PROHIBITIN3 Forms Complexes with ISOCHORISMATE SYNTHASE1 to Regulate Stress-Induced Salicylic Acid Biosynthesis in Arabidopsis

Abstract

Salicylic acid (SA) is a major defense signal in plants. In Arabidopsis (Arabidopsis thaliana), the chloroplast-localized isochorismate pathway is the main source of SA biosynthesis during abiotic stress or pathogen infections. In the first step of the pathway, the enzyme ISOCHORISMATE SYNTHASE1 (ICS1) converts chorismate to isochorismate. An unknown enzyme subsequently converts isochorismate to SA. Here, we show that ICS1 protein levels increase during UV-C stress. To identify proteins that may play roles in SA production by regulating ICS1, we analyzed proteins that coimmunoprecipitated with ICS1 via mass spectrometry. The ICS1 complexes contained a large number of peptides from the PROHIBITIN (PHB) protein family, with PHB3 the most abundant. PHB proteins have diverse biological functions that include acting as scaffolds for protein complex formation and stabilization. PHB3 was reported previously to localize to mitochondria. Using fractionation, protease protection, and live imaging, we show that PHB3 also localizes to chloroplasts, where ICS1 resides. Notably, loss of PHB3 function led to decreased ICS1 protein levels in response to UV-C stress. However, ICS1 transcript levels remain unchanged, indicating that ICS1 is regulated posttranscriptionally. The phb3 mutant displayed reduced levels of SA, the SA-regulated protein PR1, and hypersensitive cell death in response to UV-C and avirulent strains of Pseudomonas syringae and, correspondingly, supported increased growth of P. syringae The expression of a PHB3 transgene in the phb3 mutant complemented all of these phenotypes. We suggest a model in which the formation of PHB3-ICS1 complexes stabilizes ICS1 to promote SA production in response to stress.

Figure 1.

PHB3/4 form complexes with ICS1-V5 under normal and stress conditions. A, Plants grown in soil were treated with UV-C light for 45 min and assayed 24 h later (+). Untreated plants were used as controls (−). Total proteins were extracted, and ICS1-containing complexes were immunoprecipitated with V5 antibody. The presence of ICS1-V5 and PHB3/4 proteins in the total protein extracts (Input; left) and in the immunoprecipitated ICS1-containing complexes (IP αV5; right) was confirmed by immunoblotting (IB) using V5 and PHB3/4 antibodies. Coomassie Blue-stained membranes (bottom) show similar loading. Representative blots from three independent experiments (two from total extracts and one from chloroplast extracts) are shown. WT, Wild type. B and C, Quantitation of ICS1-V5 (B) and PHB3/4 (C) protein levels from three immunoprecipitation experiments as in A, using densitometry with the ImageJ program. RU, Relative units with respect to an average signal of untreated samples for each antibody. Error bars show se. Statistical analyses using Student’s t tests showed that no significant differences were detected (P = 0.78 for B and P = 0.97 for C).

Figure 2.

ICS1 protein levels are induced by UV-C treatment and constitutively elevated in sid2-2/ICS1-V5 plants. Plants grown on plates (A, B, C, and F) or soil (D) were treated with UV-C for 20 min (A, B, C, and F) or 45 min (D), and assayed 0, 8, and 24 h post treatment (hpt). Untreated plants were used as controls (–). A, ICS1 transcript levels were measured by RT-qPCR in wild-type (WT; black bars) and sid2-2/ICS1-V5 (gray bars) plants. Expression is relative (R.) to the YLS8 gene (n = 3 biological replicates). B, Immunoblot analysis showing that ICS1 is induced by UV-C treatment in the wild type and is not detectable in sid2-2 plants. C, ICS1 protein levels detected by immunoblot in wild-type and sid2-2/ICS1-V5 plants using ICS1 or V5 antibodies. D, PR1 and ICS1-V5 protein levels in wild-type and sid2-2/ICS1-V5 plants. In sid2-2/ICS1-V5 plants, basal PR1 is elevated and UV-C further induces the PR1 level. In B to D, representative results from three (B and C) or two (D) independent experiments are shown. Coomassie Blue-stained membranes (bottom) show similar loading. E and F, Free SA (gray bars) and glycosylated SA (SAG; white bars) levels in the indicated plants. E, Untreated plants grown in soil, showing higher SA levels in sid2-2/ICS1-V5 plants than in the wild type (n = 5). Similar results were found in an independent trial. F, Free SA and SAG levels are induced by UV-C in plants with constitutive ICS1 levels (n = 3). FW, Fresh weight. Statistical analyses in A and F were performed using ANOVA/Fisher’s lsd test and in E using Student’s t test. Each letter group differs from other letter groups at P < 0.05. Error bars show se.

Figure 3.

