title: Internucleosomal DNA fragmentation in wild emmer wheat is catalyzed by S1-type endonucleases translocated to the nucleus upon induction of cell death.
abstract: Leaves of cereal plants display nucleosomal fragmentation of DNA attributed to the action of nucleases induced during program cell death (PCD). Yet, the specific nuclease activity responsible for generating double strand DNA breaks (DSBs) that lead to DNA fragmentation has not been fully described. Here, we characterized a Ca2+/Mg2+-dependent S1-type endonuclease activity in leaves of wild emmer wheat (Triticum dicoccoides Köern.) capable of introducing DSBs as demonstrated by the conversion of supercoiled plasmid DNA into a linear duplex DNA. In-gel nuclease assay revealed a nuclease of about 35 kDa capable of degrading both single stranded DNA and RNA. We further showed that the endonuclease activity can be purified on Concanavalin A and treatment with peptide-N-glycosidase F (PNGase F) did not abolish its activity. Furthermore, ConA-associated endonuclease was capable of generating nucleosomal DNA fragmentation in tobacco nuclei. Since S1-type endonucleases lack canonical nuclear localization signal it was necessary to determine their subcellular localization. To this end, a cDNA encoding for a putative 34 kDa S1-type nuclease, designated TaS1-like (TaS1L) was synthesized based on available sequence data of Triticum aestivum and fused with RFP. Introduction into protoplasts showed that TaS1L-RFP is cytoplasmic 24h post transformation but gradually turn nuclear at 48 h concomitantly with induction of cell death. Our results suggest that DNA fragmentation occurring in leaves of wild emmer wheat may be attributed to S1-type endonuclease(s) that reside in the cytoplasm but translocate to the nucleus upon induction of cell death.
authors.affiliation: French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, Israel.
authors.forename: Gila
authors.initials: G
authors.lastname: Granot
authors.affiliation: French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, Israel.
authors.forename: Yaakov
authors.initials: Y
authors.lastname: Morgenstern
authors.affiliation: French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, Israel.
authors.forename: Asif
authors.initials: A
authors.lastname: Khan
authors.affiliation: French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, Israel.
authors.forename: Yemima Givaty
authors.initials: YG
authors.lastname: Rapp
authors.affiliation: French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, Israel.
authors.forename: Anat
authors.initials: A
authors.lastname: Pesok
authors.affiliation: Institute of Evolution, University of Haifa, 199 Aba-Hushi Avenue Mount Carmel, Haifa 3498838, Israel.
authors.forename: Eviatar
authors.initials: E
authors.lastname: Nevo
authors.affiliation: French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, Israel. Electronic address: ggrafi@bgu.ac.il.
authors.forename: Gideon
authors.initials: G
authors.lastname: Grafi
mesh_terms: D002467:Cell Nucleus / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D053938:DNA Fragmentation; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009707:Nucleosomes / Q000201:enzymology; D018515:Plant Leaves / Q000201:enzymology; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000235:genetics*; D014908:Triticum / Q000201:enzymology
chemical_list: D009707:Nucleosomes; D004720:Endonucleases
keywords: Cell death; Cytoplasmic-nuclear translocation; DNA fragmentation; Endonucleases; Glycosylation; Wild emmer wheat