Auxin-Growth Relationships in Maize Coleoptiles and Pea Internodes and Control by Auxin of the Tissue Sensitivity to Auxin

Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied 3H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants.

Dim-Red-Light-Induced Increase in Polar Auxin Transport in Cucumber Seedlings1 : I. Development of Altered Capacity, Velocity, and Response to Inhibitors

We have developed and characterized a system to analyze light effects on auxin transport independent of photosynthetic effects. Polar transport of [3H]indole-3-acetic acid through hypocotyl segments from etiolated cucumber (Cucumis sativus L.) seedlings was increased in seedlings grown in dim-red light (DRL) (0.5 μmol m−2 s−1) relative to seedlings grown in darkness. Both transport velocity and transport intensity (export rate) were increased by at least a factor of 2. Tissue formed in DRL completely acquired the higher transport capacity within 50 h, but tissue already differentiated in darkness acquired only a partial increase in transport capacity within 50 h of DRL, indicating a developmental window for light induction of commitment to changes in auxin transport. This light-induced change probably manifests itself by alteration of function of the auxin efflux carrier, as revealed using specific transport inhibitors. Relative to dark controls, DRL-grown seedlings were differentially less sensitive to two inhibitors of polar auxin transport, N-(naphth-1-yl) phthalamic acid and 2,3,5-triiodobenzoic acid. On the basis of these data, we propose that the auxin efflux carrier is a key target of light regulation during photomorphogenesis.

Alteration of Hormone Levels in Transgenic Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1

The rice (Oryza sativa L.) homeobox gene OSH1 causes morphological alterations when ectopically expressed in transgenic rice, Arabidopsis thaliana, and tobacco (Nicotiana tabacum L.) and is therefore believed to function as a morphological regulator gene. To determine the relationship between OSH1 expression and morphological alterations, we analyzed the changes in hormone levels in transgenic tobacco plants exhibiting abnormal morphology. Levels of the plant hormones indole-3-acetic acid, abscisic acid, gibberellin (GA), and cytokinin (zeatin and trans-zeatin [Z]) were measured in leaves of OSH1-transformed and wild-type tobacco. Altered plant morphology was found to correlate with changes in hormone levels. The more severe the alteration in phenotype of transgenic tobacco, the greater were the changes in endogenous hormone levels. Overall, GA1 and GA4 levels decreased and abscisic acid levels increased compared with wild-type plants. Moreover, in the transformants, Z (active form of cytokinin) levels were higher and the ratio of Z to Z riboside (inactive form) also increased. When GA3 was supplied to the shoot apex of transformants, internode extension was restored and normal leaf morphology was also partially restored. However, such GA3-treated plants still exhibited some morphological abnormalities compared with wild-type plants. Based on these data, we propose the hypothesis that OSH1 affects plant hormone metabolism either directly or indirectly and thereby causes changes in plant development.

Hormonal Control of Parthenocarpic Ovary Growth by the Apical Shoot in Pea1

The role of the apical shoot as a source of inhibitors preventing fruit growth in the absence of a stimulus (e.g. pollination or application of gibberellic acid) has been investigated in pea (Pisum sativum L.). Plant decapitation stimulated parthenocarpic growth, even in derooted plants, and this effect was counteracted by the application of indole acetic acid (IAA) or abscisic acid (ABA) in agar blocks to the severed stump. The treatment of unpollinated ovaries with gibberellic acid blocked the effect of IAA or ABA applied to the stump. [3H]IAA and [3H]ABA applied to the stump were transported basipetally, and [3H]ABA but not [3H]IAA was also detected in unpollinated ovaries. The concentration of ABA in unpollinated ovaries increased significantly in the absence of a promotive stimulus. The application of IAA to the stump enhanced by 2- to 5-fold the concentration of ABA in the inhibited ovary, whereas the inhibition of IAA transport from the apical shoot by triiodobenzoic acid decreased the ovary content of ABA (to approximately one-half). Triiodobenzoic acid alone, however, was unable to stimulate ovary growth. Thus, in addition to removing IAA transport from the apical shoot, the accumulation of a promotive factor is also necessary to induce parthenocarpic growth in decapitated plants.

