Salicylic Acid-Dependent Plant Stress Signaling via Mitochondrial Succinate Dehydrogenase

Abstract

Mitochondria are known for their role in ATP production and generation of reactive oxygen species, but little is known about the mechanism of their early involvement in plant stress signaling. The role of mitochondrial succinate dehydrogenase (SDH) in salicylic acid (SA) signaling was analyzed using two mutants: disrupted in stress response1 (dsr1), which is a point mutation in SDH1 identified in a loss of SA signaling screen, and a knockdown mutant (sdhaf2) for SDH assembly factor 2 that is required for FAD insertion into SDH1. Both mutants showed strongly decreased SA-inducible stress promoter responses and low SDH maximum capacity compared to wild type, while dsr1 also showed low succinate affinity, low catalytic efficiency, and increased resistance to SDH competitive inhibitors. The SA-induced promoter responses could be partially rescued in sdhaf2, but not in dsr1, by supplementing the plant growth media with succinate. Kinetic characterization showed that low concentrations of either SA or ubiquinone binding site inhibitors increased SDH activity and induced mitochondrial H₂O₂ production. Both dsr1 and sdhaf2 showed lower rates of SA-dependent H₂O₂ production in vitro in line with their low SA-dependent stress signaling responses in vivo. This provides quantitative and kinetic evidence that SA acts at or near the ubiquinone binding site of SDH to stimulate activity and contributes to plant stress signaling by increased rates of mitochondrial H₂O₂ production, leading to part of the SA-dependent transcriptional response in plant cells.

Figure 1.

GSTF8:luc induction in sdhaf2 and dsr1 after SA treatment compared to wild type. Average of total fluorescence signal generated by each seedling (n = 10) per hour after treatment of 7 mm SA in the presence of 0 mm (A), 5 mm (B), and 10 mm (C) malonate (mal) in the growth media. Two-factor ANOVA between genotypes (P ≤ 0.01), posthoc Tukey test comparing signal induction to time point zero within genotype *P ≤ 0.05; **P ≤ 0.01.

Figure 2.

Lower succinate affinity and catalytic efficiency in dsr1. Concentrations of 0.1 to 10 mm of succinate were used to calculate maximal SDH activity, measured as absorbance change of DCPIP at 600 nm. K_m was calculated using Hanes-Plot and Brook Kinetics Software. A, Scheme of SDH showing electron transfer from succinate to UQ binding site. B, Correlation of SDH activity and succinate concentrations of the wild type, sdhaf2, and dsr1. C, Maximal enzyme velocity (V_max). D, Calculated K_m of succinate using Brooks kinetic software. E, enzymatic efficiency (V_max/K_m) for sdhaf2 and dsr1. se of six biological replicates. Two-factor ANOVA comparing SDH activity between genotypes (B) P ≤ 0.01 (dsr1 compared to the wild type and sdhaf2). Single-factor ANOVA comparing catalytic efficiency and succinate affinity (D and E) between genotypes. Different letters indicate significant differences (P ≤ 0.05) between genotypes. n.d., Not detected.

Figure 3.

IC₅₀ of SDH competitive inhibitors malonate and oxaloacetate are higher in dsr1. Inhibition of SDH was measured using increasing amounts of malonate and OAA together with the K_m concentration of succinate (0.5 mm for wild type and sdhaf2; 1 mm for dsr1). IC₅₀ was calculated using Brooks Kinetic Software. A, Percentage inhibition of SDH activity in the presence of malonate and OAA. B, Calculated IC₅₀ of malonate (left) and OAA (right). se of four biological replicates. Single-factor ANOVA comparing IC₅₀ between genotypes. Different letters indicate significant differences, P ≤ 0.07.

Figure 4.

SA-induced GSTF8 signal can be rescued in sdhaf2 using high concentrations of succinate. Average of total fluorescence signal generated by each seedling (n = 10) per hour after treatment of 7 mm SA in the presence of 0 (top) and 20 mm succinate (succ, bottom) in the growth media. Error bars: se, posthoc Tukey test comparing signal induction to time point zero within genotype, *P ≤ 0.05, **P ≤ 0.01.

Figure 5.

Low concentrations of SA increase SQR activity. A, SDH activity measured at the succinate binding site (PMS + DCPIP) in the presence of SA. B, SQR activity measured at UQ binding site (Q₁ [80 µm] + DCPIP) in the presence of SA. As a negative control, activity was measured in the absence of Q₁ in wild-type mitochondria (yellow bars). In both cases, SDH activity was measured in µmol DCPIP/min/mg Mit. in the presence of 5 mm succinate and SA concentrations ranging from 0.01 to 0.05 mm. C and D, Succinate-dependent oxygen consumption was measured using a Clark type oxygen electrode in the presence of 5 mm succinate and SA concentrations ranging from 0.01 to 1 mm. Fisher’s lsd test was used to determine differences (different letters indicate significant differences; for P values and letter distribution, see Supplemental Table S1 and Supplemental Fig. S3); P ≤ 0.05.

Figure 6.

mtH₂O₂ production is lower in dsr1 and sdhaf2. mtH₂O₂ production was measured using DCFDA with excitation/emission wavelengths of 490/520 nm. Succinate (5 mm), 0.5 mm ATP, 5 µm AA, and 0.03 mm SA were added to freshly isolated mitochondria immediately before the measurement. Fluorescence intensity was measured over 10 min and the rate of fluorescence/min was calculated. se of eight biological replicates. Wilcoxon signed rank test between genotypes, different letters indicate significant differences, P ≤ 0.05.