Biochemical analysis of a cytosolic small heat shock protein, NtHSP18.3, from Nicotiana tabacum

Small heat shock proteins (sHSPs) are widely distributed, and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as 70 degrees . Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.     

Nuclear-mitochondrial cross-talk during heat shock in Arabidopsis cell culture

Apart from energy generation, mitochondria perform a signalling function determining the life and death of a cell under stress exposure. In the present study we have explored patterns of heat-induced synthesis of Hsp101, Hsp70, Hsp17.6 (class I), Hsp17.6 (class II) and Hsp60, and the development of induced thermotolerance in Arabidopsis thaliana cell culture under conditions of mitochondrial dysfunction. It was shown that treatment by mitochondrial inhibitors and uncouplers at the time of mild heat shock downregulates HSP synthesis, which is important for induced thermotolerance in plants. The exposure to elevated temperature induced an increase in cell oxygen consumption and hyperpolarization of the inner mitochondrial membrane. Taken together, these facts suggest that mitochondrial functions are essential for heat-induced HSP synthesis and development of induced thermotolerance in A. thaliana cell culture, suggesting that mitochondrial-nuclear cross-talk is activated under stress conditions. Treatment of Arabidopsis cell culture at 50 degrees C initiates a programmed cell death determined by the time course of viability decrease, DNA fragmentation and cytochrome c release from mitochondria. As treatment at 37 degrees C protected Arabidopsis cells from heat-induced cell death, it may be suggested that Hsp101, Hsp70 and small heat-shock proteins, the synthesis of which is induced under these conditions, are playing an anti-apoptotic role in the plant cell. On the other hand, drastic heat shock upregulated mitochondrial Hsp60 synthesis and induced its release from mitochondria to the cytosol, indicating a pro-apoptotic role of plant Hsp60.     

Involvement of osmoregulation,glyoxalase, and non-glyoxalase systems in signaling molecule glutamic acid-boosted thermotolerance in maize seedlings

Glutamic acid (Glu) is not only an important protein building block, but also a signaling molecule in plants. However, the Glu-boosted thermotolerance and its underlying mechanisms in plants still remain unclear. In this study, the maize seedlings were irrigated with Glu solution prior to exposure to heat stress (HS), the seedlings’ thermotolerance as well as osmoregulation, glyoxalase, and non-glyoxalase systems were evaluated. The results manifested that the seedling survival and tissue vitality after HS were boosted by Glu, while membrane damage was reduced in comparison with the control seedlings without Glu treatment, indicating Glu boosted the thermotolerance of maize seedlings. Additionally, root-irrigation with Glu increased its endogenous level, reinforced osmoregulation system (i.e., an increase in the levels of proline, glycine betaine, trehalose, and total soluble sugar, as well as the activities of pyrroline-5-carboxylate synthase, betaine dehydrogenase, and trehalose-5-phosphate phosphatase) in maize seedlings under non-HS and HS conditions compared with the control. Also, Glu treatment heightened endogenous methylglyoxal level and the activities of glyoxalase system (glyoxalase I, glyoxalase II, and glyoxalase III) and non-glyoxalase system (methylglyoxal reductase, lactate dehydrogenase, aldo-ketoreductase, and alkenal/alkenone reductase) in maize seedlings under non-HS and HS conditions as compared to the control. These data hint that osmoregulation, glyoxalase, and non-glyoxalase systems are involved in signaling molecule Glu-boosted thermotolerance of maize seedlings.

     

Ethanol treatment triggers a heat shock-like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.     

Effects of salinity and heat-shock on wheat seedling growth and content of carbohydrates,proteins and amino acids

