Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/25/2010
Publication Date: N/A
Citation: N/A Interpretive Summary: To increase the oil content of soybean, attempts are being made to divert the assimilation of fixed carbon from starch accumulation to that of fatty acid synthesis. To be successful in this approach, one would need a thorough understanding of starch metabolism in soybean seed. Beta-amylase, a key enzyme involved in starch degradation, accumulates abundantly in soybean seeds. We have utilized two soybean near isogenic lines, one containing normal ß-amylase activity (Altona Sp1b) and the other with undetectable ß-amylase activity (Altona sp1) to understand the role of this enzyme in soybean seeds. We demonstrate that a 1.2 kB deletion in the 5’ region of the ß-amylase gene is responsible for the lack of ß-amylase activity in soybean Altona sp1. Additionally, we propose an indirect role of ß-amylase in lipid breakdown during soybean seed germination. Information obtained from this indepth biochemical study will help biotechnologists to design strategies that will aid the development of soybeans with enhanced oil content. Research focused on increasing the quantity of oil in soybean seed as important from the perspective of meeting the competition from other oil sources, especially palm oil.
Technical Abstract: Previous studies have identified near-isogenic soybean lines, one containing normal beta-amylase activity (Altona Sp 1b) and the other with undetectable beta-amylase activity (Altona sp 1). The molecular basis for the absence of beta-amylase activity in the mutant has not been investigated. In this study, we report the characterization of this beta-amylase mutant. SDS-PAGE analysis of 50% isopropanol extracted proteins from soybean seeds revealed a prominent 56 kDa protein in the wild-type. This protein, which was absent in Altona sp 1, was identified as beta-amylase by MALDI-TOF mass spectrometry and immunoblot analysis using antibodies generated against Arabidopsis beta-amylase. Northern blot analysis revealed the accumulation of 1.6 kB RNA transcript in developing seeds of the wild-type but not in Altona sp 1. Southern blot analysis under stringent hybridization conditions revealed at least two copies of this gene in the soybean genome and differences in the sizes of the DNA fragments hybridizing to beta-amylase probe between the near-isogenic soybean lines. We have isolated the beta-amylase gene by screening a genomic library of wild-type soybean and determined its nucleotide sequence. Analysis of the nucleotide sequence of the beta-amylase gene revealed a complete open reading frame that was interrupted by six introns. In contrast, the beta-amylase gene from Altona sp 1 had a 1207 bp deletion near the 5’ region that included the second and third exon regions. Transmission electron microscopic examination of germinating seeds revealed that the beta-amylase mutant mobilized the storage lipids at a faster rate than the wild-type genotype. The rapid mobilization of lipid bodies in the beta-amylase mutant was also associated with a higher amount of lipase activity in the germinating seeds.