soy polar lipid mixture  (Avanti Inc)

Journal: The Plant cell

Article Title: Phosphatidic acid modulates MPK3- and MPK6-mediated hypoxia signaling in Arabidopsis.

doi: 10.1093/plcell/koab289

Figure Lengend Snippet: Figure 1 Submergence induces PLDa1- and PLDd-derived PA accumulation, which triggers the nuclear localization of RAP2.12. A, Amounts of membrane lipids (PG, PC, PE, PI, PS, and PA) in the rosettes of 4-week-old WT Col-0 plants under light submergence treatment (Sub) and after re- covery (R) for the indicated times. B, Various PA and PE species in the rosettes of 4-week-old WT, plda1, pldd, and plda1 pldd plants before light submergence treatment (air) and after 2 days of submergence treatment (submergence). C, Exogenous application of PA, but not PC, PE, or PS, induces the translocation of RAP2.12-GFP from the plasma membrane to the nucleus. Detached leaves of 3-week-old RAP2.12-GFP transgenic plants were treated with 50-mM liposomes prepared from PA, PC, PE, or PS (natural lipid mixtures purified from soy, Avanti Polar Lipids) for 3 h. Leaves similarly treated with dilution buffer were set as mock controls (Mock). The GFP fluorescence was detected by confocal microscopy. Red arrows indicate nuclear signal induced by PA application. Bars, 20 lm. All experiments were performed on three biological replicates with similar results. Values represent means ± SD (n = 4) of four independent technical replicates, and each replicate was collected from the rosettes of at least seven plants. Asterisks with “H” or “L” indicate significantly higher or lower levels than in control plants (A) or in WT (B) at each time point (*P 5 0.05, **P 5 0.01 by Student’s t test).

Article Snippet: Detached leaves of 3-week-old RAP2.12-GFP transgenic plants were treated with 50-mM liposomes prepared from PA, PC, PE, or PS (natural lipid mixtures purified from soy, Avanti Polar Lipids) for 3 h. Leaves similarly treated with dilution buffer were set as mock controls (Mock).

Techniques: Derivative Assay, Membrane, Translocation Assay, Clinical Proteomics, Transgenic Assay, Liposomes, Confocal Microscopy, Control

Journal: The Plant cell

Article Title: Phosphatidic acid modulates MPK3- and MPK6-mediated hypoxia signaling in Arabidopsis.

doi: 10.1093/plcell/koab289

Figure Lengend Snippet: Figure 3 PA binds to MPK3 and MPK6 and enhances submergence-induced MPK3 and MPK6 activity. A, Lipid binding specificity of recombinant MPK3 and MPK6 proteins on membrane filters. About 50 lM of various lipids (PA, PC, PE, PI, PG, and PS dissolved in chloroform; natural lipid mixtures purified from soy, Avanti Polar Lipids) were spotted onto nitrocellulose membrane and incubated with 10 lg of purified GST-MPK3, GST-MPK6, or GST protein. Binding was detected by immunoblotting using an anti-GST antibody. Equal volume of chloroform was spotted as negative control (Blank). B, Pull-down assay showing the physical interaction between PA and recombinant MPK3 and MPK6 proteins. Recombinant proteins were incubated with PA beads, and the precipitated GST-MPK3 and GST-MPK6 were detected with anti-GST antibody. GST-PYL4 was used as a negative control. C, Dissociation constant (Kd) for the binding of recombinant MPK3 and MPK6 proteins to liposomes of PA, PC, PI, and PS (natural lipid mixtures purified from soy, Avanti Polar Lipids). A serial dilution of various liposomes ranging from 1.5 nM to 50 lM was prepared for mixing with the labeled proteins, and their binding affinities were measured by MST analysis. Kd, dissociation constant. ND, not detected. D and E, MPK3 and MPK6 are activated by submergence. Ten-day-old WT seedlings were exposed to light submergence (LS, D) or in the dark submergence (DS, E). MPK3/MPK6 kinase activities were detected with anti-pTEpY antibody. MPK3 and MPK6 proteins were detected with anti-MPK3 and anti-MPK6 antibodies, respectively. Actin, detected with an anti-actin antibody, was used as loading control. F, Quantification of MPK phosphorylation activity shown in (D) and (E). Data were calculated according to relative intensity from three indepen- dent experiments and the average values ± SD are shown. G, MPK3 and MPK6 kinase activities in WT and plda1 pldd plants and following submer- gence for 0.5, 1, 3, and 6 h. Total proteins were extracted and immunoblotting assays were performed using anti-pTEpY, anti-MPK3, and anti-MPK6 antibodies, with Ponceau S-stained total protein as loading control. hpt, hour posttreatment. Relative intensity of each p-MPK3 or pMPK6 band normalized to the loading control is shown below. H, PA induces MPK3 and MPK6 activity in planta. Ten-day-old WT seedlings were treated without (Mock) or with 50 lM PA or PS liposomes (natural lipid mixtures purified from soy, Avanti Polar Lipids) for 0.5, 1, and 3 h, and immunoblotting assays were performed using anti-pTEpY, anti-MPK3, anti-MPK6, and anti-actin antibodies, with actin as loading control. Relative intensity of each p-MPK3 or pMPK6 band normalized to the loading control is shown below. All experiments were performed on three biological replicates with similar results. Data in (C) and (F) are means ± SD of three biological replicates. Asterisks indicate significant differences from WT at 0 h (*P 5 0.05 by Student’s t test).

