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Molecular Chaperones: Key Players of Abiotic Stress Response in Plants
Abstract
Plants counter an array of stresses by generation of a group of stress-related proteins, often referred to as the chaperones. Expression of these chaperones is induced in response to almost all kinds of stress. However, there are numerous evidences showing that these chaperones are vital for survival even under normal physiological conditions. They act as key modulators in physiological stress response and acquired tolerance. Research carried out over the past several years has clearly established that these chaperones are involved in diverse cellular functions such as folding, accumulation, translocation and degradation of proteins. Thus, these evolutionary conserved proteins affect a broad array of cellular processes. Gaining knowledge about this cellular chaperone machinery is of immense significance to understand the mechanism of interdependent stress-related cross talk in plants and ultimately, for the crop improvement programs.
... The interaction of HSP70 with other proteins prevents them from aggregating together, facilitates changes in their folding to reach their final shape, and controls how the proteins perform (Usman et al., 2017;Waudby et al., 2019). Many HSPs have been extracted from several organelles like plastids, the cytosol, and the endoplasmic reticulum of many different plant species under various abiotic stresses (Roy et al., 2019). These HSPs act as chaperones, proving their role in tolerance against heat and other stresses (Tang et al., 2016). ...
Human activities and climate change have resulted in frequent and intense weather fluctuations, leading to diverse abiotic stresses on crops which hampers greatly their metabolic activities. Heat stress, a prevalent abiotic factor, significantly influences cotton plant biological activities resulting in reducing yield and production. We must deepen our understanding of how plants respond to heat stress across various dimensions, encompassing genes, RNAs, proteins, metabolites for effective cotton breeding. Multi-omics methods, primarily genomics, transcriptomics, proteomics, metabolomics, and phenomics, proves instrumental in studying cotton’s responses to abiotic stresses. Integrating genomics, transcriptomics, proteomics, and metabolomic is imperative for our better understanding regarding genetics and molecular basis of heat tolerance in cotton. The current review explores fundamental omics techniques, covering genomics, transcriptomics, proteomics, and metabolomics, to highlight the progress made in cotton omics research.
... The interaction of HSP70 with other proteins prevents them from aggregating together, facilitates changes in their folding to reach their final shape, and controls how the proteins perform (Usman et al., 2017;Waudby et al., 2019). Many HSPs have been extracted from several organelles like plastids, the cytosol, and the endoplasmic reticulum of many different plant species under various abiotic stresses (Roy et al., 2019). These HSPs act as chaperones, proving their role in tolerance against heat and other stresses (Tang et al., 2016). ...
Human activities and climate change have resulted in frequent and intense weather fluctuations, leading to diverse abiotic stresses on crops which hampers greatly their metabolic activities. Heat stress, a prevalent abiotic factor, significantly influences cotton plant biological activities resulting in reducing yield and production. We must deepen our understanding of how plants respond to heat stress across various dimensions, encompassing genes, RNAs, proteins, metabolites for effective cotton breeding. Multi-omics methods, primarily genomics, transcriptomics, proteomics, metabolomics, and phenomics, proves instrumental in studying cotton’s responses to abiotic stresses. Integrating genomics, transcriptomics, proteomics, and metabolomic is imperative for our better understanding regarding genetics and molecular basis of heat tolerance in cotton. The current review explores fundamental omics techniques, covering genomics, transcriptomics, proteomics, and metabolomics, to highlight the progress made in cotton omics research.
... Among these genes, some were found sHSP, HSP70 (both Heat Shock Protein), and chaperone DNAj which inhibit the action of ethylene in fruits, and therefore delay their ripening (Chirinos et al., 2023). The role of chaperones in preventing protein aggregation due to oxidative stress has been profoundly described in many organisms (Roy et al., 2019;Jacob et al., 2017). HSPs are widely known for their role in plant response to HS, but in recent years the role of HSPs in the ripening process has also been well documented (Bertero et al., 2021;Upadhyay et al., 2020;Shukla et al., 2017). ...
... Another study with drought tolerance common bean cultivars highlights that the accumulation of different osmoprotectants and antioxidants parallel with the maintenance of photosynthesis has an impact on common bean yield after drought stress [11]. Considering that molecular chaperons are expressed in response to almost all kinds of stress in plants and involved in diverse cellular functions such as folding, accumulation, translocation and degradation of proteins [12,13], their accumulation in different tolerant bean genotypes represent particular interest for breeding programs as well. ...
Drought compromises edible vegetable production worldwide, including common bean (Phaseolus vulgaris L.) an economically important crop that is highly dependent on optimum rainfall or abundant irrigation. In the present study, phenotypic data of 26 Bulgarian common bean mutant lines and cultivars subjected to drought stress has been summarized, and drought stress reaction was evaluated by chlorophyll fluorescence and proteomics approaches. Several basic photosyn-thetic parameters were examined during treatment to evaluate the drought stress response, and the mutant lines showed different responses. Subsequently, a relationship was found between productivity and photosynthetic performance with the expression of ribulose-1,5-bisphosphate carboxylase through comparative 2D-gel based electrophoresis; accumulation of the well-known stress-related proteins markers dehydrins and small heat shock proteins was established as well. These findings support the further selection of drought tolerant common bean lines for a sustainable agriculture.
... TRINITY_DN425597_c0_g2_i2 (heat shock protein 83-like) belongs to the heat shock protein group, which are well known for their protective role in response to elevated temperatures in almost all organisms, including plants. These proteins perform multiple functions, such as protein folding, translocation across membranes, facilitation of protein-protein interaction, and preventing protein aggregation and regulation of synthesis of other stress-related genes during heat stress conditions (Åkerfelt et al., 2010;Roy et al., 2019). Conner et al. (1990) reported the heat-inducible expression of AtHS83 in Arabidopsis thaliana. ...
... TRINITY_DN425597_c0_g2_i2 (heat shock protein 83-like) belongs to the heat shock protein group, which are well known for their protective role in response to elevated temperatures in almost all organisms, including plants. These proteins perform multiple functions, such as protein folding, translocation across membranes, facilitation of protein-protein interaction, and preventing protein aggregation and regulation of synthesis of other stress-related genes during heat stress conditions (Åkerfelt et al., 2010;Roy et al., 2019). Conner et al. (1990) reported the heat-inducible expression of AtHS83 in Arabidopsis thaliana. ...
Heat stress is one of the significant constraints affecting wheat production worldwide. To ensure food security for ever-increasing world population, improving wheat for heat stress tolerance is needed in the presently drifting climatic conditions. At the molecular level, heat stress tolerance in wheat is governed by a complex interplay of various heat stress-associated genes. We used a comparative transcriptome sequencing approach to study the effect of heat stress (5°C above ambient threshold temperature of 20°C) during grain filling stages in wheat genotype K7903 (Halna). At 7 DPA (days post-anthesis), heat stress treatment was given at four stages: 0, 24, 48, and 120 h. In total, 115,656 wheat genes were identified, including 309 differentially expressed genes (DEGs) involved in many critical processes, such as signal transduction, starch synthetic pathway, antioxidant pathway, and heat stress-responsive conserved and uncharacterized putative genes that play an essential role in maintaining the grain filling rate at the high temperature. A total of 98,412 Simple Sequences Repeats (SSR) were identified from de novo transcriptome assembly of wheat and validated. The miRNA target prediction from differential expressed genes was performed by psRNATarget server against 119 mature miRNA. Further, 107,107 variants including 80,936 Single nucleotide polymorphism (SNPs) and 26,171 insertion/deletion (Indels) were also identified in de novo transcriptome assembly of wheat and wheat genome Ensembl version 31. The present study enriches our understanding of known heat response mechanisms during the grain filling stage supported by discovery of novel transcripts, microsatellite markers, putative miRNA targets, and genetic variant. This enhances gene functions and regulators, paving the way for improved heat tolerance in wheat varieties, making them more suitable for production in the current climate change scenario.
