Environmental Stresses Induce Misfolded Protein Aggregation in Plant Cells in a Microtubule-Dependent Manner

Misfolded protein aggregation in mammalian cells is one of the cellular responses to environmental stresses. However, the aggregation of misfolded proteins in plant cells exposed to environmental stresses is still poorly understood. Here, we report the misfolded protein aggregation in plant cells in response to environmental stresses, including ultraviolet (UV) radiation, heat stress and cold stress. Treatment of grape and tobacco cultured cells with MG-132, a proteasome inhibitor, induced misfolded protein aggregation. All of the environmental stresses examined induced the endoplasmic reticulum (ER) stress response in the cells. The cells under ER stress showed aggregation of misfolded proteins. The misfolded protein aggregation was completely inhibited by treatment of the cells with trichostatin A or colchicine, suggesting that the misfolded proteins might be aggregated in plant cells in a microtubule-dependent manner. Detected aggregates were initially observed immediately after exposure to the environmental stresses (1 min after UV radiation, 5 min after heat stress exposure, and 15 min after cold stress exposure). Based on these findings, we hypothesize that environmental stresses induce misfolded protein aggregation in plant cells in a microtubule-dependent manner

Magnesium and zinc stable isotopes as a new tool to understand Mg and Zn sources in stream food webs

Non-traditional stable isotopes of metals were recently shown as new dietary tracers in terrestrial and marine mammals. Whether these metal stable isotopes can be used to understand feeding habits in stream food webs is not known yet. In this study, we explored the potential of stable isotopes of essential Mg (δ²⁶Mg) and Zn (δ⁶⁶Zn) as a new tool in stream ecology. For this purpose, we determined δ²⁶Mg and δ⁶⁶Zn values of stream organisms and their potential metal sources in upper and lower reaches of two streams in the Lake Biwa catchment, Central Japan. Our goals were (1) to explore variations in δ²⁶Mg and δ⁶⁶Zn across organisms of different feeding habits and (2) to understand Mg and Zn sources to stream organisms. Overall, δ²⁶Mg and δ⁶⁶Zn values of organisms were neither related to each other, nor to δ¹³C and δ¹⁵N values, indicating different elemental sources and factors controlling isotopic fractionation depending on element and taxa. Low δ²⁶Mg values in filter-feeding caddisfly larvae and small gobies indicated aqueous Mg uptake. Higher δ²⁶Mg values in leaf-shredding crane fly and grazing mayfly larvae suggested Mg isotopic fractionation during Mg uptake from the diet. While the δ²⁶Mg values of stonefly nymphs reflected those of caddisfly larvae as a potential prey, the highest δ²⁶Mg values found in dobsonfly nymphs can be explained by ²⁶Mg enrichment during maturing. δ⁶⁶Zn values of caddisfly and mayfly larvae indicated Zn was a mixture of aqueous and dietary available Zn, while higher δ⁶⁶Zn values in crane fly larvae pointed to Zn isotopic fractionation during Zn uptake from plant litter. δ⁶⁶Zn values in stonefly and dobsonfly nymphs were often in the range of those of caddisfly larvae as their prey, while dragonfly nymphs and small goby were depleted in ⁶⁶Zn relative to their dietary Zn sources. We conclude that δ²⁶Mg is a promising indicator to assess Mg sources in stream ecology depending on taxa, while the use of δ⁶⁶Zn is limited due to the complexity in Zn sources

Magnesium and zinc stable isotopes as a new tool to understand Mg and Zn sources in stream food webs

Non‐traditional stable isotopes of metals were recently shown as new dietary tracers in terrestrial and marine mammals. Whether these metal stable isotopes can be used to understand feeding habits in stream food webs is not known yet. In this study, we explored the potential of stable isotopes of essential Mg (δ26Mg) and Zn (δ66Zn) as a new tool in stream ecology. For this purpose, we determined δ26Mg and δ66Zn values of stream organisms and their potential metal sources in upper and lower reaches of two streams in the Lake Biwa catchment, Central Japan. Our goals were (1) to explore variations in δ26Mg and δ66Zn across organisms of different feeding habits and (2) to understand Mg and Zn sources to stream organisms. Overall, δ26Mg and δ66Zn values of organisms were neither related to each other, nor to δ13C and δ15N values, indicating different elemental sources and factors controlling isotopic fractionation depending on element and taxa. Low δ26Mg values in filter‐feeding caddisfly larvae and small gobies indicated aqueous Mg uptake. Higher δ26Mg values in leaf‐shredding crane fly and grazing mayfly larvae suggested Mg isotopic fractionation during Mg uptake from the diet. While the δ26Mg values of stonefly nymphs reflected those of caddisfly larvae as a potential prey, the highest δ26Mg values found in dobsonfly nymphs can be explained by 26Mg enrichment during maturing. δ66Zn values of caddisfly and mayfly larvae indicated Zn was a mixture of aqueous and dietary available Zn, while higher δ66Zn values in crane fly larvae pointed to Zn isotopic fractionation during Zn uptake from plant litter. δ66Zn values in stonefly and dobsonfly nymphs were often in the range of those of caddisfly larvae as their prey, while dragonfly nymphs and small goby were depleted in 66Zn relative to their dietary Zn sources. We conclude that δ26Mg is a promising indicator to assess Mg sources in stream ecology depending on taxa, while the use of δ66Zn is limited due to the complexity in Zn sources

