Higher extracellular pH suppresses tracheary element differentiation by affecting auxin uptake

In an optimized liquid medium containing auxin and cytokinin, mesophyll cells isolated from Zinnia elegans L. seedlings can be induced to differentiate into tracheary elements (TEs) at high frequency. However, it is known that buffering the medium at neutral pH severely suppresses TE differentiation. In the process of modifying the medium, we found that excessive administration of auxin restored the suppression. Based on this finding, we physiologically characterized auxin actions involved in TE differentiation by focusing on the influence of extracellular pH. First, dose/response relationships between auxin [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] concentrations and differentiated cell ratios were determined under various extracellular pH conditions. Secondly, intracellular concentrations of free forms and metabolites of auxin species were determined by analyzing extracts from cells cultured with radiolabeled NAA and 2,4-D under different extracellular pH conditions with liquid scintillation counting and thin-layer chromatography autoradiograms. Higher extracellular pH was found to reduce both the auxin potency for inducing TE differentiation and intracellular auxin accumulation. Reduction levels correlatively varied depending on the auxin species. These results suggest that the weakening in auxin potency at higher extracellular pH is ascribed to lower auxin uptake, which leads to decreased intracellular perception of the auxin signal. A model to predict auxin action that considers membrane transport, metabolism, and the perception of auxin is also presented.     

Inhibited polar auxin transport results in aberrant embryo development in Norway spruce

Current hypotheses concerning the role of polar auxin transport in embryo development are entirely based on studies of angiosperms, while little is known about how auxin regulates pattern formation in gymnosperms. In this study, different developmental stages of somatic embryos of Norway spruce (Picea abies) were treated with the polar auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Effects of the treatments on auxin content, embryo differentiation and programmed cell death (PCD) were analysed. During early embryo development, NPA-treatment led to increased indole-3-acetic acid (IAA) content, abnormal cell divisions and decreased PCD, resulting in aberrant development of embryonal tube cells and suspensors. Mature embryos that had been treated with NPA showed both apical and basal abnormalities. Typically the embryos had abnormal cotyledon formation and irregular cell divisions in the area of the root meristem. Our results show that polar auxin transport is essential for the correct patterning of both apical and basal parts of conifer embryos throughout the whole developmental process. Furthermore, the aberrant morhologies of NPA-treated spruce embryos are comparable with several auxin response and transport mutants in Arabidopsis. This suggests that the role of polar auxin transport is conserved between angiosperms and gymnosperms.     

Loss of Tonoplast Integrity Programmed in Tracheary Element Differentiation

A tracheary element (TE) is a typical example of a cell type that undergoes programmed cell death in the developmental processes of vascular plants. The loss of the selective permeability of the tonoplast, which corresponds to tonoplast disintegration, occurred after the cells commenced secondary wall thickening and played a pivotal role in the programmed cell death of TEs in a zinnia (Zinnia elegans L.) cell culture. A search for events specifically associated with the TE vacuole provided an important clue to the understanding of the cell death mechanism. The transport of fluorescein, a fluorescent organic anion, across the tonoplast declined drastically in differentiating TEs. The capacity of the vacuole to accumulate the probe was also impaired. Treatment with probenecid, an inhibitor of organic anion transport, caused rapid cell death of TEs and led to the ultimate disruption of the vacuole even in other types of cultured cells. These changes in vacuolar properties during TE development were suppressed by cycloheximide. Specific mRNA accumulation in cells cultured in a TE differentiation-inductive condition was abolished by probenecid. These results suggest that a change in vacuolar membrane permeability promotes programmed cell death in TEs.     

