Primary root growth in the absence or presence of exogenous $\mathrm{N}{\mathrm{O}}_{3}^{-}$ was studied by a quantitative genetic approach in a recombinant inbred line (RIL) population of Medicago ...truncatula. A quantitative trait locus (QTL) on chromosome 5 appeared to be particularly relevant because it was seen in both N-free medium (LOD score 5.7; R 2 =13.7) and medium supplied with $\mathrm{N}{\mathrm{O}}_{3}^{-}$ (LOD score, 9.5; R 2 =21.1) which indicates that it would be independent of the general nutritional status. Due to its localization exactly at the peak of this QTL, the putative NRT1- $\mathrm{N}{\mathrm{O}}_{3}^{-}$ transporter (Medtr5g093170.1), closely related to Arabidopsis AtNRT1.3, a putative low-affinity nitrate transporter, appeared to be a significant candidate involved in the control of primary root growth and $\mathrm{N}{\mathrm{O}}_{3}^{-}$ sensing. Functional characterization in Xenopus oocytes using both electrophysiological and ^{15}\mathrm{N}{\mathrm{O}}_{3}^{-}$ uptake approaches showed that Medtr5g093170.1, named MtNRT1.3, encodes a dual-affinity $\mathrm{N}{\mathrm{O}}_{3}^{-}$ transporter similar to the AtNRT1.1 'transceptor' in Arabidopsis. MtNRT1.3 expression is developmentally regulated in roots, with increasing expression after completion of germination in N-free medium. In contrast to members of the NRT1 superfamily characterized so far, MtNRT1.3 is environmentally up-regulated by the absence of $\mathrm{N}{\mathrm{O}}_{3}^{-}$ and down-regulated by the addition of the ion to the roots. Split-root experiments showed that the increased expression stimulated by the absence of $\mathrm{N}{\mathrm{O}}_{3}^{-}$ was not the result of a systemic signalling of plant N status. The results suggest that MtNRT1.3 is involved in the response to N limitation, which increases the ability of the plant to acquire $\mathrm{N}{\mathrm{O}}_{3}^{-}$ under N-limiting conditions.
ABSTRACT
Seed vigour is important for successful establishment and high yield, especially under suboptimal environmental conditions. In legumes, raffinose oligosaccharide family (RFO) sugars have ...been proposed as an easily available energy reserve for seedling establishment. In this study, we investigated whether the composition or amount of soluble sugars (sucrose and RFO) is part of the genetic determinants of seed vigour of Medicago truncatula using two recombinant inbred line (RIL) populations. Quantitative trait loci (QTL) mapping for germination rate, hypocotyl and radicle growth under water deficit and nutritional stress, seed weight and soluble sugar content was performed using RIL populations LR1 and LR4. Seven of the 12 chromosomal regions containing QTL for germination rate or post‐germinative radicle growth under optimal or stress conditions co‐located with Suc/RFO QTL. A significant negative correlation was also found between seed vigour traits and Suc/RFO. In addition, one QTL that explained 80% of the variation in the ratio stachyose/verbascose co‐located with a stachyose synthase gene whose expression profile in the parental lines could explain the variation in oligosaccharide composition. The correlation and co‐location of Suc/RFO ratio with germination and radicle growth QTL suggest that an increased Suc/RFO ratio in seeds of M. truncatula might negatively affect seed vigour.
Legume seeds accumulate large amount of oligosaccharides (RFO) at the expense of sucrose during their development. The role of these RFO remains enigmatic, ranging from a protection to survive in the dry state to a source of easily available carbon during germination. Using a QTL approach on the model legume Medicago truncatula on two RIL populations, we demonstrate a negative correlation and co‐location (6 out of 8 QTL) between the ratio Suc/RFO and the speed of germination and radicle growth. It is suggested that the conversion of Suc to RFO rather than the amount of RFO should be considered in order to understand the role of RFO in vigour of legume seeds.
Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid (ABA) but how this hormone controls ...seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.
A gene MtPPRD1, encoding a protein of 132 amino acids containing a proline-rich domain (PRD), has been revealed by suppressive subtractive hybridization (SSH) with two mRNA populations of embryo axes ...harvested immediately before and after radicle emergence. Although at the protein level MtPPRD1 showed low homology with plant lipid transfer proteins (LTPs), it did exhibit the eight cysteine residues conserved in all plant LTPs, a characteristic signature that allows the formation of a hydrophobic cavity adapted for loading hydrophobic molecules. Expression studies of MtPPRD1 have been carried out by quantitative real time RT-PCR throughout germination and post-germination processes in control seeds and seeds in which germination was delayed by abscisic acid (ABA) or the glutamine synthetase inhibitor methionine sulphoximine (MSX) treatments. The results showed that MtPPRD1 expression is developmentally regulated, induced in the embryo axis immediately before radicle emergence, reaches its maximum expression and declines during the early post-germination phase. Organ specificity studies showed that, except for a low and probably constitutive expression in roots, MtPPRD1 is specifically expressed in the embryo axis. Based on both experimental and in silico studies several putative roles are proposed for MtPPRD1 in Medicago truncatula, this protein can intervene (i) as an LTP in membrane biogenesis and regulation of the intracellular fatty acid pool by binding and transferring fatty acids and phospholipids between membranes, (ii) in the control of a developmental process specific to late germination and to early phases of post-germination, and (iii) and/or pathogen defence.
