Purification and some properties of two forms of chitinase from mycelial cells of Mucor rouxii

Chitinase activity was measured in extracts of mycelial cells of Mucor rouxii as a function of the culture age. There was a peak of specific activity at the mid-exponential phase of growth (10 h), which paralleled chitin synthase activity. An additional peak of chitinase with higher specific activity was detected in 4 h cultures, which coincided with the onset of germination. Purification of chitinase activities from the cytoplasm revealed two enzymes, I and II, with different molecular mass and ionic charge. Antibodies induced with chitinase I did not cross-react with chitinase II. Both enzymes digested nascent chitin preferentially over preformed chitin, yielding diacetylchitobiose as the sole product of hydrolysis.     

Two calcium dependent protein kinases are differently regulated by light and have different activity patterns during seedling growth in <Emphasis Type="Italic">Pharbitis nil</Emphasis>

In this study using biochemical approaches we identified two calcium-dependent protein kinases (CDPKs) named PnCDPK52 and PnCDPK56 in soluble protein extracts from seedlings of Pharbitis nil. Both enzymes phosphorylated the specific substrate histone III-S in the presence of Ca²⁺ and cross-reacted with antibodies against the CDPK. PnCDPKs exhibited quite different activity and protein levels during germination and successive stages of seedling growth. PnCDPK52 protein level was high in seeds and during germination, whereas PnCDPK56 increased in the next stages of seedling growth, being the dominant enzymes in mature seedlings, of the light- and dark-grown plant. In all cases both activity and accumulation of protein PnCDPK56 was higher in dark grown plants whereas exposure to light reduced both factors. When etiolated cotyledons were exposed to light, the activity of PnCDPK56 was reduced to the basal level within 5 h. Conversely, increasing activity of PnCDPK56 in cotyledons of green plants shifted to darkness was extremely rapid, reaching the maximum level after just 1 h of darkness and then gradually decreased. Further lengthening of the darkness to 16 h resulted in a strong increase in activity at 12 h. These data indicate that at least two isoforms of CDPK are involved in germination and seedling growth of P. nil. The differences in PnCDPKs strongly argue for the pleiotropic role of these isoforms. It seems that PnCDPK52 is associated with the germination process and PnCDPK56 with seedling growth. Moreover it suggests that activity of PnCDPK56 is controlled by light via the photoreceptor-dependent pathway.     

Developmental regulation of NAD+ kinase in Neurospora crassa

The specific activity of NAD⁺ kinase (ATP:NAD⁺ 2-phosphotransferase, EC 2.7.1.23) from Neurospora crassa shows sharp peaks when the organism enters a new developmental stage of the asexual life cycle: the peaks are observed during hydration and germination of conidia, at the transition from exponential to stationary growth and at the photostimulated conidiation. As stimulation of NAD⁺ kinase activity by light in conidiating mycelium is not sensitive to translation inhibitors, the activiation of pre-existing molecules, rather than induction of protein synthesis de novo may be supposed. Enzyme electrophoresis revealed the presence of four forms of NAD⁺ kinase having different apparent molecular weights (I=333,000; II=306,000; III=229,000 and IV=203,000). Manifestation of the activity of individual forms of NAD⁺ kinase is developmentally controlled: form III is most abundant during vegetative growth, forms I and II prevail in conidia. At the conidial germination the increase of NAD⁺ kinase activity is associated with the activation of form III, whereas during photostimulation of conidiation form II is the most activated one. Therefore, certain molecular forms of the enzyme may be regarded as biochemical markers for different developmental stages of N. crassa.     

The Occurrence of Lipid Hydroperoxide-Decomposing Activities in Rice and the Relationship of Such Activities to the Formation of Antifungal Substances

Two fractions that decomposed lipid hydroperoxide (lipid hydroperoxide-decomposingactivities), were isolated from rice seeds by ion-exchange chromatographyon DEAE-Toyopearl. Thin-layer chromatography showed that bothfractions generated two products (I and II) when 9-linoleatehydroperoxide (9-LOOH) was used as a substrate. Structural analysisby gas chromatography/mass spectrometry showed that productsI and II were 9-hydroxyoctadecadienoic acid and 9,12,13-trihydroxyoctadec-lO-enoicacid, respectively, which have been isolated from rice leavesas antifungal substances that are active against rice blastfungus. The antifungal activity of 9-LOOH was examined againstthe blast fungus and compared with that of product I. Both compoundsstrongly inhibited conidial germination, growth of germ tubesand formation of appressoria at 50ppm. Product I was more activein this assay than 9-LOOH. (Received October 18, 1989; Accepted September 4, 1990)     

