Auxin-Promoted Transport of Metabolites in Stems of Phaseolus vulgaris L.: EFFECTS REMOTE FROM THE SITE OF HORMONE APPLICATION

Phloem transport in stems of Phaseolus vulgaris was found tobe sensitive to treatment with the auxin transport inhibitor,2,3,5-triidobenzoic acid (TIBA). The response was dependenton the concentration of TIBA applied. A concentration of TIBA(0?5% in lanolin) which did not interfere with normal phloemtransport proved inhibitory to both basipetal transport of IAAand the acropetal component of IAA-promoted metabolite transport.In contrast, both acropetal IAA transport and basipetal IAA-promotedmetabolite transport were unaffected by TIBA treatment. Theinhibitory effect of TIBA on acropetal IAA-promoted transportwas overcome by providing IAA below the point of TIBA application.Both acropetal and basipetal IAA-promoted transport in stemsegments were unaccompanied by any corresponding changes inthe accumulation of [¹⁴C]sucrose by the segments.     

表油菜素内酯促进小麦胚芽鞘伸长的作用

BR促进小麦胚芽鞘伸长的生理活性大于IAA,但高浓度的促进现象不如IAA明显。BR刺激乙烯生成与浓度相关。BR和IAA混合处理,对芽鞘切段的伸长、乙烯释放和H~+分泌都表现了加成作用。这二种激素在作用时间上有明显的差别,BR作用的滞后期更为清楚。BR有拮抗ABA对小麦胚芽鞘切段伸长的抑制作用。     

Auxin-Promoted Transport of Metabolites in Stems of Phaseolus vulgaris L.: Auxin Dose-Response Curves and Effects of Inhibitors of Polar Auxin Transport

Transport of ¹⁴C-photosynthate in decapitated stems of Phaseolusvulgaris explants was dependent on the concentration of indole-3-aceticacid (IAA) applied to the cut surfaces of the stem stumps. Thephysiological age of the stem influenced the nature of the transportresponse to IAA with stems that had ceased elongation exhibitinga more pronounced response with a distinct optimum. Increasednutrient status of the explants had little influence on theshape of the IAA dose-response curve but increased, by two ordersof magnitude, the IAA concentration that elicited the optimalresponse. Applications of the inhibitor of polar auxin transport,1-(2-carboxyphenyl)-3-phenylpropane-1, 3-dione (CPD), affectedIAA-promoted transport of ¹⁴C-photosynthates. At sub-optimalIAA concentrations, CPD inhibited transport, whereas at supra-optimalIAA concentrations, ¹⁴C-photosynthate transport was marginallystimulated by CPD. Treatment with CPD resulted in a significantreduction in stem levels of [¹⁴C]IAA below the site of inhibitorapplication, while above this point, levels of [¹⁴C]1AA remainedunaltered. The divergent responses of auxin-promoted transportto CPD treatment are most consistent with a remote action ofIAA on photosynthate transport in the decapitated stems. Key words: Auxin, photosynthate, transport     

Distribution and transport of IAA in tomato plants

We determined as to indole-3-acetic acid (IAA), endogenous levels byliquid chromatography-mass spectrometry using ¹³C₆-IAA, diffusible levels byfluorometric detection using indole-propionic acid, and polar transportactivityby radioactive IAA in 3-month-old tomato plants (stems, leaves or roots). TheIAA concentration in the apoplast (AP) solution was higher than those in thesymplast (SP) solution in both the upper and lower parts of stems, showing thatIAA analysis of AP solution is important. Younger leaves exported much morediffusible IAA than older leaves. The IAA concentration in the main roots wasalmost at the same level as in the stems. The results suggested that thetransport capacity of IAA is probably the limiting factor for the amount of IAAtransported in stems and the amount of polar IAA transport might be only 19% ofthe endogenous IAA amount in stems.     

