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 TiO₂ 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 described previously. The phosphorylation of AtPIP2;1 at two C-terminal sites (Ser²⁸⁰ and Ser²⁸³) was monitored by an absolute quantification method and shown to be altered in response to treatments of plants by salt (NaCl) and hydrogen peroxide. The two treatments are known to strongly decrease the water permeability of Arabidopsis roots. To investigate a putative role of Ser²⁸⁰ and Ser²⁸³ phosphorylation in aquaporin subcellular trafficking, AtPIP2;1 forms mutated at either one of the two sites were fused to the green fluorescent protein and expressed in transgenic plants. Confocal microscopy analysis of these plants revealed that, in resting conditions, phosphorylation of Ser²⁸³ is necessary to target AtPIP2;1 to the plasma membrane. In addition, an NaCl treatment induced an intracellular accumulation of AtPIP2;1 by exerting specific actions onto AtPIP2;1 forms differing in their phosphorylation at Ser²⁸³ to induce their accumulation in distinct intracellular structures. Thus, the present study documents stress-induced quantitative changes in aquaporin phosphorylation and establishes for the first time a link with plant aquaporin subcellular localization.

Tomato (Solanum lycopersicum) is a model plant for studying fleshy fruit development. Several genetic and molecular approaches have been developed to increase our knowledge about the physiological basis of fruit growth, but very few data are yet available at the proteomic level. The main stages of fruit development were first determined through the dynamics of fruit diameter and pericarp cell number. Then, total proteins were extracted from pericarp tissue at six relevant developmental stages and separated by two-dimensional gel electrophoresis. Protein patterns were markedly different between stages. Proteins showing major variations were monitored. We identified 90 of 1,791 well-resolved spots either by matrix-assisted laser-desorption ionization time-of-flight peptide mass fingerprinting or liquid chromatography-mass spectrometry sequencing and expressed sequence tag database searching. Clustered correlation analysis results pointed out groups of proteins with similar expression profiles during fruit development. In young fruit, spots linked to amino acid metabolism or protein synthesis were mainly expressed during the cell division stage and down-regulated later. Some spots linked to cell division processes could be identified. During the cell expansion phase, spots linked to photosynthesis and proteins linked to cell wall formation transiently increased. In contrast, the major part of the spots related to C compounds and carbohydrate metabolism or oxidative processes were up-regulated during fruit development, showing an increase in spot intensity during development and maximal abundance in mature fruit. This was also the case for spots linked to stress responses and fruit senescence. We discuss protein variations, taking into account their potential role during fruit growth and comparing our results with already known variations at mRNA and metabolite-profiling levels.

Root NO3 − efflux to the outer medium is a component of NO3 − net uptake and can even overcome influx upon various stresses. Its role and molecular basis are unknown. Following a functional biochemical approach, NAXT1 (for NITRATE EXCRETION TRANSPORTER1) was identified by mass spectrometry in the plasma membrane (PM) of Arabidopsis thaliana suspension cells, a localization confirmed using a NAXT1–Green Fluorescent Protein fusion protein. NAXT1 belongs to a subclass of seven NAXT members from the large NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER family and is mainly expressed in the cortex of mature roots. The passive NO3 − transport activity (K m = 5 mM) in isolated root PM, electrically coupled to the ATP-dependant H+-pumping activity, is inhibited by anti-NAXT antibodies. In standard culture conditions, NO3 − contents were altered in plants expressing NAXT-interfering RNAs but not in naxt1 mutant plants. Upon acid load, unidirectional root NO3 − efflux markedly increased in wild-type plants, leading to a prolonged NO3 − excretion regime concomitant with a decrease in root NO3 − content. In vivo and in vitro mutant phenotypes revealed that this response is mediated by NAXT1, whose expression is upregulated at the posttranscriptional level. Strong medium acidification generated a similar response. In vitro, the passive efflux of NO3 − (but not of Cl−) was strongly impaired in naxt1 mutant PM. This identification of NO3 − efflux transporters at the PM of plant cells opens the way to molecular studies of the physiological role of NO3 − efflux in stressed or unstressed plants.

Aquaporins are channel proteins that facilitate the diffusion of water across cell membranes. The genome of Arabidopsis thaliana encodes 35 full-length aquaporin homologues. Thirteen of them belong to the plasma membrane intrinsic protein (PIP) subfamily and predominantly sit at the plasma membrane (PM). In the present work we combine separations of membrane proteins (by one- and two-dimensional gel electrophoresis) with identification by MS (matrix-assisted laser-desorption ionization–time-of-flight and electrospray-ionization tandem MS) to take an inventory of aquaporin isoforms expressed in the PM of Arabidopsis thaliana roots. Our analysis provides direct evidence for the expression of five PIPs (PIP1;1, PIP1;5, PIP2;1, PIP2;2 and PIP2;7) in the root PM and suggests the presence of at least three other PIP isoforms. In addition, we show that the same PIP isoform can be present under several forms with distinct isoelectric points. More specifically, we identify phosphorylated aquaporins in the PIP1 and PIP2 subgroups and suggest the existence of other post-translational modifications. Their identification should provide clues to reveal novel molecular mechanisms for aquaporin regulation.

A proteomic approach was used to analyze protein changes during nitrogen mobilization (N mobilization) from leaves to filling seeds in pea (Pisum sativum). First, proteome reference maps were established for mature leaves and stems. They displayed around 190 Coomassie Blue-stained spots with pIs from 4 to 7. A total of 130 spots were identified by mass spectrometry as corresponding to 80 different proteins implicated in a variety of cellular functions. Although the leaf proteome map contained more abundant spots, corresponding to proteins involved in energy/carbon metabolism, than the stem map, their comparison revealed a highly similar protein profile. Second, the leaf proteome map was used to analyze quantitative variations in leaf proteins during N mobilization. Forty percent of the spots showed significant changes in their relative abundance in the total protein extract. The results confirmed the importance of Rubisco as a source of mobilizable nitrogen, and suggested that in pea leaves the rate of degradation of Rubisco may vary throughout N mobilization. Correlated with the loss of Rubisco was an increase in relative abundance of chloroplastic protease regulatory subunits. Concomitantly, the relative abundance of some proteins related to the photosynthetic apparatus (Rubisco activase, Rubisco-binding proteins) and of several chaperones increased. A role for these proteins in the maintenance of a Rubisco activation state and in the PSII repair during the intense proteolytic activity within the chloroplasts was proposed. Finally, two 14-3-3-like proteins, with a potential regulatory role, displayed differential expression patterns during the massive remobilization of nitrogen.