... TRINITY_DN425597_c0_g2_i2 (heat shock protein 83-like) belongs to the heat shock protein group, which are well known for their protective role in response to elevated temperatures in almost all organisms, including plants. These proteins perform multiple functions, such as protein folding, translocation across membranes, facilitation of protein-protein interaction, and preventing protein aggregation and regulation of synthesis of other stress-related genes during heat stress conditions (Åkerfelt et al., 2010;Roy et al., 2019). Conner et al. (1990) reported the heat-inducible expression of AtHS83 in Arabidopsis thaliana. ...
... Exposure to abiotic stresses such as salinity and drought affects growth, development, and, subsequently, the yield of crop plants (Jeyasri et al., 2021). Genes providing salinity tolerance in plants include those encoding stress-responsive transcriptional regulators, molecular chaperones, antioxidant enzymes, or the proteins that aid in posttranscriptional modifications (Mirdar Mansuri et al., 2020;Roy et al., 2019). Previous work from our laboratory has established that Pokkali, a native salt-tolerant wild rice, has a repertoire of such stressadaptive functional genes (Kumari et al., 2009;Mishra et al., 2021). ...
OsCYP2-P is an active cyclophilin (having peptidyl-prolyl cis/trans isomerase activity, PPIase) isolated from the wild rice Pokkali having a natural capacity to grow and yield seeds in coastal saline regions of India. Transcript abundance analysis in rice seedlings showed the gene is inducible by multiple stresses, including salinity, drought, high temperature, and heavy metals. To dissect the role of OsCYP2-P gene in stress response, we raised overexpression (OE) and knockdown (KD) transgenic rice plants with >2-3 folds higher and ~2-fold lower PPIase activity, respectively. Plants overexpressing this gene had more favourable physiological and biochemical parameters (K+ /Na+ ratio, electrolytic leakage, membrane damage, antioxidant enzymes) than wild type, and the reverse was observed in plants that were knocked down for this gene. We propose that OsCYP2-P contributes to stress tolerance via maintenance of ion homeostasis and thus prevents toxic cellular ion buildup and membrane damage. OE plants were found to have a higher harvest index and higher number of filled grains under salinity and drought stress than wild type. OsCYP2-P interacts with calmodulin, indicating it functions via the Ca-CaM pathway. Compared to the WT, the germinating OE seeds exhibited a substantially higher auxin level, and this hormone was below the detection limits in the WT and KD lines. These observations strongly indicate that OsCyp2-P affects the signaling and transport of auxin in rice.
... The molecular signaling is observed as the freshly synthesized proteins come from the ribosomes, the substrate binding domain HSP70 recognizes and interacts with sequences of hydrophobic amino acid residues (Rosenzweig et al. 2019). HSP proteins are isolated from different organelles such as cytosol, endoplasmic reticulum and plastid of several plant species under multiple abiotic stresses (Roy et al. 2019). These HSPs are involved in the heat and other stresses' tolerance (Tang et al. 2016) in diverse plant species such as soybean (Fuganti-Pagliarini et al. 2017), Arabidopsis (Chen et al. 2019), pearl millet (Divya et al. 2019), tobacco (Song et al. 2019) and Agave sisalana (Batcho et al. 2020a, b). ...
Genetic improvement of local cotton for tolerance against heat and other abiotic stresses is urgently needed due to climatic changes. Agrobacterium mediated transformation of G. hirsutum was done with heat shock protein gene (AsHSP70) and plants were analyzed for transgene expression at molecular, physiological and biochemical levels under heat stress. Genetic transformation revealed 1.9 % transformation efficiency. PCR amplified 1800 bp fragment of AsHSP70 in genomic DNA of transgenic plants. Polyamine oxidase induced the relative expression of AsHSP70 from 1.02 to 9.58 in transgenic plants as the duration of heat stress was increased. Expression of AsHSP70 was relatively higher in the leaves of transgenic plant as compared to root and stem under combined stress of heat/drought. Cell electrolyte leakage was 39.83 μS cm−1 and 63.43 μS cm−1 in transgenic and control plants respectively. Relative conductivity (4.05 %) was correlated with the membrane stability index (MSI) (97.19 %) in transgenic plants while it is 7.28 % when MSI is 90.19 % in control plants. Membrane injury was 19–52 % in control and 15.6–29 % in transgenic plants over the time of exposure to heat stress. Significant variation in chlorophyll, proline and soluble sugar content was observed in transgenic plants as compared to controls. Performance of transgenic plants was better for boll formation in the field. Single copy of transgene was detected in T1 progeny. Hence, transgenic cotton plants are tolerating heat and other abiotic stresses at physiological and biochemical levels. Single analyses of progeny may lead the selection of pure heat tolerant lines to be used in breeding program for cotton improvement under heat stress.
Proteomics of wild and cultivated tomato species challenged with Alternaria solani revealed altered protein profile with 1827 proteins in challenged susceptible plants (KTr), 1867 in non-challenged plants (KNTr), 1721 in challenged wild (CTr) and 1715 in non-challenged plants (CNTr). PLS-DA and heatmap analysis highlighted differences in protein composition and abundance as differential response species to pathogen. Compared to 321 differentially expressed proteins (DEPs) in wild tomato, cultivated plants showed 183 DEPs. Key upregulated proteins in wild tomato included defense-related t-SNARE, glucan endo-1,3-beta-D-glucosidase, pathogenesis-related protein P2, stress responsive DEK domain containing protein, heat shock 70 kDa protein 17, SHSP chaperone, signaling linked DAG, SCP domain-containing protein, Cutin-deficient protein, immunity-related translation initiation factor and RRM domain-containing protein. Protein-protein interaction (PPI) network analysis clustered defense related up-regulated chaperonins and other proteins into three distinct clusters in wild tomato. Prominent subcellular locations of up-regulated proteins were extracellular and intracellular regions, cytoplasm and membrane bound organelles. Compared to cultivated species, majority of plant defense, stress response and growth-related protein biomarkers were found up-regulated in wild tomato, suggesting its tolerance against pathogen due to stronger response. We conclude that significant up-regulation of defense, signaling and plant growth-related proteins enabled wild species to mount stronger response against the pathogen A. solani. Higher compositional protein diversity in the wild plants likely provided metabolic plasticity to modulate intrinsic defense mechanisms more effectively. This study enhances our understanding of the proteome-related molecular mechanisms underlying differential responses of wild and cultivated tomato species to this devastating pathogen.
Main conclusion
Plant Biomarkers are objective indicators of a plant’s cellular state in response to abiotic and biotic stress factors. They can be explored in crop breeding and engineering to produce stress-tolerant crop species.
Abstract
Global food production safely and sustainably remains a top priority to feed the ever-growing human population, expected to reach 10 billion by 2050. However, abiotic and biotic stress factors negatively impact food production systems, causing between 70 and 100% reduction in crop yield. Understanding the plant stress responses is critical for developing novel crops that can adapt better to various adverse environmental conditions. Using plant biomarkers as measurable indicators of a plant’s cellular response to external stimuli could serve as early warning signals to detect stresses before severe damage occurs. Plant biomarkers have received considerable attention in the last decade as pre-stress indicators for various economically important food crops. This review discusses some biomarkers associated with abiotic and biotic stress conditions and highlights their importance in developing stress-resilient crops. In addition, we highlighted some factors influencing the expression of biomarkers in crop plants under stress. The information presented in this review would educate plant researchers, breeders, and agronomists on the significance of plant biomarkers in stress biology research, which is essential for improving plant growth and yield toward sustainable food production.
