978 1 62100 769 2 - ch1

In: Droughts: New Research
Editors: D. F. Neves and J. D. Sanz
ISBN: 978-1-62100-769-2
2012 Nova Science Publishers, Inc.
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Chapter 1
THE RESPONSE OF PLANTS TO DROUGHT STRESS:

THE ROLE OF DEHYDRINS, CHAPERONES,
PROTEASES AND PROTEASE INHIBITORS
IN MAINTAINING CELLULAR
PROTEIN FUNCTION
I. Vaseva,1 J. Saboti,2 J. utar-Vozli,3 V. Megli,3 M. Kidri,2
K. Demirevska,1 and L. Simova-Stoilova1
1
Department of Plant Stress Molecular Biology,

Institute of Plant Physiology and Genetics,
Bulgarian Academy of Sciences, Sofia, Bulgaria
2
Department of Biotechnology, Joef Stefan Institute, Ljubljana, Slovenia
3
Crop and Seed Production Department,
Agricultural Institute of Slovenia, Ljubljana, Slovenia
ABSTRACT
Abiotic stresses with a dehydration component (drought, salt, and freezing) involve,
as a common feature, increased numbers of inactive proteins denatured, aggregated or
oxidatively damaged. Maintaining proteins in their functional conformation, preventing
aggregation of non-native proteins, refolding of denatured proteins to their native
conformation and removal of non-functional and potentially harmful polypeptides are all
vital for cell survival under dehydration stress. To achieve this, plants respond to drought
by synthesis of protective proteins such as dehydrins and chaperones and by degradation
of irreversibly damaged proteins by proteases. Here we review the important cellular
functions of dehydrins, chaperones, proteases and protease inhibitors, together with their
role in the response to drought, that make them potential biochemical markers for
assessing drought tolerance.
Corresponding author, email: lsimova@mail.bg.
I. Vaseva, J. Saboti, J. utar-Vozli, et al.
1. INTRODUCTION
Drought is the most widespread abiotic stress in the plant world. It reduces yields and
threatens the survival of many field crops (Passioura, 2007, Xoconostle-Czares et al., 2011).
The possibility of increasing drought tolerance in crop plants has therefore been actively
pursued (Blum 1996, Lafitte et al., 2007). Of the various approaches that have been used to
address the problem, plant breeding, either conventional or involving genetic engineering is
an effective and economic means of tailoring crops to grow successfully in water-sparse
environments (Ashraf, 2010). For their execution, however, such approaches require basic
knowledge, at the structural and molecular levels, of how drought affects plants. Furthermore,
markers of drought tolerance in plants are required to guide the breeding processes (Tuberosa
and Salvi, 2006; Chaves et al., 2009).
Plants perceive and respond rapidly to changes in water status by several morphological,
physiological, cellular, and molecular changes occurring in parallel (Ingram and Bartels,
1996; Pinheiro and Chaves 2011). Photosynthesis, together with cell growth, is among the
primary processes that are affected by water stress. At the physiological and metabolic levels,
drought causes inhibition of shoot growth, adjustment of leaf area, stomatal closure and
reduction of transpiration, inhibition of photosynthesis, shifts in carbon and nitrogen
metabolism, synthesis of compatible solutes, and secondary oxidative stress (XoconostleCzares et al., 2011). In parallel with these alterations, plants perceive and respond to stress
by rapidly altering their gene expression; this can occur even under mild to moderate stress
conditions (Chaves et al. 2009). Investigation of the model plant Arabidopsis thaliana
revealed several hundred identified genes expressed at the transcriptional level in response to
drought stress (Seki et al. 2002; Shinozaki and Yamaguchi-Shinozaki 2007). Importantly, in
the context of such studies, attention should be drawn to the fact that change in expression of
a gene cannot necessarily be interpreted in terms of change in activity of the corresponding
protein. Post-transcriptional and/or post-translational events may prevent a gene response
from being translated into a functional protein response (Chaves et al., 2003 Watson et al.
2003).
Changes in protein expression and post-translational modification of proteins are an
important part of the perception of and response to abiotic stress (Hashiguchi et al., 2010).
Proteins serve as important components of the major signalling and biochemical pathways.
Some plant stress proteins are expressed intensively in response to water deficit, especially
those involved in avoiding stress, in repairing damage and in protecting cellular machinery
from the damaging effects of the stress (Bray 1997). But abiotic stresses with a dehydration
component (drought, salt, and freezing) are an important threat to proteins, since diminution
in cellular volume, macromolecular crowding and oxidative injury can increase the number of
inactive proteins denatured, aggregated or oxidatively damaged (Hoekstra et al., 2001).
Such molecules must be either renatured or broken down and replaced by native ones.
Maintaining proteins in their functional conformations, preventing aggregation of non-native
proteins, refolding of denatured proteins to their native conformation and removal of nonfunctional and potentially harmful polypeptides are vital functions for cell survival under
dehydration stress. For these reasons drought tolerance has to be considered as a complex trait
achieved by modulation of gene expression and accumulation of specific protective proteins
and metabolites.
The Response of Plants to Drought Stress
This chapter is focused on features and mechanisms that are known to be important in the
growth of plants and that account for their sensitivity to drought and/or enable them to resist
or recover from drought stress. We describe the interplay of different proteins, including
dehydrins and chaperones, that preserve the integrity of other proteins and enable their
recovery from drought-induced damage, together with the proteolytic enzymes that are
responsible for the breakdown and recycling of damaged proteins (Figure 1).
2. DEHYDRINS
Dehydrins are a subgroup of the Late Embryogenesis Abundant (LEA) proteins. Special
attention is therefore paid to them as the most widely studied and well characterized LEA
proteins in plants under dehydration stress. This is preceded by an overview of all the groups
of LEAs.
2.1. LEA Proteins Structural and Functional Diversity

Hydrophilic proteins, which form an important part of the plant response to water
shortage, were first characterized in cotton during the late stages of embryogenesis, and
termed LEA proteins (Dure et al., 1989). On the basis of their predicted structure,
accumulation in response to drought, salt and cold stress, and in vitro activity, LEA proteins
are considered to participate in protecting cellular components from dehydration (Reyes et al.,
2005). They are characterized by relatively high glycine content and hydrophilicity and low
secondary structure content. Generally they remain soluble upon boiling, a property that is
used as an initial step in their purification.
Several nomenclature systems have been reported to classify LEA proteins. Initially, they
were classified according to their molecular weight and thus named D7, D11, D19, D29, D34,
D73, D95, D113 (Hughes and Galau, 1991). In response to the growing number of newly
identified LEA proteins, a new classification system, based on protein amino acid
composition and sequence motifs, was introduced (Battaglia et al., 2008). LEA proteins have
been classified into six groups by different nomenclature systems on the basis of conserved
sequence motifs.
Group 1 LEA proteins are only found in plants. They contain a hydrophilic 20-mer motif
TRKEQ[L/M]G[T/E]EGY[Q/K]EMGRKGG[L/E] (Baker et al., 1995) and exhibit a high
proportion of random coil configuration in aqueous solution (Soulages et al., 2002). Group 1
LEA proteins accumulate preferentially during embryo development, and some of them are
responsive to abscisic acid (ABA) and to water-limiting conditions, mainly in embryos and in
vegetative tissues of young seedlings (Vicient et al., 2000).
Dehydrins belong to group 2 of the LEA proteins. All dehydrins have a conserved,
lysine-rich 15-amino acid domain, EKKGIMDKIKEKLPG, named the K-segment (Close,
1997) and usually present near the C-terminus. Other typical dehydrin features are a track of
Ser residues (the S-segment), a consensus motif, T/VDEYGNP (the Y-segment) located near
the N-terminus, and less conserved regions, usually rich in polar amino acids (the segments). Dehydrins do not display well-defined secondary structure. The expression pattern
of group 2 LEA genes is frequently associated with higher tolerance of crop plants to abiotic
stresses such as cold (Zhu et al., 2000) and drought (Lopez et al., 2003; Suprunova et al.,
2004, Vaseva et al., 2010).
Group 3 LEA proteins are characterized by a repeated 11-mer amino acid motif whose
consensus sequence has been broadly defined as E/QXKE/QKXE/D/Q, where
represents a hydrophobic residue (Dure, 1993). Proteins homologous to the group 3 LEAs
have been discovered in organisms other than plants, including nematodes and prokaryotes.
They are natively unfolded in solution, but appear to be more structured when the water
content of the environment is lower (Tolleter et al., 2007).
Group 4 LEA proteins accumulate in mature seeds, and are also induced in leaves by
plant hormones, abiotic elicitors and environmental stresses (Dure et al., 1989). The proteins
of this family are conserved in their N-terminal portion, which is 70 to 80 residues long and is
predicted to form amphipathic -helices, while the less conserved C-terminal portion differs
in size (Dure, 1993). A characteristic motif in the proteins in this group is motif 1, located at
the N-terminal region with the consensus sequence: AQEKAEKMTA[R/H] DPXKEMA
HERK[E/K][A/E][K/R]. LEA D113 gene from G. hirsutum L. is a typical representative of
this group (Luo et al., 2008).
LEA 5 proteins (hydrophobic or atypical LEA proteins) contain a significantly higher
proportion of hydrophobic residues and include non-homologous proteins. These proteins are
not soluble after boiling, which suggests that they adopt a globular conformation (Singh et al.,
2005). LEA 5 transcripts accumulate during the late stage of seed development and in
response to stress conditions, such as drought, UV light, salinity, cold, and wounding (Park et
al., 2003; Kim et al., 2005).
Proteins belonging to LEA 6 family have been identified in a variety of vascular plant
species. Their molecular mass is relatively low (714 kDa) and they exhibit high sequence
conservation. Four motifs distinguish this group, two of which are highly conserved
(sequence LEDYK present in motif 1 and the Pro and Thr residues in motif 2). In general,
these proteins are highly hydrophilic, lack Cys and Trp residues, and do not coagulate upon
exposure to high temperatures. PvLEA18 protein from bean (Phaseolus vulgaris) was the first
protein described from the LEA 6 group (Colmenero-Flores et al., 1997). It is present at high
levels in dry seeds and pollen grains and responds to water deficit and ABA treatment. There
is evidence that the molecular targets of these proteins differ from those of other LEA
proteins (Reyes et al., 2005).
2.2. Protective Functions of Dehydrins on Proteins and Cell Structures

Dehydrins are distributed over a wide range of organisms including the higher plants,
algae, yeast and cyanobacteria. They accumulate late in embryogenesis and in nearly all the
vegetative tissues during normal growth conditions and in response to stress leading to cell
dehydration (e.g. drought, low temperature and salinity). It is suggested that the dehydrins
interact with membranes in the interior of cells and reduce dehydration induced damage
(Danyluk et al., 1998). The mechanism of these interactions could be explained by their
ability to replace water and, through their hydroxyl groups, to solvate cytosolic structures
(Baker et al., 1995). Other possible explanations are that dehydrins prevent interactions
between membrane bilayers or that they are able to chelate ions, alleviating the damaging
effect of increased ion concentrations (Danyluk et al., 1998).
Structural and biochemical studies indicate that dehydrins are intrinsically disordered
proteins (IDPs), i.e. in their functional state they are devoid of a single and stable tertiary
structure (Tompa, 2009). The molecular function of dehydrins is still poorly characterized,
although several mechanisms have been proposed by which the consequences of
environmental stresses could be mitigated, such as membrane stabilization, resistance to
osmotic pressure and protection of proteins the so-called chaperone function (Agoston et
al., 2011). It was suggested that this latter effect is based on a molecular shield
mechanism, rather than typical chaperone activity. According to this concept dehydrins are
able to inhibit the interaction between denatured protein molecules, preventing the formation
of aggregates. The structure/function relationship of dehydrins, as IDPs, is much less well
established than that of globular proteins. They are proposed to function either as entropic
chains or by molecular recognition (Tompa, 2005). It is very probable that dehydrins, being
typical IDPs, are able to bind their partner molecules via short recognition elements. During
this interaction, dehydrin molecules could participate in a structurally adaptive process termed
disorder-to-order transition or induced folding (Fuxreiter et al., 2004; Agoston et al., 2011).
The number and order of the Y-, S- and K-segments define different dehydrin subclasses: YnSKn, YnKn, SKn, Kn and KnS. Each dehydrin structural type may possess a
specific function and tissue distribution. Their precise function has not been established, but
in vitro experiments indicate that some dehydrins (YSKn-type) bind to lipid vesicles that
contain acidic phospholipids, and others (KnS) bind metals and are able to scavenge hydroxyl
radicals (Asghar et al., 1994, Alsheikh et al., 2003), protect lipid membranes against
peroxidation and are cryoprotective towards enzymes sensitive to freezing. Dehydrins of the
SKn and K sub-classes appear to be directly involved in cold acclimation processes (Houde et
al., 1995; Danyluk et al.,1998; Zhu et al., 2000; Allagulova et al., 2007). Those of the YnSKn
type are usually low molecular weight, alkaline proteins that are induced by drought (Xiao
and Nassuth, 2006; Vaseva et al., 2010).
Biochemical analyses of dehydrins have shown that spinach COR85, maize G50, wheat
WSC120 and peach PCA60 have cryoprotective activity (Houde et al., 1995; Wisniewski et
al., 1999). PCA60 also exhibits antifreeze activity by modifying the normal growth of ice and
exhibiting thermal hysteresis (Wisniewski et al., 1999).
2.3. Subcellular and Tissue Localization of Dehydrins

