teaching and research institutions in France or abroad, or from public or private research centers.


Nolwenn Guéguen
email: nolwenn.gueguen@cea.fr

Eric Maréchal
email: eric.marechal@cea.fr

Affiliation

Origin of Cyanobacteria thylakoids via a non-vesicular glycolipid phase transition and impact on the Great Oxygenation Event Authors

Keywords: Cyanobacteria, thylakoid, Gloeobacter, monoglucosyldiacylglycerol, monogalactosyldiacylglycerol, Hexagonal II lipid, phosphatidylglycerol, sulfoquinovosyldiacylglycerol, MGD, Great Oxygenation Event PtdCho, phosphatidylcholine PtdEtn, phosphatidylethanolamine PtdGro, phosphatidylglycerol

The quest for thylakoid origin resists vesicular-based scenarios. An unexplored hypothesis is based on Hexagonal II-forming glycolipids, turned within nanoseconds and without any external energy source into membrane multilayers.

Introduction

The appearance of oxygenic photosynthesis is a major event in the early evolution of Life and had an irreversible impact on our planet. This bioenergetic and trophic innovation, coupling H 2 O splitting upon exposure to sun light with the reduction of atmospheric CO 2 , occurred in the Cyanobacteria ancestral lineage [START_REF] Sanchez-Baracaldo | On the origin of oxygenic photosynthesis and Cyanobacteria[END_REF][START_REF] Cardona | Thinking twice about the evolution of photosynthesis[END_REF][START_REF] Cardona | Early Archean origin of heterodimeric Photosystem I[END_REF][START_REF] Cardona | Early Archean origin of Photosystem II[END_REF]. It deeply modified ecosystems, positioning Cyanobacteria as prominent primary producers at the base of trophic networks, and allowed the development of a tremendous biodiversity of secondary and tertiary consumers. At geological scale, the production of O 2 as a byproduct of water splitting enabled the oxygenation of the atmosphere during the Great Oxygenation Event (GOE), 2.4-2.32 billion years ago (b.y.a.) [START_REF] Bekker | Dating the rise of atmospheric oxygen[END_REF][START_REF] Lyons | The rise of oxygen in Earth's early ocean and atmosphere[END_REF].

Later, oxygenic photosynthesis was acquired by Eukaryota of the Archaeplastida phylum, by integration of an intracellular organelle called the 'chloroplast', deriving from the engulfment of a close relative of the deep-branching Gloeomargarita cyanobacterium, combined with components acquired by gene transfers from a variety of other sources [START_REF] Ponce-Toledo | Horizontal and endosymbiotic gene transfer in early plastid evolution[END_REF][START_REF] Sato | Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis[END_REF][START_REF] Sato | Diverse origins of enzymes involved in the biosynthesis of chloroplast peptidoglycan[END_REF][START_REF] Marechal | Primary Endosymbiosis: Emergence of the Primary Chloroplast and the Chromatophore, Two Independent Events[END_REF]. Following this unique endosymbiotic event, the expansion of marine eukaryotic phytoplankton correlated with the oxygenation of the oceans during the Neoproterozoic Oxygenation Event (NOE), 800-600 million years ago. This so-called 'rise of Algae' is thought to have created food webs with even more efficient nutrient and energy transfers, driving ecosystems towards larger and more complex organisms, including animals [START_REF] Brocks | The rise of algae in Cryogenian oceans and the emergence of animals[END_REF].

Access to increasing genomic data and refine reconstructions of molecular phylogenies have improved our understanding of the early evolution of photosynthetic reaction center proteins [START_REF] Sanchez-Baracaldo | On the origin of oxygenic photosynthesis and Cyanobacteria[END_REF][START_REF] Cardona | Thinking twice about the evolution of photosynthesis[END_REF][START_REF] Cardona | Early Archean origin of heterodimeric Photosystem I[END_REF][START_REF] Cardona | Early Archean origin of Photosystem II[END_REF]. Schematically, oxygenic photosynthesis requires two sets of dimeric reaction centers, one of type II (in photosystem II, PSII) and one of type I (in PSI), all deriving from a unique origin in an ancestral anoxygenic photosynthetic prokaryote. Whereas single PSII-like or PSI-like reaction centers have evolved independently in various clades of anoxygenic phototrophic Bacteria [START_REF] Hohmann-Marriott | Evolution of photosynthesis[END_REF], crown group Cyanobacteria inherited both an heterodimeric versions of PSII, where water-splitting occurs, and an heterodimeric PSI [START_REF] Sanchez-Baracaldo | On the origin of oxygenic photosynthesis and Cyanobacteria[END_REF]. Recent estimates of the age of PSII and PSI gene families coincide with geological evidence of oxygen traces throughout a division of Precambrian time called the Archean eon (4-2.5 b.y.a.). This implies that oxygenic photosynthesis was established by 3.0 b.y.a., long before the GOE [START_REF] Cardona | Early Archean origin of heterodimeric Photosystem I[END_REF][START_REF] Cardona | Early Archean origin of Photosystem II[END_REF]. It is still unresolved whether a 'protocyanobacteria' acquired the two sets by gene duplication of anoxygenic photosystems [START_REF] Martin | A physiological perspective on the origin and evolution of photosynthesis[END_REF] or via gene transfers [START_REF] Raymond | Wholegenome analysis of photosynthetic prokaryotes[END_REF]. Amongst plausible scenarios, a versatile protocyanobacteria containing two reaction centers, i.e. an H 2 S splitting PSI (photolithotrophy) with an expression controlled by fluctuating hydrogen sulfide concentrations, and a PSII (photoorganotrophy), might have developed in shallow water at the vicinity of temporary hydrothermal springs. This protocyanobacteria having a flexible metabolism adapted to alternating redox conditions, might have acquired oxygenic photosynthesis by mutations in the direction of a constitutive expression of both Types I and II reaction centers, and an extraction of electrons, firstly from manganese and secondarily from water. This hypothesis was termed the 'Redox switch hypothesis for the first Cyanobacteria' [START_REF] Allen | A Proposal for Formation of Archaean Stromatolites before the Advent of Oxygenic Photosynthesis[END_REF][START_REF] Allen | Origin of Oxygenic Photosynthesis from Anoxygenic Type I and Type II Reaction Centers[END_REF].

Since oxygenic photosynthesis predated the GOE by about 0.5 to 1 b.y., what was the actual driver of this major atmospheric upheaval if not photosynthesis appearance per se? The extant diversity of Cyanobacteria evolved after the GOE (Sanchez- [START_REF] Sanchez-Baracaldo | On the origin of oxygenic photosynthesis and Cyanobacteria[END_REF]. Most ancient Cyanobacteria are likely to have populated low salinity habitats and dry environments (Blank and Sanchez-Baracaldo, 2010) as small unicellular forms close to present-day Gloeobacter (Sanchez- [START_REF] Sanchez-Baracaldo | On the origin of oxygenic photosynthesis and Cyanobacteria[END_REF][START_REF] Mares | Evolutionary Patterns of Thylakoid Architecture in Cyanobacteria[END_REF][START_REF] Raven | Gloeobacter and the implications of a freshwater origin of Cyanobacteria[END_REF]. The GOE was proposed to be the consequence of the ability of Cyanobacteria to form multicellular filaments, resembling extant Pseudanabaena, facilitating the formation of microbial mats, a feature supposed to increase their ecological dominance to such an extent that atmospheric O 2 underwent this dramatic increase [START_REF] Sanchez-Baracaldo | On the origin of oxygenic photosynthesis and Cyanobacteria[END_REF]).

