Protoplasts as a tool to study Alternative Splicing in Arabidopsis

GODOY HERZ, MICAELA A; PETRILLO, EZEQUIEL; KORNBLIHTT, ALBERTO

Congreso; SAIB, 48th Annual Meeting, Argentine Society for Biochemistry and Molecular Biology; 2012

Arabidopsis protoplasts are cells without cell walls obtained from leaves after an enzymatic treatment. Protoplasts maintain many of the physiological properties of the plants which they come from. We have established the experimental conditions to obtain Arabidopsis mesophyl protoplasts in order to asess alternative splicing responses to light/darkness treatments as previously observed in Arabidopsis seedlings. We use the RS31 transcriptional unit as a model. In seedlings, light treatment causes an increase of the functional mRNA isoform. Protoplasts seem to show a light/darkness effect, only slightly lower than in seedlings. Furthermore, we have constructed a series of alternative splicing reporter minigenes to be introduced into the protoplasts through PEG-mediated transformation. These minigenes combine two different alternative splicing events ?exon cassette and intron retention or alternative 5? splicing sites- with three different promoters. The idea is to evaluate if promoters affect alternative splicing in protoplasts as it was described in animal cells. Our protoplast transformation controls using a GFP reporter include detection of the expressed protein by epifluorescence microscopy and of expressed RNA by RT-PCR. Our results indicate that protoplast isolation and transformation are a useful new tool to study alternative splicing in plants.