A light-responsive in vitro transcription system from evacuolated parsley protoplasts.
Evacuolated protoplasts (EP) retain transcriptional activity responding, after UV-light treatment, to the expression of chalcone synthase (CHS); this behaviour is similar to the parsley cell culture and protoplasts from which the EP were derived. Chemical lysis of the EP with low concentrations of a detergent in a minimal volume had no negative effect on the inducibility of CHS by UV-containing white light. Based on these observations a new method is presented here for establishing a light-responsive in vitro transcription system from evacuolated protoplasts of a parsley (Petroselinum crispum) cell culture. A 615 bp promoter fragment of the CHS gene, fused to the beta-Glucuronidase (GUS) reporter gene, was accurately transcribed as in transiently transformed protoplasts and the reaction was highly sensitive to alpha-amanitin. A 226 bp CHS promoter/GUS reporter construct with mutated cis-acting elements was unable to stimulate GUS transcription, whilst a 341 bp 35S-promoter from the cauliflower mosaic virus (CaMV) was constitutively expressed in dark- or light-treated lysates. The expression of the CHS full-length promoter/GUS construct in the cell-free system was three-fold increased by white or red light compared with the dark level. These results demonstrate that within this in vitro system there must exist a largely intact signal transduction chain between photoreceptor(s) and the CHS promoter. As such it will be a valuable tool for elucidating signalling mechanisms and functional assays of trans-acting factors acting at the end of the pathway.
Frohnmeyer
H
H
Institut für Biologie II, Universität Freiburg, Germany.
Hahlbrock
K
K
Schäfer
E
E
D000217:Acyltransferases / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002474:Cell-Free System / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000528:radiation effects*; D017930:Genes, Reporter; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D011523:Protoplasts; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014466:Ultraviolet Rays; D014617:Vacuoles; D014675:Vegetables / Q000235:genetics*
D012333:RNA, Messenger; D000217:Acyltransferases; C020141:flavanone synthetase; D005966:Glucuronidase