An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate.

We isolated and characterized a 2.8-kb, full-length, Arabidopsis thaliana cDNA clone encoding a lipoxygenase. DNA sequence analysis showed that the deduced amino acid sequence of the Arabidopsis protein is 72 to 78% similar to that of legume seed lipoxygenases. DNA blot analysis indicated that Arabidopsis contains a single gene, LOX1, with appreciable homology to the cDNA clone. RNA blot analysis showed that the LOX1 gene is expressed in Arabidopsis leaves, roots, inflorescences, and young seedlings. LOX1 expression levels were highest in roots and young seedlings. In mature plants, LOX1 mRNA levels increased upon treatment with the stress-related hormones abscisic acid and methyl jasmonate and remained high for at least 96 h. Expression of the LOX1 gene was examined following infiltration of leaves with virulent (Psm ES4326) and avirulent (Pst MM1065) strains of Pseudomonas syringae. LOX1 mRNA levels were induced approximately 6-fold by both virulent and avirulent strains; however, the response to avirulent strains was much more rapid. Infiltration of leaves with Pst MM1065 resulted in maximal induction within 12 h, whereas maximal induction by Psm ES4326 did not occur until 48 h. When a cloned avr gene, avrRpt2, was transferred to Psm ES4326, LOX1 mRNA accumulated in a pattern similar to that observed for the avirulent strain Pst MM1065.     

Coupled, but not uncoupled, fluxes in a neuronal glutamate transporter can be activated by lithium ions

In the central nervous system a family of related (Na(+)-K(+))-coupled glutamate transporters remove the transmitter from the cleft and prevent its neurotoxic actions. In addition to this coupled uptake, these transporters also mediate a sodium- and glutamate-dependent uncoupled anion conductance. Most models assume that the initial steps for both processes are the same, leading to the anticipation that both may exhibit a similar requirement for cations. In this study we have tested this idea in the neuronal glutamate transporter EAAC-1. We report that in this transporter lithium can replace sodium in the coupled uptake. Strikingly, the glutamate-dependent gating of the uncoupled conductance mediated by EAAC-1 has a strict requirement for sodium; lithium cannot substitute for it. Moreover, we describe two mutants, T370S and G410S, in which the cation selectivity of the two processes is affected differently. In both mutants sodium, but not lithium, can support coupled transport. On the other hand, the sodium selectivity of the gated anion conductance in oocytes expressing the mutant transporters is not affected. Our observations indicate that although both the coupled and the uncoupled fluxes are sodium-dependent, the conformation gating the anion conductance is different from that during substrate translocation.     

Effects of abscisic acid analogues on abscisic acid-induced gene expression in barley aleurone protoplasts: Relationship between structure and function of the abscisic acid molecule

The plant hormone abscisic acid (ABA) mediates gene expression in barley aleurone protoplasts. In order to elucidate the essential functional groups of the ABA molecule, the specificity of a number of ABA analogues for inducing ABA-regulated gene (e.g., RAB, BASI) expression in barley aleurone protoplasts was studied. These analogues have modifications at three different positions of the ABA molecule: (a) the 1-hydroxyl group (1-deoxy ABA), (b) the carboxyl group (ABA-methyl ester or ABA-glucose ester), and (c) both the 1-hydroxyl and 4-carbonyl groups (-ionylidene acetic acid). The importance of the different putative functional groups was analyzed. The dose-response analysis of ABA analogues upon the induction gene expression showed the following order: ABA > ABA methyl ester > 1-deoxy ABA > ABA glucose ester > -ionylidene acetic acid > --ionone.     

Regulation of alcohol dehydrogenase gene expression in barley aleurone by gibberellin and abscisic acid

The expression of the Adh1 gene (alcohol dehydrogenase, EC 1.1.1.1) was studied in the aleurone layer of barley ( Hordeum vulgare cv. Himalaya). Expression increased markedly during grain development at the levels of activity, enzyme protein and mRNA. mRNA content, but not enzyme activity, could be increased further by exogenous abscisic acid (ABA) when isolated, de-embryonated developing grains were pre-treated with gibberellic acid (GA₃) or fluridone. In isolated mature aleurone layers incubated with exogenous hormones, ADH mRNA was strongly up-regulated by ABA and down-regulated by GA₃ within 6 h. With ABA, this increase in mRNA was followed by an increase in ADH protein and activity, peaking at 18 h. With GA₃, the decrease in mRNA was accompanied by simultaneous decreases in protein and activity. In general, GA₃ counteracted the effect of ABA and vice versa. In the aleurone of germinating grain, ADH activity decayed in a distal direction from the embryo, consistent with down-regulation by gibberellin(s) diffusing from it. It was concluded that ADH gene expression in the aleurone of the intact grain is regulated by an ABA/gibberellin interaction.     

Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines

Streptococcus equisimilis hyaluronan (HA) synthase (SeHAS) contains four cysteines (C226, C262, C281 and C367) that are conserved in the mammalian HAS family. Previous studies of single Cys-to-Ser and all possible Cys-to-Ala mutants of SeHAS found that: the Cys-null mutant is active, Cys modification inhibits HAS activity and the conserved cysteines are clustered at the membrane-enzyme interface in substrate-binding sites (Kumari K, Weigel PH. 2005. Identification of a membrane-localized cysteine cluster near the substrate binding sites of the Streptococcus equisimilis hyaluronan synthase. Glycobiology. 15:529-539). We re-examined these Cys mutants using a single technique (size exclusion chromatography-multi-angle laser light scattering) that allows simultaneous assays on the same sample for both HA synthesis activity and HA product size. Among 18 mutants compared with wild type, 4 showed no change in either function and 3 showed changes in both (decreased activity and HA size). Only one of the two functions was altered in 11 other mutants, which showed either decreased polymerizing activity or product size. No mutants made larger HA, 8 made smaller HA and 10 showed no change in HA size. Nine mutants showed no change in activity and nine were less active. The mutants fell into four of nine possible groups in terms of changes in HA size or synthesis rate (i.e. none, increased or decreased). Specific Cys residues were associated with each mutant group and the pattern of effects on both functions. Thus, the four conserved Cys residues, individually and in specific combinations, influence the rate of sugar assembly by HAS and HA product size, but their participation in one function is independent of the other.     

Light and abscisic acid signalling are integrated by MIZ1 gene expression and regulate hydrotropic response in roots of Arabidopsis thaliana

Plant roots undergo tropic growth in response to environmental cues, and each tropic response is affected by several environmental stimuli. Even its importance, molecular regulation of hydrotropism has not been largely uncovered. Tropic responses including hydrotropism were impacted by other environmental signal. We found that hydrotropism was reduced in dark-grown seedling. Moreover, we found that the expression of MIZ1, an essential gene for hydrotropism, was regulated by light signal. From our genetic analysis, phytochrome A (phyA)-, phyB- and HY5-mediated blue-light signalling play curial roles in light-mediated induction of MIZ1 and hydrotropism. In addition, we found that abscisic acid (ABA) also induced MIZ1 expression. ABA treatment could recover weak hydrotropism and MIZ1 expression level of hy5, and ABA synthesis inhibitor, abamineSG, further reduced hydrotropic curvature of hy5. In contrast, ABA treatment did not affect ahydrotropic phenotype of miz1. These results suggest that ABA signalling regulates MIZ1 expression independently from light signalling. Our results demonstrate that environmental signals, such as light and stresses mediated by ABA signalling, are integrated into MIZ1 expression and thus regulate hydrotropism. These machineries will allow plants to acquire sufficient amounts of water.     

Occurrence and identification of jasmonic acid and its amino acid conjugates induced by osmotic stress in barley leaf tissue

