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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.

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.

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 (β-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: β-glucosidase 21; BIP3: binding protein 3; BZIP: basic leucine zipper; DAPI: 4ʹ, 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.

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.

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.

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.

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.

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.