Inhibition of carotenoid biosynthesis

In addition to their light-harvesting role, carotenoids protect chlorophyll from attack by active oxygen species by quenching both triplet chlorophyll and singlet oxygen dissipating this energy as heat (Figure 6.4) . How thermal energy dissipation is achieved has challenged researchers for several decades. In 2000, Li et al. reported a breakthrough using a mutant of the plant Arabidopsis thaliana that contained normal concentrations of zeaxanthin but was unable to dissipate thermal energy. They used molecular and genetic markers to show that a photosystem II (PS II) protein known as CP22 (see Figure 5.4. a chlorophyll-binding protein with a relative molecular mass of 22,000) was

heat

Figure 6.4 Protection of chlorophyll (Chll) by carotenoids (car) (after Britton et al. 1989). © Cambridge University Press.

heat

Figure 6.4 Protection of chlorophyll (Chll) by carotenoids (car) (after Britton et al. 1989). © Cambridge University Press.

absent in the mutant. On reintroduction into the mutant plant of a normal copy of the gene encoding CP22, the ability to dissipate thermal energy was regained. CP22 is one of over 30 proteins thought to be involved in light harvesting and its precise function remains unclear. Li and colleagues imply that a conformational change in the thylakoid takes place via CP22 to achieve the dissipation, which requires zeaxanthin and a trans-t hylakoid pH gradient. Interestingly, these workers have also suggested that CP22 synthesis may increase in leaves exposed to excess light on a daily basis, implying an important physiological role for this PS II protein.

Surplus reductive capacity in the chloroplasts (i.e. production of NADP.2H in excess of that required for carbon fixation and other biosynthetic reactions) is dissipated by the xanthophyll cycle (Figure 6.5) which involves three different carotenoids: violaxanthin (a di - epoxide), antheraxanthin (a mono - epoxide) and zeaxanthin (epoxide - -ree). These three carotenoids are reversibly interconvertible by the addition or removal of an epoxide group, as shown in Figure 6.5 , In strong light, violaxanthin is converted to zeaxanthin via antheraxanthin. This conversion, which is catalysed by a de- epoxidase enzyme, is optimal at low pH (around 5.1). The conversion of violaxanthin to zeaxanthin takes place within a few minutes in the presence of high-intensity light. During irradiation of chloroplasts, the de-epoxidase enzyme is activated by the drop in pH within the thylakoid due to photosynthetic electron transport. Under the conditions of a proton gradient across

Figure 6.5 The xanthophyll cycle.
2NADP.2H + 202

the thylakoid membrane, violaxanthin is reduced to zeaxanthin with the participation of the redox systems glutathione / oxidized glutathione and ascorbic acid / dehydroascorbic acid. Reconversion of zeaxanthin to violaxanthin is catalysed by an epoxidase enzyme, requires NADP.2H and uses oxygen. This reaction occurs rapidly in low-intensity light or darkness and is optimal at higher pH (around 7.5; see Figure 6.5). Thus, under high-intensity light conditions, the xanthophyll cycle favours production of zeaxanthin, thereby increasing the capacity for dissipating light energy as heat and protecting against photooxidative damage.

The atomic structure of the major light-harvesting antenna protein, LHC II, has been determined by X-iay crystallography. It can exist in different reversible states, operating in either light- harvesting or in energy-dissipation mode. In addition to chlorophyll molecules, the LHC II also contains the carotenoid lutein. The formation of the quenched antenna state is controlled by the carotenoids of the xanthophyll cycle. Thus de-epoxidation of violaxanthin to zeaxanthin, which stimulates energy dissipation, is thought to modulate structural changes in LHC II. In this way Pascal et al. (2005) consider that the LHC II molecule behaves as a 'natural nanoswitch' to control the emission or transfer of incoming light quanta.

it follows from the above that if carotenoids biosynthesis is inhibited, free^adical attack and lipid peroxidation will rapidly ensue under high lightiintensity conditions, as previously described.

