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Proceedings of the Indiana (1991) Volume Academy of Science j 29 99, p. 129-136 THE EFFECT OF CHLORO-DERIVATIVES OF INDOLEACETIC ACID ON PLASMA MEMBRANE ELECTRON TRANSPORT AND PROTON EXCRETION Michael Bottger 2 and Frederick L. Crane Rita Barr 1 , 1 Department of Biological Sciences 1 Purdue University West Lafayette, Indiana 47907 and Institut fur Allgemeine Botanik 2 University of Hamburg Ohnhorststr. 18 D 2000 Hamburg 52 Federal Republic of ABSTRACT: As shown Germany by Bottger and Hilgendorf [Plant Physiol. 86, 1038-1043 (1988)], the natural auxin indoleacetic acid in high concentrations (10 |xM) inhibited both e and proton efflux by corn roots of undamaged plants measured with a pH-stat combined with a redoxstat. we show study inhibited that of various chloro-substituted indoleacetic acid derivatives NADH oxidation and several derivatives stimulated trans-membrane hexacyanoferrate reduction in concentrations from 0.1 these cells was nM to 10 jxM in cultured carrot cells. Proton excretion by also stimulated by 5-, 6- and 5,7-chloro substituted indoleacetic acid, but the 4- chloro derivative inhibited in In this only 4-chloro-IAA H+ excretion by the plasma membrane H + -ATPase, while H + excretion presence of hexacyanoferrate was stimulated. These divergent effects of chloro-substituted indoleacetic acid derivatives proton movement through Abbreviations. HCF III show the that the membrane trans-membrane redox system in a is not directly related to relationship. 1:1 potassium hexacyano ferrate or potassium ferricyanide. INTRODUCTION Indole-3-acetic acid (IAA) sponses in plant cells is a growth (Brummell and Hall, hormone which induces rapid cellular re1987). A hypothesis for the mechanism of auxin action according to Brummell and Hall (1987) to a receptor in the plasma membrane, an signal cascade (Boss and Morre, 1989) in the stimulation act initiation as follows: First, the auxin binds which promotes the by which cellular Ca 2+ of protein kinases, which prepare an with auxin. The protein-auxin complex then induces cell wall is is unknown m-RNA whereas the addition of auxin to protein to synthesis, loosening and leads to growth. These responses require and could, therefore, not be considered as the inositol trisphosphate released. This results first complex which promotes at least 10-20 min for plant response to auxin, measurement of plasma membrane electron transport Indiana 130 Academy of Science Vol. 99 (1991) NADH reactions gives an immediate response in terms of stimulation of hexacyano oxidation or ferrate reduction with associated increase in proton release. Chlorinated auxins were chosen for the study of their effects on plasma electron transport because they all show high position 7 on the benzene ring unlike membrane of plasma biological activity, unless substituted in 1984). Since stimulation itself (Pless et al., IAA and electron transport by chloro derivatives its mechanism for inducing cascade proposed by Brummell and Hall, (1987). response, signal IAA it could provide an membrane is a fast the inositol trisphosphate initial MATERIALS AND METHODS Carrot cells were grown in suspension culture as previously described (Barr et 1985; and Barr et a DW-2A 1987). al., NADH oxidation was assayed spectrophotometrically with beam mode following a decrease wavelength set at 430nm (Barr et al., 1985). double beam spectrophotometer of absorbance 340nm at Reactions were run with reference in the dual (lOmM KC1, lOmM NaCl sucrose-salts solution lOmM CaCl and (0.005gdrywt.). After a 3-min incubation period, 100|ulM NADH IAA or its magnetic 24°C (Barr cell at et al., pH electrode in a 5- The assembly was gently 1987). stirred on IAA or its chloro derivatives added + in ethanol. ferricyanide The basal rate of H excretion was monitored for 5min, then 100|xM was added to measure the contribution of protons from plasma membrane electron transport with ferricyanide as the HC1 to start the during assays. The reaction mixture contained carrot cells stirrer (0.005gdrywt.), 25fxM phosphate buffer, pH7, and cells. cells chloro derivatives were added in concentrations indicated in figures. ml water-jacketed of and carrot 2) was added Proton excretion was measured with a Corning combination a Tris-Mes, pH7, Reaction rates were calculated using a millimolar extinction coefficient of reaction. 6.22. 