Plant Growth Regulation

, Volume 85, Issue 2, pp 267–279 | Cite as

LjCOCH interplays with LjAPP1 to maintain the nodule development in Lotus japonicus

  • Yu-Chen Liu
  • Ya-Wen Lei
  • Wei Liu
  • Lin Weng
  • Ming-Juan Lei
  • Xiao-He Hu
  • Zhicheng Dong
  • Da Luo
  • Jun Yang
Original paper


Legume plants develop nodules during their symbiotic interaction with rhizobia, and much progress has been made towards understanding Nod factor perception and downstream signaling pathways, while our knowledge about the maintenance of nodule organogenesis was limited. We report here the knockdown mutants of LjCOCH, an ortholog of COCHLEATA in Pisum sativum, cause severe defects in nodule organogenesis in Lotus japonicus. The mature nodule number was drastically decreased accompanied with abnormal lenticel and vascular bundle developmental defects, but not produce roots from nodules in both Ljcoch mutants and LjCOCH-RNAi transgenic hairy roots. LjAPP1, a membrane-associated soluble aminopeptidase P1, was identified to interact with LjCOCH through yeast two-hybrid screening. Unlike that of Ljcoch mutants, insertion mutants of LjAPP1 and LjAPP1-RNAi transgenic hairy roots showed increased nodule number, while the lenticel and vascular development were not affected. Gene expression analysis indicated that LjCOCH and LjAPP1 were differentially upregulated by rhizobia inoculation, and LjNF-YA1 was the major downstream target of LjCOCH and LjAPP1. Our findings suggested that LjCOCH acts as a key factor involved in determinate nodule development through direct interaction with LjAPP1 to regulate the expression of LjNF-YA1, opposite effects of LjCOCH and LjAPP1 provide a dynamic regulation of nodule development in L. japonicus.


Nodule Organogenesis LjCOCH LjAPP1 Lenticel LjNF-YA1 Lotus japonicus 



This research was funded by the National Key R & D Program of China (2016YFA0500502), National 973 Project (2010CB126501) and the Ministry of Agriculture of the People’s Republic of China for Transgenic Research (Grant No. 2014ZX0800943B). We thank Prof. Nan Yao in Sun Yat-sen University for providing ER-RB plasmids and Prof. Ertao Wang in Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences for his kind help during our LORE1 mutants’ phenotyping. We also thank Prof. Yanzhang Wang in Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences for providing M. loti strain MAFF303099 DsRed and M. loti strain NZP2235.

Author contributions

Y.C. Liu, D. Luo and J. Yang designed the experiments. Y.C. Liu performed the hairy roots, yeast two-hybrids, in situ hybridization in nodules and Co-IP experiments. Y.W. Lei performed Ljcoch and Ljapp1 LORE1 mutants’ analysis and qPCR experiments. W. Liu performed the experiment on subcellular localization of the proteins. L. Weng and M.J. Lei cloned LjCOCH gene and did the in situ hybridization of COCH and LjCOCH in vegetative and reproductive SAM. X.H. Hu prepared the seeds of Lotus japonicus. Y.C. Liu, Y.W. Lei, Z.C. Dong, D. Luo and J. Yang wrote the paper.

Supplementary material

10725_2018_392_MOESM1_ESM.xls (45 kb)
Supplementary material 1 (XLS 45 KB)
10725_2018_392_MOESM2_ESM.pdf (8.1 mb)
Supplementary material 2 (PDF 8321 KB)


  1. Arrighi JF, Barre A, Ben AB, Bersoult A, Soriano LC, Mirabella R, de Carvalho-Niebel F, Journet EP, Gherardi M, Huguet T, Geurts R, Denarie J, Rouge P, Gough C (2006) The Medicago truncatula lysin motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes. Plant Physiol 142:265–279. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Boot KJM, Brussel AANV., Tak T, Spaink HP, Kijne JW (1999) Lipochitin oligosaccharides from rhizobium leguminosarum bv. viciae reduce auxin transport xapacity in Vicia sativa subsp. nigra roots. Mol Plant Microbe Interact 12:839–844. CrossRefGoogle Scholar
  3. Brewin NJ (1991) Development of the leguem root nodule. Annu Rev Cell Dev Biol 7:191–226. CrossRefGoogle Scholar
  4. Chen T, Zhu H, Ke D, Cai K, Wang C, Gou H, Hong Z, Zhang Z (2012) A MAP kinase kinase interacts with SymRK and regulates nodule organogenesis in Lotus japonicus. Plant Cell 24:823–838. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Couzigou JM, Zhukov V, Mondy S, Abu el Heba G, Cosson V, Ellis TH, Ambrose M, Wen J, Tadege M, Tikhonovich I, Mysore KS, Putterill J, Hofer J, Borisov AY, Ratet P (2012) NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes. Plant Cell 24:4498–4510. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Couzigou JM, Magne K, Mondy S, Cosson V, Clement J, Ratet P (2016) The legume NOOT-BOP-COCH-LIKE genes are conservedregulators of abscission, a major agronomical trait in cultivatedcrops. New Phytol 209:228–240. CrossRefPubMedGoogle Scholar
  7. Crespi M, Frugier F (2008) De novo organ formation from differentiated cells: root nodule organogenesis. Sci Signal 1:1–8. CrossRefGoogle Scholar
  8. Ferguson BJ, Reid JB (2005) Cochleata: getting to the root of legume nodules. Plant Cell Physiol 46:1583–1589. CrossRefPubMedGoogle Scholar
  9. Ferguson BJ, Indrasumunar A, Hayashi S, Lin MH, Lin YH, Reid DE, Gresshoff PM (2010) Molecular analysis of legume nodule development and autoregulation. J Integr Plant Biol 52:61–76. CrossRefPubMedGoogle Scholar
  10. Flemetakis E, Kavroulakis N, Quaedvlleg NEM, Spaink HP, Dimou M, Roussis A, Katinakis P (2000) Lotus japonicus contains two distinct ENOD40 genes that are expressed in symbiotic, nonsymbiotic, and embryonic tissues. Mol Plant Microbe Interact 13:987–994. CrossRefPubMedGoogle Scholar
  11. Fobert PR, Coen ES, Murphy GJP, Doonan JH (1994) Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants. EMBO J 13:616–624PubMedPubMedCentralGoogle Scholar
  12. Fu ZQ, Yan S, Saleh A, Wang W, Ruble J, Oka N, Mohan R, Spoel SH, Tada Y, Zheng N, Dong X (2012) NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature 486:228–232. PubMedPubMedCentralGoogle Scholar
  13. Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Curr Opin Plant Biol 9:436–442. CrossRefPubMedGoogle Scholar
  14. 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. CrossRefPubMedGoogle Scholar
  15. Gronlund M, Gustafsen C, Roussis A, Jensen D, Nielsen LP, Marcker KA, Jensen EQ (2003) The Lotus japonicus ndx gene family is involved in nodule function and maintenance. Plant Mol Biol 2003:303–316. CrossRefGoogle Scholar
  16. Hayashi T, Banba M, Shimoda Y, Kouchi H, Hayashi M, Imaizumi-Anraku H (2010) A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts. Plant J 63:141–154. PubMedPubMedCentralGoogle Scholar
  17. Hirsch AM, Bhuvaneswari TV, Torrey JG, Bisselling T (1989) Early nodulin genes are induced in alfalfa root outgrowths elicited by auxin transport inhitors. Proc Natl Acad Sci USA 86:1244–1248. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kumagal H (2003) Gene silencing by expression of Hairpin RNA. Mol Plant Microbe Interact 16:663–668. CrossRefGoogle Scholar
