Research ArticlePLANT SCIENCES

The plant cuticle regulates apoplastic transport of salicylic acid during systemic acquired resistance

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Science Advances  06 May 2020:
Vol. 6, no. 19, eaaz0478
DOI: 10.1126/sciadv.aaz0478
  • Fig. 1 ACP4 and MOD1 are required for distal transport of SA.

    (A) SA and SAG levels in local tissues after mock (10 mM MgCl2) and pathogen (avrRpt2) inoculations. The leaves were sampled 48 hours after treatments, and the experiment was repeated two times with similar results. Asterisks denote a significant difference with respective mock-inoculated samples (t test, P < 0.0001). Columbia (Col-0) and Nössen (Nö) are wild-type ecotypes for mod1 and acp4, respectively. (B) G3P levels in local tissues after mock and pathogen (avrRpt2) inoculations. The leaves were sampled 24 hours after treatments, and the experiment was repeated two times with similar results. Asterisks denote a significant difference with respective mock-inoculated samples (t test, P < 0.0005). (C) G3P levels in PEX collected from mock (PEXMgCl2)– and avrRpt2 (PEXavrRpt2)–inoculated plants. The experiment was repeated three times with similar results. Asterisks denote a significant difference with respective mock-inoculated samples (t test, P < 0.0007). (D) Size of foci measured as numbers of rings of cells containing P30-2XGFP punctae around a transformed cell 48 hours after treatment in wild-type (Col-0 or Nö) or acp4 and mod1 leaves. (E) SA levels in PEX collected from mock (PEXMgCl2)– and avrRpt2 (PEXavrRpt2)–inoculated plants. Results are representative of four independent experiments. Single (t test, P < 0.0001) and double (t test, P < 0.004) asterisks denote a significant difference with respective mock-inoculated samples or between indicated pairs, respectively. (F) Quantification of radioactivity transported to distal tissues of mock- and avrRpt2-inoculated plants. Leaves were infiltrated with 20 μM solution of 14C-SA and sampled 48 hours after mock (MgCl2) or avrRpt2 inoculations. The error bars indicate SD. Asterisks denote a significant difference with respective mock-inoculated samples (t test, P < 0.006). NS indicates data not significantly different. (G) Autoradiograph of TLC plate showing transport of 14C-SA from the local to distal leaves. 14C-SA (20 μM) was mixed with MgCl2 (mock) or avrRpt2 and infiltrated into the local leaves of wild type (Nö) and acp4. Both local and distal leaves were sampled 48 hours after treatment and analyzed on a silica TLC plate using toluene/methanol/acetic acid (45:8:4, by volume) solvent system. Arrowhead indicates position of the 14C-SA. Vertical arrow indicates direction of the run. Numbers below the bands indicate relative intensity of bands in mock versus avr samples quantified using ImageQuant TL image analysis software.

  • Fig. 2 MOD1 is required for normal cuticle development.

    (A) Morphological phenotype of 4-week-old Col-0 and mod1 plants (scale, 0.5 cm). Photo credit: P.K., University of Kentucky. (B) Toluidine blue–stained leaves from 4-week-old leaves of Col-0, mod1, and acp4 plants. The leaves were stained on their adaxial surface for 5 min. Photo credit: P.K., University of Kentucky. (C) Transmission electron micrographs showing cuticle layer on adaxial surface of Col-0 and mod1 plants. Arrows indicate cuticle, which is electron dense in Col-0 leaves. Scale bars, 50 nm. (D) Scanning electron micrographs showing abaxial surface of Col-0 or mod1 leaves. Scale bars, 200 μm.

  • Fig. 3 Exogenous application of SA, G3P, or Pip is unable to confer SAR on mod1 or acp4 plants.

