title: CYTOKININ OXIDASE/DEHYDROGENASE3 Maintains Cytokinin Homeostasis during Root and Nodule Development in Lotus japonicus.
abstract: Cytokinins are required for symbiotic nodule development in legumes, and cytokinin signaling responses occur locally in nodule primordia and in developing nodules. Here, we show that the Lotus japonicus Ckx3 cytokinin oxidase/dehydrogenase gene is induced by Nod factor during the early phase of nodule initiation. At the cellular level, pCkx3::YFP reporter-gene studies revealed that the Ckx3 promoter is active during the first cortical cell divisions of the nodule primordium and in growing nodules. Cytokinin measurements in ckx3 mutants confirmed that CKX3 activity negatively regulates root cytokinin levels. Particularly, tZ and DHZ type cytokinins in both inoculated and uninoculated roots were elevated in ckx3 mutants, suggesting that these are targets for degradation by the CKX3 cytokinin oxidase/dehydrogenase. The effect of CKX3 on the positive and negative roles of cytokinin in nodule development, infection and regulation was further clarified using ckx3 insertion mutants. Phenotypic analysis indicated that ckx3 mutants have reduced nodulation, infection thread formation and root growth. We also identify a role for cytokinin in regulating nodulation and nitrogen fixation in response to nitrate as ckx3 phenotypes are exaggerated at increased nitrate levels. Together, these findings show that cytokinin accumulation is tightly regulated during nodulation in order to balance the requirement for cell divisions with negative regulatory effects of cytokinin on infection events and root development.
authors.affiliation: Centre for Carbohydrate Recognition and Signalling (CARB), Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, Aarhus C, 8000, Denmark (D.E.R., A.B.H., S.K., J.S.); and Laboratory of Growth Regulators and Department of Chemical Biology and Genetics, Palacký University and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, CZ-78371, Olomouc, Czech Republic (O.N.).
authors.forename: Dugald E
authors.identifier: http://orcid.org/0000-0001-9291-9775
authors.initials: DE
authors.lastname: Reid
authors.affiliation: Centre for Carbohydrate Recognition and Signalling (CARB), Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, Aarhus C, 8000, Denmark (D.E.R., A.B.H., S.K., J.S.); and Laboratory of Growth Regulators and Department of Chemical Biology and Genetics, Palacký University and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, CZ-78371, Olomouc, Czech Republic (O.N.).
authors.forename: Anne B
authors.initials: AB
authors.lastname: Heckmann
authors.affiliation: Centre for Carbohydrate Recognition and Signalling (CARB), Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, Aarhus C, 8000, Denmark (D.E.R., A.B.H., S.K., J.S.); and Laboratory of Growth Regulators and Department of Chemical Biology and Genetics, Palacký University and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, CZ-78371, Olomouc, Czech Republic (O.N.).
authors.forename: Ondřej
authors.identifier: http://orcid.org/0000-0003-3452-0154
authors.initials: O
authors.lastname: Novák
authors.affiliation: Centre for Carbohydrate Recognition and Signalling (CARB), Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, Aarhus C, 8000, Denmark (D.E.R., A.B.H., S.K., J.S.); and Laboratory of Growth Regulators and Department of Chemical Biology and Genetics, Palacký University and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, CZ-78371, Olomouc, Czech Republic (O.N.).
authors.forename: Simon
authors.initials: S
authors.lastname: Kelly
authors.affiliation: Centre for Carbohydrate Recognition and Signalling (CARB), Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, Aarhus C, 8000, Denmark (D.E.R., A.B.H., S.K., J.S.); and Laboratory of Growth Regulators and Department of Chemical Biology and Genetics, Palacký University and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, CZ-78371, Olomouc, Czech Republic (O.N.) stougaard@mbg.au.dk.
authors.forename: Jens
authors.identifier: http://orcid.org/0000-0002-9312-2685
authors.initials: J
authors.lastname: Stougaard
mesh_terms: D000483:Alleles; D002454:Cell Differentiation; D003583:Cytokinins / Q000378:metabolism*; D017343:Genes, Plant; D006706:Homeostasis; D000070116:Lotus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D018519:Meristem / Q000166:cytology / Q000254:growth & development; D009154:Mutation / Q000235:genetics; D009566:Nitrates / Q000378:metabolism; D009586:Nitrogen Fixation / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D055170:Plant Root Nodulation / Q000235:genetics; D011401:Promoter Regions, Genetic; D053204:Root Nodules, Plant / Q000235:genetics / Q000254:growth & development*
chemical_list: D003583:Cytokinins; D009566:Nitrates; D010940:Plant Proteins; D010088:Oxidoreductases; C019984:cytokinin oxidase