2013 Annual Report
1a.Objectives (from AD-416):
The goal of this project is to facilitate development of new genetic lines of fruits and vegetables that are superior with respect to sensory quality, storage life, and betterment of human health by providing breeders with the knowledge and molecular tools they require. Research over the next 5 years will pursue the following two broad objectives: Objective.
1)Determine molecular mechanisms governing natural and stress-induced deterioration of fresh produce quality during postharvest storage and shelf life; and Objective.
2)Identify, clone, and manipulate key genes regulating accumulation or loss of phenylpropanoids and other health-beneficial secondary metabolites in stored whole and fresh-cut fruits and vegetables. The initial phase under objective 1 will aim to identify and clone both regulatory and metabolic genes potentially involved in ripening, senescence, and responses to stress (e.g. low temperature) in fresh fruits and vegetables. Primary focus will be on genes and encoded proteins regulated by calcium, which is known to retard senescence and mitigate certain stress disorders, and on genes/enzymes directly involved in degradation of cell membranes. Gene silencing and other molecular strategies will then be used to confirm the critical role of specific genes/enzymes in ripening, senescence, and stress responses. This will provide target genes for manipulation or germplasm screening to yield new lines of produce with extended storage life and resistance to stress disorders. Under objective 2 there will be two foci. The first is to identify and clone genes that can be manipulated to enhance accumulation, retention, and/or bioavailability of anthocyanins (red pigments) and other flavonoids in sweet cherry and strawberry. Dietary intake of this group of plant chemicals is known to confer protection against cardiovascular disease, diabetes, cancer, and stroke. The second pursuit under objective 2 will be to identify, clone, and manipulate key genes in biosynthesis of health-beneficial hydroxycinnamic acid conjugates in wild germplasm of eggplant and tomato that can be transferred to commercial lines to yield new functional foods.
1b.Approach (from AD-416):
Immature green and breaker tomato fruit pericarp tissue discs will be dipped in 2% calcium chloride solution or water, and then frozen in liquid nitrogen 0–6 hours after treatment. Extracted total RNA will be hybridized to a gene chip for the tomato genome, and genome-wide expression profiles will be studied by microarray analysis. Major genes regulated by calcium will be determined by bioinformatic analysis and possible crosstalk with other signaling pathways. Expression patterns of selected key genes during all phases of fruit development, and in response to calcium treatment, will be studied using real-time RT-PCR. Gene expression in response to ethylene, phosphatidic acid, methyl jasmonate, chilling, and fungal elicitors will also be tested to find if there is interplay with calcium-regulated genes that control fruit ripening and senescence. Key calcium-regulated genes will be subjected to further functional studies. Primary focus will be on several genes/proteins already known to be regulated by Ca/calmodulin and believed to play roles in enhancement or loss of produce quality, including the novel transcription factor SR and phospholipase D (PLD) families. All SR genes in tomato will be isolated by screening a cDNA library using full length LeSR1 as a probe. Expression profiles will be determined for all LeSR and LePLDa family genes in response to various treatments by microarray analysis. The promoter of the apple a-farnesene synthase gene AFS1 will be analyzed for ethylene responsive elements (EREs) involved in ethylene-mediated activation. Promoter deletion fragment-GUS fusion constructs will be used in transformation studies to identify functional EREs. A yeast one-hybrid system will be used to screen a cDNA library for transcription factors that bind to AFS1 EREs. Five bioactive compounds identified by chemical genomic screening will be applied to mature fruiting sweet cherry trees, and the harvested ripe fruit evaluated for treatment effects on key quality attributes over time. Subtractive cloning methods will be used both to determine genes specifically associated with quality enhancement of strawberries treated with bioactive compounds, and to identify and isolate key genes involved in synthesis of nutraceutical hydroxycinnamic acid-polyamine amides in fruit of a wild eggplant relative. Ca/calmodulin regulation and the functions of UDP-glucosyltransferases related to synthesis and bioavailability of anthocyanins and flavonoids will be studied in strawberry fruit. Stable or transient transformation with silencing or over-expression gene constructs driven by constitutive or fruit-specific promoters will be used to assess the function of specific genes or gene families in various aspects of fruit physiology and metabolism, including ripening, senescence, responses to stress, and accumulation and/or retention of health-beneficial secondary metabolites from the phenylpropanoid pathway. Quality traits such as flavor, color, firmness, stress tolerance, and phytonutrient content will be analyzed in the transgenic lines.
This report covers year 3 of a 5-year project aimed at improvement of fresh produce quality, including appearance, texture, flavor, and nutrition. Objective 1 entails characterization of genes involved in the beneficial effects of calcium on fruit firmness and shelf life in tomato, and efforts to turn off (silence) genes involved in loss of tissue integrity in tomato and melon or peel browning in apple fruit. Objective 2 focuses on genetic means to increase or introduce new health-beneficial compounds in eggplant, strawberry, and other fruits.
