Legume mutants have shown the requirement for receptor-mediated cytokinin signaling in symbiotic nodule organogenesis. While the receptors are central regulators, cytokinin also is accumulated during early phases of symbiotic interaction, but the pathways involved have not yet been fully resolved. To identify the source, timing, and effect of this accumulation, we followed transcript levels of the cytokinin biosynthetic pathway genes in a sliding developmental zone of Lotus japonicus roots. LjIpt2 and LjLog4 were identified as the major contributors to the first cytokinin burst. The genetic dependence and Nod factor responsiveness of these genes confirm that cytokinin biosynthesis is a key target of the common symbiosis pathway. The accumulation of LjIpt2 and LjLog4 transcripts occurs independent of the LjLhk1 receptor during nodulation. Together with the rapid repression of both genes by cytokinin, this indicates that LjIpt2 and LjLog4 contribute to, rather than respond to, the initial cytokinin buildup. Analysis of the cytokinin response using the synthetic cytokinin sensor, TCSn, showed that this response occurs in cortical cells before spreading to the epidermis in L. japonicus While mutant analysis identified redundancy in several biosynthesis families, we found that mutation of LjIpt4 limits nodule numbers. Overexpression of LjIpt3 or LjLog4 alone was insufficient to produce the robust formation of spontaneous nodules. In contrast, overexpressing a complete cytokinin biosynthesis pathway leads to large, often fused spontaneous nodules. These results show the importance of cytokinin biosynthesis in initiating and balancing the requirement for cortical cell activation without uncontrolled cell proliferation.

Centre for Carbohydrate Recognition and Signaling Department of Molecular Biology and Genetics Aarhus University Aarhus C 8000 Denmark

Laboratory of Growth Regulators Faculty of Science Palacký University and Institute of Experimental Botany Czech Academy of Sciences CZ 78371 Olomouc Czech Republic

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Bauer P, Ratet P, Crespi MD, Schultze M, Kondorosi A (1996) Nod factors and cytokinins induce similar cortical cell division, amyloplast deposition and MsEnod12A expression patterns in alfalfa roots. Plant J 10: 91–105

Boivin S, Kazmierczak T, Brault M, Wen J, Gamas P, Mysore KS, Frugier F (2016) Different cytokinin histidine kinase receptors regulate nodule initiation as well as later nodule developmental stages in Medicago truncatula. Plant Cell Environ 39: 2198–2209 PubMed

Broughton WJ, Dilworth MJ (1971) Control of leghaemoglobin synthesis in snake beans. Biochem J 125: 1075–1080 PubMed PMC

Cerri MR, Frances L, Kelner A, Fournier J, Middleton PH, Auriac MC, Mysore KS, Wen J, Erard M, Barker DG, et al. (2016) The symbiosis-related ERN transcription factors act in concert to coordinate rhizobial host root infection. Plant Physiol 171: 1037–1054 PubMed PMC

Cerri MR, Frances L, Laloum T, Auriac MC, Niebel A, Oldroyd GED, Barker DG, Fournier J, de Carvalho-Niebel F (2012) Medicago truncatula ERN transcription factors: regulatory interplay with NSP1/NSP2 GRAS factors and expression dynamics throughout rhizobial infection. Plant Physiol 160: 2155–2172 PubMed PMC

Cerri MR, Wang Q, Stolz P, Folgmann J, Frances L, Katzer K, Li X, Heckmann AB, Wang TL, Downie JA, et al. (2017) The ERN1 transcription factor gene is a target of the CCaMK/CYCLOPS complex and controls rhizobial infection in Lotus japonicus. New Phytol 215: 323–337 PubMed

Chang L, Ramireddy E, Schmülling T (2015) Cytokinin as a positional cue regulating lateral root spacing in Arabidopsis. J Exp Bot 66: 4759–4768 PubMed PMC

Chen Y, Chen W, Li X, Jiang H, Wu P, Xia K, Yang Y, Wu G (2014) Knockdown of LjIPT3 influences nodule development in Lotus japonicus. Plant Cell Physiol 55: 183–193 PubMed

