Hormonal control of cambial activity and vessel differentiation in Quercus robur

Cambial activity and vessel differentiation of the Quercus robur stem were investigated in relation to concentration of growth regulators and sucrose, seasonal changes in the sensitivity of cambial cells, and axial polarity of the stem. Basipetal efflux of natural auxin was measured in the oak stem cambial region. IAA, GA₃, kinetin and sucrose affected cambial activity and/or initiation of vessel differentiation differently, depending upon concentration. Depending upon the season, kinetin increased or reduced the stimulation of cambial activity caused by IAA and GA₃, but it did not affect the differentiation of vessels. Supply of sucrose in higher concentrations reduced the number of differentiated vessels but did not decrease the stimulation of cambial divisions.Unlike stimulation of cambial activity by GA₃, auxin stimulation of cambial divisions and differentiation of vessels were highly dependent upon stem polarity, 2,3,5-triiodobenzoic acid (TIBA) inhibited formation of vessels, but not cambial activity. The oscillations in basipetal efflux of natural auxin from the cambial stem region of successive 6 mm long sections substantiate the hypothesis that the histogenesis of xylem tissue in ring-porous species is under control of the vectoriat field that is associated with oscillatory phenomena in polar auxin transport.     

Hormonal Modulation of the Oscillatory System Involved in Polar Transport of Auxin

The amount of natural auxin collected in agar as a result of basipetal efflux from the cambial region of successive short sections of pine stem varies so that a wave-like pattern is formed. The wave-length is several times longer than the cell length in the cambial region, suggesting the existence of a supracellular oscillatory system, which forms a morphogenic field in the stem tissues, The amplitude of the auxin wave is amplified by apical application of IAA to the longer stem sections, particularly at she time of spring initiation of cambial activity. The wave of auxin disappears after simultaneous apical application of IAA and ABA. The modulatory effects of IAA and ABA are translocated along the investigated stem sections faster than known transport velocities of IAA molecules. This fact is considered as evidence of apical control of the morphogenic field by way of influence upon a supracellular system of conjugated oscillators in the tissue.     

Auxin Stimulation of Cambial Activity in Pinus silvestris II. Dependence upon Basipetal Transport

Natural auxin content has been determined in the cambial region of large Pinus silvestris L. trees at various dates during the year. The tissue was collected from the stem of intact or ring-barked trees and from stumps remaining after the trees were cut down at breast height in early summer or late autumn. No seasonal decrease of concentration of the extractable auxin in the cambial region could be detected. Decapitation or ring-barking produced severe reduction in auxin content and arrested cambial division. In the next season the auxin level and the cambial activity remained completely depressed. It is concluded that without tissue continuity in the region external to xylem and without basipetal supply of substances, no mechanism operated by roots or remaining stem tissue near the tree base can ensure a high level of auxin in the cambial region or activate and maintain the cambial division. The activity of extracted pine auxin was found not to be identical with the stimulatory potential of authentic IAA determined by standard bioassays. The possibility of interaction with other extracted substances is discussed.     

Auxin Stimulation of Cambial Activity in Pinus silvestris I. The Differential Cambial Response

Isolated stem segments of Pinus silvestris L. produce new xylem in sterile culture for 5 weeks if sucrose and IAA are present in the medium. The response of cambium varies in the course of the season and along the tree stem. The cambium is more sensitive in spring and in the stem portion closer to tree apex than later in the season and closer to the stem base. Spring initiation of cambial activity in adult pine trees under natural conditions could not be correlated with any consistent concentration gradient of natural auxin extracted from the cambial region. Thus, the relation between concentration of auxin and the activity of cambium is complex and involves changes of cambial responsivity. Interaction with gibberellic acid or kinetin and changing concentration of sucrose were studied during the season, but none of these substances alone appeared to be responsible for the observed variation in cambial response to auxin.     

Modulation of the oscillatory system involved in polar transport of auxin by other phytohormones

A wave-like pattern of the basipetal efflux of natural auxin from the cambial region of a series of consecutive short sections of stems of Larix decidua Mill., Acer pseudoplatanus L. and Picea abies (L.) Karst. has been demonstrated as it was earlier reported for Pinus silvestris L. Apical application of ABA suppressed the IAA-stimulated increase of the auxin-wave amplitude, and zeatin or GA₃ prevented this repression in stem segments of Pinus silvestris . All the exogenously applied substances were highly effective in physiological concentrations. Already 20-min of exposure to IAA or ABA at the apical end produced modulations of the auxin-wave along the whole 6.6 cm long stem segment. Application of 2, 3, 5-triiodobenzoic acid (TIBA) caused suppression of the wave-like pattern of auxin efflux similarly as ABA, supporting the association of the modulatory effects of ABA with the phenomena involved in polar transport of auxin. Abscisic acid applied to the basal end of the stem segment also reduced the auxin-wave amplification caused by simultaneous supply of IAA to the apical end. This finding additionally confirms the hypothesis that: 1) the supracellular auxin-wave generation is associated with the functioning of a system of oscillators coupled at the cellular level and 2) the auxin-wave modulations can be propagated acropetally, that is against the main direction of the auxin molecular transport.     

