{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T09:10:18Z","timestamp":1789031418898,"version":"build-2803163510"},"reference-count":26,"publisher":"Oxford University Press (OUP)","license":[{"start":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T00:00:00Z","timestamp":1786924800000},"content-version":"am","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/pages\/standard-publication-reuse-rights"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["#2414183"],"award-info":[{"award-number":["#2414183"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["#2425989"],"award-info":[{"award-number":["#2425989"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"name":"United States Department of Agriculture National Institute of Food and Agriculture","award":["#2022-67013-36144"],"award-info":[{"award-number":["#2022-67013-36144"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Probe-based spatial transcriptomics platforms use predefined oligonucleotide panels to detect selected RNAs in tissue sections while preserving transcript spatial coordinates. Accurate cell segmentation is required for reliable transcript-to-cell assignments. This analytical process is affected in plant tissues by cell walls, large vacuoles, and strong autofluorescence, which often reduce boundary contrast and elevate background. Nucleus-only segmentation with fixed-distance expansion can be an alternative approach, but it underestimates cellular area and morphology and reduces the number of assignable transcripts per cell. Here, we present a practical workflow for segmentation and downstream processing in plant probe-based spatial transcriptomics. Using the soybean nodule, soybean seed, rice root, and wheat inflorescence, we demonstrate the applicability of our workflow across species, tissues, and technological platforms. In brief, candidate cell masks are generated from available fluorescence signals and then selected and corrected using two napari plugins. Transcript-informed refinement with Baysor is included as an optional step. Upon benchmarking our approach using a collection of metrics (assignment yield, background\/negative controls, and per-cell transcript\/gene distributions) and linked segmentation choices to expression-matrix quality and downstream clustering, we demonstrate the potential of our workflow to support the analysis of plant probe-based spatial transcriptomics.<\/jats:p>","DOI":"10.1093\/jxb\/erag403","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T11:12:06Z","timestamp":1786705926000},"source":"Crossref","is-referenced-by-count":0,"title":["Optimizing cell segmentation and downstream processing for plant probe-based spatial transcriptomics"],"prefix":"10.1093","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0418-9819","authenticated-orcid":false,"given":"Yaohua","family":"Li","sequence":"first","affiliation":[{"name":"Division of Plant Science and Technology, College of Agriculture, Food, and Natural Resources, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]},{"name":"Interdisciplinary Plant Group of Missouri-Columbia, Christopher S. Bond Life Sciences Center, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9837-0397","authenticated-orcid":false,"given":"Erik J","family":"Am\u00e9zquita","sequence":"additional","affiliation":[{"name":"Division of Plant Science and Technology, College of Agriculture, Food, and Natural Resources, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]},{"name":"Interdisciplinary Plant Group of Missouri-Columbia, Christopher S. Bond Life Sciences Center, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]},{"name":"Department of Mathematics, College of Arts and Science, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Avinash","family":"Shrestha","sequence":"additional","affiliation":[{"name":"Division of Plant Science and Technology, College of Agriculture, Food, and Natural Resources, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]},{"name":"Interdisciplinary Plant Group of Missouri-Columbia, Christopher S. Bond Life Sciences Center, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sutton","family":"Tennant","sequence":"additional","affiliation":[{"name":"Division of Plant Science and Technology, College of Agriculture, Food, and Natural Resources, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]},{"name":"Interdisciplinary Plant Group of Missouri-Columbia, Christopher S. Bond Life Sciences Center, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7419-9129","authenticated-orcid":false,"given":"Marc","family":"Libault","sequence":"additional","affiliation":[{"name":"Division