Cellular differentiation in root caps of Zea mays that do not secrete mucilage

We quantified the structural changes accompanying cellular differentiation in root caps of Zea mays cv. Ageotropic to determine the developmental basis for the nongraviresponsiveness of their primary roots. Cells of the calyptrogen and columella of primary roots of the ageotropic mutant have structures indistinguishable from those of caps of primary roots of Z. mays cv. Kys the graviresponsive, wild-type parent of Z. mays cv. Ageotropic. However, the relative volumes of dictyosomes, dictyosome-derived vesicles and starch in the outermost peripheral cells of wild-type roots were significantly lower than were those in peripheral cells of mutant roots. This corresponds to a dramatic accumulation of starch and mucilage-filled vesicles in peripheral cells of mutant roots. Cellular differentiation in root caps of graviresponsive seminal roots of the Ageotropic mutant resembled that of primary and seminal roots of the wild-type cultivar, and differed significantly from that of primary roots of the mutant. We conclude that the mutation that blocks secretion of mucilage from peripheral cells of Ageotropic roots: (1) expresses itself late in cellular differentiation in root caps; (2) is expressed only in primary (but not seminal) roots of the Ageotropic mutant; and (3) is consistent with malfunctioning dictyosomes and dictyosome-derived vesicles being the cellular basis for agravitropism of primary roots of this mutant.     

Cytochalasin B,but not colchicine,inhibits migration of secretory vesicles in root tips of maize

Summary The effects of colchicine and cytochalasin B on the structure of dictyosomes of maize root tips were studied. Colchicine did not significantly affect dictyosome structure or change the distribution of dictyosome-derived secretory vesicles. Cytochalasin B did not significantly change dictyosome structure or intercisternal fibers, but did alter markedly the distribution of the secretory vesicles in both the epidermal and outer cap cells. With cytochalasin B, the vesicles accumulated in a region close to their site of formation and did not migrate to the cell surface. The results show that a cytochalasin B-sensitive subcellular component is involved in the vectorial movement of secretory vesicles from sites of formation at dictyosomes to sites of fusion at the cell surface.     

Heterogeneity of auxin-accumulating membrane vesicles from Cucurbita and Zea: a possible reflection of cell polarity

When microsomes from hypocotyls of Cucurbita pepo L. or coleoptiles of Zea mays L. were centrifuged on dextran-sucrose gradients a heterogeneity of auxin-accumulating vesicles was observed. Vesicles from the top part of the gradient showed saturable, specific accumulation of indole-3-acetic acid with only a small stimulation by phytotropins, and with very few binding sites for 1-N-naphthylphthalamic acid. In the vesicles from the lower part of the gradient, net accumulation of indole-3-acetic acid could be strongly increased by addition of phytotropins; binding of 1-N-naphthylphthalamic acid was high in this region. After two-phase partitioning, both kinds of vesicles were found in the upper-phase membrane fraction considered to be purified plasma membrane. The hypothesis is discussed that vesicles can be separated from the apical and basal parts of the cell's plasmalemma.Abbreviations CCO cytochrome-c oxidase - CCR KCN-insensitive NADH-dependent cytochrome-c reductase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IDPase inosine 5-diphosphatase - ION3 ionophore mixture of carbonylcyanide-3-chlorophenylhydrazone, nigericin and valinomycin - 1-NAA 1-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - UDPG uridine diphosphoglucose     

The effect of cytochalasin B on the rate of growth and ultrastructure of wheat coleoptiles and maize roots

Cytochalasin B (CB) inhibits the elongation growth of maize roots, and that of wheat coleoptile segments incubated in indolyl-3-acetic acid, by over 30% after a lag period of about 60 min. This long lag is not due to poor tissue penetration by the inhibitor, but seems to reflect a property of the process inhibited by CB. The only visible ultrastructural change accompanying growth inhibition is the accumulation of secretory vesicles in the vicinity of dictyosomes, which occurs between 90 and 300 min. However, a massive accumulation of vesicles is seen after 120 min in root cap cells which possess very active dictyosomes. The results indicate that CB does not inhibit elongation growth by interfering with cytoplasmic streaming. Instead, they indicate that the drug acts to inhibit the secretion of cell wall components at some stage after vesicle production, but prior to their transport.Abbreviations CB cytochalasin B - IAA indolyl-3-acetic acid - DMSO dimethyl sulphoxide     