    (A) SAR response in distal leaves of Col-0, mod1, and lacs2 plants treated locally with MgCl2 or avrRpt2. The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. CFU indicates colony forming units. Asterisks denote a significant difference with respective mock-inoculated samples (t test, P < 0.0001). (B) SA levels in PEX collected from mock (PEXMgCl2)– and avrRpt2 (PEXavrRpt2)–inoculated Col-0 and lacs2 plants. Single and double asterisks denote a significant difference with respective mock-inoculated samples (t test, P < 0.0001) or indicated pair (t test, P < 0.0001), respectively. (C) SA levels in PEX collected from mock (PEXMgCl2)– and avrRpt2 (PEXavrRpt2)–inoculated plants. The local leaves were abraded to damage the cuticle 12 hours before inoculation. Results are representative of two independent experiments. Single and double asterisks denote a significant difference with respective mock-inoculated samples (t test, P < 0.0006) or indicated pair (t test, P < 0.0001), respectively. (D) SAR response in distal leaves of untreated or cuticle abraded plants shown in (C). The virulent pathogen (DC3000) was inoculated 48 hours after mock or avr inoculations. Asterisks denote a significant difference with respective mock-inoculated samples (t test, P < 0.0001). NS indicates “not significant.” (E) SAR response in distal leaves of wild-type (Col-0 or Nö), acp4, or mod1 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 with mock (t test, P < 0.0002). The experiment was repeated three times with similar results. (F and G) Pip levels in local (F) and distal (G) tissues of wild-type (Col-0 or Nö), acp4, or mod1 plants after mock and avrRpt2 inoculations. The leaves were sampled 48 hours after treatments. Asterisks denote a significant difference with mock (t test, P < 0.0001). “a” denotes significant difference between avrRpt2-inoculated wild type and mutants (P < 0.0005). The experiment was repeated three times with similar results. (H) SAR response in distal leaves of wild-type (Col-0 or Nö), acp4, or mod1 plants treated locally with water or Pip (1000 μM). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Asterisks denote a significant difference with mock (t test, P < 0.0001). The experiment was repeated two times with similar results. (I) SAR response in distal leaves of wild-type (Col-0 or Nö), acp4, or mod1 plants treated locally with water, G3P (100 μM), or AzA (1000 μM). The virulent pathogen (DC3000) was inoculated 48 hours after local treatments. Asterisks denote a significant difference with mock (t test, P < 0.0005). The experiment was repeated three times with similar results.

  • Fig. 4 NahG plants are competent in SA transport.

    (A) SA levels in PEX collected from mock (PEXMgCl2)– or pathogen (avrRpt2 or TMV)–infected Arabidopsis (Di-3) or tobacco (Samsung N background) wild-type and nahG plants. Results are representative of three independent experiments. Asterisks denote significant differences from respective mock-inoculated samples (t test, P < 0.001). (B) Levels of 14C-SA in distal tissue of Arabidopsis wild-type (Di-3 ecotype) or Di-3 nahG plants. Arabidopsis leaves were coinfiltrated with 20 μM 14C-SA and MgCl2 or Pst avrRpt2. Forty-eight hours later, amount of radiolabel in SA extracts from the leaves was quantified. Results are representative of three independent experiments. Asterisks denote significant differences (t test, P < 0.004). (C) Percentage of 14C-SA transported from local to distal tissue of Di-3 and Di-3 nahG plants shown in (B). Asterisks denote significant differences (t test, P < 0.02). (D and E) Pip levels in local and distal tissue of Arabidopsis (D) or tobacco (E) plants after mock and pathogen inoculations. The Arabidopsis and tobacco distal leaves were sampled 48 or 140 hours after inoculation, respectively. Asterisks denote significant differences from mock (t test, P < 0.0001). The experiment was repeated three times with similar results. (F) Protein immunoblot showing PR-1 levels in the scion (distal) of tobacco grafts that were inoculated with TMV in the rootstock (local). W and N indicate wild type and nahG. Scion leaf samples were collected 5 days after TMV inoculation. Ponceau-S staining of the immunoblot was used as the loading control. The experiment was repeated twice with similar results.

  • Fig. 5 Distal transport of SA is associated with water potential.

    (A) SA levels in PEX collected from mock (PEXMgCl2)– and avrRpt2 (PEXavrRpt2)–inoculated Nö and acp4 plants. A week before inoculations, one set of plants was covered with a transparent dome to increase humidity and reduce transpiration. Results are representative of three independent experiments. Asterisks denote a significant difference with respective mock-inoculated samples (t test, P < 0.001). (B) SAR response in distal leaves of Nö and acp4 that were grown in open or covered with a transparent dome. 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 < 0.0001). The experiment was repeated two times with similar results. (C) Leaf water potential of indicated genotypes. Values are means ± SD (n = 15). Asterisks denote a significant difference with mock (t test, P < 0.001). (D) Stomatal apertures of indicated genotype. Values are means ± SD (n = 20). Asterisks denote a significant difference with mock (t test, P < 0.0001). (E) WUE for Nö and acp4. Values are means ± SD (n = 4). Asterisks denote a significant difference with mock (t test, P < 0.006).

  • Fig. 6 SA exists in a deprotonated form at cytosolic pH and is exported into cuticular waxes.

