Annual Progress Report on Malting Barley Research March, 2007

germination that is known to stimulate expression of many genes in the aleurone layers
surrounding the endosperm.
To analyze expression of the proteins encoded by the four abundantly expressed
Agl genes, we prepared four antisera. Each antiserum was designed to be specific to
only one of the four proteins encoded by these four genes. The specificities of these
antisera were determined by testing against extracts of E. coli transformed with one of
each of the four Agl genes we identified. Each of these cultures expressed only one of
the recombinant isozymes. Each antiserum recognized only the isozyme containing
the peptide to which it was designed. Again, in this report we will focus on comparing
expression of the proteins α-glucosidase1 and α-glucosidase2, which are encoded by
Agl1 with Agl2, respectively. Western blot analyses of germinated seed extracts
probed with antibodies that recognized only the α-glucosidase1 protein showed that this
antibody reacted with seed proteins having molecular masses of approximately 100, 95,
75 and 65 kD. These are data in agreement with those reported by Tibbot et al., 1998.
The anti-α-glucosidse1 probe cross reacted with proteins extracted from seedling
leaves with approximate molecular masses of 100, 95 and 80 kD. The Western blots
using the anti-α-glucosidase2 probe detected cross reacting proteins of approximately
100 and 80 kD in seedling leaves but no cross reacting proteins were detected in
germinated seeds. Possible explanations of why the Agl2 gene is transcribed yet the
protein is not detected in the seed extracts include: 1) the protein may be present but
below the limits of detection with the antibody; 2) the seed protein may be processed
differently from the leaf protein such that the epitope is cleaved from the seed protein; 3)
the seed protein may be insoluble possibly via membrane associations or aggregations;
or 4) RNA silencing may be occurring in the seeds that is not occurring in the leaves.
Purification and characterization of Agl2 demonstrated that it not only had
maltase activity (the definitive test for Agl), but also had α-xylosidase activity.
Xyloglucans were found to be better substrates than maltose for Agl2. This indicates
that although Agl2 can contribute to starch degradation, it could have a major role in cell
wall degradation during germination. Xyloglucans are the primary hemicelluloses
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