Date of Award

2018

Document Type

Honors Thesis (Open Access)

Department

Colby College. Chemistry Dept.

Advisor(s)

Russell R. Johnson

Abstract

The wheat transcription factor TaABF1 plays an important role in hormone-mediated regulation of seed dormancy and germination of cereal grains. Evidence shows that TaABF1 activity is regulated by phosphorylation, and previous work in our lab showed that when serine residues in its conserved regions; S36, S37, S113, S115 were altered to phosphomimetic aspartate, the 4xD TaABF1 mutant had increased activity as a transcription factor. However, when only S113 was altered, TaABF1’s activity was greatly reduced. The work presented here explored whether the S36D/S37D/S115D mutant would have stronger activity than the 4xD mutant. Using the particle bombardment technique to introduce the TABF1 effector and reporter constructs in aleurone cells, we found that the 3xD mutant was able to enhance TaABF1 activity, but not to a stronger degree than the 4xD. This suggest that the phosphorylation effect of these residues is not additive, and individual residues have different roles in regulating TaABF1 activity. We also complemented this work by attempting to identify TaABF1 phosphorylation sites in untreated wheat grains by exposing purified TaABF1 to aleurone kinases and analyzing the resulting phosphorylation patterns using LC-MS/MS. We successfully detected phosphorylatable TaABF1 peptides at high intensities, but we did not detect any TaABF1 phosphopeptides. Determining sites of TaABF1 phosphorylation under different conditions will give insights in what role specific residues play in regulating TaABF1 activity, as well as the TaABF1 mechanism of action in regulating the ABA and GA pathways in imbibing cereal grains.

Keywords

abscisic acid; aleurone; barley; gibberellin; particle bombardment; phosphorylation

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