RESUMEN
Heat stress induces misfolded protein accumulation in endoplasmic reticulum (ER), which initiates the unfolded protein response (UPR) in plants. Previous work has demonstrated the important role of a rice ER membrane-associated transcription factor OsbZIP74 (also known as OsbZIP50) in UPR. However, how OsbZIP74 and other membrane-associated transcription factors are involved in heat stress tolerance in rice is not reported. In the current study, we discovered that OsNTL3 is required for heat stress tolerance in rice. OsNTL3 is constitutively expressed and up-regulated by heat and ER stresses. OsNTL3 encodes a NAC transcription factor with a predicted C-terminal transmembrane domain. GFP-OsNTL3 relocates from plasma membrane to nucleus in response to heat stress and ER stress inducers. Loss-of-function mutation of OsNTL3 confers heat sensitivity while inducible expression of the truncated form of OsNTL3 without the transmembrane domain increases heat tolerance in rice seedlings. RNA-Seq analysis revealed that OsNTL3 regulates the expression of genes involved in ER protein folding and other processes. Interestingly, OsNTL3 directly binds to OsbZIP74 promoter and regulates its expression in response to heat stress. In turn, up-regulation of OsNTL3 by heat stress is dependent on OsbZIP74. Thus, our work reveals the important role of OsNTL3 in thermotolerance, and a regulatory circuit mediated by OsbZIP74 and OsNTL3 in communications among ER, plasma membrane and nucleus under heat stress conditions.
Asunto(s)
Oryza , Termotolerancia , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Oryza/metabolismo , Termotolerancia/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada/genéticaRESUMEN
Plants coordinate their growth and developmental programs with various endogenous signals and environmental challenges. Phytochrome interacting factor 4 (PIF4) plays a critical positive role in thermoresponsive gene expression and hypocotyl growth in Arabidopsis, whereas early flowering 3 (ELF3) negatively regulates the activity of PIF4 at elevated temperatures. However, it is unknown how ELF3 activity is regulated at warm temperatures. Here, we report the identification of B-box 18 (BBX18) and BBX23 as important thermomorphogenesis regulators in Arabidopsis. BBX18 and BBX23 mutations result in reduced thermoresponsive hypocotyl elongation. In contrast, BBX18 overexpression promotes hypocotyl growth at elevated temperatures, which depends on either PIF4 or constitutive photomorphogenic 1 (COP1). BBX18 and BBX23 interact with ELF3 or COP1. Knocking out BBX18 and BBX23 increases ELF3 abundance under normal and warm temperature conditions. The expression of multiple thermoresponsive genes is impaired in both a PIF4 mutant and a BBX18/BBX23 double mutant. Thus, our findings reveal an important role of B-box proteins during thermomorphogenesis and provide insights into our understanding of how warm temperature signals regulate ELF3 activity and PIF4-dependent genes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Morfogénesis , Temperatura , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción/genéticaRESUMEN
Grain size and shape are important determinants of grain weight and yield in rice. Here, we report a new major quantitative trait locus (QTL), qTGW3, that controls grain size and weight in rice. This locus, qTGW3, encodes OsSK41 (also known as OsGSK5), a member of the GLYCOGEN SYNTHASE KINASE 3/SHAGGY-like family. Rice near-isogenic lines carrying the loss-of-function allele of OsSK41 have increased grain length and weight. We demonstrate that OsSK41 interacts with and phosphorylates AUXIN RESPONSE FACTOR 4 (OsARF4). Co-expression of OsSK41 with OsARF4 increases the accumulation of OsARF4 in rice protoplasts. Loss of function of OsARF4 results in larger rice grains. RNA-sequencing analysis suggests that OsARF4 and OsSK41 repress the expression of a common set of downstream genes, including some auxin-responsive genes, during rice grain development. The loss-of-function form of OsSK41 at qTGW3 represents a rare allele that has not been extensively utilized in rice breeding. Suppression of OsSK41 function by either targeted gene editing or QTL pyramiding enhances rice grain size and weight. Thus, our study reveals the important role of OsSK41 in rice grain development and provides new candidate genes for genetic improvement of grain yield in rice and perhaps in other cereal crops.
Asunto(s)
Glucógeno Sintasa Quinasa 3/genética , Oryza/genética , Sitios de Carácter Cuantitativo , Factores de Transcripción/metabolismo , Clonación Molecular , Grano Comestible , Regulación de la Expresión Génica de las Plantas , Oryza/anatomía & histología , Oryza/enzimología , FosforilaciónRESUMEN
Two salt hypersensitive mutants she1 and she2 were identified through genetic screening. SHE1 encodes a cellulose synthase CESA6 while SHE2 encodes a cellulose synthase-interactive protein CSI1. Both of them are involved in cellulose deposition. Our results demonstrated that the sustained cellulose synthesis is important for salt stress tolerance in Arabidopsis.
