Download The evolution, function, structure, and expression

Journal of Experimental Botany, Vol. 64, No. 2, pp. 391–403, 2013
doi:10.1093/jxb/ers355 Advance Access publication 18 November, 2012
Darwin Review
The evolution, function, structure, and expression of the
plant sHSPs
Elizabeth R. Waters*
Department of Biology, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, USA
* To whom correspondence should be addressed. E-mail: [email protected]
Received 13 March 2012; Revised 19 November 2012; Accepted 20 November 2012
Abstract
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.
Key words: Abiotic stress, chaperones, evolution, gene families, heat shock proteins, molecular evolution.
Introduction
All organisms respond to high temperatures or heat stress by
altering their gene expression patterns and turning on the heat
shock genes. These events are referred to as the heat shock
response (Linquist and Craig, 1988; Nover, 1991; Vierling,
1991; Kotak et al., 2007a; Richter et al., 2010). Heat stress activates the heat shock transcription factors (HSFs) which then
bind to heat shock elements or promoters. In this way the HSFs
turn on or greatly up-regulate the heat shock genes (Schoffl
et al., 1998; von Koskull-Döring et al., 2007; Scharf et al.,
2012). The products of the heat shock genes are the heat shock
proteins or HSPs. The HSPs include a number of conserved
protein families: the HSP100s, HSP90s, HSP70s, HSP60s, and
the HSP20s or the small HSPs. Each of these families is found
across a wide diversity of organisms. The HSPs are chaperones,
and as such they assist in protein folding and prevent irreversible protein aggregation (Becker and Craig, 1994; Hartl, 1996;
Liberek et al., 2008; Tyedmers et al., 2010). Despite their close
working relationship in the cell, the individual HSP families are
evolutionarily unrelated to each other. The HSPs were first identified based on their up-regulation during heat shock, but we
now know that many of the HSPs are also found in unstressed
cells. Therefore, these proteins are both a necessary component
Abbreviations: ACD, α-crystallin domain; C, cytoplasmic/nuclear; CP, chloroplast; ER, endoplasmic reticulum; HSP, heat shock protein; MT, mitochondria; PX,
peroxisome; sHSP: small heat shock protein
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392 | Small heat shock evolution
of unstressed cells and a crucial part of the cell’s response to
stress. During stress, the presence of HSPs within the cell prevents cell death, and this then confers organismal tolerance to
high temperatures. The plant small heat shock proteins (sHSPs)
are particularly diverse and are a crucial component of the plant
heat shock response. In recent years, considerable progress has
been made in elucidating the structure, function, and evolution
of the sHSPs. This review brings together the most recent findings in the study of these important and diverse proteins.
Structure and function of sHSPs
The small heat shock proteins defined
The sHSPs are a large and ancient family of proteins. They have
been found in all domains of life, Achaea, Eukarya, and Bacteria
(Plesofsky-Vig et al., 1992; Waters and Vierling, 1999a; Kappe
et al., 2002; Franck et al., 2004; Fu et al., 2006; Kim et al., 1998;
Aevermann and Waters, 2007; Waters and Rioflorido, 2007;
Kriehuber et al., 2010; Poulain et al., 2010). These ubiquitous
proteins are required for life and are found in even the smallest
genomes (Waters et al., 2003). As the name suggests, the monomers of these proteins are small and range in size from 12 kDa
to 42 kDa. Most sHSPs are in the range of 15–22 kDa; thus,
these proteins are also sometimes referred to as the HSP20 family. The primary sHSP amino acid sequence includes a variable
N-terminal region, a more conserved C-terminal region, and a
C-terminal extension (Fig. 1). The highly conserved C-terminal
region is often referred to as the α-crystallin domain, or the
HSP20 domain. The sHSPs that function in specific cellular
organelles or compartments have N-terminal transit, leader, or
signal sequences needed to get the sHSP to the proper cellular
compartment. The C-terminal extension can be quite variable
in both length and sequence, and may also contain organellespecific retention amino acid motifs.
As more complete genomes have become available and are
analysed, it has become clear that the C-terminal region or
HSP20 region has become incorporated into a large number
of other proteins. Some authors have used the term ACD for
these α-crystallin domain-containing proteins (Scharf et al.,
2001; Bondino et al., 2012). However, in some cases, the ACDs
may be multidomain proteins with other unrelated conserved
domains present (Scharf et al., 2001). At this time, very little is
known of the structure, function, or evolutionary history of the
ACD proteins, and for this reason they are excluded from this
review. Future work on these proteins should shed light on their
evolutionary relationships to the sHSPs and will determine if
they share any structural or functional features with the sHSPs.
Small heat shock proteins form large oligomers
The first two high-resolution crystal structures determined for
sHSPs were Hsp16.5 from an archaea, Methanocaldococcus
(Methanococcus) jannaschii (Kim et al., 1998), and Hsp16.9
from wheat (Triticum aestivum) (van Montfort et al., 2002).
