Download Possible roles of LEA proteins and sHSPs in seed protection: a short

LEA PROTEINS AND sHSPs IN SEED PROTECTION
3
BIOLOGICAL LETT. 2007, 44(1): 3–16
Available online at http://www.biollett.amu.edu.pl
Possible roles of LEA proteins and sHSPs in seed protection:
a short review
EWA M. KALEMBA and STANIS£AWA PUKACKA
Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland
e-mail: [email protected]
(Received on 11 May 2007; Accepted 27 September 2007)
Abstract: Late embryogenesis abundant (LEA) proteins and small heat shock proteins (sHSPs) are
produced in seeds during maturation and under various stress conditions. Their expression is
developmentally regulated or induced by drought, salinity, cold, oxidative stress, and abscisic acid (ABA).
Protective functions of sHSPs and LEA proteins have been widely analysed in many plant seeds, including
seeds of the orthodox, recalcitrant and intermediate category. Moreover, both the sHSPs and LEA proteins
are intensively synthesized during seed development, as a part of the embryogenesis program. Numerous
investigations have proved that LEA proteins are involved in complex processes that permit seeds to survive
in the dry state, such as glass formation and cryoprotection mechanisms. Considering all LEA groups,
dehydrins are major proteins associated with conditions affecting the water status in cells. Either LEA
proteins or sHSPs are strongly correlated with seed longevity. Due to the ability of sHSPs to interact
with other chaperone proteins, they can attach to denatured proteins and therefore stabilize their
conformation, but they also assist in protein folding and refolding, intracellular transportation, marking
proteins for degradation, and possibly glassy state formation. Thus the presence of protective proteins
and their proper action as an essential factor for seed protection is discussed here.
Keywords: seed, abiotic stress, LEA proteins, dehydrin, small heat shock proteins
INTRODUCTION
Plant species diversity is important for preserving the natural relations in ecosystems. Currently in seed genebanks various seeds can be stored for decades or even
centuries, but tree propagation from seeds is not easy, especially for species that
produce seeds irregularly or rarely. It is even more difficult if the seeds cannot be
stored for a long time, or at least no effective storage conditions have been identified yet. There is usually a reserve of dormant seeds in the soil in habitats where the
plants grow. However, seed production can be seriously limited by various environmental stresses.
Some environmental stresses, such as drought stress, cold stress and salinity
stress, result in cellular dehydration. Water is one of the factors that limit seed de-
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Ewa M. Kalemba and Stanis³awa Pukacka
velopment, storage, and germination, so seeds must have some mechanisms that allow them to withstand the loss of water. It is more interesting if we consider that
storage is not the only situation when seeds are exposed to limited water availability.
The shortage of water in seeds activates specific processes and can lead to a loss of
viability and decreased vigour or even death. Metabolism reduction, distribution of
amphiphilic elements in the cellular membrane, and glass structure formation, enable cells to survive the time of desiccation.
Seeds are able to survive dehydration, but to a varied extent. Two types of
tolerance can be distinguished on the basis of moisture content: drought tolerance
and desiccation tolerance (HOEKSTRA et al. 2001). Drought tolerance is the tolerance
to dehydration down to 23% of fresh weight, which is about 0.3 g H 2O per 1 g of
dry weight. That means a lack of bulk water in the cytoplasm, so the mechanisms
that stabilize intracellular structures by preferential hydration are launched. Desiccation tolerance can be defined as the ability to survive stronger dehydration and to
restore normal function when rehydrated. Then mechanisms connected with water
replacement by compounds able to form hydrogen bonds are activated. The organisms resistant to desiccation have structural, physiological and molecular mechanisms
enabling survival at the time of water deficit. But the mechanisms are still not fully
explained. Morphological changes formed as a result of the water loss, cause physiological changes and hence modifications at the molecular level: accumulation of
protective compounds, changes in enzymatic activity and in gene expression (CLOSE
1996). Considering the classification of seeds into orthodox, recalcitrant and intermediate categories, predominantly orthodox seeds are resistant to desiccation, and
recalcitrant ones are only drought-tolerant (ROBERTS 1973, ELLIS et al. 1990). The
resistance to desiccation is possible in the presence of specific mono-, di- and oligosaccharides, late embryogenesis abundant (LEA) proteins, and small heat shock
proteins (sHSPs) (DEROCHER & VIERLING 1994), soluble sugars (KOSTER & LEOPOLD
1998), and mechanisms related to ROS removal (HENDRY 1993) and glass formation (BUITINK et al. 1998).
Investigations with various mutants of species that produce orthodox seeds
(mainly Arabidopsis thaliana (L.) Heynn) have proved that modification of several
genes can be sufficient to lose the resistance to desiccation (OOMS et al. 1993).
However, no species with recalcitrant seeds was reported to produce orthodox seeds
after any mutation. Nevertheless, the resistance to desiccation can be induced externally. Embryonic axes from the normally recalcitrant seeds of silver maple (Acer
saccharinum L.) can be made more tolerant to desiccation (moisture content 10%)
and low temperature (-196°C) by pretreatment with abscisic acid (ABA) and the
growth-retardant tetcyclacis, which enhances endogenous ABA concentrations and
induces synthesis of some LEA proteins (B EARDMORE & WHITTLE 2005). Considering that desiccation tolerance is a complex multigene process, the mechanisms by
which various organisms can survive the time of desiccation must have some common, universal elements.
Both LEA proteins and sHSPs appear and accumulate during seed development, and then gradually disappear during germination, indicating that desiccation
tolerance is at first achieved and then lost. Moreover, their expression can be induced
or enhanced by various stress conditions. Desiccation-tolerant organisms have the
LEA PROTEINS AND sHSPs IN SEED PROTECTION
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ability to form glasses. Below 0.1 g H2O per 1 g of dry weight, the cytoplasm undergoes glass transition. The glass is a metastable and amorphous structure, which thermodynamically behaves as a liquid. In that state the diffusion of chemical compounds
and reactions are slowed down (BUITINK et al. 2000). Tg, the temperature of transition from the liquid to glassy state, depends on the moisture content, temperature
and chemical composition of the cytosol. Carbohydrates (such as sucrose, raffinose
and stachyose) can effectively help in osmotic pressure adaptation during water loss
and participate in glass formation. They generate the highly reactive hydrogen bonds,
so the mechanical properties of glass are similar to those of solid bodies. Tg values
correlate with the molecular mass of carbohydrates: the higher the molecular mass,
the higher the Tg value. In seeds, LEA proteins (WOLKERS et al. 2001), together with
oligosaccharides and possibly sHSPs (VIERLING 1997), participate in glass formation and stabilization. Research on functions of LEA proteins and sHSPs in seeds is
important and will be discussed here.
