Col-0: P = 4.9E-05. 103 (a) 50 µM JA Relative root length (%) 60 50 40 30 20 10 0 Col-0 pin5 (b) ire1 ire1 pin5 ACC Relative root length (%) 120 Col-0 100 ire1 80 pin5 60 ire1pin5 40 20 0 0 1 10 Figure 4.7. ire1 and ire1 pin5 display comparable sensitivity to JA, ACC, and ABA. 104 Figure 4.7 (cont’d) ABA (c) Relative root length (%) 120 Col-0 100 ire1 80 pin5 60 ire1pin5 40 20 0 0 5 10 (µ M) (a-c) Relative primary root length of 10-day-old Col-0, pin5, ire1, and ire1 pin5 Arabidopsis seedlings grown in the presence of 50 µM JA (a) or 1, 10, 100 µM ACC (b), or 5, 10 µM ABA (c) compared with those grown in the absence of the chemicals. Error bars represent standard error of the mean (SEM), n > 30. 105 Relative expression level 4 BiP1/2 3 PDI6 2 1 0 IAA NPA Tm Figure 4.8. The UPR target genes were not altered under IAA or NPA treatment. qRT-PCR analyses of BiP1/2 and PDI6 in 10-day-old Col-0 Arabidopsis seedlings relative to DMSO or EtOH mock control after a 1-h treatment with 10 µM IAA, 50 µM NPA, or 5 µg/ml Tm. Error bars represent standard error of the mean (SEM) from three independent biological replicates. 106 Free IAA (pmol per g FW) 160 DMSO 120 Tm 80 40 0 0.5 1 4 h (Tm) Figure 4.9. The free auxin level is unchanged on ER stress. Free IAA measurement in the roots of 10-day-old Col-0 after treatment with 5 µg/ml Tm or DMSO for 0.5, 1, or 4 h. Error bars represent standard error of the mean (SEM) from three independent biological replicates. 107 Relative expression level OxPIN1 aux1 pin7 pin3 pin4 pin7 1 0.5 1.5 phenotype. 108 0 OxPIN1 aux1 BiP1/2 YUC tri1 afb pils2 pils5 abp1 pils5 pin6 pils2 Col-0 pin5 0.0 pin3 pin4 pin7 0.5 pin3 pin4 pin7 1.0 pin2 1.5 2.5 Relative expression level YUC tri1 afb abp1 2.0 pin1 pin6 pils2 pin5 BiP1/2 Col-0 1.5 pils5 pils2 pils5 (b) pin3 pin4 pin2 pin1 2.5 Relative expression level 0 Col-0 0.0 Col-0 Relative expression level (a) PDI6 2.0 1.5 1.0 0.5 PDI6 1 0.5 Figure 4.10. Mutants impaired in intracellular auxin transport display a defective UPR Relative expression level 0 YUC tri1 afb pils2 pils5 abp1 pils5 0 109 YUC tri1 afb pils2 pils5 abp1 pils5 0.5 1.5 pin6 pils2 1 Relative expression level BiP1/2 Col-0 pin5 1.5 pin6 pils2 Col-0 pin5 Figure 4.10 (cont’d) (c) PDI6 1 0.5 Figure 4.10 (cont’d) (a) qRT-PCR analyses of BiP1/2 and PDI6 in 10-day-old pin5-5 (pin5), pin6-4 (pin6), pils2-2 (pils2), pils5-2 (pils5), pils2-2 pils5-2 (pils2 pils5), abp1-5 (abp1), YUC, and tir1 afbs(tir1 afb) relative to wild-type Col-0 Arabidopsis seedlings after a 1-h treatment with 5 µg/ml Tm. Error bars represent standard error of the mean (SEM) from three independent biological replicates. P-values are relative to Col-0: pin5 (P = 0.00029), pin6 (P = 0.00095), pils2 (P = 0.00093), pils5 (P = 0.00067), pils2 pils5 (P = 0.00089), abp1 (P = 0.00215), YUC (P = 0.00014), tir1 afb (P = 0.00026). (b) qRT-PCR analyses of BiP1/2 and PDI6 in 10-day-old pin1-1 (pin1), eir1-1 (pin2), pin3-4 (pin3), pin4-3 (pin4), pin7-2 (pin7), pin3-4 pin4-3 pin7-2 (pin3 pin4 pin7), OxPIN1, and aux1-22 (aux1) relative to wild-type Col-0 Arabidopsis seedlings after a 1-h treatment with 5 µg/ml Tm. Error bars represent SEM from three independent biological replicates. (c) qRT-PCR analyses of BiP1/2 and PDI6 in 10-day-old Col-0, pin5-5 (pin5), pin6-4 (pin6), pils2-2 (pils2), pils5-2 (pils5), pils2-2 pils5-2 (pils2 pils5), abp1-5 (abp1), YUC, and tir1 afbs (tir1 afb) Arabidopsis seedlings without Tm treatment. Error bars represent standard error of the mean (SEM) from three independent biological replicates. 110 (a) Free IAA (pmol per g FW) 120 Relative root length (%) (b) Root length 100 80 * 60 40 20 0 Col-0 pin5 Free IAA 350 300 250 * 200 150 100 50 0 ire1 ire1 pin5 Col-0 pin5 ire1 ire1 pin5 ire1 pin5 pin5 c 0 50 100 200 NAA (nM) Relative root length (%) (c) 120 100 80 60 40 Col-0 ire1 pin5 ire1 pin5 20 0 0 50 100 NAA (nM) 200 Figure 4.11. pin5 enhances the auxin and ER stress response phenotype in ire1. 111 Figure 4.11 (cont’d) (d) (e) 120 Col-0 ire1 pin5 ire1 pin5 100 80 Relative root length (%) Relative root length (%) 120 100 60 40 20 0 0 1 2 3 4 IAA (µM) 80 60 40 20 0 5 0 2.5 5 NPA (µM) (f) 3.5 3.0 Relative expression level Relative expression level 3.5 BiP1/2 2.5 * 2.0 1.5 1.0 0.5 0.0 Col-0 pin5 ire1 3.0 2.5 112 * 2.0 1.5 1.0 0.5 0.0 ire1 pin5 PDI6 Col-0 pin5 ire1 ire1 pin5 Figure 4.11 (cont’d) (a) pin5-5 (pin5) enhances the short-root phenotype of ire1. Relative primary root length of pin5, ire1, and ire1 pin5 compared with Col-0 under unstressed conditions. Error bars represent standard error of the mean (SEM), n > 30. P-value is ire1 pin5 relative to ire1: *P = 0.00226. (b) Free IAA measurement in the roots of 10-day-old Col-0, pin5, ire1, and ire1 pin5 Arabidopsis seedlings. Error bars represent SEM from three independent biological replicates. P-value is ire1 pin5 relative to ire1: *P = 0.00182. (c-e) Relative primary root length of 10-day-old Col-0, pin5, ire1, and ire1 pin5 Arabidopsis seedlings grown in the presence 50, 100, 200 nM NAA (c), or 1, 2, 3, 4, 5 µM IAA (d), or 2.5, 5 µM NPA (e) compared with those grown in the absence of the chemicals. Error bars represent SEM, n > 30. P-values are ire1 pin5 relative to ire1: 50, 100 or 200 nM NAA (P < 0.00032), 1, 2, 3, 4, or 5 µM IAA (P < 0.00075), 2.5, 5 µM NPA (P < 0.00149). Scale bars = 1 cm. (f) pin5 enhances the UPR defects in ire1 under ER stress. qRT-PCR analyses of BiP1/2 and PDI6 in 10-day-old Col-0, pin5, ire1, and ire1 pin5 Arabidopsis seedlings relative to DMSO mock control after a 1-h treatment with 5 µg/ml Tm. Error bars represent standard error of the mean (SEM) from three independent biological replicates. 