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Miroslav Strnad - One of the best experts on this subject based on the ideXlab platform.
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Antibody-mediated modulation of Cytokinins in tobacco: organ-specific changes in Cytokinin homeostasis.
Journal of experimental botany, 2017Co-Authors: Z. Gelova, Václav Motyka, Markéta Pernisová, Ondřej Novak, Petra Ten Hoopen, Siarhei Dabravolski, Vojtěch Didi, Isolde Tillack, Jana Oklešťková, Miroslav StrnadAbstract:Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of Cytokinin metabolism are partially known, the role of individual Cytokinin types in the maintenance of Cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated Cytokinin ribosides, the proposed transport forms of Cytokinins. We observed asymmetric activity of Cytokinin biosynthetic genes and Cytokinin distribution in wild-type tobacco seedlings with higher Cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of Cytokinin ribosides further enhanced the relative Cytokinin abundance in the roots and induced Cytokinin-related phenotypes in an organ-specific manner. The activity of Cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the Cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root Cytokinin levels. In contrast, a significant decrease of Cytokinins occurred in the shoot. We suggest the roots as the main site of Cytokinin biosynthesis in tobacco seedlings. Conversely, Cytokinin levels in the shoot seem to depend largely on long-range transport of Cytokinin ribosides from the root and their subsequent metabolic activation.
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The role of Cytokinins in clubroot disease
European Journal of Plant Pathology, 2016Co-Authors: Robert Malinowski, Miroslav Strnad, Ondřej Novak, M. Hossein Borhan, Lukáš Spíchal, Stephen A. RolfeAbstract:Clubroot (Plasmodiophora brassicae) is a pathogen of Brassicaceae that causes significant reductions in yield as a consequence of gall formation in the root and hypocotyl of infected plants. The pathogen hijacks host vascular cambium development, and Cytokinins are implicated in this process. This paper uses transcriptomics and metabolomics to investigate changes in Cytokinin metabolism during gall formation of clubroot-infected Arabidopsis thaliana. RNASeq analysis of infected tissue showed that host Cytokinin metabolism was strongly down-regulated both at the onset and late stages of gall formation. Expression of host genes associated with Cytokinin biosynthesis, signalling, degradation and conjugation was strongly repressed. Analysis of Cytokinin precursors, active components and conjugates by microanalytical techniques was consistent with these transcriptional responses. Two isopentenyltransferase genes associated with Cytokinin biosynthesis are present in the P. brassicae genome and are expressed throughout gall formation. The impact of pathogen-derived Cytokinins on the total Cytokinin content of infected tissue and host gene expression was minimal in wild type plants. However, infection of ipt1;3;5;7 mutants that are severely restricted in their ability to synthesise active Cytokinins led to an increase in expression of host Cytokinin-responsive genes. We interpret these results as indicating that P. brassicae can synthesise small amounts of Cytokinin, but this has little impact on the host plant as the ipt1;3;5;7 phenotype is not rescued. Intriguingly, plasmodial development was slowed and spore viability reduced in these mutants indicating a potential role for Cytokinins in plasmodial development.
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CHASE domain-containing receptors play an essential role in the Cytokinin response of the moss Physcomitrella patens.
Journal of Experimental Botany, 2016Co-Authors: Klaus Von Schwartzenberg, Miroslav Strnad, Ondrej Novák, Martine Gonneau, Ann-cathrin Lindner, Njuscha Gruhn, Fabien Nogué, Alexander HeylAbstract:While the molecular basis for Cytokinin action is quite well understood in flowering plants, little is known about the Cytokinin signal transduction in early diverging land plants. The genome of the bryophyte Physcomitrella patens (Hedw.) B.S. encodes three classical Cytokinin receptors, the CHASE domain-containing histidine kinases, CHK1, CHK2, and CHK3. In a complementation assay with protoplasts of receptor-deficient Arabidopsis thaliana as well as in Cytokinin binding assays, we found evidence that CHK1 and CHK2 receptors can function in Cytokinin perception. Using gene targeting, we generated a collection of CHK knockout mutants comprising single (Δchk1, Δchk2, Δchk3), double (Δchk1,2, Δchk1,3, Δchk2,3), and triple (Δchk1,2,3) mutants. Mutants were characterized for their Cytokinin response and differentiation capacities. While the wild type did not grow on high doses of Cytokinin (1 µM benzyladenine), the Δchk1,2,3 mutant exhibited normal protonema growth. Bud induction assays showed that all three Cytokinin receptors contribute to the triggering of budding, albeit to different extents. Furthermore, while the triple mutant showed no response in this bioassay, the remaining mutants displayed budding responses in a diverse manner to different types and concentrations of Cytokinins. Determination of Cytokinin levels in mutants showed no drastic changes for any of the Cytokinins; thus, in contrast to Arabidopsis, revealing only small impacts of Cytokinin signaling on homeostasis. In summary, our study provides a first insight into the molecular action of Cytokinin in an early diverging land plant and demonstrates that CHK receptors play an essential role in bud induction and gametophore development.
