Gravitational compensation and the phototropic response of oat coleoptiles

Avena seedlings were germinated and grown while continuously rotated on the horizontal axis of a clinostat. The coleoptiles of these gravity-compensated plants were phototropically more responsive than those of plants rotated on a vertical axis. When the plants were compensated after unilateral irradiation, phototropic curvature of the shoot progressed for the next 6 hours, with the rate of curving decreasing about 3 hours after irradiation. The decrease in rate was less in the plants gravity-compensated before irradiation than in those vertically rotated. In the period 70 to 76 hours after planting, the growth rate of the compensated coleoptiles was significantly less than that of the vertically rotated seedlings. The greater phototropic curvature, the decreased growth rate, and the slower rate of straightening of the curved, compensated shoot can be correlated with several consequences of compensation: an increase in sensitivity to auxin, a lowering of auxin content in the coleoptile tip, and possibly, from an interaction between compensation and phototropic stimulation, an enhanced difference in auxin transport between the illuminated and shaded halves of the unilaterally irradiated shoot.

The phototropic response of the vertically rotated seedling was significantly different from that of the vertical stationary, indicating the importance of vertically rotated controls in clinostat experiments.

     

Simulated Low-gravity Environments and Respiratory Metabolism in Avena Seedlings

Using horizontal and vertical axis clinostats and sand-grown oat seedlings (Avena sativa), it was found that horizontal clinostat rotation at 2 rpm increased respiration and inorganic and organic phosphorus content of seedlings. Increased coleoptile geotropism and root growth are attributed to rotational nullification of the directional component of the gravitational stimulus. These growth modifications are mechanistically explicable by the relationship between plant metabolism and auxin concentration in these organs.     

Role of calcium in the polar secretion of indoleacetic Acid

The rate of auxin transport in sunflower hypocotyls (Helianthus annuus L. cv `Russian mammoth') or corn coleoptiles (Zea mays L. cv `WF9 × 38') was less in seedlings grown in Ca-deficient medium than in controls. The rate of IAA transport depended on the concentration of Ca in the root medium up to 1 millimolar. Further increases in auxin transport were observed when the isolated segments were incubated in medium containing up to 30 millimolar Ca. We suggest that the rate of auxin transport in plant tissue is dependent on the pool of ionic Ca in the extracellular space.

Segments from Ca-deficient seedlings exhibited a high specific requirement for Ca²⁺ in auxin transport. Magnesium, strontium, and several other divalent cations tested for their ability to replace Ca²⁺ in restoring auxin transport showed no effect; partial replacement by lanthanum was observed.

Auxin transport, or auxin flux through the segment, which is the result of IAA secretion by individual cells, was reduced in the low Ca²⁺ segments due both to lowered velocity and to reduced capacity of transport. The requirement for Ca²⁺ in the secretion of auxin is believed to be equivalent to the phenomenon observed in animal cell secretion, where the influx of Ca²⁺ serves as a link between an external stimulus and the secretion response.

     

Transport and Degradation of Auxin in Relation to Geotropism in Roots of Phaseolus vulgaris

The movement of auxin in Phaseolus vulgaris roots has been examined after injection of IAA^?3H into the basal root/hypocotyl region of intact, dark-grown seedlings. Only a portion of the applied IAA^?3H was transported unchanged to the root tip. The major part of the chromatographed, labelled compounds translocated to the roots was indole-3-acetylaspartic acid (IAAsp) and an unidentified compound running near the front in isopropanol, ammonia, water. The velocity of the auxin transport (7.2 mm per hour) was calculated from scintillation countings of methanol extracts from serial sections of the root. An accumulation of radioactive compounds in the extreme root tip, was observed 5 h after the injection of IAA. The influence of exogenous IAA on the geotropical behaviour of the bean seedling roots was examined. Pretreated roots were stimulated for 5 min in the horizontal position and then rotated parallel to the horizontal axis of the klinostat for 60 or 90 min. The resulting geotropic curvature of IAA-injected and control roots showed significantly different patterns of development. When the stimulation was started 5 h after application of the auxin, the geotropic curvature became larger in roots of the injected plants than in the controls. If, however, the translocation period was extended to 20 h the geotropic curvature was significantly smaller in the roots of the injected plants. The auxin injection did not significally affect the rate of root elongation. The change in geotropical behaviour of the roots is interpreted as a result of the influence of the conversion products of the applied IAA on the geotropical responsiveness.     

