As the climate warms, mechanisms of thermal tolerance will become increasingly important for both crop and wild plants. Lobel & Asner (2003)
demonstrate a 17% yield decline for each 1 °C growing season temperature increase for both corn and soybean, compared to historical long-term averages in North America. In natural populations, models of habitat shifts expected to accompany temperature increases suggest that the rate of movement of suitable habitat can exceed the maximum dispersal rate of many species (Malcolm et al. 2002
, but see also Pearson & Dawson 2003
). For many species of plants, adapting to higher temperatures rather than moving with shifting climate zones may be the only alternative to extirpation.
Heat shock proteins (HSPs), so named because their transcription is up-regulated upon exposure to elevated temperatures, are prime candidates for targets of selection as plants experience increasingly frequent episodes of stress-inducing high temperatures. In this study, we examine genetic variation in HSP expression, its fitness consequences and its pleiotropic effects in Arabidopsis thaliana
, a species that grows in environments that are thermally moderate during its growing season (Hoffmann 2002
). Our investigation of the functional significance of HSPs and the evolvability of HSP expression in this model organism is intended to provide insight for both crop breeders and those interested in the evolutionary ecology of natural populations in changing environments.
In response to high temperature, plants rapidly produce proteins belonging to five HSP families: Hsp100/ClpB (eukaryotic/Escherichia coli nomenclature), Hsp90, Hsp70/DnaK, Hsp60/GroEL and small HSPs. HSPs are proposed to act primarily as molecular chaperones, refolding misfolded proteins and/or preventing accumulation and/or aggregation of nonfunctional proteins in stressed cells.
Despite their up-regulation in response to elevated temperature, no direct role in survival at high temperature has been demonstrated for most HSPs. This may result in part from genetic redundancy and consequent limits on the utility of reverse genetics for elucidating gene function. The apparently essential nature of HSP function even in nonstressful environments can further complicate the description of function, since complete gene knockouts may be lethal. For three of the four large HSP families, this is the case. Fourteen Hsp70 genes have been identified in A. thaliana
. It has proven difficult to alter expression levels and maintain viability (see Larkindale et al. 2005
). Hsp90, found in four cytosolic copies, is up-regulated in response to heat, but no direct role in acquired thermal tolerance has been demonstrated (Larkindale et al. 2005
). A null mutant of the chloroplast Hsp60β does show increased heat sensitivity, although expression does not appear to be heat induced (Ishikawa et al. 2003
Here, we focus on the best-studied HSP in plants, AtHsp101 (ClpB1, At1g73410; hereafter Hsp101). This HSP, a member of the Hsp100/ClpB family of proteins, is thought to be involved in disaggregation of misfolded proteins and their hand-off for either refolding by Hsp90 or Hsp70, or degradation (Wang et al. 2004
). Three additional ClpB proteins have been identified in A. thaliana
: ClpB2 – ClpB4. ClpB2 has recently been re-annotated as encoding a 68.8-kDa protein. There is no evidence of expression of a ClpB-sized mRNA or protein from this locus (Lee et al. 2007
). ClpB3 is localized in chloroplasts, while ClpB4 is associated with mitochondria (Rottgers et al. 2002
; Lee et al. 2007
). Thus Hsp101/ClpB1 is the only cytosolic ClpB in Arabidopsis
. ClpB3 and ClpB4 apparently diverged from Hsp101 early in the evolution of life, since their gene sequences are derived from cyanobacterial ClpBs in an ancient split from the lineage leading to Arabidopsis
Hsp101, and there appears to have been substantial divergence in function as well (Lee et al. 2007
). Indeed, genetic analysis has shown that Hsp101 is absolutely essential for thermal tolerance in both Arabidopsis
and maize (Hong & Vierling 2000
; Queitsch et al. 2000
; Nieto-Sotelo et al. 2002
), while ClpB3 and ClpB4 null mutants are indistinguishable from wild type in their ability to acquire thermal tolerance.
Under optimal growth conditions, Hsp101 is present in vegetative tissues in such low-copy number that it is difficult to detect by standard biochemical technique. However, increased Hsp101 transcript and protein production begins essentially instantaneously on exposure of plants to temperatures 5–10 °C above optimal growth temperature and increases dramatically in heat-stressed plants (30 °C and above in Arabidopsis
) (Hong et al. 2001
; Young et al. 2001
). Expression levels increase with the degree of thermal stress until lethal temperatures are reached (Chen et al. 1990
). Hsp101 protein expression therefore acts as a virtual thermometer. Hong et al. (2001)
show that a Hsp101 null mutant lacks thermal tolerance, but is visually normal under standard (non-heat-stressed) growth conditions. Quantitative analysis of growth and reproductive output were not performed.
Together, the rapid response of Hsp101 to temperature changes and its absolute requirement for survival in extreme heat stress suggest that Hsp101 will be an essential component of plant adaptation to thermal stress in future climates. Across all population studies of HSPs in animals, the evidence suggests that the HSP expression response to thermal stress evolves in natural populations (Krebs & Loeschke 1995
; Otsuka et al. 1997
; McColl & McKechnie 1999
; Bettencourt et al. 2002
; Frydenberg et al. 2003
). However, the evolutionary responses in animals are not simple and suggest that there are both costs and benefits to HSP expression. These include effects on longevity and fecundity that result in the evolution of reduced HSP induction in populations that experience chronic thermal stress (reviewed in Sørensen et al. 2003
). We know almost nothing about HSP adaptation in plants.
