Kinetic studies of septum synthesis,erosion and nuclear migration in a growing B-mutant of Schizophyllum commune

Summary Microscopic measurements of apical growth and primary branch elongation were compared with nuclear movements, septum synthesis and erosion in a growing B-mutant of Schizophyllum commune. Apical growth, mitosis, septum formation, and intercalary cell division were similar to wild-type hyphae. Nuclear replication and new cross-wall formation also occurred in either apical cells bounded by eroding septa or in subterminal cells adjoined by eroded septa. An anucleate subterminal unit of the B-mutant hypha was invaded by a migrant nucleus which subsequently replicated and laid down a new septum in this region. Septum erosion occurred as early as 1 h following synthesis. Cellular granules and filaments were implicated in both septum synthesis and erosion.     

墨兰菌根的结构及酸性磷酸酶定位研究

利用光学显微镜、电子显微镜及细胞化学方法,对墨兰菌根的结构和酸性磷酸酶定位进行了初步研究。结果表明墨兰具有典型的兰科植物根结构,发现该兰花的根的外皮层不具薄壁通道细胞,菌根真菌通过破坏部分根被和外皮层细胞而侵入根的皮层细胞并在细胞内形成菌丝结,侵入的菌丝被染菌皮层细胞质膜和电子透明物质包围,进一步被消化并聚集成衰败菌丝团块。酸性磷酸酶在染菌皮层细胞及包围菌丝的皮层细胞质膜和衰败菌丝细胞壁上有强烈的酶反应,衰败菌丝周围分布有许多单层膜的含酶小泡,它们可相互愈合形成大的含酶泡或与包围菌丝的质膜融合,类似于兰科植物共生原球茎中观察到的现象。说明皮层细胞可主动释放水解酶参与对菌丝的消化     

蜜环菌侵染天麻皮层过程中酸性磷酸酶的细胞化学研究

被蜜环菌(Armillaria mellea)侵染的天麻(Gastrodia elata B1.)皮层中,由外至内形成三种类型的染菌细胞:菌丝结细胞、空腔细胞和消化细胞。外部两类细胞中的酸性磷酸酶定位显示,一些位于空腔细胞或衰老的菌丝结细胞中的菌丝内部逐渐产生大量酸性磷酸酶,随后菌丝发生自溶。这两类细胞中未发现明显的释放水解酶消化菌丝的现象。当菌丝进入消化细胞以后,情况与此不同,大量包含酸性磷酸酶的微小颗粒出现在菌丝周围,随后这些酶颗粒相互融合,形成包围菌丝的消化泡,菌丝被溶酶体水解酶所消化。最后消化泡变为包含代谢废物的残体。     

Study on Structure and Localization of Acid Phosphatase of Mycorrhizal Root of Cymbidium sinense (Orchidaceae)

The microstructure and ultrastructure of mycorrhizal root of Cymbidum sinenese (Andr.) Wild were studied. The results showed that this species possesses the typical root structure of orchids. There is no passage cells in the exodermis of root. Mycorrhizal fungi invade into the cortex by destroying the velamen and exodermal cells, and form pelotons in cortical cells. The hyphae colonizing cortical cells were separated from the cortical cells by electron- lucent material and cortical cell plasma membrane and digested. They often gathered to form clumps. Localization of acid phosphatase revealed that this enzyme possessed higher activity in the cortical cells containing hyphae. Many products of it also occurred on cortical cell plasma membrane surrounding hyphae and degenerated hyphae cell wall. Higher acid phosphatase activity was observed in many vesicles in the cortical cells infected by fimgi. These enzyme vesicles gathered around the invaded hy-phae and often fused with each other, or with cortical cell plasma membrane surrounding hyphae to digest these hyphae. It means cortical cells were able to release hydrolytic enzyme to digest the invaded hyphae.     

Acid phosphatase and esterase activity in orchid mycorrhiza

Summary A cytochemical study was made to examine the possibility that acid phosphatase may be specifically involved in the digestion of endophytic hyphae in orchid mycorrhiza. Esterase activity was studied for comparison. Frozen sections of unfixed or glutaraldehyde-fixed protocorms of Dactylorhiza purpurella infected by Thanatephorus cucumeris (Rhizoctonia solani) were reacted for acid naphthol AS BI phosphatase, acid -glycerophosphatase or naphthol AS D esterase.A marked increase in particulate acid naphthol AS BI phosphatase activity was observed during infection of host, central, parenchyma cells shortly before hyphae lysed; a diffuse reaction of high activity was localised on lysed fungus. Acid -glycerophosphatase was present at particulate sites only in fungal cytoplasm and as a diffuse reaction on lysed fungus.Naphthol AS D esterase showed highest activity at hyphal apices. Esterase seems to be associated with growth and differentiation of hyphae in orchid cells, rather than lysis of the fungus.     

