Prebiotic chemo-osmosis

From Wikiversity
Jump to: navigation, search
Nuvola apps biology.png Subject classification: this is a biology resource.
Emblem-extra-cool.svg Completion status: this resource is considered to be ready for use.
38254-new folder-12.svg Resource type: this resource is an article.
Nuvola apps edu science.svg Educational level: this is a research resource.


Applying the theory of chemo-osmosis of Peter Mitchell*, to a system of liposomes and ionophores in abiotic environment, the reflections in this work**, claims to the formation of functional membrane proteins and the initialization of metabolism within the liposome by this system.

The metabolism can't be more conceived then as a set of chemical reactions in a synchronous network, subject to the laws of thermodynamics, but as two coupled networks of protons and electrons subject to electromagnetic laws and whose structures are located in the membrane created and maintained by the chemiosmotic process.

The concomitant changes in metabolism, structure and chemiosmotic process mutually reinforcing and should result in an organism that evolves consistently.

Earlier molecular evolution each part of the system can reproduce independently of one another. Liposomes can incorporate abiotic phospholipids or those synthesized by the new metabolism and split in half without damaging the islets of membrane proteins.

Oligo-nucleotides can self-duplicate by matching nucleics acids bases.

Two copies of oligo-nucleotides can bind by hydrogen bonds to two groups of amino acids almost identical, integrated into the membrane, on the inner surface of the liposome and positioned by the chemiosmotic process. This reproduction of groups of amino acids, through copies of oligo-nucleotides, initiates the translation process that we know in living organisms.

Reproduction of this 3 parts in a coordinated manner should be considered further in depth study of ribosomes and translation.

The hypothesis of the geochemical formation of abiotic pocket oil is considered in this reflection as prebiotic environment for prebiotic chemo-osmosis. This hypothesis follows works, in laboratory and in real conditions, on the origins of life at hydrothermal vents on mid-ocean ridges.

 *Peter Mitchell (1961). "Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism". Nature 191(4784):144–148.
 ** français

Note on 14.03.2015: This article is part of the summary of my work until 2014, published in Origins of Life and Evolution of Biospheres, March 2015.
Reference: Prebiotic Petroleum; Mekki-Berrada Ali, Origins of Life and Evolution of Biospheres, 2015, DOI 10.1007/s11084-015-9416-7.[1]

Molecular evolution hypothesis from mineral to living: the prebiotic chemo-osmosis[edit]

The chemo-osmosis's Peter Mitchell[2] theory account for ATP synthesis by the electro-chemical potential and by protons circulation accross the membrane at ATP synthase and ionics transporters. The electro-chemical potential is done by a lot of membrane proteins and coenzymes. If we ignore proteins and nucleics acids, it remains 3 prebiotics kinds of coenzymes: hemes, ubiquinones and complexes FeS; The electro-chemical potential is no more done from inside the liposome, but may be due to environment liposome ionics changes against inside; On the contrary, there is no more ions circulation accross the membrane, because the membrane is impermeable to the ions.

The prebiotic chemo-osmosis, origin of biotic chemo-osmosis , can work only if the ions pass through the membrane. This is possible with prebiotics kinds of molecules: the ionophores, that transport ions accross the membrane, specialy monovalents cations including protons. They are specifics to one cation, but may transport others.

Inspit of they are orginated often from bacteria,this molecules are very interesting from theorytical view:

  • They are short, simple, cyclic and oligomeric with few monomers.
  • The monomers may be L or D alpha-amino-acids, L or D alpha-hydroxyl-acids analogs kinds of biotic amino-acids.

    ionophores examples, (x differents monomers, repeated n times):

        depsipeptides ionophores : valinomycin(4,3), beauvericin(2,3);

        Oligopeptide : gramicidin, 5 different amino-acids in configuration L and D alternately ending with ethanolamine, a total of 15 amino-acids;

        Ionophore without amino-acid: nonactin (1,4).

  • Between 2 hydroxyl-acids or, between one amino-acid and one hydroxyl-acid, there is ester bond like those between phospholipides monomers (ethanolamine, phosphate, glycerol, faty acids). There is only esters bonds in nonactin.
  • This features make it closer to the oligomers of walls bacteria. And particularly, because they remain into the membrane, they are like porins. Just one sample by liposome enables the chemo-osmosis process permanently.
  • Some ionophores chelate metalic cations, that make it closer also to the membranes enzymes.

