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In plants, the effective mobilization of seed nutrient reserves is crucial during germination and for seedling establishment. The Arabidopsis H+-PPase-loss-of-function fugu5 mutants exhibit a reduced number of cells in the cotyledons. This leads to enhanced post-mitotic cell expansion, also known as compensated cell enlargement (CCE). While decreased cell numbers have been ascribed to reduced gluconeogenesis from triacylglycerol, the molecular mechanisms underlying CCE remain ill-known. Given the role of indole 3-butyric acid (IBA) in cotyledon development, and because CCE in fugu5 is specifically and completely cancelled by ech2, which shows defective IBA-to-indoleacetic acid (IAA) conversion, IBA has emerged as a potential regulator of CCE. Here, to further illuminate the regulatory role of IBA in CCE, we used a series of high-order mutants that harbored a specific defect in IBA-to-IAA conversion, IBA efflux, IAA signaling, or vacuolar type H+-ATPase (V-ATPase) activity and analyzed the genetic interaction with fugu5-1. We found that while CCE in fugu5 was promoted by IBA, defects in IBA-to-IAA conversion, IAA response, or the V-ATPase activity alone cancelled CCE. Consistently, endogenous IAA in fugu5 reached a level 2.2-fold higher than the WT in 1-week-old seedlings. Finally, the above findings were validated in icl-2, mls-2, pck1-2 and ibr10 mutants, in which CCE was triggered by low sugar contents. This provides a scenario in which following seed germination, the low-sugar-state triggers IAA synthesis, leading to CCE through the activation of the V-ATPase. These findings illustrate how fine-tuning cell and organ size regulation depend on interplays between metabolism and IAA levels in plants., Competing Interests: The authors have declared that no competing interests exist.

Adventitious roots form only at the proximal cut surface (PCS) but not at the distal cut surface (DCS) of mango cotyledon segments. In this study, mango embryos treated with indole-3-butyric acid (IBA) showed significantly increased adventitious root formation, while those treated with 2, 3, 5-triiodobenzoic acid (TIBA) demonstrated complete inhibition of adventitious rooting. Mango embryos treated with auxin influx inhibitors demonstrated lower inhibition of adventitious roots than those treated with TIBA. The endogenous indol-3-acetic acid (IAA) content on the PCS and DCS was similar at 0 h, then increased on both surfaces after 6 h, and IAA content on the PCS were always higher than those on the DCS. We cloned three genes encoding auxin efflux carriers (i.e., MiPIN2-4) and examined their temporal and spatial expression patterns under different treatments. Relative expression of all MiPINs studied was very low at 0 h but significantly increased on both PCS and DCS from 1 d to 10 d, to varying degrees. We overexpressed MiPIN1-4 in Arabidopsis plants and found a significant increase in adventitious root quantity in MiPIN1 and MiPIN3 transgenic lines. Immunofluorescence results showed that MiPIN1 and MiPIN3 are primarily localized in the vascular tissues and the cells adjacent to abaxial surface. In conclusion, we propose that in mango cotyledon segments, wounding stimulates IAA biosynthesis, the transcription levels of PIN genes were significantly increased in different magnitudes on the PCS and DCS, resulting in polar IAA transport from the DCS to PCS via the vascular tissues, thereby triggering adventitious root formation., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Hormesis, which describes the stimulatory effect of low doses of toxic substances on growth, is a well-known phenomenon in the plant and animal kingdoms. However, the mechanisms that are involved in this phenomenon are still poorly understood. We performed preliminary studies on corn coleoptile sections, which showed a positive correlation between the stimulation of growth by Cd or Pb and an increase in the auxin and H 2 O 2 content in the coleoptile sections. Subsequently, we grew corn seedlings in hydroponic culture and tested a wide range of Cd or Pb concentrations in order to determine hormetic growth stimulation. In these seedlings the gas exchange and the chlorophyll a fluorescence, as well as the content of chlorophyll, flavonol, auxin and hydrogen peroxide, were measured. We found that during the hormetic stimulation of growth, the response of the photosynthetic apparatus to Cd and Pb differed significantly. While the application of Cd mostly caused a decrease in various photosynthetic parameters, the application of Pb stimulated some of them. Nevertheless, we discovered that the common features of the hormetic stimulation of shoot growth by heavy metals are an increase in the auxin and flavonol content and the maintenance of hydrogen peroxide at the same level as the control plants.

