Query (optional)   in Class  All Allele Author Colleague Collection Assembly Gene Gene Class Gene Product Germplasm Image Journal Keyword Library Locus Map Map Data Marker Pathology Polymorphism Probe Protein QTL Rearrangement Reference Sequence Species Trait Trait Study Two Point Data  

---

GrainGenes Reference Report: PPS-102-279

[Submit comment/correction]

Reference	PPS-102-279
Title	Visualizing enzyme secretion from individual barley (Hordeum vulgare) aleurone protoplasts
Journal	Plant Physiology
Year	1993
Volume	102
Pages	279-286
Author	Hillmer S Gilroy S Jones R
Abstract	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.
Keyword	[ Hide all but 1 of 11 ] aleurone-layer alpha-amylase enzyme activity gene expression genetic regulation hordeum vulgare hydrolysis plant composition protoplast starch transcription

GrainGenes is a product of the Agricultural Research Service of the US Department of Agriculture.