Sample size (n), mean value (in AU = arbitrary units), standard deviation (SD) and coefficient of variation (CV%) for the relative level of three heat shock proteins, Hsp70, Hsp90a, and Hsp90b, measured during spring and summer in different
As Hochachka and Somero emphasized in their seminal book “Biochemical Adaptation: Mechanism and Process in Physiological Evolution“, the key question to be posed in the study of biochemical adaptation is: “How have living systems, which are based on a common set of biochemical structures and processes and subject to a common set of physical-chemical lows, been able to adapt to the enormously wide spectrum of environmental conditions found in the biosphere?“ (Hochachka & Somero, 2002). Given that the biosphere encompasses habitats with tremendously diverse combinations of physical, chemical, and biotic environmental factors, it seems reasonably to believe that the diversity of life forms that are observable in these habitats is the outcome of
1.1. Conceptual approaches to study adaptation
Evolutionary biologists are well conscious that the term adaptation refers to both a process and its product; however, there still exists the controversy about the perception of that biological phenomenon. Amudson pointed out that “Natural selection, the mechanism universally regarded as the primary causal influence on phenotypic evolutionary change, is first and foremost an explanation of adaptation“(Amudson, 1996). In this context, adaptation is a process, which creates the
Gould & Vrba (1982) called attention to the presence of another two distinct concepts of adaptation, one designated as
Vermeij (1996) advocated a
1.1.1. Biochemical adaptation
Any biological entity that satisfies three necessary conditions for natural selection to operate: has the ability to vary, has continuity (heritability), and differs in its success relative to another co-occurring entity, can be adapted and can produce adaptations (Frank, 1996; Vermeij, 1996). In line with this statement, a frequently addressed question is whether the amazing adaptive diversity discovered in morphology, habitat preferences, mode of life and other attributes of contemporary organisms is comparable with the degree of their adaptive differentiation at the biochemical level. Unfortunately, the literature data indicate that the answer to that question is ambivalent (i.e., a mixture of “yes” and “no”).
Because natural habitats occupied by different life forms exhibit a great diversity in physicochemical parameters, biochemical systems must confront with numerous environmental challenges during the process of adaptation. All of these challenges are focused exclusively to the two most important sources: (i) the essential biochemical constituents of every organism, including nucleic acids, enzymatic and structural proteins, and lipoprotein structures, and (ii) the competence of cells to maintain an adequate level of energy turnover to sustain life. Given that the core biochemical structures (and the interactions among them) are highly susceptible to direct perturbations from external physical and chemical factors, each of them must remain a delicate balance between stability and instability to preserve its functional uniqueness, even in the face of environmental forces that may danger their integrity. However, many environmental factors can damage the cell indirectly by influencing its ability to maintain sufficient rates of energy turnover. In such cases, cells can modulate activities of the existing biochemical systems by redirecting metabolic flux in the manner that compensates for particular environmental insult (Hochachka & Somero, 2002). The unity of cellular biochemical design was found in all organisms and in all environments, presumably as the result of adaptive processes that allow conservation of the core set of structures and processes that form their biochemical architecture. Moreover, there is consensus that genes encoding the core components of cellular function and structure are highly conserved in most species as well, including genes for direct sensing the extracellular environmental changes, and those ones for transdusing these informations to corresponding intraracellular targets. However, the latter kind of genes is fundamental for the evolution of physiological diversity and species specificity. In this context, Hochachka and Somero pointed out “Underlying biochemical unity is preserved at the same time that diversity is generated“(Hochachka & Somero, 2002).
