Salt stress-induced chloroplastic hydrogen peroxide stimulates pdTPI sulfenylation and methylglyoxal accumulation

Abstract

High salinity, an adverse environmental factor affecting about 20% of irrigated arable land worldwide, inhibits plant growth and development by causing oxidative stress, damaging cellular components, and disturbing global metabolism. However, whether and how reactive oxygen species disturb the metabolism of salt-stressed plants remain elusive. Here, we report that salt-induced hydrogen peroxide (H2O2) inhibits the activity of plastid triose phosphate isomerase (pdTPI) to promote methylglyoxal (MG) accumulation and stimulates the sulfenylation of pdTPI at cysteine 74. We also show that MG is a key factor limiting the plant growth, as a decrease in MG levels completely rescued the stunted growth and repressed salt stress tolerance of the pdtpi mutant. Furthermore, targeting CATALASE 2 into chloroplasts to prevent salt-induced overaccumulation of H2O2 conferred salt stress tolerance, revealing a role for chloroplastic H2O2 in salt-caused plant damage. In addition, we demonstrate that the H2O2-mediated accumulation of MG in turn induces H2O2 production, thus forming a regulatory loop that further inhibits the pdTPI activity in salt-stressed plants. Our findings, therefore, illustrate how salt stress induces MG production to inhibit the plant growth.

Figure 1

pdTPI confers plant salt stress tolerance. A, The sugar-phosphate metabolism pathway associated with TPI. FBP, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; G-3-P, glycerol-3- phosphate; MG, methylglyoxal; SLG, S-d-lactoylglutathione; GSH, glutathione; PEP, phosphoenolpyruvate; TPI, triose phosphate isomerase; GLY I/II, glyoxalase I/II. B, pdTPI activity of chloroplasts isolated from the leaves of 7-day-old wild-type, 35S:pdTPI, as-pdTPI, and pdtpi seedlings treated with no (Mock) or 100 mM NaCl (NaCl) for 12 h. C, MG contents of leaves from 7-day-old wild-type, 35S:pdTPI, as-pdTPI, and pdtpi seedlings treated with no or 100 mM NaCl for 12 h. D–F, Phenotypes (D), fresh weight (E), and chlorophyll contents (F) of 7-day-old wild-type, 35S:pdTPI, as-pdTPI, and pdtpi seedlings treated with no or 100 mM NaCl for 10 days. Scale bar, 1 cm. G–I, Phenotypes (G), fresh weight (H), and chlorophyll contents (I) of 10-day-old wild-type, 35S:pdTPI, and as-pdTPI plants treated with no or 200 mM NaCl for 2 weeks. Scale bar, 5 cm. FW, fresh weight. B, C, E, F, H, and I, Data are presented as means ± Sd of three independent biological replicates, and different letters indicate significant differences at P < 0.05 by two-way analysis of variance with Tukey's multiple comparison test.

Figure 2

pdTPI affects DHAP, GAP, and g-3-p levels in salt-stressed plants. A–C, DHAP (A), GAP (B), and G-3-P (C) contents of leaves from 7-day-old wild-type, 35S:pdTPI, as-pdTPI, and pdtpi seedlings treated with no (Mock) or 100 mM NaCl (NaCl) for 12 h. FW, fresh weight. Data are presented as means ± Sd of three independent biological replicates. Different letters indicate significant differences at P < 0.05 by two-way analysis of variance with Tukey's multiple comparison test.