解淀粉芽胞杆菌HZ-12中ysnE参与吲哚-3-乙酸合成的代谢途径研究

【目的】吲哚-3-乙酸是调控植物生长发育和生理活动的重要激素,吲哚-3-乙酸N-乙酰转移酶YsnE在吲哚-3-乙酸合成中发挥重要作用,本研究拟解析解淀粉芽胞杆菌中YsnE参与吲哚-3-乙酸合成的代谢途径。【方法】通过基因ysnE缺失和强化表达,分析ysnE对吲哚-3-乙酸合成影响,结合吲哚-3-乙酸合成中间物(吲哚丙酮酸、吲哚乙酰胺、色胺和吲哚乙腈)添加和体外酶转化实验,解析ysnE参与吲哚-3-乙酸合成的代谢途径。【结果】明确了YsnE在解淀粉芽胞杆菌HZ-12吲哚-3-乙酸合成中发挥重要作用。发现ysnE缺失菌株中的吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈利用显著降低,揭示了YsnE主要发挥吲哚丙酮酸脱羧酶YclB和吲哚乙酰胺水解酶/腈水解酶/腈水合酶YhcX的功能,并通过参与吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径来影响吲哚-3-乙酸合成。【结论】初步揭示了YsnE通过影响吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径参与吲哚-3-乙酸合成的代谢机理,为吲哚-3-乙酸合成途径解析和代谢工程育种构建吲哚-3-乙酸高产菌株奠定了基础。     

In-vitro auxin transport in membrane vesicles from maize coleoptiles

When membrane vesicles from maize (Zea mays L.) coleoptiles are extracted at high buffer strength, a pH-driven, saturable association of [¹⁴C] indole-3-acetic acid is found, similar to the in-vitro auxin-transport system previously described for Cucurbita hypocotyls. The phytotropins naphthylphthalamic acid and pyrenoylbenzoic acid increase net uptake, pressumably by inhibiting the auxin-efflux carrier.Abbreviations IAA indole-3-acetic acid - ION3 ionophore mixture of carbonylcyanide-3-chlorophenylhydrazone, nigericin and valinomycin - 1-NAA, 2-NAA 1-, 2-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid     

In vitro root differentiation and essential-oil accumulation in Angelica archangelica

Summary We studied the relationship between root differentiation and the accumulation of essential oils in Angelica archangelica in in vitro cultures and in the intact plant. Root regeneration was obtained using stem and leaf explants subjected to treatment with the auxins indole-3-butyric acid, indole-3-acetic acid and α-naphthaleneacetic acid. In both stem and leaf explants, treatment with indole-3-butyric acid induced the highest rhizogenic response in terms of both percentage of explants with roots and number of roots per explant. Independently of hormonal treatment, stem explants produced a higher average number of roots per explant. Root meristemoids were already visible at day 7 of culture in the treatments with indole-3-butyric acid and indole-3-acetic acid; they were formed directly by cambial-cell division. In vitro-regenerated roots retained primary root structure and differentiated only two primary ephemeral ducts in the pericycle; no accumulation of essential oils was detected. Same-size roots taken from the intact plant showed secondary structure and essential-oil accumulation. The results of this study suggest that the synthesis and accumulation of essential oils in Angelica archangelica is closely linked to the differentiation of secondary secretory ducts.     

An <Emphasis Type="Italic">ipdC</Emphasis> gene knock-out of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion

The indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of IAA biosynthesis in Azospirillum brasilense SM was functionally disrupted in a site-specific manner. This disruption was brought about by group II intron-based Targetron gene knock-out system as other conventional methods were unsuccessful in generating an IAA-attenuated mutant. Intron insertion was targeted to position 568 on the sense strand of ipdC, resulting in the knock-out strain, SMIT568s10 which showed a significant (∼50%) decrease in the levels of indole-3-acetic acid, indole-3-acetaldehyde and tryptophol compared to the wild type strain SM. In addition, a significant decrease in indole-3-pyruvate decarboxylase enzyme activity by ∼50% was identified confirming a functional knock-out. Consequently, a reduction in the plant growth promoting response of strain SMIT568s10 was observed in terms of root length and lateral root proliferation as well as the total dry weight of the treated plants. Residual indole-3-pyruvate decarboxylase enzyme activity, and indole-3-acetic acid, tryptophol and indole-3-acetaldehyde formed along with the plant growth promoting response by strain SMIT568s10 in comparison with an untreated set suggest the presence of more than one copy of ipdC in the A. brasilense SM genome.     

