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Ying Jia, Dianren Xia, and E.S. Louzada

A cDNA coding for a putative terpene synthase (Grtps) was isolated from `Rio Red' grapefruit (Citrus paradisi Macf.) mature fruit by differential display RT-PCR and the corresponding full-length cDNA and genomic clone were subsequently obtained. The isolated cDNA clone was 1644 bp in length encoding a protein of 548 amino acids with a predicted molecular mass of 64 kDa and of pI 5.38. The genomic clone was 3203 bp in length with 6 introns and 7 exons. This Grtps appears to be a sesquiterpene synthase based on molecular weight, genomic organization, and similarity with the other terpene synthases. Both RT-PCR and Northern blot expression analysis indicated that Grtps is not expressed in immature fruits, roots, or leaves, but only in mature fruits. Southern blot analysis of genomic DNA demonstrated that Grtps is one of the members in the family of terpene synthases.

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José A. Narváez, Patricia Flores-Pérez, Virginia Herrera-Valencia, Fernando Castillo, Roberto Ku-Cauich, Blondy B. Canto-Canché, Nancy Santana Buzzy, and Renata Rivera-Madrid

The tropical plant Bixa orellana L. (annatto) is the sole source of bixin, the most frequently employed natural pigment in the food industry. Little is known about the physiology, biochemistry, and molecular genetics of this crop. Our purpose was to establish a set of analytical tools that could be applied in the genetic improvement of B. orellana, particularly for the screening of characteristics such as bixin content and resistance to diseases or pests. Some preliminary results on the study of carotenoid synthesis are presented. In vitro cultures from several B. orellana tissues were established and DNA, RNA, and proteins were extracted from them and analyzed. Similarly, bixin and total carotenoids were quantified.

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Cristián Vela-Hinojosa, Héctor B. Escalona-Buendía, José A. Mendoza-Espinoza, Juan M. Villa-Hernández, Ricardo Lobato-Ortíz, Juan E. Rodríguez-Pérez, and Laura J. Pérez-Flores

their importance in understanding the regulatory cross-talk that contributes to the nutritional quality of tomato fruit ( Almeida et al., 2014 ; Quadrana et al., 2013 ). Carotenoids, tocopherols, and chlorophylls are isoprenoid-derived compounds that

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Rufino Perez and Randolph M. Beaudry

Volatile production is known to change with stages of plant organ development. Research has primarily focused on ripening-related volatiles; however, the potential exists to use volatiles as markers of organ damage and senescence. We have employed gas chromatography/mass spectrometry to establish stages of senescence based on volatile profiles of whole and lightly processed broccoli and carrot. An air-tight chopping apparatus was used as a flow-through chamber system and the exit gas stream analyzed for each commodity with and without tissue disruption. For carrot, isoprenoid pathway volatiles, such as 3-carene, caryophellene, α-caryophellene, and β-pinene, increase with damage and tissue senescence. Similar trends were obtained for broccoli with volatiles characteristic of β-oxidation and shikimic acid pathways. Time and condition-related volatile profile changes will be presented for carrot, broccoli, and strawberry.

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Megan J. Bowman, David K. Willis, and Philipp W. Simon

Carotenoids are isoprenoid compounds synthesized in plants that serve as photoprotectants essential for photosynthesis and provide plant tissues with red, orange, and yellow pigmentation. These compounds are important in human health, because they serve as both vitamin A precursors as well as having antioxidant properties. Carrot (Daucus carota ssp. sativus) provides an important source of carotenoids in the human diet, providing up to 30% of provitamin A in the United States. Although essential to human health, very little is currently understood about the accumulation of carotenoids in carrot. To better understand the molecular mechanism for carotenoid accumulation in carrot, we used reverse-transcription quantitative polymerase chain reaction (PCR) to evaluate the expression of nine genes in the carotenoid biosynthetic pathway in storage root tissue. No significant difference was found among white, yellow, orange, and dark orange carrot roots in seven of the nine genes evaluated. However, increased phytoene synthase 1 (PSY1) and phytoene synthase 2 (PSY2) expression was observed in orange and dark orange carrot roots compared with yellow and white carrots. Increased PSY1 and PSY2 expression was not observed in the leaf tissue of these genotypes, indicating a different mechanism for carotenoid accumulation in the leaf tissue of carrot. This study is the first to demonstrate that naturally occurring mutations that dramatically increase carotenoid accumulation in orange carrot are associated with increased PSY1 and PSY2 expression and it provides insights into the mechanism underlying the biosynthesis of these important photoprotectants and nutrients.

