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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

2017 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 largely meeting or exceeding goals for fiscal year (FY) 2017 with no substantive setbacks and only one milestone partially met but originally proposed as a two year milestone that would not be completed until the end of FY18. 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. In the latter regard, with collaborators in France who showed that a tomato DEMETER-like gene SlDML2 regulates the ripening-related genome DNA demethylation, we have recently demonstrated through whole-genome cytosine methylation analysis that most but not all ripening methylation dynamics is mediated through DML2. Recent data we have generated on melon fruit ripening suggests a similar methylation dynamics phenomena as we described in tomato and further suggests a role for ethylene in mediating these changes. Finally, 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 (manuscript under review). In addition, newly identified interacting proteins (e.g. with Or) 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. We recently identified an unanticipated target for enhancement of carotenoid content, phytoene desaturase, which we showed becomes limiting when phytoene synthase is elevated (for example during ripening) and is the target for multiple points of endogenous regulatory control. We also demonstrated that expression of a heterologous protein in tomato bypassed endogenous regulatory circuitry yielding substantially elevated carotenoid content. Simultaneous manipulation of a carotenoid isomerase resulted in accumulation of a more bioavailable form of this additional carotenoid. 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 de novo genome sequence of the tomato wild species S. lycopersicoides as a collaborative effort with labs in Belgium and in the Boyce Thompson Institute. 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 fully implemented an improved protocol (developed the year prior) to determine carotenoid concentrations in plant tissues by incorporating a Super Critical Fluid (SCF) chromatography-based protocol. 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 databases operated and supported in conjunction with colleagues at the Boyce Thompson Institute (Tomato Functional Genomics Database; http://ted.bti.cotnell.edu and Tomato Epigenome Database http://ted.bti.cornell.edu/epigenome/) has been updated to house and disseminate all of the information reported here either directly or via linkage with the Solgenomics (http://solgenomics.net).


Accomplishments
1. Tomatoes are among the most widely consumed fruit in the U.S. and are central components of many home gardens. They are important sources of dietary carotenoids including the antioxidant lycopene (which give tomatoes their characteristic red color) and beta-carotene, which our bodies convert to the necessary nutrient vitamin A. ARS researchers in Ithaca, New York identified an important regulatory step limiting the synthesis of carotenoids in tomatoes and genetic solutions to both elevate fruit carotenoid levels and convert the accumulated lycopene to a form more accessible for uptake in the human digestive system. These findings will help the breeding of tomatoes for higher nutritional value.


Review Publications
Li, L., Yuan, H., Zeng, Y., Xu, O. 2016. Plastids and carotenoid accumulation. In: Stange, C., editor. Subcellular Biochemistry. Carotenoids in Nature - Biosynthesis, Regulation and Function. 1st edition. Switzerland: Springer International Publishing. p. 273-293.
Liu, C., Long, J., Zhu, K., Liu, L., Yang, W., Zhang, H., Li, L., Xu, Q., Deng, X. 2016. Characterization of a citrus R2R3-MYB transcription factor that regulates the flavonol and hydroxycinnamic acid biosynthesis. Scientific Reports. 6:25352.
Chayut, N., Yuan, H., Ohali, S., Meir, A., Yeselson, Y., Portnoy, V., Fei, Z., Lewinsohn, E., Katzir, N., Schaffer, A., Gepstein, S., Burger, J., Li, L., Tadmor, Y. 2015. A bulk segregant transcriptome analysis reveals metabolic and cellular processes associated with melon orange allelic variation and fruit B-carotene accumulation. Biomed Central (BMC) Plant Biology. 15:274.
Yin, X., Xie, X., Xia, X., Furgeson, I., Giovannoni, J.J., Chen, K. 2016. Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening. Plant Journal. 86:403-412.
Elitzur, T., Yakir, E., Fei, Z., Vrebalov, J., Giovannoni, J.J., Freidman, H. 2016. Banana MaMADS transcription factors are necessary for fruit ripening and molecular tools to promote shelf-life and food security. Plant Physiology. 171:390-391.
Elitzur, T., Yakir, E., Fei, Z., Vrebalov, J., Giovannoni, J.J., Freidman, H. 2016. Harnessing epigenome modifications for better crops. Journal of Experimental Botany. 67:2535-2537.
Leisso, R.S., Gapper, N.E., Mattheis, J.P., Sullivan, N.L., Watkins, C.B., Giovannoni, J.J., Schaffer, R.J., Johnston, J.W., Hanrahan, I., Hertog, M.L., Nicolai, B.M., Rudell Jr, D.R. 2016. Gene expression and metabolism preceding soft scald, a delayed chilling injury of ‘Honeycrisp’ apple fruit. BMC Genomics. 17:798.
Gapper, N.E., Hertog, M., Lee, J., Buchanan, D.A., Leisso, R.S., Giovannoni, J.J., Johnston, J.W., Shaffer, R.J., Micolai, B.M., Mattheis, J.P., Watkins, C.B., Rudell Jr, D.R. 2017. Delayed response to cold stress is characterized by successive metabolic shifts culminating in apple fruit peel necrosis. Biomed Central (BMC) Plant Biology. 17:77. https://doi.org/10.1186/s12870-017-1030-6.
Chialva, M., Zouari, I., Salvioli, A., Novero, M., Cao, Z., Vrebalov, J., Giovannoni, J.J., Bonfante, P. 2016. Gr and hp-1 tomato mutants unveil unprecedented interactions between arbuscular mycorrhizal symbiosis and fruit ripening. Planta. 244(1):155-165.
Pankratov, I., Mcquinn, R., Schwartz, J., Bar, E., Fei, Z., Zamir, D., Giovannoni, J.J., Hirschberg, J. 2016. Fruit color mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis. Plant Journal. doi: 10.1111/tpj13232.
Martin, L., Scherwood, R., Nicklay, J., Yang, Y., Muratore, S., Anderson, E., Thannhauser, T.W., Rose, J., Zhang, S. 2016. Application of wide selected-ion monitoring data-independent acquisition to identify tomato fruit proteins regulated by the CUTIN DEFICIENT2 transcription factor. Proteomics. 16:2081-2094.
Zhu, Y., Hui, L., Bhatti, S., Zhou, S., Yang, Y., Fish, T., Thannhauser, T.W. 2016. Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots. Horticulture Research. 3:16026.
Zhou, S., Okekeogbu, I., Sangireddy, S., Yi, Z., Hui, L., Bhatti, S., Hui, D., Yang, Y., Howe, K.J., Fish, T., Thannhauser, T.W. 2016. Proteome modification in tomato plants upon long-term aluminum treatment. Journal of Proteome Research. 15:1670-1684.
Ye, Z., Sangireddy, S., Okekeogbu, I., Zhou, S., Yu, C., Hui, D., Howe, K.J., Fish, T., Thannhauser, T.W. 2016. Drought-induced leaf proteome changes in switchgrass seedlings. International Journal of Molecular Sciences. 17:1251-1269.