Physiological and cellular effects of TENin1, a novel small molecule inhibitor of endomembrane protein trafficking

Gravitropic response is required for proper orientation of plant growth and development. One of the factors that influence gravitropism in plants is the polar distribution of the plant hormone auxin, which is maintained by auxin transporters at the plasma membrane (PM). These endomembrane proteins are transported to the PM via the secretory pathway and undergo constitutive endocytic recycling from the PM. This thesis characterises an inhibitor of endomembrane protein trafficking, Trafficking & ENdocytosis inhibitor 1 TENin1 (TE1), that reduces gravitropic response in Arabidopsis thaliana seedlings. Short term TE1 treatment causes intracellular accumulation of membrane proteins including brassinosteroid receptor BRI1, aquaporin PIP2a, and auxin transporters PIN2 and PIN7. All the accumulated experimental evidence gained throughout the duration of this project also suggest that TE1 interferes with the endomembrane recycling to the TGN both from the pre-vacuolar compartment (PVC) and the PM therefore causing accumulation of PIN2-GFP at the PVC, which is eventually re-directed to the vacuole. The long term effects of TE1 were also characterised and revealed dose-dependent growth inhibition of whole plants and reduction in organelle dynamics. In a separate study in the laboratory two Arabidopsis thaliana accessions that displayed resistance to the effects of TE1 were identified. A library of Arabidopsis thaliana recombinant inbred lines (RILs) generated by crossing a TE1 resistant accession with the sensitive Columbia accession was commercially available. Therefore, 174 and 117 RILs were screened for different traits to identify a region of the genome responsible for the resistance to the effects of TE1. The data generated from the RIL screens revealed a major resistant locus lies within 9 to 16 Mb in the chromosome 5. A further study is now required to map the target gene(s) responsible for the resistance to TE1

Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana

Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure-activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants

The life of the peroxisome: from birth to death

Peroxisomes are dynamic and metabolically plastic organelles. Their multiplicity of functions impacts on many aspects of plant development and survival. New functions for plant peroxisomes such as in the synthesis of biotin, ubiquinone and phylloquinone are being uncovered and their role in generating reactive oxygen species (ROS) and reactive nitrogen species (RNS) as signalling hubs in defence and development is becoming appreciated. Understanding of the biogenesis of peroxisomes, mechanisms of import and turnover of their protein complement, and the wholesale destruction of the organelle by specific autophagic processes is giving new insight into the ways that plants can adjust peroxisome function in response to changing needs

An inhibitor of oil body mobilization in Arabidopsis

Fatty acid β-oxidation is an essential process in many aspects of plant development, and storage oil in the form of triacylglycerol (TAG) is an important food source for humans and animals, for biofuel and for industrial feedstocks. In this study we characterize the effects of a small molecule, diphenyl methylphosphonate, on oil mobilization in Arabidopsis thaliana. Confocal laser scanning microscopy, transmission electron microscopy and quantitative lipid profiling were used to examine the effects of diphenyl methylphosphonate treatment on seedlings. Diphenyl methylphosphonate causes peroxisome clustering around oil bodies but does not affect morphology of other cellular organelles. We show that this molecule blocks the breakdown of pre-existing oil bodies resulting in retention of TAG and accumulation of acyl CoAs. The biochemical and phenotypic effects are consistent with a block in the early part of the β-oxidation pathway. Diphenyl methylphosphonate appears to be a fairly specific inhibitor of TAG mobilization in plants and whilst further work is required to identify the molecular target of the compound it should prove a useful tool to interrogate and manipulate these pathways in a controlled and reproducible manner