Wall morphogenesis in diatoms: Deposition of silica by cytoplasmic vesicles

Summary Several TEM and SEM techniques were applied to examine developing structures in valves of the centric diatomThalassiosira eccentrica (Ehrenb.) Cleve after cytokinesis. It was possible to confirm that in each stage of the silicification process there is a distinction between a growing zone with a loose assemblage of silica spheres and a compacting zone in an older phase of development. The spherical structure of the silica in the growing zone results from the addition of silica by small cytoplasmic vesicles of about 300 to 400 Å in diameter. The vesicle membrane fuses with the silicalemma and the vesicle content is released into the silica-deposition vesicle. The origin of these vesicles, named STV, is still unknown.     

Characterizing the silica deposition vesicle of diatoms

Summary The present study on a centric diatom,Ditylum brightwellii, includes two parts: detection of sugars in the silica deposition vesicle (SDV) with lectins and labeling the developing siliceous cell wall in the SDV with rhodamine 123. Cells with developing valves are treated with SDS to remove all the cytoplasmic contents, then either stained with fluorescein labeled lectins or thin-sectioned and stained with colloidal gold labeled lectins. The results show that mannose is part of the organic matrix in the SDV. Rhodamine 123, a non-toxic fluorescent laser dye, enters the cell immediately and is trapped in the SDV probably by the high reducing potential of the SDV. Silica is co-deposited with rhodamine 123 in the SDV, and the resulting valves and girdle bands become fluorescent. Implications of this study for the mechanism of silicification are discussed.Abbreviation SDV Silica deposition vesicle     

Effect of blue-green light on growth rate and chemical composition of three diatoms

The diatomsChaetoceros sp.,Skeletonema costatum andThalassiosira pseudonana were grown with different irradiances of white and of blue-green light, and with a mixture of blue-green plus 6.5 mol m^–2 s^–1 of white light. Exponential growth rates were higher in mixed blue for the first two, whileT. pseudonana grew faster in white light but, in all cases, mean cell division rates did not differ with increasing irradiances. Harvesting in stationary, rather than in late exponential growth phase, resulted in higher protein contents forChaetoceros sp. andS. costatum, but forT. pseudonana the highest value was in the exponential phase. The highest protein content was in blue-green light for the three species and it increased with irradiance. As to other fractions, the three strains showed different responses, related to quality and quantity, as well as to culture ages.     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary The sequential wall formation in the centric diatom,Ditylum brightwellii (West) Grunow, is described. The silica deposition vesicle is formed by the coalescence of small vesicles. Silicification of the new valve starts from the central labiate process area prior to the completion of cytokinesis, and the developing valve grows in a centrifugal direction. The initiation of the structures on the valve follows the sequence: labiate process, marginal ridge, and rota. A novel labiate process apparatus, which is situated in the cytoplasm close to the developing labiate process, appears prior to the initiation of the labiate process and disappears upon its maturation. Segments of the girdle bands are formed in individual silica deposition vesicles after the valve matures and is exocytosed. Three morphological forms of deposited silica have been determined: thin base layers, microfibrils, and hexagonal columns. The involvement of cytoplasmic structures in the patterning of the siliceous wall is discussed.     

Biosynthesis of long-chain polyamines by crenarchaeal polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum

Polyamines are ubiquitously present in all organisms. In addition to the common polyamines, thermophilic archaea synthesize long-chain polyamines. In the present study polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum were cloned and their substrate specificity was analyzed. The polyamine synthase HbSpeE II from H. butylicus synthesized long-chain polyamines with high activity using the same mechanism that is used by a wide range of organisms to synthesize common polyamines, in which the aminopropyl residue derives from decarboxylated S-adenosylmethionine. This is the first polyamine synthase described that synthesizes a polyamine longer than a tetramine with high activity.     

