N-Sulfonated IAA was discovered as a novel auxin metabolite in Urtica where it is biosynthesized de novo utilizing inorganic sulfate. It showed no auxin activity in DR5::GUS assay, implying possible inactivation/storage mechanism. A novel auxin derivative, N-sulfoindole-3-acetic acid (IAA-N-SO3H, SIAA), was discovered in stinging nettle (Urtica dioica) among 116 sulfonated metabolites putatively identified by a semi-targeted UHPLC-QqTOF-MS analysis of 23 plant/algae/fungi species. These sulfometabolites were detected based on the presence of a neutral loss of sulfur trioxide, as indicated by the m/z difference of 79.9568 Da in the MS2 spectra. The structure of newly discovered SIAA was confirmed by synthesizing its standard and comparing retention time, m/z and MS2 spectrum with those of SIAA found in Urtica. To study its natural occurrence, 73 species in total were further analyzed by UHPLC-QqTOF-MS or targeted UHPLC-MS/MS method with a limit of detection of 244 fmol/g dry weight. However, SIAA was only detected in Urtica at a concentration of 13.906 ± 9.603 nmol/g dry weight. Its concentration was > 30 times higher than that of indole-3-acetic acid (IAA), and the SIAA/IAA ratio was further increased under different light conditions, especially in continuous blue light. In addition to SIAA, structurally similar metabolites, N-sulfoindole-3-lactic acid, 4-(sulfooxy)phenyllactic acid and 4-(sulfooxy)phenylacetic acid, were detected in Urtica for the first time. SIAA was biosynthesized from inorganic sulfate in seedlings, as confirmed by the incorporation of exogenous 34S-ammonium sulfate (1 mM and 10 mM). SIAA exhibited no auxin activity, as demonstrated by both the Arabidopsis DR5::GUS assay and the Arabidopsis phenotype analysis. Sulfonation of IAA may therefore be a mechanism for IAA deactivation and/or storage in Urtica, similar to sulfonation of the jasmonates in Arabidopsis.

Czech Advanced Technology and Research Institute Palacký University Olomouc Šlechtitelů 27 CZ 77900 Olomouc Czech Republic

Department of Chemical Biology Palacký University Olomouc Šlechtitelů 27 CZ 77900 Olomouc Czech Republic

Department of Experimental Biology Palacký University Olomouc Šlechtitelů 27 CZ 77900 Olomouc Czech Republic

Laboratory of Growth Regulators Institute of Experimental Botany The Czech Academy of Sciences and Faculty of Science Palacký University Olomouc Czech Republic

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Abdullah Y, Schneider B, Petersen M (2008) Occurrence of rosmarinic acid, chlorogenic acid and rutin in Marantaceae species. Phytochem Lett 1:199–203

Arcos M, Olivera ER, Arias S, Naharro G, Luengo JM (2010) The 3,4-dihydroxyphenylacetic acid catabolon, a catabolic unit for degradation of biogenic amines tyramine and dopamine in Pseudomonas putida U. Environ Microbiol 12:1684–1704 PubMed

Baek D, Pathange P, Chung JS, Jiang JF, Gao LQ, Oikawa A, Hirai MY, Saito K, Pare PW, Shi HZ (2010) A stress-inducible sulphotransferase sulphonates salicylic acid and confers pathogen resistance in Arabidopsis. Plant, Cell Environ 33:1383–1392 PubMed

Bialek K, Cohen JD (1986) Isolation and partial characterization of the major amide-linked conjugate of indole-3-acetic-acid from phaseolus-vulgarIS L. Plant Physiol 80:99–104 PubMed PMC

Boerjan W, Cervera MT, Delarue M, Beeckman T, Dewitte W, Bellini C, Caboche M, Vanonckelen H, Vanmontagu M, Inze D (1995) Superroot, a recessive mutation in arabidopsis, confers auxin overproduction. Plant Cell 7:1405–1419 PubMed PMC

