The plant hormone cytokinin controls various processes in plant development and responses to environmental stresses. Cytokinin degradation is catalyzed by a group of CKX enzymes. Cellular levels of these proteins significantly impact the cytokinin homeostasis in plants and it is, therefore, important to understand the mechanisms regulating their activities. In this study, several CKX-interacting proteins, belonging to a plant-unique protein family (HIPP), were molecularly characterized and their biological function elucidated, particularly in respect to the regulation of cytokinin homeostasis. In the first part of this work, the molecular basis of the CKX-HIPP interaction, such as the essential interaction motifs, interaction specificity, and the subcellular compartmentation of the CKX-HIPP complex, were investigated. Interaction assays performed in yeast and in planta revealed protein-protein interactions between specific members of the CKX and HIPP protein families. CKX1 interacted with HIPP proteins from the phylogenetic cluster I and III, but not other members of the family. The analyzed cluster I-HIPP proteins interacted additionally with most CKX proteins targeted to the secretory system but did not interact with the cytosolic CKX7 isoform. The CKX1-HIPP7 interaction required the prenylation motif at the C-terminus of HIPP7, implying that the lipid modification mediates the CKX-HIPP interaction. In addition, the tested HIPP proteins were found to form homodimers, which required both the functional prenylation and HMA domains, suggesting that metal binding could mediate the HIPP homodimerization. The Arabidopsis CKX1 protein, a case example in this study, has been shown to be a type II membrane protein that localizes predominantly to the ER. However, the subcellular localization studies in this work revealed that HIPP1, HIPP5 and HIPP7 are localized apparently outside of the secretory system, predominantly in the cytosol and nucleus. To address this discrepancy, bimolecular fluorescence complementation (BiFC) assays were performed. The fluorescence of the BiFC CKX1/HIPP7 complex clearly showed that the interaction occur at the cortical and perinuclear ER. Moreover, a strong BiFC signal mainly localized in the nucleus and cytosol was detected for the CKX1/HIPP1 pair. These results suggest that CKX1 in the detected complexes represents a protein form that was relocated to the cytosolic site of the ER membrane. The second part of this work aimed to uncover the biological function of the identified HIPP proteins, especially in respect to their potential role in regulating CKX protein levels and cytokinin responses in Arabidopsis. The HIPP-overexpressing plants displayed cytokinin-related phenotypic changes and were hypersensitive to cytokinin. This was correlated with an increased cytokinin activity in these plants. It could be further shown that HIPP proteins differentially affected the abundance of the CKX1 protein. Given that CKX1 has been previously shown to be an ERAD substrate protein, it is proposed that the analyzed HIPP proteins might play a role during the retrotranslocation of CKX proteins from ER into the cytosol or during their cytosolic proteasomal degradation. It is hypothesized that the increased cytokinin activity displayed by HIPP-overexpressing plants is due to reduced levels of CKX proteins in the ER, which results in more cytokinin being sensed by the AHK cytokinin receptors localized in this compartment. Analysis of the concentration of other phytohormones or key genes determining their biosynthesis revealed that HIPP-overexpression resulted in the accumulation of stress-related hormones, i.e. ABA and SA, and downregulation of genes related to GA biosynthesis. These changes probably accounted for the enhanced drought tolerance and delayed onset of flowering of the HIPP-overexpressing plants. These data suggest that HIPPs may have broader biological activity beyond the regulation of CKX. Additionally, the effects of hipp loss-of-function mutations and the expression patterns of the analyzed HIPP genes were investigated in this work. In comparison to the phenotypic changes of the HIPP-overexpressing plants, hipp single and double mutants did not display obvious phenotypic changes, suggesting a higher degree of functional redundancy among the cluster I HIPP genes in controlling cytokinin responses and plant development. The HIPP genes were found to be expressed in distinct tissues, including mainly root and shoot apical meristems, and vascular tissues. Interestingly, HIPP transcript levels were repressed by exogenous cytokinin applications, suggesting a regulatory feedback loop between cytokinin and analyzed HIPP genes.