Oxidation of Indole-3-acetic Acid and Oxindole-3-acetic Acid to 2,3-Dihydro-7-hydroxy-2-oxo-1H Indole-3-acetic Acid-7′-O-β-d-Glucopyranoside in Zea mays Seedlings

Radiolabeled oxindole-3-acetic acid was metabolized by roots, shoots, and caryopses of dark grown Zea mays seedlings to 2,3-dihydro-7-hydroxy-2-oxo-1H indole-3-acetic acid-7′-O-β-d-glycopyranoside with the simpler name of 7-hydroxyoxindole-3-acetic acid-glucoside. This compound was also formed from labeled indole-3-acetic acid supplied to intact seedlings and root segments. The glucoside of 7-hydroxyoxindole-3-acetic acid was also isolated as an endogenous compound in the caryopses and shoots of 4-day-old seedlings. It accumulates to a level of 4.8 nanomoles per plant in the kernel, more than 10 times the amount of oxindole-3-acetic acid. In the shoot it is present at levels comparable to that of oxindole-3-acetic acid and indole-3-acetic acid (62 picomoles per shoot). We conclude that 7-hydroxyoxindole-3-acetic acid-glucoside is a natural metabolite of indole-3-acetic acid in Z. mays seedlings. From the data presented in this paper and in previous work, we propose the following route as the principal catabolic pathway for indole-3-acetic acid in Zea seedlings: Indole-3-acetic acid → Oxindole-3-acetic acid → 7-Hydroxyoxindole-3-acetic acid → 7-Hydroxyoxindole-3-acetic acid-glucoside.     

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme

Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole-3-acetonitrile to the plant growth hormone, indole-3-acetic acid in vitro. To probe the capacity of this enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV-35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg^?1 protein with indole3-acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1-[¹³C]-indole-3-acetonitrile to 1-[¹³C]-indole-3-acetic acid in vivo as determined by HPLC/ GC-MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole-3-acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole-3-acetonitrile, a strong auxin-overproducing phenotype developed resulting in increased lateral root formation (at 10 µM indole-3-acetonitrile) or stunted shoot growth, excessive lateral root initiation, inhibition of root out-growth and callus formation at the root/shoot interface (at 100 µM indole-3-acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole-3-acetonitrile to indole-3-acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high-auxin phenotype upon transgenic plants. Thus, the A. thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.     

Catabolism of indole-3-acetic acid in chloroplast fractions from light-grown Pisum sativum L. seedlings

Abstract The catabolism of indole-3-acetic acid was investigated in chloroplast preparations and a crude enzyme fraction derived from chloroplasts of Pisum sativum seedlings. Data obtained with both systems indicate that indole-3-acetic acid undergoes decarboxylative oxidation in pea chloroplast preparations. An enhanced rate of decarboxylation of [1′-¹C]indole-3-acetic acid was obtained when chloroplast preparations were incubated in the light rather than in darkness. Results from control experiments discounted the possibility of this being due to light-induced breakdown of indole-3-acetic acid. High performance liquid chromatography analysis of [2′-¹⁴C]indole-3-acetic acid-fed incubates showed that indole-3-methanol was the major catabolite in both the chloroplast and the crude enzyme preparations. The identification of this reaction product was confirmed by gas chromatography-mass spectrometry when [²H₅]indole-3-methanol was detected in a purified extract derived from the incubation of an enzyme preparation with ³²H₅]indole-3-acetic acid.     