Calcium and strontium stable isotopes reveal similar behaviors of essential Ca and nonessential Sr in stream food webs

Recent studies showed the potential of stable isotopes of the macronutrient calcium (δ⁴⁴/⁴⁰Ca) and nonessential strontium (δ⁸⁸/⁸⁶Sr) as new trophic level indicators in terrestrial vertebrates and marine teleost fishes. In this study, we tested whether similar Ca and Sr isotopic fractionation trends existed in macroinvertebrate-dominated stream food webs compared to vertebrates despite their physiological differences. We have determined the δ⁴⁴/⁴⁰Ca and δ⁸⁸/⁸⁶Sr values as well as the ⁸⁷Sr/⁸⁶Sr ratios of stream macroinvertebrates and small gobies and their potential metal sources (stream water, periphyton, and terrestrial plant litter) in upper and lower reaches of two streams in the Lake Biwa catchment, central Japan. The ⁸⁷Sr/⁸⁶Sr ratios revealed that stonefly nymphs, crustacea, and gobies mostly relied on aquatic Sr sources. Higher ⁸⁷Sr/⁸⁶Sr ratios of some crane fly and caddisfly larvae, mayfly, dobsonfly, and dragonfly nymphs indicated greater terrestrial contributions via plant litter. Positive correlations between the δ⁴⁴/⁴⁰Ca and δ⁸⁸/⁸⁶Sr values implied that similar Ca and Sr sources existed, and that Ca and Sr stable isotopes underwent similar fractionation trends although Sr was not essential. The δ⁴⁴/⁴⁰Ca and partly the δ⁸⁸/⁸⁶Sr values were positively correlated with Sr/Ca ratios and negatively with δ¹⁵N values indicating trophic effects on Ca and Sr stable isotopes. The enrichment of ⁴⁴Ca and ⁸⁸Sr in large filter-feeding caddisfly larvae was a notable exception from these trophic trends. Our data confirm that the trophic ⁴⁴Ca and ⁸⁸Sr depletion observed for marine teleost fishes and terrestrial vertebrates also applied to macroinvertebrate-dominated stream food webs despite their different physiologies indicating that shared mechanisms of Ca and Sr isotopic fractionation may exist at the cellular or molecular level between these taxa

Effects of inorganic mercury and methylmercury on osteoclasts and osteoblasts in the scales of the marine teleost as a model system of bone

To evaluate the effects of inorganic mercury (InHg) and methylmercury (MeHg) on bone metabolism in a marine teleost, the activity of tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) as indicators of such activity in osteoclasts and osteoblasts, respectively, were examined in scales of nibbler fish (Girella punctata). We found several lines of scales with nearly the same TRAP and ALP activity levels. Using these scales, we evaluated the influence of InHg and MeHg. TRAP activity in the scales treated with InHg (10-5 and 10-4 M) and MeHg (10-6 to 10-4 M) during 6 hrs of incubation decreased significantly. In contrast, ALP activity decreased after exposure to InHg (10-5 and 10-4 M) and MeHg (10-6 to 10-4 M) for 18 and 36 hrs, although its activity did not change after 6 hrs of incubation. As in enzyme activity 6 hrs after incubation, mRNA expression of TRAP (osteoclastic marker) decreased significantly with InHg and MeHg treatment, while that of collagen (osteoblastic marker) did not change significantly. At 6 hrs after incubation, the mRNA expression of metallothionein, which is a metal-binding protein in osteoblasts, was significantly increased following treatment with InHg or MeHg, suggesting that it may be involved in the protection of osteoblasts against mercury exposure up to 6 hrs after incubation. To our knowledge, this is the first report of the effects of mercury on osteoclasts and osteoblasts using marine teleost scale as a model system of bone. © 2014 Zoological Society of Japan

Alteration of the bZIP60/IRE1 Pathway Affects Plant Response to ER Stress in Arabidopsis thaliana

