Purification and Characterization of NADP+-Linked Isocitrate Dehydrogenase from Scots Pine1 : Evidence for Different Physiological Roles of the Enzyme in Primary Development

NADP+-isocitrate dehydrogenase (NADP+-IDH; EC 1.1.1.42) is involved in the supply of 2-oxoglutarate for ammonia assimilation and glutamate synthesis in higher plants through the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Only one NADP+-IDH form of cytosolic localization was detected in green cotyledons of pine (Pinus spp.) seedlings. The pine enzyme was purified and exhibited molecular and kinetic properties similar to those described for NADP+-IDH from angiosperm, with a higher catalytic efficiency (105 m−1 s−1) than the deduced efficiencies for GS and GOGAT in higher plants. A polyclonal antiserum was raised against pine NADP+-IDH and used to assess protein expression in the seedlings. Steady-state levels of NADP+-IDH were coordinated with GS during seed germination and were associated with GS/GOGAT enzymes during chloroplast biogenesis, suggesting that NADP+-IDH is involved in the provision of carbon skeletons for the synthesis of nitrogen-containing molecules. However, a noncoordinated pattern of NADP+-IDH and GS/GOGAT was observed in advanced stages of cotyledon development and in the hypocotyl. A detailed analysis in hypocotyl sections revealed that NADP+-IDH abundance was inversely correlated with the presence of GS, GOGAT, and ribulose-1,5-bisphosphate carboxylase/oxygenase but was associated with the differentiation of the organ. These results cannot be explained by the accepted role of the enzyme in nitrogen assimilation and strongly suggest that NADP+-IDH may have other, as-yet-unknown, biological functions.

Hexose Transport in Growing Petunia Pollen Tubes and Characterization of a Pollen-Specific, Putative Monosaccharide Transporter1

We investigated the molecular and physiological processes of sugar uptake and metabolism during pollen tube growth and plant fertilization. In vitro germination assays showed that petunia (Petunia hybrida) pollen can germinate and grow not only in medium containing sucrose (Suc) as a carbon source, but also in medium containing the monosaccharides glucose (Glc) or fructose (Fru). Furthermore, high-performance liquid chromatography analysis demonstrated a rapid and complete conversion of Suc into equimolar amounts of Glc and Fru when pollen was cultured in a medium containing 2% Suc. This indicates the presence of wall-bound invertase activity and uptake of sugars in the form of monosaccharides by the growing pollen tube. A cDNA designated pmt1 (petunia monosaccharide transporter 1), which is highly homologous to plant monosaccharide transporters, was isolated from petunia. Pmt1 belongs to a small gene family and is expressed specifically in the male gametophyte, but not in any other vegetative or floral tissues. Pmt1 is activated after the first pollen mitosis, and high levels of mRNA accumulate in mature and germinating pollen. A model describing the transport of sugars to the style, the conversion of Suc into Glc and Fru, and the active uptake by a monosaccharide transporter into the pollen tube is presented.

Synthesis and Phosphorylation of Maize Acidic Ribosomal Proteins1 : Implications in Translational Regulation

The objective of this research was to determine the role of acidic ribosomal protein (ARP) phosphorylation in translation. Ribosomes (Rbs) from germinated maize (Zea mays L.) axes had four ARP bands within 4.2 to 4.5 isoelectric points when analyzed by isoelectric focusing. Two of these bands disappeared after alkaline phosphatase hydrolysis. During germination a progressive change from nonphosphorylated (0 h) to phosphorylated ARP (24 h) forms was observed in the Rbs; a free cytoplasmic pool of nonphosphorylated ARPs was also identified by immunoblot and isoelectric focusing experiments. De novo ARP synthesis initiated very slowly early in germination, whereas ARP phosphorylation occurred rapidly within this period. ARP-phosphorylated versus ARP-nonphosphorylated Rbs were tested in an in vitro reticulocyte lysate translation system. Greater in vitro mRNA translation rates were demonstrated for the ARP-phosphorylated Rbs than for the non-ARP-phosphorylated ones. Rapamycin application to maize axes strongly inhibited S6 ribosomal protein phosphorylation, but did not interfere with the ARP phosphorylation reaction. We conclude that ARP phosphorylation does not depend on ARP synthesis or on ARP assembly into Rbs. Rather, this process seems to be part of a translational regulation mechanism.

Organ-specific and Agamous-regulated expression and glycosylation of a pollen tube growth-promoting protein.

