Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study.

Cytokinin oxidase/dehydrogenase (CKO) is a flavoenzyme, which irreversibly degrades the plant hormones cytokinins and thereby participates in their homeostasis. Several synthetic cytokinins including urea derivatives are known CKO inhibitors but structural data explaining enzyme-inhibitor interactions are lacking. Thus, an inhibitory study with numerous urea derivatives was undertaken using the maize enzyme (ZmCKO1) and the crystal structure of ZmCKO1 in a complex with N-(2-chloro-pyridin-4-yl)-N'-phenylurea (CPPU) was solved.

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor.

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N(6)-(buta-2,3-dienyl)adenine (HA-8) and N(6)-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme.

High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica.

Cytokinin oxidase/dehydrogenase (CKO/CKX) is a flavoenzyme, which irreversibly inactivates cytokinins by severing the isoprenoid side chain from the adenine/adenosine moiety. There are several genes coding for the enzyme in maize (Zea mays). A Z.

The Arabidopsis COW1 gene encodes a phosphatidylinositol transfer protein essential for root hair tip growth.

Root hairs are a major site for the uptake of water and nutrients into plants, and they form an increasingly important model system for the study of development in higher plants. We now report on the molecular genetic analysis of the srh1 mutant in Arabidopsis thaliana impaired in root hair tip growth. We show that srh1 is a new allele of cow1 (can of worms1) and we identified the COW1 gene using a positional cloning strategy.

Maize cytokinin oxidase genes: differential expression and cloning of two new cDNAs.

Cytokinin oxidases (CKOs) play a major role in the regulation of hormone levels in plants by irreversibly degrading cytokinins. Two new cDNAs from maize (CKO2 and CKO3) were cloned and CKO activity of a recombinant CKO3 enzyme was demonstrated. CKO2 and CKO3 encode flavoproteins with 93% identity among each other compared with 45% identity with CKO1.

Purification, crystallization and preliminary X-ray diffraction study of a recombinant cytokinin oxidase from Zea mays.

Cytokinins are hormones that are involved in plant growth and development. They are irreversibly degraded by cytokinin oxidases/dehydrogenases, flavoenzymes which contain a covalently bound flavine adenine dinucleotide (FAD) cofactor. Cytokinin oxidase from Zea mays (ZmCKO1) was overexpressed in the yeast Yarrowia lipolytica, purified (molecular weight 69 kDa) and crystallized using the hanging-drop method.

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency.

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants.

Photoaffinity labelling with the cytokinin agonist azido-CPPU of a 34 kDa peptide of the intracellular pathogenesis-related protein family in the moss Physcomitrella patens.

As in higher plants, the development of the moss Physcomitrella patens is regulated by environmental signals and phytohormones. At the protonema level transition from chloronema to caulonema cells is under auxin control. The formation on second sub-apical caulonema cells of buds that will give rise to the leafy gametophore requires cytokinins.

Cytokinin overproducing ove mutants of Physcomitrella patens show increased riboside to base conversion.

Ove mutants in the moss Physcomitrella patens can arise from different recessive mutations. These mutants display a much larger number of buds than the wild type (wt) due to a dramatic overproduction of cytokinins (Cks), which are released into the culture medium (T.L.

Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts.

Cytokinins are degraded by cytokinin oxidases (CKOs) which catalyse cleavage of the N6-(isopent-2-enyl)-side chain resulting in formation of adenine-type compounds. CKO activity has been recorded in many plants and is thought to play a key role in controlling cytokinin levels in plants. Several partially purified CKOs have been characterised but no genes have been isolated yet.

Cloning and characterization of an adenosine kinase from Physcomitrella involved in cytokinin metabolism.

Adenosine kinase (adk) from the moss Physcomitrella patens (Hedw.) B.S.

Male sterility associated with APRT deficiency in Arabidopsis thaliana results from a mutation in the gene APT1.

Four mutants of Arabidopsis thaliana that are deficient in adenine phosphoribosyl transferase (APRT) activity have been isolated by selecting for germination of seeds and growth of the plantlets on a medium containing 2,6-diaminopurine (DAP), a toxic analog of adenine. In all mutants, DAP resistance is due to a recessive nuclear mutation at a locus designated apt. The mutants are male sterile due to pollen abortion after meiosis.

Isolation of cDNAs encoding two purine biosynthetic enzymes of soybean and expression of the corresponding transcripts in roots and root nodules.

Soybean nodule cDNA clones encoding glycinamide ribonucleotide (GAR) synthetase (GMpurD) and GAR transformylase (GMpurN) were isolated by complementation of corresponding Escherichia coli mutants. GAR synthetase and GAR transformylase catalyse the second and the third steps in the de novo purine biosynthesis pathway, respectively. One class of GAR synthetase and three classes of GAR transformylase cDNA clones were identified.

A second form of adenine phosphoribosyltransferase in Arabidopsis thaliana with relative specificity towards cytokinins.

Adenine phosphoribosyltransferase (APRTase) is an important enzyme for its ability to convert adenine, a byproduct of many biochemical reactions, into AMP. By functional complementation of an Escherichia coli mutant, cDNAs encoding two APRTases have been cloned from Arabidopsis thaliana. One of the cDNAs (ATapt1) has been previously identified while the second (ATapt2) is of a previously unknown type.

