Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity.

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 subtilisin-like 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.

Suspension Cell Culture of Polyscias fruticosa (L.) Harms in Bubble-Type Bioreactors-Growth Characteristics, Triterpene Glycosides Accumulation and Biological Activity.

Polyscias fruticosa (L.) Harms, or Ming aralia, is a medicinal plant of the Araliaceae family, which is highly valued for its antitoxic, anti-inflammatory, analgesic, antibacterial, anti-asthmatic, adaptogenic, and other properties. The plant can be potentially used to treat diabetes and its complications, ischemic brain damage, and Parkinson's disease. Triterpene glycosides of the oleanane type, such as 3-O-[ß-D-glucopyranosyl-(1→4)-ß-D-glucuronopyranosyl] oleanolic acid 28-O-ß-D-glucopyranosyl ester (PFS), ladyginoside A, and polysciosides A-H, are mainly responsible for biological activities of this species. In this study, cultivation of the cell suspension of P. fruticosa in 20 L bubble-type bioreactors was attempted as a sustainable method for cell biomass production of this valuable species and an alternative to overexploitation of wild plant resources. Cell suspension cultivated in bioreactors under a semi-continuous regime demonstrated satisfactory growth with a specific growth rate of 0.11 day-1, productivity of 0.32 g (L · day)-1, and an economic coefficient of 0.16 but slightly lower maximum biomass accumulation (~6.8 g L-1) compared to flask culture (~8.2 g L-1). Triterpene glycosides PFS (0.91 mg gDW-1) and ladyginoside A (0.77 mg gDW-1) were detected in bioreactor-produced cell biomass in higher concentrations compared to cells grown in flasks (0.50 and 0.22 mg gDW-1, respectively). In antibacterial tests, the minimum inhibitory concentrations (MICs) of cell biomass extracts against the most common pathogens Staphylococcus aureus, methicillin-resistant strain MRSA, Pseudomonas aeruginosa, and Escherichia coli varied within 250-2000 µg mL-1 which was higher compared to extracts of greenhouse plant leaves (MIC = 4000 µg mL-1). Cell biomass extracts also exhibited antioxidant activity, as confirmed by DPPH and TEAC assays. Our results suggest that bioreactor cultivation of P. fruticosa suspension cell culture may be a perspective method for the sustainable biomass production of this species.

Intracellular targeting of a hordeiviral membrane-spanning movement protein: sequence requirements and involvement of an unconventional mechanism.

The membrane-spanning protein TGBp3 is one of the three movement proteins (MPs) of Poa semilatent virus. TGBp3 is thought to direct other viral MPs and genomic RNA to peripheral bodies located in close proximity to plasmodesmata. We used the ectopic expression of green fluorescent protein-fused TGBp3 in epidermal cells of Nicotiana benthamiana leaves to study the TGBp3 intracellular trafficking pathway. Treatment with inhibitors was used to reveal that the targeting of TGBp3 to plasmodesmata does not require a functional cytoskeleton or secretory system. In addition, the suppression of endoplasmic reticulum-derived vesicle formation by a dominant negative mutant of small GTPase Sar1 had no detectable effect on TGBp3 trafficking to peripheral bodies. Collectively, these results suggested the involvement of an unconventional pathway in the intracellular transport of TGBp3. The determinants of targeting to plasmodesmata were localized to the C-terminal region of TGBp3, including the conserved hydrophilic and terminal membrane-spanning domains.

Evidence of Strict Stereospecificity in the Structure of sn-1,2-Diacyl-3-Acetyl-Glycerols from Euonymus maximowiczianus Seeds Using Nuclear Magnetic Resonance Spectroscopy.

Asymmetric, optically active sn-1,2-diacyl-3-acetyl-glycerols (AcDAG) have been known to scientists for several decades. However, to date, the problem of their structure has not been definitely resolved, which has led to a vast diversity of terms used for their designation in the literature. Using two-dimensional nuclear magnetic resonance, we have investigated AcDAG from the mature seeds of Euonymus maximowiczianus, from which we have been able to both identify a correlation of the methyl group in acetic acid residue with protons at the carbon atom at sn-3 position in the glycerol residue of the AcDAG molecule and, for the first time, demonstrate that this correlation is observed exclusively with one carbon atom at the α-position, but not with two as would have been expected in case of a racemic mixture. Moreover, results of our analysis of AcDAG isolated from the seeds of E. maximowiczianus directly confirm that diacylglycerol-3-acetyl-transferase is responsible for their biosynthesis, which reveals a strict specificity not only to acetyl-CoA as one of the substrates but also to the sn-3-position of the glycerol residue in sn-1,2-diacylglycerol during their biosynthesis.

Formation of protein complexes containing plant virus movement protein TGBp3 is necessary for its intracellular trafficking.

Cell-to-cell movement of Poa semilatent virus (genus Hordeivirus) in infected plants is mediated by three viral 'triple gene block' (TGB) proteins. One of those termed TGBp3 is an integral membrane protein essential for intracellular transport of other TGB proteins and viral genomic RNA to plasmodesmata. TGBp3 targeting to plasmodesmata-associated sites is believed to involve an unconventional mechanism which does not employ endoplasmic reticulum-derived transport vesicles. Previously TGBp3 has been shown to contain a composite transport signal consisting of the central hydrophilic protein region which includes a conserved pentapeptide YQDLN and the C-terminal transmembrane segment. This study demonstrates that these TGBp3 structural elements have distinct functions in protein transport. The YQDLN-containing region is essential for TGBp3 incorporation into high-molecular-mass protein complexes. In transient expression assay formation of such complexes is necessary for entering the TGBp3-specific pathway of intracellular transport and protein delivery to plasmodesmata-associated sites. In virus-infected plants TGBp3 is also found predominantly in the form of high-molecular-mass complexes. When the complex-formation function of YQDLN-containing region is disabled by a mutation, targeting to plasmodesmata-associated sites can be complemented by a heterologous peptide capable of formation multimeric complexes. The C-terminal transmembrane segment is found to be an essential signal of TGBp3 intracellular transport to peripheral sites.