The basic prebiotic chemo-osmosis process[edit]

As for the hypothesis of biotic process, it is based on an electro-chemical potential, with an electric potential created locally and temporarily. But here the osmotic gradient varies and is dictated by the external environment. As the biotic process, the electric field is reinforced by the ionic asymmetry due to the presence of serine on the inside, instead of ethanolamine. This electro-chemical potential is converted into work that is not only cations transport to restore osmotic and ionic balance, but also in the temporary structuring of local electric fields. It is a physical remote action. This structure affects mainly polar molecules in contact with internal and external surfaces of the liposome, but also polar molecules chelated within the membrane. These remote actions move closer and position molecules with affinities until to cause the creation or destruction of a covalent bond.

At the beginning of the integration of the ionophore(s) in the membrane the effect may be very weak, due to disorder. But as time passes, the order grew and grew over the effect. It is a process analogous to crystallization, involving covalent bonds, with mobility of molecules formed, instead of positioning of the ions in a crystal in a fixed geometry.

Establishment of structures for the biotic chemo-osmosis[edit]

The prebiotic chemiosmotic process, even if his action may seem much lower than that of the biotic process, however it evolves in a reactive medium structured by and with the liposome and they reinforce mutually. Its effects will then be guided by this environment. The effects will be very different from one region to another region. Thus we must consider the external environment, the external surface of the liposome, the hydrophobic region of the liposome, the inner surface of the liposome and the internal medium.

The external environment[edit]

On the outside environment, the prebiotic process has no influence. The extent of the latter does not exceed the thickness of a membrane, and any molecule released into the environment will be lost by diffusion. On the contrary, for this first stage of molecular evolution, the surroundings must have physical and chemical conditions and composition to support this evolution. I will detail this medium below, in the section of the geochemical origin of the initial molecules.

Evolution of the external surface of the liposome[edit]

  • Ionophores formation and their penetration into the membrane : I consider that the hydrophilic head consists of ethanolamine on the outside. This head is a zwitterion such as an amino acid and can pair with by two ionic bonds. The α-hydroxy acids can also pair with phospholipid by hydrogen bonding (between OH of the alcohol and phosphate) and by an ionic bond.

   The liposome surface, bearing millions of zwiterrions heads (107), promote the formation of depsipeptide ionophores and their penetration into the membrane.

  • However, the steric hindrance of the hydrophilic heads, imposed by the combination of hydrophobic aliphatic chains, opposes a strong constraint to penetration. And this steric hindrance is reinforced by the length of ethanolamine which fasten a phospholipid to his neighbor. This is also what makes the strength and consistency of the liposome. Methylamine in place of ethanolamine would not allow the stowage and propylamine would make it too loose.

   The steric hindrance of the hydrophilic heads should impose a steric configuration to amino acids and α-hydroxyl acids that penetrate the membrane, even contained in a cyclic oligomer. This configuration concern the length of the carbon chain and the chirality.
   Thus, among the 20 biotic amino acids, 13 have the functional group on the 3rd C, 4 on the 4th C, 1 on the 5th C (Arg) and 1 on the 6th C (Lys).
   The steric configuration concern the chirality also. It is imposed by the glycerol chirality of the hydrophilic head, self-imposed by the steric hindrance. The two chiralities should be complementary to further reduce steric hindrance. As the glycerol configuration is D, the chirality of amino acids and α-hydroxyl acids must be L.
   The phosphate ion also has a chirality. But it is changing because of the delocalized negative charge over its two free oxygens. This charge may be attracted by a positive charge outside, creating chirality. This flexibility allows to organize and to penetrate into the membrane somes kinds of molecules including amino acids, α-hydroxy acids and ionophores.
   We thus see that in the outer surface, the liposome acts by its very structure, regardless of the chemiosmotic process to which it adds. Added to these 2 processes there is the osmotic pressure of the molecules that can not cross the membrane, pressure that enhances the penetration of ionophores, amino acids and α-hydroxy acids. The minimal chemical reactions on the outer surface, in addition to ester bonds that join the hydrophilic heads to the fatty acids are ester and peptide bonds between monomers of ionophores.
   Once an ionophore puted in the membrane, it can chelate a metal and become a coenzyme. This is the case of chelation complex FeS we have seen, when we ignored proteins. Ionophores can also evolve in porins and enzymes such as ATPase.

