RESUMO
We have developed a one-credit semester-long research experience for undergraduate students that involves the use of CRISPR/Cas9 to edit genes in zebrafish. The course is available to students at all stages of their undergraduate training and can be taken up to four times. Students select a gene of interest to edit as the basis of their semester-long project. To select a gene, exploration of developmental processes and human disease is encouraged. As part of the course, students use basic bioinformatic tools, design guide RNAs, inject zebrafish embryos, and analyze both the molecular consequences of gene editing and phenotypic outcomes. Over the 10 years we have offered the course, enrollment has grown from less than 10 students to more than 60 students per semester. Each year, we choose a different gene editing strategy to explore based on recent publications of gene editing methodologies. These have included making CRISPants, targeted integrations, and large gene deletions. In this study, we present how we structure the course and our assessment of the course over the past 3 years.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Animais , Edição de Genes/métodos , Peixe-Zebra/genética , RNA Guia de Sistemas CRISPR-Cas , EstudantesRESUMO
Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFPis86 line to examine the behavior and interactions between cells during vascular formation in zebrafish.
Assuntos
Sistemas CRISPR-Cas , Peixe-Zebra , Animais , Peixe-Zebra/genética , Proteína Vermelha Fluorescente , Biotina/genética , Células Endoteliais , RNA Guia de Sistemas CRISPR-CasRESUMO
The accumulation of misfolded proteins in the endoplasmic reticulum (ER) defines a condition called ER stress that induces the unfolded protein response (UPR). The UPR in mammalian cells attenuates protein synthesis initiation, which prevents the piling up of misfolded proteins in the ER. Mammalian cells rely on Protein Kinase RNA-Like Endoplasmic Reticulum Kinase (PERK) phosphorylation of eIF2α to arrest protein synthesis, however, plants do not have a PERK homolog, so the question is whether plants control translation in response to ER stress. We compared changes in RNA levels in the transcriptome to the RNA levels protected by ribosomes and found a decline in translation efficiency, including many UPR genes, in response to ER stress. The decline in translation efficiency is due to the fact that many mRNAs are not loaded onto polyribosomes (polysomes) in proportion to their increase in total RNA, instead some of the transcripts accumulate in stress granules (SGs). The RNAs that populate SGs are not derived from the disassembly of polysomes because protein synthesis remains steady during stress. Thus, the surge in transcription of UPR genes in response to ER stress is accompanied by the formation of SGs, and the sequestration of mRNAs in SGs may serve to temporarily relieve the translation load during ER stress.
RESUMO
The unfolded protein response (UPR) and the heat shock response (HSR) are two evolutionarily conserved systems that protect plants from heat stress. The UPR and HSR occur in different cellular compartments and both responses are elicited by misfolded proteins that accumulate under adverse environmental conditions such as heat stress. While the UPR and HSR appear to operate independently, we have found a link between them in maize (Zea mays) involving the production of the BASIC LEUCINE ZIPPER60 (bZIP60) transcription factor, a pivotal response of the UPR to heat stress. Surprisingly, a mutant (bzip60-2) knocking down bZIP60 expression blunted the HSR at elevated temperatures and prevented the normal upregulation of a group of heat shock protein genes in response to elevated temperature. The expression of a key HEAT SHOCK FACTOR TRANSCRIPTION FACTOR13 (HSFTF13, a HEAT SHOCK FACTOR A6B [HSFA6B] family member) was compromised in bzip60-2, and the HSFTF13 promoter was shown to be a target of bZIP60 in maize protoplasts. In addition, the upregulation by heat of genes involved in chlorophyll catabolism and chloroplast protein turnover were subdued in bzip60-2, and these genes were also found to be targets of bZIP60. Thus, the UPR, an endoplasmic-reticulum-associated response, quite unexpectedly contributes to the nuclear/cytoplasmic HSR in maize.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Resposta ao Choque Térmico/fisiologia , Proteínas de Plantas/genética , Resposta a Proteínas não Dobradas/fisiologia , Zea mays/fisiologia , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Clorofila/metabolismo , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico/genética , Proteínas de Plantas/metabolismo , Splicing de RNA , Resposta a Proteínas não Dobradas/genética , Zea mays/crescimento & desenvolvimentoRESUMO
