ReviewEvolution of galactoglycerolipid biosynthetic pathways – From cyanobacteria to primary plastids and from primary to secondary plastids
Introduction
Photosynthetic eukaryotes (algae, plants and some protists) are characterized by the presence of a chlorophyll-containing organelle, the chloroplast, whose origin dates back to a primary endosymbiotic event, when an ancestral cyanobacterium was engulfed within or invaded a primary eukaryotic host (for review, [1], [2], [3], [4], [5], [6], [7], [8]). The membrane architecture of cyanobacteria and primary chloroplasts are similar: both are delimited by a two-membrane envelope and contain flattened membrane sacs, or thylakoids, in which the photosynthetic complexes are embedded. These membranes have a lipid composition, which has been remarkably well conserved through evolution. In particular, they are characterized by a very high content in galactoglycerolipids, i.e., mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively). The anomery of the terminal galactosyl groups differs in these two lipids: in MGDG, the galactose is in β conformation, forming the 1,2-diacyl-3-O-(β-D-galactopyranosyl)-sn-glycerol structure, whereas in DGDG, the second galactose is in α conformation, forming 1,2-diacyl-3-O-(α-D-galactopyranosyl-(1→6)-O-β-D-galactopyranosyl)-sn-glycerol [9], [10] (Fig. 1). In this review, we shall refer to these structures as β−MGDG and αβ−DGDG. The transfer of galactose from one galactolipid to another, which occurs in Angiosperms during certain environmental stresses including exposure to ozone or cold, and leading to the production of ββ−DGDG, βββ−triGDG and ββββ−tetraGDG [11], [12] shall not be discussed here.
MGDG and DGDG were first isolated from the benzene extract of wheat flour (Triticum aestivum) by Carter et al. in 1956 [9]. The systematic inventory of lipids in photosynthetic organisms was initiated a decade later, taking advantage of the thin-layer chromatography separation methods developed by Nichols in 1963 [13] and Allen et al. in 1966 [14]. The ubiquity of galactolipids in all photosynthetic organisms emerged as they were discovered successively in cyanobacteria, e.g., Anacystis nidulans and Anabaena variabilis [15], various green algae, firstly Chlorella vulgaris [16], [17] and then Chlamydomonas reinhardtii [18], [19], various embryophyta (plants), e.g., the moss Hypnum cupressiforme [20], the fern Adiantum capillus-veneris [21], the gymnosperm Pinus sylvestris [22] and the angiosperm Spinacia oleracea [23], and eventually to various photosynthetic protists deriving from green algae, such as Euglena gracilis [24] or deriving from red algae, such as the diatom Phaeodactylum tricornutum [25]. The presence of MGDG and DGDG was recognized as a hallmark of all oxygen-evolving photosynthetic organisms [10], and consequently as the most abundant lipid classes on Earth [26]. Analytical technologies (mass spectrometry, NMR) have increased in sensitivity and throughput the last 15 years. Lipidomic characterization of numerous photosynthetic microorganisms in a large variety of genetic backgrounds, growth conditions and stresses have been made available [27] and provide precious information to start reconstructing the evolution of glycerolipid metabolism.
Together with phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG), MGDG and DGDG make up the lipid matrix hosting the photosystems [29]. Besides their role as a membrane component, galactoglycerolipids are also known to fulfill specific molecular functions. They stabilize photosystem subunits [29], [30], bind to the plastid protein import machinery [31], are a source of polyunsaturated fatty acids for various purposes and, in some eukaryotes like plants, DGDG was furthermore shown to be exported to extra-plastidial membrane compartments [32], [33], [34], where it could substitute for phosphoglycerolipids [32], [35], [36], [37]. These roles shall not be detailed here and the reader is invited to refer to some recent reviews [12], [28], [29].
Considering the ubiquity of MGDG and DGDG, one might expect that the enzymes involved in their biosynthesis have been conserved through evolution. Surprisingly, this is not the case: the biosynthetic machinery producing MGDG and DGDG in eukaryotes has strongly diverged from that found in extant cyanobacteria.
Here, we summarize the evolution of galactolipid metabolism beginning with the cyanobacteria, although this was not the first system to be elucidated. Indeed, genes coding for galactolipid synthesis enzymes were initially characterized in angiosperms by Shimojima et al. in 1997 [38] and Dörman et al. in 1999 [39]. Subsequently, other organisms were explored based on sequence similarity. Frustratingly, bioinformatic searches for galactolipid orthologs of plant genes in cyanobacteria provided no candidates, and their identification was eventually achieved by Awai et al. in 2006 [40] and 2007 [41] and by Sakurai et al. in 2007 [42] through more classical means. We shall therefore describe the evolution of these pathways, regardless of the timeline of scientific discovery.
