Applied indol-3yl-acetic acid on the cap and auxin movements in gravireacting maize roots.

The graviresponsiveness of intact and primary maize roots kept horizontally in darkness and humid air is analysed. A precise local application of IAA is possible when using resin beads (diameter: 0.45 +/- 0.05 mm) loaded with IAA. The beads are placed on the upper or lower sides of the caps. They significantly change the root gravireaction. The effect of IAA is discussed in terms of its possible level in the growing and gravibending zones and its transport (acropetal, lateral and basipetal) respectively in the stele, the cap and the cortex of the elongating root.

Simultaneous gas chromatography-mass spectrometry quantification of endogenous [C]- and applied [C]indole-3yl-acetic Acid levels in growing maize roots.

The use of stable indole-3yl-acetic acid (IAA) labeled by 6 atoms of (13)C allowed, after [(13)C]IAA treatment, simultaneous gas chromatography-mass spectrometry quantifications of both endogenous [(12)C]IAA and applied [(13)C]IAA levels in Zea mays L. roots. Root material was immersed for 1 hour in a buffered (pH 6.0) solution without or with [(13)C]IAA at 10(-7) molar. Both applied and endogenous IAA were thus measured for three zones of the roots (apical, elongating, differentiating) directly after treatment and also 2 hours later. Growth was followed over a 4 hour period. Roots not immersed elongated more than control roots (immersed in buffer), which grew more than IAA-treated roots. Immersion in buffer induced a large decrease (-68%) of [(12)C]IAA in the apical part of control roots, whereas immersion in [(13)C]IAA prevented most of it. No significant difference between control and treated roots occurred in the two other zones. Two hours after treatment, [(13)C]IAA had completely disappeared from the elongating zone even though [(12)C]IAA level was essentially stable. A direct relationship occurred between the level of IAA in the elongating zone and the growth of the root. This relationship was strongly disturbed if unmetabolized [(13)C]IAA was present. However, the relationship returned to its initial state when significant amounts of free [(13)C]IAA were no longer detectable. These results are discussed in terms of the stability of both types of compounds and the utility of the method of using stable isotopes of hormones, for the understanding of hormonal regulation of plant growth.

Local treatment with indole-3-acetic acid induces differential growth responses in Zea mays L. roots.

Differential growth responses of primary Zea mays L. roots were induced after asymmetrical applications of IAA-loaded beads along their elongating zone (1.0-4.0 mm from tip) and were modulated according to the initial growth rates of the roots. The amplitude and location (midpoint) of curvature, the elongation of the roots and the uptake of IAA were measured after 4 h. The amount of curvature depended on the location of the bead and upon the amount of IAA taken up. Curvature occurred towards the site of IAA application and the zone (midpoint) of curvature was always found to be basipetal to the IAA-loaded bead, indicating the transport of a growth signal in a basipetal direction from the zone of IAA application towards the zone of curvature. Two regions within the elongation zone were thus characterized: an acropetal region (between 1.0-3.5 mm from the tip) in which IAA induced a growth signal and a basipetal region (3.5-6.5 mm from the tip) which displayed the growth response; neither of these regions were able to carry out both physiological functions. The acropetal region was extended in fast-growing roots or when the amount of applied IAA was increased. No significant growth stimulation of fast-growing roots was induced regardless of the amount of IAA supplied, but the growth of slow-growing roots was stimulated with very low amounts of IAA ([Symbol: see text]100 pg/root after 4 h). Inhibition of growth of both classes of roots increased with amounts of IAA between 100-600 pg/root after 4 h, but no further inhibition of growth appeared after treatment with greater amounts of IAA. A model based on these data shows a possible relationship between the uptake of IAA and root growth and indicates a clear relationship between an optimal level of IAA and root growth. The responses of the elongating cells of primary maize roots to applied IAA depend on their developmental stage and on the initial growth rates of the root.

