The barley limit dextrinase inhibitor: Gene expression, protein location and interaction with 14-3-3 protein

The barley (Hordeum vulgare L.) limit dextrinase inhibitor (LDI) binds to and prevents the enzymic action of the starch debranching enzyme limit dextrinase (E.C. 3.2.1.142), and reduced levels of LDI have pronounced effects on starch biosynthesis including a loss of the small B-type starch granules. In this work, the time and place of LDI gene expression, and also the expression of LDI protein and inhibitory activity, and binding of LDI to the regulatory protein 14-3-3 were investigated. It was found that LDI is encoded for by a small multigene family, and the LDI gene is expressed at 2–4 weeks post anthesis in the endosperm. The LDI protein is present and active from 4 weeks post anthesis onward only in the starchy endosperm, not in the aleurone layer or other tissues, and is broken down during germination. Immunolocalisation shows the LDI protein to accumulate predominantly in the outer regions of the starchy endosperm, which coincides with the region where the small B-type granules are found. Additionally, the LDI protein sequence contains a 14-3-3 binding motif, and LDI binds to the barley 14-3-3A and 14-3-3C proteins in a phosphorylation-dependent manner, although addition of full length 14-3-3 proteins had no effect on LDI or LD activities in vitro. These findings are consistent with an important role for LDI (and by inference limit dextrinase) in the regulation of starch biosynthesis and starch granule formation.