Towards the profiling of the Arabidopsis thaliana plasma membrane transportome by targeted proteomics

Abstract

Plant membranes bear a variety of transporters belonging to multigene families that are affected by environmental and nutritional conditions. In addition, they often display high-sequence identity, making difficult in-depth investigation by current shot-gun strategies. In this study, we set up a targeted proteomics approach aimed at identifying and quantifying within single experiments the five major proton pumps of the autoinhibited H(+) ATPases (AHA) family, the 13 plasma membrane intrinsic proteins (PIP) water channels (PIPs), and ten members of ammonium transporters (AMTs) and nitrate transporter (NRT) families. Proteotypic peptides were selected and isotopically labeled heavy versions were used for technical optimization and for quantification of the corresponding light version in biological samples. This approach allowed to quantify simultaneously nine PIPs in leaf membranes and 13 PIPs together with three autoinhibited H(+) ATPases, two ammonium transporters, and two NRTs in root membranes. Similarly, it was used to investigate the effect of a salt stress on the expression of these latter 20 transporters in roots. These novel isoform-specific data were compared with published transcriptome information and revealed a close correlation between PIP isoforms and transcripts levels. The obtained resource is reusable and can be expanded to other transporter families for large-scale profiling of membrane transporters.

Regulation of Arabidopsis Leaf Hydraulics Involves Light-Dependent Phosphorylation of Aquaporins in Veins

“…Expression profiling with macroarrays carrying gene-specific tags (see Supplemental Figure 1 online) identified four PIP genes (PIP1;2, PIP2;1, PIP2;6, and PIP2;7) as highly expressed in the Arabidopsis rosette. Each of these PIPs ranks among the three most highly expressed isoforms in at least one previous gene or protein expression study of Arabidopsis leaves (Alexandersson et al, 2005;Monneuse et al, 2011). T-DNA insertion lines corresponding to knockouts for each of these PIPs in a Columbia-0 (Col-0) (PIP1;2, PIP2;1, and PIP2;6) or Landsberg erecta (Ler) (PIP2;7) background were available from previous studies (Da Ines et al, 2010;Postaire et al, 2010;Péret et al, 2012) or were molecularly characterized in this work (see Supplemental Figure 2 online).…”

“…To investigate the mechanisms of K ros regulation by the light regime, we profiled PIP expression in rosettes of L-and Dgrown plants using multiple reaction monitoring (MRM) of proteotypic aquaporin peptides in microsomal extracts (Monneuse et al, 2011). This proteomic approach is based on a comparison of the native aquaporin peptides to chemically synthesized and heavy isotope-labeled reference peptides.…”

“…A microsomal fraction was isolated (Gerbeau et al, 2002), stripped from extrinsic membrane proteins , and digested overnight with endolysin-C (Monneuse et al, 2011). MRM analyses (Monneuse et al, 2011) were performed on three transitions per peptide (see Supplemental Table 1 online). Corresponding heavy isotope labeled peptides were chemically synthesized and used for absolute quantification (Monneuse et al, 2011).…”

“…MRM analyses (Monneuse et al, 2011) were performed on three transitions per peptide (see Supplemental Table 1 online). Corresponding heavy isotope labeled peptides were chemically synthesized and used for absolute quantification (Monneuse et al, 2011). The identification of phosphorylated forms of the C-terminal tail of PIP2;1 was performed with a quantitative time-of-flight mass spectrometer (Maxis; Bruker Daltonik), interfaced with a nano-HPLC Ultimate 3000 (Dionex).…”

Selected Reaction Monitoring to Determine Protein Abundance in Arabidopsis Using the Arabidopsis Proteotypic Predictor

“…This combination of filters gives SRM approaches their power in complex samples and allows the quantification of many different proteins over 4 orders of magnitude in crude whole-protein extracts from plant tissue samples (Picotti and Aebersold, 2012). SRM MS, also referred to as mass westerns, has previously been used in plants to quantify a number of proteins, including Suc phosphate synthase isoforms in Arabidopsis (Lehmann et al, 2008), Suc synthase isoforms and nitrogen metabolism enzymes in Medicago species , a basic amino acid carrier involved in Arg metabolism in rice (Oryza sativa; Taylor et al, 2010), cytosolic and organelle markers in Arabidopsis (Ito et al, 2011), and the plasma membrane transportome in Arabidopsis (Monneuse et al, 2011). Label-free quantitation in this manner requires reproducibility in sample extraction, digestion, liquid chromatography, and ionization and has been widely reviewed (Lange et al, 2008;Picotti and Aebersold, 2012).…”