Inhibition of lipid biosynthesis

The first hint of a specific target process for graminicide inhibition came from the studies of Hoppe (1980) . He found that although diclofop-methyl did not interfere with photosynthesis, respiration, protein synthesis or nucleic acid synthesis, the inhibition of acetate incorporation into fatty acids could be demonstrated in susceptible species. Lichtenthaler and Meier (1984) later reported that the CHDs also disrupted de novo lipid biosynthesis in developing barley seedlings, and in 1987-88 four different research teams independently reported that the enzyme ACCase was the target of inhibition by both graminicide classes (see Lichtenthaler et al., 1989 and Secor et al., 1989 for further details).

Plant membranes contain unique fatty acids that have crucial structural and biochemical roles. For example, at least 70% of the total leaf fatty acids consist of the unsaturated a-linolenic acid (18:3), which itself makes up between 40% and 80% of the lipid fraction in the chloroplast. Indeed, the unique functioning of the thylakoid membrane to permit the movement of protons, electrons, and their carriers is considered by many workers to result from the property of membrane fluidity conferred by this unsaturated fatty acid. Trans-A3-hexadecanoic acid and linoleic acid (18:2) are additional examples of important thylakoid fatty acids. The synthesis of fatty acids in plants involves two major enzymes; acetyl-CoA carboxylase (ACCase) and fatty acid synthase. Fatty acids are synthesised both in the chloroplast stroma and the cytoplasm (Figure 8.1). Essentially, malonyl-CoA is formed from acetyl-CoA and converted to the saturated palmitate (16:0) by the action of a soluble stromal enzyme complex, termed fatty acid synthetase. This complex contains seven enzymes covalently bound to an acyl carrier protein (ACP), which transfers intermediates between the seven enzymes. Thus, seven enzyme cycles are needed for the condensation of seven additional C2 units into one palmitate. Two metabolic routes are now possible from palmitate. On the one hand, a soluble condensing enzyme and

CO Acetyl-CoA

aryloxyphenoxy-propionates, cyclohexanediones and phenylpyrazolines

Malonyl-CoA

acetyl CoA carboxylase

Acetyl-CoA (primer) v

CO, fatty acid synthetase

Palmitate (16:O-ACP)

Ag desaturase

frans-A3-Hexadecanoic acid

Malonyl-CoA CO,

Malonyl-CoA CO,

Condensing enzyme (soluble)

Stearate (18:O-ACP)

Condensing enzyme (soluble)

Stearate (18:O-ACP)

Ag desaturase thiocarbamates elongase

Oleic acid desaturase

Linoleic acid

Linoleic acid

18 : 3 a-Linolenic acid

18 : 3 a-Linolenic acid

Epicuticular waxes and suberin

Figure 8.1 Fatty acid biosynthesis in plants. ACP, acyl carrier protein.

elongases bound to the endoplasmic reticulum are able to add further C2 units in the cytoplasm to yield the long-chain saturated fatty acids found in suberin and the epicuticu-lar waxes on plant surfaces, and desaturases are present in the chloroplast to form the unsaturated fatty acids mentioned earlier (Harwood, 1988).

Acetyl-CoA carboxylase (acetyl-coenzyme A: bicarbonate ligase [ATP], E.C. 6.4.1.2.), or ACCase, is the first committed step for fatty acid biosynthesis in plants, and catalyses the formation of malonyl- CoA (Harwood, 1989) . ACCase is a high molecular weight, multifunctional protein with three distinct functional regions (two enzymic regions and one a carrier protein region), which involves biotin as an essential cofactor that functions as a CO2 carrier (Figure 8.2a). Initially, a carboxyl group is donated from a bicarbonate anion, and ATP hydrolysis is used to allow the formation of a carboxybiotin intermediate

(b)

ACCase (homomeric form) i malonyl-CoA

flavonoids, anthocyanins malonated amino acids very-long-chain fatty acids malonated ethylene precursor

Figure 8.2 (a) Diagrammatic representation of the three functional domains of ACCase (BC, BCCP, a-CT/p-CT) (reproduced from Ohlrogge and Browse, 1995). Reproduced with permission of American Society of Plant Biologists via Copyright Clearance Center. (b) Composition and compartmentalisation of the two forms of ACCase found in higher plants. See text for abbreviations (reproduced from Sasaki and Nagano, 2004, with permission).

