Large-scale analysis of 73 329 physcomitrella plants transformed with different gene disruption libraries: production parameters and mutant phenotypes.

Gene targeting in the moss Physcomitrella patens has created a new platform for plant functional genomics. We produced a mutant collection of 73 329 Physcomitrella plants and evaluated the phenotype of each transformant in comparison to wild type Physcomitrella. Production parameters and morphological changes in 16 categories, such as plant structure, colour, coverage with gametophores, cell shape, etc., were listed and all data were compiled in a database (mossDB). Our mutant collection consists of at least 1804 auxotrophic mutants which showed growth defects on minimal Knop medium but were rescued on supplemented medium. 8129 haploid and 11 068 polyploid transformants had morphological alterations. 9 % of the haploid transformants had deviations in the leaf shape, 7 % developed less gametophores or had a different leaf cell shape. Other morphological deviations in plant structure, colour, and uniformity of leaves on a moss colony were less frequently observed. Preculture conditions of the plant material and the cDNA library (representing genes from either protonema, gametophore or sporophyte tissue) used to transform Physcomitrella had an effect on the number of transformants per transformation. We found correlations between ploidy level and plant morphology and growth rate on Knop medium. In haploid transformants correlations between the percentage of plants with specific phenotypes and the cDNA library used for transformation were detected. The number of different cDNAs present during transformation had no effect on the number of transformants per transformation, but it had an effect on the overall percentage of plants with phenotypic deviations. We conclude that by linking incoming molecular, proteome, and metabolome data of the transformants in the future, the database mossDB will be a valuable biological resource for systems biology.

Detection and tissue distribution of potato spindle tuber viroid in infected tomato plants by tissue print hybridization.

Potato spindle tuber viroid (PSTVd) was detected in two cultivars of tomato (Lycopersicon esculentum Mill.) by tissue print hybridization of cross-sections of stem and rhachis, using a 35S-labeled PSTVd RNA probe. PSTVd was detectable in the viroid-sensitive and symptom-developing cv "Rutgers" 2 weeks p.i., and in the viroid-tolerant and practically symptomless cv "Goldkugel" 3 weeks p.i. In both tomato cultivars, PSTVd accumulated in the upper parts of the plants newly grown after inoculation. It was predominantly found in association with the ring formed by the vascular tissue. The final accumulation of PSTVd as well as its spatial distribution were similar in the sensitive and in the tolerant tomato cultivar, as estimated from the tissue print autoradiographs. Thus, tissue print hybridization provides a rapid and sensitive means for viroid diagnosis and for the assessment of tissue-specific localization of the viroid RNA.

Detection and tissue distribution of potato spindle tuberviroid in infected tomato plants by tissue print hybridization.

Potato spindle tuber viroid (PSTVd) was detected in two cultivars of tomato (Lycopersicon esculentum Mill.) by tissue print hybridization of cross-sections of stem and rhachis, using a 35S-labeled PSTVd RNA probe. PSTVd was detectable in the viroid-sensitive and symptom-developping cv "Rutgers" 2 weeks p.i., and in the viroid-tolerant and practically symptomless cv "Goldkugel" 3 weeks p.i. In both tomato cultivars, PSTVd accumulated in the upper parts of the plants newly grown after inoculation. It was predominantly found in association with the ring formed by the vascular tissue. The final accumulation of PSTVd as well as its spatial distribution were similar in the sensitive and in the tolerant tomato cultivar, as estimated from the tissue print autoradiographs. Thus, tissue print hybridization provides a rapid and sensitive means for viroid diagnosis and for the assessment of tissue-specific localization of the viroid RNA.

Isolation of viroid-RNA-binding proteins from an expression library with nonradioactive-labeled RNA probes.

The detection and isolation of cDNAs of tomato proteins that are able to bind to viroid RNA molecules are described. They were found by screening of a lambda gt11 cDNA expression library using a modification of the previously established ligand-blotting procedure to detect DNA- and RNA-binding proteins. The essentials of our modifications are the use of (i) digoxigenin-labeled viroid RNA, (ii) low concentration of the labeled probes and (iii) an expression library that allows the direct isolation of cDNA clones. The analysis of various isolated clones showed that this method is reliable for RNA-ligand screening and North-Western blotting. Applied to viroid RNA, these experimental tools provide the precondition for further studies on the specificity of the isolated proteins.

Photosynthetically active suspension cultures of potato spindle tuber viroid infected tomato cells as tools for studying viroid - host cell interaction.

