Expression and interaction of small heat shock proteins (sHsps) in rice in response to heat stress

The inherent immobility of rice (Oryza sativa L.) limited their abilities to avoid heat stress and required them to contend with heat stress through innate defense abilities in which heat shock proteins played important roles. In this study, Hsp26.7, Hsp23.2, Hsp17.9A, Hsp17.4 and Hsp16.9A were up-regulated in Nipponbare during seedling and anthesis stages in response to heat stress. Subsequently, the expressing levels of these five sHsps in the heat-tolerant rice cultivar, Co39, were all significantly higher than that in the heat-susceptible rice cultivar, Azucena. This indicated that the expressive level of these five sHsps was positively related to the ability of rice plants to avoid heat stress. Thus, the expression level of these five sHsps can be regarded as bio-markers for screening rice cultivars with different abilities to avoid heat stress. Hsp18.1, Hsp17.9A, Hsp17.7 and Hsp16.9A, in the three rice cultivars under heat stress were found to be involved in one protein complex by Native-PAGE, and the interactions of Hsp18.1 and Hsp 17.7, Hsp18.1 and Hsp 17.9A, and Hsp17.7 and Hsp16.9A were further validated by yeast 2-hybridization. Pull down assay also confirmed the interaction between Hsp17.7 and Hsp16.9A in rice under heat stress. In conclusion, the up-regulation of the 5 sHsps is a key step for rice to tolerate heat stress, after that some sHsps assembled into a large hetero-oligomeric complex. In addition, through protein–protein interaction, Hsp101 regulated thiamine biosynthesis, and Hsp82 homology affected nitrogen metabolism, while Hsp81-1 were involved in the maintenance of sugar or starch synthesis in rice plants under heat stress. These results provide new insight into the regulatory mechanism of sHsps in rice.     

Differential expression pattern of heat shock protein 70 gene in tissues and heat stress phenotypes in goats during peak heat stress period

It has been established that the synthesis of heat shock protein 70 (Hsp70) is temperature-dependent. The Hsp70 response is considered as a cellular thermometer in response to heat stress and other stimuli. The variation in Hsp70 gene expression has been positively correlated with thermotolerance in Drosophila melanogaster, Caenorhabditis elegans, rodents and human. Goats have a wide range of ecological adaptability due to their anatomical and physiological characteristics; however, the productivity of the individual declines during thermal stress. The present study was carried out to analyze the expression of heat shock proteins in different tissues and to contrast heat stress phenotypes in response to chronic heat stress. The investigation has been carried out in Jamunapari, Barbari, Jakhrana and Sirohi goats. These breeds differ in size, coat colour and production performance. The heat stress assessment in goats was carried out at a temperature humidity index (THI) ranging from 85.36–89.80 over the period. Phenotyping for heat stress susceptibility was carried out by combining respiration rate (RR) and heart rate (HR). Based on the distribution of RR and HR over the breeds in the population, individual animals were recognized as heat stress-susceptible (HSS) and heat stress-tolerant (HST). Based on their physiological responses, the selected animals were slaughtered for tissue collection during peak heat stress periods. The tissue samples from different organs such as liver, spleen, heart, testis, brain and lungs were collected and stored at ?70 °C for future use. Hsp70 concentrations were analyzed from tissue extract with ELISA. mRNA expression levels were evaluated using the SYBR green method. Kidney, liver and heart had 1.5–2.0-fold higher Hsp70 concentrations as compared to other organs in the tissue extracts. Similarly, the gene expression pattern of Hsp70 in different organs indicated that the liver, spleen, brain and kidney exhibited 5.94, 4.96, 5.29 and 2.63-fold higher expression than control. Liver and brain tissues showed the highest gene expression at mRNA levels as compared to kidney, spleen and heart. HST individuals had higher levels of mRNA level expression than HSS individuals in all breeds. The Sirohi breed showed the highest (6.3-fold) mRNA expression levels as compared to the other three breeds, indicating the better heat stress regulation activity in the breed.     

