Saffron, the processed stigma of Crocus sativus, is characterized by the presence of several apocarotenoids that contribute to the color, flavor, and aroma of the spice. However, little is known about the synthesis of aroma compounds during the development of the C. sativus stigma. The developing stigma is nearly odorless, but before and at anthesis, the aromatic compound ␤-ionone becomes the principal norisoprenoid volatile in the stigma. In this study, four carotenoid cleavage dioxygenase (CCD) genes, CsCCD1a, CsCCD1b, CsCCD4a, and CsCCD4b, were isolated from C. sativus. Expression analysis showed that CsCCD1a was constitutively expressed, CsCCD1b was unique to the stigma tissue, but only CsCCD4a and -b had expression patterns consistent with the highest levels of ␤-carotene and emission of ␤-ionone derived during the stigma development. The CsCCD4 enzymes were localized in plastids and more specifically were present in the plastoglobules. The enzymatic activities of CsCCD1a, CsCCD1b, and CsCCD4 enzymes were determined by Escherichia coli expression, and subsequent analysis of the volatile products was generated by GC/MS. The four CCDs fell in two phylogenetically divergent dioxygenase classes, but all could cleave ␤-carotene at the 9,10(9 ,10 ) positions to yield ␤-ionone. The data obtained suggest that all four C. sativus CCD enzymes may contribute in different ways to the production of ␤-ionone. In addition, the location and precise timing of ␤-ionone synthesis, together with its known activity as a fragrance and insect attractant, suggest that this volatile may have a role in Crocus pollination.Crocus sativus is a triploid sterile plant characterized by its long red stigmas. These stigmas, when desiccated, constitute the spice known as saffron. Saffron is considered a complex mixture of volatile and non-volatile compounds that contribute to its overall aroma and flavor. The main components of saffron responsible for coloring strength are the derived carotenoids cis-and trans-crocins, picrocrocin, and its degradation product, the odor-active safranal that composes up to 70% of total volatiles (1). In addition to safranal, 150 volatile compounds are estimated to be present in the spice, and ϳ60 constituents have been identified (2). The structures of several of these reveal an isoprenoid-based origin like safranal, and are assumed to be the products of the oxidative cleavage of carotenoids.In recent years, a family of enzymes that cleave carotenoid substrates at different double bond positions have been described in plants. This family, the carotenoid cleavage dioxygenases (CCDs), 2 are specific for the location of the double bond in the molecule they cleave, but many are promiscuous in their carotenoid substrate choice (3, 4). The founding member of the CCD family was Vp14, a 9-cis-epoxycarotenoid dioxygenase (NCED). This and other closely related dioxygenases catalyze the cleavage at the 11,12 double bond of both 9-cis-violaxanthin and 9-cis-neoxanthin to produce xanthoxin (5-7), which is subsequently conv...

We undertook this study to understand how the transcription factor Sox2 contributes to the malignant phenotype of glioblastoma multiforme (GBM), the most aggressive primary brain tumor. We initially looked for unbalanced genomic rearrangements in the Sox2 locus in 42 GBM samples and found that Sox2 was amplified in 11.5% and overexpressed in all the samples. These results prompted us to further investigate the mechanisms involved in Sox2 overexpression in GBM. We analyzed the methylation status of the Sox2 promoter because high CpG density promoters are associated with key developmental genes. The Sox2 promoter presented a CpG island that was hypomethylated in all the patient samples when compared to normal cell lines. Treatment of Sox2-negative glioma cell lines with 5-azacitidine resulted in the re-expression of Sox2 and in a change in the methylation status of the Sox2 promoter. We further confirmed these results by analyzing data from GBM cases generated by The Cancer Genome Atlas project. We observed Sox2 overexpression (86%; N = 414), Sox2 gene amplification (8.5%; N = 492), and Sox 2 promoter hypomethylation (100%; N = 258), suggesting the relevance of this factor in the malignant phenotype of GBMs. To further explore the role of Sox2, we performed in vitro analysis with brain tumor stem cells (BTSCs) and established glioma cell lines. Downmodulation of Sox2 in BTSCs resulted in the loss of their self-renewal properties. Surprisingly, ectopic expression of Sox2 in established glioma cells was not sufficient to support self-renewal, suggesting that additional factors are required. Furthermore, we observed that ectopic Sox2 expression was sufficient to induce invasion and migration of glioma cells, and knockdown experiments demonstrated that Sox2 was essential for maintaining these properties. Altogether, our data underscore the importance of a pleiotropic role of Sox2 and suggest that it could be used as a therapeutic target in GBM.

Supplementary data are available at Bioinformatics online.