Current technologies have changed biology into a data-intensive field and significantly increased our understanding of signal transduction pathways in plants. However, global defense signaling networks in plants have not been established yet. Considering the apparent intricate nature of signaling mechanisms in plants (due to their sessile nature), studying the points at which different signaling pathways converge, rather than the branches, represents a good start to unravel global plant signaling networks. In this regard, growing evidence shows that the generation of reactive oxygen species (ROS) is one of the most common plant responses to different stresses, representing a point at which various signaling pathways come together. In this review, the complex nature of plant stress signaling networks will be discussed. An emphasis on different signaling players with a specific attention to ROS as the primary source of the signaling battery in plants will be presented. The interactions between ROS and other signaling components, e.g., calcium, redox homeostasis, membranes, G-proteins, MAPKs, plant hormones, and transcription factors will be assessed. A better understanding of the vital roles ROS are playing in plant signaling would help innovate new strategies to improve plant productivity under the circumstances of the increasing severity of environmental conditions and the high demand of food and energy worldwide.
Protein disulfide isomerases (PDIs) play critical roles in protein folding by catalyzing the formation and rearrangement of disulfide bonds in nascent secretory proteins. There are six distinct PDI subfamilies in terrestrial plants. A unique feature of PDI-C subfamily members is their homology to the yeast retrograde (Golgi-to-endoplasmic reticulum) cargo receptor proteins, Erv41p and Erv46p. Here, we demonstrate that plant Erv41p/Erv46p-like proteins are divided into three subfamilies: ERV-A, ERV-B and PDI-C, which all possess the N-proximal and C-proximal conserved domains of yeast Erv41p and Erv46p. However, in PDI-C isoforms, these domains are separated by a thioredoxin domain. The distribution of PDI-C isoforms among eukaryotes indicates that the PDI-C subfamily likely arose through an ancient exon-shuffling event that occurred before the divergence of plants from stramenopiles and rhizarians. Arabidopsis has three PDI-C genes: PDI7, PDI12, and PDI13. PDI12- and PDI13-promoter: β-glucuronidase (GUS) gene fusions are co-expressed in pollen and stipules, while PDI7 is distinctly expressed in the style, hydathodes, and leaf vasculature. The PDI-C thioredoxin domain active site motif CxxS is evolutionarily conserved among land plants. Whereas PDI12 and PDI13 retain the CxxS motif, PDI7 has a CxxC motif similar to classical PDIs. We hypothesize that PDI12 and PDI13 maintain the ancestral roles of PDI-C in Arabidopsis, while PDI7 has undergone neofunctionalization. The unusual PDI/cargo receptor hybrid arrangement in PDI-C isoforms has no counterpart in animals or yeast, and predicts the need for pairing redox functions with cargo receptor processes during protein trafficking in plants and other PDI-C containing organisms.
Peroxisomes are present in almost all plant cells. These organelles are involved in various metabolic processes, such as lipid catabolism and photorespiration. A notable feature of plant peroxisomes is their flexible adaptive responses to environmental conditions such as light. When plants shift from heterotrophic to autotrophic growth during the post-germinative stage, peroxisomes undergo a dynamic response, i.e., enzymes involved in lipid catabolism are replaced with photorespiratory enzymes. Although the detailed molecular mechanisms underlying the functional transition of peroxisomes have previously been unclear, recent analyses at the cellular level have enabled the unveiling of this detailed machinery. During the functional transition, obsolete enzymes are degraded inside peroxisomes by Lon protease, while newly-synthesized enzymes are transported into peroxisomes. In parallel, mature and oxidized peroxisomes are eliminated via autophagy; this functional transition occurs in an efficient manner. Moreover, it has become clear that quality control mechanisms are important for the peroxisomal response to environmental stimuli. In this review, we highlight recent advances in elucidating the molecular mechanisms required for the regulation of peroxisomal roles in response to changes in environmental conditions.
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The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals , and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit pep-tides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.
We isolated cDNA clones for two nuclear-encoded, organellar members of the Arabidopsis hsp70 gene family, mtHsc70-2(AF217458) and cpHsc70-2 (AF217459). Together with the completion of the genome sequence, the hsp70 family in Arabidopsis consists of 14 members unequally distributed among the five chromosomes. To establish detailed expression data of this gene family, a comprehensive reverse transcriptase-polymerase chain reaction analysis for 11 hsp70s was conducted including analysis of organ-specific and developmental expression and expression in response to temperature extremes. All hsp70s showed 2- to 20-fold induction by heat shock treatment except cpHsc70-1 andmtHsc70-1, which were unchanged or repressed. The expression profiles in response to low temperature treatment were more diverse than those evoked by heat shock treatment. Both mitochondrial and all cytosolic members of the family except Hsp70bwere strongly induced by low temperature, whereas endoplasmic reticulum and chloroplast members were not induced or were slightly repressed. Developmentally regulated expression of the heat-inducibleHsp70 in mature dry seed and roots in the absence of temperature stress suggests prominent roles in seed maturation and root growth for this member of the hsp70 family. This reverse transcriptase-polymerase chain reaction analysis establishes the complex differential expression pattern for the hsp70s in Arabidopsis that portends specialized functions even among members localized to the same subcellular compartment.
Heat stress in plants elevates the potential across the inner mitochondrial membrane (mtΔψ) and activates the expression of heat shock proteins (HSPs). The treatment of Saccharomyces cerevisiae cells with amiodarone (AMD) elevated the cytosolic Ca2+ level ([Ca2+]cyt) in parallel with (mtΔψ) increase and led to the induction of Hsp104 synthesis. The hyperpolarization was presumably due to the increase in [Ca2+]cyt. In the present study the effects of AMD (0–100 μM) on cell viability, HSP expression, mtΔψ, and [Ca2+]cyt were investigated using the cell culture of Arabidopsis thaliana (L.) Heynh. The treatment of cultured cells with AMD led to the elevation of [Ca2+]cyt, which was accompanied by the increase in mtΔψ and by activation of HSP101 expression. The increase in [Ca2+]cyt and expression of HSP101 were also observed upon the treatment with the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone, 4 μM) known to diminish mtΔψ. The results suggest that plant cell mitochondria modulate the cytosolic Ca2+ level by changing the potential at the inner mitochondrial membrane and, thereby, participate in the retrograde regulation of HSP101 expression.
The present study forms part of a program investigating the role of small heat
shock proteins (sHSPs) in the acquired and transgenic thermotolerance of the
cyanobacterium Synechococcus PCC7942. The genes for
three minimally related sHSPs, OsHSP from
Oryza sativacytoplasm, tom111 from
Lycopersicon esculentumchloroplasts, and 6803 HSP from
Synechocystis sp. PCC6803, were cloned into the
Escherichia coli vector pTrcHisA, so as to produce an
N-terminal polyhistidine tag. The genes were transformed into
E. coli and overexpressed. The tagged HSPs were purified
(not completely in the case of tom111) by immobilised metal affinity
chromatography. The native proteins exhibited a high degree of oligomerisation
when analysed by size-exclusion chromatography. All three proteins were able
to protect malate dehydrogenase (MDH) from in vitro
thermal aggregation. They could also protect several soluble proteins in
E. coli extracts from thermal aggregation
in vitro, as well as protecting phycocyanin in extracts
from Synechococcus sp. PCC7942. None of the proteins
were able to protect photosystem II (measured as ΦPSII, the effective
quantum fluorescence yield of PSII) of thylakoids isolated from
Synechococcus sp. PCC7942 from heat damage
in vitro, although in vivo, after
acclimation, photosystem II did exhibit acquired thermotolerance.
