Analyzing Ppi1 gene expression using promoter-GUS fusions

PPI1 (PROTON PUMP INTERACTOR 1) is a protein identified in a two hybrid screen whose Nterminus binds to the Arabidopsis thaliana plasma membrane proton pump (PM H+-ATPase). The entire PPI1 protein, or fragments thereof, expressed as fusion protein in E. coli, are able to stimulate the activity of the proton pump in vitro (Morandini et al., 2002 Plant J. 31:487-97). To study the pattern of Ppi1 expression we produced transgenic reporter lines with the GUS gene under the control of Ppi1 promoter. Since the gene presents a large intron before the beginning of the coding region (a so-called ‘leader intron’), we decided to test three different constructs. The first contains both the promoter and the leader intron; the second one is lacking the leader intron but still contains the Ppi1 5’UTR; the last one is missing the intron and the 5’UTR derives from the TMV. In an alternative approach to study the function of the PPI1 protein we make use of Arabidopsis KO lines bearing a T-DNA insertion. Two lines were characterized in detail for Ppi1: an insertion in intron I (line N93) and one in exon VII (line F09) at aa 525. These KO lines were confirmed with RT-PCR and western analysis. The Ppi1 KO lines do not show evident phenotype when grown in pots. In contrast, KO culture cells seems to grow faster, to release a brown compound and to form tracheary elements at higher frequencies than the WT.