Arabidopsis thaliana in Culture: A Powerful Tool to Decipher the Mode of Action/Target Sites of Herbicides with Antimetabolite Activity

Abstract

Arabidopsis thaliana in culture is a powerful tool to determine the mode of action of herbicides with antimetabolite activity. The culture media can be manipulated to identify various biosynthetic pathways blocked by probe compounds. For example, inhibition of Arabidopsis in culture caused by four standards, viz., asulam, glyphosate, sulcotrione, and pyrithiobac-sodium (PTB) were specifically reversed by p-aminobenzoate (PABA), aromatic amino acids, homogentisic acid and branched chain amino acids, respectively. These standards are known inhibitors of 7,8-dihydropteroate synthase (DPT synthase, in the folic acid biosynthesis pathway), 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, in the shikimate pathway), p-hydroxyphenylpyruvate dioxygenase (HPPD, in the plastoquinone biosynthesis pathway) and acetolactate synthase (ALS, in the pathway for branched chain amino acids), respectively. This technique was used to investigate two compounds with previously unknown modes of action. Inhibition of Arabidopsis growth by hydantocidin (Hy), and 6-methylanthranilate (MA), was specifically reversed by adenosine-5′-monophosphate (AMP, for Hy), and anthranilate or tryptophan (for MA). Hy was thus suspected to block purine biosynthesis, while MA was proposed to block the biosynthesis of tryptophan. Follow-up studies revealed that Hy and MA are proherbicides. The herbicidal forms were identified as hydantocidin-5′-phosphate (HP) and 4-methyltryptophan (4MT), respectively. Target sites for HP and 4MT were found to be adenylosuccinate synthetase (ADSS) in the purine biosynthesis pathway and anthranilate synthase (AS) in the tryptophan biosynthesis pathway. Observations made with Hy were also confirmed with a known inhibitor of ADSS, hadacidin (Ha). ADSS was inhibited competitively by both Ha and HP, but with respect to aspartate and inosine-5′-monophosphate (IMP), respectively. HP was found to be about two orders of magnitude more potent an inhibitor than Ha. Further, HP was co-crystallized with ADSS complexed to its substrates. A detailed analysis of the crystal structure should help in the design of new inhibitors as possible herbicides.