Lead (Pb) is an important environmental contaminant due to its widespread use over many centuries. While it affects primarily every organ system of the body, the most pernicious effects of Pb are on ...the central nervous system leading to cognitive and behavioral modification. Despite decades of research, the mechanisms responsible for Pb toxicity remain poorly understood. Recent work has suggested that Pb exposure may have consequences on chromosomal integrity as it was shown that Pb exposure leads to the generation of γH2Ax foci, a well-established biomarker for DNA double stranded break (DSB formation). As the chromosomal localization of γH2Ax foci plays an important role in determining the molecular mechanism responsible for their formation, we examined the localization of Pb-induced foci with respect to telomeres. Indeed, short or dysfunctional telomeres (uncapped or damaged telomeres) may be recognized as DSB by the DNA repair machinery, leading to "telomere-Induced Foci" (TIFs). In the current study, we show that while Pb exposure did not increase intra-chromosomal foci, it significantly induced TIFs, leading in some cases, to chromosomal abnormalities including telomere loss. The evidence suggests that these chromosomal abnormalities are likely due to perturbation of telomere replication, in particular on the lagging DNA strand. We propose a mechanism by which Pb exposure leads to the loss of telomere maintenance. As numerous studies have demonstrated a role for telomere maintenance in brain development and tissue homeostasis, our results suggest a possible mechanism for lead-induced neurotoxicity.
•We evaluate rapid global γH2AX fluorescence methods as emergency biodosimetry tools.•Global γH2AX fluorescence measurements were well correlated with γH2AX foci scoring.•Inter-individual variability ...was undetectable with global methods hours after exposure.•We propose the use of flow cytometry for rapid triage in an emergency setting.•We define triage categories using flow cytometry in fibroblasts and lymphocytes.
The phosphorylation of the H2AX histone to form γH2AX foci has been shown to be an accurate biomarker of ionizing radiation exposure. It is well established that there is a one-to-one correlation between the number of γH2AX foci and radiation-induced double strand breaks in cellular DNA, which can be translated to the received dose. However, manual counting of foci is time-consuming, and cannot accommodate high throughput analysis required to obtain rapid results for medical triage purposes in the case of large-scale accidental exposure. Furthermore, the accuracy of γH2AX measurements could potentially be compromised by delays between the time of exposure and analysis of results, as well as inter-cellular and inter-individual variability of this biological response. To evaluate more rapid approaches of quantifying γH2AX for use in an emergency situation, and to determine the impact of inter-individual variability, we compared two methods of global γH2AX fluorescence quantification (low magnification immunofluorescence microscopy and flow cytometry) to the well-established γH2AX foci scoring method in human primary fibroblasts. All three approaches were well correlated, indicating that global γH2AX fluorescence measurements are suitable for dose estimation. For rapid triage in an emergency situation, we propose the use of flow cytometry, as it is more highly correlated with foci scoring and because of the speed and ease of the method. Dose response curves (0.25–6Gy) using flow cytometry measurements showed that inter-individual variability in global γH2AX fluorescence is statistically insignificant at 4h post-irradiation. Based on these data, we propose calibration curves that can be applied to populations exposed to moderate radiation doses to estimate individual received doses, independent of individual radiosensitivity, at this specific time point post-irradiation using human fibroblasts and lymphocytes. Furthermore, we define three triage categories that could facilitate immediate and follow-up care in the case of a radiological accident.
The phosphorylation of the H2AX histone to form ...H2AX foci has been shown to be an accurate biomarker of ionizing radiation exposure. It is well established that there is a one-to-one correlation ...between the number of ...H2AX foci and radiation-induced double strand breaks in cellular DNA, which can be translated to the received dose. However, manual counting of foci is time-consuming, and cannot accommodate high throughput analysis required to obtain rapid results for medical triage purposes in the case of large-scale accidental exposure. Furthermore, the accuracy of ...H2AX measurements could potentially be compromised by delays between the time of exposure and analysis of results, as well as inter-cellular and inter-individual variability of this biological response. To evaluate more rapid approaches of quantifying ...H2AX for use in an emergency situation, and to determine the impact of inter-individual variability, we compared two methods of global ...H2AX fluorescence quantification (low magnification immunofluorescence microscopy and flow cytometry) to the well-established ...H2AX foci scoring method in human primary fibroblasts. All three approaches were well correlated, indicating that global ...H2AX fluorescence measurements are suitable for dose estimation. For rapid triage in an emergency situation, we propose the use of flow cytometry, as it is more highly correlated with foci scoring and because of the speed and ease of the method. Dose response curves (0.25-6 Gy) using flow cytometry measurements showed that inter-individual variability in global ...H2AX fluorescence is statistically insignificant at 4 h post-irradiation. Based on these data, we propose calibration curves that can be applied to populations exposed to moderate radiation doses to estimate individual received doses, independent of individual radiosensitivity, at this specific time point post-irradiation using human fibroblasts and lymphocytes. Furthermore, we define three triage categories that could facilitate immediate and follow-up care in the case of a radiological accident. (ProQuest: ... denotes formulae/symbols omitted.)
Studies suggest strong underreporting of occupational diseases in industrialized countries. We studied physician-related barriers to reporting of occupational sciatica (with herniated disk). We ...conducted a telephone survey of 391 general practitioners (GPs) and 96 rheumatologists in southeastern France with a standardized questionnaire and case-vignette of a patient with occupational sciatica. Our results show that 71% of GPs and 78% of rheumatologists would not recommend that the case-vignette patient file a workers' compensation claim, for the following reasons: possible role of nonwork-related activities (77%, 74%), should be reported as an occupational accident (67%, 42%), recommendation should be made by occupational physicians (60%, 60%), and risk of patient's losing his job (47%, 38%). Our results suggest that occupational sciatica is underreported in France. Physicians' lack of knowledge of reporting principles and procedures and their ethical dilemma regarding job loss may be barriers to reporting.
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