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First published online July 18, 2008; 10.1104/pp.108.123372 Plant Physiology 148:580-592 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Monogalactosyldiacylglycerol Deficiency in Arabidopsis Affects Pigment Composition in the Prolamellar Body and Impairs Thylakoid Membrane Energization and Photoprotection in Leaves1,[W],[OA]Department of Plant and Environmental Sciences, University of Gothenburg, SE–405 30 Gothenburg, Sweden (H.A., C.S., S.K.); Max-Planck-Institute of Molecular Plant Physiology, 14476 Golm, Germany (M.A.S., P.D.); Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE–106 91 Stockholm, Sweden (A.A.K.); and Department of Biology, University of Leicester, Leicester LE1 7RH, United Kingdom (P.J.)
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.
Galactolipids are the most abundant nonproteinaceous constituents of plastid membranes. Two galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are abundant in plant plastid membranes, where they account for as much as approximately 50 and approximately 20 mol%, respectively, of total lipids (Douce and Joyard, 1990  ). Because of the different sizes of their head groups, MGDG and DGDG differ in shape and biophysical properties. DGDG has a cylindrical shape that is typical for most plastid lipids and is considered a bilayer-prone lipid. In contrast, MGDG has a conical shape due to its smaller head group and high content of unsaturated fatty acids, giving it nonbilayer-forming characteristics (Webb and Green, 1991; Bruce, 1998).
The synthesis of MGDG predominantly takes place in the inner envelope of chloroplasts (Block et al., 1983
We previously identified an MGDG-deficient mutant (mgd1-1) that carries a T-DNA insertion in the promoter region of MGD1 (Jarvis et al., 2000
Chloroplast protein import studies using the Arabidopsis dgd1 mutant, which is approximately 90% deficient in DGDG, revealed a significant decrease in import efficiency (Chen and Li, 1998
The prolamellar bodies (PLBs) of etioplasts have a high lipid to protein ratio compared to thylakoids. While MGDG is the dominant lipid, NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the most abundant protein in PLBs (Selstam and Sandelius, 1984
Several photosynthesis-related reactions are thought to be dependent on galactolipids. For example, the Arabidopsis dgd1 mutation, which causes DGDG deficiency, affects the composition of the water-oxidizing complex (Reifarth et al., 1997
The aim of this study was to investigate the in vivo roles of MGDG in various processes in which it is putatively involved, as discussed above. The recently characterized mgd1 null mutant (mgd1-2) has extremely severe defects in chloroplast and plant development (Kobayashi et al., 2007
Protein Targeting Into and Across Chloroplast Membranes Is Not Affected in the mgd1 Mutant
Based on in vitro studies, MGDG has been suggested to play an important role in protein import into chloroplasts and in the insertion of chloroplast outer envelope membrane proteins (van't Hof et al., 1991
Next, we assessed the possibility that protein insertion into the outer envelope membrane is affected by mgd1 because MGDG has been suggested to facilitate protein recognition during such targeting processes (Pinnaduwage and Bruce, 1996 ). We used two different outer envelope membrane proteins, atToc33 and atToc34, both of which lack a transit peptide (like most outer envelope proteins; Hofmann and Theg, 2005). However, we again observed no defect in protein targeting efficiency in the mgd1 mutant. Both proteins inserted equally well into the outer envelope membrane of wild-type and mutant chloroplasts (data not shown), and the inserted proteins withstood extraction by alkaline treatment to the same extent (Fig. 1C, top) such that no protein was extracted to the soluble supernatant fraction (Fig. 1C, bottom), clearly demonstrating that insertion efficiency was not affected by the mgd1 mutation. These results are similar to those reported previously for OEP14 for the dgd1 mutant (Chen and Li, 1998), suggesting that galactolipids may not play an important role in the insertion of outer envelope proteins in vivo. However, OEP14 is a signal-anchored protein, whereas atToc33 and atToc34 are tail-anchored proteins, and it is presently not clear if these different protein classes use the same or different insertion mechanisms (Hofmann and Theg, 2005).
