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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Plant, Soil and Nutrition Research » Research » Research Project #424885

Research Project: Genetic and Genomic Basis of Vegetable and Fruit Biology, Quality and Nutrient Content

Location: Plant, Soil and Nutrition Research

2016 Annual Report


Objectives
1: Analyze the genome, transcriptome, metabolite and protein components that drive fruit and vegetable development, maturation, ripening, nutrient content and quality traits. 1A: Develop comprehensive systems-based gene expression, proteomic and metabolic data for fruit ripening and development from a range of fruit tissues and sub-tissues. 1B: Determine the regulatory control of chromoplast development and carotenoid accumulation using the Or gene as a model. 1C: Identify genes and proteins affecting Or-regulated chromoplast development and carotenoid accumulation. 1D: Continue efforts toward an improved tomato reference genome sequence. 2: Identify the genes and quantitative trait loci (QTL) that underlie variation in crop traits associated with vegetable and fruit biology, including processes that determine nutrient content, quality and shelf life. 2A: Analyze tomato genetic and phenotypic variation and associated gene expression changes resulting from defined introgressions of wild species DNA so as to identify loci and genes underlying fruit quality and nutrient content. 2.B: Elucidate how the epigenome contributes to regulating tomato fruit ripening and quality. 3: Determine the molecular function and utility of genes that contribute to target fruit and vegetable traits. 4: Evaluate the translatability of validated nutrient quality gene sequence activities in additional crop species.


Approach
A number of general themes will be followed to secure progress toward all four primary objectives and associated sub-objectives described below. 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 loci and corresponding genes underlying fruit and vegetable quality and nutritional content loci. Candidate genes will be isolated and 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. For example, while chromoplasts can be generated from fully developed chloroplasts, as is most commonly observed during fruit ripening of, for example, tomato and pepper, chromoplasts can also be derived from proplastids or other non-colored plastids as is the case in melon, watermelon, and carrot (Burger et al., 2008; Kim et al., 2010; Tadmor et al., 2005). Better understanding of such 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.


Progress Report
The project has continued on track meeting or exceeding goals for fiscal year 2016 with no substantive setbacks and only one milestone partially met but near completion at the writing of this report. Three basic strategies continue to be employed to identify and functionally characterize genes associated with crop nutrient quality. The first is identification of genes known through mutation to influence nutrient or other quality parameters. These activities address NP301 component 301.3.C.2010 and 301.4.2010 as DNA markers were developed that are being used by seed companies to accelerate their breeding programs for nutrient, ripening, and shelf-life traits. Second, gene targets and/or interacting proteins of these regulators have been identified and analyzed in addition to homologs of these genes in other species (suggesting potential for broad applicability to diverse crops species). We have completed whole genome characterization of DNA (promoter) interactions between the RIN, NOR and EIN3 transcription factors and their respective target genes. This analysis has been complemented with deep whole transcriptome expression studies in addition to methylome analyses to provide multiple independent perspectives on regulation of important ripening, shelf-life and nutritional quality associated genes. Prior efforts leading to characterization of the tomato UNIFORM RIPENING GLK1 and GLK2 genes has been leveraged to develop pre-breeding lines and molecular tools to facilitate introgression of the alternative alleles of this gene into elite germplasm for either color uniformity or elevated sugars/carotenoids as targeted. In addition, the newly identified interacting proteins provide us new genetic tools and new directions for in-depth elucidation of the complex regulatory networks in controlling carotenoids and nutritional quality of food crops. The last year has resulted in development of numerous transgenic lines for functional analysis of multiple candidate genes for nutrient quality and shelf-life improvement. The tomato genome sequence continues to be leveraged and improved. In the last year, sequence assembly and annotation versions have been developed and released (solgenomics.net) as the result of an international consortium to which this group has contributed. Continued collaborative efforts in the last year have resulted in a draft genome sequence of the tomato wild species S. lycopersicoides. The third track toward nutrient-associated gene identification is through comparative gene and/or protein expression analysis on a range of germplasm from cauliflower and tomato varieties with altered nutrient composition in an effort to understand regulation of these pathways which are important in human nutrition and crop quality. In the last year we have expanded use of mass spectrometry into our analysis pipeline which enhances both sensitivity and selectivity of the analysis. In the last year we have developed an improved protocol to determine carotenoid concentrations in plant tissues by incorporating a Super Critical Fluid (SCF) chromatography-based protocol (see accomplishments below). These improvements enhance both the sensitivity and selectivity of the assays and reduce the assay time by more than a factor of three; and lastly, we have developed assays to identify metabolite biomarkers from tomato fruit tissues through the use of ultra-performance liquid chromatography (UPLC) and high resolution mass spectrometry leading to enhanced coverage of the metabolome significantly increasing the probability of identifying biologically important biomarkers. With regard to data dissemination, our public database (Tomato Functional Genomics Database; http://ted.bti.cotnell.edu) has been updated to house and disseminate all of the information reported here either directly or via linkage with the Solgenomics (http://solgenomics.net) in addition to integrated data recovered from the public domain.


