Location: Plant, Soil and Nutrition Research
2023 Annual Report
Objectives
Objective 1: Identify loci and functionally characterize underlying genes that contribute to fruit and vegetable shelf-life, appearance, flavor, texture, and nutritional quality by characterizing cultivated and wild species diversity so as to develop a better understanding of corresponding trait biology and to develop new molecular tools for breeding. (See uploaded postplan for sub-objectives)
Objective 2: Generate genome-scale DNA sequence data, gene expression profiles, proteomic and metabolite data of fruit and vegetable crops for facilitating trait discovery and trait improvement. (See uploaded postplan for sub-objectives)
Objective 3: Develop and test models for the regulation of fruit and vegetable development and quality traits at the genome level that incorporate epigenome dynamics and epigenetic regulators. (See uploaded postplan for sub-objectives)
Objective 4: Develop and utilize new advanced analytical approaches to characterize fruit and vegetable proteins and chemical metabolites, their modifications and interactions, via targeted and genome-scale methodologies. (See uploaded postplan for sub-objectives)
Objective 5: Develop, test, and thoroughly analyze at the whole genome level gene editing technologies in tomato for use in enhancing nutrient levels and shelf-life, and in selected high value crops for use in breeding and research. (See uploaded postplan for sub-objectives)
Approach
The overall approach of this project will be use of molecular, genetic and genomics approaches to address our objectives centered on advancing our understanding of fruit and vegetable quality and deploying said knowledge toward crop improvement. We will take advantage of existing germplasm in the form of mutant/variant lines and segregating populations and/or wild species introgression lines to identify genes underlying fruit and vegetable quality and nutritional content. Candidate genes will be isolated, sequenced, and characterized for gene expression attributes in addition to allelic variation that will be correlated with trait and/or metabolic outputs. Functional analyses will be undertaking for candidate quality and nutrition impacting genes through identification and development, respectively, of chemical/natural or transgenic mutations. In some instances, we will test potential for translation of insights from model and crop systems studies to additional crop and stable crop species. Better understanding of processes underlying fruit and vegetable quality will facilitate design of molecular strategies to improve crop quality attributes in both primary experimental crop systems and targets of translational biology. Through these undertakings, we will develop transgenic and gene edited lines to address gene function. We will further utilize said lines and additional lines developed as controls to assess the nature and degree of genome changes resulting from transformation or gene editing and the extent of possible biotechnological risk, if any.
Progress Report
Over the course of the project, we have explored tomato genetic diversity as it relates to ripening, regulation of carotenoid accumulation via Or and interacting genes, developed streamlined procedures for efficient extraction and characterization of secondary metabolites, explored the role of the epigenome in fruit development and assess genome effects of gene-editing. Key activities included: 1) eQTL mapping studies in immortalized introgression line (IL) mapping populations derived from crosses between the cultivated tomato (S. lycopersiocum) and interfertile wild species S. pennellii, S. habrochaites and S. lycopersicoides. Through mapping of RNA-seq reads we have also defined the gene level resolution of all introgressions in each population and identified numerous “ghost” introgressions – small introgressions that eluded prior low resolution mapping efforts.
