Source: UNIV OF MARYLAND submitted to NRP
INVESTIGATION OF SS-CAROTENE ACCUMULATION AND STABLE STORAGE IN ENDOSPERM TOWARD ENGINEERING NEXT-GENERATION GOLDEN RICE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030353
Grant No.
2023-67013-39628
Cumulative Award Amt.
$647,000.00
Proposal No.
2022-08447
Multistate No.
(N/A)
Project Start Date
Jun 1, 2023
Project End Date
May 31, 2026
Grant Year
2023
Program Code
[A1103]- Foundational Knowledge of Plant Products
Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
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%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2061530101050%
2061530104050%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1530 - Rice;

Field Of Science
1040 - Molecular biology; 1010 - Nutrition and metabolism;
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.

Progress 06/01/24 to 05/31/25

Outputs
Target Audience:This project will provide cutting-edge training on plant genome editing technology and metabolic engineering for two post-doc researchers, as well as a few undergraduate and high school interns. The new genome engineering strategies established in rice for enhancing β-carotene accumulation may be directly transferred to other crops to achieve similar goals. The next generation Golden Rice engineered in this study may allow breeders to breed Golden Rice 3 in different local or elite varieties. So, this project has a diverse audience and stakeholders, including researchers in both academic and industrial sectors, rice breeders, government agencies on developing regulatory frameworks for genetically modified or genome edited crops, and those who are interested in seeking solutions for fighting vitamin A deficiency with a biofortification approach. Changes/Problems:Our approach has relied on the use of Golden Rice 2.1 lines. We have spent more effort characterizing these parental lines before using the seeds for plant transformation. Although this has slowed down our progress toward plant transgenesis, other molecular vector construction and analysis work went smoothly. Due to the slight project setback, we will seek for non-cost extension to fully catch up and finish the objectives as planned. What opportunities for training and professional development has the project provided?This project so far has provided training opportunities for one postdoc, Aytug Tuncel, in the Qi lab. How have the results been disseminated to communities of interest?We are still in the process of generating the results, which will be shared with the communities in conferences and eventually as peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?The primary set of T0 plants including overexpression, multiplexing and overexpression and multiplexing, were generated. We plan to propagate the selected lines, based on genotyping and transgene expression, to T1 generation and obtain the homozygous T2 seeds which will be biochemically characterized for carotenoid profiles and β-carotene retention capacity. We also plan to complement the primary set of transgenic lines by generating knock-out lines in which the target genes are individually disrupted (for example, BCH1, CCD1, or LOX1) in the GR2 background to determine the contribution of each gene to β-carotene accumulation.

