Visualizing enzyme secretion from individual barley (Hordeum vulgare) aleurone protoplasts

• Published in: Plant physiology (Bethesda) 1993-05, Vol.102 (1), p.279-286
• Main Authors: Hillmer, S, Gilroy, S, Jones, R.L
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Aleurone cells; Aleurone layer; ALFA AMILASA; ALMIDON; ALPHA AMYLASE; AMIDON; Barley; Biological and medical sciences; Cell Biology and Signal Transduction; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Enzymes; EXPRESION GENICA; EXPRESSION DES GENES; Fundamental and applied biological sciences. Psychology; Gels; GENETICA; GENETIQUE; HIDROLISIS; HORDEUM VULGARE; Hormones; HYDROLYSE; Metabolism; Plant cells; Plant physiology and development; PROTOPLASTE; PROTOPLASTOS; Protoplasts; Secretion; Starches; TEJIDOS VEGETALES; TISSU VEGETAL
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.102.1.279

A method was developed to detect alpha-amylase gene expression and alpha-amylase secretion from individual barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts. Protoplasts are incubated in liquid media with or without hormones and embedded in a thin film of agarose and starch, where they remain viable for up to 24 h. alpha-Amylase secreted by individual protoplasts digests the starch, and starch hydrolysis is visualized after 45 min by staining the preparation with I2KI. After I2KI staining, secreting protoplasts are surrounded by a clear, starch-free halo visible by light microscopy. The formation of starch-free halos is dependent on the synthesis and secretion of alpha-amylase and is not caused by carry-over of preformed enzyme from incubation media. Treating protoplasts with inhibitors of protein synthesis or exposing them to anaerobic conditions for 2 h before embedding them in agarose prevents the formation of halos. When alpha-amylase secretion is observed by counting the percentage of secreting protoplasts, the data are comparable to that obtained by measuring alpha-amylase secretion from a population of cells. The response of individual protoplasts to gibberellic acid (GA3) and abscisic acid measured by the thin-film method is almost identical to the response of populations of protoplasts to these hormones, validating the utility of this method. Although not generally practical for quantifying secretion, the thin-film method is uniquely useful in distinguishing secreting from nonsecreting protoplasts. In none of our experiments did more than 60% of the protoplasts secrete alpha-amylase when exposed to GA3, even though more than 95% of the protoplasts in the preparations were viable. Similar results were obtained when the response to GA3 was assayed at the level of gene transcription by visualizing the transient expression of a plasmid containing the promoter from alpha-amylase fused to the reporter gene glucuronidase in single protoplasts. The thin-film secretion assay also revealed that the response of a population of protoplasts to GA3 was not uniform with time. The effect of GA3 treatment was to gradually increase the percentage of responding protoplasts up to a maximum of 50 to 60%. Abscisic acid, which inhibits alpha-amylase secretion by GA3-treated protoplasts, reduced the proportion of protoplasts that secrete the enzyme