The question of the evolution of galactoglycerolipid metabolism has to be formulated in the context of photosynthetic eukaryote evolution, which is characterized by dramatic transitions in subcellular architecture. Important evolutionary transitions comprise primary and secondary endosymbiotic events, from an ancestral cyanobacterium to a primary chloroplast (Fig. 2A), and then from a symbiotic unicellular alga to a secondary plastid (Fig. 2B), respectively (for reviews, [1], [2], [3], [4], [5], [6], [7]).
If we first consider the simplest situation observed in plants and algae (the Archaeplastida kingdom, Fig. 2A), cells contain “simple plastids” and molecular evidence supports the view that all these plastids trace back to a single event of endosymbiosis [1]. An envelope comprising two membranes (the inner envelope membrane, IEM, and the outer envelope membrane, OEM) delineates the chloroplasts and derives from the two limiting membranes of the Gram-negative cyanobacterial ancestor. Based on photosynthetic pigments, storage material and cell walls, three lineages of these primary plastid bearing eukaryotes have diverged: a blue lineage (Glaucophyta), a red lineage (Rhodophyta), and a green lineage (green algae and plants) (Fig. 2A). In the “blue lineage”, in which chlorophyll a is associated to phycocyanin and allophycocyanin, there is a small group of unicellular organisms (Glaucophyta), including Cyanophora paradoxa, in which the chloroplast still contains a peptidoglycan cell wall between the inner and outer envelopes. The “red lineage”, in which chlorophyll a is energetically coupled to phycobilin, includes the red algae or Rhodophyta, such as Cyanidioschyzon merolae. Lastly, the “green lineage”, in which chlorophyll a is associated to chlorophyll b, contains green algae or Chlorophyta, such as C. reinhardtii, and plants, or Streptophyta, such as Arabidopsis thaliana.
Recently it has emerged that the primary endosymbiotic creation of plastids was not a unique event. Reduced endosymbiotic cyanobacteria within cells of the amoeba Paulinellia (in the Rhizaria taxon) are now recognized as a second, independent origin of plastids [43]. This organelle, also called the chromatophore, is therefore derived from a cyanobacterium, but is not ontogenetically related to chloroplasts found in all other species examined to date.
It is more difficult to understand how unicellular organisms may contain plastids limited by more than two membranes. Fig. 2B gives some examples of reasonable scenarios (adapted from [1]). Protists originating from a secondary endosymbiosis belong to at least three lineages: two independent green lineages (Chlorarachniophytes and Euglenids), and a red lineage (Chromalveolates). The Chlorarachniophytes, such as Bigelowiella natans, have a plastid surrounded by four membranes. They have retained a relic of the endosymbiont algal nucleus, called a nucleomorph, between the two innermost and the two outermost membranes of their plastid [44] (Fig. 2B). On the other hand, some Euglenozoa such as E. gracilis contain a plastid limited by three membranes, and they lack a nucleomorph. Parasites of the Trypanosomatidae phylum, such as Trypanosoma bruceii, belong to Euglenozoa, but have no plastid.
The “red lineage”, in which a red alga is believed to have been engulfed by another eukaryote, is thought to account for all the other plastid-bearing protists. Significant biodiversity is represented in this lineage, including Cryptomonads, such as Guillardia theta, which have conserved a nucleomorph, Haptophytes, such as Nannochloropsis gaditana, Heterokonts, such as the diatoms P. tricornutum and Thalassiosira pseudonana, Chromerida, such as Chromera velia, and the closely related phylum of Apicomplexa, comprising human parasites such as Toxoplasma gondii and Plasmodium falciparum. Cavalier-Smith has proposed that most secondary endosymbionts of the red lineage can be grouped as Chromalveolata [45], a super-group which might not be monophyletic [46], [47]. They were also shown to have been subjected to large transfers of genes from a green algal origin, proposed to be from a secondary endosymbiosis involving a green alga prior to the red algal endosymbiosis that is believed to be common to all Chromalveolates [48]. The nuclear genomes of Chromalveolata are therefore chimeric, a feature we shall consider further later on. Alveolata, including Apicomplexa and Chromerida, and Heterokontophyta, including Diatoms and Eustigmatophytes discussed here, seem to be monophyletic within the Chromalveolata.
