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Journal of Experimental Botany, Vol. 58, No. 10, pp. 2391–2407, 2007 doi:10.1093/jxb/erm089 Advance Access publication 1 June, 2007 REVIEW ARTICLE Protein dynamics and proteolysis in plant vacuoles Klaus Müntz* Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany Received 1 November 2006; Revised 25 March 2007; Accepted 26 March 2007 Abstract Plant cells cannot live without their vacuoles. The tissues and organs of a plant contain a wide variety of differentiated and specialized vacuoles—even a single plant cell can possess two or more types of vacuoles. Vacuolar proteins are encoded by nuclear genes and synthesized in the cytoplasm. Their transport into the vacuolar compartment is under cytoplasmic control. Transcription seems to be a major control level for differential protein supply to the vacuoles. It is at this level that vacuole differentiation and functions are mainly integrated into cellular processes. Recycling amino acids generated by protein degradation is a major function of the vacuole. This is most evident when storage proteins are mobilized in storage tissues of generative or vegetative organs in order to nourish the embryo of germinating seeds or sprouting buds. When specific proteins are transferred to the vacuole for immediate degradation this compartment contributes to the adaptation of protein complexes in response to changes in developmental or environmental conditions. Vacuolar proteases are involved in protein degradation during reversible senescence and programmed cell death, which is also called irreversible senescence. Vacuoles contribute to defence against pathogens and herbivores by limited and unlimited proteolysis. Our present knowledge on functions and processes of vacuolar protein dynamics in plants is reviewed. Research perspectives are deduced. Key words: Complete protein breakdown, differential protein supply to vacuoles, limited proteolysis, lytic vacuoles, PCD, protein storage vacuoles, vacuolar protein mobilization, vacuolar proteins, vacuolar protein transfer, vacuolar proteolysis in defence. Introduction Plant cells cannot survive without their extra-cytoplasmic vacuolar compartment (Rojo et al., 2001; Surpin and Raikhel, 2004). Since protein synthesis does not take place in vacuoles (for reviews on vacuoles and their functions, see Leigh and Sanders, 1997; De, 2000; Robinson and Rogers, 2000) all vacuolar proteins have to be imported from the cytoplasm. One major life-supporting function of vacuoles is that of protein breakdown. Special sets of proteins are responsible for the structure and function of vacuoles. When vacuoles undergo developmental and environmental changes some of these proteins are replaced by new imports from the cytoplasm. So far export of proteins from vacuoles has not been observed. However, it is unknown whether all substituted proteins are broken down by vacuolar proteases or whether some, or at least their fragments, are degraded outside the vacuole. The vacuolar compartment represents a major site of amino acid recycling in plant cells. This function is closely related to plant development and plant response to environmental effects. In this context various proteins are imported from the cytoplasm to be degraded in the vacuole. Breakdown takes place either immediately after arrival in the vacuole or after short- to long-term storage. For a long time vacuoles were believed to be the only site of protein breakdown in the plant cell. Vacuolar * E-mail: [email protected] This paper is dedicated with gratitude to Prof. Dr Ulrich Wobus on the occasion of his 65th birthday and his retirement after 15 years as director of IPK, Gatersleben. Abbreviations: CCV, clathrin-coated vesicles; CP, carboxypeptidase; DV, dense vesicles; ER, endoplasmic reticulum; ESCRT, endosomal sorting complex required for transport; HR, hypersensitivity response; H+-V-ATPase, proton-translocating vacuolar ATPase; H+-V-PP, proton-translocating vacuolar pyrophosphatase; I-CliP, intra-membrane-cleaving protease; LV, lytic vacuole; MVB, multi-vesicular bodies; PAC, precursor-accumulating vesicle; PB, protein body; PCD, programmed cell death; PI, protease inhibitor; PM, plasma membrane; PSV, protein storage vacuole; PVC, pre-vacuolar compartment; rER, rough endoplasmic reticulum; SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; TGN, trans-Golgi network; TI, trypsin inhibitor; TIP, tonoplast intrinsic protein; VPE, vacuolar processing enzyme; VSP, vegetative storage protein. ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected] 2392 Müntz proteases were the first proteases to be detected, isolated, and analysed. During the past 20 years, however, our knowledge of proteolytic systems in other compartments and of vacuolar proteins and protein import has increased. It is now known that different vacuoles can exist simultaneously in a single plant cell. Therefore, the role of plant vacuoles in protein dynamics and proteolysis has to be re-evaluated. Vacuolar proteins Recent proteomic analysis revealed the presence of 402 proteins in isolated and purified lytic vacuoles (LV) of Arabidopsis thaliana leaves (Fig. 1), 46.5% of which could be identified (Carter et al., 2004). Some of these proteins were typical of other non-vacuolar organelles, which may reflect contamination or, more probably, an autophagic uptake into the vacuole. Vacuolar proteins can be classified, according to their localization, into luminal and tonoplast proteins, and the latter further into integral membrane and membrane peripheral proteins (e.g. cytoskeleton proteins at the outer and lectins at the inner surface). The proteomic analysis of vacuolar proteins has been further refined by separating tonoplast from luminal fractions (Carter et al., 2004; Shimaoka et al., 2004; Szponarski et al., 2004). Tonoplast preparations from LV of Arabidopsis leaves contained 38 transporters belonging to the integral membrane proteins (Carter et al., 2004). Among them carrier proteins, ion channels, and ion pumps, which have several trans-membrane domains, play a role in the exchange of inorganic and organic compounds between cytoplasm and vacuole. For example, proton-translocating vacuolar ATPase (H+-V-ATPase) and the protontranslocating vacuolar pyrophosphatase (H+-V-PP) catalyse energy-dependent proton transfer, and generate the acidic pH inside vacuoles and the energy gradients needed for ion transfer. Furthermore, they contribute to maintaining the cytoplasmic pH in narrow neutral limits. Mg2+and Ca2+-ATPases are active transporters of inorganic cations, whereas ABC-transporters of the tonoplast catalyse the ATP-dependent transfer of organic compounds. Tonoplast intrinsic proteins (TIPs, also called aquaporins), which are water channels that facilitate the exchange of water molecules between cytoplasm and vacuole, are very abundant. TIPs and transporters are components of a vital cellular function. By controlling ion concentrations and the amount of water in the vacuole, the cell can control its osmotic and turgor pressure and thereby contribute to cell growth and mechanical stability of the plant itself. Finally SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex proteins have Fig. 1. Schematic overview of the plant vacuole and functional categorization of the vacuolar proteins identified [Fig. 4 in Carter et al. (2004), reproduced with permission, which is gratefully acknowledged, of ASPB as copyright holder and the authors]. The proteins identified are grouped into eight major categories, and the number of proteins belonging to each group is shown in parentheses. Some of the specific activities of each group and a model of the tonoplast SNARE-pin complex are shown. Vacuolar protein dynamics 2393 been identified among tonoplast proteins, providing direct evidence for vesicular protein trafficking into vacuoles. The luminal fraction of LV is characterized by its high percentage of hydrolytic enzymes, such as glucosidases, proteases, chitinases, RNases, glucanases, invertase, and myrosinase, as well as protease inhibitors (PIs) and lectins. These proteins indicate the other important functions of LV, the turnover of biological macromolecules and their role in defence against pathogens and herbivores. Comprehensive lists of proteins found in the vacuolar lumen were published by De (2000) and Carter et al. (2004). Protein