Lateral Auxin Transport Research Articles

Some aspects of geotropism in coleoptiles

Auxin transport was studied in coleoptile sections that were stimulated geotropically. The early time course of auxin-transport asymmetry was measured. An initial phase in which more IAA was delivered into the receptor for the upper half was found after 5 min of horizontal exposure. After about 15 min this was followed by the expected known asymmetry in which more auxin flows in the lower side of the coleoptile. Upon return of the coleoptile to a vertical position, this asymmetry disappeared within 30 min.Earlier correlations of geosensitivity of the auxin transport system with sedimentation of amyloplasts in comparisons of wild type corn and an amylomaize mutant were confirmed and extended. It was also shown that, in contrast to the geotropic effect, phototropically induced lateral auxin asymmetry was not significantly different in wild type and amylomaize. Eleven other single-gene endosperm starch mutants of corn were compared to their corresponding normals. In all pairs, if a difference in geosensitivity of lateral auxin transport was present, it was correlated with a parallel difference in amyloplast sedimentation: e.g., sugary 1 ("67") had an amyloplast asymmetry index of 0.32 and a 13% gravity effect on auxin transport; the paired wild-type had both a greater amyloplast asymmetry (0.61) and a greater gravity effect on transport (23%).Correlations between gravity effects on auxin transport and amyloplasts were also shown in comparisons of apical and basal sections of corn, oat and Sorghum coleoptiles.Further results, confirming the increased effect of centrifugal acceleration greater than 1xg on lateral auxin transport and on curvature, are in agreement with the hypothesis that the pressure exerted by amyloplasts, acting as statoliths, locally stimulates the auxin transport system in the individual cells.

Geotropism and the lateral transport of auxin in the corn mutant amylomaize

In coleoptiles of the amylomaize corn mutant (AM), the amyloplasts are much reduced in size in comparison with the wild type corn (WT), permitting a comparison of geotropic responsiveness as related to lateral displacement of amyloplasts and lateral transport of auxin. The amyloplasts of AM showed 30-40% lesser lateral redistribution in response to horizontal exposure in comparison with WT. With geotropic stimulation, the lateral transport of auxin in the direction of growth was 40-80% less, and the geotropic curvature by the coleoptiles was also significantly less in the mutant as compared with WT. These correlations support the hypothesis that the starch plastids serve as gravity sensors in the geotropic responses of coleoptiles.

Second positive phototropic response patterns of the oat coleoptile

1.During second positive irradiation, bending increases steadily with time. Under optimal conditions, the lag between onset of illumination and beginning of parabolic bending behavior is about 3 min. - 2. Shortly after irradiation ceases, bending becomes linear with time. On a clinostat, bending continues for about 2.5 hr. Auxanometric measurements show that the ultimate cessation of bending is not due to failing growth rate. - 3. The second positive response shows a striking dependence on intensity of irradiation. Inactivation occurs when irradiation approaches the intensity of full daylight. - 4. Induction is linear with duration of illumination, both at purely activating intensities and at partially inactivating intensities. - 5. Induction at 2°, while somewhat slower than at 25°, retains linear dependence on exposure duration. This suggests that the reactions immediately following light reception are slowed but not stopped at low temperature. - 6. Growth, which drops to about 0.5 μ/min at 2°, resumes at about 18 μ min(-1) as soon as plants are warmed to 25°. Curvature does not seem to begin for about 10 min. Combined with information about lag time for primary auxin action, this suggests that lateral auxin transport, as well as growth, is strongly inhibited at near-freezing temperatures. - 7. The induced transport system is highly stable at 2°. - 8. Under optimal conditions, the lag between onset of irradiation and induction of capacity to produce measurable curvature is only a few seconds. The length of the lag is dependent on the rate of induction. The lag is thought to be due to the requirement that enough induction be accumulated to overcome resistance of the coleoptile. - 9. Induction is dependent on the gradient of light across the coleoptile, whether measured for purely activating or partially inactivating intensities. The light received is probably integrated either across individual cells or across the entire width of the coleoptile.

