Jaroslav Matoušek

The Release and Some Properties of Nuclease from Various Pollen Species

Summary Mature pollen grains of all 15 species examined release nuclease within the first minutes of contact with a liquid medium. These species include plants with both binucleate and trinucleate pollen and with biotic and abiotic modes of pollination. The enzyme of Pinus nigra pollen exhibits similar characteristics as the earlier described enzyme of tobacco pollen classified as plant nuclease I (E.C. 3.1.30.x). It is shown to be a sugar-unspecific endonuclease with maximal activity at acidic pH and preference for single-stranded molecules. The enzyme produces oligonucleotides and 5-mononucleotides. Nuclease of the other pollen species also has more or less pronounced preference for denatured as opposed to native DNA. A pH in the range of 5.0-5.9 and 6.8-7.0 is optimal for the enzyme of dicotyledons and cereal species, respectively. Different pollen species release different amounts of nuclease with different specific activities. The enzyme also exhibits species-related variation in electrophoretic mobility and in the number of molecular forms. Molecular sieving of pine pollen diffusate produced one nuclease peak with an apparent molecular mass of 29.5 x 10 3 .

The release of nucleases from tobacco pollen

Abstract Sugar-unspecific nucleases diffuse out of intact and ungerminated pollen grains of Nicotiana tabacum within 2 min of suspension in pollen medium. No further increase in nuclease activity of the medium was detected during the subsequent pollen cultivation. Two fractions of the nuclease activity are separable by diethylaminoethyl-cellulose (DEAE-C) chromatography differing in relative activities towards native DNA, denatured DNA and RNA. The pH optimum for hydrolysis of the different substrates range from 5.0 to 5.7.

Developmental Changes in Nuclease and other Phosphohydrolase Activities in Anthers of Nicotiana tabacum L.

Summary Nuclease activity in maturing tobacco anthers is mainly confined to the extracellular fraction and is the lowest in pollen extract. The extracellular fraction further contains phosphodiesterase (exonuclease), 3- and 5-nucleotidase and phosphatase activities. The level of enzyme activities exhibits different changes during anther development from the stage of microspore to the maturity. An overall rise occurring after completion of microspore division is followed by a fall of phosphodiesterase, a relative constancy in nucleotidase and a continuous rise of nuclease activities till the stage of almost mature anthers. The nuclease is similar in electrophoretic pattern and in substrate specificity to the earlier described extracellular pollen nuclease classified as plant nuclease I (E.C. 3.1.30.x). The diffusate of mature pollen exhibits as much as 28 % of nuclease activity but less than 4 % of the other phosphohydrolytic activities, with respect to their highest levels in the extracellular anther fraction. Polynucleotide molecules present in this fraction are heterogeneous and of low molecular mass, and the RNA component has high G and low U content, corresponding to the preference of nuclease for poly(U) sequences and to its inability to hydrolyze poly(dG) homopolymers. High nuclease and nucleotidase activities in the extracellular anther fraction coincide with the earlier observed high RNA synthesis and an increase of uptake capacity for uridine in developing pollen. The function of extracellular anther phosphohydrolases is discussed in relation to anther senescence and to pollen maturation.

Analysis of Progeny of Arabidopsis thaliana Plants Regenerated from Crown Gall Tumors

Summary Seeds of Arabidopsis thaliana plants, differentiated from nopaline and octopine crown galls, were harvested and sown on agar medium in bulk. Approximately 5% of the seedlings of both types showed an abnormal phenotype (dwarf type of growth and/or curled leaves). When their calli were subcultured on medium without growth regulators and cultivated in the light, 47 clones expressed opine synthesizing activities which became detectable after 2–5 subcultivations. Seed progeny of a single nopaline teratoma was tested separately. The differentiated floral axes were completly or partly sterile due to the alternations in floral development. In F 1 and F 2 generations, NpDH activity was found in some of the plants; undifferentiated calli appeared occasionally on roots and stems. These calli showed NpDH activity and they prove the transmission of tumor markers to the F 2 generation.

Acid nucleases in PSTV-infected tomato (Lycopersicum esculentum L.) I. Levels of Acid Nuclease Activity in Healthy and PSTV-Infected Tomato Leaves and Callus Tissues

Summary Nuclease activities at pH 5.2 were investigated in potato spindle tuber viroid (PSTV)-infected tomato leaves and calluses. DNase activity per g fresh mass and per mg soluble protein was, approximately, two times higher in infected leaves at «early» stage of pathogenesis than in healthy controls. The activity remained enhanced also when symptoms related to PSTV infection expressed to a greater degree. The nuclease activity was higher with heat denatured ssDNA than with native dsDNA. The ratio of activities did not change significantly upon viroid infection. The activity with RNA in healthy leaves was about forty-fold higher than with dsDNA, but was enhanced only by 19% in infected leaves at the «early» stage of pathogenesis. The nuclease activity with dsDNA was several times higher in tomato calluses than in tomato leaves. Examination of the DNase activities in healthy and PSTV-infected calluses grown at different temperatures showed significant differences and their relation to PSTV pathogenesis.

