Hydration-state-responsive proteins link cold and drought stress in spinach

Spinach (Spinacia oleracea L.) seedlings exposed to low nonfreezing temperatures (0–10° C) that promote cold acclimation, synthesize a variety cold-acclimation proteins and at the same time acquire a greater ability to withstand cellular dehydration imposed by the freezing of tissue water. Two of these proteins (160 and 85 kDa) become more abundant over time at low temperature. In addition, a small decline in tissue water status from a maximally hydrated state also appears to be associated with an initiation of the accumulation of these proteins at a noninductive temperature. Imposing a severe water stress on young seedlings grown at 25° C by withholding water leads to substantial accumulation of the 160- and 85-kDa proteins, and maximal induction of freezing tolerance. This evidence implies that responses to cold acclimation and water stress involve common mechanisms, and further establishes the linkage of these two proteins with stresses having an osmotic component.Abbreviations ABA abscisic acid - CAP cold-acclimation protein - kDa kilodaltons We thank T. Sinclair and K. Cline for critical reading and discussions, N. Denslow for assistance with protein sequencing methods, and L. Greene, S. Henry for preparing the monoclonal antibodies. The work was made possible by support from the USDA Competitive Grants Program No. 90-37280-5527, the Institute for Food and Agricultural Sciences, and through access to the protein sequencing and hybridoma facilities of the Interdisciplinary Center for Biotechnology Research at the University of Florida. Florida Agricultural Experiment Station Journal Series R-02399.     

The role of heat shock proteins in inflammatory injury induced by cold stress in chicken hearts

The aim of this study was to investigate the effects of cold stress on the expression levels of heat shock proteins (Hsps90, 70, 60, 40, and 27) and inflammatory factors (iNOS, COX-2, NF-κB, TNF-α, and PTGEs) and oxidative indexes in hearts of chickens. Two hundred forty 15-day-old male chickens were randomly divided into 12 groups and kept at the temperature of 12 ± 1 °C for acute and chronic cold stress. There were one control group and five treatment groups for acute cold stress, three control groups, and three treatment groups for chronic cold stress. After cold stress, malondialdehyde level increased in chicken heart; the activity of superoxide dismutase and glutathione peroxidase in the heart first increased and then decreased. The inflammatory factors mRNA levels were increased in cold stress groups relative to control groups. The histopathological analysis showed that heart tissues were seriously injured in the cold stress group. Additionally, the mRNA levels of Hsps (70, 60, 40, and 27) increased significantly (P < 0.05) in the cold stress groups relative to the corresponding control group. Meanwhile, the mRNA level and protein expression of Hsp90 decreased significantly (P < 0.05) in the stress group, and showed a gradually decreasing tendency. These results suggested that the levels of inflammatory factors and Hsps expression levels in heart tissues can be influenced by cold stress. Hsps commonly played an important role in the protection of the heart after cold stress.     

ExoCarta: A compendium of exosomal proteins and RNA

Exosomes, membrane microvesicles (40–100 nm) secreted by most cell types, can be isolated in several ways while characterizing them is heavily based on electron microscopy and, most importantly, the identification of exosome marker proteins. Researchers rely on the identification of certain exosomal marker proteins including Alix, CD9 and CD63 to confirm the presence of exosomes in their preparations. An evolutionary‐conserved set of protein molecules have been identified in most exosomes studied to date. However, with the complexity of tissue/cell type‐specific proteins being incorporated in the exosomes, some of these so‐called exosomal markers are not always present in all the exosomes. The presence of tissue/cell type‐specific proteins in exosomes allows researchers to isolate them using immunoaffinity capture methods. A compendium for exosomal proteomes will aid researchers in identifying proteins that were more commonly found in various exosomes (exosome markers) and those that are specific to certain tissue/cell type‐derived exosomes. Here, we describe ExoCarta, a compendium for proteins and RNA molecules identified in exosomes. ExoCarta is first of its kind and the resource is freely available to the scientific community through the web ( http://exocarta.ludwig.edu.au ). We believe that this community resource will be of great biological importance for any future exosome analyses.     

Effects of cold stress on glutathione and related enzymes in rat erythrocytes

Effects of acute and chronic cold stress on glutathione and related enzymes in rat erythrocytes were investigated. Blood from both cold-acclimated (CA) and cold-adapted (CG) rats had significantly lower concentrations of glutathione than blood from control animals. Superoxide dismutase activity was increased significantly in CA rats and tended to rise in CG rats. Activity of glutathione peroxidase in erythrocytes was inconsistent in that it tended to increase in CA rats but decreased significantly in CG rats. The results may imply that CG rats suffered deleterious effects of hydrogen peroxide. On the other hand, there were marked decreases in glutathione peroxidase and glutathione reductase activities in acutely cold-exposed rats in conjunction with unchanged levels of glutathione. In all treatments the state of riboflavin metabolism was estimated to be adequate, since no increases were observed in the erythrocyte glutathione reductase activity coefficient.     

