The microtubule cytoskeleton does not integrate auxin transport and gravitropism in maize roots

The Cholodny-Went hypothesis of gravitropism suggests that the graviresponse is controlled by the distribution of auxin. However, the mechanism of auxin transport during the graviresponse of roots is still unresolved. To determine whether the microtubule (MT) cytoskeleton is participating in auxin transport, the cytoskeleton was examined and the movement of 3 H-IAA measured in intact and excised taxol, oryzalin, and naphthylphthalamic acid (NPA)-treated roots of Zea mays cv. Merit. Taxol and oryzalin did not inhibit the graviresponse of roots but the auxin transport inhibitor NPA greatly inhibited both auxin transport and graviresponse. NPA had no effect on MT organization in vertical roots, but caused MT reorientation in horizontally placed roots. Regardless of treatment, the organization of MTs in intact roots differed from that in root segments. The MT inhibitors, taxol and oryzalin had opposite effects on the MTs, namely, depolymerization (oryzalin) and stabilization and thickening (taxol), but both treatments caused swelling of the roots. The data indicate that the MT cytoskeleton does not directly interfere with auxin transport or auxin-mediated growth responses in maize roots.     

The Lazy Mutation in Rice Affects a Step between Statoliths and Gravity-Induced Lateral Auxin Transport

Abstract: Compared to wild type, the lazy mutant in Oryza sati-va L. shows a reduced gravitropic response. In order to locate the lesion in the stimulus-response chain, coleoptile segments of lazy rice were investigated with respect to auxin transport. Gravity-induced lateral movement of radiolabelled indoleacetic acid (IAA) was strongly inhibited by the lazy mutation compared to wild type while uptake and longitudinal transport of IAA, as well as amyloplast sedimentation, were not significantly affected. These findings suggest that LAZY controls a step in the signalling chain between statoliths and auxin secretion.     

Starch Synthetase, Phosphorylase, ADPglucose Pyrophosphorylase, and UDPglucose Pyrophosphorylase in Developing Maize Kernels

Three types of whole plant experiments are presented to substantiate the concept that an important function of ethylene in abscission is to reduce the transport of auxin from the leaf to the abscission zone. (a) The inhibitory effect of ethylene on auxin transport, like ethylene-stimulated abscission, persists only as long as the gas is continuously present. Cotton (Gossypium hirsutum L. cv. Stoneville 213) and bean (Phaseolus vulgaris L. cv. Resistant Black Valentine) plants placed in 14 μl/l of ethylene for 24 or 48 hours showed an increase in leaf abscission and a reduced capacity to transport auxin; but when returned to air, auxin transport gradually increased and abscission ceased. (b) Ethylene-induced abscission and auxin transport inhibition show similar sensitivities to temperature. A 24-hour exposure of cotton plants to 14 μl/l of ethylene at 8 C resulted in no abscission and no significant inhibition of auxin transport. Increasing the temperature during ethylene treatment resulted in a progressively greater reduction in auxin transport with abscission occurring at [unk]27 C where auxin transport was inhibited over 70%. (c) Auxin pretreatment reduced both ethylene-induced abscission and auxin transport inhibition. No abscission occurred, and auxin transport was inhibited only 18% in cotton plants which were pretreated with 250 mg/l of naphthalene acetic acid and then placed in 14 μl/l of ethylene for 24 hours. In contrast, over 30% abscission occurred, and auxin transport was inhibited 58% in the corresponding control plants.     

In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.     

Some aspects of geotropism in coleoptiles

Summary Auxin transport was studied in coleoptile sections that were stimulated geotropically. The early time course of auxin-transport asymmetry was measured. An initial phase in which more IAA was delivered into the receptor for the upper half was found after 5 min of horizontal exposure. After about 15 min this was followed by the expected known asymmetry in which more auxin flows in the lower side of the coleoptile. Upon return of the coleoptile to a vertical position, this asymmetry disappeared within 30 min.Earlier correlations of geosensitivity of the auxin transport system with sedimentation of amyloplasts in comparisons of wild type corn and an amylomaize mutant were confirmed and extended. It was also shown that, in contrast to the geotropic effect, phototropically induced lateral auxin asymmetry was not significantly different in wild type and amylomaize. Eleven other single-gene endosperm starch mutants of corn were compared to their corresponding normals. In all pairs, if a difference in geosensitivity of lateral auxin transport was present, it was correlated with a parallel difference in amyloplast sedimentation: e.g., sugary 1 (67) had an amyloplast asymmetry index of 0.32 and a 13% gravity effect on auxin transport; the paired wild-type had both a greater amyloplast asymmetry (0.61) and a greater gravity effect on transport (23%).Correlations between gravity effects on auxin transport and amyloplasts were also shown in comparisons of apical and basal sections of corn, oat and Sorghum coleoptiles.Further results, confirming the increased effect of centrifugal acceleration greater than 1xg on lateral auxin transport and on curvature, are in agreement with the hypothesis that the pressure exerted by amyloplasts, acting as statoliths, locally stimulates the auxin transport system in the individual cells.with participation by Charles steele and Vicky fan     

