Progress 01/15/24 to 01/14/25
Outputs
Target Audience:The target audiences for this project include the general scientific community as well as the local and national agriculturecommunity and plant agriculture and biotechnologyindustry. Undergraduate and graduatestudents are also target audiences for the project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1 PhD student (Jonathan Lomas) received training in bioinformatics under this project 1 PhD student (Fan Huang) has received training in crop biotechnology research, including crop transformation, gene editing, and metabolite analysis 1 undergraduate student (Genesis Thomas) from North Carolina A&T State University, was mentored during the summer of 2024 receiving hands-on training in carrot transgenic techniques (CRISPR design, tissue culture) and GC-MS metabolite analysis. How have the results been disseminated to communities of interest?Publications Lomas J, QuanH, Kosma DK, ZhangX, Tang H, CushmanJC, Yim WC(2024, submitted) Sylvan: a whole genome annotation pipeline and gene model filter using semi-supervised random forests.GigaScience. Other Products Invited Seminar Presentation Kosma DK, Santos P, Yim WC, Cahoon E et al. (2024). Specialized Lipid Metabolism: Understanding the Biosynthesis and Transcriptional Regulation of Protective Plant Lipid-Derived Natural Products. Professor Baeta Neves Center for Applied Ecology (CEABN) seminar series. Center for Forest Studies (CEF). University of Lisbon, Portugal. Presentation Type: Invited Public Talk/Seminar. Scope/Audience: International. Kosma DK, Santos P, Yim WC, Cahoon E et al. (2024). Specialized Lipid Metabolism: Understanding the Biosynthesis and Transcriptional Regulation of Protective Plant Lipid-Derived Natural Products. Institute of Biological Chemistry Seminar Series. Washington State University. Presentation Type: Invited Public Talk/Seminar. Poster Presentation Huang F, Trinh B, Mayer M, Lee B, Trotta D, Busta L, Santos P, Yim WC, Cahoon E, & Kosma DK (2024). Understanding the Biosynthesis of Health-Promoting Polyacetylenes in Carrot. InternationalSymposium on Plant Lipids. University of Nebraska, Lincoln (UNL). Presentation Type: Poster. Scope/ Audience: International What do you plan to do during the next reporting period to accomplish the goals? Our multi-pronged approach integrates genomics, metabolomics, and genome editing to dissect falcarin biosynthesis. Immediate priorities include: Advancing FAD2/CER1/CER3 carrot and Arabidopsis overexpression lines to subsequent generations to evaluate heritable metabolic traits Moving CRISPR-Cas9 knockout lines to advanced generations to clarify hypothesized gene functions Metabolite profiling of multiple tissues from multiple developmental stages of advanced generations of overexpression and gene-edited carrot and Arabidopsis lines to refine hypothesized metabolic pathway models. Completing the gene expression component of the falcarin chemical atlas to provide a comprehensive understanding of the dynamics of falcarin synthesis and accumulation throughout carrot plant development Correlating transcritpome data (public and in house generated) with falcarin metabolite data sets to get a better understanding of the transcriptional dynamics underlying falcarin biosynthesis.
Impacts
What was accomplished under these goals?
