Sporopollenin biosynthesis in an OsTKPR2-containg metabolon is required for rice pollen exine formation.

Sporopollenin polymer is a major component of pollen exine. Fatty acid derivatives synthesized in tapetum are among the precursors of sporopollenin. Progresses have been made to understand sporopollenin metabolism in rice, however, the underlying molecular mechanisms remain elusive. We found that OsTKPR2 and OsTKPR1 share similar expression pattern, and their coding proteins have similar subcellular localization and enzyme activities towards reduce tetraketide α-pyrone and hydroxylated tetraketide α-pyrone. Unexpectedly, OsTKPR1pro:OsTKPR2-eGFP could not rescue the phenotype of ostkpr1-4. Three independent ostkpr2 mutant lines generated by CRISPR/Cas9 displayed reduced male fertility to various extents which were correlated with the severity of gene disruptions. Notably, anther cuticle, Ubisch bodies, and pollen development were affected in ostkpr2-1 mutant, where thinner pollen exine was noticed. OsTKPR1 and OsTKPR2 were integrated into a metabolon including OsACOS12 and OsPKS2, which resulted in a significant increased enzymatic efficiency when both OsTKPR1 and OsTKPR2 were present, indicating the mutual dependence of OsTKPR2 and OsTKPR1 for their full biochemical activities. Thus, our results demonstrated that OsTKPR2 is required for anther and pollen development where an OsTKPR2-containing metabolon is functional during rice sporopollenin synthesis. Furthermore, the cooperation and possible functional divergence between OsTKPR2 and OsTKPR1 is also discussed.