ZmAGA1 enhances seed germination through raffinose hydrolysis and ZmSnRK1α-mediated starch mobilization in maize

Raffinose family oligosaccharides are key endogenous factors influencing seed germination, with raffinose as the sole member in maize. However, the mechanisms by which raffinose regulates germination remain largely unexplored.

A metabolite-based genome-wide association study using a maize CUBIC population identifies six candidate genes involved in raffinose metabolism in maize kernel. Among these, ZmAGA1, encoding an alkaline α-galactosidase, is localized in the cytosol and exhibits high expression levels in the embryo during seed germination. CRISPR-Cas9-mediated knockout of ZmAGA1 leads to delayed germination, reduced germination rates, and impaired seedling growth. Integrative spatial metabolomics and single-nucleus transcriptomics analyses at 18, 24, 36, and 48 h after imbibition in germinating seeds reveal that ZmAGA1 not only mediates raffinose catabolism and spatiotemporal sugar dynamics, but also positively correlates with ZmαAMY2 and ZmαAMY3 expression in aleurone cells and elevates α-amylase activity. Moreover, ZmAGA1 physically interacts with the energy-sensing kinase ZmSnRK1α in the cytosol, and both genes are co-expressed across embryonic tissues. In zmaga1 protoplasts, ZmSnRK1α shifts toward nuclear enrichment, where it interacts with the transcription factor ZmMYBS1, which directly activates ZmαAMY3 transcription. Consistently, ZmαAMY3 transcript levels are reduced in zmaga1 and zmsnrk1α protoplasts. These findings support a ZmAGA1-ZmSnRK1α-ZmMYBS1 cascade linking raffinose hydrolysis to α-amylase activation during seed germination.

Our integrative spatial-omics analyses reveal that ZmAGA1 facilitates seed germination by modulating sugar metabolism through the hydrolysis of raffinose and via ZmSnRK1α-dependent regulation of starch mobilization, with both processes providing essential energy for seed germination and early seedling establishment in maize.