Auxin Transport Inhibitors and the Rooting of Hypocotyl Cuttings from Etiolated Mung-bean Vigna radiata (L.) Wilczek Seedlings

The auxin transport inhibitors 2, 3, 5-triiodobenzoic acid (TIBA)and naphthylphthalamic acid (NPA) inhibited adventitious rootformation (ARF) induced by indol-3-butyric acid (IBA) on cuttingsfrom etiolated mung-bean seedlings floated on solutions of thegrowth regulators. The concentrations of TIBA and NPA requiredfor a 25 per cent reduction in ARF with 10 µM IBA wereestimated by linear interpolation to be 11.3 µm and 0.42µM respectively. NPA is a particularly potent inhibitorof IBA-induced ARF. The inhibitory effect of either compoundwas reversible by higher concentrations of IBA. NPA had no effectwhen applied after the auxin treatment. The inhibitory effects of TIBA or NPA could not be explainedby effects on the uptake or metabolism of [2-^14C]IAA. Consideringthis and other evidence, it is suggested that NPA and possiblyTIBA are acting as specific antagonists of auxin in the inductionof ARF. Vigna radiata (L.), mung-bean, root induction, hypocotyl cuttings, auxin inhibitors, indol-3-butyric acid, 2,3,5-triiodobenzoic acid, naphthylphthalamic acid, auxin uptake, auxin metabolism, adventitious roots     

Role of basipetal auxin transport and lateral auxin movement in rooting and growth of etiolated lupin hypocotyls

The involvement of polar auxin transport (PAT) on the growth of light-grown seedlings and rooting is generally accepted, while the role of auxin and PAT on the growth of dark-grown seedlings is subject to controversy. To further investigate this question, we have firstly studied the influence of NPA, a known inhibitor of PAT, on the rooting and growth of etiolated Lupinus albus hypocotyls. Rooting was inhibited when the basal ends of de-rooted seedlings were immersed in 100 micro m NPA but was partially restored after immersion in NPA + auxin. However, NPA applied to de-rooted seedlings or the roots of intact seedlings did not inhibit hypocotyl growth. It was taken up and distributed along the organ, and actually inhibited the basipetal transport of ((3)H)-IAA applied to isolated hypocotyl sections. Since the apex is the presumed auxin source for hypocotyl growth and rooting, and the epidermis is considered the limiting factor in auxin-induced growth, the basipetal and lateral auxin movement (LAM) after application of ((3)H)-IAA to decapitated seedlings were studied, in an attempt to evaluate the role of PAT and LAM in the provision of auxin to competent cells for growth and rooting. Local application of ((3)H)-IAA to the stele led to the basipetal transport of auxin in this tissue, but the process was drastically reduced when roots were immersed in NPA since no radioactivity was detected below the apical elongation region of the hypocotyl. LAM from the stele to the cortex and the epidermis occurred during basipetal transport, since radioactivity in these tissues increased as transport time progressed. Radioactivity on a per FW basis in the epidermis was 2-4 times higher than in the cortex, which suggests that epidermal cells acted as a sink for LAM. NPA did not inhibit LAM along the elongation region. These results suggest that while PAT was essential for rooting, LAM from the PAT pathway to the auxin-sensitive epidermal cells could play a key role in supplying auxin for hypocotyl elongation in etiolated lupin seedlings.     

Flavonoids and flavonoid sulphates as probes of auxin-transport regulation in Cucurbita pepo hypocotyl segments and vesicles

The accumulation of IAA by sealed microsomal vesicles prepared from hypocotyls of dark-grown Cucurbita pepo L. (zucchini) seedlings was stimulated by N-1-naphthylphthalamic acid (NPA: an inhibitor of carrier-mediated auxin efflux and hence of polar auxin transport) as well as by quercetin and certain other flavonoids with a specificity pattern similar to that previously shown for their NPA-like effects on auxin transport and inhibition of NPA binding to saturable sites. In contrast, putatively nonpenetrant negatively charged quercetinsulphate esters did not stimulate such auxin accumulation although they were able to oppose stimulation by NPA or quercetin itself. However, the binding of NPA to hypocotyl microsomes was 30- to 80-fold more strongly inhibited by the quercetin sulphates than by unsubstituted quercetin. As with vesicles, net IAA uptake by hypocotyl segments (2 mm) from dark-grown zucchini was stimulated less effectively by quercetin-sulphate esters than by quercetin itself. We discuss the implications of these observations for the accessibility of the NPA receptor from cell wall or cytoplasm and for the coupling of its occupancy to inhibition of the auxin efflux carrier.Abbreviations ION3 mixture of 4 M carbonylcyanide m-chlorophenylhydrazone, nigericin and valinomycin - NPA N-1-naphthylphthalamic acid - PMSF phenylmethylsulphonyl fluoride This work was supported by a Studentship (I.J.F.) from the Science and Engineering Research Council and by the Gatsby Charitable Foundation. We are particularly grateful to Dr. W. Michalke for a preprint and permission to use his method of microsome preparation in advance of publication.     