An alternative hypothesis based on studies reviewed here is that such an important atmospheric change as the GOE requires an extraordinary multiplier effect that multicellularity, introducing no additive effect, is not sufficient to explain. The transition from Gloeobacter-type to other Cyanobacteria, is marked by the emergence of internal membranes sacks, known as 'thylakoids', allowing a significant gain in the area of the 'solar panels' accommodating photosystems [START_REF] Mares | Evolutionary Patterns of Thylakoid Architecture in Cyanobacteria[END_REF]. This review highlights the role of lipid components in Cyanobacteria membranes, disregarded until now in the determinant processes at the origin of thylakoids.

2

Emergence of thylakoids in early Cyanobacteria had a multiplier effect on the photosynthetic surface per cell.

Thylakoids are evidently not required for oxygenic photosynthesis since Gloeobacter hosts its photosystems in its cytoplasmic (or inner) membrane [START_REF] Raven | Gloeobacter and the implications of a freshwater origin of Cyanobacteria[END_REF]. The evolution of thylakoids starts with the appearance of continuous linear sacks with several concentric, uninterrupted layers [START_REF] Mares | Evolutionary Patterns of Thylakoid Architecture in Cyanobacteria[END_REF]. Based on a spherical cell with a 1 µm diameter (volume = 0.53 µm 3 ), a Gloeobacter membrane surface would be ~3.14 µm 2 . Taking into account that in Gloeobacter, photosynthetic proteins are concentrated in domains representing less than a third of the cytosolic membrane [START_REF] Rexroth | The plasma membrane of the cyanobacterium Gloeobacter violaceus contains segregated bioenergetic domains[END_REF], the actual photosynthetic surface is in the 1 µm 2 range. By contrast, in a Cyanobium-like coccal cell of same size [START_REF] Mares | Evolutionary Patterns of Thylakoid Architecture in Cyanobacteria[END_REF], with four concentric thylakoids lining the plasma membrane (so-called parietal thylakoids), the photosynthetic surface would reach ~20 µm 2 . Parietal thylakoid fascicules encountered in Synechocystis-like coccal or Leptolyngbya-like filamentous cyanobacteria [START_REF] Mares | Evolutionary Patterns of Thylakoid Architecture in Cyanobacteria[END_REF] with four layers would reach about the same photosynthetic surface per cell. Cyanobacteria containing parallel or radial arrangement of thylakoids [START_REF] Mares | Evolutionary Patterns of Thylakoid Architecture in Cyanobacteria[END_REF] can increase the photosynthetic surface even further. An Arthrospira-like cell with 50 radial thylakoids or a Cyanothece-like cell with 50 parallel thylakoids can reach a photosynthetic surface in the range of 50-70 µm 2 , based on the summed area of flattened cisternae, within the same volume. In the course of evolution, following chloroplast integration in Archaeplastida, thylakoids evolved in the direction of an increased surface and organization in functional domains with the development of stacks and grana, which do not form in Cyanobacteria. In the cells of eukaryotic microalgae, the volume of the plastid is commonly higher than 10 µm 3 [START_REF] Uwizeye | Morphological bases of phytoplankton energy management and physiological responses unveiled by 3D subcellular imaging[END_REF] whereas in vascular plants, such as maize, the volume of mesophyll cell chloroplasts can reach volumes higher than 100 µm 3 [START_REF] Kutik | Ultrastructure and dimensions of chloroplasts in leaves of three maize (Zea mays L.) inbred lines and their F-1 hybrids grown under moderate chilling stress[END_REF]. Based on a 10-100 µm 3 chloroplast containing 50 thylakoids, a gross calculation gives a photosynthetic surface of 10 -3 -10 -2 mm 2 per chloroplast. Overall, a population of 1 million Gloeobacter-type cells harbors a global photosynthetic surface of about 1 mm 2 , whereas 1 million of Arthrospira-or Cyanothece-like cells can easily exceed 50-70 mm 2 . Because of this obvious multiplier effect, the appearance of thylakoids seems therefore the most plausible determinant in the GOE.

One would naturally examine extant protein machinery(ies) involved in thylakoid biogenesis to trace back its origin and look for evidence supporting this hypothesis. The difficulty lies in the still unresolved process(es) governing the biogenesis of this membrane system, most notably because this question is mainly posed considering protein components only. We advocate here that spontaneous organization of lipids, following an appropriate biosynthetic sequence, would be sufficient to produce very large amounts of membrane surfaces in nanoto microseconds. This process does not require vesicles budding from the Cyanobacteria plasma membrane or the chloroplast inner envelope membrane. Actually, the traditional hypothesis for a 'vesicular-based' biogenesis of thylakoids finds little support in ultrastructural analyses of Cyanobacteria and chloroplasts, where the observation of inner membrane invaginations is extremely rare [START_REF] Mares | Evolutionary Patterns of Thylakoid Architecture in Cyanobacteria[END_REF]Botte et al., 2011a;Boudiere et al., 2012;[START_REF] Bastien | New Insights on Thylakoid Biogenesis in Plant Cells[END_REF][START_REF] Gupta | Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity[END_REF]. Some reports highlight the presence of vesicles in eukaryotic proplastids prior their conversion into chloroplasts [START_REF] Mechela | A brief history of thylakoid biogenesis[END_REF], but the lack of such vesicular compartment in Cyanobacteria disqualifies this mechanism at the origin of thylakoids in ancient clades. Likewise, the focus on Synechocystis 'thylakoid convergence zones' where thylakoids merge into high-curvature membranes in close contact with the plasma membrane, also observed in some chloroplasts [START_REF] Gupta | Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity[END_REF], do not account for the biogenesis of more primitive parietal thylakoids organized in concentric and uninterrupted layers [START_REF] Mares | Evolutionary Patterns of Thylakoid Architecture in Cyanobacteria[END_REF]. Here, we do not disregard the fact that protein components evolved in the direction of helping, organizing, guiding, orchestrating the process of thylakoid biogenesis, leading to the very sophisticated and efficient processes observed today, but we assume that no evidence support their determinant role in the early stages.

Thylakoids emerged from a mixture of lipids allowing a non-vesicular biogenesis of membranes

Membrane bilayers are made of polar lipids, harboring a 'polar head group' exposed to the surface, and hydrophobic 'tails' inside its core. How can a lipid bilayer form without any source of vesicles, protruding or budding from a parent membrane, merging to produce large surfaces? An answer developed here comes from the biophysical properties of polar lipids. Depending on the equilibrium between polar head interactions and repulsive forces between hydrophobic tails, each of the polar lipid can self-organize and form so called 'phases', either with a positive curvature (e.g. micellar tubules, also known as 'hexagonal I', or HexI), or no curvature ('lamellar', or Lm) or with a negative curvature ('inverted hexagonal', or HexII) [START_REF] Jouhet | Importance of the hexagonal lipid phase in biological membrane organization[END_REF]. These properties have attracted the attention for long, with a focus on lipids promoting an Lm  HexII transition, since the disturbance introduced by inverted hexagonal phase was considered essential for the promotion of highly bent and fused domains [START_REF] Jouhet | Importance of the hexagonal lipid phase in biological membrane organization[END_REF][START_REF] Gawrisch | Energetics of a Hexagonal Lamellar Hexagonal-Phase Transition Sequence in Dioleoylphosphatidylethanolamine Membranes[END_REF][START_REF] Toombes | Determination of L-alpha-H-II phase transition temperature for 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine[END_REF][START_REF] Kuzmin | A quantitative model for membrane fusion based on low-energy intermediates[END_REF]. Little thermodynamic studies are available to evaluate all possible phase transitions in the different lipid mixtures found in nature. Our knowledge relies on model lipids such as phosphatidylethanolamine (PtdEtn), which organization in Lm or HexII phases depends on its chemical structure, i.e. the length and desaturation level of acyl chains; physical properties, such as hydration or temperature; and the presence of other lipids [START_REF] Gawrisch | Energetics of a Hexagonal Lamellar Hexagonal-Phase Transition Sequence in Dioleoylphosphatidylethanolamine Membranes[END_REF][START_REF] Toombes | Determination of L-alpha-H-II phase transition temperature for 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine[END_REF][START_REF] Mannock | An analysis of the relationship between fatty acid composition and the lamellar gel to liquidcrystalline and the lamellar to inverted nonlamellar phase transition temperatures of phosphatidylethanolamines and diacyl-alpha-D-glucosyl glycerols[END_REF][START_REF] Harper | X-ray diffraction structures of some phosphatidylethanolamine lamellar and inverted hexagonal phases[END_REF].