Figure 3

MG is involved in pdTPI-mediated plant salt stress response. A, MG contents of leaves from 7-day-old wild-type, 35S:GLYI2 35S:GLYII4, pdtpi, and pdtpi 35S:GLYI2 35S:GLYII4 seedlings treated with no (Mock) or 100 mM NaCl (NaCl) for 12 h. B–D, Phenotypes (B), fresh weight (C), and chlorophyll contents (D) of 7-day-old wild-type, 35S:GLYI2 35S:GLYII4, pdtpi, and pdtpi 35S:GLYI2 35S:GLYII4 seedlings treated with no or 100 mM NaCl for 10 days. Scale bar, 1 cm. E–G, Phenotypes (E), fresh weight (F), and chlorophyll contents (G) of 10-day-old wild-type, 35S:GLYI2 35S:GLYII4, pdtpi, and pdtpi 35S:GLYI2 35S:GLYII4 plants treated with 0 or 200 mM NaCl for 2 weeks. Scale bar, 5 cm. FW, fresh weight. A, C, D, F, and G, Data are presented as means ± Sd of three independent biological replicates, and different letters indicate significant differences at P < 0.05 by two-way analysis of variance with Tukey's multiple comparison test.

Figure 4

H₂O₂ sulfenylates pdTPI and inhibits its activity. A, Immunodetection of pdTPI in leaves from 7-day-old wild-type seedlings treated with no (Mock), or 100 mM NaCl, or 10, 100, or 1000 μM H₂O₂ for 12 h, using the anti-pdTPI antibody. Anti-actin served as a loading control. B, Flowchart showing the procedure for the isolation of sulfenylated proteins using the biotin-switch method. C, Sulfenylation of pdTPI as assayed by immunoblotting after the protein was purified from E. coli and treated with 0, 10, 100, or 1000 μM H₂O₂ for 30 min. D, Activity of purified pdTPI treated with 0, 10, 100, or 1000 μM H₂O₂ for 30 min. E, Immunodetection of sulfenylated pdTPI in leaves from 7-day-old wild-type seedlings treated with 0, 10, 100, or 1000 μM H₂O₂ for 12 h, using the anti-pdTPI antibody. Anti-actin served as a loading control. F, pdTPI activity of chloroplasts isolated from leaves of 7-day-old wild-type seedlings treated with 0, 10, 100, or 1000 μM H₂O₂ for 12 h. G, MG contents of leaves from 7-day-old wild-type seedlings treated with 0, 10, 100, or 1000 μM H₂O₂ for 12 h. FW, fresh weight. A, C, and E, The intensity of each band was measured using an image-processing and analysis software package (ImageJ), and relative protein levels were normalized against those in untreated controls, which were set to 1. A, C, D–G, Data are presented as means ± Sd of three independent biological replicates, and different letters indicate significant differences at P < 0.05 by one-way analysis of variance with Tukey's multiple comparison test.

Figure 5

H₂O₂ induced by salt stress sulfenylates pdTPI and inhibits its activity. A, Degree of chl-roGFP2-Orp1 OxD in the leaves from 7-day-old 35S:chl-roGFP2-Orp1 seedlings treated with 10 μM MV or 10 μM ATZ for 12 h. Data are presented as means ± Sd; n = 10. B, Immunodetection of sulfenylated pdTPI in leaves from 7-day-old wild-type seedlings treated with 10 μM MV or 10 μM ATZ for 12 h, using the anti-pdTPI antibody. Anti-actin served as a loading control. C, pdTPI activity of chloroplasts isolated from leaves of 7-day-old wild-type seedlings treated with 10 μM MV or 10 μM ATZ for 12 h. D, MG contents of leaves from 7-day-old wild-type seedlings treated with 10 μM MV or 10 μM ATZ for 12 h. E, Degree of chl-roGFP2-Orp1 OxD in the leaves from 7-day-old 35S:chl-roGFP2-Orp1 and rbohD rbohF 35S:chl-roGFP2-Orp1 seedlings treated with no (Mock) or 100 mM NaCl (NaCl) for 12 h. Data are presented as means ± Sd; n = 10. F, Immunodetection of sulfenylated pdTPI in leaves from 7-day-old wild-type and rbohD rbohF seedlings treated with no or 100 mM NaCl for 12 h, using the anti-pdTPI antibody. Anti-actin served as a loading control. G, pdTPI activity of chloroplasts isolated from leaves of 7-day-old wild-type and rbohD rbohF seedlings treated with no or 100 mM NaCl for 12 h. H, MG contents of leaves from 7-day-old wild-type and rbohD rbohF seedlings treated with no or 100 mM NaCl for 12 h. FW, fresh weight. B and F, the intensity of each band was measured using ImageJ, and relative protein levels were normalized against those in untreated wild-type plants, which were set to 1. B–D and F–H, Data are presented as means ± Sd of three independent biological replicates. Different letters indicate significant differences at P < 0.05 by one-way analysis of variance (ANOVA) (A–D) or two-way ANOVA (E–H) with Tukey's multiple comparison test.