Plant growth processes in Arabidopsis under microgravity conditions simulated by a clinostat.

The life cycle of Arabidopsis plants was examined by growing them on a horizontal clinostat. Seeds on agar media were allowed to germinate and seedlings were grown under a simulated microgravity on a horizontal clinostat. Clinorotation (3 rpm) did not appear to interfere with germination of plant seeds and development of cotyledons and leaves. Stress relaxation parameters of the cell wall, the minimum relaxation time and the relaxation rate did not appear to be affected by clinostat rotation. On the other hand, the length of inflorescences was reduced to 61-62% by clinostat rotation. Rotation was found to inhibit the polar transport of auxin, although inflorescence growth and auxin transport were not completely inhibited. From these facts, it is possible that the life cycle in Arabidopsis plants could be accomplished in space, although growth phenomena involving auxin transport and its action may be disturbed. Plants may have a capacity to grow in space and we may be able to cultivate crops in space.     

Effect of Transverse Gravity Stimulation, Gibberellin and Indoleacetie Acid upon Polar Transport of IAAC14 in the Stem of Helianthus annuus

Using IAA^C14, polar transport has been studied in Helianthus annuus shoots in which stem elongation was inhibited by a transverse gravity stimulus induced by horizontal orientation of the plant with daily rotation. Marked inhibition of polar transport of IAA ^C14 occurred in the treated plants. A similar degree of inhibition occurred in the upper and lower halves of non-rotated horizontally trained shoots. Horizontal orientation of stem segments during the transport test had no consistent effect upon IAA transport. Pretreatment of erect plants with gibberellin greatly enhanced IAA^C14 transport and also reduced the inhibitory effect of horizontal orientation. Pretreatment of erect plants with non-radioactive IAA or ethylene inhibited transport of IAA ^C14 and induced the same symptoms in the shoot as the transverse gravity stimulus. The similarities between the response of the auxin transport system to gravity stimulation, IAA and ethylene are discussed.     

Disruption of the Polar Auxin Transport System in Cotton Seedlings following Treatment with the Defoliant Thidiazuron

The effect of the defoliant thidiazuron (TDZ) on basipetal auxin transport in petiole segments isolated from cotton (Gossypium hirsutum L. cv LG102) seedlings was examined using the donor/receiver agar block technique. Treatment of intact seedlings with TDZ at concentrations of 1 micromolar or greater resulted in a dose-dependent inhibition of ¹⁴C-IAA transport in petiole segments isolated 1 or 2 days after treatment. Using 100 micromolar TDZ, the inhibition was detectable 19 hours after treatment and was complete by 27 hours. Both leaves and petiole segments exhibited a marked increase in ethylene production following treatment with TDZ at concentrations of 0.1 micromolar or greater. The involvement of ethylene in this TDZ response was evaluated by examining the effects of two inhibitors of ethylene action: silver thiosulfate, 2,5-norbornadiene. One day after treatment, both inhibitors effectively antagonized the TDZ-induced inhibition of auxin transport. Two days after TDZ treatment both inhibitors were ineffective. The decrease in IAA transport in TDZ treated tissues was associated with increased metabolism of IAA. The transport of ¹⁴C-2,4-dichlorophenoxyacetic acid was also inhibited by TDZ treatment. This inhibition was not accompanied by increased metabolism. Incorporation of TDZ into the receiver blocks had no effect on auxin transport. The ability of the phytotropin N-1-naphthylphthalamic acid to stimulate IAA uptake from a bathing medium was reduced in TDZ-treated tissues. This reduction is thought to reflect a decline in the auxin efflux system following TDZ treatment.     

