TRANSIENT TRANSFORMATION OF ARABIDOPSIS LEAF PROTOPLASTS - A VERSATILE EXPERIMENTAL SYSTEM TO STUDY GENE-EXPRESSION

An-improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protop lasts support high levels of expression of the bacterial reporter gene coding for beta-glucuronidase (GUS), under the control of the caulifl ower mosaic virus (CaMV) 35S promoter. Transient expression of GUS act ivity was monitored spectrophotometrically and reached a maximum betwe en 18 and 48 h after polyethyleneglycol (PEG)-mediated DNA uptake. His tochemical staining for GUS activity revealed reproducible transformat ion frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expressio n system, the subcellular localization of GUS proteins tagged with dif ferent nuclear polypeptides was studied in transfected mesophyll proto plasts, revealing nuclear compartmentalization of the chimeric GUS enz ymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin-med iated induction of chloramphenicol acetyltransferase (CAT) activity wh en transfected with a transcriptional fusion between the CAT reporter gene and the early auxin-inducible PS-IAA4/5 promoter. Hence, the meth od allows in vivo analysis of promoter activity and subcellular locali zation of fusion proteins in a homologous transformation system.