1.Elucidate the falcarin biosynthetic pathway in carrot as well as the developmental dynamics of falcarin biosynthesis using biochemical and genomics-based approaches Last year (2023), the Yim lab developed a comprehensive reannotation of the carrot genome by incorporating new sequencing data from both public repositories and in-house sources. This reannotation process is now complete and has been submitted to Gigascience (Lomas et al., 2024, submitted). As a result, the number of FAD2 and FAD2 variant genes identified in the genome increased from 24 to 42. In carrot, the functions of only 5 have been experimentally validated so far. The Cahoon lab is currently trying to understand the relevance of FAD2 gene family expansion to the development of the falcarin biosynthetic pathway in carrots and other species. FAD2 expansion via tandem duplications appears to be common across several species of Euasterids. For example, the tomato genome contains tandemly arranged FAD2 genes, with the amino acid sequences of ACET1a and ACET1b (both FAD2s) being identical (Jeon et al., 2020). In carrot, we find a quintuplication of FAD2 genes on Chr5 and a sextuplication of FAD2 genes on Chr4, both representing potential, partial biosynthetic gene clusters (BGCs). The first and last genes of this potential BGC on Chr4, DCAR_013548 and DCAR_013552, differ by only one amino acid at the C-terminus. The key functional regions are completely identical. Interchromasomal instances of ~100% AA identity can be found in multiple instances throughout the carrot genome.These results suggest that the expansion of the FAD2 gene family may have occurred only recently. It also implies that the biosynthesis of falcarins in these species is a relatively recent development. However, whether different species synthesize falcarins due to convergent evolution, or whether their common ancestor already synthesized falcarins and divergences occurred more recently, requires further research. The Kosma lab has completed a spatiotemporal chemical "atlas" of falcarin accumulation spanning 5 developmental stages (10, 30, 45, 60, and 75 days after germination) encompassing a total of 42 tissue types. We have recently identified several novel falcarins in these data sets and are currently revising these data to provide a more comprehensive picture of the diversity of falcarins that accumulate throughout development. Furthermore, we have obtained and are testing probes for TaqMan-based Real Time qPCR in key developmental stages of carrots. The necessity of using probes for profiling the expression of FAD2 genes, given their high sequence similarity, was predicated on the Yim lab's carrot genome reannotation efforts. This, and parallel efforts by the Cahoon lab, hase established baseline falcarin levels for multiple stages of development and tissues types providing a reference for transgenic comparisons.To gain a clearer understanding of the specific FAD2 genes involved in the synthesis of falcarins in carrots, it will be necessary to collect different tissue samples from carrot seedlings, re-assess the falcarin content, and correlate the data with transcriptome information. This is part of the subsequent plan for our project. The Cahoon and Yim labs have further employed a transcriptome mining approach to better understand the developmental dynamics of FAD2 expression in carrots. The Yim lab developed a comprehensive coexpression matrix using both publicly available data (NCBI SRA) and a tissue-specific transcriptome data set that was developed through a multi-lab effort (PI and Co-PIs of this grant). Analysis of this coexpression data set using our identified gene candidates as bait to identify coexpressed genes and better understand the gene regulatory networks of falcarin synthesis is ongoing. Cahoon lab efforts with transcriptome data sets from different tissues and different carrot cultivars indicates that most FAD2 genes reduce their expression during the later stages of carrot development. It remains to be seen if this holds true for the Danvers Half Long variety used in our laboratories; this will be validated by Kosma lab efforts to profile FAD2 expression levels in key developmental stages using TaqMan probe-based RT-qPCR. 2.Develop carrot germplasm that is enhanced or deficient in falcarins using biotechnological methodologies, including CRISPR/Cas9 gene editing, as tools for future studies of falcarin-mediated health promotion, flavor properties, and pathogen resistance To better decipher the falcarin biosynthetic pathway (Obj. 1) and create carrot germplasm enhanced or deficient in falcarins (Obj. 2), the Cahoon lab has been developing transgenic overexpression lines and CRISPR-Cas9 gene-edited lines in carrot. We have expanded our efforts to include potential decarbonylase/decarboxylase genes (CER1 and CER3). CER1 & CER3 constitue a complex thought to synthesize odd chain alkanes from even chain acyl CoAs. Falcarins are C17 compounds derived from C18 acyl chain precursors necessitating some form decarbonylase/decarboxylase activity for their formation. We took advantage of a tissue-specifc RNA-seq data set that we generated, wherein we mapped reads to our newly reannotated carrot genome, to identify candidate CER1 and CER3 genes. We employed recent knowledge that major QTLs for falcarin content can be found on chromosome 9 (Dunemann et al., 2022). Combigning this with our knowledge that falcarins