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H2O2-triggered retrograde signaling from chloroplasts to nucleus plays specific role in response to stress - PubMed

  • ️Sun Jan 01 2012

H2O2-triggered retrograde signaling from chloroplasts to nucleus plays specific role in response to stress

Takanori Maruta et al. J Biol Chem. 2012.

Abstract

Recent findings have suggested that reactive oxygen species (ROS) are important signaling molecules for regulating plant responses to abiotic and biotic stress and that there exist source- and kind-specific pathways for ROS signaling. In plant cells, a major source of ROS is chloroplasts, in which thylakoid membrane-bound ascorbate peroxidase (tAPX) plays a role in the regulation of H(2)O(2) levels. Here, to clarify the signaling function of H(2)O(2) derived from the chloroplast, we created a conditional system for producing H(2)O(2) in the organelle by chemical-dependent tAPX silencing using estrogen-inducible RNAi. When the expression of tAPX was silenced in leaves, levels of oxidized protein in chloroplasts increased in the absence of stress. Microarray analysis revealed that tAPX silencing affects the expression of a large set of genes, some of which are involved in the response to chilling and pathogens. In response to tAPX silencing, the transcript levels of C-repeat/DRE binding factor (CBF1), a central regulator for cold acclimation, was suppressed, resulting in a high sensitivity of tAPX-silenced plants to cold. Furthermore, tAPX silencing enhanced the levels of salicylic acid (SA) and the response to SA. Interestingly, we found that tAPX silencing-responsive genes were up- or down-regulated by high light (HL) and that tAPX silencing had a negative effect on expression of ROS-responsive genes under HL, suggesting synergistic and antagonistic roles of chloroplastic H(2)O(2) in HL response. These findings provide a new insight into the role of H(2)O(2)-triggered retrograde signaling from chloroplasts in the response to stress in planta.

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Figures

FIGURE 1.
FIGURE 1.

Estrogen-dependent silencing of tAPX in the transgenic plants. Seventeen-day-old IS-GUS-2-17 and IS-tAPX-19-23 plants, grown under NL, were sprayed with a 100 μ

m

estrogen. A, shown is the transcript levels of APX genes. Semiquantitative RT-PCR was performed using specific primers for APX genes and Actin8 on total RNA from transgenic plants. PCR amplification was performed with 18–28 cycles of 95 °C for 60 s, 55 °C for 60 s, and 72 °C for 60 s. Aliquots of the products were analyzed on 2% agarose gel. B, shown are Western blots of tAPX, sAPX, and APX1 proteins using anti-tAPX antibody. C, activities of soluble and insoluble APXs in crude extracts of Arabidopsis leaves are shown. Seventeen-day-old wild-type and KO-tAPX plants were also used in this study. Error bars indicate S.D. (n = 3). Values without a common letter are significantly different according to t tests (p < 0.05).

FIGURE 2.
FIGURE 2.

Effect of tAPX silencing on cellular redox state and photosynthesis under NL. Seventeen-day-old IS-GUS-2-17 and IS-tAPX-19-23 plants were sprayed with a 100 μ

m

estrogen and kept under NL for indicated period. A, H2O2 was detected by DAB staining. A detached leaf at 48 h after the estrogen treatment was vacuum-infiltrated with DAB solution and incubated under NL for 3 h. The leaf was then decolorized by incubation in 70% ethanol. The same results were obtained in five independent experiments. Results of representative leaves were photographed. B, total proteins were extracted from leaves of IS-GUS-2-17 and IS-tAPX-19-23 plants. Oxidized proteins were detected using a protein gel blot assay with an OxyBlot protein oxidation kit as described under “Experimental Procedures.” C and D, levels of AsA, dehydroascorbate (DHA), GSH, and GSSG were measured. White bars (showing dehydroascorbate and GSSG) are started on top of gray bars (showing AsA and GSH). E and F, quantum yields of photosystem II, Fv/Fm and ΦPSII, were also measured using a Closed FluorCam 800MF as described under “Experimental Procedures.”

FIGURE 3.
FIGURE 3.

Protein oxidation in chloroplasts isolated from tAPX-silenced leaves. Seventeen-day-old IS-GUS-2-17 and IS-tAPX-19-23 plants were sprayed with a 100 μ

m

estrogen and kept under NL. At 48 h after estrogen treatment, the chloroplasts were isolated from leaves of IS-GUS-2-17 and IS-tAPX-19-23 plants. A, oxidized proteins in the crude extracts and chloroplasts were detected by Western blotting. The same results were obtained in five independent experiments. Two representative results are shown as Exp. 1 and 2. CBB, Coomassie Brilliant Blue. B, shown are Western blots of tAPX, sAPX, and APX1 for Exp. 1. The blot of APX1 indicated that there was no contamination of the cytosolic fraction in the chloroplasts. In the figure, IS-GUS-2-17 and IS-tAPX-19-23 plants are shown as IS-GUS and IS-tAPX, respectively.

FIGURE 4.
FIGURE 4.

Effects of treatment with AsA or dark on the induction of RTS genes by tAPX-silencing. A, seventeen-day-old IS-GUS-2-17 and IS-tAPX-19-23 plants were sprayed with a 100 μ

m

estrogen and kept under NL or dark. At 45 h after the treatment with estrogen, the leaves of IS-GUS-2-17 and IS-tAPX-19-23 plants were sprayed with 10 m

m

AsA or water and kept under NL. The transcript levels of five RTS genes (GRFP, JLFP, ULT2, CYP72A14, and PK) were measured by q-PCR as described under “Experimental Procedures.” Error bars indicate S.D. (n = 3). Values without a common letter are significantly different (p < 0.05). B, transcript levels of five RTS genes (described in Fig. 4A) in the wild-type and KO-tAPX plants, grown under light for 17 days, were measured by q-PCR. Error bars indicate S.D. (n = 3). Significant differences: *, p < 0.05 versus the value for wild-type plants.