These two structures from very distantly related species share
a large number of similarities (van Montfort et al., 2001,
2002). Both are composed of a β-sandwich of two antiparallel sheets and form a hollow ball. The building blocks of both
oligomers are dimers. However, the oligomers are of different
sizes: the plant Hsp16.9 forms a dodecamer, and the oligomer
of Hsp16.5 has 24 subunits. Hsp16.9 the dodecamer has three
tetramers that form two six-membered rings (hexameric double disks) (Fig. 2) (van Montfort et al., 2001). A model for the
structure of Hsp21 from Arabidopsis indicates that this plant
sHSP is also a dodecamer with two hexameric disks (Lambert
et al., 2011). Recently it has become clear that the sHSPs are
polydisperse, meaning that more than one oligomeric state is
found (Baldwin, 2011). Stengel et al. (2010) showed that the
dodecamer form of Hsp18.1CI from Pisum sativum is in equilibrium with Hsp18.1 dimers, monomer, and higher order
oligomers. This information increases our understanding of
how sHSPs act in vivo. It also raises both our understanding
of the complexity of the sHSPs and our appreciation of how
difficult it is to study the structure of sHSPs.
There are now 10 published high-resolution sHSP crystal
structures (see review by Basha et al., 2012). However, none
of these is from plants, and a clear need for more structural
data for the diverse plant sHPSs exists. It is very interesting
that there is lack of high-resolution structural information
for the N-terminal region of the sHSPs. This may be due
to dynamic changes or a lack of stability of the N-terminal
regions (Basha et al., 2012; McDonald et al., 2012). It has
been suggested that the N-terminal region is important in
substrate binding (Giese et al., 2005; Basha et al., 2006; Jaya
et al., 2009; McDonald et al., 2012).
Mature Protein sHSP
N-terminal
Transit/Target/Signal
HSP20
C-Terminal Extension
Fig. 1. Small heat shock protein domains. The sHSP monomer contains a variable N-terminal domain (light blue), the conserved
HSP20 or α-crystallin domain (red), and a variable C-terminal extension (grey). The sHSPs that localize to the organelles, chloroplast,
mitochondrion, endoplasmic reticulum, and peroxisome, have the necessary transit, targeting, or signal sequences (dark blue) needed
for successful entry into those individual organelles. These sequences are not part of the mature sHSP. The C-terminal extension is
variable in length and contains the endoplasmic reticulum (KDEL) or peroxisome (SKL) retention signals in those sHSPs that function in
those cellular compartments.
Waters | 393
Fig. 2. Crystal structure of Hsp16.9 from Triticum aestivum. (A) The structure of Hsp16.9 from the T. aestivum monomer. The strands
are in yellow and the helices are in red. The loops are grey. (B) The oligomeric structure of Hsp16.9. The colour scheme is the same as
for the monomer. Hsp16.9 is a dodecamer that forms a β-sandwich.
The sHSPs prevent irreversible protein aggregation
The HSPs are known to be molecular chaperones. Specifically,
they prevent irreversible protein aggregation (Lee et al., 1995;
Boston et al., 1996; Buchner, 1996; Hartl, 1996; Dobson, 2003;
Basha et al., 2004; Liberek et al., 2008; Saibil, 2008; McHoaurab
et al., 2009; Tyedmers et al., 2010; Hilario et al., 2011; Hilton
et al., 2012). It has been noted that the sHSPs are often the
first line of defence in the cell when proteins begin to misfold
and as such have been referred to as ‘paramedics of the cell’
(Hilton et al., 2012). Much still needs to be learned about the
details of the specific interactions between the HSPs and their
protein substrates, but in recent years a much more detailed and
nuanced model has developed showing how the HSPs work
both individually and together (Dobson, 2003; Liberek et al.,
2008; Saibil, 2008; Eyles and Gierasch, 2010; Tyedmers et al.,
2010). The current models (Fig. 3) of HSP chaperone function
in the cell suggest that the sHSPs work with other chaperones
Fig. 3. Model of the role of sHSPs in the chaperone network. The sHSPs are an important part of the chaperone network. The sHSPs
bind denaturing proteins, protecting them from irreversible aggregation. The substrate proteins can then be refolded by other parts of
the chaperone network.
394 | Small heat shock evolution
to prevent irreversible aggregation and to re-solubilize proteins that have already aggregated. Two functional features of
the sHSPs stand out among other HSP chaperones: first, they
do not require ATP to bind substrate proteins, and, secondly,
they have a very high capacity for binding denatured substrates
(Haslbeck et al., 2005; Nakamoto and Vigh, 2007; McHaourab
et al., 2009; Eyles and Gierasch, 2010; Tyedmers et al., 2010).
It is notable that small HSPs can bind an almost equal weight
of substrate protein. It is thought that they do this by exposing
hydrophobic surfaces (Nakamoto and Vigh, 2007).
The plant sHSPs: a diverse and complex
family of proteins
We know that plant sHSPs are a part of the larger sHSP family
present in all organisms, and as such they share many aspects
of the conserved sHSP structure and function described above.
However, plants have many more sHSPs than do other eukaryotes, including algae (Waters and Rioflorido, 2007). The number of sHSPs varies across individual plant species: Arabidopsis
has 19 sHSPs (Scharf et al., 2001), rice (Oryza sativa) has 23,
and poplar (Populus trichocarpa) has 36 (Waters et al., 2008a).