Dehydrins may have multiple functions and are specialized for different growth
conditions, tissues, cells and stresses. Their predominant localization in the plant vascular
system and the apical meristems is crucial for plant growth and survival. Their accumulation
in root tips may promote water influx into the actively dividing parenchymal cells of the root
meristem. Similarly, in cells surrounding the xylem vessels, dehydrins may function as water
attractants during the transport of water from xylem vessels to sink tissue.
Extensive localization studies revealed that dehydrins accumulate in specific tissues and
cells in Arabidopsis under normal growth conditions. Immunohistochemical localization
demonstrated tissue and cell type specificity in unstressed plants (Nylander et al., 2001).
Arabidopsis dehydrins ERD14 and LTI29 were detected in the root tip of plants grown under
normal conditions. ERD14, LTI29 and RAB18 are expressed in vascular tissues and RAB18
in stomatal guard cells. Accumulation of ERD14, LTI29 and RAB18 was detected in most
cells upon stress treatment, most intensely in cells surrounding vascular tissues. Nylander et
al. (2001) also found that dehydrin LTI30 was absent in unstressed plants, but accumulated
upon stress treatment, mainly in vascular tissues and pollen sacks, probably being both
transcriptionally and posttranscriptionally regulated.
Dehydrins have been associated with most cellular organelles, and post translational
modifications, such as phosphorylation, can affect their localization. At the subcellular level
dehydrins are localized in various cell compartments, such as the cytosol, nucleus,
mitochondria, vacuole, and in the vicinity of the plasma membrane, but primarily in the
cytosol and nucleus. The wheat dehydrin WCOR410, however, was found to accumulate
preferentially in the vicinity of the plasma membrane of cells, in the vascular transition area
(Danyluk et al., 1998). In some cases dehydrins are associated with cellular organelles. For
example, peach PCA60, in addition to the cytosol and nucleus, is associated with chloroplasts
(Wisniewski et al., 1999). Dehydrin-like proteins have been shown to be associated with
mitochondria in winter wheat, winter rye and maize (Borovskii et al., 2000) and in
cauliflower, Arabidopsis and yellow lupin plants submitted to cold stress (Rurek, 2010).
2.4. Relation of Expression of Dehydrins to Their Function

The differences in stress specificity and spatial distribution of dehydrins suggest a
functional specialization for the members of this protein family. Dehydrins, as well as being
important for cell survival during stress, may play an osmoregulatory role in certain cell-types
in the absence of stress. The presence of constitutively expressed dehydrins has been reported
in various plant species. Examples are pea B61, which is an SK2 type, and Craterostigma
plantagineum DSP16, which is a YSK2 type dehydrin (Robertson and Chandler, 1994;
Schneider et al., 1993). Constitutive dehydrin-like proteins have also been reported in birch
(16 kDa), poplar (50 kDa) and dogwood (60 kDa) (Rinne et al., 1999; Wisniewski et al.,
1999). Some constitutively expressed dehydrins are also stress-responsive. For example,
Arabidopsis RAB18 (YSK-type) dehydrin, present in the nuclei of guard cells under normal
conditions, is also present in the cytosol under stress. Similarly, stress-induced promoter
activity in stomatal guard cells has been reported for C. plantagineum DSP16 (Taylor et al.,
1995) and tomato TAS14 (Parra et al., 1996), both of which are also YSK-type dehydrins.
Stress-induced accumulation of five dehydrins in Arabidopsis (COR47, LTI29, ERD14,
LTI30 and RAB18), after treatments with low temperature, ABA and high salt concentration,
was characterized immunologically with protein-specific antibodies (Nylander et al., 2001).
The dehydrins exhibited clear differences in their accumulation patterns in response to
different stressors. LTI30 dehydrin was not detected in unstressed plants. ERD14 (SK2)
accumulated in unstressed plants, although the protein level was up-regulated by ABA,
salinity and low temperature. LTI29 (SK3) accumulated mainly in response to low
temperature, but was also found in ABA- and salt-treated plants. LTI30 (K6) and COR47
(SK3) accumulated primarily in response to low temperature, whereas RAB18 (Y2SK2) was
only found in ABA-treated plants and was the only dehydrin in this study that accumulated in
dry seeds.
2.5. Involvement of Dehydrins in the Response to Drought

The accumulation of dehydrins in plants is a common feature of the response to drought
(Close, 1997). Earlier studies indicated that the accumulation of certain dehydrin proteins in
seedlings was associated with drought tolerance in adult plants (Lopez et al., 2003).
Overproduction of a wheat dehydrin (DHN5) in Arabidopsis enhanced the tolerance to
osmotic stress. When compared to wild type plants, the dehydrin-5 transgenic plants exhibited
stronger growth under water deprivation and more rapid recovery (Brini et al., 2007).
Some genes coding for mainly low-molecular weight and some alkaline YnSK-type
dehydrins tend to be induced only by drought (Close et al., 2000). Recent results have
confirmed that, under water deprivation, white clover synthesized an alternatively spliced
Y2SK transcript, which was absent after recovery of plant upon rehydration (Vaseva et al.
2011).
Two isoforms of YSK type dehydrin gene have been reported in expression studies of
Vitis riparia and Vitis vinifera (Xiao and Nassuth 2006), and of winter wheat (Vaseva et al.
2010). Wheat WZY1-2 gene encodes a 262 amino acid alkaline YSK2-type dehydrin with a
predicted molecular weight of 28 kDa. It contains a serine-rich segment that can be
phosphorylated and is thought to participate in nuclear localisation (Close, 1997).
Quantitative differences in accumulation of WZY2 transcripts have been observed soon after
withholding water, in winter wheat cultivars with different drought tolerance (Vaseva et al.,
2010). The drought-tolerant variety showed a considerable increase in WZY2 transcript
levels, accompanied by the presence of a second, high-density band, absent in the control
samples. This alternatively spliced WZY2 transcript occurred only in drought-stressed plants
and its expression levels were related to the drought-tolerance of the cultivars. The tolerant
variety tends to accumulate WZY2 alternatively spliced transcripts earlier than the less
tolerant ones. This makes WZY2 dehydrin a suitable candidate for a molecular marker of
drought tolerance.
3. MOLECULAR CHAPERONES
Molecular chaperones are a large group of structurally conserved and genetically diverse
protein families unified by their main functions - to facilitate folding and assembly of proteins
into macromolecular structures, to facilitate protein transport across membranes, to inhibit
misfolding and to prevent and/or reverse aggregation of proteins (Feder and Hofmann 1999).
Initially this group of proteins was discovered as heat shock proteins (Hsps) by Ritossa
(1962) in his study on the gene expression in Drosophila after exposure to heat. They are thus
often referred to as Hsps/chaperones. Chaperones not belonging to Hsps some enzymes
called foldases which catalyse covalent reactions involved in the protein folding at the
endoplasmic reticulum are not considered here. Rather, a comprehensive overview of the
families of molecular chaperones and their involvement in drought response is provided.
3.1. General Characteristics

Chaperones are essential for the proper functioning of cells and are distributed
ubiquitously in all living organisms, in almost all cell compartments (Ellis, 1990).
Chaperones are among the most abundant cell proteins for example Hsp 90 accounts for
about 1-2 % of total cell protein (Vierling, 1991; Leidhold and Voos, 2007, Patterson and
Hhfeld, 2006) and are particularly abundant in cytosol, endoplasmic reticulum, mitochondria
and chloroplasts. They are highly structured and undergo conformational changes linked to
their function, recognizing and binding to exposed hydrophobic patches in the polypeptide
chains, thus stabilizing proteins and promoting folding to the native state if possible or
assisting recognition and removal by the proteasome systems (Liberek et al., 2008). To
accomplish their essential functions, chaperones usually work in close cooperation with each
other and with other proteins like co-chaperones and components of the proteasome systems
(Leidhold and Voos 2007). Various chaperones act in concert in protecting proteins from
stress, forming a network of chaperone machinery. There is a tight interplay between the
cellular protein-folding and protein-degradation systems mediated by the chaperone network
(Patterson and Hhfeld, 2006). They have essential functions in protein homeostasis in
normal conditions and are highly responsive to various stresses (Wang et al., 2004).
3.2. Structural and Functional Diversity

The striking molecular diversity of chaperones is related to the diversity of their functions
in general and their different roles in non-stress and stress conditions. Each of the five major
families of chaperones is characterised by amino acid sequence homology, approximate
molecular weight, structure, mode of action and specific function - 12-40 kDa small Hsps
which are widely present in plants; chaperonins (GroEL and Hsp60), the Hsp70/DnaK family,
the Hsp90 family, and the Hsp100/Clp family. Except for the small Hsps, all other chaperones
use ATP for processing client proteins. ATP binding and hydrolysis (the ATPase cycle)
controls binding and release of the substrate protein through conformational changes in the
chaperone (Wang et al., 2004; Patterson and Hhfeld, 2006).
3.2.1. Small Hsp Families

Higher plants possess at least 20 types of small Hsp (sHsp) families in the range of 12-40
kDa, all encoded by nuclear multigene families and divided into 6 classes three (CI, CII and
CIII) coding for sHsps located in the cytosol and nucleus and three (CIV, CV and CVI) in the
endoplasmic reticulum, plastids and mitochondria (Mahmood et al., 2010). Cytosolic CI
sHsps are the most abundant sHsps in plants. Sequence similarity in one class is up to 93%
for different plant species while sHsps belonging to different classes in one species have only
50-75% sequence similarity (Vierling, 1991; Mahmood et al., 2010).
Chaperones of this group share a conserved 90 amino acid C-terminal domain the crystallin domain or heat shock domain that contains several -strands forming two -sheets.
This domain is involved in the oligomerization of sHsps to dimers and trimers, forming a
dodecamer double ring (Liberek et al., 2008). In vivo, plant sHSPs form large spherical or
cylindrical oligomeric structures of 200-300 kDa. Complex formation is ATP-independent
but appears to be regulated by temperature and phosphorylation (Chang, 2009). The

oligomeric complex is thought to be a storage form that continuously exchanges constantly
dimerized subunits. Dissociation may be necessary for effective chaperoning activity, the
active form of sHsps being a dimer (Patterson and Hhfeld, 2006). Small Hsps have the
ability to bind strongly to exposed hydrophobic regions in non-native proteins through
hydrophobic interaction in an ATP-independent manner, thus stabilizing them and preventing
irreversible aggregation. It appears that temperature-activated sHsps associate with heatdestabilized proteins in the aggregates, conferring different physicochemical properties on the
aggregates (Liberek et al., 2008). Although sHsps are not able to refold non-native proteins,
they could provide immediate protection under unfavourable conditions, maintaining a pool
of substrates for subsequent refolding by the ATP-dependent chaperones of the Hsp70 and
Hsp100 families.
3.2.2. Chaperonins (Hsp60 Family)