are enriched in the skin or periderm of mature carrot taproots, we identified three CER1 genes and a single CER3 gene as candidates for enzymes encoding decarbonylation/decarboxylation activities. To this end, the Cahoon lab has generated carrot T0 lines overexpressing core FAD2 genes and FAD2-CER1-CER3 combinations to probe their roles in polyacetylene metabolism. Phenotypic screening of T0 plants is ongoing. So far, seeds and seedlings of various transgenic events targeting different gene combinations have been obtained. See tables below. Construct transformed into CarrotGeneration DCAR_013552 (FAD2) T1 DCAR_017011 (FAD2) T1 DCAR_019786 (FAD2) T1 DCAR_013552 (FAD2) + DCAR_19786 (FAD2) T1 DCAR_017011 (FAD2) + DCAR_19786 (FAD2) T1 CER1-1 T0 CER1-2 T0 CER1-3 T0 CER3 T0 CER1-1 + CER3 T1 CER1-2 + CER3 T0 CER1-3 + CER3T1 To explore desaturation reactions required for falcarin synthesis beyond what we have previously characterized, functional testing of candidate FAD2 genes is also ongoing in Arabidopsis. Construct transformed into ArabidopsisGeneration DCAR_017011 (FAD2)T1 DCAR_25697 (FAD2)T1 DCAR_25697 (FAD2)T1 DCAR_25933 (FAD2)T1 DCAR_013552 (FAD2) + DCAR_19786 (FAD2) + DCAR_017010T1 DCAR_013552 (FAD2) + DCAR_19786 (FAD2) + DCAR_025967T1 DCAR_017011 (FAD2) + DCAR_19786 (FAD2) + DCAR_025967T1 DCAR_017011 (FAD2) + DCAR_19786 (FAD2) + DCAR_017010T1 DCAR_017011 (FAD2) + DCAR_19786 (FAD2)T1 DCAR_017010 (FAD2) + DCAR_19786 (FAD2)T1 To generate carrot lines deficient in falcarins, we have targerted three FAD2 genes known to function in the initial steps of falcarin biosynthesis using CRISPR-Cas9 to simulatenously knock out all three of these FAD2 genes in carrot.T1 seeds were obtained; genotyping and homozygous line purification is underway. Citations DunemannFet al.The genetic control of polyacetylenes involved in bitterness of carrots (Daucus carotaL.): Identification of QTLs and candidate genes from the plant fatty acid metabolism.BMC Plant Biol22, 92 (2022). Jeon JEet al.. A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato. Cell. (2020) 180:176-187 3. Investigate the biosynthesis and tissue level distribution of falcarin-type molecules in an agronomically relevant crop from the Asteraceae family, sunflower The reviewers of our grant proposal felt that it was unnecessary to investigate species in addition to carrot. This was indicated in our initiation report. As such, little effort has gone into this aim.
Publications
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Progress 01/15/23 to 01/14/24
Outputs
Target Audience:The target audiences for this project include the general scientific community as well as the local and national agriculturecommunity and plant agriculture and biotechnologyindustry. Undergraduate and graduatestudents are also target audiences for the project. A special emphasis has been placed on recruiting individuals from groups traditionallyunderrepresented in STEM. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? 1 PhD student received training in bioinformatics under this project 1 PhD student has received training in crop biotechnology research, including crop transformation and gene editing 1 MS student received training in metabolite analysis and molecular biology under this project 2 technicians have received training in metabolite analysis and molecular biology under this project How have the results been disseminated to communities of interest? Invited Seminar Presentation Kosma DK.(2023) Specialized Lipid Metabolism: Understanding the Biosynthesis and Transcriptional Regulation of Protective Plant Lipid-Derived Natural Products. Department of Biochemistry. University of Missouri. Columbia, MO. Poster Presentation Kosma DK, Huang F, Trinh B, Chincilla A, LaBrant E, Lee B, So Wonmi, Mayer M, Santos S, Busta L, Yim W, Cahoon E. (2023)Biosynthesis of Falcarin-Type Polyacetylenic Lipids. Gordon Research Conference, Plant Lipids: Structure, Metabolism and Function. Galveston, TX. The updated genome annotation will be published in leading scientific journals relevant to plant genomics and bioinformatics. These publications will provide detailed genome annotation methodologies, results, and interpretations, ensuring the research is accessible to the scientific community. The updated genome annotation data will be uploaded to publicly accessible databases such as Phytozome, Ensembl Plants, and other genomic repositories. This ensures that researchers worldwide can access and utilize the data for their studies. What do you plan to do during the next reporting period to accomplish the goals? We will complete the gene expression component of the spatiotemporal atlas of falcarin accumulation using TaqMan probe-based Real Time qPCR assays Efforts will be focused at advancing and generation of additional stable transgenic carrots deficient in, or replete with, falcarins We will evaluate falcarin concentrations in engineered carrot lines and advance those with the highest and lowest falcarin levels to test for fungal resistance We will focus on generating a co-expression network to validate candidate genes based on their expression patterns and interactions within the system. This will involve collecting gene expression data from carrot tissue types using RNA-Seq and constructing the network using Pearson Correlation Coefficient. We will then perform functional analyses, including Gene Ontology (GO) enrichment and pathway analysis.
Impacts
What was accomplished under these goals?
1.Elucidate the falcarin biosynthetic pathway in carrot as well as the developmental dynamics of falcarin biosynthesis using biochemical and genomics-based approaches The Kosma lab has employed constructs from both the Kosma and Cahoon labs to establish a platform for pathway assembly in the model plant speciesNicotiana benthamiana. Candidate FAD2 genes and CYP450s have been tested for function in falcarin biosynthesis using this system. Whereas we have been able to recapitulate the first three steps of the falcarin biosynthetic pathway, to date, none of the CYP450s tested were identified to participate in falcarin biosynthesis. Transient expression of multi-gene constructs containing FAD2 variants not only resulted in production of dehydrocrepenynic acid, but also led to alterations in fatty acid composition including reductions in the proportions of total fatty acids comprised of palmitoleic and oleic acid (18:1) with concomitant increases in the proportion of total fatty acids comprised of linolenic acid (18:3). During our utilization of the existing carrot genome annotation, several limitations were identified that impacted its effectiveness. Specifically, the 5' and 3' untranslated regions (UTRs) of transcripts and tandemly duplicated genes were inadequately annotated or missing. This inadequacy hindered the precise identification of transcriptome abundance. To address these issues, the Yim lab undertook a comprehensive reannotation of the carrot genome by incorporating new sequencing data from both public repositories and in-house sources. This reannotation process is now complete. As a result, the number of FAD2 and FAD2 variant genes identified in the genome increased from 24 to 42. The Kosma lab utilized this updated annotation to design primers and probes for the gene expression component of the falcarin spatiotemporal atlas. Furthermore, the updated annotation was employed to analyze RNA-seq data from the transcriptomes of five carrot tissue types (leaf, phloem, petiole, periderm, and xylem). This analysis yielded critical insights into the expansion of gene families and identified previously unannotated genes potentially involved in falcarin biosynthesis. Consequently, our list of candidate genes has significantly expanded. The Kosma lab has completed a spatiotemporal "atlas" of falcarin accumulation spanning 5 developmental stages (10, 30, 45, 60, and 75 days after germination) encompassing a total of 42 tissue types. Efforts to profile the expression of falcarin biosynthesis-related genes in key developmental stages using TaqMan probe-based Real Time qPCR have begun. 2.Develop carrot germplasm that is enhanced or deficient in falcarins using biotechnological methodologies, including CRISPR/Cas9 gene editing, as tools for future studies of falcarin-mediated health promotion, flavor properties, and pathogen resistance Efforts were directed at developing stably transformed lines of carrot enhanced or deficient in falcarin production. During this reporting period we successfully established a transformation protocol using the Danver's Half-Long genotype. Transformation was confirmed by genomic PCR, RT-PCR measurement of transgene expression, and production of novel fatty acids from introduced transgenes. Previously, delta12 acetylenases (DCAR_013548 and DCAR_013552) and bifunctional delta12/14 desaturases (DCAR_019786), identified in yeast andArabidopsis thaliana(Busta et al., 2018), were introduced using 35S CaMV and parsley ubiquitin promoter-mediated constitutive expression vectors. Seeds have been collected from six different overexpression constructs. Kosma lab validated elevated dehydrocrepenynic acid levels in leaves of multiple transgenic carrot lines expressing FAD2 gene variants generated by the Cahoon lab. These lines exhibited 62 - 95% increases in dehydrocrepenynic acid content. Fatty acid composition was also altered in these transgenic lines. All lines exhibited reductions in oleic acid (18:1). Select lines exhibited elevated amounts of linolenic acid (18:3) as well. In terms of falcarin accumulation, two lines were shown to contain 68% and 137% higher total falcarins than empty vector controls. These lines contained elevated amounts of falcarintriol-8-acetate that accounted for the overall increase in total falcarins. Two additional lines were shown to possess only slight increases in total falcarin content (5 - 9%) yet contained 47 - 112% more falcarindiol than empty vector controls. Collectively, our team was able to generate transgenic carrot plants with elevated falcarin contents and altered falcarin compositions. To develop carrot germplasm deficient in falcarins, we employed a CRISPR/Cas9 construct with guide RNAs targeting ?12 acetylenase (DCAR_013548, DCAR_013552, and DCAR_017011). Seeds have been collected from six independent transgenic events, and 11 lines are currently growing in the greenhouse. From 90 seeds planted from one gene-edited line, 27 germinated, resulting in a germination rate of 30%. TheCER1andCER3gene families were predicted to be involved in the falcarin biosynthesis pathway. Thus,CER3, together with three differentCER1paralogous genes, mediated by the 35S CaMV promoter, were introduced into Danver's Half-Long. We have generated eight independent events for the combination ofCER3andCER1-2, and 14 independent events for another combination ofCER3andCER1-2. As for the other combination vectors, includingCER1-1,CER1-2,CER1-3,CER3, and the combination ofCER1-1withCER3, they have been induced and are being selected and cultured in MS medium containing kanamycin. 3. Investigate the biosynthesis and tissue level distribution of falcarin-type molecules in an agronomically relevant crop from the Asteraceae family, sunflower The reviewers of our grant proposal felt that it was unnecessary to investigate species in addition to carrot. This was indicated in our initiation report. As such, little effort has gone into this aim.
Publications
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience:The target audiences for this project include the general scientific community as well as the local and national agriculturecommunity and plant agriculture and biotechnologyindustry. Undergraduate and graduatestudents are also target audiences for the project. A special emphasis has been placed on recruiting individuals from groups traditionallyunderrepresented in STEM. Changes/Problems: During our utilization of the existing iteration of the carrot genome annotation, we identified certain limitations that affected its usefulness. Specifically, the 5' and 3' untranslated regions (UTRs) of transcripts and tandemly duplicated genes were not adequately annotated and were absent from the currently available genome annotation. To address this issue, Co-PI Yim's laboratory has undertaken the task of reannotating the carrot genome by incorporating new sequencing data from both public sources and in-house generated data. The reannotation process is nearing completion, and the findings will be published in an upcoming publication. What opportunities for training and professional development has the project provided? 3 postdoctoral scholars have received training in biochemistry, molecular biology, and tissue culture under this project 2 PhD students have received training in bioinformatics under this project. 1 PhD student has received training in crop biotechnology research, including crop transformation and gene editing. 2 MS students have received training biochemistry, molecular biology, and tissue culture under this project 2 technicians have received training in in biochemistry, molecular biology, and tissue culture under this project How have the results been disseminated to communities of interest? Scott S, Cahoon EB, Busta L. (2022) Variation on a theme: the structures and biosynthesis of specialized fatty acid natural products in plants. The Plant Journal. 111:954-65.https://doi.org/10.1111/tpj.15878 Santos P, Busta L, Yim WC, Cahoon EB, Kosma DK (2022). Structural Diversity, Biosynthesis, and Function of Plant Falcarin-type Polyacetylenic Lipids. Journal of Experimental Botany. Volume 73: 2889-2904,https://doi.org/10.1093/jxb/erac006 Presentation at the 2023 Plant Lipid Structure and Function Gordon Research Conference. What do you plan to do during the next reporting period to accomplish the goals? We will continue to identify and characterize enzymes from the falcarin biosynthetic pathway using our transient, heterologous expression system in Nicotiana benthamiana. We will complete the spatiotemporal atlas of falcarin accumulation and pursue identification of falcarin precursors (e.g. crepenynic acid) as well as measure the transcript abundance of known falcarin-related biosynthesis genes at key developmental stages. Efforts will be focused at advancing and generation of additional stable transgenic carrots deficient in, or replete with, falcarins We will evaluate falcarin concentrations in engineered carrot lines and advance those with the highest and lowest falcarin levels to test for fungal resistance. Hairy root transformation efforts will continue. Our attempts with hypocotyl-based transformation have not proven successful. We will now employ a carrot taproot disk-based method. We will focus on functional annotation for the reannotated carrot genome and generating a co-expression network to validate candidate genes based on their expression patterns and interactions within the system.
Impacts
What was accomplished under these goals?
