Functional Diversification of ER Stress Responses in Arabidopsis.

The endoplasmic reticulum (ER) is responsible for the synthesis of one-third of the cellular proteome and is constantly challenged by physiological and environmental situations that can perturb its homeostasis and lead to the accumulation of misfolded secretory proteins, a condition referred to as ER stress. In response, the ER evokes a set of intracellular signaling processes, collectively known as the unfolded protein response (UPR), which are designed to restore biosynthetic capacity of the ER. As single-cell organisms evolved into multicellular life, the UPR complexity has increased to suit their growth and development. In this review, we discuss recent advances in the understanding of the UPR, emphasizing conserved UPR elements between plants and metazoans and highlighting unique plant-specific features.

The UPR regulator IRE1 promotes balanced organ development by restricting TOR-dependent control of cellular differentiation in Arabidopsis.

Proteostasis of the endoplasmic reticulum (ER) is controlled by sophisticated signaling pathways that are collectively called the unfolded protein response (UPR) and are initiated by specialized ER membrane-associated sensors. The evidence that complete loss-of-function mutations of the most conserved of the UPR sensors, inositol-requiring enzyme 1 (IRE1), dysregulates tissue growth and development in metazoans and plants raises the fundamental question as to how IRE1 is connected to organismal growth. To address this question, we interrogated the Arabidopsis primary root, an established model for organ development, using the tractable Arabidopsis IRE1 mutant ire1a ire1b, which has marked root development defects in the absence of exogenous stress. We demonstrate that IRE1 is required to reach maximum rates of cell elongation and root growth. We also established that in the actively growing ire1a ire1b mutant root tips the Target of Rapamycin (TOR) kinase, a widely conserved pro-growth regulator, is hyperactive, and that, unlike cell proliferation, the rate of cell differentiation is enhanced in ire1a ire1b in a TOR-dependent manner. By functionally connecting two essential growth regulators, these results underpin a novel and critical role of IRE1 in organ development and indicate that, as cells exit an undifferentiated state, IRE1 is required to monitor TOR activity to balance cell expansion and maturation during organ biogenesis.

NADPH oxidase activity is required for ER stress survival in plants.

In all eukaryotes, the unfolded protein response (UPR) relieves endoplasmic reticulum (ER) stress, which is a potentially lethal condition caused by the accumulation of misfolded proteins in the ER. In mammalian and yeast cells, reactive oxygen species (ROS) generated during ER stress attenuate the UPR, negatively impacting cell survival. In plants, the relationship between the UPR and ROS is less clear. Although ROS develop during ER stress, the sources of ROS linked to ER stress responses and the physiological impact of ROS generation on the survival from proteotoxic stress are yet unknown. Here we show that in Arabidopsis thaliana the respiratory burst oxidase homologs, RBOHD and RBOHF, contribute to the production of ROS during ER stress. We also demonstrate that during ER stress RBOHD and RBOHF are necessary to properly mount the adaptive UPR and overcome temporary and chronic ER stress situations. These results ascribe a cytoprotective role to RBOH-generated ROS in the defense from proteotoxic stress in an essential organelle, and support a plant-specific feature of the UPR management among eukaryotes.

Maintaining the factory: the roles of the unfolded protein response in cellular homeostasis in plants.

Much like a factory, the endoplasmic reticulum (ER) assembles simple cellular building blocks into complex molecular machines known as proteins. In order to protect the delicate protein folding process and ensure the proper cellular delivery of protein products under environmental stresses, eukaryotes have evolved a set of signaling mechanisms known as the unfolded protein response (UPR) to increase the folding capacity of the ER. This process is particularly important in plants, because their sessile nature commands adaptation for survival rather than escape from stress. As such, plants make special use of the UPR, and evidence indicates that the master regulators and downstream effectors of the UPR have distinct roles in mediating cellular processes that affect organism growth and development as well as stress responses. In this review we outline recent developments in this field that support a strong relevance of the UPR to many areas of plant life.

Programmed cell death regulator BAP2 is required for IRE1-mediated unfolded protein response in Arabidopsis.

