Increased glutamine in leaves of poplar transgenic with pine GS1a caused greater anthranilate synthetase α-subunit (ASA1) transcript and protein abundances: an auxin-related mechanism for enhanced growth in GS transgenics?

The initial reaction in the pathway leading to the production of indole-3-acetic acid (IAA) in plants is the reaction between chorismate and glutamine to produce anthranilate, catalysed by the enzyme anthranilate synthase (ASA; EC 4.1.3.27). Compared with non-transgenic controls, leaves of transgenic poplar with ectopic expression of the pine cytosolic glutamine synthetase (GS1a; EC 6.3.1.2) produced significantly greater glutamine and significantly enhanced ASA α-subunit (ASA1) transcript and protein (approximately 130% and 120% higher than in the untransformed controls, respectively). Similarly, tobacco leaves fed with 30 mM glutamine and 2 mM chorismate showed enhanced ASA1 transcript and protein (175% and 90% higher than controls, respectively). Furthermore, free IAA was significantly elevated both in leaves of GS1a transgenic poplar and in tobacco leaves fed with 30 mM glutamine and 2 mM chorismate. These results indicated that enhanced cellular glutamine may account for the enhanced growth in GS transgenic poplars through the regulation of auxin biosynthesis.

Tryptophan-dependent indole-3-acetic acid biosynthesis by 'IAA-synthase' proceeds via indole-3-acetamide.

Plants are suggested to produce their major growth promoting phytohormone, indole-3-acetic acid (IAA), via multiple redundantly operating pathways. Although great effort has been made and plenty of possible routes have been proposed based on experimental evidence, a complete pathway for IAA production has yet to be demonstrated. In this study, an in-vitro approach was taken to examine the conversion of l-tryptophan (l-trp) to IAA by gas chromatography-mass spectrometry (GC-MS). Especially the influence of putative reaction intermediates on the enzymatic conversion of l-trp to IAA was analyzed. Among the substances tested only indole-3-acetamide (IAM) showed a pronounced effect on the l-trp conversion. We additionally report that IAM is synthesized from l-trp and that it is further converted to IAA by the utilized cell free Arabidopsis extract. Together, our results underscore the functionality of an IAM-dependent auxin biosynthesis pathway in Arabidopsis thaliana.

Phytochrome A requires jasmonate for photodestruction.

The plant photoreceptor phytochrome is organised in a small gene family with phytochrome A (phyA) being unique, because it is specifically degraded upon activation by light. This so called photodestruction is thought to be important for dynamic aspects of sensing such as measuring day length or shading by competitors. Signal-triggered proteolytic degradation has emerged as central element of signal crosstalk in plants during recent years, but many of the molecular players are still unknown. We therefore analyzed a jasmonate (JA)-deficient rice mutant, hebiba, that in several aspects resembles a mutant affected in photomorphogenesis. In this mutant, the photodestruction of phyA is delayed as shown by in vivo spectroscopy and Western blot analysis. Application of methyl-JA (MeJA) can rescue the delayed phyA photodestruction in the mutant in a time- and dose-dependent manner. Light regulation of phyA transcripts thought to be under control of stable phytochrome B (phyB) is still functional. The delayed photodestruction is accompanied by an elevated sensitivity of phytochrome-dependent growth responses to red and far-red light.

A hydraulic signal in root-to-shoot signalling of water shortage.

Photosynthesis and biomass production of plants are controlled by the water status of the soil. Upon soil drying, plants can reduce water consumption by minimizing transpiration through stomata, the closable pores of the leaf. The phytohormone abscisic acid (ABA) mediates stomatal closure, and is the assigned signal for communicating water deficit from the root to the shoot. However, our study does not support ABA as the proposed long-distance signal. The shoot response to limited soil water supply is not affected by the capacity to generate ABA in the root; however, the response does require ABA biosynthesis and signalling in the shoot. Soil water stress elicits a hydraulic response in the shoot, which precedes ABA signalling and stomatal closure. Attenuation of the hydraulic response in various plants prevented long-distance signalling of water stress, consistent with root-to-shoot communication by a hydraulic signal.