Cloning of new members of heat shock protein HSP101 gene family in wheat (Triticum aestivum (L.) Moench) inducible by heat, dehydration, and ABA(1)

We have cloned two cDNAs, TaHSP101B and TaHSP101C, encoding two heat stress-inducible members of HSP101/ClpB family in bread wheat (Triticum aestivum (L.) Moench.). Proteins encoded by these cDNAs are highly similar at the primary sequence level and diverged from the previously reported TaHSP101 (designated TaHSP101A) both in the consensus ATP/GTP-binding region II and in the carboxy terminal region. The HSP101 gene was determined to be a single copy gene or a member of a small gene family in hexaploid wheat. Messages encoding HSP101 proteins were inducible by heat stress treatments in both wheat leaves and roots. Accumulation of the TaHSP101C mRNA was less abundant than that of TaHSP101B mRNA. We are showing for the first time that in addition to heat stress, expression of HSP101 mRNAs in wheat leaves was induced by a 2-h dehydration and a treatment with 5x10(-5)M ABA, but not affected by chilling or wounding, indicating that HSP101 proteins may be involved in both heat and drought responses in wheat.     

Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress

Hsp101 is a molecular chaperone that is required for the development of thermotolerance in plants and other organisms. We report that Arabidopsis thaliana Hsp101 is also regulated during seed development in the absence of stress, in a pattern similar to that seen for LEA proteins and small Hsps; protein accumulates during mid-maturation and is stored in the dry seed. Two new alleles of the locus encoding Hsp101 (HOT1) were isolated from Arabidopsis T-DNA mutant populations. One allele, hot1-3, contains an insertion within the second exon and is null for Hsp101 protein expression. Despite the complete absence of Hsp101 protein, plant growth and development, as well as seed germination, are normal, demonstrating that Hsp101 chaperone activity is not essential in the absence of stress. In thermotolerance assays hot1-3 shows a similar, though somewhat more severe, phenotype to the previously described missense allele hot1-1, revealing that the hot1-1 mutation is also close to null for protein activity. The second new mutant allele, hot1-2, has an insertion in the promoter 101 bp 5' to the putative TATA element. During heat stress the hot1-2 mutant produces normal levels of protein in hypocotyls and 10-day-old seedlings, and it is wild type for thermotolerance at these stages. Thus this mutation has not disrupted the minimal promoter sequence required for heat regulation of Hsp101. The hot1-2 mutant also expresses Hsp101 in seeds, but at a tenfold reduced level, resulting in reduced thermotolerance of germinating seeds and underscoring the importance of Hsp101 to seed stress tolerance.     

BiP, HSP70, NDK and PDI in wheat endosperm. II. Effects of high temperature on protein and mRNA accumulation

The effects of high temperature on accumulation of the 70‐kDa heat shock protein (HSP70) and nucleoside diphosphate kinase (NDK) as well as two other proteins that have roles in the biosynthesis of storage proteins were examined during grain development. An HSP70 homolog and a 17‐kDa NDK were co‐purified from wheat endosperm, their identity verified, and a cDNA for an HSP70 expressed in endosperm was isolated. Wheat plants ( Triticum aestivum , cvs Butte and Vulcan) were heat shocked at 40°C or exposed to maximum daily temperatures of 37 or 40°C during early or mid‐grain fill. Antibodies and cDNA probes for BiP, HSP70, NDK and PDI were used to examine the effect of high temperatures on the accumulation of protein and mRNA in the endosperm. HSP70 mRNA levels increased substantially when plants were exposed to heat shock or to a 1‐day gradual increase to 40°C. The effects of a 5‐day heat treatment on mRNA levels were more complicated and depended on the developmental stage of the grain. A treatment that began at 7 days post‐anthesis (DPA) decreased the level of mRNA for HSP70, BiP, PDI and NDK, whereas a treatment that began at 14 DPA slightly increased mRNA levels. The same treatments increased the accumulation of HSP70 but did not affect BiP, PDI, or NDK protein levels. This is the first detailed report on the effects of heat on mRNA and protein levels for HSP70 in a developing seed storage tissue.     