The effect of osmotically active substances on the alteration of endogenous jasmonates was studied in barley (Hordeum vulgare L. cv. Salome) leaf tissue. Leaf segments were subjected to solutions of d-sorbitol, d-mannitol, polyethylene glycol 6000, sodium chloride, or water as a control. Alterations of endogenous jasmonates were monitored qualitatively and quantitatively using immunoassays. The structures of jasmonates isolated were determined on the basis of authentic substances by capillary gas chromatography-mass spectrometry. The stereochemistry of the conjugates was confirmed by high performance liquid chromatography with diastereoisomeric references. In barley leaves, jasmonic acid and its amino acid conjugates, for example, with valine, leucine, and isoleucine, are naturally occurring jasmonates. In untreated leaf segments, only low levels of these native jasmonates were found. After treatment of the leaf tissues with sorbitol, mannitol, as well as with polyethylene glycol, an increase of both jasmonic acid and its conjugates could be observed, depending on the stress conditions used. In contrast, salt stress was without any stimulating effect on the levels of endogenous jasmonates. From barley leaf segments exposed to sorbitol (1m) for 24 h, jasmonic acid was identified as the major accumulating compound. Jasmonic acid-amino acid conjugates increased likewise upon stress treatment.Abbreviations JM methyl jasmonate - JA jasmonic acid - JIP(s) jasmonate-induced protein(s) - PEG polyethylene glycol - RIA radioimmunoassay - ELISA enzyme-linked immunosorbent assay - HPLC high performance liquid chromatography - GC-MS gas chromatography-mass spectrometry - R _t retention time - IAA indole-3-acetic acid     

Control of protein synthesis in barley aleurone layers by the plant hormones gibberellic acid and abscisic acid

The patterns of protein synthesis in barley aleurone layers treated with gibberellic acid (GA₃) and abscisic acid (ABA) are compared with the patterns observed in wheat germ in vitro translation assays directed by RNA isolated from similarly treated layers. When used alone, GA₃ and ABA both induce the formation of new translatable mRNAs and cause new proteins to be synthesized. The effects of GA₃ are more dramatic than those of ABA. In GA₃-treated tissues, overall protein synthesis is redirected to produce large quantities of α-amylase and a few other GA₃-induced proteins, while other protein synthesis is reduced or stopped. Large amounts of new translatable mRNA for α-amylase are also induced such that the dominant in vitro translation product is α-amylase. These changes are blocked by the simultaneous addition of ABA to the tissue. In GA₃ plus ABA-treated layers, few changes in protein synthesis in vivo are observed when compared to protein synthesis in untreated tissue, although the induction of mRNA for α-amylase and the other GA₃-induced mRNAs does occur. This indicates that ABA does not interfere with GA₃ induction of translatable mRNAs but prevents the translation of these mRNAs in vivo. Thus ABA and potentially GA₃ regulate the translation of proteins in vivo in barley aleurone layers.     

Derivatives of glutamic acid and trehalose which can be transformed into long-living free radicals by the loss of an electron are identified in some bacteria

A derivative of glutamic acid (ammonigenin) and a trisaccharide named lysodektose which are converted into long-living free radicals by the loss of one electron were isolated from Brevibacterium ammoniagenes and Micrococcus lysodeikticus. Structural formulae suggested for both substances based on ESR-, NMR- and mass spectra, isotopic substitution experiments and other data are: lactone of N-hydroxy-N-(2-carbamoylethyl)-glutamyl-4-amino-2-hydroxybutyric amide and 6-O-[2-deoxy-2-(N-methyl)-hydroxylamino-beta-D-glucopyranosyl]- alpha, alpha-trehalose. Radical forms appear on reversible oxidation of hydroxylamino groups to nitroxyl groups. Participation in the protection of bacterial cells and regulation of their metabolism is suggested for these compounds.     

Induction of hydroxycinnamic acid amides and tryptophan by jasmonic acid, abscisic acid and osmotic stress in barley leaves

The effects of jasmonic acid (JA) and abscisic aid (ABA) on secondary metabolism in barley (Hordeum vulgare L.) were investigated. Treatment with JA at 100 microM for 48 h induced accumulation of four compounds in barley primary leaves. The accumulation of these compounds was also observed after treatment with ABA at 100 microM. The induced compounds were identified as p-coumaroylputrescine, p-coumaroylagmatine, p-coumaroyl-3-hydroxyagmatine and tryptophan by spectroscopic methods. The profiles of compounds induced by application of JA and ABA were different. JA exhibited stronger inducing activity for hydroxycinnamic acid amides than ABA, while ABA was more active in tryptophan accumulation. The major hydroxycinnamic acid amides in JA- and ABA-treated leaves were p-coumaroylagmatine and p-coumaroyl-3-hydroxyagmatine, respectively. These differences suggested that JA and ABA act in distinct modes. The induction of these compounds was also observed in leaf segments treated with 1 M sorbitol and glucose. These findings suggested that JA and ABA are involved in accumulation of hydroxycinnamic acid amides and tryptophan in response to osmotic stress in barley.     

