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In this study, we investigated the role of tRNA thiolation in thermospermine-dependent regulation of xylem development in Arabidopsis thaliana. Using a suppressor screen of the thermospermine-deficient mutant acaulis5 (acl5), which exhibits dwarfism and excessive xylem differentiation, we identified suppressor-of-acl502 (sac502) as a recessive loss-of-function allele of CTU2, a gene encoding a key enzyme in the biosynthesis of the wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine. Mutations in other components of the same modification pathway, including ROL5 and TRM9, similarly suppressed the acl5 phenotype. Translational analyses using 5\u2032 leader-GUS reporter constructs revealed that the ctu2 mutation did not enhance translation of the mRNA containing a thermospermine-responsive uORF of SAC51, but instead significantly reduced translation of that of SACL3, a member of the SAC51 family, and that of LONESOME HIGHWAY (LHW), which contains another conserved uORF in the 5\u2032 leader region. Polysome profiling further demonstrated decreased association of SACL3 and LHW mRNAs with actively translating ribosomes in ctu2. Genetic interaction analyses supported the conclusion that the suppression of excessive xylem formation in acl5 by ctu2 is attributable to reduced LHW activity. In addition, ctu2 mutants displayed increased sensitivity to exogenous thermospermine, resembling the response of lhw mutants. Together, our results reveal that tRNA thiolation contributes to uORF-mediated translational regulation of key developmental regulators and identify tRNA modification as an important regulatory layer controlling vascular development.<\/jats:p>","DOI":"10.1093\/pcp\/pcag055","type":"journal-article","created":{"date-parts":[[2026,4,24]],"date-time":"2026-04-24T11:42:20Z","timestamp":1777030940000},"page":"1346-1355","source":"Crossref","is-referenced-by-count":0,"title":["Involvement of tRNA thiolation in uORF-mediated translational regulation during Xylogenesis in\n                    <i>Arabidopsis thaliana<\/i>"],"prefix":"10.1093","volume":"67","author":[{"given":"Yuichi","family":"Nishii","sequence":"first","affiliation":[{"name":"Graduate School of Environmental, Life, Natural Science and Technology, Okayama University , 3-1-1 Tsushimanaka, Kitaku, Okayama 700-8530 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daichi","family":"Araki","sequence":"additional","affiliation":[{"name":"Graduate School of Environmental, Life, Natural Science and Technology, Okayama University , 3-1-1 Tsushimanaka, Kitaku, Okayama 700-8530 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mitsuru","family":"Saraumi","sequence":"additional","affiliation":[{"name":"Graduate School of Environmental, Life, Natural Science and Technology, Okayama University , 3-1-1 Tsushimanaka, Kitaku, Okayama 700-8530 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8879-0718","authenticated-orcid":false,"given":"Taku","family":"Takahashi","sequence":"additional","affiliation":[{"name":"Graduate School of Environmental, Life, Natural Science and Technology, Okayama University , 3-1-1 Tsushimanaka, Kitaku, Okayama 700-8530 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,4,28]]},"reference":[{"key":"2026090803063587000_ref1","doi-asserted-by":"publisher","first-page":"537","DOI":"10.1080\/15476286.2017.1356562","article-title":"Celebrating wobble decoding: half a century and still much is new","volume":"15","author":"Agris","year":"2018","journal-title":"RNA Biol."},{"key":"2026090803063587000_ref2","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1080\/15476286.2025.2515663","article-title":"Epitranscriptomic modifications in plant RNAs","volume":"22","author":"Bhat","year":"2025","journal-title":"RNA Biol."},{"key":"2026090803063587000_ref3","doi-asserted-by":"publisher","first-page":"263","DOI":"10.1146\/annurev.bi.56.070187.001403","article-title":"Transfer RNA modification","volume":"56","author":"Bj\u00f6rk","year":"1987","journal-title":"Annu. 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