title: Arabidopsis cell-free extract, ACE, a new in vitro translation system derived from Arabidopsis callus cultures.
abstract: The analysis of post-transcriptional regulatory mechanisms in plants has benefited greatly from the use of cell-free extract systems. Arabidopsis as a model system provides extensive genetic resources; however, to date a suitable cell-free translation system from Arabidopsis has not been available. In this study, we devised an Arabidopsis cell-free extract (ACE) to be used for in vitro translation studies. Protoplasts were prepared from callus cultures derived from Arabidopsis seedlings, and cell-free extracts were prepared after evacuolation of the protoplasts by Percoll gradient centrifugation. The new ACE system exhibits translation activity comparable with that of the wheat germ extract system. We demonstrated that ACE prepared from the 5'-3' exoribonuclease-deficient mutant of Arabidopsis, xrn4-5, exhibited increased stability of an uncapped mRNA as compared with that from wild-type Arabidopsis. We applied the ACE system to study post-transcriptional regulation of AtCGS1. AtCGS1 codes for cystathionine γ-synthase (CGS) that catalyzes the first committed step of methionine and S-adenosyl-l-methionine (AdoMet) biosynthesis in plants, and is feedback regulated by mRNA degradation coupled with translation elongation arrest. The ACE system was capable of reproducing translation elongation arrest and subsequent AtCGS1 mRNA degradation that are induced by AdoMet. The ACE system described here can be prepared in a month after seed sowing and will make it possible to study post-transcriptional regulation of plant genes while taking advantage of the genetics of Arabidopsis.
authors.affiliation: Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan.
authors.forename: Katsunori
authors.initials: K
authors.lastname: Murota
authors.forename: Yuka
authors.initials: Y
authors.lastname: Hagiwara-Komoda
authors.forename: Keisuke
authors.initials: K
authors.lastname: Komoda
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Onouchi
authors.forename: Masayuki
authors.initials: M
authors.lastname: Ishikawa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Naito
mesh_terms: D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D005091:Exons / Q000235:genetics; D005095:Exoribonucleases / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010936:Plant Extracts / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D012315:RNA Caps / Q000235:genetics; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000494:pharmacology; D046509:Tissue Culture Techniques / Q000379:methods*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D010936:Plant Extracts; D010940:Plant Proteins; D012315:RNA Caps; D012333:RNA, Messenger; C418137:Xrn1 protein, plant; D012436:S-Adenosylmethionine; D005095:Exoribonucleases