Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance

• Published in: The Plant cell 2009-12, Vol.21 (12), p.4044-4058
• Main Authors: Barkla, Bronwyn J, Vera-Estrella, Rosario, Hernández-Coronado, Marcela, Pantoja, Omar
• Format: Article
• Language: English
• Subjects: Arabidopsis - enzymology; Arabidopsis - genetics; ATP; Electrophoresis; Electrophoresis, Gel, Two-Dimensional; Enzymes; Fructose-Bisphosphate Aldolase - genetics; Fructose-Bisphosphate Aldolase - metabolism; Gels; Gene expression regulation; Gene Expression Regulation, Plant; Mesembryanthemum - enzymology; Mesembryanthemum - genetics; Microsomes; Phosphopyruvate Hydratase - genetics; Phosphopyruvate Hydratase - metabolism; Physiological regulation; Plants; Proteome - metabolism; Proteomics; Salt tolerance; Salt-Tolerant Plants - enzymology; Salt-Tolerant Plants - genetics; Salts; Sodium Chloride - metabolism; Spectrometry, Mass, Electrospray Ionization; Tonoplast; Vacuolar Proton-Translocating ATPases - genetics; Vacuolar Proton-Translocating ATPases - metabolism; Vacuoles; Vacuoles - metabolism; Yeasts
• ISSN: 1040-4651; 1532-298X
• DOI: 10.1105/tpc.109.069211

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na⁺ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H⁺-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H⁺-pump activity.