Cultivating rice in wet or water direct seeding systems is simple and time and labor efficient. Rice (Oryza sativa) seeds are a unique cereal that can germinate not only when submerged, but also in anoxic conditions. Many complicated hormone signals interact in submerged seed germination. Ethylene is involved in rice coleoptile elongation, but little is known regarding the role of auxin signaling under submergence. This study demonstrated that the coleoptile is shorter and curlier when submerged with 2,3,5-triiodobenzoic acid (TIBA). In transcriptomic analysis, 3448 of the 31,860 genes were upregulated, and 4360 genes were downregulated with submergence and TIBA treatment. The Gene Ontology function classification results demonstrated that upregulated differentially expressed genes (DEGs) were mainly involved in redox, stress, and signal transduction, whereas the down-regulated DEGs were mainly involved in RNA transcription, stress, and development. Furthermore, auxin signaling involved in the carbohydrate metabolism pathway was demonstrated while using transcriptomic analysis and confirmed in a quantitative real-time polymerase chain reaction. In addition, the transcript levels of development-related genes and mitochondria-electron- transport-related genes were regulated by auxin signaling under submergence. Auxin signaling was not only involved in regulating rice coleoptile elongation and development, but also regulated secondary metabolism, carbohydrate metabolism, and mitochondria electron transport under submergence. Our results presented that auxin signaling plays an important role during rice coleoptile elongation upon the submergence condition and improving the advance of research of direct rice seeding system., Competing Interests: The authors declare that they have no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Naphthoquinones, plants secondary metabolites are known for their antibacterial, antifungal, anti-inflammatory, anti-cancer and anti-parasitic properties. The biological activity of naphthoquinones is connected with their ability to generate reactive oxygen species and to modify biological molecules at their nucleophilic sites. In our research, the effect of naphthazarin (DHNQ) combined with 2-hydroxy-1,4-naphthoquinone (NQ-2-OH) or 1,4-naphthoquinone (1,4-NQ) on the elongation growth, pH changes of the incubation medium, oxidative stress and redox activity of maize coleoptile cells were investigated. This paper describes experiments performed with maize ( Zea mays L.) coleoptile segments, which is a classical model system to study plant cell elongation growth. The data presented clearly demonstrate that lawsone and 1,4-naphthoquinone combined with naphthazarin, at low concentrations (1 and 10 nM), reduced the endogenous and IAA-induced (Indole-3-Acetic Acid) elongation growth of maize coleoptile segments. Those changes in growth correlated with the proton concentration in the incubation medium, which suggests that the changes in the growth of maize coleoptile segments observed in the presence of naphthoquinones are mediated through the activity of PM H⁺-ATPase. The presence of naphthoquinones induced oxidative stress in the maize coleoptile tissue by producing hydrogen peroxide and causing changes in the redox activity. Moreover, the incubation of maize segments with both naphthoquinones combined with naphthazarin resulted in lipid peroxidation and membrane damage. The regulation of PM H⁺-ATPase activity, especially its inhibition, may result from two major types of reaction: first, a direct interaction between an enzyme and naphthoquinone, which leads to the covalent modification of the protein thiols and the generation of thioethers, which have been found to alter the activity of the PM H⁺-ATPases; second, naphthoquinones induce reactive oxygen species (ROS) production, which inhibits PM H⁺-ATPases by increasing cytosolic Ca 2+ . This harmful effect was stronger when naphthazarin and 1,4-naphthoquinone were added together. Taking these results into account, it can be suggested that by combining naphthoquinones in small quantities, an alternative to synthetic pesticides could be developed.

Key Message: Enhancement of endoreduplication in dark-grown hypocotyl is a common feature in dicotyledonous polysomatic plants, and TIBA-mediated inhibition of the endoreduplication is partially due to abnormal actin organization. Many higher plant species use endoreduplication during cell differentiation. However, the mechanisms underlying this process have remained elusive. In this study, we examined endoreduplication in hypocotyls and cotyledons in response to light in some dicotyledonous plant species. Enhancement of endoreduplication was found in the dark-grown hypocotyls of all the polysomatic species analyzed across five different families, indicating that this process is a common feature in dicotyledonous plants having polysomatic tissues. Conversely, endoreduplication was enhanced in the light-grown cotyledons in four of the five species analyzed. We also analyzed the effect of a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA) on endoreduplication in hypocotyl and cotyledon tissues of radish (Raphanus sativus L. var. longipinnatus Bailey). TIBA was found to inhibit and promote endoreduplication in hypocotyls and cotyledons, respectively, suggesting that the endoreduplication mechanism differs in these organs. To gain insight into the effect of TIBA, radish and spinach (Spinacia oleracea L.) seedlings were treated with a vesicle-trafficking inhibitor, brefeldin A, and an actin polymerization inhibitor, cytochalasin D. Both of the inhibitors partially inhibited endoreduplication of the dark-grown hypocotyl tissues, suggesting that the prominent inhibition of endoreduplication by TIBA might be attributed to its multifaceted role.

Background and Aims: The mechanism of auxin action on ion transport in growing cells has not been determined in detail. In particular, little is known about the role of chloride in the auxin-induced growth of coleoptile cells. Moreover, the data that do exist in the literature are controversial. This study describes experiments that were carried out with maize (Zea mays) coleoptile segments, this being a classical model system for studies of plant cell elongation growth., Methods: Growth kinetics or growth and pH changes were recorded in maize coleoptiles using two independent measuring systems. The growth rate of the segments was measured simultaneously with medium pH changes. Membrane potential changes in parenchymal cells of the segments were also determined for chosen variants. The question of whether anion transport is involved in auxin-induced growth of maize coleoptile segments was primarily studied using anion channel blockers [anthracene-9-carboxylic acid (A-9-C) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS)]. In addition, experiments in which KCl was replaced by KNO3 were also performed., Key Results: Both anion channel blockers, added at 0·1 mm, diminished indole-3-acetic acid (IAA)-induced elongation growth by ~30 %. Medium pH changes measured simultaneously with growth indicated that while DIDS stopped IAA-induced proton extrusion, A-9-C diminished it by only 50 %. Addition of A-9-C to medium containing 1 mm KCl did not affect the characteristic kinetics of IAA-induced membrane potential changes, while in the presence of 10 mm KCl the channel blocker stopped IAA-induced membrane hyperpolarization. Replacement of KCl with KNO3 significantly decreased IAA-induced growth and inhibited proton extrusion. In contrast to the KCl concentration, the concentration of KNO3 did not affect the growth-stimulatory effect of IAA. For comparison, the effects of the cation channel blocker tetraethylammonium chloride (TEA-Cl) on IAA-induced growth and proton extrusion were also determined. TEA-Cl, added 1 h before IAA, caused reduction of growth by 49·9 % and inhibition of proton extrusion., Conclusions: These results suggest that Cl(-) plays a role in the IAA-induced growth of maize coleoptile segments. A possible mechanism for Cl(-) uptake during IAA-induced growth is proposed in which uptake of K(+) and Cl(-) ions in concert with IAA-induced plasma membrane H(+)-ATPase activity changes the membrane potential to a value needed for turgor adjustment during the growth of maize coleoptile cells., (© The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Indole-3-acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole-3-pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC-expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1-His suggested that yucasin strongly inhibited YUC1-His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over-expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss-of-function mutant of TAA1, sav3-2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l-kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin-treated sav3-2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

