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Here, we used MALDI-MSI-based spatial metabolomics to systematically profile tobacco leaves during Pseudomonas syringae infection. Multidimensional analysis of 1,399 annotated metabolites revealed distinct spatiotemporal regulation patterns. Temporally, early infection (12\u2005h postinfection (hpi)) was characterized by increased organic acids and terpenoids, followed by a mid-stage shift toward phenolic acids and quinones (24 hpi) and a late-stage enrichment of alkaloids by 60 hpi. Spatially, constrained clustering produced anatomy-aligned segmentation maps and revealed cell type preferences across epidermal, mesophyll, and vascular regions, with directional redistribution of differentially expressed metabolites as infection progressed. Defense hormones, including salicylic acid (SA) and jasmonic acid (JA), preferentially accumulated in vascular bundles and varied dynamically over time. Functional validation through exogenous application of representative metabolites (eg calystegine C1 and L-phenylalanine) and hormones (JA and SA), together with genetic manipulation of JA-biosynthetic genes, confirmed their roles in reducing lesion development and suppressing bacterial proliferation. Notably, epidermal enrichment of alkaloids\u2014especially nicotine\u2014and amino acid derivatives showed a decrease-then-increase pattern consistent with early consumption and later replenishment; nicotine's defensive contribution was further supported using a low-nicotine mutant. Collectively, P. syringae infection orchestrates a coordinated, cell type-compartmentalized defense metabolic program in tobacco, providing a resource for mechanistic studies and metabolic engineering of disease resistance. This time- and tissue-resolved atlas links metabolite remodeling to hormone-associated signaling and chemical barrier formation during wildfire disease progression.<\/jats:p>","DOI":"10.1093\/plphys\/kiag623","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T11:43:57Z","timestamp":1787139837000},"source":"Crossref","is-referenced-by-count":0,"title":["Spatiotemporal metabolomics of tobacco leaves in response to\n                    <i>Pseudomonas syringae<\/i>\n                    infection"],"prefix":"10.1093","volume":"202","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-9732-4948","authenticated-orcid":false,"given":"Xinhua","family":"Tian","sequence":"first","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]},{"name":"Beijing Life Science Academy , Beijing 102200 ,","place":["China"]},{"name":"Department of Plant Pathology, College of Plant Protection, Shenyang Agricultural University , No. 120 Dongling Road, Shenhe District, Shenyang, Liaoning 110866 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-8875-1038","authenticated-orcid":false,"given":"Zechao","family":"Qu","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaqi","family":"Wang","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]},{"name":"Beijing Life Science Academy , Beijing 102200 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lijun","family":"Meng","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-9540-7168","authenticated-orcid":false,"given":"Huan","family":"Su","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]},{"name":"Beijing Life Science Academy , Beijing 102200 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4650-0257","authenticated-orcid":false,"given":"Qian","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Plant Pathology, College of Plant Protection, Shenyang Agricultural University , No. 120 Dongling Road, Shenhe District, Shenyang, Liaoning 110866 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4998-1279","authenticated-orcid":false,"given":"Jiemeng","family":"Tao","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]},{"name":"Beijing Life Science Academy , Beijing 102200 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Lu","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]},{"name":"Beijing Life Science Academy , Beijing 102200 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-2195-1274","authenticated-orcid":false,"given":"Qiao","family":"Wang","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]},{"name":"Beijing Life Science Academy , Beijing 102200 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"He","family":"Li","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-2405-1414","authenticated-orcid":false,"given":"Jianfeng","family":"Zhang","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]},{"name":"Beijing Life Science Academy , Beijing 102200 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9991-423X","authenticated-orcid":false,"given":"Peijian","family":"Cao","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]},{"name":"Beijing Life Science Academy , Beijing 102200 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-3001-7065","authenticated-orcid":false,"given":"Yuanhu","family":"Xuan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Elemento-Organic Chemistry and Department of Plant Protection, National Pesticide Engineering Research Center, Nankai University , 94 Weijin Road, Tianjin 300071 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9683-4402","authenticated-orcid":false,"given":"Jingjing","family":"Jin","sequence":"additional","affiliation":[{"name":"China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 ,","place":["China"]},{"name":"Beijing Life Science Academy , Beijing 102200 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"2026090102370467800_kiag623-B1","doi-asserted-by":"publisher","first-page":"1414","DOI":"10.1111\/ppa.13244","article-title":"Phylogenetic, genetic, and phenotypic diversity of pseudomonas syringae pv. syringae strains isolated from citrus blast and black pit in Tunisia","volume":"69","author":"Abdellatif","year":"2020","journal-title":"Plant 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