Location: Plant, Soil and Nutrition Research
2024 Annual Report
Objectives
Objective 1. Develop new knowledge of the genes and regulatory interactions influencing the ripening and nutritional pathways of fruits and vegetables for consumers.
Sub-objective 1A: Identify novel genes regulating fruit ripening, nutrition, and shelf-life traits using tissue-focused characterization.
Sub-objective 1A.1: Phenotyping, transcriptional profiling, and sequencing of a tomato diversity core collection as a resource for exploring questions of fruit biology.
Sub-objective 1A.2: Identify gene structural variations associated with gene expression.
Sub-objective 1A.3: Functionally validate putative regulatory SVs via gene editing.
Sub-objective 1B: Define the role of transcriptional regulators and their homologs in the control of ripening, nutrition, shelf-life, and quality traits.
Sub-objective 1B.1: Genetic analysis of transcription factor homolog function.
Sub-objective 1B.2: Development of a tomato predicted protein structure database and analysis of protein interaction potential.
Sub-objective 1C: Isolation of the genes that control carotenoid accumulation in fruit and vegetables
Sub-objective 1D: Regulatory mechanisms underlying carotenoid and other nutrient accumulation
Objective 2. Optimize techniques for the extraction and quantitation of nutritional metabolites from micro-scale crop tissue samples.
Objective 3. Characterize at the whole genome level off-targeting of genome editing and methylome editing technologies.
Sub-objective 3.1: Assessment of off-targeting in gene-edited tomato lines.
Sub-objective 3.2: Development of targeted methylation edited tomato lines.
Sub-objective 3.3: Assessment of proteome changes in gene-edited tomato lines.
Approach
Molecular, genetic, and genomics strategies will be deployed to advance understanding of the molecular basis of fruit and vegetable quality, nutrition, and storability. Resulting genes, genetic diversity and metabolic insights related to these traits will be applied toward crop improvement. We will take advantage of existing germplasm in the form of mutant/variant lines, diversity panels, recombinant inbred and wild species introgression lines to identify genes underlying fruit and vegetable quality, storability 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 of genes will be undertaken for candidate quality, storage and nutrition impacting genes through gene editing and via correlation with trait variation resulting from natural genetic diversity. We will explore potential for translation of insights from model crop systems to additional crop species. Better understanding of processes underlying fruit and vegetable quality will facilitate design of targeted molecular strategies to improve crop quality attributes in both primary experimental crop systems and targets of translational biology. In developing gene edited and transgenic lines we will also create a resource to explore at the genome, epigenome and metabolome levels biotechnology risk, with an emphasis on gene-editing approaches that are of wide experimental, commercial and consumer interest.
Progress Report
Objective 1. During this first year of the project, we have secured seed for our diversity collection and expanded it from our original target of 140 accessions to 190, mainly through the inclusion of additional wild species accessions particularly from the closest wild relatives of the cultivated tomato – S. pimpinellifolium, S. cheesmaniae, S. galapagensi. Comparative genome analyses among these wild relatives as compared to the cultivated tomato genome suggest that prior determinations of which genome is the true progenitor of cultivated tomato may be incorrect. As such we are expanding the population to include more wild species accessions that will also be subject to long-read genome sequencing to clarify the genetic origin of the cultivated tomato and provide greater insight into the available natural genetic diversity of interfertile tomato species. Triplicate RNA-seq leaf transcriptome analysis has been completed for all population accessions and tissue has been harvested and cryogenically stored for flowers (combined buds through anthesis) and fruit at the mature green, breaker, and red-ripe stages. Transcriptome analysis on these tissues has been initiated. These tissues were harvested from a greenhouse trial and a field trial for phenotypic analysis is in progress. Existing genome sequences are being aligned to identify structural variants (SVs) and an initial version of alignments is being developed in jbrowse2.
Recent advances in AI have accelerated the ability to predict protein structure from DNA/a.a. sequence, in particular, using Alphafold. While the project plan included generation of a tomato alphafold database of all predicted proteins, Alphafold has recently released predicted structures based on DNA sequences of many species including tomato. We are currently using Alphafold 2 to generate a tomato predicted structure database, but are also redirecting some effort to model specific interactions of interest including those among specific transcription factors with existing interaction data (e.g. from Y2H) and interaction of folate biosynthesis and putative folate storage proteins with biological folate molecules.
