Analysis of transcripts differentially expressed between fruited and deflowered 'Gala' adult trees: a contribution to biennial bearing understanding in apple.

BACKGROUND: The transition from vegetative to floral state in shoot apical meristems (SAM) is a key event in plant development and is of crucial importance for reproductive success. In perennial plants, this event is recurrent during tree life and subject to both within-tree and between-years heterogeneity. In the present study, our goal was to identify candidate processes involved in the repression or induction of flowering in apical buds of adult apple trees. RESULTS: Genes differentially expressed (GDE) were examined between trees artificially set in either 'ON' or 'OFF' situation, and in which floral induction (FI) was shown to be inhibited or induced in most buds, respectively, using qRT-PCR and microarray analysis. From the period of FI through to flower differentiation, GDE belonged to four main biological processes (i) response to stimuli, including response to oxidative stress; (ii) cellular processes, (iii) cell wall biogenesis, and (iv) metabolic processes including carbohydrate biosynthesis and lipid metabolic process. Several key regulator genes, especially TEMPRANILLO (TEM), FLORAL TRANSITION AT MERISTEM (FTM1) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) were found differentially expressed. Moreover, homologs of SPL and Leucine-Rich Repeat proteins were present under QTL zones previously detected for biennial bearing. CONCLUSIONS: This data set suggests that apical buds of 'ON' and 'OFF' trees were in different physiological states, resulting from different metabolic, hormonal and redox status which are likely to contribute to FI control in adult apple trees. Investigations on carbohydrate and hormonal fluxes from sources to SAM and on cell detoxification process are expected to further contribute to the identification of the underlying physiological mechanisms of FI in adult apple trees.

Live imaging of DORNRÖSCHEN and DORNRÖSCHEN-LIKE promoter activity reveals dynamic changes in cell identity at the microcallus surface of Arabidopsis embryonic suspensions.

KEY MESSAGE : Transgenic DRN::erGFP and DRNL::erGFP reporters access the window from explanting Arabidopsis embryos to callus formation and provide evidence for the acquisition of shoot meristem cell fates at the microcalli surface. The DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL) genes encode AP2-type transcription factors, which are activated shortly after fertilisation in the zygotic Arabidopsis embryo. We have monitored established transgenic DRN::erGFP and DRNL::erGFP reporter lines using live imaging, for expression in embryonic suspension cultures and our data show that transgenic fluorophore markers are suitable to resolve dynamic changes of cellular identity at the surface of microcalli and enable fluorescence-activated cell sorting. Although DRN::erGFP and DRNL::erGFP are both activated in surface cells, their promoter activity marks different cell identities based on real-time PCR experiments and whole transcriptome microarray data. These transcriptome analyses provide no evidence for the maintenance of embryogenic identity under callus-inducing high-auxin tissue culture conditions but are compatible with the acquisition of shoot meristem cell fates at the surface of suspension calli.

Gene expression profiling of Arabidopsis meiocytes.

Meiosis is a special type of cell division present in all organisms that reproduce by sexual reproduction. It ensures the transition between the sporophytic and gametophytic state and allows gamete production through meiotic recombination and chromosome number reduction. In this paper, we describe a technique for the isolation of Arabidopsis thaliana male meiocytes. From this cellular material, it was then possible to develop large-scale transcriptome studies using CATMA microarrays and thus to obtain an overview of genes expressed during Arabidopsis meiosis. The expression profiles were studied with either stringent statistical criteria or by performing clustering. Both methods resulted in gene clusters enriched in meiosis-specific genes (from 14- to 55-fold). Analysis of these data provided a unique set of genes that will be pivotal to further analysis aimed at understanding the meiotic process.

Sublethal cadmium intoxication in Arabidopsis thaliana impacts translation at multiple levels.

To study the impact of translational regulation during heavy metal poisoning, Arabidopsis thaliana cell cultures were submitted to sublethal cadmium stress. At the concentration used, cadmium had a minimal impact on the growth of the culture but induced an accumulation of high molecular weight polysomes without de novo production of new ribosomes together with a reduction of protein synthesis. In addition, cadmium stress induces phosphorylation of eukaryotic initiation factor 2α by GCN2 and, in planta, gcn2 mutants are more sensitive to cadmium stress, suggesting a role for this translational regulation mechanism in the response to cadmium stress. Microarray analysis of total and polysomal RNAs in control and cadmium-treated cells reveals a large class of genes for which a variation in total RNA abundance is not linked to a variation in polysomal loading, suggesting that transcription and translation are uncoupled and that these genes are not recruited at the initiation step of translation.

Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots.

Transcriptional regulation in response to cadmium treatment was investigated in both roots and leaves of Arabidopsis, using the whole genome CATMA microarray containing at least 24,576 independent probe sets. Arabidopsis plants were hydroponically treated with low (5 microM) or high (50 microM) cadmium concentrations during 2, 6, and 30 hours. At each time point, Cd level was determined using ICP-AES showing that both plant tissues are able to accumulate the heavy metal. RT-PCR of eight randomly selected genes confirmed the reliability of our microarray results. Analyses of response profiles demonstrate the existence of a regulatory network that differentially modulates gene expression in a tissue- and kinetic-specific manner in response to cadmium. One of the main response observed in roots was the induction of genes involved in sulfur assimilation-reduction and glutathione (GSH) metabolism. In addition, HPLC analysis of GSH and phytochelatin (PC) content shows a transient decrease of GSH after 2 and 6 h of metal treatment in roots correlated with an increase of PC contents. Altogether, our results suggest that to cope with cadmium, plants activate the sulfur assimilation pathway by increasing transcription of related genes to provide an enhanced supply of GSH for PC biosynthesis. Interestingly, in leaves an early induction of several genes encoding enzymes involved in the biosynthesis of phenylpropanoids was observed. Finally, our results provide new insights to understand the molecular mechanisms involved in transcriptional regulation in response to cadmium exposure in plants.

MRI virtual biopsies: analysis of an explanted endovascular device and perspectives for the future.

Information that can be obtained by magnetic resonance imaging (MRI) of explanted endovascular devices must be validated as this method is non-destructive. Histology of such a device together with its encroached tissues can be elegantly performed after polymethymethacrylate (PMMA) embedding, but this approach requires destruction of the specimen. The issue is therefore to determine if the MRI is sufficient to fully validate an explanted device based upon the characterization of an explanted specimen. An AneuRx device deployed percutaneously 25 months earlier in a 75-year-old patient was removed en bloc at autopsy together with the surrounding aneurysmal sac and segments of the upstream and downstream arteries. Macroscopic pictures were taken and a slice of the cross-section was processed for histology after polymethylmethacrylate (PMMA) embedding. For the magnetic resonance imaging investigation, the device was inserted in a Biospec 4.7 T MRI system with a 20 mm diameter birdcage resonator used for both emission and reception. A Spin-Echo (SE) was used to acquire both T1 proton density (PD) and T2 weighted images. A gradient-echo (GE) sampling of a free induction decay (GESFID) was used to generate multiple GE images using a single excitation pulse so that four images at different TE were obtained in the same acquisition. The selected explanted device was outstandingly well-healed compared to most devices harvested from humans. No inflammatory process was observed in contact or at distance of the materials. In MRI T1 images display no specific contrast and were homogeneous in the different tissues. The contrast was improved on proton density weighed images. On the T2 weighed images, the different areas were well identified. The diffusion images displayed in the surrounding B region had the greatest diffusion coefficient and the greatest anisotropy. The MRI analysis of the explanted AneuRx device illustrates the possibilities of this technique to characterize the interaction of the endovascular graft with the surrounding tissues. MRI is a breakthrough to investigate explanted medical devices but it also can be advantageously used in vivo to obtain virtual biopsies, because real biopsies to determine the 3 Bs (biocompatibility, biofunctionality and bioresilience) cannot be carried out as they could obviously initiate infection and degradation of the foreign materials.

Effect of flour minor components on bubble growth in bread dough during proofing assessed by magnetic resonance imaging.

Fermentation of dough made from standard flour for French breadmaking was followed by nuclear magnetic resonance imaging at 9.4 T. The growth of bubbles (size > 117 microm) was observed for dough density between 0.8 and 0.22 g cm(-3). Cellular structure was assessed by digital image analysis, leading to the definition of fineness and rate of bubble growth. Influence of composition was studied through fractionation by extraction of soluble fractions (6% db), by defatting (< 1% db) and by puroindolines (Pin) addition (< or = 0.1%). Addition of the soluble fraction increased the dough specific volume and bubble growth rate but decreased fineness, whereas defatting and Pin addition only increased fineness. The role of molecular components of each fraction could be related to dough elongational properties. A final comparison with baking results confirmed that the crumb cellular structure was largely defined after fermentation.

