Flavodiiron protein Flv2/Flv4-related photoprotective mechanism dissipates excitation pressure of PSII in cooperation with phycobilisomes in Cyanobacteria

Abstract

Oxygenic photosynthesis evolved with cyanobacteria, the ancestors of plant chloroplasts. The highly oxidizing chemistry of water splitting required concomitant evolution of efficient photoprotection mechanisms to safeguard the photosynthetic machinery. The role of flavodiiron proteins (FDPs), originally called A-type flavoproteins or Flvs, in this context has only recently been appreciated. Cyanobacterial FDPs constitute a specific protein group that evolved to protect oxygenic photosynthesis. There are four FDPs in Synechocystis sp. PCC 6803 (Flv1 to Flv4). Two of them, Flv2 and Flv4, are encoded by an operon together with a Sll0218 protein. Their expression, tightly regulated by CO2 levels, is also influenced by changes in light intensity. Here we describe the overexpression of the flv4-2 operon in Synechocystis sp. PCC 6803 and demonstrate that it results in improved photochemistry of PSII. The flv4-2/OE mutant is more resistant to photoinhibition of PSII and exhibits a more oxidized state of the plastoquinone pool and reduced production of singlet oxygen compared with control strains. Results of biophysical measurements indicate that the flv4-2 operon functions in an alternative electron transfer pathway from PSII, and thus alleviates PSII excitation pressure by channeling up to 30% of PSII-originated electrons. Furthermore, intact phycobilisomes are required for stable expression of the flv4-2 operon genes and for the Flv2/Flv4 heterodimer-mediated electron transfer mechanism. The latter operates in photoprotection in a complementary way with the orange carotenoid protein-related nonphotochemical quenching. Expression of the flv4-2 operon and exchange of the D1 forms in PSII centers upon light stress, on the contrary, are mutually exclusive photoprotection strategies among cyanobacteria.

Figure 1.

Construction and characterization of the flv4-2/OE mutant. A, Schematic representation of the insertion of flv4-2 operon into the Synechocystis chromosome under the control of the psbA2 gene promoter. B and C, Western blots were performed with protein samples isolated from cultures grown at air-level CO₂ (LC; B) and at 3% CO₂ (HC; C). Twenty micrograms of total protein from the cell extracts were loaded into each lane (= 100%), if not otherwise indicated. WT, wild type. [See online article for color version of this figure.]

Figure 2.

Growth phenotype of wild-type and mutant strains. A and B, Growth curves were obtained with cells cultivated at 500-HL (A) or at 1,500-HL (B). C, The difference in color of cells grown for 7 d at corresponding light intensities. WT, wild type.

Figure 3.

Car/Chl a ratio measured from cultures cultivated at standard GL conditions (black bars) and at high light intensity of 500-HL (gray bars). The cultures were adjusted to an OD₇₅₀ = 0.6 before the measurements. Values are means ± sd of three independent experiments. WT, wild type.

Figure 4.

Production of singlet oxygen in wild-type, ΔpsbA2, flv4-2/OE, and Δflv4 strains in the presence of 5 mm His upon illumination of the cells at 2,300 μmol photon m⁻² s⁻¹. The cultures were adjusted to a chlorophyll concentration of 5 µg mL⁻¹. To demonstrate that the O₂ uptake signal indeed arises from ¹O₂, 10 mm sodium azide, which is specific quencher of ¹O₂, was also applied. Because sodium azide inhibits O₂ evolution, its ¹O₂ quenching effect was tested in the presence of DCMU in which the artifact that would arise from the decreased of O₂ evolution rate could be avoided. The results are a mean ± sd of three independent experiments. NaN₃, sodium azide; WT, wild type.

Figure 5.

PSII photoinhibition kinetics in wild-type, ΔpsbA2, and flv4-2/OE strains. The strains were illuminated with white light intensity of 500 µmol photons m⁻² s^-1in the presence of lincomycin (300 µg mL⁻¹). Oxygen evolution rates were measured in the presence of 2 mm DMBQ as an artificial electron acceptor. The cultures were adjusted to a chlorophyll concentration of 5 µg mL⁻¹. Values are means ± sd of three independent experiments. WT, wild type.

Figure 6.

Fluorescence induction curves from wild-type, ΔpsbA2, flv4-2/OE, and Δflv4 strains. Cells were dark adapted and then exposed to red actinic light of 120 µmol photons m⁻² s⁻¹. The cultures were adjusted to a chlorophyll concentration of 10 µg mL⁻¹. AL, actinic light; WT, wild type. [See online article for color version of this figure.]

Figure 7.

Decrease of maximal fluorescence induced by OCP-mediated NPQ upon illumination of cells with strong blue light intensity (750 µmol photons m⁻² s⁻¹). The cultures were adjusted to a chlorophyll concentration of 10 µg mL⁻¹. The values shown are the mean ± sd of three independent experiments. WT, wild type. [See online article for color version of this figure.]

Figure 8.

A, Protein immunoblots showing the content of ApcD, PC, and OCP in wild-type, ΔpsbA2, flv4-2/OE, and Δflv4 strains. Ten micrograms of total protein from the cell extract were loaded in each lane. B, Protein immunoblots showing the content of Flv4, Sll0218, and Flv2 in wild-type, PAL, CK, ΔApcDF, and ΔOCP mutants. Twenty micrograms of total protein from the cell extract were loaded in each lane (= 100%), if not otherwise stated. This is a representative picture of three independent immunoblots. C, Transcript accumulation of flv4-2 operon in the wild-type, PAL, CK, ΔApcDF, and ΔOCP mutants analyzed by real-time quantitative RT-PCR. Transcript abundance is shown as relative units. The transcript level of the rnpB gene is used as a reference. The results are the mean from three independent experiments ± sd. ApcD allophycocyanin D; PC, phycocyanin; rel unit, relative unit; WT, wild type.

Figure 9.

Flash-induced chlorophyll fluorescence relaxation curves in darkness from wild-type, ΔpsbA2, flv4-2/OE, and Δflv4 strains, grown in LC conditions. The curves were recorded in the absence (closed symbols) and in the presence of 20 µM DBMIB (open symbols). The cultures were adjusted to a chlorophyll concentration of 5 µg mL⁻¹. To facilitate the comparison of the curves, F₀ and F_mD values were normalized to 0 and 1, respectively. The fluorescence traces are the mean of three independent experiments. rel unit, relative unit; WT, wild type. [See online article for color version of this figure.]

Figure 10.

The 77K fluorescence emission spectra of wild-type, ΔpsbA2, flv4-2/OE, and Δflv4 strains excited at 580 nm. The spectra were averaged from three independent experiments. Each spectrum was normalized to PSI fluorescence peak at 723 nm (set as 1). The cultures were adjusted to a chlorophyll concentration of 5 µg mL⁻¹. WT, wild type. [See online article for color version of this figure.]