VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation

Plant embryogenesis is intimately associated with programmed cell death. The mechanisms of initiation and control of programmed cell death during plant embryo development are not known. Proteolytic activity associated with caspase-like proteins is paramount for control of programmed cell death in animals and yeasts. Caspase family of proteases has unique strong preference for cleavage of the target proteins next to asparagine residue. In this work, we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity in the regulation of programmed cell death during plant embryogenesis. We demonstrate that VEIDase is a principal caspase-like activity implicated in plant embryogenesis. This activity increases at the early stages of embryo development that coincide with massive cell death during shape remodeling. The VEIDase activity exhibits high sensitivity to pH, ionic strength and Zn(2+) concentration. Altogether, biochemical assays show that VEIDase plant caspase-like activity resembles that of both mammalian caspase-6 and yeast metacaspase, YCA1. In vivo, VEIDase activity is localised specifically in the embryonic cells during both the commitment and in the beginning of the execution phase of programmed cell death. Inhibition of VEIDase prevents normal embryo development via blocking the embryo-suspensor differentiation. Our data indicate that the VEIDase activity is an integral part in the control of plant developmental cell death programme, and that this activity is essential for the embryo pattern formation.     

Possible involvement of the cytoskeleton in the regulation of barley caryopsis dormancy and germination

This study was conducted on barley cv. Ars. caryopses collected at full ripeness and divided into two batches. From one batch (dormant caryopses) polysomes were isolated from embryos immediately after harvesting and after two days of germination. From the other batch (non-dormant caryopses) the same was done after eight months storage in a dry state. A low ionic strength cytoskeleton-stabilizing buffer was used for the isolation of polysomes. Four different fractions of polysomes were examined: free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP). In germs grown from non-dormant caryopses, the first two fractions (FP + MBP) made up about 78 % of the total ribosomal material, whereas in embryos of dormant, imbibed caryopses, two last fractions (CBP + CMBP) made up about 71 %. The percentage of polysomes after 48 hours of imbibition of dormant caryopses in the FP, MBP and CBP was only about 13 % (i.e., 87 % monosomes), whereas a greater proportion (19.4 %) was found in the CMBP. The highest incorporation of ³H-uridine and ¹⁴C-amino acids (after 48 hours of germination and 0.5, 3 and 6 hrs incubation with precursors) took place in trhc CMBP both in dormant and non-dormant caryopses The major amount of the two polysome fractions associated with the cytoskeleton (CBP and CMBP) and the higher activity of CMBP in protein synthesis in embryos of dormant, imbibed triticale caryopses may indicate a significant role for polysomes associated with the cytoskeleton in the control of protein synthesis in dormant and germinating caryopses.     

Characterisation and functional analysis of two barley caleosins expressed during barley caryopsis development

Two full-length cDNA sequences homologous to caleosin, a seed-storage oil-body protein from sesame, were identified from a series of barley grain development cDNA libraries and further characterised. The cDNAs, subsequently termed HvClo1 and HvClo2, encode proteins of 34 kDa and 28 kDa, respectively. Real-time RT-PCR indicated that HvClo1 is expressed abundantly during the later stages of embryogenesis and is seed-specific, accumulating in the scutellum of mature embryos. HvClo2 is expressed mainly in the endosperm tissues of the developing grain. We show that HvClo1 and HvClo2 are paralogs that co-segregate on barley chromosome 2HL. Transient expression of HvClo1 in lipid storage and non-storage cells of barley using biolistic particle bombardment indicates that caleosins have different subcellular locations from the structural oil-body protein oleosin, and by inference participate in different sorting pathways. We observe that caleosin sorts via small vesicles, suggesting a likely association with lipid trafficking, membrane expansion and oil-body biogenesis.     

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.     

Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae.

An alpha-glucosidase activity (SAG) occurs in a/alpha Saccharomyces cerevisiae cells beginning at about 8 to 10 h after the initiation of sporulation. This enzyme is responsible for the rapid degradation of intracellular glycogen which follows the completion of meiosis in these cells. SAG differs from similar activities present in vegetative cells and appears to be a sporulation-specific enzyme. Cells arrested at various stages in sporulation (DNA replication, recombination, meiosis I, and meiosis II) were examined for SAG activity; the results show that SAG appearance depends on DNA synthesis and some recombination events but not on the meiotic divisions.     

