Enhanced JAGN1 expression Modulates ER Stress Signaling and Promotes Survival in IRE1-Deficient Arabidopsis

Enhanced JAGN1 expression Modulates ER Stress Signaling and Promotes Survival in IRE1-Deficient Arabidopsis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enhanced JAGN1 expression Modulates ER Stress Signaling and Promotes Survival in IRE1-Deficient Arabidopsis Regina Bedgood, Karolina Pajerowska-Mukhtar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8893802/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Jagunal homolog 1 (JAGN1) is essential for endoplasmic reticulum (ER) organization and function in animals, where mutations cause severe congenital neutropenia in humans. Although JAGN1 function is conserved across animal species, its role in plants has not been examined. Here, we investigated JAGN1 function in Arabidopsis using a transgenic line with elevated JAGN1 expression exposed to ER stress inducers. Increased JAGN1 expression in Inositol Requiring Enzyme 1 (ire1) double knockout plants partially restored survivability under ER stress and enhanced expression of unfolded protein response (UPR) genes, including BiP3 , PDI9 , and SAR1a , indicating an IRE1-independent mechanism. In contrast, crossing the JAGN1 overexpression line with Basic Leucine Zipper 28 ( bZIP28 ) mutants reduced survival and failed to alter downstream gene expression. These results provide the first evidence that JAGN1 function is conserved across plant and animal kingdoms and establish Arabidopsis as a model system to study JAGN1 biology and disease-associated mutations. Biological sciences/Biotechnology Biological sciences/Genetics Biological sciences/Molecular biology Biological sciences/Plant sciences Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The Unfolded Protein Response (UPR) is a cellular level stress response conserved across eukaryotes and is triggered by accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) 1 , 2 . The UPR in plants is triggered by stressors such as pathogen infection, drought, or heat, but is also important for certain developmental stages such as root and reproductive development 3 – 5 . Vascular plants and metazoans share two arms of the UPR –the Inositol Requiring Enzyme 1 ( IRE1 ) pathway and the Basic Leucine Zipper 28/17 ( bZIP28 / bZIP17 ) pathway, which acts as the functional equivalent of the Activating Transcription Factor 6 ( ATF6 ) pathway in metazoans 2 . IRE1 is an ER membrane protein that interacts with Binding Immunoglobulin Protein (BiP) at its luminal domain during homeostasis, keeping it in its inactive state. Under stress, misfolded proteins replace BiP which triggers dimerization and autophosphorylation of IRE1 thereby activating the cytosolic ribonuclease domain. In plants this activity splices bZIP60 mRNA, whereas in metazoans the homologous transcript is XBP1 mRNA. The resulting bZIP60 and XBP1 proteins are translated into functional transcription factors that translocate to the nucleus 2 , 4 . In plants, IRE1 knockouts cause deficits in response to drought, pathogen infection, salt tolerance, and chemically induced ER stress 4 , 6 . The bZIP28/bZIP17 pathway consists of two transcription factors retained at the ER membrane via association with BiP. Accumulation of unfolded or misfolded proteins causes BiP dissociation, allowing bZIP28/17 to relocate to the Golgi where cleavage releases the active transcription factor to the nucleus 2 , 7 . In plants, mutations in bZIP28 cause deficits in response to heat stress and negatively affect root development 5 , 8 . Despite our understanding of the two canonical arms of the UPR, there are ancillary genes involved in the UPR whose contributions or mode of action are not fully understood—one of these genes is called Jagunal Homolog 1. Jagunal was first discovered in Drosophila melanogaster during a screening of lethal mutations in the 3rd chromosome specific for a small egg phenotype, hence the name jagunal - meaning “small egg” in Korean 9 . Drosophila is used as a model to study vesicular transport because there are specific stages of oocyte development involving major ER reorganization and large amounts of exocytic activity to increase the surface area of the plasma membrane, which can be observed with microscopy 9 . Oocytes with a jagn knockout mutation had abnormal ER reorganization evidenced by lack of ER clusters, a decrease of exocytosis of certain proteins, and a decrease in cell surface area, while distribution of Golgi complex was normal suggesting Golgi apparatus function is not effected by JAGN 9 . Somatic cell clone lines with mutated jagn1 had thinner, shorter, bristles and defected surface structure which further supports the idea that JAGN is necessary for proper exocytosis of proteins and endomembrane to the cell membrane 9 . JAGN1 is proposed to play a role in protein sorting between the ER and Golgi. It is compared to two well established families of ER membrane resident proteins important for homeostatic control of protein movement through the endomembrane system, tetraspanins (TSP) and endoplasmic reticulum vesicle (Erv) proteins. TSPs are highly conserved across eukaryotes playing key roles in cellular trafficking of proteins, lipids, miRNA, and motility and fusion of cells 10 , 11 . The N-terminal domain, C-terminal domain, and one short loop expand into the cytoplasm and two larger loops expand into the extracellular space. Conserved regions on non-membrane domains allow for dimerization of TSPs forming tetraspanin-enriched microdomains (TEMs) while less conserved regions allow for specific protein-protein interactions to guide cellular trafficking of endosomes, pathogen identification, and can dictate reactive oxygen species (ROS) generation 10 – 12 . While some Ervs have four transmembrane domains, it is not a defining characteristic of the proteins, they are instead categorized based on function 13 . Ervs aid in anterograde movement of molecules from the ER to Golgi via coatomer protein complex II (COPII) coated vesicles and are recycled back to the ER via coatomer protein complex I (COPI) coated vesicles 11 , 13 . Jagunal Homolog 1 ( JAGN1 ) is a gene encoding an ER membrane resident protein unique to its own family due to its lack of sequence with either TSPs or Ervs. However, it has motifs for interaction with clathrin coats and contains four membrane spanning domains with N and C terminal domains both extending into the cytoplasm, as do TSPs 11 . JAGN1 also interacts with three members of the COPI complex, important for retrograde movement from the Golgi to the ER, coatomer subunit alpha (COPA), coatomer subunit beta 2 (COPB2), and coatomer subunit gamma 2 (COPG2), as do Ervs 11 , 14 . These morphological and functional similarities to Ervs and TSPs, suggest that JAGN1 plays a role in the sorting and trafficking of proteins between the ER and Golgi 11 . It is highly conserved across kingdoms with homologs in animals and plants with amino acid sequences particularly well conserved at the NH2 and COOH terminals of animals suggesting these regions are particularly important for functionality 11 . Since its original discovery in Drosophila, mutations to the homologous gene in humans, called Jagunal Homolog 1 (JAGN1), have been identified to cause a primary immunodeficiency called severe congenital neutropenia (SCN) 14 , 15 . SCN patients struggle with life threatening infections due to a lack of functional neutrophils 16 . Neutrophils are a type of white blood cell that play an important role in the innate immune system via creation and secretion of antimicrobial proteins by the endomembrane system 17 . Neutrophil differentiation is heavily influenced by ER stress and the resulting UPR. HL-60 cells stimulated to become neutrophils and subsequently treated to inhibit the IRE1 or ATF6 pathways exhibited increased apoptosis, indicating that functional UPR is essential for survival of differentiating neutrophils 18 . HL-60 cells stimulated to become neutrophils and treated to block UPR display lower expression of CD11b and decreased segmentation of nuclei, two indicators which would measure successful differentiation into functionally mature neutrophils 18 , suggesting UPR is also essential for their function once differentiated. Likewise, JAGN1 is necessary for differentiation of human neutrophils 14 . Electron microscopy of ER in myeloid progenitor cells, the precursors to neutrophils, showed JAGN1 mutation resulted in an enlarged ER and granules that were severely underdeveloped 14 . Additionally, JAGN1 mutant cells had elevated levels of binding immunoglobulin protein (BiP, also known as Grp78) ,a chaperone protein that plays an important role in the UPR, which is consistent with ER stress 14 . Coimmunoprecipitation experiments showed a protein level interaction between JAGN1 and Grp78/BiP, 19 . Moreover, treatment of neutrophils with the protein kinase inhibitor staurosporine, an inducer of ER stress, lead to increased apoptosis in JAGN1 deficient cells compared to controls 14 , suggesting a role of JAGN1 in ER stress. In insulinoma cells treated with tunicamycin, an inducer of ER stress, expression of JAGN1 mRNA was increased, again, indicating its role in ER stress 20 . In mice plasmablasts, cells with Jagn1 loss showed higher gene transcription, for stress response, apoptosis, protein folding, and UPR 21 , suggesting compensation for the absence of functional JAGN1 . Mice plasmablasts with jagn1 mutations displayed a reduction in immunoglobulin production 21 , a function which is heavily dependent on functional UPR 22 . Additionally, in JAGN1 deficient cells, there was in increase in XBP1 splicing 21 , which is the metazoan equivalent of bZIP60 splicing in plants, both of which are spliced by IRE1 during UPR 2 , suggesting that increased function of the IRE1 pathway is compensating for lack of JAGN1 function. Collectively, this indicates that JAGN1 and the UPR are tightly correlated in animal models. Because of the necessity of JAGN1 for ER organization, for response to ER stress, and the conservation of function from fruit flies to zebrafish to humans, an additional model with a novel experimental approach will increase the broad understanding of JAGN1 function during ER stress. Arabidopsis is an opportune model for understanding the role of JAGN1 in the UPR because unlike in mice 23 and fruit flies 24 , knockout mutations of IRE1 are survivable in plants 6 . Thus, Arabidopsis provides a unique reverse genetics framework to investigate if JAGN1 can compensate for defects in each canonical UPR pathway— IRE1 and bZIP28 , which would not be possible in animal models. Using this approach, our data demonstrate that JAGN1 contributes to the UPR of plants and modulates the effects of ER stress inducing chemicals, particularly in mutants of the UPR pathways. Here, we provide the first experimental evidence of functional conservation of JAGN1 across plants and animals which not only contributes to the broader knowledge of the UPR in plants and to the broader knowledge of JAGN1 function, it also provides a new model to study the role of JAGN1 in basic cellular biology. Results Cross-Kingdom Sequence and Structural Conservation of JAGN1 in Plants and Animals Across plants and animals, all JAGN1 homologs share the characteristic four membrane spanning domains with carboxy and amino terminals facing into the cytoplasm. The predicted aligned error for each protein suggests there is a singular domain (Fig. 1 A-F). While there is considerable amino acid sequence conservation of JAGN1 among animals, i.e. Homo sapiens compared to the following: to Mus musculus has a percent identity of 97.81% and E-value of 6e-136; to Danio rerio (1a) 74.32% with an E-value of 2e-109; to D. rerio (1b) a percent identity of 71.32% with an E-value of 6e-104; to Drosophila melanogaster a percent identity of 30.46% with an E-value of 4e-28, and likewise among plants, i.e. Arabidopsis thaliana compared to the following: to Acer yangbiense a percent identity of 55.49% with an E-value of 8e-60; to Prunus persica a percent identity of 48.26% with an E-value of 93 − 53; and Spinacia oleracea a percent identity of 42.44%, the sequence conservation between A. thaliana and animals is comparatively lower, i.e. A. thaliana compared to the following: to D. melanogaster percent identity of 26.02% and E-value of 8e-08; to Homo sapiens 27.27% with an E-value of 4e-07; to M. musculus a percent identity of 28.57% and an E-value of 5e-07; to Danio rerio (1a) percent identity of 24.73% and E-value of 1e-06; and D. rerio (1b) with a percent identity of 32.56% and an E-value of 1e-05. However, the NH 2 terminus of A. thaliana has a 7 amino acids with very high sequence homology to humans, mice, zebrafish, and fruit flies (Fig. 1 G). Interestingly, this sequence, amino acids 11–17 of the human sequence, is a concentrated region of documented mutations resulting in SCN 11 . Both the sequence conservation and the high concentration of disease causing mutations in this region suggests an important role for this area of the protein and potential functional conservation of this gene into the plant kingdom. Additionally, while the sequence similarity between Arabidopsis and animals is around 25–30%, the structure of JAGN1 between Arabidopsis and animals is noticably similar and is more structurally similar to human JAGN1 than Drosophila. Characterization of JAGN1 T-DNA insertion line To investigate JAGN1 function in Arabidopsis, we queried the SIGnAL (Salk Institute Genomic Analysis Laboratory) T-DNA database, identified a single line for the JAGN1 ortholog AT5G51510 : SALK_065212C and confirmed homozygosity of the T-DNA insertion (Supplemental Fig. 1). The SIGnAL website predicts that the SALK_065212C line has the T-DNA insertion in an intron between the 5th and 6th exon, while the original, not confirmed stock line, SALK_065212, where the confirmed stock line originated, is predicted to have the T-DNA insertion in the 6th exon. Typically, T-DNA insertions in an exon result in a knockdown, knockout, or truncation of the gene, however our RT-qPCR analysis revealed a higher level of JAGN1 expression in the T-DNA insertion line compared to the Columbia-0 wild-type control (Fig. 2 A). This enhaced expression of JAGN1 was checked with three primer combinations and the expression was consistent across all primers (Figure This was corroborated with semi-quantitative PCR to ensure there was a single band, and the amplicons were of the expected size (242 bp for cDNA, 693 for genomic DNA) (Fig. 2 B). Because SIGnAL predicted the insertion to be a homozygous knockout line, this enhanced expression was not expected. To investigate further the reason for this overexpression, Sanger sequencing identified the location of the T-DNA insertion (Supplemental Fig. 2) to be 28 bases into the 3’ UTR (Fig. 2 C). It is important to distinguish this T-DNA insertion line, which exhibits elevated JAGN1 transcript levels, from classical “overexpression lines,” which are typically transgenic plants carrying a strong constitutive promoter such as the cauliflower mosaic virus 35S (CaMV 35S) promoter. Traditionally, such lines are treated as constitutive overexpressors of a gene of interest. However, growing evidence indicates that gene expression driven by the CaMV 35S promoter can vary substantially with photoperiod, abiotic stress, temperature, tissue type, and developmental stage 25 , 26 . Thus, even in a classical overexpressor line there can still be variability in the extent of overexpression depending on the age of the plant, tissue type of interest, and experimental conditions. Although the T-DNA insertion line used here is not driven by an introduced promoter, it shows increased JAGN1 transcript abundance relative to wild type. For clarity, this 3′-UTR T-DNA insertion line will therefore be referred to as a JAGN1 overexpression line ( jagn1-OX ). This line will be used to test whether elevated JAGN1 transcript levels enhance plant tolerance to ER stress. Elevated JAGN1 expression does not enhance disease resistance to pathogen infection but does increase plant growth under Tunicamycin stress Because animal lines with JAGN1 mutations show strong changes in immune phenotypes, we first set out to determine if increased expression of JAGN1 causes a phenotypical difference in pathogen infection response, 4-week-old leaves were infiltrated with Pseudomonas syringae pv. tomato DC3000 at a concentration of 0.001 OD 600 . After three days of pathogen growth, leaf tissue was collected and homogenized, and number of bacterial colonies per leaf disc was measured. Additionally, to test if JAGN1 overexpression changes cell death in response to pathogens, 4-week-old leaves were infiltrated with Pseudomonas syringae pv. maculicola ES4326 at a concentration of 0.1 OD 600 and cell death was measured as percentage of ions leaked from leaves over 8 hours. For both experiments, there was