The Unfolded Protein Response (UPR) is elicited under cellular and environmental stress conditions that disrupt protein folding in the endoplasmic reticulum (ER). Through the transcriptional induction of genes encoding ER resident chaperones and proteins involved in folding, the pathway contributes to alleviating ER stress by increasing the folding capacity in the ER. Similarly to other eukaryotic systems, one arm of the UPR in Arabidopsis is set off by a non-conventional splicing event mediated by ribonuclease kinase IRE1b. The enzyme specifically targets mature bZIP60 RNA for cleavage, which results in a novel splice variant encoding a nuclear localized transcription factor. Although it is clear that this molecular switch widely affects the transcriptome, its exact role in overall plant response to stress has not been established and mutant approaches have not provided much insight. In this study, we took a transgenic approach to manipulate the pathway in positive and negative fashions. Our data show that the ER-resident chaperone BiP accumulates differentially depending on the level of activation of the pathway. In addition, phenotypes of the transgenic lines suggest that BiP accumulation is positively correlated with plant tolerance to chronic ER stress

The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco

The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought

Salt stress responses in Arabidopsis utilize a signal transduction pathway related to endoplasmic reticulum stress signaling

We describe a signaling pathway that mediates salt stress responses in Arabidopsis. The response is mechanistically related to endoplasmic reticulum (ER) stress responses described in mammalian systems. Such responses involve processing and relocation to the nucleus of ER membrane-associated transcription factors to activate stress response genes. The salt stress response in Arabidopsis requires a subtilisin-like serine protease (AtS1P), related to mammalian S1P and a membrane-localized b-ZIP transcription factor, AtbZIP17, a predicted type-II membrane protein with a canonical S1P cleavage site on its lumen-facing side and a b-ZIP domain on its cytoplasmic side. In response to salt stress, it was found that myc-tagged AtbZIP17 was cleaved in an AtS1P-dependent process. To show that AtS1P directly targets AtbZIP17, cleavage was also demonstrated in an in vitro pull-down assay with agarose bead-immobilized AtS1P. Under salt stress conditions, the N-terminal fragment of AtbZIP17 tagged with GFP was translocated to the nucleus. The N-terminal fragment bearing the bZIP DNA binding domain was also found to possess transcriptional activity that functions in yeast. In Arabidopsis, AtbZIP17 activation directly or indirectly upregulated the expression of several salt stress response genes, including the homeodomain transcription factor ATHB-7. Upregulation of these genes by salt stress was blocked by T-DNA insertion mutations in AtS1P and AtbZIP17. Thus, salt stress induces a signaling cascade involving the processing of AtbZIP17, its translocation to the nucleus and the upregulation of salt stress genes

Overexpression of the Endoplasmic Reticulum Chaperone BiP3 Regulates XA21-Mediated Innate Immunity in Rice

Recognition of pathogen-associated molecular patterns by pattern recognition receptors (PRRs) activates the innate immune response. Although PRR-mediated signaling events are critical to the survival of plants and animals, secretion and localization of PRRs have not yet been clearly elucidated. Here we report the in vivo interaction of the endoplasmic reticulum (ER) chaperone BiP3 with the rice XA21 PRR, which confers resistance to the Gram negative bacterium, Xanthomonas oryzae pv. oryzae (Xoo). We show that XA21 is glycosylated and is primarily localized to the ER and also to the plasma membrane (PM). In BiP3-overexpressing rice plants, XA21-mediated immunity is compromised, XA21 stability is significantly decreased, and XA21 proteolytic cleavage is inhibited. BiP3 overexpression does not affect the general rice defense response, cell death or brassinolide-induced responses. These results indicate that BiP3 regulates XA21 protein stability and processing and that this regulation is critical for resistance to Xoo

Transcriptional Analysis Implicates Endoplasmic Reticulum Stress in Bovine Spongiform Encephalopathy

Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. To date, the disease process is still poorly understood. In this study, brain tissue samples from animals naturally infected with BSE were analysed to identify differentially regulated genes using Affymetrix GeneChip Bovine Genome Arrays. A total of 230 genes were shown to be differentially regulated and many of these genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response and transcription. Seventeen genes are associated with the endoplasmic reticulum (ER) and 10 of these 17 genes are involved in stress related responses including ER chaperones, Grp94 and Grp170. Western blotting analysis showed that another ER chaperone, Grp78, was up-regulated in BSE. Up-regulation of these three chaperones strongly suggests the presence of ER stress and the activation of the unfolded protein response (UPR) in BSE. The occurrence of ER stress was also supported by changes in gene expression for cytosolic proteins, such as the chaperone pair of Hsp70 and DnaJ. Many genes associated with the ubiquitin-proteasome pathway and the autophagy-lysosome system were differentially regulated, indicating that both pathways might be activated in response to ER stress. A model is presented to explain the mechanisms of prion neurotoxicity using these ER stress related responses. Clustering analysis showed that the differently regulated genes found from the naturally infected BSE cases could be used to predict the infectious status of the samples experimentally infected with BSE from the previous study and vice versa. Proof-of-principle gene expression biomarkers were found to represent BSE using 10 genes with 94% sensitivity and 87% specificity