Coordination of plant development requires modulation of growth responses that are under control of the phytohormone auxin. PIN-FORMED plasma membrane proteins, involved in intercellular transport of the growth regulator, are key to the transmission of such auxin signals and subject to multilevel surveillance mechanisms, including reversible post-translational modifications. Apart from well-studied PIN protein modifications, namely phosphorylation and ubiquitylation, no further post-translational modifications have been described so far. Here, we focused on root-specific Arabidopsis PIN2 and explored functional implications of two evolutionary conserved cysteines, by a combination of in silico and molecular approaches. PIN2 sequence alignments and modeling predictions indicated that both cysteines are facing the cytoplasm and therefore would be accessible to redox status-controlled modifications. Notably, mutant pin2C-A alleles retained functionality, demonstrated by their ability to almost completely rescue defects of a pin2 null allele, whereas high resolution analysis of pin2C-A localization revealed increased intracellular accumulation, and altered protein distribution within plasma membrane micro-domains. The observed effects of cysteine replacements on root growth and PIN2 localization are consistent with a model in which redox status-dependent cysteine modifications participate in the regulation of PIN2 mobility, thereby fine-tuning polar auxin transport.

Department of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences Vienna Muthgasse 18 1190 Wien Austria

Department of Experimental Plant Biology Faculty of Science Charles University Vinicna 5 128 44 Prague 2 Czech Republic

Institute of Experimental Botany of the Czech Academy of Sciences Rozvojová 263 165 02 Praha 6 Czech Republic

School of Life Sciences University of Warwick Gibbet Hill Road Coventry CV4 7AL UK

Zobrazit více v PubMed

Lavy M., Estelle M. Mechanisms of auxin signaling. Development. 2016;143:3226–3229. doi: 10.1242/dev.131870. PubMed DOI PMC

Enders T.A., Strader L.C. Auxin activity: Past, present, and future. Am. J. Bot. 2015;102:180–196. doi: 10.3732/ajb.1400285. PubMed DOI PMC

Leyser O. Auxin signaling. Plant Physiol. 2017 doi: 10.1104/pp.17.00765. PubMed DOI PMC

Adamowski M., Friml J. PIN-dependent auxin transport: Action, regulation, and evolution. Plant Cell. 2015;27:20–32. doi: 10.1105/tpc.114.134874. PubMed DOI PMC

Borghi L., Kang J., Ko D., Lee Y., Martinoia E. The role of ABCG-type ABC transporters in phytohormone transport. Biochem. Soc. Trans. 2015;43:924–930. doi: 10.1042/BST20150106. PubMed DOI PMC

Zazimalova E., Murphy A.S., Yang H., Hoyerova K., Hosek P. Auxin transporters—Why so many? Cold Spring Harb Perspect Biol. 2010;2:a001552. doi: 10.1101/cshperspect.a001552. PubMed DOI PMC

Luschnig C., Vert G. The dynamics of plant plasma membrane proteins: PINs and beyond. Development. 2014;141:2924–2938. doi: 10.1242/dev.103424. PubMed DOI

Huang F., Zago M.K., Abas L., van Marion A., Galvan-Ampudia C.S., Offringa R. Phosphorylation of conserved PIN motifs directs Arabidopsis PIN1 polarity and auxin transport. Plant Cell. 2010;22:1129–1142. doi: 10.1105/tpc.109.072678. PubMed DOI PMC

Zourelidou M., Absmanner B., Weller B., Barbosa I.C., Willige B.C., Fastner A., Streit V., Port S.A., Colcombet J., de la Fuente van Bentem S., et al. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID. Elife. 2014;3 doi: 10.7554/eLife.02860. PubMed DOI PMC

Michniewicz M., Zago M.K., Abas L., Weijers D., Schweighofer A., Meskiene I., Heisler M.G., Ohno C., Zhang J., Huang F., et al. Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux. Cell. 2007;130:1044–1056. doi: 10.1016/j.cell.2007.07.033. PubMed DOI

Korbei B., Luschnig C. Plasma membrane protein ubiquitylation and degradation as determinants of positional growth in plants. J. Integr. Plant Biol. 2013;55:809–823. doi: 10.1111/jipb.12059. PubMed DOI

Isono E., Kalinowska K. ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants. Curr. Opin. Plant Biol. 2017;40:49–55. doi: 10.1016/j.pbi.2017.07.003. PubMed DOI

Korbei B., Moulinier-Anzola J., De-Araujo L., Lucyshyn D., Retzer K., Khan M.A., Luschnig C. Arabidopsis TOL proteins act as gatekeepers for vacuolar sorting of PIN2 plasma membrane protein. Curr. Biol. 2013;23:2500–2505. doi: 10.1016/j.cub.2013.10.036. PubMed DOI

