Signalling pathways in pollen germination and tube growth

Summary. Signalling is an integral component in the establishment and maintenance of cellular identity. In plants, tip-growing cells represent an ideal system to investigate signal transduction mechanisms, and among these, pollen tubes (PTs) are one of the favourite models. Many signalling pathways have been identified during germination and tip growth, namely, Ca²⁺, calmodulin, phosphoinositides, protein kinases, cyclic AMP, and GTPases. These constitute a large and complex web of signalling networks that intersect at various levels such as the control of vesicle targeting and fusion and the physical state of the actin cytoskeleton. Here we discuss some of the most recent advances made in PT signal transduction cascades and their implications for our future research. For reasons of space, emphasis was given to signalling mechanisms that control PT reorientation, so naturally many other relevant works have not been cited. Correspondence and reprints: Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.     

The effect of glucose-6-phosphate isomerase genotype onin vitro specific activity andin vivo flux inMytilus edulis

Four samples of the musselMytilus edulis were taken between 1984 and 1987 from Stony Brook, New York, and used to study the glucose-6-phosphate isomerase (GPI) polymorphism in this species.In vitro specific activity andin vivo flux measured in the same animals were found to be significantly correlated. A significant effect of GPI genotype on flux was observed in one of the samples; overall, significant evidence of effect of genotype on enzyme activity was also obtained. GPI activities of common genotypes tend to deviate less from the population mean than those of rare (frequency less than 5%) genotypes. This suggests the possibility that rare GPI genotypes are rare as a consequence of having biochemical properties that deviate from an optimum level and, therefore, having a lower fitness. In support of this hypothesis, we found in one of our samples that shell length is a concave function of GPI activity with an intermediate optimum activity level. The financial support provided to P.J.N.S. by the Luso-American Educational Commission (Fulbright Program), the Instituto Nacional de Investigacao Científica (Portugal), and the Faculdade de Ciências da Universidade de Lisboa during several stages of this research is gratefully acknowledged. Financial support from the Ministerio de Educatión y Ciencia (Spain) in the form of a postdoctoral Fulbright/MEC fellowship to M.S. is also gratefully acknowledged. Research was supported by National Science Foundation Grant BSR-8415060 to R.K.K. This is contribution No. 736 from the Program in Ecology and Evolution, State University of New York at Stony Brook. On leave from Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Campo Grande C2, Lisboa, Portugal.     

Modulation of endocytosis in pollen tube growth by phosphoinositides and phospholipids

Summary. In plants, tip-growing cells represent an ideal system to investigate signal transduction mechanisms, and among those, pollen tubes are one of the favourite models. Many signalling pathways have been identified during germination and tip growth, namely, Ca²⁺, calmodulin, phosphoinositides, cyclic AMP, and GTPases. Not surprisingly, the apical secretory machinery, essential for tip growth, seems to be an intersection point for all these pathways. Recently, the phospholipid phosphatidic acid was also suggested to actively participate in the control of endo- and exocytosis and to interfere with the correct positioning of the actin cytoskeleton. Phosphatidic acid seems to act concertedly with the phosphoinositides phosphatidylinositol 4,5-bisphosphate and D-myo-inositol 1,4,5-trisphosphate. Here we review previous data and discuss additional evidence that these three molecules have a combined action modulating both the actin cytoskeleton and the apical secretory machinery. We further discuss how these findings can be integrated into a working model for pollen tube apical secretion that contemplates the existence of a rapid endocytosis mechanism. Correspondence and reprints: Instituto de Ciência Aplicada e Tecnologia, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal.     

Quantification of trehalose in biological samples with a conidial trehalase from the thermophilic fungus Humicola grisea var. thermoidea

The partially-purified, thermally-stable trehalase from conidia of Humicola grisea was highly specific for trehalose and was free of potentially interfering activities. The enzyme was fully stable when stored in solution at -15°C for at least 6 months. This preparation could be used to quantify trehalose from 0.05 to 1.25 mol/ml either in carbohydrate mixtures or in complex biological materials.M.J. Neves, H.F. Terenzi and J.A. Jorge are with Departmento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirao Preto, Universidade de São Paulo, 14040-901-Ribeirão Preto, São Paulo, Brazil; F.A. Leone is with Departamento de Quimica, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901-Ribeirão Preto, São Paulo, Brazil.     