A Theory for Circumnutations in Helianthus annuus

AbstractA theory is given for circumnutations in plants, especially hypocotyls of Helianthus annuus, which were used as experimental materialThe theory is based on the lateral auxin transport, which arises when a gravitational force component acts on the plant. With a suitable time delay between stimulus and response, oscillations or circumnutations should arise. It is possible to describe these oscillation phenomena by the solutions of a differential equation, derived in this paper. The time delay has a central role in this equation.The time delay is assumed to be identical with the geotropic reaction time for the hypocotyls. The ratio between the periodic time for the circumnutations and the reaction time for geotropic curvatures was found to be approximately constant in the temperature region investigated (namely 15–40°C), which supports the theory. Different methods of recording the circumnutations were used, 8 mm film camera technique being the most frequently employed.The introduction of a weighting function for describing the plants “memory” of the stimulation makes it possible to relate the periodic time of the circumnutations to the reaction time for geotropic curvatures. The necessity of this weighting function as well as of the time delay in the equations is emphasized. An explanation of the “Fünfphasen‐bewegungen”, reported in the literature, is presented.

Lateral Auxin Transport in Stems and Roots

The lateral movement of auxin has been sttudied with bioassays and also by application of '4C-IAA. Both techniques have demonstrated that the upper half of a horizontally positioned stem or root contains at least 30( to 45 % of the total auxin present in the tissue (2). However, such an auxin gradient is difficult to reconcile with the Cholodny-Weit theory of root geotropism because it is too small to account for the growth inhibition which occurs in the underside of a root experiencing a geotropic curvatture (2). We shall present evidence that the auxin differential actulally is considerably larger than these experiments indicate, and discuss several problems which have obscured this fact and hindered the measurement of lateral auixin transport. Previously we reported (3) that in 8 experiments, each using 20 sections and 1 ,um 14C-IAA, the specific activity of the upper half of horizontally positioned etiolated pea stem segments was 37.5 + 2.7 % that of the lower half. In an equal number oif experiments the specific activities of the upper quarter, middle half, and lower quarter relative to that of the intact section were 0.58 ? 0.07, 0.99 + 0.07, and 1.46 + 0.10 respectively (3). From these values the distribution of 14C-IAA across the diameter of the stem has been calculated (fig 1). The data show the specific activity to be fairly uniform throughout the middle of the stem, only rising or falling markedly at the ouiter suirfaces. Apparently each cell passes the auxin it receives to the next lower cell so that IAA is displaced from the upper to the lower surface with little change in concentration in the center of the section where most of the tissuie mass resides. Consequently when sections are halved the large differential between the upper and lower suirfaces is obscured by the lack of a gradient in the central builk of tisstue included in each sample. The magnitude of the gradient between the surfaces can be es-timated by extrapolating the curves (dotted lines, fig 1) and may be as large as 10:1. The problem of the central tissue mass can be circumvented by utilizing hollow cylinders such as Avena an(l corn coleoptiles, but this approach introduces new difficulties. It is well established that auxin does not spread laterally in these tissues exceept under the influence of gravity; otherwise the Avena and corn -curvature tests would not work. Consequently when coleoptiles are positioned horizontally, IAA should only migrate at the flanks from the upper to the lower half of the coleoptile (see fig 2). In addition a net movement of IAA from U1 to U2 and L1 to Lo would be expected, giving rise to the final distribution illtistrated in fi'gure 2. If this interpretation is correct the lower half of a split coleoptile must contain a mass of flank cells in which there is no gradient as well as a zone (,1) partially depleted of auxin, whereas the upper half shotuld contain an auxin rich zone (U,). Therefore, we believe that auxin gradients U1 :U9 an( I1 :L2 are greater than the differential measured between the upper and lower halves of sulit coleoD-

