RESUMO
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 beta-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 beta-carotene and emission of beta-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 beta-carotene at the 9,10(9',10') positions to yield beta-ionone. The data obtained suggest that all four C. sativus CCD enzymes may contribute in different ways to the production of beta-ionone. In addition, the location and precise timing of beta-ionone synthesis, together with its known activity as a fragrance and insect attractant, suggest that this volatile may have a role in Crocus pollination.
Assuntos
Crocus/enzimologia , Dioxigenases/metabolismo , Genes de Plantas/fisiologia , Norisoprenoides/biossíntese , Proteínas de Plantas/metabolismo , beta Caroteno/metabolismo , Crocus/genética , Citosol/enzimologia , Dioxigenases/genética , Norisoprenoides/genética , Filogenia , Proteínas de Plantas/genética , Polinização/fisiologia , beta Caroteno/genéticaRESUMO
Nucleotide sugar transporters (NST) mediate the transfer of nucleotide sugars from the cytosol into the lumen of the endoplasmatic reticulum and the Golgi apparatus. Because the NSTs show similarities with the plastidic phosphate translocators (pPTs), these proteins were grouped into the TPT/NST superfamily. In this study, a member of the NST-KT family, AtNST-KT1, was functionally characterized by expression of the corresponding cDNA in yeast cells and subsequent transport experiments. The histidine-tagged protein was purified by affinity chromatography and reconstituted into proteoliposomes. The substrate specificity of AtNST-KT1 was determined by measuring the import of radiolabelled nucleotide mono phosphates into liposomes preloaded with various unlabelled nucleotide sugars. This approach has the advantage that only one substrate has to be used in a radioactively labelled form while all the nucleotide sugars can be provided unlabelled. It turned out that AtNST-KT1 represents a monospecific NST transporting UMP in counterexchange with UDP-Gal but did not transport other nucleotide sugars. The AtNST-KT1 gene is ubiquitously expressed in all tissues. AtNST-KT1 is localized to Golgi membranes. Thus, AtNST-KT1 is most probably involved in the synthesis of galactose-containing glyco-conjugates in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Complexo de Golgi/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Transporte Biológico/genética , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
SUMMARY The cDNA-amplified fragment length polymorphism approach was used to identify differentially expressed transcripts from cassava infected by Xanthomonas axonopodis pv. manihotis (Xam). Approximately 3600 transcript-derived fragments (TDFs) were screened of which 340 were isolated. The nucleotide sequences of 250 TDFs were analysed and assembled into contigs and singletons. The amino acid sequences of their predicted products were compared with entries in databases and 63 of these clones showed homology to known plant genes. Of these, 32 showed similarity to plant defence proteins. Fifty-one TDFs corresponded to proteins of unknown function and 106 did not match any sequence in the public databases. Quantitative reverse transcription PCR was carried out with a selected set of gene transcripts that demonstrated an increase of expression during the infection. These results point out candidate genes that are associated with cassava resistance to Xam and reinforce the idea of a complex process occurring during this plant-pathogen interaction.