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Comparison of endogenous Cytokinins and Cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes
Journal of plant physiology, 2012Co-Authors: Wendy A. Stirk, Václav Motyka, Miroslav Strnad, Ondřej Novak, Eva Žižková, Johannes Van StadenAbstract:Abstract Tagetes minuta L. achenes are thermoinhibited at temperatures above 35 °C and have accelerated radicle emergence (germination) when subsequently transferred to an optimal temperature (25 °C). Endogenous Cytokinins and Cytokinin oxidase/dehydrogenase (CKX) activity were compared in normally germinating (25 °C) and thermoinhibited (72 h at 36 °C then transferred to 25 °C) T. minuta achenes. Following imbibition, endogenous Cytokinin concentrations changed in normally germinating T. minuta achenes, with a gradual decrease in dihydrozeatin-type (DHZ) Cytokinins, a large increase in cis - z eatin-type ( c Z) Cytokinins, a smaller increase in N 6 -(2-isopentenyl)adenine-type (iP) Cytokinins and a peak of trans -zeatin-type ( t Z) Cytokinins at 13 h. These changes in the isoprenoid Cytokinin profile were similar in the thermoinhibited achenes imbibed at 36 °C, despite the thermal block preventing radicle emergence. The exception was the iP-type Cytokinins that only increased when transferred to 25 °C. Profiles of the physiologically active free bases showed an increase in t Z prior to radical emergence in both normally germinating (13 h) and thermoinhibited achenes. A large transient peak in aromatic Cytokinins [ N 6 -benzyladenine-type (BA)] occurred during early seedling establishment in normally germinating achenes (40 h) while a transient maximum in BA-type Cytokinins was found prior to radicle emergence in the thermoinhibited achenes (24 h). The CKX activity was enhanced in normally germinating achenes as the Cytokinin concentration increased following imbibition. In thermoinhibited achenes, an elevated temperature negatively affected the CKX activity that only increased when the achenes were transferred to 25 °C, corresponding to an increase in iP-type Cytokinins. However, the favored Cytokinin deactivation pathway in T. minuta appears to be 9-glycosylation, as 9-glucosides accounted for over 50% of the total Cytokinin pool in both normal and thermoinhibited achenes.
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Cytokinins mediate resistance against pseudomonas syringae in tobacco through increased antimicrobial phytoalexin synthesis independent of salicylic acid signaling
Plant Physiology, 2011Co-Authors: Dominik K Groskinsky, Miroslav Strnad, Ondřej Novak, Muhammad Naseem, Usama Ramadan Abdelmohsen, Nicole Plickert, Thomas Engelke, Thomas Griebel, Jurgen Zeier, Hartwig W PfeifhoferAbstract:Cytokinins are phytohormones that are involved in various regulatory processes throughout plant development, but they are also produced by pathogens and known to modulate plant immunity. A novel transgenic approach enabling autoregulated Cytokinin synthesis in response to pathogen infection showed that Cytokinins mediate enhanced resistance against the virulent hemibiotrophic pathogen Pseudomonas syringae pv tabaci. This was confirmed by two additional independent transgenic approaches to increase endogenous Cytokinin production and by exogenous supply of adenine- and phenylurea-derived Cytokinins. The Cytokinin-mediated resistance strongly correlated with an increased level of bactericidal activities and up-regulated synthesis of the two major antimicrobial phytoalexins in tobacco (Nicotiana tabacum), scopoletin and capsidiol. The key role of these phytoalexins in the underlying mechanism was functionally proven by the finding that scopoletin and capsidiol substitute in planta for the Cytokinin signal: phytoalexin pretreatment increased resistance against P. syringae. In contrast to a Cytokinin defense mechanism in Arabidopsis (Arabidopsis thaliana) based on salicylic acid-dependent transcriptional control, the Cytokinin-mediated resistance in tobacco is essentially independent from salicylic acid and differs in pathogen specificity. It is also independent of jasmonate levels, reactive oxygen species, and high sugar resistance. The novel function of Cytokinins in the primary defense response of solanaceous plant species is rather mediated through a high phytoalexin-pathogen ratio in the early phase of infection, which efficiently restricts pathogen growth. The implications of this mechanism for the coevolution of host plants and Cytokinin-producing pathogens and the practical application in agriculture are discussed.
