Distribution of growth regulators in relation to the light-induced geotropic responsiveness in Zea roots

Growth regulators were measured in extracts from the upper and lower halves of 7-mm apical segments of horizontally oriented, red-light-irradiated and non-irradiated roots of Zea mays L. cv. Golden Cross Bantam 70 which exhibit a georesponse only after an exposure to light. Abscisic acid (ABA) was measured by gas-liquid chromatography, auxin (indole-3-acetic acid, IAA) by the Avena straight-growth assay, and an unidentified growth inhibitor by a Zea root-growth assay. The ratio of ABA in the upper and lower halves was 1.6 in the irradiated roots and 1.0 in the non-irradiated ones. The total amount of ABA after irradiation was increased by a factor of ca. 1.8. The ratio of IAA in the upper and lower halves of irradiated and non-irradiated roots was 1:3.4 and 1:2.9, respectively. The content (or activity) of an unidentified growth inhibitor was highest in the lower halves of horizontally oriented roots which had been irradiated with red light. The unidentified growth inhibitor, rather than IAA or ABA, may be the major factor in the light-induced geotropic responsiveness in Zea roots.     

Abscisic acid distribution in horizontal maize root segments

Using gas chromatography-mass spectrometry (GC/MS) techniques of analyses, it has been found that endogenous abscisic acid (ABA) becomes asymmetrically distributed in the elongation zone of horizontal Zea mays (cv. LG 11) roots which are showing a positive gravitropic response. There is a relative increase in the ABA content of the lower half and a concomitant decrease for the upper half in such roots. Asymmetric distribution of ABA is also detected in the elongation zone of half-decapped roots.Abbreviations IAA indoleacetic acid - ABA abscisic acid - GC/MS gas chromatography-mass spectrometry     

The role of the epidermis and cortex in gravitropic curvature of maize roots

In order to determine the role of the epidermis and cortex in gravitropic curvature of seedling roots of maize (Zea mays L. cv. Merit), the cortex on the two opposite flanks was removed from the meristem through the growing zone; gravitropic curvature was measured with the roots oriented horizontally with the cut flanks either on the upper and lower side, or on the lateral sides as a wound control. Curvature was slower in both these treatments (53° in 5 h) than in intact roots (82°), but there was no difference between the two orientations in extent and rate of curvature, nor in the latent time, showing that epidermis and cortex were not the site of action of the growth-regulating signal. The amount of cortex removed made no difference in the extent of curvature. Curvature was eliminated when the endodermis was damaged, raising the possibility that the endodermis or the stele-cortex interface controls gravitropic curvature in roots. The elongation rate of roots from which just the epidermis had been peeled was reduced by 0.01 mM auxin (indole-3-acetic acid) from 0.42 to 0.27 mm h^-1, contradicting the hypothesis that only the epidermis responds to changes in auxin activity during gravistimulation. These observations indicate that gravitropic curvature in maize roots is not driven by differential cortical cell enlargement, and that movement of growth regulator(s) from the tip to the elongating zone is unlikely to occur in the cortex.Abbreviations df degrees of freedom - IAA indole-3-acetic acid     

Distribution of Gibberellins and Abscisic Acid in Geotropically Stimulated Vicia faba Roots

The occurrence of gibberellins and abscisic acid (ABA) in extracts of roots of Vicia faba was demonstrated by gas-liquid chromatography (GLC) of the methylated eluates from the relevant zones of thin-layer chromatograms (TLC) of purified extracts. Quantitative determination of the hormone contents in extracts from upper and lower halves of roots which had been kept in the horizontal position for 30 min indicated a redistribution of the hormones during the geotropic stimulation. Gibberellins whose methyl esters appeared at the retention time of methylated gibberellic acid (GA₃), used as a standard, occurred in higher concentration in the upper than in the lower halves (ratio 2.08:1), whereas the concentration of ABA was highest in the lower halves (ratio 3.08:1). The ratio of the hormones in right and left halves of vertical roots was close to 1:1. Indoleacetic acid (IAA) and ABA were found to retard the elongation of roots of Vicia faba and Lepidium during the first 24 h. Additional experiments with Lepidium showed that this retardation occurs within the first hour after application. Low concentrations of GA₃, when applied to germinating seeds just after the radicles had broken the seed coat, stimulated root elongation in Vicia faba within 24 h and in Lepidium within 36 h. When applied to Lepidium seedlings with 20 mm long roots, GA₃ showed a stimulatory trend within the first 2 h, and distinct stimulation in the subsequent hours, particularly at the lowest concentrations, 0.01 and 0.001 mg/1. These results suggest the possibility of a participation of ABA and gibberellins (in addition to IAA) in the development of the positive geotropic curvature.     