Stems of the <Emphasis Type="Italic">Arabidopsis pin1-1</Emphasis> mutant are not deficient in free indole-3-acetic acid

The pin1-1 mutant of Arabidopsis thaliana has been pivotal for studies on auxin transport and on the role of auxin in plant development. It was reported previously that when whole shoots were analysed, levels of the major auxin, indole-3-acetic acid (IAA) were dramatically reduced in the mutant, compared with the WT (Okada et al. 1991). The cloning of PIN1, however, provided evidence that this gene encodes a facilitator of auxin efflux, raising the question of how the pin1-1 mutation might reduce overall IAA levels as well as IAA transport. We therefore re-examined IAA levels in individual parts of pin1-1 and WT plants, focusing on inflorescence stems. Our data show that there is in fact no systemic IAA deficiency in the mutant. The previously reported difference between mutant and WT may have been due to the inclusion of reproductive structures in the WT harvest: we show here that the inflorescence itself contains high levels of IAA. We reconcile the normal IAA levels of pin1-1 inflorescence stems with their (previously-reported) reduced ability to transport IAA by presenting evidence that the auxin in mutant stems is not imported from their apical portion. Our data also indicate that levels of another auxin, indole-3-butyric acid (IBA), are very low in stems of the genotypes used in this study.     

Involvement of indole-3-acetic acid in the circadian growth of the first internode of Arabidopsis

The extension rate of the first inflorescence node of Arabidopsis was measured during light/dark or continuous light exposure and was found to exhibit oscillations which showed a circadian rhythmicity. Decapitation induced a strong inhibition of stem extension. Subsequent application of IAA restored growth and the associated extension–rate oscillations. In addition, IAA treatments, after decapitation, re-established the circadian rhythmicity visible in the intact plants during free run. This indicates that the upper zone of the inflorescence has a major influence on the extension rate of floral stems and implies a role for auxin. Application of N-(1-naphthyl)phthalamic acid, an IAA transport inhibitor, to an intact floral stem inhibited growth and the rhythmicity in the extension rate oscillations, indicating that IAA polar transport may play a role in the dynamics of stem elongation. Furthermore, IAA-aspartate application, after decapitation, did not restore growth and rhythmicity. Nevertheless, biochemical analysis of IAA and IAA-aspartate demonstrated circadian fluctuations of the endogenous levels of both compounds. These observations suggest that IAA metabolism is an essential factor in the regulation of the circadian growth rhythm of Arabidopsis floral stems. Received: 21 September 1998 / Accepted: 23 January 1999     

The role of aspterric acid in auxin-regulated reproductive growth of Arabidopsis thaliana

Application of 100 microM aspterric acid (AA), a pollen growth inhibitor, with different concentrations of indole-3-acetic acid (IAA) results in the recovery of normal pollen development of Arabidopsis thaliana. Treatment with 100 microM AA plus 5 mM IAA significantly induced the normal seed production. Treatment with 100 microM N-1-naphthylphthalamic acid (NPA), a polar auxin transport inhibitor, did not reduce the pollen growth but inhibited seed production. 100 microM NPA plus 5 mM IAA did not induce any seed production. The endogenous level of IAA in stems and leaves of A. thaliana treated with 100 microM AA was similar to that of the untreated control. In contrast to AA treatment, the IAA level by the treatment with 100 microM NPA was about twice as much as that of the untreated control. These results suggest that AA affects the Arabidopsis reproductive growth without inhibiting IAA biosynthesis and transport.     

