SummaryLONELY GUY (LOG) genes encode cytokinin riboside 5′-monophosphate phosphoribohydrolases and are directly involved in the activation of cytokinins.To assess whether LOG proteins affect the influence of cytokinin on nodulation, we studied two LOG genes of Medicago truncatula.Expression analysis showed that MtLOG1 and MtLOG2 were upregulated during nodulation in a CRE1-dependent manner. Expression was mainly localized in the dividing cells of the nodule primordium. In addition, RNA interference revealed that MtLOG1 is involved in nodule development and that the gene plays a negative role in lateral root development. Ectopic expression of MtLOG1 resulted in a change in cytokinin homeostasis, triggered cytokinininducible genes and produced roots with enlarged vascular tissues and shortened primary roots. In addition, those 35S:LOG1 roots also displayed fewer nodules than the wild-type. This inhibition in nodule formation was local, independent of the SUPER NUMERIC NODULES gene, but coincided with an upregulation of the MtCLE13 gene, encoding a CLAV-ATA3/EMBRYO SURROUNDING REGION peptide.In conclusion, we demonstrate that in M. truncatula LOG proteins might be implicated in nodule primordium development and lateral root formation.

Cytokinin dehydrogenase (CKX; EC 1.5.99.12) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in plant cells. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a translocation signal and presumably functions in the cytosol. The only extensively characterized maize CKX is the apoplastic ZmCKX1; a maize gene encoding a non-secreted CKX has not previously been cloned or characterized. Thus, the aim of this work was to characterize the maize non-secreted CKX gene (ZmCKX10), elucidate the subcellular localization of ZmCKX10, and compare its biochemical properties with those of ZmCKX1. Expression profiling of ZmCKX1 and ZmCKX10 was performed in maize tissues to determine their transcript abundance and organ-specific expression. For determination of the subcellular localization, the CKX genes were fused with green fluorescent protein (GFP) and overexpressed in tomato hairy roots. Using confocal microscopy, the ZmCKX1-GFP signal was confirmed to be present in the apoplast, whereas ZmCKX10-GFP was detected in the cytosol. No interactions of ZmCKX1 with the plasma membrane were observed. While roots overexpressing ZmCKX1-GFP formed significantly more mass in comparison with the control, non-secreted CKX overexpression resulted in a small reduction in root mass accumulation. Biochemical characterization of ZmCKX10 was performed using recombinant protein produced in Pichia pastoris. In contrast to the preference for 2,6-dichlorophenolindophenol (DCPIP) as an electron acceptor and trans-zeatin, N(6)-(Delta(2)-isopentenyl)adenine (iP) and N(6)-(Delta(2)-isopentenyl)adenosine (iPR) as substrates for ZmCKX1, the non-secreted ZmCKX10 had a range of suitable electron acceptors, and the enzyme had a higher preference for cis-zeatin and cytokinin N-glucosides as substrates.

(P.G., L.S., P.T., O.P., M.S., P.J.)In vitro shoot regeneration is implemented in basic plant research and commercial plant production, but for some plant species, it is still difficult to achieve by means of the currently available cytokinins and auxins. To identify novel compounds that promote shoot regeneration, we screened a library of 10,000 small molecules. The bioassay consisted of a two-step regeneration protocol adjusted and optimized for high-throughput manipulations of root explants of Arabidopsis (Arabidopsis thaliana) carrying the shoot regeneration marker LIGHT-DEPENDENT SHORT HYPOCOTYLS4. The screen revealed a single compound, the cytokinin-like phenyl-adenine (Phe-Ade), as a potent inducer of adventitious shoots. Although Phe-Ade triggered diverse cytokinin-dependent phenotypical responses, it did not inhibit shoot growth and was not cytotoxic at high concentrations. Transcript profiling of cytokinin-related genes revealed that Phe-Ade treatment established a typical cytokinin response. Moreover, Phe-Ade activated the cytokinin receptors ARABIDOPSIS HISTIDINE KINASE3 and ARABIDOPSIS HISTIDINE KINASE4 in a bacterial receptor assay, albeit at relatively high concentrations, illustrating that it exerts genuine but weak cytokinin activity. In addition, we demonstrated that Phe-Ade is a strong competitive inhibitor of CYTOKININ OXIDASE/DEHYDROGENASE enzymes, leading to an accumulation of endogenous cytokinins. Collectively, Phe-Ade exhibits a dual mode of action that results in a strong shoot-inducing activity.

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