Elizabeth R. Waters

The evolution, function, structure, and expression of the plant sHSPs

Small heat shock proteins are a diverse, ancient, and important family of proteins. All organisms possess small heat shock proteins (sHSPs), indicating that these proteins evolved very early in the history of life prior to the divergence of the three domains of life (Archaea, Bacteria, and Eukarya). Comparing the structures of sHSPs from diverse organisms across these three domains reveals that despite considerable amino acid divergence, many structural features are conserved. Comparisons of the sHSPs from diverse organisms reveal conserved structural features including an oligomeric form with a β-sandwich that forms a hollow ball. This conservation occurs despite significant divergence in primary sequences. It is well established that sHSPs are molecular chaperones that prevent misfolding and irreversible aggregation of their client proteins. Most notably, the sHSPs are extremely diverse and variable in plants. Some plants have >30 individual sHSPs. Land plants, unlike other groups, possess distinct sHSP subfamilies. Most are highly up-regulated in response to heat and other stressors. Others are selectively expressed in seeds and pollen, and a few are constitutively expressed. As a family, sHSPs have a clear role in thermotolerance, but attributing specific effects to individual proteins has proved challenging. Considerable progress has been made during the last 15 years in understanding the sHSPs. However, answers to many important questions remain elusive, suggesting that the next 15 years will be at least equally rewarding.

Comparative analysis of the small heat shock proteins in three angiosperm genomes identifies new subfamilies and reveals diverse evolutionary patterns

The small heat shock proteins (sHSPs) are a diverse family of molecular chaperones. It is well established that these proteins are crucial components of the plant heat shock response. They also have important roles in other stress responses and in normal development. We have conducted a comparative sequence analysis of the sHSPs in three complete angiosperms genomes: Arabidopsis thaliana, Populus trichocarpa, and Oryza sativa. Our phylogenetic analysis has identified four additional plant sHSP subfamilies and thus has increased the number of plant sHSP subfamilies from 7 to 11. We have also identified a number of novel sHSP genes in each genome that lack close homologs in other genomes. Using publicly available gene expression data and predicted secondary structures, we have determined that the sHSPs in plants are far more diverse in sequence, expression profile, and in structure than had been previously known. Some of the newly identified subfamilies are not stress regulated, may not posses the highly conserved large oligomer structure, and may not even function as molecular chaperones. We found no consistent evolutionary patterns across the three species studied. For example, gene conversion was found among the sHSPs in O. sativa but not in A. thaliana or P. trichocarpa. Among the three species, P. trichocarpa had the most sHSPs. This was due to an expansion of the cytosolic I sHSPs that was not seen in the other two species. Our analysis indicates that the sHSPs are a dynamic protein family in angiosperms with unexpected levels of diversity.

Comparative genomic analysis of the Hsp70s from five diverse photosynthetic eukaryotes

Abstract We have identified 24 members of the DnaK subfamily of heat shock 70 proteins (Hsp70s) in the complete genomes of 5 diverse photosynthetic eukaryotes. The Hsp70s are a ubiquitous protein family that is highly conserved across all domains of life. Eukaryotic Hsp70s are found in a number of subcellular compartments in the cell: cytoplasm, mitochondrion (MT), chloroplast (CP), and endoplasmic reticulum (ER). Although the Hsp70s have been the subject of intense study in model organisms, very little is known of the Hsp70s from early diverging photosynthetic lineages. The sequencing of the complete genomes of Thalassiosira pseudonana (a diatom), Cyanidioschyzon merolae (a red alga), and 3 green algae (Chlamydomonas reinhardtii, Ostreococcus lucimarinus, Ostreococcus tauri) allow us to conduct comparative genomics of the Hsp70s present in these diverse photosynthetic eukaryotes. We have found that the distinct lineages of Hsp70s (MT, CP, ER, and cytoplasmic) each have different evolutionary histories. In general, evolutionary patterns of the mitochondrial and endoplasmic reticulum Hsp70s are relatively stable even among very distantly related organisms. This is not true of the chloroplast Hsp70s and we discuss the distinct evolutionary patterns between “green” and “red” plastids. Finally, we find that, in contrast to the angiosperms Arabidopsis thaliana and Oryza sativa that have numerous cytoplasmic Hsp70, the 5 algal species have only 1 cytoplasmic Hsp70 each. The evolutionary and functional implications of these differences are discussed.

