BCH/PLS/PPA 609 -- Plant Biochemistry
Lecture Nineteen
Lipids -- Introduction, Synthesis of Fatty Acids, Waxes and Cutin

Purpose and Outline

The purpose of today's lecture/discussion is introduce some aspects of the biological chemistry of plant glycerolipids you likely didn't study previously. We will also review saturated fatty acid biosynthesis with emphasis on aspects unique to plants. Finally we will briefly cover the synthesis of waxes and cutin in plants.

Introduction

In biology the term lipid can refer to almost any molecule hydrophobic enough such that it would partition into an organic solvent from an aqueous solution.  Major lipids in plants include the glycerolipids and other fatty acid derivatives, isoprenoids and some phenylpropanoids.  In the narrow sense lipids only include glycerolipids.  In the next 3 lectures we will discuss the metabolism of glycerolipids and some other important fatty acid derivatives.

Glycerolipids are molecules in which in glycerol is esterified to 1-3 fatty acids.  Monoglycerides, having a fatty acid only esterified to the sn-1, or -2 position of glycerol, are rare in biological systems.  Triglycerides (TG) are the principal storage lipids and diglycerides are the main membrane lipids.  Most diglycerides have polar head groups including either a phosphate group for the phospholipids (PL) or a sugar or sugar derivative for the glycolipids (GL).  The main phospholipids in plants are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL).  The principal glycolipids are monogalactosyldiacylglycerol (MGD), digalactosyldiacylglycerol (DGD) and sulfoquinovosyldiacylglycerol (SL).  These are illustrated in Fig. 1 of Ohlrogge and Browse (1995) The Plant Cell 7, 957-970 and Tables 10.2 & 10.3 and Figure 10.5 of the class text.

Plant cell diagram

Fatty acids (FAs) are organic acids of the general formula RCOOH where R is an alkyl or alkane group -- pure hydrocarbon or a substituted hydrocarbon.  Thus FAs are alkyl acids = CH3(CH2)nCOOH.  Such alkyl acids are not sufficiently hydrophobic to generally be considered fatty acids unless n>4.  Some short and medium chain fatty acids and related acids with chemical names and common names where available are given in the following table:

Table 1. Short and medium chain fatty acids.

Formula

(chemical name)
common name

# Carbons

HCOOH

formic acid

1

CH3COOH

acetic acid

2

CH3CH2COOH

proprionic

3

CH3(CH2)2COOH

butyric

4

CH3(CH2)3COOH

(pentanoic)
valeric

5

CH3(CH2)4COOH

(hexanoic)
caproic

6

CH3(CH2)5COOH

heptanoic

7

CH3(CH2)6COOH

(octanoic)
caprylic

8

CH3(CH2)7COOH

nonanoic

9

CH3(CH2)8COOH

(decanoic)
capric

10

CH3(CH2)9COOH

undecanoic

11

CH3(CH2)10COOH

(____________)
lauric

12

CH3(CH2)11COOH

tridecanoic

13

CH3(CH2)12COOH

(____________)
myristic

14

CH3(CH2)13COOH

pentadecanoic

15

According to standard chemical nomenclature, the double bonds of unsaturated fatty acids are numbered from the carboxylic acid end of the molecule.  Thus linolenic acid is actually cis, cis, cis D9, D12, D15-octadecatrienoic acid. 

Table 2. Important long-chain fatty acids:
(hexadecanoic)
palmitic
16:0 ->		 (cis 7-hexadecenoic) [palmitoleic(D9)]
16:1 ->		 7,10-hexadecadienoic

16:2 ->		 7,10,13-hexadecatrienoic
(roughanic)
16:3
heptadecanoic
17:0
(_______________)
stearic
18:0 ->		 (9-octadecenoic)
oleic acid
18:1 ->		 (9,12-octadecadienoic)
linoleic
18:2 ->		 (9,12,15-octadecatrienoic)
linolenic acid
18:3
nonadecanoic
19:0
(eicosanoic)
arachidic
20:0 ->		 ->->-> (5,8,11,14-eicosatetraenoic)
arachidonic
20:4 [->]		 (5,8,11,14,17-eicosapentaenoic)
timnodonic; EPA
20:5
(docosanoic)
behenic
22:0 ->		 (13-docosenoic)
erucic
22:1 [->]		 ->->-> (4,7,10,13,16,19-docosahexaenoic)
cervonic; DHA
22:6
(tetracosanoic)
lignoceric
24:0 ->		 (15-tetracosenoic)
nervonic
24:1
v

