Vacuolar Processing Enzyme Activity Research Articles

Genome-wide isolation and expression analysis of vacuolar processing enzyme (VPE) gene family in four apple cultivars with different storability

ABSTRACT This research was undertaken to determine expression patterns of vacuolar processing enzyme (VPE) genes in four apple (Malus domestica (Suckow) Borkh) cultivars ‘Royal Gala (RG)’, ‘Golden Delicious (GD)’, ‘Ralls Janet (RJ)’ and ‘Fuji (FJ)’ during ripening. The results showed that GD and RG had higher respiration rate, ethylene production, content of malondialdehyde (MDA), relative electrical conductivity (REC) and VPE activity than RJ and FJ during ripening. A total of 17 MdVPEs were found to be expressed in four cultivars. These MdVPEs exhibited different expression patterns during ripening of four cultivars. Generally, MdVPEβ1, MdVPEγ2, MdVPEγ4 and MdVPEδ7 might be closely associated with ripening of all cultivars. MdVPEα6 might have a role in the early period of ripening of ACS1–2/-2 (1-Aminocyclopropane-1-carboxylate synthase) homozygous cultivars (RG, RJ and FJ). Our data here would be helpful in understanding the associations between MdVPEs and storage properties of different apple cultivars.

1-Methylcyclopropene (1-MCP) treatment differentially mediated expression of vacuolar processing enzyme (VPE) genes and delayed programmed cell death (PCD) during ripening and senescence of apple fruit

• 1-MCP reduced VPE activity and delayed vacuolar-mediated PCD in apple fruit. • MdVPE s had varying expression patterns in response to 1-MCP during ripening. • MdVPE s were differentially expressed after transient over-expression of MdERF2. The main purpose of this research was to evaluate the impacts of 1-methylcyclopropene (1-MCP) treatment on vacuolar processing enzyme ( VPE ) genes and programmed cell death (PCD) during ripening and senescence of apple fruit ( Malus × domestica Borkh.). The ethylene antagonists 1-MCP (1 μL L −1 ) was applied to apples after harvest. Both treated and control fruit were stored at 20 ± 2°C. The data showed that 1-MCP treatment maintained flesh firmness, delayed climacteric peaks of respiration and ethylene, reduced VPE activity, relative electrical conductivity (REC), malondialdehyde (MDA) content and reactive oxygen species (ROS) accumulation during ripening. Transmission electron microscopy (TEM) and DNA ladder assay revealed that 1-MCP delayed vacuolar-mediated PCD in ripening apple fruit. Generally, expression of MdVPEγ1, MdVPEγ2, MdVPEδ4 and MdVPEδ7 was down-regulated by 1-MCP treatment, while expression of MdVPEα4 and MdVPEβ2 was enhanced by it during ripening compared with controls. 1-MCP treatment differentially reduced expression of MdVPEα1, MdVPEα2 and MdVPEα5 at the early ripening stage but promoted their expression at the late ripening stage, while it had the opposite effects on MdVPEβ1, MdVPEβ3 and MdVPEγ4 expression. After transient overexpression of MdERF2, MdVPEs displayed varying expression patterns, among which MdVPEα2 transcripts increased greater than the other genes. Our results indicated that MdVPEs expression and vacuolar-mediated PCD were mediated by ethylene signal during ripening and senescence of apple fruit.

Vacuolar processing enzyme translocates to the vacuole through the autophagy pathway to induce programmed cell death

ABSTRACT The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Under conditions of carbon starvation, VPE activity and PCD symptoms strongly increased in BY-2 cells, but to a much lesser extent in VPE-RNAi BY-2 cells. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole through tonoplast engulfment. Exposure of BY-2 culture to the macroautophagy/autophagy inhibitor concanamycin A led to, along with an accumulation of autophagic bodies, accumulation of StVPE1-GFP in the cell vacuole. This accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing RFP-StATG8IL, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD. Abbreviations: ATG: autophagy related; CLP: caspase-like protease; HR: hypersensitive response; PCD: programmed cell death; St: Solanum tuberosum; VPE: vacuolar processing enzyme.