Because MGDG is the major lipid constituent of thylakoid membranes and because CF0II targeting follows the spontaneous thylakoid insertion pathway that does not appear to depend on any proteinaceous factors (Michl et al., 1994
Immunoblots were prepared to study the levels of proteins involved in the chloroplast import mechanism (atToc33 and atToc75-III), as well as of some substrates of the import machinery (atToc75-III, Lhcb2, PsaD, PsbS, plastocyanin [PC], and VDE; note that D1, PsbB, PsaC, and β-ATPase are encoded by the plastome and so do not need to be imported), in wild-type and mgd1 plants (Fig. 2
). The chloroplast import machinery consists of several different components; the atToc33 and atToc75-III proteins tested here contribute to the receptor and channel-forming functions, respectively, in the outer envelope membrane (Bédard and Jarvis, 2005
Thus, our data suggest that MGDG may not be as important for chloroplast protein targeting as was suggested on the basis of earlier, in vitro studies. The many proteins normally present in chloroplast membranes are missing in the artificial membrane systems used for such in vitro experiments, and so the MGDG dependency observed in those studies may have been an overestimation. Alternatively, our negative results may be related to the fact that mgd1-1 contains only 42% less MGDG than the wild type, or indicate that some of the other lipids compensate for the loss of MGDG in the mutant (Jarvis et al., 2000 ). It is conceivable that a larger MGDG reduction would cause significant targeting defects. However, in the extreme case of the mgd1-2 null mutant (Kobayashi et al., 2007), plant and chloroplast development are so badly affected that analysis and interpretation would both be extremely problematic.
Our results (in particular, Fig. 1, A and B) contrast with those reported previously for the dgd1 mutant (Chen and Li, 1998
An interaction between POR and MGDG has been proposed to stabilize the formation of the etioplast structure in darkness and to increase the fluorescence of Pchlide at around 655 nm (Klement et al., 1999 The hook of dark-grown seedlings was subjected to rapid freezing in liquid nitrogen (77 K), and then fluorescence emission was recorded after excitation at 440 nm. Measurements of wild-type samples revealed a lower emission peak for Pchlide-F656 than for Pchlide-F631. By contrast, the mgd1 mutant had a higher peak at 656 nm, significantly increasing the ratio of Pchlide-F656 to Pchlide-F631 relative to the wild type (Fig. 3A ). This pattern was even more pronounced when an excitation wavelength of 460 nm was used (data not shown).
Pchlide-F631 is the first form of the pigment to be produced during plastid development, and it is referred to as nonphotoactive Pchlide because it cannot participate in Chl formation (it is in a free or unbound state). By contrast, Pchlide-F656 corresponds to photoactive Pchlide bound to POR. Previous work has suggested that Pchlide-F631 (or an equivalent species) is a photosensitizer, rendering plants susceptible to photooxidative damage (Runge et al., 1996 ; Sperling et al., 1997; Erdei et al., 2005).
The photoactivity of Pchlide-F656 was verified by flash illumination. The dark-grown 5-d-old seedlings stored at 77 K were rewarmed to 253 K (–20°C), flashed with strong light, and then immediately frozen in liquid nitrogen (77 K); thereafter, the emission spectra were recorded (Fig. 3B). The 2-fold higher levels of the chlorophyllide (Chlide)-F688 fluorescence peak in mgd1 can be taken as further evidence for the increased amount of photoactive Pchlide-F656 in the mutant (Fig. 3B). Measurements of dark-grown seedlings of different ages (5, 7, and 8 d old) always revealed an increase in the Pchlide-F656 form in the mutant, excluding the possibility that the differences in fluorescence were due to developmental stage differences (data not shown). To further verify the observed increase in formation of Chlide in mgd1, the degree of phototransformation was determined. A predetermined fluorescence ratio between Pchlide and Chlide of 1.09 (Ryberg and Sundqvist, 1982
Previous work demonstrated that overexpression of POR increases the Pchlide F656 to F631 ratio (equivalent to the F655 to F632 ratio; Franck et al., 2000
The observed differences in pigment-protein composition in dark-grown mgd1 plants (Fig. 3, A and B) are interesting because etioplasts in 5-d-old mgd1 plants were reported to have normal ultrastructure (Jarvis et al., 2000 We also investigated the role of MGDG in determining the composition of pigment-protein complexes in thylakoids of light-grown plants (Fig. 3C). No obvious differences in the fluorescence emission for PSII and PSI were observed in plants grown at low light intensity (approximately 100 µmol m–2 s–1; data not shown). However, when plants were grown at an elevated light intensity of approximately 200 µmol m–2 s–1, the fluorescence at around 735 nm was decreased in mgd1 (Fig. 3C). The relative fluorescence intensity at 692 nm, which reflects PSII, was similar for mgd1 and wild type at the light intensity used (Fig. 3C). Because the ratio of the peaks at 692 and 735 nm is a measure for the antenna cross sections of the two photosystems relative to each other, these data suggest that either the accumulation of PSI reaction centers relative to PSII is reduced or that the distribution of light-harvesting complex (LHC) proteins between PSI and PSII is altered in mgd1 at elevated light intensities.