In addition to being numerous, sHSPs are also very diverse both
in sequence and in where they function in the cell. One of the
biggest differences between the plant sHSPs and sHSPs in other
organisms is the presence of distinct subfamilies of plant sHSPs.
Only in plants are there evolutionarily related sHSP subfamilies
that cross multiple species boundaries. Some of these subfamilies are >400 million years old (Waters and Vierling, 1999a, b)
and others have evolved more recently (Waters et al., 2008a).
Angiosperms have 11 diverse sHSP subfamilies
Using analysis of the available angiosperm complete genome
sequences, researchers have identified 11 sHSP subfamilies,
as shown in Table 1 (Scharf et al., 2001; Siddique et al., 2008;
Waters et al., 2008a; Sarkar et al., 2009; Bondino et al., 2012).
These include six subfamilies that are cytoplasmic/nuclear
localized (CI–CVI) and five sHSP subfamilies that localize
to organelles. The organelle subfamilies include one subfamily that localizes to the endoplasmic reticulum (ER), another
that localizes to the peroxisome (PX), one subfamily that
localizes to the chloroplast (CP), and two subfamilies that
localize to the mitochondria (MTI and MTII). Ten of these
subfamilies are present in both monocots and eudicots (CP,
MTI, MTII, ER, PX, CI, CII, CIII, CIV, and CV), and one is
found only in eudicots (CVI). It is important to note that not
all sHSPs belong to these 11 conserved subfamilies (Siddique
et al., Waters et al., 2008a; Sarkar 2009; Bondino et al., 2012).
These unique sHSPs may represent newer gene duplications
and could represent incipient subfamilies.
The extent of functional variation among the plant sHSP
subfamilies is not yet known. However, there is evidence that
the plant sHSPs are in fact quite functionally diverse. A multiple alignment of representatives of each of the 11 plant
sHSPs subfamilies found in Arabidopsis thaliana (Fig. 4)
demonstrates that the plant sHSP subfamilies share with all
sHSPs the conserved C-terminal or HSP20 region and a more
variable N-terminal region. While there are a few amino acids
conserved across all the sHSPs, what is most important is the
conserved structural features in the C-terminal domain. The
HSP20 region of the A. thaliana sHSP subfamilies contains
the nine highly conserved β-sheets (Fig. 4) found in all sHSPs.
This region is present in sHSPs across all domains of life,
and has been suggested to be important for oligomerization
(Basha et al., 2012).
Despite the considerable conservation across the plant
sHSPs in the HSP20 region, some interesting differences exist
in this region among plant sHSP subfamilies. Two plant subfamilies (CIV and CVI) lack the crucial β6 sheet. This is notable because it has been shown that the β6 sheet is important
Table 1. Expression patterns and cellular locations of the Arabidopsis thaliana sHSP members of the angiosperm sHSP subfamilies
Information presented is based on published data (Siddique et al., 2008; Waters et al., 2008a).
Subfamily
Abbreviation
A. thaliana sHSP
Stress-induced expression
Developmental expression
1
Cytoplasmic/nuclear I
CI
Cytoplasmic/nuclear II
CII
3
4
5
6
7
8
Cytoplasmic/nuclear III
Cytoplasmic/nuclear IV
Cytoplasmic/nuclear V
Cytoplasmic/nuclear VI
Chloroplast
Mitochondrial I
CIII
CIV
CV
CVI
CP
MTI
9
10
11
Mitochondrial II
Endoplasmic reticulum
Peroxisome
MTII
ER
PX
H, Os, S, D, Ox, UV-B, W
H, Os, S, D, Ox, UV-B, W
H, Os, S, D, Ox, UV-B, W
H, Os, S, D, Ox, UV-B, W
H, Os, S, Ox, UV-B, W
H, Ox, S, D, Ox, UV-B
Constitutive, H
Constitutive, H, W
Constitutive
Constitutive, H
H, Os, S
H, Ox, UV-B
H, Ox, UV-B
H, Os, S
H, Os
H, Os, S, Ox, B
Pollen, seeds
Pollen, seeds
Pollen, seeds
Pollen, seeds
2
Hsp17.4CI
Hsp17.6ACI
Hsp17.6BCI
Hsp17.6CI
Hsp17.6CII
Hsp17.7CII
Hsp17.4CIII
Hsp15.4CIV
Hsp21.7CV
Hsp18.5CVI
Hsp21CP
Hsp23.5MTI
Hsp23.6MTI
Hsp23.5MTII
Hsp22ER
Hsp15.7PX
None
Apex, flowers, pollen, siliques, seeds
Apex, flowers, siliques
Flowers, pollen, siliques, seeds
None
Seeds
Seeds
Waters | 395
Fig. 4. Alignment of the sHSPs of Arabidopsis thaliana. One representative from each of the 11 sHSP subfamilies from A. thaliana
were aligned using the Promals3D structural alignment program. Each predicted α-helix is in red and β-pleated sheets are in blue. The
conserved β-pleated sheets found in the HSP20 domain are numbered according to Triticum aestivum Hsp16.9 (Van Montfort et al.,
2001b). There are no conserved amino acids or motifs in the N-terminal region (residues 1–140).
