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.