cis-Cinnamic Acid Is a Novel, Natural Auxin Ef Acid Is a Novel, Natural Auxin...

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  • cis-Cinnamic Acid Is a Novel, Natural Auxin EffluxInhibitor That Promotes Lateral Root Formation1[OPEN]

    Ward Steenackers, Petr Klma, Mussa Quareshy, Igor Cesarino, Robert P. Kumpf, Sander Corneillie,Pedro Arajo, Tom Viaene, Geert Goeminne, Moritz K. Nowack, Karin Ljung, Jir Friml,Joshua J. Blakeslee, Ondrej Novk, Eva Zazmalov, Richard Napier, Wout Boerjan2*, andBartel Vanholme2*

    Department of Plant Systems Biology, VIB, B-9052 Gent, Belgium (W.S., I.C., R.P.K., S.C., P.A., T.V., G.G.,M.K.N., W.B., B.V.); Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Gent,Belgium (W.S., I.C., R.P.K., S.C., P.A., T.V., G.G., M.K.N., W.B., B.V.); Institute of Experimental Botany, CzechAcademy of Sciences, CZ-16502 Prague, Czech Republic (P.K., E.Z.); School of Life Sciences, University ofWarwick, Coventry CV4 7AL, United Kingdom (M.Q., R.N.); Department of Botany, Institute of Biosciences,University of So Paulo, Butant, So Paulo 03178-200, Brazil (I.C.); Ume Plant Science Centre, Department ofForest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Ume, Sweden(K.L., O.N.); Institute of Science and Technology, Austria, 3400 Klosterneuburg, Austria (J.F.); Department ofHorticulture and Crop Science, The Ohio State University/Ohio Agricultural Research and DevelopmentCenter, Wooster, Ohio 44691 (J.J.B.); and Laboratory of Growth Regulators, Centre of the Region Han forBiotechnological and Agricultural Research, Institute of Experimental Botany CAS and Faculty of Science ofPalack University, CZ-78371 Olomouc, Czech Republic (O.N.)

    ORCID IDs: 0000-0001-5678-2919 (P.K.); 0000-0001-8115-9803 (M.Q.); 0000-0002-6789-2432 (I.C.); 0000-0002-4046-6935 (R.P.K.);0000-0002-0337-2999 (G.G.); 0000-0003-2901-189X (K.L.); 0000-0002-8302-7596 (J.F.); 0000-0002-0605-518X (R.N.);0000-0003-1495-510X (W.B.); 0000-0002-7214-7170 (B.V.).

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

    Plant growth and development are tightly regulatedby a plethora of signaling compounds, which are pre-sent within the plant at extremely low concentrations.Although the molecular working mechanism for sev-eral of these compounds has been described in detail(phytohormones, such as auxin and cytokinin, beingamong the best studied), for others the underlyingmode of action is still unknown. Cinnamic acid (CA) isone of them, and whereas the first report on its bio-logical activity dates back to 1935 (Haagen-Smit andWent, 1935; Hitchcock, 1935), little additional researchhas been performed on this compound.

    CA is found in planta, both as trans (t)- and cis(c)-isomers, though not in equal concentrations (Yanget al., 1999; Yin et al., 2003). t-CA is synthesized through

    the deamination of Phe by PHE AMMONIA-LYASE,after which it is hydroxylated to p-coumaric acid byCINNAMIC ACID-4-HYDROXYLASE (C4H; Boerjanet al., 2003). These are the first steps of the generalphenylpropanoid pathway that lead toward a plethoraof secondary metabolites such as flavonoids, stilbenes,tannins, andmonolignols (Vogt, 2010; Supplemental Fig.S1). Besides being a crucial intermediate of an importantpathway, t-CA itself has also been described as a bioac-tive compound, though its exact activity has remained amatter of debate. Depending on the experiment, t-CAhas been described as inactive, anta-, or agonistic toauxin or an inhibitor of polar auxin transport (VanOverbeek et al., 1951; berg, 1961; Letham, 1978; Liuet al., 1993). c-CA is a photoisomerization product of

    552 Plant Physiology, January 2017, Vol. 173, pp. 552565, www.plantphysiol.org 2017 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.orgon May 19, 2018 - Published by Downloaded from

    Copyright 2017 American Society of Plant Biologists. All rights reserved.

