The source and lateral transport of growth inhibitors in geotropically stimulated roots of Zea mays and Pisum sativum

Summary The positive geotropic responses of the primary roots of Zea mays and Pisum sativum seedlings depend upon at least one growth inhibiting factor which arises in the root cap and which moves basipetally through the apex into the extending zone. The root apex (as distinct from the cap) and the regions more basal to the extending zone are not sources of growth regulators directly involved in the geotropic response. A difference in the concentration or effectiveness of the inhibitory factor(s) arising in the cap must be established between the upper and lower halves of a horizontal root. Positive geotropic curvature in a horizontal root is attributable, at least in part, to a downward lateral transport of inhibitor(s) from the upper to the lower half of the organ.     

Effects of Light on the Georeaction and Growth Inhibitor Content of Roots

The positive geotropic response of the apical segments prepared from the primary roots of Zea mays depends upon at least one growth inhibitor, produced by the root cap, moving basipetally into the extending zone of the root in which it accumulates in the lower part. Anjou maize reacts in both darkness and light while Kelvedon maize is, for the first few hours, geotropic only in light. The production (or activity) of the growth-inhibiting substance — tested by using vertical half-decapitated root segments — is quite similar to the georeaction. This finding provides strong evidence that, in the case of Kelvedon maize roots, the inhibitory substance may depend on light. Observations related to the root segment of Anjou and Kelvedon maizes of which the tips are exchanged, are in agreement with the above results.     

Abscisic acid and the response of the roots of Zea mays L. seedlings to gravity

Summary Exogeneous application of abscisic acid (ABA) to intact roots of LG 11 maize seedlings inhibits root elongation and induces bending of the root in response to gravity in darkness, even though the roots of these seedlings are not normally positively geotropic in the dark. ABA cannot, however, induce geotropic curvature in dark-exposed decapped roots, thus confirming that the root cap is the site of graviperception in the intact root.Abbreviation ABA abscissic acid     

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.     

Effect of indoleacetic acid,abscisic acid,root tips and coleoptile tips on growth and curvature of maize roots

The effect of externally applied indoleacetic acid (IAA) and abscisic acid (ABA) on the growth of roots of Zea mays L. was measured. Donor blocks of agar with IAA or ABA were placed laterally on the roots and root curvature was measured. When IAA was applied to vertical roots, a curvature directed toward the donor block was observed. This curvature corresponded to a growth inhibition at the side of the root where the donor was applied. When IAA was applied to horizontal roots from the upper side, normal geotropic downward bending was delayed or totally inhibited. The extent of retardation and the inhibition of curvature were found to depend on the concentration of IAA in the donor block. ABA neither induced curvature in vertical roots nor inhibited geotropic curvature in horizontal roots; thus the growth of roots was not inhibited by ABA. However, when, instead of donor blocks, root tips or coleoptile tips were placed onto vertical roots, a curvature of the roots was observed.Abbreviations ABA abscisic acid - IAA 3-indoleacetic acid     

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.     

Growth inhibitor from the root cap of Zea mays

Summary The downward lateral transport of at least one inhibitor produced in or released by the cap of a maize root partly explains differences in growth of the upper and lower sides of roots placed in the horizontal position.The relative activity or level of such a regulator increases with the increase in root length.     

Georeaction and elongation of the flower stalk in a poppy, Papaver Rhoeas L.

The geotropic response of a poppy flower stalk was studied andthe following results were obtained.

1. After formation, the stalk first grows upright showing negativegeotropic behavior, then positive behavior by growing downward,and finally after about 10 days negative behavior by standingupright followed by the opening of the flower.
2. The bendingzone of the stalk showing negative geotropic curvaturemovesacropetally from the base as the stalk ages, and finallythewhole stalk acquires negative geotropic behavior.
3. Curvatureoccurs due to enhanced elongation of the convex (upper)sideof the stalk compared with that of the concave (lower)side,and this appears to be due to differential rates of cellelongationbetween the upper and lower sides. The epidermalcell wall wasloosened more in the upper side according to stress-relaxationanalysis.
4. When the curvature disappears and the stalk growsupright again,the concave (lower) side of the zones of curvatureelongatesfaster than the convex (upper) side.
5. If the flowerbud is decapitated, the stalk quickly becomesstraight, standingupright. However, if a lanolin paste containingIAA (1 mg/plant)is applied to the cut end, the growth and movementsproceedin a manner similar to that of the control plants havingflowerbuds. On the other hand, with GA (3 mg/plant) application,thecurvature is not retained although elongation of the decapitatedstalk is restored more than 50%.

(Received October 20, 1978; )     

Recovery of Geotropism after Removal of the Root Cap

Removal of the cap from the primary roots of Zea mays and Triticumaestivum renders the roots unresponsive to gravity. In bothspecies a geotropic response is recovered before a new cap hasstarted to regenerate. Immediately after decapping amyloplastsstart to develop in cells of the root apex and it is proposedthat as the development of amyloplasts continues so they becomefunctional as gravity sensors. It is also suggested that theamyloplasts may be the source of an inhibitor that has beenpostulated to be the intermediary between the perception ofgravity and the geotropic response.     

