Phosphatidic acid interacts with a MYB transcription factor and regulates its nuclear localization and function in Arabidopsis

Abstract

Phosphatidic acid (PA) has emerged as a class of cellular mediators involved in various cellular and physiological processes, but little is known about its mechanism of action. Here we show that PA interacts with werewolf (WER), a R2R3 MYB transcription factor involved in root hair formation. The PA-interacting region is confined to the end of the R2 subdomain. The ablation of the PA binding motif has no effect on WER binding to DNA, but abolishes its nuclear localization and its function in regulating epidermal cell fate. Inhibition of PA production by phospholipase Dζ also suppresses WER's nuclear localization, root hair formation, and elongation. These results suggest a role for PA in promoting protein nuclear localization.

Figure 1.

PA Binding to WER. (A) Immunoblotting of His-WER, His-CPL3, and His-ETC2 expressed in E. coli Rosetta (DE3). Two concentrations of each protein were used for SDS-PAGE, followed by immunoblotting with anti-His-tag antibodies. (B) Lipid-protein blotting of various lipids with WER, CPL3, or ETC2; 0.5 µg of each lipid was spotted on nitrocellulose strips. PA, PC, PE, PG, PI, PS, LysoPC, and LysoPA were from egg yolk, and the synthetic PA with defined acyl species was as specified. (C) Lipid-protein blotting assay of PA, PI, PI4P, and PI4,5P₂ with WER and mutated WER; 0.5 µg of each lipid was spotted on nitrocellulose strips. PA and PI were from egg yolk, and PI4P and PI4,5P₂ were from porcine brain. Purified protein (WER and mutated WER, 0.5 µg/ml) was used, followed by immunoblotting with anti-His-tag antibodies. (D) Liposomes were made up of di18:1-PC only or di18:1-PA/PC (1/3 mole ratio). 1× and 10× refer to the concentration gradient of PC or PA/PC liposomes used, with 10× indicating a 10 times higher concentration than 1×. NL, no liposome was used and only 25% of input WER used for liposomal binding was loaded. (E) Representative SPR sensorgram of PA binding to WER. His-WER (2 μM) was immobilized on the NTA chip and 200 μM PC only or PA/PC (1/3 mole ratio) liposomes were injected. Each binding assay was repeated three times. Kinetic constants of PA binding to WER were calculated based on one sensorgram.

Figure 2.

Identification of the PA Binding Motif and Amino Acid Residues Involved in WER Binding to PA. (A) Schematic diagram showing serial deletions of WER. The WER fragments were expressed in E. coli and used for defining the PA binding domain. (B) Binding of His-WER fragments to PA/PC liposomes. Liposomes were made up of di18:1-PC only or di18:1-PA/PC (1/3 mole ratio). 1× and 10× refer to the concentration gradient of PC or PA/PC liposomes used. NL, no liposome was used and only 25% of input WER fragment proteins used for liposomal binding was loaded. (C) Sequence alignment of the PA binding fragment of WER with that of the PA binding motifs in chicken Raf1, abscisic acid insensitive 1 (ABI1), and constitutive triple response1 (CTR1) from Arabidopsis. The residues in bold are basic, potentially involved in PA binding and were mutated to Ala. (D) Representative SPR sensorgram of PA binding to WER and its single and double mutants. Full-length His-WER or mutant WERs (2 μM) were immobilized on the NTA chip, followed by injection of PA/PC (1/3 mole ratio) liposomes. [See online article for color version of this figure.]

Figure 3.

Subcellular Localization of WER, WER_K51AR52A, and WER_R58AR60A (A) Longitudinal view (median section) of transgenic roots by confocal microscopy. P35S:WER, P35S:WER_K51AR52A, and P35S:WER_R58AR60A were fused with YFP and PWER:WER, PWER:WER_K51AR52A, and PWER:WER_R58AR60A were fused with GFP. The constructs were transformed into Arabidopsis wer mutants. GFP fluorescence in PWER:WER wer plants was mainly observed in the nucleus of the root epidermal cells (as marked by arrowheads). Roots were counterstained with propidium iodide to view cell boundaries. Bar = 100 µm. (B) Immunoblotting of WER and mutant WER proteins expressed in Arabidopsis. P and S refer to proteins from nuclear pellets and soluble cytosol fractions, respectively. The same amounts of proteins (10 μg) were separated by SDS-PAGE and transferred onto a filter. WER proteins were detected using an anti-flag antibody conjugated with horseradish peroxidase. The proteins were immunoblotted with anti-PEPC and anti-histone H3 as cytosolic and nuclear markers, respectively. WT, wild type.

