Naturwissenschaften (2007) 94:791–812
DOI 10.1007/s00114-007-0254-y
REVIEW
The continuing conundrum of the LEA proteins
Alan Tunnacliffe & Michael J. Wise
Received: 10 January 2007 / Revised: 27 March 2007 / Accepted: 11 April 2007 / Published online: 4 May 2007
# Springer-Verlag 2007
Abstract Research into late embryogenesis abundant
(LEA) proteins has been ongoing for more than 20 years
but, although there is a strong association of LEA proteins
with abiotic stress tolerance particularly dehydration and
cold stress, for most of that time, their function has been
entirely obscure. After their initial discovery in plant seeds,
three major groups (numbered 1, 2 and 3) of LEA proteins
have been described in a range of different plants and plant
tissues. Homologues of groups 1 and 3 proteins have also
been found in bacteria and in certain invertebrates. In this
review, we present some new data, survey the biochemistry,
biophysics and bioinformatics of the LEA proteins and
highlight several possible functions. These include roles as
antioxidants and as membrane and protein stabilisers during
water stress, either by direct interaction or by acting as
molecular shields. Along with other hydrophilic proteins
and compatible solutes, LEA proteins might also serve as
“space fillers” to prevent cellular collapse at low water
activities. This multifunctional capacity of the LEA proteins
is probably attributable in part to their structural plasticity,
as they are largely lacking in secondary structure in the
fully hydrated state, but can become more folded during
water stress and/or through association with membrane
surfaces. The challenge now facing researchers investigating these enigmatic proteins is to make sense of the
various in vitro defined functions in the living cell: Are
A. Tunnacliffe (*)
Institute of Biotechnology, University of Cambridge,
Cambridge, UK
e-mail: [email protected]
M. J. Wise
Biomolecular, Biomedical and Chemical Sciences,
University of Western Australia,
Perth, Austria
the LEA proteins truly multi-talented, or are they still just
misunderstood?
Keywords Anhydrobiosis . Desiccation tolerance .
Drought stress . Cold stress . Water stress
...a riddle wrapped in a mystery inside an enigma...
Sir Winston Churchill
Introduction
Churchill was describing the possible actions of Russia
during the early days of the Second World War, but we
might, with scientific license, adopt his phrase for a
description of our understanding of the burgeoning family
of LEA proteins. Despite being discovered more than
20 years ago, the precise function of these intriguing
macromolecules is still unclear, and worse, as we learn
more about them, we uncover further interesting but
puzzling properties so that much of the information
obtained provides more questions than answers. Potentially,
the experience of attempting to understand LEA protein
function could act as an object lesson for post-genomics
projects. Genome sequencing has uncovered many novel
genes in all species studied, which have no or little
similarity to genes of known function. For example, in the
summary of statistics for the coverage of different proteomes by InterPro descriptions, 25.3% of the human
proteome is not covered by InterPro terms (http://www.
ebi.ac.uk/integr8). If each novel gene or gene family
requires even a fraction of the time and effort so far
devoted to the LEA proteins, then we are in for a very long
792
haul before we can claim a comprehensive understanding of
even a single genome.
LEA stands for late embryogenesis abundant, as coined by
Galau et al. (1986), the discoverers of the LEA proteins in the
cotton plant Gossypium hirsutum (Dure et al. 1981; Galau and
Dure 1981). As the first reports, many similar proteins and
their genes or complementary DNAs (cDNAs) have been
described in other plant species (Dure et al. 1989; catalogued
in Wise 2003). Their name reflects the fact that the proteins
originally described are expressed at high levels during the
later stages of embryo development (post-abscission) in plant
seeds. As, at this stage in the development process, orthodox
seeds acquire the ability to withstand extreme dehydration,
LEA proteins have been associated with desiccation tolerance
(reviewed in Cuming 1999).
However, as further studies were carried out in plants
and more recently other types of organism, including
invertebrates and microorganisms, the concept of what
constitutes an LEA protein has become increasingly
blurred. Classifying proteins solely on the basis of
expression profile is fraught with danger, as it is negated
each time an exception is discovered. Indeed, in plants,
many exceptions are known where, for example, LEA
protein expression is not restricted to embryonic tissues or
where expression is associated with other stresses besides
desiccation, chiefly cold stress; a few LEA protein genes
are even apparently constitutively expressed [for a fuller
discussion of this point, and many examples, see Wise
(2003)]. While the nomenclature is unsatisfactory in plants,
the nature and categorisation of LEA proteins became even
more problematic when they were discovered in organisms
outside the plant kingdom. Clearly, the idea of defining a
nematode protein, for example, by reference to expression
of plant seed proteins, is nonsensical.
This difficulty is, at least in part, a reflection of our poor
understanding of LEA protein function. If we had greater
insight into the role of LEA proteins, then we could choose
a more appropriate name for them. An analogous situation
is that of the large group of proteins known as heat shock
proteins. The heat shock response began to be investigated
in the fruit fly Drosophila melanogaster in the early 1960s
and was soon recognised as an important stress response in
essentially all organisms, where multiple genes and their
cognate proteins are up-regulated on thermal stress.
However, more than 20 years after the first studies, the
role of the various heat shock proteins was still not clear,
despite much information on their sequence and regulation
(Lindquist 1986).
It is instructive to read Lindquist’s (1986) review of the
heat shock proteins in the light of our current understanding
of the LEA proteins. There are distinct parallels in that
much of the heat shock protein literature at that time was
phenomenological. Very soon, however, experiments
Naturwissenschaften (2007) 94:791–812
demonstrating function became prevalent, such that several
families of heat shock proteins were characterised as
molecular chaperones and their roles in protein folding
defined (Ellis and Vandervies 1991; Gething and Sambrook
1992). It would seem then that we could make a similar
rationalisation of LEA protein terminology if we could
determine their function. Although we have not yet reached
a position where a new nomenclature for LEA proteins can
be put in place, we have perhaps taken some steps recently
towards this goal. This review outlines the current picture
of the field and measures some of the progress towards
functional characterisation of the LEA proteins: We first
analyse the characteristics of the LEA proteins, then
examine the literature on their function, and finally give a
brief outline of what still needs to be done to further our
understanding of the role of LEA proteins in water stress
tolerance.
The characteristics of LEA proteins
Sequence motifs and peptide profiles
Several nomenclatures have been developed. LEA protein
sequences, derived from their cDNAs and genes, were first
described in cloning experiments with cotton seeds (Baker
et al. 1988), but it was soon realised that similar genes were
being found in other plant species and that these fell into a
few groups of related sequences. LEA protein groups 1, 2
and 3 were identified, and members of each group were
categorised by the presence of particular sequence motifs
(Dure et al. 1989): Group 1 proteins are characterised by a
hydrophilic 20-amino-acid motif; group 2 proteins have at
least two of three distinct sequence motifs (named Y, S and
K by Close 1997); and members of group 3 contain
multiple copies of an 11-amino-acid motif (Table 1). A
more sophisticated version of this idea has been the recent
appearance of Pfam motifs for the respective LEA protein
groups, each defined by a hidden Markov model based in
the first instance on a curated multiple sequence alignment
(Bateman et al. 2004). The Pfam families corresponding to
the different LEA protein groups are also shown in Table 1.
Other sequence-based evidence can also be helpful. For
example, the group 3 LEA protein sequences have runs of
regularly spaced lysines appearing with period 11 (i.e.
another Lys appearing every 11 aa). Group 3 LEA protein
sequences also have regularly spaced Ala and Asp and/or
Glu, all with period 11. On the other hand, regularly spaced
Gly and Glu with period 20 supports inclusion among the
group 1 LEA proteins. By contrast, group 2 LEA protein
sequences have periodic repeats with a range of periods
(beyond the well-known poly-Ser stutters or the somewhat
less common poly-Lys stutters).
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Table 1 LEA group motifs and corresponding Pfam families
Group
1
2
2
2
2
3
4
6
Lea5
Motif Name
Y
S
K
Motif Sequence
Reference
Pfam
GGQTRREQLGEEGYSQMGRK
Cuming 1999
PF00477
PF00257
DEYGNP
Sn
EKKGIMDKIKEKLPG
TAQAAKEKAXE
Close 1997
Close 1997
Close 1997
Cuming 1999
PF02987
PF03760
PF04927
PF03242
Group 2 LEA proteins were initially characterised on the basis of having at least two of three motifs, labeled Y, S and K. There is a single Pfam
family encompassing the three motifs. The Pfam database can be accessed at http://www.sanger.ac.uk/Software/Pfam or several mirrors
worldwide.
An alternative naming scheme, proposed by Dure
(1993), labelled the groups according to the cottonseed
prototype, i.e. D-19 for group 1, D-11 for group 2 and D-7
for group 3. However, most workers use group 1, group 2,
etc., first introduced by Dure et al. (1989) and further
developed by Bray (1993, 1994) and Cuming (1999).
Group 2 proteins are also called dehydrins (dehydrationinduced proteins; Close et al. 1989) by many researchers,
but this term seems rather arbitrary, as it could equally
apply to the other major LEA protein groups. Although
most LEA proteins fall into the three main categories, a few
other minor groups have also been proposed (Bray 1993,
1994): group 4 (D-113), group 5 (D-29) and group 6
(D-34). There are also two unnumbered groups named after
particular exemplars: Lea5 (D-73) and Lea14 (D-95; Galau
et al. 1993). As they are discovered, new examples of LEA
proteins are usually assigned to one of these groups
depending on sequence relatedness and the presence of
the characteristic motifs, but Wise (2003) also highlights a
few cases not assigned to any group.
Wise (2003) has refined the group nomenclature based on
newly-developed bioinformatics tools, chiefly peptide profile
(POPP) analysis. This has led to the definition of superfamilies (SFs) of LEA proteins, with one or more SFs
comprising each of the main groups (Table 2). Using this
information, group 2 can be split into groups 2a and 2b,
corresponding to SF1/10 and SF3, respectively, and these
subsets seem to fit with known properties of the proteins.
Members of group 2a show a canonical expression pattern,
i.e. literally late in embryogenesis, and are likely to have a Y
motif, while Group 2b proteins are associated with cold
tolerance, or at least are not produced late in embryogenesis
(Wise and Tunnacliffe 2004), and are unlikely to have the Y
motif (especially those most associated with cold tolerance).
Group 2a proteins are neutral or basic in overall charge, with
over-representation of glycine; group 2b proteins have
similar levels of basic residues to group 2a but, in addition,
have increased levels of acidic residues and correspond to
the acidic subset of dehydrins first noted by Danyluk et al.
