Distribution of activated calmodulin in the chloronema tip cells of the moss Funaria hygrometrica

Auxin (indole-3-acetic acid) regulates caulonema differentiation as a result of gradual transitional events in the chloronema tip cells in moss protonema. This auxin action in the moss Funaria hygrometrica involves a rapid influx of calcium ions from the extracellular medium. This investigation demonstrates spatial and temporal changes in calmodulin (CaM) activation (formation of Ca(2+)-CaM complex) in the chloronema tip cells subjected to auxin treatment. Photomicroscopic localisation of the fluorescence (excitation at 365 nm and emission of 397 nm) from the tricomplex of Ca(2+)-CaM with trifluoperazine (TFP, a blocker of Ca(2+)-CaM action) shows a tip to base (tip high) gradient of Ca(2+)-CaM in the chloronema tip cells. Comparison of Ca(2+)-CaM-TFP fluorescence over time in the chloronema tip cells of wild type Funaria with the response in an auxin overproducer mutant (86.1) and an auxin deficient mutant (87.13) reveals the involvement of auxin in calmodulin activation as a rapid response prior to cell differentiation.     

Unique Tissue-Specific Cell Cycle in Physcomitrella

Abstract: The moss Physcomitrella patens (Hedw.) B.S.G. is a novel tool in plant functional genomics as it has an inimitable high gene targeting efficiency facilitating the establishment of gene/function relationships.
Here we report, based on flow cytrometric (FCM) data, that the basic nuclear DNA content per cell of Physcomitrella is 0.53 pg, equating to a genome size of 1 C = 511 Mbp. Furthermore, we describe a unique tissue-specific cell cycle change in this plant. Young plants consisting of only one cell type (chloronema) displayed one single peak of fluorescence in FCM analyses. As soon as the second cell type (caulonema) developed from chloronema, a second peak of fluorescence at half the intensity of the previous one became detectable, indicating that caulonema cells were predominantly at the G1/S transition, whereas chloronema cells were mainly accumulating at the G2/M transition. This conclusion was validated by further evidence: i) The addition of ammonium tartrate arrested Physcomitrella in the chloronema state and in G2/M. ii) Two different developmental mutants, known to be arrested in the chloronema/caulonema transition, remained in G2/M, regardless of age and treatment. iii) The addition of auxin or cytokinin induced the formation of caulonema, as well as decreasing the amount of cells in G2/M phase. Additionally, plant growth regulators promoted endopolyploidisation.
Thus, cell cycle and cell differentiation are closely linked in Physcomitrella and effects of plant hormones and environmental factors on both processes can be analysed in a straight forward way. We speculate that this unique tissue-specific cell cycle arrest may be the reason for the uniquely high rate of homologous recombination found in the Physcomitrella nuclear DNA.     

Bound auxin metabolism in cultured crown-gall tissues of tobacco

Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [¹⁴C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [¹⁴C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66-transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [¹⁴C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [¹⁴C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation.     

A saturable site responsible for polar transport of indole-3-acetic acid in sections of maize coleoptiles

The velocity of transport and shape of a pulse of radioactive indole-3-acetic acid (IAA) applied to a section of maize (Zea mays L.) coleoptile depends strongly on the concentration of nonradioactive auxin in which the section has been incubated before, during, and after the radioactive pulse. A pulse of [³H]IAA disperses slowly in sections incubated in buffer (pH 6) alone; but when 0.5–5 M IAA is included, the pulse achieves its maximum velocity of about 2 cm h^-1. At still higher IAA concentrations in the medium, a transition occurs from a discrete, downwardly migrating pulse to a slowly advancing profile. Specificity of IAA in the latter effect is indicated by the observation that benzoic acid, which is taken up to an even greater extent than IAA, does not inhibit movement of [³H]IAA. These results fully substantiate the hypothesis that auxin transport consists of a saturable flux of auxin anions (A^-) in parallel with a nonsaturable flux of undissociated IAA (HA), with both fluxes operating down their respective concentration gradients. When the anion site saturates, the movement of [³H]IAA is nonpolar and dominated by the diffusion of HA. Saturating polar transport also results in greater cellular accumulation of auxin, indicating that the same site mediates the cellular efflux of A^-. The transport inhibitors napthylphthalamic acid and 2,3,5-triiodobenzoic acid specifically block the polar A^- component of auxin transport without affecting the nonsaturable component. The transport can be saturated at any point during its passage through the section, indicating that the carriers are distributed throughout the tissue, most likely in the plasmalemma of each cell.Abbreviations A^- auxin anion - HA undissociated auxin - IAA indole-3-acetic acid - NPA N-1-napthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Connection between the Synthesis of Differentiation Specific Proteins and the Capacity of Cells to Respond to Cytokinin in the Moss Funaria

