p-Chlorophenoxyisobutyric acid (PCIB) is known as a putative antiauxin and is widely used to inhibit auxin action, although the mechanism of PCIB-mediated inhibition of auxin action is not characterized very well at the molecular level. In the present work, we showed that PCIB inhibited BA::␤-glucuronidase (GUS) expression induced by indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid, and 1-naphthaleneacetic acid. PCIB also inhibited auxin-dependent DR5::GUS expression. RNA hybridization and quantitative reverse transcriptase-polymerase chain reaction analyses suggested that PCIB reduced auxin-induced accumulation of transcripts of Aux/IAA genes. In addition, PCIB relieved the reduction of GUS activity in HS::AXR3NT-GUS transgenic line in which auxin inhibits GUS activity by promoting degradation of the AXR3NT-GUS fusion protein. Physiological analysis revealed that PCIB inhibited lateral root production, gravitropic response of roots, and growth of primary roots. These results suggest that PCIB impairs auxin-signaling pathway by regulating Aux/IAA protein stability and thereby affects the auxin-regulated Arabidopsis root physiology.The plant hormone auxin (indole-3-acetic acid [IAA]) plays an important role in every aspect of plant growth and development (Thimann, 1977; Davies, 1995). Despite its physiological significance, the molecular mechanism of auxin action is not fully understood yet. One of the earliest events in the auxin action involves the changing of expression pattern of some specific genes with a lag period of 5 to 30 min (Abel and Theologis, 1996). Auxin-dependent degradation of Aux/IAA proteins is a key event for early auxin-dependent gene induction (Leyser, 2002). Aux/IAA proteins interact with auxin response factors (ARFs) that bind to auxin-responsive elements (AuxREs) in the auxin-responsive promoters (Guilfoyle, 1998). Aux/IAA proteins are short-lived proteins, and their stability is regulated by auxin through the ubiquitin-mediated protein degradation pathway (Leyser, 2002). In the absence of auxin, large amounts of Aux/IAA proteins are present, bind to ARFs, and prevent ARFs to activate transcription of auxin-induced genes from AuxREs. In the presence of auxin, degradation of Aux/IAA proteins is promoted, and then ARF proteins are released from Aux/IAA proteins and activate transcription of auxin-responsive genes (Tiwari et al., 2003). Although detailed molecular mechanism of the role of ARF and Aux/IAA proteins and their regulation has been revealed in these recent years, the mechanisms of the very early step of auxin signaling, i.e. how auxin is recognized by plant cell and how ubiquitin proteolysis system is activated, still remains unknown (Benfey, 2002).p-Chlorophenoxyisobutyric acid (PCIB), also called ␣-(4-chlorophenoxy) isobutyric acid, 2-(p-chlorophenoxy)-2-methylpropionic acid, or clofibric acid, has been most widely used to inhibit auxin action (e.g. Kim et al., 2000;Xie et al., 2000). Because of the structural similarity of PCIB with a synthetic auxin 4-chlorophenoxyac...

Summary 2,4-dichlorophenoxyacetic acid (2,4-D), a chemical analogue of indole-3-acetic acid (IAA), is widely used as a growth regulator and exogenous source of auxin. Because 2,4-D evokes physiological and molecular responses similar to those evoked by IAA, it is believed that they share a common response pathway. Here, we show that a mutant, antiauxin resistant1 (aar1), identified in a screen for resistance to the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), is resistant to 2,4-D, yet nevertheless responds like the wild-type to IAA and 1-napthaleneacetic acid in root elongation and lateral root induction assays. That the aar1 mutation alters 2,4-D responsiveness specifically was confirmed by analysis of GUS expression in the DR5:GUS and HS:AXR3NT-GUS backgrounds, as well as by real-time PCR quantification of IAA11 expression. The two characterized aar1 alleles both harbor multi-gene deletions; however, 2,4-D responsiveness was restored by transformation with one of the genes missing in both alleles, and the 2,4-D-resistant phenotype was reproduced by decreasing the expression of the same gene in the wild-type using an RNAi construct. The gene encodes a small, acidic protein (SMAP1) with unknown function and present in plants, animals and invertebrates but not in fungi or prokaryotes. Taken together, these results suggest that SMAP1 is a regulatory component that mediates responses to 2,4-D, and that responses to 2,4-D and IAA are partially distinct.