A LIGHT-RESPONSIVE IN-VITRO TRANSCRIPTION SYSTEM FROM EVACUOLATED PARSLEY PROTOPLASTS

Evacuolated protoplasts (EP) retain transcriptional activity respondin g, after UV-light treatment, to the expression of chalcone synthase (C HS); this behaviour is similar to the parsley cell culture and protopl asts from which the EP were derived. Chemical lysis of the EP with low concentrations of a detergent in a minimal volume had no negative eff ect on the inducibility of CHS by UV-containing white light. Based on these observations a new method is presented here for establishing a l ight-responsive in vitro transcription system from evacuolated protopl asts of a parsley (Petroselinum crispum) cell culture. A 615 bp promot er fragment of the CHS gene, fused to the beta-Glucuronidase (GUS) rep orter gene, was accurately transcribed as in transiently transformed p rotoplasts and the reaction was highly sensitive to a-amanitin. A 226 bp CHS promoter/GUS reporter construct With mutated cis acting element s was unable to stimulate GUS transcription, whilst a 341 bp 35S-promo ter from the cauliflower mosaic virus (CaMV) was constitutively expres sed in dark- or light-treated lysates. The expression of the CHS full- length promoter/GUS construct in the cell free system was three-fold i ncreased by white or red light compared with the dark level. These res ults demonstrate that within this in vitro system there must exist a l argely intact signal transduction chain between photoreceptor(s) and t he CHS promoter. As such it will be a valuable tool for elucidating si gnalling mechanisms and functional assays of trans-acting factors acti ng at the end of the pathway.