Regions of differential cell elongation and mitosis, and root meristem morphology in different tissues of geotropically stimulated maize root apices

We examined cell length, mitosis, and root meristem “cuticle” in different tissues of geostimulated, red light-exposed primary roots of corn (Zea Mays, Wisconsin hybrid 64A × 22R). The examination was done at 15-minute intervals for a period of 240 minutes. Differences in cell elongation between the upper and lower sides were most prominent between 1.5 and 2.5 mm from the root meristem; the outer cortex had the greatest elongation growth, and the upper cells showed a significant increase in length compared to the lower. A differential mitosis was also found, with the lower tissue being greater. We infer that the mitotic activity is indicative of cell division, and this division occurs strictly in the first 1.5 mm of the root meristem. The combined effect of differential cell elongation and cell division results in the localization of the geotropic curvature in the 1.5- to 2.5-mm region from the root meristem. Mitosis that occurs primarily in the cortex and stele were asynchronous; the peak of cortical division preceded that of the stele. Both peaks occurred before the peak of geotropism. A densely stained layer separates the cap from the root meristem. This layer is thinner at the apex of the root meristem. The area of the thin region increased with time and peaked at 180 minutes after geostimulation, which was coincidental with the peak of the geotropic response.     

Root Growth Inhibitors from Root Cap and Root Meristem of Zea mays L.

A micro-assay based on the growth inhibition of root segmentsof the seminal roots of Zea mays has been used to investigatethe root-growth-inhibiting substances in root caps and meristemsrespectively of the roots of Zea mays. This micro-assay is sensitiveto 50 pg of IAA or less. Paper chromatography of the acid fractionof methanolic extracts shows the presence of one main inhibitorin root caps and a different main inhibitor in root meristems.Neither is IAA, whose presence in meristems is sometimes indicatedby small inhibitions (or stimulations) at the characteristicRf of IAA. A Commelina leaf-epidermis assay shows the presenceof one stomata-closing ABA-like substance in root caps and onein meristems, one corresponding in Rf to the main root-growthinhibitor from the root cap. The implications of these findingsfor the geotropic responses of roots is briefly discussed.     

Gas chromatography-mass spectrometric determinations of abscisic acid levels in the cap and the apex of maize roots

Quantitative analyses of abscisic acid (ABA) in different parts of maize root tips (Zea mays L. cv. Kelvedon 33) were performed by mass fragmentography using the hexadeuterated analog of ABA as internal standard. It was found that the cap and the apex contained 36.1 g and 66.5 g ABA kg^–1 fresh weight, respectively. The possibility that the growth regulator formed in the cap and inhibiting the elongation of the extending zone of the root is ABA is discussed.Abbreviations ABA abscisic acid - ABA-D₆ hexadeuterated ABA - ABA-Me and ABA-D₆-Me methyl esters of ABA and ABA-D₆, respectively - GC-MS gas chromatograph(y)-mass spectrometry/spectrometer - IAA indol-3-yl-acetic acid - MF mass fragmentography - TMS trimethylsilyl     

Abscisic acid rescues the root meristem defects of the Medicago truncatula latd mutant

The LATD gene of the model legume, Medicago truncatula, is required for the normal function of three meristems, i.e. the primary root, lateral roots and nitrogen-fixing nodules. In latd mutants, primary root growth eventually arrests, resulting in a disorganized root tip lacking a presumptive meristem and root cap columella cells. Lateral root organs are more severely affected; latd lateral roots and nodules arrest immediately after emerging from the primary root, and reveal a lack of organization. Here we show that the plant hormone, abscisic acid (ABA), can rescue the latd root, but not nodule, meristem defects. Growth on ABA is sufficient to restore formation of small, cytoplasm-rich cells in the presumptive meristem region, rescue meristem organization and root growth and formation of root cap columella cells. In contrast, inhibition of ethylene synthesis or signaling fails to restore latd primary root growth. We find that latd mutants have normal levels of ABA, but exhibit reduced sensitivity to the hormone in two other ABA-dependent processes: seed germination and stomatal closure. Together, these observations demonstrate that the latd mutant is defective in the ABA response and indicate a role for LATD-dependent ABA signaling in M. truncatula root meristem function.     

An explanation of the inhibition of root growth caused by indole-3-acetic Acid

Low concentrations of indole-3-acetic acid inhibit the growth of pea root sections by inducing the formation of the growth regulator, ethylene gas. Ethylene is produced within 15 to 30 minutes after indole-3-acetic acid is applied and roots begin to swell immediately after they are exposed to the gas. Carbon dioxide competitively inhibits ethylene action in roots, impedes their geotropic response, and partially reinstates auxin inhibited growth. It is concluded that ethylene participates in the geotropic response of roots, but not that of stems.     

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.     