The differentiation stage of the caulonema in Funaria hygrometrica protonema is distinguished from the chloronema stage by three additional protein bands (CSP) in the soluble protein fraction. During the change of caulonema to chloronema, which is induced by isolation of single filaments (regeneration), the CSP disappear. This is not the consequence of an accelerated degradation or turnover but of a gradual termination in the de novo synthesis of CSP during regeneration as demonstrated by pulse-chase experiments with l -[4,5–³H] leucine. Cytokinin inhibits the termination of the synthesis of CSP. The decrease in synthesis parallels the decrease in ability of the isolated caulonema cells to respond to cytokinin via bud formation. It is therefore concluded that the CSP are involved in the competence of caulonema cells to respond to cytokinins.     

pH-Dependent accumulation of indoleacetic acid by corn coleoptile sections

The uptake of auxin by 1-mm slices of corn (Zea mays L.) coleoptiles, a tissue known to transport auxin polarly, depends on the pH of the medium. Short-term uptake of indole-3-acetic acid (IAA) in coleoptiles increases with decreasing pH of the buffer as would be expected if the undissociated weak acid, IAA·H, were more permeable than the auxin anion, IAA^-, and IAA^- accumulates in the tissues because of the higher pH of the cytoplasm. Although uptake of [³H]IAA is reduced in neutral buffers, it is greater than expected if it were limited to just the extracellular space of the tissue. The radioactivity accumulated by the tissue can be quantitatively extracted by organic solvents and identified as IAA by thin-layer chromatography. The tissue radioactivity is freely mobile and can efflux from the tissue. Thus these cells in pH 5 buffer are able to retain an average internal concentration of mobile IAA that is at least several times greater than the external concentration. A prominent feature of auxin uptake from acidic buffers is enhanced accumulation at high auxin concentration. This indicates that, in addition to fluxes of IAA·H, a saturable site is involved in auxin uptake. Whenever the auxin-anion gradient is directed outward, saturating the efflux of auxin anions increases accumulation. Furthermore, the observed slowing of short-term uptake of radioactive IAA by increasing concentrations of IAA or K⁺ indicates either an activation of the presumptive auxin leak or saturation of another carrier-mediated uptake system such as a symport of auxin anions with protons. By contrast in neutral buffers, effects of concentration on uptake rates disappear. This implies that at neutral pH the anion leak is decreased and influx depends on the symport.     

Characteristics of spore germination and protonemal development in Hypnum pacleseens

The spore germination, protonemal development, and gametophyte differentiation of Hypnum pacleseens were observed in cultivation. Photomicrographs showed that spore germination of Hypnum pacleseens occured within the exospore. Its protonema is massive with filamentous chloronema formed inside. The terminal part of the chloronema differentiated into filamentous caulonema and its rhizoid was derived from the apical cell of the filamentous chloronema. The initial cell of gametophyte differentiated from chloronema and caulonema. Sporeling-type of Hypnum pacleseens is developmentally similar to Glyphmitrium-type.     

Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophan-resistant cell lines of wild carrots

A 5-methyltryptophan(5-MT)-resistant cell line of wild carrot (Daucus carota L.), W001, that exhibited auxin-independent callus growth, was found to accumulate indole-3-acetic acid (IAA) and tryptophan (trp). Anthranilate-synthetase activity in W001 cell extract was less sensitive to feedback inhibition by trp than in the original 5-MT-sensitive cell lines. It is hypothesized that the resistant enzyme allowed more trp synthesis and accumulation which, in turn, affected the IAA concentration in the cell. Since carrot cultures cannot regenerate in the presence of exogenous auxin, the elevated IAA concentration in W001 may be responsible for its drastically reduced capacity to regenerate. The relationship between trp and IAA levels was further investigated by examining the effect of 2,4-dichlorophenoxy acetic acid (2,4-D) on the endogenous concentration of trp and IAA. In general, the IAA level was reduced but the trp concentration was elevated when 2,4-D was present in the culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - 5-MT^r 5-MT-resistant - 5-MT^s 5-MT-sensitive - trp tryptophan     

Quantitative studies of embryogenesis in normal and 5-methyltryptophan-resistant cell lines of wild carrot

The frequency of embryo formation was determined in normal and 5-methyltryptophan-resistant (5-MT^r) cell lines of wild carrot (Daucus carota L.) grown in the presence or absence of 2-isopentenyladenine (2-ip) and 2,4-dichlorophenoxyacetic acid (2,4-D). 2-ip stimulated the intitation of embryo formation and also accelerated embryo development. 2.4-D inhibited embryo differentiation at several stages: at 0.1 mg/l, it stopped regeneration at the earliest stage, resulting in callus growth instead of embryo formation; at 0.04 mg/l 2,4-D, some globular embryos were produced, but they did not develop into more advanced embryos. Variant cell lines with higher levels of auxin (indole-3-acetic acid, IAA) were used to study the effect of an elevated endogenous concentration of auxin on embryogenesis. IAA at these concentrations suppressed regeneration in the same manner as the exogenous auxin, 2,4-D, did. This result confirms the hypothesis that high levels of IAA are responsible for the suppression of regeneration in the 5-MT^r cell lines.     

On the action mechanism of cytokinins in mosses: Caulonema specific proteins

Soluble proteins extracted with Tris-buffer pH 8.8 from both differentiation stages of the protonema of Funaria hygrometrica (chloronema and caulonema) were separated by microgel electrophoresis. 4x10^-3 mg protein/gel was applied. The caulonema, which is the only part of the protonema able to form buds following cytokinin treatment, contained 3 protein bands, which were absent in chloronema. We designate them as caulonema specific proteins (CSP, approximate molecular weight 500,000). The CSP disappear when the caulonema is isolated and its cells regenerate to chloronema. The CSP bind kinetin (6 Furfurylamino [8-¹⁴C]purine) or BAP (6-benzylamino[8-¹⁴C]purine) about 10 times stronger than the remaining protein bands in the gel. The greatest part of the cytokinin is metabolized in a short time and consequently a part of the activity in the gel is incorporated into RNA and removable with RNase. Simultaneous application of adenosine and cytokinin reduced the incorporation of radioactivity into RNA and enhanced the specific activity in one of the CSP bands. In all other bands it remained unchanged.From the results it can be suggested that the CSP are probably involved in the early reactions to cytokinin of the target cells.Abbreviations CSP Caulonema specific proteins - BAP 6-benzylaminopurine - GA₁ gibberellin A₁     

Expression of AMIDASE1 (AMI1) is suppressed during the first two days after germination

