Regulation and function of Arabidopsis thaliana secreted ribonucleases

While secreted ribonucleases (RNases) have been well studied at the enzymatic and structural levels, little is known regarding their biological functions. One family of secreted RNases, the RNase T2 family, is particularly widespread, with members throughout various kingdoms. In recent years, many plant members of this superfamily have been identified. Gametophytic self-incompatibility in several plant groups involves the activity of S-RNases, one subfamily of plant T2 RNases. Another subfamily, the S-like RNases, are found in self-compatible as well as self-incompatible plant species, indicating that they have different functions than have S-RNases. Expression patterns of S-like RNases in many species led to the suggestion that these enzymes are involved in remobilization and recycling of phosphate from nucleic acid sources. The project described in this thesis involves the study of secreted RNases in the model plant Arabidopsis thaliana, which has five members of the S-like subfamily. I found that several Arabidopsis RNases are induced in wounded tissues through an as-yet unidentified signaling mechanism. One S-like member, RNS1, is induced both locally and systemically by wounding through a novel signal transduction pathway. The regulation of RNS1 expression was studied using several reporter constructs. I found that transcriptional regulation accounts for the majority of the wound response of this gene. Several other factors were shown to affect RNS1 transcript levels, including abscisic acid, salt stress, and heat shock.; In addition to regulatory mechanisms controlling RNS1 transcription, the function of RNS1 was also studied. Tools used for these studies include plants with constitutively high RNS1 activity and T-DNA mutant lines with insertions in the RNS1 gene. One of these mutants, rns1-2, has little or no RNS1 activity. Conditions that affect RNS1 transcript accumulation provided a starting point for the analysis of the mutant and overexpressor. The observation that the overexpressors grew better than wild type when supplied with RNA as the sole source of P i supports the hypothesis that RNS1 is involved in Pi remobilization. Analysis of the mutant and overexpressor also revealed that RNS1 activity levels affect root length in Arabidopsis. This effect was not dependent on Pi concentration and indicated that secreted RNases are involved in unexpected processes. Certain phenotypes of the rns1-2 plants resembled those of the rny1Delta mutant. Rny1, the only RNase T2 enzyme found in Saccharomyces cereviseae , has been proposed to function in regulating aspects of membrane stability and permeability. Therefore, I propose that RNS1 may also be involved in similar processes.; Finally, the analysis of an Arabidopsis mutant with altered activity levels of several RNA-degrading enzymes is presented. Although one of the increased activities resembles RNS1, I demonstrate that this activity is not RNS1. The altered activities are specific to stems of the mutant, as are its morphological phenotypes. This mutant, arp1, provides an insight into the involvement of RNases in stem-associated processes and stem development.