LATE EMBRYOGENESIS ABUNDANT (LEA) PROTEINS
Water-protein interactions are one of the main forces in protein folding and
stabilizing the conformation, but also in determining the dynamics and properties of
bonds in proteins and their catalytic activity. LEA proteins were first identified in
seeds, when mRNA and proteins that occur in large quantities were investigated
(DURE 1993). LEA proteins are located in the cytoplasm, nucleus, mitochondria,
vacuoles (HOUDE et al. 1995, EGERTON-WARBURTON et al. 1997), near the cellular
membrane (DANYLUK et al. 1998), as well as in amyloplasts (RINNE et al. 1999), but
primarily they accumulate in the cytoplasm and nucleus. After synthesis they are
transported to cell organelles and membranes, where they stabilize cell structures and
molecules. The partition of LEA proteins into various cell compartments determines
their possible function in the protection or regulation of essential biochemical processes, like replication or respiration.
Classification of LEA proteins
LEA proteins can be classified into 3 or 6 groups. The classification is based
on sequence similarity and properties (CUMING 1999, WISE 2003). Proteins of group
1 contain a 20-amino-acid (aa) region, which was first identified in the wheat Em
protein (osmoprotective molecule) (CUMING 1999). LEA proteins of group 2 are widespread and widely analysed in respect of water stress. Group 3 of LEA proteins is
characterized by the 11-aa motive ΦΦE/QXΦKE/QKΦXE/D/Q, where Φ represents
a hydrophobic aa. It has been predicted that this region forms an amphipathic α-helix,
which probably participates in structural interactions (BAKER et al. 1988). LEA proteins of group 3 are involved in response to cold stress. Usually they are located in
the cytoplasm, nucleus and mitochondria, where they can be associated with membranes. Groups 4 and 5 have a less conserved structure and are possibly involved in
the protection of membrane stability.
Group 2 of LEA proteins is also called the D-11 family (DURE et al. 1989) or
dehydrins (CLOSE 1997). They are glycine- and lysine-rich proteins with molecular
weight of 9–200 kDa. Glycine, histidine, lysine and threonine comprise 56% of all
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Ewa M. Kalemba and Stanis³awa Pukacka
aa in LTI30 (water stress-responsive protein). K, S and Y segments are characteristic of dehydrins (CLOSE 1996). The K segment sequence is conserved and consists
of 14 aa: EKKGIMDKIKELPG. Substitutes and structural modifications of aa are
possible in the K segment, which can be repeated 1–12 times in a single protein and
always is situated near the C end (CLOSE 1997). Many dehydrins contain a serinerich S segment, which might be the phosphorylation site of the protein. The S segment phosphorylation might be an important posttranslational modification, which
is required for proper protein location, as it is observed during Rab 17 placing in the
nucleus (GODAY et al. 1994), and a signal in stress transduction pathway.
The classification of dehydrins is based on the presence of conserved segments
(CLOSE 1997). Five classes of dehydrins can be distinguished: YnSK2, Kn, KnS, SKn,
and Y2Kn. The YnSK2 dehydrins consist of 1–3 Y segments, a serine-rich segment,
and 2 K segments. Their expression is induced mainly by ABA (RAB type) and
drought, less often by low temperature. Some YnSK2 dehydrins have an ability to bind
lipid vesicles containing acidic phospholipids (KOAG et al. 2003). In Kn dehydrins,
the occurrence of 1–9 serine-rich segments is observed. They are acidic or neutral
proteins, mainly induced by low temperature and somewhat by ABA and drought.
The K segment of class KnS begins with sequence (H/Q) KEG instead of EKKG.
Their expression is induced by ABA and dehydration. KnS dehydrins can bind various metal ions or scavenge the hydroxyl radicals (A SHGAR et al. 1994). Y2Kn proteins usually consist of 2 Y and 2 K segments. They are expressed during seed germination and related with resistance to low temperature. The SKn dehydrins contain
1 S and 1–3 K segments and are preferentially induced by low temperature. Moreover, they can be induced by drought, salinity, injury and jasmonate treatment. Dehydrins lacking the Y segment were identified in many other plant tissues, such as stem
tissue, vegetative and reproductive buds, needle tissue, unstressed leaf and root tissue. Its expression was induced by water, salt, cold and osmotic stress (CARUSO et
al. 2002). The K segment was considered to appear in all classes of dehydrins (C LOSE
1997), but recently a protein lacking the K segment (ROD 25 from cold acclimated
Cornus sericea, syn. C. stolonifera) was detected (SARNIGHAUSEN et al. 2002). The
role of the conserved segments is to exert their biological function upon recognition
and interaction with specific biological targets. The combination and number of repeated segments is related with the possible protein functions displayed in seeds under
stress conditions.
LEA gene expression and protein structure
LEA genes are sensitive to ABA because of the presence of ABREs (ABAresponsive elements), i.e. regulatory elements in promoter regions, which contain the
ACGT sequence called cassette G. Sensitivity to ABA also depends on the presence
of MYC elements that include the sequence CACCTG, and MYB elements that include the TAACTG motive. ABA-independent expression of dehydrins is based on
activation of DREs (drought-responsive elements), also known as CRT (C-repeat)
elements (STOCKINGER et al. 1997). The ABREs can interact with the bZIP transcription factors. ABRE motifs are also present in the promoter regions in drought-stress–
inducible regulatory genes that are involved in modulation of the expression of
multiple genes related to plant response to stress.
