Changes in the Expression of Carbohydrate Metabolism Genes during Three Phases of Bud Dormancy in Leafy Spurge

Authors: Chao, Wun; SERPE, MARCELO - Boise State University

Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/30/2009
Publication Date: 4/9/2010
Citation: Chao, W.S., Serpe, M.D. 2010. Changes in the expression of carbohydrate metabolism genes during three phases of bud dormancy in leafy spurge. Plant Molecular Biology. 73:227-239.

Interpretive Summary: Underground adventitious buds of leafy spurge undergo three well-defined phases of dormancy, para-, endo-, and ecodormancy, throughout the year. In this study, relationships between carbohydrate metabolism and bud dormancy were examined and real-time PCR was used to determine if shifts in carbohydrate contents correlate with the expression levels of some carbohydrate metabolism genes. Our results indicated that many carbohydrate metabolism genes were differentially-regulated after paradormancy release and in response to seasonal signals. Among these genes, a specific ß-amylase transcript increased 100-fold after growth induction and increased 16,000-fold from July to December. This ß-amylase was represented by two genes, Ee-BAM1 and Ee-BAM2. The deduced amino acid sequences of these two genes are very similar at the N-terminal end but are disparate at the C-terminal. Both contain a nearly identical, predicted 48-amino acid plastid transit peptide. Immunoblot analyses identified a 29-kD and a 35-kD protein; the 29-kD protein could be the mature Ee-BAM1 where the transit peptide was cleaved. Unlike transcript expressions, both 35-kD and 29-kD proteins were constitutively expressed in growth-induced and seasonal samples. Immunolocalization indicated that Ee-BAM1 is in the cytosol of cells constituting leaf primordium and procambium at the tip of the bud. Ee-BAM1 also surrounds the amyloplasts in mature cells toward the base of the bud. These observations implicate that Ee-BAM1 may have dual functions; it serves as nutrient reserve in the cytosol and acts as degrading enzyme at the surface of amyloplasts.

Technical Abstract: Underground adventitious buds of leafy spurge (Euphorbia esula) undergo three well-defined phases of dormancy, para-, endo-, and ecodormancy. In this study, relationships among genes involved in carbohydrate metabolism and bud dormancy were examined after paradormancy release (growth induction) by decapitation and in response to seasonal signals. Real-time PCR was used to determine the expression levels of carbohydrate metabolism genes at different phases of bud dormancy. Among differentially-regulated genes, expression of a specific Euphorbia esula ß-amylase gene (EeBAM1) increased 100-fold after growth induction and 16,000-fold from July (paradormancy) to December (ecodormancy). Sequence data analysis indicated that two genes, Ee-BAM1 and Ee-BAM2, could encode this ß-amylase. However, real-time PCR using gene-specific primer pairs only amplified Ee-BAM1, indicating that Ee-BAM2 is either specific to other organs or not abundant. The deduced amino acid sequences of these two genes are very similar at the N-terminal but differ at the C-terminal. Both contain a nearly identical, predicted 48-amino acid plastid transit peptide. Immunoblot analyses identified a 29 kD (mature Ee-BAM1 after cleavage of the transit peptide) and a 35 kD (unprocessed EeBAM1) protein. Both 35 kD and 29 kD proteins were constitutively expressed in growth-induced and seasonal samples. Immunolocalization indicated that Ee-BAM1 is in the cytosol of cells at the shoot tip of the bud. Ee-BAM1 also surrounds the amyloplasts in mature cells toward the base of the bud. These observations suggests that Ee-BAM1 may have dual functions; serving as reserve protein in the cytosol and as a degrading enzyme at the surface of amyloplasts.