MECHANISMS OF UNFOLDED PROTEIN RESPONSE SIGNALING IN PLANTS By Ya-Shiuan Lai A DISSERTATION Submitted to Michigan State University in partial fulfillment of requirements for the degree of Cell and Molecular Biology-Doctor of philosophy 2018 ABSTRACT MECHANISMS OF UNFOLDED PROTEIN RESPONSE SIGNALING IN PLANTS By Ya-Shiuan Lai The endoplasmic reticulum (ER) is responsible for the synthesis of one third of the cellular proteome. A potentially lethal condition termed as ER stress occurs when environmental and physiological stimuli alter the protein-folding and glycosylation activities of the ER. A complex system of signaling pathways known as the unfolded protein response (UPR) is in place to alleviate stress. Unresolved ER stress due to inefficient or dysfunctional UPR leads to cell death. In plants, there are two distinct UPR signaling pathways. The first pathway involves the ER-membrane- bound transcription factor basic Leucine Zipper 28 (bZIP28), which is activated by proteolysis in the Golgi with consequent relocation of the active transcription factor domain to the nucleus. The second pathway is controlled by the Inositol Requiring Enzyme-1 (IRE1), an ER-anchored membrane kinase and ribonuclease (RNase). In conditions of ER stress, IRE1 oligomerizes, triggering autophosphorylation and activation of its RNase domain in the cytosol. This in turn splices the mRNA of the transcription factor bZIP60. The active forms of bZIP28 and bZIP60 translocate to the nucleus, resulting in the regulation of downstream genes that are involved in protein folding and ER quality control (ERQC). The UPR response could be induced by the defects of ER morphology that affect the proper ER functionality, or by environmental stresses. To follow up the investigation on these two aspects, first the ER ability to trigger UPR in the various ER defective mutants was determined in model plant, Arabidopsis. A specific mutation in the formation of ER tubules caused by the loss of function of RHD3 showing attenuated RNase activity of IRE1 regulated UPR reduction was characterized. It also identified RHD3 as a new UPR modulator in plants. UPR also get activated in response to environmental stresses. In these conditions, plants switch on distinct stress signaling pathways, which frequently coordinate with each other through shared signaling molecules. Upon pathogen-caused biotic stress, the elevated salicylic acid (SA) initiates the stress signaling in which the activation of the UPR machinery serves as a part of defense response to ensure the ER’s capability to cope with the rapid demand of antimicrobial protein production. Therefore, the involvement of SA and SA-mediated signaling molecules in the ER stress response was further explored by testing the UPR response in mutants defective in SA production and SA-responsive cofactor, respectively, under ER stress. Subsequently, a SA- independent role of nonexpressor of PR1 genes 1 (NPR1) —a master regulator in SA-mediated defense—in the UPR was identified. In response to ER stress-induced cytosolic reduction, NPR1 translocates to the nucleus, whereby it interacts with bZIP28 and bZIP60 and negatively regulates UPR activation. Ultimately, this study uncovered another novel UPR transducer involved in the ER surveillance system. UPR signaling throughout the whole organism is a complicated regulation network and intensive research were done at a cellular level. However, UPR signaling transmitted at an organismal level in a cell non-autonomous manner in metazoans and it is unknown in plants. Consequently, the existence of long-distance UPR signaling was evaluated through genetic approaches in combination with multiple molecular reporters and conventional micro-grafting. Toward the end, the systemic UPR regulation mediated by intracellular movement of bZIP60 was established which further extends current knowledge on the mechanism of UPR signal transduction. ACKNOWLEDGEMENTS First, I would like to thank my advisor Federica Brandizzi. She has been supportive since the day I joined the lab. I appreciate her time, contribution of thoughts and financial support to make my Ph.D career productive and inspiring. She is a very determined mentor. During the long waiting process for the paper review, she was the only person who had faith in me and encouraged another attempt. After I was more experienced, she allowed me to pursue new directions and design my projects independently. Additionally, she always reminded me to criticize my work from the point of view of a reviewer, an attitude that definitely will benefit me in my research. I would also like to thank the members of the guidance committee, Dr. Christoph Benning, Dr. Sheng Yang He, Dr. Eva Farre and Dr. Curtis Wilkerson for their stimulating discussions and insightful suggestions. My Ph.D career would not be fruitful without their expertise, support, and encouragement. I also would like to thank all Brandizzi lab members for their companionship. They treated me very nice and offered enormous help and valuable suggestions on my projects. I especially thank Dr. Giovanni Stefano, Dr. Luciana Renna, Dr. Christina Ruberti and Dr. Sang-Jin Kim for their helpful discussions and advice, Dr. Noelia Pastor Cantizano for the comfort and encouragement during the preparation period for graduation, and Elena Michel, Jack Yarema and Liz Gibbons for their assistance in experiments in the past six years. I was recruited by Cell and Molecular Biology department and DOE-Plant Research Laboratory program. I would like to acknowledge the CMB director Sue Conrad for career advice; the past CMB secretary Becky Mansel, present secretary Alaina Burghardt, and PRL staff Carol Wood for their help in making my Ph.D life smooth. iv At the end, my gratitude goes to my family and friends. I appreciate my parents for many years of love, and financial support. I owe them so many thanks for their understanding and support, regardless of the decisions I made in both my career and life. Meanwhile, I am grateful for the precious time spent with my friends, which became my biggest spiritual support and enriched my life in MSU. v TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x CHAPTER I .................................................................................................................................... 1 INTRODUCTION ....................................................................................................................... 1 The impacts of environmental stimuli and physiological alterations on elicitation of ER stress .................................................................................................................................... 2 Structure and function of the endoplasmic reticulum ...................................................... 2 Overview of ER stress- Effects of environmental milieu and physiological change ...... 6 Summary of UPR signaling from metazoans to plants .................................................... 8 Significance of dysregulated UPR on plant growth and development .......................... 11 The cross-regulations between UPR signaling and other stress signaling ........................ 13 The chemical drugs commonly used as ER stress inducers........................................... 13 The surveillance system in the UPR .............................................................................. 14 Examples of functional sharing between the UPR and distinct stress-signaling routes 15 Role of systemic signaling in plants and an overview of systemic UPR .......................... 17 Outline and significance of long-distance (systemic) signaling in plants ..................... 17 The significance of vascular tissue in long-distance signaling ...................................... 19 The regulation of plasmodesma (PD) involved in cell-to-cell communication ............. 23 Instances of systemic UPR in multicellular organisms ................................................. 25 Rationale for study ............................................................................................................ 26 APPENDIX ............................................................................................................................... 28 REFERENCES .......................................................................................................................... 36 CHAPTER II ............................................................................................................................... 50 A specific defect of ER architecture disrupts ER function to invoke UPR ................................ 50 ABSTRACT .............................................................................................................................. 51 INTRODUCTION ..................................................................................................................... 52 MATERIALS AND METHODS .............................................................................................. 56 Plant Materials, Growth Conditions, and Tm treatment ................................................... 56 AGI numbers ..................................................................................................................... 56 RNA Extraction and quantitative RT–PCR (qRT–PCR) analysis .................................... 57 Phenotypical Analyses ...................................................................................................... 57 RESULTS ................................................................................................................................. 58 Defects in ER architecture negatively influence UPR activation in a background specific- manner............................................................................................................................... 58 RHD3 and AtIRE1 work dependently in the UPR ........................................................... 60 RHD3 is necessary for efficient splicing of bZIP60 mRNA ............................................ 61 The involvement of RHD3 in ER stress responses and in physiological growth is uncoupled .......................................................................................................................... 62 DISCUSSION ........................................................................................................................... 63 The UPR signaling function of AtIRE1 depends on the presence of RHD3 in the ER vi membrane .......................................................................................................................... 63 Among the Arabidopsis RHD3 isoforms, RHD3 likely functions as a dominant gene in the UPR signaling ................................................................................................................... 65 The dependence of AtIRE1 on RHD3 for UPR signaling is uncoupled from their roles in organ growth .................................................................................................................... 65 A causative relationship between loss of RHD3 genes and defective UPR signaling may explain the lethality of high order RHD3 mutations ....................................................... 66 ACKNOWLEDGEMENTS ...................................................................................................... 68 APPENDIX ............................................................................................................................... 69 REFERENCES .......................................................................................................................... 81 CHAPTER III ............................................................................................................................... 87 A salicylic acid-independent role of NPR1 is required for protection from proteotoxic stress in the plant endoplasmic reticulum .................................................................................................. 87 ABSTRACT .............................................................................................................................. 88 INTRODUCTION ..................................................................................................................... 89 MATERIALS AND METHODS .............................................................................................. 93 Lines and Plant Growth Conditions .................................................................................. 93 Tm, SA and AZC Treatment ............................................................................................. 93 RT-PCR and qRT-PCR Expression Analyses .................................................................. 94 Plasmid Construction ........................................................................................................ 94 Confocal Laser Scanning Microscopy Imaging and Ratiometric Assays ......................... 95 Isolation of Nuclei ............................................................................................................. 96 Protein Extraction and Western Blot analyses .................................................................. 96 FRET Assay ...................................................................................................................... 97 Yeast two-Hybrid Analyses .............................................................................................. 97 Salicylic Acid Measurements ........................................................................................... 98 Hoechst Staining ............................................................................................................... 98 RESULTS ................................................................................................................................. 99 The Loss of NPR1 Increases Resistance to Chronic ER Stress ........................................ 99 NPR1 Negatively Modulates UPR Activation under ER stress...................................... 100 NPR1-mediated Attenuation of the Plant UPR is Independent of Endogenous SA Levels and Partially Intersects with the TBF1 signaling ............................................................ 101 ER Stress Causes Reduction of the Redox Potential of the Cytosol .............................. 103 ER Stress Induces Translocation of NPR1 from the Cytosol to the Nucleus ................ 106 NPR1 Physically and Genetically Interacts with bZIP28 and bZIP60 .......................... 107 NPR1 Functions as a Negative Regulator of the UPR under ER stress ........................ 109 DISCUSSION ......................................................................................................................... 111 ACKNOWLEDGEMENTS .................................................................................................... 116 APPENDIX ............................................................................................................................. 117 REFERENCES ........................................................................................................................ 139 CHAPTER IV ............................................................................................................................. 147 A systemic signaling contributes to the unfolded protein response of the plant endoplasmic reticulum… ................................................................................................................................ 147 ABSTRACT ............................................................................................................................ 148 vii INTRODUCTION ................................................................................................................... 149 MATERIALS AND METHODS ............................................................................................ 152 Lines and Plant Growth Condition ................................................................................. 152 Shoot-Root Split Culture System .................................................................................... 152 qRT-PCR Expression Analyses ...................................................................................... 152 Tunicamycin Measurements ........................................................................................... 153 Grafting Experiment ....................................................................................................... 153 Plasmid Construction ...................................................................................................... 154 Confocal Laser Scanning Microscopy Imaging .............................................................. 154 Quantification of Protein Colocalization at PD .............................................................. 155 Propidium Iodide Staining .............................................................................................. 155 GUS staining ................................................................................................................... 155 Aniline Blue staining ...................................................................................................... 155 RESULTS ............................................................................................................................... 157 Spliced bZIP60 Translocates Transcellularly ................................................................. 157 Systemic Induction of the UPR genes ............................................................................ 160 ER Stress Response Acts Systemically in a Shoot-ward Direction ................................ 162 Root-Derived Signals are Involved in the Induction of UPR genes in Unchallenged Tissues............................................................................................................................. 165 Systemic UPR Signaling is Plasmodesmata-Dependent ................................................. 167 DISCUSSION ......................................................................................................................... 170 The Plant UPR Constitutes a Systemic Signal ................................................................ 170 The Intercellular Movement of bZIP60 is a component of Long-distance UPR Signaling ......................................................................................................................................... 171 A Plasmodesma-Regulated Symplastic Transport Contributes to Long-distance UPR Signaling ......................................................................................................................... 173 Long-Distance ER Stress Signaling may Aid Stress Anticipation ................................. 175 ACKNOWLEDGEMENTS .................................................................................................... 177 APPENDIX ............................................................................................................................. 178 REFERENCES ........................................................................................................................ 198 CHAPTER V .............................................................................................................................. 205 Future perspectives ..................................................................................................................... 205 RHD3 in ER stress signaling .......................................................................................... 206 The multiple roles of NPR1 in biotic and abiotic stress ................................................. 209 The systemic UPR response in plants and animals ......................................................... 211 REFERENCES ........................................................................................................................ 215 viii LIST OF TABLES Table 2. 1. Primers used in this study ........................................................................................... 70 Table 3. 1. Primers used in this study ......................................................................................... 118 Table 4. 1. Primers used in this study ......................................................................................... 179 ix LIST OF FIGURES Figure 1. 1. Different structural subdomains of the Endoplasmic Reticulum ..............................29 Figure 1. 2. Unfolded protein response (UPR). ...........................................................................30 Figure 1. 3. The features and topology of UPR modulators ........................................................31 Figure 1. 4. UPR signaling pathway in mammals ...................................................................... 32 Figure 1. 5. UPR signaling pathway in plants. .............................................................................33 Figure 1. 6. The systemic response in plants: SAR (systemic acquired resistance) and SAA (systemic acquired acclimation). ..................................................................................................34 Figure 1. 7. Study overview. ........................................................................................................35 Figure 2. 1. Mutants with defects in ER organization and/or function show an ER stress response similar to wild type ........................................................................................................72 Figure 2. 2. RHD3 has compromised UPR ..................................................................................73 Figure 2. 3. RHD3 and IRE1 work dependently in UPR signaling .............................................74 Figure 2. 4. Loss of rhd3 results in reduced splicing of bzip60 mRNA under ER stress. ...........75 Figure 2. 5. Analyses of ire1/ rhd3 mutant support a synergistic interaction between RHD3 and IRE1 in the control of primary root elongation. ...........................................................................76 Figure 2. 6. Loss of RHD3 does not affect the basal levels of the UPR ......................................78 Figure 2. 7. Loss of RHD3 does not affect the mRNA abundance of genes encoding secretory and cytosolic proteins in conditions of ER stress .........................................................................79 Figure 2. 8. The compromised UPR induction phenotype is specific to the loss of RHD3 ........80 Figure 3. 1. The loss of NPR1 confers resistance to prolonged ER stress .................................122 Figure 3. 2. NPR1 negatively modulates UPR activation under ER stress ................................123 Figure 3. 3. Reduction of roGFP2 in the cytosol during ER stress .......................................... 124 Figure 3. 4. ER stress promotes NPR1 nuclear translocation .................................................. 125 Figure 3. 5. NPR1 interacts with bZIP28 and bZIP60 ............................................................. 127 Figure 3. 6. NPR1 antagonizes the UPR .................................................................................. 128 x Figure 3. 7. The loss of NPR1 does not affect the aerial growth under ER stress .....................130 Figure 3. 8. NPR1 represses the expression of ER resident genes that are shared partially with the SA/TBF1 signaling ...............................................................................................................131 Figure 3. 9. ER stress does not influence SA-dependent signaling and SA content ..................132 Figure 3. 10. TBF1 does not modulate late-adaptive UPR activation upon ER stress...............133 Figure 3. 11. UPR activation causes cytosolic reduction ...........................................................134 Figure 3. 12. NPR1 trans-localizes to the nucleus under ER stress ...........................................136 Figure 3. 13. NPR1 interacts with bZIP28 and bZIP60 genetically in the UPR signaling ........137 Figure 3. 14. Lines overexpressing NPR1. ................................................................................138 Figure 4. 1. Intercellular translocation of sbZIP60 induces BiP3 expression in systemic tissues ....................................................................................................................................................181 Figure 4. 2. Root-expressed sbZIP60 transcripts are translocated to the shoot .........................183 Figure 4. 3. Local induction of ER stress ignites the UPR systemically, mostly in a shoot-ward direction .................................................................................................................................... 184 Figure 4. 4. Reciprocal micro-grafting analyses with UPR-able and UPR-deficient backgrounds support the existence of endogenous systemic signals in the plant UPR ...................................186 Figure 4. 5. Long-distance of UPR signaling relies on the PD availability ...............................188 Figure 4. 6. Subcellular localization of GFP-bZIP60 in the root under physiological conditions and induced ER stress conditions ...............................................................................................189 Figure 4. 7. Stele-expressed sbZIP60 distributes throughout the root ..................................... 190 Figure 4. 8. ER stress-induced BiP3 expression occurs throughout the root ............................191 Figure 4. 9. pRoot drives expression of GLYT specifically in the root ..................................... 192 Figure 4. 10. Distribution of Tm in bzip28/60 ...........................................................................193 Figure 4. 11. Transcriptomic kinetic response of systemic UPR signaling ...............................194 Figure 4. 12. Assessment of endogenous systemic transcripts in the plant UPR .......................195 Figure 4. 13. PD-associated bZIP60 is a mobile transcription factor moving intercellularly ....197 xi KEY TO ABBREVIATIONS AMPK- AMP-activated protein kinase AP1- APETALA1 ASK1- Apoptosis signal-regulating kinase 1 ATF4- Activating transcription factor 4 ATF6- Activating Transcription Factor 6 ATL- ATLASTIN AzA- azelaic acid AZC- azetidine-2-carboxylate Bax- BCL2-associated X protein BFA- brefeldin A BI-1- Bax inhibitor BiP-binding protein bZIP-basic leucine zipper CALS- callose synthase cAMP- Cyclic adenosine monophosphate CC- companion cells CEP- c terminally encoded peptide CEPR1- CEP receptor CHOP- transcription factor C/EBP homologous protein CK- cytokinin COP- coat protein CRAC- Ca2+ release-activated channels DAG- diacyl-glycerol DFHO- 3,5-difluoro-4-hydroxybenzylidene imidazolinone-2-oxime xii DTT- dithiothreitol eIF−eukaryotic initiation factor 2 ER- endoplasmic reticulum ERAD- ER-associated degradation ERGIC- ER-Golgi intermediate compartment ERK- Extracellular signal-regulated protein kinases ERSE- ER stress response element ETI- effector-triggered immunity FT- FLOWERING LOCUS T G3P- glycerol-3-phosphate GlcNAc-1-P- N-acetylglucosamine-1-phosphate GPCR- G protein-coupled receptor GPT- GlcNAc phosphotransferase HSR- heat-shock response HY5- hypocotyl 5 INM- inner nuclear membrane IP3- inositol 1,4,5-triphosphate IP3R-IP3 receptors IRE- Inositol-Required Enzyme ISR- Induced Systemic Resistance JA-jasmonic acid JNK- Jun N-terminal kinases KASH- Klarsicht, ANC-1, Syne homology KN1- KNOTTED1 Lnp- Lunapark MAPK- Mitogen-Activated Protein Kinase MP- movement protein NE- nuclear envelope xiii NF- nuclear factor NPC- nuclear pore complex OCTR1- octopamine G-protein-coupled catecholamine receptor ONM- outer nuclear membrane PCD- programmed cell death PD- plasmodesma PERK- protein kinase RNA-like ER kinase PGC1− peroxisome proliferator-activated receptor gamma, coactivator 1  PHO2- phosphate 2 PI- phosphatidylinositol PiP- pipecolic acid PIP2- phosphatidylinositol 4,5-bisphosphate PIS1- CDP-diacylglycerol--inositol 3- phosphatidyltransferase 1 PLC- phospholipase C POMC- pro-opiomelanocortin PR1- pathogenesis-related protein 1 PTM- posttranslational modification RFP- red fluorescent protein RHD3- root hair defective 3 RIDD- IRE1 dependent RNA decay RL2- RHD3-LIKE 2 ROS- reactive oxygen species RTNLB13- reticulon like protein B13 RyRs- ryanodine receptors S1P- site-1 protease S2P- site-2 protease xiv SA- salicylic acid SAA- systemic-acquired acclimation SAM- shoot apical meristem SAR- systemic acquired resistance SCR- SCARECROW SD- short day SE- sieve tube elements SERSS- secreted ER stress signal Sey1p- synthetic enhancer of yop1p SHR- SHORT-ROOT SP- signal peptide SRP- signal recognition particle STIM1- Stromal interaction molecule 1 SUN- Sad1p, UNC-84 TBF1- TL1-binding TF TF-transcription factor TG- thapsigargin Tm- tunicamycin TMH- tandem transmembrane hairpin TMV- tobacco mosaic virus TRAF2- tumor necrosis factor receptor-associated factor 2 TSF- TWIN SISTER OF FT TTG1- TRANSPARENT TESTA GLABRA1 UNC-13- uncoordinated 13 protein UPR- unfolded protein response WFS1- Wolfram syndrome 1 XBP1- X-box binding protein 1 xv XIP1- xylem intermixed with phloem 1 XSP10- xylem sap protein 10 kDa xvi CHAPTER I INTRODUCTION 1 The impacts of environmental stimuli and physiological alterations on elicitation of ER stress Structure and function of the endoplasmic reticulum In eukaryotes, the endoplasmic reticulum (ER) is the largest organelle within the cell and functions as a base for critical cellular processes such as protein synthesis, protein folding, lipid biogenesis, calcium storage, and interaction with other organelles (Braakman & Hebert, 2013; Clapham, 2007; Fagone & Jackowski, 2009a; Rapoport, 2007; Reid & Nicchitta, 2015; Westrate, Lee, Prinz, & Voeltz, 2015). To fulfill these multiple roles, the ER has a complex physical architecture of interconnected continuous membranes, including the nuclear envelope (NE) and the peripheral ER. Figure 1.1 illustrates these structures (Shibata, Hu, Kozlov, & Rapoport, 2009). The two-lipid-bilayer nuclear envelope allows RNAs and proteins of certain sizes to pass through the nuclear pores. The nuclear envelope extends to flat sheets forming part of the peripheral ER. The primary location for protein synthesis, folding, and post-translational modification is the cytosolic face of sheets with stuffed ribosomes, which is defined as rough sheets (Shibata et al., 2010; West, Zurek, Hoenger, & Voeltz, 2011). Conversely, fewer ribosomes bind to the membrane surface of ER tubules, defined as smooth tubules with curvatures, which constitute a dynamic structure of three-way junctions (West et al., 2011). In addition, calcium signaling, which contributes to muscle contractions, occurs in specialized cortical ER of the peripheral ER that exhibits an intermediate structure with highly curved and flat membranes (Block, Imagawa, Campbell, & Franzini-Armstrong, 1988; Takeshima, Komazaki, Nishi, Iino, & Kangawa, 2000). These examples illustrate the close correlation between function and morphology in the ER. 2 The morphology of the ER is tightly regulated in response to environmental cues and growth conditions. The ratio and distribution of ER tubules and sheets vary, based on the different cell types (Shibata, Voeltz, & Rapoport, 2006; West et al., 2011). Formation of the ER tubule network and the ER sheet network are coded for different sets of ER-shaping proteins. The mystery of the process of tubule formation was first uncovered by the discovery of reticulon proteins. Reticulon proteins possess two hydrophobic domains spanning the ER membrane, and they maintain the curvature of ER tubules (Voeltz, Prinz, Shibata, Rist, & Rapoport, 2006). In addition, the following tandem transmembrane hairpin (TMH)-containing proteins contribute to the tubule structure that mark the growing point of tubules: (1) ATLASTIN (ATL)/synthetic enhancer of yop1p (Sey1p)/root hair defective (RHD3) (metazoan/yeast/plants); (2) Lunapark (Lnp); and (3) Protrudin (Chen et al., 2015; Chen, Novick, & Ferro-Novick, 2012; Hu et al., 2009). Furthermore, the role of ATL/Sey1p/RHD3 in tubule formation has been characterized in which GTP binding induces protein dimerization followed by GTP hydrolysis that induces tubular membranes to merge (Bian et al., 2011; L. J.Byrnes & Sondermann, 2011; Laura J.Byrnes et al., 2013; Yan et al., 2015). Such membrane fusion is a critical step in maintaining the continuity of ER membranes and the tubule network during the dynamic process of ER morphogenesis. Given that maintenance of the shape of the ER is necessary for its primary function of protein synthesis, intensive research has focused on the formation of ER sheets. Thus, the establishment of the luminal space and the curvature at the edges of sheets has been explored, identifying the regulators that are partly overlapped with those involving in formation of the tubule network. For example, sheet-enriched proteins like Climp-63, kinectin, and p180 function as luminal spacers to the apposed membranes and they maintain the flatness of the ER sheets 3 (Klopfenstein et al., 2001; Shibata et al., 2010). In addition, the inner nuclear (INM) and outer nuclear membrane (ONM) are composed of partial ER sheets that rely on the nuclear pore complex (NPC) to keep curvature. Proteins localized in the nuclear membrane that are functionally similar to Climp-63, such as KASH and SUN, maintain the luminal space (Starr & Fridolfsson, 2010). The proper functioning of the ER depends on the successful development of the ER architecture. One major function of ER is synthesis, folding, and modification of secreted and integral membrane proteins. These processes rely on the canonical mechanism of co- translational event that initiates from docking the ribosome-mRNA complex to the cytosolic phase of the ER. During this process, the polypeptides of secreted and integral membrane proteins are synthesized, starting at the cytosol. These polypeptides are then recruited to the ER when the amino terminus signal peptide (SP) of the polypeptide is recognized by the signal recognition particle (SRP) on the ER membrane (Walter, Ibrahimi, & Blobel, 1981). After the mRNA:ribosome:peptide:SRP complex docks on the SRP receptor, the translation resumes on the ER and the mature polypeptide with the SP cleaved off enters the ER lumen through the translocon that spans the lipid bilayers (Deshaies, Sanders, Feldheim, & Schekman, 1991; Gilmore, Blobel, & Walter, 1982). Integral membrane proteins containing hydrophobic domains or stop-transfer membrane anchor sequences will be paused by translocons leading to the formation of single or multiple transmembrane proteins (Blobel, 1980), Instead, the proteins destined to enter the secretory pathway or luminal space of membrane-bound organelles will begin the transport process. The successful completion of translation allows either a release of ribosomes or a release of mRNA to the cytosol (Potter & Nicchitta, 2000; Seiser & Nicchitta, 2000). Following protein synthesis 4 and transport into the ER lumen, secretory proteins require folding and further modification, which is performed with the assistance of molecular chaperones and folding enzymes. ER luminal proteins such as chaperones need to be folded properly; however, the secretory proteins may proceed for particular modifications, such as N-linked glycosylation, disulfide bond formation, and oligomerization (Braakman & Hebert, 2013). The post-modified proteins undergo translocation to the Golgi, then to the final destination such as peroxisomes and plasma membrane whereby they proceed with their function (Van DerZand, Gent, Braakman, & Tabak, 2012). Nonetheless, under certain condition some proteins fail to be appropriately folded, forming misfolded proteins or aggregates (Hartl & Hayer-Hartl, 2009). Such proteins are either retained in the ER or are removed by a canonical ER-associated degradation (ERAD) pathway mediated by proteasome in the cytosol (Ruggiano, Foresti, & Carvalho, 2014). Scavenging the aggregates is tightly controlled to maintain ER homeostasis. Defects in the scavenging machinery activate the ER stress-response pathway, and may cause neurodegenerative protein misfolded disease, inflammatory bowel disease, and cancers. Investigation of the components of the ER stress-response pathway can thus help guide discovery of potential therapeutic targets (Ryno, Wiseman, & Kelly, 2013). Another function of ER is lipid biogenesis (Fagone & Jackowski, 2009b). Proteins and phospholipids, primary components of lipid membranes, are synthesized in the ER and Golgi and then modified in the ER-Golgi intermediate compartment (ERGIC). After modification, these lipids are dispersed throughout the cell through organelle contacts and vesicles (Appenzeller-Herzog, 2006). 