for dimer formation and for oligomerization (von Montfort
et al., 2001a, b). Despite the lack of the β6 sheet, it has been
demonstrated that AtHsp18.5 CVI can form dimers (Siddique
et al., 2008). In addition, these authors (Siddique et al., 2008)
have also demonstrated that AHsp18.5 (CVI) can function as a
chaperone. These findings are interesting because some animal
sHSPs also lack the β6 sheet. In the case of the human proteins,
the β7 sheet plays a crucial role in dimer formation (Bagneris
et al., 2009). The loss of the β6 sheet in some plant sHSPs and
in some animals is a convergent event, and further comparative
studies of the structure and function of the plant CIV and CVI
sHSPs and the animal sHSPs should prove informative.
In contrast to the conservation seen within the HSP20
C-terminal region, the N-terminal region of the plant sHSP
subfamilies is highly diverse across subfamilies, and no conserved motifs or structural regions across all sHSPs are found
(Fig. 4). Outside of the targeting, signal, or leader sequences
needed for localization of the organelle-targeted sHSPs (CP,
MTI, MTII, ER, and PX) into the proper cellular compartments, the most notable feature in the N-terminal domain is
the complete lack of sequence conservation across the plant
sHSP subfamilies.
Despite the lack of conservation in the N-terminal
region across sHSP subfamilies the keys to understanding
functional divergence among the different plant sHSP subfamilies may be found in this region. This is due to the considerable conservation in the N-terminal region that is seen
within plant subfamilies (Waters, 1995; Waters and Vierling,
396 | Small heat shock evolution
1999a, b; Siddique et al., 2008; Waters et al., 2008a; Bondino
et al., 2012). The presence of subfamily-specific N-terminal
motifs is intriguing in light of recent studies indicating that
the N-terminal region is functionally important and is crucial for substrate binding (Giese et al., 2005; Basha et al.,
2006; Jaya et al., 2009; McDonald et al., 2012). The CP
sHSPs have a methionine-rich amphipathic α-helix that is
highly conserved across angiosperms (Chen and Vierling,
1991; Waters, 1995; Waters and Vierling, 1999b). This
methionine-rich region has been demonstrated to be important in substrate binding (Harndahl et al., 2001) and is sensitive to the chloroplast redox state (Gustavsson et al., 2001;
Sundby et al., 2005). The CII subfamily also has a conserved
N-terminal amino acid motif (Waters et al., 1995; Waters
and Vierling, 1999a) (DA-AMAATP) that is not found in
the other cytoplasmic/nuclear sHSPs. The role of this particular motif in CII structure and function is not yet known.
However, it is unlikely that natural selection would maintain
these subfamily-specific motifs in the absence of functional
importance.
In a very interesting study, Basha et al. (2010) examined
the structural and functional differences between the CI and
CII subfamilies. These researchers reported that both the CI
and CII subfamilies form dodecamers and both have chaperone activity. However, the CII proteins are more efficient
than the CI proteins at preventing irreversible aggregation.
The same authors reported that the CII sHSPs maintain their
dodecmeric structure at higher temperatures than do the CI
proteins. Finally, it is interesting that while the CI and CII
proteins are both found in the cytosol, they do not form
hetero-oligomers (Basha et al., 2010). However, when studies
of other species are examined, the role of the CII proteins
in the protection of proteins becomes less clear. In a study
of tomato protoplasts, Tripp et al. (2009) generated knockdowns of the CI and CII genes and demonstrated that the
CI sHSPs have a clear thermoprotective role but that the CII
sHSPs do not. Further evidence that the CII sHSPs may have
non-thermoprotective roles in the cell comes from a report
that the tomato sHSP Hsp17.5CII is important in regulating the activity of HsfA2C (Port et al., 2004). In summary,
the patterns of N-terminal sequence conservation within but
not across subfamilies are consistent with the evidence from
select sHSPs that biochemical and biological differences exist
between the subfamilies. Continued studies of the N-terminal
region and how sequence differences here reflect functional
changes will be crucial to understanding the relationship
between sequence and function in the sHSPs.
Complex expression patterns of
plant sHSPs
As their name implies, almost all the plant sHSPs are heat
induced. However, the sHSP gene expression data reported in
the literature indicate that there is no single pattern of gene
expression for all the plant sHSP genes. In fact it is now clear
that not even all the members of the same sHSP subfamily
have the same expression patterns.
In addition to being heat induced, many, but not all, sHSPs
are expressed at specific developmental stages. Small HSPs
are expressed during embryogenesis and seed maturation
(Almoguera and Jordano, 1992; DeRocher and Vierling,
1994; Gifford and Taleisnik, 1994; Wehmeyer et al., 1996;
Wehmeyer and Vierling, 2000; Lubaretz and Zur Nieden,
2002; Kotak et al., 2007b). They are also expressed during
pollen development and fruit maturation (Zarsky et al., 1995;
Carranco et al., 1997; Neta-Sharir et al., 2005; Volkov et al.,
2005; Mu et al., 2011). Recently, our understanding of sHSP
gene expression has expanded greatly due to the availability
of genome-wide studies of a large number of conditions in all
plant tissues. (Swindell, 2006; Qin et al., 2008; Swindell, et al.,
2007; Siddique et al., 2008; Waters et al., 2008a; Sarkar et al.,
2009; Weston et al., 2011). These studies have confirmed previous work and have greatly expanded the conditions that are
known to induce sHSP expression.