storage vacuoles (PSV) in cells of vegetative (root, stem parenchyma, tubers, pods) and generative (endosperm, cotyledon mesophyll, embryonic axis) organs are sites of short- or long-term deposition of protein reserves. An extensive proteome analysis of PSV has not yet been conducted but, using immunohistochemistry, many proteins have been found to be localized there. Besides large amounts of polymorphic storage proteins (Staswick, 1994; Müntz, 1998; Shewry and Casey, 1999) PSV often contain proteases (Müntz et al., 2001) and other hydrolytic enzymes, even at the time of protein deposition. Protein dynamics How do proteins get into the vacuole? Little is known about the mechanisms of vacuole formation. Early during mitosis of root tip cells, clusters of pro-vacuolar vesicles of similar appearance have been observed in both daughter cells, suggesting their allocation from the mother cell and a persistence of the vacuolar system through cell division (Marty, 1999). However, a very early de novo generation of pro-vacuolar vesicles in the emerging daughter cells cannot be excluded. De novo generation of a vacuole was even observed in specializing cells after cell division ceased (Hoh et al., 1995). It is not clear whether de novo formation of vacuoles starts from the trans-Golgi network (TGN) as suggested by Marty (1999) or from the endoplasmic reticulum (ER). In any case, vesicular biogenesis of vacuoles takes place (Fig. 2). During cell differentiation the numerous pro-vacuolar vesicles merge into a few larger vacuoles. These grow by material transfer in vesicles that are generated from the ER or Golgi apparatus. Several different types of vesicles for vacuolar protein transfer are known (Hara-Nishimura et al., 2004; Herman and Schmidt, 2004; Vitale and Hinz, 2005; Neuhaus and Paris, 2006). If pro-vacuolar vesicles persist through cell division as suggested by Marty (1999) then the primary sets of vacuolar membrane and lumen proteins are derived from the mother cell. In the case of de novo formation of vacuoles, the primary protein sets are provided by the biosynthetic pathway (Herman, 1994). Biosynthesis of vacuolar proteins takes place at the rough endoplasmic reticulum (rER). The nascent precursors of vacuolar proteins are sequestered co-translationally into the ER lumen. Concomitantly the N-terminal signal peptide is cleaved from the precursor and it enters the biosynthetic pathway (also called the secretion pathway). Large segments of the precursor correspond to the mature vacuolar protein. Precursors can contain more than one subunit of the mature protein (e.g. 2S and 11S storage proteins; Müntz, 1998) or even multiple copies of a protein (e.g. proteinase inhibitors and cyclotides; see sections below entitled ‘Proteinase inhibitors’ and ‘Cyclotides’, respectively). Precursor segments corresponding to mature vacuolar proteins can be flanked by pro-peptides, and the different segments can be connected by linker peptides. Pro- and linker peptides act in maintaining a conformational state of the precursor that makes it compatible with the structural demands of intra-cellular protein traffic (Müntz, 1998) and/or maintains enzyme precursors in an inactive state (e.g. precursors of proteases; see Müntz and Shutov, 2002). In addition, precursors of several proteins become glycosylated in the ER and their glycosyl side chains are further trimmed and modified in the Golgi apparatus (e.g. 7S globulin of Phaseolus vulgaris; Müntz, 1998). Precursors of vacuolar proteins contain sorting and targeting signals which direct the interaction with sorting receptors when transfer vesicles are generated at the ER or Golgi apparatus (Jiang and Rogers, 2003; Robinson et al., 2005; Vitale and Hinz, 2005). After they have completed their function, pro- and linker peptides, as well as in some cases N- or C-terminal peptides containing targeting signals, are detached by site-specific limited proteolysis in the vacuole (Müntz, 1998) or even already in the prevacuolar compartment (PVC) (Otegui et al., 2006). This proteolytic tailoring transforms precursors into mature vacuolar proteins at their destination. It is, for example, in the vacuole that storage proteins assume the conformation which is compatible with their vacuolar deposition, and inactive enzyme precursors are activated. From the ER, tonoplast and luminal proteins are transported to the vacuole either directly or after passage through the Golgi apparatus. In cotyledons of developing pumpkin seeds, precursor-accumulating vesicles (PAC) transfer storage proteins from the ER directly to the PSV (Hara-Nishimura et al., 1998). Direct ER-to-vacuole transfer has also been observed for the sorting receptor AtSRC2 (Oufattole et al., 2005). Transfer of proteins from the ER to the cis-Golgi takes place in coat protein complex II vesicles (Aniento et al., 2003; Hanton et al., 2006; Matheson et al., 2006). At the cis-Golgi, dense vesicles (DV) are generated that contain storage globulins destined for the PSV (Hillmer et al., 2001), whereas from the TGN clathrin-coated vesicles (CCV) bud that carry proteins destined for the LV (Robinson et al., 1998a; Hinz et al., 1999), for example, acid hydrolases. On their way 2394 Müntz Fig. 2. Scheme of mechanisms and routes of protein transfer to the vacuole. Upper part: vacuole generation during cell division by de novo generation or apportionment from mother cells (Marty, 1999). Lower part: transfer of proteins into the vacuole. Proteins can arrive by endomembrane progression when they are transported by vesicular transfer via the Golgi apparatus (Hara-Nishimura et al., 2004; Herman and Schmidt, 2004; Vitale and Hinz, 2005; Neuhaus and Paris, 2006). Vesicular transfer of proteins from the ER to the Golgi is indicated by white circles. On their way from the TGN to the vacuole many proteins with sorting determinants are contained in transfer vesicles, such as DV or CCV, which discharge their cargo to MVB/PVC or might even bypass this endosomal compartment. The ER generates several vesicles (PAC, ER bodies, PB) where proteins can be transported to the vacuole without progression through the Golgi. A specific variant of this mechanism represents the direct formation of PB from the ER. Autophagy transfers cytoplasmic proteins or organelles into the vacuole. PM proteins can be transported to the vacuole by endocytosis. CCV, clathrin-coated vesicles; ctVSD, C-terminal vacuolar sorting determinant; DV, dense vesicles; ER, endoplasmic reticulum; LV, lytic vacuole; MVB, multi-vesicular bodies; PB, protein bodies; PM, plasma membrane; PSV, protein storage vacuole; PVC, pre-vacuolar compartment; PAC, precursoraccumulating vesicles; ssVSD, sequence-specific vacuolar sorting determinant. Greek letters indicate characteristic isoforms of TIPs of different types of vacuoles. to the vacuole both types of vesicles enter multi-vesicular bodies (MVB) which have been identified as endosomal pre-vacuolar compartments (PVC) (Robinson et al., 2000; Jiang et al., 2002, Tse et al., 2006). Storage proteins have been found in the luminal matrix of MVB (Robinson et al., 1998a). The presence of sorting receptors for vacuolar proteins in MVB (Li et al., 2002; Miao et al., 2006) is additional proof of vesicular transfer of vacuolar proteins from the Golgi to this compartment. According to Jiang et al. (2002) the intra-luminal vesicles contain acid hydrolases known to be transferred in CCV. In the acidic environment, MVB receptors involved in vesicular protein transfer to vacuoles dissociate from their cargo protein before the latter are further transferred into vacuoles. The recycling of receptors involved in vesicular protein transfer to vacuoles starts from MVB (Lam et al., 2006). Correct storage protein transfer to vacuoles was shown to depend on intact receptor recycling to the Golgi. Recent experimental results indicate that similarly to yeast (reviewed by Bowers and Stevens, 2005) a retromer complex must also be involved in this receptor recycling to the Golgi in plants. In Arabidopsis, mutation of a gene encoding a protein homologous to a component of the yeast retromer complex interferes with vacuolar storage protein transfer (Shimada et al., 2006). In the MVB/PVC of the same plant species, components of a retromer complex that interact with a vacuolar sorting receptor were found (Oliviusson et al., 2006). It is unclear whether DV and CCV can