Dexamethasone impairs muscle energetics, studied by (31)P NMR, in rats.

AIMS/HYPOTHESIS: Glucocorticoid treatments are associated with increased whole-body oxygen consumption. We hypothesised that an impairment of muscle energy metabolism can participate in this increased energy expenditure. METHODS: To investigate this possibility, we have studied muscle energetics of dexamethasone-treated rats (1.5 mg kg(-1) day(-1) for 6 days), in vivo by (31)P NMR spectroscopy. Results were compared with control and pair-fed (PF) rats before and after overnight fasting. RESULTS: Dexamethasone treatment resulted in decreased phosphocreatine (PCr) concentration and PCr:ATP ratio, increased ADP concentration and higher PCr to gamma-ATP flux but no change in beta-ATP to beta-ADP flux in gastrocnemius muscle. Neither 4 days of food restriction (PF rats) nor 24 h fasting affected high-energy phosphate metabolism. In dexamethasone-treated rats, there was an increase in plasma insulin and non-esterified fatty acid concentration. CONCLUSIONS/INTERPRETATION: We conclude that dexamethasone treatment altered resting in vivo skeletal muscle energy metabolism, by decreasing oxidative phosphorylation, producing ATP at the expense of PCr.

[Natural polymers according to NMR data: cross-relaxation in hydrated collagen macromolecules from two connective tissues]. / Prirodnye polimery po dannym IaMR: krossrelaksatsiia v makromolekulakh kollagena iz dvukh soedinitel'nykh tkanei.

Spin-lattice relaxation and cross-relaxation in oriented and randomly oriented collagen fibers from two connective tissues (15-month-old calf and 8-year-old steer) at a water content of 0.6 g H2O/g dry matter were studied. Collagens were chosen according to different numbers of covalent nonreducible cross-links, which increase during the life of the animal. The spin-lattice relaxation curves for all the collagens after a 180 degree-tau-90 degree pulse sequence were described by two exponential components. The dependences of two components of spin lattice relaxation time and their populations on the length of the 180 degree-pulse were obtained. On the basis of data of Goldman-Shen sequence and the two-phase model, the populations of proton fractions (p(w) and p(c)) as well as the rates of transfer of magnetization between water protons and collagen protons (k(w) and k(c)) were calculated. No significant difference between k(w) (k(c)) in oriented and randomly oriented fibers as well as in fibers with different cross-linking was found. The estimates of the cross-relaxation times for low cross-link collagen and high cross-link one were done. The correlation times of dipole-dipole interactions for both connective tissues were calculated using the cross-relaxation theory.

Brain GABA editing by localized in vivo (1)H magnetic resonance spectroscopy.

Editing of GABA by (1)H MRS in a specific brain area is a unique tool for in vivo non-invasive investigation of neurotransmission disorders. Selective GABA detection is achieved using sequences based on double quantum coherence (DQC). Our pulse sequence makes accurate measurements without artefacts due to spatial localization. The sequence was tested on a phantom solution. The effect of vigabatrin, a specific inhibitor of GABA transaminase, was measured in rat brain and GABA detection was performed in vivo in monkey brain using this procedure. Rats were split into two groups. In the control group, the rats had access to water and, in the other group (vigabatrin, VGB, rats), animals were allowed free access to drinking water containing vigabatrin. After 3 weeks of treatment, rats were anesthetized for in vivo NMR spectroscopy investigation. At the end of the experiment, brains were quickly removed, freeze-clamped and extracted with 4% perchloric acid. One part of the acid extract was used for GABA concentrations assessment by ion exchange chromatography with ninhydrin detection. The second was used for high-resolution NMR analysis. By chromatography measurements, the GABA concentration was 1.23+/-0.06 micromol/g for controls, while for vigabatrin-treated rats the GABA concentration was 4.89+/-1.60 micromol/g. The NMR in vivo results were closely correlated with the NMR ex vivo (r=0.99, p<0.01) and chromatography results (r=0.98, p<0.01). The correlation between ex vivo results and chromatography results was also high (r=0.99, p<0.001). This pulse sequence performed GABA editing from a 376 microl voxel located on the right basal ganglia area in a non-human primate brain. This in vivo GABA editing scheme can thus be proposed for accurate measurement of brain GABA concentrations.

Lack of effects of hyperkalemia on the metabolism of normoxic or anoxic rabbit triceps brachii muscle.