MhMAPK4 from Malus hupehensis Rehd. decreases cell death in tobacco roots by controlling Cd2+ uptake

Cadmium (Cd) induces cell death in plant roots. Mitogen-activated protein kinase (MAPK) plays a role in the regulation of cell death induced by Cd in plant roots. In this study, MhMAPK4 was isolated from the roots of Malus hupehensis. Subcellular localization showed that the MhMAPK4 protein was located in the cell membrane and cytoplasm and is a transmembrane protein that is characterized by hydrophily. The expression of MhMAPK4 in the roots of M. hupehensis was up-regulated by Cd sulfate and Cd chloride. Phenotypic comparison under Cd stress showed that the growth of wild-type (WT) tobacco was lower than the transgenic lines overexpressing MhMAPK4. The fresh weight and the root length of WT also was lower than that of the transgenic tobacco. The net Cd2+ influx in the tobacco roots was decreased by the overexpression of MhMAPK4, as was root Cd accumulation. The recovery time of the Cd2+ influx to stable state in the transgenic tobacco was also shorter than the WT. The expression of iron-regulated transporter 1 (NtIRT1) and natural resistance associated macrophage protein 5 (NtNRAMP5) was relatively low in the transgenic lines under Cd stress. Cell death and apoptosis in the tobacco roots was reduced following the overexpression of MhMAPK4. The activity of vacuolar processing enzyme (VPE) and the transcript level of VPE in the transgenic tobacco was lower than that of WT under Cd stress. In addition, the electrolyte leakage and malondialdehyde and hydrogen peroxide contents in the transgenic tobacco were lower than those of WT, whereas the antioxidant enzyme activity and expression were higher. These results suggest that MhMAPK4 regulates Cd accumulation by mediating Cd2+ uptake by the roots, and controls Cd-caused cell death by adjusting VPE activity.

Measurement of the Caspase-1-Like Activity of Vacuolar Processing Enzyme in Plants.

Caspase-like activities are essential to regulate programed cell death in plants. Although no caspase orthologous enzymes with aspartic acid specificity have been identified in plants, vacuolar processing enzyme (VPE) exhibits a caspase-1-like activity. In this chapter, we introduce two methods for the measurement of the caspase-1-like/VPE activity. These methods are based on the cleavage of caspase-1 specific synthetic substrates and on monitoring the active forms of VPE using a biotinylated-inhibitor blot analysis. Both methods are also adaptable to other plant caspase-like activities.

Two proteases with caspase-3-like activity, cathepsin B and proteasome, antagonistically control ER-stress-induced programmed cell death in Arabidopsis.

Summary Programmed cell death (PCD) induced by endoplasmic reticulum (ER) stress is implicated in various plant physiological processes, yet its mechanism is still elusive. An activation of caspase‐3‐like enzymatic activity was clearly demonstrated but the role of the two known plant proteases with caspase‐3‐like activity, cathepsin B and proteasome subunit PBA1, remains to be established.Both genetic downregulation and chemical inhibition were used to investigate the function of cathepsin B and PBA1 in ER‐stress‐induced PCD (ERSID). Transcript level and activity labelling of cathepsin B were used to assess activation. To study tonoplast rupture, a plant PCD feature, both confocal and electronic microscopies were used.Cathepsin B downregulation reduced reactive oxygen species (ROS) accumulation and ERSID without affecting the induction of the unfolded protein response (UPR), but downregulation of PBA1 increased UPR and ERSID. Tonoplast rupture was not altered in the cathepsin B mutant and cathepsin B activation was independent of vacuolar processing enzyme (VPE). VPE activity was independent of cathepsin B. ERSID is regulated positively by cathepsin B and negatively by PBA1, revealing a complex picture behind caspase‐3‐like activity in plants. Cathepsin B may execute its function after tonoplast rupture and works in parallel with VPE.

