Plant Hormones and their Receptors
Development (Jenkins, G. 1. & Schuch, W., eds.), pp. 129-148, The Company of Biologists, Cambridge 29. Schroeder, J. I. & Hagiwara, S. (1990) Proc. Natl. Acad. Sci. U.S.A. 87.9305-9309 30. Alexandre, J., I,assalles, J. P. & Kado, R. T. (1990) Nature (London) 343, 567-570 31. Drobak, B. K.. Watkins, P. A. C.. Chattaway, J. A., Roberts, K.. & Dawson, A. 1’. (199 1) Plant Physiol. 95,412-419
32. Ma, H., Yanofsky, M. F. & Meyerowitz, E. M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3821-3825 33. Lawton, M. A., Yamamoto, R. T., Hanks, S. K. & Lamb, C. J. (1989) Proc. Natl. Acad. Sci. U S A . 86, 3 140-3144 34. Lee, Y. & Assmann, S. M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88,2127-2 131 Received 10 September 1991
~~~
Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts Helene Barbier-Brygoo, Genevieve Ephritikhine, Christophe Maurel and Jean Guern lnstitut des Sciences Vegetates, C.N.R.S., B i t 22, Avenue de la Terrasse, F-9 I I98 Gif-sur-Yvette Cedex, France
Introduction Considerable progress has been made in the last few years towards the understanding of plant hormone action (see recent reviews in [ 1, 2 ] ) ,although the precise molecular mechanisms by which phytohormones control plant development remain to be elucidated. T h e most important breakthroughs concerned the analysis of auxin action, and led to the molecular characterization of auxin-binding proteins (ARPs) which may constitute auxin receptors. As to their binding characteristics and subcellular localization, the diversity of these proteins, arising in early studies (reviewed in [ 3 ] ) , can be further illustrated by a few examples selected in the most recent studies. In maize coleoptiles, the major auxin-binding moiety (ARP 1), located at the lumen of the endoplasmic reticulum, is a 22 kDa protein whose cDNA has been cloned and sequenced [4-61, and mapped to chromosome 3 [ 7 ] .This protein probably corresponds to the auxin-binding site I described previously [8,93. Minor isoforms of ARPI, in the same molecular range, have still to be located within the cell [4, lo]. The application of the photoaffinity labelling technique using azido-auxins resulted in the identification of proteins ranging from 40 up to 60 kDa in plasma membraneenriched fractions from zucchini [ 111, tomato [ 121 or maize [ 131. Most recently, Prasad & Jones [ 141 Abbreviations used: ABP, auxin-binding protein; Em, transmembrane electrical potential difference; NAA, 1-naphthaleneacetic acid; IAA, indoleacetic acid; 2.4-D. 2,4-dichlorophenoxyacetic acid; 2-NAA, 2-naphthaleneacetic acid pHPAA, p-brornophenylacetic acid NPA, naphthylphthalamic acid; L A . indolelactic acid; tARP, tobacco auxin-responsive protein; TP, transmembrane protein; GUS,j3-glucuronidase gene.
using anti-idiotypic antibodies characterized in soybean a 65 kDa protein localized in the nucleus. An homologous protein was detected in seedling extracts from a wide range of species including Arubidopsis, maize and tobacco [14]. Facing such a diversity, the major difficulty remains how to identify, among these ARPs, those which really function as hormonal receptors [ 15-17]. Such an assessment is most often hindered by the lack of suitable assay systems for monitoring early auxin effects at the cell level. W e developed such an assay system based on the auxin-induced modifications of the transmembrane potential difference of protoplasts isolated from tobacco leaves [ 181. The characterization of this response led us to propose that it involves auxin-responsive proteins located at the plasma membrane and immunologically related to maize ABP1. W e investigated also other responses to auxin in mesophyll protoplasts or protoplastderived cells: the auxin-dependent activity of the rol B gene promoter from Agrobucterium rhizogenes [ 191 and the auxin-controlled cell division [20]. The finding that the sensitivities of these cellular responses to auxin can be differentially affected by Ri transformation raises the possibility of multiple reception-transduction pathways for auxin in plant cells.
