2012 Annual Report 1a.Objectives (from AD-416): 1. To determine when bioactive GAs reach the aleurone tissue in the superior malting barley cultivar, Morex. Results will be related to the temporal decline in the the GA-response inhibitor protein, DELLA, and the appearance of alpha-amylase messenger RNA. 2. To determine the source, within the barley seed, of these GAs. 3. To determine whether this temporal bracketing can be used to assess potential malting quality of other new barley varieties. 1b.Approach (from AD-416): The gibberellin (GA) hormone(s) control hydrolytic enzyme production in the seeds aleurone layer. The origin of these GAs is still unknown. Seed tissues from the Morex malting barley will be carefully dissected and pooled for hormone analysis. The tissue extracts will be spiked with various internal control GAs and analyzed by GC-Mass spectrometry. The deduced GA types will indicate the biosynthesis pathway used by the aleurone, if GA synthesis indeed occurs in the aleurone. Alternatively, GA intermediates produced only in the root/shoot axis or in the scutellum will determine whether the GA comes from either of these tissues. The temporal profile of active GA accumulation in these tissues will be correlated with the rise in high-pI alpha-amylase messenger RNA production in the aleurone. 3.Progress Report: We examined barley seeds to determine the source of active gibberellin (GA) hormone and its site of accumulation. Dogma has it that a GA is produced in the embryo of the germinating seed; this then migrates to the aleurone, where it turns on the genes encoding enzymes that degrade large storage molecules (e.g., starch, proteins, glucans). We prepared microdissected samples of the aleurone, subaleurone, root/shoot axis, and scutella from seeds that were 0, 8, 24, and 48 hours old (from the beginning of imbibition). Bioactive GA1 did not occur in the aleurone at the right time and concentration to account for the pattern of alpha-amylase accumulation in the seed. In each of the 3 repetitions, various GAs differed considerably in distribution in the seed organs - especially in the aleurone. However, in each repetition, alpha-amylase messenger ribonucleic acid production in the aleurone started at exactly the same time. A specific gene transcription factor may be responsible for this. Relevance to Parent Project: Objectives: Objective 3 – Identify components of the GA response that can be used as predictors of malting quality. Linkage: Differences in the gid genes for Morex (which is a cultivar of superior malting quality) and Steptoe (an inferior cultivar) have been found. The production of malting enzymes produced in the aleurone should correlate with the production or arrival of specific GAs. This should lead to differences in timing with regard to loss of the Della protein, which is dependent upon gid binding to specific GAs. Thus, knowing whether bioactive GAs are actually present in alpha-amylase releasing tissue(s) is crucial to the interpretation of these findings.