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 ofLotus japonicusroots.LjIpt2andLjLog4were 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 ofLjIpt2andLjLog4transcripts occurs independent of theLjLhk1receptor during nodulation. Together with the rapid repression of both genes by cytokinin, this indicates thatLjIpt2andLjLog4contribute 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 inL. japonicusWhile mutant analysis identified redundancy in several biosynthesis families, we found that mutation ofLjIpt4limits nodule numbers. Overexpression ofLjIpt3orLjLog4alone 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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