Article Snippet: Detached leaves of 3-week-old RAP2.12-GFP transgenic plants were treated with 50-mM liposomes prepared from PA, PC, PE, or PS (natural lipid mixtures purified from soy, Avanti Polar Lipids) for 3 h. Leaves similarly treated with dilution buffer were set as mock controls (Mock).

Techniques: Activity Assay, Binding Assay, Recombinant, Membrane, Incubation, Protein Binding, Western Blot, Negative Control, Pull Down Assay, Liposomes, Serial Dilution, Labeling, Control, Phospho-proteomics, Staining

Journal: The Plant cell

Article Title: Phosphatidic acid modulates MPK3- and MPK6-mediated hypoxia signaling in Arabidopsis.

doi: 10.1093/plcell/koab289

Figure Lengend Snippet: Figure 5 PA enhances MPK3- and MPK6-mediated phosphorylation of RAP2.12 to activate its transcriptional activity. A, Co-IP assay showing the interac- tion between MPK3/MPK6 and RAP2.12. Constructs encoding MPK3-FLAG and MPK6-FLAG, and RAP2.12-HA were transiently transfected in WT Arabidopsis protoplasts and immunoprecipitated with anti-FLAG beads. B, Immunoblot analyses showing RAP2.12 protein levels when co-expressed with MPK3 or MPK6. RAP2.12-HA was co-transfected with or without MPK3-FLAG or MPK6-FLAG in WT Arabidopsis protoplasts overnight. pUC119-eGFP-HA was co-transfected to determine transfection efficiency for each sample. Anti-HA and anti-FLAG antibodies were used for immunoblotting. Relative inten- sity of each protein band normalized to the GFP-HA control is shown below. C, Dual-LUC reporter assay showing RAP2.12-activated transcription of ADH1 in the absence (mock) or presence of PA. When indicated, protoplasts were treated with 10-lM PA liposomes (natural lipid mixture purified from soy, Avanti Polar Lipids) for 16h. “a” indicates significantly higher or lower levels than in control; “b” indicates significantly higher or lower levels than with RAP2.12 alone; “c” indicates significantly higher or lower levels than in mock-treated protoplasts. D, Dual-LUC reporter assays showing RAP2.12-activated transcription of ADH1 in WT and plda1 pldd protoplasts. “a” indicates significantly higher or lower levels than controls not co-transfected with RAP2.12 (CK), “b” indicates significantly higher or lower levels than RAP2.12 in WT. E, Activated MPK3 and MPK6 phosphorylate RAP2.12 in vitro. Phosphorylated recombinant MPK3, MPK6, MKK5DD, as well as RAP2.12 were detected with anti-thiophosphate ester rabbit monoclonal antibodies after gel electrophore- sis (top), recombinant MKK5DD, MPK3, and MPK6 were detected with anti-His antibody (middle), and recombinant RAP2.12 was detected with anti-GST antibody (bottom). Reactions lacking the specified components (–) were used as controls. Recombinant proteins were separated by 10% SDS–PAGE after incubation in protein kinase buffer containing ATPcS and PNBM. F, MPK6-mediated RAP2.12 phosphorylation is enhanced by the application of PA. Phosphorylated recombinant MPK6 and MKK5DD, as well as RAP2.12 were detected with anti-thiophosphate ester rabbit monoclonal antibodies after gel electrophoresis (top), recombinant MKK5DD and MPK6 were detected with anti-His antibody (middle), and recombinant RAP2.12 was detected with anti- GST antibody (bottom). Reactions lacking the specified components (–) were used as controls. Recombinant proteins were separated by 10% SDS–PAGE after incubation in protein kinase buffer containing ATPcS and PNBM. Relative intensity of phosphorylated proteins normalized to the control is shown below. G, Phosphorylation of RAP2.12 by MPK6 in vivo. Constructs encoding MKK5DD-HA and MPK6-HA, and RAP2.12-FLAG were transiently transfected in Arabidopsis protoplasts. Proteins were extracted 16h after incubationto allow protein accumulation. The phosphorylation of RAP2.12 was confirmed by incubation with phosphatase and phosphatase inhibitor, and the immunoblots were probed with anti-HA and anti-FLAG antibodies. All experiments were performed on three biological replicates with similar results. For all blots, numbers on the left indicate the molecular weight (kDa) of each band. For the LUC reporter assay, data are means± SD of three independent experiments. Asterisks indicate significant differences from WT (**P5 0.01 by Student’s t test).

Article Snippet: Detached leaves of 3-week-old RAP2.12-GFP transgenic plants were treated with 50-mM liposomes prepared from PA, PC, PE, or PS (natural lipid mixtures purified from soy, Avanti Polar Lipids) for 3 h. Leaves similarly treated with dilution buffer were set as mock controls (Mock).

Techniques: Phospho-proteomics, Activity Assay, Co-Immunoprecipitation Assay, Construct, Transfection, Immunoprecipitation, Western Blot, Control, Reporter Assay, Liposomes, In Vitro, Recombinant, Bioprocessing, SDS Page, Incubation, Nucleic Acid Electrophoresis, In Vivo, Molecular Weight