Role of Leaves in Phototropism

Experiments with green seedlings of sunflower (Helianthus annuns L.) indicate the existence of a phototropic mechanism which involves the leaves or cotyledons, and which can produce an asymmetry of auxin content without the involvement of lateral auxin transport, the classic explanation of phototropism in etiolated seedlings. The basic lines of evidence for the leaf-mediated tropism are: 1) darkening of one cotyledon will cause curvature of the stem toward the lighted cotyledon: 2) the darkened cotyledon sustains an enhanced growth rate in the stem below it: 3) conversely, light suppresses the growth-stimulating effects of a single cotyledon: and 4) more diffusible auxin is obtained from the stem below darkened cotyledons than below lighted ones.

Mediation of Phototropic Responses of Corn Coleoptiles by Lateral Transport of Auxin

Mediation of Phototropic Responses of Corn Coleoptiles by Lateral Transport of Auxin

Auxin Factor in Branch Epinasty

Until specific growth regulators were known to control the enlargement phase of the growth process in plant organs, the angular position of the plagiotropic branch could be analyzed only as the resultant of negative geotropism and an opposing epinasty. Epinasty of branches was first reported in 1872 by De Vries (3) and confirmed by Baranetzsky (1) in 1901. Rawitscher reviewed (13) subsequent studies by a few workers before 1932 but they had done little more than interpret what seemed to be an inherent property of the plagiotropic branch. The dependence of negative geotropism on a lateral transport of auxin to the lower side of a horizontal organ was demonstrated in 1930 by Dolk (4). More recently his work has been verified by Gillespie and Briggs (5), with supplementary proof supplied by Gillespie and Thimann (6) with IAA-C14. Horizontal coleoptiles of Avena develop a gradient of diffusible auxin from 35 to 40 % in the upper half to 65 to 60 % in the lower half of the organ. The mechanism for this lateral transport of auxin is still unknown but the associated geoelectric effect of Brauner (2) has recently been verified by Grahm and Hertz (8). It is logical to suspect a corresponding but reversed imbalance of auxin in lateral branches when the effect of gravity on its lateraltransport is removed by turning the plant on a horizontal clinostat, as in most demonstrations of epinastic curvatures. S6ding (17) seems to favor this basis for the resulting greater growth of the upper side of the branch, as suggested by the inconclusive evidence of Uyldert (19), and von Witsch (20) with shoots of Tradescantia and by the work of Munch (12) and of Snow (15,16) with auxin pastes. A recent note by Leike and von Guttenberg (11) describes an epinastic response of a defoliated Coleus branch to exogenous auxin applied to the disbudded tip or to the lower side of the youngest internode. This strongly suggests some lateral transport of the growth regulator to the upper side but in no work thus far has it been possible to demonstrate an excess of auxin in the upper (convex) tissues of an epinastic curvature produced in any way. This paper includes an independent confirmation and extension of the points reported by Leike and von Guttenberg, based on work done before their brief note was published. It has also been possible to measure by two methods the distribution of indoleacetic acid (IAA) within the growth zone of a lateral branch when the transport effects of gravity are eliminated.

Transport & Distribution of Auxin during Tropistic Response. I. The Lateral Migration of Auxin in Geotropism

