Location: Food Quality Laboratory2015 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 5 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. Calcium regulated genes, calmodulins, are critical for tomato fruit development. The expression of six camodulin genes during fruit development and ripening was analyzed. Two peaks were identified: one was in the early stage of fruit development, and another one was in a later stage between orange and red stages. At mature green and breaker stages, the expression of all genes was lowest. A gene SlCaM2 showing the highest expressions during peak times was selected for further analysis. Tomato fruits were produced for SlCaM2 in which the gene was turned off (silenced) or turned up (over-expressed). Fruit ripening and softening were evaluated. Transiently expressing SlCaM2 in mature green fruit delayed fruit ripening, whereas reducing SlCaM2 expression accelerated ripening. This work will identify target genes for genetic improvement of tomato fruit quality. Calmodulin is often involved in the stress response. In response to mechanical injury and Botrytis cinerea infection, expression of all calmodulin genes was stimulated. SlCaM2 was the most responsive gene to both treatments. Furthermore, all calmodulin genes were upregulated by salicylic acid and methyl jasmonate. Overexpression of SlCaM2 in tomato fruit enhanced resistance to Botrytis-induced decay, whereas reducing its expression resulted in increased lesion development. These results indicate that calmodulin is a positive regulator of plant defense in fruit by activating defense pathways including salicylate- and jasmonate- signaling pathways, and SlCaM2 is the major calmodulin gene responsible for this event. Strawberry fruits are rich in anthocyanins and related bioactive compounds. FvMYB10, the key transcriptional activator of anthocyanin biosynthetic genes, was a calcium-regulated gene/protein. Mutation studies revealed that the calmodulin-binding site was critical for the functions of this gene in anthocyanin biosynthesis. The knowledge of the structure/functions of this gene could be used to improve the fruit anthocyanin biosynthesis and quality. Hydroxycinnamic acid amides (HCAAs) are a class of conjugates of hydrocinnamic acid and polyamines catalyzed by polyamine hydroxycinnamoyl transferases (HT). Like most plants, the predominant HCAA in cultivated egg plant fruit (Solanum. melongena) is hydroxycinnamoyl-spermidine (HCSPD). However, wild eggplant fruit (S. richardii) is rich in hydroxycinnamoyl-spermidine (HCSPM). It has been shown that HCSPM confers greater biological and medicinal values. Two novel HTs, SrSHT and SrSpmHT, responsible for the biosyntheses of HCSPD and HCSPMs were identified in S. richardii. SrSHT conjugated hydroxycinnamoyl moieties to spermidine, spermine and putrescine, whereas SrSpmHT exclusively catalyzed one hydroxycinnamoyl moiety to spermine. The results provide a platform for metabolic engineering of HCAA pathway to generate HCSPM rich eggplant fruits.
1. Temporal expression suppression of calmodulin at the mature green stage accelerates tomato fruit ripening. Nearly a quarter of all fresh fruits and vegetables in the U.S. market are lost after harvest. Calcium has been shown to be important in controlling fruit ripening and quality through regulating calcium-binding proteins, such as calmodulin. However, the function of calmodulin remains elusive for fleshy fruit development. ARS scientists in Beltsville, Maryland, found that all calmodulins showed repressed expression at mature green and breaker, the critical stages before fruit ripening. Transiently overexpressing SlCaM2 in mature green fruit delayed ripening, while reducing SlCaM2 expression accelerated ripening. This study provides important information for industry and breeders to enable manipulation of calmodulin levels in order to control fruit ripening and improve overall fruit quality.
2. Increased expression of calmodulin at the mature green stage enhances fruit resistance to pathogens. Tomato is the second most important vegetable crop worldwide. However, nearly 25% of tomatoes are lost after harvest due to decay and quality deterioration. Pre- and post- harvest application of calcium has been shown to reduce decay, delay ripening and maintain firmness in fruits. Yet, the underlying molecular mechanisms involved in these beneficial effects remained unclear. ARS scientists in Beltsville, Maryland, found that overexpression of one calmodulin gene in tomato fruit enhanced resistance to Botrytis-induced decay, whereas reducing its expression resulted in increased lesion development. Therefore calmodulin is a positive regulator of plant defense in fruit. These results provide new knowledge that can be used by plant breeders or growers to improve fruit postharvest quality and reduce decay.
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