Adverse environmental conditions such as heat and salt stress create endoplasmic reticulum (ER) stress in maize and set off the unfolded protein response (UPR). A key feature of the UPR is the upregulation of ZmbZIP60 and the splicing of its messenger RNA. We conducted an association analysis of a recombinant inbred line (RIL) derived from a cross of a tropical founder line, CML52 with a standard temperate line, B73. We found a major QTL conditioning heat-induced ZmbZIP60 expression located cis to the gene. Based on the premise that the QTL might be associated with the ZmbZIP60 promoter, we evaluated various maize inbred lines for their ability to upregulate the expression of ZmbZIP60 in response to heat stress. In general, tropical lines with promoter regions similar to CML52 were more robust in upregulating ZmbZIP60 in response to heat stress. This finding was confirmed by comparing the strength of the B73 and CML52 ZmbZIP60 promoters in transient maize protoplast assays. We concluded that the upstream region of ZmbZIP60 is important in conditioning the response to heat stress and was under selection in maize when adapted to different environments. Summary: Heat stress has large negative effects on maize grain yield. Heat stress creates ER stress in maize and sets off the UPR. We searched for factors conditioning heat induction of the UPR in maize seedlings by conducting an association analysis based on the upregulation of unspliced and spliced forms of ZmbZIP60 mRNA (ZmbZIP60u and ZmbZIP60s, respectively). ZmbZIP60u was upregulated more robustly by heat stress in the tropical maize line, CML52, than in B73, and a major QTL derived from the analysis of RILs from a cross of these two lines mapped in the vicinity of ZmbZIP60. We conducted a cis/trans test to determine whether the QTL was acting as a cis regulatory element or in trans, as might be expected for a transcription factor. We found that the QTL was acting in cis, likely involving the ZmbZIP60 promoter. ZmbZIP60 promoters in other temperate and tropical lines similar to CML52 showed enhanced expression of ZmbZIP60u by heat. The contribution of the CML52 promoter to heat induction of ZmbZIP60 was confirmed by analyzing the CML52 and B73 promoters linked to a luciferase reporter and assayed in heat-treated maize protoplasts.
RESUMO
Macroautophagy/autophagy is a conserved process in eukaryotes that contributes to cell survival in response to stress. Previously, we found that endoplasmic reticulum (ER) stress induces autophagy in plants via a pathway dependent upon AT5G24360/IRE1B (INOSITOL REQUIRING 1-1), an ER membrane-anchored factor involved in the splicing of AT1G42990/BZIP60 (basic leucine zipper protein 60) mRNA. IRE1B is a dual protein kinase and ribonuclease, and here we determined the involvement of the protein kinase catalytic domain, nucleotide binding and RNase domains of IRE1B in activating autophagy. We found that the nucleotide binding and RNase activity of IRE1B, but not its protein kinase activity or splicing target BZIP60, are required for ER stress-mediated autophagy. Upon ER stress, the RNase activity of IRE1B engages in regulated IRE1-dependent decay of messenger RNA (RIDD), in which mRNAs of secreted proteins are degraded by IRE1 upon ER stress. Twelve genes most highly targeted by RIDD were tested for their role in inhibiting ER stress-induced autophagy, and 3 of their encoded proteins, AT1G66270/BGLU21 (ß-glucosidase 21), AT2G16005/ROSY1/ML (MD2-related lipid recognition protein) and AT5G01870/PR-14 (pathogenesis-related protein 14), were found to inhibit autophagy upon overexpression. From these findings, IRE1B is posited to be a 'licensing factor' linking ER stress to autophagy by degrading the RNA transcripts of factors that interfere with