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International audience; Biotechnologies offer breeders good opportunities for breakthrough genetic improvements of bread wheat, one of mankind's main food crops. Since the production of the first transgenic wheat, one of the major concerns has been the removal of selective markers, first because of societal concerns about the antibiotic resistance of some of these genes, and second because removal of a selective marker was the first step toward retransformation using the same selection system. Site-directed nucleases are enzymes that cut genomic DNA in vivo at predefined sites. Among them, meganucleases cut DNA at predefined, long DNA (up to 24 nt) sites, thereby enabling single cuts on large genomes including the bread wheat genome (17 Gbp). In this paper, we describe for the first time the use of a customized meganuclease to cut wheat DNA in vivo. We show that double cuts provoked the deletion of previously inserted DNA cassettes containing the DsRed reporter gene, and that in many cases, the meganuclease target site was correctly reconstituted, offering opportunities for subsequent insertion of stacked transgenes to replace the gene of selection. Moreover, perfect deletions were observed not only in the callus after transient expression of the meganucleases, but also in T0 transgenic wheat after stable retransformation with the meganuclease. Future prospects for the removal of selective markers and transgene stacking are discussed.

International audience; Plant cell walls are complex structures critical for plant fitness and valuable for human nutrition as dietary fiber and for industrial uses such as biofuel production. The cell wall polysaccharides in wheat endosperm consist of two major polymers, arabinoxylans and beta-glucans, as well as other minor components. Most of these polysaccharides are synthesized in the Golgi apparatus but the mechanisms underlying their synthesis have yet to be fully elucidated and only a few of the enzymes involved have been characterized. To identify actors involved in the wheat endosperm cell wall formation, we used a subcellular fractionation strategy to isolate Golgi-enriched fractions from endosperm harvested during active cell wall deposition. The proteins extracted from these Golgi-enriched fractions were analyzed by LC-MS/MS. We report the identification of 1135 proteins among which 64 glycosyltransferases distributed in 17 families. Their potential function in cell wall synthesis is discussed. In addition, we identified 63 glycosylhydrolases, some of which may be involved in cell wall remodeling. Several glycosyltransferases were validated by showing that when expressed as fusion proteins with a fluorescent reporter, they indeed accumulate in the Golgi apparatus. Our results provide new candidates potentially involved in cell wall biogenesis in wheat endosperm.

Improving genetic transformation efficiency is a major concern in plant genetic engineering. While various strategies have been investigated, the enhancement of selectable marker gene expression has not been tried extensively. We used maize matrix attachment regions (MARs) to bracket an herbicide resistance transgene, bar. MARs have been reported to enhance transgene expression level and stability. We show here that MARs not only enhance transformation efficiency by 50%, but are also able to increase or decrease relative efficiencies of each step of the regeneration process depending on MAR sequence combinations. Furthermore, we assessed the trans-effect of MARs in co-bombardment experiments with two independent plasmids, one including the MAR sequences and the other one the bar gene. As for simple bombardment, MARs enhanced transformation efficiency by having a positive influence on organogenesis step in the regeneration process.

International audience; The feasibility of map-based cloning in wheat has been demonstrated recently, opening new perspectives for a better understanding of wheat plant biology and for accelerating wheat improvement in the coming decades. To validate the function of candidate genes, an efficient transformation system is needed. Here, we have performed two methods for wheat transformation using particle bombardment that ensures the production of transgenic plants with simple integration patterns for research purposes and stable transgene expression for accurate and rapid validation of gene function. To establish this method, we used the bar and pmi selectable genes either as part of whole plasmids, gene cassettes (obtained by PCR or purified on agarose gels), or as dephosphorylated cassettes. The analysis of about 300 transgenic plants showed that the use of gene cassettes or dephosphorylated gene cassettes leads to a majority (50-60 %) of simple integration events. This is significantly higher than the number of simple events obtained with whole plasmids (9-25 %). Moreover, the decrease of the quantity of DNA from 500 to 5 ng/mu l for PCR-amplified cassettes used for transformation increased the number of single integration events. The transformation efficiency remained stable at 2.5 %, and a higher number of plants expressing the transgenes were obtained with the dephosphorylated cassette. No correlation was observed between the complexity of the events and stability of expression of the transgene, suggesting that plasmid sequences could be involved on transgene silencing. The inheritability of the transgene was demonstrated in T1 and T2 generations. These results show that biolistic transformation of dephosphorylated gene cassettes provides an easy and efficient route to produce backbone vector-free transgenic wheat carrying and expressing intact and single transgenes.