Cooper JB, Long SR (1994) Morphogenetic rescue of Rhizobium meliloti nodulation mutants by trans-zeatin secretion. Plant Cell 6: 215–225 PubMed PMC

Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139: 5–17 PubMed PMC

Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE 4: e5553. PubMed PMC

Faiss M, Zalubìlová J, Strnad M, Schmülling T (1997) Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants. Plant J 12: 401–415 PubMed

Fang Y, Hirsch AM (1998) Studying early nodulin gene ENOD40 expression and induction by nodulation factor and cytokinin in transgenic alfalfa. Plant Physiol 116: 53–68 PubMed PMC

Fukai E, Soyano T, Umehara Y, Nakayama S, Hirakawa H, Tabata S, Sato S, Hayashi M (2012) Establishment of a Lotus japonicus gene tagging population using the exon-targeting endogenous retrotransposon LORE1. Plant J 69: 720–730 PubMed

Galichet A, Hoyerová K, Kamínek M, Gruissem W (2008) Farnesylation directs AtIPT3 subcellular localization and modulates cytokinin biosynthesis in Arabidopsis. Plant Physiol 146: 1155–1164 PubMed PMC

Gavrilovic S, Yan Z, Jurkiewicz AM, Stougaard J, Markmann K (2016) Inoculation insensitive promoters for cell type enriched gene expression in legume roots and nodules. Plant Methods 12: 4. PubMed PMC

Gonzalez-Rizzo S, Crespi M, Frugier F (2006) The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti. Plant Cell 18: 2680–2693 PubMed PMC

Handberg K, Stougaard J (1992) Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J 2: 487–496

Hansen J, Jørgensen JE, Stougaard J, Marcker KA (1989) Hairy roots: a short cut to transgenic root nodules. Plant Cell Rep 8: 12–15 PubMed

Hayashi T, Shimoda Y, Sato S, Tabata S, Imaizumi-Anraku H, Hayashi M (2014) Rhizobial infection does not require cortical expression of upstream common symbiosis genes responsible for the induction of Ca2+ spiking. Plant J 77: 146–159 PubMed PMC

Heckmann AB, Lombardo F, Miwa H, Perry JA, Bunnewell S, Parniske M, Wang TL, Downie JA (2006) Lotus japonicus nodulation requires two GRAS domain regulators, one of which is functionally conserved in a non-legume. Plant Physiol 142: 1739–1750 PubMed PMC

Heckmann AB, Sandal N, Bek AS, Madsen LH, Jurkiewicz A, Nielsen MW, Tirichine L, Stougaard J (2011) Cytokinin induction of root nodule primordia in Lotus japonicus is regulated by a mechanism operating in the root cortex. Mol Plant Microbe Interact 24: 1385–1395 PubMed

Held M, Hou H, Miri M, Huynh C, Ross L, Hossain MS, Sato S, Tabata S, Perry J, Wang TL, et al. (2014) Lotus japonicus cytokinin receptors work partially redundantly to mediate nodule formation. Plant Cell 26: 678–694 PubMed PMC

Hirsch S, Kim J, Muñoz A, Heckmann AB, Downie JA, Oldroyd GED (2009) GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula. Plant Cell 21: 545–557 PubMed PMC

Jardinaud MF, Boivin S, Rodde N, Catrice O, Kisiala A, Lepage A, Moreau S, Roux B, Cottret L, Sallet E, et al. (2016) A laser dissection-RNAseq analysis highlights the activation of cytokinin pathways by Nod factors in the Medicago truncatula root epidermis. Plant Physiol 171: 2256–2276 PubMed PMC

Kakimoto T. (2001) Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases. Plant Cell Physiol 42: 677–685 PubMed

Kaló P, Gleason C, Edwards A, Marsh J, Mitra RM, Hirsch S, Jakab J, Sims S, Long SR, Rogers J, et al. (2005) Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science 308: 1786–1789 PubMed

Kawaharada Y, James EK, Kelly S, Sandal N, Stougaard J (2017) The Ethylene Responsive Factor Required for Nodulation 1 (ERN1) transcription factor is required for infection-thread formation in Lotus japonicus. Mol Plant Microbe Interact 30: 194–204 PubMed

