Research ArticlePLANT SCIENCES

Pipecolic acid confers systemic immunity by regulating free radicals

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Science Advances  30 May 2018:
Vol. 4, no. 5, eaar4509
DOI: 10.1126/sciadv.aar4509
  • Fig. 1 Pip confers SAR in a dose-dependent manner.

    (A) SAR response in distal leaves of WT Col-0 plants treated locally with MgCl2, methanol (0.01%), avirulent pathogen (avrRpt2), or Pip (1000 μM). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Error bars indicate SD (n = 4). CFU, colony-forming units. (B) SAR response in distal leaves of Col-0 and ald1 plants treated locally with MgCl2, methanol (0.01%), avirulent pathogen (avrRpt2), or Pip (1000 μM). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Error bars indicate SD (n = 4). (C) SAR response in distal leaves of WT Col-0 plants treated locally with MgCl2, methanol (0.01%), avirulent pathogen (avrRpt2), and different concentrations of Pip (100 to 2000 μM). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Error bars indicate SD (n = 4). (D) SAR response in distal leaves of Col-0 plants treated locally with MgCl2, avirulent pathogen (avrRpt2), or Pip (1000 μM each). The virulent pathogen (DC3000) was inoculated at indicated hours after local treatments with Pip. Error bars indicate SD (n = 4). For (A) to (D), asterisks and “*a” denote significant differences with mock-treated plants (t test, P < 0.05) or plants treated with 1 and 2 mM Pip, respectively. The percentage of methanol for control experiments was determined on the basis of dilution prepared from Pip stock solutions. The results are representative of three to five independent experiments.

  • Fig. 2 Pip increases NO levels, and Pip-conferred SAR is dependent on NOA1 NIA proteins.

    (A) Venn diagrams showing the number of genes up-regulated (left) or down-regulated (right) in local tissues of Col-0 plants treated with Pip or water. (B) Protein immunoblot showing NOA1 levels in local and distal tissues of Col-0 and ald1 plants treated with MgCl2 (mock), avrRpt2 bacteria, or Pip. Leaves were sampled 24 hours after inoculations. Ponceau S staining of the immunoblot was used as the loading control. The experiment was repeated four times with similar results. (C) Confocal micrograph showing pathogen-and Pip-induced NO accumulation in Col-0 plants at 24 hours after treatments. The leaves were inoculated with MgCl2 (mock) or avrRpt2 Pst, or treated with Pip, and at least 10 independent leaves were analyzed in four experiments with similar results. Chloroplast autofluorescence (red) was visualized using Ds-Red2 channel. Scale bar, 10 μm. DAF-FM DA, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. (D) SAR response in distal leaves of Col-0 and noa1 nia2 plants treated locally with MgCl2, methanol (0.01%), avirulent pathogen (avrRpt2), or Pip (1000 μM). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Error bars indicate SD (n = 4). Asterisks denote significant differences with mock-treated plants (t test, P < 0.05), and results are representative of three independent experiments.

  • Fig. 3 Pip increases ROS, AzA, and G3P levels, and Pip-mediated SAR is dependent on RBOHD/F, MGD1 DGD1, and GLY1 GLI1 proteins.

    (A) H2O2 levels in local tissues after mock and pathogen (avrRpt2) inoculations or Pip treatments of Col-0 and rbohD plants. The leaves were sampled 24 hours after treatments. The error bars represent SD. H2O2 was quantified from the tissue extracts prepared as described in Materials and Methods. Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated twice with similar results. (B) SAR response in distal leaves of Col-0, rbohD, and rbohF plants treated locally with MgCl2, methanol (0.01%), avirulent pathogen (avrRpt2), or Pip (1000 μM). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Error bars indicate SD (n = 4). Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated four times with similar results for Col-0 and rbohF plants. The rbohD plants showed a nominal SAR after avrRpt2 inoculation in two of the four repeats. (C) AzA levels in local tissues after mock and pathogen (avrRpt2) inoculations or Pip treatments of Col-0 plants. The error bars represent SD. Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated three times with similar results. (D) G3P levels in local tissues after mock and pathogen (avrRpt2) inoculations or Pip treatments of Col-0 plants. The error bars represent SD. Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated three times with similar results. (E) SAR response in distal leaves of Col-0 and mgd1 dgd1 plants treated locally with MgCl2, methanol (0.01%), avirulent pathogen (avrRpt2), or Pip (1000 μM). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Error bars indicate SD (n = 4). Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated two times with similar results. (F) SAR response in distal leaves of Col-0, gly1, gli1, or gly1 gli1 plants treated locally with MgCl2, methanol (0.01%), avirulent pathogen (avrRpt2), or Pip (1000 μM). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Error bars indicate SD (n = 4). Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated four times with similar results for Col-0, gli1, and gly1 gli1 plants. The gly1 plants showed a nominal SAR after Pip treatment in two of the four repeats. FW, fresh weight.

  • Fig. 4 A defect in Pip biosynthesis in ald1 plants impairs accumulation of ROS, AzA, and G3P but not SA.