The last stage of protein folding, the "endgame," involves the ordering of amino acid side-chains into a well defined and closely packed configuration. We review a number of topics related to this process. We first describe how the observed packing in protein crystal structures is measured. Such measurements show that the protein interior is packed exceptionally tightly, more so than the protein surface or surrounding solvent and even more efficiently than crystals of simple organic molecules. In vitro protein folding experiments also show that the protein is close-packed in solution and that the tight packing and intercalation of side-chains is a final and essential step in the folding pathway. These experimental observations, in turn, suggest that a folded protein structure can be described as a kind of three- dimensional jigsaw puzzle and that predicting side-chain packing is possible in the sense of solving this puzzle. The major difficulty that must be overcome in predicting side-chain packing is a combinatorial "explosion" in the number of possible configurations. There has been much recent progress towards overcoming this problem, and we survey a variety of the approaches. These approaches differ principally in whether they use ab initio (physical) or more knowledge-based methods, how they divide up and search conformational space, and how they evaluate candidate configurations (using scoring functions). The accuracy of side-chain prediction depends crucially on the (assumed) positioning of the main-chain. Methods for predicting main-chain conformation are, in a sense, not as developed as that for side-chains. We conclude by surveying these methods. As with side-chain prediction, there are a great variety of approaches, which differ in how they divide up and search space and in how they score candidate conformations.
The last stage of protein folding, the "endgame," involves the ordering of amino acid side-chains into a well defined and closely packed configuration. We review a number of topics related to this process. We first describe how the observed packing in protein crystal structures is measured. Such measurements show that the protein interior is packed exceptionally tightly, more so than the protein surface or surrounding solvent and even more efficiently than crystals of simple organic molecules. In vitro protein folding experiments also show that the protein is close-packed in solution and that the tight packing and intercalation of side-chains is a final and essential step in the folding pathway. These experimental observations, in turn, suggest that a folded protein structure can be described as a kind of three-dimensional jigsaw puzzle and that predicting side-chain packing is possible in the sense of solving this puzzle. The major difficulty that must be overcome in predicting side-chain packing is a combinatorial "explosion" in the number of possible configurations. There has been much recent progress towards overcoming this problem, and we survey a variety of the approaches. These approaches differ principally in whether they use ab initio (physical) or more knowledge-based methods, how they divide up and search conformational space, and how they evaluate candidate configurations (using scoring functions). The accuracy of side-chain prediction depends crucially on the (assumed) positioning of the main-chain. Methods for predicting main-chain conformation are, in a sense, not as developed as that for side-chains. We conclude by surveying these methods. As with side-chain prediction, there are a great variety of approaches, which differ in how they divide up and search space and in how they score candidate conformations.
Environmental stress conditions such as drought, heat, salinity, cold, or pathogen infection can have a devastating impact on plant growth and yield under field conditions. Nevertheless, the effects of these stresses on plants are typically being studied under controlled growth conditions in the laboratory. The field environment is very different from the controlled conditions used in laboratory studies, and often involves the simultaneous exposure of plants to more than one abiotic and/or biotic stress condition, such as a combination of drought and heat, drought and cold, salinity and heat, or any of the major abiotic stresses combined with pathogen infection. Recent studies have revealed that the response of plants to combinations of two or more stress conditions is unique and cannot be directly extrapolated from the response of plants to each of the different stresses applied individually. Moreover, the simultaneous occurrence of different stresses results in a high degree of complexity in plant responses, as the responses to the combined stresses are largely controlled by different, and sometimes opposing, signaling pathways that may interact and inhibit each other. In this review, we will provide an update on recent studies focusing on the response of plants to a combination of different stresses. In particular, we will address how different stress responses are integrated and how they impact plant growth and physiological traits.
Contents
Summary
32
I.
Introduction
32
II.
Effects of stress combination on growth, yield and physiological traits in plants and crops
34
III.
The complexity of stress response signaling during stress combination
38
IV.
Conclusions
39
Acknowledgements
41
References
41
The proteome responses to heat stress have not been well understood. In this study, alfalfa (Medicago sativa L. cv. Huaiyin) seedlings were exposed to 25°C (control) and 40°C (heat stress) in growth chambers, and leaves were collected at 24, 48 and 72 h after treatment, respectively. The morphological, physiological and proteomic processes were negatively affected under heat stress. Proteins were extracted and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), and differentially expressed protein spots were identified by mass spectrometry (MS). Totally, 81 differentially expressed proteins were identified successfully by MALDI-TOF/TOF. These proteins were categorized into nine classes: including metabolism, energy, protein synthesis, protein destination/storage, transporters, intracellular traffic, cell structure, signal transduction and disease/defence. Five proteins were further analyzed for mRNA levels. The results of the proteomics analyses provide a better understanding of the molecular basis of heat-stress responses in alfalfa.
Plants are unique among eukaryotes in having evolved organelles:
the protein storage vacuole, protein body, and chloroplast. Disulfide transfer pathways that function in the endoplasmic reticulum (ER) and chloroplasts of plants play critical roles in the development of protein storage organelles and the biogenesis of chloroplasts, respectively. Disulfide bond formation requires the cooperative function of disulfide-generating enzymes (e.g., ER oxidoreductase 1), which generate disulfide bonds de novo, and disulfide carrier proteins (e.g., protein disulfide isomerase), which transfer disulfides to substrates by means of thiol-disulfide exchange reactions. Selective molecular communication between disulfide-generating enzymes and disulfide carrier proteins, which reflects the molecular and structural diversity of disulfide carrier proteins, is key to the efficient transfer of disulfides to specific sets of substrates. This review focuses on recent advances in our understanding of the mechanisms and functions of the various disulfide transfer pathways involved in oxidative protein folding in the ER, chloroplasts, and mitochondria of plants.
Background
The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homoeologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species.
Results
Eight new non-homoeologous wheat genes were cloned and characterized. The nine PDI and PDI-like sequences of wheat were located in chromosome regions syntenic to those in rice and assigned to eight plant phylogenetic groups. The nine wheat genes differed in their sequences, genomic organization as well as in the domain composition and architecture of their deduced proteins; conversely each of them showed high structural conservation with genes from other plant species in the same phylogenetic group. The extensive quantitative RT-PCR analysis of the nine genes in a set of 23 wheat samples, including tissues and developmental stages, showed their constitutive, even though highly variable expression.
Conclusions
The nine wheat genes showed high diversity, while the members of each phylogenetic group were highly conserved even between taxonomically distant plant species like the moss Physcomitrella patens. Although constitutively expressed the nine wheat genes were characterized by different expression profiles reflecting their different genomic organization, protein domain architecture and probably promoter sequences; the high conservation among species indicated the ancient origin and diversification of the still evolving gene family. The comprehensive structural and expression characterization of the complete set of PDI and PDI-like wheat genes represents a basis for the functional characterization of this gene family in the hexaploid context of bread wheat.
Rabbit reticulocyte lysate contains a multiprotein system that assembles steroid receptors into a heterocomplex with hsp90.