Auxin control of the sensitivity to auxin of the proton translocation catalyzed by the tobacco plasma membrane H+-ATPase

The sensitivity to indole-3-acetic acid of the proton translocation catalyzed by the plasma membrane proton pump from tobacco cells was determined in vitro, on plasma membrane vesicles, according to the 2,4-dichlorophenoxyacetic acid concentration during cell culture. The sensitivity was shown to increase by 20-fold along with the 2,4-dichlorophenoxyacetic acid concentration (between 0.05 µM and 0.25 µM). Treatment of cells with indole-3-acetic acid prior to membrane purification promoted an increase in the sensitivity up to 100-fold. This increase was observed after treatment with micromolar indole-3-acetic acid for cells cultured in the presence of 0.05 µM 2,4-dichlorophenoxyacetic acid, but required sub-millimolar indole-3-acetic acid concentrations for cells cultured in the presence of 0.25 µM 2,4-dichlorophenoxyacetic acid. On the other hand, the increase in sensitivity occured within 20 min irrespective of the 2,4-dichlorophenoxyacetic acid concentration during cell culture. It is proposed that such increase in the sensitivity could constitute a progressive amplification process favouring the stimulation of proton translocation by limited changes in auxin concentration.     

Indole-3-methanol is the main product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases from Lupinus

The nature of the products of the auxin catabolism mediated by both basic and acidic isoperoxidases has been studied. While indole-3-methanol is only a minor product of the oxidation of indole-3-acetic acid catalyzed by extracellular acidic isoperoxidases, it is the only product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases (EC 1.11.1.7) from lupin (Lupinus albus L.) hypocotyls. The putative indole-3-methanol formed by these latter isoperoxidases was isolated and then characterized by mass spectrometry and ¹H-nuclear magnetic resonance spectrometry. These results are discussed with respect to the diversity and compartmentation of the catabolism of indole-3-acetic acid in plant tissues.Abbreviations DCP 2,4-dichlorophenol - IAA indole-3-acetic acid - IM indole-3-methanol     

Indole-3-acetic acid catabolism in Zea mays seedlings. Metabolic conversion of oxindole-3-acetic acid to 7-hydroxy-2-oxindole-3-acetic acid 7'-O-beta-D-glucopyranoside

A new metabolite of the plant growth substance indole-3-acetic acid has been extracted from Zea mays seedlings and characterized as the 7'-O-beta-D-glucopyranoside of 7-hydroxy-2-oxindole-3-acetic acid. This compound was the major product formed from [5-3H] 2-oxindole-3-acetic acid, incubated with intact plants or root and coleoptile sections. Identification was by gas chromatography-mass spectrometry of the trimethylsilyl derivative and by analysis of the hydrolysis products. A synthesis is reported for 7-hydroxy-2-oxindole-3-acetic acid. These results and prior work demonstrate the following catabolic route for indole-3-acetic acid in Zea: indole-3-acetic acid----2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid glucoside.     

The role of the shoot apex in controlling rhizogenesis in vitro

This paper reports that rhizogenesis in woody plant species in vitro was mediated through the basipetal transport of auxin from the shoot apex. This can directly induce roots in easy-to-root species such as Betula pendula, but was dependent upon an interaction with exogenous auxin in more difficult-to-root species such as Daphne cneorum, and to a lesser extent in Quercus robur. Shoot apex removal reduced rhizogenesis in Quercus, and inhibited it in Daphne, even in the presence of exogenous auxin, whereas rooting in Betula was unaffected. That basipetally transported auxin modulates rhizogenesis was demonstrated by the inhibition of root induction in Betula shoots by the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA), and by the substitution of indole-3-acetic acid (IAA) for a bud in Betula internodal sections.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog medium - WPM woody plant medium     

Role of Auxin in Induction of Polarity in Fucus vesiculosus Zygotes

We studied the effects of auxin (indole-3-acetic acid) on formation of the primary polarity axis in zygotes of the brown algae Fucus vesiculosusL. Within the first 2.5 h after fertilization, the zygotes release this phytohormone in the ambient medium. The treatment of developing zygotes with the inhibitor of indole-3-acetic acid transport from the cell 2,3,5-triiodobenzoic acid at 5 mg/l arrests the auxin secretion and leads to its accumulation in the cells. This causes a significant delay in zygote polarization. The treatment of zygotes with the exogenous indole-3-acetic acid at 1 mg/l stimulates cell polarization and formation of a rhizoid protuberance. When auxin was added to the medium with triiodobenzoic acid, the inhibitory effect of the latter was eliminated. It has been proposed that the content of indole-3-acetic acid in the ambient medium is a key factor in the induction of polarity of the F. vesiculosus zygotes.     