Developmental and organ-specific expression of an ABA- and stress-induced protein in barley

An mRNA species, HVA1, has been shown to be rapidly induced by abscisic acid (ABA) in barley aleurone layers (Hong, Uknes and Ho, Plant Mol Biol 11: 495–506, 1988). In the current work we have investigated the expression of HVA1 in other organs of barley plants. In developing seeds, HVA1 mRNA is not detected in starchy endosperm cells, yet it accumulates in aleurone layers and embryo starting 25 days after anthesis, and its level remains high in these organs in dry seeds. Although the levels of HVA1 mRNA are equivalent in the dry embryos of dormant and nondormant barley seeds, upon imbibition HVA1 mRNA declines much slower in the dormant than in the nondormant embryos. The HVA1 mRNA and protein levels are highly induced by ABA treatment in all organs of 3-day-old seedlings. However, the induction in the leaf of 7-day-old seedlings is less than one tenth the level observed in the leaf of 3-day-old seedlings. In the leaf, HVA1 mRNA and protein are induced mainly at the base. These observations indicate that the expression of HVA1 is under developmental regulation. Besides the HVA1 protein, a smaller protein (p20) of approximately 20 kDa cross-reacting with anti-HVA1 polyclonal antibodies, is induced by ABA in barley seedlings but not in seeds. HVA1 mRNA is induced by drought, NaCl, cold or heat treatment. Similar to ABA treatment, the drought induction of HVA1 occurs in all the tissues of 3-day-old seedling, but the induction decreases dramatically in the leaf of 7-day-old plants. The significance of organ-specific, developmentally regulated, and stress-induced expression of HVA1 is discussed.     

Expression and localization of Hsps in the heart and blood vessel of heat-stressed broilers

The objective of this study was to investigate the kinetics of Hsp60, Hsp70, Hsp90 protein, and messenger RNA (mRNA) expression levels and to correlate these heat shock protein (Hsp) levels with tissue damage resulting from exposure to high temperatures for varying amounts of time. One hundred broilers were heat-stressed for 0, 2, 3, 5, and 10 h, respectively, by rapidly increasing the ambient temperature from 22 +/- 1 degrees C to 37 +/- 1 degrees C. Obvious elevations of plasma creatine kinase indicate damage to myocardial cells after heat stress. Hsp70 and Hsp90, and their corresponding mRNAs in the heart tissue of heat-stressed broilers, elevated significantly after 2 h of heat exposure and decreased quickly with continued heat stress. However, the levels of hsp60 mRNA in the heart of heat-stressed broilers increased sharply (P < 0.01) at 2 h of heat stress but then decreased quickly after 3 h, while the level of Hsp60 protein in the heart increased (P < 0.01) at 2 h of heat stress and maintained a high level throughout heat exposure. The results indicate that the elevation of the three Hsps, especially Hsp60 in heart, may be important markers at the beginning of heat stress and act as protective proteins in adverse environments. The reduction of Hsp signals in the cytoplasm of myocardial cells implies that myocardial cell lesions may have an adverse impact on the function of Hsps during heat stress. Meanwhile, the localization of Hsp70 in blood vessels of broiler hearts suggests another possible mechanism for protection of the heart after heat exposure.     

An Arabidopsis heat shock protein complements a thermotolerance defect in yeast.

The heat shock protein Hsp104 of the yeast Saccharomyces cerevisiae plays a key role in promoting survival at extreme temperatures. We found that when diverse higher plant species are exposed to high temperatures they accumulate proteins that are antigenically related to Hsp104. We isolated a cDNA corresponding to one of these proteins from Arabidopsis. The protein, AtHSP101, is 43% identical to yeast Hsp104. DNA gel blot analysis indicated that AtHSP101 is encoded by a single- or low-copy number gene. AtHsp101 mRNA was undetectable in the absence of stress but accumulated to high levels during exposure to high temperatures. When AtHSP101 was expressed in yeast, it complemented the thermotolerance defect caused by a deletion of the HSP104 gene. The ability of AtHSP101 to protect yeast from severe heat stress strongly suggests that this HSP plays an important role in thermotolerance in higher plants.     

