Involvement of ethylene signalling in a non-climacteric fruit: new elements regarding the regulation of ADH expression in grapevine

Although grape berries have been classified as non climacteric fruits, ongoing studies on grape ethylene signalling lead to challenge the role of ethylene in their ripening. One of the significant molecular changes in berries is the up regulation of ADH (alcohol dehydrogenase, EC. 1.1.1.1) enzyme activity at the inception of fruit ripening and of VvADH2 transcript levels. This paper shows that the ethylene signal transduction pathway could be involved in the control of VvADH2 expression in grapevine berries and in cell suspensions. The induction of VvADH2 transcription, either in berries at the inception of ripening or in cell suspensions, was found to be partly inhibited by 1 methylcyclopropene (1 MCP), an inhibitor of ethylene receptors. Treatment of cell suspensions with 2 chloroethylphosphonic acid (2-CEPA), an ethylene releasing compound, also resulted in a significant increase of ADH activity and VvADH2 transcription under anaerobiosis, showing that concomitant ethylene and anaerobic treatments in cell suspensions could result in changes of VvADH2 expression. All these results, associated with the presence in the VvADH2 promoter of regulatory elements for ethylene and anaerobic response, suggest that ethylene transduction pathway and anaerobic stress could be in part involved in the regulation of VvADH2 expression in ripening berries and cell suspensions. These data open new aspects of the expression control of a ripening-related gene in a non climacteric fruit

Effect of different 3’ flanking regions on the activity of the Vitis vinifera alcohol dehydrogenase 2 promoter

3’untranslated regions (3' UTR) are isogene specific regions which contain sequences likely playing an important role in gene expression. To evaluate the importance of these regions on Vitis vinifera alcohol dehydrogenase 2 (VvAdh2) expression, we designed expression cassettes containing the luciferase reporter gene under the VvAdh2 or CaMV 35S promoters and flanked by different 3’ UTRs. Luciferase activity monitoring was performed through transient expression experiments, using biolistic on Cabernet Sauvignon suspension cells. Results showed that absence of the 3’ region had a strong down-regulating effect on the VvAdh2 promoter activity (but not on the CaMV 35S promoter activity). The nature of the flanking 3' UTR was shown to influence expression cassette activity. Whatever the promoter, VvAdh1 and VvAdh2 terminators had similar effect on expression of luciferase in air leading to an activity level very close to that of CaMV 35S terminator. Under anaerobiosis, luciferase expression was strongly increased with all terminators, VvAdh6 3'-end inducing the highest level of expression. Functional constructs with VvAdh2 promoter and VvAdh terminators designed in this study could be used wherever grapevine-homologous, stress-stimulated cassettes should be of interest

Yeast cell death caused by nutrient desequilibrium during alcoholic fermentation is impacted by nitrogen sources

Nutrients availability is a key factor for controlling wine alcoholic fermentation. Among them, nitrogen has been identified as an essential parameter, controlling both the fermentation rate and the durationof the fermentation. However, nitrogen is not sufficient to ensure a correct fermentation and other nutrients such as vitamins and lipids, present in lower quantities, are required. Furthermore, we showed in a previous study that an excess of nitrogen combined with a depletion in certain micronutrients can lead to cell death and sluggish or stuck fermentation. In this study, we provide evidence of the mechanism controlling cell death and we show that all the nitrogen sources are not equivalent in the initiation of this phenomenon.Fermentations limited in oleic acid, pantothenic acid and nicotinic acid showed yeast cell death linked to a high nitrogen content. In each case, lowering the nitrogen level restored yeast viability. We evidenced that yeast cell lack of a correct stress response to those micronutrient starvations in presence of high levels of nitrogen. A transcriptional analysis showed a correct stress response suggesting that the lack of resistance originates from a post-transcriptional control mechanism. We then provide evidence that the nitrogen Tor/Sch9 signaling pathway is involved in triggering cell death.Yeast cell viability was then monitored and compared during fermentation starting at different nitrogen levels, with the addition of different nitrogen sources (19 amino acids and NH4+) and two different timing of NH4+ addition. We observed that cell death was triggered with different intensities.Yeast cell death associated to disequilibrium between micronutrients and nitrogen has been evidenced and its implication on fermentations highlighted. We showed a strong impact of both the nature of the nitrogen source and time of addition on yeast cell death and fermentation outcome

Genetic bases of nitrogen requirement in wine yeast assessed trhrough QTL analysis