Environmental and physiological situations can challenge the balance between protein synthesis and folding capacity of the endoplasmic reticulum (ER) and cause ER stress, a potentially lethal condition. The unfolded protein response (UPR) restores ER homeostasis or actuates programmed cell death (PCD) when ER stress is unresolved. The cell fate determination mechanisms of the UPR are not well understood, especially in plants. Here, we integrate genetics and ER stress profiling with natural variation and quantitative trait locus analysis of 350 natural accessions of the model species Arabidopsis thaliana. Our analyses implicate a single nucleotide polymorphism to the loss of function of the general PCD regulator BON-ASSOCIATED PROTEIN2 (BAP2) in UPR outcomes. We establish that ER stress-induced BAP2 expression is antagonistically regulated by the UPR master regulator, inositol-requiring enzyme 1 (IRE1), and that BAP2 controls adaptive UPR amplitude in ER stress and ignites pro-death mechanisms in conditions of UPR insufficiency.

Relevance of the Unfolded Protein Response to Spaceflight-Induced Transcriptional Reprogramming in Arabidopsis.

Plants are primary producers of food and oxygen on Earth and will likewise be indispensable to the establishment of large-scale sustainable ecosystems and human survival in space. To contribute to the understanding of how plants respond to spaceflight stress, we examined the significance of the unfolded protein response (UPR), a conserved signaling cascade that responds to a number of unfavorable environmental stresses, in the model plant Arabidopsis thaliana. To do so, we performed a large-scale comparative transcriptome profiling in wild type and various UPR-defective mutants during the SpaceX-CRS12 mission to the International Space Station. We established that orbital culture substantially alters the expression of hundreds of stress-related genes compared with ground control conditions. Although expression of those genes varied in the UPR mutants on the ground, it was largely similar across the genotypes in the spaceflight condition. Our results have yielded new information on how plants respond to growth in orbit and support the hypothesis that spaceflight induces the activation of signaling pathways that compensate for the loss of UPR regulators in the control of downstream transcriptional regulatory networks.

AtIRE1C, an unconventional isoform of the UPR master regulator AtIRE1, is functionally associated with AtIRE1B in Arabidopsis gametogenesis.

The unfolded protein response (UPR), a highly conserved set of eukaryotic intracellular signaling cascades, controls the homeostasis of the endoplasmic reticulum (ER) in normal physiological growth and situations causing accumulation of potentially toxic levels of misfolded proteins in the ER, a condition known as ER stress. During evolution, eukaryotic lineages have acquired multiple UPR effectors, which have increased the pliability of cytoprotective responses to physiological and environmental stresses. The ER-associated protein kinase and ribonuclease IRE1 is a UPR effector that is conserved from yeast to metazoans and plants. IRE1 assumes dispensable roles in growth in yeast but it is essential in mammals and plants. The Arabidopsis genome encodes two isoforms of IRE1, IRE1A and IRE1B, whose protein functional domains are conserved across eukaryotes. Here, we describe the identification of a third Arabidopsis IRE1 isoform, IRE1C. This protein lacks the ER lumenal domain that has been implicated in sensing ER stress in the IRE1 isoforms known to date. Through functional analyses, we demonstrate that IRE1C is not essential in growth and stress responses when deleted from the genome singularly or in combination with an IRE1A knockout allele. However, we found that IRE1C exerts an essential role in gametogenesis when IRE1B is also depleted. Our results identify a novel, plant-specific IRE1 isoform and highlight that at least the control of gametogenesis in Arabidopsis requires an unexpected functional coordination of IRE1C and IRE1B. More broadly, our findings support the existence of a functional form of IRE1 that is required for development despite the remarkable absence of a protein domain that is critical for the function of other known IRE1 isoforms.

Production of (15)N-labeled α-amanitin in Galerina marginata.

α-Amanitin is the major causal constituent of deadly Amanita mushrooms that account for the majority of fatal mushroom poisonings worldwide. It is also an important biochemical tool for the study of its target, RNA polymerase II. The commercial supply of this bicyclic peptide comes from Amanita phalloides, the death cap mushroom, which is collected from the wild. Isotopically labeled amanitin could be useful for clinical and forensic applications, but α-amanitin has not been chemically synthesized and A. phalloides cannot be cultured on artificial medium. Using Galerina marginata, an unrelated saprotrophic mushroom that grows and produces α-amanitin in culture, we describe a method for producing (15)N-labeled α-amanitin using growth media containing (15)N as sole nitrogen source. A key to success was preparing (15)N-enriched yeast extract via a novel method designated "glass bead-assisted maturation." In the presence of the labeled yeast extract and (15)N-NH4Cl, α-amanitin was produced with >97% isotope enrichment. The labeled product was confirmed by HPLC, high-resolution mass spectrometry, and NMR.

Profiling of amatoxins and phallotoxins in the genus Lepiota by liquid chromatography combined with UV absorbance and mass spectrometry.

