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 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.

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 (López-Otín 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 (López-Otín 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).

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; López-Otín 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 (Simões 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.

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 (Antão and Malcata, 2005).

Recently, several serine proteases have been isolated from different organs of various plant species (Antão 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 β-sheet sandwiched between two layers of helices, with the 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 α/β 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).

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 caspase-like 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 α/β/α 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.

Cysteine proteases are involved in many different processes, particularly those associated with degradation of storage proteins (Müntz, 2007) and programmed cell death (Lam and del Pozo, 2000).

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 β-strands and very little α-helix. The molecules are bilobal, with the active site having two catalytic Asp residues, located in a large cleft between the two domains (Simões 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 (Simões 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 (Simões and Faro, 2004), responses to pathogen attack (Xia et al., 2004) and abiotic stress (Timotijevic et al., 2010) have been suggested.

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.

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.

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).

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 (Müntz, 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; Müntz, 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 sasaran 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 sasaran the proteins and/or proteases of herbivores and phytopathogens (Müntz, 2007). Wound-inducible 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 membrane-associated 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 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 stroma-exposed 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; López-Otín and Bond 2008).

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, post-translational 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, non-competitive, uncompetitive, su1cide 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 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 (α₂-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 α₂-macroglobulin of family I39) that sasaran 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 metallo-proteases, 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 sasaran 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).

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 Kidrič, 1995).

Proteases and Protease Inhibitors Involved in the Response to Drought

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 Kidrič, 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-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 (Hausühl et al. 2001; Hieng et al. 2004; Pinheiro et al., 2005; Dramé 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-García 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 (Hausühl 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 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.

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-Rodrıguez 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 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).

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.

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