Chloroplast ultrastructure was previously reported to be altered in mgd1 (Jarvis et al., 2000 To assess this possibility, we first measured photosynthetic pigment composition in light-adapted plants. Pigments levels were analyzed by HPLC and are presented as nanomoles per gram fresh weight in Table I . The total amount of pigments is reduced by approximately 29% in mgd1 relative to wild type due to a 30% reduction in total Chl and a 23% reduction in carotenoids. In mgd1, Chl a is reduced by approximately 28%, and an even stronger reduction is observed for Chl b (35%); these changes are reflected in an increased Chl a to b ratio in the mutant (3.3 in wild type; 3.6 in mgd1). The increased Chl a to b ratio suggests a preferred maintenance of Chl a-rich reaction centers at the expense of Chl b-rich antenna complexes (which may be a mechanism to enable more efficient use of light energy with limited pigment resources). The quantification of photosynthetic complexes and low-temperature Chl a fluorescence measurements is certainly supportive of this interpretation (see below).
The major carotenoid pigment, lutein, was reduced in abundance in mgd1 by 33.7% (on a per fresh weight basis; Table I). Moreover, the amounts of all xanthophyll cycle pigments were reduced in mgd1 (Table I). V was reduced by 8.5%, Z was reduced by 37.5%, and neoxanthin was reduced by 33.7% (all on a per fresh weight basis). When the pigment concentrations were normalized to the amount of Chl a (mmol pigment/mol Chl a; Table I), most pigment values were still reduced in mgd1 relative to wild type.
To characterize the function of the electron transport chain, the quantum yield of PSII, a measure of photosynthetic efficiency, was determined in dark-adapted leaves (Fv/Fm). Measurements of Fv/Fm in wild type and mgd1 were indistinguishable (data not shown), indicating that a reduction in MGDG content of approximately 40% has no impact on photosynthetic efficiency in low-light-grown plants. Next, we determined light saturation curves of linear electron transport from PSII yield measurements. Because the absorption properties of wild-type and mgd1 leaves were unaltered, despite the approximately 30% reduction in pigments, and because quantification of the photosynthetic complexes indicated only a minor change in the stoichiometry of the photosystems and their antenna cross sections (see below), it is legitimate to calculate linear electron flux from PSII quantum efficiency (Genty et al., 1989
While the diminished linear electron flux and assimilation rate per leaf area point to a reduced efficiency of photosynthesis in mgd1 (Fig. 4A), the functional organization of the electron transport chain does not seem to be strongly affected by MGDG deficiency in mgd1; when electron transport rates are recalculated on a Chl basis (e.g. per photosynthetic unit), they were comparably high in mgd1 and the wild type. This was confirmed by quantifications of the photosynthetic complexes by means of immunoblotting (Fig. 2) and difference absorption spectroscopy (Table II ).
