Barley aleurone layer

Hanson, A.D. Jacobsen, J.V. (1984). Control of lactate dehydrogenase, lactate glycolysis and a-amylase of O2 deficit in barley aleurone layers. Plant Physiology, 75, 566-72. 

A similar time course for translatable mRNA has been reported (lA) in barley aleurone layers, in response to glbberel-lic acid. Total poly(A) RNA was found to Increase dramatically in the first 12 hr after hormone application, followed by a rapid decline to 25% of the maximum at 18 hr. On the other hand, the accumulation of ovalbumin mRNA in response to progesterone in chick oviducts (15) is an example that does not appear to behave in this manner. 

Fig. 3. Translation products of barley aleurone layer mRNAs. Lanes 1-4, total products from layers treated in water (control), 1 jxm ABA, 1 pM GA3, or both hormones, respectively. The rab 6 protein is marked with an arrow. Lanes 5-8, immunoprecipitation of a-amylase (upper bands) and the a-amylase/subtilisin inhibitor (lower bands) from the translation products shown in lanes 1-4. mRNA extraction, translation and immunoprecipitation were performed as previously described (Mundy etal., 1986).

Hong, B., Uknes, S.J. Ho, T.-H.D. (1988). Cloning and characterization of a cDNA encoding mRNA rapidly induced by ABA in barley aleurone layers. Plant Molecular Biology 11, 495-506. 

Roles of Metabolites of Abscisic Acid. Nothing is known about the physiological role of PA and DPA in plants, although these two metabolites of ABA have been tested in several bioassays recently. In the cotton explant abscission assay PA had one-tenth of the activity of ABA (19). PA and DPA were equally effective in inhibiting a-amylase secretion by barley aleurone layers treated with glbberellin A3 DPA had approximately one-tenth of the activity of ABA in this system (74). The effect of PA on growth of bean embryos was negligible (75). 

Ho, D. T. Response of barley aleurone layers to abscisic acid. Plant Physiol., 1976, 57, 175-178. 

Jacobsen, J. V. Know, R. B. The proteins released by isolated barley aleurone layers before and after gibberellic-acid treatment. Planta, 1974, 115, 193-206. 

Jacobsen, J. V. Zwar, J. A. Gibberellic acid and RNA synthesis in barley aleurone layers metabolism of rRNA and tRNA and of RNA containing polyadenylic acid sequences. 

Chrispeels, M. J. Varner, J. E. Gibberellic acid-enhanced synthesis and release of -amylase and ribonuclease by isolated barley aleurone layers. Plant Physiol., 1967, 42(3), 398-406. 

Locy, R. Kende, H. The mode of secretion of -amylase in barley aleurone layers. Planta, 1978, 143, 89-99. 

Musgrave, A. Kays, S. E. Kende, H. Uptake and metabolism of radioactive gibberellins by barley aleurone layers. 

Murthy, P.P.N., Renders, J.M., and Keranen, L.M., 1989, Phosphoinisitides in barley aleurone layers and gibberellic acid-induced changes in metabolism. Plant Physiol. 91 1266-1269. 

A range of methods have been described in the literature, and we shall consider them in the following order (1) the methods developed at the Food Research Institute-Norwich (FRIN), to isolate cell walls from a range of vegetables and fruits (runner beans, potatoes, cabbage and apples), cereals (oats, wheat and rye) and cereal products (wheat bran and rye biscuits), and lignified tissues (parchment layers of runner bean pods) (2) the special techniques, which may include wet sieving steps, used for the isolation of cell walls from potatoes, wheat endosperm, and wheat and barley aleurone layers (3) alternative methods for the isolation of cell walls from starch and protein-rich products (rice) and (4) methods used for the isolation of cell walls from suspension-cultured tissues. 

Judged by enzyme induction, Menary and Jones (1972) indicated that nitrate was unable to move from a storage pool (vacuole) to the cytoplasm of ripe paw paw fruit. Using a modified in vivo assay, Ferrari et al. (1973) estimated sizes of active (metabolic) and inactive (storage) pools of nitrate in tobacco cells, barley aleurone layers and maize leaves. The location and nature of the pools was not defined. 

When applied to barley aleurone layers, gibberellic acid stimulated the incorporation of [met/iy/- CJcholine into PC (Evins and Varner, 1971). Absci-sic acid prevented this effect of gibberellic acid. Johnson and Kende (1971) showed that gibberellin stimulated both PC cytidyltransferase and choline phosphotransferase and that these stimulations could be prevented by absci-sic acid, cycloheximide, and actinomycin D. 

Regulation of Gene Expression by Abscisic Acid in Barley Aleurone Layers... 

The metabolism of ABA in isolated barley aluerone layers follows the ABA PA- DPA pathway that has been shown to exist in many plant tissues [3, 23]. The biological activities of isolated PA and DPA have been tested in this system. Although DPA has little or no biological activity, PA is as active as ABA, i.e. the GAg-induced a-amylase is effectively inhibited by either PA and ABA, but not by DPA [3]. It has been reported that in animal tissues vitamin D has to be metabolized by hydroxylation in order to become biologically active [4]. Thus, the observation that PA is biologically active has raised the question of whether PA is the active component in ABA action. The metabolism from ABA to PA is catalyzed by a cytochrome P450 type monooxygenase with a short-lived intermediate, 6 -hy-droxymethyl ABA [23]. A pretreatment of barley aleurone layers with 10 M ABA for 24 h enhances the tissue s ability to convert [ H]ABA to [ H]PA by 3- to 5-fold... 