Auxin efflux carrier activity and auxin accumulation regulate cell division and polarity in tobacco cells

Division and growth of most types of in vitro-cultured plant cells require an external source of auxin. In such cultures, the ratio of external to internal auxin concentration is crucial for the regulation of the phases of the standard growth cycle. In this report the internal concentration of auxin in suspension-cultured cells of Nicotiana tabacum L., strain VBI-0, was manipulated either (i) by increasing 10-fold the normal concentration of 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid in the external medium; or (ii) by addition 1-N-naphthylphthalamic acid (NPA; an inhibitor of auxin efflux and of auxin efflux carrier traffic). Both treatments delayed the onset of cell division for 6-7 days without loss of cell viability. In both cases, cell division activity subsequently resumed coincident with a reduction in the ability of cells to accumulate [(3)H]NAA from an external medium. Following renewed cell division, a significant proportion of the NPA-treated cells but not those grown at high auxin concentration, exhibited changes in the orientation of new cell divisions and loss of polarity. We conclude that cell division, but not cell elongation, is prevented when the internal auxin concentration rises above a critical threshold value and that the directed traffic of auxin efflux carriers to the plasma membrane may regulate the orientation of cell divisions.     

Triiodobenzoic acid, an auxin polar transport inhibitor, suppresses somatic embryo formation and postembryonic shoot/root development in Eleutherococcus senticosus

The effect of auxin polar transport inhibitor on somatic embryo development and postembryonic growth in Siberian ginseng (Eleutherococcus senticosus) was examined. In the presence of 2,3,5-triiodobenzoic acid (TIBA), an auxin polar transport inhibitor, embryo formation from embryogenic cells was suppressed, while cell division was not affected. When globular embryos at different stages were transferred onto medium containing TIBA, development of axial and bilateral polarity was suppressed in a stagespecific manner. In abnormal embryos induced by TIBA, further development of shoot and root apical meristems and vascular differentiation was also suppressed. Thus, abnormal development of embryos induced by inhibition of auxin polar transport resulted in plantlets without shoots and roots.     

Both induction and morphogenesis of cyst nematode feeding cells are mediated by auxin

Various lines of evidence show that local changes in the auxin concentration are involved in the initiation and directional expansion of syncytia induced by cyst nematodes. Analysis of nematode infections on auxin-insensitive tomato and Arabidopsis mutants revealed various phenotypes ranging from complete inhibition of syncytium development to a decrease in hypertrophy and lateral root formation at the infection site. Specific activation of an auxin-responsive promoter confirmed the role of auxin and pointed at a local accumulation of auxin in developing syncytia Disturbance of auxin gradients by inhibiting polar auxin transport with N-(1-naphthyl)phtalamic acid (NPA) resulted in abnormal feeding cells, which were characterized by extreme galling, massive disordered cell divisions in the cortex, and absence of radial expansion of the syncytium initial toward the vascular bundle. The role of auxin gradients in guiding feeding cell morphogenesis and the cross-talk between auxin and ethylene resulting in a local activation of cell wall degrading enzymes are discussed.     

The action of specific inhibitors of auxin transport on uptake of auxin and binding of N-1-naphthylphthalamic acid to a membrane site in maize coleoptiles

Using both 1-mm segments of corn (Zea mays L.) coleoptiles and a preparation of membranes isolated from the same source, we have compared the effectiveness of several inhibitors of geotropism and polar transport in stimulating uptake of auxin (indole-3-acetic acid, IAA) into the tissue and in competing with N-1-naphthylphthalamic acid (NPA) for a membrane-bound site. Low concentrations of 2,3,5-triiodobenzoic acid (TIBA), NPA, 2-chloro-9-hydroxyfluorene-9-carboxylic acid (morphactin), and fluorescein, eosin, and mercurochrome all stimulated net uptake of [³H]IAA by corn coleoptile tissues while higher concentrations reduced the uptake of both [³H]IAA and another lipophilic weak acid, [¹⁴C]benzoic acid. Since low concentrations of fluorescein and its derivatives competed for the same membrane-bound site in vitro as did morphactin and NPA, the basis for both the specific stimulation of auxin accumulation and the inhibition of polar auxin transport by all these compounds may be their ability to interfere with the carrier-mediated efflux of auxin anions from cells. At higher concentrations, the decrease in accumulation of weak acids was nonspecific and thus may be the result of acidification of the cytoplasm and a general decrease in the driving force for uptake of the weak acids. Triiodobenzoic acid was an exception. Low concentration of TIBA (0.1–1 M) were much less effective than NPA in competing for the NPA receptor in vitro, but little different from NPA in ability to stimulate auxin uptake. One possibility is that TIBA, a substance which is polarly transported, may compete with auxin for the polar transport site while NPA, morphactin, and the fluorescein derivatives may render this site inactive.Abbreviations C1-NPA 2,3,4,5-tetrachloro-N-1-naphthylphthalamic acid - IAA indole-3-acetic acid - -NAA -naphthaleneacetic acid - -NAA -naphthalenacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Jasmonic acid modulates xylem development by controlling polar auxin transport in vascular tissues