Asunto(s)
Adaptación Fisiológica/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas Portadoras/genética , Celulosa/biosíntesis , Genes de Plantas , Glucosiltransferasas/genética , Estrés Fisiológico/genética , Adaptación Fisiológica/efectos de los fármacos , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Glucosiltransferasas/metabolismo , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacosRESUMEN
Abscisic acid plays important roles in maintaining seed dormancy while gibberellins (GA) and other phytohormones antagonize ABA to promote germination. However, how ABA signaling is desensitized during the transition from dormancy to germination is still poorly understood. We functionally characterized the role of membrane-associated transcription factor peptidase, site-2 protease (S2P), in ABA signaling during seed germination in Arabidopsis. Genetic analysis showed that loss-of-function of S2P conferred high ABA sensitivity during seed germination, and expression of the activated form of membrane-associated transcription factor bZIP17, in which the transmembrane domain and endoplasmic reticulum (ER) lumen-facing C-terminus were deleted, in the S2P mutant rescued its ABA-sensitive phenotype. MYC and green fluorescent protein (GFP)-tagged bZIP17 were processed and translocated from the ER to the nucleus in response to ABA treatment. Furthermore, genes encoding negative regulators of ABA signaling, such as the transcription factor ATHB7 and its target genes HAB1, HAB2, HAI1 and AHG3, were up-regulated in seeds of the wild-type upon ABA treatment; this up-regulation was impaired in seeds of S2P mutants. Our results suggest that S2P desensitizes ABA signaling during seed germination through regulating the activation of the membrane-associated transcription factor bZIP17 and therefore controlling the expression level of genes encoding negative regulators of ABA signaling.
Asunto(s)
Ácido Abscísico/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Péptido Hidrolasas/metabolismo , Semillas/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Arabidopsis/enzimología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Transporte Biológico , Regulación del Desarrollo de la Expresión Génica , Germinación/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Péptido Hidrolasas/genética , Desarrollo de la Planta/genética , Latencia en las Plantas/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Semillas/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
Accumulation of unfolded or misfolded proteins causes endoplasmic reticulum (ER) stress, which activates a set of ER membrane-associated transcription factors for protein homeostasis regulation. Previous genome-wide chromatin immunoprecipitation analysis shows a strong correlation between histone H3K4 trimethylation (H3K4me3) and active gene expression. However, how the histone modification complex is specifically and timely recruited to the active promoters remains unknown. Using ER stress responsive gene expression as a model system, we demonstrate that sequence-specific transcription factors interact with COMPASS-like components and affect H3K4me3 formation at specific target sites in Arabidopsis. Gene profiling analysis reveals that membrane-associated basic leucine zipper (bZIP) transcription factors bZIP28 and bZIP60 regulate most of the ER stress responsive genes. Loss-of-functions of bZIP28 and bZIP60 impair the occupancy of H3K4me3 on promoter regions of ER stress responsive genes. Further, in vitro pull-down assays and in vivo bimolecular fluorescence complementation (BiFC) experiments show that bZIP28 and bZIP60 interact with Ash2 and WDR5a, both of which are core COMPASS-like components. Knockdown expression of either Ash2 or WDR5a decreased the expression of several ER stress responsive genes. The COMPASS-like complex is known to interact with histone methyltransferase to facilitate preinitiation complex (PIC) assembly and generate H3K4me3 during transcription elongation. Thus, our data shows that the ER stress stimulus causes the formation of PIC and deposition of H3K4me3 mark at specific promoters through the interaction between transcription factor and COMPASS-like components.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Histonas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas Portadoras/genética , Estrés del Retículo Endoplásmico/fisiología , Histonas/genética , Metilación , Complejos Multiproteicos , Regiones Promotoras Genéticas/fisiología , Iniciación de la Transcripción Genética/fisiologíaRESUMEN
Many sources of stress cause accumulation of unfolded or misfolded proteins in endoplasmic reticulum (ER), which elicits the unfolded protein response (UPR) to either promote cell survival or programmed cell death depending on different developmental context or stress severity. The Arabidopsis membrane-associated transcription factor, bZIP28, is the functional equivalent of mammalian ATF6, which relocates from the ER to the Golgi where it is proteolytically processed and released from the membrane to the nucleus to mediate the UPR. Although the canonical site-1 protease (S1P) cleavage site on the ER lumen-facing domain is well conserved between bZIP28 and ATF6, the importance of S1P cleavage on bZIP28 has not been experimentally demonstrated. Here we provide genetic evidence that the RRIL(573) site, but not the RVLM(373) site, on the lumen-facing domain of bZIP28 is critical for the biological function of bZIP28 under ER stress condition. Further biochemistry and cell biology studies demonstrated that the RRIL(573) site, but not the RVLM(373) site, is required for proteolytic processing and nuclear relocation of bZIP28 in response to ER stress. Our results reveal that S1P cleavage site plays a pivotal role in activation and function of bZIP28 during UPR in plants.
