Research Project: Genetics, Epigenetics, Genomics, and Biotechnology for Fruit and Vegetable Quality

Location: Plant, Soil and Nutrition Research

2022 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
Exploring the potential of tomato wild species genetic diversity: The project involves assessment of tomato genetic diversity toward understanding fruit development, ripening and nutritional quality while simultaneously using resulting materials for biotechnology risk assessment with an emphasis on emerging gene editing approaches. In the current reporting period, we have completed 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. Though mapping of RNA-seq reads we have also defined to gene level resolution 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 (estimated to be 5% of natural genetic diversity). During this year a de novo genome sequence of Solanum lycopersicoides, which is adapted to extreme cold and drought in the Andes mountain range, was completed and published. The genome sequence was a collaboration with researchers at the Boyce Thompson Institute and in Belgium. This resource provides insights into tomato evolution and molecular tools for discovery and tomato improvement. We have further optimized our informatics pipeline allowing in silico creation of reference genomes for each member of a population based on parental SNP data which minimizes misleading transcriptome data resulting from use of a single parent reference genome. Understanding control of carotenoid accumulation in fruit and vegetable crops: 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 or interactions and some reside in close proximity to carotenoid quatitative trail locus (QTL)s. Efforts to functionally test these genes in transgenic plants have bene initiated. Tomato QTLs contributing to carotenoid accumulation have also been mapped and those residing at pathway gene loci defined. Efforts to fine map non-pathway loci are underway as these may be important regulators or as yet undefined carotenoid metabolic enzymes. Finally, efforts to understand cell wall metabolism changes associated with ripening resulting in an unexpected carotenoid induction phenotype when a cell wall-associated transcription factor was repressed by RNAi or edited to a non-functional allele. Efforts are underway to clarify whether carotenoid accumulation is responsive to this otherwise cell wall-associated gene or whether carotenoid accumulation is influenced by cell wall derived signaling molecules. Regulation of fruit ripening and nutrient quality: Efforts to characterize transcription factors regulating tomato fruit ripening are ongoing and continue to result in characterization of additional regulatory genes. In the current year, we continued to characterize, the Solanum lycopersicum LATERAL ORGANS BOUNDRY 1 (SlLOB1) gene, which is primarily responsible for influencing cell wall remodeling during fruit maturation as it regulates 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. Efforts have been initiated to edit SlLOB1 in high quality heirloom and breeding lines that suffer from poor texture and associated shelf-life traits as a proof-of-concept for the ability to deploy gene editing for addition of target traits to improve otherwise elite germplasm. 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. Protocols are being modified for analysis of folate and other metabolites. In the last year segregating populations resulting from crosses with ILs showing elevated fruit folate were used to fine-map folate loci. Folate is a necessary nutrient synthesized in plants and acquired though diet. It is a difficult nutritional metabolite to measure as it can be unstable in certain forms or absent interaction with folate binding proteins. Using AlphaFold2 efforts have been initiated to model putative folate binding proteins and their interactions with folic acid and its glutamated 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. 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. In the last reporting period, 12 tomato genomes of gene edited and non-edited control lines were created. Assessment of off-targeting and degree of variation is underway. Genetic modifications in addition to tissue culture and regeneration steps involved in gene editing can elicit stress responses which in term can have genomic consequences. The same genomes have been subject to bisulfite genome sequencing and small RNA analyses to assess genomic effects beyond DNA sequence modification that may result from gene editing. Bilsufite genome libraries have been synthesized and are currently being sequenced. 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. Furthermore, we have joined a consortium of 1890 Universities including TSU, Virginia State University (VSU) and Delaware State University (DSU) to develop a proposal to the NIFA CBP entitled “Extending the self-life of strawberries by focusing on fruit surfaces exposed to essential oils” (log #: 0071904), which will be submitted soon. We are excited about expanding our outreach efforts to include two additional 1890 Universities and helping to fulfill our obligation to support the 1890 Universities and the under-represented minority groups they serve.

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 carotenoid 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 analysis of tomato fruit samples. They have applied it to quantify carotenoids in tomato tissues from unripe to ripe using samples of less than 10 nano liters (nL) of cells. This workflow is being adapted for the analysis of diverse metabolites, cell types, and crops.

2. Identification of a gene regulating fruit and vegetable nutrient quality and heat tolerance. Fruits and vegetables are important sources of carotenoids – pigments that give plants color, attract pollinators and assist with photosynthesis and protection against high light intensities. Carotenoids are important sources of antioxidants and vitamin A in the human diet. ARS researchers in Ithaca, New York, demonstrated that the OR gene, which is expressed in many carotenoid producing tissues, is also a master regulator of chlorophyll biosynthesis and regulator of how many chloroplasts (site of photosynthesis) are produced. This discovery revealed a conserved mechanism in green plants tissues coordinating chlorophyll and carotenoid biosynthesis for photosynthesis and nutritional quality. Moreover, this work showed that enhanced OR gene activity can safeguard photosynthetic pigment synthesis and enhance plant temperature tolerance, making the OR gene a target for developing climate-resilient crops with enhanced crop nutritional value.

