Inhibition of chlorophyll biosynthesis

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Chlorophyll is the principal pigment in photosynthesis. In addition to a light-harvesting function, chlorophyll is located within reaction centres, and so it plays a pivotal role in the movement of electrons from low energy (H-O) to high energy (NADP+). The

Herbicides and Plant Physiology, Second Edition Andrew H. Cobb and John P.H. Reade © 2010 A.H. Cobb and J.P.H. Reade. ISBN: 978-1-405-12935-0

CHLOROPHYLLS

CHLOROPHYLLS

Glutamate Chlorophyll

biosynthesis of porphyrins and tetrapyrroles begins with the formation of 8-aminolaevulinic acid (8-ALA) and its conversion to porphobilinogen (PBG) (Figure 6.2). In animals, 8-ALA is formed from glycine and succinyl-CoA and in plants 8-ALA is synthesised via glutamate and a pyridoxal phosphate-linked transaminase. Gabaculine inhibits the transaminase by covalent binding to the pyridoxal phosphate cofactor, whereas 4-amino-5-fluropentanoic acid (AFPA; Gardner et al., 1988) can attack the transaminase active site to form a stable complex that inactivates the enzyme. Laevulinic acid, dioxovaleric acid and dioxoheptanoic acid all compete with 8 -ALA to inhibit the synthesis of PBG. A succinyl moiety (COOH-(CH2)2-C=O) appears to be an essential prerequisite for this competitive inhibition of 8-ALA dehydratase (Figure 6.2 ).

Since these two reactions are common to the biosynthesis of all tetrapyrroles their inhibition also results in the cessation of phytochrome, cytochrome, peroxidase and cata-lase synthesis. The inhibitors just described clearly show some potential as non-selective herbicides, but since terapyrroles have a central role in the metabolism of all organisms, a lack of mammalian toxicity would need to be conclusively demonstrated.

In Animals In Plants

COOH

In Animals In Plants

COOH

Figure 6.2 Biosynthesis of 8-aminolaevulinic acid (ALA) and porphobilinogen (PBG).

The chlorophyll biosynthesis inhibitors are also referred to as 'peroxidising herbicides' because their action results from the peroxidising effect of active oxygen species. The pioneering examples are the p-nitrophenyl ethers (Table 6.1) and the cyclic imides (Table 6.2), developed between 1965 and 1980, and the most recent examples (such as fluthiacet-methyl, Table 6.2) have been developed in the last decade to be active at very low rates of 1-10 g ha-1.

The diphenyl ethers and cyclic imides are an important group of broad-spectrum herbicides that have been widely used for the selective control of annual grasses and broadleaf weeds in major world crops, including soybean, peanut, cotton and rice. Successful active ingredients include acifluoren, oxyflurorfen and bifenox (Table 6.1), and the generation of singlet oxygen produces phytotoxicity. It was initially thought that the phytotoxic species was generated in the thylakoid - as is the case with the inhibitors of photosynthetic electron flow - but research has conclusively demonstrated that photosynthesis is not directly involved in diphenyl ether action, and that the chloroplast envelope is an initial site of action (see Figure 5.14 ; Derrick et al., 1988).

Observations that diphenyl ether action was particularly sensitive to low energy blue light led to the suggestion that carotenoids could mediate singlet oxygen generation. However, Matringe and Scalla (1988) have shown typical phytotoxic symptoms in carotenoid-free cell lines and instead reported the accumulation of protoporphyrin IX in treated plants. These authors have since conclusively demonstrated that protopor-phyrinogen oxidase is the target enzyme (Matringe et al., 1989).

I t is currently envisaged that protoporphyrinogen IX synthesis and its oxidation to protoporphyrin IX are chloroplast envelope membrane- bound reactions, and that pro-toporphyrinogen IX spontaneously and non- enzymatically oxidises to form the potent photosensitiser, protoporphyrin IX, often referred to as PROTOGEN. This molecule cannot be further metabolised to porphyrins and so accumulates, possibly in the stroma, where it generates toxic singlet oxygen and lipid photoperoxidation ensues (Figure 6.3).

Diphenyl ethers are now known to be potent inhibitors of protoporphyrinogen IX oxidase, with concentrations as low as 4nM aciflurofen-methyl inhibiting this enzyme

Table 6.1 Structure of some diphenyl ether peroxidising herbicides.