  19. Kuppusamy KT, Ivashuta S, Bucciarelli B, Vance CP, Gantt JS, VandenBosch KA (2009) Knockdown of CELL DIVISION CYCLE16 reveals an inverse relationship between lateral root and nodule numbers and a link to auxin in Medicago truncatula. Plant Physiol 151:1155–1166. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Laloum T, Baudin M, Frances L, Lepage A, Billault-Penneteau B, Cerri MR, Ariel F, Jardinaud M-F, Gamas P, de Carvalho-Niebel F, Niebel A (2014) Two CCAAT-box-binding transcription factors redundantly regulate early steps of the legume-rhizobia endosymbiosis. Plant J 79:757–768. CrossRefPubMedGoogle Scholar
  21. Li XL, Lei MJ, Yan ZY, Wang Q, Chen AM, Sun J, Luo D, Wang YZ (2014) The REL3-mediated TAS3 ta-siRNA pathway integrates auxin and ethylene signaling to regulate nodulation in Lotus japonicus. New Phytol 201:531–544. CrossRefPubMedGoogle Scholar
  22. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408. CrossRefPubMedGoogle Scholar
  23. Madsen EB, Madsen LH, Radutolu S, Olbryt M, Rakwalska M, Szczyglowski K, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J (2003) A receptor kinase gene of the LysM type is involved in legumeperception of rhizobial signals. Nature 425:637–640. CrossRefPubMedGoogle Scholar
  24. 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. CrossRefPubMedGoogle Scholar
  25. Małolepszy A, Mun T, Sandal N, Gupta V, Dubin M, Urbański D, Shah N, Bachmann A, Fukai E, Hirakawa H, Tabata S, Nadzieja M, Markmann K, Su J, Umehara Y, Soyano T, Miyahara A, Sato S, Hayashi M, Stougaard J, Andersen SU (2016) The LORE1 insertion mutant resource. Plant J 88:306–317. CrossRefPubMedGoogle Scholar
  26. Mao G, Turner M, Yu O, Subramanian S (2013) miR393 and miR164 influence indeterminate but not determinate nodule development. Plant Signal Behav. Google Scholar
  27. Mathesius U, Schlaman HRM, Spaink HP, Sautter C, Rolfe BG, Djordjevic MA (1998) Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides. Plant J 14:23–34. CrossRefPubMedGoogle Scholar
  28. Matvienko M, Van De Sande K, Yang WC, Kammen AV, Bisseling T, Fransen H (1994) Comparison of soybean and pea ENOD40 cDNA clones representing genes expressed during both early and late stages of nodule development. Plant Mol Biol 26:487–493. CrossRefPubMedGoogle Scholar
  29. Mishra NS, Tuteja R, Tuteja N (2006) Signaling through MAP kinase networks in plants. Arch Biochem Biophys 452:55–68. CrossRefPubMedGoogle Scholar
  30. Miwa H, Sun JH, Oldroyd G, Downie A (2006) Analysis of nod factor induced calcium signaling in root hair of symbiotically defective mutants of lotus japonicus. Mol Plant Microbe Interact 19:914–923. CrossRefPubMedGoogle Scholar
  31. Murphy A, Taiz L (1999a) Localization and characterization of soluble and plasma membrane aminopeptidase activities in Arabidopsis seedlings. Plant Physiol Biochem 37:431–443. CrossRefGoogle Scholar
  32. Murphy A, Taiz L (1999b) Naphthylphthalamic acid is enzymatically hydrolyzed at the hypocotyl-root transition zone and other tissues of Arabidopsis thaliana seedlings. Plant Physiol Biochem 37:413–430. CrossRefGoogle Scholar
  33. Murphy A, Peer WA, Taiz L (2000) Regulation of auxin transport by aminopeptidases and endogenous flavonoids. Planta 211:315–324. CrossRefPubMedGoogle Scholar
  34. Murphy AS, Hoogner KR, Peer WA, Taiz L (2002) Identification, purification, and molecular cloning of N-1-naphthylphthalmic acid-binding plasma membrane-associated aminopeptidases from Arabidopsis. Plant Physiol 128:935–950. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136. CrossRefPubMedGoogle Scholar
  36. Pitzschke A, Schikora A, Hirt H (2009) MAPK cascade signalling networks in plant defence. Curr Opin Plant Biol 12:421–426. CrossRefPubMedGoogle Scholar
