May 2, 1983 - The plateau of IAA concentration-neutral growth is seen from 0.4 to 4.0 ... whole plants are not linearly related to auxin concentration (20).
Plant Physiol. (1984) 74, 335-339 0032-0889/84/74/0335/05/$01.00/0
Auxin Concentration/Growth Relationship for Avena Coleoptile Sections from Seedlings Grown in Complete Darkness1 Received for publication May 2, 1983 and in revised form October 19, 1983
JAMES R. SHINKLE* AND WINSLOW R. BRIGGS Department ofPlant Biology, Carnegie Institution of Washington, Stanford, California 94305 ABSTRACI A biphasic auxin dose-response curve has been obtained for indoleacetic acid (IAA)-stimulated growth of subapical sections of coleoptiles from totally dark-grown oats (Arena sativa L. cv Lodi). The curve for growth at 6 h is composed of a log-linear phase and a modified bellshaped phase separated by a plateau. The curve is log-linear from 0.003 to 0.4 micromolar IAA when sections are incubated in pH 5.9 buffer. The plateau of IAA concentration-neutral growth is seen from 0.4 to 4.0 micromolar UIA. Further increase in growth occurs from 4.0 to 10 micromolar IAA. Changing the pH of the buffer from 5.9 to 5.5 or 6.2 changes the shape of the curve, shifting the plateau to lower IAA concentration, or abolishing it, respectively. The synthetic auxin 2,4dichlorophenoxyacetic acid also shows a biphasic dose-response curve, but the synthetic auxin 1-naphthalene acetic acid does not. The plateau is not affected by the auxin-transport inhibitor 2,3,5-triiodobenzoic acid. The plateau is eliminated by taking sections from coleoptiles grown under continuous dim red light. We advance a model to account for these results based on two modes of auxin uptake into the cell: carrier-mediated uptake and uptake via chemiosmotic diffusion.
There have been several interpretations of the dose-response curve for auxin-stimulated growth in oat (Avena sativa) coleoptiles. Over the last 50 years, these have ranged from analyses of the mode of auxin interaction with a putative auxin receptor/ enzyme (4), to assertions that changes in growth rates seen in whole plants are not linearly related to auxin concentration (20). In addition, the shape of the auxin dose-response curve for growth has been variously reported as: linear with concentration (24); linear with the log of auxin concentration (11); bell-shaped on a log auxin concentration scale (4); and sigmoidal on a log auxin concentration scale (1). One feature common to all of these studies was that the response of the tissue to varying auxin concentration yielded a single phase continuous dose-response curve. This common feature resulted in models of growth responses to auxin which postulated only single step/single receptor transduction as the mechanism of auxin action. We report here conditions yielding a biphasic dose-response curve for IAA-stimulated growth in oat coleoptile segments. We believe that the two phases can be interpreted in terms of two different processes in the tissue response to auxin: two modes of uptake of IAA into the cell; and the transduction of internal IAA into growth. Responses which are composites of multiple processes involving hormones are probably common. The analysis of
these composite responses into component parts may resolve certain concerns which have arisen pertaining to interpretations of the mode of action of plant growth substances (20).
MATERIALS AND METHODS Unhusked seeds of Avena sativa cv Lodi were prepared for imbibition and growth as in Mandoli and Briggs (14). Seeds were imbibed in complete darkness and the seedlings were grown in plastic boxes in a totally dark growth chamber at 26C and 85% humidity for 72 to 76 h. All manipulations were carried out in the same growth chamber in complete darkness except where otherwise noted. At 72 to 76 h after imbibition, seedlings were selected for uniform size by touch and were removed by hand from the seeds. The coleoptiles ranged from 1 to 1.5 cm in length. The coleoptiles, with some mesocotyl attached, were placed in a specially designed two-blade cutter assembly. The cutter removed a 3-mm tip section, leaving the next 5.7 mm between the blades. Primary leaves were not removed. These sections were then placed in 9 to 12 ml of 5 mm K-phosphate buffer containing 80 gM chloramphenicol and the appropriate test substances in a Petri dish on ice. Sections were collected for up to seven treatments (seven dishes) in a given harvest. By rotating delivery of 4 to 5 sections around all Petri dishes, each treatment dish received the same distribution of sections with respect to time after cutting. Each harvest took less than 1 h, and yielded between 18 and 30 sections/dish. Two or more harvests, performed in series, were used for experiments requiring more than seven treatments. When each harvest was concluded, dishes were removed from ice and placed in a completely dark cabinet on a gyratory shaker running at 100 rpm. Incubation time was counted from the time