1.Elucidate the falcarin biosynthetic pathway in carrot as well as the developmental dynamics of falcarin biosynthesis using biochemical and genomics-based approaches The Kosma lab has employed constructs from the Cahoon lab to establish a platform for pathway assembly in the model plant speciesNicotiana benthamiana. To date, both Kosma and Cahoon labs have been able to recapitulate the first three steps of the falcarin biosynthetic pathway using this system. Both groups are well poised to assemble the pathway with this heterologous expression system in a stepwise manner. All candidate genes for these experiments have been cloned or synthesized by Kosma and Cahoon. The Kosma lab has nearly completed a spatiotemporal "atlas", spanning multiple developmental stages and tissue types, of falcarin content and composition. All extractions are complete and ~85% of data have been analyzed. Select tissues were tested for the presence of the falcarin precursors crepenynic and dehydrocrepenynic acids which were identified in some, but not all, tissues tested. The Kosma lab is pursuing the characterization of these same tissues and developmental stages for crepenynic and dehydrocrepenynic acid content which employs a different form of extraction than falcarins. Kosma's group will extend this work to profile the expression of falcarin biosynthesis-related genes in key developmental stages. Cahoon and collaborator Patricia Santos are developing a publication on transient expression systems for functional analysis of falcarin biosynthesis genes, and other genes, in carrot and celery. The Yim laboratory is presently immersed in a scientific pursuit aimed at augmenting the currently available carrot genome through the identification of the gene cluster accountable for the falcarin biosynthetic pathway. This undertaking entails the reannotation of the genome and the production of a scholarly publication elucidating the discoveries made. Furthermore, the Yim lab is leveraging publicly accessible RNA-Seq data to carry out co-expression network analysis, with the objective of exploring and assembling the pathway using falcarin biosynthetic pathway candidate genes. A comprehensive assessment of 14 studies, comprising 158 paired-end Illumina RNA-Seq datasets, will be conducted alongside the reannotated genome. 2.Develop carrot germplasm that is enhanced or deficient in falcarins using biotechnological methodologies, including CRISPR/Cas9 gene editing, as tools for future studies of falcarin-mediated health promotion, flavor properties, and pathogen resistance Efforts were directed at developing stably transformed lines of carrot for enhanced or deficient in falcarin production. During this reporting period we successfully established a transformation protocol using the Danver's Half-Long genotype. Transformation was confirmed by genomic PCR, RT-PCR measurement of transgene expression, and production of novel fatty acids from introduced transgenes. A total of 10 overexpression transgenic constructs were introduced, and 91 total independent events have been generated. These include 35S CaMV- and parsley ubiquitin promoter-mediated constitutive expression of combinations of?12 acetylenases and bifunctional?12/14 desaturases. These are intended to increase flux of linoleic acid into the falcaring pathway. For developing carrot germplasm deficient in falcarins, a construct with CRISPR/Cas9 guide RNAs targeting?12 acetylenase and bifunctional?12/14 desaturase transgenes. We currently have 19 lines that we are now regenerating in tissue culture. 3. Investigate the biosynthesis and tissue level distribution of falcarin-type molecules in an agronomically relevant crop from the Asteraceae family, sunflower The reviewers of our grant proposal felt that it was unnecessary to investigate species in addition to carrot. This was indicated in our initiation report. As such, little effort has gone into this aim.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Santos P, Busta L, Yim WC, Cahoon EB, Kosma DK (2022). Structural Diversity, Biosynthesis, and Function of Plant Falcarin-type Polyacetylenic Lipids. Journal of Experimental Botany. Volume 73: 28892904, https://doi.org/10.1093/jxb/erac006
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Scott S, Cahoon EB, Busta L. (2022) Variation on a theme: the structures and biosynthesis of specialized fatty acid natural products in plants. The Plant Journal. 111:954-65. https://doi.org/10.1111/tpj.15878
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Kosma DK, Huang F, Trinh B, Chincilla A, LaBrant E, Lee B, So Wonmi, Mayer M, Santos S, Busta L, Yim W, Cahoon E. Biosynthesis of Falcarin-Type Polyacetylenic Lipids. Gordon Research Conference, Plant Lipids: Structure, Metabolism and Function. Galveston, TX. (2023)