A novel plant-fungal mutualism associated with fire

Bromus tectorum, or cheatgrass, is native to Eurasia and widely invasive in western North America. By late spring, this annual plant has dispersed its seed and died; its aboveground biomass then becomes fine fuel that burns as frequently as once every 3-5 y in its invaded range. Cheatgrass has proven to be better adapted to fire there than many competing plants, but the contribution of its fungal symbionts to this adaptation had not previously been studied. In sampling cheatgrass endophytes, many fire-associated fungi were found, including Morchella in three western states (New Mexico, Idaho, and Washington). In greenhouse experiments, a New Mexico isolate of Morchella increased both the biomass and fecundity of its local cheatgrass population, thus simultaneously increasing both the probability of fire and survival of that event, via more fuel and a greater, belowground seed bank, respectively. Re-isolation efforts proved that Morchella could infect cheatgrass roots in a non-mycorrhizal manner and then grow up into aboveground tissues. The same Morchella isolate also increased survival of seed exposed to heat typical of that which develops in the seed bank during a cheatgrass fire. Phylogenetic analysis of Eurasian and North American Morchella revealed that this fire-associated mutualism was evolutionarily novel, in that cheatgrass isolates belonged to two phylogenetically distinct species, or phylotypes, designated Mel-6 and Mel-12 whose evolutionary origin appears to be within western North America. Mutualisms with fire-associated fungi may be contributing to the cheatgrass invasion of western North America.     

Multi-gene analysis and morphology reveal novel Ilyonectria species associated with black foot disease of grapevines

Black foot is an important disease of grapevines, which has in recent years been recorded with increased incidence and severity throughout the world, affecting grapevines both in nurseries and young vineyards. In the past the disease has been associated with infections by Ilyonectria macrodidyma, Ilyonectria liriodendri, Campylocarpon fasciculare, and Campylocarpon pseudofasciculare. Based on published data, a high level of genetic diversity was detected among isolates of I. macrodidyma. To resolve this issue, we employed a multigene analysis strategy (based on the β-tubulin, histone H3, translation elongation factor 1-α, and the internal transcribed spacers on both sides of the 5.8S nuclear ribosomal RNA gene) along with morphological characterisation to study a collection of 81 I. macrodidyma-like isolates from grapevine and other hosts. Morphological characters (particularly conidial size) and molecular data (highest resolution achieved with histone H3 nucleotide sequence) enabled the distinction of six monophyletic species within the I. macrodidyma complex, four of which (Ilyonectria alcacerensis, Ilyonectria estremocensis, Ilyonectria novozelandica, and Ilyonectria torresensis) are described here. This work forms part of an effort by the International Council on Grapevine Trunk Diseases to resolve the species associated with black foot disease, which we believe will clarify their taxonomy, and therefore help researchers to devise control strategies to reduce the devastating impact of this disease.     

Gibbsiella greigii sp. nov., a novel species associated with oak decline in the USA

In 2010, cream-coloured, Gram-negative staining, facultatively anaerobic enterobacteria were isolated from a single black oak tree (Quercus kelloggii) exhibiting decline symptoms in southern California, USA. These 12 isolates were tentatively identified as Gibbsiella quercinecans based on partial gyrB sequencing. Closer examination of the strains using multilocus sequence analysis, based on partial sequences of gyrB, rpoB, infB and atpD genes, and almost complete 16S rRNA gene sequencing suggested that the isolates belong to a novel taxon within the genus Gibbsiella with G. quercinecans as their closest phylogenetic relative. DNA–DNA relatedness studies confirmed that the strains belong to a single taxon in Gibbsiella, which can be differentiated from other members of the genus by several phenotypic traits. Therefore, the name Gibbsiella greigii sp. nov. is proposed for this novel species isolated from symptomatic Q. kelloggii in the USA with FRB 224^T (=LMG 27716^T = NCPPB 4583^T) as the type strain.     

Protein expression during heat stress in thermo-intolerant and thermo-tolerant diatoms