that the dishes were placed on the shaker. Section lengths were measured after 6-h incubation. Sections were removed from buffer, blotted dry, placed on a clear glass plate, and photocopied. The section images were measured individually by a computer-assisted digitizer as described in Mandoli and Briggs (14). The program used provides mean and standard error determinations for each treatment population. Auxins were added to test solutions in ethanol, with ethanol content neyer exceeding 0.2%. IAA, I-NAA,2 2,4-D, and TIBA were obtained from Sigma, and used without further purification. Auxin stocks were maintained at 0.1 M in 100% ethanol, and were stored at -20°C. Buffer pH was adjusted by titration of mono- and dibasic 5 mM K-phosphate solutions. Unless otherwise noted, pH 5.9 buffer was used. For some experiments, the seeds prepared as above were imbibed and grown under continuous red light at 25°C, in a different growth room. Red light was obtained from Sylvania R40/RED fluorescent tubes filtered by 1 layer each of nos. 1 and
'Carnegie Institution of Washington-Department of Plant Biology 'Abbreviations: I-NAA, l-naphthalene acetic acid; TIBA, 2,3,5-triioPublication 825. dobenzoic acid. 335
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14 cinemoid. The fluence rate was 0.2 ,umol m-2 s-' at bench level. Plants were grown for 72 to 76 h in translucent white plastic boxes with lids of the same material to maintain high humidity. Lids and boxes attenuated light by approximately 50%. Harvesting and incubation of sections were performed as above, except that all procedures were performed under red light, instead of total darkness. Coleoptiles used were 2 to 2.5 cm in length.
Plant Physiol. Vol. 74, 1984
obtained at pH 5.9 with curves obtained at pH 5.5 and 6.2. The plateau was absent at pH 6.2, and was shifted to a lower concentration range at pH 5.5, with respect to pH 5.9. At pH 5.5, the dose-response curve above 1 gM appears distinctly bellshaped. At pH 5.9 and 6.2, sections showed suboptimal growth at IAA concentrations higher than 30 Mm (not shown). The curves in Figure 2 were obtained on different days for the different pH values. To determine whether the different values for maximum growth were a consequence of pH treatment or RESULTS simply the result of day to day variability, the growth response Figure 1 shows a representative IAA dose-response curve from to IAA at all three pH values was tested on the same day (Table sections incubated at pH 5.9 for 6 h. The curve is the average of I). At the three pH values studied, there were no significant two replicate experiments. The curve appeared to be log-linear differences in growth observed at IAA concentrations between 1 below 0.6 Mm IAA. A discontinuity in the curve is apparent above and 10 Mm, the region of maximal stimulation. At 1 and 0.3 Mm this concentration. As shown in the inset, a more detailed ex- IAA, growth at pH 5.5 and 5.9 showed a significant difference. amination of the region around the discontinuity revealed a Growth at pH 6.2 was the same as pH 5.5 and 1 Mm and the plateau in the growth response to increasing IAA concentration same as growth at pH 5.9 at 0.3 uM IAA. All three pH values showed similar growth stimulation at IAA concentrations above extending from 0.4 to 4 Mm IAA. The curves shown in Figure 2 compare the IAA dose-response the pH 5.9 plateau range, and decreasing pH caused increased growth at IAA concentrations below the plateau range. I~~~~~~~~~~~~~~~~~ The dose-response curves for growth are quite different when 45 the applied auxin is the synthetic hormone 1-NAA. In Figure 3, no suggestion of a plateau was seen. Experiments similar to those used in the inset in Figure 1 with 1-NAA as the applied auxin showed no dose-neutral concentration range, with respect to increase in growth (see inset in Fig. 3). Figure 3 also indicated +, that the threshold concentration for stimulation of growth is 10 times higher for 1-NAA than it is for IAA. Maximal stimulation * of growth, however, was reached at the same concentration for both IAA and 1-NAA. The maximal growth induced by 1-NAA is lower than the growth induced by IAA at plateau concentra*E: tions in the full scale figure, and higher in the inset figure. Each curve is derived from five separate replicate experiments. The differences result from day to day variability in the magnitude II / of the growth response measured. This variability never affected 0 0.01 0.1 1.0 10.0 the presence or range of the plateau in the IAA dose-response IAA, yM curve
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FIG. 1. Biphasic IAA dose-response curve at pH 5.9 for dark-grown Avena coleoptile sections, harvested in complete darkness. Curve shown is generated from the means of two replicate experiments. Error bars represent the average SE for the two replicates. Inset shows another set of experiments with more concentrations in the plateau region of the pH 5.9 IAA dose-response curve. Results are the mean of three replicate experiments. Error bars represent the average SE for the three replicates. 60
