GSNOR | S-nitrosoglutathione reductase

AtCaM4 binds to GSNOR in response to salt stress.(A) In planta BiFC assay of the CaM4 interaction with GSNOR in tobacco leaves. The C-terminal half of YFP was fused to CAM4 (CaM4-YC) and GSNOR (GSNOR-YC), and the N-terminal half of YFP was fused to GSNOR (GSNOR-YN) and CAM4 (CaM4-YN). The expression of CaM4-YC/YN and CaM4-YN/YC was used as a control. Bar = 50 ?m. (B) Model of the GSNOR N- and C-terminal deletions for the overlay assay. (C) In vitro overlay assay of the CaM4 interaction with GSNOR. GST and GST-GSNOR fusion proteins were expressed in E. coli. Recombinant proteins were analyzed by Western blotting (WB) using anti-GST antibodies. The overlay assay was performed using CaM4-His in the presence of 0.1 mM CaCl2 or 0.1 mM EGTA. (D) Co-IP assays showing the interaction between CaM4 and GSNOR. CaM4-GFP was expressed from the native CaM4 promoter. Extracted proteins were incubated with anti-GFP agarose beads. Total and immunoprecipitated proteins were analyzed by immunoblotting using anti-GSNOR and -GFP antibodies.

AtCaM4 decreases GSNOR activity in response to salt stress.(A, E) GSNOR activity of total protein (A) or purified protein (E) from 7-day-old wild-type, cam1-1, cam1-2, cam4, cam1/4-1, cam1/4-2, 4COM1 and 4COM2 seedlings grown in 0.5 × MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to 0 mM NaCl (Student’s t-test, *P < 0.05 and **P < 0.01). (B) Immunoblot analysis of GSNOR expression in the seedlings. Tubulin was used as an internal control. The experiments were repeated three times; the results indicate similar trends in protein accumulation. (C) SNO content in the seedlings grown in 0.5× MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). The numbers at the top indicate the fold change in SNO level after the salt challenge. (D) GSNOR activity in 7-day-old cam1-1, cam1-2, cam4, cam1/4-1, and cam1/4-2 seedling after addition of 0.1 mM EGTA, 0.02 or 0.04 mM CaM4-GST in the reaction mixture. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to 100 mM NaCl (Student’s t-test, *P < 0.05). (F) Immunoblot analysis of purified GSNOR from total protein with anti-GSNOR. gsnor seedlings was used as a negative control. The experiments were repeated three times; the results indicate similar trends in protein accumulation.

AtCaM4 decreases GSNOR activity in response to salt stress.(A, E) GSNOR activity of total protein (A) or purified protein (E) from 7-day-old wild-type, cam1-1, cam1-2, cam4, cam1/4-1, cam1/4-2, 4COM1 and 4COM2 seedlings grown in 0.5 × MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to 0 mM NaCl (Student’s t-test, *P < 0.05 and **P < 0.01). (B) Immunoblot analysis of GSNOR expression in the seedlings. Tubulin was used as an internal control. The experiments were repeated three times; the results indicate similar trends in protein accumulation. (C) SNO content in the seedlings grown in 0.5× MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). The numbers at the top indicate the fold change in SNO level after the salt challenge. (D) GSNOR activity in 7-day-old cam1-1, cam1-2, cam4, cam1/4-1, and cam1/4-2 seedling after addition of 0.1 mM EGTA, 0.02 or 0.04 mM CaM4-GST in the reaction mixture. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to 100 mM NaCl (Student’s t-test, *P < 0.05). (F) Immunoblot analysis of purified GSNOR from total protein with anti-GSNOR. gsnor seedlings was used as a negative control. The experiments were repeated three times; the results indicate similar trends in protein accumulation.

GSNOR acts as a negative regulator of salt tolerance.(A) RT-qPCR and immunoblot analyses of GSNOR expression in wild-type, gsnor, 2COM1, 2COM2, 2OE1 and 2OE2 plants. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05, **P < 0.01, and ***P < 0.001). (B) NO accumulation in the roots of 7-day-old seedlings. The experiments were repeated three times with similar results. Bar = 50 ?m. (C) Relative DCF fluorescence densities in the roots. Each data point represents the mean ± SE (n = 20). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05 and **P < 0.01). (D) Salt stress sensitivity of the seedlings in 0.5× MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. (E) Survival ratios of the seedlings after salt treatment. Each data point represents the mean ± SE (n = 30). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05 and **P < 0.01). (F) Root lengths of the seedlings with or without 100 mM NaCl. Each data point represents the mean ± SE (n = 30). The numbers at the top indicate the fold change in root length after the salt challenge.

GSNOR alleviates Fe-dependent NO and H2O2 toxicities to promote root meristem growth. a Box plots for root growth upon high Fe and nitric oxide (NO) donor (sodium nitroprusside, SNP) at day 7 (n?=?16 biologically independent samples). Root length of hot5-2 relative to WT is shown in the right panel. b Fluorescence signal of root NO stained with DAF-FM at day 6. White arrows denote cortex cells in meristems. c Fluorescence signal of root expressing pGSNOR:GSNOR-GFP at day 6 under control conditions. d Root meristems stained by propidium iodide (PI) at day 6. e Box plots for root meristem of experiments depicted in (d) (n?=?10 biologically independent samples). f Root meristems stained by PI upon H2O2 and Fe treatments at 7 days. White arrows indicate the border of elongation zone, while region outlined in white line and asterisk indicates cell death in the root meristem (d, f). g Box plots for root meristem length of experiments depicted in (f) (n?=?9 biologically independent samples). Different letters indicate the significant differences according to Tukey’s HSD test (p?

GSNOR alleviates Fe-dependent NO and H2O2 toxicities to promote root meristem growth. a Box plots for root growth upon high Fe and nitric oxide (NO) donor (sodium nitroprusside, SNP) at day 7 (n?=?16 biologically independent samples). Root length of hot5-2 relative to WT is shown in the right panel. b Fluorescence signal of root NO stained with DAF-FM at day 6. White arrows denote cortex cells in meristems. c Fluorescence signal of root expressing pGSNOR:GSNOR-GFP at day 6 under control conditions. d Root meristems stained by propidium iodide (PI) at day 6. e Box plots for root meristem of experiments depicted in (d) (n?=?10 biologically independent samples). f Root meristems stained by PI upon H2O2 and Fe treatments at 7 days. White arrows indicate the border of elongation zone, while region outlined in white line and asterisk indicates cell death in the root meristem (d, f). g Box plots for root meristem length of experiments depicted in (f) (n?=?9 biologically independent samples). Different letters indicate the significant differences according to Tukey’s HSD test (p?

GSNOR alleviates Fe-dependent NO and H2O2 toxicities to promote root meristem growth. a Box plots for root growth upon high Fe and nitric oxide (NO) donor (sodium nitroprusside, SNP) at day 7 (n?=?16 biologically independent samples). Root length of hot5-2 relative to WT is shown in the right panel. b Fluorescence signal of root NO stained with DAF-FM at day 6. White arrows denote cortex cells in meristems. c Fluorescence signal of root expressing pGSNOR:GSNOR-GFP at day 6 under control conditions. d Root meristems stained by propidium iodide (PI) at day 6. e Box plots for root meristem of experiments depicted in (d) (n?=?10 biologically independent samples). f Root meristems stained by PI upon H2O2 and Fe treatments at 7 days. White arrows indicate the border of elongation zone, while region outlined in white line and asterisk indicates cell death in the root meristem (d, f). g Box plots for root meristem length of experiments depicted in (f) (n?=?9 biologically independent samples). Different letters indicate the significant differences according to Tukey’s HSD test (p?