Time course of programmed cell death, which included autophagic features, in hybrid tobacco cells expressing hybrid lethality

PCD with features of vacuolar cell death including autophagy-related features were detected in hybrid tobacco cells, and detailed time course of features of vacuolar cell death were established. A type of interspecific Nicotiana hybrid, Nicotiana suaveolens×N. tabacum exhibits temperature-sensitive lethality. This lethality results from programmed cell death (PCD) in hybrid seedlings, but this PCD occurs only in seedlings and suspension-cultured cells grown at 28°C, not those grown at 36°C. Plant PCD can be classified as vacuolar cell death or necrotic cell death. Induction of autophagy, vacuolar membrane collapse and actin disorganization are each known features of vacuolar cell death, but observed cases of PCD showing all these features simultaneously are rare. In this study, these features of vacuolar cell death were evident in hybrid tobacco cells expressing hybrid lethality. Ion leakage, plasma membrane disruption, increased activity of vacuolar processing enzyme, vacuolar membrane collapse, and formation of punctate F-actin foci were each evident in these cells. Transmission electron microscopy revealed that macroautophagic structures formed and tonoplasts ruptured in these cells. The number of cells that contained monodansylcadaverine (MDC)-stained structures and the abundance of nine autophagy-related gene transcripts increased just before cell death at 28°C; these features were not evident at 36°C. We assessed whether an autophagic inhibitor, wortmannin (WM), influenced lethality in hybrid cells. After the hybrid cell began to die, WM suppressed increases in ion leakage and cell deaths, and it decreased the number of cells containing MDC-stained structures. These results showed that several features indicative of autophagy and vacuolar cell death were evident in the hybrid tobacco cells subject to lethality. In addition, we documented a detailed time course of these vacuolar cell death features.

Specific capture of the hydrolysate on magnetic beads for sensitive detecting plant vacuolar processing enzyme activity

Conventional plant protease detection always suffers from high background interference caused by the complex coloring metabolites in plant cells. In this study, a bio-modified magnetic beads-based strategy was developed for sensitive and quantitative detection of plant vacuolar processing enzyme (VPE) activity. Cleavage of the peptide substrate (ESENCRK-FITC) after asparagine residue by VPE resulted in the 2-cyano-6-amino-benzothiazole (CABT)-functionalized magnetic beads capture of the severed substrate CRK-FITC via a condensation reaction between CABT and cysteine (Cys). The catalytic activity was subsequently obtained by the confocal microscopy imaging and flow cytometry quantitative analysis. The sensor system integrated advantages of (i) the high efficient enrichment and separation capabilities of magnetic beads and (ii) the catalyst-free properties of the CABT-Cys condensation reaction. It exhibited a linear relationship between the fluorescence signal and the concentration of severed substrate in the range of 10–600pM. The practical results showed that, compared with normal growth conditions, VPE activity was increased by 2.7-fold (307.2±25.3μMmin−1g−1) upon cadmium toxicity stress. This platform effectively overcame the coloring metabolites-caused background interference, showing fine applicability for the detection of VPE activity in real samples. The strategy offers great sensitivity and may be further extended to other protease activity detection.

Response of vacuolar processing enzyme in Malus hupehensis and MhVPEγ-overexpressing Arabidopsis to high temperature stress

Malus hupehensis Rehd. is often used as rootstock of apple trees. Vacuolar processing enzyme (VPE) is one type of cysteine protease with substrate specificity to residues of asparagine and aspartic acid. The VPE activity, MhVPEγ gene (GenBank number: FJ891065) expression and the amount of cell death in seedlings of M. hupehensis Rehd. all gradually increased with the increase of temperature from 26 to 42 °C. To explore the function of VPE response to temperature stress, the overexpressing vector of MhVPEγ was constructed and transformed into wild-type Arabidopsis thaliana Columbia. The responses of VPE, MhVPEγ and apoptosis to high temperature stress were determined in the Arabidopsis seedlings of wild-type, MhVPEγ-overexpressing lines and the VPE-null mutant. The leaves of MhVPEγ-overexpressing lines were smaller and VPE activity and MhVPEγ expression level were both obviously higher than the levels found in the wild-type and VPE-null mutant seedlings. Under high temperature treatment, VPE activity and MhVPEγ expression level significantly increased accompanied by leaf withering, shorter roots, lipid peroxidation and increased cell death and apoptosis rate in all types of plants. The changes of these indicators were highest in MhVPEγ-overexpressing transgenic plants, followed by the wild-type and were lowest in VPE-null mutant plants. These results suggest that MhVPEγ overexpression in Arabidopsis accelerated cell membrane lipid peroxidation, promoted cell death and reduced the tolerance to high temperature stress compared with wild-type plants.

