The Type III secretion system of Xanthomonas fuscans subsp. fuscans is involved in the phyllosphere colonization process and in transmission to seeds of susceptible beans.

Understanding the survival, multiplication, and transmission to seeds of plant pathogenic bacteria is central to study their pathogenesis. We hypothesized that the type III secretion system (T3SS), encoded by hrp genes, could have a role in host colonization by plant pathogenic bacteria. The seed-borne pathogen Xanthomonas fuscans subsp. fuscans causes common bacterial blight of bean (Phaseolus vulgaris). Directed mutagenesis in strain CFBP4834-R of X. fuscans subsp. fuscans and bacterial population density monitoring on bean leaves showed that strains with mutations in the hrp regulatory genes, hrpG and hrpX, were impaired in their phyllospheric growth, as in the null interaction with Escherichia coli C600 and bean. In the compatible interaction, CFBP4834-R reached high phyllospheric population densities and was transmitted to seeds at high frequencies with high densities. Strains with mutations in structural hrp genes maintained the same constant epiphytic population densities (1 x 10(5) CFU g(-1) of fresh weight) as in the incompatible interaction with Xanthomonas campestris pv. campestris ATCC 33913 and the bean. Low frequencies of transmission to seeds and low bacterial concentrations were recorded for CFBP4834-R hrp mutants and for ATCC 33913, whereas E. coli C600 was not transmitted. Moreover, unlike the wild-type strain, strains with mutations in hrp genes were not transmitted to seeds by vascular pathway. Transmission to seeds by floral structures remained possible for both. This study revealed the involvement of the X. fuscans subsp. fuscans T3SS in phyllospheric multiplication and systemic colonization of bean, leading to transmission to seeds. Our findings suggest a major contribution of hrp regulatory genes in host colonization processes.

Secondary ion mass spectrometry imaging of the fixation of 15N-labelled NO in pollen grains.

We used secondary ion mass spectrometry to image cellular targets of nitrogen oxides (widespread air pollutants) in pollen grains of birch (Betula verrucosa Ehrh.) and cockfoot (Dactylis glomerata L.). The pollen samples were exposed to air supplemented with high doses of 15NO. The pollen grains were then fixed, dehydrated using a newly developed 'vapour phase' preparation method and embedded in LRW resin. Semithin sections were then analysed. Imaging was performed in scanning mode. As usual, the two isotopes 14N and 15N were imaged as 12C14N- and 12C15N-, respectively. The isotopic percentages of 15N were quantitatively determined either by image processing or by direct analysis. We show that the preferential areas of NO fixation in the pollen cell are the sporoderm and discrete intracytoplasmic structures that we tentatively describe as globoid-like structures similar to those encountered in seeds.

Hypothesis: hyperstructures regulate bacterial structure and the cell cycle.

A myriad different constituents or elements (genes, proteins, lipids, ions, small molecules etc.) participate in numerous physico-chemical processes to create bacteria that can adapt to their environments to survive, grow and, via the cell cycle, reproduce. We explore the possibility that it is too difficult to explain cell cycle progression in terms of these elements and that an intermediate level of explanation is needed. This level is that of hyperstructures. A hyperstructure is large, has usually one particular function, and contains many elements. Non-equilibrium, or even dissipative, hyperstructures that, for example, assemble to transport and metabolize nutrients may comprise membrane domains of transporters plus cytoplasmic metabolons plus the genes that encode the hyperstructure's enzymes. The processes involved in the putative formation of hyperstructures include: metabolite-induced changes to protein affinities that result in metabolon formation, lipid-organizing forces that result in lateral and transverse asymmetries, post-translational modifications, equilibration of water structures that may alter distributions of other molecules, transertion, ion currents, emission of electromagnetic radiation and long range mechanical vibrations. Equilibrium hyperstructures may also exist such as topological arrays of DNA in the form of cholesteric liquid crystals. We present here the beginning of a picture of the bacterial cell in which hyperstructures form to maximize efficiency and in which the properties of hyperstructures drive the cell cycle.