Action of malformin A1 on gravitropic curvature in primary roots of maize (Zea mays L.)

Malformins, a small family of cyclic pentapeptides, are active plant growth regulators isolated from the fungusAspergillus niger. We purified malformin A₁ from the crude malformin A mixture, and studied its action in the gravitropic response of maize roots. Intact primary roots that had been pretreated vertically with malformin A₁ were placed in a humidified box in the horizontal position. Positive curvature (downward) was inhibited in the pretreated roots compared with the control. In addition, we measured the lateral transport of IAA in primary roots. Roots pretreated with malformin A, did not show asymmetric distribution of IAA between the upper and lower sides of the elongation zone. Malformin A, also stimulated ethylene production in maize root segments. Our results had suggested that malformin A₁ might inhibit the lateral transport of IAA across the roots from the upper to the lower side because of an increased level of ethylene. Therefore, we placed more IAA on the upper side at the initial phase of gravistimulation. These results were consistent with malformin A₁-pretreated roots showing inhibited positive gravitropic curvature.     

Redistribution of Phosphorus, Sulfur, Potassium and Calcium in Relation to Light-Induced Gravitropic Curvature in Zea Roots

Phosphorus, sulfur, potassium and calcium were measured by particle-inducedX-ray emission (PIXE) in horizontally oriented, light-irradiatedor non-irradiated primary roots of Zea mays L., cv. Golden CrossBantam 70 which exhibit gravitropic response only after exposureto light. The content of the four elements increased in thelower half of horizontally oriented roots which had been brieflyexposed to white or red light, while there were no marked differencesin distribution between the upper and lower halves of non-irradiatedroots. The increase of each element in the lower half was observed15–30 min after irradiation in root caps and 30–60min after irradiation in the elongation zones. The effect ofred light was not reversed by far-red light given immediatelyafter the red irradiation. Ethylene glycol-bis(ß-aminoethylether)-N, N, N',N'-tetraacetic acid (EGTA) treatment of roottips inhibited the gravitropic curvature of roots, and the additionof Ca reduced this inhibition. The meaning of Ca redistributionin root caps and elongation zones during light-induced gravitropiccurvature of maize roots is discussed. (Received December 4, 1985; Accepted March 22, 1986)     

Hormone-Induced Gene Expression During Gravicurvature of <Emphasis Type="Italic">Brassica</Emphasis> Roots

To investigate the spatial and temporal dependence of hormonal regulation during gravitropism, we compared the effects of root cap application of indole-3-acetic acid (IAA) and abscisic acid (ABA) with gene expression changes occurring naturally during gravitropic reaction of Brassica rapa roots. The expression of auxin, ABA, and metabolism-related genes in the tip, elongation zone, and maturation zone varied with time, location, and hormone concentration and characterized polar auxin transport. IAA was transported readily shootward and inhibited growth more than ABA. Expression of PIN3 and IAA5 in the elongation zone showed downregulation on the convex but upregulation on the concave side. Both PIN7 and IAA5 responded near maximally to 10^?8 M IAA within 30 min, suggesting that auxin activates its own transport system. Ubiquitin 1 (UBQ1) responded after a lag time of more than 1 h to IAA. The metabolic control gene Phosphoenolpyruvate carboxylase 1 (PEPC1) was more sensitive to ABA but upregulated by high concentrations of either hormone. The time course and duration of gene activation suggests that ABA is not involved in gravitropic curvature, differential elongation is not simply explained by IAA-induced upregulation, and that reference genes are sensitive to auxin.     