The effects of salinity (0, 50, 100, 150, and 200 mM NaCl) and heat-shock (42°C) and their interactions on germination, seedling growth, and some relevant metabolic changes of two cultivars (cv. Giza 155 and cv. Stork) of wheat (Triticum vulgaris L.) were studied. Germination studies indicate that plants tolerated salinity up to 100 mM NaCl. The lengths of roots and shoots and their water content, as well as fresh and dry matter yield of cv. Giza 155 seedlings remained more or less unchanged up to 100 mM NaCl and of cv. Stork up to 50 mM NaCl. Salinity induced progressive increase in soluble carbohydrates, soluble proteins and proline in cv. Giza 155 and in soluble proteins, proline and other free amino acids in cv. Stork. However, under the higher salinity levels, in cv. Giza 155 increase in soluble carbohydrates was accompanied by lose in other free amino acids, whereas in cv. Stork an opposite effect was obtained. Heat-shock treatment (42°C for 24 h) induced a significant decrease in the final germination percentage, the shoot and root lengths, fresh matter yield and the water content. The dry matter yield of the two cultivars was considerably increased as compared with the corresponding treatments with NaCl only. Heat-shock treatment resulted in a significant increase, in the amount of soluble carbohydrates and proline in salt treated seedlings of both cultivars. The pattern of changes in amino acids was opposite to that of soluble proteins, indicating that the increase in soluble proteins was at the expense of other amino acids in cv. Giza 155 andvice versa in cv. Stork.     

Cloning and characterisation of a Primula heat shock protein gene, PfHSP17.1, which confers heat, salt and drought tolerance in transgenic Arabidopsis thaliana

Small heat shock proteins (sHSPs) are the critical components of responses to various environmental stresses. However, few have been functionally characterised in Primula. In this study, we cloned a sHSP gene, PfHSP17.1, which is highly up-regulated in the leaves of Primula forrestii exposed to thermal stress (42 °C for 2 h). Sequence alignment and phylogenetic analysis indicated that PfHSP17.1 is a member of the plant cytosolic class I sHSPs. This gene was basally and ubiquitously expressed in different plant organs. The expression of PfHSP17.1 was also triggered remarkably by salt, drought and oxidative stress conditions but was only slightly induced by abscisic acid. Transgenic Arabidopsis thaliana constitutively expressing PfHSP17.1 displayed increased thermotolerance and higher resistance to salt and drought compared with wild-type plants. These results highlight the important role that PfHSP17.1 plays in diverse physiological and biochemical processes related to adverse conditions.     

Heat shock-induced acquisition of thermotolerance at the levels of cell survival and translation in Xenopus A6 kidney epithelial cells.

In this study we have investigated the acquisition of thermotolerance in a Xenopus laevis kidney A6 epithelial cell line at both the level of cell survival and translation. In cell survival studies, A6 cells were incubated at temperatures ranging from 22 to 35 degrees degrees C for 2 h followed by a thermal challenge at 39 degrees degrees C for 2 h and a recovery period at 22 degrees C for 24 h. Optimal acquisition of thermotolerance occurred at 33 degrees degrees C. For example, exposure of A6 cells to 39 degrees degrees C for 2 h resulted in only 3.4% survival of the cells whereas prior exposure to 33 degrees C for 2 h enhanced the survival rate to 69%. This state of thermotolerance in A6 cells was detectable after 1 h at 33 degrees C and was maintained even after 18 h of incubation. Cycloheximide inhibited the acquisition of thermotolerance at 33 degrees C suggesting the requirement for ongoing protein synthesis. The optimal temperature for the acquisition of translational thermotolerance also occurred at 33 degrees C. Treatment of A6 cells at 39 degrees C for 2 h resulted in an inhibition of labeled amino acid incorporation into protein which recovered to approximately 14% of control after 19 h at 22 degrees C whereas cells treated at 33 degrees C for 2 h prior to the thermal challenge recovered to 58% of control levels. These translationally thermotolerant cells displayed relatively high levels of the heat shock proteins hsp30, hsp70, and hsp90 compared to pretreatment at 22, 28, 30, or 35 degrees C. These studies demonstrate that Xenopus A6 cells can acquire a state of thermotolerance and that it is correlated with the synthesis of heat shock proteins.     

Proline accumulation induced by weak acids and IAA in coleoptiles of wheat seedlings

Proline accumulation in coleoptiles of wheat seedlings or in excised coleoptile segments incubated under shaking for a 24 h period was studied. There was no increase of proline content of coleoptiles after incubation of the seedlings in 5 mM citric acid (a relatively strong and slowly penetrating organic acid) in a pH range from 4.5 to 7.0 and only a slight increase of proline content after incubation in phosphate buffer at pH 7.0 to 7.5 duo to the higher osmotic concentration of phosphate buffer in this pH range. Quite different results were obtained with seedlings incubated in 10 mM acetic acid, a weak and easily penetrating organic acid. With increasing proton concentrations, proline accumulation increased. Application of 400 mM mannitol or higher concentrations of IAA (more than 10⁻⁵M) additionally increased proline accumulation in the presence of 10 mM acetic acid in the pH range from 6.0 to 7.5 in which acetic acid alone was loss effective. It is suggested that a decrease of cytosolic pH causes stress—induced proline accumulation.     

