The MADS transcription factor CmANR1 positively modulates root system development by directly regulating CmPIN2 in chrysanthemum

Abstract

Plant root systems are essential for many physiological processes, including water and nutrient absorption. MADS-box transcription factor (TF) genes have been characterized as the important regulators of root development in plants; however, the underlying mechanism is largely unknown, including chrysanthemum. Here, it was found that the overexpression of CmANR1, a chrysanthemum MADS-box TF gene, promoted both adventitious root (AR) and lateral root (LR) development in chrysanthemum. Whole transcriptome sequencing analysis revealed a series of differentially expressed unigenes (DEGs) in the roots of CmANR1-transgenic chrysanthemum plants compared to wild-type plants. Functional annotation of these DEGs by alignment with Gene Ontology (GO) terms and biochemical pathway Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that CmANR1 TF exhibited “DNA binding” and “catalytic” activity, as well as participated in “phytohormone signal transduction”. Both chromatin immunoprecipitation–polymerase chain reaction (ChIP-PCR) and gel electrophoresis mobility shift assays (EMSA) indicated the direct binding of CmPIN2 to the recognition site CArG-box motif by CmANR1. Finally, a firefly luciferase imaging assay demonstrated the transcriptional activation of CmPIN2 by CmANR1 in vivo. Overall, our results provide novel insights into the mechanisms of MADS-box TF CmANR1 modulation of both AR and LR development, which occurs by directly regulating auxin transport gene CmPIN2 in chrysanthemum.

References

[1] Nibau, C., Gibbs, D. J. & Coates, J. C. Branching out in new directions: the control of root architecture by lateral root formation. New Phytol. 179, 595-614 (2008).

[2] Giehl, R. F. & von Wirén, N. Root nutrient foraging. Plant Physiol. 166, 509-517 (2014).

[3] Bellini, C., Pacurar, D. I. & Perrone, I. Adventitious roots and lateral roots: similarities and differences. Annu. Rev. Plant. Biol. 65, 639-666 (2014).

[4] Coudert, Y. et al. Genetic control of root development in rice, the model cereal. Trends Plant. Sci. 15, 219-226 (2010).

[5] Yu, P., Gutjahr, C., Li, C. J. & Hochholdinger, F. Genetic control of lateral root formation in Cereals. Trends Plant. Sci. 21, 11 (2016).

[6] Hochholdinger, F. et al. From weeds to crops: genetic analysis of root development in cereals. Trends Plant. Sci. 9, 42-48 (2004).

[7] Geiss, G., Gutierrez, L. & Bellini, C. Adventitious root formation: new insights and perspectives. Annu. Rev. Plant. Biol. 37, 127-156 (2009).

[8] Li, S. W. et al. Mediators, genes and signaling in adventitious rooting. Bot. Rev. 75, 230-247 (2009).

[9] Casimiro, I. et al. Auxin transport promotes Arabidopsis lateral root initiation. Plant Cell 13, 843-852 (2001).

[10] Fukaki, H., Okushima, Y. & Tasaka, M. Auxin-mediated lateral root formation in higher plants. Int. Rev. Cytol. 256, 111-137 (2007).

[11] Gutierrez, L. et al. Auxin controls Arabidopsis adventitious root initiation by regulating jasmonic acid homeostasis. Plant Cell 24, 2515-2527 (2012).

[12] Pacurar, D. I. et al. Auxin is a central player in the hormone cross-talks that control adventitious rooting. Physiol. Plant. 151, 83-96 (2014).

[13] Du, Y. J. & Scheres, B. Lateral root formation and the multiple roles of auxin. J. Exp. Bot. 69, 155-167 (2018).

[14] Niemi, K. et al. Ectomycorrhizal fungi and exogenous auxins influence root and mycorrhiza formation of Scots pine hypocotyl cuttings in vitro. Tree Physiol. 22, 1231-1239 (2002).

