Heat-Stable Proteins and Abscisic Acid Action in Barley Aleurone Cells

Abstract

[35S]Methionine labeling experiments showed that abscisic acid (ABA) induced the synthesis of at least 25 polypeptides in mature barley (Hordeum vulgare) aleurone cells. The polypep-tides were not secreted. Whereas most of the proteins extracted from aleurone cells were coagulated by heating to 100°C for 10 minutes, most of the ABA-induced polypeptides remained in solution (heat-stable). ABA had little effect on the spectrum of polypeptides that were synthesized and secreted by aleurone cells, and most of these secreted polypeptides were also heat-stable. Coomassie blue staining of sodium dodecyl sulfate poly-acrylamide gels indicated that ABA-induced polypeptides already occurred in high amounts in mature aleurone layers having accumulated during grain development. About 60% of the total protein extracted from mature aleurone was heat stable. Amino acid analyses of total preparations of heat-stable and heat-labile proteins showed that, compared to heat-labile proteins, heat-stable intracellular proteins were characterized by higher glu-tamic acid/glutamine (Glx) and glycine levels and lower levels of neutral amino acids. Secreted heat-stable proteins were rich in Glx and proline. The possibilities that the accumulation of the heat-stable polypeptides during grain development is controlled by ABA and that the function of these polypeptides is related to their abundance and extraordinary heat stability are considered. The aleurone layer of barley has played an important role in the study of the actions of ABA and GA3 in regulating cellular processes. In this tissue, GA3 promotes and ABA inhibits the synthesis of a number of hydrolytic enzymes, in particular a-amylase, and studies of this interaction at the levels of gene transcription, mRNA and protein synthesis have shown that ABA antagonizes all of the GA3-promoted events (for review see ref. 14). Associated with its antagonistic role in aleurone, ABA promotes the synthesis of a number of polypeptides (1 1, 12, 15, 18, 23). The synthesis of these polypeptides is antagonized by GA3 (18). The number of polypeptides has been estimated to be between 9 and 16, and they have a broad mol wt range (18). Little is known about the identity and function of these polypeptides. Some may be involved in a self-induced catal-ysis of ABA to PA' (38), one is an a-amylase/subtilisin inhibitor (24), and one is recognized by a barley lectin anti-body although it has not been shown to have lectin activity 'Abbreviations: PA, phaseic acid; LEA, late embryogenesis-abundant. (18). There is no evidence yet which bears on the question of whether or not ABA-induced polypeptides are related to the mechanism by which ABA antagonizes GA3 action. The object of this study was to further characterize the ABA-induced polypeptides and we focus on their heat stability (resistance to coagulation by heat). It has been found previously (17) that heat shock proteins are resistant to coagulation by heat. Because of the association of ABA with the response of plant tissues to water stress (for review see ref. 8), it seemed worthwhile to consider the possibility that ABA-induced poly-peptides might also be stress-related and that they might also be resistant to coagulation by heat. This report shows that many of the ABA-induced polypeptides in barley aleurone are not coagulated by heat and using this property in a purification step, we have been able to examine the polypep-tides more closely that has hitherto been possible. Our results indicate that many of the ABA-induced polypeptides are abundant, that they already exist in mature aleurone, and that they exhibit a range of solubility characteristics. MATERIALS AND METHODS Preparation and Incubation of Aleurone Layers Aleurone layers were prepared from grains of Hordeum vulgare L. cv Himalaya grain grown in Pullman, WA (1985 harvest) and incubated as described (6). The aleurone layers (10 per flask) were incubated in 2.0 mL of medium containing 10 mm CaCl2 and 25 AM cis-trans-ABA (± enantiomer mixture , Sigma Chemical Co.) usually for 24 or 48 h. If required, 30 ,uCi of L-[35S]methionine (Amersham, approximately 1400 Ci/mmol) were added for the last 6 h. Preparation of Protein Extracts and Electrophoresis After incubation, the medium was removed and each lot of aleurone layers was rinsed off with two aliquots of 4 mL distilled water. The layers were then ground in a total of 1.5 mL of 20 mm Tes KOH (pH 8.0) containing 0.5 M NaCl in a mortar and pestle with the aid of acid-washed sand. Aleu-rone extracts and media were clarified by centrifugation, aliquots were taken, and the protein was precipitated by adding four volumes of cold acetone. Other aliquots were heated at various temperatures, usually 100°C for 10 min, allowed to cool on ice, and centrifuged, and the protein remaining in solution was precipitated with acetone. Precipitated protein was pelleted by centrifugation, dried, dissolved in SDS-PAGE sample buffer, and the polypeptides were sep-1520