Movement of Cell Organelles and the Geotropic Curvature in Roots of Norway Spruce (Picea abies)

The geotropic development in roots of Norway spruce [(Picea abies (L.)] H. Karst, has been followed by light and electron microscopy and compared with the movement of cell organelles (statoliths) in the root cap cells. The geotropic curvature develops in two phases: (a) an initial curvature in the root cap region, which results in an asymmetry in the extreme root tip and which appears after about 3 h stimulation in the horizontal position; and (b) the geotropic curvature in the basal parts of the root tip, which after 8 h is distributed over the entire elongation zone. A graphic extrapolation, based on measurements of the root curvatures after various stimulation periods, indicates a presentation time in the range of 8 to 10 min. The root anatomy and ultrastructure have been examined in detail in order to obtain information as to which organelles may act as gravity receptors. The root cap consists of a central core (columella) distinct from the peripheral part. The core contains three to four rows of parenchymatic cells each consisting of 15 to 18 storeys of statocyte cells with possibly mobile cell organelles. Amyloplasts and nuclei have been found to be mobile in the root cap cells, and the movement of both types of organelles has been followed after inversion of the seedlings and stimulation in the horizontal position for various periods of time at 4°C and 21°C. Three-dimensional reconstructions of spruce root cap cells based on serial sectioning and electron microscopy have been performed. These demonstrate that the endoplasmic reticulum (ER)-system and the vacuoles occupy a considerable part of the statocyte cell. For this reason the space available for free movement of single statolith particles is highly restricted.     

Role of cytokinin in the regulation of root gravitropism

The models explaining root gravitropism propose that the growth response of plants to gravity is regulated by asymmetric distribution of auxin (indole-3-acetic acid, IAA). Since cytokinin has a negative regulatory role in root growth, we suspected that it might function as an inhibitor of tropic root elongation during gravity response. Therefore, we examined the free-bioactive-cytokinin-dependent ARR5::GUS expression pattern in root tips of transformants of Arabidopsis thaliana (L.) Heynh., visualized high cytokinin concentrations in the root cap with specific monoclonal antibodies, and complemented the analyses by external application of cytokinin. Our findings show that mainly the statocytes of the cap produce cytokinin, which may contribute to the regulation of root gravitropism. The homogenous symmetric expression of the cytokinin-responsive promoter in vertical root caps rapidly changed within less than 30 min of gravistimulation into an asymmetrical activation pattern, visualized as a lateral, distinctly stained, concentrated spot on the new lower root side of the cap cells. This asymmetric cytokinin distribution obviously caused initiation of a downward curvature near the root apex during the early rapid phase of gravity response, by inhibiting elongation at the lower side and promoting growth at the upper side of the distal elongation zone closely behind the root cap. Exogenous cytokinin applied to vertical roots induced root bending towards the application site, confirming the suspected inhibitory effect of cytokinin in root gravitropism. Our results suggest that the early root graviresponse is controlled by cytokinin. We conclude that both cytokinin and auxin are key hormones that regulate root gravitropism.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-004-1381-8     

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.     

The rôle of abscisic acid in root growth and gravireaction: A critical review

A brief account is given of the discovery of abscisic acid (ABA) in roots and root caps of higher plants as well as the techniques by which ABA may be demonstrated in these tissues. The remainder of the review is concerned with examining the rôle of ABA in the regulation of root growth. In this regard, it is well established that when ABA is supplied to roots their elongation is usually inhibited, although at low external concentrations a stimulation of growth may also be found. Fewer observations have been directed at exploring the connection between root growth and the level of naturally occurring, endogenous ABA. Nevertheless, the evidence here also suggests that ABA is an inhibitory regulator of root growth. Moreover, ABA appears to be involved in the differential growth that arises in response to a gravitational stimulus. Recent reports that deny a rôle for ABA in root gravitropism are considered inconclusive. The response of roots to osmotic stress and the changes in ABA levels which ensue, are summarised; so are the interrelations between ABA and other hormones, particularly auxin (e.g. indoleacetic acid); both are considered in the context of the root growth and development. Quantitative changes in auxin and ABA levels may together provide the root with a flexible means of regulating its growth.     

Abscisic acid,xanthoxin and violaxanthin in the caps of gravistimulated maize roots

The occurrence and distribution of abscisic acid (ABA), xanthoxin (Xa) and the carotenoid violaxanthin (Va) were investigated in root tips of maize (Zea mays L. cv. Merit). In roots grown in the dark, Va and ABA were present in relatively high amounts in the root cap and in low amounts in the adjacent terminal 1.5 mm of the root. Xanthoxin was present in equal concentrations in both regions. In roots exposed to light, the ABA distribution was reversed, with relatively low levels in the root cap and high levels in the adjacent 1.5-mm segment. Light also caused a decrease in Va in both regions of the root and an increase in Xa, especially in the cap. In the maize cultivar used for this work, light is necessary for gravitropic curving. This response occurs within the same time frame as the light-induced ABA redistribution as well as the changes in the levels of Va and Xa. These data are consistent with a role for ABA in root gravitropism and support the proposal that Xa may arise from the turnover of Va.Abbreviations ABA abscisic acid - GC gas chromatography - HPLC high-performance liquid chromatography - GC-MS gas chromatography-mass spectroscopy - V_a violaxanthin - Xa xanthoxin