[15] Wynne, J. & McDonald, M. Adventitious root formation in woody plant tissue: influence of light and indole-3-butyric acid (IBA) on adventitious root induction in Betula pendula. In. Vitr. Cell Dev. Biol. Plant 38, 210-212 (2002).

[16] Lanteri, M. L., Pagnussat, G. C. & Lamattina, L. Calcium and calcium-dependent protein kinases are involved in nitric oxide- and auxin-induced adventitious root formation in cucumber. J. Exp. Bot. 57, 1341-1351 (2006).

[17] Yang, J. et al. OsCBL1 modulates lateral root elongation in rice via affecting endogenous indole-3-acetic acid biosynthesis. JGG 42, 331-334 (2015).

[18] Gibbs, D. J. et al. AtMYB93 is a novel negative regulator of lateral root development in Arabidopsis. New Phytol. 203, 1194-1207 (2014).

[19] Lucas, M. et al. SHORT-ROOT regulates primary, lateral, and adventitious root development in Arabidopsis. Plant Physiol. 155, 384-398 (2011).

[20] Zhao, Y. et al. The interaction between rice ERF3 and WOX11 promotes crown root development by regulating gene expression involved in cytokinin signaling. Plant Cell 27, 2469-2483 (2015).

[21] Negi, S., Ivanchenko, M. G. & Muday, G. K. Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana. Plant J. 55, 175-187 (2008).

[22] Ivanchenko, M. G., Muday, G. K. & Dubrovsky, J. G. Ethylene-auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana. Plant J. 55, 335-347 (2008).

[23] Mauriat, M. et al. Gibberellins inhibit adventitious rooting in hybrid aspen and Arabidopsis by affecting auxin transport. Plant J. 78, 372-384 (2014).

[24] Feng, S. H. et al. Arabidopsis CAND1, an unmodified CUL1-interacting protein, is involved in multiple developmental pathways controlled by ubiquitin/proteasome-mediated protein degradation. Plant Cell 16, 1870-1882 (2004).

[25] Gutierrez, L. et al. Phenotypic plasticity of adventitious rooting in Arabidopsis is controlled by complex regulation of AUXIN RESPONSE FACTOR transcripts and microRNA abundance. Plant Cell 21, 3119-3132 (2009).

[26] Lavenus, J. et al. Lateral root development in Arabidopsis: fifty shades of auxin. Trends Plant Sci. 18, 450-458 (2013).

[27] Yu, L. H. et al. MADS-box transcription factor AGL21 regulates lateral root development and responds to multiple external and physiological signals. Mol. Plant 7, 1653-1669 (2014).

[28] Benková, E. et al. Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115, 591-602 (2003).

[29] Swarup, R. & Bennett, M. Auxin transport: the fountain of life in plants?. Dev. Cell 5, 824-826 (2003).

[30] Rashotte, A. M. et al. Basipetal auxin transport is required for gravitropism in roots of Arabidopsis. Plant Physiol. 122, 481-490 (2000).

[31] Yang, Y. et al. High-affinity auxin transport by the AUX1 influx carrier protein. Curr. Biol. 16, 1123-1127 (2006).

[32] Swarup, R. & Péret, B. AUX/LAX family of auxin infux carriers-an overview. Front. Plant Sci. 3, 225 (2012).

[33] Weijers, D. et al. Maintenance of embryonic auxin distribution for apical-basal patterning by PIN FORMED-dependent auxin transport in Arabidopsis. Plant Cell 17, 2517-2526 (2005).

[34] Wisniewska, J. et al. Polar PIN localization directs auxin flow in plants. Science 312, 883 (2006).

[35] Gälweiler, L. et al. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science 282, 2226-2230 (1998).

[36] Blilou, I. et al. The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature 433, 6 (2005).

[37] Feraru, E. & Friml, J. PIN polar targeting. Plant Physiol. 147, 1533-1559 (2008).

[38] Dal Bosco, C. et al. The endoplasmic reticulum localized PIN8 is a pollen-specific auxin carrier involved in intracellular auxin homeostasis. Plant J. 71, 860-870 (2012).