by biotin carboxylase (BC). Carboxybiotin is attached to an e-amino group of a lysine residue on the biotin carboxyl carrier protein (BCCP). Carboxybiotin then functions as a CO2 donor in malonyl-CoA formation (Figure 8.2a). Malonyl-CoA is a substrate for fatty acid synthetase and also for fatty acid elongation (and the subsequent production of a number of important secondary metabolites, including flavonoids and phytoalexins). It is therefore likely that inhibition of ACCase affects a number of malonyl-CoA-requiring metabolic pathways. ACCase activity can be regulated at the transcription and posttranscription level but is also regulated by a number of metabolic factors. ACCase is most active when a plant is in light. During photosynthesis the stromal pH rises from 7 to 8 and Mg2+ concentration rises from approximately 1 mM to 3 mM. Plastid ACCase activity reaches a maximum at pH 8 and at Mg-+ concentrations of 2-5 mM. This ensures that ACCase is most active in the light, when photosynthesis is producing ATP, reductant and photosynthate, all of which are necessary for lipid biosynthesis. Laboratory studies by Kozaki and Sasaki (1999) have demonstrated that reducing agents will also activate ACCase, specifically the carboxytransferase (CT) domain of the enzyme. This appears to be due to the formation of a disulfide bridge between 2 cysteine residues located in the a and P-CT regions of ACCase (Kozaki et al., 2001). During photosynthesis concentration of reductant rises in the plastid and this represents a further light-mediated regulation of ACCase activity. It has also been postulated that ACCase may undergo phosphorylation, and this may also regulate its activity, although the mechanisms involved have yet to be fully elucidated (Savage and Ohlrogge, 1999).

Rendina and colleagues (1990) have characterised the kinetics of ACCase inhibition by graminicides. AOPPs and CHDs appear to be non-competitive inhibitors with respect to Mg2+ ATP, HCO- and acetyl-CoA. It therefore seems likely that this class of herbicides inhibits the carboxytransferase rather than the carboxylation step of ACCase activity. It also appears that both AOPPs and CHDs compete for the same site on the enzyme and that the inhibition is reversible. The inhibition of ACCase is both rapid and concentration dependent so that, for example, about 1 |im haloxyfop or tralkoxydim can inhibit the enzyme in vitro by 50% within 20 min. Furthermore, haloxyfop-acid is more than 100-fold more potent than the methyl-ester and only the R(+- enantiomer is herbicidally active (Secor et al., 1989). Recent observations that amino acids in the CT region are responsible for ACCase inhibition may lead to further information on the mechanism of herbicide binding to the enzyme. Indeed, the observation that Ile1781^Leu results in resistance to all fops and most dims but that Ile2041^Asn only results in resistance to fops, clearly indicates the importance of Ile2041 in fop but not dim binding (Dé-ye et al., 2003). Additionally, a homozygous herbicide-resistant black-grass biotype that contains Gly at position 2078 shows decreased fitness in the absence of herbicide, highlighting the importance of position 2078 in ACCase activity (Menchari et al., 2008).

Two forms of ACCase are found in higher plants (Figure 8.2b) and this plays an important role in the selectivity demonstrated by AOPPs and CHDs. In dicotyledons a hetero-meric form (termed the prokaryotic form) of ACCase, located in plastids, is insensitive to AOPPs and CHDs. The heteromeric form is composed of BCCP, BC, a-CT and P-CT polypeptides. It has been postulated that this form of ACCase is BCCP4 BC2 a-CT2 P-CT2, similar to bacterial ACCase (Choi-Rhee and Cronan, 2003). This form of ACCase is absent from the monocotyledon grasses and evidence suggests that this is due to the absence of the accD gene that encodes the P-CT polypeptide (Konishi and Sasaki, 1994). A homomeric

Table 8.2 Forms of ACCase found in higher plants (after Sasaki et al., 1995). Reproduced with permission of American Institute of Biological Sciences via Copyright Clearance Center.

Prokaryotic form

Eukaryotic form

Structure

Heteromeric (separate BCC, BC and CT subunits)

Homomer; single multifunctional polypeptide

Grasses

Absent

Plastids and cytosol

Dicotyledonous species

Plastids

Cytosol

Sensitivity to 'fops' and ' dims'

Insensitive

Sensitive (plastidic) Insensitive (cytosolic)

BCC, biotin carboxyl carrier; BC, biotin carboxylase; CT, carboxytransferase.

BCC, biotin carboxyl carrier; BC, biotin carboxylase; CT, carboxytransferase.

Table 8.3 Sensitivity of ACCase I and II from Lolium multiflorum biotypes to diclofop (from Evenson et al., 1997).