113 (a) Lateral root density Hypocotyl length ( cm ) Lateral number / cm 2 (b) 1.5 1 0.5 0 Col-0 pin5 ire1 ire1 pin5 Hypocotyl length 1.5 1 0.5 0 Col-0 pin5 ire1 ire1 pin5 Figure 4.12. ire1 and ire1 pin5 display normal root density and hypocotyl elongation. (a) Lateral root density of 10-day-old Col-0, pin5, ire1, and ire1 pin5 Arabidopsis seedlings Lateral root density is calculated as the number of lateral roots per cm of primary root Error bars represent standard error of the mean (SEM), n > 30. (b) Quantification of the hypocotyl length of five-day-old Col-0, pin5, ire1, and ire1 pin5 Arabidopsis seedlings grown under dark condition. Error bars represent standard error of the mean (SEM), n > 30. 114 (a) Auxin response and homeostasis IRE1 ERlocalized PINs ER membrane PMlocalized PINs (b) ER stress UPR activation ERlocalized PINs ER membrane IRE1 TIR1/AFBs PMlocalized PINs Figure 4.13. Working model. 115 Figure 4.13 (cont’d) (a) IRE1 is required for the auxin responses upon external auxin application. IRE1, ERand PM-localized PINs are involved in the maintenance of auxin homeostasis without chemical induction of ER stress. (b) ER stress triggers down-regulation of auxin receptors TIR1/AFBs, ER- and PMlocalized PINs. IRE1 and ER-localized PINs are required for the optimal induction of UPR target genes. 116 Table 4.1. DNA primers used in this study Primers Sequence (5’–3’) WiscDsLox420D09 RP TATCTCCGATCCATCGTTGAC WiscDsLox420D09 LP CAAAATCTTCAGTGCTAGCGG WiscDsLox LP AACGTCCGCAAT GTGTTATTAAGTTG SAIL_238_F07 RP GAAGGAAAACGGACATCCTTC SAIL_238_F07 LP CCTCTCGAACCCTTCAGGTAC SAIL_LB2 GCTTCCTATTATATCTTCCCAAATTACCAATACA SM_3_28638 RP CTCCTGAGTCCCTGATCACAC SM_3_28638 LP TAGAATCATATGCCACGGCAC Spm32 TACGAATAAGAGCGTCCATTTTAGAGTGA TIR1 qP For TTCCGTCCGAGCCTTTTG TIR1 qP Rev AAGCCCCTGTTCCGTCAAT AFB1 qP For GGTCAGTCCTGCTGCGGTTA AFB1 qP Rev CCCCTTCAAAGTCAAAGATCTCA AFB2 qP For GAGTCTGAAGCTAAATCGTGCAGTA AFB2 qP Rev GCGCACACGCCATTAACC AFB3 qP For GGGCTTTGGTGCAATCGTA AFB3 qP Rev CGGAGACAGAGAGCCGTCTT 117 Table 4.1 (cont’d) PIN1 qP For CGGTGGGAACAACATAAGCA PIN1 qP Rev CACACTTGTTGGTGGCATCAC PIN2 qP For AACGCAAGCAAAGCTCCAA PIN2 qP Rev GCGCCGCCGTAGCTATTA PIN3 qP For CGGAGCACCTGACAACGAT PIN3 qP Rev CGGATCTCTTTAGCACCTTGGT PIN4 qP For GTTGCTGGGATTGCCATTG PIN4 qP Rev GCAGCCTGAACGATGGCTAT PIN5 qP For CCATGGCCATCGGCTCTAT PIN5 qP Rev TGGCTACGCGGAGGACAT PIN6 qP For CGCCGGCGCAATGT PIN6 qP Rev GGACTGGGACGGCGAAA PIN7 qP For CATAAACCGCTTCGTCGCTAT PIN7 qP Rev TTGAGGAGATGAAGTGGAAAGAGA BiP1/2 qP For CCACCGGCCCCAAGAG BiP1/2 qP Rev GGCGTCCACTTCGAATGTG PDI6 qP For CGAAGTGGCTTTGTCATTCCA PDI6 qP Rev GCGGTTGCGTCCAATTTT 118 Table 4.1 (cont’d) IAA3 qP For TCTTTTTATCTTCTCCTGCAATTCTTG IAA3 qP Rev CCAGCCTCAGCTCTGTTTCC IAA5 qP For GTCCATCTCCGGGAAGAAGAG IAA5 qP Rev CGCCGGTTCACATTTCAAAT IAA19 qP For TGGCCACCGGTTTGTTCTT IAA19 qP Rev TTCGTGGTCGAAGCTTCCTT IAA20 qP For AATGGCTACCGCGACTTGAT IAA20 qP Rev CCAGAGAATGGATGCGTTGA GH3.6 qP For GCTCGTGACATTAGAACCGGTACT GH3.6 qP Rev GCCTCACGAACCGAAGAATC 119 Table 4.1 (cont’d) ETR1 qP For AACCGTGGCGCTTGTGAT ETR1 qP Rev CACACGAGACAACAGCGGTTA VSR1 qP For CTCCCAACTCTTGTCGTGAACA VSR1 qP Rev AGCACTGCCCCCTTTTCC SCAMP3 qP For AGGCGGGAGCAGGAAATC SCAMP3 qP Rev ACAATTCCGGCCTGTGCTAT RAN2 qP For TCAGCCTCTCCCCGATGAT RAN2 qP Rev CACAGCAGACATTTTTGGAAGATT ABH1 qP For GGCGCTGTTCGTGCACTA ABH1 qP Rev GACGCGTCCTGGATTCCAT FIB1 qP For CAGCCACCACCGCACAT FIB1 qP Rev CGAAAGACGAGAACCGAAAAA AHK2 qP For GGTTCCGGCAGATCATTACAA AHK2 qP Rev CCCCTTTCCTGTGTGAATTTG AHK3 qP For TCCAATGCAACGCCAAAA AHK3 qP Rev CAAACACTCCCCCGAGATACC IPP2 For ATTTGCCCATCGTCCTCTGT IPP2 Rev GAGAAAGCACGAAAATTCGGTAA 120 Methods Plant material and growth conditions Arabidopsis thaliana ecotype Columbia (Col-0) plants were used. Surfacesterilized seeds were plated directly onto petri dishes containing half-strength Linsmaier and Skoog (LS) medium, 1.5% w/v sucrose, and 0.4% Phytagel (Sigma). For normal growth conditions, plants were grown at 21°C under a 16-h light/8-h dark cycle. Tm treatment Seeds were germinated and grown on half-strength LS medium for 10 days, and then transferred to half-strength LS medium containing 5 µg/ml Tm (Sigma) for the indicated periods of time. RNA extraction and quantitative RT-PCR analysis Total RNA was extracted from whole seedlings using an RNeasy Plant Mini Kit (Qiagen) and treated with DNase I (Qiagen). All samples within an experiment were reverse-transcribed simultaneously using SuperScript® VILO™ Master Mix, (Invitrogen). A no-RT reaction, in which RNA was subjected to the same conditions of cDNA synthesis but without reverse transcriptase, was included as a negative control in all real-time quantitative PCR (qRT-PCR) assays. qRT-PCR with SYBR Green detection was performed in triplicate using the Applied Biosystems 7500 Fast Real-Time 7500 PCR system. Data were analyzed by the summary of efficiency (DDCT) method. The values presented are the mean of three independent biological replicates. Primers used are listed in Supplementary Table S1. 