Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene Navot Galpaz a , Yosi Burger a , Tamar Lavee a , Galil Tzuri a , Amir Sherman b , Tal Melamed b , Ravit Eshed b , Ayala Meir a , Vitaly Portnoy a , Einat Bar a , Einav Shimoni-Shor a , Ari Feder a , Yuval Saar a , Uzi Saar a , Fabian Baumkoler a , Efraim Lewinsohn a , Arthur A. Schaffer c , Nurit Katzir a , Yaakov Tadmor a,⇑ a Department of Vegetable Crops & Plant Genetics, Agricultural Research Organization, Newe Ya’ar Research Center, P.O. Box 1021, Ramat Yishay 30-095, Israel b Department of Fruit Science, Agricultural Research Organization, Volcani Center, P.O. Box 6, Bet Dagan 50-250, Israel c Department of Vegetable Crops & Plant Genetics, Agricultural Research Organization, Volcani Center, P.O. Box 6, Bet Dagan 50-250, Israel article info Article history: Available online xxxx Keywords: Cucumis melo Ethyl methanesulfonate Carotenoid isomerase Pro-lycopene Apocarotenoids Gene expression abstract In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh ‘Charentais’ type melon line that accumulates b-carotene. One mutag- enized M 2 family segregated for a novel recessive trait, a yellow–orange fruit flesh (‘yofI’). HPLC analysis revealed that ‘yofI’ accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of ‘yofI’ is associated with a significant change of the fruit aroma since cleavage of b-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to b-carotene in melon fruit. Cloning and sequencing of ‘yofI’ CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A–T transver- sion in ‘yofI’ which leads to a premature STOP codon. The early carotenoid pathway genes were up reg- ulated in yofI fruit causing accumulation of other intermediates such as phytoene and f-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon. Ó 2013 Elsevier Inc. All rights reserved. Introduction Carotenoids are C 40 essential compounds, localized in plastids of all photosynthetic organisms. Carotenoids assist in light energy harvesting and protect the photosynthetic apparatus against harm- ful reactive oxygen species that are produced by over-excitation of chlorophyll [19]. Carotenoids furnish flowers and fruits with dis- tinctive yellow, orange, and red colors. Enzymatic oxidative cleav- age of carotenoids gives rise to volatile apocarotenoid compounds, giving many plants species their typical aroma [20]. Carotenoids based colors and flavors are utilized for the attraction of pollinators and seed dispersers. Carotenoids also serve as the precursors of the phytohormones abscisic acid and strigolactones and as stimulants for symbiotic and parasitic organisms [41]. Additionally, carote- noids are phytonutrients with a widely acclaimed range of health-benefiting activities, including prevention of major mala- dies such as cancer, coronary diseases and age-related eye mal- functions [23]. a and b-carotene are the major sources of dietary pre-vitamin A in human and animals. Carotene-poor diets lead to vitamin A deficiency, affecting an estimated 250 million children under 5 years of age. Approximately 250,000–500,000 malnour- ished children in the developing world go blind each year from vitamin A deficiency (http://www.who.int/nutrition/topics/vad/ en/). The non-cyclic carotene lycopene has been found to possess antioxidant and anti-proliferative properties in animal and labora- tory studies and numerous studies have shown that ingesting lyco- pene-rich foods can result in health benefits [40]. In plants, carotenoids are synthesized within the plastids through the deoxy-xylulose phosphate isoprenoid pathway [18,8]. The first carotenoid in the pathway is the colorless phytoene, which is pro- duced by the enzyme phytoene synthase (PSY) 1 through a head to head condensation of two molecules of geranylgeranyl diphosphate 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.08.006 ⇑ Corresponding author. Fax: +972 4 983693. E-mail address: tadmory@agri.gov.il (Y. Tadmor). 1 Abbreviations used: PSY, phytoene synthase; GGPP, geranylgeranyl diphosphate; PDS, phytoene desaturase; ZDS, f-carotene desaturase; Z-ISO, f-carotene isomerase; CRTISO, carotenoid isomerase gene; CCD, carotenoid cleavage dioxygenases; EMS, ethyl methanesulfonate; MTBE, methyl tert-butyl ether; BAC, bacterial artificial chromosome. Archives of Biochemistry and Biophysics xxx (2013) xxx–xxx Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi Please cite this article in press as: N. Galpaz et al., Arch. Biochem. Biophys. (2013), http://dx.doi.org/10.1016/j.abb.2013.08.006