Cambium pre-activation in beech correlates with a strong temporary increase of calcium in cambium and phloem but not in xylem cells.

Using secondary ion mass spectrometry (SIMS), calcium was imaged in cambium cells and in the adjacent secondary phloem and xylem cells during the different phases of cambium functioning in beech (Fagus sylvatica L.). At the end of the period of quiescence, immediately before the resumption of cell divisions (i.e. at the cambium pre-activation phase), a strong temporary increase of calcium concentration was observed to take place in cambium and phloem but not in xylem cells.

Localization of methyltransferase activities throughout the endomembrane system of flax (Linum usitatissimum L) hypocotyls.

A microsomal fraction from flax hypocotyls (Linum usitatissimum L) showed a methylation ability from S-adenosyl-methionine on to the cell wall polysaccharides. Two kinds of methylation were found: (i) a methyl esterification of uronic acids in the oxalate extracts and (ii) an O-methylation of the hydroxyl groups in the NaOH extracts. The methyltransferase study showed a rapid decrease of the methyl esterification abilities, whereas the O-methylation on to the hydroxyl groups was maintained throughout the culture duration. The localization of such activities in the flax endomembrane system was performed using isopycnic centrifugation. Enzymic marker tests allowed us to identify the different membrane types. Methyltransferase activities in the different enriched fractions appeared to be associated with the Golgi apparatus for the O-methylation, and with the plasma membrane, Golgi apparatus and endoplasmic reticulum compartments for the carboxymethyl esterification.

Differential extractability of calcium and pectic substances in different wall regions of epicotyl cells in young flax plants.

We applied the simultaneous use of a subtractive method and two imaging techniques (secondary ion mass spectrometry and electron microscopy after PATAg staining) to correlate the distribution of Ca2+ to pectic substances in cell walls of young flax plants. The calcium images were compared with the structural electron microscopy images. This suggests that the linkage of the pectic substances within the wall is mainly by calcium bridges in the intercellular junctions of most types of cells under study (epidermis, subepidermis, fiber layer, and endodermis) and in the outer part (close to the cuticle) of the wall of the epidermal cells. In the primary walls of the various types of cells under study and in the inner part (close to the cytoplasm) of the wall of the epidermal cells, the linkage of the pectic substances would be mainly by covalent bonds. In the middle lamellae of the various cells, and in the intercellular junctions within the cortical parenchyma, both types of linkages apparently coexist. The mechanism of "ionic condensation" may provide an interpretation for the chemical status of the Ca2+ ions which are associated with the pectic components solubilized in boiling water, and which do not seem to contribute to the linkage of these components within the wall.

[Demonstration of pectin-lyase in Bacillus subtilis]. / Mise en évidence d'une activité pectine-lyase chez Bacillus subtilis.

After a screening performed on pectinolytic micro-organisms, a strain of B. subtilis was isolated. This strain has a pectic enzyme capable of beta-elimination on highly methylated pectin (degree of esterification = 85%), without the action of a pectinesterase. This activity corresponds to that of a pectin-lyase.

Compartmental analysis of sulphate transport in Lemna minor L., taking plant growth and sulphate metabolization into consideration.

The compartmental analysis of sulphate transport in cells of Lemna plants has been performed, taking into account the growth of the samples and the metabolization of sulphate into organic thiocompounds during the course of the experiment. The results obtained form efflux and influx experiments are fully consistent with one another. Both unidirectional fluxes between the external medium and the cell wall are very large (order of magnitude of 1 MUMOL/h per g fresh weight of plants). All the other unidirectional fluxes, including the flux of sulphate metabolization, are much smaller (from about 10 to 60 nmol/h per g). Over 70% of the total sulphur of the plant corresponds to that incorporated into organic thio compounds, and over 25% to free sulphate in the vacuola. The pool of free sulphate in the cytoplasm is only about 1% of the total sulphur, and the sulphate content of the cell wall (free spaces) is also about 1%. Two remarks of general relevance have been made concerning the influx curves. First, these curves exhibit a long (several hours), quasi-stationary phase after the first few minutes of absorption, though the slope of this straight line does not correspond to the unidirectional flux of sulphate entry through the plasmalemma (from cell wall to cytoplasm). Second, the Lemna plants seem to be sensitive to the effect of "gas shock'.

