Oral Presentation 24th Australian Conference on Microscopy and Microanalysis 2016

Using caged cytokinin to investigate root development at the single-cell level (#92)

Lachlan Dow 1 2 , Ulrike Mathesius 2 , Russell A. Barrow 1 , Rosemary G. White 3
  1. Research School of Chemistry, Australian National University, Canberra, ACT, Australia
  2. Research School of Biology, Australian National University, Canberra, ACT, Australia
  3. CSIRO Agriculture, Canberra, ACT, Australia

Cytokinins are a class of small molecules that regulate nearly every aspect of plant growth, and shape plant organs, including roots. Below ground, cytokinins can inhibit root elongation, and will arrest lateral root development and growth of primary roots. To understand their involvement in root development we need precise spatial and temporal information on cytokinin activities, but current techniques are lacking in this regard, thus many cell-type specific effects of cytokinins remain unknown. This work aimed to develop a method to investigate the effects of increasing cytokinin levels within single root cells.

We synthesised 9(2-nitrobenzyl)-benzyladenine, a caged form of a commonly used synthetic cytokinin, N‑benzyladenine (BA), following a protocol of Hayashi et al. (2012, Bioorganic and Medicinal Chemistry Letters 22: 5663-5667). We also generated caged adenine as a control for the uncaging process. Upon irradiation with UV in vitro, the photosensitive chemical cages were cleaved, producing either free BA or free adenine, respectively.

We used roots of Arabidopsis thaliana to assess whether the typical growth inhibition response, as seen with applied BA, was detected after uncaging. We further assessed local release of caged BA using multiphoton (MP) confocal microscopy with the cytokinin reporter line TCSn::GFP in which GFP expression is induced with increased tissue cytokinin levels.

Free BA was released upon UV-illumination of the caged BA, as determined by HPLC assays. In root growth assays (in Arabidopsis thaliana), caged BA produced a cytokinin phenotype only after UV irradiation. UV illumination also released caged adenine, which produced no growth response in roots. Multiphoton confocal microscopy also produced a detectable cytokinin response, indicated by induction of GFP, within illuminated tissues or cells of the cytokinin reporter line TCSn::GFP incubated in caged BA. Details about uncaging BA in specific tissues and cells, and their physiological responses to the locally-released cytokinin, will be discussed.