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Photosynthetic membranes of plants primarily contain non-phosphorous glycolipids. The exception is phosphatidylglycerol (PG), which is an acidic/anionic phospholipid. A second major anionic lipid in chloroplasts is the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). It is hypothesized that under severe phosphate limitation, SQDG substitutes for PG, ensuring a constant proportion of anionic lipids even under adverse conditions. A newly constructed SQDG and PG-deficient double mutant supports this hypothesis. This mutant, sqd2 pgp1-1, carries a T-DNA insertion in the structural gene for SQDG synthase (SQD2) and a point mutation in the structural gene for phosphatidylglycerolphosphate synthase (PGP1). In the sqd2 pgp1-1 double mutant, the fraction of total anionic lipids is reduced by approximately one-third, resulting in pale yellow cotyledons and leaves with reduced chlorophyll content. Photoautotrophic growth of the double mutant is severely compromised, and its photosynthetic capacity is impaired. In particular, photosynthetic electron transfer at the level of photosystem II (PSII) is affected. Besides these physiological changes, the mutant shows altered leaf structure, a reduced number of mesophyll cells, and ultrastructural changes of the chloroplasts. All observations on the sqd2 pgp1-1 mutant lead to the conclusion that the total content of anionic thylakoid lipids is limiting for chloroplast structure and function, and is critical for overall photoautotrophic growth and plant development.
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The two major anionic/acidic lipids of photosynthetic membranes are phosphatidylglycerol (PG) and the non-phosphorous glycolipid sulfoquinovosyldiacylglycerol (SQDG). The phospholipid PG is common in the biological membranes of animals, plants, and microorganisms and was found to be essential in photosynthetic organisms (Babiychuk et al., 2003; Hagio et al., 2000, 2002; Sato et al., 2000). In vitro experiments showed that PG is enriched in light-harvesting pigment–protein complexes of photosystem II (PSII; Murata et al., 1990; Tremolieres et al., 1994), and that it is essential for the dimerization of the PSII reaction center core pigment–protein complex (Kruse et al., 2000). Furthermore, the recently published crystal structure of PSI contained three molecules of PG in tight co-ordination with the core and the antenna complex (Jordan et al., 2001). A PG-deficient mutant of the cyanobacterium Synechocystis sp. PCC6803 is no longer able to grow photoheterotrophically, and PSII activity was severely compromised (Hagio et al., 2000; Sato et al., 2000). In addition, a pale green mutant of Arabidopsis, pgp1-1, carrying a leaky mutation in the gene for phosphatidylglycerolphosphate synthase (PGP1) was recently described by Xu et al. (2002). In this mutant, a reduction in the content of PG of only 30% resulted in the impairment of photosynthesis. Moreover, complete inactivation of the PGP1 gene by T-DNA insertion led to the abolishment of chloroplasts and loss of photoautotrophy (Babiychuk et al., 2003; Hagio et al., 2002). These results suggested that PG was essential for chloroplast development and function in seed plants.
The second anionic lipid found in thylakoid membranes, the sulfolipid SQDG, is not essential under normal growth conditions. Mutants of photosynthetic bacteria and Arabidopsis completely lacking this lipid showed only subtle impairments in photosynthesis and growth unless they were phosphate starved (Benning et al., 1993; Güler et al., 1996; Yu et al., 2002). As a common phenomenon in photosynthetic organisms, the relative amount of total anionic thylakoid lipids is maintained by reciprocally adjusting SQDG and PG contents as phosphate availability changes. Typically, the relative content of PG decreases and that of SQDG increases following phosphate limitation. In SQDG-deficient mutants, the proportion of PG does not decrease under phosphate limitation and these mutants become phosphate starved sooner than the respective wild type. Based on these observations, it was suggested that one of the main functions for SQDG is to substitute for PG under phosphate limitation to maintain the proper balance of anionic charge in the thylakoid membrane (Benning et al., 1993; Güler et al., 1996; Yu et al., 2002). To further test this SQDG/PG substitution hypothesis by endogenously manipulating the total amount of anionic/acidic thylakoid lipids, we constructed an sqd2 pgp1-1 double mutant of Arabidopsis lacking SQDG completely and PG partially. The sqd2 mutant carries a T-DNA insertion into the sulfolipid synthase gene SQD2 (Yu et al., 2002). The weak allele, pgp1-1, for the PGP synthase gene PGP1 was preferred in this study because plants carrying stronger pgp1 homozygous alleles are already completely non-photosynthetic (Babiychuk et al., 2003; Hagio et al., 2002) and additive effects of SQDG deficiency cannot be revealed. Here, we describe the construction and characterization of the sqd2 pgp1-1 double mutant.