Figure 6

Chloroplast-localized CAT2 restricts pdTPI sulfenylation. A and B, Detection of CAT2 protein (A) and catalase activity (B) in chloroplasts isolated from leaves of 7-day-old wild-type and 35S:chl-CAT2 seedlings. Anti-cpHSC70 and anti-GAPC (cytosolic glyceraldehyde-3-phosphate dehydrogenase) served as loading controls. C, Degree of chl-roGFP2-Orp1 OxD in the leaves from 7-day-old 35S:chl-roGFP2-Orp1 and 35S:chl-CAT2 35S:chl-roGFP2-Orp1 seedlings treated with no (Mock) or 100 mM NaCl (NaCl) for 12 h. Data are presented as means ± Sd; n = 10. D, Immunodetection of sulfenylated pdTPI in leaves from 7-day-old wild-type and 35S:chl-CAT2 seedlings treated with no or 100 mM NaCl for 12 h, using the anti-pdTPI antibody. Anti-actin served as a loading control. A and D, The intensity of each band was measured using ImageJ, relative protein levels were normalized against those in untreated wild-type or 35S:chl-CAT2 #2 plants, which was set to 1. A, B, and D, Data are presented as means ± Sd of three independent biological replicates. Different letters indicate significant differences at P < 0.05 by one-way analysis of variance (ANOVA) (A and B) or two-way ANOVA (C and D) with Tukey's multiple comparison test.

Figure 7

Chloroplast-localized CAT2 confers plant salt stress tolerance. A–C, Phenotypes (A), fresh weight (B), and chlorophyll content (C) of 7-day-old wild-type, 35S:chl-CAT2, as-pdTPI, and 35S:chl-CAT2 as-pdTPI seedlings treated with no (Mock) or 100 mM NaCl (NaCl) for 10 days. Scale bar, 1 cm. D–F, Phenotypes (D), fresh weight (E), and chlorophyll content (F) of 10-day-old wild-type, 35S:chl-CAT2, as-pdTPI, and 35S:chl-CAT2 as-pdTPI seedlings treated with o or 200 mM NaCl for 2 weeks. Scale bar, 5 cm. G, pdTPI activity of chloroplasts isolated from leaves of 7-day-old wild-type, 35S:chl-CAT2, as-pdTPI, and 35S:chl-CAT2 as-pdTPI seedlings treated with no or 100 mM NaCl for 12 h. H, MG contents of leaves from 7-day-old wild-type, 35S:chl-CAT2, as-pdTPI, and 35S:chl-CAT2 as-pdTPI seedlings treated with no or 100 mM NaCl for 12 h. FW, fresh weight. B, C, and E–H, Data are presented as means ± Sd of three independent biological replicates, and different letters indicate significant differences at P < 0.05 by two-way analysis of variance with Tukey's multiple comparison test.