Various experimental methods and image analysis procedures have been used to underscore the microporosity evolution of the Charroux-Civray tonalite. Throat pore effect is the dominant geometrical parameter, which controls permeability and determines the transport process. Indeed, in unaltered tonalite, flowing is not possible in spite of the fact that diffusion is made possible by a connected random network. Fluid pathways are located along the feldspar clusters joining quartz grains. Alteration effects are seen on the increased mean aperture of microcracks (feldspar dissolution). This paper raises the problem of the experimental limits in the study of very low permeable rocks.

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Matrix attachment regions (MARs) have been used to enhance transgene expression and to reduce transgene expression instability in various organisms. In plants, contradictory data question the role of MAR sequences. To assess the use of MAR sequences in maize, we have used two well-characterized MARs from the maize adh-1 region. The MARs have been cloned either 5' to or at both sides of a reporter gene expression cassette to reconstitute a MAR-based domain. Histochemical staining revealed a new transgene expression pattern in roots of regenerated plants and their progeny. Furthermore, MARs systematically induced variegation. We show here that maize adh-1 MARs are able to modify transgene expression patterns as a heritable trait, giving a new and complementary outcome following use of MARs in genetic transformation.

Retinol mobilization from retinyl esters stores of hepatic stellate cells (HSCs) is a key step in the regulation of mammalian retinol homeostasis, but the precise mechanisms of such a mobilization are still poorly understood. Using primary cultures of HSCs, we first demonstrated that HSCs expressed immunoreactivity against retinol-binding-protein (RBP) when cultured in a medium containing RBP but were unable to synthesize RBP transcripts and proteins. Using pulse and chase-type experiments, we demonstrated that radioactive retinol was released in culture medium without binding proteins. Inhibition of protein secretion by brefeldin A did not modify quantitatively retinol release. This data ruled out, for the first time, the direct involvement of RBP in retinol mobilization from HSCs. Moreover, HSCs co-cultured with primary isolated hepatocytes displayed an increase of retinol transfer from HSCs to hepatocytes when they established direct physical contacts, as compared with co-cultures without contact. Based on this latter data, a mechanism of retinol mobilization from HSCs via the hepatocytes using retinol transfer through cellular membranes is proposed.

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International audience; To assess the influence of age on vitamin A intestinal and liver metabolism in humans, the postprandial plasma concentrations of intestinal-originated vitamin A, i.e., retinyl esters, and liver-originated vitamin A, i.e., retinol, were compared in eight young (20–30 years old) and eight elderly (64–72 years old) healthy men. Plasma and chylomicron retinyl esters and retinol concentrations were measured for up to 24 h following the intake of a test meal that contained 23,300 RE retinyl palmitate. The chylomicron retinyl palmitate response (area under the curve) was not significantly different between the two groups, but its peak was slightly delayed (1 h) in the elderly men. The proportion of the different retinyl esters secreted in the chylomicrons was not significantly different between the two groups. The postprandial plasma retinol concentration did not change in the young participants, whereas it significantly increased in the elderly. These results suggest that vitamin A intestinal absorption and retinol intestinal esterification processes are not markedly modified in the elderly, whereas the chylomicron clearance and the regulation of postprandial plasma retinol concentrations are apparently altered in these subjects.

The effect of ageing on vitamin E bioavailability in humans was assessed by comparing chylomicron and plasma alpha-tocopherol postprandial concentrations after a dose of vitamin E (432 or 937 IU as d1-alpha-tocopherol acetate), in eight young (20-30 years old) and eight healthy elderly men (64-72 years old). The fasting plasma alpha-tocopherol concentration was significantly higher in the elderly (33 +/- 2 mumol L-1) than in the young (22 +/- 2 mumol L-1). In both groups, the plasma and chylomicron alpha-tocopherol postprandial concentrations were significantly, approximately twofold, higher after the 937-IU meal than after the 432-IU meal. For both test meals, the chylomicron alpha-tocopherol areas under the curve were significantly lower in the elderly than in the young subjects: 98.9 +/- 16.5 (young group) vs. 55.3 +/- 7.8 (elderly group) mumol L-1 h for the 937-IU test meal and 60.4 +/- 14.1 (young group) vs. 26.0 +/- 7.6 (elderly group) mumol L-1 h for the 432-IU test meal, whereas the plasma alpha-tocopherol area under the curve was significantly higher in elderly than in young subjects: 337.56 +/- 16.11 (937-IU test meal) vs. 159.81 +/- 35.55 (432-IU test meal) mumol L-1 h in the young group and 709.55 +/- 69.33 (937-IU test meal) vs. 436.39 +/- 41.08 (432-IU test meal) mumol L-1 h in the elderly group. We concluded that (a) the amount of vitamin E appearing in plasma is proportional to the dose ingested (up to 937 IU); (b) the intestinal absorption of vitamin E is not increased, even possibly decreased, in the elderly; and (c) the amount of vitamin E transported by non-chylomicron lipoproteins is apparently higher in the elderly. This suggests that vitamin E postprandial transport is affected by ageing, mainly as the consequence of age-related modifications of lipoprotein metabolism.