Induction of phenylalanine ammonia-lyase (PAL) in germinating lettuce seeds (Lactuca sativa)

Dry lettuce seeds (achenes of Lactuca sativa L. cv. Grand Rapids) contain no detectable phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity. Enzyme activity could be detected in these seeds within 4 h of imbibition under white light. The specific activity of PAL increased rapidly during the next 12–16 h of imbibition. Far-red light completely suppressed germination as well as the development of PAL. Gibberellic acid (GA₃, 0.1 m M ), although effective in causing almost 100% germination in dark, did not induce proportionate increases in PAL. Seed germination as well as PAL activity were substantially inhibited by cis -4-cyclohexene-l, 2-dicarboximide (CHDC, 1.0 m M ) both in light and dark. Both GA₃ and benzyladenine (BA, 0.1 m M ) retarded radicle elongation in light. Concomitantly, a decrease in PAL activity was observed. Benzyladenine was able to reverse the effects of CHDC on germination but PAL activity was still highly reduced, probably due to the inhibitory effects of BA on elongation of the radicle. More than 95% of the extractable PAL was found to be present in the radicle. When seeds incubated in white light for 10 h were transferred to FR, further increases in PAL activity as well as the growth of the radicle were severely inhibited. It is suggested that the induction of PAL in light-sensitive lettuce seeds is coincidental with the germination of seeds, and the amount of PAL per germinated seed is related to the extent of elongation of the embryonic axes.     

Increase of seed germination,growth and membrane integrity of wheat seedlings by exposure to static and a 10-KHz electromagnetic field

There is a large body of experimental data demonstrating various effects of magnetic field (MF) on plants growth and development. Although the mechanism(s) of perception of MF by plants is not yet elucidated, there is a possibility that like other stimuli, MF exerts its effects on plants by changing membrane integrity and conductance of its water channels, thereby influencing growth characteristics. In this study, the seeds of wheat (Triticum aestivum L. cv. Kavir) were imbibed in water overnight and then treated with or without a 30-mT static magnetic field (SMF) and a 10-kHz electromagnetic field (EMF) for 4 days, each 5 h. Water uptake of seeds reduced 5 h of the treatment with EMF but did not show changes in SMF treatment. Exposure to both magnetic fields did not affect germination percent of the seeds but increased the speed of germination, compared to the control group. Treatment with EMF significantly reduced seedling length and subsequently vigor index I, while SMF had no effects on these parameters. Both treatments significantly increased vigor index II, compared to the control group. These treatments also remarkably increased catalase activity and proline contents of seedlings but reduced the activity of peroxidase, the rate of lipid peroxidation and electrolyte leakages of membranes. The results suggest promotional effects of EMFs on membrane integrity and growth characteristics of wheat seedlings.     

Control of cell-cycle-associated tetrahydrobiopterin synthesis in rat thymocytes.

The cell-cycle progression of rat thymocytes from G0 through G1 to DNA synthesis is associated with a transient synthesis of H4biopterin, the concentration of which reaches a maximum at the time of S-phase entry and then decreases. This synthesis of H4biopterin is controlled by the specific activity of GTP cyclohydrolase I, which peaks in G1/S cells. In contrast, the catalytic activity of sepiapterin reductase remains constant throughout the cell-cycle. At G0 the steady state mRNA levels specific for GTP cyclohydrolase I and sepiapterin reductase, respectively, are below the limits of detection. Both accumulate as the thymocytes progress through the cell-cycle but lack cyclic down regulation. The data indicate that the variations in H4biopterin synthesis during the cell-cycle are caused by growth regulated increase in GTP cyclohydrolase I mRNA expression, with subsequent post-translational inactivation. This latter is likely due to the degree of enzyme phosphorylation.     