In the case of the glucocorticoid receptor (GR), the receptor must be bound to hsp90 to bind steroid, and assembly of the
GR·hsp90 complex restores the hormone binding domain of the receptor to the steroid binding conformation. Using both direct
assay of heterocomplex assembly by Western blotting and indirect assay of assembly by steroid binding, it has previously been
determined that the assembly system is both ATP/Mg2+-dependent and K+-dependent and that hsp70 and an acidic 23-kDa protein (p23) are required to form a functional GR·hsp90 complex. It is also
thought that a 60-kDa protein (p60) may be required for progesterone receptor·hsp90 heterocomplex assembly, but a complete
heterocomplex assembly system has never been reconstituted from individual components. In this work, we separate the proteins
of rabbit reticulocyte lysate into three fractions by DEAE chromatography and then reconstitute the GR·hsp90 heterocomplex
assembly system in a manner that requires the presence of each fraction. Fraction A contains most of the hsp70 and all of
the p60 in lysate, and elimination of p60 by immunoadsorption inactivates this fraction, with bioactivity being restored by
the addition of bacterially expressed human p60. The activity of fraction A is replaced by a combination of highly purified
rabbit hsp70 and lysate from p60-expressing bacteria. Fraction B contains hsp90, and its activity is replaced by purified
rabbit hsp90. Fraction C contains p23, and its activity is replaced in the recombined system by highly purified bacterially
expressed human p23. A minimal GR·hsp90 heterocomplex assembly system was reconstituted with purified rabbit hsp70 and hsp90
and bacterially expressed human p23 and p60. This reports the first reconstitution of this apparently ubiquitous protein folding/heterocomplex
assembly system.
Arabidopsis thaliana, the first plant for which the entire genome sequence is available, was also among the first plant species from which Hsp100 proteins were characterized. The Athsp101 complementary DNA (cDNA) corresponds to the gene identification At1g74310 in the Arabidopsis genome sequence. Analysis of the genome revealed 7 additional proteins that are variably homologous with At1g74310 throughout the entire amino acid sequence and significant similarities or identities in the signature sequences conserved among Hsp100 proteins. Although AtHsp101 is cytoplasmic, 5 of the 7 related proteins have predicted plastidial localization signals. This complete description of the AtHsp100 family sets the stage for future research on expression and function.
The 90-kDa heat shock protein (Hsp90) is an essential molecular chaperone in eukaryotic cells, with key roles in the folding and activation of proteins involved in signal transduction and control of the cell cycle. A search for Hsp90 sequences in the Arabidopsis thaliana genome revealed that this family includes 7 members. The AtHsp90-1 through AtHsp90-4 proteins constitute the cytoplasmic subfamily, whereas the AtHsp90-5, AtHsp90-6, and AtHsp90-7 proteins are predicted to be within the plastidial, mitochondrial, and endoplasmic reticulum compartments, respectively. The deduced amino acid sequences of each of the cytoplasmic proteins contains the highly conserved C-terminal pentapeptide MEEVD. All of the AtHsp90 sequences include a conserved adenosine triphosphate-binding domain, whereas only the cytoplasmic and endoplasmic reticulum-resident sequences include an adjacent charged linker domain that is common in mammalian and yeast sequences. The occurrence of multiple AtHsp90 proteins in the cytoplasm and of family members in other subcellular compartments suggests a range of specific functions and target polypeptides.
Protein quality control systems protect cells against the accumulation of toxic misfolded proteins by promoting their selective degradation. Malfunctions of quality control systems are linked to aging and neurodegenerative disease. Folding of polypeptides is facilitated by the association of 70 kDa Heat shock protein (Hsp70) molecular chaperones. If folding cannot be achieved, Hsp70 interacts with ubiquitylation enzymes that promote the proteasomal degradation of the misfolded protein. However, the factors that direct Hsp70 substrates toward the degradation machinery have remained unknown. Here, we identify Fes1, an Hsp70 nucleotide exchange factor of hitherto unclear physiological function, as a cytosolic triaging factor that promotes proteasomal degradation of misfolded proteins. Fes1 selectively interacts with misfolded proteins bound by Hsp70 and triggers their release from the chaperone. In the absence of Fes1, misfolded proteins fail to undergo polyubiquitylation, aggregate, and induce a strong heat shock response. Our findings reveal that Hsp70 direct proteins toward either folding or degradation by using distinct nucleotide exchange factors.
Plant steroid hormones, brassinosteroids, are essential for growth, development and responses to environmental stresses in plants. Although BR signaling proteins are localized in many organelles, i.e., the plasma membrane, nuclei, endoplasmic reticulum and vacuole, the details regarding the BR signaling pathway from perception at the cellular membrane receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) to nuclear events include several steps. Brz (Brz220) is a specific inhibitor of BR biosynthesis. In this study, we used Brz-mediated chemical genetics to identify Brz-insensitive-long hypocotyls 2-1D (bil2-1D). The BIL2 gene encodes a mitochondrial-localized DnaJ/Heat shock protein 40 (DnaJ/Hsp40) family, which is involved in protein folding. BIL2-overexpression plants (BIL2-OX) showed cell elongation under Brz treatment, increasing the growth of plant inflorescence and roots, the regulation of BR-responsive gene expression and suppression against the dwarfed BRI1-deficient mutant. BIL2-OX also showed resistance against the mitochondrial ATPase inhibitor oligomycin and higher levels of exogenous ATP compared with wild-type plants. BIL2 participates in resistance against salinity stress and strong light stress. Our results indicate that BIL2 induces cell elongation during BR signaling through the promotion of ATP synthesis in mitochondria.
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The heat shock protein 90 (Hsp90) family mediates stress signal transduction, and plays important roles in the control of normal growth of human cells and in promoting development of tumor cells. Hsp90s have become a currently important subject in cellular immunity, signal transduction, and anti-cancer research. Studies on the physiological functions of Hsp90s began much later in plants than in animals and fungi. Significant progress has been made in understanding complex mechanisms of HSP90s in plants, including ATPase-coupled conformational changes and interactions with cochaperone proteins. A wide range of signaling proteins interact with HSP90s. Recent studies revealed that plant Hsp90s are important in plant development, environmental stress response, and disease and pest resistance. In this study, the plant HSP90 family was classified into three clusters on the basis of phylogenetic relationships, gene structure, and biological functions. We discuss the molecular functions of Hsp90s, and systematically review recent progress of Hsp90 research in plants.
Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction.
Background: Hsp100 chaperones cooperate with Hsp70 chaperones to disaggregate and reactivate heat-denatured proteins.
Results: Mutations in the interface region between NBD1 and M domains of Hsp100 result in a hyperactive protein toxic to the cell.
Conclusion: The interaction between M and NBD1 domains is crucial for regulation of Hsp100 activity.
Significance: A novel important aspect of the Hsp100 mechanism of action is described.
Cellular chaperones and folding enzymes play central roles in the formation of positive-strand and negative-strand RNA virus infection. This article examines the key cellular chaperones and discusses evidence that these factors are diverted from their cellular functions to play alternative roles in virus infection. For most chaperones discussed, their primary role in the cell is to ensure protein quality control. They are system components that drive substrate protein folding, complex assembly or disaggregation. Their activities often depend upon co-chaperones and ATP hydrolysis. During plant virus infection, Hsp70 and Hsp90 proteins play central roles in the formation of membrane-bound replication complexes for certain members of the tombusvirus, tobamovirus, potyvirus, dianthovirus, potexvirus, and carmovirus genus. There are several co-chaperones, including Yjd1, RME-8, and Hsp40 that associate with the bromovirus replication complex, pomovirus TGB2, and tospovirus Nsm movement proteins. There are also examples of plant viruses that rely on chaperone systems in the endoplasmic reticulum (ER) to support cell-to-cell movement. TMV relies on calreticulin to promote virus intercellular transport. Calreticulin also resides in the plasmodesmata and plays a role in calcium sequestration as well as glycoprotein folding. The pomovirus TGB2 interacts with RME-8 in the endosome. The potexvirus TGB3 protein stimulates expression of ER resident chaperones via the bZIP60 transcription factor. Up-regulating factors involved in protein folding may be essential to handling the load of viral proteins translated along the ER. In addition, TGB3 stimulates SKP1 which is a co-factor in proteasomal degradation of cellular proteins. Such chaperones and co-factors are potential targets for antiviral defense.
