Monogalactosyldiacylglycerol deficiency in Arabidopsis affects pigment composition in the prolamellar body and impairs thylakoid membrane energization and photoprotection in leaves

Abstract

Monogalactosyldiacylglycerol (MGDG) is the major lipid constituent of chloroplast membranes and has been proposed to act directly in several important plastidic processes, particularly during photosynthesis. In this study, the effect of MGDG deficiency, as observed in the monogalactosyldiacylglycerol synthase1-1 (mgd1-1) mutant, on chloroplast protein targeting, phototransformation of pigments, and photosynthetic light reactions was analyzed. The targeting of plastid proteins into or across the envelope, or into the thylakoid membrane, was not different from wild-type in the mgd1 mutant, suggesting that the residual amount of MGDG in mgd1 was sufficient to maintain functional targeting mechanisms. In dark-grown plants, the ratio of bound protochlorophyllide (Pchlide, F656) to free Pchlide (F631) was increased in mgd1 compared to the wild type. Increased levels of the photoconvertible pigment-protein complex (F656), which is photoprotective and suppresses photooxidative damage caused by an excess of free Pchlide, may be an adaptive response to the mgd1 mutation. Leaves of mgd1 suffered from a massively impaired capacity for thermal dissipation of excess light due to an inefficient operation of the xanthophyll cycle; the mutant contained less zeaxanthin and more violaxanthin than wild type after 60 min of high-light exposure and suffered from increased photosystem II photoinhibition. This is attributable to an increased conductivity of the thylakoid membrane at high light intensities, so that the proton motive force is reduced and the thylakoid lumen is less acidic than in wild type. Thus, the pH-dependent activation of the violaxanthin de-epoxidase and of the PsbS protein is impaired.

Figure 1.

Chloroplast protein translocation and insertion assays. A, Import of different precursor proteins into wild-type and mgd1 mutant Arabidopsis chloroplasts. Import reaction time points are defined as follows: a, 3 min (1 min for pL11); b, 6 min (2 min for pL11); c, 10 min (3 min for pL11). Note that for pL11, a shorter time series was used (1–3 min instead of 3–10 min for the other precursors) because pL11 is known to have a faster import rate (Aronsson and Jarvis, 2002). TM, Translation mixture; p, precursor protein; m, mature protein. B, Quantification of the import data shown in A. The amount of imported, mature protein in mgd1 chloroplasts is expressed as percentage of the amount of imported protein in wild-type chloroplasts (set to 100%). Values shown are means (±sd) derived from at least three assays. C, Direct insertion into the outer envelope membrane. In vitro-translated atToc33 and atToc34 proteins were incubated with wild-type and mgd1 chloroplasts for 30 min under import conditions and then subjected to treatment with Na₂CO₃, pH 11.5. The treated samples were then divided into a soluble (S) and an envelope-enriched (E) fraction prior to analysis. D, Direct insertion into the thylakoid membrane by the spontaneous pathway. In vitro-translated pCF₀II was incubated with wild-type and mutant chloroplasts under import conditions for 30 min. At the end of the import reaction, thermolysin treatment was used to remove any unimported preprotein, and then thylakoid membranes were isolated.

Figure 2.

Immunodetection of different chloroplast proteins. Chloroplasts were isolated from 2-week-old wild-type and mgd1 mutant plants and used to prepare immunoblots. Equal (10 μg or 1 μg for PsbS) samples of total chloroplast protein were loaded per lane, and the blots were used to compare levels of several different proteins, as indicated. Antibodies were detected using an ECL system (Amersham Biosciences).

Figure 3.

Low-temperature (77 K) fluorescence emission spectra. Fluorescence emission spectra were recorded using the following wild-type and mgd1 mutant samples: A, the cotyledons of 5-d-old dark-grown plants; B, the cotyledons of 5-d-old flash-irradiated, dark-grown plants; C, the leaves of 28-d-old plants grown at approximately 200 μmol m⁻² s⁻¹. The spectra were normalized at 631 nm (A and B) or 705 nm (C). Excitation wavelength was 440 nm in each case, as indicated. The inset in A shows an immunoblot performed on total protein samples isolated from 5-d-old dark-grown plants and indicates the level of total POR protein. In B, a flash lamp (Braun F 800 Professional) with the effect of 165 W and a duration of 1 ms of the flash was used for phototransformation.

Figure 4.

Light response curves for linear electron transport and qN. A, Linear electron transport was calculated from PSII quantum yield in wild-type and mgd1 mutant plants according to Genty et al. (1989). Five-week-old plants grown in normal light (150 μmol m⁻² s⁻¹) were measured without further treatment. Data shown are means (±sd) derived from measurements of five different plants per genotype. Black symbols, wild type; white symbols, mgd1. B, qN was calculated using the data shown in A according to Krause and Weis (1991).

Figure 5.

Xanthophyll cycle measurements. Dark-adapted plants (0 min) were moved to a high-light chamber (1,000–1,100 μmol m⁻² s⁻¹), and xanthophyll cycle pigments were measured by HPLC at the indicated time points. The relative concentrations of V, A, and Z in wild-type (A) and mgd1 mutant (B) plants are shown. Values for each pigment are expressed as a percentage of the total xanthophyll pool in each sample. Data shown are means (±sd) derived from at least five measurements. C, The DES of the wild-type and mutant plants was calculated for each time point, using the data shown in A and B. Values were calculated as follows: DES = (A + Z)/(V + A + Z).

Figure 6.

Thylakoid membrane energization is strongly reduced in the mgd1 mutant at high light intensities. A, Light response curve of the total ECS, which is proportional to the steady-state pmf across the thylakoid membrane. The pmf across the thylakoid membrane during steady-state photosynthesis was estimated from the amplitude of the ECS during short intervals (15 s) of dark relaxation. Leaves of 5-week-old plants grown in normal light (150 μmol m⁻² s⁻¹) were preilluminated at each light intensity for 10 min to allow photosynthesis to reach steady state, and the total ECS was normalized to the Chl content of the leaf segment being measured. B, Contribution of the ΔpH to the total pmf across the thylakoid membrane. Estimates of the partitioning of pmf between its ΔpH and ΔΨ components were derived from measurements obtained during the slowly relaxing phase of the ECS. Data shown in A and B are means (±sd) of measurements of six different plants per genotype. Black symbols, wild type; white symbols, mgd1.