Species in the mushroom genus Lepiota can cause fatal mushroom poisonings due to their content of amatoxins such as α-amanitin. Previous studies of the toxin composition of poisonous Lepiota species relied on analytical methods of low sensitivity or resolution. Using liquid chromatography coupled to UV absorbance and mass spectrometry, we analyzed the spectrum of peptide toxins present in six Italian species of Lepiota, including multiple samples of three of them collected in different locations. Field taxonomic identifications were confirmed by sequencing of the internal transcribed spacer (ITS) regions. For comparison, we also analyzed specimens of Amanita phalloides from Italy and California, a specimen of A. virosa from Italy, and a laboratory-grown sample of Galerina marginata. α-Amanitin, ß-amanitin, amanin, and amaninamide were detected in all samples of L. brunneoincarnata, and α-amanitin and γ-amanitin were detected in all samples of L. josserandii. Phallotoxins were not detected in either species. No amatoxins or phallotoxins were detected in L. clypeolaria, L. cristata, L. echinacea, or L. magnispora. The Italian and California isolates of A. phalloides had similar profiles of amatoxins and phallotoxins, although the California isolate contained more ß-amanitin relative to α-amanitin. Amaninamide was detected only in A. virosa.

Peptide macrocyclization catalyzed by a prolyl oligopeptidase involved in α-amanitin biosynthesis.

Amatoxins are ribosomally encoded and posttranslationally modified peptides that account for the majority of fatal mushroom poisonings of humans. A representative amatoxin is the bicyclic octapeptide α-amanitin, formed via head-to-tail macrocyclization, which is ribosomally biosynthesized as a 35-amino acid propeptide in Amanita bisporigera and in the distantly related mushroom Galerina marginata. Although members of the prolyl oligopeptidase (POP) family of serine proteases have been proposed to play a role in α-amanitin posttranslational processing, the exact mechanistic details are not known. Here, we show that a specific POP (GmPOPB) is required for toxin maturation in G. marginata. Recombinant GmPOPB catalyzed two nonprocessive reactions: hydrolysis at an internal Pro to release the C-terminal 25-mer from the 35-mer propeptide and transpeptidation at the second Pro to produce the cyclic octamer. On the other hand, we show that GmPOPA, the putative housekeeping POP of G. marginata, behaves like a conventional POP.

Factors contributing to the recalcitrance of herbaceous dicotyledons (forbs) to enzymatic deconstruction.

BACKGROUND: Many different feedstocks are under consideration for the practical production of biofuels from lignocellulosic materials. The best choice under any particular combination of economic, agronomic, and environmental conditions depends on multiple factors. The use of old fields, restored prairie, or marginal lands to grow biofuel feedstocks offers several potential benefits including minimal agronomic inputs, reduced competition with food production, and high biodiversity. However, a major component of such landscapes is often herbaceous dicotyledonous plants, also known as forbs. The potential and obstacles of using forbs as biofuel feedstocks compared to the more frequently considered grasses and woody plants are poorly understood. RESULTS: The factors that contribute to the yield of fermentable sugars from four representative forbs were studied in comparison with corn stover. The forbs chosen for the study were lamb's quarters (Chenopodium album), goldenrod (Solidago canadensis), milkweed (Asclepias syriaca), and Queen Anne's lace (Daucus carota). These plants are taxonomically diverse, widely distributed in northern temperate regions including the continental United States, and are weedy but not invasive. All of the forbs had lower total glucose (Glc) content from all sources (cell walls, sucrose, starch, glucosides, and free Glc) compared to corn stover (range 16.2 to 23.0% on a dry weight basis compared to 39.2% for corn stover). When digested with commercial enzyme mixtures after alkaline pretreatment, yields of Glc as a percentage of total Glc were lower for the forbs compared to corn stover. Enzyme inhibition by water-extractable compounds was not a significant contributor to the lower yields. Based on experiments with optimized cocktails of pure glycosyl hydrolases, enzyme imbalance probably accounted for much of the lower yields. Addition of xyloglucanase and α-xylosidase, two enzymes targeting Glc-containing polysaccharides that are more abundant in dicotyledonous plants compared to grasses, enhanced Glc yields from lamb's quarters, but Glc yields were still lower than from corn stover. CONCLUSION: The potential utilization of forb-rich plant communities as biofuel feedstocks must take into account their lower Glc content compared to grasses such as corn stover. Furthermore, new enzyme mixtures tailored to the different cell wall composition of forbs will have to be developed.