A proteomic analysis of 14‐3‐3 binding proteins from developing barley grains

14‐3‐3 proteins are important eukaryotic regulatory proteins. Barley (Hordeum vulgare L.) 14‐3‐3A was over‐expressed, immobilised and used to affinity purify 14‐3‐3 binding proteins from developing barley grains. Binding was shown to be phosphorylation‐dependent. These proteins were fractionated by PAGE and identified by MALDI‐TOF MS. In total, 54 14‐3‐3 binding proteins were identified, 49 of these interactions are novel to plants. These proteins fell into a number of functional categories. The largest category was for carbohydrate metabolism, including plastidic enzymes for starch synthesis and modification. 14‐3‐3 was shown to be present in isolated plastids. Four of five enzymes involved in sucrose biosynthesis from triose phosphates were identified, suggesting co‐ordinated regulation of this pathway. Invertase and sucrose synthase, which break down sucrose to hexoses, were found. Sucrose synthase activity was shown to be inhibited by exogenous 14‐3‐3 in a dosage‐dependent manner. The second‐largest functional group was for proteins involved in stress and defence responses; for example, RGH2A, closely related to the MLA powdery mildew resistance protein, was found. This work illustrates the broad range of processes in which 14‐3‐3 may be involved, and augments previous data demonstrating key roles in carbohydrate metabolism and plant defence.