English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

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.

Visualizing Enzyme Secretion from Individual Barley (Hordeum vulgare) Aleurone Protoplasts