KIPase activity is a novel caspase-like activity associated with cell proliferation.

A novel caspase-like activity, which is directly regulated with cell proliferation is a candidate to regulate the abundance of the cyclin-dependent kinase inhibitor, p27(KIP1), in human lymphoid cells. This activity, which we term KIPase activity, can also cleave a subset of caspase substrates. Here we demonstrate that KIPase is a novel enzyme distinct from any of the previously characterized human caspases. We show that KIPase is active in a variety of cell lineages, its activity is associated with the proliferation of the human T-cell line, Jurkat, and is not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. Gel filtration analysis revealed that KIPase has a native molecular mass of approximately 100-200 kDa. Furthermore, the activity of KIPase does not change during apoptosis induced by either ligation of FAS or exposure of cells to etoposide. The uniqueness of KIPase is demonstrated by the fact that none of the human caspases tested (1-10) are able to cleave a specific KIPase substrate (Ac-DPSD-AMC) and that an aldehyde modified derivative of the DPSD tetra peptide is unable to inhibit caspases, but is a good inhibitor of KIPase activity. This supports a hypothesis whereby KIPase is a currently unidentified caspase-like enzyme which regulates the abundance of p27(KIP1) in a proliferation-dependent manner.     

Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae

A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.     

Developmental regulation of phenylalanine hydroxylase activity in Drosophila melanogaster

Herein we demonstrate that Drosophila larvae possess a synthetic activity capable of converting phenylalanine to tyrosine. This system is readily extractable and displays many characteristics of phenylalanine hydroxylase systems described in other organisms, the most notable being that a tetrahydropteridine is required for full expression of activity. The level of phenylalanine hydroxylase activity present in the organism varies with the stage of development: from an undetected level of activity at the first larval instar, there is a rapid increase in phenylalanine hydroxylase activity which reaches a peak at the time of puparium formation, after which there is a rapid decrease again to an undetected level.     

Post-translational regulation and proteolytic activity of the metalloproteinase ADAMTS8

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs (ADAMTS)8 is a secreted protease, which was recently implicated in pathogenesis of pulmonary arterial hypertension (PAH). However, the substrate repertoire of ADAMTS8 and regulation of its activity are incompletely understood. Although considered a proteoglycanase because of high sequence similarity and close phylogenetic relationship to the proteoglycan-degrading proteases ADAMTS1, 4, 5, and 15, as well as tight genetic linkage with ADAMTS15 on human chromosome 11, its aggrecanase activity was reportedly weak. Several post-translational factors are known to regulate ADAMTS proteases such as autolysis, inhibition by endogenous inhibitors, and receptor-mediated endocytosis, but their impacts on ADAMTS8 are unknown. Here, we show that ADAMTS8 undergoes autolysis at six different sites within its spacer domain. We also found that in contrast to ADAMTS4 and 5, ADAMTS8 levels were not regulated through low-density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytosis. Additionally, ADAMTS8 lacked significant activity against the proteoglycans aggrecan, versican, and biglycan. Instead, we found that ADAMTS8 cleaved osteopontin, a phosphoprotein whose expression is upregulated in PAH. Multiple ADAMTS8 cleavage sites were identified using liquid chromatography–tandem mass spectrometry. Osteopontin cleavage by ADAMTS8 was efficiently inhibited by TIMP-3, an endogenous inhibitor of ADAMTS1, 4, and 5, as well as by TIMP-2, which has no previously reported inhibitory activity against other ADAMTS proteases. These differences in post-translational regulation and substrate repertoire differentiate ADAMTS8 from other family members and may help to elucidate its role in PAH.     

VPEgamma exhibits a caspase-like activity that contributes to defense against pathogens

BACKGROUND: Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants. RESULTS: We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens. CONCLUSIONS: We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.     