  37. Popp C, Ott T (2011) Regulation of signal transduction and bacterial infection during root nodule symbiosis. Curr Opin Plant Biol 14:458–467. CrossRefPubMedGoogle Scholar
  38. Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y, Gronlund M, Sato S, Nakamura Y, Tabata S, Sandal N, Stougaard J (2003) Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425:585–592. CrossRefPubMedGoogle Scholar
  39. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Stiller J, Martirani L, Tuppale S, Chian RJ, Chiurazzi M, Gresshoff PM (1997) High frequency transformation and regeneration of transgenic plants in the model legume Lotus japonicus. J Exp Bot 48:1357–1365. CrossRefGoogle Scholar
  41. Subramanian S, Stacey G, Yu O (2006) Endogenous isoflavones are essential for the establishment of symbiosis between soybean and Bradyrhizobium japonicum. Plant J 48:261–273. CrossRefPubMedGoogle Scholar
  42. Suzaki T, Yano K, Ito M, Umehara Y, Suganuma N, Kawaguchi M (2012) Positive and negative regulation of cortical cell division during root nodule development in Lotus japonicus is accompanied by auxin response. Development 139:3997–4006. CrossRefPubMedGoogle Scholar
  43. Takanashi K, Sugiyama A, Yazaki K (2011) Involvement of auxin distribution in root nodule development of Lotus japonicus. Planta 234:73–81. CrossRefPubMedGoogle Scholar
  44. Tena G, Asai T, Chiu WL, Sheen J (2001) Plant mitogen-activated protein kinase signaling cascades. Curr Opin Plant Biol 4:392–400. CrossRefPubMedGoogle Scholar
  45. Turner M, Nizampatnam NR, Baron M, Coppin S, Damodaran S, Adhikari S, Arunachalam SP, Yu O, Subramanian S (2013) Ectopic expression of miR160 results in auxin hypersensitivity, cytokinin hyposensitivity, and inhibition of symbiotic nodule development in soybean. Plant Physiol 162:2042–2055. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 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. CrossRefPubMedGoogle Scholar
  47. van Noorden GE, Ross JJ, Reid JB, Rolfe BG, Mathesius U (2006) Defective long-distance auxin transport regulation in the Medicago truncatula super numeric nodules mutant. Plant Physiol 140:1494–1506. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Wasson AP, Pellerone FI, Mathesius U (2006) Silencing the flavonoid pathway in Medicago truncatula inhibits root nodule formation and prevents auxin transport regulation by rhizobia. Plant Cell 18:1617–1629. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572. CrossRefPubMedGoogle Scholar
  50. Yuan S, Zhu H, Gou H, Fu W, Liu L, Chen T, Ke D, Kang H, Xie Q, Hong Z, Zhang Z (2012) A ubiquitin ligase of symbiosis receptor kinase involved in nodule organogenesis. Plant Physiol 160:106CrossRefPubMedPubMedCentralGoogle Scholar
  51. Zhang J, Subramanian S, Stacey G, Yu O (2009) Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti. Plant J 57:171–183. CrossRefPubMedGoogle Scholar
  52. Zhang B, Holmlund M, Lorrain S, Norberg M, Bakó L, Fankhauser C, Nilsson O (2017) BLADE-ON-PETIOLE proteins act in an E3 ubiquitin ligase complex to regulate PHYTOCHROME INTERACTING FACTOR 4 abundance. eLife 6:e26759. PubMedPubMedCentralGoogle Scholar
  53. Zipfel C, Oldroyd GE (2017) Plant signalling in symbiosis and immunity. Nature 543:328–336. CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life SciencesSun Yat-sen UniversityGuangzhouChina
  2. 2.National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiChina
  3. 3.Key Laboratory of South China Agriculture Plant Molecular Analysis and Genetic Improvement, South China Botanical GardenChinese Academy of SciencesGuangzhouChina

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