Asunto(s)
Arabidopsis/metabolismo , Retículo Endoplásmico/metabolismo , Proproteína Convertasas/metabolismo , Serina Endopeptidasas/metabolismo , Estrés Fisiológico , Factores de Transcripción/metabolismo , Humanos , Proteolisis , Fracciones Subcelulares/metabolismoRESUMEN
The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) triggers a well conserved pathway called the unfolded protein response (UPR) in eukaryotic cells to mitigate ER stress. Two signaling pathways, S2P-bZIP28 and IRE1-bZIP60, play important roles in transmitting ER stress signals from the ER to the nucleus in Arabidopsis (Arabidopsis thaliana). It is not known whether other components in the secretory pathway also contribute to the alleviation of ER stress. Here we report the identification of a plasma membrane-associated transcription factor, NAC062 (also known as ANAC062/NTL6), as another important UPR mediator in Arabidopsis plants. NAC062 relocates from the plasma membrane to the nucleus and regulates the expression of ER stress responsive genes in Arabidopsis. Knock-down of NAC062 in the wild-type background confers ER stress sensitivity, while inducible expression of a nucleus-localized form of NAC062, NAC062D, in the bZIP28 and bZIP60 double mutant (zip28zip60) background increases ER stress tolerance. Knock-down of NAC062 impairs ER-stress-induced expression of UPR downstream genes while over-expression of NAC062D-MYC induces the expression of UPR downstream genes under normal growth condition. CHIP-qPCR reveals that NAC062D-MYC is enriched at the promoter regions of several UPR downstream genes such as BiP2. Furthermore, NAC062 itself is also up-regulated by ER stress, which is dependent on bZIP60 but not on bZIP28. Thus, our results have uncovered an alternative UPR pathway in plants in which the membrane-associated transcription factor NAC062 relays ER stress signaling from the plasma membrane to the nucleus and plays important roles in regulating UPR downstream gene expression.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Estrés del Retículo Endoplásmico , Regulación de la Expresión Génica de las Plantas , Transducción de Señal , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Dimerización , Retículo Endoplásmico/metabolismo , Expresión Génica , Modelos Biológicos , Mutación , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Plantones/citología , Plantones/genética , Plantones/metabolismo , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
The unfolded protein response (UPR) is activated to sustain cell survival by reducing misfolded protein accumulation in the endoplasmic reticulum (ER). The UPR also promotes programmed cell death (PCD) when the ER stress is severe; however, the underlying molecular mechanisms are less understood, especially in plants. Previously, two membrane-associated transcriptions factors (MTFs), bZIP28 and bZIP60, were identified as the key regulators for cell survival in the plant ER stress response. Here, we report the identification of another MTF, NAC089, as an important PCD regulator in Arabidopsis (Arabidopsis thaliana) plants. NAC089 relocates from the ER membrane to the nucleus under ER stress conditions. Inducible expression of a truncated form of NAC089, in which the transmembrane domain is deleted, induces PCD with increased caspase 3/7-like activity and DNA fragmentation. Knock-down NAC089 in Arabidopsis confers ER stress tolerance and impairs ER-stress-induced caspase-like activity. Transcriptional regulation analysis and ChIP-qPCR reveal that NAC089 plays important role in regulating downstream genes involved in PCD, such as NAC094, MC5 and BAG6. Furthermore, NAC089 is up-regulated by ER stress, which is directly controlled by bZIP28 and bZIP60. These results show that nuclear relocation of NAC089 promotes ER-stress-induced PCD, and both pro-survival and pro-death signals are elicited by bZIP28 and bZIP60 during plant ER stress response.
Asunto(s)
Proteínas de Arabidopsis/genética , Estrés del Retículo Endoplásmico/genética , Proteínas de la Membrana/genética , Factores de Transcripción/genética , Respuesta de Proteína Desplegada/genética , Apoptosis/genética , Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Supervivencia Celular/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de la Membrana/metabolismo , Factores de Transcripción/aislamiento & purificación , Activación TranscripcionalRESUMEN
The unfolded protein response (UPR) plays important roles in plant development and plant-pathogen interactions, as well as in plant adaptation to adverse environmental stresses. Previously bZIP28 and bZIP60 have been identified as important UPR regulators for mitigating the endoplasmic reticulum (ER) stress in Arabidopsis thaliana. Here we report the biological function of NAC103 in a novel transcriptional regulatory cascade, connecting bZIP60 to the UPR downstream genes in Arabidopsis. Expression of NAC103 was induced by ER stress, and was completely abolished in the bZIP60 null mutant. A new ER stress-responsive cis-element UPRE-III (TCATCG) on the NAC103 promoter was identified, and trans-activation of UPRE-III by bZIP60 was confirmed in both yeast cells and Arabidopsis protoplasts. The direct binding of bZIP60 to UPRE-III-containing DNA was also demonstrated in an electrophoretic mobility shift assay. NAC103 formed homodimers in yeast two-hybrid and bimolecular fluorescence complementation assays. It had transcriptional activation activity and was localized in the nucleus. Over-expression of NAC103 had pleiotropic effects on plant growth, and induced expression of several UPR downstream genes in Arabidopsis under normal growth conditions. The activation of UPR gene promoters by NAC103 was also confirmed in effector/reporter protoplast assays. Thus, our study demonstrates a transcriptional regulatory cascade in which NAC103 relays ER stress signals from bZIP60 to UPR downstream genes through a newly identified ER stress cis-element (UPRE-III) and transcriptional activation activity of its encoded protein NAC103.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Estrés del Retículo Endoplásmico , Redes Reguladoras de Genes , Respuesta de Proteína Desplegada , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Activación Transcripcional , Técnicas del Sistema de Dos HíbridosRESUMEN
The membrane-associated transcription factor, bZIP28, is relocated from the endoplasmic reticulum (ER) to the Golgi and proteolytically released from the membrane mediated by two proteases, S1P and S2P, in response to ER stress in Arabidopsis. The activated N-terminal domain recruits nuclear factor Y (NF-Y) subunits in the nucleus to regulate ER stress downstream genes. Little is known about the functions of the bZIP28 C-terminal lumen-facing domain. Here, we provide novel insights into how the ER lumen-facing domain affects the biological function and organelle-to-organelle movement of bZIP28 in the ER stress response. First, we demonstrated the functional redundancy of bZIP28 and bZIP60 by generation and analysis of the bZIP28 and bZIP60 double mutant zip28zip60. Subsequent genetic complementation experiments in zip28zip60 background with deletions on bZIP28 lumen-facing domain highlighted the importance of lumen-facing domain for its in vivo function of bZIP28 in the ER stress response. The protein subcellular localization and Western blotting results further revealed that the bZIP28 lumen-facing domain contains ER retention signal which is important for the proteolytic activation of bZIP28. Thus, the bZIP28 lumen-facing C-terminus plays important roles in the ER-to-Golgi movement of bZIP28, which may contribute to the sensing of the ER stress.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Estrés del Retículo Endoplásmico , Orgánulos/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Prueba de Complementación Genética , Modelos Biológicos , Mutación/genética , Fenotipo , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Relación Estructura-Actividad , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Tunicamicina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genéticaRESUMEN
Protein folding in the endoplasmic reticulum (ER) is a fundamental process in plant cells that is vulnerable to many environmental stresses. When unfolded or misfolded proteins accumulate in the ER, the well-conserved unfolded protein response (UPR) is initiated to mitigate the ER stress by enhancing the protein folding capability and/or accelerating the ER-associated protein degradation. Here, we report the conservation of the activation mechanism of OsbZIP74 (also known as OsbZIP50), an important ER stress regulator in monocot plant rice (Oryza sativa L.). Under normal conditions, OsbZIP74 mRNA encodes a basic leucine-zipper transcription factor with a putative transmembrane domain. When treating with ER stress-inducing agents such as tunicamycin and DTT, the conserved double stem-loop structures of OsbZIP74 mRNA are spliced out. Thereafter, the resulting new OsbZIP74 mRNA produces the nucleus-localized form of OsbZIP74 protein, eliminating the hydrophobic region. The activated form of OsbZIP74 has transcriptional activation activity in both yeast cells and Arabidopsis leaf protoplasts. The induction of OsbZIP74 splicing is much suppressed in the OsIRE1 knock-down rice plants, indicating the involvement of OsIRE1 in OsbZIP74 splicing. We also demonstrate that the unconventional splicing of OsbZIP74 mRNA is associated with heat stress and salicylic acid, which is an important plant hormone in systemic acquired resistance against pathogen or parasite.