    http://orcid.org/0000-0001-5678-2919http://orcid.org/0000-0001-8115-9803http://orcid.org/0000-0002-6789-2432http://orcid.org/0000-0002-4046-6935http://orcid.org/0000-0002-0337-2999http://orcid.org/0000-0003-2901-189Xhttp://orcid.org/0000-0002-8302-7596http://orcid.org/0000-0002-0605-518Xhttp://orcid.org/0000-0003-1495-510Xhttp://orcid.org/0000-0002-7214-7170http://www.plantphysiol.org/cgi/content/full/pp.16.00943/DC1http://www.plantphysiol.org/cgi/content/full/pp.16.00943/DC1http://www.plantphysiol.org

  • t-CA and, in contrast to the latter, is detected only intrace amounts in plants (Yin et al., 2003; Wong et al.,2005). However, it has been suggested to have higherbiological activity compared to t-CA (Haagen-Smit andWent, 1935). c-CA inhibits the gravitropic response ofetiolated tomato (Lycopersicon esculentum) seedlings andyoung tomato plants (Yang et al., 1999) and promotescell-elongation in Pisum sativum (Haagen-Smit andWent, 1935; Koepfli et al., 1938; Went, 1939) and epi-nastic curvature of tomato plants (Yang et al., 1999).Although these effects resemble, to some extent, thephysiological effects caused by perturbed auxin or

    ethylene homeostasis, further studies claimed that themode of action of c-CA might be different from that ofauxin and independent of ethylene-signaling (Yanget al., 1999; Wong et al., 2005).

    In addition to this inconsistent view on the physio-logical role of CA in plants, an adequate explanationconcerning the molecular mechanism by which bothisomers independently affect plant growth and devel-opment is lacking. We evaluated the working mecha-nism of CA and demonstrate that t-CA is inactive as amolecular signal, consistent with its role as a primaryintermediate in the general phenylpropanoid pathway.In contrast, its c-isomer is biologically active and acts asa natural inhibitor of cellular auxin efflux, promotinglateral root formation.

    RESULTS

    CA Affects Plant Development

    An evolutionary diverse set of plant species wasgrown on tissue culture medium supplemented withcommercially available CA and analyzed for aberrantgrowth phenotypes. In the higher land plants tested,CA inhibited primary root growth and inducedthe proliferation of adventitious and lateral roots in adose-dependentmanner (Fig. 1A; Supplemental Fig. S2,AD). In the Pteridophyte Selaginella moellendorffii, CAaffected root apical meristem bifurcation, thickening ofthe root and root hair proliferation, resulting in a moredense root architecture (Supplemental Fig. S2E). InPhyscomitrella patens, representing the Bryophytes, noclear effect on rhizoid growth was observed, but CAdid stimulate cell and leaf elongation in the gameto-phores (Supplemental Fig. S2, F and G). These resultsindicate that the addition of CA to the growth mediumaffects plant growth and development throughout theplant lineage.

    To study the underlying molecular working mecha-nism of this compound, we focused on Arabidopsis(Arabidopsis thaliana). In this model plant, the IC50-rootvalue (i.e. the CA concentration needed to reduce theprimary root length by 50%) was determined to be9.2mM under the conditions tested (Fig. 1B). Lateral rootformation and adventitious rooting were stimulated,and the overall increase in number of emerged lateralroots combined with the reduction in primary rootlength resulted in a considerable increase in lateral rootdensity (LRD). A 1.4- and 2.5-fold increase in LRD wasobtained at applied CA concentrations of 2.5 and 5 mM,respectively (Fig. 1C). Concentrations above 10 mMresulted in the outgrowth of fasciated lateral rootsalong the primary root and a significant increase in thenumber of adventitious roots (Fig. 1, D and E). Besides,an increase in root hair number and length was ob-served not only on the primary root (Fig. 1F), but alsoon the lateral roots (Fig. 1G). Finally, a root-wavingphenotype was observed in CA-treated plants (Fig.1A), indicating gravitropism defect. This was confirmed

    1 This work has been supported by grants from the Hercules Foun-dation for the Synapt Q-Tof (grant no. AUGE/014), by the Multidis-ciplinary Research Partnership Biotechnology for a SustainableEconomy (01MRB510W) of Ghent University, and by the StanfordUniversity Global Climate and Energy Project (Lignin management:optimizing yield and composition in lignin-modified plants); S.C. issupported by the Research Foundation Flanders for a predoctoral(3G032912) fellowship; W.S.