(1994). Some authors identify phosphorylation-dependent
metal-ion binding properties with the acidic group 2b LEA
proteins (Alsheikh et al. 2005), but others show metal-ion
binding without phosphorylation (Hara et al. 2005), while a
third group use metal ion binding to purify both groups 2a
and 2b proteins [Svensson et al. 2000; note: Wise 2003
originally defined three subgroups of group 2 proteins, but
subsequent analysis shows that the original group 2c can be
included in group 2b (MW, unpublished data)]. Group 2a
sequences have periodic repeats of Gly, particularly with
period 3, while the group 2b sequences (especially those
most implicated in cold tolerance) have few if any periodic
repeats of Gly but more periodic repeats involving Lys or
Glu. Subgroups of groups 1 and 3 proteins can also be
defined by the new bioinformatics, but at this time, the
subgroups do not appear to correlate with known functional
or structural characteristics. A more radical result of the
POPP analysis has been the elimination of groups 4 and 5,
whose members redistribute to groups 2 and 3. The other
minor groups, i.e. group 6, and the Lea5 and Lea14 groups
retain their integrity, by and large (Table 2), but it is
debatable whether, in fact, they should be categorised as
LEA proteins (see below).
Perhaps the most significant outcome of this approach is
that the POPPs characterising each SF can be compared with
peptide profiles generated from proteins of known function.
Where similarities are detected, it is hypothesised that this
reflects similarity of function, and therefore, putative LEA
protein functions can be proposed and subsequently tested
(Wise 2003; Wise and Tunnacliffe 2004). We have used this
facility to investigate a novel activity for some LEA proteins
(Goyal et al. 2005a; see below).
LEA protein phylogeny
Establishing the phylogeny of the LEA proteins with any
certainty is very problematic. On the one hand, there is
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Table 2 LEA group, POPs superfamily and consensus POPP for anchor family
Group
SF
Anchor Family Consensus POPP
1
1
2
2
2
2
2
3
3
4
6
1
3
8
9
10
2
5
6
Lea5
Lea14
7
(299)
(297)
+E, +G, +EG, +GE, +GG, +KG, +QE, +RK, +GGE, +KGG
+E, +G, +DE, +EG, +ES, +GG, +GQ, +RE, +RK, +ARE, +DES, +REG
+G, −L, +EK, +GG, +GT, +EKL, +IKE, +KEK, +KIK, +KKG, +KLP, +LPG
−F, −I, −L, −R, −W, +DK, +EK, +KK, +KL, +LP, +TH, +EKK, +KEK, +KLP, +LPG
+EK, +SS, +EKI, +KEK, +KIK, +SSS
−F, +G, −I, −L, −V, +AG, +EK, +GG, +GH, +GT, +TA, +TG, +GGT, +GTG, +TAG, +TGG
−F, +G, −I, +AG, +EK, +GG, +GQ, +KE, +SS, +EKL, +GAG, +IKE, +KEK, +KLP, +LPG, +SSS
+A, −C, +E, −F, −I, +K, −L, −P, +AE, +AK, +EK, +ET, +GE, +GK, +KE, +AAE, +AKD, +EKA
+A, −I, +K, −L, −P, +Q, +T, −V, +AA, +AQ, +EK, +KE, +KT, +QA, +QQ, +QS, +QT, +TQ, +AAK, +AQA, +EKT, +QAA,
+TQQ
+A, −F, −L, +AA, +AE, +MQ, +QS, +VA, +AAA, +GVA, +QSA, +SAA
+A, +R, +S, +AM, +GA, +GY, +RP, +SF, +SS, +YS
+D, −R, +AS, +IP, +KV, +VS, +TIP
POPPs representing the protein sequences are clustered into families and superfamilies (SF). The superfamilies closely mirror the structure of the
LEA groups, with the exception of groups 4 and 5. Against each LEA group are listed the superfamilies that contain proteins from that group
(column 2) and the peptides forming the consensus POPP of the anchor (i.e. most typical) family in the superfamily. Plus sign before a peptide
indicates significant over-representation; minus sign indicates significant under-representation. Note that SF8 and SF9 only contain a single
family but are included for completeness. The minor groups Lea5 and Lea14 have too few representatives to form a family and are here
represented by a single cluster each (Table adapted from Table 13 of Wise 2003).
much sequence diversity even within groups despite
sufficient conservation for Pfam HMM models to have
been created. There is also a level of similarity between the
three principal groups. For example, Wise (2003) notes that
when POPP clustering thresholds are reduced groups 2 and
3 are clustered together. To gain some idea of how the
world of principal LEA protein groups may be structured, a
draft phylogenetic tree was created based on the set of
proteins for LEA groups 1 to 4 found in Wise (2003),
together with the group 1 sequence from Bacillus subtilis
and the group 3 sequence from Deinococcus radiodurans
(non-plant LEA proteins are discussed further below). The
results are shown in Fig. 1, whose caption outlines the
methods used. Examples have been found of non-plant
groups 1 and 3 LEA proteins, but no examples have yet
been found of group 2 proteins outside the plant kingdom.
Placing that in the context of the tree, it seems possible that
LEA proteins derive from ancestral bacterial proteins.
These ancestral proteins would be found on the phylogenetic tree somewhere between the points where groups 1
and 3 LEA proteins branch off. Given the more complex
domain structure of group 2 LEA proteins, of whose three
motifs only the K motif in any way resembles those of
groups 1 or 3, it is likely that group 2 LEA proteins are an
innovation unique to plants. More definitive statements,
however, must be postponed until a systematic, criteriareferenced search has been undertaken.
Expression profile
The original definition of the LEA proteins involved two
characteristics: gene expression at a specific stage of plant
seed embryogenesis and, de facto, sequence similarity to
canonical LEA proteins. Hughes and Galau (1989) proposed reserving the Lea designation for only those genes
expressing post-abscission and using the term LeaA to
describe genes that also have a smaller expression spike
during the earlier maturation stage (associated with abscisic
acid) in addition to the much larger post-abscission peak.
While the proposal never caught on, induction by abscisic
acid came to be one of the other expression hallmarks for
LEA protein genes. Response to desiccation stress, salt
stress and cold stress have also been noted (see discussion
in Bray 2000 and Wise 2003), and improved stress
tolerance often correlates with LEA protein expression.
For example, Blackman et al. (1995) showed that when
abscisic acid was used to activate LEA protein gene
expression prematurely in immature soybean seeds, a
corresponding improvement in cell integrity was noted
after desiccation stress.
Over time, many new examples have been defined as
LEA proteins on the basis of only one of the above
characteristics, i.e. according to either expression pattern or
sequence similarity alone. In the former case, where no
sequence relatedness to previously recognised LEA proteins was evident, such proteins have led to the designation
of new groups (e.g. Lea5 or Lea14; Galau et al. 1993), or at
least to a recognition that they fall outside the existing
categories. Where the expression profile has proved
different to that of the original prototypes, these proteins
are sometimes referred to as “LEA-like” proteins. For
example, dehydrin Xero1 (XERO1_ARATH; P25863) has
often been included among the group 2 LEA proteins.
However, neither is it found in seeds, nor is it inducible by
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795
Fig. 1 Draft phylogenetic tree for LEA protein groups 1, 2 and 3 and
former group 4. The set of LEA protein sequences used in this
analysis, representing groups 1, 2, 3 and former group 4, was
essentially the same as the sets used in the analyses reported in Wise
(2003), except that almost all the identifiers have changed in the
intervening time. A multidomain group 3 LEA protein, Q41060_PEA,
was removed from the set; group 3 LEA protein UB72_DEIRA from
(Deinococcus radiodurans) and putative group 1 LEA GSIB_BACSU
(from Bacillus subtilis) were added. The set of proteins was processed
using nrdb90.pl (Holm and Sander 1998) so that no two sequences
were more that 90% similar. A multiple-sequence alignment was
created using Muscle (Edgar 2004) and, after conversion to Phylip
format, was passed to the maximum likelihood phylogenetic tree
construction application (proml; Felsenstein and Churchill 1996) from
the Phylip phylogenetic analysis suite (Felsenstein 2004). The slow/
accurate feature was specified together with global rearrangements so
that the best single tree would be returned. In addition, the input set
was jumbled three times and the best tree was returned (bootstrapping
has not been done, however). The resulting tree was visualised using
the drawtree application (also from the Phylip suite), with the output
further processed by hand using the Xfig application to colour the
protein identifiers for the different groups and to separate labels that
were covering each other
desiccation or cold stress or by application of abscisic acid;
rather, it appears to be constitutively expressed (Welin et al.
1994). Similarly, Q06431_BETVE, the BP8 protein from
birch, a homologue of the group 3 LEA protein
LEAD8_DAUCA, is itself only constitutively expressed
and at low levels (Puupponen-Pimia et al. 1993).
More recently, global analyses of transcriptomes and
proteomes have become technically possible. For example,
in Arabidopsis, whose genome contains more than 50 LEA
protein or LEA-like genes (Hundertmark and Hincha,
unpublished data), expression of the whole set can be
queried in different tissues and at developmental stages
under a variety of conditions. The LEA protein gene set
divides roughly into those with seed-specific expression
and those expressed in vegetative tissues, with surprisingly
little overlap (Illing et al. 2005; Hundertmark and Hincha,
unpublished data). This confirms that LEA proteins also have
a role to play in vegetative tissues of plants like Arabidopsis
thaliana, which are not desiccation tolerant. Intriguingly, at
least one Lea gene expressed in seeds of A. thaliana has an
orthologue that is up-regulated in desiccating leaves of the
resurrection plant Xerophyta humilis (Illing et al. 2005). This
is consistent with the hypothesis that resurrection plants
acquired systemic desiccation tolerance by reprogramming
seed-specific gene sets, although this presumes that LEA
proteins normally expressed in seeds are more potent
desiccation protectants than those expressed in non-seed
tissues. LEA protein gene expression also figured promi-
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protein isoforms (Röhrig et al. 2006; Irar et al. 2006) and has
been associated with functional characteristics of some
proteins (Riera et al. 2004; Brini et al. 2007), including
metal-ion binding (see below).
