Regulated trafficking of cellulose synthases.

New findings reveal that proteins involved in cellulose biosynthesis undergo regulated trafficking between intracellular compartments and the plasma membrane. The coordinated secretion and internalization of these proteins involve both the actin and cortical microtubule cytoskeletons. This regulated trafficking allows the dynamic remodeling of cellulose synthase complex (CSC) secretion during cell expansion and differentiation. Several new actors of the cellulose synthesis machinery have been recently identified.

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. Using [3H]azidoCPPU (1-(2-azido-6-chloropyrid-4-yl)-3-(4-[3H])phenylurea), a photoactivatable cytokinin agonist, we have specifically photolabelled a soluble 34 kDa protein of P. patens. Urea derivatives were very efficient competitors of photolabelling while purine-type cytokinins were poor competitors. The protein UBP34 was purified by affinity chromatography and the sequences of six internal peptides obtained. A cDNA encoding UBP34 was cloned by screening a P. patens protonema cDNA library with a probe amplified by PCR using degenerate primers designed from the peptide sequences. The UBP34 amino acid sequence shows an average sequence identity of 42% with both intracellular PR proteins and the BetV1-related family of plant allergens. Recombinant UBP34 expressed in Escherichia coli was confirmed to bind azidoCPPU.

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.

Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana.

Abscisic acid (ABA) is a plant hormone which plays an important role in seed development and dormancy and in plant response to environmental stresses. An ABA-deficient mutant of Nicotiana plumbaginifolia, aba2, was isolated by transposon tagging using the maize Activator transposon. The aba2 mutant exhibits precocious seed germination and a severe wilty phenotype. The mutant is impaired in the first step of the ABA biosynthesis pathway, the zeaxanthin epoxidation reaction. ABA2 cDNA is able to complement N.plumbaginifolia aba2 and Arabidopsis thaliana aba mutations indicating that these mutants are homologous. ABA2 cDNA encodes a chloroplast-imported protein of 72.5 kDa, sharing similarities with different mono-oxigenases and oxidases of bacterial origin and having an ADP-binding fold and an FAD-binding domain. ABA2 protein, produced in Escherichia coli, exhibits in vitro zeaxanthin epoxidase activity. This is the first report of the isolation of a gene of the ABA biosynthetic pathway. The molecular identification of ABA2 opens the possibility to study the regulation of ABA biosynthesis and its cellular location.

Identification by mutational analysis of four critical residues in the molybdenum cofactor domain of eukaryotic nitrate reductase.

The nucleotide sequence of the nitrate reductase (NR) molybdenum cofactor (MoCo) domain was determined in four Nicotiana plumbaginifolia mutants affected in the NR apoenzyme gene. In each case, missense mutations were found in the MoCo domain which affected amino acids that were conserved not only among eukaryotic NRs but also in animal sulfite oxidase sequences. Moreover an abnormal NR molecular mass was observed in three mutants, suggesting that the integrity of the MoCo domain is essential for a proper assembly of holo-NR. These data allowed to pinpoint critical residues in the NR MoCo domain necessary for the enzyme activity but also important for its quaternary structure.

Genetic and biochemical analysis of intragenic complementation events among nitrate reductase apoenzyme-deficient mutants of Nicotiana plumbaginifolia.

Intragenic complementation has been observed between apoenzyme nitrate reductase-deficient mutants (nia) of Nicotiana plumbaginifolia. In vivo as in vitro, the NADH-nitrate reductase (NR) activity in plants heterozygous for two different nia alleles was lower than in the wild type plant, but the plants were able to grow on nitrate as a sole nitrogen source. NR activity, absent in extracts of homozygous nia mutants was restored by mixing extracts from two complementing nia mutants. These observations suggest that NR intragenic complementation results from either the formation of heteromeric NR or from the interaction between two modified enzymes. Complementation was only observed between mutants retaining different partial catalytic activities of the enzyme. Results are in agreement with molecular data suggesting the presence of three catalytic domains in the subunit of the enzyme.

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia.

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.

Sensitivity to nikkomycin Z in Candida albicans: role of peptide permeases.

The uptake of tritiated nikkomycin Z, a potent inhibitor of chitin synthetase, is mediated by a peptide transport system in Candida albicans. Kinetic transport assays with radioactive di- and tripeptides and competition studies suggest that two distinct systems operate in this yeast. Nikkomycin Z was transported through one of these systems, common to di- and tripeptides. A peptide transport-deficient mutant was isolated on the basis of its resistance to nikkomycin Z. The mutant lost most of its capacity to take up dipeptides but simultaneously increased its ability to transport tripeptides. These results indicate that C. albicans handles peptides through multiple transport systems and adjusts their expression to environmental conditions.

Photoaffinity inhibition of peptide transport in yeast.

A photoaffinity label, 4-azidobenzoyltrimethionine has been synthesized. It competitively inhibits trimethionine uptake in the yeast C. albicans. Upon UV irradiation it irreversibly and specifically blocks oligopeptide uptake. These results give the first example of photoinhibition of peptide uptake in yeast.