The monocot coleoptile tip region has been generally supposed to be the source of IAA to supply IAA to basal parts by the polar IAA transport system, which results in gravi- and phototropic curvature of coleoptiles. Based on this IAA transport system and gravitropism of maize coleoptiles, we have developed two screening methods to identify small molecules from a large chemical library that inhibit IAA transport. The methods detect molecules that affect (i) gravitropic curvature of coleoptiles; and (ii) the amount of IAA transported from the tip. From 10,000 chemicals, eight compounds were identified and categorized into two groups. Four chemicals in group A decreased IAA transport from the tip, and increased endogenous IAA levels in the tip. The structures of two compounds resembled that of 1-N-naphthylphthalamic acid (NPA), but those of the other two differed from structures of known IAA transport inhibitors. Four chemicals in group B strongly inhibited IAA transport from the tip, but IAA levels at the tip were only slightly affected. At higher concentrations, group B compounds inhibited germination of Arabidopsis, similarly to brefeldin A (BFA). Analysis of the cellular distribution of PIN2-green fluorescent protein (GFP) and PIN1-GFP in Arabidopsis revealed that one of the four chemicals in group B induced internalization of PIN1 and PIN2 proteins into vesicles smaller than BFA bodies, suggesting that this compound affects cellular vesicle trafficking systems related to PIN trafficking. The eight chemicals identified here will be a useful tool for understanding the mechanisms of IAA transport in plants.

In axial organs of juvenile plants, the phytohormone auxin (indole-3-acetic acid, IAA) rapidly mediates cell wall loosening and hence promotes turgor-driven elongation. In this study, we used rye (Secale cereale) coleoptile sections to investigate possible effects of IAA on the proteome of the cells. In a first set of experiments, we document that IAA causes organ elongation via promotion of expansion of the rigid outer wall of the outer epidermis. A quantitative comparison of the proteome (membrane-associated proteins), using two-dimensional difference gel electrophoresis (2-D DIGE), revealed that, within 2 h of auxin treatment, at least 16 protein spots were up- or down-regulated by IAA. These proteins were identified using reverse-phase liquid chromatography electrospray tandem mass spectrometry. Four of these proteins were detected in the growth-controlling outer epidermis and were further analysed. One epidermal polypeptide, a small Ras-related GTP-binding protein, was rapidly down-regulated by IAA (after 0.5 h of incubation) by -35% compared to the control. Concomitantly, a subunit of the 26S proteasome was up-regulated by IAA (+30% within 1 h). In addition, this protein displayed IAA-mediated post-translational modification. The implications of these rapid auxin effects with respect to signal transduction and IAA-mediated secretion of glycoproteins (osmiophilic nano-particles) into the growth-controlling outer epidermal wall are discussed., (© 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.)

The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) is a useful compound for investigating the role of auxin flow during plant growth and development. In Arabidopsis lines, applications of TIBA during the induction phase of somatic embryogenesis inhibit embryo development and induce the differentiation of the meristematic cells of the shoot apical meristem (SAM), leading to the fusion of the cotyledons. These abnormalities were associated to changes in the expression levels of auxin transporter genes (PINs) and endogenous distribution of IAA. Treatments of TIBA caused a rapid accumulation of IAA within the epidermal and cortical root cells of the explants (bent-cotyledon zygotic embryos), as well as in the apical and sub-apical cells of the callus generated by the surface of the cotyledons of the explants. Within the callus only a few cells acquired meristematic characteristics, and this was associated to low expression levels of genes involved in embryogenic cell fate acquisition, such as WUSCHEL (WUS), LEAFY COTYLEDON 1 and 2. All these deleterious effects were attenuated when TIBA was administered to lines over-expressing SHOOT MERISTEMLESS (STM) isolated from Brassica oleracea (Bo), B. napus (Bn), and B. rapa (Br). Of interest, TIBA-treated explants of Arabidopsis lines over-expressing the Brassica STM were able to produce a large number of embryogenic cells and somatic embryos which exhibited a normal morphology and two distinct cotyledons. A proposed reason for this behaviour was ascribed to the ability of the transformed tissue to retain a normal distribution of auxin in the presence of TIBA. Proper localization of auxin might be required for the normal expression of several genes needed for the acquisition of embryogenic competence and formation of somatic embryos., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Nine genotypes of melon (Cucumis melo L.) were selected for the investigation of regeneration. Most of the tested varieties showed regeneration ability on medium containing 0.5 mg l⁻¹ or 1 mg l⁻¹ BA, but following the appearance of shoot buds, only six varieties produced leafy shoots. The effect of combinations of BA with different auxins (IAA, NA, 2,4-D) and ABA in the culture medium on shoot regeneration was tested on cotyledon explants of 'Hógolyó' and 'Hale's Best'. To establish optimal conditions for the adventitious shoot induction six types of seedling-derived explants were prepared from seedlings of four different ages. The best results for shoot forming capacity were achieved with cotyledons followed by decapitated seedlings and hypocotyls derived from 4-day-old seedlings. Cotyledon segments of 'Hógolyó' and 'Hale's Best' were also cultivated on media with different concentrations of IAA and BA supplemented with 0.26 mg l⁻¹ ABA. The highest number of well-formed plantlets was counted for 'Hógolyó' on the medium supplemented with 0.9 mg l⁻¹ BA+ 0.6 mg l⁻¹ IAA+ 0.26 mg l⁻¹ ABA. This is the first report on the in vitro regeneration of 'Hógolyó' from decapitated seedling and hypocotyl explants and of 'JavÍtott Zentai', 'Muskotály', 'Hógolyó', 'Tétényi csereshéjú' and 'Magyar Kincs' from cotyledon explants.

