Characterization of cluster-III HIPP proteins targeted to plasmodesmata in Arabidopsis thaliana

Abstract

The plant hormone cytokinin plays essential regulatory roles in many aspects of plant growth and development. Cellular cytokinin concentrations are controlled by numerous interconnected metabolic reactions, such as hormone biosynthesis, metabolic activation and inactivation. The irreversible cytokinin degradation is mediated by the cytokinin oxidase/dehydrogenase (CKX) enzymes. Several heavy metal-associated isoprenylated plant proteins (HIPP), which belong to two distinct phylogenetic clusters have been identified to specifically interact with CKX proteins. These plant-unique proteins are defined by the combination of one or two heavy metal-binding domains (HMA) and a C-terminal prenylation site. The physiological function and molecular activity of HIPP proteins are largely unknown, and only few HIPP genes have been characterized so far. This current thesis studied the highly homologue HIPP genes constituting together the phylogenetic cluster III. Phenotypic analysis of the hipp knockouts indicate that the respective genes regulate different aspects of plant growth, such as root development, leaf formation, as well as floral organ development and gametogenesis. Furthermore, the activity of HIPP genes is required for pattern formation and maintenance of the shoot and root apical meristems during embryogenesis. In agreement, gene promoter-reporter constructs showed highest activity in regions of active growth such as root apical meristem, young leaf primordia and inflorescence. Consistent with the pleiotropic phenotypes of hipp mutant plants, the transcriptional profiling by RNA-Seq in hipp mutants revealed the differential expression of hundreds of genes involved in various processes of plant development. Cluster-III HIPP proteins were shown to localize specifically to plasmodesmata (PD). Symplastic transport assays performed in generated hipp mutant plants suggest that HIPP proteins function in restricting PD-mediated macromolecular trafficking. It is currently still unclear whether HIPP proteins influence size exclusion limit of PD, PD abundancy or PD architecture. Notably, symplasmic trafficking in the root meristem of hipp mutants was not affected by iron treatment, suggesting that HIPP proteins might also be involved in PD regulation in response to iron stress. Co-IP experiments performed in this study confirmed the interactions between CKX1 and HIPP proteins from cluster III originally identified in yeast. However, the molecular mechanisms underlying the interaction between CKX1 and HIPP proteins remain to be clarified. Interestingly, TCSn:GFP reporter revealed attenuated cytokinin activity in hipp roots, reflecting the redundant function of the cluster-III HIPP genes in positively regulating the cytokinin signaling output. Moreover, transcriptome analysis performed in hipp mutants revealed that HIPP genes are required for the transcriptional response to cytokinin.