Increase in cytotoxic lignans production after smut infection in sugar cane plants.

Smut infection alters the transcription of dirigent proteins (DIR) by sugarcane plants. Here, we show that these alterations are associated to an elevated production of cytotoxic lignans. Smut-resistant sugarcane varieties display a fivefold increase in pinoresinol and also produce elevated amounts of secoisolariciresinol. Conversely, smut-sensitive varieties do not produce pinoresinol or secoisolariciresinol upon infection, synthesizing instead small amounts of matairesinol. Our data indicate that commercial pinoresinol and secoisolariciresinol seem to prevent smut teliospore germination and sporidia release from sprouted teliospores. Consistently, we observed abundant morphological alterations of sporidia incubated in the presence of these lignans. However, commercial lignans do not block the development of the pathogen in a definitive way. Additional experiments demonstrate that only the extracts from healthy or smut-exposed resistant plants inhibit sporidia growth in vitro, indicating that a specific mixture of lignans from resistant plants is necessary to constitute an effective defense mechanism.

Differential expression of SofDIR16 and SofCAD genes in smut resistant and susceptible sugarcane cultivars in response to Sporisorium scitamineum.

Proteomic profiling of the stalk of a smut resistant and a susceptible sugarcane cultivars revealed the presence of dirigent and dirigent-like proteins in abundance in the pool of high molecular mass (HMMG) and mid-molecular mass (MMMG) glycoproteins, produced as part of the defensive response to the fungal smut pathogen. Quantitative RT-PCR analysis showed that expression levels of SofDIR16 (sugarcane dirigent16) and SofCAD (sugarcane cinnamyl alcohol dehydrogenase) were higher in the smut resistant My 55-14 cultivar than in the sensitive B 42231 cultivar prior to infection. Inoculation with fungal sporidia or water decreased the level of SofCAD transcripts in My 55-14, indicating that regulation of SofCAD expression does not take part of the specific response to smut infection. In contrast, SofDIR16 expression was almost nullified in My 55-14 after inoculation with fungal sporidia, but not after water injection. It is proposed that the decreased expression of dirigent proteins induces the formation of lignans, which are involved in the defense response of the smut resistant My 55-14 cultivar.

Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia.

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD.