Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors

Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca²⁺ concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and β-glucan elicitors caused a rapid increase in cytosolic Ca²⁺ concentration, which peaked within 2–2.5 min of treatment. The Ca²⁺ concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca²⁺ concentration with a maximum occurring after about 7–8 min was observed in the case of β-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca²⁺ concentration from resting concentrations as low as 0.1 lM to highest levels of about 2 lM. Dose-response experiments showed that the concentration of elicitors giving a Ca²⁺ response at the 50% level was 0.4 nM for the chitin fragment and 28 lM and 72 lM, respectively, for a synthetic hepta-β-glucoside and a fungal β-glucan fraction. The β-glucan- or N,N′,N′′,N′′′-tetraacetyl chitotetratose (CH4)-induced Ca²⁺ signals were inhibited by both the Ca²⁺ chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and by the Ca²⁺-channel inhibitor La³⁺. Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca²⁺ curve following β-glucan treatment. Bacterial cyclic β-glucans known to suppress β-glucan-induced phytoalexin production were also found to function as a suppressor for the Ca²⁺ response that was elicited by the fungal β-glucans. The results clearly show that the increase in the cytosolic Ca²⁺ concentration is an early and rapid event in the elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses. Received: 23 July 1998 / Accepted: 16 October 1998     

Purification and properties of a thermoactive and thermostable pullulanase from Thermococcushydrothermalis, a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent

The extremely thermophilic archaeon Thermococcus hydrothermalis, isolated from a deep-sea hydrothermal vent in the East Pacific Rise at 21°N, produced an extracellular pullulanase. This enzyme was purified 97-fold to homogeneity from cell-free culture supernatant. The purified pullulanase was composed of a single polypeptide chain having an estimated molecular mass of 110 kDa (gel filtration) or 128 kDa (sodium dodecyl sulfate/polyacryl amide gel electrophoresis). The enzyme showed optimum activity at pH 5.5 and 95 °C. The thermostability and the thermoactivity were considerably increased in the presence of Ca²⁺. The enzyme was activated by 2-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and α-cyclodextrin were inhibitors. This enzyme was able to hydrolyze, in addition to the α-1,6-glucosidic linkages in pullulan, α-1,4-glucosidic linkages in amylose and soluble starch, and can therefore be classified as a type II pullulanase or an amylopullulanase. The purified enzyme displayed Michaelis constant (K _m) values of 0.95 mg/ml for pullulan and 3.55 mg/ml for soluble starch without calcium and, in the presence of Ca²⁺, 0.25 mg/ml for pullulan and 1.45 mg/ml for soluble starch. Received: 19 November 1997 / Received revision: 9 March 1998 / Accepted: 14 March 1998     

Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw

The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg²⁺ and Ca²⁺ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by CelA. Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998     

The SR Ca2+ ATPase of the Antarctic scallop Adamussium colbecki: cold adaptation and heavy metal effects

Cell calcium is accumulated in intracellular stores by sarco-endoplasmic reticulum Ca²⁺ ATPases functionally interacting with the membrane lipid environment. Cold adaptations of membrane lipids in Antarctic Sea organisms suggest possible adaptive effects also on sarco-endoplasmic reticulum Ca²⁺ ATPases. We investigated the SR Ca²⁺ ATPase of an Antarctic scallop, Adamussium colbecki, by characterising the enzyme activity and studying temperature effects. Ca²⁺ ATPase, assayed by following ATP hydrolysis, was thapsigargin- and vanadate-sensitive, showed maximum activity under 2 μM Ca²⁺, 200 mM KCl and pH 7.2, and had a K _M for ATP of 22 ± 7 μM. Temperature effects showed an Arrhenius inversion between −1.8 and 0°C, indicating cold adaptation, an Arrhenius break at 10°C, and a collapse above 20°C. A. colbecki accumulates high amounts of cadmium in the digestive gland; heavy metal effects on sarco-endoplasmic reticulum Ca²⁺ ATPases were therefore tested, finding an IC₅₀ = 0.9 μM for Hg²⁺ and 3 μM for Cd²⁺. Finally, SDS-PAGE analysis showed a main band at about 100 kDa, which was identified as sarco-endoplasmic reticulum Ca²⁺ ATPase after trypsin digestion, and accounted for 60% total protein. Accepted: 10 December 1998     

Isolation and characterization of highly (R)-specific N-acetyl-1-phenylethylamine amidohydrolase, a new enzyme from Arthrobacter aurescens AcR5b

A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8 and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity was inhibited by Cu²⁺, Co²⁺, Ni²⁺, and Zn²⁺, and this inhibition was reversed by EDTA.M Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996     

Ion antagonism in phytochrome-mediated calcium-dependent germination of turions of Spirodela polyrhiza (L.) Schleiden

The light-dependent germination response of turions (resting fronds) is mediated by phytochrome and requires the presence of Ca²⁺ in the medium (K.-J. Appenroth and H. Augsten, 1990, Photochem. Photobiol. 52: 61–65). The Ca²⁺ requirement of germination is apparent only in the presence of exogenous Mg²⁺. A competitive ion antagonism was demonstrated between Ca²⁺ and Mg²⁺ in this physiological response; Mg²⁺ could also be replaced by Ba²⁺ or Sr²⁺. Without exog-enous Mg²⁺, a Ca²⁺ concentration as low as 0.9 μM fulfilled the Ca²⁺ requirement. This type of ion antagonism resembled the competitive Ca/Mg interaction reported previously for calcium-binding proteins. The physiological response was blocked by inhibitors of Ca²⁺ uptake (verapamil, La³⁺). It was concluded that uptake of Ca²⁺ from the external medium is an essential step in the phytochrome-mediated germination of turions. The results are in agreement with the assumption that the uptake of Ca²⁺ is blocked at the side of entry by other alkaline earth ions. Treatment of turions with Mg²⁺ (1 mM) for 24 h at varying times after the red light pulse in otherwise virtually Ca²⁺-free KNO₃ solution resulted in a response similar to a Ca²⁺ step-down treatment. This is in agreement with the assumption that the Ca²⁺- and the Mg²⁺-sensitive periods coincide. The ion interaction described here represents the first photophysiological example in plants of an antagonistic effect between Ca²⁺ and Mg²⁺ similar to that which occurs in vitro with calmodulin. Received: 12 June 1998 / Accepted: 28 December 1998     

Blue light but not red light induces a calcium transient in the moss Physcomitrella patens (Hedw.) B., S. & G.

In caulonemal filaments of the moss, Physcomitrella patens, which had been incubated in darkness, 3 s irradiation with blue light (λ_max 450 nm) at fluence rates of 100 μmol m⁻² s⁻¹ and above caused a transient␣increase in cytosolic calcium ion concentration, [Ca²⁺]_cyt, which was both intensity- and time-dependent. Measurements of [Ca²⁺]_cyt were made using moss transformed with the cDNA for apoaequorin and reconstituting the Ca²⁺-dependent photoprotein aequorin in the cytosol by incubation in coelenterazine.␣In response to blue light at fluence rates of 100–1000 μmol photons m⁻² s⁻¹, [Ca²⁺]_cyt increased transiently from a basal level of approximately 50 nM to between 200 and 700 nM. Irradiation with red light did not evoke any measurable change in [Ca²⁺]_cyt. The presence of calcium in the incubating medium was not required for the increase in [Ca²⁺]_cyt to occur. A mutant strain, gad-139, was identified which required an irradiance of only 1 s to evoke a response. The kinetics showed a delay of approximately 6 s from the beginning of illumination before the beginning of the increase in [Ca²⁺]_cyt. The data suggest that the activation of a photoreceptor rather than the direct opening of calcium channels is involved in this blue-light response. Received: 4 December 1997 / Accepted: 4 May 1998     

Free calcium ion concentration in the sieve-tube sap of Ricinus communis L. seedlings

