Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds

Authors: SUN, TIANHU - Cornell University; ZHU, QINLONG - Cornell University; WEI, ZIQING - Cornell University; OWENS, LAUREN - Cornell University; Fish, Tara; KIM, HYOJIN - University Of Nebraska; Thannhauser, Theodore - Ted; CAHOON, EDGAR - University Of Nebraska; Li, Li

Submitted to: aBIOTECH
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/26/2021
Publication Date: 5/17/2021
Citation: Sun, T., Zhu, Q., Wei, Z., Owens, L.A., Fish, T., Kim, H., Thannhauser, T.W., Cahoon, E.B., Li, L. 2021. Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds. aBIOTECH. https://doi.org/10.1007/s42994-021-00046-1.
DOI: https://doi.org/10.1007/s42994-021-00046-1

Interpretive Summary: Carotenoids are critically important for human nutrition and health as they provide dietary sources of vitamin A and antioxidants. Seeds are the main organ for carotenoid biofortification in crops. However, carotenoid degradation during seed maturation and post-harvest storage is a serious problem. Here, we utilized Arabidopsis as a model to establish multi-plex genetic engineering strategies for carotenoid biofortification in seeds. We demonstrate that seed-specific modification of a number genes not only increased provitamin A and total carotenoid accumulation during seed maturation, but also enhanced their stability during post-harvest storage without significantly affecting seed germination. Our results document an effective multi-strategy approach of simultaneously boosting biosynthetic activity, increasing storage sink capacity, and reducing carotenoid turnover, which is likely applicable to provitamin A carotenoid enrichment in seeds of various staple crops.

Technical Abstract: Staple grains with low levels of provitamin A carotenoids contribute to the global prevalence of vitamin A deficiency and therefore are main targets for provitamin A biofortification. However, carotenoid stability during both seed maturation and postharvest storage is a serious concern for the full benefits of carotenoid biofortified grains. In this study, we utilized Arabidopsis as a model to establish carotenoid biofortification strategies in seeds. We discovered that manipulation of carotenoid biosynthetic activity by seed-specific expression of Phytoene synthase (PSY) increases both provitamin A and total carotenoid levels but the increased carotenoids are prone to degradation during seed maturation and storage, consistent with previous studies of provitamin A biofortified grains. In contrast, stacking with Orange (ORHis), a gene that initiates chromoplast biogenesis, dramatically enhances provitamin A and total carotenoid content and stability. Up to 65- and 10-fold increases of ß-carotene and total carotenoids, respectively, with provitamin A carotenoids composing over 63% were observed in the seeds containing ORHis and PSY. Co-expression of Homogentisate geranylgeranyl transferase (HGGT) with ORHis and PSY further increases carotenoid accumulation and stability during seed maturation and storage. Moreover, knocking-out of ß-carotene hydroxylase 2 (BCH2) by CRISPR/Cas9 not only potentially facilitates ß-carotene accumulation, but also minimizes the negative effect of carotenoid over-accumulation on seed germination. Our findings provide new insights into various processes on carotenoid accumulation and stability in seeds and establish a multiplexed strategy to simultaneously target carotenoid biosynthesis, turnover, and stable storage for carotenoid biofortification in crop seeds.