The Ethylene-insensitive sickle mutant of Medicago truncatula shows altered auxin transport regulation during nodulation

We studied the ethylene-insensitive, hypernodulating mutant, sickle (skl), to investigate the interaction of ethylene with auxin transport during root nodulation in Medicago truncatula. Grafting experiments demonstrated that hypernodulation in skl is root controlled. Long distance transport of auxin from shoot to root was reduced by rhizobia after 24 h in wild type but not in skl. Similarly, the ethylene precursor 1-amino cyclopropane-1-carboxylic acid inhibited auxin transport in wild type but not in skl. Auxin transport at the nodule initiation zone was significantly reduced by rhizobia after 4 h in both wild type and skl. After 24 h, auxin transport significantly increased at the nodule initiation zone in skl compared to wild type, accompanied by an increase in the expression of the MtPIN1 and MtPIN2 (pin formed) auxin efflux transporters. Response assays to different auxins did not show any phenotype that would suggest a defect of auxin uptake in skl. The auxin transport inhibitor N-1-naphthylphtalamic acid inhibited nodulation in wild type but not skl, even though N-1-naphthylphtalamic acid still inhibited auxin transport in skl. Our results suggest that ethylene signaling modulates auxin transport regulation at certain stages of nodule development, partially through PIN gene expression, and that an increase in auxin transport relative to the wild type is correlated with higher nodule numbers. We also discuss the regulation of auxin transport in skl in comparison to previously published data on the autoregulation mutant, super numerary nodules (van Noorden et al., 2006).     

Modulation of P-glycoproteins by auxin transport inhibitors is mediated by interaction with immunophilins

The immunophilin-like FKBP42 TWISTED DWARF1 (TWD1) has been shown to control plant development via the positive modulation of ABCB/P-glycoprotein (PGP)-mediated transport of the plant hormone auxin. TWD1 functionally interacts with two closely related proteins, ABCB1/PGP1 and ABCB19/PGP19/MDR1, both of which exhibit the ability to bind to and be inhibited by the synthetic auxin transport inhibitor N-1-naphylphtalamic acid (NPA). They are also inhibited by flavonoid compounds, which are suspected modulators of auxin transport. The mechanisms by which flavonoids and NPA interfere with auxin efflux components are unclear. We report here the specific disruption of PGP1-TWD1 interaction by NPA and flavonoids using bioluminescence resonance energy transfer with flavonoids functioning as a classical established inhibitor of mammalian and plant PGPs. Accordingly, TWD1 was shown to mediate modulation of PGP1 efflux activity by these auxin transport inhibitors. NPA bound to both PGP1 and TWD1 but was excluded from the PGP1-TWD1 complex expressed in yeast, suggesting a transient mode of action in planta. As a consequence, auxin fluxes and gravitropism in twd1 roots are less affected by NPA treatment, whereas TWD1 gain-of-function promotes root bending. Our data support a novel model for the mode of drug-mediated P-glycoprotein regulation mediated via protein-protein interaction with immunophilin-like TWD1.     

Actomyosin mediates gravisensing and early transduction events in reoriented cut snapdragon spikes