It is essential to keep in mind that so-called "HexII lipids" are also found in membrane bilayers, either because in their pure state they can form both HexII or Lm phases depending on temperature, hydration, etc., or because when mixed with Lm lipids, the lamellar phase is the more stable organization. In past studies, Lm  HexII was the direction examined in the search for plausible membrane fusion mechanisms. The enthalpy of dioleoyl-PtdEtn phase transition (H) measured by differential scanning calorimetry had a positive value (+0.3 kcal.mol -1 = 1.255 kJ.mol -1 ), meaning that energy external to the system had to be provided [START_REF] Gawrisch | Energetics of a Hexagonal Lamellar Hexagonal-Phase Transition Sequence in Dioleoylphosphatidylethanolamine Membranes[END_REF]. It was further showed that neither the Lm  HexII nor the HexII  Lm kinetics had any significant deviation from a power-law relationship: a relative change in the Lm phase results in a proportional relative change in HexII and vice versa [START_REF] Toombes | Determination of L-alpha-H-II phase transition temperature for 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine[END_REF][START_REF] Garab | Self-assembly and structural-functional flexibility of oxygenic photosynthetic machineries: personal perspectives[END_REF]. The Lm  HexII transition is therefore remarkable for the symmetry between forward and reverse dynamics [START_REF] Toombes | Determination of L-alpha-H-II phase transition temperature for 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine[END_REF]. A corollary is that the HexII  Lm transition for this pure lipid species may release some energy, and therefore occur spontaneously. Same spontaneous HexII  Lm transition of dioleoyl-PtdEtn could occur by changing the hydration level [START_REF] Gawrisch | Energetics of a Hexagonal Lamellar Hexagonal-Phase Transition Sequence in Dioleoylphosphatidylethanolamine Membranes[END_REF]. The HexII  Lm phase transition provides a solution to the unresolved question of the emergence of early membrane systems, especially with such extended surface as thylakoids.

Let us first consider the lipid composition of Cyanobacteria in a simplified evolutionary context. A variety of chemical structures can produce impermeable membranes delineating cells [START_REF] Jia | Membraneless polyester microdroplets as primordial compartments at the origins of life[END_REF]. We have evidence of two distinct options for the structure of polar lipids in primordial membranes, based on their conservation in the three domains of life. These primordial lipids are all built on a three-carbon glycerol scaffold (numbered sn-1, sn-2, sn-3). On the one hand, Archaea are bounded by membranes made of isoprenoid hydrocarbon chains linked via ether bonds to an sn-2,3-glycerol phosphate backbone, and multiple derivatives with two polar heads, which can form lipid monolayers [START_REF] Salvador-Castell | Characterisation of a synthetic Archeal membrane reveals a possible new adaptation route to extreme conditions[END_REF][START_REF] Imachi | Isolation of an archaeon at the prokaryote-eukaryote interface[END_REF]. On the other hand, Bacteria and Eukaryota are primarily bounded by membranes made of acyl-glycerolipids, deriving from a precursor containing fatty acids linked via ester bonds to an sn-1,2-glycerol phosphate backbone [START_REF] Imachi | Isolation of an archaeon at the prokaryote-eukaryote interface[END_REF]. It is established that in Archaea, the presence of phytanyl chains, ether bonds and bipolar lipids confer a high stability to their membranes [START_REF] Guler | Effects of ether vs. ester linkage on lipid bilayer structure and water permeability[END_REF][START_REF] Garcia-Arribas | Effects of ether groups and acyl-chain branching on lipids tested by Atomic Force Microscopy[END_REF][START_REF] Balleza | Ether-versus Ester-Linked Phospholipid Bilayers Containing either Linear or Branched Apolar Chains[END_REF]. By contrast, in Bacteria and Eukarya, acyl-glycerolipids may confer a higher flexibility, especially via the controlled addition of double bonds to the acyl chains, tuning the lateral fluidity of membranes, but also with the balance between HexII and Lm lipids [START_REF] Garab | Self-assembly and structural-functional flexibility of oxygenic photosynthetic machineries: personal perspectives[END_REF][START_REF] De Kruijff | Biomembranes. Lipids beyond the bilayer[END_REF]. Increasing evidence support that Eukaryogenesis originated from the merger between an archaeal host and a bacterial endosymbiont related to modern -proteobacteria [START_REF] Imachi | Isolation of an archaeon at the prokaryote-eukaryote interface[END_REF]. This raises the puzzling question of the loss of Archaea-type lipids in Eukarya. An hypothesis is that the integrated -proteobacteria gave rise to a proto-mitochondrion, producing ATP by specific protein machineries preferring Bacteria-type membranes, which were subsequently conserved [START_REF] Sojo | Why the Lipid Divide? Membrane Proteins as Drivers of the Split between the Lipids of the Three Domains of Life[END_REF]. In these initial membrane matrices, other lipidic structures and hydrophobic polypeptides were integrated, contributing to the multitude of functions harbored by cell membranes.

Acyl-glycerolipids with a polar head containing a phosphate group, such as the HexII lipid PtdEtn, or phosphatidylcholine (PtdCho), an Lm lipid, are conserved in the majority of Bacteria and Eukarya clades, with the notable exception of crown Cyanobacteria who only contain one phospholipid, phosphatidylglycerol (PtdGro) [START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF][START_REF] Yuzawa | Phylogeny of galactolipid synthase homologs together with their enzymatic analyses revealed a possible origin and divergence time for photosynthetic membrane biogenesis[END_REF][START_REF] Sato | Diversity in Biosynthetic Pathways of Galactolipids in the Light of Endosymbiotic Origin of Chloroplasts[END_REF]. PtdGro is anionic and its proportion is regulated in response to phosphate availability (Boudiere et al., 2014;[START_REF] Frentzen | Phosphatidylglycerol and sulfoquinovosyldiacylglycerol: anionic membrane lipids and phosphate regulation[END_REF]. The conservation of PtdGro is consistent with its role in the assembly and functioning of photosynthesis components (Boudiere et al., 2014), supported by the lethality of PtdGro-less mutants of Cyanobacteria [START_REF] Kopecna | Lack of Phosphatidylglycerol Inhibits Chlorophyll Biosynthesis at Multiple Sites and Limits Chlorophyllide Reutilization in Synechocystis sp. Strain PCC 6803[END_REF][START_REF] Hagio | Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis[END_REF]Sakurai et al., 2003a, b;[START_REF] Kobori | Phosphatidylglycerol is implicated in divisome formation and metabolic processes of cyanobacteria[END_REF] and plants [START_REF] Babiychuk | Arabidopsis phosphatidylglycerophosphate synthase 1 is essential for chloroplast differentiation, but is dispensable for mitochondrial function[END_REF][START_REF] Xu | The pgp1 mutant locus of Arabidopsis encodes a phosphatidylglycerolphosphate synthase with impaired activity[END_REF][START_REF] Xu | Phosphatidylglycerol biosynthesis in chloroplasts of Arabidopsis mutants deficient in acyl-ACP glycerol-3phosphate acyltransferase[END_REF]. Such membrane composition parsimonious in phospholipids supports the idea that early Cyanobacteria populated an environment where phosphorous was limiting, at least to meet their basal metabolic demand.