Since Darwins' pioneering experiments, monocot coleoptiles have been used to investigate indole-3-acetic acid (IAA) production and polar transport. In a recent study, using maize coleoptiles, we first showed that the asymmetric IAA flow from the tip in response to gravistimulus directly affects the TIR/AFBs-mediated auxin signaling pathway, which results in tropic curvature. In this work, we also showed that IAA is synthesized from tryptophan (Trp) in the apical 1 mm region, and from there the synthesized IAA moves to the basal part via polar transport by ZmPIN1(s). These results clearly show the importance of the tip region in perception of gravistimulus and in transmitting the perceived information to the lower region using IAA as a messenger signal. Thus, it is concluded that IAA production and transport from the tip are key factors controlling the cell elongation rate in the lower part of the coleoptiles, by making a regulated and dynamic IAA flow net work in the coleoptiles.

The use of the grass coleoptile for the elucidation of the mechanism of cell elongation is a legacy of the classic experiments of Charles Darwin, who described this organ in 1880 as a "reddish sheath". In this study we quantified the growth of intact, etiolated rye (Secale cereale L.) seedlings and selected 3-day-old (growing) vs. 4-day-old (pierced) coleoptiles for a comparative analysis. Upon emergence of the reddish primary leaf on day 4 after sowing, growth slowed down by 70% and the sensitivity of the coleoptile to auxin (Indole-3-acetic acid) was lost, but turgor pressure was maintained. A quantitative comparison of the proteome (microsomal- and cytoplasmic protein fractions, respectively), using the two-dimensional difference gel electrophoresis (2-D DIGE)-technique, revealed that at least 28 proteins (spots) were differentially up- or down-regulated more than 1.5-fold. Eight of these proteins were identified by reverse-phase liquid chromatography-electrospray tandem mass spectrometry. Cessation of coleoptile growth was associated with the down-regulation (- 81 %) of subunit E of the vacuolar H(+)-ATPase (V-ATPase) and the up-regulation of enzymes involved in lignification (phenylalanine ammonia lyase) and wounding responses (xylanase inhibitor; two lipoxygenases). We conclude that the degradation of the V-ATPases, electrogenic proton pumps on the tonoplast and the membranes of the Golgi- dependent secretory pathway, may be the cause for the cessation of growth in turgid coleoptiles and the associated loss of auxin sensitivity. However, the intracellular signals that cause these proteomic changes have not yet been identified.

The light- and indole-3-acetic acid (IAA)-regulated 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene (PnACS) from Pharbitis nil was isolated. Here, it was shown that the gene was expressed in cotyledons, petioles, hypocotyls, root and shoot apexes both in light- and dark-grown seedlings. The highest expression level of PnACS was found in the roots. IAA applied to the cotyledons of P. nil seedlings caused a clear increase of PnACS messenger accumulation in all the organs examined. In this case, the most IAA-responsive were the hypocotyls. Our studies revealed that the PnACS transcript level in the cotyledons exhibited diurnal oscillations under both long-day (LD) and short-day (SD) conditions. IAA applied at the beginning of inductive darkness caused a dramatic increase in the expression of PnACS, suggesting that the inhibitory effect of IAA on P. nil flowering may result from its stimulatory effect on ethylene production.

Gravity-controlled transport of auxin was studied for a negative gravitropic response in the early growth stage of etiolated pea (Pisum sativum L. cv. Alaska) seedlings, in which epicotyl bending was observed near the cotyledon nodes of the seedlings grown continuously from seeds germinated in a horizontal or an inclined position. Increased expression of an auxin-inducible gene, PsIAA4/5, was observed in the elongated side of epicotyls grown in a horizontal or an inclined position. Regardless of the conditions of seed germination, polar auxin transport in the proximal side of the first internodes of the seedlings was significantly higher than in the distal side. Polar auxin transport in the proximal side of epicotyls grown in an inclined position was significantly lower than in those grown in a horizontal position. In contrast, lateral auxin distribution from the proximal to distal sides in epicotyls grown in an inclined position was significantly higher than in epicotyls grown in a horizontal position. Accumulation of PsPIN1 mRNA encoding a putative auxin efflux facilitator, which was observed in vascular tissue, cortex and epidermis in the proximal and distal sides of epicotyls, was markedly influenced by gravistimulation. These results strongly suggest that gravistimulation induces changeable polar auxin transport and one-way lateral auxin distribution in epicotyls as well as asymmetric auxin accumulation in the proximal and distal sides of epicotyls, resulting in a negative gravitropic response of epicotyls in the early growth stage of pea seedlings.

