Caspases are cysteine-dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase-specific proteolytic activity. Nevertheless, plants do display caspase-like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase-like proteases. Here, we report the identification and characterisation of a novel PCD-related subtilisinlike protease from tobacco and rice named phytaspase (plant aspartate-specific protease) that possesses caspase specificity distinct from that of other known caspase-like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD-related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re-imported into the cell during PCD providing insights into how phytaspase operates.

Activities displaying caspase cleavage specificity have been well documented in various plant programmed cell death (PCD) models. However, plant genome analyses have not revealed clear orthologues of caspase genes, indicating that enzyme(s) structurally unrelated yet possessing caspase specificity have functions in plant PCD. Here, we review recent data showing that some caspase-like activities are attributable to the plant subtilisin-like proteases, saspases and phytaspases. These proteases hydrolyze a range of tetrapeptide caspase substrates following the aspartate residue. Data obtained with saspases implicate them in the proteolytic degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) during biotic and abiotic PCD, whereas phytaspase overproducing and silenced transgenics provide evidence that phytaspase regulates PCD during both abiotic (oxidative and osmotic stresses) and biotic (virus infection) insults. Like caspases, phytaspases and saspases are synthesized as proenzymes, which are autocatalytically processed to generate a mature enzyme. However, unlike caspases, phytaspases and saspases appear to be constitutively processed and secreted from healthy plant cells into the intercellular space. Apoplastic localization presumably prevents enzyme-mediated protein fragmentation in the absence of PCD. In response to death-inducing stimuli, phytaspase has been shown to re-localize to the cell interior. Thus, plant PCD-related proteases display both common (D-specific protein fragmentation during PCD) and distinct (enzyme structure and activity regulation) features with animal PCD-related proteases. Plants, like animals, employ programmed cell death (PCD) for a variety of purposes including development (e.g., xylem formation, seed germination and senescence), stress responses (e.g., salt, cold, heat) and defence (e.g., hypersensitive response (HR)). Although it is becoming increasingly evident that multiple forms of PCD exist in both plants and animals, 1,2 certain forms of PCD in both kingdoms share a number of hallmarks, such as DNA fragmentation, cytochrome c release from mitochondria, cell shrinkage, and so on.3 However, despite extensive similarities, the equivalence of the molecular mechanisms regulating plant PCD is less clearly understood. Among the more surprising findings is an absence of caspase orthologues in plants. Because caspases, a family of cysteine-dependent aspartate-specific proteases, are known to have important functions in the initiation and execution of PCD in animals through cleavage of a number of protein targets, their apparent absence in plants poses a dilemma. Either the principle of specific protein fragmentation at aspartate residues does not operate during PCD in plants or the function of caspases is assumed by other protease(s) that are structurally unrelated to caspases.Evidence accumulated during the past decade indicates that, in at least some cases, the second scenario is correct and that caspase-like (in a functional rather than a structural sense) prote...

Peptide hormones are implicated in many important aspects of plant life and are usually synthesized as precursor proteins. In contrast to animals, data for plant peptide hormone maturation are scarce and the specificity of processing enzyme(s) is largely unknown. Here we tested a hypothesis that processing of prosystemin, a precursor of tomato (Solanum lycopersicum) wound hormone systemin, is performed by phytaspases, aspartate-specific proteases of the subtilase family. Following the purification of phytaspase from tomato leaves, two tomato phytaspase genes were identified, the cDNAs were cloned and the recombinant enzymes were obtained after transient expression in Nicotiana benthamiana. The newly identified tomato phytaspases hydrolyzed prosystemin at two aspartate residues flanking the systemin sequence. Site-directed mutagenesis of the phytaspase cleavage sites in prosystemin abrogated not only the phytaspase-mediated processing of the prohormone in vitro, but also the ability of prosystemin to trigger the systemic wound response in vivo. The data show that the prohormone prosystemin requires processing for signal biogenesis and biological activity. The identification of phytaspases as the proteases involved in prosystemin maturation provides insight into the mechanisms of wound signaling in tomato. Our data also suggest a novel role for cell death-related proteases in mediating defense signaling in plants.

The majority of known nuclear proteins are highly mobile. The molecular mechanisms by which they accumulate inside stable compartments that are not separated from the nucleoplasm by membranes are obscure. The compartmental retention of some proteins is associated with their biological function; however, some protein interactions within distinct nuclear structures may be non-specific. The non-specific retention may lead to the accumulation of proteins in distinct structural domains, even if the protein does not function inside this domain. In this study, we have shown that histone H2B-EGFP initially accumulated in the nucleolus after ectopic expression, and then gradually incorporated into the chromatin to leave only a small amount of nucleolus-bound histone that was revealed by removing chromatin-bound proteins with DNase I treatment. Nucleolar histone H2B had several characteristics: (i) it preferentially bound to granular component of the nucleolus and interacted with RNA or RNA-containing nucleolar components; (ii) it freely exchanged between the nucleolus and nucleoplasm; (iii) it associated with the nuclear matrix; and (iv) it bound to interphase prenuclear bodies that formed after hypotonic treatment. The region in histone H2B that acts as a nucleolar localization/retention signal (NoRS) was identified. This signal overlapped with a nuclear localization signal (NLS), which appears to be the primary function of this region. The NoRS activity of this region was non-specific, but the molecular mechanism was probably similar to the NoRSs of other nucleolar proteins. All known NoRSs are enriched with basic amino acids, and we demonstrated that positively charged motifs (nona-arginine (R9) and nona-lysine (K9)) were sufficient for the nucleolar accumulation of EGFP. Also, the correlation between measured NoRS activity and the predicted charge was observed. Thus, NoRSs appear to achieve their function through electrostatic interactions with the negatively charged components of the nucleolus. Though these interactions are non-specific, the functionally unrelated retention of a protein can increase the probability of its interaction with specific and functionally related binding sites.