Carotenoids are synthesised in the chloroplast via the mevalonic acid pathway (Figure 6.6). In essence, this pathway involves the condensation of five carbon units (isopentenyl pyrophosphate) to yield many molecules of both physiological (e.g. natural plant growth regulators) and biotechnological (e.g. terpenes) importance. In carotenoid biosynthesis two molecules of the 20-carbon geranylgeranyl pyrophosphate combine to yield the carotenoid precursor 15-cis-phytoene which undergoes a series of desaturation steps to all-trans-iycopene. Cyclisation followed by ring hydroxylation yields the major higher plant carotenoids, namely P-carotene and lutein. The inhibition of isopentenyl pyrophosphate or geranylgeranyl pyrophosphate biosynthesis would affect the production of all plant terpenoids and could present a target for future herbicide action, although no such inhibitors are currently known. The desaturation steps between phytoene and lycopene, however, have been successfully exploited agrochemically. These enzymes are present as a multi-enzyme complex situated at the thylakoid membrane.

Any enzyme involved in the reaction sequence to P-carotene is a potential herbicide target to induce bleaching symptoms, though all commercial examples are phytoene desaturase inhibitors, with in vitro I50 values of 0.01-0.1 ^M. In plants, both phytoene desaturase (PDS) and Z-carotene desaturase (ZDS) (Figure 6.6) catalyse the desaturation sequence from phytoene to lycopene, proceeding via hydrogen abstraction forming double bonds with NAD and NADP as potential hydrogen acceptors, although plastoquinone has a 20-fold higher affinity than NADP. This role of plastoquinone in phytoene desaturation also explains why the presence of inhibitors of plastoquinone biosynthesis leads to phytoene accumulation.

While the herbicidal inhibition of phytoene desaturase is non-competitive with respect to the substrate phytoene, competition has been observed for the cofactors.

Phytoene desaturase inhibitors are numerous and have been intensively studied during the last two decades. Commercial examples include norflurazon, flurochloridone and

"/3-Carotene Figure 6.6 Carotenoid biosynthesis.

diflufenican (Figure 6.7), which are used as pre-planting or pre-emergent herbicides. The persistent activity of diflufenican has been effectively exploited in long-term weed control in paths and drives. Since germinating weeds lack photoprotective carotenoids, lipid peroxidation rapidly follows seedling emergence; weeds appear bleached and quickly die.

Flurochloridone

Norflurazon cf3

Flurochloridone

Diflufenican

Figure 6.7 Commercial examples of herbicides that inhibit phytoene desaturase.

Diflufenican

Figure 6.7 Commercial examples of herbicides that inhibit phytoene desaturase.

Fewer compounds are known to interfere with ^-carotene desaturase or lycopene cyclase, and none appears to have been commercially developed as yet.

Efficient bleaching herbicides possess a central five-or-six-membered heterocycle, carrying one or two substituted phenyl rings, implying a common binding site at the phytoene desaturase protein (Sandmann, 2002).

Fedtke et al. (2001) have reported the inhibition of lycopene cyclase by some novel diethy lamines. Lycopene accumulates in treated plants with marked reductions in the concentrations of neoxanthin, violaxanthin, lutein and P-carotene. Those workers demonstrated that an observed inhibition of photosynthesis was due to interference with the turnover of the D1 protein in the PS II reaction centre. The reassembly process requires the continuous biosynthesis of two reaction-centre P-carotene molecules, without which protein D1 disappears, especially at high light- flux densities. Interestingly, this depletion of PS II precedes the bleaching process, which may imply a new mechansim of herbicidal activity shared by both the lycopene cyclase and phytoene desaturase inhibitors.