Effects of Cations on Hormone Transport in Primary Roots of Zea mays

We examined the influence of aluminum and calcium (and certain other cations) on hormone transport in corn roots. When aluminum was applied unilaterally to the caps of 15 mm apical root sections the roots curved strongly away from the aluminum. When aluminum was applied unilaterally to the cap and ³H-indole-3-acetic acid was applied to the basal cut surface twice as much radioactivity (assumed to be IAA) accumulated on the concave side of the curved root as on the convex side. Auxin transport in the apical region of intact roots was preferentially basipetal, with a polarity (basipetal transport divided by acropetal transport) of 6.3. In decapped 5 mm apical root segments, auxin transport was acropetally polar (polarity = 0.63). Application of aluminum to the root cap strongly promoted acropetal transport of auxin reducing polarity from 6.3 to 2.1. Application of calcium to the root cap enhanced basipetal movement of auxin, increasing polarity from 6.3 to 7.6. Application of the calcium chelator, ethylene-glycol-bis-(β-aminoethylether)-N,N,N′, N′-tetraacetic acid, greatly decreased basipetal auxin movement, reducing polarity from 6.3 to 3.7. Transport of label after application of tritiated abscisic acid showed no polarity and was not affected by calcium or aluminum. The results indicate that the root cap is particularly important in maintaining basipetal polarity of auxin transport in primary roots of corn. The induction of root curvature by unilateral application of aluminum or calcium to root caps is likely to result from localized effects of these ions on auxin transport. The findings are discussed relative to the possible role of calcium redistribution in the gravitropic curvature of roots and the possibility of calmodulin involvement in the action of calcium and aluminum on auxin transport.     

Endogenous gibberellin transport and biosynthesis in relation to geotropic induction of excised sunflower shoot-tips

Summary Surgical experiments on Helianthus annuus and Phaseolus multiflorus seedlings involving the application of auxin and gibberellin to decapitated plants, suggested that internode extension growth occurs under the controlling influence of apically synthesised gibberellin rather than auxin. Studies were made of diffusible gibberellins from sunflower apical buds in relation to geotropic stimulation. Approximately ten times as much gibberellin was obtained from lower than from upper tissues of horizontal shoot-tips, whereas approximately equal quantities were obtained from the two halves of upright tips. Evidence was obtained suggesting both lateral transport of gibberellin in the young internode, and also enhanced gibberellin synthesis in buds maintained in a horizontal position during the collection of diffusible gibberellins into agar. The results are discussed in relation to current concepts of the role of auxin in geotropism.     

Gravity-controlled asymmetrical transport of auxin regulates a gravitropic response in the early growth stage of etiolated pea (Pisum sativum) epicotyls: studies using simulated microgravity conditions on a three-dimensional clinostat and using an agravitropic mutant, ageotropum

Increased expression of the auxin-inducible gene PsIAA4/5 was observed in the elongated side of epicotyls in early growth stages of etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown in a horizontal or an inclined position under 1 g conditions. Under simulated microgravity conditions on a 3D clinostat, accumulation of PsIAA4/5 mRNA was found throughout epicotyls showing automorphosis. Polar auxin transport in the proximal side of epicotyls changed when the seedlings were grown in a horizontal or an inclined position under 1 g conditions, but that under clinorotation did not, regardless of the direction of seed setting. Accumulation of PsPIN1 and PsPIN2 mRNAs in epicotyls was affected by gravistimulation, but not by clinorotation. Under 1 g conditions, auxin-transport inhibitors made epicotyls of seedlings grown in a horizontal or inclined position grow toward the proximal direction to cotyledons. These inhibitors led to epicotyl bending toward the cotyledons in seedlings grown in an inclined position under clinorotation. Polar auxin transport, as well as growth direction, of epicotyls of the agravitropic mutant ageotropum did not respond to various gravistimulation. These results suggest that alteration of polar auxin transport in the proximal side of epicotyls regulates the graviresponse of pea epicotyls.     

Orientation of wheat seedling organs in relation to gravity

Seedlings of wheat (Triticum aestivum L.) were grown in special holders that permitted the coleoptile and early roots to develop in moist air. The orientation of the organs of seedlings erect to gravity was compared with that of organs produced on a horizontal clinostat. Orientation was described by the angular position of each organ tip with reference to the axis of the embryo. Comparative tests were also made with barley, rye, and oat seedlings.

The coleoptile of all species developed curvatures in 3 dimensions when geotropic responses were eliminated. The primary root was not precise in its positive geotropism. Seedlings grew on clinostats with much greater variations in the lateral orientation of the central root and with a tendency for it to curve away from the endosperm to a greater degree than in erect seedlings.