不同浓度外源IAA处理对杉木茎部基因表达的影响

为了揭示吲哚-3-乙酸(Indole-3-acetic acid,IAA)参与杉木木材发育调控的遗传机制,文章分别以0、3mg.IAA/g.lanolin处理不同阶段的杉木截顶茎秆作为驱动方(Driver)和测试方(Tester),利用抑制消减杂交技术(Suppression substractive hybridization,SSH),对其中差异表达的目的基因进行了分离和克隆。共获得332个Unigenes,其潜在的功能分别涉及到细胞组织和生物合成、发育进程调控、电子传递、逆境应答以及信号传导等方面;进一步地表达鉴定发现ClHIRA、ClPGY1和ClARF4等集中于茎部近轴区域表达的基因,能够积极地响应外源IAA刺激的维管形成层分裂和管胞分化活动;而ClSMP1、ClTCTP1和ClTRN2等集中于茎部远轴区域表达的基因,则在转录水平上对外源IAA的处理水平及近轴次生维管的发育变化表现出负相关的关系。这一结果表明特异性定位的发育基因对木材形成组织中内源IAA水平变化的差异性识别和响应很可能是生长素参与林木维管形成层次生发育调节的重要分子机制。     

Novel use of positively charged nylon transfer membranes for trapping indoleacetic acid or other small anions during efflux from plant tissues.

Positively charged nylon blotting membranes were used as an anion binding medium to trap [14C]indoleactic acid (IAA) as it exited cells at the basal ends of Coleus blumei L. stem and Zea mays L. coleoptile segments. Autoradiography was used to visualize where the [14C] that moved out of the cut ends was localized on the nylon membrane. Diffusion of [14C]IAA from the initial point of contact with the nylon membrane was minimal. Comparison of the autoradiograms with anatomical tissue prints of the cut ends of the segments was used to determine what tissues participate in IAA movement. The results of these initial studies were consistent with other reports suggesting that [14C]IAA movement was primarily associated with vascular tissues in both C. blumei stems and corn coleoptiles, but the resolution was not sufficient to identify which vascular tissues were involved in IAA transport. With further refinements, this technique could also be used for studying the movement of other small charged molecules through plant tissues.     

Kinetin-promoted transport of assimilates in stems of Phaseolus vulgaris L.

Kinetin, applied as a dispersion in aqueous lanolin to the stumps of decapitated stems of P. vulgaris plants with their roots removed, was found to promote the transport of ¹⁴C- and ³²P-labelled assimilates to the site of hormone application. Measurement of photosynthetic rate of, and assimilate export rate from the source leaves, indicated that kinetin was not acting to promote assimilate transport by stimulating these processes. Moreover, it was found that the time between kinetin application and detection of an enhanced transport flux was independent of the distance over which kinetin would need to move to be present throughout the length of the transport pathway. These observations, together with the finding that lateral applications of kinetin to the stems resulted in an enhanced localized accumulation of assimilates, provided evidence that kinetin acted locally at its point of application to stimulate assimilate transfer.Abbreviations GA₃ gibberellic acid - IAA indol-3yl-acetic acid     

An assessment of auxin-promoted transport in decapitated stems and whole shoots of Phaseolus vulgaris L.

¹⁴C-photosynthate transfer in decapitated stems of P. vulgaris plants, treated with IAA (indol-3yl-acetic acid), appeared, as ascertained by microautoradiography, to be restricted to cells of sieve-element appearance. The IAA-induced promotion of photosynthate transport was found not to depend on any artifacts caused by the decapitation procedure. Rather, decapitation primarily served the purpose of removing photosynthate sources above the point of hormone application which otherwise suppressed the expression of the IAA effect on acropetal photosynthate transport. Furthermore, by manipulating stem levels of endogenous auxins with the inhibitor of polar auxin transport, 1-(2¹-carboxyphenyl)-3-phenylpropane-1,3-dione (ACP1.55), evidence was obtained indicating that photosynthate transfer to the shoot apex depended, at least in part, on endogenous levels of auxins at site(s) remote from the apical sink (i.e. shoot apex).Abbreviations ACP1.55 1-(2¹-carboxyphenyl)-3-phenylpropane-1,3-dione - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - IAA indol-3yl-acetic acid     

