Natural occurrence and production of tenuazonic acid in wine grapes in Argentina.

A survey was carried out to determine natural occurrence of tenuazonic acid (TA) in healthy and rotten wine grapes samples from different varieties (n = 37) collected during 2016 vintage in the region of DOC San Rafael (Argentina). In addition, inoculation experiments with three Alternaria alternata strains in wine grapes were done to elucidate TA production and its major influencing factors. The 16.2% (6/37) of total wine grape samples showed TA contamination with 4% (1/25) of incidence in healthy samples (77 µg·kg-1) and 42% (5/12) in rotten samples (10-778 µg·kg-1). Malbec, Cabernet Sauvignon, and Syrah varieties showed TA contamination, whereas Bonarda, Ancelota, Torrontés, Semillón, and Chenin did not. During inoculation experiments in wine grapes, two of three strains were able to produce TA among the evaluated conditions and the highest TA production was observed at 15°C and 25°C after 24 days of incubation. Nutritional composition of grapes results appropriate for A. alternata infection and TA production and, together with the adequate field conditions, favors TA natural occurrence in wine grapes.

Isolation, identification and selection of antagonistic yeast against Alternaria alternata infection and tenuazonic acid production in wine grapes from Argentina.

Epiphytic isolates with yeast characteristics from grapes of the Malbec cultivar were obtained in order to find antagonists against Alternaria alternata. From a total of 111 isolates, 82% corresponded to the yeast-like organism Aureobasidium pullulans and the rest to the non-Saccharomyces yeasts Hanseniaspora uvarum (6.3%), Metschnikowia pulcherrima or spp. (5.4%), Cryptoccocus laurentti II (2.7%), Starmerella bacilaris or Candida zemplinina (2.7%) and Rhodotorula spp. (0.9%). The 22.4% (15 out of 67) of epiphytic yeasts and yeast-like organisms evaluated were able to reduce A. alternata infection from 0.0 to 4.4% when applied 2h previous to pathogen inoculation on wounds of grape berries. From these selected strains, 14 out of 15 strains completely prevented A. alternata infection (0.0%), which implies potential for field application. All Metschnikowia (pulcherrima or spp.), S. bacillaris and almost all H. uvarum evaluated strains showed antagonist capability against A. alternata. Meanwhile, none of the lesser nutritional requirement strains belonging to A. pullulans, Cr. laurenti II and Rhodotorula spp. did. All the yeasts with capacity to prevent A. alternata infection also reduced tenuazonic acid (TA) production by 81.2 to 99.8%, finding TA levels similar to negative controls. Therefore, the epiphytic yeasts selected are promising as biological control agents against Alternaria infection and toxin production in grapes for winemaking.

Visualization by BiFC of different C/EBPß dimers and their interaction with HP1α reveals a differential subnuclear distribution of complexes in living cells.

How the co-ordinated events of gene activation and silencing during cellular differentiation are influenced by spatial organization of the cell nucleus is still poorly understood. Little is known about the molecular mechanisms controlling subnuclear distribution of transcription factors, and their interplay with nuclear proteins that shape chromatin structure. Here we show that C/EBPß not only associates with pericentromeric heterochromatin but also interacts with the nucleoskeleton upon induction of adipocyte differentiation of 3T3-L1 cells. Different C/EBPß dimers localize in different nuclear domains. Using BiFC in living cells, we show that LAP (Liver Activating Protein) homodimers localize in euchromatin and heterochromatin. In contrast, LIP (Liver Inhibitory Protein) homodimers localize exclusively in heterochromatin. Importantly, their differential subnuclear distribution mirrors the site for interaction with HP1α. HP1α inhibits LAP transcriptional capacity and occupies the promoter of the C/EBPß-dependent gene c/ebpα in 3T3-L1 preadipocytes. When adipogenesis is induced, HP1α binding decreases from c/ebpα promoter, allowing transcription. Thus, the equilibrium among different pools of C/EBPß associated with chromatin or nucleoskeleton, and dynamic changes in their interaction with HP1α, play key roles in the regulation of C/EBP target genes during adipogenesis.

A segment containing a G-box and an ACGT motif confers differential expression characteristics and responses to the Arabidopsis Cytc-2 gene, encoding an isoform of cytochrome c.

Sequences required for the expression of Cytc-2 (At4g10040), one of two cytochrome c genes from Arabidopsis thaliana, were characterized using plants transformed with deleted and mutagenized promoter fragments fused to gus. These studies indicated that a region containing a G-box and an ACGT motif is essential for expression. Mutation of the ACGT motif causes a complete loss of expression, while mutation of the G-box causes decreased expression in aerial parts and abolishes expression in roots and induction by environmental factors. Upstream located site II elements are required for maximal expression, mainly in reproductive tissues, and maximal induction by different factors. One-hybrid screenings allowed the identification of transcription factors from the bZIP and bHLH families that interact mainly with the G-box. Four of these factors were able to bind to the Cytc-2 promoter in vitro and in transactivation assays in Arabidopsis. Analysis of available microarray data indicated that the bZIP transcription factors share expression characteristics with the Cytc-2 gene, suggesting that they act as mediators of its response to tissue-specific, environmental, and metabolic conditions. Site II elements interact with a TCP family protein and may co-ordinate the expression of the Cytc-2 gene with that of other respiratory chain components. A model is proposed for the evolution of the Cytc-2 gene through the incorporation of a segment containing a G-box and an ACGT motif into an ancestral gene that contained site II elements. This may have reduced the importance of site II elements for basal expression and conferred new responses to environmental factors.