cyclo-Oxylipin-galactolipids in plants: occurrence and dynamics.

cyclo-Oxylipin-galactolipids (cGL) are mono- or digalactosyldiglycerides carrying a cyclo-oxylipin in the sn1- and/or sn2-position or esterified to the galactose moiety. These compounds were recently identified in Arabidopsis thaliana. We provide evidence that cGL are mainly, if not exclusively, part of the thylakoid and can be hydrolysed by lipolytic activities associated with photosynthesis-related protein complexes in vitro. Using HPLC/ESI-mass spectrometry, cGL are shown to be restricted in occurrence to the genus Arabidopsis, they do not occur in other plants tested. A. thaliana cGL are rapidly and transiently formed upon wounding with characteristic changes in composition of the cGL-fraction. While the biological role of cGL is not understood, the genus Arabidopsis may present a model-case of chemical evolution of a novel class of regulatory molecules.

Alpha-tocopherol may influence cellular signaling by modulating jasmonic acid levels in plants.

Most studies on the function of tocopherols in plants have focused on their photo-protective and antioxidant properties, and it has been recently suggested, though not yet demonstrated, that they may also play a role in cellular signaling. By using vte1 mutants of Arabidopsis thaliana, with an insertion in the promoter region of the gene encoding tocopherol cyclase, we demonstrate here for the first time that tocopherol deficiency may alter endogenous phytohormone levels in plants, thereby reducing plant growth and triggering anthocyanin accumulation in leaves. In plants grown under a combination of high light and low temperature conditions to induce anthocyanin accumulation, we evaluated age-dependent changes in tocopherols, indicators of photo-oxidative stress, phytohormone levels, plant growth and anthocyanin levels in wild type and vte1 mutants. These mutants showed lower tocopherol levels, reduced growth and enhanced anthocyanin accumulation compared with the wild type, while both the maximum and relative efficiencies of PSII, chlorophylls, and carotenoids were not significantly altered. Analyses of phytohormone levels revealed that reduced growth and enhanced anthocyanin accumulation in tocopherol-deficient plants were preceded by increased jasmonic acid levels. This is the first study suggesting a direct effect of tocopherols on phytohormones levels in plants and will undoubtedly help us to better understand the multiple functions tocopherols play in plants, as well as the cellular signaling mechanisms responsible for the phenotypes thus far described in tocopherol-deficient plants.

Preparative enzymatic solid phase synthesis of cis(+)-12-oxo-phytodienoic acid - physical interaction of AOS and AOC is not necessary.

The pathway of jasmonic acid (JA) biosynthesis was established in the 1980s by Vick and Zimmerman but, until now, the preparative biosynthesis of the jasmonic acid precursors 12-oxo-phytodienoic acid (OPDA) and 3-oxo-2-[2'-pentenyl]-cyclopentan-1-octanoic acid (OPC-8:0) in their endogenous and biologically relevant cis(+)-configuration was only possible in small amounts and had to put up with high costs. This was mainly due to the lack of high amounts of pure and enzymatically active allene oxide cyclase (AOC), which is a key enzyme in the biosynthesis of jasmonates in that it releases, in a coupled reaction with allene oxide synthase (AOS), the first cyclic and biological active metabolite - OPDA. We describe here the expression and purification of AOS and AOC and their subsequent coupling to solid matrices to produce an enantioselective, reusable bioreactor for octadecanoid production. With the method described here it is possible to produce optically pure enantiomers of octadecanoids in high amounts in a cost- and time-efficient manner. Furthermore, it could be demonstrated that a physical interaction of AOS and AOC, hitherto postulated to be required for substrate channeling from AOS to AOC, is not necessary for the in vitro cyclization of the unstable epoxide generated by the AOS reaction.

Hormone profiling in Arabidopsis.