Václav Motyka - One of the best experts on this subject based on the ideXlab platform.
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Antibody-mediated modulation of Cytokinins in tobacco: organ-specific changes in Cytokinin homeostasis.
Journal of experimental botany, 2017Co-Authors: Z. Gelova, Václav Motyka, Markéta Pernisová, Ondřej Novak, Petra Ten Hoopen, Siarhei Dabravolski, Vojtěch Didi, Isolde Tillack, Jana Oklešťková, Miroslav StrnadAbstract:Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of Cytokinin metabolism are partially known, the role of individual Cytokinin types in the maintenance of Cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated Cytokinin ribosides, the proposed transport forms of Cytokinins. We observed asymmetric activity of Cytokinin biosynthetic genes and Cytokinin distribution in wild-type tobacco seedlings with higher Cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of Cytokinin ribosides further enhanced the relative Cytokinin abundance in the roots and induced Cytokinin-related phenotypes in an organ-specific manner. The activity of Cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the Cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root Cytokinin levels. In contrast, a significant decrease of Cytokinins occurred in the shoot. We suggest the roots as the main site of Cytokinin biosynthesis in tobacco seedlings. Conversely, Cytokinin levels in the shoot seem to depend largely on long-range transport of Cytokinin ribosides from the root and their subsequent metabolic activation.
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Comparison of endogenous Cytokinins and Cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes
Journal of plant physiology, 2012Co-Authors: Wendy A. Stirk, Václav Motyka, Miroslav Strnad, Ondřej Novak, Eva Žižková, Johannes Van StadenAbstract:Abstract Tagetes minuta L. achenes are thermoinhibited at temperatures above 35 °C and have accelerated radicle emergence (germination) when subsequently transferred to an optimal temperature (25 °C). Endogenous Cytokinins and Cytokinin oxidase/dehydrogenase (CKX) activity were compared in normally germinating (25 °C) and thermoinhibited (72 h at 36 °C then transferred to 25 °C) T. minuta achenes. Following imbibition, endogenous Cytokinin concentrations changed in normally germinating T. minuta achenes, with a gradual decrease in dihydrozeatin-type (DHZ) Cytokinins, a large increase in cis - z eatin-type ( c Z) Cytokinins, a smaller increase in N 6 -(2-isopentenyl)adenine-type (iP) Cytokinins and a peak of trans -zeatin-type ( t Z) Cytokinins at 13 h. These changes in the isoprenoid Cytokinin profile were similar in the thermoinhibited achenes imbibed at 36 °C, despite the thermal block preventing radicle emergence. The exception was the iP-type Cytokinins that only increased when transferred to 25 °C. Profiles of the physiologically active free bases showed an increase in t Z prior to radical emergence in both normally germinating (13 h) and thermoinhibited achenes. A large transient peak in aromatic Cytokinins [ N 6 -benzyladenine-type (BA)] occurred during early seedling establishment in normally germinating achenes (40 h) while a transient maximum in BA-type Cytokinins was found prior to radicle emergence in the thermoinhibited achenes (24 h). The CKX activity was enhanced in normally germinating achenes as the Cytokinin concentration increased following imbibition. In thermoinhibited achenes, an elevated temperature negatively affected the CKX activity that only increased when the achenes were transferred to 25 °C, corresponding to an increase in iP-type Cytokinins. However, the favored Cytokinin deactivation pathway in T. minuta appears to be 9-glycosylation, as 9-glucosides accounted for over 50% of the total Cytokinin pool in both normal and thermoinhibited achenes.