5 In addition to providing biogenesis and transport of biomolecules, ER is also a critical site for calcium (Ca2+) storage to maintain cellular Ca2+ homeostasis (Eisen & Reynolds, 1985; Jaffe, 1983). At steady state, the Ca2+ concentration is around 100 nM in the cytosol and about 100–800 M in the ER lumen (Clapham, 2007; Samtleben et al., 2013). In response to certain stressors, the Ca2+ flux is rerouted from the ER to the cytosol through a serious of ER-associated Ca2+ channels coordinated with ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate (IP3) receptors (IP3R) (Clapham, 2007). During the process of Ca2+ release, the phospholipase C (PLC) is initiated through the activation of G protein-coupled receptor (GPCR) in the plasma membrane, followed by the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into IP3 and DAG (diacyl-glycerol). IP3 bound to IP3R stimulates release of Ca2+. This promotes an increase in intracellular Ca2+ levels (Clapham, 2007). RyRs works on Ca2+-induced Ca2+ release while cytoplasmic Ca2+ is increased (Endo, 2009). Once the ER luminal Ca2+ is depleted, the pathway regulated by STIM1 proteins and Ca2+ release-activated channels (CRAC) pumps extracellular Ca2+ back to the ER lumen, maintaining Ca2+ homeostasis (Feske et al., 2006; Zhang et al., 2006). Ca2+ functions as a cellular signaling molecule involved in multiple cellular responses. For instance, the salt stress-induced Ca2+ wave triggers rapid, root-to-shoot signaling in plants (Choi, Hilleary, Swanson, Kim, & Gilroy, 2016a). Additionally, disruption of Ca2+ signaling in both ER and cytosol caused by the loss of nutrients or energy triggers activation of the ER stress response (Krebs, Agellon, & Michalak, 2015). Overview of ER stress- Effects of environmental milieu and physiological change To maintain cellular homeostasis, the ER plays a crucial role in the balance between protein synthesis and protein folding, ensuring a functional protein network. Under stress conditions in the ER resulting from adverse environmental circumstances or physiological 6 growth transitions, the protein-folding machinery fails to meet the rapid enhanced demand for protein synthesis. This leads to the overaccumulation of misfolded or unfolded proteins in the ER lumen. The unfolded protein response (UPR) is a communication pathway between the ER and the nucleus to alleviate ER stress. The biological activities in the ER and the UPR are illustrated in Figure 1.2. Ultimately, if the UPR is unable to solve the overaccumulation of misfolded or unfolded proteins, the cell goes through cell-death. Therefore, the UPR plays dual roles in both pro-survival and pro-cell death programs (Tabas & Ron, 2011). Hostile environmental conditions triggering ER stress include abiotic stressors such as rising temperature, salinity stress, and oxidative stress. For example, rats exposed to heat get ER stress signals, but the heat-shock response (HSR) in the cortex and cortical neurons is blocked (Liu et al., 2012). Similarly, heat treatment also activates ER stress signaling in plants (Deng et al., 2011). Salinity stress, common in agricultural crop plants, induces both ER stress and cell- death (J.-X.Liu, Srivastava, Che, & Howell, 2007). Biotic stressors such as bacterial and viral infections activate ER stress to meet the corresponding demand of anti-pathogen secretory proteins (Moreno et al., 2012; D.Wang, Weaver, Kesarwani, & Dong, 2005). Under conditions of physiological alteration, cells also stimulate the ER stress response to cope with transition phases. For instance, through mediating and coordinating various cellular processes, ER helps tumor cells to adapt to many challenges, including dysregulated proliferation, hypoxia, oxidative stress, nutrient and lipid deprivation, and extracellular acidic PH (Mohamed, Cao, & Rodriguez, 2017). Physiological processes like aging also influence the protein-folding capacity of cells, inducing ER stress (Brown & Naidoo, 2012; Macario & DeMacario, 2002). In the reproductive phase of plants, appropriate ER-stress activation facilitates pollen growth and protects plants from unfavorable environments (Deng et al., 2016). 7 Summary of UPR signaling from metazoans to plants The UPR signaling pathway is evolutionarily conserved among metazoans, yeast, and plants, so research of the UPR pathway in any of these groups can offer insights into potential applications in a range of different organisms. The first identified UPR transducer, Ire1p (Inositol-Required Enzyme 1), is a dual ER transmembrane protein in yeast that carries the following: (1) an amino-terminus sensing domain facing the ER lumen and (2) carboxy-terminus ribonuclease/protein kinase domains facing the cytosol. With the advent of ER stress, the luminal domain of Ire1p bound by unfolded proteins initiates homo-oligomerization, and then enhances protein kinase activity that causes autophosphorylation (Shamu & Walter, 1996). The clustering of Ire1p on the ER membrane fully activates the ribonuclease, which performs unconventional splicing on the HAC1 pre-mRNA with 252 base pair (bp) intron excision (Cox & Walter, 1996). The spliced HAC1 escapes from translational inhibition, then Hac1ps enters into the nucleus, turning on the transcription of UPR target genes that encode for chaperones (Kar2p), folding enzymes, and proteins that are required for secretory pathways addressing the overload of unfolded proteins in the ER (Okamura, Kimata, Higashio, Tsuru, & Kohno, 2000). In the course of evolution, UPR signaling expanded to three transducers in metazoans: IRE1, activating transcription factor 6 (ATF6), and double-stranded RNA activated protein kinase (PKR)-like ER kinase. The structures of UPR modulators are shown in Figure 1.3. The most conserved transducer, IRE1, has two homologs: IRE1 and IRE1 Unlike IRE1, which has a broadly expression pattern, IRE1 express only in the epithelia of the intestines (Bertolotti, 2001; Wang et al., 1998). The activation of endoribonuclease of IRE1 cleaves the 26 bp intron from the XBP1 mRNA, Hac1p homolog, generating a frameshift variant, sXBP1. Then, sXBP1 acts as a transcription factor (TF). In the nucleus, sXBP1 works as a homodimer collaborating 8 with other co-regulators, such as the nuclear factor (NF) family to regulate the expression of UPR genes (Lee et al., 2002). IRE1 is also involved in the late-phase UPR. The IRE1 dimer interacts with an adaptor called tumor necrosis factor receptor-associated factor 2 (TRAF2), acting to turn on the signal regulating kinase (ASK1), and then activating the phosphorylation cascade (the cJUN-JNK kinase pathway). Through phosphorylation, JNK regulates downstream genes involved in the stimulation of pro-apoptotic signaling (Urano et al., 2000). Whereas IRE1 has dual roles with both pro-survival and pro-apoptotic phases, ATF6 functions as a pro-survival factor during ER stress. The N-terminus Golgi target sequence allows the translocation of ATF6 from the ER membrane to the Golgi, followed by the proteolysis by serine protease site-1 protease (S1P) and metalloprotease site-2 protease (S2P). The released transcriptional domain of ATF6 translocates to the nucleus in conjunction with other bZIP TFs and NF, where it regulates the expression of UPR genes (Ye et al., 2000). In addition, ATF6 induces the expression of XBP1 to enhance chaperone activity (Tsuru, Imai, Saito, & Kohno, 2016). A type-I ER transmembrane protein, PERK, possesses the luminal stress sensor and cytosolic kinase domains. ER stress initiates the dimerization and autophosphorylation of kinase domains, followed by the phosphorylation of the  subunit of eukaryotic initiation factor 2 (eIF) The phosphorylated eIF2 remains bound with eIF2B, leading to general translational block to attenuate the protein influx into the ER (Wek & Cavener, 2007a). Meanwhile, such translation attenuation enhances the degradation of unfolded proteins through the ERAD pathway. However, PERK activates the expression of ATF4, which encodes for the cAMP response element-binding TF to further increase the expression of pro-survival genes, such as genes involved in amino acid synthesis and transport, redox reactions, and secretory 9 pathways (Harding et al., 2000; Vattem & Wek, 2004; Wek & Cavener, 2007b). Additionally, PERK stimulates pro-cell death through ATF4 to induce the TF C/EBP homologous protein (CHOP) primary, repressing pro-survival gene expression (Harding et al., 2000; Marciniak et al., 2004). A schematic of the UPR signaling pathway in mammals is shown in Figure 1.4. In research to date, the PERK/ATF4 pathway has not been discovered in plants. Thus far two main UPR signalings have been identified, involved in both pro-survival and pro-cell death. Plants have two IRE1 homologs, IRE1a and IRE1b. These homologs sense the stress by the luminal sensor and then enhance the activity by oligomerization and autophosphorylation. The splicing target of IRE1, bZIP60 mRNA, is the HAC1/ XBP1 functional homolog splicing out a 23 bp intron in response to stress (Deng et al., 2011; Nagashima et al., 2011). The spliced bZIP60 (sbZIP60) eliminates the transmembrane domain localized at the nucleus to turn on expression of UPR target genes. Under normal conditions, the function of low expressed unspliced bZIP60 (ubZIP60) remains unknown. In addition to its splicing function, IRE1 also degrades transcripts encoding secretory pathway proteins, a process called regulated-IRE1 dependent RNA decay (RIDD). This process reduces the protein loading into the ER (Mishiba et al., 2013). The ATF6 homolog, bZIP28, senses the accumulation of unfolded proteins in the ER lumen, translocates to the Golgi going through proteolysis by Golgi localized proteases, S2P (Iwata et al., 2017). The cytoplasmic bZIP28 triggers the transcription of UPR targets to promote a pro-survival program by enhancing the protein-folding machinery (Liu & Howell, 2010b). A schematic of the UPR signaling pathway in plants is shown in Figure 1.5. Recent study further identified the independent role of bZIP28 in the recovery of the ER stress process (Ruberti, Lai, & Brandizzi, 2018). 10 Contrary to metazoans and yeast, the UPR signaling pathway in plants is still largely unmapped. Investigations to uncover new components in the UPR can further deepen our understanding of ER stress signaling, which could lead to a discovery of potential targets for treating UPR-dysregulated diseases. Significance of dysregulated UPR on plant growth and development Research on the UPR recently has been extended to plant physiological growth under normal conditions. The UPR-deficient mutant exhibits defective root growth, and the UPR is involved in both vegetative and reproductive development. Most studies of vegetative growth focus on the root phenotype supported by the short root phenotype of ire1a/ire1b double mutants under physiological conditions (Chen & Brandizzi, 2012). Both IRE1a and IRE1b have certain levels of overlapping function; however, they have nonoverlapping expression patterns. IRE1a expresses more widely, but it is not generally believed to express in the vascular tissue of young seedlings, leaves, and roots. IRE1b expression is more tissue and developmental-stage limited in the apical meristem, leaf margin, and cotyledons immediately after germination (Koizumi et al., 2001). These tissue-dependent expression patterns suggest the presence of unique functions for IRE1a and IRE1b. Further study identified that ribonuclease activity of IRE1b is required for normal root growth; however, complementation assays using site-specific mutation knock out mutants indicate that the catalytic activity of protein kinase is unnecessary (Deng, Srivastava, & Howell, 2013). The bzip60, the splicing target of IRE1, displayed a similar root phenotype to wild type plants under chronic ER stress (Humbert, Zhong, Deng, Howell, & Rothstein, 2012). This indicates that the influence of IRE1 on root growth only relies on ribonuclease activity instead of depending on the downstream target, bZIP60 mRNA. Nonetheless, recent work uncovered 11 the role of bZIP60 on root growth during recovery from the ER stress response (Ruberti et al., 2018). Additionally, because bzip28/ ire1b exhibited a short root phenotype under unstressed conditions, this suggests that both UPR signaling pathways are involved in vegetative development (Deng et al., 2013). Deng et al. (2013) reported that the UPR also plays a key role in protecting against thermodynamic stress during reproductive development, because growth of the male gametophyte is vulnerable to high temperatures. The double mutants ire1a/ire1b and bzip60 are infertile in response to heat stress (Deng et al., 2016). Further genetic studies through reciprocal crosses suggest such heat-sensitive sterility is due to defective pollen production (Deng et al., 2016). In response to rapid increases in temperature, mitotic and meiotic cells have a decreased ability to mount appropriate protection machinery to safeguard the process of pollen tube elongation (Deng et al., 2016). To build up the pollen tube, allowing for successful delivery of sperm cells into the female gametophyte, a corresponding increase in membrane trafficking is required. This increased trafficking causes the UPR to assist with the high demand of protein synthesis for pollen development. IRE1 immediate splicing target, bZIP60, has been identified to regulate the expression of downstream genes that encode for pollen coat/wall components (Deng et al., 2016). In addition, the complementation examination adopting site-specific mutation mutants revealed that both ribonuclease and kinase activities of IRE1 are critical for male gametophyte development (Deng et al., 2013). Furthermore, the downstream target of bZIP60 and bZIP28, molecular chaperone BiPs, are also required for male gametophyte development and fertilization. Previous studies determined that BiP3 expression is up-regulated at the stage of pollen tube growth. In addition, the bip1/bip2 double mutants have viable pollens but reduced tube growth. 12 Knock outs of all BiPs in Arabidopsis leads to aborted pollens at the bicellular stage after pollen mitosis indicating that BiPs are involved in the later development (Maruyama, Sugiyama, Endo, & Nishikawa, 2014). Theoretically, UPR activation occurs under stress conditions, but remains quiescent under normal conditions. Nevertheless, the basal level of UPR activity may be sufficient for supporting plant development. Activation of IRE1 may be caused by the temporary high demands of protein synthesis and secretion. However, the putative pathway to actuate such machinery is still a mystery. The cross-regulations between UPR signaling and other stress signaling The chemical drugs commonly used as ER stress inducers ER stress is triggered chemically through different mechanisms by various drugs, such as tunicamycin (Tm), dithiothreitol (DTT), brefeldin A (BFA), thapsigargin (TG), and azetidine- 2-carboxylate (AZC). Tm belongs to a group of nucleoside antibiotics that block N- glycosylation in the ER. Tm blocks N-glycosylation in the ER by inhibiting the GlcNAc phosphotransferase (GPT) activity that catalyzes the transfer of N-acetylglucosamine-1- phosphate (GlcNAc-1-P) from UDP-GlcNAc to dolichol-P (Yoo et al., 2018). Another relatively unstable chemical inducer, DTT, functions as a strong reducing agent, blocking the disulfide- bond formation in the ER and quickly inducing ER stress. Even though DTT works within a minute, it also causes the same effects in the cytosol as a non-specific inducer (Oslowski & Urano, 2011). BFA produced from fungi was commonly used for studying membrane trafficking. It inhibits COPI vesicle budding from the Golgi, COPII vesicle from the ER, as well as later post-Golgi steps (Sciaky et al., 1997). Given the inhibition of protein transport from ER to Golgi, BFA causes the over-accumulation of proteins inside the ER, leading to ER stress. In mammals, 13 TG functions as a common inhibitor of the ER Ca2+ ATPase pump. This induces ER stress by depleting ER calcium storage (Treiman, Caspersen, & Christensen, 1998). In addition, the L- proline analogue, AZC, triggers ER stress by competing with L-proline incorporating into newly synthesized proteins that mimic the condition of accumulation of unfolded or misfolded proteins in the ER lumen (Costa et al., 2008). The above chemical compounds target different components in the UPR signaling pathway and are easily manipulated in the laboratory. The surveillance system in the UPR During ER stress, the activation of the UPR works as a first layer of cyto-protective mechanism. It monitors and recovers proteostasis by increasing the expression of protein folding and ERAD-related genes. If the damage exceeds the limits and the UPR is unable to restore the balance in the ER, cells undergo cell death (Hetz, 2012; Iwata & Koizumi, 2012; Ruberti, Kim, Stefano, & Brandizzi, 2016; Watanabe & Lam, 2008). Therefore, to avoid irreversible cell impairment, plants evolved an elaborate surveillance system in their cells in response to the fluctuation of ER homeostasis. This surveillance system relies on the balance between pro-survival and pro-cell death programs, which are regulated through a series of anti- and pro-cell death proteins in the ER. The anti-cell death proteins, such as bZIP TFs (XBP1/Hac1p/bZIP60/bZIP28), have been widely studied as positive regulators activating UPR signaling (Deng et al., 2011; Moreno et al., 2012; Nagashima et al., 2011). However, recent work has uncovered signaling molecules that modulate the UPR negatively. For instance, in mammalian cells, the Bax inhibitor (BI-1) serves as a negative regulator suppressing UPR activation by downregulating the ribonuclease activity of IRE1 (Bailly-Maitre et al., 2006). Moreover, another well-defined negative regulation mechanism has been established. By enhancing the E3 ubiquitin ligase HRD1-modulated 14 proteasome degradation machinery under physical conditions, the ER transmembrane protein, WFS1, downregulates the following: (1) expression of ATF6-mediated UPR target genes and (2) the protein level of ATF6 (Fonseca et al., 2010). In yeast, the phosphorylation cascade in UPR signaling has been found to involve negative regulation, as follows. The Snf1 complex, an ortholog of AMP-activated protein kinase (AMPK), represses the expression of Ssk1 at the transcriptional level. Next, the Hog1-regulated MAPK pathway and the ER stress response are down-regulated (Mizuno, Masuda, & Irie, 2015). Nonetheless, such a negative regulator was not identified in plants until 2017. Research revealed that the positive regulator in light signaling, elongated hypocotyl 5 (HY5), is a negative regulator in the UPR, competing with bZIP28 TF for binding to the ER stress response element (ERSE). This binding leads to down-regulation of the UPR response (Nawkar et al., 2017a). Although the UPR is a well-conserved mechanism among eukaryotes, the plants are considered to have developed unique mechanisms to handle ER stress. Therefore, discovery of negative regulation mechanisms is a main focus in this field, as investigators seek to better understand the surveillance system in the UPR. Examples of functional sharing between the UPR and distinct stress-signaling routes Cellular processes are operated by a complicated network with distinct signal transduction pathways. Most signal transduction pathways collaborate with one or more signaling pathways to address stress or physical alteration. The interaction between signaling pathways termed as cross-regulation is classified into three groups: (1) Primary cross-regulation: two or more separate signaling pathways co-regulate the shared transduction factor in a positive or negative way; (2) Secondary cross-regulation: the output of one signaling pathway enhances/suppresses the perception of signal or input level of the other signaling pathway; and 15 (3) Tertiary cross-regulation: the outputs of two or more separate signaling pathways affect one another in a positive or negative way. Cells switch on an integrated signaling system to restore homeostasis and function in response to various environmental stresses; ER stress signaling is involved in such cross- regulation. For example, in the development of triple negative breast carcinoma cells, the caspase-dependent apoptosis is enhanced by activation of the ER stress response in conjunction with inhibition of the MEK/ERK pathway, indicating the potential interaction between ER stress signaling and the MEK/ERK pathway (Ghosh et al., 2015). In addition, the ubiquitin ligase Parkin, a critical modulator involving mitochondria dynamics, bioenergetics, and mitophagy has been demonstrated to be regulated by the UPR transducer, ATF4. In turn, activated Parkin enhances one of the UPR arms, the IRE1/XBP1 pathway, exemplifying that a regulatory node underlies the cross-regulation between UPR and mitochondria signaling (Pellegrino & Haynes, 2015). Furthermore, another UPR arm regulated by ATF6 is also associated with a metabolic program in which ATF6 activates the PGC1 (peroxisome proliferator-activated receptor gamma, coactivator 1 ), a master regulator of mitochondria biogenesis (Daniela Senft & Ronai, 2016). In plants, both growth and development rely on light; therefore, light signaling cross- communicates with other stress signaling such as UPR signaling. The ER stress sensitivity of plants is enhanced by an increase in light intensity, and HY5 functions as a shared TF regulated by both light signaling and UPR signaling pathways (Nawkar et al., 2017b). The UPR TF, bZIP60, exemplifies another shared factor involved in multiple stress-signaling pathways. Recent studies identified the potential role of the un-spliced form of bZIP60 in the salt stress pathway. Specifically, the transcript levels of bZIP60 and Bip2 are induced in response to salt 16 stress (M.Wang, Xu, Yu, & Yuan, 2010), and over-expression of bZIP60 enhances plant-stress tolerance (Fujita et al., 2007). Another role of the IRE1/bZIP60 arm in the plant defense response has been well defined, suggesting additional cross-communication between abiotic stress signaling (ER stress) and biotic stress signaling (pathogen stress). This hypothesis is supported by the observation that the expression level of IRE1a and IRE1b are significantly increased, and the more susceptible phenotype of ire1a/ire1b responding to the pathogen treatment (Moreno et al., 2012). Additionally, in response to salicylic acid (SA) treatment that mimics the pathogen attack condition, the two UPR arms, including the splicing of bZIP60 and processing of bZIP28, are stimulated followed by the up-regulation of UPR gene expression (Nagashima, Iwata, Ashida, Mishiba, & Koizumi, 2014). The biological significance of such UPR activation upon pathogen infection is to enhance the protein folding machinery, facilitating the secretion of anti- pathogen proteins. Eventually, the investigation of the cross-regulation and the shared regulators between ER stress and other stress signalings can provide a fresh perspective on the molecular mechanism of the surveillance system in the UPR. Role of systemic signaling in plants and an overview of systemic UPR Outline and significance of long-distance (systemic) signaling in plants The life pattern of plants is very different than that of most animals because plants do not have the capacity for locomotion. Immobility requires that plants perceive external stimuli efficiently so that they can survive. Plants are constantly challenged by outside stimuli, and through physiological and developmental responses, they tailor themselves to adjust to fluctuations in the environment. The typical stimuli are commonly categorized as abiotic and biotic stresses. Abiotic stresses are triggered by physiological conditions such as light, salt, 17 temperature fluctuation, and osmotic stresses. The plant’s ability to react rapidly and appropriately to these stressors, and to survive in ever-changing conditions, relies on collaborations between defense mechanisms and signaling pathways that enhance the plant’s stress tolerance. In nature, biotic stresses can be caused by herbivory or pathogens and the interactions between plants and herbivores or pathogens are highly specific based on the plant species and stressor type. Some abiotic stressors such as elevated air temperature, and the transition between day and night, are sensed by the whole aerial portion of plants simultaneously. Other stressors, such as attacks by herbivores or pathogens are perceived locally at a restricted area of the plant, eliciting a response that is circulated throughout the whole plant body. To achieve the systemic response, the tissue not perceiving the stimulus directly senses the long-distance signal arising from the site of perception. Such long-distance signals can spread through the plant cell to cell, tissue to tissue and/or plant to plant to prepare the whole plant to cope with future challenges. Thus far, three distinct long-distance signaling types have been discovered: (1) chemical signals, including reactive oxygen species (ROS), ions (Ca2+/K+), volatiles, phytohormones, MAP kinase, and inositol triphosphate (Choi, Hilleary, Swanson, Kim, & Gilroy, 2016b); (2) hydraulic signals, which can be triggered by the alteration of turgor pressure and mass flow (Christmann, Grill, & Huang, 2013); and (3) electrical signals, which can be stimulated by action potentials, slow wave potentials, and system potentials (Trebacz, Dziubinska, & Krol, 2006). These three types of long-distance signals are distinguishable in terms of both chemical properties and propagation speed. These systemic responses conducted by long-distance signals can be divided into two major categories: (1) systemic acquired resistance (SAR), mostly triggered by pathogen attacks 18 (Conrath, 1994) and (2) systemic acquired acclimation (SAA), elicited by abiotic stressors like salt, heat, osmotic conditions, and high light conditions (Mittler & Blumwald, 2015). The schematic diagram of SAR and SAA are shown in Figure 1.6. An efficient and functional network of such systemic signal responses provides plants with improved fitness. For instance, when Arabidopsis plants with prior SAR induction are exposed to bacterial infection, this can lead to increases in total biomass and seed production. The plant’s ability to mount profound defensive SAR after perceiving the initial signal applies to both biotic and abiotic stress conditions, and it can even be passed to the next generation (Boyko & Kovalchuk, 2011). The significance of vascular tissue in long-distance signaling Rapid signal propagation has been proposed to be achieved through symplastic (cytoplasmic) and apoplastic (extracellular) pathways. Previous studies have unraveled the involvement of the plant vascular system, xylem and phloem, in symplastic-regulated long- distance signaling. Xylem tissue consists of dead thickened cell walls serving as a water conduit from root to shoot (Fukuda, 1996). Phloem tissue, composed of a complex of living cells, sieve tube elements (SE), and nearby companion cells (CC), is responsible for translocating the photosynthetic assimilates from adult tissues to developing young tissues (Lucas et al., 2013). Such basic structures of xylem vessels and phloem sieve tubes are well-designed for transport of water and nutrients because they run through the entire plant body. In addition, intensive studies on the content of vascular flow fluids have revealed the possibility that various forms of gene products such as nucleic acids, peptides, and proteins that are potential signaling molecules are transported through the xylem and phloem. This supports the possible role of vascular tissue in systemic signaling pathway. 