Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.     

Patterns of auxin and abscisic acid movement in the tips of gravistimulated primary roots of maize

Because both abscisic acid (ABA) and auxin (IAA) have been suggested as possible chemical mediators of differential growth during root gravitropism, we compared with redistribution of label from applied ³H-IAA and ³H-ABA during maize root gravitropism and examined the relative basipetal movement of ³H-IAA and ³H-ABA applied to the caps of vertical roots. Lateral movement of ³H-ABA across the tips of vertical roots was non-polar and about 2-fold greater than lateral movement of ³H-IAA (also non-polar). The greater movement of ABA was not due to enhanced uptake since the uptake of ³H-IAA was greater than that of ³H-ABA. Basipetal movement of label from ³H-IAA or ³H-ABA applied to the root cap was determined by measuring radioactivity in successive 1 mm sections behind the tip 90 minutes after application. ABA remained largely in the first mm (point of application) whereas IAA was concentrated in the region 2–4 mm from the tip with substantial levels found 7–8 mm from the tip. Pretreatment with inhibitors of polar auxin transport decreased both gravicurvature and the basipetal movement of IAA. When roots were placed horizontally, the movement of ³H-IAA from top to bottom across the cap was enhanced relative to movement from bottom to top whereas the pattern of movement of label from ³H-ABA was unaffected. These results are consistent with the hypothesis that IAA plays a role in root gravitropism but contrary to the idea that gravi-induced asymmetric distribution of ABA contributes to the response.     

Changes in IAA responsiveness in the elongation region of graviresponding mung bean roots

IAA responsiveness of sections of root tissue taken from the top and bottom of mung bean roots was assessed prior to and at varying times following gravistimulation. Prior to gravistimulation, root tissue sections from the sides of the elongation zone responded similarly to IAA. After gravistimulation (within 5 min), root sections from the bottom of the elongation zone became more responsive to IAA than sections collected from the upper side of the elongation zone. The change in IAA responsiveness of these tissue sections was transient with root sections from both the top and bottom of the elongation zone again exhibiting similar responsiveness to IAA following 15 minutes of gravistimulation.These studies also examined if the root tip is required for the gravity-induced shift in IAA responsiveness in the tissues of the elongation zone. The IAA responsiveness of top and bottom sections of the elongation zone from decapped mung bean roots was assessed at varying times following gravistimulation. The responsiveness to IAA of top and bottom sections changed rapidly in decapped roots, just as had been previously found for intact roots. Although the alteration in responsiveness was transient in decapped roots (just as intact roots), the time it took for the sections to recover previous responsiveness to IAA was extended.These results suggest that the initial growth response of graviresponding roots may be due to a change in the IAA responsiveness of tissues in the elongation zone and not an asymmetric accumulation of IAA on the lower side of the elongation zone. The results also indicate that the gravity-induced shift in IAA responsiveness in the elongation zone occurs independently of the root cap, suggesting that the cells in the elongation region can perceive and respond to gravity independently of the root cap during the intial phases of the gravity response.     

Distribution of IAA and ABA in gravistimulated primary roots ofZea mays. L.

The transport of¹⁴C-IAA and¹⁴C-ABA applied exogenously to root cap toward the elongation zone was investigated in gravi- and light-stimulated primary roots ofZea mays L. cv. Golden Cross Bantam 70. No significant difference of either IAA or ABA in radioactivities was observed between upper and lower halves of elongation zones during the latent period (0–60 min after the stimulation) of gravitropic response. When quantitative analysis of endogenous IAA and ABA by an internal standard method was carried out 60 min after gravi- and/or light-stimulation, no asymmetric redistribution of either IAA or ABA was observed between upper and lower halves of elongation zones. Light irradiation increased by 20% the contents of ABA in elongation zones. These results suggest that although both IAA and ABA are basipetally transportable and can transmit their information to the elongation zone during a latent period we cannot explain the gravitropic curvature by their redistributions between the two (upper and lower) halves of primary roots ofZea. On the basis of results from the present work and previous papers, the distribution of IAA and ABA in gravistimulatedZea roots is discussed. A part of this study was reported at the Eighth Annual Meeting of the IUPS Commission on Gravitational Physiology at Tokyo 1986.     

