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...

stimulus of Lp r in Arabidopsis. Since both NaCl and SA treatments increased the accumulation of ROS in roots, it was hypothesized that H 2 O 2 or other ROS may have a central role in the regulation of root water transport in response to various biotic or abiotic stimuli. When Arabidopsis roots were treated with mM concentrations of exogenous H 2 O 2 , Lp r was inhibited within minutes by up to 90%. These findings are consistent with previous reports showing that ROS can downregulate water transport in cucumber and maize roots or in the algae Chara corallina. 4-7 H 2 O 2 and possibly other derived ROS may modulate the Lp r through signaling mechanisms or by a direct oxidative gating of aquaporins. The latter hypothesis, which has been favored in previous studies by Steudle and colleagues,6,7 was investigated by Boursiac et al., by functionally expressing aquaporins in Xenopus oocytes and by testing their sensitivity to external H 2 O 2 . The results show that Arabidopsis aquaporins are insensitive to direct oxidation by H 2 O 2 or hydroxyl radicals. Thus, these and complementary pharmacological analyses on excised roots rather support a role for H 2 O 2 as a second messenger that connects environmental stimulus perception to water transport regulation in plant roots. The additional finding that H 2 O 2 can be transported by aquaporins 8,9 opens the possibility of intricate loop mechanisms whereby these proteins may interfere with their own regulation. For example, active PIP aquaporins could facilitate the diffusion within the cell of NADPH-oxidase derived apoplastic H 2 O 2 , which in turn would activate signaling pathways acting on PIP activity and/or subcellular localization.In a previous study, we monitored the subcellular localization of AtPIP1;2 and AtPIP2;1, two of the most abundant PIPs in roots, by expression in transgenic Arabidopsis of fusions with the green fluorescent protein (GFP). 10 We observed that a 100 mM NaCl treatment induced in 2-4 hours an increased intracellular labeling which was interpreted as an intracellular relocalization of the two aquaporins. 10 In our more recent study, both a 150 mM NaCl and a 0.5 mM SA treatments induced an intracellular labeling by GFP-PIP1;2 and PIP2;1-GFP fusions, with a "fuzzy" pattern or at the level of spherical bodies. Preventing the NaCl-or SA-dependent accumulation of ROS with exogenous catalase was able to almost completely counteract the effects of the two stimuli on the localization pattern Aquaporins, which facilitate the diffusion of water across biological membranes, are key molecules for the regulation of water transport at the cell and organ levels. We recently reported that hydrogen peroxide (H 2 O 2 ) acts as an intermediate in the regulation of Arabidopsis root water transport and aquaporins in response to NaCl and salicylic acid (SA). 1 Its action involves signaling pathways and an internalization of aquaporins from the cell surface. The present addendum connects these findings to another recent work which describes multiple phosphorylations...