Molybdate uptake and molybdenum cofactor (Moco) biosynthesis were investigated in detail in the last few decades. The present study critically reviews our present knowledge about eukaryotic molybdate transporters (MOT) and focuses on the model plant Arabidopsis thaliana, complementing it with new experiments, filling missing gaps, and clarifying contradictory results in the literature. Two molybdate transporters, MOT1.1 and MOT1.2, are known in Arabidopsis, but their importance for sufficient molybdate supply to Moco biosynthesis remains unclear. For a better understanding of their physiological functions in molybdate homeostasis, we studied the impact of mot1.1 and mot1.2 knock-out mutants, including a double knock-out on molybdate uptake and Moco-dependent enzyme activity, MOT localisation, and protein–protein interactions. The outcome illustrates different physiological roles for Moco biosynthesis: MOT1.1 is plasma membrane located and its function lies in the efficient absorption of molybdate from soil and its distribution throughout the plant. However, MOT1.1 is not involved in leaf cell imports of molybdate and has no interaction with proteins of the Moco biosynthesis complex. In contrast, the tonoplast-localised transporter MOT1.2 exports molybdate stored in the vacuole and makes it available for re-localisation during senescence. It also supplies the Moco biosynthesis complex with molybdate by direct interaction with molybdenum insertase Cnx1 for controlled and safe sequestering.

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AbstractAn ever-increasing number of protein complexes participating in metabolic pathways and of multi-protein intracellular networks is identified in plant cells. Split-GFP based protein-protein interaction assays combine the advantages of in vivo interaction studies in native environment with additional visualisation of protein complex localisation. However, fluorescence proteins have several drawbacks for 3D- imaging and super-resolution microscopy: high photobleaching rate during long-term observations, correlation of fluorescence intensity to expression level, blinking behaviour and the tendency to form oligomers. The HaloTag® system was shown to overcome these drawbacks. This reporter is able to form covalent irreversible bonds with synthetic chloralkane ligands. Several stable fluorescence ligands are available like TMR or Oregon Green, which can be used in variable concentrations for optimal fluorescence intensity. Therefore, we established the new Split-HaloTag® imaging assay to enable advanced fluorescence imaging such as single molecule-, subdiffractional polarisation imaging or super-resolution microscopy of protein-protein interactions. This is demonstrated via studies of molybdenum cofactor biosynthesis complex anchoring to filamentous actin. Specific interactions were visualised with 3D- and advanced subdiffractional polarisation microscopy in distinctive manner. Therefore, this assay is a promising new tool for 3D-imaging and super-resolution microscopy of protein-protein interactions in plant cells.HighlightsThe Split-HaloTag® assay combine for the first time in planta a flexible and photostable fluorescence system with a split-reporter system, which enables protein complex analyses with advanced microscope technologies.