Acid phosphatase activity in Pisolithus arrhizus mycelium treated with cadmium dust

The influence of cadmium dust (containing lead, cadmium, copper, zinc, silicium and other elements) on acid phosphatase activity of Pisolithus arrhizus was observed by means of electron microscopy. Dust-treated mycelium showed increased activity of the enzyme, especially on the surface of the cell wall. There was an increase in abundance of autophagic vacuoles marked by a strong phosphatase reaction. An increase in the number of hyphae with diffuse enzyme activity within the cytoplasm coincided with a decrease of lifespan of the fungus, rapid changes in the mictoplasm stage, earlier closing of the dolipori and presumably the earlier autolysis of cell cytoplasm. Hyphae showing strong autolytic activity were separated from other hyphae by the material deposited within the doliporus and this whole area was devoid at that stage of acid phosphatase activity. The role of the enzyme in the mechanism of resistance to toxic elements is discussed.     

有机碳和无机碳对3种真菌胞外酸性磷酸酶和蛋白酶活性的影响

多数研究表明外生菌根真菌能够促进植物养分吸收并提高植物生长,但是对其发生的原因研究较少。本文在室内控制条件下,研究了真菌菌丝分泌N、P相关胞外酶及其受土壤有机碳（胡敏酸）和无机碳（碳酸钙）添加的影响,结果表明：1）3种真菌——松乳菇(Lactarius deliciosus)、变色红菇（Russula integra）、铆钉菇（Gomphidius viscidus）菌丝均能够分泌酸性磷酸酶和蛋白酶,而且多数情况下,MMN培养基培养14 d时,各个酶活性较高,而不同菌的胞外酶活性存在较大的差异,平均值来看铆钉菇酸性磷酸酶活性最低而蛋白酶活性最高,其它2个真菌菌丝的胞外酶活性差异不大;2）添加胡敏酸后,3种菌丝的酸性磷酸酶活性都是随着胡敏酸添加量的增加而逐渐增加;但蛋白酶活性存在差异：松乳菇的蛋白酶活性随着胡敏酸添加量的增加而逐渐增加;变色红菇的蛋白酶活性对胡敏酸不敏感,受其影响不大;铆钉菇的蛋白酶活力在少量的胡敏酸作用下最强,但浓度过高反而抑制其蛋白酶的活性。3）添加碳酸钙后,总体来看,3种菌丝胞外酸性磷酸酶和蛋白酶活性都是添加少量碳酸钙时酶活性最强,随着浓度的增加(如0.1 g),其酶活性开始受到抑制。综上所述,真菌菌丝能够分泌酸性磷酸酶和蛋白酶,这可能是因为这些外生菌根真菌能够促进植物养分吸收和快速生长的原因;有机碳和无机碳的加入可以直接影响真菌菌丝胞外酶的分泌,进而影响土壤内有机磷和有机氮化合物的分解,显示其在土壤碳循环中的作用。     

Study on Ultrastructure and Localization of Acid Phosphatase of Gastrodia elata Cortical Cells in Their Digestive Process of Armillaria mellea

In order to study the mechanism of the digestive process of Armillaria mellea in Castrodia data, electron microscopy and cytochemical method for determination of acid phosphatase activity was employed. The provacuoles were formed by means of expanded or convoluted ER under the stimulation of cortical cells and large cells of Gastrodia data by Armillaria mellea. A product of acid phosphatase (lead phosphate deposits) occured on the tonoplast. The papillae were produced in the cell wall of cortex in Gastrodia data when Armillaria mellea penetrated into its cortex. Our results showed that the enzyme was not released from cell of Armillaria mellea. A number of small vacuoles in the cortical cells disappeared. At the same time, lead phosphate deposits on the Armillaria mellea hyphae wall were observed and than Armillaria mellea hyphae wall was going to be digested, and the hyphae lost their structure. The activity of Armillaria mellea hyphae was not observed in the large cell of Gastrodia data. A great deal of small vacuoles and mitochondria were produced, at the same time the renewable nuclei and nuclolar vacuoles etc. appeared in the large cells of Gastrodia data under the stimulation of Armillaria mellea.     