We present here a transient expression system that allows the response of actin microfilaments to physiological stimuli (changes in auxin content, light) to be observed in single cells in vivo. Etiolated, intact rice seedlings are attached to glass slides, transfected biolistically with talin fused to yellow-fluorescent protein to visualize actin microfilaments, and either treated with auxin or irradiated. The talin marker labels distinct populations of actin that are differentially expressed depending on the physiological state of the coleoptile (active elongation versus ceased elongation). Whereas longitudinal transvacuolar bundles prevail in cells that have ceased to elongate, fine cortical strands are characteristic for elongating cells. The visualized actin structures remain dynamic and responsive to signals. Exogenous auxin triggers a loosening of the bundles and an extension of the cortical strands, whereas irradiation reorientates cortical strands into longitudinal arrays. These responses correspond in quality and timing to the signal responses inferred previously from fixed specimens and biochemical studies. In big advantage over those methods it is now possible to observe them directly at the single cell level. Thus, the rice coleoptile system can be used as a convenient model to study actin dynamics in vivo, in response to physiologically relevant stimuli.

An early auxin-induced gene was isolated from rice (Oryza sativa L. subsp. japonica cv Nihonmasari) coleoptiles by a fluorescent-labeled differential display screen. The full-length gene contains conserved domains characteristic for the cytochrome p450 superfamily. This gene, designated as CYP87A3, was weakly expressed in dark-grown coleoptiles but was up-regulated rapidly and transiently when coleoptile segments were incubated in 5 microm indole-3-acetic acid. This induction by auxin could not be suppressed by cycloheximide. Depletion of segments from endogenous auxin reduced the amount of CYP87A3 transcripts. The CYP87A3 transcript level was rapidly, although transiently, up-regulated in response to light as well. The observed pattern of gene regulation might indicate a role in the suppression of auxin-induced coleoptile growth. The role of CYP87A3 is discussed with respect to auxin signaling in the regulation of coleoptile growth.

The plant hormone auxin affects cell elongation in both roots and shoots. In roots, the predominant action of auxin is to inhibit cell elongation while in shoots auxin, at normal physiological levels, stimulates elongation. The question of whether the primary receptor for auxin is the same in roots and shoots has not been resolved. In addition to its action on cell elongation in roots and shoots, auxin is transported in a polar fashion in both organs. Although auxin transport is well characterized in both roots and shoots, there is relatively little information on the connection, if any, between auxin transport and its action on elongation. In particular, it is not clear whether the protein mediating polar auxin movement is separate from the protein mediating auxin action on cell elongation or whether these two processes might be mediated by one and the same receptor. We examined the identity of the auxin growth receptor in roots and shoots by comparing the response of roots and shoots of the grass Zea mays L. and the legume Vigna mungo L. to indole-3-acetic acid, 2-naphthoxyacetic acid, 4,6-dichloroindoleacetic acid, and 4,7-dichloroindoleacetic acid. We also studied whether or not a single protein might mediate both auxin transport and auxin action by comparing the polar transport of indole-3-acetic acid and 2-naphthoxyacetic acid through segments from Vigna hypocotyls and maize coleoptiles. For all of the assays performed (root elongation, shoot elongation, and polar transport) the action and transport of the auxin derivatives was much greater in the dicots than in the grass species. The preservation of ligand specificity between roots and shoots and the parallels in ligand specificity between auxin transport and auxin action on growth are consistent with the hypothesis that the auxin receptor is the same in roots and shoots and that this protein may mediate auxin efflux as well as auxin action in both organ types.

We screened for auxin-induced genes with an expression correlated to the auxin-induced growth response from rice coleoptiles by fluorescent differential display. A rice homologue of the auxin response factor (ARF) family of transcriptional regulators, OsARF1, was identified. An OsARF1:GFP fusion protein was localized to the nucleus. Steady-state levels of OsARF1 mRNA correlated positively with auxin-dependent differential growth: gravitropic stimulation enhanced the amount of OsARF1 transcript in the lower, faster-growing flank accompanied by a decrease in the upper flank of gravitropically stimulated rice coleoptiles. Exogenous auxin up-regulated the steady-state level of OsARF1 mRNA within 15-30 min. This up-regulation is independent of de novo protein synthesis. Thus, OsARF1 is the first ARF that classifies as an early auxin-responsive gene. The observed auxin-dependent regulation comprises a new level of regulation in auxin-induced gene expression and is discussed as a possible feedback mechanism in plant growth control.

The physiological activity of exogenous 4-Cl-IAA, as compared to IAA, was examined in maize coleoptile segments. It was found that in this model system 4-Cl-IAA is much more active in the stimulation of elongation than IAA. Simultaneous measurements of growth and external pH indicated that administration of either IAA or 4-Cl-IAA resulted in medium acidification. The kinetics of the pH changes, however, were faster after the addition of 4-Cl-IAA. In contrast to IAA, the coleoptile segments treated with chlorinated auxin were not able to increase medium pH after its initial drop. The re-addition of IAA after 5 h further enhanced growth over the next 2 h by 31%. By contrast, the re-addition of 4-Cl-IAA at the same time protocol as IAA did not cause an additional effect. The administration of 10 microM IAA induced in maize coleoptile cells a transient depolarization followed by a slow hyperpolarization of their membrane potential. In contrast to IAA, 4-Cl-IAA at 1 microM caused an immediate hyperpolarization of the membrane potential which, on average, was 2-fold greater than for IAA. The results reported here provide further evidence that 4-Cl-IAA is much more active, as compared to IAA, in stimulating the growth of maize coleoptile segments. Although it has not been directly demonstrated here, a plausible interpretation for the high 4-Cl-IAA activity is that, at least in part, it might be caused via a reduced metabolism of 4-Cl-IAA. Furthermore, for the first time, the data show that membrane potential responds to 4-Cl-IAA in a qualitatively different fashion than to IAA. These findings may, in turn, suggest a specific signal transduction pathway to 4-Cl-IAA in maize coleoptile cells.