The response to auxin of rapeseed (Brassica napus L.) roots displaying reduced gravitropism due to transformation by Agrobacterium rhizogenes

It has recently been documented that, compared to untransformed controls, the roots of oilseed rape (Brassica napus L. CV CrGC5) seedlings transformed by Agrobacterium rhizogenes A4 show a reduced gravitropic reaction (Legué et al. 1994, Physiol Plant 91: 559–566). After stimulation at 90°C or 135°, the transformed root tips curve, but never reach a vertical orientation. In the present study, we investigated the causes of reduced gravitropic bending observed in stimulated transformed root tips. First, we localized the gravitropic curvature in normal and in transformed roots after 1.5 h of stimulation. The cells involved in root curvature (target cells) corresponded at the cellular level to the apical part of the zone of increasing cell length. In transformed roots grown in the vertical position, these cells showed a reduction in cell length compared to controls. Because auxin is considered to be the gravitropic mediator, the response of normal and transformed roots to exogenous auxin was studied. Indole-3-acetic acid (IAA) was applied along the first 3 mm using resin beads loaded with the hormone. In comparison to normal roots, transformed roots showed reduced bending toward the bead at all points of bead application. Moreover, the cells which responded to IAA corresponded to the target cells involved in the gravitropic reaction. The level of endogenous IAA was lower in transformed roots. Thus, it was concluded that the modified behavior of transformed roots during gravitropic stimulation could be due to differences either in IAA levels or in reactivity of the target cells to the message from the cap.Abbreviations DEZ distal elongation zone - ELISA enzymelinked immunosorbent assay - T-DNA DNA transferred from Agrobacterium rhizogenes to the plant genome This work was supported by the Centre National d'Etudes Spatiales.     

Redistribution of Endogenous Indoleacetic Acid, Abscisic Acid and Gibberellins in Geotropically Stimulated Ribes nigrum Shoots

The presence of IAA, ABA and gibberellins in extracts of shoots of Ribes nigrum was demonstrated by gas-liquid chroma-tography (GLC) for both IAA and ABA and by the lettuce hypocotyl assay for gibberellins. Quantitative estimation of the three substances in extracts from upper and lower halves of shoots which had been kept horizontal, and which showed negative geotropic response after 4 h, indicated a redistribution of hormones during the geotropic stimulation. The ratio of the hormones in lower and upper halves was 3.8:1 and 2.8:1 for IAA and giberellins respectively, whereas the ratio of ABA in upper and lower halves was 2.1:1. There is, however, no evidence for the participation of gibberellins and ABA in the early development of negative geotropic curvatures, since shoots of intact Vicia faba seedlings treated with 100 mg/1 solution of GA₃, ABA and 10 mg/1 IAA for 30 min prior to geotropic stimulation, developed negative geotropic curvatures, although shoots pretreated with 50 and 100 mg/1 IAA did not develop curvature.     

Graviresponsiveness and abscisic-acid content of roots of carotenoid-deficient mutants of Zea mays L.

The abscisic-acid (ABA) content of roots of the carotenoid-deficient w-3, vp-5, and vp-7 mutants of Z. mays was analyzed using gas chromatography-mass spectrometry with an analysis sensitivity of 6 ng ABA g^–1 fresh weight (FW). Roots of normal seedlings of the same lines were characterized by the following amounts of ABA (as ng ABA g^–1 FW,±standard deviation): w-3, 279±43; vp-5, 237±26; vp-7, 338±61. We did not detect any ABA in roots of any of the mutants. Thus, the lack of carotenoids in these mutants correlated positively with the apparent absence of ABA. Primary roots of normal and mutant seedlings were positively gravitropic, with no significant differences in the curvatures of roots of normal as compared with mutant seedlings. These results indicate that ABA 1) is synthesized in maize roots via the carotenoid pathway, and 2) is not necesary for positive gravitropism by primary roots of Z. mays.Abbreviation ABA abscisic acid     

The lateral transport of IAA in intact coleoptiles of Avena sativa L. and Zea mays L. during geotropic stimulation

