Advancements in the Analysis of the Arabidopsis Plasma Membrane Proteome

The plasma membrane (PM) regulates diverse processes essential to plant growth, development, and survival in an ever-changing environment. In addition to maintaining normal cellular homeostasis and plant nutrient status, PM proteins perceive and respond to a myriad of environmental cues. Here we review recent advances in the analysis of the plant PM proteome with a focus on the model plant Arabidopsis thaliana. Due to membrane heterogeneity, hydrophobicity, and low relative abundance, analysis of the PM proteome has been a special challenge. Various experimental techniques to enrich PM proteins and different protein and peptide separation strategies have facilitated the identification of thousands of integral and membrane-associated proteins. Numerous classes of proteins are present at the PM with diverse biological functions. PM microdomains have attracted much attention. However, it still remains a challenge to characterize these cell membrane compartments. Dynamic changes in the PM proteome in response to different biotic and abiotic stimuli are highlighted. Future prospects for PM proteomics research are also discussed

<i>BoEWR1</i> over-expression enhances Arabidopsis resistance to Verticillium wilt.

<p>(A). Typical disease symptoms caused by <i>V. dahliae</i> on the wild-type (WS) and three independent <i>BoEWR1</i> over-expressing plants (BoEWR1-1, BoEWR1-2 and BoEWR1-3) at 21 days post inoculation (dpi). The experiment was repeated at least three times and representative of the three independent biological replications is shown. (B) <i>Verticillium</i>-induced stunting of wild-type (WS), three independent <i>BoEWR1</i> over-expressing plants (BoEWR1-1, BoEWR1-2 and BoEWR1-3) at 21 dpi. Rosette diameters of inoculated plants were compared with those of mock-inoculated plants. The bars represent averages of three independent experiments with standard deviation and asterisks indicate significant differences (Dunnett t-test at <i>P = 0.05</i>). (C) Relative quantification (RQ) by real-time PCR of <i>Verticillium</i> colonization by comparing levels of the <i>V. dahliae</i> internal transcribed spacer (ITS) region of the ribosomal DNA (as measure for fungal biomass) relative to levels of the large subunit of the Arabidopsis <i>RubisCo</i> gene (for equilibration) at 21 dpi. Bars represent averages with standard deviation of four technical replicates. A representative of three independent experiments is shown. (D) Relative quantification (RQ) of <i>EWR1</i> transcription in wild-type (WS) and three independent <i>BoEWR1</i> over-expressing plants (BoEWR1-1 and BoEWR1-2, and BoEWR1-3). Bars represent averages with standard deviation of three biological replicates</p

<i>EWR1</i> over-expressing Arabidopsis plants are resistant to <i>V. dahliae</i>.

<p>(A) Typical symptoms of <i>V. dahliae</i> on the wild-type (Col-0), three <i>EWR1</i> expressing lines (EWR1-1, EWR1-2, and EWR1-3) and <i>EWR1</i> knock out line (<i>ewr1</i>). Picture was taken at 21 days post inoculation and a representative of three experimental replicates is shown. (B) Disease severity score for the wild-type (Col-0), the three <i>EWR1</i> expressing lines (EWR1-1, EWR1-2, and EWR1-3) and <i>EWR1</i> knock out line (<i>ewr1</i>) at 14 (white bar) and 21 (grey bar) days post inoculation (dpi). The total number of rosette leaves and the number of rosette leaves that showed Verticillium symptoms were counted at least from eight plants and percentage of the diseased leaves were calculated as an indication of disease severity. The bars represent the average of three independent experiments with standard deviation and asterisks indicate significance differences (Dunnett t-test at <i>P = 0.05</i>). (C) Relative quantification (RQ) by real-time PCR of Verticillium colonization by comparing levels of the <i>V. dahliae</i> internal transcribed spacer (ITS) region of the ribosomal DNA (as measure for fungal biomass) relative to levels of the large subunit of the Arabidopsis <i>RubisCo</i> gene (for equilibration) at 21 dpi. Bars represent averages with standard deviation of four technical replicates. A representative of three independent experiments is shown.</p

The activation-tagged Arabidopsis mutant A2 is more resistant to <i>V. dahliae</i> and <i>V. albo-atrum</i>.