121 Phenotypic analysis Root length and hypocotyl elongation measurements were averaged from 30 plants for each genotype. Data were analyzed by Student’s two-tailed t-test, assuming equal variance; differences with a P-value < 0.05 were considered significant. Immunoblotting and confocal microscopy analyses Fifty milligrams of fresh root tissues was ground in plastic tubes with plastic pestles using liquid nitrogen and 500 ml of SDS-containing extraction buffer (60 mM Tris-HCL (pH 8.8), 2% SDS, 2.5% glycerol, 0.13 mM EDTA (pH 8.0), and 1X Protease Inhibitor Cocktail Complete (Roche)). The tissue lysates were vortexed for 30 s, heated at 70°C for 10 minutes, and then centrifuged at 13,000 g twice for 5 minutes at room temperature. The supernatants were then transferred to new tubes. For SDS-PAGE analysis, 5 µl of the extract in 1x NuPAGE LDS Sample Buffer (Invitrogen) was separated on 4-12% NuPage gel (Invitrogen) and then transferred to PVDF (polyvinyl difluoride) membrane. The membrane was incubated with 3% BSA in 1x TBST (50 mM Tris-base, 150 mM NaCl, 0.05% Tween 20, pH 8.0) overnight at 4°C, and was then probed with antibody (α-GFP, 1:20,000; Abcam) diluted in the blocking buffer (1:20,000) at room temperature for 1 h. The probed membrane was washed three times with 1x TBST for 5 min and then incubated with secondary antibody (goat anti-rabbit IgG for αGFP, 1:20,000; Abcam) at room temperature for 1 h. Finally, the membrane was washed four times with 1x TBST for 10 min before the signals were visualized with SuperSignal® West Dura Extended Duration Substrate (Pierce Biotechnology). To 122 visualize YFP fluorescence, an inverted laser scanning confocal microscope Zeiss LSM510 was used to detect the DII expression. Free IAA analysis Approximately 20 roots were cut from 10-day-old seedlings and transferred into an Eppendorf tube containing 1 ml of methanol. Internal standard of [2H5] IAA was added to the sample at amount of 100 fmol per root. Acknowledgements We thank Teva Vernoux for sharing the DII-VENUS seeds and Jürgen KleineVehn for sharing the pils2-2, pils5-2, and pils2-2 pils5-2 seeds and the Arabidopsis Biological Resource Center (ABRC) for seed stocks. This study was supported by grants from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. DOE (DE-FG02-91ER20021) for the infrastructure, National Institutes of Health (R01 GM101038-01), and NASA (NNX12AN71G). 123 CHAPTER 5 Conclusion and Future Perspectives 124 The UPR maintains the integrity and dynamics of the secretory pathway in eukaryotic cells. Establishing the UPR signaling network is fundamental to illuminating molecular mechanisms underlying maintenance of the secretory pathway. This dissertation aims to identify regulators and biological roles of the UPR in Arabidopsis. When I started the plant UPR studies, functional roles of plant IRE1 in the UPR were not established in vivo. Regulatory relationships between the UPR and other cellular processes were largely unexplored. Plant IRE1 is a Functional ER Stress Sensor and Involved in Root Growth In Chapter 3, I present in vivo evidences that the two plant IRE1 homologs, IRE1A and IRE1B, are functional ER stress sensors in Arabidopsis. Based on the phenotypic analyses, IRE1A and IRE1B have at least partially overlapping function in ER stress tolerance and UPR activation. In addition, ire1 displays a short-root phenotype, indicating a regulatory role of plant IRE1 in root growth. By contrast, a mutant of the splicing substrate of IRE1, bzip60, shows normal primary root growth. The difference of root phenotype of ire1 and bzip60 indicates that IRE1 mediates root growth regulation in a manner not completely dependent on the bZIP60 pathway. One possibility is that IRE1 might have additional substrate(s) other than bZIP60 and the substrate(s) has a role in the root regulation. It is also plausible that IRE1 relies on its RNA decay activity to regulate root growth. Another explanation is that IRE1 mediates root growth through protein-protein interaction. I also showed that a mutation of a component of G-protein complex, AGB1, enhances the ER stress and the short-root 125 phenotype in ire1, suggesting that the AGB1 regulation on the plant UPR does not completely rely on IRE1. Future directions The mutant analyses of ire1 agb1 triple mutant demonstrate that the functional relationships between UPR transducers can be established in vivo in plants. To further investigate the plant UPR signaling