We investigated the involvement of the actomyosin network in the early events of the gravitropic response of cut snapdragon (Antirrhinum majus L.) spikes. The effects of the actin-modulating drug, cytochalasin D (CD) and/or the myosin inhibitor, 2,3-butanedione-2-monoxime (BDM) on amyloplast displacement, lateral auxin transport and consequently on stem bending were examined. The inhibitory effect on cytoskeleton integrity was studied by using indirect immunofluorescence double-labeling of actin and myosin. Our results demonstrate that no organizational changes in actin filaments occurred in cortical and endodermal cells of the stem bending zone during reorientation. These results suggest that actin depolymerization is not required for amyloplast sedimentation. Unlike the chloroplasts in the cortex, the amyloplasts in the endodermis were surrounded by actin and myosin, indicating that amyloplasts may be attached to the actin filaments via the motor protein, myosin. This suggests the involvement of myosin as part of the actomyosin complex in amyloplast movement in vertical as well as in reoriented stems. This suggestion was supported by the findings showing that: (a) BDM or CD disrupted the normal organization of actin either by altering characteristic distribution patterns of myosin-like protein in the cortex (BDM), or by causing actin fragmentation (CD); (b) both compounds inhibited the gravity-induced amyloplast displacement in the endodermis. Additionally, these compounds also inhibited lateral auxin transport across the stem and stem gravitropic bending. Our study suggests that during stem reorientation amyloplasts possibly remain attached to the actin filaments, using myosin as a motor protein. Thus, gravisensing and early transduction events in the gravitropic response of snapdragon spikes, manifested by amyloplast displacement and lateral auxin transport, are mediated by the actomyosin complex.     

Tomato root growth, gravitropism, and lateral development: correlation with auxin transport.

Tomato (Lycopersicon esculentum, Mill.) roots were analyzed during growth on agar plates. Growth of these roots was inhibited by the auxin transport inhibitors naphthylphthalamic acid (NPA) and semicarbazone derivative I (SCB-1). The effect of auxin transport inhibitors on root gravitropism was analyzed by measurement of the angle of gravitropic curvature after the roots were reoriented 90 degrees from the vertical. NPA and SCB-1 abolished both the response of these roots to gravity and the formation of lateral roots, with SCB-1 being the more effective at inhibition. Auxins also inhibited root growth. Both auxins tested has a slight effect on the gravity response, but this effect is probably indirect, since auxins reduced the growth rate. Auxins also stimulated lateral root growth at concentration where primary root growth was inhibited. When roots were treated with both IAA and NPA simultaneously, a cumulative inhibition of root growth was found. When both compounds were applied together, analysis of gravitropism and lateral root formation indicated that the dominant effect was exerted by auxin transport inhibitors. Together, these data suggest a model for the role of auxin transport in controlling both primary and lateral root growth.     

Role of Indole-3-acetic Acid in Modification of Geotropic Responses in Clinostat Rotated Avena Seedlings

Oat seedlings were grown in a sand medium on clinostats with horizontal axes of rotation to nullify the directional component of the gravity-force vector. Coleoptile segments from such seedlings showed an enhanced absorption of apically applied exogenous auxin (indole-3-acetic acid), compared to segments from vertically rotated or stationary controls. Absorption of basally applied auxin and auxin transport were unaffected by the gravity treatments. Horizontal rotation did not materially change the amount of auxin produced and transported from excised coleoptile tips: however, plants so rotated showed an enhanced curvature response to unilaterally applied auxin.

Collectively, these experiments indicate that enhanced plant responses to horizontal clinostat rotation, where rates of rotation are sufficient to nullify the directional component of the gravity-force vector, are caused primarily by increases in metabolism and not by a modification of auxin availability. These data do not support recently advanced hypotheses that the polarity of auxin transport is based on gravitational sedimentation of cell inclusions.

     

The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport

Arabidopsis pinoid mutants show a strong phenotypic resemblance to the pin-formed mutant that is disrupted in polar auxin transport. The PINOID gene was recently cloned and found to encode a protein-serine/threonine kinase. Here we show that the PINOID gene is inducible by auxin and that the protein kinase is present in the primordia of cotyledons, leaves and floral organs and in vascular tissue in developing organs or proximal to meristems. Overexpression of PINOID under the control of the constitutive CaMV 35S promoter (35S::PID) resulted in phenotypes also observed in mutants with altered sensitivity to or transport of auxin. A remarkable characteristic of high expressing 35S::PID seedlings was a frequent collapse of the primary root meristem. This event triggered lateral root formation, a process that was initially inhibited in these seedlings. Both meristem organisation and growth of the primary root were rescued when seedlings were grown in the presence of polar auxin transport inhibitors, such as naphthylphtalamic acid (NPA). Moreover, ectopic expression of PINOID cDNA under control of the epidermis-specific LTP1 promoter provided further evidence for the NPA-sensitive action of PINOID. The results presented here indicate that PINOID functions as a positive regulator of polar auxin transport. We propose that PINOID is involved in the fine-tuning of polar auxin transport during organ formation in response to local auxin concentrations.     