Kurakawa T, Ueda N, Maekawa M, Kobayashi K, Kojima M, Nagato Y, Sakakibara H, Kyozuka J (2007) Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445: 652–655 PubMed

Lévy J, Bres C, Geurts R, Chalhoub B, Kulikova O, Duc G, Journet EP, Ané JM, Lauber E, Bisseling T, et al. (2004) A putative Ca2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses. Science 303: 1361–1364 PubMed

Lohar DP, Schaff JE, Laskey JG, Kieber JJ, Bilyeu KD, Bird DM (2004) Cytokinins play opposite roles in lateral root formation, and nematode and rhizobial symbioses. Plant J 38: 203–214 PubMed

Lohar DP, Sharopova N, Endre G, Peñuela S, Samac D, Town C, Silverstein KAT, VandenBosch KA (2006) Transcript analysis of early nodulation events in Medicago truncatula. Plant Physiol 140: 221–234 PubMed PMC

Lohmann GV, Shimoda Y, Nielsen MW, Jørgensen FG, Grossmann C, Sandal N, Sørensen K, Thirup S, Madsen LH, Tabata S, et al. (2010) Evolution and regulation of the Lotus japonicus LysM receptor gene family. Mol Plant Microbe Interact 23: 510–521 PubMed

Madsen LH, Tirichine L, Jurkiewicz A, Sullivan JT, Heckmann AB, Bek AS, Ronson CW, James EK, Stougaard J (2010) The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus. Nat Commun 1: 10. PubMed PMC

Maekawa T, Maekawa-Yoshikawa M, Takeda N, Imaizumi-Anraku H, Murooka Y, Hayashi M (2009) Gibberellin controls the nodulation signaling pathway in Lotus japonicus. Plant J 58: 183–194 PubMed

Małolepszy A, Mun T, Sandal N, Gupta V, Dubin M, Urbański D, Shah N, Bachmann A, Fukai E, Hirakawa H, et al. (2016) The LORE1 insertion mutant resource. Plant J 88: 306–317 PubMed

Marsh JF, Rakocevic A, Mitra RM, Brocard L, Sun J, Eschstruth A, Long SR, Schultze M, Ratet P, Oldroyd GED (2007) Medicago truncatula NIN is essential for rhizobial-independent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase. Plant Physiol 144: 324–335 PubMed PMC

Miri M, Janakirama P, Held M, Ross L, Szczyglowski K (2016) Into the Root: how cytokinin controls rhizobial infection. Trends Plant Sci 21: 178–186 PubMed

Mohd-Radzman NA, Laffont C, Ivanovici A, Patel N, Reid D, Stougaard J, Frugier F, Imin N, Djordjevic MA (2016) Different pathways act downstream of the CEP peptide receptor CRA2 to regulate lateral root and nodule development. Plant Physiol 171: 2536–2548 PubMed PMC

Mortier V, Wasson A, Jaworek P, De Keyser A, Decroos M, Holsters M, Tarkowski P, Mathesius U, Goormachtig S (2014) Role of LONELY GUY genes in indeterminate nodulation on Medicago truncatula. New Phytol 202: 582–593 PubMed

Müller B, Sheen J (2008) Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis. Nature 453: 1094–1097 PubMed PMC

Mun T, Bachmann A, Gupta V, Stougaard J, Andersen SU (2016) Lotus Base: An integrated information portal for the model legume Lotus japonicus. Sci Rep 6: 39447. PubMed PMC

Murakami Y, Miwa H, Imaizumi-Anraku H, Kouchi H, Downie JA, Kawaguchi M, Kawasaki S (2006) Positional cloning identifies Lotus japonicus NSP2, a putative transcription factor of the GRAS family, required for NIN and ENOD40 gene expression in nodule initiation. DNA Res 13: 255–265 PubMed

Murray JD, Karas BJ, Sato S, Tabata S, Amyot L, Szczyglowski K (2007) A cytokinin perception mutant colonized by Rhizobium in the absence of nodule organogenesis. Science 315: 101–104 PubMed

Novák O, Hauserová E, Amakorová P, Dolezal K, Strnad M (2008) Cytokinin profiling in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry. Phytochemistry 69: 2214–2224 PubMed