network, future experimental approaches are presented here. In addition of IRE1, two membrane-anchored transcription factors, bZIP17 and bZIP28, regulate the plant UPR. How these two distinct UPR signaling pathways are coordinated is not clear yet. The proteolytic cleavage of bZIP17/bZIP28 and bZIP60 mRNA splicing are the molecular indicators of bZIP17/bZIP28 and IRE1 activation respectively. To determine whether bZIP17/bZIP28 or IRE1 has differential contributions on the UPR, their activated status can be monitored under various intensities and periods of ER stress treatment. Also, regulatory roles of bZIP17/bZIP28 and IRE1 in ER-stress triggered cell death have not been examined yet. To do so, an experimental system for a time course of prolonged ER stress treatment should be established. Once a reliable experimental setting is available, monitor of bZIP17/bZIP28 and IRE1 activation over the time course will shed lights on their involvement in regulation of cell death. Also, comparison of UPR target gene induction and cell death activation in bzip17/bzip28 and ire1 will potentially determine whether bZIP17/bZIP28 or IRE1 functions as a molecular switch of cell fate on severe ER stress. To identify mediators of ER stress-triggered cell death, transcriptomic analyses at time points covering prior and 126 after cell death initiation can be performed in wild-type Col-0, bzip17/bzip28, and ire1. The studies can also determine that whether bZIP17/bZIP28 or IRE1 transcriptionally regulates identified cell death mediators. Finally, functional relationships between the two types of plant ER stress sensors have not to be established yet. Generation of higher-order mutants of bZIP17/bZIP28 and IRE1 and perform phenotypic analyses in the above proposed experiments will define regulatory relationships between two UPR signaling arms. In case disruption of the two parallel UPR pathways leads to lethality in plants, an artificial miRNA method can be adopted to generate knock-down mutants of bZIP17/bZIP28 or IRE1. The observation that ire1 displays only the short-root phenotype but shows comparable shoot morphology with wild-type Col-0 indicates that IRE1 has tissuespecific role in root growth. More specifically, IRE1 is likely involved in the regulation of rapid elongation of root cells. How IRE1 senses the specialized secretory activity is unknown. One possibility is existence of cell type- or stage-specific IRE1-interacting proteins. To identify IRE1-interacting proteins in a context-specific manner, generation of epitope-tagged IRE1 under its native promoter will enable in vivo screening using specific tissues and growth stages. Another potential in vivo approach to detect IRE1interacting proteins is to use Arabidopsis transgenic lines expressing IRE1 under control of an inducible promoter. After induction of IRE1 expression, blue native/SDS gel electrophoresis can be performed to detect whether IRE1 forms a protein complex. Alternatively, a yeast two-hybrid system using tissue-specific prey libraries may overcome challenges regarding potentially low expression of IRE1-interacting proteins. 127 In addition, how IRE1 activates the appropriate downstream response to support specific secretory activity is still unclear. One tempting model is that plant IRE1 may also process multiple types of RNA substrates according to need. To identify additional IRE1 substrates in a context-specific manner, a genome-wide screening of IRE1interacting RNA can be performed in a tissue- or stage-specific manner. RNAimmunoprecipitation using IRE1 as bait and quantitative measurements of transcriptomes by deep RNA sequencing methods may be able to identify tissuespecific substrates of plant IRE1. Because interaction between IRE1 and its RNA substrates might be transient, an RNase inactive form of IRE1 can be used as a bait to potentially increase the duration of their physical interaction. The Inter-regulation of UPR and Auxin Signaling In Chapter 4, I established the inter-regulation of the UPR and auxin signaling. I found that ER stress modulates the transcription of auxin receptors and transporters. In addition, intra-cellular auxin transporters are required for the optimal induction of UPR target genes. The results support the suggestion that plants may regulate the auxin response to coordinate the growth regulation and stress adaption. Moreover, the requirement of IRE1 in the auxin homeostasis and response implies that the auxin signaling at least partially relies on the UPR-dependent secretory pathway. Future directions Because IRE1 and TIR1/AFBs are not essential for ER stress-triggered repression of auxin regulators, novel ER stress mediators involved in the auxin 128 regulation may exist. To identify genes involved in repression of auxin signaling on ER stress, ethyl methanesulfonate (EMS) mutagenesis can be performed using Arabidopsis expressing auxin reporters. Screening of mutants showing unaltered auxin signaling on ER stress may identify novel regulators of the UPR and auxin