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

Identification of auxins by a chemical genomics approach

Thirteen auxenic compounds were discovered in a screen of 10 000 compounds for auxin-like activity in Arabidopsis roots. One of the most potent substances was 2-(4-chloro-2-methylphenoxy)-N-(4-H-1,2,4-triazol-3-yl)acetamide (WH7) which shares similar structure to the known auxenic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). A selected set of 20 analogues of WH7 was used to provide detailed information about the structure–activity relationship based on their efficacy at inhibiting and stimulating root and shoot growth, respectively, and at induction of gene expression. It was shown that WH7 acts in a genetically defined auxin pathway. These small molecules will extend the arsenal of substances that can be used to define auxin perception site(s) and to dissect subsequent signalling events

The Microphenotron: a robotic miniaturized plant phenotyping platform with diverse applications in chemical biology

Background Chemical genetics provides a powerful alternative to conventional genetics for understanding gene function. However, its application to plants has been limited by the lack of a technology that allows detailed phenotyping of whole-seedling development in the context of a high-throughput chemical screen. We have therefore sought to develop an automated micro-phenotyping platform that would allow both root and shoot development to be monitored under conditions where the phenotypic effects of large numbers of small molecules can be assessed. Results The ‘Microphenotron’ platform uses 96-well microtitre plates to deliver chemical treatments to seedlings of Arabidopsis thaliana L. and is based around four components: (a) the ‘Phytostrip’, a novel seedling growth device that enables chemical treatments to be combined with the automated capture of images of developing roots and shoots; (b) an illuminated robotic platform that uses a commercially available robotic manipulator to capture images of developing shoots and roots; (c) software to control the sequence of robotic movements and integrate these with the image capture process; (d) purpose-made image analysis software for automated extraction of quantitative phenotypic data. Imaging of each plate (representing 80 separate assays) takes 4 min and can easily be performed daily for time-course studies. As currently configured, the Microphenotron has a capacity of 54 microtitre plates in a growth room footprint of 2.1 m², giving a potential throughput of up to 4320 chemical treatments in a typical 10 days experiment. The Microphenotron has been validated by using it to screen a collection of 800 natural compounds for qualitative effects on root development and to perform a quantitative analysis of the effects of a range of concentrations of nitrate and ammonium on seedling development. Conclusions The Microphenotron is an automated screening platform that for the first time is able to combine large numbers of individual chemical treatments with a detailed analysis of whole-seedling development, and particularly root system development. The Microphenotron should provide a powerful new tool for chemical genetics and for wider chemical biology applications, including the development of natural and synthetic chemical products for improved agricultural sustainability

Chloroplast-to-nucleus communication: Current knowledge, experimental strategies and relationship to drought stress signaling

In order for plant cells to function efficiently under different environmental conditions, chloroplastic processes have to be tightly regulated by the nucleus. It is widely believed that there is inter-organelle communication from the chloroplast to the nucleus, called retrograde signaling. Although some pathways of communication have been identified, the actual signals that move between the two cellular compartments are largely unknown. This review provides an overview of retrograde signaling including its importance to the cell, candidate signals, recent advances and current experimental systems. In addition, we highlight the potential of using drought stress as a model for studying retrograde signaling