Summary Movement of IAA was studied in excised coleoptile apices and whole seedlings of Zea mays L. and Avena sativa L. during geotropic stimulation. A micropipette technique permitted the application of [5-³H]IAA at predetermined points on the coleoptiles with minimal tissue damage.When [5-³H]IAA was applied to the upper side of a horizontal excised Zea coleoptile, about 60% of the recoverable radioactivity had moved into the lower half after 2 h. In contrast, when application was made to the lower side of a horizontal excised coleoptile, only 4% of the radioactivity migrated to the upper half. There was, thus, a net downward movement of 56%. Similar patterns of distribution were found for radioactivity in both the tissue and the basal receiver blocks. In horizontal shoot tissues of intact Zea seedlings a net downward movement of about 30% of the recoverable radioactivity occurred after 1 h of geotropic stimulation. Comparable experiments with Avena indicated a net downward movement of 6–12% in excised apices of coleoptiles and in the intact shoot. In both Zea and Avena chromatographic analyses of tissue and receiver blocks indicated that the movement of radioactivity reflected that of IAA.In Zea coleoptiles, the lateral migration of radioactivity after 2 h was 3 to 4 times greater in the apical tissues than in the basal tissues. A significant net downward movement of radioactivity was detected after 10 min of geotropic stimulation in the extreme apex of Zea coleoptiles but not in the more basal regions.These experiments show that downward lateral transport of IAA occurs in intact shoots of Zea and Avena seedlings upon geotropic stimulation. Lateral transport of IAA had previously been demonstrated only in sub-apical segments of Zea coleoptiles.     

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.     

Computer-based video digitizer analysis of surface extension in maize roots

We used a video digitizer system to measure surface extension and curvature in gravistimulated primary roots of maize (Zea mays L.). Downward curvature began about 25 +/- 7 min after gravistimulation and resulted from a combination of enhanced growth along the upper surface and reduced growth along the lower surface relative to growth in vertically oriented controls. The roots curved at a rate of 1.4 +/- 0.5 degrees min-1 but the pattern of curvature varied somewhat. In about 35% of the samples the roots curved steadily downward and the rate of curvature slowed as the root neared 90 degrees. A final angle of about 90 degrees was reached 110 +/- 35 min after the start of gravistimulation. In about 65% of the samples there was a period of backward curvature (partial reversal of curvature) during the response. In some cases (about 15% of those showing a period of reverse bending) this period of backward curvature occurred before the root reached 90 degrees. Following transient backward curvature, downward curvature resumed and the root approached a final angle of about 90 degrees. In about 65% of the roots showing a period of reverse curvature, the roots curved steadily past the vertical, reaching maximum curvature about 205 +/- 65 min after gravistimulation. The direction of curvature then reversed back toward the vertical. After one or two oscillations about the vertical the roots obtained a vertical orientation and the distribution of growth within the root tip became the same as that prior to gravistimulation. The period of transient backward curvature coincided with and was evidently caused by enhancement of growth along the concave and inhibition of growth along the convex side of the curve, a pattern opposite to that prevailing in the earlier stages of downward curvature. There were periods during the gravitropic response when the normally unimodal growth-rate distribution within the elongation zone became bimodal with two peaks of rapid elongation separated by a region of reduced elongation rate. This occurred at different times on the convex and concave sides of the graviresponding root. During the period of steady downward curvature the elongation zone along the convex side extended farther toward the tip than in the vertical control. During the period of reduced rate of curvature, the zone of elongation extended farther toward the tip along the concave side of the root. The data show that the gravitropic response pattern varies with time and involves changes in localized elongation rates as well as changes in the length and position of the elongation zone. Models of root gravitropic curvature based on simple unimodal inhibition of growth along the lower side cannot account for these complex growth patterns.     

Effect of ethylene antagonists on auxin-induced inhibition of intact primary root elongation in maize (Zeamays L.)