The lipid composition of Gloeobacter membranes is simple, containing three lipid classes in addition to PtdGro: monoglucosyldiacylglycerol (MGlcDG), monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) [START_REF] Rexroth | The plasma membrane of the cyanobacterium Gloeobacter violaceus contains segregated bioenergetic domains[END_REF][START_REF] Yuzawa | Phylogeny of galactolipid synthase homologs together with their enzymatic analyses revealed a possible origin and divergence time for photosynthetic membrane biogenesis[END_REF][START_REF] Sato | Diversity in Biosynthetic Pathways of Galactolipids in the Light of Endosymbiotic Origin of Chloroplasts[END_REF][START_REF] Sato | Is Monoglucosyldiacylglycerol a Precursor to Monogalactosyldiacylglycerol in All Cyanobacteria?[END_REF]. MGlcDG and MGDG are HexII lipids, whereas DGDG and PtdGro are Lm lipids [START_REF] Jouhet | Importance of the hexagonal lipid phase in biological membrane organization[END_REF]. It is interesting to note that the addition of HexIIforming MGlcDG could rescue a bacterial Escherichia coli mutant lacking HexII-forming PtdEtn [START_REF] Wikstrom | Monoglucosyldiacylglycerol, a foreign lipid, can substitute for phosphatidylethanolamine in essential membrane-associated functions in Escherichia coli[END_REF], indicating that an HexII lipid could replace another HexII lipid, thus filling a vital role governed by its biophysical properties. The plasma membrane of Gloeobacter has only traces of MGlcDG [START_REF] Sato | Diversity in Biosynthetic Pathways of Galactolipids in the Light of Endosymbiotic Origin of Chloroplasts[END_REF][START_REF] Sato | Is Monoglucosyldiacylglycerol a Precursor to Monogalactosyldiacylglycerol in All Cyanobacteria?[END_REF]. It was shown to contain segregated bioenergetic domains, some with low-chlorophyll content being MGDGrich (~70% MGDG, ~22% DGDG and ~8% PtdGro), whereas high-chlorophyll domains were more balanced in their lipid composition (~55% MGDG, ~35% DGDG, ~10% PtdGro) [START_REF] Rexroth | The plasma membrane of the cyanobacterium Gloeobacter violaceus contains segregated bioenergetic domains[END_REF]. These latter domains also contain photosynthetic components, i.e. PSII, PSI and ATP-synthetase. It is very important to note that the relative MGDG/DGDG ratio differs in the two membrane domains: this indicates that although DGDG is produced from MGDG, some regulatory mechanisms uncouple its final proportion from the available amount of substrate. This is consistent with the multitude of roles played by DGDG which need to be finely controlled, including the stability of the photosynthetic machinery (Boudiere et al., 2014;[START_REF] Apdila | Configuration of the sugar head of glycolipids in thylakoid membranes[END_REF][START_REF] Sakurai | Digalactosyldiacylglycerol is required for stabilization of the oxygen-evolving complex in photosystem II[END_REF][START_REF] Awai | Digalactosyldiacylglycerol is required for better photosynthetic growth of Synechocystis sp PCC6803 under phosphate limitation[END_REF]. The MGDG-rich domains at the plasma membrane appear therefore as ideal platforms to promote HexII  Lm phase transition. Nevertheless, the HexII (MGlcDG, MGDG) / Lm (DGDG, PtdGro) balance is still insufficient in Gloeobacter to promote the formation of thylakoids. In addition, because PtdGro is essential for the stability of photosystems [START_REF] Kopecna | Lack of Phosphatidylglycerol Inhibits Chlorophyll Biosynthesis at Multiple Sites and Limits Chlorophyllide Reutilization in Synechocystis sp. Strain PCC 6803[END_REF][START_REF] Hagio | Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis[END_REF]Sakurai et al., 2003a, b;[START_REF] Kobori | Phosphatidylglycerol is implicated in divisome formation and metabolic processes of cyanobacteria[END_REF][START_REF] Babiychuk | Arabidopsis phosphatidylglycerophosphate synthase 1 is essential for chloroplast differentiation, but is dispensable for mitochondrial function[END_REF][START_REF] Xu | The pgp1 mutant locus of Arabidopsis encodes a phosphatidylglycerolphosphate synthase with impaired activity[END_REF][START_REF] Xu | Phosphatidylglycerol biosynthesis in chloroplasts of Arabidopsis mutants deficient in acyl-ACP glycerol-3phosphate acyltransferase[END_REF], a minimal quantity of this anionic lipid should be included in thylakoids, and the production of exceeding amounts of PtdGro in the bulk of membrane lipids appears difficult to maintain in a phosphate-limiting environment.

How could thylakoids emerge from an HexII  Lm transition process? An absolute prerequisite is a topological segregation of domains producing lipids in HexII phase from those promoting Lm bilayers. MGDG-rich domains in Gloeobacter plasma membrane makes this condition plausible. The enzymes synthesizing MGlcDG and MGDG should be distant enough from those consuming them to produce the Lm forming DGDG. The galactolipid biosynthesis pathway in Gloeobacter was shown to be similar to that in all other Cyanobacteria. Firstly MGlcDG is produced by a monoglucosyldiacylglycerol synthase (MgdA) [START_REF] Apdila | Configuration of the sugar head of glycolipids in thylakoid membranes[END_REF][START_REF] Awai | Comparative genomic analysis revealed a gene for monoglucosyldiacylglycerol synthase, an enzyme for photosynthetic membrane lipid synthesis in cyanobacteria[END_REF], then the glucosyl polar head is epimerized by an unknown mechanism into a galactosyl residue, thus forming MGDG; eventually DGDG is formed by the action of a digalactosyldiacylglycerol synthase (DgdA) similar to that identified in other Cyanobacteria [START_REF] Apdila | Configuration of the sugar head of glycolipids in thylakoid membranes[END_REF][START_REF] Sakurai | Digalactosyldiacylglycerol is required for stabilization of the oxygen-evolving complex in photosystem II[END_REF][START_REF] Awai | Digalactosyldiacylglycerol is required for better photosynthetic growth of Synechocystis sp PCC6803 under phosphate limitation[END_REF]. The activity converting MGlcDG into MGDG in Gloeobacter was measured [START_REF] Sato | Is Monoglucosyldiacylglycerol a Precursor to Monogalactosyldiacylglycerol in All Cyanobacteria?[END_REF], but the corresponding enzyme is still uncharacterized molecularly. It was tentatively called MgdX [START_REF] Apdila | Configuration of the sugar head of glycolipids in thylakoid membranes[END_REF]. MGlcDG does not accumulate and this lipid is barely detected in Gloeobacter, in support of a physical proximity between MgdA and MgdX in vivo. With a few exceptions, major Cyanobacteria clades who have radiated from a Gloeobacter-like ancestor contain another epimerase, called MgdE shown to convert MGlcDG into MGDG [START_REF] Awai | Oxygenic photosynthesis without galactolipids[END_REF]. Since MGlcDG can accumulate, even in small but detectable amounts in MgdE-containing Cyanobacteria [START_REF] Sato | Diversity in Biosynthetic Pathways of Galactolipids in the Light of Endosymbiotic Origin of Chloroplasts[END_REF][START_REF] Apdila | Configuration of the sugar head of glycolipids in thylakoid membranes[END_REF], one can suppose that MgdE is topologically separated from MgdA. The galactolipid pathway thus evolved in the direction of a topological separation of each step synthesizing each of its components, MGlcDG, MGDG and DGDG. Based on membrane fractionation and enzymatic measurements, MgdA activity was detected in both thylakoid and plasma membranes of Anacystis [START_REF] Omata | Glucolipid synthesis activities in cytoplasmic and thylakoid membranes from the cyanobacterium Anacystis nidulans[END_REF] and Synechocystis [START_REF] Selao | Subcellular localization of monoglucosyldiacylglycerol synthase in Synechocystis sp. PCC6803 and its unique regulation by lipid environment[END_REF], whereas DgdA appears restricted to the plasma membrane [START_REF] Selao | Subcellular localization of monoglucosyldiacylglycerol synthase in Synechocystis sp. PCC6803 and its unique regulation by lipid environment[END_REF]. This topological segregation of MGlcDG and MGDG synthesizing enzymes from DGDG synthesizing ones may have enabled a transient accumulation of HexII lipids.

