Pierrette Fleurat-Lessard

Lipid-Induced ER Stress: Synergistic Effects of Sterols and Saturated Fatty Acids

Stress within the endoplasmic reticulum (ER) induces a coordinated response, namely the unfolded protein response (UPR), devoted to helping the ER cope with the accumulation of misfolded proteins. Failure of the UPR plays an important role in several human diseases. Recent studies report that intracellular accumulation of saturated fatty acids (SFAs) and cholesterol, seen in diseases of high incidence, such as obesity or atherosclerosis, results in ER stress. In the present study, we evaluated the effects of perturbations to lipid homeostasis on ER stress/UPR induction in the model eukaryote Saccharomyces cerevisiae. We show that SFA originating from either endogenous (preclusion of fatty acid desaturation) or exogenous (feeding with extracellular SFA) sources trigger ER stress and that ergosterol, the major sterol in yeast, acts synergistically with SFA in this process. This latter effect is connected to ergosterol accumulation within microsomal fractions from SFA‐accumulating cells, which display highly saturated phospholipid content. Moreover, treating the cells with the molecular chaperone 4‐phenyl butyrate abolishes UPR induction, suggesting that lipid‐induced ER stress leads to an overload of misfolded protein that acts, in turn, as the molecular signal for induction of the UPR. The present data are discussed in the context of human diseases that involve lipid deregulation.

Plasma Membrane Aquaporins Are Involved in Winter Embolism Recovery in Walnut Tree

In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity. Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels. Low water potential leads to water movement from the parenchyma cells into the xylem vessels. The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity. We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia). First, we established that xylem parenchyma starch is converted to sucrose in the winter months. Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins. The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes. The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period. In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose. Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels. Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels.

Increased Expression of Vacuolar Aquaporin and H+-ATPase Related to Motor Cell Function in Mimosa pudica L.

Mature motor cells of Mimosa pudica that exhibit large and rapid turgor variations in response to external stimuli are characterized by two distinct types of vacuoles, one containing large amounts of tannins (tannin vacuole) and one without tannins (colloidal or aqueous vacuole). In these highly specialized cells we measured the abundance of two tonoplast proteins, a putative water-channel protein (aquaporin belonging to the [gamma]-TIPs [tonoplast intrinsic proteins]) and the catalytic A-subunit of H+-ATPase, using either high-pressure freezing or chemical fixation and immunolocalization. [gamma]-TIP aquaporin was detected almost exclusively in the tonoplast of the colloidal vacuole, and the H+-ATPase was also mainly localized in the membrane of the same vacuole. Cortex cells of young pulvini cannot change shape rapidly. Development of the pulvinus into a motor organ was accompanied by a more than 3-fold increase per length unit of membrane in the abundance of both aquaporin and H+-ATPase cross-reacting protein. These results indicate that facilitated water fluxes across the vacuolar membrane and energization of the vacuole play a central role in these motor cells.

Immunolocalization of the plasma membrane H+-ATPase in minor veins of Vicia faba in relation to phloem loading

The immunolocalization of the plasma membrane H+ -ATPase, which generates a proton motive force energizing the uptake of inorganic and organic solutes, was studied by electron microscopy. The cells studied were in minor veins of Vicia faba L. exporting leaves, where photosynthates are supposed to be absorbed from the apoplast by phloem transfer cells. Immunologically detectable H+ -ATPase varied among the different cell types and was considerably denser in the transfer cells than in the other cell types, particularly in the sieve tube. Moreover, the distribution of the H+ -ATPase was not homogeneous in transfer cells, that pump being more concentrated in the region adjacent to the bundle sheath, phloem parenchyma, and xylem vessels than along the smooth part of the wall bordering the sieve tube. These results show that the plasma membrane infoldings of transfer cells possess the proton-pumping machinery required to energize an efficient uptake of photosynthates from the phloem apoplast and an efficient retrieval of nitrogenous compounds from the vascular sap.

Early events induced by chitosan on plant cells

Chitosan (a polymer of beta-1,4-glucosamine residues) is a deacetylated derivative of chitin which presents antifungal properties and acts as a potent elicitor of plant resistance against fungal pathogens. Attention was focused in this study on the chitosan-induced early events in the elicitation chain. Thus, it was shown that chitosan triggered in a dose-dependent manner rapid membrane transient depolarization of Mimosa pudica motor cells and, correlatively, a transient rise of pH in the incubation medium of pulvinar tissues. By using plasma membrane vesicles (PMVs), it was specified that a primary site of action of the compound is the plasma membrane H(+)-ATPase as shown by its inhibitory effect on the proton pumping and the catalytic activity of the enzyme up to 250 microg ml(-1). As a consequence, chitosan treatment modified H(+)-mediated processes, in particular it inhibited the uptake of the H(+)-substrate co-transported sucrose and valine, and inhibited the light-induced H(+)/K(+)-mediated turgor reaction of motor cells. The present data also allowed the limit of the cytotoxicity of the compound to be established close to a concentration of 100 microg ml(-1) at the plasma membrane level. As a consequence, chitosan could be preferably used in plant disease control as a powerful elicitor rather than a direct antifungal agent.

