2013 Annual Report
1a.Objectives (from AD-416):
The primary objective of this research project is to identify genes and define molecular mechanisms that regulate nutritional content, quality and availability of plant-based foods with a primary emphasis on carotenoids. The value of such research will be in expanding the knowledge base of molecular biology related to crop nutrient quality and more specifically, to understanding of primary and secondary biochemical pathways and associated genetic regulatory systems that influence nutritional characteristics of plant-derived foods. Discoveries resulting from activities pursued through this project will lead to molecular tools for testing biochemical and molecular regulatory hypotheses and eventually for manipulating crop nutrient profiles and\or content. Implementation of said discoveries will be through both creation of genetically modified crops plants and indirect genetic manipulation via DNA markers associated with target nutrient traits. Resulting genetically modified plants will further be useful in testing not only nutrient levels but also availability to humans through diet within the context of a given crop tissue or derived food. Specific broad objectives of this project include:
Objective 1: Define genetic regulatory mechanisms that control endogenously regulated and environmentally influenced synthesis and accumulation of carotenoids in plant-based foods.
Objective 2: Develop and characterize genetic and biochemical plant attributes contributing to regulation of accumulation of carotenoids with exploratory efforts toward additional plant-derived nutrients.
1b.Approach (from AD-416):
Efforts will focus on the use of tomato and cauliflower for identification and characterization of nutrient-related genes with primary emphasis on carotenoids. We propose expansion of the tomato model to include analysis of genome-wide expression patterns during fruit development and ripening. We will perform comparative expression profiling of pre-ripening and ripening fruit from normal, ancestral and mutant varieties, combined with HPLC analysis of carotenoid accumulation to identify candidate transcription factors impacting fruit carotenoid accumulation. Additional insights into transcriptional control of carotenoid accumulation will be developed through analysis of protein accumulation profiles in addition to (and in some cases in support of) transcription data. A major goal of this project is to identify novel genes involved in or regulating a specific metabolite pathway using correlation analysis between genotype, metabolite and gene expression data. We will develop both biology-driven and statistics-driven integration tools that will be presented to the research community and broader public via the world wide web. The secondary model for our activities will be cauliflower as both a source of unique genetic variation related to carotenoids and a member of the Brassicaceae which includes many important vegetable species. Previously, we have demonstrated that expression of the Or genomic DNA allele in transgenic cauliflower induced carotenoid accumulation. To begin to shed light on the nature of the Or mutation and endogenous OR protein function, we propose to generate both "knockout" and over-expression lines in cauliflower. We will employ a range of proteomics approaches including 2-hybrid, gel filtration and mass spectrometry to identify proteins that interact with Or. Finally, as a relatively minor activity and in an effort to identify future areas of promise, we will begin to improve our understanding of Se metabolism in plants for enhancing the biosynthesis of functional forms of organoselenium compounds. We will employ molecular and genomics approaches to identify, isolate and characterize important genes controlling Se metabolism.
The project has continued on track and at or exceeding goals for FY13. The overall project objective is to elucidate mechanisms of nutrient accumulation in crop plant tissues so that the underlying genes can be used as breeding markers or transgenes to develop more healthy and nutritious crops. 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). For example, we have found that the tomato rin gene is a master regulator of ripening at the level of both transgene effects on multiple crop species and via analysis of RIN binding to numerous fruit ripening gene promoters. These results confirm RIN is an optimal target for manipulating the complete fruit ripening process to optimize both production traits (i.e., uniform ripening and shelf-life) and consumer traits (flavor, texture and nutritional quality). In the last year we have completed development and characterization of transgenic plants altered in expression of the tomato UNIFORM RIPENING (u) gene and showed that this gene and its homolog (SlGLK1) are capable of influencing fruit chloroplast development and photosynthesis, and thus directly impacts sugar accumulation, appearance and quality of ripe fruit. The tomato genome sequence has been completed by an international consortium of plant scientists and led by ARS scientists at the Robert Holley Center and is a powerful resource for the identification of key genes regulating nutritional content, ripening and shelf-life. In the last year this project has also continued work on refinement of the tomato genome sequence to bring it to the highest quality standard possible with available resources. 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 on identification of genes and proteins influenced by the cauliflower OR and tomato RIN and U proteins. Finally, whole genome level characterization of RIN binding sites indicates that RIN regulates numerous ripening genes in a manner dependent on epigenome dynamics which are substantially altered during fruit development and ripening.
With regard to data dissemination, our public database has been updated to house and disseminate all of the information reported here in addition to integrated data recovered from the public domain.
Identified proteins that interact with the ORANGE (Or) protein regulating carotenoid accumulation in plant tissues. Carotenoid is a class of secondary metabolite which are important to human health and nutrition. ARS researchers at Ithaca, New York have investigated the general regulatory mechanism of carotenoid accumulation via Or gene activity. In the last year they have identified proteins that interact with Or to mediate enhanced carotenoid accumulation. A key finding was the interaction of Or with phytoene synthase (PSY), the rate limiting step in carotenoid synthesis in most tissues. This interaction stabilized the PSY protein resulting in increased activity and provides a mechanistic explanation for Or activity. These findings have the potential to increase crop carotenoid accumulation through manipulation of Or and/or its associated protein partners.
Defining the role of epigenome dynamics in the regulation of fruit development and ripening. Plant development can be affected by DNA methylation status. In the last year ARS researchers at Ithaca, New York, demonstrated that changes in DNA cytosine methylation are dynamic during fruit development and ripening and are specifically associated with binding of transcription factors necessary for ripening. This work opens an entire new frontier in possibilities for identification of and selection for genetic variation based on epigenome modification in addition to specific changes in DNA sequence with benefit to crop improvement.
Completion of the tomato genome sequence. The 950 Mb tomato genome sequence was accomplished under the prior project with the ARS researchers at Ithaca, New York leading the US contribution toward a large multinational effort. The tomato genome sequence is of exceptional quality and was reported in Nature in 2012 with extensive world-wide media attention. The tomato genome sequence will drive future crop improvement activities for tomato and serve as a reference sequence to facilitate crop improvement for many related and important crops including pepper, potato, eggplant and coffee. In the last year we and collaborators have utilized PacBio sequencing and optical mapping to close the majority of gaps in the tomato genome sequence making it one of the most complete and accurate plant genome sequences available.
Lui, J., Li, H., Miao, M., Tang, X., Giovannoni, J.J., Xiao, F., Liu, Y. 2012. The tomato UV-damaged DNA-binding protein-1 (DDB1) is implicated in pathogenesis-related (PR) gene expression and resistance to Agrobacterium tumefaciens. Molecular Plant Pathology. 13:123-134.
Barry, C., Aldridge, G., Herzog, G., Ma, Q., Mcquinn, R., Hirschberg, J., Giovannoni, J.J. 2012. Altered chloroplast development and delayed fruit ripening caused by mutations in a zinc metalloprotease at the lutescent 2 locus of tomato. Plant Physiology. 159:1086-1098.
Liu, J., Tang, X., Gao, L., Sun, X., Miao, M., Guo, X., Niu, X., Giovannoni, J.J., Xiao, F., Liu, Y. 2012. The tomato UV-damaged DNA binding protein 1 (DDB1) plays a role in organ size control via an epigenetic manner. PLoS One. 7(8):e42621.
Osorio, S., Alba, R., Nikoloski, Z., Kochevenko, A., Fernie, A., Giovannoni, J.J. 2012. Integrative comparative analyses of transcript and metabolite profiles from pepper and tomato ripening and development stages uncovers species-specific patterns of network regulatory behavior. Plant Physiology. 159:1713-1729.