Evolution of the hydrophobic region of the liposome[edit]

During its formation, and once formed, the liposome may incorporate long aliphatic chains, unsaturated and without ionic charge. This is the case of quinones, which can span the entire thickness of the membrane, such as ubiquinone. The liposome can also integrate heme wich has chelated, or may chelate, a metal. The heme is hydrophobic and carries a long aliphatic unsaturated chain.
Chemiosmotic process will guide the electrons of delocalized double bonds and facilitate, where appropriate, the transition of redox electrons from one heme to another. Adding some amino acids in the membrane and around the hemes by the chemiosmotic process, hemes gradually evolve into cytochromes increasingly powerful. On the external side of liposome, the head of a quinone may be surrounded by amino acids and act as a dehydrogenase may be initiating the oxidation-reduction chain. The methanol dehydrogenase EC. of a Gram-negative bacteria, for example, uses pyrroloquinoline quinone instead of NAD +.

Evolution of the inner surface of the liposome and the internal medium[edit]

Continuously with the evolution of the 2 previous parts the inner surface is enriched by the amino acids that have been subjected to the steric hindrance as they pass through the membrane, alone or as ionophores. They were selected for their length and they are homochiral.
Here we find the behavior of amino acids in relation to the hydrophilic heads we saw on the outside: grouping by pairing with zwiterrions may have a function of dehydrogenase by surrounding coenzyme of quinone kind, then initializing the metabolism. Again the chemical reactions take place on a poly-ion surface. However the evolution of the inner surface is very specific and even represent the origin of biological order as we know it:

  • In addition to the prebiotic chemiosmotic process that it is subjected to like other parts, the inner surface is in contact with the internal medium that is closed, and whose chemical reactivity is triggered remotely by the osmotic pressure of global external anions that can not penetrate the membrane.
  • The internal medium is closed, it keeps any organization created by the inner face, limiting molecular diffusion.The changes in chemical species are under the control of the membrane. The composition of these species is very specific: these are small nonpolar or polarized molecules, but not ionized. They are weakly acidic or basic, because they were able to cross the membrane in neutral form. They will serve as a substrate for reactions occurring on the inner surface: methane, methanol, glycerol, H2S, CO2, H2, adenine, uracil, urea ...
  • Anions and large external molecules can not penetrate the membrane. These anions are facing the repulsion of the phosphate anion of the hydrophilic head, repulsion that is stronger than that opposed to cation by the amine group of ethanolamine. And apparently for anions, the equivalents of ionophores for cation transport must be rare, in any case in the literature. On contrary some weak acids, those who have crossed the membrane, can dissociate into anions and protons, thus reducing the overall osmotic pressure due to external anions. Which increases the chemical reactivity within and on the inner surface.
  • However internal surface will make the most important: the release of phosphate in the internal medium. The global osmotic pressure of external anions and that of the phosphate ion itself, will promote on the internal surface the hydrolysis of the ester bond between glycerol and phosphate. Phospho-serine or phospho-ethanolamine becomes free. They provide potential anion and phosphate. This is equivalent to a phosphate anion transport. What is not valid for other inorganic anions such as chlorate and sulfate. The spontaneous process of flip-flop resets the glycerol naked and tied to the fatty acid on the outer surface where it is esterified by a phosphate. With the fact that the internal environment maintains the organization generated by the inner surface, the chemistry within becomes the chemistry of phosphate. This is illustrated by the fact that the nucleotides are not found either outside, or even in the periplasm when this latter is narrow; the anions being in this case between two hydrophilic heads. In addition there are two types of phosphate ion transport in living organisms, every two much more complex than those of cations.

The following steps of molecular evolution.[edit]

  • Metabolism initialization and sequestration of phosphate.

Gradually, the chemiosmotic process helping, amino acids arrange themselves around the coenzymes (hemes, quinones, complex FeS) on the internal and external surfaces to complete the oxidation-reduction chain. Some ionophores will chelate metals to form coenzymes or enzymes. Other ionophores evolve to porins, ion channels and proton pumps such as ATPase.
Quickly the first enzymes of the inner side will initiate the metabolism of 3 carbons sugar phosphates already foreshadowed by free glycerol, phospho-serine and phospho-ethanolamine. This is the sequestration of phosphate. It will become increasingly irreversible with the synthesis of nucleotides whose bases arrive by passive diffusion.

