Michel Laloue

MALE STERILITY ASSOCIATED WITH APRT DEFICIENCY IN ARABIDOPSIS THALIANA RESULTS FROM A MUTATION IN THE GENE APT1

Abstract 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. Furthermore, it has been shown that metabolism of cytokinins is impaired in the mutant BM3, which has the lowest level of APRT activity among the mutants tested. However, three different cDNAs encoding APRT have been isolated in A. thaliana and this raised the question of the nature of the mutation which results in low APRT activity. The mutation was genetically mapped to chromosome I and lies within 6 cM of the phenotypic marker dis2, indicating that the mutation affects the APT1 gene, a result confirmed by sequencing of mutant alleles. The mutation in the allele apt1-3 is located at the 5′ splicing site of the third intron, and eliminates a BstNI restriction site, as verified by Southern blotting and PCR fragment length analysis.

Kinetics of cytokinin production and bud formation in Physcomitrella : Analysis of wild type, a developmental mutant and two of its ipt transgenics

Summary Cytokinins that are known to induce bud formation in mosses were quantified during the course of development of Physcomitrella patens (Hedw.) B.S.G. Analyses were carried out on wild type, the developmental mutant PC22 and two ipt -transgenic strains of PC22. The major cytokinins detected were isopentenyladenine (iP) and isopentenyladenosine ([9R]iP). The cytokinin overproducing ipt -strains released large amounts of iP into the culture medium (up to 32 nmol/L). For Physcomitrella wild type an iP maximum at day 9 preceded bud formation, which occurred at day 13. In the developmental mutant PC22 iP maxima were found at day 9 and at day 21; however, bud formation was not observed within this time. Two transgenics of this mutant, carrying the Agrobacterium ipt gene under control of its own promoter, released up to 34 and 372-fold more iP into the culture medium and continuously produced malformed buds beginning from the first days of culture. The time courses correlating the onset of bud formation with extracellular iP show for all 4 genotypes that iP concentration does not continuously increase but is fluctuating.

Cytokinin metabolism in Physcomitrella patens: differences and similarities to higher plants

Cytokinins play an important role in plant development and occur informs with different hormonal activity. As the nucleotide forms of cytokininsare considered to have little or no biological activity, the conversion ofcytokinin bases and ribosides to their nucleotides can contribute to the tuningof cytokinin activity in plant cells. Cytokinin metabolism was monitoredin vivo by feeding either radiolabelledisopentenyladenosine (3H-[9R]iP) or isopentenyladenine(3H-iP) to liquid grown chloronema tissue ofPhyscomitrellapatens (Hedw.) B.S.G. wild type. The riboside 3H-[9R]iPwas rapidly converted to 3H-iP, which was released into the culturemedium. The intracellular concentration of the 3H-iP was twice ashigh as extracellular. From the overall amount of 3H-iP about 95%were present in the medium. Cytokinin nucleotides occurred as tritiated mono-,di- and triphosphates of 3H-[9R]iP. When feeding the base3H-iP however, its main metabolic fate was degradation and nosignificant amounts of radiolabelled cytokinin nucleotides were detected. Forthe cytokinin metabolism in P. patens it is concludedthat,in contrast to higher plants nucleotides are mainly formed from ribosidesvia the adenosine kinase pathway and not byribophosphorylation of the cytokinin base via adeninephosphoribosyltransferase.

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. Northern blot analysis clearly shows that these purine biosynthetic genes are highly expressed in young and mature nodules but weakly expressed in roots and leaves. Expression levels of GMpurD and GMpurN mRNAs were not enhanced when ammonia was provided to non-nodulated roots.

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. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 A resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.

Specific photoaffinity labelling of a thylakoid membrane protein with an azido-cytokinin agonist

A cytokinin-binding peptide (CBP) of 46 kDa (Thy46) has been identified in thylakoid membranes of pea chloroplasts, by photoaffinity labelling with tritiated 1-(2-azido-6-chloropyrid-4-yl)-3-phenylurea ([3H]azidoCPPU), a urea-type cytokinin agonist. The labelled peptide is also detected in Nicotiana plumbaginifolia, Nicotiana tabacum and spinach thylakoid membranes, but is absent in thylakoid membranes of Chlamydomonas reinhardtii. A pharmacological study of the interaction of this peptide with different cytokinin agonist molecules has been achieved. Urea derivatives are the most efficient competitors of photolabelling, and this efficiency is in good agreement with the cytokinin activity of these compounds. A quantitative analysis of the displacement of the photoaffinity labelling of the peptide by increasing concentrations of CPPU indicates an apparent dissociation constant of 1 μM for this ligand. Purine-type cytokinins are weaker competitors than urea-type molecules, but the efficiency of the competition is also correlated to their respective cytokinin activity. A partial purification of Thy46 by a protocol involving ion exchange chromatography and 2D-gel electrophoresis is described.

The response of a cytokinin resistant mutant is highly specific and permits a new cytokinin bioassay

We have previously isolated six independent cytokinin-resistant mutants of Nicotiana plumbaginifolia which define three complementation groups, zeal, zea2 and zea3. We report here the characterization of the phenotypic response to cytokinin treatment of the mutant 1–64, belonging to the zeal group, and the result of the study of the specificity of this response. The phenotype of this mutant grown in the presence of cytokinin concentrations higher than 0.1 μM is characterized by a hypertrophy of the cotyledons and hypocotyl which results in an increase of plantlet fresh weight. This hypertrophy is correlated to cytokinin concentration in a range between 0.01 to 10 μM. The specificity of this response has been verified by using adenine and urea type cytokinins, as well as enantiomers of methylzeatin and methylbenzyladenine which differ widely in their cytokinin activities. We show that the high specificity of the hypertrophic response to cytokinins can be used as a convenient bioassay to screen the cytokinin activity of adenine or urea type molecules.

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. Like other 2,6-disubstituted pyridylurea analogs, it exhibits high cytokinin activity, and it is easily photolysable. Thus it appears to be a good candidate as a photoaffinity labeling reagent for cytokinin-binding proteins, receptors in particular. In the absence of the 6-chloro substituent, the tetrazole form was the only existing tautomer. The corresponding compound does not exhibit cytokinin activity and is not photolysable.

Identification of Physcomitrella patens genes specific of bud and gametophore formation

The simple morphology and ease of gene knockout makes the moss Physcomitrella patens an attractive model for studies in plant physiology and developmental biology. In this study, we looked for genes expressed during bud formation, an essential step during moss development. Differential expression of genes was analyzed during bud formation, in response to cytokinin, using suppression subtractive hybridization (SSH). Six genes expressed during bud formation were identified. Their expression during the time course of bud development, at the gametophore stage, and in mutants defective in bud formation was characterized using semi-quantitative reverse transcription (RT)-PCR analysis.

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. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.