qualitative heightened resistance to pathogen infection in jagn1-OX compared to Col-0 , evidenced by the lower mean concentration of bacterial growth (Fig. 3 A), the lower amount of chlorosis in infected leaves (Fig. 3 B), and the lower average conductivity (Fig. 3 C) but not drastic enough for statistical significance. Based on this result and because pathogen infection is a broad inducer of ER stress, we assessed whether a more targeted approach to induce ER stress via the chemical inducer, Tunicamycin (TM), would potentiate the effects of JAGN1 overexpression. Thus, we tested if overexpression of JAGN1 would result in a phenotype rescue under sustained, targeted ER stress by subjecting 5-day old seedlings to two concentrations of Tunicamycin for 4 days (Fig. 3 D). TM is a naturally occurring antibiotic which interrupts Asparagine-linked, also known as, N-linked glycosylation of membrane proteins, resulting in unfolded proteins accumulating in the ER, leading to ER stress 27 , 28 . Protein glycosylation is a covalent post translational modification that occurs in the ER, and increases the specificity of proteins by tagging them for certain fates within the cell 29 . Importantly for this assay, glycosylated proteins display higher solubility, lower degradation via proteases, and increased stability all due to the large hydrophilic nature of glycans 30 – 32 . Thus, by blocking N-linked glycosylation, therefore destabilizing proteins in the ER, TM serves as a pointed chemical inducer of ER stress. Fresh seedling weight was measured on day 9 of growth. Across the three genotypes, there was no weight difference in the control condition, indicating no phenotype change in plant weight under homeostatic conditions. The lower mean seedling weight of all three genotypes when exposed to TM compared to control shows both concentrations of TM were high enough to induce ER stress which negatively affected seedling growth (Fig. 3 D). An IRE1 double mutant in two isoforms of the gene, called ire1a-2/ire1b-4 was used as negative control for a proper ER stress response. This double mutant of IRE1 has been previously established to be an expressional knockout of both isoforms 6 . For convenience, this mutant will be referred to as a2b4 moving forward. At both TM concentrations, there was a lower mean seedling weight of a2b4 compared to Col-0 indicating that nonfunctional UPR in the presence of TM due to lack of functional IRE1 causes increased stress to the seedling which compounded the negative effect on growth seen in TM treated Col-0 . At 0.75 µg/mL of TM, there was a substantially higher mean weight of the jagn1-OX genotype compared to Col-0 , which indicated that higher transcript levels of JAGN1 increased seedlings’ capacity to withstand ER stress (Fig. 3 D). There are two arms of the UPR in plants and the observation that seedlings overexpressing JAGN1 are more capable of withstanding ER stress suggests that JAGN1 may participate in or act parallel to the two canonical arms of the UPR. IRE1 double knockout survival in the presence of ER stressors is partially rescued when crossed with JAGN1 overexpression mutant To further elucidate the role of JAGN1 during ER stress and to place its function into one of the two arms of the UPR, jagn1-OX was crossed into a2b4 , the mutant deficient in the IRE1 pathway, and bzip28 , a mutant deficient in the bZIP28 pathway, yielding jagn1-OX/a2b4 and jagn1-OX/bzip28 , respectively. While bZIP17 and bZIP28 have overlapping functions for cell growth, bZIP28 is the primary player of the UPR 5 , and is used here as the representative gene of that arm of the UPR. Importantly, this approach also accounts for the possibility of an off-site T-DNA insertion in the original jagn1-OX line affecting the phenotypes seen in Fig. 3 . By crossing jagn1-OX into two other Col-0 backgrounds, a2b4 and bzip28 , we have effectively eliminated that risk and can assume the phenotypes observed are based on the enhanced expression of JAGN1 . To examine if the increased capacity of jagn1-OX to withstand ER stress can be replicated and reach significance with other elicitors of ER stress, survivability assays of multiple ER stressors were performed using the new crossed genotypes (Fig. 4 ). To allow for a higher n number and therefore higher power, a survivability assay of germination of individual seedlings was used in place of pooled samples of weighted plants to observe changes in growth during prolonged ER stress. For all elicitors, 7 plates were grown for each condition (treatment and control) with each plate containing 30 seeds of each genotype ( Col-0, a2b4, bzip28, jagn1-OX, jagn1-OX/a2b4, and jagn1-OX/bzip28 ). Seedlings that germinated successfully and on day 10 had grown to the stage of at least 2 green cotyledons and displayed primary root growth were survivors. Seeds that failed to break dormancy or attempted germination but had severe chlorosis and/or lack of primary root growth on day 10 were non-survivors. In addition to TM, two more chemical inducers of ER stress, Monensin (Mon) and 2-Deoxyglucose (2DG) were used. Mon induces ER stress by allowing leakage of hydrogen protons from the Golgi apparatus altering the necessary relatively low pH, and allows Na + to leak inside leading to swelling of the Golgi 33 , 34 . The increased pH of the Golgi prevents proper glycosylation, disrupts protein and lipid trafficking and causes loss of organelle organization 35 . While the transport of proteins in the Golgi is inhibited by Mon, it does not affect protein synthesis 36 . When used to stress Arabidopsis, Mon has been shown to reduce germination and induce expression of bZIP60s 37 . 2-DG is an analog of glucose with a hydrogen in place of a hydroxyl group on the second carbon and induces ER stress due to its disruption of glycosylation of proteins. 2-DG is an analog of D-mannose which is a key player in glycosylation, therefore in the presence of 2-DG, there is abnormal N-linked glycosylation of proteins resulting in ER stress 38 , 39 . Across all genotypes, there was no observable phenotype difference in control conditions (Fig. 4 ). As expected, TM, Mon, 2-DG, and heat, all direct ER stressors, showed a severe decrease in germination for the a2b4 mutant compared to Col-0 (Fig. 4 A-D). In this assay, the jagn1-OX line did not show an increased ability to withstand ER stress from any of the elicitors when compared to Col-0 . However, the jagn1-OX/a2b4 cross showed a phenotype rescue compared to a2b4 for TM, Mon, heat, and PQ (Fig. 4 A-B, D-E). Treatment with 2-DG resulted in no change in phenotype for jagn1-OX crossed into a2b4 or bzip28 compared to the corresponding mutant (Fig. 4 C). For the jagn1-OX/bzip28 line, there was a decrease in survival when compared to bzip28 for TM, Mon, heat, and PQ stress (Fig. 4 A-B, D-E). The increased survival of a2b4 plants crossed with jagn1-OX and decreased survival of bzip28 plants crossed with jagn1-OX suggests that JAGN1 is participating in one or both pathways to affect the response to ER stress, however this experiment does not provide the resolution to determine how. [Here let’s also mention that crossing jagn1-OX into other mutant backgrounds effectively eliminates the concern that a background T-DNA in the jagn1-OX plants could be responsible for some or all of the phenotypes presented in Fig. 3 .] Expression of genes downstream of the IRE1 pathway are increased in jagn1-OX/a2b4 line compared to IRE1 double knockout in response to Tunicamycin treatment To identify with more precision the mechanism by which overexpression of JAGN1 changes seedling ability to withstand ER stress, RT-qPCR was performed for gene expression downstream of each arm of the UPR after treatment with TM. One week old seedlings were treated with 5 µg/mL of Tunicamycin dissolved in DMSO for treatment and sterile MQ water with an equivalent volume of DMSO for the control. After 6 hours of treatment, samples were collected for RNA extraction and RT-qPCR. To confirm the UPR was active, bZIP60 unspliced and spliced transcripts were measured (Supplementary Fig. 3 and showed that under control conditions there was no expression of spliced bZIP60 for all genotypes. Under TM stress, there was an increase in bZIP60S transcript for all genotypes with an intact IRE1 pathway (Supplementary Fig. 3). The jagn1-OX/a2b4 mutant had no spliced bZIP60 indicating that overexpression of JAGN1 does not directly affect the beginning of the IRE1 pathway, the splicing of bZIP60 (Supplementary Fig. 3). For all genotypes, the 4 genes measured downstream of the IRE1 pathway, BiP3 , PDI9 , SAR1a , and NAC103 had consistently very low expression under control conditions (Fig. 5 ). Col-0 showed increased expression of each downstream gene under TM treatment (Fig. 5 ). jagn1-OX also showed increased expression of each downstream gene under TM treatment; however, it was statistically lower than Col-0, indicating that overexpression of JAGN1 alone does not cause an automatic increase in the expression of the IRE1 arm of the UPR in response to TM (Fig. 5 ). The a2b4 mutant showed no increase in expression of SAR1a , suggesting expression of IRE1 is necessary for UPR induction of SAR1a expression (Fig. 5 ). However, the jagn1-OX/a2b4 mutant had a significant increase in SAR1a expression compared to a2b4 , bringing it up to the same expression level as jagn1-OX (Fig. 5 ). Similarly, PDI9 expression is also rescued in the jagn1-OX/a2b4 line compared to a2b4 (Fig. 5 ). The increased expression of SAR1a and PDI9 in the presence of JAGN1 overexpression and lack of IRE1 expression points to a possible mechanism for increased survival of the jagn1-OX/a2b4 line compared to a2b4 under TM stress (Figure V3). The a2b4 mutant had no change in expression of BiP3 and NAC103 in response to TM. Crossing jagn1-OX into the a2b4 line caused no change in expression indicating a lack of phenotype rescue for BiP3 and NAC103 in the jagn1-OX/a2b4 line under TM stress (Fig. 5 ). To see if there was any phenotype rescue associated with the bZIP28 arm of UPR after TM treatment, gene expression of CRT2 , SDF2 , and P58IPK were analyzed. Across all genotypes, CRT2 , SDF2 , and P58IPK were very low under control conditions (Fig. 5 ). Col-0 showed an increase in CRT2 , SDF2 , and P58IPK expression under TM treatment showing the bZIP28 arm was activated. The bzip28 mutant line had no increase in expression of the three genes investigated and was also not rescued by crossing jagn1-OX into the bzip28 line, indicating a lack of a phenotype rescue for each of the measured genes in the bZIP28 pathway of the UPR. Collectively this points towards a closer correlation between JAGN1 and the IRE1 pathway than JAGN1 and the bZIP28 pathway. Expression of genes downstream of the IRE1 pathway are increased in jagn1-OX/a2b4 line compared to IRE1 double knockout in response to Monensin treatment One week old seedlings were treated with 100 µM Monensin dissolved in ethanol for treatment and sterile MQ water with an equivalent volume of ethanol as the control. After 6 hours of treatment, samples were collected for RNA extraction. To determine that the UPR was active, bZIP60 unspliced and spliced transcripts were measured (Supplementary Fig. 3) and showed that under control conditions there was no expression of spliced bZIP60 for all genotypes. Under Mon stress, there was an increase in bZIP60S transcript for all genotypes with an intact IRE1 pathway. The jagn1-OX/a2b4 mutant had no spliced bZIP60 indicating that overexpression of JAGN1 does not directly affect the beginning of the IRE1 pathway, the splicing of bZIP60 (Supplementary Fig. 3). For all genotypes, the 4 genes measured downstream of the IRE1 pathway, BiP3 , PDI9 , SAR1a , and NAC103 had consistently very low expression under control conditions (Fig. 6 ). Col-0 showed increased expression of each downstream gene under Mon treatment (Fig. 6 ). jagn1-OX also showed increased expression of each downstream gene under Mon treatment, however it was statistically lower than Col-0 for SAR1a , PDI9 , and NAC103 and was the same expression level as Col-0 for BiP3 , indicating that, as with TM, overexpression of JAGN1 alone does not cause a baseline increase in the expression of the IRE1 arm of the UPR in response to Mon (Fig. 6 ). The a2b4 mutant had a very small increase in the expression of BiP3 under Mon stress, but was still the lowest out of all genotypes which suggests IRE1 function is necessary for a complete BiP3 transcriptional response to Mon. However, the jagn1-OX/a2b4 line had a significant increase in the expression of BiP3 compared to a2b4 showing that increased expression of JAGN1 in the absence of a functional IRE1 pathway results in increased expression of certain UPR genes. The a2b4 mutant had no change in expression of PDI9 , SAR1a , or NAC103 in response to Mon (Fig. 6 C, D, E). The jagn1-OX/a2b4 line did not show any increase in expression of these genes compared to a2b4 indicating a lack of phenotype rescue for PDI9 , SAR1a , NAC103 in response to Mon. There was an increase in bZIP28 downstream gene expression for Col-0 in the treatment. However, there was no change in expression of CRT2 , SDF2 , or P58IPK for jagn1-OX/bzip28 compared to bzip28 . As with TM, this too points towards a closer correlation between JAGN1 and the IRE1 pathway than JAGN1 and the bZIP28 pathway Expression of genes downstream of the IRE1 pathway are increased in jagn1-OX/a2b4 line compared to IRE1 double knockout in response to 2-deoxyglucose treatment One week old seedlings were treated with 10 mM 2-DG for treatment and sterile MQ water as the control. After 6 hours of treatment, samples were collected for RNA extraction. To determine that the UPR was active, bZIP60 unspliced and spliced transcripts were measured (Supplementary Fig. 3) and showed that under control conditions there was no expression of spliced bZIP60 for all genotypes. Under 2-DG stress, there was an increase in bZIP60S transcript for all genotypes with an intact IRE1 pathway. The jagn1-OX/a2b4 mutant had no spliced bZIP60 indicating that, as would be expected, overexpression of JAGN1 does not directly affect the beginning of the IRE1 pathway, the splicing of bZIP60 (Supplementary Fig. 3).There was a significant increase in all IRE1 downstream genes in Col-0 in the presence of 2-DG, likewise there was a significant increase in all IRE1 downstream genes in jagn1-OX in response to 2-DG, however they were all either equal to or less than Col - 0 expression levels indicating overexpression of JAGN 1 alone does not cause a baseline increase in the expression of IRE1 downstream genes in the presence of 2-DG. a2b4 showed no change expression with 2-DG treatment for BiP3 , SAR1a , or NAC103 indicating the need for IRE1 to be functional for expression of those downstream genes. PDI9 did have increased expression in a2b4 treated with 2-DG but was lower than that of Col-0 . For all four genes tested downstream in the IRE1 pathway, jagn1-OX/a2b4 had significantly increased expression compared to a2b4 . For all bZIP28 pathway downstream genes tested, Col-0 had increased expression under 2-DG stress compared to the control treatment (Fig. 7 E,F,G). The bzip28 mutant did not have any increase of genes under 2-DG treatment showing functional bZIP28 is necessary for their expression. The jagn1-OX/bzip28 mutant did not have any change of expression compared to the bzip28 mutant. As with TM and Mon, this again points towards a closer correlation between JAGN1 and the IRE1 pathway than JAGN1 and the bZIP28 pathway. H 2 O 2 production does not appear to be affected by JAGN1 overexpressor crossed into IRE1 double knockout or bZIP28 knockout As a secondary experiment for PQ stress, a leaf infiltration of 3 µM PQ, followed by DAB staining for H 2 O 2 production was conducted and found no observable phenotypic difference in H 2 O 2 production across genotypes (Supplemental Fig. 4) that would explain the decrease in survival of the jagn1-OX/bzip28 line seen in Fig. 4 E. This means that the mechanism for change in survival in response to PQ is most likely not centered around H 2 O 2 production. Discussion This study investigated the functional conservation of JAGN1 in plants. While the function of this gene has been explored in fruit flies 9 , 40 , humans/human cell lines 14 , 19 , 20 , mice 16 , 21 , and zebrafish 41 , to our knowledge, this is the first evidence of functional conservation of JAGN1 across animal to plant kingdoms. We found that increased expression of JAGN1 in IRE1 knockouts of Arabidopsis partially rescues survival and partially rescues expression of UPR genes in response to ER stress. While many animal models looking at JAGN1 function focus on immune related phenotypes such as neutrophil maturation 19 , 41 , or antibody production 21 , we did not find immunity to be an obvious phenotype associated with JAGN1 in Arabidopsis. The lack of an extreme phenotype seen with pathogen infection in plants is likely because of the inherent differences in plant and animal immunity. While animals have specialized immune cells such as neutrophils that produce a large number of antimicrobial proteins in response to infection, plants lack specialized immune cells and instead, all cells are capable of mounting an immune response 21 , 42 , 43 . Because there are no specialized immune cells in plants, there is not a specific cell type that can be targeted for observing how JAGN1 affects immune response that would be directly comparable to that of animal research. Upon targeted UPR stress, noticeable phenotype changes due to JAGN1 expression level became evident. The increased survival for an IRE1 deficient plant with overexpression of JAGN1 leads to a few possibilities. 