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:The target audiences for this project include the general scientific community as well as the local and national agriculturecommunity and plant agriculture and biotechnologyindustry. Undergraduate and graduatestudents are also target audiences for the project. A special emphasis has been placed on recruiting individuals from groups traditionallyunderrepresented in STEM. Changes/Problems:There was a slight delay in hiring scholars to work on this project related with restrictions imposed on travel and lab occupancy that resulted from the pandemic. As such, our initial progress has not proceeded as rapidly as we'd hoped. We are now fully staffed and do not anticipate any further delays. What opportunities for training and professional development has the project provided? 2 postdoctoral scholars have received training in biochemistry, molecular biology, and tissue culture under this project 2 PhD students have received training in bioinformatics under this project 1 technician has received training in in biochemistry, molecular biology, and tissue culture under this project These individuals are self-identified members of groups traditionally underrepresented in STEM Individual development plans (IDPs) are currently being developed with these individuals How have the results been disseminated to communities of interest?1 refereed publication was produced from this work so far. Santos P, Busta L, Yim WC, Cahoon EB, Kosma DK (2022). Structural Diversity, Biosynthesis, and Function of Plant Falcarin-type Polyacetylenic Lipids. Journal of Experimental Botany. Volume 73: 2889-2904, https://doi.org/10.1093/jxb/erac006 What do you plan to do during the next reporting period to accomplish the goals? We will continue to identify and characterize enzymes from the falcarin biosynthetic pathway Efforts will be focused on obtaining stable transgenic carrots deficient in, or replete with, falcarins Hairy root transformation efforts will continue Our newly developed transient expression approaches will continue to be pursued for full assembly of the falcarin pathway An emphasis will be placed on characterizing CYP450s involved hydroxylation of falcarin backbones
Impacts
What was accomplished under these goals?
1.Elucidate the falcarin biosynthetic pathway in carrot as well as the developmental dynamics of falcarin biosynthesis using biochemical and genomics-based approaches Sequences for 95% of candidate genes have been cloned or synthesized. These candidate genes have been subcloned into varioussingle-gene expression vectors for either stable or transient expression. Several multi-gene constructs have been assembled by the Cahoon lab.The Kosma lab is developing a new Gateway-compatible vector with a fluorescent marker protein for hairy-root transformation. The Cahoon lab has developed a transient expression system for functional analysis of candidate falcarin biosynthesis genes in carrot that will be employed for functional characterization of candidate FAD2 and FAD2 variant genes. Furthermore, using seed-specific expression, Cahoon has two more FAD2 variants that appear participate in falcarin biosynthesis. The Kosma lab has made progress on understanding the tissue-level distribution of falcarins in carrot.Tissues from acomplete developmental series of carrot plants spanning multiple timepoints and multiple tissues have been harvested and archived.Tissues are currently being extracted for falcarins and data are beinganalyzed. The Yim lab developed a co-expression network utilizing in-house generated and publicly available RNA-seq data that has permitted the identification of additional falcarin biosynthesiscandidate genes. 2.Develop carrot germplasm that is enhanced or deficient in falcarins using biotechnological methodologies, including CRISPR/Cas9 gene editing, as tools for future studies of falcarin-mediated health promotion, flavor properties, and pathogen resistance The Cahoon lab has developed constructs that employ a Ubiquitin gene promoter from Parsley (PcUbi4-2) and has generated what appear to be stably-transformed callus tissues. Transformation efforts using constructs for the overexpression of candidate acetylenases and desaturases are underway. Initial attempts by the Kosma lab to generate transgenic hairy roots have been unsuccessful. Bothin vitroandex vitromethods are being investigated and refined. 3. Investigate the biosynthesis and tissue level distribution of falcarin-type molecules in an agronomically relevant crop from the Asteraceae family, sunflower The reviewers of our grant proposal felt that it was unnecessary to investigate species in addition to carrot. This was indicated in our initiation report. As such, little effort has gone into this aim.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Santos P, Busta L, Yim WC, Cahoon EB, Kosma DK (2022). Structural Diversity, Biosynthesis, and Function of Plant Falcarin-type Polyacetylenic Lipids. Journal of Experimental Botany. Volume 73: 28892904, https://doi.org/10.1093/jxb/erac006
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