Progress 04/15/24 to 04/14/25
Outputs
Target Audience:Our primary audience again was other scientists and plant breeders. We reached them through presentations at scientific conferences and industry field days. Given the technical nature of the work, the results are most relevant to those seeking to understand the mechanisms of carotenoid accumulation to apply our results to their systems and plant breeders seeking tools to track the genes underlying the key phenotypes. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Six undergraduates, two graduate students, and two postdoctoral research associates were involved in this project. The project provided the training opportunity in plant genetics, plant molecular biology and bioinformatics analyses. It also offered the opportunity for developing skills for scientific communications by presenting findings in writing and verbal presentations. How have the results been disseminated to communities of interest?Results have been disseminated to the scientific community through conference, field day and seminar presentations as noted in publications and other products. What do you plan to do during the next reporting period to accomplish the goals?We will continue our ongoing experiments to further explore B and L2 interaction in affecting carotenoid accumulation and characterize the impact of Bmax in C. moschata as well as exploring its role in C. maxima towards carotenoid accumulation in fruit flesh. The mechanism of Bmax for affecting chlorophyll accumulation will also be explored.
Impacts
What was accomplished under these goals?
Significant progress has been made in characterizing B and L-2 synergistic effects in promoting carotenoid accumulation during the reporting periods. Analysis unique lines with various combinations of B and L2 revealed that lines with both B and L-2 alleles exhibited significantly higher total carotenoids at young (<5 DAP), mid-mature (15 DAP), and mature (50 DAP) stages compared to b/b L2/L2 or B/B l-2/l-2 line, indicating that B and L2 synergize to enhance carotenoid accumulation throughout fruit development. To functionally verify b, VIGS targeting to the b gene was performed in b/b L2/L2 plants. Positive lines, confirmed by reduced b transcript and protein levels, exhibited green-to-yellow fruit skin with increased carotenoids in yellow regions at all developmental stages. L2 was isolated using QTL-seq and genetic mapping. Functional validation using VIGS in a B/B L2/L2 background led to yellow-to-green fruit color changes and reduced carotenoids. Interestingly, b protein was detectable in green but not yellow regions of VIGS-treated fruit, suggesting a possible recovery mechanism. To understand how CpL2 and B promote carotenoid accumulation, we assessed carotenoid pathway gene expression across 13 lines and CpL2-VIGS lines. Most genes showed no significant change except for PSY, which was positively correlated with L2 expression and total carotenoids. Higher PSY protein levels and activity in B/B L2/L2 than b/b L2/L2 backgrounds were confirmed, suggesting that L2 and B together enhance PSY transcription, protein accumulation, and enzymatic function to enhance carotenoid levels. Moreover, molecular studies revealed that in the B background, L2 activates PSY activity to promote carotenoid biosynthesis, whereas in the b background, L2 activity is inhibited by b binding, causing reduced PSY activity and carotenoid production. For Objective 3, high resolution mapping was performed on the previously identified Bmax QTL and mapped the Bmax to a 96 Kbp region containing 17 genes. Analysis of genes within this region support that Bmax does not encode a paralog of Bpepo in C. maxima. The sequencing of transcripts from squash fruit with and without the Bmax phenotype suggests Bmax is caused by a small intron retention in a gene influencing protein formation inside plastids. The inclusion of this partial intron forms a truncated protein that is predicted to have none or negligible function. We hypothesize a negligible function disrupts the formation of chloroplasts leading to the miniscule chlorophyll accumulation in young Bmax fruit tissues. At fruit maturity, we determined Bmax fruits contain significantly higher levels of total carotenoids with a particularly strong increase in B-carotene compared to mature fruits without Bmax. To support our candidate as Bmax, we are currently in the process of silencing the gene in squash and tobacco as well as overexpressing it in Arabidopsis mutants which display a disruption in chlorophyll accumulation in their leaves.
Publications
- Type:
Other Journal Articles
Status:
Published
Year Published:
2025
Citation:
Wrightstone, E., Xu, L., Rao, S., Hazra, A., & Li, L. (2025). ORANGE family proteins: multifunctional chaperones shaping plant carotenoid level, plastid development, stress tolerance, and more. Molecular horticulture, 5(1), 43.
