2012 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 2 of a 5-year project aimed at retention and enhancement of fresh produce quality, including appearance, texture, flavor, aroma, 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 novel health-beneficial compounds in eggplant, strawberry, and other fruits.
Three members of the SR gene family in tomato, which are involved in metabolic regulation by calcium, were further characterized with respect to their roles in fruit ripening. Results indicated that SR genes operate downstream of the important ripening regulatory gene RIN, and function as part of both the developmentally-regulated and ethylene-mediated signaling pathways that control tomato ripening. Tomato plants were transformed to yield lines in which each SR gene is turned off (silenced) or turned up (over-expressed). Fruit from these lines will be evaluated for ripening, softening, and responses to chilling and wounding. This work, in concert with studies of calcium effects on gene expression in tomatoes, is aimed at identification of target genes for genetic improvement of fruit quality.
Anthocyanins, the red to blue pigments present in many fruits, are known to promote human health, and work progressed toward genetic enhancement of these compounds in strawberries. The profiles of anthocyanins and related flavonoids were compared in fruit of wild and cultivated strawberry. Several new flavonoids identified in wild strawberry were absent from cultivated fruit. Two growth regulators known to alter anthocyanin accumulation were used to treat strawberry fruit, and genes affected by the treatments were identified. One of those genes, UGT1, was expressed only in fruit and encodes a calcium-regulated enzyme that performs a last step in anthocyanin synthesis. Expression of UGT1 was detected in red but not in yellow fruit, indicating a key role in anthocyanin accumulation. Future studies will find if increased UGT1 gene expression results in higher, health-beneficial anthocyanin levels in ripe fruit.
Levels of HCAA, a family of antioxidant phenolic compounds with reported health benefits, were determined in fruits of cultivated Asian eggplant and eight related wild Solanum species. A wide variation among species was observed in HCAA content and composition. A family of at least five genes encoding putative SHT enzymes that perform the last step in HCAA synthesis was mined from Solanum genetics databases. Comparison of the relative expression of these genes with accumulation of HCAA in various Solanum fruits identified SHTs 1, 3B, and 4 as important enzymes for HCAA production. These three SHT genes and their encoded enzymes will be studied further to find how production of HCAA is regulated, with the long-range goal of increasing or enabling accumulation of these compounds in eggplant and tomato fruits to enhance their disease preventive potency.
Gene required for anthocyanin production in strawberry fruit is identified. Dietary intake of health-promoting phytonutrients such as anthocyanins, the red and blue pigments found in many fruits, is recommended by USDA as a means to combat chronic inflammatory diseases and certain cancers. One way to increase the intake of these phytonutrients is to develop new “super fruit” lines with higher concentrations of the compounds. A last step in biosynthesis of anthocyanins involves the attachment of a sugar catalyzed by an enzyme called UGT. The gene encoding strawberry UGT1 was cloned, and its expression was shown to be fruit specific and directly correlated with anthocyanin accumulation. This finding provides strawberry breeders and plant geneticists a single target gene that can be manipulated to increase accumulation of health-beneficial anthocyanins in strawberry fruit. 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.
A new family of genes involved in regulation of tomato fruit development, ripening, and quality. Rapid softening and over-ripening as well as chilling injury and storage decay are problems that limit the shelf life and increase losses of tomato fruit. It is well documented that fruit treatment with calcium can delay and extend ripening and softening, but the mechanism is unclear. A family of seven tomato SR genes was cloned and characterized, providing a good explanation for the benefits of calcium on fruit quality. The encoded SRs are calcium-regulated proteins called transcription factors that control developmental processes like ripening as well as defense responses to decay organisms and stress. Research results indicated that tomato SR genes serve as a nexus for signaling pathways controlling ripening and stress responses. This knowledge can be used by plant scientists to elucidate the complex interactions of regulators of fruit ripening, and by plant geneticists and breeders for development of new tomato lines that better retain fruit quality after harvest. Such new tomato lines will reduce the estimated 10-15% post-harvest losses annually, saving millions of dollars for growers, packers, and retailers.
Ma, C., Dastmalchi, K., Whitaker, B.D., Kennelly, E. 2011. Two new antioxidant malonated caffeoylquinic acid isomers in fruits of wild eggplant relatives. Journal of Agricultural and Food Chemistry. 59:9645-9651.
Baker, C.J., Kovalskaya, N.U., Mock, N.M., Owens, R.A., Deahl, K.L., Whitaker, B.D., Roberts, D.P., Hammond, R., Averyanov, A.A. 2012. An internal standard technique for improved quantitative analysis of apoplastic metabolites in tomato leaves. Physiological and Molecular Plant Pathology. 78:31-37.
Yang, T., Peng, H., Whitaker, B.D., Conway, W.S. 2012. Characterization of a calcium/calmodulin-regulated SR/CAMTA gene family during tomato fruit development and ripening. Biomed Central (BMC) Plant Biology. 12:19.