Jay Taylor
Small Heat Shock Proteins
It has long been known that certain plants acquire thermotolerance if exposed to a gradual increase in temperature, or a brief thermal shock, prior to a long incubation at high temperature. Exposure to high temperatures normally result in a reduced growth rate through the differential denaturation of enzymes with different heat sensitivities. At extremely high temperatures denatured enzymes may form insoluble aggregates, via interaction between the exposed hydrophobic regions, hampering cell recovery to thermal shock (Martin and Hartl 1997). Typical thermal acclimation involves the inhibition of normal protein synthesis, while the cellular machinery increases the expression of molecular chaperones and foldases, which may or may not be present before the stress conditions (Fig 1). These chaperones are believed prevent the formation of irreversible protein aggregates and may mediate protein refolding via the controlled binding and release of misfolded protein intermediates.
Molecular chaperones are molecules that bind to protein folding intermediates blocking off-pathway folding and mediate the correct folding of proteins into their active states. Protein foldases are molecules that include the cis-trans proline isomerases (PPIases) and the protein disulfide isomerases (PDIases). PPIases have been shown biochemically to catalyze the cis-trans isomeration around the N-terminal of proline in a polypeptide, which is the rate limiting step in many protein folding pathways. While PDIases enzymatically catalyze the hydration/condensation of disulfide bonds. Numerous good articles have been written discussing the biochemical activity of foldases (Fisher 1994), therefore they will not be discussed here in detail.
Tolerance and acclimation to thermal stress are of particular importance in plants, which are sessile and only able to control temperature via transpiration, which may be water limited. The expression of heat shock proteins in plants show both developmental and tissue specificity, with expression elevated at life stages and tissues particularly prone to thermal stress. Unlike animals which usually have only 2-4 types of smHSPs plants tend to have from 20-40 smHSPs, which are encoded by the nucleus and later associate with specific cellular compartments (Yeh et al. 1994). Studies of plant smHSP gene families show that they exhibit differential rates of evolution, and therefore different selective pressures, suggesting that they have diversified in function (Waters 1995). The acquisition of thermotolerance is important in the evolution of plants, for they are subject to a diurnal cycle with brief periods of high temperatures midday. Furthermore, thermal tolerance in crop plants may become increasingly important with the possible elevation of mean global temperatures, due to increased levels of greenhouse gasses. The purpose of this review is to study the biochemical and molecular evidence supporting the biological role of HSPs in plant thermotolerance, with emphasis on HSP expression and the plant smHSPs.
Correlation between HSP production and thermal tolerance
Plant in the field exhibit a wide range of thermal tolerances that directly reflect the selective pressures of the environment from which they evolved. This would automatically suggest that there are heritable adaptations related to thermal tolerance and heat acclimation. It has been shown with thermotolerant tropical cereals that abrupt changes in temperature are correlated with a shift from normal protein synthesis to the production of large and small HSPs (Howarth 1991). It has also been shown that Glycine max seedling incubated at 28o exposed to a brief 10 minute heat shock of 45oC produced HSPs, and could survive an otherwise lethal long term exposure to 45oC (Yeh et al. 1994). Furthermore the presence of heat shock mRNAs have been shown to be elevated during times of heat stress and recovery (Howarth1991). These facts would suggest that the ability of a plant to survive near lethal heat exposures is directly related to the presence or absence of heat shock proteins. In addition to these studies, mutant plants that produce partially or totally dysfunctional HSPs have been found, and prove to lack thermal tolerance (Downs et al. 1998)
Genetics provides further proof of HSP function
The transcription of HSPs is regulated by a series of proteins known as heat shock factors (HSFs). HSFs contain a conserved helix-turn-helix DNA binding motif at the N terminus and a leucine-zipper like motif at the C-terminus (Lyke et al. 1997). Band shift assays coupled with site specific mutagenesis of HSFs have shown that the conserved N-terminus domain is responsible for DNA binding, while deletional analysis coupled immunoflourescent antibody staining has shown that the C-terminus domain is necessary for nuclear import (Lyke et al. 1997).
