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.

To isolate novel auxin-responsive mutants in Arabidopsis (Arabidopsis thaliana), we screened mutants for root growth resistance to a putative antiauxin, p-chlorophenoxyisobutyric acid (PCIB), which inhibits auxin action by interfering the upstream auxinsignaling events. Eleven PCIB-resistant mutants were obtained. Genetic mapping indicates that the mutations are located in at least five independent loci, including two known auxin-related loci, TRANSPORT INHIBITOR RESPONSE1 and Arabidopsis CULLIN1. antiauxin-resistant mutants (aars) aar3-1, aar4, and aar5 were also resistant to 2,4-dichlorophenoxyacetic acid as shown by a root growth assay. Positional cloning of aar3-1 revealed that the AAR3 gene encodes a protein with a domain of unknown function (DUF298), which has not previously been implicated in auxin signaling. The protein has a putative nuclear localization signal and shares homology with the DEFECTIVE IN CULLIN NEDDYLATION-1 protein through the DUF298 domain. The results also indicate that PCIB can facilitate the identification of factors involved in auxin or auxin-related signaling.

A semi-dominant mutant suppressor of hy2 (shy2-1D) of Arabidopsis thaliana, originally isolated as a photomorphogenesis mutant, shows altered auxin responses. Recent molecular cloning revealed that the SHY2 gene is identical to the IAA3 gene, a member of the primary auxin-response genes designated the Aux/IAA gene family. Because Aux/IAA proteins are reported to interact with auxin response factors, we investigated the pattern of expression of early auxin genes in the iaa3/shy2-1D mutant. RNA hybridization analysis showed that levels of mRNA accumulation of the early genes were reduced dramatically in the iaa3/shy2-1D mutants, although auxin still enhanced gene expression in the iaa3/shy2-1D mutant. Histochemical analysis using a fusion gene of the auxin responsive domain (AuxRD) and the GUS gene showed no IAA-inducible GUS expression in the root elongation zone of the iaa3/shy2-1D mutant. On the other hand, ectopic GUS expression occurred in the hypocotyl, cotyledon, petiole and root vascular tissues in the absence of auxin. These results suggest that IAA3/SHY2 functions both negatively and positively on early auxin gene expression.

We have constructed transgenic Arabidopsis lines that contain a gene for green fluorescent protein (GFP) under the control of auxin-responsive domains A and B of the promoter from the pea PS-IAA4/5 gene. The chimeric transgene was named BA-mgfp5-ER. GFP was detected after the application of indole-3-acetic acid at concentrations as low as 100 nM in epidermal cells in the root elongation zone. The induction of the reporter gene was highly specific to auxin and was correlated with the auxin-induced change in epidermal cell shape. No GFP accumulation was observed in the lateral root meristem that was formed as a result of exogenous auxin application. These results suggest that auxin signals were transmitted through several distinct pathways depending on the cell type. The intensity of the GFP signal was strong enough to be observed through the plastic lid of the culture dish using a dissecting microscope, thereby enabling GFP expression to be monitored in an aseptic environment. Thus, the BA-mgfp5-ER transgenic plant can be a powerful tool for screening mutants that are defective in auxin signaling and the expression of early auxinresponse genes.