ABSTRACT
To investigate the mechanism of phytohemagglutinin (PHA) susceptibility or resistance to the action of proteolytic enzymes, its in vitro proteolysis by trypsin was studied. It was found that Ca (2+) gives resistance to the native PHA molecule to trypsin proteolysis. In the absence of Ca (2+) trypsin performs a thorough hydrolysis of PHA. At the first stage of trypsin hydrolysis of PHA the formation of a relatively stable high molecular mass product occurs (PHA-T) as a result of non-co-operative proteolysis. At the second stage, the degradation of PHA-T occurs, and this degradation is performed by parallel co-operative proteolysis. This type of proteolysis differs from the action of trypsin on phaseolin, the main storage protein from common bean ( Phaseolus vulgaris L.). The implications of Ca (2+)influence of PHA hydrolysis by trypsin are discussed.
Subject(s)
Calcium/pharmacology , Phytohemagglutinins/metabolism , Trypsin/metabolism , Calcium Chloride/pharmacology , Cations, Divalent , Edetic Acid/pharmacology , Hydrolysis , Plant Proteins/metabolismABSTRACT
Programmed cell death (PCD) is a genetically controlled cell death that is regulated during development and activated in response to environmental stresses or pathogen infection. The degree of conservation of PCD across kingdoms and phylum is not yet clear; however, whereas caspases are proteases that act as key components of animal apoptosis, plants have no orthologous caspase sequences in their genomes. The discovery of plant and fungi metacaspases as proteases most closely related to animal caspases led to the hypothesis that metacaspases are the functional homologues of animal caspases in these organisms. Arabidopsis thaliana has nine metacaspase genes, and so far it is unknown which members of the family if any are involved in the regulation of PCD. We show here that metacaspase-8 (AtMC8) is a member of the gene family strongly up-regulated by oxidative stresses caused by UVC, H(2)O(2), or methyl viologen. This up-regulation was dependent of RCD1, a mediator of the oxidative stress response. Recombinant metacaspase-8 cleaved after arginine, had a pH optimum of 8, and complemented the H(2)O(2) no-death phenotype of a yeast metacaspase knock-out. Overexpressing AtMC8 up-regulated PCD induced by UVC or H(2)O(2), and knocking out AtMC8 reduced cell death triggered by UVC and H(2)O(2) in protoplasts. Knock-out seeds and seedlings had an increased tolerance to the herbicide methyl viologen. We suggest that metacaspase-8 is part of an evolutionary conserved PCD pathway activated by oxidative stress.
Subject(s)
Apoptosis/physiology , Arabidopsis Proteins/genetics , Caspase 8/genetics , Cysteine Endopeptidases/genetics , Hydrogen Peroxide/pharmacology , Ultraviolet Rays , Apoptosis/drug effects , Apoptosis/radiation effects , Arabidopsis , Arabidopsis Proteins/drug effects , Arabidopsis Proteins/radiation effects , Caspase 8/drug effects , Caspase 8/radiation effects , Cell Death , Cysteine Endopeptidases/deficiency , Cysteine Endopeptidases/drug effects , Cysteine Endopeptidases/radiation effects , DNA Primers , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/radiation effects , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/radiation effects , Oxidative Stress , Plants, Genetically Modified/metabolism , Protoplasts/drug effects , Protoplasts/physiology , Protoplasts/radiation effects , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Plants, animals, and several branches of unicellular eukaryotes use programmed cell death (PCD) for defense or developmental mechanisms. This argues for a common ancestral apoptotic system in eukaryotes. However, at the molecular level, very few regulatory proteins or protein domains have been identified as conserved across all eukaryotic PCD forms. A very important goal is to determine which molecular components may be used in the execution of PCD in plants, which have been conserved during evolution, and which are plant-specific. Using Arabidopsis thaliana, we have shown that UV radiation can induce apoptosis-like changes at the cellular level and that a UV experimental system is relevant to the study of PCD in plants. We report here that UV induction of PCD required light and that a protease cleaving the caspase substrate Asp-Glu-Val-Asp (DEVDase activity) was induced within 30 min and peaked at 1 h. This DEVDase appears to be related to animal caspases at the biochemical level, being insensitive to broad-range cysteine protease inhibitors. In addition, caspase-1 and caspase-3 inhibitors and the pan-caspase inhibitor p35 were able to suppress DNA fragmentation and cell death. These results suggest that a YVADase activity and an inducible DEVDase activity possibly mediate DNA fragmentation during plant PCD induced by UV overexposure. We also report that At-DAD1 and At-DAD2, the two A. thaliana homologs of Defender against Apoptotic Death-1, could suppress the onset of DNA fragmentation in A. thaliana, supporting an involvement of the endoplasmic reticulum in this form of the plant PCD pathway.