v
waxes & cuticular lipids
Five fatty acids common in most plant tissues: 16:0, 18:0, 18:1, 18:2 and 18:3.  The double bonds for natural unsaturated fatty acids are all cis except for chloroplast PG which is ~30% 16:1-trans D3 exclusively at the sn-2 position.  Certain plants, known as 16:3 plants, accumulate significant levels (~10% of total leaf lipids) of a tri-unsaturated 16 carbon fatty acid mainly in plastid MGD, but also in DGD to a lesser extent (see Table 2 above).

Fatty Acid Biosynthesis

The biosynthesis and metabolic function of polyunsaturated fatty acids (PUFAs) are usually dependent on the distance of the double bonds from the methyl or  ω end of the molecule.  Common polyunsaturated fatty acids are either ω-6 or ω-6 fatty acids.  The double bonds of these fatty acids are also methylene (-C=C-C-C=C-) interrupted.  Fatty acids with conjugated dienes (-C=C-C=C-) can have quite distinct properties.  Cis unsaturated fatty acids have very different properties than trans unsaturated or saturated fatty acids.  For example the melting point of stearic acid (18:0) is 70°C, oleic acid (cis 18:1) 6°C, linoleic acid (cis, cis 18:2) -5 °C and linolenic acid (cis, cis, cis 18:2) -11°C.  Plants adapted for growth in temperate climates change the degree of unsaturation of membrane lipids in order to maintain membrane fluidity under different temperature conditions. The melting point of membrane lipids is determined by the combined fatty acid melting points.  At temperatures below their melting points membranes are in the gel phase and in the liquid crystalline phase at temperatures above their melting points (see Jakubowski's online text for a model).  Plants unable to rapidly increase desaturation of membrane fatty acids undergo chilling injury at moderately low temperatures (e.g. < ~10°C). 

In plants primary saturated and monounsaturated fatty acids are made in plastids.  Much of the polyunsaturated fatty acids are synthesized in the ER.  Triglycerides accumulate in structures known as oil bodies.  A general scheme of fatty acid biosynthesis in plant cells is depicted in the following figure:

[Image of fatty acid biosynthesis] KAS =

TE =

DS =

AT = As mentioned above, synthesis of primary saturated and monounsaturated fatty acid in plants occurs in plastids.  In the first step of fatty acid biosynthesis acetyl-CoA + CO2 -> malonyl-CoA is catalyzed by acetyl-CoA carboxylase.

What is the source of the acetyl-CoA?

This reaction actually occurs in 2 steps and the acetyl-CoA carboxylase has 3 functional domains.  The initial reaction involves an ATP-dependent transfer of CO2 from HCO3- to an N of the biotin prosthetic group of acetyl-CoA carboxylase.  This activated CO2 is transferred from biotin to acetyl-CoA forming malonyl-CoA in the 2nd step.  A model of the acetyl-CoA carboxylase complex is shown in Fig. 2 of Ohlrogge and Browse (1995) The Plant Cell 7, 957-970 and Figure 10.10 of the class text:


[Image of acetyl-CoA carboxylase complex]

The biotin, which "activates" the CO2, is attached to an epsilon-amino of the biotin carboxyl carrier protein by the biotin carboxylase functional region.  Carboxyltransferase transfers the CO2 from biotin to acetyl-CoA forming malonyl-CoA.  Plants have been found to have 2 forms of acetyl-CoA carboxylase.  A tri-functional single protein > 200 kD and a multi-subunit heteromeric complex.  The multi-subunit form is found in plastids of most plants except members of the Gramineae.  Plastids and cytosol of grasses and cytosol of other plants contain the single multi-functional polypeptide form.

The synthesis of saturated fatty acids is generally similar in all organisms.  However, fatty acid biosynthesis in plants has 2 fundamental differences from all other eukaryotic organisms.  First, in plants this occurs in the stroma of plastids whereas in other eukaryotes it occurs in the cytosol.  Second, saturated fatty acid synthesis, which requires at least 6 different enzymatic reactions, requires 6 different enzymes in plants in contrast to one hexa-functional protein in other higher eukaryotes (e.g. Fig 1 of Maier, Leibundgut and Ban, 2008).