The specificity of the auxin transport system

Summary In an effort to examine the specificity of the auxin transport system, the movement of a variety of growth substances and of auxin analogues through corn coleoptile sections was measured in both the basipetal and acropetal directions. In contrast to the basipetal, polar transport of the auxins indoleacetic acid (IAA) and 2,4-dichlorophenoxyacetic acid, no such movement was found for benzoic acid or for gibberellin A₁. A comparison of the - and -isomers of naphthaleneacetic acid showed that the growth-active -form is transported, but not the inactive -analogue. Both the dextro (+) and leavo (-) isomer of 3-indole-2-methylacetic acid showed the basipetal movement characteristic of IAA, the dextro isomer being more readily transported than the (-)-form. In this instance, too, the transport was roughtly proportional to the growth promoting activity. The antiauxin p-chlorophenoxyisobutyric acid inhibited auxin transport as it inhibited auxin-induced growth. These results agree with the hypothesis that processes involved in auxin transport are closely linked to or even identical with the primary auxin action.     

Auxin and the response of pea roots to auxin transport inhibitors: morphactin

The auxin transport inhibitor methyl-2-chloro-9-hydroxyfluorene-9-carboxylate (CFM), a morphactin, inhibits negative geotropism, causes cellular swelling, and induces root hair formation in roots of intact Pisum sativum L. seedlings. In excised pea root tips, CFM inhibits elongation more than increase in fresh weight (swell ratio = 1.3 at 20 mum CFM). CFM growth inhibition was expressed in the presence of ethylene. Indoleacetic acid (IAA) prevented the expression of CFM growth inhibition possibly because IAA inhibited the accumulation of CFM into the tissue sections. CFM inhibited the accumulation of IAA and 2,4-dichlorophenoxyacetic acid into excised root tips. Applying Leopold's (1963. Brookhaven Symp. Biol. 16: 218-234) model for polar auxin transport, this result suggests a possible explanation for CFM inhibition of geotropism in pea roots, i.e. disruption of auxin transport by interfering with auxin binding.     

Auxin Response in Arabidopsis under Cold Stress: Underlying Molecular Mechanisms

To understand the mechanistic basis of cold temperature stress and the role of the auxin response, we characterized root growth and gravity response of Arabidopsis thaliana after cold stress, finding that 8 to 12 h at 4°C inhibited root growth and gravity response by ∼50%. The auxin-signaling mutants axr1 and tir1, which show a reduced gravity response, responded to cold treatment like the wild type, suggesting that cold stress affects auxin transport rather than auxin signaling. Consistently, expression analyses of an auxin-responsive marker, IAA2-GUS, and a direct transport assay confirmed that cold inhibits root basipetal (shootward) auxin transport. Microscopy of living cells revealed that trafficking of the auxin efflux carrier PIN2, which acts in basipetal auxin transport, was dramatically reduced by cold. The lateral relocalization of PIN3, which has been suggested to mediate the early phase of root gravity response, was also inhibited by cold stress. Additionally, cold differentially affected various protein trafficking pathways. Furthermore, the inhibition of protein trafficking by cold is independent of cellular actin organization and membrane fluidity. Taken together, these results suggest that the effect of cold stress on auxin is linked to the inhibition of intracellular trafficking of auxin efflux carriers.     

Effects of ethylene on auxin transport

The effect of ethylene on the uptake, distribution and polar transport of C¹⁴ from indole-3-acetic acid-2-C¹⁴ and naphthalene acetic acid-1-C¹⁴ in tissue sections was studied. Test species were cotton (Gossypium hirsutum, L.) and cowpea (Vigna sinensis, Endl.). Generally, incubation of tissue or intact plants with ethylene reduced the degree of polar auxin transport. Ethylene inhibited the movement of both auxins in stem tissue and IAA in petiole tissue of cotton. The effect of ethylene on auxin movement in cow-peas was more complex. Ethylene apparently inhibited transport in younger petiole and stem tissue, but stimulated the process to a small but significant degree in basal petiole segments.

Ethylene, in some experiments, reduced C¹⁴ (auxin) uptake. This reduction was consistently smaller than the inhibition of transport. Effects upon transport were observed when uptake was not different. Differences in uptake declined as the period of incubation with auxin was lengthened, but transport was inhibited for up to 23 hours.