Phytaspases are Asp-specific subtilisin-like plant proteases that have been likened to animal caspases with respect to their regulatory function in programmed cell death (PCD). We identified twelve putative phytaspase genes in tomato that differed widely in expression level and tissue-specific expression patterns. Most phytaspase genes are tandemly arranged on tomato chromosomes one, four, and eight, and many belong to taxon-specific clades, e.g. the P69 clade in the nightshade family, suggesting that these genes evolved by gene duplication after speciation. Five tomato phytaspases (SlPhyts) were expressed in N. benthamiana and purified to homogeneity. Substrate specificity was analyzed in a proteomics assay and with a panel of fluorogenic peptide substrates. Similar to animal caspases, SlPhyts recognized an extended sequence motif including Asp at the cleavage site. Clear differences in cleavage site preference were observed implying different substrates in vivo and, consequently, different physiological functions. A caspase-like function in PCD was confirmed for five of the seven tested phytaspases. Cell death was triggered by ectopic expression of SlPhyts 2, 3, 4, 5, 6 in tomato leaves by agro-infiltration, as well as in stably transformed transgenic tomato plants. SlPhyts 3, 4, and 5 were found to contribute to cell death under oxidative stress conditions.

Proteases with an aspartate cleavage specificity are known to contribute to programmed cell death (PCD) in animals and plants. In animal cells this proteolytic activity belongs to caspases, a well-characterized family of cysteine-dependent death proteases. Plants, however, lack caspase homologs and thus the origin of this type of proteolytic activity in planta was poorly understood. Here, we review recent data demonstrating that a plant serine-dependent protease, phytaspase, shares cleavage specificity and a role in PCD analogous to that of caspases. However, unlike caspases, regulation of phytaspase-mediated cleavage of intracellular target proteins appears to be attained not at the level of proenzyme processing/activation, which occurs, in the case of phytaspase, autocatalytically and constitutively. Rather, the mature phytaspase is excluded from healthy cells into the apoplast and is allowed to re-enter cells upon the induction of PCD. Thus, PCD-related proteases in animals and plants display both common features and important distinctions.

The modern concepts of programmed cell death (PCD) in plants are reviewed as compared to PCD (apoptosis) in animals. Special attention is focused on considering the potential mechanisms of implementation of this fundamental biological process and its participants. In particular, the proteolytic enzymes involved in PCD in animals (caspases) and plants (phytaspases) are compared. Emphasis is put on elucidation of both common features and substantial differences of PCD implementation in plants and animals.

BackgroundCutting edge research of human microbiome diversity has led to the development of the microbiome-gut-brain axis concept, based on the idea that gut microbes may have an impact on the behavior of their human hosts. Many examples of behavior-altering parasites are known to affect members of the animal kingdom. Some prominent examples include Ophiocordyceps unilateralis (fungi), Toxoplasma gondii (protista), Wolbachia (bacteria), Glyptapanteles sp. (arthropoda), Spinochordodes tellinii (nematomorpha) and Dicrocoelium dendriticum (flat worm). These organisms belong to a very diverse set of taxonomic groups suggesting that the phenomena of parasitic host control might be more common in nature than currently established and possibly overlooked in humans.Presentation of the hypothesisSome microorganisms would gain an evolutionary advantage by encouraging human hosts to perform certain rituals that favor microbial transmission. We hypothesize that certain aspects of religious behavior observed in the human society could be influenced by microbial host control and that the transmission of some religious rituals could be regarded as the simultaneous transmission of both ideas (memes) and parasitic organisms.Testing the hypothesisWe predict that next-generation microbiome sequencing of samples obtained from gut or brain tissues of control subjects and subjects with a history of voluntary active participation in certain religious rituals that promote microbial transmission will lead to the discovery of microbes, whose presence has a consistent and positive association with religious behavior. Our hypothesis also predicts a decline of participation in religious rituals in societies with improved sanitation.Implications of the hypothesisIf proven true, our hypothesis may provide insights on the origin and pervasiveness of certain religious practices and provide an alternative explanation for recently published positive associations between parasite-stress and religiosity. The discovery of novel microorganisms that affect host behavior may improve our understanding of neurobiology and neurochemistry, while the diversity of such organisms may be of interest to evolutionary biologists and religious scholars.ReviewersThis article was reviewed by Prof. Dan Graur, Dr. Rob Knight and Dr. Eugene Koonin