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The ER (endoplasmic reticulum) is the largest membrane-bound multifunctional organelle in eukaryotic cells, serving particularly important in protein synthesis, modification, folding and transport. UPR (unfolded protein response) is one of the systematized strategies that eukaryotic cells employ for responding to ER stress, a condition represents the processing capability of ER is overwhelmed and stressed. UPR is usually triggered when the protein folding capacity of ER is overloaded, and indeed, mounting studies were focused on the stress responding side of UPR. In plants, beyond stress response, accumulating evidence suggests that UPR is essential for growth and development, and more importantly, the necessity of UPR in this regard requires its endogenous basal activation even without stress. Then plants must have to fine tune the activation level of UPR pathway for balancing growth and stress response. In this review, we summarized the recent progresses in plant UPR, centering on its role in controlling plant reproduction and root growth, and lay out some outstanding questions to be addressed in the future. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 (basic leucine zipper 60) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro, the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta. Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues.

The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 (basic leucine zipper 60) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro , the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta. Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is the starting point for protein secretion and lipid biosynthesis in eukaryotes. ER homeostasis is precisely regulated by the unfolded protein response (UPR) to alleviate stress, involving both transcriptional and translational regulators. Autophagy is an intracellular self-eating process mediated by the double-membrane structure autophagosome for the degradation of cytosolic components and damaged organelles to regenerate nutrient supplies under nutrient-deficient or stress conditions. A recent study has revealed that besides serving as a membrane source for phagophore formation, the ER is also tightly regulated under stress conditions by a distinct type of autophagosome, namely ER-phagy. ER-phagy has been characterized with receptors clearly identified in mammals and yeast, yet relatively little is known about plant ER-phagy and its receptors. Here, we will summarize our current knowledge of ER-phagy in yeast and mammals and highlight recent progress in plant ER-phagy studies, pointing towards a possible interplay between ER-phagy and ER homeostasis under ER stress responses (ERSRs) in plants.

The endoplasmic reticulum (ER) is the starting point for protein secretion and lipid biosynthesis in eukaryotes. ER homeostasis is precisely regulated by the unfolded protein response (UPR) to alleviate stress, involving both transcriptional and translational regulators. Autophagy is an intracellular self-eating process mediated by the double-membrane structure autophagosome for the degradation of cytosolic components and damaged organelles to regenerate nutrient supplies under nutrient-deficient or stress conditions. A recent study has revealed that besides serving as a membrane source for phagophore formation, the ER is also tightly regulated under stress conditions by a distinct type of autophagosome, namely ER-phagy. ER-phagy has been characterized with receptors clearly identified in mammals and yeast, yet relatively little is known about plant ER-phagy and its receptors. Here, we will summarize our current knowledge of ER-phagy in yeast and mammals and highlight recent progress in plant ER-phagy studies, pointing towards a possible interplay between ER-phagy and ER homeostasis under ER stress responses (ERSRs) in plants. [ABSTRACT FROM AUTHOR]

The function of human Golgi antiapoptotic proteins (GAAPs) resembles that of BAX inhibitor-1, with apoptosis inhibition triggered by intrinsic and extrinsic stimuli. However, little is known about the function of GAAP-related proteins in plants. Here, we studied Arabidopsis GAAP1 and GAAP3 and found that they were localized on the cellular membrane, including the endoplasmic reticulum (ER) membrane. The function of GAAP1/GAAP3 in ER-stress response was tested, and results showed that single or double mutation in GAAP1 and GAAP3 reduced plant survival and enhanced cell death under ER stress. The expression of both genes was induced by various abiotic stress signals. Quantitative real-time polymerase chain reaction analysis showed that GAAP1/GAAP3 level affected the expression pattern of the unfolded-protein response (UPR) signaling pathway genes upon prolonged ER stress. The mutation in both GAAP1 and GAAP3 genes promoted and enhanced UPR signaling when confronted with mild ER stress. Moreover, GAAP1/GAAP3 inhibited cell death caused by ER stress and promoted plant-growth recovery by turning down inositol-requiring enzyme 1 (IRE1) signaling after ER stress had been relieved. Co-immunoprecipitation (Co-Ip) and BiFC assays showed that GAAP1/GAAP3 interacted with IRE1. These data suggested that GAAP1/GAAP3 played dual roles in the negative regulation of IRE1 activity and anti-programmed cell death.

Maintenance of homeostasis of the endoplasmic reticulum (ER) ensures the balance between loading of nascent proteins and their secretion. Certain developmental conditions or environmental stressors affect protein folding causing ER stress. The resultant ER stress is mitigated by upregulating a set of stress-responsive genes in the nucleus modulating the mechanism of the unfolded protein response (UPR). In plants, the UPR is mediated by two major pathways; by the proteolytic processing of bZIP17/28 and by the IRE1-mediated splicing of bZIP60 mRNA. Recent studies have shown the involvement of plant-specific NAC transcription factors in UPR regulation. The molecular mechanisms activating plant-UPR transducers are only recently being unveiled. This review focuses on important structural features involved in the activation of the UPR transducers like bZIP17/28/60, IRE1, BAG7, and NAC017/062/089/103. Also, we discuss the activation of the UPR pathways, including BAG7-bZIP28 and IRE1-bZIP60, in detail, together with the NAC-TFs, which adds a new paradigm to the plant UPR.