Higher Activity of an Aldehyde Oxidase in the Auxin-Overproducing superroot1 Mutant of Arabidopsis thaliana1

Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1–3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings.

Dendrimer-supported combinatorial chemistry.

A new methodology for the construction of combinatorial libraries is described. The approach, termed dendrimer-supported combinatorial chemistry (DCC), centers on the use of dendrimers as soluble supports. Salient features of DCC include solution phase chemistry, homogeneous purification, routine characterization of intermediates, and high support loadings. To demonstrate the feasibility of DCC, single compounds and a small combinatorial library were prepared via the Fischer indole synthesis. Excellent product yields and purities were obtained, and dendrimer-protected intermediates could be routinely analyzed by 1H and 13C NMR and by mass spectrometry. The results indicate that DCC is a general and efficient strategy for the generation of combinatorial libraries.

Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesicle-like structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.

Auxin as a positional signal in pattern formation in plants.

By using a novel, extremely sensitive and specific gas chromatography-mass spectrometry technique we demonstrate in Pinus sylvestris (L.) trees the existence of a steep radial concentration gradient of the endogenous auxin, indole-3-acetic acid, over the lateral meristem responsible for the bulk of plant secondary growth, the vascular cambium. This is the first evidence that plant morphogens, such as indole-3-acetic acid, occur in concentration gradients over developing tissues. This finding gives evidence for a regulatory system in plants based on positional signaling, similar to animal systems.

Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate.

A minor groove binder (MGB) derivative (N-3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate tripeptide; CDPI3) was covalently linked to the 5' or 3' end of several oligodeoxyribonucleotides (ODNs) totally complementary or possessing a single mismatch to M13mp19 single-stranded DNA. Absorption thermal denaturation and slot-blot hybridization studies showed that conjugation of CDPI3 to these ODNs increased both the specificity and the strength with which they hybridized. Primer extension of the same phage DNA by a modified form of phage T7 DNA polymerase (Sequenase) was physically blocked when a complementary 16-mer with a conjugated 5'-CDPI3 moiety was hybridized to a downstream site. Approximately 50% of the replicating complexes were arrested when the blocking ODN was equimolar to the phage DNA. Inhibition was unaffected by 3'-capping of the ODN with a hexanol group or by elimination of a preannealing step. Blockage was abolished when a single mismatch was introduced into the ODN or when the MGB was either removed or replaced by a 5'-acridine group. A 16-mer with a 3'-CDPI3 moiety failed to arrest primer extension, as did an unmodified 32-mer. We attribute the exceptional stability of hybrids formed by ODNs conjugated to a CDPI3 to the tethered tripeptide binding in the minor groove of the hybrid. When that group is linked to the 5' end of a hybridized ODN, it probably blocks DNA synthesis by inhibiting strand displacement. These ODNs conjugated to CDPI3 offer attractive features as diagnostic probes and antigene agents.

Transition metal ion activation of DNA binding by the diphtheria tox repressor requires the formation of stable homodimers.

The diphtheria tox repressor (DtxR) is a transition metal ion-dependent regulatory element that controls the expression of diphtheria toxin and several genes involved in the synthesis of siderophores in Corynebacterium diphtheriae. In the presence of transition metal ions apo-DtxR becomes activated and specifically binds to its target DNA sequences. We demonstrate by glutaraldehyde cross-linking that monomeric apo-DtxR is in weak equilibrium with a dimeric form and that upon addition of activating metal ions to the reaction mixture a dimeric complex is stabilized. Addition of the DNA-binding-defective mutant apo-DtxR(delta 1-47) to apo-DtxR in the absence of transition metal ions inhibits conversion of the apo-repressor to its activated DNA-binding form. We also show that the binding of Ni2+ to both apo-DtxR and apo-DtxR(delta 1-47) is cooperative and that upon ion binding there is a conformational change in the environment of the indole ring moiety of Trp-104. For the wild-type repressor the consequences of this conformational change include a shift in equilibrium toward dimer formation and activation of target DNA binding by the repressor. We conclude that the formation of DtxR homodimers is mediated through a protein-protein interaction domain that is also activated on metal ion binding.

Grasshopper crop and midgut extract effects on plants: an example of reward feedback.