In vitro oxidation of indoleacetic Acid by soluble auxin-oxidases and peroxidases from maize roots.

Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl(2) and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [(3)H] indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol.

Target Molecular Size and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis of the ATP-and Pyrophosphate-Dependent Proton Pumps from Maize Root Tonoplast.

Tonoplast-enriched membranes were prepared from maize (Zea mays L. cv LG 11) primary roots, using sucrose nonlinear gradients. The functional molecular size of the tonoplast ATP-and PPi-dependent proton pumps were analyzed by radiation inactivation. Glucose-6-phosphate dehydrogenase (G6PDH) was added as an internal standard. Frozen samples (-196 degrees C) of the membranes were irradiated with (60)Co for different periods of time. After thawing the samples, the activities of G6PDH, ATPase, and PPase were tested. By applying target theory, the functional sizes of the ATPase and PPase in situ were found to be around 540 and 160 kilodaltons, respectively. The two activities were solubilized and separated by gel filtration chromatography. The different polypeptides copurifying with the two pumps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands (around 59 and 65 kilodaltons) were associated with the ATPase activity, whereas a double band (around 40 kilodaltons) was recovered with the PPase activity.

Endogenous ABA in Growing Maize Roots: Light Effects.

The growth of intact maize (Zea mays L.) roots and the abscisic acid (ABA) content (measured by gas chromatography-mass spectrometry of the root tip were analyzed after a white-light treatment. The decrease of the elongation rate due to the illumination corresponded to a concomitant increase in the ABA found in the root. When selecting roots, on the basis of their growth rate, it was possible to show that the relation between growth and ABA content, previously reported in darkness was conserved after light treatments. Therefore, light decreased the root growth rate while it simultaneously increased the ABA content in the roots. This increase was higher than expected, demonstrating the complexity of the involvement of ABA on root growth.

Differential growth and hormone redistribution in gravireacting maize roots.

When growing roots are placed in a horizontal position gravity induces a positive curvature. It is classically considered to be the consequence of a faster elongation rate by the upper side compared to the lower side. A critical examination indicates that the gravireaction is caused by differential cell extension depending on several processes. Some of the endogenous regulators which may control the growth and gravitropism of elongating roots are briefly presented. The growth inhibitors produced or released from the root cap move preferentially in a basipetal direction and accumulate in the lower side of the elongation zone of horizontally maintained roots. The identity of these compounds is far from clear, but one of these inhibitors could be abscisic acid (ABA). However, indol-3y1 acetic acid (IAA) is also important for root growth and gravitropism. ABA may interact with IAA. Two other aspects of root cell extension have also to be carefully considered. An elongation gradient measured from the tip to the base of the root was found to be important for the growth of both vertical and horizontal gravireactive roots. It was changed significantly during the gravipresentation and can be considered as the origin of the differential elongation. Sephadex beads have been used as both growth markers and as monitors of surface pH changes when they contain some pH indicator. This technique has shown that the distribution of cell extension along the main root axis is related to a pH gradient, the proton efflux being larger for faster growing parts of roots. A lateral movement of calcium is obtained when Ca2+ is applied across the tips of horizontally placed roots with a preferential transport towards the lower side. Endogenous calcium, which may accumulate inside the endoplasmic reticulum of some cap cells, may also act in the gravireception. These observations and several others strongly suggest that calcium may play an essential role in controlling root growth and several steps of the root gravireaction.

Changes in the level of free and ester indol-3yl-acetic Acid in growing maize roots.

The levels of free and ester-linked indole-3-acetic acid (IAA) in different parts of the maize root were measured using gas chromatography-mass spectrometry (selected ion monitoring). In roots of 2-day-old plants, the distribution of free and ester IAA differed both along the root and between stele and cortex. The levels of IAA and IAA esters were then measured in whole roots and in the elongation zone using roots of different ages. The level of ester IAA decreased steadily with time. In contrast, the level of free IAA in the elongation zone was found to increase after a few days of culture at which time the rate of root growth was decreasing.