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5.5 altered neither the shape of the dose response curve, nor the threshold concentration for stimulation of growth (data not shown). However, at pH 5.5, the decline in growth seen at supraoptimal 1-NAA concentrations was steeper than at pH 5.9, as was also the case for IAA (Fig. 2). The dose-response curve for another synthetic auxin, 2,4-D, did exhibit a discontinuity over the same concentration range as was seen for IAA at pH 5.9 (data not shown). However, there was considerably less stimulation of growth by concentrations of 2,4,-D below the plateau range, when compared to growth caused by IAA at the same concentrations. Correspondingly greater stimulation of growth was caused by concentrations of 2,4-D above 1 uM, such that the maximal stimulation of growth by 2,4D was similar to the maximal stimulation of growth caused by 1-NAA. The concentration of 2,4-D eliciting the maximal stimulation of growth was 3 to 10 times higher than that found for IAA or 1-NAA. The effect of the auxin transport inhibitor TIBA on the IAA dose-response curve was studied. As shown in Figure 4, TIBA at 3.33 uM, the concentration shown to be optimal for inhibition of effiux of IAA in vitro (8, 10), caused increased growth of the zero IAA controls, and also increased the threshold concentration
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for stimulation of growth. The plateau in the pH 5.9 doseresponse curve was unchanged, as was the rest of the curve. Figure 5 shows a comparison between sections taken from dark-gown plants (and experiments performed in the dark), and sections from plants grown and experimented on under continuous red light. The dose-response curve for IAA at pH 5.9 for red light-grown plant tissue was monophasic. The shape of the
337
AUXIN AND A VENA SECTION GROWTH Table I. Simultaneous Experiments Comparing the Effects ofpH 5.5, 5.9, and 6.2 Buffers on IAA-Induced Growth Final length at 6 h is shown. Dark-grown Avena coleoptiles were harvested and sections were cut as in Figure 1. The IAA concentration series was applied to all 3 pH values in the same experiment. Results are given as ±SE and are the means of two replicate experiments. n = total number of sections for each treatment. Significance was determined by z statistic as in Johnson (12). 3.0 Mm 1.0 MM 10 Mm 0 0.3 uM 0.6 Mm [IAA] pH 5.5 6.4 ± 0.06a 8.33 ± 0.08' 8.45 ± 0.09' 8.6 ± 0.09& 8.75 ± 0.10a 8.87 ± 0.10a (51) (52) (50) (43) (51) (49) pH 5.9 6.4 ± 0.06a 8.05 ± 0.10b 8.38 ± 0.108 8.38 ± 0.09b 8.78 ± 0.09a 8.85 ± 0.11a pH 6.2
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significant difference between results. b Differs significantly from (a): P < 0.03 or better. c Differs significantly from (b): 0.09 > P > 0.08; no significant difference from (a).
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FIG. 3. Comparison of dose-response curves for IAA and 1-NAA, for Avena coleoptile sections as in Figure 1. IAA and I-NAA were tested in pH 5.9 K-phosphate buffer. The IAA dose-response curve is the pH 5.9 curve from Figure 2. The inset is a comparison of IAA- and l-NAAinduced growth over the pH 5.9 dose-response curve plateau region seen in Figure 1. Each curve is generated from the means of five separate experiments. Error bars represent average SE for each set of five replicates.
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FIG. 5. Comparisons of IAA dose-response curves for sections from dark-grown and red light-grown Avena seedlings, tested at pH 5.9. Curve for sections from dark-grown seedlings is the pH 5.9 curve from Figure 3. Sections from red light-grown plants were prepared as in 'Materials and Methods." Inset shows the IAA dose-response curve and the 1-NAA dose-response curve for red light-grown plants. Each curve is generated from the mean of five replicate experiments. Error bars represent average SE for the five replicates. curve appears sigmoidal, but the range of the response is broader than 2 orders of magnitude, unlike Cleland's results (1). The inset shows 1-NAA dose-response curves obtained for redgrown plants, compared to the similar IAA dose-response curve from the full scale figure. The value for threshold is 10 times higher than that found for red-grown tissue treated with IAA, but the saturation occurs at almost the same concentration for 1-NAA and IAA. The maximum stimulation of growth was significantly lower for 1-NAA than for IAA. In contrast, sections cut from dark-grown plants which were then incubated for 6 h under room light, displayed a discontinuous LAA dose-response curve, similar to dark-incubated sections from dark-grown plants (results not shown). The 1-NAA dose response was always similar whether the plants were dark- or red-grown, and whether the sections were incubated with or without light (not shown).