In non-plant organisms, of course, expression profiles of
Lea-like genes and LEA-like proteins cannot relate to seed
maturation, and examples from microorganisms or invertebrates have been identified on the basis of relatedness to
plant sequences (Table 3). To date, all non-plant LEA
proteins have been categorised as Group 3, with the single
exception of an apparently Group 1 protein from B. subtilis
(Stacy and Aalen 1998) [preliminary reports suggest that
the brine shrimp Artemia franciscana contains a group 1
protein, however (M Sharon, JS Clegg, A Warner, personal
communication)]. A link to tolerance of water stress is
maintained in invertebrates, as in the few examples studied
that gene expression is induced by dehydration. In the
anhydrobiotic nematode Aphelenchus avenae, the gene
Aav-lea-1, encoding group 3 LEA protein AavLEA1, is
up-regulated by desiccation and osmotic upshift but not by
cold, heat or oxidative stresses (Browne et al. 2002, 2004).
Unlike A. avenae, the entomopathogenic nematode
Steinernema feltiae is only partially desiccation tolerant, yet
it expresses Lea-like gene Sf-LEA-1 in response to dehydration (Gal et al. 2003). The model nematode, Caenorhabditis
elegans, can produce a so-called dauer juvenile larval stage
nently in the transcriptome of desiccation-tolerant bryophytes
undergoing rehydration, suggesting a role for these proteins
in recovery from desiccation, as well as the drying process
per se (Oliver et al. 2004). Boudet et al. (2006) performed a
global analysis of proteins whose expression is associated
with desiccation tolerance. These authors compared the heat
stable proteomes of radicles of germinating Medicago
trunculata seeds both before and after emergence: the
former are desiccation tolerant, the latter are sensitive. In
addition, desiccation tolerance can be reinduced in emergent radicles by an osmotic treatment. Boudet et al. looked
for proteins that were abundant during desiccation tolerant
states but present at low levels when rootlets exhibited
sensitivity. They found 15 such polypeptides on 2D protein
gels and identified 11 of them as LEA proteins by mass
spectrometry [arguably, though, one of these, MtPM25, is
not a true LEA protein, belonging to the D-34 group,
variously called group 5 (Cuming 1999) or group 6 (Bray
1994); see below]. These were shown to represent just six
different proteins (or five, if MtPM25 is excluded) from all
the main groups, some of which were present as different
isoforms. The presence of isoforms is intriguing but might
be explained by a susceptibility of some LEA proteins to
spontaneous modification by deamidation and oxidation
(Tolleter et al. 2007) or by other modifications. Phosphorylation is likely to be responsible for at least some LEA
Table 3 Non-plant LEA proteins with expression evidence
LEA group
Prokaryotes
1
3
3
3
3
3
3
3
Eukaryotes
3
3
3
3
3
3
3
3
3
3
3
ID
AC
Seq Len
Expr
Species
GSIB_BACSU
Q3EHT1_ACTSC
UB72_DEIRA
Q3XWV0_ENTFC
Q39ZB1_GEOMG
Y1339_HAEIN
Q11Z91_POLSJ
Q2LPL8_SYNAS
P26907
Q3EHT1
Q9RV58
Q3XWV0
Q39ZB1
P71378
Q4AXW6
Q2LPL8
122
173
298
200
94
129
195
113
?
?
D
?
?
?
?
?
Bacillus subtilis
Actinobacillus succinogenes 130Z
Deinococcus radiodurans
Enterococcus faecium DO
Geobacter metallireducens (strain GS-15)
Haemophilus influenzae
Polaromonas sp. JS666
Syntrophus aciditrophicus (strain SB)
LEA1_APHAV
AfrLEA1
AfrLEA2
Q61VH9_CAEBR
O16527_CAEEL
Q19790_CAEEL
Q54VG7_DICDI
Q8SVY9_ENCCU
Q1XI26_9DIPT
Q1XI25_9DIPT
Q1XI24_9DIPT
Q95V77
*
*
Q61VH9
O16527
Q19790
Q54VG7
Q8SVY9
Q1XI26
Q1XI25
Q1XI24
143
357
364
775
733
780
319
166
742
180
484
D
D
D
?
D
?
?
?
D
D
D
Aphelenchus avenae
Artemia franciscana
Artemia franciscana
Caenorhabditis briggsae
Caenorhabditis elegans
Caenorhabditis elegans
Dictyostelium discoideum
Encephalitozoon cuniculi
Polypedilum vanderplanki
Polypedilum vanderplanki
Polypedilum vanderplanki
ID denotes SwissProt identifier; AC, accession number; Seq Len, protein sequence length. The Expr column indicates whether expression has been
tested under conditions of desiccation stress (D) or otherwise (?). The sequences for the two proteins denoted by asterisks can be found in Hand
et al. (2006).
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under conditions where food is limiting. The dauer form
exhibits increased desiccation resistance compared to adult
worms, which are themselves considered desiccation sensitive. The desiccation resistance of dauer larvae is associated
with the expression of Ce-lea-1, a gene encoding a LEA-like
protein. Moreover, when Ce-lea-1 expression is reduced by
RNA interference, dauer juveniles exhibit significantly
increased mortality on dehydration (Gal et al. 2004). In the
anhydrobiotic larva of the chironomid Polypedilum vanderplanki, there are several proteins that are expressed during
desiccation or osmotic stress (Kikawada et al. 2006). While
the authors found three of these to be group 3 LEA proteins,
two of them have unusual properties: PvLEA1
(Q1XI26_9DIPT) and PvLEA3 (Q1XI24_9DIPT) have
periodic repeats involving leucine (not seen in other group
3 LEA proteins) and are predicted to be transmembrane
proteins. Group 3 LEA proteins are also found in
desiccation tolerant bdelloid rotifers (Tunnacliffe et al.
2005; Pouchkina-Stantcheva et al., in preparation), and
McGee (2006) found Western blot evidence for group 3
proteins in several other invertebrates. Finally, group 3 LEA
proteins have also recently been described in Artemia
franciscana (Hand et al. 2006).
Understanding expression of invertebrate LEA proteins
is complicated somewhat by the finding that corresponding
messenger RNA (mRNA) and protein levels do not
necessarily correlate in a simple manner. Thus, although
Aav-lea-1 is up-regulated in dehydrating Aphelenchus
avenae, its protein is already present in unstressed animals,
and on drying, AavLEA1 is processed from its full-length
form of 16.5 kDa into smaller polypeptides of 8–9 kDa. A
desiccation-dependent processing activity was detected in
protein extracts, which directed comparable cleavage of
recombinant AavLEA1 in vitro (Goyal et al. 2005b). The
out-of-phase expression patterns of mRNA and protein
have not been reported in other organisms to date, but
similar protein processing activities have been observed in
P. vanderplanki (Kikawada et al. 2006) and the bdelloid
rotifer, Adineta ricciae (McGee 2006). Processing probably
does not take place with all invertebrate LEA proteins,
however (Tunnacliffe et al. 2005; McGee 2006), so its
significance is unclear. One possibility is that the specific
activity of LEA protein is increased in this way (Goyal et
al. 2005b). Processing activity has not been reported for
plant LEA proteins during drying, or at least not commented on. Some Western blot data might be consistent
with processing, however. For example, group 3 LEA
protein HVA1 from barley (Hordeum vulgare) seems to
exist as two forms in seedlings (Hong et al. 1992) and in
transgenic rice (Oryza sativa) seeds but not leaves (Xu et al.
1996). Controlled proteolytic cleavage of group 1 Em
proteins has been associated with degradation of these
proteins during germination (Taylor and Cuming 1993a, b).
797
However, Bies et al. (1998) showed that proteolysis occurs
before germination proper, during inhibition of Arabidopsis
thaliana seeds, and it is possible that these cleavage events
could relate to Em function during water influx.
To summarise, there is a strong association in both plants
and animals between expression of the various categories of
LEA protein gene and tolerance to water stress, or at least
the anticipation of water stress, whether this expression is
developmentally programmed, as in the case of the
maturing plant seed, or is a response to environmental
stress. There are, of course, exceptions where Lea genes
might be constitutively expressed or expressed in response
to stresses other than decreased water activity, for example,
but these examples are relatively few in number. In
addition, expression of LEA proteins is clearly not
sufficient to confer desiccation tolerance, as they have been
found in desiccation-sensitive seeds (Finch-Savage et al.
1994). It is also true that at present we have little
information that is more than correlative, and therefore,
we must exercise caution.
There are, however, some reports that offer genetic
evidence for a causative link between LEA protein (gene)
expression and resistance to water stress. We have already
mentioned the experiments of Gal et al. (2004) in C.
elegans dauer larvae, but Battista et al. (2001) also showed
that a deletion mutant of a Group 3 Lea-like gene (DR1172)
in the desiccation tolerant bacterium D. radiodurans was
less able to withstand drying. In plants, elegant experiments
were performed with near-isogenic strains of cowpea
(Vigna unguiculata) differing at Dhn1, which encodes a
∼35 kDa group 2 LEA protein (dehydrin): In strains where
DHN1 protein is expressed, emerging seedlings show
improved chilling tolerance over those lacking the group
2 LEA protein (Ismail et al. 1999a). Intriguingly, although
V. unguiculata seeds are clearly viable in the absence of
DNH1, which would seem to rule out an essential role for
this protein in desiccation tolerance, the chilling sensitivity
of the Dhn1 variants is more pronounced when seeds are
dried to 6% moisture content compared to the 12%
moisture content more typical of commercial seed (Ismail
et al. 1997, 1999a). This might reflect increased “fitness” of
seeds containing DHN1 under extreme desiccation conditions. Recently, a knockout mutant of a dehydrin gene in
the moss Physcomitrella patens was reported, which
showed poor recovery from salt and osmotic stress
compared to wild type (Saavedra et al. 2006). However, it
should be pointed out that the sequence for the latter protein
is most unusual compared with other group 2 LEA proteins:
It has ten Y segments (compared to the usual one or two).
One problem with the genetic approach is that functional
overlap of multiple LEA proteins could obscure their role.
Carles et al. (2002) identified regulatory mutants of
Arabidopsis thaliana lacking, or severely reduced in, both
798
Naturwissenschaften (2007) 94:791–812
group 1 LEA proteins encoded in the genome but whose
seeds nevertheless remained desiccation tolerant. A similar
problem will pertain for other LEA proteins in Arabidopsis,
and therefore, multiple gene knockouts or knockdowns
might be necessary to demonstrate a phenotype. Despite
potential problems of this kind, more genetic experiments
will be necessary to increase confidence in the importance
of LEA proteins for water stress tolerance.
Subcellular localisation
Both computer predictions using TMHMM v.2 (Krogh et
al. 2001; MW, unpublished data) and experimental observations suggest that LEA proteins are not transmembrane
proteins. Rather, they are expressed in a number of
subcellular compartments, including chloroplasts, mitochondria, the nucleus, and in the cytoplasm (Table 4).
