Generation and Suppression of Microsomal Ribonuclease Activity after Treatments with Auxin and Cytokinin

Abstract

plant tissues, but effects are often opposite in different species or growth situations and they do not bear any consistent relationship to total RNA levels. It has been suggested (19,27) that the ability of auxins and cytokinins to stimulate or to inhibit RNA metabolism depends in part on relative hormone concentrations. Another source of confusion may be the fact that, in most studies to date where RNase activity has been studied during plant development, only total levels are measured or, in cases where tissue is fractionated, only one fraction is assayed. This fails to take into account the likelihood that more than one RNase exists in growing tissues and that the various activities are associated with either soluble or membrane-bound fractions (18,21,28,30). A thorough investigation of the effects of different growth regulators on total and subcellular levels of RNA and RNase activity and their relation to growth in a well characterized tissue has not been reported. This paper describes effects on these parameters mainly of the auxin indoleacetic acid (IAA) and the cytokinin benzyladenine, applied alone or together to growing regions of decapitated pea epicotyls. In this semi-intact system, auxin treatment has been shown (5,8,16) to result in lateral cell expansion and massive increases in RNA and protein in various cell subfractions, including microsomes. Growing regions of peas are well known to yield RNase activity in soluble and microsomal fractions (11, 15, 18).Many plant and animal growth hormones evoke synthesis of messenger and ribosomal RNA, and the evidence is convincing that new polysomal species are needed for synthesis of enzymes that help to bring about particular morphogenetic events (6,9,13,22,25 growth regulator, and seedlings were allowed to continue growth in darkness at 22 C. Three days later, at least 50 segments per treatment were detached, lanolin was wiped from the surface, and length and fresh weight were recorded (8).Isolation of "Light" and "Heavy" Microsomes. Segments (approximately 5 g fresh weight per treatment) were ground in a mortar at 4 C in 2 volumes of extraction medium consisting of 0.4 M sucrose (RNase free), 5 mm Mg acetate, 100 mM tris-HCl (pH 7.5 at 22 C), 20 mm KCl, and 5 mM 8t-mercaptoethanol. The brei was squeezed through Miracloth (Calbiochem) to remove cell walls and debris (wall fraction) and the exudate was centrifuged at 16,000g for 10 min in order to sediment mitochondria and nuclei (particulate fraction). The supernatant was divided into two 6-ml aliquots and layered over 6 ml 50% (w/v)

Polyribosomes from Peas

“…Besides Trewavas (8), who used NH4Cl rather than sucrose as an osmoticum, the only investigators who have published profiles of polyribosomes isolated from etiolated pea stem segments are Birmingham and Maclachlan (2). In addition, the most thorough examination of the effects of DEP on plant polyribosomes was published by Anderson and Key (1).…”

“…The relative distribution between total polyribosomes, large polyribosomes (hexamers and greater), and monosomes is reported in Table I 8.5 (Fig. 2); D and F: ground in buffer containing 100 mM tris-HCl, pH 7.5 (Birmingham and Maclachlan [2] [ Fig. 1]).…”

“…These include assay of polyribosome-associated enzymes such as RNase itself (2), assay of other enzymes which may be DEP1This research was supported in part by a Biomedical Sciences Support Grant RT-07055 through the University of Nebraska Research Council, and a National Science Foundation Fellowship to B.A.L.…”

“…Because the proportion of polyribosomes increased when tissue was ground in the presence of the ribonuclease inhibitor, diethyl pyrocarbonate, Weeks and Marcus (9) and Anderson and Key (1) suggested that such an inhibitor should be employed routinely for work involving polyribosome distribution in plants. This inhibitor might be used profitably with peas, as they have been shown to contain high levels of endogenous RNase (5), some of which is firmly associated with the microsomes (2,6).…”

Polyribosomes from Peas

“…Accordingly, epicotyls were decapitated and treated in such a way that different growth patterns ensued. Two of these treatments, IAA and IAA plus BA,2 cause expansion (3,10,16): one, GA, causes elongation (8); and one, lanolin alone, pro-motes little growth (8,10). All hormones were applied at 0.5% (w/w) in lanolin.…”