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Characterization of Arabidopsis thaliana mutant ror-1 (roscovitine-resistant) and its utilization in understanding of the role of Cytokinin N-glucosylation pathway in plants
Plant Growth Regulation, 2010Co-Authors: Somya Dwivedi, Václav Motyka, Radomira Vankova, Eva Žižková, Carmen Herrera, Carol AuerAbstract:Cytokinin analogue roscovitine exhibits a strong inhibitory effect on Cytokinin N -glucosylation, one of the most important pathways of Cytokinin inactivation in plants. R oscovitine - resistant mutant. ( ror - 1 ) was isolated using T-DNA tagged lines of Arabidopsis thaliana (L.) Heynh in order to find a gene putatively involved in Cytokinin N -glucosylation. The amount of Cytokinin N -glucosides of trans -zeatin- and isopentenyladenine-type was elevated by 20% in ror - 1 mutant compared to WT. The Cytokinin oxidase/dehydrogenase activity exhibited a mild elevation in ror - 1 compared to WT in basal media. Additionally, ror - 1 plants showed slightly enhanced resistance to exogenously supplied aromatic Cytokinins (benzyladenine). Incubation with exogenous Cytokinin (5 μM BA for 24 h) resulted in significant up-regulation of ROR -1 gene expression in ror - 1 mutant. In silico analysis showed that ROR - 1 gene encoded for a protein consisting of GRAM (Glycosyltransferases Rab-like GTPase activators and Myotubularins) and C2 domains. Here, we report on the role of ROR - 1 gene in metabolism of bioactive Cytokinins in the plants.
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Evidence for Importance of tRNA-Dependent Cytokinin Biosynthetic Pathway in the Moss Physcomitrella patens
Journal of Plant Growth Regulation, 2008Co-Authors: Natalya A. Yevdakova, Jiri Malbeck, Václav Motyka, Miroslav Strnad, Alena Trávníčková, Ondrej Novák, Klaus SchwartzenbergAbstract:To study Cytokinin biosynthesis, we characterized a temperature-sensitive Cytokinin-overproducing mutant, ove ST25, of the moss Physcomitrella patens with respect to changes in Cytokinin content during thermal induction in comparison to wild type. Our findings, based on combined liquid chromatography-mass spectrometry (LC-MS) analyses, show that thermoinduction caused a strong increase of extracellular N ^6-(Δ^2-isopentenyl)adenine (iP), N ^6-(Δ^2-isopentenyl)adenosine (iPR), cis -zeatin (cZ), cis -zeatin riboside (cZR) and its O-glucoside cZROG in ove ST25. In contrast, no significant changes were measured in the wild type. To investigate the relevance of tRNA for Cytokinin production in Physcomitrella , we determined Cytokinins in tissue and culture medium as well as in tRNA hydrolysates. The analysis of Cytokinins from whole-culture extracts of wild type revealed 56% of iP-type, 32% of cZ-type, and 11% of trans -zeatin (tZ)-type forms. In tRNA, 90% of Cytokinins were represented by cZ-type and 8% by iP-type forms; tZ-type Cytokinins were found only in trace amounts. The finding that the major free Cytokinins are, albeit with altered proportions, also major forms in tRNA is compatible with the hypothesis of a strong tRNA-mediated biogenesis of Cytokinins in this plant. Our RT-PCR-based studies on the expression of the tRNA-IPT gene, PpIPT1 , revealed enhanced transcription levels in the Cytokinin-overproducing ove ST25 mutant at the inducing temperature of 25°C, but not at noninducing conditions (15°C). A wild-type transgenic line with Cytokinin deficiency due to heterologous Cytokinin oxidase/dehydrogenase overexpression ( AtCKX2 ) also exhibited enhanced PpIPT1 expression levels, indicating that Cytokinin deficiency might upregulate tRNA-mediated Cytokinin biosynthesis. The evidence that the tRNA-mediated pathway might be mainly responsible for biosynthesis of isoprenoid Cytokinins in Physcomitrella is strongly supported by the recent release of the Physcomitrella genomic sequence where only tRNA-IPTs but no adenylate-IPTs are present.