19 In addition to the well-known functions of physical support toward the aerial tissue and transport of water and essential nutrients from the environment, xylem vessels are also involved in transporting various signaling molecules that mediate tissue-to-tissue communication. For example, upon the resupply of nitrogen to nitrogen-depleted maize plants, the trans-zeatin (Z)- type cytokinin (CK) is enriched in the xylem sap, indicating that the abundance of CK reflects the status of nitrogen nutrients (Takei, 2001a). Further analysis of exudation rate, CK concentration, and content in xylem sap during the recovery from nitrogen starvation indicates that there is nitrogen-dependent accumulation, translocation of CK from root to shoot, followed by the induction of ZmPRs expression by Z-type CK (Takei, 2001b). Using grafting experiments, another study provided additional evidence that Z-type is the signaling molecule and the main regulator for cambium development (Matsumoto-Kitano et al., 2008). This suggests a crucial role of a small phytohormone in plant root-to-shoot coordination. In addition to phytohormone, the proteins involved in the structure of the cell wall and pathogen-related responses have also been identified in xylem exudates from various plant species. The protein XSP10, a secreted protein similar to lipid transfer protein, is detected in the xylem sap of tomato plants as part of a fungus defense response (Krasikov, Dekker, Rep, & Takken, 2011). In recent years, genes encoding small secreted peptides have been widely identified in many seed plants, including A. thaliana and M. truncatula. In legume plants, to control the numbers of nodules formed during the process of nitrogen fixation, the root- derived signal, CLE-RS2 peptides, travels to the shoot via xylem vessels to bind to the shoot-derived HAR1 receptor. This leads to the suppression of nodulation (Okamoto, Shinohara, Mori, Matsubayashi, & Kawaguchi, 2013). Interestingly, the Arabidopsis genome encodes over 900 such peptides, 20 some of which function as signaling molecules in long-distance signaling (Matsubayashi, 2011). For example, a group of small-secreted peptides named CEPs has been implicated in the root- to-shoot signaling response to nitrogen status. It has been reported that some CEPs expression is induced, followed by up-regulation of the nitrogen transporter, NRT2.1, in response to nitrogen depletion, and its encoded product is also detectable in the xylem sap. Moreover, the identification of shoot CEPs receptors, XIP1/CEPR1 and CEPR2, further suggests that plants adopt the CEP as a systemic root-to-shoot signal, coordinating the root response dealing with the nitrogen fluctuation at whole plant level (Ohkubo, Tanaka, Tabata, Ogawa-Ohnishi, & Matsubayashi, 2017). The phloem SE network forms a conduit for long-distance allocation of phytosynthates and signaling molecules via two pathways: transport either from the apoplasmic to the intracellular compartment or from SE cells to neighboring CC via interconnecting specialized secondary plasmodesma (PD) (Lucas et al., 2013). Phloem flow carries small molecules such as metabolites, photosynthate sugars, and phytohormone as well as macromolecules like proteins and RNAs. As in xylem, various phytohormones have been detected in the phloem sap, including salicylic acids (SA), auxins, gibberellins, CK, and jasmonic acids (JA) as well as their derivatives, which are proposed to be involved in the defense response (Hoad, 1995; Vlot, Klessig, & Park, 2008). Additionally, the concentration of hormones in the phloem alters in response to environmental and developmental conditions; this suggests that mobile hormones have a role in the modulation of plant physiological processes (Hoad, 1995). Besides phytohormones, phloem flow contains huge amount of proteins, and numerous forms of transcripts such as mRNAs, siRNAs, and long-noncoding RNA. The roles of these molecules in long-distance signaling has been broadly explored in the last decade. The well- 21 established case of mobile protein in Arabidopsis phloem, FT, which serves as a florigenic signal to promote flowering and the initial outgrowth of dormant buds and lateral shoot primordia (Böhlenius et al., 2006; Hiraoka, Yamaguchi, Abe, & Araki, 2013; Niwa et al., 2013). Under long-day photoperiod conditions, the expression of FT and its paralog, TWIN SISTER OF FT (TSF), are induced in the phloem CC of cotyledons and leaves (FT and TSF) and hypocotyl (TSF). However, the autonomous and vernalization negatively regulate their expression. The proteins of FT and TSF are transported to shoot apical meristem (SAM), whereby they recruit another bZIP TF FD, forming a florigenic complex to up-regulate the transcription of MADS- domain genes like APETALA1 (AP1). This reverses the inhibitory effects of floral repressors, and then leads to floral initiation and development (Andres et al., 2015). It is also evident that cellular transcripts proceed to the destinations across the phloem and interact with their targets. Among them, the most well-studied instance is the regulation of siRNA. The mobility of siRNA is clearly demonstrated by reciprocal grafting in which the dicer mutants as recipients failed to produce the siRNAs. Such movement facilitates the SAR to act against pathogen infection by switching on post-transcriptional silencing and transcriptional silencing (Molnar et al., 2010). In addition, most mobile miRNAs have been discovered to be involved in the balance of nutrition status. For example, miRNA399 travels from shoots to root through the phloem to suppress PHO2, further enhancing Pi uptake and redistribution during the onset of Pi depletion (Pant, Buhtz, Kehr, & Scheible, 2008). In grafting experiments, miRNA395 has been reported to move down towards the roots, repressing the target in the rootstock under sulfate deficient conditions (Buhtz, Pieritz, Springer, & Kehr, 2010). The function and action of phloem-mobile mRNA are also determined by grafting. The first documented example, a maize homeobox TF, KNOTTED1 (KN1), is able to move from cell to cell or layer to layer as both 22 protein and mRNA forms via the phloem tube system. KN1 modulates meristem growth and wide organogenesis (Kim, Yuan, Cilia, Khalfan-Jagani, & Jackson, 2002). These findings strongly suggest the critical involvement of plant xylem and phloem systems in systemic signaling pathways. We continue to investigate the biological relevance of yet-to-be identified signaling molecules and underlying mechanisms. The regulation of plasmodesma (PD) involved in cell-to-cell communication In plants, to achieve long-distance signaling, the cell has to overcome the local distance between cells. Therefore, cell-to-cell communication plays a crucial role in sexual reproduction, morphology, physical homeostasis, defense, and stress tolerance. Unlike animal cells, plant cells possess a cell wall that acts as a physical barrier inhibiting the translocation of particular signaling molecules between cells. For instance, the cell wall is mainly composed of polysaccharides that limit the transport of extracellular vesicles. Moreover, the high levels of Ca2+ ions in the cell wall maintain a low pH environment compared with that of the cytosol, allowing the movement of certain signaling molecules. Therefore, plants evolved a symplastic route for transport of ions, proteins, metabolites, nucleic acids, hormones, and other signaling molecules through the specialized plasma membrane-lined cytoplasmic tunnels named plasmodesmata (PD). PDs are highly dynamic, versatile nano-tunnels connecting adjacent cells with the basic ultrastructure. They consist of primary PDs, which comprise the central axial rod formed by an appressed ER tubule termed a desmotubule (Ghoshroy, Lartey, Sheng, & Citovsky, 1997). Surrounding the PD, neck regions are specialized cell walls rich in non-esterified pectins and callose (-1, 3-glucan) instead of regular cellulose and hemicellulose. Therefore, callose turnover and biogenesis play critical roles in part of the regulation of PD conductivity (Turner, 23 Wells, & Roberts, 1994). Consistent with this, enzymes that participate in callose biogenesis (callose synthase AtCALS3) and degradation (-1, 3-glucanase, AtBG_ppap) are involved in modulating the PD aperture and intercellular trafficking (Levy, Erlanger, Rosenthal, & Epel, 2007). Similarly, callose deposition and degradation is known to influence the movement of plant virus proteins to neighboring cells in tobacco (Bucher et al., 2001; Rinne et al., 2005). In response to alteration of physiological stage, mechanical injury, and pathogen attack, plant PDs change their morphology and architecture. Upon cell differentiation, the primary simple PDs can turn into specialized deltoid forms presented in the middle of the companion cell and sieve elements. In addition, secondary PDs may be formed at special developmental events, such as the connections of graft and host-pathogen interfaces (Faulkner, Akman, Bell, Jeffree, & Oparka, 2008). PD-established cell-to-cell trafficking allows the passage of both non-selective molecules—i.e., below the size-exclusion limits (SEL). SELs are typically measured between ~30 kDa and ~60–70 kDa, but vary among cell types and developmental stages. Selective large molecules possibly contain trafficking signal peptide. Thus far, several molecular signals have been characterized as transporting through PD, including metabolites, protein/peptides, small RNAs, and ions. In plants, SHORT-ROOT (SHR) transcription factor serves as a mobile signal from stele to endodermis, which turns on the downstream SCARECROW (SCR) gene expression that modulates cell divisions in the root apex (root patterning) (Nakajima, Sena, Nawy, & Benfey, 2001). Moreover, trichome patterning depends mainly on trichome promoting factor, TRANSPARENT TESTA GLABRA1 (TTG1), acting cell non-autonomously and capable of moving between cells (Bouyer et al., 2008). With regard to the role of PD mediated 24 cell-to-cell trafficking involved in plant defense response, viral movement proteins (MPs) generate the passage for intact virions passing through via modifying the PDs. Posttranslational modifications (PTM) of NCAPs and viral MPs, such as phosphorylation and glycosylation, further modulate the cell-to-cell trafficking of such proteins. In the case of tobacco mosaic virus (TMV), the interaction of viral MPs with the plasmodesmata facilitates the distribution of TMV between neighboring cells. The phosphorylation at C- terminal residues (Ser/ Thr) of viral MPs leads to abolishment in the ability of TMV to spread from cell to cell in plants. Further, it was shown that the pumpkin NCAP Cm-PP16 requires both phosphorylation and glycosylation for its interaction with NCAPP receptor, and in turn, for its passage through the PDs. These observations underscore the importance of PTM in cell-to-cell movement (J. Y.Lee et al., 2003; Xoconostle-Cázares et al., 1999). Instances of systemic UPR in multicellular organisms In multicellular organism, cells have evolved delicate mechanisms to maintain the integrity of their proteome and cell homeostasis in the face of ever-altering environments. That is to say, to cope with the proteotoxic stress that possibly occurs in distinct cellular compartments, cells have developed a specialized UPR machinery in a cell non-autonomous manner via intercellular communication that facilitates the surveillance system and the modulation of proteostasis. A recent study in the multicellular model, C. elegans, revealed the cell non-autonomous and systemic regulation of the UPR, which is achieved by the nervous system. The OCTR-1, an octopamine G-protein-coupled catecholamine receptor, presents in the sensory neurons mediating the constitutive XBP1-regulated UPR activation. This UPR activation responds to the increased demand of protein synthesis and folding during the C. elegans transition from 25 embryotic and post-embryonic phases of worm into adulthood (Sun, Liu, & Aballay, 2012). Further study indicated that ectopic expression of the active form of XBP1 in C. elegans neurons induces XBP1-regulated signaling, leading to the expression of chaperones, BiP, in the intestines. This action is operated by a secreted ER stress signal (SERSS) that belongs to the neurotransmitter group mediated by the UNC-13 protein (Taylor & Dillin, 2013). Although the underlying mechanism is yet-to-be identified, such systemic regulation is undoubtedly capable of enhancing the stress tolerance and longevity for C. elegans. A similar scenario has been discovered in mammals, indicating that cell-non- autonomous regulation is likely to be seen more extensively among eukaryotes. In mice, constitutive activation of the transcriptional active form of XBP1 in the POMC, pro- opiomelanocortin neuron, stimulates the splicing of XBP1 and activation of downstream UPR in remote hepatocytes. This leads to improved sensitivity to insulin and homeostasis of blood glucose levels, protecting against diet-induced obesity (Williams et al., 2014). Rationale for study The ER stress response is a conserved mechanism among the eukaryotes. Although the UPR is extensively-studied in mammals and yeast, there are certain signaling pathways modulated by particular molecules that have been identified only in mammals. These mammal- only pathways include PERK/ATF4 signaling (Harding et al., 2000; Wek & Cavener, 2007b) and apoptosis signal regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) signaling cascade (Urano et al., 2000). This indicates that there are differences in these pathways between mammalian and plant cells. In addition, unlike mammals, the specific hormones and second- metabolites generated by plants also play significant roles in the regulation of stress signaling routes. Such evidence that there is a lack of particular signaling pathways and established 26 hormone-regulation suggests the possibility that plants may develop unique mechanisms to handle ER stress. In my dissertation research, I attempted to study the underlying mechanism of the UPR from the subcellular level to the organismal level, as illustrated in Figure 1.7. First, I unraveled the role of the integrity of ER structure on the UPR, as described in Chapter II. Second, I explored the identification of novel UPR transducers, as described in Chapter III. Third, I investigated the mechanism of UPR signal transduction, as described in Chapter VI. These investigations will further expand the current knowledge of how plants respond to ER stress and mediate growth and development, as well as advancing knowledge of the underlying mechanisms involved in regulating these processes. These studies hold promise for unraveling new regulatory components of UPR and the underlying mechanisms to deal with stresses leading to important potential applications, including manipulation of traits in economically important crops to enhance their stress tolerance and productivity. 27 APPENDIX 28 Figure 1. 1. Different structural subdomains of the Endoplasmic Reticulum Based on the presence or absence of ribosomes, the endoplasmic reticulum (ER) is divided into two categories, rough sheets (rough ER) and smooth tubules (Smooth ER). Rough sheets are composed of flatted sac-like structures with both ends sealed. They form the connective structures extending from nuclear envelopes and are stuffed with ribosomes at their cytosolic side. The absence of ribosomes on the structures is why smooth tubules are called smooth ER. The smooth ER can be made up of flatten sheets, but tubules are more common in nature. Modified image of Encyclopaedia Britannica/UIG/Getty. 29 Figure 1. 2. Unfolded protein response (UPR) Polypeptides translated by ER membrane-associated ribosomes enter the ER lumen. Glucans synthesized and attached to the ER membrane are co-translationally transferred to polypeptides. Binding protein (BiP) binds to nascent polypeptides to assist folding. Protein disulfide isomerase (PDI) further facilitates the formation of disulfide bonds. Calnexin (CNX) recognizes glucans attached to proteins to retain unfolded proteins in the ER. Correctly folded and assembled proteins are transported to the Golgi apparatus for advanced modifications before arriving at their final destinations, such as the lysosome (the vacuole in plants), the plasma membrane, and the extracellular space. Unfolded/misfolded proteins are scavenged through the ER-associated degradation (ERAD) pathway in the cytoplasm. Upon accumulation of unfolded proteins caused by ER stress, a signal transduction pathway termed the unfolded protein response (UPR) from the ER to the nucleus is activated. This enhances the protein folding machinery by up-regulating UPR gene expression. 30 Figure 1. 3. The features and topology of UPR modulators IRE1 and PERK possess the N-terminal ER lumenal domain (stress-sensing domain), sensing ER stress and the C-terminal Kinase and RNase domains for its auto-phosphorylation and cytoplasmic splicing of mRNA of UPR transcription factor, respectively. Conversely, ATF6 is composed of C-terminal stress-sensing domain and N-terminal basic leucine zipper domain (bZIP) for DNA targeting and transcriptional activation in the nucleus. TM: transmembrane domain. Arrows indicate the cleavage sites of ATF6 by Golgi resident protease, S1P (site-1 protease) and S2P (site-2 protease). 31 Figure 1. 4. UPR signaling pathway in mammals Upon ER stress, ER membrane-associated transcription factor (TF), ATF6 is transported to the Golgi and undergoes proteolysis by S1P and S2P proteases. The released cytoplasmic form of TF goes into the nucleus to activate the downstream transcription of UPR genes. The PERK kinase reduces protein translation by phosphorylating the translation initiation factor 2. The activated IRE1 splices the mRNA of XBP1, which encodes bZIP transcription factors. The spliced active form of bZIP TF enters the nucleus to turn on transcription of UPR genes. IRE1 RNase can also degrade unstable mRNA encoding secretory pathway proteins termed RIDD to attenuate protein synthesis. Meanwhile, the Kinase domain of IRE1 interacts with TRAF2 protein to switch on the ASK1/ JNK phosphorylation cascade, leading to autophagy activation. 32 Figure 1. 5. UPR signaling pathway in plants In plants, there is no PERK protein identified thus far. Upon the advent of ER stress, IRE1 promotes unconventional splicing of XBP1 homolog, bZIP60. With the removal of membrane- association domain, the spliced active form of bZIP60 goes into the nucleus to turn on UPR gene expression. The ATF6 functional homolog, bZIP28, is transported to the Golgi and is activated through proteolysis. Then the released cytoplasmic bZIP28 turns on the UPR genes in the nucleus. 33 Figure 1. 6. The systemic response in plants: SAR (systemic acquired resistance) and SAA (systemic acquired acclimation) Long-distance signals generated by pathogen/insect biotic stress moving from the infected leaf to distant leaves where the SAR is induced to protect against second infection with limited symptoms. SAA is induced by abiotic stressors such as drought, high light, and nutrient depletion, enabling acclimation across the entire plant. 34 Figure 1. 7. Study overview This study focused first on the investigation of the underlying mechanisms of the UPR at the cellular level: i.e., examining the influence of ER shape on its ability to trigger the UPR, and identifying UPR transducers. The study then examined the UPR at the whole organism level, investigating UPR signaling through local and long-distance mechanisms. 35 REFERENCES 36 REFERENCES Andres, F., Romera-Branchat, M., Martínez-Gallegos, R., Patel, V., Schneeberger, K., Jang, S., …Coupland, G. (2015). Floral induction in Arabidopsis thaliana by FLOWERING LOCUS T requires direct repression of BLADE-ON-PETIOLE genes by homeodomain Plant protein pp.00960.2015. https://doi.org/10.1104/pp.15.00960 169(November), PENNYWISE. Physiology, Appenzeller-Herzog, C. (2006). 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Here we have queried the ability of mutants with defective structure of the endoplasmic reticulum (ER) to invoke the unfolded protein response (UPR), an essential ER signaling pathway. Through molecular and genetic approaches, we show that loss of the ER- shaping GTPase Root Hair Defective 3 (RHD3) specifically disrupts UPR by interfering with the mRNA splicing function of the master regulator IRE1. These findings establish a novel role for RHD3 in the ER and support specificity of the effects of ER-shaping mutations on ER function. 51 INTRODUCTION The ER is responsible for the synthesis, modification, and quality control of most of the cell’s building blocks that traffic through the Golgi to reach their final destination (Matheson et al., 2006; Vitale & Denecke, 1999). The unique reticulated architecture of the ER mainly relies on a dynamic remodeling of interconnected sheets and tubules, as well as tubule initiation, growth and fusion (Chen et al., 2013a; Sparkes et al., 2011). Whether the tubular and sheet forms of the ER have different functions and whether their inter-conversion is essential to the life of a cell is unknown. However, examples in animal cells suggest that ER shape and function are interlinked. For instance, in specialized secretory cells, such as pancreatic B cells, the sheet form predominates (Marchetti et al., 2007), suggesting that this ER form may facilitate the synthesis of secretory proteins. Nonetheless, the extent to which disruption of ER architecture affects the function of this essential organelle is largely unknown. While ER movement mostly depends on the cytoskeleton (Friedman et al., 2010; Sparkes et al., 2009; Ueda et al., 2011), ER structure depends on its lipid composition as well as ER- structuring proteins. For example, genetic disruption of the ER phospholipid biosynthetic pathway in the model plant species Arabidopsis leads to a drastic modification of the ER shape with replacement of tubules by sheets (Eastmond et al., 2010). Similarly, crucial roles in ER structure have been attributed to proteins such as the ER tubule-forming proteins (reticulons and DP1/Yop1p) and atlastins (ATLs). While reticulons are required and sufficient to form an ER tubular network, most likely by stabilizing the high curvature of the tubules (Hu et al., 2008; Park et al., 2010; Voeltz et al., 2006), ATLs mediate the process of ER tubule fusion in metazoans (Hu et al., 2009; McNew et al., 2013; Orso et al., 2009). ATLs are GTPases of the dynamin superfamily of proteins involved in diverse cellular processes, including membrane 52 remodeling, organelle fusion and division (Heymann & Hinshaw, 2009; Praefcke & McMahon, 2004). Yeast and plants do not have sequence homologs of ATLs, but they have proteins called Sey1p in S. cerevisiae and RHD3 in A. thaliana (Hu et al., 2012; Hu et al., 2009), with similar signature motifs of ATLs, which include a large GTPase domain, a helical bundle domain, two closely spaced trans-membrane segments and a short C-terminal tail. Similarly to ATLs and Sey1p, RHD3 has a role in ER architecture (Chen et al., 2011; Stefano et al., 2012) and facilitates membrane fusion (Zhang et al., 2013). A recently discovered lunapark protein (Lnp1p) has also been shown to interact with reticulon possibly to antagonize Sey1p function in ER remodeling (Chen et al., 2012), supporting that additional proteins besides reticulons and ER-associated GTPases have critical roles in ER structure. In animals and plants, defects in ER structure due to loss of atlastins or RHD3 have been implicated in severe growth and developmental phenotypes, including abnormal tissue growth and embryonic lethality (Audhya et al., 2007; Chen et al., 2011; Park et al., 2010), underscoring that maintenance of an optimal architecture of the ER due to the presence of these proteins has important implications for the life of the organism. Nonetheless, how a loss of function of ER- shaping proteins translates into growth defects at cell and tissue levels is unknown. The smaller size of the aerial and root tissues in RHD3 loss-of-function mutants compared to wild type (Chen et al., 2011; Hu et al., 2003; Stefano et al., 2012; Wang et al., 1997) is most likely linked to reduced cell elongation (Wang et al., 1997). The evidence that ER export of membrane and soluble fluorescent protein markers is not affected in RHD3 loss-of-function mutants (Chen et al., 2011) implies that the plant phenotype of rhd3 is linked to yet-to-be discovered causes that are unrelated to defects in bulk flow from the ER. 53 Intriguingly, loss of the reticulon Rntl1 in Drosophila has been shown to alter cause elevated levels of ER stress (O'Sullivan et al., 2012), which is a condition that cells generally experience when the ability of the ER to balance protein synthesis demand and capacity is compromised. In conditions of ER stress, a largely conserved cytoprotective signaling pathway, known as the unfolded protein response (UPR), is activated (Liu & Howell, 2010; Ron & Walter, 2007). Compromised UPR leads to serious conditions and even death in animals and plants (Chen & Brandizzi, 2012; Chen & Brandizzi, 2013; Deng et al., 2011; Iwawaki et al., 2009). The evidence that loss of an ER-shaping protein in Drosophila activates the UPR suggests that the homeostasis of ER-shaping proteins influences not only ER morphology but also a critical cellular response that is related to the function of this organelle. In this work we aimed to establish whether loss of proper shape affects functional aspects of organelles by querying the ability of the ER to respond to stress in genetic backgrounds that have defects in ER network integrity. Amongst the mutants tested, we found that the loss of RHD3 negatively affects the UPR activation arm mediated by the major ER stress sensor, IRE1. Our data demonstrate a novel requirement of RHD3 in cell physiology besides a known role in ER architecture (McNew et al., 2013; Stefano et al., 2012; Zhang et al., 2013), and show that ER network integrity can be correlated to a function of the ER although the phenotype is linked to specific ER architecture mutations. Furthermore, by analyzing mutants with defects in the expression of RHD3 and IRE1, we established that the loss of RHD3 compromises the IRE1- UPR signaling arm and that this phenotype does not account for the similar organ growth defects reported for the RHD3 and AtIRE1 mutants (Chen et al., 2012; Hu et al., 2003; Stefano et al., 2012). Therefore, our results also support that the RHD3 growth phenotype is uncoupled from 54 UPR defects, and that defects in the UPR due to ER architecture mutations cannot inevitably be associated with cell growth phenotypes. 55 MATERIALS AND METHODS Plant Materials, Growth Conditions, and Tm treatment Plant material used in this work was Arabidopsis thaliana ecotype Columbia (Col-0). The Arabidopsis transfer-DNA insertion mutants were atire1a atire1b (ire1) (Chen & Brandizzi, 2012), the bzip60-1 knockout (bzip60) (Moreno et al., 2012), and rhd3-7 knockout (Stefano et al., 2012). In the ire1 mutant, atire1a is a knockout and atire1b is a knockdown (Chen & Brandizzi, 2012). The ire1/rhd3 triple mutant was obtained through crosses of ire1 with rhd3-7, followed by genotyping of the F2 generation and isolation of homozygous lines at the F3 generation. The RHD3 EMS allele used in this work gom8, which encodes a non-functional RHD3 protein (Stefano et al., 2012). Surface-sterilized seeds were plated directly onto ½ Linsmaier and Skoog's (LS) medium, 1.5% (w/v) sucrose. For UPR induction, Tm (Sigma, T7765; dissolved in DMSO) was directly added in the medium as follows: 0.5 µg/ml for UPR induction analyses, and 0.05 µg/mL for testing sensitivity to ER stress; as mock control, Tm volume was replaced by the same volume of DMSO. AGI numbers ACT3: At3g53750; ACT8: At1G49240; AtIRE1A: At2g17520; AtIRE1: At5g24360; BiP3: At1g09080; BP80: At3g52850; CNX1: At5g61790; ERDJ3A: At3g08970; G92/Sec24A: At3g07100; Gold36: At1g54030; Pah1: At3g09560; Pah2: At5g42870; PDIL: At1g21750; RHD3: At3g13870; RHD3-L1: At5g45160; RHD3-L2: At1g72960; TIP4: At2g25810; TUB4: At5g44340; UBQ10: At4g05320; PVA12: At2g45140. 56 RNA Extraction and quantitative RT–PCR (qRT–PCR) analysis The RNeasy Plant Mini Kit (Qiagen) and treatment with DNaseI (Qiagen) were used for total RNA extraction. All samples within an experiment were reverse transcribed at the same time using High Capacity RNA-to-cDNA Master Mix Kit (ABI 4390712). To ensure the purity of RNA sample, 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. Real-time qRT-PCR with SYBR green detection was performed in triplicate using the Applied Biosystems 7500 Fast Real-Time PCR System as described earlier (Chen & Brandizzi, 2012). Relative expression levels were normalized to that of an internal control, either UBQ10 or Actin8. Values are representative averages from three technical replicates. Similar patterns of expression were observed in three independent biological replicates. Statistical significance was established with the Student’s two-tailed t-test, assuming equal variance. Primers are provided in Table 2. 1. Phenotypical Analyses Standard protocols were followed for root length measurements of seedlings germinated on vertical square plates (Weigel & Glazebrook, 2002). Statistical analyses included the Student’s two-tailed t-test, assuming equal variance. To establish the position of the transition zone, the Cell-o-Tape macro (open source ImageJ/Fiji) was used (French et al., 2012). Briefly, along a root cell row the macro computes the cell number versus size to establish significant changes in cell length. The occurrence of significant changes marks the transition between the elongation and division zones. The CellSeT software was used to build the root segmentation based on the propidium iodide staining (1 μg/ml), as described earlier (Pound et al., 2012) 57 RESULTS Defects in ER architecture negatively influence UPR activation in a background specific- manner To establish whether defects in the ER structure could affect the ER ability to evoke the UPR, we analyzed various mutant backgrounds with marked ER architecture defects. Specifically, we adopted a mutant in which the ER tubules are converted into sheets known as pah1 pah2 (herein referred as to pah1/2). This is a double knock out of two phosphohydrolase genes involved in the biosynthesis of phospholipids (Eastmond et al., 2010). We also used mutants with deformed ER network in which non-cortical ER tubules are intertwined into large globular structures. Specifically, we used: gold36/MVP1/ERMO3 (herein referred as to gold36), which is linked to a loss of function mutant of a pseudo-lipase (Agee et al., 2010; Marti et al., 2010; Nakano et al., 2012), and g92/ERMO2 (herein referred as to g92), which is a partial loss of function of the COPII coat component AtSEC24A (Faso et al., 2009; Nakano et al., 2009). Unlike gold36, which has compromised ER export, the g92 mutant does not have general ER export defects (Agee et al., 2010; Faso et al., 2009; Marti et al., 2010; Nakano et al., 2012; Nakano et al., 2009). In addition, we adopted mutants of RHD3, specifically a null allele (rhd3- 7) (herein referred as to rhd3) as well as a mutant bearing a non-silent missense mutation (gom8) (Stefano et al., 2012), which have long unbranched ER tubular structures similar to mutations linked in atlastins and Sey1p in metazoans and yeast, respectively (McNew et al., 2013; Orso et al., 2009; Zhang et al., 2013). Similar to g92, rhd3 does not have general ER export defects (Chen et al., 2011). To test whether the mutants have defects in the UPR, we followed a standard assay based on the analysis of variations in mRNA abundance of well-established UPR molecular markers 58 such as BiP3, protein disulphide isomerase (PDIL) and the heat shock protein ERDJ3A during ER stress (Chen et al., 2013b; Chen & Brandizzi, 2012). To evoke the UPR we used tunicamycin (Tm), an ER stress agent that inhibits glycosylation in the ER and leads to transcriptional up- regulation of UPR genes. Upon Tm treatment in the pah1/2, gold36 and g92 backgrounds the levels of the UPR gene transcripts were similar to wild type (Figure 2. 1.). We next analyzed the UPR induction in the rhd3 loss-of-function mutants and found that in conditions of tunicamycin treatment, the levels of UPR genes were significantly lower compared to wild type (Figure 2. 2.), despite the basal levels of UPR gene transcript being similar in control samples (Figure 2. 6.). Together these data indicate that defects in ER architecture and in UPR activation can be simultaneously verified, although this phenotype is not a general feature of mutants with abnormal ER morphology. We next aimed to dissect the verified UPR-defective phenotype of the RHD3 alleles by testing whether loss of RHD3 could lead to general transcription attenuation in conditions of ER stress. Quantitative RT-PCR analyses showed no significant differences in the transcript levels of other genes encoding either secretory or cytosolic proteins in wild type and rhd3 mutants treated with Tm (Figures 2. 7.). Together with the evidence that loss of RHD3 attenuates the induction of UPR gene transcription in conditions of ER stress (Figure 2. 2.), these data show that RHD3 is specifically required for the expected increase of UPR gene transcripts in conditions of induced ER stress. The Arabidopsis genome encodes three RHD3 isoforms: RHD3, RHD3-like 1 (RHD3- L1) and RHD3-like 2 (RHD3-L2). To test whether the UPR defects were linked exclusively to the loss of RHD3, we analyzed the expression of UPR indicators in RHD3-L1 and RHD3-L2 loss-of-function backgrounds (Chen et al., 2011). We found that BiP3 transcript levels were 59 unaffected in the rhd3-l1 and rhd3-l2 knock-outs compared to wild type (Figure 2. 8.). Although we cannot exclude that RHD3-like proteins and RHD3 may share partially overlapping roles in the UPR, these data indicate that the loss of RHD3 has a predominant impact on the plant’s ability to respond to ER stress compared to the other RHD3-like genes. RHD3 and AtIRE1 work dependently in the UPR In animals and plants, several sensors and transducers initiate the UPR; however, IRE1, an ER-associated protein kinase and ribonuclease, functions as a major ER stress sensor and plays a key role in the UPR signaling by controlling expression of a conserved suite of UPR genes (Chen & Brandizzi, 2012; Tirasophon et al., 2000; Urano et al., 2000). The Arabidopsis genome encodes two sequence homologues of IRE1, AtIRE1A and AtIRE1B (Chen & Brandizzi, 2012; Koizumi et al., 2001; Noh et al., 2002). To understand how RHD3 could affect the UPR, we tested whether the loss of RHD3 could compromise AtIRE1 signaling in ER stress using a genetic approach. We generated an rhd3-7 atire1a atire1b triple mutant, hereby named ire1/rhd3, and tested its sensitivity to a Tm concentration in the medium that inhibits growth of ire1 for comparison to wild type, rhd3 and ire1 (Chen et al., 2013b; Chen & Brandizzi, 2012). We found that rhd3 grew similar to Col-0, while the triple mutant showed sensitivity to Tm similar to ire1 mutant (Figure 2. 