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Characterizing the role of cytokinin transport in Lotus japonicus nodule development

Research output: Book/anthology/dissertation/reportPh.D. thesisResearch

The identification of several classes of cytokinin transporters indicates that both short and long-distance transport are likely required for regulating cytokinin localization and signalling. Legume nodule development provides a good system to study cytokinin transport, as cytokinin is both necessary and sufficient for the cortical cell divisions observed during this process. This study aims to characterize the role of transporters of the Purine Permease (PUP) family during symbiotic nodule development in Lotus japonicus. Transcript profiling and promoterPUP:GUS reporter gene studies showed several LjPUP genes are expressed during nodule development, especially LjPUP1 and LjPUP3, which have prominent expression in nodules.Single mutants, Ljpup1, Ljpup2 and Ljpup3 with Lotus Endogenous Retrotransposon1 (LORE1) insertions were isolated and subsequently used for phenotypic and functional analyses and for generating double mutants. Overexpressing studies with PUP genes (LjUbi:PUP) have also been undertaken for additional phenotypic analyses. The significant reductions of nodule numbers observed both in Ljpup alleles and overexpression (OE) lines, indicated that an appropriate content of endogenous cytokinin is required for
nodulation. Accordingly, the expressions of LjNIN in both mutants and OE lines were reduced relative to their expression in WT. Further TCSn:reporter analyses indicated altered cytokinin signalling responses in Ljpup and OE lines. Moreover, the IT numbers in Ljpup1 were significantly decreased. Some alteration in ethylene production prior to inoculation indicates this may play a role in the phenotype. We also identified the subcellular localization of these LjPUP proteins through fusion proteins composed of LjPUP-GFP which showed that LjPUP1 and LjPUP3 are localized at the plasma membrane (PM). These constructs were additionally used for transport assays in tobacco leaves, and trans Zeatin (tZ) exporter function of LjPUP1 has been identified. Taken together, we predict that LjPUP1 influences the cytokinin sink by exporting tZ on the PM in cortical cells during nodule formation, which further positively regulate IT establishment and nodule organogenesis in an intricate signalling pathway in L. japonicus. These studies add a further regulatory mechanism of cytokinin transport in maintaining cytokinin homeostasis in plant development.
Original languageEnglish
Publication statusSubmitted - 14 Dec 2018

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