One could consider that the most important HexII lipid in this process would be the most abundant one, i.e. MGDG, but Cyanobacteria genetic studies do not support this strict requirement. The knock-out of mgdA was lethal in Synechocystis [START_REF] Apdila | Configuration of the sugar head of glycolipids in thylakoid membranes[END_REF][START_REF] Awai | Comparative genomic analysis revealed a gene for monoglucosyldiacylglycerol synthase, an enzyme for photosynthetic membrane lipid synthesis in cyanobacteria[END_REF], probably because this enzyme is the committing step for the formation of three lipid classes, i.e. MGlcDG, MGDG and DGDG. The knock-out of the second enzyme, mgdE, led to the hyper-accumulation of MGlcDG in Synechocystis, reaching nearly 70% of the total proportion of lipids, replacing all MGDG and DGDG which could not be produced.

The Synechocystis mgdE mutant was viable and contained thylakoids [START_REF] Awai | Comparative genomic analysis revealed a gene for monoglucosyldiacylglycerol synthase, an enzyme for photosynthetic membrane lipid synthesis in cyanobacteria[END_REF][START_REF] Apdila | Configuration of the sugar head of glycolipids in thylakoid membranes[END_REF]. This indicated that MGDG was not strictly required for thylakoid biogenesis, but that MGlcDG or MGDG, as HexII lipids, may be indistinctly usable. Synechocystis mgdE mutation also indicated that thylakoid bilayers, if generated from MGlcDG instead of MGDG, did not strictly require DGDG as a necessary Lm forming lipid.

Altogether, thylakoids might have emerged from a Gloeobacter-type Cyanobacteria, in which a specific biosynthetic platform allowed the accumulation of an HexII lipid, most likely MGDG, although MGlcDG might have been appropriate as well, reminiscent of the complementation of viable E. coli PtdEtn mutants by external supply of MGlcDG [START_REF] Wikstrom | Monoglucosyldiacylglycerol, a foreign lipid, can substitute for phosphatidylethanolamine in essential membrane-associated functions in Escherichia coli[END_REF].

A 'trigger' should then be added to this system, to promote the transition of an HexII phase into an Lm one. We discussed above that the model lipid PtdEtn could operate such transition by discrete changes of its chemical structure, in particular the level of its acyl chains' desaturation, by modulating temperature or hydration and that the enthalpy of Lm  HexII transition was positive [START_REF] Gawrisch | Energetics of a Hexagonal Lamellar Hexagonal-Phase Transition Sequence in Dioleoylphosphatidylethanolamine Membranes[END_REF]. Having in mind the mechanism of membrane fusions, the modeling of the Lm  HexII transition showed that, given an initial multilamellar configuration and with appropriate provision of external energy (increasing temperature), a formation of so called 'stalks' (elongated structures) between bilayers could be observed in a nanosecond timescale. The stalks were not stable and they subsequently elongated in a cooperative manner, thus leading to the formation of an inverted HexII phase [START_REF] Marrink | Molecular view of hexagonal phase formation in phospholipid membranes[END_REF]. Thylakoids may have simply emerged from the reverse process, from a MGlcDG/MGDG HexII matrix converted within nano-to microseconds into piled multilamellar membranes. It was reported that a decrease of double bonds per molecule of MGDG was sufficient to promote the conversion from HexII structure to Lm phase [START_REF] Gounaris | Monogalactosyldiacylglycerol -the Most Abundant Polar Lipid in Nature[END_REF]. The study of MGDG mixed with other thylakoid lipids has also shown that the presence of various Lm lipids to MGDG triggered a spontaneous HexII to Lm transition upon increasing hydration [START_REF] Deme | Contribution of galactoglycerolipids to the 3-dimensional architecture of thylakoids[END_REF]. The trigger could therefore simply be an Lm lipid, such as any of this type present in Cyanobacteria membranes. One should now identify which of the Lm forming lipid would play this role at the beginning.

Another difference between Gloeobacter-type and subsequent Cyanobacteria is the absence of a fifth lipid class, i.e. the anionic sulfoquinovosyldiacylglycerol (SQDG). The absence of the equipment to synthesize SQDG in Gloeobacter, i.e. the sqdB and sqdX genes, was interpreted as an ancestral trait (Sato et al., 2016), although a secondary loss was also suggested [START_REF] Sato | Diversity in Biosynthetic Pathways of Galactolipids in the Light of Endosymbiotic Origin of Chloroplasts[END_REF]. Here, it may be interesting to remind one of the scenarios reported above for the origin of oxygenic photosynthesis, starting from a protocyanobacteria containing two reaction centers, one being an H 2 S splitting PSI controlled by fluctuating hydrogen sulfide concentrations (redox switch hypothesis for the first Cyanobacteria) [START_REF] Allen | A Proposal for Formation of Archaean Stromatolites before the Advent of Oxygenic Photosynthesis[END_REF][START_REF] Allen | Origin of Oxygenic Photosynthesis from Anoxygenic Type I and Type II Reaction Centers[END_REF]. It may be that, in addition to living in a phosphorus-limiting environment, the protocyanobacteria were exposed to reduced forms of sulfur such as hydrogen sulfide (H 2 S), rather than oxidized ones such as sulfate (H 2 SO 4 ). The acquisition of the sulfolipid synthesis pathway by horizontal gene transfer would then be a key event in the radiation of early Cyanobacteria. In thylakoid-containing Cyanobacteria, mutants lacking SQDG are either affected, such as in Synechocystis, or with no apparent phenotype under optimal growth condition, such as in Synechococcus [START_REF] Apdila | Configuration of the sugar head of glycolipids in thylakoid membranes[END_REF], leading to the common idea that this lipid could be to some extent dispensable (Sato et al., 2016). It is now established in Cyanobacteria, but also in chloroplasts of vascular plants that SQDG function is linked to that of PtdGro, replacing temporarily this anionic phospholipid in case of phosphate shortage in the environment (Boudiere et al., 2014;[START_REF] Frentzen | Phosphatidylglycerol and sulfoquinovosyldiacylglycerol: anionic membrane lipids and phosphate regulation[END_REF][START_REF] Shimojima | Biosynthesis and functions of the plant sulfolipid[END_REF]. The gain is clear: Cyanobacteria with SQDG have acquired an anionic surrogate to replace PtdGro, and can overcome stronger depletions of phosphorus in the environment. Thanks to SQDG, anionic membrane lipids are no more limiting. This gain is an advantage when competing with SQDG-missing Cyanobacteria, and in any case, when competing with Bacteria with phospholipid-rich membranes in a phosphorus-deprived context. This also allows the availability of sufficient amounts of an anionic Lm-forming lipid for the expensive formation of an additional membrane system. Together, SQDG and PtdGro, and to some extent DGDG, can be used as lipid triggers, some kind of catalysts, for the nanosecond-scale HexII (MGlcDG, MGDG)  Lm (MGlcDG, MGDG, DGDG, PdtGro, SQDG) transition.