signaling. It is also possible that specific transcriptional regulators are responsible for down-regulation of auxin receptors and transporters on ER stress. To identify proteins that bind to promoters of auxin regulators on ER stress, a yeast one-hybrid screening system using the PIN5 promoter and Arabidopsis cDNA or transcription factor libraries can be performed. Although yeast fails to survive on severe ER stress, experimental conditions allowing yeast survival and certain extent of UPR activation can be established by testing appropriate ER stress intensities. The observation that the PIN5 is involved in the optimal induction of UPR target genes hints the possibility that ER stress regulates the PIN5 activity. ER stress may influences protein modifications, localization, or protein-protein interaction of PIN5. To monitor PIN5 protein, antibodies against PIN5 endogenous protein or epitope-tagged PIN5 under control of its native promoter can be generated. To determine whether PIN5 associates or disassociates with a protein complex in vivo, similar approaches mentioned above for identification of IRE1-interacting proteins can be conducted. Also, how PIN5 transmits stress signals to impact the UPR activation is not clear. One tempting hypothesis is that PIN5 induces auxin transport between subcellular compartments on ER stress. Because detection assays of auxin levels in the ER have not been established yet, an indirect but achievable approach is using a yeast efflux assay to test whether ER stress triggers auxin transport in vitro. 129 Using a strong ER stress inducer Tm, all the tested mutants of ER-localized auxin regulators show compromised UPR activation phenotype. Because the ERlocalized auxin regulators differentially express, it is possible that individual ER-localized auxin regulator has a unique role in UPR-related stress adaption or growth regulation. It would be interesting to explore that whether ER-localized auxin regulators are involved in specific UPR-related regulations, such as heat and biotic stress. To investigate molecular mechanisms how IRE1 mediates the auxin response and levels, IRE1-interacting proteins or RNA substrates under exogenous auxin treatment can be screened using the approaches mentioned above. Another possible mechanism underlying the IRE1 regulation on the auxin response is that IRE1 mediates the transcriptional reprogramming of auxin regulators. Comparison of transcriptomic profiling between wild-type Col-0 and ire1 under exogenous auxin treatment will determine whether IRE1 controls auxin regulators at the transcriptional levels. Also, to learn whether IRE1 mediates the auxin response by regulation of mobilization of auxin regulators, fluorescently tagged auxin regulators under control of their native promoters can be introduced into ire1. Thus, mobilization of auxin regulators can be compared between wild-type Col-0 and ire1. The Significance of Plant UPR in Cellular Function My studies support the suggestion that the plant UPR is involved in primary root growth and auxin regulation. Because the UPR is fundamental for the secretory pathway, it is plausible that other biological processes dependent on the secretory pathway also closely associate with the UPR. 130 Future directions The ER not only supports synthesis of secretory proteins, but also maintains many essential metabolic processes such as calcium storage and lipid/membrane production. As the UPR is essential for ER function, ER-dependent cellular regulation may also rely on the UPR. It would be interesting to explore functional relationships between ER-related biological processes and the plant UPR. For instance, whether lipid biosynthesis is mediated by IRE1- or bZIP17/bZIP28-dependent UPR has not reported yet. One straightforward approach is to examine the lipid compositions in mutants of UPR transducers. Also, comparison of lipid profiling between mock and ER stresstreated wild-type plants will determine that whether regulation of lipid metabolism is one of mechanisms underlying the plant UPR. Likewise, as calcium signaling is vital for physiological and environmental responses, it would be interesting to test whether ER stress also regulates calcium homeostasis to coordinate stress adaption and growth regulation. Because calcium signaling is controlled by multiple protein kinases, one straightforward approach is to monitor the transcriptional and translational regulation of protein kinases important for calcium signaling on ER stress. If ER stress regulates components of calcium signaling pathways, phenotypic analyses of ER stress can be performed in mutants defective in calcium signaling. Likewise, calcium-signaling transmission can be monitored in mutants of UPR transducers to test whether the plant UPR is required for the calcium signaling regulation. 