25mM 24°C. The reaction mixture contained at al., impermeable electron acceptor on the outside Proton excretion rates were calculated from the addition of known amounts of as described in (Barr et al., 1987). IAA and NAA were purchased from the Sigma Chemical Co. Louis, (St. MO). The chloro derivatives of IAA were obtained from Dr. Bottger's laboratory, University of Hamburg, F.R.G. RESULTS AND DISCUSSION (NAA) Indole-3-acetic acid (IAA) and a-naphthalene acetic acid promote growth action was is not clear and to see if IAA and it its electron transport and chloro derivatives, as well as H + excretion. A Waldrum and Davies, 1981), with NAA and only twofold by 0.01 |xM concentration had a greater effect on IAA H+ NAA, NAA, the plasma membrane H membrane which cannot be oxidized, shows NADH (Figs. 1 oxidation -ATPase may lead is stimulated tenfold IAA A, 1C). However, excretion than NAA be significant, considering that the acid growth hypothesis, + affected plasma 1984; Reinecke and Bandurski, et al., differences on cultured carrot cells: the rate of by to comparison of the effects of IAA, an auxin which can be oxidized by plant tissue (Pless 1988; and known are (Brummell and Hall, 1987), but the mechanism of auxin may differ from species to species. The purpose of this study in plant cells in which to cell wall loosening H + same may excreted by and increased growth (Hayashi, 1989; and Rayle and Cleland, 1982). The chloro derivatives of IAA, 7-chloro IAA and 5,7-dichloro IAA) were in the (Figs. IB, ID). This tested to find out if IAA (5-chloro they could act as Botany: Vol. 99(1991) 8 9 Barr, Crane 1. The ; nfifl of H+ C-rate of cells. A-rate of NADH oxidation at NADH oxidation IAA concentrations; Bvarious IAA concentrations; various excretion without HCFIII (O) and with HCFIII (O) in presence of NADH 8 cmD" K (IAA) and naphthalene acetic acid (NAA) on effect of indole-3-acetic acid and proton excretion by cultured carrot rate ^9~ iO _ mac no* Figure 131 NAA concentrations; D-rate of H + excretion without HCFIII various NAA concentrations. NADH oxidation was assayed oxidation with various and with HCFIII (#) in spectrophotometrically, presence of H+ excretion with a pH electrode, as described in Materials and Methods. (O) Indiana 132 Academy of Science ^CHlOftOIAflCmD 5-CHlOFtOlAH CfH]" IAA) on The effect of 5-chloro-indole-3-acetic acid NADH (5-C1-IAA) and 7-chIoro-indole-3-acetic acid (7-C1- oxidation and proton excretion by cultured carrot cells. A-rate of various 5-C1-IAA concentrations; B-rate of H+ 7-CI-IAA concentrations. H+ NADH oxidation at excretion wihtout HCFIII (O) and with HCFIII NADH NADH oxidation at excretion without HCFIII (O) and with HCFIII (•) in presence of various 5-C1-IAA concentrations; C-rate of concentrations; D-rate of K 7-CHL0R0-mflCfTD"H 7-CHlORO-IAACrTD Figure 2. Vol. 99(1991) various 7-C1-IAA (•) oxidation was assayed spectrophotometrically, electrode as described in Materials and Methods. in H + presence of various excretion with a pH Botany: Vol. 99(1991) Barr, Crane 133 5,7-DICHLORO-lflflCfTD Figure 3. The carrot cells. effect of 5,7-dichloro-indole-3-acetic acid NADH (5,7-CMAA) on oxidation was assayed spectrophotometrically as described in Materials and Methods. at NADH oxidation by cultured various 5,7-Cl-IAA concentrations — V Indiana 134 Academy of Science Vol. 99 (1991) O BflSfll • AHCfUE h 3 — t rH 5,7-DICHLORO-lflfl Cm: Figure 4. The effect of 5,7-dichloro-indole-3-acetic acid (5,7-Cl-IAA) on proton excretion by cultured carrot cells. H+ excretion measured without HCFIII (O) and with HCFIII concentrations, using a pH (•) electrode as described in Materials and Methods. at various 5,7-Cl-IAA Botany: Vol. 99 (1991) growth regulators as noted mode in their by carrot cells, but in IAA was A IAA H + cells in H+ NADH oxidation less effective than 7-chloro or 5,7-dichloro maximum to achieve IAA stimulation of NADH oxidation (Fig. excretion were most notable in case of 7-chloro (Fig. 2D) and IAA (Fig. 3B), 5-chloro IAA showed a 2B). The Here, again, differences were itself. thousand times higher concentration (0.1 (xM vs. 0.1 mM) was both of which inhibited the basal rate of 5,7-dichloro while 135 of action. All three chloro derivatives stimulated 5-chloro necessary for 5-chloro 2A). Differences compound, IAA their parent 2A, 2C, 3A). (Figs. Barr, Crane of ATPase-mediated slight stimulation membrane excretion mediated through plasma H + H+ excretion, excretion (Fig. electron transport by carrot presence of ferricyanide was not remarkable, showing only slight stimulation or inhibition. Previous studies by our group on cultured carrot cells (Crane transformed versus non-transformed tobacco cells (Barr transplasma membrane HCF electron transport with et al., III et al., 1983) or 1985) have shown that as the impermeable electron acceptor respond to plant growth regulators and that an increase in proton excretion, H+ aside from membrane H + -ATPase, is seen when low conused (< |jlM). Until the discovery of NADH oxidase excreted by the plasma centrations of plant hormones are from soybean