Showing papers by "Institut national de la recherche agronomique published in 1993"

Integration of hydraulic and chemical signalling in the control of stomatal conductance and water status of droughted plants

Abstract: We describe here an integration of hydraulic and chemical signals which control stomatal conductance of plants in drying soil, and suggest that such a system is more likely than control based on chemical signals or water relations alone. The determination of xylem [ABA] and the stomatal response to xylem [ABA] are likely to involve the water flux through the plant. (1) If, as seems likely, the production of a chemical message depends on the root water status (Ψr), it will not depend solely on the soil water potential (Ψs) but also on the flux of water through the soil-plant-atmosphere continuum, to which are linked the difference between Ψr and Ψs. (2) The water flux will also dilute the concentration of the message in the xylem sap. (3) The stomatal sensitivity to the message is increased as leaf water potential falls. Stomatal conductance, which controls the water flux, therefore would be controlled by a water-flux-dependent message, with a water-flux-dependent sensitivity. In such a system, we have to consider a common regulation for stomatal conductance, leaf and root water potentials, water flux and concentration of ABA in the xylem. In order to test this possibility, we have combined equations which describe the generation and effects of chemical signals and classical equations of water flux. When the simulation was run for a variety of conditions, the solution suggested that such common regulation can operate. Simulations suggest that, as well as providing control of stomatal conductance, integration of chemical and hydraulic signalling may also provide a control of leaf water potential and of xylem [ABA], features which are apparent from our experimental data. We conclude that the root message would provide the plant with a means to sense the conditions of water extraction (soil water status and resisance to water flux) on a daily timescale, while the short-term plant response to this message would depend on the evaporative demand.

High-efficiency gene inactivation and replacement system for gram-positive bacteria.

Abstract: A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.

Prediction of digestibility of nutrients and energy values of pig diets from chemical analysis.

Abstract: The DE and ME values and digestible nutrient contents of 114 diets were measured in 45-kg growing pigs (four to five animals per diet) fed approximately 500 kcal of ME/kg BW60 Diets differed widely with regard to their chemical characteristics and their ingredients Chemical composition of each diet was measured by at least four laboratories The results were used to establish prediction equations of DE or ME values, digestible nutrient contents, and digestibility coefficients of energy and nutrients from chemical characteristics Digestibility coefficients of energy (range: 65 to 95%) and CP (range: 64 to 94%) were highly dependent on dietary fiber and mineral contents The digestibility coefficient of ether extract increased curvilinearly (from 2 to 84%) with the dietary fat content The digestibility of fiber was lower (45%, for NDF) than for the other chemical consti- tuents The ME:DE ratio averaged 963% and was negatively correlated to the dietary protein content The DE and ME values could be accurately predicted (R2 > 90 and CV < 2%) from chemical characteristics; the best equations were obtained when the following predictors were combined in a linear model: ash, ether extract, crude protein, and an estimate of dietary fiber The accuracy of the prediction was higher with NDF than with ADF or Weende crude fiber The results suggest that even a rather large proportion of dietary fiber (approximately 50%) is degraded in the digestive tract, the amount of available energy from fiber digestion is negligible in connection with in- creased endogenous protein and fat losses The equa- tions obtained in the present, study represent a basis for the prediction of the energy values of mixed diets with a composition of unknown ingredients

(CT) n and (GT) n microsatellites: a new class of genetic markers for Salmo trutta L. (brown trout)

Abstract: Thirteen (GT)n and four (CT)n microsatellite loci (n = 10 or more and n = 20 or more, respectively) have been isolated from a partial genomic library of brown trout and sequenced. On average, a (GT)n repeat sequence occurs approximately every 23 kb and a (CT)n repeat sequence every 76 kb in brown trout genome. Primers for DNA amplifications using the polymerase chain reaction (PCR) were synthesized for three single locus microsatellites. Mendelian inheritance of the observed polymorphisms was confirmed in full-sib families. Four brown trout populations (10 unrelated individuals per population) were screened for polymorphism with these three microsatellite loci. The total number of alleles detected in the four populations is five at one locus, six at the other two microsatellite loci and is three, on average, per population. Heterozygosities range from 0.18 to 0.74. The largest differences in allelic frequencies occurred between the Mediterranean and the Atlantic populations: this result is congruent with previous allozymic data. The gene-centromere distances of the three microsatellite markers were determined on gynogenetic lines: post-reduction rates range from 0.17 to 0.60. For all the three microsatellite loci, the primers designed from brown trout sequences can be used in another closely related species of salmonid, the rainbow trout (Oncorhynchus mykiss). This last aspect supports the view that microsatellite markers may have wide application in genetic studies in salmonid species and fishes in general.

