Interações e efeitos fisiológicos de bactérias diazotróficas e fungos micorrízicos arbusculares na mandioca.

Visando conhecer as interações entre fungos micorrízicos arbusculares e bactérias diazotróficas na cultura da mandioca (Manihot esculenta Crantz), foram conduzidos vários experimentos para avaliar os efeitos de exsudatos de mandioca e de bactérias nos fungos micorrízicos, e a produção de ácido indolacético (AIA) in vitro pelas bactérias diazotróficas. Os experimentos evidenciaram que as bactérias diazotróficas estimularam a colonização micorrízica de Glomus clarum a partir do 30o dia. Os exsudatos de mandioca e das bactérias não apresentaram efeito na germinação de Gigaspora gigantea, mas influenciaram seu crescimento micelial. A adição de exsudatos de mandioca estimulou o crescimento das bactérias diazotróficas in vitro, evidenciando que podem existir, nos exsudatos, substâncias que atuariam como sinais moleculares ou estimulantes do crescimento, e não como fatores nutricionais. As bactérias diazotróficas apresentam ainda capacidade de produzir AIA in vitro. Azospirillum lipoferum isolado da mandioca produziu até 130 mM de AIA após 48 horas de incubação, ao passo que Klebsiella sp. produziu cerca 60 mM e a Bactéria E, aproximadamente 20 mM.

An investigation into differences between indoleacetic acid and fusicoccin in their influence on RNA synthesis, protein synthesis and growth in Avena coleoptile tissue

Growth stimulation of Avena coleoptile tissue by indoleacetic acid (IAA) and fusicoccin (FC) was compared by measuring both their influence on RNA and protein synthesis during IAA or FC stimulated growth. FC stimulated growth more than IAA during the initial four hour exposure, after which the growth rate gradually declined to the control rate. FC, but not IAA, increased the uptake of 3H-Ieucine into tissue and the specific radioactivity of extracted protein. Cycloheximide inhibited the incorporation of 3H-Ieucine into protein by approximately 60% to 70% in all cases. In the presence of cycloheximide 3H-radioactivity accumulated in FC-treated tissue, whereas IAA did not seem to influence 3H-accumulation. These results suggest that FC stimulated leucine uptake into the tissue and that increased specific activity of coleoptile protein is due to increased leucine uptake, not an increased rate of protein synthesis. There was no measurable influence of IAA and/or FC on RNA and protein synthesis during the initial hours of a growth stimulation. Inhibitors of RNA and protein synthesis, actinomycin D and cycloheximide, respectively, severely inhibited IAA enhanced growth but only partially inhibited FC stimulated growth. The data are consistent with suggestions that a rapidly turning over protein participates in IAA stimulated growth, and that a continual synthesis of RNA and proteins is an absolute requirement for a long term growth response to IAA. On the contrary, FC-stimulated growth exhibited less dependency on the transcription and translation processes. The data are consistent with proposals suggesting different sites of action for FC and IAA stimulated growth. l?hen compared to CO2-free air, CO2 at 300 ppm had no significant influence on coleoptile growth and protein synthesis in the presence or absence of lAA or FC. Also, I mM malate, pH 6.0 did not influence growth of coleoptiles in the presence or absence of lAA. This result was obtained despite reports indicating that 300 ppm CO2 or I mM malate stimulates growth and protein synthesis. This lack of difference between CO2-treated and untreated tissue could indicate either that the interstitial space CO2 concentration is not actually different in the two treatments due to significant endogenous respiratory CO2 or else the data would suggest a very loose coupling between dark CO2 fixation and growth. IAA stimulated the in vivo fixation of 14c-bicarbonate (NaHI4c03) by about 25% and the addition of cycloheximide caused an inhibition of bicarbonate fixation within 30 min. Cycloheximide has also been reported to inhibit IAA-stimulated H+ excretion. These data are consistent with the acid growth theory and suggest that lAA stimulated growth involves dark CO2 fixation. The roles of dark CO2 fixation in lAA-stimulated growth are discussed.