  • The periplasm:

If the population of liposomes in a given environment, becomes dense, one liposome can include another. The space between the two is called periplasm in bacteria. If we have the same configuration as in bacteria, that is to say that the external liposome contains only ionophores and the second is much more evolved vis-à-vis the chemo-osmosis, then the evolution of the latter will accelerate because the periplasm will retain cations including protons, which enhances the electro-chemical potential.

  • Phospholipid synthesis and multiplication of liposomes:

At first time the multiplication of liposomes can be produced by an intake of fatty acid chains that insert into the membrane and fixe glycerol, phosphate and ethanolamine by esterification. This is the very process of liposomes formation that will be studied further in the chapipter of their geochemical origins.
Another way that liposomes multiply is that they synthesize fatty acids themselves. It is quite likely in the geochemical context raised at their abiotic synthesis. This possibility is the chelation by amino acids of the catalytic group which synthesizes the fatty acids in the external medium and its integration into the membrane .
In all cases the incorporation of fatty acids do not destroy structures scaffolded by the chemiosmotic process. Construction of these structures resembles the formation of a crystal. The multiplication of liposomes allows thus increasing the number of these structures.

  • Nucleotide synthesis, ribosomes and RNA:

At initialization of metabolism, the mononucleotides may be formed very early in molecular evolution. They will serve as coenzymes and substrate. They will be incorporated into groups of amino acids by hydrogen bonds. If there is synthesis of oligonucleotides by these pseudo-membrane-attached enzymes, they will remain linked by hydrogen bonds.
We see here the birth of ribosomes. In my opinion, before the appearance of the RNA world where RNA strands are free, this group of amino acids and oligo-nucleotides attached to the membrane certainly must outline the reproduction of the amino acid sequences pre-existing. And there would appear a relationship between nucleotide sequence and amino acid.
Duplicating a sequence of nucleotides should be done first by the process of base pairing that involves hydrogen bonds. This process is simple, flexible and reversible and does not involve only 4 bases at most. Two identical sequences of nucleotides will combine two sets of nearly identical amino acids by hydrogen bonds: This is a reproduction of groups of amino acids through the duplication of oligo-nucleotides. This is the beginning of the translation as we know among the living.
I stop here the scenario of molecular evolution, because it would further investigate the complexity of ribosomes and RNA. Otherwise it would consolidate the scenario developed until now by experiment.

The conceptual consequences of the prebiotic chemo-osmosis hypothesis.[edit]

  • The metabolic network of chemical reactions with small free molecules, is reduced through the chemiosmotic process applied to the liposome, to two electrochemical networks of cations and electrons.
  • The alkali cations are as important as C H O N P S chimical elements for living including prebiotic chemo-osmosis.
  • The liposome incorporating ionophores is the minimal living organism since it defines a closed space that is evolving and that instantly communicates with the outside through chemo-osmosis.
  • Prebiotic chemo-osmosis acts on small abiotic molecules which amino acids and α-hydroxy acids to form the first ionophores and membrane enzymes that initiate the metabolism inside by the 3-carbon sugar phosphates. With amino acids produced by this metabolism, prebiotic chemo-osmosis will evolve into biotic chemo-osmosis.
  • Chemistry in the membrane and inside the liposome is very specific and can not be done outside. This specificity is due to sequestration of phosphate within the liposome.
  • Chemical reactions are no longer under the laws of thermodynamics, but according to the laws of the electronics of the liposomal membrane and proteins. Enzymes are not catalysts, as conceived by the catalysis with metal atoms, much smaller than themselves. Enzymes are electronic structures that shape the local electric field, which attracts and directs small molecules to the catalytic site. Instead of the concept of biochemistry underlying chemistry and thermodynamics therefore, we must put the concept of organic electronics. For small molecules, upon contact with the membrane, go from one electromagnetic space defined by a protein to another electromagnetic space defined by another protein. They follow, certainly not linear paths, but physical corridors. Metabolic pathways of biochemistry are merely mnemonic patterns.
  • Functional proteins appeared before RNA that could recreate them.
  • RNA once formed, it forks itself by a matching process between its nucleic bases. Duplicated sequences attract amino acids by hydrogen bonds. This constitutes almost identical acid groups. The evolution of this process will lead to the genetic code and translation.
  • Reproduction of liposomes is made by self-assembly of abiotic phospholipids then by phospholipid produced by a limited metabolism of fatty acids, ethanolamine, serine and glycerol.
  • The proliferation of the first proteins is made by nucleation on the liposomes in the manner of nucleation of mineral crystals.