Cyclophilins constitute a subgroup of large family of proteins called immunophilins, which also include FKBPs and Parvulins. They are remarkably conserved in all genera, highlighting their pivotal role in important cellular processes. Most cyclophilins display PPIase enzymatic activity, multiplicity, diverse cellular locations and active role in protein folding which render them to be included in the class of diverse set of proteins called molecular chaperones. Due to their distinct PPIase function, besides protein disulfide isomerases and protein foldases, cyclophilins have been deemed necessary for in vivo chaperoning activity. Unlike other cellular chaperones, these proteins are specific in their respective targets. Not all cyclophilin proteins possess PPIase activity, indicating a loss of their PPIase activity during the course of evolution and gain of function independent of their PPIase activity. The PPIase function of cyclophilins is also compensated by their functional homologs, like FKBPs. Multiple cyclophilin members in plants like Arabidopsis and rice have been reported to be associated with diverse functions and regulatory pathways through their foldase, scaffolding, chaperoning or other unknown activities. Although many functions of plant cyclophilins were reported or suggested, the physiological relevance and molecular basis of stress-responsive expression of plant cyclophilins is still largely unknown. However, their wide distribution and ubiquitous nature signifies their fundamental importance in plant survival. Several of these members have also been directly linked to multiple stresses. This review attempts to deal with plant cyclophilins with respect to their role in stress response.
Deregulated expression of an Arabidopsis H(+)/Ca(2+) antiporter (sCAX1) in agricultural crops increases total calcium (Ca(2+)) but may result in yield losses due to Ca(2+) deficiency-like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca(2+) binding protein located at endoplasmic reticulum) in sCAX1-expressing tobacco and tomato plants mitigated these adverse effects while maintaining enhanced Ca(2+) content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca(2+) transporters and binding proteins at different intracellular compartments can alter the content and distribution of Ca(2+) within the plant matrix.
Root-knot nematodes are obligate biotrophic parasites that settle close to the vascular tissues in roots, where they induce the differentiation of specialized feeding cells and maintain a compatible interaction for three to eight weeks. Transcriptome analyses of the plant response to parasitic infection have shown that plant defenses are strictly controlled during the interaction. This suggests that, similar to other pathogens, RKN secrete effectors that suppress host defenses. We show here that Mi-CRT, a calreticulin secreted by the nematode into the apoplasm of infected tissues, plays an important role in infection success, as Mi-CRT knockdown by RNA interference affected the ability of the nematodes to infect plants. Stably transformed Arabidopsis thaliana plants producing the secreted form of Mi-CRT were more susceptible to nematode infection than wild-type plants. They were also more susceptible to infection with another root pathogen, the oomycete Phytophthora parasitica. Mi-CRT overexpression in A. thaliana suppressed the induction of defense marker genes and callose deposition after treatment with the PAMP elf18. Our results show that Mi-CRT secreted in the apoplasm by the nematode has a role in the suppression of plant basal defenses during the interaction.
Chloroplast small (low-molecular-weight) heat-shock proteins (csHsps) can protect photosynthetic electron transport (P et), and quantitative variation in csHsps is correlated with thermotolerance of net photosynthesis and Photosystem II. However, the functional (i.e. protective) consequence of natural variation in csHsps is unknown. To investigate this, we used an in vitro assay to determine the contribution of csHsps to the tolerance of P et to high temperatures in five ecotypes of Chenopodium album collected from habitats ranging from cool to warm, and we partitioned total P et thermotolerance into basal and induced P et components (without and with a pre-heat treatment, respectively, to induce csHsps). The ecotypes varied in total P et thermotolerance and this was correlated with habitat temperature. Variation in total P et thermotolerance was associated primarily with variation in induced P et thermotolerance, and not with basal P et thermotolerance. Variation in induced P et was highly correlated with csHsp protection of P et . Variation in csHsp function was associated with variation in csHsp content among ecotypes. These results are the first to demonstrate the direct functional consequences for natural variation in Hsps in plants, and show that functional variation is associated with evolutionary adaptation to specific habitats among ecotypes.
We have determined that one small heat shock protein gene, encoding Hsp17.7, plays an important role in the ability of carrot cells and plants to survive thermal stress. Transgenic cells and regenerated plants were generated in which the carrot Hsp17.7 gene was either constitutively expressed (denoted CaS lines) or expressed as a heat inducible antisense RNA (denoted AH lines). Thermotolerance measurements demonstrated that CaS lines were more thermotolerant than vector controls and AH antisense lines were less thermo- tolerant than vector controls. RNA analysis demonstrated that Hsp17.7 mRNA was detectable, but not abundant, prior to heat shock in CaS cells, but not in vector control cells. Conversely, RNA analysis of antisense cells showed that, after heat shock, the amounts of mRNA for Hsp17.7 was moderately less abundant in AH cells than in vector controls. Analysis of protein synthesis in CaS cells did not indicate substantial synthesis or accumulation of Hsp17.7, or any small Hsp, at 23°C. However, in the most thermotolerant line, protein synthesis was maintained at a higher rate than in other cell lines at a more extreme heat shock (42°C). In contrast, antisense AH cells showed reduced synthesis of many Hsp, large and small. These results suggest that the Hsp17.7 gene plays a critical, although as yet not understood, role in thermotolerance in carrot. This represents the first demonstration of the ability to both increase and decrease thermotolerance by the manipulation of expression of a single gene.
Calnexin and calreticulin are lectin‐like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N‐linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N‐linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N‐glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin‐like interaction, and not a protein‐protein interaction, played the central role in stabilizing RNase‐calnexin/calreticulin complexes.
We identified a T-DNA-generated mutation in thechaperonin-60α gene of Arabidopsis that produces a defect in embryo development. The mutation, termedschlepperless (slp), causes retardation of embryo development before the heart stage, even though embryo morphology remains normal. Beyond the heart stage, theslp mutation results in defective embryos with highly reduced cotyledons. slp embryos exhibit a normal apical-basal pattern and radial tissue organization, but they are morphologically retarded. Even though slp embryos are competent to transcribe two late-maturation gene markers, this competence is acquired more slowly as compared with wild-type embryos.slp embryos also exhibit a defect in plastid development–they remain white during maturation in planta and in culture. Hence, the overall developmental phenotype of theslp mutant reflects a lesion in the chloroplast that affects embryo development. The slp phenotype highlights the importance of the chaperonin-60α protein for chloroplast development and subsequently for the proper development of the plant embryo and seedling.
In the yeast Saccharomyces cerevisiae, only a subset of preproteins that are translocated across the ER membrane require the function of the signal recognition particle (SRP), suggesting that an alternative, SRP-independent pathway must exist (Hann, B.C., and P. Walter. 1991. Cell. 67:131-144). We have established that the two targeting pathways function in parallel. Mutant alleles of SEC62 and SEC63 were isolated that specifically impaired the translocation of SRP-independent preproteins in vivo and in vitro, whereas SRP-dependent preproteins were unaffected. Based on this analysis, preproteins fall into three distinct classes: SRP dependent, SRP independent, and those that can use both pathways. Pathway specificity is conferred by the hydrophobic core of signal sequences. Our studies show a previously unrecognized diversity in ER-directed signal sequences, that carry structural information that serves to identify the route taken.
