ZmDREB2A regulates ZmGH3.2 and ZmRAFS, shifting metabolism towards seed aging tolerance over seedling growth

Seed aging tolerance and rapid seedling growth are important agronomic traits for crop production; however, how these traits are controlled at the molecular level remains largely unknown. The unaged seeds of two independent maize DEHYDRATION-RESPONSIVE ELEMENT-BINDING2A mutant (zmdreb2a) lines, with decreased expression of GRETCHEN HAGEN3.2 (ZmGH3.2, encoding indole-3-acetic acid [IAA] deactivating enzyme), and increased IAA in their embryo, produced longer seedling shoots and roots, than the null segregant (NS) controls. However, the zmdreb2a seeds, with decreased expression of RAFFINOSE SYNTHASE (ZmRAFS) and less raffinose in their embryo, exhibit decreased seed aging tolerance, than the NS controls. Overexpression of ZmDREB2A in maize protoplasts increased the expression of ZmGH3.2, ZmRAFS genes and that of a Rennila LUCIFERASE reporter (Rluc) gene, which was controlled by either the ZmGH3.2- or ZmRAFS-promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assay quantitative polymerase chain reaction showed that ZmDREB2A directly binds to the DRE motif of the promoters of both ZmGH3.2 and ZmRAFS. Exogenous supplementation of IAA to the unaged, germinating NS seeds increased subsequent seedling growth making them similar to the zmdreb2a seedlings from unaged seeds. These findings provide evidence that ZmDREB2A regulates the longevity of maize seed by stimulating the production of raffinose while simultaneously acting to limit auxin-mediated cell expansion.