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.

• MeSH
• Models, Biological MeSH
• Cytokinins biosynthesis   MeSH
• Root Nodules, Plant cytology   genetics   growth & development   physiology   MeSH
• Plant Roots cytology   genetics   growth & development   physiology   MeSH
• Lotus cytology   genetics   growth & development   physiology   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators biosynthesis   MeSH
• Rhizobiaceae physiology   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Signal Transduction * MeSH
• Symbiosis MeSH
• Plant Root Nodulation MeSH
• Gene Expression Regulation, Developmental genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytokinins MeSH
• Plant Growth Regulators MeSH
• Plant Proteins MeSH

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.

• MeSH
• Alleles MeSH
• Cell Differentiation MeSH
• Cytokinins metabolism   MeSH
• Nitrates metabolism   MeSH
• Phenotype MeSH
• Nitrogen Fixation genetics   MeSH
• Phylogeny MeSH
• Homeostasis * MeSH
• Root Nodules, Plant genetics   growth & development   MeSH
• Lotus enzymology   genetics   growth & development   MeSH
• Meristem cytology   growth & development   MeSH
• Mutation genetics   MeSH
• Oxidoreductases genetics   metabolism   MeSH
• Promoter Regions, Genetic MeSH
• Genes, Plant MeSH
• Plant Proteins genetics   metabolism   MeSH
• Plant Root Nodulation genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• cytokinin oxidase MeSH Browser
• Cytokinins MeSH
• Nitrates MeSH
• Oxidoreductases MeSH
• Plant Proteins MeSH