Effect of calmodulin antagonists on the growth and graviresponsiveness of primary roots of maize

We examined the effect of calmodulin (CaM) antagonists applied at the root tip on root growth, gravity-induced root curvature, and the movement of calcium across the root tip and auxin (IAA) across the elongation zone of gravistimulated roots. All of the CaM antagonists used in these studies delayed gravity-induced curvature at a concentration (1 M) that did not affect root growth. Calmodulin antagonists ( 1M) inhibited downward transport of label from ⁴⁵Ca²⁺ across the caps of gravistimulated roots relative to the downward transport of ⁴⁵Ca²⁺ in gravistimulated roots which were not treated with CaM antagonists. Application of CaM antagonists at the root tip ( 1M) also decreased the relative downward movement of label from ³H-IAA applied to the upper side of the elongation zone of gravistimulated roots. In general, tip application of antagonists inhibited neither the upward transport of ⁴⁵Ca²⁺ in the root tip nor the upward movement of label from ³H-IAA in the elongation zone of gravistimulated roots. Thus, roots treated with CaM antagonists ( 1 M) become less graviresponsive and exhibit reduced or even a reversal of downward polarity of calcium transport across the root tip and IAA transport across the elongation zone. The results indicate that calmodulin-regulated events play a role in root gravitropism.     

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     

Calcium-dependent asymmetric movement of 3H-indole-3-acetic acid across gravistimulated isolated root caps of maize

There is evidence that the cap is the initial site of lateral auxin redistribution during the gravitropic response of roots. We tested this further by comparing asymmetric auxin redistribution across the tips of gravistimulated intact roots, decapped roots, isolated root caps and isolated apical sections taken from decapped roots. Gravistimulation caused asymmetric (downward) auxin movement across the tips of intact roots and isolated root caps but not across the tips of decapped roots or across isolated apical root segments. Naphthylphthalamic acid and pyrenoylbenzoic acid, inhibitors of polar auxin transport, inhibited asymmetric auxin redistribution across gravistimulated isolated root caps and across the tips of gravistimulated intact roots. For intact roots there was a positive correlation between the extent of inhibition of assymmetric auxin redistribution by polar auxin transport inhibitors and the extent of inhibition of asymmetric calcium chelating agent, ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, also caused parallel inhibition of asymmetric auxin redistribution and gravitropic curvature and this effect was reversed by subsequent treatment with calcium. The results support the hypothesis that the cap is a site of early development of auxin asymmetry in gravistimulated roots and that calcium plays an important role in the development of lateral auxin redistribution.     

Rootless with undetectable meristem 1 encodes a monocot-specific AUX/IAA protein that controls embryonic seminal and post-embryonic lateral root initiation in maize

The maize (Zea mays L.) rum1‐R (rootless with undetectable meristems 1‐Reference) mutant does not initiate embryonic seminal roots and post‐embryonic lateral roots at the primary root. Map‐based cloning revealed that Rum1 encodes a 269 amino acid (aa) monocot‐specific Aux/IAA protein. The rum1‐R protein lacks 26 amino acids including the GWPPV degron sequence in domain II and part of the bipartite NLS (nuclear localization sequence). Significantly reduced lateral root density (approximately 35%) in heterozygous plants suggests that the rum1‐R is a semi‐dominant mutant. Overexpression of rum1‐R under the control of the maize MSY (Methionine SYnthase) promoter supports this notion by displaying a reduced number of lateral roots (31–37%). Functional characterization suggests that Rum1 is auxin‐inducible and encodes a protein that localizes to the nucleus. Moreover, RUM1 is unstable with a half life time of approximately 22 min while the mutant rum1‐R protein is very stable. In vitro and in vivo experiments demonstrated an interaction of RUM1 with ZmARF25 and ZmARF34 (Z. mays AUXIN RESPONSE FACTOR 25 and 34). In summary, the presented data suggest that Rum1 encodes a canonical Aux/IAA protein that is required for the initiation of embryonic seminal and post‐embryonic lateral root initiation in primary roots of maize.     