There is also at least one example (Ukaji et al. 2001) of two
closely related group 3 LEA proteins located in the
endoplasmic reticulum (ER) and two others that have an
N-terminal signal peptide suggesting translocation of the
ER membrane; the latter proteins were shown to be reduced
in Mr slightly when produced in the presence of microsomes, consistent with cleavage of the signal peptide
(Hsing et al. 1995). Other signal peptides are likely to
control targetting to mitochondria, plastids, etc., as appropriate. In the case of the maize group 2 protein, DHN1/
Rab17, distribution between nucleus and cytoplasm is
controlled by phosphorylation of its serine stutter: removal
of this sequence results in lack of phosphorylation and
retention in the cytoplasm (Jensen et al. 1998). Using
bioinformatics applications that are rather conservative
(Predotar and PredictNLS), subcellular locations for a small
number of other proteins can be predicted (Table 5). The
more liberal LOCTarget application (Nair and Rost 2004)
also classifies all of the group 1 LEA proteins as nuclear,
Table 4 LEA proteins with known subcellular localisation
ID
LEA group
Species
Location
Reference
DHN1_MAIZE
ERD14_ARATH
DHR18_ARATH
COR47_ARATH
Q9ZR21_CITUN
TAS14_SOLLC
DHR21_ORYSA
CS120_WHEAT
CO410_WHEAT
VCaB45b
LEA13_GOSHI, LEAD7_GOSHI
Q06540_WHEAT
Q8S385_SECCE
DRPF_CRAPL
Q42512_ARATH
LEAD8_DAUCA
Q39873_SOYBN
LEA1_HORVU
Q93Y63_9ROSA
Q41060_PEA
Q5NJL5_PEA
LEA1_APHAV
2
2
2
2
2
2
2
2
2
2
2,3
3
3
3
3
3
3
3
3
3
3
3
Zea mays (maize)
Arabidopsis thaliana (mouse-ear cress)
Arabidopsis thaliana (mouse-ear cress)
Arabidopsis thaliana (mouse-ear cress)
Citrus unshiu (satsuma orange)
Solanum lycopersicum (tomato)
Oryza sativa (rice)
Triticum aestivum (wheat)
Triticum aestivum (wheat)
Apium gravolens (celery)
Gossypium hirsutum (cotton)
Triticum aestivum (wheat)
Secale cereale (rye)
Craterostigma plantagineum (resurrection plant)
Arabidopsis thaliana (mouse-ear cress)
Daucus carota (carrot)
Glycine max (soybean)
Hordeum vulgare (barley)
Morus bombycis (mulberry tree)
Pisum sativum (garden pea)
Pisum sativum (garden pea)
Aphelenchus avenae (nematode)
Nuc/Cyt
Clp/Per
Nuc/Cyt
Nuc/Cyt
Mita
Nuc/Cyt
Cyt
Nuc/Cyt
PlMem
Vac
Cyt
Clp
Clp
Clp
Clp
Cyt
ER
PSV/Cyt
ER
Cyt
Mit
Cyt
Goday et al. 1994
SUBA At1g76180
Nylander et al. 2001
SUBA At1g20440
Hara et al. 2003
Godoy et al. 1994
Mundy and Chua 1988
Houde et al. 1995
Danyluk et al. 1998
Heyen et al. 2002
Roberts et al. 1993
NDong et al. 2002
NDong et al. 2002
Iturriaga et al. 1992
Lin and Thomashow 1992b
Franz et al. 1989
Hsing et al. 1995
Marttila et al. 1996
Ukaji et al. 2001
Alban et al. 2000
Grelet et al. 2005
Goyal et al. 2005b
The LEA proteins for which subcellular localisations have been determined experimentally: Clp Choroplast, Per (peroxisome), Nuc nucleus, Cyt
cytoplasm, Mit mitochondria, PlMem plasma membrane, ER endoplasmic reticulum/secreted, PSV protein storage vacuole, Vac vacuole. It
should be noted that localisation to the cytoplasm does not preclude further refinement to more specific sub-compartments. The references are to
the literature, apart from SUBA, which is followed by the TAIR gene name corresponding to the respective Arabidopsis thaliana LEA proteins.
SUBA results (Heazlewood et al. 2005; http://www.suba.bcs.uwa.edu.au/) are based on GFP tagging or cell fractionation and mass spectrometry
data. Note that Q41060_PEA is a multidomain protein. Some papers report localisation of LEA proteins by immunoelectron microscopy or
Western blotting using cross-species active antisera, e.g. Rinne et al. (1999) identify three different group 2 proteins in various subcellular
compartments in Betula pubescens (birch) using an antiserum raised against a Craterostigma plantagineum protein. Such examples have not
been included in Table 4 because a specific protein sequence has not been confirmed.
a
In transgenic tobacco
b
The sequence is given in Heyen et al. (2002).
Naturwissenschaften (2007) 94:791–812
799
Table 5 LEA proteins with predicted subcellular localisation
ID
LEA group
Species
Location
Predictor
ERD10_ARATH
ERD14_ARATH
Q9ZTR5_HORVU
Q39058_ARATH
Q42386_BRANA
Q39660_CHLVU
DRPF_CRAPL
Q39873_SOYBN
Q40869_PICGL
Q5NJL5_PEA
2
2
2
3
3
3
3
3
3
3
Arabidopsis thaliana
Arabidopsis thaliana
Hordeum vulgare
Arabidopsis thaliana
Brassica napus
Chlorella vulgaris
Craterostigma plantagineum
Glycine max
Picea glauca
Pisum sativum
Nuc
Nuc
Nuc
Clp?
Clp
Mit?
Clp
ER
Mit
Mit
PredictNLS
PredictNLS
PredictNLS
Predotar
Predotar
Predotar
Predotar
Predotar
Predotar
Predotar
The subcellular localisation predictor Predotar (Small et al. 2004) examines protein sequences for the presence of N-terminal signals, which allow
the sequences to be categorized as either mitochondrial, ER/secreted, plastid (for plant sequences) or “other”. The overwhelming majority of
LEA proteins are classified by Predotar as “other”. By contrast, the subcellular localization predictor PredictNLS (Nair and Rost 2004) looks for
a number of patterns principally based on Lys, appearing in the sequences, which are indicative of nuclear localization.
and all bar one of these as DNA-binding. This mirrors the
keyword analysis for this group reported in Wise (2003).
Overall, there is a strong inference that LEA proteins from
the principal groups are ubiquitous within cells and their
respective tissues, suggesting that their function is required
in all cellular compartments during water stress.
Structure
The first structural characterisation of a LEA protein was
published as early as 1985 by McCubbin et al., who used a
variety of biophysical techniques to study the wheat
(Triticum aestivum) group 1 protein, Em. Sedimentation
analysis and gel filtration gave a Stokes radius which was
higher than expected for a typical globular protein with the
molecular mass of Em, indicating a lack of compactness.
Viscosity measurements suggested that the protein has an
asymmetrical or flexible conformation, and far-UV circular
dichroism (CD) revealed little secondary structure (i.e. αhelix or β-sheet), with as much as 70% of the protein
behaving as random coil. The authors attributed the largely
unstructured nature of Em to its high hydration potential,
which in turn is due to an unusual amino acid composition,
a high proportion of Gly, Glu and Gln residues.
These early findings turn out to be largely typical for the
three major groups of LEA proteins. A group 1 LEA
protein (p11) from pea (Pisum sativum) was also shown to
be almost entirely unstructured by CD, with only an
estimated 2% of the protein folding as α-helix in water.
This was not changed appreciably by incubation at 80°C,
which is in keeping with a largely unfolded protein being
resistant to denaturation by heating and the general
observation that LEA proteins are heat soluble (Russouw
et al. 1995, 1997). Similarly, the Em homologue, EMB-1,
from carrot (Daucus carota) showed no secondary or
tertiary structure when examined by proton nuclear magnetic resonance (1H-NMR; Eom et al. 1996). The group 1
LEA protein, MtEm6, from M. trunculata is >50%
unstructured according to Fourier transform infrared spectroscopy (FTIR), although significant amounts of α-helix
(37%) and β-sheet (10%) were also found (Boudet et al.
2006). For the group 2 LEA proteins, examples from maize,
the resurrection plant Craterostigma plantagineum, cowpea, Citrus and Arabidopsis are also predominantly
unfolded (Ceccardi et al. 1994; Lisse et al. 1996; Ismail et
al. 1999b; Hara et al. 2001; Mouillon et al. 2006), as are
group 3 LEA proteins from bullrush pollen (Wolkers et al.
2001), the anhydrobiotic nematode Aphelenchus avenae
(Goyal et al. 2003) and soybean (Shih et al. 2004) [note: the
latter protein, GmPM16 from Glycine max, is termed group
4 by the authors, but would be categorised as group 3 under
the scheme of Wise (2003); see above]. It is worth
recording that many authors have supposed group 3 LEA
proteins to be largely α-helical based on analyses
performed using secondary-structure prediction programs.
However, such programs have usually been designed with
reference to PDB (http://www.pdb.org), a database of
experimentally determined protein structures, which is
largely made up of globular proteins (Gerstein 1998). Such
proteins are folded around a hydrophobic core, and their
folding is at least partially driven by the “hydrophobic
effect” (Scheef and Fink 2003). With highly hydrophilic
proteins such as the LEA proteins, this effect is absent and
interactions with solvent, i.e. water, predominate. By
summation of Gibbs energies of hydration for constituent
amino acids, McCubbin et al. (1985) calculated the relative
hydration potential of the group 1 LEA protein Em and
showed that this was “conspicuously higher than [...] for
most other proteins”. For the nematode group 3 LEA
protein, AavLEA1, Goyal et al. (2003) calculated that the
800
amount of associated water was >20-fold more than for a
typical globular protein of equivalent size. Using a novel
solid-state NMR relaxation technique, Bokor et al. (2005)
showed that a group 2 LEA protein also binds significantly
more water than globular proteins. The result is that little
organised secondary structure is observed.
Lack of conventional secondary structure means that
members of the major LEA protein groups are included in
the large class of proteins variously called “natively
unfolded”, “intrinsically disordered” or “intrinsically unstructured” (Uversky et al. 2000; Dunker et al. 2001;
Tompa 2002), and it is thus not surprising that attempts to
crystallise purified LEA proteins for X-ray crystallography
have reportedly been unsuccessful (e.g. McCubbin et al.