131 APPENDICES 132 APPENDIX A Analysis of unfolded protein response in Arabidopsis This section has been previously published in Methods in Molecular Biology YA-NI CHEN and FEDERICA BRANDIZZI (2013) Methods in Molecular Biology 1043:73-80 133 Abstract The unfolded protein response (UPR) is essential for development and adaption in eukaryotic cells. Arabidopsis has become one of the best model systems to uncover conserved regulatory mechanisms of the UPR in multicellular eukaryotes as well as unique UPR regulation in plants. Monitoring the UPR in planta is a fundamental approach to identifying regulatory components and to revealing molecular mechanisms of the plant UPR. In this chapter, we provide protocols for the plant UPR induction as well as the UPR activation analyses at a molecular level in Arabidopsis. Three kinds of ER stress treatment methods and quantitation of the plant UPR activation are described here. Introduction The unfolded protein response (UPR) is a collection of conserved signaling pathways aiming to maintain endoplasmic reticulum (ER) protein folding homeostasis in eukaryotic cells (UPR) [1, 2]. There is approximately one-third of protein initially folded and modified in the ER lumen. Environmental or physiological factors that cause an imbalance between ER protein folding demand and capability lead to ER stress. To relieve the ER stress, eukaryotic cells activate the UPR to increase the ER protein folding ability. Wide ranges of stimuli trigger the activation of the UPR. To experimentally examine the UPR, chemicals disturbing the ER protein folding homeostasis are applied to activate the UPR. One of the most frequently used UPR 134 inducers is a glycosylation inhibitor, tunicamycin (Tm). The majority of secretory proteins are glycosylated in the ER as the glycosylation is critical for protein structure formation and for protein targeting to cellular compartments. As Tm blocks the first step of N-linked glycosylation, it can efficiently lead to an accumulation of unfolded proteins in the ER lumen and therefore activate the UPR [163-165]. To observe the plant UPR at different plant stages, we describe three experimental approaches to perform ER stress treatment. To test long-term ER stress tolerance, seeds are directly germinated on medium containing a relatively low concentration of Tm. Tm can also been infiltrated into leaves to specifically monitor the UPR on ground tissues. Finally, to examine the early outputs of the plant UPR, a shortterm Tm treatment using a liquid method is conducted to observe a more direct effect from ER stress. To cope with dynamic ER protein folding demands, the UPR adjusts the transcription of regulators involved in assembling protein structure, degrading misfolded protein, and determining cell fates [19, 166]. Hence, the upregulation of wellestablished UPR target genes, such as BiP3 in Arabidopsis [39, 40, 167], is considered a molecular indicator of UPR activation. To introduce the quantitative method of reading UPR outputs, real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis of UPR target genes induction is included in this chapter. 135 Materials 1. Basic reagents and equipment for plant sterile tissue culture and RNA work handling. 2. Plant growth medium: Linsmaier and Skoog (LS) with Buffer and Sucrose (Caisson LSP04); Phytagel (Sigma P8169). 3. Growth chamber: temperature set to 21°C, 16 h light/ 8 h dark cycle, 100 m Einstein/m2 s and 65% humidity. 4. Tunicamycin (Sigma T7765). 5. Dimethyl sulfoxide (DMSO) solvent. 6. 1 ml needleless syringes. 7. Liquid nitrogen. 8. RNeasy plant mini kit (Qiagen 74904). 9. RNase-Free DNase Set (Qiagen 79254). 10. SuperScript® VILO™ Master Mix (Invitrogen 11755500). 11. Reagents for real-time PCR: MicroAmp® Fast optical 96-well reaction plate (ABI 4346936); optical adhesive cover (ABI 4311971); FAST SYBR Master Mix (ABI 4385612). 136 Methods ER stress tolerance assay To examine the tolerant ability of plants in coping with different intensities of ER stress, seeds are directly germinated on medium containing Tm concentrations ranging from 10-50 ng/ml. Comparison of phenotype between wild-type plants and mutants of interest can reveal whether the mutants display over-sensitive or resistant growth phenotype under ER stress conditions. The Tm infiltration assay enables the observation of the plant UPR using adult plants. 1. Sterilize seeds and store at 4 ° C for two days (see Note 1). 2. Prepare ½ LS with 0.4 % Phytagel medium. 3. Autoclave the ½ LS medium on liquid cycle program for 25-40 minutes. 4. Dissolve Tm powder in DMSO to prepare 10 mg/ml Tm stock solution (see Note 2). 5. Prepare 10, 20, 30, 40 and 50 µg/ml Tm stock solutions by 1000, 500, 333, 250, and 200X dilution of 10 mg/ml Tm stock solution respectively using ½ LS liquid medium. 6. Cool the autoclaved ½ LS medium to 50 ° C. 7. Add 10, 20, 30, 40, and 50 µg/ml Tm stock solutions respectively to cooled ½ LS medium (50 ° C) by 1000X dilution to make ½ LS medium containing 10, 20, 30, 40, and 50 ng/ml Tm (see Note 3). 137 8. Swirl to mix and use a pipet to pour equal amount of Tm-containing medium per plate in sterile tissue culture hood. Prepare Tm-containing medium freshly right before the Tm germination assay (see Notes 4). 