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
• biologické modely MeSH
• cytokininy biosyntéza   MeSH
• kořenové hlízky rostlin cytologie   genetika   růst a vývoj   fyziologie   MeSH
• kořeny rostlin cytologie   genetika   růst a vývoj   fyziologie   MeSH
• Lotus cytologie   genetika   růst a vývoj   fyziologie   MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin biosyntéza   MeSH
• Rhizobiaceae fyziologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• signální transdukce * MeSH
• symbióza MeSH
• tvorba kořenových hlízek MeSH
• vývojová regulace genové exprese genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokininy MeSH
• regulátory růstu rostlin MeSH
• rostlinné proteiny 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
• alely MeSH
• buněčná diferenciace MeSH
• cytokininy metabolismus   MeSH
• dusičnany metabolismus   MeSH
• fenotyp MeSH
• fixace dusíku genetika   MeSH
• fylogeneze MeSH
• homeostáza * MeSH
• kořenové hlízky rostlin genetika   růst a vývoj   MeSH
• Lotus enzymologie   genetika   růst a vývoj   MeSH
• meristém cytologie   růst a vývoj   MeSH
• mutace genetika   MeSH
• oxidoreduktasy genetika   metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• rostlinné geny MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tvorba kořenových hlízek genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• dusičnany MeSH
• oxidoreduktasy MeSH
• rostlinné proteiny MeSH