A highly sensitive and accurate multiplex gas chromatography-mass spectrometry (GC-MS/MS)-technique is reported for the quantitative analysis of acidic phytohormones in Arabidopsis thaliana and other plant species. The optimized setup allows the routine processing and analysis of between 20 mg and 5 g of tissue. The protocol was designed and the equipment used was chosen to facilitate implementation of the method into other laboratories and to provide access to state-of-the-art analytical tools for the acidic phytohormones and related signaling molecules.

Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana.

Amidase 1 (AMI1) from Arabidopsis thaliana converts indole-3-acetamide (IAM), into indole-3-acetic acid (IAA). AMI1 is part of a small isogene family comprising seven members in A. thaliana encoding proteins which share a conserved glycine- and serine-rich amidase-signature. One member of this family has been characterized as an N-acylethanolamine-cleaving fatty acid amidohydrolase (FAAH) and two other members are part of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) or mitochondria (Tom complex) and presumably lack enzymatic activity. Among the hitherto characterized proteins of this family, AMI1 is the only member with indole-3-acetamide hydrolase activity, and IAM is the preferred substrate while N-acylethanolamines and oleamide are not hydrolyzed significantly, thus suggesting a role of AMI1 in auxin biosynthesis. Whereas the enzymatic function of AMI1 has been determined in vitro, the subcellular localization of the enzyme remained unclear. By using different GFP-fusion constructs and an A. thaliana transient expression system, we show a cytoplasmic localization of AMI1. In addition, RT-PCR and anti-amidase antisera were used to examine tissue specific expression of AMI1 at the transcriptional and translational level, respectively. AMI1-expression is strongest in places of highest IAA content in the plant. Thus, it is concluded that AMI1 may be involved in de novo IAA synthesis in A. thaliana.

Cholodny-Went revisited: a role for jasmonate in gravitropism of rice coleoptiles.

Gravitropism is explained by the Cholodny-Went hypothesis: the basipetal flow of auxin is diverted laterally. The resulting lateral auxin gradient triggers asymmetric growth. However, the Cholodny-Went hypothesis has been questioned repeatedly because the internal auxin gradient is too small to account for the observed growth asymmetry. Therefore, an additional gradient in indolyl-3-acetic acid (IAA) sensitivity has been suggested (Brauner and Hager in Planta 51:115-147, 1958). We challenged the Cholodny-Went hypothesis for gravitropism of rice coleoptiles (Oryza sativa L.) and found it to be essentially true. However, we observed, additionally, that the two halves of gravitropically stimulated coleoptiles responded differentially to the same amount of exogenous auxin: the auxin response is reduced in the upper flank but normal in the lower flank. This indicates that the auxin-gradient is amplified by a gradient of auxin responsiveness. Hormone contents were measured across the coleoptile by a GC-MS/MS technique and a gradient of jasmonate was detected opposing the auxin gradient. Furthermore, the total content of jasmonate increased during the gravitropic response. Jasmonate gradient and increase persist even when the lateral IAA gradient is inhibited by 1-N-naphtylphtalamic acid. Flooding with jasmonate delays the onset of gravitropic bending. Moreover, a jasmonate-deficient rice mutant bends more slowly and later than the wild type. We discuss a role of jasmonate as modulator of auxin responsiveness in gravitropism.

Tocopherol content and activities of tyrosine aminotransferase and cystine lyase in Arabidopsis under stress conditions.

Tocopherols are presumed to be important antioxidants and scavengers of lipid radicals and reactive oxygen species in plants. Age is known to be a condition under which oxidative stress increases. In leaves of aging Arabidopsis thaliana plants, the content of alpha-tocopherol as well as of gamma-tocopherol increased significantly. The activity of tyrosine aminotransferase, which supplies the biosynthetic pathway with 4-hydroxyphenylpyruvate, was increased as well. On the other hand, coronatine, a phytotoxin mimicking octadecanoids and leading to symptoms of senescence, caused a moderate increase in alpha-tocopherol as well as some enhancement of gamma-tocopherol.

Pulsed magnetic-field therapy: a new concept to treat tinnitus?