The regulation of cellular auxin levels is a critical factor in determining plant growth and architecture, as indole-3-acetic acid (IAA) gradients along the plant axis and local IAA maxima are known to initiate numerous plant growth responses. The regulation of auxin homeostasis is mediated in part by transport, conjugation and deconjugation, as well as by de novo biosynthesis. However, the pathways of IAA biosynthesis are yet not entirely characterized at the molecular and biochemical level. It is suggested that several biosynthetic routes for the formation of IAA have evolved. One such pathway proceeds via the intermediate indole-3-acetamide (IAM), which is converted into IAA by the activity of specific IAM hydrolases, such as Arabidopsis AMIDASE1 (AMI1). In this article we present evidence to support the argument that AMI1-dependent IAA synthesis is likely not to be used during the first two days of seedling development.Key words: Arabidopsis thaliana, auxin biosynthesis, AMIDASE1, indole-3-acetic acid, indole-3-acetamide, LEAFY COTYLEDON1, seed developmentAuxins are versatile plant hormones that play diverse roles in regulating many aspects of plant growth and development.¹ To enable auxins to develop their activity, a tight spatiotemporal control of cellular indole-3-acetic acid (IAA) contents is absolutely necessary since it is well-documented that auxin action is dose dependent, and that high IAA levels can have inhibitory effects on plant growth.² To achieve this goal, plants have evolved a set of different mechanisms to control cellular hormone levels. On the one hand, plants possess several pathways that contribute to the de novo synthesis of IAA. This multiplicity of biosynthetic routes presumably facilitates fine-tuning of the IAA production. On the other hand, plants are equipped with a variety of enzymes that are used to conjugate free auxin to either sugars, amino acids or peptides and small proteins, respectively, or on the contrary, that act as IAA-conjugate hydrolases, releasing free IAA from corresponding conjugates. IAA-conjugates serve as a physiologically inactive storage form of IAA from which the active hormone can be quickly released on demand. Alternatively, conjugation of IAA can mark the first step of IAA catabolism. In general, conjugation and deconjugation of free IAA are ways to positively or negatively affect active hormone levels, which adds another level of complexity to the system. Additionally, IAA can be transported from cell to cell in a polar manner, which is dependent on the action of several transport proteins. All together, these means are used to form auxin gradients and local maxima that are essential to initiate plant growth processes, such as root or leaf primordia formation.³     

Is Cell Elongation Regulated by Extracellular Auxin?

Experiments with isolated epidermal strips of maize coleoptiles, pretreated with auxin and further incubated on sucrose agar containing different concentrations of auxin (indole-3-acetic acid, IAA or naphthalene-1-acetic acid, NAA) and/or naphthylphthalamic acid (NPA), are described. Preincubation for 2h with 2 . 10^?4M IAA or 10^?5M NAA in buffer, followed by 30 min wash in buffer results in measurable cell elongation during a subsequent incubation for 6 h on sucrose agar. Addition of 10^?4M NPA inhibited the response to auxin and this inhibition could be reversed by providing IAA in addition to NPA. Inner tissue fragments (without outer epidermis) did not respond to external IAA. These results lead to the conclusion that auxin secretion at the outer epidermis may be an essential step in auxin-regulated coleoptile growth.     

Physiological evidence that the primary site of auxin action in maize coleoptiles is an intracellular site

To locate functionally the primary site of auxin action in growing cells, the pool of auxin relevant to induction of growth in maize (Zea mays L.) coleoptile sections was determined. A positive correlation was consistently noted between growth and intracellular levels of indole-3-acetic acid (IAA), i.e. growth appears to be relatively independent of the external level of IAA. N-1-Naphthylphthalamic acid (NPA), a potent inhibitor of auxin transport, was used to enhance accumulation of IAA in coleoptile cells. From the use of NPA, it is shown that: 1) increasing the accumulation of IAA in cells, while the external concentration is held constant, resulted in a concomitant increase in growth, and 2) blocking the exit of IAA from cells with NPA sustained an IAA-induced growth response in the absence of externally applied IAA. Furthermore, the absence of any alterations in auxin binding to microsomal fractions by NPA indicates that the action of NPA in causing enhancement of auxin-induced growth is based upon its inhibition of efflux of IAA from the cells. This research was supported by National Science Foundation grant No. DMB 8515925. The careful assistance of Laurie Brulport is gratefully acknowledged.     