Cloning and expression of cytochrome p450 genes controlling flower colour

Abstract: BLUE and violet flowers generally contain derivatives of delphinidin; red and pink flowers generally contain derivatives of cyanidin or pelargonidin1.Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. Here we report on the isolation of complementary DNA clones of two different flavonoid 3',5'-hydroxylase genes that are expressed in petunia flowers. Restriction-fragment length polymorphism mapping and complementation of mutant petunia lines showed that the flavonoid 3',5'-hydroxylase genes correspond to the genetic loci Hfl and Hf2.

Regulation of nitrate and nitrite reductase expression in Nicotiana plumbaginifolia leaves by nitrogen and carbon metabolites.

Abstract: Nitrate (NR) and nitrite reductase (NiR) catalyse the reduction of nitrate to ammonium. The regulation of NR and NiR gene expression by carbohydrates (C) and nitrogen (N) metabolites was studied using detached leaves. In the dark, glucose fructose and sucrose supplied to detached green leaves of dark-adapted Nicotiana plumbaginifolia plants resulted in NR mRNA and protein accumulation and the loss of circadian rhythmicity in the size of the transcript pool. The characterization of transgenic plants expressing either a NR cDNA controlled by the 35S CaMV promoter or a transcriptional fusion between the tobacco nia1 (NR structural gene) promoter and the beta-glucuronidase reporter gene, led us to conclude that C metabolite control is taking place at the transcriptional level. Under low light conditions (limiting photosynthetic conditions), the supply of glutamine or glutamate resulted in a drop in the level of NR mRNA. Exogenously supplied carbohydrates partially antagonized this inhibitory effect suggesting that the availability of N and C metabolites affects the expression of the NR gene. The effects of carbohydrates and glutamine on NiR expression were also studied. NiR mRNA levels in the dark were relatively insensitive to feeding with glucose. Glutamate and glutamine were less efficient at decreasing NiR mRNA than NR mRNA levels. In contrast to NR, NiR mRNA levels were significantly increased by light treatments, indicating that NiR display regulatory characteristics reminiscent of photosynthetic genes such as the small subunit of ribulose bisphosphate carboxylase than to NR.

Plantaricins S and T, Two New Bacteriocins Produced by Lactobacillus plantarum LPCO10 Isolated from a Green Olive Fermentation.

Abstract: Twenty-six strains of Lactobacillus plantarum isolated from green olive fermentations were tested for cross-antagonistic activities in an agar drop diffusion test. Cell-free supernatants from four of these strains were shown to inhibit the growth of at least one of the L. plantarum indicator strains. L. plantarum LPCO10 provided the broadest spectrum of activity and was selected for further studies. The inhibitory compound from this strain was active against some gram-positive bacteria, including clostridia and propionibacteria as well as natural competitors of L. plantarum in olive fermentation brines. In contrast, no activity against gram-negative bacteria was detected. Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. Since the inhibitory activity of the active supernatant was lost after treatment with various proteolytic enzymes, this substance could be classified as a bacteriocin, designated plantaricin S. Plantaricin S was also sensitive to glycolytic and lipolytic enzymes, suggesting that it was a glycolipoprotein. It exhibited a bactericidal and nonbacteriolytic mode of action against indicator cells. This bacteriocin was heat stable (60 min at 100 degrees C), active in a pH range of 3.0 to 7.0, and also stable in crude culture supernatants during storage. Ultrafiltration studies indicated that plantaricin S occurred as multimolecular aggregates and that the size of the smallest active form is between 3 and 10 kDa. In sodium dodecyl sulfate-polyacrylamide gels, plantaricin S migrated as a peptide of ca. 2.5 kDa. Maximum production of plantaricin S was obtained in a fermentor system in unregulated pH and log-phase cultures of L. plantarum LPCO10 in MRS broth plus 4% NaCl. In these culture conditions, a second bacteriocin (designated plantaricin T) was produced in late-stationary-phase cultures of L. plantarum LPCO10. On the basis of its biological activity, its sensitivity to various enzymes, and its molecular weight (lower than that of plantaricin S) as assessed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, plantaricin T appeared different from plantaricin S. Curing experiments with L. plantarum LPCO10 resulted in the appearance of variants that no longer produced either of the two bacteriocins but that were still immune to both of them.

Drought‐induced leaf shedding in walnut: evidence for vulnerability segmentation

Abstract: Trees of Juglans regia L. shed leaves when subjected to drought. Before shedding (when leaves are yellow), the petioles have lost 87% of their maximum hydraulic conductivity, but stems have lost only 14% of their conductivity. This is caused by the higher vulnerability of petioles than stems to water-stress induced cavitation. These data are discussed in the context of the plant segmentation hypothesis.

