Effect of auxin on acropetal auxin transport in roots of corn

Acropetal [¹⁴C]indoleacetic acid (IAA) transport was investigated in roots of corn. At least 40 to 50% of this movement is dependent on activities in the root apex. Selective excision of various populations of cells comprising the root apex, e.g. the root cap, quiescent center, or proximal meristem show that the proximal meristem is the critical region in the apex with regard to influencing IAA movement. The quiescent center has no influence and the root cap has only a minor effect. Excision and replacement of the proximal meristem with an exogenous supply of 10⁻⁸ to 10⁻⁹ molar IAA prevents the reduction in acropetal IAA transport which would normally occur in the absence of this meristem. Substituting 10⁻⁹ molar IAA for the excised root cap brings about a significant increase in the amount of IAA moved acropetally, as compared to intact roots with the root cap still in place. From this and previous work, it is concluded that IAA synthesis occurring in the proximal meristem stimulates the movement of IAA from the basal to apical end of the root.     

Narciclasine modulates polar auxin transport in Arabidopsis roots

Plant development displays an exceptional plasticity and adaptability that involves the dynamic, asymmetric distribution of the phytohormone auxin. Polar auxin flow, which requires transport facilitators of the PIN family, largely contributes to the establishment and maintenance of auxin gradients and mediates multiple developmental processes. Here, we report the effects of narciclasine (NCS), an Amaryllidaceae alkaloid isolated from Narcissus tazetta bulbs, on postembryonic development of Arabidopsis roots. Arabidopsis seedlings grown on NCS showed defects in root gravitropism which correlates with a reduction in auxin transport in roots. Expressions of auxin transport genes were affected and the polar localization of PIN2 protein was altered under NCS treatment. Taken together, we propose that NCS modulates auxin transport gene expression and PIN2 localization, and thus affects auxin transport and auxin distribution necessary for postembryonic development of Arabidopsis roots.     

Active transport of ions across sunflower roots

Summary The electrical potential difference across exuding roots of Helianthus annuus in two strengths of complete culture solution was measured. The determination of the concentration of the major nutrient ions in the outside solution and the xylem sap enabled the Nernst potential for each ion to be calculated. A comparison of the measured and calculated potentials indicated that the anions NO₃, SO₄, H₂PO₄ and HPO₄ were actively transported into the sap against the electrochemical potential gradient. The cations Ca and Mg, on the other hand, appeared to move passively into the sap. The behaviour of potassium depended on its concentration in the medium. With a relatively low external concentration (0.75 mM) it appeared to be actively tansported into the sap, whilst at higher outside concentrations (7.5 mM) it was apparently moving passively into the xylem down the electrochemical potential gradient. The possibility of potassium being pumped out of the sap with relatively high external concentrations is discussed.     

Polar calcium flux in sunflower hypocotyl segments : I. The effect of auxin

The flux of Ca²⁺ at the apical or basal ends of short sunflower (Helianthus annuus L.) hypocotyl segments was monitored using a Ca²⁺-specific electrode. A higher Ca²⁺ efflux was observed at the apical end relative to the basal end, indicating a net polar flux of Ca²⁺. The extreme low mobility of Ca²⁺ in the isolated segment makes it likely that the observed Ca²⁺ fluxes are of localized origin, that is, from the parenchyma cells close to the exposed cut ends and may represent acropetal transport of Ca²⁺ at the cellular level. The rate of Ca²⁺ efflux depended on the concentration of Ca in the seedling medium. Incubation of hypocotyl segments in 10 mm CaCl₂ for 24 h did not eliminate the net acropetal flux of Ca²⁺ at the apical end.     

Components of auxin transport in stem segments of Pisum sativum L.