Asymmetric distribution of the plasma-membrane H+-ATPase in embryos of Vicia faba L. with special reference to transfer cells

The ultrastructural localization of the plasma-membrane H+ -ATPase by immunocytochemistry was studied in Vicia faba embryos which absorb nutrients from the maternal organism through the transfer cells of their external epidermis. The samples were embedded in LR White resin and the specificity of immunolabelling was checked by inhibition in the presence of purified H+-ATPase. The following results were obtained: (i) The H+-ATPase density varied according to the cell type, being higher in transfer cells than in other cell types, especially the non-modified cells of the internal epidermis. (ii) There was a marked polarity in transfer cells as proton pumps were more numerous in the area of plasmalemma infoldings where active nutrient uptake is assumed to take place, (iii) No clear immunolabelling occurred on the plasma membrane of plasmodesmata. These results demonstrate that in transfer cells the area of plasmalemma infoldings is highly specialized for active solute transport; they also support the idea of specific structural properties of the plasmalemma in plasmodesmata.

Distribution and Activity of the Plasma Membrane H+-ATPase in Mimosa pudica L. in Relation to Ionic Fluxes and Leaf Movements.

Plasma membrane H+-ATPase was immunolocalized in several cell types of the sensitive plant Mimosa pudica L., and transmembrane potentials were measured on cortical cells. In comparison with the nonspecialized cortical cells of the petiole or stem, the proton pump was highly expressed in motor cells. These immunological data are in close agreement with electrophysiological data, because the active component of the transmembrane potential was low in the nonspecialized cortical cells and high in motor cells. Therefore, motor cells contain the plasma membrane H+-ATPase required to mediate the ionic fluxes that are involved in circadian leaf movements and that are necessary to recover the turgor potential that is considerably affected by the large K+ and Cl- efflux associated with seismonastic movement. With the exception of sieve tubes, the phloem also had a high density of H+-ATPase. This suggests that the recovery of the transmembrane ionic gradients (K+ and Cl-), which is affected by various stimuli, is more energized by the companion and parenchyma cells than by the sieve elements. In addition, at the phloem/cortex interface collocytes displayed the required properties for lateral transduction of the action potential toward the pulvinal motor cells.

Control of Vascular Sap pH by the Vessel-Associated Cells in Woody Species (Physiological and Immunological Studies)

In Robinia wood, the vessel-associated cells form a continuous sleeve around the vessels. Variations in pH of the solution perfused through the vessels during the annual cycle and the opposing effects of carbonyl cyanide-m-chlorophenylhydrazone and fusicoccin on this pH value indicate that some living cells of the wood are involved in the control of vascular sap pH and that this control fluctuates with the seasons. The immunolocalization of the plasma membrane HT+-ATPase in Robinia wood was studied by the immunogold-silver-staining technique using an antibody raised against a conserved stretch of the cytoplasmic domain of the H+-ATPase. The immunostaining is much stronger in vessel-associated cells than in other living cell types (ray and axial parenchyma elements) of the secondary xylem. Our data show an efficient involvement of this cell type in the control of vascular sap pH.

STRUCTURAL AND ULTRASTRUCTURAL FEATURES OF CORTICAL CELLS IN MOTOR ORGANS OF SENSITIVE PLANTS

(1) The movements are only expressed in motor cells, regardless of the nature of the stimulation or its point of application. Therefore, these cells have structures capable of traducing the different stimulation‐induced messages which are received in parts incapable of movement.

Structural and ultrastructural characteristics of the vascular apparatus of the sensitive plant (Mimosa pudica L.)

Summary1. In motor organs ofMimosa pudica xylem contains living fibriform elements limited by a thick lignified highly pitted wall, whereas in other parts of the plant (stem, petiole, rachis), xylem and protoxylem vessels are closely associated with parenchyma cells which possess wall ingrowths. These ingrowths, at the apex of which the plasmalemma and the tonoplast touch, are localized like those of “transfer cells” of C type described byGunning andPate. Nevertheless, xylem parenchyma cells differ from cells of C type in several characteristics. Moreover, in motor organs, phloem contains cells characterized by wall ingrowths, less abundant on the parts adjacent to the sieve tubes; these cells which are localized near collenchyma cells of primary phloem, look like “transfer cells” of A type defined byGunning andPate; they are absent from internodes, petioles and rachides. 2. In motor organs, three types of vascular cells (companion cells, living xylem fibriform elements and protoxylem parenchyma cells) are characterized by reduced vacuolar volumes and well developed membrane systems, as compared with homologuous cells belonging to other parts of the plant. 3. A symplastic continuity holds from the middle of motor organs to their cortex: it is provided by the presence, in xylem and phloem respectively, of living fibriform elements and collenchyma cells bearing numerous pit fields containing large numbers of plasmodesmata. Several ultrastructural features suggest that the vascular apparatus ofMimosa pudica would be the site of intensive lateral transfer at different levels, specially in motor organs. Possible functions of certain structures observed are discussed in relation to some hypotheses relative to excitatory conduction pathways.