There are 2 subfamilies of chaperonins - Group I, E. coli GroEL and the homologous
Cpn60 in chloroplasts and mitochondria, and Group II, the CCT chaperonins (containing tcomplex polypeptide 1) in Archea and the cytosol of eukaryotes. Folding of the most
abundant chloroplast enzyme Rubisco is assisted by Rubisco binding protein (RBP) which
belongs to the chaperonin family. Plastid Cpn60 in Arabidopsis is encoded by 7 genes, and 9
are predicted to encode the distinct CCT subunits in Arabidopsis (Wang et al., 2004).
The main function of this group is to assist the proper folding of newly synthesized and
membrane-translocated proteins into their native conformation. Cpn60 molecules form a
multisubunit structure of nearly one million Daltons, composed of double back-to-back rings,
each of 7 subunits of about 60 kDa, surrounding a central cavity large enough to refuge a
protein of up to 60-70 kDa (Bukau and Horwich, 1998). The central cavity functions in two
states binding-active and closed. The former is open at the end, exposing a flexible
hydrophobic lining that binds the non-native protein, provoking additional partial unfolding,
in that way removing the bound protein from a kinetic trap. The closed state is formed by
complexing with a co-chaperone (Hsp10 or Hsp20), resulting in dramatic en bloc upstream
movements of the hydrophobic parts, enlarging the central cavity and exposing the now
hydrophilic inner surface, thus promoting the transition of the trapped protein into its native
state (Bukau and Horwich, 1998). Chaperonins thus appear to function through alternating
exposure of hydrophobic and hydrophilic inner surfaces. The complex with co-chaperone is
stable with bound ATP and unstable with bound ADP. ATP hydrolysis triggers substrate
release, enabling the next cycle of chaperone action (Bukau and Horwich, 1998). Under heat
shock conditions the folding capacity of the chaperonins is suppressed while their binding
affinity towards the unfolded client proteins is increased (Llorca et al., 1998). That way
chaperonins could protect their client proteins from unfavourable conditions by sequestering
them, releasing them only when the stress is relieved. CCT chaperonins form 8-9 subunit
rings, each subunit being encoded by a distinct but related gene. They assist the folding of
actin and tubulin in the cytoplasm (Wang et al., 2004).
3.2.3. Hsp70 Family
This family is highly evolutionarily conserved (about 50% identity between E.coli and
eukaryotic Hsp70s) and includes two subfamilies: Hsp70/Hsp40 and Hsp110/SSE (Grigorova
2010). The Arabidopsis genome contains 18 genes coding for Hsp70 chaperones 14 of the
10
DnaK subfamily and 4 of the Hsp110/SSE subfamily (Wang et al., 2004). Some members of
this family are phosphorylated and/or methylated (Chang, 2009).
Hsp70 family is engaged in assisting folding of newly synthesized proteins, enabling
translocation of proteins across membranes of organelles, disassembling oligomeric protein
structures, refolding misfolded and aggregated proteins, facilitating proteolytic degradation of
unstable proteins by targeting them to proteasomes, controlling the activity of regulatory
proteins (Bukau and Horwich, 1998). Hsp70 chaperones exhibit common structural features a highly conserved N-terminal ATPase domain (nucleotide binding domain, NBD) of 44 kDa,
a central peptide-binding cleft, and a C-terminus that forms a lid over the peptide-binding
cleft. Crystallographic analysis has revealed the formation of a substrate-binding hydrophobic
pocket. Hsp70 binds with high affinity to short hydrophobic segments in extended
conformation. The consensus motif recognized in the client proteins consists of 4-5
hydrophobic residues, flanked at each side by basic residues. Such a motif occurs frequently
in buried -strands and is exposed in non-native proteins. ATP binding to the N-terminal
domain of Hsp70 drives conformational changes in the C-terminal domain the substrate
binding pocket is open allowing rapid exchange of substrates. In the ADP-bound state the
substrate binding site is closed, with high affinity and slow exchange rate for substrates.
Hsp70 prevents protein aggregation and promotes proper folding by shielding hydrophobic
segments in the protein substrate. ATP hydrolysis is the rate limiting step in the ATPase cycle
of Hsp70 and this step is subjected to control by co-chaperones (Bukau and Horwich, 1998).
Co-chaperones of the Hsp40 family stimulate the ATP hydrolysis step and promote substrate
binding. The Hsp70-interacting protein Hip stabilizes the ADP-bound conformation, whereas
the co-chaperone BAG-1 stimulates nucleotide exchange.
3.2.4. Hsp90 Family

The Hsp90 family is distinct from other families by its active involvement in signal
transduction networks and cell cycle control. Besides managing protein folding of signal
transduction proteins, Hsp90 interacts with 26S proteasome and plays a principal role in its
assembly and maintenance (Wang et al., 2004). Plant Hsp90s exhibit 63-71% amino acid
identity with yeast and animal Hsp90s (Krishna and Gloor, 2001). In Arabidopsis, 7 genes
code for members of the Hsp90 family 4 for cytoplasmic Hsp90 proteins, and 3 for Hsp90s
located in plastids, mitochondria and the endoplasmic reticulum (Krishna and Gloor, 2001;
Wang et al., 2004).
This group of chaperones binds to substrates at a late stage of folding to assist activation
of signalling proteins, by accepting partially folded proteins from Hsp70 for further
processing (Pearl et al., 2008). The functional form of Hsp90 is a phosphorylated dimer
containing 2 to 3 covalently bound phosphate molecules per monomer. Biochemical and
electron microscopic studies indicate that Hsp90 contains two clearly distinguishable
domains, attached to each other by a relatively flexible, highly charged loop. The C-terminal
domain itself may also have a bilobal structure (Krishna and Gloor, 2001). The N-terminal
ATP-binding domain is a highly twisted, eight-stranded -sheet covered on one side by helices, with a deep pocket site for ATP/ADP binding at the centre of the helical side. This
domain is involved in binding target proteins. The central, highly charged region of Hsp90
contains alternating lysine and glutamic acid residues (KEKE-motifs) which may be
involved in protein-protein interactions and serve as a binding site for the proteasome. The Cterminal domain has a binding site for calmodulin and is engaged in constitutive Hsp90
11
dimerization. ATP binding and hydrolysis are coupled to transient N-domain dimerization in
an ATP-driven molecular clamp mechanism. The N-terminal and central domains, which both
bind protein-kinase client protein (bivalent interaction), move relative to each other during the
ATPase cycle, thus forcing a change of the mutual orientation of the domains in the substrate
protein for its activation (Saibil, 2008). Besides various structurally unrelated proteins, Hsp90
client proteins are mostly protein kinases and transcription factors (Pearl et al., 2008). That
way Hsp 90 integrates multiple regulatory signals in signalling networks.
Members of the Hsp70 and 90 families are able to cooperate with the ubiquitinproteasome system (described in section 5.1) with the mediation of the CHIP protein, which
is an E3 ligase that interacts with molecular chaperones through its N-terminal tetratricopeptide domain (Murata et al., 2003). In this way the CHIP protein assists interaction between
the chaperone system and the ubiquitin-proteasome system.
3.2.5. Hsp100/Clp Family

Chaperones of this family are members of the large AAA ATPase superfamily of
enzymes that catalyze mechanical processes such as locomotion, unwinding, disassembly and
unfolding of other macromolecules (Maurizi and Xia, 2004). Hsp100/Clp chaperones are
divided into two classes. Class 1 contains proteins with two AAA modules including Hsp104,
ClpB, mitochondrial Hsp78, plant Hsp101, ClpA and ClpC. Class 2 comprises proteins
having only one AAA module, such as ClpX. Class 1 is divided into 2 subfamilies ClpB/Hsp104, which possesses disaggregating activity coupled with the refolding activity of
Hsp70 chaperones, and the ClpA subfamily whose unfolding activity is coupled with ATPdependent proteolysis by ClpP subunits. The Arabidopsis genome contains 8 genes coding for
Hsp100 proteins, 5 of which have predicted plastidial localization signals (Agarwal et al.,
2001). Five Hsp100 genes are present in rice, 4 of them predicted to have chloroplast transit
peptides (Batra et al., 2007).
All members of this family have a conserved structural core the AAA module that
consists of 2 subdomains a large / domain consisting of five-stranded parallel -sheets,
flanked by -helices and connected by a mobile linker to a smaller helical C-terminal domain.
ATP binds between the two domains in a crevice containing Walker A and B motifs (the
catalytic residues for ATP hydrolysis) and sensors 1 and 2 which respond to the nucleotide
state (Maurizi and Xia, 2004). ClpA and ClpB have two AAA modules in tandem (NBD1 and
NBD2) linked to a large helical N-domain, as well as a ClpP-binding loop in the C-terminal
module. ClpB also possesses an additional intermediate or middle domain (I-domain or Mdomain) situated near the junction of NBD1 and NBD2, and composed of two coiled-coils
running in opposite directions from the point of attachment, like a two-bladed propeller. The
I-domain undergoes displacements in response to nucleotide hydrolysis by NBD1 and plays a
substantial role in the re-solubilization of protein aggregates (Barends et al., 2010).
Hsp100/Clp chaperones form hexameric rings with a central channel of 25 in Clp proteins
(Doyle and Wickner, 2008). ClpA and ClpB, which contain two AAA domains, form a
bilayered structure with two homomeric rings formed by NBD1 and NBD2, both acting in the
same direction for vectorial translocation of the unfolded protein substrate through the central
channel. The channel is lined with highly dynamic pore loops containing conserved tyrosine
residues, implicated in binding unfolded substrate and translocating it through the channel
(Saibil, 2008; Barends et al., 2010). Hsp100 chaperones act as unfoldases hydrolysis of
ATP is coupled to the mechanical action of unravelling the folded structure, beginning from a
12
loosely folded region of the polypeptide substrate. Members of the Hsp100/Clp family
function as chaperone parts of the ATP-dependent Clp proteases (see section 5.2) in plant
organelles (Leidhold and Voos, 2007).
3.3. Relation of Expression to Function

Some chaperones are constantly expressed and are referred as heat shock cognates (Hsc)
a term that indicates Hsps expressed constitutively to serve protein homeostasis in the
absence of stress. They are involved in folding of de novo synthesized polypeptides and the
import/translocation of precursor proteins. Proper folding is mediated by Hsc70 for about 1020% of the nascent proteins; for more complex proteins, especially in organelles (10-15% of
the cases), the assistance of the chaperonins is necessary (Liberek et al., 2008). While, in
prokaryotes, Hsp70 and Hsp60 chaperones have both housekeeping and stress-responsive
roles, in eukaryotes a distinction exists between chaperones linked to protein synthesis and
those induced by stress (Liberek et al., 2008). Chaperone concentrations are highly responsive
to diverse stresses. The induction of chaperones is regulated by heat shock transcription
factors (HSFs) which interact with the heat shock element sequence in the promoter region of
the HSP genes. HSFs are expressed constitutively but are stored in inactive form in the
cytoplasm. Stress provokes oligomerization of HSFs, with translocation to the nucleus
(Grigorova, 2010). Some Hsps are expressed predominantly under stress, their major function
being to assist refolding/ degradation of stress modified proteins. Hsp100/Clp proteins
perform essential functions in plants and are constitutively expressed, however their
expression is also developmentally regulated and induced by environmental stresses such as
heat, cold, drought, salinity, dark-induced etiolation, as well as in the post-stress phase during
recovery from stress (Wang et al., 2004). Hsp100 chaperones act in collaboration with Hsp70
and sHSPs to rescue proteins from stress-induced aggregation (Liberek et al., 2008).
3.4. Involvement of Chaperones in Response to Drought