To further prove the function of heat shock proteins Prandl et al. (1995) studied the developmental and tissue specific regulation of HSPs in vivo. Heat shock inducible Gus reporter genes (hsGus-17.3) were synthesized by taking the promoter of the Glycine max 17.3 HSP (Gmhsp17.3) and fusing it to the Gus gene, the chimeric gene was then inserted into Nicotiana tobaccum. Gus staining with X-gluc (5-bromo-4-chloro-3-indonyl-(-D-glucuronide) showed that the hsGus-17.3 construct was constitutionally expressed in tobacco seeds and seedlings(Prandl et al. 1995). Constitutional expression of HSP genes in seed and seedlings further supports a role of HSPs in thermal tolerance, because seeds and seedlings are highly susceptible to thermal stress in hot soil. Seedlings also lack the transpiration surface area utilized by plants as a cooling mechanism. Experiments using Agrobacterium transformed Arabidopsis with the same gene construct failed to further support these findings however. Possible problems leading to this discrepancy, could be an inability of Arabidopsis HSFs to recognize the soy promoter or result of post-transcriptional modifications.
In situ hybridization of RNA to mRNA from Arabidopsis coding for a 17.6 kDa HSP showed that HSP expression was localized in the meristematic and provascular tissues of embryos, after a mild heat shock (Prandl et al. 1995). Hydration of seeds upon germination and the rapid growth of seedlings is necessary for plant establishment, it is therefore imperative that plants defend meristematic and provascular tissues from the deleterious effects of thermal stress. All of this data suggests a correlation between HSP production and thermotolerance, but does not provide any clue about the mechanism of HSP activity.
In vitro analysis of the smHSPs
Probably the best information about plant responses to thermal stress comes from biochemical studies of smHSP activity. The small HSPs are a class of proteins with a molecular weight of ~15-30 kDa and a conserve C-terminus domain similar to the (-crystalline domain of the vertebrate eye lens (Waters et al. 1995). HPLC studies of the smHSPs show that the tend to exist as multimeric structures ranging from 200-400 kDa in size. In thermal stress conditions plants produce more smHSPs than any other variety of protein.
In one study of smHSP function Lee et al. (1995) took purified Pisum savitum cytosolic HSP18.1 and HSP17.7 added seperately added each to heat denatured cytrate synthetase (CS) and lactate dehydrogenase (LD) at 38oC. Both heat shock proteins were subsequently found to prevent aggregation and irreversible inactivation of CS and LD in an ATP independant fashion. Chaperone activity was only exhibited when HSP18.1 & HSP17.7 was added to the in vitro CS and LD during the 38oC incubation, not after, suggesting that the smHSPs can only bind and prevent misfolding and aggregation but cannot themselves mediate protein refolding. When HSP18.1 and HSP17.7 were added to CS denatured at 45oC the smHSPs failed to prevent CS reactivation in vitro. Irreversibly inactivated CS was however prevented from aggregating, suggesting that the smHSPs maintain the solubility of irreversibly misfolded polypeptides (Lee et al. 1995). All of this data suggests a mechanism where smHSPs bind to misfolded protein intermediates to prevent there aggregation during stressed conditions. Once favorable conditions are again prevalent smHSPs may then release misfolded polypeptides for refolding by the larger ATP-dependant HSPs. Irreversibly folded polypeptides are maintained in soluble form and targeted for proteolytic degradation (Fig II).
Studies of the animal truncated C-terminus of HSP16-2 a cytosolic smHSP from C. elegans further support this hypothesis. Leroux et. al. (1997) found that the truncated C-terminus of HSP16-2 prevented the thermally induced aggregation of CS in animals. Interestingly in vitro immunoflourescent staining studies showed that HSP16-2 bound stringently to heat denatured actin and tubulin early in the aggregation pathway, but did not bind to normal cytoskeletal components (Leroux et al 1997). Molecular evidence supporting a role for cytosolic smHSPs in protecting heat denatured cytoskeletal elements have not been found in plants however.
smHSPs may be diversified in plants
The smHSPs of plants unlike those of other kingdoms show great diversity in sequence and cellular localization. Plant smHSP exist in six nuclear encoded gene families each of which is targeted to a particular cellular compartment. Of the smHSP families two are cytosolic, one mitochondrial, one is localized in the chloroplast, one is retained in the ER (Waters et al. 1995). The location of the sixth HSP family has not yet been discovered. All animal smHSPs are cytosolic and differential targeting of plant smHSPs may be proof of functional divergence (Table I).
Comparison of animal and plant smHSP DNA and amino acid sequences suggest that the plant smHSPs diversified after animal and plant lines diverged (Waters 1995). Computerized models comparing the amino acid sequences of smHSP families from various organisms suggests that gene duplication, gene conversion, and sequence divergence all contributed to the evolutionary diversification of plant smHSPs (Waters 1995). More intricate comparisons using computers suggest that the different plant smHSP families are evolving at different rates, showing that smHSP families are subject to differential selection, providing further evidence of functional divergence.
