Fungi, as well as the rest of living organisms must deal with environmental challenges such as stressful stimuli. Fungi are excellent models to study the general mechanisms of the response to stress, because of their simple, but conserved, signal-transduction and metabolic pathways that are often equivalent to those present in other eukaryotic systems. A factor that has been demonstrated to be involved in these responses is polyamine metabolism, essentially of the three most common polyamines: putrescine, spermidine and spermine. The gathered evidences on this subject suggest that polyamines are able to control cellular signal transduction, as well as to modulate protein-protein interactions. In the present review, we will address the recent advances on the study of fungal metabolism of polyamines, ranging from mutant characterization to potential mechanism of action during different kinds of stress in selected fungal models.

Polyamines are essential metabolites present in all living organisms, and this subject has attracted the attention of researchers worldwide interested in defining their mode of action in the variable cell functions in which they are involved, from growth to development and differentiation. Although the mechanism of polyamine synthesis is almost universal, different biological groups show interesting differences in this aspect that require to be further analyzed. For these studies, fungi represent interesting models because of their characteristics and facility of analysis. During the last decades fungi have contributed to the understanding of polyamine metabolism. The use of specific inhibitors and the isolation of mutants have allowed the manipulation of the pathway providing information on its regulation. During host-fungus interaction polyamine metabolism suffers striking changes in response to infection, which requires examination. Additionally the role of polyamine transporter is getting importance because of its role in polyamine regulation. In this paper we analyze the metabolism of polyamines in fungi, and the difference of this process with other biological groups. Of particular importance is the difference of polyamine biosynthesis between fungi and plants, which makes this process an attractive target for the control of phytopathogenic fungi.

To analyze the role of spermidine in cell growth and differentiation of Ustilago maydis, the gene encoding spermidine synthase (Spe) was isolated using PCR. We found that the enzyme is encoded by a chimeric bifunctional gene (Spe-Sdh) that also encodes saccharopine dehydrogenase (Sdh), an enzyme involved in lysine biosynthesis. The gene contains a 5' region encoding Spe, followed, without a termination signal or a second initiation codon, by a 3' region encoding Sdh, and directs the synthesis of a single transcript that hybridizes with 3' or 5' regions' probes of the gene. The gene could not be disrupted in a wild-type strain, but only in a mutant defective in the gene encoding ornithine decarboxylase (Odc). Single spe-sdh mutants were isolated after sexual recombination in planta with a compatible wild-type strain. Mutants were auxotrophic for lysine and spermidine, but not for putrescine, and contained putrescine and spermidine, but not spermine. Putrescine in double mutants is probably synthesized from spermidine by the concerted action of polyamine acetyl transferase and polyamine oxidase. spe-sdh mutants were sensitive to stress, unable to carry out the yeast-to-mycelium dimorphic transition, and showed attenuated virulence to maize. These phenotypic alterations were reverted by complementation with the wild-type gene.

In previous communications the essential role of spermidine in Ustilago maydis was demonstrated by means of the disruption of the genes encoding ornithine decarboxylase (ODC) and spermidine synthase (SPE). However, the assignation of specific roles to each polyamine in different cellular functions was not possible because the spermidine added to satisfy the auxotrophic requirement of odc/spe double mutants is partly back converted into putrescine. In this study, we have approached this problem through the disruption of the gene-encoding polyamine oxidase (PAO), required for the conversion of spermidine into putrescine, and the construction of odc/pao double mutants that were unable to synthesize putrescine by either ornithine decarboxylation or retroconversion from spermidine. Phenotypic analysis of the mutants provided evidence that putrescine is only an intermediary in spermidine biosynthesis, and has no direct role in cell growth, dimorphic transition, or any other vital function of U. maydis. Nevertheless, our results show that putrescine may play a role in the protection of U. maydis against salt and osmotic stress, and possibly virulence. Evidence was also obtained that the retroconversion of spermidine into putrescine is not essential for U. maydis growth but may be important for its survival under natural conditions.

Objective. To determine the effect of pH, and exposure time over the inactivation of aflatoxin B 1 (AFB 1 ) during the tortilla making process as well as the degradative molecules generated. Materials and methods. Inactivation of AFB 1 in maize-dough with alkaline pH and in alkaline methanolic solutions was determined by HPLC. Kinetics of time exposure of AFB 1 in methanolic solution and the degradative products were analyzed by direct injection electrospray mass spectometry (DIESI-MS). Results. The alkaline pH of the maize-dough after nixtamalización between 10.2, and 30-40 minutes of resting at room temperature allows the 100% reduction of AFB 1 . DIESI-MS analysis of the extracts indicated the presence of two degradation molecules from AFB 1 . Conclusion. The alkaline pH of maize-dough and resting time are the principal factors involved in diminishing AFB 1 levels in tortillas. A procedure to the tortilla making process is proposed, which allows the reduction of remnant AFB 1 , avoiding the accumulative effect over consumers. ResumenObjetivo. Determinar el efecto del pH alcalino de la masa de maíz y el tiempo de exposición sobre la aflatoxina B 1 (AFB 1 ) durante la producción de tortillas e identificar los posibles productos de degradación mediante DIESI-MS. Material y métodos. La inactivación de la AFB 1 a pH alcalino y diferentes tiempos de exposición en masa nixtamalizada y en soluciones metanólicas fueron determinadas por HPLC. La cinética de degradación de AFB 1, y los productos de degradación en soluciones metanólicas se determinaron por DIESI-MS. Resultados. El pH alcalino de la masa y 30 a 40 minutos de reposo redujeron en 100% la AFB 1 adicionada. Se identificaron dos moléculas de degradación. Conclusión. Los principales factores involucrados en la disminución de la AFB 1 durante la producción de tortillas son la hidrólisis alcalina y el tiempo de reposo. Se propone un procedimiento para la producción de tortilla que reducirá la AFB 1 residual evitando el efecto acumulativo en los consumidores.