Both clinical and neurophysiological data suggest that chronic tinnitus is characterized by focal brain activation. In the study reported here, pulsed magnetic-field therapy induced a highly significant increase of average total power for the delta, theta, and alpha frequency bands, predominantly within the frontal regions of the brain. We conclude that pulsed magnetic-field therapy induces changes of the electroencephalography pattern that correlated with a decrease in tinnitus symptoms.

Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyze the final step in the biosynthesis of the glucosinolate core structure.

The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions. To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed. Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in coronatine-treated plants, and the corresponding cDNA was found to encode the sulfotransferase AtST5a. Likewise, upon wounding, an immediate and transient increase in AtST5a mRNA levels could be observed in both locally wounded and unwounded (systemic) leaves. Furthermore, application of octadecanoids and ethylene as compounds involved in plant wound defense reactions resulted in AtST5a gene activation, whereas pathogen defense-related signals (yeast elicitor and salicylic acid) were inactive. AtST5a and its close homologs AtST5b and AtST5c were purified as His6-tagged proteins from Escherichia coli. The three enzymes were shown to catalyze the final step in the biosynthesis of the glucosinolate (GS) core structure, the sulfation of desulfoglucosinolates (dsGSs). They accept a broad range of dsGSs as substrates. However, in a competitive situation, AtST5a clearly prefers tryptophan- and phenylalanine-derived dsGSs, whereas long chain dsGSs derived from methionine are the preferred substrates of AtST5b and AtST5c. Treatment of Arabidopsis plants with low concentrations of coronatine resulted in an increase in the amounts of specific GSs, primarily glucobrassicin and neoglucobrassicin. Hence, it is suggested that AtST5a is the sulfotransferase responsible for the biosynthesis of tryptophan-derived GSs in vivo.

Quantitative electroencephalography patterns in patients suffering from tinnitus.

We conducted this study in an attempt to determine whether the electroencephalographic activity in patients suffering from tinnitus exhibits tinnitus-typical electroencephalography features. Our results indicated a significant increase in the average total power in female tinnitus patients and a significant decrease in average total power in male tinnitus patients. Furthermore, we noted a suppression of the alpha peaks or a split alpha band (or both). The reactivity of the alpha frequency was employed to evaluate the efficacy of noise generators, a critical component of tinnitus-retraining therapy. In conclusion, tinnitus-typical electroencephalography features can be extracted from the electroencephalogram.

Impaired induction of the jasmonate pathway in the rice mutant hebiba.

The elongation of rice (Oryza sativa) coleoptiles is inhibited by light, and this photoinhibition was used to screen for mutants with impaired light response. In one of the isolated mutants, hebiba, coleoptile elongation was stimulated in the presence of red light, but inhibited in the dark. Light responses of endogenous indolyl-3-acetic acid and abscisic acid were identical between the wild type and the mutant. In contrast, the wild type showed a dramatic increase of jasmonate heralded by corresponding increases in the content of its precursor o-phytodienoic acid, whereas both compounds were not detectable in the mutant. The jasmonate response to wounding was also blocked in the mutant. The mutant phenotype was rescued by addition of exogenous methyl jasmonate and o-phytodienoic acid. Moreover, the expression of O. sativa 12-oxophytodienoic acid reductase, an early gene of jasmonic acid-synthesis, is induced by red light in the wild type, but not in the mutant. This evidence suggests a novel role for jasmonates in the light response of growth, and we discuss a cross-talk between jasmonate and auxin signaling. In addition, hebiba represents the first rice mutant in which the induction of the jasmonate pathway is impaired providing a valuable tool to study the role of jasmonates in Graminean development.

Arabidopsis mutants affected in the transcriptional control of allene oxide synthase, the enzyme catalyzing the entrance step in octadecanoid biosynthesis.

