Transcriptional Responses of the Heat Shock Protein 20 (Hsp20) and 40 (Hsp40) Genes to Temperature Stress and Alteration of Life Cycle Stages in the Harmful Alga Scrippsiella trochoidea (Dinophyceae).
ABSTRACT: The small heat shock protein (sHsp) and Hsp40 are Hsp members that have not been intensively investigated but are functionally important in most organisms. In this study, the potential roles of a Hsp20 (StHsp20) and a Hsp40 (StHsp40) in dinoflagellates during adaptation to temperature fluctuation and alteration of different life stages were explored using the representative harmful algal blooms (HABs)-causative dinoflagellate species, Scrippsiella trochoidea. We isolated the full-length cDNAs of the two genes via rapid amplification of cDNA ends (RACE) and tracked their differential transcriptions via real-time qPCR. The results revealed StHsp20 and StHsp40 exhibited mRNA accumulation patterns that were highly similar in response to heat stress but completely different toward cold stress, which implies that the mechanisms underlying thermal and cold acclimation in dinoflagellates are regulated by different sets of genes. The StHsp20 was probably related to the heat tolerance of the species, and StHsp40 was closely involved in the adaptation to both higher and lower temperature fluctuations. Furthermore, significantly higher mRNA abundance of StHsp40 was detected in newly formed resting cysts, which might be a response to intrinsic stress stemmed from encystment. This finding also implied StHsp40 might be engaged in resting cyst formation of S. trochoidea. Our findings enriched the knowledge about possible cross-talk of different Hsp members in dinoflagellates and provided clues to further explore the molecular underpinnings underlying resting cyst production and broad temperature tolerance of this group of HABs contributors.
Project description:Due to the vital importance of resting cysts in the biology and ecology of many dinoflagellates, a transcriptomic investigation on Scrippsiella trochoidea was conducted with the aim to reveal the molecular processes and relevant functional genes regulating encystment and dormancy in dinoflagellates. We identified via RNA-seq 3,874 (out of 166,575) differentially expressed genes (DEGs) between resting cysts and vegetative cells; a pause of photosynthesis (confirmed via direct measurement of photosynthetic efficiency); an active catabolism including ?-oxidation, glycolysis, glyoxylate pathway, and TCA in resting cysts (tested via measurements of respiration rate); 12 DEGs encoding meiotic recombination proteins and members of MEI2-like family potentially involved in sexual reproduction and encystment; elevated expressions in genes encoding enzymes responding to pathogens (chitin deacetylase) and ROS stress in cysts; and 134 unigenes specifically expressed in cysts. We paid particular attention to genes pertaining to phytohormone signaling and identified 4 key genes regulating abscisic acid (ABA) biosynthesis and catabolism, with further characterization based on their full-length cDNA obtained via RACE-PCR. The qPCR results demonstrated elevated biosynthesis and repressed catabolism of ABA during the courses of encystment and cyst dormancy, which was significantly enhanced by lower temperature (4 ± 1°C) and darkness. Direct measurements of ABA using UHPLC-MS/MS and ELISA in vegetative cells and cysts both fully supported qPCR results. These results collectively suggest a vital role of ABA in regulating encystment and maintenance of dormancy, akin to its function in seed dormancy of higher plants. Our results provided a critical advancement in understanding molecular processes in resting cysts of dinoflagellates.
Project description:Dinoflagellates are a major component of marine phytoplankton and many species are recognized for their ability to produce harmful algal blooms (HABs). Scrippsiella trochoidea is a non-toxic, marine dinoflagellate that can be found in both cold and tropic waters where it is known to produce "red tide" events. Little is known about the genomic makeup of S. trochoidea and a transcriptome study was conducted to shed light on the biochemical and physiological adaptations related to nutrient depletion. Cultures were grown under N and P limiting conditions and transcriptomes were generated via RNAseq technology. De novo assembly reconstructed 107,415 putative transcripts of which only 41% could be annotated. No significant transcriptomic response was observed in response to initial P depletion, however, a strong transcriptional response to N depletion was detected. Among the down-regulated pathways were those for glutamine/glutamate metabolism as well as urea and nitrate/nitrite transporters. Transcripts for ammonia transporters displayed both up- and down-regulation, perhaps related to a shift to higher affinity transporters. Genes for the utilization of DON compounds were up-regulated. These included transcripts for amino acids transporters, polyamine oxidase, and extracellular proteinase and peptidases. N depletion also triggered down regulation of transcripts related to the production of Photosystems I & II and related proteins. These data are consistent with a metabolic strategy that conserves N while maximizing sustained metabolism by emphasizing the relative contribution of organic N sources. Surprisingly, the transcriptome also contained transcripts potentially related to secondary metabolite production, including a homolog to the Short Isoform Saxitoxin gene (sxtA) from Alexandrium fundyense, which was significantly up-regulated under N-depletion. A total of 113 unique hits to Sxt genes, covering 17 of the 34 genes found in C. raciborskii were detected, indicating that S. trochoidea has previously unrecognized potential for the production of secondary metabolites with potential toxicity.