Variation in indole-3-acetic acid transport and its relationship with growth in etiolated lupin hypocotyls

The relationship between the variation in polar auxin transport (PAT) and elongating growth in etiolated Lupinus albus hypocotyls was investigated. Parameters of auxin transport, such as the amount transported, intensity of the transport and sensitivity to 1-N-naphthylphthalamic acid (NPA) inhibition were measured in isolated sections from different sites (apical, middle and basal) along the hypocotyls in seedlings of different ages. Auxin transport was studied by applying radioactive indole-3-acetic acid (IAA) to upright and inverted sections. Basipetal transport was much higher than acropetal and very sensitive to NPA inhibition, which indicates that transport is polarized. Polarity was expressed as the NPA-induced inhibition and the basipetal/acropetal ratio. As a rule, both the amount of IAA transported and the polarity varied with the age of the seedlings, with values increasing from 3 to 5d and then decreasing. Both parameters were higher in apical (where most growth is localized) than in middle and basal regions, although this longitudinal gradient tended to disappear with aging as hypocotyl growth slowed and finally ceased. The application of NPA did not modify hypocotyl elongation in 5-d-old intact seedlings. Derooting of the seedlings drastically reduced elongation in the control, while NPA partially restored the growth, which suggests that NPA induces an increase in auxin in the elongation region. These results suggest that a basipetally decreasing gradient in PAT along the hypocotyl, which changes with age, may be responsible for auxin distribution pattern controlling growth.     

Effect of synthetic and natural protein tyrosine kinase inhibitors on auxin efflux in zucchini (Cucurbita pepo) hypocotyls

Basipetal auxin transport along the zucchini hypocotyl requires the cell-to-cell translocation of this hormone. Several authors have demonstrated that naphthylphthalamic acid (NPA) blocks auxin efflux. It is believed that NPA binds to a regulatory protein that modulates the activity of the carrier rather than to the carrier itself. A few regulatory mechanisms have been suggested but all lack strong supporting experimental evidence. Four classes of protein tyrosine kinase inhibitors (PTKIs) have been used to investigate the NPA perception pathway. First, six synthetic tyrphostins, inhibitors of the epidermal growth factor receptor (EGF-R), were shown to displace NPA on plasmalemma fractions. The most potent compound was (3,4-dihydroxybenzylidene)-thiocyanoacetamide (tyrphostin A47). The inactive counterpart of tyrphostin A47, (4-methoxybenzylidene)-malononitrile (tyrphostin A1), was unable to displace NPA. Tyrphostins did not inhibit auxin efflux but were antagonists of the inhibition of auxin efflux by NPA. Again, tyrphostin A47 was the most effective and tyrphostin A1 was inactive. Second, the flavonoid genistein, also an EGF-R inhibitor, showed an effect on NPA binding and NPA antagonism similar to the one for the tyrphostins. Daidzein, the inactive counterpart of genistein, was ineffective in displacing NPA and in antagonizing its inhibition of auxin efflux. Two other PTKIs, curcumin and lavendustin A, displayed similar characteristics. Calmodulin antagonists, protein serine/threonine kinase inhibitors, and phosphatase inhibitors were inactive. These results suggest that the NPA binding protein may be related to mammalian tyrosine kinases and may regulate the auxin efflux carrier by phosphorylation.     

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m⁻² s⁻¹ of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.     