also bypass MVB/PVC and go directly to the PSV as hypothesized by Vitale and Hinz (2005). The mechanism of protein transfer from MVB into vacuoles has still to be verified experimentally but fusion with pre-existing vacuoles is assumed to be most probable (Robinson et al., 2000). In Arabidopsis cells, two different populations of endosomes have been observed, one of which seems to act as an early endosome in endocytosis, Vacuolar protein dynamics 2395 whereas the other seems to act as a PVC in vacuolar protein transfer (Ueda et al., 2004). Possibly, docking of different transfer vesicles and receptor-recycling retromer vesicles at the MVB leads to modifications, turning the MVB into a PVC that finally fuses with a vacuole. In this PVC, precursors of storage proteins are already processed by limited proteolysis before entering the PSV (Otegui et al., 2006). Protein traffic to the vacuole seems to be a one-way passage. In yeast, receptors and other components of the vesicular protein traffic machinery undergo a retrograde transport from the vacuole to the MVB and TGN (Bryant et al., 1998; Bowers and Stevens, 2005). The docking of transfer vesicles at the membrane boundary of LV in Arabidopsis leaves is reflected by the presence of SNARE proteins in its tonoplast membranes (Carter et al., 2004). Secretion of vacuolar luminal hydrolases was observed in suspension-cultured tobacco cells (Kunze et al., 1998), but the authors could not rule out that the secreted enzymes came from a PVC, in which at least precursors of storage globulins can undergo maturation by limited proteolysis (see above). In conclusion, so far no retrograde traffic of resident tonoplast or other vacuolar proteins has been conclusively shown for plants. Another pathway of protein (and other cargo) supply to the vacuole is endocytosis (Fig. 2) (Murphy et al., 2005; Šamaj et al., 2005, 2006). Endocytotic vesicles which are generated at the plasma membrane (PM) can transfer PM proteins to the LV (Takano et al., 2005). On their way to the LV, the PM proteins first pass through MVB or the TGN (Lam et al., 2006). In yeast, heteromeric protein complexes have been detected that are required for the vesicular transport of membrane proteins to MVB. The Arabidopsis genome contains genes encoding proteins that are homologous to the components of the yeast endosomal sorting complex required for transport (ESCRT) to MVB (Winter and Hauser, 2006). It remains to be shown whether in the plant the encoded proteins have functions similar to the yeast ESCRT machinery. The intra-luminal vesicles within MVB originate, at least in part, from invagination of endocytotic vesicles. The endocytotic pathway from the PM to the LV and the biosynthetic pathway (secretory pathway) of protein transfer from the Golgi apparatus to the LV and, partially, the PSV converge in MVB. It is unclear, however, whether proteins from the endocytic pathway and those of the biosynthetic pathway can enter the same MVB. In yeast, some proteins are known to be transferred directly from the cytosol into the vacuole (Noda et al., 2000). The yeast vacuole is often used as a model for plant vacuoles, but so far no similar mechanism has been observed in plant cells. Aside from the targeted transfer so far described, cytoplasmic material, including proteins, is taken up into the vacuole by autophagic processes (Robinson et al., 1998b; Herman and Schmidt, 2004; see also section below entitled ‘Programmed cell death’). According to Marty’s hypothesis about vacuole generation from the TGN (Marty, 1997, 1999), cytoplasmic material is trapped autophagically by the emerging vacuole and is afterwards degraded by lytic enzymes. Material uptake by autophagy (Fig. 2) was observed in cells of germinating bean seed cotyledons (Herman et al., 1981; Toyooka et al., 2001). As a result of this process remnants of plastids and mitochondria are transiently present in vacuoles. Recently autophagy has been shown to be a basic process involved in amino acid recycling that continuously takes place in young as well as in stressed and senescing plant cells (Yashimoto et al., 2004; Slávikova et al., 2005). Changes in vacuolar protein composition The vacuolar compartment of a plant cell undergoes development- and environment-dependent structural and functional changes which are accompanied by alterations in its protein composition. Best known is the formation of PSV from LV during seed development and the transformation of PSV into LV during seed germination and seedling growth. Analogous processes take place during storage and mobilization of vegetative storage proteins (VSP) (Staswick, 1994). Different vacuoles can be formed in leaf cells during senescence (Otegui et al., 2005) and in salt-stressed ice plants (Epimashko et al., 2004). The transformation of one type of vacuole into another adapts the vacuolar compartment to different functions. The simultaneous presence of different vacuoles generates a physical separation of different physiological functions in a cell. A structural separation of different vacuolar functions has even been attributed to vacuolar subcompartments which are observed in PSV (Jiang and Rogers, 2002; see section below entitled ‘Intra-vacuolar compartments’). It is disputed whether already existing vacuoles undergo structural and functional transformation or whether vacuoles are formed de novo in order to substitute preceding ones and to form additional new ones. Independent of the mechanism that underlies vacuole differentiation in a cell, this process has to be accompanied by proteolytic elimination of specific proteins and the insertion of new ones. Deposition of proteins in PSV: Two mechanisms have been described for PSV generation: transformation of LV during early seed development or substitution of LV by a newly formed PSV (Herman, 1994; Robinson and Hinz, 1997; Vitale and Hinz, 2005). In both cases the newly formed PSV contain a-TIP instead of c-TIP, which is characteristic of the tonoplast of the LV, and the lumen of the vacuole is progressively filled with storage proteins. Several tonoplast and luminal proteins have to be substituted or added during PSV formation. There is evidence that membrane proteins such as TIPs and luminal proteins 2396 Müntz such as storage proteins use independent trafficking mechanisms to the PSV. They are probably transported in different vesicles from their site of biosynthesis at the rER to the PSV (Gomez and Chrispeels, 1993; Park et al., 2004) independently of whether the proteins pass or bypass the Golgi apparatus and MVB/PVC. However, it is disputed whether a-TIP and storage proteins are transferred separately in different vesicles, since Hinz et al. (1999) co-localized both by immuno-cytochemical methods in DV (see also Robinson and Hinz, 1999). Different mechanisms participate in the sorting of storage proteins into transfer vesicles, including their reversible binding to receptors (Robinson et al., 2005). Electron microscopic observations of developing pea cotyledons indicated an autophagocytic elimination of the old vacuole whereby tonoplast and luminal proteins of the original LV have to be degraded in de novo generated PSV (Hoh et al., 1995; Robinson et al., 1998a). Proteases of this PSV either derive from the preceding LV or arrive by vesicle transport from the rER. In order to accumulate, storage proteins must either be resistant against or physically separated from these proteases, otherwise the elimination of the LV components has to be completed and the proteases involved eliminated before storage proteins can accumulate in the PSV. During LV–PSV transformation, when c-TIP is substituted by a-TIP, the former has to be eliminated by degradation in the membrane (membrane proteases?), by degradation in the cytoplasm, or/and in the vacuolar lumen after detachment from the membrane. Mature storage proteins accumulate to high levels in cells of developing storage tissue, which indicates that their turnover is low or at least is vastly exceeded by their deposition in the PSV. Half lives of 2 weeks and >5 weeks have been measured for storage proteins in developing soybean and pea seeds, respectively (Madison et al., 1981). Storage proteins are synthesized as transportcompatible precursors that are transferred by vesicles into PSV (Fig. 2) where they are processed into mature vacuolar proteins by limited proteolysis (see section