Generally Hsps are expressed under normal conditions and accumulate in response to
high temperature, nevertheless they could be induced or increasingly expressed to protect
against various other stresses including drought (Shinozaki and Yamaguchi-Shinozaki, 1996;
Campalans et al., 2001). Feder and Hofmann (1999) postulated that all known stresses, if
sufficiently intense, induce Hsp expression. It has been demonstrated that Hsps can be
induced by water deficit, preventing protein aggregation and denaturation during the stress;
they may also play a role in desiccation tolerance (Almoguera and Jordano, 1992; Alamillo et
al., 1995). The drought induced Hsps with chaperone function include different classes of
Hsps - Hsp100 or Clp, Hsp90, Hsp70, Hsp60 and sHsps below 30 kDa (Wang et al., 2004).
The effect of drought on gene expression of some Hsps has been analyzed in tobacco
plants and Arabidopsis thaliana by Rizhsky et al. (2002, 2004). The highest Hsp expression
was established under combined drought and heat stress in wheat plants (Grigorova et al.,
2011a, 2011b). During drought stress, alone or combined with high temperature stress, the
drought-tolerant cultivar exhibited higher Hsp70 and sHsp contents than the sensitive cultivar.
Recently, Akashi et al. (2011) studied dynamic changes in the leaf proteome of a wild
13
watermelon. Using PCR and immunoblot analyses they found that 15 of the 23 up-regulated
proteins under water deficit (65% of annotated up-regulated proteins) were Hsps. Moreover,
10 out of the 15 up-regulated Hsps belonged to the sHsp family. Other stress-induced proteins
included those related to anti-oxidative defence and carbohydrate metabolism. According to
the authors these observations suggest that the defence response of wild watermelon may
involve orchestrated regulation of a diverse array of functional proteins related to cellular
defence and metabolism, of which Hsps may play a pivotal role in protecting the plant under
water deficit (Akashi et al., 2011). The protective chaperone activities of Hsp70 help to
confer tolerance to heat, glucose deprivation, and drought. Overexpression of Hsp70 in many
organisms correlates with enhanced thermotolerance, altered growth, and development. In
antisense transgenic Nicotiana tabacum plants subjected to heat stress and drought, the results
suggested the indirect functions of Nt-Hsp70 in defence mechanisms (Cho and Choi, 2009).
Later, Cho and Hong (2006) considered that over-expression of tobacco NtHsp70-1
contributes to drought-stress tolerance. Alvim et al. (2001) also detected enhanced
accumulation of Hsp70 in transgenic tobacco plants after a reduction of its relative water
content to 65%, which confers tolerance to water stress.
Pareek et al. (1997) found two proteins from the Hsp90 family that were expressed on
exposure of rice seedlings to water stress and elevated salinity. Three At-Hsp90 isoforms cytosolic AtHsp90.2, chloroplast-located At-Hsp90.5 and endoplasmic reticulum (ER)located At-Hsp90.7 - were characterized by constitutively overexpressing their genes in
Arabidopsis thaliana (Song et al., 2009). It was concluded that the overexpression of AtHsp90 isoforms enhances plant sensitivity to salt and drought stresses. The overexpression of
At-Hsp90 isoforms may shift the equilibrium of Hsp90s with their client-bound states, disrupt
ABA-dependent or Ca2+ pathways, and thus impair plant tolerance to abiotic stresses,
suggesting that proper homeostasis of Hsp90 is critical for a cellular stress response and/or
tolerance in plants.
Sato and Yokoya (2008) suggested that overproduction of sHsp17.7 could increase
drought tolerance in transgenic rice seedlings. The authors observed that, although no
significant difference was found in water potential of seedlings between transgenic lines and
wild-type plants at the end of drought treatments, only transgenic seedlings with higher
expression levels of sHsp17.7 protein could resume growth after rewatering. Overproduction
of At-Hsp17.6A could increase salt and drought tolerance in Arabidopsis thaliana (Weining
et al., 2001). Wehmeyer and Vierling (2000) proposed that the expression of sHsps suggests a
general protective role in desiccation tolerance.
4. PROTEASES AND PROTEASE INHIBITORS

Protein breakdown has been recognized as essential for the adaptation of plants to
environmental conditions (Vierstra, 1996). The main players in carrying out and regulating
protein breakdown are proteases, together with specific endogenous inhibitors that regulate
their activities. A very large number of different proteases are present in all living organisms.
We present a general overview of the structure and function of plant proteases and protease
inhibitors, as well as of their involvement in the response of plants to drought. Some of the
proteases involved in specific, targeted proteolysis require metabolic energy in the form of
14
ATP for their activity and are termed ATP-dependent (Adam, 2007). In general, such energy
coupling is typical of the large multi-subunit protease complexes that operate in the
proteinaceous milieu of the cytoplasm and in close cooperation with molecular chaperones
(Vierstra, 1996). They are described separately in section 5.
4.1. Structural and Functional Diversity of Plant Proteases

Proteases are enzymes that catalyse hydrolysis of peptide bonds. They are also referred to
as proteolytic enzymes or peptidases. The latter term is broader, since it denotes the ability to
cleave any peptide bond, while the terms protease and proteolytic enzyme signify enzymes
whose natural substrates are proteins. Further, peptidases that cleave peptide bonds within the
polypeptide chain are called endopeptidases, for which the older term proteinase is still used
by some authors. Peptidases that cleave peptide bonds at the termini of polypeptide chains are
called exopeptidases; they can be aminopeptidases, acting on N-terminal, and carboxypeptidases, acting on C-terminal peptide bonds. They can act by cleaving off only one amino
acid or a small peptide fragment. Recently, a global approach to the proteolytic system,
termed degradomics, has been introduced. It covers the application of genomic and proteomic
approaches to identifying the protease and protease-substrate repertoires in an organism in
order to disclose the specific functions of these enzymes (Lpez-Otn and Overall, 2002).
Generally, proteases are synthesized as inactive pre-pro-polypeptides with auto-inhibitory
pro-domains and undergo strictly controlled maturation to result in active enzymes (LpezOtn and Bond, 2008).
The classification of proteases is based on the mechanism of catalysis. According to their
catalytic type they are usually classified into aspartic, cysteine, serine and threonine
peptidases, based on the amino acid residue at the active site that is directly involved in
peptide bond hydrolysis, and metalloproteases that require a catalytic divalent metal ion
within the active site. There are also proteases of unknown catalytic type for which no active
site residues have yet been determined, either biochemically or by site-directed mutagenesis.
The IUBMB Enzyme Nomenclature system (http://www.chem.qmul.ac.uk/iubmb)
classifies peptidases as hydrolases acting on peptide bonds with the number EC 3.4 and, in
further sub-classification, combines the position of the cleaved peptide bond and the catalytic
type. For example, EC 3.4.11 are aminopeptidases, EC 3.4.13 are dipeptidases, EC 3.4.15
peptidyl-dipeptidases, EC 3.4. 16 serine-type carboxypeptidases, EC 3.4.21 serine
endopeptidases, EC 3.4.22 cysteine endopeptidases, etc., and EC 3.4.99 includes endopeptidases of unknown catalytic mechanism.
The MEROPS database (http://merops.sanger.ac.uk/) accurately reflects the growth of
knowledge of new proteases and is thus very useful for research in the field of peptidases.
The classification used by MEROPS takes into account catalytic type and, in addition to the
above mentioned catalytic types, glutamic and asparagine peptidases are included. Peptidases
are further classified into families according to similarities in their amino acid sequences.
Families are classified into clans according to similarities of their tertiary structures that
provide evidence of their evolutionary relationships. Until recently (Release 9.5) 224
peptidase families have been described in the MEROPS data base. Those with available
primary and tertiary structure data are classified into more than 60 clans (Rawlings et al.,
2010).
15
Plants are no exception from other living organisms in possessing numerous proteases
within almost every cell compartment, as well as extracellularly. A large number of proteases
have already been isolated from plants, as seen in the MEROPS database. They belong to all
catalytic types except glutamic and asparagine; among the classified members of
corresponding families there are so far no plant peptidases. The catalytic type is still not
known for several proteases of plant origin (Rawlings et al. 2010). In addition, based on
homology with genes known to code for proteases, gene sequence studies have revealed
genes that may code for proteases, usually termed putative proteases. The number of putative
protease genes also supports the great diversity of these enzymes, even though, at this point in
evolution, they may not be transcribed. For example, the Arabidopsis genome contains over
800 of such genes, which are distributed into 60 families belonging to 30 clans and amount to
almost 3 % of the proteome. This reflects the diversity in their functions that regulate the fate
of many different proteins. Most of these proteases are likely to have overlapping functions
(Van der Hoorn, 2008).
The occurrence of proteases in all living organisms indicates their important metabolic
and regulatory functions (Rao et al., 1998; Lpez-Otn and Bond 2008) but the specific
biological functions of many plant proteases are still not known (Schaller, 2004). One of the
main problems is that their physiological substrates are not known. The great number of
different plant proteases, often with rather similar molecular characteristics, complicates the
study of their roles. Their physiological function is often supported by indirect proofs
originating from investigations of different tissues or expression patterns specific for different
stages of development. It is clear nevertheless that proteases are involved in many processes
essential for plant development and their response to stressful changes in their environment
(Vierstra, 1996). Proteases are essential for both building up and breaking down seed storage
proteins during seed germination and, importantly, for protein remobilization on organ
senescence. Proteolysis is a crucial part of many developmental processes such as
embryogenesis, chloroplast biogenesis, photomorphogenesis, hormone signalling, flower
development and pollen-pistil interaction. In addition, plant proteases are important actors in
defence against pathogens and herbivores (Simes and Faro, 2004; Salas et al., 2008;
Schaller, 2004; van der Hoorn, 2008). As will be discussed later (section 4.4), there is also
increasing evidence of their involvement in the response of plants to drought.
4.1.1. Serine Proteases

Serine proteases constitute a large class of proteolytic enzymes in plants (Rawlings et al.
2010). It is estimated that plant genomes encode for more than 200 serine proteases belonging
to different families and clans. It is interesting that genomes that are evolutionarily unrelated
and differ greatly in size, such as those of Arabidopsis thaliana and rice, contain very similar
numbers of genes for putative serine proteases: 205 in Arabidopsis and 222 in rice (Tripathi
and Sowdhamini, 2006). The majority of serine proteases have the characteristic catalytic
triad of serine - the nucleophile that attacks the peptide bond - and aspartate and histidine
residues, which are together essential for the hydrolytic process (Anto and Malcata, 2005).
Recently, several serine proteases have been isolated from different organs of various
plant species (Anto and Malcata, 2005; Rawlings et al., 2010). The largest family is S8 from
clan SB which comprises proteases homologous to the subtilisins (Rawlings et al., 2010;
Tripathi and Sowdhamini, 2006). They are often termed subtilisin-like proteases or subtilases.
They contain a seven-stranded -sheet sandwiched between two layers of helices, with the
16
Asp, His, and Ser residues (in sequence order) forming their catalytic triad in an arrangement
characteristic of subtilisins from Bacillus species (Dodson and Wlodawer, 1998). The optimal
pH for activity of the majority of serine proteases is within the neutral to alkaline region. The
gene family of putative subtilases in Arabidopsis thaliana comprises 56 members
(Rautengarten et al., 2005), 63 in Oryza sativa (Tripathi and Sowdhamini, 2006) and at least
15 in Lycopersicon esculentum (Meichtry et al., 1999). The majority of reported subtilases
can cleave numerous substrates and therefore be involved in non-selective protein
degradation (Schaller, 2004). Some, however, probably cleave highly specific peptide bonds
and thus, for example by processing protein precursors, regulate plant growth and
development (Janzik et al. 2000; Coffeen and Wolpert 2004). They could be also involved in
the response to pathogen attack (Golldack et al., 2003).
Another important group of plant serine proteases comprises members of the S1, S26 and
S14 families, which are localised in plastids (see section 5), and carboxypeptidases from
family S10. The catalytic triad of the latter is, in the primary sequence order, Ser, Asp and His
(van der Hoorn, 2008). They possess the / hydrolase fold. Carboxypeptidases from this
family are distinct from other serine proteases, in that they are active only at acidic pH. Till
now only a few serine aminopeptidases have been reported, among them those from the
family S33 which preferentially cleave off N-terminal proline. All plant serine aminopeptidases described to date belong to this family. In the Arabidopsis genome there are 53
genes and in the rice genome 22 genes that code for putative proline aminopeptidases
(Rawlings et al., 2010).
4.1.2. Cysteine Proteases

Cysteine proteases are the best characterised plant proteases, with a great number of
isolated and characterised members (Rawlings et al., 2010). The extensively studied plant
protease papain has even become a symbol for this class of proteolytic enzymes. In proteases
of this catalytic type the SH group of a cysteine residue is the nucleophile that attacks a
peptide bond.
Plant genomes encode approximately 140 putative cysteine proteases that belong to 15
families in 5 clans, each clan having a different structural fold (van der Hoorn, 2008). Clans
CA and CE contain proteases with a papain-like fold, whereas CD proteases have a caspaselike fold. Papain-like proteases from family C1 of clan CA contain catalytic residues in the
sequence order Cys, His, Asn. Their fold consists of two domains with the catalytic site
located between them. Papain-like proteases are mostly active at acidic pH. Family C1
contains the well-known plant protease aleurain, which preferably acts as aminopeptidase,
although it can also show endopeptidase activity.
Meta-caspases (family C14) and vacuolar processing enzymes (also called legumains,
family C13), both classified in clan CD, are also cysteine proteases. They are highly selective
in cleaving after specific residues: Arg for meta-caspases and Asn for vacuolar processing
enzymes (Rawlings et al., 2010). Caspase-like enzymes are folded as an // sandwich.
Phytocalpain is a cysteine protease belonging to the calpains, which are members of
family C2 from clan CA, and thus evolutionarily related to papain. They are Ca2+ activated
neutral cysteine proteases (Vierstra, 1996). Plant genomes contain only one gene for this type
of protease.
17
Cysteine proteases are involved in many different processes, particularly those associated
with degradation of storage proteins (Mntz, 2007) and programmed cell death (Lam and del
Pozo, 2000).
4.1.3. Aspartic Proteases