FIGURE 5.
FIGURE 5.

Effect of tAPX silencing on cold acclimation. A, 17-day-old IS-GUS-2-17 and IS-tAPX-19-23 plants were sprayed with a 100 μ

m

estrogen and kept under NL. At 48 h after the estrogen treatment, the transcript levels of RTS genes (CBF1/DREB1B, CBF2/DREB1C, COR6.6, COR15B, COR414-TM1, and COR414-TM2), known to be involved in cold acclimation, were measured by q-PCR. Error bars indicate S.D. (n = 3). Significant differences: *, p < 0.05 versus the value for IS-GUS-2-17 plants. B and C, 17-day-old IS-GUS-2-17 and IS-tAPX-19-23 plants were sprayed with a 100 μ

m

estrogen solution or water (Mock) and transferred to cold stress conditions (100 μmol photons m−2 s−1, 4 °C) for 2 weeks. The treatment with estrogen was performed every 3 days to maintain the tAPX silencing. B, 14 days after cold stress, the IS-GUS-2-17 and IS-tAPX-19-23 plants were photographed. The same results were obtained in four independent experiments. C, Fv/Fm values in the leaves of IS-GUS-2-17 and IS-tAPX-19-23 10 days after cold stress were measured using a Closed FluorCam 800MF. Error bars indicate S.D. (n = 3). Significant differences: *, p < 0.05 versus the value of IS-GUS-2-17 plants.

FIGURE 6.
FIGURE 6.

Effect of tAPX silencing on the transcript levels of disease-resistance genes. Seventeen-day-old IS-GUS-2-17 and IS-tAPX-19-23 plants were sprayed with a 100 μ

m

estrogen and kept under NL. At 48 h after the estrogen treatment, the transcript levels of RTS genes (ICS2, TolB, TIR, RLP7, RLP23, RLP34, RLP39, RLP41, NIMIN3, NUDX6, LCR68, LCR70), PR1, and PR2, known to be involved in disease resistance, were measured by q-PCR. Error bars indicate S.D. (n = 3). Significant differences: *, p < 0.05 versus the value for IS-GUS-2-17 plants. TIR, Toll-interleukin resistance.

FIGURE 7.
FIGURE 7.

Effect of tAPX silencing on the response to SA. Seventeen-day-old IS-GUS-2-17 and IS-tAPX-19-23 plants were sprayed with a 100 μ

m

estrogen and kept under NL for 48 h. A, levels of free and total SA in the IS-GUS-2-17 and IS-tAPX-19-23 plants before and after estrogen treatment were measured as described under “Experimental Procedures.” B, 48 h after treatment with estrogen, IS-GUS-2-17 and IS-tAPX-19-23 plants were sprayed with a 100 μ

m

SA. The transcript levels of PR1 and PR2, SA-responsive genes, were measured by q-PCR. Error bars indicate S.D. (n = 3). Significant differences: *, p < 0.05 versus the value for IS-GUS-2-17 plants.

FIGURE 8.
FIGURE 8.

Effect of HL on the expression of RTS genes. Nineteen-day-old wild-type plants were exposed to HL (1000 μmol of photons m−2 s−1). The transcript levels of RTS genes (GRFP, JLFP, ULT2, CYP72A14, and PK) were measured by q-PCR. Error bars indicate S.D. (n = 3). Values without a common letter are significantly different according to t tests (p < 0.05).

FIGURE 9.
FIGURE 9.

Effect of tAPX silencing on the expression of RTS genes under ML. One-week-old IS-GUS-2-17 and IS-tAPX-19-23 plants grown under NL were further grown for 10 days under ML (400 μmol of photons m−2 s−1). The plants were then sprayed with estrogen and kept under ML for 72 h. A, 48 h after estrogen treatment the IS-GUS-2-17 and IS-tAPX-19-23 plants were photographed. B, Fv/Fm values in the leaves of IS-GUS-2-17 and IS-tAPX-19-23 after estrogen treatment were measured using a Closed FluorCam 800MF. Error bars indicate S.D. (n = 3). C, 48 h after estrogen treatment, the transcript levels of RTS genes (GRFP, JLFP, ULT2, CYP72A14, and PK) were measured by q-PCR. Error bars indicate S.D. (n = 3). Error bars indicate S.D. (n = 3). Significant differences: *, p < 0.05 versus the value for IS-GUS-2-17 plants.

FIGURE 10.
FIGURE 10.

Effect of tAPX silencing on expression of ROS-responsive genes under HL. Seventeen-day-old IS-GUS-2-17 and IS-tAPX-19-23 plants were sprayed with a 100 μ

m

estrogen and kept under NL. At 48 h after treatment with estrogen, IS-GUS-2-17 and IS-tAPX-19-23 plants were exposed to HL (1000 μmol photons m−2 s−1). The transcript levels of ROS-responsive genes (HsfA2, APX2, and HSP18.1-C1) were measured by q-PCR. Error bars indicate S.D. (n = 3). Significant differences: *, p < 0.05 versus the value for IS-GUS-2-17 plants.

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