Nagel M.K., Kalinowska K., Vogel K., Reynolds G.D., Wu Z.X., Anzenberger F., Ichikawa M., Tsutsumi C., Sato M.H., Kuster B., et al. Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3. Proc. Natl. Acad. Sci. USA. 2017;114:E7197–E7204. doi: 10.1073/pnas.1710866114. PubMed DOI PMC

Leitner J., Petrasek J., Tomanov K., Retzer K., Parezova M., Korbei B., Bachmair A., Zazimalova E., Luschnig C. Lysine63-linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth. Proc. Natl. Acad. Sci. USA. 2012;109:8322–8327. doi: 10.1073/pnas.1200824109. PubMed DOI PMC

Giles N.M., Watts A.B., Giles G.I., Fry F.H., Littlechild J.A., Jacob C. Metal and redox modulation of cysteine protein function. Chem. Biol. 2003;10:677–693. doi: 10.1016/S1074-5521(03)00174-1. PubMed DOI

Xia X.J., Zhou Y.H., Shi K., Zhou J., Foyer C.H., Yu J.Q. Interplay between reactive oxygen species and hormones in the control of plant. J. Exp. Bot. 2015;66:2839–2856. doi: 10.1093/jxb/erv089. PubMed DOI

Rouhier N., Cerveau D., Couturier J., Reichheld J.P., Rey P. Involvement of thiol-based mechanisms in plant development. Biochim. Biophys. Acta. 2015;1850:1479–1496. doi: 10.1016/j.bbagen.2015.01.023. PubMed DOI

Bashandy T., Guilleminot J., Vernoux T., Caparros-Ruiz D., Ljung K., Meyer Y., Reichheld J.P. Interplay between the NADP-linked thioredoxin and glutathione systems in Arabidopsis auxin signaling. Plant Cell. 2010;22:376–391. doi: 10.1105/tpc.109.071225. PubMed DOI PMC

Yu X., Pasternak T., Eiblmeier M., Ditengou F., Kochersperger P., Sun J., Wang H., Rennenberg H., Teale W., Paponov I., et al. Plastid-localized glutathione reductase2-regulated glutathione redox status is essential for Arabidopsis root apical meristem maintenance. Plant Cell. 2013;25:4451–4468. doi: 10.1105/tpc.113.117028. PubMed DOI PMC

Koprivova A., Mugford S.T., Kopriva S. Arabidopsis root growth dependence on glutathione is linked to auxin transport. Plant Cell Rep. 2010;29:1157–1167. doi: 10.1007/s00299-010-0902-0. PubMed DOI

Feechan A., Kwon E., Yun B.W., Wang Y., Pallas J.A., Loake G.J. A central role for S-nitrosothiols in plant disease resistance. Proc. Natl. Acad. Sci. USA. 2005;102:8054–8059. doi: 10.1073/pnas.0501456102. PubMed DOI PMC

Shi Y.F., Wang D.L., Wang C., Culler A.H., Kreiser M.A., Suresh J., Cohen J.D., Pan J., Baker B., Liu J.Z. Loss of GSNOR1 function leads to compromised auxin signaling and polar auxin transport. Mol. Plant. 2015;8:1350–1365. doi: 10.1016/j.molp.2015.04.008. PubMed DOI

Fernandez-Marcos M., Sanz L., Lewis D.R., Muday G.K., Lorenzo O. Nitric oxide causes root apical meristem defects and growth inhibition while reducing PIN-FORMED 1 (PIN1)-dependent acropetal auxin transport. Proc. Natl. Acad. Sci. USA. 2011;108:18506–18511. doi: 10.1073/pnas.1108644108. PubMed DOI PMC

Luschnig C., Gaxiola R.A., Grisafi P., Fink G.R. EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes Dev. 1998;12:2175–2187. doi: 10.1101/gad.12.14.2175. PubMed DOI PMC

Krecek P., Skupa P., Libus J., Naramoto S., Tejos R., Friml J., Zazimalova E. The PIN-FORMED (PIN) protein family of auxin transporters. Genome Biol. 2009;10:249. doi: 10.1186/gb-2009-10-12-249. PubMed DOI PMC

Bennett T., Brockington S.F., Rothfels C., Graham S.W., Stevenson D., Kutchan T., Rolf M., Thomas P., Wong G.K., Leyser O., et al. Paralogous radiations of PIN proteins with multiple origins of noncanonical PIN structure. Mol. Biol. Evol. 2014;31:2042–2060. doi: 10.1093/molbev/msu147. PubMed DOI PMC