Fig. 1. Effect of pretreatment of barley aleurone layers with ABA on the metabolism of pH]ABA. Barley aleurone layers were incubated with or without lO " M ABA for 24 h. After incubation, fresh medium containing 1 juCi pH]ABA/ml was added and the tissue was incubated for an additional 4 h before ABA and its metabolites were extracted and analyzed by TLC. The cpm values were plotted against the distance travelled in cm (Quenching was constant among the TLC fractions). The negative values on the distance travelled are the preadsorbent area on the TLC plate. 0 is the junction between the preadsorbent area and the silica gel. The shaded area indicates the amount of pH]PA. A Control, no ABA pretreatment B + ABA, pretreated with 10 ...

Fig. 1 21]. An ABA concentration as low as 10 M is sufficient to enhance its own metabolism, and this effect can be observed within 2 h of ABA treatment [21]. The formation of the next stable metabolite, DPA, is not enhanced by pretreatments with either ABA or PA [21]. Thus, the enhanced PA formation is unlikely a scavenging mechanism to remove excessive ABA because the tissue would have to enhance the formation of DPA in order to eliminate the biological activities. The self-induction of ABA metabolism can be prevented by transcription and translation inhibitors, suggesting that ABA induces the monooxygenase (or a cofactor for this enzyme) responsible for PA formation. The regulation of ABA metabolism in barley aleurone layers is similar to the induction of nitrate reductase by its substrate, nitrate. In this latter case, treatment of a plant tissue with nitrate enhances its ability to metabolize nitrate. 

Fig. 3. Effect of the transcription inhibitor, cordycepin, on the action of ABA in barley aleurone layers. Northern blot analysis is the same as described under Fig. 2. Aleurone layers were incubated for 25 h in the absence of GA3 (lane 7), 25 h in the presence of lO M GA, (lane 2), 24 h in the presence of GA, and 2 X lO M ABA with a second addition of ABA at 12 h (lane i), 25 h in the presence of GA3 with ABA added 10 h after GA3 (lane 4), 25 h in the presence of GA3 with ABA added 20 h after GA3 (lane 5), or 25 h in the presence of GA3 with ABA and 10 " M cordycepin added 20 h after GA3 (lane 6). Note that cordycepin addition prevented the reduction of a-amylase mRNA induced by ABA (cf lanes 5 and 6). From Nolan et al. [19]...

Fig. 4. Immunoprecipitation of AB A-induced proteins in barley aleurone layers with antibodies against barley lectin and a-amylase inhibitor. Aleurone layers were incubated with or without 2 X 10 M ABA for 24 h. Labeling with 50 jaCi/ml p S] methionine took place in the final h of incubation. In A proteins were extracted with 0.1 M Na acetate (pH 5.5) in B proteins were extracted with 2% Triton X-100 at 40 C for 30 min. Aliquots of the extracted proteins were reacted with the antibodies and then precipitated with Staphylococcus aureus Cowan strain 1. The pellets were washed and analyzed by SDS-PAGE. Fluo-rograms of the gels are shown. Mol wt marker are indicated by bars at left. L.-S. Lin and T.H.D. Ho, unpublished...

Thr-Glu-Ala-Ala-Lys-Gln-Lys-Ala-Ala-Glu-Thr. A similar sequence has been observed in one of the ABA-induced proteins in cotton embryogenesis (Lea 7) [1]. ABA-induced proteins with a repeating sequence and unusually high content of specific amino acids have been observed in several plant systems [ 1,5,16]. In barley aleurone layers, pHV Al as well as several other ABA-induced proteins can be induced by osmotic or salt stress (LS Lin and THD Ho, unpublished observation). At least in osmotic stress, the level of ABA is increased, and the osmotic stress induction of ABA proteins is prevented by an ABA biosynthesis inhibitor, fluridone. Thus, the stress-induction of these proteins is most likely the consequence of stress-induced synthesis of ABA. 

The function of these ABA-induced proteins remains unclear. Since the a-amylase inhibitor is also synthesized during seed development, it is likely to play a mop-up role in getting rid of unwanted a-amylase activity [15,22]. Some of these ABA proteins may be related to a plant s tolerance to water stress, yet a definite proof is still lacking. In barley aleurone layers, some of these proteins could be... 

Ho THD (1979) Hormonal control of enzyme formation in barley aleurone layers. In Rubenstein I (ed) Plant molecular biology. Academic Press, London New York, pp 217-240... 

Sussex IM (1972) Somatic embryos in long-term carrot tissue cultures Histology, cytology, and development. Phytomorphology 22 50-59 Taiz L, Jones RL (1970) Gibberellic acid, -1,3-glucanase and the cell walls of barley aleurone layers. Planta (Berl) 92 73-84... 

Finally, a brief word about cyclic AMP. Activation of the enzyme adenyl cyclase which catalyses cyclic 3, 5 -adenosine monophophate synthesis constitutes the initial molecular event in the target cells of several mammalian hormones. The nucleotide then initiates a series of events leading to a final response characteristic of the hormone. Despite numerous studies on the barley aleurone layer there is no convincing evidence that GA action is mediated via cyclic AMP [73] in fact the weight of evidence is against this nucleotide playing an important role in any plant tissue [7]. 

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