Restoration of cis-acting elements in the rice PSY1 promoter combined with CrtI expression enables carotenoid accumulation in seeds and reveals distinct promoter methylation states

Rice endosperm does not normally accumulate provitamin A carotenoids such as β-carotene. Our previous work showed that restoration of six near-miss cis -acting elements in the rice PSY1 promoter could support carotenoid accumulation in callus when combined with a plant phytoene desaturase (PDS), but this strategy did not lead to carotenoid accumulation in mature seeds. Here, we tested whether combining the corrected 6M-OsPSY1 cassette with the bacterial carotene desaturase CrtI, which bypasses multiple endogenous desaturation and isomerization steps, could overcome this limitation. Co-transformed callus lines accumulated high levels of carotenoids, and regenerated plants produced mature seeds containing β-carotene, reaching 3.57 μg g -1 DW. Across independent callus lines, carotenoid accumulation did not scale proportionally with PSY1 or CrtI transcript abundance; PSY1 transcript abundance showed a positive association with carotenoid accumulation, whereas no positive association was observed for CrtI . In seeds, phenotypic classes differing in color and carotenoid content were associated with distinct methylation states of the engineered 6M-OsPSY1 promoter. These findings show that CrtI can overcome pathway limitations that remained in the previous 6M-OsPSY1/PDS system, enabling carotenoid accumulation in mature rice seeds. They further reveal an association between promoter methylation and phenotypic variation, although the present data do not establish a causal relationship between methylation and carotenoid accumulation.