HSP101 belongs to the ClpB protein subfamily whose members promote the renaturation of protein aggregates and are essential for the induction of thermotolerance. We found that maize HSP101 accumulated in mature kernels in the absence of heat stress. At optimal temperatures, HSP101 disappeared within the first 3 days after imbibition, although its levels increased in response to heat shock. In embryonic cells, HSP101 concentrated in the nucleus and in some nucleoli. Hsp101 maps near the umc132 and npi280 markers on chromosome 6. Five maize hsp101-m-::Mu1 alleles were isolated. Mutants were null for HSP101 and defective in both induced and basal thermotolerance. Moreover, during the first 3 days after imbibition, primary roots grew faster in the mutants at optimal temperature. Thus, HSP101 is a nucleus-localized protein that, in addition to its role in thermotolerance, negatively influences the growth rate of the primary root. HSP101 is dispensable for proper embryo and whole plant development in the absence of heat stress.
Plants are sessile organisms, and their ability to adapt to stress is crucial for survival in natural environments. Many observations suggest a relationship between stress tolerance and heat shock proteins (HSPs) in plants, but the roles of individual HSPs are poorly characterized. We report that transgenic Arabidopsis plants expressing less than usual amounts of HSP101, a result of either antisense inhibition or cosuppression, grew at normal rates but had a severely diminished capacity to acquire heat tolerance after mild conditioning pretreatments. The naturally high tolerance of germinating seeds, which express HSP101 as a result of developmental regulation, was also profoundly decreased. Conversely, plants constitutively expressing HSP101 tolerated sudden shifts to extreme temperatures better than did vector controls. We conclude that HSP101 plays a pivotal role in heat tolerance in Arabidopsis. Given the high evolutionary conservation of this protein and the fact that altering HSP101 expression had no detrimental effects on normal growth or development, one should be able to manipulate the stress tolerance of other plants by altering the expression of this protein.
Bag (Bcl2-associated athanogene) domains occur in a class of cofactors of the eukaryotic chaperone 70-kilodalton heat shock
protein (Hsp70) family. Binding of the Bag domain to the Hsp70 adenosine triphosphatase (ATPase) domain promotes adenosine
5′-triphosphate–dependent release of substrate from Hsp70 in vitro. In a 1.9 angstrom crystal structure of a complex with
the ATPase of the 70-kilodalton heat shock cognate protein (Hsc70), the Bag domain forms a three-helix bundle, inducing a
conformational switch in the ATPase that is incompatible with nucleotide binding. The same switch is observed in the bacterial
Hsp70 homolog DnaK upon binding of the structurally unrelated nucleotide exchange factor GrpE. Thus, functional convergence
has allowed proteins with different architectures to trigger a conserved conformational shift in Hsp70 that leads to nucleotide
exchange.
The analysis of protein sorting signals responsible for the retention of reticuloplasmins (RPLs), a group of soluble proteins that reside in the lumen of the endoplasmic reticulum (ER), has revealed a structural similarity between mammalian and plant ER retention signals. We present evidence that the corresponding epitope is conserved in a vast family of soluble ER resident proteins. Microsequences of RPL60 and RPL90, two abundant members of this family, show high sequence similarity with mammalian calreticulin and endoplasmin. RPL60/calreticulin cofractionates and costains with the lumenal binding protein (BiP). Both proteins were detected in the nuclear envelope and the ER, and in mitotic cells in association with the spindle apparatus and the phragmoplast. Immunoprecipitation of proteins from in vivo-labeled cells demonstrated that RPL60/calreticulin is associated with other polypeptides in a stress- and ATP-dependent fashion. RPL60/calreticulin transcript levels increased rapidly in abundance during the proliferation of the secretory apparatus and the onset of hydrolase secretion in gibberellic acid-treated barley aleurone cells. This induction profile is identical to that of the well-characterized ER chaperones BiP and endoplasmin. However, expression patterns in response to different stress conditions as well as tissue-specific expression patterns indicate that these genes are differentially regulated and may not act in concert.
Cyclophilins are a set of ubiquitous proteins present in all subcellular compartments, involved in a wide variety of cellular processes. Comparative bioinformatics analysis of the rice and Arabidopsis genomes led us to identify novel putative cyclophilin gene family members in both the genomes not reported previously. We grouped cyclophilin members with similar molecular weight and subtypes together in the phylogenetic tree which indicated their co-evolution in rice and Arabidopsis. We also characterized a rice cyclophilin gene, OsCyp2-P (Os02g0121300), isolated from a salinity-tolerant landrace, Pokkali. Publicly available massively parallel signature sequencing (MPSS) and microarray data, besides our quantitative real time PCR (qRT-PCR) data suggest that transcript abundance of OsCyp2-P is regulated under different stress conditions in a developmental and organ specific manner. Ectopic expression of OsCyp2-P imparted multiple abiotic stress tolerance to transgenic tobacco plants as evidenced by higher root length, shoot length, chlorophyll content, and K+/Na+ ratio under stress conditions. Transgenic plants also showed reduced lipid peroxidase content, electrolyte leakage, and superoxide content under stress conditions suggesting better ion homeostasis than WT plants. Localization studies confirmed that OsCyp2-P is localized in both cytosol and nucleus, indicating its possible interaction with several other proteins. The overall results suggest the explicit role of OsCyp2-P in bestowing multiple abiotic stress tolerance at the whole plant level. OsCyp2-P operates via reactive oxygen species (ROS) scavenging and ion homeostasis and thus is a promising candidate gene for enhancing multiple abiotic stress tolerance in crop plants.
We chose a class I cytosolic small HSP gene, TLHS1, whose protein showed a stronger molecular chaperone activity among the class I cytosolic small HSPs in tobacco, and constitutively expressed it in tobacco. Bioassay of the transgenic tobacco seedlings at T2 generation for thermotolerance was performed as the rates of cotyledon opening after heat-stress. Transgenic tobacco seedlings with the constitutively expressed TLHS1 showed up to two times higher cotyledon opening rates compared to the transgenic tobacco seedlings carrying the TLHS1 gene in antisense orientation and the expression vector only after the high temperature stresses of 40 °C and 45 °C for 1 to 4 h. When the correlation between the level of TLHS1 accumulated in the seedling before the heat-stress and the level of thermotolerance was examined, a strong correlation between them could be observed. Together these results support the thermoprotective function of a class I small HSP in plants.
The hypothesis of protein self-assembly arose from observations that some purified proteins could refold to an active form in vitro following the removal of denaturing agents. Such results prove that all the information necessary for folding into the correct final conformation is present within the primary amino acid sequence. However, in vitro folding is slow and yields are low, especially under physiological conditions. The reduction in yield is typically caused by the aggregation of partially folded intermediates. The need to reconcile the in vitro and in vivo differences in protein folding rate and efficiency led to a modification of the self-assembly hypothesis - the molecular chaperone concept. Molecular chaperones are a specific group of proteins that assist in the assembly of other proteins by suppressing nonproductive side reactions.
Molecular chaperones are synthesized and accumulated under a variety of unfavorable conditions in all organisms. Heat shock protein 90 (Hsp90) and Hsp60, which are classified into the major classes of molecular chaperones, play important roles in cellular stress responses. In this study, we characterized sterile Ulva pertusa Hsp90 (UpHsp90) and UpHsp60 genes which may be involved in tolerance to thermal and heavy metal stresses in this alga. The UpHsp90 cDNA consisting of 2,118 nucleotides encoded a polypeptide of 705 amino acids (AA). On the other hand, the UpHsp60 cDNA consisting of 1,722 nucleotides encoded a protein whose predicted length was 573 AA. The AA sequence alignment and phylogenetic analyses showed that the UpHsp90 and UpHsp60 proteins were more similar to cytoplasmic Hsp90s and mitochondrial Hsp60s, respectively, than to other types of the respective Hsps. Southern blot analysis indicated that the sterile U. pertusa genome had at least two cytoplasmic Hsp90-encoding genes and two mitochondrial Hsp60-encoding genes. The UpHsp90 and UpHsp60 mRNA levels were significantly affected by diurnal and temperature changes, and slightly affected by exposure to heavy metals. These results suggest that UpHsp90 and UpHsp60 genes play particularly important roles in adaptation to diurnal and temperature changes.