Effect of carbon source on the β-glucosidase system of the thermophilic fungus Humicola grisea

H. grisea produced an extracellular -glucosidase (EC 3.2.1.21) at high activity in media supplemented with carboxymethyl cellulose (CMC) or cellobiose. Cellobiose-induced -glucosidase was insensitive to glucose repression whereas that of CMC-supplemented cultures was partially repressed. Molecular sieving revealed three main active components (M_r 50, 128 and 240 kDa). Glucose competitively inhibited -glucosidase activities with K_i values of 0.9mM and 3.3mM (extracellular) and 10.2mM and 22.6mM (cytosolic), induced in the presence of CMC or cellobiose respectively.The authors are with the Departamento de Biologia, Faculdade de Filosofia. Ciências e Letras de Ribeirão Preto, Universidade de São Paulo-14040-901 Ribeirão Preto, São Paulo, Brasil;     

Oxypetalum banksii subsp. banksii: a taxon of Asclepiadaceae with an extragynoecial compitum

 The floral morphology of seven Oxypetalum species and, in particular, the spatial relationship between the five stigmatic chambers and two separate ovaries of their flowers with respect to transmission of the pollen tube are studied. In all species, except O. banksii subsp. banksii, floral morphology is similar to that in other Asclepiadeae, and the flowers pollinated with one pollinium develop only one follicle, which means compitum absence. In O. banksii subsp. banksii flowers, the secretory interstaminal tissue lines the inner walls of the stigmatic chambers as in the other species studied, but it also reaches the upper part of the inner surface of the filament tube, where it surrounds the styles, an unprecedented feature for Asclepiadaceae. This tissue secretes nectar and mucilage; the latter acts as transmitting medium for the growth of pollen tubes from pollinia inserted and hydrated in stigmatic chambers (“hyperstigmas”). Mucilage also functions as an extragynoecial compitum: in flowers pollinated with one pollinium both carpels develop into a follicle. Received August 28, 2001; accepted April 9, 2002 Published online: October 14, 2002 Addresses of the authors: Milene Faria Vieira (e-mail: mfvieira@mail.ufv.br), Departamento de Biologia Vegetal, Universidade Federal de Vi?osa, 36571-000, Vi?osa, Minas Gerais, Brazil. George John Shepherd, Departamento de Botanica, Instituto de Biologia, Universidade Estadual de Campinas, C.P. 6109, 13083-970, Campinas, S?o Paulo, Brazil.     

Molecular differentiation of two vicariant neotropical tree species, Plathymenia foliolosa and P. reticulata (Mimosoideae), inferred using RAPD markers

 Plathymenia foliolosa and P. reticulata are two tropical tree species economically important for their high quality wood. P. foliolosa occurs mainly in areas of Atlantic Forest (a tropical forest) whereas P. reticulata occurs mainly in the Cerrado region (a savanna vegetation), both Brazilian biomes classified as hotspots for conservation priorities. In the present study RAPD markers were used to differentiate P. foliolosa from the vicariant species P. reticulata. Using 10 random primers it was possible to obtain 84 markers, of which 22 (26.2%) were present in just one of the two species. Nine of the ten primers were successful in discriminating between the species through the detection of diagnostic markers or band frequency differences. One of the populations of P. foliolosa, located in a region where the two species co-occur, presented several markers that were characteristic of P. reticulata, suggesting the occurrence of gene flow between the two species. Received September 3, 2001; accepted August 19, 2002 Published online: November 22, 2002 Addresses of the authors: Daniela Rodrigues Lacerda (dlacerda@dedalus.lcc.ufmg.br), Maria Bernadete Lovato (lovatomb@icb.ufmg.br), Maria Dolores Porto Acedo (acedomd@icb.ufmg.br), Dept. Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CP: 486, Belo Horizonte, MG, 31270-901, Brasil. José Pires de Lemos Filho (lemos@icb.ufmg.br), Dept. Botanica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CP: 486, Belo Horizonte, MG, 31270-901, Brasil.     