LEA PROTEINS AND sHSPs IN SEED PROTECTION
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Only one spatial structure of dehydrin is deposited in the PDB (Protein Data
Bank) database. It is the 1YYC Arabidopsis thaliana protein, whose structure was
solved by using the NMR method (SINGH et al. 2005). Analyses of the wheat Em
protein and maize G50 protein suggest that up to 75% of the native protein can be
unordered (MCCUBBIN et al. 1985). The lack of an ordered structure results in resistance to high temperature. The 3-dimensional structure can be achieved after protein-target interaction. The role of the conserved segments in the promotion of tertiary structure is not clear (MOUILLON et al. 2006). Formation of the secondary structure
of LEA proteins was examined during gradual water removal from solutions. Formation of α-helix structures was detected in the dehydrin-related DSP16 protein from
Craterostigma plantagineum Hochst. (LISSE et al. 1996) and another dehydrin-related protein from Vigna unguiculata L. in the presence of sodium dodecyl sulfate (ISMAIL et at. 1999). In water solutions, LEA proteins do not display any spatial structure, but various secondary structures appear depending on the solvent type the
proteins are put in. In saline solutions, α-helices are always formed. In sucrose solutions, both α-helices and β-sheets are created during slow drying, but only α-helices
during rapid drying (GOYAL et al. 2003). Various stress conditions seem to be a signal for protein folding and for achieving the tertiary structure.
Seed development, desiccation and storage – apparent functions of LEA proteins
LEA proteins have been widely analysed in plants. They play an important role
in seed maturation and osmotic stress. LEA proteins in seeds are related to desiccation
tolerance (XU et al. 1996). In seeds the germination rate (KOSTER & LEOPOLD 1998),
electrolyte leakage (SUN & LEOPOLD 1993), presence of heat-stable proteins and LEA
accumulation (CLOSE 1997) can be measured to detect the moment of desiccation tolerance acquisition. The mRNA of LEA genes appears in maturing seeds and becomes
the most abundant mRNA species in the dry seed, but disappears shortly after imbibition. LEA proteins are hydrophilic. In drought-tolerant seeds, numerous polar groups
situated on the surface of LEA proteins participate in preferential hydration. During
water deficit in desiccation-tolerant seeds, they can interact with other groups and replace water in the cell. LEA proteins occur mostly in orthodox but also in recalcitrant
and intermediate seeds, indicating their key functions, and possible protective roles could
be displayed in all categories. Constitutive expression of some LEA genes was detected in A. thaliana, e.g. dhnX (WELIN et al. 1994) and ERD14 (NYLANDER et al. 2001),
and in pea (ROBERTSON & CHANDLER 1992). LEA gene expression can be induced
both in desiccation-resistant and sensitive tissues after ABA treatment, except some
tropical species that produce orthodox seeds (FARRANT et al. 1996, KERMODE 1997).
Dehydrins or dehydrin-related proteins were detected in many species, with
seeds classified into various categories: recalcitrant (Quercus robur L., Castanea
sativa L., Acer saccharinum L.), orthodox (Acer platanoides L., Pisum sativum L.,
Zea mays L.), and intermediate (Coffea arabica L.) (ROBERTSON & CHANDLER 1992,
FINCH-SAVAGE et al. 1994, CAMPBELL et al. 1998, HINNIGER et al. 2006). Differences
observed in dehydrin occurrence in embryonic axes and cotyledons of mature and
immature seeds of diverse organisms show differential desiccation sensitivity of seed
tissues and variations in desiccation tolerance between species (FINCH-SAVAGE et al.
1994, CLOSE 1997).
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Ewa M. Kalemba and Stanis³awa Pukacka
The glass matrix formed in sucrose solutions containing LEA proteins is characterized by higher Tg values and stronger hydrogen bonds than those observed in a
sucrose solution without LEA proteins. Such properties are typical of glass structure
formed in seeds during storage (OLIVER et al. 2001). The glass structure assures cell
stability at the time of dehydration. Intracellular glass, due to its high viscosity, can
prevent the crystallization of chemical compounds (in spite of their high concentrations), membrane fusion, and conformational changes of proteins. There is a linear
dependence between the limited mobility of the cytoplasm and cell life span, therefore intracellular glass has a significant effect on the storage life of seeds (LEOPOLD
et al. 1994, BUITINK et al. 2000). The existence of glassy state was detected in many
orthodox seeds, e.g. in Pisum sativum L. (BUITINK et al. 1998), Lactuca sativa L.
(BUITINK et al. 2000), Zea mays L. (WILLIAMS & LEOPOLD 1989), Glycine max (L.)
Merr. (SUN & LEOPOLD 1993), and also in the intermediate seeds of Fagus sylvatica
L. (PUKACKA et al. 2003). Glassy state is essential for seeds to stay alive during storage
and grow when they are needed.
LEA proteins in relation to environmental stresses
Various LEA proteins have been found to accumulate in plants during cold
acclimation. Therefore, LEA proteins might participate in cryoprotection mechanisms,
by accumulation in the tissues where primary ice nucleation occurs. Enzyme protection is important during long-term seed storage in seedbanks (usually low temperature) and in the soil during winter. In various plant tissues LEA proteins are able to
prevent the inactivation of cyclic frozen and unfrozen lactate dehydrogenase (HOUDE
et al. 1995), so they probably display the same protective role in seeds. Moreover,
during germination, LEA proteins may regulate α-amylase activity and starch grain
degradation throughout the time of water deficit (RINNE et al. 1999). The regulatory
role in relation to enzymatic activity also can be detected at the reaction level, because in dry seeds and whole plants LEA proteins can store the water molecules
needed to catalyse biochemical reactions, especially in wintertime. A high salt concentration affects many important processes, such as seed development and germination. Due to saline stress, diverse compatible solutes (osmotically active compounds)
and polyamines are produced, and the antioxidant defence mechanism, ion transport
and compartmentalization of toxic ions are initiated. The expression of LEA proteins
of all groups is responsive to salinity, as it was shown many times in salt-stressed
seedlings and mature plants (NAOT et al. 1995). During osmotic stress, dehydrins
can act as antioxidants scavenging the toxic quantities of metal ions in whole plants
(HARA et al. 2005).
