The ability of plants to sense and orient their root growth towards gravity is studied in many laboratories. It is known that manual analysis of image data is subjected to human bias. Several semi-automated tools are available for analysing images from flatbed scanners, but there is no solution to automatically measure root bending angle over time for vertical-stage microscopy images. To address these problems, we developed ACORBA, which is an automated software that can measure root bending angle over time from vertical-stage microscope and flatbed scanner images. ACORBA also has a semi-automated mode for camera or stereomicroscope images. It represents a flexible approach based on both traditional image processing and deep machine learning segmentation to measure root angle progression over time. As the software is automated, it limits human interactions and is reproducible. ACORBA will support the plant biologist community by reducing labour and increasing reproducibility of image analysis of root gravitropism.

Department of Experimental Plant Biology Faculty of Sciences Charles University Prague Czech Republic

See more in PubMed

Band, L. R. , Wells, D. M. , Larrieu, A. , Sun, J. , Middleton, A. M. , French, A. P. , Brunoud, G. , Sato, E. M. , Wilson, M. H. , Peret, B. , Oliva, M. , Swarup, R. , Sairanen, I. , Parry, G. , Ljung, K. , Beeckman, T. , Garibaldi, J. M. , Estelle, M. , Owen, M. R. , … Bennett, M. J. (2012). Root gravitropism is regulated by a transient lateral auxin gradient controlled by a tipping-point mechanism. Proceedings of the National Academy of Sciences, 109, 4668–4673. 10.1073/pnas.1201498109 PubMed DOI PMC

Basu, P. , Pal, A. , Lynch, J. P. , & Brown, K. M. (2007). A novel image-analysis technique for kinematic study of growth and curvature. Plant Physiology, 145, 305–316. 10.1104/pp.107.103226 PubMed DOI PMC

Bennett, M. J. , Marchant, A. , Green, H. G. , May, S. T. , Ward, S. P. , Millner, P. A. , Walker, A. R. , Schulz, B. , & Feldmann, K. A. (1996). Arabidopsis AUX1 gene: A permease-like regulator of root gravitropism. Science, 273, 948–950. 10.1126/science.273.5277.948 PubMed DOI

Bernotas, G. , Scorza, L. C. T. , Hansen, M. F. , Hales, I. J. , Halliday, K. J. , Smith, L. N. , Smith, M. L. , & McCormick, A. J. (2019). A photometric stereo-based 3D imaging system using computer vision and deep learning for tracking plant growth. GigaScience, 8, giz056. 10.1093/gigascience/giz056 PubMed DOI PMC

Clark, R. T. , Famoso, A. N. , Zhao, K. , Shaff, J. E. , Craft, E. J. , Bustamante, C. D. , Mccouch, S. R. , Aneshansley, D. J. , & Kochian, L. V. (2013). High-throughput two-dimensional root system phenotyping platform facilitates genetic analysis of root growth and development: Root phenotyping platform. Plant, Cell & Environment, 36, 454–466. 10.1111/j.1365-3040.2012.02587.x PubMed DOI

Cséplő, Á. , Zsigmond, L. , Andrási, N. , Baba, A. I. , Labhane, N. M. , Pető, A. , Kolbert, Z. , Kovács, H. E. , Steinbach, G. , Szabados, L. , Fehér, A. , & Rigó, G. (2021). The AtCRK5 protein kinase is required to maintain the ROS NO balance affecting the PIN2-mediated root Gravitropic response in Arabidopsis. International Journal of Molecular Sciences, 22, 5979. 10.3390/ijms22115979 PubMed DOI PMC

Fendrych, M. , Akhmanova, M. , Merrin, J. , Glanc, M. , Hagihara, S. , Takahashi, K. , Uchida, N. , Torii, K. U. , & Friml, J. (2018). Rapid and reversible root growth inhibition by TIR1 auxin signalling. Nature Plants, 4, 453–459. 10.1038/s41477-018-0190-1 PubMed DOI PMC

Fischer, C. A. , Besora-Casals, L. , Rolland, S. G. , Haeussler, S. , Singh, K. , Duchen, M. , Conradt, B. , & Marr, C. (2020). MitoSegNet: Easy-to-use deep learning segmentation for analyzing mitochondrial morphology. IScience, 23, 101601. 10.1016/j.isci.2020.101601 PubMed DOI PMC