3. Genome sequence of a wild species that can contribute to tomato improvement. Solanum lycopersicum 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. ARS researchers in Ithaca, New York, partnered with scientists at the Boyce Thompson Institute and in Belgium to sequence the genome of Solanum lycopersicum. 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.

Review Publications
Yano, R., Ariizumi, T., Nonaka, S., Kawazu, Y., Zhong, S., Mueller, L., Giovannoni, J.J., Rose, J., Ezura, H. 2020. Comparative genomics of muskmelon reveals a potential role for retrotransposons in the modification of gene expression. Communications Biology. 3:432. https://doi.org/10.1038/s42003-020-01172-0.
Shi, Y., Vrebalov, J., Zheng, H., Xu, Y., Yin, X., Liu, W., Liu, Z., Sorensen, I., Su, G., Ma, Q., Evanich, D., Rose, J., Fei, Z., Van Eck, J., Thannhauser, T.W., Chen, K., Giovannoni, J.J. 2021. A tomato LATERAL ORGAN BOUNDARIES transcription factor, SlLOB1, predominantly regulates cell wall and softening components of ripening. Proceedings of the National Academy of Sciences (PNAS). 118(33):e2102486118. https://doi.org/10.1073/pnas.2102486118.
Yuan, H., Pawlowski, E., Yang, Y., Sun, T., Thannhauser, T.W., Mazourek, M., Schnell, D., Li, L. 2020. Arabidopsis OR protein regulates plastid preprotein import through interacting with Tic proteins. Journal of Experimental Botany. 2(4):1059-1072. https://doi.org/10.1093/jxb/eraa528.
Sun, T., Zhu, Q., Wei, Z., Owens, L.A., Fish, T., Kim, H., Thannhauser, T.W., Cahoon, E.B., Li, L. 2021. Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds. aBIOTECH. https://doi.org/10.1007/s42994-021-00046-1.
Casajus, V., Civello, P., Martinez, G., Howe, K.J., Fish, T., Yang, Y., Thannhauser, T.W., Li, L., Lobato, M.G. 2021. Effect of continuous white light illumination on glucosinolate metabolism during postharvest storage of broccoli. LWT - Food Science and Technology. 145:111302. https://doi.org/10.1016/j.lwt.2021.111302.
Mclean, P.E., Lee, R., Howe, K.J., Osborne, C., Grimwood, J., Levy, S., Haugrud, A.P., Plott, C., Robinson, M., Skiba, R.M., Tanha, T., Zamani, M., Thannhauser, T.W., Glahn, R.P., Schmutz, J., Osorno, J., Miklas, P.N. 2022. The common bean V gene encodes flavonoid 3'5' hydroxylase: A major mutational target for flavonoid diversity in angiosperms. Frontiers in Plant Science. 13:869582. https://doi.org/10.3389/fpls.2022.869582.
Chayut, N., Yuan, H., Saar, Y., Zheng, Y., Sun, T., Zhou, X., Hermanns, A., Oren, E., Faigenboim, A., Hui, M., Fei, Z., Mazourek, M., Burger, J., Tadmor, Y., Li, L. 2021. Comparative transcriptome analyses shed light on carotenoid production and plastid development in melon fruit. Horticulture Research. 8:112. https://doi.org/10.1038/s41438-021-00547-6.
Hermanns, A., Zhou, X., Xu, Q., Tadmor, Y., Li, L. 2020. Carotenoid pigment accumulation in horticultural plants. Horticultural Plant Journal. 6(6):343-360. https://doi.org/10.1016/j.hpj.2020.10.002.
Zhou, X., Rao, S., Wrightstone, E., Lui, A., Welsch, R., Li, L. 2022. Phytoene synthase: the key rate-limiting enzyme of carotenoid biosynthesis in plants. Frontiers in Plant Science. 13:884720. https://doi.org/10.3389/fpls.2022.884720.
Ramsey, J.S., Fish, T., Thannhauser, T.W., Giovannoni, J.J. 2022. Laser capture of tomato pericarp tissues for microscale carotenoid analysis by supercritical fluid chromatography. Methods in Enzymology. 670:213-233. https://doi.org/10.1016/bs.mie.2022.01.014.
Zhu, F., Jadhav, S., Toghe, T., Salem, M., Li, J., Giovannoni, J.J., Cheng, Y., Alseekh, S., Fernie, A. 2022. Comparative transcriptomics and eQTL approach identified SlWD40 as a novel tomato fruit ripening regulator. Plant Physiology. https://doi.org/10.1093/plphys/kiac200.
Powell, A., Feder, A., Li, J., Schmidt, M., Courtney, L., Alseekh, S., Jobson, E., Vogel, A., Xu, Y., Lyon, D., Dumschott, K., Mchale, M., Suplice, R., Bao, K., Lal, R., Dunhan, A., Halab, A., Denton, A., Bolger, M., Fernie, A., Hind, S., Mueller, L., Martin, G., Fei, Z., Martin, C., Giovannoni, J.J., Stickler, S., Usedal, B. 2022. A solanum lycopersicoides reference genome facilitates insights into tomato specialized metabolism and immunity. The Plant Journal. 110(6):1791-1810. https://doi.org/10.1111/tpj.15770.