Aspartic proteases are optimally active at acidic pH. In contrast to serine and cysteine
proteases, the nucleophile is an activated water molecule, whose ligands are the two catalytic
aspartic acid residues. Families of this catalytic type are so far known to contain only
endopeptidases, with no exopeptidases (Rawlings et al., 2010). The best known aspartic
protease is pepsin, member of family A1. Characteristic for this family is structure that in its
mature form contains two-chain polypeptides, with -strands and very little -helix. The
molecules are bilobal, with the active site having two catalytic Asp residues, located in a large
cleft between the two domains (Simes and Faro, 2004).
Plant aspartic proteases, often termed phytepsins, are so far classified into three large
families (Rawlings et al., 2010). There are several structural and functional features that make
them unique among aspartic proteases in general (Simes and Faro, 2004). The great majority
contain an exclusive sequence of approximately 100 amino acids at the C-terminal region,
termed the plant-specific insert. The Arabidopsis genome encodes approximately 70 aspartic
proteases, which can be divided into five subfamilies (Faro and Gal, 2005). The biological
roles of these enzymes in plants are still unclear, although involvement in processes such as
programmed cell death (Simes and Faro, 2004), responses to pathogen attack (Xia et al.,
2004) and abiotic stress (Timotijevic et al., 2010) have been suggested.
4.1.4. Metalloproteases
Metalloproteases appear to have more diverse structures and functions than other
catalytic types (Schaller, 2004). As in the case of aspartic proteases, nucleophilic attack on a
peptide bond by metallopeptidases is mediated by a water molecule. But in proteases of this
catalytic type, the water molecule is activated by a metal cation, usually zinc, sometimes
cobalt, manganese, nickel or copper. The metal ion is in most cases held in place by three
amino acid ligands, often by histidine, glutamic acid, and an aspartic acid or lysine residue.
The majority of known aminopeptidases belong to this catalytic type (Rawlings et al., 2010).
Plant metalloproteases are rare and less studied. Their families contain fewer than 20
members. Plant genomes code for approximately 100 putative metalloproteases that belong to
19 families of 11 clans (van der Hoorn, 2008). One of the well studied enzymes is the
multimeric leucine aminopeptidase, a homo- or heterohexameric protein (Gu and Walling,
2000). Endopeptidases of this type have been even less well investigated. They are suggested
to be involved in meiosis, regulation of root and shoot meristem size, sensitivity to auxin
conjugates, plastid differentiation, nodulation and thermo-tolerance. The best known are those
from chloroplasts (Adam and Clarke, 2002), which are described in section 5.2.
4.1.5. Threonine Proteases
Plant threonine proteases have so far been classified into four families. Family T1
contains peptidases forming the complex oligomeric structure of the proteasome and related
compound peptidases (Rawlings et al., 2010). The nucleophile in catalysis is an N-terminal
threonine. The structure and function of the proteasome is described in section 5.1.
18
The Arabidopsis genome encodes 21 T1 family homologues, and putative proteases from
this family have been found in many plant species (Rawlings et al., 2010). Much less is
known about other families of threonine proteases in plants (T2, T3 and T5), with only 1 to 4
homologues per family present in the Arabidopsis genome (Rawlings et al., 2010).
4.2. Subcellular Localization of Plant Proteases

It is well documented that there are distinct proteolytic pathways in all compartments of
plant cells. The proteases involved are adapted to specific functions and the local
environments of the cytosol and cellular organelles.
Most of the proteolytic activity measured in crude plant extracts originates from vacuoles
the lytic compartment of the plant cell. Vacuoles contain proteases of all mechanistic
classes, optimally active at acidic pH (Mntz, 2007; Carter et al., 2004). Bulk non-specific
cellular protein degradation takes place in lytic vacuoles, whose main function is turnover of
macromolecules and defence against pathogens and herbivores, and in storage vacuoles,
which serve for deposition and subsequent, temporarily regulated degradation of reserve
proteins (Otegui et al., 2005, Ishida et al., 2008). The amino acids released are reused in other
plant parts during certain developmental stages such as germination and senescence, and
under different environmental stress conditions that induce nutrient starvation, oxidative
stress and premature senescence (Feller, 2004; Mntz, 2007; Bassham et al., 2006; Liu et al.,
2009). Cysteine proteases, such as legumains and papain-like proteases, and aspartic
proteases are most common, but other catalytic types are also present. The acidic environment
in the vacuoles may facilitate unfolding of the target proteins. Autophagic uptake of proteins
into the vacuole for bulk degradation is well documented (Otegui et al., 2005; Ishida et al.,
2008). Autophagy is a nonspecific protein degradation pathway induced under multiple
environmental stress conditions and at certain stages of development in plants, such as
nutrient starvation, oxidative stress, senescence and osmotic stress (Liu et al., 2009).
Vacuoles are also the storage place of an array of proteases and protease inhibitors which
target the proteins and/or proteases of herbivores and phytopathogens (Mntz, 2007). Woundinducible proteinase inhibitors reside in vacuoles; however, for other inhibitors different
locations has been reported cell wall, cytosol, nuclei (Chye et al., 2006). Proteases acting in
the vacuoles or extracellular space are generally energy independent; however, vacuolar
degradation may also require energy for trafficking substrates to this lytic compartment
(Vierstra, 1996).
The cytosol is dominated by components related to protein biosynthesis and the
degradation machinery (Ito et al., 2011). In the cytosol and nucleus mainly selective
proteolysis occurs that eliminates misfolded, damaged and/or regulatory proteins. The main
proteolytic system is the ubiquitin/26S proteasome pathway, involving threonine proteases
(see section 5.1). Cleavage of a restricted set of numerous membrane-bound or membraneassociated proteins, carried out by the cysteine protease phytocalpain, also occurs in the
cytosol (Croall and Ersfeld, 2007).
Mitochondria and chloroplasts possess their own conserved proteolytic systems very
similar to those of the prokaryotes, while non-functional proteins of the endoplasmic
reticulum are directed by chaperones for degradation in the cytosol (Leidhold and Voos,
2007). Each of the major chloroplast compartments contains defined proteases, some
19
involved in non-selective degradation, several of them probably functioning as highly specific

processing peptidases (Adam and Clarke, 2002). The ATP-dependent Clp (serine-type)
proteases occur in stroma, and the ATP-dependent FtsH proteases (metallo-type) in stromaexposed thylakoid membranes. They are described in more detail in section 5.2. DegP
proteases (serine-type), involved in the ATP-independent proteolytic pathway, are found
within the thylakoid lumen and on both sides of thylakoid membranes, and the SppA protease
(serine-type) on the stromal side of the thylakoid (Adam and Clarke, 2002).
Proteases are also present in peroxisomes (Palma et al., 2002). About 70% of the total
proteolytic activity in these organelles can be assigned to serine endopeptidases. It is
suggested that, together with cysteine and metalloproteases, they participate not only in the
turnover of peroxisomal proteins but also in the turnover of proteins from other cell
compartments in advanced stages of senescence (Distefano et al., 1997).
The apoplast is the site of the first line of proteolytic defence against pathogens.
Extracellular proteases then catalyse the hydrolysis of proteins into smaller peptides and
amino acids for subsequent absorption into the cell and constitute a very important step in
nitrogen metabolism (Vierstra, 1996; Lpez-Otn and Bond 2008).
4.3. Regulation of Proteolysis, with Emphasis on Protease Inhibitors

Because the roles of proteases in plant physiology are essential and because peptide bond
hydrolysis is irreversible, the activity of proteases has to be tightly regulated. This is achieved
through regulation at several levels, including regulation of gene expression at transcriptional
and post-transcriptional levels, synthesis as inactive zymogens (pre-pro-polypeptides),
blockade by endogenous inhibitory proteins, spatial and temporal compartmentalization, posttranslational modification (glycosylation, phosphorylation, co-factor binding), limited
proteolysis and degradation (Lopez-Otin and Bond, 2008).
In addition to various mechanisms of regulation at the protease level, structural changes
in protease substrates may also contribute to their overall regulation. The susceptibility of
proteins to proteolytic attack can be changed if their environment changes in a way that
affects their overall structure or oligomerization. Changes of pH or solute concentration
(cations, substrates, co-substrates in the case of enzymes attacked by proteases) in cell
compartments are often a consequence of water deficit. In addition, modifications linked to
protein stability, such as oxidation of amino acid residues (also often the consequence of
drought), phosphorylation and acetylation may render protease substrates more susceptible to
proteolysis (Callis, 1995). Ubiquitination is another protein modification affecting the
susceptibility of substrate proteins to protease attack and is addressed in section 5.
Protease inhibitors constitute a very important mechanism of regulating proteolytic
activity. They can be classified, according to their reaction mechanism (competitive, noncompetitive, uncompetitive, suicide protease inhibitors) or according to their specificity, into
those that inhibit different classes of proteases, one class of proteases, one family of proteases
or a single protease. There are two general mechanisms of protease inhibition, namely
irreversible trapping reactions and reversible tight-binding reactions. The latter is
widespread and the best known is the standard or Laskowski mechanism, where the
inhibitor has a reactive site peptide bond that interacts with the peptidase active site in a
substrate-like manner. Other reversible tight-binding protease inhibitors physically block the
20
protease active site by high-affinity binding to sites on either side of the active site. There are
also some inhibitors that block the exosites, to which substrate binds as well as to the active
site in some proteases. The irreversible trapping reactions work only on endopeptidases and
are the result of a conformational change of the inhibitor triggered by cleavage of an internal
peptide bond. Only two families of protease inhibitors found in plants utilize a trapping
mechanism: I4 (serpins) and I39 (2-macroglobulin)(Christeller, 2005; Rawlings, 2010,
Rawlings et al., 2004).
Protease inhibitors are often roughly classified according to the class of protease they
inhibit (for example: cysteine or serine protease inhibitors) (Salvesen and Nagase, 1989;
Rawlings et al., 2004). However, protease inhibitors composed of multiple inhibitor units and
pan-inhibitors (such as 2-macroglobulin of family I39) that target proteases of different
catalytic classes preclude unambiguous classification. A more detailed classification is
included in the MEROPS database (http://merops.sanger.ac.uk/), (Rawlings, 2010). It follows
a hierarchy similar to that for the classification of proteases. Protein protease inhibitors are
grouped into families based on sequence homology and into clans based on protein tertiary
structure. As of 4th July 2011, the MEROPS database (release 9.5) lists 71 families of
protease inhibitors, and those with available three-dimensional structural data have been
assigned to 38 different clans. Of the 71 families, 21 that have been assigned into 17 clans
include members of plant origin. Of these, 10 families include protease inhibitors exclusively
of plant origin (I3, I6, I7, I12, I18, I20, I37, I67, I73 and I90). Families of serine protease
inhibitors predominate, followed by a few families of inhibitors of cysteine and metalloproteases, while aspartic protease inhibitors are rare and dispersed in different families. The
two largest families described are of serine protease inhibitors in I3 (Kunitz-P family) and I12
(Bowman-Birk family), while phytocystatins (family I25) and propeptide-like protease
inhibitors (family I29) are the largest families of cysteine proteases.
The soybean trypsin inhibitor (STI) was the first isolated plant protease inhibitor (Kunitz,
1945), and similar proteins subsequently characterized were named Kunitz trypsin inhibitors
(KTI). They are widespread in plants and encoded by families of genes that are expressed in
all plant tissues, but mostly in the seeds of leguminous plants of taxonomic subfamilies
Mimosoideae, Caesalpinioideae and Papilionoideae. KTIs inhibit mostly serine proteases
belonging to family S1, but some members also inhibit aspartic proteases of family A1 or
cysteine proteases of family C1 (Oliva et al., 2010; Rawlings, 2010; Ma et al., 2011).
Protease inhibitors belonging to the Bowman-Birk family (BBI) are named after the two
scientists who isolated (Bowman, 1946) and characterized (Birk et al., 1963) the first
member from soya beans. They have been found in leguminous plants (Fabaceae) and
grasses (Poaceae), where they are expressed in all plant tissues and are encoded by small
families of genes. BBIs are compound inhibitors, containing one to six inhibitor units, which
often have different specificities, but all target serine proteases of clan PA (mainly families
S1 and S3). The soybean Bowman-Birk protease inhibitor is, for example, a double-headed
inhibitor of trypsin and chymotrypsin (Qi et al., 2005; Rawlings, 2010).
The largest family of cysteine protease inhibitors in plants is the cystatin family, also
called phytocystatins. The first one isolated was oryzacystatin from rice (Abe et al., 1987).
They are widespread in the plant kingdom and are expressed in all plant tissues.
Phytocystatins inhibit papain-like cysteine proteases of family C1. They can be compound
inhibitors comprised of up to 8 inhibitor units, which are then called multicystatins
(Rawlings, 2010; Benchabane et al., 2010).
21
The second largest family of cysteine protease inhibitors in plants comprises proteins that
are homologous to the proregions of papain-like cysteine proteinases. They are widespread
throughout the plant kingdom and inhibit papain-like proteases (family C1) with higher
selectivity for individual proteases than do cystatins (Rawlings, 2010; Wiederanders, 2003;
Yamamoto et al., 2002).
In general, protease inhibitors can be divided into those directed against endogenous
proteases and those directed against exogenous proteases. They control endogenous plant
proteases performing essential physiological regulatory functions at the cellular level,
affecting cell growth and differentiation, cell cycle, response to different stress conditions,
misfolded protein response, and programmed cell death (Lopez-Otin and Bond, 2008; Rao et
al., 1998). Plant protease inhibitors also control proteases involved in metabolic processes,
including build-up and breakdown of seed storage proteins, protein remobilization upon organ
senescence, as well as many developmental processes such as embryogenesis, chloroplast
biogenesis, photomorphogenesis, hormone signalling and flower development. (Simoes and
Faro, 2004; Salas et al., 2008; Schaller, 2004; van der Hoorn, 2008). The great abundance of
protease inhibitors found in storage organs (seeds and tubers) suggests that they serve a triple
function, as defence proteins, as storage proteins, and as regulators of endogenous proteases
during seed dormancy. During germination, proteolytic activity increases and the content of
protease inhibitors declines, along with other storage proteins. Plant protease inhibitors have,
however, been studied more extensively as an important part of the plant defence system
against pests, parasites and pathogens. Their defensive role is based either on inhibition of
digestive proteases, as in herbivorous arthropods (insects and mites), causing critical shortage
of essential amino acids important for growth and development, or on inhibition of
pathogenesis related proteases or virulence factors of plant pathogens and parasites.
Expression of many protease inhibitors is induced under various biotic and abiotic stress
conditions, including wounding, insect herbivory, infection by phytopathogenic nematodes,
fungi, bacteria or viruses, anaerobiosis, low or high temperature, insufficient illumination,
high salinity, drought, etc. (Habib and Fazili, 2007; Haq et al., 2004; Fan and Wu, 2005;
Mosolov and Valueva, 2005; Jongsma and Beekwilder, 2008; Brzin and Kidri, 1995).
4.4. Proteases and Protease Inhibitors Involved in the Response to Drought

4.4.1. Proteases
Abiotic stresses such as drought affect various classes of proteases. The involvement of
proteolytic enzymes in response to drought has been shown by induction of genes coding for
putative proteases and by detection of changes of levels of proteolytic activities of different
specificities (Brzin and Kidri, 1995; Ingram and Bartels, 1996; Bartels and Sunkar, 2005).
However, our knowledge is still fragmentary, especially having in mind the great number and
diversity of plant proteases that could be involved in response to water deficit. In addition, as
already noted, changes in gene expression do not necessarily lead to changes in actual levels
and/or activities of proteases and, conversely, the latter may change without change in gene
expression. It has yet to be proved that expression of a gene supposed, on the basis of
homology only, to code for a protease actually leads to synthesis of the corresponding protein.
Studies on the influence of drought on protease activities have been carried out, mainly
on wheat (i.e. Zagdanska and Wisniewski, 1996; Srivalli and Khanna-Chopra, 1998; Simova-
22
Stoilova et al., 2009) and legume plants (Roy-Macauley et al., 1992; Cruz de Carvalho et al.,
2001; Hieng et al., 2004), while increased expression of genes for putative proteases was
shown also in Arabidopsis (Seki et al. 2002; Bartels and Sunkar, 2005). Cysteine
endopeptidases were the first, and still the most frequently reported proteases to be influenced
by drought, usually induced (Ingram and Bartels, 1996; Simova-Stoilova et al., 2010). A few
reports concern aspartic proteases (Cruz de Carvalho et al., 2001; Timotijevic et al., 2010),
serine endopeptidases (Haushl et al. 2001; Hieng et al. 2004; Pinheiro et al., 2005; Dram et
al., 2007) and aminopeptidases (Wun et al. 1999; Hieng et al., 2004).
Most proteases shown to be influenced by drought are probably vacuolar enzymes,
judged on the basis of the optimal pH for their activities or of gene homology. Under drought
stress the proteolytic potential of Phaseolus and Vigna bean leaf cells increases, particularly
in the vacuolar sap (Roy-Macauley et al., 1992). Increase in acidic proteolytic activity in
leaves of plants subjected to prolonged drought stress is well documented in plants at the
vegetative growth stage (Zagdanska and Wisniewski, 1996; Cruz de Carvalho et al., 2001;
Simova-Stoilova et al., 2010) as well as at the reproductive stage when accelerated
senescence is observed (Srivalli and Khanna-Chopra, 1998; Simova-Stoilova et al., 2009).
Some difference is observed between the composition of drought-induced proteases and of
those up-regulated in natural senescence (Khanna-Chopra et al., 1999). The induced proteases
are predominantly cysteine (Koizumi et al., 1993; Martinez et al., 2008; Esteban-Garca et al.,
2010), aspartic (Contour-Ansel et al., 2010; Timotijevic et al., 2010) or serine (Hieng et al.,
2004; Drame et al., 2007) types, depending on the species studied.
Some of the affected proteases may be active in other cell compartments. Increased
proteolytic activities were observed in purified chloroplast fractions from Phaseolus and
Vigna leaf cells (Roy-Macauley et al., 1992). Maximal activity at alkaline pH indicated an
extravacuolar site of action of one serine endopeptidase whose activity increased in certain
cultivars of common bean under drought (Hieng et al., 2004). However, the subcellular
localization of these proteolytic activities remains to be determined. It was also found that
drought affected gene expression of the chloroplast homologue of the prokaryotic trypsin in
Arabidopsis thaliana (Haushl et al. 2001). Furthermore, proteome analysis showed an effect
of drought on the putative subtilisin-like serine endopeptidase from the stem of white lupin
Lupinus albus (Pinheiro et al., 2005). It appears that up-regulation of the proteolytic response
to drought occurs at both transcript and post-transcriptional levels (Contour-Ansel et al.,
2010).
The above discussed proteases are endopeptidases. But increase in aminopeptidase
activities in response to water deficit has also been observed, as in the cases of leucine
aminopeptidase, a metalloprotease from tomato (Wun et. al., 1999), aminopeptidases of
metallo- and serine catalytic type from common beans (Hieng et al., 2004) and aminopeptidase activities from wheat (Miazek and Zagdanska, 2008).
However, despite the generally observed increase of proteolytic activity accompanied by
diminution in protein content, in several plant species described above, there is increasing
evidence that tolerant species or cultivars show little increase in proteolytic enzymes under
drought, at either protein or transcript level (Cruz de Carvalho et al., 2001; Hieng et al., 2004;
Drame et al., 2007; Simova-Stoilova et al., 2010).
The influence of drought stress on ATP-dependent proteolytic pathways will be discussed
in section 5.4. Here it is relevant to record that some studies are in favour of cross-talk
between ATP-dependent and ATP-independent protein degradation and of compensation of
23
the lower activity of vacuolar proteases by increased ATP-dependent activity, especially in

acclimation to dehydration stresses and drought tolerance (Wisniewski and Zagdanska, 2001;
Grudkowska and Zagdanska, 2010).
None of the proteases shown to be influenced by drought have been characterised in more
detail and we are far from having a complete picture. It can be assumed that, as in the case of
common bean (Hieng et al., 2004), the response to drought involves different types of
endopeptidase and aminopeptidase having complex and probably specific functions. Although
the specific roles of various proteases are still not understood, it is clear that all changes in
metabolism under drought need the active involvement of regulated proteolysis. It may have
several functions: i) rearrangement of metabolism through selective degradation of key
enzymes and/or degradation of short-lived proteins involved in cell signalling; ii) removal of
oxidatively damaged, improperly folded or irreversibly denatured proteins; iii) recycling of
carbon-starvation-related amino acids and hastening of senescence under source-sink
regulation; iv) protection against potential biotic stress (Vierstra, 1996; Feller, 2004).
Regulated proteolysis is therefore vital for cell survival under dehydration stress. On the
other hand, the so-called uncontrolled proteolysis, resulting mainly from disruption of cellular
membranes provoked by water deficit, is damaging for cells since it leads to random
breakdown of the majority of cellular proteins and to premature senescence of the plant. It is
important to note that, under unfavourable environmental conditions, senescence may be
induced before the appropriate stage in development in order to accelerate flower and seed
formation. However, in the case of crops this often leads to decreased yield. Regulation of
proteolysis thus appears, first, to be an important.
4.4.2. Protease Inhibitors

There is increasing evidence that plant protease inhibitors are multifunctional proteins
involved not only in plant protection against pathogens and herbivores but implicated also in
the control of endogenous proteolysis under abiotic stress conditions (Brzin and Kidric, 1995;
Martinez and Diaz, 2008; Benchabane et al., 2010). In contrast to the numerous publications
on plant protease inhibitors involved in defence against insect pests over the past three
decades (Ryan, 1990, Habib and Fazili, 2007, Lawrence and K.R., 2002, Ferry and
Gatehouse, 2010), research on their involvement in abiotic stress responses has emerged only
in the past ten years. Transcriptome and proteome analyses under conditions of drought have
revealed the involvement of protease inhibitors in stress response in Arabidopsis thaliana
(Seki et al., 2002), legume crops Lupinus albus (Pinheiro et al., 2005) and horsegram
Macrotyloma uniflorum (Reddy et al., 2008), peanut (Luo et al., 2005), and oilseed rape
Brassica napus (Desclos et al., 2008).
Cystatin gene expression is among the early top up-regulated genes in a comparative
drought and salt stress profiling in grapevine, and cystatin transcripts accumulated further
with stress prolongation (Cramer et al., 2007). Levels of plant cystatins are highly responsive
to various abiotic stresses (Valdes-Rodrguez et al., 2007; Zhang et al., 2008). Induction of
cystatin expression in chestnut by cold and salt stress has been reported, as well as by
wounding, fungal infection and exogenous jasmonic acid treatment. The induced cystatin
could inhibit the endogenous cysteine type proteinase activity (Pernas et al., 2000). Drought
stress and exogenous ABA application resulted in accumulation of two multicystatin
transcripts in two cowpea cultivars differing in tolerance to drought (an earlier response in the
tolerant one), followed by rapid decrease in transcript levels following rewatering. At the
24
protein level, 25 and 39 kDa cystatin bands were enhanced on intensification of stress (Diop
et al., 2004). A multicystatin in winter wheat (Triticum aestivum L. cv. Chihoku), whose
expression is induced during cold acclimation, has also been induced by drought, mostly in
roots (Christova et al., 2006). Expression of two cystatin genes (AtCYSa and AtCYSb) was
strongly induced in Arabidopsis thaliana by multiple abiotic stresses, including high salt and
drought. In addition, overexpression of these genes in transgenic Arabidopsis plants increased
their resistance to high salt, drought, oxidative and cold stresses (Zhang et al., 2008). It
appears that adaptation to drought involves control of protein degradation through the pool of
endogenous proteinase inhibitors. Massonneau et al. (2005) observed down-regulation of
some cystatin genes in response to severe water deficit in maize, and linked the observed data
with higher activities of cysteine proteases under severe drought stress. It has been shown
that different cystatin genes in A. thaliana show different patterns of expression during
development and in its response to abiotic stresses, indicating that individual cystatins may
have distinct functions in response to abiotic stresses (Hwang et al., 2010).
The involvement of serine protease inhibitors in the response to drought is also
supported by experiment (Downing et al., 1992; Gosti et al., 1995; Kang et al., 2002; Huang
et al., 2007). Expression of a Kunitz-type serine protease inhibitor (BnD22) was induced in
young leaves of oilseed rape (Brassica napus) in response to drought. Since the senescence of
young leaves occurred later than in old and mature leaves, it is hypothesized that BnD22
protects the younger leaves by inhibiting proteases and maintaining metabolic activity and
growth. In addition, BnD22 moonlights as a water-soluble chlorophyll binding protein
(WSCP), which may contribute to the photoprotection mechanism and the delay of
senescence of young leaves under adverse conditions (Desclos et al., 2008; Downing et al.,
1992; Ilami et al., 1997). Similarly, expression of the trypsin specific protease inhibitor
SPLTI from sweet potato (Ipomoea batatas), belonging to family I13 of MEROPS
classification (potato inhibitor 1 family), is regulated differently in young and old leaves.
Constitutive expression of this inhibitor in unexpanded young leaves may allow them to
respond rapidly to water deficiency and, in expanded mature leaves, where expression is
induced upon decrease in relative water content, it may play an early role in the response to
water deficiency in sweet potato leaves (Wang et al., 2003). Three different trypsin inhibitors
are expressed in leaves of amaranth (Amaranthus hypochondriacus), as well as their
accumulation in seeds. One trypsin inhibitor (29 kDa) is constitutively expressed and
probably represents a constitutive defence mechanism against various biotic and abiotic
stresses. Expression of the two smaller trypsin inhibitors (2 and 8 kDa) is induced by water
and salt stresses (Sanchez-Hernandez et al., 2004). The expression of OCPI1, a chymotrypsin
inhibitor from rice (Oryza sativa), was strongly increased in rice plants subjected to
dehydration and treatment with ABA. Furthermore, over-expression of OCPI1 in rice
increased the inhibition of endogenous chymotrypsin activity, and transgenic plants showed
less protein degradation under severe drought conditions than non-transformed plants, which
resulted in significantly improved drought resistance in terms of yield loss in the field (Huang
et al., 2007). In a wheat cultivar with improved salt and drought tolerance, a Bowman-Birk
inhibitor of trypsin was one of the main differentially expressed proteins and it showed
increased levels of expression in roots exposed to salt, drought or oxidative stress (Shan et al.,
2008). Serine protease inhibitor was induced in drought-tolerant sugarcane cultivars together
with up-regulation of ubiquitin genes (Jangpromma et al., 2010).
25
The variety of physiological functions of proteolytic enzymes under stress conditions

