Shot-Gun Proteomic Analysis on Roots of Arabidopsis pldα1 Mutants Suggesting the Involvement of PLDα1 in Mitochondrial Protein Import, Vesicular Trafficking and Glucosinolate Biosynthesis

Abstract

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.

Keywords: Arabidopsis; cytoskeleton; mitochondrial protein import; phospholipase D alpha1; proteomics; quality control; vesicular transport.

Figure 1

Overview of differential root proteomes of pldα1 mutants. (A) Numbers of proteins with increased and decreased abundances in pldα1-1 and pldα1-2 mutant. (B) Venn diagram showing difference between differential proteomes the pldα1-1 and pldα1-2.

Figure 2

Functional classification of differentially abundant proteins found collectively in roots of pldα1-1 and pldα1-2 mutants using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis.

Figure 3

Distribution of protein families, in differentially abundant proteins found collectively in roots of pldα1-1 and pldα1-2 mutants, as evaluated by InterPro application of Blast2Go software. HAD = haloacid dehydrogenase; ADF = actin depolymerization factor; TIM = mitochondrial import inner membrane translocase; NAC = nascent polypeptide-associated complex; SGNH = serin, glycin, asparagine, histidin.

Figure 4

Functional classification of differentially abundant proteins found collectively in roots of pldα1-1 and pldα1-2 mutants based on published information, as presented in Table S1.

Figure 5

Immunoblotting analysis of ironic superoxide dismutase 1 (FeSOD1), syntaptotagmin 1 (SYT1) and mitochondrial uncoupling protein 1 (UCP1) in the roots of the Arabidopsis wild type and pldα1 mutants. (A,C,E) Immunoblots probed with anti-FeSOD (A), anti-SYT1 (B) and anti-UCP1 (C) antibodies and visualization of proteins transferred on nitrocellulose membranes using Ponceau S. (B,D,F) Optical density quantification of the respective bands in (A,C,E). Stars indicate significant differences between mutants and wild type at p ≤ 0.05 according to the Student t-test. Error bars represent standard deviations.

Figure 6

Immunolocalization of synaptotagmin (SYT1) in root epidermal cells of wild type (A), pldα1-1 (C) and pldα1-2 (E). (B,D,F) Fluorescence intensity profiles of immunolabeled synaptotagmin distributions in wild type (B), pldα1-1 (D) and pldα1-2 (F). Arrows indicate positions of measured cells for fluorescence intensity profiles. Asterisks indicate peaks of highest fluorescence intensities in measured cells. Note that fluorescence intensities in pldα1 mutants are much higher in comparison to the wild type, indicating overabundance of SYT1 in these mutants. Scale bar = 10 μm.

Figure 7

Immunolocalization of mitochondrial uncoupling protein 1 (UCP1) in root epidermal cells of wild type (A), pldα1-1 (C) and pldα1-2 (E). (B,D,F) Fluorescence intensity profiles of immunolabeled synaptotagmin distributions in wild type (B), pldα1-1 (D) and pldα1-2 (F). Arrows indicate positions of measured cells for fluorescence intensity profiles. Asterisks indicate peaks of highest fluorescence intensities in measured cells. Note that fluorescence intensities in pldα1 mutants are much higher in comparison to the wild type, indicating an overabundance of UCP1 in these mutants. Scale bar = 10 μm.