<p>(A) Typical symptoms of <i>Verticillium</i> on the wild-type (WS) and the activation-tagged mutant A2. Picture was taken at 21 days post inoculation (dpi) and a representative of three independent experimental replicates is shown. (B) Relative quantification (RQ) by real-time PCR of Verticillium colonization by comparing levels of the <i>V. dahliae</i> (white bars) and <i>V. albo-atrum</i> (grey bars) internal transcribed spacer (ITS) region of the ribosomal DNA (as measure for fungal biomass) relative to levels of the large subunit of the Arabidopsis <i>RubisCo</i> gene (for equilibration) at 14 and 21 dpi. Bars represent averages with standard deviation of four technical replicates. A representative of three independent experiments is shown. (C) Relative quantification (RQ) of <i>EWR1</i> transcription level in the wild-type WS and the activation-tagged mutant A2. The bar represents the average of three independent experiments and standard deviation of the means and asterisks indicate significant differences (Dunnett t-test at <i>P = 0.01</i>) compared to the wild-type WS.</p

<i>EWR1</i> is highly conserved in <i>Brassicaceae</i>.

<p>(A) Schematic representation of the full-length genomic DNA sequence of <i>EWR1</i> gene. (B) Nucleotide sequence alignment of <i>AtEWR1</i> and its homologs from <i>Arabidopsis lyrata</i> (<i>AlEWR1</i>), <i>Brassica oleracea</i> var. <i>gemmifera</i> (<i>BoEWR1</i>), <i>Brassica rapa (BrEWR1)</i>, and <i>Sisymbrium irio</i> (<i>SiEWR1</i>).</p

Analysis of the genes flanking the activation-tag insertion site in mutant A2.

1<p>Gene expression in mutant A2 relative to the expression in wild-type.</p>2<p>Phenotype of knock-out alleles upon <i>V. dahliae</i> inoculation when compared to wild-type plants.</p

Deletion of <i>EWR1</i> enhances Arabidopsis susceptibility to Verticillium wilt.

<p>(A) Typical symptoms of <i>V. dahliae</i> on the wild-type (Col-0) and <i>EWR1</i> knock out (<i>ewr1</i>) plants. Picture was taken at 21 days post inoculation (dpi) and a representative of three independent experimental replicates is shown. (B) Disease severity score for the wild-type (Col-0) and <i>ewr1</i> at 14 (white bar) and 21 (grey bar) days post inoculation (dpi). The total number of rosette leaves and the number of rosette leaves that showed Verticillium symptoms were counted at least from eight plants and percentage of the diseased leaves were calculated as an indication of disease severity. The bars represent averages of three independent experiments with standard deviation and asterisks indicate significance differences (Dunnett t-test at <i>P = 0.05</i>). (C) Relative quantification (RQ) by real-time PCR of Verticillium colonization by comparing levels of the <i>V. dahliae</i> internal transcribed spacer (ITS) region of the ribosomal DNA (as measure for fungal biomass) relative to levels of the large subunit of the Arabidopsis <i>RubisCo</i> gene (for equilibration) at 21 dpi. Bars represent averages with standard deviation of four technical replicates. A representative of three independent experiments is shown.</p

<i>AtEWR1</i> over-expressing plants are tolerant to drought stress.

<p>Three weeks-old wild-type Col-0, <i>AtEWR1</i> expressing line (EWR1-2) and <i>AtEWR1</i> knock out line (<i>ewr1</i>) plants were exposed to drought stress and picture was taken at 14 days post drought treatment. The assay was repeated three times and a representative of the replicates is shown.</p