Expression of an Aspartate Kinase Homoserine Dehydrogenase Gene Is Subject to Specific Spatial and Temporal Regulation in Vegetative Tissues,Flowers, and Developing Seeds

Although the regulation of amino acid synthesis has been studied extensively at the biochemical level, it is still not known how genes encoding amino acid biosynthesis enzymes are regulated during plant development. In the present report, we have used the [beta]-glucuronidase (GUS) reporter gene to study the regulation of expression of an Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase (AK/HSD) gene in transgenic tobacco plants. The polypeptide encoded by the AK/HSD gene comprises two linked key enzymes in the biosynthesis of aspartate-family amino acids. AK/HSD-GUS gene expression was highly stimulated in apical and lateral meristems, lateral buds, young leaves, trichomes, vascular and cortical tissues of growing stems, tapetum and other tissues of anthers, pollen grains, various parts of the developing gynoecium, developing seeds, and, in some transgenic plants, also in stem and leaf epidermal trichomes. AK/HSD-GUS gene expression gradually dimished upon maturation of leaves, stems, floral tissues, and embryos. GUS expression was relatively low in roots. During seed development, expression of the AK/HSD gene in the embryo was coordinated with the initiation and onset of storage protein synthesis, whereas in the endosperm it was coordinated with the onset of seed desiccation. Upon germination, AK/HSD-GUS gene expression in the hypocotyl and the cotyledons was significantly affected by light. The expression pattern of the A. thaliana AK/HSD-GUS reporter gene positively correlated with the levels of aspartate-family amino acids and was also very similar to the expression pattern of the endogenous tobacco AK/HSD mRNA as determined by in situ hybridization.     

A soybean 101-kD heat shock protein complements a yeast HSP104 deletion mutant in acquiring thermotolerance.

A cDNA clone encoding a 101-kD heat shock protein (HSP101) of soybean was isolated and sequenced. Genomic DNA gel blot analysis indicated that the corresponding gene is a member of a multigene family. The mRNA for HSP101 was not detected in 2-day-old etiolated soybean seedlings grown at 28 degrees C but was induced by elevated temperatures. DNA sequence comparison has shown that the corresponding gene belongs to the Clp (caseinolytic protease) (or Hsp100) gene family, which is evolutionarily conserved and found in both prokaryotes and eukaryotes. On the basis of the spacer length between the two conserved ATP binding regions, this gene has been identified as a member of the ClpB subfamily. Unlike other Clp genes previously isolated from higher plants, the expression of this soybean Hsp101 gene is heat inducible, and it does not have an N-terminal signal peptide for targeting to chloroplasts. Transformation of the soybean Hsp101 gene into a yeast HSP104 deletion mutant complemented restoration of acquired thermotolerance, a process in which cells survive an otherwise lethal heat stress after they are given a permissive heat treatment.     

DNA content and expression of genes related to cell cycling in developing Gossypium hirsutum (Malvaceae) fibers

The cell cycle in cotton (Gossypium hirsutum) fibers is poorly understood. The objective of this study was to evaluate the cell cycle status and DNA content in developing cotton fibers. The DNA content and cell cycle distribution in fiber and hypocotyl cells were determined by flow cytometry. Expression levels of minichrosomal maintenance protein (mcm), cyclin B, and a retinoblastoma-like protein (rb) genes were determined with real-time PCR in fibers and dividing and nondividing tissues. No endoreduplication occurred, nor did genome size or percentage of G1-phase nuclei differ between hypocotyls and fibers. Approximately 13 and 17% of fiber nuclei were in the S phase in 14 days after anthesis (d) fibers and 25 d fibers, respectively. The mcm and cyclin B were expressed at higher levels in fibers than in mature leaves, but expression levels in fibers were less than 15% of meristematic tissues. Rb was expressed in fibers at levels less than 50% of mature leaves or meristematic tissues. Based on an apparent increase in S-phase cells as fibers mature and the low level of expression of genes associated with cell cycle progression, we conclude that S-phase arrest occurs in developing cotton fiber.     

Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress

Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. To accurately investigate the mRNA expression profiles of multiple Hsp70s in rainbow trout Oncorhynchus mykiss, we isolated full-length cDNA clones encoding Hsp70 from the fish and investigated their mRNA expression profiles during heat stress. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells. The induction levels of total Hsp70b mRNAs were consistently higher than Hsp70a counterparts during heat stress, although the expression profiles of the two genes were similar to each other in temperature shift and time course experiments. Interestingly, an mRNA species with a larger molecular size was expressed only under severe heat stress not less than 28 degrees C irrespective of Hsp70a and Hsp70b. These results suggest that the comprehensive identification of duplicated genes is a prerequisite to examining the gene expression profiles for tetraploid species such as rainbow trout.