A novel mechanism of aluminium-induced cell death involving vacuolar processing enzyme and vacuolar collapse in tobacco cell line BY-2

The role of vacuole in the cell death mechanism induced by aluminium (Al) was investigated in tobacco (Nicotiana tabacum L.) cell line BY-2. Cells at logarithmic phase of growth were treated without (control) or with Al (up to 150μM) in a treatment medium containing CaCl2, sucrose and 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH5.0). After 18h treatment, both the integrity of the plasma membrane (estimated by Evans blue uptake) and growth capacity (estimated by post-Al treatment growth in nutrient medium) were decreased, while the activity of vacuolar processing enzyme (VPE) was increased, in the Al dose-dependent manner. The activity of the vacuole (estimated by neutral red uptake) was slightly increased at 50μM then decreased with an increase in Al concentration. Direct observation of morphological changes of vacuole in a transgenic BY-2 expressing GFP-AtVam3p fusion protein localized on tonoplast indicated Al-induced collapse of vacuole. Time-course experiments indicated that both an increase in VPE activity and a loss of growth capacity were clearly observed at 6h of the treatment time, prior to the loss of plasma membrane integrity. The presence of Ac-YVAD-CHO (an inhibitor effective to VPE) during Al treatment suppressed a loss of plasma membrane integrity. The expression of VPE genes (VPE-1a, VPE-1b) were significantly enhanced by Al treatment. Taken together, we conclude that an enhancement of VPE activity by Al is controlled at transcriptional level, and is a key factor leading to a loss of integrity of the plasma membrane and a loss of growth capacity.

MAP Kinase 6‐mediated activation of vacuolar processing enzyme modulates heat shock‐induced programmed cell death in Arabidopsis

• Vacuolar processing enzyme (VPE), a cysteine protease, has been intensively studied in plant hypersensitive cell death, but the role and molecular mechanism of VPEs in response to abiotic stresses remain unclear. This work investigated the involvement of VPEs in Arabidopsis response to heat stress. • Under heat shock (HS), Arabidopsis VPE activity and the transcript level of γVPE were both upregulated, and γVPE deficiency suppressed vacuolar disruption and delayed caspase-3-like activation in HS-induced programmed cell death (PCD). Moreover, the change of VPE activity generally paralleled the alteration of caspase-1-like activity under HS treatment, indicating that HS-induced VPE activity might exhibit the caspase-1-like activity. • Further studies showed that MAP Kinase 6 (MPK6) activity was increased after HS treatment, and experiments with inhibitors and mutants suggested that MPK6 was responsible for the γVPE activation after HS treatment. In response to HS stress, reactive oxygen species (ROS) production, increase of cytoplasmic calcium concentration ([Ca(2+) ](cyt)) and the upregulation of calmodulin 3 (CaM3) transcript level occurred upstream of MPK6 activation. • Our results suggested that activation of Arabidopsis γVPE was mediated by MPK6 and played an important role in HS-induced Arabidopsis PCD, providing new insight into the mechanistic study of plant VPEs.

Vacuolar Processing Enzyme plays an Essential Role in the Crystalline Structure of Glutelin in Rice Seed

To identify the function of genes that regulate the processing of proglutelin, we performed an analysis of glup3 mutants, which accumulates excess amounts of proglutelin and lack the vacuolar processing enzyme (VPE). VPE activity in developing seeds from glup3 lines was reduced remarkably compared with the wild type. DNA sequencing of the VPE gene in glup3 mutants revealed either amino acid substitutions or the appearance of a stop codon within the coding region. Microscopic observations showed that alpha-globulin and proglutelin were distributed homogeneously within glup3 protein storage vacuoles (PSVs), and that glup3 PSVs lacked the crystalline lattice structure typical of wild-type PSVs. This suggests that the processing of proglutelin by VPE in rice is essential for proper PSV structure and compartmentalization of storage proteins. Growth retardation in glup3 seedlings was also observed, indicating that the processing of proglutelin influences early seedling development. These findings indicate that storage of glutelin in its mature form as a crystalline structure in PSVs is required for the rapid use of glutelin as a source of amino acids during early seedling development. In conclusion, VPE plays an important role in the formation of protein crystalline structures in PSVs.

