Location: Plant, Soil and Nutrition Research2015 Annual Report
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.
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.
The project has continued on track meeting or exceeding goals for FY15 with no substantive setbacks other than a delay in acquisition of a subset of targeted antibodies. 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. 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 have resulted in the generation of over 300 tomato genotypes reported in a 2015 manuscript for which we provided assembly input, quality assessment and transcriptome data. We have also initiated a collaborative effort to sequence the genome of the most distant inter-crossable wild tomato relative, S. lycopersicoides, which harbors numerous alleles potentially contributing to fruit quality and abiotic stress tolerance. 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. Proteome analysis in the last year has focused especially on developing and improving analysis techniques, leveraging resources and expertise to acquire new equipment and subsequent characterization of protein and proteome level modifications that provide additional avenues of regulatory control and potential targets for crop improvement. As examples, we have 1) developed improved protocols for the determination of glucosinolate concentrations in plant tissues. These compounds impact nutrient quality and flavor of plant products and these improvements involved alterations in isolation protocols which increase data reliability. We further introduced a mass spectrometry into our analysis pipeline which enhances both sensitivity and selectivity of the analysis; 2) we are developing improved protocols to determine carotenoid concentrations in plant tissues incorporating ultra-performance liquid chromatography (UPLC) and high performance mass spectrometry. 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.
1. Understanding melon fruit coloration. ARS researchers at Ithaca, New York, collaborating with colleagues in Israel, demonstrated that a protein originally identified by the Ithaca lab interacts directly with a major regulator of plant carotenoid accumulation. Carotenoids are plant derived chemicals with high antioxidant activity, in some cases are precursors to necessary nutrients, and often contribute to appearance and aroma of plant tissues and plant-derived products. Interaction with a key carotenoid pathway regulator provides insight into the mechanisms through which this protein operates to manifest differences in fruit and vegetable carotenoid levels. Furthermore, variation in this protein (termed OR or "ORANGE") was shown to be the basis of determining orange versus green flesh coloration in melons (e.g. orange cantaloupe types versus green honeydew varieties). Revelation of this interaction and discovery of the effects of the melon version of this gene will provide opportunities to develop tools facilitating more efficient color selection during melon breeding and lead to more rapid development of attractive and nutritious (higher carotenoid content) melon varieties.
2. Method to elevate carotenoids in fruits and vegetables. ARS researchers at Ithaca, New York, have demonstrated a molecular strategy to increase carotenoid accumulation in fruits and vegetables. Carotenoids are plant derived chemicals with high antioxidant activity, in some cases are precursors to necessary nutrients, and contribute to appearance and aroma of plant tissues and plant-derived products. Prior efforts to increase plant carotenoid levels through genetic means have been confounded by the complex internal mechanisms that regulate carotenoid accumulation, often resulting in little, if any, effect. In some instances, such as golden rice, elevated carotenoids have been achieved after much effort including the use of genes from non-plant sources and through targeting multiple pathway steps via multiple genes. ARS researchers have identified a critical carotenoid synthesis step in tomato that is not subject to strong internal regulatory constraint when a gene from a different plant (Arabidopsis thaliana) is used. Over-expression of this single plant gene in tomato resulted in significant elevation of fruit carotenoids. This proof of concept can be deployed on the same carotenoid pathway step in different plant species to improve carotenoid levels of fruits and vegetables effecting fruit quality, nutritional content and with associated food security effects especially important for developing countries.
Avila, F., Yang, Y., Faquin, V., Ramos, S., Guilherme, L., Thannhauser, T.W., Li, L. 2014. Impact of selenium supply on se-methylselenocysteine and glucosinolates accumulation in selenium-biofortified brassica sprouts. Food Chemistry. 165:578-586.
Zhou, S., Palmer, M., Zhou, J., Bhatti, S., Howe, K.J., Fish, T., Thannhauser, T.W. 2013. Differential root proteome expression in tomato genotypes with contrasting drought tolerance exposed to dehydration. Journal of the American Society for Horticultural Science. 138(2):131-141.