the induction of autophagy. ABBREVIATIONS: ACT2: actin 2; ATG: autophagy-related; BGLU21: ß-glucosidase 21; BIP3: binding protein 3; BZIP: basic leucine zipper; DAPI: 4', 6-diamidino-2-phenylindole; DTT: dithiothreitol; ER: endoplasmic reticulum; ERN1: endoplasmic reticulum to nucleus signaling 1; IRE1: inositol requiring 1; GFP: green fluorescent protein; MAP3K5/ASK1: mitogen-activated protein kinase kinase kinase 5; MAPK8/JNK1: mitogen-activated protein kinase 8/c-Jun N-terminal kinase 1; MDC: monodansylcadaverine; PR-14: pathogenesis-related protein 14; RIDD: Regulated IRE1-Dependent Decay of Messenger RNA; ROSY1/ML: interactor of synaptotagmin1/MD2-related lipid recognition protein; Tm: tunicamycin; UPR: unfolded protein response; WT: wild-type.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Autofagia , Estresse do Retículo Endoplasmático , Proteínas Quinases/metabolismo , Estabilidade de RNA , Arabidopsis/genética , Autofagia/genética , Regulação para Baixo/genética , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Mutação/genética , Raízes de Plantas/metabolismo , Protoplastos/metabolismo , Estabilidade de RNA/genética , Plântula/metabolismoRESUMO
The unfolded protein response (UPR) is a highly conserved response that protects plants from adverse environmental conditions. The UPR is elicited by endoplasmic reticulum (ER) stress, in which unfolded and misfolded proteins accumulate within the ER. Here, we induced the UPR in maize (Zea mays) seedlings to characterize the molecular events that occur over time during persistent ER stress. We found that a multiphasic program of gene expression was interwoven among other cellular events, including the induction of autophagy. One of the earliest phases involved the degradation by regulated IRE1-dependent RNA degradation (RIDD) of RNA transcripts derived from a family of peroxidase genes. RIDD resulted from the activation of the promiscuous ribonuclease activity of ZmIRE1 that attacks the mRNAs of secreted proteins. This was followed by an upsurge in expression of the canonical UPR genes indirectly driven by ZmIRE1 due to its splicing of Zmbzip60 mRNA to make an active transcription factor that directly upregulates many of the UPR genes. At the peak of UPR gene expression, a global wave of RNA processing led to the production of many aberrant UPR gene transcripts, likely tempering the ER stress response. During later stages of ER stress, ZmIRE1's activity declined, as did the expression of survival modulating genes, Bax inhibitor1 and Bcl-2-associated athanogene7, amid a rising tide of cell death. Thus, in response to persistent ER stress, maize seedlings embark on a course of gene expression and cellular events progressing from adaptive responses to cell death.
Assuntos
Morte Celular/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Zea mays/citologia , Zea mays/metabolismo , Morte Celular/genética , Estresse do Retículo Endoplasmático/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resposta a Proteínas não Dobradas/genética , Zea mays/genéticaRESUMO
The Arabidopsis (Arabidopsis thaliana) genome encodes homologs of the Guided Entry of Tail (GET)-anchored protein system for the posttranslational insertion of tail-anchored (TA) proteins into endoplasmic reticulum (ER) membranes. In yeast, TA proteins are loaded onto the cytosolic targeting factor Get3 and are then delivered to the membrane-associated Get1/2 complex for insertion into ER membranes. The role of the GET system in Arabidopsis was investigated by monitoring the membrane insertion of a tail-anchored protein, SYP72, a syntaxin. SYP72 bound to yeast Get3 in vitro, forming a Get3-SYP72 fusion complex that could be inserted into yeast GET1/2-containing proteoliposomes. The Arabidopsis GET system functioned in vivo to insert TA proteins into ER membranes as demonstrated by the fact that the YFP-tagged SYP72 localized to the ER in wild-type plants but accumulated as cytoplasmic inclusions in get1, get3, or get4 mutants. The GET mutants get1 and get3 were less tolerant of ER stress agents and showed symptoms of ER stress even under unstressed conditions. Hence, the GET system is responsible for the insertion of TA proteins into the ER in Arabidopsis, and mutants with GET dysfunctions are more susceptible to ER stress.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Qa-SNARE/metabolismo , Mutação/genética , Fenótipo , Proteolipídeos/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Especificidade por SubstratoRESUMO