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H.P.V. Rupasinghe, G. Paliyath, and D.P. Murr

α-Farnesene is an acyclic sesquiterpene hydrocarbon that is a constituent of the volatile components and the surface wax of apples (Malus ×domestica Borkh.). Although oxidation products of α-farnesene have been implicated in the development of superficial scald in apples, the relation between α-farnesene biosynthesis and scald development is not well understood. In vivo labeling studies using isolated tissue segments showed that α-farnesene is derived from trans,trans-[1,2-14Cor 1-3H]-farnesyl pyrophosphate (FPP) mostly in the skin rather than cortex tissue. Among other labeled products, farnesol was >100-fold higher compared to α-farnesene. However, HPLC analysis of hexane-extractable components from apple skin revealed farnesol is not a predominant natural constituent of apple skin tissue. In addition, trans,trans-[1-3H]-farnesol was not converted to α-farnesene by apple skin tissue. Our results indicate that biosynthesis of α-farnesene in apple tissue occurs through the isoprenoid pathway, and the conversion of FPP to α-farnesene is catalyzed by a single sesquiterpene synthase enzyme, trans,trans-α-farnesene synthase, rather than via farnesol as an intermediate. A comparison of α-farnesene biosynthesis between scald-developing and scald-free regions of the same apple showed that incorporation of radiolabel into α-farnesene from trans,trans-[1-3H]-FPP was nearly 3-fold lower in scald-developing skin tissue than in scald-free skin tissue.

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Zhenyong Wang and David R. Dilley

We are investigating alternative strategies to control scald on apples. Ethanol vapors were applied to `Law Rome' and `Red Delicious' apples in the storage chambers by ventilating air through aqueous solutions of ethanol at different concentrations, and in modified atmosphere packages by adding various initial concentrations of ethanol vapor. Fruits in storage chambers treated with ethanol vapor at 1600 ppm for about 2 months showed no scald when stored for an additional period in air storage whereas the scald index in control was up to 2.33 (the highest is 3). The similar results in the modified atmosphere experiments confirmed that ethanol vapor could prevent apple scald. Ethanol vapor treatment was also correlated with a reduction of α-farnesene production by the fruits. α-farnesene is an isoprenoid metabolite in the pathway to carotenoid synthesis that has been implicated indirectly as a factor in scald development. Evidence for this based on diphenylamine (DPA) reducing the level of a conjugated terpene product of α-farnesene oxidation. Our results suggested that the control of scald by ethanol vapor treatment may be related to the reduction of α-farnesene production and its subsequent oxidation. Ethanol vapor treatment resulted in accumulation of ethanol in the fruits in direct proportion to the ethanol concentration administered and reduced the rate of ethylene production, and the internal ethanol levels dropped rapidly when fruits were returned to air without ethanol vapor.

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Susan Lurie, Amnon Lers, Zohar Shacham, Lilian Sonego, Shaul Burd, and Bruce Whitaker

Untreated control, 1-methylcyclopropene (1-MCP)-treated, and heated fruit of the superficial scald-susceptible `Granny Smith' cultivar of apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] were compared with respect to scald incidence, internal ethylene concentration (IEC), α-farnesene metabolism, expression of the genes AFS1, which encodes α-farnesene synthase, the final, rate-limiting enzyme in the α-farnesene biosynthetic pathway, and HMG2 and HMG3, which encode isozymes of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the proposed rate-limiting enzyme in the mevalonate pathway of isoprenoid synthesis. The incidence of scald in untreated `Granny Smith' apples after 16 weeks at 0 °C plus 1 week at 20 °C was 100%; 1-MCP treatment prevented scald development, whereas heat treatment delayed and reduced scald development. 1-MCP also inhibited both α-farnesene and IEC, suggesting that ethylene induces transcription of key genes involved in α-farnesene biosynthesis. Heat treatment reduced levels of α-farnesene and and its oxidation products, conjugated trienols (CTols), but not to the extent of 1-MCP. Internal ethylene concentrations in heated apples did not differ from those in the controls. In both control and heated fruit, a sharp increase in AFS1 mRNA during the first 4 weeks of storage preceded an increase in α-farnesene and a subsequent increase in CTols. AFS1 transcript was absent from 1-MCP-treated apples for the first 10 weeks of storage, and even at 16 weeks was lower than in heated and untreated control fruit. Levels of the HMG2 and HMG3 transcripts varied during storage and among treatments, and were not correlated with the incidence of scald. HMG2 mRNA transcript accumulation was low at harvest and increased in abundance during storage in all treatments, with the greatest increase occurring in 1-MCP-treated fruit. In contrast, HMG3 transcript was constitutively present at all storage times, although it too was slightly more abundant in 1-MCP-treated fruit.

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Dong Sik Yang, Ki-Cheol Son, and Stanley J. Kays

also release a diverse cross-section of volatiles into the surrounding environment. Three primary pathways (isoprenoid, shikimic acid, and the oxidative cleavage and decarboxylation of various fatty acids) are responsible for the synthesis of many of

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T. Casey Barickman, Dean A. Kopsell, and Carl E. Sams

stress response in the plant. The increased activities of these enzymes require an available source of isoprenoid substrates, which leads to the production of carotenoids. Previous research has demonstrated that abiotic stress-induced ABA formation leads