Location: Plant, Soil and Nutrition Research2014 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 and generally meeting or exceeding goals for FY14 with only a few minor setbacks. Three basic strategies continue to be employed to identify 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). As examples, we have found in the last year that the tomato EIN3 and NOR genes represent master regulators of ripening and ethylene response, respectively. These results confirm NOR and EIN3 as optimal targets for manipulating the complete fruit ripening process to optimize both production traits (shelf-life) and consumer traits (flavor, texture and nutritional quality). We also have completed development and characterization of transgenic plants altered in expression of the tomato UNIFORM RIPENING GLK1 family member gene and showed that this gene is capable of influencing fruit chloroplast development and photosynthesis, and thus directly impacts sugar accumulation, appearance and quality of ripe fruit. Thus the genes GLK1 or GLK2 can be targeted for fruit quality improvement. The tomato genome sequence has been completed by an international consortium of plant scientists led by ARS scientists in Ithaca and is a powerful resource for the identification of key genes regulating nutritional content, ripening and shelf-life. In the last year, refinement of the tomato genome sequence was done to bring it to the highest quality standard possible with available resources. New annotation and assembly has also been completed. This sequencing has also been the stepping stone toward contributions to the pepper and wild tomato (S. pennellii) genome sequences (both published in FY 2014). 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. Proteomic and gene expression profiling in the last year has focused especially on characterization of protein and proteome level modifications that provide additional avenues of regulatory control and potential targets for crop improvement. As an example, we have previously developed methods to enrich glycoproteins (proteins modified by sugar attachments), an essential first step in studies to identify protein glycosylation. This year we have enhanced this approach by developing a system that makes use of multiple lectin affinity chromatography (MLAC). Using this approach, we were able to identify 318 putative glycosylation sites on a total of 230 N-glycoproteins. This demonstrates a vast array of modifications with possible impact on traits of interest and provides new targets and strategies for molecular crop improvement. 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. Genome sequencing of important vegetable crops. ARS researchers at Ithaca, New York contribute to international efforts developing genome sequences of pepper and a wild tomato species. Genome sequences of important crops and their relatives are critical tools for guiding breeding activities toward increased yield, stress tolerance and food quality. For example, the wild tomato species whose genome was sequenced, Solanum pennellii, is a breeding source of resistance to many tomato pathogens. The genome sequence allows for efficient selection of such genes while retaining the majority of desirable tomato genes during traditional breeding. Such breeding approaches lead to varieties with natural pathogen resistance allowing reduced pesticide application. In the cases of both the wild tomato and pepper, ARS researchers developed and contributed information on expression of all the genes in each genome and from various tissues. Such data is necessary for both defining gene expression patterns and validating the genes predicted from the genome sequences. Understanding the presence and location of genes of important crops allows breeders to more efficiently select for desired genes and the traits they impact, promoting higher yield (and thus more efficient land use) and increased quality while reducing the needs for space and chemical inputs promoting more sustainable production. Both genome sequences were reported in separate reports appearing in the journal Nature Genetics.
2. Improved methods for understanding plant proteins. ARS researchers at Ithaca, New York developed methods that facilitate the study of protein modifications important for crop performance and quality. Proteins are the products of gene expression and have both catalytic and structural functions in living organisms. The function of proteins is influenced by chemical modifications that can alter protein stability, localization and/or activity. Protein glycosylation (the addition of sugar molecules to a protein) is one of the most common modifications potentially influencing numerous crop traits (e.g. yield, stress tolerance, water use efficiency, shelf-life, flavor and nutritional content), yet its importance in crop biology is poorly understood due to the lack of methodology for efficiently identifying and characterizing protein glycosylation. In the past year Ithaca ARS researchers developed new procedures for efficient glycoprotein analysis fostering the rapid and accurate analysis of numerous crop proteins and the traits they impact. This work will provide crop biologists and breeders with a new set of targets associated with important crop traits for selection during crop breeding and improvement.
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Gapper, N., Mcquinn, R., Giovannoni, J.J. 2013. Molecular and genetic regulation of fruit ripening. Plant Molecular Biology. 82:575-591.