French, A. , Ubeda-Tomás, S. , Holman, T. J. , Bennett, M. J. , & Pridmore, T. (2009). High-throughput quantification of root growth using a novel image-analysis tool. Plant Physiology, 150, 1784–1795. 10.1104/pp.109.140558 PubMed DOI PMC

Goh, T. (2019). Long-term live-cell imaging approaches to study lateral root formation in Arabidopsis thaliana . Microscopy, 68, 4–12. 10.1093/jmicro/dfy135 PubMed DOI

Grossmann, G. , Guo, W.-J. , Ehrhardt, D. W. , Frommer, W. B. , Sit, R. V. , Quake, S. R. , & Meier, M. (2011). The RootChip: An integrated microfluidic Chip for plant science. The Plant Cell, 23, 4234–4240. 10.1105/tpc.111.092577 PubMed DOI PMC

Hamidinekoo, A. , Garzón-Martínez, G. A. , Ghahremani, M. , Corke, F. M. K. , Zwiggelaar, R. , Doonan, J. H. , & Lu, C. (2020). DeepPod: A convolutional neural network based quantification of fruit number in Arabidopsis. GigaScience, 9, giaa012. 10.1093/gigascience/giaa012 PubMed DOI PMC

Jost, A. P.-T. , & Waters, J. C. (2019). Designing a rigorous microscopy experiment: Validating methods and avoiding bias. The Journal of Cell Biology, 218, 1452–1466. 10.1083/jcb.201812109 PubMed DOI PMC

Kingma, D. P. , & Ba, J. (2017). Adam: A method for stochastic optimization. ArXiv:1412.6980 [Cs]. http://arxiv.org/abs/1412.6980

Lazic, S. E. (2018). The quarterly review of biology. In Experimental design for laboratory biologists: Maximising information and improving reproducibility (Vol. 93, p. 131). Cambridge University Press. 10.1086/698030 DOI

Lee, J.-Y. , & Kitaoka, M. (2018). A beginner’s guide to rigor and reproducibility in fluorescence imaging experiments. Molecular Biology of the Cell, 29, 1519–1525. 10.1091/mbc.E17-05-0276 PubMed DOI PMC

Lindsey, B. E. , Rivero, L. , Calhoun, C. S. , Grotewold, E. , & Brkljacic, J. (2017). Standardized method for high-throughput sterilization of Arabidopsis seeds. Journal of Visualized Experiments, 128, 56587. 10.3791/56587 PubMed DOI PMC

Liu, C.-J. (2012). Deciphering the enigma of lignification: Precursor transport, oxidation, and the topochemistry of lignin assembly. Molecular Plant, 5, 304–317. 10.1093/mp/ssr121 PubMed DOI

Luschnig, C. , Gaxiola, R. A. , Grisafi, P. , & Fink, G. R. (1998). EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana . Genes & Development, 12, 2175–2187. 10.1101/gad.12.14.2175 PubMed DOI PMC

Marquès-Bueno, M. M. , Armengot, L. , Noack, L. C. , Bareille, J. , Rodriguez, L. , Platre, M. P. , Bayle, V. , Liu, M. , Opdenacker, D. , Vanneste, S. , Möller, B. K. , Nimchuk, Z. L. , Beeckman, T. , Caño-Delgado, A. I. , Friml, J. , & Jaillais, Y. (2021). Auxin-regulated reversible inhibition of TMK1 signaling by MAKR2 modulates the dynamics of root gravitropism. Current Biology, 31, 228–237.e10. 10.1016/j.cub.2020.10.011 PubMed DOI PMC

Naeem, A. , French, A. P. , Wells, D. M. , & Pridmore, T. P. (2011). High-throughput feature counting and measurement of roots. Bioinformatics, 27, 1337–1338. 10.1093/bioinformatics/btr126 PubMed DOI

Nickerson, R. S. (1998). Confirmation bias: A ubiquitous phenomenon in many guises. Review of General Psychology, 2, 175–220. 10.1037/1089-2680.2.2.175 DOI

Oliva, M. , & Dunand, C. (2007). Waving and skewing: How gravity and the surface of growth media affect root development in Arabidopsis . New Phytologist, 176, 37–43. 10.1111/j.1469-8137.2007.02184.x PubMed DOI

Ovečka, M. , Vaškebová, L. , Komis, G. , Luptovčiak, I. , Smertenko, A. , & Šamaj, J. (2015). Preparation of plants for developmental and cellular imaging by light-sheet microscopy. Nature Protocols, 10, 1234–1247. 10.1038/nprot.2015.081 PubMed DOI