Growth of the sqd2 pgp1-1 double mutant is reduced
The sqd2 pgp1-1 double mutant was constructed by crossing the two respective single mutant lines (Xu et al., 2002; Yu et al., 2002). These lines were of different ecotypes (sqd2, Wassilewskija and pgp1-1, Columbia-2) – a fact that might have posed a serious problem for the interpretation of comparative data – as discussed below. To create nearly isogenic lines for the four genotypes compared in this study, sqd2/sqd2 PGP1/PGP1 (sqd2 mutant), SQD2/SQD2 pgp1-1/pgp1-1 (pgp1-1 mutant), SQD2/SQD2 PGP1/PGP1 (wild type), and sqd2/sqd2 pgp1-1/pgp1-1 (homozygous double mutant), a recombinant inbred line was generated by repeated selfing of single double heterozygous descendants from the original cross through eight generations. According to the theory by Reiter et al. (1992), the four different homozygous lines isolated in the F8 generation should be nearly (99.2%) isogenic, eliminating effects from genetic variations at loci different from those under direct consideration. In agreement with recessive mutations, the F1 progeny were phenotypically wild type. Thin-layer chromatography (TLC) of lipid extracts from 315 segregating F2 plants showed that 64 (approximately 3 out of 16) had a reduced proportion of PG (homozygous for pgp1-1), 60 were SQDG deficient (homozygous for sqd2), and 20 (approximately 1 out of 16) lacked SQDG and showed a reduced proportion of PG. The latter were the expected homozygous sqd2 pgp1-1 double mutants.
The cotyledons and true leaves of sqd2 pgp1-1 double mutants were paler in color than those of the pgp1-1 mutants, as shown in Figure 1. The true leaves of the double mutant were smaller, and its growth rate was reduced. As shown in Figure 2(a), the FW of the aerial part of the double mutant was reduced to approximately one-third compared to the other lines at 3 weeks of growth on phosphate-replete medium. The double mutant was able to survive on soil and on agar-solidified medium lacking sucrose, but the growth rate was greatly reduced compared to plants grown in the presence of sucrose (data not shown). These results suggested that the sqd2 pgp1-1 double mutant is capable of photoautotrophic growth, but its growth is severely limited by a compromised photosynthetic apparatus. Under phosphate-limited conditions, the growth of the double mutant was reduced compared to the wild type and the pgp1 mutant but to nearly the same extent as that observed for the sqd2 mutant (Figure 2b).