Synthesis of spermidine involves the action of two enzymes, spermidine synthase (Spe) and S-adenosylmethionine decarboxylase (Samdc). Previously we cloned and disrupted the gene encoding Spe as a first approach to unravel the biological function of spermidine in Ustilago maydis. With this background, the present study was designed to provide a better understanding of the role played by Samdc in the regulation of the synthesis of this polyamine. With this aim we proceeded to isolate and delete the gene encoding Samdc from U. maydis, and made a comparative analysis of the phenotypes of samdc and spe mutants. Both spe and samdc mutants behaved as spermidine auxotrophs, and were more sensitive than the wild-type strain to different stress conditions. However, the two mutants displayed significant differences: in contrast to spe mutants, samdc mutants were more sensitive to LiCl stress, high spermidine concentrations counteracted their dimorphic deficiency, and they were completely avirulent. It is suggested that these differences are possibly related to differences in exogenous spermidine uptake or the differential location of the respective enzymes in the cell. Alternatively, since samdc mutants accumulate higher levels of S-adenosylmethionine (SAM), whereas spe mutants accumulate decarboxylated SAM, the known opposite roles of these metabolites in the processes of methylation and differentiation offer an additional attractive hypothesis to explain the phenotypic differences of the two mutants, and provide insights into the additional roles of polyamine metabolism in the physiology of the cell. INTRODUCTIONPolyamines are organic polycations required by all living organisms (Pegg & McCann, 1982;Tabor & Tabor, 1984, 1985Cohen, 1998). They have drawn interest because they are essential for cell growth and differentiation, one model of which is fungal dimorphism, which provides a useful system to study their role (Ruiz-Herrera & Calvo-Méndez, 1987;Ruiz-Herrera, 1993, 1994Guevara-Olvera et al., 1993;Herrero et al., 1999; Jiménez-Bremont et al., 2001; Blasco et al., 2002). The most common polyamines in eukaryotes are putrescine, spermidine and spermine, but some fungi lack spermine, and contain only putrescine and spermidine (Nickerson et al., 1977; Valdés-Santiago et al., 2009). Putrescine, the smallest of the polyamines and precursor of the others, is the result of decarboxylation by ornithine decarboxylase (ODC). In Ustilago maydis, we observed that odc mutants were unable to carry out the pH-dependent dimorphic transition, even using concentrations of putrescine that were high enough to satisfy their growth requirements (Guevara-Olvera et al., 1997). A similar behaviour was displayed by Yarrowia lipolytica (Jiménez-Bremont et al., 2001) and Candida albicans (Herrero et al., 1999) odc mutants. These mutants were able to carry out the yeast-to-mycelium dimorphic transition only in the presence of an exceedingly high concentration of putrescine. These results clearly demonstrated the role of polyamines in fungal di...

Trehalose is an important disaccharide that can be found in bacteria, fungi, invertebrates and plants. In some Ascomycota fungal plant pathogens, the role of trehalose was recently studied and shown to be important for conferring protection against several environmental stresses and for virulence. In most of the fungi studied, two enzymes are involved in the synthesis of trehalose: trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2). To study the role of trehalose in virulence and stress response in the Basidiomycota maize pathogen Ustilago maydis, Δtps2 deletion mutants were constructed. These mutants did not produce trehalose as confirmed by HPLC analysis, showing that the single gene disruption impaired its biosynthesis. The mutants displayed increased sensitivity to oxidative, heat, acid, ionic and osmotic stresses as compared to the wild-type strains. Virulence of Δtps2 mutants to maize plants was extremely reduced compared to wild-type strains, possibly due to reduced capability to deal with the hostile host environment. The phenotypic traits displayed by Δtps2 strains were fully restored to wild-type levels when complemented with the endogenous UmTPS2 gene, or a chimeric construct having the Saccharomyces cerevisiae TPS2 ORF. This report demonstrates the presence of a single biosynthetic pathway for trehalose, and its importance for virulence in this model Basidiomycota plant pathogen.

The inhibitory effect of recombinant amaranth cystatin (AhCPI) on the spore germination and growth of the mycotoxigenic fungus Aspergillus parasiticus and Aspergillus niger was investigated. AhCPI showed a concentration-dependent antifungal activity against both fungi. Differential effects were observed when fungi were treated with cystatin in two developmental stages. When AhCPI was added to young mycelium cultures of A. niger, it had a dramatic effect on mycelial growth compared with old mycelium cultures. On the contrary, there was no differential effect of AhCPI addition to either old or young mycelium of A. parasiticus. Furthermore, electron microscopic observations showed that cystatin caused important effects at the level of cell morphology and organelle integrity of both fungi. Additionally, A. parasiticus spores treated with AhCPI presented sensitivity to oxidative, osmotic and ionic stresses; in opposition, under same conditions, A. niger did not show sensitivity to any stressful agent. These results suggest that AhCPI antifungal activity might be related with damage to cell integrity, affecting the survival of the fungi. In addition, our evidences showed that fungal species respond dissimilarly to cystatin; however, such disparities can be used to the control of unwanted fungi.