In the last 20 years, these populations have been used by breeders and seed companies to bring new genetic variation to the narrow germplasm of cultivated tomato; 2) We developed a de novo genome sequence of Solanum lycopersicoides, which is adapted to extreme cold and drought in the Andes mountain range. This resource provides insights into tomato evolution and molecular tools for discovery and tomato improvement; 3) Several genes have been identified in melon that physically interact with the OR gene. OR plays an important role in regulating carotenoid accumulation as a chaperone that brings pathway enzymes into physical proximity with each other and has a role in plastid numbers and chromoplast development. Said genes additionally have expression patterns suggestive of interactions and some reside in close proximity to carotenoid quantitative trail locus (QTL)s. Tomato QTLs contributing to carotenoid accumulation have also been mapped and those residing at pathway gene loci defined including an unanticipated lipid metabolism enzyme which surprisingly influences carotenoid volatile accumulation and flavor; 4) Efforts to characterize transcription factors regulating tomato fruit ripening resulted in characterization of additional regulatory genes. The Solanum lycopersicum LATERAL ORGANS BOUNDRY 1 (SlLOB1) gene was of particular interest as it is primarily responsible for influencing cell wall remodeling during fruit maturation as it specifically regulates many genes involved in cell wall synthesis, breakdown and associated textural features. This gene is unique in ripening control as most regulators described to date influence numerous ripening phenomena while SlLOB1 is highly specific to textural changes and will provide an opportunity to manipulate this important storage and consumer trait absent effects on color, flavor and ripening time; 5) Protocols for carotenoid analysis have been substantially modified to allow analysis of samples derived from laser capture microdissection (LCM). LCM has been used to localize transcriptional activity in developing fruit and these analysis improvements allow correlation of nutritional metabolites with high resolution transcription data. Progress on protocols for analysis of folate and flavonoids also advanced substantially; 6) Using AI and specifically AlphaFold2, efforts have been undertaken to model putative folate binding proteins and their interactions with folic acid and its glutamate and polyglutamated forms as a prelude to modifications that will be tested in bioavailability studies necessary to target genetic modifications supporting human health and nutrition in the next project; 7) Biotechnology risk assessment - Enormous opportunities exist for the modification of genes and creation of useful genetic diversity via gene editing. Consumers understandably have concerns with any new technology. 12 tomato genomes of gene edited and non-edited control lines were created and sequenced for analysis of off-targeting and changes in epigenome state and phenotypic changes (especially metabolites); 8) Outreach and diversity: We have continued our long-standing collaboration with Tennessee State University (TSU), one of the 1890 Universities by submitting a collaborative grant proposal under the auspices of the NIFA Capacity Building Program (BP) entitled “Strengthen Plant Biotech Program by Integrating Genome Editing and Artificial Intelligence Technologies in Tomato Projects.” This proposal was funded (Grant #: 2022-38821-3739) and will bring funding to the TSU Biotech Program.
Accomplishments
1. Improved measurements of dietary nutrients from crops. Tomato is the world's most valuable fruit crop and is an important source of health-promoting dietary nutrients, including antioxidants, vitamin C and carotenoids which our bodies convert to vitamin A. Tomato tissues are complex and include an array of differing cell types that do not ripen uniformly. Thus, our ability to analyze and understand the synthesis, metabolism, and accumulation of these plant derived nutrients requires improved methods for cell- and tissue-specific analysis. ARS scientists in Ithaca, New York, have developed a new experimental protocol to perform cell-type-specific carotenoid, flavonoid and folate analysis of laser captured samples defining limited tissues and cell types. They have also advanced extraction protocols that minimize use of organic solvents reducing hazards to staff and the environment.
2. Identification of genes influencing crop yield. Yield is among the most important traits in crop breeding as it is essential to sustainably meeting the food security needs of a growing population with limited land and resource inputs. Efforts to study important traits including crop nutrient content and response to growth regulators used in crop management and production often include yield assessments beyond specific analysis of the targeted traits of interest to ensure that any proposed enhancement of these traits do not adversely impact yield. ARS researchers in Ithaca, New York, working with collaborators at Boyce Thompson Institute (BTI) and the Federal University of Vicosa, Brazil, demonstrated that two genes involved in carotenoid accumulation and ethylene response had significant effects on tomato yield. In both cases, specific amino acid modifications are associated with the positive yield increase suggesting that altered rather than loss-of-function in these genes is responsible for this important change in yield. While tomato is an important crop world-wide, as both genes are conserved across diverse plant and crop species, it is likely that positive yield effects can be attained in crops beyond tomato though modification of these genes.
3. Genome sequence of a wild species that can contribute to tomato improvement. Solanum lycopersicoides is a wild relative that can be crossed to tomato and has many desirable characteristics. Its natural home ranges from sea level to high mountains in South America and as such is adapted to heat, cold and drought environments. This species was used by tomato breeders to develop a wild-species introgression population where each line in the 100 accession population carries approximately 2 – 5% of the wild species genome enabling use in breeding where desirable wild species alleles can be brought into elite tomato germplasm absent large amounts of non-desirable wild species DNA. Unfortunately, the introgressions were originally defined using restriction fragment length polymorphism (RFLP) technology which provides very low resolution insight into what wild species DNA segments are in a particular introgression. ARS researchers in Ithaca, New York, partnered with scientists at the Boyce Thompson Institute and in Belgium to sequence the genome of Solanum lycopersicoides. Availability of a complete genome sequence will allow breeders to use this species in crosses with tomato to transfer desirable traits (e.g. lower water needs, tolerance to heat and cold, improved storability) more quickly due to their ability to monitor and select for desired genome sequences directly from S. lycopercoides or from the introgression lines which are now mapped at high resolution due to this genome sequence.
Review Publications
Oliveira, N., Namorato, F., Rao, S., Cardoso, A., Renendeluiz, P., Guiherme, L., Liu, J., Li, L. 2023. Iron counteracts zinc-induced toxicity in soybeans. Plant Physiology and Biochemistry. 194:335-344. https://doi.org/10.1016/j.plaphy.2022.11.024.
Garcia, J., Gannett, M., Wei, L., Cheng, L., Hu, S., Sparks, J., Giovannoni, J.J., Kao-Kniffin, J. 2022. Selection pressure on the rhizosphere microbiome can alter nitrogen use efficiency in seed yield in Brassica rapa. Communications Biology. https://doi.org/10.1038/s42003-022-03860-5.
Nicolas, P., Shinozaki, Y., Powell, A., Philippe, G., Snyder, S., Gao, K., Zheng, Y., Xu, Y., Courtney, L., Vrebalov, J., Casteel, C., Mueller, L., Fei, Z., Giovannoni, J.J., Rose, J., Catala, C. 2022. Spatiotemporal dynamics of the tomato fruit transcriptome under prolonged water stress. Plant Physiology. https://doi.org/10.1093/plphys/kiac445.
Welsch, R., Li, L. 2022. Golden rice—lessons learned for inspiring future metabolic engineering strategies and synthetic biology solutions. In: Wurtzel, E., editor. Methods in Enzymology. Frontiers in Plant Science. Amsterdam, Netherlands: Elsevier, Inc. (671):1-29. https://doi.org/10.1016/bs.mie.2022.03.014.
Sun, T., Rao, S., Zhou, X., Li, L. 2022. Plant carotenoids: recent advances and future perspectives. Molecular Horticulture. https://doi.org/10.1186/s43897-022-00023-2.
Sun, T., Zhou, X., Rao, S., Liu, J., Li, L. 2022. Protein–protein interaction techniques to investigate post-translational regulation of carotenogenesis. In: Wurtzel, E., editor. Methods in Enzymology. Amsterdam, Netherlands: Elsevier, Inc. 167:301-325. https://doi.org/10.1016/bs.mie.2022.02.001.
Cardoso, A., Gomes, F., Antonio, J., Guilherme, L., Liu, J., Li, L., Silva, M. 2022. Sulfate availability and soil selenate adsorption alleviate selenium toxicity in rice plants. Environmental and Experimental Botany. 201: Article e104971. https://doi.org/10.1016/j.envexpbot.2022.104971.
Cardoso, A., Gomes, F., Antonio, J., Guilherme, L., Liu, J., Li, L., Silva, M. 2022. Phytoene synthase: The key rate-limiting enzyme of carotenoid biosynthesis in plants. Environmental and Experimental Botany. 201:e104971. https://doi.org/10.1016/j.envexpbot.2022.104971.
Jaramillo, A., Sierra, S., Chavarriaga-Aguirre, P., Castillo, D., Gkanogiannis, A., Lopez-Lavalle, L., Acriniegas, J., Sun, T., Li, L., Welsch, R., Boy, E., Alvarez, D. 2022. Characterization of cassava orange proteins and their capability to increase provitamin A carotenoids accumulation. PLoS ONE. 17(1):Article e0262412. https://doi.org/10.1371/journal.pone.0262412.
He, J., Xu, Y., Huang, D., Fu, J., Liu, Z., Wang, L., Zhang, Y., Xu, R., Li, L., Deng, X., Xu, Q. 2022. Triptychon-like regulates aspects of both fruit flavor and color in citrus. Journal of Experimental Botany. 73(11):3610-3624. https://doi.org/10.1093/jxb/erac069.
Ramsey, J.S., Ammar, D., Mahoney, J.E., Rivera, K., Johnson, R., Igwe, D.O., Thannhauser, T.W., Maccoss, M.J., Hall, D.G., Heck, M.L. 2022. Host plant adaptation drives changes in Diaphorina citri proteome regulation, proteoform expression and transmission of Candidatus Liberibacter asiaticus, the citrus greening pathogen. Phytopathology. 112:101-115. https://doi.org/10.1094/phyto-06-21-0275-r.
