BOTANY VOLUME 2 - PLANT PHYSIOLOGY - 2007

6. METABOLIC PHYSIOLOGY

6.11. Biosynthesis of Structural and Storage Lipids

The construction of PLANT Cells AND the maintenance of compartmentalization require the continuous synthesis of structural Lipids, which serve as the fundamental Building Blocks of membranes. In addition, plant cells store reduced carbon in the form of lipids, reaching up to 50% in fat-storing seeds. Compared to Reserve Polysaccharides, carbon storage in the form of lipids halves the mass of storage reserves and thereby facilitates the dispersal of airborne seeds. Other structural lipids (Waxes) are deposited in extracellular layers on the outer cuticle or impregnate the walls of specific plant cells: cutin (e.g., Casparian strips) or suberin (the primary substance found in cork). Cutin is also an integral component of the cuticle. Little is known about The Biosynthesis of waxes, cutin, and suberin, and therefore this topic will not be pursued further.

Membrane Lipids and storage lipids are glycerolipids (see 1.5, Fig. 1.12). They are complex esters consisting of the trihydric alcohol glycerol and three acyl residues (storage lipids are triacylglycerols or triglycerides). Membrane lipids correspondingly contain two acyl residues, while the third hydroxyl group bears a polar substituent (Phospholipids, Glycolipids). Lipid METABOLISM IN The plant Cell is a complex series of reactions involving Plastids, the Cytoplasm, and The Endoplasmic reticulum (Fig. 6.101). These reactions are discussed in more detail in the following chapters.

6.11.1. Biosynthesis of Fatty Acids

According to current concepts, de novo biosynthesis of Fatty acids in plants occurs exclusively in plastids—in Chloroplasts in green cells, and in chromoplasts, leukoplasts, or proplastids in non-green cells. In some Algae (e.g., Euglena gracilis), a cytoplasmic fatty acid synthetase Functions alongside the plastid one, whereas Fungi synthesize fatty acids in the cytoplasm. The starting molecule for synthesis is acetyl-CoA, to which two-carbon units provided by malonyl-CoA are successively added via Condensation. Acetyl-CoA in plastids arises either from Pyruvate through the action of a plastid pyruvate dehydrogenase isoform or (mostly) from acetate, whose cytoplasmic origin is not yet fully elucidated. Acetyl-CoA synthetase (see Fig. 6.101) transfers an adenylate residue to acetate with the Cleavage of pyrophosphate from AMP, resulting in The formation of a mixed phosphoric-carboxylic anhydride with acetic acid. In the second step, the adenylate residue is replaced by coenzyme A. Malonyl-CoA is produced by the carboxylation of acetyl-CoA via a multienzyme complex of acetyl-CoA carboxylase using biotin as a prosthetic group (Fig. 6.102).

Class="center">Fig. 6.101. Scheme of Fatty acid and glycerolipid metabolism in a plant cell. Individual reactions are described in the text; the structures of various lipid classes and their Abbreviations can be found in Fig. 1.21. Reactions occurring on membranes are shown on a gray Background. (1) acetyl-CoA synthetase; (2) acetyl-CoA carboxylase; (3) acyl-CoA synthetase; (4) glycerol-3-phosphate dehydrogenase; (5) Acyltransferases. Gal = galactose; Ch = Choline; ACP-SH = acyl carrier protein. Fatty acid designation: 18:1 — 18 carbon atoms, 1 double bond; X:n — any fatty acid. Acyl residues indicate that glycerolipid synthesis proceeded either via the prokaryotic pathway (16:n at the sn-2 position, as in blue-green algae) or the eukaryotic pathway (18:n at the sn-2 position). Glycerol-3-phosphate is, by definition, written in the L-configuration (the OH group is located to the left of the symmetrically substituted middle carbon atom) and numbered by analogy with the structurally similar 3-phosphoglyceraldehyde. This refers to stereospecific numbering (sn), with carbon atoms designated as sn-1, sn-2, and sn-3. Semi-schematic formulas preserve the traditional spatial depiction of acyl residue arrangement, which was omitted in Fig. 1.21. The spatial arrangement of substituents shown there provides a clearer picture of the actual three-dimensional Structure of glycerolipids, thereby facilitating the understanding of Membrane Structure formation. The C- and N-termini of oleosin are located on the cytoplasmic side of the half-membrane and form the hydrophilic HEAD domain, whereas the large central part of the protein forms a lipophilic domain that presumably assists in the loading of oleosomes with triglycerides.