The proportion of total anionic lipids in the double mutant is decreased
Total leaf lipid analysis of the sqd2 pgp1-1 double mutant as shown in Figure 3(a) revealed that the proportion of PG was approximately 11 mol% higher than that in the pgp1-1 mutant. As PG is also found in extraplastidic membranes, this result might reflect a decrease in the absolute amount of chloroplast membranes. Indeed, the proportion of the exclusively extraplastidic lipid phosphatidylethanolamine (PE) was also increased in the double mutant compared to the pgp1-1 mutant, in agreement with an overall increase in the ratio of extraplastidic to plastidic membrane lipids. Comparing the lipid composition of isolated chloroplasts from the four different lines as shown in Figure 3(b), a similar decrease in the relative amount of PG was observed for the pgp1-1 mutant and the sqd2 pgp1-1 double mutant corroborating the hypothesis that the ratio of extraplastidic to plastidic lipids is increased in the double mutant. Summing up the amount of the anionic lipids SQDG and PG in chloroplast extracts (Figure 3b, inset), it was apparent that the total amount of anionic lipids in the two single mutants remained similar to that of the wild type, but was reduced in the sqd2 pgp1-1 double mutant by approximately one-third (99% confidence level based on Student's t-test). The relative increase in SQDG in the pgp1-1 mutant and the relative increase in PG in the sqd2 single mutant appeared to reciprocally compensate the respective deficiency in the alternate anionic lipid. These compensatory mechanisms were also visible when total leaf lipid extracts were examined following phosphate-limited growth (Figure 3c). The relative amount of PG in the two SQDG-deficient lines was similarly maintained under these conditions, explaining the similar growth reduction of the sqd2 and sqd2 pgp1-1 double mutant following phosphate deprivation (Figure 2b). In addition to these changes in relative anionic lipid content, the proportion of the galactolipid digalactosyldiacylglycerol (DGDG) increased following phosphate limitation, as was previously observed for Arabidopsis (Härtel et al., 2000).
Photosynthetic activity is compromised in the double mutant
The pale green leaves of the sqd2 pgp1-1 double mutant suggested a reduction in chlorophyll content and a possible impairment in photosynthetic activity. Indeed, the amounts of chlorophylls a and b and carotenoids were decreased in the double mutant to approximately one-third that observed for the wild type and sqd2 mutant and to one-half that of the pgp1-1 mutant (Table 1). The chlorophyll a/b ratio of the double mutant was higher than that for the wild type and sqd2, but was similar to that of the pgp1-1 mutant. Taken together, these results suggested that the ratio of light-harvesting antenna to reaction core complex was similarly altered in the pgp1-1 single and the sqd2 pgp1-1 double mutants, but that the total amount of pigment–protein complexes and therefore the extent of total thylakoid membrane in the double mutant was strongly reduced. Photosynthesis in plants of the four lines included in this study was investigated in greater detail using pulse amplitude-modulated chlorophyll fluorescence analysis, a sensitive and non-invasive method (Krause and Weis, 1991). This method provides an indication of the overall photosynthetic competence of leaves and indirectly permits conclusions with regard to the electron-transport reactions at the thylakoid membranes. As shown in Figure 4(a,b), there was no obvious difference in the maximum intrinsic efficiency of PSII in the light-adapted state (FV′/FM′) over a wide range of photosynthetic photon flux densities (PPFDs), but the quantum yield of linear electron transfer through PSII (φPSII) was reduced in the double mutant (95% confidence level based on t-test), especially in the low PPFD range as compared to the wild type, sqd2, and pgp1-1. The deduced fluorescence parameter 1–qP, which reflects the fraction of ‘open’ PSII reaction centers, provides an indication of the reduction state of the primary electron acceptor of PSII, QA. This parameter was increased in the sqd2 pgp1-1 double mutant (99% confidence level), while no obvious differences were visible for the wild type and the two single mutant lines sqd2 and pgp1-1. Therefore, it seemed likely that PSII is more reduced in the sqd2 pgp1-1 double mutant in accordance with a decreased quantum yield of PSII (φPSII). To verify this hypothesis using an independent approach, we tested the effect of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the growth of plants. This herbicide binds at the secondary electron acceptor (QB) site of PSII, and blocks the electron transfer from QA to QB (Oettmeier and Soll, 1983). As shown in Figure 5, the sqd2 pgp1-1 double mutant was considerably more sensitive to DCMU than the two single mutant lines and the wild type, in agreement with an increased reduction pressure at the QA site of PSII in the double mutant. After 1 week in the presence of 13.5 nm DCMU, the relative growth of the double mutant was reduced to approximately 50% (99% confidence level), while the other three lines were still growing normally. A concentration of 405 nm DCMU led to a complete cessation of growth of the sqd2 pgp1-1 double mutant. At even higher concentrations of DCMU, the single mutants also showed increased sensitivity to the herbicide compared to the wild type, suggesting that complete SQDG deficiency or a decrease of the PG content by 30% was sufficient to cause subtle changes in PSII activity, however, with no apparent effect on growth under normal laboratory conditions (Figure 2a).
