A chloroplast light-regulated oxidative sensor for moderate light intensity in Arabidopsis

Abstract

The transition from dark to light involves marked changes in the redox reactions of photosynthetic electron transport and in chloroplast stromal enzyme activity even under mild light and growth conditions. Thus, it is not surprising that redox regulation is used to dynamically adjust and coordinate the stromal and thylakoid compartments. While oxidation of regulatory proteins is necessary for the regulation, the identity and the mechanism of action of the oxidizing pathway are still unresolved. Here, we studied the oxidation of a thylakoid-associated atypical thioredoxin-type protein, ACHT1, in the Arabidopsis thaliana chloroplast. We found that after a brief period of net reduction in plants illuminated with moderate light intensity, a significant oxidation reaction of ACHT1 arises and counterbalances its reduction. Interestingly, ACHT1 oxidation is driven by 2-Cys peroxiredoxin (Prx), which in turn eliminates peroxides. The ACHT1 and 2-Cys Prx reaction characteristics in plants further indicated that ACHT1 oxidation is linked with changes in the photosynthetic production of peroxides. Our findings that plants with altered redox poise of the ACHT1 and 2-Cys Prx pathway show higher nonphotochemical quenching and lower photosynthetic electron transport infer a feedback regulatory role for this pathway.

Figure 1.

Reaction Scheme of the Two-Step Dithiol Disulfide Transfer Reaction of ACHT1. (A) Initially, the nucleophilic N-terminal Cys of the ACHT1 active site (s⁻) attacks the disulfide bond of its redox protein partner (RP), resulting in an intermolecular-disulfide reaction intermediate between ACHT1 and RP. Next, an attack of the C-terminal Cys of the ACHT1 active site on the intermolecular-disulfide bond releases oxidized ACHT1 and reduced RP. (B) In a redox-active site mutant of ACHT1, in which the C-terminal Cys is replaced with a Ser, the resolving step is inhibited, resulting in increased stabilization of the intermolecular disulfide intermediate. [See online article for color version of this figure.]

Figure 2.

ACHT1 Reoxidizes in Plants after Illumination. (A) The two Cys residues of the ACHT1 active site engage in vivo in a disulfide exchange reaction. Immunoblot assay showing the in vivo redox state of ACHT1 Cys residues captured in plants expressing either the catalytic active ACHT1 (dithiol) or the ACHT1_C35S mutant (monothiol). The number of oxidized Cys residues was determined by the increased molecular mass of ACHT1 after labeling with mPEG (indicated by arrows). Two Cys residues were oxidized in the catalytic active ACHT1 and one in ACHT1_C35S, indicating that the two Cys residues of the ACHT1 active site engage in vivo in disulfide exchange reactions (schematically depicted on the left). NEM treatment blocked reduced thiols. DTT reduced disulfides prior to mPEG labeling. (B) ACHT1 reoxidizes in plants shortly after illumination. Immunoblot assay showing the in vivo oxidized state of ACHT1 active-site Cys residues captured in plants at the end of the night and at 1 min (m), 5 min, 30 min, 1 h, and 2 h after illumination. Equal loading was verified by the stained level of ribulose-1,5-bis-phosphate carboxylase/oxygenase (RBCL). The results shown are representative of five independent experiments. (C) ACHT1 amount remains constant throughout the day. Immunoblot assay showing the total amount of ACHT1 in the plants at the indicated time points. Equal loading was verified by the stained level of ribulose-1,5-bis-phosphate carboxylase/oxygenase. [See online article for color version of this figure.]

Figure 3.

ACHT1 Reduction Requires Photosynthetic Reducing Equivalents. Immunoblot assay showing the in vivo oxidized state of ACHT1 active site Cys residues captured in plants that were left in the dark (A) or illuminated but treated with 50 μM of the photosynthetic transport inhibitor DCMU (B). Equal protein loading was verified as in Figure 2. The results shown are representative of three independent experiments.

Figure 4.

ACHT1 Reacts with 2-Cys Prx in Plants. (A) Intermolecular disulfide complexes of ACHT1 and its redox protein partners. Immunoblot assay showing the intermolecular disulfide complexes trapped in plants expressing the catalytic active ACHT1 (DT) or the monothiol mutant ACHT1_C35S (MT). Proteins were electrophoresed under nonreducing (NR) or reducing (R) conditions, and complexes containing ACHT1 were identified by immunoblot assays using anti-HA-specific antibodies. The major intermolecular disulfide complexes of ACHT1, which were identified by MS, are marked with an asterisk. (B) Reciprocal immunoprecipitation verified the ACHT1–2-Cys Prx interaction. Immunoblot assay of proteins immunoprecipitated with anti-HA (ACHT1 IP) or anti-2-Cys Prx (Prx IP) affinity matrixes or with nonspecific matrix (Control IP) from plants expressing ACHT1_C35S. Purified proteins were run under reducing conditions and blotted with antibodies specific to the HA-tag of ACHT1 (αHA) or to 2-Cys Prx (αPrx).

Figure 5.

2-Cys Prx Is Reduced by ACHT1 in Planta. Immunoblot assay showing the oxidized state of 2-Cys Prx Cys residues in plants after 5 min of illumination. The number of oxidized Cys residues (ox-Cys) was determined by the increased molecular mass of 2-Cys Prx after labeling with mPEG (indicated by arrows) as detected by 2-Cys Prx-specific sera. The assay showed that the 2-Cys Prx pool was more reduced in plants with increased expression of ACHT1 (OE) relative to wild-type plants (WT). Equal protein loading was verified as in Figure 2. The results shown are representative of three independent experiments. RBCL, ribulose-1,5-bis-phosphate carboxylase/oxygenase.