10.0
IM, pM
FIG. 4. Effect of 3.33 Mm TIBA on IAA-induced growth in coleoptile sections as in Figure 1. Results minus TIBA are from Figure 2 curve for pH 5.9. Results for plus TIBA are from three replicate experiments. Error bars represent average SE for each set of replicates.
DISCUSSION Our working hypothesis to explain the plateau in the IAA dose-response curve for dark-incubated coleoptile sections from dark-grown plants is that the plateau represents the transition
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between two modes of IAA uptake. Below the plateau, we hypothesize that carrier-mediated uptake of IAA via the postulated proton/IAA symport (6, 8, 16) determines the internal IAA level. Above the plateau, increased IAA uptake occurs via chemiosmotic diffusion of IAAH. The carrier is saturable (16), and we propose that with carrier saturation the equilibrium internal IAA concentration does not increase further with external IAA concentration. The growth response is neutral to increasing external IAA until the applied IAA concentration is high enough to drive significant IAA uptake via diffusion of IAAH. We postulate that chemiosmotic diffusion does not contribute to net IAA uptake at external IAA concentrations below the plateau because the carrier functions as an active IAA pump driven by the pH gradient. The uptake of IAA via the carrier produces internal IAA levels higher than would be produced by diffusion in response to a pH gradient. Lomax et al. (13) have shown that pH gradient-dependent IAA uptake into sealed plasma membrane vesicles from zucchini produces IAA levels inside the vesicles at least five times the concentration predicted by the equilibrium distribution of IAA as a weak acid. For the transition from carrier-mediated uptake of IAA to diffusion-mediated uptake to be visible in an IAA dose-response curve for growth, as in Figure 1, the growth response must be proportional to internal IAA concentration, and transduced by a single IAA receptor. The IAA symport carrier has a very limited affinity for 1-NAA (10, 16, 19). Thus, the 10-fold difference in the threshold concentration growth stimulation between IAA and 1-NAA is seen in Figure 3 because 1-NAA uptake probably occurs essentially by chemiosmotic diffusion. The carrier produces higher internal IAA levels than diffusion up to the external IAA concentration at which the carrier becomes saturated. The plateau exists over the external IAA concentration range between the saturation of the carrier and concentrations at which an inward concentration gradient for diffusion is reached. At pH 5.9, this range is almost a 10-fold increase in external concentration (Fig. 1, inset). By this principle, the low affinity of the carrier for 1-NAA accounts for the absence of a plateau in the 1-NAA dose-response curve. The absence of the plateau compresses the 1-NAA dose-response curve to a narrower range of concentrations over which growth is concentration-dependent, relative to the IAA dose-response curve. The 2,4-D dose-response curve shows the plateau over the same concentration range as IAA at pH 5.9, and the broader range of concentration-dependent growth, relative to 1-NAA. The symport is presumably active in taking up 2,4-D, but the
concentration dependence for growth induced by internal 2,4-D is different from that for IAA. The shift of the plateau range to lower IAA concentrations with increasing proton concentration as seen in comparing pH 5.9 to 5.5 (Fig. 3) suggests that with a higher external proton concentration, it takes less external IAA to saturate the symport. The shift of the upper limit of the plateau to a lower IAA concentration can result from the lower pH causing an increase in the external IAAH concentration. Although the plateau is not evident at pH 6.2, the unchanged threshold for IAA stimulation of growth indicates that the carrier is functioning. At the lower external proton concentration, the uptake carrier becomes saturated at a higher external IAA concentration, while the final internal IAA concentration remains the same as established at carrier saturation at pH 5.9 and 5.5. The saturation of the proton-mediated uptake occurs at an external IAA concentration high enough that any increase in external IAA concentration above carrier saturation will drive diffusion-mediated uptake. It is also possible that carrier function is reduced to the point where it never becomes saturated, and the smooth curve results from the function of the carrier and
Plant Physiol. Vol. 74, 1984
diffusion of IAA combined. The external pH determines the rate of carrier function, which in turn sets the external IAA concentration required to establish the stable internal level of IAA. The pH of the cytoplasm is probably higher than the external pH, and the cytoplasmic pH tends to be stable (15). Therefore, altering the external pH changes the pH gradient across the plasma membrane. At the lower pH, the IAA-/IAAH ratio declines, even as the external IAA required to saturate the carrier is reduced (Fig. 2). Thus, we hypothesize that the proton/IAA- symport is more strongly dependent on the proton concentration gradient than on external IAA- levels. The hypothesis assumes that the growth measured represents the response to equilibrium levels of IAA established by the external pH and IAA concentration. The validity of this assumption is indicated by IAA uptake experiments which show equilibrium internal IAA levels being reached within a 60-min incubation (3), and our