Genetic dissection of regulatory domains and signalling interactions of PRL1 WD-protein in Arabidopsis

The Arabidopsis PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) encodes a nuclear WD40 protein. Plants carrying a prl1 insertion mutation are smaller than wild type, have shorter roots, leaves with serrated leaf margins and shorter petioles. In photosynthetic tissues of the prl1 mutant elevated glucose, sucrose, fructose, starch, anthocyanin and chlorophyll levels are detected. Loss of the PRL1 function results in hypersensitivity to glucose, sucrose and plant hormones, including cytokinin, ethylene, abscisic acid and auxin. To study the regulatory function of PRL1, we first developed molecular tools for characterization of PRL1 gene expression. A full length (4.2 kb) PRL1 promoter driving the expression of β-glucuronidase (GUS) reporter gene in fusion with coding sequences for the first 69 amino acids of PRL1 protein was found to be expressed in most tissues. To identify regulatory regions in the PRL1 gene, a set of promoter deletions was generated and propagated in cell suspension and in planta. Our results demonstrated that intragenic sequences are crucial for correct gene expression as PRL1 constructs lacking the second intron showed only low activity. A short promoter containing an alternative TATA-box, 5�-UTR and 0.5 kb coding region with the first two introns of PRL1 showed GUS activity similar to the previously characterized full-length promoter. Whereas upstream regulatory sequences (~3.5 kb) alone were not sufficient for correct mRNA expression, constructs containing the second intron proved to be fully functional in genetic complementation assays in the prl1 mutant background. Sequence analysis suggested the importance of a TC-rich region in the second intron. However, deletion of this sequence did not affect gene expression dramatically. When PRL1 was tagged with green fluorescent protein (GFP), it was found to be expressed in most cell types and localized mainly in the nucleus. To differentiate the role of PRL1 protein in organ development, a PRL1 genomic fragment and cDNA were misexpressed using the heterologous promoters of AtKNAT1; AtSTM; AtUFO; AtAS1; AtSUC2, At4CL1 and TobRB7 genes. Whereas genomic constructs complemented the prl1 phenotype in all cases, only cDNA construct driven by the AtAS1 and AtSUC2 promoters were able to complement the prl1 mutant leaf phenotype. Posttranslational regulation for PRL1 was suggested by our result indicating that the protein is unstable. The reversible proteasome inhibitor MG132 stabilized PRL1 suggesting that PRL1 is degraded in a proteasome-dependent manner. In the N-terminal region of PRL1, a destruction box (D box) motif was identified, which represents a putative degron for the Anaphase Promoting Complex/Cyclosome (APC) E3 ligase complex. Point mutations were introduced to disrupt the D box consensus sequence, in order to stabilize PRL1 protein. In cell suspension higher level of GFP-tagged PRL1 protein could be detected as potential result of stabilizing mutations, but this was not the case in three-week-old seedlings. Therefore, full understanding the control of PRL1 degradation requires further investigations. Biochemical studies revealed that PRL1 is present in a large nuclear protein complex associated with the spliceosome component AtCDC5 protein. Our data indicate that AtCDC5 interacts with various elements of the protein degradation system, such as the 20S core and 19S lid particles of the proteasome, the CSN5 subunit of the COP9 signalosome and the SCF E3 ubiqutin ligase subunit CULLIN 1. In addition, ubiquitinated proteins were detected in pull-down assays with AtCDC5 suggesting that substrates targeted to the proteasome are potentially also associated to this complex. These results indicate potential roles for PRL1 through its interaction with AtCDC5 in mRNA splicing and proteasome-dependent protein degradation. Ongoing projects aim at further understanding of the PRL1 function using genetic and site-specific mutagenesis approaches.