Polarity of Xylem Formation in Isolated Stem Segments of Pinus silvestris

The culture of isolated stem sogments was used as a technique to study polarity of xylem formation in Pinus silvestris L. Cambial activity was greatest at the apical or the basal end, whichever received the complete fresh medium, but decreased more abruptly with increasing distance when the basal end was so supplied. Whether gradients of activity increased basipetally or acropetally also depended upon the end to which the auxin and the sugar components were supplied. Ring-barking of segments, then cultur-ing them with apical supply of medium to the xylem, resulted in inhibition of xylem production below, and above, the ring. Cambial activity at the apical ends of such segments was expressible as a logarithmic function of the uninterrupted length of contiguous extraxylary tissues. The involvement of polarity phenomena in control of cambial activity in isolated stem segments is confirmed.     

P-glycoprotein4 displays auxin efflux transporter-like action in Arabidopsis root hair cells and tobacco cells

ATP binding cassette (ABC) transporters transport diverse substrates across membranes in various organisms. However, plant ABC transporters have only been scantily characterized. By taking advantage of the auxin-sensitive Arabidopsis thaliana root hair cell and tobacco (Nicotiana tabacum) suspension cell systems, we show here that Arabidopsis P-glycoprotein4 (PGP4) displays auxin efflux activity in plant cells. Root hair cell-specific overexpression of PGP4 (PGP4ox) and known auxin efflux transporters, such as PGP1, PGP19, and PIN-FORMEDs, decreased root hair elongation, whereas overexpression of the influx transporter AUXIN-RESISTANT1 enhanced root hair length. PGP4ox-mediated root hair shortening was rescued by the application of auxin or an auxin efflux inhibitor. These results indicate that the increased auxin efflux activity conferred by PGP4 reduces auxin levels in the root hair cell and consequently inhibits root hair elongation. PGP4ox in tobacco suspension cells also increased auxin efflux. PGP4 proteins were targeted to the plasma membrane of Arabidopsis root hair cells and tobacco cells without any clear subcellular polarity. Brefeldin A partially interfered with the trafficking of PGP4 reversibly, and this was rescued by pretreatment with auxin. These results suggest that PGP4 is an auxin efflux transporter in plants and that its trafficking to the plasma membrane involves both BFA-sensitive and -insensitive pathways.     

Maintenance of Asymmetric Cellular Localization of an Auxin Transport Protein through Interaction with the Actin Cytoskeleton

In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.     

Perception of gravity expressed by vascular differentiation

Abstract. The differentiation of new vessels was induced by local applications of auxin to isolated pieces of turnip storage roots. This differentiation was compared in varied relative orientations of the location of auxin application, the shoot-root polarity and the direction of gravity. Vessel differentiation was restricted to the cambial region and its course was determined primarily by the location of auxin application and the original, determined polarity of the tissues. Within this framework, the course of vessel differentiation was significantly modified by gravity: individual vessels tended to curve downwards and to be concentrated on the lower half of the tissue. The results can be understood as an observable expression of an influence of gravity on auxin transport.     

The role of auxin stored in scots pine trunk during spring activation of cambial activity

In a 9-year-old pine girdled during the winter cambial activity was observed below the girdle in the next spring. This indicates that cambial activity was initiated without auxin produced in the spring by buds. The auxin produced in apical shoots successively flows down the stem, where as a result of periodic restriction in transport it remains over the winter till the next year. This auxin of apical origin but locally stored over the winter in the stem is responsible for the activation of cambium before the new flow of auxin produced in the apical meristems arrives. Calculations based on seasonal changes in auxin levels can explain both, earlier spring activation of cambium in the crown and the temporary cambial divisions below the girdle, without assumption of direct auxin synthesis in the lateral meristems.     

Seasonal Changes in the Effects of Auxin on Rooting in Stem Cuttings of Ficus infectoria

Ficus infectoria stem cuttings were treated with 10 and 100 μg/ml each of IAA, IBA, 2,4, -D and NAA at monthly intervals and planted to study their rooting response after recording morphophysiological status and cambial activty of the parent branches. Attempts were also made to surgically expose the cambium before auxin treatment to determine the relationship of seasonal variation in auxin effectivity to cambial activity. The results show that: (1) there are two distinct phases in the sensitivity of Ficus infectoria stem cuttings to auxin-induced rooting; (2) the high rooting phase coincides with renovation of growth and high cambial activity starting in March and lasting through August and the low rooting phase coincides with winter dormancy and low cambial activity; (3) roots emerge in longitudinal rows in slitted auxin-treated cuttings; (4) slitted auxin-treated cuttings root profusely in June when cambial activity is high but not in October when cambial activity is low suggesting a close correspondence of seasonal variation between the rooting activity of auxin and cambial activity.     

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.     