Influencia da ativaçao acida na estabilidade termica de hibridos vermiculita/epoxido

ALVES, Ana Paula de M. ; GERMANO, A. F. S. ; ARAKAKI, L N H ; Ferreira, U. V. S. ; ARAUJO, A. S. Influencia da ativaçao acida na estabilidade termica de hibridos vermiculita/epoxido. In: CONGRESSO NACIONAL DE ANLISETERMICA E CALORIMETRIA, 7., 2010, São Pedro, SP. Anais... Sao Pedro, SP: ABRATEC, 2010

Produção de biodiesel através da hidroesterificação do óleo de andiroba (Carapa guianensis, Aubl.) via Catalise Heterogenea Acida

O processo de hidroesterificação (hidrolise seguida de esterificação) se constitui como uma alternativa ao processo convencional de produção de biodiesel, pois permite o uso de matérias-primas de qualquer teor de água e de ácidos graxos. O biodiesel foi gerado a partir da esterificação do óleo de andiroba com alto teor de ácidos graxos sendo utilizado o oxido de nióbio em pó da CBMM (HY-340) como catalisador. Para execução dos experimentos foi utilizado um reator autoclave (batelada). A reação de hidrolise foi conduzida na temperatura de 300°C, ~1200psi e razão molar água/óleo de 20. Nas reações de esterificação foram observados os efeitos da razão molar metanol/acido graxo (1,2; 2,1 e 3,0), da temperatura (150, 175 e 200oC) e da concentração de catalisador (0, 10 e 20% m/m) tendo como resposta a conversão e a taxa inicial da reação. Os dados foram conduzidos segundo um planejamento experimental (fatorial com 23 e adição de 3 pontos centrais) analisado pelo programa Statistica. A conversão das reações de esterificação foi monitorada a partir de medidas titulometricas de acidez das alíquotas, retiradas nos tempos de 5, 10, 15, 20, 25, 30, 45 e 60 min. Nos experimentos de esterificação a maior conversão obtida foi 96,1% com temperatura de 200oC, 20% m/m de catalisador e razão molar metanol/acido igual a 3,0. Eliminando o catalisador e considerando os mesmos niveis para as outras duas variáveis analisadas (temperatura e razão molar), foi obtida uma conversão de 95,3%, porem foi observada uma taxa de conversão menor tendo-se um aumento de 35 minutos no tempo de reação comparando com a reação catalisada.

Valorizzazione di molecole da materie prime rinnovabili: la trasformazione del 2,5-diidrossimetilfurano con catalisi acida

The present work is part of a larger project aimed at obtaining compounds of industrial interest from renewable sources. The work is particularly aimed to investigate the reactivity of 2,5-bis-hydroxymethylfuran (BHMF), an important building block of organic nature easily obtainable from biomass, with acid catalysis. Through the study of the reactivity of BHMF in water, in the presence of an heterogeneous acid catalyst (Amberlyst 15), has been developed a new synthetic method for the preparation of α-6-hydroxy-6-methyl-4-enyl-2H-pyran-3-one a derivative whose molecular skeleton is similar to that of natural products which are used in pharmaceutical chemistry. The product is obtained in milder conditions and with better selectivity with respect to the strongly oxidizing conditions with which it is prepared in the literature starting from different precursors containing furan ring.

Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces

We investigated the spatial pattern of expression of ipdC, a plant inducible gene involved in indoleacetic acid biosynthesis in Erwinia herbicola, among individual cells on plants to gain a better understanding of the role of this phenotype in the epiphytic ecology of bacteria and the factors involved in the regulation of ipdC. Nonpathogenic E. herbicola strain 299R harboring a transcriptional fusion of ipdC to gfp was inoculated onto bean plants, recovered from individual leaves 48 h after inoculation, and subjected to fluorescence in situ hybridization using a 16S rRNA oligonucleotide probe specific to strain 299R. Epifluorescence images captured through a rhodamine filter were used to distinguish the 5carboxytetramethylrhodamine-labeled cells of strain 299R from other leaf microflora. Quantification of the green fluorescence intensity of individual cells by analysis of digital images revealed that about 65% of the 299R cells recovered from bean leaves had higher ipdC expression than in culture. Additionally, 10% of the cells exhibited much higher levels of green fluorescence than the median fluorescence intensity, indicating that they are more heterogeneous with respect to ipdC expression on plants than in culture. Examination of 299R cells in situ on leaf surfaces by confocal laser scanning microscopy after fluorescence in situ hybridization of cells on leaf samples showed that even cells that were in close proximity exhibited dramatically different green fluorescence intensities, and thus, were in a physical or chemical microenvironment that induced differential expression of ipdC.

Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5.

The plant growth hormone indole-3-acetic acid (IAA) transcriptionally activates expression of several genes in plants. We have previously identified a 164-bp promoter region (-318 to -154) in the PS-IAA4/5 gene that confers IAA inducibility. Linker-scanning mutagenesis across the region has identified two positive domains: domain A (48 bp; -203 to -156) and domain B (44 bp; -299 to -256), responsible for transcriptional activation of PS-IAA4/5 by IAA. Domain A contains the highly conserved sequence 5'-TGTCCCAT-3' found among various IAA-inducible genes and behaves as the major auxin-responsive element. Domain B functions as an enhancer element which may also contain a less efficient auxin-responsive element. The two domains act cooperatively to stimulate transcription; however, tetramerization of domain A or B compensates for the loss of A or B function. The two domains can also mediate IAA-induced transcription from the heterologous cauliflower mosaic virus 35S promoter (-73 to +1). In vivo competition experiments with icosamers of domain A or B show that the domains interact specifically and with different affinities to low abundance, positive transcription factor(s). A model for transcriptional activation of PS-IAA4/5 by IAA is discussed.

Dissertatio medico-chymica, quâ demonstratur sanguinem humanum nullo acido vitiari : accedit appendix quâ inquiritur an equidem in primis viis contineatur acidum, ulterius considerantur remedia anti-acida, praecipue pulveres absorbentes de quorum tam usu salutari quàm abusu medicum instructum esse oportet /

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Tissue culture of forest trees: Clonal multiplication of Eucalyptus grandis L

Axillary shoot proliferation was obtained using explants of Eucalyptus grandis L. juvenile and mature stages on a defined medium. Murashige and Skoog medium (MS) supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and additional thiamine. Excised shoots were induced to root on a sequence of three media: (1) White's medium containing indoleacetic acid (IAA), NAA and indole butyric acid; (IBA), (2) half-strength MS medium with charcoal and (3) half-strength MS liquid medium. The two types of explants differed in rooting response, with juvenile-derived shoots giving 60% rooting and adult-derived ones only 35%. Thus, the factors limiting cloning of selected trees in vitro are determined to be those controlling rooting of shoots in E. grandis.

Morphogenesis and plant regeneration from cotyledonary cultures of Eucalyptus

Callus cultures were established from hypocotyls and cotyledons derived from young seedlings of Eucalyptus citriodora. Successful plantlet production from cotyledonary callus was achieved within 6 weeks on Murashige and Skoog's basal medium supplemented with zeatin (1 mg/l) and indoleacetic acid (0.2 mg/l). Leaf and shoot callus obtained from one-year-old plants did not differentiate. Results reported contribute to defining optimal conditions for callus growth and plantlet formation

Rapid burst of H2O2 by plant growth regulators increases intracellular Ca2+ amounts and modulates CD4(+) T cell activation

The identification of small molecules that affect T cell activation is an important area of research. Three molecules that regulate plant growth and differentiation, but not their structurally similar analogs, were identified to enhance primary mouse CD4(+) T cell activation in conjunction with soluble anti-CD3 stimulation: Indoleacetic acid (natural plant auxin), 1-Napthaleneacetic acid (synthetic plant auxin) and 2,4-Dichlorophenoxyacetic acid (synthetic plant auxin and herbicide). These effects are distinct in comparison to Curcumin, the well known phenolic immunomodulator, which lowers T cell activation. An investigation into the mechanisms of action of the three plant growth regulators revealed a rapid induction of reactive oxygen species (ROS), mainly comprising H2O2 . In addition, these three molecules synergize with soluble anti-CD3 signaling to enhance intracellular Ca2+ concentrations Ca2+](i), leading to greater T cell activation, e.g. induction of CD25 and IL-2. Enhanced production of TNF alpha and IFN gamma by CD4+ T cells is also observed upon plant growth regulator treatment with soluble anti-CD3. Interestingly, maximal IL-2 production and CD4(+) T cell cycle progression are observed upon activation with soluble anti-CD3 and phorbol 12-myristate 13-acetate (PMA), a phorbol ester. Additionally, stimulation with PMA and Ionomcyin (a Ca2+ ionophore), which activates T cells by circumventing the TCR, and plant growth regulators also demonstrated the role of the strength of signal (SOS): T cell cycle progression is enhanced with gentle activation conditions but decreased with strong activation conditions. This study demonstrates the direct effects of three plant growth regulators on CD4(+) T cell activation and cycling. (C) 2010 Elsevier B.V. All rights reserved.