The auxin-induced hyperpolarization of tobacco protoplasts involves plasma membrane auxin-responsive proteins related to maize ABPI Auxin induces variations in the transmembrane electrical potential difference (Em) of protoplasts from tobacco leaves [ 181 and roots (Y. Le Roux & H. Barbier-Brygoo, unpublished work), or from
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roots of different species [21, 221. This response displays a bell-shaped dose-response curve from which the sensitivity of protoplasts to auxin can be deduced [18, 21, 221. Fig. 1 illustrates the specificity, towards active and inactive auxins, of the response of mesophyll protoplasts isolated from wild-type tobacco plants (Nicotiana tabacum cv Xanthi). The activity of the different molecules tested was expressed relative to that of l-naphthaleneacetic acid (NAA). Indoleacetic acid (IAA), the natural auxin, and 2,4-dichlorophenoxyacetic acid (2,443) evoked non-monotonous dose-response curves similar to the one induced by NAA, 2,4-D being less efficient (optimal concentration 30 p ~ ) Fig. I
Specificity of the auxin-induced plasma membrane hyperpolarization in tobacco mesophyll protoplasts Mesophyll protoplasts were isolated from tobacco plants (Nicotiono tobocurn cv Xanthi, clone XHFD8) as described by Caboche [ZS] The transmembrane electrical potential difference (Em) of protoplasts was measured by the microelectrode technique and the auxin-induced Em variations (AEm) were evaluated as described in Barbier-Brygoo et a/ [27] Dose-response curves for Em were established for active and inactive auxin analogues Effector-induced membrane responses are expressed as a percentage of the (control) N M induced response Protoplasts were treated by each effector alone (closed symbols) or were incubated 5 min at room temperature with 3 ,uM-NM t o induce the maximal response before the addition of increasing amounts of effector (open symbols) Data are given from one representative experiment for each effector NAA. IAA, 2,4-D. picloram, 2-NAA. p-BPAA. NPA and ILA h
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than NAA and IAA (optimal concentration 3 p ~ ) . Picloram induced protoplast hyperpolarization, but no relative depolarization was observed in the concentration range used, up to 100 p ~ Non-func. tional auxin analogues: 2-naphthaleneacetic acid (2-NAA), p-bromophenylacetic acid (p-HPAA), naphthylphthalamic acid (NPA) and indolelactic acid ( L A ) had no significant effect on Em. However, 2-NAA and p-HPAA were able to reverse the hyperpolarizing effect of 3 ~ M - N A Awhich , suggested that these analogues, although inactive per se, could compete with NAA at the sites involved in the membrane response. NPA and ILA did not interfere with the NAA-induced response. The absence of effect of NPA suggests that the auxin eMux carrier located at the plasma membrane is not involved in the electrical response of protoplasts to NAA. This specificity pattern of the membrane response of protoplasts closely resembles that of the auxin-controlled proliferation of cells derived from the same mesophyll protoplast preparations [23]. Moreover, in protoplasts from an auxin-resistant mutant, the sensitivities of both responses towards a set of auxin analogues were similarly affected [ 18, 231. These results provide evidence for a specific action of auxin at the membrane level related to its biological activity. This contrasts with the recent results of Felle et al. [24] who investigated the electrical response of maize coleoptiles to auxins, and found that all tested compounds, growth-promoting auxins as well as growth-inactive structural analogues, evoked a hyperpolarization, making this response unspecific on coleoptiles. The fact that non-permeating auxins (proteincoupled NAA derivatives) induced protoplast hyperpolarization within less than 1 min, the same time course as NAA, suggested that the recognition of the auxin signal occurred at the outer face of the plasma membrane [ 251, probably involving tobacco auxin-responsive proteins (tARPs). Other evidence for this localization was provided, showing that antibodies raised to ABPs from maize coleoptiles, or specifically directed against ARP1, inhibited the electrical response to auxin when applied exogenously to tobacco protoplasts [26, 271. This suggested immunological relations between tARP and maize ABP. The progressive inactivation of this pool of tARP by increasing amounts of anti-AHP antibodies resulted in a decrease in auxin sensitivity, read through the shift of the dose-response curves of Em towards higher NAA concentrations [27]. Conversely, incubation of tobacco protoplasts with purified maize AHP led to an enhancement of their sensitivity to auxin, together with an increased
Plant Hormones and their Receptors
number of sites immunoreactive to the anti-ABP antibody; homogeneous maize ARPl evoked a similar effect [27]. It was assumed in that case that treatment of protoplasts with ABP increased the number of active perception units at the plasma membrane, thus inducing an increase in auxin sensitivity. These results point to the existence, at the plasma membrane of protoplasts, of tARF’s immunologically and functionally related to maize ABP1, and suggest that auxin sensitivity detected by the membrane electrical response correlates with the level of these tARPs. They also raise puzzling questions as to the organization of the reception-transduction units involved in the membrane response to auxin. As to the change in sensitivity of the membrane response induced by exogenous maize ARP1, we proposed that AHP1, a nonintegral membrane protein [4-61, could interact with a transmembrane protein (TP) of tobacco plasma membrane essential for the transmission of the auxin signal [27]. In such a model, an auxinperception unit could be formed by the homologous association of a tARP with a TP, or by the heterologous interaction between maize AHP and the tARP-binding site of TP. To assess the relevance of this model, binding studies using labelled exogenous ARP on protoplasts should assist the search for evidence of the putative membrane protein interacting with ABP.