6.4 Inhibition of plastoquinone biosynthesis

Plastoquinone is an electron acceptor in carotenoid biosynthesis in addition to its key role in photosynthetic electron transport (Figure 6.8). Inhibitors of plastoquinone biosynthesis are also herbicides and cause typical bleaching symptoms. Plants synthesise plastoquinone from the aromatic amino acid tyrosine via the intermediate homogentisic acid (Figure 6.9).

Phytoene

Phytoene

Phytoene desaturase

Phytofluene

Phytoene desaturase

Phytofluene

Plastoquinol-9

Plastoquinol-9

Photosynthetic electron flow

Plastoquinone-9

Plastoquinone-9

Figure 6.8 The role of plastoquinone in carotenoid biosynthesis and its regeneration by photosynthetic electron flow.

COOH

Figure 6.8 The role of plastoquinone in carotenoid biosynthesis and its regeneration by photosynthetic electron flow.

COOH

tyrosine

4-hydroxyphenyl-pyruvic acid (HPP)

homogentisic acid

COOH

tyrosine

4-hydroxyphenyl-pyruvic acid (HPP)

Figure 6.9 Synthesis of homogentisic acid from tyrosine.

homogentisic acid

The herbicides that inhibit the activity of hydroxyl phenylpyruvate dioxygenase (HPPD) are shown in Figure 6.10.

Isoxaflutole is a pro-herbicide and its metabolic byproduct, a diketonitrile, acts as the inhibitor. Similarly, a metabolite of the herbicide pyrazolate, termed detosyl-pyrozolate, is a potent HPPD inhibitor. Intriguingly, inhibition of HPPD activity by the graminicide sethoxydim has also been claimed, in addition to its inhibitory activity against acetyl-CoA carboxylase (Lin and Young, 1999).

The benzoyl-cyclohexanediones, sometimes termed the herbicidal triketones, were first patented in 1985 as a new group of bleaching herbicides. They are particularly effective when applied pre-emergence in maize at doses as low as 60-100 g ha-1 and provide an effective low-dose alternative to atrazine.

SO2CH3

isoxaflutole (pro-herbicide)

SO2CH3

o SO2CH3

o SO2CH3

CF, diketonitrile (inhibitor)

CF, isoxaflutole (pro-herbicide)

diketonitrile (inhibitor)

sulcotrione; mesotrione; NTBC;

sulcotrione; mesotrione; NTBC;

OH O

OH O

benzoylresorcinols

A = Cl, B = SO2CH3 A = NO2, B = SO2CH3 A = NO2, B = CF3

c2h5s

benzoylresorcinols

O Cl c2h5s n-oc2h5

sethoxydim

c3h7

n-oc2h5

sethoxydim

O Cl

detosyl-pyrazolate Figure 6.10 Structures of HPPD inhibitors.

detosyl-pyrazolate Figure 6.10 Structures of HPPD inhibitors.

Barta and Böger (1996) isolated HPPD from maize and demonstrated potent competitive inhibition by several experimental benzoyl-cyclohexanediones with I50 values in the range of 3-23 nm. Viviani et al. (1998) performed a detailed kinetic study of HPPD inhibition by diketonitrile and other triketones and concluded that the herbicides acted as tightly binding inhibitors that dissociate extremely slowly from enzyme-inhibitor complex.

HPPD (E.C. 1.13.11.27; E.C. 1.14.2.2) is a monomeric polypeptide of molecular mass 43 kDa (Barton and Böger, 1996) in maize, whereas it behaves as a homodimer of 48-kDa subunits in cultured carrot cells. Amino acid sequences are known from plant, animal and microbial HPPDs. Highly conserved regions at the C-terminus suggest an involvement of this region in the catalytic process, perhaps including sites for the binding of the substrate and an iron atom at specific histidine and glutamate residues.

As indicated by Pallett (2000) , the prospects of further HPPD inhibitors are enhanced by the resolution of the crystal structure of the enzyme. Similarly, an examination of other enzymes in the biosynthesis of quinones and tocopherols should be evaluated as potential targets for herbicide development.

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