[39] Ding, Z. et al. ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis. Nat. Commun. 3, 941 (2012).

[40] Sawchuk, M. G., Edgar, A. & Scarpella, E. Patterning of leaf vein networks by convergent auxin transport pathways. PLoS Genet. 9, e1003294 (2013).

[41] Ganguly, A. et al. Functional analysis of the hydrophilic loop in intracellular trafficking of Arabidopsis PIN-FORMED proteins. Plant Cell 26, 1570-1585 (2014).

[42] Simon, S. et al. PIN6 auxin transporter at endoplasmic reticulum and plasma membrane mediates auxin homeostasis and organogenesis in Arabidopsis. New Phytol. 211, 65-74 (2016).

[43] Xu, M. et al. A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice. Plant Cell Physiol. 46, 1674-1681 (2005).

[44] Chen, Y. N. et al. Over-expression of OsPIN2 leads to increased tiller numbers, angle and shorter plant height through suppression of OsLAZY1. Plant Biotechnol. J. 10, 139-149 (2012).

[45] Ding, Z. J. et al. Transcription factor WRKY46 modulates the development of Arabidopsis lateral roots in osmotic/salt stress conditions via regulation of ABA signaling and auxin homeostasis. Plant J. 84, 56-59 (2015).

[46] Zhu, L. et al. Chrysanthemum transcription factor CmLBD1 direct lateral root formation in Arabidopsis thaliana. Sci. Rep. 6, 20009 (2015).

[47] Michaels, S. D. et al. AGL24 acts as a promoter of flowering in Arabidopsis and is positively regulated by vernalization. Plant J. 33, 867-874 (2003).

[48] de Folter, S. et al. A Bsister MADS-box gene involved in ovule and seed development in petunia and Arabidopsis. Plant J. 47, 934-946 (2006).

[49] Dong, T. et al. A tomato MADS-box transcription factor, SlMADS1, acts as a negative regulator of fruit ripening. Plant Physiol. 163, 1026-1036 (2013).

[50] Huang, B. W. et al. Overexpression of the class D MADS-box gene Sl-AGL11 impacts fleshy tissue differentiation and structure in tomato fruits. J. Exp. Bot. 68, 4869-4884 (2017).

[51] Zhang, H. & Forde, B. G. An Arabidopsis MADS box gene that controls nutrient induced changes in root architecture. Science 16, 407-409 (1998).

[52] Tapia-Lopez, R. et al. An AGAMOUS-related MADS-box gene, XAL1 (AGL12), regulates root meristem cell proliferation and flowering transition in Arabidopsis. Plant Physiol. 146, 1182-1192 (2008).

[53] Garay-Arroyo, A. et al. The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression. EMBO J. 32, 2884-2895 (2013).

[54] Yu, C. Y. et al. MADS-box transcription factor OsMADS25 regulates root development through affection of nitrate accumulation in rice. PLoS One 8, e0135196 (2015).

[55] Liu, W. L. et al. A novel sucrose-regulatory MADS-box transcription factor GmNMHC5 promotes root development and nodulation in soybean (Glycine max [L.] Merr.). Int. J. Mol. Sci. 16, 20657-20673 (2015).

[56] Sun, C. H. et al. Chrysanthemum MADS-box transcription factor CmANR1 modulates lateral root development via homo-/heterodimerization to influence auxin accumulation in Arabidopsis. Plant Sci. 266, 27-36 (2017).

[57] Hong, B. et al. Expression of Arabidopsis DREB1A gene in transgenic chrysanthemum enhances tolerance to low temperature. J. Hortic. Sci. Biotechnol. 81, 1002-1008 (2006).

[58] Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644-652 (2011).

[59] Zheng, Y. et al. iAssembler: a package for de novo assembly of Roche-454/Sanger transcriptome sequences. BMC Bioinforma. 12, 453 (2011).

[60] Yang, Y. J. et al. A zinc finger protein regulates flowering time and abiotic stress tolerance in chrysanthemum by modulating gibberellin biosynthesis. Plant Cell 26, 2038-2054 (2014).

