Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans

The study of plant ribonuclease (RNase) functions is complicated by a complex profile of RNase activities detected in tissues. Thus, isolation of individual RNase genes will be desirable for the further understanding of function of each RNase. Here, we describe the isolation of cDNAs encoding two RNases, ZRNaseI and ZRNaseII, in differentiating tracheary elements (TEs) induced from isolated mesophyll cells of Zinnia elegans. Both the ZRNaseI and ZRNaseII exhibit putative secretion signal sequences at the amino-terminal ends with predicted molecular masses of 24 247 Da and 22 448 Da as mature proteins, respectively. DNA gel blot analysis showed that both RNases in Zinnia appear to be encoded by a small gene family. RNA gel blot analysis showed that the expression of the ZRNaseI gene was associated with the late stage of in vitro TE differentiation, whereas the ZRNaseII gene was mainly induced in response to stress. Neither RNase gene was induced in response to phosphate starvation, or to H2O2 challenge in the cultured mesophyll cells, or to senescence in the leaves. In young leaves, the ZRNaseI gene was not induced in response to wounding. But the ZRNaseII gene was markedly induced by 6 h after wounding. Tissue print hybridization showed that the expression of the ZRNaseI gene was preferentially associated with the differentiation TEs in Zinnia stems, while the ZRNaseII mRNA was not detected in unwounded Zinnia organs. Taken together, the results indicated that the ZRNaseI gene is expressed during the process of xylogenesis both in vitro and in the plant, whereas the ZRNaseII gene is predominantly induced in response to wounding. The identification of these RNase genes provides molecular tools for the dissection of the process of autolysis during xylogenesis, and for the dissection of the role of RNase in wounding response.