Phytochromes and cryptochromes regulate the differential growth of Arabidopsis hypocotyls in both a PGP19-dependent and a PGP19-independent manner

Photoreceptors, phytochromes and cryptochromes regulate hypocotyl growth under specific conditions, by suppressing negative gravitropism, modulating phototropism and inhibiting elongation. Although these effects seem to be partially caused via the regulation of the phytohormone auxin, the molecular mechanisms underlying this process are still poorly understood. In our present study, we demonstrate that the flabby mutation enhances both phytochrome- and cryptochrome-inducible hypocotyl bending in Arabidopsis. The FLABBY gene encodes the ABC-type auxin transporter, PGP19, and its expression is suppressed by the activation of phytochromes and cryptochromes. Our current results therefore indicate that the phytochromes and cryptochromes have at least two effects upon the tropic responses of the hypocotyls in Arabidopsis: the enhancement of hypocotyl bending through the suppression of PGP19, and a PGP19-independent mechanism that induces hypocotyl bending. By the using an auxin polar transport assay and DR5:GUS expression analysis, we further find that the phytochromes inhibit basipetal auxin transport, and induce the asymmetric distribution of auxin in the hypocotyls. These data suggest that the control of auxin transport by phytochromes and cryptochromes is a critical regulatory component of hypocotyl growth in response to light.     

Genetic Analysis of the Effects of Polar Auxin Transport Inhibitors on Root Growth in Arabidopsis thaliana

Polar auxin transport inhibitors, including N-1-naphthylphthalamicacid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), have variouseffects on physiological and developmental events, such as theelongation and tropism of roots and stems, in higher plants.We isolated NPA-resistant mutants of Arabidopsis thaliana, withmutations designated pir1 and pir2, that were also resistantto TIBA. The mutations specifically affected the root-elongationprocess, and they were shown ultimately to be allelic to aux1and ein2, respectively, which are known as mutations that affectresponses to phytohormones. The mechanism of action of auxintransport inhibitors was investigated with these mutants, inrelation to the effects of ethylene, auxin, and the polar transportof auxin. With respect to the inhibition of root elongationin A. thaliana, we demonstrated that (1) the background levelof ethylene intensifies the effects of auxin transport inhibitors,(2) auxin transport inhibitors might act also via an inhibitorypathway that does not involve ethylene, auxin, or the polartransport of auxin, (3) the hypothesis that the inhibitory effectof NPA on root elongation is due to high-level accumulationof auxin as a result of blockage of auxin transport is not applicableto A. thaliana, and (4) in contrast to NPA, TIBA itself hasa weak auxin-like inhibitory effect. (Received April 12, 1996; Accepted September 2, 1996)     

Do phytotropins inhibit auxin efflux by impairing vesicle traffic?

Phytotropins such as 1-N-naphthylphthalamic acid (NPA) strongly inhibit auxin efflux, but the mechanism of this inhibition remains unknown. Auxin efflux is also strongly decreased by the vesicle trafficking inhibitor brefeldin A (BFA). Using suspension-cultured interphase cells of the BY-2 tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line, we compared the effects of NPA and BFA on auxin accumulation and on the arrangement of the cytoskeleton and endoplasmic reticulum (ER). The inhibition of auxin efflux (stimulation of net accumulation) by both NPA and BFA occurred rapidly with no measurable lag. NPA had no observable effect on the arrangement of microtubules, actin filaments, or ER. Thus, its inhibitory effect on auxin efflux was not mediated by perturbation of the cytoskeletal system and ER. BFA, however, caused substantial alterations to the arrangement of actin filaments and ER, including a characteristic accumulation of actin in the perinuclear cytoplasm. Even at saturating concentrations, NPA inhibited net auxin efflux far more effectively than did BFA. Therefore, a proportion of the NPA-sensitive auxin efflux carriers may be protected from the action of BFA. Maximum inhibition of auxin efflux occurred at concentrations of NPA substantially below those previously reported to be necessary to perturb vesicle trafficking. We found no evidence to support recent suggestions that the action of auxin transport inhibitors is mediated by a general inhibition of vesicle-mediated protein traffic to the plasma membrane.     