3. A). Next, we measured the induction levels of UPR genes in conditions of ER stress in wild type, rhd3, ire1 and ire1/rhd3 mutants (Figure 2. 3. B). As expected, we found a reduction of UPR transcript genes in ire1 and in rhd3; notably however, in ire1/rhd3 the reduction of transcript levels of UPR genes was similar to ire1 (Figure 2. 3. B). The oversensitive phenotype of ire1 to Tm treatment compared to wild type and rhd3 highlights that, contrarily to RHD3, AtIRE1 is essential to respond to ER stress. Together with the evidence that the induction 60 of UPR genes in the ire1/rhd3 mutant exposed to Tm is comparable to ire1 and significantly different from rhd3, the data also support that RHD3 acts upstream AtIRE1 in ER stress responses. RHD3 is necessary for efficient splicing of bZIP60 mRNA Given the established genetic interaction of RHD3 and AtIRE1 in UPR activation (Figure 2. 3.), we next investigated how RHD3 loss could interfere with proper activation of the UPR. In all eukaryotes, the IRE1 RNAse domain initiates spliceosome-independent splicing of mRNAs encoding bZIP transcription factors, namely XBP1 in mammalian cells, HAC1 in yeast and bZIP60 in plants (Cox et al., 1997; Deng et al., 2011; Kawahara et al., 1997; Moreno et al., 2012; Plongthongkum et al., 2007; Shen et al., 2001; Sidrauski & Walter, 1997). Similarly to the effect of RHD3 deletion on the UPR, loss of bZIP60 splicing interferes with UPR activation (Moreno et al., 2012). However, although bZIP60 is downstream AtIRE1 in the UPR signaling, a bZIP60 loss-of-function mutant does not show the ER stress oversensitive phenotype that is typical of an atire1a atire1b double mutant (Chen & Brandizzi, 2012; Deng et al., 2013) (Figure 2. 4. A), indicating that in addition to the splicing of bZIP60 mRNA, AtIRE1 has other roles that are essential to cope with ER stress. Therefore, a lack of a ER stress oversensitive phenotype of RHD3 and bZIP60 mutants [Figures 2. 4., (Deng et al., 2013)], and the evidence of a genetic interaction between RHD3 and AtIRE1 for UPR induction (Figure 2. 3.) suggested to us that loss of RHD3 could affect the AtIRE1-mediated splicing of bZIP60 mRNA. To test this, we measured the abundance of spliced bZIP60 (SbZIP60) mRNAs as a readout of AtIRE1 activity in the UPR in wild type and rhd3 alleles in normal conditions of growth as well as in the presence of Tm. Indeed, quantitative RT-PCR analyses demonstrated that in the rhd3 alleles spliced 61 bZIP60 transcript was detectable but at significantly reduced levels compared to wild type (Figure 2. 4. B). These data support that the proper UPR gene induction verified in the RHD3 loss-of-function backgrounds during ER stress is linked to interference with the splicing of bZIP60 mRNA in the AtIRE1 signaling pathway. The involvement of RHD3 in ER stress responses and in physiological growth is uncoupled Mutants with defective expression of either RHD3 or AtIRE1 show an obvious phenotype in the elongation of primary root (Chen et al., 2011; Chen & Brandizzi, 2012; Hu et al., 2003; Stefano et al., 2012; Wang et al., 1997) (Figure 2. 5. A), but the mechanisms underlying this phenotype are still unknown. In order to investigate whether RHD3 and AtIRE1 interact in the control of organ growth in physiological conditions of growth, we analyzed the roots of the ire1/rhd3 mutant for comparison to wild type, rhd3 and ire1. The length of the primary root was shorter in ire1/rhd3 than in the respective rhd3 and ire1 mutants, supporting an addictive interaction between these two genes in root growth. Further support to this observation was provided by quantitative semi-automated cell segmentation analyses (French et al., 2012) specifically designed for the unbiased identification of the transition zone, which marks the boundaries of the division zone and the elongation zone (Figure 2. 5. C,D). Consistent with the length measurements of the primary root, in rhd3 and ire1 the division zone was shorter than in wild type and even shorter in the ire1/rhd3 mutant (Figure 2. 5. C), further demonstrating that the triple mutant has addictive phenotypic defects of ire1 and rhd3 mutations. Together these data indicate that even if the function of AtIRE1 in UPR signaling largely depends on the cellular availability of RHD3, AtIRE1 and RHD3 have independent roles in physiological organ growth. 62 DISCUSSION Our results couple architecture and function of the ER by providing evidence that the ability of the ER to respond to induced stress is connected with the cellular availability of the ER-shaping protein RHD3. We demonstrate that RHD3 is necessary for proper UPR signaling through the ribonuclease activity of the major UPR sensor AtIRE1. The evidence that RHD3 acts upstream AtIRE1 in the UPR and that the UPR is unaffected in mutants with disrupted ER network integrity that are unlinked to RHD3 supports that the verified UPR defects of RHD3 are not a general feature of ER mutants. These results imply specificity of the UPR defects directly to RHD3 or to the ER defects caused by loss of RHD3 in the ER membrane. The UPR signaling function of AtIRE1 depends on the presence of RHD3 in the ER membrane High-order IRE1 oligomerization is required for the regulation of IRE1 function in mammalian and yeast cells (Korennykh et al., 2009; Li et al., 2010). Furthermore, interactions of IRE1 with other proteins, including the membrane associated BAX-Inhibitor 1 (BI-1) and the cytosolic adaptor protein TRAF2 (Lisbona et al., 2009; Yoneda et al., 2001) have a demonstrated role in the signaling pathway of IRE1 in animal cells. Specifically, it has been shown that in animal cells BI-1 physically interacts with IRE1 and that loss of BI-1 leads to upregulation of XBP1 mRNA splicing, supporting an inhibitory regulatory role of BI-1 on the UPR signaling function of IRE1/XBP1 pathway (Lisbona et al., 2009). Given the large conservation of the UPR across eukaryotes, it is plausible to hypothesize that similarly to non-plant IRE1 molecules, AtIRE1 interacts with other proteins to modulate its own activity. Therefore, similarly to BI-1, RHD3 may interact with IRE1, although in this model RHD3 would functions as an activator 63 for AtIRE1. The genetic interaction between RHD3 and AtIRE1 in UPR signaling demonstrated in this work and the subcellular localization of both proteins to the ER membranes suggest the occurrence of interactions of RHD3 with AtIRE1 in modulating the activity of this UPR sensor in conditions of ER stress. An alternative explanation for the epistatic relationship between AtIRE1 and RHD3 poses that the defects of the ER morphology due to loss of RHD3 are associated with disruption of the signaling mechanism of AtIRE1 in the UPR independently from heterotypic protein-protein interactions. For example, upon UPR activation in yeast and in mammalian cells, IRE1, which is distributed over the ER network in physiological conditions of growth, forms oligomers that are necessary for its RNAse activity, and localizes into dynamic clusters in the ER, which have been proposed to function as specialized molecular microenvironments for IRE1 signaling in the UPR (Li et al., 2010). Whether AtIRE1 also undergoes dynamic clustering in the UPR is unknown but this possibility is likely given the conservation of IRE1 at protein and functional levels (Chen & Brandizzi, 2013). Although the biological meaning of the dynamic rearrangements of the ER network is unknown, ER tubule fusion mediated by RHD3 may represent a cellular strategy to facilitate mixing of ER lumenal and membrane tubule content as a necessary step to enable protein functions at the ER, including the activity of membrane-anchored signaling proteins. In this view, it is possible that the disruption of homotypic membrane fusion due to loss of RHD3 may reduce the ability of AtIRE1 either to traffic through the ER membrane to form signaling clusters or interact efficiently with its bZIP60 mRNA substrate. 64 Among the Arabidopsis RHD3 isoforms, RHD3 likely functions as a dominant gene in the UPR signaling We have shown that the reduction of UPR activation in rhd3 is not phenocopied in the rhd3-l1 and rhd3-l2 backgrounds. Recent evidence supports that RHD3-L1 and RHD3-L2 share partially overlapping functions with RHD3. Specifically, genetic analyses have shown that rhd3/rhd3-l1 is lethal and rhd3/rhd2-l2 plants are male sterile (Zhang et al., 2013); however, unlike loss of RHD3, deletion of either RHD3-L1 or RHD3-L2 does not affect plant growth (Chen et al., 2011). Furthermore, overexpression of mutant RHD3-L2 proteins bearing mutations in the GTPase domain leads to defects in ER morphology (Zhang et al., 2013), posing that although RHD3-L2 is functional and likely shares overlapping role with RHD3 in ER morphology, RHD3 is a dominant isoform. It is therefore possible that the limited UPR induction verified in rhd3 is linked to a partially overlapping function of RHD3-L1 and RHD3-L2 in the UPR. The dependence of AtIRE1 on RHD3 for UPR signaling is uncoupled from their roles in organ growth Loss of function of RHD3 and AtIRE1 causes defects in primary root elongation (Chen et al., 2011; Chen & Brandizzi, 2012; Hu et al., 2003; Stefano et al., 2012; Wang et al., 1997). Here we confirmed this notion and we also showed that these defects are, at least partially, linked to reduced elongation of cells in the division zone. Moreover, we demonstrated the absence of a functional relationship between ire1 and rhd3 in root growth, given that the triple ire1/rhd3 mutant exhibits an addictive combination of the root growth phenotypes of either ire1 or rhd3 mutants. Under ER stress condition, we instead demonstrated epistasis between RHD3 and 65 AtIRE1 in UPR signaling. Taken together, these data strongly support that the role of RHD3 and AtIRE1 in UPR signaling is uncoupled from the role of the two proteins in organ growth. Nonetheless, it cannot be excluded that RHD3 and AtIRE1 may share the control of some critical components in pathways necessary for root growth. The ER produces one-third of the cellular proteome including enzymes necessary for the biosynthesis of the cell wall, which are then exported to the Golgi for modification and sorting (Rojo & Denecke, 2008). Therefore, during physiological growth, which requires enhanced production of the building blocks of the cell and cell wall, the loss of AtIRE1 is likely to impede with the optimal protein synthesizing capacity of the ER. On the other hand, loss of RHD3 function is known to interfere with the subcellular positioning and movement of the Golgi apparatus (Chen et al., 2011; Stefano et al., 2012). Therefore, AtIRE1 and RHD3 may be controlling convergent pathways in organ growth through functionally parallel routes whereby loss of AtIRE1 may disrupt the production of proteins necessary for organ growth, while disruption of RHD3 may lead to aberrant subcellular distribution of such proteins due to a spatial disorganization of the critical sorting organelles such as the Golgi. A causative relationship between loss of RHD3 genes and defective UPR signaling may explain the lethality of high order RHD3 mutations Independently from the underlying causes for the attenuation of AtIRE1 signaling in the UPR due to loss of RHD3, our results demonstrate a functional connection between RHD3 and AtIRE1 in the ER stress responses. The pathway components of the UPR are essential to support the ability of the cell to promote protein folding and maturation of newly synthesized proteins and to dispose of misfolded proteins. When the cell’s adaptive responses to stress are insufficient, 66 cells enter apoptotic death. The evidence that a concomitant loss of RHD3 and RHD3-like proteins leads to cell death strongly implies that the formation of the tubular ER network is of general physiological significance (Zhang et al., 2013) but the causes underlying lethality due to high-order mutations in the RHD3 gene family are unknown. Insufficient or deregulated UPR contributes to the pathology of several important human diseases, including diabetes, neurodegeneration and cancer, and to death in early development in mouse (Ron & Walter, 2007; Rutkowski & Hegde, 2010). Since in the absence of RHD3 cells fail to actuate the UPR properly, we suggest that the reported lethality of higher order mutations within the RHD3 family (Zhang et al., 2013) may be a consequence of the inability of cells to evoke the UPR efficiently during growth which imposes physiological stress on the ER. Given the functional conservation of ER- associated dynamin-like proteins in ER homotypic fusion as well as of the general mechanisms of UPR signaling, it will be interesting to test whether the UPR is compromised also in atlastin and Sey1p mutants and define whether the etiology of genetic mutations linked loss of functional atlastins in animals (Blackstone et al., 2010; Chen et al., 2013a) are linked to inefficient UPR. 67 ACKNOWLEDGEMENTS We thank 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 NSF (MCB 1243792). 68 APPENDIX 69 Table 2. 1. Primers used in this study Primer name Sequence Purpose UBQ10 For 5'-GGCCTTGTATAATCCCTGATGAATAAG-3' qRT-PCR UBQ10 Rev 5'-AAAGAGATAACAGGAACGGAAACATAGT- 3' qRT-PCR bZIP60us_FWD 5'-GGAGACGATGATGCTGTGGCT-3' qRT-PCR bZIP60s_REV 5'-CAGGGAACCCAACAGCAGACT-3' qRT-PCR BiP3 For1 BiP3 Rev1 5'-CGAAACGTCTGATTGGAAGAA-3' qRT-PCR 5'-GGCTTCCCATCTTTGTTCAC-3' qRT-PCR PDIL For 5'-AAGTGGTCCTGCTTCTGTTGAA-3' qRT-PCR PDIL Rev 5'-TTGAACAGCCTCACTGCAGGT-3' qRT-PCR ERdj3A For 5'-GTGAAAGCGAAGAGCGTTGAT-3' qRT-PCR ERdj3A Rev 5'-TCACGCTGCTTTGCATCCT-3' IRE1a_FWD 5'-GCTTCAGACCTCATATCCCG-3 qRT-PCR qRT-PCR IRE1a_REV 5'-AGCATCACGAAGGAAAGACAG-3' qRT-PCR IRE1b_FWD 5'-GGTGGGATGAGAAACTGGATAG-3' qRT-PCR IRE1b_REV 5'-AGTTTGTTCCGTATGACCCG-3' qRT-PCR BP80 For1 5'-GCTCTTTCCGGTGGTGATATG-3' qRT-PCR BP80 Rev1 5'-CAGGATGTGGAACCGACTCA-3' qRT-PCR PVA12 For1 5'-CAAGGTTAAGACGACGAATCCAA-3' qRT-PCR PVA12 Rev1 5'-TCTGGGATGAACAACACCAGTATT-3' qRT-PCR Tip4 For2 5'-GGACTCGCCGGTTTCATCTA-3' qRT-PCR Tip4 Rev2 5'-GTCAGCGACTGGGACATGTG-3' qRT-PCR Actin3 For2 5'-CAGGCCCGTCGATTGTTC-3' qRT-PCR 70 Table 2. 1. (cont’d) Actin3 Rev2 5'-TCCGGAAGCAGACTTAACTTCAA-3' qRT-PCR Tub4 For2 5'-TGGATCCCAAACAACGTCAA-3' qRT-PCR Tub4 Rev2 5'-GCCATTTTCAAACCCTTTGGT-3' qRT-PCR 71 Figure 2. 1. Mutants with defects in ER organization and/or function show an ER stress response similar to wild type 14-day-old seedlings of the indicated genotypes were transferred to growth medium containing Tm (0.5 µg/ml) for 1 day. Samples were analyzed by quantitative RT-PCR for transcriptional levels of the UPR indicators BIP3, PDIL and ERDJ3A. Transcription of ACTIN8 was used as internal control. No significant differences were established among DMSO values within each group. Error bars represent SE of three replicates. NS, not significant; Student’s t- test. 72 Figure 2. 2. RHD3 has compromised UPR Quantitative RT-PCR of various UPR indicators in wild type and rhd3 mutants. cDNA was synthesized using 14-day old seedlings treated with Tm (0.5 µg/ml) for 1 day. Values are presented relative to indicated DMSO control, which was set to 1. Transcription of UBQ10 was used as internal control. Error bars represent SE of three replicates. Data significantly different from the corresponding controls are indicated (mutant versus the wild type under treatment) (*P < 0.05, **P < 0.01; Student’s t- test). 73 Figure 2. 3. RHD3 and IRE1 work dependently in UPR signaling (A) To examine Tm sensitivity of Col-0, ire1, rhd3, ire1/ rhd3, seeds were sown on solid medium containing Tm (0.05 µg/mL). Images show the appearance of two-week-old seedlings grown on control (DMSO) or Tm plates. (B) Quantitative RT-PCR of different UPR indicators. cDNA was synthesized using 14-day old seedlings of each genotype treated with Tm (0.5 µg/mL) for 1 day. Values are presented relative to indicated DMSO control, which was set to 1. Transcription of UBQ10 was used as internal control. Error bars represent SE among three replicates. Data significantly different are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, NS: not significant; Student’s t- test.) 74 Figure. 2. 4. Loss of rhd3 results in reduced splicing of bzip60 mRNA under ER stress (A) To examine Tm sensitivity of bzip60, ire1, rhd3 and gom8, approximately 36 seeds of each genotype were sown on growth medium containing Tm (0.05 µg/mL). The images shown in the figure were captured at 10 day after germination. Col-0 was used as control. Note that wild-type, bzip60, rhd3 and gom8 did not show the strong Tm sensitivity of ire1. (B) 14-day-old seedlings were transferred into liquid growth medium containing Tm (0.5 µg/mL) for 2 hrs. Abundance of spliced bZIP60 mRNA in two independent rhd3 alleles was established by qRT-PCR in non-treated samples (0 hrs) or in Tm-treated samples (2 hrs). No significant differences were established among DMSO values. Errors bar represent SE. (*P<0.05; Student’s t-test). 75 Figure 2. 5. Analyses of ire1/ rhd3 mutant support a synergistic interaction between RHD3 and IRE1 in the control of primary root elongation 76 (A) 10-day-old seedlings of wild-type Col-0, ire1, rhd3 and ire1/ rhd3 were grown on solid growth medium on vertical plates. (B) Measurements of the primary root length of wild-type Col-0, bzip60, ire1, rhd3 and ire1/ rhd3. Primary root length of 30-35 seedlings was measured for each genotype. Errors bar represent SE.(****P<0.0001, NS, not significant; Unpaired t-test). (C-D) Landscape analyses of root defects. Images of primary roots stained with propidium iodide to counterstain the cell wall (C) were segmented using CellSeT software, and the TSZ was identified using the Cell-o-Tape macro (see methods section). In C, a dotted line marks the transition zone (TSZ) based on the coordinates established in panel D. From these analyses it appears that the division zone is shorter in the ire1/ rhd3 mutant compared to rhd3 and ire1. EZ: elongation zone; DS: division zone; CS: cell size; CN: cell number. 77 Figure 2. 6. Loss of RHD3 does not affect the basal levels of the UPR Quantitative RT-PCR of different UPR indicators. cDNA was synthesized using 14-day old seedlings exposed to medium containing DMSO, which serves as ER stress treatment control, for 1 day. The experiment shows that the basal levels of UPR indicators in wild-type and rhd3 are low and similar. Transcription of UBQ10 was used as internal control. Error bars represent SE among three replicates. (NS, not significant; Student’s t-test). 78 Figure 2. 7. Loss of RHD3 does not affect the mRNA abundance of genes encoding secretory and cytosolic proteins in conditions of ER stress 14-day-old seedlings were transferred either to solid medium containing Tm (0.5 µg/ml) or to control plates (DMSO) for 1 day. Samples were analyzed by qRT-PCR for transcriptional levels of secretory proteins such as the prevacuolar sorting receptor, BP80, the ER associated VAMP- like protein, PVA12, and the tonoplast intrinsic protein, TIP4, and AtIRE1 isoforms IRE1A, IRE1B, as well as cytosolic proteins actin (ACT3) and tubulin (TUB4). Transcription of UBQ10 was used as internal control. Errors bar represent SE. (NS, not significant; Student’s t-test). 79 Figure 2. 8. The compromised UPR induction phenotype is specific to the loss of RHD3 Quantitative RT-PCR of various UPR indicators in wild type and RHD3 -like mutants. cDNA was synthesized using 14-day old seedlings treated with Tm (0.5 µg/ml) for 1 day. Values are presented relative to indicated DMSO control, which was set to 1. Transcription of UBQ10 was used as internal control. Error bars represent SE of three replicates. No significant differences were found from the corresponding controls (mutant versus wild type under treatment). 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J Biol Chem 276, 13935-40. Zhang, M., Wu, F., Shi, J., Zhu, Y., Zhu, Z., Gong, Q. & Hu, J. (2013). RHD3 family of dynamin- like GTPases mediates homotypic endoplasmic reticulum fusion and is essential for Arabidopsis development. Plant Physiol 163, 713-720. Zheng, H., Kunst, L., Hawes, C. & Moore, I. (2004). A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus. Plant J 37, 398-414. 86 CHAPTER III A salicylic acid-independent role of NPR1 is required for protection from proteotoxic stress in the plant endoplasmic reticulum The work presented in this chapter has been published in Proceedings of the National Academy of Sciences (10.1073/pnas.1802254115) Lai Y-S, Renna L, Yarema J, Ruberti C, He SY, Brandizz F. A salicylic acid-independent role of NPR1 is required for protection from proteotoxic stress in the plant endoplasmic reticulum. 87 ABSTRACT The unfolded protein response (UPR) is an ancient signaling pathway designed to protect cells from the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). Because mis-regulation of the UPR is potentially lethal, a stringent surveillance signaling system must be in place to modulate the UPR. The major signaling arms of the plant UPR have been discovered and rely on the transcriptional activity of the transcription factors bZIP60 and bZIP28 and on the kinase and ribonuclease activity of IRE1, which splices mRNA to activate bZIP60. Both bZIP28 and bZIP60 modulate UPR gene expression to overcome ER stress. In this study, we demonstrate at a genetic level that the transcriptional role of bZIP28 and bZIP60 in ER stress responses is antagonized by nonexpressor of PR1 genes 1 (NPR1), a critical redox- regulated master regulator of salicylic acid (SA)-dependent responses to pathogens, independently from its role in SA-defense. We also establish that the function of NPR1 in the UPR is concomitant with ER stress-induced reduction of the cytosol and translocation of NPR1 to the nucleus where it interacts with bZIP28 and bZIP60. Our results support a novel cellular role of NPR1 as well as a new model for plant UPR regulation whereby SA-independent ER stress-induced redox activation of NPR1 suppresses the transcriptional role of bZIP28 and bZIP60 in the UPR. 88 INTRODUCTION The ER is the power hub for the synthesis and folding of proteins of the secretory pathway (Stefano et al., 2015; Vitale & Denecke, 1999). In the ER, the newly synthesized proteins are folded and modified with the assistance of several proteins, including BiP, calnexin and protein disulfide isomerase (PDI) (Mori, 2000). Although the ER functions to maintain a balance between synthesis capacity and the cell’s demand for secretory proteins, altered physiological conditions and/or environmental stimuli can overload the ER protein synthesis capacity and cause accumulation of unfolded and secretory misfolded proteins. This leads to a potentially-lethal condition known as ER stress (Kaufman et al., 2002; Rutkowski & Kaufman, 2004). To overcome ER stress, cells execute cytoprotective signaling programs, collectively known as the unfolded protein response (UPR) (Walter & Ron, 2011). The UPR functions at the transcriptional and translational levels by enhancing the ER protein-folding ability and removing unfolded/misfolded proteins through the production of degradation-associated factors of the ER quality control (ERQC) and ER associated degradation (ERAD) pathways, respectively (Jian- Xiang Liu & Howell, 2010b; Walter & Ron, 2011). In yeast, the UPR relies on inositol-requiring enzyme-1 (Ire1p), an ER membrane resident kinase and ribonuclease (Sidrauski & Walter, 1997). Upon sensing ER stress, Ire1p releases the translational inhibition of HAC1 mRNA through cytoplasmic splicing (Aragón et al., 2009; Sidrauski & Walter, 1997). The spliced HAC1 mRNA encodes a basic leucine zipper domain (bZIP) transcription factor (TF) that translocates into the nucleus to modulate transcription of UPR target genes. In metazoans, the UPR has expanded to three signaling arms operated by distinct UPR sensors and transducers: IRE1, activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK). IRE1 mediates cytoplasmic splicing of the Hac1 orthologue, 89 XBP1, resulting in the activation of downstream UPR target genes. ATF6, an ER membrane- tethered bZIP TF, is activated by proteolytic cleavage in the Golgi, which is followed by transport of the active TF domain into the nucleus where it regulates UPR gene expression (Ye et al., 2000). The third arm of the UPR is controlled by the ER membrane protein kinase PERK and it attenuates general translation but selectively promotes expression of ATF4 and C/EBP- homologous protein (CHOP) TFs for regulation of UPR genes(B’Chir et al., 2013). The plant UPR relies on two pathways that are similar to the metazoan IRE1 and ATF6- mediated signaling. The Arabidopsis genome encodes two plant IRE1 isoforms, IRE1A and IRE1B, as well as the TF bZIP28. Upon ER stress, AtIRE1 splices the mRNA of bZIP60 causing a frameshift and synthesis of a potent bZIP-TF, spliced bZIP60 (sbZIP60). SbZIP60 is imported into the nucleus where it modulates the expression of UPR genes(Deng et al., 2011; Iwata, Fedoroff, & Koizumi, 2008). Analogously to ATF6, bZIP28 is an ER-associated membrane bZIP-TF. Upon translocation from the ER to the Golgi in response to ER stress, bZIP28 is activated by proteolysis actuated by site-1 and site-2 proteases. Activated bZIP28 is translocated into the nucleus where it modulates expression of ER stress responsive genes (Jian-Xiang Liu, Srivastava, Che, & Howell, 2007a; Jian-Xiang Liu et al., 2007b). BZIP60 and bZIP28 share largely overlapping roles at the onset of ER stress and in prolonged ER stress conditions. Indeed, loss of both TFs leads to an inability of cells to modulate expression of ER stress responsive genes and can also lead to plant death in conditions of unresolved ER stress (Deng et al., 2013). Because of the critical role of the UPR in cell homeostasis, a stringent surveillance system must be in place to regulate its amplitude and duration. For example, the yeast Snf1, an AMP-activated protein kinase (AMPK), is involved in the negative regulation of ER stress signaling through the Hog1 MAPK cascade (Mizuno et al., 2015). In metazoans, the Wolfram 90 syndrome 1 (WFS1) protein physically interacts with ATF6 and boosts ATF6 proteasome- mediated degradation causing the suppression of ATF6-mediated signaling in the UPR (Fonseca et al., 2010). Furthermore, the ER associated Bax-inhibitor 1 (BI-1) is believed to attenuate UPR signaling through protein-protein interaction with IRE1 (Lisbona et al., 2009). Because of the essential roles of the UPR, it is plausible that also plants harness a UPR surveillance system; however, such modulators of the plant UPR transducers are unknown. The evidence that plants lack the metazoan PERK pathway and that there is limited conservation of the UPR transducers at protein sequence levels suggests the existence of plant-unique mechanisms for UPR signaling and for UPR surveillance. In this work, we demonstrate that nonexpressor of PR1 genes (NPR1) acts as a component of the UPR monitoring system in plants. NPR1 is a known master regulator of the salicylic acid (SA)-controlled signaling pathway in pathogen infection. Pathogen attack activates SA defense in which antimicrobial proteins, such as the pathogenesis-related protein 1 (PR1), are produced in the secretory pathway for delivery into the apoplast (Wang et al., 2005). During SA defense, changes in the cytosolic redox state convert NPR1 oligomers into monomers. In turn, monomeric NPR1 regulates expression of PR1 through interaction with TGA TFs(Fan & Dong, 2002). Elevated levels of endogenous or exogenous SA have also been shown to activate the UPR (Nagashima et al., 2014; Pajerowska-Mukhtar et al., 2012). NPR1 is required for endogenous SA-mediated up-regulation of the genes encoding proteins that mitigate ER stress through the TL1-binding transcription factor 1 (TBF1) (Pajerowska-Mukhtar et al., 2012). However, in other studies, UPR activation by exogenous SA appears to occur through the signaling of the IRE1/bZIP60 and bZIP28 UPR arms, but independently from NPR1, as supported by the evidence that the UPR signaling in a NPR1 loss-of-function mutant is 91 indistinguishable from wild type in the presence of exogenous SA (Moreno et al., 2012; Nagashima et al., 2014). The ambiguous role of NPR1 in the UPR activation responding to SA raises the question whether SA signaling is cross-regulated with ER stress signaling. Here we address this question directly and we demonstrate that NPR1 is a critical regulator for ER stress responses induced by tunicamycin (Tm; a well-established ER stress inducer) through mechanisms that are independent from the availability of endogenous SA and that rely on redox-induced nuclear translocation of NPR1 upon ER stress-prompted reduction of the cytosolic environment. Consistent with these findings, we uncover that the loss of functional NPR1 exhibits a more robust Tm-induced UPR, resulting in increased tolerance to chronic ER stress and upregulation of UPR target genes compared to wild type, while an overabundance of NPR1 has opposite effect on the plant’s ER stress tolerance and UPR gene expression. Based on these results and the evidence of a physical interaction of NPR1 with bZIP28 and bZIP60 demonstrated in this work, we propose that NPR1 functions as a negative regulator of bZIP28 and bZIP60 to optimize their cytoprotective role in the UPR. Together, our findings demonstrate a novel role of NPR1 in stress responses that relies on SA-independent NPR1-activation mechanisms shared by a functionally-distinct stress signaling route. 92 MATERIALS AND MATHODS Lines and Plant Growth Conditions Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as a wild-type reference genotype in this study. The mutant lines used in this work are: ire1a (Col-0; WISCDSLOX420D09), ire1b (Col-0; SAIL_238_F07), bzip28 (Col-0; SALK_132285), bzip60- 1 (Col-0; SALK_050203), npr1-6 (Col-0; SAIL_708F09), tbf1 (Col-0; SALK_104713). Seeds were stratified at 4 °C for 2 days and plated on half-strength Linsmaier Skoog (LS) medium (1/2x LS salts, 1.5% sucrose, 1.2% Agar). For standard growth condition, plants were grown in a 16h-light/ 8h-dark cycle at 21 °C. Agrobacterium (GV3101)-mediated transformation of Arabidopsis thaliana and Nicotiana tabacum plants was performed by the floral dip and infiltration methods, respectively (Batoko, Zheng, Hawes, & Moore, 2000; Clough & Bent, 1998). Tm, SA and AZC Treatment Tunicamycin resistance experiments were performed by growing seedlings directly on plates containing 25 nM or 50 nM Tm. Tm was replaced by the same volume of DMSO in control treatment. For each treatment, the primary root length of 35 seedlings was measured. For qRT-PCR analyses, seedlings were grown on 1/2 LS salts, 1.5% sucrose, 1.2% Agar medium for 14 days followed by transfer of seedlings to 0.5 M Tm plates for indicated time periods. For roGFP2 and subcellular localization assays, 14-day-old seedlings were transferred to plates containing 0.5 M Tm, 0.5 mM SA, 100 M AZC or 500 M AZC for one day, followed by confocal microscopy analyses. 93 Reduction of primary root growth and shoot growth were determined by comparing the 14-day-old primary root length or shoot weight grown on mock medium versus 50 nM Tm medium using the formula, (Root mock-Root Tm/ Root mock)*100 or (Shoot mock-Shoot Tm/ Shoot mock)*100, respectively. The analyses were repeated at least with three biological replicate with at least two technical replicates in each experiment. RT-PCR and qRT-PCR Expression Analyses Total RNA was extracted using Macherey-Nagel NucleoSpin RNA Plant kit (www.mn- net.com) and used for RT-PCR and qRT-PCR with triplicates as described previously (Lai, Stefano, & Brandizzi, 2014). UBQ10 was utilized as a loading control of RT-PCR and an internal control in normalization of qRT-PCR. Similar patterns of expression were observed in the three independent biological replicates. Primers used in this study are listed in Table 3. 1. Plasmid Construction The Phusion high-fidelity DNA polymerase (NEB, USA) was employed to amplify all DNA sequences using the primer sets in the Table 3. 1. and the Gateway system (Invitrogen, USA) was used to generate expression plasmids. The coding sequences of SEC22 N, bZIP28 N, bZIP28 T, bZIP60 S, bZIP1, NPR1 and CPY* were amplified using Arabidopsis cDNA as templates. With the exception of cloning of SEC22 N coding sequence into roGFP2 expressing vector through enzyme digestion method, all amplified coding sequences were recombined into indicated gateway vectors via LR reaction (Invitrogen, USA). To create the npr1 complementing construct, the full length coding sequence of NPR1 and a 2.3kb DNA fragment upstream of NPR1 encompassing the NPR1 promoter were amplified 94 using NPR1_F and NPR1_R primers and pNPR1_F and pNPR1_R primers (Table 3. 