HYPOTrace: Image Analysis Software for Measuring Hypocotyl Growth and Shape Demonstrated on Arabidopsis Seedlings Undergoing Photomorphogenesis

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Abstract
Analysis of time series of images can quantify plant growth and development, including the effects of genetic mutations (phenotypes) that give information about gene function. Here is demonstrated a software application named HYPOTrace that automatically extracts growth and shape information from electronic gray-scale images of Arabidopsis (Arabidopsis thaliana) seedlings. Key to the method is the iterative application of adaptive local principal components analysis to extract a set of ordered midline points (medial axis) from images of the seedling hypocotyl. Pixel intensity is weighted to avoid the medial axis being diverted by the cotyledons in areas where the two come in contact. An intensity feature useful for terminating the midline at the hypocotyl apex was isolated in each image by subtracting the baseline with a robust local regression algorithm. Applying the algorithm to time series of images of Arabidopsis seedlings responding to light resulted in automatic quantification of hypocotyl growth rate, apical hook opening, and phototropic bending with high spatiotemporal resolution. These functions are demonstrated here on wild-type, cryptochrome1, and phototropin1 seedlings for the purpose of showing that HYPOTrace generated expected results and to show how much richer the machine-vision description is compared to methods more typical in plant biology. HYPOTrace is expected to benefit seedling development research, particularly in the photomorphogenesis field, by replacing many tedious, error-prone manual measurements with a precise, largely automated computational tool.