Pectinase production by Penicillium frequentans

High activities of extracellular pectinase with viscosity-diminishing and reducing groups-releasing activities were produced by Penicillium frequentans after 48 h at 35°C, in agitated cultures supplemented with 0.5% citrus pectin and initial pH of 2.5. Under these conditions the fungus also produced high activity of pectinesterase. At an initial pH of 7.0 or 8.0, pectin lyase activity was also detected. Enzyme activity releasing reducing sugars was more stable at 50°C than viscosity-diminishing activity. Both activities were maximal at pH 2.5 to 5.2 and at 55°C.The authors are from the Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Avenida do Café, s/n^o, Bairro Monte Alegre, 14.049 Ribeirão Preto, S.P., Brazil.     

Effects of anoxia on root ultrastructure of four neotropical trees

Summary. The root ultrastructure of seedlings grown in anaerobic conditions was investigated in four neotropical species: Sesbania virgata, Erythrina speciosa, Sebastiania commersoniana (all present in waterlogged or flooded areas), and Schizolobium parahyba (that occupies mainly dry areas). Anaerobiosis induced an increase in the size of mitochondria, dilatation of cristae and of the endoplasmic reticulum, and fragmentation or concentric arrangement of reticulum saccules. The ultrastructural alterations were reversible only for S. virgata and E. speciosa. The seedlings of S. parahyba and S. commersoniana were more sensitive to oxygen deprivation and presented extensive cell disruption. The results are discussed in terms of energy supply.Correspondence and reprints (present address): Departamento de Biologia Animal e Vegetal, Centro de Ciências Biológicas, Universidade Estadual de Londrina, P.O. Box 6001, 86051-970, Londriná, Paraná, Brazil.     

Structure, morphology, and composition of silicon biocomposites in the palm tree Syagrus coronata (Mart.) Becc.

Summary.  Syagrus coronata is an economically important palm tree grown as an ornament, for the oil extracted from its seeds, and the wax from its leaves which has several applications in industry. Silicon biocomposites were analyzed in leaves of S. coronata. Silica bodies were found as extracellular silica masses between the hypodermal-layer cell walls and in granules present in the vacuoles of palisade cells. Scanning electron microscopy of the hypodermal layer of cells showed a collection of spherical bodies embedded in enveloping cavities that outlined the general structure of the bodies. Globular subunits with sharp edges formed the spherical bodies that ranged from 6 to 10 μm in diameter (average, 7.8 μm). X-ray microanalysis detected only silicon and oxygen homogeneously distributed throughout the bodies. Vacuoles of palisade cells contained a large number of granules ranging from 20 nm to 1.2 μm in size (average, 300 nm). Transmission electron microscopy associated with electron spectroscopic imaging and electron energy loss spectroscopy were used to determine the elemental composition of the granules. Vacuolar granules were amorphous and composed of silicon and oxygen, suggesting they consist of amorphous silica biominerals. No nitrogen, indicative of organic matter, was detected in the granules. Received November 26, 2001; accepted July 1, 2002; published online October 31, 2002 RID="*" ID="*" Correspondence and reprints: Departamento de Microbiologia Geral, Instituto de Microbiologia Professor Paulo de Góes, Centro de Ciências da Saude, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil.     

Determination of the enzymatic activity of pectinases from different microorganisms

The decrease in viscosity is widely used to estimate the activity of pectinolytic enzymes. This method is shown to be influenced by the production strain and this prevents an accurate comparison between the activities of different microorganisms, especially under different conditions.A.E. Maiorano and Y. Ogaki are with the Divisão de Quimica, with the Agrupamento de Biotecnologia, Instituto de Pesquisas Tecnológicas do Estado de São Paulo S/A.-IPT, Cidade Universitária, Caixa Postal 7141, CEP 01064-970, São Paulo, SP, Brazil; W. Schmidell is with the Departamento de Engenharia Quimica, Escola Politécnia, Universidade de São Paulo, Caixa Postal 61548, CEP 05424-970, São Paulo, SP, Brazil.     