hypocotyls (Barr 1 1985), the stimulation of et al., HCF III reduction by NADH oxidation isolation of a hormone-sensitive NADH 1988), the stimulation of NADH oxidation growth regulators was relatively small (about 25%), but stimulation of can be 10X, as seen 1C. Since the in Fig. oxidase by Brightman etal. (Brightmanetal., by IAA and its shown here can chloro derivatives in the future be related to plant growth in general. The stimulation by IAA and its chloro derivatives of excretion of cultured carrot cells seen here and Hilgendorf, 1988), who found is NADH oxidation and contrary to Bottger and Hilgendorf (Bottger inhibition of HCFIII reduction and H+ excretion by intact maize seedling roots, when treated with IAA. HCFIII reduction by carrot in this study was not significantly affected by These differences may be due may be H+ IAA or its cells derivatives (data not presented). to using tissue culture cells versus intact seedlings, or it a species-related difference. In an earlier study, Bottger and Luthen (1986) found a correspondence between NADH oxidation and proton excretion by roots of Zea mays. In NADH summary, it is concluded NAA, IAA and its chloro derivatives stimulate oxidation by cultured carrot cells in low concentrations (0.1 whereas only the H + -ATPase Unlike oxidation products of mM to 1(jlM), generated protons are affected by these growth regulators. IAA metabolism, such as oxindole-3-acetic acid (Ernstsen 1987; Henderson and Patel, 1972; and Nonhebel and Bandurski, 1984), which et al., were found to be inactive as growth promoters, the chloro derivatives of no inhibition of NADH oxidase activity. by 7-chloro and 5,7-dichloro is that IAA may The inhibition of also be significant, H if + IAA showed -ATPase-excreted protons the acid growth hypothesis correct. LITERATURE CITED Barr, R., T.A. Craig and F.L. Crane. 1985. Transmembrane ferricyanide reduction in carrot cells. Biochim. Biophys. Acta 812:49-54. Barr, R., F.L. J. Plant Crane and T.A. Craig. 1984. Transmembrane ferricyanide reduction Growth Regul. 2:243-249. in tobacco callus cells. Academy of Science Indiana 136 Barr, R., O. Martin, Jr. Vol. 99 (1991) and F.L. Crane. 1987. Redox-induced proton excretion by cultured carrot affected by protonophores and inhibitors of ATPase. Indiana Acad. cells is Sci. 96:139-144. Barr, R., A.S. Sandelius, F.L. Crane and D.J. Morre. 1985. Oxidation of reduced pyridine nucleotides by plasma membranes of soybean hypocotyl. Biochem. Biophys. Res. Boss, W.F. and D.J. Morre, eds. Liss, Bottger, New York, 348 M. and 1989. Second Messengers in Plant Commun. 131, 943-948. Growth and Development. Alan R. p. F. Hilgendorf. Hormone 1988. H+ action on transmembrane electron and transport. Plant Physiol. 86:1038-1043. Bottger, M. and H. Luthen. mays L. roots. J. 1986. Possible linkage between NADH-oxidation and proton secretion in Zea Exp. Bot. 37:666-675. Brightman, A.O., R. Barr, F.L. Crane and D.J. Morre. 1988. Auxin-stimulated NADH oxidase purified from plasma membrane of soybean. Plant Physiol. 86:1264-1269. Brummell, D.A. and J.L. Hall. 1987. Rapid article. Plant, Cell cellular responses to auxin and the regulation of growth: review and Environment 10:523-543. Crane, F.L., T.A. Craig, P.C. Misra and R. Barr. 1983. Hormonal control of transplasma transport and proton flux. Proceed. 10th membrane Annu. Meeting Plant Growth Regul. Soc. of America, electron vol. 10 (H. R. Cooke, ed.), p.70-75. Ernstsen, A., G. Sandberg and K. Lundstrom. 1987. Identification of oxindole-3-acetic acid, and metabolic conversion of indole-acid to oxindole-3-acetic acid Hayashi, T. 1989. Xyloglucans primary in the in cell wall. Pinus sylvestris seeds. Planta 172:47-52. Ann. Rev. Plant Physiol. Plant Molecular Biol. 40:139-168. Henderson, J.H.M. and C.S. Patel. 1972. Oxindole-3-acetic acid: physical properties and lack of influence on growth. Physiol. Plant. 27:441-442. Nonhebel, H.M. and R.S. Bandurski. 1984. Oxidation of indole-3-acetic acid and oxindole-3-acetic acid 2,3-dihydro-7-hydroxy-2-oxo-l H indole-3-acetic acid-7'-0-3-D-glucopyranoside in Zea mays to seedlings. Plant Physiol. 76, 979-983. Pless, T., M. Bottger, P. and correlation of its Hedden and J. Graebe. 1984. Occurrence of 4-Cl-indole acetic acid level with seed in broad beans development. Plant Physiol. 74:320-323. Rayle, D.L. and R. Cleland. 1972. The in vitro acid-growth response: relation to in vivo growth responses and auxin action. Planta 104:282-296. D.M. and R. S. Bandurski. 1988. Oxidation of an enzyme preparation from Zea mays. Plant Physiol. Reinecke, Waldrum, J.D. and 1307. indole-3-acetic acid to oxindole-3-acetic acid by 86, 868-872. E. Davies. 1981. Subcellular localization of IAA oxidase in peas. Plant Physiol. 68:1303-