Impacts
What was accomplished under these goals? GR is a genetically modified variety of rice that has been engineered to produce β-carotene, the main provitamin A, to address the hidden hunger of vitamin A deficiency, which affects millions of people in developing countries and can lead to severe health problems, including blindness and increased mortality rates in children. By incorporating β-carotene directly into a staple food crop, Golden Rice provides a sustainable and cost-effective solution to improve the nutritional status of vulnerable populations, potentially saving lives and enhancing overall health outcomes. CRISPR-Cas mediated genome editing can further enhance the development of novel GR varieties by enabling more precise genetic modifications. These modifications not only improve the nutritional quality of GR but also ensure that the provitamin A content is more stable in the seeds. In this project, we aim to address the shortcomings of current GR2 varieties. In objective 1, we will improve β-carotene storage capacity in GR2 endosperm by regulating chromoplast biogenesis and number. In objective 2, we will increase β-carotene availability in GR 2 endosperm by limiting its metabolic conversion. In objective 3, we will enhance post-harvest stability of β-carotene in GR2 seeds by alleviating its degradation. This project aligns well with the goals of 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. The data generated from this work will broaden our understanding of carotenoid metabolism in rice endosperm and will most likely lead to the development of new generations of GR varieties with improved provitamin A content and stability. Objective 1: Improve β-carotene storage capacity in GR2 endosperm by regulating chromoplast biogenesis and number. (1) Major activities completed: Two plasmids were constructed, overexpressing OrHis alone (pAT102) or together with the AtPDV1 gene (pAT105), under control of the endosperm specific rice prolamin promoter. These constructs were transformed into GR2 and, 10 and 12 independent T0 events were generated for OrHis and OrHis + AtPDV1 overexpression lines, respectively (Table 1). The plants are now being grown in the greenhouse. (2) Ongoing and future research: These lines will be grown to maturity and pre-screened qualitatively on the basis of orange color enhancement indicating an increase in β-carotene accumulation. We will also examine the presence and number of chromoplasts in developing seeds using microscopic techniques. Selected lines will be propagated to T1 generation. (3) Expected key outcomes: Overexpression of the OrHis gene is expected to result in chromoplast formation in the GR2 seeds, significantly enhancing the β-carotene accumulation. Overexpression of the AtPDV1 gene, in addition to OrHis, is expected to further enhance β-carotene content by increasing the number of chromoplasts in the endosperm. Objective 2: Increase β-carotene availability in Golden Rice 2 endosperm by limiting its metabolic conversion. (1) Major activities completed: Plasmids for targeting the different isoforms of β-carotene hydroxylases (BCHs) 1, 2, and 3 and carotenoid cleavage dioxygenases (CCDs) 1, 4a, and 4b were constructed and are being transformed into GR2. Additional target genes include lipoxygenase (LOXs) 1, 2, and 3 that cause non-specific oxidative degradation of β-carotene. Select genes (BCH1, CCD, CCD4a, LOX1, and LOX3) that have the most potential to increase β-carotene availability in the endosperm upon their disruption are targeted in two different multiplexing constructs namely pAT188 and pAT189. These two constructs were transformed into GR2 and, 23 and 28 T0 lines were generated which are now being grown in the green house (Table 1). As an alternative approach we have also generated 13 independent T0 lines overexpressing the AtDXS1 gene (under control of the rice glutelin A-3 promoter) to increase precursor supply for the carotenoid pathway (Table 1). (2) Ongoing and future research: T0 lines harboring the multiplexing constructs are being genotyped to characterize the editing outcomes. Mutants containing homozygous or biallelic mutations in the target genes will be selected and propagated to T1 generation for further biochemical characterization. Lines with partial gene disruptions (for example, editing in all targets except the BCH1 gene) will also be propagated and analyzed to determine the contribution of each gene to β-carotene accumulation. (3) Expected key outcomes: We expect to observe effective disruption of the targeted genes in T0 plants as our tRNA based Cas9 editing system has been proven to be highly efficient. Disruption of the BCH1 is expected to reduce metabolic conversion of β-carotene to downstream xanthophylls, while disruption of CCD1 and CCD4a is expected to reduce conversion of β-carotene to apocarotenoids. Similarly, the disruption of LOX 1 and 3 is expected to enhance post-harvest stability of β-carotene. Objective 3: Enhance post-harvest stability of β-carotene in Golden Rice 2 seeds by alleviating its degradation. (1) Major activities completed: We transformed the GR2 with the plasmid (pAT110) overexpressing the barley HvHGGT gene under control of the rice Glutelin B-4 promoter. 11 T0 lines were generated, and the plants are being grown in the greenhouse (Table 1). (2) Expected key outcomes: Overexpression of the HvHGGT is expected to increase tocotrienol content in the rice endosperm, increasing the antioxidant capacity of the seeds thereby enhancing the post-harvest stability of β-carotene. Combinatorial approaches to further enhance β-carotene accumulation and stability in GR2. (1) Major activities completed: In addition to the overexpression and multiplexing constructs described in objectives 1, 2, and 3 we also constructed the following plasmids: i) the ultimate overexpression plasmid that expresses AtDXS1, ORHis, AtPDV1, and the HvHGGT (pAT111), and ii) two separate constructs (pAT191 and pAT193) that share the AtDXS1, ORHis, AtPDV1, and the HvHGGT overexpression cassette from pAT111, and carry the multiplexing modules from either pAT188 or pAT189. These three additional constructs were also transformed into GR2 and 14, 26, and 27 T0 lines were generated carrying the plasmids pAT111, pAT191, and pAT193, respectively. (2) Ongoing and future research: The T0 plants are now being grown in the greenhouse. Lines selected based on seed color enhancement and editing outcomes will be propagated to T1 generation for further biochemical analyses. (3) Expected key outcomes: We expect to see the most dramatic increases in β-carotene accumulation and post-harvest stability in seeds from the lines carrying the pAT191 and pAT193 plasmids. Summary of the constructs transformed into GR2: Overexpression constructs pAT97: AtDXS1 pAT102: OsOrHis pAT105: OsOrHis + AtPDV1 pAT110: HvHGGT pAT111: AtDXS1 + OsOrHis + AtPDV1 + HvHGGT Multiplexing constructs: pAT188: BCH1 + CCD1 + CCD4a + LOX1 + LOX3 pAT189: BCH1 + CCD1 + CCD4a + LOX1 + LOX3 Multiplexing and overexpression constructs: pAT191 = pAT111 + pAT188 pAT193 = pAT111 + pAT189 Control construct and lines: pAT194 (contains only the hygromycin selection cassette) transformed into GR2 and WT Kitaake.