Was DGDG necessary at the origin of thylakoids? This could make sense since SQDG and PtdGro are anionic, and their use, although essential for photosystems, can thus introduce repulsive electrostatic forces between membrane bilayers, which may prevent the formation of tightly stacked membranes. Some arguments contradict this idea though, most importantly since the mgdE mutant of Synechocystis can still make thylakoids whereas it has lost all of its DGDG [START_REF] Awai | Comparative genomic analysis revealed a gene for monoglucosyldiacylglycerol synthase, an enzyme for photosynthetic membrane lipid synthesis in cyanobacteria[END_REF][START_REF] Apdila | Configuration of the sugar head of glycolipids in thylakoid membranes[END_REF]. One should admit that Synechocystis thylakoids can organize as stacks in spite of the strong anionic charges harbored by the polar head groups of its Lm forming lipids. Protein components may contribute to this stable architecture in Synechocystis, but these proteins may have been missing in the original steps. Indeed, DGDG was shown to stabilize membrane stacks, including in lipid mixtures mimicking that of photosynthetic membranes [START_REF] Deme | Contribution of galactoglycerolipids to the 3-dimensional architecture of thylakoids[END_REF][START_REF] Kanduc | Tight cohesion between glycolipid membranes results from balanced waterheadgroup interactions[END_REF]. Thus, we cannot assess with certainty the potential role of DGDG at the origin, but this lipid would have been advantageous in the formation of stacked thylakoid primordia.

The hypothesis defended here relies on the fact that all the lipid actors but one, SQDG, were present in Gloeobacter-type early Cyanobacteria. SQDG introduces a more flexible and robust membrane lipid metabolism by the tuning of the SQDG/PtdGro balance in response to phosphorus in the environment. HexII lipids, mostly MGDG, could be produced concentrically toward the inside of the cell, followed by the transfer of Lm lipids, i.e. anionic PtdGro and SQDG and neutral DGDG. These Lm lipids would then act as triggers of an HexII  Lm phase transition. This gives an explanation to the non-vesicular biogenesis of thylakoids, and to the initial formation of concentric, uninterrupted layers [START_REF] Mares | Evolutionary Patterns of Thylakoid Architecture in Cyanobacteria[END_REF].

Role of proteins

An HexII  Lm phase transition is of 'first-order', i.e. involving a latent heat or phase transition enthalpy, releasing or absorbing a fixed and typically large amount of energy, thus differing from a 'second order', or 'continuous phase transition'. The dynamics of the dramatic lipid rearrangements occurring during this transition, although very rapid, has limiting rate steps in topological transitions [START_REF] Toombes | Determination of L-alpha-H-II phase transition temperature for 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine[END_REF]. Protein actors could have played a role at the origin of thylakoid formation, by acting in the dynamics of Lm lipids' transfers to an HexII matrix and the 'nucleation' of HexII  Lm transition.

Gloeobacter contains a homolog of Vipp1, found in all other oxygenic photosynthetic organisms [START_REF] Raven | Gloeobacter and the implications of a freshwater origin of Cyanobacteria[END_REF]. The Vipp1 protein has been functionally associated to thylakoid biogenesis in a multitude of biological models, by various genetic, structural or biochemical studies [START_REF] Mechela | A brief history of thylakoid biogenesis[END_REF], but the precise understanding of its role, at molecular level, still remains puzzling in spite of recent advances in the resolution of its structural organization [START_REF] Gupta | Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity[END_REF]. Gloeobacter Vipp1 lacks a ~30 amino acid extension found in thylakoid-containing organisms [START_REF] Raven | Gloeobacter and the implications of a freshwater origin of Cyanobacteria[END_REF], and this protein plays a primitive function which is still elusive. In thylakoid-containing Synechocystis or in the chloroplast of the green alga Chlamydomonas reinhardtti, vipp1 mutations lead to the formation of aberrant thylakoid convergence zones [START_REF] Gupta | Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity[END_REF][START_REF] Nordhues | Evidence for a role of VIPP1 in the structural organization of the photosynthetic apparatus in Chlamydomonas[END_REF]. This has led to the idea that Vipp1 had gained a new role in the course of Evolution, acting at the level of highly bent regions of thylakoids (which are absent in Cyanobacteria containing concentric thylakoids with uninterrupted layers). Of interest here, Vipp1 has a strong affinity for lipids, particularly anionic lipids including but not restricted to PtdGro, in vitro and in vivo [START_REF] Otters | The first alphahelical domain of the vesicle-inducing protein in plastids 1 promotes oligomerization and lipid binding[END_REF][START_REF] Li | Molecular cloning of a chloroplastic protein associated with both the envelope and thylakoid membranes[END_REF][START_REF] Kroll | VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation[END_REF][START_REF] Mcdonald | Structure and function of PspA and Vipp1 N-terminal peptides: Insights into the membrane stress sensing and mitigation[END_REF][START_REF] Heidrich | Organization into Higher Ordered Ring Structures Counteracts Membrane Binding of IM30, a Protein Associated with Inner Membranes in Chloroplasts and Cyanobacteria[END_REF]. Structural studies suggest a possible role of Vipp1 in guiding some lipids from a donor membrane to thylakoid primordia [START_REF] Gupta | Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity[END_REF]. If Vipp1 played this function for all thylakoid lipids, one should expect a detection of innumerable complexes connecting the inner membrane, Vipp1 and thylakoids, but these are instead very rarely observed [START_REF] Gupta | Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity[END_REF]. Little evidence are therefore available to support that Vipp1 would act in the transfer of the bulk of thylakoid lipids, and in any case, a role in a massive vesicular-based process is not supported. Vipp1 may have acquired a new function in early Cyanobacteria, guiding some anionic lipids toward the light of the cell, thus 'helping' the nucleation of thylakoid bilayer formation. The Vipp1 ancestral protein in Gloeobacter might therefore have evolved in the direction of a guidance of thylakoid biogenesis based on its initial biophysical properties, interacting with other lipids or proteins.

In this paper, we did not elaborate any scenario for the evolution of Vipp1 or others proteins [START_REF] Raven | Gloeobacter and the implications of a freshwater origin of Cyanobacteria[END_REF]. We simply suggest that proteins present in early Cyanobacteria, completed with others acquired later on, following gene transfers, might have been integrated based on their intrinsic biophysical properties. This is supported by studies showing that Cyanobacteria Vipp1 C-terminal domain associates with lipid bilayers and modulates membrane fusion and fluxes of lipids and proteins between membranes [START_REF] Saur | A Janus-Faced IM30 Ring Involved in Thylakoid Membrane Fusion Is Assembled from IM30 Tetramers[END_REF][START_REF] Hennig | The IM30/Vipp1 C-terminus associates with the lipid bilayer and modulates membrane fusion[END_REF][START_REF] Hennig | IM30 triggers membrane fusion in cyanobacteria and chloroplasts[END_REF]. Here, we did not address the question of the loading of thylakoids with all kinds of other components, proteins and cofactors, including the photosystems and ATP-synthetase, also requiring guiding machineries. This could maybe rely on HexII glycolipids, which are found in association with reaction centers from anoxygenic photosynthetic Bacteria to Cyanobacteria, all algae and plants [START_REF] Mizoguchi | A variety of glycolipids in green photosynthetic bacteria[END_REF]. A photosynthetic reaction center from Blastochlorus viridis, a species containing HexII glycolipid, was thus shown to form a so-called 'lipidic sponge phase' crystal structure [START_REF] Wohri | Lipidic sponge phase crystal structure of a photosynthetic reaction center reveals lipids on the protein surface[END_REF]. It is known for long that in energy-transducing membranes, HexII lipids are preferentially associated with that part of the bilayers in which the coupling factors are embedded, i.e. MGDG in the outer layer of plant thylakoids, PtdEtn on the cytosolic face of Rhodobacter sphaeroides chromatophores, and both PtdEtn and cardiolipin in the inner monolayer of internal mitochondrial membrane [START_REF] Rawyler | The transmembrane distribution of galactolipids in chloroplast thylakoids is universal in awide variety of temperate climate plants[END_REF]. In this context, a parsimonious hypothesis is that at the origin, photosynthetic components were loaded during the HexII  Lm phase transition.