Components analyzed by immunoblotting were as follows. For PSII, we analyzed the antenna complex polypeptide, Lhcb2, the two essential core complex proteins, D1 and PsbB, and PsbS localized at the periphery of PSII. For PSI, we analyzed PsaC and PsaD, which are localized at the periphery of PSI but which nevertheless represent reasonable indicators of PSI abundance, as in the absence of PsaC, no stable accumulation of PSI is possible (Takahashi et al., 1991 ). We also analyzed two proteins (PC and the β-subunit of the ATP synthase) not directly associated with either PSII or PSI but that are nonetheless important to maintain optimal photosynthesis. As shown in Figure 2, none of these proteins was expressed at significantly different levels in mgd1 relative to wild type. The results presented in Figure 1 are also consistent with this conclusion. These conclusions are supported by difference absorption spectroscopy data (Table II). While the content of PSII was slightly increased in the mutant (approximately 120% of the wild-type level), the contents of the cytochrome b6f complex, PSI, and PC were similar in the wild type and mutant. The slight elevation of PSII content, which is below the resolution of our immunoblot analysis, may explain the increased Chl a to b ratio of the mutant; because the PSI content was unaffected, the ratio increase can only be explained by the loss of LHC antenna proteins of the photosystems, as alluded to earlier. This conclusion is in line with 77-K Chl a fluorescence emission signals determined on leaves of 28-d-old plants; the PSI fluorescence emission signal at 735-nm wavelength is slightly smaller in the wild type than in the mutant, which is in agreement with a slightly elevated PSII to PSI ratio in mgd1 thylakoids (Fig. 3C).
In addition to having lower photosynthesis capacity per unit leaf area (Fig. 4A), Chl a fluorescence light response curves revealed that mgd1 has a much lower capacity for qN (qN is approximately 0.8 and 0.55 in wild-type and mgd1 leaves, respectively, at light intensities exceeding 1,000 µmol m–2 s–1; Fig. 4B). Consequently, the mgd1 mutant was clearly more susceptible to light stress. After leaves had been exposed to 1,000 µmol m–2 s–1 of actinic light for 1 h, the proportion of photoinhibited PSII was calculated from the dark-relaxation kinetics of qN. The qI component, the fraction of qN relaxing with a half-time >15 min (which correlates with PSII photoinhibition; Krause and Weis, 1991
Reductions in the capacity of qN could be due to any of several possible defects in the mgd1 mutant because qE is essentially induced by acidification of the thylakoid lumen (which is itself influenced by numerous factors). Under standard growth conditions used during this study, the pH of the thylakoid lumen is predicted to be in the region of 6.5 (Takizawa et al., 2007
To assess the different factors possibly contributing to alterations in qN and qE in mgd1, we first checked for changes in the levels of PsbS and VDE by immunoblotting. However, we detected no change in the abundance of either protein in the mutant (Fig. 2). Next, to compare in vivo parameters of the xanthophyll cycle in the mutant and wild type, we measured the kinetics of V de-epoxidation in leaves of plants that had first been adapted to complete darkness for 2 h and which were then exposed to strong light (approximately 1,000 µmol m–2 s–1) for up to 60 min. The measured amounts of V, A, and Z were normalized to the total pool size of xanthophyll cycle pigments and then plotted against the duration of exposure to high light (Fig. 5 ). Following dark adaptation, wild-type and mutant plants both contained high amounts of V, very low amounts of A, and no detectable Z (see 0-min data points; Fig. 5, A and B), due to the inactivation of VDE in darkness. Immediately after the onset of light, the level of V rapidly declined in both genotypes, while the levels of A and Z both increased (Z to a much higher value than A).