We tested that the hypothesis that root elongation might be controlled by altering the level of ethylene in intact primary roots of maize(Zea mays L.). We measured root elongation in a short period using a computerized root auxanometer. Compounds which regulate ethylene production were applied to intact primary roots in different time periods. Root elongation was stimulated by the treatment with ethylene antagonists such as Co²⁺, aminoethoxyvinylglycine (AVG) and L-canaline. This result suggested that root elongation was closely related to ethylene level of intact primary roots. Furthermore, IAA- and 1-aminocyclopropane-1-carboxylic acid (ACC)-induced inhibition of root elongation was reversed by treatment with Co²⁺. The application of ACC to roots which have been exposed to IAA and Co²⁺ have no significant effect on root elongation. However, the inhibition of root elongation by ACC in roots previously treated with IAA and AVG became manifest when the applied IAA concentrations were lower. These results were consistent with the hypothesis that the level of ethylene in intact roots functions to moderate root elongation, and suggested that auxin-induced inhibition of root elongation results from auxin induced promotion of ethylene production.     

An auxin-responsive 1-aminocyclopropane-1-carboxylate synthase is responsible for differential ethylene production in gravistimulated<Emphasis Type="Italic"> Antirrhinum majus</Emphasis> L. flower stems

The regulation of gravistimulation-induced ethylene production and its role in gravitropic bending was studied in Antirrhinum majus L. cut flower stems. Gravistimulation increased ethylene production in both lower and upper halves of the stems with much higher levels observed in the lower half. Expression patterns of three different 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) genes, an ACC oxidase (ACO) and an ethylene receptor (ETR/ERS homolog) gene were studied in the bending zone of gravistimulated stems and in excised stem sections following treatment with different chemicals. One of the ACS genes (Am-ACS3) was abundantly expressed in the bending zone cortex at the lower side of the stems within 2 h of gravistimulation. Am-ACS3 was not expressed in vertical stems or in other parts of (gravistimulated) stems, leaves or flowers. Am-ACS3 was strongly induced by indole-3-acetic acid (IAA) but not responsive to ethylene. The Am-ACS3 expression pattern strongly suggests that Am-ACS3 is responsible for the observed differential ethylene production in gravistimulated stems; its responsiveness to IAA suggests that Am-ACS3 expression reflects changes in auxin signalling. Am-ACS1 also showed increased expression in gravistimulated and IAA-treated stems although to a much lesser extent than Am-ACS3. In contrast to Am-ACS3, Am-ACS1 was also expressed in non-bending regions of vertical and gravistimulated stems and in leaves, and Am-ACS1 expression was not confined to the lower side cortex but evenly distributed over the diameter of the stem. Am-ACO and Am-ETR/ERS expression was increased in both the lower and upper halves of gravistimulated stems. Expression of both Am-ACO and Am-ETR/ERS was responsive to ethylene, suggesting regulation by IAA-dependent differential ethylene production. Am-ACO expression and in vivo ACO activity, in addition, were induced by IAA, independent of the IAA-induced ethylene. IAA-induced growth of vertical stem sections and bending of gravistimulated flowering stems were little affected by ethylene or 1-methylcyclopropene treatments, indicating that the differential ethylene production plays no pivotal role in the kinetics of gravitropic bending.     

Translocation of14C-abscisic acid from roots into the aboveground part of pea (pisum sativum L.) seedlings

The translocation of¹⁴C-ABA from roots into other parts of the plant was followed in intact and decapitated pea seedlings. In intact plants ABA from roots was translocated above all into the apical part of epicotyl. In decapitated plants the regulative ability of intact apex can be partly simulated by exogenous IAA. The growth of lateral buds occurring after decapitation was associated with an intensive flow of¹⁴C-ABA from roots into released lateral buds as late as 72 h after decapitation,i.e. in the stage of intensive elongation growth of buds.     

Involvement of IAA in the interaction betweenAzospirillum brasilense andPanicum miliaceum roots