1. IRE1 and JAGN1 compete for function and in the absence of IRE1 , JAGN1 can function to a higher capacity, i.e. an antagonistic relationship between the two genes. Or, 2. IRE1 and JAGN1 have some level of functional redundancy so in the absence of IRE1 , JAGN1 compensates, i.e., a compensatory relationship. Previous studies in animal models have shown that there is an interplay between JAGN1 and the IRE1 pathway. In mice plasmablasts with mutated jagn, there is increased splicing of XBP1 21 , in insulin producing cells, JAGN1 transcription increased after TM treatment 20 , and in zebrafish embryos with either mutated or silenced JAGN1b , there was an increase in UPR gene expression 41 all indicating that lack of JAGN1 increased ER stress. This trend of decreased JAGN1 function resulting in heightened ER stress is in line with our observations that increased JAGN1 expression led to decreased ER stress. When looking at gene expression, there was a trend of increased expression of genes downstream of the IRE1 pathway in the jagn1-OX/a2b4 line compared to a2b4 suggesting that overexpression of JAGN1 causes an IRE1 -independent change in IRE1 pathway gene expression (Figures U,T,S). Specifically, there was a statistically significant increase in expression of PDI9 and SAR1a in the presence of TM, BiP3 in the presence of Mon, and PDI9 , SAR1a , and BiP3 in the presence of 2-DG for jagn1 - OX / a2b4 compared to a2b4 . IRE1 double knockouts exposed to TM, have drastically decreased BiP3 and SAR1a compared to wild type 44 which is recapitulated in our results. Thus, the increased expression of BiP3 and SAR1a in the jagn1-OX/a2b4 line points towards a novel observation of the involvement of JAGN1 in the UPR of plants. BiP is an important chaperone protein within the ER and in unstressed states, BiP binds to the luminal domain of IRE1 keeping it in the non-active monomeric state 45 . BiP3 expression is highly induced during abiotic or ER stress as a target of the active bZIP60 transcription factor 4 , 7 . Protein disulfide isomerase 9 ( PDI9) is another chaperone protein that resides in the ER where it catalyzes disulfide bond formation and isomerization, thus increasing the protein folding capacity of the ER 46–48 . PDI9 is upregulated during UPR and is a target of the bZIP60 TF, placing it in the IRE1 arm of UPR in plants 47 . PDI9 also participates in a negative feedback loop where a low demand for catalysis via PDI9 allows for high association with IRE1 , diminishing IRE1 pathway activation but when PDI9 chaperone function is needed, it dissociates from IRE1 thereby increasing activation of the IRE1 pathway 46 . Because PDI9 and BiP3 are both ER resident chaperone proteins 7 , 47 , the increased expression of each in jagn1-OX/a2b4 versus a2b4 would increase the protein folding capacity of the ER and is, at least in part, the reason for the increased survival of jagn1-OX/a2b4 compared to a2b4 under ER stress. While PDI9 expression is primarily regulated by the IRE1 pathway, the observed increased PDI9 expression in jagn1-OX/a2b4 compared to a2b4 aligns with a previous finding that bZIP60 is largely, but not solely responsible for PDI9 expression in response to stress 49 . Therefore, JAGN1 overexpression is interacting with a separate mechanism to increase PDI9 expression in response to stress, independently of IRE1. This finding supports the idea that JAGN1 and IRE1 are behaving in a compensatory manner in this experiment. Secretion-associated and RAS superfamily-related protein 1a ( SAR1a) is a small GTPase and part of the COPII protein complex responsible for cargo transport from the ER to the Golgi 50 . SAR1-GTP anchors the COPII complex to the membrane via its amphipathic helix 13 . Mutations of the Sar1 protein in yeast have been shown to block vesicles from reaching the Golgi, highlighting the important role of SAR1 in ER to Golgi traffic 51 . SAR1a expression in response to ER stress is most closely associated with bZIP60, thus placing it downstream of the IRE1 pathway 5 . The correlation between higher JAGN1 expression and higher SAR1a expression suggests that like ERVs, JAGN1 could be participating in anterograde movement of cargo from the ER to the Golgi. Like BiP3 and PDI9 , SAR1a expression must have alternative pathways capable of increasing expression independently of bZIP60 , and although our data cannot identify the exact pathway responsible. Regardless of the mechanism by which it happens, the increased expression of SAR1a in jagn1-OX/a2b4 compared to a2b4 should increase the amount of protein trafficking able to advance from ER to Golgi via COPII movement. Increased movement of proteins from the ER to the Golgi reduces the overall protein burden of the ER, and is likely another explanation for the increased survival seen in the IRE1 knockdown plants with JAGN1 overexpression. Overall, it seems that JAGN1 and IRE1 both work to increase a cell’s ability to withstand ER stress and have at least partially overlapping pathways. Therefore, when one pathway is nonfunctional, in this case IRE1 , the other pathway, i.e., overexpression of JAGN1 , can partially compensate for its absence. The decreased survival of jagn1-OX/bzip28 compared to bzip28 with TM, Mon, heat, and especially PQ, suggests that 1. bZIP28 and JAGN1 either work independently to increase cell survival, i.e., they are additive. Or, 2. they interact in such a way that both need to be functional simultaneously for a proper stress response to occur, i.e. they are complementary to one another (Fig. 4 ). Contrary to IRE1 , there was no change in gene expression of examined genes downstream of the bZIP28 pathway for jagn1 -OX/bzip28 compared to bzip28 upon treatment with TM, Mon, or 2-DG. Thus, based on the genes investigated here, JAGN1 does not interact with the bZIP28 pathway on the level of gene expression. CRT2 (calreticulin 2) is an ER resident chaperone that regulates intracellular calcium levels 52 , 53 . CRT2 expression in response to ER stress is regulated by the bZIP28 pathway of the UPR 5 . SDF2 (stromal cell-derived factor 2) is conserved across plants and animals and is activated during UPR 54 , 55 . It is an ER-resident protein that has been demonstrated to be integral in Arabidopsis seedling response to UPR eliciting chemicals 54 . While the exact mechanism by which SDF2 functions in UPR is still not known, it is thought to participate in the quality control of glycoproteins within the ER 54,56 . P58IPK (58 kDa inhibitor of protein kinase) is primarily regulated by the bZIP28 pathway of the UPR 5 . Though this data cannot explain the reason for decreased survival of jagn1-OX/bzip28 compared to bzip28 , this is an interesting finding and points towards a need to explore this relationship further in animal models where a similar interaction could exist. While the list of genes analyzed here for expression changes is not all inclusive, it can still be concluded that overexpression of JAGN1 in an IRE1 knockout causes expression changes in genes downstream of IRE1 independently of IRE1 function. On the other hand, overexpression of JAGN1 in a bZIP28 mutant does not cause expression changes of bZIP28 downstream genes independently of bZIP28 function. In IRE1 and bZIP28 , as with many functionally redundant pathways, if there is an absence of one, the other pathway will compensate. This is displayed well in Fig. 6 E-G, where the a2b4 mutant shows a marked increase in bZIP28 pathway gene expression under Mon stress. Importantly, overexpression of JAGN1 in each respective UPR pathway mutant does not induce changes in gene expression for the opposite UPR pathway. Thus, it seems that overexpression of JAGN1 does not simply enhance the innate compensation of one UPR pathway for the absence of another, there is a more delicate interaction at play. This study focuses primarily on mRNA expression and phenotype analysis and is lacking protein level analysis. Localization of the JAGN1 protein in Arabidopsis to confirm it is an ER membrane protein would be helpful in further confirming the conservation of this gene across kingdoms. Additionally, a Co-IP experiment looking at interacting partners of JAGN1 would help to further delineate the function of the protein in plant cells. If the interacting partners were orthologs of the interacting proteins in animal cells, this would again further confirm the functional conservation. A knock-down or knock-out of JAGN1 in plants would allow for additional reverse genetics approaches to determine the function of the gene and/or whether a JAGN1 knockout is a survivable phenotype in plants. Another interesting future approach could be expressing known SCN mutations of JAGN1 in humans in Arabidopsis. Expression of human proteins (Human beta defensin 2 and three antiapoptotic genes) in plant systems has been shown effective at increasing resistance to pathogens, which shows that expression of human proteins in plant systems can uncover shared functionality in cellular responses 57 , 58 . Especially with the convenience of Arabidopsis providing a unique platform to look at the implications of IRE1 double knockouts alongside JAGN1 mutations, this could help uncover cellular phenotypes without the limitation of IRE1 knockout lethality present in animal models. Conclusions In summary, our data demonstrates that JAGN1 plays a role in the ER stress response of plants, which indicates functional conservation of this gene across plant and animal kingdoms. This suggests that JAGN1 is a player in the UPR where it decreases the protein folding burden of the ER Overexpression of JAGN1 in an IRE1 double knockout plant under ER stress partially rescues survival of plants experiencing ER stress and increases expression of the IRE1 downstream genes, SAR1a , PDI9 , and BiP3 in an IRE1 independent manner. This increased gene expression of two chaperone proteins, PDI9 and BiP3 , and a component of the COPII pathway, SAR1a , can be attributed to the increased survival of plants during ER stress. JAGN1 overexpression in a bZIP28 mutant decreases survival of stressed plants and does not change expression of the bZIP28 downstream genes, CRT2 , SDF2 , or P58IPK . Materials and Methods Plant growth: Soil grown plants were grown in growth chambers at 22°C, 12-hour light/dark cycle at 220 µmol/m 2 s.Tissue culture plants were surface sterilized and grown on ½ MS media with 1% sucrose, 50 µg/mL ampicillin, and 1% agar. Plates were sealed with micropore tape and placed in growth chambers with the same conditions as soil grown plants. All seeds were stratified at 4°C in the dark for 1 week before being put under light. Genotyping JAGN1: To confirm homozygosity of T-DNA insertion, gene specific (JLP geno F/ JLP geno R) and insertion specific (LBb1.3/JLP geno R) primers were used. To determine JAGN1 expression in T-DNA insertion line, three combinations of 4 primers were used, (F1/R1, F1/R2, F2,R2) for RT-qPCR of 2-week-old seedlings grown on ½ MS media. For sequencing, JLP Geno R/LBb1.3 were used to amplify genomic DNA from the JAGN1 T-DNA insertion line with a sequence of ~ 800–900 bp beginning at the 4’th exon. This template with the JLP geno R primer was sent for sequencing. Sequence alignment with documented gene sequence was used to determine the location of the T-DNA insertion. Plant genetic lines used: All genotypes in this paper are in the Columbia-0 ecotype background, thus Col-0 was used as wild type control. The JAGN1 T-DNA insertion line used is SALK_065212C. The IRE1 double knockout used is ire1a-2/ire1b-4 . The ire1a-2 mutant line is SALK_018112 and the ire1b-4 mutant line is SAIL_238_F07. The bZIP28 mutant used is SALK_132285. Plant crosses: Crosses were performed via cross pollination of jagn1-OX to ire1a-2b-4 and crossing jagn1-OX to bzip28 . F 1 plants were genotyped for successful crossing and F 2 plants were genotyped for homozygosity of all individual genes. For jagn1-OX/a2b4 this meant homozygosity of jagn1-OX , ire1a-2 , and ire1b-4 . For jagn1-OX/bzip28 this meant homozygosity of jagn1-OX and bzip28 . Pathogen assay: Four-week-old plants, 3 leaves per plant, were syringe infiltrated with 0.001 OD600 of Pseudomonas syringae Pst DC3000 suspended in 10 mM MgCl2. Protocol from [paper about syringe infiltration and dilutions] was followed. Graph is representative of 3 biological replicates with each replicate containing 6 plants, 3 leaves per plant. Ion leakage assay: Four-week-old plants, 3 leaves per plant, were removed and vacuum infiltrated with 0.1 OD 600 of Pseudomonas syringae Psm ES4326 in sterile MQ water. Leaves were added to 50 mL conical tubes containing 15 uL of sterile MQ water and conductivity was measured every 30 minutes for 8 hours. Each tube was then autoclaved to leak all remaining ions, and this was used to compare each measurement as a percentage of total ions leaked. RT-qPCR: RNA extraction was performed using Zymo Quick RNA mini-prep kit (R1055). Tissue was homogenized while frozen with liquid nitrogen and the first step of Trizol/chloroform extraction was used. After centrifugation at 4°C for 15 minutes, the top layer was added to the RNA extraction kit and followed the protocol as written. cDNA synthesis was performed using AB Clonal Kit (RK20400). qPCR was run using AB Clonal Sybr Green Master Mix (RK2103) on a Quant Studio 3. Ubiquitin 5 was used as a housekeeping gene for relative expression quantification. Each biological sample had three technical replicates per plate. Plant weight Tunicamycin stress: Seedlings were grown on ½ MS media for 5 days before being transferred to liquid ½ MS in a 12-well plate with 10 seedlings per well. Low tunicamycin concentration was 0.75 µg/mL and high concentration was 1.5 µg/mL. Seedlings were left to grow for 4 more days, then fresh weight of each pooled sample was measured. Analyzed via ANOVA followed by Tukey’s HSD represented with letters. Survivability Assay: All seeds were surface sterilized then stratified in sterile MQ water for 7 days at 4°C before being placed on freshly prepared ½ MS media with the stressor added in then placed under light. For Tunicamycin 0.1 µg/mL, Monensin 2.5 µM, 2-deoxyglucose 3 mM, paraquat 1 µM. Heat treated seeds were treated in a 1.5 mL tube filled with sterile MQ water in a benchtop tube warmer at 50°C for 1 hour before being placed on freshly prepared ½ MS media and put directly to light. TM, Mon, and 2-DG were all plated on the same day and used the same control plate for individual comparison. Likewise, paraquat and heat seeds were plated on the same day and used the same control plates for individual comparisons. Each plate had all 6 genotypes, 30 seeds per genotype. All conditions had 7 plates. Plates were imaged on day 10 of growth. Survivability was measured as number of seedlings surviving on treatment divided by number of seedlings surviving on control plate represented as a percentage. Analyzed via two-way ANOVA followed by a Tukey’s HSD represented as letters. ER stressors for gene expression analysis Seedlings were grown vertically on ½ MS media for 12 days, then transferred to 6 well plates with sterile MQ water and dissolved stressors. Each well had 10 seedlings. Tunicamycin treatment was 5 µg/mL dissolved in DMSO and control was the equivalent concentration of DMSO. Monensin treatment was 100 µM dissolved in ethanol and control was the equivalent concentration of ethanol. 2-deoxyglucose treatment was 10 mM dissolved in water and control was just MQ water. For each treatment there were 3 biological replicates of pooled samples of each genotype in each condition. All treatments lasted 6 hours and samples were flash frozen in liquid nitrogen for RNA extraction preparation. Analyzed via two-way ANOVA followed by a Tukey’s HSD represented as letters. Declarations Author contributions: Conceptualization, R.B. and K.P.M.; experiments, data analysis and figure preparation, R.B., writing-original draft preparation, R.B.; writing-review and editing, K.P.M.; supervision, K.P.M.; project coordination and administration, K.P.M.; funding acquisition, R.B. and K.P.M. Both authors have agreed to the published version of the manuscript. Acknowledgements: The authors wish to thank Dr. Elizabeth Sztul for helpful discussions. This study was funded by This work was funded under NSF Award #IOS-2038872 to K.P.M and a Sigma Xi Grant in Aid of Research G2022315-2135 awarded to R.B. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Competing interests: All authors declare no financial or non-financial competing interests. 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Arabidopsis thaliana plants expressing human beta-defensin-2 are more resistant to fungal attack: functional homology between plant and human defensins. Plant Cell Rep 26, 1391–1398 (2007). Dickman, M. B. et al. Abrogation of disease development in plants expressing animal antiapoptotic genes. Proceedings of the National Academy of Sciences 98, 6957–6962 (2001). Additional Declarations No competing interests reported. Supplementary Files JAGNSupplementaryFile.docx image1.png Graphical Abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8893802","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":598607072,"identity":"292d1ece-e2f6-4383-afd1-62799fff3b54","order_by":0,"name":"Regina Bedgood","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Regina","middleName":"","lastName":"Bedgood","suffix":""},{"id":598607073,"identity":"37ff4cf9-6bb7-4a89-9277-45681619cc4f","order_by":1,"name":"Karolina Pajerowska-Mukhtar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIie3RsQrCMBCA4bi0y5WukYC+wpVCHCz0NRwrQl3q7i508gE6CH2FPoIQaJeCa6Sj4OTmoihq6Kqkujnkn++Du4QQk+kPQ5lw5jzowM3cY/+yFCTM16gl3ubEfcDApxLsMdQHgrTWE3944jPAeFpMwL710rMiiZ5wlnABKOYFBav5igQsGa3uKBYFdcv9NW1I+BWBloAlHUW6F6PtYs85KkKddNdNfEXU+SLCSUuqbuJlcaweWXiZBItBXQHSMtISlLOSKTJ08137lYMwX2215C34bdxkMplMH3sB+ExMV1DUELcAAAAASUVORK5CYII=","orcid":"","institution":"Clemson University","correspondingAuthor":true,"prefix":"","firstName":"Karolina","middleName":"","lastName":"Pajerowska-Mukhtar","suffix":""}],"badges":[],"createdAt":"2026-02-16 14:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8893802/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8893802/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103751980,"identity":"f785b748-a6ab-46e6-afdd-87b27b09aeaf","added_by":"auto","created_at":"2026-03-02 13:12:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":924008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequence alignment and predicted membrane topology of JAGN1\u003c/strong\u003e. (a-f) Left to right: Alpha fold protein folding prediction, predicted aligned error, TOPCONS prediction of membrane topology. (a) Arabidopsis thaliana average pLDDT 81.23 (b) Danio rerio (1a) average pLDDT 92.2 (c) Danio rerio (1b) average pLDDT 91.43 (d) Homo sapiens average pLDDT 91.56 (e) Mus musculus average pLDDT 91.79 (f) Drosophila melanogaster average pLDDT 91.56 (g) Protein sequence alignment of Arabidopsis thalianato animals. Dark purple indicates 100% sequence homology while light purple represents at least 67% sequence homology. (h) Protein sequence alignment of Arabidopsis thaliana and plants –Spinacia oleracea, Acer yangbiense, Prunus persica. Dark purple indicates 100% sequence homology while light purple is 75% sequence homology.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8893802/v1/772e5c5aaf9a68ce622917ee.png"},{"id":103751988,"identity":"a160fb0f-801c-44f5-9553-3092cb703a63","added_by":"auto","created_at":"2026-03-02 13:12:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1980599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of a T-DNA insertion line in JAGN1\u003c/strong\u003e. (a) Gene expression analysis of JAGN1 in A. thaliana. RT-qPCR of expression using genotypes atjagn1-OX and Columbia-0 as a wild-type control. Student’s t-test P-value for Col-0 compared tojagn1for each primer set: F1R1 p= 4.36e-03, F1R2 p=2.12e-07, F2R2 p=1.91e-04 (b) Semi-quantitative PCR utilizing the same cDNA as in Figure 1a \u0026nbsp;with primers F1/R2, run on a gel to ensure the amplification of a single, on target band for each genotype. (c) Depiction of the structure of the JAGN1 gene in wild-type (top) and T-DNA insertion line (bottom). Tall boxes represent \u0026nbsp;exon locations, green is 5’ UTR and 3’UTR, purple is translated regions of exons, pink is location of T-DNA insertion, short boxes represent introns.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8893802/v1/829a9afdbd40aa38c017aed5.png"},{"id":103752015,"identity":"7bc43390-a067-42b5-ae13-bf60b4007d3e","added_by":"auto","created_at":"2026-03-02 13:12:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":538484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of JAGN1 overexpression on pathogen infection, cell death, and response to ER stress.\u003c/strong\u003e (a) Quantification of Pseudomonas syringae pv. tomato DC3000 growth 3 days post infiltration in 4-week-old leaves. Whiskers on boxplots are representative the range of data within 1.5 times the interquartile range from the upper and lower box boundaries.(b) image of a representative sample of infected leaves at time of sample collection (c) cell death measured by ion leakage of 4-week-old leaves infiltrated with infiltration Pseudomonas syringae pv. maculicola ES4326 (d) 5-day old seedlings were treated with 0 μg/mL, 0.75 μg/mL, or 1.5 μg/mL of TM for 4 days and fresh plant weight was measured on day 9 of seedling growth; each genotype and condition had three biological replicates of a pooled sample of 10 seedlings.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8893802/v1/237defcc2dda8b97f56142d7.png"},{"id":103752014,"identity":"653b3c3e-976b-4bf7-b60d-26c39de8d174","added_by":"auto","created_at":"2026-03-02 13:12:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1576350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival phenotypes of JAGN1 and UPR mutants exposed to a UPR elicitor.\u003c/strong\u003e Seeds were surface sterilized and stratified in sterile water for 7 days at 4°C, then placed on freshly prepared ½ MS media in the presence or absence of a UPR elicitor (A-C, E) or heat treated then placed on freshly prepared ½ MS media (D) and moved straight to light. Each plate has 30 seeds per genotype. One representative plate for each condition is shown, statistics are based on 7 plates. Images were taken after 10 days of growth. Tunicamycin concentration was 0.1 μg/mL (A). Monensin concentration was 2.5 μM (B). 2-Deoxyglucose concentration was 3 mM (C). Seeds were heat treated in sterile water at 55°C for 1 hour (D). Paraquat concentration was 1 μM (E). Error bars are one standard error above and below the mean.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8893802/v1/a93b3962b3d4babf81a1e88a.png"},{"id":103752021,"identity":"2b3996a2-c07a-4295-8a19-5cfa38a606e4","added_by":"auto","created_at":"2026-03-02 13:12:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2304465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDownstream expression changes in response to Tunicamycin treatment.\u003c/strong\u003e Seeds were surface sterilized and stratified for 7 days at 4°C, then grown on ½ MS media. Seeds were grown vertically under 12 hr light/dark cycles. On day 12, seedlings were transferred to sterile MQ water with 5μg/mL of tunicamycin for 6 hours. qRT-PCR was performed on three biological replicates per genotype per condition. Each biological replicate consisted of a pooled sampled of 8-10 seedlings. All qPCR samples were conducted with 3 technical replicates. Values are relative gene expression compared to Ubiquitin5 housekeeping gene. Genes measured downstream in the IRE1 pathway include: BiP3, PDI9, SAR1a, and NAC103. Genes measured downstream of the bZIP28 pathway include: CRT2, SDF2, and P58IPK. Whiskers on boxplots are representative the range of data within 1.5 times the interquartile range from the upper and lower box boundaries.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8893802/v1/21e474cc4103cef06a463952.png"},{"id":103751904,"identity":"7aeb943f-6b22-4510-9e1e-6ff1e5df15b6","added_by":"auto","created_at":"2026-03-02 13:12:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2368495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDownstream expression changes in response to Monensin treatment.\u003c/strong\u003e Seeds were surface sterilized and stratified in sterile water for 7 days at 4°C, then grown on ½ MS media. Seeds were grown vertically under 12 hr light/dark cycles. On day 12, seedlings were transferred to sterile MQ water with 100 μM of monensin for 6 hours. qRT-PCR was performed on three biological replicates per genotype per condition. Each biological replicate consisted of a pooled sampled of 8-10 seedlings. All qPCR samples were conducted with 3 technical replicates. Values are relative gene expression compared to Ubiquitin5 housekeeping gene. Genes measured downstream in the IRE1 pathway include: BiP3, PDI9, SAR1a, and NAC103. Genes measured downstream of the bZIP28 pathway include: CRT2, SDF2, and P58IPK. Whiskers on boxplots are representative the range of data within 1.5 times the interquartile range from the upper and lower box boundaries.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8893802/v1/bab2e615e95c1be30d493311.png"},{"id":103751907,"identity":"0f77b966-18b6-431d-bf1a-0e2e99333cdd","added_by":"auto","created_at":"2026-03-02 13:12:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":617963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDownstream expression changes in response to 2-Deoxyglucose treatment.\u003c/strong\u003e Seeds were surface sterilized and stratified in sterile water for 7 days at 4°C, then grown on ½ MS media. Seeds were grown vertically under 12 hr light/dark cycles. On day 12, seedlings were transferred to sterile MQ water with 10 mM 2-DG for 6 hours. qRT-PCR was performed on three biological replicates per genotype per condition. Each biological replicate consisted of a pooled sampled of 8-10 seedlings. All qPCR samples were conducted with 3 technical replicates. Values are relative gene expression compared to Ubiquitin5 housekeeping gene. Genes measured downstream in the IRE1 pathway include: BiP3, PDI9, SAR1a, and NAC103. Genes measured downstream of the bZIP28 pathway include: CRT2, SDF2, and P58IPK. Whiskers on boxplots are representative the range of data within 1.5 times the interquartile range from the upper and lower box boundaries.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8893802/v1/fc1418f86b429670be6f8493.png"},{"id":105957378,"identity":"7a9e45ca-cc2b-448d-b1b3-593a06201370","added_by":"auto","created_at":"2026-04-01 20:55:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11211001,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8893802/v1/3c404f1d-8d02-4102-91fb-bdb85d95aa31.pdf"},{"id":103752010,"identity":"f6a14116-ea81-459c-8acd-2eae8907bb0c","added_by":"auto","created_at":"2026-03-02 13:12:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18649838,"visible":true,"origin":"","legend":"","description":"","filename":"JAGNSupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8893802/v1/47cc110e4bfbc207c83d7432.docx"},{"id":103751983,"identity":"67dda8ed-f5cb-4e87-a58d-293c4a3445e6","added_by":"auto","created_at":"2026-03-02 13:12:42","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2698843,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8893802/v1/b540acb6c5f136f789765b55.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced JAGN1 expression Modulates ER Stress Signaling and Promotes Survival in IRE1-Deficient Arabidopsis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Unfolded Protein Response (UPR) is a cellular level stress response conserved across eukaryotes and is triggered by accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The UPR in plants is triggered by stressors such as pathogen infection, drought, or heat, but is also important for certain developmental stages such as root and reproductive development \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Vascular plants and metazoans share two arms of the UPR \u0026ndash;the Inositol Requiring Enzyme 1 (\u003cem\u003eIRE1\u003c/em\u003e) pathway and the Basic Leucine Zipper 28/17 (\u003cem\u003ebZIP28\u003c/em\u003e /\u003cem\u003ebZIP17\u003c/em\u003e) pathway, which acts as the functional equivalent of the Activating Transcription Factor 6 (\u003cem\u003eATF6\u003c/em\u003e) pathway in metazoans \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. IRE1 is an ER membrane protein that interacts with Binding Immunoglobulin Protein (BiP) at its luminal domain during homeostasis, keeping it in its inactive state. Under stress, misfolded proteins replace BiP which triggers dimerization and autophosphorylation of IRE1 thereby activating the cytosolic ribonuclease domain. In plants this activity splices \u003cem\u003ebZIP60\u003c/em\u003e mRNA, whereas in metazoans the homologous transcript is \u003cem\u003eXBP1\u003c/em\u003e mRNA. The resulting bZIP60 and XBP1 proteins are translated into functional transcription factors that translocate to the nucleus \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In plants, \u003cem\u003eIRE1\u003c/em\u003e knockouts cause deficits in response to drought, pathogen infection, salt tolerance, and chemically induced ER stress \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003ebZIP28/bZIP17\u003c/em\u003e pathway consists of two transcription factors retained at the ER membrane via association with BiP. Accumulation of unfolded or misfolded proteins causes BiP dissociation, allowing bZIP28/17 to relocate to the Golgi where cleavage releases the active transcription factor to the nucleus \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In plants, mutations in \u003cem\u003ebZIP28\u003c/em\u003e cause deficits in response to heat stress and negatively affect root development \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Despite our understanding of the two canonical arms of the UPR, there are ancillary genes involved in the UPR whose contributions or mode of action are not fully understood\u0026mdash;one of these genes is called Jagunal Homolog 1.\u003c/p\u003e \u003cp\u003eJagunal was first discovered in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e during a screening of lethal mutations in the 3rd chromosome specific for a small egg phenotype, hence the name jagunal - meaning \u0026ldquo;small egg\u0026rdquo; in Korean \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Drosophila is used as a model to study vesicular transport because there are specific stages of oocyte development involving major ER reorganization and large amounts of exocytic activity to increase the surface area of the plasma membrane, which can be observed with microscopy \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Oocytes with a \u003cem\u003ejagn\u003c/em\u003e knockout mutation had abnormal ER reorganization evidenced by lack of ER clusters, a decrease of exocytosis of certain proteins, and a decrease in cell surface area, while distribution of Golgi complex was normal suggesting Golgi apparatus function is not effected by \u003cem\u003eJAGN\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Somatic cell clone lines with mutated \u003cem\u003ejagn1\u003c/em\u003e had thinner, shorter, bristles and defected surface structure which further supports the idea that JAGN is necessary for proper exocytosis of proteins and endomembrane to the cell membrane \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eJAGN1 is proposed to play a role in protein sorting between the ER and Golgi. It is compared to two well established families of ER membrane resident proteins important for homeostatic control of protein movement through the endomembrane system, tetraspanins (TSP) and endoplasmic reticulum vesicle (Erv) proteins. TSPs are highly conserved across eukaryotes playing key roles in cellular trafficking of proteins, lipids, miRNA, and motility and fusion of cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The N-terminal domain, C-terminal domain, and one short loop expand into the cytoplasm and two larger loops expand into the extracellular space. Conserved regions on non-membrane domains allow for dimerization of TSPs forming tetraspanin-enriched microdomains (TEMs) while less conserved regions allow for specific protein-protein interactions to guide cellular trafficking of endosomes, pathogen identification, and can dictate reactive oxygen species (ROS) generation \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. While some Ervs have four transmembrane domains, it is not a defining characteristic of the proteins, they are instead categorized based on function \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Ervs aid in anterograde movement of molecules from the ER to Golgi via coatomer protein complex II (COPII) coated vesicles and are recycled back to the ER via coatomer protein complex I (COPI) coated vesicles \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eJagunal Homolog 1\u003c/em\u003e (\u003cem\u003eJAGN1\u003c/em\u003e) is a gene encoding an ER membrane resident protein unique to its own family due to its lack of sequence with either TSPs or Ervs. However, it has motifs for interaction with clathrin coats and contains four membrane spanning domains with N and C terminal domains both extending into the cytoplasm, as do TSPs \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. JAGN1 also interacts with three members of the COPI complex, important for retrograde movement from the Golgi to the ER, coatomer subunit alpha (COPA), coatomer subunit beta 2 (COPB2), and coatomer subunit gamma 2 (COPG2), as do Ervs \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. These morphological and functional similarities to Ervs and TSPs, suggest that JAGN1 plays a role in the sorting and trafficking of proteins between the ER and Golgi \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. It is highly conserved across kingdoms with homologs in animals and plants with amino acid sequences particularly well conserved at the NH2 and COOH terminals of animals suggesting these regions are particularly important for functionality \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSince its original discovery in Drosophila, mutations to the homologous gene in humans, called Jagunal Homolog 1 (JAGN1), have been identified to cause a primary immunodeficiency called severe congenital neutropenia (SCN) \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. SCN patients struggle with life threatening infections due to a lack of functional neutrophils \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Neutrophils are a type of white blood cell that play an important role in the innate immune system via creation and secretion of antimicrobial proteins by the endomembrane system \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Neutrophil differentiation is heavily influenced by ER stress and the resulting UPR. HL-60 cells stimulated to become neutrophils and subsequently treated to inhibit the IRE1 or ATF6 pathways exhibited increased apoptosis, indicating that functional UPR is essential for survival of differentiating neutrophils \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. HL-60 cells stimulated to become neutrophils and treated to block UPR display lower expression of CD11b and decreased segmentation of nuclei, two indicators which would measure successful differentiation into functionally mature neutrophils \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, suggesting UPR is also essential for their function once differentiated. Likewise, \u003cem\u003eJAGN1\u003c/em\u003e is necessary for differentiation of human neutrophils \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Electron microscopy of ER in myeloid progenitor cells, the precursors to neutrophils, showed \u003cem\u003eJAGN1\u003c/em\u003e mutation resulted in an enlarged ER and granules that were severely underdeveloped \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Additionally, \u003cem\u003eJAGN1\u003c/em\u003e mutant cells had elevated levels of binding immunoglobulin protein (BiP, also known as Grp78) ,a chaperone protein that plays an important role in the UPR, which is consistent with ER stress \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Coimmunoprecipitation experiments showed a protein level interaction between JAGN1 and Grp78/BiP, \u003csup\u003e19\u003c/sup\u003e. Moreover, treatment of neutrophils with the protein kinase inhibitor staurosporine, an inducer of ER stress, lead to increased apoptosis in JAGN1 deficient cells compared to controls \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, suggesting a role of JAGN1 in ER stress. In insulinoma cells treated with tunicamycin, an inducer of ER stress, expression of \u003cem\u003eJAGN1\u003c/em\u003e mRNA was increased, again, indicating its role in ER stress \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In mice plasmablasts, cells with \u003cem\u003eJagn1\u003c/em\u003e loss showed higher gene transcription, for stress response, apoptosis, protein folding, and UPR \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, suggesting compensation for the absence of functional \u003cem\u003eJAGN1\u003c/em\u003e. Mice plasmablasts with jagn1 mutations displayed a reduction in immunoglobulin production \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, a function which is heavily dependent on functional UPR \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Additionally, in \u003cem\u003eJAGN1\u003c/em\u003e deficient cells, there was in increase in \u003cem\u003eXBP1\u003c/em\u003e splicing \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, which is the metazoan equivalent of \u003cem\u003ebZIP60\u003c/em\u003e splicing in plants, both of which are spliced by IRE1 during UPR \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, suggesting that increased function of the \u003cem\u003eIRE1\u003c/em\u003e pathway is compensating for lack of \u003cem\u003eJAGN1\u003c/em\u003e function. Collectively, this indicates that JAGN1 and the UPR are tightly correlated in animal models.\u003c/p\u003e \u003cp\u003eBecause of the necessity of \u003cem\u003eJAGN1\u003c/em\u003e for ER organization, for response to ER stress, and the conservation of function from fruit flies to zebrafish to humans, an additional model with a novel experimental approach will increase the broad understanding of \u003cem\u003eJAGN1\u003c/em\u003e function during ER stress. Arabidopsis is an opportune model for understanding the role of JAGN1 in the UPR because unlike in mice \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and fruit flies \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, knockout mutations of \u003cem\u003eIRE1\u003c/em\u003e are survivable in plants \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Thus, Arabidopsis provides a unique reverse genetics framework to investigate if \u003cem\u003eJAGN1\u003c/em\u003e can compensate for defects in each canonical UPR pathway\u0026mdash;\u003cem\u003eIRE1\u003c/em\u003e and \u003cem\u003ebZIP28\u003c/em\u003e, which would not be possible in animal models. Using this approach, our data demonstrate that \u003cem\u003eJAGN1\u003c/em\u003e contributes to the UPR of plants and modulates the effects of ER stress inducing chemicals, particularly in mutants of the UPR pathways. Here, we provide the first experimental evidence of functional conservation of \u003cem\u003eJAGN1\u003c/em\u003e across plants and animals which not only contributes to the broader knowledge of the UPR in plants and to the broader knowledge of \u003cem\u003eJAGN1\u003c/em\u003e function, it also provides a new model to study the role of \u003cem\u003eJAGN1\u003c/em\u003e in basic cellular biology.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCross-Kingdom Sequence and Structural Conservation of JAGN1 in Plants and Animals\u003c/h2\u003e \u003cp\u003eAcross plants and animals, all JAGN1 homologs share the characteristic four membrane spanning domains with carboxy and amino terminals facing into the cytoplasm. The predicted aligned error for each protein suggests there is a singular domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-F). While there is considerable amino acid sequence conservation of JAGN1 among animals, i.e. \u003cem\u003eHomo sapiens\u003c/em\u003e compared to the following: to \u003cem\u003eMus musculus\u003c/em\u003e has a percent identity of 97.81% and E-value of 6e-136; to \u003cem\u003eDanio rerio (1a)\u003c/em\u003e 74.32% with an E-value of 2e-109; to \u003cem\u003eD. rerio (1b)\u003c/em\u003e a percent identity of 71.32% with an E-value of 6e-104; to \u003cem\u003eDrosophila melanogaster\u003c/em\u003e a percent identity of 30.46% with an E-value of 4e-28, and likewise among plants, i.e. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e compared to the following: to \u003cem\u003eAcer yangbiense\u003c/em\u003e a percent identity of 55.49% with an E-value of 8e-60; to \u003cem\u003ePrunus persica\u003c/em\u003e a percent identity of 48.26% with an E-value of 93\u0026thinsp;\u0026minus;\u0026thinsp;53; and \u003cem\u003eSpinacia oleracea\u003c/em\u003e a percent identity of 42.44%, the sequence conservation between \u003cem\u003eA. thaliana\u003c/em\u003e and animals is comparatively lower, i.e. \u003cem\u003eA. thaliana\u003c/em\u003e compared to the following: to \u003cem\u003eD. melanogaster\u003c/em\u003e percent identity of 26.02% and E-value of 8e-08; to \u003cem\u003eHomo sapiens\u003c/em\u003e 27.27% with an E-value of 4e-07; to \u003cem\u003eM. musculus\u003c/em\u003e a percent identity of 28.57% and an E-value of 5e-07; to \u003cem\u003eDanio rerio (1a)\u003c/em\u003e percent identity of 24.73% and E-value of 1e-06; and \u003cem\u003eD. rerio (1b)\u003c/em\u003e with a percent identity of 32.56% and an E-value of 1e-05. However, the NH\u003csub\u003e2\u003c/sub\u003e terminus of \u003cem\u003eA. thaliana\u003c/em\u003e has a 7 amino acids with very high sequence homology to humans, mice, zebrafish, and fruit flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Interestingly, this sequence, amino acids 11\u0026ndash;17 of the human sequence, is a concentrated region of documented mutations resulting in SCN \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Both the sequence conservation and the high concentration of disease causing mutations in this region suggests an important role for this area of the protein and potential functional conservation of this gene into the plant kingdom. Additionally, while the sequence similarity between Arabidopsis and animals is around 25\u0026ndash;30%, the structure of JAGN1 between Arabidopsis and animals is noticably similar and is more structurally similar to human JAGN1 than Drosophila.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCharacterization of JAGN1 T-DNA insertion line\u003c/h3\u003e\n\u003cp\u003eTo investigate \u003cem\u003eJAGN1\u003c/em\u003e function in Arabidopsis, we queried the SIGnAL (Salk Institute Genomic Analysis Laboratory) T-DNA database, identified a single line for the \u003cem\u003eJAGN1\u003c/em\u003e ortholog \u003cem\u003eAT5G51510\u003c/em\u003e: SALK_065212C and confirmed homozygosity of the T-DNA insertion (Supplemental Fig.\u0026nbsp;1). The SIGnAL website predicts that the SALK_065212C line has the T-DNA insertion in an intron between the 5th and 6th exon, while the original, not confirmed stock line, SALK_065212, where the confirmed stock line originated, is predicted to have the T-DNA insertion in the 6th exon. Typically, T-DNA insertions in an exon result in a knockdown, knockout, or truncation of the gene, however our RT-qPCR analysis revealed a higher level of \u003cem\u003eJAGN1\u003c/em\u003e expression in the T-DNA insertion line compared to the \u003cem\u003eColumbia-0\u003c/em\u003e wild-type control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This enhaced expression of \u003cem\u003eJAGN1\u003c/em\u003e was checked with three primer combinations and the expression was consistent across all primers (Figure This was corroborated with semi-quantitative PCR to ensure there was a single band, and the amplicons were of the expected size (242 bp for cDNA, 693 for genomic DNA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Because SIGnAL predicted the insertion to be a homozygous knockout line, this enhanced expression was not expected. To investigate further the reason for this overexpression, Sanger sequencing identified the location of the T-DNA insertion (Supplemental Fig.\u0026nbsp;2) to be 28 bases into the 3\u0026rsquo; UTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). It is important to distinguish this T-DNA insertion line, which exhibits elevated \u003cb\u003eJAGN1\u003c/b\u003e transcript levels, from classical \u0026ldquo;overexpression lines,\u0026rdquo; which are typically transgenic plants carrying a strong constitutive promoter such as the cauliflower mosaic virus 35S (CaMV 35S) promoter. Traditionally, such lines are treated as constitutive overexpressors of a gene of interest. However, growing evidence indicates that gene expression driven by the CaMV 35S promoter can vary substantially with photoperiod, abiotic stress, temperature, tissue type, and developmental stage\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Thus, even in a classical overexpressor line there can still be variability in the extent of overexpression depending on the age of the plant, tissue type of interest, and experimental conditions. Although the T-DNA insertion line used here is not driven by an introduced promoter, it shows increased JAGN1 transcript abundance relative to wild type. For clarity, this 3\u0026prime;-UTR T-DNA insertion line will therefore be referred to as a JAGN1 overexpression line (\u003cem\u003ejagn1-OX\u003c/em\u003e). This line will be used to test whether elevated JAGN1 transcript levels enhance plant tolerance to ER stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eElevated\u003c/b\u003e \u003cb\u003eJAGN1\u003c/b\u003e \u003cb\u003eexpression does not enhance disease resistance to pathogen infection but does increase plant growth under Tunicamycin stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBecause animal lines with \u003cem\u003eJAGN1\u003c/em\u003e mutations show strong changes in immune phenotypes, we first set out to determine if increased expression of \u003cem\u003eJAGN1\u003c/em\u003e causes a phenotypical difference in pathogen infection response, 4-week-old leaves were infiltrated with \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003etomato\u003c/em\u003e DC3000 at a concentration of 0.001 OD\u003csub\u003e600\u003c/sub\u003e. After three days of pathogen growth, leaf tissue was collected and homogenized, and number of bacterial colonies per leaf disc was measured. Additionally, to test if \u003cem\u003eJAGN1\u003c/em\u003e overexpression changes cell death in response to pathogens, 4-week-old leaves were infiltrated with \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003emaculicola\u003c/em\u003e ES4326 at a concentration of 0.1 OD\u003csub\u003e600\u003c/sub\u003e and cell death was measured as percentage of ions leaked from leaves over 8 hours. For both experiments, there was qualitative heightened resistance to pathogen infection in \u003cem\u003ejagn1-OX\u003c/em\u003e compared to \u003cem\u003eCol-0\u003c/em\u003e, evidenced by the lower mean concentration of bacterial growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), the lower amount of chlorosis in infected leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and the lower average conductivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) but not drastic enough for statistical significance. Based on this result and because pathogen infection is a broad inducer of ER stress, we assessed whether a more targeted approach to induce ER stress via the chemical inducer, Tunicamycin (TM), would potentiate the effects of \u003cem\u003eJAGN1\u003c/em\u003e overexpression. Thus, we tested if overexpression of \u003cem\u003eJAGN1\u003c/em\u003e would result in a phenotype rescue under sustained, targeted ER stress by subjecting 5-day old seedlings to two concentrations of Tunicamycin for 4 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). TM is a naturally occurring antibiotic which interrupts Asparagine-linked, also known as, N-linked glycosylation of membrane proteins, resulting in unfolded proteins accumulating in the ER, leading to ER stress \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Protein glycosylation is a covalent post translational modification that occurs in the ER, and increases the specificity of proteins by tagging them for certain fates within the cell \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Importantly for this assay, glycosylated proteins display higher solubility, lower degradation via proteases, and increased stability all due to the large hydrophilic nature of glycans \u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Thus, by blocking N-linked glycosylation, therefore destabilizing proteins in the ER, TM serves as a pointed chemical inducer of ER stress. Fresh seedling weight was measured on day 9 of growth. Across the three genotypes, there was no weight difference in the control condition, indicating no phenotype change in plant weight under homeostatic conditions. The lower mean seedling weight of all three genotypes when exposed to TM compared to control shows both concentrations of TM were high enough to induce ER stress which negatively affected seedling growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). An \u003cem\u003eIRE1\u003c/em\u003e double mutant in two isoforms of the gene, called \u003cem\u003eire1a-2/ire1b-4\u003c/em\u003e was used as negative control for a proper ER stress response. This double mutant of \u003cem\u003eIRE1\u003c/em\u003e has been previously