One of the classical examples of biochemical adaptation defined in the restrictive sense of the word (Gould & Vrba, 1982) is molecular chaperones. Molecular chaperones are a highly conserved group of proteins that occur universally in all prokaryotes and eukaryotes (Pearl & Prodromou, 2006). Chaperones assist in various cellular functions, such as
The 70-kDa heat shock proteins (Hsp70s) are a large family of chaperones that plays diverse role in the cell. In addition to the folding of
In addition to Hsp70s, the 90-kDa heat shock proteins (Hsp90s) are also essential components of the chaperone system in all eukarya and eubacteria, with exception of the archaea, were they are no detected (Pearl & Prodromou, 2006; Wegele et al., 2004). In contrast to Hsp70, which is less selective to its substrates, Hsp90 is restricted to the conformational regulation of highly specific cell-cycle- and developmental regulators, including cyclin-dependent kinases, tyrosine kinases, steroid hormone receptors, transcription factors or mitochondrial membrane components (Buchner, 1999; Mc Clellan et al., 2007; Picard, 2006; Rutherford, 2003; Young et al., 2004). It is likely that Hsp90 recognizes its client proteins on the basis of their hydrophobic surface features that are found on nearly mature proteins in normal condition (Richter & Bucher, 2001) or at the initial stage of unfolding on proteins damaged by stress (Freeman & Morimoto, 1996). Although each of the two Hsps is indispensable for viability, they fulfill non-overlapping functions in the cell (Frydman, 2001; Young et al., 2004). Moreover, regardless of an elevated content of both Hsps during environmental stress, there is no data to demonstrate that the Hsp90 chaperone is required for thermotolerance and/or disaggregation of heat-denatured proteins, as was found for Hsp70.
To elucidate the functions of Hsp90 more deeply, Mc Clellan et al. (2007) have applied a genome-wide chemical-genetic screen in
2. Testing hypotheses about local adaptation to environmental conditions
The phrase “
2.1. Reciprocal transplant experiments
Experimental biologists interested in testing hypotheses about local adaptation to native environments have used reciprocal transplant experiments for more than a half-century (see Emms & Arnold, 1997; O'Hara et al., 2004; Schluter, 2000). The method requires reciprocally transplanting two or more populations among their natural habitats in the wild and, within a single generation, comparing the relative performance of a sample of genotypes from the “local“ population and those one from the “foreign“ population(s) under the same environmental conditions,
2.2. Chaperone Hsps as biochemical adaptation
There is growing evidence on adaptive variation in thermal resistance in natural populations, indicating that stressful environmental conditions select for adaptations in natural populations (Hoffmann et al., 2003). For example, in laboratory experiments, very small heat induction of Hsps results in measurable effects on development, life span, fecundity, and stress resistance in plants and animals (Queitsch et al., 2002; Rutherford & Lindquist, 1998). In the field, adaptive changes in the Hsp level over days (Nguyen et al., 1994) and/or over season (Hoffmann & Somero, 1995; Manitašević et al., 2007) appeared to be the ecologically significant for natural populations as well. Hovewer, in contrast to laboratory conditions where the importance of Hsps for survival following heat shock is evident, under field conditions their ecological significance is less explicit and rarely directly explored (Gehring & Wehner, 1995; Manitašević et al., 2007). Recently, the ecological significance of Hsps for adaptation in natural populations have been confirmed using data from latitudinal and climatic clines (Frydenberg et al., 2003; Hemmer-Hansen et al. 2007; Sørensen et al., 2009), which documented that natural selection affects
To this end, the goals of this study were to corroborate the statements: (i) that variation in the endogenous relative level of Hsp70 and Hsp90 chaperones is a biochemical adaptation to fluctuating environmental conditions, and (ii) that the amount of plasticity in the relative level of these two chaperones is protein-specific and dependent on the habitat type that the examined plants were derived from. The model-organism used was
3. Material and methods
3.1. Studied are and species
The Deliblato Sands is an isolated district of sand masses located between the western Carpathian slopes and the Danube River in southern Banat (Serbia). The relief of this area is of eolian origin and therefore has an undulating dune shape. The sand dunes extend in a straight south-east-north-west direction, as does the complete sandy district (44o 47' 39'' N / 21o 20' 00'' E to 45 o 13' 10" N / 28 o 26' 08”E). Surface water streams are completely absent in the Deliblato Sands. The uniqueness of its relief combined with shortage of ground water generated the specific ecological conditions and, as a result, the diversity of habitats and wildlife therein.