The unfolded protein response (UPR) is activated by various stresses during vegetative development in Arabidopsis, but is constitutively active in anthers of unstressed plants. To understand the role of the UPR during reproductive development, we analyzed a double mutant, ire1a ire1b. The double mutant knocks out the RNA-splicing arm of the UPR signaling pathway. It is fertile at room temperature but male sterile at modestly elevated temperature (ET). The conditional male sterility in the mutant is a sporophytic trait, and when the double mutant was grown at ET, defects appeared in the structure of the tapetum. As a result, the tapetum in the double mutant failed to properly deposit the pollen coat at ET, which made pollen grains clump and prevented their normal dispersal. IRE1 is a dual protein kinase/ribonuclease involved in the splicing of bZIP60 mRNA, and through complementation analysis of various mutant forms of IRE1b it was demonstrated that the ribonuclease activity of IRE1 was required for protecting male fertility from ET. It was also found that overexpression of SEC31A rescued the conditional male sterility in the double mutant. SEC31A is involved in trafficking from the endoplasmic reticulum to Golgi and a major target of the IRE1-mediated UPR signaling in stressed seedlings. Thus, IRE1, a major component of the UPR, plays an important role in protecting pollen development from ET.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Retículo Endoplasmático/metabolismo , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Complexo de Golgi/metabolismo , Temperatura Alta , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/genéticaRESUMO
NHL (NDR1/HIN1-like) genes play crucial roles in pathogen induced plant responses to biotic stress. Here, we report the possible function of NHL6 in plant response to abscisic acid (ABA) and abiotic stress. NHL6 was highly expressed in non-germinated seeds, and its expression was strongly induced by ABA and multiple abiotic stress signals. Loss-of-function of NHL6 decreased sensitivity to ABA in the early developmental stages including seed germination and post-germination seedling growth of the nhl6 mutants. However, overexpression of NHL6 increased sensitivity to ABA, salt and osmotic stress of the transgenic plants. Further studies indicated that the increased sensitivity in the 35S::NHL6 overexpressing plants could be a result of both ABA hypersensitivity and increased endogenous ABA accumulation under the stress conditions. It was also seen that the ABA-responsive element binding factors AREB1, AREB2 and ABF3 could regulate NHL6 expression at transcriptional level. Our results indicate that NHL6 plays an important role in the abiotic stresses-induced ABA signaling and biosynthesis, particularly during seed germination and early seedling development in Arabidopsis.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Membrana/metabolismo , Sementes/fisiologia , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação/efeitos dos fármacos , Germinação/genética , Proteínas de Membrana/genética , Mutação , Pressão Osmótica , Plantas Geneticamente Modificadas , Plântula/genética , Plântula/crescimento & desenvolvimento , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Fatores de Transcrição/genéticaRESUMO
Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum, and is caused by conditions such as heat or agents that cause endoplasmic reticulum stress, including tunicamycin and dithiothreitol. Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in a manner dependent on inositol-requiring enzyme 1b (IRE1b), and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin- or dithiothreitol-induced autophagy, but not autophagy caused by unrelated stresses. Similarly, over-expression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heat-induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b-dependent manner. Moreover, zeolin and CPY* partially co-localized with the autophagic body marker GFP-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress.