In grape must, nitrogen content is ofteninsufficient for the completion of alcoholic fermentation by yeast. For Saccharomyces cerevisiae, response to nitrogen deficiency is strain-dependent, some strains being able to complete fermentation despite nitrogen deficiency whereas others are not and result in sluggish or stuck fermentation. Thus, it is of high interest to study the mechanisms behind those different responses and exploit them to improve yeast strain for wine fermentation when nitrogen content is low. Previous study highlighted different genomic regions involved in nitrogen requirement through BSA (Bulk Segregant Analysis), and the contributions of three genes: MDS3, GCN1, and ARG81 have been shown (1). However, many other large genomic regions were also defined for which we could not find evident candidate genes. In addition, BSA did not provide any information on possible interactions between loci. In order to explore further the genetic bases of nitrogen requirement, we applied a QTL analysis to the fermentation rate in nitrogen deficient medium, on a population of 131 individually genotyped segregants obtained from the same cross as (1). The dense genetic map available for the segregant population (3727 markers) enabled us to perform single and multiple map QTL and thus define genomic regions which could be implied in low nitrogen requirement. In order to further validate the impact of candidate genes on the phenotype, alleles were “swapped” by CRISPR-Cas9 technique and phenotype was evaluated in comparison with haploid parent strains.Several regions with high LOD scores were identified, some above the significance threshold, and others below, among which the regions containing the genes identified by (1), probably in relation with the multigenic character of the trait. In the region with the highest LOD score, two candidate genes in relation with nitrogen metabolism (namely, Target of Rapamycin (TOR) pathwayand lifespan regulation) were identified. In addition, in order to reveal possible interaction between genes, strains carrying different combinations of GCN1 and MDS3 parental alleles (implied in TOR pathway) have been evaluated. These constructions confirm their role on the fermentation rate in low-nitrogen conditions and indicate dependence on the genetic background. These results confirm the complexity of mechanisms involved in nitrogen requirement during alcoholic fermentation and will permit to optimise wine yeast strain selection in response to winemaking industry demands

Analysis of the transcript levels of VvAdh1, VvAdh2 and VvGrip4, three genes highly expressed during Vitis vinifera L. berry development

Well defined promoter sequences are required for the targeted expression of genes in transgenic grapevines. This paper describes a detailed study of the expression of three genes with potentially useful promoters. We have used real-time RT-PCR to evaluate the transcript levels of VvAdh1, VvAdh2 and VvGrip4 genes in various tissues (root, bud, tendril, inflorescence, fruit and embryogenic callus) of Vitis vinifera L. at different stages of development. Transcript levels of the three genes were highest in berry tissues but each had a distinct pattern of expression. VvAdh1 showed higher transcript levels during the early stages of berry development, while levels of VvAdh2 and VvGrip4 were higher during ripening. However, none of these genes was expressed in a strictly fruit-specific manner. In particular, significant levels of VvAdh1 and VvGrip4 transcripts were observed during late tendril development and in the inflorescence, respectively. Transcript levels of all three genes were similar in both flesh and skin, indicating no tissue-specificity within the berry. Promoter sequences of the VvAdh1, VvAdh2 and VvGrip4 genes could be very useful to drive ectopic gene expression in berries of transgenic plants

Ethylene and other stimuli affect expression of the UDP glucose-flavonoid 3-O-glucosyltransferase in a non-climacteric fruit

The UDP glucose-flavonoid 3-O-glucoslyltransferase (UFGT) is a key enzyme for biosynthesis and stability of anthocyanin pigments of red grapes. Understanding factors affecting expression of this enzyme is thus important for the control of grape colour. A 1640 bp promoter region of the grapevine ufgt gene was cloned and sequenced. Sequence analysis revealed seven putative ethylene-responsive cis-elements and others related to three major signals known to induce anthocyanin accumulation in plant tissues: light, sugar, and abscisic acid. In order to evaluate the ability of ethylene and other signals to drive expression from the ufgt promoter, we ran transient expression experiments using an anthocyanin-rich grape cell culture, with very low green auto-fluorescence. After biolistic bombardment, the cells were treated with various combinations of the four signals on gfp expression (green fluorescent protein). The comparison of fluorescent light intensity in cells subjected to the various treatments showed that ethylene better stimulates expression of the ufgt promoter in the dark than under light. In addition, results showed that there may be a positive interaction between ethylene and abscisic acid. This system, a promoter of interest driving the gfp expression in cells with low auto-fluorescence, may be a good tool for studies about synergistic or antagonist roles of transcription factors. Moreover, treatment of grape berries with a specific inhibitor of ethylene receptors (1-methylcyclopropene) inhibited ufgt mRNA accumulation. This confirms that the ethylene signal is likely a regulator of grape UFGT expression in a non-climacteric fruit.

