A small HSP gene is not responsible for diapause and cold tolerance acquisition in Chilo suppressalis

Abstract:  The cDNA sequence of a small heat shock protein ( hsp19.7 ) was cloned and sequenced from the rice stem borer, Chilo suppressalis Walker. The cDNA encoded a protein of 177 amino acids with a calculated molecular weight of 19.7 kDa. The deduced amino acid sequence showed the highest identity of 90% to Bombyx mori hsp19.9 . Expression levels of hsp19.7 were similar between diapausing and non-diapausing larvae. In non-diapausing larvae, but not in diapausing ones, hsp19.7 expression was upregulated by cold acclimation. Involvement of hsp19.7 in larval diapause and cold tolerance in C. suppressalis is discussed.     

Asymmetric life-history decision-making in butterfly larvae

In temperate environments, insects appearing in several generations in the growth season typically have to decide during the larval period whether to develop into adulthood, or to postpone adult emergence until next season by entering a species-specific diapause stage. This decision is typically guided by environmental cues experienced during development. An early decision makes it possible to adjust growth rate, which would allow the growing larva to respond to time stress involved in direct development, whereas a last-minute decision would instead allow the larva to use up-to-date information about which developmental pathway is the most favourable under the current circumstances. We study the timing of the larval pathway decision-making between entering pupal winter diapause and direct development in three distantly related butterflies (Pieris napi, Araschnia levana and Pararge aegeria). We pinpoint the timing of the larval diapause decision by transferring larvae from first to last instars from long daylength (inducing direct development) to short daylength conditions (inducing diapause), and vice versa. Results show that the pathway decision is typically made in the late instars in all three species, and that the ability to switch developmental pathway late in juvenile life is conditional; larvae more freely switched from diapause to direct development than in the opposite direction. We contend that this asymmetry is influenced by the additional physiological preparations needed to survive the long and cold winter period, and that the reluctance to make a late decision to enter diapause has the potential to be a general trait among temperate insects.     

Proteomic analysis of chicory root identifies proteins typically involved in cold acclimation

Chicory (Cichorium intybus) roots contain high amounts of inulin, a fructose polymer used as a storage carbohydrate by the plant and as a human dietary and prebiotic compound. We performed 2‐D electrophoretic analysis of proteins from root material before the first freezing period. The proteins were digested with trypsin and the peptides analyzed by MS (MALDI‐TOF/TOF). From the 881 protein spots analyzed, 714 proteins corresponded to a database accession, 619 of which were classified into functional categories. Besides expected proteins (e.g. related to metabolism, energy, protein synthesis, or cell structure), other well‐represented categories were proteins related to folding and stability (49 spots), proteolysis (49 spots), and the stress response (67 spots). The importance of abiotic stress response was confirmed by the observation that 7 of the 21 most intense protein spots are known to be involved in cold acclimation. These results suggest a major effect of the low temperature period that preceded root harvesting.     

Supercooling point plasticity during cold storage in the freeze‐tolerant sugarbeet root maggot Tetanops myopaeformis

Abstract The sugarbeet root maggot Tetanops myopaeformis (Röder) overwinters as a freeze‐tolerant third‐instar larva. Although most larvae are considered to overwinter for only 1 year, some may exhibit prolonged diapause in the field. In the laboratory, they can live for over 5 years using a combination of diapause and post‐diapause quiescence. In the present study, the cold survival strategies of these larvae during storage is investigated by measuring their supercooling points in combination with survival data. Supercooling points (SCPs) change significantly during storage, highlighted by a marked increase in the range of SCPs recorded, although the ability to tolerate freezing is not affected. Additionally, a freezing event ‘re‐focuses’ the SCPs of aged larvae to levels similar to those seen at diapause initiation. This change in SCPs is dependant not only on the initial freezing event, but also on the parameters of the incubation period between freezing events. Finally, the temperatures of larval overwintering microhabitats are monitored during the 2007–2008 boreal winter. The results indicate that, although overwintering larva are physiologically freeze‐tolerant, they may essentially be freeze avoidant during overwintering via microhabitat selection.     