Fig. 6.102. The process of de novo fatty acid synthesis in the plastid stroma. The prosthetic group of acetyl-CoA carboxylase (biotin) is linked to the apoenzyme via a Lysine residue. The structure of biotin and carboxybiotin is shown in the box on the left. The Formation of Acetyl-CoA and malonyl-CoA from their respective coenzyme A precursors proceeds without energy consumption or generation; it is a reversible reaction. Decarboxylation coupled with the condensation of two two-carbon units (the 3-ketoacyl synthase reaction) proceeds with the release of a large amount of energy, making this reaction irreversible. Both this and subsequent decarboxylation reactions drive the direction of the biosynthetic pathway, since the remaining Enzymes catalyze only reversible reactions in each case. ACP-bound metabolites are present as thioesters. The thiol group belongs to a pantetheine residue (just as in coenzyme A, see Fig. 6.93), which is linked via a phosphate group to a Serine residue of the apoenzyme through Esterification (see box at the bottom). ACP = acyl carrier protein; ACP-SH = free acyl carrier protein with an unoccupied thiol group (-SH).

The fatty acid synthetase (FAS) complex, which in this case is also a multienzyme complex, consists of individual enzymes that—unlike the FAS systems in fungi and animals—can be isolated in pure form and clearly separated by function, along with a free soluble acyl carrier protein (ACP) (10–14 kDa, English: acyl carrier protein, ACP). It attaches both starting compounds (acetate or malonate) and intermediate products arising from carbon chain elongation (acyl residues). FAS reacts exclusively with ACP-bound metabolites. Plant ACP and FAS components are structurally very similar to the corresponding bacterial structures. Consequently, plant FAS has a prokaryotic Organization. The reaction sequence is shown in Fig. 6.102. Synthesis terminates when the carbon chain reaches a length of 16 or 18 C atoms, yielding either a palmitoyl-ACP complex (16:0-ACP) or a stearoyl-ACP complex (18:0-ACP). In this notation, the number before the colon indicates the number of C atoms in the fatty acid molecule, and the number after the colon indicates the number of double bonds.

Still within the plastid stroma, a soluble desaturase converts stearoyl-ACP into oleoyl-ACP (18:1-ACP), which contains one unsaturated bond. Some of the products of plastid fatty acid synthesis are used to build the plastid's own membrane lipids, while the rest are exported to the cytoplasm (see Fig. 6.102). In the process, immediately before or during the passage of the fatty acid-acyl carrier protein complex through the plastid envelope membrane, ACP is cleaved off by acyl-ACP thioesterase. However, sufficiently large amounts of free fatty acids do not accumulate in the cytoplasm, because an acyl-CoA synthetase localized on the outer envelope membrane converts them into acyl-CoA using ATP.

Thus, the newly synthesized acyl-coenzyme A molecules (palmitoyl-CoA, stearoyl-CoA, and oleoyl-CoA) can enter further reactions in various ways (see Fig. 6.101).

✵ In the endoplasmic reticulum, carbon chains are elongated with the help of membrane-bound elongases. This is how fatty acids with 20 or more C atoms are synthesized, which are found particularly in storage lipids.

✵ In addition, the ER is responsible for incorporation into membrane or storage lipids.

✵ Unsaturated fatty acids with two or more double bonds (e.g., linoleic (18:2) and linolenic

(18:3), which are not synthesized in The Human Body, are essential and must be obtained from the diet) are often formed in the ER only at the stage of glycerolipid synthesis with the participation of membrane-bound desaturases, and are released via the acyl exchange of oleoyl-CoA (18:1-CoA) for linoleyl-CoA (18:2-CoA) or linolenyl-CoA (18:3-CoA) (see Fig. 6.101).

6.11.2. Biosynthesis of membrane Lipids

As mentioned earlier, the synthesis of membrane lipids (see Fig. 6.101) takes place on the plastid envelope membranes and in the ER. The structural backbone, glycerol, is generated in the cytoplasm via the reduction of dihydroxyacetone phosphate into the final product glycerol-3-phosphate (mediated by glycerol-3-phosphate dehydrogenase). Acyl residues are transferred by an acyltransferase either from acyl-ACP (in the case of plastid synthesis) or from acyl-CoA (in the case of ER synthesis). Enzyme Specificity varies. Glycerolipids of plastid origin are characterized by the mandatory presence of a C₁₆ acyl residue at the sn-2 position, whereas glycerolipids synthesized in the ER constantly feature a C₁₈ acyl residue at this position.

