Hartwig Lüthen

In an era of genomics, proteomics, and metabolomics a large number of mutants are available. The discovery of their phenotypes is fast becoming the bottleneck of molecular plant physiology. This crisis can be overcome by imaging-based phenotyping, an emerging, rapidly developing and innovative approach integrating plant and computer science. A tremendous amount of digital image data are automatically analysed using techniques of ‘machine vision’. This minireview will shed light on the available imaging strategies and discuss standard methods for the automated analysis of images to give the non-bioinformatic reader an idea how the new technology works. A number of successful platforms will be described and the prospects that image-based phenomics may offer for elucidating hormonal cross-talk and molecular growth physiology will be discussed.

Next generation phenotyping of auxin response mutants will be greatly facilitated by the ability to record rapid growth responses in roots and hypocotyls at high throughput and at high temporal resolution. As Arabidopsis seedlings are very tiny and fragile, imaging is the only adequate way for data acquisition. As camera-based systems described before have a limited throughput, we used commercial flatbed scanners to record a large number of simultaneous experiments. We developed Hansa Trace, software for automatically detecting and measuring hypocotyl segments and roots in the images. We validated this system by measuring some well-characterized growth responses to auxins, non-auxins, ATPase activators and apoplastic acidification. The method can be shared on a cooperation basis and is able to perform measurements with minimal user intervention.

We investigated the relation between auxin-induced gene expression and the rapid auxin-induced growth inhibition in Arabidopsis thaliana roots. The natural auxin indole-3-acetic acid (IAA) induced a strong activation of gene expression as visualized by the DR5rev::GFP reporter gene technique. This effect was specific for active auxins and was abolished in knockout mutants of the F-box auxin receptors. We measured the IAA-induced growth inhibition at high time resolution and show that the F-box auxin receptor mutants failed to display this effect. We conclude that the F-box auxin receptors are needed for the response. In hypocotyls, auxin induces an increase in elongation growth, and this effect has been earlier shown to be independent of the F-box receptors. Based on these findings, we discuss differences in the growth control modes in roots and shoots. We demonstrate that the rapid auxin-induced root growth inhibition, unlike the induction of growth in hypocotyls, requires the presence of the F-box auxin receptors.

The diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) is known to lack a number of typical auxin responses. Here we show that rapid auxin-induced growth of seedling hypocotyls is completely abolished by the mutation over the full range of auxin concentrations tested, and also in early phases of the time course. Protoplasts isolated from wild-type hypocotyls respond to auxin by a rapid increase in cell volume, which we measured by image analysis at a high temporal resolution. A similar swelling could be triggered by antibodies directed against a part of the putative auxin-binding domain (box-a) of the auxin-binding protein 1 (ABP1). Induction of swelling both by auxin and by the antibody was not observed in the protoplasts isolated from the dgt mutant. However, dgt protoplasts are able to respond to the stimulator of the H⁺-ATPase, fusicoccin, with normal swelling. We propose that dgt is a signal-transduction mutation interfering with an auxin-signalling pathway that uses ABP1 as a receptor.