Quantification of abscisic Acid in a single maize root.

Quantitative analyses of abscisic acid in the elongating zone of a single maize root (Zea mays L. cv LG 11) were performed by gas chromatography-mass spectrometry using negative chemical ion ionization. Data showed that the more abscisic acid, the slower the growth, but a large dispersion of individual values was observed. We assume that abscisic acid is perhaps not correlated only to the growth rate.

Maize root growth and localized indol-3yl-acetic Acid treatment: a new methodological approach.

Resin beads loaded with indol-3yl-acetic acid (IAA) were used as asymmetrical donors along the elongation zone of intact primary Zea mays L. roots. A strong curvature, towards and above the bead, occurred when IAA was applied at a mean distance of 2.20 mm from the tip. No curvature was detected after applications at 3.89 and 5.71 mm from the tip. Correspondence analysis, a new methodological approach in plant hormone studies, permitted the evaluation of the relative influence of several factors on the curvature observed for each root. The parameters considered were the initial growth rate, the exact location of the bead (1.64-2.73 millimeters from tip) and the quantity of IAA absorbed. Roots which grew rapidly bent earlier than slowly growing ones and the more basal the treatment was, the less curvature occurred. Surprisingly, the amount of IAA taken up (between 1.2 and 2.2 times the endogenous IAA content) was found to have no influence on either the time-course or the magnitude of this growth inhibition (curvature). The usefulness of this multivariate analysis is also discussed.

Localization in sucrose gradients of the pyrophosphate-dependent proton transport of maize root membranes.

A maize (Zea mays L. cv LG 11) root homogenate was prepared and centrifuged to sediment the mitochondria. The pellet (6 KP) and the supernatant (6 KS) were collected and fractionated on linear sucrose density gradients. Marker enzymes were used to study the distribution of the different cell membranes in the gradients. The distribution of the ATP- and pyrophosphate-dependent proton pumping activities was similar after 3 hours of centrifugation of the 6 KS or the 6 KP fraction. The pumps were clearly separated from the mitochondrial marker cytochrome c oxidase and the plasmalemma marker UDP-glucose-sterolglucosyl-transferase. The pyrophosphate-dependent proton pump might be associated with the tonoplast, as the ATP-dependent pump, despite the lack of a specific marker for this membrane. However, under all the conditions tested, the two pumps overlapped the Golgi markers latent UDPase and glucan synthase I and the ER marker NADH-cytochrome c reductase. It is therefore not possible to exclude the presence of proton pumping activities on the Golgi or the ER of maize root cells. The two pumps (but especially the pyrophosphate-dependent one) were more active (or more abundant) in the tip than in the basal part of maize roots, indicating that these activities might be important in growth processes.

Effect of pH on IAA Uptake by Maize Root Segments.

The uptake of [5-(3)H]indoleacetic acid (IAA) by Zea mays L. root segments involves nonsaturable and saturable processes. The pH optimum of the saturable component was found to be 5.0. The proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone inhibited at 100 micromolar the saturable component of IAA uptake but had no effect on non-saturable uptake. This indicates that the saturable component of IAA uptake is dependent on the proton gradient across the plasmalemma. The high level of proton extrusion in the elongation zone of the root will stimulate nonsaturable and saturable uptake of IAA in that zone.

Effect on Root Growth of Endogenous and Applied IAA and ABA: A Critical Reexamination.

