Terrestrialization depended on the evolution of biosynthetic pathways for biopolymers including lignin, cutin and suberin, which were concentrated in specific tissues, layers or organs such as the xylem, cuticle and roots on the submillimetre scale. However, it is often difficult, or even impossible especially for individual cells, to resolve the biomolecular composition of the different components of fossil plants on such a scale using the well-established coupled techniques of gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Here, we report the application of techniques for surface analysis to investigate the composition of Rhynia gwynne-vaughanii X-ray photoelectron spectroscopy of two different spots (both 300 µm × 600 µm) confirmed the presence of carbon. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed 'chemical maps' (imaging mode with 300 nm resolution) of aliphatic and aromatic carbon in the intact fossil that correlate with the vascular structures observed in high-resolution optical images. This study shows that imaging ToF-SIMS has value for determining the location of the molecular components of fossil embryophytes while retaining structural information that will help elucidate how terrestrialization shaped the early evolution of land plant cell wall biochemistry.This article is part of a discussion meeting issue 'The Rhynie cherts: our earliest terrestrial ecosystem revisited'.

Abstract—Soil samples were collected from and around Alkomos cement factory, Alkomos town, Libya. Soil physiochemical properties were determined. In addition, olive leaves were scanned for their fungal content. This work can conclude that the results obtained for the examined physiochemical characteristics of soil in the area studied prove that cement dust from the Alkomos cement factory in Libya has had a significant impact on the soil. The affected soil properties are pH and total calcium content. These characteristics were found to be higher than those in similar soils from the same area. The increment of soil pH in the same area may be a result of precipitation of cement dust over the years. Different responses were found in each season and each site. For instance, the dominance of fungi of soil and leaves was lowest at 100 m from the factory and the evenness and diversity increased at this site compared to the control area and 250 m from the factory.

The genus Pseudoalteromonas constitutes an ecologically significant group of marine Gammaproteobacteria with potential biotechnological value as producers of bioactive compounds and of enzymes. Understanding their roles in the environment and bioprospecting for novel products depend on efficient ways of identifying environmental isolates. Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) biotyping has promise as a rapid and reliable method of identifying and distinguishing between different types of bacteria, but has had relatively limited application to marine bacteria and has not been applied systematically to Pseudoalteromonas. Therefore, we constructed a MALDI-TOF MS database of 31 known Pseudoalteromonas species, to which new isolates can be compared by MALDI-TOF biotyping. The ability of MALDI-TOF MS to distinguish between species was scrutinized by comparison with 16S rRNA gene sequencing. The patterns of similarity given by the two approaches were broadly but not completely consistent. In general, the resolution of MALDI-TOF MS was greater than that of 16S rRNA gene sequencing. The database was tested with 13 environmental Pseudoalteromonas isolates from UK waters. All of the test strains could be identified to genus level by MALDI-TOF MS biotyping, but most could not be definitely identified to species level. We conclude that several of these isolates, and possibly most, represent new species. Thus, further taxonomic investigation of Pseudoalteromonas is needed before MALDI-TOF MS biotyping can be used reliably for species identification. It is, however, a powerful tool for characterizing and distinguishing among environmental isolates and can make an important contribution to taxonomic studies.

Accurate and defendable taxonomic identification of microalgae strains is vital for culture collections, industry and academia; particularly when addressing issues of intellectual property. We demonstrate the remarkable effectiveness of Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS) biotyping to deliver rapid and accurate strain separation, even in situations where standard molecular tools prove ineffective. Highly distinctive MALDI spectra were obtained for thirty two biotechnologically interesting Dunaliella strains plus strains of Arthrospira , Chlorella , Isochrysis, Tetraselmis and a range of culturable co-occurring bacteria. Spectra were directly compared with genomic DNA sequences (internal transcribed spacer, ITS). Within individual Dunaliella isolates MALDI discriminated between strains with identical ITS sequences, thereby emphasising and enhancing knowledge of the diversity within microalgae culture collections. Further, MALDI spectra did not vary with culture age or growth stage during the course of the experiment; therefore MALDI presents stable and accurate strain-specific signature spectra. Bacterial contamination did not affect MALDI’s discriminating power. Biotyping by MALDI-TOF-MS will prove effective in situations wherein precise strain identification is vital, for example in cases involving intellectual property disputes and in monitoring and safeguarding biosecurity. MALDI should be accepted as a biotyping tool to complement and enhance standard molecular taxonomy for microalgae.