Infraclinical vitamin A deficiencies may be health-threatening for elderly people, yet they are difficult to assess unequivocally in this population. In this study, we evaluate the vitamin A status of an elderly institutionalized population (49 subjects, 83.6 +/- 6.1 years of age), by examining four different criteria: the dietary vitamin A intake, the retinol concentration in serum, the relative dose-response test and the impression cytology with transfer. The incidence of infra-clinical deficiencies was estimated to be 55% by examining dietary vitamin A intake, 21% by using the RDR test, 6% by the ICT and 2% from serum retinol values. These variations are not due to the choice of threshold values for each of the methods, yet rather to poor correlations between the results given by these methods. Canonical correlation analyses indicate that some parameters related to retinol secretion from the liver, including Zn, prealbumin and retinol-binding protein, can affect individual patient response towards the different methods. Validation of the RDR test in this elderly population was not successful because of poor reproducibility and moderate correction of RDR-detected vitamin A deficiencies by vitamin A supplementation. The method chosen for the determination of vitamin A status in elderly people must be carefully evaluated to account for possible age-related changes in the patient response to the method employed. In the institutionalized elderly population examined in this study, we observed a low vitamin A intake, whereas serum retinol and ICT are within normal ranges and while RDR test's responses appear too variable to draw any conclusion.

International audience; Objective: Assessing the effect of the dose of dietary triglycerides on preformed vitamin A (retinyl esters) bioavailability in humans. Design: Four test meals containing 15 000 RE retinyl-palmitate and either 0, 15, 30 or 40 g added triglycerides were ingested by eight healthy volunteers, at different days and in a randomized order. Setting: The study was done in the Hospital Sainte Marguerite, Marseille, France. Subjects: Eight healthy male volunteers were recruited by advertisement. Intervention: Blood samples were collected every hour for seven hours after the test meals intake. Serum and chylomicron (Svedberg flotation unit > 1000) were prepared by centrifugation and retinyl esters were measured by HPLC. Results: The serum retinyl ester response was not significantly lower after the intake of the meal without added triglycerides (7944±3262 nmol/L h) than after the intake of the fat meals (10012±2182, 7869±3157 and 10777±2067 nmol/L h for the 15, 30 and 40 g-fat meal, respectively), indicating that the serum retinyl ester response was not related to the amount of meal triglycerides. Chylomicron retinyl linoleate response stepwise increased when the amount of meal triglycerides increased while retinyl palmitate and retinyl stearate responses reached a maximum since 15 g triglycerides. Postprandial serum retinol concentration did not change whatever the meal ingested. Conclusions: (i) a significant amount of preformed vitamin A is apparently absorbed when ingested with trace amount of meal triglycerides only; (ii) meal triglycerides, up to 40 g/meal, do not increase preformed vitamin A bioavailability; (iii) the retinyl ester pattern recovered in the chylomicrons, and probably the esterification process of retinol, is affected by the amount of meal triglycerides; (iv) postprandial retinol homeostasis is not affected by dietary triglycerides

Data on the physico-chemical properties of carotenoids in biological emulsions are essential to our knowledge of carotenoid metabolism. Therefore, we determined the behavior of carotenoids in phospholipid-stabilized triglyceride emulsions, a model for biological emulsions such as dietary emulsions, triglyceride-rich lipoproteins, and intracellular storage droplets. The solubility of beta-carotene (a model for apolar carotenoids, carotenes) in pure bulk triglycerides (0.112 to 0.141 wt % according to triglycerides) was significantly higher than zeaxanthin (a model for polar carotenoids, xanthophylls) (0.022 to 0.088 wt %). The solubility of both carotenoids increased when the chain-length of the triglycerides' fatty acids decreased. The amount of zeaxanthin associated with lipid droplet dramatically increased in phospholipid-triglyceride droplets as compared to the pure corresponding triglyceride droplets, whereas the amount of beta-carotene associated with lipid droplets increased only slightly beta-Carotene distributed almost exclusively in the core of triolein-lecithin-carotenoid droplets, while zeaxanthin distributed preferentially at the droplet's surface. A significant percentage (8.3%) of zeaxanthin was spontaneously transferred from lipid droplets to aqueous phase and the remaining part was transferred during triglyceride hydrolysis catalysed by pancreatic lipase, while beta-carotene absolutely required triglyceride lipolysis to be transferred to the aqueous phase. Our results show that polar and apolar carotenoids behave differently in biological emulsions. They further our understanding of the bioavailability of polar and apolar carotenoids and of their distribution between lipoprotein particles.