1. The uptake of indol-3-yl acetic acid ([1-¹⁴C]IAA, 0–2.0 M) into light-grown pea stem segments was measured under various conditions to investigate the extent to which mechanisms of auxin transport in crown gall suspension culture cells (Rubery and Sheldrake, Planta 118, 101–121, 1974) are also found in a tissue capable of polar auxin transport. — 2. IAA uptake increased as the external pH was lowered. IAA uptake was less than that of benzoic acid (BA), naphthylacetic acid (NAA) or 2,4 dichlorophenoxyacetic acid (2,4D) under equivalent conditions. TIBA enhanced net IAA uptake through inhibition of efflux, and to a lesser extent, also increased uptake of NAA and 2,4D while it had no effect on BA uptake. — 3. Both DNP and, at higher concentrations, BA, reduced IAA uptake probably because of a reduction of cytoplasmic pH. However, low concentrations of both BA and DNP caused a slight enhancement of IAA net uptake, possibly through a reduction of carrier-mediated IAA efflux. In the presence of TIBA, the inhibitory effects of DNP and BA were more severe and there was no enhancement of uptake at low concentrations. — 4. Non-radioactive IAA (10 M) reduced uptake of labelled IAA but further increases in concentration up to 1.0 mM produced first an inhibition (0–10 min) of labelled IAA uptake, followed by a stimulation at later times. Non-radioactive 2,4 D decreased, but was not observed to stimulate, uptake of labelled IAA. In the presence of TIBA labelled IAA uptake was inhibited by non-radioactive IAA regardless of its concentration. — 5. Sulphydryl reagents PCMB and PCMBS promoted or inhibited IAA uptake depending, respectively, on whether they penetrated or were excluded from the cells. The penetrant PCMB also reduced the promotion of labelled IAA uptake by TIBA or by high concentrations of added non-labelled IAA. — 6. Our findings are interpreted as being consistent with the diffusive entry of unionised IAA into cells together with some carrier-mediated uptake. Auxin efflux from the cells also appears to have a carrier-mediated contribution, at least part of which is inhibited by TIBA, and which has a capacity at least as great as that of the uptake carrier. The data indicate that pea stem segments contain cells whose mechanisms of trans-membrane auxin transport fit the model of polar auxin transport proposed from experiments with crown gall suspension cells, although differences, particularly of carrier specificity, are apparent between the two systems.Abbreviations IAA indol-3-yl acetic acid - BA benzoic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - DNP 2,4-dinitrophenol - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzene sulphonic acid This work was performed in Cambridge during the tenure of a sabbatical leave by P.J.D. Supported by a grant for supplies from the American Philosophical Society to P.J.D.     

Basipetal auxin transport is required for gravitropism in roots of Arabidopsis

Auxin transport has been reported to occur in two distinct polarities, acropetally and basipetally, in two different root tissues. The goals of this study were to determine whether both polarities of indole-3-acetic acid (IAA) transport occur in roots of Arabidopsis and to determine which polarity controls the gravity response. Global application of the auxin transport inhibitor naphthylphthalamic acid (NPA) to roots blocked the gravity response, root waving, and root elongation. Immediately after the application of NPA, the root gravity response was completely blocked, as measured by an automated video digitizer. Basipetal [(3)H]IAA transport in Arabidopsis roots was inhibited by NPA, whereas the movement of [(14)C]benzoic acid was not affected. Inhibition of basipetal IAA transport by local application of NPA blocked the gravity response. Inhibition of acropetal IAA transport by application of NPA at the root-shoot junction only partially reduced the gravity response at high NPA concentrations. Excised root tips, which do not receive auxin from the shoot, exhibited a normal response to gravity. The Arabidopsis mutant eir1, which has agravitropic roots, exhibited reduced basipetal IAA transport but wild-type levels of acropetal IAA transport. These results support the hypothesis that basipetally transported IAA controls root gravitropism in Arabidopsis.     