    (A to D) H2O2 (A), AzA (B), G3P (C), or SA (D) levels in local tissues after mock and pathogen (avrRpt2) inoculations of Col-0 and ald1 plants. The leaves were sampled 24 hours after treatments. The error bars represent SD. Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated three times with similar results. (E to H) SAR response in distal leaves of Col-0 and ald1 plants treated locally with MgCl2, avirulent pathogen (avrRpt2), or H2O2 (500 μM; E), AzA (1000 μM; F), G3P (100 μM; G), and SA (500 μM; H). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Error bars indicate SD (n = 4). Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated three times with similar results. (I) SAR response in Col-0 and ald1 plants infiltrated with PEX collected from Col-0 or ald1 plants that were treated either with MgCl2 (PEXMgCl2) or avrRpt2 (PEXavrRpt2). The distal leaves were inoculated with virulent pathogen at 48 hours after infiltration of primary leaves. Error bars indicate SD (n = 4). Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated twice with similar results.

  • Fig. 5 Induction of Pip in the distal tissues is associated with transport of SA and G3P.

    (A) Pip levels in local and distal tissues of Col-0 plants after mock and pathogen (avrRpt2) inoculations or localized application of methanol (0.01%) or Pip. The leaves were sampled 48 hours after treatments. The error bars represent SD. Asterisks denote a significant difference from mock treatment (t test, P < 0.05). The experiment was repeated three times with similar results. (B) Pip levels in PEX collected from Col-0 plants after Pip treatment. The leaves were treated with 1 mM Pip, and the infiltrated leaves (~2 g per sample) were sampled 48 hours after treatment. The error bars represent SD. Asterisks denote a significant difference from mock treatment (t test, P < 0.05). The experiment was repeated two times with similar results. (C) Autoradiograph of TLC plate showing transport of 14C-Pip from the local to distal leaves. 14C-Pip (26 μM) was mixed with MgCl2 (mock) or avrRpt2 and infiltrated in the local leaves of Col-0 plants. Both local and distal leaves were sampled 24 hours after treatment and analyzed on a silica TLC plate using a butanol/acetic acid/water (3:1:1, v/v) solvent system. The arrowhead indicates the position of the 14C-Pip. The vertical arrow indicates the direction of the run. (D) Pip levels in local and distal tissues of Col-0 and gly gli1 plants after mock and pathogen (avrRpt2) inoculations. The leaves were sampled 48 hours after treatments. The error bars represent SD. Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated two times with similar results. (E) Pip levels in local and distal tissues of Col-0, mgd1 dgd1, rbohD, or rbohF plants after mock and pathogen (avrRpt2) inoculations. The leaves were sampled 48 hours after treatments. The error bars represent SD. Asterisks denote a significant difference from mock treatment (t test, P < 0.05). The experiment was repeated two times with similar results. (F) SAR response in distal leaves of Col-0, sid2, and gly1 gli1 plants treated locally with water or SA (500 μM). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Error bars indicate SD (n = 4). Asterisks denote a significant difference from mock treatment (t test, P < 0.05). The experiment was repeated two times with similar results. (G) Pip levels in Col-0 and sid2 plants after water or SA treatment. The local leaves were sampled 48 hours after treatments. The error bars represent SD. Asterisks denote a significant difference with mock (t test, P < 0.05). The experiment was repeated two times with similar results.

  • Fig. 6 A simplified model illustrating the relationship between SA, G3P, and Pip in local and distal leaves.

    Inoculation of avirulent pathogen triggers independent signaling events that lead to accumulation of SA and NO in the local leaves. NO triggers synthesis of ROS, which catalyze oxidation of free C18-unsaturated FAs that are released from membrane lipids (4). NO and ROS operate in a feedback loop. Oxidation of C18 FAs generates AzA, which triggers biosynthesis of G3P via up-regulation of genes encoding G3P biosynthetic enzymes. Of these, chemical signals SA, G3P, AzA, and Pip are detected in the PEX collected from leaves inoculated with avirulent pathogen. SA and G3P are required for synthesis of Pip in the distal leaves. Exogenous G3P, but not SA, can overcome a requirement for Pip and confer SAR on ald1 plants that are unable to synthesize Pip. Dashed green lines indicate transport of SA, G3P, and Pip from local to distal tissues.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaar4509/DC1

    fig. S1. The ald1 plants accumulate basal levels of Pip.

    fig. S2. Pip-induced NO is dependent on ALD1.

    fig. S3. Pip-induced ROS is dependent on ALD1 and RBOHD.

    fig. S4. The ald1 plants show reduced ion leakage.

    fig. S5. Pip is unable to confer SAR on mutants impaired in the SA pathway.

    fig. S6. Induction of Pip is associated with ALD1 transcript levels.

    fig. S7. Induction of Pip in the distal tissues is associated with transport of SA and G3P.

    table S1. Gene expression analysis in response to methanol (0.001%) and Pip treatments in Arabidopsis thaliana.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. The ald1 plants accumulate basal levels of Pip.
    • fig. S2. Pip-induced NO is dependent on ALD1.
    • fig. S3. Pip-induced ROS is dependent on ALD1 and RBOHD.
    • fig. S4. The ald1 plants show reduced ion leakage.
    • fig. S5. Pip is unable to confer SAR on mutants impaired in the SA pathway.
    • fig. S6. Induction of Pip is associated with ALD1 transcript levels.
    • fig. S7. Induction of Pip in the distal tissues is associated with transport of SA and G3P.

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    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). Gene expression analysis in response to methanol (0.001%) and Pip treatments in Arabidopsis thaliana.

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