Counteraction of Salinity Stress on Wheat Plants by Grain Soaking in Ascorbic Acid, Thiamin or Sodium Salicylate

The interactive effects of salinity stress (40, 80, 120 and 160 mM NaCl) and ascorbic acid (0.6 mM), thiamin (0.3 mM) or sodium salicylate (0.6 mM) were studied in wheat (Triticum aestivum L.). The contents of cellulose, lignin of either shoots or roots, pectin of root and soluble sugars of shoots were lowered with the rise of NaCl concentration. On the other hand, the contents of hemicellulose and soluble sugars of roots, starch and soluble proteins of shoots, proline of either shoots or roots, and amino acids of roots were raised. Also, increasing NaCl concentration in the culture media increased Na⁺ and Ca²⁺ accumulation and gradually lowered K⁺ and Mg²⁺ concentration in different organs of wheat plant. Grain soaking in ascorbic acid, thiamin or sodium salicylate could counteract the adverse effects of NaCl salinity on the seedlings of wheat plant by suppression of salt stress induced accumulation of proline.     

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.     

Chemically induced resistance to heat treatment and stress protein synthesis in cultured mammalian cells

Short exposure (1-2 h) of cultured cells, derived from a transplantable murine mammary carcinoma, to sodium arsenite, 2,4-dinitrophenol (DNP), carbonylcyanide-3-chlorophenylhydrazone (CCP) or disulfiram, induced resistance to a subsequent heat treatment, similar to heat-induced thermotolerance. Optimum resistance to a test heat treatment of 45 min at 45 degrees C after sodium arsenite exposure was obtained at a concentration of 300 microM, after DNP exposure at 3mM, after CCP at 300 microM and after disulfiram exposure in the range 1-30 microM. Exposure of cells to CCP, sodium arsenite or disulfiram led to enhanced synthesis of some proteins with the same molecular weight as 'heat shock' proteins. The pattern of enhanced synthesis of these proteins was agent specific. We could not detect significantly enhanced synthesis of the proteins after DNP using one-dimensional gel electrophoresis. These results suggest that enhanced stress protein synthesis is not a prerequisite for the development of thermal resistance.     

Constitutive Expression of a Tomato Small Heat Shock Protein Gene <Emphasis Type="Italic">LeHSP21</Emphasis> Improves Tolerance to High-Temperature Stress by Enhancing Antioxidation Capacity in Tobacco

It is well established that small heat shock proteins (sHSPs) play an important role in thermotolerance in various organisms due to their abundance and diversity. In the present study, a chloroplast small heat shock protein gene (LeHSP21) from tomato (Lycopersicon esculentum cv PKM-1) was constitutively expressed in tobacco (Nicotiana tabacum L. cv Wisconsin 38) plants via Agrobacterium-mediated transformation. When compared to wild-type control plants, transgenic tobacco plants constitutively expressing LeHSP21, driven by the cauliflower mosaic virus 35S promoter, exhibited improved tolerance to both high temperature and oxidative stress. Furthermore, when the seedlings were subjected to high temperature treatment, the activities of anti-oxidative enzymes and the content of proline were significantly higher in transgenic plants than those in the wild-type plants. Our results presented here demonstrate the feasibility of improving high temperature and oxidative stress tolerance in plants through the expression of LeHSP21 gene.     

Exogenous salicylic acid improves salinity tolerance of <Emphasis Type="Italic">Nitraria tangutorum</Emphasis>