1.) following by cloned into pENJAZ9C, a Gateway compatible entry vector individually to generate pNPR1::NPR1. Then, the pNPR1::NPR1 construct was LR recombined into the expression vector pGWB540 to produce the pNPR1::NPR1-YFP construct (Huot et al., 2017). For CPY*-RFP construct, the coding sequence of CPY* with a G227A mutation was generated by mutagenesis followed by the LR recombination into pGWB654 vector. Confocal Laser Scanning Microscopy Imaging and Ratiometric Assays Confocal imaging was performed with an inverted laser scanning confocal microscope Nikon A1RSi. For subcellular localization assays, leaf tissues were mounted on a slide in a drop of tap water and viewed with the confocal microscope. YFP and monomeric RFP fluorescences were monitored at excitation wavelength of 513 nm/561 nm and a bandpass 520-550 nm/ 575 nm long pass emission filter, respectively. For the ratiometric analyses, we used leaves of transgenic A. thaliana plants or tobacco plants transiently expressing roGFP2-SEC22 and followed an established protocol (Brach et al., 2009). Briefly, the confocal images were collected with a 40 x lens in multi-track mode of the microscope with line switching. The roGFP2 was excited at 405 nm and 488 nm and the emitted fluorescence was collected at 505-530 nm. To ensure consistency in the data collection across samples, we used identical confocal microscope settings (i.e., laser, laser intensity, pinhole size and photomultiplier settings) for all the samples. For limiting the contribution of noise to the final ratio image and enabling optimal ratiometric analysis of roGFP2, only cells in which the fluorescence excited at 405 nm was at least five times higher than the background were used for the analysis. The pixel fluorescence intensity was analyzed using the Nikon microscope software (NIS-Element AR 4.30). To analyze images, 95 we adopted the default settings of the microscope software, which adopts on a broad fluorescence intensity scale (0-4095.grey levels for 12 bits images). The 405 nm fluorescence intensity values were divided by the 488 nm fluorescence intensity values to estimate a ratio image, which was then used for generation of histograms. A Gaussian fit was carried out on the histograms to produce the mean of ratio value. For the imaging analyses, the presented results are representative of at least three biological replicates. Isolation of Nuclei Fourteen-day-old seedlings were treated either in mock conditions (DMSO), with Tm or SA for one day prior to collection and subjected to nuclear extraction using CelLytic PN isolation/extraction Kit (www.sigmaaldrich.com). Protein Extraction and Western Blot analyses Fourteen-day-old seedlings were treated with either in mock conditions (DMSO), Tm or SA for one day prior to collection and subjected to total protein extraction in lysis buffer (1x SDS buffer, 0.125M Tris-HCl pH 7.5, 2% SDS, 5% -mercaptoethanol). After centrifugation at 14000 rpm for 5 min, the supernatants containing 65g total protein were loaded to SDS-PAGE gel followed by transferring to a nitrocellulose membrane. The membrane was blocked with 1x PBS, 5% milk, 0.05% Tween 20 and 0.02% sodium azide for 2h and incubated with primary anti-NPR1 (1:1000) overnight or anti-GFP (1:3000; ab290, www.abcam.com), anti-UGPase (1:3000; cytosol, www.agrisera.com)and anti-Histone3 (1:10000; nucleus, www.agrisera.com) for 4h at 4°C, followed by secondary goat anti-rabbit: HRP (1:20000) for 2h at room temperature. Further steps in the analysis were performed as described in Crofts et al. (1999) (Crofts, 1999). 96 The Western blot presented in the work is representative of at least three independent biological replicates that showed similar pattern of protein fractionation. FRET Assay The coding sequences of NPR1 and UPR genes, bZIP28 N and bZIP60s, and bZIP1 were cloned into pEarleyGate104 and pVKcCFP-GW to generate YFP and CFP fusion proteins (Invitrogen, USA). After co-transformation with indicated sets of constructs into Nicotiana tabacum for two days, sensitized emission FRET was examined with a Nikon A1RSi confocal microscope. The images in donor at excitation 443 nm; emission 465-505 nm, acceptor at excitation 514 nm; emission 525-600 nm and FRET at emission 525-600 nm were captured. The fluorescence levels of CFP and YFP channels were scanned as for sensitized emission FRET before and after photo-bleaching. The energy transfer efficiency (EFRET) between the paired proteins was calculated based on the alteration in fluorescence intensity of the donor before and after photo-bleaching. Yeast two-Hybrid Analyses Yeast two-hybrid analyses were performed using the matchmaker LexA system following the manufacturer’s manual (Clontech Yeast protocol No. PT3024-1). The coding sequence (CDS) of NPR1 was cloned into pGilda-GW bait vector and bZIP1 and UPR genes, bZIP28 and bZIP60 CDS deleting AD and TMD were cloned into pB42AD-GW prey vectors using LR reaction (Invitrogen, USA) individually. Each bait-prey combination was co- transformed into yeast strain EGY48 following by determination of protein-protein interaction 97 using -galactosidase assay. The picture was taken after two days of incubation at 30 °C. This experiment was repeated three times with consistent results. Salicylic Acid Measurements Two-week-old plate-grown seedlings (~100 mg) of seedlings were harvested and ground in liquid nitrogen. The samples were extracted at 4 °C overnight using 1 ml of ice cold methanol:water (80:20 v/v) containing 0.1% formic acid; 0.1 g L-1 butylated hydroxytoluene (BHT) spiked with propyl 4-hydroxybenzoate as an internal standard for quantification of SA. Then samples were vortexed and centrifuged at 12000 xg 4 °C for 10 min, after which the flow throughs were transferred to HPLC vials for the measurement of endogenous concentration of free and glucosylated SA by LC-MS according to an established protocol (Zeng et al., 2011). The results are from three biological replicates. Hoechst Staining The nuclei in cotyledons from two-week-old seedlings were stained for one hour with 1 g/ml Hoechst33342 solution in modified Dulbecco’s PBS; 8 mM sodium phosphate, 2 mM potassium phosphate, 140 mM sodium chloride, 10 mM potassium chloride, pH 7.4. The stained nuclei were excited with 402 nm diode laser using confocal laser scanning microscope (Nikon A1RSi). 98 RESULTS The Loss of NPR1 Increases Resistance to Chronic ER Stress Because exogenous SA induces the UPR, as demonstrated by enhanced splicing of bZIP60 and proteolysis of bZIP28 in conditions of exogenous SA treatment(Nagashima et al., 2014), we tested whether SA biosynthesis and SA signaling factors could be involved in the UPR activation in response to ER stress. To assay plants’ ER stress response, we adopted the customary approach to measure root length and aerial growth in conditions of physiological growth and chronic ER stress conditions (Chen & Brandizzi, 2012; Liu et al., 2007a). The latter conditions are known to cause reduction of primary root length and shoot weight in wild type (wild-type Col-0), a response that is enhanced by defective UPR signaling due to the loss of IRE1 (Deng et al., 2013) or to the combined loss of bZIP28 and bZIP60 (Deng et al., 2013; L. Sun et al., 2013). Therefore, we used this approach to establish the responses of wild type, UPR signaling mutants and SA-mutants in conditions of chronic ER stress, and monitored the primary root elongation and shoot growth of seedlings growing on media containing the well-established ER stress inducer, tunicamycin (Tm) or Tm-solvent (DMSO, control) (Iwata & Koizumi, 2005). We adopted wild type (Col-0), an ire1a/ire1b double mutant (hereafter ire1) (Chen & Brandizzi, 2012), the bzip28/bzip60 (Deng et al., 2013) double mutant (hereafter bzip28/60), the SA induction-deficient mutant sid2-2 (hereafter sid2) (Wildermuth, Dewdney, Wu, & Ausubel, 2001) and the npr1-6 null mutant (hereafter npr1) (Xin et al., 2016) as genetic backgrounds. Consistent with previous studies (Deng et al., 2013), the ire1 and bzip28/60 showed a significant decline of primary root elongation and shoot growth in the presence of increasing concentrations of Tm (Figures 3. 1. A, B, Figure 3. 7. A, B). We also found that the sid2 mutant exhibited similar primary root elongation and shoot growth compared to Col-0 under mock and 99 Tm conditions (Figures 3. 1. A, B, Figure 3. 7. A). However, both the root length and shoot weight of the npr1 mutant were reduced in response to 25 nM and 50 nM Tm treatments compared to mock conditions (Figure 3.1. B, Figure 3. 7. A). To quantify the Tm-sensitivity for each genetic background, we also estimated the ratio values of root length and shoot weight in mock versus Tm 50 nM conditions for each genetic background, and found that the reduction of primary root length was significantly higher for Col-0 compared to npr1 (Figure 3. 1. C) and no difference on reduction ratio of shoot weight between Col-0 and npr1 (Figure 3. 7. B). While the Tm sensitivity assay on ire1 and bzip28/60 confirms previous findings(Deng et al., 2013), these results indicate that compared to Col-0 and sid2, the npr1 mutant displays enhanced resistance to chronic ER stress in the root growth. We therefore hypothesize that the homeostasis of Tm-induced ER stress response in plants is independent from endogenous SA, as demonstrated by the wild type-like response of sid2 to Tm, but it requires NPR1, as demonstrated by a reduced sensitivity of npr1 to Tm compared to wild type in root growth. NPR1 Negatively Modulates UPR Activation under ER stress Based on our results of a reduced sensitivity of the npr1 mutant to chronic ER stress (Figures. 3. 1., 3. 7.), we hypothesized that the loss of NPR1 could be linked to dis-regulation of UPR gene expression under ER stress. To test this, we aimed to determine the UPR activation in Col-0, bzip28/60 and npr1 by measuring the transcript levels of well-known UPR marker genes, such as unspliced bZIP60 (unbZIP60), spliced bZIP60 (sbZIP60), Binding Protein 1 (BiP1), Protein Disulfide Isomerase Like 1-2 (PDL1-2) and Calnexin 1 (CNX1), which are induced at the onset of ER stress and mark the activation of adaptive UPR (Chen & Brandizzi, 2012; Chen Y, Aung K, Rolčík J, Walicki K, Friml J, 2014). Using quantitative RT-PCR (qRT- 100 PCR), we found that the UPR activation was significantly reduced in bzip28/60 mutant plants compared to Col-0 (Figure. 3. 2., Figure 3. 8. A, B). In sharp contrast, the induction of UPR genes in npr1 was significantly higher relative to Col-0 (Figure. 3. 2., Figure 3. 8. A, B), indicating that NPR1 negatively modulates adaptive UPR activation. NPR1-mediated Attenuation of the Plant UPR is Independent of Endogenous SA Levels and Partially Intersects with the TBF1 signaling Because NPR1 functions downstream of SA (Kinkema, Fan, & Dong, 2000), we next aimed to test whether endogenous SA levels could contribute to NPR1-mediated suppression of the UPR activation. To do this, we assessed the transcriptional induction of UPR marker genes in sid2 plants compared to Col-0 in the presence of Tm, and found no significant differences (Figure 3. 2.). We also found no effect of Tm treatment on the accumulation of free and conjugated SA (Figure 3. 9. A), and we established that the transcript levels of genes involved in the SA signaling pathway (i.e., CBP60g, SID2 and PR1) (Chen et al., 2009; Mou, Fan, & Dong, 2003; Wang et al., 2009) displayed no significant changes in response to Tm treatment in Col-0 (Figure 3. 9. B). Based on these results and on the similar sensitivity of sid2 and Col-0 to chronic ER stress (Figure 3. 1.), we deduce that the UPR activation does not depend on elevation of endogenous SA levels. These data indicate that ER stress does not trigger the SA signaling pathway and support the possibility that NPR1 is involved in UPR gene activation independently from SA. It is well established that in localized acquired resistance (LAR) and systemic acquired resistance (SAR) NPR1 serves as a transcriptional cofactor of TL1-binding factor (TBF1) to 101 facilitate protein secretion and induce expression of several UPR genes (Pajerowska-Mukhtar et al., 2012; Dong Wang et al., 2005). Therefore, we next sought to test whether TBF1 is necessary for mounting the UPR activation in ER stress conditions. First we assessed whether ER stress could alter the expression of TBF1-dependent SA-induced genes, such as TGA3, PAD4, CRT3, CNX1, BiP2 and PDIL (Pajerowska-Mukhtar et al., 2012). Using qRT-PCR analyses in Col-0, bzip28/60 and npr1, we found no significant changes in the transcript levels of TGA3, PAD4 and CRT3 in a time course treatment with 0.5 M Tm compared to mock-treated plants (Figure 3. 8. B). These results indicate that ER stress conditions do not influence the transcriptional levels of these genes in Col-0 and that canonical UPR transcription factors, bZIP28 and bZIP60, as well as NPR1 are not involved in the transcriptional regulation of these SA-dependent genes. However, the transcript levels of CNX1, BiP2 and PDIL showed enhanced induction in npr1 compared to Col-0 (Figure 3. 8. B) suggesting that these SA/TBF1 responsive genes are regulated by NPR1 during ER stress. We next tested the induction of genes that are independent of the SA/TBF1 modulation (i.e., sbZIP60, BiP1, PDIL1-2 and CRT1 (Pajerowska-Mukhtar et al., 2012)). We found that these genes were significantly up-regulated in the npr1 mutant compared to Col-0 (Figure 3. 8. A). Together these results suggest that under ER stress NPR1 regulates UPR genes that are only partially overlapping with those modulated by the SA/TBF1 signaling. To further examine TBF1 dependency on NPR1-regulated UPR activation, we determined the transcript levels of TBF1 in Col-0 during ER stress and observed no significant induction (Figure 3. 10. A), indicating that TBF1 does not respond to ER stress at a transcriptional level. Additionally, the transcript levels of several UPR markers were analyzed in a time course of Tm-treatment in the tbf1 null mutant (Pajerowska-Mukhtar et al., 2012) for 102 comparison to Col-0 and bzip28/60. The expected induction of the UPR genes exhibited no significant change in tbf1 compared to Col-0 at 24 and 36 hours of Tm treatment (Figure 3. 10. B). However, the tbf1 mutant displayed significant induction of UPR genes compared to Col-0 at 12 hours of Tm treatment. These results combined with the evidence that the UPR markers were significantly induced in npr1 compared to Col-0 upon ER stress (Figure 3. 2.) support that NPR1 suppresses late-adaptive UPR signaling (i.e., 24 hours) independently of TBF1 regulation. Taken together, the results indicate that the involvement of NPR1 in attenuation of UPR activation is independent of elevated endogenous SA and may function largely in parallel to TBF1 regulation. ER Stress Causes Reduction of the Redox Potential of the Cytosol ER stress is known to lead to alteration of the redox potential of the ER lumen and cytosol. Specifically, the UPR activation leads to hyper-oxidation of the ER lumen due to enhanced activity of the ER lumenal oxidoreductase ERO1 (Cabibbo et al., 2000; Sevier et al., 2007). Conversely, as demonstrated in Pichia pastoris (Delic et al., 2012), induction of the UPR can also cause reduction of the cytosolic redox potential. Analogously to the effect of ER stress on the cellular redox potential, in plant cells SA accumulation following pathogen attack alters cytosol redox potential resulting in conformational change of NPR1, from an oligomeric form to a monomeric form; such a conformational change is critical for NPR1 activation, nuclear translocation, and function in transcriptional reprogramming (Mou et al., 2003). Because in conditions of ER stress we verified a negative role of NPR1 in resistance to chronic ER stress (Figure 3. 1.), UPR homeostatic gene regulation (Figure 3. 2.), and established a SA-independent activation of the UPR (Figures 3. 2., 3. 9.), we hypothesized that ER stress could cause redox 103 changes in the cytosol of the plant cell. To test this, we first monitored the cytosolic redox status in vivo by adopting the reduction-oxidation sensitive green fluorescent protein (roGFP2), a genetically-encoded redox sensor (Dooley et al., 2004; Hanson et al., 2004). The chromophore orientation of roGFP2 changes in response to the environmental redox status leading to alternation in fluorescence excitability between two maximum peaks: 405 and 488 nm. Upon oxidation, excitation at 405 nm rises and excitation at 488 nm diminishes; upon reduction, the opposite effect occurs. Hence, the ratio of excitation from two wavelengths serves as an indicator of the GFP redox state (Dooley et al., 2004; Hanson et al., 2004). To test whether ER stress could change redox state of the cytosol, we generated Arabidopsis transgenic plants expressing an established roGFP2 fusion to the membrane domain of the ER-Golgi SNARE, SEC22 (roGFP2-SEC22 (Brach et al., 2009; Chatre, Brandizzi, Hocquellet, Hawes, & Moreau, 2005); herein dubbed roGFP2). The orientation of the protein fusion is such that the fluorescent protein is exposed into the cytosol (Brach et al., 2009) and can therefore report on the redox potential of the cytosol. As a positive control, we treated the transgenic plants with SA, which is known to induce reduction of the cytosol (Mou et al., 2003). Confocal laser scanning microscopy (CLSM) analyses indicated that compared to untreated plants (DMSO), roGFP2 was reduced in the cytosol of SA-treated plants, as reflected by a significant decrease in the 405/488 nm fluorescence ratio (Figures 3. 3. A, B, D). Importantly, Tm-treatment of the roGFP2-SEC22 transgenics also led to a significant reduction of the cytosolic redox potential compared to mock- treated samples (Figures 3. 3. C, D). The 405/488 ratio levels were not statistically different in cells treated with SA or Tm (Figure 3. 3. D). To rule out the possibility that potential side effects of Tm treatment rather than the ER stress could lead to a cytosolic reduction, we adopted the L- proline analog, azetidine-2-carboxylic acid (AZC), which causes ER stress by incorporating in 104 competition with D-proline into newly synthesized proteins, causing protein misfolding (J. H. Kim et al., 2017). We used two concentrations of AZC, and found that the AZC treatment also resulted in a cytosolic reduction of roGFP2 compared to untreated conditions in a dose dependent manner (Figure 3. 11. A). To further support our findings, we introduced the misfolded vacuolar carboxypeptidase from Arabidopsis fused to the monomeric red fluorescent protein (At.CPY*-RFP). CPY* is misfolded and retained in the ER, eliciting ER stress in transient expression in Arabidopsis protoplasts (Yamamoto, Kawanabe, Hayashi, Endo, & Nishikawa, 2010; Yang, Srivastava, Howell, & Bassham, 2016). As a control for the experimental set up, we used SEC-RFP (Renna et al., 2013), a secreted monomeric RFP protein. The folding of RFP does not require catalysts and, unlike wild-type CPY, is biologically inactive (Alerting, 2007; Campbell et al., 2002; Renna et al., 2013; Yamamoto et al., 2010). We expressed roGFP2 alone or in combination with either At.CPY*-RFP or SEC-RFP into tobacco leaf epidermal cells, and determined the redox potential in the cytosol as performed above. We found that compared to the roGFP2 basal level and roGFP2+SEC-RFP level, the overexpression of At.CPY*-RFP exhibited a cytosolic reduction with a significantly diminished 405/488 ratio (Figure 3. 11. B). Notably, such cytosolic redox status corresponded to an intrinsic level of UPR activation, i.e. the expression of CPY*-RFP caused significant induction of UPR genes such as Nt.BiP, Nt.PDI and Nt.CRT compared to cells expressing either roGFP2 alone or in combination with SEC-RFP (Figure 3. 11. C). Taken together, these results indicate that a reduction of roGFP2 occurs in the cytosol in conditions of ER stress and support that ER stress induces reduction of the cytosolic redox potential analogously to SA accumulation, a condition known to induce nuclear translocation of NPR1 (Mou et al., 2003). 105 ER Stress Induces Translocation of NPR1 from the Cytosol to the Nucleus Based on the evidence that NPR1 is involved in UPR regulation in chronic and adaptive UPR (Figures. 3. 1., 3. 2.), and that ER stress leads to a reduction of cytosolic redox state (Figure 3. 3.), which is required for monomerization of NPR1 and translocation to the nucleus where NPR1 controls gene expression (Kinkema et al., 2000), we hypothesized that NPR1 could be translocated to the nucleus during ER stress. To test this hypothesis, we examined the subcellular localization of NPR1 during ER stress. We expressed a yellow fluorescent protein (YFP) fusion to NPR1 (NPR1-YFP) under the control of the NPR1 promoter in npr1 (npr1; pNPR1::NPR1- YFP). A similar fusion is known to be functional as a NPR1 overexpression line tagged with GFP was shown to complement the loss of pathogen resistance phenotype of npr1 (Kinkema et al., 2000). Consistently, we found that NPR1-YFP expression complements the ER stress resistance of npr1 (npr1 Y1; Figure 3. 6. A). Two independent transgenic lines, herein dubbed npr1 Y1 and npr1 Y2, were selected for our analyses (Huot et al., 2017). Compared to Col-0, these lines showed a slight increase in NPR1 transcript levels, with npr1 Y2 having more elevated levels of NPR1 transcripts compared to npr1 Y1 (Figure 3. 4. C). Consistent with earlier findings (Mou et al., 2003), the fluorescence of NPR1-YFP was undetectable in physiological conditions of growth; however, treatment with SA led to visible accumulation of NPR1 in the nucleus (Figures 3. 4. A, B). We next analyzed NPR1-YFP in npr1 Y1 and npr1 Y2 upon treatment with Tm. We found that, although to lower levels compared to the SA treatment, Tm treatment led to accumulation of NPR1-YFP in the nucleus (Figures. 3. 4. A, B). The nucleus localization of NPR1-YFP was further assayed with Hoechst33342, a well-established fluorescent nuclear marker and primary cell wall stain (Hernandez & Palmer, 1988; Tamura, Fukao, Iwamoto, Haraguchi, & Hara-Nishimura, 2010), which confirmed localization of NPR1- 106 YFP in nuclei under both SA and Tm treatments (Figure 3. 12. A). This was also reflected in Western blot analyses of nuclear extracts using anti-GFP serum (Kinkema et al., 2000) (Figure 3. 4. D). As expected from earlier results showing induction of NPR1 production by SA (Kinkema et al., 2000), NPR1-YFP abundance was induced in the cytosol under SA treatment; however, it was not detectable under both DMSO and Tm conditions (Figure 3. 4. E). The evidence that Tm does not affect NPR1 expression and protein level compared to untreated control (Figures 3. 4. C, 3. 12. B) argues that the nuclear accumulation of NPR1-YFP into the nuclei of Tm-challenged plants is due to concentrative accumulation of an existing pool of NPR1-YFP in the nuclei rather than de novo synthesis and nuclear accumulation of the protein. More importantly, the results also indicate that ER stress causes NPR1 nuclear translocation. NPR1 Physically and Genetically Interacts with bZIP28 and bZIP60 Because NPR1 is a co-factor that binds transcriptional regulators (Kinkema et al., 2000; Mou et al., 2003), and because of its newly demonstrated requirement for UPR gene modulation (Figure 3. 2.), we hypothesized that NPR1 could bind the UPR TFs, bZIP28 and bZIP60. To test this hypothesis, we aimed to assay protein-protein interactions between NPR1 and bZIP28 or bZIP60. We first used an in vivo approach based on a fluorescence resonance energy transfer (FRET) assay, which is commonly used to assay protein proximity through the measurement of the energy that is transferred non-radiatively from an excited fluorophore (the donor) to another (the acceptor) (Sieben, Mikosch, Brandizzi, & Homann, 2008). In this approach, photo- bleaching of the donor leads to increased fluorescence intensity of the acceptor. For our assay, we expressed YFP-NPR1 and CFP fusions to the transcriptionally-active form of bZIP28 (bZIP28ΔTMD) or bZIP60 (sbZIP60), as donor/acceptor pairs in tobacco leaf epidermal cells, 107 and photobleached the acceptor (YFP-NPR1) in the nuclei. We then measured donor (CFP) fluorescence after the YFP photobleaching in the nuclei and calculated EFRET as percentage of CFP intensity levels compared to the prebleaching levels of CFP. As a negative controls, in place of YFP-NPR1 we used cytosolic YFP, which diffuses into the nuclei (daSilva et al., 2004), and bZIP1, a TF not related to the UPR (Gene, Price, Lin, Chan, & Jang, 2010). FRET analyses indicated both bZIP28 and bZIP60 individually interact with NPR1, as supported by significantly higher FRET efficiency values compared to the respective negative controls in combination with NPR1 (Figure 3. 5. A). To confirm these findings, we pursued an independent line of evidence by assaying the interaction of NPR1 with bZIP28 or bZIP60 through yeast two- hybrid analyses (Y2-H). For these assays, we fused the LexA Binding Domain (LexA-BD), and the LexA Activation Domain (LexA-AD) to NPR1 and transcriptionally-active bZIP28 (bZIP28ΔTMD) or bZIP60 (sbZIP60) or bZIP1, respectively. The AD/BD constructs where then transformed into yeast, which was grown on selective medium containing X-Gal. In the case of interaction, the galactosidase reporter gene would be expressed and the yeast cells would turn blue. We found that NPR1/bZIP28 and NPR1/bZIP60 combinations activated the LacZ reporter indicating physical interaction between NPR1 and bZIP28 and bZIP60 (Figure 3. 5. B). Such an interaction was not verified when NPR1-BD was co-expressed with LexA-AD alone, bZIP28ΔTMD or sbZIP60 were co-expressed with LexA-BD or when NPR1-BD was co- expressed with bZIP1-AD. Therefore, these results support that NPR1 forms a complex with transcriptionally-active forms of bZIP28 and bZIP60 in the nucleus. Next, to validate these findings at a genetic level, we isolated and characterized an npr1/bzip28/60 triple mutant in physiological conditions of growth and chronic ER stress. We found that the triple mutant displayed similar primary root elongation to bzip28/60 while are significantly reduced compared 108 to Col-0 and npr1 under ER stress (Figure 3. 13.). These genetic interaction results support our interaction data of NPR1 with bZIP28 and bZIP60. The observed phenotypic responses that the triple mutant phenocopies the bzip28/60 mutant in ER stress conditions also argue that NPR1 operates in the bZIP28 and bZIP60 signaling pathways rather than in additional signaling routes in the UPR. NPR1 Functions as a Negative Regulator of the UPR under ER stress The evidence that NPR1 interacts with the two critical UPR TFs, bZIP28 and bZIP60 (Figure 3. 5.), and that the loss of NPR1 causes an increase in the transcript levels of UPR genes that are controlled by these TFs (Figure 3. 2.) prompted the hypothesis that NPR1 could function as a functional repressor of these UPR TFs. To test this hypothesis, we aimed to assay the ER stress responses in conditions of NPR1 overexpression. We expected that overexpression of NPR1 would lead to opposite phenotype to its loss-of-function mutation and therefore to an increased susceptibility to chronic ER stress and reduction of UPR gene activation in adaptive UPR. As NPR1 over-expressing backgrounds, we adopted npr1 Y2, which showed significantly enhanced levels of NPR1 transcripts compared to Col-0 and npr1 Y1 (Figure 3. 4. C) as well as an independent line expressing NPR1-GFP under the control of the strong constitutive 35S CaMV promoter (oxnpr1) (Kinkema et al., 2000) (Figure 3. 14.). Consistent with our hypothesis and expectations, we found that the growth of NPR1-overexpressors on Tm was affected to a larger extent compared to Col-0 in conditions of chronic ER stress (Figures 3. 6. A, B). Furthermore compared to Col-0, the activation of the UPR was reduced in the NPR1 overexpressors, as supported by the evidence that the transcriptional levels of UPR marker genes were significantly lower in NPR1 overexpressors compared to Col-0 and npr1 Y1 in conditions 109 of ER stress (Figure 3. 6. C). These results indicate that NPR1 functions as a repressor of the UPR. Based on the demonstrated interaction of NPR1 with bZIP28 and bZIP60, we propose that NPR1 suppresses the transcriptional activity of these TFs in ER stress responses. 110 DISCUSSION In response to stress, common transducers can be adopted in diverse signaling pathways whereby shared transduction components may exert positive or negative modulating roles in alternative pathways. For example, the ER-localized UPR TFs, bZIP17 and bZIP60, are involved in ER stress-induced UPR signaling pathway (Henriquez-Valencia et al., 2015; Iwata et al., 2008). On the other hand, both bZIP17 and bZIP60 also participate in specific cellular response to salt stress (Henriquez-Valencia et al., 2015). Therefore, bZIP17 and bZIP60 are shared by diverse pathways and possibly facilitate cross-signaling between salt stress and ER stress responses. Identification of such shared signaling factors is one of the main challenges in understanding the dynamic nature of and the complexity in stress biology. In our study, we demonstrated a novel role of NPR1, a well-known master regulator of SA-mediated plant defense signaling, in the UPR. Specifically, we established the role of NPR1 as a negative regulator of the plant UPR (Figures 3. 1, 3. 2., 3. 6.). Indeed, we showed that the loss of function of NPR1 led to a compromised actuation of efficient adaptive and chronic UPR modulated through two main UPR arms, ire1/bZIP60 and bZIP28, as demonstrated by an enhanced induction of UPR marker genes and increased tolerance to prolonged ER stress respectively (Figures 3. 1., 3. 2.). Consistently with these observations, a gain of function of NPR1 due to overexpression led to a reduction in plants’ adaptation to chronic ER stress and in the induction UPR gene expression in adaptive UPR (Figure 3. 6.). It has been demonstrated that the UPR can be triggered by exogenous SA applied in high concentrations to the seedlings (0.5 mM), whereby the two UPR signaling arms are activated and several UPR genes are induced (Nagashima et al., 2014). These findings led to the suggestion of the existence of mechanisms underlying UPR activation by SA (Nagashima et al., 111 2014). However, the findings that an SA induction-deficient mutant (sid2) exhibits ER stress responses that are indistinguishable from wild type (Figure 3. 1.) and that ER stress does not induce SA-accumulation or the activation of SA-dependent signaling pathways (Figure 3. 9.) implies that endogenous elevated SA does not participate in the Tm-mediated plant UPR activation. These data also indicate that SA does not play a role in potentiating UPR signal transduction under Tm-mediated ER stress conditions. TBF1 has been identified as a transcription factor triggering the NPR1-dependent UPR genes to promote PR1 secretion during plant defense signaling (Pajerowska-Mukhtar et al., 2012). In addition, the compromised UPR phenotype of a tbf1 mutant was further confirmed by enhanced Tm resistance in transgenic plants overexpressing TBF1 gene (Hossain, Henríquez- valencia, & Gómez-páez, 2016; Pajerowska-Mukhtar et al., 2012). In other reports, however, the tbf1 mutant displayed similar sensitivity to Tm as well as induction profiles of UPR marker genes to Col-0 (Nagashima et al., 2014). Therefore, we tested the possibility of TBF1 participation to the UPR modulation in conditions of ER stress. In our experimental conditions, we found that tbf1 mutant displayed unaltered UPR activation compared to Col-0 in response to adaptive ER stress at 24 and 36 hours post Tm treatment, suggesting that TBF1 does not regulate the UPR in late stages of adaptive UPR. Nevertheless, the verified induction of UPR genes in tbf1 compared to Col-0 at 12 hours of Tm treatment (Figure 3. 10. B) indicates a time-dependent involvement of TBF1 and a role of this TF in early adaptive UPR. The sensitivity of NPR1 to the cellular redox status plays a pivotal role in plant defense responses as SA-induced reduction of the cytosolic redox potential causes NPR1 monomerization followed by translocation into the nucleus where NPR1 exerts its function as a transcription cofactor (Kinkema et al., 2000; Mou et al., 2003). Our study reveals that a similar 112 scenario occurs under ER stress conditions, as NPR1 translocates into the nucleus in response to ER stress-induced reduction of the cytosolic redox potential (Figures 3. 3., 3. 4., 3. 12. A). These results provide new significant insights into plant cell responses to stress. First, our findings show that a reduction of the cytosolic redox potential occurs in plant cells after Tm or AZC treatment or in the ER stress mimic condition triggered by expression of At.CPY*, which indicates that ER stress causes alterations not only in the ER but also in the homeostasis of the plant cell milieu. Second, our results also indicate that NPR1 has multiple cellular roles, as it is not only involved in SA-mediated plant defense but also in ER stress responses independently from SA. We speculate that a convergence of signaling pathways onto common regulating factors may be beneficial to plant cells as it could facilitate economization of resource allocation for the production, and eventual disposal, of signaling factors that are in common to multiple signaling pathways. A nuclear localization of NPR1 triggered by SA facilitates NPR1 function in transcriptional reprogramming for the expression of SA-responsive genes through interaction with basic leucine zipper TFs (e.g., TGAs) (Fan & Dong, 2002; Gatz, 2013; Rochon, Boyle, Wignes, Fobert, & Després, 2006). In the plant-pathogen defense signaling pathway, NPR1 either represses or activates the transcription of the secreted pathogen-related protein 1 (PR1) through the formation of either repressor or activator complexes with different groups of protein factors (Gatz, 2013; Weigel, Pfitzner, & Gatz, 2005), supporting dual roles of NPR1 in transcriptional regulation. Additionally, the SA- induced clade I TGA transcription factors, TGA1 and TGA4, which interact with NPR1, have been involved in the ER stress-related secretion pathways during defense response, suggesting a potential role of NPR1 in the UPR through interaction with transcription factors (Lipu Wang & Fobert, 2013). Our studies provide 113 evidence that NPR1 plays a negative role in attenuating the UPR amplitude in conditions of ER stress (Figure 3. 