Auxin steers numerous physiological processes in plants, making the tight control of its endogenous levels and spatiotemporal distribution a necessity. This regulation is achieved by different mechanisms, including auxin biosynthesis, metabolic conversions, degradation, and transport. Here, we introduce cis-cinnamic acid (c-CA) as a novel and unique addition to a small group of endogenous molecules affecting in planta auxin concentrations. c-CA is the photo-isomerization product of the phenylpropanoid pathway intermediate trans-CA (t-CA). When grown on c-CA-containing medium, an evolutionary diverse set of plant species were shown to exhibit phenotypes characteristic for high auxin levels, including inhibition of primary root growth, induction of root hairs, and promotion of adventitious and lateral rooting. By molecular docking and receptor binding assays, we showed that c-CA itself is neither an auxin nor an anti-auxin, and auxin profiling data revealed that c-CA does not significantly interfere with auxin biosynthesis. Single cell-based auxin accumulation assays showed that c-CA, and not t-CA, is a potent inhibitor of auxin efflux. Auxin signaling reporters detected changes in spatiotemporal distribution of the auxin response along the root of c-CA-treated plants, and long-distance auxin transport assays showed no inhibition of rootward auxin transport. Overall, these results suggest that the phenotypes of c-CA-treated plants are the consequence of a local change in auxin accumulation, induced by the inhibition of auxin efflux. This work reveals a novel mechanism how plants may regulate auxin levels and adds a novel, naturally occurring molecule to the chemical toolbox for the studies of auxin homeostasis.

Department of Botany Institute of Biosciences University of São Paulo Butantã São Paulo 03178 200 Brazil

Department of Botany Institute of Biosciences University of São Paulo Butantã São Paulo 03178 200 Brazil ;

Department of Horticulture and Crop Science The Ohio State University Ohio Agricultural Research and Development Center Wooster Ohio 44691 ; and

Department of Plant Biotechnology and Bioinformatics Ghent University B 9052 Gent Belgium

Department of Plant Biotechnology and Bioinformatics Ghent University B 9052 Gent Belgium ;

Department of Plant Systems Biology VIB B 9052 Gent Belgium

Department of Plant Systems Biology VIB B 9052 Gent Belgium ;

Institute of Experimental Botany Czech Academy of Sciences CZ 16502 Prague Czech Republic

Institute of Experimental Botany Czech Academy of Sciences CZ 16502 Prague Czech Republic ;

Institute of Science and Technology Austria 3400 Klosterneuburg Austria

Institute of Science and Technology Austria 3400 Klosterneuburg Austria ;

Laboratory of Growth Regulators Centre of the Region Haná for Biotechnological and Agricultural Research Institute of Experimental Botany CAS and Faculty of Science of Palacký University CZ 78371 Olomouc Czech Republic

School of Life Sciences University of Warwick Coventry CV4 7AL United Kingdom

School of Life Sciences University of Warwick Coventry CV4 7AL United Kingdom ;

Umeå Plant Science Centre Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences SE 901 83 Umeå Sweden

Umeå Plant Science Centre Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences SE 901 83 Umeå Sweden ;

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Åberg B. (1961) Studies on plant growth regulator XVIII. Some β-substituted acrylic acids. Ann Roy Agric Coll Sweden 27: 99–123

Adamowski M, Friml J (2015) PIN-dependent auxin transport: action, regulation, and evolution. Plant Cell 27: 20–32 PubMed PMC

Beeckman T, Engler G (1994) An easy technique for the clearing of histochemically stained plant tissue. Plant Mol Biol Rep 12: 37–42

Benková E, Michniewicz M, Sauer M, Teichmann T, Seifertová D, Jürgens G, Friml J (2003) Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115: 591–602 PubMed

Blilou I, Xu J, Wildwater M, Willemsen V, Paponov I, Friml J, Heidstra R, Aida M, Palme K, Scheres B (2005) The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature 433: 39–44 PubMed

Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54: 519–546 PubMed

Brown DE, Rashotte AM, Murphy AS, Normanly J, Tague BW, Peer WA, Taiz L, Muday GK (2001) Flavonoids act as negative regulators of auxin transport in vivo in arabidopsis. Plant Physiol 126: 524–535 PubMed PMC

Brunoud G, Wells DM, Oliva M, Larrieu A, Mirabet V, Burrow AH, Beeckman T, Kepinski S, Traas J, Bennett MJ, et al. (2012) A novel sensor to map auxin response and distribution at high spatio-temporal resolution. Nature 482: 103–106 PubMed

Buer CS, Kordbacheh F, Truong TT, Hocart CH, Djordjevic MA (2013) Alteration of flavonoid accumulation patterns in transparent testa mutants disturbs auxin transport, gravity responses, and imparts long-term effects on root and shoot architecture. Planta 238: 171–189 PubMed

Calderón Villalobos LI, Lee S, De Oliveira C, Ivetac A, Brandt W, Armitage L, Sheard LB, Tan X, Parry G, Mao H, et al. (2012) A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin. Nat Chem Biol 8: 477–485 PubMed PMC

Casimiro I, Marchant A, Bhalerao RP, Beeckman T, Shooge S, Swarup R, Graham N, Inzé D, Sandberg G, Casero PJ, Bennet M (2001) Auxin transport promotes Arabidopsis lateral root initiation. Plant Cell 13: 843–852 PubMed PMC

Chen YL, Huang ST, Sun FM, Chiang YL, Chiang CJ, Tsai CM, Wang CJ (2011) Transformation of cinnamic acid from trans- to cis-form raises a notable bactericidal and synergistic activity against multiple-drug resistant Mycobacterium tuberculosis. Eur J Pharm Sci 43: 188–194 PubMed

Colón-Carmona A, You R, Haimovitch-Gal T, Doerner P (1999) Technical advance: spatio-temporal analysis of mitotic activity with a labile cyclin-GUS fusion protein. Plant J 20: 503–508 PubMed

De Rybel B, Vassileva V, Parizot B, Demeulenaere M, Grunewald W, Audenaert D, Van Campenhout J, Overvoorde P, Jansen L, Vanneste S, et al. (2010) A novel aux/IAA28 signaling cascade activates GATA23-dependent specification of lateral root founder cell identity. Curr Biol 20: 1697–1706 PubMed

Delbarre A, Muller P, Imhoff V, Guern J (1996) Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells. Planta 198: 532–541 PubMed

Dharmasiri N, Dharmasiri S, Weijers D, Lechner E, Yamada M, Hobbie L, Ehrismann JS, Jürgens G, Estelle M (2005) Plant development is regulated by a family of auxin receptor F box proteins. Dev Cell 9: 109–119 PubMed

Ding Z, Galván-Ampudia CS, Demarsy E, Łangowski Ł, Kleine-Vehn J, Fan Y, Morita MT, Tasaka M, Fankhauser C, Offringa R, et al. (2011) Light-mediated polarization of the PIN3 auxin transporter for the phototropic response in Arabidopsis. Nat Cell Biol 13: 447–452 PubMed

Friml J, Vieten A, Sauer M, Weijers D, Schwarz H, Hamann T, Offringa R, Jürgens G (2003) Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426: 147–153 PubMed

Fukaki H, Tameda S, Masuda H, Tasaka M (2002) Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis. Plant J 29: 153–168 PubMed

Geisler M, Blakeslee JJ, Bouchard R, Lee OR, Vincenzetti V, Bandyopadhyay A, Titapiwatanakum B, Peer WA, Bailly A, Richards EL, et al. (2005) Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1. Plant J 44: 179–194 PubMed

González-Carranza ZH, Elliott KA, Roberts JA (2007) Expression of polygalacturonases and evidence to support their role during cell separation processes in Arabidopsis thaliana. J Exp Bot 58: 3719–3730 PubMed