Occurrence and expression of acid phosphatase of Hymenoscyphus ericae (Read) Korf & Kernan,in isolation or associated with plant roots

Summary The activity of acid phosphatase produced in pure culture by the endomycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan (H. ericae LPA 2) was inhibited by high phosphorus levels, alkaline pH, fluoride, molybdate and mannosidase, and activated by concanavalin A. Over 80% of the enzyme activity was due to two wall-bound acid phosphatase isozymes with the characteristics of mannose-rich glycoproteins. Antiserum was raised against the major, low-molecular-weight wall isozyme and its activity tested by immunoblotting and ELISA. The antiserum cross reacted 100% with exocellular (excreted) and 28% with cytoplasmic cellular fractions of H. ericae (LPA 2) cultures, and showed high reactivity with other strains of H. ericae but not with fungal isolates from Erica hispidula L. or E. mauritanica L. Ultrastructural localization of acid phosphatase by cytoenzymology and indirect immunogold labelling confirmed its association with the fungal wall in pure culture and showed that the influence of a high phosphorus level, fluoride and molybdate is through inactivation of the enzyme. Intense acid phosphatase activity, sensitive to the latter inhibitors, was also present on external hyphae growing over a host or non-host root but it was weak or absent from intracellular hyphae where these developed within a host root. Indirect immunolabelling confirmed that this acid phosphatase was of fungal origin and that the specific inhibitory effect of host cells is due to inactivation of the enzyme rather than repression of its synthesis. Possible implications of fungal acid phosphatase in ericoid endomycorrhizal infection processes are discussed together with mechanisms that may be regulating the enzyme activity.     

A Cytochenfical Study of Acid Phosphatas in the Mycorrhizal Cells of Gastrodia elata Seedling

A cytochemical study has been made to examine the activity of acid β-glycerophosphatase in the mycorrhizal cells of the seedling of Gastrodia elata BI. using thin sectioning technique in which sections were embedded in glycol mathacrylate (GMA). After the seedling was invaded by the hyphae of Mycena osmundicola Lange, two different kinds of infected cells were formed in its root cortex.the outer 1–2 cell layers namely the hyphae-containing cells (or host cells) contained many coiled hyphae pelotons; the inner comparativly large cell layer or fungus-digesting cells contained a few straight hyphae. Localization of acid phosphatase in hyphae-containing cells showed that only a few senescent hyphae retained the enzyme activity and the plant cells did not release hydrolytic enzyme. So it is considered that the hyphal lysis in hyphae-containing cell may be due to autolysis. In contrast, higher acid phosphatase activity was visualized in many vesicles and small vacuoles of the fungus-digesting cells. When a hypha entered a fungus-digesting cell through a hyphae-containing cell, a number of enzyme granules (i. e, enzymecontaining vesicles) gathered around it. Later on the enzyme granules expanded gradually and became small enzyme vacuoles of 1.6–2.0 μm in diameter. Still later the small enzyme vacuoles fused with each other to form a large vacuole in which a part of an invading hypha was enclosed and gradually digested by hydrolytic enzymes. Finally,the digesting vacuole changed into a residual body containing some metabolic waste. The above results suggest that fungus-digesting cells can actively release hydrolytic enzymes by lysosomal vesicles to digest the invading hyphae, but such function is not present in the hyphae-containing cells,the role of which may be attributed to attracting and controling the invading hyphae.     

Metabolic activity of Glomus intraradices in Arum- and Paris-type arbuscular mycorrhizal colonization

Colonization of two plant species by Glomus intraradices was studied to investigate the two morphological types (Arum and Paris), their symbiotic interfaces and metabolic activities. Root pieces and sections were stained to observe the colonization and metabolic activity of all mycorrhizal structures. There were no growth responses observed in the plants caused by mycorrhizal symbiosis. The two morphological types had a similar percentage of root colonized, but the Arum-type had higher metabolic activity. Most of the mycorrhizal structures (88%) showed succinate dehydrogenase activity; about half showed acid phosphatase activity; and a small percentage showed alkaline phosphatase activity. Phosphatase activity was highest in arbuscules and low in intercellular hyphae in the Arum-type colonization. In the Paris-type, hyphal coils and arbusculate coils showed a similar intermediate percentage of phosphatase activity. We conclude that acid phosphatase is more important than alkaline phosphatase in both colonization types. We discuss the possibility that, whereas arbuscules in Arum-type are the main site for phosphorus release to the host plant, both the hyphal and arbusculate coils may be involved in the Paris-type.     