Geochemical origins of the initial molecules.[edit]

Hydrothermal vents on mid-ocean ridges[edit]

Currently most research on the origins of life are under the conditions of hydrothermal vents on mid-ocean ridges: around 300 °C and 300 bar. It has been demonstrated in these sites that the methane produced was abiotic ( Charlou [3] 2002, Proskurowski [4] 2008 ).
   Since then, laboratory work in conditions of similar temperature and pressure showed that we can synthesize abiotically:

  • Fatty acids and long chain alcohols from the Fischer-Tropsch process: Rushdi[5] 2001, McCollom 1999,[6] 2006 [7].
  • Esters of fatty acids with glycerol: Rushdi[8] 2006.
  • amino acids Ser, Ala, Asp et éthanolamine: A.D. Aubrey 2009 [9].
  • adenine: Michael Franiatte 2008 [10].

If one adds works done under near hydrothermal conditions or even standard, is approached almost the chemo-osmosis prebiotic hypothesis:

  • Oligonucleotide synthesis from amino acids in the presence of liposomes: Hideaki Tsukahara 2002 [11].
  • Synthesis of hemes, Lindsey [12] 2009.
  • Synthesis of 3-carbon sugars, including glyceraldehyde, by hydroformylation at 120 bar and 100-140 °C [13].
  • Hydrogenation of glyceraldehyde to glycerol.

Apart from works on the abiotic methane, all these works are laboratory experiments. On site, they had measured traces of long chain hydrocarbons but the aquatic environment in principle disperse all products by diffusion.

The pocket of abiotic oil[edit]

On the contrary, it is accepted by the specialists of this research (Charlou, Ifremer), that large quantities of gas produced at mid-ocean ridges can form gas clathrates. These clathrates may be covered with sediments and thus evolve over geological time. This is another hypothesis of abiotic oil formation.

  • Under pressure from a few kilos bars of sediments , much higher pressure than the pressure of hydrothermal vents, gas clathrates, H2 CO2 N2 CH4 H2S as reducers, can turn into oil and abiotic organic molecules. At these pressures the reactions of Fischer-Tropsch process for hydrocarbon formation and the reactions of the Haber-Bosch process for ammonia synthesis, see their balances move to hydrocarbons and ammonia, while their velocities decrease. In the case of the Tupi oil field discovered off Brazil, in an area without subduction under 2 km of water and 5 km of sediments, pressure is estimated at 1.5 kbar and temperature at 150 °C. By comparison, the pressure and temperature of a pocket of fossil oil harboring prokaryotes [14] cover a range of 400-800 bar and 55 °C, respectively.
  • For the intake of phosphate that did not mention it until now, there is a problem solubility for the most common mineral form of the phosphate, the apatite. Arrhenius's works (1997) [15] have shown that phosphates made ​​by erosion on the surface and brought on deep oceans floor, precipitate to hydrogenphosphates. The latter, at standard pressure and heated between 100 and 550 °C release oligophosphates. I guess the sediments that cover and mix with the clathrates of the above hypothesis, bring by this process the phosphate necessary for the formation of liposomes of the prebiotic chemo-osmosis hypothesis.

The ester bonds of phospholipids[edit]

These ester bonds are: glycerol-fatty acid, glycerol-phosphate, ethanolamine phosphate.
Abiotically and without mineral catalyst, such reactions are very slow, athermal and reversible. Therefore well suited for geochemical processes. Furthermore in the abiotic pocket oil, in the oil phase, aqueous vesicles concentrate ionic and polar substrates that act on fatty acid carboxyl heads on the surface of the vesicle. The phospholipids were formed by abiotic esterification of these molecules themselves formed abiotically.
To works of esterification of fatty acids with glycerol (Rushdi 2006), cited above, we must add works of L. Orgel 1996 [16] and 2004 [17]. He gave a summary of the phosphorylation of glyceric acid and α-hydroxy aldehydes by trimetaphosphate under standard temperature and pressure.


