These authors contributed equally to this work. SummaryThe plant hormone, auxin, regulates many aspects of growth and development. Despite its importance, the molecular mechanisms underlying the action of auxin are largely unknown. To gain a more comprehensive understanding of the primary responses to auxin, we analyzed the expression of genes in Arabidopsis seedlings treated with indole-3-acetic acid (IAA) for 15 min. We identified a single gene that is downregulated early, and 29 genes that are upregulated early. Several types of typical transcription factors are identified as early upregulated genes, suggesting that auxin signals are mediated by a master set of diverse transcriptional regulators. Of the genes that responded to auxin, the expression of the homeobox gene, HAT2, was induced rapidly. Furthermore, we show that the expression of HAT2 is induced by auxin, but not by other phytohormones. To analyze the function of HAT2 in the plant's response to auxin, we generated 35S::HAT2 transgenic plants. These produced long hypocotyls, epinastic cotyledons, long petioles, and small leaves, which are characteristic of the phenotypes of the auxin-overproducing mutants, superroot1 (sur1) and superroot2 (sur2). On the other hand, 35S::HAT2 plants showed reduced lateral root elongation, and reduced auxin sensitivity compared to wild-type plants. Together with the results of RNA blotting and biochemical analyses, these findings suggest that HAT2 plays opposite roles in the shoot and root tissues in regulating auxin-mediated morphogenesis.

Despite numerous physiological studies addressing the interactions between brassinosteroids (BRs) and auxins, little is known about the underlying molecular mechanisms. We studied the expression of IAA5 and IAA19 in response to treatment with indole acetic acid (IAA) or brassinolide (BL), the most active BR. Exogenous IAA induced these genes quickly and transiently, whereas exogenous BL induced them gradually and continuously. We also found that a fusion of DR5, a synthetic auxin response element, with the GUS (␤-glucuronidase) gene was induced with similar kinetics to those of the IAA5 and IAA19 genes in response to both IAA and BL treatment of transgenic plants. These results suggest that the IAA genes are induced by BL, at least in part, via the activation of the auxin response element. Endogenous IAA levels per gram fresh weight did not increase when seedlings of Arabidopsis wild type (WT) or the BR-deficient mutant det2 were treated with BL. Furthermore, the levels of IAA transcripts were lower in the det2 mutant than in the WT, even though endogenous IAA levels per gram fresh weight were higher in the det2 mutant than in the WT. In conclusion, the lack of evidence for auxin-mediated activation of early auxin-inducible genes in response to BL suggests that the BR and auxin signaling pathways independently activate the transcriptional system of the IAA and DR5-GUS genes.Exogenous application of brassinosteroids (BRs) to plants at nanomolar to micromolar concentrations produces a wide spectrum of physiological effects. These include promotion of cell elongation and division, enhancement of tracheary element differentiation, delaying of abscission, enhancement of gravityinduced bending, promotion of ethylene biosynthesis, and enhancement of stress resistance, as reviewed by Clouse and Sasse (1998) andSasse (1999). A number of BR-deficient mutants have been identified in Arabidopsis, pea (Pisum sativum), and tomato (Lycopersicon esculentum; for review, see Clouse and Feldmann, 1999;Clouse, 2002; Fujioka and Yokota, 2003). These mutants exhibit dwarfism when grown in either light or dark conditions. Many of these mutants also have dark-green leaves, reduced fertility, a prolonged life span, and abnormal skotomorphogenesis. BR-insensitive mutants have been identified in Arabidopsis, pea, tomato, and rice (Oryza sativa; for review, see Clouse and Feldmann, 1999;Clouse, 2002; Fujioka and Yokota, 2003). The molecular mechanisms of BR action, however, remain unclear.It has been suggested that the actions of BRs are related to auxin action (Mandava, 1988;Sasse, 1999). Synergistic interactions between BRs and auxins occur in elongating tissues and cells in dicots (Yopp et al., 1981;Katsumi, 1985;Sala and Sala, 1985) and in monocots (Yopp et al., 1981). Such synergism is also found in the bending responses of dicots (Yopp et al., 1981; Cohen and Meudt, 1983;Meudt, 1987) and monocots (Takeno and Pharis, 1982; Fujioka et al., 1998). Several authors have proposed that BRinduced effects might be mediated via auxin, wi...

SummaryAuxin response factor (ARF) family genes play a central role in controlling sensitivity to the plant hormone auxin. We characterized the function of ARF8 in Arabidopsis by investigating a T-DNA insertion line (arf8-1) and overexpression lines (ARF8 OX) of ARF8. arf8-1 showed a long-hypocotyl phenotype in either white, blue, red or far-red light conditions, in contrast to ARF8 OX that displayed short hypocotyls in the light. Stronger and weaker apical dominance, and promotion and inhibition of lateral root formation were observed in arf8-1 and ARF8 OX respectively. Sensitivity to auxin was unaltered in arf8-1 hypocotyls with respect to growth inhibition caused by exogenously applied auxin and growth promotion induced by higher temperatures. ARF8 expression was observed constitutively in shoot and root apexes, and was induced in the light condition in hypocotyls. Free IAA contents were approximately 30% reduced in light-grown hypocotyls of ARF8 OX, but were similar between those of arf8-1 and wild type. Expression of the three GH3 genes was reduced in arf8-1 and increased in ARF8 OX, indicating that they are targets of ARF8 transcriptional control. Because the three GH3 proteins may be involved in the conjugation of IAA as suggested by Staswick et al. (2002), and because two of the three GH3 genes are auxin inducible, ARF8 may control the free IAA level in a negative feedback fashion by regulating GH3 gene expression. ARF family genes seem to control both auxin sensitivity and homeostasis in Arabidopsis.

In most flowering plants, the female gametophyte develops in an ovule deeply embedded in the ovary. Through double fertilization, the egg cell fuses with the sperm cell, resulting in a zygote, which develops into the embryo. In the present study, we analyzed egg cell lysates by polyacrylamide gel electrophoresis and subsequent mass spectrometry-based proteomics technology, and identified major protein components expressed in the egg cell. The identified proteins included three cytosolic enzymes of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and triosephosphate isomerase, two mitochondrial proteins, the ATP synthase beta-subunit and an adenine nucleotide transporter, and annexin p35. In addition, expression levels of these proteins in the egg cell were compared with those in the early embryo, the central cell and the suspension cell. Annexin p35 was highly expressed only in the egg cell, and glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and the adenine nucleotide transporter were expressed at higher levels in egg cells than in central and cultured cells. These results indicate that annexin p35 in the egg cell and zygote is involved in the exocytosis of cell wall materials, which is induced by a fertilization-triggered increase in cytosolic Ca2+ levels, and that the egg cell is rich in an enzyme subset for the energy metabolism.

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.