In wild-type leaves, the molar proportion of V initially dropped in the first 10 min to less than 40% and then remained constant throughout the remainder of the experiment (Fig. 5A). The proportion of V in mgd1 leaves also declined to approximately 40%, during the first 5 min, but in contrast to wild-type leaves, it progressively increased thereafter, reaching 55% after 60 min (Fig. 5B). For both genotypes, the molar proportion of A increased from 5% to approximately 20% to 25% during the first 10 min but then slightly decreased to approximately 15% to 20% after 60 min (Fig. 5, A and B). The changes in molar proportions of Z appeared to be directly opposite to, and were presumably due to, alterations in the abundance of V; levels of Z initially increased rapidly in both wild-type and mgd1 leaves but then remained at a higher level (approximately 45%–50%) in wild-type leaves, while they declined in mgd1 leaves before reaching a plateau during the latter part of the experiment (Fig. 5B). Thus, prolonged illumination with high light over a period of 60 min had a severe impact on the ratio of V to Z in mgd1 plants. These changes were reflected in differences in de-epoxidation status (DES; =[A + Z]/[V + A + Z]) between wild-type and mutant plants (Fig. 5C). The reduced steady-state capacity of the xanthophyll cycle in mgd1 could be due to either impaired enzyme activity or reductions in the proton motive force (pmf) across the thylakoid membrane. However, the former possibility seems to be highly unlikely because reductions in enzyme activity should have affected both the rapid generation of A and Z during the first 5 min of light stress (Fig. 5) and the steady-state DES. Because no delay in V de-epoxidation was observed in mgd1 plants, a general impairment of VDE activity is unlikely to be the cause of the reduction in Z levels during the latter period of the light stress experiment (Fig. 5). Nevertheless, we examined VDE enzyme activities in wild-type and mgd1 samples in vitro under pH-induced dark conditions. However, as expected, no significant differences in VDE enzyme activities were detected between the wild-type and mutant samples (Supplemental Fig. S1). Because the lower qE and reduced levels of Z under light-stress conditions could not be explained by changes in PsbS or VDE protein levels, or by alterations in VDE activity, we next assessed the possibility that mgd1 causes alterations in the steady-state pmf across the thylakoid membranes.
To obtain information on the thylakoidal pmf, we measured electrochromic signals (ECS); these are carotenoid absorption shifts with kinetic properties that are proportional to the relative contributions of the electric (
Reduced qN Capacity in mgd1 Is Due to Increased Conductivity of the Thylakoid Membranes Next, we examined the relationship between pmf and actinic light intensity in wild-type and mutant leaves (Fig. 6A). The results showed that the pmf of the mutant leaves was almost as high as in wild-type leaves under light-limited conditions (200 µmol m–2 s–1 light). However, at higher light intensities, when qN becomes more important as a photoprotective mechanism, the increase in pmf was much weaker in mgd1 than in the wild type, resulting in the approximately 40% reduction in maximum pmf.
The reduced pmf under high light intensities indicates that the thylakoid lumen might be considerably less acidic in mgd1 than in wild type, impeding full activation of the VDE and PsbS proteins. This hypothesis could explain the mutant's reduced qN capacity. However, the induction of qN is not dependent on total pmf but only on the
The lumenal pH at maximum acidification is believed to be approximately 5.7 (Takizawa et al., 2007 Alteration of the steady-state energization of the thylakoid membrane may also explain the declines in Z content and DES observed in mgd1 after 5 min of high-light stress (Fig. 5). Immediately after illumination of dark-adapted leaves, the pmf is dramatically higher than under steady-state conditions because both the Calvin cycle (which consumes ATP) and the ATP synthase are still inactive, and their reductive activation by thioredoxin takes a few minutes in the light; a lower steady-state pmf is established only after ATP synthase activation because part of the pmf is then used for ATP synthesis. Therefore, in both wild-type and mgd1 mutant leaves, pmf is highest during the activation phase of photosynthesis and then subsequently declines to steady-state levels within 10 min following the onset of illumination. Hence, the pmf in the mgd1 mutant is only high enough to fully activate VDE during the first few minutes of illumination; later, under steady-state conditions, the pmf in mgd1 is below the optimal level for VDE, and so de-epoxidation rates are retarded and the equilibrium between Z and V starts to shift slightly toward V. In wild-type leaves, lower thylakoidal proton conductivity means that under 1,000 µmol m–2 s–1 actinic light (i.e. conditions used in Fig. 5), the steady-state pmf stays at a much higher level than in mgd1 (Fig. 6), and so the lumen retains sufficient acidity to maintain high VDE activity.