Glycerolysis of olive oil: batch operational stability of Candida rugosa lipase immobilized in hydrophilic polyurethane foams

The operational stability of the Candida rugosa lipase immobilized in a hydrophilic polyurethane foam was evaluated in consecutive batches for the glycerolysis of olive oil in n-hexane, aimed at the production of monoglycerides.Glycerol controlled the glycerolysis in the system under study, since it is both a substrate and a powerful water binder that reduces the water activity of the reaction medium and of the microenvironment. Two sets of experiments were carried out under different glycerol/triglyceride ratios. After 345 hours of consecutive 23 hours batches no lipase inactivation was observed.List of Symbols a_w thermodynamic activity of water - DG diglyceride (s) - FAME fatty acid methyl ester (s) - FFA free fatty acid (s) - FID flame ionization detector Gly glycerol - MG monoglyceride (s) - TG triglyceride (s) - TLC thin layer chromatography The authors are grateful to Profs. P. Adlercreutz, Technical University of Lund, Sweden, and J.M.S. Cabral, Instituto Superior Técnico, Lisbon, Portugal, for inspiring discussions and advice, to Prof. Helena Pereira, Instituto Superior de Agronomia (ISA), Lisbon, Portugal, for the use of GC equipment and to Mrs. Marlene Dionísio, ISA, for invaluable help with some of the experimental work.     

Liquid-liquid extraction of a recombinant cutinase from fermentation media with AOT reversed micelles

This work reports the extraction and back-extraction of an intracellular recombinant cutinase from complex biological media using AOT reversed micelles in isooctane. Cutinase was recovered from different complex media namely, fermentation broths and supernatants after cell disruption by osmotic shock and sonication. The application of the AOT reversed micellar system to the extraction of cutinase allowed activity yields and purification factors ranging from about 5% to 50% and 1.2 to 10.2, respectively, depending on the biological medium.Maria das Graças Carneiro da Cunha, from ITEP-Instituto Tecnológico do Estado de Pernambuco, acknowledges a Ph.D fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Centro de Pesquisa Aggeu Magalhães, Recife — PE — Brasil. E. P. Melo thanks Junta Nacional de Investigação Científica, Lisboa, Portugal, for providing a Ph.D. fellowship. The scientific support given by Prof. Sílvia M. B. Costa for the spectroscopic data and further discussions are particularly acknowledged.This work was partly financed by the BRIDGE and BIOTECHNOLOGY Programmes (Contracts BIOT-CT91-0274(DTEE) and BIOT 2 CT-943016).     

Morphohistological analysis and histochemistry of Feijoa sellowiana somatic embryogenesis

Summary. Morphohistological analysis and histochemical studies were carried out during the induction and development of Feijoa sellowiana somatic embryos. Zygotic embryos were cultured on LPm medium containing 2,4-dichlorophenoxyacetic acid (20µM) and glutamine (8mM). Somatic embryogenesis could be induced from embryogenic cells that originated in meristematic centers or from clusters of cells. The presence of few starch grains and abundant protein bodies was observed in the globular and early torpedo stages, while in torpedo and cotyledonary-stage somatic embryos an enhanced synthesis of starch grains was associated with the accumulation of reserves to be used in the conversion of the embryos to plantlets. Proteins were predominantly observed in protoderm cells, as well as in the meristematic apical region of torpedo and cotyledonary-stage somatic embryos.Correspondence and reprints: Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, C.P. 476, 88034-001 Florianópolis, SC, Brazil.     

Two new species of Drosophila (Diptera,Drosophilidae) associated with inflorescences of Goeppertia monophylla (Marantaceae) in the city of São Paulo,state of São Paulo,Brazil

Two new Brazilian species of Drosophila (subgenus Drosophila) are described and illustrated: Drosophila asymmetrica sp. nov. and Drosophila peixotoi sp. nov. Both species were collected, and emerged, from inflorescences of Goeppertia monophylla (Marantaceae) in the urban Forest Reserve of the Instituto de Biociências da Universidade de São Paulo and their types will be deposited in the Museu de Zoologia da USP. The former species, which could not be assigned to any known group, has a conspicuously asymmetric aedeagus and a narrow oviscapt valve. The latter species belongs to the guarani group and is closely related to D. guaru, D. ornatifrons and D. subbadia, from which it can be distinguished by the presence of just one conspicuous large black spine at inner lower tip of cercus instead of two spines.     