Op den Camp RHM, De Mita S, Lillo A, Cao Q, Limpens E, Bisseling T, Geurts R (2011) A phylogenetic strategy based on a legume-specific whole genome duplication yields symbiotic cytokinin type-A response regulators. Plant Physiol 157: 2013–2022 PubMed PMC

Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, Raitskin O, Leveau A, Farré G, Rogers C, et al. (2015) Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol 208: 13–19 PubMed

Penmetsa RV, Uribe P, Anderson J, Lichtenzveig J, Gish JC, Nam YW, Engstrom E, Xu K, Sckisel G, Pereira M, et al. (2008) The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations. Plant J 55: 580–595 PubMed

Plet J, Wasson A, Ariel F, Le Signor C, Baker D, Mathesius U, Crespi M, Frugier F (2011) MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula. Plant J 65: 622–633 PubMed

R Core Team (2015) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna

Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y, Grønlund M, Sato S, Nakamura Y, Tabata S, Sandal N, et al. (2003) Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425: 585–592 PubMed

Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339: 62–66 PubMed

Rasband WS. (2016) ImageJ. US National Institutes of Health, Bethesda, MD, http://imagej.nih.gov/ij/ (August 5, 2016)

Reid DE, Heckmann AB, Novák O, Kelly S, Stougaard J (2016) CYTOKININ OXIDASE/DEHYDROGENASE3 maintains cytokinin homeostasis during root and nodule development in Lotus japonicus. Plant Physiol 170: 1060–1074 PubMed PMC

Rodpothong P, Sullivan JT, Songsrirote K, Sumpton D, Cheung KWJT, Thomas-Oates J, Radutoiu S, Stougaard J, Ronson CW (2009) Nodulation gene mutants of Mesorhizobium loti R7A-nodZ and nolL mutants have host-specific phenotypes on Lotus spp. Mol Plant Microbe Interact 22: 1546–1554 PubMed

Sakakibara H. (2006) Cytokinins: activity, biosynthesis, and translocation. Annu Rev Plant Biol 57: 431–449 PubMed

Sasaki T, Suzaki T, Soyano T, Kojima M, Sakakibara H, Kawaguchi M (2014) Shoot-derived cytokinins systemically regulate root nodulation. Nat Commun 5: 4983. PubMed

Sato S, Nakamura Y, Kaneko T, Asamizu E, Kato T, Nakao M, Sasamoto S, Watanabe A, Ono A, Kawashima K, et al. (2008) Genome structure of the legume, Lotus japonicus. DNA Res 15: 227–239 PubMed PMC

Schauser L, Roussis A, Stiller J, Stougaard J (1999) A plant regulator controlling development of symbiotic root nodules. Nature 402: 191–195 PubMed

Sieberer BJ, Chabaud M, Timmers AC, Monin A, Fournier J, Barker DG (2009) A nuclear-targeted cameleon demonstrates intranuclear Ca2+ spiking in Medicago truncatula root hairs in response to rhizobial nodulation factors. Plant Physiol 151: 1197–1206 PubMed PMC

Singh S, Katzer K, Lambert J, Cerri M, Parniske M (2014) CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development. Cell Host Microbe 15: 139–152 PubMed

Smit P, Raedts J, Portyanko V, Debellé F, Gough C, Bisseling T, Geurts R (2005) NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription. Science 308: 1789–1791 PubMed

Soyano T, Hirakawa H, Sato S, Hayashi M, Kawaguchi M (2014) Nodule inception creates a long-distance negative feedback loop involved in homeostatic regulation of nodule organ production. Proc Natl Acad Sci USA 111: 14607–14612 PubMed PMC

Soyano T, Kouchi H, Hirota A, Hayashi M (2013) Nodule inception directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus. PLoS Genet 9: e1003352. PubMed PMC

Stougaard J. (1995) Agrobacterium rhizogenes as a vector for transforming higher plants. In Jones H, ed, Plant Gene Transfer and Expression Protocols. Springer, Totowa, NJ, pp 49–61

Stougaard J, Abildsten D, Marcker KA (1987) The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants. Mol Gen Genet 207: 251–255