As yet, we can only speculate about possible reasons why the maximum pmf is reduced in mgd1. There are no apparent differences in pmf between wild-type and mutant plants under low-light intensities (up to 200 µmol m–2 s–1; Fig. 6A). This seems to preclude the possibility that the changes in the mutant are due to constitutive increases in the conductivity of the thylakoid membranes caused by alterations in levels of MGDG, because such changes would be expected to alter pmf under limiting light intensities as well as at high light intensities. Instead, structural changes of the thylakoids in response to increasing membrane energization may increase the conductivity of the mutant's thylakoid membranes to protons. Interestingly, the increased conductivity becomes apparent under conditions that initiate qE, which is known to involve major structural rearrangements of the PSII-LHCII supercomplexes (Horton et al., 2008
The availability of the mgd1 mutant enabled us to analyze the involvement of MGDG in various processes in vivo. The lack of a measurable effect of the mgd1 mutation on various chloroplast protein targeting pathways suggested that the residual MGDG in mgd1 is sufficient to maintain efficient protein transport (Fig. 1). Thus, while the possibility that MGDG is directly involved in chloroplast protein traffic at some level cannot be excluded at this stage, it is possible that the importance of the lipid in these processes has been somewhat overestimated. The low-temperature fluorescence measurements on dark-grown plants revealed the activation of a putative photoprotective mechanism in mgd1 (Fig. 3, A and B), suggesting that the mutation affects the plant's ability to deal with high light intensities. In accordance with this notion, we confirmed that MGDG deficiency has a negative effect on photosynthetic efficiency at elevated light intensities (Figs. 3C and 4). This is due to a reduction in qN capacity that we ascribe to increased conductivity of the thylakoid membranes, which reduces the maximum pmf in mgd1 by approximately 40%. Consequently, the threshold pH for initiating qE is barely reached, and neither PsbS nor VDE becomes fully activated in the mutant. Impairment of thylakoid membrane energization under high-light conditions may be due to structural rearrangements that occur during the onset of qE. Consideration of our results alongside those obtained using the DGDG-deficient mutant dgd1 clearly indicates that MGDG and DGDG do not simply provide a membrane environment to physically support the photosynthetic complexes. Rather, these two galactolipids contribute directly to various photosynthesis-related processes with individual contributions that are quite distinct.
Plant Material and Growth Conditions
Arabidopsis (Arabidopsis thaliana) seeds (wild type and mgd1-1; both Columbia-0 ecotype) were surface-sterilized, sown on petri plates containing Murashige-Skoog medium, and cold-treated at 4°C (Aronsson and Jarvis, 2002 For the analysis of pigment-protein complexes (Fig. 3), plants were grown on soil either in complete darkness for 5 to 8 d or in the light regime given above for 28 d. Plants used for all other measurements were 35 to 42 d old. Plants used for xanthophyll cycle measurements were kept in darkness for 2 h before applying high-light stress. Light stress was performed for 0, 2, 5, 10, 30, or 60 min at 1,000 to 1,100 µmol m–2 s–1.
Chloroplasts from 14-d-old plants were isolated according to Aronsson and Jarvis (2002)
For alkaline treatment, import reactions were scaled up three times (3 x 107 plastids) and conducted for 30 min prior to treatment with Na2CO3, pH 11.5. Thereafter, envelope membranes were enriched according to Baldwin et al. (2005)
For the thylakoid assays, a 30-min import reaction using 3 x 107 plastids was performed, followed by treatment with thermolysin (final concentration 0.1 mg mL–1) for 30 min at 4°C. Thylakoids were then isolated as described by Rawyler et al. (1992)
Concentrations of chloroplast (Fig. 2) and total protein (Fig. 3A, inset; isolated from 5-d-old dark-grown seedlings according to Kovacheva et al. [2005]
Fluorescence emission spectra from cotyledons of dark-grown plants or leaves from light-grown plants were measured at 77 K using a Fluorolog-3 spectrofluorometer (Spex Instruments S.A.). The emission was measured as photon emission per unit interval of wavelength. Emission spectra were recorded with an integration time of 0.5 s and the excitation wavelength set to 440 nm, as indicated in the figures. Both excitation and emission monochromators were used with a slit width of 3 nm. The spectra were corrected for the spectral sensitivity of the photomultiplier. Samples were inserted in cylindrical glass cuvettes and stored in liquid nitrogen prior to measurements. Small parts of the hypocotyl accompanied the cotyledons, but the fluorescence from the hypocotyl is negligible compared to the fluorescence from the cotyledons (data not shown). All spectra were smoothed 10 times using a fixed-bandwidth, sharp-cutoff, three-point, low-pass linear digital filter. The spectra shown are averages of five to 10 spectra.