Tomato fruit were treated with calcium and consequent global changes in gene expression were determined using a powerful new technique. Altered expression of over 1,000 genes was induced by calcium treatment. On the basis of putative importance in fruit ripening, 20 of those genes were selected for detailed expression analysis. Three were also selected for study of gene function, with the aim of identifying functional markers for use in tomato breeding programs.
A family of SR genes in tomato encodes calcium-regulated proteins involved in responses to stressors such as low temperature, physical injury, and pathogens. Tomato lines were produced for each SR gene in which the gene was turned off (silenced) or turned up (over-expressed). Fruit from these lines are being evaluated for ripening, softening, and responses to chilling, wounding and pathogen infection. This work in concert with studies of calcium effects on gene expression will identify target genes for genetic improvement of tomato fruit quality.
Sales of microgreens (vegetable and herb seedlings) have steadily risen in recent years because their vibrant color, intense flavor, and concentrated vitamins, minerals, and antioxidants appeal to consumers. However, microgreen marketing is limited by short shelf-life resulting from high perishability. Application of calcium was tested as a means to maintain quality and extend shelf-life of broccoli microgreens. The treatment increased yield by over 50% and calcium content by threefold. Moreover, shelf-life of calcium-treated microgreens was extended by one week compared with untreated controls. Thus, this treatment could be implemented by the industry to substantially increase the productivity, nutritional value, and marketing period of microgreens.
Last year it was discovered that expression of the gene UGT1 is essential for accumulation of anthocyanin pigments in strawberry fruit. Work this year showed that increased expression of UGT1 alone did not result in higher anthocyanin levels in ripe fruit because anthocyanin synthesis requires coordinated expression of several genes in the pathway. Moreover, it was found that a point mutation in a calcium-regulated master gene that controls anthocyanin biosynthesis abolishes pigment production, resulting in yellow fruit. Further studies will focus on modification of the master gene’s structure and function to boost levels of anthocyanins and other health-promoting phenolics in strawberries.
Two new antioxidant compounds were identified in fruit of a wild eggplant relative. They will be tested for biological activity related to human disease prevention.
New Solanum metabolic database holds promise for nutritional enrichment of major vegetable crops. The economically important plant genus Solanum includes the valuable crop species tomato, potato, and eggplant as well as numerous wild relatives that are generally regarded as noxious weeds. While the major Solanum vegetables do not entirely lack human health-promoting metabolites, their levels and arrays of beneficial phytonutrients could be substantially improved by introducing selected genes from related wild species. Using cutting-edge analytical tools, a Solanum metabolic database was generated representing 62 compounds of interest from 24 species. The new database enabled creation of a detailed biosynthetic pathway of metabolites in Solanum, which is essential to determine how the abundant and diverse bioactive chemicals are produced in wild and cultivated solanums. This knowledge will aid public and private plant breeders in developing new lines of Solanum crops with superior nutritive value. A long-range benefit of this research is a reduced incidence of diabetes, cardiovascular disease, and other chronic health problems in the U.S. that cost many billions of dollars annually.
Stress tolerance and decay resistance in stored tomato fruit may rely on a family of calcium-regulated genes. Calcium is known to ameliorate many postharvest maladies, but the molecular mechanisms underlying these effects are unclear. The possible roles of members of the calcium-regulated SlSR gene family were explored in responses of tomato fruit to wounding, chilling, and infection by a decay-causing fungus, as well as hormones that activate defenses against various types of stress. The results showed distinct patterns of gene expression among the seven SlSRs and further indicated that individual SlSRs have unique roles in responses to specific stress signals. This research is aimed at understanding the mechanisms of tomato fruit stress tolerance and decay resistance. Knowledge of genes controlling these mechanisms will help plant geneticists and breeders, and their industry partners, develop new tomato lines bearing fruit that maintain good quality after storage and shelf life. Such new tomato lines will reduce the estimated 10-15% postharvest losses annually, saving millions of dollars for growers, packers, and retailers.
Wu, S., Meyers, R.S., Whitaker, B.D., Litt, A., Kennelly, E.J. 2012. Antioxidant glucosylated caffeoylquinic acid derivatives in the invasive tropical soda apple, Solanum viarum. Journal of Natural Products. 75:2246-2250.
Baker, C.J., Kovalskaya, N.Y., Mock, N.M., Deahl, K.L., Owens, R.A., Whitaker, B.D., Roberts, D.P., Hammond, R., Averyanov, A.A. 2013. Real-time monitoring of the extracellular redox potential of cell suspensions during plant/bacterial interactions. Physiological and Molecular Plant Pathology. 82:20-27.
Prohens, J., Whitaker, B.D., Vilanova, S., Hurtado, M., Blasco, M., Plazas, M., Gramazio, P., Stommel, J.R. 2013. Genetic diversity in morphological characters and phenolic acids content resulting from an interspecific cross between eggplant (Solanum melongena) and its wild ancestor (S. incanum). Annals of Applied Biology. 162:242-257.
Yang, T., Peng, H., Whitaker, B.D., Jurick II, W.M. 2013. Differential expression of calcium-regulated SlSRs in response to abiotic and biotic stresses in tomato fruit. Physiologia Plantarum. 148(3):445-455.