Project description:Cochlodinium polykrikoides is a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated that Cochlodinium forms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution of C. polykrikoides cysts in coastal ecosystems. Here, we report on the development of a fluorescence in situ hybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) of C. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction with C. polykrikoides and minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates of C. polykrikoides but not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and map C. polykrikoides cysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities of C. polykrikoides cysts (>100 cm(-3)) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification of C. polykrikoides rDNA. This study mapped C. polykrikoides cysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species.
Project description:Molecular chaperones function in various important physiological processes. Null mutants of genes for the molecular chaperone ClpB (Hsp104), and those that encode J-domain proteins (DnaJ, CbpA, and DjlA), which may act as Hsp40 co-chaperones of DnaK (Hsp70), were constructed from Pseudomonas putida KT2442 (KT) to elucidate their roles. The KT?clpB mutant showed the same heat shock response (HSR) as the wild-type, both in terms of heat-shock protein (Hsp) synthesis (other than ClpB) and in hsp gene expression; however, the mutant was quite sensitive to high temperatures and was unable to disaggregate into thermo-mediated protein aggregates, indicating that ClpB is important for cell survival after heat stress and essential for solubilization of protein aggregates. On the other hand, the KT?dnaJ mutant was temperature-sensitive, and formed more protein aggregates (especially of high molecular weight) upon heat stress than did KT. P. putida CbpA, a probable Hsp, partially substituted the functions of DnaJ in cell growth and solubilization of thermo-mediated protein aggregates, and might be involved in the HSR which was regulated by a fine-tuning system(s) that could sense subtle changes in the ambient temperature and control the levels of ?(32) activity and quantity, as well as the mRNA levels of hsp genes.
Project description:Background: Heat shock proteins (HSPs) are molecular chaperones known to bind and sequester client proteins under stress. Methods: To identify and better understand some of these proteins, we carried out a computational genome-wide survey of the bovine genome. For this, HSP sequences from each subfamily (sHSP, HSP40, HSP70 and HSP90) were used to search the Pfam (Protein family) database, for identifying exact HSP domain sequences based on the hidden Markov model. ProtParam tool was used to compute potential physico-chemical parameters detectable from a protein sequence. Evolutionary trace (ET) method was used to extract evolutionarily functional residues of a homologous protein family. Results: We computationally identified 67 genes made up of 10, 43, 10 and 4 genes belonging to small HSP, HSP40, HSP70 and HSP90 families respectively. These genes were widely dispersed across the bovine genome, except in chromosomes 24, 26 and 27, which lack bovine HSP genes. We found an uncharacterized outer dense fiber ( ODF1) gene in cattle with an intact alpha crystallin domain, like other small HSPs. Physico-chemical characteristic of aliphatic index was higher in HSP70 and HSP90 gene families, compared to small HSP and HSP40. Grand average hydropathy showed that small HSP (sHSP), HSP40, HSP70 and HSP90 genes had negative values except for DNAJC22, a member of HSP40 gene family. The uniqueness of DNAJA3 and DNAJB13 among HSP40 members, based on multiple sequence alignment, evolutionary trace analysis and sequence identity dendrograms, suggests evolutionary distinct structural and functional features, with unique roles in substrate recognition and chaperone functions. The monophyletic pattern of the sequence identity dendrograms of cattle, human and mouse HSP sequences suggests functional similarities. Conclusions: Our computational results demonstrate the first-pass in-silico identification of heat shock proteins and calls for further investigation to better understand their functional roles and mechanisms in Bovidae.