Table 1. Pigment content (mg g−1 FW)a of wild-type and mutant lines
Samples were taken from fully expanded leaves of 20–30-day-old plants grown on agar-solidified medium containing 1 mm phosphate. Pigment values represent means (±SE) of at least four independent determinations.
0.86 ± 0.04
0.99 ± 0.07
0.61 ± 0.07
0.32 ± 0.03
0.29 ± 0.01
0.36 ± 0.02
0.18 ± 0.02
0.09 ± 0.01
Chlorophyll a + b
1.14 ± 0.05
1.34 ± 0.1
0.80 ± 0.08
0.42 ± 0.04
2.98 ± 0.03
2.76 ± 0.03
3.3 ± 0.1
3.54 ± 0.16
0.25 ± 0.01
0.27 ± 0.02
0.18 ± 0.02
0.10 ± 0.08
Leaf structure and chloroplast ultrastructure are altered in the double mutant
The biochemical analysis of the sqd2 pgp1-1 double mutant described above provided indirect evidence for an increased ratio of extraplastidic to plastidic lipids. To corroborate this hypothesis in more direct ways, the leaf structure of the different lines was compared by light microscopy of thick sections (Figure 6). The sqd2 pgp1-1 double mutant showed larger intercellular spaces and a reduction in mesophyll cell numbers. The epidermal cells and mesophyll cells were enlarged. Mesophyll cells with chloroplasts were only present adjacent to vascular structures. Chloroplast numbers per cell cross-section were reduced in the sqd2 pgp1-1 double mutant to approximately 50% of the wild type. Averaging at least 30 sections per sample, numbers of chloroplasts per cell cross-section ± SD were for: the wild type, 8.0 ± 0.3; sqd2, 7.9 ± 0.2; pgp1-1, 8.0 ± 0.3, and sqd2 pgp1-1, 3.9 ± 0.3. The ultrastructures of chloroplasts in emerging (not yet expanded) leaves of the wild type and the double mutant are shown in Figure 7(a,b). Wild-type chloroplasts contained well-developed thylakoid membranes contrary to the double mutant chloroplasts, which nearly lacked grana stacks. Figure 7(c–e) depicts chloroplasts of the expanded first true leaves from the wild type, sqd2, and pgp1-1. Chloroplasts in these samples were generally indistinguishable. However, in chloroplasts of the double mutant, the thickness of the thylakoid grana stacks (Figure 7f) appeared to be reduced (wild type, 0.22 ± 0.03 µm; sqd2, 0.20 ± 0.03 µm; pgp1-1, 0.21 ± 0.03 µm; sqd2 pgp1-1, 0.11 ± 0.02 µm; n = 25).
The analysis of genetic mutants with biochemical defects provides a powerful tool to understand the function of the affected pathway and the molecule in question. As more biochemical mutants of Arabidopsis become available, opportunities arise to combine these mutants in single lines, thereby revealing the interaction of two and more pathways and compounds. Combining mutations in different pathways in a single line might reveal redundancies and limit the plant's adaptive responses. An example is the dgd1 act1 double mutant, which is unable to grow photoautotrophically, while the parental mutants are much less affected in their growth (Dörmann et al., 1999). The phenotype was interpreted as a reduction of flux through the endoplasmic reticulum and plastid-based pathways of thylakoid lipid biosynthesis, which are partially redundant in Arabidopsis (Klaus et al., 2002).