Whether or not Hsp101 contributes to an adaptive response to increased thermal stress clearly depends on how Hsp101 expression influences fitness. In addition, we need to know that Hsp101 expression is evolvable. Evolvability has been defined as the ability to acquire and maintain new, potentially adaptive phenotypes through genetic change (after Wagner 2005
and Hansen 2006
). The conventional approach to assessing evolvability has been to measure heritability within populations, defined as the ratio of additive genetic variance to total genetic variance (Fisher 1930
). However, heritability measures often depend strongly on environmental conditions, and response to selection depends on an interaction between the complexities of genetic effects (additive vs. interactive) and mating structure (Whitlock et al. 1995
). In addition, genetic correlations between traits, caused by pleiotropy and linkage, can constrain or eliminate adaptive response despite high heritabilities (Roff 1997
), and their values can change nonproportionally with selection (Roff & Mousseau 1999
). Finally, the standing genetic variation underlying heritabilities may not be qualitatively similar to the variation that underlies adaptive evolution, consisting hypothetically of mildly deleterious mutations in mutation–selection balance that contribute little to adaptive evolution (Barton & Keightly 2002
). Indeed, Nordborg et al. (2005)
showed that much of the observed DNA sequence polymorphism in A. thaliana
is best explained in this way. Therefore, we suggest assessing evolvability by examining extant genetic variation among
populations. This approach is of particular value when a cline or contrast in environmental factors is of interest as an agent of natural selection across the geographical areas sampled. Used in this way, variation among populations quantifies a sort of realized evolvability. Furthermore, it tells us how much the evolvability, together with selection and drift, has been capable of producing genetic differentiation among populations, taking into account both the availability of mutationally derived trait variation and any negative pleiotropic constraints on its evolution. In this study, we assess evolvability of Hsp101 expression by comparing a set of wild-collected genotypes of A. thaliana
from contrasting portions of the species’ range.
In plants, despite considerable progress at the biochemical and genetic levels, we know little about naturally occurring variation in HSP or any resulting phenotypic or fitness effects in any species. Barua & Heckathorn (2006)
demonstrate an interaction between light and temperature in field Hsp70 induction responses in Solidago altissima
(Asteraceae). Manitasevic et al. (2007)
quantify Hsp70 and Hsp90 expression variation in response to season and habitat in Iris pumila
(Iridaceae). Knight & Ackerly (2003)
provide the only published support that HSP expression evolves in natural plant populations, showing differential responses of coastal and desert congeneric species in small HSP expression in field and controlled environments. Queitsch et al. (2002)
used pharmacological suppression of Hsp90 function to uncover developmental abnormalities in A. thaliana
. They suggest that dysfunction in Hsp90 expression can uncover hidden genetic variation on which selection might act in periods of high stress, hypothesizing that HSPs may serve as ‘evolutionary capacitors’. Despite the broad interest generated by this hypothesis, there is little information on among-population, within-species variation in HSP expression in plants.
Functionally, our understanding of the integrated phenotypic effects of HSP and their evolvability is limited. Functional genomic studies strongly suggest that pleiotropy links multiple plant functions, sometimes in surprising ways. That HSPs seem to be associated with stress responses in general provides a strong argument that we should expect pleiotropic effects for genes associated with HSP expression and function, since they are likely to influence multiple plant pathways and traits. Pleiotropic effects can include trade-offs and hence represent constraints on adaptive responses (Waxman & Peck 1998
; Tonsor et al. 2005
). In animal systems, chronic exposure to high temperature has been associated with reduced high temperature HSP response, implying a cost to chronic HSP expression (Bettancourt et al. 1999
). Functional studies comparing HSP-functional wild types with HSP-knockout mutants can provide a direct estimate of the fitness effects of HSP functionality and benefit/cost. When such studies also measure multiple traits known to be of ecological importance, key pleiotropic effects can be described. Comparative studies of naturally occurring variation in HSP expression and correlated variation in ecologically important traits can elucidate the extent to which multiple plant functions covary and have co-evolved with HSP expression. The results can guide strategies for plant improvement, and provide increased understanding of the mechanistic basis of plant metabolism and development in a thermally varying world.
In this study, we addressed four hypotheses aimed at a broad characterization of evolvability in Hsp101 expression in the model plant A. thaliana. The hypotheses:
|H1||Populations of diverse geographical origin vary in Hsp101 expression. We grew 10 genotypes from the ‘ecotypes’ collection at www.arabidopsis.org and quantified Hsp101 expression in each ecotype across a gradient of Hsp101 induction temperatures. We further characterized the geographical pattern by regressing Hsp101 expression on latitude of origin.|
|H2||Hsp101 expression affects fitness. We first test for the effect of complete lack of Hsp101 expression on lifetime fecundity by comparing standard laboratory genotypes to their Hsp101 null mutants. We then test how the differences in quantity of Hsp101 expressed by six genotypes influences fecundity.|
|H3||The fitness effects of Hsp101 depend on genetic background. We addressed this hypothesis by assessing the effects of the Hsp101 null mutation on fecundity in two different genetic backgrounds.|
|H4||Hsp101 expression has pleiotropic effects on multiple plant traits. We examined the effect of varying Hsp101 expression on a suite of 12 plant traits, including fecundity. For this hypothesis, we measured all traits on the standard laboratory genotypes used in hypotheses 2 and 3.|