2.) and that it interacts with bZIP28 and bZIP60 (Figure 3. 5.), which are similar to TGA TFs in possessing a basic leucine zipper motif. Based on our results that NPR1 represses the UPR, as supported by a verified enhancement of ER stress responses in a npr1 loss-of-function mutant, and a demonstrated reduction of efficient UPR in conditions of NPR1 overexpression, combined with the evidence that NPR1 interacts with critical plant UPR TFs, we propose that NPR1 causes transcriptional deactivation of bZIP28 and bZIP60 in the nucleus where it relocates upon ER stress-induced reduction of cytosolic redox potential. We propose two potential mechanisms to explain how NPR1 participates in the control of the UPR (Figure 3. 6. D): NPR1 may suppress the UPR TFs through either formation of a repressive complex or by hindrance of transcriptional activation. Specifically, in conditions of ER stress the existing cytosolic fraction of NPR1 is activated by reduction of the cytosol redox potential and is translocated into the nucleus; in the nucleus, NPR1 interacts with bZIP28 and bZIP60, which are known to heterodimerize (Liu & Howell, 2010a), leading to transcriptional suppression of UPR-responsive genes either through formation of a NPR1/bZIP28/bZIP60 repressive complex or by blocking the specific binding of bZIP28 and bZIP60 onto the promoter regions of UPR genes. bZIP28 has been shown to interact with CCAAT box binding factors, which are heterotrimeric factors composed of NF-Y subunits (Liu & Howell, 2010a). Although the biological relevance of such an interaction is yet unknown, future analyses may establish whether the NF-Y factors may be involved in the NPR1 role in the UPR. The established mechanism in plant defense responses in which NPR1 forms a ternary repressive complex with TGA2, NIMIN1 (NPR1/NIM1-interacting protein 1) on the promoter of PR1 leading to transcriptional repression in the absence of SA (Després, Delong, Glaze, Liu, 114 & Fobert, 2000; Fan & Dong, 2002; Gatz, 2013; Rochon et al., 2006; Weigel et al., 2005) lends support to the possibility of a formation of NPR1/bZIP28/bZIP60 repressive complex in ER stress responses. A second plausible explanation for the dual role of NPR1 in response to different types of stress is that a specific yet-unidentified factor may exist either under SA or Tm condition and collaborates with NPR1 to switch its property as a positive or negative regulator of gene expression. Similar to JAsmonate-Zim domain (JAZ) proteins that repress the transcription of MYC2 to fine-tune JA signaling responding to fluctuate environmental stress (Campos, Kang, & Howe, 2014), the biological significance of such suppression by NPR1 may contribute to a negative feedback loop involved in the balance of energy consumption and serve to maintain basal cellular homeostasis during ER stress signaling. Independently from the specific mechanisms of action, the establishment of NPR1 as a novel regulator of the UPR signaling pathway provided in this work offers new insights into the cellular mechanisms that enable plants to cope with ER stress and identifies a potential cross-regulation between stress responses mediated by NPR1 as a common signaling effector. 115 ACKNOWLEDGEMENTS We thank the following colleagues: Dr. B. Huot for npr1 complemented line seeds; Dr. J. Glazebrook for sid2-2 seeds; Dr. X. Dong for the seeds of oxnpr1 and NPR1 antibody; Dr. C. Wilkerson for critical comments on the manuscript; Dr. G. Stefano for kindly assisting with FRET experiments; Mr. S. Vaitkevicius for helping with genotyping of tbf1; Ms. E. M. Gibbons for helping with the nuclear fractionation; Dr. L. Chen in the Mass spectrometry and Metabolomics Core Facility at Michigan State University for LC-MS analysis. This work was supported by primarily by the National Institutes of Health (GM101038) with contributing support from 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 AgBioResearch. 116 APPENDIX 117 Table 3. 1. Primers used in this study Primer name Sequence Purpose UBQ10 For 5'-GGCCTTGTATAATCCCTGATGAATAAG-3' qRT-PCR UBQ10 Rev 5'-AAAGAGATAACAGGAACGGAAACATAGT-3' qRT-PCR bZIP60us_FWD 5'-GGAGACGATGATGCTGTGGCT-3' qRT-PCR bZIP60u_REV 5'-CAGGGATTCCAACAAGAGCACAG-3' qRT-PCR bZIP60s_REV 5'-CAGGGAACCCAACAGCAGACT-3' qRT-PCR BiP1 For1 5'-TCAGTCCTGAGGAGATTAGTGCT-3' qRT-PCR BiP1 Rev1 5'-TGCCTTTGAGCATCATTGAA-3' qRT-PCR PDIL1-2 For 5'-AAGTGGTCCTGCTTCTGTTGAA-3' qRT-PCR PDIL1-2 Rev 5'-TTGAACAGCCTCACTGCAGGT-3' CNX1 For 5'-CGCTGGATCGTTTCGAAGAA-3' qRT-PCR qRT-PCR CNX1 Rev 5'-CACACTCAAGTCCTTCCTGGAA-3' qRT-PCR AtNPR1 qF 5'-TGCATCAGAAGCAACTTTGG-3' AtNPR1 qR 5'-GGCCTTTGAGAGAATGCTTG-3' qRT-PCR qRT-PCR CBP60g For 5'-AAGAAGAATTGTCCGAGAGGAG-3' qRT-PCR CBP60g Rev 5'-GGCGAGTTTATGAAGCACAG-3' SID2 For 5'-TCCGTGACCTTGATCCTTTC-3' SID2 Rev 5'-ACAGCGATCTTGCCATTAGG-3' qRT-PCR qRT-PCR qRT-PCR PR1 For 5'-CATACACTCTGGTGGGCCTTAC-3' qRT-PCR PR1 Rev 5'-CGAGTCTCACTGACTTTCTCCA-3' TBF1-qPCR-F 5'-GTTGGTTCGCCTTCTG-3' TBF1-qPCR-R 5'-CCACACCCCAAACAAT-3' TGA3-qPCR-F 5'-TCTTGATGTCGGGAATGTGG-3' qRT-PCR qRT-PCR qRT-PCR qRT-PCR 118 Table 3. 1. (cont’d) TGA3-qPCR-R 5'-AGTTGCTGATCGGTTAAGGG-3' PAD4-qPCR-F 5'-AAGATCCATGACATCGCCG-3' PAD4-qPCR-R 5'-AGGTAGAGGTTCATCGGAGG-3' CRT3-qPCR-F 5'-ATGACCCCAACGATGT-3' CRT3-qPCR-R 5'-CCTTGTAGTTCGGGTTCT-3' qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR HSP70-qPCR-F 5'-AATGGCTGGTAAAGGAGAAGG-3' qRT-PCR HSP70-qPCR-R 5'-CTATCAGTGAAGGCGACGTAAG-3' qRT-PCR BiP2 qRT-F 5'-AGTGGACGCCAACGGTAT-3' BiP2 qRT-R 5'-AAACTCCTCTGCCTCCTTCA-3' PDIL-qPCR-F 5'-GCCATTTTCAAACCCTTTGGT-3' qRT-PCR qRT-PCR qRT-PCR PDIL-qPCR-R 5'-CTTGGAACTATCACCCTCGATC-3' qRT-PCR BIP3 For 5'-CGAAACGTCTGATTGGAAGAA-3' BIP3 Rev 5'-GGCTTCCCATCTTTGTTCAC-3' CRT1-qPCR-F 5'-CTGTGGTGGTGGCTAC-3' CRT1-qPCR-R 5'-GTCTCACATGGGACCT-3' qRT-PCR qRT-PCR qRT-PCR qRT-PCR NtBIP For 5'-AGCTTTGAGCAGTCAACACCAAGT-3' qRT-PCR NtBIP Rev 5'-AAAACGTGCCCGAGTAAGTGGTTC-3' qRT-PCR NtPDI For 5'-TCCAAAGGGATCACTGGAGCCAAA-3' qRT-PCR NtPDI Rev 5'-TCTGGAGATAGCACCACAACGCTT-3' qRT-PCR NtCRT For 5'-TCTGGAGATAGCACCACAACGCTT-3' qRT-PCR NtCRT Rev 5'-TCTGGCTTCTTGGCATCAGGATCA-3' qRT-PCR NPR1-EcoRI_F 5'-agaattcATGGACACCACCATTGATGGA-3' CDS amplification for construct in complemented line (Restriction sites are underlined) 119 Table 3. 1. (cont’d) NPR1-XhoI_R 5'-agtcgacCCGACGACGATGAGAGARTTTAC-3' CDS amplification for construct in complemented line (Restriction sites are underlined) pNPR1_F 5'-cgcggccgcTCGTTTGTTTTCCGTTTTGTTCTGA-3' Promoter amplification for construct in complemented line (Restriction sites are underlined) Promoter amplification for construct in complemented line (Restriction sites are underlined) CDS amplification for FRET construct and RT- PCR CDS amplification for FRET construct and RT- PCR CDS amplification for FRET construct CDS amplification for FRET construct CDS amplification for FRET construct CDS amplification for FRET and yeast two hybrid construct CDS amplification for yeast two hybrid construct CDS amplification for yeast two hybrid construct CDS amplification for yeast two hybrid construct CDS amplification for FRET and yeast two hybrid construct CDS amplification for FRET and yeast two hybrid construct pNPR1_R 5'-agaattcCAACAGGTTCCGATGAATTGAAAT-3' NPR1 F 5'-ATGGACACCACCATTGATGGAT-3' NPR1 R 5'-TCACCGACGACGATGAGAGAG-3' bZIP28 F 5'-ATGACGGAATCAACATCCGTGGTTG-3' bZIP28 N R 5'-TCACTTCTTGAGCTTACTTTTACCCTCA-3' bZIP60 s F1 5'-ATGGCGGAGGAATTTGGAAG-3' bZIP60 s R2 5'-TCACGCCGCAAGGGTTAAGAT-3' Y2H bZIP28 F 5'-gaCGCATCATCCTCCCCTGAATCA-3' Y2H bZIP28 R 5'-TCACTTCTTGAGCTTACTTTTACCC-3' bZIP60 s F4 5'-CCAACTAGCGATTCTGGCTC-3' bZIP1 F 5'-ATGGCAAACGCAGAGAAGAC-3' bZIP1 R 5'-TCATGTCTTAAAGGACGCCATTG-3' 120 Table 3. 1. (cont’d) CPY F1 5'-CACCATGGAGAAACTAACTTTTCTCAG-3' CDS amplification for mutated construct in transient expression CPY R1 5'-CCCAGCATATGACTCTCGAGTAATATAAAAG-3' CDS amplification for CPY F2 5'-GAGTCATATGCTGGGCACTATATCCC-3' CPY R2 5'-ACATCTGAGCAACCAGTTCCTCTC-3' mutated construct in tobacco transient expression CDS amplification for mutated construct in tobacco transient expression CDS amplification for mutated construct in tobacco transient expression 121 Figure 3. 1. The loss of NPR1 confers resistance to prolonged ER stress. (A) To determine the ER stress sensitivity of wild-type Col-0, ire1, bzip28/60, sid2 and npr1, the seeds of the respective genotypes were plated on solid medium containing DMSO (0 nM Tm) or Tm (25 nM and 50 nM). Images show the appearance of 14-day-old seedlings grown on mock (0 nM Tm) or Tm-containing plates. (B) Measurements of the primary root length of wild-type Col-0, ire1, bzip28/60, sid2 and npr1. n>30 seedlings were measured for each genotype. Errors bars represent SEM. Data significantly different from the corresponding control (Col-0) are indicated (mutant versus Col-0 under treatment, **P < 0.01, ****P<0.0001, NS, non significant; Unpaired t-test). (C) Inhibition of primary root growth of wild-type Col-0, ire1, bzip28/60, sid2 and npr1 on Tm plates (50 nM). Root length was assayed for the relative reduction of root growth on mock versus Tm treatment in the Tm sensitivity experiment (B). Errors bars represent SEM (mutant versus Col-0, ****P<0.0001, NS, non significant; Unpaired t-test). 122 Figure 3. 2. NPR1 negatively modulates UPR activation under ER stress. Quantitative RT-PCR analyses of various UPR indicators in wild-type Col-0, bzip28/60, sid2 and npr1. cDNA was synthesized from 14-day-old seedlings treated with Tm (0.5 M) for 1 day. Values are presented relative to indicated DMSO control, which was set to 1. Transcription of UBQ10 was used as internal control. The bzip60-1 allele used to generate bzip28/60 is a knockdown mutant with a partial bZIP60 transcript (L. Zhang, Chen, Brandizzi, Verchot, & Wang, 2015), which is detectable in qRT-PCR analyses. Error bars represent SEM among three biological replicates. Data significantly different from the corresponding control are indicated (mutant versus the Col-0 under treatment, **P < 0.01, ***P<0.001, ****P<0.0001, NS, non significant; Unpaired t-test). 123 Figure 3. 3. Reduction of roGFP2 in the cytosol during ER stress. (A-C) Confocal images of A. thaliana leaf epidermal cells expressing roGFP2-SEC22. Samples were collected from 14-day-old seedlings treated with DMSO as mock control (A), 0.5 m SA (B) and 0.5 M Tm (C) for 1 day. Stacks of images were collected by CLSM with excitation at 405 and 488nm wavelengths. Maximum projections of these stacks were used to calculate the ratio images (Ratio 405/488). Golgi are indicated by arrows. The color scale of 405/488 ratio represents the levels of oxidation of roGFP2. Scale bars = 5 m. (D) The ratio values were calculated from CLSM images during corresponding treatments. ***P<0.001, ****P<0.0001, NS, non significant; Unpaired t-test. 10 images were analyzed for each combination. Error bars represent SEM. 124 Figure 3. 4. ER stress promotes NPR1 nuclear translocation. (A) 14-day-old seedlings of indicated A. thaliana genetic backgrounds were treated for 1 day on solid medium with DMSO (mock), 0.5 mM SA or 0.5 M Tm. Nuclei are indicated by arrows. Scale bars = 100 m. Scale bar of insets = 10 m. (B) Fluorescence intensity values of nuclei of npr1 Y1 and npr1 Y2 were measured by the Nikon NIS-Element AR 4.30 software. At least 15 nuclei were analyzed for each treatment. Error bars represent SEM. (C) Quantitative RT-PCR analyses of NPR1 mRNA levels in wild-type Col-0, npr1, npr1 Y1 and npr1 Y2. cDNA was synthesized from 14-day-old seedlings treated with Tm (0.5 M) for 1 day. Transcription of UBQ10 was used as internal control. The primers allow amplification of a partial transcript as they were designed to anneal before the T-DNA insertion site indicating the presence of truncated form of NPR1 transcript. Error bars represent SEM among three biological replicates. Data significantly different from the corresponding controls are indicated. **P < 0.01; Unpaired t-test. (D-E) Protein levels of NPR1-YFP in the nuclear fractions (D) and cytosolic fractions (E) isolated from the indicated genetic backgrounds. Proteins extracted from seedlings treated as described in (A) were subjected to Western blot analysis with anti-GFP antibodies. The UDP- glucose pyrophosphorylase (UGPase) and Histone3 (HIS3) served as cytosolic and nuclear 125 controls, respectively, and were detected with specific antibodies. The experiment was repeated three times with similar results. 126 Figure 3. 5. NPR1 interacts with bZIP28 and bZIP60. (A) Interaction of NPR1 with either bZIP28 or bZIP60 was assayed by FRET, and the FRET efficiency (EFRET) was measured by acceptor photobleaching. Fluorescence intensity of CFP- bZIP28/bZIP60, cytosolic YFP (cYFP) and YFP-NPR1 was recorded. EFRET was calculated as relative increase of CFP-bZIP28/bZIP60 fluorescence intensity (%) in the nucleus after photobleaching of either YFP-NPR1 or cYFP negative control FRET acceptor. The experiment with either bZIP1 or cYFP with NPR1 served as a negative control. At least 10 nuclei were analyzed for each combination. Error bars represent SEM. (B) The FRET results were confirmed by Y2H analyses. The N-terminal fragments of bZIP28 and bZIP60, full-length of bZIP1 and full-length of NPR1 were tested as bait and prey by fusing with DNA binding domain (pGilda) and activation domain (pB42AD) in lexA the Y2-H system. Development of blue colonies indicates a positive interaction. The images were taken 2 days after interaction determined through activation of -galactosidase gene. The positive control (PC) and negative control (NC) were determined using pLexA-T and pB42AD-p53 and empty vectors individually. 127 Figure 3. 6. NPR1 antagonizes the UPR. (A) To determine the Tm sensitivity of wild-type Col-0, bzip28/60, npr1 complemented lines (npr1 Y1 and npr1 Y2) and an npr1 overexpression line (oxnpr1). The seeds of the corresponding genotypes were plated on solid medium containing DMSO (0 nM Tm) or Tm (25 nM and 50 nM). Images show the appearance of 14-day-old seedlings grown on mock or Tm plates. (B) Measurements of the primary root length of wild-type Col-0, bzip28/60, npr1 Y1, npr1 Y2 and oxnpr1. n>30 seedlings were measured for each genotype. Errors bars represent SEM. (mutant versus Col-0 under treatment, *P<0.05, **P < 0.01, ****P<0.0001, NS, non significant; Unpaired t-test). (C) Quantitative RT-PCR analyses of various UPR indicators in wild-type Col-0, bzip28/60, npr1 Y1, npr1 Y2 and oxnpr1. cDNA was synthesized by using the 14-day-old seedlings grown on Tm (50 nM). Values are presented relative to indicated DMSO control which was set to 1. Transcription of UBQ10 was used as internal control. Error bars represent SEM among three biological replicates. Data significantly different from the corresponding controls are indicated (mutant versus Col-0 under treatment, *P<0.05**P < 0.01 , ***P<0.001; Unpaired t-test). (D) Schematic model illustrating the molecular mechanisms by which NPR1 may attenuate the induction of selective UPR genes upon ER stress. A conformational change of NPR1 responding to ER stress-triggered redox alternation leads to nuclear translocation followed by interaction with bZIP28 and bZIP60. Such interactions may repress UPR induction by interfering with the 128 availability of bZIP28 and bZIP60 to bind DNA or through the formation of a transcriptional repressive complex. TBF1 may be involved in the early stage of UPR induction. 129 Figure 3. 7. The loss of NPR1 does not affect the aerial growth under ER stress. (A) Measurements of the aerial weight of wild-type Col-0, ire1, bzip28/60, sid2 and npr1. n>30 seedlings were measured for each genotype. Errors bars represent SEM. Data significantly different from the corresponding control (Col-0) are indicated (mutant versus the Col-0 under treatment, **P < 0.01, ****P<0.0001, NS, non significant; Unpaired t-test). (B) Inhibition of aerial growth of wild-type Col-0, ire1, bzip28/60, sid2 and npr1 on Tm plates (50 nM). Root length was assayed for the relative reduction of root growth on mock versus Tm treatment in the Tm sensitivity experiment (A). Errors bars represent SEM (mutant versus the Col-0, ****P<0.0001, NS, non significant; Unpaired t-test). 130 Figure 3. 8. NPR1 represses the expression of ER resident genes that are shared partially with the SA/TBF1 signaling. (A and B) 14-day-old seedlings were transferred to solid medium containing Tm (0.5 µM) for the indicated times. Samples were analyzed by qRT-PCR for transcriptional levels of UPR genes that are independent of SA/TBF1 signaling (A) and UPR marker genes that are responsive to SA/TBF1 regulation (B). Values are presented relative to the expression of the internal control UBQ10. Error bars represent SEM among three biological replicates (npr1 mutant versus the Col- 0, *P < 0.05, **P < 0.01, ***P<0.001; Unpaired t-test). 131 Figure 3. 9. ER stress does not influence SA-dependent signaling and SA content. (A) Endogenous SA was extracted from 14-day-old wild-type Col-0 and sid2 seedlings treated with DMSO (Mock) or 0.5 M Tm for 1 day. The samples were analyzed for both free SA and conjugated SA glucosides (SAG) with LC/MS. Error bars represent SEM. Data significantly different from the corresponding controls are indicated (treated seedlings versus the non-treated seedlings, NS, non significant; Unpaired t-test). (B) 14-day-old seedlings were transferred to liquid medium containing Tm (5 µM) for the indicated times. Samples were analyzed by qRT-PCR for the transcriptional levels of SA markers CBP60g, SID2 and PR1. Values are presented relative to the expression of the internal control UBQ10. Error bars represent SEM among three biological replicates. 132 Figure 3. 10. TBF1 does not modulate late-adaptive UPR activation upon ER stress. (A and B) 14-day-old seedlings were transferred to solid medium containing Tm (0.5 µM) for the indicated times. RNA samples were analyzed by qRT-PCR for transcriptional levels of TBF1 (A) or UPR marker genes that are either independent on TBF1 (unbZIP60, sbZIP60, BiP1 and PDIL1-2) or dependent on TBF1 (CNX1) (B). Values are presented relative to the expression of the internal control UBQ10. Error bars represent SEM among three biological replicates (tbf1 mutant versus the Col- 0, *P < 0.05, **P < 0.01; Unpaired t-test). 133 Figure 3. 11. UPR activation causes cytosolic reduction. (A) The ratio values were calculated from CLSM images of A. thaliana leaf epidermal cells expressing roGFP2-SEC22 treated with DMSO (mock), 100 M AZC and 500 M AZC for 1 day. Statistically significant differences between group means as determined by Unpaired t-test, separately (***P<0.001, ****P<0.0001). 20 images were analyzed for each combination assayed in a single experiment. Error bars represent SEM. The experiment was repeated in at least three biological replicates. (B) The ratio values were calculated from CLSM images of N. tabacum leaf epidermal cells expressing indicated construct combinations, respectively. Statistically significant differences between group means as determined by Unpaired t-test, separately (****P<0.0001). 20 images were analyzed for each combination assayed in a single experiment. Error bars represent SEM. The experiment was repeated at least in three biological replicates. (C) Quantitative RT-PCR analyses of various UPR indicators in N. tabacum expressing roGFP2, roGFP2+SEC-RFP and roGFP2+CPY*-RFP, respectively. cDNA was synthesized by using leaves infiltrated with above combination constructs after two days. Values are presented relative to non-infiltrated control which was set to 1. Transcription of L25 was used as internal control. Error bars represent SEM among three biological replicates. Data significantly different 134 from the corresponding controls are indicated (combination versus the roGFP2, **P < 0.01 , ***P<0.001, ****P<0.0001 NS, non significant; Unpaired t-test). 135 Figure 3. 12. NPR1 trans-localizes to the nucleus under ER stress. (A) Subcellular localizations of NPR1-YFP in npr1 Y1 and npr1 Y2 under treatment with solid medium containing 0.5 mM SA or 0.5 M Tm for 1 day were co-localized with the nuclei marker Hoechst33342. Nuclei are indicated by arrows. The other fluorescent structure highlighted by Hoechst 33342 in the images are the primary cell wall that are also stained by the dye. Scale bars = 10 m. (B) Protein levels of NPR1 in the indicated genetic backgrounds. 60 g of total protein extracted from seedlings treated with DMSO (mock), 0.5 mM SA or 0.5 M Tm for 1 day was subjected to Western blot analysis with anti-NPR1 antibodies. Positions of NPR1-YFP (F) and endogenous NPR1 (E) are indicated. 136 Figure 3. 13. NPR1 interacts with bZIP28 and bZIP60 genetically in the UPR signaling. (A) To determine the ER stress sensitivity of wild-type Col-0, bzip28/60, npr1 and npr1/bzip28/60, the seeds of respective genotype were plated on solid medium containing DMSO (0 nM Tm) or Tm (25 nM and 50 nM). Images show the appearance of 14-day-old seedlings grown on mock (0 nM Tm) or Tm plates. (B) Measurements of the primary root length of wild-type Col-0, bzip28/60, npr1 and npr1/bzip28/60. n>30 seedlings were measured for each genotype. Errors bars represent SEM. Data significantly different from the corresponding control (Col-0) are indicated (mutant versus the Col-0 under treatment, *P < 0.05, **P < 0.01, ***P<0.001 ,****P<0.0001, NS, non significant; Unpaired t-test). 137 Figure 3. 14. Lines overexpressing NPR1. (A) RT-PCR analyses of NPR1 expression in 14-day-old seedlings of wild-type Col-0, npr1 and overexpression NPR1 (oxnpr1) lines seedlings under normal condition of growth. 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PLoS Genetics, 11(4), 1–37. http://doi.org/10.1371/journal.pgen.1005164 146 CHAPTER IV A systemic signaling contributes to the unfolded protein response of the plant endoplasmic reticulum The work presented in this chapter has been accepted in Nature Communications: Lai Y-S, Stefano G, Zemeliss S, Ruberti C, Gibbon L and Brandizz F. A systemic signaling contributes to the unfolded protein response of the plant endoplasmic reticulum. 147 ABSTRACT The unfolded protein response (UPR) of the endoplasmic reticulum constitutes a conserved and essential cytoprotective pathway designed to survive biotic and abiotic stresses that alter the proteostasis of the endoplasmic reticulum. The UPR is typically considered cell- autonomous and it is yet unclear whether it can also act systemically through non-cell autonomous signaling. We have addressed this question using a genetic approach coupled with micro-grafting and a suite of molecular reporters in the model plant species Arabidopsis thaliana. We show that the UPR has a non-cell autonomous component, and we demonstrated that this is partially mediated by the intercellular movement of the UPR transcription factor bZIP60 facilitating systemic UPR signaling. Therefore, in multicellular eukaryotes such as plants, non- cell autonomous UPR signaling relies on the systemic movement of at least a UPR transcriptional modulator. 148 INTRODUCTION In physiological conditions of growth and in disease, eukaryotic life depends on the biosynthetic ability of the endoplasmic reticulum (ER) to synthesize correctly folded secretory proteins. Conditions that alter the ER proteostasis and induce accrual of misfolded proteins in the ER lead to a potentially lethal condition known as ER stress (Kaufman et al., 2002; Rutkowski & Kaufman, 2004). At the onset of ER stress, cells activate cell-intrinsic UPR signaling pathways mediated by specialized ER stress sensors whose function is to reprogram gene expression for the synthesis of effectors that attenuate ER stress and restore the biosynthetic ability of the ER (Walter & Ron, 2011). If the UPR is ineffective to initiate proper cytoprotective mechanisms to attenuate ER stress, it induces programmed cell death (Liu & Howell, 2010b). The main branch of the UPR is mediated by the ER-associated kinase and ribonuclease inositol-requiring protein 1 (IRE1) through largely conserved mechanisms. During ER stress, upon oligomerization and trans-autophosphorylation for self-activation, IRE1 splices the mRNA of a basic leucine zipper (bZIP) transcription factor, namely HAC1 in yeast, XBP1 in metazoans and bZIP60 in plants. This step removes the coding region for a transmembrane domain (TMD), releasing the translational inhibition of a potent UPR transcriptional factor. The newly synthesized transcription factor is translocated to the nucleus where it modulates the expression of nuclear UPR target genes for restoration of ER proteostasis (Aragón et al., 2009; Deng et al., 2011; Iwata et al., 2008; Sidrauski & Walter, 1997). Multicellular eukaryotes also harness another UPR branch, which is mediated by ER membrane tethered transcription factors (MTTFs), such as ATF6 in metazoans and bZIP28 in plants (Liu et al., 2007a). Upon ER stress sensing, these MTTFs translocate to the Golgi where the transcription factor domain is cleaved 149 off the transmembrane anchor and is then transported to the nucleus to regulate transcription of UPR target genes (Liu et al., 2007a; Ye et al., 2000). In metazoans, most of the knowledge on IRE1 has been gathered from analyses of single cells and cells in culture. Evidence is also emerging that in addition to cell-intrinsic signaling, the metazoan UPR may actuate non-cell autonomous signaling for the activation of stress responses in tissues and cell types that are different from those where the ER stress signal is originated. Specifically, in C. elegans intercellular signaling of the UPR has been induced through the overexpression of spliced (i.e. active) XBP1 in neuron cells, which elicits UPR activation in non-stressed intestine cells (Taylor & Dillin, 2013). Similarly, in mice overexpression of active XBP1 in hypothalamic proopiomelanocortin (POMC) neurons is followed by non-cell autonomous splicing of XBP1 and UPR activation in the liver (Yanes & Reckelhoff, 2014). Although the existence of secreted stress signals to actuate transcellular UPR has been hypothesized (Taylor & Dillin, 2013), the identity of the effectors that act downstream XBP1 in intercellular communication of the UPR in metazoans is currently unknown. It is yet also unknown whether the systemic UPR signaling occurs in experimental conditions that do not rely on tissue-specific overexpression of XBP1. Plants show cell-intrinsic UPR signaling (Ruberti et al., 2016); however, whether they also execute non-cell autonomous UPR signaling is still an open question. Earlier findings in tobacco that a local (i.e. proximal) induction of ER stress in a leaf did not alter the UPR in a systemic (i.e., distal) leaf within an eight hour-period from local ER stress initiation supported the hypothesis that plants may not actuate non-cell autonomous UPR signaling (Jelittovandooren, Vidal, & Denecke, 1999). However, plants respond to constantly changing environmental conditions by coordinating developmental and physiological programs at organismal level via 150 the redistribution of various chemical modulators such as protein/peptides, regulatory RNAs, ions, hormones and metabolites (Choi, Toyota, Kim, Hilleary, & Gilroy, 2014; Mousavi, Chauvin, Pascaud, Kellenberger, & Farmer, 2013). Therefore, during growth and in challenging environmental conditions non-cell autonomous UPR signaling in plants could potentially facilitate ER stress surveillance across tissues as well as stress resolution strategies through optimization of resource allocation. In this study, we addressed whether the plant UPR involves non-cell autonomous stress signaling using a genetic approach combined with micro-grafting analyses, and molecular reporters for tissue-specific and ectopic gene expression in wild type and high-order UPR knockout mutants. We provide strong evidence that in response to ER stress the plant UPR functions systemically predominantly in a shoot-ward direction mediated by root-originated non-cell autonomous signals. Consistently, we report that bZIP60 can target to plasmodesmata, which are plant-unique structures allowing intercellular communication (Xu & Jackson, 2010), and that this transcription factor moves transcellularly thus inducing in systemic tissues the expression of BiP3, the main transcriptional UPR target modulated by bZIP60. Based on these results, we propose that in plants, in addition to cell-autonomous signaling, the UPR extends to systemic tissues by non-cell autonomous signaling through the contribution of the mobile UPR transcription factor bZIP60. Our findings indicate that in eukaryotes non-cell autonomous UPR signaling can directly rely on the translocation of at least one UPR transcriptional regulator. 151 MATERIALS AND METHODS Lines and Plant Growth Condition Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as a wild-type genotype in this study. The mutant lines used in this work are: bzip28 (Col-0; SALK_132285), bzip60-1 (Col-0; SALK_050203) (Moreno et al., 2012). Sterilized seeds were stratified at 4 °C for 2 days and plated on half-strength Linsmaier Skoog (LS) medium (1/2x LS salts, 1.5% sucrose, 1.2% Agar). Plants were grown in a 16h-light/8h-dark cycle at 21 °C. Agrobacterium (GV3101)- mediated transformation of Arabidopsis thaliana and Nicotiana tabacum plants was performed by the floral dip and infiltration methods, respectively (Clough & Bent, 1998; Sparkes, Runions, Kearns, & Hawes, 2006) Shoot-Root Split Culture System Plants were grown vertically on the half-strength LS medium for 14 days and transferred onto a 9 cm Petri dish equipped with two compartments (Kord-Valmark #2903, USA) containing medium with either DMSO in one side or 0.5M Tm, 10M 17--estradiol and 0.5M Tm in combination with 10M 17--Estradiol in the other side and cultured horizontally for an additional 1 day. Data were acquired on at least three biological replicates. qRT-PCR for Gene Expression Analyses Total RNA was extracted using Macherey-Nagel NucleoSpin RNA Plant kit (www.mn- net.com) and used for qRT-PCR with triplicates, as described previously (Lai et al., 2014). UBQ10 was utilized as an internal control in normalization of qRT-PCR, unless otherwise stated 152 in Figure 4. 9. Similar patterns of expression were observed in the three independent biological replicates. Primers used in this study are listed in Table 4. 1. Tunicamycin Measurements Two-week-old plate-grown seedlings (~100 mg) exposed to the shoot-root split culture system as detailed above were harvested and ground in liquid nitrogen. The samples were extracted at 4 °C overnight using 1 ml of ice cold methanol:water (80:20 v/v) containing 0.1% formic acid; 0.1 g L-1 tunicamycin spiked with propyl 4-hydroxybenzoate as an internal standard for quantification of Tm levels. Then samples were vortexed and centrifuged at 12000xg 4 °C for 10 min, after which the flow-through samples were transferred to HPLC vials for the measurement of endogenous concentration of Tm by LC-MS with Quattro Premier XE (Waters company) instrument according to an established protocol (Zeng et al., 2011). The experiments were repeated at least two times with three biological replicates each time showing similar results. Grafting Experiment Hypocotyl micro-reciprocal grafting was performed as described in (Marsch-Martínez et al., 2013), with minor modifications. Briefly, micro-grafting was conducted using ten-day- old seedlings grown vertically that were first grown in 16h-light/8h-dark 120 µmoles/m2/s for 6 days and then in dark for 4 additional days. Grafts were generated by transverse sectioning of the aerial and root portions followed by conjunction on growth medium. After the grafts were made, samples were grown in dark vertically for 2 days, moved to light but covered with fine mesh for one day and then uncovered to grow in light. Adventitious roots were removed every 153 2-4 days. Samples were harvested by removing the whole hypocotyl portion avoiding the potential contamination at grafted junction and processed four weeks after micro-grafting for further analysis. Lack of contamination of adventitious roots in grafted plants was confirmed by genotyping with PCR. Analyses were performed on at least three biological replicates. Plasmid Construction The Phusion high-fidelity DNA polymerase (NEB, USA) was employed to amplify all DNA sequences using the primer sets in Table 4. 