Development of xylem cells is affected by environmental stresses such as drought and oxidative stress, and recent findings suggested that jasmonic acid (JA) mediates this process through interaction with other phytohormones such as cytokinin. In this study, we showed that polar auxin transport regulated by PIN3 and PIN7 is involved in the JA-mediated xylem development in vascular tissues. The mutant plants that lack the activity of PIN3 and PIN7 responsible for the auxin transport developed extra xylems in vascular tissues such as the JA-treated wild-type plants. Visualization of auxin response and xylem development in the roots treated with NPA, an inhibitor of polar auxin transport, suggested that disruption of polar auxin transport is involved in the xylem phenotype of pin3 pin7 double mutants. We also found that cytokinin increases expressions of PIN3 and PIN7 responsible for the auxin transport while JA decreases only PIN7. These suggested that PIN7-mediated polar auxin transport system modulates xylem development in response to JA. The finding that JA affects auxin distribution in root vascular tissues further supported this. Collectively, these suggest that JA promotes xylem development by disrupting auxin transport in vascular tissues, and the auxin efflux genes, more especially PIN7 whose expression is suppressed by JA mediates this process.     

A possible role of glutathione and glutathione disulfide in tracheary element differentiation in the cultured mesophyll cells of Zinnia elegans.

We investigated the relationship between the cellular redox state of GSH or GSSG and tracheary element (TE) differentiation using a Zinnia experimental system, in which isolated mesophyll cells transdifferentiate to TEs. TE differentiation was suppressed by the application of L-buthionine sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at the early stage of cell culture. Application of GSSG at the early culture stage promoted the differentiation, but that of GSH or GSSG at an advanced period of culture suppressed the differentiation. Application of GSH and GSSG nullified the TE differentiation-suppressing effect of BSO. The results suggest that changes in the redox states of GSH and GSSG have a role in TE differentiation.     

Early embryo development in Fucus distichus is auxin sensitive

Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [(3)H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development.     

Tracheary element differentiation in Zinnia mesophyll cell cultures

Mechanically isolated mesophyll cells of Zinnia elegans differentiate into tracheary elements (TEs) when cultured in a medium containing adequate auxin and cytokinin. Differentiation in this culture system is relatively synchronous, rapid (occuring within 3 days of cell isolation) and efficient (with up to 65% of the mesophyll cells differentiating into TEs), and does not require prior mitosis. The Zinnia system has been used to investigate (a) cytological and ultrastructural changes occurring during TE differentiation, such as the reorganization of microtubules controlling secondary wall deposition, (b) the influences of calcium and of various plant hormones and antihormones on TE differentiation, and (c) biochemical changes during differentiation, including those occurring during secondary wall deposition, lignification and autolysis. This review summarizes experiments in which the Zinnia system has served as a model for the study of TE differentiation.     