PHB3-GFP localizes to chloroplasts and mitochondria. A, The morphological phenotype of the phb3-3 mutant is complemented by the PHB3-GFP transgene. Images show 3-week-old wild-type (WT), phb3-3, and phb3-3/PHB3-GFP #6.5 plants grown in soil. B to E, Untransformed (B) and transiently transformed (C and E) N. benthamiana leaves, and stably transformed Arabidopsis cotyledons (D), were imaged by confocal microscopy in green (PHB3-GFP), red (OEP7-RFP and COX4-mCherry), chlorophyll autofluorescence (chl; blue), and differential interference contrast (DIC; bright-field) channels. Maximum intensity projections of Z series images are shown. C, Coexpression of PHB3-GFP and OEP7-RFP (chloroplast outer envelope marker). D, Expression of PHB3-GFP in Arabidopsis (phb3-3/PHB3-GFP #6.5 plants). E, Coexpression of PHB3-GFP and COX4-mCherry (mitochondrial marker). Insets show higher magnification. Colocalization with COX4-dsRed also was performed with similar results. White arrowheads show examples of PHB3-GFP localization in chloroplasts (C–E). Experiments were done two (D) or three (B, C, and E) times with similar results. Bars = 20 µm.

Figure 4.

PHB3 is a chloroplast membrane protein. A, Immunodetection of PHB3/4 in total and chloroplast protein extracts obtained from wild-type (WT) and sid2/ICS1-V5 plants, grown in soil, treated (+) or not (–) with UV-C as described in Figure 1. ICS1-V5 was detected with V5 antibody, and PHBs were detected with a PHB3/4 antibody (n = 2). The purity of the chloroplast fraction was evaluated by immunodetection of markers for specific subcellular localizations (AtpB for chloroplast, Prx II F for mitochondria, cytosolic Fru-1,6-bisphosphatase [cFBPase] for cytoplasm, and H⁺-ATPase for plasma membrane). B, PHB3 localizes to chloroplast membranes. Spinach (So), Arabidopsis (At), and N. benthamiana (Nb) chloroplasts were partitioned into membrane and soluble fractions. PHB was detected with a PHB3/4 antibody, and fractions were confirmed with antibodies against pea Tic110 (membrane) and spinach cpHsp70 (soluble). Coomassie Blue (A) and Ponceau S (B) staining of proteins show similar loading. C and D, PHB is mostly protected from thermolysin digestion in N. benthamiana (C) and Arabidopsis (D) chloroplasts. Chloroplasts isolated from N. benthamiana transiently expressing AtPHB3-GFP (C; top four gels), control N. benthamiana (C; bottom two gels), or Arabidopsis leaves (D) were incubated without (−) or with (+) thermolysin (Therm). Sensitivity to digestion was evaluated for PHB (PHB3/4 antibody), AtPHB3-GFP (GFP antibody and PHB3/4 antibody; C), the inner envelope Tic110, the outer envelope SFR2 (D), the thylakoid-associated AtpB, and LHCII and the stromal cpHsp70 (C). Black arrowheads indicate AtPHB3-GFP, and the white arrowhead shows NbPHB (C). Similar results were obtained for two to four chloroplast preparations.

Figure 5.

phb3-3 mutant plants have lower SA levels and PR1 accumulation after UV-C treatment. A to C, Leaf tissue from plants grown on plates treated with UV-C light for 20 min and harvested at 2, 8, and 24 h post treatment (hpt). Untreated plants were used as controls (–). A, Levels of free SA (gray bars) and glycosylated SA (SAG; white bars). FW, Fresh weight; n/d, not detected. Each bar represents the mean of three independent experiments; error bars indicate se. Statistical analysis was performed using ANOVA/Fisher’s test. Different letters denote statistically significant differences at P < 0.05. B and C, PR1 protein was detected by immunoblot analysis using a PR1 antibody. A nonspecific band in the immunoblot shows similar loading in each lane. Gels show representative results from three independent experiments. D, Two-week-old wild-type (WT), phb3-3, and sid2-2 plants grown on plates were treated with liquid Murashige and Skoog (MS) growth medium alone (Control) or MS medium supplemented with 0.5 mm SA for 2, 8, and 24 h. – indicates untreated plants. PR1 protein was detected by immunoblot analysis as in B and C. An independent trial of this experiment showed similar results.

Figure 6.

phb3-3 mutant plants have reduced SA accumulation and PR1 protein levels after infection with Pst/AvrRpm1. Two-week-old plants grown on plates were flooded with Pst/AvrRpm1 bacteria at a concentration of 1 × 10⁶ colony-forming units (CFU) mL⁻¹. Samples were taken at 0, 12, 24, 48, and 72 h post infection (hpi). A, The levels of free SA (gray bars) and glycosylated SA (SAG; white bars) are shown; each bar represents the average of three independent experiments. Error bars indicate se. Statistical analysis was performed using ANOVA/Fisher’s test. Different letters denote statistically significant differences at P < 0.05 for free SA and P < 0.001 for SAG. B, The levels of PR1 protein were detected by immunoblot analysis using a PR1 antibody. The membrane was stained with Coomassie Blue to show similar protein loading. Similar results were seen in two additional experiments. FW, Fresh weight; WT, wild type.

