Briefly, layers were ground in nondenaturing gel buffer on ice in a mortar and pestle, TCA-precipitable cpm were determined for the tissue extracts, and equal ...
Plant Physiol. (1988) 00, 588-593
0032-0889/88/88/0588/06/$0l1.00/0
Hormonal Regulation of a-Amylase Expression in Barley Aleurone Layers' THE EFFECTS OF GIBBERELLIC ACID REMOVAL AND ABSCISIC ACID AND PHASEIC ACID TREATMENTS Received for publication December 16, 1987 and in revised form June 10, 1988
RANDALL C. NOLAN2 AND TUAN-HUA DAVID Ho*
Plant Biology Program, Division ofBiology and Biomedical Sciences, Washington University, St. Louis, Missouri 63130 here that other transcription inhibitors, and also the translation inhibitor cycloheximide, block the effects of ABA on a-amylase The expression of a-amylase isozymes in barley (Hordeum vulgare L.) expression. Since Uknes and Ho (28) have shown that the aleurone layers is known to be maximally induced between 12 and 20 conversion of ABA to its metabolite PA is blocked by cordycepin hours after addition of the phytohormone, gibberellic acid (GA3). Addition and cycloheximide, it was thought that PA, rather than of another hormone, abscisic acid (ABA), or its metabolite, phaseic acid, might be the active inhibitory compound in aleurone layersABA, and during this time period resulted in reduced a-amylase expression. Expres- that cordycepin might prevent ABA action by simply preventing sion of the high isoelectric point a-amylase isozyme group was affected the enzymatic conversion of ABA to PA. In this work, the effects much more by both of these treatments than was expression of the low of PA on a-amylase expression were further investigated. isoelectric point a-amylase isozyme group. Addition of either the transSince GA3 and ABA are antagonistic regulators of a number lation inhibitor cycloheximide or the transcription inhibitor cordycepin of responses in barley aleurone layers (12, 15, 21), it prevented the decrease in a-amylase mRNA levels after ABA treatment. possible that the effects of ABA on a-amylase expressionseemed might Cordycepin also prevented the decreases in a-amylase expression that be analogous to the removal of GA3. It has, in fact, been reported result from phaseic acid treatment. Midcourse GA3 removal experiments that removal of GA3 reduces the production of a-amylase in were performed to determine whether ABA treatment and the removal barley aleurone layers (4). GA3 removal experiments were perof GA3 have analogous effects on a-amylase expression. It was found formed, in which layers were incubated in the presence of GA3 that cordycepin treatment also prevented decreases in a-amylase mRNA for 8 or 12 h and then washed and incubated in the absence of levels resulting from GA3 removal. We conclude that the suppression of GA3. The effects of cordycepin were also investigated in these a-amylase expression caused by ABA or midcourse GA3 removal is GA3 removal experiments to characterize further the effect of dependent on continuous RNA and protein synthesis. RNA synthesis inhibition on a-amylase expression in barley aleurone layers. ABSTRACT
The stimulation of a-amylase expression in barley aleurone layers by GA3 and the inhibition of this effect by ABA have been well documented (11, 12, 18, 22). However, the mechanisms by which a-amylase expression is regulated are not well understood. a-Amylase in barley aleurone layers consists of two groups of isozymes, termed high pI3 and low pl because of their different isoelectric points (3). We have shown that these two isozyme groups are differentially regulated by GA3 and ABA treatments (20). This report is a continuation of that work, with the aim of more specifically determining the regulation of a-amylase isozymes in barley aleurone layers. Previously, we have shown that ABA treatment reduces aamylase mRNA levels, even after these mRNA species have been fully induced by GA3. The RNA synthesis inhibitor cordycepin (3'-deoxyadenosine) prevents this action of ABA (20). We report
'Supported by National Science Foundation Grant DCB-8702299 to T-H. D. H. 2 Present address: Department of Biology, Indiana University, Bloomington, IN 47405. 3Abbreviations: pI, isoelectric point; PA, phaseic acid; PAPI, putative a-amylase protease inhibitor.
MATERIALS AND METHODS Preparation of Aleurone Layers. Barley aleurone layers (Hordeum vulgare L. cv Himalaya) were prepared using seeds from the 1981 harvest from Washington State University, Pullman, WA, as described previously (1). For protein synthesis experiments, 10 aleurone layers were incubated in 2 mL of 20 ,uM sodium succinate buffer (pH 5.0) and 20 uM CaCl2 and 10 ,g/ mL chloramphenicol at 25°C in a shaking water bath at 120 oscillations/min. In the GA3 removal (washing) experiments, the layers were washed by first rinsing them two times in 5 mL of the appropriate buffer. These steps were repeated for a total of 15 wash steps over a 2-h period. The layers were then incubated in 2 mL of buffer. The wash buffers were the same as the final incubation buffers. Incubations of aleurone layers for RNA extraction were performed the same way, except that 50 layers were incubated in 10 mL of buffer, and 10 mL of buffer were used for the washing steps. PA was generously provided by Dr. M. Brenner, University of Minnesota, St. Paul, MN, and Dr. B. Loveys, Commonwealth Scientific and Industrial Research Organization, Canberra, Australia. Other chemicals for aleurone layer treatments were purchased from Sigma Chemical Co., St. Louis, MO. Protein Synthesis and RNA Analyses. For a-amylase synthesis determinations, aleurone layers were pulsed with 25 ,MCi of [35S] methionine/cysteine (Trans-label, ICN-Irvine, CA) during the
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REGULATION OF a-AMYLASE EXPRESSION IN BARLEY ALEURONE LAYERS
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