PsbP protein, but not PsbQ protein, is essential for the regulation and stabilization of photosystem II in higher plants

Abstract

PsbP and PsbQ proteins are extrinsic subunits of photosystem II (PSII) and participate in the normal function of photosynthetic water oxidation. Both proteins exist in a broad range of the oxygenic photosynthetic organisms; however, their physiological roles in vivo have not been well defined in higher plants. In this study, we established and analyzed transgenic tobacco (Nicotiana tabacum) plants in which the levels of PsbP or PsbQ were severely down-regulated by the RNA interference technique. A plant that lacked PsbQ showed no specific phenotype compared to a wild-type plant. This suggests that PsbQ in higher plants is dispensable under the normal growth condition. On the other hand, a plant that lacked PsbP showed prominent phenotypes: drastic retardation of growth, pale-green-colored leaves, and a marked decrease in the quantum yield of PSII evaluated by chlorophyll fluorescence. In PsbP-deficient plant, most PSII core subunits were accumulated in thylakoids, whereas PsbQ, which requires PsbP to bind PSII in vitro, was dramatically decreased. PSII without PsbP was hypersensitive to light and rapidly inactivated when the repair process of the damaged PSII was inhibited by chloramphenicol. Furthermore, thermoluminescence studies showed that the catalytic manganese cluster in PsbP-deficient leaves was markedly unstable and readily disassembled in the dark. The present results demonstrated that PsbP, but not PsbQ, is indispensable for the normal PSII function in higher plants in vivo.

Figure 1.

RNAi constructs and suppression of the psbP and psbQ genes. A, RNAi vectors used to down-regulate the psbP and psbQ genes. El2, Doubled enhancer-like elements; 35Sp, cauliflower mosaic virus 35S promoter; Ω, Ω sequence of Tobacco mosaic virus; NOSt, nopaline synthase terminator; OCSt, octopine synthase terminator. B, Northern analysis for high-molecular-weight (HMW) and low-molecular-weight (LMW) RNAs. RNAs (5 μg) extracted from leaves of wild type and transformants were separated on agarose/formaldehyde gel and hybridized with the indicated [³²P]-labeled DNA probes.

Figure 2.

Phenotypes of wild-type, ΔPsbP, and ΔPsbQ plants. Tobacco plants precultivated on 0.5× LS agar medium supplemented with 1.5% Suc were transplanted into soil and grown at 25°C under continuous light (100 μE m⁻² s⁻¹) for 3 weeks.

Figure 3.

Hypersensitivity of ΔPsbP tobacco to light. A, Time course of photoinactivation in wild-type (circle), ΔPsbP (square), and ΔPsbQ (triangle) leaves exposed to 100 μE m⁻² s⁻¹ for 150 min with (black symbols) or without (white symbols) the vacuum infiltration of chloramphenicol (200 μg mL⁻¹). The results are expressed as percentage of the initial F_v/F_m values (0.82 for wild type and ΔPsbQ, and 0.53 for ΔPsbP) prior to light incubation. B, Chronic photodamage observed in ΔPsbP. The ΔPsbP plants were grown under moderate light conditions (approximately 50 μE m⁻² s⁻¹) for 2 months. The plant on the right was then transferred to standard light conditions (150 μE m⁻² s⁻¹) and grown for another week.

Figure 4.

Immunoblot analyses of thylakoid membrane proteins. Proteins prepared from leaves and thylakoid membranes of tobacco plants grown under moderate light conditions (approximately 50 μE m⁻² s⁻¹) were subjected to SDS-PAGE or Tricine-SDS-PAGE, and then detected by immunoblotting using specific antibodies. To detect degraded fragments, total proteins from leaves were used to analyze the extrinsic proteins (PsbO, PsbP, and PsbQ). Each lane contained 5 μg of chl. WT, Wild-type tobacco; ΔP, ΔPsbP tobacco; ΔQ, ΔPsbQ tobacco.

Figure 5.

Chl fluorescence induction and parameters obtained from wild-type and ΔPsbP plants. A, Fluorescence decay kinetics in wild type (WT) and ΔPsbP. The black and white arrowheads indicate the application of measuring light and a saturating light pulse, respectively. B, Values of 1 − qP under different light intensities in wild type (circle) and ΔPsbP (square). The value of 1 − qP represents the accumulation of the reduced quinone acceptor (Q_A⁻) within PSII and was determined 2 min after the application of actinic light at different intensities. The F_o′ level was determined with the application of far-red light after each saturating light pulse. The plants were adapted to the dark for 2 h before measurements.

Figure 6.

TL glow curves for the wild-type and ΔPsbP leaves. A, The TL band observed in the wild-type leaves. B, The ability of the TL band to decay in the dark in ΔPsbP leaves. The leaves were incubated in the dark at 20°C for the indicated period and then illuminated by a single flash at 0°C. B, The development of TL B-band capability during weak light illumination in dark-adapted ΔPsbP leaves. ΔPsbP leaves were dark adapted for 5 h and then incubated under dim light (0.5 μE m⁻² s⁻¹) at 20°C for the indicated period. The sample leaves were excited with a single flash at 0°C after 10 min of incubation in the dark. The TL B-band originates from recombination of the Q_B⁻/S₂ charge pair in PSII.