Biological Roles of Cytokinins

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1 Direct Control of Shoot Meristem Activity by a Cytokinin-Activating Enzyme By Kurakawa et. Al. Published in Nature Presented by Boyana Grigorova Biological Roles of Cytokinins Cytokinins are positive regulators of cell division in the shoot apical meristem. Reduction of the endogenous cytokinin levels results in strong retardation of shoot development due to a reduction in the rate of cell proliferation in the shoot apical. Cytokinins regulate specific components of the cell cycle and induce passage of the cells through the restriction point of the cell cycle in G1. Cytokinins delay leaf senescence Cytokinins promote chloroplast development Cytokinins promote cell expansion in leaves and cotyledons Cytokinins reduce rooting of stem cuttings and the root growth rate. CBMG 688R Trans-Zeatin is the Active Form of Cytokinin Model of Cytokinin Signaling Trans-zeatin Zeatin Isomerase Cis-zeatin Zeatin Riboside? Wuschel Some Facts 1. What is known The growth of plants depends on continuous function of the Cytokinin is required for meristem identity. 2. What is new: The paper reports a novel regulation of meristem activity which is executed by the meristem-specific activation of cytokinins. The LONELY GUY (LOG) gene of rice encodes a novel cytokinin-activating enzyme that works in the final step of bioactive cytokinin synthesis. lemma glume stamens Structures of the Rice Plant lodicule panicle

2 Identification and Characteristics of LOG gene mutants Wild type rice flower Fig1a): Panicles of wild type (left), log-1 (centre) and log-5 (right). The gene was discovered in a screen for defects in maintenance of shoot meristems. ppb spb st pi eg lodicule Wild type rice flower exhibits normal flower organs and all organs are present Log mutants exhibit abnormal or lacking flower organs Inner flower organs of LOG mutants are more severely affected than outer organs st Fig. 1c): log-1 flower with a weak phenotype, which has only one stamen and no pistil. eg Fig. 1d): log-1 flower with a strong phenotype, which has only a pair of empty glumes. Fig. 1e): Reduction of the floral organ number in log mutants Log mutants exhibit floral meristem retardation log mutants show decreased expression H4 and OSH1 Fig1h): In situ hybridization of H4 in wild type plant Fig1j): In situ hybridization of OSH1 in wild type plant Fig. 1f) and 1g): Scanning electron micrograph of an immature flower of wild type (f) and log-1 (g) at the carpel initiation site. Three stamen primordia are seen in the wild type flower. Fig1i): In situ hybridization of H4 in log mutant Fi1k): In situ hybridization of OSH1 in log mutants.

3 Conclusion Positional Cloning of LOG The results outlined in the previous slides strongly support the idea that meristem activity is not properly maintained in log mutants. Fig2 a): Fine mapping of LOG locus. The region of the LOG locus was narrowed within a 35kb region on chromosome 1, which contained 4 predicted genes. 2b) Exon/ intron structure of mutant alleles of the LOG gene containing 7 exons( balck box) and 6 introns. Wild Type LOG gene rescues the Mutant log-1 Fig2c): Complementation test. The mutant phenotype of log-2. Fig2d): Complementation test. Defects in log-2 were rescued by the introduction of LOG cdna driven by the LOG gene promoter. Conclusion On the basis of the results form the positional cloning of the LOG gene and the complementation test, it can be concluded that the Lonely Guy Genes has been indeed cloned to chromosome 1 of the rice genome. LOG mrna Expression Pattern SAM Fig3a): LOG expression in the SAM of wild type meristems. Fig3c): LOG expression in the axillary Expression of LOG mrna Fig3e): A closer view of the expression of LOG in a primary panicle branch P1 Fig3b): LOG expression in the SAM of wild type meristems. P1 indicates a young leaf in the P1 stage. Fig3d): LOG expression in the developing panicle at the primary branch initiation stage. Fig3f): A closer view of the LOG expression in the floral

4 Comparison of Expression Pattern of LOG, OSH1 and OsCLV3 Conclusion LOG expression defines a new region in the meristem and this region may play a novel part in the maintenance of meristem activity. Supplementary Figure 4. Comparison of mrna distributions of LOG, OsCLV3 andosh1. a, d, Expression pattern of LOG observed by in situ hybridization (a), and its drawing shown in yellow (d). b, e, Expression pattern of OsCLV3 (b), and its drawing shown in red (e). c, f, Expression pattern of OSH1 (c), and its drawing shown in gray (f). OsCLV3 is expressed in a small number of cells at the tip of the floral meristem,resembling stem cell-specific CLV3 expression in Arabidopsis1, and OSH1 expression is observed in the whole region of the LOG expression is restricted to a subset of cells in the meristem, and includes the entire region of presumptive stem cells marked bythe OsCLV3 expression. LOG is Involved in Cytokinin Metabolism LOG has Phosphoribohydrolase Activity IPT P450 Fig4a): Schematic representation of cytokinin biosynthesis and activating pathway. DMAPP, dimethylallyl diphosphate; ipr, IP riboside; tzr, tz ribozide; 1, IPT; 2, CYP735A; 3, nucleotodase; 4, nucleotidase. Black arrows indicate reactions whose genes for the enzyme are identified, whereas grey arrows indicate that the genes are not identified. Fig4b): Detection and identification of reaction products. Standards of iprmp and ip, reaction products of iprmp without LOG (-LOG) and with LOG (+LOG) were separated by HPLC. Standards of ip and Ribose 5 - monophosphate and the reaction products in +LOG (product) are analyzed by liquid chromatography/ mass spectrometry monitoring at massto-charge ratio (m/z) 204 for ip, and at 229 for Rib-5 -P (in the right panels). LOG reacts with Nucleoside 5 - Monophosphates LOG Functions in the Cytosol Fig4c): Substrate specificity of LOG for various nucleotides. DZRMP, dihydrozeatin riboside 5 - monophosphate; czrmp, cis-zeatin riboside 5 - monophosphate. The Km values for iprmp and tzrmp are shown above the bars. Fig4d): Analysis of subcellular localization of LOG protein. Chimeric constructs containing CaMV35S::LOG-GFP (LOG-GFP), CaMV35S::GFP-LOG (GFP-LOG) or CaMV35S::GFP (GFP) were transiently expressed in onion epidermal cells.

5 Active Cytokinin Levels Are Lower in Shoot Meristems of log Mutants There is a Correlation Between Severity of log Phenotype and Activity of LOG Protein Fig4e): Cytokinin responsiveness of OsRR1 and OsRR5 in the shoot meristem was analyzed by PCR with RT-PCR. RNA was isolated from shoot apices collected from plants that were treated with 10µM BAP for 60 minutes. Fig4f): In situ hybridization analysis of OsRR1 and OsRR5 expression of floral meristem of WT and log-6. Fig4g): Enzymatic Activity of LOG mutant proteins. Recombinant enzyme activity was assayed with iprmp, and the activities are relative to LOG as 100. Conclusions Questions To Ponder Cytokinin activity is finely regulated by the LOG protein. Its regulation controls shoot meristem activity in a positive way. It functions as a short signal rather than a transported signal in the cytosol of the shoot meristem cells. LOG protein confers the conversion of the inactive to active forms of cytokinin through a one-step process. How is LOG gene function incorporated into the bigger scheme of cytokinin regulation throughout the plant? Since cytokinin is mainly transported from the roots to the other organs, is it still possible that LOG protein might also be transported throughout the plant? What is the regulatory system that controls the spatial distribution of LOG gene? How do we rule out every possibility that a two-step reaction does not occur in the cytokinin activation?