Cloning of heat shock protein genes (hsp70, hsc70 and hsp90) and their expression in response to larval diapause and thermal stress in the wheat blossom midge,Sitodiplosis mosellana

Sitodiplosis mosellana Géhin, one of the most important pests of wheat, undergoes obligatory diapause as a larva to survive unfavorable temperature extremes during hot summers and cold winters. To explore the potential roles of heat shock proteins (hsp) in this process, we cloned full-length cDNAs of hsp70, hsc70 and hsp90 from S. mosellana larvae, and examined their expression in response to diapause and short-term temperature stresses. Three hsps included all signature sequences of corresponding protein family and EEVD motifs. They showed high homology to their counterparts in other species, and the phylogenetic analysis of hsp90 was consistent with the known classification of insects. Expression of hsp70 and hsp90 were highly induced by diapause, particularly pronounced during summer and winter. Interestingly, hsp70 was more strongly expressed in summer than in winter whereas hsp90 displayed the opposite pattern. Abundance of hsc70 mRNA was comparable prior to and during diapauses and was highly up-regulated when insects began to enter the stage of post-diapause quiescence. Heat-stressed over-summering larvae (⩾30 °C) or cold-stressed over-wintering larvae (⩽0 °C) could further elevate expression of these three genes, but temperature extremes i.e. as high as 45 °C or as low as −15 °C failed to trigger such expression patterns. Notably, hsp70 was most sensitive to heat stress and hsp90 was most sensitive to cold stress. These results suggested that hsp70 and hsp90 play key roles in diapause maintenance and thermal stress; the former may be more prominent contributor to heat tolerance and the latter for cold tolerance. In contrast, hsc70 most likely is involved in developmental transition from diapause to post-diapause quiescence, and thus may serve as a molecular marker to predict diapause termination.     

The chloroplast lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short- and long-term exposure to low temperature

Cold acclimation and over-wintering by herbaceous plants are energetically expensive and are dependent on functional plastid metabolism. To understand how the stroma and the lumen proteomes adapt to low temperatures, we have taken a proteomic approach (difference gel electrophoresis) to identify proteins that changed in abundance in Arabidopsis chloroplasts during cold shock (1 day), and short- (10 days) and long-term (40 days) acclimation to 5 degrees C. We show that cold shock (1 day) results in minimal change in the plastid proteomes, while short-term (10 days) acclimation results in major changes in the stromal but few changes in the lumen proteome. Long-term acclimation (40 days) results in modulation of the proteomes of both compartments, with new proteins appearing in the lumen and further modulations in protein abundance occurring in the stroma. We identify 43 differentially displayed proteins that participate in photosynthesis, other plastid metabolic functions, hormone biosynthesis and stress sensing and signal transduction. These findings not only provide new insights into the cold response and acclimation of Arabidopsis, but also demonstrate the importance of studying changes in protein abundance within the relevant cellular compartment.     

Proteomic and metabolomic profiles of larval hemolymph associated with diapause in the cotton bollworm,Helicoverpa armigera

Background

Diapause is programmed developmental arrest coupled with the depression of metabolic activity and the enhancement of stress resistance. Pupal diapause is induced by environmental signals and is prepared during the prediapause phase. In the cotton bollworm, Helicoverpa armigera, the prediapause phase, which contains two sub-phases, diapause induction and preparation, occurs in the larval stage. Here, we performed parallel proteomic and metabolomic analyses on H. armigera larval hemolymph during the prediapause phase.

Results

By two-dimensional electrophoresis, 37 proteins were shown to be differentially expressed in diapause-destined larvae. Of these proteins, 28 were successfully identified by MALDI-TOF/TOF mass spectrometry. Moreover, a total of 22 altered metabolites were found in diapause-destined larval hemolymph by GC-MS analysis, and the levels of 17 metabolites were elevated and 5 were decreased.

Conclusions

The proteins and metabolites with significantly altered levels play different roles in diapause-destined larvae, including diapause induction, metabolic storage, immune response, stress tolerance, and others. Because hemolymph circulates through the whole body of an insect, these differences found in diapause-destined larvae most likely correspond to upstream endocrine signals and would further influence other organ/tissue activities to determine the insect’s fact: diapause or development.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-751) contains supplementary material, which is available to authorized users.     