Seasonal changes of plasma membrane H(+)-ATPase and endogenous ion current during cambial growth in poplar plants

The plasma membrane H(+)-ATPase (PM H(+)-ATPase), potassium ions, and endogenous ion currents might play a fundamental role in the physiology of cambial growth. Seasonal changes of these parameters were studied in twigs of Populus nigra and Populus trichocarpa. Monoclonal and polyclonal antibodies against the PM H(+)-ATPase, x-ray analysis for K(+) localization and a vibrating electrode for measurement of endogenous ion currents were used as probes. In dormant plants during autumn and winter, only a slight immunoreactivity against the PM H(+)-ATPase was found in cross sections and tissue homogenates, K(+) was distributed evenly, and the density of endogenous current was low. In spring during cambial growth, strong immunoreactivity against a PM H(+)-ATPase was observed in cambial cells and expanding xylem cells using the monoclonal antibody 46 E5 B11 F6 for fluorescence microscopy and transmission electron microscopy. At the same time, K(+) accumulated in cells of the cambial region, and strong endogenous current was measured in the cambial and immature xylem zone. Addition of auxin to dormant twigs induced the formation of this PM H(+)-ATPase in the dormant cambial region within a few days and an increase in density of endogenous current in shoot cuttings within a few hours. The increase in PM H(+)-ATPase abundance and in current density by auxin indicates that auxin mediates a rise in number and activity of an H(+)-ATPase in the plasma membrane of cambial cells and their derivatives. This PM H(+)-ATPase generates the necessary H(+)-gradient (proton-motive force) for the uptake of K(+) and nutrients into cambial and expanding xylem cells.     

Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 (AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1. AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from Arabidopsis pgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors. Expression of AtPGP1 in yeast and in the standard mammalian expression system used to analyze human MDR-type proteins results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1-naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is sensitive to auxin efflux and ABC transporter inhibitors. As is seen in planta, AtPGP1 also appears to mediate some efflux of IAA oxidative breakdown products associated with apical sites of high auxin accumulation. However, unlike what is seen in planta, some additional transport of the benzoic acid is observed in yeast and mammalian cells expressing AtPGP1, suggesting that other factors present in plant tissues confer enhanced auxin specificity to PGP-mediated transport.     

Vesicular cycling mechanisms that control auxin transport polarity

The polar transport of auxin controls many important plant growth and developmental processes. The polarity of auxin movement has long been suggested to be mediated by asymmetric distribution of auxin transport proteins, yet, until recently, little was known about the mechanisms that establish protein asymmetry in auxin-transporting cells. Now, a recent paper provides significant insight into the mechanism by which the GNOM protein controls the cycling of an auxin efflux carrier protein, PIN1, between the endosome and the plasma membrane. The dynamic movement of auxin transport proteins between internal compartments and the plasma membrane suggests mechanisms for alterations in auxin transport polarity in response to changing developmental or environmental regulation.     

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.     

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.     

Cellular Modelling of Secondary Radial Growth in Conifer Trees: Application to Pinus Radiata (D. Don)

The radial growth of conifer trees proceeds from the dynamics of a merismatic tissue called vascular cambium or cambium. Cambium is a thin layer of active proliferating cells. The purpose of this paper was to model the main characteristics of cambial activity and its consecutive radial growth. Cell growth is under the control of the auxin hormone indole-3-acetic. The model is composed of a discrete part, which accounts for cellular proliferation, and a continuous part involving the transport of auxin. Cambium is modeled in a two-dimensional cross-section by a cellular automaton that describes the set of all its constitutive cells. Proliferation is defined as growth and division of cambial cells under neighbouring constraints, which can eliminate some cells from the cambium. The cell-growth rate is determined from auxin concentration, calculated with the continuous model. We studied the integration of each elementary cambial cell activity into the global coherent movement of macroscopic morphogenesis. Cases of normal and abnormal growth of Pinus radiata (D. Don) are modelled. Abnormal growth includes deformed trees where gravity influences auxin transport, producing heterogeneous radial growth. Cross-sectional microscopic views are also provided to validate the model's hypothesis and results.     

Cell polarity and PIN protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1 function

Plants have many polarized cell types, but relatively little is known about the mechanisms that establish polarity. The orc mutant was identified originally by defects in root patterning, and positional cloning revealed that the affected gene encodes STEROL METHYLTRANSFERASE1, which is required for the appropriate synthesis and composition of major membrane sterols. smt1(orc) mutants displayed several conspicuous cell polarity defects. Columella root cap cells revealed perturbed polar positioning of different organelles, and in the smt1(orc) root epidermis, polar initiation of root hairs was more randomized. Polar auxin transport and expression of the auxin reporter DR5-beta-glucuronidase were aberrant in smt1(orc). Patterning defects in smt1(orc) resembled those observed in mutants of the PIN gene family of putative auxin efflux transporters. Consistently, the membrane localization of the PIN1 and PIN3 proteins was disturbed in smt1(orc), whereas polar positioning of the influx carrier AUX1 appeared normal. Our results suggest that balanced sterol composition is a major requirement for cell polarity and auxin efflux in Arabidopsis.