Herbicide pyrazolate causes cessation of carotenoids synthesis in early watergrass by inhibiting 4-hydroxyphenylpyruvate dioxygenase

In aqueous solution, the herbicide pyrazolate [4-(2,4-dichlorobenzoyl)-1,3-dimethyl-5-pyrazolyl p-toluenesulfonate] is rapidly hydrolyzed to destosyl pyrazolate (DTP), 4-(2,4-dichlorobenzoyl)-1,3-dimethyl-5-hydroxypyrazole, which is an active form of the herbicide. The objective of this study was to examine the effect of pyrazolate and DTP on carotenoids synthesis in susceptible weed, early watergrass (Echinochloa oryzicola Vasing.). Furthermore, their in vitro effect on 4-hydroxyphenylpyruvate dioxygenase (HPPD) was determined. Roots of the plants at the two-leaf stage were soaked for 24 h into pyrazolate (5 × 10–5 mol L−1) or norflurazon (10–6 mol L−1) solution containing 0.5% volume of acetone. At the first sampling time (3 days after treatment: 3 DAT), the chlorophyll content in the third leaves of pyrazolate-treated plants were not different compared with the untreated control, but it was decreased between 3 and 6 DAT. The declining pattern of β-carotene in the third leaf of early watergrass was very similar to that of chlorophyll. Both herbicides induced greater accumulation of phytoene in the third leaves of early watergrass 3 DAT, and the levels were kept until 9 DAT. However, feeding of homogentisate reduced the phytoene accumulation only in pyrazolate-treated plants, suggesting the site of action of the herbicide located in the pathway of plastoquinone synthesis. In a HPPD assay, DTP revealed to inhibit the enzyme with an IC50 value of 13 nmol L−1 and that of pyrazolate was 52 nmol L−1. In the pyrazolate solution used in the assay, some of the herbicide possibly has been hydrolyzed to DTP. From the all results obtained, it is strongly suggested that pyrazolate inhibits carotenoids synthesis and causes bleaching on the developing leaves by the similar mechanism with norflurazon, but its action site is not phytoene desaturase and is HPPD.