温度刺激对斑马鱼仔鱼基因转录表达的影响

许多优良鱼类养殖品种不耐低温或高温的特点给水产养殖业带来诸多限制和困难,这些鱼类在胚胎和仔鱼等早期阶段的抗寒和抗热能力比成体更差,育苗过程中很容易受到温度突然变化的影响。虽然目前利用基因芯片技术已研究了温度刺激对几种鱼类成体组织中基因表达的影响,但温度刺激对仔鱼基因转录表达的影响还未见报道。研究以斑马鱼受精后96h的出膜仔鱼为实验材料,分别在低温(16℃)和高温(34℃)条件下处理12h和24h,用基因芯片技术检测温度刺激对其基因表达的影响。与培养在28℃的对照相比,低温和高温处理后共有3633个基因发生差异表达,其中低温处理后差异表达基因数目多于高温处理,而且低温抑制基因数目多于诱导表达基因的数目。生物信息学分析结果表明,低温诱导基因主要参与RNA加工和核糖体生物发生等生物学过程,高温诱导基因则主要参与应激反应和未折叠蛋白结合。低温抑制基因主要参与蛋白质水解、视觉感知以及铁离子结合等生物学功能,高温抑制基因参与的生物学功能包括DNA复制、神经系统过程和类固醇激素生物合成等。除了已报道的温度刺激响应基因外,研究鉴定出了大量尚未报道与温度刺激相关的基因,如参与RNA加工的rnmtl1a和pus3基因,以及参与转录调控的twistnb和aebp2基因等。研究结果为进一步揭示鱼类冷或热适应的分子机理和培养耐寒或耐热的养殖新品种提供理论基础。     

Differential proteomic response to heat stress in thermal Agrostis scabra and heat‐sensitive Agrostis stolonifera

Knowledge of heat‐responsive proteins is critical for further understanding of the molecular mechanisms of heat tolerance. The objective of this study was to compare proteins differentially expressed in two C₃ grass species contrasting in heat tolerance, heat‐tolerant thermal Agrostis scabra and heat‐sensitive Agrostis stolonifera L., and to identify heat‐responsive proteins for short‐ and long‐term responses. Plants were exposed to 20/15°C (day/night, control) or 40/35°C (day/night, heat stress) in growth chambers. Leaves were harvested at 2 and 10 days after temperature treatment. Proteins were extracted and separated by fluorescence difference gel electrophoresis (DIGE). Thermal A. scabra had superior heat tolerance than A. stolonifera, as indicated by the maintenance of higher chlorophyll content and photochemical efficiency under heat stress. The two‐dimensional difference electrophoresis detected 68 heat‐responsive proteins in the two species. Thermal A. scabra had more protein spots either down‐ or up‐regulated at 2 days of heat stress, but fewer protein spots were altered at 10 days of heat stress compared with A. stolonifera. Many protein spots exhibited transient down‐regulation in thermal A. scabra (only at 2 days of heat treatment), whereas down‐regulation of many proteins was also found at 10 days of heat treatment in A. stolonifera, which suggested that protein metabolism in thermal A. scabra might acclimate to heat stress more rapidly than those in A. stolonifera. The sequences of 56 differentially expressed protein spots were identified using mass spectrometry. The results suggest that the maintenance or less severe down‐regulation of proteins during long‐term (10 days) heat stress may contribute to the superior heat tolerance in thermal A. scabra, including those involved in photosynthesis [RuBisCo, RuBisCo activase, chloroplastic glyceraldehydes‐3‐phosphate dehydrogenase (GAPDH), chloroplastic aldolase, oxygen‐evolving complex, photosystem I subunits], dark respiration (cytosolic GAPDH, cytoplasmic aldolase, malate dehydrogenase, hydroxypyruvate reductase, sedoheptulose‐1,7‐bisphosphatase), photorespiration [(hydroxypyruvate reductase, alanine aminotransferase (AlaAT), hydroxymethyltransferase (SHMT), glycine decarboxylase (GDC)], as well as heat and oxidative stress protection [heat shock cognate (HSC) 70 and FtsH‐like protein].     

