Embryo-independent mobilization of endosperm starch in cereal seeds
Creators: name: Konesky, David William
Publisher: name: University of British Columbia
Year: 2010

description: Endosperm mobilization studies in cereal seeds are typically based on the view that α-amylase synthesis in the aleurone tissue is controlled by gibberellins from the germinating embryo. However, α-amylase is often produced by de-embryonated endosperm segments in the absence of added gibberellins. Two specific systems exhibiting this phenomenon were examined; 1) the ability of amino acids to promote α-amylase production in de-embryonated wild oat segments and, 2) autonomous starch hydrolysis (in the absense of exogenous GA₃ or amino acids) in de-embryonated barley endosperm halves. Specific technical problems were addressed prior to the onset of these studies. Captan (66 μM) controlled fungal contamination in the incubation medium without inhibiting GA₃-induced sugar release, which occurs following seed sterilization in hypochlorite and ethanol. The Nelson-Somogyi reducing sugar assay was not suitable for quantifying sugar levels in incubation solutions containing amino acids as cysteine, cystine, serine, tryptophan and tyrosine interfered with the assay. Absorbance (540 nm) increased as concentrations increased from 0.1 to 1 mM; simultaneous additions of amino acids with glucose resulted in absorbance values higher than glucose alone. MnCl₂ (0.5 mM) inhibited absorbance in the presence of glucose and the amino acids serine, cystine, and tryptophan. The ability of individual amino acids to promote α-amylase production in wild oat endosperm halves is yet unclear. Although certain amino acids were shown to enhance enzyme production, the level was often quite different between replicated experiments. However, incubation of endosperm halves in a mixture of 18 amino acids consistently promoted α-amylase synthesis; enzyme production was further enhanced if a level of GA₃ (10⁻⁹ M), which was too low to promote α-amylase synthesis alone, was included within the amino acid mixture. Autonomous endosperm mobilization (AEM) was variable in different barley cultivars. High sugar release correlated well with α-amylase production; the levels were similar among individual cultivars harvested in two different seasons. The onset of AEM was delayed, as the majority of sugar was released in the second day of incubation. AEM was greatly reduced by inhibitors of RNA (6-methyl purine) and protein (cycloheximide) synthesis suggesting that AEM was a result of the de novo synthesis of α-amylase. Incubation conditions greatly affected AEM. Although AEM was high at acidic pH (4.6-5.6), it was greatly reduced at basic pH (7.6-8.6). AEM increased as the temperature was raised to 28°C. Low levels of Ca²⁺ (0.25-0.5 mM) enhanced AEM whereas higher amounts (0.5-1 mM) were inhibitory. Incubation of wild oat endosperm halves in solutions of pre-emergence herbicides affected GA₃-induced sugar release to varying extents when applied at field application levels. Only triallate (22% reduction) and trifluralin (21% reduction) prevented sugar release (only - 5 at 3x10⁻⁵ M); no inhibition was seen following incubation in EPTC (5xl0⁻⁵, 5xl0⁻⁶ M), metribuzin (5xl0⁻⁶, 1x10⁻⁶M) and oryzalin (3xl0⁻⁵, 3x10⁻⁶M). However, the higher concentrations of each herbicide effectively inhibited the development of wild oat seedlings.