Plant Nucleases

Abstract

Isoenzymes of lRNase were detected in plant extracts after polyacrylamide gel electrophoresis with a new buffer system. The gels were incubated in an RNA solution, then dipped for 30 seconds into 0.2%9o toluidine blue. The method is rapid and is sensitive to very small amounts of RNase. The Plants contain a number of enzymes which degrade RNA. The enzymes found in corn fall into three general classes: (a) RNase I (ribonucleate nucleotido-2'-transferase (cyclizing) E.C. 2.7.7.17), which has a pH optimum near 5, produces 2'. 3'-cyclic nucleotides, and is a soluble enzyme; (b) RNase II (also E.C. 2.7.7.17), which has a pH optimum at 6 or above, produces 2', 3'-cyclic nucleotides, and is associated with crude microsomes; and (c) Nuclease I (E.C. 3.1.4.x), which has a pH optimum near 6, produces 5'-nucleotides from both RNA and DNA, and is associated with large cell particles (20, 21). Pure RNase I has been obtained from corn endosperm (22), while partially purified RNase II and Nuclease I have been obtained from corn roots (20).Changes in RNase levels in plants have been associated with injury (2, 7), infection (7, 12, 13), growth or growth inhibition (1,14), RNA synthesis (9), senescence (11,15), and endosperm maturation (8,5 (1) found that a light-mediated increase in RNase of lupine hypocotyls was associated with polysomes and ribosomes. This RNase appeared to be either RNase I or II.I have reported that mixtures of corn nucleases may be partially separated by differential centrifugation and by gel filtration, and that further information on the levels of different nucleases may be gained by determining the activity on RNA at pH 5 and pH 6.2 as well as the activity on DNA (20, 21).Wolf (24) detected a number of RNases from bean, wheat, and sugar beet after electrophoresis, using a methylene blue stain technique. An assay for phosphodiesterase in the gels (10) showed that some of these plant RNases were also capable of hydrolyzing the a-naphthyl ester of uridine-5'-phosphate (24). Randles (12) detected changes in RNase isoenzymes in virus-infected Chinese cabbage after polyacrylamide gel electrophoresis, but had to first inhibit the RNase with copper and later restore activity with EDTA. A more rapid method was developed which separated and detected the different partially purified corn RNases after polyacrylamide gel electrophoresis, but it was not suitable for crude preparations (18). This method has now been improved so that crude enzyme preparations from corn and other plants may be separated and detected by gel electrophoresis. The use of specific inhibitors gives further information on the types of enzymes involved. These methods are able to detect the differences in the RNA-degrading enzymes associated with crude mitochondrial and microsomal preparations from corn. MATERIALS AND METHODSThe Preparation of Enzymes. The corn (Zea mays L., "WF9 X M14") RNase I, RNase II, and Nuclease I enzymes have been described previously (20,22

Plant Nucleases

“…The polyacrylamide gel electrophoresis test for RNase detected only RNase I in extracts from 50-day endosperms, and the RNase from all of the inbreds used in this work migrated to the same position (29). The enzyme pattern produced by the 20-day M14 extract (Table I) is shown in Figure 1, where the incubation conditions were selected to favor RNase II and Nuclease I (29).…”

“…The major enzyme found in corn endosperm in the later stages of development is RNase I (29), but the types of enzymes occurring in the earlier stages of cell division and endosperm enlargement have not been examined before. Table I gives the RNase activities at two different pH values, and also the DNase activity, for three genotypes which varied widely in total activity 20 DAP.3 Calculations from the activity ratios determined for purified enzymes (28) suggest that from one-fifth to one-third of the activity in the M 14 endosperm may be other than RNase I (i.e.…”

“…The sharp peak at 4 mm is an artifact produced by a strong protein band. The two minor peaks in between were not inhibited by EDTA (data not shown) and, thus, may be isoenzymes of RNase 11 (29), but they were not further examined. The extracts from the other genotypes of Table I, with higher total activities, showed higher activities for the RNase I band, but only minor differences in the other bands on the gels (not shown).…”

“…Polyacrylamide gel electrophoresis and detection of RNase activity in the gels were performed as previously described (29). The data presented here are from the 1975 crop, but the discussion of the patterns of growth and enzyme activity takes into account the results of previous years (32).…”

Polynucleotide‐acrylamide gel electrophoresis of soluble nucleases from tobacco leaves

“…[4] ) and because the nucleic acids used are not necessarily uniformly degraded, leaving blurred zones on the gels instead of sharp, colourless bands.…”