Form and function of arabinogalactans and arabinogalactan-proteins

The occurrence, isolation, chemistry and physico-chemistry of plant arabino-3,6-galactans and arabino-3,6-galactan-proteins is reviewed. The structural relationships between arabino-3,6-galactans from gymnosperm wood, gum exudates of Acacia and other trees, and from plant callus cells and whole tissues are discussed. The nature of these proteoglycans is compared with the arabinose and galactose containing cell wall glycoproteins. Interactions of the arabino-3,6-galactan proteoglycans with carbohydrate binding proteins and with Yariv antigens are described. The utility of these reactions for both cellular and subcellular localization of the proteoglycans is discussed. The possible biological roles of the arabinogalactans and the arabinogalactan-proteins are reviewed.     

A decade of progress in understanding vitamin E synthesis in plants

The chloroplasts of higher plants contain and elaborate many unique biochemical pathways that produce an astonishing array of compounds that are vital for plastid function and are also important from agricultural and nutritional perspectives. One such group of compounds is the tocochromanols (more commonly known as Vitamin E), which is a class of four tocopherols and four toctorienols, lipid-soluble antioxidants that are only synthesized by plants and other oxygenic, photosynthetic organisms. Though the essential nature of tocopherols in mammalian diets was recognized over 80 years ago and the biosynthetic pathway in plants and algae elucidated in the late 1970s and early 80s, it has only been in the past decade that the genes and proteins for tocopherol synthesis have finally been isolated and characterized. The use of model plant and cyanobacterial systems has driven this gene discovery to the point that manipulation of tocopherol levels and types in various plant tissues and crops is becoming a reality. This article reviews progress since 1996 in the molecular and genetic understanding of tocopherol synthesis in the model photosynthetic organisms Arabidopsis thaliana and Synechocystis PCC6803 as a primer for current and future efforts to manipulate the levels of this essential nutrient in food crops by breeding and transgenic approaches.     

The Cell Wall of Fusarium oxysporum

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50–60% of the total mass of the wall. X-ray diffraction studies showed the presence of α-1,3-glucan in the alkali-soluble cell wall fraction and of β-1,3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.     

The prolonged depolarizing afterpotential and its contribution to the understanding of photoreceptor function

Comparison of flies bred on vitamine A-poor and vitamine A-rich diets show the latter to exhibit, after blue illumination, 1) slight deviation from the linear relationship between stimulus intensity and receptor sensitivity and, 2) after intense blue illumination the phenomenon of the PDA. Both these effects could result from reduced pigment distances in such membranes. Maximum PDA was produced after about 20 s of illumination with blue light, and following this the resistance of the membrane was seen to stay low, returning to the resting value at the same rate as the PDA decline. The response to test flashes, repressed during illumination, gradually returned during the decline of the PDA, similar to the way the photoreceptor would respond to the sum of two stimuli: the test flash and a decreasing background illumination. Red light immediately following blue abolished the PDA and white light produced a small PDA. All these experiments corroborate a new model (without resorting to the concept of inhibitors) which links the photopigments with receptor excitation, the assumptions for which are the following: 1) PDA is produced after abnormally high primary quantum absorption by rhodopsin molecules, 2) PDA is a retarded membrane excitation by a substance in stored form, 3) the store is built up when production of this substance is larger than its consumption, and 4) time and energy are necessary for the regeneration of excitatory rhodopsin molecules.This work was supported by the DFG (Ha 258/10) and by the SFB 114Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

The role of calmodulin in the gravitropic response of the Arabidopsis thaliana agr-3 mutant

Calmodulin, a primary plant calcium receptor, is known to be intimately involved with gravitropic sensing and transduction. Using the calmodulin-binding inhibitors trifluoperazine, W7 and calmidazolium, gravitropic curvature of Arabidopsis thaliana (L.) Heynh, ecotype Landsberg, roots was separable into two phases. Phase I was detected at very low concentrations (0.01 μM) of trifluoperazine and calmidazolium, did not involve growth changes, accounted for about half the total curvature of the root and may represent the specific contribution of the cap to gravity sensing. Phase II commenced around 1.0 μM and involved inhibition of both growth and curvature. The agr-3 mutant exhibited a reduced gravitropic response and was found to lack phase I curvature, suggesting that the mutation alters either use or expression of calmodulin. The sequences of wild-type and agr-3 calmodulin (CaM-1) cDNAs, which are root specific were completely determined and found to be identical. Upon gravitropic stimulation, wild-type Arabidopsis seedlings increased calmodulin mRNA levels by threefold in 0.5 h. On the other hand, gravitropic stimulation of agr-3 decreased calmodulin mRNA accumulation. The possible basis of the two phases of curvature is discussed and it is concluded that agr-3 has a lesion located in a general gravity transmission sequence, present in many root cells, which involves calmodulin mRNA accumulation.     