不同株龄日本落叶松插穗的内源激素含量与生根的关系

研究了日本落叶松母株年龄、插穗内源激素含量与生根之间的关系,以及外源IBA对插穗内源激素含量的影响及其对插穗生根的促进作用。结果表明：不同株龄插穗生根性状及插穗茎和叶中激素含量差异均达极显著水平,叶中激素含量对插穗生根力没有直接影响;插穗茎中生根抑制激素（ABA）含量随株龄增长而增加,生根促进激素与抑制激素的比值（IAA＋GA＋ZR）/ABA却随株龄的增长而递减,与生根力随株龄的变化趋势一致,且该比值与生根性状紧密相关,因此可作为评价母株（无性系）生根力的指标;插后13～32d是插穗愈伤组织形成和不定根诱导的关键期,此期生根促进激素消耗量大,茎中含量大幅度降低,进入根伸长生长阶段,含量上升;外源IBA促进插穗生根的机制在于通过外源激素的刺激,在不定根诱导期,插穗茎中ABA含量大幅度降低,从而有利于不定根的发生和发育。     

A Microautoradiographic Study of Auxin Transport in the Stem of Intact Pea Seedlings (Pisum sativum L.)

Soluble-compound microautoradiography was used to determinethe distribution of radioactivity in transverse sections ofintact dwarf pea stems (Pisum sativum L.) following the applicationof [³H]IAA to the apical bud. Near the transport front labelwas confined to the cambial zone of the axial bundles, includingthe differentiating secondary vascular elements. Fully differentiatedphloem and xylem elements remained unlabelled and no radioactivitywas detected in the leaf or stipule traces. Similar resultswere obtained in experiments with Vicia faba L. plants. Nearerthe labelled apical bud of the pea there was a more generaldistribution of label and evidence was found of free-space transportof radioactive material in the pith. When [³H]IAA was applied to mature foliage leaves the greatestconcentration of label was found in the differentiated phloemelements of the appropriate leaf trace and in the phloem ofthe adjacent axial bundles. Both basipetal and acropetal transportwas detected in this case. These results are consistent with the conclusions drawn fromearlier transport experiments which indicated that in the intactplant the long-distance basipetal transport of auxin from theapical bud takes place in a system which is separated from thephloem transport system and suggests that the vascular cambiumand its immediate derivatives may function as the normal pathwayfor the longdistance movement of auxin in the plant. The physiologicalsignificance of such a transport system for auxin is discussed.     

The role of plant hormones in higher plant cellular differentiation. II. Experiments with the vascular cambium, and sclereid and tracheid differentiation in the pine, Pinus contorta

In sterile-cultured explants of stems of the pine Pinus contorta Dougl., fusiform cambial cells differentiated entirely into axial parenchyma cells when exogenous indol-3yl-acetic acid (IAA) was omitted. The normal appearance of the cambial zone was maintained when IAA was included in the medium. The IAA-maintained stability of cambial structure suggests physiological rather than epigenetic control over vascular cambium structure. IAA was essential for the occurrence of callus growth in stem explants. Callus growth was similar in appearance and extent in winter- and summer-explanted material. Tracheids differentiated in explants only when actively differentiating tracheids were already present at the moment of explanting, suggesting the absence of factors necessary for tracheid differentiation in over-wintering tissues. Sclereid differentiation, which normally does not occur in phloem or xylem development in P. contorta, occurred in callus derived from active cambial explants. The sclereids were identical to sclereids which differentiated in pith of intact stems. The possibility that sclereid and tracheid differentiation may be fundamentally similar types of gene expression is discussed. Growth of P. contorta trees in continuous darkness resulted in extensive compression-wood tracheid differentiation in the upright main stem. Normal-wood tracheids differentiated in similar trees grown in light. More tracheids differentiated in light than in darkness. This apparently is the first report of induction of compression-wood tracheid differentiation in the absence of hormone treatment or tilting of trees. Different types and numbers of tracheids differentiated at different position in two-year-old disbudded defoliated stem cuttings of P. contorta in response to apically supplied IAA. No evidence for new tracheid differentiation was seen in control cuttings; however, the results suggest that neither cambial cell division nor tracheid differentiation were actually initiated by IAA. Directed transport of additional regulatory factors toward areas of high IAA concentration is formulated as a hypothesis to explain these observations. Gibberellic acid, (S)-abscisic acid and IAA inhibited tracheid differentiation when individually supplied to basal ends of P. contorta cuttings predisposed to differentiate new tracheids. Experiments with single intact needles on Pinus cembroides var. monophylla cuttings confirmed a previous interpretation that the mature pine needle, rather than the short-shoot apical meristem at its base, promotes tracheid differentiation in the stem.     