Modulation of germination of embryos isolated from dormant and nondormant barley grains by manipulation of endogenous abscisic acid

Dormant and non-dormant barley (Hordeum distichum L.) grains with identical genetic backgrounds were obtained by maturing grains under different climate conditions. When isolated embryos from dormant grains were incubated in a well containing a fixed volume of water (300 l), the germination rate and percentage were dependent on the embryo number per well. A higher embryo number per well was correlated with a lower germination rate and percentage. However, this was not the case for the embryos isolated from nondormant grains. During germination, the endogenous cis-abscisic acid (ABA) in isolated embryos from both dormant and nondormant grains was analyzed. The inhibitory effect on germination of a higher number per well of isolated dormant embryos was due to diffusion of endogenous ABA out of the embryos and accumulation of ABA in the incubation medium. Moreover, there was de-novo synthesis of ABA in embryos isolated from dormant grains during incubation but not in embryos isolated from nondormant grains. The inhibitory effect of ABA on germination of embryos isolated from dormant grains could be mimicked by addition of ABA or the medium in which dormant embryos had been placed. Embryos isolated from nondormant grains were insensitive to addition of ABA and medium from dormant embryos. Our results demonstrate that diffusion of endogenous ABA, de-novo ABA synthesis and ABA sensitivity play a role in the control of germination. It is proposed that dormancy-breaking treatments act via changes to these processes.Abbreviations ABA cis-abscisic acid - E/W embryo(s) per well Prof. K.R. Libbenga (Institute of Molecular Plant Sciences, Leiden University) is thanked for fruitful discussions. B.V.D. was partly supported by E.E.C. BIOTECH program PL 920175.     

Regulation of dormancy in barley by blue light and after-ripening: effects on abscisic acid and gibberellin metabolism

White light strongly promotes dormancy in freshly harvested cereal grains, whereas dark and after-ripening have the opposite effect. We have analyzed the interaction of light and after-ripening on abscisic acid (ABA) and gibberellin (GA) metabolism genes and dormancy in barley (Hordeum vulgare 'Betzes'). Analysis of gene expression in imbibed barley grains shows that different ABA metabolism genes are targeted by white light and after-ripening. Of the genes examined, white light promotes the expression of an ABA biosynthetic gene, HvNCED1, in embryos. Consistent with this result, enzyme-linked immunosorbent assays show that dormant grains imbibed under white light have higher embryo ABA content than grains imbibed in the dark. After-ripening has no effect on expression of ABA biosynthesis genes, but promotes expression of an ABA catabolism gene (HvABA8'OH1), a GA biosynthetic gene (HvGA3ox2), and a GA catabolic gene (HvGA2ox3) following imbibition. Blue light mimics the effects of white light on germination, ABA levels, and expression of GA and ABA metabolism genes. Red and far-red light have no effect on germination, ABA levels, or HvNCED1. RNA interference experiments in transgenic barley plants support a role of HvABA8'OH1 in dormancy release. Reduced HvABA8'OH1 expression in transgenic HvABA8'OH1 RNAi grains results in higher levels of ABA and increased dormancy compared to nontransgenic grains.     

Age-dependent accumulation of protein residues which can be hydrolyzed to D-aspartic acid in human erythrocytes

We have measured the rate of accumulation of amino acid residues in human erythrocyte membrane and cytosolic proteins which give D-aspartic acid upon acid hydrolysis. These residues would include D-aspartic acid, D-asparagine, as well as the beta-transpeptidation product, D-isoaspartic acid. Measurements made using age (density) fractionated cells indicate that racemization at these residues occurs on membrane proteins with a t1% (the time required to convert 1% to the D configuration) of about 38.6 days. Fractionation of membrane components revealed a faster rate of racemization for intrinsic proteins than for extrinsic proteins. On the other hand, significant age-dependent racemization was not detected for cytosolic proteins, and the calculated t1% value for these proteins is at least 4 times larger. These results suggest that in the 120-day life span of an erythrocyte, significant racemization of membrane (but not cytosolic) proteins can occur. We have also determined that the rates of accumulation of these residues for erythrocyte membrane and cytosolic proteins incubated in vitro are similar to those observed in vivo. These observations are discussed in terms of the possible cellular metabolism of racemized proteins.