Reactive oxygen intermediates, i.e. the superoxide radical (O*-)(2), hydrogen peroxide (H2O2) and the hydroxyl radical (*OH), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry may also play a useful role in polymer breakdown leading to wall loosening during extension growth of plant cells controlled by the phytohormone auxin. Backbone cleavage of cell wall polysaccharides can be accomplished in vitro by (*OH) produced from H2O2 in a Fenton reaction or in a reaction catalyzed by peroxidase supplied with O2 and NADH. Here, we show that coleoptile growth of maize seedlings is accompanied by the release of reactive oxygen intermediates in the cell wall. Auxin promotes release of (O*-)(2) and subsequent generation of (*OH)when inducing elongation growth. Experimental generation of (*OH) in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of (O*-)(2), H2O2 or (*OH), or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which (*OH), produced from (O*-)(2) and H2O2 by cell wall peroxidase, acts as a wall-loosening agent.

We studied the comparative effects of the phytohormone indolylacetic acid (IAA) and chitooligosaccharides (5-10 kDa, degree of deacetylation 35%) on the growth of coleoptile fragments of 3-day wheat germlings. IAA (10 mg/l) stimulated elongation of coleoptile fragments by 80%, as compared to the control (water). Chitooligosaccharides at 0.01-1500 mg/l or higher did not exert auxin-like effects, but at 100 mg/l or higher, suppressed elongation of the coleoptile fragments, as compared to the control (water). Incubation of coleoptile fragments in solutions containing both IAA and chitooligosaccharides (100 mg/l or higher) suppressed their IAA-induced elongation, which correlated with the inhibition of growth of 3-day wheat germlings after wetting seeds in solutions of chitooligosaccharides. It has been proposed that the phytohormone-like properties of chitooligosaccharides can be related to changes in the endogenous balance of phytohormones in plants.

In epidermal cells of Zea mays coleoptiles, actin microfilaments are organized in fine strands during cell elongation, but are bundled in response to signals that inhibit growth. This bundling response is accompanied by an increased membrane association of extracted actin. Brefeldin A, an inhibitor of vesicle secretion, increases the membrane association of actin, causes a bundling of cortical actin microfilaments, and reduces the sensitivity of cell elongation to auxin. A model is proposed where auxin controls the dynamics of an actin subpopulation that guides vesicles loaded with components of the auxin-signaling machinery towards the cell poles.

In Arabidopsis, SHY2 encodes IAA3, a member of the auxin-induced Aux/IAA family. Gain-of-function mutations in SHY2/IAA3 cause enlarged cotyledons, short hypocotyls, and altered auxin-regulated root development. Here we show that the gain-of-function mutation shy2-2 decreases both the induction and repression of auxin-regulated genes, suggesting that SHY2/IAA3 acts as a negative regulator in auxin signaling. shy2-2 affects auxin induction of many previously characterized primary response genes, implying that it might repress primary auxin responses. In addition, shy2-2 also affects expression of multiple auxin-nonresponsive genes. Light regulates expression of SHY2/IAA3, suggesting a possible link between light and auxin response pathways.

A semi-dominant mutant suppressor of hy2 (shy2-1D) of Arabidopsis thaliana, originally isolated as a photomorphogenesis mutant, shows altered auxin responses. Recent molecular cloning revealed that the SHY2 gene is identical to the IAA3 gene, a member of the primary auxin-response genes designated the Aux/IAA gene family. Because Aux/IAA proteins are reported to interact with auxin response factors, we investigated the pattern of expression of early auxin genes in the iaa3/shy2-1D mutant. RNA hybridization analysis showed that levels of mRNA accumulation of the early genes were reduced dramatically in the iaa3/shy2-1D mutants, although auxin still enhanced gene expression in the iaa3/shy2-1D mutant. Histochemical analysis using a fusion gene of the auxin responsive domain (AuxRD) and the GUS gene showed no IAA-inducible GUS expression in the root elongation zone of the iaa3/shy2-1D mutant. On the other hand, ectopic GUS expression occurred in the hypocotyl, cotyledon, petiole and root vascular tissues in the absence of auxin. These results suggest that IAA3/SHY2 functions both negatively and positively on early auxin gene expression.

Protoplasts of corn coleoptiles and Arabidopsis hypocotyls respond to the plant hormone auxin with a rapid change in volume. We checked the effect of antibodies directed against epitopes of auxin-binding protein 1 from Arabidopsis thaliana (AtERabp1) and Zea mays (ZmERabp1), respectively. Antibodies raised against the C-terminus of AtERabp1 inhibited the response to auxin, while antibodies raised against a part of box a, the putative auxin-binding domain, induced a swelling response similar to that caused by auxin treatment. Synthetic C-terminal oligopeptides of ZmERabp1 also caused a swelling response. These effects occurred regardless of whether the experiments were carried out with homologous (anti-AtERabp1 antibodies on Arabidopsis protoplasts or anti-ZmERabp1 antibodies in maize protoplasts) or heterologous immunological tools. The results indicate that the auxin signal for protoplast swelling is perceived by extracellular ABP1.