above entitled ‘How do proteins get into the vacuole?’). When this transport pathway includes passage through MVB/PVC the separate pathways of storage protein precursors and processing proteases converge there and the first processing steps can take place in this compartment (Otegui et al., 2006). Excised targeting, pro- and linker peptide fragments are degraded in the PSV at the latest. It has been suggested that vacuolar degradation of mis-structured proteins forms part of the cellular protein qualitycontrol system (Tamura et al., 2004; Vitale and Ceriotti, 2004; Pimpl et al., 2006) but there is still no proof that this process occurs in vivo in PSV. Highly cleavagespecific proteases catalyse limited proteolysis while proteases with broad cleavage specificity mediate complete polypeptide breakdown. The intact storage protein molecules have to be protected against degradation by structural inaccessibility or a differential subcompartmentation. According to Shutov et al. (2003) and Jiang et al. (2001) both mechanisms act in PSV. Controlled peptide degradation concomitant to storage protein deposition indicates that even PSV exhibit some lytic functions at that stage. In the starchy endosperm of developing cereal grains storage proteins are deposited directly in ER-derived protein bodies (PB) (e.g. zeins of maize, prolamins of rice) or in PSV (e.g. some wheat prolamins, oat globulins, globulin-like rice glutelins). Autophagic processes (Fig. 2) can be involved in the transfer of wheat prolamins to the PSV (Levanony et al., 1992). At the end of grain maturation, the cells of the starchy endosperm undergo programmed cell death (PCD, see section below entitled ‘Senescence and cell death’) leaving only the cells of the aleurone layer alive (Young and Gallie, 2000). In aleurone cells, at least two types of vacuoles have been found (Swanson et al., 1998): PSV where 7S globulins and lectins are stored and LV containing various hydrolases. Plants store protein reserves in vegetative propagation organs too, such as shoot (e.g. patatin in potato) and root tubers (e.g. sporamin in sweet potato). In addition, deciduous trees accumulate VSP in bark parenchyma cells during autumn for mobilization in spring. Transient deposition of protein reserves occurs even in leaves, shoot axes, and pods of various grain legumes, such as soybean VSP (Staswick, 1990; Herman, 1994), or in roots of alfalfa (Cunningham and Volonec, 1996). VSP are a heterogeneous group of polypeptides, unrelated in amino acid sequences and without evolutionary relationship to seed storage proteins. Nevertheless, they are also deposited in vacuoles. In soybeans and related legumes, VSP are deposited in a single large central LV, whereas tubers contain several vacuoles with VSP aggregations. In soybean leaves, non-coated transfer vesicles containing VSP have been observed that bud from the Golgi apparatus. These vesicles seem to discharge their cargo directly into the vacuole (Klauer and Franceschi, 1997). This may be interpreted as Golgi-to-vacuole transport of VSP which bypasses MVB/PVC, although the involvement of the endosomal compartment has not been excluded by these experiments. The tonoplast of VSPaccumulating leaf vacuoles contains d- but not a-TIP (Jauh et al., 1998). However, Park et al. (2004) found a-TIP in the tonoplasts of seed storage proteinaccumulating leaf cell PSV of transformed plants, as well as in tonoplasts of leaf PSV of untransformed Phaseolus beans. It is not clear whether PSV with d-TIP and PSV with a-TIP can both occur concomitantly in the same leaf cell. Multiplicity and transformation of vacuoles in vegetative organs: In the aleurone cells of barley seeds lectin is localized in PSV, whereas aleurain, a cysteine protease, is contained in a different vacuole, probably an LV. In root Vacuolar protein dynamics 2397 tip cells of barley and pea as well as in the plumula of pea, lectin and aleurain co-localized with a-TIP and c-TIP, respectively, indicating that, at certain developmental stages, vegetative cells can contain at least two different types of vacuoles simultaneously, the PSV and LV (Paris et al., 1996). Developmental diversification of vacuoles was observed in young root tip cells (Flückiger et al., 2003). In the youngest stage, only one type of small vacuole is present. The tonoplast of early stage vacuoles contains a- and sometimes a- and d-TIP. Later on, tonoplasts normally contain a-, c-, or d-TIP (Fig. 2), though the tonoplast of some vacuoles contains a- and c- or c- and d-TIP (Jauh et al., 1998, 1999). A hypothetical scheme for vacuole diversification has been proposed by Jauh et al. (1999). It remains to be shown whether the different types of vacuoles are generated de novo or whether vacuole transformation takes place. It seems conceivable that vacuoles with two different TIPs in their tonoplast are the result of fusion of two ancestor vacuoles (Fig. 2). Double labelling of vacuoles with TIP- and lumen protein-specific antibodies has seldom been reported (Hoh et al., 1995). If only TIPs or lumenal proteins are labelled, their assignment to different types of vacuoles is problematic. Nevertheless, several studies confirm that the plant cell uses different vacuoles to compartmentalize different vacuolar functions and that vacuolar diversity varies depending on the developmental stage of a cell and environmental effects. This was shown in the regeneration of two different vacuoles in evacuolated tobacco leaf cell protoplasts (Di Sansebastiano et al., 2001; Tamura et al., 2003) and senescent leaves of soybean and Arabidopsis thaliana (Otegui et al. 2005; see section below entitled ‘Vacuolar proteolysis in defence against herbivores and pathogens’). Besides the differential distribution of different TIP subfamily members, multiplicity and diversification of vacuoles are reflected in other tonoplast proteins as well. When maize endosperm cells differentiate into aleurone and starchy endosperm, an H+-V-PP is only inserted into vacuoles of the aleurone cells but not in starchy endosperm cells (Wisniewski and Rogowsky, 2004). In petal cells of opening flower buds of morning glory (Ipomea tricolor L.), anthocyans in the vacuole change colour from purple to blue due to a pH shift from weakly acidic (pH 6.6) to weakly basic (pH 7.7). This process is regulated by a Na(K)-antiporter protein which by active Na+ and/or K+ transport into the vacuole mediates the pH shift (Yoshida et al., 2005). Under induction conditions in tomato and petunia cells, anthocyans accumulate in vacuoles bounded by d-TIP-containing tonoplasts (Jauh et al., 1998). Environment-dependent changes in tonoplast proteins and vacuole functions have been documented for salt stress. In tomato [Lycopersicum esculentum (¼Solanum lycopersicum)], isoform A1 of the catalytic subunit of the + H -V-ATPase prevails in leaf cell vacuoles, whereas A2 is characteristic for root cell vacuoles. Under salt stress, A1 transcript and protein concentration doubled in leaves, whereas in the root cells transcript and protein levels of A2 did not change (Bageshwar et al., 2005). Differential vacuole formation also occurs in the ice plant (Mesembryanthemum crystallinum), where two different vacuoles stabilize the cytoplasmic pH in response to salt stress (Epimashko et al., 2004). Intra-vacuolar compartments: The PSV of storage tissue cells in seeds of pumpkin, castor bean, tobacco, and several other plants are substructured. Phytate globoids and protein crystalloids embedded in an amorphous PSV matrix can be identified under the microscope. For PSV from pumpkin cotyledons it has been shown that crystalloids and PSV matrix contain identical storage globulins. The phytate globoid cavity seems to be delimited by a one-unit membrane (Jiang et al., 2001). Interestingly, the tonoplast components H+-V-PP and c-TIP, which are characteristic of LV, were detected in the globoid membrane (Jiang et al., 2001), whereas the PSV tonoplast only showed d-TIP-specific immunolabelling. Using immuno-electron microscopy, the same authors localized DIP (dark intrinsic protein), which is also a member of the d-TIP subfamily of tonoplast proteins, to purified crystalloids from pumpkin and tobacco PSV (Jiang et al., 2000). In tobacco crystalloids, a new receptor-like membrane protein was detected, which shares the receptor domain for vacuolar protein targeting with BP-80. These