Three RNases in Senescent and Nonsenescent Wheat Leaves1

We have described three RNases in wheat leaves (Triticum aestlvum L. cv Chinese Spring) and developed assays for measuring each RNase individually in crude leaf extracts. We initially used activity staining in sodium dodecyl sulfate-polyacrylamide gels to characterize RNases in extracts of primary and flag leaves. We thus identified acid RNase (EC 3.1.27.1, here designated RNase WLA), and two apparently novel enzymes, designated RNases WLb and WLc. RNase WL. activity displays a distinctive isozyme pattem, a molecular mass of 26 kilodaltons (major species), a broad pH range with an optimum near neutrality, insensitivity to EDTA, and stimulation by moderate concentrations of KCI and by MgCI2. RNase WLc activity exhibits a molecular mass of 27 kilodaltons, a neutral pH optimum, insensitivity to EDTA, and inhibition by KCI, MgCI2, and tri(hydroxymethyl)aminomethane. Based on distinctive catalytic properties established in gels, we designed conventional solution assays for seiective quantitation of each RNase activity. We used the assays to monitor the individual RNases after gel filtration chromatography and native gel electrophoresis of extracts. In accompanying work, we used the assays to monitor RNases WLA, WLM, and WLc, which are present in senescent and nonsenescent leaves, during the course of leaf senescence. A net loss of RNA, encompassing differential turnover of particular RNA species, is associated with leaf senescence (7, 9). However, the enzymology of this process is not defined and the mechanisms that regulate nucleic acid turnover during senescence are not known. We recently observed that the activity of SSP-nuclease3 increases markedly during senescence of flag leaves in wheat, and during dark-induced senescence of wheat seedlings (3). In the present work, we describe RNases in wheat leaves that may also mediate RNA turnover during senescence. Our experimental approach exploited activity staining in 'Research supported in part by the T.W. Edminster Award from the Agricultural Research Service, U.S. Department of Agriculture. 2Present address: Department of Pathology SM-30, University of Washington, Seattle, WA 98195. 3Abbreviations: SSP-nuclease, single-strand-preferring nuclease; WL, wheat leaf; VE/VO, relative elution volume. SDS-polyacrylamide gels (1) to separate and characterize each of the individual RNase species found in wheat leaves. By initially using gels to determine the catalytic properties of each RNase, we were able to characterize two apparently novel RNases in addition to the ubiquitous acid RNase and to establish solution assays that measure each enzyme individually in leaf extracts. This approach circumvented the longstanding problems posed by the multiplicity of RNases found in plant tissues (7, 13). MATERIALS AND METHODS