Nodzynski T., Vanneste S., Zwiewka M., Pernisova M., Hejatko J., Friml J. Enquiry into the topology of plasma membrane-localized PIN auxin transport components. Mol. Plant. 2016;9:1504–1519. doi: 10.1016/j.molp.2016.08.010. PubMed DOI PMC

Sali A., Blundell T.L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 1993;234:779–815. doi: 10.1006/jmbi.1993.1626. PubMed DOI

Case D.A., Cerutti D.S., Cheatham I.T.E., Darden T.A., Duke R.E., Giese T.J., Gohlke H., Goetz A.W., Greene D., Homeyer N., et al. AMBER 2017. University of California; San Francisco, CA, USA: 2017.

Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin T.E. UCSF chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 2004;25:1605–1612. doi: 10.1002/jcc.20084. PubMed DOI

Grabov A., Ashley M.K., Rigas S., Hatzopoulos P., Dolan L., Vicente-Agullo F. Morphometric analysis of root shape. New Phytol. 2005;165:641–651. doi: 10.1111/j.1469-8137.2004.01258.x. PubMed DOI

Kleine-Vehn J., Wabnik K., Martiniere A., Langowski L., Willig K., Naramoto S., Leitner J., Tanaka H., Jakobs S., Robert S., et al. Recycling, clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane. Mol. Syst. Biol. 2011;7:540. doi: 10.1038/msb.2011.72. PubMed DOI PMC

Martiniere A., Lavagi I., Nageswaran G., Rolfe D.J., Maneta-Peyret L., Luu D.T., Botchway S.W., Webb S.E., Mongrand S., Maurel C., et al. Cell wall constrains lateral diffusion of plant plasma-membrane proteins. Proc. Natl. Acad. Sci. USA. 2012;109:12805–12810. doi: 10.1073/pnas.1202040109. PubMed DOI PMC

Gustafsson N., Culley S., Ashdown G., Owen D.M., Pereira P.M., Henriques R. Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations. Nat. Commun. 2016;7:12471. doi: 10.1038/ncomms12471. PubMed DOI PMC

Chevalier A.S., Chaumont F. Trafficking of plant plasma membrane aquaporins: Multiple regulation levels and complex sorting signals. Plant Cell Physiol. 2015;56:819–829. doi: 10.1093/pcp/pcu203. PubMed DOI PMC

Bienert G.P., Cavez D., Besserer A., Berny M.C., Gilis D., Rooman M., Chaumont F. A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers. Biochem. J. 2012;445:101–111. doi: 10.1042/BJ20111704. PubMed DOI

Yoo Y.J., Lee H.K., Han W., Kim D.H., Lee M.H., Jeon J., Lee D.W., Lee J., Lee Y., Lee J., et al. Interactions between transmembrane helices within monomers of the aquaporin AtPIP2;1 play a crucial role in tetramer formation. Mol. Plant. 2016;9:1004–1017. doi: 10.1016/j.molp.2016.04.012. PubMed DOI

Mou Z., Fan W., Dong X. Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell. 2003;113:935–944. doi: 10.1016/S0092-8674(03)00429-X. PubMed DOI

Tada Y., Spoel S.H., Pajerowska-Mukhtar K., Mou Z., Song J., Wang C., Zuo J., Dong X. Plant immunity requires conformational changes [corrected] of NPR1 via S-nitrosylation and thioredoxins. Science. 2008;321:952–956. doi: 10.1126/science.1156970. PubMed DOI PMC

Wu Y., Zhang D., Chu J.Y., Boyle P., Wang Y., Brindle I.D., De Luca V., Despres C. The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid. Cell Rep. 2012;1:639–647. doi: 10.1016/j.celrep.2012.05.008. PubMed DOI

Feraru E., Feraru M.I., Kleine-Vehn J., Martiniere A., Mouille G., Vanneste S., Vernhettes S., Runions J., Friml J. PIN polarity maintenance by the cell wall in Arabidopsis. Curr. Biol. 2011;21:338–343. doi: 10.1016/j.cub.2011.01.036. PubMed DOI

Mittler R., Vanderauwera S., Suzuki N., Miller G., Tognetti V.B., Vandepoele K., Gollery M., Shulaev V., Van Breusegem F. ROS signaling: The new wave? Trends Plant Sci. 2011;16:300–309. doi: 10.1016/j.tplants.2011.03.007. PubMed DOI

Abas L., Benjamins R., Malenica N., Paciorek T., Wisniewska J., Moulinier-Anzola J.C., Sieberer T., Friml J., Luschnig C. Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat. Cell Biol. 2006;8:249–256. doi: 10.1038/ncb1369. PubMed DOI