1-N-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid

Summary Auxin transport in corn coleoptile sections was inhibited by 2,3,5-triiodobenzoic acid (TIBA) as well as by 1-N-naphthylphthalamic acid (NPA); this inhibition was effected within 1 min of application.A particulate cell fraction-presumably plasma-membrane vesicles-specifically binds NPA and properties of these binding sites were studied using ³H-NPA and a pelletting technique. The saturation kinetics of the physiological NPA effect, i.e. the inhibition of auxin transport, is similar to that of the specific in-vitro NPA binding. Half saturation of the inhibitory effect was found with about 5×10^-7 M TIBA and with 10^-7 M NPA. Both substances also decreased the speed of movement of auxin pulses within coleoptile sections.NPA dissociates from its binding site when the particulate cell material is centrifuged through an NPA-free cushion. The NPA that is washed from its binding site can be used in another binding test without any apparent change and is chromatographically unaltered. Therefore, the NPA binding is probably reversible and non-covalent. Inhibition of auxin transport by TIBA or NPA could also be reversed when the coleoptile sections were washed in buffer.The movement of ¹³¹I-TIBA in corn coleoptiles appears to be polar in a basipetal direction. Higher concentrations of indoleacetic acid or TIBA inhibited this polar movement, suggesting that TIBA moves in the same channels as auxin. With ³H-NPA, however, no polar transport could be detected. Together with the in-vitro binding results, these data indicate that TIBA acts directly at the auxin receptor while NPA has a different receptor site.The effect of TIBA and NPA on elongation, with or without auxin, is neglegible in comparison to their effects on auxin transport.     

Effects of Inhibitors of Protein Synthesis on Transmembrane Auxin Transport in Cucurbita pepo L. Hypocotyl Segments

Pretreatment of 2?0 mm segments of etiolated zucchini (Cucurbitapepo L.) hypocotyl with cycloheximide (CH) or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide(MDMP) eliminated the stimulation by N-1-naphthylphthalamicacid (NPA) of net uptake of [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA),but had relatively little effect on the net uptake of IAA inthe absence of NPA. The efflux of [1-¹⁴C]IAA from preloadedsegments was not substantially affected by inhibitor pretreatmentin the absence of NPA, but CH pretreatment significantly inhibitedthe reduction of efflux caused by NPA. Pretreatment with CHor MDMP did not affect net uptake by segments of the pH probe[2-¹⁴C]5,5-dimethyl-oxazolidine-2,4-dione ([2-¹⁴C]DMO), or thenet uptake of [¹⁴C]-labelled 3-O-methylglucose ([¹⁴C]3-0-MeGlu),suggesting that neither inhibitor affected intracellular pHor the general function of proton symporters in the plasma membrane.Both compounds reduced the incorporation of label from [³⁵S]methionineinto trichloroacetic acid (TCA)-insoluble fractions of zucchinitissue, confirming their inhibitory effect on protein synthesis. The steady-state association of [³H]IAA with microsomal vesiclesprepared from zucchini hypocotyl tissue was enhanced by theinclusion of NPA in the uptake medium. The stimulation by NPAof [³H]IAA association with microsomes was substantially reducedwhen the tissue was pretreated with CH. However, CH pretreatmentdid not affect the level of high affinity NPA binding to themembranes indicating that treatments did not result in lossof NPA receptors. It is suggested that the auxin transport site on the effluxcarrier system and the receptor site for NPA may reside on separateproteins linked by a third, rapidly turned-over, transducingprotein. Key words: Auxin carriers, auxin efflux, Cucurbita pepo, phytotropin receptors     

Identification of dehydrocostus lactone and 4-hydroxy-β-thujone as auxin polar transport inhibitors

The survey of naturally occurring of auxin polar transport regulators in Asteraceae was investigated using the radish (Raphanus sativus L.) hypocotyl bioassay established in this study. Significant auxin polar transport was observed when radiolabeled indole-3-acetic acid (IAA) was applied at the apical side of radish hypocotyl segments, but not when it was applied at the basal side of the segments. Almost no auxin polar transport was observed in radish hypocotyl segments treated with synthetic auxin polar transport inhibitors of N-(1-naphthyl)phthalamic acid (NPA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) at 0.5 μg/plant. 2,3,5-Triiodobenzoic acid (TIBA) at 0.5 μg/plant was less effective than NPA and HFCA, and p-chlorophenoxyisobutyric acid (PCIB) at 0.5 μg/plant had almost no effect on auxin polar transport in the radish hypocotyl bioassay. These results strongly suggest that the radish hypocotyl bioassay is suitable for the detection of bioassay-derived auxin polar transport regulators. Using the radish hypocotyl bioassay and physicochemical analyses, dehydrocostus lactone (decahydro-3,6,9-tris-methylene-azulenol(4,5-b)furan-2(3H)-one) and 4-hydroxy-β-thujone (4-hydroxy-4-methyl-1-(1-methylethyl)-bicyclo[3.1.0]hexan-3-one) were successfully identified as auxin polar transport inhibitors from Saussurea costus and Arctium lappa, and Artemisia absinthium, respectively. About 50 and 40 % inhibitions of auxin polar transport in radish hypocotyl segments were observed at 2.5 μg/plant pre-treatment (see “Materials and methods”) of dehydrocostus lactone and 4-hydroxy-β-thujone, respectively. Although the mode of action of these compounds in inhibiting auxin polar transport has not been clear yet, their possible mechanisms are discussed.     