Endogenous extractable factors associated with auxin action in plant tissues were investigated, especially their effects on elongation of 1-mm coleoptile segments of maize (Zea mays L.), in the presence of saturating 10 microM indole-3-acetic acid (IAA). The relative growth response, to auxin alone, was much smaller in segments shorter than 2-3 mm compared to 10-mm segments. Fusicoccin-induced elongation, however, was less affected by shortening the segments. A reduced auxin response may result from the depletion through cut surfaces of a substance required for IAA-mediated growth. Sucrose, phenolics like flavonoids, and vitamins were ruled out as the causal factors. A partially purified methanol extract of maize coleoptiles supported longterm, auxin-controlled elongation. The active material was also found among substances bleeding from scrubbed maize coleoptiles. The active factor from maize was further purified by HPLC and characterised by the UV spectrum and its pH shift. This factor was identified as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by mass spectroscopy. Activity tests confirmed that pure DIMBOA from other sources sustained auxin-induced elongation of short maize coleoptile segments. However, DIMBOA only partially restored the activity lost from short segments. This indicates that an additional factor, other than DIMBOA, is required. Extracts from Avena or Cucurbita did not contain the factor DIMBOA; it was active on maize elongation, but not on Avena coleoptiles or Cucurbita hypocotyls. This narrow specificity and the lack of DIMBOA action in short-term tests with maize indicate that DIMBOA is not the general auxin cofactor but may specifically "spare" the co-auxin in maize.

The dependence of growth induced by the fungal toxin fusicoccin (FC) on the K+ content of the incubation medium was investigated in abraded maize coleoptiles. If the divalent ion Ca2+ was included in the bathing medium, no FC-induced growth occurred in the absence of K+, whereas a strong response was detected in presence of K+. The optimal K+ concentration was in the range of 1-10 mM. With the exception of Rb+, none of the other alkali ions (Na+, Li+, Cs+) could replace for K+ in sustaining FC-induced growth. The potassium channel blocker tetraethylammonium (TEA) reversibly inhibited FC-induced growth. As shown earlier for auxin-induced growth, no strict potassium dependence of FC-triggered elongation was observed in Ca2+ -free media. However, TEA abolished this apparently K+ independent FC-induced growth. It is concluded that FC-induced growth, like auxin-induced growth, requires K+ uptake through K+ channels.

When auxin stimulates rapid cell elongation growth of cereal coleoptiles, it causes a degradation of 1,3:1,4-beta-glucan in hemicellulosic polysaccharides. We examined gene expressions of endo-1,3:1,4-beta-glucanase (EI) and exo-beta-glucanase (ExoII), of which optimum pH are about 5, and molecular distribution of hemicellulosic polysaccharides in barley (Hordeum vulgare L.) coleoptile segments treated with or without IAA. IAA (10(-5) M) stimulated the gene expression of EI, while it did not affect that of ExoII. IAA induced gene expression of EI after 4 h and increased wall-bound glucanase activity after 8 h. The molecular weight distribution of hemicellulosic polysaccharides from coleoptile cell walls was shifted to lower molecular weight region by 2 h of IAA treatment. Fusicoccin (10(-6) M) mimicked IAA-induced elongation growth and the decrease in molecular weight of hemicellulosic 1,3:1,4-beta-glucan of coleoptiles in the first 4 h, but it did not promote elongation growth thereafter. These facts suggest that acidification of barley cell walls by IAA action enhances pre-existing cell wall-bound glucanase activity in the early first phase of IAA-induced growth and the late second phase involves the gene expression of EI by IAA.

As part of molecular recognition studies on the phytohormone indole-3-acetic acid (IAA) a series of alkylated IAAs has been examined. Phenyl-ring substitution (alkyl = methyl and ethyl) at positions 4-, 6- or 7- as well as pyrrole substitution at the 2-site resulted in the six compounds which are analyzed: 2-Me-IAA, 4-Me-IAA, 6-Me-IAA, 7-Me-IAA, 4-Et-IAA and 6-Et-IAA. The structure-activity relationships investigated include those between the geometrical parameters of the molecular structures determined by X-ray analysis, the growth-promoting activities in the Avena coleoptile straight-growth bioassay and relative lipophilicities calculated from retention times on a reversed-phase HPLC column and from R(F) values in reversed-phase TLC. Lipophilicities are correlated with the moments of inertia, average polarizability, molecular mass, and the van der Waals radii of the ring substituents. The influence of substitution on the electronic properties of the indole ring and its geometry is discussed on the basis of the UV and 1H NMR spectra.

Auxin-induced growth of coleoptiles depends on the presence of potassium and is suppressed by K+ channel blockers. To evaluate the role of K+ channels in auxin-mediated growth, we isolated and functionally expressed ZMK1 and ZMK2 (Zea mays K+ channel 1 and 2), two potassium channels from maize coleoptiles. In growth experiments, the time course of auxin-induced expression of ZMK1 coincided with the kinetics of coleoptile elongation. Upon gravistimulation of maize seedlings, ZMK1 expression followed the gravitropic-induced auxin redistribution. K+ channel expression increased even before a bending of the coleoptile was observed. The transcript level of ZMK2, expressed in vascular tissue, was not affected by auxin. In patch-clamp studies on coleoptile protoplasts, auxin increased K+ channel density while leaving channel properties unaffected. Thus, we conclude that coleoptile growth depends on the transcriptional up-regulation of ZMK1, an inwardly rectifying K+ channel expressed in the nonvascular tissue of this organ.