Effects of norflurazon, an inhibitor of carotenogenesis, on abscisic acid and xanthoxin in the caps of gravistimulated maize roots

Maize seeds were germinated in the dark in the presence of the carotenoid synthesis inhibitor norflurazon and the teveis of abscisic acid, xanthoxin and total carotenoids were measured in the root cap and in the adjacent 1.5 mm segment. In norflurazon-treated roots abscisic acid levels were markedly reduced, but an increase occurred in the levels of xanthoxin, a compound structurally and physiologically similar to abscisic acid. In the cultivar of maize ( Zea mays L. cv. Merit) used for this work, brief illumination of the root is required for gravitropic curving. Following illumination both control and norflurazon-treated roots showed normal gravitropic curvature, however, the rate of curvature was delayed in norflurazon-treated roots. Our data from norflurazon-treated roots are consistent with a role for xanthoxin in maize root gravitropism. The increase in xanthoxin in the presence of an inhibitor of carotenoid synthesis suggests that xanthoxin and abscisic acid originate, at least in part, via different metabolic pathways.     

Plagiogravitropism of maize roots

The direction of root growth can be studied by analyzing the trajectories of roots growing in soil. Both the primary seminal root and nodal roots of maize attain a preferred, or liminal, angle of growth that deviates from the vertical. These roots are said to be plagiogravitropic. Experiments using plants grown in soil-filled boxes revealed that the primary seminal root is truly plagiogravitropic. It shows both positive and negative gravitropism in response to gravity stimuli and tends to maintain its direction even after growing around obstacles. These are experimental results suggesting that plagiogravitropic growth is controlled by internal factors. The orientation of the grain affects the establishment of the liminal angle of the primary seminal root, and both the position of their node of origin and the root diameter are closely related to the plagiogravitropic behaviour of nodal roots. Several external factors are also known to influence plagiogravitropism. Low soil water content causes a decrease in the angle of growth and soil mechanical resistance suppresses the gravitropic curvature. Plagiogravitropic behaviour of both seminal and nodal roots plays a significant role in shaping the root system.     

Amyloplast sedimentation kinetics in gravistimulated maize roots

Amyloplast sedimentation in gravistimulated maize (Zea mays L.) roots was measured using the change in angle from the center of the cell to each amyloplast as an index of sedimentation. Using tissue fixed after gravistimulation, the relationship between mean amyloplast angle and the duration of gravistimulation was found to be linear when plotted on a logarithmic time scale. Extrapolated values for the onset of angular change are 5.9 s after the start of gravistimulation for the entire population of amyloplasts and 11.8 s for lead amyloplasts. By multiplying the instantaneous angular velocity (in radians) by the cell center to amyloplast radius, it is possible to calculate the initial sedimentation velocity to be 19.1 m min^-1 at 5.9 s. During sedimentation, the mean amyloplast angles surpass the calculated cell corner angle of 123° at 2.2 min for all amyloplasts and at 19 s for lead amyloplasts near the new lower wall. Thus, substantial sedimentation occurs within the presentation time, calculated to be 4.1 min. These kinetics are consistent with several hypotheses of graviperception.Symbol t_p presentation time     

ARL2, ARG1 and PIN3 define a gravity signal transduction pathway in root statocytes

ALTERED RESPONSE TO GRAVITY1 (ARG1) and its paralog ARG1-LIKE2 (ARL2) are J-domain proteins that are required for normal root and hypocotyl gravitropism. In this paper, we show that both ARL2 and ARG1 function in a gravity signal transduction pathway with PIN3, an auxin efflux facilitator that is expressed in the statocytes. In gravi-stimulated roots, PIN3 relocalizes to the lower side of statocytes, a process that is thought to, in part, drive the asymmetrical redistribution of auxin toward the lower flank of the root. We show that ARL2 and ARG1 are required for PIN3 relocalization and asymmetrical distribution of auxin upon gravi-stimulation. ARL2 is expressed specifically in the root statocytes, where it localizes to the plasma membrane. Upon ectopic expression, ARL2 is also found at the cell plate of dividing cells during cytokinesis, an area of intense membrane dynamics. Mutations in ARL2 and ARG1 also result in auxin-related expansion of the root cap columella, consistent with a role for ARL2 and ARG1 in regulating auxin flux through the root tip. Together these data suggest that ARL2 and ARG1 functionally link gravity sensation in the statocytes to auxin redistribution through the root cap.     