Using the POPP program, additional possible functions of LEA proteins have
been proposed. LEA proteins may have enzymatic or chaperone activity and act as
nucleus proteins that unwind or repair DNA, regulate transcription, and might be
associated with chromatin or cytoskeleton (WISE & TUNNACLIFE 2004).
SMALL HEAT SHOCK PROTEINS (sHSPs)
Eukaryotic organisms have 5 classes of heat shock proteins: Hsp90, Hsp70
(DnaK), GroEL and Hsp60, Hsp100 (Clp), and sHSPs (WANG et al. 2004). Various
LEA PROTEINS AND sHSPs IN SEED PROTECTION
9
sHSPs are widespread in all kingdoms, but are absent in several pathogenic organisms. Bacteria and unicellular eukaryotes contain 1 or 2 sHSP genes, while 4 genes
are present in Drosophila melanogaster, 16 in the nematode C. elegans, and 19 in A.
thaliana (HASLBECK et al. 2005). In plants, sHSPs are a numerous and diverse protein group. Classification into 6 groups is based on the similarity of gene sequence,
immunological reactivity, and intracellular location (WATERS et al. 1996). These
proteins usually are undetectable in vegetative tissues, but in seeds their expression
is induced by stress conditions and development stage (WEHMEYER & VIERLING
2000). Classes CI, CII and CIII are represented by cytosolic and nuclear proteins,
CIV are found in plastids (VIERLING 1997), CV are associated with endoplasmic
reticulum (HELM et al. 1993), and CVI are mitochondrial (LENNE et al. 1995). Their
broad distribution implicates that these proteins can protect practically all cellular
compartments (KOTAK et al. 2007). Specific organelle localization of sHSPs is characteristic for plants, except for HSP22, which occurs also in other eukaryotic mitochondria. Proteins that belong to the same class show a high sequence similarity, even
if they come from different plant species. Conserved elements of protein sequences
render almost identical core structures and therefore the same functions. The structural organization of sHSPs is evolutionarily conserved. They contain an α-crystalline domain, which consists of 90 aa and can undergo dimerization, and conserved
N and C ends. The α-crystalline domain functions like a molecular chaperone, in
preventing the aggregation of various proteins under a wide range of stress conditions by selective interactions of its hydrophobic surface with non-native proteins
(REDDY et al. 2006). The spatial structure of sHSPs is very dynamic and plastic. The
crystal structure of several sHSPs is known, mainly bacterial and fungal. They all
can associate in oligomeric structures with various numbers of subunits: 24 in yeasts
(Hsp26), and 12 in wheat (Hsp16.9) (VAN MONTFORT et al. 2001).
Induction of sHSP gene expression and protein synthesis in seeds
Expression of some sHSPs can be induced by osmotic stress. In sunflower
(Helianthus annuus L.) seeds, the HaHSP17.6 (CI) and HaHSp17.9 (CII) gene expression is induced by dehydration, and the mRNA level correlates with the rate of
dehydration. Also mannitol and ABA can induce HaHsp17.9 expression (ALMOGUERA
et al. 1993). HaHsp17.6 and HaHsp17.9 are similar to cytosolic sHSPs of C. plantagineum and accumulate after dehydration (ALAMILLO et al. 1995).
In Arabidopsis leaves, sHSPs of class I do not appear after dehydration, but in
seeds the expression of AtHsp17.7 (CII) and AtHsp17.6 (CII) is induced by osmotic
stress (WEHMEYER et al. 1996). Some cytosolic CI and CII sHSPs (ALAMILLO et al.
1995), mitochondrial sHSPs (BANZET et al. 1998) and chloroplast sHSPs (LEE &
VIERLING 2000) can be induced by oxidative stress. Besides, sHSPs can be induced
by cold stress (SABEHAT et al. 1998), heavy metals (GYÖRGYEY et al. 1991), ozone
(ECKEY-KALTENBACH et al. 1997), UV and γ radiation (BANZET et al. 1998). This
suggests that sHSPs can be associated with the general mechanism of cell response
to abiotic stress. Changes in sHSPs expression are similar to those in LEA proteins
at the time of dehydration and crucial developmental stages. sHSPs are synthesized
during embryogenesis, germination, pollen production, and fruit maturation (WATERS
et al. 1996, SUN et al. 2002). In A. thaliana embryos, the cytosolic AtHsp17.4 (CI),
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Ewa M. Kalemba and Stanis³awa Pukacka
AtHsp17.6 (CI) and AtHsp17.7 (CII) accumulate during the middle phase of seed
maturation. Their concentration remains high during the late phase and in dry mature seeds (WEHMEYER et al. 1996). Similarly, the accumulation of class I and class
II sHSPs is observed in pea and sunflower seeds (ALMOGUERA et al. 1993, DEROCHER
& VIERLING 1994). Moreover Oshsp16.9A abundantly accumulates in mature dry rice
seeds (GUAN et al. 2004). The synthesis of sHSPs during seed maturation indicates
their probable role in cell component protection mechanisms. Mutants sensitive to
desiccation contain smaller amounts of sHSPs during maturation (WEHMEYER et al.
1996, WEHMEYER & VIERLING 2000). In seeds the appearance of sHSPs is speciesspecific, and an increase in their content is always observed before the acquisition
of desiccation tolerance (ZUR NIEDEN et al. 1995). During germination, sHSPs are
present in large quantities for several days, and next disappear rapidly (D EROCHER
& VIERLING 1994, WEHMEYER et al. 1996). Additionally, during germination the
sHSPs disappearance is parallel to storage protein degradation (LUBARETZ & ZUR
NIEDEN 2002). Their activity is induced and does not depend on the presence of ATP.
After activation by stress conditions or phosphorylation, sHSPs cooperate with other
chaperone proteins that have ATPase activity (H ASLBECK et al. 2005, VAN MONTFORT et al. 2001).
