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

Abstract

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.

Proteomic analysis of Mesembryanthemum crystallinum leaf microsomal fractions finds an imbalance in V-ATPase stoichiometry during the salt-induced transition from C3 to CAM

“…plants that perform C3 photosynthesis. The results of this analysis are listed in Table 1 and confirm previous transcriptional studies on responses of M. crystallinum to salt stress, for example the stress-related increases in transcript levels of enolase [12,21], glucose 6-phosphate translocator [12,47], cysteine proteinase [12] and phosphoenolpyruvate carboxylase 1 [12,]. The decrease in the transcript levels of triose phosphate/phosphate translocator [47] results, in the present study, in a corresponding change in the respective protein representation.…”

“…Indeed, MS-based quantification has rapidly made an impact on the analysis of differential protein expression and during the last few years several quantification methods have been proposed [17][18][19][20]. For M. crystallinum only a few proteomic studies have so far been conducted [21]. None of the work has up to now has reported a systematic investigation by means of high-throughput proteomic technologies.…”

Effect of salinity stress on plants and its tolerance strategies: a review

“…An increased relative abundance of alcohol dehydrogenase has been found in hypocotyls of salt-stressed soybean (Sobhanian et al 2010a) and grasspea leaves (Chattopadhyay et al 2011). Moreover, it has been hypothesized that FBP aldolase could play a role in salt ion vacuolar compartmentation since it can directly physically interact with tonoplast H + -ATPase and activates its transport function (Barkla et al 2009;Tada and Kashimura 2009). It can thus be concluded that in glycophytes, photosynthesis (CO 2 assimilation) is decreased by salt stress.…”

iTRAQ Analysis Reveals Mechanisms of Growth Defects Due to Excess Zinc in Arabidopsis

“…Aldolase, which catalyzes an aldol cleavage of Fru 1,6-bisP to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, interacts with the V-ATPase B subunit (Barkla et al, 2009). It has been proposed that aldolase regulates cell elongation mediated by V-ATPase, because root growth was inhibited in aldolaseantisense transgenic rice (Oryza sativa; Konishi et al, 2005).…”