Plants are well known for their broad biochemical repertoire, enabling them to adapt to and survive under adverse environmental conditions. A mutation in a single biochemical pathway of mostly conditional importance might present no phenotype at all, or might have only mild effects on the development or physiology of the plant under standard laboratory conditions. Sulfolipid-deficient mutants are a particularly good example. When the first sulfolipid-deficient mutants of bacteria became available, their growth was generally not affected by the mutation under optimal growth conditions (Benning et al., 1993; Güler et al., 1996). In the case of the cyanobacterium Synechococcus sp. PCC7942, subtle changes in PSII photochemistry were observed. However, these changes were not growth-limiting (Güler et al., 2000). Arabidopsis completely lacking sulfolipid also did not show a growth impairment under standard laboratory growth conditions (Yu et al., 2002), which may not necessarily duplicate conditions encountered by wild plants in nature. However, when additional metabolic pressure was applied to sulfolipid-deficient mutants, they began to lag behind the wild type in growth. Phosphate limitation brings about a metabolic impasse, to which bacteria and plants respond by decreasing the ratio of phosphorous to non-phosphorous lipids (Benning et al., 1995; Härtel et al., 2000). Sulfolipid deficiency limits the ability of photosynthetic organisms to remodel their thylakoid membrane, partially replacing PG with SQDG. This inability to adjust was proposed as the primary cause of reduced growth of sulfolipid-deficient mutants under phosphate limitation (Benning et al., 1993; Güler et al., 1996; Yu et al., 2002).
A different independent way of demonstrating the conditional importance of sulfolipid is to study SQDG deficiency in a double mutant impaired also in the biosynthesis of PG. Two issues had to be addressed for the construction of such a double mutant and the corresponding control lines. First, severe PG deficiency leads to a complete lack of photosynthesis making it impossible to study the more subtle effects of SQDG in such a line (Hagio et al., 2002). This issue was overcome by choosing the weak allele pgp1-1, which causes only a 30% reduction in PG content. However, the choice of this allele posed a second problem, namely that the sulfolipid-deficient mutant of Arabidopsis, sqd2, carrying a null allele caused by T-DNA insertion was of a different ecotype. As the conclusions of this study were expected to be based on subtle differences between the double mutant, the two single mutants, and the wild type, it was prudent to generate nearly isogenic lines by inbreeding through eight generations. This strategy resulted in four strains with the required homozygous sqd2 or pgp1-1 alleles in a 99.2% identical genetic background in all four lines.
The sqd2 pgp1-1 double mutant analysis demonstrated that SQDG deficiency can be growth-limiting if the PG-based compensatory mechanism is eliminated. The two biochemical defects in this mutant caused a reduction in the overall anionic thylakoid lipid content. This was not the case for the single mutants, which compensate by adjusting the amount of the alternate anionic lipid (Figure 3b). It is proposed that the overall reduction in anionic lipids limits the development of fully functional photosynthetic membranes and therefore photoautotrophic growth. Different independent phenotypes of the double mutant were consistent with a limitation in thylakoid membrane lipid biosynthesis leading to an increase in the ratio of extraplastidic to plastidic lipids in the sqd2 pgp1-1 double mutant: (i) the proportions of PE, which exclusively occurs in extraplastidic membranes, and of PG present in plastidic and extraplastidic membranes were increased (Figure 3); and (ii) pigments exclusively associated with the photosynthetic membranes were reduced on a FW basis (Table 1), and most directly, mesophyll cell numbers, chloroplasts per cell, and the amount of thylakoid membranes within chloroplasts were reduced (Figures 6 and 7).
