Regulation of starch accumulation by granule-associated plant 14-3-3 proteins

In higher plants the production of starch is orchestrated by chloroplast-lo calized biosynthetic enzymes, namely starch synthases, ADP-glucose pyrophos phorylase, and starch branching and debranching enzymes. Diurnal regulation of these enzymes, as well as starch-degrading enzymes, influences both the levels and composition of starch, and is dependent in some instances upon phosphorylation-linked regulation. The phosphoserine/threonine-binding 14-3 -3 proteins participate in environmentally responsive phosphorylation-relat ed regulatory functions in plants, and as such are potentially involved in starch regulation. We report here that reduction of the epsilon subgroup of Arabidopsis 14-3-3 proteins by antisense technology resulted in a 2- to 4- fold increase in leaf starch accumulation. Dark-governed starch breakdown w as unaffected in these "antisense plants," indicating an unaltered starch-d egradation pathway and suggesting a role for 14-3-3 proteins in regulation of starch synthesis. Absorption spectra and gelatinization properties indic ate that the starch from the antisense plants has an altered branched gluca n composition. Biochemical characterization of protease-treated starch gran ules from both Arabidopsis leaves and maize endosperm showed that 14-3-3 pr oteins are internal intrinsic granule proteins. These data suggest a direct role for 14-3-3 proteins in starch accumulation. The starch synthase III f amily is a possible target for 14-3-3 protein regulation because, uniquely among plastid-localized starch metabolic enzymes, ail members of the family contain the conserved 14-3-3 protein phosphoserine/threonine-binding conse nsus motif, This possibility is strengthened by immunocapture using antibod ies to DU1, a maize starch synthase III family member, and direct interacti on with biotinylated 14-3-3 protein, both of which demonstrated an associat ion between 14-3-3 proteins and DU1 or DU1-like proteins.