5

The endosymbiotic origin of chloroplasts supports the conserved role of HexII lipids in the formation of their thylakoids

A vast array of evidence support an origin of the chloroplast in Archaeplastida cells (Glaucophyta, Rhodophyta and Chlorophyta) following a unique primary endosymbiosis event, by engulfment of a Cyanobacteria-like ancestor related to extant Gloeomargarita inside an unknown eukaryotic host cell [START_REF] Ponce-Toledo | Horizontal and endosymbiotic gene transfer in early plastid evolution[END_REF][START_REF] Sato | Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis[END_REF][START_REF] Sato | Diverse origins of enzymes involved in the biosynthesis of chloroplast peptidoglycan[END_REF][START_REF] Marechal | Primary Endosymbiosis: Emergence of the Primary Chloroplast and the Chromatophore, Two Independent Events[END_REF][START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF]. This view is however simplistic, since numerous genes coding for chloroplast proteins find no ancestor in crown Cyanobacteria, but rather in other clades not discussed here [START_REF] Ball | Metabolic effectors secreted by bacterial pathogens: essential facilitators of plastid endosymbiosis?[END_REF][START_REF] Cenci | Biotic Host-Pathogen Interactions As Major Drivers of Plastid Endosymbiosis[END_REF][START_REF] Sato | Characterization and biosynthesis of lipids in Paulinella micropora MYN1. Evidence for efficient integration of chromatophores into cellular lipid metabolism[END_REF]. It is extremely important to note that all Cyanobacteria lipids have been conserved, except one, MGlcDG [START_REF] Sato | Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis[END_REF][START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF][START_REF] Sato | Diversity in Biosynthetic Pathways of Galactolipids in the Light of Endosymbiotic Origin of Chloroplasts[END_REF]Boudiere et al., 2014). The four lipids MGDG, DGDG, PtdGro and SQDG are thus present in all prokaryotic and eukaryotic thylakoids. Only when photosynthesis was lost secondarily, could plastids 'lose' their glycolipids [START_REF] Botte | Plastids with or without galactoglycerolipids[END_REF][START_REF] Botte | Isolating the Plasmodium falciparum Apicoplast Using Magnetic Beads[END_REF]Botte et al., 2011b;[START_REF] Botte | Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites[END_REF].

How could galactolipids be synthesized in chloroplasts, if the committing step of MGlcDG synthesis is not present? Chloroplast MGDG is not synthesized by a Cyanobacteria pathway, but in one-step by the action of a monogalactosyldiacylglycerol synthase, or MGD acquired secondarily by horizontal gene transfer from a bacterium [START_REF] Sato | Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis[END_REF]Petroutsos et al., 2014;[START_REF] Yuzawa | Phylogeny of galactolipid synthase homologs together with their enzymatic analyses revealed a possible origin and divergence time for photosynthetic membrane biogenesis[END_REF]. The production of DGDG is catalyzed by a Cyanobacteriatype enzyme in Glaucophyta and one branch of Rhodophyta, but, again, by another type of digalactosyldiacylglycerol synthase, called DGD, also acquired secondarily [START_REF] Sato | Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis[END_REF][START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF]. Some well-studied Archaeplastida models, like Arabidopsis show clear topological separation between the MGD enzyme involved in thylakoid biogenesis, called MGD1 in the inner envelope membrane, and the DGD acting at the outer envelope membrane (Froelich et al., 2001;[START_REF] Boudiere | Galvestine-1, a novel chemical probe for the study of the glycerolipid homeostasis system in plant cells[END_REF][START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF][START_REF] Awai | Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana[END_REF], supporting that HexII lipids can transiently accumulate. In models where such topological separation is not fully demonstrated, like Chlamydomonas chloroplasts, most representations show MGD in the inner envelope separated from DGD in the outer envelope membrane [START_REF] Hurlock | Lipid trafficking in plant cells[END_REF][START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF]. In Chlamydomonas, other strategies were also shown to ensure an accumulation of a MGDG pool, by the rapid desaturation of MGDG fatty acids, generating molecular species, which cannot be used by DGD enzymes [START_REF] Yang | Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism[END_REF]. Such 'locking' of specifically unsaturated MGDG species, more prone to form HexII structures (Gounaris et al., 1983) is also observed in Arabidopsis and other photosynthetic eukaryotes [START_REF] Dolch | Inventory of fatty acid desaturases in the pennate diatom Phaeodactylum tricornutum[END_REF]. Thus, all chloroplasts present in cells of the immense biodiversity of photosynthetic Eukaryota conserved Cyanobacteria lipids, without the corresponding Cyanobacteria genes, and the new biosynthesis pathway retained initial important features, even improved, like mechanisms ensuring that HexII MGDG can accumulate.

The molecular characterization of plant chloroplast MGD1 is remarkably coherent with the hypothesis elaborated here. In biomimetic membranes, it was shown that Arabidopsis MGD1 had unique biophysical properties. MGD1 is soluble and its membrane binding is promoted by the presence of its product, MGDG [START_REF] Sarkis | The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts[END_REF] supporting the existence of a positive-feedback loop leading to a local over-accumulation of HexII lipids. MGD1 binding is inhibited by DGDG, reinforcing this initial segregation of MGDG from DGDG detailed above [START_REF] Sarkis | The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts[END_REF]. The activity of MGD1 requires the presence of anionic lipids, including PtdGro and phosphatidic acid [START_REF] Makshakova | Unraveling the complex enzymatic machinery making a key galactolipid in chloroplast membrane: a multiscale computer simulation[END_REF][START_REF] Dubots | Activation of the chloroplast monogalactosyldiacylglycerol synthase MGD1 by phosphatidic acid and phosphatidylglycerol[END_REF][START_REF] Dubots | Role of phosphatidic acid in plant galactolipid synthesis[END_REF], reminiscent of the activation Cyanobacteria MgdA enzyme by another anionic lipid, SQDG [START_REF] Selao | Subcellular localization of monoglucosyldiacylglycerol synthase in Synechocystis sp. PCC6803 and its unique regulation by lipid environment[END_REF]. This stresses the likely role of anionic lipids as triggers for thylakoid biogenesis. When lowering MGD1 activity with a specific inhibitor, the proportion of HexII MGDG decreases and some membrane invagination appear at the inner envelope membrane (Botte et al., 2011a), not indicating a vesicular-based formation of thylakoids, but rather an excess of Lm forming lipids. Similar results were obtained in a null MGD1 mutant of Arabidopsis, germinating as small albinos only in the presence of sucrose, with seedlings containing traces of galactolipids [START_REF] Kobayashi | Galactolipid synthesis in chloroplast inner envelope is essential for proper thylakoid biogenesis, photosynthesis, and embryogenesis[END_REF]. Invaginations of the inner envelope, which were not seen in mature WT chloroplasts, were also observed in this mutant, simply due to an excess of Lm lipids over HexII ones [START_REF] Kobayashi | Galactolipid synthesis in chloroplast inner envelope is essential for proper thylakoid biogenesis, photosynthesis, and embryogenesis[END_REF]. Although this phenomenon was initially suggested as an evidence for formation of thylakoids from the budding of inner envelope membrane, it fits with the non-vesicular model developed here, reflecting an uncontrolled excess of Lm lipids in this membrane. Consistently, the growth of the MGD1 mutant in low phosphate conditions, activating the expression of the MGDG synthase isoforms MGD2 and MGD3, allowed a synthesis of HexII lipids in chloroplasts and the formation of thylakoid structures [START_REF] Kobayashi | Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis[END_REF]. Nevertheless, the level of MGDG remained low under these conditions, and photosynthetic activity was still abolished, [START_REF] Kobayashi | Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis[END_REF], indicating that the production of the right amount of MGDG at the right place, by MGD1 located in the inner envelope membrane, could not be reached by the action of MGD2 and MGD3 located at the outer envelope membrane.