Auxin Transport in Secondary Tissues1

Auxin transport was investigated in excised stem segments ofNicotiana tabacum L. by the agar block technique using [1-¹⁴C]indol-3yl-acetic acid (IAA). The ability of the stems to transportauxin basipetally increased as secondary development proceeded;by contrast the ability of the pith to transport auxin declinedwith age. By separation of the stem tissues it was shown thatthe great majority of auxin transport took place in cells associatedwith the internal phloem and in cells close to the cambium;in both cases similar velocities of transport were found (c.5.0 mm h^–1 at 22°C). The effects of osmotic gradientson auxin transport through the internal phloem were investigated.IAA was found by chromatography to account for practically allthe radioactivity in receiver blocks and other extracts of stemsegments. The significance of these results is discussed.     

Transfer of exogenous auxin from the phloem to the polar auxin transport pathway in pea (Pisum sativum L.)

When [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA) was applied to the upper surface of a mature foliage leaf of garden pea (Pisum sativum L. cv. Alderman), ¹⁴C effluxed basipetally but not acropetally from 30-mm-long internode segments excised 4 h after the application of [1-¹⁴C]IAA. This basipetal efflux was strongly inhibited by the inclusion of 3.10^–6 mol· dm³ N-1-naphthylphthalamic acid (NPA) in the efflux buffer. In contrast, when [¹⁴C] sucrose was applied to the leaf, the efflux of label from stem segments excised subsequently was neither polar nor sensitive to NPA. The [1-¹⁴C]IAA was initially exported from mature leaves in the phloem — transport was rapid and apolar; label was recovered from aphids feeding on the stem; and label was recovered in exudates collected from severed petioles in 20 mM ethylenediaminetetraacetic acid. No ¹⁴C was detected in aphids feeding on the stems of plants to which [1-¹⁴C]IAA had been applied apically, even though the internode on which they were feeding transported considerable quantities of label. Localised applications of NPA to the stem strongly inhibited the basipetal transport of apically applied [1-¹⁴C]IAA, but did not affect transport of [1-¹⁴C]IAA in the phloem. These results demonstrate for the first time that IAA exported from leaves in the phloem can be transferred into the extravascular polar auxin transport pathway but that reciprocal transfer probably does not occur. In intact plants, transfer of foliar-applied [1-¹⁴C]IAA from the phloem to the polar auxin transport pathway was confined to immature tissues at the shoot apex. In plants in which all tissues above the fed leaf were removed before labelling, a limited transfer of IAA occurred in more mature regions of the stem.Abbreviations IAA indol-3yl-acetic acid - EDTA ethylenediaminetetraacetic acid - NPA N-1-naphthylphthalamic acid We are grateful to the Nuffield Foundation for supporting this research under the NUF-URB95 scheme and for the provision of a bursary to A.J.C. We thank Professor Dennis A. Baker for constructive comments on a draft of this paper and Mrs. Rosemary Bell for her able technical assistance.     

