Tobacco mesophyll protoplasts were previously shown to respond to naphthaleneacetic acid by modifying their transmembrane potential difference. In the present work, evacuolated protoplasts were used to show that this response resides only at the plasmalemma. This electrical response was investigated by using polyclonal antibodies directed against plasma membrane antigens presumably involved in the reception and transduction of the auxin signal. An IgG fraction from an antiserum directed against the membrane auxin-binding protein from maize coleoptile completely inhibited the naphthaleneacetic acid-induced response of tobacco protoplasts. The suppression of the auxin-induced variation in the transmembrane potential difference by an IgG preparation directed against the plasmalemma ATPase from yeast demonstrated the involvement of the ATPase in the electrical response. Variation induced by fusicoccin in the transmembrane potential difference of tobacco protoplasts was unaffected by the anti-auxin-binding protein IgG fraction but was completely suppressed by the anti-ATPase IgG preparation. These results demonstrate the presence of a membrane receptor for auxin at the plasmalemma, the binding of the hormone to this receptor leading to the activation of the proton-pumping ATPase. They also show that at least the primary steps of activation by naphthaleneacetic acid are distinct from those of the fusicoccin-induced response.
SummaryAuxin-induced variations of transmembrane potential difference have been shown to be a useful tool for analyzing hormone sensitivity in tobacco protoplasts.Using this technique, we demonstrated that protoplasts derived from wild-type, an auxin-resistant mutant and Agrobacterium-rhizogenes transformed plants differed widely in the sensitivity of their electrical response to naphthalene acetic acid. We have used different antibodies, raised to auxin binding proteins (ABP) from maize coleoptiles, or to the axr' gene product (ABPI), to test whether changes in auxin sensitivity can be correlated with the presence of tobacco proteins immunologically related to this ABP. Titrations indicated that 0.4 nM anti-ABP IgG inhibited 50% of the auxin-specific response of wild-type protoplasts, whereas 0.04 nM or 4 nM anti-ABP IgG were necessary to inhibit the response of mutant and transformed protoplasts, respectively, to the same extent. On wild-type protoplasts, blocking part of the immunoreactive sites with anti-ABP antibodies resulted in a decrease in auxin sensitivity of the electrical response (0.4 nM anti-ABP IgG inducing a 10-fold decrease), whereas addition of maize ABP increased this auxin sensitivity (1 pM ABPl raised the sensitivity more than 1000-fold). The results obtained suggest that the auxin sensitivity detected by our assay system correlates with the amount of tobacco proteins immunologically related to the axr' gene product from maize. A hypothesis accounting for the presence of these proteins at the external surface Of tobacco protoplasts and for the effects of heterologous maize ABP on auxin sensitivity is proposed.
to be glycosylated by a high-mannose-type oligosaccharide. Northern hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed a single mRNA species of approximately the same size as the cDNA isolated. The axrl gene is differentially expressed in RNA isolated from different organs of a maize plant, showing the highest level of expression in maize styles and ears.
Corynebacterium fascians derives its name from the characteristic symptoms of disease which infections by this organism cause in plants. '-4 These symptoms can be imitated by applications of kinetin, alone or in combinations with auxin, to seedlings,5 and the bacterium is known to produce cytokinin, as measured by release of bud inhibition or the retention of chlorophyll in excised tissues.6This report is on the isolation of cytokinins from liquid cultures of C. Purification Procedures and Results.-Preliminary tests showed that the bacterial suspensions or crude extracts were toxic to the tobacco tissue and could not be bioassayed for cytokinin activity without prior treatment. Bacterial suspensions, therefore, were filtered through Celite. The filter cake was lyophylized and extracted with about 10OX its volume of chloroform. The filtrate was passed through a Dowex-50 column (described in Fig. 1) which was thoroughly washed with water and then eluted with 5 N NH40H. Both the chloroform extract and the eluate were evaporated to dryness, and each was dissolved in a volume of water equal to one tenth the bacterial suspension. Results of one such experiment (Table 1) show a total activity of ca. 500,ug/liter KE which was about equally distributed between the cells and medium, but some toxic material was still present. Other 52
Auxin-binding protein 1 (ABP1) is a unique hormone receptor because it resides primarily in the lumen of the endoplasmic reticulum (ER); however, two lines of evidence presented here suggest that ABP1 does not bind auxin within the endoplasmic reticulum, despite its predominant location there. First, ABP1 cannot be photolabeled in intact cells that have accumulated the auxin and photolabeling reagent 5- [7-3 H]azidoindole-3-acetic acid, indicating either that auxin is excluded from the ER and is not available for photolabeling to ABP1 or that binding conditions within the ER lumen are insufficient for photolabeling. Second, at the pH of the ER lumen, auxin binding to ABP1 is not detectable. The pH estimate of the ER lumen is based on an indirect assay, which indicates that the pH is closer to pH 7 than to the binding optimum of pH 5.5. These results indicate that ABP1 does not bind auxin within the ER and point to a site of action that is post-ER. The effect of auxin on its trafficking from the ER was tested in an animal expression system. ABP1 expressed at high levels in COS7 cells is efficiently retained in the ER lumen and is not secreted even in the presence of 190 M indole-3-acetic acid, an auxin concentration that is 40 times above the K d for indole-3-acetic acid binding to ABP1. Hertel et al. (1972) reported auxin-binding in microsomes isolated from corn coleoptile cells and later designated this activity Site I. Several groups (Löbler and Klä mbt, 1985;Shimomura et al., 1986;Napier et al., 1988) purified the protein responsible for this Site I activity (cf. Table I in Jones (1994)), and it has been shown directly that this protein binds auxin .Several lines of evidence indicate that ABP1 1 in maize is an auxin receptor that acts at the plasma membrane. First, among a series of 45 auxins or similar compounds where binding affinity and growth induction was compared, there is a correlation between K d and pC 50 , except with some of the substituted phenoxypropionic acids . A molecular model based on these data, in conjunction with data on the identification of residues in the binding site, point out that auxin binding to ABP1 involves specific molecular interactions, as expected for a receptor (Edgerton et al., 1994;Brown and Jones, 1994). Second, a synthetic peptide encoding the terminal 13 residues of ABP1 significantly modulate the ion current across the plasma membrane of Vicia faba guard cells (Thiel et al., 1993), while synthetic peptides from other regions of ABP1 do not modulate current activity. This suggests that there is a specific interaction between this domain of ABP1 and a plasma membrane component. The behavior of this ABP1 peptide mimics part of the behavior of auxin in the V. faba protoplast (Blatt and Thiel, 1994). Third, antisera directed against ABP1 blocks auxin-induced polarization of the plasma membrane on tobacco mesophyll protoplasts, indicating that ABP1 or an immunochemically similar protein mediates auxin-regulated ion movement (Barbier-Brygoo et al., 1989, 1991Rü ck et al., 19...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.