Titration of Isolated Cell Walls of Lemna minor L.

A theoretical model has been built to bypass the equation of titration of the cell wall. This equation, which is an extension of the Henderson-Hasselbach equation, underlines the importance of the exchange constant, the ionic strength as well as the rate of neutralization. The model is restricted to the case when the ionization degree is equal to the neutralization degree. The shape of the titration curve is shown to be strongly dependent on the valency of the base used.Experimental results have shown that isolated cell walls bear at least two kinds of sites. The first sites which are titrated after a short time of equilibration are attributed to polyuronic acids (capacity: 0.3 milliequivalents per gram fresh cell walls). The second sites, are obtained after a long time of equilibration (capacity: 1.2 to 1.3 milliequivalents per gram, fresh cell walls). Titrations have been performed with different bases [KOH, NaOH, and Ca(OH)(2)] and under different ionic strengths.The results obtained with NaOH and KOH do not exhibit any difference of selectivity. Conversely, the sites have a much bigger affinity for the Ca(2+) ions than for the monovalent ones. The apparent pKa of the uronic acids was estimated to lie between 3.0 and 3.4; this is consistent with the values obtained with polyuronic acid solutions.

Borate Exchanges of Lemna minor L. as Studied with the Help of the Enriched Stable Isotopes and of a (n,alpha) Nuclear Reaction.

Despite the lack of a convenient radioisotope of boron, it is possible to measure unidirectional fluxes of borate between cellular systems and their external medium. It was accomplished by using the two purified stable isotopes ((10)B and (11)B), with (10)B specifically detected by a (n,alpha) nuclear reaction. The method was applied to compartmental analysis of borate with intact plants of Lemna minor L. Four compartments were suggested. Three of them apparently correspond to the three classical ones: free space (including easily dissociable borate monoesters), cytoplasm, and vacuole. The fourth one was interpreted as corresponding to very stable borate diesters in the cell walls. The method allows the determination of the borate capacities of the various compartments and of the borate unidirectional fluxes between the different compartments, at borate flux equilibrium. Other physicochemical data (mono and diester mass action constants, turn over numbers) were evaluated. The results are consistent with what is known of pure substances.

Exchange Properties of Isolated Cell Walls of Lemna minor L.

From our theoretical treatment which is an extension of the classical Donnan theory, we have estimated the rational selectivity coefficients of the carboxylic groups of the walls during exchanges of divalent ions against monovalent ones (i.e. calcium and potassium or calcium and sodium ions). These coefficients express the interactions between the different ions, those between the counter-ions and the ionized groups of the wall, and the influence of water. These quantitative values are consistent with the great affinity of the carboxylic groups for the calcium ions. They vary with the experimental conditions, showing a purely physicochemical mechanism of "regulation" of the exchanges in the cells walls of Lemna minor L.

[Regulation and electric excitation of enzyme membranes in vitro]. / Régulation et excitation électriques de membranes d'enzymes in vitro.

Enzyme membranes can be activated or inhibited by applying continuous or alternating electrical fields. The field can modify the transport or reaction term of the transport-reaction by action on the displacement of charged species including those giving pH effects or inducing volume flows. A first experimental example is given: the progressive supression of the inhibition of hexokinase by the product when increasing alternating fields are applied. In the same way the apparent optimal pH approaches that of the soluble enzyme. In addition to its theoretical and practical implications electrical regulation can lead to the monitoring of enzyme reaction-driven mechanochemical fibers.