Melatonin promotes adventitious- and lateral root regeneration in etiolated hypocotyls of Lupinus albus L

Melatonin is a well-known animal substance, which has recently been detected in plant tissues. However, there are only a few studies concerning its possible physiological role in plants. In this paper, we investigate the possible effect of melatonin on the regeneration of lateral and adventious roots in etiolated hypocotyls of Lupinus albus L. compared with the effect of indole-3-acetic acid. We performed this study by measuring both molecules in roots. Six-day-old derooted lupin hypocotyls immersed in several melatonin or indole-3-acetic acid concentrations were used to induce roots. A macro- and microscopic study of the histological origin of the adventitious and lateral roots was made, while melatonin and indole-3-acetic acid in the roots were quantified using liquid chromatography with fluorescence detection. The data show that both melatonin and indole-3-acetic acid induced the appearance of root primordia from pericicle cells, modifying the pattern of distribution of adventitious or lateral roots, the time-course, the number and length of adventitious roots, and the number of lateral roots. Melatonin and indole-3-acetic acid were detected and quantified in lupin primary roots, where both molecules were present in similar concentrations. The physiological effect of exogenous melatonin as root promoter was demonstrated, its action being similar to that of indole-3-acetic acid.     

Photoaffinity labeling of indole-3-acetic acid-binding proteins in maize

The photoaffinity labeling agent 5-azidoindole-3-acetic acid, an analog of the endogenous plant hormone indole-3-acetic acid (an auxin), was used to identify indole-3-acetic acid-binding proteins in maize. Two peptides with subunit molecular masses of 24 and 22 kilodaltons are specifically labeled in a saturable manner. Both peptides are slightly acidic and behave as dimers under nondenaturing conditions. The possibility that one of these peptides is the auxin receptor that mediates cell elongation in maize is discussed.     

Molecular characterization of two cloned nitrilases from Arabidopsis thaliana: key enzymes in biosynthesis of the plant hormone indole-3-acetic acid.

As in maize [Wright, A.D., Sampson, M. B., Neuffer, M. G., Michalczuk, L., Slovin, J. P. & Cohen, J. D. (1991) Science 254, 998-1000], the major auxin of higher plants, indole-3-acetic acid, is synthesized mainly via a nontryptophan pathway in Arabidopsis thaliana [Normanly, J., Cohen, J. D. & Fink, G. R. (1993) Proc. Natl. Acad. Sci. USA 90, 10355-10359]. In the latter species, the hormone may be accessible from the glucosinolate glucobrassicin (indole-3-methyl glucosinolate) and from L-tryptophan via indoleacetaldoxime under special circumstances. In each case, indole-3-acetonitrile is the immediate precursor, which is converted into indole-3-acetic acid through the action of nitrilase (nitrile aminohydrolase, EC 3.5.5.1). The genome of A. thaliana contains two nitrilase genes. Nitrilase I had been cloned earlier in our laboratory. The cDNA for nitrilase II (PM255) was cloned and encodes an enzyme that converts indole-3-acetonitrile to indole-3-acetic acid, the plant hormone. We show that the intracellular location as well as the expression pattern of the two A. thaliana nitrilases are distinctly different. Nitrilase I is soluble and is expressed throughout development, but at a very low level during the fruiting stage, while nitrilase II is tightly associated with the plasma membrane, is barely detectable in young rosettes, but is strongly expressed during bolting, flowering, and especially fruit development. The results indicate that more than one pathway of indole-3-acetic acid biosynthesis via indole-3-acetonitrile exists in A. thaliana and that these pathways are differentially regulated throughout plant development.     

Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues.

Indole-3-acetic acid is the major auxin in most plants. In Cruciferae, including Brassicaceae, indole-3-acetic acid is synthesized from indole-3-acetonitrile by nitrilase, after indole-3-acetonitrile is formed from tryptophan via indole-3-acetaldoxime or indole glycosinolates as the intermediate. We cloned and sequenced the gene for nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of indole-3-acetonitrile to indole-3-acetic acid, from Alcaligenes faecalis JM3. The amino acid sequence deduced from the nucleotide sequence of the nitrilase gene shows 34.7% identity with that of Klebsiella ozaenae nitrilase. A DNA clone containing the nitrilase gene expressed the active enzyme in Escherichia coli with excellent yield. Among five cysteine residues (Cys-40, Cys-115, Cys-162, Cys-163, and Cys-218) in the Alcaligenes nitrilase, only Cys-163 was conserved at the corresponding position in the Klebsiella nitrilase. Two mutant enzymes, in which Cys-162 and Cys-163 were replaced with Asn and Ala, respectively, were constructed by site-directed mutagenesis. A 35% increase of the specific activity and a large reduction of the Km for thiophene-2-acetonitrile (which was used as a standard substrate for the nitrilase) were observed in the Cys-162-->Asn mutant enzyme. The Cys-163-->Ala mutation resulted in complete loss of nitrilase activity, clearly indicating that Cys-163 is crucial for the activity and Cys-162 could not provide the catalytic function of Cys-163.     

