2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Tissue softening and deterioration in harvested fresh fruits and vegetables due to ripening, senescence, and physiological disorders result in huge economic loss, including the expense of growing, harvesting, handling, shipping, and storing the produce. In addition, as fresh-cut products claim an increasing percentage of the market share, there are the added costs and concerns of peeling and cutting, sanitation, and greatly decreased shelf-life and resistance to decay. Molecular-genetic means are being developed to reduce the rate or extent of fruit ripening, softening and senescence. The biochemical mechanism of the costly storage disorder superficial scald in apple and pear fruits is being elucidated in order to understand the genetic basis for natural resistance to the disorder and ultimately eliminate the need for prestorage application of diphenylamine plus a fungicide. Studies of the influence of modified or controlled atmospheres, chemical and physical treatments, packaging, and temperature management on the shelf-life, quality, and safety of fresh-cut fruits and vegetables will provide the fresh-cut industry with valuable information that ultimately will benefit consumers. The far-reaching health benefits of fresh fruits and vegetables in the diet are now well documented and it is recommended that people consume 5 to 10 servings per day. The extent to which the public follows these recommendations depends on the ability of the industry to provide high quality fresh produce, with good color, texture, flavor, aroma and nutritional value, which is free of pesticides and other harmful residues. There is also an increasing desire by consumers for the convenience of fresh-cut products that can be served as is or incorporated into salads and other meal preparations. Problematically, the added handling and inherent perishability of fresh-cut fruits and vegetables render them much more prone to proliferation of human pathogenic bacteria such as Listeria, Salmonella and Escherichia coli 0157:H7. Hence, poor sanitation and temperature management can result in fruit and vegetable products that pose a public health risk at least as great as that known to exist with poultry and meat products. Research must be conducted to meet the needs of the fresh produce industry and the desires of the consumer. This research project falls within Component 1 of NP-306 (Quality Characterization, Preservation, and Enhancement). The project plan focuses on Category 1 (Fruits, Vegetables, Tree Nuts, and Sugar Crops) and addresses Problem Area 1a (Definition and Basis for Quality) and Problem Area 1c (Factors and Processes that Affect Quality). The research will generate transgenic lines of tomato and evaluate the fruit for superior quality and shelf life, develop storage technologies for adoption by the fresh-cut produce industry identify strategies to prevent the storage disorder superficial scald in apples without use of chemical treatments that restrict export to foreign markets, pose consumer health and environmental risks, and are costly for apple growers. 2.List the milestones (indicators of progress) from your Project Plan. Milestones from Project Plan 1275-43000-009-00D, which began on 07-08-2004 and replaced Project Plan 1275-43000-007-00D. Year 1 (FY 2005) Complete tomato beta-galactosidase 4 (TBG4) suppression constructs Year 2 (FY 2006) Complete analyses of TBG1, TBG4, & TBG5 substrate specificities Complete cloning and bacterial expression of LePLDa2 cDNA Year 3 (FY 2007) Complete TBG4 transformation in 'Ailsa Craig' and publish results Complete characterization of TBG4 transformants Complete evaluation of fruit quality for E8 promoter-LEPLD2 antisense lines Optimize bacterial expression of LEPLD2 & LePLDa2 and purify native PLDa(s) Year 4 (FY 2008) Establish expression systems for TBGs 2, 3, 6, & 7 that yield active enzymes Characterize phenotypes of crosses of TBG4-PG-EXP1 suppressed lines Generate & propagate transformed tomato lines over-expressing galactokinase Complete cloning, analysis, & GUS fusion constructs of LEPLD2 & LePLDa2 promoters Year 5 (2009) Complete analyses of substrates for all TBGs Determine phenotypes of galactokinase over-producing tomato lines Generate transgenics with suppression of