With respect to lipid metabolism it is well known that cyanobacteria, primary and secondary plastids all contain a machinery to generate fatty acids in their stroma, the dissociated fatty acid synthase of type II (FASII) [49]. These fatty acids are used as building blocks for glycerolipids, including the galactoglycerolipids discussed here. Fatty acids are successively esterified to the sn-1 and sn-2 positions of glycerol-3-phopshate to generate phosphatidic acid (PA), which is then dephosphorylated to form diacylglycerol (DAG), the universal precursor for galactoglycerolipids (for review, [50], [51]). In green algae and in plants, the assembly of galactolipids has progressively evolved from a utilization of PA/DAG precursors synthesized de novo within the plastid, like in cyanobacteria (the so called “prokaryotic” pathway), to the utilization of diacyl-moieties imported from the endoplasmic reticulum (the “eukaryotic” pathway) [52], [53], [54]. We shall therefore focus on the evolution of the synthesizing enzymes per se, which is of relevance for the first transition (cyanobacteria to primary chloroplasts), and also on the evolution of the upstream pathways, which generate the substrates for galactoglycerolipids.
Concerning the second transition (primary to secondary plastids), information is scarce and the field of research is still open for a range of fundamental investigations. Little is known regarding the localization of galactoglycerolipids in the secondary plastid membranes, or about their biosynthetic machinery and their integration into the general scheme of cellular metabolism. We have thus compared pathways based on available molecular and biochemical data, highlighting enzymatic reactions that appear to be conserved and some that may have diverged or been lost.
Section snippets
Biosynthesis of galactoglycerolipids in cyanobacteria
Cyanobacteria have been primarily classified as Gram-negative bacteria [55]. Their cell envelope is composed of an outer and a plasma membrane, separated by a peptidoglycan layer [56]. In addition to MGDG, DGDG, PG and SQDG, cyanobacteria were shown to also contain a very low proportion of a monoglucosyldiacylglycerol, or MGlcDG [55]. The glucosyl group is in β conformation, forming 1,2-diacyl-3-O-(β-D-glucopyranosyl)-sn-glycerol, or β−MGlcDG (see Fig. 1). This lipid has been detected in major
From cyanobacteria to primary plastids: emergence of a new galactolipid synthetic pathway
The galactolipid biosynthetic pathway in primary chloroplasts has been mainly characterized enzymatically in angiosperms and in a few green algal models. Once MGDG synthase (MGD) and DGDG synthase (DGD) genes had been identified in angiosperms [38], [39], it was rapidly evident that this pathway did not derive from the ubiquitous cyanobacterial system, and that this innovation was shared by all eukaryotic plastids, from Archaeplastida (Glaucophyta, Red algae, Green algae, plants) to secondary
The puzzling question of the lipidome of secondary plastids
Although the presence of MGDG and DGDG was confirmed decades ago in a range of important phyla including secondary endosymbionts deriving from green algae, e.g., E. gracilis [24], or Chromalveolata deriving from red algae, e.g., the non-photosynthetic diatom Nitzschia alba [108], the photosynthetic diatom P. tricornutum [25] and the eustigmatophyte Nannochloropsis [109], [110], we still lack clear information about (i) the precise localization of galactoglycerolipids in the three to four
Conclusion and perspectives
In conclusion, the evolution of galactoglycerolipid metabolism is marked by important transitions. In cyanobacteria, all species rely on a stepwise synthesis via a glucosyl intermediate, catalyzed by a MGlcDG synthase, incorporating glucose from UDP-Glc, an unknown epimerase converting glucose into galactose, and a dgdA-type DGDG synthase. As an exception to this rule, some cyanobacteria like Gloeobacter sp. have acquired a bacterial MGD enzyme by a recent horizontal transfer, enabling them to
Acknowledgements
The authors were supported by Agence Nationale de la Recherche (ANR-10-BLAN-1524, ReGal; ANR-12-BIME-0005, DiaDomOil; ANR-12-JCJC, ChloroMitoLipid and ApicoLipid), ATIP-Avenir-FINOVI (C.Y.B.), Région Rhône-Alpes, the Labex GRAL (Grenoble Alliance for Integrated Structural Cell Biology), Investissement d’Avenir OCEANOMICS, the EU-funded Diatomite and MicroB3 projects and the Australian Research Council.
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