Identification of genetic diversity and mutations in higher plant acquired thermotolerance

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.     

Circular dichroism and the secondary structure of the ROF2 protein from Arabidopsis thaliana

The protein ROF2 from the plant Arabidopsis thaliana acts as a heat stress modulator, being involved in the long-term acquired thermotolerance of the plant. Here we investigate the relationship between the biological function and the structure of ROF2, inferred by circular dichroism (CD) spectroscopy. The far-UV CD spectra, analyzed with the CDPro and DICHROWEB program packages, yield the percentages of α-helices, β-sheets, unordered regions, turns and poly(Pro)II-helices in the secondary structure of ROF2. According to the analysis, the percentages of the structural elements of ROF2 are about 40% for β-sheets, 30% for unordered regions, 17% for turns, 10% for poly(Pro)II-helices and 3% for α-helices. The near-UV CD spectra suggest that ROF2 proteins can associate, forming super-secondary structures. Our CD experiments performed at temperatures between 5 °C and 97 °C indicate that the thermal denaturation of ROF2 caused by a raise in temperature up to 55 °C is followed by a thermal refolding of the protein as the temperature is raised further. The new secondary structure, acquired around 65 °C, remains stable up to 97 °C. The structural stability of ROF2 at high temperatures might play an important role in the experimentally observed thermotolerance of Arabidopsis thaliana.     

Novel structure of a high molecular weight FK506 binding protein fromArabidopsis thaliana

We have isolated clones of an Arabidopsis gene (ROF1, forrotamaseFKBP) encoding a high molecular weight member of the FK506 binding protein (FKBP) family. The deduced amino acid sequence of ROF1 predicts a 551-amino acid, 62 kDa polypeptide which is 44% identical to human FKBP59 — a 59 kDa FKBP which binds to the 90 kDa heat shock protein and is associated with inactive steroid hormone receptors. ROF1 contains three FKBP12-like domains in the N-terminal portion of the protein (in contrast to two domains in mammalian FKBP59), an internal repeat structure associated with protein-protein interactions (tetratricopeptide repeats), and a putative calmodulin binding domain near the C-terminal region of the protein. No sequences associated with protein translocation out of the cytosol were found in ROF1.ROF1 mRNA was found at equivalent low levels in light-grown roots, stems, and flowers and at slightly higher levels in leaves. The abundance ofROF1 mRNA increased several-fold under stress conditions such as wounding or exposure to elevated NaCl levels.     

Molecular characterization of a heat inducible rice gene,OsHSP1, and implications for rice thermotolerance

Higher plants have acquired complex molecular mechanisms to withstand heat stress through years of natural evolutionary processes. Although physiological responses to elevated temperatures have been well studied, thermotolerance mechanisms at the molecular level are poorly understood in rice plants. In order to identify the genes involved in the thermotolerance of rice, we used a publicly available microarray dataset and identified a number of heat stress-responsive genes. Herein, we report details of the rice gene OsHSP1, which is upregulated by heat stress. In addition, OsHSP1 is highly expressed when exposed to salt and osmotic treatments but not cold treatment. Sequence analysis indicated that OsHSP1 belongs to the heat shock protein 90 family of genes. The biological function of OsHSP1 was investigated by heterologous overexpression in Arabidopsis. Transgenic Arabidopsis overexpressing the OsHSP1 gene exhibited enhanced thermotolerance but was hypersensitive under salt and osmotic stresses. Subcellular localization analysis indicated that the OsHSP1 protein is predominantly targeted to the cytosol and nucleus under heat stress. The coexpression network showed 39 interactions for the functionally interacting genes of OsHSP1. Taken together, these findings suggest that OsHSP1 is a heat-inducible gene that may play an important role in the thermotolerance of rice.     

Characterization of the Arabidopsis thermosensitive mutant atts02 reveals an important role for galactolipids in thermotolerance

