Salicylic Acid Targets Protein Phosphatase 2A to Attenuate Growth in Plants

Abstract

Plants, like other multicellular organisms, survive through a delicate balance between growth and defense against pathogens. Salicylic acid (SA) is a major defense signal in plants, and the perception mechanism as well as downstream signaling activating the immune response are known. Here, we identify a parallel SA signaling that mediates growth attenuation. SA directly binds to A subunits of protein phosphatase 2A (PP2A), inhibiting activity of this complex. Among PP2A targets, the PIN2 auxin transporter is hyperphosphorylated in response to SA, leading to changed activity of this important growth regulator. Accordingly, auxin transport and auxin-mediated root development, including growth, gravitropic response, and lateral root organogenesis, are inhibited. This study reveals how SA, besides activating immunity, concomitantly attenuates growth through crosstalk with the auxin distribution network. Further analysis of this dual role of SA and characterization of additional SA-regulated PP2A targets will provide further insights into mechanisms maintaining a balance between growth and defense.

Keywords: NPR1; PIN; PP2A; auxin; auxin transport; gravitropism; immunity; phosphorylation; protein phosphatase 2A; salicylic acid.

Graphical abstract

Figure 1

Pathogen-Induced SA Response in Roots, Revealed by the pPR1::eYFP-NLS Reporter (A) SA contents in the roots of 5- or 10-day-old seedlings of Col-0, cpr6, and sid2 (sid2-3) measured by LC/MS-MS. n = 4 replicates, with multiple seedlings for each. Dots represent individual values, and lines indicate mean ± SD. Different letters represent significant difference; p < 0.05; by one-way ANOVA with a Tukey multiple comparison test. (B–E) Induced pPR1::eYFP-NLS expression by P. syringae DC3000 (B and D) or SA (C and E) in roots. (B and D) 5-day-old pPR1::eYFP-NLS seedlings were treated with P. syringae DC3000 (optical density 600 [OD₆₀₀] = 0.01, ∼5 × 10⁶ colony-forming units [CFUs]/mL) or with resuspension buffer (control) for 48 h and were then imaged by confocal laser scanning microscope (CLSM). (C and E) For SA treatment, 5-day-old pPR1::eYFP-NLS seedlings were transferred to plates with DMSO or 40 μM SA for 24 h and were then imaged by CLSM. Scale bars, 10 μm. For quantification, the average GFP florescence of 5–10 representative cells from 10 seedlings for each treatment was measured by Fiji. The data points were shown as dot plots. Dots represent individual values, and lines indicate mean ± SD. p values were calculated by a two-tailed t test. See also Figure S1.

Figure 2

SA Regulates Root Growth and Development in a NPR1-Independent Manner (A) Representative images showing the morphological changes of Col-0 and npr1 under SA treatment. DMSO is the solvent control. Scale bars, 2 cm. (B) SA inhibited the primary root elongation in a NPR1-independent manner. Root length of 7-day-old Col-0 and npr1 seedlings grown on MS plates with different concentrations of SA was measured. Relative length was calculated by dividing the values with the root length at SA = 0. Boxplots show the first and third quartiles, with whiskers indicating maximum and minimum, the line for median, and the black dot for mean. n = 11–28; p values were calculated by a two-tailed t test for indicated pairs of Col-0 and npr1 at a certain concentration of SA. (C–H) SA interfered with root gravitropism independently of NPR1. Root tip angles of 7-day-old Col-0 (C–E) and npr1 (F–H) seedlings were measured and shown as polar bar charts. Two-tailed t tests were performed to indicate the difference of mean value, and F-tests indicate the difference of variances. For Col-0, SA treatments were compared with the DMSO control, and the npr1 groups were compared with Col-0 under the same SA treatment, respectively. (I) Inhibition of lateral root formation by SA does not involve NPR1. The number of emerged lateral roots for 10-day-old plants was counted. n = 20–25. p values were calculated by a two-tailed t test. See also Figure S1.