Gravity signal transduction in primary roots

AIMS: The molecular mechanisms that correlate with gravity perception and signal transduction in the tip of angiosperm primary roots are discussed. SCOPE: Gravity provides a cue for downward orientation of plant roots, allowing anchorage of the plant and uptake of the water and nutrients needed for growth and development. Root gravitropism involves a succession of physiological steps: gravity perception and signal transduction (mainly mediated by the columella cells of the root cap); signal transmission to the elongation zone; and curvature response. Interesting new insights into gravity perception and signal transduction within the root tip have accumulated recently by use of a wide range of experimental approaches in physiology, biochemistry, genetics, genomics, proteomics and cell biology. The data suggest a network of signal transduction pathways leading to a lateral redistribution of auxin across the root cap and a possible involvement of cytokinin in initial phases of gravicurvature. CONCLUSION: These new discoveries illustrate the complexity of a highly redundant gravity-signalling process in roots, and help to elucidate the global mechanisms that govern auxin transport and morphogenetic regulation in roots.     

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.     

A novel root gravitropism mutant of Arabidopsis thaliana exhibiting altered auxin physiology

A root gravitropism mutant was isolated from the DuPont Arabidopsis thaliana T-DNA insertional mutagenesis collection. This mutant has reduced root gravitropism, hence the name rgrl. Roots of rgrl are shorter than those of wild-type, and they have reduced lateral root formation. In addition, roots of rgrl coil clockwise on inclined agar plates, unlike wild-type roots which grow in a wavy pattern. The rgrl mutant has increased resistance, as measured by root elongation, to exogenously applied auxins (6-fold to indole-3-acetic acid, 3-fold to 2,4-dichlorophenoxyacetic acid, and 2-fold to napthyleneacetic acid). It is also resistant to polar auxin transport inhibitors (2-fold to triiodobenzoic acid and 3- to 5-fold lo napthyleneacetic acid). The rgrl mutant does not appear to be resistant to other plant hormone classes. When grown in the presence of 10^?2 M 2.4-dichlorophenoxyacetic acid, rgrl roots have fewer root hairs than wild type. All these rgrl phenotypes are Mendelian recessives. Complementation tests indicate that rgrl is not allelic to previously characterized agravitropic or auxin-resistant mutants. The rgrl locus was mapped using visible markers to 1.4 ± 0.6 map units from the CHI locus at 1–65.4. The rgrl mutation and the T-DNA cosegregate, suggesting that rgrl was caused by insertional gene inactivation.     

Early wrong-way response occurs in orthogravitropism of maize roots treated with lithium

Millet, B. and Pickard, B. G. 1988. Early wrong-way response occurs in orthogravitropism of maize roots treated with lithium. - Physiol. Plant. 72: 555–559.
Application of lithium ions to tips of roots of Zea mays L. cv. Silver Queen shifts the direction of initial orthogravitropic curvature from downward to upward. The production of this putatively incidental perturbation of orthogravitropic bending kinetics by a pharmacological agent might provide insight into both ortho- and plagiogravitro-pism. Additionally, the protocol of the experiments bears on recent claims that mucilage external to the root cap plays an essential role in gravitropism. External mucilage was removed before roots were stimulated, yet they reached about 50 degrees gravitropic curvature in an hour.     

Radioimmunoassay for pmol-quantities of indole-3-acetic acid for use with highly stable [125I]- and [3H]IAA derivatives as radiotracers

A radioimmunoassay for the detection of as little as 0.5–1 pmol indole-3-acetic acid (IAA) in unpurified or partially purified plant extracts is described. The assay makes use of either IAA[¹²⁵I]tyrosine methyl ester or [³H]IAA methyl ester as radioactive antigens and IAA methyl ester as the assay standard (measuring range: 1–200 pmol). Levels of extractable IAA in a number of biological samples have been estimated.Abbreviations BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxy acetic acid - DMF dimethyl formamide - GC-MS gas chromatography-mass spectroscopy - IAA indole-3-acetic acid - RIA radioimmunoassay - SICM selected ion current monitoring - TLC thin layer chromatography - TME tyrosine methyl ester Part 18 in the series: Use of immunoassay in plant science