Ampofo, B., Chapman, N., Seymour, G., Giovannoni, J.J. 2013. Regulatory Networks Controlling Ripening. In: Graham B. Seymour, Mervin Poole, James J. Giovannoni, Gregory A. Tucker, editors. The Molecular Biology and Biochemistry of Fruit Ripening. New Delhi, India:Wiley-Blackwell. p189-200.
Zhong, S., Fei, Z., Chen, Y., Vrebalov, J., Mcquinn, R., Gapper, N., Giovannoni, J.J. 2013. Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nature Biotechnology. 31:154-159.
Li, L., Yuan, H. 2013. Chromoplast biogenesis and carotenoid accumulation. Archives Of Biochemistry and Biophysics. 539:102-109.
Chiu, L., Li, L. 2012. Characterization of the regulatory network of BoMYB2 in controlling anthocyanin biosynthesis in purple cauliflower. Planta. 236:1153-1164.
Souza, G., Carvalho, J., Rutzke, M., Albrecht, J., Guilherme, L., Li, L. 2013. Evaluation of germplasm effect on Fe, Zn and Se content in wheat seedlings. Plant Science. 210:206-213.
Zhang, J., Li, H., Zhang, M., Hui, M., Wang, Q., Li, L., Zhang, L. 2013. Fine mapping and identification of candidate Bo-or gene controlling orange head of Chinese cabbage (Brassica rapa L. ssp. Pekinensis). Molecular Breeding. 32:799-805.
Avila, F., Faquin, V., Yang, Y., Ramos, S., Guilherme, L., Thannhauser, T.W., Li, L. 2013. Assessing the anticancer compounds Se-methylselenocysteine and glucosinolates in Se-biofortified broccoli (brassica oleracea L. var. italica) sprouts and florets. Journal of Agricultural and Food Chemistry. 61:6216-6223.
Ruiz-May, E., Hucko, S., Howe, K.J., Zhang, S., Sherwood, R.W., Thannhauser, T.W., Rose, J.K. 2014. A comparative study of lectin affinity based plant n-glycoproteome profiling using tomato fruit as a model. Proteomes. 13:566-579.
Bowen, J., Ireland, H.S., Crowhurst, R., Luo, Z., Schaffer, R.J., Watson, A.E., Foster, T., Gapper, N., Watkins, C., Giovannoni, J.J., Mattheis, J.P., Rudell Jr, D.R., Johnston, J.W. 2014. Selection of low-variance expressed Malus x domestica (apple)genes for use as quantitative PCR reference genes (housekeepers). Tree Genetics and Genomes. 10:751-759.
Thannhauser, T.W., Shen, M., Sherwood, R., Howe, K.J., Fish, T., Yang, Y., Chen, W., Zhang, S. 2013. A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry. Electrophoresis. DOI: 10.1002/elps.201200656.
Pinheiro, P., Bereman, M., Burd, J., Pals, M.A., Armstrong, J.S., Howe, K.J., Thannhauser, T.W., Maccoss, M., Gray, S.M., Cilia, M. 2014. Evidence for the biochemical basis of host virulence in the greenbug aphid, Schizaphis graminum (Homoptera: Aphididae). Journal of Proteome Research. 13(4):2094–2108.
Zhang, S.S., Scherwood, R.W., Yang, Y., Fish, T., Chen, W.W., Mccardle, J.A., Jones, M., Yusibov, V., Ruiz-May, E., Rose, J., Thannhauser, T.W. 2012. Comparative characterization of the glycosylation profiles of an influenza hemagglutinin produced in plant and insect hosts. Proteomics. 12:1269-1288.
Okekeogbu, I., Ye, Z., Sangireddy, S.R., Li, H., Bhatti, S., Zhou, S., Howe, K.J., Fish, T., Yang, Y., Thannhauser, T.W. 2014. Effect of aluminum treatment on proteomes of radicles of seeds derived from Al-treated tomato plants. Proteomes. 2(2):169-190.