Assuntos
Arabidopsis/fisiologia , Autofagia/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Fenilbutiratos/farmacologia , Ácido Tauroquenodesoxicólico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Autofagia/efeitos dos fármacos , Ditiotreitol/farmacologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Desdobramento de Proteína , Tunicamicina/farmacologia , Vacúolos/metabolismoRESUMO
Two classes of ER stress sensors are known in plants, membrane-associated basic leucine zipper (bZIP) transcription factors and RNA splicing factors. ER stress occurs under adverse environmental conditions and results from the accumulation of misfolded or unfolded proteins in the ER lumen. One of the membrane-associated transcription factors activated by heat and ER stress agents is bZIP28. In its inactive form, bZIP28 is a type II membrane protein with a single pass transmembrane domain, residing in the ER. bZIP28's N-terminus, containing a transcriptional activation domain, is oriented towards the cytoplasm and its C-terminal tail is inserted into the ER lumen. In response to stress, bZIP28 exits the ER and moves to the Golgi where it is proteolytically processed, liberating its cytosolic component which relocates to the nucleus to upregulate stress-response genes. bZIP28 is thought to sense stress through its interaction with the major ER chaperone, binding immunoglobulin protein (BIP). Under unstressed conditions, BIP binds to intrinsically disordered regions in bZIP28's lumen-facing tail and retains it in the ER. A truncated form of bZIP28, without its C-terminal tail is not retained in the ER but migrates constitutively to the nucleus. Upon stress, BIP releases bZIP28 allowing it to exit the ER. One model to account for the release of bZIP28 by BIP is that BIP is competed away from bZIP28 by the accumulation of misfolded proteins in the ER. However, other forces such as changes in energy charge levels, redox conditions or interaction with DNAJ proteins may also promote release of bZIP28 from BIP. Movement of bZIP28 from the ER to the Golgi is assisted by the interaction of elements of the COPII machinery with the cytoplasmic domain of bZIP28. Thus, the mobilization of bZIP28 in response to stress involves the dissociation of factors that retain it in the ER and the association of factors that mediate its further organelle-to-organelle movement.
RESUMO
The unfolded protein response (UPR) endows plants with the capacity to perceive, respond, and protect themselves from adverse environmental conditions. The UPR signaling pathway in Arabidopsis has two "arms," one arm involving the bifunctional protein kinase (PK)/ribonuclease, IRE1, a RNA splicing enzyme, and another involving membrane-associated transcription factors, such as basic leucine zipper transcription factor 28 (bZIP28). Because of functional redundancies, single gene mutations in the plant UPR signaling pathway generally have not resulted in prominent phenotypes. In this study we generated multiple mutations in the UPR signaling pathway, such as an ire1a ire1b double mutant, which showed defects in stress tolerance and vegetative growth and development. Complementation of ire1a ire1b with constructs containing site-specific mutations in the PK or RNase domains of IRE1b demonstrated that a functional RNase domain is required for endoplasmic reticulum stress tolerance, and that both the PK and RNase domains are required for normal vegetative growth under unstressed conditions. Root growth under stress conditions was dependent on the splicing target of IRE1b, bZIP60 mRNA, and on regulated IRE1-dependent decay of target genes. However, root and shoot growth in the absence of stress was independent of bZIP60. Blocking both arms of the UPR signaling pathway in a triple ire1a ire1b bzip28 mutant was lethal, impacting pollen viability under unstressed conditions. Complementation with IRE1b constructs showed that both the PK and RNase domains are required for normal gametophyte development, but bZIP60 is not. Hence, the UPR plays a critical role in stress tolerance, and in normal vegetative growth and reproductive development in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas Quinases/metabolismo , Ribonucleases/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Arabidopsis/crescimento & desenvolvimento , Primers do DNA/genética , Perfilação da Expressão Gênica , Fosforilação , Raízes de Plantas/crescimento & desenvolvimento , Pólen/crescimento & desenvolvimento , Reprodução/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The endoplasmic reticulum (ER) stress response is a highly conserved mechanism that results from the accumulation of unfolded or misfolded proteins in the ER. The response plays an important role in allowing plants to sense and respond to adverse environmental conditions, such as heat stress, salt stress and pathogen infection. Since the ER is a well-controlled microenvironment for proper protein synthesis and folding, it is highly susceptible to stress conditions. Accumulation of unfolded or misfolded proteins activates a signaling pathway, called the unfolded protein response (UPR), which acts to relieve ER stress and, if unsuccessful, leads to cell death. Plants have two arms of the UPR signaling pathway, an arm involving the proteolytic processing of membrane-associated basic leucine zipper domain (bZIP) transcription factors and an arm involving RNA splicing factor, IRE1, and its mRNA target. These signaling pathways play an important role in determining the cell's fate in response to stress conditions.