previous finding that the IAA dose-response curves for growth after 12-h incubations show the same pH effects as are seen in Figure 2 (18). Polar transport of auxin is probably not required for the two modes of uptake to produce the results seen. Treating the sections with TIBA should eliminate auxin polar transport and increase the internal steady state level of IAA by blocking efflux (2, 8). The effect of TIBA is seen in the increase in growth in the absence of added IAA, and the higher threshold concentration for stimulation of growth by exogenous IAA. Maximal IAAinduced growth is not reduced by TIBA so the IAA is still getting to all of the cells (Fig. 4), and the plateau in the dose-response curve is essentially unchanged. Further, 1-NAA is transported through sections almost as well as IAA while 2,4-D is not (7). The dose-response curve for 1-NAA does not show a plateau (Fig. 3) while the curve for 2,4-D does, so the relative similarity to IAA is reversed with respect to that found for polar auxin transport. The structure/activity requirements for the uptake carrier as determined in crown gall suspension cultures (16), pea stem segments (2), corn coleoptiles (19), and in sealed plasma membrane vesicles isolated from zucchini hypocotyls (8, 10) are the same as reported here (Fig. 3 and "Results") regarding the relative activity of IAA, 2,4-D, and 1-NAA. The function of the postulated symport carrier may not be completely absent in our red light-grown plants. The threshold concentration for auxin-stimulated growth is still 10-fold higher for 1-NAA than for IAA in sections from red light-grown plants, as was the case for dark-grown tissue. Other elements of the auxin-induced growth have been shown to respond to red light (22, 23). Red light-induced changes in these elements could account for the changes seen in the auxin dose-response curve between red-grown and dark-grown plants. The details of the rigorous model developed by Rubery, Goldsmith and others (2, 3, 5, 6, 8, 16, 17, 19) should be mentioned relative to our results. Rubery (16) and Edwards and Goldsmith (3) have presented data on the pH dependence of IAA uptake in crown gall suspension cultures and corn coleoptiles, respectively. Their results and conclusions specify proton-dependent carriermediated uptake of IAA with characteristics consistent with our explanation of our results. Edwards and Goldsmith (3) showed results on the IAA concentration dependency for IAA uptake in 1-mm corn coleoptile slices which under some conditions suggest a carrier which operates at a concentration range similar to that indicated by our results for IAA-induced growth. In 20 mm MS, the net uptake of IAA was found to become nonlinear with applied IAA concentration above 1 AMm. This effect was not seen in 20 mM K-phosphate. The shift in net IAA uptake in the MS system occurs at the same IAA concentration range as the plateau in the dose-response curve for IAA-induced growth. The plateau
AUXIN AND A VENA SECTION GROWTH in the growth dose-response curve was unchanged in 5 mm MS (data not shown). The TIBA effect on growth in the absence of added IAA suggests that at low internal IAA the TIBA-sensitive anion carrier contributes significantly to the total IAA efflux, but does not affect the net efflux at higher internal IAA concentrations. This latter efflux may be the IAA- leak postulated by Goldsmith and Goldsmith (6). There are a few minor differences in experimental technique used in this report relative to standard practice for dose-response curves for IAA-induced growth (1). Because sections cut in the dark were placed directly into treatment solutions, no preincubation of sections in plain buffer was possible. The preincubation allows the endogenous auxin to diffuse out of cells, while avoiding the spontaneous growth response seen in cut tissue 1 to 3 h after cutting (21). Direct measurements of IAA levels in corn coleoptile sections show that over the first 30 min the LAA level declines, but reaches a steady value of approximately half the original level (9), so the preincubation probably does not eliminate the endogenous IAA supply. The dark-grown plants used in this study showed for the first time a plateau in the IAA dose-response curve. The similarity between the discontinuity in the concentration dependence of IAA uptake (3) and the discontinuity in the concentration dependence of IAA-induced growth is suggestive of a common mechanism. Our study of this mechanism indicates that direct measurement of IAA uptake (as in 2, 3, 16, 17, 19) also measures physiologically active IAA. Trewavas (20) has argued that the wide range of concentration dependency in dose-response curves for many types of plant hormone action makes the behavior of these hormones very unlike that of animal hormones, and makes the hormonal control of these responses by changes in hormone concentration very inefficient. We have shown here that the auxin dose-response curve for growth can be seen as a composite of the response of the auxin uptake system, itself complex, and a separate growth transduction system. The regime used in our experiments measures both the uptake process, and the actual action of the hormone. In whole plants, the uptake process is part of the means of controlling hormone concentration, not part of the process of responding to it. Many plant hormone dose-response curves probably measure such composite responses, and this
339
possibility can explain the large concentration dependency ranges found.