The symmetry of root system in wheat was found to depend on a specific mechanism. Under the influence of gravity the earliest lateral roots were oriented in a plane at characteristic angles of about 57.5° with the ideal primary root. The corresponding angles for lateral roots growing on clinostats were greater by about 47.5° as a result of epinasty not previously reported in roots. This force also appeared to be active in the seminal roots of barley and rye but not of oats.

The curvatures in coleoptiles grown without the directional effects of gravity correspond to the results of growth imbalance in Coleus stems in the absence of lateral transport of their auxin by gravity. Root epinasty appears to be based on auxin imbalance. Curvatures in the primary root are also interpreted as results of asymmetrical distribution of growth hormone.

     

Dim-Red-Light-Induced Increase in Polar Auxin Transport in Cucumber Seedlings : I. Development of Altered Capacity, Velocity, and Response to Inhibitors

We have developed and characterized a system to analyze light effects on auxin transport independent of photosynthetic effects. Polar transport of [³H]indole-3-acetic acid through hypocotyl segments from etiolated cucumber (Cucumis sativus L.) seedlings was increased in seedlings grown in dim-red light (DRL) (0.5 μmol m⁻² s⁻¹) relative to seedlings grown in darkness. Both transport velocity and transport intensity (export rate) were increased by at least a factor of 2. Tissue formed in DRL completely acquired the higher transport capacity within 50 h, but tissue already differentiated in darkness acquired only a partial increase in transport capacity within 50 h of DRL, indicating a developmental window for light induction of commitment to changes in auxin transport. This light-induced change probably manifests itself by alteration of function of the auxin efflux carrier, as revealed using specific transport inhibitors. Relative to dark controls, DRL-grown seedlings were differentially less sensitive to two inhibitors of polar auxin transport, N-(naphth-1-yl) phthalamic acid and 2,3,5-triiodobenzoic acid. On the basis of these data, we propose that the auxin efflux carrier is a key target of light regulation during photomorphogenesis.     

Requirement for the gravity-controlled transport of auxin for a negative gravitropic response of epicotyls in the early growth stage of etiolated pea seedlings

Gravity-controlled transport of auxin was studied for a negative gravitropic response in the early growth stage of etiolated pea (Pisum sativum L. cv. Alaska) seedlings, in which epicotyl bending was observed near the cotyledon nodes of the seedlings grown continuously from seeds germinated in a horizontal or an inclined position. Increased expression of an auxin-inducible gene, PsIAA4/5, was observed in the elongated side of epicotyls grown in a horizontal or an inclined position. Regardless of the conditions of seed germination, polar auxin transport in the proximal side of the first internodes of the seedlings was significantly higher than in the distal side. Polar auxin transport in the proximal side of epicotyls grown in an inclined position was significantly lower than in those grown in a horizontal position. In contrast, lateral auxin distribution from the proximal to distal sides in epicotyls grown in an inclined position was significantly higher than in epicotyls grown in a horizontal position. Accumulation of PsPIN1 mRNA encoding a putative auxin efflux facilitator, which was observed in vascular tissue, cortex and epidermis in the proximal and distal sides of epicotyls, was markedly influenced by gravistimulation. These results strongly suggest that gravistimulation induces changeable polar auxin transport and one-way lateral auxin distribution in epicotyls as well as asymmetric auxin accumulation in the proximal and distal sides of epicotyls, resulting in a negative gravitropic response of epicotyls in the early growth stage of pea seedlings.     

Increase in the gibberellin response by low temperature pretreatment of azuki bean epicotyls

Epicotyl segments cut from azuki bean (Vigna angularis) seedlings grown at 27 C for 5 days and then treated at 15 C for 2 days were compared with those cut from seedlings not receiving the 15-C treatment, for their response to auxin and auxin plus gibberellin. The segments cut from 15C-treated seedlings showed greater elongation than the segments cut from untreated seedlings both in the absence and in the presence of gibberellin. The 15-C treatment increased the elongation caused by auxin plus gibberellin to a greater extent than it did the elongation caused by auxin alone; the gibberellin response of epicotyl was increased by the treatment. Diffusates from leaves and buds inhibited both the elongation caused by auxin and that caused by auxin plus gibberellin. The diffusates inhibited the latter more strongly than the former; the gibberellin response was decreased by the diffusates. Leaves or buds appear to supply epicotyls with a substance which decreases the gibberellin response. The supply of this substance was found to be temperature dependent. The diffusates obtained at 15 C caused no inhibition, while those obtained at 27 C decreased the gibberellin response. The lack of the supply of this substance at 15 C may account for the increase in the gibberellin response by the 15-C treatment.     