Different cellular responses to auxin might involve distinct reception-transduction pathways for the hormonal signal The auxin sensitivity of the membrane response of protoplasts from A. rhizogenes (Ri)-transformed plants was shown to be greatly enhanced, in association with an increased amount of plasma membrane sites recognized by anti-ABP antibodies [27]. Single genes from the Ri-T-DNA, the rolA, rolB and rol C genes, conferred an increased sensitivity on transformed protoplasts, rolB being by far the most efficient [20]. Using transgenic plants containing a gene fusion product consisting of the promoter of the rolB gene and the coding sequence of the B-glucuronidase gene ( rol B:GUS), Maurel et al. [19] demonstrated that the expression of the rolB promoter, as revealed by GUS activity, was activated by auxin. This activation was specific for auxin-type growth regulators and occurred in the plant in different tissues as well as in isolated mesophyll protoplasts [19], constituting in this latter case a cellular response to auxin monitored after 15 to
2 4 h of protoplast culture in the presence of hormone. Tobacco plants containing both the rol B: GUS chimeric gene and a functional rol B gene were used for simultaneous monitoring of rol B expression and physiological effects at the protoplast level. The sensitivity to auxin of the hyperpolarization response and the rol B promoter activation were compared in rol B:GUS and rol B-rol B: GUS plants (Fig. 2). Protoplasts from rol B:GUS plants were hyperpolarized by auxin with an inverted bell-shaped dose-response curve, a maximal hyperpolarization being observed at the concentration of 3 ~ M - N A A (Fig. 2a), as in protoplasts from wild-type plants [27]. The dose-response curve of protoplasts from rol B-rol B: GUS plants, expressing the rol B structural gene, was shifted to lower NAA concentrations, producing an increase of sensitivity of more than 100000-fold (Fig. 2a). As shown in Fig. 2b, GUS activity was strongly enhanced in protoplasts treated with auxin. Surprisingly, maximal GUS expression driven by the 7018 promoter was obtained at the same NAA concentration of 10 ~ U M in the absence (ro1B:GUS) or in the presence (To1B-rol B:GUS) of the functional rol B gene. Two other responses to auxin were also investigated in protoplasts from untransformed or rol Btransformed plants: the occurrence of the first division of protoplasts cultured for 5 days in the presence of the hormone (Fig. 2c) and the proliferation of protoplast-derived cells after 4 weeks (Fig. 2 4 . For both division responses, it was found again that the optimal NAA concentrations were identical in untransformed and rol B-transformed cells. Transformation by the roZB gene thus increases the auxin sensitivity of the membrane response, but does not affect the auxin sensitivity of other responses. Such selective effects of Ri-transformation were also observed in protoplasts from plants containing the whole T-DNA [ZO]. These results point to a distinct functioning of various cellular responses to the hormone. Although metabolization of auxin is likely to alter the dose-dependency of responses expressed after a relatively long hormone treatment (see for example Fig. 2c and 2 4 , differences in the dose-dependency of various auxin responses might reflect the existence of distinct reception-transduction pathways to control membrane hyperpolarization on one hand, and rolB expression and cell division on the other hand. Such a possibility has already been proposed by Nakamura et al. “281. For the electrical response of protoplasts, we demonstrated that auxin perception occurs at the outer face of the plasma mem-
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Fig. 2
Cellular responses to auxin Protoplasts were isolated from tobacco plants containing the rol 6:GUS fusion gene either alone (BGUS) or associated with a functional rol6 gene (BBGUS). The NAA-induced Em variations were measured on freshly isolated protoplasts as described in Barbier-Brygoo et 01. [27] (20). for the study of ro1B:GUS expression, protoplasts were isolated in the absence of auxin. They were then cultured for 24 h at a density of 5 X lo4 protoplastdml in the presence of various concentrations of NAA. GUS activity in protoplasts was determined by a fluorimetric assay as described by Maurel et 01. [ 191 (2b). Axenically prepared mesophyll protoplastsfrom wild-type plants (W)or plants containing the rolB gene (6) were plated at a density of 5 X lo4 protoplastdml in the presence of various concentrations of NAA. After 5 days of incubation in the dark at 26°C the occurrence of the first division was estimated by light microscopy (2c). Protoplast-derivedcells were then plated at a density of 100 celldml in the presence of various NAA concentrationsand developing colonies were counted after 4 weeks. Results were expressed as relative plating efficiency, i.e. the proportion of cells which developed colonies among the cells which had already divided at the time of low-density plating ( 2 d ) .
