Date of Award
Bachelor of Science (BS)
Department of Biology
Starch, a polymer of glucose, is a source of stored energy and carbon for plants. Starch accumulates in chloroplasts during the day and is broken down at night. Many enzymes are involved in this degradation, but the main players are in the β-amylase family of enzymes. In Arabidopsis, the β-amylase (BAM) family consists of nine proteins, six of which are plastid targeted. One of these plastidic BAMs is BAM1, which was suggested to be catalytically active during the day in guard cells to aid in the opening of stomata. Also, when exposed to osmotic stress, plants close their stomata, slowing water loss but also preventing photosynthesis. In addition to BAM1’s function during the day in guard cells, BAM1 is expressed in mesophyll cells to alleviate the loss of available photosynthate. BAM5 is an active, cytosolic BAM that can contribute most of the activity in leaves during the day. This high activity masks the activity of the other active BAMs, making it difficult to compare their activities with an amylase assay. We used T-DNA-insertion mutants to genetically eliminate BAM5 in several single and double mutants that also lack BAM1, -2, -3, and/or -6, to remove this high background activity. Amylase assays without this high background activity showed that BAM1 and -3 contribute a majority of the activity in mature leaves while BAM2 and -6 contribute very little activity. This allowed us to compare mutants that lack BAM genes and learn about their activity and expression under various conditions. During the day, the pH of the stroma is 8.0 and it drops to 7.0 at night. BAM1, due to its location in the stroma, is subject to diurnal pH fluctuations. It was previously observed that assays using a synthetic substrate produced a narrow activity curve in which BAM1 was nearly inactive at pH 8.0, suggesting that BAM1 would not be active during the day. We thought that the synthetic substrate might be misrepresenting the activity of BAM1 at higher pHs so we used soluble starch as the substrate, which is similar to amylopectin, the natural substrate of BAM1 and -3. Pure BAM1 and -3 expressed in E. coli were assayed and compared to assays with extracts made from bam53 and bam51. With starch as the substrate, the pH curves of both BAM1 and BAM3 are broader. Moreover, we observed that at pH 8, BAM3 is less active while BAM1 is more active. The similarities between the pH curves of the crude extracts and corresponding BAMs expressed in E. coli confirmed that bam53 and bam51 contain primarily BAM1 and BAM3. Knowing that bam53 mutants only contained BAM1, we wanted to see how much BAM1 is expressed in mesophyll cells during osmotic stress. To see the effect of osmotic stress on the expression of BAM1, we exposed mutants that lack BAM1 and -5 to osmotic stress and measured their activities with amylase assays. The activity in plants that contained BAM1 was 40% higher under osmotic stress than plants that lacked BAM1. We also monitored the health of these plants over time and observed that, under osmotic stress, bam1 mutants experienced more chlorosis and wilting than the wild-type. We reasoned that due to the activity and location of BAM1 in mesophyll cells, BAM1 may hydrolyze starch, providing carbon skeletons for the production of osmolytes. These osmolytes help plants take up water through osmosis. The increased activity of plants containing BAM1 combined with bam1 plants experiencing less chlorosis, anthocyanin production, and wilting as the wild-type suggests that BAM1 plays a crucial role in the survival of osmotic-stress.
Schmitz, Jonathan Michael, "Role of ß-amylase1 (BAM1) in the recovery of Arabidopsis plants from osmotic stress" (2014). Senior Honors Projects, 2010-current. 474.