Progress of lignification mediated by intercellular transportation of monolignols during tracheary element differentiation of isolated Zinnia mesophyll cells

Tracheary element (TE) differentiation is a typical example of programmed cell death (PCD) in higher plants, and maturation of TEs is completed by degradation of all cell contents. However, lignification of TEs progresses even after PCD. We investigated how and whence monolignols are supplied to TEs which have undergone PCD during differentiation of isolated Zinnia mesophyll cells into TEs. Higher densities of cell culture induced greater lignification of TEs. Whereas the continuous exchanging of culture medium suppressed lignification of TEs, further addition of coniferyl alcohol into the exchanging medium reduced the suppression of lignification. Analysis of the culture medium by HPLC and GC-MS showed that coniferyl alcohol, coniferaldehyde, and sinapyl alcohol accumulated in TE inductive culture. The concentration of coniferyl alcohol peaked at the beginning of secondary wall thickening, decreased rapidly during secondary wall thickening, then increased again. These results indicated that lignification on TEs progresses by supply of monolignols from not only TEs themselves but also surrounding xylem parenchyma-like cells through medium in vitro.     

PIS1, a negative regulator of the action of auxin transport inhibitors in Arabidopsis thaliana

In order to clarify the mechanism underlying the polar auxin transport system, the pis1 mutant in Arabidopsis thaliana that is hypersensitive to N -1-naphthylphthalamic acid (NPA), an auxin transport inhibitor was isolated and characterized. Whereas the pis1 mutant is normally sensitive to phytohormones, auxins, cytokinin and ethylene precursor, this mutant is hypersensitive to NPA over the broad spectrum of its effects such as growth of seedlings, root elongation, root gravitropism, root phototropism and root curling. This result indicates that the pis1 mutant is specifically affected in the polar auxin transport system. This result also defines a genetic factor controlling both gravitropism and phototropism, and strongly indicates the involvement of auxin transport during both tropic responses. NPA, 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) represent different classes of auxin transport inhibitors. The pis1 mutation conferred hypersensitivity to both NPA and TIBA but not to HFCA. These results show the genetic separation of the actions of NPA/TIBA and of HFCA. The PIS1 gene product might be specifically involved in the response pathway of NPA/TIBA, leading to interference with auxin-efflux carriers, and might act as a negative regulator of the action of NPA/TIBA.     

The auxin transport inhibitor response 3 (tir3) allele of BIG and auxin transport inhibitors affect the gibberellin status of Arabidopsis

The Arabidopsis gene BIG (formerly DOC1/TIR3/UMB1/ASA1) is known to encode a huge calossin-like protein that is required for polar auxin transport (PAT). Mutations at this locus, in addition to reducing PAT, can alter the sensitivity of plants to several hormones and light. The tir3-1 allele of BIG reduces the response of plants to application of the gibberellin (GA) precursors ent-kaurenoic acid and GA12 and its semidwarf phenotype is partially reversed by C19-GAs. The effects of auxin transport inhibitors (ATIs) on GA 20-oxidation was examined in wild-type and tir3-1 seedlings. 1-N-naphthylphthalamic acid (NPA) and triiodobenzoic acid lead to overexpression of the GA-biosynthetic gene AtGA20ox1 comparable in magnitude to the overexpression observed in seedlings treated with paclobutrazol, a GA biosynthesis inhibitor. In contrast to that of AtGA20ox1, overexpression of AtGA20ox2 is pronounced only in paclobutrazol-treated Col and Ler, and is less in tir3-1 and in all NPA-treated seedlings. Thus the effects of BIG and ATIs on the expression of genes encoding GA 20-oxidases are complex, and suggest that at least in some tissues ATIs, directly or indirectly, may reduce the level of bioactive GA and/or alter GA signal transduction.     

The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance

The phytohormone auxin plays a critical role in plant growth and development, and its spatial distribution largely depends on the polar localization of the PIN‐FORMED (PIN) auxin efflux carrier family members. In this study, we identify a putative auxin efflux carrier gene in rice, OsPIN3t, which acts in auxin polar transport but is also involved in the drought stress response in rice. We show that OsPIN3t–GFP fusion proteins are localized in plasma membranes, and this subcellular localization changes under 1‐N‐naphthylphthalamic acid (NPA) treatment. The tissue‐specific expression patterns of OsPIN3t were also investigated using a β‐glucuronidase (GUS) reporter, which showed that OsPIN3t was mainly expressed in vascular tissue. The GUS activity in OsPIN3tpro::GUS plants increased by NAA treatment and decreased by NPA treatment. Moreover, knockdown of OsPIN3t caused crown root abnormalities in the seedling stage that could be phenocopied by treatment of wild‐type plants with NPA, which indicated that OsPIN3t is involved in the control of polar auxin transport. Overexpression of OsPIN3t led to improved drought tolerance, and GUS activity significantly increased when OsPIN3tpro::GUS plants were subjected to 20% polyethylene glycol stress. Taken together, these results suggest that OsPIN3t is involved in auxin transport and the drought stress response, which suggests that a polar auxin transport pathway is involved in the regulation of the response to water stress in plants.     