Li, L., Tadmor, Y., Qiang, X. 2014. Approaches for vegetable and fruit quality trait improvement. In: Ricroch, A., Chopra, S., Fleischer, S., editors. Plant Biotechnology - Experience and Future Prospects. Switzerland: Springer International Publishing. 18:227-243.
Zhang, M., Zhang, M., Mazourek, M., Tadmor, Y., Li, L. 2014. Regulatory control of carotenoid accumulation in winter squash during storage. Planta. 240:1063-1074.
Souza, G., Hart, J., Carvalho, J., Rutzke, M., Albrecht, J., Guilherme, L., Kochian, L.V., Li, L. 2014. Genotypic variation of zinc and selenium content in grains of Brazilian wheat lines. Plant Science. 224:27-35.
Mellidou, I., Buts, K., Hatoum, D., Ho, Q.T., Mattheis, J.P., Johnston, J.W., Watkins, C.B., Schaffer, R.J., Gapper, N.E., Giovannoni, J.J., Rudell Jr, D.R., Hertog, M.L., Nicolai, B.M. 2014. Transcriptomic events associated with internal browning of apple during postharvest storage. Biomed Central (BMC) Plant Biology. 14:328.
Nazia, N., Li, L., Lu, S., Khin, N., Pogson, B.J. 2015. Carotenoid metabolism in plants. Molecular Plant. 6:68-82.
Moyle, R., Koia, J., Vrebalov, J., Giovannoni, J.J., Botella, J. 2014. The pineapple AcMADS1 promoter confers high level expression in tomato and arabidopsis flowering and fruiting tissues, but AcMADS1 does not complement the tomato LeMADS-RIN (rin) mutant. Plant Molecular Biology. 86(4-5):395-407.
Klie, S., Osorio, S., Tohge, T., Drinkovich, M., Fiat, A., Giovannoni, J.J., Fernie, A., Nikoloski, Z. 2014. Conserved changes in dynamics of metabolic processes during fruit development and ripening across species. Plant Physiology. 164(1):55-68.
Nguyen, C., Vrebalov, J., Gapper, N., Zheng, Y., Zhong, S., Fei, Z., Giovannoni, J.J. 2014. Tomato golden 2-like (GLK) transcription factors reveal molecular gradients that function during fruit development and ripening. The Plant Cell. 26(2):585-601.
Cohen, S., Itkin, M., Yesselson, Y., Portnoi, V., Fei, Z., Xu, Y., Giovannoni, J.J., Tadmor, Y., Katzir, N., Burger, Y., Schaffer, A. 2014. The PH gene determines fruit acidity and contributes to the evolution of sweet melons. Nature Communications. 5:4026.
Lin, T., Zhu, G., Zhang, J., Xu, X., Yu, Q., Zheng, Z., Fei, Z., Giovannoni, J.J., Yi, Z., Huang, S. 2014. A brief genomic history of tomato breeding. Nature Genetics. 46:1220-1226.
Tzuri, G., Zhou, X., Chayut, N., Yuan, H., Portnoy, V., Meir, A., Saar, U., Baumkoler, F., Mazoureck, M., Lewinsohn, E., Fei, Z., Schaffer, A., Li, L., Burger, J., Katzir, N., Tadmor, Y. 2015. A golden SNP in CmOr governs fruit flesh color of melon (cucumis melo). Plant Journal. 82:267-279.
Zhou, X., Welsch, R., Yang, Y., Riediger, M., Alvarez, D., Yuan, H., Fish, T., Liu, J., Thannhauser, T.W., Li, L. 2015. Arabidopsis OR proteins are the major post-transcriptional regulators of phytoene synthase in mediating carotenoid biosynthesis. Proceedings of the National Academy of Sciences. 112:3558-3563.