Acid extracts and a resultant fraction from solid-phase extraction (SPE) of Romalea guttata crop and midgut tissues induce sorghum (Sorghum bicolor var. Rio) coleoptile growth in 24-h incubations an average of 49% above untreated controls. When combined with plant auxin, indole-3-acetic acid (IAA), the SPE fraction shows a synergistic reaction, yielding increases in coleoptile growth that average 295% above untreated controls and 8% above IAA standards. The interaction lowered the point of maximum sensitivity of IAA 3 orders of magnitude, resulting in a new IAA physiological set point at 10(-7) g/ml. This synergism suggests that contents in animal regurgitants making their way into plant tissue during feeding may produce a positive feedback in plant growth and development following herbivory. Such a process, also known as reward feedback, may exert major controls on ecosystem-level relationships in nature.

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.

Estudo da cinética da tirosinase imobilizada em nanopartícula de sílica com obtenção de revestimento de eumelanina

Melanina é um polímero constituído por uma grande heterogeneidade de monômeros tendo como característica comum a presença de grupos indóis. Por outro lado, a eumelanina produzida pela oxidação enzimática da tirosina é um polímero mais simples constituído principalmente de monômeros 5,6-dihidroxindol (DHI) e de indol-5,6-quinona (IQ). Tirosinase é a enzima chave na produção de melanina, sendo que a sua atividade cinética é medida em função da formação do intermediário dopacroma. Nanopartículas (NPs) de sílica são partículas nanométricas compostas de oxido de silício e são obtidas pelo processo sol-gel desenvolvido por Stöber de hidrólise e condensação de tetraetilortosilicato (TEOS), usando etanol como solvente em meio alcalino. As NPs foram funcionalizadas com 3-Aminopropiltrietoxissilano (ATPES) e depois com glutaraldeído. Este último permitiu a imobilização da tirosinase na superfície da sílica. Caracterizamos as NPs antes e após a reação da enzima, a atividade catalítica da enzima ligada à NP e o mecanismos de formação de melanina na superfície da sílica. As NPs foram caracterizadas por espectrofotometria de absorção e de reflectância, termogravimetria e microscopia eletrônica. A síntese da NP de sílica retornou partículas esféricas com 55nm de diâmetro e a funcionalização da partícula mostrou modificar eficientemente a sua superfície. A imobilização da tirosinase por ligação covalente foi de 99,5% contra 0,5% da adsorção física. A atividade da tirosinase foi caracterizada pela formação de dopacroma. O Km da enzima imobilizada não sofreu alteração em comparação com a tirosinase livre, mas a eficiência catalítica - que considera a eficiência recuperada - foi de apenas 1/3 para a enzima ligada covalentemente, significando que 2/3 das enzimas ligadas não estão ativas. Obtivemos NPs revestidas com melanina a partir de oxidação de tirosina solubilizada em duas preparações: NP com tirosinase ligada covalentemente na superfície e NP funcionalizada com glutaraldeido dispersa em solução de DHI e IQ. O revestimento de melanina foi na forma de um filme fino com espessura ~1,9nm, conferindo perfil de absorção luminosa equivalente ao da própria melanina. Mostramos que o mecanismo de polimerização passa pela oxidação da tirosina pela tirosinase, que gera intermediários oxidados (principalmente DHI e IQ) que vão para solução (mesmo quando a tirosinase está ligada covalentemente na sílica). Estes intermediários ligam-se ao glutaraldeido e a superfície da sílica passa a funcionar como ambiente de polimerização da melanina.

Análise química e atividades biológicas de Guatteria elliptica R. E. Fries (Annonaceae)