Figure 3

SA Regulates Auxin Transport via Modulating PIN2 Phosphorylation (A) SA inhibited the relocation of DR5-n3GFP. 5-day-old DR5v2 and eir1-4 DR5v2 seedlings were transferred to different plates with DMSO, 40 μM SA, 40 μM 3-OH-BA, or 40 μM 4-OH-BA, respectively, and then turned 90 degrees for gravistimulation. After 4 h, the roots were imaged by CLSM. The GFP channel (DR5-n3GFP) was shown. Scale bars, 10 μm. (B) The ratio of fluorescence between the upper side and the lower side was measured, as shown in (A). n = 34, 30, 35, 24, 11, 29, 19, and 12, respectively. p values are calculated by a two-tailed t test, comparing different datasets with the DR5-n3-GFP DMSO control (t = 4 h), as shown with the horizontal line in the case of DR5-n3-GFP SA 4 h. (C) SA treatment increased the accumulation of [³H]-NAA in tobacco BY-2 cells, suggesting a decrease in auxin export. DMSO and 200 μM SA were added to the cell culture and then the radioactivity inside of cells was measured at indicated time points after the addition of [³H]-NAA to the DMSO- and SA-treated cell cultures. n = 3. (D–G) SA treatment impaired the polar localization and promoted the internalization of PIN2-GFP in root epidermis (D and E). pPIN2::PIN2-GFP seedlings were grown on plates with DMSO and 40 μM SA for 4 days and were then imaged by CLSM. Scale bars, 20 μm (D) and 10 μm (E), respectively. Arrowheads in (D) indicate the beginning of root transition zone. (F) The intensity ratio of apical/lateral was measured by Fiji to assess PIN2 polarity. (G) Quantification of the PIN2-GFP intensity ratio of intracellular/PM. (F and G) Dots represent individual values, and lines indicate mean ± SD. p values are calculated by a two-tailed t test. (H) SA treatment enhanced the phosphorylation of PIN2. Roots of 7-day seedlings were treated with DMSO or 40 μM SA for 15 min and 60 min and then analyzed by western blot with an anti-PIN2 antibody (upper panel). Phosphorylation of the multiple phosphorylation sites in PIN2 causes slower migrating species. The more highly phosphorylated, the slower the migration. The same membrane was stripped and detected by anti-PIN1 (second panel) and anti-PIP2;1 (third panel) antibodies, sequentially. The molecular weight (MW) of PIN2 and PIN1 is 69 and 67 kDa, respectively. For unknown reasons, PIN2 runs faster than expected, perhaps due to incomplete denaturing when heated only at 50°C. The shifted bands indicate the phosphorylated PIN proteins. Bottom panel: Ponceau staining is shown. Asterisk indicates partial contribution by a non-specific band (see also in Figure 4A). (I) SA treatment increased the phosphorylation of His-PIN2-HL in plant extracts. Roots of 7-day seedlings were treated with DMSO or 40 μM SA for 15 min and 60 min, respectively, and then were subject to protein extraction. Crude plant extracts were incubated with recombinant His-PIN2-HL for 60 min with ³²P-ATP and MgCl₂. The first lane was without His-PIN2-HL as negative control. Reaction samples were analyzed by SDS-PAGE and the subsequent autoradiography. Upper panel: autoradiography is shown; lower panel: Coomassie Brilliant Blue (CBB) staining is shown. See also Figures S2 and S3.

Figure 4

SA Functions through PP2A in Regulating Root Development (A) SA treatment promoted the phosphorylation of PIN2 in Col-0 to a similar degree as that in pp2aa1-6. Roots of 7-day-old Col-0 and pp2aa1-6 seedlings were treated with DMSO or 40 μM SA for 60 min and were then sampled for protein isolation and western blot. The shifted bands indicate the phosphorylated PIN proteins (upper panel). Asterisk indicates a non-specific band that contributes partially to the signal. The same membrane was stripped and probed with a PIP2;1 antibody to indicate the loading (upper panel). Ponceau staining is shown in the bottom panel. (B) Phosphorylation with ³²P-ATP revealed that SA treatment increased the phosphorylation of His-PIN2-HL in Col-0, whereas this increase was attenuated in pp2aa1-6. Upper panel: autoradiography is shown; lower panel: CBB is shown. (C) Representative images revealing the hypersensitivity of pp2aa1-6 to SA. Col-0 and pp2aa1-6 seedlings were grown on plates with SA. Scale bars, 2 cm. (D) pp2aa1-6 was hypersensitive to SA in root growth inhibition. Col-0 and pp2aa1-6 seedlings grew on plates with SA for 7 days and then the primary root length was measured. n = 11–28. p values were calculated by a two-tailed t test for indicated pairs of Col-0 and pp2aa1-6 at a certain concentration of SA. (E and F) pp2aa1-6 was hypersensitive to SA in terms of interfering with root gravitropism. Col-0 (E) and pp2aa1-6 (F) seedlings grew on plates containing different concentrations of SA for 7 days, and the root tip angles were measured by ImageJ and shown as polar bar charts. p values were calculated by a two-tailed t test in (E) and (F) and indicate differences of variances by a further F-test in (F). (G) The pp2aa1, a3 double mutant exhibited decreased sensitivity to SA. Col-0 and pp2aa1, a3 seedlings grew on plates with SA for 7 days and then the primary root length was measured. n = 11–25. p values were calculated by a two-tailed t test for indicated pairs of Col-0 and pp2aa1, a3 at the given concentration of SA. (H) The pp2ac3, c4 double mutant exhibited decreased sensitivity to SA. Col-0 and pp2ac3, c4 seedlings grew on plates with SA for 7 days and then the primary root length was measured. n = 10–21. p values were calculated by a two-tailed t test for indicated pairs of Col-0 and pp2ac3, c4 at the given concentration of SA. See also Figures S3 and S4.