Evolutionary analysis of the small heat shock proteins in five complete algal genomes.

Small heat shock proteins (sHSPs) are chaperones that are crucial in the heat shock response but also have important nonstress roles within the cell. sHSPs are found in all three domains of life (Bacteria, Archaea, and Eukarya). These proteins are particularly diverse within land plants and the evolutionary origin of the land plant sHSP families is still an open question. Here we describe the identification of 17 small sHSPs from the complete genome sequences of five diverse algae: Chlamydomonas reinhardtii, Cyanidioschyzon merolae, Ostreococcus lucimarinus, Ostreococcus tauri, and Thalassiosira pseudonana. Our analysis indicates that the number and diversity of algal sHSPs are not correlated with adaptation to extreme conditions. While all of the algal sHSPs identified are members of this large and important superfamily, none of these sHSPs are members of the diverse land plant sHSP families. The evolutionary relationships among the algal sHSPs and homologues from bacteria and other eukaryotes are consistent with the hypothesis that the land plant chloroplast and mitochondrion sHSPs did not originate from the endosymbionts of the chloroplast and mitochondria. In addition the evolutionary history of the sHSPs is very different from that of the HSP70s. Finally, our analysis of the algal sHSPs sequences in light of the known sHSP crystal structures and functional data suggests that the sHSPs possess considerable structural and functional diversity.

Lowly Expressed Genes in Arabidopsis thaliana Bear the Signature of Possible Pseudogenization by Promoter Degradation

Pseudogenes are defined as nonfunctional DNA sequences with homology to functional protein-coding genes, and they typically contain nonfunctional mutations within the presumptive coding region. In theory, pseudogenes can also be caused by mutations in upstream regulatory regions, appearing as open reading frames with attenuated expression. In this study, we identified 1,939 annotated protein-coding genes with little evidence of expression in Arabidopsis thaliana and characterized their molecular evolutionary characteristics. On average, this set of genes was shorter than expressed genes and evolved with a 2-fold higher rate of nonsynonymous substitutions. The divergence of upstream sequences, based on ortholog comparisons to A. lyrata, was also higher than expressed genes, suggesting that these lowly expressed genes could be examples of pseudogenization by promoter disablement, often due to transposable element insertion. We complemented our empirical study by extending the models of Force et al. (Force A, Lynch M, Pickett FB, Amores A, Yan YL, Postlethwait J. 1999. Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151:1531-1545.) to derive the probability of promoter disablements after gene duplication.

An Unusual Dimeric Small Heat Shock Protein Provides Insight into the Mechanism of This Class of Chaperones

Small heat shock proteins (sHSPs) are virtually ubiquitous stress proteins that are also found in many normal tissues and accumulate in diseases of protein folding. They generally act as ATP-independent chaperones to bind and stabilize denaturing proteins that can be later reactivated by ATP-dependent Hsp70/DnaK, but the mechanism of substrate capture by sHSPs remains poorly understood. A majority of sHSPs form large oligomers, a property that has been linked to their effective chaperone action. We describe AtHsp18.5 from Arabidopsis thaliana, demonstrating that it is dimeric and exhibits robust chaperone activity, which adds support to the model that suboligomeric sHSP forms are a substrate binding species. Notably, like oligomeric sHSPs, when bound to substrate, AtHsp18.5 assembles into large complexes, indicating that reformation of sHSP oligomeric contacts is not required for assembly of sHSP-substrate complexes. Monomers of AtHsp18.5 freely exchange between dimers but fail to coassemble in vitro with dodecameric plant cytosolic sHSPs, suggesting that AtHsp18.5 does not interact by coassembly with these other sHSPs in vivo. Data from controlled proteolysis and hydrogen-deuterium exchange coupled with mass spectrometry show that the N- and C-termini of AtHsp18.5 are highly accessible and lack stable secondary structure, most likely a requirement for substrate interaction. Chaperone activity of a series of AtHsp18.5 truncation mutants confirms that the N-terminal arm is required for substrate protection and that different substrates interact differently with the N-terminal arm. In total, these data imply that the core α-crystallin domain of the sHSPs is a platform for flexible arms that capture substrates to maintain their solubility.