Figure 4.

Inhibition of PLDζ Activity and Its Effect on Subcellular Association of WER. (A) In vitro inhibition of PLDζ1 with PLD inhibitors, using Arabidopsis PLDζ1 produced in E. coli. The concentration response curves for each inhibitor are presented as a percentage of total PLD activity without inhibitor. Values are means ± sd (n = 3). (B) Immunoblotting of WER expressed in Arabidopsis. Proteins (10 µg) extracted from roots were separated by SDS-PAGE and transferred onto a filter. WER was detected using an anti-flag antibody conjugated with horseradish peroxidase. WT, wild type. (C) Longitudinal view (median section) of the root tip region of transgenic plants (transformed with P35S:WER-YFP, PWER:WER-GFP) treated with DMSO or PLD inhibitors (300 nM). YFP/GFP signals (green). Roots were counterstained with propidium iodide to view cell boundaries (red). Bar = 100 µm. [See online article for color version of this figure.]

Figure 5.

Root Hair Patterning as Affected by Transforming WER and Non-PA Binding Mutants to Arabidopsis. (A) Root hair morphology of WER-KO (wer), wild type (WT), and both wer and wild-type plants harboring P35S:WER, P35S:WER_K51AR52A, P35S:WER_R58AR60A, PWER:WER, PWER:WER_K51AR52A, or PWER:WER_R58AR60A. Bar = 100 µm. (B) Percentage of root epidermal cells that give rise to root hairs in wild-type, wer, and transgenic plants harboring WER or double mutants under the control of the 35S or WER native promoter. (C) Root hair density of wild-type, wer and transgenic plants harboring WER or its double mutants under the control of the 35S or WER native promoter. Values are means ± sd (n = 5).

Figure 6.

Effect of PLD2 Inhibitor on Decreasing Root Hair Density in Wild Type, wer, and PLDζ-KOs. (A) Dose-dependence of PLD2 inhibitor on root hair density. *P < 0.05 difference from the corresponding genotype with DMSO treatment as assessed by the Student’s t test. (B) Root hair density of the wild type (WT), PLDζ1-KO, PLDζ2-KO, and PLDζ1ζ2-double KO, as affected by different concentrations of PLD2-inibitors *P<0.05 difference from the wild-type control under same treatment as assessed by the Student’s t test. Values are means ± sd from root hairs of 10 seedlings.

Figure 7.

Subcellular Localization and Root Hair Patterning of an NLS-Fused GFP-WER-NLS, GFP-WER_K51AR52A-NLS, and GFP-WER_R58AR60A-NLS. (A) Schematic illustration of the complementation constructs of WER and mutated WERs with a NLS fused at the C terminus and GFP fused at the N terminus under the control of WER native promoter (PWER). (B) Longitudinal view (median section) of root tip region of PWER:GFP-WER-NLS, PWER:GFP-WER_K51AR52A-NLS, or PWER:GFP-WER_R58AR60A-NLS by confocal microscopy. The nuclear signal was found in epidermal cells and lateral root cap cells of all tested genotypes (as arrowheads). Bar = 100 µm. (C) Root hair density of wer and transgenic complementation plants harboring WER or its double mutants fused with N-terminal GFP and C-terminal NLS. Values are means ± sd (n = 5). [See online article for color version of this figure.]

Figure 8.

A Proposed Model Depicting the Role of the PA-WER Interaction in Feedback Attenuation of Root Hair Formation. PA binding of WER acts as a feedback regulator to modulate root hair patterning. PLDs produce PA that binds to WER and promotes its nuclear localization, leading to the establishment of nonhair cells. In root hair cells, PLDζ1 catalyzes the formation of PA and promotes root hair formation and growth. [See online article for color version of this figure.]