Cl

,R2

R —

fy°-

\ y—no2

Ri

r2

Comman name

cf3

COOH

acifluorfen

cf3

OC2H5

oxyfluorfen

Cl

H

c itrofen

Cl

COOH

bifenox

Table 6.2 Structures of some cyclic imide peroxidising herbicides.

Table 6.2 Structures of some cyclic imide peroxidising herbicides.

Classifica Das Bacterias

by 50% in corn etioplasts (Matringe et al. , 1989) . However, this enzyme from either mammalian and yeast sources appears equally sensitive, which suggests that the toxicological properties of these herbicides may need to be closely re-examined.

Peroxidising herbicides inhibit protoporhyrinogen oxidase, commonly abbreviated to PROTOX (E.C. 1.3.3.4). Recent studies suggest that this activity is located in several parts of the cell apart from the plastids, including at the mitochondrial inner membrane for haem production, the endoplasmic reticulum and possibly at the plasmalemma, in addition to soluble forms in the chloroplast stroma.

The membrane-bound protox activity shows very high sensitivity to peroxidising herbicides with I50 values as low as 10-10M. Since all eukaryotic protox enzymes are sensitive to these inhibitors, their safety in animals and humans has been questioned. Some authors cite evidence that these compounds can alter porphyrin metabolism in animals, with an accumulation of porphyrin intermediates. The literature suggests, however, that no health problems associated with the consumption of crops treated with protox inhibitors have been reported and that these herbicides are rapidly metabolised in animals.

5-ALA

-►Uroporphyrinogen I

4C02

Coproporphyrinogen I

2C02

non-enzymic oxidation

Protoporphyrinogen IX--------► Protoporphyrin IX (P IX)

light \

Protoporphyrin IX MJ/^chelatases chlorophylls cytochromes

> singlet oxygen lipid peroxidation membrane and pigment breakdown *

bleaching

Figure 6.3 An overview of porphyrin biosynthesis in the presence (dashed line) and absence (solid line) of diphenyl ether and cyclic imide herbicides.

Peroxidising herbicides act as rapid, reversible, competitive inhibitors of protox activity and the most potent structures show close resemblance to the geometric shape and electronic characteristics of one-half of the protogen molecule. Since this molecule is a pho-todynamic pigment in its own right, it generates highly active oxygen species in the stroma and the cytoplasm.

Protox catalyses the six-electron oxidation of protoporphyrinogen IX to protoporphyrin IX, a known and potent photosensitiser. Flavoproteins are closely involved in the activity. Photoaffinity labelling of radioligands has been used to study herbicide binding, with one herbicide binding site suggested for each FAD molecule, with the active site at the C-terminal domain of the protein. The enzyme protein appears highly resistant to protease degradation and it is thought that acylation stabilises its conformation.

Genes involved in protox activity have now been isolated from bacteria, yeasts and higher plants. The Arabidopsis protox gene encodes a protein of 537 amino acid residues with little structural homology to the enzymes encoded in human or mouse genes. There have been two cDNA sequences found in tobacco and other plants, which share only 27% sequence similarity, one located in the chloroplast and the other in the mitochondrion. These two isoforms have molecular masses of 60 kDa and 55 kDa, respectively.

Several approaches have been used to obtain plants that are resistant to the protox inhibitors. This is an active area of herbicide research and development, and new protox inhibitors continue to emerge (Matsumoto, 2002) . For example, Grossmann and colleagues (2010) have announced saflufenacil for the pre-emergent control of dicot weeds in several crops, including maize.

Mutant cell cultures have been obtained by several groups by incorporating the herbicides into the growth medium, followed by later studies to identify the gene sequence that confers resistance. This strategy has generated resistance to pyraflufen - ethyl in tobacco. A mutation of Val 389 to Met in the plastidic isoform has also generated resistance in Chlamydomonas reinhardtii. Expression of the bacterial enzyme in plants has generated tolerance to the diphenyl ether oxyfluorfen. Overexpression of the Arabidopsis plastidic form has resulted in resistance to acifluorfen. Clearly, several companies are now pursuing the development of peroxidising herbicide resistance in crops and further details are anticipated.

Readers are referred to the multi-authored text Peroxidising Herbicides, edited by Boger and Wakabayashi (1999) for a detailed treatise on these important herbicides, including their chemistry, physiology, mode of action and toxicology.

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