Initially, diacylglycerol phosphate (phosphatidic acid) is formed, from which plastids produce a specialized glycolipid—monogalactosyldiacylglycerol (MGDG — see Fig. 1.21). When necessary, after the Introduction of double bonds into the acyl residues, the latter serves as a precursor for the formation of glycolipids, sulfolipids, and phospholipids within plastids (see Fig. 6.101; Fig. 1.21, Table 1.4). However, only a portion of plastid membrane lipids is synthesized directly within the organelle; another part is formed through the metabolism of glycerolipids (specifically phosphatidylcholine) imported from the ER.

In the ER, Phosphatidic acid is also initially formed from glycerol-3-phosphate via the twofold transfer of acyl residues, and from it, phosphatidylcholine (a phospholipid) is synthesized by The addition of a head group (choline phosphate derived from cytidine diphosphocholine). When necessary, following the action of desaturases, other membrane lipids of the endoplasmic reticulum are produced from phosphatidylcholine (see Table 1.4). A portion of phosphatidylcholine, predominantly dilinoleylphosphatidylcholine (containing two linoleic acid residues (18:2)), is transported by lipid transfer Proteins to the plastid envelope membranes, where it is converted into MGDG and, if required after desaturase action, used for the synthesis of remaining membrane lipids. Lipid transfer proteins may also participate in delivering lipids to other membranes where these lipids are not synthesized (thylakoid, mitochondrial, glyoxysomal, and peroxisomal membranes).

The fatty acid composition of membrane lipids influences the Physical Properties of membranes (e.g., fluidity at a given Temperature). This is presumably an important factor in plant cold tolerance or sensitivity to low temperatures. A special role here is assigned to phosphatidylcholine, which exhibits different compositions in cold-tolerant and cold-sensitive plants. In the former, the proportion of unsaturated fatty acids is increased, whereas in the latter, saturated fatty acids predominate. By altering membrane Lipid Composition through Genetic Engineering, researchers have successfully modified the cold tolerance of the model plant Arabidopsis thaliana (thale cress, see Section 7.1).

6.11.3. Biosynthesis of Storage Lipids

All cells store some amount of triacylglycerols (triglycerides, neutral fats), though most cells do so in very small quantities. In fat-storing seeds, triglyceride content can reach up to 50% of the seed mass (peanut, flax). Additionally, the fruit Tissues of certain plant species (olive, avocado) contain high amounts of neutral fats; however, these serve not for reuse, but to increase the palatability of the fruits to consumers, thereby aiding seed dispersal. In many species, large amounts of triglycerides are produced by the tapetum, which upon dissolution enters the anther locule and forms an extracellular lipid layer around the mature pollen grains. Pollen grains may contain up to 20–30% (of their dry mass) of intracellular storage lipids. Triacylglycerols that contain a high percentage of saturated Fatty Acids and remain solid at room temperature are called fats. Triacylglycerols containing a high percentage of unsaturated fatty acids that are liquid at room temperature are called oils.

The biosynthesis of storage lipids occurs in the ER and begins with acyl-CoA and glycerol-3-phosphate. There are two pathways for this biosynthesis. The first pathway starts with the formation of phosphatidic acid, its dephosphorylation to diacylglycerol, and concludes with The transfer of a third acyl residue to the remaining free hydroxyl group. The second pathway involves The conversion of phosphatidylcholine into diacylglycerol and subsequently into triglyceride (see Fig. 6.101). The second pathway is presumed to drive the synthesis primarily of those storage lipids that contain polyunsaturated fatty acids with two or more double bonds.

It should be noted that nonpolar triacylglycerols, accumulating between the membrane surfaces of the ER lipid bilayer, separate them from one another until the lipid droplet, surrounded by a "half-unit" elementary membrane (a single lipid monolayer), is finally isolated (see Fig. 6.101). The completed lipid-storing organelle is called an oleosome (sometimes a spherosome). Oleosomes of highly desiccation-tolerant seeds contain A large number of amphipathic proteins, oleosins, which are synthesized in the ER and localize to the half-membrane during oleosome isolation (see Fig. 6.101). Oleosins are absent in the oleosomes of lipid

rich fruit tissues, recalcitrant seeds, and pollen oleosomes. Oleosins apparently prevent the "coalescence" of oleosomes into larger structures during the germination of dry seeds when Water uptake occurs, thereby facilitating the mobilization of storage lipids (by maintaining a large surface area).