9. For Mock control, the same preparation procedure is carried out with the exception of replacing the Tm in the ½ LS medium with 0.0005% DMSO. 10. Germinate Arabidopsis seeds on ½ LS medium containing 0.0005% DMSO, 10, 20, 30, 40, and 50 ng/ml Tm. Place a single seed on the medium at similar distance between each other (see Notes 5). At least three individual plates for each Tm concentration and mock control should be used. 11. Grow the plants under these conditions: 21°C, 16 h light/8 h dark cycle, 100 mEinstein/m2 s and 65% humidity. 12. Observe the growth phenotype 7-14 days after germination. (see Note 6) 13. Using Col-0 ecotype wild-type Arabidopsis, the plants shows more pronounced growth defects starting from 30 ng/ml Tm (see Figure. A.1) Tm infiltration into leaf tissues 1. Dissolve Tm powder in DMSO to prepare 10 mg/ml Tm stock solution (see Note2). 138 2. Prepare 15µg/ml Tm stock solution by 666 X dilution of 10 mg/ml Tm stock solution using ½ LS liquid medium. Prepare Tm-containing medium freshly right before the Tm infiltration assay (see Note 4). 3. Use a needleless syringe to infiltrate ½ LS liquid medium containing 15 µg/ml Tm into abaxial sides of five week-old rosette leaves (see Note 7). 4. For Mock control, the same treatment procedure is performed with the exception of replacing the Tm in the ½ LS liquid medium with 0.0015% DMSO (see Note 8). 5. Observe the leaves phenotype 1-4 days after infiltration. Short period of ER stress treatment While the ER stress tolerance assay can examine whether mutants of interest show plant phenotype under ER stress, even if the mutants display similar visible plant phenotype to wild-type plants, it is possible that the defects of UPR in mutants of interest do not reflect on the plant growth morphology. One of the examples is the mutant of AtbZIP60, a transcription factor confirmed as a UPR regulator [39, 76](10-11). To verify whether genes of interest are involved in the UPR, short-term ER stress treatment coupled with analyses of UPR target genes induction are performed to monitor the UPR phenotype at a molecular level. 1. Sterilize seeds and store at 4 ° C for two days (see Note 1). 139 2. Germinate seeds in vertical plates for ten days. Medium: half-strength LS with 0.4 % Phytagel. Place ten seeds, evenly spaced, per small round plate (100 X 15 mm) or square plate. Seal bottom part of plates with parafilm and upper part of plates with 3M surgical tape (see Figure A.2 and Note 9). 3. Dissolve Tm powder in DMSO to prepare 10 mg/ml Tm stock solution (see Note2). 4. Prepare 5µg/ml Tm-containing medium by 2000 X dilution of 10 mg/ml Tm stock solution using half-strength LS liquid medium. Prepare Tm-containing medium freshly right before the Tm treatment (see Note2). 5. Gently transfer ten day-old vertically grown seedlings to 5 µg/ml Tm-containing medium for an appropriate time period (see Notes 10 and 11). 6. Collect 10-20 individual Tm-treated seedlings per sample using liquid nitrogen (see Notes 12 and 13). 7. For Mock control, the same treatment procedure is performed with the exception of replacing the Tm in the half-strength LS liquid medium with 0.05% DMSO. Quantitative measurement of UPR activation The major output of the plant UPR identified so far is the regulation of UPR target genes transcription. Hence, measurement of UPR gene induction under ER stress is the most reliable method to quantify the plant UPR activation. 140 1. Extract RNA from Tm-treated seedlings using an RNeasy plant mini kit and RNaseFree DNase Set. 2. Synthesize CDNA from RNA using a SuperScript® VILO™ Master Mix. 3. Perform real-time PCR with SYBR Green detection in triplicate using the Applied Biosystems 7500 fast real-time PCR system. The primer sequence of UPR target genes is listed in Table A.1 [39]. 4. Analyze Data by the DDCT method. 141 Figure A.1. Plant phenotype under tunicamycin treatments. Wild-type Col-0 plants were germinated on half-strength LS medium containing DMSO, 10, 20, 30, 40, or 50 ng/ml Tm for 2 week. 142 Sealed with 3M seeds ! surgical tape ! ! ! ! ! ! ! ! ! ! ! Sealed with ! parafilm ! Figure A.2. Vertical growth of plant seedlings. The 3M surgical tapes and parafilm are used respectively to seal the upper and bottom part of vertical plates. 143 Table A.1. DNA primers of UPR target genes primers Sequence (5'–3') Gene BiP1/2-qP For ccaccggccccaagag AT5G28540/AT5G42020 BiP1/2-qP Rev ggcgtccacttcgaatgtg AT5G28540/AT5G42020 BiP3-qP For aaccgcgagcttggaaaat At1g09080 BiP3-qP Rev tcccctgggtgcaggaa At1g09080 AtERdj3A-qP For tcaagtggtggtggtttcaact AT3G0890 AtERdj3A-qP Rev cccaccgcccatattttg AT3G0890 AtERdj3B-qP For gaggaggcggcatgaatatg At3g62600 AtERdj3B-qP Rev ccatcgaacctccaccaaaa At3g62600 PDI6-qP For cgaagtggctttgtcattcca AT1G77510 PDI6-qP Rev gcggttgcgtccaatttt AT1G77510 PDI9-qP For ggccctgttgaagtgactgaa AT2G32920 PDI9-qP Rev cagcagaaccacacttcttttcc AT2G32920 CNX1-qP For gtgtcctcgtcgccattgt AT5G61790 CNX1-qP Rev ttgccaccaaagataagcttga AT5G61790 CRT1-qP For gatcaagaaggaggtcccatgt AT1G56340 CRT1-qP Rev gacggaggacgaaggtgtaca AT1G56340 ATERDJ2A-qP For tgggcttgtaggcgctctt At1g79940 ATERDJ2A-qP Rev aacccaatagttttcctccttgtg At1g79940 ATERDJ2B-qP For tgaaacgtcccaatggactca At4g21180 144 Table A.1 (cont’d) ATERDJ2B-qP Rev cctctttgtggaaaggaaagtaagg At4g21180 ATP58IPK-qP For gcgttatagtgatgccctcgat AT5G03160 ATP58IPK-qP Rev gaaagcgcagggtctgctt AT5G03160 145 Notes 1. The quality of seed stock is very important for ER stress related assays. Using seeds freshly harvested from healthy plants is one of key points to get reproducible and consistent results. 