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Cytokinins in the bryophyte physcomitrella patens analyses of activity distribution and Cytokinin oxidase dehydrogenase overexpression reveal the role of extracellular Cytokinins
Plant Physiology, 2007Co-Authors: Klaus Von Schwartzenberg, Václav Motyka, Petre I Dobrev, Ondřej Novak, Marta Fernandez Nunez, Hanna Blaschke, Miroslav StrnadAbstract:Ultra-performance liquid chromatography-tandem mass spectrometry was used to establish the Cytokinin profile of the bryophyte Physcomitrella patens (Hedw.) B.S.G.; of 40 analyzed Cytokinins, 20 were detected. cis-Zeatin-riboside-O-glucoside, N 6 -(D 2 -isopentenyl)adenosine-5#-monophosphate (iPRMP), and trans-zeatin-riboside-O-glucoside were the most abundant intracellular Cytokinins. In addition, the aromatic Cytokinins N 6 -benzyladenosine (BAR), N 6 -benzyladenine, meta-, and orthotopolin were detected. Unexpectedly, the most abundant extracellular Cytokinin was the nucleotide iPRMP, and its identity was confirmed by quadrupole time-of-flight mass spectrometry. The effects of overexpressing a heterologous Cytokinin oxidase/ dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) gene (AtCKX2 from Arabidopsis [Arabidopsis thaliana]) on the intracellular and extracellular distribution of Cytokinins was assessed. In cultures of CKX-transformed plants, ultra-performance liquid chromatography-tandem mass spectrometry measurements showed that there were pronounced reductions in the extracellular concentrations of N 6 -(D 2 -isopentenyl)adenine (iP) and N 6 -(D 2 -isopentenyl)adenosine (iPR), but their intracellular Cytokinin concentrations were only slightly affected. In vitro and in vivo measured CKX activity was shown to be strongly increased in the transformants. Major phenotypic changes observed in the CKX-overexpressing plants included reduced and retarded budding, absence of sexual reproduction, and abnormal protonema cells. In bud-induction bioassays with wild-type Physcomitrella, the nucleotides iPRMP, trans-zeatin-riboside-5#-monophosphate, BAR monophosphate, and the cis-zeatin forms cZ and cZR had no detectable effects, while the activities displayed by other selected Cytokinins were in the following order: iP . tZ . N 6 -benzyladenine . BAR . iPR . tZR . meta-topolin . dihydrozeatin . ortho-topolin. The results on wild type and CKX transgenics suggest that extracellular iP and iPR are the main Cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of Cytokinin biosynthetic pathways.
Ondřej Novak - One of the best experts on this subject based on the ideXlab platform.
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Cell-surface receptors enable perception of extracellular Cytokinins
bioRxiv, 2019Co-Authors: Ioanna Antoniadi, Z. Gelova, Ondřej Plíhal, Thomas Vain, R. Simersky, Michal Karady, Alexander A. T. Johnson, Ondřej Novak, Markéta PernisováAbstract:Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience 1,2. Although Cytokinin receptors are substantially localised to the endoplasmic reticulum 3-5, the cellular sites of Cytokinin perception continue to be debated 1,6,7. Several Cytokinin types display bioactivity 8,9 and recently a cell-specific Cytokinin gradient was reported in roots 10. Yet, the importance of spatially heterogeneous Cytokinin distribution and the specific Cytokinin(s) that account for the different responses remain unclear. Here we show that Cytokinin perception by plasma membrane receptors is an effective path for Cytokinin response in root cells. Readout from a Two Component Signalling Cytokinin-specific reporter (TCSn::GFP; 11) is closely matched to intracellular Cytokinin content, yet a proportion of bioactive Cytokinins are detected in the extracellular fluid. Using Cytokinins covalently linked to beads that could not pass the plasma membrane, we demonstrate that strong TCSn activation still occurs and that this response is greatly diminished in Cytokinin receptor mutants. Although intracellular receptors play significant roles, we argue for a revision of concepts of Cytokinin perception to include the spatial dimensions. In particular, selective ligand-receptor affinities, cellular localisation and tissue distribution of bioactive Cytokinins, their receptors, ransporters and inactivation enzymes appear all to be components of the signalling regulatory mechanisms.
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Antibody-mediated modulation of Cytokinins in tobacco: organ-specific changes in Cytokinin homeostasis.
Journal of experimental botany, 2017Co-Authors: Z. Gelova, Václav Motyka, Markéta Pernisová, Ondřej Novak, Petra Ten Hoopen, Siarhei Dabravolski, Vojtěch Didi, Isolde Tillack, Jana Oklešťková, Miroslav StrnadAbstract:Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of Cytokinin metabolism are partially known, the role of individual Cytokinin types in the maintenance of Cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated Cytokinin ribosides, the proposed transport forms of Cytokinins. We observed asymmetric activity of Cytokinin biosynthetic genes and Cytokinin distribution in wild-type tobacco seedlings with higher Cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of Cytokinin ribosides further enhanced the relative Cytokinin abundance in the roots and induced Cytokinin-related phenotypes in an organ-specific manner. The activity of Cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the Cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root Cytokinin levels. In contrast, a significant decrease of Cytokinins occurred in the shoot. We suggest the roots as the main site of Cytokinin biosynthesis in tobacco seedlings. Conversely, Cytokinin levels in the shoot seem to depend largely on long-range transport of Cytokinin ribosides from the root and their subsequent metabolic activation.
