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.

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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.Winter embolism, the generation of air bubbles in xylem vessels induced by freeze-thaw cycles, often leads to a loss of hydraulic conductivity of the vessels (Cochard and Tyree, 1990; Améglio et al., 2001; Ewers et al., 2001). Vulnerability to winter embolism is related to the anatomy and vessel diameter of woody plants (Cochard and Tyree, 1990) and affects the ability of plants to survive cold climates and the geographic distribution of species (Tyree and Cochard, 1996; Pockman and Sperry, 1997; Lemoine et al., 1999).Detailed physiological studies of the responses of temperate woody plants to winter embolism have been made. Plants minimize the impact of winter embolism by replacing embolized vessels by new functional vessels every year and/or by refilling embolized vessels by generating positive xylem pressures (Holbrook and Zwieniecki, 1999; Tyree et al., 1999; Améglio et al., 2002). Although making new vessels is common to all the plants that exhibit secondary growth, the generation of xylem pressures has only been reported in a few species such as maple (Acer pseudoplatanus; O'Malley and Milburn, 1983; Tyree, 1983; Sperry et al., 1987 Sperry et al., , 1994, grapevine (Vitis vinifera; Sperry et al., 1987), birch (Betula alleghaniensis) (Sperry et al., 1994; Zhu et al., 2000), and walnut (Juglans regia; Améglio et al., 1995Améglio et al., , 2001 Ewers et al., 2001).In walnut trees, depending on the temperature, two types of positive xylem pressures have been ...

One of the most striking features of mature plant cells is the presence of a large central vacuole that can occupy more than 80% of the total cell volume. The constituents of the vacuole are mainly inorganic salts and water (Martinoia et al., 1981; Boller and Wiemken, 1986;Martinoia, 1992). The vacuole, therefore, enables the plant to attain a large size and surface area by accumulating salts from the environment that osmotically drive further water uptake, resulting in minimal energy expenditure for metabolite synthesis (Matile, 1987). Furthermore, the vacuole is also a temporary store for metabolites and nutrients and it plays an important role in cytosolic homeostasis (Matile, 1978(Matile, , 1987 Boller and Wiemken, 1986;Martinoia, 1992). In contrast to primary metabolites that are stored only temporarily in the vacuole, many compounds of secondary metabolism (Matile, 1987) or modified, potentially toxic

In flowering plants, development of the haploid male gametophytes (pollen grains) takes place in a specialized structure called the anther. Successful pollen development, and thus reproduction, requires high secretory activity in both anther tissues and pollen. In this paper, we describe a novel member of the eukaryotic type V subfamily (P 5 ) of P-type ATPase cation pumps, the MALE GAMETOGENESIS IMPAIRED ANTHERS (MIA) gene. MIA protein is highly abundant in the endoplasmic reticulum and small vesicles of developing pollen grains and tapetum cells. T-DNA insertional mutants of MIA suffer from imbalances in cation homeostasis and exhibit a severe reduction in fertility. Mutant microspores fail to separate from tetrads and pollen grains are fragile with an abnormal morphology and altered cell wall structure. Disruption of MIA affects expression of genes essential for secretion as well as a high number of genes encoding cell wall proteins and membrane transporters. MIA functionally complements a mutant in the P 5 ATPase homolog SPF1 from Saccharomyces cerevisiae, suggesting a common function for P 5 ATPases in single and multicellular organisms. Our results suggest that MIA is required in the secretory pathway for proper secretion of vesicle cargo to the plasma membrane.[Keywords: P-type ATPase; secretory pathway; pollen; male gametogenesis] Supplemental material is available at http://www.genesdev.org.

The accumulation of sugars in grape berries requires the co-ordinate expression of sucrose transporters, invertases, and monosaccharide transporters. A monosaccharide transporter homologue (VvHT1, Vitis vinifera hexose transporter 1) has previously been isolated from grape berries at the veraison stage, and its expression was shown to be regulated by sugars and abscisic acid. The present work investigates the function and localization of VvHT1. Heterologous expression in yeast indicates that VvHT1 encodes a monosaccharide transporter with maximal activity at acidic pH (pH 4.5) and high affinity for glucose (K(m)=70 muM). Fructose, mannose, sorbitol, and mannitol are not transported by VvHT1. In situ hybridization shows that VvHT1 transcripts are primarily found in the phloem region of the conducting bundles. Immunofluorescence and immunogold labelling experiments localized VvHT1 in the plasma membrane of the sieve element/companion cell interface and of the flesh cells. The expression and functional properties of VvHT1 suggests that it retrieves the monosaccharides needed to provide the energy necessary for cell division and cell growth at an early stage of berry development.

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.

The presence of an alpha4-fucosyltransferase in plants was first deduced from the characterization of Lewis-a glycoepitopes in some N-glycans. The first plant gene encoding an alpha4-fucosyltransferase was recently cloned in Beta vulgaris. In the present paper we provide evidence for the presence of an alpha4-fucosyltransferase in A. thaliana by measurement of this glycosyltransferase activity from a purified microsomal preparation and by immunolocalization of Le(a) epitopes on glycans N-linked to glycoproteins located to the Golgi apparatus and on the cell surface. The corresponding gene AtFT4 (AY026941) was characterized. A unique copy of this gene was found in A. thaliana genome, and a single AtFT4 transcript was revealed in leaves, in roots, and at a lower extent in flowers. The coding sequence of AtFT4 gene is interrupted by two introns spanning 465 bp and 84 bp, respectively. The putative 393-amino-acid protein (44 kDa, pI: 6.59) contains an N-terminal hydrophobic region and one potential N-glycosylation site, but AtFT4 has poor homology (less than 30%) to the other alpha3/4-fucosyltransferases except for motif II. When expressed in COS 7 cells the protein is able to transfer Fuc from GDP-Fuc to a type 1 acceptor substrate, but this transferase activity is detected only in the culture medium of transfected cells