). † These authors contributed equally to this work. SummaryThe water uptake capacity of plant roots (i.e. their hydraulic conductivity, Lp r ) is determined in large part by aquaporins of the plasma membrane intrinsic protein (PIP) subfamily. In the present work, we investigated two stimuli, salicylic acid (SA) and salt, because of their ability to induce an accumulation of reactive oxygen species (ROS) and an inhibition of Lp r concomitantly in the roots of Arabidopsis plants. The inhibition of Lp r by SA was partially counteracted by preventing the accumulation of hydrogen peroxide (H 2 O 2 ) with exogenous catalase. In addition, exogenous H 2 O 2 was able to reduce Lp r by up to 90% in <15 min. Based on the lack of effects of H 2 O 2 on the activity of individual aquaporins in Xenopus oocytes, and on a pharmacological dissection of the action of H 2 O 2 on Lp r , we propose that ROS do not gate Arabidopsis root aquaporins through a direct oxidative mechanism, but rather act through cell signalling mechanisms. Expression in transgenic roots of PIP-GFP fusions and immunogold labelling indicated that external H 2 O 2 enhanced, in <15 min, the accumulation of PIPs in intracellular structures tentatively identified as vesicles and small vacuoles. Exposure of roots to SA or salt also induced an intracellular accumulation of the PIP-GFP fusion proteins, and these effects were fully counteracted by co-treatment with exogenous catalase. In conclusion, the present work identifies SA as a novel regulator of aquaporins, and delineates an ROS-dependent signalling pathway in the roots of Arabidopsis. Several abiotic and biotic stress-related stimuli potentially share this path, which involves an H 2 O 2 -induced internalization of PIPs, to downregulate root water transport.

A proteomic analysis was performed on the heat stable protein fraction of imbibed radicles of Medicago truncatula seeds to investigate whether proteins can be identified that are specifically linked to desiccation tolerance (DT). Radicles were compared before and after emergence (2.8 mm long) in association with the loss of DT, and after reinduction of DT by an osmotic treatment. To separate proteins induced by the osmotic treatment from those linked with DT, the comparison was extended to 5 mm long emerged radicles for which DT could no longer be reinduced, albeit that drought tolerance was increased. The abundance of 15 polypeptides was linked with DT, out of which 11 were identified as late embryogenesis abundant proteins from different groups: MtEm6 (group 1), one isoform of DHN3 (dehydrins), MtPM25 (group 5), and three members of group 3 (MP2, an isoform of PM18, and all the isoforms of SBP65). In silico analysis revealed that their expression is likely seed specific, except for DHN3. Other isoforms of DNH3 and PM18 as well as three isoforms of the dehydrin Budcar5 were associated with drought tolerance. Changes in the abundance of MtEm6 and MtPM25 in imbibed cotyledons during the loss of DT and in developing embryos during the acquisition of DT confirmed the link of these two proteins with DT. Fourier transform infrared spectroscopy revealed that the recombinant MtPM25 and MtEm6 exhibited a certain degree of order in the hydrated state, but that they became more structured by adopting a helices and b sheets during drying. A model is presented in which DT-linked late embryogenesis abundant proteins might exert different protective functions at high and low hydration levels.

Aquaporins form a family of water and solute channel proteins and are present in most living organisms. In plants, aquaporins play an important role in the regulation of root water transport in response to abiotic stresses. In this work, we investigated the role of phosphorylation of plasma membrane intrinsic protein (PIP) aquaporins in the Arabidopsis thaliana root by a combination of quantitative mass spectrometry and cellular biology approaches. A novel phosphoproteomics procedure that involves plasma membrane purification, phosphopeptide enrichment with TiO 2 columns, and systematic mass spectrometry sequencing revealed multiple and adjacent phosphorylation sites in the C-terminal tail of several AtPIPs. Six of these sites had not been de- Aquaporins form a family of channel proteins that mediate the transport across membranes of water, small neutral solutes, and occasionally ions (1-3). Aquaporins are present in all living kingdoms and in plants. Aquaporins exhibit a characteristically high multiplicity of forms with for instance 35 members in Arabidopsis (4, 5). Based upon their amino acid sequence homology, plant aquaporins can be classified into four subfamilies (4 -6). One of these corresponds to the plasma membrane intrinsic proteins (PIPs).1 The PIPs with 13 members in Arabidopsis represent the most abundant aquaporins in the plasma membrane (PM) and can be further divided into two sequence homology groups (AtPIP1 and AtPIP2). Aquaporins are 25-35-kDa proteins that share a typical organization with six transmembrane ␣-helices interrupted by five connecting loops (loops A-E) (7,8). In PM aquaporins, the N and C termini as well as loops B and D are exposed in the cytosol, whereas loops A, C, and E face the cell wall.Plants need to continuously adjust their water status in response to changing environmental conditions, and aquaporins play an important role in these processes (3, 9, 10). In particular, physiological and genetics studies have provided compelling evidence for a role of aquaporins in the regulation, in response to abiotic stresses, of root water transport, i.e. root hydraulic conductivity (Lp r ) (10, 11). For instance, exposure of Arabidopsis plants to salt (100 mM NaCl) induced a rapid (half-time, 45 min) and significant decrease (Ϫ70%) in Lp r that was maintained for at least 24 h (12). Whereas the long term effect of this NaCl stress can be accounted for by an overall transcriptional downregulation of aquaporins, the molecular mechanisms involved in the early inhibition of Lp r by NaCl are not fully understood yet. These mechanisms involve a slight decrease in overall abundance of AtPIP1 proteins as soon as 30 min after exposure to NaCl and a trafficking of AtPIP1 and AtPIP2 isoforms between the PM and intracellular compartments that may contribute to reducing the abundance of AtPIPs at the PM and therefore the hydraulic conductivity of salt-stressed root cells (12). Chilling is another stress that leads to inhibition of Lp r , and a relationship From the ‡Biochimie et Physiologie Molé c...