Photoaffinity labelling with the cytokinin agonist azido-CPPU of a 34 kDa peptide of the intracellular pathogenesis-related protein family in the moss Physcomitrella patens

As in higher plants, the development of the moss Physcomitrella patens is regulated by environmental signals and phytohormones. At the protonema level transition from chloronema to caulonema cells is under auxin control. The formation on second sub-apical caulonema cells of buds that will give rise to the leafy gametophore requires cytokinins. Using [³H]azidoCPPU (1-(2-azido-6-chloropyrid-4-yl)-3-(4-[³H])phenylurea), a photoactivatable cytokinin agonist, we have specifically photolabelled a soluble 34 kDa protein of P. patens. Urea derivatives were very efficient competitors of photolabelling while purine-type cytokinins were poor competitors. The protein UBP34 was purified by affinity chromatography and the sequences of six internal peptides obtained. A cDNA encoding UBP34 was cloned by screening a P. patens protonema cDNA library with a probe amplified by PCR using degenerate primers designed from the peptide sequences. The UBP34 amino acid sequence shows an average sequence identity of 42% with both intracellular PR proteins and the BetV1-related family of plant allergens. Recombinant UBP34 expressed in Escherichia coli was confirmed to bind azidoCPPU.     

Auxin in scapes,flower buds,flowers, and fruits of daffodil (Narcissus pseudonarcissus L.)

Summary Diffusates from flower buds, flower fruits, and scape segments, and extracts of flower stalks of Narcissus pseudonarcissus contain an auxin active in the Avena geo-curvature test. The auxin behaved like indole-3-acetic acid (IAA) in thin-layer chromatography (TLC) with neutral and basic solvents on different adsorbents. After TLC, the auxin of the extracts showed chromogenic reactions identical with those of IAA; in gas-liquid chromatography on two different columns, the purified substance, after methylation, appeared at the retention time of IAA methyl ester. The auxin content of the extracts has been estimated to be equivalent to ca. 10 g IAA kg^–1 fresh weight. Diffusates, collected at the basal end of excised flowering apices and of scape segments at different developmental stages, showed highest auxin activity when collected from old buds and young flowers, and from the basal, rapidly elongating scape regions. The diffusible auxin obtained from scape segments was very likely produced by the segments themselves. Thus, the shoot of Narcissus appears to possess two different sites of auxin production, namely, the apical region represented by the flower bud, the flower or the fruit, and the scape.Abbreviations IAA indole-3-acetic acid - IAA-OMe indole-3-acetic-acid methyl ester - TLC thin-layer chromatography - GLC gas-liquid chromatography     

NPA Inhibits Secretion of Amylases by Barley Aleurone Cells and Auxins can Overcome this Inhibition

Polar cell growth is regulated by auxin (Klämbt et al., 1992). It is essentially dependent upon exocytosis. Is exocytosis directly regulated by auxin? Aleurone cells secrete amylases, as well as other hydrolases, by exocytosis. In preliminary experiments, with 22 h of incubation, increasing concentration of N-1-naphthylphthalamic acid (NPA) parallels the inhibition of amylase secretion. A model system for secretion was designed. Barley aleurone layers, previously incubated in 1 μM GA₃ for 16 h, then washed extensively in the presence of 10 μM NPA for 10 min, were incubated finally for 2 h in GA₃, NPA and/or auxins. NPA inhibits amylase secretion. Auxins completely abolished secretion inhibition at the optimal concentration of 1 μM. Aleurone layers contain 1.5 to 3 μM endogenous indole-3-acetic acid (IAA) and have a high capacity to accumulate and retain IAA. A 7-fold accumulation occurred within 3 h after application of 20 μM radiolabeled IAA. A loss of about 10% could be measured within the following 4 h period in fresh media, independent of the presence of NPA. The inhibition of amylase secretion by NPA is not due to an inhibition of protein biosynthesis which was tested by the use of [³⁵S]-methionine.     