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brane, but the cellular location of the auxin receptors triggering rolB expression and cell division is unknown. Recently, Prasdd & Jones [ 141 suggested that auxin-regulated gene expression might be mediated by the 65 kDa ARP they localized at the nucleus. However, a complete indepen-
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dence of perception pathways for both the shortterm cell-surface and the long-term cell-division responses would stand in contrast with the fact that the auxin-resistant mutant was affected in a strikingly similar manner in the electrical and the celldivision responses. Further evidence based on more
Plant Hormones and their Receptors
direct approaches is needed to explore the independence or relationship between the perception pathways mediating the variety of auxin effects in plant cells. 1. Napier, R. M. & Venis, M. A. (1990) J. Plant Regul. 9, 113-126 2. Klee, H. & Estelle. M. (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 529-55 1 3. Libbenga, K. K..van der Linde, P. C. G. & Mennes, A. M. (1986) in Hormones, Receptors and Cellular Interactions in Plants (Chadwick, C. M. & Garrod. D. R., eds.), pp. 61-70, Cambridge University Press, Cambridge 4. Hesse, T., Feldwisch, J., Halshusernann, D., Hauw, G., Puype, M., Vandekerckhove, J., Lobler, M., Klarnbt, D., Schell, J. & I’alme. K. (1989) EMBO J. 8, 2453-246 1 5. Inohara, N., Shimomura. S., Fukui, T. & Futai, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3564-3568 6. Tillmann. U., Viola, G., Kayser, H., Siemeister, G., Hesse, T., I’alme, K., I,obler, M. & Klambt, D. (1989) EMBO J. 8,2463-2467 7. Lobler, M. & Hirsch. A. M. (1 990) Plant Mol. Biol. 15, 5 13-516 8. Dohrmann, U., Hertel, K. & Kowalik, H. (1978) Planta 140,97-106 9. Jones, A. M., Lamerson, 1.’ & Venis, M. A. (1989) Planta 179,409-413 10. Palme, K., Feldwisch, J.? Hesse, T., Bauw, G., Puype, M., Vandekerckhove, J. & Schell, J. (1990) in Hormonal Perception and Signal Transduction in Animals and Plants (Roberts, J., Kirk, C. & Venis, M. A., eds.), pp. 299-313, The Company of Biologists Ltd, Cambridge 11. Hicks, G. R.. Rayle, D. I,.. Jones, A. M. & Lomax, T. I,. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4948-4952 12. Hicks, G. R., Rayle, D. I,. & Lomax, T. I,. (1989) Science 245,52-54
13. Palme, K.. Hesse, T., Moore, I., Campos, N., Feldwisch, J., Garbers, C., Hesse, F. & Schell, J. (1991) Mech. Develop. 33,97-106 14. Prasad, P. V. &Jones, A. M. (1991) Proc. Natl. Acad. Sci. USA. 88, 5479-5483 15. Jones, A. M. (1990) Physiol. Plant 80, 154- 158 16. Klarnbt, D. (1990) Plant Mol. Biol. 14, 1045-1050 17. Napier, R. M. & Venis, M. A. (1991) Trends Riochem. Sci. 16,72-75 18. Ephritikhine, G., Barbier-Brygoo, H., Muller, J.-F. & Guern, J. (1987) Plant Physiol. 83,801-804 19. Maurel, C., Brevet, J., Barbier-Brygoo, H., Guern, J. & TempC, J. (1990) Mol. Gen. Genet. 223, 58-64 20. Maurel, C., Barbier-Brygoo, H., Spena, A., Temp&,J. & Guern, J. ( 1991) Plant Physiol. 97,2 12-2 I6 21. Shen, W. H., Petit, A., Guern, J. & Tempd, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,3417-3421 22. Shen, W. H., Davioud, E., David, C., Barbier-Brygoo, H., Tempi.. J. & Guern, J. (1990) Plant Physiol. 94, 554-560 23. Caboche, M., Muller, J.-F., Chanut, F., Aranda, G. & Cirakoglu, S.(1987) Plant Physiol. 83,795-800 24. Felle, H.. Peters, W. & Palme, K. (1991) Biochim. Riophys. Acta 1064, 199-204 25. Venis, M. A.. Thomas, M. W., Barbier-Brygoo, H., Ephritikhine, G. & Guern, J. (1990) Planta 182, 232-235 26. Barbier-Brygoo, H., Ephritikhine, G., Klambt, D., Ghislain, M. & Guern, J. (1989) Proc. Natl. Acad. Sci. USA. 86,891-896 27. Barbier-Brygoo. H., Ephritikhine, G., Klambt, D., Maurel, C., Palme, K., Schell, J. & Guern, J. (1991) Plant J. 1,83-93 28. Nakamura, C., van Telgen, H.-J., Mennes, A. M., Ono, H. & Libbenga, K. R. (1988) Plant Physiol. 88, 845-849 29. Caboche, M. (1980) Planta 149,7-18
Received 10 September 1991