1.; the Gateway system (Invitrogen, USA) was used to generate expression plasmids. The promoter sequences of GLYT, SHR and BiP3 and the coding sequences of spliced bZIP60 and PDLP1 were amplified using Arabidopsis genomic and cDNA as templates, respectively. The amplified coding sequences were recombined into indicated Gateway vectors via LR reaction (Invitrogen, USA) and confirmed by sequencing. Confocal Laser Scanning Microscopy Imaging Confocal imaging was performed with an inverted laser scanning confocal microscope Nikon A1RSi. For subcellular localization assays, leaf tissues were mounted on a slide in a drop of tap water and viewed with the confocal microscope. GFP fluorescence was monitored at excitation wavelength of 488 nm and a bandpass 500-550 nm emission filter. Propidium iodide was monitored with a 560 nm excitation wavelength and a 570-620 nm bandpass emission filter. YFP fluorescence was monitored with a 513 nm excitation wavelength and 520-550 nm bandpass emission filter. CFP was monitored with a 443 nm excitation wavelength and a 465- 505 nm emission filter. 154 Quantification of Protein Colocalization at PD Agrobacterium cultures at a cell density A600: 0.025 and 0.5, containing the constructs for cytosolic YFP and YFP-sbZIP60, respectively, were infiltrated through stomata into Nicotiana tabacum leaves using needles syringe. The images were acquired 48 hrs after infiltration. Pearson Correlation Coefficient (PCC) values were calculated using the colocalization tool with default settings of the Nikon microscope software (NIS-Element AR 4.30). Areas with CFP and/or YFP signals at PD puncta were selected for the coefficient calculations. The coefficient calculations were performed on 170 of 3.4 µm2 areas. Propidium Iodide Staining Arabidopsis transgenic seedlings were stained in the 10 g/ml working solution of propidium iodide which was prepared by diluting with half LS liquid medium from 1 mg/ml stock solution (Sigma-Aldrich P4864). GUS Staining β-Glucuronidase activity in transgenic roots carrying pBIP3-GUS was visualized by X- Gluc as substrate using a conventional protocol (Dhondt et al., 2010). Staining was performed three times in three independent lines with consistent results. Aniline Blue Staining The vital dye Aniline Blue diammonium salt (Sigma-Aldrich 415049) dissolved in 1M Glycine (pH=9.5) (0.1% working solution) was adopted for PD-associated callose staining upon stoma infiltration with a needless syringe into Nicotiana tabacum leaves for confocal 155 microscopy analyses. Staining was performed three times in three independent lines with consistent results. 156 RESULTS Spliced bZIP60 Translocates Transcellularly To test whether systemic UPR signaling may take place in plants, we adopted the approach that led to the discovery of non-cell autonomous UPR in metazoans, which was based on the tissue-specific expression of a constitutively active bZIP transcriptional regulator of the UPR (i.e., XBP1) and detection of UPR activation in a systemic tissue (Taylor & Dillin, 2013). To translate the approach to intact plants, we first adopted a cell-type specific expression assay in Arabidopsis transgenic roots. The well-established organization of the root and the available suite of tissue-specific promoters make this organ an exquisite model to investigate gene expression at a sub-organ resolution (Vragović et al., 2015). For our experiments we used the short-root (SHR) promoter, which is exclusively active in the stele, the central tissue of the root, and drives the expression of SHR (Nakajima et al., 2001). The latter is a nucleus-localized transcription factor that moves from the stele, where it is synthesized, to the endodermis, a tissue layer surrounding the stele; notoriously, SHR does not reach the cortex and epidermis, which envelope the endodermis(Nakajima et al., 2001). We used the SHR promoter to drive expression of cytosolic green fluorescent protein (GFP) (pSHR-GFP) (Helariutta et al., 2000), and GFP fused either to SHR (pSHR-SHR-GFP)(Nakajima et al., 2001) or to a constitutively active form of bZIP60, spliced bZIP60-GFP (pSHR-sbZIP60-GFP). As a genetic background we used wild type Col-0 (hereafter Col-0), an SHR knockout(Levesque et al., 2006) (shr-2 hereafter shr), and bzip28/60-1 (Deng et al., 2013) (hereafter bzip28/60). In the bzip28/60 mutant both UPR branches (i.e., bZIP60 and bZIP28) are inactive in conditions inducing ER stress (i.e., Tunicamycin (Tm) treatment), and the expression of UPR genes, including BiP3, the major 157 target of sbZIP60(Iwata et al., 2008), is not actuated (Ruberti et al., 2018). This set up was therefore designed to test movement of bZIP60 across tissues. A GFP fusion of bZIP60 driven by the native promoter in a bzip60 mutant (Parra-Rojas, Moreno, Mitina, & Orellana, 2015) is localized throughout the root tissues in control conditions and in conditions of ER stress (Figure 4. 6.) hampering the possibility to assess systemic movement of this transcription factor. However, in our experimental setup, we expected that cytosolic GFP would be detected exclusively in the stele, while SHR-GFP would be localized in the stele and the endodermis. Conversely, if sbZIP60 moved transcellularly, then sbZIP60-GFP expression in the stele would result in the accumulation of sbZIP60-GFP in the stele as well as in other cell layers. Confocal imaging of cytosolic GFP and SHR-GFP in the root of the respective Col-0 and shr transgenic lines showed a diffuse distribution of cytosolic GFP in the stele, and a localization of SHR-GFP in the nuclei of the stele and endodermis (Figure 4. 1. A). These results are consistent with earlier findings (Koizumi & Gallagher, 2013) and indicate that stele-expressed cytosolic GFP accumulates only in the stele, while SHR-GFP, which is produced in the stele, moves to the endodermis (Helariutta et al., 2000; Kimberly L. Gallagher, Paquette, Nakajima, & Benfey, 2004). When we analyzed bzip28/60; pSHR-sbZIP60-GFP roots, we found accumulation of sbZIP60-GFP in the nuclei and cytoplasm of cells in the stele and endodermis, as well as cortex and epidermis (Figure 4. 1. A), which is comparable with the localization of GFP-bZIP60 driven by the bZIP60 native promoter in conditions of ER stress (Parra-Rojas et al., 2015) (see also Figure 4. 6.). In addition, such distribution pattern was visible throughout the division, elongation and differentiation zones of roots with graded level of fluorescence from the younger regions of the root upward (Figure 4. 7.). In light of the restricted accumulation of cytosolic GFP to the stele and of SHR-GFP to the stele and endodermis, these results strongly support that 158 sbZIP60 can move transcellularly from the stele to the epidermis through the endodermis and cortex. Next, we aimed to test whether the transcellular movement of sbZIP60 could play a role in UPR signaling in the tissues in which it is translocated. To do so, we developed a genetically- encoded reporter for UPR activation by sbZIP60 in systemic tissues. Specifically, we fused - glucuronidase (GUS) under the control of the BiP3 promoter (pBiP3-GUS) and introduced it into either Col-0 (Col-0; pBiP3-GUS; positive control) or bzip28/60. In the latter background we introduced pBiP3-GUS alone (bzip28/60; pBiP3-GUS; negative control) or in combination with pSHR-sbZIP60-GFP (bzip28/60; pSHR-sbZIP60-GFP/pBiP3-GUS). Using these genetic backgrounds, we tested where the BiP3-promoter could be activated systemically in the root by stele-expressed sbZIP60. We expected that if sbZIP60 activated the BiP3 promoter in a systemic manner, then the tissue labelling by GUS would mirror the verified tissue distribution of sbZIP60-GFP (Figure 4. 1. A) and would show staining intensity levels above background. We analyzed three different developmental regions of the root (a: upper maturation zone, b: middle maturation zone, and c: lower maturation zone) (Figures 4. 1. B, C). As expected, we found no expression of pBiP3-GUS throughout the bzip28/60 roots (Figure 4. 1. B, C), owing to the lack of functional bZIP60 and bZIP28 in this genetic background. In wild type, BiP3 is generally expressed in the absence of induced ER stress (Liu et al., 2007a; L. Sun et al., 2013). Consistently with this, in Col-0; pBiP3-GUS we verified GUS expression, which was visible in the endodermis of the upper maturation zone, and predominantly in the stele and endodermis of the middle maturation and lower maturation zones (Figure 4. 1. B, C), in agreement with earlier high-resolution tissue-specific transcriptomics analyses of the root (Brady et al., 2007). However in conditions of ER stress, the BiP3 expression was robustly enhanced in the stele, endodermis, 159 cortex and epidermis layers throughout all the root zones (Figure 4. 8.), consistently with previous findings (Cho & Kanehara, 2017). We then analyzed bzip28/60; pSHR-sbZIP60-GFP/ pBiP3-GUS transgenic plants and found a strong GUS expression in the stele and endodermis but also in the cortex and epidermis of all the root zones under analysis (Figure 4. 1. B, C). We deduce that the strong GUS activity in the bzip28/60; pSHR-sbZIP60-GFP/pBiP3-GUS line is likely linked to the overabundance of sbZIP60 driven by pSHR in these tissues compared to Col- 0; pBiP3-GUS. Importantly also, the GUS activity in bzip28/60; pSHR-sbZIP60-GFP/pBiP3- GUS mirrors the verified distribution of pSHR-sbZIP60-GFP in the cortex and epidermis as well as BiP3 expression pattern under stress condition (Figures 4. 1. A, 4. 8.). These results lend support to our original observations that sbZIP60-GFP can translocate systemically from a tissue where it is specifically expressed and activate transcription of a target gene in systemic tissues. Taken together, these data indicate that, similar to metazoans, in conditions of overexpression of a constitutively active UPR modulator the UPR signaling is executed systemically in plants. The results also indicate that overexpressed sbZIP60 can act as transcellular mobile transcription factor, triggering UPR gene expression in a systemic manner. Systemic Induction of the UPR genes Having established that sbZIP60 can move transcellularly in the root, we next aimed to test a systemic signaling of sbZIP60 from the root to a different organ. Therefore, we aimed to assess the expression of UPR marker genes in the shoot and root of bzip28/60 lines that express sbZIP60 exclusively in the root. We expected that if a UPR signal moved shoot-ward from the root, we would detect expression of sbZIP60, and possibly of BiP3, not only in the roots but also 160 in the shoots. To express sbZIP60 specifically in the root, we generated transgenic bzip28/60 lines expressing sbZIP60 under the control of the pRoot promoter (Vijaybhaskar, Subbiah, Kaur, Vijayakumari, & Siddiqi, 2008) and selected two independent lines for analyses. pRoot drives the Arabidopsis glycosidase-transferase GLYT expression specifically in the roots (Vijaybhaskar et al., 2008), both in unchallenged and Tm-challenged seedlings, as supported by the evidence that the expression of GLYT in the shoots is proximal to the detection limit of quantitative RT- PCR (qRT-PCR) and not statistically different in both experimental conditions (Figure 4. 9.). We selected two independent transgenic lines that we named bzip28/60; pRoot::sbZIP60. For our assay, we adopted an Arabidopsis shoot-root split culture system used earlier to assay long- distance signaling in plants (Kronzucker, Li, Li, Song, & Shi, 2015). In this system, 2-week old intact seedlings grown on vertical plates are transferred to Petri dishes that are subdivided by a sealed plate divider to separate growth media with different composition (Tabata et al., 2014). The shoot and root portions of intact seedlings are laid across the plate divider and are therefore exposed to the medium contained in each plate sub-compartment separately. We adopted this method in order to apply Tm or DMSO (Tm solvent) for 24 hrs to the medium exposed to the root. We first compared the levels of UPR gene transcripts in shoot and root of seedlings challenged by Tm at the root by qRT-PCR. We used Col-0 and bzip28/60 as positive and negative controls, respectively. As expected, in Col-0 the levels of the UPR gene transcripts sbZIP60 and BiP3 were more abundant in Tm-treated seedlings compared to DMSO control (Figures 4. 2. A, B). Conversely, the bzip28/60 mutant did not show elevation of either UPR marker gene in the absence and in the presence of Tm (Figures 4. 2. A, B), despite its ability to absorb Tm (Figure 4. 10.). However, the bzip28/60; pRoot::sbZIP60 lines showed a presence of sbZIP60 and BiP3 transcripts in the root at levels that were significantly higher compared to 161 bzip28/60 (Figures 4. 2. A, B), indicating that the pRoot promoter is functional for the expression of these genes. Next, analyses of sbZIP60 and BiP3 transcripts in the shoot of bzip28/60; pRoot::sbZIP60 lines showed significantly higher levels of sbZIP60 and BiP3 transcripts compared to bzip28/60. Unlike in Col-0, in bzip28/60; pRoot::sbZIP60 the sbZIP60 and BiP3 transcript levels were largely unchanged by Tm treatment, as it would be expected for genes normally controlled by ER stress responsive promoters. Together with the evidence that pRoot is unresponsive to Tm and that it is expressed specifically in roots (Figure 4. 9.), as well as the consideration that the experiments were conducted in a genetic background normally lacking endogenous expression of the UPR bZIP-transcription factors and, consequently of their target genes under ER stress, these data indicate that the root-generated bZIP60 and BiP3 transcripts are found in tissues in which their expression is not driven locally by an ER stress responsive promoter. These findings therefore support the hypothesis that the plant UPR signaling may be non-cell autonomous and imply that the transcellular translocation of at least sbZIP60 transcripts from root to shoot may be involved in long-distance transduction of the UPR. ER Stress Response Acts Systemically in a Shoot-ward Direction Having acquired evidence that a systemic UPR could take place in conditions of tissue- specific overexpression of sbZIP60, we next sought to test the occurrence of endogenous systemic UPR. To do so, we adopted the shoot-root split culture system in order to apply Tm for 24 hrs either to the medium at the shoot or the root of 2-week old wild-type Col-0 seedlings (Figure 4. 3. A); we then monitored the UPR signaling in each portion of the seedling by qRT- PCR (Figure 4. 3. B). As UPR reporters, we used sbZIP60 and BiP3. We first monitored the 162 mRNA levels of these markers in seedlings treated with Tm at the roots and found increased levels of sbZIP60 and BiP3 compared to the root of a mock control (Figure 4. 3. B; D/T vs 0 hr). When we analyzed the untreated shoot of the root-treated seedlings, we observed a significant raise in the transcript levels of sbZIP60 and BiP3 compared to mock control (Figure 4. 3. B; D/T vs 0 hr). These results indicate that Tm-treatment of the root leads to UPR signaling activation both in the Tm-treated root as well as in the untreated shoot. Next, we analyzed the UPR gene transcripts in seedlings treated with Tm at the shoots (Figure 4. 3. A; T/D). We found that both sbZIP60 and BiP3 transcript levels increased in the shoot compared to mock control (Figure 4. 3. B; T/D). Furthermore, in net contrast to the D/T seedlings, no significant induction of sbZIP60 and only a slightly induction of BiP3 were detected in the untreated root upon shoot treatment compared to mock control (Figure 4. 3. B; T/D vs 0 hr). These results point towards the possibility of a systemic actuation of ER stress responses mainly in a shoot-ward direction. Indeed, it has been previously shown that the splicing of bZIP60 occurs very quickly in response to heat-induced ER stress (Deng et al., 2011). To monitor the time required for systemic UPR response in our experimental condition, we tested the kinetic profiles of bZIP60 transcription and splicing as well as the following activation of BiP3 expression in response to ER stress within 24 hrs. We found that in the Tm-treated roots, a significant induction of unspliced bZIP60 (unbZIP60) occurs at 3 hrs (Figure 4. 11. A). Meanwhile, the emergence of sbZIP60 transcripts started at 3 hrs and reached peak expression at 6 hrs post treatment followed by a decline of induction levels at 12 hrs and 24 hrs post treatment (Figure 4. 11. B). The BiP3 expression displayed a similar trend like sbZIP60 but reached the peak at 12 hrs post treatment (Figure 4. 11. C), in line with a time correlation of the sbZIP60-driven BiP3 activation in response to the ER stress. In the untreated shoots, the significant inductions of unbZIP60 andsbZIP60 were 163 detected at 3 hrs post treatment and BiP3 expression was predominantly detected at 6 hrs post treatment (Figures 4. 11. A-C) supporting a roughly 3 hrs-requirement for systemic UPR signals, including bZIP60 transcripts translocation, from treated roots to shoots to elicit the downstream UPR gene BiP3 in our experimental conditions. To exclude the possibility that the UPR signaling could be ignited in the untreated tissue in response to Tm translocation from the Tm- treated tissue to a distal tissue, we monitored Tm levels throughout plant body. To do so, we assayed Tm levels in the shoot and root of seedlings treated with Tm in the shoot-root split culture system, using HPLC/MS. As controls, we used seedlings exposed to either DMSO (D/D) or Tm (T/T) at both the shoot and the root. As expected, we observed a significant Tm accumulation in the treated shoot and root compared to the respective DMSO (mock control)- only treated tissues (Figure 4. 3. C; D/T and T/D vs D/D), indicating that the seedlings absorbed Tm from the growth medium. Furthermore, the root of seedlings with Tm-treated shoot showed accumulation of Tm (Figure 4. 3. C; T/D vs D/D), indicating that Tm can translocate from the shoot to the root. Importantly however, we found no significant increase of Tm levels in the DMSO only-treated shoot of seedlings with Tm-treated root compared to mock seedlings (Figure 4. 3. C; D/T vs D/D) indicating that Tm is not transported from the root to the shoot in the shoot-root split culture system to a detectable level. Taken together, these results support that an endogenous UPR signaling acts systemically mainly in a shoot-ward direction that is independent of Tm transport from the root to the shoot. 164 Root-Derived Signals are Involved in the Induction of UPR genes in Unchallenged Tissues We next aimed to acquire a parallel line of evidence for the existence of endogenous systemic UPR. To do so, we performed reciprocal micro-grafting analyses in which the aerial tissue (scion) is grafted onto the root (rootstock) of a different plant. Successful grafting leads to the development of graft unions that can be used for molecular assays. For the grafting, we used wild-type seedlings (Col-0) and bzip28/60 (Figure 4. 4.). We expected that if an endogenous transcellular UPR signal existed as so far supported by our experiments (Figures 4. 3., 4. 11.), we would observe UPR gene transcripts in the bzip28/60 tissues grafted with Col-0 tissues. Because our results indicate that a UPR signaling moves mainly in a shoot-ward direction (Figures 4. 3. B, 4. 11.), to monitor the UPR signaling at tissue-specific level we compared the abundance of sbZIP60, BiP3 and bZIP28 transcripts in self-grafts (same genetic background) and hetero-grafts (different genetic background) of scion and rootstock from independent seedlings treated with Tm at the rootstock. As reference, we used untreated (i.e., DMSO only) micro-grafted seedlings with the same genetic combination as the respective Tm- treated micro-grafted seedlings. As expected, upon Tm treatment the self-grafted bzip28/60 (scion/rootstock combination indicated as bzip28/60/bzip28/60) did not show a significant increase of sbZIP60, BiP3 and bZIP28 transcripts compared to the same untreated background both in the scion and in the rootstock (Figures 4. 4. A-C). In net contrast, compared to the bzip28/60/bzip28/60 self-grafts, the Col-0 self-grafts (Col-0/Col-0) displayed significant induction of the UPR marker genes, sbZIP60 and BiP3, in both scion and rootstock but non- induced levels of bZIP28, consistent with a bZIP28 signaling in ER stress mainly mediated at a protein level (J.-X. Liu, Srivastava, Che, & Howell, 2007) (Figures 4. 4. A-C). These controls indicate that the micro-grafting approach does not hamper the response of the grafted unions to 165 ER stress. We then tested the bzip28/60 scion grafted on Col-0 rootstock (bzip28/60/Col-0). Compared to Col-0 self-grafts, in the rootstock of bzip28/60/Col-0 we found similar levels of UPR gene transcripts (Figures 4. 4. A-C), indicating that the Col-0 rootstock in the bzip28/60/Col-0 grafts can respond to ER stress as the self-grafted Col-0/Col-0 rootstock. In the scion of bzip28/60/Col-0 hetero-grafts, the UPR gene transcript levels were significantly higher compared to the scions of bzip28/60 self-grafts albeit lower when compared to the Col-0/Col-0 scion (Figures 4. 4. A-C). Because the bzip28/60 background is unable to evoke the UPR, these results indicate that the scions of bzip28/60/Col-0 hetero-grafts contain UPR gene transcripts originated from the Col-0 rootstock. These results are consistent with our observations that a shoot-ward signal originated from a Tm-treated tissue can induce the UPR in an unchallenged systemic tissue (Figure 4. 3. B) and findings that the bZIP28 mRNA can move intracellularly in unstressed conditions (Thieme et al., 2015). Next, we aimed to test whether UPR signaling other than the canonical bZIP60 and bZIP28 arms could be involved in the systemic UPR response. To do so, we conducted a separated reciprocal grafting by adopting Col-0 as the scion and bzip28/60 as the rootstock (Col- 0/bzip28/60). We expected that, if other shoot-ward UPR signaling were in place beside the bZIP28 and bZIP60 arms, then the levels of BiP3 would be affected in the scion of the Col- 0/bzip28/60 hetero-graft. We found that similar to bzip28/60 self-grafts, there was no significant induction of sbZIP60, BiP3 and bZIP28 in the rootstocks of Col-0/bzip28/60 hetero-grafts upon Tm treatment (Figures 4. 12. A-C). Also, we found that in the scions of the Col-0/bzip28/60 hetero-grafts the sbZIP60, BiP3 and bZIP28 were not induced and their respective mRNA levels were not significantly different compared to the scions of bzip28/60 self-grafts (Figures 4. 12. A-C). While the lack of BiP3 induction in the scions is likely due to the absence of a functional 166 UPR machinery in the rootstocks of Col-0/bzip28/60 hetero-grafts, these data indicate that the systemic UPR signaling requires the function of the canonical UPR bZIP-arms. Systemic UPR Signaling is Plasmodesmata-Dependent Direct intercellular communication between plant cells, including cells of the stele and the endodermis, occurs via plasmodesmata (PD), which are connecting micro-channels between adjacent cells and the main route for certain signaling molecules in cell-to-cell trafficking (Xu & Jackson, 2010). Therefore, our results that sbZIP60 is translocated from the stele to distal root tissues (Figure 4. 1.) raise the possibility that the systemic UPR could rely on PD-mediated traffic. To test this, we first aimed to establish whether sbZIP60 could target the PD. To do so, we generated a yellow fluorescent protein (YFP) fusion to sbZIP60 driven by a constitutive promoter for confocal microscopy analyses. For confocal imaging, we used leaves because the periphery of epidermal cells is more clearly distinguishable compared to root tip cells. We verified a nuclear localization of YFP-sbZIP60 as well as a diffused distribution with conspicuous punctate reminiscent of PD at the cell periphery (Figure 4. 13. A). The PD localization of sbZIP60 was confirmed through co-localization analyses with the cyan fluorescent protein (CFP) fused to the established PD localized receptor-like transmembrane protein, PDLP1 (Lim et al., 2016) (Figure 4. 13. A). In contrast, a cytosolic YFP (cYFP) control did not appear to have a marked co-localization with PDLP1-CFP (Figure 4. 13. A). To confirm these observations, we performed integrated density measurements of the YFP signal at the PD using Aniline Blue (AB), a vital PD dye (M. G. Kim et al., 2005), and estimated the Pearson Correlation Coefficient (Yuan, Lazarowitz, & Citovsky, 2016), as a measure of the association 167 of fluorescence intensity between the YFP and AB signals. We found that YFP-sbZIP60 colocalized at PD at significantly higher levels compared to cYFP (Figure 4. 13. B) supporting our qualitative observations that sbZIP60 is targeted to PD more visibly than cYFP control (Figure 4. 13. A). The evidence that sbZIP60-GFP moves away from the stele (Figures 4. 1. A, 4. 7.), a process that is mediated exclusively by PD, and the coincidental distribution of YFP- sbZIP60 and at PD (Figure 4. 13.) support that sbZIP60 is translocated via PD in conditions of ectopic expression. Therefore, to further test a PD requirement for systemic UPR, we tested the PD dependency of endogenous systemic UPR response by detecting the abundance of endogenous UPR marker genes in the shoot-root spilt culture system using the conditional PD blockage mutant, pMDC7-icals3m (Curtis & Grossniklaus, 2003; Wu et al., 2016). Compared to wild type, in this mutant the PD passage is reduced by an enhanced accumulation of callose upon gene induction by estrogen treatment (Curtis & Grossniklaus, 2003; Vatén et al., 2011). We found that in the Col-0 plants in the presence of estrogen, the expression of sbZIP60 and BiP3 was similar to the mock condition, indicating that exogenous estrogen does not elicit ER stress (Figure 4. 5. A). We also found that both UPR markers exhibited similar expression profiles in shoots and roots in conditions of Tm-treatment only, or in condition of Tm-treatment in conjunction with estrogen application to the roots (Figure 4. 5. A), further supporting that the addition of exogenous estrogen does not affect the systemic UPR response. We next monitored the systemic UPR response in the conditional PD blockage mutant. In this mutant, there were no significant differences in the transcript levels of UPR markers in Tm+estrogen treated roots compared to Tm only treated roots, indicating no effect of estrogen on the UPR in this mutant (Figure 4. 5. B). Most importantly, we found that the expression of both UPR markers was significantly reduced in the shoots of seedlings treated with Tm+estrogen at the roots compared 168 to the shoots of seedlings with Tm-only treated roots (Figure 4. 5. B). These results indicate that an induction of PD closure compromises the systemic UPR signaling. These data elucidate that such systemic UPR signaling relies on the PD passage possibly through translocation of signaling molecules such as sbZIP60 to elicit UPR in the distal tissues. 169 DISCUSSION The Plant UPR Constitutes a Systemic Signal Long-distance signaling is required for plants to successfully actuate physiological processes and thrive in response to environmental challenges. For example, flowering is a well- established process regulated by systemic signaling whereby the transcriptional factor CONSTANS is expressed in response to photoperiod followed by activation of FLOWERING LOCUS T (FT) in companion cells of the phloem. The FT protein is translocated systemically through phloem and interacts with the transcription factor FD at the shoot apical meristem further promoting floral fate at lateral primordial (Corbesier et al., 2007), (Li et al., 2011). Meanwhile, the transposable FT mRNA, independent of the FT protein, also contributes to systemic floral signaling (Li et al., 2011). Furthermore, during pathogen attack, plants actuate systemic responses to enhance overall pathogen resistance throughout the entire organism and build a stress immunity response known as systemic acquired resistance (SAR) (Fu & Dong, 2013). Therefore, a systemic signaling plays significant roles in plant development but also stress responses. Earlier investigations on the ability of ER stress to trigger a systemic response used a tobacco system, where a tobacco leaf of an intact plant was challenged by Tm. The study led to no indication of UPR induction in a distal leaf upon eight hours from Tm treatment (Jelittovandooren et al., 1999). Based on these observations, the authors concluded that locally applied ER stress is unable to activate a systemic signal to turn on BiP expression in distal tissues in plants. Nevertheless, in our study we demonstrated that during a 24 hr-time course upon local application of Tm to wild-type seedlings, the UPR markers can be detected in systemic tissues. These results indicate that ER stress in plants evokes a long-distance signal transduction of the 170 UPR. While it is possible that the differences between our work and Jelitto-Van Dooren et al. (1999) (Jelittovandooren et al., 1999) may be linked to the use of a different experimental system, our conclusions are further corroborated by the evidence gathered from reciprocal grafting and ectopic expression assays showing significant transcript levels of sbZIP60 and BiP3 distally from the Tm-treated tissues. Therefore, our work demonstrates that in addition to the well- established existence of a cell-intrinsic UPR signaling, plants harness long distance signaling to communicate the occurrence of ER stress in a tissue to systemic tissues. The Intercellular Movement of bZIP60 is a component of Long-distance UPR Signaling In C. elegans and mice, the expression of active XBP1 in one cell type activates the UPR in distal tissues (Taylor & Dillin, 2016), (Yanes & Reckelhoff, 2014), but the molecular mechanisms underlying the cell-to-cell communication have not been elucidated. In our work, we demonstrated not only that sbZIP60 moves transcellularly but also that its movement causes downstream UPR activation in distal tissues (Figures 4. 1., 4. 2.). Based on these results, we propose that sbZIP60 participates as a non-cell autonomous factor to actuate distal UPR signaling directly through its movement across cells. It is worth noticing that previous studies in C. elegans and mice relied on overexpression of transcriptionally active XBP1 to infer the existence of systemic UPR signaling (Taylor & Dillin, 2013; Yanes & Reckelhoff, 2014). In our work, we have used overexpression of transcriptionally active bZIP60 and reached similar conclusions to the studies in metazoans. Specifically, we have provided evidence that systemic UPR was achieved in distal tissues by 171 local expression of sbZIP60 proteins leading to the activation of a downstream target gene (Figures 4. 1., 4. 2.). Noticeably however, we have also established that an endogenous signal is actuated to evoke a systemic UPR (Figures 4. 3., 4. 4.). This is supported by the detection of endogenous sbZIP60 and BiP3 transcripts in the shoot of wild-type seedlings exposed to Tm at the root in the split-plate system (Figure 4. 3. B) and the micro-grafting experiments using the bzip28/60/Col-0 hetero-grafts in which we detected the endogenous sbZIP60 and BiP3 transcripts (i.e., Col-0-originated) in the scion (Figures 4. 4. A, B). The lack of genetic information in the bzip28/60/Col-0 scions that is necessary to express normally sbZIP60 and BiP3 in the aerial tissues supports that the presence of transcripts of these genes in the scion is the result of endogenous systemic UPR signaling. In plants, there have been identified three types of mobile RNA: (i) viral pathogenic RNA (ii) small RNAs including siRNA and miRNA (iii) mRNA(Kehr & Buhtz, 2008). Transcriptomic analyses in Arabidopsis phloem also reported on the existence of cellular mRNA, suggesting the potential role of these mRNA as signaling molecules in the long-distance trafficking (Deeken et al., 2008). However, also proteins can contribute to systemic signaling, as it occurs for the well-established floral systemic signaling mediated not only by FT proteins but also by the FT mRNA (Li et al., 2011). These results indicate that both mRNA and its gene product are able to participate in the same systemic response. This may be also the case for sbZIP60 protein and sbZIP60 mRNA, which both could function as transposable molecules eliciting systemic UPR signaling. The evidence that bZIP60 is localized at the PD (Figure 4. 13.) supports the possibility that the bZIP60 protein moves systemically, but it also possible that the intercellular translocation of bZIP60 mRNA leads to translation of an active transcription factor in systemic tissues. 172 bZIP28 is another master UPR modulator contributing to the activation of ER stress response genes, including BiP3, ERDJ3A and TIN1 in the plant UPR (Ruberti et al., 2018; Song et al., 2015). While the results obtained using bZIP60 as transgene in the bzip28/60 background provide evidence for a role of bZIP60 in systemic UPR signaling (Figures 4. 1., 4. 2.), the presence of bZIP28 in the wild-type background may facilitate to some extent the expression of BiP3 in the wild-type shoot of seedlings challenged with Tm at the root on the split-plate system (Figure 4. 