This paper reports a laboratory study where multiple variables have been monitored in parallel as a function of white-rot incubation time. A model system with Pleurotus ostreatus growing on unamended wheat (Triticum aestivum) straw under solid-state cultivation conditions was used to investigate changes in lignin oxidation, amounts of the fungal biomarker ergosterol and activity of manganese peroxidase (MnP) at regular intervals over 84days. Lignin oxidation was monitored with thermally assisted hydrolysis and methylation (THM) using tetramethylammonium hydroxide (TMAH) where 3,4-dimethoxybenzoic acid, methyl ester (G6) to 3,4-dimethoxybenzaldehyde (G4) [Ac/Al]G and 3,4,5-trimethoxybenzoic acid, methyl ester (S6) to 3,4,5-trimethoxybenzaldehyde (S4) [Ac/Al]S were used as relative lignin decomposition state proxies. [Ac/Al]G, [Ac/Al]S and ergosterol production show little change during the first 21days of incubation. MnP activity, however, rises rapidly and peak activity is reached during the same time interval. This is followed by an increase in both [Ac/Al]G, [Ac/Al]S and ergosterol formation where most of the increase in these variables takes place after 21days. Therefore a rapid early rise in manganese-dependent peroxidase activity precedes significant changes both in lignin oxidation and fungal growth. We have demonstrated that it is possible to analyse lignin oxidation using THM in the presence of TMAH and, in the same system, measure enzyme activity and amounts of fungal biomarkers as a function of incubation time. This approach will be useful when investigating compositional changes in litter and soil layers.

The phylogenetic placement of four isolates of Botryococcus braunii Kützing and of Botryococcus sudeticus Lemmermann isolate UTEX 2629 was investigated using sequences of the nuclear small subunit (18S) rRNA gene. The B. braunii isolates represent the A (two isolates), B, and L chemical races. One isolate of B. braunii (CCAP 807/1; A race) has a group I intron at Escherichia coli position 1046 and isolate UTEX 2629 has group I introns at E. coli positions 516 and 1512. The rRNA sequences were aligned with 53 previously reported rRNA sequences from members of the Chlorophyta, including one reported for B. braunii (Berkeley strain). Phylogenetic trees were constructed using distance, weighted maximum parsimony, and maximum likelihood, and their reliability was estimated using bootstrap analysis for distance and parsimony and Bayesian inference for likelihood. All methods showed, with high bootstrap or credibility support, that the four isolates of B. braunii form a monophyletic group whose closest relatives are in the genus Choricystis in the Trebouxiophyceae, whereas the previously reported B. braunii sequence is from a member of the Chlamydomonadales in the Chlorophyceae and isolate UTEX 2629 is a member of the Sphaeropleales in the Chlorophyceae. Polyphyly of these sequences was confirmed by Kishino‐Hasegawa tests on artificial trees in which sequences were moved to a single lineage.

Two types of xylanase gene, XYN11A ( XYL1) and XYN11B ( XYL2), were amplified by PCR and partially sequenced in four phytopathogenic species of the ascomycete fungal genus Cochliobolus (anamorph genus Bipolaris). Three of the species, C. heterostrophus ( B. maydis), C. sativus ( B. sorokiniana), and Bipolaris sorghicola (no teleomorph known), are interrelated; the fourth, C. spicifer ( B. spicifera), was found, through analysis of the 5.8S RNA and internal transcribed spacer (ITS) sequences of its ribosomal DNA, to be more distantly related to the other three. Isolates from all four species contain orthologous XYN11A and XYN11B genes, but a set of laboratory strains of C. heterostrophus gave no product corresponding to the XYN11B gene. The patterns of evolution of the two xylanase genes and ribosomal DNA sequences are mutually consistent; the results indicate that the two genes were present in the common ancestor of all Cochliobolus species and are evolving independently of each other.

A xylanase gene, XYL2, was identified and characterised in Cochliobolus sativus (anamorph Bipolaris sorokiniana), a necrotrophic cereal pathogen that attacks both shoots and roots. The fungus was grown on a xylanase inducing medium containing mineral salts, oat spelt xylan, cellulose, and peptone, RNA was isolated, and a complementary DNA (cDNA) library constructed. The library was screened with a xylanase (XYLI) cDNA clone from the maize pathogen Cochliobolus carbonum. Xylanase cDNA clones, all representing a single gene, were identified. Corresponding genomic DNA was amplified by PCR. Sequencing of the cDNA and the PCR products gave a nucleotide sequence of 2211 bp containing two introns in an open reading frame of 693 bp that codes for a xylanase from glycosyl hydrolase family II. The most similar sequences to this gene in nucleotide sequence databases are the XYL2 gene of C, carbonum and a xylanase (XYLI) cDNA from a saprophytic fungus, Humicola insolens. Northern blot analysis and reverse transcription PCR (RT-PCR) showed expression of the gene when the fungus was grown on xylan or cellulose, but not when peptone or sucrose was the only carbon source. Expression of XYLI in inoculated barley seedlings was detected by RT-PCR.

