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

High Resolution Crystal Structures of the Wild Type and Cys-55 right-arrow Ser and Cys-59 right-arrow Ser Variants of the Thioredoxin-like [2Fe-2S] Ferredoxin from Aquifex aeolicus

The [2Fe-2S] ferredoxin (Fd4) from Aquifex aeolicus adopts a thioredoxin-like polypeptide fold that is distinct from other [2Fe-2S] ferredoxins. Crystal structures of the Cys-55 right-arrow Ser (C55S) and Cys-59 right-arrow Ser (C59S) variants of this protein have been determined to 1.25 Å and 1.05 Å resolution, respectively, whereas the resolution of the wild type (WT) has been extended to 1.5 Å. The improved WT structure provides a detailed description of the [2Fe-2S] cluster, including two features that have not been noted previously in any [2Fe-2S] cluster-containing protein, namely, pronounced distortions in the cysteine coordination to the cluster and a Calpha -H-Sgamma hydrogen bond between cluster ligands Cys-55 and Cys-9. These features may contribute to the unusual electronic and magnetic properties of the [2Fe-2S] clusters in WT and variants of this ferredoxin. The structures of the two variants of Fd4, in which single cysteine ligands to the [2Fe-2S] cluster are replaced by serine, establish the metric details of serine-ligated Fe-S active sites with unprecedented accuracy. Both the cluster and its surrounding protein matrix change in subtle ways to accommodate this ligand substitution, particularly in terms of distortions of the Fe2S2 inorganic core from planarity and displacements of the polypeptide chain. These high resolution structures illustrate how the interactions between polypeptide chains and Fe-S active sites reflect combinations of flexibility and rigidity on the part of both partners; these themes are also evident in more complex systems, as exemplified by changes associated with serine ligation of the nitrogenase P cluster

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.

Somatic embryogenesis and plant regeneration of Vitis vinifera cultivars 'Macabeo' and 'Tempranillo'

Different experimental conditions have been compared to achieve a high efficiency in embryogenic calli initiation from 'Macabeo' and 'Tempranillo' anthers. Specifically, two stages of anther development were tested (corresponding to tetrad cells or uninucleate pollen), and direct culture of anthers was compared to culture after a cold treatment of inflorescences (4 °C during 48 h). In addition, two induction media (C1 P and B2), mainly differing by microelement and cytokinin levels, were evaluated. Experiment repeatability was also examined with a repetition of anther culture one week later. Callus initiation was similar in all media and treatments for both cultivars, usually starting from the anther filament. A simple protocol for efficient induction of embryogenesis in 'Macabeo' and 'Tempranillo' consisted in:selecting the first inflorescence from hardwood cutting,excising anthers at uninucleate pollen stage without cold treatment of the inflorescences,incubating anthers on C1 P medium.The procedure used for embryo germination and plant regeneration, allowed to obtain a conversion rate up to 75 % in 'Macabeo' and 60 % in 'Tempranillo'. The protocol proposed represents the first regeneration system developed for the Spanish cultivars 'Macabeo' and 'Tempranillo'.

Fossil evidence for low gas exchange capacities for Early Cretaceous angiosperm leaves

The photosynthetic gas exchange capacities of early angiosperms remain enigmatic. Nevertheless, many hypotheses about the causes of early angiosperm success and how angiosperms influenced Mesozoic ecosystem function hinge on understanding the maximum capacity for early angiosperm metabolism. We applied structure-functional analyses of leaf veins and stomatal pore geometry to determine the hydraulic and diffusive gas exchange capacities of Early Cretaceous fossil leaves. All of the late Aptian-early Albian angiosperms measured possessed low vein density and low maximal stomatal pore area, indicating low leaf gas exchange capacities in comparison to modern ecologically dominant angiosperms. Gas exchange capacities for Early Cretaceous angiosperms were equivalent or lower than ferns and gymnosperms. Fossil leaf taxa from Aptian to Paleocene sediments previously identified as putative stem-lineages to Austrobaileyales and Chloranthales had the same gas exchange capacities and possibly leaf water relations of their living relatives. Our results provide fossil evidence for the hypothesis that high leaf gas exchange capacity is a derived feature of later angiosperm evolution. In addition, the leaf gas exchange functions of austrobaileyoid and chloranthoid fossils support the hypothesis that comparative research on the biology of living basal angiosperm lineages reveals genuine signals of Early Cretaceous angiosperm ecophysiology

Spiral2 cryomodules B tests results

MOP010International audienceAssembly and tests of the SPIRAL2 superconducting linac's cryomodules at CEA/Saclay and IPN/Orsay have now reached cruising speed after having faced a series of problems, among them contamination. 19 cryomodules are composing the whole Linac and IPN Orsay is in charge of the 7 cryomodules B, housing two 88 MHz, beta 0.12 Quarter-Wave Resonators. Threecryomodules have been assembled and successfullytested up to the nominal gradient of 6.5 MV/m for all cavities with also cryogenic losses withinspecifications. Two of them are fully qualified and already delivered to GANIL. The thirdone showed misalignment ofone cavity which could lead to partial disassembly if needed. This paper presents the results of those cryomodules tests as well as the status of the remaining ones

Tests at 2K of the beta 0.35 spoke cryomodule prototype with the MTCA.4-based Low Level RF system prototype for the MYRRHA R&D

Within the framework of the first phase of MYRRHA (Multi-purpose hYbrid Research Reactor for High-tech Applications) project, called MINERVA, IJCLab was in charge of a fully equipped Spoke cryomodule prototype development, tested at 2K. It integrates two superconducting single spoke cavities, the RF power couplers and the Cold Tuning Systems associated. On the control side, a MTCA.4-based Low Level Radio Frequency (LLRF) system prototype and the Software/EPICS developments has been realized by IJCLab and the SCK CEN in collaboration with the company IOxOS Technologies. The final version of the global system and the results of the tests at 2K will show with some perspectives.Comment: Poster pr\'esent\'e au LLRF Workshop 2023 (LLRF2023, arXiv : 2310.03199