Changes in free Ca²⁺ in sieve-tube sap have been proposed to be important in the regulation of phloem transport, and Ca²⁺-activated protein kinase activity has been described in phloem exudate (S.A. Avdiushko et al. 1997 J Plant Physiol 150: 552–559). Using atomic absorption spectrometry, we have determined that the total Ca²⁺ concentration in sieve-tube sap from Ricinus seedlings containing the endosperm is about 100 μM (range 80–150 μM). We used three independent methods to determine the free calcium ion concentration in the phloem sap ([Ca²⁺]_p). The first method was to calculate [Ca²⁺]_p from the total Ca²⁺ concentration, in combination with the binding constants and concentrations of the ionic solutes in phloem sap. The resultant estimate of [Ca²⁺]_p was 63 μM. The second method used the Ca-specific fluorescent dye 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic-acid (FURAPTRA) on exuded sieve-tube sap. Although the sap interfered severely with the fluorescence properties of the dye, Ca²⁺ titrations enabled a value of [Ca²⁺]_p = 20 μM to be deduced. The third method used Ca²⁺-selective microelectrodes on exuded sap samples, which gave an average value for [Ca²⁺]_p = 13 μM. No significant change in this value was observed during the sap exudation period. The Ca²⁺ buffer capacity was determined and the result of about 0.6 mmol · l⁻¹ · pCa⁻¹ displayed excellent agreement with the measured values of free and total Ca²⁺ concentration in sieve-tube sap. Since the measured values for free Ca²⁺ are 20- to 100-fold higher than those usually reported for the cytosol of a range of plant cells in resting conditions, it is concluded that either regulation of [Ca²⁺]_p is of limited physiological importance, or that the Ca²⁺-dependent proteins respond only to relatively high [Ca²⁺]_p. The implications for regulation of cytosolic free Ca²⁺ in symplastically connected companion cells is discussed. Received: 15 February 1998 / Accepted: 14 March 1998     

An ATP-inhibited endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence: purification and characterization

In our studies on the role of enzymes in plant DNA replication, recombination, and repair, we isolated from cauliflower (Brassica oleracea L. var. botrytis) inflorescences a single-stranded DNA-specific endonuclease that was inhibited by ATP. The endonuclease, designated cauliflower nuclease II, was purified to near homogeneity through six successive column chromatographies. The enzyme is a single polypeptide with a molecular mass of 70 kDa as judged by the results of sodium dodecyl sulfate-polyacry amide gel electrophoresis, activity gel, and gel-filtration column chromatography. The enzyme can cleave a linear or a circular single-stranded DNA but cannot cut or nick a double-stranded DNA. The mode of activity of the nuclease is endonucleolytic and non-processive. Interestingly, the endonuclease activity is strongly inhibited by less than 0.1 mM ATP, although the role of this inhibition is thus far unclear. While ATPγS and GTP can also inhibit the activity, other ribonucleoside triphosphates are much less effective. The optimum pH of the enzyme is 5.6. The enzyme requires an exceptionally high ionic strength, 0.2 M KCI for optimum activity, and without these ions no activity can be detected. The endonuclease activity is stimulated by Ca²⁺, which cannot be replaced by Mg²⁺ or Mn²⁺. The features of the enzyme and its relation to plant DNA metabolism are discussed. Received: 26 March 1998 / Accepted: 4 June 1998     

Production, purification, and characterization of a highly enantioselective (S )-N-acetyl-1-phenylethylamine amidohydrolase from Rhodococcus equi Ac6

Rhodococcus equi Ac6 was found to express an inducible (S )-specific N-acetyl-1-phenylethylamine amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed around pH 6.5. Purification of the enzyme to a single band in a Coomassie-blue-stained sodium dodecyl sulfate/polyacrylamide gel (SDS-PAGE) was achieved by ammonium sulfate precipitation of R. equi Ac6 crude extract and column chromatographies on Fractogel TSK Butyl-650(S) and Superose 12HR. At pH 7.0 and 30 °C the amidohydrolase had a half-life of around 350 days; at 44 °C it was only 10 min. Except for Ni²⁺ and, to some extent, Zn²⁺ and Co²⁺, the enzyme was neither strongly influenced by metal cations nor by chelating agents, but was inhibited by 95% at 0.1 mM phenylmethylsulfonyl fluoride. The molecular mass of the native enzyme was estimated to be 94 kDa by gel filtration and 50 kDa by SDS-PAGE, suggesting a dimeric structure. Specificity experiments revealed a spectrum of related N-acetylated compounds being hydrolyzed with variable enantiomeric selectivities. Received: 20 September 1996 / Received revision: 23 December 1996 / Accepted: 30 December 1996