Physiological and biochemical modes of action of the diphenylether aclonifen
Introduction
Since, the work of Matsunaka [1], it has been demonstrated that numerous organic compounds, having a diphenylether (DPE)1 nucleus, are potent herbicides that act in the light. This typical mode of action seems to be associated to the presence of specific substituents in the 2nd or 6th position of the DPE nucleus. The rapid photodependent destruction of isolated chloroplasts in the presence of very low concentrations of the DPE acifluorfen-methyl has been shown by Orr and Hess [2]. Furthermore, these authors have demonstrated the high effectiveness of DPE on isolated cucumber cotyledons. In the light, these organs present a rapid cell bleaching and necrosis.
The precise biochemical mode of action of acifluorfen-methyl and of most of the DPE compounds has first been elucidated by Matringe and Scalla [3]. The major target of this class of herbicides is protoporphyrinogen oxidase (Protox) located in the pathway leading from α-amino-levulinic acid to chlorophyll [3], [4], [5], [6]. Protox from various plant or animal organelles (chloroplasts, etioplasts and mitochondria) is inhibited by DPE [4], [5]. Surprisingly, the inhibition of Protox in these organelles leads to the accumulation of protoporphyrin IX in cells [3], [4], [7], [8], which is thought to result from a translocation of the Protox substrate protoporphyrinogen IX from the organelle outward [9]. This translocation is followed by non-enzymatic oxidation reactions or by herbicide-insensitive oxidases mainly in the plasma membrane [4], [10], [11], allowing the accumulation of protoporphyrin IX, which reacts with oxygen in the presence of light through a well-known photosensitization mechanism which gives rise to singlet oxygen (1O2) and probably to the superoxide anion (O2−) [12]. It is well-documented that these particular reactive oxygen species (ROS) cause lipid peroxidation and membrane damage leading to cell death [10].
A mechanism of action of DPE herbicides has been suggested by Ensminger and Hess [13]. In Chlamydomonas eugametos, these authors have shown that cell death occurs after acifluorfen-methyl herbicide treatment under light at two wavelengths, 450 and 650 nm, suggesting an effect on both chlorophyll and carotene. This might be due either to an energy transfer from the excited pigments to protoporphyrin IX (λmax: 366 nm) or to the fact that the excited state of the thylakoid membrane is much more unstable. The photosynthetic membranes of chloroplasts of higher plants and green algae collect light energy thanks to an antenna system containing chlorophyll and carotenoids. Carotenoids contribute to harvest and transfer light energy to maintain the function of reaction centers and light harvesting complexes (LHC) in the photosystems, as well as to provide an effective protective function by quenching chlorophyll triplet states, scavenging singlet oxygen species, and dissipating the excess energy absorbed by antenna chlorophyll [14], [15]. The inhibition of carotenoid synthesis by pyridazinone-type herbicides such as norflurazon leads to the destruction of antenna chlorophyll and to a rapid bleaching of young leaves but not to a rapid necrosis.
Chemically, aclonifen is a DPE herbicide which has been used for years in several types of cultures. It shows an evident structural analogy with other DPE herbicides such as acifluorfen or bifenox (Fig. 1). However, its herbicidal symptoms on plant seedlings remain unclear and are generally not the typical photodestruction of young leaves. It was therefore interesting to find out its precise biochemical activities in plants. Two pathways were taken into account in the elucidation of aclonifen biochemical activities leading to the herbicide effect, first the typical DPE mode of action and secondly the interference with carotenoid biosynthesis as suggested by bleaching symptoms observed in several plants.
Section snippets
Plant material
Seeds of corn (Zea mays L.) were soaked for 8 h in aerated tap water, 25 °C, or in tap water containing, respectively, 10−4 M norflurazon, 10−4 M aclonifen obtained from Bayer CropScience and 10−4 M acifluorfen. The imbibed seeds were planted in 200 ml plastic pots containing a mixture of sand and vermiculite and grown in darkness for 7 days at 24 °C, 80% relative humidity [16].
Extraction, chromatography and determination of photosynthetic pigments in etiolated corn seedlings
Control and treated plantlets (2 g fresh weight) were extracted in cold acetone. The crude extracts were filtered and
Results
One of the visible symptoms of aclonifen action on corn seedlings was a bleaching of young leaves with no rapid necrosis under a light of low intensity. Carotenoid biosynthesis was therefore investigated.
Discussion
Results presented here show that aclonifen was able to act as an herbicide on two separate biochemical pathways, chlorophyll synthesis and carotenoid synthesis at quite similar concentration. This indicates that aclonifen could not be physiologically considered only either as a typical DPE or as a typical inhibitor of carotenoid biosynthesis. This point was established in several plants, for instance in corn. Under a light of reduced intensity, the inhibition of carotenoid biosynthesis could be
Acknowledgment
This investigation was supported by Bayer CropScience.
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