established to be an expressional knockout of both isoforms \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. For convenience, this mutant will be referred to as \u003cem\u003ea2b4\u003c/em\u003e moving forward. At both TM concentrations, there was a lower mean seedling weight of \u003cem\u003ea2b4\u003c/em\u003e compared to \u003cem\u003eCol-0\u003c/em\u003e indicating that nonfunctional UPR in the presence of TM due to lack of functional \u003cem\u003eIRE1\u003c/em\u003e causes increased stress to the seedling which compounded the negative effect on growth seen in TM treated \u003cem\u003eCol-0\u003c/em\u003e. At 0.75 \u0026micro;g/mL of TM, there was a substantially higher mean weight of the \u003cem\u003ejagn1-OX\u003c/em\u003e genotype compared to \u003cem\u003eCol-0\u003c/em\u003e, which indicated that higher transcript levels of \u003cem\u003eJAGN1\u003c/em\u003e increased seedlings\u0026rsquo; capacity to withstand ER stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). There are two arms of the UPR in plants and the observation that seedlings overexpressing \u003cem\u003eJAGN1\u003c/em\u003e are more capable of withstanding ER stress suggests that \u003cem\u003eJAGN1\u003c/em\u003e may participate in or act parallel to the two canonical arms of the UPR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIRE1\u003c/b\u003e \u003cb\u003edouble knockout survival in the presence of ER stressors is partially rescued when crossed with\u003c/b\u003e \u003cb\u003eJAGN1\u003c/b\u003e \u003cb\u003eoverexpression mutant\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further elucidate the role of \u003cem\u003eJAGN1\u003c/em\u003e during ER stress and to place its function into one of the two arms of the UPR, \u003cem\u003ejagn1-OX\u003c/em\u003e was crossed into \u003cem\u003ea2b4\u003c/em\u003e, the mutant deficient in the \u003cem\u003eIRE1\u003c/em\u003e pathway, and \u003cem\u003ebzip28\u003c/em\u003e, a mutant deficient in the \u003cem\u003ebZIP28\u003c/em\u003e pathway, yielding \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e and \u003cem\u003ejagn1-OX/bzip28\u003c/em\u003e, respectively. While bZIP17 and bZIP28 have overlapping functions for cell growth, \u003cem\u003ebZIP28\u003c/em\u003e is the primary player of the UPR \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, and is used here as the representative gene of that arm of the UPR. Importantly, this approach also accounts for the possibility of an off-site T-DNA insertion in the original \u003cem\u003ejagn1-OX\u003c/em\u003e line affecting the phenotypes seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. By crossing \u003cem\u003ejagn1-OX\u003c/em\u003e into two other Col-0 backgrounds, \u003cem\u003ea2b4\u003c/em\u003e and \u003cem\u003ebzip28\u003c/em\u003e, we have effectively eliminated that risk and can assume the phenotypes observed are based on the enhanced expression of \u003cem\u003eJAGN1\u003c/em\u003e. To examine if the increased capacity of \u003cem\u003ejagn1-OX\u003c/em\u003e to withstand ER stress can be replicated and reach significance with other elicitors of ER stress, survivability assays of multiple ER stressors were performed using the new crossed genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). To allow for a higher n number and therefore higher power, a survivability assay of germination of individual seedlings was used in place of pooled samples of weighted plants to observe changes in growth during prolonged ER stress. For all elicitors, 7 plates were grown for each condition (treatment and control) with each plate containing 30 seeds of each genotype (\u003cem\u003eCol-0, a2b4, bzip28, jagn1-OX, jagn1-OX/a2b4, and jagn1-OX/bzip28\u003c/em\u003e). Seedlings that germinated successfully and on day 10 had grown to the stage of at least 2 green cotyledons and displayed primary root growth were survivors. Seeds that failed to break dormancy or attempted germination but had severe chlorosis and/or lack of primary root growth on day 10 were non-survivors.\u003c/p\u003e \u003cp\u003eIn addition to TM, two more chemical inducers of ER stress, Monensin (Mon) and 2-Deoxyglucose (2DG) were used. Mon induces ER stress by allowing leakage of hydrogen protons from the Golgi apparatus altering the necessary relatively low pH, and allows Na\u0026thinsp;+\u0026thinsp;to leak inside leading to swelling of the Golgi \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The increased pH of the Golgi prevents proper glycosylation, disrupts protein and lipid trafficking and causes loss of organelle organization \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. While the transport of proteins in the Golgi is inhibited by Mon, it does not affect protein synthesis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. When used to stress Arabidopsis, Mon has been shown to reduce germination and induce expression of bZIP60s \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. 2-DG is an analog of glucose with a hydrogen in place of a hydroxyl group on the second carbon and induces ER stress due to its disruption of glycosylation of proteins. 2-DG is an analog of D-mannose which is a key player in glycosylation, therefore in the presence of 2-DG, there is abnormal N-linked glycosylation of proteins resulting in ER stress \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAcross all genotypes, there was no observable phenotype difference in control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As expected, TM, Mon, 2-DG, and heat, all direct ER stressors, showed a severe decrease in germination for the \u003cem\u003ea2b4\u003c/em\u003e mutant compared to \u003cem\u003eCol-0\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). In this assay, the \u003cem\u003ejagn1-OX\u003c/em\u003e line did not show an increased ability to withstand ER stress from any of the elicitors when compared to \u003cem\u003eCol-0\u003c/em\u003e. However, the \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e cross showed a phenotype rescue compared to \u003cem\u003ea2b4\u003c/em\u003e for TM, Mon, heat, and PQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B, D-E). Treatment with 2-DG resulted in no change in phenotype for \u003cem\u003ejagn1-OX\u003c/em\u003e crossed into \u003cem\u003ea2b4\u003c/em\u003e or \u003cem\u003ebzip28\u003c/em\u003e compared to the corresponding mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). For the \u003cem\u003ejagn1-OX/bzip28\u003c/em\u003e line, there was a decrease in survival when compared to \u003cem\u003ebzip28\u003c/em\u003e for TM, Mon, heat, and PQ stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B, D-E). The increased survival of \u003cem\u003ea2b4\u003c/em\u003e plants crossed with \u003cem\u003ejagn1-OX\u003c/em\u003e and decreased survival of \u003cem\u003ebzip28\u003c/em\u003e plants crossed with \u003cem\u003ejagn1-OX\u003c/em\u003e suggests that \u003cem\u003eJAGN1\u003c/em\u003e is participating in one or both pathways to affect the response to ER stress, however this experiment does not provide the resolution to determine how.\u003c/p\u003e \u003cp\u003e[Here let\u0026rsquo;s also mention that crossing jagn1-OX into other mutant backgrounds effectively eliminates the concern that a background T-DNA in the jagn1-OX plants could be responsible for some or all of the phenotypes presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of genes downstream of the\u003c/b\u003e \u003cb\u003eIRE1\u003c/b\u003e \u003cb\u003epathway are increased in\u003c/b\u003e \u003cb\u003ejagn1-OX/a2b4\u003c/b\u003e \u003cb\u003eline compared to\u003c/b\u003e \u003cb\u003eIRE1\u003c/b\u003e \u003cb\u003edouble knockout in response to Tunicamycin treatment\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify with more precision the mechanism by which overexpression of \u003cem\u003eJAGN1\u003c/em\u003e changes seedling ability to withstand ER stress, RT-qPCR was performed for gene expression downstream of each arm of the UPR after treatment with TM. One week old seedlings were treated with 5 \u0026micro;g/mL of Tunicamycin dissolved in DMSO for treatment and sterile MQ water with an equivalent volume of DMSO for the control. After 6 hours of treatment, samples were collected for RNA extraction and RT-qPCR. To confirm the UPR was active, \u003cem\u003ebZIP60\u003c/em\u003e unspliced and spliced transcripts were measured (Supplementary Fig.\u0026nbsp;3 and showed that under control conditions there was no expression of spliced \u003cem\u003ebZIP60\u003c/em\u003e for all genotypes. Under TM stress, there was an increase in \u003cem\u003ebZIP60S\u003c/em\u003e transcript for all genotypes with an intact \u003cem\u003eIRE1\u003c/em\u003e pathway (Supplementary Fig.\u0026nbsp;3). The \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e mutant had no spliced \u003cem\u003ebZIP60\u003c/em\u003e indicating that overexpression of \u003cem\u003eJAGN1\u003c/em\u003e does not directly affect the beginning of the \u003cem\u003eIRE1\u003c/em\u003e pathway, the splicing of \u003cem\u003ebZIP60\u003c/em\u003e (Supplementary Fig.\u0026nbsp;3). For all genotypes, the 4 genes measured downstream of the IRE1 pathway, \u003cem\u003eBiP3\u003c/em\u003e, \u003cem\u003ePDI9\u003c/em\u003e, \u003cem\u003eSAR1a\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e had consistently very low expression under control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). \u003cem\u003eCol-0\u003c/em\u003e showed increased expression of each downstream gene under TM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). \u003cem\u003ejagn1-OX\u003c/em\u003e also showed increased expression of each downstream gene under TM treatment; however, it was statistically lower than Col-0, indicating that overexpression of \u003cem\u003eJAGN1\u003c/em\u003e alone does not cause an automatic increase in the expression of the \u003cem\u003eIRE1\u003c/em\u003e arm of the UPR in response to TM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The \u003cem\u003ea2b4\u003c/em\u003e mutant showed no increase in expression of \u003cem\u003eSAR1a\u003c/em\u003e, suggesting expression of \u003cem\u003eIRE1\u003c/em\u003e is necessary for UPR induction of \u003cem\u003eSAR1a\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e mutant had a significant increase in \u003cem\u003eSAR1a\u003c/em\u003e expression compared to \u003cem\u003ea2b4\u003c/em\u003e, bringing it up to the same expression level as \u003cem\u003ejagn1-OX\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similarly, \u003cem\u003ePDI9\u003c/em\u003e expression is also rescued in the \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e line compared to \u003cem\u003ea2b4\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The increased expression of \u003cem\u003eSAR1a\u003c/em\u003e and \u003cem\u003ePDI9\u003c/em\u003e in the presence of \u003cem\u003eJAGN1\u003c/em\u003e overexpression and lack of \u003cem\u003eIRE1\u003c/em\u003e expression points to a possible mechanism for increased survival of the \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e line compared to \u003cem\u003ea2b4\u003c/em\u003e under TM stress (Figure V3). The \u003cem\u003ea2b4\u003c/em\u003e mutant had no change in expression of \u003cem\u003eBiP3\u003c/em\u003e and \u003cem\u003eNAC103\u003c/em\u003e in response to TM. Crossing \u003cem\u003ejagn1-OX\u003c/em\u003e into the \u003cem\u003ea2b4\u003c/em\u003e line caused no change in expression indicating a lack of phenotype rescue for \u003cem\u003eBiP3\u003c/em\u003e and \u003cem\u003eNAC103\u003c/em\u003e in the \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e line under TM stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). To see if there was any phenotype rescue associated with the \u003cem\u003ebZIP28\u003c/em\u003e arm of UPR after TM treatment, gene expression of \u003cem\u003eCRT2\u003c/em\u003e, \u003cem\u003eSDF2\u003c/em\u003e, and \u003cem\u003eP58IPK\u003c/em\u003e were analyzed. Across all genotypes, \u003cem\u003eCRT2\u003c/em\u003e, \u003cem\u003eSDF2\u003c/em\u003e, and \u003cem\u003eP58IPK\u003c/em\u003e were very low under control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). \u003cem\u003eCol-0\u003c/em\u003e showed an increase in \u003cem\u003eCRT2\u003c/em\u003e, \u003cem\u003eSDF2\u003c/em\u003e, and \u003cem\u003eP58IPK\u003c/em\u003e expression under TM treatment showing the \u003cem\u003ebZIP28\u003c/em\u003e arm was activated. The \u003cem\u003ebzip28\u003c/em\u003e mutant line had no increase in expression of the three genes investigated and was also not rescued by crossing \u003cem\u003ejagn1-OX\u003c/em\u003e into the \u003cem\u003ebzip28\u003c/em\u003e line, indicating a lack of a phenotype rescue for each of the measured genes in the \u003cem\u003ebZIP28\u003c/em\u003e pathway of the UPR. Collectively this points towards a closer correlation between \u003cem\u003eJAGN1\u003c/em\u003e and the \u003cem\u003eIRE1\u003c/em\u003e pathway than \u003cem\u003eJAGN1\u003c/em\u003e and the \u003cem\u003ebZIP28\u003c/em\u003e pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of genes downstream of the\u003c/b\u003e \u003cb\u003eIRE1\u003c/b\u003e \u003cb\u003epathway are increased in\u003c/b\u003e \u003cb\u003ejagn1-OX/a2b4\u003c/b\u003e \u003cb\u003eline compared to\u003c/b\u003e \u003cb\u003eIRE1\u003c/b\u003e \u003cb\u003edouble knockout in response to Monensin treatment\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOne week old seedlings were treated with 100 \u0026micro;M Monensin dissolved in ethanol for treatment and sterile MQ water with an equivalent volume of ethanol as the control. After 6 hours of treatment, samples were collected for RNA extraction. To determine that the UPR was active, \u003cem\u003ebZIP60\u003c/em\u003e unspliced and spliced transcripts were measured (Supplementary Fig.\u0026nbsp;3) and showed that under control conditions there was no expression of spliced \u003cem\u003ebZIP60\u003c/em\u003e for all genotypes. Under Mon stress, there was an increase in \u003cem\u003ebZIP60S\u003c/em\u003e transcript for all genotypes with an intact \u003cem\u003eIRE1\u003c/em\u003e pathway. The \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e mutant had no spliced \u003cem\u003ebZIP60\u003c/em\u003e indicating that overexpression of \u003cem\u003eJAGN1\u003c/em\u003e does not directly affect the beginning of the \u003cem\u003eIRE1\u003c/em\u003e pathway, the splicing of \u003cem\u003ebZIP60\u003c/em\u003e (Supplementary Fig.\u0026nbsp;3). For all genotypes, the 4 genes measured downstream of the \u003cem\u003eIRE1\u003c/em\u003e pathway, \u003cem\u003eBiP3\u003c/em\u003e, \u003cem\u003ePDI9\u003c/em\u003e, \u003cem\u003eSAR1a\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e had consistently very low expression under control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). \u003cem\u003eCol-0\u003c/em\u003e showed increased expression of each downstream gene under Mon treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). \u003cem\u003ejagn1-OX\u003c/em\u003e also showed increased expression of each downstream gene under Mon treatment, however it was statistically lower than \u003cem\u003eCol-0\u003c/em\u003e for \u003cem\u003eSAR1a\u003c/em\u003e, \u003cem\u003ePDI9\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e and was the same expression level as \u003cem\u003eCol-0\u003c/em\u003e for \u003cem\u003eBiP3\u003c/em\u003e, indicating that, as with TM, overexpression of \u003cem\u003eJAGN1\u003c/em\u003e alone does not cause a baseline increase in the expression of the \u003cem\u003eIRE1\u003c/em\u003e arm of the UPR in response to Mon (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The \u003cem\u003ea2b4\u003c/em\u003e mutant had a very small increase in the expression of \u003cem\u003eBiP3\u003c/em\u003e under Mon stress, but was still the lowest out of all genotypes which suggests \u003cem\u003eIRE1\u003c/em\u003e function is necessary for a complete \u003cem\u003eBiP3\u003c/em\u003e transcriptional response to Mon. However, the \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e line had a significant increase in the expression of \u003cem\u003eBiP3\u003c/em\u003e compared to \u003cem\u003ea2b4\u003c/em\u003e showing that increased expression of \u003cem\u003eJAGN1\u003c/em\u003e in the absence of a functional \u003cem\u003eIRE1\u003c/em\u003e pathway results in increased expression of certain UPR genes. The \u003cem\u003ea2b4\u003c/em\u003e mutant had no change in expression of \u003cem\u003ePDI9\u003c/em\u003e, \u003cem\u003eSAR1a\u003c/em\u003e, or \u003cem\u003eNAC103\u003c/em\u003e in response to Mon (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D, E). The \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e line did not show any increase in expression of these genes compared to \u003cem\u003ea2b4\u003c/em\u003e indicating a lack of phenotype rescue for \u003cem\u003ePDI9\u003c/em\u003e, \u003cem\u003eSAR1a\u003c/em\u003e, \u003cem\u003eNAC103\u003c/em\u003e in response to Mon. There was an increase in \u003cem\u003ebZIP28\u003c/em\u003e downstream gene expression for \u003cem\u003eCol-0\u003c/em\u003e in the treatment. However, there was no change in expression of \u003cem\u003eCRT2\u003c/em\u003e, \u003cem\u003eSDF2\u003c/em\u003e, or \u003cem\u003eP58IPK\u003c/em\u003e for \u003cem\u003ejagn1-OX/bzip28\u003c/em\u003e compared to \u003cem\u003ebzip28\u003c/em\u003e. As with TM, this too points towards a closer correlation between \u003cem\u003eJAGN1\u003c/em\u003e and the \u003cem\u003eIRE1\u003c/em\u003e pathway than \u003cem\u003eJAGN1\u003c/em\u003e and the \u003cem\u003ebZIP28\u003c/em\u003e pathway\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of genes downstream of the\u003c/b\u003e \u003cb\u003eIRE1\u003c/b\u003e \u003cb\u003epathway are increased in\u003c/b\u003e \u003cb\u003ejagn1-OX/a2b4\u003c/b\u003e \u003cb\u003eline compared to\u003c/b\u003e \u003cb\u003eIRE1\u003c/b\u003e \u003cb\u003edouble knockout in response to 2-deoxyglucose treatment\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOne week old seedlings were treated with 10 mM 2-DG for treatment and sterile MQ water as the control. After 6 hours of treatment, samples were collected for RNA extraction. To determine that the UPR was active, \u003cem\u003ebZIP60\u003c/em\u003e unspliced and spliced transcripts were measured (Supplementary Fig.