To better understand the complexity of the expression patterns among the many different sHSPs, it is useful to summarize what we know from the best-studied ‘model’ plant
species, A. thaliana. Exploring the A. thaliana expression
pattern in depth not only gives us a detailed view of sHSP
expression, it also provides a framework with which to examine the differences in sHSP expression in other species. The
most common expression pattern seen among the A. thaliana
sHSP genes is no expression in unstressed shoot tissues and
high levels of gene expression during heat stress (Table 1). For
example, the gene for Hsp18.1CI, a member of the A. thaliana CI subfamily, is highly expressed under heat shock and
has either no expression or a much lower level of expression in response to other stressors. In addition, this gene is
not developmentally expressed (Table 1). This pattern of
high heat shock expression with no constitutive or developmental expression is shared with the CP and ER subfamilies (Table 1). Notably, this pattern of gene expression is not
shared with the other cytoplasmic/nuclear-localized subfamilies, or, most importantly, not even with other members of the
CI subfamily. The other A. thaliana CI sHSPs (Hsp17.4CI,
Hsp17.6ACI, Hsp17.6BCI, and Hsp17.6CCI) are also highly
expressed during heat stress, but they are also expressed in
response to a wide range of other stressors including osmotic
stress, oxidative stress, and UV-B exposure (Table 1). These
findings, that the CI sHSPs are expressed during a range of
stress responses, are similar to what has been found in wholegenome studies that seek to understand the overlap of gene
expression patterns between heat shock and other abiotic
stressors (Swindell et al., 2007; Hu et al., 2009). In addition
to being stress induced, all four of these A. thaliana CI sHSPs
(Hsp17.4CI, Hsp17.6ACI, Hsp17.6BCI, and Hsp17.6CI)
are also expressed in pollen and seeds (Table 1). The gene
for Hsp17.8CI is the only A. thaliana sHSP gene not on the
Affymetrix Genechip, and thus the same level of comparative data is not available for this gene. However, it has been
reported that Hsp17.8I is heat induced (Kant et al., 2008).
Interestingly there are data that show that in non-stressed
cells Hsp17.8I acts as an ankyrin repeat protein 2A (AKR2A)
cofactor and is thus important in the targeting of proteins
to the chloroplast (Kim et al., 2011). These authors also
Waters | 397
suggest that other A. thaliana CI sHSPs play a role in chloroplast protein targeting. Despite the functional differences
reviewed above, the CI and CII subfamilies have very similar
expression patterns. The two members of the A. thaliana CII
subfamily are induced by the same stressors as is the CI subfamily. However, Hsp17.6CII is expressed only in pollen, and
Hsp17.7CII is expressed in both seeds and pollen (Table 1).
It is interesting to note here that the genes Hsp17.6CII and
Hsp17.7CII are in a tandem duplication. They are the only
sHSPs to be tandemly duplicated, and all other A. thaliana
sHSPs are dispersed throughout the genome (Waters et al.,
2008a).
The CP and ER expression pattern (shared with
Hsp18.1CI) is not shared with the other organelle-localized
subfamilies (Table 1). Both of the A. thaliana MI sHSPs
(Hsp23.5MI and Hsp23.6MI) are highly expressed during
heat shock and are absent from pollen and seeds, but, unlike
the CP and ER sHSPs, these sHSP genes are also expressed
in response to oxidative stress and exposure to UV-B light.
There is a second mitochondrial-localized subfamily (MTII),
and Hsp26.5MTII is highly expressed during heat shock
and is expressed during osmotic and salt stress (Table 1). In
addition, Hsp26.5MII is expressed in seeds. Hsp15.7PX, the
A. thaliana sHSP found in the peroxisome, is expressed during HS, osmotic, salt, and oxidative stress, as well as in seeds
(Table 1).
The diversity of sHSP expression patterns is even greater
when all the cytoplasmic/nuclear subfamilies are examined.
While it is clear that there is no single expression pattern for
the CI and CII subfamilies, both of these sHSP subfamilies
are stress induced and are not expressed in unstressed shoot
tissue. In contrast the CIII–CVI subfamilies are constitutively
expressed in shoot tissue (Table 1). It is interesting that the
CIII, CIV, and CV subfamilies are also heat regulated and
have higher expression levels in response to heat shock and
a number of other stresses (Table 1). Another difference
between these subfamilies and the CI and CII subfamilies is
that the CIII, CIV, and CVI subfamilies are developmentally
expressed in the shoot apex, flowers, and siliques (Table 1).