1985; Goyal et al. 2003) [there is one report of the solution
structure of Lea14 from Arabidopsis thaliana (Singh et al.
2005), but as argued below, this minor group protein is
atypical and can be considered to be outside the LEA
protein family]. Disorder prediction programs (e.g. at http://
www.pondr.com) suggest that 27–41% of all eukaryotic
proteins contain unstructured regions ≥50 residues long,
and that 6–17% of polypeptides are wholly disordered
(Dunker et al. 2001); therefore, many proteins are natively
unfolded or have natively unfolded domains.
A search of a database of LEA protein sequences using
the FoldIndex unfolded-protein prediction tool (Prilusky et
al. 2005) reveals that LEA proteins from groups 1, 2, 3 and
the former group 4 are at least 50% unfolded; many of the
smaller proteins are predicted to be totally unfolded (MW,
unpublished data). Such lack of structure clearly has
implications for LEA protein function: if they are almost
entirely unfolded, it is unlikely that they have catalytic
function, for example, unless they are induced to fold by
co-factor or substrate binding. Nevertheless, some of the
examples cited above show some secondary structure in the
LEA proteins studied. Moreover, there is a growing
realisation that many natively unfolded proteins are not
simply highly mobile random coils but have some localised
structural elements that are in equilibrium with unstructured
states. 1H-NMR studies on a group 2 protein (Lisse et al.
1996) uncovered transient extended substructures, for
example, and two papers from Soulages et al. (2002,
2003) describe polyproline-type II (PPII) extended, lefthanded helices in groups 1 and 2 LEA proteins, respectively, from soybean. PPII helices are highly hydrated and
are thought to be stabilised by the interaction of the peptide
backbone with water molecules that bridge adjacent
carbonyl groups, rather than direct intra-helix hydrogen
bonding (Bochicchio and Tamburro 2002). Mouillon et al.
(2006) also found variable content of PPII helices in
otherwise disordered structures of four group 2 LEA
proteins from Arabidopsis thaliana. One indicator of PPII
helices, in this case in equilibrium with either disordered or
Naturwissenschaften (2007) 94:791–812
β-turn conformations, is an isodichroic point at 208 nm in
temperature-dependent CD spectra (Bochicchio and
Tamburro 2002), and therefore, the data of Goyal et al.
(2003; Fig. 5 therein) are consistent with the presence of
PPII helices in group 3 LEA proteins also. It seems likely
then that localised PPII structures are found in all LEA
proteins of the major groups in solution.
This is not the whole story, however. Many natively
unfolded proteins are known to undergo increased folding
under some conditions, usually when they bind a partner
molecule or cation (Uversky et al. 2000; Dyson and Wright
2002). Environmental conditions can also affect folding,
and several LEA proteins become more structured when
dried. Thus, Wolkers et al. (2001) showed by FTIR that a
small (8 kDa) group 3 LEA protein from Typha latifolia
became largely α-helical when dried rapidly; slow drying
resulted in intermolecular β-sheet formation, as well as αhelix. Only α-helix formation was observed in the presence
of sucrose, however, regardless of rate of drying, which is
perhaps significant, given the presence of non-reducing
disaccharides in many desiccation tolerant systems. Similarly, the group 3 LEA proteins AavLEA1 from nematode
Aphelenchus avenae (Goyal et al. 2003; McGee 2006;
Zibaee et al., unpublished data) and LEAM from pea
mitochondria (Tolleter et al. 2007) also gain structure on
drying. Boudet et al. (2006) used FTIR to study both group
1 (MtEm6) and group 6 (MtPM25) proteins from M.
trunculata and found them both to have increased folding
in the dry state. The latter proteins also showed significant
secondary structure in the hydrated state, however, rather in
contrast to the results of other workers. Nevertheless, the
consensus seems to be that drying increases folding of at
least some LEA proteins, and it is tempting to speculate that
such desiccation-induced conformational changes are related to function. Interestingly, Boudet et al. (2006) showed
that drying needed to progress below an equilibrium
relative humidity of 85%, equivalent to a hydration level
of 0.2 to 0.3 g H2O/g dry weight where the hydration shell
of proteins begins to be lost, before conformational shifts
were observed. This is consistent with water binding to
LEA proteins being favoured over the intramolecular
interactions required to stabilise secondary structure, thus
inhibiting folding. Unpublished data of K. Goyal, showing
that high concentrations of Ficoll or dextran were unable to
effect folding in a nematode group 3 LEA protein in
solution, support the idea of water loss being the most
important factor rather than macromolecular crowding.
Koag et al. (2003) have also reported gain of structure
when cowpea DHN1 is incubated with small unilamellar
vesicles (SUVs): here, the group 2 LEA protein becomes
associated with SUVs and develops increased α-helical
character as measured by CD. However, this only occurs
when vesicles are prepared from phospholipids with acidic
Naturwissenschaften (2007) 94:791–812
head groups and not with phospholipids from cowpea tissue
itself. Similar results have been obtained for the group 3
LEA protein, AavLEA1, but the wheat group 1 protein,
Em, does not fold in the presence of SUVs (Zibaee et al.
unpublished data), although Em does show increased
structure on drying (Walton 2005). Both Koag et al.
(2003) and Zibaee et al. (unpublished data) emphasise the
similarity of this vesicle-associated folding behaviour to
that of α-synuclein (Jao et al. 2004), the vertebrate protein
implicated in the development of degenerative brain
conditions such as Parkinson’s disease and point out the
amphipathic nature of the α-helices formed by LEA
proteins and α-synuclein. It is proposed that this allows
the folded protein to “snorkel” in the phospholipid bilayer
(Mishra et al. 1994), with the more hydrophobic face of the
helix dipping into the fatty acid tail region and the charged,
hydrophilic face contacting the phospholipid head groups
on the surface. In the case of the LEA proteins at least, the
hypothesis is that association with the membrane stabilises
it in some way, particularly during periods of water stress.
Recent work from Tolleter et al. provides evidence for a
membrane stabilising role for a group 3 LEA protein
(Tolleter et al. 2007; see below). As the above studies on
conformational shifts in LEA proteins on drying and in
association with phospholipid vesicles have all been
performed in vitro, another critical question is how this
compares with the in vivo situation. For α-synuclein, for
which research is further advanced, ultrastructural investigations have shown its location close to, but not at,
intracellular vesicle surfaces (Clayton and George 1999). A
group 2 LEA protein has also been found close to
membrane surfaces (Danyluk et al. 1998), but a nematode
group 3 protein seems to be distributed homogeneously
throughout the cytoplasm (Goyal et al. 2005b). A similar
cytoplasmic distribution of the nematode protein expressed
in a human cell line was not apparently modified greatly by
dehydration (Walton 2005).
801
hydrophilic space on the hydropathy plot; this is markedly
different from the result with a “conventional” globular
protein like bovine serum albumin (BSA), where different
parts of the sequence are distributed between both
hydrophilic and hydrophobic space. This observation is
given some statistical underpinning in Table 6.
One consistent property of major group LEA proteins,
then, is hydrophilicity, and this is likely to be responsible
for their lack of conventional secondary structure in the
hydrated state, noted earlier. Similarly, the ability of LEA
proteins to remain soluble at elevated temperatures can be
attributed to their hydrophilic, unstructured nature: Heatinduced aggregation of proteins results from partial denaturation and association through exposed hydrophobic
regions, something that cannot occur in a protein which is
hydrophilic and natively unfolded. However, it is not clear
that other overarching properties can be identified for the
major group LEA proteins as a whole. Thus, they are
variable in size (e.g. the group 3 LEA protein from T.
latifolia is 8 kDa, whereas the group 3 protein in C. elegans
encoded by Ce-lea-1 is predicted to be 77 kDa) and net
charge (e.g. wheat group 3 proteins can be basic [LEA3_WHEAT, pI 9.4] or acidic [Q03967_WHEAT, pI 4.7]).
There are one or two observations that we can make within
individual groups, however. For example, all the group 1
proteins, in the set of Wise (2003) at least, are acidic to
(near) neutral (pI range 5.0 to 7.7); there are no basic
proteins. For group 2, the overwhelming majority of
proteins designated as SF3, predominantly group 2b (Wise
2003) or SK3 (Close 1997) proteins, are acidic and are most
closely associated with cold tolerance; this might correlate
with metal binding, perhaps enhanced by phosphorylation
Table 6 Counts of positive and negative average hydrophobicity per
residue
LEA group
Biochemical properties
Most of the biochemical properties of LEA proteins have
been alluded to in the sections above and result from their
hydrophilic nature. This is simply demonstrated by running
a hydropathy plot, as originally defined by Kyte and
Doolittle (1982), where a sliding window, typically between 7 and 21 amino acid residues in width, is used to
score hydrophilicity/hydrophobicity of side chains along
the length of the protein. Programs are available at several
websites and in sequence analysis packages (e.g. the SDSC
Biology Workbench, http://www.workbench.sdsc.edu, or
the ExPASy Proteomics Server, http://www.expasy.org).
For the LEA proteins of all the three major groups, a large
part of the, and often the whole, sequence falls into
1
2
3
4
6
Lea5
Lea14
GRAVY per residue
Count <0
Count ≥0
14
48
29
3
1
3
0
0
2
0
1
4
0
4
p value
3.34 e-6
8.14 e-17
4.79 e-12
0.19
0.46
0.067
0.124
For each LEA group, including former group 4, counts are recorded of
the numbers of sequences with grand average hydrophobicity
(GRAVY) per residue greater than or equal to 0, signifying a
hydrophobic protein, or less than 0, signifying a hydrophilic protein,
based on the Consensus scale (Eisenberg 1984). A p value for each
observation has been computed based on similar counts of all the
protein sequences in the UniProt-SwissProt and UniProt-TrEMBL
databases.
802
(see below). In general, based on percentage amino acid
composition, group 2b LEA proteins have more Lys
residues and more Glu residues than group 2a; for group
2b, the Glu residues predominate. There are exceptions,
however, such as Q9XEL3 from Picea glauca, which is a
group 2b member, with the SKN motif arrangement, but
which has a pI of approximately 7. In this case, the
percentage composition of Glu, although high, is still less
than that of Lys.
Are group 6, Lea5 and Lea14 members really LEA
proteins?