Hsp24 is a small heat-shock protein (sHSP). Such proteins are important endogenous cytoprotection factors involved in defense. A 1116-bp full-length cDNA of the Hsp24, with a 645-bp open reading frame nucleotide encoding a 24-kDa polypeptide consisting of 214 amino acid residues, was isolated from Trichoderma harzianum. Sequence analysis revealed that Hsp24 gene has more than 42–58% amino acid sequence homology with those of other fungi. The Hsp24 gene was integrated into pYES2 by inserting into a single site for recombination, yielding the recombinant of pYES2/Hsp24. Hsp24 expressed by pYES2/Hsp24 was induced by galactose. We tested whether Hsp24 could confer abiotic stress resistance when it was introduced into yeast cells. A transgenic yeast harboring T. harzianum Hsp24 was generated under the control of a constitutively expressed GAL promoter. The results indicated that Hsp24 yeast transformants had significantly higher resistance to salt, drought and heat stresses.
Chloroplast small (low-molecular-weight) heat-shock proteins (csHsps) can protect photosynthetic electron transport (Pet), and quantitative variation in csHsps is correlated with thermotolerance of net photosynthesis and Photosystem II. However, the functional (i.e. protective) consequence of natural variation in csHsps is unknown. To investigate this, we used an in vitro assay to determine the contribution of csHsps to the tolerance of Pet to high temperatures in five ecotypes of Chenopodium album collected from habitats ranging from cool to warm, and we partitioned total Pet thermotolerance into basal and induced Pet components (without and with a pre-heat treatment, respectively, to induce csHsps). The ecotypes varied in total Pet thermotolerance and this was correlated with habitat temperature. Variation in total Pet thermotolerance was associated primarily with variation in induced Pet thermotolerance, and not with basal Pet thermotolerance. Variation in induced Pet was highly correlated with csHsp protection of Pet. Variation in csHsp function was associated with variation in csHsp content among ecotypes. These results are the first to demonstrate the direct functional consequences for natural variation in Hsps in plants, and show that functional variation is associated with evolutionary adaptation to specific habitats among ecotypes.
Mature Nicotiana benthamiana shows strong resistance to the potato late blight pathogen Phytophthora infestans. By screening using virus-induced random gene silencing, we isolated a gene for plant-specific calreticulin NbCRT3a as a required gene for resistance of N. benthamiana against P. infestans. NbCRT3a encodes an ER quality-control (ERQC) chaperone for the maturation of glycoproteins, including glycosylated cell-surface receptors. NbCRT3a-silenced plants showed no detectable growth defects, but resistance to P. infestans was significantly compromised. Defense responses induced by the treatment with INF1 (a secretory protein of P. infestans), such as production of reactive oxygen species (ROS) and accumulation of phytoalexins, were suppressed in NbCRT3a-silenced N. benthamiana. Expression of an ethylene-regulated gene for phytoalexin biosynthesis, NbEAS, was reduced in NbCRT3a-silenced plants, whereas the expression of salicylic acid-regulated NbPR-1a was not affected. Consistently, induction of ethylene production by INF1 was suppressed in NbCRT3a-silenced plants. Resistance reactions induced by hyphal wall components (HWC) elicitor prepared from P. infestans were also impaired in NbCRT3a-silenced plants. However, cell death induced by active MAPKK (NbMEK2(DD)) was not affected by the silencing of NbCRT3a. NbCRT3a is thus required for the initiation of resistance reactions of N. benthamiana in response to elicitor molecules derived from P. infestans.
The widely distributed and highly conserved Ca-binding protein calreticulin has been suggested to play a role as a Ca storage protein of intracellular Ca stores. To test this hypothesis, we have generated a mouse L fibroblast cell line stably transfected with a calreticulin
expression vector. The calreticulin content of the overexpressers was increased by 1.6 ± 0.2-fold compared with mock-transfected
cells. The total cellular Ca content of calreticulin-overexpressing and control cells, as assessed by equilibrium Ca uptake, was 141 ± 8 and 67 ± 6 pmol of Ca/106 cells, respectively (i.e. a 2.1 ± 0.2-fold increase in the Ca content of calreticulin-overexpressing cells). Over 80% of the increased Ca content was found within thapsigargin-sensitive Ca stores. The pattern of calreticulin distribution, revealed by immunofluorescence microscopy, showed an endoplasmic reticulum-like
pattern and was identical in overexpressers and control cells. In overexpressers, cytosolic free [Ca] elevations due to Ca release were enhanced when either ATP or a combination of ionomycin and thapsigargin was used as a stimulus. In contrast,
thapsigargin-induced Ca and Mn influxes from the extracellular space were markedly diminished in calreticulin-overexpressing cells, suggesting an active
involvement of calreticulin in the regulation of store-operated Ca influx.
Plants synthesize four classes of small heat shock proteins (sHSPs); two classes are targeted to the plastid and endoplasmic reticulum, respectively, and two are found in the cytoplasm. In this paper, we describe a new role for the two classes of cytoplasmic HSPs in maturing embryos of developing seeds. The expression of each class of sHSPs was examined in pea seeds grown under non-stress conditions using Western and Northern analysis. Class I and class II cytoplasmic sHSPs are coordinately expressed in the embryo and accumulate to levels seen in moderately heat-stressed leaves. Their induction in cotyledons coincides with the mid-maturation phase of seed development, and induction in axes roughly coincides with abscission of the seed from the ovary wall. Both classes of sHSPs persisted in cotyledons for 4 days after the onset of imbibition, but disappeared from axes shortly after germination. Neither class of cytoplasmic sHSP is expressed in non-embryonic organs associated with the seed. The timing and organ specificity of sHSP expression is paralleled by the expression of the corresponding mRNAs. Neither the plastid nor the endoplasmic reticulum sHSPs were consistently expressed during seed development, but both could be induced by heat-stressing the developing seed. Developmental regulation of the cytoplasmic sHSPs is evidence that these proteins function not only in responding to heat-stress but also during seed development and/or germination.
Plants experience high air and soil temperatures during periods of drought and when fields receive limited irrigation. Elevated plant temperatures that occur under these conditions negatively impact plant health and productivity. Plants, like all organisms, respond to an elevation in temperature by the synthesis of heat shock proteins (HSP). The appearance of plant HSP is strongly correlated to the development of a condition termed ‘acquired thermotolerance’. Acquired thermotolerance is induced by pre-exposure to elevated but non-lethal temperatures and leads to enhanced protection of plant cells from subsequent heat induced injury. Although the correlation between the development of acquired thermotolerance and the appearance of HSP is strong, a cause-and-effect relationship between the two has been difficult to demonstrate. To understand the relationship between HSP and acquired thermotolerance, mutations would be required that result in a coordinate change in the expressions of HSP. This paper describes research efforts leading to the development of a screening procedure for the isolation and characterization of acquired thermotolerance mutants. This method for identifying mutants is based on the inhibition of chlorophyll accumulation in etiolated tissue following challenges at lethal temperatures and the prevention of this inhibition by pre-incubation at a non-lethal elevated temperature; i.e. acquired thermotolerance. Arabidopsis thaliana mutants deficient in varying levels of acquired thermotolerance have been identified from both the RLD and Columbia ecotypes and these mutants are currently undergoing a detailed characterization at both the protein and molecular levels.