of Plant Science and Technology, College of Agriculture, Food, and Natural Resources, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]},{"name":"Interdisciplinary Plant Group of Missouri-Columbia, Christopher S. Bond Life Sciences Center, University of Missouri-Columbia , Columbia, MO 65211 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,8,17]]},"reference":[{"key":"2026091004412590600_erag403-B1","volume-title":"Xenium in situ multimodal cell segmentation: workflow and data highlights. Technical Note CG000750 Rev B","author":"10x Genomics","year":"2025"},{"key":"2026091004412590600_erag403-B2","doi-asserted-by":"crossref","first-page":"1018","DOI":"10.1016\/j.tplants.2024.03.010","article-title":"Lost in space: what single-cell RNA sequencing cannot tell you","volume":"29","author":"Adema","year":"2024","journal-title":"Trends in Plant Science"},{"key":"2026091004412590600_erag403-B3","doi-asserted-by":"crossref","DOI":"10.1002\/bies.201900221","article-title":"Spatially resolved transcriptomes-next generation tools for tissue exploration","volume":"42","author":"Asp","year":"2020","journal-title":"BioEssays"},{"key":"2026091004412590600_erag403-B4","doi-asserted-by":"crossref","DOI":"10.1016\/j.xplc.2024.100984","article-title":"Single-cell transcriptome atlases of soybean root and mature nodule reveal new regulatory programs that control the nodulation process","volume":"5","author":"Cervantes-Perez","year":"2024","journal-title":"Plant 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Zenodo. 10.5281\/zenodo.3555620"},{"key":"2026091004412590600_erag403-B20","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1038\/s41592-020-01018-x","article-title":"Cellpose: a generalist algorithm for cellular segmentation","volume":"18","author":"Stringer","year":"2021","journal-title":"Nature Methods"},{"key":"2026091004412590600_erag403-B21","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1186\/s13059-025-03920-z","article-title":"The differential subcellular localization of soybean transcripts, an additional regulatory mechanism of gene activity","volume":"27","author":"Tennant","year":"2026","journal-title":"Genome Biology"},{"key":"2026091004412590600_erag403-B22","doi-asserted-by":"publisher","author":"Weigert","year":"2022","DOI":"10.1109\/ISBIC56247.2022.9854534"},{"key":"2026091004412590600_erag403-B23","doi-asserted-by":"crossref","first-page":"6387","DOI":"10.1038\/s41467-024-50737-8","article-title":"Mapping the molecular landscape of Lotus japonicus nodule organogenesis through spatiotemporal transcriptomics","volume":"15","author":"Ye","year":"2024","journal-title":"Nature Communications"},{"key":"2026091004412590600_erag403-B24","doi-asserted-by":"crossref","first-page":"1571","DOI":"10.1111\/tpj.16437","article-title":"Spatial transcriptomics drives a new era in plant research","volume":"116","author":"Yin","year":"2023","journal-title":"The Plant Journal"},{"key":"2026091004412590600_erag403-B25","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1016\/j.cell.2024.10.050","article-title":"A spatially resolved multi-omic single-cell atlas of soybean development","volume":"188","author":"Zhang","year":"2025","journal-title":"Cell"},{"key":"2026091004412590600_erag403-B26","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1038\/s41586-025-08941-z","article-title":"Single-cell transcriptomics reveal how root tissues adapt to soil stress","volume":"642","author":"Zhu","year":"2025","journal-title":"Nature"}],"container-title":["Journal of Experimental Botany"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/jxb\/advance-article-pdf\/doi\/10.1093\/jxb\/erag403\/70660329\/erag403.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/jxb\/advance-article-pdf\/doi\/10.1093\/jxb\/erag403\/70660329\/erag403.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T08:41:55Z","timestamp":1789029715000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jxb\/advance-article\/doi\/10.1093\/jxb\/erag403\/8762125"}},"subtitle":[],"editor":[{"given":"John","family":"Lunn","sequence":"additional","affiliation":[{"name":"Max-Planck-Institut f\u00fcr Molekulare Pflanzenphysiologie ,","place":["Germany"]}],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,8,17]]},"references-count":26,"URL":"https:\/\/doi.org\/10.1093\/jxb\/erag403","relation":{},"ISSN":["0022-0957","1460-2431"],"issn-type":[{"value":"0022-0957","type":"print"},{"value":"1460-2431","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,17]]},"article-number":"erag403"}}