Saturated fatty acid synthesis proceeds 2 carbon units per cycle usually 8 or 9 times producing 16 or 18 carbon products.  The donor for the 2 carbon units for each cycle is malonyl-ACP (acyl carrier protein) and the product is also an ACP-thioester.  Malonyl-ACP is formed from ACP and malonyl-CoA by a transacylase.  The next step of the fatty acid synthesis cycle involves condensation of
malonyl-ACP + acetyl-CoA -> 3-ketobutyryl-ACP + CO2 + CoASH.

This is driven by loss of CO2 making this pathway unidirectional.  The next step involves NADPH-dependent reduction of the 3-ketobutyryl-ACP to 3-hydroxybutyryl-ACP.  A dehydratase subsequently dehydrates this to the unsaturated product trans-delta²-butenoyl-ACP.  The last step of the cycle is the NADPH-dependent reduction of the double bond producing the 4C saturated fatty acid thioester, butyryl-ACP.  This cycle is repeated 7 or 8 times producing palmitoyl-ACP or stearoyl-ACP.

The condensation reactions are catalyzed by enzymes known as 3-ketoacyl-ACP synthases or KASs.  The 1st condensation reaction going from acetyl-CoA to 3-ketobutyrate is catalyzed by KAS III, the reaction from butyryl-ACP (C4) to palmitoyl-ACP (C16) by KAS I and from palmitoyl-ACP to stearoyl-ACP (C18) by KAS II.  The saturated fatty acid synthesis cycle is illustrated in Fig. 3 of Ohlrogge and Browse (1995) The Plant Cell 7, 957-970 and Fig. 10.8 of the class text:



See pcfig3.jpg

Why is it that the reaction stops at C16 and C18 fatty acids? 

The answer appears to be in the action of thioesterases (TEs) which hydrolyze the acyl-S-ACP thioester bonds.  In other words TEs stop the reaction at the appropriate chain length.  Some plants have unusual TEs, known as medium chain TEs, which stop the reaction at C8, C10, C12 or C14 fatty acid chain lengths. 

Once a TE releases the fatty acids, they are either incorporated into plastid glycerolipids by acyltransferases (ATs) or they are transferred to the cytosol and esterified to CoA.  This is illustrated in the following figure:

[Image of CoA transfer]

Saturated Fatty Acid Thioesterase genes are known as FatBs and that for the predominate unsaturated fatty acid-ACP synthesized in plastids FatA:

The fatty acids transferred to the cytoplasm enter the acyl-CoA pool providing fatty acids for glycerolipid synthesis (see lecture 15) or further elongation in the cytosol.  The further elongated fatty acids (C20 to ~C32), known as very long chain fatty acids (VLCFAs), can occasionally be put into triacylglycerol or used in synthesis of wax, cutin or suberin.

Wax synthesis

In wax synthesis some of the VLCFAs (as CoA thioesters), e.g. C30 and C32 are decarboxylated into C29 and C31 pure hydrocarbons.  Some of these long chain hydrocarbons are hydroxylated in the middle to alcohols, some of which can be further oxidized to ketones.  Others of the VLCFAs have the carboxylic acid reduced to aldehydes, some of which are further reduced to a-hydroxy alcohols.  The a-hydroxy alcohols can be esterified to VLCFA-CoAs forming esters usually 44-52 carbons long.  All 7 of these fatty acid derivatives are components of wax.  Wax synthesis is illustrated in the following figure:


[Image of wax synthesis]

Cutin biosynthesis

Cutin synthesis, the other major cuticular component, involves modification of some palmitoyl- and oleoyl-CoAs to hydroxy, epoxy and dihydroxy derivatives all of which (~8 monomers) are inter-esterified to form complex polyesters.  Suberin biosynthesis is similar but we do not have time to cover it here.  Cutin synthesis is illustrated in the following figures:


[Image of cutin synthesis, part 1]

[Image of cutin synthesis, part 2]

Cutin and wax biosynthetic pathways - Fig. 2 of Yeats & Rose (2013)

Wax crystals on the surface of pea leaves (Gniwotta et al., 2005; Fig. 3):

Regulation of cuticle biosynthesis - Fig. 4 of Yeats & Rose (2013)