Rabbit triceps brachii muscle was perfused with bovine red cells medium. Changes in phosphorus compounds and intracellular pH were followed using (31)P NMR during 15 min in the perfused muscle and during 50 min in muscle made anoxic by perfusion stop. Potassium levels in perfusate was maintained at 4 mM (normal plasma concentration at rest) during all perfusion in one muscle and at 4 mM for 10 min then 10 mM during 5 min before perfusion stop in the contralateral muscle. The intracellular pH and phosphorylated compounds content remained stable in the perfused muscle whatever the potassium concentration of the perfusate. Five min after perfusion stop a decrease in phosphocreatine (P<0.05) and pH (P<0.01) and an increase in sugar-phosphates (P<0.01) were observed independently of potassium concentration. The lack of effect of increasing circulating potassium indicates that hyperkalemia does not affect, by itself the muscles energetic metabolism.

Water transfer analysis in pork meat supported by NMR imaging.

NMR proton density imaging was used to study isothermal and unidirectional drying of pork semi membranosus muscle samples at temperatures of 12, 16 and 20 °C. An independent calibration of the transversal relaxation time T(2) as a function of the moisture content was carried out to convert the signal amplitude into moisture content. Due to spatial heterogeneity in drying, 2D images were needed to assess the evolution of 1D moisture profiles. The relationship between the effective water diffusivity (D) was calculated in function of water content (X) using the Boltzman transformation which needs no a priori on the relationship D=f(X); the effect of lipid content, temperature and fibre direction on this relationship were also studied. In all cases a decrease in water content brought about a decrease in D. A slight increase in lipid content led to a dramatic decrease in D. The fibre direction relative to water movement had a negligible effect. No significative differences in D between the three temperatures were observed, due to variability in the chemical composition of the samples.

Measurements of extracellular volume fraction and capillary permeability in tissues using dynamic spin-lattice relaxometry: studies in rabbit muscles.

Dynamic MR longitudinal R(1) relaxometry after administration of a gadolinium contrast bolus (Gd-DTPA) has been used for in vivo measurements of the extracellular volume fraction (v) and the capillary permeability (k min(-1)) in rabbit muscles to distinguish between red slow- and white fast-twitch muscle fiber types. For this purpose a protocol imaging sequence has been used which allows fast R(1) measurements during the contrast agent uptake. Physiological tissue parameters, k and v, were obtained by computing procedures assuming a simplified monoexponential plasma model. These were shown to be about twice as large in the slow-twitch semimembranosous proprius muscle (SP), containing 100% oxidative type-I fiber, that in the fast-twitch rectus femorus muscle (RF), containing only 6% type-I fiber type. The capillary permeability has been found to be 0.25 +/- 0.02 min(-1) for the (SP) and 0.10 +/- 0.01 min(-1) for the (RF). Similarly, the extracellular volume fractions were 0.189 +/- 0.015 and 0.082 +/- 0.006 respectively, in close agreement with literature data and experimental results obtained by invasive radionuclide measurements. For the pool of the 10 studied animals, no significant variation among animals was observed in the extracellular volume fraction and the capillary permeability for the different muscle fiber types. The dynamic relaxometry method used is easy to implement on conventional MR imagers and has potential applications in muscle diseases. The method has also potential applications for tissue characterization based on extracellular volume and capillary permeability quantification. In particular, the method can be used for the evaluation of tumors and their responses to therapies.

Contrast optimization of Macaca mulatta basal ganglia in magnetic resonance images at 4.7 Tesla.

To determine whether high field MRI could distinguish among the different regions of the basal ganglia, the brains of two Macaca mulatta monkeys were explored in vivo using a 4.7 T MR imager. Gradient-echo (GE) and spin-echo images were acquired with proton-density, T1 and T2* weightings. Five GE images with increased susceptibility effects were generated using a GESFID sequence, from which T2* maps were also reconstructed. The first echo of the GESFID sequence (TE = 12.6 ms) produced the best contrast-to-noise ratio (C/N) between the pallidum and the putamen, the pallidum and the thalamus, the substantia nigra and the surrounding white matter, and the substantia nigra and the subthalamic nucleus. An increased T2*-weighting (TE = 37.2 ms) was necessary to maximize C/N between the putamen and the surrounding white matter, and between the subthalamic nucleus and the surrounding white matter. A dual GE sequence with a short TE ( approximately 10 ms) and a longer one ( approximately 30 ms) thus effectively localizes basal ganglia subregions at 4.7 T.