Figure 6.

Characteristics of the ACHT1-2-Cys Prx Reaction in Plants. (A) Reaction scheme of 2-Cys Prx reduction by ACHT1. Initially, the N-terminal Cys of the ACHT1 active site (s⁻) attacks the disulfide bridge linking the two monomers in a 2-Cys Prx dimer, resulting in an intermolecular disulfide reaction intermediate between ACHT1 and 2-Cys Prx. The rate constants of the forward and reverse reactions in this step are K1 and K-1, respectively. Next, an attack of the second Cys of the ACHT1 active site on the intermolecular disulfide bond releases oxidized ACHT1 and a reduced 2-Cys Prx. The rate constants of the forward and reverse reactions for this step are K2 and K-2, respectively. It is generally assumed that K2 is higher than K1 and that K-2 is much smaller than K2, favoring the forward reaction. If K-1 is smaller than K-2, then when the rates of the reverse reactions are high (i.e., when oxidized ACHT1 and reduced 2-Cys Prx concentrations are high), the intermediate will accumulate. (B) 2-Cys Prx redox state after illumination. Immunoblot assay showing the oxidized state of 2-Cys Prx Cys residues in wild-type plants sampled at the indicated time points. The number of oxidized Cys residues (ox-Cys) was determined by the increased molecular mass of 2-Cys Prx after labeling with mPEG (indicated by numbers and arrows), as detected by 2-Cys Prx-specific sera. The assay showed that, in contrast with ACHT1, the pool of 2-Cys Prx did not go through major redox state changes during illumination of dark-adapted plants. Equal protein loading was verified as in Figure 2. The results shown are representative of four independent experiments. RBCL, ribulose-1,5-bis-phosphate carboxylase/oxygenase (C) ACHT1-2-Cys Prx intermolecular disulfide complexes accumulate in plants when the ACHT1 pool oxidizes. Immunoblot assay showing the intermolecular disulfide complexes trapped in plants expressing the catalytic active ACHT1. Protein complexes were separated under nonreducing conditions and blotted with antibodies specific to the HA-tag of ACHT1. The heterodimer and heterotrimer ACHT1-2-Cys Prx complexes are indicated by arrows. Equal loading was verified as in Figure 2. The results shown are representative of five independent experiments. [See online article for color version of this figure.]

Figure 7.

ACHT1 Redox State Is Dependent on Light Intensity. Immunoblot assay showing the in vivo oxidized state of ACHT1 active site Cys residues captured in plants grown under 80 μE/m²s for 7 d and then illuminated by the same light intensity or by higher light intensity (200 μE/m²s) for the indicated time periods. Equal protein loading was verified as in Figure 1. The results shown are representative of four independent experiments.

Figure 8.

Elevated ACHT1 Expression Results in Altered NPQ and ETR but Not in Fv/Fm Values or Chlorophyll and Anthocyanin Content. (A) Time course of NPQ was calculated according to Maxwell and Johnson (2000) from traces of fluorescence induction in 16-h dark-adapted wild-type (WT), ACHT1 OE, and ACHT1 KD lines illuminated with 80 μE/m²s actinic light. (B) ETR was calculated from traces of fluorescence induction as in (A). (C) The maximal quantum yield (Fv/Fm) of the plants was measured before light induction. (D) Anthocyanins content was determined from the absorbance at 530 and 657 nm of extracts of 14-d-old plants, and the results are expressed as (A₅₃₀ – A₆₃₇) × 1000 per milligram of plant weight (relative units [RU]). (E) Chlorophyll a, chlorophyll b, and chlorophyll a+b concentrations were determined in extracts of 14-d-old plants as described (Porra et al., 1989). FW, fresh weight. Plants were grown at 20°C/18°C under 8/16 h light/dark cycle using fluorescent white light at approximately 80 μE/m²s. Results from two KD lines (SALK_089128 and SALK_144456) were pooled together as well as the results from two OE lines (2-25 and 2-28). Data represent mean and se (n = 20 for photosynthetic parameter measurements and n = 6 for anthocyanin and chlorophyll determination). Means sharing the same letter are not significantly different based on the Tukey-Kramer honestly significant difference test using a P value cutoff of 0.05.

Figure 9.

Working Model of ACHT1 in the Light. Depending on environmental conditions, the photosynthetic reducing equivalents may be diverted from the productive reaction of (1) NADP⁺ reduction via ferredoxin (Fd) and ferredoxin NADP-reductase (FNR) to the nonproductive (2) Mehler reaction, producing hydrogen peroxide (H₂O₂) as an intermediate. A portion of the equivalents is used, (3) via ferredoxin Trx reductase (FTR), to reduce chloroplast Trxs, such as ACHT1, for regulatory purposes. The redox state of the chloroplast ACHT1 pool in the light is determined by its reduction rate by ferredoxin Trx reductase (3) and by its oxidation rate by 2-Cys Prx (Prx). 2-Cys Prx itself is oxidized by peroxides produced by nonproductive photosynthesis (2). ACHT1 reduction rate is limiting and leads to net oxidation of ACHT1 shortly after illumination. The redox state of ACHT1 is used for redox-based feedback regulation of the membranal electron transfer reactions.