BCKGROUND: New modes of action are needed for herbicides. The flavonoid synthesis intermediate t-chalcone causes apoptosis-like symptoms in roots and bleaching of shoots of Arabidospsis, suggesting a unique mode of action as a phytotoxin. RESULTS: Using RNA-Seq, transcriptome changes were monitored in Arabidopsis seedlings during the first 24 h of exposure (at 1, 3, 6, 12 and 24 h) to 21 µM t-chalcone (I50 dose), examining effects on roots and shoots separately. Expression of 892 and 1000 genes was affected in roots and shoots, respectively. According to biological function, many of the affected genes were transcription factors and genes associated with oxidative stress, heat shock proteins, xenobiotic detoxification, ABA and auxin biosynthesis, and primary metabolic processess. These are secondary effects found with most phytotoxins. Potent phytotoxins usually act by inhibiting enzymes of primary metabolism. KEGG pathway analysis of transcriptome results from the first 3 h of t-chalcone expsoure indicated several potential primary metabolism target sites for t-chalcone. Of these, p-hydroxyphenylpyruvate dioxygenase (HPPD) and tyrosine amino transferase were consistent with the bleaching effect of the phytotoxin. Supplementation studies with Lemna paucicostata and Arabidiopsis supported HPPD as the target, although in vitro enzyme inhibition was not found. CONCLUSIONS: t-Chalcone is possibly a protoxin that is converted to a HPPD inhibitor in vivo. Overall design: Arabidopsis seedlings (14 days-old) were treated with t-chalcone at IC50 concentration (21 µM). After treatment, plants were incubated for 0, 1, 3, 6, 12 and 24 h. Three replicate samples were collected from control plants (0.1% EtOH) and treated plants for each time point.