SELECTIVE INHIBITION OF CELL CYCLE STAGES IN THE ALLIUM ROOT MERISTEM BY COLCHICINE AND GROWTH REGULATORS

The treatment of root tips of Allium carinatum, Allium cepa, and Allium flavum with colchicine, abscisic acid, kinetin, and indole-3-acetic acid, applied in appropriate concentrations, combinations, and durations, makes possible the selective blockade of the cell cycle in G₁, G₂, any mitotic stage, and between karyokinesis and cytokinesis. Moreover, treatment with abscisic acid followed by a recovery period stimulates polyploid nuclei in mature tissues to divide. Colchicine, kinetin, and indole-3-acetic acid applied together cause end-to-end association of metaphase chromosomes. These results together with earlier findings suggest that any step of the cell cycle is independently controlled both by specific balance of the growth regulators and by specific synthesis of the nucleic acids.     

The effect of calcium antagonists and inhibitors of secretory processes on auxin-induced elongation and fine structure ofPisum sativum stem segments

Summary The treatment of dark grown pea stem segments with chelators of divalent cations (EGTA, EDTA, CTC), various Ca²⁺ antagonists (LaCl₃, A-23187, verapamil) and inhibitors of secretory processes (monensin, CB) reduced elongation in the presence of indole-3-acetic acid (IAA). Generally the inhibition increased with increasing concentrations of the substances. The timing of the responses can be correlated with maximum auxin-stimulated secretion of cell wall material. Examination of cell ultrastructure showed that changes in dictyosome activity could explain a reduced deposition of cell wall material and so cause inhibition of elongation.The inhibitors affected the morphology and vesiculation of the dictyosomes, and the appearance of the plasma membrane, ER and mitochondria in different ways. The most pronounced effects on ultrastructure resulted from monensin and LaCl₃ treatments with the dictyosomes being most affected; large vesicles appeared in the cytoplasm. Less pronounced effects on cell structure were seen in EGTA, A-23187 and verapamil treated tissue. The effects on the dictyosomes are considered to be due to disturbances of Ca²⁺ and other ionic levels within the cells.Abbreviations ATPase adenosine triphosphatase - CB cytochalasin B - CTC chlorotetracycline - DMSO dimethyl sulphoxide - EDTA ethylene diamine tetraacetic acid - EGTA ethyleneglycol-bis-(B-amino ethyl ether)N,N¹-tetraacetic acid - ER endoplasmic reticulum - ver verapamil     

Phytohormones,Rhizobium mutants, and nodulation in legumes. IV. Auxin metabolites in pea root nodules

High specific activity [³H]indole-3-acetic acid (IAA) was applied directly to root nodules of intact pea plants. After 24 h, radioactivity was detected in all plant tissues. In nodule and root tissue, only 2–3% of³H remained as IAA, and analysis by thin layer chromatography suggested that indole-3-acetyl-L-aspartic acid (IAAsp) was a major metabolite. The occurrence of IAAsp in pea root and nodule tissue was confirmed unequivocally by gas chromatography-mass spectrometry (GC-MS). The following endogenous indole compounds were also unequivocally identified in pea root nodules by GC-MS: IAA, indole-3-pyruvic acid, indole-3-lactic acid, indole-3-propionic acid, indole-3-butyric acid, and indole-3-carboxylic acid. Evidence of the occurrence of indole-3-methanol was also obtained. With the exception of IAA and indole-3-propionic acid, these compounds have not previously been unequivocally identified in a higher plant tissue.     

Translocation of Radiolabeled Indole-3-Acetic Acid and Indole-3-Acetyl-myo-Inositol from Kernel to Shoot of Zea mays L.

Either 5-[³H]indole-3-acetic acid (IAA) or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[³H]indole-3-acetic acid or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.