Role of sHSPs
These proteins may act as protective proteins in vitro (LEE et al. 1997) and in
vivo (LÖW et al. 2000). Their universal protective role, low tissue specificity, and
participation in glass structure formation (HSP 17.4 in Arabidopsis) have been proposed (WEHMEYER & VIERLING 2000). They can attach to denatured proteins and
thus stabilize their conformation, also can help in protein folding, oligomer formation, intracellular transportation, and marking for degradation (HENDRICK & HARTL
1995). Conformation changes, such as dimerization, oligomer formation (HASLBECK
et al. 1999), and alternations in hydrophobic interactions on the surface of proteins
(LEE & VIERLING 2000), are essential for binding the substrate.
Relations of sHSPs with oxidative stress
Chloroplasts and mitochondria are the main organelles where ROS production
is observed both in normal and stress conditions, so sHSPs localized in those organelles might be associated with their response to oxidative stress. The unique structure of chloroplast sHSPs, which have the methionine-rich amphipathic α-helix, and
their ability to change the conformation, might be crucial in preventing oxidative
effects (LEE & VIERLING 2000, HÄRNDAHL et al. 1999). The expression of cytosolic
and mitochondrial sHSPs can also be induced by oxidative stress (BANZET et al. 1998).
A 22-kDa sHSP, called HSP22, is located in mitochondria and can prevent damages
that might be caused by oxidative stress and ageing. HSP22 accumulation appears
also after H2O2 and γ radiation treatment (LEE & VIERLING 2000). Moreover, for
Qshsp10.4-CI, the CI class sHSP that was obtained from Quercus suber L. (cork oak),
a possible protective role was proposed due to its accumulation in cork and other
oxidatively stressed tissues (JOFRÉ et al. 2003). Reactive oxygen species (ROS) attack DNA, proteins and membranes, cause lipid peroxidation and deesterification,
as well as accumulation of carbonyl derivatives (POTTS 1994). Therefore, increased
LEA PROTEINS AND sHSPs IN SEED PROTECTION
11
ROS scavenging is required during desiccation. The metabolism shut down is also
necessary to avoid oxidative stress. In seeds, during drying and storage, ROS content increases and is related to ageing. Seeds of individual species have characteristic potential life spans. The variability of seed longevity during storage is correlated
with the mechanisms of damage and protection during oxidative and ageing stress.
Beside results of HSP gene expression, analyses in transgenic seeds indicate that sHSP
are positively correlated with seed longevity (PRIETO-DAPENA et al. 2006). A detoxifying system of enzymes (superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, and catalase) might be insufficient during water
deficit (ELSTNER & OSSWALD 1994). In that case, ROS are removed by glutathione,
ascorbic acid, polyols, peroxiredoxins, tocopherol, ß-carotene, proline, polyamines
and flavonoids (SMIRNOFF 1993, HOEKSTRA et al. 2001). Oxidative stress is the main
force that causes a decrease in vitality and loss of seed vigour during storage, hence
ROS removal is needed to avoid oxidative damage of proteins – so that the native
structure of stress proteins could be sustained to play their protective role. In addition, oligomeric sHSPs can exchange the thiol and disulfide groups with glutathione
and synergistically act as protective elements regulating redox homeostasis (ZAVYALOV
et al. 1998).
Interactions of sHSPs with proteins and other compounds
In the cell, proteins are continuously degraded to aa and replaced by newly
synthesized proteins. Eukaryotic cells have a mechanism of protein degradation, in
which ubiquitin and HSPs are involved. Denatured proteins aggregate because of the
tendency of joining damaged hydrophobic domains to other such domains. Similarly, if badly folded proteins are dominant in a cell and the mechanisms of degradation
do not keep up with their removal, then damaged polypeptides form aggregates before they are removed (GOLDBERG 2003). Large amounts of damaged proteins are
noticed under stress conditions. Various sHSPs have the ability to bind the nonnative proteins and therefore prevent their aggregation (W ATERS et al. 1996,
HASLBECK et al. 1999, LEE & VIERLING 2000). Probably the hydrophobic interactions are responsible for binding denatured proteins and then protective proteins, like
DnaK or ClpB/DnaK complex (LEE et al. 1997, LEE & VIERLING 2000, REDDY et al.
2006). Recently a mechanism of native protein structure restoration, involving cooperation of sHSPs and other HSPs, has been proposed (HASLBECK et al. 2005).
In plants, sHSPs can be associated into granules, and in a fully hydrated state
are connected with membranes, where they can stabilize lipids and membrane fluidity (TSVETKOVA et al. 2002). This could have significant implications in seeds, as
sHSPs might participate in preserving membrane integrity during thermal fluctuations in winter, and also might act as a factor that lowers electrolyte leakage during
seed desiccation.
CONCLUSIONS
In this short review we summarized the possible roles of LEA proteins and
sHSPs in seed protection. Both groups of stress proteins are essential for proper seed
development, maturation, storage, and germination. They also function during vari-
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Ewa M. Kalemba and Stanis³awa Pukacka
ous environmental stresses, helping in stabilization of cell structures and compounds.
The proteins are associated with the response to drought and desiccation and strongly affect seed viability, mostly by preferential hydration of diverse molecules and water
replacement in dehydrated cells. Similarly, membrane integrity and protein conformation are assured by the action of sHSPs, which assist in protein folding and molecular interactions. Despite all experimentally investigated, theoretically predicted
or suggested functions of LEA proteins and sHSPs, further analyses are needed to
explain fully their exact roles and possible interactions with other protective compounds.
Acknowledgements: This research was supported by the Polish Ministry of Science and Information
Technology (grant No. 2P06L02927).
REFERENCES
ALAMILLO J., ALMOGUERA C., BARTELS D., JORDANO J. 1995. Constitutive expression of small heat
shock proteins in vegetative tissues of the resurrection plant Craterostigma plantagineum. Plant
Mol. Biol. 29: 1093–1099.
ALMOGUERA C., COCA M. A., JORDANO J. 1993. Tissue-specific expression of sunflower heat shock
proteins in response to water stress. Plant J. 4: 947–958.
ASGHAR R., FENTON R. D., DEMASON D. A., CLOSE T. J. 1994. Nuclear and cytoplasmic localization of maize embryo and aleurone dehydrin. Protoplasma 177: 87–94.