Figure 8

The Cys-74 residue of pdTPI is sulfenylated in vitro and in vivo. A, Activities of recombinant pdTPI, pdTPI-C74S, pdTPI-C148S, pdTPI-C186S, pdTPI-C203S, and dithiothreitol (DTT)-pretreated pdTPI treated with no or 100 μM H₂O₂ for 30 min. B and C, Sulfenylation of recombinant pdTPI, pdTPI-C74S, pdTPI-C148S, pdTPI-C186S, and pdTPI-C203S as assayed by immunoblotting after a treatment with no or 10, 100, or 1000 μM H₂O₂ for 30 min. The intensity of each band was measured using ImageJ, and relative protein levels were normalized against those in untreated controls, which were set to 1. D, Identification of dimedone-labeled Cys-74 of recombinant pdTPI treated with 100 μM H₂O₂ for 30 min. E, Number of reduced and dimedone-labeled Cys residues of recombinant pdTPI treated with 100 μM H₂O₂ for 30 min. Data shown are the results of two biological experiments. F, Identification of dimedone-labeled Cys-74 of pdTPI purified from wild-type seedlings treated with 100 μM H₂O₂ for 12 h. G, Number of reduced and dimedone-labeled Cys residues of pdTPI purified from wild-type seedlings treated with 100 μM H₂O₂ for 12 h. Data shown are the results of four biological experiments. A and C, Data are presented as means ± Sd of three independent biological replicates. Asterisks indicate a significant difference from the mock-treated proteins (Student's t-test): **P < 0.01; ***P < 0.001. ns, no significance. Different letters indicate significant differences at P < 0.05 by one-way analysis of variance with Tukey's multiple comparison test.

Figure 9

Cys-74 is the pdTPI sulfenylation target in salt-stressed plants. A, pdTPI activity in chloroplasts isolated from leaves of 7-day-old wild-type, pdtpi 35S:pdTPI-C74S, pdtpi pdTPIpro:pdTPI, and pdtpi seedlings treated with no (Mock) or 100 mM NaCl (NaCl) for 12 h. B–D, Phenotypes (B), fresh weight (C), and chlorophyll contents (D) of 7-day-old wild-type, pdtpi 35S:pdTPI-C74S, pdtpi pdTPIpro:pdTPI, and pdtpi seedlings treated with no or 100 mM NaCl for 12 h. FW, fresh weight. Scale bar, 1 cm. E, Immunodetection of pdTPI protein and sulfenylated pdTPI in leaves from 7-day-old wild-type, pdtpi 35S:pdTPI-C74S #12, and pdtpi pdTPIpro:pdTPI #7 seedlings treated with no or 100 mM NaCl for 12 h, using the anti-pdTPI antibody. Anti-actin served as a loading control. A, C, and D, The percentages indicate pdTPI activity, fresh weight, and chlorophyll contents of the NaCl-treated seedlings relative to their levels in the untreated seedlings. A and C–E, Data are presented as means ± Sd of three independent biological replicates, and different letters indicate significant differences at P < 0.05 by two-way analysis of variance with Tukey's multiple comparison test.

Figure 10

MG promotes H₂O₂ accumulation. A and B, Representative images (A) and relative DAB staining (B) of 7-day-old wild-type seedlings treated with no, or 50, 100, or 200 μM MG for 12 h. C, Degree of chl-roGFP2-Orp1 OxD in the leaves from 7-day-old 35S:chl-roGFP2-Orp1 seedlings treated with no, 50, 100, or 200 μM MG for 12 h. Data are presented as means ± Sd; n = 10. D, pdTPI activity of chloroplasts isolated from leaves of 7-day-old wild-type seedlings treated with no, 50, 100, or 200 μM MG for 12 h. E and F, Representative images (E) and relative DAB staining (F) of 7-day-old wild-type, 35S:GLYI2 35S:GLYII4, and glyI2 seedlings treated with no or 100 mM NaCl for 12 h. G, Degree of chl-roGFP2-Orp1 OxD in the leaves from 7-day-old 35S:chl-roGFP2-Orp1, 35S:chl-roGFP2-Orp1 35S:GLYI2 35S:GLYII4, and glyI2 35S:chl-roGFP2-Orp1 seedlings treated with no or 100 mM NaCl for 12 h. Data are presented as means ± Sd; n = 10. H, pdTPI activity of chloroplasts isolated from leaves of 7-day-old wild-type, 35S:GLYI2 35S:GLYII4, and glyI2 seedlings treated with no or 100 mM NaCl for 12 h. Scale bars, 0.1 cm. B, D, F, and H, Data are presented as means ± Sd of three independent biological replicates. Different letters indicate significant differences at P < 0.05 by one-way analysis of variance (ANOVA) (B–D) or two-way ANOVA (F–H) with Tukey's multiple comparison test.