Figure 7.

phb3-3 mutant plants are more susceptible to infection by Pst strains DC3000/AvrRpm1 and DC3000/AvrRpt2. A, Two-week-old plate-grown plants were inoculated by flooding the plants with Pst/AvrRpm1 at a concentration of 1 × 10⁵ CFU mL⁻¹. Bacteria were enumerated at 0, 2, and 3 d post inoculation (dpi). B, Three-week-old plants grown in soil were inoculated with Pst/AvrRpt2 by infiltration using a concentration of 1 × 10⁵ CFU mL⁻¹. Bacteria were counted at 0 and 3 dpi. For A and B, bars represent average values of 12 replicates; error bars indicate se. Statistical analysis was performed using ANOVA/Fisher’s test. Different letters denote statistically significant differences at P values as follows: A, 2 dpi (P < 0.0001) and 3 dpi (P ≤ 0.0005); B, 3 dpi (P < 0.0001). C and D, Cell death was evaluated using Trypan Blue staining in leaf tissue of 4-week-old plants grown in soil 10 h after infiltration with Pst/AvrRpm1 or Pst/AvrRpt2 (1 × 10⁸ CFU mL⁻¹). Cell death was scored as occurring if at least six blue-stained cells were visible in the area of tissue shown and if similar staining occurred throughout the inoculated leaf. Numbers indicate the total number of leaves showing cell death out of the total number scored in three independent experiments. Arrowheads indicate examples of the collapsed, Trypan Blue-stained cells. These experiments were repeated three times with similar results. WT, Wild type. Bars = 200 μm.

Figure 8.

Expression of PHB3 complements phb3-3 mutant phenotypes. A, Phenotypes of 3-week-old plants grown in soil. B, Immunodetection of the PHB3-V5 protein using V5 antibody in extracts from phb3-3 and complemented phb3-3/PHB3-V5 #15 and #17 2-week-old plants grown in soil. C, Leaf cross sections of wild-type (WT), phb3-3, and complemented phb3-3/PHB3-V5 #15 plants. Bar = 100 µm. D, Levels of free SA (gray bars) and glycosylated SA (SAG; white bars) in plants grown on plates, treated with UV-C light for 20 min, and harvested at 24 h post treatment (+). Untreated plants were used as controls (–). SA levels are expressed as μg g⁻¹ fresh weight (FW). Each bar represents the mean value of three independent experiments; error bars indicate se. Statistical analysis was performed using ANOVA/Fisher’s test. Different letters denote statistically significant differences at P < 0.02 for free SA and P < 0.04 for SAG. E, PR1 protein was detected by immunoblot using PR1 antibody in wild-type, phb3-3, and phb3-3/PHB3-V5 complemented line plants grown on plates, treated with UV-C light for 20 min, then harvested at 8 and 24 h post treatment (hpt). The gel shows a representative result from three independent experiments. F, Bacterial proliferation was quantified in 3-week-old plants grown in soil at 0 and 3 dpi with 1 × 10⁵ CFU mL⁻¹ Pst/AvrRpt2. Each bar represents the average of 12 replicates; error bars indicate se. Statistical analysis was performed using ANOVA/Fisher’s test. Different letters denote statistically significant differences at P < 0.05. This experiment was repeated three times with similar results. In B and E, membranes were stained with Coomassie Blue to show similar protein loading.

Figure 9.

PHB3 regulates ICS1 protein levels. A, ICS1 transcript levels measured by RT-qPCR in 2-week-old plants grown on plates and treated with UV-C light for 20 min. Whole seedlings were harvested at 2, 8, and 24 h post treatment (hpt). – indicates untreated plants. ICS1 transcript levels are relative (R.) to YLS8, which was used as an endogenous control. Bars show means of three biological replicates; error bars show se. Statistical analysis was performed using ANOVA/Fisher’s test. Different letters denote statistically significant differences at P < 0.05. B, ICS1 was detected by immunoblot analysis with ICS1 antibody in extracts of plants treated as in A. C, PHB3 does not affect other chloroplast protein levels. The chloroplast proteins Tic110, NOA1, AtpB, and LOX2 were detected by immunoblot analysis with specific antibodies. Representative data from three (B) or two (C) independent experiments are shown. Coomassie Blue (B) and Ponceau S (C) staining of proteins in the membranes show similar loading. The LOX2 immunoblot shows the same membrane as the ICS1 blot in B (bottom). WT, Wild type.