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Development (Jenkins, G. 1. & Schuch, W., eds.), pp. 129-148, The Company of Biologists, Cambridge 29. Schroeder, J. I. & Hagiwara, S. (1990) Proc. Natl. Acad. Sci. U.S.A. 87.9305-9309 30. Alexandre, J., I,assalles, J. P. & Kado, R. T. (1990) Nature (London) 343, 567-570 31. Drobak, B. K.. Watkins, P. A. C.. Chattaway, J. A., Roberts, K.. & Dawson, A. 1’. (199 1) Plant Physiol. 95,412-419
32. Ma, H., Yanofsky, M. F. & Meyerowitz, E. M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3821-3825 33. Lawton, M. A., Yamamoto, R. T., Hanks, S. K. & Lamb, C. J. (1989) Proc. Natl. Acad. Sci. U S A . 86, 3 140-3144 34. Lee, Y. & Assmann, S. M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88,2127-2 131 Received 10 September 1991
~~~
Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts Helene Barbier-Brygoo, Genevieve Ephritikhine, Christophe Maurel and Jean Guern lnstitut des Sciences Vegetates, C.N.R.S., B i t 22, Avenue de la Terrasse, F-9 I I98 Gif-sur-Yvette Cedex, France
Introduction Considerable progress has been made in the last few years towards the understanding of plant hormone action (see recent reviews in [ 1, 2 ] ) ,although the precise molecular mechanisms by which phytohormones control plant development remain to be elucidated. T h e most important breakthroughs concerned the analysis of auxin action, and led to the molecular characterization of auxin-binding proteins (ARPs) which may constitute auxin receptors. As to their binding characteristics and subcellular localization, the diversity of these proteins, arising in early studies (reviewed in [ 3 ] ) , can be further illustrated by a few examples selected in the most recent studies. In maize coleoptiles, the major auxin-binding moiety (ARP 1), located at the lumen of the endoplasmic reticulum, is a 22 kDa protein whose cDNA has been cloned and sequenced [4-61, and mapped to chromosome 3 [ 7 ] .This protein probably corresponds to the auxin-binding site I described previously [8,93. Minor isoforms of ARPI, in the same molecular range, have still to be located within the cell [4, lo]. The application of the photoaffinity labelling technique using azido-auxins resulted in the identification of proteins ranging from 40 up to 60 kDa in plasma membraneenriched fractions from zucchini [ 111, tomato [ 121 or maize [ 131. Most recently, Prasad & Jones [ 141 Abbreviations used: ABP, auxin-binding protein; Em, transmembrane electrical potential difference; NAA, 1-naphthaleneacetic acid; IAA, indoleacetic acid; 2.4-D. 2,4-dichlorophenoxyacetic acid; 2-NAA, 2-naphthaleneacetic acid pHPAA, p-brornophenylacetic acid NPA, naphthylphthalamic acid; L A . indolelactic acid; tARP, tobacco auxin-responsive protein; TP, transmembrane protein; GUS,j3-glucuronidase gene.
using anti-idiotypic antibodies characterized in soybean a 65 kDa protein localized in the nucleus. An homologous protein was detected in seedling extracts from a wide range of species including Arubidopsis, maize and tobacco [14]. Facing such a diversity, the major difficulty remains how to identify, among these ARPs, those which really function as hormonal receptors [ 15-17]. Such an assessment is most often hindered by the lack of suitable assay systems for monitoring early auxin effects at the cell level. W e developed such an assay system based on the auxin-induced modifications of the transmembrane potential difference of protoplasts isolated from tobacco leaves [ 181. The characterization of this response led us to propose that it involves auxin-responsive proteins located at the plasma membrane and immunologically related to maize ABP1. W e investigated also other responses to auxin in mesophyll protoplasts or protoplastderived cells: the auxin-dependent activity of the rol B gene promoter from Agrobucterium rhizogenes [ 191 and the auxin-controlled cell division [20]. The finding that the sensitivities of these cellular responses to auxin can be differentially affected by Ri transformation raises the possibility of multiple reception-transduction pathways for auxin in plant cells.