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 transport of indole-3-acetic Acid in boron- and calcium-deficient sunflower hypocotyl segments

Transfer of sunflower (Helianthus annuus L. cv Russian Mammoth) seedlings from complete nutrient solution to solutions deficient in either boron or calcium resulted in a steady decline in the rate of auxin transport, compared to seedlings that remained in the complete solution. In seedlings transferred to solutions deficient in both B and Ca, the decline in auxin transport was greater than seedlings deficient in only one element. The transfer of B- or Ca-deficient seedlings back to the complete solution prevented further decline in auxin transport, but auxin transport did not increase to the same level as seedlings maintained in complete solution. The significant reduction in auxin transport during the early stages of B or Ca deficiency was not related to (a) reduced growth rate of the hypocotyl, (b) increased acropetal movement of auxin, or (c) lack of respiratory substrates in the hypocotyl. In addition, no difference was found in the water-extractable total and ionic Ca in B-deficient and control nondeficient hypocotyls, indicating a direct effect of B on auxin transport, rather than indirectly by affecting Ca absorption. The rate of auxin transport in hypocotyls deficient in either B or Ca, was inversely correlated with K⁺ leakage and rate of respiration. The data presented strongly support the view that there are separate sites for B and Ca in the basipetal transport of the plant hormone indoleacetic acid.     

Potassium carrier TRH1 is required for auxin transport in Arabidopsis roots

Disruption of the TRH1 potassium transporter impairs root hair development in Arabidopsis, and also affects root gravitropic behaviour. Rescue of these morphological defects by exogenous auxin indicates a link between TRH1 activity and auxin transport. In agreement with this hypothesis, the rate of auxin translocation from shoots to roots and efflux of [3H]IAA in isolated root segments were reduced in the trh1 mutant, but efflux of radiolabelled auxin was accelerated in yeast cells transformed with the TRH1 gene. In roots, Pro(TRH1):GUS expression was localized to the root cap cells which are known to be the sites of gravity perception and are central for the redistribution of auxin fluxes. Consistent with these findings, auxin-dependent DR5:GUS promoter-reporter construct was misexpressed in the trh1 mutant indicating that partial block of auxin transport through the root cap is associated with upstream accumulation of the phytohormone in protoxylem cells. When [K+] in the medium was reduced from 20 to 0.1 mm, wild type roots showed mild agravitropic phenotype and DR5:GUS misexpression in stelar cells. This pattern of response to low external [K+] was also affected by trh1 mutation. We conclude that the TRH1 carrier is an important part of auxin transport system in Arabidopsis roots.     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: inhibition of polar auxin transport in intact plants and stem segments

The transport of [¹⁴C]phenylacetic acid (PAA) in intact plants and stem segments of light-grown pea (Pisum sativum L. cv. Alderman) plants was investigated and compared with the transport of [¹⁴C]indiol-3yl-acetic acid (IAA). Although PAA was readily taken up by apical tissues, unlike IAA it did not undergo long-distance transport in the stem. The absence of PAA export from the apex was shown not to be the consequence of its failure to be taken up or of its metabolism. Only a weak diffusive movement of PAA was observed in isolated stem segments which readily transported IAA. When [1-¹⁴C]PAA was applied to a mature foliage leaf in light, only 5.4% of the ¹⁴C recovered in ethanol extracts (89.6% of applied ¹⁴C) had been exported from the leaf after 6.0 h. When applied to the corresponding leaf, [¹⁴C]sucrose was readily exported (46.4% of the total recovered ethanol-soluble ¹⁴C after 6.0 h). [1-¹⁴C]phenylacetic acid applied to the root system was readily taken up but, after 5.0 h, 99.3% of the recovered ¹⁴C was still in the root system.When applied to the stem of intact plants (either in lanolin at 10 mg·g^-1, or as a 10^-4 M solution), unlabelled PAA blocked the transport through the stem of [1-¹⁴C]IAA applied to the apical bud, and caused IAA to accumulate in the PAA-treated region of the stem. Applications of PAA to the stem also inhibited the basipetal polar transport of [1-¹⁴C]IAA in isolated stem segments. These results are consistent with recent observations (C.F. Johnson and D.A. Morris, 1987, Planta 172, 400–407) that no carriers for PAA occur in the plasma membrane of the light-grown pea stem, but that PAA can inhibit the carrier-mediated efflux of IAA from cells. The possible functions of endogenous PAA are discussed and its is suggested that an important role of the compound may be to modulate the polar transport and-or accumulation by cells of IAA.Abbreviations IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - IIBA 2,3,5-triiodobenzoic acid     

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.     