[61] Karp, P. D., Paley, S. & Romero, P. The pathway tools software. Bioinformatics 18, S225-S232 (2002).

[62] Xu, Y. J. et al. Transcriptome sequencing and whole genome expression profiling of chrysanthemum under dehydration stress. BMC Genom. 14, 662 (2013).

[63] Hu, D. G. et al. MdMYB1 regulates anthocyanin and malate accumulation by directly facilitating their transport into vacuoles in apples. Plant Physiol. 170, 1315-1330 (2016).

[64] Chen, Q. et al. The basic helix-loop-helix transcription factor MYC2 directly represses PLETHORA expression during jasmonate-mediated modulation of the root stem cell niche in Arabidopsis. Plant Cell 23, 3335-3352 (2011).

[65] An, J. P. et al. The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple. Hort. Res. 4, 17023 (2017).

[66] Lin, W. H. et al. At5PTase13 modulates cotyledon vein development through regulating auxin homeostasis. Plant Physiol. 139, 1677-1691 (2005).

[67] Wynne, J. & Treisman, R. SRF and MCMl have related but distinct DNA binding specificities. Nucleic Acids Res. 20, 3297-3303 (1992).

[68] Lee, H. et al. The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev. 14, 2366-2376 (2000).

[69] Theißen, G. Development of floral organ identity: stories from the MADS house. Curr. Opin. Plant Biol. 4, 75-85 (2001).

[70] Ripoll, J. J. et al. A novel role for the floral homeotic gene APETALA2 during Arabidopsis fruit development. Development 138, 5167-5176 (2011).

[71] Koo, S. C. et al. Control of lateral organ development and flowering time by the Arabidopsis thaliana MADS-box gene AGAMOUS-LIKE6. Plant J. 62, 807-816 (2010).

[72] Sang, X. C. et al. CHIMERIC FLORAL ORGANS1, encoding a monocot-specific MADS box protein, regulates floral organ identity in rice. Plant Physiol. 160, 788-807 (2012).

[73] Tripathi, V. et al. CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants. Plant J. 58, 778e790 (2009).

[74] Robles, L., Stepanova, A. & Alonso, J. Molecular mechanisms of ethylene-auxin interaction. Mol. Plant 6, 1734-1737 (2013).

[75] Tsuchisaka, A. & Theologis, A. Unique and overlapping expression patterns among the Arabidopsis 1-amino-cyclopropane-1-carboxylate synthase gene family members. Plant Physiol. 136, 2982-3000 (2004).

[76] Rahman, A. et al. Auxin is a positive regulator for ethylene-mediated response in the growth of Arabidopsis roots. Plant Cell Physiol. 42, 301-307 (2001).

[77] Stepanova, A. N. et al. Multilevel interactions between ethylene and auxin in Arabidopsis roots. Plant Cell 19, 2169-2185 (2007).

[78] Ruzicka, K. et al. Ethylene regulates root growth through effects on auxin biosynthesis and transport-dependent auxin distribution. Plant Cell 19, 2197-2212 (2007).

[79] Lewis, D. R. et al. Ethylene inhibits lateral root development, increases IAA transport and expression of PIN3 and PIN7 auxin efflux carriers. Development 138, 3485-3495 (2011).

[80] Cai, X. T. et al. Arabidopsis ERF109 mediates cross-talk between jasmonic acid and auxin biosynthesis during lateral root formation. Nat. Commun. 5, 5833 (2014).

[81] Staswick, P. E., Su, W. & Howell, S. H. Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant. Proc. Natl. Acad. Sci. USA 89, 6837-6840 (1992).

[82] Remans, T., Nacry, P. & Pervent, M. The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches. Proc. Natl. Acad. Sci. USA 103, 19206-19211 (2006).

[83] Ho, C. H. et al. CHL1 functions as a nitrate sensor in plants. Cell 138, 1184-1194 (2009).