Resolution and Brain Regional Distribution of Cysteine Sulfinate Decarboxylase Isoenzymes from Hog Brain

Abstract: Cysteine sulfinate decarboxylase (CSD; EC 4.1.1.29) activity from porcine brain was resolved into three peaks by hydroxylapatite chromatography. The first two peaks (I and II) did not decarboxylate and were not inhibited by glutamate. The third peak (III) cochromatographed with glutamate decarboxylase (GAD; EC 4.1.1.15) activity. The K_m values of cysteine sulfinate for peaks I, II, and III were 5.5 × 10⁻⁴ m , 1.3 × 10⁻⁴ m , and 4.5 × 10⁻³ m , respectively. The possibility that the same enzyme was responsible for peak III CSD and GAD activities was suggested by several findings: (1) Mutual competitive inhibition was observed between glutamate and cysteine sulfinate for these activities. (2) Similar first-order heat-inactivation curves were obtained for peak III CSD and GAD when incubated at 55xBOC. (3) Both activities were inhibited similarily by ATP and chloride ion. High concentrations of glutamate (0. l m ) inhibited peak III CSD activity more than 90% but had no effect on either peak I or II CSD activities. This difference in sensitivity of the isoenzymes to inhibition by glutamate was used to examine the relative regional distributions and the relative contributions to total activity of the glutamate-sensitive (peak III CSD, GAD) and glutamate-insensitive (peaks I and II CSD) isoenzymes. Glutamate-insensitive CSD activity contributed only part of the total activity in all brain regions tested (ranging from 23% in the superior colliculus to 64% in the pons). However, the specific activity of glutamate-insensitive CSD was more constant than the total or glutamate-sensitive specific activities among the brain regions tested. The results indicate that GAD is responsible for a significant proportion of the total CSD activity in porcine brain.     

Extracellular inulinases from Penicillium janczewskii, a fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae)

Extracellular inulinases from Penicillium janczewskii were obtained from the filtrate of 12 day-old cultures supplemented with inulin from Vernonia herbacea. Crude filtrates and partially-purified enzyme preparations (peaks I and II) were active on inulin, sucrose and raffinose. The apparent M(r) of the enzymes from peaks I and II were 48 and 66 kDa, respectively. The apparent K(m) (mmol l-1) values of peak I were 0.43 for inulin and 18.7 for sucrose; for peak II they were 0.87 and 18.5 for inulin and sucrose, respectively. Their temperature and pH optima were 55 degrees C and 5.0, respectively. Both peaks catalysed the hydrolysis of beta-(2,1) fructans more rapidly than beta-(2,6) fructans. Free fructose was the predominant product released from inulin, indicating that these enzymes display exo-inulinase activity. In view of these characteristics, the yield and the high specific activity towards beta-(2,1) fructans, inulinases from P. janczewskii can be utilized for the preparation of fructose syrup from inulin.     

Changes in the content of cyclic AMP and cyclic GMP during the development of Xenopus laevis.

The levels of the three major DNA-dependent RNA polymerases (enzymes I, II and III) present in the dimorphic fungus Mucor rouxii have been investigated during the transition from yeast-like cells to mycelial growth. Increases in the specific activity of crude extracts were observed at 2 h and at 6 h after induction of mycelium formation by aeration of yeast-like cells. These increases could be attributed to changes in the specific activities of enzymes I and II. Alterations were also found in the relative amounts of enzymes I and II: prior to aeration, 31% of the total polymerase activity of crude extracts was present as enzyme I; after 2 h of aeration, the specific activity of this enzyme doubled and the relative amount increased to 64% of the total activity. After 6 h of aeration, the relative amounts of enzymes I and II were 25 and 65%, respectively, and the specific activity of enzyme II had nearly doubled. The amounts and specific activities of enzyme III did not change significantly during the transition.     

Proteinase activities in resting and germinating seeds of Scots pine, Pinus sylvestris

Resting seeds of Scots pine contained a moderate amount of acid proteinase activity, about 90% of which was inhibited by pepstatin A and about 10% by p-hydroxymer-curibenzoate. In gel chromatography on Sephacryl S-200 the proteinase activity showed a complex elution pattern with poorly separated peaks at positions corresponding to mol. wts. 100,000 and 30,000 and several shoulders. The results suggested that pine proteinases I and II, which are the main proteinases in the endosperms of germinating seeds (Salmia 1981: Physiol. Plant. 51: 253–258), were not present in the resting seeds.—Seedling extracts showed a low level of acid proteinase activity, which separated into several peaks in chromatography on Sephacryl S-200. As none of the peaks had the catalytic properties of proteinase I or II, it seems that these endospermal enzymes are also lacking in the seedling tissues.—In the endosperms of germinating seeds the activity of the pepstatin-sensitive acid proteinase(s) remained at a constant level throughout the period of reserve protein mobilization (lasting up to the stage when the length of dark-grown seedlings was 60 mm). Proteinases I and II were absent from resting seeds, showed a small increase up to the 20-mm stage, and then increased rapidly up to the 60-mm stage.—Resting embryos contained relatively higher acid proteinase activity than resting endosperms, and again about 90% of it was inhibited by pepstatin A and about 10% by p-hy-droxymercuribenzoate. During germination the former activity decreased, the latter activity remained at approximately the same level, and the activity of the other acid proteinases increased continuously with the growth of the seedling.—It is concluded that the pepstatin-sensitive proteinase(s), which is not affected by endogenous proteinase inhibitors, plays a central role in the initiation of reserve protein mobilization in both the embryo and the endosperm. Proteinases I and II, on the other hand, seem to account for the greater part of reserve protein breakdown in the main protein storage tissue, the endosperm.     