both PLDas, PLDa + LAH, and/or PLDa + TBGs Complete biochemical characterization of tomato PLDa isozymes Complete analysis of GUS-LEPLD promoter tissue-specific expression 3a.List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. Complete tomato beta-galactosidase 4 (TBG4) suppression constructs Progress: Both multi-copy 3'UTR and RNAi constructs of TBG4 were made and cloned into a binary vector. Milestone Fully Met Complete analyses of TBG1, TBG4, & TBG5 substrate specificities Progress: Activities with both synthetic and plant cell wall derived substrates were determined for TBG1, TBG4, & TBG5 expressed in yeast. Milestone Fully Met Complete cloning and bacterial expression of LePLDa2 cDNA Progress: Full-length LePLDa2 cDNA was cloned and the open reading frame was expressed in E. coli, yielding a 92-kDa immunoreactive PLDa protein. Milestone Fully Met 3b.List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006 Complete TBG1, TBG4, & TBG5 substrate analyses and publish results Transform 'Ailsa Craig' tomato plants with TBG4 suppression constructs Generate transformed tomato lines over-expressing galactokinase Optimize bacterial expression of LEPLD2 Complete evaluation of fruit quality for E8 promoter-LEPLD2 antisense lines FY 2007 Complete TBG4 transformation in 'Ailsa Craig' and publish results Complete characterization of TBG4 suppressed transformants Establish expression systems for TBGs 2, 3, 6, & 7 that yield active enzymes Publish results on fruit quality of LEPLD2 E8-antisense tomato lines Determine substrates and inhibitors of bacterially-expressed LEPLD2 Optimize bacterial expression of LePLDa2 Isolate native LEPLD2 and/or LePLDa2 and purify to homogeneity Clone LEPLD2 & LePLDa2 promoters and make GUS fusion constructs FY 2008 Complete crosses of 'Ailsa Craig' TBG4-PG-EXP1 knockouts Complete analyses of TBG 2, 3, 6, & 7 substrate specificity Characterize phenotypes of galactokinase over-expressing lines Determine substrates and inhibitors of bacterially-expressed LePLDa2 Determine substrates and inhibitors of native fruit PLDa(s) Complete study of LEPLD2 promoter-GUS tissue-specific expression 4a.What was the single most significant accomplishment this past year? The enzymes phospholipase D (PLD) and lipoxygenase (LOX) have been implicated to play a role in ripening, softening and senescence in melon fruit. CmPLDa1 and CmLOX1, a pair of cDNAs encoding a PLD-alpha and a 13-LOX, were cloned from a hybrid honeydew melon cultivar using RNA isolated from leaf and fruit tissues. Quantitative analysis of CmPLDa1 and CmLOX1 transcript levels showed that expression of both genes was relatively high in fruit mesocarp tissues, as well as root and flower tissues. Regardless of the tissue or stage of fruit development, CmPLDa1 transcript was much more abundant than that of CmLOX1. During fruit development from 20 to 55 days post-pollination (dpp), with 55-day fruit being fully ripe, distinct patterns of expression were observed for both genes in hypodermal compared with middle mesocarp tissues. The former tissue is important for control of water loss, while the latter includes much of the edible fruit flesh. The expression profiles of CmPLDa1 and CmLOX1 in both mesocarp tissues were consistent with a role in ripening and senescence, with maximum transcript abundance at 50-55 dpp. In particular, maximum levels of CmPLDa1 mRNA were observed at 50 dpp in middle mesocarp and at 55 dpp in hypodermal mesocarp, which matched the maximum PLDa protein levels determined by immunoblot analysis. The goals of this research are to understand the roles of PLD and LOX enzymes in melon fruit ripening and senescence, and to use that knowledge to develop transgenic lines bearing high quality fruit with long shelf life both as a whole and fresh-cut product. 