The recent evolution of a pseudogene: diversity and divergence of a mitochondria-localized small heat shock protein in Arabidopsis thaliana

In this study we examined the evolution of the genes for three organelle-localized small heat shock proteins in Arabidopsis thaliana: the chloroplast-localized (CP) protein HSP21 and two mitochondria-localized (MT) proteins, HSP23.5 and HSP23.6. We found that the CP protein and one of the MT proteins, HSP23.6, are evolving under purifying selection to maintain function. In contrast, the gene for HSP23.5, the other MT protein, is highly variable within A. thaliana, and in some accessions or ecotypes this gene may be a pseudogene. HSP23.5 and HSP23.6 are related via a segmental duplication event, and the presence of orthologs of each gene in other species within the Brassicaceae indicates that the duplication generating HSP23.5 and HSP23.6 may have occurred as much as 20 million years ago. This is considerably longer than the 4 million year half-life of gene duplicates (functional genes as well as pseudogenes) reported by some studies. Our results are consistent with the prediction that after gene duplication one gene duplicate can be maintained for some time under relaxed selection while it accumulates random mutations. By capturing a pseudogene in the making our study provides important information on how pseudogenes are formed.

Triticum aestivum cDNAs homologous to nuclear-encoded mitochondrion-localized small heat shock proteins.

Two cDNAs, TaHSP23.5 and TaHSP23.6, encoding proteins with homology to mitochondrion-localized (MT) small heat shock proteins (sHSPs) were isolated from a heat shock cDNA library from Triticum aestivum (wheat). TaHSP23.5 specified a 214 amino acid protein and TaHSP23.6 specified a 216 amino acid protein. Amino acid sequence identity was only 45.7% between the two proteins. However, both proteins showed greater identity to MT sHSPs of other plant species than to any other sHSPs from wheat. Amino acid sequence alignments with other MT sHSPs identified the putative amino terminus of the mature proteins and consensus regions specific to this class of sHSPs. Transcripts of both genes were absent from control tissues, but strongly induced by heat stress. Phylogenetic analysis indicates that these two wheat genes arose by duplication after the divergence of monocots and dicots.

Boechera Species Exhibit Species-Specific Responses to Combined Heat and High Light Stress

As sessile organisms, plants must be able to complete their life cycle in place and therefore tolerance to abiotic stress has had a major role in shaping biogeographical patterns. However, much of what we know about plant tolerance to abiotic stresses is based on studies of just a few plant species, most notably the model species Arabidopsis thaliana. In this study we examine natural variation in the stress responses of five diverse Boechera (Brassicaceae) species. Boechera plants were exposed to basal and acquired combined heat and high light stress. Plant response to these stresses was evaluated based on chlorophyll fluorescence measurements, induction of leaf chlorosis, and gene expression. Many of the Boechera species were more tolerant to heat and high light stress than A. thaliana. Gene expression data indicates that two important marker genes for stress responses: APX2 (Ascorbate peroxidase 2) and HsfA2 (Heat shock transcription factor A2) have distinct species-specific expression patterns. The findings of species-specific responses and tolerance to stress indicate that stress pathways are evolutionarily labile even among closely related species.

Patterns of alternative splicing vary between species during heat stress

Alternative splicing (AS) can generate significant protein variation from a single gene. While the basic process of AS has been known for some time, little is known about how AS varies across species. In addition, there are many unanswered questions about the role of AS in plant responses to abiotic stress. In this study of the model plant species, Arabidopsis thaliana, and a native California mustard, Boechera depauperata, researchers report that the genes that undergo AS are distinct in each species. This provides evidence that AS plays an important and species-specific role in the plant stress response.