A new race of Venturia inaequalis virulent to apples with resistance due to the Vf gene

Abstract: The gene Vf from Malus floribunda 821 for resistance to scab (Venturia inaequalis) has been used successfully for 50 years in apple breeding programs. Since 1984, scab symptoms have been observed in the field at Ahrensburg, Germany, on seedlings of apple cv. Prima that have been selected as resistant in the greenhouse. In 1988, small scab lesions were found on some Vf selections in the same orchard. The inoculum from Ahrensburg was compared with the inoculum currently used at Angers, France, for selecting apple seedlings for resistance to V. inaequalis. All Vf-gene cvs. or selections tested were susceptible to the Ahrensburg inoculum, whereas M. floribunda 821 itself and the ornamental crabapple Evereste were resistant. The progeny from a cross between a resistant (Vf) and a susceptible cv. segregate into the 5 expected classes to Angers inoculum, but were completely susceptible to Ahrensburg inoculum. These results indicate the urgency of diversifing the sources of resistance to V. inaequalis in new breeding strategies. The distinction should be made between the resistance of M. floribunda 821, that is resistant to Ahrensburg inoculum, and that of the named cv. and selections, that are susceptible. The new race of V. inaequalis is named race 6.

An inventory of 1152 expressed sequence tags obtained by partial sequencing of cDNAs from Arabidopsis thaliana

Abstract: As part of the goal to generate a detailed transcript map for Arabidopsis thaliana, 1152 single run sequences (expressed sequence tags or ESTs) have been determined from cDNA clones taken at random in libraries prepared from different sources of plant material: developing siliques, etiolated seedlings, flower buds, and cultured cells. Eight hundred and ninety-five different genes could be identified, 32% of which showed significant similarity to existing sequences in Arabidopsis and an array of other organisms. These sequences in combination with their positioning on the Arabidopsis genetic map will not only constitute a new set of molecular markers for genome analysis in Arabidopsis but also provide a direct route for the in vivo analysis of their gene products. The sequences have been made available to the public databases.

Recent advances in the biological control of Fusarium wilts.

Abstract: Studies of soils suppressive to fusarium wilts have indicated that the phenomenon is fundamentally microbiological in nature, resulting from complex microbial interactions between the pathogen and all, or a part of, the saprophytic microflora. Many groups of micro-organisms have been proposed as having a role in this process. The most consistent results show that non-pathogenic Fusarium oxysporum and fluorescent pseudomonads are the main agents for biological control. Each group has been effective in reducing the severity of fusarium diseases in several crops under experimental conditions. All the problems associated with application of antagonists have not yet been solved, but special situations exist where biological control could be applied soon. In Europe, the present development of soil-less systems to grow tomatoes, cucumbers, carnations and a few other species of flowers and vegetables in greenhouses, offers a unique opportunity to use antagonists. A strain of non-pathogenic Fusarium isolated from soil from Chǎteaurenard can be established in the soil-less substrates, or in the potting mixtures, before transplanting the plants and in these conditions can prevent colonization of the substrates by the pathogens. It gave good control of fusarium wilts of tomatoes, carnations and cyclamens, and of crown and root rot of tomatoes due to F. oxysporum f.sp.radicis lycopersici. This non-pathogenic strain, produced by liquid fermentation and mixed with talc, is easy to introduce into soil-less substrates after having been suspended in water or in liquid fertilizer. Experiments have been under way in commercial greenhouses for several years, but it will be necessary to wait at least two more years to complete all the tests required for the registration of the product as a biopesticide. Similar experiments have been conducted with a strain of fluorescent pseudo-monad also isolated from the soil of Chǎteaurenard, which shows poor antagonistic effect when applied alone but consistently improves disease control when applied in combination with the non-pathogenic Fusarium. In fact, application of both antagonists together always gave total control of the diseases, the yield in the treated plots being equal to that in the healthy control. These experiments show that combining antagonists can improve the consistency of biological control.

Root-induced release of interlayer potassium and vermiculitization of phlogopite as related to potassium depletion in the rhizosphere of ryegrass

Abstract: SUMMARY The ability of roots to weather phlogopite as shown by the release of interlayer-K and by the concomitant vermiculitization of the mica was investigated in the rhizosphere of Italian ryegrass (Lolium multiflorum), grown for 1 to 32 d on 2–105 μm mica as sole source of both K and Mg. The K-balance showed that ryegrass was able to induce a significant release of interlayer-K from phlogopite after 8 d of cropping. After 32 d, this root-induced release of K amounted to 191 g Kg−1 of total-K and contributed a major part of plant nutrition. Concurrently, the concentration of K in solution decreased markedly in the rhizosphere and X-ray diffractometry showed a strong vermiculitization of the phlogopite. The transformation of phlogopite into vermiculite was detected when the K-concentration in the rhizosphere decreased below a threshold of about 80 μmol dm−3. That suggests that the roots act by depleting the rhizosphere-K, and thus shifting the exchange equilibrium, so as to release interlayer-K and expand the interlayer space of the phyllosilicate leading to the transformation of the phlogopite into vermiculite.

