Silicon Supply Improves Nodulation and Dinitrogen Fixation and Promotes Growth in <i>Trifolium incarnatum</i> Subjected to a Long-Term Sulfur Deprivation

In many crops species, sulfur (S) deprivation negatively affects growth, seed yield quality and plant health. Furthermore, silicon (Si) is known to alleviate many nutritional stresses but the effects of Si supply on plants subjected to S deficiency remain unclear and poorly documented. The objective of this study was to evaluate whether Si supply would alleviate the negative effects of S deprivation on root nodulation and atmospheric dinitrogen (N2) fixation capacity in Trifolium incarnatum subjected (or not) to long-term S deficiency. For this, plants were grown for 63 days in hydroponic conditions with (500 µM) or without S and supplied (1.7 mM) or not with Si. The effects of Si on growth, root nodulation and N2 fixation and nitrogenase abundance in nodules have been measured. The most important beneficial effect of Si was observed after 63 days. Indeed, at this harvest time, a Si supply increased growth, the nitrogenase abundance in nodules and N2 fixation in S-fed and S-deprived plants while a beneficial effect on the number and total biomass of nodules was only observed in S-deprived plants. This study shows clearly for the first time that a Si supply alleviates negative effects of S deprivation in Trifolium incarnatum

Impact of T161, Y318 and S363 alanine mutations on regulation of the human delta-opioid receptor (hDOPr) induced by peptidic and alkaloid agonists

Previously, we showed a differential regulation of the human delta-opioid receptor (hDOPr) by etorphine and [D-Pen(2), D-Pen(5)] enkephalin (DPDPE). To understand the molecular basis of such differences, we introduced 3 alanine mutations at the residues T161. Y318 and S363. Both wild type (WT) and hDOPr mutants were expressed in HEK cells containing endogenous arrestins or CFP-tagged arrestin 3, then desensitization, internalization, recycling and phosphorylation were studied. In a context of endogenous arrestin expression, a major difference in DOPr desensitization was observed between agonists that was modified with the T161A mutation upon etorphine and with the S363A substitution upon DPDPE exposure. While both agonists induced a major receptor internalization, T161A and S363A impaired DOPr sequestration only for etorphine. However, similar level of S363 phosphorylation was measured between agonists. When CFP-tagged arrestin 3 was over-expressed, a similar profile of desensitization was measured for both agonists. In this context, all the 3 alanine mutations decreased etorphine-induced receptor desensitization. Using FRET, we showed similar interactions between WT hDOPr and arrestin 3 under DPDPE and etorphine stimulation which were delayed by both the Y318A and the S363A substitutions for etorphine. Finally, hDOPr recycling was qualitatively evaluated by microscopy and showed neither arrestin 3/hDOPr colocalization nor major impact of alanine mutations except for the S363A which impaired internalization and recycling for etorphine. The T161, Y318 and S363 residues of hDOPr could underlie the differential regulation promoted by DPDPE and etorphine

Effects of insulin on glucose consumption and lactate production.

HL-1 cells were seeded at similar density in T75 flasks, cultured in 25 mmol/l glucose and then exposed or not to 3 IU insulin for 24h. Glucose and lactate concentrations were determined at 0 and 24h in the culture medium to calculate glucose consumption (A) and lactate production (B). Data are the means ± S.E.M of 7 independent experiments. Statistical analysis was conducted using the Wilcoxon test. *, P < 0.05.</p

Evaluation of glucose consumption and lactate production in culture medium of HL-1 cells exposed to LG, NG, HG and IHG during 12 and 72h.

HL-1 cells were cultured at least during 3 weeks with normal glucose (5.5 mmol/l) then submitted to LG, NG, HG or IHG for 12 or 72h. Glucose (A and C) and lactate (B and D) concentrations were measured in the culture medium at different times (see Fig 1) to calculate glucose consumption and lactate production. Data are the means ± S.E.M of 4–6 independent experiments. Two-way ANOVA followed by the Tukey’s multiple comparisons test when evaluating the effect of glucose and time treatment. *, P $, P$ $, P$ $$, P §, P §§§, P treatment (3, 48) = 23.85, Ptime (3, 48) = 7.03, P = 0.0005; FtreatmentXtime (9, 48) = 6.92, Ptreatment (3, 48) = 38.90, Ptime (3, 48) = 3.43, P = 0.0242; FtreatmentXtime (9, 48) = 1.63, P = 0.132. For 72h treatment and glucose consumption, Ftreatment (3, 79) = 49.66, Ptime (3, 79) = 3.602, P = 0.017; FtreatmentXtime (9, 79) = 12.26, Ptreatment (3, 79) = 22.69, Ptime (3, 79) = 4.308, P = 0.007; FtreatmentXtime (9, 79) = 2.819, P = 0.006. (PPTX)</p

Comparison of the MMP in HL-1 cells exposed to LG, NG, HG and IHG between short- (12h) and long-time (72h) glucose treatment.