and other results indicate that the crystalloid also has a membrane boundary (Jiang et al., 2000). To explain the differing protein compositions of the membrane boundaries of PSV and intrinsic crystalloids and globoids, Jiang and Rogers (2002) published a model to show how PSV subcompartments might be generated and how proteins characteristic of the different PSV subcompartments are transported from the ER to the PSV on separate pathways that can be traced back to the MVB/PVC. Under the electron microscope, PSV of Brassicaceae seeds appear to contain only globoids and no crystalloids. However, in PSV studies from Brassica napus seeds the isolation of a crystalloid-like fraction has been reported (Gillespie et al., 2005). A new protein, BPEP (Brassicaceae PSV-embedded protein), was isolated from this globoid fraction. In immunofluorescence studies, BPEP- and DIPspecific antibodies label the same substructures in B. napus and A. thaliana PSV. Since peptide fragments responsible for protein–protein interactions were found in BPEP the authors suggest that it may act as a scaffolding protein forming an internal membrane network in globoids and crystalloid-like structures as well. There appears to be no subcompartmentation in LV and in vacuoles that store VSP. In PSV of seeds, however, subcompartmentation could explain how lytic enzymes 2398 Müntz can be stored separately from storage proteins. At the time of germination when amino acids have to be mobilized in order to nourish the embryo, controlled fusion of subcompartments could give the proteases access to their substrates. Proteolysis in vacuoles Controlled proteolysis of tonoplast proteins? Transfer vesicles (Fig. 2) fusing with the MVB/PVC or vacuoles have to be recycled. As mentioned in the section above entitled ‘How do proteins get into the vacuole?’ indication for MVB/PVC-to-Golgi retrograde protein transport in plant cells has been obtained by analysing appropriate mutants of Arabidopsis thaliana. However, so far no export of a tonoplast ‘resident’ protein from vacuoles has been found. It is therefore possible that removal and substitution of these vacuolar proteins may only occur via protein degradation. During seed germination and seedling growth the PSV is transformed into an LV (see section below entitled ‘Mobilization of storage proteins in PSV’). Concurrently with this transformation a- and d-TIP are removed and substituted by c-TIP. Other tonoplast proteins may also undergo controlled substitution (see section above entitled ‘Multiplicity and transformation of vacuoles in vegetative organs’). Complete proteins or fragments generated by limited proteolysis might be removed. Their subsequent proteolysis could occur in the cytosol or vacuolar lumen. Intra-membranecleaving proteases (I-CliP) are known from animal and bacterial cells. I-CliP-encoding genes have also been found in plants (Weihofen and Martoglio, 2003). I-CliP are localized in membranes and catalyse the release of extra-membranous domains and intra-membrane peptides from membrane proteins. In bacterial and animal cells this generates regulatory poly- or oligopeptides or results in the degradation of cleavage products. So far, intramembrane proteases have not been reported for tonoplasts of plant vacuoles. Proteolysis in the lumen of vacuoles Many proteases reside in the lumen of plant vacuoles (Table 1). Protein degradation (Fig. 3) occurs either immediately after proteins have entered the lumen of LV or, in the case of storage proteins, after temporary deposition in PSV when these are transformed into LV. Amino acids are recycled for metabolic processes that take place outside the vacuole. Peptide and amino acid transporters of the tonoplast catalyse the export of small protein degradation products (Waterworth et al., 2001). Immediate protein degradation in LV: The acidic environment of an LV might facilitate conformational changes of proteins and thus make them more accessible to resident lumen proteases. After endocytosis (Figs 2, 3) PM proteins can be transferred to the vacuole and degraded. In pericycle cells of A. thaliana, boron deficiency leads to the formation of a specific boron transporter protein (BOR1) which is inserted into the plasmalemma. This is followed by increased boron transfer into the xylem for supply to shoot organs. When normal boron nutrition is restored the BOR1 protein is removed by endocytosis and degraded in the vacuole (Takano et al., 2005). Posttranslational control processes act outside the vacuole, which in this case is just a non-specific site of proteolysis. Dismantling and degradation of complex cell structures after autophagic uptake into the vacuole are described in the section above entitled ‘Changes in vacuolar protein composition and the section below entitled ‘Senescence and cell death’. Mobilization of storage proteins in PSV: At the onset of mobilization, the mechanisms that protect stored proteins against degradation by stored proteases have to be overcome; for example, structural inaccessibility of proteins (Shutov et al., 2003), membrane boundaries separating stored proteins from proteases (Jiang and Rogers, 2002), or pH values that maintain proteases inactive (Fath et al., 2000). In addition, de novo biosynthesis of proteases and the activation of protease precursors Table 1. List of vacuolar proteases known to be involved in storage protein metabolism of seeds (Müntz, 2003) Protease Type Function Plant Legumains (syn. VPE): Asn-specific CPR Endopeptidase Precursor processing, protein breakdown Papain-like CPR Endopeptidase Protein breakdown Aspartic proteinase Endopeptidase Subtilisin-like serine proteinase Metalloproteinase Carboxpeptidase Endopeptidase Endopeptidase Exopeptidase Precursor processing, protein breakdown Protein breakdown Protein breakdown Protein breakdown Arabidopsis thaliana, Nicotina tabaccum, Ricinus communis, Vicia sativa, Vigna radiata, and others Glycine max, Hordeum vulgare, Ricinus communis, Vigna radiata, Vicia sativa, and many others Arabidopsis thaliana, Brassica napus CPR, Cysteine proteinase; VPE, Vacuolar processing enzyme. Glycine max Fagopyrum esculentum Hordeum vulgare, Triticum sativum, Vigna radiata, and various others Vacuolar protein dynamics 2399 Fig. 3. Schematic overview of the functions of vacuolar proteolysis in cellular protein breakdown and amino acid recycling. Upper part: precursors of vacuolar proteases are synthesized at the ER and transferred in vesicles to the vacuole either via endomembrane progression through the Golgi and MVB (e.g. CCV) (for references see legend to Fig. 2) or bypassing the Golgi (PAC, precursor protease vesicles, ER bodies, riconosomes) (Chrispeels and Herman, 2000; Gietl and Schmidt, 2001; and references in the legend to Fig. 2). Lower part: in MVB and vacuoles (LV, PSV) precursors of processing enzymes (e.g. VPE) are activated auto-catalytically by limited proteolysis (precursor processing). Activated processing proteases process other precursor polypeptides and thereby activate enzymes and generate polypeptides involved in defence processes. Proteins to be degraded get into vacuoles as shown in Fig. 2. Only vacuolar protein transfer by variants of autophagy is indicated here because of its specific contribution to senescence and PCD. Limited and complete vacuolar proteolysis is a major process of cellular amino acid recycling after long-term storage of protein reserves, when selected proteins are broken down immediately after getting into the vacuole or during senescence. Mega-autophagy represents the final step in PCD when the release of proteases leads to cell corpse degradation. (Okamoto and Minamikawa, 1998) contribute to the initiation of protein reserve mobilization. Protease action and protein mobilization are spatially and temporally highly regulated (Müntz et al., 2001). When globulin precursors arrive in PSV at the time of protein deposition, exposed Asn-flanked processing sites are cleaved by Asn-specific vacuolar processing enzymes (VPEs or legumains) yet the majority of Asn-flanked peptide bonds remain uncleaved in these globulins (Shutov et al., 2003). This shows that certain protective conformations exist in the stored proteins that have to change before breakdown can start. As described in the section above entitled ‘Intra-vacuolar compartments’, fusion of vacuolar subcompartments could give the proteases access to