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The biogenesis of photosynthetic membranes in plants relies largely on lipid import from the endoplasmic reticulum (ER) and this lipid transport process is mediated by TGD proteins in Arabidopsis. Such a dependency of chloroplast biogenesis on ER-to-plastid lipid transport was recently exemplified by analyzing double mutants between tgd1-1 or tgd4-3 and fad6 mutants. The fad6 mutants are defective in the desaturation of membrane lipids in chloroplasts and therefore dependent on import of polyunsaturated lipid precursors from the ER for constructing a competent thylakoid membrane system. In support of a critical role of TGD proteins in ER-to-plastid lipid trafficking, we showed that the introduction of the tgd mutations into fad6 mutant backgrounds led to drastic reductions in relative amounts of thylakoid lipids. Moreover, the tgd1-1 fad6 and tgd4-3 fad6 double mutants were deficient in polyunsaturated fatty acids in chloroplast membrane lipids, and severely compromised in the biogenesis of photosynthetic membrane systems. Here we report that these double mutants are severely impaired in chloroplast division. The possible role of membrane lipids in chloroplast division is discussed.
Intracellular lipid transfer is essential for organelle biogenesis, membrane proliferation and lipid homeostasis and signaling,1,2 but the underlying mechanisms remain elusive. The biosynthesis of glycolipids characteristic of photosynthetic membranes in plants encompasses two parallel pathways involving the endoplasmic reticulum (ER) and plastids.3–5 This necessitates the extensive transfer of lipids and lipid precursors between these two organelles, within and across their membranes. During the last decade, much progress has been made in our understanding of the enzymes and pathways involved in lipid biosynthesis in plants,6 and even the molecular components involved in ER-to-plastid lipid trafficking are beginning to be unrevealed,3 particularly in the model plant Arabidopsis.
The discovery of Arabidopsis TGD proteins has led to new insights into lipid-trafficking phenomena involving plastids.7–10 Among the four TGD proteins so far identified, TGD1, TGD2 and TGD3 presumably form a multipartite ABC lipid transporter localized in the inner chloroplast envelope membrane, and are responsible for the transfer of phosphatidic acid (PA) across this membrane. Genetic ablation of this transporter blocks lipid trafficking between the ER and the plastid. Recently, a novel Arabidopsis protein, TGD4, was identified by a genetic approach.9 The current working hypothesis is that TGD4 mediates the transfer of phosphatidylcholine (PC) assembled at the ER to the outer plastid envelope.3 Hydrolysis of PC by an unknown phospholipase D generates PA, the proposed substrate of the TGD1, TGD2 and TGD3 complex. Alternatively, TGD4 may be directly involved in PA transfer from the ER to the outer envelope of chloroplasts.11
To provide further genetic evidence for the role TGD proteins in lipid trafficking, we constructed the double mutants of tgd1-1,8 or tgd4-3,9 with the fad6-1 mutant12 defective in the chloroplast 16:1/18:1 desaturase. Because the fad6-1 mutant is dependent upon import of polyunsaturated lipid precursors from the ER for constructing a competent photosynthetic membrane system,12,13 the double mutants between tgds and fad6-1 were expected to be compromised in chloroplast biogenesis. Indeed, lipid analysis revealed that the plastid-specific galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were strongly reduced in the tgd1-1 fad6-1 and tgd4-3 fad6-1 double mutants whereas the extraplastidic membrane lipid phosphatidylethanolamine (PE) was markedly increased,14 suggesting a decreased ratio of plastid-to-extraplastidic membranes in the double mutants. In support, ultrastructural analysis revealed that the thylakoid membrane systems in double mutants were severely underdeveloped with almost no grana stacking, particularly in the tgd4-3 fad6-1 double mutant. In addition, analysis of leaf cross sections revealed that the double mutants displayed a large reduction in the number of chloroplasts per cell compared with the wild type, fad6 and tgd1-1 mutants (Fig. 1). Most of mesophyll cells in both fad6-1 tgd1-1 and fad6-1 tgd4-3 double mutants appeared to contain only one, sometimes two enlarged chloroplast(s) (Fig. 1D and E) as was observed in the double mutant of tgd4-1 with the ats1-1 mutant defective in plastidic glycerol-3-phosphate acyltransferase thereby deficient in thylakoid lipids derived from the plastid.9
One major question arose from these observation concerns the role of individual membrane lipids in chloroplast division. Because the chloroplast division defects were observed in both tgds fad6-1 and tgd4-1 ats1-1 double mutants and the latter double mutant contained abundant polyunsaturated fatty acids in thylakoid lipids,9 it seems unlikely that deficiency in the level of fatty acid unsaturation in chloroplast membrane lipids is one of the major factors affecting chloroplast division. Likewise, the facts that the number of chloroplasts per cell cross-section in both ats1,15 and tgd1-1 (Fig. 1C) mutants were comparable to that observed in wild type suggest that the chloroplast division phenotypes observed in tgds fad6 and tgd4-1 ats1-1 double mutants are not due to lack of either ER- or plastid-derived thylakoid lipids. The possible involvement of individual thylakoid lipids in chloroplast division is also not supported by genetic studies showing that disrupting the biosynthesis of DGDG,15 in dgd1, phosphatidylglycerol in pgp1,16 or sulfoquinovosyldiacylglycerol in sqd2,17 mutant does not confer defects in chloroplast division. In both tgds fad6-1 and tgd4-1 ats1-1 double mutants, there were more than 50% reductions in levels of MGDG with concomitant increases in phospholipids, particularly PC.9,14 Whether these lipid changes are the cause of the chloroplast division defects in the double mutants can not be ascertained at this time. However, it has been shown that a similar extent of reduction in the level of MGDG in the pgp1 mutant does not compromise chloroplast division.16 Thus, a more likely possibility is that the chloroplast defects in the double mutants are due to an inadequate lipid supply for membrane proliferation during chloroplast division. In support of this view, it has recently shown that inactivation of de novo fatty acid synthesis in the plastid also suppress chloroplast division.18 Further experiments are needed to understand the exact role of membrane lipids in chloroplast division.
This work was supported by a Laboratory Directed Research and Development Award at the Brookhaven National Laboratory under contract with the United States Department of Energy (to C.X.).