In the present study, the physiological responses of Nitraria tangutorum Bobr. seedlings to NaCl stress and the regulatory function of exogenous application of salicylic acid (SA) were investigated. NaCl in low concentration (100 mM) increased while in higher concentrations (200–400 mM) decreased the individual plant dry weights (wt) of seedlings. Decreased relative water content (RWC) and chlorophyll content were observed in the leaves of seedlings subjected to salinity stress (100–400 mM NaCl). Furthermore, NaCl stress significantly increased electrolyte leakage and malondialdehyde (MDA) content. The levels of osmotic adjustment solutes including proline, soluble sugars, and soluble protein were enhanced under NaCl treatments as compared to the control. In contrast, exogenous application of SA (0.5–1.5 mM) to the roots of seedlings showed notable amelioration effects on the inhibition of individual plant dry wt, RWC, and chlorophyll content. The increases in electrolyte leakage and MDA content in the leaves of NaCl-treated seedlings were markedly inhibited by SA application. The SA application further increased the contents of proline, soluble sugars, and soluble protein. The activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were up-regulated by NaCl stress and the activities of SOD, POD, and CAT were further enhanced by SA treatments. Application of SA in low concentration (0.5 mM) enhanced while in higher concentrations (1.0 and 1.5 mM) inhibited APX activities in leaves of NaCl-treated seedlings. These results indicate that SA effectively alleviated the adverse effects of NaCl stress on N. tangutorum.     

Salicylic acid-induced salinity tolerance in corn grown under NaCl stress

Effects of salicylic acid on some physiological and biochemical characteristics of maize ( Zea mays L.) seedlings under NaCl stress were studied. Pre-soaking treatments of NaCl (0, 50, 100 and 200 mM) were given to maize seeds in the presence as well as in the absence of 0.5 mM salicylic acid. Two-week-old maize seedlings exhibited significant decrease in dry weight, root length, shoot length and leaf area on 6 h exposure of 100 and 200 mM NaCl stress. Photosynthetic pigments and NR activity in leaves decreased sharply with increasing stress levels. Both proline content and lipid peroxidation (measured in terms of MDA) levels increased significantly under saline conditions. However, seedlings pretreated with 0.5 mM salicylic acid along with the salinity levels showed enhancement in growth parameters, photosynthetic pigments, NR activity while, free proline and MDA levels decreased. The results showed that salt-induced deleterious effects in maize seedlings were significantly encountered by the pretreatment of salicylic acid. It is concluded that 0.5 mM salicylic acid improves the adaptabilities of maize plants to NaCl stress.     

The <Emphasis Type="Italic">Populus trichocarpa PtHSP17.8</Emphasis> involved in heat and salt stress tolerances

Key message

PtHSP17.8 was regulated by various abiotic stresses. Overexpression of PtHSP17.8 enhanced the tolerance to heat and salt stresses through maintain ROS homeostasis and cooperate with stress-related genes in Arabidopsis.

Abstract

Small heat shock proteins (sHSPs) play important roles in response to diverse biotic and abiotic stresses, especially in heat tolerance. However, limited information is available on the stress tolerance roles of sHSPs in woody species. To explore the function of sHSPs in poplar, we isolated and characterized PtHSP17.8 from Populus trichocarpa. Phylogenetic analysis and subcellular localization revealed that PtHSP17.8 was a cytosolic class I sHSP. The gene expression profile of PtHSP17.8 in various tissues showed that it was significantly accumulated in stem and root, which was consistent with the GUS expression pattern driven by promoter of PtHSP17.8. The expression of PtHSP17.8 could be induced by various abiotic stresses and significantly activated by heat stress. Overexpression of PtHSP17.8 enhanced the tolerance to heat and salt stresses in Arabidopsis. The seedling survival rate, root length, relative water content, antioxidative enzyme activities, proline, and soluble sugar content were increased in transgenic Arabidopsis under heat and salt stresses, but not in normal condition. The co-expression network of PtHSP17.8 were constructed and demonstrated many stress responsive genes included. The stress-related genes in the co-expression network were up-regulated in the PtHSP17.8 overexpression seedlings. These results suggest that PtHSP17.8 confers heat and salt tolerances in plants.

     

<Emphasis Type="Italic">NnHSP17.5</Emphasis>, a cytosolic class II small heat shock protein gene from <Emphasis Type="Italic">Nelumbo nucifera</Emphasis>, contributes to seed germination vigor and seedling thermotolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.     

Small heat shock proteins and adaptation of various Drosophila species to hyperthermia

The dynamics and the level of accumulation of small heat shock proteins (sHSP group 21–27) after a heat exposure were studied in three Drosophila species differing in thermotolerance. The southern species Drosophila virilis, having the highest thermotolerance, surpassed thermosensitive D. lummei and D. melanogaster in the level of sHSPs throughout the temperature range tested. The results suggest an important role of sHSPs in the molecular mechanisms of adaptation to adverse environmental conditions, particularly to hyperthermia.