A central tenet of the theory of tropisms is that shoots are enabled to bend toward light and away froml the earth by a migration of auxin across the stimiulated plant. Following Cholodny's proposal of such a lateral migration (12), Went in 1928 (31) shoX-ed directly that when the tips of Avena coleoptiles are illuminated from one side, more auxin diffuses out of the darker than out of the lighter side. The evidence was almost simultaneously extended to geotropism by Dolk, whose doctoral thesis (15) provi(lel a careful demonstration that when Avena coleoptile tips are held horizontal more auxin diffuses out of the lower than out of the upper side. Dolk's experiments further showed that when auxin is externally applied to horizontal sections cut from decapitated coleoptiles, it undergoes a similar asymmetric distribution. For both phototropism and geotropism the asymmetric distribution of endogenous auxin was soon confirmed by other workers, and the observations were extended both by diffusion and by extraction to dicotyledonous seedlings as well (see Went & Thimann (33) and Thimann & Curry (27) for reviews). Subsequently a large aggregate of concepts, though actually not many additional data, have crystallized around this simple notion of lateral auxin transport. In recent years, however, the lateral transport hypothesis has been seriously questioned, primarily because a number of workers failed to find any asymmetric distribution within the tissue when radioactive indoleacetic acid was applied to stimulated plant parts, and radioactivity, instead of growthpromoting activity, was the criterion. Negative results with plants placed horizontally or illuminated unilaterally were reported for more than one species by several groups of workers, notably Bunning et al. (10), Gordlon and Eib (19), Reisener (21, 22), Reisener and Simon (23), and Ching and Fang (11). On the other hand the original measurements, based on lioassay of endogenouts auxin diffusing from the basal surface of stimulated plant sections into agar blocks, have been confirmed and extended; Briggs et al. (9) and Gillespie and Briggs (16) have found clear-cut asymmetric distribution. Briggs (8) presents still more evidence for the reproducibility of these results. Much of this work has recently been reviewed by Anker (2). Such striking disagreement between two methods of measurement calls for explanation. If a plant indeed translocates its native indoleacetic acid, it must without discrimination move exogenous indoleacetic acid in the same way, providing that the applied auxin is made accessible to the transport system. It is of fundamental importance that when IAA-C14 is applied to oat coleoptile sections, the radioactivity transported out into agar blocks is indeed biologically active as auxin (18). On the other hand, several interpretations of the discrepancy may be considered. In the first place, the endogenous auxin which becomes asymmetrically distributed might not be indoleacetic acid (IAA). This is, however, made improbable by the repeated demonstrations that IAA is the auxin produced by Avena coleoptile tips (34, 20, 25) and by the fact that it does restore geotropic sensitivity to decapitated Avena coleoptile sections placed horizontally in solutions (1). As a second alternative, the material which becomes asymmetrically distributed might not be the auxin itself, but an auxin precursor or some other factor controlling the synthesis of auxin. Thirdly, it is important to note that the measurements of radioactivity have invariably been made on the tissue itself or on tissue extracts, while most of the measurements of biological activity have been made on agar blocks receiving the transported auxin. Thus gravity or light might conceivably cause the lateral migration of a factor controlling polar transport, so that although the amounts of auxin on the two sides of the plant remained the same, the amounts transported through the tissue into receiver blocks might be different. It would, indeed, be surprising if careful determinations of radioactivity in the tissue failed to reveal some change, but it is not impossible. Mechanisms of this and otlher kinds involving changes in auxin production. release, transport, or utilization on one side (without change in the anmounts present at any moment) have been postulated 1 Received Nov. 5, 1962. 2 This work was supported in part by a grant from the National Science Foundation, No. G9084, to Professor K. V. Thimann, and by predoctoral fellowships to B. Gillespie from the National Science Foundation (1959-61) and from the Public Health Service, Division of General Medical Sciences (No. CPM-8662-C1, 1961-63).