Doubts have been mounting for some time about whether it
makes sense to include among the LEA proteins those that
now comprise group 6, Lea5 and Lea14. It was already
noted in Wise (2003) that proteins from these three groups
have fewer polar residues and higher average hydrophobicity than sequences from the three principal LEA protein
groups. Furthermore, in contrast to groups 1, 2 and 3
proteins, which are largely natively unfolded, a Lea14
protein from Arabidopsis thaliana is structured, as determined by NMR (PDB entry 1xo8; Singh et al. 2005). It
seems likely that the Lea5 and group 6 proteins will also
be structured. Recently developed programs that predict
whether a protein is natively unfolded support this view.
When O03983_ARATH, the Lea14 sequence corresponding to 1xo8 (above), the canonical group 6
protein LE34_GOSHI and the Group 3 LEA sequence
LEA1_APHAV are submitted to FoldIndex (Prilusky et al.
2005), the first is judged to be completely folded, the
second is predicted to have an unstructured N-terminal
region of 39 aa, but to be otherwise completely structured,
while the group 3 LEA protein is found to be totally
unstructured, the latter prediction fitting experimental
observations (Goyal et al. 2003).
Recalling that LEA proteins have been recognised by
comparison with a canonical set of sequences and
recognising both that many other (i.e. non-LEA) proteins
are expressed in plants during embryogenesis and that
Lea5, Lea14 and group 6 (D34) proteins are very different
from groups 1, 2, and 3 LEA proteins, we believe it is now
time to regard these proteins as being something other than
in the LEA protein family.
Summary
The information currently available on LEA protein
sequence, expression, structure and biochemistry suggests
that we can offer a fairly clear picture of what an LEA
protein is: a highly hydrophilic protein from one of three
groups with characteristic sequence motifs, which is largely
unstructured in the hydrated state. In general, LEA proteins
Naturwissenschaften (2007) 94:791–812
are associated with tolerance of water stress and/or cold
stress in plants, invertebrates and prokaryotes, although
much of the data are correlative at present and we require
more in vivo genetic or molecular evidence to strengthen
this conclusion. Several studies have demonstrated that
drying LEA proteins can increase their degree of secondary
structure, and a few reports suggest that groups 2 and 3
proteins form α-helix in contact with certain phospholipid
membranes. A propensity for gain of structure under some
conditions might also therefore be a general property of at
least two of the main groups.
Finally, LEA proteins are usually considered to be single
domain proteins, although perhaps with a single domain
appearing multiple times. It is therefore worth noting that a
likely LEA protein has been found with LEA domains
appearing in a true multi-domain architecture. In sequence
Q41060_PEA, SBP65, the C-terminal region has several
clear repetitions of the group 3 motif, but the first 175
amino acids are not related to LEA proteins, and indeed the
function of this domain remains obscure beyond its ability
to act as a sink for biotin (Alban et al. 2000). At this stage,
it is not clear how LEA protein domains might function in
multi-domain proteins.
LEA protein function
Transgenic studies
Besides the extensive correlative data linking the expression of LEA proteins or their genes with stress resistance,
the few genetic studies mentioned above imply a role for
LEA proteins in desiccation tolerance of prokaryotes
(Battista et al. 2001), in dehydration resistance of C.
elegans dauer larvae (Gal et al. 2004) and in chilling
tolerance of cowpea seedlings (Ismail et al. 1999a). There
are numerous additional studies where heterologous LEA
protein genes have been introduced into plants or microorganisms, which then showed a degree of increased stress
tolerance. Thus, several groups have used bakers’ yeast
(Saccharomyces cerevisiae) to test LEA protein function.
The tomato (Solanum lycopersicum/Lycopersicon esculentum) group 4 protein LE25 (reassigned to group 3 by Wise
2003) improved salt tolerance when expressed in yeast:
Transformants grew significantly better than wild type in
media supplemented with 1.2 M NaCl, although rather
worse in 2 M sorbitol, indicating that the protection
conferred is against salt stress, rather than osmotic stress
per se. Freezing tolerance was also improved in the LE25containing yeast strain (Imai et al. 1996), as it was in yeast
expressing an algal protein (from Chlorella vulgaris)
related to group 3 LEA proteins (Honjoh et al. 1999).
Swire-Clark and Marcotte (1999) reported somewhat
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different results with yeast expressing the wheat group 1
protein, Em, where no protection against freeze stress was
apparent; growth in medium supplemented with both salt
(1 M NaCl or KCl) and polyol (1.5 M sorbitol) osmotica
was improved, however. Zhang’s group (Zhang et al. 2000)
extended their earlier investigations by testing tomato LE4
(group 2; now called TAS14, a homologue of DHN1/
Rab17) and barley group 3 LEA protein HVA1, in
comparison to LE25, in S. cerevisiae. They found that
HVA1-expressing yeast behaved similarly to those containing LE25 and displayed improved salt tolerance to both
NaCl and KCl but that LE4/TAS14 only conferred
increased tolerance to KCl and not NaCl. Again, none of
the LEA proteins afforded protection when sorbitol was
used as osmoticum. Zhang et al. (2000) attributed the
different behaviour of the various yeast transformants to
functional divergence among the LEA proteins used.
Less is known about how LEA proteins might affect
prokaryotic stress tolerance, but Liu and Zheng (2005) have
shown that Escherichia coli expressing recombinant PM2, a
group 3 LEA protein from soybean (Glycine max), were
more tolerant of salt (0.5 M NaCl or KCl), but not a polyol
osmoticum (1.1 M sorbitol), than control bacteria. This is
reminiscent of the results of Zhang et al. (2000) using yeast
as the recipient cell type, suggesting that group 3 LEA
proteins are effective against a salt stress rather than a
purely osmotic stress. E. coli expressing a modified
recombinant PM2 with a duplicated 22-mer repeat region
(a variant of the 11-mer motif typical of group 3 proteins)
exhibited higher salt tolerance levels than bacteria containing proteins with a single repeat region, implicating the
repeat region as the functional domain of the LEA protein.
This approach was extended to analyse the protective effect
of soybean LEA proteins from all three major groups:
group 1 (PM11; Em homologue) and group 3 (PM30)
proteins offered protection against both salt stress and cold
stress, but the group 2 protein, ZLDE-2, was ineffective
(Lan et al. 2005). Intriguingly, Campos et al. (2006) have
presented evidence suggesting that certain LEA proteins are
growth inhibitory in E. coli. Thus, group 2 (ERD10) and
group 4 (LEA4-D113), but not group 1 (Em6) and group 3
(LEA3-76), proteins from Arabidopsis thaliana prevented
bacterial cell growth when expressed as recombinant
proteins. Using partial deletion mutants, the authors were
able to localise the inhibitory activity to the K-stutter and
K-segments in the group 2 protein and to the N-terminal 32
amino acids of the group 4 protein. Campos et al.
hypothesise that the cationic nature of these sequences
might be responsible for the antibiotic effect. Many other
groups have produced recombinant group 2 LEA proteins
in E. coli but have not reported an effect on bacterial
growth. However, as, invariably, inducible plasmid expression systems are used and recombinant gene expression is
803
induced after most bacterial growth has taken place, this
effect would not perhaps be noticed. Nevertheless, if the
phenomenon is confirmed, it would mean that bacteria or at
least E. coli might be of limited use for investigating the
physiological role of some LEA proteins.
Other workers have constructed transgenic plants
expressing LEA protein genes; e.g. the barley HVA1 gene
(encoding group 3 LEA protein sequence LEA1_HORVU)
conferred enhanced tolerance of water stress and salt stress
in transgenic rice (Xu et al. 1996; Rohila et al. 2002). Such
transgenic plants were able to maintain higher relative
water content in their leaves than non-transgenic controls
and suffered less electrolyte leakage from cells (Babu et al.
2004; Rohila et al. 2002), suggesting that the HVA1 protein
might protect cell membranes from injury during drought.
Groups 1 and 2 LEA protein genes from wheat were also
able to offer rice similar protection against dehydration
stress (Cheng et al. 2002) and the same barley HVA1 gene
introduced into wheat endowed better growth and water use
under water stress conditions (Sivamani et al. 2000).
Another group 3 LEA protein, from rapeseed (Brassica
napus), was used to make transgenic lines of Chinese
cabbage (B. campestris), resulting in improved salt and
drought tolerance (Park et al. 2005).
Results reported using group 2 LEA proteins have been
somewhat variable. Strains of transgenic tobacco (Nicotiana tabacum) constitutively expressing a Citrus unshiu
group 2 LEA protein showed better germination and growth
than controls at low temperature (15°C), correlating with
level of protein expression, and reduced electrolyte leakage
and lipid peroxidation on chilling and freezing. The authors
show that the group 2 protein is localised to mitochondria
in the transgenic tobacco (although they have not confirmed
its localisation in C. unshiu) and acts as an antioxidant there
(Hara et al. 2003). Others have also reported enhanced cold
tolerance in transgenic plants expressing group 2 LEA
proteins. Thus, Houde et al. (2004) introduced wheat
COR410 into strawberry and found a 5°C improvement in
freeze tolerance of leaves expressing the protein compared
to controls, although this was only apparent after cold
acclimation. Maize DHN1/Rab17 overexpression in
Arabidopsis thaliana improves osmotic stress tolerance
(Figueras et al. 2004). Yin et al. (2006) introduced a potato
(Solanum sogarandinum) group 2 protein, DHN24
(Q7Y1A0_SOLSG), into cucumber (Cucumis sativus) and
demonstrated enhanced chilling tolerance in most transgenic strains and improved freeze tolerance in one. However,
there have also been reports of only slight or no effect on
cold stress tolerance in transgenic plants expressing group 2
proteins. For example, introduction of two dehydrins and a
group 3 LEA protein from Craterostigma plantagineum did
not improve drought tolerance of transgenic tobacco
(Iturriaga et al. 1992). Similarly, overexpression of
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RAB18 (DHR18_ARATH) in Arabidopsis thaliana did not
improve freeze or drought tolerance (Lång 1993). With the
latter study in mind, Puhakainen et al. (2004) generated
Arabidopsis strains overexpressing pairs of group 2 LEA
proteins, i.e. RAB18 and COR47, and LTI29 (ERD10) and
LTI30 (Xero2). Both sets of modified Arabidopsis lines
outperformed wild type in freeze tolerance assays (survival,
electrolyte leakage), with the LTI29/LTI30 lines better than
those overexpressing RAB18 and COR47. No differences
in drought tolerance were observed, however. The implication of the experiments of Puhakainen et al. (2004) is that
two group 2 proteins are better than one, but it would be
interesting to know how the single gene overexpressing
lines [other than for RAB18 (Lång 1993)] would perform.