    (A) NMR spectra of SA at pH 2.5, 4.5, and 7.0. The samples were made in DMSO-d6 containing 400 μl of buffered solution of SA. The transmitter offset frequency was placed at the corresponding water peak (4.506 ppm for pH 2.5 and 4.5 and 4.513 ppm for pH 7). All samples were then referenced to DMSO (2.5 ppm). (B) Top: Uptake assays showing percentage of 14C-SA transported into isolated protoplasts or chloroplasts. Fresh or 24-hour-old protoplasts (106/ml) or chloroplast (107/ml) were incubated with 2 μM 14C-SA for 1 hour, analyzed microscopically before and after four washes, and quantified for the amount of radiolabel. NC indicates data not considered since chloroplasts were damaged after 1-hour incubation at pH 4.5. The experiment was repeated three times with similar results. Bottom: Uptake assays showing percentage of 14C-SA transported into isolated protoplasts in the absence (control; Cnt) or presence of proton pump inhibitors sodium orthovanadate (OV) and omeprazole (OM). The experiment was repeated two times with similar results. (C) SA levels in cuticular wax fraction of mock- and avrRpt2-inoculated leaves. Leaves were sprayed (108 CFU/ml) or infiltrated (105 CFU/ml) with avrRpt2 and sampled 48 hours after inoculation. (D) Relative SA levels in cuticular wax fraction of indicated plants. The experiment was repeated three times with similar results. (E) SA levels in cuticular wax fraction of mock- and avrRpt2-inoculated leaves. Leaves were sprayed (108 CFU/ml) with avrRpt2 and sampled 48 hours after inoculation. Asterisks denote a significant difference (t test, P < 0.0015). (F) Real-time quantitative RT-PCR showing relative expression levels of PR-1 in mock- and avrRpt2-inoculated plants at 18 and 48 hours after inoculation. The error bars indicate SD (n = 3). Results are representative of two independent experiments. Single and double asterisks denote a significant difference between mock-inoculated samples or indicated pairs (t test, P < 0.003). (G) Protein immunoblot showing PR-5 levels in mock- and avrRpt2-inoculated leaves at 18 hours after infection. Ponceau-S staining of the immunoblot was used as the loading control. The experiment was repeated three times with similar results.

  • Fig. 7 Cuticular SA regulates stomatal opening.

    (A) Total wax levels in Col-0 and sid2 plants treated with water or 500 μM SA for 24 hours before wax extraction. The experiment was repeated two times with similar results. (B) Analysis of wax components from leaves of 4-week-old Col-0 and sid2 plants that were treated with water or 500 μM SA for 24 hours before wax extraction. 16:0 to 24:0 are FAs, C25 to C33 are alkanes, and C26-OH to C30-OH are primary alcohols. The experiment was repeated two times with similar results. (C) Stomatal apertures in mock- and avrRpt2-inoculated leaves of Col-0 and sid2 plants 24 hours after infection. Values are means ± SD (n = 20). Single and double asterisks denote a significant difference between Col-0 and sid2 and between indicated pairs, respectively (t test, P < 0.003). (D) Microscopic images showing representative stomata of mock-, pathogen (avrRpt2)–, SA-, or ABA-treated leaves from Col-0 and sid2 plants. (E) Leaf water potential of Col-0 and sid2 plants treated with MgCl2 (mock), SA (500 μM), or avrRpt2 for 24 hours. Values are means ± SD (n = 15). Single and double asterisks denote a significant difference between Col-0 and sid2 and between indicated pairs, respectively (t test, P < 0.0001). (F) WUE for Col-0 and sid2 plants. Values are means ± SD (n = 4). Asterisks denote a significant difference (t test, P < 0.005). (G) Stomatal apertures in mock-, avrRpt2-, or SA (500 μM)–treated leaves of Nö and acp4 plants. Values are means ± SD (n = 20). Asterisks denote a significant difference (t test, P < 0.0001). (H) Microscopic images showing representative stomata of mock-, SA (500 μM)–, or avrRpt2-inoculated leaves from Nö and acp4 plants.

Supplementary Materials

  • Supplementary Materials

    The plant cuticle regulates apoplastic transport of salicylic acid during systemic acquired resistance

    Gah-Hyun Lim, Huazhen Liu, Keshun Yu, Ruiying Liu, M. B. Shine, Jessica Fernandez, Tessa Burch-Smith, Justin K. Mobley, Nicholas McLetchie, Aardra Kachroo, Pradeep Kachroo

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