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Iterative subtraction facilitates automated, quantitative analysis of multiple pollen tube growth features

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Abstract

In flowering plants, successful reproduction and generation of seed depends on the delivery of immotile sperm to female gametes via the pollen tube. As reproduction in flowering plants is the cornerstone of our agricultural industry, there is a need to uncover the genes, small molecules, and environmental conditions that affect pollen tube growth dynamics. However, methods for measuring pollen tube phenotypes are labor intensive, and suffer from a tradeoff between workload and resolution. To approach these problems, we use an image analysis technique called Automated Stack Iterative Subtraction (ASIST). Our tool converts growing pollen tube tips into closed particles, making the automated simultaneous extraction of multiple pollen tube phenotypes from hundreds of individual cells tractable via existing particle identification technology. Here we use our tool to analyze growth dynamics of pollen tubes in vitro, and semi in vivo. We show that ASIST provides a framework for robust, high throughput analysis of pollen tube growth behaviors in populations of cells, thus facilitating pollen tube phenomics.

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Acknowledgements

We thank Judith Bender, Daniel Damineli, Alison DeLong, Jennifer Forcina, and Jenna Kotak for helpful discussions and Sheila McCormick for providing Tomato (VF-36) LAT52:GFP seeds. M.A.J. and N.P. were supported by National Science Foundation (NSF) Grant IOS- IOS-1540019 (M.J.) and National Institutes of Health Training Grant #T32-GM007601 (N.P.).

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  1. Alexander Leydon

    Present address: Department of Biology, University of Washington, Seattle, WA, 98195, USA

Authors and Affiliations

  1. Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, 02912, USA

    Nathaniel Ponvert, Jacob Goldberg & Mark A. Johnson

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  1. Nathaniel Ponvert
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Corresponding author

Correspondence to Mark A. Johnson.

Additional information

Communicated by Thomas Dresselhaus.

A contribution to the special issue ‘Cellular Omics Methods in Plant Reproduction Research’.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure 1

ASIST preprocessing for DIC tip identification. ASIST is capable of defining tip extension in spite of variable tip definition from Differential Interference Contrast (DIC) timelapse movies. A1-5. Five representative frames from a DIC timelapse of tomato pollen tube growth.  B1-5. Frames from series A after minimum pixel intensity filtering over five pixels. C1-5. Frames from series B after contrast enhancement.  D1-5. Frames from series C after median pixel intensity filtering over eight pixels.  E1-5. Frames from series D after inversion.  F1-5. Frames from series E after contrast enhancement.  G1-5. Frames from series F after iterative subtraction.  H1-5.  Frames from series G (false colored in cyan) overlayed onto original DIC frames (PNG 613 kb)

Supplementary Figure 2

Growth rate comparison between Semi-in vivo and in vitro grown pollen tubes. A. Comparison between pollen tube total displacement. S.I.V. pollen tubes grow significantly longer than in vitro pollen (ANOVA, α = 0.05, Bonferroni post hoc). B. Comparison between average growth rate of S.I.V. and in vitro pollen tubes. S.I.V. tubes grow significantly faster than in vitro grown pollen (PNG 32 kb)

Supplementary material 3 (ZIP 933904 kb)

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Ponvert, N., Goldberg, J., Leydon, A. et al. Iterative subtraction facilitates automated, quantitative analysis of multiple pollen tube growth features. Plant Reprod 32, 45–54 (2019). https://doi.org/10.1007/s00497-018-00351-8

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