LITERATURE CITED 1. CLELAND R 1972 The dosage-response curve for auxin-induced cell elongation: a reevaluation. Planta 104: 1-9 2. DAVIES PJ, PH RUBERY 1978 Components of auxin transport in stem segments
of Pisum sativum L. Planta 142: 211-219 3. EDWARDS KL, MHM GOLDSMITH 1980 pH dependent accumulation of indoleacetic acid by corn coleoptile sections. Planta 147: 457-466 4. FoSTER RJ, DH McRAE, J BONNER 1952 Auxin-induced growth inhibition, a natural consequence of two-point attachment. Proc Natl Acad Sci USA 38: 1014-1022 5. GOLDSMITH MHM 1977 The polar transport of auxin. Annu Rev Plant Physiol 28: 439478 6. GOLDSMITH MHM, TH GOLDSMITH 1981 Quantitative predictions for the chemiosmotic uptake of auxin. Planta 153: 25-33 7. HERTEL R, R FLORY 1968 Auxin movement in corn coleoptiles. Planta 82: 123-144 8. HERTEL R, TL LOMAX, WR BiuGGS 1983 Auxin transport in membrane vesicles from Cucurbita pepo L. Planta 157: 193-201 9. IINO M, DJ CARR 1982 Sources of free IAA in the mesocotyl of etiolated maize seedlings. Plant Physiol 69: 1109-1112 10. JACOBS M, R HERTEL 1978 Auxin binding to subcellular fractions from Cucurbita hypocotyls: in vitro evidence for an auxin transport carrier. Planta 142: 1-10 11. JACOBS WP 1979 Plant Hormones and Plant Development. Cambridge University Press, Cambridge, pp 17-23 12. JOHNSON RR 1980 Statistics, 3rd ed. Duxbury Press, Belmont, pp 349-350 13. LOMAX TL, RJ MEHLHORN, WR BRIGGS 1983 Quantitation of sealed vesicle volume, pH gradient, and auxin uptake of zucchini hypocotyl membrane preparations. Cam Inst Wash Yrbk 82: 34-37 14. MANDOLI DF, WR BRIGGS 1981 Phytochrome control of two low-irradiance responses in etiolated oat seedlings. Plant Physiol 67: 733-739 15. ROBERTS JKM, PM RAY, N WADE-JARDEZY, 0 JARDETZKY 1981 Extent of intracellular pH changes during H+ extrusion by maize root tip cells. Planta 152: 74-78 16. RUBERY PH 1977 The specificity of carrier-mediated auxin transport by suspension-cultured crown gall cells. Planta 135: 275-283 17. RUBERY PH 1978 Hydrogen ion dependence ofcarrier-mediated auxin uptake by suspension-cultured crown gall cells. Planta 142: 203-206 18. SHINKLE JR, WR BRIGGS 1982 Aspects of red light and auxin effects on sections from etiolated oat coleoptiles. Cam Inst Wash Yrbk 81: 28-31 19. SUSSMAN MR, MHM GOLDSMITH 1981 Auxin uptake and the action of N-1naphthylphthalamic acid in corn coleoptiles. Planta 150: 15-25 20. TREWAVAS A 1981 How do plant growth substances work? Plant Cell Environment 4: 203-228 21. VESPER MJ, ML EVANS 1978 Time-dependent changes in the auxin sensitivity of coleoptile segments. Plant Physiol 61: 204-208 22. WALTON JD, PM RAY 1981 Evidence for receptor function of auxin binding sites in maize. Red light inhibition of mesocotyl elongation and auxin binding. Plant Physiol 68: 1334-1338 23. WALTON JD, PM RAY 1982 Inhibition by light of growth and Golgi-localized glucan synthetase in the maize mesocotyl. Planta 156: 302-308 24. WENT FW, KV THIMANN 1937 Phytohormones. MacMillan, New York