HL-1 cells were cultured at least during 3 weeks with normal glucose (5.5 mmol/l) then submitted to LG, NG, HG or IHG for 12h with medium change every 2h or 72h with medium change every 12h (see Fig 1). The MMP was measured using TMRE in the absence (basal, A) or in the presence of either succinate (B), palmitate (C) or pyruvate (D). Results were normalized to the NG condition. Data are the means ± S.E.M of 3–7 independent experiments. Two-way ANOVA followed by the Tukey’s multiple comparisons test when evaluating the effect of glucose and time treatment *, P $, P$ $$, P §, P §§§, P 12 vs 72h (1, 20) = 41.98, Pglucose treatment (3, 20) = 3.239, P = 0.0438; F 12 vs 72hXglucose treatment (3, 20) = 10.56, P = 0.0002; For succinate, F 12 vs 72h (1, 32) = 3.722, P = 0.0626; Fglucose treatment (3, 32) = 7.572, P = 0.0006; F 12 vs 72hXglucose treatment (3, 32) = 0.7248, P = 0.5447; For palmitate, F 12 vs 72h (1, 36) = 2.257, P = 0.1417; Fglucose treatment (3, 36) = 1.543, P = 0.2200; F 12 vs 72hXglucose treatment (3, 36) = 2.886, P = 0.0489; For pyruvate, F 12 vs 72h (1, 28) = 0.4816, P = 0.4934; Fglucose treatment (3, 28) = 2.624, P = 0.0701; F 12 vs 72hXglucose treatment (3, 28) = 3.349, P = 0.0331.</p

Measurement of mitochondrial superoxide anion production in HL-1 cells exposed for 12 or 72h to LG, NG, HG or IHG.

HL-1 cells were cultured at least during 3 weeks with normal (5.5 mmol/l) glucose then submitted to 4 different regimens (LG, NG, HG or IHG) either for 12 (A) or 72h (B). The mitochondrial superoxide anion production was measured using MitoSox under basal or in the presence of the specific complex III inhibitor, antimycin A. Results were normalized to the NG condition. Data are the means ± S.E.M of 3–4 independent experiments. Two-way ANOVA followed by the Tukey’s multiple comparisons test when evaluating the effect of glucose treatments and antimycin A. *, P $$, P $$ $, P §, P §§, P antimycin A (1, 24) = 29.42, Ptreatment (3, 24) = 63.82, Pantimycin AXtreatment (3, 24) = 3.914, P = 0.0208. For 72h treatment, Fantimycin A (1, 16) = 3.569, P = 0.0771; Ftreatment (3, 16) = 239.6, Pantimycin AXtreatment (3, 16) = 4.047, P = 0.0256. (PPTX)</p

A 2 bp deletion in the mitochondrial ATP 6 gene responsible for the NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome

International audienceMitochondrial (mt) DNA-associated NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) syndrome is due to mutation in the MT-ATP6 gene. We report the case of a 18-year-old man who presented with deafness, a myoclonic epilepsy, muscle weakness since the age of 10 and further developed a retinitis pigmentosa and ataxia. The whole mtDNA analysis by next-generation sequencing revealed the presence of the 2 bp microdeletion m.9127-9128 del AT in the ATP6 gene at 82% heteroplasmy in muscle and to a lower load in blood (10-20%) and fibroblasts (50%). Using the patient's fibroblasts, we demonstrated a 60% reduction of the oligomycin-sensitive ATPase hydrolytic activity, a 40% decrease in the ATP synthesis and determination of the mitochondrial membrane potential using the fluorescent probe tetramethylrhodamine, ethyl ester indicated a significant reduction in oligomycin sensitivity. In conclusion, we demonstrated that this novel AT deletion in the ATP6 gene is pathogenic and responsible for the NARP syndrome

Schematic representation of short- and long-time treatment of HL-1 cells with different glucose concentrations.

Cells were exposed to low (2.8 mmol/l, LG), normal (5.5 mmol/l, NG), high (25 mmol/l, NG), and intermittent high glucose (25 followed by 2.8 mmol/l, IHG) either during 12 or 72h. The culture medium was changed either every 2h or every 12h for short- and long-time treatment, respectively. Arrows indicate glucose and lactate measurements.</p