Publications


    Progress 06/01/23 to 05/31/24

    Outputs
    Target Audience:This project will provide cutting-edge training on plant genome editing technology and metabolic engineering for two post-doc researchers, as well as a few undergraduate and high school interns. The new genome engineering strategies established in rice for enhancing β-carotene accumulation may be directly transferred to other crops to achieve similar goals. The next generation Golden Rice engineered in this study may allow breeders to breed Golden Rice 3 in different local or elite varieties. So, this project has a diverse audience and stakeholders, including researchers in both academic and industrial sectors, rice breeders, government agencies on developing regulatory frameworks for genetically modified or genome-edited crops, and those who are interested in seeking solutions for fighting vitamin A deficiency with a biofortification approach. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project so far has provided training opportunities for one postdoc, Aytug Tuncel, in the Qi lab. How have the results been disseminated to communities of interest?We are still in the process of generating the results, which will be shared with the communities in conferences and eventually as peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?As we have finalized our target gene selections and plasmid designs for gene editing and overexpression, the next steps are to construct these plasmids, test them in the transient protoplast assays, and then transform them into the GR2. We plan to progress, at the very least, up to the T0 plants (or T1 seeds) within the next year for all the three objectives proposed.

    Impacts
    What was accomplished under these goals? Golden Rice (GR) is a genetically modified variety of rice that has been engineered to produce β-carotene, the main provitamin A, to address the hidden hunger of vitamin A deficiency, which affects millions of people in developing countries and can lead to severe health problems, including blindness and increased mortality rates in children. By incorporating β-carotene directly into a staple food crop, Golden Rice provides a sustainable and cost-effective solution to improve the nutritional status of vulnerable populations, potentially saving lives and enhancing overall health outcomes. CRISPR-Cas-mediated genome editing can further enhance the development of novel GR varieties by enabling more precise genetic modifications. These modifications not only improve the nutritional quality of GR but also ensure that the provitamin A content is more stable in the seeds. In this project, we aim to address the shortcomings of current GR2 varieties. In objective 1, we will improve β-carotene storage capacity in GR2 endosperm by regulating chromoplast biogenesis and number. In objective 2, we will increase β-carotene availability in GR 2 endosperm by limiting its metabolic conversion. In objective 3, we will enhance post-harvest stability of β-carotene in GR2 seeds by alleviating its degradation. This project aligns well with the goals of the USDA-NIFA-AFRI program area "1b. Foundational Knowledge of Plant Products" (A1103), where it calls for studying the biosynthesis of plant-derived, high-value biomolecules for use in foods, pharmaceuticals, and other products. The data generated from this work will broaden our understanding of carotenoid metabolism in rice endosperm and will most likely lead to the development of new generations of GR varieties with improved provitamin A content and stability. Objective 1: Improve β-carotene storage capacity in GR2 endosperm by regulating chromoplast biogenesis and number. (1) Major activities completed: To introduce the R115H (C344T) mutation in the rice Orange protein we tested four different constructs consisting of different combinations of sgRNAs (sgRNA-1 or -2) and base editing systems (hA3A/Y130F or PmCDA deaminases) and PAM flexible Cas variants (SpRY-zCas9 or NG-zCas9). As a positive control, we overexpressed the ORHis gene under the control of the ZmUbi promoter. (2) Data collected: Preliminary results obtained from the transgenic T0 plants transformed with these plasmids revealed that the hA3A/Y130F-SpRY-zCas9-sgRNA1 construct was the most efficient in