A very recent work has shown that MGD enzymes could also play roles outside of the chloroplast, in the cytosol. During the development of Arabidopsis pollen tube, it was thus shown that MGD2 accumulated in the cytosol, following the expression of mRNAs controlled by a specific RNA-binding protein [START_REF] Billey | LARP6C orchestrates posttranscriptional reprogramming of gene expression during hydration to promote pollen tube guidance. Plant Cell[END_REF]. This raises questions, since pollen tubes do not contain chloroplasts. Nevertheless pollen tubes are known to accumulate galactolipids (Botte et al., 2011a;[START_REF] Nakamura | Activation of galactolipid biosynthesis in development of pistils and pollen tubes[END_REF]. In green tissues, Arabidopsis MGD2 had been localized at the outer envelope membrane of the chloroplast, but some cytosolic localization had also been observed based on the imaging of proteins fused with a fluorescent reporter [START_REF] Awai | Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana[END_REF]. It is therefore plausible that in addition to its role in chloroplast architecture, the soluble eukaryotic MGD enzymes may have introduced a powerful system for integrating lipid metabolism from different compartments. One possibility is that MGD2 might be able to bind its diacylglycerol substrate from one cytosolic membrane and cargo it to another one, including the chloroplast where MGDG synthesis should occur or possibly other cell compartment.

Altogether, the chloroplast MGD biosynthesis system performs much better than the primitive MgdA/MgdE system of the Gloeomargarita-type cyanobacterium integrated following primary endosymbiosis. It may be one of the reasons for the substitution of the MgdA/MgdE/DgdA pathway by a MGD/DGD pathway early in the radiation of Archaeplastida. An HexII  Lm phase transition is therefore the most likely scenario for the non-vesicular biogenesis of thylakoids in chloroplasts as well [START_REF] Bastien | New Insights on Thylakoid Biogenesis in Plant Cells[END_REF]. Protein actors, such as VIPP1, have become more and more important in the orchestration of thylakoid biogenetic processes, possibly guiding anionic lipids, including chloroplastic lipids such as PtdGro, but also extraplastidial lipids such as phosphoinositides [START_REF] Gupta | Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity[END_REF].

The increase in size of chloroplasts compared to Cyanobacteria, the development of larger photosynthetic surfaces per cell, some structural innovations like the appearance of pyrenoids or grana, the bioenergetic coupling of chloroplast within eukaryotic metabolism, etc, led to a supplementary multiplier effect, likely determinant for the 'rise of Algae' during the Neoproterozoic period, and the oxygenation of oceans known as the NOE.

Concluding remarks

The origin of internal membrane compartments is one of the most fascinating and difficult questions in the evolution of cells. Textbooks highlight that one of the differences between the three Domains of Life, Archae and Bacteria on the one hand, and Eukaryota on the other hand, lies in the level of subcellular compartmentalization, absent or poor in the former, prolific, sophisticated and dynamic in the latter. Such statement suggests that cell compartmentalization should be accomplished the way it is described in a model Eukaryota, i.e. either by vesicles budding from one parent membrane merging to form a novel compartment, and/or by various membrane protrusions, fusion mechanisms, etc. The biophysical rearrangements of membrane lipids during a fusion process were shown to have a positive enthalpy and require an external source of energy, usually provided by ATP or GTP. Such reorganizations of membrane architecture are not spontaneous and involve dedicated protein machineries.

The formation of thylakoids in Cyanobacteria resists vesicular-based scenarios, firstly because of the lack of any ultrastructural observation of vesicles supposed to protrude from the plasma membrane toward the inside of the cell, but also because primitive thylakoids are concentric, and we do not see where thylakoid biogenesis could be initiated. Here, we reviewed studies converging into a simple hypothesis based on the stepwise production of HexII lipids, at the inner periphery of the cytosolic membrane of an early cyanobacterium, turned within nanoseconds and without any external source of energy into multilayered pro-thylakoids.

Comparison of lipid biosynthetic pathways suggests that most ancient Cyanobacteria could not achieve thylakoid biogenesis due to the lack of anionic Lm lipids, only represented by a minor phospholipid with a proportion controlled by phosphorus level in the environment. The acquisition of the biosynthesis pathway producing a sulfolipid seemed critical for a regular provision of sufficient amounts of anionic lipids, allowing the HexII  Lm phase transition to occur thus forming parietal thylakoids. We designed this model before the very recent description of the spatio-temporal biogenesis process of thylakoid membranes in the rodshaped cyanobacterium Synechococcus elongatus PCC 7942 [START_REF] Huokko | Probing the biogenesis pathway and dynamics of thylakoid membranes[END_REF]. In this cyanobacterium, the plasma membrane and concentric thylakoid layers have no physical connections and newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids [START_REF] Huokko | Probing the biogenesis pathway and dynamics of thylakoid membranes[END_REF]. This is strikingly reminiscent of the model described in Figs. 2A and4 and illustrates the power of the HexII-to-Lm phase transition not only at the origin of early thylakoids, but also in biogenetic processes observed today. With this non-vesicular lipid-phase transition, a framework is therefore also available to reexamine the role of companion proteins, such as Vipp1, which characterization in a hypothetic vesicular-based biogenetic process remains unsatisfying in both Cyanobacteria and chloroplasts. It is time to address these questions by considering lipids and proteins together, with additional challenging issues, such as the integration of membrane proteins and cofactors in the course of thylakoid biogenesis. Eventually, first-order phase transitions are not restricted to changes in lipid phases. They are also involved in RNA and protein structural changes between liquid phases [START_REF] Hardenberg | Widespread occurrence of the droplet state of proteins in the human proteome[END_REF][START_REF] Guo | RNA and liquid-liquid phase separation[END_REF][START_REF] Rhine | RNA Droplets[END_REF], which might provide clues on extremely rapid, nearly instant processes, which cannot be comprehended yet with more classical biochemical, structural or cell biology approaches.

Taking into consideration all these phase transitions might improve our understanding of other steps in cell evolution.

Acknowledgements

Authors are supported by the National Research Agency (Alpalga ANR-20-CE02-0020, Oceanomics ANR-11-BTBR-0008, GlycoAlps ANR-15-IDEX-02, GRAL Labex ANR-10-LABEX-04, EUR CBS ANR-17-EURE-0003) and Institut Carnot 3BCAR. NG is supported by a PhD grant from INRAE. EM is supported by the Kilian Jornet Foundation.

Author Contributions

NG and EM contributed to the development of the hypothesis developed in this paper. NG and EM contributed to the writing of the article.