The growth of tomato (Lycopersicon esculentum Mill.) hypocotyls in the light and in darkness differentially involves auxin.

Light and auxin antagonistically regulate hypocotyl elongation. We have investigated the physiological interactions of light and auxin in the control of tomato (Lycopersicon esculentum Mill.) hypocotyl elongation by studying the auxin-insensitive mutant diageotropica (dgt). The length of the hypocotyls of the dgt mutant is significantly reduced when compared to the wild type line Ailsa Craig (AC) in the dark and under red light, but not under the other light conditions tested, indicating that auxin sensitivity is involved in the elongation of hypocotyls only in these conditions. Similarly, the auxin transport inhibitor naphthylphthalamic [correction of naphtylphtalamic] acid (NPA) differentially affects elongation of dark- or light-grown hypocotyls of the MoneyMaker (MM) tomato wild type. Using different photomorphogenic mutants, we demonstrate that at least phytochrome A, phytochrome B1 and, to a much lesser extent [correction of extend], cryptochrome 1, are necessary for a switch from an auxin transport-dependent elongation of hypocotyls in the dark to an auxin transport-independent elongation in the light. Interestingly, the dgt mutant and NPA-treated seedlings exhibit a looped phenotype only under red light, indicating that the negative gravitropism of hypocotyls also differentially involves auxin in the various light conditions.     

Cadmium interferes with maintenance of auxin homeostasis in Arabidopsis seedlings

Auxin and its homeostasis play key roles in many aspects of plant growth and development. Cadmium (Cd) is a phytotoxic heavy metal and its inhibitory effects on plant growth and development have been extensively studied. However, the underlying molecular mechanism of the effects of Cd stress on auxin homeostasis is still unclear. In the present study, we found that the root elongation, shoot weight, hypocotyl length and chlorophyll content in wild-type (WT) Arabidopsis seedlings were significantly reduced after exposure to Cd stress. However, the lateral root (LR) formation was markedly promoted by Cd stress. The level and distribution of auxin were both greatly altered in primary root tips and cotyledons of Cd-treated plants. The results also showed that after Cd treatment, the IAA content was significantly decreased, which was accompanied by increases in the activity of the IAA oxidase and alteration in the expression of several putative auxin biosynthetic and catabolic genes. Application of the auxin transport inhibitor, 1-naphthylphthalamic acid (NPA) and 1-naphthoxyacetic acid (1-NOA), reversed the effects of Cd on LR formation. Additionally, there was less promotion of LR formation by Cd treatment in aux1-7 and pin2 mutants than that in the WT. Meanwhile, Cd stress also altered the expression of PINs and AUX1 in Arabidopsis roots, implying that the auxin transport pathway is required for Cd-modulated LR development. Taken together, these findings suggest that Cd stress disturbs auxin homeostasis through affecting auxin level, distribution, metabolism, and transport in Arabidopsis seedling.     