Formation of early-light-inducible-protein complexes and status of xanthophyll levels under high light and cold stress in barley (Hordeum vulgare L.)

In our previous work we found considerable accumulation of early light-inducible proteins (ELIPs) in barley during adaptation to combined high light and cold stress, an accumulation which occurred preferentially in the apical part of the leaves (M.-H. Montané et al., 1997, Planta 202: 293–302). Here we studied, under the same conditions, the effect of adaptation on the composition of thylakoid membrane proteins and pigments, particularly xanthophylls and chlorophyll, and their distribution within the barley leaf. It was observed that high light fluxes appeared to favour the trimerization of the light-harvesting complex of photosystem II (LHC II) whereas cold appeared to favour the monomers of LHC II. High light, cold or the combination of both factors had only a small effect on the protein composition of the thylakoid membranes except for the proteins of LHC II which were found to decrease under high light to a greater extent at 25 °C than at 5 °C. The total xanthophyll-cycle carotenoid content increased linearly with cellular development, the highest amount being observed in the apical part of the leaf. Cold and high light acted synergistically to induce less than a doubling in the amount of total xanthophylls, while chlorophylls a and b remained nearly constant. The fraction consisting of antheraxanthin plus zeaxanthin was up to 4- to 5-fold higher at 5 °C than at 25 °C. As determined previously (Montané et al. 1997), the same conditions caused a 15-fold increase in the accumulation of ELIPs. Consequently, neither the distribution of total xanthophylls nor that of antheraxanthin plus zeaxanthin along the leaf followed the same pattern as ELIP. Thus, the accumulation of xanthophylls cannot be stoichiometrically correlated with that of ELIPs. Using electrophoresis in the presence of decylmaltoside, we could demonstrate for the first time that ELIPs of 13.5 kDa are contained in high-molecular-mass complexes of >100 kDa, which are located in the unstacked stroma lamellar region of the thylakoid membranes. Received: 6 April 1998 / Accepted: 26 January 1999     

Effect of cyclical cold stress during embryonic development on aspects of physiological responses and HSP70 gene expression of chicks

The objective of the present study was to evaluate the effects of cyclical lower incubation temperature at different embryonic ages on the hatchability, body and organs weights, thyroid hormones, and liver HSP70 gene expression of newly hatched chicks. In a completely randomized design, fertile eggs of a broiler breeder (34 weeks of age) were assigned to three treatment groups with six replicates and 145 eggs per each. The treatment groups were as: control group (C) that eggs were incubated at 37.6 °C during the whole incubation period; incubation temperature was decreased to 36 °C for 3 h per day at embryonic age from 12 to 14 (T1); and incubation temperature was decreased to 36 °C for 3 h per day at embryonic age from 15 to 17 (T2). No significant difference was found among treatments for hatchability (P>0.05). There were no differences (P>0.05) among treatments for body weight and liver weight, while heart weight of chicks in T1 and T2 groups were significantly higher than the control group (P<0.05). There were no differences (P>0.05) among treatments for the levels of thyroid hormones, however, the levels of both hormones tended to increase in chicks exposed to cold stress (T1 and T2). Chicks in T2 group had higher liver HSP70 gene expression compared with those in T1 and the control group (P<0.05). Cold stress in both incubation periods had no significant effect on the plasma levels of aspartate aminotransferase and alanine aminotransferase. Treatments had no effect on the plasma levels of glucose, cholesterol and triglyceride. The results of this study suggest that cyclical lower incubation temperatures (36 °C) at the embryonic age from day 15–17 could induce the liver HSP70 gene expression, without negative effects on the hatchability and body weight of hatched chicks.     

Heat induced stress proteins and the concept of molecular chaperones

Heat stress proteins can be assigned to eleven protein families conserved among bacteria, plants and animals. Most of them aid other proteins to maintain or regain their native conformation by stabilizing partially unfolded states. Hence, they are called molecular chaperones. Experimental data indicate that many of them form heterooligomeric complexes, so-called chaperone machines, interacting with each other to generate a network for maturation, assembly and intracellular targeting of proteins. In this review we summarize the essential information on the structure and function of chaperone and chaperone complexes. In addition we present a compilation ofin vivo andin vivo test systems used in the preceding ten years of chaperone research.     

Induction of stress proteins by sodium chloride treatment in Bacillus subtilis

In Bacillus subtilis, heat shock proteins can be classified into two main groups: specific heat shock proteins (about 5) and general stress proteins (at least 14). Salt stress was very effective in the induction of general stress proteins (5 to 50-fold stimulation), but the synthesis of heat-specific stress proteins was not stimulated. Furthermore there were some proteins whose synthesis was accelerated only by salt stress.     

A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2)

The gene expression changes produced by moderate hypothermia are not fully known, but appear to differ in important ways from those produced by heat shock. We examined the gene expression changes produced by moderate hypothermia and tested the hypothesis that rewarming after hypothermia approximates a heat-shock response. Six sets of human HepG2 hepatocytes were subjected to moderate hypothermia (31°C for 16 h), a conventional in vitro heat shock (43°C for 30 min) or control conditions (37°C), then harvested immediately or allowed to recover for 3 h at 37°C. Expression analysis was performed with Affymetrix U133A gene chips, using analysis of variance-based techniques. Moderate hypothermia led to distinct time-dependent expression changes, as did heat shock. Hypothermia initially caused statistically significant, greater than or equal to twofold changes in expression (relative to controls) of 409 sequences (143 increased and 266 decreased), whereas heat shock affected 71 (35 increased and 36 decreased). After 3 h of recovery, 192 sequences (83 increased, 109 decreased) were affected by hypothermia and 231 (146 increased, 85 decreased) by heat shock. Expression of many heat shock proteins was decreased by hypothermia but significantly increased after rewarming. A comparison of sequences affected by thermal stress without regard to the magnitude of change revealed that the overlap between heat and cold stress was greater after 3 h of recovery than immediately following thermal stress. Thus, while some overlap occurs (particularly after rewarming), moderate hypothermia produces extensive, time-dependent gene expression changes in HepG2 cells that differ in important ways from those induced by heat shock.     

Role of HSF activation for resistance to heat, cold and high-temperature knock-down

Regulation of heat shock proteins (Hsps) by the heat shock factor (HSF) and the importance of these proteins for resistance to heat stress is well documented. Less characterized is the importance of Hsps for cold stress resistance although Hsp70 is known to be induced following long-term cold exposure in Drosophila melanogaster. In this study, a temperature-sensitive HSF mutant line was used to investigate the role of HSF activation following heat hardening, rapid cold hardening (RCH) and long-term cold acclimation (LTCA) on heat and cold resistance, and this was correlated with Hsp70 expression. In addition, the effect of HSF activation on high-temperature knock-down resistance was evaluated. We found a significantly decreased HSF activation in the mutant line as compared to a corresponding control line following heat hardening, and this was correlated with decreased heat resistance of the mutant line. However, we did not find this difference in HSF activity to be important for resistance to cold stress or high-temperature knock-down. The findings indicate that induction of stress genes regulated by HSF, such as Hsps, although occurring following LTCA, are not of major importance for cold stress resistance and neither for RCH nor high-temperature knock-down resistance in D. melanogaster.     

A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation

A novel cDNA clone, Tad1, was isolated from crown tissue of winter wheat after differential screening of cold acclimation-induced genes. The Tad1 cDNA encoded a 23kDa polypeptide with a potential N-terminal signal sequence. The putative mature sequence showed striking similarity to plant defensins or gamma-thionins, representing low molecular size antipathogenic polypeptides. High levels of Tad1 mRNA accumulation occurred within one day of cold acclimation in crown tissue and the level was maintained throughout 14 days of cold acclimation. Similar rapid induction was observed in young seedlings treated with low temperature but not with exogenous abscisic acid. In contrast to defensins from other plant species, neither salicylic acid nor methyl jasmonate induced expression of Tad1. The recombinant mature form of TAD1 polypeptide inhibited the growth of the phytopathogenic bacteria, Pseudomonas cichorii; however, no antifreeze activity was detected. Collectively, these data suggested that Tad1 is induced in cold-acclimated winter wheat independent of major defense signaling(s) and is involved in low temperature-induced resistance to pathogens during winter hardening.     

A rice protein library: a data-file of rice proteins separated by two-dimensional electrophoresis

Proteins extracted from embryos, endosperms and leaves of rice were separated by two-dimensional electrophoresis and relative molecular weights and isoelectric points were determined. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 85 electroblotted proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino-acid sequences of 27 out of 85 proteins were determined in this manner. The N-terminal regions of the remaining proteins could not be sequenced and they were inferred to have a blocking group at the N-terminus. Among proteins, 11 could be sequenced after deblocking by in situ treatment with pyroglutamyl peptidase. The internal amino-acid sequences of 23 proteins were determined by sequence analysis of peptides obtained by Cleveland peptide mapping. The amino-acid sequences determined here were compared with those of known plant and animal proteins. The concanavalin A-peroxidase method was used to determine whether the 85 proteins were glycosylated and the diagonal electrophoresis method was used to determine whether they contained disulphide bonding. Finally, we constructed a data-file of rice proteins including information on relative molecular weight, isoelectric point, amino-acid sequence, sequence homology, glycosylation, and the presence of disulphide bonding.     

Induction of water-stress proteins in cyanobacteria exposed to matric- or osmotic-water stress

Abstract: Specific stress polypeptides were detected in three drought-resistant cyanobacteria ( Phormidium autumnale , LPP₄ and Chroococcidiopsis sp.) subjected to matric- and osmotic-water stress. Drought stress caused the induction of at least 2–3 new polypeptides of apparent molecular masses of 30 kDa and 40–45 kDa. The polypeptide of 30 kDa was located in the thylakoid membranes, and the 45-kDa polypeptide in the cytoplasm. When these cyanobacteria were exposed to salt stress polypeptides of similar size appeared.