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Automated motion estimation of root responses to sucrose in two Arabidopsis thaliana genotypes using confocal microscopy

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Abstract

Root growth is a highly dynamic process influenced by genetic background and environment. This paper reports the development of R scripts that enable root growth kinematic analysis that complements a new motion analysis tool: PlantVis. Root growth of Arabidopsis thaliana expressing a plasma membrane targeted GFP (C24 and Columbia 35S:LTI6b-EGFP) was imaged using time-lapse confocal laser scanning microscopy. Displacement of individual pixels in the time-lapse sequences was estimated automatically by PlantVis, producing dense motion vector fields. R scripts were developed to extract kinematic growth parameters and report displacement to ±0.1 pixel. In contrast to other currently available tools, Plantvis-R delivered root velocity profiles without interpolation or averaging across the root surface and also estimated the uncertainty associated with tracking each pixel. The PlantVis-R analysis tool has a range of potential applications in root physiology and gene expression studies, including linking motion to specific cell boundaries and analysis of curvature. The potential for quantifying genotype × environment interactions was examined by applying PlantVis-R in a kinematic analysis of root growth of C24 and Columbia, under contrasting carbon supply. Large genotype-dependent effects of sucrose were recorded. C24 exhibited negligible differences in elongation zone length and elongation rate but doubled the density of lateral roots in the presence of sucrose. Columbia, in contrast, increased its elongation zone length and doubled its elongation rate and the density of lateral roots.

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Abbreviations

CLSM:

Confocal laser scanning microscopy

DAG:

Days after germination

DVF:

Displacement vector field

EGR:

Elemental growth rate

PIV:

Particle image velocimetry

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Acknowledgments

Prof. Jim Haseloff (Cambridge University) for the kind donation of A. thaliana. D. White (University of Western Australia) for access to the Cambridge University PIV code. This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), UK. SCRI receives grant-in-aid from the Scottish Government Rural and Environmental Research and Analysis Directorate (SG-RERAD). Dr. Kath Wright, Dr. Lionel Dupuy and Prof. Philip White for helpful comments on this manuscript.

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Author notes

  1. Nathalie Wuyts

    Present address: Earth and Life Institute, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

Authors and Affiliations

  1. Scottish Crop Research Institute, Dundee, DD2 5DA, UK

    Nathalie Wuyts, A. Glyn Bengough & Tracy A. Valentine

  2. School of Computing, University of Dundee, Dundee, DD1 4HN, UK

    Timothy J. Roberts, Chengjin Du & Stephen J. McKenna

  3. Division of Civil Engineering, University of Dundee, Dundee, DD1 4HN, UK

    A. Glyn Bengough & M. Fraser Bransby

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  1. Nathalie Wuyts
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  2. A. Glyn Bengough
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Corresponding author

Correspondence to Tracy A. Valentine.

Electronic supplementary material

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425_2011_1435_MOESM1_ESM.tif

Fig. S1 Motion estimation in a time-lapse image sequence of an A. thaliana primary root. PlantVis was applied on a CLSM image sequence (1 min between frames), acquired using transmission settings, of an A. thaliana C24 LTI6b-EGFP primary root (10 DAG) at 10× magnification. Results were imported into R for analysis and visualisation. a Confocal image. b Absolute velocity (μm min−1) (TIFF 1,869 kb)

425_2011_1435_MOESM2_ESM.mpg

Fig. S2 Video of a time-lapse CLSM image sequence (1 min between frames) of an A. thaliana LTI6b-EGFP primary root (8 DAG) at 10× magnification. a C24. b Col (MPG 186 kb)

Supplementary material 3 (MPG 222 kb)

425_2011_1435_MOESM4_ESM.tif

Fig. S3 Scatterplots of growth parameters for individual A. thaliana primary roots. PlantVis was applied on CLSM image sequences (1 min between frames) of A. thaliana C24 LTI6b-EGFP primary roots (5, 8 and 11 DAG) at 10× magnification. Growth parameters are plotted against longitudinal velocity for three individual roots (black, dark grey, light grey) at 5, 8 and 11 DAG, including root length (a), growth zone length (b), elongation zone length (c), division zone length (d), maximum elemental growth rate (e), and position of maximum elemental growth rate (f) along the root axis (as distance from the quiescent centre) (TIFF 2,472 kb)

Supplementary Table S1 (RTF 42 kb)

Supplementary Table S2 (DOC 36 kb)

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Wuyts, N., Bengough, A.G., Roberts, T.J. et al. Automated motion estimation of root responses to sucrose in two Arabidopsis thaliana genotypes using confocal microscopy. Planta 234, 769–784 (2011). https://doi.org/10.1007/s00425-011-1435-7

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