Diatoms (Chrysophyta) are photosynthetic microorganisms that are abundant in the natural environment and often associated with specific habitat and water quality conditions. Their significance as bioindicators and as exploitable sources of fine chemicals makes them desirable candidates for the study of stress responses. The protein expression of a thermo-intolerant (Phaeodactylum tricornutum) and thermo-tolerant (Chaetoceros muelleri) diatom following exposure to elevated temperature was investigated using one- and two-dimensional gel electrophoresis and Western blot analysis. It was determined using SDS PAGE with ³⁵S-methionine labeled proteins and Western blot analysis using pea HSP70 antisera that higher temperatures and longer duration treatment were required to cause a noticeable stress response in C. muelleri compared to P. tricornutum. This may be explained by C. muelleri possessing higher amounts of constitutively expressed heat shock proteins, which allows these cells to rapidly adjust to temperature increases. Two-dimensional gel electrophoresis revealed that putative small heat shock proteins (smHSPs) may appear to play a role during heat stress in both diatoms, which is similar to the response in plants. SDS PAGE data are also presented characterizing the recovery of P. tricornutum after heat shock. These results suggest that there is a lag period between heat shock and stress protein synthesis in these thermo-intolerant cells. This supports the hypothesis that cells without higher amounts of constitutively expressed stress proteins have a greater sensitivity to increased temperature. Work is underway to identify particular stress proteins responsible for conveying thermo-tolerance and to determine if overexpression of these genes in thermo-intolerant diatoms affects their temperature sensitivity.     

Somatic embryogenesis of Panax ginseng in liquid cultures: a role for polyamines and their metabolic pathways

A callus with embryogenic capacity was generated fromroot sections of Panax ginseng and used as aninoculum source for embryogenic liquid cultures in athree-step process: – a suspension culture of cellaggregates in the presence of an auxin/cytokininmixture, – an induction medium containing auxin only(for 5 to 30 days), – a regeneration medium containingcytokinin only (for one month). Up to 25 embryos wererecovered per 2.5 g of aggregates in these conditions.Incorporation of polyamines or their precursorsarginine and ornithine into either the induction orregeneration media increased the number of embryosproduced by up to 4 times. Inhibitors of bothbiosynthesis and biodegradation of polyamines reducedthe number of embryos. These results support earlierfindings of the role of polyamines in the process ofsomatic embryogenesis. The success of these liquidcultures opens up the possibility of producing somaticembryos of Panax ginseng in bioreactors.     

Ectomycorrhizal fungal species and strains differ in their ability to produce free and conjugated polyamines

Production of free and conjugated polyamines by one strain of Laccaria proxima (Boud.) Maire, three strains (H, O, K) of Paxillus involutus (Batsch) Fr., and one strain of Pisolithus tinctorius was studied in vitro. Spermidine (Spd) was the main polyamine in the 4-week-old mycelium of all the fungi. It was mainly present in the free form, but it also occurred in conjugated forms. Paxillus involutus strain H released large amounts of free putrescine (Put), and the Pisolithus tinctorius released a compound probably related to cadaverine (Cad). On the other hand, these two fungi contained less conjugated polyamines than the other fungi. In addition to the amounts, the forms (perchloric acid soluble and insoluble) of conjugated polyamines in the mycelium varied between species and strains. L. proxima contained nearly as much insoluble conjugated Spd as free Spd, whereas Paxillus involutus strains O and K contained relatively large amounts of soluble conjugated Spd. The results suggest that ectomycorrhizal fungal species and strains differ in their ability and need to produce conjugated polyamines. The small amounts of soluble conjugated polyamines found in the culture filtrates indicate that some specific conjugated polyamines may be involved in polyamine translocation across the plasma membrane.     

Cell damage and recovery in cryopreserved microphytobenthic diatoms

Two pennate microphytobenthic diatoms, Amphora coffeaeformis (Agardh) Kutzing and Navicula transitans var. derasa f. delicatula Heimdal, were cryopreserved and monitored on thawing to track the mechanical injuries and their post-preservation recovery. Cells were subjected to (1) direct freezing in liquid nitrogen and (2) two-step cooling with and without the cryoprotectant, dimethyl sulfoxide (Me(2)SO). Mechanical injury due to exposure to low temperature differed between the two species. While A. coffeaeformis cells were intact and could survive even direct freezing without a cryoprotectant, N. delicatula cell chloroplasts were damaged. However, the two-step cooling along with a cryoprotectant minimized the mechanical injury to cells of both species thereby enhancing the post-thaw viability.     