Biotype

Isoform

Source

Diclofop conc. (|M)

Inhibition (%)

Susceptible

ACCase I

Plastid

0.2

50

ACCase II

Cytosol

125

42

Resistance

ACCase I

Plastid

7

50

ACCase II

Cytosol

127

31

form of ACCase, located in plastids, is found in grasses and is sensitive to AOPPs and CHDs. This homomeric form is also found in the cytosol of both dicotyledons and grasses. It is a large polyeptide (~ 250 kDa) which contains BCCP, BC, a-CT and P-CT regions. It appears to be active as a dimer. These forms of ACCase are summarised in Table 8.2 and Figure 8.2b. This explains the selectivity of ACCase inhibitors between dicotyledons and grasses, as the presence of the herbicide-insensitive heterodimeric ACCase in dicotyledons allows the synthesis of fatty acids in the presence of these herbicides.

In the monocot maize two isoforms of ACCase are reported. The plastid form (ACCase I) predominates and is inhibited by AOPPs and CHDs. A cytosolic form (ACCase II) is 2000-fold less sensitive to these herbicides. Further studies have found a similar situations in other grasses, and it appears that some naturally occurring resistant biotypes of Lolium multiflorum possess an altered form of the plastid ACCase that is less sensitive to herbicides (Evenson et al., 1997; Table 8.3). Plant ACCases have recently been reviewed by Sasaki and Nagano (2004).

ACCase is not the only site of graminicide action. Weisshaar et al. (1988) have demonstrated that micromolar concentrations of the chloroacetamides also inhibit fatty acid biosynthesis by preventing the elongation of palmitate and the desaturation of oleate in the green microalga Scenedesmus acutus. It has subsequently been confirmed that both the elongation and desaturation steps of very long-chain fatty acid biosynthesis are targets for a number of herbicides.

Very-long-chain fatty acids (VLCFAs) are important components of the plant cell plasma membrane (plasmalemma) and are enriched in the leaf epicuticular waxes. Here they are embedded in a matrix and ensure the hydrophobicity of the leaf surface (Chapter 3.2).

Indeed, the cuticle is the main barrier against invasion by external agents and microorganisms, while preventing the loss of water and solutes from the leaves. Disrupting the plasma membrane will lead to a loss of permeability, transport and hormone receptor functions. It therefore follows that inhibition of VLCFA biosynthesis is a valuable target for herbicide action and development.

Members of several herbicide groups are now known to act as specific inhibitors of VLCFA biosynthesis, with alkyl chains longer than Cj8. They inhibit elongase activity in reactions taking place outside the chloroplast. Chemical groups include the chloroaceta-mides (e.g. alachlor), oxyacetamides (e.g. mefenacet), carbomylated five-membered nitrogen heterocycles (e.g. cafenstrole), oxiranes (e.g. indanofan) and miscellaneous others (such as ethofumesate). Some examples of herbicide structures acting as inhibitors of VLCFA biosynthesis are shown in Figure 8.3.

Alachlor

(chloroacetamide)

C2H5

C2H5

CH2OCH

COCH2O

Mefenacet (oxyacetamide)

COCH2O

Cafenstrole

(triazole)

"N

C2H5 C2H5

Indanofan (oxirane)

Ethofumesate

(thiocarbamate)

CH3 CH3

CH3 CH3

OC2H5

OC2H5

Figure 8.3 Structures of a selection of herbicides that inhibit very-long-chain fatty acid biosynthesis.

The chloracetamides have been successfully used in maize, soybean and rice for several decades, and remain today an important means of weed control, especially in maize. They are persistent herbicides, taken up from the soil, and several safeners have been developed for use in mixtures to extend their range of use. While germination is unaffected, early seedling growth is typically inhibited and the seedlings do not emerge from the soil or are severely stunted. Cell division and expansion are both inhibited.

The enzymes of acyl elongation are membrane- bound and thought to be associated with the endoplasmic reticulum and the Golgi apparatus in the cytoplasm, and perhaps with the plasma membrane itself. There are 21 genes encoding VLCFA elongases in Arabidopsis thaliana. Trentkamp and colleagues (2004) investigated the expression and activity of six gene products in the presence of VLCFA biosynthesis inhibiting herbicides and found wide substrate specificity. They suggest that such complex patterns of substrate specificity may explain why resistance to these herbicides is rare.

The substituted pyridazinones have been found to inhibit lipid biosynthesis in addition to their known inhibition of carotenoid biosynthesis (see section 6.3). Norflurazon can inhibit the A15-desaturase to prevent a-linolenic acid synthesis, and metflurazon may additionally prevent the formation of trans-A3-hexadecanoate by the inhibition of the A9-desaturase. These herbicides are classed as carotenoid biosynthesis inhibitors and their effects on lipid biosynthesis probably represent a secondary mode of action. That they have two points of action should not be surprising as they inhibit a desaturase enzyme in both metabolic pathways.

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