4b.List other significant accomplishments, if any. Activity of yeast-expressed tomato beta-galactosidases 1, 4, & 5 was determined with both synthetic and plant-derived cell wall substrates. During development and ripening, the most prominent change in tomato fruit pericarp cell wall composition is the loss of galactosyl residues. To understand the role of galactosyl turnover in fruit softening, three recombinant tomato beta-galactosidase/exo-galactanase (TBG) fusion proteins were produced in yeast. TBG1, 4 and 5 enzyme properties were assessed, including pH & temperature optima, Kms, and substrate specificities. TBG1 & TBG5 released galactosyl residues from 1-4 linkages in synthetic and plant-derived substrates. TBG4 released galactosyl residues from a wide range of plant-derived oligosaccharides and polysaccharides. Using tomato fruit cell wall material, TBG1, TBG4 and TBG5 released galactosyl residues from a number of cell wall fractions derived from fruit at several ripening stages. TBG4 activity was greatest with the acid-soluble pectin (ASP) fraction, particularly with the ASP fraction from Turning stage fruit. Although cell walls from Turning fruit contain fewer total galactosyl residues than walls from Mature Green fruit, TBG4 released 3 to 4-fold more galactose from pectic fractions isolated from Turning fruit, suggesting that structural changes in wall pectic polysaccharides prior to the Turning stage cause the wall to become more prone to hydrolysis by TBG4. The aim of this work is to develop transgenic tomato lines that bear fruit with longer shelf life and improved quality as both a fresh market and fresh-cut product. The gene PcAFS1, encoding alpha-farnesene synthase (AFS), was cloned from d'Anjou pear using RT-PCR with peel tissue RNA and primers based on the equivalent apple gene AFS1. Treatment of d'Anjou pears prior to cold storage with 1-MCP, a potent inhibitor of ethylene action, delayed up-regulation of PcAFS1, and inhibited alpha-farnesene production and oxidation. After 6 months at -1C, only 13% of the 1-MCP-treated pears developed the costly storage disorder superficial scald, whereas scald incidence was 100% in untreated fruit after 3 months or more at -1C. These results strongly support the hypothesis that the biochemical mechanism of scald is the same in susceptible apple and pear fruits, involving oxidation products of alpha-farnesene. The current limitation of treatment of d'Anjou pears with a dose of 1-MCP sufficient to prevent scald is the coincident inhibition of ripening. With further refinement of the treatment and storage regime, it may be possible to overcome this problem. Alternatively, transgenic suppression of PcAFS1 could be used to block alpha-farnesene production. The goal of this work is to extend storage life and prevent scald development in pears without pre-storage chemical treatment. The most serious postharvest problem in bell peppers is water (weight) loss and consequent shriveling. The cause of water loss is not known, but cuticle thickness and composition are thought to be determining factors. To address this problem we examined different harvesting and storage methods using four commercial cultivars (cvs). Our results over two growing seasons showed that neither the harvesting method (tearing versus cutting) nor the storage temperature had a significant impact on fruit water loss, although temperatures below 10C resulted in increased chilling damage. In contrast, large differences in water loss were noted among fruit of the four cvs. One cv widely grown in Maryland had the highest rate of water loss and also exhibited the most chilling injury. Fruit from the other three cvs lost water weight much more slowly, and the cv with the slowest rate of weight loss was also resistant to chilling injury. Wax samples extracted from fruit of the four cvs are currently being analyzed to assess the role of the natural waxy coating in limiting water loss. Our research will help plant breeders to identify superior bell pepper cvs and to map the trait of water loss. This knowledge can then be used in traditional breeding efforts to improve bell pepper quality, without resorting to transgenic modification. 