Over-wintering stage polymorphism of a bruchine beetle: geographical variation in optimal diapause strategy

Abstract.  1. Most temperate insects undergo diapause at a specific developmental stage to overcome severe winters. The bruchine beetle Bruchidius dorsalis in a warmer region in Japan, however, has diverse over-wintering stages – non-diapausing larvae, diapausing larvae, and diapausing adults, whereas in a cooler region, it over-winters only as the final instar larva or adult in diapause.
2. The geographical pattern of over-wintering stages in 12 populations was investigated over a wide range along the mainland of Japan. It revealed that a distinct geographical pattern of over-wintering stages exists in relation to temperature conditions. In regions with warmer climates, the proportion of non-diapausing larvae increased and B. dorsalis had a more complex over-wintering stage structure.
3. Life cycles were also compared between two areas of Japan by field experiments. In the cooler area, the first generation over-wintered in the diapausing larval or adult stage. Conversely, in the warmer area, diapause was induced later and some of the first-generation adults produced second-generation offspring before over-wintering.
4. Based on the geographical cline of climates and the differences in cold hardiness among stages, we can demonstrate that the over-wintering stage variation among and within populations results from an adaptive timing of diapause induction in each region, because the late larval or adult diapauses protect pupae or eggs – which unlike other stages are not cold hardy – from being produced late in the season.     

Plant defense in response to chewing insects: proteome analysis of Arabidopsis thaliana damaged by Plutella xylostella

The interactions between Arabidopsis thaliana and Plutella xylostella have been considered as a model system to unravel the responses of plants to herbivorous insects. Here, we use a 2-DE proteome approach to detect protein expression changes in the leaves of Arabidopsis plants exposed to P. xylostella larval infestation at 27°C within 8?h. Approximately 450 protein spots were reproducibly detected on gels. Of these, comparing healthy and infested leaves, we identified 18 differentially expressed protein spots. Thirteen proteins were successfully identified by MALDI-TOF/MS and LC-ESI-MS/MS. Functional classification analysis indicated that the differentially identified proteins were associated with amino acid, carbohydrate, energy, lipid metabolism, and photosynthesis. In addition, their relative abundances were assessed according to larval pest feeding on Arabidopsis leaves. These data provide valuable new insights for further works in plant-biotic and environmental stress interaction.     

A proteomic analysis to identify cold acclimation associated proteins in wild wheat (Triticum urartu L.)

To gain a better understanding of cold acclimation process in wheat, we applied a 2-DE based proteomic approach to discover changes in proteome profile of a diploid wild wheat (Triticum urartu L.) during prolonged cold stress treatment. To this end, plants were grown in pots and the growing seedlings (4-leaf stage) were exposed to cold stress. After 4 weeks of cold acclimation (4–6 °C) and subsequent treatment for 12 h at ?2 °C, samples were collected from control and stressed plants and were subjected to proteome pattern analysis. Among approximately 450 reproducible protein spots displayed in each given 2-DE gels, 34 proteins changed significantly in abundance in response to cold stress. Among them, 25 and 9 proteins were up and down-regulated under stress condition, respectively. Analysis by matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry coupled with non-redundant protein database search allowed the identification of 20 cold-induced proteins. Integrated proteomic and database survey resulted in identification of several cold stress related proteins such as pathogenesis related protein, cold regulated protein, cold-responsive LEA/RAB-related COR protein, oxygen-evolving enhancer protein and oxalate oxidase. The presumed functions of the identified proteins were mostly related to cold acclimation, oxidative stress and photosynthesis. The possible implications of differentially accumulated proteins in acquiring systemic tolerance to freezing stress following exposure to prolonged low temperature will be discussed.     

Comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid F1 line under drought stress

Rapeseed (Brassica napus L.), which is the third leading source of vegetable oil, is sensitive to drought stress during the early vegetative growth stage. To investigate the initial response of rapeseed to drought stress, changes in the protein expression profiles of drought-sensitive (RGS-003) and drought-tolerant lines (SLM-003), and their F1 hybrid, were analyzed using a proteomics approach. Seven-day-old rapeseed seedlings were treated with drought stress by restricting water for 7 days, and proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. In the sensitive rapeseed line, 35 protein spots were differentially expressed under drought stress, and proteins related to metabolism, energy, disease/defense, and transport were decreased. In the tolerant line, 32 protein spots were differentially expressed under drought stress, and proteins involved in metabolism, disease/defense, and transport were increased, while energy-related proteins were decreased. Six protein spots in F1 hybrid were common among expressed proteins in the drought-sensitive and -tolerant lines. Notably, tubulin beta-2 and heat shock protein 70 were decreased in the drought-sensitive line and hybrid F1 plants, while jasmonate-inducible protein and 20S proteasome subunit PAF1 were increased in the F1 hybrids and drought-tolerant line. These results indicate that (1) V-type H⁺ ATPase, plasma-membrane associated cation-binding protein, HSP 90, and elongation factor EF-2 have a role in the drought tolerance of rapeseed; (2) The decreased levels of heat shock protein 70 and tubulin beta-2 in the drought-sensitive and hybrid F1 lines might explain the reduced growth of these lines in drought conditions.     

Adaptation to cold and proteomic responses of the psychrotrophic biopreservative Lactococcus piscium strain CNCM I-4031

There is considerable interest in the use of psychrotrophic bacteria for food biopreservation and in the understanding of cold adaptation mechanisms. The psychrotrophic biopreservative Lactococcus piscium strain CNCM I-4031 was studied for its growth behavior and proteomic responses after cold shock and during cold acclimation. Growth kinetics highlighted the absence of growth latency after cold shock, suggesting a very high promptness in cold adaptation, a behavior that has never been described before for lactic acid bacteria (LAB). A comparative proteomic analysis was applied with two-dimensional gel electrophoresis (2-DE), and upregulated proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both cold shock and cold acclimation triggered the upregulation of proteins involved in general and oxidative stress responses and fatty acid and energetic metabolism. However, 2-DE profiles and upregulated proteins were different under both conditions, suggesting a sequence of steps in cold adaptation. In addition, the major 7-kDa Csp protein was identified in the L. piscium CNCM I-4031 genome but was not cold regulated. The implication of the identified cold shock proteins and cold acclimation proteins in efficient cold adaptation, the possible regulation of a histidyl phosphocarrier protein, and the roles of a constitutive major 7-kDa Csp are discussed.     

Comparative proteomic analysis of human placenta derived from assisted reproductive technology

The aim of this study was to use proteomics-based approach to examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Using 2-DE we found that, compared with the control group, 12 spots in standard in vitro fertilization group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differentially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, alpha-SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot and immunohistochemistry analysis. Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.     

Identification of changes in Triticum aestivum L. leaf proteome in response to drought stress by 2D-PAGE and MALDI-TOF/TOF mass spectrometry

Drought is an abiotic stress that strongly influences plant growth, development and productivity. To gain a better understanding of the drought-stress responses at physiological and molecular level in wheat plants (Triticum aestivum cv. KTC86211), we performed a comparative physiological and proteomics analysis. Eight-day-old wheat seedlings were treated with polyethylene glycol-simulated drought stress for 0, 24, 48 and 72 h. Drought treatment resulted in alterations of morphology, increased relative electrolyte leakage and reduced length and weight on leaf and root. Stress-induced proteome changes were analyzed by two-dimensional gel electrophoresis in conjunction with MALDI-TOF/TOF. Twenty-three spots differed significantly between control and treated plants following 48 h of drought stress, with 19 upregulated, and 4 downregulated, in leaf tissues. All of the differentially expressed protein spots were identified, revealing that the majority of proteins altered by drought treatment were involved in reactive oxygen species scavenging enzymes and photosynthesis. Other proteins identified were involved in protein metabolism, cytoskeleton structure, defense response, acid metabolism and signal transduction. All proteins might contribute cooperatively to reestablish cellular homeostasis under drought stress. The present study not only provides new insights into the mechanisms of acclimation and tolerance to drought stress in wheat plants, but also provides clues for improving wheat’s drought tolerance through breeding or genetic engineering.