However, like the CI subfamily, the CIII and CIV subfamilies are also found in pollen and seeds. The CV subfamily has
the most distinctive expression pattern. It is constitutively
expressed in shoots but it is not up-regulated by heat or any
other stress. It is also notable that Hsp21.7CV is absent from
pollen and seeds.
Examinations of sHSP expression patterns in other species
are largely congruent with the patterns seen in A. thaliana.
A pattern clearly shared across many species is the diversity
of expression patterns among the CI sHSPs. For example, in
a study of two tandemly duplicated CI sHSP genes, Rampino
et al. (2010) report that these two closely related and highly
similar genes are both expressed during heat shock but do not
share similar responses to other abiotic stressors. However,
some expression patterns do differ across species. In a study
of the rice sHSPs, Sarkar et al. (2009) reported similar but
not always identical patterns of gene expression for the
O. sativa homologues of the A. thaliana sHSPs. In contrast
to what is known for A. thaliana, these authors reported that
members of the rice CP and ER subfamily are expressed in
seeds (Sarkar et al., 2009). Species-level differences in gene
expression were also reported by Weston et al. (2011) in their
examination of the HSP20 gene network during heat shock
in three different species (A. thaliana, P. trichocharpa, and
Glycine max).
While it has been known for some time that sHSPs are
important in the plant cell response to abiotic stress, it has
also been reported that biotic stress can induce the gene
expression of some but not all sHSPs (Siddique et al., 2008;
Waters et al., 2008a; Sarkar et al., 2009). It is also important
to note here that two recent studies of rice and A. thaliana
report that sHSPs have distinct patterns of gene expression
in the shoots and roots (Siddique et al., 2008; Sarkar et al.,
2009). Most other studies on sHSP function and expression have been conducted with only shoot tissues, and further studies to determine specific root function should prove
informative.
Studies with a range of different species have indicated
that sHSPs are associated with thermotolerance (Knight and
Ackerly, 2001; Stout and Al-Niemi, 2002; Knight and Ackerly,
2003; Wang and Luthe, 2003; Amano et al., 2012). An interesting recent study indicated that leaf cooling in agave (Lujan
et al., 2009) is also associated with sHSP expression. It is
clear from these studies that sHSPs have very important roles
in organismal thermotolerance and plant adaptation to the
environment (Sorensen et al., 2003). It is therefore not surprising that researchers target the manipulation of the sHSPs
in studies that seek to generate plants with increased thermotolerance. However, the relationship between the expression of any one sHSP and organismal thermotolerance is still
unclear because modification of sHSP expression only sometimes impacts thermotolerance (Sun and MacRae, 2005). The
large number of sHSPs present in plants and the likely overlap
or redundancy of function of individual sHSPs may explain
the lack of a phenotype of single gene knockouts reported in
the literature (Sun and MacRae, 2005). Dafyny-Yellin et al.
(2008) created multiple gene knockouts in Arabidopsis to
examine the functional specialization of the CI sHSPs. They
reported that the three sHSPs they examined (Hsp17.4CI,
Hsp17.6ACI, and Hsp18.1CI) have non-redundant functions
in acquired thermotolerance, but that in early embryogenesis the three CI proteins are redundant. This study indicates
both the rewards and challenges of performing multiple gene
knockout studies. It is likely that complex knockout studies
are needed in order to gain a detailed understanding of the
roles of individual sHSP proteins and subfamilies. However,
with some plant species possessing ≥30 sHSPs, other methods
to determine sHSP in vivo function will be needed.
Hilton et al. (2012) have called the sHSPs the ‘paramedics’ of the cell. As a group it is clear that at least one sHSP
is expressed in response to most stresses, but not all sHSPs
respond to all cellular emergencies. Most sHSPs have their
highest expression levels during heat stress and some (e.g.
AtHSP18.1CI) are only expressed during heat stress. It is
important to mention again that A. thaliana Hsp21.7CV, the
sHSP with the most distinctive gene expression pattern (constitutive expression, not induced by heat stress or any other
398 | Small heat shock evolution
stress, and absent from pollen and seeds), is to date the only
sHSP that has been demonstrated to lack chaperone activity
(Siddique et al., 2008). This suggests that the chaperone function of the sHSPs is linked to their stress and developmental
expression.
Evolution of sHSPs
The evolutionary processes that generate complex gene families include gene duplication, recombination (including gene
conversion), and gene loss. These processes are reviewed in
detail elsewhere (Nei and Rooney, 2005; Flagel and Wendel,
2009). Gene duplication in plants can occur via single-gene
duplication events and whole-genome duplication events.
Once genes duplicate they can have a number of fates, loss
being the most likely. The less frequent but more significant
possibility is that genes can evolve a new function, a process
driven by natural selection and termed neofunctionalization.
Neofunctionalization and a related process called subfunctionalization are the processes that have generated the diverse
plant sHSP subfamilies. However, not all gene duplicates
undergo neofunctionalization, and gene duplicates can also
be maintained in the genome and retain the original function. This process generates new members of individual subfamilies. For gene duplicates that have been maintained, gene
conversion plays an important role in maintaining sequence
similarity and thus function within gene families. It is clear
that all these evolutionary forces, gene duplication, gene conversion, neofunctionalization, and genome duplication, have
played a role in the evolution of the sHSPs.