Beyond the inability of the sqd2 pgp1-1 double mutant to assemble thylakoid membranes at a rate required for normal growth, lack of SQDG, and reduction in PG appeared to have also more direct effects on the photosynthetic apparatus. The double mutant clearly showed altered chlorophyll fluorescence consistent with a reduced electron transport rate through PSII in the double mutant, as compared to the single mutants or the wild type (Figure 4). This effect was further aggravated by additions of the herbicide DCMU, which blocks the electron transfer from PSII to the QB plastoquinone acceptor (Oettmeier and Soll, 1983). Contrary to the control lines, growth of the double mutant was three orders of magnitude more sensitive to DCMU (Figure 5). Studying a sulfolipid-deficient mutant of Chlamydomonas reinhardtii, Sato et al. (2003) and Minoda et al. (2002) have observed that this mutant is sensitive to DCMU and shows other signs of photosynthetic impairment. In the case of Arabidopsis as studied here, there is only a slight increase in sensitivity toward DCMU in the SQDG-deficient and PG-deficient mutants, suggesting that the sensitivity of the double mutant is not specifically because of SQDG deficiency, but a consequence of the reduction of anionic lipids in general. The alga C. reinhardtii contains a relatively high proportion (approximately 12 mol%) of SQDG (Sato et al., 1995), compared to Arabidopsis (approximately 3 mol%), and SQDG deficiency may sufficiently reduce the amount of anionic thylakoid lipids to cause an impairment in photosynthesis by itself. This effect became more aggravated at elevated temperature (Sato et al., 2003), suggesting structural roles for SQDG in association with pigment–protein complexes of the photosynthetic membrane. In general, the findings made with the C. reinhardtii mutant and the Arabidopsis sqd2 pgp1-1 double mutant agree, suggesting a role of SQDG as an anionic lipid in the assembly and proper function of the photosynthetic membrane. In the case of Arabidopsis, which contains only small amounts of SQDG, it takes the additional reduction in PG content to clearly bring about the effects of sulfolipid deficiency under normal growth conditions. However, under phosphate-limited growth conditions, when the relative amount of SQDG is increased in the wild type, the inability to synthesize SQDG as a substitute for PG quickly becomes limiting for growth in an SQDG-deficient mutant. At first glance, it seemed puzzling that under phosphate-limited conditions, the sqd2 pgp1-1 double mutant showed no additional reduction in growth in comparison to sqd2 (Figure 2). Upon closer examination of the lipid composition in the double mutant and sqd2 (Figure 3), it is apparent that the relative PG content in these two lines under phosphate limitation was similar, and therefore no further reduction of anionic lipids in the double mutant occurred. Presumably, the PG-biosynthetic activity provided by the enzyme encoded by the pgp1-1 allele was sufficient under these conditions to keep up with the demand of PG biosynthesis.
Whatever the specific biochemical or molecular roles that anionic lipids might fill in the photosynthetic membrane, i.e. providing a proton-conducting pathway at the surface of the thylakoid membrane (Haines, 1983), or as boundary lipids and integral components of photosynthetic complexes (Barber and Gounaris, 1986), the double mutant analysis described here led to the conclusion that the total amount of the anionic lipids PG and SQDG is most critical. These findings also support the general concept of substituting one anionic lipid for another under phosphate-limited conditions. While SQDG and PG both play an important role in this adaptive mechanism to maintain the proper balance of anionic lipids in the thylakoid membrane, PG might have additional and more specific functions, as the greater impairment of the PG-deficient single mutants suggests (Babiychuk et al., 2003; Hagio et al., 2002).