offers multiple functions for proteins that inhibit them. Plant protease inhibitors may confer
any of the following potential physiological functions during conditions of drought, some
even simultaneously, including i) inhibition of proteases activated on water deficit, ii)
osmoprotection, resulting from their highly hydrophilic nature, and iii) defence against biotic
stress caused by viral, bacterial or fungal pathogens, nematodes or herbivorous arthropods
during the period of reduced growth under drought conditions. The drought responsiveness of
proteases and protease inhibitors makes them potential biochemical markers for assessing
drought tolerance.
5. ATP-DEPENDENT PROTEASE COMPLEXES

ATP-dependent protease complexes are large, oligomeric complexes composed of
chaperone and proteolytic subunits (Clp and proteasome families). In some cases the
chaperone and proteolytic parts are located in the same polypeptide chain (Lon and FtsH
families). These proteases are built on the principle of self-compartmentalization the
proteolytic active sites are buried inside a narrow channel in the oligomeric structure; only
substrates unfolded and translocated inside by the chaperone part are accessible to
degradation, thus the surrounding native proteins are protected from breakdown (Schmidt et
al., 1999). This structural organization, in which the protease active sites are hidden into the
interior chamber, allows only processive degradation of unfolded polypeptide chains - gradual
degradation to small peptides (5-10 amino acids) and to amino acids, coupled to ATPdependent, chaperone-assisted unfolding and translocation of the unfolded polypeptide.
Energy-dependent proteases, in cooperation with the chaperone system, act as protein
quality control systems in maintaining the functional state of cell proteins in essential
compartments such as cytosol, endoplasmic reticulum, mitochondria and chloroplasts
(Leidhold and Voos 2007). As peptide bond hydrolysis is an exergonic reaction, ATP
hydrolysis is most probably linked to specific recognition, regulation and control over
proteolysis. Besides ATP coupling, these proteases are also able to degrade poorly structured
or oxidatively modified proteins in an ATP-independent manner (Kurepa et al., 2009).
5.1. The Proteasome

The main proteolytic degradation system of eukaryotes, operating in the cytoplasm and
nucleus, is the ubiquitin/26S proteasome pathway. The proteasome plays a crucial role in the
turnover of regulatory proteins, cellular house-keeping and stress tolerance (Kurepa and
Smalle, 2008). The structure and activities of proteasomes are highly conserved among
eukaryotes, suggesting essential functions in protein homeostasis. This system is an extremely
large and complex route for protein degradation, accounting for nearly 6% of the Arabidopsis
thaliana transcriptome (Vierstra, 2009).
Proteins targeted for degradation are covalently tagged with the highly conserved 76
residue peptide ubiquitin (Ub) by a cascade of three types of enzymes E1 (ubiquitinactivating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) that
26
catalyse the conjugation of Ub monomer to a lysine residue of the target protein. E3 controls
the specificity of Ub conjugation. In the Arabidopsis genome, 2 E1s, at least 37 E2s and over
1,400 different E3s are expressed (Vierstra, 2009). Ub conjugation is a reversible reaction detachment of Ub is catalysed by Ub-specific proteases that form a group of multiple
enzymes, controlling ubiquitination and recycling Ub for reuse. While mono-ubiquitination of
proteins facilitates interaction with ubiquitin binding domains in specific target proteins and
has a regulatory function, polyubiquitinated proteins (at least four Ubs linked to target
proteins by the residue lysine 48 of Ub) are recognized by specific receptors within the 26S
proteasome or by adaptor proteins associated with the proteasome and are degraded in an
ATP-dependent manner (Miura and Hasegava, 2010).
The 26S proteasome controls proteolysis of key components of numerous signalling
pathways, regulated proteolysis of functional proteins and removal of misfolded and damaged
proteins (Smalle and Vierstra, 2004; Vierstra, 2009). Besides ubiquitin, there is a set of other
ubiquitin-like proteins involved in posttranslational modification of proteins such as
RUB1/Nedd8, SUMO, HUB, ISG15, and ATG (Catala et al., 2007). SUMO (small ubiquitinrelated modifiers) tagging of proteins proceeds in a pathway similar to that for ubiquitination,
with sequential action of analogous E1, E2 and E3 enzymes. SUMO-specific proteases cleave
off SUMO for re-use (Novatchkova et al., 2004). SUMOylation regulates protein degradation
and localization, proteinprotein interactions, transcriptional activity and counteracts the Ubmediated degradation by the proteasome (Novatchkova et al., 2004).
The 26S proteasome is a 32-subunit, multicatalytic protease that degrades polyubiquitinated protein targets (Smalle and Vierstra 2004; Vierstra, 2009). It is composed of
two subcomplexes - the barrel-shaped, proteolytically active core 20S proteasome (about 700
kDa in size) and the 19S regulatory particle (700 kDa) that recognizes, unfolds and channels
Ub-targeted proteins into the 20S proteasome for degradation (Kurepa and Smalle, 2008). In
the absence of ATP the 26S proteasome dissociates into 20S and 19S subcomplexes (Vierstra,
1996). The proteolytic core belongs to the threonine-class of proteases and possesses trypsinlike, chymotrypsin-like and peptidyl glutamyl-peptide-hydrolase activities. The core particle
is built of four rings: two inner rings of seven beta-subunits each, three of them proteolytically active (the proteolytic chamber), and two outer rings of seven alpha-subunits (the
entrance to the proteolytic chamber) which interact with the regulatory particle. The
regulatory particle contains two subcomplexes, the lid and the base. The base contains six
different AAA ATPases that form a ring-like structure and uses ATP hydrolysis to unfold
target proteins, together with non-ATPase subunits that function as docking sites for proteins.
The lid, an 8-subunit, non-ATPase assembly linked to the core particle by the base, is
required for ubiquitin-conjugate degradation and is closely related to the signalosome
(Schmidt et al., 1999). The structural composition of the proteasome is heterogeneous and
some subunits are subjected to phosphorylation, acetylation and other post-translational
modifications. The main functions of Ub-dependent proteolysis are degradation of misfolded
or damaged proteins and of inherently unstable proteins that carry specific degradation
signals, quality control by removal of proteins with translational errors, and inactivation of
regulatory proteins. Phosphorylation and dephosphorylation in the signal transduction
cascades often alter protein stability and affect the affinity of E3 for the respective target
proteins. Further, the proteasome exerts control on metabolic fluxes by degrading key
enzymes whose stability may depend on changes in metabolite concentrations (Kurepa and
Smalle, 2008). Ub-independent proteolysis is involved in degradation of oxidized proteins -
27
thus 20S may play an important role in tolerance to the secondary oxidative stress
accompanying many primary abiotic stresses. In addition, plant proteasomes have RNAse
activity, which is important in protection against pathogenic viruses.
5.2. Rotease Complexes in Chloroplasts and Mitochondria

Chloroplasts and mitochondria contain their own proteolytic systems, which are less
complex than the ubiquitin/26S proteasome pathway. They are homologous to the bacterial
proteases and have evolved in plants from single genes into multigene families (Adam, 2007;
Janska et al., 2010). In Arabidopsis chloroplasts, at least 11 different types of protease
families, encoded by more than 50 genes, have been found, amounting to about 2.3% of the
chloroplast proteins (Sakamoto, 2006). The Clp complex is an abundant proteolytic system in
chloroplast stroma. Its proteolytic subunits are encoded in Arabidopsis by 6 different genes,
one of the products being targeted to mitochondria and 5 to chloroplasts. One of these genes
is in the plastome and the others in the nuclear genome. Additional genes code for 4 noncatalytic ClpP-related ClpR subunits. The chaperone part of Clp in Arabidopsis is coded by
three genes for chloroplasts (ClpC1, ClpC2, ClpD) and three for mitochondria (ClpX). There
are an additional two copies of ClpS (unique for land plants) and one ClpT copy (Adam,
2007).
The structure of the Clp protease complex resembles that of the 26S proteasome. It
consists of two functional elements: one, a barrel-shaped, hetero-oligomeric proteolytic core
complex of 325-350 kDa, composed of two stacked heptameric rings of ClpP, ClpR and ClpS
subunits with narrow (about 10 ) axial openings, forming a chamber which hides inside
serine-type proteolytic active sites (catalytic triad Ser-His-Asp), and the other, two hexameric
rings at the openings of the proteolytic core, composed of chaperones of the AAA+
superfamily of ATPases (Clp/Hsp100). The mitochondrial Clp core complex is a 320 kDa
homotetradecamer of ClpP2 associated with ClpX chaperones (Peltier et al., 2004). ClpC and
ClpX chaperones appear to be constitutively expressed, while ClpD is responsive to drought
stress and senescence (Sakamoto, 2006).
Plant organelles also have FtsH and Lon ATP-dependent proteases in which the
proteolytic active site and chaperone domain are parts of the same polypeptide chain. The
FtsH gene family in Arabidopsis contains 12 members, 4 of whose products are targeted to
mitochondria and 9 to chloroplasts. FtsH proteases have a 400-450 kDa hexameric ring-like
structure formed by identical or closely related subunits. These provide an internal channel
harbouring the proteolytic sites, access to which is controlled by the chaperone part. Each 74
kDa FtsH monomer consists of two trans-membrane helices at the N-terminus which anchor
the protein to membranes. The stroma-facing AAA+ATPase domain and the proteolytic
domain possess a conserved zinc-binding motif (His-Glu-X-X-His) at the C-terminus, typical
of metalloproteases. FtsH complexes are heteromeric and its subunits are partially
functionally redundant (Sakamoto, 2006).
Arabidopsis possesses four genes for Lon protease which are targeted to mitochondria,
chloroplasts and peroxisomes (Adam, 2007; Janska et al., 2010). Lon is a hexameric ATPdependent serine protease with a Ser-Lys dyad in the active site. Chaperone and catalytic
domains are located in the same polypeptide.
28
The interesting hexameric DegP serine protease, with 16 paralogs, is coded for in the
Arabidopsis genome. It is not involved in the ATP-dependent proteolytic pathway but
switches between chaperoning and proteolytic activity under the influence of high
temperature (Adam, 2007). The DegP monomers of 48 kDa are composed of a proteolytic
domain at the N-terminus with the catalytic triad Ser-His-Asp, and two tandem PDZ domains
at the C-terminus which regulate protein-protein interactions and recognition of the substrate
proteins. In the chaperone form the active site of the protease is blocked by an auto-inhibitory
segment. The temperature-induced conformational change pulls apart this segment, making
the active centre accessible to substrates (Adam, 2007).
The main functions ascribed to the processive ATP-dependent proteases in organelles are
i) to degrade excess subunits of proteins, ii) to eliminate photo-oxidatively damaged proteins,
and iii) to exert protein quality control (Sakamoto, 2006). The involvement of FtsH and DegP
in the repair cycle of PSII in chloroplasts under high light, by degradation of D1 protein, is
well documented (Kato and Sakamoto, 2009). Mitochondrial Lon and FtsH proteases are
involved in the biogenesis and maintenance of the oxidative phosphorylation system (Janska
et al., 2010).
5.3. Relation of Expression of ATP-Dependent Protease