The auxin-induced hyperpolarization of tobacco protoplasts involves plasma membrane auxin-responsive proteins related to maize ABPI Auxin induces variations in the transmembrane electrical potential difference (Em) of protoplasts from tobacco leaves [ 181 and roots (Y. Le Roux & H. Barbier-Brygoo, unpublished work), or from
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roots of different species [21, 221. This response displays a bell-shaped dose-response curve from which the sensitivity of protoplasts to auxin can be deduced [18, 21, 221. Fig. 1 illustrates the specificity, towards active and inactive auxins, of the response of mesophyll protoplasts isolated from wild-type tobacco plants (Nicotiana tabacum cv Xanthi). The activity of the different molecules tested was expressed relative to that of l-naphthaleneacetic acid (NAA). Indoleacetic acid (IAA), the natural auxin, and 2,4-dichlorophenoxyacetic acid (2,443) evoked non-monotonous dose-response curves similar to the one induced by NAA, 2,4-D being less efficient (optimal concentration 30 p ~ ) Fig. I
Specificity of the auxin-induced plasma membrane hyperpolarization in tobacco mesophyll protoplasts Mesophyll protoplasts were isolated from tobacco plants (Nicotiono tobocurn cv Xanthi, clone XHFD8) as described by Caboche [ZS] The transmembrane electrical potential difference (Em) of protoplasts was measured by the microelectrode technique and the auxin-induced Em variations (AEm) were evaluated as described in Barbier-Brygoo et a/ [27] Dose-response curves for Em were established for active and inactive auxin analogues Effector-induced membrane responses are expressed as a percentage of the (control) N M induced response Protoplasts were treated by each effector alone (closed symbols) or were incubated 5 min at room temperature with 3 ,uM-NM t o induce the maximal response before the addition of increasing amounts of effector (open symbols) Data are given from one representative experiment for each effector NAA. IAA, 2,4-D. picloram, 2-NAA. p-BPAA. NPA and ILA h
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than NAA and IAA (optimal concentration 3 p ~ ) . Picloram induced protoplast hyperpolarization, but no relative depolarization was observed in the concentration range used, up to 100 p ~ Non-func. tional auxin analogues: 2-naphthaleneacetic acid (2-NAA), p-bromophenylacetic acid (p-HPAA), naphthylphthalamic acid (NPA) and indolelactic acid ( L A ) had no significant effect on Em. However, 2-NAA and p-HPAA were able to reverse the hyperpolarizing effect of 3 ~ M - N A Awhich , suggested that these analogues, although inactive per se, could compete with NAA at the sites involved in the membrane response. NPA and ILA did not interfere with the NAA-induced response. The absence of effect of NPA suggests that the auxin eMux carrier located at the plasma membrane is not involved in the electrical response of protoplasts to NAA. This specificity pattern of the membrane response of protoplasts closely resembles that of the auxin-controlled proliferation of cells derived from the same mesophyll protoplast preparations [23]. Moreover, in protoplasts from an auxin-resistant mutant, the sensitivities of both responses towards a set of auxin analogues were similarly affected [ 18, 231. These results provide evidence for a specific action of auxin at the membrane level related to its biological activity. This contrasts with the recent results of Felle et al. [24] who investigated the electrical response of maize coleoptiles to auxins, and found that all tested compounds, growth-promoting auxins as well as growth-inactive structural analogues, evoked a hyperpolarization, making this response unspecific on coleoptiles. The fact that non-permeating auxins (proteincoupled NAA derivatives) induced protoplast hyperpolarization within less than 1 min, the same time course as NAA, suggested that the recognition of the auxin signal occurred at the outer face of the plasma membrane [ 251, probably involving tobacco auxin-responsive proteins (tARPs). Other evidence for this localization was provided, showing that antibodies raised to ABPs from maize coleoptiles, or specifically directed against ARP1, inhibited the electrical response to auxin when applied exogenously to tobacco protoplasts [26, 271. This suggested immunological relations between tARP and maize ABP. The progressive inactivation of this pool of tARP by increasing amounts of anti-AHP antibodies resulted in a decrease in auxin sensitivity, read through the shift of the dose-response curves of Em towards higher NAA concentrations [27]. Conversely, incubation of tobacco protoplasts with purified maize AHP led to an enhancement of their sensitivity to auxin, together with an increased
Plant Hormones and their Receptors
number of sites immunoreactive to the anti-ABP antibody; homogeneous maize ARPl evoked a similar effect [27]. It was assumed in that case that treatment of protoplasts with ABP increased the number of active perception units at the plasma membrane, thus inducing an increase in auxin sensitivity. These results point to the existence, at the plasma membrane of protoplasts, of tARF’s immunologically and functionally related to maize ABP1, and suggest that auxin sensitivity detected by the membrane electrical response correlates with the level of these tARPs. They also raise puzzling questions as to the organization of the reception-transduction units involved in the membrane response to auxin. As to the change in sensitivity of the membrane response induced by exogenous maize ARP1, we proposed that AHP1, a nonintegral membrane protein [4-61, could interact with a transmembrane protein (TP) of tobacco plasma membrane essential for the transmission of the auxin signal [27]. In such a model, an auxinperception unit could be formed by the homologous association of a tARP with a TP, or by the heterologous interaction between maize AHP and the tARP-binding site of TP. To assess the relevance of this model, binding studies using labelled exogenous ARP on protoplasts should assist the search for evidence of the putative membrane protein interacting with ABP.