One consistent feature with the dehydrin studies is that
where a group 2b protein is used, particularly those
identified with SF3, chilling and freeze tolerance seem to
be improved in the recipient plant; where the transgene
encodes a group 2a protein (e.g. RAB18), little or no effect
is seen.
systems such as those of chloroplasts and that lamellar to
hexagonal II phase transitions can promote damaging
fusion events between the respective membranes.
COR15am seems able to decrease these freeze-induced
phase transitions, perhaps by altering the curvature of the
chloroplast inner membrane (Steponkus et al. 1998).
Q42512_ARATH (COR15a) was identified as a group 3
LEA protein by Wise (2003), but its inclusion in this
category is rather marginal (the mature protein, COR15am,
lacks amino acids with period 11, other than Ala; in
particular, no periodicity of Lys is seen. The match with
Pfam motif LEA_4 is weak, with an e-value of 1.2; other
non-LEA protein motifs score better. Finally, a BLAST
search with the COR15a sequence finds similar low
temperature responsive proteins, but more typical group 3
LEA proteins only appear with poor e-values). Overall, this
example could be an object lesson for in vitro studies with
LEA proteins.
In vitro studies: a caveat
A number of groups have shown that LEA proteins, most
often from group 2, can protect proteins such as LDH
against freeze damage (Kazuoka and Oeda 1994; Houde et
al. 1995; Wisniewsk et al. 1999; Honjoh et al. 2000; Hara et
al. 2001; Bravo et al. 2003; Momma et al. 2003; SanchezBallesta et al. 2004; Goyal et al. 2005a). More recently,
studies have also been performed to evaluate protection
against desiccation (without freezing), and examples from
all main LEA protein groups seem able to offer protection
to sensitive enzymes such as LDH, malate dehydrogenase,
fumarase and citrate synthase (CS; Sanchez-Ballesta et al.
2004; Grelet et al. 2005; Reyes et al. 2005; Goyal et al.
2005a). Intriguingly, other highly hydrophilic proteins
which cannot be assigned to LEA protein groups seem to
possess similar protective activity (Reyes et al. 2005;
McGee 2006).
Goyal et al. (2005a) have provided evidence that at least
part of the protective function of LEA proteins is due to an
ability to prevent aggregation of dehydration-sensitive
proteins. This is reminiscent of the role of molecular
chaperones as stress protectants: in fact, the POPP analysis
of Wise (2003), which suggests shared peptide profiles of
LEA proteins with some chaperones, prompted this study.
The LEA proteins tested (the group 1 member, Em, from
wheat and the nematode group 3 protein AavLEA1) did not
prevent CS aggregation due to heat stress, like classical
heat shock proteins, but could be functioning as dehydration-specific molecular chaperones. However, chaperones
not only prevent inappropriate protein aggregation but form
specific, transient complexes with their client proteins,
typically (although not exclusively) through interaction of
hydrophobic patches (Ellis 2004). It is not clear whether
If we accept that LEA proteins play a role in tolerance to
water stress and confer some protection against stressimposed damage, we must then ask how they might do this.
A number of in vitro studies have attempted to provide
some answers, but it is clear that care must be taken in
interpretation of the results obtained. This is illustrated by
experiments with COR15a (Q42512_ARATH), an LEAlike protein identified as strongly cold-induced in Arabidopsis thaliana. Full length (15 kDa) native COR15a
protein was produced by in vitro transcription/translation
and was able to protect lactate dehydrogenase (LDH) from
freeze-thaw damage better than other proteins such as BSA
or RNaseA by several orders of magnitude (Lin and
Thomashow 1992a). However, COR15a is actually located
in the stroma of chloroplasts in vivo, and once the
chloroplast import sequence is removed, as in the mature
9.4 kDa protein, COR15am, cryoprotection of LDH is no
better than BSA (unpublished data cited in Artus et al.
1996). Instead, subsequent work showed that a membrane
protection function is more likely: When the COR15a gene
was overexpressed in Arabidopsis, resulting in elevated
levels of COR15am in chloroplasts, chloroplast freezing
tolerance in leaves was significantly increased in nonacclimated plants; protoplasts isolated from the leaves of
transgenic strains were also more tolerant of freeze stress
than wild type (Artus et al. 1996). The latter observation
raised the interesting possibility that a chloroplast protein
could influence stability of the whole plant cell. Later work
suggested that this was because during freezing, plasma
membrane can come into contact with endomembrane
Protein stabilisation and molecular shield function
Naturwissenschaften (2007) 94:791–812
LEA proteins also form specific complexes with clients,
and it is likely to be difficult to demonstrate this in the dry
state, as close proximity will be forced on proteins as they
are dried from mixed solutions. The reality of LEA protein
activity might well be simpler, where they behave as
“molecular shields” (Wise and Tunnacliffe 2004; Goyal et
al. 2005a). As unstructured, so-called entropic chains
(Tompa 2002), highly hydrophilic proteins will exert an
excluded volume effect that, in the increasingly crowded
environment of the dehydrating cytoplasm, could serve to
decrease interaction between partially denatured polypeptides with the potential to aggregate. An analogous effect is
well known to physical chemists interested in polymer
stabilisation of colloidal dispersions (i.e. “depletion stabilisation”; Napper 1983). Molecular shield function is also
similar to that of the entropic bristles of MAP2, tau and
neurofilament side arms, which serve as spacers to prevent
close association of microtubules and neurofilaments
(Mukhopadhyay et al. 2004), except that shield proteins
are not necessarily tethered to a surface. Association of
molecular shields with the surface of other proteins, and
even membrane surfaces (see below) is also a possibility,
however, and would lead to a steric stabilisation effect,
again familiar to colloid scientists. Finally, shield proteins
might have a broader space-filling role and help to prevent
collapse of the cell as its water is lost. Close (1996) has
pointed out early studies by Siminovitch and Briggs (1953)
who correlate the accumulation of water-soluble (presumably, hydrophilic) proteins with frost hardiness in the black
locust tree and hypothesise a role as “plasticizers or
mechanical buffers in the cell”. Still earlier work noted
the contribution of these proteins to the “high non-solvent
space” in frost-hardy cells (Levitt and Scarth 1936; Asai
1943). We suggest that molecular shields are an important
protective mechanism in many organisms, which tolerate
water stress.
Ion binding and antioxidant function
One consequence of dehydrating the cell is an increase in
concentration of intracellular components, including ions.
This has potentially damaging effects on macromolecular
structure and function, and it has been proposed that LEA
proteins, because of their many charged amino acid
residues, might act to sequester ions (Dure 1993; Danyluk
et al. 1998), and POPP analysis (Wise 2003) suggests Ca2+
binding as a possible property of both groups 2b and 3a
proteins (Wise and Tunnacliffe 2004). A protein loosely
related to the dehydrins was described by Heyen et al.
(2002) in celery (Apium graveolens). This protein binds
Ca2+ when phosphorylated and is located in the vacuole; in
seedlings, although not in fully-grown plants, its expression
is increased by water stress. Three acidic Group 2b
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proteins, ERD10, COR47 and ERD14, have also been
shown to exhibit phosphorylation-dependent Ca2+ binding
(Alsheikh et al. 2003, 2005). Mass spectrometry data
suggest that the phosphorylation sites for these three
proteins are located in the Ser stutter motif. By the same
token, the basic group 2 protein Xero2, lacking a poly-Ser
segment, is not phosphorylated and does not bind Ca2+.
Furthermore, although the neutral group 2 protein DHR18
does carry the poly-Ser motif and can be phosphorylated,
it does not bind Ca2+, indicating that an environment of
acidic residues may also play a role. By analogy, if
phosphorylation-dependent Ca2+ binding is present in
group 3 LEA proteins, as predicted in Wise (2003), the
precondition of an acidic protein is only met in a third of
the group 3 LEA proteins. However, Glu is significantly
over-represented in over half the sequences, so there may
be locally acidic moieties. Phosphorylation in that case is
also likely to be dependent on Thr rather than Ser, as the
former is significantly over-represented in two thirds of the
sequences.
Group 2 LEA proteins are also known to bind a number
of other metal ions, but the mechanism is rather different
from that used for Ca2+. Svensson et al. (2000) showed that
an affinity for divalent metal ions could be exploited as part
of a purification protocol for four different recombinant
Arabidopsis thaliana group 2 LEA proteins. In a refinement
of this strategy, native group 2 LEA proteins from several
plant species were purified with a Cu2+-chromatography
step (Herzer et al. 2003). Krüger et al. (2002) used a similar
protocol to purify an iron-binding protein from castor bean
(Ricinus communis) with some similarities to group 2 LEA
proteins. In this study, binding affinities across a range of
transition metal ions are ranked, with Fe3+ having the
highest affinity, followed by Cu2+, Zn2+, Mn2+ and Fe2+.
Svensson et al (2000) noted in passing that all of the
sequences in question are rich in His and proposes that this
indicates the binding mechanism. Direct evidence for the
role of His in binding first row transition metal ions comes,
however, from a peptide used to study a very different
protein. The study, reported in Grossoehme et al. (2006), is
of an iron-regulated transporter from Arabidopsis thaliana.
The authors synthesised the peptide PHGHGHGHGP and
discovered that it has a high affinity for Fe3+ and then a
similar list of metal ions (but also including Co2+, Ni2+ and
Cd2+) in the same descending order as that reported by
Krüger et al. (2002). The importance of this study is that
although the specific peptide sequence used is not found
in dehydrins, His is over-represented in 46 out of 50
group 2 LEA protein sequences and Gly is overrepresented in 42 of them. The biochemistry of the
Grossoehme peptide is that of a natively unfolded peptide
dominated by His. The implication is that in the group 2
LEA proteins, the flexibility imbued by a high Gly
806
content allows multiple His residues to make contact with
metal ions. Other recent work (Tompa et al. 2006), using
1
H-NMR and differential scanning calorimetry measurements, suggests that group 2 proteins, as exemplified by
ERD10, have a more general propensity for binding of
solute ions, which is in keeping with the broader concept of
LEA proteins as ion sinks.
Other workers report upregulation of group 2 proteins or
their transcripts after exposure of algae (Chlorella zofingensis) to selenium (Pelah and Cohen 2005) or of bean
(Phaseolus vulgaris) to heavy metals such as mercury,
cadmium and copper (Zhang et al. 2006). In the latter case,
the PvSR3 gene did not respond to drought stress or ABA.
This, together with the reports of Krüger et al. (2002) and
Heyen et al. (2002), suggests that there has been diversification of function among the group 2 proteins.
