2011 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.
Three members of the SR gene family in tomato, which is involved in metabolic regulation by calcium, were identified as the most likely to play roles in fruit ripening, senescence, and resistance to stress. 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 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 will identify target genes for genetic improvement of fruit quality.
Effects on honeydew melon quality and storage life were evaluated for fruit from plants in which genes encoding PLDa and LOX, enzymes thought to play key roles in water loss and senescence, were turned off. LOX silenced fruit did not differ from normal fruit, while PLDa knock-outs showed a promising extension of storage life. Work is underway to confirm that reduced PLDa gene expression and enzyme activity result in better keeping quality of melons.
Anthocyanins, the red to blue pigments in fruit, are known to benefit human health, and work progressed toward enhancement of these compounds in strawberries and sweet cherries. Field tests were completed evaluating novel chemical treatments that delayed harvest and thereby prolonged anthocyanin accumulation. In addition, UGT genes thought to play a key role in anthocyanin synthesis and accumulation were isolated from strawberry. Future studies will determine if increased UGT gene expression results in higher anthocyanin levels in ripe fruit.
A family of compounds (HCAA), including two regarded as beneficial to human health (hydroxycinnamic acids and polyamines), were shown to be present at low levels in eggplant, absent in tomato, and present at very high levels in fruit of a wild eggplant relative. Homologues of a key gene (SHT1) involved in biosynthesis of HCAA were isolated from each species. The SHT1 genes will be studied to find how production of HCAA is regulated, with the long-range goal of genetically increasing or enabling accumulation of these compounds in eggplant and tomato fruits.
Two new antioxidant compounds were identified in fruit of wild eggplant lines. These will be tested for biological activity related to prevention of chronic diseases.
Identification of new antioxidant compounds found in fruit of wild eggplant relatives. Eggplant fruit are rich in antioxidant chemicals that are potentially beneficial to human health. In most cultivated eggplant lines, these so-called nutraceuticals comprise a small number of known compounds, but in fruits of some wild relatives there is an abundance of rare and/or unknown compounds that may have novel beneficial biological activity. Two such previously unidentified antioxidant compounds were isolated from fruit of two wild eggplant species and their chemical structures were elucidated. Antioxidant assays of the compounds revealed two modes of action that both eliminate damaging free radicals and limit their formation. These natural chemicals will also be tested for biological activity that could block or delay the advent of chronic diseases. The end goal of this work is to develop new commercial eggplant lines bearing fruit with a broader array of nutraceuticals that combat various cancers, diabetes, and cardiovascular disease.
Ma, C., Whitaker, B.D., Kennelly, E. 2010. New 5-O-caffeoylquinic acid derivatives in fruit of the wild eggplant relative Solanum viarum. Journal of Agricultural and Food Chemistry. 58:11036-11042.
Baker, C.J., Owens, R.A., Whitaker, B.D., Mock, N.M., Deahl, K.L., Roberts, D.P., Orlandi, E., Averyanov, A.A. 2011. Detection of bacterial aggregation in cell suspensions treated with pathogenic bacteria. Physiological and Molecular Plant Pathology. 75:170-175.
Rudell Jr, D.R., Buchanan, D.A., Leisso, R.S., Whitaker, B.D., Mattheis, J.P., Zhu, Y., Varanasi, V. 2011. Ripening, storage temperature, ethylene action, and oxidative stress alter apple peel phytosterol metabolism. Phytochemistry. 72:1328-1340.