To investigate the mechanism of auxin action during pattern formation in dicot embryos, we tested the effects of the natural auxin indole-3-acetic acid (IAA), the auxin transport inhibitor N-(1-naphthyl)thalamic acid (NPA) and the antiauxin p-chlorophenoxyisobutyric acid (PCIB). In vitro treatments of isolated zygotic Brassica juncea embryos with these substances led to a wide range of morphogenetic alterations. Treatment of globular embryos with exogenous auxin (10-40 microM) either completely inhibited morphogenesis, resulting in ball-shaped embryos, or caused the development of egg- and cucumber-shaped embryos, which only consisted of a shortened hypocotyl without any apical structures. Axis duplication was observed sometimes after inhibition of auxin transport in globular embryos, and led to the development of twin embryos. During the transition from globular to heart stage, changes in auxin distribution or activity frequently caused the development of either split-collar or collar-cotyledons. Antiauxin inhibited cotyledon growth, leading to embryos with single or no cotyledons, or inhibited the development of the hypocotyl and the radicle. Inhibition of auxin transport in transition embryos sometimes led to axis broadening, which resulted in the development of two radicles. The described changes in embryo shapes represent arrests in different auxin-regulated developmental steps and phenocopy some Arabidopsis morphogenetic mutants.

The rice mutant Yin-Yang has been selected during a screen for resistance to cytoskeletal drugs and is characterized by alterations in epidermal cell length and a precocious onset of gravitropism. The elongation response of coleoptile segments to auxin does not reveal changes of auxin sensitivity in Yin-Yang. However, in contrast to the wild type, cell elongation in Yin-Yang is highly sensitive to the actin-polymerisation blocker cytochalasin D. This increased sensitivity to cytochalasin D requires optimal concentrations of auxin to become manifest. The auxin response of actin microfilaments in epidermal cells differs between wild type and mutant. In the wild type, the longitudinal microfilament bundles become loosened in response to auxin. In the mutant, these bundles disintegrate partially and are replaced by a network of short filaments surrounding the nucleus. Several aspects of the mutant phenotype can be mimicked in the wild type by treatment with cytochalasin D. The mutant phenotype is discussed in terms of signal-dependent changes of actin dynamics and the putative role of actin during cell elongation.

The literature indicates that the tip of maize (Zea mays L.) coleoptiles has the localized functions of producing auxin for growth and perceiving unilateral light stimuli and translocating auxin laterally for phototropism. There is evidence that the auxinproducing function of the tip is restored in decapitated coleoptiles. We examined whether the functions for phototropism are also restored by using blue-light conditions that induced a first pulse-induced positive phototropism (fPIPP) and a time-dependent phototropism (TDP). When the apical 5 mm, in which photosensing predominantly takes place, was removed, no detectable fPIPP occurred even if indole-3-acetic acid (lanolin mixture) was applied to the cut end. However, when the blue-light stimulation was delayed after decapitation, fPIPP became inducible in the coleoptile stumps supplied with indole-3-acetic-acid/lanolin (0.01 mg g-1), indicating that phototropic responsiveness was restored. This restoration progressed 1 to 2 h after decapitation, and the curvature response became comparable to that of intact coleoptiles. The results for TDP were qualitatively similar, but some quantitative differences were observed. It appeared that the overall TDP was based on a major photosensing mechanism specific to the tip and on at least one additional mechanism not specific to the tip, and that the tip-specific TDP was restored in decapitated coleoptiles with kinetics similar to that for fPIPP. It is suggested that the photoreceptor system, which accounts for fPIPP and a substantial part of TDP, is regenerated in decapitated coleoptiles, perhaps together with the mechanism for lateral auxin translocation.

We report improved method for white clover (Trifolium repens) transformation using Agrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. The bar gene and phosphinothricin selection was shown to be a more effective selection system than nptII (kanamycin selection) or aadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (beta-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.

An indirect enzyme immunoassay for detection of as little as 10-50 pmole IAA is described for the first time. The assay is based on the development of highly specific polyclonal antibodies against the carboxyl site of IAA. The binding specificity is nearly as high as the radioimmunoassay and the titre of the specific antibody was also remarkably high (1:40,000 of the primary antibody). Such an easy, rapid, specific and highly sensitive assay would be extremely useful in gaining more information on the mode of action of phytohormones, and their effects on physiological processes.

A study has been made of the prolonged growth of Avena coleoptile sections in response to fusicoccin (FC), a phytotoxin that promotes apoplastic acidification. The final amount of FC-induced growth is a function of the FC concentration. Removal of the epidermis speeds up the initial rate of elongation and shortens the duration of the response, without affecting the total amount of extension. A suboptimal FC concentration (7 x 10(-8) M) which induces the same rate of proton excretion as does optimal indoleacetic acid (IAA) (1 x 10(-5) M), causes elongation which is 60-75% of that induced by IAA in 4 h or 50-65% in 7 h. This suggests that acid-induced extension could make a major contribution to auxin-induced growth for at least 7 h.

Galactose inhibits auxin-induced growth of Avena coleoptiles by at least two mechanisms. First, it inhibits auxin-induced H(+)-excretion needed for the initiation of rapid elongation. Galactose cannot be doing so by directly interfering with the ATPase since fusicoccin-induced H(+)-excretion is not affected. Secondly, galactose inhibits long-term auxin-induced growth, even in an acidic (pH 4.5) solution. This may be due to an inhibition of cell wall synthesis. However, galactose does not reduce the capacity of walls to be loosened by H+, given exogenously or excreted in response to fusicoccin.

According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport.