Alice Agasse

Aquaporins are multifunctional water and solute transporters highly divergent in living organisms

Aquaporins (AQPs) are ubiquitous membrane proteins whose identification, pioneered by Peter Agres team in the early nineties, provided a molecular basis for transmembrane water transport, which was previously thought to occur only by free diffusion. AQPs are members of the Major Intrinsic Protein (MIP) family and often referred to as water channels. In mammals and plants they are present in almost all organs and tissues and their function is mostly associated to water molecule movement. However, recent studies have pointed out a wider range of substrates for these proteins as well as complex regulation levels and pathways. Although their relative abundance in plants and mammals makes it difficult to investigate the role of a particular AQP, the use of knock-out and mutagenesis techniques is now bringing important clues regarding the direct implication of specific AQPs in animal pathologies or plant deficiencies. The present paper gives an overview about AQP structure, function and regulation in a broad range of living organisms. Emphasis will be given on plant AQPs where the high number and diversity of these transport proteins, together with some emerging aspects of their functionalities, make them behave more like multifunctional, highly adapted channels rather than simple water pores.

Sugar-regulated expression of a putative hexose transport gene in grape.

Different lengths of the promoter of grape (Vitis vinifera) VvHT1 (Hexose Transporter 1) gene, which encodes a putative hexose transporter expressed during the ripening of grape, have been transcriptionally fused to the β-glucuronidase reporter gene. In transgenic tobacco (Nicotiana tabacum) transformed with these constructs,VvHT1 promoters were clearly responsible for the sink organ preferential expression. The potential sugar effectors ofVvHT1 promoter were studied in tobacco cv Bright-Yellow 2 cells transformed with chimeric constructs. Glucose (56 mm), sucrose (Suc; 58 mm), and the non-transported Suc isomer palatinose doubled the β-glucuronidase activity conferred by the VvHT1 promoter, whereas fructose did not affect it. These effects were the strongest with the 2.4-kb promoter, which contains all putative sugar-responsive elements (activating and repressing), but they were also significant with the 0.3-kb promoter, which contains only activating sugar boxes. The induction of VvHT1 expression by both Suc and palatinose was confirmed in the homologous grape berry cell culture. The data provide the first example of a putative sugar transporter, which is induced by both glucose and Suc in higher plants. Although induction ofVvHT1 expression by Suc does not require transport, the presence of glucosyl moiety is necessary for Suc sensing. These results provide new insights into sugar sensing and signaling in plants.

Pathways of Glucose Regulation of Monosaccharide Transport in Grape Cells

Grape (Vitis vinifera) heterotrophic suspension-cultured cells were used as a model system to study glucose (Glc) transport and its regulation. Cells transported d-[14C]Glc according to simple Michaelis-Menten kinetics superimposed on first-order kinetics. The saturating component is a high-affinity, broad-specificity H+-dependent transport system (Km = 0.05 mm). Glc concentration in the medium tightly regulated the transcription of VvHT1 (Vitis vinifera hexose transporter 1), a monosaccharide transporter previously characterized in grape berry, as well as VvHT1 protein amount and monosaccharide transport activity. All the remaining putative monosaccharide transporters identified so far in grape were poorly expressed and responded weakly to Glc. VvHT1 transcription was strongly repressed by Glc and 2-deoxy-d-Glc, but not by 3-O-methyl-d-Glc or Glc plus mannoheptulose, indicating the involvement of a hexokinase-dependent repression. 3-O-Methyl-d-Glc, which cannot be phosphorylated, and Glc plus mannoheptulose induced a decrease of transport activity caused by the reduction of VvHT1 protein in the plasma membrane without affecting VvHT1 transcript levels. This demonstrates hexokinase-independent posttranscriptional regulation. High Glc down-regulated VvHT1 transcription and Glc uptake, whereas low Glc increased those parameters. Present data provide an example showing control of plant sugar transporters by their own substrate both at transcriptional and posttranscriptional levels. VvHT1 protein has an important role in the massive import of monosaccharides into mesocarp cells of young grape berries because it was localized in plasma membranes of the early developing fruit. Protein amount decreased abruptly throughout fruit development as sugar content increases, consistent with the regulating role of Glc on VvHT1 expression found in suspension-cultured cells.

Mannitol transport and mannitol dehydrogenase activities are coordinated in olea europaea under salt and osmotic stresses

The intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) was previously characterized as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in cells growing in mannitol, at 25°C and pH 9.0, were as follows: K(m), 54.5 mM mannitol; and V(max), 0.47 μmol h⁻¹ mg⁻¹ protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport in mannitol- and sucrose-grown cells. Furthermore, sucrose-grown cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence that OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and polyethylene glycol (PEG) treatments, in both mannitol- and sucrose-grown cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Taken together, these studies support that olive trees cope with salinity and drought by coordinating mannitol transport with intracellular metabolism.

The grape aquaporin VvSIP1 transports water across the ER membrane

Water diffusion through biological membranes is facilitated by aquaporins, members of the widespread major intrinsic proteins (MIPs). In the present study, the localization, expression, and functional characterization of a small basic intrinsic protein (SIP) from the grapevine were assessed. VvSIP1 was expressed in leaves and berries from field-grown vines, and in leaves and stems from in vitro plantlets, but not in roots. When expressed in tobacco mesophyll cells and in Saccharomyces cerevisiae, fluorescent-tagged VvSIP1 was localized at the endoplasmic reticulum (ER). Stopped-flow spectroscopy showed that VvSIP1-enriched ER membrane vesicles from yeast exhibited higher water permeability and lower activation energy for water transport than control vesicles, indicating the involvement of protein-mediated water diffusion. This aquaporin was able to transport water but not glycerol, urea, sorbitol, glucose, or inositol. VvSIP1 expression in Xenopus oocytes failed to increase the water permeability of the plasma membrane. VvSIP1-His-tag was solubilized and purified to homogeneity from yeast ER membranes and the reconstitution of the purified protein in phosphatidylethanolamine liposomes confirmed its water channel activity. To provide further insights into gene function, the expression of VvSIP1 in mature grapes was studied when vines were cultivated in different field conditions, but its transcript levels did not increase significantly in water-stressed plants and western-exposed berries. However, the expression of the aquaporin genes VvSIP1, VvPIP2;2, and VvTIP1;1 was up-regulated by heat in cultured cells.