BAKER J., STEELE C., DURE L. III 1988. Sequence and characterization of 6 Lea proteins and their
genes from cotton. Plant Mol. Biol. 11: 277–291.
BANZET N., RICHAUD C., DEVEAUX Y., KAZMAIZER M., GAGNON J., TRIANTAPHYLIDÈS C. 1998.
Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells. Plant J. 13: 519–527.
BEARDMORE T., WHITTLE C. A. 2005. Induction of tolerance to desiccation and cryopreservation
in silver maple (Acer saccharinum) embryonic axes. Tree Physiol. 25: 965–972.
BUITINK J., CLAESSENS M. M. A. E., HEMMINGA M. A., HOEKSTRA F. A. 1998. Influence of water
content and temperature on molecular mobility and intracellular glasses in seed and pollen.
Plant Physiol. 118: 531–541.
BUITINK J., HOEKSTRA F. A., HEMMINGA M. A. 2000. Molecular mobility in the cytoplasm of lettuce radicles correlates with longevity. Seed Sci. Res. 10: 285–292.
CAMPBELL S. A., CRONE D. E., CECCARDI T. L., CLOSE T. J. 1998. A ca. 40 kDa maize (Zea mays
L.) embryo dehydrin is encoded by the dhn2 locus on chromosome 9. Plant Mol. Biol. 38:
417–423.
CARUSO A., MORABITO D., DELMOTTE F., KAHLEM G., CARPIN S. 2002. Dehydrin induction during drought and osmotic stress in Populus. Plant Physiol. Biochem. 40: 1033–1042.
CLOSE T. J. 1996. Dehydrins: emergence of a biochemical role of a family of plant dehydration
proteins. Physiol. Plant. 97: 795–803.
CLOSE T. J. 1997. Dehydrins: a commonalty in the response of plants to dehydration and low temperature. Physiol. Plant. 100: 291–296.
CUMING A. C. 1999. LEA proteins. In: Seed Proteins, pp. 753–780, Kluwer Academic Publishers,
Dordrecht.
DANYLUK J., PERRON A., HOUDE M., LIMIN A., FOWLER B., BENHAMOU N., SARHAN F. 1998.
Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. Plant Cell 10: 623–638.
LEA PROTEINS AND sHSPs IN SEED PROTECTION
13
DEROCHER A. E., VIERLING E. 1994. Developmental control of small heat shock protein during
pea seed maturation. Plant J. 5: 93–102.
DURE L. III 1993. A repeating 11-mer amino acid motif and plant desiccation. Plant J. 3: 363–369.
DURE L. III, CROUCH M., HARADA J., HO T. H. D., MUNDY J., QUATRANO R., THOMAS T., SUNG
Z. R. 1989. Common amino acid sequence domains among the LEA proteins of higher plants.
Plant Mol. Biol. 12: 475–486.
ECKEY-KALTENBACH H., KIEFER E., GROSSKOPF E., ERNST D., SANDERMANN H. JR 1997. Differential transcript induction of parsley pathogenesis-related proteins and of a small heat shock
protein by ozone and heat shock. Plant Mol. Biol. 33: 343–350.
EGERTON-WARBURTON L. M., BALSAMO R. A., CLOSE T. J. 1997. Temporal accumulation and
ultrastructural localization of dehydrins in Zea mays. Physiol. Plant. 101: 545–555.
ELLIS R. H., HONG T. D., ROBERTS E. H. 1990. An intermediate category of seed storage behaviour? J. Exp. Bot. 41: 1167–1174.
ELSTNER E. F., OSSWALD W. 1994. Mechanisms of oxygen activation during plant stress. Proc. R.
Soc. Edinb. 102: 131–154.
FARRANT J. M., PAMMENTER N. W., BERJAK P., FARNSWORTH E. J., VERTUCCI C. W. 1996. Presence of dehydrin-like proteins and levels of abscisic acid in recalcitrant (desiccation sensitive) seeds may be related to habitat. Seed Sci. Res. 6: 175–82.
FINCH-SAVAGE W. E., PRAMANIK S. K., BEWLEY J. D. 1994. The expression of dehydrin proteins
in desiccation-sensitive seeds of temperate trees. Planta 193: 478–485.
GODAY A., JENSEN A. B., CULIANEZ-MACIA F. A, ALBA M. M., FIGUERAS M., SERRATOSA J.,
TORRENT M., PAGES M. 1994. The maize abscisic acid-responsive protein RAB17 is located
in the nucleus and interacts with nuclear localization signals. Plant Cell 6: 351–360.
GOLDBERG A. L. 2003. Protein degradation and protection against misfolded or damaged proteins.
Nature 426: 895–899.
GOYAL K., TISI L., BASRAN A., BROWNE J., BURNELL A., ZURDO J., TUNNACLIFFE A. 2003. Transition from natively unfolded to folded state induced by desiccation in an anhydrobiotic nematode protein. J. Biol. Chem. 278: 12977–12984.
GUAN J. C., JINN T. L., YEH C. H., FENG S. P., CHEN Y. M., LIN C. Y. 2004. Characterization
of the genomic structures and selective expression profiles of nine class I small heat shock
protein genes clustered on two chromosomes in rice (Oryza sativa L.). Plant Mol. Biol. 56:
795–809.
GYÖRGYEY J., GARTNER A., NEMETH K., MAGYAR Z., HIRT H., HEBERLE-BORS E., DUDITS D. 1991.
Alfalfa heat shock genes are differentially expressed during somatic embryogenesis. Plant Mol.
Biol. 16: 999–1007.
HASLBECK M., FRANZMANN T., WEINFURTNER D., BUCHNER J. 2005. Some like it hot: the structure and function of small heat-shock proteins. Nat. Struct. Mol. Biol. 12: 842–846.
HASLBECK M., WALKE S., STROMER T., EHRNSPERGER M., WHITE H. E., CHEN S., SAIBIL H. R.,
BUCHNER J. 1999. Hsp26: a temperature-regulated chaperone. EMBO J. 18: 6744–6751.