Developmental regulation of proteolytic activities and subunit pattern of 20 S proteasome in chick embryonic muscle

The proteolytic activities of the 20 S proteasome were found to change in their levels during the development of chick embryonic muscle. The peptide-cleaving activities against N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-beta-naphthylamide gradually decreased with the time of development. On the other hand, the casein-degrading activity in the presence of poly-L-lysine markedly increased from embryonic day 11 and reached a maximal level by day 17. These changes appeared to be tissue-specific because little or no change in any of the proteolytic activities was observed with developing embryonic brain, while dramatic alterations occurred in the extents of the peptide hydrolyses in liver. Furthermore, a number, but not all, of the proteasome subunits in embryonic muscle were changed in their amounts during the development. These results suggest that the alterations in the proteasome activities and subunit pattern are developmentally regulated and may be correlated.     

Developmental regulation of locomotive activity in Xenopus primordial germ cells

Primordial germ cells (PGCs) arise in the early embryo and migrate toward the future gonad through species‐specific pathways. They are assumed to change their migration properties dependent on their own genetic program and/or environmental cues, though information concerning the developmental change in PGC motility is limited. First, we re‐examined the distribution of PGCs in the endodermal region of Xenopus embryos at various stages by using an antibody against Xenopus Daz‐like protein, and found four stages of migration, namely clustering, dispersing, directionally migrating and re‐aggregating. Next, we isolated living PGCs at each stage and directly examined their morphology and locomotive activity in cell cultures. PGCs at the clustering stage were round in shape with small blebs and showed little motility. PGCs in both the dispersing and the directionally migrating stages alternated between the locomotive phase with an elongated morphology and the pausing phase with a rugged morphology. The locomotive activity of the elongated PGCs was accompanied by the persistent formation of a large bleb at the leading front. The duration of the locomotive phase was shortened gradually with the transition from the dispersing stage to the directionally migrating stage. At the re‐aggregating stage, PGCs became round in shape and showed no motility. Thus, we directly showed that the locomotive activity of PGCs changes dynamically depending upon the migrating stage. We also showed that the locomotion and blebbing of the PGCs required F‐actin, myosin II activity and RhoA/Rho‐associated protein kinase (ROCK) signaling.     

Occurrence of proteolytic inhibitors in various tissues of barley

Summary The three groups of proteolytic inhibitors present in resting barley grains, namely, trypsin inhibitors, Aspergillus-proteinase inhibitors, and inhibitors of endogenous proteinases, occur in both the embryo and the two endosperm tissues. There are pronounced quantitative differences, however. The three inhibitor activities in the embryo are, respectively, 6-, 0.1-, and 6-fold of those in the endosperm.During germination at 20° all inhibitor activities disappear from the endosperms in 4–5 days. Young rootlets and coleoptiles contain inhibitors of trypsin and Aspergillus proteinase, but these disappear after 4–5 days' germination. However, the trypsin inhibitor content per seedlings remains roughly constant through the whole period. The Aspergillus-proteinase inhibitors, in contrast, exhibit a pronounced increase of activity per seedling.No inhibitor activities were detected in leaves and roots at later stages of growth.The trypsin inhibitor which we have earlier purified from resting grains occurs exclusively in the two endospermal tissues and is immunologically entirely different from the trypsin inhibitors present in embryos and young seedlings.     

Proteasome-associated proteins: regulation of a proteolytic machine

The proteasome is a compartmentalized, ATP-dependent protease composed of more than 30 subunits that recognizes and degrades polyubiquitinated substrates. Despite its physiological importance, many aspects of the proteasome's structural organization and regulation remain poorly understood. In addition to the proteins that form the proteasome holocomplex, there is increasing evidence that proteasomal function is affected by a wide variety of associating proteins. A group of ubiquitin-binding proteins assist in delivery of substrates to the proteasome, whereas proteasome-associated ubiquitin ligases and deubiquitinating enzymes may alter the dynamics of ubiquitin chains already associated with the proteasome. Some proteins appear to influence the overall stability of the complex, and still others have the capacity to activate or inhibit the hydrolytic activity of the core particle. The increasing number of interacting proteins identified suggests that proteasomes, as they exist in the cell, are larger and more diverse in composition than previously assumed. Thus, the study of proteasome-associated proteins will lead to new perspectives on the dynamics of this uniquely complex proteolytic machine.     