4c.List any significant activities that support special target populations. None 4d.Progress report. None 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Project Plan 1275-43000-009-00D, initiated on 07-08-04, bridged with the prior Project Plan 1275-43000-007-00D, for which the major accomplishments are described. Discovered and characterized a family of seven genes coding for beta-galactosidases, key enzymes involved in tomato fruit development, ripening and softening. Antisense tomato plants were constructed which produced fruit 40% firmer than wild-type fruit, indicating a critical role for this enzyme in cell wall alterations leading to fruit softening. The clones have been patented and are being distributed to other researchers who will elucidate the role of beta-galactosidases in a variety of plants and plant processes. A potential commercial use of this family of genes would be to increase viscosity and quality of tomato products such as ketchup, sauce and paste. Found that antisense suppression of TBG6, the most abundantly expressed beta-galactosidase gene in tomato fruit, caused a marked increase in cracking and deformation of internal structure. In normal wild-type fruit, TBG6 is turned on during early development and completely off at the onset of ripening. Taken together, these findings indicate that during fruit development the TBG6 gene product is required for proper cell expansion or enhances tissue elasticity. A practical application of this discovery will be to generate transgenic plants bearing fruit that continue to express TBG6 during ripening. It is expected that these transgenic fruit would be much less prone to cracking during transport, which is a currently a serious problem for the tomato industry. Cloned and characterized a gene encoding a phospholipase D-alpha (PLDa) from tomato fruit. PLDa degrades membrane lipids and is involved in senescence and in responses to stress and plant pathogens. Expression of the PLDa gene was shown to increase 8-fold over the course of fruit development and ripening, suggesting a role of the enzyme in ripening and senescence. Tomato plants were transformed with an antisense construct of the PLDa gene, and fruit from several transformants were shown to have 4- to 5-fold lower gene expression as well as markedly reduced PLDa enzyme activity. The aim of this work is to develop transgenic tomato lines that bear fruit with a longer shelf life at the red-ripe stage, both as a whole and fresh-cut product, thus allowing fuller development of color and flavor prior to harvest and processing. Cloned genes encoding two key enzymes of the farnesene synthesis pathway in apple fruit: hydroxymethylglutaryl CoA reductase (HMG isogenes 1, 2, &. 3)and farnesene synthase (FS). Farnesene oxidation products have been linked with development of superficial scald in apples and pears, a costly storage disorder that is currently controlled by expensive and potentially unsafe chemical treatments. We found that accumulation of farnesene in apple peel tissue during the initial weeks of storage coincides with increased expression of the FS gene, and that these increases do not occur if tissue response to the plant hormone ethylene is blocked. Characterization of the FS and HMGR genes and their promoters and enzyme products will elucidate how ethylene promotes farnesene synthesis in stored apples and form the basis of a molecular genetic strategy to prevent scald without postharvest chemical treatments. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A biotechnology corporation in the U.S. has expressed interest in use of the tomato beta-galactosidase genes to produce enzymes for new industrial processes such as synthesis of biopolymeric films. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). None Review Publications Gapper, N.E., Whitaker, B.D., Bai, J. 2005. Inhibition of ethylene-induced alpha-farnesene synthase gene expression in d'anjou pears with 1-mcp delays alpha-farnesene synthesis and oxidation and prevents superficial scald [abstract]. Plant Biology 2005 Meeting. p.84. Ishimaru, M., Smith, D.L., Gross, K.C. 2005. Yeast expressed tomato beta-galactosidases 1, 4 and 5 have activity against synthetic and plant-derived cell wall substrates [abstract]. Hortscience. 40(4):1092. Lurie, S., Lers, A., Shaham, Z., Sonego, L., Burd, S., Whitaker, B.D. 2005. Expression of a-farnesene synthase and hmg-co a reductase genes in relation to levels of a-farnesene and conjugated trienols in peel tissue of 'granny smith' apples treated with heat or 1-mcp to prevent scald. Journal of the American Society for Horticultural Science. 130(2):232-236. Pechous, S.W., Watkins, C.B., Whitaker, B.D. 2005. Expression of alpha-farnesene synthase gene afs1 in relation to levels of alpha-farnesene and conjugated trienols in peel tissue of scald-susceptible 'law rome' and scald-resistant 'idared' apple fruit. Postharvest Biology and Technology. 35:125-132.