RED LIGHT, AUXIN RELATIONSHIPS, AND THE PHOTOTROPIC RESPONSES OF CORN AND OAT COLEOPTILES

Briggs, Winslow R. (Stanford U., Stanford, Calif.) Red light, auxin relationships, and the phototropic responses of corn and oat coleoptiles. Amer. Jour. Bot. 50(2): 196–207. Illus. 1963.— Red light decreases the phototropic sensitivity of corn (Zea mays Burpee ‘Golden Cross Bantam’) and oat (Avena saliva ‘Victory’) coleoptiles. The decrease is reflected by a shift of the curve ploting log dosage vs. response to higher dosages, as described in the literature. In the absence of red light treatment, 1,000 meter‐candle‐seconds (mcs) white light induces first negative curvature in oats and almost no curvature in corn, which appears to lack the mechanism for first negative curvature. Immediately following a 2‐hr red light treatment, the same white light dosage induces almost maximum first positive curvature both in corn and in oat coleoptiles. The increase in curvature obtained reflects the decreased phototropic sensitivity of both plants shown by the dosage‐response curve shift. After red treatment, the effect of red light remains maximal for an hour, decaying to the level of non‐red‐treated plants within another 2 hr. Red light suppresses auxin production by corn coleoptiles. The effect decays after the end of red treatment. Both changes follow time courses parallel to those for the phototropic sensitivity changes. The 1,000 mcs light dosage induces lateral transport of auxin both in red‐treated and untreated corn coleoptiles, despite the lack of curvature of the latter. Red light does not induce a circadian rhythm for the phototropic sensitivity changes in oats, is not effective if administered after phototropic induction, and its effect is probably mediated by phytochrome. The hypothesis, not original with this paper, that red light induces an increase in the amount of pigment mediating second positive curvature most closely accounts for the results obtained. Pertinent literature is discussed.

Mediation of geotropic response by lateral transport of auxin

A suggestion concerning the mechanism of geotropic response was offered independently by Cholodny in 1924 (12) and by Went in 1926 (27). It was known by this time that a growth promoting hormone (an auxin) is produced in tips of coleoptiles and seedlings and miioves in a continual streamii toward their bases. Cholodny and \Went postulated that auxin moves also from the upper to the lower side when a shoot is placed horizontally. As a result, the growth rates of the upper and lower halves would decrease and increase, respectively, and the shoot would curve upward. Tn 1927 Cholodny (13) generalized his hypothesis to cover the phototropic response, and in 1928 Went (28), again independently, arrived at the same explanation. The concept of hormonal coordination of plant tropisms has come to be called the Cholodny-Went Theory, in honor of both men. The development of the theory has been reviewed by Went and Thimann (29). Several workers performedl experiments which demonstrate that geotropic curvature of a shoot is the result of a decrease in growth of the uoper lalf accompanied by an increase in growth of the lower half. Among these, the careful measurements of Navez and Robinson (20) on the Avena coleoptile seem particularly reliable. Direct studies of auxin dlistribution followving phototropic induction were first performed by Went in 1928 (28). He placed agar blocks basipetal to each half of a coleoptile tip which had been longitudlinally split at the base. He illuminated the coleoptile unilaterally, perpendicular to the split, and, after a suitable lelngth of time, measured the relative amounts of auxin in the two blocks by means of the Avena test. He found that the illuminated side released much less auxin than did the shaded side. Shortly afterward, Dolk (15) performed similar experiments on both Avena sativia and Zca niays coleoptiles which had been induced by gravity. More auxin was obtained from halves which had been lowermost than from those whiclh lhad been uppermost, while the total auxin yield of each organ was unaffected by stimulation. A number of workers repeated Dolk's experiment with other plant organs, and found similar distributions of auxin betweeni the top and bottom halves (4, 14, 19, 20, 25). Other workers obtained similar distributions when auxin was obtained by extraction (5, 26). Buinning et al (9), Gor(lon and Eib (18). Reisener (21), Reisener and Sinmon (22), and Ching and Fang (10), applying carbon 14-labeled indoleacetic acid to oat coleoptiles and to other plant parts, have recently been unable to demonstrate lateral mlovement of the labeled hormone (luring or following either geotropic or phototropic stimulation. Numerous other vorkers have expressed lack of confidence that lateral auxin transport occurs (1, 2, 3, 5, 11, 23). and some have proposed other mechanisms to explain the differential growth of the two sides (2, 7, 9). In view of the recent experiments of Briggs et al (8) suggesting that lateral transport of auxin may indeed mediate the phototropic response of coleoptiles of Zea ways to certain dosages of unilateral light, it seemed wortlh while to repeat these experiments, using gravity instea(d of light as the stimulus.