Applications of indole-3yl-acetic acid (IAA) and abscisic acid (ABA) were done on two-day-old intact maize (cv LG 11) roots. The effect of the treatment on the root growth depends on their initial elongation rate. The slow growing roots were all inhibited by exogenous IAA and ABA at any concentrations used whereas for the fast growing roots their elongation was promoted by these two hormones at low concentrations. Quantitative analyses of endogenous IAA and ABA were performed using the gas chromatography-mass spectrometry technique. Detection and quantification of endogenous IAA and ABA were done on the zone of the root implicated in elongation. These techniques were achieved by electron impact on the IAA-Me-heptafluorobutyryl derivative and by negative ion chemical ionization with NH(3) on the ABA-Me ester derivative. A negative correlation between the growth and the endogenous content of these two hormones was obtained. ABA presented a larger range of endogenous level than IAA on the whole population of roots tested. When using applied IAA and ABA at different concentrations the same differentiating effect on the growth was observed. This allowed us to conclude that for identical concentrations, IAA has a more powerful effect on root elongation than ABA. Present results are discussed in relation to previous data related to the role of IAA and ABA in the growth and gravireaction of maize roots.

Saturable uptake of indol-3yl-acetic Acid by maize roots.

The uptake of 5-[(3)H]indol-3yl-acetic acid (IAA(*)) by segments of Zea mays L. roots was measured in the presence of nonradioactive indol-3yl-acetic acid (IAA degrees ) at different concentrations. IAA uptake was found to have a nonsaturable component and a saturable part with (at pH 5.0) an apparent K(m) of 0.285 micromolar and apparent V(max) 55.0 picomoles per gram fresh mass per minute. These results are consistent with those which might be expected for a saturable carrier capable of regulating IAA levels. High performance liquid chromatography analyses showed that very little metabolism of IAA(*) took place during 4 minute uptake experiments. Whereas nonsaturable uptake was similar for all 2 millimeter long segments prepared within the 2 to 10 millimeter region, saturable uptake was greatest for the 2 to 4 millimeter region. High levels of uptake by stelar (as compared with cortical) segments are partly attributable to the saturable carrier, and also to a high level of uptake by nonsaturable processes. The carrier may play an essential role in controlling IAA levels in maize roots, especially the accumulation of IAA in the apical region. The increase in saturable uptake toward the root tip may also contribute to the acropetal polarity of auxin transport.

Local application of indole-3-acetic acid, by resin beads to intact growing maize roots.

Five types of anion-exchanger resin beads which had adsorbed indole-3-acetic acid (IAA) were tested as IAA donors. The rate of IAA-uptake by beads was a function of time and pH. The release was relatively steady during 6 h application on vertical maize roots. No IAA degradation occurred in the beads (Amberlite IRA 400 type) but 45.8% was metabolised in the roots during treatment. Beads loaded with IAA and placed on one side of the root (at 2.20±0.03 mm from the tip) induced a curvature towards and above the bead (23.3±1.1 degrees after 5.25 h application). In contrast, control beads (without IAA) did not change the axial growth rate. Applied IAA seemed to move differently from endogenous IAA. The use of resin beads loaded with IAA offers a technique to study the effects of local IAA application on intact growing roots.

Importance of the cap in maize root growth.

A large population of primary roots of Zea mays (cv. LG 11) was selected for uniform length at zero time. Their individual growth rates were measured over an 8-h period in the vertical position (in humid air, darkness). Three groups of these roots with significantly different growth rates were then chosen and their cap length was measured. It was found that slowly growing roots had long caps whereas rapidly growing roots had short caps. The production by the cap cells of basipetally transported growth inhibitors was tested (biologically by the curvature of half-decapped roots) and found to be significantly higher for longer root caps than that for shorter ones.

Distribution of growth and proton efflux in gravireactive roots of maize (Zea mays L.).

Roots of Zea mays were maintained in a vertical orhorizontal position and the local elongation rate and H(+) fluxes were measured using Sephadex beads containing a pH indicator. When the roots were kept horizontally, the growth of the lower side was strongly inhibited and that of the upper side slightly stimulated as compared with vertical roots. The H(+) extrusion, which was greatest in the elongation zone, was strongly inhibited on the lower side and slightly stimulated on the upper side as compared with vertical roots.