- Type:
Other
Status:
Other
Year Published:
2024
Citation:
Wrightstone, E. Genetic mapping of Bmax: a crucial gene for carotenoid accumulation in squash . Plant Breeding & Genetics Graduate Student Seminars, December 3, 2024
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2025
Citation:
Wrightstone, E., Zhou, X., Myers, J., Mazourek, M., Li, L. From green to orange: boosting carotenoid production in squash through the Bmax locus . Carotenoids Gordon Research Seminar, Ventura, CA, January 4, 2025
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2025
Citation:
Wrightstone, E., Zhou, X., Myers, J., Mazourek, M., Li, L. From green to orange: the unique way of Bmax in promoting carotenoid accumulation of crops . Carotenoids Gordon Research Conference, Ventura, CA, January 7, 2025
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Wrightstone, E., Zhou, X., Myers, J., Mazourek, M., Li, L. Genetic mapping of Bmax: a major locus facilitating carotenoid production in squash . Plant & Animal Genome Conference 32, San Diego, CA, January 10, 2025
- Type:
Other
Status:
Other
Year Published:
2025
Citation:
Wrightstone, E. Elucidating the carotenoid boosting Bmax locus from squash . USDA-ARS All-Center Meeting, Ithaca, NY, April 1, 2025
- Type:
Other
Status:
Other
Year Published:
2025
Citation:
Wrightstone, E. To be or not to be orange: discovering carotenoid genes in crops . Georgia Gosnell Seminar Series at Rochester Institute of Technology, Rochester, NY, April 28, 2025
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Progress 04/15/23 to 04/14/24
Outputs
Target Audience:Our primary audience again was other scientists and plant breeders. We reached them through presentations at scientific conferences and industry field days. Given the technical nature of the work, the results are most relevant to those seeking to understand the mechanisms of carotenoid accumulation to apply our results to their systems and plant breeders seeking tools to track the genes underlying key phenotypes. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Casey Wilson earned his MS under the mentorship of Jim Myers at OSU. Xuesong Xu served as a postdoctoral researcher and provided mentorship in Li Li's lab. Emalee Wrightstone and Jack Fabrizio performed a portion of their doctoral research and presented on their findings. Both mentored undergraduates in projects connected to this grant. An undergrad was trained in molecular biology by working on the project: Kyle Gisworld, 2024 Plant Science Major (undergraduate at Cornell), August 2023 - May 2024 How have the results been disseminated to communities of interest?Results have been disseminated through presentations and publications. These have included a dissertation, graduate student presentations and presentations to seed industry plant breeders at field days. What do you plan to do during the next reporting period to accomplish the goals?We will continue to identify interacting partners of Bpepo and its mechanism of expression. L-2 candidate genes will be tested for function. To date we have relied on introgression lines for Bmax characterisation, taking advantage of early ectopic expression in seedling stems as a proxy for expression in C. moschata. We now have the opportunity to leverage other projects in C. maxima to work with the native Bmax allele. CucCAP resequenced our MicroMax panel representing the diversity of the C. maxima species in a minimalist way. A Hatch award is allowing us to explore the species more deeply which will result in an opportunity to examine the genomic context of Bmax without interspecific recombination suppression.
Impacts
What was accomplished under these goals?
Objective 1. During this reporting period, we characterized the B gene in different varieties of C. pepo. The B gene is a chlorophyll biosynthesis pathway gene and hypothesized to be non-functional in chlorophyll biosynthesis to produce yellow squash fruit. We found that the B gene shows much lower expression in yellow squash than green ones. The B gene has a larger size of transcript from yellow squash fruit than green fruit due to intron retention. As a result, the B gene encodes a truncated enzyme protein. Consistently, western blot analysis of B protein reveals that the yellow fruit has non-detectable protein band but the green fruit has detectable band. These results support the role of the B gene function in conferring carotenoid accumulation by impairing chlorophyll biosynthesis in squash fruit. We are in the process to examine whether other genes associated with B in chlorophyll biosynthesis also exhibit the same function as B in promoting carotenoid accumulation in chromoplasts. Objective 2. 2.1. To identify L-2, we performed QTL-seq analysis that combines bulked segregant analysis and high-throughput whole-genome re-sequencing of pale yellow l-2/l-2 and dark orange L-2/L-2 plants. This analysis detected a single QTL associated with L-2. 2.2. To validate the identified QTL, genetic mapping was carried out. InDel markers equally distributed within the QTL region were developed and used to generate a low-resolution genetic linkage map. Two flanking markers closely linked to L2 were utilized to screen ~500 F2 plants for recombinants. Additional InDel markers were developed and used to genotype the recombinants. A high-resolution linkage map was constructed and the L-2 was delimited in a physical interval of 55 Kbp. There are nine predicted genes in this region. Among them, only one gene shows a high level of expression in fruit tissue, which represents the best candidate for L-2 gene. 2.3. To functional confirm the candidate gene for L-2, we first cloned it from the L-2 and l-2 backgrounds. Sequence and protein structure analyses show differences for the candidate from L-2 and l-2 backgrounds. We are performing functional confirmation in squash plants now. 2.4. We are also in the process to characterize the candidate and found that L-2 has higher activity in affecting carotenoid levels while l-2 has much weaker function. Analysis of various squash inbred lines shows that L-2 can greatly increase the carotenoid level in the B background. Objective 3. Allelism tests of Bmax and Bpepo suggests that Bmax is a second potentially superior B locus. DNA bulks created from a F2 population were used for QTL-seq analysis. The analysis identified a single QTL, which is localized in a different chromosome from the Bpepo studied in Objective 1, supporting that Bmax and Bpepo are different B genes. We developed six InDel markers closely linked to Bmax and constructed a low-resolution linkage map. The Bmax locus was flanked by two markers with a genetic distance of 6.4 cM. Currently we are performing high-resolution mapping to narrow down Bmax interval to a region with a small number of candidate genes.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2023
Citation:
Wilson, Casey. 2023. "Inheritance and Allelism Among the B genes in Cucurbita moschata". Thesis.