their substrates. Activation of stored proteinase precursors in PSV has not been described so far. In seedlings of cereals, as well as those of dicotyledonous plants, de novo formation of proteinases is under transcriptional control. This is reflected by the coincidence of histo-patterns of immuno-localization of proteinases and in situ hybridization of corresponding mRNAs in cotyledons of legume seedlings (Tiedemann et al., 2001). Precursors of new proteinases are synthesized at the rER and transferred via the PVC into the vacuole where they are activated by proteolytic processing. The mobilization of proteins does not take place simultaneously in all storage tissue cells but proceeds in a layer, a few cells wide, moving through the storage tissue, leaving behind cells with LV free of storage proteins (Müntz et al., 2001; Tiedemann et al., 2001). Protein mobilization in storage tissue is under the control of the growing embryo which, in seedlings, acts as the major amino acid sink. In cereal aleurone cells, control is exhibited by the antagonistically acting phytohormones gibberellic acid and abscisic acid, and mediated mainly by transcription regulation (Bethke et al., 2006). So far no conclusive evidence has been presented that a similar hormonal control acts in seedlings of dicotyledonous plants. It cannot be excluded that there is a feedback control of protease activities mediated by concentration gradients of amino acids between embryo and storage tissue. This would allow a fine tuning of amino acid provision from the source according to the demand in the sink represented by the growing embryo. 2400 Müntz PSV contain stored proteinases that start protein mobilization within a few hours of seed imbibition (Müntz et al., 2001; Schlereth et al., 2001). During germination of dicotyledonous seeds, protein degradation begins in radicle tips, prevascular strands, and in subepidermal cell layers where growth and differentiation are initiated. Vacuoles dispose of families of proteases (Table 1) which undergo compositional changes during germination and seedling growth. At the end of germination the protein reserves of the embryonic axis are nearly exhausted and functional vascular strands have been established between axis and storage cotyledons. In the cotyledons of seedlings, the major mobilization of storage proteins is catalysed by de novo formed proteases. Storage protein mobilization proceeds in a species-specific histological pattern through the storage cotyledons. The emptying PSV merge and transform into a few or a single central LV. In Phaseolus beans it has been found that, concomitantly with changes in the luminal protein composition during this fusion process, aTIP is substituted by c-TIP in the tonoplast. In cereal grains, 7S globulins are stored mainly in PSV of the embryo and aleurone layers. The mobilization of these storage proteins is initiated by an aspartic protease, called phytepsin, stored in PSV (Bethke et al., 1996). Later during germination, phytepsin is supplemented by newly formed cysteine proteases, which were found in PSV as well as in LV, that have been called secondary lytic vacuoles by Swanson et al. (1998). It is unclear whether these LV are similar to another type of LV where the cysteine protease called aleurain was detected in aleurone cells of barley (Holwerda and Rogers, 1993). Prior to germination the stored phytepsin is inactive due to the near basic pH of PSV in mature grains. Acidification of the vacuole which is controlled by gibberellic acid (Swanson and Jones, 1996) activates phytepsin and leads to the activation of cysteine proteinases which then mediate protein breakdown. Phytepsin was shown to process the barley lectin precursor by limited proteolysis and it is highly probable that it similarly activates cysteine proteases (Fath et al., 2000). The amino acids generated in this way are used for de novo biosynthesis of hydrolases, including cysteine proteinases, which are secreted into the dead starchy endosperm in order to mobilize the proteins stored there. Vacuoles of the aleurone cells undergo developmental changes related to storage protein mobilization (Bethke et al., 1998). PSV of aleurone layer cells are transformed into LV that finally contribute to PCD (Jones, 2001; see section below entitled ‘Programmed cell death’). Despite many experimental attempts, no conclusive evidence has so far been presented that in germinating dicotyledonous seeds a pH-dependent control of protease activation contributes to the initiation of protein mobilization in a way similar to cereal grains. Mobilization of vegetative protein reserves follows similar patterns to those of generative organs, although a detailed analysis of these processes and the contributing proteolytic enzymes is still to be done. Vacuolar proteolysis in defence against herbivores and pathogens Although conclusive evidence is rare there is no doubt that vacuolar proteases are involved in the defence of plants against pathogens and herbivores (Mosolov et al., 2001; van der Hoorn and Jones, 2004). The first line of proteolytic defence against pathogens occurs in the apoplast. Herbivorous attack destroys cells mechanically (wounding) and thus confronts insects or nematodes directly with the proteases of the vacuole. Since pathogen attack frequently induces local and systemic hypersensitivity response (HR), vacuolar proteolysis forms part of the defence against pathogens. An additional major defence mechanism is the inhibition of proteases of pathogens and herbivores. Precursors of the proteases and PIs are synthesized at membrane-bound polysomes, co-translationally imported into the ER lumen, and transferred to the vacuole. Vacuolar processing proteinases, which catalyse sitespecific limited proteolysis, can transform precursors into active enzymes and enzyme inhibitors. Other vacuolar proteins involved in defence (reviewed by Shewry and Lucas, 1997), for example lectins (Peumans et al., 1999) and cyclotides (Craik et al., 2004), are also synthesized as precursors and activated by limited proteolysis. Proteinase inhibitors: Plants contain inhibitors against all classes of proteases known (Mosolov et al., 2001; Birk, 2003). Endogenous plant proteases usually remain unaffected by these PIs which mainly act on proteases of pathogens and/or animals. Herbivorous insects have several proteases in their gut against which vacuolar PIs act. This can retard the development of or even kill the attacking insect (Ryan, 1990; Koiwa et al., 1997). The constitutive and induced expression of PI genes therefore forms an important tool in the defence of plants against pathogens and herbivores. As an effective mechanism preventing PIs from affecting endogenous cellular processes, most known plant PIs are synthesized as inactive precursor polypeptides and become active only after arrival in the vacuole where they are converted into mature active inhibitors by limited proteolysis. Precursors of serine proteinase inhibitors with one and multiple inhibitor domains have been found in Lycopersicum esculentum (¼Solanum lycopersicum) (Graham et al., 1985), Nicotiana alata (Atkinson et al., 1993), Capsicum annuum (Moura and Ryan, 2001), and Nicotiana attenuata (Horn et al., 2005), respectively. In N. attenuata the multi-domain precursor of trypsin inhibitors (TIs) comprises six homologous inhibitors of ;6 kDa in tandem (Horn et al., 2005). A seventh is formed by the fusion of the precursor fragment that Vacuolar protein dynamics 2401 precedes the first inhibitor domain with the linker peptide that follows the sixth domain. These inhibitor domains are excised by a legumain (VPE) and a subtilisin-like serine proteinase. The formation of the seventh TPI includes a transpeptidation reaction similar to that which contributes to the formation of mature concanavalin A in PSV of developing Canavalia ensiformis seeds (Sheldon et al., 1996). After mechanical wounding by herbivores, methyl jasmonate acts in the signalling pathway to induce proteinase inhibitor production for defence (Bergey et al., 1996). In N. attenuata, methyl jasmonate induces an increased production of the complex of TI and alters the quantitative ratios between some of the seven components by differential effects on the expression of the two processing proteinases. Whereas an increased inhibitor production is controlled transcriptionally, the compositional