introducing the desired mutation though the editing efficiency was low (one out of seven transgenic lines investigated had a heterozygous mutation at the target site). The NG-zCas9-gRNA2 constructs did not produce the desired mutation as the targeted cytosine (C344) is at position C17 and thus out of the editing window. We also observed that overexpression of the rice ORHis mutant gene results in yellow-orange colored rice callus. (3) Key outcomes and discussion of the results: These results demonstrate that the ORHis mutation can be introduced in the GR2 genome and that homozygous mutations can be obtained provided that the sufficient number of lines are screened. As an alternative approach, we will overexpress ORHis under the control of the endosperm-specific prolamin promoter. Since the ORHis limits the number of chromoplasts we will also overexpress the Arabidopsis Plastid division 1 (PDV1) gene under the same promoter to promote chromoplast division, thus enhancing β-carotene reservoirs in the endosperm. These constructs are designed and will be transformed into GR2. Objective 2: Increase β-carotene availability in Golden Rice 2 endosperm by limiting its metabolic conversion. (1) Major activities completed: We also successfully introduced a stop codon using the above-mentioned base editing systems in the first exon of BCH1 to knock out this gene and therefore prevent the conversion of β-carotene to downstream metabolites. As an alternative approach to improve β-carotene availability in the rice endosperm, we targeted the upstream open reading frames (uORFs) of the β-carotene synthesis genes including 1-deoxy-D-xylulose 5-phosphate synthase 3 (DXS3), phytoene synthase 1 (PSY1), phytoene desaturase 1 (PDS1), and zeta-carotene desaturase (ZDS) in the wildtype Kitaake cultivar. We targeted thirteen different uORFs, either experimentally or computationally determined, using four different constructs each expressing different combinations of genome editing elements (7 to 8 sgRNAs, Cas9, Cas12a, or NG-zCas9 fused to C-to-T or dual base editing deaminases). (2) Data collected: Preliminary results obtained from the protoplast assays of the uORF editing constructs showed that the editing efficiencies of the SpCas9 and LbCas12a (RRV variant) were higher than those of the base editing constructs as expected. However, we do expect to achieve better results with the stable lines. More than 200 T0 plants were generated and are being analyzed to determine the most promising edited lines. (3) Ongoing and future research: The uORF mutant pool will be reduced to 20-30 lines representing different types of mutations at different target regions and will be propagated further to obtain homozygous plants. β-carotene content of the seeds from these lines will then be characterized. Objective 3: Enhance post-harvest stability of β-carotene in Golden Rice 2 seeds by alleviating its degradation. (1) Ongoing and future research: We designed the HvHGGT overexpression plasmid to be expressed in the rice endosperm under the control of the rice Glutelin B-4 promoter. To improve the stability of the β-carotene even further we also designed the constructs for knocking the carotenoid cleavage dioxygenase (CCD) 1, 4a, and 4b, and lipoxygenase (LOX) r9-LOX1, LOX2, and LOX3 genes. We will use the conventional SpCas9 along with two sgRNAs to ensure full disruption of the gene activities. We will knock out these genes either individually or employ multiplex editing in case disruption of the single isoform is not sufficient. These constructs will be delivered into GR2 and will be further propagated to obtain homozygous lines. (2) Expected key outcomes: Since CCDs catalyze specific enzymatic cleavage of β-carotene to apocarotenoids and LOXs catalyze non-specific enzymatic oxidation of β-carotene, reducing the activity of these enzymes is expected to significantly improve β-carotene post-harvest stability as determined by carotenoid retention assays.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2023 Citation: Genome-edited foods Aytug Tuncel, Changtian Pan, Thorben Sprink, Ralf Wilhelm, Rodolphe Barrangou, Li Li, Patrick M. Shih, Rajeev K. Varshney, Leena Tripathi, Joyce Van Eck, Kranthi Mandadi & Yiping Qi Nature Reviews Bioengineering volume 1, pages799816 (2023)