Plant material, growth experiments, and DCMU treatment
Arabidopsis wild type and mutants were grown under a PPFD of 70–80 µmol m−2 sec−1 at 22/18°C (day/night) with a 14-h light/10-h dark period. Surface-sterilized seeds were germinated on 0.8% (w/v) agar-solidified Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 1% sucrose. Seedlings were grown for 8 days on agar before transfer to soil or plates containing Arabidopsis medium (half strength without sucrose) (Estelle and Somerville, 1987). For phosphate limitation experiments, KH2PO4 was omitted from the medium and substituted by 2-(N-morpholino) ethane sulfonic acid (MES) buffer (Härtel et al., 2000). Quantitative growth experiments were carried out as described by Yu et al. (2002). Briefly, plants were grown under a uniform PPFD of 80 µmol m−2 sec−1. At each time point, 7–10 plants per line were harvested and their aerial parts were weighed. For DCMU treatment, half-strength Arabidopsis medium without sucrose was used with addition of different concentrations of DCMU as indicated. Plants were grown on MS medium for 8 days and then transferred to the DCMU-containing agar plates. After 7 days of growth, the FW of four to five plants was averaged and relative growth was plotted as the ratio of the FW per plant of DCMU untreated to that of DCMU treated samples.
Construction of the sqd2 pgp1-1 double mutant
To generate the sqd2 pgp1-1 double mutant, sqd2 (Yu et al., 2002) was used as the pollen donor in a cross to pgp1-1 (Xu et al., 2002). The ecotypes were Wassilewskija for the sqd2 mutant and Columbia-2 for pgp1-1. Nearly isogenic lines were generated by inbreeding through eight generations. Following each round of selfing, seeds of 20 individual plants were collected, 100–200 seeds per plant were plated, and the segregation of young seedlings was phenotypically scored. This was accomplished by visual examination of the plants, as homozygous pgp1-1 seedlings, double homozygous mutants, and the wild type were clearly distinguishable from each other and other genotypes based on their tint and size (Figure 1). Descendants from a double heterozygous line were analyzed again in the next generation until the four required lines were recovered from a single F7 double heterozygous plant.
Analysis of lipids from leaves and chloroplasts
Leaves were harvested and frozen immediately in liquid nitrogen, and lipids were extracted as previously described by Dörmann et al. (1995). Lipid extracts were analyzed on activated, ammonium sulfate-impregnated silica gel TLC plates (Härtel et al., 2000) using a running solvent of acetone:toluene:water (93 : 30 : 8 v/v/v). Lipids were visualized with iodine vapor and identified by co-chromatography with lipid extracts of known composition. For quantitative analysis, methyl esters were prepared and quantified by GLC (Rossak et al., 1997). For chloroplast lipid analysis, plastids were isolated as described by Härtel et al. (2000). The chloroplasts were immediately re-suspended in 200 μl chloroform:methanol:formic acid (10 : 10 : 1, v/v/v), followed by the addition of 100 μl of 0.2 m H3PO4 and 1 m KCl. In all quantitative experiments, Student's t-test was used to assess the significance of differences between samples.
Transmission electron microscopy and light microscopy
Three leaves from each line were prepared for light and transmission electron microscopy. The leaves were fixed with 2.5% (v/v) glutaraldehyde and 2% paraformaldehyde (v/v) in 0.1 m sodium phosphate buffer (pH 7.4) by infiltration under vacuum and continually incubated at ambient pressure and 4°C for 24 h. The samples were post-fixed with 1% osmium tetroxide in the same buffer for 2 h at room temperature. This was followed by a serial dehydration with 50, 70, 80, 88, 95, and 100% (v/v) of acetone in water. The specimens were infiltrated with a series of 33, 50, 75, and 100% (v/v) of poly/bed resin (Polyscience Inc., Warrington, PA, USA) in acetone. After embedding in poly/bed resin, three blocks each of the wild type and mutants were sectioned and the sections were stained with 1% toluidine blue and examined with a Zeiss Pascal confocal laser scanning microscope (Carl Zeiss, Germany). The thin sections were stained with 2% uranyl acetate and lead citrate before viewing in a JEOL 100CX transmission electron microscope (JEOL Inc., Peabody, MA, USA).
We thank Dr Alicia Pastor for her help with light and transmission electron microscopy and Dr Changcheng Xu for the sqd2 pgp1-1 F1 seeds. This work was supported in parts, by grants from the US Department of Energy Bioscience Program and the MSU Center for Novel Plant Products.