  2. Physiology and biochemistry of procaryotes 2nd edition, David White, Indiana university, Oxford university press 2000. Pages 66-102
  3. Charlou J.L., Donval J.P., Fouquet Y., Jean-Baptiste P., Holm N., « Geochemistry of high H2 and CH4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field », Chemical Geology, vol. 191, 2002, p. 345-359. sciencedirect
  4. Giora Proskurowski, Marvin D. Lilley, Jeffery S. Seewald, Gretchen L. Früh-Green, Eric J. Olson,1 John E. Lupton, Sean P. Sylva, Deborah S. Kelley: Abiogenic Hydrocarbon Production at Lost City Hydrothermal Field . Science vol 319, 1 février 2008 sciencemag
  5. Ahmed I. Rushdi, and Bernd R.T. Simoneit: Lipid formation by aqueous fischer-tropsch-type synthesis over a temperature range of 100 to 400°C. Origins of Life and Evolution of Biospheres (2001) 31: 103–118.
  6. T.M.McCollom et al. 1999: Lipid synthesis under hydrothermal conditions by fischer-tropsch-type reactions . Origins of Life and Evolution of the Biosphere 29: 153–166, 1999
  7. T.M.McCollom et al. 2006:Carbon isotope composition of organic compounds produced by abiotic synthesis under hydrothermal conditions. Earth and Planetary Science Letters Volume 243, Issues 1-2, 15 March 2006, Pages 74-84
  8. Ahmed I. Rushdi, and Bernd R.T. Simoneit : Abiotic condensation synthesis of glyceride lipids and wax esters under simulated hydrothermal conditions . Origins of Life and Evolution of Biospheres (2006) 36: 93–108 . DOI: 10.1007/s11084-005-9001-6 sciencedirect
  9. A.D. Aubrey, H.J. Cleaves, Jeffrey L. Bada: The Role of Submarine Hydrothermal Systems in the Synthesis of Amino Acids. Orig Life Evol Biosph (2009) 39:91–108.sciencedirect
  10. Michael Franiatte, Laurent Richard, Marcel Elie, Chinh Nguyen-Trung, Erwan Perfetti, Douglas E. LaRowe: Hydrothermal Stability of Adenine Under Controlled Fugacities of N2, CO2 and H2. Orig Life Evol Biosph (2008) 38:139–148.sciencedirect
  11. Hideaki Tsukahara, Ei-Ichi Imai, Hajime Honda, Kuniyuki Hatori and Koichiro Matsuno: Prebiotic oligomerization on or inside lipid vesicles in hydrothermal environments.Origins of Life and Evolution of the Biosphere 32: 13–21, 2002. sciencedirect
  12. Jonathan S. Lindsey, Marcin Ptaszek, Masahiko Taniguchi: Simple Formation of an Abiotic Porphyrinogen in Aqueous Solution. Orig Life Evol Biosph (2009) 39:495–515. DOI 10.1007/s11084-009-9168-3.
  13. Zoltán Györgydeák,István F. Pelyvás. Monosaccharide sugars: chemical synthesis by chain elongation, degradation...(Page 8). Academic Press 1998.
  14. Dorota Wolicka, Andrzej Borkowski, and Dariusz Dobrzynski: Interactions between Microorganisms, Crude Oil and Formation Waters. Geomicrobiology Journal, 27:43–52, 2010.
  15. G. Arrhenius, B. Sales, S. Mojzsis and T. Lee : Entropy and Charge in Molecular Evolution-the Case of Phosphate Journal of Theoretical Biology Volume 187, Issue 4, 21 August 1997, Pages 503-522 sciencedirect
  16. Vera Kolb and Leslie E. Orgel:Phosphorylation of glyceric acid in aqueous solution using trimetaphosphate. Origins of Life and Evolution of Biospheres Volume 26, Number 1, 7-13, DOI: 10.1007/BF01808156.
  17. Leslie E. Orgel: Prebiotic Chemistry and the Origin of the RNA World. Critical Reviews in Biochemistry and Molecular Biology, 39:99–123, 2004

External Links[edit]