Auxin-promoted Transport of Metabolites in Stems of Phaseolus vulgaris L.: SOME CHARACTERISTICS OF THE EXPERIMENTAL TRANSPORT SYSTEMS

Indol-3yl-acetic acid (IAA) applied to sterns of Phaseolus vulgarisseedlings, decapitated above primary leaves, enhanced the mobilizationof ¹⁴C-metabolites to the treated stumps and this effect wasapparent within 3–6 h of applying the hormone. More than90 per cent of the total ¹⁴C-activity transported to the stumpswas detected in the alcohol-soluble extracts. In all treatments,less than 5 per cent of the ¹⁴C-photosynthate exported fromthe primary leaves was translocated upwards. Accumulation of¹⁴C-activity was also increased when the IAA was applied laterallyto intact internodes. This effect was obtained when ¹⁴C wassupplied either above or below the point of hormone application.By selective heat girdling, it was shown that the auxin affected¹⁴C transport when either the root ‘sink’ was removedor transpiratory flow of water through the treated internodewas maintained. Decapitated stems treated with plain lanolinfor 3 d were found to retain their responsiveness to auxin interms of enhanced metabolite transport. Heat-girdling experimentsand estimates of ¹⁴C transport velocity suggested that mostof the ¹⁴C movement was restricted to the phloem of treatedstumps. Similar effects of IAA on a transport in excised stemsegments of Phaseolus vulgaris were observed.     

Control of Negative Gravitropism and Tension Wood Formation by Gibberellic Acid and Indole Acetic Acid in Fraxinus mandshurica Rupr. var. japonica Maxim Seedlings

In the present study, we investigated the role of gibberellic acid (GA3) and indole acetic acid (IAA) in the gravity response of stems and tension wood formation using two-year-old stems of Fraxinus mandshurica Rupr. var.japonica Maxim seedlings. Forty-five seedlings were used and divided into nine groups that included five seedlings in each group. Seedlings were treated with applications of GA3 alone at concentrations of 2.89 × 10-8and 2.89 × 10-7 μmol/L, IAA alone at concentrations of 5.71×10-8 and 5.71 ×10-7 μmol/L, or their combination to the apical bud of the stem using a micropipette. Seedlings were positioned horizontally after the first treatment.The same treatments were repeated six times per week. At the end of the experiment, all seedlings were harvested. Then, stem segments were cut under a light microscope. Application of exogenous GA3 at the higher concentration stimulated the upward bending of stems, whereas exogenous IAA had no effect. A synergistic effect of GA3 and IAA on upward stem bending was observed following application of the two combinations of GA3 and IAA. Moreover, application of exogenous GA3 at the higher dose stimulated wood formation on both the upper and lower sides of the stems, whereas the mixture of GA3 and IAA had a synergistic effect on wood formation in horizontal stems. Application of exogenous IAA alone at the lower concentration (5.71×10-8 μ mol/L) or application of a mixture of the higher concentrations of GA3 (2.89 × 10-7 μmol/L) and IAA (5.71×10-7 μmol/L) inhibited the development of gelatinous fibers (the G-layer) of tension wood on the upper side of the horizontal stems. The differentiation of gelatinous fibers of tension wood was not inhibited by GA3when it was applied alone, whereas the development of the gelatinous fibers of tension wood was strongly affected by the application of IAA. The findings of the present study suggest that the development of the G-layer is not related to the dose of GA3, but needs a relatively lower concentration of IAA.     

Stimulatory effect of cytokinins and interaction with IAA on the release of lateral buds of pea plants from apical dominance

Lateral buds of pea plants can be released from apical dominance and even be transformed into dominant shoots when repeatedly treated with synthetic exogenous cytokinins (CKs). The mechanism of the effect of CKs, however, is not clear. The results in this work showed that the stimulatory effects of CKs on the growth of lateral buds and the increase in their fresh weights in pea plants depended on the structure and concentration of the CKs used. The effect of N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) was stronger than that of 6-benzylaminopurine (6-BA). Indoleacetic acid (IAA) concentration in shoot, IAA export out of the treated apex and basipetal transport in stems were markedly increased after the application of CPPU or 6-BA to the apex or the second node of pea plant. This increase was positively correlated with the increased concentration of the applied CKs. These results suggest that the increased IAA synthesis and export induced by CKs application might be responsible for the growth of lateral shoots in intact pea plants.