Accomplishments
1. Developing a Super Critical Fluid (SCF)-based chromatographic method for the analysis of carotenoids in plants. Vitamin A deficiency (VAD) is the leading cause of preventable blindness in children, increases the risk of disease and death from infection and, in pregnant women, it increases the risk of maternal mortality. With a view towards developing crop plants enriched in pro-vitamin A precursors, extensive and accurate data on the carotenoid composition of plants is needed. However, carotenoid analysis is inherently complicated and current methods involving high performance or ultra-performance liquid chromatography are time consuming and generate large volumes of toxic waste. ARS scientists working in Ithaca, New York have developed a novel SCF-based chromatographic method that reduces analysis time by a factor of 4 while reducing the volume of hazardous waste generated by over 80%.

2. Discovery of genes regulating tomato fruit firmness. Fruit texture is an important trait related to a consumer liking of fruit and fruit products. Over softening of fruit is generally considered a negative consumer trait with additional deleterious consequences on fruit storage and shipping. Researchers at the Robert W. Holley Center in Ithaca, New York identified and characterized two genes contributing to the proportion of tomato fruit that is pericarp (flesh) versus locule (jelly surrounding the seeds). Reduced expression of these genes resulted in increased flesh and reduced jelly while elevated expression had the inverse effect. A natural mutant in one gene was identified and which resulted in nearly complete replacement of the jelly component of the tomato fruit with flesh tissue. Fruit with greater flesh versus jelly content have superior shipping quality, have advantages in fresh products such as sandwich slices, and have superior processing quality for production of paste, sauce and salsas. Discovery of these genes provides a path toward improved fruit quality via use of the natural mutation of direct genetic intervention.


An ARS researcher in Ithaca, New York, became an Adjunct Professor in the School of Graduate Studies, College of Agriculture, Human and Natural Sciences, at Tennessee State University (an 1890 University) where he provide guidance to and mentored two PhD candidate graduate students of the University with interests in protein chemistry and proteomics by providing advice on experimental design, data interpretation and serving as an extramural advisor on their special committees. ARS researchers in Ithaca, New York, actively participated in the presidential initiative “My Brother’s Keeper” (MBK) by organizing a day long STEM Laboratory Tour of facilities located at the R.W. Holley Center, Cornell University and the Boyce Thompson Institute for 12 young men (9th-12th grade) and their mentors from the inner city of Rochester, New York (March 4, 2016). Through this program, these individuals were exposed to state-of-the-art technologies involving plant science, mass spectrometry, microscopy, imaging and DNA sequencing. ARS researchers hosted a total of five research thesis students and school-year or summer interns in their laboratories in fiscal year 2016, including two under-represented minorities, two women and two military veterans.