Isolation and characterization of peroxisomes from diatoms

Peroxisomes were isolated by gradient centrifugation from two different diatoms: Nitzschia laevis (subgroup of Pennales) and Thalassiosira fluviatilis (subgroup of Centrales). In neither of these organelles could catalase or any H₂O₂-forming oxidase be demonstrated. The glycolate-oxidizing enzyme present in the peroxisomes is a dehydrogenase capable of oxidizing l-lactate as well. The peroxisomes also contain the glyoxysomal markers isocitrate lyase and malate synthase. However, enzymes of the fatty-acid -oxidation pathway are located exclusively in the mitochondria. The mitochondria additionally possess glutamate-glyoxylate aminotransferase and a glycolate dehydrogenase which differs from the peroxisomal glycolate dehydrogenase since it preferably utilizes d-lactate as an alternative substrate. Hydroxypyruvate reductase and glyoxylate carboligase were not found in the cells of either diatom. By culturing Nitzschia laevis it could be demonstrated that decreasing the CO₂ concentration in the aeration mixture from 2% to 0.03% and increasing the irradiance from 40 to 250 mol quanta · m^–2 · s^–1 resulted in an increase of all peroxisomal enzyme activities. In addition, enzyme activities of the -oxidation pathway were increased. However, mitochondrial glycolate dehydrogenase and aminotransferase did not alter their activities under these conditions. Summarizing all results, it is postulated that there are two different pathways for the metabolism of glycolate in the diatoms.This work was supported by the Deutsche Forschungsgemeinschaft.     

Molecular cloning and characterization of dullard: a novel gene required for neural development

In a screen for genes expressed in neural tissues and pronephroi, we isolated a novel gene, named dullard. Dullard protein contains the C-terminal conserved domain of NLI-IF (Nuclear LIM Interactor-Interacting Factor), a protein whose function is not yet characterized. Dullard mRNA was maternally derived and localized to the animal hemisphere. At neurula stages, the expression was in neural regions and subsequently localized to neural tissues, branchial arches, and pronephroi. Using antisense morpholino oligonucleotide-mediated inhibition, we showed that dullard was required for neural development. The translational knock-down of dullard resulted in failure of neural tube development and the embryos consequently showed a reduction of head development. Expression of neural marker genes in dullard-inhibited embryos was also suppressed. These results suggest that dullard is necessary for neural development.     

Purification and characterization of a novel glutathione S-transferase from Atactodea striata

A novel GST isoenzyme was purified from hepatopancreas cytosol of Atactodea striata with a combination of affinity chromatography and reverse-phase HPLC. The molecular weight of the enzyme was determined to be 24 kDa by SDS-PAGE electrophoresis and 48 kDa by gel chromatography, in combination with GST information from literature revealed that the native enzyme was homodimeric with a subunit of M(r) 24 kDa. The purified enzyme, exhibited high activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a K(m) of 0.43 mM and V(max) of 0.24 micromol/min/mg and a specific activity of 108.9 micromol/min/mg. The isoelectric point of the enzyme was 5.5 by isoelectric focusing and its optimum temperature was 38 degrees C and the enzyme had a maximum activity at approximately pH 8.0. The amino acid composition was also determined for the purified enzyme.     

Relationship between polyamines and paraquat toxicity in sunflower leaf discs

Polyamines have been reported as efficient antioxidantcompounds in plants. Sunflower leaf discs, treatedwith 100 µM paraquat (PQ), a well known oxidativestress inducer, showed decreased levels of putrescine(Put), spermidine (Spd) and spermine (Spm) (between33% and 80% with respect to the controls). Argininedecarboxylase (ADC) and ornithine decarboxylase (ODC)activities decreased 42% and 33% respectively. Amongthe markers of oxidative stress measured after PQtreatment, chlorophyll and glutathione content werereduced (30% and 49% respectively) andthiobarbituric acid reactive substances (TBARS)content increased (60%). Superoxide dismutase (SOD)activity declined 60% with respect to the control andlipoxygenase (LOX) increased 25% when leaf-discs weretreated with the herbicide. Pretreatment withexogenous polyamines (1 mM) reversed paraquat toxicityto different degrees according to the polyamine and/orthe tested parameter. Spermidine was able to inhibitchlorophyll loss, while Spm reverted the effect of PQon the level of TBARS almost completely and alsorestored SOD activity close to control values.Putrescine was the least effective as an oxidantprotectant. These results provide support for theargument that polyamines are effective antioxidantsthrough their ability to act as radical scavengers.