Takei K, Sakakibara H, Sugiyama T (2001) Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana. J Biol Chem 276: 26405–26410 PubMed

Takei K, Yamaya T, Sakakibara H (2004) Arabidopsis CYP735A1 and CYP735A2 encode cytokinin hydroxylases that catalyze the biosynthesis of trans-zeatin. J Biol Chem 279: 41866–41872 PubMed

Thykjær T, Schauser L, Danielsen D, Finnemann J, Stougaard J (1998) Transgenic plants: Agrobacterium-mediated transformation of the diploid legume Lotus japonicus. In Celis JE, ed, Cell Biology: A Lab Handbook, Ed 2 Academic Press, New York, pp 518–525

Tirichine L, Imaizumi-Anraku H, Yoshida S, Murakami Y, Madsen LH, Miwa H, Nakagawa T, Sandal N, Albrektsen AS, Kawaguchi M, et al. (2006) Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development. Nature 441: 1153–1156 PubMed

Tirichine L, Sandal N, Madsen LH, Radutoiu S, Albrektsen AS, Sato S, Asamizu E, Tabata S, Stougaard J (2007) A gain-of-function mutation in a cytokinin receptor triggers spontaneous root nodule organogenesis. Science 315: 104–107 PubMed

Urbański DF, Małolepszy A, Stougaard J, Andersen SU (2012) Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus. Plant J 69: 731–741 PubMed

van Spronsen PC, Grønlund M, Pacios Bras C, Spaink HP, Kijne JW (2001) Cell biological changes of outer cortical root cells in early determinate nodulation. Mol Plant Microbe Interact 14: 839–847 PubMed

van Zeijl A, Op den Camp RHM, Deinum EE, Charnikhova T, Franssen H, Op den Camp HJM, Bouwmeester H, Kohlen W, Bisseling T, Geurts R (2015) Rhizobium lipo-chitooligosaccharide signaling triggers accumulation of cytokinins in Medicago truncatula roots. Mol Plant 8: 1213–1226 PubMed

Verdier J, Torres-Jerez I, Wang M, Andriankaja A, Allen SN, He J, Tang Y, Murray JD, Udvardi MK (2013) Establishment of the Lotus japonicus Gene Expression Atlas (LjGEA) and its use to explore legume seed maturation. Plant J 74: 351–362 PubMed

Vernié T, Kim J, Frances L, Ding Y, Sun J, Guan D, Niebel A, Gifford ML, de Carvalho-Niebel F, Oldroyd GED (2015) The NIN transcription factor coordinates diverse nodulation programs in different tissues of the Medicago truncatula root. Plant Cell 27: 3410–3424 PubMed PMC

Warner CA, Biedrzycki ML, Jacobs SS, Wisser RJ, Caplan JL, Sherrier DJ (2014) An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy. Plant Physiol 166: 1684–1687 PubMed PMC

Xiao TT, Schilderink S, Moling S, Deinum EE, Kondorosi E, Franssen H, Kulikova O, Niebel A, Bisseling T (2014) Fate map of Medicago truncatula root nodules. Development 141: 3517–3528 PubMed

Yano K, Aoki S, Liu M, Umehara Y, Suganuma N, Iwasaki W, Sato S, Soyano T, Kouchi H, Kawaguchi M (2017) Function and evolution of a Lotus japonicus AP2/ERF family transcription factor that is required for development of infection threads. DNA Res 24: 193–203 PubMed PMC

Yoro E, Suzaki T, Toyokura K, Miyazawa H, Fukaki H, Kawaguchi M (2014) A positive regulator of nodule organogenesis, NODULE INCEPTION, acts as a negative regulator of rhizobial infection in Lotus japonicus. Plant Physiol 165: 747–758 PubMed PMC

Zürcher E, Tavor-Deslex D, Lituiev D, Enkerli K, Tarr PT, Müller B (2013) A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta. Plant Physiol 161: 1066–1075 PubMed PMC

GmPIN-dependent polar auxin transport is involved in soybean nodule development

Soil nutrient status of KwaZulu-Natal savanna and grassland biomes causes variation in cytokinin functional groups and their levels in above-ground and underground parts of three legumes