Haagen-Smit SAJ, Went FW (1935) A physiological analysis of the growth substance. Proceedings. Koninklijke Akademie van Wetenschappen te Amsterdam 38: 852–857

Hitchcock AE. (1935) Indole-3-n-propionic acid as a growth hormone and quantitative measurement of plant response. In Contributions of the Boyce Thompson Institute, Vol 7 Boyce Thompson Institute for Plant Research, Yonkers, NY, pp 87–95

Hocking MB. (1969) Photochemical and thermal isomerizations of cis- and trans-cinnamic acids, and their photostationary state. Can J Chem 47: 4567–4576

Kim JY, Henrichs S, Bailly A, Vincenzetti V, Sovero V, Mancuso S, Pollmann S, Kim D, Geisler M, Nam HG (2010) Identification of an ABCB/P-glycoprotein-specific inhibitor of auxin transport by chemical genomics. J Biol Chem 285: 23309–23317 PubMed PMC

Kang J, Park J, Choi H, Burla B, Kretzschmar T, Lee Y, Marinoia E (2011) Plant ABC transporters. The Arabidopsis Book 9: e0153. PubMed PMC

Koepfli JB, Thimann KB, Went FW (1938) Plant hormones: structure and physiological activity. I. J Biol Chem 122: 763–780

Kumpf RP, Shi CL, Larrieu A, Stø IM, Butenko MA, Péret B, Riiser ES, Bennett MJ, Aalen RB (2013) Floral organ abscission peptide IDA and its HAE/HSL2 receptors control cell separation during lateral root emergence. Proc Natl Acad Sci USA 110: 5235–5240 PubMed PMC

Lee S, Sundaram S, Armitage L, Evans JP, Hawkes T, Kepinski S, Ferro N, Napier RM (2014) Defining binding efficiency and specificity of auxins for SCF(TIR1/AFB)-Aux/IAA co-receptor complex formation. ACS Chem Biol 9: 673–682 PubMed PMC

Letham DS. (1978) Naturally occurring plant growth regulators other than the principle hormones of higher plants. In Letham DS, Goodwin PB, Higgins TJV, eds, Phytohormones and Related Compounds: A Comprehensive Treatise, Vol 1 Elsevier/North Holland Biomedical Press, Amsterdam, pp 349–417

Lewis DR, Muday GK (2009) Measurement of auxin transport in Arabidopsis thaliana. Nat Protoc 4: 437–451 PubMed

Liu C, Xu Z, Chua NH (1993) Auxin polar transport is essential for the establishment of bilateral symmetry during early plant embryogenesis. Plant Cell 5: 621–630 PubMed PMC

Lukowitz W, Mayer U, Jürgens G (1996) Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product. Cell 84: 61–71 PubMed

Moreno-Risueno MA, Van Norman JM, Moreno A, Zhang J, Ahnert SE, Benfey PN (2010) Oscillating gene expression determines competence for periodic Arabidopsis root branching. Science 329: 1306–1311 PubMed PMC

Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 30: 2785–2791 PubMed PMC

Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell-line as the Hela-cell in the cell biology of higher-plants. Int Rev Cytol 132: 1–30

Novák O, Hényková E, Sairanen I, Kowalczyk M, Pospíšil T, Ljung K (2012) Tissue-specific profiling of the Arabidopsis thaliana auxin metabolome. Plant J 72: 523–536 PubMed

Okushima Y, Fukaki H, Onoda M, Theologis A, Tasaka M (2007) ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis. Plant Cell 19: 118–130 PubMed PMC

Péret B, De Rybel B, Casimiro I, Benková E, Swarup R, Laplaze L, Beeckman T, Bennett MJ (2009) Arabidopsis lateral root development: an emerging story. Trends Plant Sci 14: 399–408 PubMed