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Cytokinin oxidase dehydrogenase3 maintains Cytokinin homeostasis during root and nodule development in lotus japonicus
Plant Physiology, 2016Co-Authors: Dugald Reid, Ondřej Novak, Anne B Heckmann, Simon Kelly, Jens StougaardAbstract: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.
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The role of Cytokinins in clubroot disease
European Journal of Plant Pathology, 2016Co-Authors: Robert Malinowski, Miroslav Strnad, Ondřej Novak, M. Hossein Borhan, Lukáš Spíchal, Stephen A. RolfeAbstract:Clubroot (Plasmodiophora brassicae) is a pathogen of Brassicaceae that causes significant reductions in yield as a consequence of gall formation in the root and hypocotyl of infected plants. The pathogen hijacks host vascular cambium development, and Cytokinins are implicated in this process. This paper uses transcriptomics and metabolomics to investigate changes in Cytokinin metabolism during gall formation of clubroot-infected Arabidopsis thaliana. RNASeq analysis of infected tissue showed that host Cytokinin metabolism was strongly down-regulated both at the onset and late stages of gall formation. Expression of host genes associated with Cytokinin biosynthesis, signalling, degradation and conjugation was strongly repressed. Analysis of Cytokinin precursors, active components and conjugates by microanalytical techniques was consistent with these transcriptional responses. Two isopentenyltransferase genes associated with Cytokinin biosynthesis are present in the P. brassicae genome and are expressed throughout gall formation. The impact of pathogen-derived Cytokinins on the total Cytokinin content of infected tissue and host gene expression was minimal in wild type plants. However, infection of ipt1;3;5;7 mutants that are severely restricted in their ability to synthesise active Cytokinins led to an increase in expression of host Cytokinin-responsive genes. We interpret these results as indicating that P. brassicae can synthesise small amounts of Cytokinin, but this has little impact on the host plant as the ipt1;3;5;7 phenotype is not rescued. Intriguingly, plasmodial development was slowed and spore viability reduced in these mutants indicating a potential role for Cytokinins in plasmodial development.
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Cytokinin OXIDASE/DEHYDROGENASE3 maintains Cytokinin homeostasis during root and nodule development in Lotus japonicus
Plant physiology, 2015Co-Authors: Dugald Reid, Ondřej Novak, Anne B Heckmann, Simon Kelly, Jens StougaardAbstract: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.
Thomas Schmülling - One of the best experts on this subject based on the ideXlab platform.
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two Cytokinin receptors of arabidopsis thaliana cre1 ahk4 and ahk3 differ in their ligand specificity in a bacterial assay
Plant and Cell Physiology, 2004Co-Authors: Lukáš Spíchal, Miroslav Strnad, Natalia Yu Rakova, Michael Riefler, Takeshi Mizuno, Georgy A Romanov, Thomas SchmüllingAbstract:Strains of Escherichia coli that express two different Cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, were used to study the relative sensitivity of these receptors to various Cytokinins. Both receptors were most sensitive to the bases of the isoprenoid-type Cytokinins trans-zeatin and isopentenyladenine but differed significantly in the recognition of other Cytokinin compounds. In particular, CRE1/AHK4 recognized at 1 microm concentration only trans-zeatin while AHK3 recognized cis-zeatin and dihydrozeatin as well, although with a lower sensitivity. Similarly, CRE1/AHK4 was not activated by Cytokinin ribosides and ribotides, but AHK3 was. Comparisons using the ARR5::GUS fusion gene as a Cytokinin reporter in Arabidopsis showed similar relative degrees of responses in planta, except that Cytokinins with aromatic side chains showed much higher activities than in the bacterial assay. These results indicate that the diverse Cytokinin compounds might have specific functions in the numerous Cytokinin-regulated processes, which may depend in turn on different receptors and their associated signalling pathways. The importance of precise control of local concentrations of defined Cytokinin metabolites to regulate the respective downstream event is corroborated.