Auxin and Ethylene Control of Growth in Seedlings of Zea mays L. and Avena sativa L

The effects of applied ethylene on the growth of coleoptilesand mesocotyls of etiolated monocot seedlings (oat and maize)have been compared with those on the epicotyl of a dicot seedling(the etiolated pea). Significant inhibition of elongation by ethylene (10 µll^–1for 24 h) was found in intact seedlings of all three species,but lateral expansion growth was observed only in the pea internodeand oat mesocotyl tissue. The sensitivity of the growth of seedlingparts to ethylene is in the decreasing order pea internode,oat coleoptile and oat mesocotyl, with maize exhibiting theleast growth response. Although excised segments of mesocotyland coleoptile or pea internode all exhibit enhanced elongationgrowth in IAA solutions (10^–6–2 ? 10^–5 moll^–1), no consistent effects were found in ethylene. Ethyleneproduction in segments was significantly enhanced by applicationof auxin (IAA, 10^–5 mol l^–6 or less) in all tissuesexcept those of the eat mesocotyl. Segments of maize show a slow rate of metabolism of applied[2-¹⁴C]IAA (30 per cent converted to other metabolites within9 h) and a high capacity for polar auxin transport. Ethylene(10 µl l^–1 for 24 h) has little effect on eitherof these processes. The oat has a smaller capacity for polartransport than maize and the rate ef metabolism of auxin isas fast as in the pea (90 per cent metabolized in 6 h). Althoughethylene pretreatment does not change the rate of auxin metabolismin oat, there is a marked reduction in auxin transport. It is proposed that the insensitivity of maize seedlings toethylene is related to the supply and persistence of auxin whichcould protect the seedling against the effects of applied orendogenously produced ethylene. Although the mesocotyl of oatis sensitive to applied ethylene it may be in part protectedagainst ethylene in vivo by the absence of an auxin-enhancedethylene production system. The results are discussed in relationto a model for the auxin and ethylene control of cell growthin the pea.     

Defective long-distance auxin transport regulation in the Medicago truncatula super numeric nodules mutant

Long-distance auxin transport was examined in Medicago truncatula and in its supernodulating mutant sunn (super numeric nodules) to investigate the regulation of auxin transport during autoregulation of nodulation (AON). A method was developed to monitor the transport of auxin from the shoot to the root in whole seedlings. Subsequently, the transport was monitored after inoculation of roots with the nodulating symbiont Sinorhizobium meliloti. The sunn mutant showed an increased amount of auxin transported from the shoot to the root compared to the wild type. The auxin transport capacity of excised root segments was similar in wild type and sunn, suggesting that the difference in long-distance auxin transfer between them is due to loading in the shoot. After inoculation, wild-type seedlings showed decreased auxin loading from the shoot to the root; however, the sunn mutant failed to reduce the amount of auxin loaded. The time of reduced auxin loading correlated with the onset of AON. Quantification of endogenous auxin levels at the site of nodule initiation showed that sunn contained three times more auxin than wild type. Inoculation of sunn failed to reduce the level of auxin within 24 h, as was observed in the wild type. We propose a model for the role of auxin during AON of indeterminate legumes: 1) high levels of endogenous auxin are correlated with increased numbers of nodules, 2) inoculation of roots reduces auxin loading from the shoot to the root, and 3) subsequent reduction of auxin levels in the root inhibits further nodule initiation.     