\u0026nbsp;3) and showed that under control conditions there was no expression of spliced \u003cem\u003ebZIP60\u003c/em\u003e for all genotypes. Under 2-DG stress, there was an increase in \u003cem\u003ebZIP60S\u003c/em\u003e transcript for all genotypes with an intact \u003cem\u003eIRE1\u003c/em\u003e pathway. The \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e mutant had no spliced \u003cem\u003ebZIP60\u003c/em\u003e indicating that, as would be expected, overexpression of \u003cem\u003eJAGN1\u003c/em\u003e does not directly affect the beginning of the \u003cem\u003eIRE1\u003c/em\u003e pathway, the splicing of \u003cem\u003ebZIP60\u003c/em\u003e (Supplementary Fig.\u0026nbsp;3).There was a significant increase in all \u003cem\u003eIRE1\u003c/em\u003e downstream genes in \u003cem\u003eCol-0\u003c/em\u003e in the presence of 2-DG, likewise there was a significant increase in all \u003cem\u003eIRE1\u003c/em\u003e downstream genes in \u003cem\u003ejagn1-OX\u003c/em\u003e in response to 2-DG, however they were all either equal to or less than \u003cem\u003eCol\u003c/em\u003e-\u003cem\u003e0\u003c/em\u003e expression levels indicating overexpression of \u003cem\u003eJAGN\u003c/em\u003e1 alone does not cause a baseline increase in the expression of \u003cem\u003eIRE1\u003c/em\u003e downstream genes in the presence of 2-DG. \u003cem\u003ea2b4\u003c/em\u003e showed no change expression with 2-DG treatment for \u003cem\u003eBiP3\u003c/em\u003e, \u003cem\u003eSAR1a\u003c/em\u003e, or \u003cem\u003eNAC103\u003c/em\u003e indicating the need for \u003cem\u003eIRE1\u003c/em\u003e to be functional for expression of those downstream genes. \u003cem\u003ePDI9\u003c/em\u003e did have increased expression in \u003cem\u003ea2b4\u003c/em\u003e treated with 2-DG but was lower than that of \u003cem\u003eCol-0\u003c/em\u003e. For all four genes tested downstream in the \u003cem\u003eIRE1\u003c/em\u003e pathway, \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e had significantly increased expression compared to \u003cem\u003ea2b4\u003c/em\u003e. For all \u003cem\u003ebZIP28\u003c/em\u003e pathway downstream genes tested, \u003cem\u003eCol-0\u003c/em\u003e had increased expression under 2-DG stress compared to the control treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE,F,G). The \u003cem\u003ebzip28\u003c/em\u003e mutant did not have any increase of genes under 2-DG treatment showing functional \u003cem\u003ebZIP28\u003c/em\u003e is necessary for their expression. The \u003cem\u003ejagn1-OX/bzip28\u003c/em\u003e mutant did not have any change of expression compared to the \u003cem\u003ebzip28\u003c/em\u003e mutant. As with TM and Mon, this again points towards a closer correlation between \u003cem\u003eJAGN1\u003c/em\u003e and the \u003cem\u003eIRE1\u003c/em\u003e pathway than \u003cem\u003eJAGN1\u003c/em\u003e and the \u003cem\u003ebZIP28\u003c/em\u003e pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003eH\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eproduction does not appear to be affected by\u003c/b\u003e \u003cb\u003eJAGN1\u003c/b\u003e \u003cb\u003eoverexpressor crossed into\u003c/b\u003e \u003cb\u003eIRE1\u003c/b\u003e \u003cb\u003edouble knockout or\u003c/b\u003e \u003cb\u003ebZIP28\u003c/b\u003e \u003cb\u003eknockout\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs a secondary experiment for PQ stress, a leaf infiltration of 3 \u0026micro;M PQ, followed by DAB staining for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production was conducted and found no observable phenotypic difference in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production across genotypes (Supplemental Fig.\u0026nbsp;4) that would explain the decrease in survival of the \u003cem\u003ejagn1-OX/bzip28\u003c/em\u003e line seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE. This means that the mechanism for change in survival in response to PQ is most likely not centered around H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated the functional conservation of \u003cem\u003eJAGN1\u003c/em\u003e in plants. While the function of this gene has been explored in fruit flies \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, humans/human cell lines \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, mice \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and zebrafish \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, to our knowledge, this is the first evidence of functional conservation of \u003cem\u003eJAGN1\u003c/em\u003e across animal to plant kingdoms. We found that increased expression of \u003cem\u003eJAGN1\u003c/em\u003e in \u003cem\u003eIRE1\u003c/em\u003e knockouts of Arabidopsis partially rescues survival and partially rescues expression of UPR genes in response to ER stress.\u003c/p\u003e \u003cp\u003eWhile many animal models looking at \u003cem\u003eJAGN1\u003c/em\u003e function focus on immune related phenotypes such as neutrophil maturation \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, or antibody production \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, we did not find immunity to be an obvious phenotype associated with \u003cem\u003eJAGN1\u003c/em\u003e in Arabidopsis. The lack of an extreme phenotype seen with pathogen infection in plants is likely because of the inherent differences in plant and animal immunity. While animals have specialized immune cells such as neutrophils that produce a large number of antimicrobial proteins in response to infection, plants lack specialized immune cells and instead, all cells are capable of mounting an immune response \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Because there are no specialized immune cells in plants, there is not a specific cell type that can be targeted for observing how \u003cem\u003eJAGN1\u003c/em\u003e affects immune response that would be directly comparable to that of animal research.\u003c/p\u003e \u003cp\u003eUpon targeted UPR stress, noticeable phenotype changes due to \u003cem\u003eJAGN1\u003c/em\u003e expression level became evident. The increased survival for an \u003cem\u003eIRE1\u003c/em\u003e deficient plant with overexpression of \u003cem\u003eJAGN1\u003c/em\u003e leads to a few possibilities. 1. \u003cem\u003eIRE1\u003c/em\u003e and \u003cem\u003eJAGN1\u003c/em\u003e compete for function and in the absence of \u003cem\u003eIRE1\u003c/em\u003e, \u003cem\u003eJAGN1\u003c/em\u003e can function to a higher capacity, i.e. an antagonistic relationship between the two genes. Or, 2. \u003cem\u003eIRE1\u003c/em\u003e and \u003cem\u003eJAGN1\u003c/em\u003e have some level of functional redundancy so in the absence of \u003cem\u003eIRE1\u003c/em\u003e, \u003cem\u003eJAGN1\u003c/em\u003e compensates, i.e., a compensatory relationship. Previous studies in animal models have shown that there is an interplay between \u003cem\u003eJAGN1\u003c/em\u003e and the \u003cem\u003eIRE1\u003c/em\u003e pathway. In mice plasmablasts with mutated jagn, there is increased splicing of \u003cem\u003eXBP1\u003c/em\u003e \u003csup\u003e21\u003c/sup\u003e, in insulin producing cells, \u003cem\u003eJAGN1\u003c/em\u003e transcription increased after TM treatment \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and in zebrafish embryos with either mutated or silenced \u003cem\u003eJAGN1b\u003c/em\u003e, there was an increase in UPR gene expression \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e all indicating that lack of \u003cem\u003eJAGN1\u003c/em\u003e increased ER stress. This trend of decreased \u003cem\u003eJAGN1\u003c/em\u003e function resulting in heightened ER stress is in line with our observations that increased \u003cem\u003eJAGN1\u003c/em\u003e expression led to decreased ER stress.\u003c/p\u003e \u003cp\u003eWhen looking at gene expression, there was a trend of increased expression of genes downstream of the \u003cem\u003eIRE1\u003c/em\u003e pathway in the \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e line compared to \u003cem\u003ea2b4\u003c/em\u003e suggesting that overexpression of \u003cem\u003eJAGN1\u003c/em\u003e causes an \u003cem\u003eIRE1\u003c/em\u003e-independent change in \u003cem\u003eIRE1\u003c/em\u003e pathway gene expression (Figures U,T,S). Specifically, there was a statistically significant increase in expression of \u003cem\u003ePDI9\u003c/em\u003e and \u003cem\u003eSAR1a\u003c/em\u003e in the presence of TM, \u003cem\u003eBiP3\u003c/em\u003e in the presence of Mon, and \u003cem\u003ePDI9\u003c/em\u003e, \u003cem\u003eSAR1a\u003c/em\u003e, and \u003cem\u003eBiP3\u003c/em\u003e in the presence of 2-DG for \u003cem\u003ejagn1\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e/\u003cem\u003ea2b4\u003c/em\u003e compared to \u003cem\u003ea2b4\u003c/em\u003e. \u003cem\u003eIRE1\u003c/em\u003e double knockouts exposed to TM, have drastically decreased \u003cem\u003eBiP3\u003c/em\u003e and \u003cem\u003eSAR1a\u003c/em\u003e compared to wild type \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e which is recapitulated in our results. Thus, the increased expression of \u003cem\u003eBiP3\u003c/em\u003e and \u003cem\u003eSAR1a\u003c/em\u003e in the \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e line points towards a novel observation of the involvement of \u003cem\u003eJAGN1\u003c/em\u003e in the UPR of plants. BiP is an important chaperone protein within the ER and in unstressed states, BiP binds to the luminal domain of IRE1 keeping it in the non-active monomeric state \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eBiP3\u003c/em\u003e expression is highly induced during abiotic or ER stress as a target of the active \u003cem\u003ebZIP60\u003c/em\u003e transcription factor \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Protein disulfide isomerase 9 (\u003cem\u003ePDI9)\u003c/em\u003e is another chaperone protein that resides in the ER where it catalyzes disulfide bond formation and isomerization, thus increasing the protein folding capacity of the ER \u003csup\u003e46\u0026ndash;48\u003c/sup\u003e. \u003cem\u003ePDI9\u003c/em\u003e is upregulated during UPR and is a target of the \u003cem\u003ebZIP60\u003c/em\u003e TF, placing it in the \u003cem\u003eIRE1\u003c/em\u003e arm of UPR in plants \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. PDI9 also participates in a negative feedback loop where a low demand for catalysis via \u003cem\u003ePDI9\u003c/em\u003e allows for high association with \u003cem\u003eIRE1\u003c/em\u003e, diminishing IRE1 pathway activation but when PDI9 chaperone function is needed, it dissociates from IRE1 thereby increasing activation of the \u003cem\u003eIRE1\u003c/em\u003e pathway\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Because \u003cem\u003ePDI9\u003c/em\u003e and \u003cem\u003eBiP3\u003c/em\u003e are both ER resident chaperone proteins \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, the increased expression of each in \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e versus \u003cem\u003ea2b4\u003c/em\u003e would increase the protein folding capacity of the ER and is, at least in part, the reason for the increased survival of \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e compared to \u003cem\u003ea2b4\u003c/em\u003e under ER stress. While \u003cem\u003ePDI9\u003c/em\u003e expression is primarily regulated by the \u003cem\u003eIRE1\u003c/em\u003e pathway, the observed increased \u003cem\u003ePDI9\u003c/em\u003e expression in \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e compared to \u003cem\u003ea2b4\u003c/em\u003e aligns with a previous finding that \u003cem\u003ebZIP60\u003c/em\u003e is largely, but not solely responsible for \u003cem\u003ePDI9\u003c/em\u003e expression in response to stress \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Therefore, \u003cem\u003eJAGN1\u003c/em\u003e overexpression is interacting with a separate mechanism to increase \u003cem\u003ePDI9\u003c/em\u003e expression in response to stress, independently of IRE1. This finding supports the idea that \u003cem\u003eJAGN1\u003c/em\u003e and \u003cem\u003eIRE1\u003c/em\u003e are behaving in a compensatory manner in this experiment.\u003c/p\u003e \u003cp\u003eSecretion-associated and RAS superfamily-related protein 1a (\u003cem\u003eSAR1a)\u003c/em\u003e is a small GTPase and part of the COPII protein complex responsible for cargo transport from the ER to the Golgi \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. SAR1-GTP anchors the COPII complex to the membrane via its amphipathic helix \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Mutations of the Sar1 protein in yeast have been shown to block vesicles from reaching the Golgi, highlighting the important role of \u003cem\u003eSAR1\u003c/em\u003e in ER to Golgi traffic \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eSAR1a\u003c/em\u003e expression in response to ER stress is most closely associated with bZIP60, thus placing it downstream of the \u003cem\u003eIRE1\u003c/em\u003e pathway \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The correlation between higher \u003cem\u003eJAGN1\u003c/em\u003e expression and higher \u003cem\u003eSAR1a\u003c/em\u003e expression suggests that like ERVs, \u003cem\u003eJAGN1\u003c/em\u003e could be participating in anterograde movement of cargo from the ER to the Golgi. Like \u003cem\u003eBiP3\u003c/em\u003e and \u003cem\u003ePDI9\u003c/em\u003e, \u003cem\u003eSAR1a\u003c/em\u003e expression must have alternative pathways capable of increasing expression independently of \u003cem\u003ebZIP60\u003c/em\u003e, and although our data cannot identify the exact pathway responsible. Regardless of the mechanism by which it happens, the increased expression of SAR1a in \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e compared to \u003cem\u003ea2b4\u003c/em\u003e should increase the amount of protein trafficking able to advance from ER to Golgi via COPII movement. Increased movement of proteins from the ER to the Golgi reduces the overall protein burden of the ER, and is likely another explanation for the increased survival seen in the \u003cem\u003eIRE1\u003c/em\u003e knockdown plants with \u003cem\u003eJAGN1\u003c/em\u003e overexpression. Overall, it seems that \u003cem\u003eJAGN1\u003c/em\u003e and \u003cem\u003eIRE1\u003c/em\u003e both work to increase a cell\u0026rsquo;s ability to withstand ER stress and have at least partially overlapping pathways. Therefore, when one pathway is nonfunctional, in this case \u003cem\u003eIRE1\u003c/em\u003e, the other pathway, i.e., overexpression of \u003cem\u003eJAGN1\u003c/em\u003e, can partially compensate for its absence.