Electrical and Curvature Responses of the Avena Coleoptile to Unilateral Illumination

One fact that is agreed upon in the literature regarding the mechanism of phototropism is that the growth curvature results from an unequal distribution of auxin. There are at least three hypotheses as to how unilateral illumination causes the unequal distribution: 1. Photoinactivation of auxin on the lighted side of the plant. 2. A differential change in the rate of synthesis of auxin on the lighted and shaded sides. 3. Unilateral displacement of auxin toward one of the sides. It is the last of these mechanisms with which this paper will be primarily concerned. If lateral transport of auxin serves as a mechanism for phototropism, it requires a transverse polarity which is induced by the tropic stimulation. This is essentially a statement of the familiar Cholodny-Went theory, which applies to other tropisms as well as phototropism. One logical possibility is that such a transverse polarity is electrical in nature. Because of this possibility, the transverse electrical changes which occur in the Avena coleoptile as a result of most of the tropic stimuli have been established. The results of these studies have been reviewed recently by Schrank (10). Apparently only two investigators, (Oppenoorth, 6 Schrank 8), have attempted to measure the transverse electrical changes resulting from stimulation of the Avena coleoptile by unilateral illumination. Oppenoorth's findings (?) indicate that the lighted side of the Avena coleoptile becomes electropositive to the shaded side when the plant is unilaterally illuminated with a dosage of light which causes positive phototropic curvature (500 mes). Schrank (8), on the other hand, measured the changes in the transverse electrical polarity in the apex of the Avena coleoptile resulting from continuous unilateral illumination and found that the lighted side became electronegative to the shaded side. It is the purpose of this investigation to make additional measurements of the electrical changes of the Avena coleoptile when it is stimulated by a dosage of light which causes positive phototropic bending and to observe the electrical changes resulting from illumination which causes negative bending. If the transverse electrical polarity indeed serves as a polar force which directs the auxin to the side of the coleoptile which becomes convex, the orientation of the light induced polarities in these two instances should be opposite.

A Photokymograph for the Analysis of the Avena Test

1. With an automatic photokymograph, the procedure of curvature in the Avena test was investigated. In general, when auxin was applied unilaterally there was little or no effect for about 20 minutes, after which curvature set in and continued at a constant rate for about an hour, then diminished or reversed, depending on the treatment. 2. It was found that increasing the auxin concentration inside the plant decreased the sensitivity to unilaterally applied auxin. This explains the increase in sensitivity after decapitation, since the auxin content decreases after removal of the tip. 3. To study this process in detail, the effect of the i.d.p. (length of interval between decapitation and putting on of the agar block) and the number of decapitations was followed for different concentrations of auxin. 4. For all concentrations up to maximum angle concentration, the sensitivity increased rapidly for the first 30 minutes i.d.p., increased gradually until 200-240 minutes i.d.p., then gradually decreased. 5. But the maximum angle concentration fell off rapidly for the first 100 minutes i.d.p. 6. For i.d.p. longer than 100 minutes, a second decapitation just before putting on causes: (a) the reaction to maximum angle concentration to be doubled; (b) the reaction to extremely low concentration to be slightly decreased, even to the extent of sometimes giving small positive angles; (c) more uniform reactions. 7. A modified standard Avena test is recommended: (a) an i.d.p. of 3-4 hours; (b) a second decapitation 20-40 minutes before putting on; (c) photographs at 90 minutes after putting on. 8. Besides the regeneration effect which reverses the curvatures at 130 minutes after decapitation for i.d.p. of less than 50 minutes, there is also a pseudo-regeneration effect for longer i.d.p. which also balances or reverses curvature at 80-90 minutes after putting on. 9. This effect, and certain others (decreases of maximum angle concentration with increasing i.d.p., and positive angles if low concentration auxin-agar blocks are put on soon after decapitation) cannot be explained by geotropism, lateral auxin transport, or aging. Thus it is tentatively proposed that they may be due to food factor distribution.