Mature Amaranthus hypochondriacus seeds contain non-processed 11S precursors

Amaranth is a dicotyledonous plant whose major seed storage proteins are globulins and glutelins. An unique feature of amaranth seeds is the presence of a fraction named albumin-2, that is extractable with water only after an exhaustive extraction of globulins and albumin-1. In this work, we tested the hypothesis that albumin-2 fraction could be constituted by a non-processed 11S globulin (proglobulin). To this end, the gene encoding the amaranth 11S subunit was cloned and expressed in Escherichia coli. Subsequently, the recombinant proglobulin and albumin-2 purified from seeds were treated with a sunflower vacuolar processing enzyme (VPE). A 55 kDa component of albumin-2 was specifically cleaved into 38 and 17–15 kDa polypeptides, as a consequence of this endoproteolytic cleavage a change of the oligomeric state from trimeric to hexameric was observed. Amaranth 11S globulin fraction was not modified under these proteolysis conditions. Using VPE-specific antibodies, it was shown that amaranth expresses a 57 kDa VPE, and that both developing and mature amaranth seeds have VPE activity, although the increase of this activity during amaranth seed development is higher than that observed for sunflower seeds. These results confirm the presence of unprocessed 11S precursors in mature amaranth seeds; this phenomenon cannot, however, be attributed to low VPE activity during developing of amaranth seeds.

Storage protein accumulation in the absence of the vacuolar processing enzyme family of cysteine proteases.

The role(s) of specific proteases in seed protein processing is only vaguely understood; indeed, the overall role of processing in stable protein deposition has been the subject of more speculation than direct investigation. Seed-type members of the vacuolar processing enzyme (VPE) family were hypothesized to perform a unique function in seed protein processing, but we demonstrated previously that Asn-specific protein processing in developing Arabidopsis seeds occurs independently of this VPE activity. Here, we describe the unexpected expression of vegetative-type VPEs in developing seeds and test the role(s) of all VPEs in seed storage protein accumulation by systematically stacking knockout mutant alleles of all four members (alphaVPE, betaVPE, gammaVPE, and deltaVPE) of the VPE gene family in Arabidopsis. The complete removal of VPE function in the alphavpe betavpe gammavpe deltavpe quadruple mutant resulted in a total shift of storage protein accumulation from wild-type processed polypeptides to a finite number of prominent alternatively processed polypeptides cleaved at sites other than the conserved Asn residues targeted by VPE. Although alternatively proteolyzed legumin-type globulin polypeptides largely accumulated as intrasubunit disulfide-linked polypeptides with apparent molecular masses similar to those of VPE-processed legumin polypeptides, they showed markedly altered solubility and protein assembly characteristics. Instead of forming 11S hexamers, alternatively processed legumin polypeptides were deposited primarily as 9S complexes. However, despite the impact on seed protein processing, plants devoid of all known functional VPE genes appeared unchanged with regard to protein content in mature seeds, relative mobilization rates of protein reserves during germination, and vegetative growth. These findings indicate that VPE-mediated Asn-specific proteolytic processing, and the physiochemical property changes attributed to this specific processing step, are not required for the successful deposition and mobilization of seed storage protein in the protein storage vacuoles of Arabidopsis seeds.

Vacuolar Processing Enzymes Are Essential for Proper Processing of Seed Storage Proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.

