Coordinated Post-translational Responses of Aquaporins to Abiotic and Nutritional Stimuli in Arabidopsis Roots

Abstract

In plants, aquaporins play a crucial role in regulating root water transport in response to environmental and physiological cues. Controls achieved at the post-translational level are thought to be of critical importance for regulating aquaporin function. To investigate the general molecular mechanisms involved, we performed, using the model species Arabidopsis, a comprehensive proteomic analysis of root aquaporins in a large set of physiological contexts. We identified nine physiological treatments that modulate root hydraulics in time frames of minutes (NO and H 2 O 2 treatments), hours (mannitol and NaCl treatments, exposure to darkness and reversal with sucrose, phosphate supply to phosphate-starved roots), or days (phosphate or nitrogen starvation). All treatments induced inhibition of root water transport except for sucrose supply to darkgrown plants and phosphate resupply to phosphatestarved plants, which had opposing effects. Using a robust label-free quantitative proteomic methodology, we identified 12 of 13 plasma membrane intrinsic protein (PIP) aquaporin isoforms, 4 of the 10 tonoplast intrinsic protein isoforms, and a diversity of post-translational modifications including phosphorylation, methylation, deamidation, and acetylation. A total of 55 aquaporin peptides displayed significant changes after treatments and enabled the identification of specific and as yet unknown patterns of response to stimuli. The data show that the regulation of PIP and tonoplast intrinsic protein abundance was involved in response to a few treatments (i.e. NaCl, NO, and nitrate starvation), whereas changes in the phosphorylation status of PIP aquaporins were positively correlated to changes in root hydraulic conductivity in the whole set of treatments The absorption of soil water by roots is crucial in order for plants to maintain their water status. Studies in various plant species have shown that the root water permeability (root hydraulic conductivity Lp r ) is constantly adjusted depending on the developmental stage of the plant, its nutritional or hormonal status, or multiple environmental stimuli (1). Despite their importance in plant growth and adaptation, these multiple responses have not been investigated in a single plant species yet. Aquaporins form a large class of channel proteins that facilitate the diffusion of water and small neutral solutes across cell membranes and, among many other functions, contribute to root water uptake (2, 3). Aquaporins are 25-to 30-kDa proteins with six membrane-spanning domains and five connecting loops (A-E), with N-and C-terminal tails exposed to the cytosol (4, 5). Plant aquaporins show a high multiplicity of isoforms. Thirty-five homologs belonging to four homology subclasses have been identified in Arabidopsis. The plasma membrane intrinsic proteins (PIPs) 1 (with 13 isoforms further subdivided into the PIP1 and PIP2 subgroups) and the tonoplast intrinsic proteins (TIPs) with 10 homologs are the most abundant aquaporins in the plasma membrane and the tonoplast, respectivel...

Novel Aquaporin Regulatory Mechanisms Revealed by Interactomics

“…S4A, S4B). Three phosphorylation sites have been described in PIP2;1, at Ser 121 , Ser 280 , and Ser 283 (1,4,8). The P f of oocytes expressing a PIP2;1 form with Ser-to-Ala mutations at positions 280 and 283 (Ser 280 Ala-Ser 283 Ala) was almost similar to P f of oocytes expressing wild-type PIP2;1 (Fig.…”

“…Protein Digestion-In-solution reductions/alkylations were performed simultaneously with detergent removing by the filter-aided sample preparation protocol (1,36). These steps were followed by a endolysin-C (Sequencing Grade Modified, Promega, Madison, WI) digestion 3 h at 37°C followed by a trypsin (Sequencing Grade Modified, Promega) digestion overnight at 37°C.…”

“…Studies in various plant species have shown that the root water permeability (root hydraulic conductivity; Lp r 1 ) is constantly adjusted depending on the developmental stage of the plant, its nutritional or hormonal status, or multiple environmental stimuli (1,2). These adjustments depend in large part on the function and regulation of aquaporins, a large class of channel proteins that facilitate the diffusion of water and small neutral solutes across cell membranes (2,3).…”

“…For instance, following exposure of Arabidopsis roots to salt (NaCl) or hydrogen peroxide (H 2 O 2 ), AtPIP2;1 phosphorylation was decreased and increased, respectively (8). A recent quantitative phosphoproteomic work showed a strong correlation between the level of PIP phosphorylation and Lp r , under a wide range of environmental conditions (1). However, knowledge of the protein kinases (PKs) that phosphorylate aquaporins is still scarce.…”

Update: Post-translational protein modifications in plant metabolism

“…Finally, a study of aquaporins (plasma membrane intrinsic proteins [PIPs]) in Arabidopsis roots showed increased deamidation (by MS/MS analysis and spectral counting) in response to (a)biotic stress conditions. Moreover deamidation levels of PIP2 appeared coupled to its phosphorylation status, suggesting cross talk between these PTMs (di Pietro et al, 2013). It was suggested that deamidation could be involved in regulating aquaporin activity.…”