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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Food Quality Laboratory » Research » Research Project #419861


Location: Food Quality Laboratory

2014 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.

3. Progress Report:
This report covers year 4 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 which altered the expression of over 1,000 genes. Among them, three genes were selected for further functional studies. Increasing and silencing the expression of calcium-responsive gene displayed obvious effect on fruit ripening and disease resistance. Calcium-regulated SR genes/proteins are 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 ripening, softening, and responses to chilling, wounding and pathogen infection were evaluated. The lines with tolerance to wounding and pathogen infection were identified. 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. Last year, it was found that application of calcium increased yield by over 50% and shelf-life of calcium-treated microgreens was extended by one week compared with untreated controls. Work performed this year indicated that calcium treatment increased the content of glucosinolates, a group of health beneficial compounds unique to species in the Brassica family. Thus, this treatment could be implemented by the industry to substantially increase the productivity, nutritional value, and marketing period of microgreens. Strawberry is an economically important crop, and its fruit are rich in anthocyanins and related bioactive compounds. Last year, it was found that FvUGT1, a gene that encodes a fruit specific UDP-glucose:anthocyanidin 3-O-glucosyltransferase, and other genes in the anthocyanin biosynthetic pathway are regulated by calcium. Further studies of the FvUGT1 enzyme showed its activity is decreased (inhibited) at high substrate concentrations. Calcium together with a calcium-regulated protein relieved this inhibition. Similarly, it was found that FvMYB10, the key transcriptional activator of anthocyanin biosynthetic genes, is a calcium-regulated gene/protein. This work provides an opportunity to increase the anthocyanin accumulation in fruits by modifying FvUGT1 and FvMYB10 structures at the sites regulated by calcium. Arctic™ Granny apple is a transgenic “non-browning” variety wherein all polyphenol oxidase (PPO) genes are inactivated. The aim of developing Arctic™ lines of Granny Smith and other commercial apple cultivars was to eliminate flesh browning as a result of slicing or other injury. Experiments were conducted to compare the appearance of superficial scald, a skin-browning storage disorder, in Arctic™ Granny and Granny Smith control apples harvested from the same location. Fruit of each variety were removed from 1°C storage at 22 and 29 weeks, and evaluated for scald incidence and severity after 8 days at 22°C. Accumulation of conjugated trienols, scald-associated stress metabolites, was lower in peel tissue of Arctic™ Granny compared with Granny Smith apples. In accord with this finding, scald incidence and severity were reduced in Arctic™ Granny apples. Nevertheless, the PPO-suppressed fruit did develop scald symptoms. Thus, while PPO may contribute to oxidative stress that promotes scald, it plays only a minor role in the browning reactions associated with death of hypodermal and epidermal tissues.

4. Accomplishments
1. Anthocyanins are the red to blue pigments present in many fruits and vegetables that appeal visually to consumers. Anthocyanins are also recognized as compounds with potential health-benefits, such as anti-inflammatory and anti-carcinogenic activity, cardiovascular disease prevention, obesity control, and alleviation of diabetes. Strawberry is an economically important crop, and its fruit are rich in anthocyanins and related bioactive compounds. To determine which genes are most important for anthocyanin accumulation in strawberries, the expression patterns of anthocyanin synthesis pathway genes were compared in two genotypes that bear either red or yellow fruit, and those genes highly expressed in only red fruit were identified. It was further found that calcium spray treatment of strawberry plants boosted expression of the same set of anthocyanin pathway genes and increased anthocyanin accumulation in fruit. These results provide new knowledge that can be used by plant breeders or growers to enhance the color and health-benefits of strawberries.

Review Publications
Kou, L., Yang, T., Luo, Y., Liu, X., Huang, L., Codling, E.E. 2013. Pre-harvest calcium application increases biomass and delays senescence of broccoli microgreens. Postharvest Biology and Technology. 87:70-78.
Sun, J., Liu, X., Yang, T., Solvin, J., Chen, P. 2013. Profiling polyphenols of two diploid strawberry (Fragaria vesca) inbred lines using UHPLC-HRMSn. Analytical Biochemistry. 5:2945-53.