Photoaffinity labeling of Arabidopsis thaliana plasma membrane vesicles by 5-azido-[7-3H]indole-3-acetic acid: identification of a glutathione S-transferase.

We used 5-azido-[7-3H]indole-3-acetic acid (5-azido-[7-3H]IAA), a photoaffinity analogue of the plant hormone indole-3-acetic acid (IAA), to search for auxin-binding proteins in Arabidopsis thaliana membranes. We identified an auxin-binding protein with a molecular mass of 24 kDa (Atpm24) in microsomes as well as in plasma membrane vesicles. Atpm24 was solubilized by 1% Triton X-100 and partially purified. A cDNA clone (Atpm24.1) corresponding to Atpm24 was isolated. The amino acid sequence predicted from the Atpm24.1 cDNA contains 212 amino acid residues with a relative molecular mass of 24,128 Da. Data base searches revealed that the predicted protein has homology to glutathione S-transferases (GSTs; EC 2.5.1.18). When Atpm24.1 was expressed in Escherichia coli, we found a high level of GST activity in the bacterial extracts. We have analyzed the substrate specificity of this protein and found that cumene hydroperoxide and trans-stilbene oxide but not trans-cinnamic acid or IAA-CoA were substrates. A role for this GST in physiological processes of plants is discussed.     

5'-Azido-[3,6-3H2]-1-napthylphthalamic acid, a photoactivatable probe for naphthylphthalamic acid receptor proteins from higher plants: identification of a 23-kDa protein from maize coleoptile plasma membranes

1-Naphthylphthalamic acid (NPA) is a specific inhibitor of polar auxin transport that blocks carrier-mediated auxin efflux from plant cells. To allow identification of the NPA receptor thought to be part of the auxin efflux carrier, we have synthesized a tritiated, photolabile NPA analogue, 5'-azido-[3,6-3H2]NPA ([3H2]N3NPA). This analogue was used to identify NPA-binding proteins in fractions highly enriched for plasma membrane vesicles isolated from maize coleoptiles (Zea mays L.). Competition studies showed that binding of [3H2]N3NPA to maize plasma membrane vesicles was blocked by nonradioactive NPA but not by benzoic acid. After incubation of plasma membrane vesicles with [3H2]N3NPA and exposure to UV light, we observed specific photoaffinity labeling of a protein with an apparent molecular mass of 23 kDa. Pretreatment of the plasma membrane vesicles with indole-3-acetic acid or with the auxin-transport inhibitors NPA and 2,3,5-triiodobenzoic acid strongly reduced specific labeling of this protein. This 23-kDa protein was also labeled by addition of 5-azido-[7-3H]indole-3-acetic acid to plasma membranes prior to exposure to UV light. The 23-kDa protein was solubilized from plasma membranes by 1% Triton X-100. The possibility that this 23-kDa polypeptide is part of the auxin efflux carrier system is discussed.     

An Arabidopsis indole-3-butyric acid-response mutant defective in PEROXIN6, an apparent ATPase implicated in peroxisomal function

Genetic evidence suggests that plant peroxisomes are the site of fatty acid beta-oxidation and conversion of the endogenous auxin indole-3-butyric acid (IBA) to the active hormone indole-3-acetic acid. Arabidopsis mutants that are IBA resistant and sucrose dependent during early development are likely to have defects in beta-oxidation of both IBA and fatty acids. Several of these mutants have lesions in peroxisomal protein genes. Here, we describe the Arabidopsis pex6 mutant, which is resistant to the inhibitory effects of IBA on root elongation and the stimulatory effects of IBA on lateral root formation. pex6 also is sucrose dependent during early seedling development and smaller and more pale green than WT throughout development. PEX6 encodes an apparent ATPase similar to yeast and human proteins required for peroxisomal biogenesis, and a human PEX6 cDNA can rescue the Arabidopsis pex6 mutant. The pex6 mutant has reduced levels of the peroxisomal matrix protein receptor PEX5, and pex6 defects can be partially rescued by PEX5 overexpression. These results suggest that PEX6 may facilitate PEX5 recycling and thereby promote peroxisomal matrix protein import.     