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Cytokinin deficient transgenic arabidopsis plants show multiple developmental alterations indicating opposite functions of Cytokinins in the regulation of shoot and root meristem activity
The Plant Cell, 2003Co-Authors: Tomas Werner, Václav Motyka, Harry Van Onckelen, Valerie Laucou, Rafael Smets, Thomas SchmüllingAbstract:Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of Cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which Cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the Cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased Cytokinin breakdown (30 to 45% of wild-type Cytokinin content) and reduced expression of the Cytokinin reporter gene ARR5:GUS (β-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, Cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that Cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in Cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that Cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of Cytokinin functions.
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Cytokinin deficient transgenic arabidopsis plants show multiple developmental alterations indicating opposite functions of Cytokinins in the regulation of shoot and root meristem activity
The Plant Cell, 2003Co-Authors: Tomas Werner, Václav Motyka, Valerie Laucou, Rafael Smets, Harry Van Onckelen, Thomas SchmüllingAbstract:Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of Cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which Cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the Cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased Cytokinin breakdown (30 to 45% of wild-type Cytokinin content) and reduced expression of the Cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, Cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that Cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in Cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that Cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of Cytokinin functions.
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Regulation of plant growth by Cytokinin
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Tomas Werner, Václav Motyka, Miroslav Strnad, Thomas SchmüllingAbstract:Cytokinins are a class of plant-specific hormones that play a central role during the cell cycle and influence numerous developmental programs. Because of the lack of biosynthetic and signaling mutants, the regulatory roles of Cytokinins are not well understood. We genetically engineered Cytokinin oxidase expression in transgenic tobacco plants to reduce their endogenous Cytokinin content. Cytokinin-deficient plants developed stunted shoots with smaller apical meristems. The plastochrone was prolonged, and leaf cell production was only 3–4% that of wild type, indicating an absolute requirement of Cytokinins for leaf growth. In contrast, root meristems of transgenic plants were enlarged and gave rise to faster growing and more branched roots. These results suggest that Cytokinins are an important regulatory factor of plant meristem activity and morphogenesis, with opposing roles in shoots and roots.
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Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division
Planta, 1998Co-Authors: Kirsten Reutter, Thomas Schmülling, Rainer Atzorn, Birgit Hadeler, Ralf ReskiAbstract:Development of Physcomitrella patens (Hedw.) B.S.G. starts with a filamentous protonema growing by apical cell division. As a developmental switch, some subapical cells produce three-faced apical cells, the so-called buds, which grow to form leafy shoots, the gametophores. Application of Cytokinins enhances bud formation but no subsequent gametophore development in several mosses. We used the ipt gene of Agrobacterium tumefaciens, encoding a protein which catalyzes the rate-limiting step in Cytokinin biosynthesis, to transform two developmental Physcomitrella mutants. One mutant (P24) was defective in budding (bud) and thus did not produce three-faced cells, while the other one (PC22) was a double mutant, defective in plastid division (pdi), thus possessing at the most one giant chloroplast per cell, and in gametophore development (gad), resulting in malformed buds which could not differentiate into leafy gametophores. Expression of the ipt gene rescued the mutations in budding and in plastid division but not the one in gametophore development. By mutant rescue we provide evidence for a distinct physiological difference between externally applied and internally produced Cytokinins. Levels of immunoreactive Cytokinins and indole-3-acetic acid were determined in tissues and in culture media of the wild-type moss, both mutants and four of their stable ipt transformants. Isopentenyl-type Cytokinins were the most abundant Cytokinins in Physcomitrella, whereas zeatin-type Cytokinins, the major native Cytokinins of higher plants, were not detectable. Cytokinin as well as auxin levels were enhanced in ipt transgenics, demonstrating a cross-talk between both metabolic pathways. In all genotypes, most of the Cytokinin and auxin was found extracellularly. These extracellular pools may be involved in hormone transport in the non-vascular mosses. We suggest that both mutants are defective in signal-transduction rather than in Cytokinin metabolism.
Tatsuo Kakimoto - One of the best experts on this subject based on the ideXlab platform.