Brunoni F, Pencík A, Zukauskaite A, Ament A, Kopecná M, Collani S, Kopecny D, Novák O (2023) Amino acid conjugation of oxIAA is a secondary metabolic regulation involved in auxin homeostasis. New Phytol 238:2264–2270 PubMed

Cheynier V, Comte G, Davies KM, Lattanzio V, Martens S (2013) Plant phenolics: Recent advances on their biosynthesis, genetics, and ecophysiology. Plant Physiol Biochem 72:1–20 PubMed

Davies PJ (2004) The plant hormones: Their nature, occurrence, and functions. Plant Hormones: Biosynthesis, Signal Transduction, Action. 1–15

De Diego N, Fürst T, Humplík JF, Ugena L, Podlesáková K, Spíchal L (2017) An automated method for high-throughput screening of arabidopsis rosette growth in multi-well plates and its validation in stress conditions. Front Plant Sci. 10.3389/fpls.2017.01702 PubMed PMC

Di Q, Li Y, Zhang DP, Wu W, Zhang L, Zhao X, Luo L, Yu LL (2022) A novel type of phytosulfokine, PSK-ε, positively regulates root elongation and formation of lateral roots and root nodules in Medicago truncatula. Plant Signal Behav 17:2134672 PubMed PMC

Elliott MC, Stowe BB (1970) A novel sulphonated natural indole. Phytochemistry 9:1629–2000

Faulkner IJ, Rubery PH (1992) Flavonoids and flavonoid sulfates as probes of auxin-transport regulation in cucurbita-pepo hypocotyl segments and vesicles. Planta 186:618–625 PubMed

Fernández-Milmanda GL, Crocco CD, Reichelt M, Mazza CA, Köllner TG, Zhang T, Cargnel MD, Lichy MZ, Fiorucci AS, Fankhauser C, Koo AJ, Austin AT, Gershenzon J, Ballaré CL (2020) A light-dependent molecular link between competition cues and defence responses in plants. Nature Plants 6:223–230 PubMed

Gidda SK, Miersch O, Levitin A, Schmidt J, Wasternack C, Varin L (2003) Biochemical and molecular characterization of a hydroxyjasmonate sulfotransferase from Arabidopsis thaliana. J Biol Chem 278:17895–17900 PubMed

Gläser K, Kanawati B, Kubo T, Schmitt-Kopplin P, Grill E (2014) Exploring the Arabidopsis sulfur metabolome. Plant J 77:31–45 PubMed

Günal S, Hardman R, Kopriva S, Mueller JW (2019) Sulfation pathways from red to green. J Biol Chem 294:12293–12312 PubMed PMC

Halliday KJ, Fankhauser C (2003) Phytochrome-hormonal signalling networks. New Phytol 157:449–463 PubMed

Hayashi K, Arai K, Aoi Y, Tanaka Y, Hira H, Guo RP, Hu Y, Ge CN, Zhao YD, Kasahara H, Fukui K (2021) The main oxidative inactivation pathway of the plant hormone auxin. Nat Commun 12:6752 PubMed PMC

Hilbert M, Voll LM, Ding Y, Hofmann J, Sharma M, Zuccaro A (2012) Indole derivative production by the root endophyte Piriformospora indica is not required for growth promotion but for biotrophic colonization of barley roots. New Phytol 196:520–534 PubMed

Hirschmann F, Krause F, Papenbrock J (2014) The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions. Front Plant Sci. 10.3389/fpls.2014.00556 PubMed PMC

Horng AJ, Yang SF (1975) Aerobic oxidation of indole-3-acetic-acid with bisulfite. Phytochemistry 14:1425–1428

Kai K, Wakasa K, Miyagawa H (2007) Metabolism of indole-3-acetic acid in rice: Identification and characterization of N-β-D-glucopyranosyl indole-3-acetic acid and its conjugates. Phytochemistry 68:2512–2522 PubMed

Keuskamp DH, Sasidharan R, Pierik R (2010) Physiological regulation and functional significance of shade avoidance responses to neighbors. Plant Signal Behav 5:655–662 PubMed PMC