The possible involvement of IAA in the effect thatAzospirillum brasilense has on the elongation and morphology ofPanicum miliaceum roots was examined by comparing in a Petri dish system the effects of inoculation with a wild strain (Cd) with those of an IAA-overproducing mutant (FT-326). Both bacterial strains produced IAA in culture in the absence of tryptophan. At the stationary growth phase, production of IAA by FT-326 wasca. 12 times greater than that of Cd. When inoculation was made with bacterial concentrations higher than, 10⁶ colony forming units ml^–1 (CFU ml^–1), both strains inhibited root elongation to the same extent. At lower concentrations Cd enhanced elongation, by 15–20%, while FT-326 was ineffective. Both strains promoted root-hair development, and root-hairs were produced nearer the root tip the higher the bacterial concentration (e. g. root elongation region was reduced). Effects of FT-326 on root-hair development were greater than those of Cd. Acidified ether extracts of Cd and FT-326 cultures had inhibitory or promoting effects on root elongation depending on the dilution applied. At low dilutions, extracts from FT-326 were more inhibitory for elongation than those from Cd. At higher dilutions root elongation was promoted, but FT-326 extracts had to be more diluted than those from Cd. Dilutions that promoted root elongation contained supra-optimal concentrations of IAA, 1–3 orders of magnitude higher than those required for optimal enhancement by synthetic IAA. It is suggested that the bacteria produce in culture an IAA-antagonist or growth inhibitor that decreases the effectiveness of IAA action. The large variability reported for the effects ofAzospirillum on root elongation could be the result of the opposite effects on root elongation of IAA and other compounds, produced by the bacteria.     

Growth and gravitropism of corn roots in solution

Growth and early gravitropic responses of corn roots in solution have been studied using time-lapse photography. Aeration was required for both root growth and gravitropism. The optimum pH for gravitropism was in the range 5 to 6. The bending response seemed to be greater for roots in non-buffered solution than in buffered solution. Fastest growth and maximum curvature occurred with about 0.2 mol m⁻³ Ca²⁺. Under some conditions, the gravitropic response started with apparently negligible time delay after the start of the gravitropic stimulus. This may denote graviperception in or near the elongation zone itself. This mechanism for early but relatively weak gravitropism may help to explain a variety of gravitropic responses such as the ‘early wrong way’ curvature, and the behaviour of roots whose columella cells lack amyloplasts. More rapid bending appears to start at about 20 min, which is consistent with observations on roots in humid air and with the accepted statolith model of perception in the root cap.     

Zeta potential of protoplasts from gravireacting maize roots

Protoplasts were isolated from cortical cells of the elongating zone of maize (Zea mays L. cv. LG 11) roots and submitted to microelectrophoresis. Significant and transient differences in zeta potential between protoplasts from upper and lower root sides were compared with the gravireaction and the differential elongation of these roots. The maximum difference in the zeta potential was obtained between protoplasts from the upper and lower cortical cells after 90 min, exactly the time of gravipresentation for which the maximum rate of gravireaction was observed. In addition, this almost corresponded to the time for which the difference between the elongation rates of upper and lower sides of the extending zone began to increase. Consequently, the changes in the charges of the plasmalemma of the cortical cells from the growing part of roots could be more or less directly related to the root graviresponse.     

Transport and metabolism of [3H]iso-pentenyladenine following application to the tips of intact and decapitated tap roots of Pinus pinea

[³H]iso-Pentenyladenine ([³H]iP) was fed for 24 h to the tips of intact and root tip-decapitated Pinus pinea seedlings. Twelve and 24 h after application to the roots of intact plants most of the applied radioactivity (±60%) was transported to the shoot. Root tip removal increased transport of the applied radioactivity to the shoot, but the overall pattern of distribution of radioactivity in the seedling did not change. Large amounts of radioactivity were recovered from the elongation zone of the root. Some radioactivity also accumulated in the older part of the root with well-developed lateral roots. When [³H]iP was applied one day after decapitation, no significant changes in the pattern of radioactivity distribution were found between the intact and decapitated root systems. However, when applied 7 days after decapitation there was a significant increase of radioactivity in the region of the root where lateral roots were emerging. HPLC separation of extracts from the different root sections showed that [³H]iP was extensively metabolized in the root. Six peaks of radioactivity, which co-chromatographed with authentic cytokinin standards, were detected.Abbreviations ABA abscisic acid - ADE adenine - IAA indole-acetic acid - iP iso-pentenyladenine - HPLC high performance liquid chromatography - [OG]DHZ O-glycosyldihydrozeatin - [9R-MP]DHZ ribosyldihydrozeatin monophosphate - [9G]iP iso-pentenyladenine-9-glucoside - [9R]Z ribosylzeatin - [9R]iP iso-pentenyladenosine - TLC thin layer chromatography