Effects of mercury and cadmium on the activities of antioxidative enzymes in the seedlings ofPhaseolus aureus

Phaseolus aureus Roxb. was exposed to HgCl₂ and Cd(NO₃)₂ either at the germination stage in concentration 0.5, 5 and 25 μM for 48 and 96 h, or at the seedling stage (5^th day of germination) in concentration 0.5, 5 and 20 μM for 6, 24 and 48 h. The germination and the growth of roots (germination stage treatment) were less in Hg than in Cd treatment. The seedlings (seedling stage treatment) were, however, more susceptible to Cd than Hg. Both root and leaf tissues of the plant treated at the germination stage showed enhanced lipid peroxidation and activities of the antioxidative enzymes (catalase, guaiacol peroxidase and ascorbate peroxidase), except the catalase in leaf in 25 μM Cd treatment. At seedling stage the content of malondialdehyde increased significantly only in the leaf tissue, during 6 h exposure. The activities of all the enzymes exhibited an increasing trend in both the tissue of the seedlings, particularly the leaf, at least after 24 and 48 h, except the catalase whose activity declined in response to Cd. Active involvement of the guaiacol and ascorbate peroxidases, rather than catalase, in scavenging cellular H₂O₂ was indicated. It was concluded that the two metals had little primary damaging effect on membranes.     

Glyoxysomes in megagamethophyte of germinating ponderosa pine seeds

Decoated ponderosa pine (Pinus ponderosa Laws) seeds contained 40% lipids, which were mainly stored in megagametophytic tissue and were utilized or converted to sugars via the glyoxylate cycle during germination. Mitochondria and glyoxysomes were isolated from the tissue by sucrose density gradient centrifugation at different stages of germination. It was found that isocitrate lyase, malate synthase, and catalase were mainly bound in glyoxysomes. Aconitase and fumarase were chiefly localized in mitochondria, whereas citrate synthase was common for both. Both organelles increased in quantity and specific activity of their respective marker enzymes with the advancement of germination. When the megagametophyte was exhausted at the end of germination, the quantity of these organelles and the activity of their marker enzymes decreased abruptly. At the stage of highest lipolysis, the isolated mitochondria and glyoxysomes were able to synthesize protein from labeled amino acids. Both organellar fractions contained RNA and DNA. Some degree of autonomy in glyoxysomes is indicated.     

Jasmonic acid promotes germination and lipase activity in non‐stratified apple embryos

The effects of (−)jasmonic acid (JA) on germination of embryos isolated from dormant seeds of apple ( Malus domestica Borb. cv. Antonówka) cultured in darkness or at 12‐h photoperiod were studied, as well as its effects on the activity of alkaline lipase (AlkL, EC 3.1.1.3) in these embryos. The maximum sensitivity of germination to JA occurred on days 3 and 4 of embryo culture. Both germination and enzyme activity were stimulated by JA, its effect being additive to that of light. Inhibitors of lipoxygenase inhibited embryo germination and AlkL activity, both effects being partially reversed by JA treatment. We suggest that (1) JA is implicated in an endogenous complex controlling apple seed germination, and that (2) it acts independently of the mechanism triggered by light.     