The evolutionary histories of individual sHSP subfamilies
are quite diverse; some subfamilies are >400 million years
old, while others evolved much more recently (Fig. 5). None
of the subfamilies is found in algae, and three subfamilies,
CI, CII, and CP, are present in mosses (Waters and Vierling,
1999a, b) and in Selaginella moellendorffii (E.R. Waters
and B. Bharjawadji, unpublished data) (Fig. 5). The sHSPs
appear to have undergone one burst of duplications early
in land plant history and then a later one after the divergence of Selaginella, a lycophyte. This pattern is somewhat
different from what has been reported for Hsp70 and other
stress-related genes that diversified early in land plant evolution (Rensing et al., 2008). As stated earlier, the CI and CII
sHSPs are developmentally regulated in seeds and in pollen in
Origin of flowers
Origin of seeds
Origin of land plants
angiosperms. Because there is no information on the expression patterns for sHSPs in P. patens or S. moellendorffii, we
do not now know if these genes are developmentally regulated in these species that lack both pollen and seeds but do
have gametogenesis and embryogenesis.
The CI and CII sHSPs are not the only cytosolically localized sHSPs in these species. Both P. patens, which has 29
sHSPs, and S. moellendorffii, which has 16, have sHSPs that
are not members of any of the angiosperm sHSP subfamilies. Some of these ‘unique’ sHSPs may represent ancestral
genes that gave rise to the angiosperm sHSP subfamilies.
Some sHSPs angiosperm sHSPs also do not belong to one
of the 11 subfamilies (Siddique et al., 2008; Waters et al.,
2008a; Sarkar et al., 2009; Bondino et al., 2012). It is possible that these genes are recent duplicates that will eventually
be lost, but they may also represent new sHSPs subfamilies
in the very early stages of evolution. There is evidence that
there are additional grass sHSP subfamilies (Sarkar et al.,
2009; Bondino et al., 2012). Analysis of additional monocot
genomes when they become available will determine if these
subfamilies are present in non-grass species. At this time,
we can conclude that while at least some plant subfamilies
evolved very early in the land plant lineage, the processes
that generated these gene families are continuing to act to
generate new plant subfamilies.
The timing of origin of the sHSP subfamilies (CIII–CVI,
ER, MI, MII, and PX) found only in angiosperms is still
unclear, and additional genomes of members of key lineages,
especially gymnosperms, will be needed to determine the timing of their origin. It is interesting that the CP and MT sHSPs
in angiosperms are closely related to each other (Waters et al.,
2008a) and are not related to the bacterial endosymbionts
that gave rise to the chloroplast and mitochondrion (Waters
and Vierling, 1999b). Taken together, this indicates that the
CP sHSP subfamily evolved first early in land plant evolution
and that the MT sHSPs evolved later from the CP sHSPs.
This raises the possibility that the CP sHSPs may be dual-targeted in mosses and/or S. moellendorffii. It has been reported
that dual targeting of proteins to both the CP and MT is not
rare in plants (Berglund et al., 2009; Carrie et al., 2009). If
this evolutionary scenario is correct, then this would represent subfunctionalization. In subfunctionalization, the ancestral protein is found in more than once place or has more
than one function following gene duplication; each duplicate
is freed to specialize in this case in either the CP or the MT.
Monocots
10 sHSP subfamilies: CP, MT, MTII, ER, PX, C1-CV
Eudicots
11 sHSP subfamilies: CP, MT, MTII, ER, PX, C1-CVI
Gymnosperms
Seedless Vascular Plants Selaginella moellendorffii,a lycophyte: CP, CI,CII
Non-vascular Plants
Phycomitrella patens, a byryophyte: CP, CI,CII
Green Algae
Cytosolic sHSPs present but no sHSP subfamilies
Fig. 5. Presence of sHSP subfamilies among the major groups of green plants. Information on the presence of sHSP subfamilies is based
on analysis of complete genomes of algae and land plants (Waters and Rioflorido, 2007; Waters et al., 2008a; Bondino et al., 2012).
Waters | 399
This hypothesis could be tested by examining the targeting of
the CP sHSPs in P. patens and in S. moellendorffii.
Once established, most sHSPs subfamilies have a very
stable evolutionary history. That is, they usually have one
(or sometimes two) copy per genome, and the evolutionary
relationships among the members of the subfamily represent organismal relationships (i.e. they are orthologues).
This pattern is seen in the CIII, CIV, CV, PX, MI, MII, and
CP subfamilies (Waters et al., 2008a; Bondino et al., 2012).