Cellular localisation of VvRops and VvRabA5e, small GTPases developmentally regulated in grape berries

VvRops, in particular VvRop9, and VvRabA5e are small GTPases which are developmentally regulated in grape berries. In an attempt to help elucidate the role of these proteins during fruit development and ripening, we investigated their localisation in the fruit by immunocytofluorescence. These proteins were observed at a perinuclear location, at cell periphery and around vesicles. In particular VvRops were found to be located in the nucleus and likely on the plasma membrane. VvRop9 and VvRabA5e cDNAs were introduced separately into S. cerevisiae mutants with RHO1 and YPT31/YPT32 defective genes respectively. Neither cDNAs could complement these temperature-sensitive mutants, suggesting that the functions of the VvRop9 and VvRabA5e genes in grapevine likely differ from the functions of RHO1 and YPT31/YPT32 genes in yeast.

Functional characterization of SlscADH1, a fruit-ripening associated short-chain alcohol dehydrogenase of tomato

A tomato short-chain dehydrogenase-reductase (SlscADH1) is preferentially expressed in fruit with a maximum expression at the breaker stage while expression in roots, stems, leaves and flowers is very weak. It represents a potential candidate for the formation of aroma volatiles by interconverting alcohols and aldehydes. The SlscADH1 recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several volatile compounds present in tomato flavour with a strong preference for the NAD/NADH co-factors. The strongest activity was observed for the reduction of hexanal (Km = 0.175 mM) and phenylacetaldehyde (Km = 0.375 mM) in the presence of NADH. The oxidation process of hexanol and 1-phenylethanol was much less efficient (Kms of 2.9 and 23.0 mM, respectively), indicating that the enzyme preferentially acts as a reductase. However activity was observed only for hexanal, phenylacetaldehyde, (E)-2-hexenal and acetaldehyde and the corresponding alcohols. No activity could be detected for other aroma volatiles important for tomato flavour, such as methyl-butanol/methyl-butanal, 5-methyl-6-hepten-2-one/5-methyl-6-hepten-2-ol, citronellal/citronellol, neral/nerol, geraniol. In order to assess the function of the SlscADH1 gene, transgenic plants have been generated using the technique of RNA interference (RNAi). Constitutive down-regulation using the 35S promoter resulted in the generation of dwarf plants, indicating that the SlscADH1 gene, although weakly expressed in vegetative tissues, had a function in regulating plant development. Fruitspecific down-regulation using the 2A11 promoter had no morphogenetic effect and did not alter the aldehyde/alcohol balance of the volatiles compounds produced by the fruit. Nevertheless, SlscADH1-inhibited fruit unexpectedly accumulated higher concentrations of C5 and C6 volatile compounds of the lipoxygenase pathway, possibly as an indirect effect of the suppression of SlscADH1 on the catabolism of phospholipids and/or integrity of membranes

Short-course antibiotic therapy for critically ill patients treated for postoperative intra-abdominal infection: the DURAPOP randomised clinical trial

PURPOSE: Shortening the duration of antibiotic therapy (ABT) is a key measure in antimicrobial stewardship. The optimal duration of ABT for treatment of postoperative intra-abdominal infections (PIAI) in critically ill patients is unknown. METHODS: A multicentre prospective randomised trial conducted in 21 French intensive care units (ICU) between May 2011 and February 2015 compared the efficacy and safety of 8-day versus 15-day antibiotic therapy in critically ill patients with PIAI. Among 410 eligible patients (adequate source control and ABT on day 0), 249 patients were randomly assigned on day 8 to either stop ABT immediately (n = 126) or to continue ABT until day 15 (n = 123). The primary endpoint was the number of antibiotic-free days between randomisation (day 8) and day 28. Secondary outcomes were death, ICU and hospital length of stay, emergence of multidrug-resistant (MDR) bacteria and reoperation rate, with 45-day follow-up. RESULTS: Patients treated for 8 days had a higher median number of antibiotic-free days than those treated for 15 days (15 [6-20] vs 12 [6-13] days, respectively; P < 0.0001) (Wilcoxon rank difference 4.99 days [95% CI 2.99-6.00; P < 0.0001). Equivalence was established in terms of 45-day mortality (rate difference 0.038, 95% CI - 0.013 to 0.061). Treatments did not differ in terms of ICU and hospital length of stay, emergence of MDR bacteria or reoperation rate, while subsequent drainages between day 8 and day 45 were observed following short-course ABT (P = 0.041). CONCLUSION: Short-course antibiotic therapy in critically ill ICU patients with PIAI reduces antibiotic exposure. Continuation of treatment until day 15 is not associated with any clinical benefit. CLINICALTRIALS. GOV IDENTIFIER: NCT01311765