3.2. Experimental setup
To detect local adaptation in the level of Hsps in natural populations of
3.2.1. Leaf extracts preparation
We pulverized frozen leaf tissue under liquid nitrogen and resuspended in two volumes (w/v) of extraction buffer (0.1 M Tris, pH 7.6, 1 mM EDTA, 1 mM DTT and 0.1 mM PMSF). After sonification for 3 x 15 s on ice, at 1A and 50/60 Hz, with 30% amplitude (Hielscher Ultrasound Processor), the homogenates were centrifuged twice at 12000g at 4 ºC, for 15 min and the supernatants analyzed for total protein content by the method of Spector (1978) with bovine serum albumin (BSA) used as a standard.
3.2.2. SDS-polyacrilamide gel electrophoresis and Western blot analysis
Tissue extracts, mixed with equal volumes of 2X SDS-sample buffer (0.125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol), were boiled for 5 min at 100oC. Samples containing 40 µg proteins were loaded onto 7.5% SDS-PA gels (Laemmli, 1970) and separated by electrophoresis at 120 V and 4 ºC, using Mini-Protean II Electrophoresis Cell (Bio-Rad Laboratories, Hercules, CA). We applied a Page Ruler Prestained Protein Ladder (Fermentas International Inc., Canada) for precise molecular weight determinations. After electrophoresis, the separated proteins were transferred from the gels to nitrocellulose membranes (Hybond-C, Amersham) and electroblotted overnight at 135 mA and 4°C in 25 mM Tris buffer, pH 8.3, containing 192 mM glycine and 20% (v/v) methanol, using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA). Unbound sites on the membranes we blocked by incubation in PBS (1.5 mM KH2PO4, 6.5 mM Na2HPO4, 2.7 mM KCl, 0.14 M NaCl, pH 7.2) containing 1% nonfat dry milk (GE Healthcare Bio-Sciences) for 1.5 h at room temperature. Proteins of interests were detected using appropriate monoclonal antibodies; Hsp70 by SPA-820 (1:1000; StressGen, Canada) and Hsp90 by SPA-830 (1:2000; StressGen, Canada). After washing with PBS containing 0.1% Tween 20, we incubated the membranes for 1h with alkaline phosphatase-conjugated secondary antibody (1:20000). The immunoreactive bands we visualized and quantified by enhanced chemifluorescence (ECF) detection system using STORM Imager and ImageQuant image analysis software (Amersham Biosciences Limited, UK). To make quantitative comparisons between multiple immunoblots reliable, i.e. to exclude the inter-gel variation, an internal reference sample consisting of a mixture of all samples was run simultaneously on each gel. Prior to any comparison, the intensity of each analyzed immunospecific band we normalized to the intensity of the respective internal reference band on the same blot. The representative immunoblots display the presence of one immunospecific band for Hsp70 and two different bends for Hsp90, in all leaf samples of
4. Statistical analyses
All analyses were conducted using 22
where X1 is the Hsp level of a clone (c) in the open habitat, or in the spring within that (open) habitat, whereas X2 refers to the Hsp level of the same clone in the shaded habitat, or in the summer within that (shaded) habitat. The index of plasticity,
5.1. Habitat and seasonal variation in the mean relative level of Hsp70 and Hsp90 chaperones
In the sun-exposed habitat during both seasons, the mean relative level of all tree foliar chaperones, Hsp70, Hsp90a and Hsp90b, was generally greater in the local (Dune) genotypes relative to their foreign (Woods) counterparts (Table 1).
Considering individual Hsps, in spring, Hsp70 exhibited the lowest (0.63 in the Dune, and 0.29 in the Woods population), while Hsp90a had the highest mean value (0.91 in the Dune, and 0.53 in the Woods population) among all chaperones studied. Yet, when Hsp90a and Hsp90b were compared one to another, the relative level of Hsp90a was greater than that of Hsp90b in both populations of
In the shaded habitat during spring, the mean relative level of all tree foliar chaperones, Hsp70, Hsp90a and Hsp90b, was generally greater in the local (Woods) genotypes as compared to the foreign (Dune) genotypes (Table 1). Again, the mean relative level of Hsp70 chaperone appeared to be the lowest in the Dune genotypes (0.33). In contrast to the open habitat, the average relative level of Hsp90b chaperone in the Woods genotypes was appeared to be the greatest (0.79). In the summer, the foreign (Dune) genotypes produced a higher relative amount of all heat shock proteins (Hsp70, Hsp90a and Hsp90b) than the local (Woods) genotypes.