3. B). The bZIP28 transcripts are transposable between cells, at least in conditions different from ER stress(Thieme et al., 2015). Therefore, we do not exclude the possibility that bZIP28 mRNA or its gene product may be involved in the systemic UPR regulation in parallel to the bZIP60 arm. Indeed, the lack of induction of UPR transcripts in the scions of Col- 0/bzip28/60 hetero-grafts (Figure 4. 12.) exposed to Tm at the roots argues that the systemic UPR signaling relies on the canonical UPR arms. Therefore together, our results indicate that the systemic UPR signaling can occur in conditions of overexpression of a UPR transcription factor as demonstrated in metazoans; however, they also support that it occurs endogenously in plants via the canonical UPR arms. A Plasmodesma-Regulated Symplastic Transport Contributes to Long-distance UPR Signaling In plants, cell-to-cell communication takes place through three main pathways: (i) apoplast-driven transport between cells within the same tissue or different tissues over long distance via the continuum of the cell wall; (ii) symplastic-driven cytoplasmic transport between different cells within the same tissue or among tissues connected by PD; and (iii) vascular-driven 173 transport between different groups of cells or tissues utilizing conducting system composed of phloem and xylem(Gilroy et al., 2014). In our work, using subcellular localization analyses and transcriptional analyses, we have found sbZIP60 protein and sbZIP60 mRNA in ectopic tissues, respectively (Figures 4. 1.-5.); we also found that the sbZIP60 protein can associate with PD and enter the nucleus (Figure 4. 13.), implying that the movement of bZIP60 in systemic signaling may occur through PD. A requirement for PD availability for long-distance UPR signaling is supported by the evidence that in a conditional PD mutant the systemic UPR is attenuated when PD closure is induced (Figure 4. 5.). Although we have verified the presence of sbZIP60 protein at the PD, the sbZIP60 mRNA may translocate through the PD, as it occurs for the mRNA of other proteins(Li et al., 2011). Additionally, the reported existence of unspliced bZIP60 (unbZIP60) mRNA and likely colocalization with ER-associated unbZIP60 protein (Parra-Rojas et al., 2015) does not exclude a potential mobility of unbZIP60 mRNA for involvement in the systemic UPR through PD where a modified ER is present (Ghoshroy et al., 1997). The visualization of sbZIP60 protein at the PD may be facilitated by expression of sbZIP60 by the CaMV 35S promoter; however, PD protein targeting is a highly specific process (Crawford & Zambryski, 2001). Therefore, the verified subcellular localization of sbZIP60 at PD is unlikely a result of overexpression. This is further supported by a relatively lower frequency of localization of cytosolic YFP at the PD in the same experimental conditions (Figure 4. 13.). It has been demonstrated in plants that some systemic response regulators that target PD do not act directly on systemic response effectors (Ishikawa et al., 2017; Yu et al., 2013). The evidence provided in our work that transcellularly translocated sbZIP60 protein can induce the activity of the promoter of a target gene (Figure 4. 1.) indicates that the systemic movement of sbZIP60 is functional in evoking UPR gene expression. 174 Long-Distance ER Stress Signaling may Aid Stress Anticipation To deal with biotic stress like pathogen attack, plants induce SAR by which the signals generated from infected sites are translocated to distal tissues priming the defense response and eliciting immunity for subsequent infections (Fu & Dong, 2013). Our results show that sbZIP60 can traffic across cells. We propose that similar to SAR, sbZIP60 participates in long-distance stress signaling to modulate the UPR in cells placed distally from the site where ER stress occurs. In nature, ER stress is caused by a variety of challenges including pathogens, heat and salt (Deng et al., 2011; Liu et al., 2007b; Moreno et al., 2012). It is possible that the long-distance signaling of ER stress from challenged tissues helps cells anticipate incoming stress to yet-unchallenged tissues. For example, the UPR is required for plant defense response by modulating secretion of antimicrobial proteins (Wang et al., 2005). Therefore, a systemic signaling of ER stress may prepare cells of systemic tissues for responding to a potentially-upcoming ER stress by inducing the accumulation of transcripts of ER stress-attenuating proteins. In our work, we have verified significant levels of sbZIP60 and BiP3 transcript accumulation in the shoot when Tm was applied to the root; conversely, when applied to the shoot, Tm induced sbZIP60 and BiP3 transcript accumulation in the roots to much lower levels. Based on these results, we propose that a root-originated transcriptional signal may operate mainly in a shoot-ward direction for ER stress signaling. We do not exclude the possibility however that a shoot-originated signal that moves in a root-ward direction may also exist. If such a signal were in place it would operate to lower levels than the root-originated signal, which would prevent its detection by qRT-PCR. 175 Long-distance stress signaling in plants is mediated by a number of inducers. For example, several chemical inducers including salicylic acid (SA), pipecolic acid (Pip), glycerol- 3-phosphate (G3P), dehydroabietinal (DA), the free radicals nitric oxide (NO) and reactive oxygen species (ROS) have been identified facilitating SAR (Yu et al., 2013). The role of these inducers in long-distance ER stress signaling is yet to be evaluated and it is conceivable that long-distance ER stress signaling may overlay a bZIP60-mediated signaling with the action of other stress transducers. Nonetheless, it has been shown in seedlings that ER stress responses are independent from endogenous SA and that Tm does not induce accumulation of SA (Lai et al., 2018). Therefore, long-distance ER stress signaling is likely independent from SA. The evidence that micro-grafts with a bzip28/60 rootstock fail to evoke UPR signaling in a Col-0 scion (Figure 4. 12.) further supports that the systemic UPR signaling mainly relies on the canonical UPR arms. Our findings address the long-standing question – whether plant UPR constitutes an endogenous systemic signal. From our results, we conclude that this is the case. The identification of bZIP60 as a component of the long-distance UPR signal transduction is a significant step forward in the understanding of the mechanisms underlying systemic signaling transduction of ER stress responses in intact organisms. 176 ACKNOWLEDGEMENTS We thank the following colleagues: Dr. P. Benfey for pSHR-GFP and pSHR-SHR-GFP seeds; Dr. A. Orellana for the seeds of pbZIP60-GFP-bZIP60; Dr. K. Gallagher for the seeds of pMDC7-icalsm; Dr. L. Chen in the Mass spectrometry and Metabolomics Core Facility at Michigan State University for LC-MS analysis. This work was supported by primarily by the National Institutes of Health (GM101038) with contributing support from 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), DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494), National Science Foundation (MCB 1714561) and AgBioResearch. 177 APPENDIX 178 Table 4. 1. Primers used in this study Primer name UBQ10 For Sequence 5'-GGCCTTGTATAATCCCTGATGAATAAG-3' UBQ10 Rev 5'-AAAGAGATAACAGGAACGGAAACATAGT-3' ACT8 For 5'-TCAGCACTTTCCAGCAGATG-3' ACT8 Rev 5'-ATGCCTGGACCTGCTTCAT-3' IPP2 For 5'-GTATGAGTTGCTTCTCCAGCAAAG-3' IPP2 For 5'-GAGGATGGCTGCAACAAGTGT-3' bZIP60us_FWD 5'-GGAGACGATGATGCTGTGGCT-3' bZIP60s_REV 5'-CAGGGAACCCAACAGCAGACT-3' BiP3 For1 5'-CGAAACGTCTGATTGGAAGAA-3' BiP3 Rev1 5'-GGCTTCCCATCTTTGTTCAC-3' bZIP28 For1 5'-CGTCATCAGTCTCCAGCATTTC-3' bZIP28 Rev1 5'-CTTGCCGTGGGTAGTGACATT-3' GLY T For 5'-TCCGACGTTGAGACCACAGG-3' GLY T Rev 5'-GCCACGACAGGTTTCCCACA-3' pSHR F1 5'- ATGTTTTGAAAATTAGTCTGGATCTGAAATTCTTTAATT AGC-3' pSHR R1 5'-AAATTCCTCCGCCATTGAATAGAAGAAAGGGA-3' sbZIP60 F1 5'-TCCCTTTCTTCTATTCAATGGCGGAGGAATTT-3' Purpose qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR Promoter amplification for construct in pSHR- sbZIP60-GFP line Promoter amplification for construct in pSHR- sbZIP60-GFP line CDS amplification for construct in pSHR- sbZIP60-GFP line 179 Table 4. 1. (cont’d) bZIP60 R4 5'-ACGCCGCAAGGGTTAAGATTTGGTAT-3' BiP3 pro F1 5'-TACAATTACATTATTCACGCTG-3' BiP3 pro R1 5'-TAGTTTATTTGGAAGAGTATGAAGTTC-3' CDS amplification for construct in pSHR- sbZIP60-GFP line Promoter amplification for construct in pBiP3-GUS line Promoter amplification for construct in pBiP3-GUS line 180 Figure 4. 1. Intercellular translocation of sbZIP60 induces BiP3 expression in systemic tissues (A) Confocal laser scanning microscopy of Col-0; pSHR-GFP, shr; pSHR-SHR-GFP and bzip28/60; pSHR-sbZIP60-GFP at the primary root tips of 5-day-old transgenics reveals stele (St) accumulation of GFP, and stele and endodermis (En) distribution of SHR-GFP; noticeably, sbZIP60-GFP is localized in the stele, endodermis, cortex (Co) and epidermis (Ep). Similarly to SHR-GFP, sbZIP60-GFP is localized in nuclei (arrows). As also reported earlier(Kimberly L. Gallagher et al., 2004), we did not find SHR-GFP localization in the nuclei of the cortex and epidermis. Propidium iodide (PI) was used for counterstaining. Scale bar: 50m. (B) Expression of pBIP3:GUS in bzip28/60, Col-0 and bzip28/60;pSHR-sbZIP60-GFP seedlings grown vertically on half LS agar medium for 11 days. X-Gluc was used for histochemical staining to monitor GUS activity. Scale bar: 100 m. 181 (C) Longitudinal confocal optical sections of the regions along the primary root shown in (B). Epidermis: Ep; Co: cortex; En: endodermis; St: stele. The indications upper, middle and lower refer to the a, b and c zones indicated in panel B. Scale bar: 20 m. 182 Figure 4. 2. Root-expressed sbZIP60 transcripts are translocated to the shoot (A, B) Quantitative RT-PCR analyses of sbZIP60 (A) and BiP3 (B) in 14-day-old wild-type (Col-0), bzip28/60, and bzip28/60; pRoot::sbZIP60 seedlings treated with DMSO or 0.5µM Tm at the root in the shoot-root split system for 24 hrs. Transcription of UBQ10 was used as internal control. Error bars represent SEM among three biological replicates. Data significantly different from the corresponding control are indicated by asterisks (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Unpaired t-test). Black asterisks designate differences between bzip28/60 and bzip28/60; pRoot::sbZIP60 lines under Tm non-treatment condition. Red asterisks designate differences between bzip28/60 and bzip28/60; pRoot::sbZIP60 under Tm treatment condition. 183 Figure 4. 3. Local induction of ER stress ignites the UPR systemically, mostly in a shoot- ward direction (A) Diagrams illustrating the Arabidopsis shoot-root split culture system in which the shoot and root of an intact seedling are exposed to separate growth media with different chemical conditions: mock DMSO (D) or 0.5µM Tm (T). T/D denotes shoot on Tm-containing medium and root on DMSO-containing medium; conversely, D/T denotes shoot on DMSO-containing medium and root on Tm-containing medium. (B) Quantitative RT-PCR analyses of UPR markers in 14-day-old wild-type seedlings treated with DMSO or 0.5M Tm for 24 hrs as described in (A). Values are presented relative to non- treated control (0 hr), which was set to 1. Transcription of UBQ10 was used as internal control. Error bars represent SEM among three biological replicates. Data significantly different from the corresponding control are indicated by asterisks (*P < 0.05, ****P<0.0001, NS, non significant; Unpaired t-test). 184 (C) Quantitative HPLC/MS analyses of Tm content in shoot and root of seedlings after treatments as described in (A) in a shoot-root split culture system. The numbers over the histograms express ng/g fresh weight (F.W.). Data significantly different from the corresponding control are indicated by asterisks (**P < 0.01, NS, non significant; Unpaired t-test). 185 Figure 4. 4. Reciprocal micro-grafting analyses with UPR-able and UPR-deficient backgrounds support the existence of endogenous systemic signals in the plant UPR 186 (A, B, C) Plots of the mean values (dots) of the quantitative RT-PCR analyses of the transcripts of sbZIP60 (A) BiP3 (B) and bZIP28 (C) in scion and rootstock of 4-week-old grafted seedlings of wild-type and bzip28/60 backgrounds treated with DMSO or 0.5M Tm for 48 hrs as described in Figure 3A. The average of all the values is indicated by a black line. Error bars represent mean with 95% confidence interval among all grafts. Error bars above and below indicate the 95th and 5th percentiles. Average values significantly different from the values of wild-type (Col-0) and bzip28/60 self-grafts are indicated by red asterisks (*P < 0.05, **P<0.01, ***P<0.001,****P<0.0001, NS, non significant; Mann-Whitney test). n, total number of grafted unions. 187 Figure 4. 5. Long-distance of UPR signaling relies on the PD availability (A, B) Quantitative RT-PCR analyses of sbZIP60 and BiP3 in 14-day-old wild-type (Col-0) (A) and a conditional PD block mutant (pMDC7-icals3m) (B) seedlings treated with DMSO (Mock),10M 17--estradiol (estrogen), 0.5µM Tm, and 10M 17--estradiol (estrogen) in combination with 0.5µM Tm at the root in the shoot-root split system for 24 hrs. Transcription of UBQ10 was used as internal control. Error bars represent SEM among three biological replicates. Data significantly different from the corresponding control are indicated by asterisks (*P<0.05, NS, non significant; Unpaired t-test). 188 Figure 4. 6. Subcellular localization of GFP-bZIP60 in the root under physiological conditions and induced ER stress conditions. Three-dimensional rendering of confocal optical sections of the root tips of 7-day-old Arabidopsis bzip60; pbZIP60-GFP-bZIP60(Parra-Rojas et al., 2015) seedlings treated with DMSO or 0.5M Tm for 24 hrs. 189 Figure 4. 7. Stele-expressed sbZIP60 distributes throughout the root Confocal laser scanning microscopy analyses of 5-day-old bzip28/60; pSHR-sbZIP60-GFP throughout the primary root, including division zone (DVZ), elongation zone (EZ), lower differentiation zone (LDZ) and upper differentiation zone (UDZ), reveals a subcellular localization of sbZIP60-GFP in the nuclei of stele (St), endodermis (En), cortex (Co) and epidermis (Ep). Scale bar: 50 m. 190 Figure 4. 8. ER stress-induced BiP3 expression occurs throughout the root (A) Expression of pBIP3:GUS in 11-day-old Col-0 seedlings treated with 0.5M Tm for 24 hrs. X-Gluc was used for histochemical staining to monitor GUS activity. Scale bar: 100 m. (B) Longitudinal confocal optical sections of the regions along the primary root shown in (A). Ep: epidermis; Co: cortex; St: stele. The indications upper, middle and lower refer to the a, b and c zones indicated in panel A. Scale bar: 50 m. 191 Figure 4. 9. pRoot drives expression of GLYT specifically in the root qRT-PCR analyses of GLYT expression in shoot and root of 14-day-old wild-type (Col-0) seedlings after treatment with DMSO (-) or 0.5M Tm (+) for 24 hrs. UBQ10, ACT8 and IPP2 were used as the internal controls. 192 Figure 4. 10. Distribution of Tm in bzip28/60 Quantitative HPLC/MS analyses of Tm content in shoot and root of bzip28/60 seedlings after treatment in shoot-root split culture system (Kronzucker et al., 2015) with 0.5M Tm or DMSO (Tm control) for 24 hrs. In this system, intact seedlings are laid over Petri dishes that are subdivided by a sealed plate divider. Each dish sub-compartment contains growth medium. The shoot and root portions of intact seedlings are placed across the plate divider and are therefore exposed to the medium contained in each plate sub-compartment separately. D/D denotes both shoot and root on DMSO-containing media; T/T denotes both shoot and root on Tm-containing media; T/D denotes shoot on Tm-containing medium and root on DMSO-containing medium; D/T denotes shoot on DMSO-containing medium and root on Tm-containing medium. The numbers over the histograms express ng/g fresh weight (F.W.). Data significantly different from the corresponding control are indicated (*P < 0.05, NS, non significant; Unpaired t-test). 193 Figure 4. 11. Transcriptomic kinetic response of systemic UPR signaling (A, B, C) Quantitative RT-PCR analyses of unbZIP60 (A), sbZIP60 (B) and BiP3 (C) in 14-day- old wild-type seedlings treated with DMSO or 0.5M Tm at roots only for the indicated times in the shoot-root split culture system. Values are presented relative to non-treated control (0 hr), which was set to 1. Error bars represent s.e.m among three biological replicates. Data significantly different from the corresponding control (0 hr) are indicated by asterisks (*P < 0.05, **P<0.01, ***P<0.001, ****P<0.0001; Unpaired t-test). 194 Figure 4. 12. Assessment of endogenous systemic transcripts in the plant UPR 195 (A, B, C) Plots of the mean values (dots) of the quantitative RT-PCR analyses of the transcripts of sbZIP60 (A) BiP3 (B) and bZIP28 (C) in scion and rootstock of 4-week-old grafted seedlings of wild-type and bzip28/60 backgrounds treated with DMSO or 0.5M Tm for 48 hrs as described in Figure 3A. The average of all the values is indicated by a black line. Error bars represent mean with 95% confidence interval among all grafts. Error bars above and below indicate the 95th and 5th percentiles. Average values significantly different from the values of wild-type (Col-0) and bzip28/60 self-grafts are indicated by red asterisks (**P<0.01, ****P<0.0001, NS, non significant; Mann-Whitney test). n, total number of grafted unions. 196 Figure 4. 13. PD-associated bZIP60 is a mobile transcription factor moving intercellularly (A) Subcellular distribution of cytosolic YFP (cYFP, cytosolic control) and YFP-sbZIP60 in N. tabacum leaf epidermal cells co-expressing the PD marker PDLP1-CFP. Similar to PDLP1-CFP, cYFP was distributed uniformly in the cytosol but PDLP1-CFP also accumulated at PD (arrows). The subcellular localization of YFP-sbZIP60 overlays that of PDLP1-CFP but YFP-sbZIP60 also localized to the nucleus (arrowhead). Scale bar: 10 µm. (B) Quantification analyses of PD visualized by AB with either cYFP or YFP-sbZIP60. The graph represents the extent of overlay between AB at PD and YFP fusion proteins obtained by calculating the Pearson correlation coefficient (PCC). 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Maintenance of ER homeostasis is critical not only for multi-stages of physiological growth, but it is also an adaptive strategy to changing conditions in an ever-changing environment, such as heat, salinity, and pathogenic infections. Known sensors perceive and respond to ER stress, but how their signaling is modulated is largely unknown, especially in plants. Because the UPR machinery is highly conserved among eukaryotes, functional identification of novel UPR transducers, and mechanistic study of ER stress-signal transduction in model plants, can enable investigators to manipulate valuable traits. Manipulation of these traits can lead to development of stress- resistant crops and medicines to treat illnesses caused by a dysregulated UPR response, including neurogenerative and malignant diseases. RHD3 in ER stress signaling RHD3 has been reported to have a role in the continuous ER network mediating the dynamic transition from a cisternal to a tubule structure during cell membrane expansion (Hu et al., 2009). RHD3 possesses GTPase activity functioning in membrane fusion in ER tubulation (M. Zhang et al., 2013). Subsequent research by Maneta-Peyret L. et al., 2014 measured phospholipid compositions in rhd3 mutants to study RHD3-correlated alteration of ER lipid bilayers. They identified abnormal increases in total amount of ER phospholipids and ER proteins in rhd3 mutants. These abnormal increases are potentially caused by the dysfunction of PIS1, which is responsible for synthesis of phosphatidylinositol (PI) and phosphatidic acid. This indicates that RHD3 may conduct PIS1-mediated ER membrane expansion. 206 Our study presented in Chapter II established the connection between ER structure and the ability to trigger an ER stress response. This delineated the novel role of RHD3 in UPR signaling through mediating the IRE1/bZIP60 UPR arm. The lack of mechanistic evidence of how RHD3 modulates RNase activity of IRE1 leads to another open question for the further investigation. The involvement of RHD3/PIS1 in ER membrane enlargement, and peripheral ER membrane expansion upon ER stress identified in yeast (Schuck, Prinz, Thorn, Voss, & Walter, 2009), suggests the possibility that RHD3 acts on UPR signaling by mediating a proper ER membrane expansion. Due to activation of IRE1 leans on its oligomerization and clustering at the ER membrane (H. Li, Korennykh, Behrman, & Walter, 2010; L. Zhang, Zhang, & Wang, 2016), that may be affected by the ER membrane expansion as well as the abundance of IRE1, maintains homeostasis under ER stress (Lai et al., 2014). Therefore, it is possible that RHD3/PIS1 modulates IRE1 splicing activity by achieving a proper level of ER membrane volume during ER stress. An experiment to visualize IRE1 clustering in the rhd3 mutant under ER stress can be performed by fluorescence microscopy to evaluate the function of RHD3 on IRE1 activation. To test this hypothesis, the approach used in mammalian cell lines as described in Han Li et al., 2010 (H. Li et al., 2010), in which they determined the dynamic localization and clustering of IRE1-3F6HGFP at discrete foci in the ER membrane during ER stress, could be adopted in plants. We cannot exclude that the RHD3-mediated proper ER membrane expansion may also facilitate the critical interaction between RHD3 and UPR transducers, hence regulating activity of UPR modulators. It has been shown that the seed-specific isoform of RHD3, RHD3-LIKE 2 (RL2), locates to the ER membrane and physically interacts with reticulon RTNLB13, acting together in the tubular ER shaping process (Lee et al., 2013). This is further supported by the 207 finding that RL2-RFP coimmunoprecipitated with RTNLB13-myc (Lee et al., 2013). An alternative hypothesis is that RHD3 mediates UPR signaling through networking with other factors to promote cellular response upon stress. To test this hypothesis, a protein-protein interaction experiment between RHD3 and IRE1 or bZIP60 under ER stress conditions can be performed to validate their physical interaction at the ER membrane or the test of the interaction by direct yeast-two-hybrid using cytoplasmic domains of RHD3 and IRE1/bZIP60, additionally coimmunoprecipitation assay with co-expression of tagged versions of RHD3 and IRE1/bZIP60 in transient tobacco plants could be performed. Taken together, these approaches can help to establish the underlying mechanism of RHD3-regulated UPR signaling. Post-translational modification such as phosphorylation often plays a role in regulating protein activity or protein-protein interaction. Also, phosphorylation events are involved in UPR signaling. For instance, the JNK/ASK phosphorylation cascade is well-identified in the ER stress-induced cell death process (Urano et al., 2000). A recent study revealed that C-terminal phosphorylation of RHD3 is required for its homotypic interaction, further enhancing fusion of ER tubule membranes (Ueda et al., 2016). Therefore, the linkage between RHD3 phosphorylation and UPR signaling can be further explored. An experiment to evaluate the effects of RHD3 phosphorylation under ER stress can be conducted in which bZIP60 splicing can be detected under ER stress using rhd3 transgenic mutants that express modified RHD3 lacking a C-terminal Ser cluster. This modified RHD3 has been shown to fail to enhance formation of ER tubules (Ueda et al., 2016). Taken together, the approaches discussed above can help uncover the underlying mechanisms of RHD3-mediated UPR signaling. In the future, it could potentially contribute to 208 engineering crops that are more resistant to abiotic stress, that would be extremely valuable economically. The multiple roles of NPR1 in biotic and abiotic stress In past decades, NPR1 has been studied intensively and identified to have multiple roles in the plant immunity response. SA-mediated signaling through NPR1 is required for triggering systemic acquired resistance (SAR). In turn, SAR initiates the broad-spectrum defense response throughout the plant by producing SA-responsive proteins. One such protein is PR1 antimicrobial protein, which protects against second infections (Withers & Dong, 2016). NPR1 is also involved in the induced systemic resistance (ISR) caused by the colonization of P. fluorescens, which is responsive to JA and ethylene instead of SA (Pieterse et al., 1998). Primarily, NPR1 functions as a positive transcription cofactor to reprogram the transcriptome in SAR and ISR. However, it negatively works on the effector-triggered immunity (ETI), which usually results in programmed cell death (PCD) at the infection site (Senthil-Kumar & Mysore, 2013). Therefore, NPR1 is a determinant for deciding the life or death of plant cells in ETI. A great deal has been discovered about SA/NPR1 signaling during SAR, but some questions remain. In response to SA content, NPR1 forms either repressive or active complexes with distinct transcription factors to modulate the expression of the PR1 gene. For example, NPR1/TGA2/NIMIN1 repressive complex in combination with TOPLESS and CBNAC/SSN1 co-repressors suppress PR1 transcription in the absence of SA (Boyle et al., 2009; Rochon et al., 2006). Conversely, in the presence of SA, the complexes NPR1/TGA2/TGA3 and NPR1/TGA7/SSN2 promote the expression of PR1 (Johnson, Boden, & Arias, 2003), 209 suggesting dual activity of NPR1 on transcriptional reprogramming depending on the SA concentration and interacting proteins. Our discoveries presented in Chapter III demonstrated a negative role of NPR1 in the attenuation of UPR gene activation by forming a complex with UPR transcription factors, bZIP28 and bZIP60, during ER stress. This leads to the interesting question of how such SA- independent repression can be achieved. Two plausible models are proposed: (1) NPR1 forms a repressive complex with bZIP28 and bZIP60 targeting to the promoter region of UPR genes and (2) NPR1 forms the complex with bZIP28 and bZIP60 to block their recruitment to the promoters of UPR genes. To test the predictions of the first model, the degree of promoter enrichments of UPR genes can be evaluated by conducting a chromatin immunoprecipitation (ChIP)-qPCR assay using either npr1 or oxnpr1 transgenic plants overexpressing a tagged version of bZIP28 and bZIP60 individually. A similar approach has been adopted before to estimate the effects of mutated bZIP28 and bZIP60 on the ER stress-induced H3K4 histone trimethylation at promoters of UPR genes (Song et al., 2015). If the enrichment level remains consistent in all conditions, that supports the possibility of repressive complex formation in the first model, and vice versa. During SA-mediated defense signaling, NPR1 acts together with TBF1 transcription factor activating the expression of UPR genes enhancing the secretory machinery (Pajerowska- Mukhtar et al., 2012; Wang, 2005). Given the dual roles of NPR1 as a positive or negative regulator in distinct stress responses, this suggests the likelihood of the presence of stress- specific yet-unidentified NPR1 interacting factors. An intriguing question is whether there is a SA-specific or ER stress-specific factor to switch the opposite activity of NPR1 in UPR signaling. To answer this question, an in vivo pull-down assay can be performed using nuclei- 210 enriched fractions extracted from transgenic plants that express a tagged form of NPR1 in the conditions of ER stress or SA treatment. Our study uncovered that, in addition to its well-known role in plant immunity, NPR1 plays a novel role in ER stress signaling, suggesting that plants may re-allocate such sharing signaling molecules to cope with different environmental stresses. Our research also provides new insights into the potential of engineering enhanced stress tolerance into crops. This could be particularly important in the face of global climate change, because many regions are experiencing increased temperatures, and some are experiencing increased draught. When attempting to engineer NPR1 to generate stress tolerant crops, it will be important to take the expression level and spatiotemporal expression into consideration. For instance, a promoter titration test may be possible to determine the ideal expression level of NPR1 that is suitable for maintaining the balance while dealing with ER stress and pathogen stress. In summary, the deeper understanding of the underlying mechanism of NPR1-mediated cross-regulation in response to abiotic and biotic stress sheds light on an innovative way to engineer crops to be more tolerant to multiple stresses. The systemic UPR response in plants and animals Cell non-autonomous UPR signaling was first identified in metazoans in C. elegans. It was discovered that the neuronally-derived constitutive expression of Xbp1s is able to rescue the aging-induced loss of cellular ER proteostasis and activate the UPR in distal non-neuronal cells. In addition, a neurotransmitter has been identified as a secreted ER stress signal (SERSS) functioning downstream of XBP1s to promote organismal longevity with enhanced ER stress tolerance (Taylor & Dillin, 2016). This suggests the physiological role of systemic UPR in the 211 aging stress response. Subsequently, the systemic effects of Xbp1s on whole-body energy balance, body-weight homeostasis, and blood glucose metabolism have been established in a mice model in which Xbp1s serves as a cell non-autonomous feeding sensor. The Pomc- dependent activation of Xbp1s and Xbp1s downstream target genes in the liver is sufficient to provide a signal of a fed state in a cell non-autonomous manner. This may contribute to the maintenance of liver metabolic homeostasis. Furthermore, the dysregulation of such systemic UPR results in insulin and leptin resistant type II diabetes (Yanes & Reckelhoff, 2014). Collectively, the discovery of systemic UPR regulation in response to physiological stress conditions and metabolic equilibrium suggests a new approach to the understanding of degenerative diseases. Our work presented in Chapter IV demonstrated the first instance of cell non- autonomous UPR in plants. We found that the Xbp1s homolog, sbZIP60, is involved in such systemic responses. Additionally, we observed that both mRNA and gene product of sbZIP60 are mobile to remote tissue, contributing to systemic UPR regulation. We propose two possible pathways to further clarify the biological significance of sbZIP60 mRNA and sbZIP60 protein, respectively, in the systemic UPR. The first approach would be to visualize sbZIP60 mRNA trafficking directly. In our research, we demonstrated evidence showing the mobility of sbZIP60 protein by visualizing the movement of GFP tagged-sbZIP60 recombinant between cell layers, and we indirectly examined the mobility of sbZIP60 mRNA by qRT-PCR, detecting the presence of mRNA in remote tissues. In future research, we could visualize sbZIP60 mRNA trafficking directly by adopting the latest advanced RNA imaging tool (Engelhart, 2017) using a fluorogenic sbZIP60-aptamer, sbZIP60-Corn, bound with noncytotoxic dye such as DFHO ligands. The visualization would be done with a red-shift spectrum that can track the movement 212 of sbZIP60 mRNA in living tissues. It is possible that this approach could help establish the dynamic and kinetic profile of sbZIP60 transportation in systemic UPR signaling. The second approach would be an experiment evaluating whether the transposable sbZIP60 mRNA functions independently from sbZIP60 protein contributing to long-distance UPR signaling. This could be examined by expressing ectopically a non-translatable sbZIP60 RNA. For example, a similar approach was employed to characterize the physiological relevance of the systemic movement of Arabidopsis FT mRNA in the context of long-distance florigenic signaling and identify its role in promoting early flowing under SD conditions (Li et al., 2011). Another exciting question that arises from our study is the possible involvement of other signaling molecules in such systemic UPR responses. The identification of actual signaling molecules to achieve SAR in plant immunity have been debated for years. Several molecules other than phytohormone SA have been proposed to serve as SAR mobile signals, such as AzA, NO, PiP, and G3P (Gao, Zhu, Kachroo, & Kachroo, 2015). Therefore, it is reasonable to hypothesize that more than one signal is involved in systemic UPR signaling. Additional signaling molecules can be explored by performing the conventional micro-grafting approach using different mutant backgrounds with defective potential candidates. The XBP1s and its homologue sbZIP60 both play a critical role in systemic UPR regulation individually in animals and plants. This suggests the potential of bZIP60 as a target to engineer enhanced plants, especially crop plants that are tolerant to ER stress. In addition, manipulation of the XBP1s-mounted systemic response is a likely target for developing therapeutics that could treat degenerative-related pathology. 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