Different cellular responses to auxin might involve distinct reception-transduction pathways for the hormonal signal The auxin sensitivity of the membrane response of protoplasts from A. rhizogenes (Ri)-transformed plants was shown to be greatly enhanced, in association with an increased amount of plasma membrane sites recognized by anti-ABP antibodies [27]. Single genes from the Ri-T-DNA, the rolA, rolB and rol C genes, conferred an increased sensitivity on transformed protoplasts, rolB being by far the most efficient [20]. Using transgenic plants containing a gene fusion product consisting of the promoter of the rolB gene and the coding sequence of the B-glucuronidase gene ( rol B:GUS), Maurel et al. [19] demonstrated that the expression of the rolB promoter, as revealed by GUS activity, was activated by auxin. This activation was specific for auxin-type growth regulators and occurred in the plant in different tissues as well as in isolated mesophyll protoplasts [19], constituting in this latter case a cellular response to auxin monitored after 15 to
2 4 h of protoplast culture in the presence of hormone. Tobacco plants containing both the rol B: GUS chimeric gene and a functional rol B gene were used for simultaneous monitoring of rol B expression and physiological effects at the protoplast level. The sensitivity to auxin of the hyperpolarization response and the rol B promoter activation were compared in rol B:GUS and rol B-rol B: GUS plants (Fig. 2). Protoplasts from rol B:GUS plants were hyperpolarized by auxin with an inverted bell-shaped dose-response curve, a maximal hyperpolarization being observed at the concentration of 3 ~ M - N A A (Fig. 2a), as in protoplasts from wild-type plants [27]. The dose-response curve of protoplasts from rol B-rol B: GUS plants, expressing the rol B structural gene, was shifted to lower NAA concentrations, producing an increase of sensitivity of more than 100000-fold (Fig. 2a). As shown in Fig. 2b, GUS activity was strongly enhanced in protoplasts treated with auxin. Surprisingly, maximal GUS expression driven by the 7018 promoter was obtained at the same NAA concentration of 10 ~ U M in the absence (ro1B:GUS) or in the presence (To1B-rol B:GUS) of the functional rol B gene. Two other responses to auxin were also investigated in protoplasts from untransformed or rol Btransformed plants: the occurrence of the first division of protoplasts cultured for 5 days in the presence of the hormone (Fig. 2c) and the proliferation of protoplast-derived cells after 4 weeks (Fig. 2 4 . For both division responses, it was found again that the optimal NAA concentrations were identical in untransformed and rol B-transformed cells. Transformation by the roZB gene thus increases the auxin sensitivity of the membrane response, but does not affect the auxin sensitivity of other responses. Such selective effects of Ri-transformation were also observed in protoplasts from plants containing the whole T-DNA [ZO]. These results point to a distinct functioning of various cellular responses to the hormone. Although metabolization of auxin is likely to alter the dose-dependency of responses expressed after a relatively long hormone treatment (see for example Fig. 2c and 2 4 , differences in the dose-dependency of various auxin responses might reflect the existence of distinct reception-transduction pathways to control membrane hyperpolarization on one hand, and rolB expression and cell division on the other hand. Such a possibility has already been proposed by Nakamura et al. “281. For the electrical response of protoplasts, we demonstrated that auxin perception occurs at the outer face of the plasma mem-
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Fig. 2
Cellular responses to auxin Protoplasts were isolated from tobacco plants containing the rol 6:GUS fusion gene either alone (BGUS) or associated with a functional rol6 gene (BBGUS). The NAA-induced Em variations were measured on freshly isolated protoplasts as described in Barbier-Brygoo et 01. [27] (20). for the study of ro1B:GUS expression, protoplasts were isolated in the absence of auxin. They were then cultured for 24 h at a density of 5 X lo4 protoplastdml in the presence of various concentrations of NAA. GUS activity in protoplasts was determined by a fluorimetric assay as described by Maurel et 01. [ 191 (2b). Axenically prepared mesophyll protoplastsfrom wild-type plants (W)or plants containing the rolB gene (6) were plated at a density of 5 X lo4 protoplastdml in the presence of various concentrations of NAA. After 5 days of incubation in the dark at 26°C the occurrence of the first division was estimated by light microscopy (2c). Protoplast-derivedcells were then plated at a density of 100 celldml in the presence of various NAA concentrationsand developing colonies were counted after 4 weeks. Results were expressed as relative plating efficiency, i.e. the proportion of cells which developed colonies among the cells which had already divided at the time of low-density plating ( 2 d ) .
62
(0)
Mesophyll protoplasts
'4
NAA-induced
E Y
a -4
BBGUS maxSE
BGus
I min.