Assuntos
Estresse do Retículo Endoplasmático , Fenômenos Fisiológicos Vegetais , Animais , Autofagia , Degradação Associada com o Retículo Endoplasmático , Evolução Molecular , Mamíferos , Modelos Biológicos , Células Vegetais/metabolismo , Desenvolvimento Vegetal , Fenômenos Fisiológicos Vegetais/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Dobramento de Proteína , Transdução de Sinais , Resposta a Proteínas não Dobradas/genéticaRESUMO
BINDING PROTEIN (BiP) is a major chaperone in the endoplasmic reticulum (ER) lumen, and this study shows that BiP binds to the C-terminal tail of the stress sensor/transducer bZIP28, a membrane-associated transcription factor, retaining it in the ER under unstressed conditions. In response to ER stress, BiP dissociates from bZIP28, allowing it to be mobilized from the ER to the Golgi where it is proteolytically processed and released to enter the nucleus. Under unstressed conditions, BiP binds to bZIP28 as it binds to other client proteins, through its substrate binding domain. BiP dissociates from bZIP28 even when bZIP28's exit from the ER or its release from the Golgi is blocked. Both BiP1 and BiP3 bind bZIP28, and overexpression of either BiP detains bZIP28 in the ER under stress conditions. A C-terminally truncated mutant of bZIP28 eliminating most of the lumenal domain does not bind BiP and is not retained in the ER under unstressed conditions. BiP binding sites in the C-terminal tail of bZIP28 were identified in a phage display system. BiP was found to bind to intrinsically disordered regions on bZIP28's lumen-facing tail. Thus, the dissociation of BiP from the C-terminal tail of bZIP28 is a major switch that activates one arm of the unfolded protein response signaling pathway in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Transporte/metabolismo , Estresse do Retículo Endoplasmático , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Transporte/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Immunoblotting , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Mutação , Plantas Geneticamente Modificadas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Homologia de Sequência de AminoácidosRESUMO
In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress-induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress-induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Autofagia/fisiologia , Retículo Endoplasmático/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Estresse do Retículo Endoplasmático , Mutagênese Insercional , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Folhas de Planta/ultraestrutura , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão , Plântula/genética , Plântula/metabolismo , Plântula/fisiologia , Plântula/ultraestrutura , Transdução de Sinais , Vacúolos/metabolismoRESUMO
Arabidopsis bZIP28, an ER membrane-associated transcription factor, is activated in response to conditions that induce ER stress-adverse environmental conditions or exposure to ER stress agents such as tunicamycin and dithiothreitol. Upon stress treatment, bZIP28 exits the ER and moves to the Golgi, where it is proteolytically processed, releasing its transcriptional component, which relocates to the nucleus. In this study, we tracked the movement of GFP-tagged bZIP28 in an effort to understand its mobilization from the ER and release from the Golgi. We identified a small region in bZIP28 that is rich in dibasic amino acids and proximal to the transmembrane domain required for its movement from the ER. In response to ER stress, bZIP28 showed enhanced interaction with Sar1 and Sec12, components of the COPII machinery. We demonstrated that the dibasic amino acid-rich region in bZIP28 is involved in the interaction with Sar1. Upon migration to the Golgi, bZIP28 is proteolytically processed by proteases S1P and S2P. We found a putative helix-breaking residue in the transmembrane domain of bZIP28 to be crucial for its processing and liberation from Golgi bodies. Thus, in response to stress, bZIP28 moves from organelle to organelle by interaction of critical elements in the molecule with the transport and/or proteolytic machinery resident in the various organelles.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas R-SNARE/metabolismoRESUMO