A espécie endêmica G. elliptica R. E. Fries não apresentava estudos fitoquímicos e biológicos detalhados na literatura. Assim, o objetivo desse trabalho foi avaliar a composição química e as propriedades biológicas dos óleos essenciais, extratos brutos, alcaloides totais, tortas, frações das tortas, amostras isoladas dessa espécie. O material vegetal foi coletado em Paranapiacaba (Santo André, SP, Brasil). O óleo essencial extraído das folhas por destilação à vapor apresentou um rendimento de 0,2%. A análise histológica das folhas encontrou óleo em células oleíferas localizadas no parênquima esponjoso. A composição do óleo (CG-EM) indicou espatulenol e óxido de cariofileno como compostos majoritários. Os alcaloides totais foram obtidos dos extratos brutos das folhas e dos galhos e analisados por CG-EM, identificando quatro aporfinas (nornuciferina, estefarina, corituberina e asimilobina) e duas protoberberinas (discretamina e caseadina). Os alcaloides totais foram fracionados em coluna cromatográfica ou por Extração em Fase Sólida e purificados por cromatografia em camada preparativa, originando duas amostras (Amostra 9 e 10). Na Amostra 9, foram identificados dois alcaloides aporfinicos nornuciferina e asimilobina (CG-EM e RMN-1H). Na Amostra 10, foram identificados (LC-EM/EM) cinco alcaloides aporfínicos (desidronantenina, glaunidina, liriodenina, N-óxido de oliverina e telikovina) e um alcaloide protoberberínico (caseadina). Caseadina, glaunidina, N-óxido de oliverina e telikovina não foram previamente identificados em Guatteria. Os resíduos dos extratos brutos livres de alcaloides foram fracionados pelo método de partição com solventes de polaridade crescente. Os extratos brutos e as frações acetato de etila e butanólicas de folhas e galhos apresentaram flavonoides (NP-PEG). Nos ensaios biológicos, a melhor atividade antioxidante (sequestro do radical DPPH) foi encontrada para a fração clorofórmica dos galhos (EC50=24,25±1,14 µg/mL) e a torta dos galhos (EC50=26,23±4,20 µg/mL). No ensaio antimicrobiano pelo método turbidimétrico a atividade mais importante foi obtida contra Staphylococcus aureus (ATCC 6538) para os alcaloides totais dos galhos (CIM/CBM=0,12±0,01/0,26 mg/mL) e das folhas (CIM/CBM=0,21±0,01/0,28 mg/mL), e fração hexânica das folhas (CIM/CBM=0,24±0,02/>1 mg/mL). Uma alta atividade antitumoral foi observada frente a células humanas de mama (MCF-7) para Amostra 10 (IC50=2,28±0,18 µg/mL), fração de acetato de etila das folhas (IC50=4,47±0,40 µg/mL), óleo essencial (IC50=7,01±0,23 µg/mL) e os alcaloides totais das folhas (IC50=9,32±0,36 µg/mL). Para as células de próstata (PC-3), foi encontrada atividade para a Amostra 10 (IC50=1,37±0,36 µg/mL) e o óleo essencial (IC50=5,32±0,35 µg/mL). A futura aplicação dos extratos e frações de G. elliptica como um agente antitumoral parece ser segura, pois mantiveram uma viabilidade celular maior do que 90% no ensaio de citotoxicidade com culturas de fibroblasto murino (BALB/c 3T3, ATCC CCL-163) nas concentrações onde a atividade antitumoral foi promissora (<30 µg/mL) contra MCF-7 e/ou PC-3.

Biogeographical Patterns and Phenological Changes in Lapiedra martinezii Lag. Related to Its Alkaloid Diversity

The aim of this work was to investigate the alkaloid patterns of Lapiedra martinezii and their relation to biogeography and phenology focused in a phylogenetic comparison. Plants from 14 populations of L. martinezii, covering almost its entire distribution area, were subjected to morphological, ecological, and phytochemical analysis. Experiments for different alkaloid-type content are proposed as a new tool for analysis of plant distribution. Several plants were transplanted for weekly observation of their phenological changes, and alkaloids from different plant organs were extracted, listed, and compared. The alkaloid pattern of L. martinezii comprises 49 compounds of homolycorine, lycorine, tazettine, haemantamine, and narciclasine types. The populations located in the north and south margins of the distribution area displayed alkaloid patterns different from those of the central area. Changes in these patterns during their phenological cycle may be related to a better defence for plant reproduction. L. martinezii is an old relict plant, and it has maintained some of the more primitive morphological features and alkaloid profiles of the Mediterranean Amaryllidaceae. The variations in alkaloid content observed could be interpreted in a phylogenetic sense, and those found in their phenological changes, in an adaptive one.