Antibody array-based proteome approach reveals proteins involved in grape seed development.

ABSTRACT

Grape (Vitis vinifera) is one of the most widely cultivated fruits globally, primarily used for processing and fresh consumption. Seedless grapes are favored by consumers for their convenience, making the study of seedlessness a subject of great interest to scientists. To identify regulators involved in this process in grape, a monoclonal antibody (mAb)-array-based proteomics approach, which contains 21,120 mAbs, was employed for screening proteins/antigens differentially accumulated in grape during development. Differences in antigen signals were detected between seeded and seedless grapes revealing the differential accumulation of 2,587 proteins. After immunoblotting validation, 71 antigens were further immunoprecipitated and identified by mass spectrometry (MS). An in planta protein-protein interaction (PPI) network of those differentially accumulated proteins was established using mAb antibody by immunoprecipitation (IP)-MS, which reveals the alteration of pathways related to carbon metabolism and glycolysis. To validate our result, a seedless-related protein, DUF642 domain-containing protein (VvDUF642), which is functionally uncharacterized in grapes, was ectopically overexpressed in tomato (Solanum lycopersicum "MicroTom") and led to a reduction in seed production. PPI network indicated that VvDUF642 interacts with pectin acetylesterase (VvPAE) in grapes, which was validated by BiFC and Co-IP. As anticipated, overexpression of VvPAE substantially reduced seed production in tomato. Moreover, S. lycopersicum colourless non-ripening expression was altered in VvDUF642- and VvPAE-overexpressing plants. Taken together, we provided a high-throughput method for the identification of proteins involved in the seed formation process. Among those, VvDUF642 and VvPAE are potential targets for breeding seedless grapes and other important fruits in the future.