Transcriptomic and metabolomic insights into the role of Rc gene in enhancing salt tolerance during rice germination

Salt stress during seed germination severely limits direct-seeded rice productivity in saline areas. The bHLH transcription factor Rc, well known for regulating proanthocyanidin biosynthesis and rice pericarp pigmentation, is also implicated in antioxidant activity and seed stress resistance; however, its role in germination-stage salt tolerance remains unclear. Here, we generated two gain-of-function Rc mutants (Rc-GOF1 and Rc-GOF2) from the white-pericarp rice variety Nongxiang132 (NX132) using CRISPR/Cas9, correcting the recessive rc allele's frameshift mutation to restore a functional Rc allele. Under normal conditions, both Rc mutants and NX132 exhibited nearly 100% germination by day 4. Under 200 mM NaCl stress, the Rc mutants achieved nearly full germination by day 9, significantly outperforming NX132 (63.3%). Transcriptomic analysis of embryos subjected to 8 h of salt stress identified 935 differentially expressed genes (DEGs), with KEGG enrichment highlighting the auxin/IAA-related signaling pathway, which was further validated by qRT-PCR. Metabolomic profiling of embryos exposed to 24 h of salt stress detected 389 differential metabolites, enriched in ATP-binding cassette (ABC) transporters and cofactor biosynthesis pathways. Integrative analysis revealed that Rc acts upstream of the aux/IAA signaling pathway; upregulation of this pathway in mutants activates downstream ABC transporters and cofactor biosynthesis pathways to promote germination. Our findings uncover a novel role for Rc beyond pericarp pigmentation, demonstrating that it enhances rice germination-stage salt tolerance via transcriptional and metabolic reprogramming. This provides valuable mechanistic insights and genetic resources for breeding salt-tolerant rice to safeguard food security in salinized areas.