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the ckh1 eer4 gene encoding a taf12 like protein negatively regulates Cytokinin sensitivity in arabidopsis thaliana
Plant and Cell Physiology, 2011Co-Authors: Minoru Kubo, Kaori Furuta, Taku Demura, Hiroo Fukuda, Daisuke Shibata, Tatsuo KakimotoAbstract:: The recessive ckh1 (Cytokinin hypersensitive 1) mutant of Arabidopsis thaliana shows hypersensitivity to Cytokinins, which promote proliferation and greening of calli. The CKH1 gene encodes a protein resembling TAF12 (TATA BOX BINDING PROTEIN ASSOCIATED FACTOR 12), which is a component of transcription factor IID (TFIID)- and histone acetyltransferase-containing complexes in yeast and animals. Microarray analyses revealed that a substantially greater number of genes responded to a low level of Cytokinins in the ckh1 mutant than in the wild type. However, expression of Cytokinin primary response genes was not significantly affected by the ckh1 mutation. These results suggest that the CKH1 protein regulates a set of genes involved in late signaling processes governing a range of Cytokinin responses, including cell proliferation and differentiation.
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The CKH1/EER4 Gene Encoding a TAF12-Like Protein Negatively Regulates Cytokinin Sensitivity in Arabidopsis thaliana
Plant and Cell Physiology, 2011Co-Authors: Minoru Kubo, Kaori Furuta, Taku Demura, Hiroo Fukuda, Daisuke Shibata, Tatsuo KakimotoAbstract:: The recessive ckh1 (Cytokinin hypersensitive 1) mutant of Arabidopsis thaliana shows hypersensitivity to Cytokinins, which promote proliferation and greening of calli. The CKH1 gene encodes a protein resembling TAF12 (TATA BOX BINDING PROTEIN ASSOCIATED FACTOR 12), which is a component of transcription factor IID (TFIID)- and histone acetyltransferase-containing complexes in yeast and animals. Microarray analyses revealed that a substantially greater number of genes responded to a low level of Cytokinins in the ckh1 mutant than in the wild type. However, expression of Cytokinin primary response genes was not significantly affected by the ckh1 mutation. These results suggest that the CKH1 protein regulates a set of genes involved in late signaling processes governing a range of Cytokinin responses, including cell proliferation and differentiation.
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identification of plant Cytokinin biosynthetic enzymes as dimethylallyl diphosphate atp adp isopentenyltransferases
Plant and Cell Physiology, 2001Co-Authors: Tatsuo KakimotoAbstract:It has been believed that the key step in Cytokinin biosynthesis is the addition of a 5-carbon chain to the N(6) of AMP. To identify Cytokinin biosynthesis enzymes that catalyze the formation of the isopentenyl side chain of Cytokinins, the Arabidopsis genomic sequence was searched for genes that could code for isopentenyltransferases. This resulted in the identification of nine putative genes for isopentenyltransferases. One of these, AtIPT4, was subjected to detailed analysis. Overexpression of AtIPT4 caused Cytokinin-independent shoot formation on calli. As shoot formation on calli normally occurs only when Cytokinins are applied, it suggested that this gene product catalyzed Cytokinin biosynthesis in plants. Recombinant AtIPT4 catalyzed the transfer of an isopentenyl group from dimethylallyl diphosphate to the N(6) of ATP and ADP, but not to that of AMP. AtIPT4 did not exhibit the DMAPP:tRNA isopentenyltransferase activity. These results indicate that Cytokinins are, at least in part, synthesized from ATP and ADP in plants.
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identification of cre1 as a Cytokinin receptor from arabidopsis
Nature, 2001Co-Authors: Tsutomu Inoue, Masayuki Higuchi, Yukari Hashimoto, Motoaki Seki, Masatomo Kobayashi, Tomohiko Kato, Satoshi Tabata, Kazuo Shinozaki, Tatsuo KakimotoAbstract:Cytokinins are a class of plant hormones that are central to the regulation of cell division and differentiation in plants. It has been proposed that they are detected by a two-component system, because overexpression of the histidine kinase gene CKI1 induces typical Cytokinin responses and genes for a set of response regulators of two-component systems can be induced by Cytokinins. Two-component systems use a histidine kinase as an environmental sensor and rely on a phosphorelay for signal transduction. They are common in microorganisms, and are also emerging as important signal detection routes in plants. Here we report the identification of a Cytokinin receptor. We identified Arabidopsis cre1 (Cytokinin response 1) mutants, which exhibited reduced responses to Cytokinins. The mutated gene CRE1 encodes a histidine kinase. CRE1 expression conferred a Cytokinin-dependent growth phenotype on a yeast mutant that lacked the endogenous histidine kinase SLN1 (ref. 10), providing direct evidence that CRE1 is a Cytokinin receptor. We also provide evidence that Cytokinins can activate CRE1 to initiate phosphorelay signalling.
