The accumulation of carotenoids such as lycopene and beta-carotene greatly influences the quality of ripe tomato (Lycopersicon esculentum) fruit because cellular levels of these compounds determine the intensity of red color. As well, lycopene has anti-cancer properties and beta-carotene is a Vitamin A precursor. Recent work has demonstrated phytochrome regulation of the carotenoid pathway but the mechanism is not completely understood. This work investigates phytochrome regulation of 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and phytoene synthase (PSY), two key enzymes of carotenogenesis. A simple procedure for the assay of PSY from crude pericarp extracts was developed and mRNA levels of DXS and PSY1 genes were measured by relative RT-PCR. Discs from mature green tomatoes were ripened in total darkness, or in darkness interrupted by brief daily treatments of red light, or red light followed by far red light. After ten days of incubation, lycopene levels of red light-treated discs had reached ≈12 mg/100 g fresh weight; nearly a 50% increase over discs ripened in total darkness. This increase was not observed in discs treated with red light followed by far red light, demonstrating the red/far red reversibility (and thus phytochrome control) of carotenoid accumulation. Similar patterns of phytochrome control are observed for PSY activity but not for DXS and PSY1 transcript levels, suggesting the mechanism of control may be at the level of post-translational modification of PSY. Potential applications of this regulation of carotenoid accumulation will be discussed.
Andrew Schofield* and Gopinadhan Paliyath
H.P. Vasantha Rupasinghe, Gopinadhan Paliyath and Dennis P. Murr
To decipher the relation between α-farnesene metabolism and the development of superficial scald in apples, trans,trans-α-farnesene synthase, the enzyme that catalyzes the conversion of farnesyl pyrophosphate to α-farnesene, was partially purified from skin tissue of `Delicious' apples (Malus ×domestica Borkh.) and characterized. Total and specific activities of the enzyme were higher in the cytosolic fraction than in membrane fractions. α-Farnesene synthase was purified 70-fold from the cytosolic fraction by ion exchange chromatography and gel permeation, and the native molecular weight was estimated to be 108,000. The enzyme had optimal activity at a pH of 5.6 and absolutely required a divalent metal ion such as Mg2+ or Mn2+ for activity. It exhibited allosteric kinetics, S(0.5) for farnesyl pyrophosphate being 84±18 μmol·L-1, and a Hill coefficient (nH) of 2.9, indicating the number of subunits to be two or three. Enzyme activity was highest between 10 and 20 °C, while 50% of the maximal activity was retained at 0 °C. In vivo α-farnesene synthase activity was minimal at harvest, then increased rapidly during 16 weeks storage in air at 0 °C, and decreased during further storage. Activity of α-farnesene synthase, α-farnesene content, and conjugated triene alcohol (the putative scald-causing oxidation product of α-farnesene) content in skin tissue were not correlated to the inherent nature of scald susceptibility or resistance in 11 apple cultivars tested.
Dineshkumar Selvaraj, Sherif Sherif, Mohd Sabri Pak Dek, Gopinadhan Paliyath, Islam El-Sharkawy and Jayasankar Subramanian
European plum fruit (Prunus domestica) are normally blue-black to dark purple. However, some genotypes remain green/yellow after ripening. We hypothesized that in such genotypes anthocyanin biosynthesis is genetically disturbed. To examine this hypothesis, six european plum genotypes with diverse fruit colors were investigated for the expression pattern of several anthocyanin biosynthetic genes (ABGs)—e.g., phenylalanine ammonia-lyase, chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase 1 and 2 (UFGT 1 and 2). Expression profiles indicated that ABGs, especially Pd-CHS and UFGT 2, were significantly downregulated in the green/yellow fruit compared with the dark-purple fruit. Furthermore, the quantification of total polyphenols and individual flavonoid compounds showed substantial differences between the off-colored and the purple genotype. To further examine the contribution of each of the ABGs in color development, the open reading frame (ORP) of Pd-CHS, Pd-DFR, Pd-ANS, and Pd-UFGT 2 was ectopically expressed in tobacco (Nicotiana tabacum). The characterization of transgenic plants showed that the petals of plants expressing Pd-CHS were darker in color and had higher anthocyanin content than control or even other transgenic types, suggesting the significant contribution of CHS in determining anthocyanin production levels and hence fruit coloration. The results of this study provides better understanding of color development in european plum, which can be rewarding in developing european plum cultivars with desired colors through classical or modern breeding tools.