The mechanisms of reception/transduction of cytokinins still remain largely unknown. We used 1‐(2‐azido‐6‐chloropyrid‐4‐yl)‐3‐(4‐[3H])phenylurea ([3H]azido‐CPPU), a new photoaffinity probe to search for cytokinin‐binding proteins. A soluble protein that binds phenylurea‐type cytokinins has been specifically photolabeled in Nicotiana plumbaginifolia (cv. Viviani line pbH1D) leaf extracts. The protein was purified to homogeneity by affinity chromatography. Its N‐terminal amino acid sequence, as well as four internal peptidic sequences are highly homologous with the theta class of the glutathione S‐transferase superfamily (GST, EC 2.5.1.18) including Hyoscyamus muticus and Arabidopsis GSTs identified as auxin‐binding proteins. The purified N. plumbaginifolia protein also possesses GST enzymatic activity. To test the possible involvement of this GST in the mechanism of action of cytokinin, we studied the binding of tritiated‐CPPU to the purified GST in the presence of various compounds, cytokinin agonists, cytokinin antagonists, or inactive molecules. Thidiazuron is a poor competitor, and neither zeatin nor the active optical isomer R‐MeBA is able to inhibit the binding of CPPU. There is no correlation between the cytokinin activity and the binding properties of the molecules tested. Our results confirmed that plant GSTs bind different compounds, especially plant hormones but probably have no specific role in the mode of action of cytokinins.

It is now generally accepted that protoporphyrinogen oxidase is the target-enzyme for diphenylether-type herbicides. Recent studies [Camadro, J-M., Matringe, M., Scalla, R. & Labbe, P. (1991) Biochem. J . 277,[17][18][19][20][21] have revealed that in maize, diphenyl ethers competitively inhibit protoporphyrinogen oxidase with respect to its substrate, protoporphyrinogen IX.In this study, we show that, in purified pea etioplast, [3H]acifluorfen specifically binds to a single class of high-affinity binding sites with an apparent dissociation constant of 6. All these results providc clear evidence that in pea etioplasts, ['Hlacifluorfen exclusively binds to prvtoporphyrinogen oxidase, that the protoporphyrinogen oxidase inhibitors tested so far bind to the same region of the enzyme and that this region overlaps the catalytic site of the enzyme.Chloropbylls and cytochromes are two major classes of plant tetrapyrroles, and their biosyntheses have a number of steps in common. Some of the relevant enzymes, however, are poorly characterized. Such is the case for protoporphyrinogen oxidase, the last enzyme at the common branch of thc two biosynthetic pathways.Protoporphyrinogen oxidase catalyses the oxidative transformation of protoporphyrinogen IX into protoporphyrin IX. The enzyme is strongly inhibited by several families of peroxidative herbicides, especially by diphenyl ethers like acifluorfen ( Fig. 1

No abstract

The specific binding of the herbicide acifluorfen 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-~tro~nzoic acid to corn etioplast membranes is competitively inhibited by protoporphyrinogen IX, the substrate of protopotphyrinogen oxidase. Three other peroxidizing molecules, oxadiazon [5-terbutyl-3-(2,4dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-one], LS 82556 [(S)3-N-(methylbenzyl)carbamoyl-5-propionyl-2,6-lutidine], and M&B 39279 [5-amino-4-cyano-l-(2,6-dichloro-4-trifluoromethylphenyl)pyr~ol], also compete with acifluorfen for its binding site. The four herbicides thus bind to the same site, or to closely located sites, on the enzyme protoporphyrinogen oxidase.