Plants are constantly challenged with various abiotic stresses in their natural environment. Elevated temperatures have a detrimental impact on overall plant growth and productivity. Many plants increase their tolerance to high temperatures through an adaptation response known as acquired thermotolerance. To identify the various mechanisms that plants have evolved to cope with high temperature stress, we have isolated a series of Arabidopsis mutants that are defective in the acquisition of thermotolerance after an exposure to 38 degrees C, a treatment that induces acquired thermotolerance in wild-type plants. One of these mutants, atts02, was not only defective in acquiring thermotolerance after the treatment, but also displayed a reduced level of basal thermotolerance in a 30 degrees C growth assay. The affected gene in atts02 was identified by positional cloning and encodes digalactosyldiacylglycerol synthase 1 (DGD1) (the atts02 mutant was, at that point, renamed dgd1-2). An additional dgd1 allele, dgd1-3, was identified in two other mutant lines displaying altered acquired thermotolerance, atts100 and atts104. Expression patterns of several heat shock proteins (HSPs) in heat-treated dgd1-2 homozygous plants were similar to those from identically treated wild-type plants, suggesting that the thermosensitivity in the dgd1-2 mutant was not caused by a defect in HSP induction. Lipid analysis of wild-type and mutant plants indicated a close correlation between the ability to acquire thermotolerance and the increases in digalactosyldiacylglycerol (DGDG) level and in the ratio of DGDG to monogalactosyldiacylglycerol (MGDG). Thermosensitivity in dgd1-2 and dgd1-3 was associated with (1) a decreased DGDG level and (2) an inability to increase the ratio of DGDG to MGDG upon exposure to a 38 degrees C sublethal temperature treatment. Our results suggest that the DGDG level and/or the ratio of DGDG to MGDG may play an important role in basal as well as acquired thermotolerance in Arabidopsis.     

Isolation of Arabidopsis mutants lacking components of acquired thermotolerance

Acquired thermotolerance is a complex physiological phenomenon that enables plants to survive normally lethal temperatures. This study characterizes the temperature sensitivity of Arabidopsis using a chlorophyll accumulation bioassay, describes a procedure for selection of acquired thermotolerance mutants, and provides the physiological characterization of one mutant (AtTS02) isolated by this procedure. Exposure of etiolated Arabidopsis seedlings to 48 degrees C or 50 degrees C for 30 min blocks subsequent chlorophyll accumulation and is eventually lethal. Arabidopsis seedlings can be protected against the effects of a 50 degrees C, 30-min challenge by a 4-h pre-incubation at 38 degrees C. By the use of the milder challenge, 44 degrees C for 30 min, and protective pretreatment, mutants lacking components of the acquired thermotolerance system were isolated. Putative mutants isolated by this procedure exhibited chlorophyll accumulation levels (our measure of acquired thermotolerance) ranging from 10% to 98% of control seedling levels following pre-incubation at 38 degrees C and challenge at 50 degrees C. The induction temperatures for maximum acquired thermotolerance prior to a high temperature challenge were the same in AtTS02 and RLD seedlings, although the absolute level of chlorophyll accumulation was reduced in the mutant. Genetic analysis showed that the loss of acquired thermotolerance in AtTS02 was a recessive trait. The pattern of proteins synthesized at 25 degrees C and 38 degrees C in the RLD and AtTS02 revealed the reduction in the level of a 27-kD heat shock protein in AtTS02. Genetic analysis showed that the reduction of this protein level was correlated with the acquired thermotolerance phenotype.     

Acquisition of thermotolerance in bay scallops, Argopecten irradians irradians, via differential induction of heat shock proteins

Many cells and organisms are rendered transiently resistant to lethal heat shock by short exposure to sublethal temperatures. This induced thermotolerance is thought to be related to increased amounts of heat shock proteins (HSPs) which, as molecular chaperones, protect cells from stress-induced damage. As part of a study on bivalve stress and thermotolerance, work was undertaken to examine the effects of sublethal heat shock on stress tolerance of juveniles of the northern bay scallop, Argopecten irradians irradians, in association with changes in the levels of cytoplasmic HSP70 and 40. Juvenile bay scallops heat-shocked at a sublethal temperature of 32 °C survived an otherwise lethal heat treatment at 35 °C for at least 7 days. As determined by ELISA, acquisition of induced thermotolerance closely paralleled HSP70 accumulation, whereas HSP40 accrual appeared less closely associated with thermotolerance. Quantification of scallop HSPs following lethal heat treatment, with or without conditioning, suggested a causal role for HSP70 in stress tolerance, with HSP40 contributing to a lesser, but significant extent. Overall, this study demonstrated that sublethal heat shock promotes survival of A. irradians irradians juveniles upon thermal stress and the results support the hypothesis that HSPs have a role in this induced thermotolerance. Exploitation of the induced thermotolerance response shows promise as a means to improve survival of bay scallops in commercial culture.     

