Hormonal Regulation of Protein and Phytic Acid Mobilization in Germinating Barley
Hammerton, Rachel Wilson
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Permalink
https://hdl.handle.net/2142/77647
Description
Title
Hormonal Regulation of Protein and Phytic Acid Mobilization in Germinating Barley
Author(s)
Hammerton, Rachel Wilson
Issue Date
1987
Department of Study
Biology
Discipline
Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, General
Language
eng
Abstract
The mobilization of endosperm reserves in germinated cereal grains has been shown to be hormonally regulated. This work examines the involvement of gibberellic acid (GA$\sb{3}$) in the hydrolysis of protein and phytic acid (myo -inositol hexakis-phosphate) reserves.
Two groups of proteases were examined in GA$\sb{3}$-treated barley aleurone (Hordeum vulgare cv. Himalaya). Carboxypeptidase and proteinase activities were investigated using the substrates N-carbobenzoxy-ala-phe and hemoglobin. GA$\sb{3}$ was required for the production of proteinase activity in the aleurone layer and for its release; both processes were inhibited by abscisic acid. Carboxypeptidase activity developed in the aleurone layers during imbibition but was released only in the presence of GA$\sb{3}$. ABA inhibited carboxypeptidase release. Carboxypeptidase activity was inhibited by diisopropylfluorophosphate and was maximal between pH 5 and 6. Proteinase activity was inhibited by bromate and leupeptin, enhanced by 2-mercaptoethanol, and utilized the storage proteins gliadin and hordein as substrates. Proteinase activity was represented by three enzymes which differed in charge but all had apparent molecular weights of 37 kD on SDS polyacrylamide gels. This 37 kD band was a newly-synthesized, GA$\sb{3}$-induced protein.
When phytic acid levels were examined in mature, dry barley grain it was determined that 90% of the phytic acid was stored in the aleurone cells, with little or none in the starchy endosperm. In barley germinated at 25$\sp\circ$C phytate levels had dropped by 50% after three days. At 10$\sp\circ$C, more than ten days were required for 50% hydrolysis. GA$\sb{3}$ was required for significant phytate hydrolysis in endosperm half-grains and the GA$\sb{3}$-stimulated rate paralleled the loss in the endosperm of germinated grains.
Phosphatases were detected in aleurone cells using native polyacrylamide gels stained for activity using $\alpha$-naphthyl phosphate. Bands 1-3 (numbered from lowest mobility) were detected in the aleurone cells of dry grains. Bands 4-8 developed when the grain was imbibed. When intracellular activities were examined in aleurone protoplasts, only bands 4-8 were found. GA$\sb{3}$ appeared to stimulate an increase in all bands but most noticeably in bands 1-3. GA$\sb{3}$ also enhanced the release of bands 1-6 from the aleurone cells.
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