The binding of metal ions by group 2 LEA proteins
might be linked to the antioxidant properties reported for
the citrus CuCOR19 protein, which protected liposomes
from peroxidation, as well as reducing cold-induced
electrolyte leakage in transgenic tobacco seedlings (Hara
et al. 2003). In vitro experiments with the same protein
showed scavenging activity for hydroxyl and peroxyl
radicals although not hydrogen peroxide or superoxide
(Hara et al. 2004). It is known that, for example, hydroxyl
radicals and singlet oxygen are generated by catalytic
metals and that these reactive oxygen species (ROS) are
highly reactive towards organic molecules. Water deficit
can lead to accumulation of catalytic metal ions in plants
(Iturbe-Ormaetxe et al. 1998). LEA proteins might therefore act to reduce oxidative stress in dehydrating cells both
directly by scavenging ROS and indirectly by sequestering
metal ions that generate ROS.
Membrane association, folding and stabilisation
The work of Steponkus et al. (1998) on Cor15am, cited
above, suggests that LEA-like proteins could associate with
and stabilise membranes. The acidic group 2 LEA protein
CO410_WHEAT (WCOR410) accumulates at the plasma
membrane during cold acclimation, particularly in tissues
more sensitive to freeze damage (Danyluk et al. 1998).
Other clear examples of membrane association in vivo have
not been described, however (Table 4), although some
group 2 proteins are localised to membrane-rich areas
within particular cellular compartments (Asghar et al. 1994;
Egerton-Warburton et al. 1997).
The results of Koag et al. (2003) and Zibaee et al.
(unpublished data), cited in “Structure” above, demonstrate
folding of groups 2 and LEA proteins on acidic SUVs but
not how this relates to function. Most recently, however,
Tolleter et al. (2007) have shown that the group 3 LEA
protein LEAM from pea mitochondria is able to inhibit
Naturwissenschaften (2007) 94:791–812
fusion of SUVs during air drying and depress their gel-liquid
crystal phase transition temperature. Intriguingly, the vesicles
comprised phosphatidylcholine and phosphatidylethanolamine, rather than acidic phospholipids; perhaps significantly, the authors report no association with these vesicles under
hydrated conditions. Tolleter et al. propose that LEAM
protects the inner mitochondrial membrane on drying and
occupies most of the protein-free area; we might speculate
that this could also impact on oxidative phosphorylation and
thus could be at least partly responsible for the metabolic
shutdown typical of dehydrating cells and organisms.
Further studies of this kind are awaited with interest.
Other potential functions
It has been suggested, based on the hydrophilicity of LEA
proteins, that they might act as hydration buffers, slowing
down the rate of water loss during dehydration (McCubbin
et al. 1985; Cuming 1999; Garay-Arroyo et al. 2000).
Ultimately, in a desiccating cell or organism, water loss is
inevitable, but under less stringent conditions such as
(partial) drought, osmotic or chilling stress, hydration buffers
might allow sufficient water activity for proteins to retain
function. Thus, Rinne et al. (1999) show that α-amylase
activity is improved at low water activity (imposed by the
addition of 20% polyethylene glycol) by the presence of
group 2 LEA proteins. Based on the phenotype of a
knockout mutant in Arabidopsis thaliana, whose seeds
exhibit premature dehydration, Manfre et al. (2006) have
also proposed a role for the group 1 LEA protein AtEm6 as a
hydration buffer. Mouillon et al. (2006) elaborate a model
based on a shift in LEA protein structure as water activity
decreases from random coil when highly hydrated, through
an increased proportion of PPII helix as water is lost from
the drying organism, to a collapsed structure enriched in αhelix or β-sheet as desiccation proceeds further. The authors
suggest that LEA proteins behave as hydration buffers in this
way before releasing their water into the cellular environment under conditions of severe dehydration.
One model for desiccation tolerance proposes that
vitrification of the cytoplasm is important (Burke 1986;
Crowe et al. 1998). The prevailing hypothesis highlights
the role of sugars, particularly non-reducing disaccharides,
in glass formation, but presumably in the complex milieu of
the cytoplasm, other components particularly proteins will
also play a role. One LEA protein has been shown to
increase the glass transition temperature (Tg) of sugar
glasses (Wolkers et al. 2001), but this stabilising function
is not unique and is also exhibited by poly-L-lysine
(Wolkers et al. 1998), BSA and other polymers such as
hydroxyethyl starch (Crowe et al. 1997). Based on the
increased folding as α-helix and possibly higher ordered
structures on drying (see “Structure” above), we have
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proposed that group 3 LEA proteins might form filaments
under water stress conditions, which could add mechanical
stability to the dehydrated cell (Goyal et al. 2003; Wise and
Tunnacliffe 2004), perhaps by increasing the tensile
strength of cytoplasmic glasses, an idea which has been
amplified eloquently by Berjak (2006). To date, using
confocal microscopy, we have not observed gross structural
reorganisation in the protein AavLEA1 when expressed in
mammalian cells, which are then subjected to water loss
(Walton 2005). This does not preclude changes at the
ultrastructural level or in a desiccation-tolerant system,
however.
LEA proteins: multi-talented or misunderstood?
From a situation only a few years ago, where there was
very little information on the role of LEA proteins, we now
find ourselves with an embarrassment of riches: Recent
research has provided data supporting several apparently
disparate functions (summarised in Table 7). To some
extent, this could reflect different roles for different groups
of LEA proteins. This has not yet been tested systematically
by experiments to compare the activity of many LEA
proteins from different groups in the same assays or
conversely by using the same protein in several different
assays; it would now be timely to perform such studies. A
related question concerns the precise role of the conserved
sequence motifs that characterise the LEA protein groups.
Mouillon et al. (2006) suggested that these sequences in
group 2 proteins are recognition motifs, driving interaction
with other cellular components. A related explanation is
that they are important for particular folding patterns such
as the amphipathic α-helices of the group 3 proteins, which
in turn might govern binding to membrane or protein
surfaces. Another alternative is that the different sequence
motifs or peptide profiles allow a function to be performed
upon the LEA protein, e.g. localisation to a particular cell
or tissue compartment, or a specific processing event, such
Table 7 Possible functions for LEA proteins based on current in vitro
data
Proposed function
Group 1
Group 2
Group 3
Hydration buffer
Molecular shield
Enzyme protectant
Metal ion binding
Antioxidant
Membrane association
?
Yes
Yes
?
?
No
Yes
?
Yes
Yes
Yes
Yes
?
Yes
Yes
?
?
Yes
A summary of potential functions of the three main groups of LEA
proteins as described in the text.
as activation or degradation. An example would be
phosphorylation of the Ser stutter motif in group 2 proteins,
which is associated with nuclear localisation or metal ion
binding in some studies. Clearly, such questions will be
easier to address when we have a better idea of LEA protein
function.
The data currently available might suggest that LEA
proteins are to some extent multi-functional, although we
have seen that some reported functions could well be
related, e.g. the protection of enzyme activity during drying
is consistent with a molecular shield function. The
antioxidant and metal-ion binding functions of some group
2 proteins could also be complementary, and indeed, as
Hara et al. (2003) showed inhibition of lipid peroxidation
by a Citrus protein, there could be a further link with
vesicle association and protection. We might even extend
the molecular shield concept to include a role in preventing
membrane fusion by blocking close approach of bilayers
during water loss, and perhaps some LEA proteins can
perform this function after associating with membrane
surfaces.
Nevertheless, should LEA proteins be confirmed as
multi-functional, multi-talented proteins, this capacity will
almost certainly be facilitated by their largely unfolded
nature in solution. Tompa et al. (2005) have argued that
“moonlighting”, the ability of some proteins to perform
dual functions, is more likely to evolve in intrinsically
unstructured proteins than in more conventional, folded
proteins. Of course, a more pessimistic interpretation of the
apparent multi-tasking of LEA proteins is that the in vitro
data do not accurately reflect in vivo function, i.e. that we
still have much to learn. There is likely to be some truth in
this sentiment, and therefore, more in vivo studies are a
matter of imperative. Certainly, we must remain alert to the
problems in interpreting in vitro results, as the case study
outlined above in “In vitro studies: a caveat” illustrates.
Other dangers include the difficulty in comparing experiments involving different methods of drying and/or
freezing; these impose diverse stress vectors, which
consequently demand different functions of any putative
protectant molecule, such as an LEA protein.
Part of the rationale for addressing the in vivo situation
more intensively in the near future is that we should not
consider the role of LEA proteins in isolation. Cells dealing
with the consequences of water loss launch a whole battery
of defence or repair mechanisms besides the LEA proteins.
One strategy, which is probably common to plants, animals
and microorganisms and which is now being recognised, is
that the LEA proteins are not the only hydrophilic proteins
involved in the response to dehydration. Examples include
CpEdi-9 from the resurrection plant C. plantagineum
(Rodrigo et al. 2004), anhydrin from the anhydrobiotic
nematode Aphelenchus avenae (Browne et al. 2004) and
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several hydrophilic proteins from yeast and bacteria (Sales
et al. 2000; Garay-Arroyo et al. 2000). Recent transcriptome studies in invertebrates suggest that half or more
of the novel sequences expressed in response to dehydration encode hydrophilic proteins (Tyson et al. 2007; N.
Pouchkina-Stantcheva and AT, unpublished data). These
proteins are likely to share many properties with LEA
proteins as a result of their hydrophilicity, e.g. anhydrin
performs well in molecular shield assays (McGee 2006).
Whether they fall into discrete sets of sequences, like the
LEA proteins, and how LEA proteins interact or cooperate
with them, awaits future research. Similarly, the interplay
between LEA and other hydrophilic proteins and the sugars
and other compatible solutes associated with water stress
tolerance in many organisms, and indeed other components
of the desiccation tolerance armoury (e.g. reviewed in
Hoekstra et al. 2001; Vicré et al. 2004; Alpert 2005; Bartels
2005), requires further study. Their potential role as benign
space fillers, “plasticizers or mechanical buffers in the cell”,
as outlined in the early work of Siminovitch and Briggs
(1953), Asai (1943) and Levitt and Scarth (1936), is
perhaps unglamorous but surely of enormous importance
to the dehydrating cell.
Despite the continuing conundrum they pose for us, the
recent research reviewed in this paper provides considerable hope that, as with the heat shock proteins 20 years ago,
we will soon develop a clearer understanding of LEA
protein function and a concomitant deeper understanding of
tolerance to dehydration and cold stress.
Acknowledgement We would like to thank Dirk Hincha, David
Macherel, Al Warner and Shahin Zibaee for permission to cite results
from their laboratories before publication.
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