SummaryTobacco leaf was used to investigate the mechanism of action of auxin-binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin-inducible growth were each determined in control tobacco leaves and leaves over-expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a de®ned window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1-induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1-induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin-inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY-2) in culture displayed two dose-dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [ 3 H]-thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidenti®ed receptor.

An auxin-binding protein was purified from membranes of maize shoots including the coleoptiles, leaf rolls and mesocotyls. The method of Ca2+-promoted sedimentation of membrane particles was adopted for large-scale preparation. The auxin-binding protein was solubilized from the acetone-washed membranes, and purified by successive chromatographies on DEAE-Sephacel, 1-naphthylacetic acid-linked AH-Sepharose 4B, and Sephadex G-100 columns. The yield of the purified protein was about 0.2 mg from 1 kg of shoots. The binding protein exists as a dimer with a subunit molecular weight of 21,000, and possesses one auxin-binding site per dimer. The preparation also contains a minor molecular form with a subunit molecular weight of 20,000. The auxin-binding protein is not a hydrophobic protein, as judged from its amino acid composition and solubility. The circular dichroic (CD) spectrum of the binding protein resembles the spectrum anticipated from the beta-structures, and shows no alpha-helix characteristic in the secondary structure. The CD spectral changes induced on the binding of auxin and its antagonist seem to be related to the receptor function. The affinity of the binding protein for auxin is dependent on pH, with an optimum at pH 5.0, while the binding protein is unstable below pH 6. We discuss here the intracellular localization of the auxin-binding protein from the view point of the controversial pH-dependence of the binding affinity and stability.

We previously purified an auxin-binding protein (ABP) from the microsomal fraction of maize shoots (Zea mays L. cv. Golden Cross Bantam). In the present study cDNA clones derived from mRNAs encoding the ABP were isolated and sequenced. The nucleotide sequence of the 822-base-pair cDNA includes a 603-base-pair open reading frame. RNA blot hybridization analysis indicated a single mRNA species of =1.0 kilobase. The predicted precursor of ABP is composed of 201 amino acid residues and has a molecular weight of 21,976. The NH2-terminal sequence of 38 residues is hydrophobic and may be a signal peptide for translocation of the ABP across the membrane of the endoplasmic reticulum. The mature ABP, composed of 163 residues with a molecular weight of 18,352, contains a potential N-glycosylation site (Asn-Thr-Thr), and the COOH-terminal tetrapeptide (Lys-Asp-Glu-Leu) may be a signal for retention of the ABP in the lumen of the endoplasmic reticulum.The plant hormone auxin (indole-3-acetic acid) in higher plants functions in regulating normal growth and development, including cell elongation, division, and differentiation (1,2). Rapid responses to auxin have been observed in organelles such as plasma membrane (3-6), Golgi apparatus (7), endoplasmic reticulum (ER) (8), and nucleus (2, 9, 10) as well as in the cell wall (11). The specificity of auxin is supposed to be due to the presence of a specific receptor(s) (12).However, it is unknown whether the diverse responses of different organelles to auxin are mediated by a single or multiple species of receptors.Auxin can enter cells because it is a membrane-permeant, lipophilic weak acid. In addition, specific transporters for auxin have been demonstrated in plasma membranes (13)(14)(15)(16). Specific and saturable auxin-binding sites are also found in other organelles such as ER, tonoplast, and nucleus and in the cytosol (12), and these should be taken into consideration as possible candidates for auxin receptors in addition to those located in the plasma membranes.We previously purified an auxin-binding protein (ABP) from maize shoot membranes (17). This ABP was found in the ER (18), and its amount seemed to be correlated with auxin-induced cell elongation of the coleoptile and mesocotyl (18,19). The specificity of the binding site for various natural and synthetic auxins was roughly parallel to that in the cell elongation response (20). Information on the structure of the ABP should help in understanding its roles in the action of auxin. Therefore, in this study we cloned a cDNA encoding the ABP. The primary structure deduced from the nucleotide sequence showed the characteristics of proteins localized in the lumen of the ER, consistent with the location of the ABP in maize shoots.t MATERIALS AND METHODSConstruction and Screening of a cDNA Library. Poly(A)+ RNA used for cDNA synthesis was isolated from the polysome fraction (21) of whole shoots of 3-day-old maize seedlings (Zea mays L. cv. Golden Cross Bantam, Fujita Seed, Osaka). cDNA was synthesized according t...