Detection of susceptibility effects using simultaneous T(2)* and magnetic field mapping.

Tracking susceptibility effects is a convenient way to detect small inclusions in a bulk tissue matrix by MRI. We propose a quantitative assessment of these susceptibility effects by simultaneously mapping T(2)* and magnetic field from the time course of magnitude and phase using a multiple GE sequence at 4.7 T. A high-pass scheme is also introduced to highlight the mesoscopic magnetic field variations due to local susceptibility differences specifically in the magnetic field map. Applying this method to muscle tissue, we demonstrate that connective tissue generates detectable susceptibility effects through concomitant local magnetic field variation and T(2)* shortening.

Expression of a yeast RNase III gene in transgenic tobacco silences host nitrite reductase genes.

When a gene encoding the Schizosaccharomyces pombe dsRNA-specific RNase III, pac1, was expressed in transgenic tobacco plants, six out of thirteen transformed plants gave progeny among which were individuals displaying a distinctive chlorotic phenotype. These chlorotic plants strongly resemble those transformed with a 35S-Nii (nitrite reductase) transgene, in which both Nii host genes and the 35S-Nii transgene are silenced by co-suppression. RNA blots showed that the host Nii genes were silenced in chlorotic 35S-pac1 plants but not in individuals with a normal green phenotype. Neither the transcript levels of the other cellular genes tested nor the transcription of Nii genes was significantly affected by the expression of pac1. This is the first observation of post-transcriptional silencing of host genes by a transgene with no apparent sequence similarity to the target gene.

1H NMR studies: dynamics of water in gelatin.

Proton magnetic resonance was used to characterize the dynamics of water in gelatin. Both sol and gel states were investigated. Transverse relaxation rates (R2) were dependent on the proton frequency measurement. (R2) measured with the Carr-Purcell-Meiboom-Gill pulse sequence was dependent on pulse spacing. These observations were interpreted in terms of chemical exchanges between water protons and those of the macromolecules in the sol state, whereas in the gel state the contribution of diffusion through microheterogeneities in the sample seems to provide an additional transverse relaxation mechanism.

Imaging of water and fat fractions in high-field MRI with multiple slice chemical shift-selective inversion recovery.

Fat and water fractions were quantified at high field using a chemical shift-selective inversion recovery (CSS-IR) sequence to address the major difficulties encountered at high field by phase-sensitive techniques used for fat/water discrimination. Water- and fat-suppressed images were perfectly registered, which is a prerequisite for quantification. Immunity of the inversion pulse to B(1) field modulations and off-resonance effects was tested for two adiabatic inversion pulses, with hyperbolic secant and asymmetric hyper-pulse waveforms. Taking into account adiabaticity, immunity to off-resonance, radiofrequency power requirements, and specific absorption rate, the former was chosen. A close correlation was found between fat content measured by CSS-IR at 4.7 T (R(2)= 0.97, P < 0.001) and 9.4 T (R(2)= 0.99, P < 0.05) and that measured by Soxhlet extraction. Different sources of bias (low signal-to-noise ratio, magnetization transfer effect) are discussed. J. Magn. Reson. Imaging 2000;12:488-496.

1H-nmr study of water dynamics in hydrated collagen: transverse relaxation-time and diffusion analysis.

Water proton transverse relaxation times (T2) and self-diffusion coefficients (D) were measured in randomly oriented hydrated collagen fibers. Three T2 relaxation times were discerned indicating the presence of at least three water fractions in the collagen sample. The D values associated with each water fraction were determined. The diffusion time dependence of D suggests water motion is restricted by macromolecular structure. The experimental results are discussed with reference to the structural properties of hydrated collagen fibers.

Lack of effect of ageing on acetate oxidation in rat skeletal muscle during starvation: a (13)C NMR study.

Acetate oxidation was examined by (13)C nuclear magnetic resonance in skeletal muscle from adult and old rats. Rats fasted for 5 days were perfused with [2-(13)C]acetate over 2 h, and muscle extracts were analyzed for [(13)C]glutamate isotopomers. This study shows that approximately 80 % of acetyl-coenzyme A entering the tricarboxylic cycle was derived from substrate infusion in both adult and old rats, and that the flux through anaplerotic pathways was approximately 21 % of the flux through citrate synthase. These data demonstrate that skeletal muscle from adult and old rats oxidizes the same proportion of exogenous acetate.