Plant growth-promoting hormones activate mammalian guanylate cyclase activity

In vivo injections of plant growth-promoting hormones increase the growth of animals as well as plants. Plant growth-promoting hormones and positive plant growth regulators are known to increase RNA and protein synthesis. Since cyclic GMP also increases RNA and protein synthesis, the object of the present investigation was to determine whether physiological levels of plant growth-promoting hormones and positive plant growth regulators have part of their mechanism(s) of action through stimulation of the guanylate cyclase (EC 4.6.1.2)-cyclic GMP system. Representatives of the three classes of growth-promoting hormones were investigated. Thus, auxins (indole-3-acetic acid, indole-3-butyric acid, beta-naphthoxyacetic acid, and 2,4,5-trichlorophenoxy acetic acid), gibberellins (gibberellic acid), and cytokinins [N6-benzyl adenine, kinetin (6-furfuryl aminopurine), and beta-(2-furyl) acrylic acid] all increased rat lung, small intestine, liver, and renal cortex guanylate cyclase activity 2- to 4-fold at the 1 microM concentration. Dose response curves revealed that maximal stimulation of guanylate cyclase by these plant growth regulators was at 1 microM; there was no augmented cyclase activity at 1 nM. The guanylate cyclase cationic cofactor manganese was not essential for augmentation of guanylate cyclase by these plant growth-promoting regulators. The antioxidant butylated hydroxytoluene did not block the enhancement of guanylate cyclase by these plant growth-promoting factors. These data suggest that guanylate cyclase may play a role in the mechanism of action of plant growth-promoting hormones and even of positive plant regulators at the cellular level.     

Simultaneous analysis of phytohormones,phytotoxins, and volatile organic compounds in plants

Phytohormones regulate the protective responses of plants against both biotic and abiotic stresses by means of synergistic or antagonistic actions referred to as signaling crosstalk. A bottleneck in crosstalk research is the quantification of numerous interacting phytohormones and regulators. The chemical analysis of salicylic acid, jasmonic acid, indole-3-acetic acid, and abscisic acid is typically achieved by using separate and complex methodologies. Moreover, pathogen-produced phytohormone mimics, such as the phytotoxin coronatine (COR), have not been directly quantified in plant tissues. We address these problems by using a simple preparation and a GC-MS-based metabolic profiling approach. Plant tissue is extracted in aqueous 1-propanol and mixed with dichloromethane. Carboxylic acids present in the organic layer are methylated by using trimethylsilyldiazomethane; analytes are volatilized under heat, collected on a polymeric absorbent, and eluted with solvent into a sample vial. Analytes are separated by using gas chromatography and quantified by using chemical-ionization mass spectrometry that produces predominantly [M+H]+ parent ions. We use this technique to examine levels of COR, phytohormones, and volatile organic compounds in model systems, including Arabidopsis thaliana during infection with Pseudomonas syringae pv. tomato DC3000, corn (Zea mays) under herbivory by corn earworm (Helicoverpa zea), tobacco (Nicotiana tabacum) after mechanical damage, and tomato (Lycopersicon esculentum) during drought stress. Numerous complex changes induced by pathogen infection, including the accumulation of COR, salicylic acid, jasmonic acid, indole-3-acetic acid, and abscisic acid illustrate the potential and simplicity of this approach in quantifying signaling crosstalk interactions that occur at the level of synthesis and accumulation.     

Grasshopper crop and midgut extract effects on plants: an example of reward feedback

Acid extracts and a resultant fraction from solid-phase extraction (SPE) of Romalea guttata crop and midgut tissues induce sorghum (Sorghum bicolor var. Rio) coleoptile growth in 24-h incubations an average of 49% above untreated controls. When combined with plant auxin, indole-3-acetic acid (IAA), the SPE fraction shows a synergistic reaction, yielding increases in coleoptile growth that average 295% above untreated controls and 8% above IAA standards. The interaction lowered the point of maximum sensitivity of IAA 3 orders of magnitude, resulting in a new IAA physiological set point at 10(-7) g/ml. This synergism suggests that contents in animal regurgitants making their way into plant tissue during feeding may produce a positive feedback in plant growth and development following herbivory. Such a process, also known as reward feedback, may exert major controls on ecosystem-level relationships in nature.     