小麦TaRanBP基因功能的初步分析

小G蛋白（small GTPases）是真核生物中广泛存在的一类调节各种生命活动的信号分子。根据结构与功能的不同，小G蛋白家族成员可分成五个亚家族，分别为Ras，Rab，Rho，Arf和Ran。五类小G蛋白通过其活化态（GTP结合态）和非活化态（GDP结合态）的相互转换行使着各种功能。Ras GTPases在酵母和哺乳动物中调节细胞增殖过程; Rho GTPases调控肌动蛋白重组过程，并参与MAP 激酶的细胞信号转导过程等; Rab GTPases和Arf GTPases分别在膜转运过程中起着不同的重要作用;而Ran GTPases则在核孔位置调节着蛋白和RNA分子的运输过程。 小G蛋白附属蛋白调节着小G蛋白活化态与非活化态之间的转换，其中鸟核苷酸交换因子(guanine nucleotide exchange factors, GEFs)可以催化小G蛋白转换为GTP结合形式，即活化态;而GTPase 激活蛋白(GTPase-activating proteins, GAPs)和小G蛋白结合蛋白(small GTPases binding proteins)可以激活小G蛋白自身的水解活性，从而将其转变成非活化态形式。 相比其它小G蛋白，Ran GTPases及其附属蛋白在真核生物中的研究相对较少。已有的成果表明它们主要在核质运输过程中及对相应的信号转导途径起调节作用。而针对Ran GTPases及其附属蛋白在真核生物尤其是高等植物个体发育过程中的作用，目前报道还很少。 为了揭示Ran结合蛋白(Ran binding protein, RanBP)在植物发育过程中的作用，本文通过转基因手段对其功能进行 了研究。在此之前，本实验室已从小麦cDNA文库中成功克隆Ran结合蛋白基因：TaRanBP。该基因cDNA全长1035 bp，编码207个氨基酸。通过农杆菌介导叶圆片法，分别用正义、反义及TaRanBP与GFP融合蛋白等表达载体转化烟草，并成功获得转基因植株。亚细胞定位观察发现TaRanBP蛋白主要定位于细胞质内，尤其是在核膜附近富集。生理学和细胞学等方面的研究分析发现，TaRanBP基因在烟草个体发育过程中产生重要作用。过量表达TaRanBP基因的转基因植株在一定数量上表现出愈合的花冠筒上出现不同程度开裂，花冠筒上有附生舌状花瓣，及带有花瓣状颜 色的花萼等异常花表型。同时，转反义基因在一定程度上促进了转基因植株初生主根的生长（为对照烟草的2.3倍），而转正义基因烟草与对照烟草的初生主根长度差异不明显。用碘化丙锭(Propidium Iodide, PI)进行根部细胞染色。观察发现，不同的转基因烟草与对照烟草之间在根的各个不同形态区域的细胞大小差异不明显，推测根长的差异可能是由于整体细胞数目变化的原因导致。向重力性实验发现，转反义基因烟草幼苗较对照烟草的向重力性反应增加，而转正义基因的则表现为降低。激素吲哚乙酸(Indoleacetic Acid, IAA)的添加处理可以恢复转反义基因烟草的向重力性异常表型，而对转正义基因烟草几乎无影响。添加激动素(Kinetin, KT)的处理发现不同转基因烟草和对照烟草的向重力性均有减弱。观测后期，转正义基因的向重力敏感性较对照烟草得到恢复。测量不同转基因株系T1代幼苗鲜重，发现不同转基因烟草和对照烟草的幼苗鲜重动态变化在各个时间点有差异，且差异情况不尽相同。而不同转基因幼苗T1代幼苗可溶性蛋白含量较对照烟草有不同程度的下降。这种下降并没有影响转基因烟草的整体生长进程，开花期和结实情况与对照烟草相比也无明显变化。

The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 protein is a phototropin signaling element that regulates leaf flattening and leaf positioning.

In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1-PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.