change results from a post-translational control of inhibitor formation (Horn et al., 2005). Transpeptidation is also involved in the formation of some members of the TI families of sunflower and squash, where head-to-tail peptide bond formation generates macrocyclic inhibitory peptides; for example, Helianthus annuus trypsin inhibitor 1 (SFTI 1) and Momordica cochinenesis TI (MCoTI) I and II. In case of SFTI 1 Asn flanks one of the processing sites in the P1 position, indicating that legumain is involved in the excision from its precursor and its subsequent macrocyclization (Korsinczky et al., 2004; Mulvenna et al., 2005a, b). McoTI I and II have an even more complicated macrocyclic (knottin) structure than SFTI1 but are similarly generated from a precursor by limited proteolysis combined with transpeptidation which results in macrcocycle formation (Chiche et al., 2004). Cyclotides: Recently, toxins called cyclotides have been discovered in plants (Craik et al., 2004; Simonsen et al., 2005) which also have a macrocyclic ‘knottin’ structure but differ from MCoTI I and II by the absence of the TI active loop. Cyclotides affect cellular membranes and are believed to be involved in defence. The macrocyclic polypeptides (28–35 amino acids long) are generated from gene-encoded precursors containing one to three cyclotide domains separated by linker regions (Dutton et al., 2004; Mulvenna et al., 2005b). Since one processing site of the domains is flanked by Asn in the P1 position, legumaincatalysed limited proteolysis may contribute to the maturation and cyclization of cyclotide in vacuoles. Proteases: A defence-related rise in the general activity of vacuolar proteolysis, as well as that of specific proteases, has been observed frequently (reviewed by Mosolov et al., 2001; van der Horn and Jones, 2004). Unfortunately, only a few detailed studies on the function of specific proteases in response to infection and wounding have been conducted. VPE (legumains) play a key role in the mechanism of HR after infection by fungal and viral pathogens (Hatsugai et al., 2004; Rojo et al., 2004). Since PCD contributes to HR, this will be discussed in more detail in the section below entitled ‘Senescence and cell death’. In addition, VPE contribute to defence by processing proenzymes (Rojo et al., 2003) or precursors of other defence-related proteins such as TI (see above). In Lycopersicum esculentum (¼Solanum lycopersicum), wounding of leaves induces a local formation of a vacuolar serine carboxypeptidase (CP) (Moura et al., 2001). Two mature CP are generated from a CP precursor by excision of a peptide linker. This involves the cleavage of an Asnflanked peptide bond which may be catalysed by a VPE. Whereas legumains (VPE) contribute directly to storage protein degradation in germinating seeds (Müntz and Shutov, 2002; Müntz et al., 2002) it is still unknown whether they also participate in the digestion of cellular proteins as part of a defence reaction. Senescence and cell death Life ends with death. The order in structures and functions needed for life breaks down and the respective cell or organism dies. With the exception of necrosis, death is preceded by genetically programmed processes called senescence or PCD. Whereas some authors use the terms PCD and senescence synonymously (van Doorn and Woltering, 2004) others see fundamental differences between both processes (Thomas et al., 2003). Senescence can be reversed experimentally during early stages, which can sometimes happen in nature. Only after reaching a point of no return does death become an unavoidable consequence of progressing senescence, which at the cellular level corresponds to PCD (Thomas et al., 2003; van Doorn, 2005). The reversible and irreversible stages of senescence show differences in the proteolytic processes taking place in the vacuoles and the proteases involved. Reversible stage of senescence: During the reversible stage of senescence the compartmentation of the cell remains intact and vacuolar proteolysis does not seem to be an important process (Matile, 1997; Buchanan-Wollaston et al., 2003). Nevertheless, at this stage mobilization of amino acids becomes a major metabolic activity. A reduction in protein biosynthesis shifts the balance towards protein breakdown. Still, transcription and translation continue and the formation of specific proteins might increase and new senescence-related proteins, including proteases, are synthesized (Buchanan-Wollaston et al., 2003; Lin and Wu, 2004). Correlations between senescence and an increase in the transcription of genes, the activity, and/or concentration of proteases, in particular vacuolar proteases, have been reported frequently. Vacuoles containing the senescence-associated cysteine proteinase SAG12 2402 Müntz are formed specifically in leaf cells of A. thaliana and soybean (Otegui et al., 2005). In mutants lacking this enzyme the progress in senescence is not inhibited, which may be due to a functional substitution of SAG12 by other proteinases. A senescence-correlated increase in legumains (a- and c-VPE) was observed in Arabidopsis leaves (Kinoshita et al., 1999). c-VPE not only processes the precursor of a CP activating this vacuolar enzyme, it is also involved in the senescence-correlated degradation of a vacuolar invertase, presumably by activating another proteinase. Initially c-VPE, the CP precursor, and the invertase are contained in cytoplasmic vesicles, called ER bodies. The pH range in these vesicles keeps the legumain inactive. Once legumain is released into the acidic environment of the LV it becomes functional and can trigger the proteolytic activation of CP and the protease that mediates invertase degradation (Rojo et al., 2003). A similar process has been proposed to regulate the maturation of cysteine proteinase RD21 observed in vacuoles of senescing A. thaliana leaf cells (Yamada et al., 2001). Senescence-correlated increases in cysteine and other vacuolar proteinases were also observed in other plants (Chen et al., 2002; Parrott et al., 2005). During after-harvest senescence in broccoli florets, antisense inhibition of the formation of the respective protease delays the senescence process (Coupe et al., 2003; Eason et al., 2005). Generally it is still unknown on which substrates the senescence-related proteases act in vivo. Not all proteases expressed during reversible senescence have a real physiological function during this stage but rather may only accumulate in preparation of vacuolar functions in the irreversible stage (PCD). Programmed cell death: PCD starts outside the vacuole but its completion depends on vacuolar proteolysis. In plants, vacuolar breakdown processes are prominent features that all variants of PCD have in common (Jones, 2001). In order to be degraded, cytoplasmic proteins arrive in the vacuole by autophagic processes. Although micro- and macro-autophagy have not been conclusively demonstrated during PCD of plant cells (Thomas et al., 2003) there is no doubt that these processes are involved in the recycling of cytoplasmic compounds during senescence (see section above entitled ‘How do proteins get into the vacuole?’). Towards the end of PCD the vacuoles accumulate high levels of hydrolases. The exact composition of these enzyme mixtures varies according to cell type and the type of PCD. This reflects the different genetic programmes of PCD. Finally the vacuole ruptures and releases the hydrolases into the cytoplasm. This process, called mega-autophagy (van Doorn and Woltering, 2004), represents a post-mortal event that eliminates the cell corpse and recycles cell material (Jones, 2001). Whereas most cellular proteins are recycled during developmental PCD, for example in leaves of deciduous trees, only a minor part is recycled during HR-related PCD. No recycling occurs during necrotic cell death. Very specific degradation processes are often involved in developmental PCD; for example, during endosperm development in cereals grains, tracheary differentiation, sieve tube differentiation, or aerenchyma formation. The role of different plant proteinases in the network of PCD-related proteolysis is largely unknown. Among the various proteinases that have been implicated so far to participate in plant PCD (Coffeen and Wolpert, 2004; Woltering, 2004; Bozhkov et al., 2005; Rotari et al., 2005; Sanmartin et al., 2005) the only vacuolar