Boron and calcium sites involved in indole-3-acetic Acid transport in sunflower hypocotyl segments

Sunflower (Helianthus annuus L. cv Russian Mammoth) hypocotyl segments deficient in either B or Ca exhibited a higher rate of potassium leakage, compared to nondeficient segments. Potassium leakage, used here as an indication of membrane integrity, was completely reversed by the addition of H₃BO₃ or Ca(NO₃)₂ to the incubation medium of the B-deficient or Ca-deficient hypocotyl segments, respectively. This role of B and Ca in membrane integrity, which may be important in the entry and exit of auxin in cells, is identified as the first site of action for each of these two essential elements in the basipetal secretion of auxin. A second site for B is postulated because auxin transport was not restored, even when K⁺ leakage has been completely reversed to the nondeficient level, when B-deficient hypocotyls were incubated in B solution. This lack of reversibility of auxin transport implied that the incubation for 2 h in B solution was not enough to restore the auxin transport process. However, since the transfer of B-deficient seedlings to B solutions prevented further deterioration of auxin transport, these observations suggest that: (a) either an intact seedling, or a longer period of incubation of the hypocotyl in B solution, is required for the synthesis or maintenance of the functional second site for B; (b) B is probably essential in the synthesis of a ligand, which may or may not be needed to bind B, but which is essential in the basipetal transport of auxin. The second site for Ca in auxin transport, is indicated by the complete reversal of its inhibition in Ca-deficient hypocotyl, when incubated in Ca solution. The second site for Ca is thought to be directly involved in the secretion of auxin, in which Ca probably plays the role of a second messenger, as in stimulus-response coupling. The two sites for Ca can be distinguished from each other by their cation specificity. The requirement for Ca in the first site can be substituted by other divalent cations, while the second site is highly specific for Ca.     

Effects of calmodulin antagonists on transmembrane auxin transport in Cucurbita pepo L. hypocotyl segments

The effects of calmodulin antagonists on indol-3-yl acetic acid (IAA) uptake by Cucurbita pepo L. hypocotyl segments have been studied. Chlorpromazine (CP) and trifluoperazine (TFP) stimulate uptake of IAA, but not in the presence of 1-naphthylphthalamic acid (NPA), which itself stimulates net IAA uptake by inhibiting the IAA efflux carrier. Maximum stimulation of IAA uptake is observed at 300 μM CP, at both pH 4.0 and 5.0, with a decrease below this maximum at 1 mM CP. CP also stimulates uptakes of abscisic acid (ABA) and 5,5-dimethyl-oxazolidine-2,4-dione (DMO) with the same concentration-dependence. Indirect evidence suggests that enhanced permeability to lipophilic molecules rather than cytoplasmic alkalinification is involved. At submaximal stimulatory concentrations, CP and NPA have apparently additive effects. However, the carrier antagonism by CP is less specific than that of NPA since it also renders the IAA uptake carrier undetectable. The local anaesthetics, dibucaine and tetracaine, and the calcium entry blocker, flunarizine, had similar effects to CP on uptake. However, only CP increased the binding of IAA (but not of ABA or DMO) to frozen-and-thawed segments. It is suggested that CP may increase membrane permeability to apolar molecules, probably non-specifically, and also interfere with auxin carrier action by hydrophobic interaction with phospholipids or membrane proteins.     

Olpidium brassicae in cabbage roots

Morphology and development of the vegetative, asexual and sexual cycle Olpidium brassicae (Woronin) Dangeard parasitic in the roots of cabbage (Brassica oleracea L. var. capitata) seedlings was studied in detail. Release of zoo-spores through an exit tube from zoosporangia, their motility and encystment on the epidermal cells and root hairs, process of infection/host entry and later thallus development were described. Motility and fusion of 2 planogametes resulting in zygote formation were observed in slide cultures and described. The process of infection by zygotes and developmental stages of resting sporangia were also studied and described.     