HARA M., FUJINAGA M., KUBOI T. 2005. Metal binding by citrus dehydrin with histidine-rich
domains. J. Exp. Bot. 56: 2695–2703.
HÄRNDAHL U., BUFFONI HALL R., OSTERYOUNG K. W., VIERLING E., BORNMAN J. F., SUNDBY C.
1999. The chloroplast small heat shock protein undergoes oxidation-dependent conformational
changes and may protect plants from oxidative stress. Cell Stress Chaperones 4: 129–138.
HELM K. W., LAFAYETTE P. R., NAGAO R. T., KEY J. L., VIERLING E. 1993. Localization of small
heat shock proteins to the higher plant endomembrane system. Mol. Cell. Biol. 13: 238–247.
HENDRICK J. P., HARTL F. U. 1995. The role of molecular chaperones in protein folding. FASEB
J. 9: 1559–1569.
HENDRY G. A. F. 1993. Oxygen, free radical processes and seed longevity. Seed Sci. Res. 3: 141–153.
HINNIGER C., CAILLET V., MICHOUX F., AMOR M. B., TANKSLEY S., LIN C., MCCARTHY J. 2006.
Isolation and characterization of cDNA encoding three dehydrins expressed during Coffea
canephora (Robusta) grain development. Ann. Bot. 97: 755–765.
14
Ewa M. Kalemba and Stanis³awa Pukacka
HOEKSTRA F. A., GOLOVINA E. A., BUITINK J. 2001. Mechanisms of plant desiccation tolerance.
Trends Plant Sci. 6: 431–438.
HOUDE M., DANIEL C., LACHAPELLE M., ALLARD F., LALIBERTE S., SARHAN F. 1995. Immunolocalization of freezing-tolerance-associated proteins in thecytoplasm and nucleoplasm of wheat
crown tissues. Plant J. 8: 583–593.
ISMAIL A. M., HALL A. E., CLOSE T. J. 1999. Purification and partial characterization of a dehydrin involved in chilling tolerance during seedling emergence of cowpea. Plant Physiol. 120:
237–244.
JOFRÉ A., MOLINAS M., PLA M. 2003. A 10-kDa class-CI sHsp protects E. coli from oxidative and
high-temperature stress. Planta 217: 813–819.
KERMODE A. R. 1997. Approaches to elucidate the basis of desiccation-tolerance in seeds. Seed
Sci. Res. 7: 75–95.
KOAG M. C., FENTON R.D., WILKENS S., CLOSE T. J. 2003. The binding of maize DHN1 to lipid
vesicles. Gain of structure and lipid specificity. Plant Physiol. 131: 309–316.
KOSTER K. L., LEOPOLD A. C. 1998. Sugars and desiccation tolerance in seeds. Plant Physiol. 88:
829–832.
KOTAK S., LARKINDALE J., LEE U., VON KOSKULL-DORING P., VIERLING E., SCHARF K. D. 2007.
Complexity of the heat stress response in plants. Curr. Opin. Plant Biol. 10: 310–316.
LEE G. J., ROSEMAN A. M., SAIBIL H. R., VIERLING E. 1997. A small heat shock protein stably
binds heat-denaturated model substrates and can maintain a substrate in folding-competent
state. EMBO J. 16: 659–671.
LEE G. J., VIERLING E. 2000. A small heat shock protein cooperates with heat shock protein 70
systems to reactivate a heat-denatured protein. Plant Physiol. 122: 189–197.
LENNE C., BLOCK M. A., GARIN J., DOUCE R. 1995. Sequence and expression of the mRNA encoding HSP22, the mitochondrial small heat-shock protein in pea leaves. Biochem. J. 311:
805–813.
LEOPOLD A. C., SUN W. Q., BERNAL-LUGO I. 1994. The glassy state in seeds: analysis and function. Seed Sci. Res. 4: 267–274.
LISSE T., BARTELS D., KALBITZER H. R., JAENICKE R. 1996. The recombinant dehydrin-like
desiccation stress protein from the resurrection plant Craterostigma plantagineum displays
no defined three-dimensional structure in its native state. Biol. Chem. 377: 555–561.
LÖW D., BRÄNDLE K., NOVER L., FORREITER C. 2000. Cytosolic heat-stress proteins Hsp17.7 class
I and Hsp17.3 class II of tomato act as molecular chaperones in vivo. Planta 211: 575–582.
LUBARETZ O., ZUR NIEDEN U. 2002. Accumulation of plant small heat-stress proteins in storage
organs. Planta 215: 220–228.
MCCUBBIN W. D., KAY C. M., LANE B. G. 1985. Hydrodynamic and optical properties of the wheat
germ Em protein. Can. J. Biochem. Cell Biol. 63: 803–811.
MOUILLON J. M., GUSTAFSSON P., HARRYSON P. 2006. Structural investigation of disordered stress
proteins. Comparison of full-length dehydrins with isolated peptides of their conserved
segments. Plant Physiol. 141: 638–650.
NAOT D., BEN-HAYYIM G., ESHDAT Y., HOLLAND D. 1995. Drought, heat, and salt stress induce
the expression of a citrus homologue of an atypical late embryogenesis Lea5 gene. Plant Mol.
Biol. 27: 619–622.
NYLANDER M., SVENSSON J., PALVA E. T., WELIN B. V. 2001. Stress-induced accumulation and
tissue specific localization of dehydrins in Arabidopsis thaliana. Plant Mol. Biol. 45: 263–
279.
OLIVER A. E., LEPRINCE O., WOLKERS W. F., HINCHA D. K., HEYER A. G., CROWE J. H. 2001.
Non-disaccharide-based mechanisms of protection during drying. Cryobiology 43: 151–167.
OOMS J. J. J., LEON-KLOOSTERZIEL K. M., BARTELS D., KOORNNEEF M., KARSEN C. M.. 1993.
Acquisition of desiccation tolerance and longevity in seeds of Arabidopsis thaliana: a comparative study using abscisic acid-insensitive abi3 mutants. Plant Physiol. 102: 1185–1191.
POTTS M. 1994. Desiccation tolerance in prokaryotes. Microbiol. Rev. 58: 755–805.