Transmitting tissue-specific (TTS) protein is a pollen tube growth-promoting and attracting glycoprotein located in the stylar transmitting tissue extracellular matrix of the pistil of tobacco. The TTS protein backbones have a deduced molecular mass of about 28 kDa, whereas the glycosylated stylar TTS proteins have apparent molecular masses ranging between 50 and 100 kDa. TTS mRNAs and proteins are ectopically produced in transgenic tobacco plants that express either a cauliflower mosaic virus (CaMV) 35S promoter-TTS2 transgene or a CaMV 35S-promoter-NAG1 (NAG1 = Nicotiana tabacum Agamous gene) transgene. However, the patterns of TTS mRNA and protein accumulation and the quality of the TTS proteins produced are different in these two types of transgenic plants. In 35S-TTS transgenic plants, TTS mRNAs and proteins accumulate constitutively in vegetative and floral tissues. However, the ectopically expressed TTS proteins in these transgenic plants accumulate as underglycosylated protein species with apparent molecular masses between 30 and 50 kDa. This indicates that the capacity to produce highly glycosylated TTS proteins is restricted to the stylar transmitting tissue. In 35S-NAG transgenic plants, NAG1 mRNAs accumulate constitutively in vegetative and floral tissues, and TTS mRNAs are induced in the sepals of these plants. Moreover, highly glycosylated TTS proteins in the 50- to 100-kDa molecular mass range accumulate in the sepals of these transgenic, 35S-NAG plants. These results show that the tobacco NAGI gene, together with other yet unidentified regulatory factors, control the expression of TTS genes and the cellular capacity to glycosylate TTS proteins, which are normally expressed very late in the pistil developmental pathway and function in the final stage of floral development. The sepals in the transgenic 35S-NAG plants also support efficient pollen germination and tube growth, similar to what normally occurs in the pistil, and this ability correlates with the accumulation of the highest levels of the 50- to 100-kDa glycosylated TTS proteins.

On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.

We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.

Extratos de Ascophyllum nodosum no tratamento de sementes de milho e soja: avaliações fisiológicas e moleculares

A obtenção de uma lavoura com população adequada de plantas depende da utilização de diferentes práticas agronômicas, estando o sucesso condicionado ao uso de sementes de boa qualidade. No entanto, a semeadura dificilmente é realizada em condições ideais o que resulta em problemas na emergência das plantas. Grande é a procura por alternativas que melhorem a germinação e o desenvolvimento inicial dos cultivos resultando em uniformidade de emergência, garantindo o estande e culminando em produção. O tratamento de sementes com agroquímicos fitossanitários é uma solução parcial no combate de agentes fitopatológicos e pragas, mas poucas são as soluções adotadas para melhorar o desenvolvimento vegetal inicial, que pode levar a desuniformidade e falhas no estande gerando prejuízos econômicos. Os extratos de algas já demonstraram em diversos estudos sua eficiência no desenvolvimento vegetal quando aplicados em plantas. Porém poucos são os estudos voltados para os efeitos dos extratos de algas na germinação e emergência. Assim, o intuito deste trabalho foi testar o extrato comercial de Ascophyllum nodosum, e diferentes fracionamentos do mesmo, no tratamento de sementes de soja e milho. Avaliou-se o efeito de diferentes doses no desenvolvimento das plântulas e as doses de melhor resposta foram utilizadas no tratamento de sementes de soja a fim de associar as respostas obtidas à expressão gênica de 9 genes relacionados ao processo germinativo em 24 e 48h de embebição. Sementes de soja tratadas com o extrato comercial resultaram em plântulas menos desenvolvidas o que pode estar relacionado ao alto teor de sais contidos no produto. O tratamento com as demais frações favoreceu o desenvolvimento das plântulas, principalmente o desenvolvimento radicular. Sementes de milho tratadas não apresentaram desenvolvimento tão satisfatório quanto as sementes de soja tratadas. A análise da expressão gênica relativa demonstrou que o tratamento com frações do extrato comercial é capaz de regular algumas vias do metabolismo hormonal, como a isopentenil transferase e a GA20 oxidase 2, e do catabolismo de reservas, como a acil-CoA oxidase. Em condições ótimas, o tratamento de sementes de soja com frações do extrato comercial de A. nodosum favoreceu o desenvolvimento inicial das plântulas de soja, no entanto não ocasionou grandes alterações no desenvolvimento de milho. Este estudo demonstrou a possibilidade de utilização de frações do extrato de A. nodosum no favorecimento do desenvolvimento inicial de plântulas de soja. Maiores estudos são necessários quanto às respostas em campo e na atenuação de estresses para viabilizar seu uso como um bioestimulante em sementes.