Quantitative predictions for the chemiosmotic uptake of auxin

1. The predictions of a general kinetic model for the chemiosmotic uptake of auxin and other weak acids are compared with experimental results for the auxin indoleacetic acid. The proposed mechanism involves diffusional flux of undissociated acid, a saturable, voltage-sensitive flux of anion (A^-), and a carrier-mediated symport of H⁺ and A^-, all operating in parallel. During much of uptake, the electrochemical gradients are such that the net symport and the net anion flux are in opposition: the symport contributes more to influx; the anion path, to efflux. The voltage-sensitive flux of A^- therefore constitutes a leak. 2. The presence of a symport, whose carrier can distribute across the membrane in response to the internal and external concentrations of auxin, can speed the rate of uptake, but does not by itself alter the accumulation of auxin at equilibrium. 3. The accumulation ratio at equilibrium is less at low concentrations of auxin than at higher concentrations, indicating the presence of a saturable anion path. The concentration dependence of the transition depends on several factors, and is not a reliable indicator of the A^--carrier binding constant. 4. Observed uptake near neutral pH appears larger than is consistent with a voltage-sensitive anion flux being the only carrier-mediated path across the membrane. This observation provides indirect evidence for the presence of an auxin-proton symport in addition to a saturable A^- carrier. 5. The change in kinetics of uptake of [³H]indole-3-acetic acid (IAA), observed as the total concentration of IAA is raised from 0.1 to 100 M, is consistent with either (i) a symport that saturates at low concentrations, or (ii) activation of an A^- efflux by intermediate concentrations of auxin. 6. The data on the concentration dependence of uptake of auxin are not consistent with a multi-proton symport.Abbreviations A^- auxin anion - HA weak acid, particularly IAA - HXA carrier in electroneutral complex with a proton and the auxin anion - H₂XA carrier in electroneutral complex with two protons and the auxin anion - IAA indole-3-acetic acid - X auxin carrier - XA carrier-auxin anion complex     

Characteristics of spore germination and protonemal development in Hypnum pacleseens

The spore germination,protonemal development,and gametophyte differentiation of Hypnum pacleseens were observed in cultivation.Photomicrographs showed that spore germination of Hypnum pacleseens occured within the exospore.Its protonema is massive with filamentous chloronema formed inside.The terminal part of the chloronema differentiated into filamentous caulonema and its rhizoid was derived from the apical cell of the filamentous chloronema.The initial cell of gametophyte differentiated from chloronema and caulonema.Sporeling type of Hypnum pacleseens is developmentally similar to Glyphmitrium-type.     

Auxin dependent growth of rhizoids of Chara globularis

Auxin greatly influences plant cell elongation, particularly in the organs of shoots but also in roots. Earlier reports are limited to organ and/or cell growth connected with a mosaic type of cell elongation. The present paper describes auxin sensitivity of polarly growing rhizoid cells of Chara globularis Thuill. where auxin-dependent growth could be observed in two different ways: (1) Auxin had no effect when applied to intact Chara explants with developed thizoids, but decapitated explants reacted to auxin with optimal growth at 1 μ M indole-3-acetic-acid (IAA). (2) N-I-Naphthylphthalamic acid (NPA) at 10 and 100 μ M caused a strong inhibition in rhizoid growth of intact Chara explants. Auxin applied at the same time abolished this inhibition but, due to lack of plant material, endogenous IAA content could not be measured. Chara explants pre-fed with 1-[¹⁴C] IAA from a 3.5 μ M solution for 8 h, then washed and transferred for 11 h to auxin free solution containing 0, 10 or 100 μ M NPA, showed an effect of NPA upon IAA accumulation. Therefore, NPA may inhibit auxin secretion in Chara , 100. Our data are in agreement with earlier results on auxin regulated cell elongation and H⁻-secretion, and show that auxin secretion may also be an essential step in endogenous regulation of polar growth in Chara rhizoids.