Kleinenkuhnen N, Büchel F, Gerlich SC, Kopriva S, Metzger S (2019) A novel method for identification and quantification of sulfated flavonoids in plants by neutral loss scan mass spectrometry. Front Plant Sci. 10.3389/fpls.2019.00885 PubMed PMC

Koprivova A, Kopriva S (2016) Sulfation pathways in plants. Chem Biol Interact 259:23–30 PubMed

Korasick DA, Enders TA, Strader LC (2013) Auxin biosynthesis and storage forms. J Exp Bot 64:2541–2555 PubMed PMC

Kurth C, Welling M, Pohnert G (2015) Sulfated phenolic acids from Dasycladales siphonous green algae. Phytochemistry 117:417–423 PubMed

Lacomme C, Roby D (1996) Molecular cloning of a sulfotransferase in Arabidopsis thaliana and regulation during development and in response to infection with pathogenic bacteria. Plant Mol Biol 30:995–1008 PubMed

Lau OL, John WW, Yang SF (1978) Inactivity of oxidation-products of indole-3-acetic-acid on ethylene production in mung bean hypocotyls. Plant Physiol 61:68–71 PubMed PMC

Li Y, Di Q, Luo L, Yu LL (2024) Phytosulfokine peptides, their receptors, and functions. Front Plant Sci. 10.3389/fpls.2023.1326964 PubMed PMC

Ljung K (2013) Auxin metabolism and homeostasis during plant development. Development 140:943–950 PubMed

Ludwig-Muller J (2011) Auxin conjugates: their role for plant development and in the evolution of land plants. J Exp Bot 62:1757–1773 PubMed

Marsolais F, Boyd J, Paredes Y, Schinas AM, Garcia M, Elzein S, Varin L (2007) Molecular and biochemical characterization of two brassinosteroid sulfotransferases from Arabidopsis, AtST4a (At2g14920) and AtST1 (At2g03760). Planta 225:1233–1244 PubMed

Matsubayashi Y, Sakagami Y (1996) Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L. Proc Natl Acad Sci USA 93:7623–7627 PubMed PMC

Miersch O, Neumerkel J, Dippe M, Stenzel I, Wasternack C (2008) Hydroxylated jasmonates are commonly occurring metabolites of jasmonic acid and contribute to a partial switch-off in jasmonate signaling. New Phytol 177:114–127 PubMed

Noleto-Dias C, Harflett C, Beale MH, Ward JL (2020) Sulfated flavanones and dihydroflavonols from willow. Phytochem Lett 35:88–93 PubMed PMC

Nzowa LK, Teponno RB, Tapondjou LA, Verotta L, Liao Z, Graham D, Zink MC, Barboni L (2013) Two new tryptophan derivatives from the seed kernels of Entada rheedei: Effects on cell viability and HIV infectivity. Fitoterapia 87:37–42 PubMed PMC

Pencík A, Casanova-Sáez R, Pilarová V, Zukauskaite A, Pinto R, Micol JL, Ljung K, Novák O (2018) Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis. J Exp Bot 69:2569–2579 PubMed PMC

Porco S, Pencík A, Rashed A, Voss U, Casanova-Sáez R, Bishopp A, Golebiowska A, Bhosale R, Swarup R, Swarup K, Penáková P, Novák O, Staswick P, Hedden P, Phillips AL, Vissenberg K, Bennett MJ, Ljung K (2016) Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis. Proc Natl Acad Sci USA 113:11016–11021 PubMed PMC

Qin GJ, Gu HY, Zhao YD, Ma ZQ, Shi GL, Yang Y, Pichersky E, Chen HD, Liu MH, Chen ZL, Qu LJ (2005) An indole-3-acetic acid carboxyl methyltransferase regulates Arabidopsis leaf development. Plant Cell 17:2693–2704 PubMed PMC

Rárová L, Ncube B, Van Staden J, Fürst R, Strnad M, Gruz J (2019) Identification of narciclasine as an in vitro anti-inflammatory component of cyrtanthus contractus by correlation-based metabolomics. J Nat Prod 82:1372–1376 PubMed