The mechanism of rhythmic ethylene production in sorghum. The role of phytochrome B and simulated shading

Mutant sorghum (Sorghum bicolor [L.] Moench) deficient in functional phytochrome B exhibits reduced photoperiodic sensitivity and constitutively expresses a shade-avoidance phenotype. Under relatively bright, high red:far-red light, ethylene production by seedlings of wild-type and phytochrome B-mutant cultivars progresses through cycles in a circadian rhythm; however, the phytochrome B mutant produces ethylene peaks with approximately 10 times the amplitude of the wild type. Time-course northern blots show that the mutant's abundance of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase mRNA SbACO2 is cyclic and is commensurate with ethylene production, and that ACC oxidase activity follows the same pattern. Both SbACO2 abundance and ACC oxidase activity in the wild-type plant are very low under this regimen. ACC levels in the two cultivars did not demonstrate fluctuations coincident with the ethylene produced. Simulated shading caused the wild-type plant to mimic the phenotype of the mutant and to produce high amplitude rhythms of ethylene evolution. The circadian feature of the ethylene cycle is conditionally present in the mutant and absent in the wild-type plant under simulated shading. SbACO2 abundance in both cultivars demonstrates a high-amplitude diurnal cycle under these conditions; however, ACC oxidase activity, although elevated, does not exhibit a clear rhythm correlated with ethylene production. ACC levels in both cultivars show fluctuations corresponding to the ethylene rhythm previously observed. It appears that at least two separate mechanisms may be involved in generating high-amplitude ethylene rhythms in sorghum, one in response to the loss of phytochrome B function and another in response to shading.     

Seasonal variations of symbiotic ultrastructure and relationships of two natural ectomycorrhizae of beech (Fagus sylvatica/Lactarius blennius var. viridis and Fagus sylvatica/Lactarius subdulcis)

This study concerned two perennial ectomycorrhizae of beech: Fagus sylvatica L./Lactarius blennius var. viridis and Fagus sylvatica L./Lactarius subdulcis, collected in a natural forest site. Nutritional exchanges existing between beech roots and fungi were evaluated over a seasonal cycle by studying the variations of ultrastructure, polyphosphate and nitrogenous granules and glycogen content (periodic acid thiocarbohydrazide silver test). The acid phosphatase activity, which is an indicator of the intensity of the physiological activity of symbiosis partners, was also revealed by transmission electron microscopy. The occurrence of the two ectomycorrhizae in the soil was not related to the climatic conditions. In winter, the number of polyphosphate and nitrogenous granules was high in the fungi, and glycogen was abundant in the cytoplasm of hyphae. In late winter, an intense acid phosphatase activity was revealed on plasmalemmas of live hyphae. It could indicate the beginning of fungal reserve mobilization, which progressively disappeared as spring progressed. In summer, glycogen was quasi non-existent and only few polyphosphate and nitrogenous granules were observed. At this period, the acid phosphatase activity was weak. Finally, in autumn and early winter, glycogen and granules were progressively restored in fungal structures. Relationships between changes of granules and glycogen content and the physiological states of each symbiont throughout the year are discussed.     

Comparison of phosphatase localization in the intraradical hyphae of arbuscular mycorrhizal fungi,Glomus spp. and Gigaspora spp.

