Phytoene Desaturase, the Essential Target for Bleaching Herbicides

Many bleaching herbicides with different core structures inhibit phytoene desaturase (PD), a membranebound enzyme in the carotenogenic pathway catalyzing the hydrogen abstraction step at the first C40 precursor of n-carotene. Prospects are good that new PD-active herbicides will be discovered by screening for bleaching activity. Accordingly, interest in PD enzymology and molecular genetics has increased. Although active carotenogenic cell-free systems are available, no isolation of PD has been achieved since the enzyme cannot be detected in its isolated form due to complete loss of activity. A portion of the Rhodobacter PD gene was incorporated into an appropriate plasmid which could be expressed in E. coli. This system was used to produce an antibody specific against PD from higher plants as well as Rhodobacter. All PDs assayed had an apparent molecular weight of 52 to 55 kDa. A Rhodobacter gene probe hybridized with a 3.1 kbBamH I fragment from Aphanocapsa which allowed us to sequence the PD gene from this cyanobacterium. Its DNA sequence matched with the apparent molecular weight of the PD band in the western blot, and a fusion-gene product was found to be immunoreactive with the Rhodobacter PD antibody, Anacystis mutants were produced exhibiting cross-resistance against norflurazon and fluorochloridone. Apparently, this resistance is due to an altered PD with concurrent decrease of inhibitor binding affinity. Cloning of the resistant gene into the wild type is in progress. Additional index words. Herbicide resistance, cell-free carotenogenic assays, phytoene desaturase. THE CAROTENOGENIC PATHWAY Chlorophylls and carotenoids are formed by plant-specific pathways, and interruption of biosynthesis by inhibitors causes bleaching of cells and plant foliage (4, 25). Carotenogenesis is affected by many agrochemicals. Carotenoids, namely carotenes and xanthophylls, play an essential role in photosynthesis by protecting chlorophyll against photooxidative destruction by singlet oxygen. This highly reactive oxygen species is formed in excess through excited chlorophyll when the photosynthetic machinery (electron transport and subsequent CO2 assimilation) operates too slowly to accommodate strong light. Protection against singlet 02 is achieved when nine or more double bonds are 'Received for publication February 22, 1990, and in revised form January 15, 1991. 2Assoc. Prof., former Grad. Student, Grad. Student, and Prof. of Plant Physiol. and Biochem., Univ. Konstanz, Konstanz, Germany. Present address of senior author Lehrstuhl fUr Physiologie und Biochemie der Pflanzen, Universitit Konstanz, D-7750 Konstanz, Germany. present in the carotenoid molecule (13) as is the case with lycopene, 5-carotene, or with the xanthophylls. These molecules physically interact with triplet state chlorophyll and dissipate its energy as heat. If a herbicide prevents carotene and xanthophyll formation, chlorophyll although formed will not accumulate either. Consequently leaves emerging after herbicide treatment will be depleted of all colored plastidic pigments. The mode of action of bleaching herbicides is inhibition of formation of carotenes. Thylakoids having an intact pigment inventory before herbicide treatment are not affected by application of the bleaching herbicide. Typically, bleaching occurs only in newly formed leaves as is well known to herbicide "screeners" in the greenhouse. This phenomenon is in contrast to peroxidative, degradative herbicides like p