The degree of phototransformation of Pchlide to Chlide in Arabidopsis seedlings was determined after irradiating the samples with three flashes and extracting the pigments into acetone to measure the fluorescence spectra at room temperature. The amount of Chlide found was used to calculate the amount of Pchlide before the flash using a ratio of 1.09 for the fluorescence yield of Pchlide:Chlide. The 1.09 ratio was determined using a PLB preparation (Ryberg and Sundqvist, 1982
Chl fluorescence was recorded with a pulse-amplitude modulation fluorometer (Dual-PAM; Heinz Walz). Plants were dark-adapted for 1 h, and fluorescence light response curves were recorded after a 5-min exposure to the photosynthetically active radiation as indicated. PSII quantum yield and linear electron flux were calculated (Genty et al., 1989
The PSII, cytochrome b6f complex, and PSI contents were determined in thylakoids, isolated according to Schöttler et al. (2004)
The relative stoichiometries of PC per P700 were determined using a PC version of the Dual-PAM spectroscope (Dual-PAM-S; Heinz Walz; Schöttler et al., 2007
The ECS peak at 517 nm was used as an in vivo measure of the proton motive force across the thylakoid membrane (Kramer et al., 2003
Leaf samples were homogenized in liquid nitrogen, and then pigments were extracted at 4°C under low-light conditions using, first, 700 µL of 80% (v/v) acetone, and second, 700 µL of 100% acetone. The two supernatants were combined, and then a 20-µL sample was used immediately for HPLC analysis according to Thayer and Björkman (1990) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AAF65066.
The following materials are available in the online version of this article.
The authors kindly appreciate technical assistance from Monica Appelgren, Markus Nordqvist, Ramesh Patel, Wolfram Thiele, Tony Wardle, and Regina Wendenburg. For antibodies provided, we kindly thank Agrisera AB (Vännäs, Sweden), Hans-Erik Åkerlund (Lund University, Sweden), Adrian Clarke (University of Gothenburg, Sweden), Lars-Gunnar Franzén (Halmstad University, Sweden), Kenneth Keegstra (Michigan State University), and Jürgen Soll (Münich University, Germany). Template DNA was helpfully provided by Dario Leister (Munich University, Germany; pL11) and the Arabidopsis Biological Resource Center (pSS, 188D4T7; pCF0II, 109L16T7; atToc33, 190I17T7; atToc34; 167B21T7). Received May 23, 2008; accepted July 14, 2008; published July 18, 2008.
1 This work was supported by the Swedish Research Council FORMAS (to H.A.) and VR (to C.S.), by the Wenner-Gren Foundation (to H.A. and A.A.K.), by the Royal Society of Arts and Sciences in Göteborg (to H.A.), by the Royal Swedish Academy of Sciences (to H.A.), by the Deutsche Forschungsgemeinschaft (grant no. SFB429, A12 to M.A.S.; and grant no. SFB429, B6 to P.D.), by the Royal Society Rosenheim Research Fellowship (to P.J.), and by the Biotechnology and Biological Sciences Research Council (to P.J.). 
2 These authors contributed equally to the article.
3 Present address: HRI/University of Warwick, Wellesbourne, Warwick CV35 9EF, United Kingdom.
4 Present address: University of Bonn, Institute of Molecular Physiology and Biotechnology of Plants, Karlrobert-Kreiten-Strasse 13, 53115 Bonn, Germany.
5 Present address: Institute of Biology, University of Tromsø, NO–9037 Tromsø, Norway. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Henrik Aronsson (henrik.aronsson{at}dpes.gu.se).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.123372 * Corresponding author; e-mail henrik.aronsson{at}dpes.gu.se.
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ABSTRACT
RESULTS AND DISCUSSION
) and osmotic (
100% B and 25 to 32 min 100% B, followed by 10 min equilibration in 100% solvent A prior to the next measurement, all at a flow rate of 0.4 mL min–1. Detection was carried out at 440 nm, and peaks were identified by comparing their UV spectra and their retention times with commercially available standards. 