It is interesting that most subfamilies have only one copy
per genome when it is well known that plant genomes have
undergone numerous polyploidy events. This suggests that
the extra copies of these subfamilies are always lost after
whole-genome duplication. In a landmark study, Blanc and
Wolfe (2004) showed that plant genes are retained after
whole-genome duplication in a non-random manner, and
that genes for organelle-localized proteins were preferentially lost while cytosolic ones were retained. Blanc and
Wolfe (2004) also examined the retention and loss of functional categories of proteins and found clear differences in
the retention and loss of genes based on function. They
noted that genes involved in protein modification and apoptosis were usually retained after whole-genome duplication
but that those involved in defence were lost. These authors
did not specifically examine chaperones. However, the fact
that most cytosolic sHSP subfamilies have just one or at
most two copies per genome suggests that natural selection
has acted to remove gene duplicates in these subfamilies
based on their function in the cell. In short, the evolutionary
patterns for most of the sHSP subfamilies reflect selection
to maintain consistent function across species and indicate
that there are clear constraints on the number of sHSPs
needed or allowed within each genome.
The plant sHSP CI subfamily, however, has a very distinct
and complex evolutionary history. In all the genomes examined, the CI subfamily is always the largest and most diverse. In
A. thaliana there are six CIs; O. sativa has nine; P. trichocarpa
has 18; and G. max has 19 (Waters et al., 2008a; Bondino
et al., 2012). Phylogenetic analysis of the CI subfamily indicates a complex pattern of orthologous (due to speciation)
and paralogous (due to gene duplication) relationships even
within this subfamily (Waters et al., 2008a; Bondino et al.,
2012). The fact that the phylogenetic relationships within the
CI sHSP subfamily do not strictly reflect orthology (organismal relationships) suggests that gene duplication and loss
processes are occurring independently across plant genomes.
Analysis of the evolutionary dynamics of the CI subfamily in the A. thaliana, P. trichocarpa, and O. sativa genome
found evidence of recent gene duplication and gene conversion in some species (O. sativa) but not in others (A. thaliana
and P. trichocarpa) (Waters et al., 2008a). This indicates that
within the CI subfamily evolutionary forces shaping this subfamily are not consistent across species.
The very clear differences in the evolutionary patterns
among the plant sHSP subfamilies suggest as yet undiscovered functional differences among subfamilies. In a very interesting paper on P450s in vertebrates, Thomas (2007) suggests
that genes that are phylogeneticaly stable have conserved core
functions while genes that display a much more varied or
unstable phylogeny are much more varied in function or possibly in substrate specificity. If this is also true of the sHSPs,
then we would expect much more diversity in substrate binding and other functional attributes within the CI sHSPs than
within the other sHSP subfamilies that demonstrate more stable evolutionary histories.
Recent studies suggest that the analysis of gene sequences
within species can also elucidate important evolutionary patterns among the sHSPs. Studies of diversity of the CP sHSPs
in the genus Rhododendron found evidence of positive selection to alter amino acids in the HSP20 domain (Wu et al.,
2007). In a study of selection acting on a CI sHSP gene in
the same genus (Rhododendron), Liao et al. (2010) reported
evidence of purifying selection but also reported the presence
of a seven amino acid insertion in the N-terminal region.
As expected, strong purifying selection was also found acting on the CP sHSP and on one of the mitochondrial sHSPs
(Hsp23.6MI) in A. thaliana (Waters et al., 2008b). In contrast,
this same study found that the gene coding for Hsp23.5MI
had become a pseudogene in some ecotypes of A. thaliana due to a deletion event that disrupted the amino acid
sequence (Waters et al., 2008b). Taken together, these studies indicate that further studies of sHSP sequence diversity
within and between closely related species will provide important insights into sHSP evolution.
Conclusions and future directions
The sHSPs are a fascinating group of proteins. They are
ancient and ubiquitous, yet highly variable. In addition, as
chaperones, they have a crucial and fundamental role in plant
cell biology. Studies of the sHSPs have a broader importance
because they connect cellular function to organismal-level
traits such as tolerance to heat stress. In addition, studies of
sHSPs further our understanding of fundamental processes
of evolution such as gene duplication. Studies of sHSPs have
clearly made great strides in recent years, but many important
questions can still be addressed. First, while we have a general
understanding of the function and structure of the sHSPs,
we do not yet know how the diversity in primary sequence
reflects differences in structure or function both within the
plant subfamilies and between subfamilies. The patterns of
sequence diversity among the sHSPs strongly suggest that
some aspects, possibly substrate specificity, are quite variable
among the sHSPs. In addition, little is known about higher
level structural variation among plant sHSPs. Additional
high-resolution structures of plant sHSPs will provide crucial
information and will elucidate the differences in structure and
function between and within the diverse plant subfamilies. In
addition, analysis of whole-genome data for plants outside
the angiosperm lineage will provide a better understanding
of the evolutionary history of the sHSPs in the embryophyte
lineage. These types of data would allow us to determine the
time or origin of the diverse sHSP subfamilies, and this in
turn would provide insight into the selective pressures that
gave rise to the individual subfamilies.
400 | Small heat shock evolution
Acknowledgements
I thank Dr Elizabeth Vierling for introducing me to the heat
shock proteins and for many hours of interesting discussions on all aspects of the small heat shock proteins. Bharath
Bharadwaj assisted with the P. patens and S. moellendorffii
sequence analysis. I thank the current and past members of
my lab for joining me in studying the small heat shock proteins. I also thank anonymous reviewers for their thoughtful
comments that greatly improved this manuscript. The preparation of this manuscript was partially supported by NSF
award IOS-0920611.
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