Project : USDA ARS

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

Location: Plant, Soil and Nutrition Research

2021 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. Tomato researchers have access to multiple populations resulting from crosses of cultivated and wild species that facilitate exploration and introgression of vast genetic diversity not found in cultivated tomato. These populations are both recombinant inbred (RIL) and introgression line (IL) populations – a key feature being that they are inbred/true breeding, allowing low resolution mapping of observed traits (facilitating eventual gene isolation) and recovery of identical lines for future analyses. This year involved continued phenotypic analysis of fruit and additional tissues from these population, especially for nutritional chemicals including carotenoids and their derivatives, folic acid and flavonoids. Flavonoid chemistry is more complex and we are continuing exploration of ways of developing/optimizing protocols to more efficiently extract and analyze these compounds. In collaboration with Cornell investigators, we are also developing a database of mass spectrometry derived signals to simplify future metabolic analyses. Primary emphasis in this year has been on a population derived from a cross between tomato and its most diverged sexually compatible relative, Solanum lycopersicoides, which is adapted to extreme cold and drought in the Andes mountain range. In the last year, we demonstrated that this wild species has genetic diversity for shelf-life, ripening initiation and carotenoid accumulation beyond that found in cultivated tomato or in previously examined wild species. Of note, in the last year, we identified an additional S. lycopercoides lycopene-beta-cyclase gene, inactive in and thus not described before in cultivated tomato genotypes. Through encoding the catalysis of lycopene to beta-carotene, this locus provides a novel tool for manipulation of fruit carotenoid, color and nutrient quality. It also helps explain the fact that S. lycopersicum, the cultivated tomato, and its wild progenitor, S. pimpinnelifolium, are the only red fruited tomato species with all others having green or orange fruit. In short, this enzyme facilitates metabolism of carotenoids to pro-vitamin A beta-carotene (orange color) and downstream colorless carotenoids and their metabolites including flavor and aroma volatiles. Tighter control of this activity in cultivated tomato results in characteristic red fruit. In collaboration with researchers at the Boyce Thompson Institute (BTI) and in Belgium, we have completed de novo sequencing of the S. lycopersicoides genome to exceptional quality using long-read sequencing and further refined the resolution of the cross-over events defining individual members of the IL population derived from S. lycopercoides and S. lycopersicum, facilitating rapid definition of candidate genes. These genomics resources will enable breeders and biologists to more effectively exploit the considerable genetic diversity found in this wild tomato relative. 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: In addition to functional characterization of an additional lycopene beta-cyclase as noted above, we have continued prior characterization of tomato TomLOX C, which encodes a fatty acid desaturase that effectively facilitates carotenoid metabolism via interactions between carotenoids and intermediates of fatty acid break down products. Both the resulting fatty acid and carotenoid derived metabolites include volatiles associated with fruit aroma, flavor and consumer quality perception. The optimal high expression allele of TomLOX C in tomato is derived from S. pimpinnelifolium and is rarely found in modern tomato genotypes. Genotyping of a large diversity panel of tomato cultivars revealed several genotypes homozygous for the high expression allele that can be deployed by breeders for enhanced fruit quality. Indeed, it is clear that while still not prevalent, this allele has been indirectly selected in modern tomato varieties presumably by breeders targeting flavor and aroma. Additional efforts on carotenoids have continued to focus on crucifers as a source of novel genes and alleles for both understanding carotenoid accumulation and as tools for breeding. In the last year, efforts have focused on directing findings toward manipulation of carotenoid seed content. Preliminary efforts in Arabidopsis demonstrated that high levels of seed carotenoids could be achieved through manipulation of genes involved in carotenoid stability resulted in seeds with substantially elevated carotenoids including pro Vitamin A beta-carotene. These findings serve as a proof-of-concept for manipulation of carotenoid content in seed crops. As most staple crops are seeds typified by low carotenoid content, these findings have potential application to influence food and nutritional security in the U.S. and the world-over. 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. One such gene, SlLOB1, 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. In the last year, it was demonstrated that SlLOB1 directly interacts with promoters of multiple cell wall genes including pectate lyase and Expansion 1, two of the genes best associated with fruit texture in tomato. 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. Repression of the latter are central in consumer dissatisfaction with commercial tomatoes and additional fruit crops. Characterization of SlLOB1 was recently published. In the last year, we have initiated work on an additional novel gene involved in ripening control through ethylene in addition to efforts to more fully define the function of the widely commercially bred tomato ripening-inhibitor (rin) MADS-box transcription factor allele in ripening control. Efforts on large scale characterization of transcription factor – promoter interactions continue through genome wide DNA affinity purification sequencing (DAP-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analyses. Several gene edited lines from the above efforts have been fully characterized in the last year and entered our whole genome sequencing pipeline to address efficiency, off-targeting, and the generation of unanticipated DNA modifications resulting from gene editing to provide information on biotechnology risk of this fast emerging approach to stable genome modification. We are also sequencing several introgression lines as examples of non-modified breeding lines for this analysis. Working with collaborators from Cornell, BTI, U.K, China, and New Zealand, we have characterized homologs of ripening transcription factors previously described under this project or its prior iterations. These collaborative efforts have resulted in the characterization of a NAC domain transcription factor as central in apple ripening control and a possible target for manipulation of fruit shelf-life and quality. Identification and functional characterization of a strawberry MADS-box gene from the same clade as the RIN transcription factor in ripening control demonstrates conservation of such transcriptional ripening regulators in both climacteric and non-climacteric species and adds further support to our assertion that MADS-box loci are logical targets for ripening and shelf-life control in numerous fruit crops. Both the apple and strawberry genes were recently published.

Accomplishments
1. Enhanced carotenoid level and stability in seeds. Staple seeds are the main target for carotenoid biofortification in crops due to their uniquity in the food supply. However, carotenoid degradation during seed maturation and post-harvest storage is a serious problem limiting seed carotenoid levels. Scientists in Ithaca, New York, established an effective multi-strategy approach to elevate seed carotenoids by simultaneously modifying biosynthetic activity, turnover, and storage capacity using Arabidopsis seeds as a model, and achieved up to 71- and 12-fold increases of b-carotene and total carotenoids, respectively, during seed maturation and 86% retention during post-harvest storage. This strategy is likely applicable to provitamin A carotenoid enrichment in seeds of various staple crops.

2. Improved fruit firmness and shelf-life while maintaining quality. A major limitation in delivery of fresh high quality fruit to consumers is the natural propensity of ripening fruit to over-soften, decreasing consumer appeal and accelerating decay and product loss. Most means of insuring firmness and shipping/shelf-life whether through early harvest, application of ethylene scavengers or inhibitors, or controlled atmosphere storage, inhibit complete ripening and diminish consumer appeal. Researchers in Ithaca, New York, collaborating with Cornell and Zhejiang University identified a tomato regulatory gene which primarily influences the softening but not additional aspects of ripening. Targeted inhibition of this gene (SlLOB1) resulted in firmer and longer shelf life fruit with little influence over desirable ripening characteristics, thus presenting a unique target for extending shelf-life and minimizing softening absent negative fruit quality outcomes associated with current fruit softening control solutions.

U.S. DEPARTMENT OF AGRICULTURE

Plant, Soil and Nutrition Research: Ithaca, NY