Adventitious root formation in relation to irradiance and auxin supply

High irradiance during treatment of mung bean cuttings favours root formation in response to supplied auxin, whether the latter is IAA or IBA. On the other hand it is inhibitory towards root formation in the absence of supplied auxin. Light promotes the uptake of¹⁴C-IAA into cuttings and its upward movement into the leaves. When¹⁴C-IAA is applied to leaves of cuttings high irradiance favours movement of radioactivity into the epicotyl and hypocotyl. This movement is also enhanced by concomitant supply of IBA to the base of the cuttings. The irradiance under which stock plants are raised also affects the extent of root formation on cuttings. When cuttings are held in darkness without a supply of exogenous auxin they root best if prepared from seedlings raised under high irradiance. However, transport of¹⁴C-IAA out of leaves of cuttings is favoured when cuttings are prepared from seedlings grown under low irradiance. These observations are discussed in relation to auxin transport, photodestruction and, possibly, metabolism.     

The effects of electric field on the growth of intact seedlings and coleoptile segments ofZea mays L.

The experiments were carried out with 96-h-old intact maize seedlings and 10 mm long coleoptile segments cut 4 mm below the tip. The electric fields were applied longitudinally along the seedlings. The electric field (15 V) caused inhibition of the elongation growth of intact seedlings which was dependent on both the polarity and the duration of the applied voltage. The growth inhibition was greater when the tip of the shoot was positive relative to the roots. The electric field also caused inhibition of indole-3-acetic acid (IAA) and fusicoccin (FC) induced growth of maize coleoptile segments excised from electrically treated seedlings. IAA-induced growth of coleoptile segments was greater when the tip of the shoot was negative to the roots (not in the case of FC-treated segments and intact seedlings). It was suggested that apart from the changes induced by electric field in transport system of auxin the electric field affected also the activity of plasmalemma proton pump.     

Polar transport of kinetin in tissues of radish

Polar transport of kinetin-8-¹⁴C occurred in segments of petioles, hypocotyls, and roots of radish (Raphanus sativus L.). The polarity was basipetal in petioles and hypocotyls and acropetal in roots. In segments excised from seedlings with fully expanded cotyledons, indole-3-acetic acid was required for polarity to develop. In hypocotyl segments isolated at this stage, basipetal and acropetal movements were equal during the first 12 hours of auxin treatment after which time acropetal movement declined. Pretreatment with auxin eliminated this delay in the appearance of polarity. In hypocotyl segments excised from seedlings with expanding cotyledons, exogenous auxin was unnecessary for polarity. Potassium cyanide abolished polarity at both stages of growth by allowing increased acropetal movement. The rate of accumulation of kinetin in receiver blocks was greater than the in vivo increase in cytokinin content of developing radish roots.     

Boron and calcium sites involved in indole-3-acetic Acid transport in sunflower hypocotyl segments

Sunflower (Helianthus annuus L. cv Russian Mammoth) hypocotyl segments deficient in either B or Ca exhibited a higher rate of potassium leakage, compared to nondeficient segments. Potassium leakage, used here as an indication of membrane integrity, was completely reversed by the addition of H₃BO₃ or Ca(NO₃)₂ to the incubation medium of the B-deficient or Ca-deficient hypocotyl segments, respectively. This role of B and Ca in membrane integrity, which may be important in the entry and exit of auxin in cells, is identified as the first site of action for each of these two essential elements in the basipetal secretion of auxin. A second site for B is postulated because auxin transport was not restored, even when K⁺ leakage has been completely reversed to the nondeficient level, when B-deficient hypocotyls were incubated in B solution. This lack of reversibility of auxin transport implied that the incubation for 2 h in B solution was not enough to restore the auxin transport process. However, since the transfer of B-deficient seedlings to B solutions prevented further deterioration of auxin transport, these observations suggest that: (a) either an intact seedling, or a longer period of incubation of the hypocotyl in B solution, is required for the synthesis or maintenance of the functional second site for B; (b) B is probably essential in the synthesis of a ligand, which may or may not be needed to bind B, but which is essential in the basipetal transport of auxin. The second site for Ca in auxin transport, is indicated by the complete reversal of its inhibition in Ca-deficient hypocotyl, when incubated in Ca solution. The second site for Ca is thought to be directly involved in the secretion of auxin, in which Ca probably plays the role of a second messenger, as in stimulus-response coupling. The two sites for Ca can be distinguished from each other by their cation specificity. The requirement for Ca in the first site can be substituted by other divalent cations, while the second site is highly specific for Ca.