The localization of acid and alkaline phosphatases in the intraradical hyphae of the arbuscular mycorrhizal fungi, Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (Gm), Gl. etunicatum Becker and Gerd. (Ge) and Gigaspora rosea Nicol. and Schenck (Gir) were compared. Marigold (Tagetes patula L.) and leek (Allium porrum L.) were inoculated with each of the three fungi. The mycorrhizal roots were harvested at 3, 4, 5 and 6 weeks after sowing (WAS), treated with a digestion solution containing cellulase and pectinase, and then stained for phosphatase activities at pH 5.0 and pH 8.5. The development of fungal structures in the host root was also examined. Gm formed fine-branched (mature) arbuscules only at the early phase of infection (3 to 4 WAS). Mature arbuscules of Ge and Gir were observed from the early phase (4 WAS) up to the end of experiment. At pH 5.0, the localization of the phosphatase activities of the three fungi were similar irrespective to host plant species. The activity appeared in mature arbuscules and intercellular hyphae, whereas the collapsed arbuscules were inactive. Ten millimolar NaF, an acid phosphatase inhibitor, inhibited the phosphatase activities of Gm and Ge but did not affect that of Gir. At pH 8.5, a difference among the fungal species was found in the localization of phosphatase activity while that between host species was not. The mature arbuscules were also the active sites in all three species. Only Gir showed the activity in the intercellular hyphae while the two Glomus spp. did not. Five millimolar EDTA inhibited the activity of Gir at pH 8.5 while the activities of Ge and Gm were not affected by either 5 mM EDTA or 10 mM KCN (both are alkaline phosphatase inhibitors).     

不同磷源对红三叶草根际和菌根际磷酸酶活性的影响

以红三叶草为研究对象,利用三室培养系统,在接种菌根真菌(Glomus mosseae)的条件下研究了不同磷源对根际和菌根际磷酸酶活性的影响,植株生长8周后收获并测定根室、菌丝室的土壤磷酸酶活性、植株干重及含磷量．结果表明,根室酸性磷酸酶活性比碱性磷酸酶活性更裔,接种条件下二者都稍有增加,特别是在供给有机磷(植酸钠)的条件下明显增加了菌丝室土壤磷酸酶活性．接种菌根真菌显著增加了植株干重、磷含量和总磷吸收．施用磷酸二氢钾(KH2PO4)时菌丝吸磷量占吸磷总量的43．1%,而施用植酸钠(Na-phytate)时菌丝吸磷量占吸磷总量的60．8%。     

Ultrastructural histochemical localization of acid phosphatase in salivary glands of Drosophila melanogaster.

The ultrastructural histochemical localization of acid phosphatase in salivary glands of third instar larvae of Drosophila melanogaster has been studied. Using Gomori's lead phosphate method for acid phosphatase detection, the optimal incubation time in the reaction medium was determined to be 30 min. When glands having wild-type acid phosphatase activity are incubated for this time, deposition of the final reaction product is observed in essentially every lysosome and artifactual staining is minimal.     

Regulation of nonspecific acid phosphatase in Salmonella: phoN and phoP genes.

Mutations in Salmonella typhimurium strains lacking nonspecific acid phosphatase mapped in two unlinked loci. One of these, phoP, was cotransducible by phage P22 with purB, whereas the second, phoN, was cotransducible by phage P1 with purA. Mutants with temperature-sensitive nonspecific acid phosphatase activity (measured in whole cells) were also isolated. A phoN mutant with thermolabile whole-cell activity was isolated directly from wild-type LT-2. Several other mutants with temperature-sensitive enzyme activity were also isolated as revertants of phoN mutants. These data suggest that phoN might be a structural locus for nonspecific acid phosphatase. The observation that a mutation resulting in high level of nonspecific acid phosphatase mapped in phoP suggests a possible regulatory role for this locus.     

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70 min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30 min, nematodes were observed adhered after 40 min and many were captured by the typical fungus traps after 70 min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.     

An electron-microscopical analysis of capture and initial stages of penetration of nematodes byArthrobotrys oligospora

A detailed analysis was made of the capture and subsequent penetration of nematodes by the nematophagous fungusArthrobotrys oligospora using different electron-microscopical techniques. Capture of nematodes by this fungus occurred on complex hyphal structures (traps) and was effectuated by an adhesive coating, present on these trap cells. The adhesive layer was largely fibrillar in nature and was absent on cells of normal hyphae. Following capture, penetration hyphae were formed at those sites where the trap cell wall was anchored to the nematode cuticle by the adhesive. New walls of these hyphae were formed underneath the original trap cell walls, which were partly hydrolysed to allow growth and development of the penetration tubes through the adhesive coating towards the cuticle. Our observations indicated that the cuticle of the nematode was subsequently penetrated by the penetration tubes by mechanical means. After penetration a large infection bulb was formed from which trophic hyphae arose. Cytochemical experiments indicated that the sites of penetration of the cuticle were intensely stained for acid phosphatase activity. At later stages of infection activity of this enzyme was present throughout the nematode contents; the enzyme was most probably secreted by complex membranous structures associated with the cytoplasmic membrane of the infection bulb and the trophic hyphae.