Auxin and Ethylene-stimulated Adventitious Rooting in Relation to Tissue 9

We have previously shown that both endogenous auxin and ethylenepromote adventitious root formation in the hypocotyls of derootedsunflower (Helianthus annuus) seedlings. Experiments here showedthat promotive effects on rooting of the ethylene precursor,1-aminocyclopropane-l-carboxylic acid (ACC) and the ethylene-releasingcompound, ethephon (2-chloro-ethylphosphonic acid), dependedon the existence of cotyledons and apical bud (major sourcesof auxin) or the presence of exogenously applied indole-3-aceticacid (IAA). Ethephon, ACC, aminoethoxyvinylglycine (an inhibitorof ethylene biosynthesis), and silver thiosulphate (STS, aninhibitor of ethylene action), applied for a length of timethat significantly influenced adventitious rooting, showed noinhibitory effect on the basipetal transport of [³H]IAA. Theseregulators also had no effect on the metabolism of [³H]IAA andendogenous IAA levels measured by gas chromatography-mass spectrometry.ACC enhanced the rooting response of hypocotyls to exogenousIAA and decreased the inhibition of rooting by IAA transportinhibitor, N-1-naphthylphthalamic acid (NPA). STS reduced therooting response of hypocotyls to exogenous IAA and increasedthe inhibition of rooting by NPA. Exogenous auxins promotedethylene production in the rooting zone of the hypocotyls. Decapitationof the cuttings or application of NPA to the hypocotyl belowthe cotyledons did not alter ethylene production in the rootingzone, but greatly reduced the number of root primordia. We concludethat auxin is a primary controller of adventitious root formationin sunflower hypocotyls, while the effect of ethylene is mediatedby auxin. Key words: Auxin, ethylene, adventitious rooting, sunflower     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

Clathrin regulates blue light‐triggered lateral auxin distribution and hypocotyl phototropism in Arabidopsis

Phototropism is the process by which plants grow towards light in order to maximize the capture of light for photosynthesis, which is particularly important for germinating seedlings. In Arabidopsis, hypocotyl phototropism is predominantly triggered by blue light (BL), which has a profound effect on the establishment of asymmetric auxin distribution, essential for hypocotyl phototropism. Two auxin efflux transporters ATP‐binding cassette B19 (ABCB19) and PIN‐formed 3 (PIN3) are known to mediate the effect of BL on auxin distribution in the hypocotyl, but the details for how BL triggers PIN3 lateralization remain poorly understood. Here, we report a critical role for clathrin in BL‐triggered, PIN3‐mediated asymmetric auxin distribution in hypocotyl phototropism. We show that unilateral BL induces relocalization of clathrin in the hypocotyl. Loss of clathrin light chain 2 (CLC2) and CLC3 affects endocytosis and lateral distribution of PIN3 thereby impairing BL‐triggered establishment of asymmetric auxin distribution and consequently, phototropic bending. Conversely, auxin efflux inhibitors N‐1‐naphthylphthalamic acid and 2,3,5‐triiodobenzoic acid affect BL‐induced relocalization of clathrin, endocytosis and lateralization of PIN3 as well as asymmetric distribution of auxin. These results together demonstrate an important interplay between auxin and clathrin function that dynamically regulates BL‐triggered hypocotyl phototropism in Arabidopsis.     

Rapid auxin-induced nitric oxide accumulation and subsequent tyrosine nitration of proteins during adventitious root formation in sunflower hypocotyls

Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25–80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development.     

Response to auxin changes during maturation-related loss of adventitious rooting competence in loblolly pine (Pinus taeda) stem cuttings

Hypocotyl cuttings (from 20- and 50-day-old Pinus taeda L. seedlings) rooted readily within 30 days in response to exogenous auxin, while epicotyl cuttings (from 50-day-old seedlings) rarely formed roots within 60 days. Responses to auxin during adventitious rooting included the induction of cell reorganization and cell division, followed by the organization of the root meristem. Explants from the bases of both epicotyl and hypocotyl cuttings readily formed callus tissue in response to a variety of auxins, but did not organize root meristems. Auxin-induced cell division was observed in the cambial region within 4 days, and later spread to the outer cortex at the same rate in both tissues. Cells at locations that would normally form roots in foliated hypocotyl cuttings did not produce callus any differently than those in other parts of the cortex. Therefore, auxin-induced root meristem organization appeared to occur independently of auxin-induced cell reorganization/division. The observation that N-(1-naphthyl)phthalamic acid (NPA) promoted cellular reorganization and callus formation but delayed rooting implies the existence of an auxin signal transduction pathway that is specific to root meristem organization. Attempts to induce root formation in callus or explants without foliage were unsuccessful. Both the cotyledon and epicotyl foliage provided a light-dependent product other than auxin that promoted root meristem formation in hypocotyl cuttings.