Genetic basis of barley caryopsis dormancy and seedling desiccation tolerance at the germination stage

The genomic regions controlling caryopsis dormancy and seedling desiccation tolerance were identified using 152 F₄ lines derived from a cross between Mona, a Swedish cultivar, and an Israeli xeric wild barley Hordeum spontaneum genotype collected at Wadi Qilt, Israel. Dormancy, the inability of a viable seed to germinate, and desiccation tolerance, the ability of the desiccated seedlings to revive after rehydration, were characterized by fitting the germination and revival data with growth curves, using three parameters: minimum, maximum, and slope of germination or revival rate derived by the least square method. The genetic map was constructed with 85 genetic markers (SSRs, AFLPs, STSs, and Dhn genes) using the multipoint-mapping algorithm. Quantitative trait loci (QTLs) mapping was conducted with the multiqtl package. Ten genomic regions were detected that affected the target traits, seven of which affected both dormancy and desiccation tolerance traits. Both the wild barley genotype and the Swedish cultivar contributed the favorite alleles for caryopsis dormancy, whereas seedling desiccation tolerance was attributed to alleles descending from the cultivar. The results indicate that some barley dormancy genes are lost during domestication and that dormancy QTLs are associated with abiotic stress tolerance.     

The inhibition of a chromatin-bound proteolytic activity

A chromatin-bound proteolytic activity found in rabbit thymus nuclear lysates has been studied. Incubation of washed chromatin fibers led to degradation of histone H1 resulting in products which ran on sodium dodecyl sulfate-gels slightly ahead of the three native H1 fractions. The activity has also been demonstrated in a chromatin preparation purified by chromatography on Sephadex G-200. Of the five histones, H1 appeared to be the most readily degraded although with extended incubation times the core histones were also affected. The proteolytic activity was not inhibited by Na₂SO₃, PMSF, STI, pepstatin, or EDTA. Effective inhibitors included leupeptin, tosyllysine chloromethyl ketone, and tosylaminophenylethylchloromethyl ketone.     

Incorporation of 3H-uridine and 3H-leucine during early embryogenesis of rice and barley in caryopsis culture

Incorporation of ³H-uridine and ³H-leucine during early embryogenesisin rice and barley was examined by culturing caryopses on White'smedium containing these isotopes. Grain counts showed that nodetectable amount of ³H-uridine was incorporated during theearly and middle periods of the globular embyo in both plants.³H-Uridine was detected in an embryo consisting of more than100 cells. ³H-Leucine, however, was incorporated from the beginningof embryogenesis. These results suggest that during early embryonicdevelopment in these plants, as in animal embryogenesis, RNAsynthesis is limited and the proteins will be synthesized byusing RNAs stored in the egg cell. The 100-cell stage is considered a critical point in early embryonicdevelopment, because at this stage cereal embryos start exponentialand dual rhythmic growth as well as uridine incorporation. (Received December 21, 1978; )     

Ethylene effects on amylase activity from isolated barley aleurone layers : possible modification by proteolytic enzymes

The effect of protease inhibitors on the response of gibberellic acid-treated barley aleurone layers to ethylene was examined. In the absence of protease inhibitors, ethylene plus gibberellic acid initially increased the production of amylase activity relative to layers incubated with gibberellic acid alone. Exposure to ethylene plus gibberellic acid for 48 hours or longer, however, led to depressed levels of amylase activity compared to samples incubated with gibberellic acid in hydrocarbon-free air.

The direct assay of proteolytic activity revealed a small increase in activity in response to ethylene. The significance of this response was probed further by including inhibitors of barley proteases in the incubation medium. When potassium bromate was introduced, ethylene did not cause any alteration in amylase activity compared to samples incubated in hydrocarbon-free air. However, in the presence of N-ethylmaleimide, ethylene treatment induced a 52% increase in amylase activity recovered from samples after 48 hours. These results suggest that proteases contribute to the loss of amylase activity in response to ethylene and thus alter the apparent effect of ethylene on amylase synthesis. The effect of protease inhibitors on other hydrolases is also discussed.

During the incubation period, the pH of the medium declined significantly. However, ethylene had no effect on the extent of this decline.