Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.
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.
Barkla
Bronwyn J
BJ
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Colonia Miraval, Cuernavaca, Morelos, Mexico. bronwyn@ibt.unam.mx
Vera-Estrella
Rosario
R
Hernández-Coronado
Marcela
M
Pantoja
Omar
O
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D035723:Mesembryanthemum / Q000201:enzymology / Q000235:genetics*; D008861:Microsomes; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D055051:Salt-Tolerant Plants / Q000201:enzymology / Q000235:genetics*; D012965:Sodium Chloride / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
D020543:Proteome; D012965:Sodium Chloride; D025262:Vacuolar Proton-Translocating ATPases; D005634:Fructose-Bisphosphate Aldolase; D010751:Phosphopyruvate Hydratase