Platre, M. P. , Bayle, V. , Armengot, L. , Bareille, J. , Marquès-Bueno, M. , del, M. , Creff, A. , Maneta-Peyret, L. , Fiche, J.-B. , Nollmann, M. , Miège, C. , Moreau, P. , Martinière, A. , & Jaillais, Y. (2019). Developmental control of plant rho GTPase nano-organization by the lipid phosphatidylserine. Science, 364, 57–62. 10.1126/science.aav9959 PubMed DOI

Prigge, M. J. , Platre, M. , Kadakia, N. , Zhang, Y. , Greenham, K. , Szutu, W. , Pandey, B. K. , Bhosale, R. A. , Bennett, M. J. , Busch, W. , & Estelle, M. (2020). Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions. eLife, 9, e54740. 10.7554/eLife.54740 PubMed DOI PMC

Retzer, K. , Akhmanova, M. , Konstantinova, N. , Malínská, K. , Leitner, J. , Petrášek, J. , & Luschnig, C. (2019). Brassinosteroid signaling delimits root gravitropism via sorting of the Arabidopsis PIN2 auxin transporter. Nature Communications, 10, 5516. 10.1038/s41467-019-13543-1 PubMed DOI PMC

Ronneberger, O. , Fischer, P. , & Brox, T. (2015). U-Net: Convolutional networks for biomedical image segmentation. ArXiv:1505.04597 [Cs]. http://arxiv.org/abs/1505.04597

Samakovli, D. , Roka, L. , Dimopoulou, A. , Plitsi, P. K. , Žukauskait, A. , Georgopoulou, P. , Novák, O. , Milioni, D. , & Hatzopoulos, P. (2021). HSP90 affects root growth in Arabidopsis by regulating the polar distribution of PIN1. The New Phytologist, 231, 1814–1831. 10.1111/nph.17528 PubMed DOI

Serre, N. B. , Kralík, D. , Yun, P. , Shabala, S. , Slouka, Z. , & Fendrych, M. (2021). The AFB1 auxin receptor controls rapid auxin signaling and root growth through membrane depolarization in Arabidopsis thaliana . Nature Plants, 7, 1229–1238. 10.1101/2021.01.05.425399 PubMed DOI PMC

Shih, H.-W. , DePew, C. L. , Miller, N. D. , & Monshausen, G. B. (2015). The cyclic nucleotide-gated channel CNGC14 regulates root gravitropism in Arabidopsis thaliana . Current Biology, 25, 3119–3125. 10.1016/j.cub.2015.10.025 PubMed DOI

Slovak, R. , Göschl, C. , Su, X. , Shimotani, K. , Shiina, T. , & Busch, W. (2014). A scalable open-source pipeline for large-scale root phenotyping of Arabidopsis . The Plant Cell, 26, 2390–2403. 10.1105/tpc.114.124032 PubMed DOI PMC

Swarup, R. , Kramer, E. M. , Perry, P. , Knox, K. , Leyser, H. M. O. , Haseloff, J. , Beemster, G. T. S. , Bhalerao, R. , & Bennett, M. J. (2005). Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal. Nature Cell Biology, 7, 1057–1065. 10.1038/ncb1316 PubMed DOI

von Wangenheim, D. , Hauschild, R. , Fendrych, M. , Barone, V. , Benková, E. , & Friml, J. (2017). Live tracking of moving samples in confocal microscopy for vertically grown roots. eLife, 6, 26792. 10.7554/eLife.26792 PubMed DOI PMC

Yasrab, R. , Atkinson, J. A. , Wells, D. M. , French, A. P. , Pridmore, T. P. , & Pound, M. P. (2019). RootNav 2.0: Deep learning for automatic navigation of complex plant root architectures. GigaScience, 8, giz123. 10.1093/gigascience/giz123 PubMed DOI PMC

Yazdanbakhsh, N. , & Fisahn, J. (2012). High-throughput phenotyping of root growth dynamics. In J. Normanly (Ed.), High-throughput phenotyping in plants (Vol. 918, pp. 21–40). Humana Press. 10.1007/978-1-61779-995-2_3 PubMed DOI

RAF-like protein kinases mediate a deeply conserved, rapid auxin response

The AUX1-AFB1-CNGC14 module establishes a longitudinal root surface pH profile

The AFB1 auxin receptor controls the cytoplasmic auxin response pathway in Arabidopsis thaliana