Complexes to Function
The proteasome is essential for proper cellular function, removing proteins containing
translation errors, improper processing and irreparable damage. It participates in protein
quality control for both cytosolic and endoplasmic located proteins via the ER-associated
degradation pathway, involving the assistance of ER chaperones and retrograde transport
back to the cytoplasm (Kurepa and Smalle, 2008). Up to 30% of the translation products are
rapidly removed by the proteasome under non-stress conditions (Smalle and Vierstra, 2004).
Various pathway mutants are lethal or hypersensitive to stresses that damage or denature cell
proteins (Smalle and Vierstra, 2004). In addition, the Ub-proteasome pathway controls
hormonal signalling, including stress signal transduction, as well as cell metabolism, by
removing short-lived regulatory proteins and enzymes that direct rate-limiting steps of
metabolite pathways (Vierstra, 2009). These indispensable housekeeping functions and the
great complexity of the proteasome favour its primarily constitutive expression and a role in
acclimation to stress rather than of rapid stress induction.
Plant cells contain 26S and 20S proteasomes which mediate Ub-dependent and Ubindependent proteolysis, respectively. It is established that increased 26S biogenesis supports
increased growth and increased tolerance to misfolded protein stresses (heat-shock stress for
example). In contrast, increase in the relative abundance of 20S leads to decreased growth and
better performance in oxidative stress conditions, since 20S degrades oxidatively modified
proteins in an ATP-independent manner (Kurepa et al., 2009).
Chloroplast Clp protease is principally a constitutively expressed enzyme that degrades
numerous stromal proteins. Among its putative substrates are enzymes involved in metabolic
pathways such as photosynthetic carbon fixation, nitrogen metabolism and chlorophyll/haem
biosynthesis. Other putative substrates of Clp have been described that are involved in
housekeeping roles such as RNA maturation, protein synthesis and maturation, and recycling
processes (Stanne et al., 2009). Constitutive expression of ClpP proteins, without significant
29
changes, has been reported under various stress conditions. It appears that plastid and
mitochondrial proteolysis is regulated, not through regulation of ClpP gene expression, but
rather through interaction of the core complex with ClpS1,2 and other chaperone-like
molecules, as well as through substrate recognition mechanisms (Peltier et al., 2004). FtsH
transcript levels in chloroplasts are highly responsive to strong light stress while temperature
stresses have no effect on FtsH transcript abundance (Adam, 2007). Expression of
mitochondrial ATP-dependent proteases is rather constant during their development, with
some changes found in flowers and seeds (Janska et al., 2010). Expression of the Deg
protease, which is not dependent on ATP, is increased under salt, light and temperature
stresses (Sakamoto, 2006).
5.4. Involvement of ATP-Dependent Protease Complexes

in the Response to Drought
Water deficit resulted in decreased total ATP-dependent proteolytic activity in wheat
genotypes (Wisniewski and Zagdanska, 2001), while acclimation to drought prevented this
decline. ATP-dependent protein degradation under drought was greater in the leaves of
acclimated than of non-acclimated stressed plants (Zagdanska and Wisniewski, 1998;
Wisniewski and Zagdanska, 2001). This activity is associated mainly with the proteasome in
the cytosol and nucleus and the ATP-dependent proteases in chloroplasts and mitochondria.
ATP consumption for energy-dependent proteolysis amounts to about half that required
for protein synthesis, and increases substantially under water deficit conditions (Zagdanska,
1995).
Regulation of proteasome activity includes adjustment of both total cellular proteolytic
potential and target specificity. Changes in total proteasome activity can be caused by altered
proteasome abundance or by posttranslational modification of subunits without affecting
proteasome content. Changes in target specificity can be brought about by subunit
modification and by the incorporation of subunit variants (Kurepa and Smalle, 2008).
Plomion et al. (2006) reported induction by drought of members of the protein degradation
machinery (i.e. 20S proteasome, polyubiquitin) in poplar trees, at the transcript level and the
proteome level (26S protease regulatory subunit). Up-regulation of the 20S proteasome
subunit was found in a proteomic study of drought-treated alfalfa plants (Aranjuelo et al.,
2011). Wan et al. (2011) described up-regulation of a ubiquitin-conjugating enzyme gene
AhUBC2 in dehydrated peanut plants; constitutive expression of this gene in Arabidopsis
resulted in improved water-stress tolerance. The AtAIRP1 gene, encoding an E3 Ub ligase,
was rapidly and significantly induced by ABA and by abiotic stresses including drought, low
temperature, and high salinity. AtAIRP1-overexpressing transgenic plants were highly
resistant to severe water stress (Ryu et al., 2010). It appears that the sumoylation system is
highly responsive to environmental cues and plays a regulatory role in plant stress responses.
Low and high temperatures, drought, salt, and oxidative stresses induce SUMO conjugation
to protein substrates (Reed et al., 2010; Miura and Hassegava, 2010). Arabidopsis plants
exposed to drought stress accumulate increased levels of sumoylated proteins by an ABAindependent pathway which is, in part, dependent on the transient increase of E3 SUMO
ligase levels in response to drought (Catala et al., 2007).
30
Transcript analysis of ClpP/ClpR genes indicates constitutive expression in roots and

leaves of A. thaliana with rather minor changes under stress and in senescence (Zheng et al.,
2002; Sinvany-Villalobo et al., 2004). Short-term moderate and severe stresses (desiccation,
high salt, cold, heat, oxidation, wounding and high light) all failed to elicit significant or rapid
increases in any chloroplast Clp protein. However, increases in mRNA and protein content
for ClpD and several ClpP isomers did occur during long-term high light and cold
acclimation of Arabidopsis plants. These results reveal the great complexity of Clp proteins
within the stroma of plant chloroplasts (Nakashima et al., 1997; Zheng et al., 2002).
Immunoblotting analysis showed an enhancement of Clp proteases in drought treated wheat
plants (Demirevska et al., 2008a).
Figure 1. Drought stress response and cross-talk between dehydrins, chaperones (Hsps), proteases and
protease inhibitors in maintaining cell protein conformation and function.
31
Both FtsH genes in maize were constitutively expressed, the expression level of
ZmFtsH2B transcripts being higher than that of ZmFtsH2A. Under polyethylene glycol, cold,
high salt and ABA treatments, ZmFtsH2B transcription in leaves was markedly up-regulated
by water deficit stress and ABA treatment, while ZmFtsH2A was constitutively expressed in
both leaves and roots. However, drought tolerance of transgenic tobaccos overexpressing
ZmFtsH2A and ZmFtsH2B was not greater than that of wild-type controls (Yue et al., 2010).
Decreased abundance of FtsH was established in a susceptible Kentucky bluegrass cultivar
under drought stress (Xu and Huang, 2010). Li et al. (2010) established that Arabidopsis Lon
protease (atlon4) mutant is more sensitive to drought stress than wild-type plants.
CONCLUSIONS
The results presented in this chapter support and underline the active participation of
dehydrins, chaperones, proteases and protease inhibitors in plant protection against
dehydration stress. Stress tolerance results from the coordinated action of a variety of stressresponsive mechanisms. Gene expression and specific protein identification studies point to
simultaneous up-regulation under drought stress of genes belonging to the LEA group, Hsps
and proteolytic systems (Cramer et al., 2007; Kavar et al., 2008; Demirevska et al., 2008b). It
appears that the so-called chemical chaperones osmolytes or compatible solutes, known
to accumulate in plants during osmotic stress, act in synergy with dehydrins and sHsps,
increasing the stability of cell proteins, thus suppressing their denaturation and aggregation
(Wang et al., 2004). Chemical chaperones and dehydrins also stabilize cell membrane
structures, thus preventing uncontrolled proteolysis. Members of different Hsps groups in the
chaperone network act in concert; they cooperate with each other and in ATP-dependent
proteolytic pathways in preventing aggregation (sHsps and Hsp70), resolubilizing aggregates
of denatured proteins (Hsp100 and Hsp70), assisting proper regain of conformation (Hsp60,
Hsp70 and Hsp90) and degrading irreversibly damaged proteins (Hsp100/Clp). Enhanced
antioxidative protection minimizes oxidative damage to proteins and preserves cell
membranes under stress, while dehydrins and chaperones maintain the native conformation of
the active oxygen scavenging enzymes. Chaperones (Hsp70 and Hsp90) and ATP-dependent
protease complexes play a key role in cellular signal transduction networks. Furthermore,
proteases in general play a key role in protein degradation linked to nutrient starvation under
prolonged drought stress, and in stress-accelerated senescence. Various protease inhibitors
appear to exert control over endogenous proteolysis, in addition to their protective role
against biotic stresses. The cross-talk between the described groups of stress-responsive
proteins in maintaining cell proteins under drought stress is summarized in Figure 1. A close
relationship and coordination of functions appears to exist between chaperonins, HSPs,
dehydrins and proteases under drought stress, as well as a certain diversity in the expression
of drought inducible proteins, based on comparison of sensitive and tolerant varieties. The
expression of HSPs, dehydrins, proteases and other stress-inducible proteins depends on the
duration and severity of the existing stresses, and on the superimposition of other seasonal
and environmental changes which occur in nature (Rizhsky et al., 2002, 2004). Although
we are a long way from a full understanding of drought tolerance and of the way in which
it could be increased in plants, this chapter shows that many of the basic actors are now
32
recognised. Thus, having in mind the diversity and the complex regulation of the distinct
groups of drought inducible proteins, further approaches combining transcriptomic, proteomic
and metabolomic analyses will be useful in understanding, manipulating and improving
drought tolerance in field crops.
ACKNOWLEDGMENTS
We are grateful to Professor Urs Feller, Institute of Plant Sciences and Oeschger Centre
for Climate Change Research, University of Bern, Switzerland for critical review of the
chapter and valuable suggestions for its improvement and to Professor Roger H. Pain for
critical reading of the manuscript and English improvement. The review has formed part of
activities under the bilateral project REPRODIMO between the Institute of Plant Physiology
and Genetics, Bulgarian Academy of Sciences in Sofia, Bulgaria, the Joef Stefan Institute
and the Agricultural Institute of Slovenia, both in Ljubljana, Slovenia.
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Reviewed by: prof. Urs Feller, Institute of Plant Sciences and Oeschger Centre for
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Switzerland.

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In: Droughts: New Research

Editors: D. F. Neves and J. D. Sanz

THE RESPONSE OF PLANTS TO DROUGHT STRESS:

Department of Plant Stress Molecular Biology,

Corresponding author, email: lsimova@mail.bg.

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

The Response of Plants to Drought Stress

2.1. LEA Proteins Structural and Functional Diversity

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

2.2. Protective Functions of Dehydrins on Proteins and Cell Structures

The Response of Plants to Drought Stress

2.3. Subcellular and Tissue Localization of Dehydrins

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

2.4. Relation of Expression of Dehydrins to Their Function

The Response of Plants to Drought Stress

2.5. Involvement of Dehydrins in the Response to Drought

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

3.1. General Characteristics

3.2. Structural and Functional Diversity

3.2.1. Small Hsp Families

The Response of Plants to Drought Stress

but appears to be regulated by temperature and phosphorylation (Chang, 2009). The

3.2.2. Chaperonins (Hsp60 Family)

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

3.2.4. Hsp90 Family

The Response of Plants to Drought Stress

3.2.5. Hsp100/Clp Family

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

3.3. Relation of Expression to Function

3.4. Involvement of Chaperones in Response to Drought

The Response of Plants to Drought Stress

4. PROTEASES AND PROTEASE INHIBITORS

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

4.1. Structural and Functional Diversity of Plant Proteases

The Response of Plants to Drought Stress

4.1.1. Serine Proteases

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

4.1.2. Cysteine Proteases

The Response of Plants to Drought Stress

4.1.3. Aspartic Proteases

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

4.2. Subcellular Localization of Plant Proteases

The Response of Plants to Drought Stress

involved in non-selective degradation, several of them probably functioning as highly specific

4.3. Regulation of Proteolysis, with Emphasis on Protease Inhibitors

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

The Response of Plants to Drought Stress

4.4. Proteases and Protease Inhibitors Involved in the Response to Drought

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

The Response of Plants to Drought Stress

the lower activity of vacuolar proteases by increased ATP-dependent activity, especially in

4.4.2. Protease Inhibitors

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

The Response of Plants to Drought Stress

The variety of physiological functions of proteolytic enzymes under stress conditions

5. ATP-DEPENDENT PROTEASE COMPLEXES

5.1. The Proteasome

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

The Response of Plants to Drought Stress

5.2. Rotease Complexes in Chloroplasts and Mitochondria

I. Vaseva, J. Saboti, J. utar-Vozli, et al.

5.3. Relation of Expression of ATP-Dependent Protease

The Response of Plants to Drought Stress