Polar auxin transport controls suspensor fate

Polar auxin transport is critical for normal embryo development in angiosperms. It has been proposed that auxin accumulates dynamically at specific positions, which in early Arabidopsis embryos correlates with developmental decisions such as specification of the apical cell lineage, specification of the hypophysis, and differentiation of the two cotyledons. In conifers, pattern formation during embryo development is different, and includes a free nuclear stage, nondividing suspensor cells, presence of tube cells, lack of hypophysis and formation of a crown of cotyledons surrounding the shoot apical meristem. We have recently shown that polar auxin transport is important for normal embryo development also in conifers. Here we suggest a model where auxin is transported from the suspensor cells to the embryonal mass during early embryogeny in conifers. This transport is essential for the developmental decisions of the tube cells and the suspensor, and affects both the amount of programmed cell death and the embryo patterning.Key words: conifer, embryo development, 1-N-naphtylphthalamic acid (NPA), patterning, polar auxin transport, programmed cell death, somatic embryogenesis, suspensorIn the model plant Arabidopsis thaliana auxin is transported, already from the first cell division of the zygote, from the basal cell to the apical cell, where it is involved in establishing the identity of the apical cell lineage. At the 32-cell stage the polar auxin transport is reversed, leading to an auxin accumulation in the uppermost suspensor cell, which occurs concomitantly with the specification of the hypophysis. During the heart stage auxin is transported towards the cotyledonary primordia, giving positional information about the cotyledon outgrowth.¹ Formation of the apical-basal embryonic pattern during early embryogeny in conifers is quite different from that in Arabidopsis and proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass, the embryonal tube cells and terminally differentiated nondividing suspensor cells.²The somatic embryo system of Picea abies (Norway spruce) includes a stereotyped sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments.^3,4 We are using this as a model system for elucidating the regulation of embryo development in conifers.² Early somatic embryos differentiate from proembryonic masses (PEMs) after withdrawal of the plant growth regulators (PGRs) auxin and cytokinin (Fig. 1A and B). We have previously shown that the organisation of the apical-basal polarity in early embryos is dependent on a gradient of PCD from the embryonal tube cells committed to death, to the cell corpses at the basal end of the suspensor.^5–7 Dysregulation of the PCD leads to aberrant apical-basal patterning.Open in a separate windowFigure 1Model for polar auxin transport control of early embryo patterning in conifers. (A) Embryogenic cultures proliferate as proembryonic masses (PEMs) in the presence of the plant growth regulators (PGRs) auxin and cytokinin. (B) Early embryos start to differentiate from PEMs after withdrawal of PGRs. Endogenous auxin is transported to the newly formed embryonal mass. (C) Early embryos are formed within two weeks in PGR-free medium. Early embryos have a distinct embryonal mass, tube cells and a suspensor. IAA is transported from the suspensor and the tube cells to the embryonal mass. (D) Fully matured cotyledonary embryos are formed after 5–6 weeks on maturation medium. (E) Treatment with NPA blocks the polar auxin transport to the embryonal mass, leading to an IAA accumulation in the suspensor cells, tube cells and perhaps also in the cells of the embryonal mass most adjacent to the tube cells. (F) Embryos with supernumerary suspensor cells are formed if polar auxin transport is inhibited only during the earliest stages of suspensor differentiation. (G) Embryos with meristematic cells in the suspensor are formed if polar auxin transport is inhibited during both differentiation and elongation of the suspensor. We assume that these abnormalities abort further development and maturation of viable embryos. em, embryonal mass; s, suspensor; tc, tube cells. Green arrows indicate polar auxin transport, T indicates blocked polar auxin transport, green shadings indicate auxin accumulation.We recently showed that in embryogenic cultures of Norway spruce treated with the polar auxin transport inhibitor NPA, the number of cells undergoing PCD decreases. As a consequence the balance between the number of cells in the embryonal mass and the number of cells in the suspensor develop abnormally, and concomitantly the endogenous free IAA content increases almost two-fold.