Tomato pollen respiration in relation to in vitro germination and pollen tube growth under favourable and stress-inducing temperatures

Tomato pollen germination, pollen tube growth and respiratory activity were recorded during incubation in a liquid medium for 7 h over a temperature range of 15–35°C. Although the initial rate of respiration was highest at 30°C, both at 30°C and 35°C respiration decreased after the first hour of incubation due to high temperature impairment of germination and pollen tube growth. The total per cent germination of pollen over the 7-h period was maximal at 15°C whereas pollen tube length was maximal at 25°C. Although the production of CO₂ measured at hourly intervals throughout the incubation period did not correlate to a statistically significant level with either the per cent pollen germination or the length of the pollen tubes alone, nevertheless from 2 h after the start of incubation, it closely correlated with the values for germination × pollen tube length, indicating that the respiratory activity of tomato pollen at a given time is a function of both the per cent germination and the pollen tube growth. We suggest therefore that the rate of respiration might be preferable to a simple germination test for the assessment of pollen germination ability since it expresses not only the pollen germination potential but also the growth vigour of the pollen tubes. In addition, where in vitro tests are designed to assess pollen germination–temperature interactions, they should employ a long incubation period (e.g. 7 h) to permit differences in sensitivity to temperature to be observed.     

Mutants in three genes affecting transport of magnesium in Escherichia coli: genetics and physiology.

Mutants in three genes affecting two Mg2+ transport systems are described. System I, for which Co2+, Mn2+, and Mg2+ are substrates, is inactive in corA mutants corB mutants express system I after growth on high (10 mM) Mg2+ but not low (0.1 mM) Mg2+. Both corA and corB mutants are resistant to Co2+ or Mn2+. corA mutants are sensitive to CA2+. Transport system II is specific for Mg2+ and is repressed by growth on 10 mM Mg2+. mgt mutations inactivate system II. Growth on mgt mutants in normal except on very low (1 muM) concentrations of Mg2+, corA mgt strains exhibit no high-affinity, energy-dependent transport of Mg2+ and require 10 mM Mg2+ for optimal growth. The three genes are not linked. The corA locus is contransducible with ilv at 75 min, corB is cotransducible with pyrB at 85 min, and mgt is cotransducible with malB and mel at 81 min on the genetic map.     

Characterization of the Amylolytic Activity of Araucana araucana (Mol.) Koch Germinating Seeds

Starch is the main reserve in Araucaria araucana (Mol.) Kochseeds. In an attempt to understand the regulation of starchmobilization during seed germination, amylase activity was characterizedand its levels were measured for the first 90 h after the startof seed imbibition. Amylase is present in both the embryo andmegagametophyte when seeds are quiescent. The enzyme activityof the embryo has two peaks during the 90 h, while amylase activityof the megagametophyte remains low and unchanged during thistime. Analysis of the products released by the enzyme and thermostabilityexperiments identify the amylase as a-amylase. Differentialcentrifugation of embryo homogenates under isoosmotic conditionsshows that the enzyme is soluble. Quantification of the endogenous gibberellins of the embryoshows a time correlation between the rise in amylase activityand the rise in specific gibberellin levels during the 40 hafter the start of imbibition. The growth retardant AMO-1618lowers enzyme activity to 37.3% of the control at this time.However, activity is almost completely restored when the gibberellinsfrom embryonic tissue are added in physiological doses to theimbibition medium containing AMO-1618. ³ Present address: Department of Biology, Washington University,St. Louis, Missouri, USA. (Received September 2, 1982; Accepted December 27, 1982)     

Differences in mycorrhizal preferences between two tropical orchids

Orchids parasitize their mycorrhizal fungi and are dependent on them for seed germination. Controversy reigns over how specific the mycorrhizal association is in tropical species. Although there is little experimental evidence to support any viewpoint, some variation is known to exist. We compared mycorrhizal specificity and performance in two phylogenetically related epiphytic orchids from Puerto Rico, Tolumnia variegata and Ionopsis utricularioides (Oncidiinae) by integrating two techniques: phylogenetic analysis of mycorrhizal fungi based on nuclear ribosomal internal transcribed spacer (ITS) sequences, and symbiotic seed germination experiments. Most of the mycorrhizal isolates from T. variegata fell into four different clades of Ceratobasidium, while most of those from I. utricularioides were restricted to a single clade of the same genus. Seeds of T. variegata germinated equally well with fungi from both T. variegata and I. utricularioides, but seeds of I. utricularioides germinated significantly better with its own isolates. Seeds of I. utricularioides germinated and developed faster than those of T. variegata. Both the molecular phylogeny and the seed germination experiments showed that T. variegata is a generalist in its association with fungal symbionts. In contrast, I. utricularioides is more specialized and more effective at exploiting a specific fungal clade. Our data are consistent with the theoretical trade-offs between specialized and generalized interactions.