Activation of Arabidopsis Vacuolar Processing Enzyme by Self-Catalytic Removal of an Auto-Inhibitory Domain of the C-Terminal Propeptide

Vacuolar processing enzyme (VPE) is a cysteine proteinase responsible for the maturation of various vacuolar proteins in higher plants. To clarify the mechanism of maturation and activation of VPE, we expressed the precursors of Arabidopsis gamma VPE in insect cells. The cells accumulated a glycosylated proprotein precursor (pVPE) and an unglycosylated preproprotein precursor (ppVPE) which might be unfolded. The N-terminal sequence of pVPE revealed that ppVPE had a 22-amino-acid signal peptide to be removed co-translationally. Under acidic conditions, the 56-kDa pVPE was self-catalytically converted to a 43-kDa intermediate form (iVPE) and then to the 40-kDa mature form (mVPE). N-terminal sequencing of iVPE and mVPE showed that sequential removal of the C-terminal propeptide and N-terminal propeptide produced mVPE. Both iVPE and mVPE exhibited the activity, while pVPE exhibited no activity. These results imply that the removal of the C-terminal propeptide is essential for activating the enzyme. Further removal of the N-terminal propeptide from iVPE is not required to activate the enzyme. To demonstrate that the C-terminal propeptide functions as an inhibitor of VPE, we expressed the C-terminal propeptide and produced specific antibodies against it. We found that the C-terminal propeptide reduced the activity of VPE and that this inhibitory activity was suppressed by specific antibodies against it. Our findings suggest that the C-terminal propeptide functions as an auto-inhibitory domain that masks the catalytic site. Thus, the removal of the C-terminal propeptide of pVPE might expose the catalytic site of the enzyme.

Vacuolar processing enzyme is up-regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions.

Vacuolar processing enzyme (VPE) has been shown to be responsible for maturation of various seed proteins in protein-storage vacuoles. Arabidopsis has three VPE homologues; betaVPE is specific to seeds and alphaVPE and gammaVPE are specific to vegetative organs. To investigate the activity of the vegetative VPE, we expressed the gammaVPE in a pep4 strain of the yeast Saccharomyces cerevisiae and found that gammaVPE has the ability to cleave the peptide bond at the carbonyl side of asparagine residues. An immunocytochemical analysis revealed the specific localization of the gammaVPE in the lytic vacuoles of Arabidopsis leaves that had been treated with wounding. These findings indicate that gammaVPE functions in the lytic vacuoles as the betaVPE does in the protein-storage vacuoles. The betaVPE promoter was found to direct the expression of the beta-glucuronidase reporter gene in seeds and the root tip of transgenic Arabidopsis plants. On the other hand, both the alphaVPE and gammaVPE promoters directed the expression in senescent tissues, but not in young intact tissues. The mRNA levels of both alphaVPE and gammaVPE were increased in the primary leaves during senescence in parallel with the increase of the mRNA level of a senescence-associated gene (SAG2). Treatment with wounding, ethylene and salicylic acid up-regulated the expression of alphaVPE and gammaVPE, while jasmonate slightly up-regulated the expression of gammaVPE. These gene expression patterns of the VPEs were associated with the accumulation of vacuolar proteins that are known to respond to these treatments. Taken together, the results suggest that vegetative VPE might regulate the activation of some functional proteins in the lytic vacuoles.

Vacuolar Processing Enzyme Responsible for Maturation of Seed Proteins

Summary A vacuolar processing enzyme responsible for maturation of seed proteins was isolated from the castor bean and soybean. The processing enzyme belongs to a novel cysteine proteinase with a molecular mass of 37 kDa for castor bean and 39 kDa for soybean. The enzyme splits a peptide bond on the C-terminal side of an exposed asparagine residue of the proprotein precursors to produce their mature seed proteins such as 11S globulin and 2S albumin. Immunocytochemical localization of the enzyme in the vacuolar matrix of maturing castor bean endosperm indicates that the maturation of the seed proteins occurs in the vacuoles. Molecular characterization revealed that the enzyme is synthesized as an inactive precursor with a larger molecular mass. The results of immunoelectron microscopic analysis suggested that the precursor is transported to vacuoles via dense vesicles together with proproteins of seed proteins. After arriving at the vacuoles, the inactive precursor is converted into an active enzyme. This suggests that a cascade for proprotein processing is involved in the maturation of seed proteins. Vacuolar processing enzyme activity was found in various plant tissues and several cDNA homologues of the enzyme were isolated from different plants. Thus a similar processing enzyme is widely distributed in plant tissues and plays a crucial role in the maturation of a variety of proteins in plant vacuoles.