I
i
brane, but the cellular location of the auxin receptors triggering rolB expression and cell division is unknown. Recently, Prasdd & Jones [ 141 suggested that auxin-regulated gene expression might be mediated by the 65 kDa ARP they localized at the nucleus. However, a complete indepen-
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dence of perception pathways for both the shortterm cell-surface and the long-term cell-division responses would stand in contrast with the fact that the auxin-resistant mutant was affected in a strikingly similar manner in the electrical and the celldivision responses. Further evidence based on more
Plant Hormones and their Receptors
direct approaches is needed to explore the independence or relationship between the perception pathways mediating the variety of auxin effects in plant cells. 1. Napier, R. M. & Venis, M. A. (1990) J. Plant Regul. 9, 113-126 2. Klee, H. & Estelle. M. (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 529-55 1 3. Libbenga, K. K..van der Linde, P. C. G. & Mennes, A. M. (1986) in Hormones, Receptors and Cellular Interactions in Plants (Chadwick, C. M. & Garrod. D. R., eds.), pp. 61-70, Cambridge University Press, Cambridge 4. Hesse, T., Feldwisch, J., Halshusernann, D., Hauw, G., Puype, M., Vandekerckhove, J., Lobler, M., Klarnbt, D., Schell, J. & I’alme. K. (1989) EMBO J. 8, 2453-246 1 5. Inohara, N., Shimomura. S., Fukui, T. & Futai, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3564-3568 6. Tillmann. U., Viola, G., Kayser, H., Siemeister, G., Hesse, T., I’alme, K., I,obler, M. & Klambt, D. (1989) EMBO J. 8,2463-2467 7. Lobler, M. & Hirsch. A. M. (1 990) Plant Mol. Biol. 15, 5 13-516 8. Dohrmann, U., Hertel, K. & Kowalik, H. (1978) Planta 140,97-106 9. Jones, A. M., Lamerson, 1.’ & Venis, M. A. (1989) Planta 179,409-413 10. Palme, K., Feldwisch, J.? Hesse, T., Bauw, G., Puype, M., Vandekerckhove, J. & Schell, J. (1990) in Hormonal Perception and Signal Transduction in Animals and Plants (Roberts, J., Kirk, C. & Venis, M. A., eds.), pp. 299-313, The Company of Biologists Ltd, Cambridge 11. Hicks, G. R.. Rayle, D. I,.. Jones, A. M. & Lomax, T. I,. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4948-4952 12. Hicks, G. R., Rayle, D. I,. & Lomax, T. I,. (1989) Science 245,52-54
13. Palme, K.. Hesse, T., Moore, I., Campos, N., Feldwisch, J., Garbers, C., Hesse, F. & Schell, J. (1991) Mech. Develop. 33,97-106 14. Prasad, P. V. &Jones, A. M. (1991) Proc. Natl. Acad. Sci. USA. 88, 5479-5483 15. Jones, A. M. (1990) Physiol. Plant 80, 154- 158 16. Klarnbt, D. (1990) Plant Mol. Biol. 14, 1045-1050 17. Napier, R. M. & Venis, M. A. (1991) Trends Riochem. Sci. 16,72-75 18. Ephritikhine, G., Barbier-Brygoo, H., Muller, J.-F. & Guern, J. (1987) Plant Physiol. 83,801-804 19. Maurel, C., Brevet, J., Barbier-Brygoo, H., Guern, J. & TempC, J. (1990) Mol. Gen. Genet. 223, 58-64 20. Maurel, C., Barbier-Brygoo, H., Spena, A., Temp&,J. & Guern, J. ( 1991) Plant Physiol. 97,2 12-2 I6 21. Shen, W. H., Petit, A., Guern, J. & Tempd, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,3417-3421 22. Shen, W. H., Davioud, E., David, C., Barbier-Brygoo, H., Tempi.. J. & Guern, J. (1990) Plant Physiol. 94, 554-560 23. Caboche, M., Muller, J.-F., Chanut, F., Aranda, G. & Cirakoglu, S.(1987) Plant Physiol. 83,795-800 24. Felle, H.. Peters, W. & Palme, K. (1991) Biochim. Riophys. Acta 1064, 199-204 25. Venis, M. A.. Thomas, M. W., Barbier-Brygoo, H., Ephritikhine, G. & Guern, J. (1990) Planta 182, 232-235 26. Barbier-Brygoo, H., Ephritikhine, G., Klambt, D., Ghislain, M. & Guern, J. (1989) Proc. Natl. Acad. Sci. USA. 86,891-896 27. Barbier-Brygoo. H., Ephritikhine, G., Klambt, D., Maurel, C., Palme, K., Schell, J. & Guern, J. (1991) Plant J. 1,83-93 28. Nakamura, C., van Telgen, H.-J., Mennes, A. M., Ono, H. & Libbenga, K. R. (1988) Plant Physiol. 88, 845-849 29. Caboche, M. (1980) Planta 149,7-18
Received 10 September 1991
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