Acclimation of Arabidopsis thaliana to the light environment: changes in photosynthetic function

Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta was grown under light regimes of differing spectral qualities, which results in differences in the stoichiometries of the two photosynthetic reaction centres. The acclimative value of these changes was investigated by assessing photosynthetic function in these plants when exposed to two spectrally distinct actinic lights. Plants grown in an environment enriched in far-red light were better able to make efficient use of non-saturating levels of actinic light enriched in long-wavelength red light. Simultaneous measurements of chlorophyll fluorescence and absorption changes at 820 nm indicated that differences between plants grown under alternative light regimes can be ascribed to imbalances in excitation of photosystems I and II (PSI, PSII). Measurements of chlorophyll fluorescence emission and excitation spectra at 77 K provided strong evidence that there was little or no difference in the composition or function of PSI or PSII between the two sets of plants, implying that changes in photosynthetic stoichiometry are primarily responsible for the observed differences in photosynthetic function.Abbreviations Chl chlorophyll - FR far-red light - HF highirradiance FR-enriched light (400 mol·m^–2·s^–1, RFR = 0.72) - HW high-irradiance white light (400 mol·m^–2 1·1 s^–1RFR = 1.40) - LHCI, LHCII light-harvesting complex of PSI, PSII - qO quenching of dark-level chlorophyll fluorescence - qN non-photochemical quenching of variable chlorophyll fluorescence - qP photochemical quenching of variable chlorophyll fluorescence - R red light - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Dr. Sasha Ruban for assistance with the 77 K fluorescence measurements and for helpful discussions. This work was supported by Natural Environment Research Council Grant GR3/7571A.     

Comprehensive approach to genes involved in cell wall modifications in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The plant cell wall is of supermolecular architecture, and is composed of various types of heterogeneous polymers. A few thousand enzymes and structural proteins are directly involved in the construction processes, and in the functional aspects of the dynamic architecture in Arabidopsis thaliana. Most of these proteins are encoded by multigene families, and most members within each family share significant similarities in structural features, but often exhibit differing expression profiles and physiological functions. Thus, for the molecular dissection of cell wall dynamics, it is necessary to distinguish individual members within a family of proteins. As a first step towards characterizing the processes involved in cell wall dynamics, we have manufactured a gene-specific 70-mer oligo microarray that consists of 765 genes classified into 30 putative families of proteins that are implicated in the cell wall dynamics of Arabidopsis. By using this array system, we identified several sets of genes that exhibit organ preferential expression profiles. We also identified gene sets that are expressed differentially at certain specific growth stages of the Arabidopsis inflorescence stem. Our results indicate that there is a division of roles among family members within each of the putative cell wall-related gene families.     

The function of male aggressive displays towards females in mountain gorillas

In groups ofGorilla g. beringei, male aggression towards females regularly takes the form of male display. This paper examines male display towards females in two Karisoke study groups (Group 5 and Group BM) in 1989, a period when none of the females were new immigrants. Results are based on 259 hr of focal observations and 121 hr ofad libitum observations on male behaviors towards females. The goal is to see if the data are compatible with four non-mutually exclusive hypotheses to explain male displays towards females: (1) demonstration of male fighting abilities to influence female long term residence decision; (2) decrease potential competitive inequities between females; (3) provision to females of an occasion to confirm their subordinance to a male; and (4) short term influence on mating. First, male-female proximity was tested against proportion of male displays, to rule out the possibility that males display towards females simply because they happen to be close by. There was no association between proximity and male display. Dominant males were responsible for a higher proportion of displays than subordinate males. This is consistent with the idea that males display to demonstrate their fighting abilities, or their qualities as protector, since dominant males are the ones offering long term protection against infanticide and predators. Females that were in a position to transfer did not receive a higher proportion of male display, however. Long term resident dominant females received a higher proportion of displays from the dominant males, which is consistent with the idea that males attempt to decrease potential competitive inequities between females. There was an association between female appeasement reactions and male displays, which suggests that males display to create occasions for the females to confirm their subordinance to them. Estrous females did not receive a higher proportion of male displays, and there was no association between male display and copulation, suggesting that male displays are not a form of courtship aggression aimed at influencing mating in the short term.     

