INVESTIGATION OF �-CAROTENE ACCUMULATION AND STABLE STORAGE IN ENDOSPERM TOWARD ENGINEERING NEXT-GENERATION GOLDEN RICE

Objective 1: Improve β-carotene storage capacity in GR2 endosperm by regulating chromoplast biogenesis and number.Chromoplasts are organelles with superb capacity to synthesize and stably store massive amounts of carotenoids. Chromoplasts as a metabolic sink for carotenoid accumulation contain carotenoid-lipoprotein sequestering substructures, which facilitate the sequestration of newly synthesized carotenoids for stable storage and stimulate continuous biosynthesis by removing end products at the site of carotenoid biosynthesis. As such, it is not surprising to find that increase in chromoplast compartment size and number strongly correlates with elevated carotenoid accumulation. Regulation of chromoplast biogenesis has been demonstrated to exert a profound effect on total carotenoid levels in crops by providing the unique metabolic sink structures. While chromoplasts are frequently observed in many colored crop organs, the genes that control chromoplast biogenesis and duplication are less known. In our previous studies, we discovered that the Orange (Or) gene represents the only known gene that acts as a bona fide molecular switch to initiate chromoplast formation. In Objective 1, we will improve β-carotene storage capacity of GR2 by introducing chromoplast formation in rice endosperm. To achieve this goal, we will employ multiple, yet parallel, approaches including introduction of the R115H point mutation in the OsOr gene along with activation or over-expression of the ORHis and PDV1 genes.Objective 2: Increase β-carotene availability in Golden Rice 2 endosperm by limiting its metabolic conversion.In general, two pairs of carotenoid hydroxylases convert α- and β-carotene into xanthophylls. These are classified as two heme-containing cytochrome P450 type hydroxylases (CYP97A and CYP97C) and two non-heme β-ring hydroxylases (BCH1 and BCH2), which convert α- and β-carotene into lutein and zeaxanthin, respectively. The strategy of blocking the conversion of β-carotene to zeaxanthin to improve β-carotene content in storage organs has been proven to be a feasible approach in many crops including potato, orange, and wheat. Ample evidence indicates that suppression of BCH activity by gnome editing alone is sufficient to boost β-carotene content in storage organs, a strategy which has not been explored in rice so far. Basic bioinformatic analysis revealed that the rice genome has three isoforms of BCH with each displaying different spatio-temporal expression pattern. While the BCH2 and BCH3 expressions appear to drop drastically during transition from mid to late developing stage (14 to 42 DAF) of endosperm, BCH1 expression follows a more stable pattern throughout the entire endosperm development. This suggests that BCH1 is the isoform in rice endosperm that is responsible for major BCH activity. In Objective 2, we will investigate whether β-carotene availability, thus its accumulation, in the Golden Rice 2 (GR2) endosperm can be boosted through reducing its metabolic conversion to downstream xanthophylls. To test this hypothesis, we will primarily target BCH genes and knock-out the isoform(s) that is highly expressed in the seeds. This will allow us to assess whether the desired mutations can be introduced and if so, whether they could improve β-carotene level and availability in the rice endosperm.Objective 3: Enhance post-harvest stability of β-carotene in Golden Rice 2 seeds by alleviating its degradation.In endosperm of monocot seeds, including oats, wheat, and rice, tocotrienols constitute >50% of the vitamin E antioxidants which scavenge reactive oxygen species. Over-expression of homogentisate geranylgeranyl transferase (HGGT), the enzyme that catalyzes the rate limiting step of tocotrienol synthesis, to improve antioxidant capacity and β-carotene stability in seeds have yielded promising results in maize, sorghum, and barley. In Objective 3, we will further enhance post-harvest stability of β-carotene in GR2 by increasing antioxidant capacity of the rice seeds. We will activate the endogenous OsHGGT gene and alternatively over-express the barley HvHGGT ortholog to increase vitamin E level in GR2 endosperm.