Clough S.J., Bent A.F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998;16:735–743. doi: 10.1046/j.1365-313x.1998.00343.x. PubMed DOI

Koncz C., Schell J. The promoter of Tl-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 1986;204:383–396. doi: 10.1007/BF00331014. DOI

Jones D.T. GenTHREADER: An efficient and reliable protein fold recognition method for genomic sequences. J. Mol. Biol. 1999;287:797–815. doi: 10.1006/jmbi.1999.2583. PubMed DOI

Jones D.T. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 1999;292:195–202. doi: 10.1006/jmbi.1999.3091. PubMed DOI

McGuffin L.J., Jones D.T. Improvement of the GenTHREADER method for genomic fold recognition. Bioinformatics. 2003;19:874–881. doi: 10.1093/bioinformatics/btg097. PubMed DOI

Lobley A., Sadowski M.I., Jones D.T. pGenTHREADER and pDomTHREADER: New methods for improved protein fold recognition and superfamily discrimination. Bioinformatics. 2009;25:1761–1767. doi: 10.1093/bioinformatics/btp302. PubMed DOI

Mongan J., Simmerling C., McCammon J.A., Case D.A., Onufriev A. Generalized Born model with a simple, robust molecular volume correction. J. Chem. Theory Comput. 2007;3:156–169. doi: 10.1021/ct600085e. PubMed DOI PMC

Nguyen H., Roe D.R., Simmerling C. Improved generalized born solvent model parameters for protein simulations. J. Chem. Theory Comput. 2013;9:2020–2034. doi: 10.1021/ct3010485. PubMed DOI PMC

Maier J.A., Martinez C., Kasavajhala K., Wickstrom L., Hauser K.E., Simmerling C. ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 2015;11:3696–3713. doi: 10.1021/acs.jctc.5b00255. PubMed DOI PMC

Jorgensen W.L., Chandrasekhar J., Madura J.D., Impey R.W., Klein M.L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983;79:926–935. doi: 10.1063/1.445869. DOI

Hornak V., Abel R., Okur A., Strockbine B., Roitberg A., Simmerling C. Comparison of multiple amber force fields and development of improved protein backbone parameters. Proteins. 2006;65:712–725. doi: 10.1002/prot.21123. PubMed DOI PMC

Ryckaert J.P., Ciccotti G., Berendsen H.J.C. Numerical-integration of Cartesian equations of motion of a system with constraints—Molecular-dynamics of N-alkanes. J. Comput. Phys. 1977;23:327–341. doi: 10.1016/0021-9991(77)90098-5. DOI

Miyamoto S., Kollman P.A. Settle—An analytical version of the Shake and Rattle algorithm for rigid water models. J. Comput. Chem. 1992;13:952–962. doi: 10.1002/jcc.540130805. DOI

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Leinonen R., Sugawara H., Shumway M., International Nucleotide Sequence Database Collaboration The sequence read archive. Nucleic Acids Res. 2011;39:D19–D21. doi: 10.1093/nar/gkq1019. PubMed DOI PMC

Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., Madden T.L. BLAST+: Architecture and applications. BMC Bioinform. 2009;10:421. doi: 10.1186/1471-2105-10-421. PubMed DOI PMC

Huang X., Madan A. CAP3: A DNA sequence assembly program. Genome Res. 1999;9:868–877. doi: 10.1101/gr.9.9.868. PubMed DOI PMC

Katoh K., Standley D.M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013;30:772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Hellemans J., Mortier G., de Paepe A., Speleman F., Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007;8:R19. doi: 10.1186/gb-2007-8-2-r19. PubMed DOI PMC

Arabidopsis flippase ALA3 is required for adjustment of early subcellular trafficking in plant response to osmotic stress

Recent insights into metabolic and signalling events of directional root growth regulation and its implications for sustainable crop production systems

The Hydrophilic Loop of Arabidopsis PIN1 Auxin Efflux Carrier Harbors Hallmarks of an Intrinsically Disordered Protein

Throttling Growth Speed: Evaluation of aux1-7 Root Growth Profile by Combining D-Root system and Root Penetration Assay

Lessons Learned from the Studies of Roots Shaded from Direct Root Illumination

No Time for Transcription-Rapid Auxin Responses in Plants

Characterization of ALBA Family Expression and Localization in Arabidopsis thaliana Generative Organs

Dissecting Hierarchies between Light, Sugar and Auxin Action Underpinning Root and Root Hair Growth

Brassinosteroid signaling delimits root gravitropism via sorting of the Arabidopsis PIN2 auxin transporter

The Nuts and Bolts of PIN Auxin Efflux Carriers