During the past year, quantitative trait locus QTL-seq analysis was performed that combines bulked segregant analysis and high-throughput whole-genome re-sequencing of squash plants with pale yellow l-2/l-2 and dark orange L-2/L-2 fruit. This analysis detected a single QTL associated with L-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 locus was delimited in a physical interval of 55 Kbp.
We identified the NUDX23 protein that regulates phytoene synthase PSY protein level. During the past year, we performed experiments showing that the Nudix domain of NUDX23 directly interacts with PSY and is sufficient to regulate PSY protein level and in turn carotenogenesis. We also generated transgenic lines overexpressing PSY in a nudx23 knockout background and demonstrated that NUDX23 is required for PSY protein complex assembly. We found that NUDX23 does not regulate OR protein activity and has no interaction with OR in yeast two-hybrid analysis.
Objective 2:
Various commercially available membrane materials including Polyvinylidene Fluoride PVDF (hydrophobic), nylon-positive (anion exchange), nitrocellulose (cation exchange) are being evaluated for imprinting cross sectional chemical images of tomato fruit. Preliminary evaluation of transfer efficiency is being evaluated by hyperspectral imaging. Protocols for the Liquid chromatography–mass spectrometry LC-MS analysis using scheduled single ion recordings (SIR) have been developed for several classes of metabolites including flavonoids, folates, tocopherols and tocotrienols and cannabinoids. While the selectivity of the SIR approach has been excellent the sensitivity of the single quadrupole mass spectrometer has been somewhat disappointing. To improved sensitivity, we have developed a different approach involving high resolution-multi-reaction monitoring (HR-MRM) on a Zeno-TOF 7600 mass spectrometer. The high mass resolution of this instrument provides even better selectivity than the SIR approach and an 8-10-fold increase in sensitivity when applied to our panel of folates. HR-MRM methods are currently being developed for the other metabolite panels.
Objective 3:
To assess off-targeting of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 CRISPR/CAS9 gene editing, genomes of tomato cultivars Ailsa Craig and M82 in addition to nearly isogenic lines in Ailsa Craig for the natural rin mutation have been grown in addition to gene edited lines targeting the rin locus in both cultivars. Additional lines in both cultivars that have gone through transformation absent editing constructs have also been generated and grown to identify the rate of mutation resulting from regeneration and distinguish such variation from gene-editing effects. Leaf tissue has been harvested and cryogenically stored and DNA has been extracted from all accessions. Oxford Nanopore sequencing on the PromethION instrument is being implemented in our host institution (Cornell University) and is currently being tested with other tomato genomes by our lab as part of the University’s initial implementation of this platform. The platform yields both long read sequences facilitating assembly and 5mC data providing insights into DNA methylation, simultaneously facilitating the epigenome analysis portion of the project plan without the need for bisulfite sequencing. Tissues have been harvested for transcriptome analysis and RNA-seq has been completed.
In collaboration with researchers at University of California, Los Angeles, (UCLA), constructs targeting methylation and demethylation of a differentially methylated ripening transcription factor locus have been developed and tomato transformation has been initiated. In addition, gene editing constructs have been developed to mutate and also remove a hypermethylated region associated with ripening control to functionally validate the published hypothesis that DNA methylation at this locus influences ripening control. Transformation of these constructs into tomato has also been initiated.
Accomplishments
1. Carotenoids are critical to human health and plant growth. However, the regulatory mechanisms and the regulators conserved among plant species remain poorly understood. ARS scientists in Ithaca New York, have discovered that a Nudix protein is a novel regulator of carotenoid biosynthesis. It controls two key enzymes of the carotenoid biosynthesis pathway and is required for their protein stability and enzyme complex formation for efficient carotenoid biosynthesis. These results reveal both a novel regulatory mechanism to modulate carotenoid biosynthesis in plants and provide promising genetic tools to produce carotenoid-enriched food crops. This work was recently published in The Plant Cell.
