A UCH4 – OsMT2b deubiquitinase–metallothionein module coordinates redox and zinc homeostasis to safeguard spikelet fertility

Spikelet abortion is a critical defect affecting rice yield. Here, we characterized an apical panicle abortion1304 (apa1304) mutant, which displays apical spikelet abortion and defective anther cuticle formation. Phenotypic and cytological analyses revealed aborted and pollen-less anthers in the apical spikelets of apa1304. Results of Map-based cloning and MutMap analyses revealed that the candidate gene controlling the phenotype of apa1304 is a deubiquitinase, OsUCH4, encoding a ubiquitin carboxyl-terminal hydrolase. RNA in situ hybridization and qRT-PCR showed that OsUCH4 is ubiquitously expressed with enrichment in young spikelets, tapetum, and male meiocytes. CRISPR/Cas9 knockout lines mimicked the mutant phenotype, while complementation rescued spikelet fertility. Protein interaction assays (Y2H, LUC complementation, and GST pull-down) demonstrated that OsUCH4 physically interacts with OsMT2b, a protein previously known for ROS-scavenging and zinc transportation to the panicle. In vitro assays confirmed that OsUCH4 can deubiquitinate polyubiquitinated proteins derived from substrates UBQ1 and UBQ10, both independently and in the presence of OsMT2b. Multi-omics analyses further highlighted disruptions in redox and zinc transportation pathways in apa1304. Collectively, our findings reveal that OsUCH4 contributes to spikelet fertility through interaction with redox-associated protein OsMT2b, providing new insights into the molecular regulation of spikelet fertility in rice.