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Universidade do Minho (2013)

Biochemical and molecular mechanisms of salt stress tolerance in and Olea europaea

Silva, Paulo Filipe Pereira de Jesus

Titre : Biochemical and molecular mechanisms of salt stress tolerance in and Olea europaea

Auteur : Silva, Paulo Filipe Pereira de Jesus

Université de soutenance : Universidade do Minho

Grade : Doctoral Thesis 2013

The current work focused in the research subject of membrane transport and plant - environment interactions and two plant models were the target of the studies : Olea europaea and Populus euphratica. Olive tree is an emblematic species and one of the most important fruit crops in the Mediterranean basin. The halophytic and salt and drought stress tolearant plant P. euphratica, which occurs naturally in semiarid areas, has recently been used as a model to study plant defense mechanisms against salt stress. In both plant species we aimed to contribute to the elucidation of the biochemical mechanisms involved in salt response, in particular those involving transmembrane transport steps of photoassimilates and tonoplast transport of protons and salt. The mechanism on how sodium is accumulated in the vacuole in response to salt in P. euphratica, and how salt stress may affect the generation and maintenance of a transmembrane proton gradient across the tonoplast were investigated. Biochemical data corroborated the involvement of Na+/H+ exchange activity in cell suspensions at the tonoplast level, whose activity increased 6-fold in NaCl-treated cells. Accordingly, confocal and epifluorescence microscopy analyses with the Na+-sensitive probe Sodium Green showed that suspension-cultured cells subjected to a salt pulse accumulated Na+ in the vacuole. In tonoplast vesicles the V-H+-PPase activity decreased with exposure to NaCl, in contrast to the observed sodium-induced increase in the activity of vacuolar H+-ATPase. The increase of both the transmembrane H+ gradient - generated by tonoplast proton pumps - and the Na+/H+ antiport activity in response to salt strongly suggested that Na+ accumulation into the vacuole contributes to salt tolerance in P. euphratica, in line with the confocal microscopy observations. In O. europaea, key biochemical and molecular steps involved in the partitioning of sugars and polyols, and how polyols may enhance salt and drought stress resistance were addressed at the protein activity and gene expression levels. Polyols are the reduced form of aldoses and ketoses, present in several species. In O. europaea leaves, mannitol was found to be the main soluble carbohydrate, followed by the monosaccharide glucose. Fructose was not detected, probably because it acted as precursor for mannitol biosynthesis. Transport experiments with [14C]mannitol showed that a polyol:H+ symport system operates in O. europaea heterotrophic cultured cells (Km = 1.3 mM). Subsequent work led to the cloning of a cDNA sequence of a mannitol carrier which was named OeMaT1 (O. europaea mannitol transporter 1). In parallel experiments, salt strongly repressed mannitol dehydrogenase activity, the first enzyme responsible for intracellular mannitol oxidation, and down-regulated OeMTD1 (O. europaea mannitol dehydrogenase 1) transcripts. This should allow for the intracellular accumulation of mannitol in order to compensate for the decrease of external water activity, thus conferring a response mechanism to salinity in O. europaea. Subsequent studies on the molecular mechanisms of glucose utilization by olive cells led to the cloning and functional characterization of the monosaccharide transporter OeMST2. Heterologous expression of this gene in Saccharomyces cerevisiae deficient in glucose transport restored its capacity to grow and to transport glucose. Transcript levels of OeMST2 increased during fruit maturation, confirming that OeMST2 catalyzes the membrane transport process of hexoses during sugar unloading in the fruits. In addition to this saturable energy dependent transport systems, in a variety of cell types, including plant cells, sugars, polyols and other solutes may be incorporated according to a diffusion-like kinetics, in spite of the real nature of this transport mechanism having been elusive. The measurement of [14C]glucose transport by cells and membrane vesicles in the presence of specific inhibitors, the measurement of activation energies of glucose uptake, among other biochemical approaches, led us to demonstrate that the low-affinity, high-capacity, diffusion-like glucose uptake in olive cells occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation. The recent publication in Nature reporting the identification and functional characterization of a new class of sugar transporters, named SWEET, which are postulated to be involved in phloem transport and plant nectar production, further strengthened the involvement of low-affinity sugar facilitators in plants


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