Project description:Biological response to rapid changing environments is an outstanding research question in ecology and evolution. Biological invasions provide excellent "natural" experiments to study such a complex response process, as invaders often encounter rapidly changing environments during biological invasions. The regulation of heat shock proteins (Hsp) is a common pathway responsible for various environmental stresses; however, the comprehensive study on Hsp system across the whole genome and potential roles in determining invasion success are still largely unexplored. Here, we used a marine invasive model ascidian, Ciona savignyi, to investigate transcriptional response of Hsp-related genes to harsh environments. We identified 32 genes, including three Hsp20, six Hsp40, ten Hsp60, eight Hsp70, three Hsp90, one Hsp100, and one heat shock transcription factor (Hsf), across the whole genome of C. savignyi. We further characterized gene structure and protein motifs, and identified potential heat shock elements (HSEs) in promoters of Hsp genes. The expression analysis showed that most Hsp genes, but not all, were involved in transcriptional response to temperature and salinity challenges in a duration- and stress-specific pattern, and the maximum amplitude of induction occurred in Hsp70-4 after 1-h of high temperature treatment. However, the Hsf gene was scarcely induced and limited interactions were predicted between Hsp and Hsf genes. Our study provide the first systematic genome-wide analysis of Hsp and Hsf family in the marine invasive model ascidian, and our results are expected to dissect Hsp-based molecular mechanisms responsible for extreme environmental adaptation using Ciona as a model system.
Project description:Heat shock protein gene (Hsp) families are thought to be important in thermal adaptation, but their expression patterns under various thermal stresses have still been poorly characterized outside of model systems. We have therefore characterized Hsp genes and their stress responses in the oriental fruit moth (OFM), Grapholita molesta, a widespread global orchard pest, and compared patterns of expression in this species to that of other insects. Genes from four Hsp families showed variable expression levels among tissues and developmental stages. Members of the Hsp40, 70, and 90 families were highly expressed under short exposures to heat and cold. Expression of Hsp40, 70, and Hsc70 family members increased in OFM undergoing diapause, while Hsp90 was downregulated. We found that there was strong sequence conservation of members of large Hsp families (Hsp40, Hsp60, Hsp70, Hsc70) across taxa, but this was not always matched by conservation of expression patterns. When the large Hsps as well as small Hsps from OFM were compared under acute and ramping heat stress, two groups of sHsps expression patterns were apparent, depending on whether expression increased or decreased immediately after stress exposure. These results highlight potential differences in conservation of function as opposed to sequence in this gene family and also point to Hsp genes potentially useful as bioindicators of diapause and thermal stress in OFM.
Project description:Heat shock proteins (Hsps) are molecular chaperones that prevent the aggregation of client proteins by facilitating their refolding, or trafficking them for degradation. The chaperone activities of Hsps are dependent on dynamic protein-protein interactions, including their oligomerisation into large multi-subunit complexes. Thus, tagging Hsps with fluorescent proteins can interfere with their chaperone activity. To overcome this limitation, we have exploited bicistronic constructs for the concurrent expression of a non-tagged Hsp and fluorescent reporter from a single mRNA in cells. We used the Hsp-encoding bicistronic constructs in a cell-based model of protein aggregation, using a destabilised (mutant) form of firefly luciferase (mFluc) that forms inclusion bodies in cells. Expression of Hsp40, Hsp70, or Hsp40 and Hsp70 in cells expressing mFluc decreased the formation of inclusion bodies by 25-46% compared to controls. Moreover, there was a concentration-dependent decrease in the proportion of cells with inclusions when Hsp70, or Hsp40 and Hsp70 were co-expressed with mFluc in cells. The Hsp-encoding bicistronic constructs enable transfection efficiencies and concentration-dependent effects of Hsp expression to be determined using fluorescence based techniques, without the need to tag the Hsp with a fluorescent protein.
Project description:In seasonal environments, strong gradients of environmental parameters can shape life cycles of phytoplankton. Depending on the rate of environmental fluctuation, specialist or generalist strategies may be favored, potentially affecting life cycle transitions. The present study examined life cycle transitions of the toxin producing Baltic dinoflagellate Alexandrium ostenfeldii and their regulation by environmental factors (temperature and nutrients). This investigation aimed to determine whether genetic recombination of different strains is required for resting cyst formation and whether newly formed cysts are dormant. Field data (temperature and salinity) and sediment surface samples were collected from a site with recurrent blooms and germination and encystment experiments were conducted under controlled laboratory conditions. Results indicate a lack of seasonal germination pattern, set by an endogenous rhythm, as commonly found with other dinoflagellates from the Baltic Sea. Germination of quiescent cysts was triggered by temperatures exceeding 10°C and combined nutrient limitation of nitrogen and phosphorus or a drop in temperature from 16 to 10°C triggered encystment most efficiently. Genetic recombination was not mandatory for the formation of resting cysts, but supported higher numbers of resistant cysts and enhanced germination capacity after a resting period. Findings from this study confirm that A. ostenfeldii follows a generalist germination and cyst formation strategy, driven by strong seasonality, which may support its persistence and possibly expansion in marginal environments in the future, if higher temperatures facilitate a longer growth season.