DSRNA DEGRADING NUCLEASES ARE DIFFERENTIALLY EXPRESSED IN TOBACCO ANTHERS

Nucleases, capable of digesting double-stranded RNAs are mainly confin ed to extracellular fractions of tobacco anthers and diffusate of matu re pollen. dsRNAse activity is about 150-fold higher in anther fractio ns than in crude nuclease extracts from tobacco leaves. The level of d sRNAse activity varies during pollen development from the microspore s tage to maturity. In the anther soluble fraction, dsRNAse activity rea ched a maximum (approx. 50 units/anther) at the end of microspore mito sis and then decreased continuously until the stage of almost mature a nthers. In contrast, the nuclease activity associated with pollen incr eased continuously reaching a maximum (5 units/ anther), during subseq uent stages of pollen maturation. Gel electrophoretic analysis reveale d four slowly migrating sugar-unspecific nucleases (active against DNA and RNA) and three faster migrating RNases which were all able to dig est dsRNA. Competition experiments showed that the sugar-unspecific nu cleases accounted for 95% of the total dsRNAse activity. Anther extrac ellular nucleases were further characterized after partial purificatio n on NADP-agarose: dsRNAse activity had a pH optimum at 5.5, was stron gly inhibited by NaCl and by 1 mM Zn2+ and was insensitive to EDTA whi ch could stimulate activity in crude preparations. Analysis of the act ivity with defined substrates showed that ssRNA is more readily degrad ed than dsRNA and that both, endo- and exonucleolytic activities are d etected.