Adverse environmental conditions produce endoplasmic reticulum (ER) stress in plants. In response to heat or ER stress agents, Arabidopsis seedlings mitigate stress damage by activating ER-associated transcription factors and a RNA splicing factor, IRE1b. IRE1b splices the mRNA-encoding bZIP60, a basic leucine-zipper domain containing transcription factor associated with the unfolded protein response in plants. bZIP60 is required for the up-regulation of BINDING PROTEIN3 (BIP3) in response to ER stress, and loss-of-function mutations in IRE1b or point mutations in the splicing site of bZIP60 mRNA are defective in BIP3 induction. These findings demonstrate that bZIP60 in plants is activated by RNA splicing and afford opportunities for monitoring and modulating stress responses in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Quinases/metabolismo , Splicing de RNA/fisiologia , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Fatores de Transcrição de Zíper de Leucina Básica/genética , Mutação Puntual , Proteínas Quinases/genética , RNA Mensageiro/genética , RNA de Plantas/genética , Plântula/genética , Plântula/metabolismoRESUMO
Arabidopsis thaliana encodes 56 subtilisin-like serine proteases (subtilases), and some are involved in the proteolytic processing of plant peptide hormones. Here, we have investigated the role of one subtilase, AtSBT5.4, in whole plant physiology by examining gain- or loss-of-function phenotypes. Knockouts of AtSBT5.4 had no apparent phenotype; however, overexpression produced a clavata-like phenotype with fasciated inflorescence stems and compounded terminal buds. Production of the phenotype depended on the enzymatic activity of the subtilase, because substitution of serine at the active site abolished the overexpression phenotype. When AtSBT5.4 was overexpressed in a clavata3 mutant background, a novel phenotype was produced suggesting that AtSBT5.4 interacts with the clavata signaling pathway. However, AtSBT5.4 did not cleave CLAVATA3 (CLV3) or a fluorogenic peptide representing the putative cleavage site in CLV3 under in vitro conditions suggesting that the interaction in vivo does not involve the cleavage of CLV3. Overexpression of AtSBT5.4 in a wuschel (wus) background suppressed the AtSBT5.4 overexpression phenotype indicating that WUS function is required for the AtSBT5.4 overexpression phenotype.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Genes de Plantas , Serina Proteases/genética , Subtilisinas/genética , Proteínas de Arabidopsis/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Mutação/genética , Especificidade de Órgãos , Fenótipo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/enzimologia , Serina Proteases/metabolismo , Frações Subcelulares/enzimologia , Subtilisinas/metabolismoRESUMO
Arabidopsis has 34 genes encoding proteins related to rapid alkalinization factor (RALF), a peptide growth factor. One of those genes (AtRALF23) is significantly downregulated by brassinolide (BL) treatment of Arabidopsis seedlings or in mutant seedlings expressing a constitutively active form of BES1, a transcriptional effector of the brassinosteroid signaling pathway. Overexpression of AtRALF23 impairs BL-induced hypocotyl elongation in seedlings, and mature overexpressing plants are shorter and bushier. Overexpression of AtRALF23 produces slower growing seedlings, with roots that have reduced capacity to acidify the rhizosphere. AtRALF23 encodes a 138-aa protein, and when an epitope-tagged form (AtRALF23-myc) was expressed in transgenic plants, the protein was processed to release a C-terminal peptide. The presumed junction between the precursor and the processed peptide contains a recognition site for site-1 protease (AtS1P), a plant subtilisin-like serine protease (subtilase). When AtRALF23-myc was expressed in the background of a site-1 protease mutant (s1p-3), or when the AtS1P recognition site (RRIL) was mutated (RR --> GG) and expressed in a wild-type background, the precursor was not cleaved, and the bushy phenotype was not produced. A fluorogenic peptide representing the presumed subtilase recognition site in AtRALF23 was cleaved in vitro by AtS1P. Thus, BL downregulates AtRALF23 expression, presumably relieving the growth-retarding effect of a peptide growth factor, which is processed from a larger precursor protein by AtS1P.