\u003c/p\u003e \u003cp\u003eThe decreased survival of \u003cem\u003ejagn1-OX/bzip28\u003c/em\u003e compared to \u003cem\u003ebzip28\u003c/em\u003e with TM, Mon, heat, and especially PQ, suggests that 1. \u003cem\u003ebZIP28\u003c/em\u003e and \u003cem\u003eJAGN1\u003c/em\u003e either work independently to increase cell survival, i.e., they are additive. Or, 2. they interact in such a way that both need to be functional simultaneously for a proper stress response to occur, i.e. they are complementary to one another (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Contrary to \u003cem\u003eIRE1\u003c/em\u003e, there was no change in gene expression of examined genes downstream of the bZIP28 pathway for \u003cem\u003ejagn1\u003c/em\u003e-OX/bzip28 compared to \u003cem\u003ebzip28\u003c/em\u003e upon treatment with TM, Mon, or 2-DG. Thus, based on the genes investigated here, \u003cem\u003eJAGN1\u003c/em\u003e does not interact with the \u003cem\u003ebZIP28\u003c/em\u003e pathway on the level of gene expression. \u003cem\u003eCRT2\u003c/em\u003e (calreticulin 2) is an ER resident chaperone that regulates intracellular calcium levels \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. CRT2 expression in response to ER stress is regulated by the \u003cem\u003ebZIP28\u003c/em\u003e pathway of the UPR \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eSDF2\u003c/em\u003e (stromal cell-derived factor 2) is conserved across plants and animals and is activated during UPR \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. It is an ER-resident protein that has been demonstrated to be integral in Arabidopsis seedling response to UPR eliciting chemicals \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. While the exact mechanism by which \u003cem\u003eSDF2\u003c/em\u003e functions in UPR is still not known, it is thought to participate in the quality control of glycoproteins within the ER \u003csup\u003e54,56\u003c/sup\u003e. \u003cem\u003eP58IPK (58 kDa inhibitor of protein kinase)\u003c/em\u003e is primarily regulated by the \u003cem\u003ebZIP28\u003c/em\u003e pathway of the UPR \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Though this data cannot explain the reason for decreased survival of \u003cem\u003ejagn1-OX/bzip28\u003c/em\u003e compared to \u003cem\u003ebzip28\u003c/em\u003e, this is an interesting finding and points towards a need to explore this relationship further in animal models where a similar interaction could exist.\u003c/p\u003e \u003cp\u003eWhile the list of genes analyzed here for expression changes is not all inclusive, it can still be concluded that overexpression of \u003cem\u003eJAGN1\u003c/em\u003e in an \u003cem\u003eIRE1\u003c/em\u003e knockout causes expression changes in genes downstream of \u003cem\u003eIRE1\u003c/em\u003e independently of \u003cem\u003eIRE1\u003c/em\u003e function. On the other hand, overexpression of \u003cem\u003eJAGN1\u003c/em\u003e in a \u003cem\u003ebZIP28\u003c/em\u003e mutant does not cause expression changes of \u003cem\u003ebZIP28\u003c/em\u003e downstream genes independently of \u003cem\u003ebZIP28\u003c/em\u003e function.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eIRE1\u003c/em\u003e and \u003cem\u003ebZIP28\u003c/em\u003e, as with many functionally redundant pathways, if there is an absence of one, the other pathway will compensate. This is displayed well in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-G, where the \u003cem\u003ea2b4\u003c/em\u003e mutant shows a marked increase in \u003cem\u003ebZIP28\u003c/em\u003e pathway gene expression under Mon stress. Importantly, overexpression of \u003cem\u003eJAGN1\u003c/em\u003e in each respective UPR pathway mutant does not induce changes in gene expression for the opposite UPR pathway. Thus, it seems that overexpression of \u003cem\u003eJAGN1\u003c/em\u003e does not simply enhance the innate compensation of one UPR pathway for the absence of another, there is a more delicate interaction at play.\u003c/p\u003e \u003cp\u003eThis study focuses primarily on mRNA expression and phenotype analysis and is lacking protein level analysis. Localization of the \u003cem\u003eJAGN1\u003c/em\u003e protein in Arabidopsis to confirm it is an ER membrane protein would be helpful in further confirming the conservation of this gene across kingdoms. Additionally, a Co-IP experiment looking at interacting partners of \u003cem\u003eJAGN1\u003c/em\u003e would help to further delineate the function of the protein in plant cells. If the interacting partners were orthologs of the interacting proteins in animal cells, this would again further confirm the functional conservation. A knock-down or knock-out of \u003cem\u003eJAGN1\u003c/em\u003e in plants would allow for additional reverse genetics approaches to determine the function of the gene and/or whether a \u003cem\u003eJAGN1\u003c/em\u003e knockout is a survivable phenotype in plants. Another interesting future approach could be expressing known SCN mutations of \u003cem\u003eJAGN1\u003c/em\u003e in humans in Arabidopsis. Expression of human proteins (Human beta defensin 2 and three antiapoptotic genes) in plant systems has been shown effective at increasing resistance to pathogens, which shows that expression of human proteins in plant systems can uncover shared functionality in cellular responses \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Especially with the convenience of Arabidopsis providing a unique platform to look at the implications of \u003cem\u003eIRE1\u003c/em\u003e double knockouts alongside \u003cem\u003eJAGN1\u003c/em\u003e mutations, this could help uncover cellular phenotypes without the limitation of \u003cem\u003eIRE1\u003c/em\u003e knockout lethality present in animal models.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our data demonstrates that \u003cem\u003eJAGN1\u003c/em\u003e plays a role in the ER stress response of plants, which indicates functional conservation of this gene across plant and animal kingdoms. This suggests that JAGN1 is a player in the UPR where it decreases the protein folding burden of the ER Overexpression of \u003cem\u003eJAGN1\u003c/em\u003e in an \u003cem\u003eIRE1\u003c/em\u003e double knockout plant under ER stress partially rescues survival of plants experiencing ER stress and increases expression of the \u003cem\u003eIRE1\u003c/em\u003e downstream genes, \u003cem\u003eSAR1a\u003c/em\u003e, \u003cem\u003ePDI9\u003c/em\u003e, and \u003cem\u003eBiP3\u003c/em\u003e in an \u003cem\u003eIRE1\u003c/em\u003e independent manner. This increased gene expression of two chaperone proteins, \u003cem\u003ePDI9\u003c/em\u003e and \u003cem\u003eBiP3\u003c/em\u003e, and a component of the COPII pathway, \u003cem\u003eSAR1a\u003c/em\u003e, can be attributed to the increased survival of plants during ER stress. \u003cem\u003eJAGN1\u003c/em\u003e overexpression in a \u003cem\u003ebZIP28\u003c/em\u003e mutant decreases survival of stressed plants and does not change expression of the \u003cem\u003ebZIP28\u003c/em\u003e downstream genes, \u003cem\u003eCRT2\u003c/em\u003e, \u003cem\u003eSDF2\u003c/em\u003e, or \u003cem\u003eP58IPK\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth:\u003c/h2\u003e \u003cp\u003eSoil grown plants were grown in growth chambers at 22\u0026deg;C, 12-hour light/dark cycle at 220 \u0026micro;mol/m\u003csup\u003e2\u003c/sup\u003es.Tissue culture plants were surface sterilized and grown on \u0026frac12; MS media with 1% sucrose, 50 \u0026micro;g/mL ampicillin, and 1% agar. Plates were sealed with micropore tape and placed in growth chambers with the same conditions as soil grown plants. All seeds were stratified at 4\u0026deg;C in the dark for 1 week before being put under light.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenotyping JAGN1:\u003c/h3\u003e\n\u003cp\u003eTo confirm homozygosity of T-DNA insertion, gene specific (JLP geno F/ JLP geno R) and insertion specific (LBb1.3/JLP geno R) primers were used. To determine JAGN1 expression in T-DNA insertion line, three combinations of 4 primers were used, (F1/R1, F1/R2, F2,R2) for RT-qPCR of 2-week-old seedlings grown on \u0026frac12; MS media. For sequencing, JLP Geno R/LBb1.3 were used to amplify genomic DNA from the JAGN1 T-DNA insertion line with a sequence of ~\u0026thinsp;800\u0026ndash;900 bp beginning at the 4\u0026rsquo;th exon. This template with the JLP geno R primer was sent for sequencing. Sequence alignment with documented gene sequence was used to determine the location of the T-DNA insertion.\u003c/p\u003e\n\u003ch3\u003ePlant genetic lines used:\u003c/h3\u003e\n\u003cp\u003eAll genotypes in this paper are in the \u003cem\u003eColumbia-0\u003c/em\u003e ecotype background, thus \u003cem\u003eCol-0\u003c/em\u003e was used as wild type control. The \u003cem\u003eJAGN1\u003c/em\u003e T-DNA insertion line used is SALK_065212C. The \u003cem\u003eIRE1\u003c/em\u003e double knockout used is \u003cem\u003eire1a-2/ire1b-4\u003c/em\u003e. The \u003cem\u003eire1a-2\u003c/em\u003e mutant line is SALK_018112 and the \u003cem\u003eire1b-4\u003c/em\u003e mutant line is SAIL_238_F07. The bZIP28 mutant used is SALK_132285.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePlant crosses:\u003c/h2\u003e \u003cp\u003eCrosses were performed via cross pollination of \u003cem\u003ejagn1-OX\u003c/em\u003e to \u003cem\u003eire1a-2b-4\u003c/em\u003e and crossing \u003cem\u003ejagn1-OX\u003c/em\u003e to \u003cem\u003ebzip28\u003c/em\u003e. F\u003csub\u003e1\u003c/sub\u003e plants were genotyped for successful crossing and F\u003csub\u003e2\u003c/sub\u003e plants were genotyped for homozygosity of all individual genes. For \u003cem\u003ejagn1-OX/a2b4\u003c/em\u003e this meant homozygosity of \u003cem\u003ejagn1-OX\u003c/em\u003e, \u003cem\u003eire1a-2\u003c/em\u003e, and \u003cem\u003eire1b-4\u003c/em\u003e. For \u003cem\u003ejagn1-OX/bzip28\u003c/em\u003e this meant homozygosity of \u003cem\u003ejagn1-OX\u003c/em\u003e and \u003cem\u003ebzip28\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePathogen assay:\u003c/h2\u003e \u003cp\u003eFour-week-old plants, 3 leaves per plant, were syringe infiltrated with 0.001 OD600 of Pseudomonas syringae \u003cem\u003ePst DC3000\u003c/em\u003e suspended in 10 mM MgCl2. Protocol from [paper about syringe infiltration and dilutions] was followed. Graph is representative of 3 biological replicates with each replicate containing 6 plants, 3 leaves per plant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIon leakage assay:\u003c/h2\u003e \u003cp\u003eFour-week-old plants, 3 leaves per plant, were removed and vacuum infiltrated with 0.1 OD\u003csub\u003e600\u003c/sub\u003e of \u003cem\u003ePseudomonas syringae Psm ES4326\u003c/em\u003e in sterile MQ water. Leaves were added to 50 mL conical tubes containing 15 uL of sterile MQ water and conductivity was measured every 30 minutes for 8 hours. Each tube was then autoclaved to leak all remaining ions, and this was used to compare each measurement as a percentage of total ions leaked.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRT-qPCR:\u003c/h2\u003e \u003cp\u003eRNA extraction was performed using Zymo Quick RNA mini-prep kit (R1055). Tissue was homogenized while frozen with liquid nitrogen and the first step of Trizol/chloroform extraction was used. After centrifugation at 4\u0026deg;C for 15 minutes, the top layer was added to the RNA extraction kit and followed the protocol as written. cDNA synthesis was performed using AB Clonal Kit (RK20400). qPCR was run using AB Clonal Sybr Green Master Mix (RK2103) on a Quant Studio 3. Ubiquitin 5 was used as a housekeeping gene for relative expression quantification. Each biological sample had three technical replicates per plate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePlant weight Tunicamycin stress:\u003c/h2\u003e \u003cp\u003eSeedlings were grown on \u0026frac12; MS media for 5 days before being transferred to liquid \u0026frac12; MS in a 12-well plate with 10 seedlings per well. Low tunicamycin concentration was 0.75 \u0026micro;g/mL and high concentration was 1.5 \u0026micro;g/mL. Seedlings were left to grow for 4 more days, then fresh weight of each pooled sample was measured. Analyzed via ANOVA followed by Tukey\u0026rsquo;s HSD represented with letters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSurvivability Assay:\u003c/h2\u003e \u003cp\u003eAll seeds were surface sterilized then stratified in sterile MQ water for 7 days at 4\u0026deg;C before being placed on freshly prepared \u0026frac12; MS media with the stressor added in then placed under light. For Tunicamycin 0.1 \u0026micro;g/mL, Monensin 2.5 \u0026micro;M, 2-deoxyglucose 3 mM, paraquat 1 \u0026micro;M. Heat treated seeds were treated in a 1.5 mL tube filled with sterile MQ water in a benchtop tube warmer at 50\u0026deg;C for 1 hour before being placed on freshly prepared \u0026frac12; MS media and put directly to light. TM, Mon, and 2-DG were all plated on the same day and used the same control plate for individual comparison. Likewise, paraquat and heat seeds were plated on the same day and used the same control plates for individual comparisons. Each plate had all 6 genotypes, 30 seeds per genotype. All conditions had 7 plates. Plates were imaged on day 10 of growth. Survivability was measured as number of seedlings surviving on treatment divided by number of seedlings surviving on control plate represented as a percentage. Analyzed via two-way ANOVA followed by a Tukey\u0026rsquo;s HSD represented as letters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eER stressors for gene expression analysis\u003c/h2\u003e \u003cp\u003eSeedlings were grown vertically on \u0026frac12; MS media for 12 days, then transferred to 6 well plates with sterile MQ water and dissolved stressors. Each well had 10 seedlings. Tunicamycin treatment was 5 \u0026micro;g/mL dissolved in DMSO and control was the equivalent concentration of DMSO. Monensin treatment was 100 \u0026micro;M dissolved in ethanol and control was the equivalent concentration of ethanol. 2-deoxyglucose treatment was 10 mM dissolved in water and control was just MQ water. For each treatment there were 3 biological replicates of pooled samples of each genotype in each condition. All treatments lasted 6 hours and samples were flash frozen in liquid nitrogen for RNA extraction preparation. Analyzed via two-way ANOVA followed by a Tukey\u0026rsquo;s HSD represented as letters.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization,\u0026nbsp;R.B. and K.P.M.; experiments, data analysis and figure preparation,\u0026nbsp;R.B., writing-original draft preparation, R.B.; writing-review and editing,\u0026nbsp;K.P.M.; supervision, K.P.M.; project coordination and administration, K.P.M.; funding acquisition, R.B. and K.P.M.\u0026nbsp;Both\u0026nbsp;authors have agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Dr. Elizabeth Sztul for helpful discussions. This study was funded by This work was funded under NSF Award #IOS-2038872 to K.P.M and a Sigma Xi Grant in Aid of Research G2022315-2135 awarded to R.B. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors declare no financial or non-financial competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBao, Y. \u0026amp; Howell, S. H. 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B. \u003cem\u003eet al.\u003c/em\u003e Abrogation of disease development in plants expressing animal antiapoptotic genes. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 98, 6957\u0026ndash;6962 (2001).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8893802/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8893802/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eJagunal homolog 1 (JAGN1) is essential for endoplasmic reticulum (ER) organization and function in animals, where mutations cause severe congenital neutropenia in humans. Although JAGN1 function is conserved across animal species, its role in plants has not been examined. Here, we investigated JAGN1 function in Arabidopsis using a transgenic line with elevated JAGN1 expression exposed to ER stress inducers. Increased JAGN1 expression in \u003cem\u003eInositol Requiring Enzyme 1 (ire1) \u003c/em\u003edouble knockout plants partially restored survivability under ER stress and enhanced expression of unfolded protein response (UPR) genes, including \u003cem\u003eBiP3\u003c/em\u003e, \u003cem\u003ePDI9\u003c/em\u003e, and \u003cem\u003eSAR1a\u003c/em\u003e, indicating an IRE1-independent mechanism. In contrast, crossing the JAGN1 overexpression line with \u003cem\u003eBasic Leucine Zipper 28\u003c/em\u003e (\u003cem\u003ebZIP28\u003c/em\u003e) mutants reduced survival and failed to alter downstream gene expression. These results provide the first evidence that JAGN1 function is conserved across plant and animal kingdoms and establish Arabidopsis as a model system to study JAGN1 biology and disease-associated mutations.\u003c/p\u003e","manuscriptTitle":"Enhanced JAGN1 expression Modulates ER Stress Signaling and Promotes Survival in IRE1-Deficient Arabidopsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 13:11:49","doi":"10.21203/rs.3.rs-8893802/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d89c27c4-3c53-4fd9-a8e2-856a79a0c5b7","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63704554,"name":"Biological sciences/Biotechnology"},{"id":63704555,"name":"Biological sciences/Genetics"},{"id":63704556,"name":"Biological sciences/Molecular biology"},{"id":63704557,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-04-01T20:54:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 13:11:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8893802","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8893802","identity":"rs-8893802","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}