dsRNA Degrading Nucleases are Differentially Expressed in Tobacco Anthers

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Abstract
Nucleases, capable of digesting double-stranded RNAs are mainly confined to extracellular fractions of tobacco anthers and diffusate of mature pollen. dsRNAse activity is about 150-fold higher in anther fractions than in crude nuclease extracts from tobacco leaves. The level of dsRNAse activity varies during pollen development from the microspore stage to maturity. In the anther soluble fraction, dsRNAse activity reached a maximum (approx. 50 units/anther) at the end of microspore mitosis and then decreased continuously until the stage of almost mature anthers. In contrast, the nuclease activity associated with pollen increased continuously reaching a maximum (5 units/anther), during subsequent stages of pollen maturation. Gel electrophoretic analysis revealed four slowly migrating sugar-unspecific nucleases (active against DNA and RNA) and three faster migrating RNases which were all able to digest dsRNA. Competition experiments showed that the sugar-unspecific nucleases accounted for 95% of the total dsRNAse activity. Anther extracellular nucleases were further characterized after partial purification on NADP-agarose: dsRNAse activity had a pH optimum at 5.5, was strongly inhibited by NaCl and by 1 mM Zn2+ and was insensitive to EDTA which could stimulate activity in crude preparations. Analysis of the activity with defined substrates showed that ssRNA is more readily degraded than dsRNA and that both, endo- and exonucleolytic activities are detected.