A Proteomic Analysis Of 14-3-3 Binding Proteins From Developing Maize Kernels

14-3-3 protein is one of the ubiquitous eukaryotic proteins with regulatorycapacity. There is clear evidence for the involvement of 14-3-3 proteins in theregulation of plant primary metabolism, cell signaling, as well as in some plantdefense mechanisms.14-3-3 binding can lead to enzyme activation or deactivation, orcan bring different proteins together over 14-3-3 dimmers and multiple target bindingsites, although the latter possibilityhas not yet been demonstrated in plants.Functions of plant 14-3-3 protein expression are diverse and its current researchfocuses on the following aspects: regulation of cell membrane transport; participationin nutrient uptake, water transport, stomata switch and other physiological processes;involvement in response to stress and pathogen; regulation of plant carbohydratemetabolism and so on. The recent researches showed that plant 14-3-3 proteins playan important role in carbohydrate metabolism and the starch synthesis. For example,14-3-3 protein isoforms were found to combine with the starch granules and thiscombination resulted in inhibitting the activity of 14-3-3 protein isoforms andincreasing starch accumulation in Arabidopsis thaliana. And in barley, it was alsoreported that more than 50 kinds of barley endosperm proteins interact with 14-3-3proteins, of which 31% of the proteins are related with starch and sucrose metabolism.All above results show that 14-3-3 proteins in starch biosynthesis play an importantrole.Progress about maize 14-3-3 proteins is slower. Recent studies have shown thatin the immature pollen grains of maize, 14-3-3 proteins combined with starch grainsand starch synthase SSIII, suggesting that 14-3-3 proteins play an important role inregulation of starch metabolism in maize endosperm and modification of starch particles.To resolve the regulatory mechanism of 14-3-3 proteins in maize kernels, wecarried out studies on discovery of 14-3-3 proteins interacting proteins in thedeveloping maize kernels. In order to achieve this purpose, tissue-specific distributionof various subtype 14-3-3 proteins were identified from NCBI database, from whichendosperm-expressing 14-3-3 protein isoform was selected to conduct theexperiments. This study used nested PCR method to acquire zmAAU93690 cloningfrom cDNAof high amylopectin maize kernels and then some 14-3-3 binding proteinsfrom maize kernels were captured by affinity chromatography Pull-down. The resultsobtained are:(1) The 14-3-3 protein affinity chromatography column was successfullyprepared by coupling 14-3-3 protein with affinity media Affi 15 and the whole activeproteins in maize kernels were successfully extracted. The 14-3-3 binding proteinsfrom maize kernels were captured by affinitychromatographyPull-down.(2) So far, a total of 14 novel 14-3-3 interacting proteins have been identifiedby 2-DE and LC-MS, and identification of other proteins is still in progress. Therewere 4 glycolytic enzymes: triosephosphate isomerase, 2-phosphoglyceratedehydratase, sorbitol dehydrogenase and Malate dehydrogenas. They all played animportant role in the glycolysis.As descriebed above, this study acquired 14 14-3-3 binding proteins and 4 ofthem were glycolytic enzymes. These novel interaction factors of 14-3-3 proteinprovided a very good research materials for the in-depth analysis of 14-3-3 protein inthe regulation of starch metabolism and construction of the maize starch synthesisregulatorynetworks.