Cell-wall-bound lytic activity in Chlorella fusca: function and characterization of an endo-mannanase

A cell-wall-degrading activity was solubilized from young cells and from mother cell walls of Chlorella fusca by treatment with LiCl. The cytoplasmic enzyme hexokinase was not detectable in these extracts. The LiCl-solubilized activity increased in the cell cycle parallel to the release of autospores. The enzyme was purified on a chromatofocusing column followed by gel filtration. Sodium dodecyl sulfate/polyacryl amide gel electrophoresis of the purified enzyme revealed a molecular weight of 44 kDa, whereas gel filtration indicated a molecular weight of 25 kDa. Cell-wall-lytic activity and -1,4-mannanase activity coeluted in gel filtration and were separated from -d-fucosidase activity. The enzyme degraded isolated cell walls and ivory nut mannan primarily to oligosaccharides with an estimated degree of polymerization 6. The soluble degradation products of the cell wall consisted of 92–96% mannose and 4–8% glucose. It is concluded that the cell-wall-lytic activity is caused by an endo-mannanase. In vivo, this enzyme probably degrades the mother cell wall and, after autospore release, remains bound to it as well as to the surface of the daughter cells by ionic forces. The identity of this bound enzyme with a soluble wall-degrading enzyme previously obtained from mother cells is discussed.     

Responses to iron deficiency in Arabidopsis thaliana: The Turbo iron reductase does not depend on the formation of root hairs and transfer cells

Arabidopsis thaliana (L.) Heynh. Columbia wild type and a root hair-less mutant RM57 were grown on iron-containing and iron-deficient nutrient solutions. In both genotypes, ferric chelate reductase (FCR) of intact roots was induced upon iron deficiency and followed a Michaelis-Menten kinetic with a K _m of 45 and 54 M Fe^III-EDTA and a V _max of 42 and 33 nmol Fe²⁺·(g FW)^–1·min^–1 for the wild type and the mutant, respectively. The pH optimum for the reaction was around pH 5.5. The approximately four fold stimulation of FCR activity was independent of formation of root hairs and/or transfer cells induced by iron deficiency. Iron-deficiency-induced chlorosis and the development of a rigid root habit disappeared when ferric chelate was applied to the leaves, while FCR activity remained unchanged. The time course of the responses to iron deficiency showed that morphological and physiological responses were controlled separately.Abbreviations FCR ferric chelate reductase - FW fresh weight Thanks are due to Klaas Sjollema (Department of Electronmicroscopy, University of Groningen, The Netherlands) for help with the electron microscopy sample preparation and especially to Dr. Uwe Santore (Heinrich-Heine-University for electron microscopy. This work was supported by the SCIENCE programm of the European community; P.R.M.) and a Personal Research Grant by the Ministerium für Wissenschaft und Forschung of Nordrhein-Westfalen (P.R.M.) and last, not least by the productive discussions in ECOTRANS B.V.     

Reduction in vacuolar volume in the tapetal cells coincides with conclusion of the tetrad stage in Arabidopsis thaliana

Although the tapetum is known for its role in the removal of the tetrad wall, a morphological change in the tapetum correlating with such a role has not been described. Here we report that in two ecotypes of Arabidopsis thaliana, Landsberg erecta and Columbia, the vacuoles in tapetal cells underwent progressive enlargement prior to the separation of tetrads but became drastically reduced when tetrads just separated from one another. Such a drastic change in vacuolar volume was not observed in later anther development. We also observed that the walls of associated tetrads were much less stained with Toluidine Blue O than the walls of separate tetrads, indicating that the tetrad walls underwent an alteration during the tetrad stage. Furthermore, we identified the N-terminal propeptide signals for sorting vacuolar proteins in 15 β-1,3-glucanases, five polygalacturonases, and two endocellulases that are expressed in Arabidopsis young floral buds; all three types of the enzymes are known to participate in degradation of the tetrad wall. These results suggest that the tapetal vacuoles might be a storage site for these enzymes prior to their secretion to the anther locule.     

The Role of GIGANTEA Gene in Mediating the Oxidative Stress Response and in Arabidopsis

The Arabidopsis GIGANTEA (GI) gene has been shown to be involved in the regulation of the oxidative stress response; however, little is known about the mechanism by which GI gene regulates the oxidative stress response. We show here that enhanced tolerance of the gi-3 mutant to oxidative stress is associated, at least in part, with constitutive activation of superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes. The gi-3 plants were more tolerant to parquart (PQ) or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of concentrations of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of gi-3 plants to oxidative stress was not due to defects in the uptake of PQ or the sequestration of PQ from its site of action, and that the gi-3 mutation alleviated oxidative damage of plant cells from PQ stress. Moreover, the gi-3 mutant showed constitutive activation of cytosolic Cu/ZnSOD and plastidic FeSOD as well as cytosolic APX1 and stromal APX genes, which at least in part contributed to constitutive increases in activities of anti-oxidative enzymes SOD and APX, respectively. To our knowledge, we demonstrate, for the first time, that GI gene regulates the oxidative stress response, at least in part, through modulation of SOD and APX genes.     