The three-way ANOVA applied to each of the three Hsps revealed a highly significant main effects of abiotic environmental conditions on all but one of these traits, the mean relative level of Hsp90a between contrasting light habitats (Table 2). These results suggest that
|H x P||1||11.39||0.0011||16.99||0.0001||11.42||0.0011|
|S x P||1||1.01||0.3183||0.74||0.3929||1.15||0.2871|
|H x S x P||2||10.89||0.0001||9.00||0.0003||5.86||0.0042|
5.2. Individual variation (CV %) in the relative level of Hsp70 and Hsp90 between habitats and season
Apart from exhibiting seasonal and habitat-specific differences in the average relative level of Hsp70 and Hsp90, our study provides evidence that the individual variation among genotypes, expressed in term of a coefficient of variation (CV %), also changed over seasons and between the populations from which they originate. In general, Hsp70 expressed the greatest individual variation in both populations across both seasons, with only exception during summer (21.17%). In addition, the lowest level of CV% was observed for Hsp90b during spring (4.46%) in the Dune population, as well (Table 1).
The individual variation (CV%) in the mean relative level of the three analyzed chaperones in the shaded habitat displayed similar trend to that revealed at the open Dune site. However, the percentage of individual variation for each chaperone analyzed appeared to be greater for the local (Woods) genotypes compared to that revealed for the foreign (Dune) genotypes.
Again, in the summer, the Dune genotypes exhibited the lowest individual variation for the mean relative level of Hsp70 (17.93%), as was revealed for the individual variation of the mean relative level of Hsp90b in the Woods genotypes (17.57%) (Table 1).
At the open habitat, the univariate ANOVAs revealed a significant difference in the endogenous level of all Hsps (Hsp70, Hsp90a and Hsp90b) between the Dune and the Woods genotypes in both seasons (all
When the mean relative level of all three Hsps (Hsp70, Hsp90a and Hsp90b) in genotypes stemming from the two populations were compared between alternative light habitats over seasons, a univariate ANOVA revealed that the Dune genotypes expressed significantly different level of all these Hsps in both season (all
We presented the reaction norm plots to habitat type for the level of three Hsps (Hsp 70, Hsp90a and Hsp90b) in leaves of
|Population Dune||Population Woods|
|Population Dune||Population Woods|
The plots of reaction norms over seasons are shown for the level of three Hsps (Hsp 70, Hsp90a and Hsp90b) in leaves of
The mean reaction norms were steep for all Hsps measured (Figs. 2 and 3), suggesting a general ability of
The plasticity means between seasons or habitats (average
A Wilcoxon 2-sample test revealed that the amount of plasticity for identical
|Spring ||Open habitat |
|Trait (n = 11)||Open habitat||Shaded habitat||Spring||Summer|
During the course of evolution, plants have evolved a variety of different biochemical mechanisms for preventing fitness reduction under adverse environmental conditions (Bazzaz, 1996; Lambers et al., 2008; Tucić et al., 2009; Vuleta et al., 2010). One of such mechanisms is molecular chaperones – a group of proteins that respond to sudden increase in temperature or exposure to other environmental stresses (Feder & Hofmann, 1999; Salathia & Queitsch 2007; Sangster et al., 2004; Sørensen et al., 2003). Molecular chaperones, particularly Hsp90, also restrict stochastic phenomena within cells, by minimizing developmental perturbations, thereby canalizing the organism’s development (Samakovli et al., 2007). Results presented in this study provide evidence that in
6.1. The role of Hsp70 chaperones for adaptation
It is well known that molecular chaperones play a crucial role at two stages in the life of a protein: throughout
Because the essential role of Hsp70 in stressful environments is to prevent aggregation, and to facilitate refolding and/or proteolytic degradation of nascent proteins (Wang et al., 2004), the elevated level of Hsp70 in sun-exposed plants, especially during summer, might be viewed as a kind of “anticipatory” phenotypic plasticity to increasing chances of heat stress in that habitat. In the forest understory, however, where thermal fluctuations are fewer and less frequent, a lower relative level of Hsp70 are sufficient to maintain native protein structure and occasional refolding of damaged proteins (Manitašević et al., 2007). Given that increased level of Hsp70 chaperone correlates well with greater thermotolerance in many plant species and that heat stress jointly occurs with high irradiance, the
6.2. The role of Hsp90 chaperones for adaptation
Contrary to Hsp70 proteins, which achieved their maximal relative level in the summer, the average level of both Hsp90 isoforms (inducible-Hsp90a and constitutive-Hsp90b) were highly suppressed in the summer compared to their spring counterpart, especially Hsp90b isoform (Table 1; Fig. 3). In the open habitat, genotypes from both populations differed significantly in the average level of the two Hsp90 isoforms (Fig. 3A and 3B). Conversely, in the shaded habitat, their relative level was similar between local and foreign genotypes in both seasons, with only exception of Hsp90b isoforms in the summer, which relative amount appeared to be greater in the Dune than in the Woods population (0.54
In the eukaryotic cytosol, Hsp70 and Hsp90 chaperones are each essential for cell viability under all growth conditions, implying that they fulfill non-overlapping function (Frydman, 2001; Young et al., 2004). Hsp90s are highly conserved group of molecular chaperones, which constitute about 1-2% of all cytosolic proteins in most cells under non-stress conditions (Parsell & Lindquist, 1993). Hsp90 is not a chaperone for newly synthesized proteins, but, instead, its cellular function is restricted to the conformational regulation of the limited group of substrates or “clients” (Mc Clellan et al., 2007). In higher eukaryotes, Hsp90 work together with a large set of co-chaperones to mediate the conformational regulation of tyrosine kinases and steroid hormone receptors (Picard, 2006), but also to prevent phenotypic variation of these signaling molecules in the face of gene mutation (Sangster et al., 2004). The current understanding of Hsp90 function in tyrosine kinase and steroid hormone receptors maturation suggests that Hsp90 binds to “clients” that are substantially folded, facilitating their conformational remodeling.
Recently, Mc Clellan et al. (2007) have used a genome-wide chemical-genetic screen combined with bioinformatic analyses to elucidate more deeply the Hsp90 functions. They identified several
Our study provides evidence that at the sun-exposed habitat the relative level of inducible and constitutive Hsp90 isoforms of Hsp90 chaperone was lower in the summer-collected leaf samples from both local and foreign
Apart from producing environmentally contingent differences between populations in the mean value of a trait, natural selection can influence the trait plasticity within the same habitat as well. We quantified the degree of plasticity in the level of Hsps in
There is a consensus among evolutionary biologists that the phenomenon of adaptation has dual meaning: as a process and as a product of that process, which mechanism is natural selection. In this context, any biological entity that satisfies three necessary conditions for natural selection to operate: has the ability to vary, has continuity (heritability), and differs in its success relative to another co-occurring entity, can be adapted and can produce adaptations. When defined in a restrictive sense of the word, the term “adaptation” refers to a “trait (i) that enhances the fitness of an organism, and (ii) whose current beneficial characteristics reflect the selective advantage of the trait at its time of origin” (Hochachka & Somero, 2002). Molecular chaperones are a highly conserved set of functionally defined proteins that are involved in the folding and degradation of stress-demaged proteins. Because the role they play in all contemporary organisms is the benefit, which is very likely to be the same as the benefit that initially favoured the evolutionary development of these proteins, molecular chaperones are viewed as “adaptations”. Among the molecular chaperones, the heat shock proteins Hsp70s are involved in the folding of newly translated and stress-denatured proteins. In addition to stress-inducible chaperone networks, eukaryotes contain a stress-repressed chaperone network that is dedicated to protein biogenesis. Although the Hsp90 molecular chaperones are highly abundant under normal conditions, they are restricted on a limited set of nearly mature, but inherently unstable, signaling proteins. Thus, under normal conditions, Hsp90 plays a key role in various aspects of the secretory pathway and cellular transport, while during environmental stress, Hsp90 is necessary for the cell cycle, meiosis and cytokinesis. In this study local adaptations for the relative level of three heat shock proteins, Hsp70, Hsp90a and Hsp90b in leaves of
The authors are grateful to Nikola Tucić for critical reading of the paper. This research was supported by a grant (No. 173007) to B. T. from the Ministry of Education and Science of the Republic of Serbia.