Review Publications
Mcquinn, R., Giovannoni, J.J., Pogson, B. 2015. More than meets the eye: from carotenoid biosynthesis to new insights into apocarotenoid signaling. Current Opinion in Plant Biology. 27:172-179.
Villarino, G., Bombarely, A., Giovannoni, J.J., Scanlon, M., Mattson, N. 2014. Analysis of petunia hybrida in response to salt stress using high throughput RNA sequencing. PLoS One. 9(4):e94651.
Shearer, L., Anderson, L., De Jong, H., Smit, S., Goicoechea, J., Giovannoni, J.J., Stack, S. 2014. Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome. Genes, Genomes, and Genomics. 4:1395-1405.
Tang, X., Miao, M., Niu, X., Zhang, D., Cao, X., Wang, A., Giovannoni, J.J., Liu, Y. 2015. UV-damaged DNA binding protein-1 and de-etiolated-1 regulate golden 2-like transcription factor by assembling a cullin 4-based ubiquitin ligase in tomato. New Phytologist. 209(3):1028-1039.
Liu, R., How-Kit, A., Stammitti, L., Teyssier, E., Rolin, D., Halle, S., Giovannoni, J.J., Gallusci, P. 2015. A DEMETER-like DNA demethylase governs tomato fruit ripening. Proceedings of the National Academy of Sciences. 112:10804-10809.
Kim, S., Park, M., Yeom, S., Kim, Y., Lee, J., Lee, H., Giovannoni, J.J., Sorensen, I., Rose, J., Choi, D. 2014. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capscicum species. Nature Genetics. 46:270-279.
Shinozaki, Y., Hao, S., Kojima, M., Zheng, Y., Fei, Z., Zhong, S., Giovannoni, J.J., Rose, J., Ariizumi, T. 2015. Ethylene suppresses tomato (solanum lycopersicum) fruit set through modification of gibberellin metabolism. Plant Journal. 83:237-251.
Boldrin, P.F., Figueiredo, M., Yang, Y., Luo, H., Giri, S., Hart, J.J., Faquin, V., Guilherme, L., Thannhauser, T.W., Li, L. 2016. Selenium promotes sulfur accumulation and plant growth in wheat (Triticum aestivum). Physiologia Plantarum. 158:80-91.
Yuan, H., Owsiang, K., Sheeja, T., Zhou, X., Rodriguez, C., Li, Y., Welsch, R., Chayut, N., Yang, Y., Thannhauser, T.W., Pathasarathy, M.V., Xu, Q., Deng, X., Fei, Z., Schaffer, A., Katzir, N., Burger, J., Tadmor, Y., Li, L. 2014. A single amino acid substitution in an ORANGE protein promotes carotenoid overaccumulation in arabidopsis. Plant Physiology. 169(1):421-431.
Mon, J., Bronson, K.F., Hunsaker, D.J., Thorp, K.R., White, J.W., French, A.N., Conley, M.M. 2016. Interactive effects of nitrogen fertilization and irrigation on grain yield, canopy temperature, and nitrogen use efficiency in overhead sprinkler-irrigated Durum Wheat. Field Crops Research. 191:54-65.
Bolger, A., Scossa, F., Bolger, M., Fei, Z., Rose, J., Zamir, D., Cararri, F., Giovannoni, J.J., Weigel, D., Fernie, A. 2014. The genome of the stress tolerant wild tomato species solanum pennellii. Nature Genetics. 46:1034-1038.
Ricardi, M., Gonzalez, R., Zhong, S., Duffy, T., Turjanski, P., Alleva, K., Carrari, F., Giovannoni, J.J., Estevez, J., Iusem, N. 2014. Genome-wide data (ChIP-seq) enabled identification of cell wall-related and aquaporin genes as targets of tomato ASR1, a drought stress-responsive transcription factor. Biomed Central (BMC) Plant Biology. 14:29.
Yuan, H., Zhang, J., Nageswaran, D., Li, L. 2015. Carotenoid metabolism and regulation in horticultural crops. Horticulture Research. 8:68-82.
Zhang, J., Yuan, H., Fei, Z., Pogson, B.J., Li, L. 2015. Molecular characterization and transcriptome analysis of orange head Chinese cabbage (brassica rapa L. ssp.pekinensis). Planta. 241:1381-1394.
Zhang, J., Yuan, H., Yang, Y., Fish, T., Lyi, S., Thannhauser, T.W., Zhang, L., Li, L. 2016. Plastid ribosomal protein S5 plays a critical role in photosynthesis, plant development, and cold stress tolerance in arabidopsis. Journal of Experimental Botany. doi: 10.1093/jxb/erw106.