Plant smHSPs exhibit functional divergence
If the plant smHSPs are subject to different selective constraints, as both cellular localization and sequence analysis suggest, then plant smHSPs should exhibit different functions. There is now increasing evidence that this is the case. It has been shown that that if plant cells are incubated at 45oC the plasma membrane becomes increasingly permeable to ions. Cells pre-acclimated by a brief heat shock at 45oC, followed by incubation at 28oC, do not increase in ion permeability when returned to 45oC (Yeh et al.1994). Subsequent labeling with antibodies raised against conserved regions of plant smHSPs, showed that a 15 kDa HSP was associated with PM proteins on the thermally stabile plasma membrane, providing evidence of a possible role in the stabilizing ion channels.
Some cytosolic smHSPs are able to produce large insoluble multimers larger than one MDa, commonly called heat shock granules (HSGs). Cofractionation experiments have shown that HSGs separate in fractions containing mRNA, leading to models where plant smHSPs protect cellular mRNA that is unable to be transcribed during heat stress (Waters et al 1995).
The most impressive proof of the divergent functions plant smHSPs comes from recent studies of the methionine rich chloroplast smHSPs. In addition to the conserved C-terminus heat shock and (-crystalline domains of the other smHSPs, the chlpHSPs contain a highly conserved methionine rich domain. Antibodies against this methionine rich domain has been shown to bind to chlpHSPs in C3, C4, CAM, monocots, dicots, members of Psilophyta, Equisetophyta, Polypodiophyta, and Ginkophyta (Downs et al 1998), proving that the methionine rich chlpHSPs are widely conserved within terrestrial plant. EM immunogold labeling of chlpHSPs has shown that they are associated with the thykaloid membrane during heat stress (Heckathorn et al. 1998). In an elegant experiment, Heckathorn et al. measured the O2 exchange of thykaloid vesicles associated with chlpHSPs, in an in vitro assay of electron transport at 47 oC. Disruption of the chlpHSPs with antibodies produced against the methionine rich (Abmet) or the (-crystalline domain (Abo) resulted in the loss of PSII e- transport in heat stressed vesicles. This in vitro assay provides the most specific example of smHSP function to date, the protection of PSII O2 evolving complex, which is often the most heat labile complex in the chloroplast e- transport chain. The ability to photosynthesize is fundamental for plant survival, and it is not surprising that plants would have evolved specific proteins that bind and protect heat sensitive complex involved in harvesting light energy.
Model systems utilized in the study of HSPs
Models systems for studying heat shock proteins include Drosophila, E. coli, and S. cerevisiae, are all well defined model systems in molecular biology. No definitive system has yet to be chosen in the study of plant smHSPs. A current search of the literature produces papers utilizing a countless number of systems to look at smHSP activity, with emphasis on soy and tobacco. The bulk of the research in plant large and small HSPs is currently being pursued in various crop plants. It is easier find grants supporting crop research, and the ultimate goal of HSP research is the production of thermal tolerant crop plants that can survive "greenhouse" induced temperature rises. There is also a small amount of work being done with tropical and arid plants, which was initiated with the idea that these plants may be the best thermally adapted plant species.
Conclusions
Evidence supporting the role of heat shock proteins in plant acclimation to thermal stress is mounting. Furthermore biochemical and evolutionary evidence suggest that the smHSPs in plants have developed distinct functions. It is necessary that further studies be done to prove the function of both large and smHSPs in vivo. Most current biochemical knowledge about the function of smHSPs and HSPs in plant and animal systems comes from in vitro biochemical analysis, and may not necessarily function that way in a fully regulated cell. Furthermore there are very little field studies examining the HSP activities and the thermal stress actually experienced by plants in the wild.
The end goal of HSP studies in plants is the bioengineering of heat tolerant plants via transformation. Transgenic tobacco plants have already been engineered to contain a soy HSP hs6831 flanked by a CaMV-35s promoter. Constitutive expression of hs6831 produced large quantities of the 17.6 kDa HSP product. Future experiments of this type should be done, coupled with assays for thermoprotection. EM coupled with immunogold labeling is a powerful too for studying protein localization in fixed cells, and protein aggregates are easily viewed via electron microscopy (Fig IV). Cytological studies of HSP localization of HSPs in respect to protein aggregates could also be a powerful tool for elucidating the in vivo function of plant HSPs. Although the evidence for large and small HSP activity in cells is convincing, it is imperative that more in vivo evidence be elucidated in the near future, and that different HSPs functions studied to allow for the specific selection of those HSPs best suited for plant transformation experiments.
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