enzymes are the legumains (VPE) (Yamada et al., 2005) with the exception of d-VPE which in A. thaliana is localized in the extracellular space (Nakaune et al., 2005). Some evidence suggests that legumains are involved in tonoplast destabilization which is a prerequisite for mega-autophagy in HR-related PCD (Hatsugai et al., 2004). In A. thaliana leaves the vacuolar c-VPE is specifically involved in HRrelated PCD (Rojo et al., 2004). Somehow conflicting with these results is that neither developmental PCDrelated nor senescence-related defects were observed in legumain knock-out mutants of Arabidopsis, including mutants in which all members of the legumain family were inactivated (Gruis et al., 2004). Developmental PCD ends with the partial or complete elimination of all cell components. Partial elimination occurs in the starchy endosperm cells of cereals during grain maturation when the membranous boundaries of PSV, PB, and other cytoplasmic components selectively disappear, while stored carbohydrates, proteins, and lipids remain unaffected (Young and Gallie, 2000). These storage compounds are mobilized during germination as described in the section above entitled ‘Mobilization of storage proteins in PSV’. During germination, the cells of the aleurone layer in the endosperm fulfil a major role in this mobilization process and also finally undergo PCD after this function has been completed (Fath et al., 2000). The aleurone cell PSVs fuse and transform into large central LVs which contain a set of at least two aspartic and three cysteine proteinases, for example a-, b- and caleurains, some of which were already present in the PSV. As long as the central vacuole is still intact it might execute lytic functions by degrading proteins of autophagically ingested organelles and cytoplasmic fragments. In the final stages of aleurone cell PCD the tonoplast is destabilized and proteases are released to eliminate the cell corpse (Bethke et al., 1998, 1999, 2001). Differences in the types of PCD during endosperm maturation and aleurone autolysis are reflected in the sets of proteases. Whereas mainly genes encoding components of the cytoplasmic ubiquitin–proteasome system and only a few vacuolar legumains (b- and c-VPE), presumably acting as processing enzymes, are expressed during partial PCD in maturing barley starchy endosperm (Sreenivasulu et al., Vacuolar protein dynamics 2403 2006), vacuolar proteases predominate during complete PCD of aleurone cells during barley germination (Fath et al., 2000). Similarly, when the pericarp tissues undergo complete PCD during grain development, genes of vacuolar proteases are mainly expressed in this tissue (Sreenivasulu et al., 2006). In contrast to the starchy endosperm of cereals, most dicotyledonous plants store proteins in tissues that remain alive during seed maturation, such as the endosperm of castor bean or cotyledons of Leguminosae. However, similarly to cereal aleurone PCD, after the storage proteins have been completely mobilized during seedling growth, the PSV in these dicotyledonous tissues fuse and are transformed into LV when the cells undergo PCD. PCDrelated proteases have been studied in more detail in castor bean. During castor bean seed maturation nucellar cells degenerate by PCD (Greenwood et al., 2005). Cells of the major storage tissue, the endosperm, remain alive but undergo PCD during seedling growth after storage proteins have been mobilized from PSV (Gietl et al., 2000; Gietl and Schmid, 2001). In both tissues, membrane-bound and ribosome-studded vesicles, called riconosomes (Fig. 3), bud from the ER during PCD (Schmid et al., 2001). The major protein component of these riconosomes is a precursor of a C-terminally KDEL-tailed cysteine proteinase (Schmid et al., 1999) which is probably kept inactive by an N-terminal pro-peptide. According to the authors, riconosomes act as ‘suicide bombs’ in the senescing endosperm cells. First the rupture of the PSV tonoplast causes an acidification of the cytoplasm which, in turn, destabilizes the riconosome boundary membrane, and finally these release their content into the cytoplasm. Autocatalytic processing in the acidic environment may then activate the cysteine proteinase which is supposed to be the major catalyst of post-mortal cell corpse degradation during castor bean endosperm cell PCD. It remains to be elucidated whether other proteases released from the collapsing PSV also contribute to cell corpse elimination. Conclusions Intact vacuoles are indispensable for protein metabolism in plant cells. Initially all vacuoles originate with similar basic functions. Their subsequent differentiation depends on changes of their protein content which varies in a tissue- and organ-specific manner and according to developmental processes and environmental effects. Isoenzymes with different dynamic characters catalyse similar reactions in vacuoles of different tissues and organs. Individual plants thus contain a wide variety of differentiated and specialized vacuoles, and even a single cell can contain two or more different types of vacuoles. Differential intra-vacuolar proteolytic processing and de- gradation may contribute to the specific protein equipment of vacuoles but, since proteins are supplied by the cytoplasm, vacuole differentiation remains under cytoplasmic control. Transcription is a major control level for differential protein supply to the vacuoles. At this level the basic integration of vacuole differentiation and functions into cellular processes is likely to take place. Nevertheless, control and regulation downstream of transcription can also contribute to the diversification of vacuolar protein furnishing. Even the timing of protease release from transfer vesicles into vacuoles and the activation of pro-proteases could constitute elements of the control system. Major tasks of future research will be: (i) to analyse the controlling gene expression network that integrates vacuole differentiation and function into the complex of other cellular processes; (ii) to investigate mechanisms that underlie differential protein transfer in the endomembrane system from the site of protein biosynthesis at the rER to the vacuole(s); and (iii) to study the substitution mechanisms for resident vacuolar proteins. Vacuoles are major sites of cellular proteolysis and contribute strongly to amino acid recycling in the cell. This specific function predominates when protein reserves are mobilized in PSV of generative and vegetative storage tissues, during senescence, and at the end of developmental and HR-related PCD when proteins of the cell corpse are degraded. When proteins are transferred from cytoplasmic compartments into vacuoles to be degraded by the resident proteases, the vacuole not only recycles the amino acids but also contributes to protein differentiation in the cytoplasm. New examples of these processes will be detected in the future and the underlying mechanisms will be further elucidated. It will also be necessary to investigate the enzymes and mechanisms of proteolysis that play a role in the substitution of resident vacuolar proteins. It is still not known which proteases degrade storage proteins in vivo, a process which is not only controlled at the level of protease formation, but which is also regulated by the interaction of tissues and by tuning the activity of individual enzymes. Differential furnishing of vacuoles with proteases has to be studied in relation to different forms of developmental PCD. Finally it has to be investigated how much and by which mechanisms vacuolar proteolysis contributes to amino acid recycling during the reversible stage of senescence and how megaautophagic protein breakdown is controlled and works as the terminal step of PCD. Acknowledgements The present paper was written when the author was a guest of IPK after retirement. He greatly appreciates the generous support given by the Council of Directors of IPK and several other colleagues in the institute. The author wants to express his special gratitude to Dr R Jung from Pioneer Hi-Bred International Inc., Johnston, IA, USA for critically reading the manuscript and suggesting many 2404 Müntz improvements. The author is especially indebted to Dr Twan Rutten for language editing and to Mrs K Lipfert and U Tiemann for carefully designing the figures. 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