We reported earlier that adding 2 g.L(-1) L-proline and raising fourfold the level of inorganic micronutrients in a modified Murashige and Skoog (MS) medium enhances shoot regeneration from primary-leaf-node explants of 7-d-old soybean (Glycine max [L.] Merr.) seedlings. These findings are here extended to regeneration from explants consisting of a cotyledon cut from a 7-d-old soybean seedling. For both kinds of explant, a synergistic, approximately threefold stimulation of shoot regeneration by these two supplements in combination is documented. Proline increased number but decreased length of regenerated shoots, whereas raising micronutrient level generally increased both shoot number and length and, thus, partly overcame the effect of proline on length. Examining the effects of singly including, omitting, raising the levels of, and lowering the levels of the seven different micronutrient elements of the original MS medium provides evidence that the supply of cobalt is near-optimal at the original MS concentration but the other six elements are in less than optimal supply. Altering the zinc supply had the strongest effects overall.

RNA isolated from etiolated seedling shoot mitochondrial of maize (Zea mays L.) with normal (N) or Texas male-sterile (T) cytoplasm stimulated the incorporation of [35S]-methionine into protein when added to a cell-free protein-synthesizing system from wheat germ. Discrete polypeptides with molecular masses of up to approximately 67 kDa were synthesized, and the pattern of bands was distinct from that obtained with total RNA. Products of translation of T-urf13 RNA were identified by immunoprecipitation, and ofatpA, coxI, andcoxII RNA by hybrid arrest of translation by the cloned gene. several polypeptides were differentially synthesized from N and T mitochondrial RNA; these differences were more extensive than those found when isolated, intact, N and T mitochondria are allowed to synthesize proteins.

The protein T-URF13 (URF13) is specific to mitochondria of maize (Zea mays L.) with Texas (T) male-sterile cytoplasm and has been implicated in causing male sterility and susceptibility to T-cytoplasm-specific fungal diseases. T-URF13 was purified from isolated mitochondria from maize (line B73) with T cytoplasm by gel filtration and a quasi two-dimensional polyacrylamide gel electrophoresis system. Antibodies to the purified and denatured protein were produced in rabbits. Anti-T-URF13 antiserum was used to show that T-URF13 is in the inner membrane of mitochondria and behaves as an integral membrane protein when mitochondria are fractionated with sodium carbonate or Triton X-114. The antiserum and protein A tagged with 20-nanometer-gold particles were used to localize T-URF13 in T mitochondria by electron microscopy of sections of isolated mitochondria from etiolated shoots and sections of roots and of tapetal cells at pre-and post-degeneration stages of microsporogenesis. The microscopic study confirms that T-URF13 is specifically localized in the mitochondrial membranes of all of the T mitochondria tested, notably those in the tapetum from the meiocyte stage to the late-microspore stage. No change in the amount of labeled T-URF13 protein in the mitochondria of aging tapetal cells was detected.

A fusion protein was expressed in transgenic tobacco and yeast cells to examine the functional conservation of mechanisms for importing precursor proteins from the cytosol into mitochondria and chloroplasts. The test protein consisted of the mitochondrial leader peptide from the yeast precursor to cytochrome oxidase subunit Va (prC5) fused to the reporter protein chloramphenicol acetyltransferase. This protein, denoted prC5/CAT, was transported into the mitochondrial interior in yeast and tobacco cells. In both organisms, the mitochondrial form of prC5/CAT was smaller than the primary translation product, suggesting that proteolytic processing occurred during the transport process. prC5/CAT also was translocated into chloroplasts in vivo, accumulating to approximately the same levels as in plant mitochondria. However, accumulation of prC5/CAT in chloroplasts relative to mitochondria varied with the conditions under which plants were grown. The chloroplast form of prC5/CAT also appeared to have been proteolytically processed, yielding a mature protein of the same apparent size as that seen in mitochondria of either tobacco or yeast. Chloramphenicol acetyltransferase lacking a mitochondrial targeting peptide did not associate with either chloroplasts or mitochondria. The results demonstrated that in plant cells a single leader peptide can interact functionally with the protein translocation systems of both chloroplasts and mitochondria, and raised the possibility that certain native proteins might be shared between these two organelles.