Subcellular trafficking of PIN auxin efflux carriers in auxin transport

The directional transport of the plant hormone auxin is a unique process mediating a wide variety of developmental processes. Auxin movement between cells depends on AUX1/LAX, PGP and PIN protein families that mediate auxin transport across the plasma membrane. The directionality of auxin flow within tissues is largely determined by polar, subcellular localization of PIN auxin efflux carriers. PIN proteins undergo rapid subcellular dynamics that is important for the process of auxin transport and its directionality. Furthermore, various environmental and endogenous signals can modulate trafficking and polarity of PIN proteins and by this mechanism change auxin distribution. Thus, the subcellular dynamics of auxin transport proteins represents an important interface between cellular processes and development of the whole plant. This review summarizes our recent contributions to the field of PIN trafficking and auxin transport regulation.     

Sites and regulation of auxin biosynthesis in Arabidopsis roots

Auxin has been shown to be important for many aspects of root development, including initiation and emergence of lateral roots, patterning of the root apical meristem, gravitropism, and root elongation. Auxin biosynthesis occurs in both aerial portions of the plant and in roots; thus, the auxin required for root development could come from either source, or both. To monitor putative internal sites of auxin synthesis in the root, a method for measuring indole-3-acetic acid (IAA) biosynthesis with tissue resolution was developed. We monitored IAA synthesis in 0.5- to 2-mm sections of Arabidopsis thaliana roots and were able to identify an important auxin source in the meristematic region of the primary root tip as well as in the tips of emerged lateral roots. Lower but significant synthesis capacity was observed in tissues upward from the tip, showing that the root contains multiple auxin sources. Root-localized IAA synthesis was diminished in a cyp79B2 cyp79B3 double knockout, suggesting an important role for Trp-dependent IAA synthesis pathways in the root. We present a model for how the primary root is supplied with auxin during early seedling development.     

Quantitative distribution and metabolism of auxin herbicides in roots

The internal concentrations of four auxin herbicides— 2,4-dichlorophenoxyacetic acid, dicamba, picloram, and naphthaleneacetic acid—were measured in the roots of treated pea seedlings. Intact seedlings were immersed in solutions of labeled herbicides at concentrations sufficient to produce toxic symptoms (inhibition of elongation, radial enlargement, and lateral root proliferation). Measurements of volume and herbicide content of segments taken sequentially along the root showed that an acropetal concentration gradient of each herbicide was established within the root immediately following treatment. Although there was a net loss of herbicide in the following 24 hours, the gradient was maintained. Initially, the concentration of herbicide in the root tips exceeded that in the external medium.     

PGP4, an ATP binding cassette P-glycoprotein, catalyzes auxin transport in Arabidopsis thaliana roots

Members of the ABC (for ATP binding cassette) superfamily of integral membrane transporters function in cellular detoxification, cell-to-cell signaling, and channel regulation. More recently, members of the multidrug resistance P-glycoprotein (MDR/PGP) subfamily of ABC transporters have been shown to function in the transport of the phytohormone auxin in both monocots and dicots. Here, we report that the Arabidopsis thaliana MDR/PGP PGP4 functions in the basipetal redirection of auxin from the root tip. Reporter gene studies showed that PGP4 was strongly expressed in root cap and epidermal cells. PGP4 exhibits apolar plasma membrane localization in the root cap and polar localization in tissues above. Root gravitropic bending and elongation as well as lateral root formation were reduced in pgp4 mutants compared with the wild type. pgp4 exhibited reduced basipetal auxin transport in roots and a small decrease in shoot-to-root transport consistent with a partial loss of the redirective auxin sink in the root. Seedlings overexpressing PGP4 exhibited increased shoot-to-root auxin transport. Heterologous expression of PGP4 in mammalian cells resulted in 1-N-naphthylthalamic acid-reversible net uptake of [3H]indole-3-acetic acid. These results indicate that PGP4 functions primarily in the uptake of redirected or newly synthesized auxin in epidermal root cells.