Petrášek J, Cerná A, Schwarzerová K, Elckner M, Morris DA, Zazímalová E (2003) Do phytotropins inhibit auxin efflux by impairing vesicle traffic? Plant Physiol 131: 254–263 PubMed PMC

Petrášek J, Friml J (2009) Auxin transport routes in plant development. Development 136: 2675–2688 PubMed

Petrášek J, Mravec J, Bouchard R, Blakeslee JJ, Abas M, Seifertová D, Wisniewska J, Tadele Z, Kubes M, Covanová M, et al. (2006) PIN proteins perform a rate-limiting function in cellular auxin efflux. Science 312: 914–918 PubMed

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612 PubMed

Romano CP, Hein MB, Klee HJ (1991) Inactivation of auxin in tobacco transformed with the indoleacetic acid-lysine synthetase gene of Pseudomonas savastanoi. Genes Dev 5: 438–446 PubMed

Santelia D, Henrichs S, Vincenzetti V, Sauer M, Bigler L, Klein M, Bailly A, Lee Y, Friml J, Geisler M, et al. (2008) Flavonoids redirect PIN-mediated polar auxin fluxes during root gravitropic responses. J Biol Chem 283: 31218–31226 PubMed PMC

Schalk M, Cabello-Hurtado F, Pierrel MA, Atanossova R, Saindrenan P, Werck-Reichhart D (1998) Piperonylic acid, a selective, mechanism-based inactivator of the trans-cinnamate 4-hydroxylase: a new tool to control the flux of metabolites in the phenylpropanoid pathway. Plant Physiol 118: 209–218 PubMed PMC

Seifertová D, Skůpa P, Rychtář J, Laňková M, Pařezová M, Dobrev PI, Hoyerová K, Petrášek J, Zažímalová E (2014) Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells. J Plant Physiol 171: 429–437 PubMed

Tan Z, Calderon-Villalobos LIA, Sharon M, Zheng C, Robinson CV, Estelle M, Zheng N (2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446: 640–645 PubMed

Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31: 455–461 PubMed PMC

Van de Wouwer D, Vanholme R, Decou R, Goeminne G, Audenaert D, Nguyen L, Höfer R, Pesquet E, Vanholme B, Boerjan W (2016) Chemical genetics uncovers novel inhibitors of lignification. Plant Physiol 172: 198–220 PubMed PMC

Van Overbeek J, Blondeau R, Horne V (1951) Trans-cinnamic acid as an anti-auxin. Am J Bot 38: 589–595

Vanneste S, Friml J (2009) Auxin: a trigger for change in plant development. Cell 136: 1005–1016 PubMed

Vogt T. (2010) Phenylpropanoid biosynthesis. Mol Plant 3: 2–20 PubMed

Went FW. (1939) Analysis and integration of various auxin effects. II. Proceedings. Koninklijke Akademie van Wetenschappen te Amsterdam 42: 731–739

Wong WS, Guo D, Wang XL, Yin ZQ, Xia B, Li N (2005) Study of cis-cinnamic acid in Arabidopsis thaliana. Plant Physiol Biochem 43: 929–937 PubMed

Yang XX, Choi HW, Yang SF, Li N (1999) A UV-light activated cinnamic acid isomer regulates plant growth and gravitropism via an ethylene receptor-independent pathway. Aust J Plant Physiol 26: 325–335 PubMed

Yin R, Han K, Heller W, Albert A, Dobrev PI, Zažímalová E, Schäffner AR (2014) Kaempferol 3-O-rhamnoside-7-O-rhamnoside is an endogenous flavonol inhibitor of polar auxin transport in Arabidopsis shoots. New Phytol 201: 466–475 PubMed PMC

Yin ZQ, Wong WS, Ye WC, Li N (2003) Biologically active cis-cinnamic acid occurs naturally in Brassica parachinensis. Chin Sci Bull 48: 555–558

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