- Type:
Other
Status:
Other
Year Published:
2024
Citation:
Wrightstone, Emalee, Li, Li. "Many ways to orange: my approaches towards uncovering novel carotenoid regulators in plants", Plant Breeding & Genetics Graduate Student Seminars, April 30, 2024
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Fabrizio, Jack. "A Genomic Approach for a More Sustainable Pumpkin". Plant and Animal Genome Conference, San Diego, CA, January 12, 2024
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Progress 04/15/22 to 04/14/23
Outputs
Target Audience:Our target audience during this period have been other Cucurbita researchers and public and private plant breeders. Until recently, Mazourek and Myers were the only public sector squash breeders in the United States (US) and Mazourek was the only breeder doing genomics work in squash. Now, there are two additional public sector faculty doing breeding and genomics and we are interacting with them frequently. Further, this project has been a bridge to our colleagues in Israel. Our progress is also shared with the seed industry at industry focused field days. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?An undergraduate student was trained on how to set up agricultural fields for genetic studies and to record accurate phenotypic data to differentiate between Bmax and Bpepo. Additional trainings included techniques for manual squash pollination and taking squash leaf samples for DNA extraction. How have the results been disseminated to communities of interest?Mazourek presented on the project at the Vegetable Breeding Institute Field Days to seed company plant breeders. We have also been in contact with collaborators in Israel that we originally connected with via a BARD project on carbohydrates. What do you plan to do during the next reporting period to accomplish the goals?We will proceed with the characterization of Bpepo as planned. Our most immediate steps will be to use RNASeq data and Western analysis to verify the nature of the mutation. We will also proceed to fine map the L-2 locus. We are hopeful that near isogenic lines generously shared by a collaborator will allow us to rapidly narrow down the target region. Depending on the timeline of QTL-seq analysis it may be necessary to grow out F3 progeny from the (CN1-7 x TG1-2 & CN2-3 x TG1-1) populations for fine mapping. In addition, select F3 progeny from the (AC1-3/CN1-5 & CN1-5/CN3) allelism test populations can be grown out for phenotypic/genotypic validation. We currently have BC3F1 individuals segregating for Bmax that are a promising alternative to the F3 families.
Impacts
What was accomplished under these goals?
Objective 1. As the first step toward the goal of elucidation of the molecular basis of the B gene in C. pepo, we have grown and collected the fruit tissues of C. pepo varieties. We examined the B gene alleles from green and yellow fruit. We also cloned one allele of the B gene for phenotypical complementation for potentially testing out whether the B gene can reduce chlorophyll biosynthesis. Objective 2. To identify L-2, the synergistic partner of the B gene, we grew a F2 population that is homozygous for B gene and segregates for L-2. Fruit rind and flesh color from three fruit per plant were visually assessed. Carotenoid levels of flesh of three fruit per plant were measured by UPC2. Phenotypic bulks were created by selecting 21 pale yellow l-2/l-2 and 25 dark orange L-2/L-2 plants. Genomic DNA from the bulks were extracted and submitted for library constructions and the whole-genome sequencing using an Illumina HiSeq 2500 system with paired-end 2 × 150 bp run. Raw sequencing reads were obtained. One region, ~1Mb, has been identified as clearly harboring the L-2 locus. Objective 3.1, to test Bmax and Bpepo allelism in a conspecific C. moschata background, was completed. From 2019-2022 C. moschata lines with Bpepo and Bmax independently introgressed were established and intermated to create F2 and backcross populations segregating for both loci. In the summer of 2022 two populations (AC1-3/CN1-5 & CN1-5/CN3) totaling 1600 plants were grown and allelism tests were performed. Plants were phenotyped for Bmax and Bpepo by visual assessment of fruit and foliage and segregating classes were self-pollinated for future phenotypic/genotypic validation. Segregation data was analyzed for both one and two gene hypothesis. In addition, we began work on Objective 3.2, to examine Bmax candidates. From 2019-2021 another C. moschata population was generated segregating for only Bmax. In the summer of 2021 two F2 populations (CN1-7 x TG1-2 & CN2-3 x TG1-1) totaling 487 plants were phenotyped for Bmax and self-pollinated for further genotyping. Phenotypic bulks were created by selecting 20 orange Bmax/Bmax plants and 20 green Bmax+/Bmax+ plants from each population. In the winter of 2023 leaf samples of these phenotypic bulks were sent to Cornell for QTL-seq analysis.
Publications
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