It is proposed that ethylene may, through its effect on transport, cause localized shortages and surpluses of auxin which in turn contribute to symptoms now associated with the response of sensitive species to ethylene.

     

Polar transport and accumulation of indole-3-acetic acid during root regeneration by Pinus lambertiana embryos

Summary The relation of indoleacetic acid (IAA) transport to accumulation of auxin at the base of cuttings and to polar root formation was investigated with small cuttings from germinating embryos of Pinus lambertiana.The transport of endogenous auxin participates in regeneration of roots. This is shown by the facts that (1) more than 40% of the cuttings rooted without addition of exogenous indoleacetic acid; (2) the first regeneration always occurred at the basal tip of a slanting cut; and (3) 2,3,5-triiodobenzoic acid (TIBA), a specific inhibitor of auxin transport, totally inhibited rooting. Addition of IAA to the medium increased the number of roots formed per rooting hypocotyl.Sections of hypocotyls excised from dormant embryos and tested immediately after 2 h hydration were capable of polar transport of IAA. This polarity increased during the first 3 days of culture because of a marked increase in basipetal transport. Culturing the cuttings in 1 M IAA for 3–5 days doubled both the basipetal transport of 1-¹⁴C-IAA by hypocotyl segments and the accumulation of radioactivity at the base of cuttings.The extent of the accumulation at the base of cuttings was similar at early (2 days, first mitoses) and late stages (5 days, organized meristem) of regeneration and was not affected by removal of the regenerating region immediately prior to uptake and transport of ¹⁴C-IAA. The accumulation was inhibited by TIBA. In terms of increase in wet and dry weight and mitotic activity, the cotyledons rather than the regenerating root meristems were the most actively growing region of the cuttings. The upper part of the hypocotyl elongated more than the region of the slanting cut where regeneration was occurring.These results provide no support for the idea that the regenerating root controls the direction of polar transport by acting as a sink. The results are consistent with the view that polar auxin transport delivers auxin to the base of the cutting and raises the local concentration to levels sufficient to promote root formation.     

Effect of glyphosate on auxin transport in corn and cotton tissues

Basipetal auxin transport in 6-day-old dark-grown corn coleoptiles was severely inhibited by increasing levels of glyphosate applied during the transport period.     

Initiation and growth of cell lines of Taxus brevifolia (Pacific yew)

Summary Conditions have been established for the induction and maintenance of callus cultures of Taxus brevifolia (Pacific yew) from bark, stem, and needle tissues. Cultures were established on a modified Gamborg's B5 medium, 1% sucrose, 0.2% casamino acids and 1 mg/L 2,4-D. There was no apparent inhibition of callus induction as a result of taxol concentration in the explant material. Cell lines derived from explants of individual trees were used to investigate growth characteristics. Although none of the cell lines contained taxol, some contained low levels of related taxanes. Variability was observed with each cell line in response to light, and auxin type and concentration. Growth index was most affected by cell line, followed by auxin type and concentration. These culturing methods may be useful for the goal of developing a highproducing cell line applicable for large-scale taxol production.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - CA casamino acids - B5CA B5 with 0.2% casamino acids - IBA indolebutryric acid; Picloram (4-amino-3,5,6-tricnloro-2-pyridinecarboxylic acid) - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - BA 6-benzylaminopurine     

Effect of ethylene on auxin transport and metabolism in midrib sections in relation to leaf abscission of woody plants

Abstract The relationship between ethylene-induced leaf abscission and ethylene-induced inhibition of auxin transport in midrib sections of the leaf blade of Citrus sinensis L. Osbeck, Populus deltoides Bart, and Eucalyptus camaldulensis Dehn. was studied. These species differed greatly in their abscission response to ethylene. The kinetic trend of abscission resembled that of the inhibition of auxin transport in all three species. It is suggested that one of the main actions of ethylene in the leaf blade is to inhibit auxin transport in the veinal tissues, thus reducing the amount of auxin transported from the leaf blade to the abscission zone. Ethylene inhibited transport of both IAA (indole-3-acetic acid) and NAA (α-naphthaleneacetic acid) in the midrib sections. However, while ethylene enhanced the conjugation of IAA with aspartic acid and glucose in the apical (absorbing) segment of the midrib sections, it had little effect on the conjugation of NAA. The data indicate that auxin destruction through conjugation does not play a major role in the inhibition of auxin transport by ethylene.