Allene oxide synthase (AOS) catalyzes the entrance reaction in the biosynthesis of the octadecanoids 12-oxophytodienoic acid (OPDA) and jasmonic acid (JA). The enzyme is feedback-regulated by JA and thus a target of the JA-signalling pathway. A fusion genetic approach was used to isolate mutants in this signalling pathway. Seeds from transgenic Arabidopsis thaliana plants expressing the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) under the control of the AOS promoter were mutagenized with ethylmethane sulfonate and the progeny was screened for individuals exhibiting constitutive expression of uidA in the absence of an added octadecanoid. From 21,000 mutagenized plants, 8 lines showing constitutive AOS expression were obtained. The mutant lines were characterized further and fell into four classes, I to IV. All showed signs of growth inhibition encompassing both shoot and root systems, and accumulated higher than normal levels of OPDA. Mutants belonging to classes I and IV failed to set seeds due to defects in flower development which prevented self-pollination. One mutant, designated cas1, was characterized in more detail and showed, in addition to elevated levels of AOS mRNA, AOS polypeptide, OPDA, and JA, constitutive expression of JA-responsive genes ( VSP2, PDF1.2). The cas1 mutation is recessive and affects a single locus. Using cleaved amplified polymorphic sequences (CAPS) and simple sequence length polymorphisms (SSLP), the mutated gene was mapped to chromosome IV next to the SSLP marker CIW7.

Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid.

Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against L-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1.

Cloning and characterization of a jasmonic acid-responsive gene encoding 12-oxophytodienoic acid reductase in suspension-cultured rice cells.

In suspension-cultured rice ( Oryza sativaL.) cells, jasmonic acid (JA) functions as a signal transducer in elicitor N-acetylchitoheptaose-induced phytoalexin production. Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with JA (10(-4) M) for 2 h yielded a cDNA for a gene that responded to exogenous JA by an increase in mRNA level. Nucleotide sequence analysis indicated that the cDNA encodes an homologue of the yeast Old Yellow Enzyme. The deduced amino acid sequence was very similar to the sequences of 12-oxophytodienoic acid reductases (OPR) 1 and 2 from Arabidopsis thaliana(AtOPR1 and AtOPR2) and OPR1 from tomato ( Lycopersicon esculentum) (LeOPR1). The cDNA-encoded protein purified from recombinant Escherichia coli cells as a hexahistidine-tagged fusion protein exhibited OPR activity similar to that of AtOPR1, AtOPR2, and LeOPR1, which catalyze reduction of (-)- cis-12-oxophytodienoic acid (OPDA) preferentially over (+)- cis-OPDA, a natural precursor of JA. Thus the rice enzyme was termed OsOPR1. The physiological roles of OsOPR1 are discussed. This is the first report of the cloning of an OPR gene from a monocot plant.

Sensory principles of higher plants.

Plants respond to a broad variety of stimuli from the interior of their body and from the outside environment. A revolution in our knowledge of the sensory capabilities of plants took place during the last decade, thanks to the consequent application of the tools of molecular genetics and the focusing of most work, in a world-wide effort, on a single, representative, higher plant: Arabidopsis thaliana. This review concentrates on the complete sensory periphery of higher plants, with focus placed on the principles rather than the details, and such systems that have been functionally identified unambiguously. Higher plants are no less fascinating, no less complex, and no less sensitive than animals, even man, with respect to their sensory capabilities. Plants constantly monitor their environment and their interior situation by using a stunning array of sensory systems, that are mostly different from those of animals or man.

Dorntherapy: its effect on electroencephalographic activity in tinnitus patients with craniocervical dysfunction.

Dorntherapy was developed in the 1980s by Dieter Dorn, a nonmedical person, to cure his and his family members' vertebral problems. This technique achieves correction of dysfunctions of the vertebrae and joints simply by using natural movements of arms and legs or by applying gentle pressure (thumb) to the spinous process. Various observations suggest that craniocervical dysfunction can lead to tinnitus, thus causing changes in the electroencephalographic patterns. This study demonstrates that the successful application of Dorntherapy induced prominent changes of the electroencephalographic activity. Data analysis revealed a significant increase in the power of the alpha (8-13 Hz) and the alpha2 (9-11 Hz). Besides electrophysiological effects, changes in the quality of tinnitus and in the intensity of the tinnitus were noted. These results suggest that Dorntherapy must be an integral part of any tinnitus therapy.