Insights into ROS-dependent signalling underlying transcriptomic plant responses to the herbicide 2,4-D

The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) functions as an agronomic weed control herbicide. High concentrations of 2,4-D induce plant growth defects, particularly leaf epinasty and stem curvature. Although the 2,4-D phenotype is associated with ROS production, little is known about ROS-dependent signalling. In this study, by using T-DNA Arabidopsis mutants to silence peroxisomal acyl CoA oxidase 1 (acx1-2), we identified ACX1 as one of the main sources of ROS production and as partly the cause of the epinasty phenotype following the application of 2,4-D. Transcriptomic analyses of WT plants after treatment with 2,4-D revealed a ROS-related peroxisomal footprint in early plant responses, while other organelles, such as mitochondria and chloroplasts, are involved in later responses. Interestingly, a group of ACX1-dependent transcripts in plant responses to 2,4-D, previously associated with epinasty, is related to auxin biosynthesis, metabolism and signalling. We found that protein AUXIN SIGNALING F-BOX 3 (AFB3), a component of SCF (ASK-cullin-F-box) E3 ubiquitin ligase complexes, which functions as an auxin receptor and mediates Aux/IAA proteasomal degradation, acts downstream of ACX1 and is involved in the epinasty phenotype induced by 2,4-D. We also found that protein degradation associated with ubiquitin E3-RING and E3-SCF-FBOX in ACX1-dependent signalling in plant responses to 2,4-D is significantly regulated over longer treatment periods. Col-0 was the background used for all the Arabidopsis seeds in the study: WT and the transgenic lines acx1-2 (kindly donated by Dr. J. León; SALK_041464). Seeds were sown in moistened soil and grown in 16 h light and 8 h dark photoperiod cycles at 22 and 20 ºC, respectively, under 120 µE and at 60 % relative humidity. The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on Arabidopsis plants was analysed by spraying 23 mM 2,4-D solution (1% dimethyl sulfoxide acid, DMSO and 1% EtOH), and plant leaves were collected after 1 and 72 h post treatment (hpt). Control plants were sprayed with 1% DMSO and 1% EtOH. Treatment times, 2,4-D concentrations and treatment techniques used in this study had been previously optimised. 2,4-D- and DMSO/EtOH (control)-sprayed leaves of similar sizes and developmental stages were sampled at 1 and 72 h. For microarray analysis, three independent biological replicates, each composed of leaves pooled from at least 5 different plants, were used per experimental condition. Time course