Performing Department
Plant science and landscape architecture
Non Technical Summary
Hidden hunger, a condition that occurs when intake and absorption of essential micronutrients are too low to maintain good health, is one of the most serious problems worldwide. Vitamin A is an essential micronutrient that plays crucial roles in human health and development. Its deficiency is one of the prevailing causes of blindness and child mortality in low-income countries where most of the population mainly depends on one type of staples as the main caloric source that is not necessarily rich in micronutrients. Biofortification of crops presents a cheap, sustainable, and easy-to-access alternative to combat micronutrient deficiencies. Over the last two decades extensive efforts have been taken to develop rice varieties enriched with provitamin A. These efforts heavily relied on genetic engineering techniques since conventional breeding is not possible as provitamin A-rich variants of rice do not exist in nature. To date, two well-known Golden Rice (GR) varieties, namely GR1 and GR2, have been developed. GR2 accumulates a high level of β-carotene, which is provitamin A. GR 2 has already received food safety approvals by the United States, Canada, Australia, and Philippines for commercial farming to mitigate vitamin A deficiency. Although GR2 can provide significant part of the daily required amount of vitamin A, it suffers from issues that adversely affect its nutritional impacts. These issues include stable accumulation and storage of β-carotene, the main provitamin. Therefore, the purpose of this work is to tackle the shortcomings of earlier generations of Golden Rice. This research project is to address the USDA-NIFA-AFRI program area "1b. Foundational Knowledge of Plant Products" (A1103), where it calls for studying biosynthesis of plant-derived, high value biomolecules for use in foods, pharmaceuticals, and other products. This project aims to mainly use CRISPR genome engineering tools to address the shortcomings of GR2 by employing three different, yet parallel, objectives to improve storage, stability, and metabolic conversion of β-carotene in the rice endosperm. First, we will improve β-carotene accumulation capacity in GR2 endosperm by regulating chromoplast biogenesis and number. Second, we will increase β-carotene availability in GR2 endosperm by limiting its metabolic conversion. Third, we will enhance post-harvest stability of β-carotene in GR2 seeds by alleviating its degradation. The proposed research will provide foundational knowledge about the carotenoid metabolism in rice endosperm. Furthermore, it will aid the engineering of next-generation Golden Rice 3 to combat global vitamin A deficiency.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
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.
Project Methods
Objective 1: Improve β-carotene storage capacity in GR2 endosperm by regulating chromoplast biogenesis and number.In Objective 1, we will introduce R115H mutation in OsOR protein through base editing and increase activity of OrHis and PDV1 in the endosperm through gene activation. R115 is located in the second exon and encoded by C343G344C345 codon. Since there are no NGG PAM sites are available near the target region, we will use CBEs based on SpRY and NG-zCas9 that enable targeting relaxed PAM sequences. Concurrently, we will increase OsOrHis expression using our recently developed CRISPR-Combo system which allows simultaneous gene editing and activation. We will also test whether SpRY- and NG-zCas9 variants can significantly activate OsOr expression. Since OrHis variant restricts chromoplast number to one or two, we will also activate OsPDV1 gene, which is the major isoform in rice endosperm, to promote chromoplast division and further improve the sink strength. The sgRNAs that provide the best activation will be determined and will then be combined with the mutagenesis and activation elements of the OsOr gene. Collectively, the final plasmid will introduce the R115H mutation in OsOr and activate both the OsOrHis mutant and OsPDV1. This construct will then be transformed into GR2 callus by Agrobacterium-mediated transformation and transgenic T0 events will be screened for biallelic OsOrHis mutation by Sanger sequencing. The biallelic events with the best activations of OrHis and PDV1 genes (as determined by qRT-PCR analyses) will be propagated to T2 generation to obtain homozygous seeds. Carotenoid profiles, along with chromoplast formation and carotenoid gene expression, of the seeds from three to five biallelic T0 and/or homozygous T2 seeds with the highest activation in OrHis and PDV1 genes will be investigated. In particular, we will investigate provitamin A content of the seeds and perform retention assays to determine stability of β-carotene after storing the seeds at elevated temperature for a certain period (e.g., 37 °C for 0, 4, 8 weeks) along with at room temperature for extended time period. We will use WT Kitaake and parental GR2 seeds as controls for these experiments.Objective 2: Increase β-carotene availability in Golden Rice 2 endosperm by limiting its metabolic conversion.In Objective 2, we will explore two different approaches to increase β-carotene in Golden Rice 2 (GR2) background by limiting its metabolic conversion. Our first approach will focus on the generation of BCH1 knock-out lines in the Golden Rice 2 (GR2) background. With this strategy, we hope to block β-carotene's conversion to zeaxanthin. Although BCH1 appears to be the dominant isoform in the endosperm, we will re-investigate expression patterns of all three BCH isoforms in different tissues (e.g., leaf and seed) at different developmental stages (e.g., early, mid, and late endosperm development) by qRT-PCR. The isoform(s) that are highly expressed in the endosperm will be targeted by Cas9 mediated mutagenesis either separately or together. First, we will test efficiency of multiple sgRNAs in rice protoplast assays. The best performing sgRNAs will be used in the Agrobacterium-mediated transformations of GR2 calli. Biallelic T0 mutants of BCH(s) that result in non-functional proteins will be identified by Sanger sequencing or NGS of PCR amplicons. Select edited T0 lines will be further propagated to T1 generation to obtain homozygous lines. β-carotene content of seeds from the biallelic T0 mutants and the homozygous T1 lines will be quantified and compared to that of the GR2. Our second approach will focus on generation of CCD1 and CCD4a knock-out lines of GR2 Rice. In this separate, yet parallel, set of experiment, we will target the CCDs to prevent β-carotene oxygenation and conversion to apocarotenoids. We will target these oxygenase genes either separately or together by generating three sets of lines: OsCCD1-edited, OsCCD4a edited, and OsCCD1&OsCCD4a-simultaneously edited. Cas9 mediated mutagenesis and analyses of seed carotenoid profiles will be performed as described in the previous section. In addition, we will examine the effects of editing these genes on rice seed development and plant growth (e.g., germination delay).Objective 3: Enhance post-harvest stability of β-carotene in Golden Rice 2 seeds by alleviating its degradation.In Objective 3, we will increase expression of HGGT through gene activation, which is expected to increase the antioxidant capacity, thus carotenoid stability, of GR2 seeds. We will achieve this by OsHGGT activation using our recently developed CRISPR-Act3.0 system. We will first screen 4 to 8 sgRNAs per gene in the 5'UTR and in 200 bp upstream of the transcription start site using protoplast assays. The sgRNAs that yield the highest activations will be combined and re-evaluated. The activation plasmid will then be transformed into the GR2 calli by Agrobacterium-mediated transformation to generate stable lines. In parallel and as a second strategy, we will over-express the HvHGGT gene under control of an endosperm specific promoter (e.g., glutelin). This over-expression plasmid will also be transformed into the GR2 calli. Tocotrienol and tocopherol levels from seeds of T0 events with the highest HGGT expression events will be determined as described in. The seeds with the highest vitamin E content will be further propagated to obtain homozygous T1 lines. Vitamin E levels of the homozygous seeds will be re-evaluated, and carotenoid retention assays will be performed to determine the effects of these antioxidants on carotenoid stability during post-harvest storage.