⁸In order to visualise the IAA accumulation within the embryos we used a -318 bp deletion of the auxin-responsive IAA4/5 promoter from Pisum sativum (pea), previously characterized by Oeller et al.,⁹ and Ballas et al.,¹⁰ fused to the GUS reporter gene.¹¹ In tobacco (Nicotiana tabacum) the promoter is expressed in rapidly elongating hypocotyls,¹² (our unpublished observations) and strong induction by auxin is clear in elongating zones of both roots and hypocotyls in transgenic pIAA4/5-GUS Arabidopsis plants.¹¹ However, to our knowledge, expression of IAA4/5 has not been reported in embryonal shoot apical meristems. Hence, the pIAA4/5-GUS may preferentially be used as a biosensor of auxin activity in non-meristematic cells during spruce embryo development. During normal somatic embryo development in spruce, pIAA4/5-GUS activity is detected in PEMs, tube cells and suspensor cells, but not in the embryonal mass. Early embryos of Norway spruce that are treated with NPA show increased pIAA4/5-GUS activity in tube cells and suspensor cells (unpublished), well in line with the increment of free IAA levels.Our results indicate that IAA under normal conditions is transported from the suspensor cells to the cells in the embryonal mass (Fig. 1B and C). NPA-treatment blocks this polar transport of endogenous IAA, which results in an accumulation of IAA and increased pIAA4/5-GUS activity in the suspensor cells, the tube cells, and perhaps also in the cells of the embryonal mass most adjacent to the tube cells (Fig. 1F and G). Blocked polar auxin transport during early differentiation of the suspensor stimulates abnormal cell divisions of the meristematic cells most adjacent to the tube cells or perhaps even of the tube cells themselves. Consequently, embryos with supernumerary tube and suspensor cells are formed (Fig. 1F). If the polar auxin transport is blocked for a longer time, i.e., during both differentiation and elongation of the suspensor, the auxin accumulation leads to maintenance of meristematic fate and a failure to undergo PCD (Fig. 1G).It has been proposed that the fate of the suspensor cells is regulated by signals from the embryo proper which impede developmental potential and initiate PCD.¹³ In accordance, we assume that the abnormal embryo morphologies formed after NPA-treatment may result from adverse inhibitory signals from the embryonal mass.     

Convergent evolution of shoots in land plants: lack of auxin polar transport in moss shoots

SUMMARY The shoot is a repeated structure made up of stems and leaves and is the basic body plan in land plants. Vascular plants form a shoot in the diploid generation, whereas nonvascular plants such as mosses form a shoot in the haploid generation. It is not clear whether all land plants use similar molecular mechanisms in shoot development or how the genetic networks for shoot development evolved. The control of auxin distribution, especially by polar auxin transport, is essential for shoot development in flowering plants. We did not detect polar auxin transport in the gametophytic shoots of several mosses, but did detect it in the sporophytes of mosses without shoot structure. Treatment with auxin transport inhibitors resulted in abnormal embryo development, as in flowering plants, but did not cause any morphological changes in the haploid shoots. We fused the soybean auxin-inducible promoter GH3 with a GUS reporter gene and used it to indirectly detect auxin distribution in the moss Physcomitrella patens . An auxin transport inhibitor NPA did not cause any changes in the putative distribution of auxin in the haploid shoot. These results indicate that polar auxin transport is not involved in haploid shoot development in mosses and that shoots in vascular plants and mosses are most likely regulated differently during development.