Maintenance of homeostasis of the endoplasmic reticulum (ER) ensures the balance between loading of nascent proteins and their secretion. Certain developmental conditions or environmental stressors affect protein folding causing ER stress. The resultant ER stress is mitigated by upregulating a set of stress-responsive genes in the nucleus modulating the mechanism of the unfolded protein response (UPR). In plants, the UPR is mediated by two major pathways; by the proteolytic processing of bZIP17/28 and by the IRE1-mediated splicing of bZIP60 mRNA. Recent studies have shown the involvement of plant-specific NAC transcription factors in UPR regulation. The molecular mechanisms activating plant-UPR transducers are only recently being unveiled. This review focuses on important structural features involved in the activation of the UPR transducers like bZIP17/28/60, IRE1, BAG7, and NAC017/062/089/103. Also, we discuss the activation of the UPR pathways, including BAG7-bZIP28 and IRE1-bZIP60, in detail, together with the NAC-TFs, which adds a new paradigm to the plant UPR. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) is a stress response conserved in eukaryotic organisms and activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Adverse environmental conditions disrupt protein folding in the ER and trigger the UPR. Recently, it was found that the UPR can be elicited in the course of plant development and defense. During vegetative plant development, the UPR is involved in normal root growth and development, the effect of which can be largely attributed to the influence of the UPR on plant hormone biology. The UPR also functions in plant reproductive development by protecting male gametophyte development from heat stress. In terms of defense, the UPR has been implicated in virus and microbial defense. Viral defense represents a double edge sword in that various virus infections activate the UPR, however, in a number of cases, the UPR actually supports viral infections. The UPR also plays a role in plant immunity to bacterial infections, again through the action of plant hormones in regulating basal immunity responses. [ABSTRACT FROM AUTHOR]

Tunicamycin sensitivity assays are a useful method for studies of endoplasmic reticulum stress and the unfolded protein response in eukaryotic cells. While tunicamycin sensitivity and tunicamycin recovery assays have been previously described, these existing methods are time-consuming, labor intensive and subjected to mechanical wounding. This study shows an improved method of testing tunicamycin sensitivity in Arabidopsis using liquid Murashige and Skoog medium versus the traditional solid agar plates. Liquid medium bypasses the physical manipulation of seedlings, thereby eliminating the risk of potential mechanical damage and additional unwanted stress to seedlings. Seedlings were subjected to comparative treatments with various concentrations of tunicamycin on both solid and liquid media and allowed to recover. Determination of fresh weight, chlorophyll contents analysis and qRT-PCR results confirm the efficacy of using liquid medium to perform quantitative tunicamycin stress assays.

The ER (endoplasmic reticulum) is the largest membrane-bound multifunctional organelle in eukaryotic cells, serving particularly important in protein synthesis, modification, folding and transport. UPR (unfolded protein response) is one of the systematized strategies that eukaryotic cells employ for responding to ER stress, a condition represents the processing capability of ER is overwhelmed and stressed. UPR is usually triggered when the protein folding capacity of ER is overloaded, and indeed, mounting studies were focused on the stress responding side of UPR. In plants, beyond stress response, accumulating evidence suggests that UPR is essential for growth and development, and more importantly, the necessity of UPR in this regard requires its endogenous basal activation even without stress. Then plants must have to fine tune the activation level of UPR pathway for balancing growth and stress response. In this review, we summarized the recent progresses in plant UPR, centering on its role in controlling plant reproduction and root growth, and lay out some outstanding questions to be addressed in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Lv, Wei, Guo, Li, Wang, Wang and Bao.)

The function of human Golgi antiapoptotic proteins (GAAPs) resembles that of BAX inhibitor-1, with apoptosis inhibition triggered by intrinsic and extrinsic stimuli. However, little is known about the function of GAAP-related proteins in plants. Here, we studied Arabidopsis GAAP1 and GAAP3 and found that they were localized on the cellular membrane, including the endoplasmic reticulum (ER) membrane. The function of GAAP1/GAAP3 in ER-stress response was tested, and results showed that single or double mutation in GAAP1 and GAAP3 reduced plant survival and enhanced cell death under ER stress. The expression of both genes was induced by various abiotic stress signals. Quantitative real-time polymerase chain reaction analysis showed that GAAP1/GAAP3 level affected the expression pattern of the unfolded-protein response (UPR) signaling pathway genes upon prolonged ER stress. The mutation in both GAAP1 and GAAP3 genes promoted and enhanced UPR signaling when confronted with mild ER stress. Moreover, GAAP1/GAAP3 inhibited cell death caused by ER stress and promoted plant-growth recovery by turning down inositol-requiring enzyme 1 (IRE1) signaling after ER stress had been relieved. Co-immunoprecipitation (Co-Ip) and BiFC assays showed that GAAP1/GAAP3 interacted with IRE1. These data suggested that GAAP1/GAAP3 played dual roles in the negative regulation of IRE1 activity and anti-programmed cell death.

The unfolded protein response (UPR) is a stress response conserved in eukaryotic organisms and activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Adverse environmental conditions disrupt protein folding in the ER and trigger the UPR. Recently, it was found that the UPR can be elicited in the course of plant development and defense. During vegetative plant development, the UPR is involved in normal root growth and development, the effect of which can be largely attributed to the influence of the UPR on plant hormone biology. The UPR also functions in plant reproductive development by protecting male gametophyte development from heat stress. In terms of defense, the UPR has been implicated in virus and microbial defense. Viral defense represents a double edge sword in that various virus infections activate the UPR, however, in a number of cases, the UPR actually supports viral infections. The UPR also plays a role in plant immunity to bacterial infections, again through the action of plant hormones in regulating basal immunity responses.