Novel structure of a high molecular weight FK506 binding protein fromArabidopsis thaliana

We have isolated clones of an Arabidopsis gene (ROF1, forrotamaseFKBP) encoding a high molecular weight member of the FK506 binding protein (FKBP) family. The deduced amino acid sequence of ROF1 predicts a 551-amino acid, 62 kDa polypeptide which is 44% identical to human FKBP59 — a 59 kDa FKBP which binds to the 90 kDa heat shock protein and is associated with inactive steroid hormone receptors. ROF1 contains three FKBP12-like domains in the N-terminal portion of the protein (in contrast to two domains in mammalian FKBP59), an internal repeat structure associated with protein-protein interactions (tetratricopeptide repeats), and a putative calmodulin binding domain near the C-terminal region of the protein. No sequences associated with protein translocation out of the cytosol were found in ROF1.ROF1 mRNA was found at equivalent low levels in light-grown roots, stems, and flowers and at slightly higher levels in leaves. The abundance ofROF1 mRNA increased several-fold under stress conditions such as wounding or exposure to elevated NaCl levels.The nucleotide sequences in this paper have been submitted to the GenBank/EMBL Data bank with accession numbers U49453 and U57838     

圆锥南芥适应高山环境极端高温的系统性耐热能力

高山生态系统的主要气候特征是温度变化幅度较大,目前研究主要集中于高山植物的抗冻机制,而很少关注其对极端高温（高于45℃）的适应性。本研究发现高山物种圆锥南芥跟拟南芥比,具有更强的基础耐热性和获得性耐热性。通过叶绿素荧光检测发现在极端高温下,圆锥南芥具有更稳定的光系统II和更有效的能量耗散机制来维持更高水平的光合效率。通过电导率和丙二醛含量的检测发现圆锥南芥的膜伤害更小,膜流动性与脂肪酸链的长度和不饱和度紧密相关,圆锥南芥脂肪酸具有更低的16∶3含量,更长的碳链,不饱和度没有明显的变化,这些可能有助于维持膜的稳定。另外,更高表达量的HSP101和HSP70可能为圆锥南芥提供了更好的保护作用。以上结果表明,圆锥南芥能利用生理生化活动的调整来适应高山环境中的极端高温,这种耐热策略与低地耐热植物相似,因此圆锥南芥具有系统性耐热能力,可以作为研究植物耐热分子机制的模式物种。     

Identification of acquired thermotolerance deficiency within the ditelosomic series of `Chinese Spring' wheat

The relative contribution of individual heat shock proteins to acquired thermotolerance was evaluated through analysis of chromosomal deletions in a ditelosomic series of the hexaploid wheat (Triticum aestivum L.) cultivar `Chinese Spring'. This study describes the identification of a line within this ditelosomic series that exhibited a reduced level of acquired thermotolerance. Changes in the temperature sensitivity of chlorophyll accumulation were used as an indicator of acquired thermotolerance. The temperature providing maximum chlorophyll accumulation was 30 °C in leaves under continuous light. A 30-min challenge temperature of 48 °C prior to the light exposure was shown to inhibit subsequent chlorophyll accumulation. Preincubation at 40 °C for 4 h before the 30-min 48 °C challenge triggered the acquired thermotolerance system of the plant resulting in chlorophyll accumulation upon exposure to light. Evaluation of the ditelosomic series revealed reductions in acquired thermotolerance levels in the DT7DS line relative to controls. Two-dimensional SDS polyacrylamide gel analysis was used to identify reduction in the level of two low molecular mass heat shock proteins in DT7DS.     

Four members of the HSP101 gene family are differently regulated in Triticum durum Desf

Heat shock proteins play an essential role in preventing deleterious effects of high temperatures. In many plants, HSP101 has a central role in heat stress survival. We report the isolation and characterization of four cDNAs corresponding to different members of the durum wheat HSP101 gene family. Expression analysis revealed differences in their induction. Accordingly, durum wheat HSP101 genes are differently regulated, therefore having distinct roles in stress response and thermotolerance acquisition. These findings are important for further dissection of the molecular mechanisms underlying the stress response and for understanding the functions of the HSP101 family members. This information could be important for the exploitation of specific alleles in marker assisted selection for abiotic stress resistance.     

Heat Shock Response in the Thermophilic Enteric Yeast Arxiozyma telluris

Heat stress tolerance was examined in the thermophilic enteric yeast Arxiozyma telluris. Heat shock acquisition of thermotolerance and synthesis of heat shock proteins hsp 104, hsp 90, hsp 70, and hsp 60 were induced by a mild heat shock at temperatures from 35 to 40°C for 30 min. The results demonstrate that a yeast which occupies a specialized ecological niche exhibits a typical heat shock response.