2. Identification of an ethylene response transcription factor critical to fruit ripening. Fruit eating quality and nutritional content are tightly linked to the ripening process which is in turn regulated by a network of plant hormones, transcription factors, developmental signals and environmental inputs. While many components of ripening control are known, how they interact is less well understood. ARS researchers in Ithaca, New York, identified a tomato transcription factor, ERF.D6 necessary for normal ripening control. This gene is a member of the ETHYLENE RESPONSE FACTOR (ERF) family and is a downstream mediator of response to the plant hormone ethylene. Gene-edited mutant tomatoes deficient in ERF.D6 activity ripen more slowly than controls while lines over-expressing the gene ripen more quickly. ERF.D6 was further shown to be part of a cascade of three transcription factors necessary for normal ripening, connecting three regulators directly in a network with the plant hormone ethylene. These findings provide greater insight into the genetic mechanism of ripening control and will allow tomato breeders to select for natural alleles within tomato germplasm conferring higher or lower expression so as to facilitate early ripening or extended shelf-life as market demands dictate. This work is currently under review for publication.
Review Publications
Frick, E., Sapkota, M., Pereira, L., Wang, Y., Hermanns, A., Giovannoni, J.J., Van Der Knaap, E., Tieman, D., Klee, H. 2023. A family of methyl esterases converts methyl salicylate to salicylic acid in ripening tomato fruit. Plant Physiology. 191(1):110-124. https://doi.org/10.1093/plphys/kiac509.
Li, B., Shi, Y., Jai, H., Yang, X., Lu, J., Giovannoni, J.J., Jiang, G., Rose, J., Chen, K. 2023. Abscisic acid mediated strawberry receptacle ripening involves the interplay of multiple phytohormone signaling networks. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2023.1117156.
Yoo, H., Chung, M., Lee, H., Lee, S., Grandillo, S., Giovannoni, J.J., Lee, J. 2023. Natural overexpression of carotenoid cleavage dioxygenase 4 in tomato alters carotenoid flux. Plant Physiology. 192(2):1289-1306. https://doi.org/10.1093/plphys/kiad049.
Mcquinn, R., Leroux, J., Sierra, J., Escobar-Tovar, L., Frusciante, S., Finnegan, E., Diretti, G., Giuliano, G., Giovannoni, J.J., Leon, P., Pogson, B. 2023. Deregulation of ¿-carotene desaturase in Arabidopsis and tomato exposes a unique carotenoid-derived redundant regulation of floral meristem identity and function. The Plant Journal. 114(4):783-804. https://doi.org/10.1111/tpj.16168.
D'Inca, E., Foresti, C., Orduna, L., Amato, A., Vandelle, E., Santiago, A., Botton, A., Cazzaniga, S., Bertini, E., Pezzotti, M., Giovannoni, J.J., Vrebalov, J., Matus, J., Tornielli, G., Zenoni, S. 2023. The transcription factor VviNAC60 regulates senescence- and ripening-related processes in grapevine. Plant Physiology. 192(3):1928-1946. https://doi.org/10.1093/plphys/kiad050.
Tu, X., Ren, S., Wei, S., Li, J., Li, Y., Li, C., Li, Y., Zhong, Z., Xie, W., Grierson, D., Fei, Z., Giovannoni, J.J., Li, P., Zhong, S. 2022. Limited conservation in cross-species comparison of GLK transcription factor binding suggested wide-spread cistrome divergence. Nature Communications. 13(1). Article 7632. https://doi.org/10.1038/s41467-022-35438-4.
Zhou, X., Sun, T., Owens, L., Yang, Y., Fish, T., Liu, A., Wrightstone, E., Yuan, H., Chayut, N., Burger, J., Tadmor, Y., Thannhauser, T.W., Guo, W., Cheng, L., Li, L. 2023. Carotenoid sequestration protein fibrillin participates in CmOr-regulated B-carotene accumulation in melon. Plant Physiology. 193:643-660. https://doi.org/10.1093/plphys/kiad312.