Identification of Acremonium isolates from grapevines and evaluation of their antagonism towards Plasmopara viticola

Some endophytic fungal genera in Vitis vinifera, including Acremonium, have been reported as antagonists of Plasmopara viticola. Endophytic Acremonium isolates from an asymptomatic grapevine cultivar Inzolia from Italy were identified by morphological features and multigene phylogenies of ITS, 18S and 28S genes, and their intra-specific genomic diversity was analyzed by RAPD analysis. Culture filtrates (CFs) obtained from Acremonium isolates were tested in vitro for their inhibitory activity against the P. viticola sporangia germination. Among 94 isolates, 68 belonged to the Acremonium persicinum and 26 to the Acremonium sclerotigenum. RAPD analysis grouped the A. persicinum isolates into 15 clusters and defined 31 different strains. The A. sclerotigenum isolates, instead, were clustered into 22 groups and represented 25 strains. All A. persicinum CFs inhibited sporangia germination of P. viticola, while not all those of A. sclerotigenum had inhibitory effect. A different degree of inhibition was observed between strains of the same species, while some strains of different species showed identical inhibitory effect. No correlation was found between RAPD groups and inhibitory activity in both Acremonium species.

The gardener's manual : containing plain instructions for the selection, preparation, and management of a kitchen garden : with practical directions for the cultivation and management of some of the most useful culinary vegetables /

Attributed to Charles F. Crosman. Cf. Richmond, M.L.H. Shaker literature.

Catalogue of seeds collected by the Kentucky Geological Survey at the Centennial Exhibition, Philadelphia, 1876 ...

Mode of access: Internet.

Seed farming in Britain : a practical treatise on the cultivation of vegetables for the production of seeds.

Mode of access: Internet.

Year book and proceedings of the annual convention

1908 report includes Topical index of annual reports from 1883 to 1907 inclusive and Proceedings of Congress of horticulture, Jamestown exposition, Sept., 1907

Gut content analysis to distinguish between seed feeding and mycophagy of a biphyllid beetle larva found on Acacia melanoxylon

In a search for potential biocontrol agents for Acacia melanoxylon R. Br. (Mimosaceae), larvae of the beetle Diplocoelus dilataticollis Lea (Coleoptera; Biphyllidae) were found within damaged seeds of A. melanoxylon. The gut contents of larvae and adults were examined to determine whether their diet included seeds, in apparent contradiction to the known mycophagous diet of members of this family of beetles. Calcofluor M2R White, a plant cell-wall staining optical brightener was used to differentiate between plant cell fragments and fungal tissue in the gut content smears. Gut contents of adults of a known seed predator of A. melanoxylon, a weevil of the genus Melanterius, were examined in the same way to provide a benchmark. The gut contents of D. dilataticollis differed from those of Melanterius sp. Fungal structures and microbes were found in the gut of D. dilataticollis, in contrast to plant cell fragments found in the gut of the weevil and from scrapes made directly from seeds. We conclude that larvae of D. dilataticollis feed primarily on fungi associated with damaged seed and therefore may not be the proximate cause of seed damage.

Structure and folding of potato type II proteinase inhibitors: Circular permutation and intramolecular domain swapping

Potato type II serine proteinase inhibitors are proteins that consist of multiple sequence repeats, and exhibit a multidomain structure. The structural domains are circular permutations of the repeat sequence.. as a result or intramolecular domain swapping. Structural studies give indications for the origins of this folding behaviour, and the evolution of the inhibitor family.

Pollination of Australian Macrozamia cycads (Zamiaceae): Effectiveness and behavior of specialist vectors in a dependent mutualism

Complementary field and laboratory tests confirmed and quantified the pollination abilities of Tranes sp. weevils and Cycadothrips chadwicki thrips, specialist insects of their respective cycad hosts, Macrozamia machinii and M. lucida. No agamospermous seeds were produced when both wind and insects were excluded from female cones; and the exclusion of wind-vectored pollen alone did not eliminate seed set, because insects were able to reach the cone. Based on enclosure pollination tests, each weevil pollinates an average 26.2 ovules per cone and each thrips 2.4 ovules per cone. These pollinators visited similar numbers of ovules per cone in fluorescent dye tests that traced insect movement through cones. Fluorescent dye granules deposited by Cycadothrips were concentrated around the micropyle of each visited ovule, the site of pollen droplet release, where pollen must be deposited to achieve pollination. In contrast, Tranes weevils left dye scattered on different areas of each visited ovule, indicating that chance plays a greater role in this system. Each weevil and 25 thrips delivered 6.2 and 5.2 pollen grains, respectively, on average, to each visited ovule per cone, based on examination of dissected pollen canals. In sum, the pollination potential of 25 Cycadothrips approximates that of one Tranes weevil.

Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by H-1 NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 Angstrom, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H-alpha protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.