Identification of the bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in plants

Stringent control mediated by the bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp) is a key regulatory process governing bacterial gene expression. By devising a system to measure ppGpp in plants, we have been able to identify ppGpp in the chloroplasts of plant cells. Levels of ppGpp increased markedly when plants were subjected to such biotic and abiotic stresses as wounding, heat shock, high salinity, acidity, heavy metal, drought, and UV irradiation. Abrupt changes from light to dark also caused a substantial elevation in ppGpp levels. In vitro, chloroplast RNA polymerase activity was inhibited in the presence of ppGpp, demonstrating the existence of a bacteria-type stringent response in plants. Elevation of ppGpp levels was elicited also by treatment with plant hormones jasmonic acid, abscisic acid, and ethylene, but these effects were blocked completely by another plant hormone, indole-3-acetic acid. On the basis of these findings, we propose that ppGpp plays a critical role in systemic plant signaling in response to environmental stresses, contributing to the adaptation of plants to environmental changes.     

Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium

The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM.     

Electrical polarity in embryos of wild carrot precedes cotyledon differentiation

Endogenous electrical currents traverse embryos of a higher plant, the wild carrot Daucus carota L. Current enters the apical pole and leaves the region near the presumptive radicle in the radially symmetric globular embryo. Current also enters the exposed surfaces of incipient globular embryos. This electrical polarity precedes differentiation of vascular tissue and cotyledon development. Localized current is observed at both growing ends of the embryos in subsequent stages of embryogenesis. Inward current is found at the cotyledons; outward current is found at the radicle/root. Exogenous indole-3-acetic acid (3 μM) reversibly inhibits these currents. Little current traverses the surface of intermediate regions of the embryo. The ionic gradients generated by these currents may be important in accumulation of metabolites and in other developmental processes within the embryo.     

Arabidopsis cytochrome P450s that catalyze the first step of tryptophan-dependent indole-3-acetic acid biosynthesis

Plants synthesize numerous secondary metabolites that are used as developmental signals or as defense against pathogens. Tryptophan (Trp)-derived secondary metabolites include camalexin, indole glucosinolates, and indole-3-acetic acid (IAA); however, the steps in their synthesis from Trp or its precursors remain unclear. We have identified two Arabidopsis cytochrome P450s (CYP79B2 and CYP79B3) that can convert Trp to indole-3-acetaldoxime (IAOx), a precursor to IAA and indole glucosinolates.     

Melatonin regulates Arabidopsis root system architecture likely acting independently of auxin signaling

Melatonin (N-acetyl-5-methoxytryptamine) is a tryptophan-derived signal with important physiological roles in mammals. Although the presence of melatonin in plants may be universal, its endogenous function in plant tissues is unknown. On the basis of its structural similarity to indole-3-acetic acid, recent studies mainly focusing on root growth in several plant species have suggested a potential auxin-like activity of melatonin. However, direct evidence about the mechanisms of action of this regulator is lacking. In this work, we used Arabidopsis thaliana seedlings as a model system to evaluate the effects of melatonin on plant growth and development. Melatonin modulated root system architecture by stimulating lateral and adventitious root formation but minimally affected primary root growth or root hair development. The auxin activity of melatonin in roots was investigated using the auxin-responsive marker constructs DR5:uidA, BA3:uidA, and HS::AXR3NT-GUS. Our results show that melatonin neither activates auxin-inducible gene expression nor induces the degradation of HS::AXR3NT-GUS, indicating that root developmental changes elicited by melatonin were independent of auxin signaling. Taken together, our results suggest that melatonin is beneficial to plants by increasing root branching and that root development processes elicited by this novel plant signal are likely independent of auxin responses.     

The main auxin biosynthesis pathway in Arabidopsis

The phytohormone auxin plays critical roles in the regulation of plant growth and development. Indole-3-acetic acid (IAA) has been recognized as the major auxin for more than 70 y. Although several pathways have been proposed, how auxin is synthesized in plants is still unclear. Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development, but these enzymes were placed in two independent pathways. In this article, we demonstrate that the TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana) by a quantification method of IPA using liquid chromatography-electrospray ionization-tandem MS. We further show that YUC protein expressed in Escherichia coli directly converts IPA to IAA. Indole-3-acetaldehyde is probably not a precursor of IAA in the IPA pathway. Our results indicate that YUC proteins catalyze a rate-limiting step of the IPA pathway, which is the main IAA biosynthesis pathway in Arabidopsis.     

Rapid change in water flux induced by auxins

Auxin (indole-3-acetic acid) enhances movement of tritiated water into and out of stem segments of etiolated peas. The time required for water in the tissue to attain one-half equilibrium with water in the surrounding solution is significantly shorter and the initial flux rate is higher in auxin-treated sections. The response is one of the most rapid to auxin, occurring well within 1 min after application of the hormone. This suggests that an action of auxin on the membrane system occurs before or coincident with auxin-induced enhancement of growth and wall elastic extensibility.