Rouleau M, Marsolais F, Richard M, Nicolle L, Voigt B, Adam G, Varin L (1999) Inactivation of brassinosteroid biological activity by a salicylate-inducible steroid sulfotransferase from Brassica napus. J Biol Chem 274:20925–20930 PubMed

Sanda S, Leustek T, Theisen MJ, Garavito RM, Benning C (2001) Recombinant arabidopsis SQD1 converts UDP-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro. J Biol Chem 276:3941–3946 PubMed

Sardar P, Kempken F (2018) Characterization of indole-3-pyruvic acid pathway-mediated biosynthesis of auxin in Neurospora crassa. PLoS ONE 13:e0192293 PubMed PMC

Shen Y, Diener AC (2013) Arabidopsis thaliana resistance to fusarium oxysporum 2 implicates tyrosine-sulfated peptide signaling in susceptibility and resistance to root infection. In Figshare. 10.1371/journal.pgen.1003525 PubMed PMC

Sprunck S, Jacobsen HJ, Reinard T (1995) Indole-3-lactic acid is a weak auxin analogue but not an anti-auxin. J Plant Growth Regul 14:191–197

Supikova K, Kosinova A, Vavrusa M, Koplikova L, François A, Pospisil J, Zatloukal M, Wever R, Hartog A, Gruz J (2022) Sulfated phenolic acids in plants. Planta 255:124 PubMed

Tarakhovskaya ER, Maslov YI, Shishova MF (2007) Phytohormones in algae. Russ J Plant Physiol 54:163–170

Torrens-Spence M, Gillaspy G, Zhao B, Harich K, White R, Li J (2012) Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme. Biochem Biophys Res Commun 418:211–216 PubMed

Ugena L, Hylová A, Podlesáková K, Humplík JF, Dolezal K, De Diego N, Spíchal L (2018) Characterization of biostimulant mode of action using novel multi-trait high-throughput screening of arabidopsis germination and rosette growth. Front Plant Sci. 10.3389/fpls.2018.01327 PubMed PMC

Walz A, Park S, Cohen JD, Momonoki YS, Slovin JP, Ludwig-Mueller J (2001) A gene encoding a protein modified by the phytohormone indoleacetic acid. Plant Biology (Rockville) 2001:82 PubMed PMC

Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT (1971) Plant antitumor agents.6. isolation and structure of taxol, a novel antileukemic and antitumor agent from taxus-brevifolia. J Am Chem Soc 93:2325–2327 PubMed

Woodward AW, Bartel B (2005) Auxin: Regulation, action, and interaction. Ann Bot 95:707–735 PubMed PMC

Xu JJ, Fang X, Li CY, Zhao Q, Martin C, Chen XY, Yang L (2018) Characterization of Arabidopsis thaliana Hydroxyphenylpyruvate reductases in the tyrosine conversion pathway. Front Plant Sci. 10.3389/fpls.2018.01305 PubMed PMC

Yi L, Dratter J, Wang C, Tunge JA, Desaire H (2006) Identification of sulfation sites of metabolites and prediction of the compounds’ biological effects. Anal Bioanal Chem 386:666–674 PubMed PMC

Zazímalová E, Murphy AS, Yang HB, Hoyerová K, Hosek P (2010) Auxin transporters - why so many? Cold Spring Harb Perspect Biol 2:a001552 PubMed PMC

Zhang S, van Duijn B (2014) Cellular auxin transport in algae. Plants-Basel 3:58–69 PubMed PMC

Zhao YD (2010) Auxin biosynthesis and its role in plant development. Annu Rev Plant Biol 61(61):49–64 PubMed PMC

Zhao YD (2012) Auxin biosynthesis: a simple two-step pathway converts tryptophan to indole-3-acetic acid in plants. Mol Plant 5:334–338 PubMed PMC

Zubieta C, Ross JR, Koscheski P, Yang Y, Pichersky E, Noel JP (2003) Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family. Plant Cell 15:1704–1716 PubMed PMC