LEA PROTEINS AND sHSPs IN SEED PROTECTION
15
PUKACKA S., HOFFMANN S. K., GOSLAR J., PUKACKI P. M., WOJKIEWICZ E. 2003. Water and lipid
relations in beech (Fagus sylvatica L.) seeds and its effect on storage behaviour. Biochim.
Biophys. Acta. 1621: 48–56.
PRIETO-DAPENA P., CASTAÑO R., ALMOGUERA C., JORDANO J. 2006. Improved resistance to controlled deterioration in transgenic seeds. Plant Physiol. 142: 1102–1112.
REDDY G. B., KUMAR P. A., KUMAR M. S. 2006. Chaperone-like activity and hydrophobicity of
alpha-crystallin. IUBMB Life 58: 632–641.
RINNE P. L., KAIKURANTA P. L., VAN DER PLAS L. H., VAN DER SCHOOT C. 1999. Dehydrins in
cold-acclimated apices of birch (Betula pubescens Ehrh.): production, localization and potential role in rescuing enzyme function during dehydration. Planta 209: 377–388.
ROBERTS E. H. 1973. Predicting the storage life of seeds. Seed Sci. Technol. 1: 499–514.
ROBERTSON M., CHANDLER P. M. 1992. Pea dehydrins: identification, characterization and expression. Plant Mol. Biol. 19: 1031–1044.
SABEHAT A., LURIE S., WEISS D. 1998. Expression of small heat-shock proteins at low temperatures: a possible role in protecting against chilling injuries. Plant Physiol. 117: 651–658.
SARNIGHAUSEN E., KARLSON D., ASHWORTH E. 2002. Seasonal regulation of a 24-kDa protein from
red-osier dogwood (Cornus sericea) xylem. Tree Physiol. 22: 423–430.
SINGH S., CORNILESCU C. C., TYLER R. C., CORNILESCU G., TONELLI M., LEE M. S., MARKLEY J.
L. 2005. Solution structure of a late embryogenesis abundant protein (LEA14) from Arabidopsis thaliana, a cellular stress-related protein. Protein Sci. 14: 2601–2609.
SMIRNOFF N. 1993. The role of active oxygen in the response of plants to water deficit and desiccation. New Phytol. 125: 27–58.
STOCKINGER E. J., GILMOUR S. J., THOMASHOW M. F. 1997. Arabidopsis thaliana CBF1 encodes
an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting
DNA regulatory element that stimulates transcription in response to low temperature and water
deficit. Proc. Natl. Acad. Sci. USA 94: 1035–1040.
SUN W. Q., LEOPOLD A. C. 1993. The glassy state and accelerated aging of soybeans. Physiol. Plant.
89: 767–774.
SUN W. Q., MOTANGU M. V., VERBRUGGEN N. 2002. Small heat shock proteins and stress tolerance in plants. Biochim. Biophys. Acta 1577: 1–9. TSVETKOVA N. M., HORVÁTH I., TÖRÖK Z., WOLKERS W. F., BALOGI Z., SHIGAPOVA N., CROWE L.
M., TABLIN F., VIERLING E., CROWE J. H., VIGH L. 2002. Small heat-shock proteins regulate
membrane lipid polymorphism. Proc. Natl. Acad. Sci. USA 99: 13504–13509.
VAN MONTFORT R. L., BASHA E., FRIEDRICH K. L., SLINGSBY C., VIERLING E. 2001. Crystal structure and assembly of a eukaryotic small heat shock protein. Nat. Struct. Biology 8: 1025–
1030.
VIERLING E. 1997. The small heat shock proteins in plants are members of an ancient family of
heat induced proteins. Acta Physiol. Plant. 19: 539–547.
WANG W., VINOCUR B., SHOSEYOV O., ALTMAN A. 2004. Role of plant heat-shock proteins and
molecular chaperones in the abiotic stress response. Trends Plant Sci. 9: 244–252.
WATERS E. R., LEE G. J., VIERLING E. 1996. Evolution, structure and function of the small heat
shock proteins in plants. J. Exp. Bot. 47: 325–338.
WEHMEYER N., HERNANDEZ L. D., FINKELSTEIN R. R., VIERLING E. 1996. Synthesis of small heatshock proteins is part of the developmental program of late seed maturation. Plant Physiol.
112: 747–757.
WEHMEYER N., VIERLING E. 2000. The expression of small heat shock proteins in seeds responds
to discrete development signals and suggest a general protective role in desiccation tolerance.
Plant Physiol. 122: 1099–1108.
WELIN B. V., OLSON A., NYLANDER M., PALVA E. T. 1994. Characterization and differential expression of dhn/lea/rab-like genes during cold acclimation and drought stress in Arabidopsis
thaliana. Plant Mol. Biol. 26: 131–144.
WILLIAMS R. J., LEOPOLD A. C. 1989. The glassy state in corn embryos. Plant Physiol. 89: 977–981.
16
Ewa M. Kalemba and Stanis³awa Pukacka
WISE M. J. 2003. LEAping to conclusions: a computational reanalysis of late embryogenesis abundant proteins and their possible roles. BMC Bioinformatics 4: 52–71.
WISE M. J., TUNNACLIFE A. 2004. POPP the question: what do LEA proteins do? Trends Plant
Sci. 9: 13–17.
WOLKERS W. F., MCCREADY S., BRANDT W. F., LINDSEY G. G., HOEKSTRA F. A. 2001. Isolation
and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro. Biochim. Biophys. Acta 1544: 196–206.
XU D., DUAN X., WANG B., HONG B., HO T.-H. D., WU R. 1996. Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress
in transgenic rice. Plant Physiol. 110: 249–257.
ZAVYALOV A. V., GAESTEL M., KORPELA T., ZAVYALOV P. 1998. Thiol/disulfide exchange between
small heat shock protein 25 and glutathione. Biochim. Biophys. Acta 1388: 123–132.
ZUR NIEDEN U., NEUMANN D., BUCKA A., NOVER L. 1995. Tissue-specific localization of heatstress proteins during embryo development. Planta 196: 530–538.