Modulation of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica by divalent cations, protons and H2O2

AbstractElectrical properties of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica were analyzed after incorporation of BCC1 into black lipid bilayers. Single channel current fluctuations were modulated by divalent cations, protons and H2O2. Whereas the channel is permeable for Ca2+, Ba2+ and Sr2+, its conductance is strongly reduced in solutions containing MgCl2. Cu2+ and Zn2+ are potent inhibitors of BCC1 in micromolar concentrations. The open channel conductance of BCC1 increases with acidification of the electrolyte solution. H2O2 shows strong inhibitory effects on BCC1. The channel is almost completely closed at submillimolar concentrations of H2O2. The effects of pH and H2O2 on channel properties are directional and affect BCC1 at the Ca exit side, but not on the entry site. Thus, cytosolic pH and H2O2 levels may play an important role in the modulation of the cytoplasmic free calcium concentration through BCC1

Occurrence and formation of indole-3-acetamide in Arabidopsis thaliana

An HPLC/GC–MS/MS technique (high-pressure liquid chromatography in combination with gas chromatography–tandem mass spectrometry) has been worked out to analyze indole-3-acetamide (IAM) with very high sensitivity, using isotopically labelled IAM as an internal standard. Using this technique, the occurrence of IAM in sterile-grown Arabidopsis thaliana (L.) Heynh. was demonstrated unequivocally. In comparison, plants grown under non-sterile conditions in soil in a greenhouse showed approximately 50% higher average levels of IAM, but the differences were not statistically significant. Thus, microbial contributions to the IAM extracted from the tissue are likely to be minor. Levels of IAM in sterile-grown seedlings were highest in imbibed seeds and then sharply declined during the first 24 h of germination and further during early seedling development to remain below 20–30 pmol g–1 fresh weight throughout the rosette stage. The decline in indole-3-aetic acid (IAA) levels during germination was paralleled by a similar decline in IAM levels. Recombinant nitrilase isoforms 1, 2 and 3, known to synthesize IAA from indole-3-acetonitrile, were shown to produce significant amounts of IAM in vitro as a second end product of the reaction besides IAA. NIT2 was earlier shown to be highly expressed in developing and in mature A. thaliana embryos, and NIT3 is the dominantly active gene in the hypocotyl and the cotyledons of young, germinating seedlings. Collectively, these data suggest that the elevated levels of IAM in seeds and germinating seedlings result from nitrilase action on indole-3-acetonitrile, a metabolite produced in the plants presumably from glucobrassicin turnover

The Agrobacterium vitis T-6b oncoprotein induces auxin-independent cell expansion in tobacco

Among the Agrobacterium T-DNA genes, rolB, rolC, orf13, orf8, lso, 6b and several other genes encode weakly homologous proteins with remarkable effects on plant growth. The 6b oncogene induces tumors and enations. In order to study its properties we have used transgenic tobacco plants that carry a dexamethasone-inducible 6b gene, dex-T-6b. Upon induction, dex-T-6b plants develop a large array of morphological modifications, some of which involve abnormal cell expansion. In the present investigation, dex-T-6b-induced expansion was studied in intact leaves and an in vitro leaf disc system. Although T-6b and indole-3-acetic acid (IAA) both induced expansion and were non-additive, T-6b expression did not increase IAA levels, nor did it induce an IAA-responsive gene. Fusicoccin (FC) is known to stimulate expansion by increasing cell wall plasticity. T-6b- and FC-induced expansion were additive at saturating FC concentrations, indicating that T-6b does not act by a similar mechanism to FC. T-6b expression led to higher leaf osmolality values, in contrast to FC, suggesting that the T-6b gene induces expansion by increasing osmolyte concentrations. Metabolite profiling showed that glucose and fructose played a major role in this increase. We infer that T-6b disrupts the osmoregulatory controls that govern cell expansion during development and wound healing

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

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