Occurrence of sugar non-specific nuclease in tobacco callus: changes of nuclease activity during callus growth and plant regeneration

It was determined using electrophoresis in polyacrylamide gels containing native DNA or RNA that sugar non-specific nuclease active at pH 5.2 was expressed in tobacco callus. The nuclease had a relative molecular mass of about 34.6 kDaltons and degraded substrates in the following order of decreasing rate: denaturated DNA>poly dA>UV-irradiated native DNA>native DNA>alkylated native DNA>apurinated native DNA>poly dG≳poly dC. The nuclease activity changed during callus growth and plant regeneration, but no developmental changes in electrophoretic patterns were detected. The increase in specific DNAse activity of nuclease was maximal in the exponential phase of callus growth on both growth and regeneration media, except for activity in the cytokinin-independent cell strain grown on growth medium. The specific DNAse activity of nuclease decreased during the bud formation period, while total DNAse activity calculated per mg of dry weight was slightly higher in vegetative buds (9.1U) than in undifferentiated tissue of callus (8.5U). Specific DNAse activity was, on the average, several hundred-fold lower in the vegetative tissues of flowering tobacco plants than in calluses in the exponential phase of growth.

Acid Nucleases in PSTV-Infected Tomato (Lycopersicum esculentum L.) II. Characterization of Sugar Non-Specific Nuclease Extracted from Healthy and PSTV-Infected Tomato Leaves

Summary The activity of acid nuclease was investigated in healthy and potato spindle tuber viroid (PSTV)-infected tomato leaves. The enzyme has some characteristics of nuclease I, exhibiting DNase, RNase and presumably 3′-nucleotidase activities. It also showed preference for heat-denatured DNA versus native DNA and was active at pH 5.2. Molecular sieving of the extract from infected tomato leaves produced one distinct nuclease peak with an apparent molecular mass of 26 kDaltons. The nuclease extracted from healthy and PSTV-infected leaves exhibited similar PAGE patterns under both native and denatured conditions. On the other hand, some quantitative and qualitative differences were observed on SDS-PAGE and from immunoelectrophoresis patterns of proteins extracted at pH 5.2 from infected and healthy leaves. Both, circular and linear forms of PSTV were found to be cleavable with the nuclease. But PSTVspecific RNA was about 900-fold more resistant substrate to cleavage with plant nucleases than highly polymerized RNA from yeast.

Some immunochemical properties of pollen extracellular nuclease

The extracellular nuclease fromPinus silvestris pollen was isolated and characterized. Molecular sieving produced only one peak of nuclease with apparent molecular mass of about 30 kDaltons, while molecular mass of the enzyme as determined by means of sodium dodecyl sulphate-DNA gel electrophoresis was 25.7 kDaltons or less. The nuclease exhibited one electrophoretic molecular form only.Specific antiserum against this nuclease was prepared and the nuclease was shown to be immunochemically cross-reactive with extracellular nuclease from Pinus nigra pollen. No immunochemical relationships were observed between pine nuclease and the pollen extracellular nucleases from the angiosperm species,Nicotiana tabacum, Corylus avellana, Alnus incana andBetula pubescens. No cross-reactivity was detected by double diffusion between the pine nuclease and the extracellular proteins from vegetative spores ofEquisetum arvense.

PSTV infection in tobacco (Nicotiana tabacum L.) transformed with PSTV cDNA

Tobacco cv. White Burley was transformed with disarmed expression vector pCB1314 containing dimeric cDNA of potato spindle tuber viroid (PSTV, severe strain) in plus orientation regulated from the mannopine promoter. Amount of PSTV specific (−) and (+) sequences and PSTV circular forms was measured in transformed tobacco stock and compared with PSTV content in untransformed tomato and tobacco grafts. It follows from the results that lower rate of accumulation of PSTV in tobacco as compared with tomato is due to less intensive viroid transportation through the cytoplasm and/or cell to cell moverment, whereas both, viroid replication and processing showed comparable characteristics in tomato and tobacco with respect to accumulation of minus and plus strands and circular forms in infected tissues. Despite of accumulation of viroid in comparable amount in both transformed tobacco and infected tomato, no expression of any morphological symptom of disease was observed in transgenic tobacco.

Differentiation of Transformed Plants from Tumors Induced by Agrobacterium rubi ATCC 13335

Summary Agrobacterium rubi ATCC 13335 induced cane gall tumors in all plant species tested: Atropa belladonna, Kalanchoe daigremontiana, Nicotiana tabacum, Petunia hybrida , and Solanum tuberosum . Tumors of all except Kalanchoe were cultivated in vitro . They differentiated teratomas, roots, and rooted plants, which often showed morphological aberrations. All tumors, teratomas, and some of the plants synthesized octopine. Octopine synthase of cane gall tobacco tumors was partially purified and shown to be similar to that of A. tumefaciens B6S3 induced crown gall tumors with regard to the M r (about 38–42 × 10 3 ) and its ability to utilize NADH as well as NADPH for octopine synthesis.