Figure 5

Genetic Analysis of pp2aa1-6 and cpr6 Mutations, and SA Inhibits PP2A Activity In Planta (A) Representative images showing the enhanced sensitivity of pp2aa1-6 to SA. Col-0, pp2aa1-6, cpr6, and pp2aa1-6 cpr6 seedlings were grown on plates with different concentrations of SA for 7 days. Scale bars, 2 cm. (B) The root growth analysis revealed that the cpr6 mutation decreased the primary root length and increased the SA sensitivity of pp2aa1-6. n = 16. Different letters represent significant difference; p < 0.05; by one-way ANOVA with a Tukey multiple comparison test. (C and D) The pp2aa1-6 mutation enhances the stunted shoot phenotype of cpr6. Col-0, pp2aa1-6, cpr6, and pp2aa1-6 cpr6 plants were grown for 38 days, and representative plants are shown (C). Scale bar, 2 cm. (D) The height of plants was measured and shown as dot plots. Dots represent individual values, and lines indicate mean ± SD. n = 16. Different letters represent significant difference; p < 0.05; by one-way ANOVA with a Tukey multiple comparison test. (E) SA treatment decreased the total PP2A activity in planta. Col-0, pp2aa1-6, and npr1 seedlings were grown on plates containing DMSO or 40 μM SA for 5 days and then sampled for protein isolation and PP2A activity measurement. n = 6. Different letters represent significant difference; p < 0.05; by one-way ANOVA with a Tukey multiple comparison test. (F) SA treatment increased the phosphorylation of the PP2A catalytic subunits (PP2Ac), suggesting the decrease in PP2A activity. 7-day-old Col-0 seedlings were treated with DMSO or 40 μM SA for 0, 15 min, 30 min, and 60 min respectively, and were then collected for protein extraction and the subsequent western blot. A pY307-PP2Ac antibody was used, 1:1,000 (upper panel). The anti-actin blot (medium panel; 1:2,000) and Ponceau staining (bottom panel) indicate the loading amounts. See also Figure S4.

Figure 6

SA Binds to the A Subunit of PP2A (A) DARTS assay suggests that PP2AA1 is potential target of SA. pPP2AA1::PP2AA1-GFP seedlings were used for the protein isolation. Samples were treated with DMSO (mock) and SA and digested by different concentrations of Pronase. Samples were further analyzed by western blot with an anti-GFP antibody. (B) DSC analysis suggesting the potential binding of SA to recombinant His-PP2AA1. 5 μM of purified His-PP2AA1 protein was analyzed by DSC with or without 50 μM SA. Tm = 48.01°C and 45.03°C for His-PP2AA1+DMSO and His-PP2AA1+SA, respectively. (C) SPR analysis of the His-PP2AA1 and SA interaction. An active synthetic SA analog (SA-f) was immobilized on a CM-5 sensor chip, and different concentrations of His-PP2AA1 were applied. The binding curve was plotted by values at the steady state, for which the sensorgram is shown is Figure S7D. A K_D value of 3.623 μM was detected. (D) SPR assay reveals the binding of His-PP2AA3 to SA. The same sensor chip as above was used, and different concentrations of His-PP2AA3 were applied. The binding curve was plotted by values at the steady state, with the data points shown in the sensorgram in Figure S7E. A K_D value of 1.916 μM was detected. See also Figures S5, S6, and S7.

Figure 7

Model for the Parallel SA Action in Immunity and Growth Regulation (A) SA plays a key role in the growth-immunity transition following pathogen attack: on one hand, SA activates the immune response, through stimulating NPR1 and repressing NPR3/4, all together increasing the expression of downstream defense genes; on the other hand, SA inhibits growth via suppressing PP2A activity and the subsequent dephosphorylation of substrates. (B) The auxin efflux carrier PIN2 is phosphorylated by different kinases, including PINOID/WAGs, D6PK/D6PKLs, and MAPKs, and dephosphorylated by PP2A. Following pathogen attack, the SA levels increase. SA binds to the A subunits of PP2A and thereafter represses its dephosphorylation activity toward PIN proteins, which leads to hyperphosphorylation of PIN, thereby a decrease in PIN activity ultimately resulting in a decrease in auxin export and attenuation of growth. (C and D) Induced stronger expression of pPR1::eYFP-NLS by SA was detected in pp2aa1-6. (C) pPR1::eYFP-NLS seedlings were constantly grown on plates with DMSO or 40 μM SA for 5 days from germination and were then imaged by CLSM. Scale bars, 10 μm. (D) For quantification, the average GFP florescence of 5–10 representative cells from 10 seedlings for each treatment was measured by Fiji. The data points were showed as dot plots, and lines indicate mean ± SD. Different letters represent significant difference; p < 0.05; by one-way ANOVA with a Tukey multiple comparison test.