Photosynthetically active callus and cell suspension cultures were established from uninfected Lycopersicon peruvianum plants and from uninfected and potato spindle tuber viroid (PSTVd) infected plants of Lycopersicon esculentum cv. Rutgers. Viroid infection was maintained in photoheterotrophic culture on media containing 3% sucrose, but during continuous photo-mixotrophic culture in low sucrose media (1% sucrose), the level of PSTVd accumulation decreased. Photoautotrophic cell suspensions could be established with uninfected, but not with viroid infected tomato cells. As compared to uninfected cells, PSTVd infected cells grew slowly, were morphologically different in size and shape, and formed tight cell aggregates. Electronmicroscopy showed that starch accumulation in chloroplasts, deformation of the chloroplast envelope and irregular plasmalemmasomes at the cell membrane were associated with PSTVd infection.

Cloning of a cDNA for the S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum.

We screened a cDNA library of the primitive eukaryote Dictyostelium discoideum constructed in the expression vector lambda gt11 with a specific antiserum directed against S-adenosyl-L-homocysteine hydrolase (AdoHcy hydrolase) and isolated cDNA clones coding for fusion proteins with beta-galactosidase. The identity of the largest of these clones was further assessed by analysis of hybrid-selected in vitro translation products. Using the cDNA as a probe, we showed that only one gene coding for AdoHcy hydrolase is present per genome and that a single transcript of 1.6 kb could be detected at various stages of differentiation. The nucleotide sequence of the cDNA was determined. It corresponds to the carboxyterminal half of the protein whose primary structure is highly homologous to the AdoHcy hydrolase from rat liver. The significance of this strong conservation of AdoHcy hydrolase in the course of evolution is discussed.

S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase variations during differentiation of Dictyostelium discoideum.

We have analyzed the level of substrate (AdoMet) and products (AdoHcy) of transmethylations throughout the developmental cycle of the primitive eukaryote Dictyostelium discoideum. The ratio AdoMet/AdoHcy varied dramatically during differentiation. The intracellular level of AdoHcy decreased sharply after the beginning of starvation reaching a value of 18% of that in vegative cells within 4 h. In contrast, there was a two-fold transient increase in AdoMet at the time of aggregation. However, these changes were not related to changes in AdoHcy hydrolase since constant levels of both the protein and the activity were found until 16 h of differentiation. In particular, there was no indication of an in vivo inactivation of the enzyme by cAMP at the time of aggregation. These results are discussed with respect to the previously postulated role of AdoHcy hydrolase in the regulation of the AdoMet/AdoHcy ratio in eukaryotic cells.

Inactivation of S-adenosyl-L-homocysteine hydrolase by cAMP results from dissociation of enzyme-bound NAD+.

S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) is inactivated by cAMP and also by 2'-deoxyadenosine, and in both cases, activity is restored by incubating the inactivated enzyme with NAD+. We have previously presented evidence that, despite these similarities, inactivation by these two ligands proceeds by different mechanisms. We have now used a fluorescence technique to quantitate enzyme-bound NAD+ and NADH on S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum, and we have confirmed that cAMP and 2'-deoxyadenosine inactivate by different mechanisms. Whereas inactivation by 2'-deoxyadenosine is due to reduction of the enzyme-bound NAD+ to NADH, incubation of S-adenosyl-L-homocysteine hydrolase with cAMP results in dissociation of the enzyme-bound NAD+. The dissociation is reversible, and reactivation likely occurs by restoration of the initial NAD+ content. This reversible inactivation by cAMP may be a mechanism of controlling biological methylation reactions by adjusting intracellular concentrations of S-adenosyl-L-homocysteine through action of S-adenosyl-L-homocysteine hydrolase.

Purification of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum: reversible inactivation by cAMP and 2'-deoxyadenosine.

S-Adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum has been purified to homogeneity. It is composed of four subunits, each with a molecular mass of 47,000. In the hydrolysis direction, the enzyme has a pH optimum of 7.5, a Km for S-adenosyl-L-homocysteine (SAH) of 6 microM, and a Vmax of 0.22 mumol min-1 mg-1. In the synthesis direction, the pH optimum is 8.0, the Km for adenosine is 0.4 microM, and the Vmax is 0.30 mumol min-1 mg-1. Although the enzyme binds beta-nicotinamide adenine dinucleotide, as well as adenosine 3',5'-cyclic monophosphate and 2'-deoxyadenosine, these ligands have no effect on enzymatic activity when added to the assay mixture. However, preincubation of SAH hydrolase with NAD+ results in a 25% activation of the enzyme. In addition, this ligand has a striking effect on subunit-subunit interactions, as shown by stabilization of quaternary structure during polyacrylamide gel electrophoresis. Preincubation with cAMP or 2'-deoxyadenosine inactivates the enzyme. Although in both cases the activity is restored upon further incubation with NAD+, we show that inactivation by these two ligands proceeds by different mechanisms. NAD+-reversible inactivation by cAMP and 2'-deoxyadenosine was also observed with the SAH hydrolase from rabbit erythrocytes. Thus, these previously unreported properties of SAH hydrolase also occur with mammalian enzymes and are not restricted to D. discoideum.