2. Aliquot Tm stock solution (10 mg/ml) into relatively small amount and store in a -20° C freezer. Avoid freezing and thawing. 3. High temperature destabilizes Tm. 4. Tm-containing medium is unstable if it is not freshly prepared. 5. For a fair comparison, the distance between seeds should be consistent. Square petri dish with Grid (Fisher 08-757-11A) is useful, as a single seed can be placed on the center of each small square area on the plates (see Fig1). 6. The growth phenotype can be more obvious at a relative early stage (within one week) or vice versa. To observe growth-stage-specific phenotypes, the Tm-treated seedlings should be check every day during the assay. 7. To fairly compare the ER tolerance between wild-type and mutant plants, choose the same stage, size, and condition of leaves for both varieties of plants. 8. The infiltration process needs be done carefully and should not lead to any damage of plants. Leaves infiltrated with DMSO (Mock control) should look similar to leaves without infiltration after one day. 146 9. To allow proper ventilation, do not wrap plates completely with parafilm. 10. If mutants of interest display normal growth phenotype as wild-type plants, choose healthy and unstressed seedlings as well as similar growth morphology for all plants to perform the treatment. 11. Using ten day-old seedlings coupled with a real time PCR system, the induction of UPR target genes can be detected from 0.5 to 16 hr. Prolong treatment is not recommended using this liquid system. 12. Using more seedlings per biological sample can reduce the standard deviation of fold change of UPR gene induction between biological replicates. 13. Sample collection should be done carefully and timely to avoid additional stress before seedlings are frozen by liquid nitrogen. Acknowledgments We acknowledge support by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy (award number DE-FG02-91ER20021) and the National Aeronautics and Space Agency (NNH08ZTT003N NRA–08-FSB_Prop-0052). 147 APPENDIX B Published manuscripts 148 1. Chen Y and Brandizzi F. IRE1: ER stress sensor and cell fate executor. Trends in cell biology 2013 (online preview) 2. Chen Y and Brandizzi F. Analysis of Unfolded Protein Response in Arabidopsis. Methods in Molecular Biology 2013; 1043:73-80 3. Chen Y, Aung K, Rolčík J, Walicki K, Friml J, and Brandizzi F. Inter-regulation of the unfolded protein response and auxin signaling. The plant journal 2013 (preview on line) 4. Chen Y and Brandizzi F. AtIRE1A/AtIRE1B and AGB1 independently control two essential unfolded protein response pathways in Arabidopsis. The plant journal 2012; 69:266-277. 5. Faso C*, Chen Y*, Tamura K*, Held M, Zemeli S, Marti L, Saravanan R, Hummel E, Kung L, Miller E, Hawes C, Brandizzi F. A missense mutation in the Arabidopsis COPII coat protein Sec24A induces the formation of clusters of the endoplasmic reticulum and Golgi apparatus. The Plant Cell 2009; 21:3655-3671. *These authors contributed equally to the work. 6. Chen Y*, Slabaugh E*, and Brandizzi F. Membrane-tethered transcription factors in Arabidopsis thaliana: novel regulators in stress response and development. Current Opinion in Plant Biology 2008; 11:695-701. *These authors contributed equally to the work. 7. Srivastava R, Chen Y, Deng Y; Brandizzi F and Howell S. Elements proximal to and within the transmembrane domain mediate the organelle-to-organelle movement of bZIP28 under ER stress conditions. The plant journal 2012; 6:1033-1044 8. Conger R, Chen Y, Fornaciari S, Faso C, Held M, Renna L and Brandizzi F. Evidence 149 for the involvement of the Arabidopsis SEC24A in male transmissions. Journal of Experimental Botany 201;62(14):4917-26 9. Moghe GD, Lehti-Shiu MD, Seddon AE, Yin S, Chen Y, Juntawong P, Brandizzi F, Bailey-Serres J, Shiu SH. Characteristics and significance of intergenic polyadenylated RNA transcription in Arabidopsis. Plant Physiology 2013; 161:210-224 10. Moreno AA, Mukhtar MS, Blanco F, Boatwright JL, Moreno I, Jordan M, Chen Y, Brandizzi F, Dong X, Orellana A, Pajerowska-Mukhtar K. IRE1/bZIP60-Mediated Unfolded Protein Response Plays Distinct Roles in Plant Immunity and Abiotic Stress Responses. PLoS ONE 2012; 7(2) 150 REFERENCES 151 REFERENCES 1 Kozutsumi, Y., et al. (1988) The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 332, 462-464 2 Schroder, M. and Kaufman, R.J. (2005) The mammalian unfolded protein response. 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