Proteomic Analysis of Glutathione S-Transferases of Arabidopsis thaliana Reveals Differential Salicylic Acid-Induced Expression of the Plant-Specific Phi and Tau Classes

Plant glutathione S -transferases (GSTs) are a large group of multifunctional proteins that are induced by diverse stimuli. Using proteomic approaches we identified 20 GSTs at the protein level in Arabidopsis cell culture with a combination of GST antibody detection, LC-MS/MS analysis of 23-30 kDa proteins and glutathione-affinity chromatography. GSTs identified were from phi, tau, theta, zeta and DHAR sub-sections of the GST superfamily of 53 members. We have uncovered preliminary evidence for post-translational modifications of plant GSTs and show that phosphorylation is unlikely to be responsible. Detailed analysis of GST expression in response to treatment with 0.01-1 mM of the plant defence signal salicylic acid (SA) uncovered some interesting features. Firstly, GSTs appear to display class-specific concentration-dependent SA induction profiles highlighting differences between the large, plant specific phi and tau classes. Secondly, different members of the same class, while sharing similar SA dose responses, may display differences in terms of magnitude and timing of induction, further highlighting the breadth of GST gene regulation. Thirdly, closely related members of the same class ( GSTF6 and GSTF7 ), arising via tandem duplication, may be regulated differently in terms of basal expression levels and also magnitude of induction raising questions about the role of subfunctionalisation within this family. Our results reveal that GSTs exhibit class specific responses to SA treatment suggesting that several mechanisms are acting to induce GSTs upon SA treatment and hinting at class-specific functions for this large and important, yet still relatively elusive gene family.     

Utility of Amplified Fragment Length Polymorphisms (AFLP) to analyse genetic structures within the Alexandrium tamarense species complex

Phylogenetic analyses of the Alexandrium tamarense species complex using ribosomal RNA sequences show a differentiation of ribotypes/clades into geographic areas and not into the three morphotypes/species A. tamarense, A. fundyense and A. catenella. Different parts of the rRNA operon have proven informative in revealing the existence and the relationships of these geographic clades, whereas even internal transcribed spacer (ITS) regions lack the resolution required to gain a deeper insight into the population structure of the species complex. Here, the utility of the DNA fingerprinting technique Amplified Fragment Length Polymorphism (AFLP) as a possible tool for such purposes was tested. A mixed sampling strategy was used in order to assess the amount of variation of AFLP banding patterns at the level of populations and geographic clades. We also describe optimized methods to achieve a good reproducibility. Our results suggest that AFLPs can provide useful information at the population level using clonal samples from a certain bloom, whereas the amount of variation that we found is too high to allow for meaningful comparisons of a few strains collected from different localities at different time points even though they belong to one geographic clade.     

Extension-growth responses and expression of flavonoid biosynthesis genes in the Arabidopsis hy4 mutant

The hy4 mutant of Arabidopsisthaliana(L.) Heynh. was previously shown to be impaired in the suppression of hypocotyl extension specifically by blue light. We report here that hy4 is altered in a range of blue-light-mediated extension-growth responses in various organs in seedlings and mature plants: it shows greater length of bolted stems, increased petiole extension and increased leaf width and area in blue light compared to the wild type. The hy4 mutant shows decreased cotyledon expansion in both red and blue light compared to the wild type. Anthocyanin formation and the expression of several flavonoid biosynthesis genes is stimulated by blue light in the wild type but to a much lower extent in hy4. The results indicate that the HY4 gene product is concerned with the perception of blue light in a range of extension-growth and gene-expression responses in Arabidopsis.Abbreviations DFR dihydroflavonol reductase - CHS chalcone synthase - CHI chalcone isomeraseWe thank the UK Agricultural and Food Research Council for supporting this work through the award of a research grant to G.I.J. We are grateful to Robert Brown for excellent technical assistance and Drs B.W. Shirley (Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, USA), C.D. Silflow (Department of Genetics and Cell Biology, University of Minnesota, St. Paul, USA) and I.E. Somssich (Department of Biochemistry, Max-Planck-Institut, Köln, Germany) for providing plasmid DNA.     

Structural genomics as an approach towards understanding the biology of tuberculosis

Tuberculosis (TB) is a devastating disease of worldwide importance. The availability of the genome sequence of Mycobacterium tuberculosis (Mtb), the causative agent, has stimulated a large variety of genome-scale initiatives. These include international structural genomics efforts which have the dual aim of characterising potential new drug targets and addressing key aspects of the biology of Mtb. This review highlights the various ways in which structural analysis has illuminated the biological activities of Mtb gene products, which were previously of unknown or uncertain function. Key information comes from the protein fold, from bound ligands, solvent molecules, ions etc. or from unexpectedly modified amino acid residues. Most importantly, the three dimensional structure of a protein permits the integration of data from many sources, both bioinformatic and experimental, to develop testable functional hypotheses. This has led to many new insights into TB biology.