Macromolecular desorientation in detached tendons

Conclusions Based on the results obtained on the grounds of the present discussion we may conclude: Under the described condition, in the detached tendons of guinea pigs and also in the implanted tendons the acid mucopolysaccharides gradually undergo a structural disorder, loosing its molecular orientation. In the referred tendons, the loss of structural orientation of the acid mucopolysaccharides proceeds the desorientation of the collagen polypeptide chains. In the tendon repair process, under the described conditions, acid mucopolysaccharides with oriented macromolecules are elaborated during the fibrillogenesis phenomenon.This paper is part of the author's Doctoral and Livre Docencia thesis published in October 1964.Professor do Departamento de Patologia da Faculdade de Farmácia e Odontologia de Piracicaba — Govêrno do Estado de São Paulo.     

Plant regeneration from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis,S. macrocephala and S. scabra

Plant regeneration from protoplasts is a prerequisite to the production of modified plants using somatic hybridization and transformation. Whole plant regeneration was achieved from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis, S. macrocephala and S. scabra, three economically important species of this tropical forage legume genus. The effects of both protoplast density and protoplast culture method on cell division and plating efficiency are presented.Abbreviations BAP 6-benzylaminopurine - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalenacetic acid On leave from: Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Brasil     

Lipolytic activity of dermatophytes

Lipolytic activity of dermatophytes was tested by the method generally used forCandida lipolytica. Most of the freshly isolated strains ofMicrosporum canis, M. gypseum, Epidermophyton floccosum andTrichophyton mentagrophytes gave positive reactions, whereas, only few strains ofT. schoenleini, T. violaceum, T. megnini, T. rubrum andT. tonsurans yielded such reactions. Among subcultured strains, it was found a decreased lipolytic activity inMicrosporum species and an increased lipolytic activity inEpidermophyton and someTrichophyton species.Part of this work was performed at Serviço de Doenças Infecto-Contagiosas (Secção de Dermatologia), Hospital de Curry Cabral - Lisboa.     

Aspergillus Gracilis Bainier var. Sartoryi (Biourge) Batista,Lima e Vital n. var. — Sua importáncia filogenética

Summary We deal with a new variety ofAspergillus which we callAspergillus gracilis Bainiervar. sartoryi (Biourge) Batista, Lima and Vital.This variety appears to be very important under the phylogenetic point of view, apparently being an intermediate form betweenAspergillus andPenicillium.Its morphology resemblesA. gracilis but the conidia characteristics are distinct; we took the epithetsartoryi fromA. sartoryi Biourge which was not described by the respective author.We suggest, too, the transfer of the seriesA. restrictus, where we put our new variety, from the groupA. glaucus, to become independant, as a connective series in the phylogenetic development betweenAspergillus andPenicillium, since the type of the series and the other elements that it embraces does not produce sclerotia or cleistothecia and the conidial apparatus offers curious similarity toPenicillium. Besides, some fungi of the series grow well in certain culture media, in complete discordance with the members of the groupA. glaucus.

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Publiçacão no 4

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Chefe do Instituto de Micologia;

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Diretor do Instituto de Antibióticos e Prof. de Microbiologia da Escola de Química;

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Assistente micologista do Instituto de Micologia — (Todos da Universidade do Recife).

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Os autores declinam o seu agradecimento ao Dr.Heraldo da Silva maia, Assistente micologista eD. Marilene Maranhão Moreira, Auxiliar-Técnico, do Instituto de Micologia, pela colaboração que lhes prestaram, durante a realização do presente trabalho.     

The golden age of Drosophila research at the Universidade de S?o Paulo (USP): A testimonial on the decades 1940–1950

This article is a testimonial written by the first author regarding the research work performed with Drosophila between 1943 and 1959, at the Departamento de Biologia Geral of the Universidade de São Paulo (USP), which permitted the building of a nucleus of excellence. This research work, focused on the systematics and population genetics of the Neotropical species, began during the first of Dobzhansky’s six visits to USP. Special attention was given to the multinational megaprojects conducted during his longer stays, from August 1948 to July 1949 and from June 1955 to July 1956. The role played by the Rockefeller Foundation is duly remembered, and so is the undeniable contribution brought by Dobzhansky, to the establishment of several laboratories dedicated to research in the field of natural population genetics and to the qualification of human resources. On the other hand, important “backstage” episodes are retrieved which were omitted in the official history and occurred from the planning to the execution of the project, carried out on the Angra dos Reis islands. Special attention was given to the relationship problems which resulted from Dobzhansky’s, the leader’s, difficult personality and contributed to the failure of the second and last multinational project.