Synthesis, azido-tetrazole equilibrium studies and biological activity of 1-(2-azido-6-chloropyrid-4-yl)-3-phenylurea, a photoaffinity labeling reagent for cytokinin-binding proteins.

1-(2-Azido-6-chloropyrid-4-yl)-3-phenylurea was synthesized using known methods. Azido-tetrazole equilibrium for this compound was studied in various solvents, and the azide tautomer was found to be largely predominant. However, in water solution, it is suspected to exist in the tetrazole form in a significant amount.

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes.

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase.

Comparison of Benzyl Adenine Metabolism in Two Petunia hybrida Lines Differing in Shoot Organogenesis.

The uptake and metabolism of the cytokinin benzyl adenine (BA) was compared in two lines of Petunia hybrida Vilm. differing in their shoot organogenic response. Leaf transfer experiments using shoot induction medium containing 4.

Rapid identification of cytokinins by an immunological method.

A method for rapid identification of bacterial cytokinins has been developed in which cultures are fed [(3)H]adenine, the cytokinins (including (3)H-labeled cytokinins) are isolated by immunoaffinity chromatography, and analyzed by HPLC with on-line scintillation counting. Analysis of Agrobacterium tumefaciens strains showed that some produced primarily trans-zeatin, whereas others produced primarily trans-zeatin riboside. Pseudomonas syringae pv savastanoi produced mixtures of trans-zeatin, dihydrozeatin, 1''-methyl-trans-zeatin riboside, and other unknown cytokinin-like substances.

Metabolism of Benzyladenine is Impaired in a Mutant of Arabidopsis thaliana Lacking Adenine Phosphoribosyltransferase Activity.

Formation of the riboside-5'-monophosphate is a general feature of the metabolism of cytokinins in plants. As part of a study of the biological significance of the nucleotide form of cytokinins, we analyzed a mutant of Arabidopsis thaliana deficient in adenine phosphoribosyltransferase (APRT) activity for its ability to metabolize N(6)-benzyladenine (BA). Formation of N(6)-benzyladenosine-5'-monophosphate (BAMP) was assayed in vivo, by feeding tritiated BA to wild-type and mutant plantlets, and in crude plantlet extracts.

Cytokinin oxidase from wheat: partial purification and general properties.

As part of the study of the possible role(s) of CBF-1, a cytokinin-binding protein abundant in wheat embryo, a cytokinin oxidase was found in wheat (Triticum aestivum L.) germ and partially purified by conventional purification techniques and high performance chromatofocusing. This preparation catalyzes conversion of N(6)-(Delta(2)-isopentenyl)adenosine to adenosine at a V(max) of 0.

Kinetics of N-(Delta-Isopentenyl)Adenosine Degradation in Tobacco Cells: EVIDENCE OF A REGULATORY MECHANISM UNDER THE CONTROL OF CYTOKININS.

Uptake and degradation of the cytokinin, N(6)-(Delta(2)-isopentenyl) adenosine, were studied in tobacco cells grown as cell suspensions. Degradation occurs by cleavage of the isopentenyl chain which gives adenylic products. Rate of N(6)(Delta(2)-isopentenyl)[8-(14)C]adenosine degradation increases several-fold after a 3- to 4-hour delay when cells have been exposed to a cytokinin.

Biological Effects of Cytokinin Antagonists 7-(Pentylamino) and 7-(Benzylamino)-3-Methylpyrazolo(4,3-d)Pyrimidines on Suspension-cultured Tobacco Cells.

We have studied the biological effects of two structural analogs of cytokinins, 3-methyl-7-(pentylamino)pyrazolo(4,3-d)pyrimidine and 3-methyl-7-(benzylamino)pyrazolo(4,3-d)pyrimidine, on tobacco cell suspension cultures. These two cytokinin analogs are highly inhibitory to cytokinin-autonomous and cytokinin-requiring tobacco cells. The growth inhibitory effect is markedly antagonized by benzyladenine, but not by adenine.

Cytokinins: Metabolism and Biological Activity of N-(Delta-Isopentenyl)adenosine and N-(Delta-Isopentenyl)adenine in Tobacco Cells and Callus.

The cytokinin, N(6)-(Delta(2)-isopentenyl)adenine, is found to be at least 3.3 times as active as N(6)-(Delta(2)-isopentenyl)adenosine in promoting the growth of cytokinin-requiring tobacco (Nicotiana tabacum) callus. Absorption rates of N(6)-(Delta(2)-isopentenyl)adenine and N(6)-(Delta(2)-isopentenyl)adenosine by tobacco cells in liquid suspension do not differ significantly.

Cytokinins: 7-glucosylation is not a prerequisite of the expression of their biological activity.

(14)C-labelled benzyladenine-7-glucoside (0.57 μM) supplied to cytokinin-requiring Nicotiana tabacum (cv. Wisconsin 38) cells cultivated in liquid medium was slowly absorbed and resulted in rather large intracellular quantities of benzyladenine-7-glucoside (30 pmol/10(5) cells) but did not show any biological activity, while 0.