Geographic structure of chloroplast DNA polymorphisms in European oaks.

Abstract: Chloroplast DNA polymorphisms have been detected by the conventional Southern-blotting hybridization method in four species of European oaks (Quercus petraea, Q. robur, Q. pubescens and Q. pyrenaica). Three polymorphisms, shared by at least three of these species, can be scored directly in ethidium bromidestained gels and were used in a broad survey of the level of differentiation of the oak species and of their pattern of genetic structure in western Europe. The highly significant geographic variation and the high genetic differentiation (Gst=0.895, SGst=0.025) indicate a low level of cytoplasmic gene flow. We conclude that cytoplasmic genomes are well suited for the reconstruction of past migrational routes of such a complex of species.

Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics.

Abstract: After labelling DNA with the specific vital fluorophore Hoechst 33342, oocytes, isolated by puncture from antral follicles in adult mice, have two essentially different configurations of their nuclear fluorescence images. These have been called SN (where the nucleolus is surrounded by chromatin) and NSN (where the nucleolus is not surrounded by chromatin). Intermediate configurations are also found, although with a lower frequency. The proportion of each class is on the average equal and depends neither on the presence of cumulus cells nor on the age of the mouse. Electron microscopy confirms several ultrastructural differences between these two nuclear configurations, namely, the structure of the nucleolus, which is vacuolated in NSN-type and compact in SN-type oocytes. Using video-enhanced fluorescence microscopy at low level of excitation light, we could follow directly in vitro the meiotic maturation of both classes, without impairing their viability. We show that in germinal vessicle (GV) state, the chromatin does not change from one configuration into the other and that both classes are able to mature to metaphase II, although the maturation has slightly different characteristics.

An M protein with a single C repeat prevents phagocytosis of Streptococcus pyogenes: use of a temperature-sensitive shuttle vector to deliver homologous sequences to the chromosome of S. pyogenes.

Abstract: The major virulence factor of the important human pathogen Streptococcus pyogenes is the M protein, which prevents phagocytosis of the bacterium. In different strains of streptococci, there are over 80 serologically different M proteins and there are additional M-like proteins, some of which bind immunoglobulins. Although the sequence of the M molecules differs among different S. pyogenes strains, all M proteins, and some of the immunoglobulin-binding molecules, have at least two copies of the C repeat region. We describe construction of a deletion mutation in S. pyogenes, which has only one C repeat copy, and show that the mutant strain is still resistant to phagocytosis. The mutation was constructed in vitro and used to replace the resident emm allele in an S. pyogenes strain. To facilitate homologous recombination into the streptococcal chromosome, we adapted a shuttle vector which is temperature sensitive for replication in Gram-positive bacteria but not in Gram-negative hosts. This new method for delivery of a homologous DNA fragment to the S. pyogenes chromosome is efficient and reproducible and should be of general use.

Organization and regulation of genes for amino acid biosynthesis in lactic acid bacteria.

Abstract: The recent description of large clusters of biosynthetic genes in the chromosome of Lactococcus lactis and, to a lesser extent, of Lactobacillus, has brought some information on gene organization and control of gene expression in these organisms. The genes involved in a given amino acid biosynthetic pathway are clustered at a single chromosomal location and form an operon. Additional genes which are not required for the biosynthesis are present within some operons. Genetic signals are, in general, similar to those found in other prokaryotes. Several systems controlling gene expression have been identified and transcription attenuation seems frequent. Among the attenuation mechanisms identified, one resembles that controlling amino acid biosynthesis in many bacteria by ribosome stalling at codons corresponding to limiting amino acid. The others are different and might be related to a new class of attenuation mechanism. Preliminary evidence for a new type of regulatory mechanism, involving a metabolic shunt, is also reviewed.

Detection of turbulent coherent motions in a forest canopy part I: Wavelet analysis

Abstract: Turbulence measurements performed at high frequencies yield data revealing intermittent and multi-scale processes Analysing time series of turbulent variables thus requires extensive numerical treatment capable, for instance, of performing pattern recognition This is particularly important in the case of the atmospheric surface layer and specifically in the vicinity of plant canopies, where largescale coherent motions play a major role in the dynamics of turbulent transport processes In this paper, we examine the ability of the recently developedwavelet transform to extract information on turbulence structure from time series of wind velocities and scalars It is introduced as a local transform performing a time-frequency representation of a given signal by a specific wavelet function; unlike the Fourier transform, it is well adapted to studying non-stationary signals After the principles and the most relevant mathematical properties of wavelet functions and transform are given, we present various applications of relevance for our purpose: determination of time-scales, data reconstruction and filtering, and jump detection Several wavelet functions are inter-compared, using simple artificially generated data presenting large-scale features similar to those observed over plant canopies Their respective behaviour in the time-frequency domain leads us to assign a specific range of applications for each