A simple and reproducible protocol for regeneration of soybean plants from explants derived from 7-day-old seedlings has been developed.Explants, each consisting of the primary leaf node (the unifoliate leaf node), the cotyledonary node, the internode between them, and one cotyledon, were cultured in vitro on a modified Murashige and Skoog (MS) agar medium containing 3% sucrose, four times the MS inorganic micronutrient level, the vitamins of B 5 medium, 2g L-1 L-proline,2mgL-1 N6-benzyladenine (BA), and 0.02mgL-1 α-naphthalene acetic acid (NAA) to induce the formation of adventitious shoots at the primary leaf node.In inducing shoot formation, this medium was superior to MS and B5 media even when all were supplemented with the same BA, NAA and proline levels.Explants from seedlings of the cultivar Peking produced an average of ca.20 shoots per explant after 4–5 weeks on this medium.Shoot formation was greater when a cotyledon remained attached to the explant.Shoot formation was also influenced by medium composition, explant orientation, age of donor seedling, and cultivar.Exogenous cytokinin (BA) was essential for shoot formation; proline and the fourfold-raised level of MS inorganic micronutrients caused a large increase in shoot number.

Mitochondria contain a protein, hsp60, that is induced by heat shock and has been shown to function as a chaperonin in the assembly of mitochondrial enzyme complexes composed of proteins encoded by nuclear genes and imported from the cytosol. To determine whether products of mitochondrial genes are also assembled through an interaction with hsp60, we looked for association between hsp60 and proteins synthesized by isolated mitochondria. We have determined by electrophoretic, centrifugal, and immunological assays that at least two of those proteins become physically associated with hsp60. In mitochondrial matrix extracts, this association could be disrupted by the addition of Mg-ATP. One of the proteins that formed a stable association with hsp60 was the alpha subunit of the multicomponent complex F1-ATPase. We have not identified the other protein. These results indicate that hsp60 can function in the folding and assembly of mitochondrial proteins encoded by both mitochondrial and nuclear genes.

The mitochondrial gene T‐urf13 from maize (Zea mays L.) with Texas male‐sterile (T) cytoplasm codes for a unique 13 kd polypeptide, T‐URF13, which is implicated in cytoplasmic male sterility and sensitivity to the insecticide methomyl and to host‐specific fungal toxins produced by Helminthosporium maydis race T (HmT toxin) and Phyllosticta maydis (Pm toxin). A chimeric gene coding for T‐URF13 fused to the mitochondrial targeting peptide from the Neurospora crassa ATP synthase subunit 9 precursor was constructed. Expression of this gene in the yeast Saccharomyces cerevisiae yielded a polypeptide that was translocated into the membrane fraction of mitochondria and processed to give a protein the same size as maize T‐URF13. Methomyl, HmT toxin and Pm toxin inhibited growth of yeast cells expressing the gene fusion on medium containing glycerol as sole carbon source and stimulated respiration with NADH as substrate by isolated mitochondria from these cells. These effects were not observed in yeast cells expressing T‐URF13 without a targeting peptide. The results show that T‐URF13 is sufficient to confer sensitivity to methomyl and the fungal toxins in a heterologous eukaryotic system, and suggest that mitochondrial localization of T‐URF13 is critical for these functions.

Proline accumulation is a well-known response to water deficits in leaves. The primary cause of accumulation is proline synthesis. Delta(1)-Pyrroline-5-carboxylate reductase (PCR) catalyzes the final reaction of proline synthesis. To determine the subcellular location of PCR, protoplasts were made from leaves of Pisum sativum L., lysed, and fractionated by differential and Percoll density gradient centrifugation. PCR activity comigrated on the gradient with the activity of the chloroplast stromal marker NADPH-dependent triose phosphate dehydrogenase. We conclude that PCR is located in chloroplasts, and therefore that chloroplasts can synthesize proline. PCR activities from chloroplasts and etiolated shoots were compared. PCR activity from both extracts is stimulated at least twofold by 100 millimolar KCl or 10 millimolar MgCl(2). The pH profiles of PCR activity from both extracts reveal two separate optima at pH 6.5 and 7.5. Native isoelectric focusing gels of sampies from etiolated tissue reveal a single band of PCR activity with a pl of 7.8.

When isolated cucumber (Cucumis sativus L.) mitochondria were treated with 14C-labelled dicyclohexylcarbodiimide (DCCD), a single polypeptide was predominantly labelled. This polypeptide was soluble in 1-butanol or chloroform: methanol (2: 1, v/v) and had an apparent molecular mass of approximately 7 kDa; it therefore had the characteristic properties of the DCCD-binding proteolipid subunit of the ATP synthase complexes of mitochondria, chloroplasts, and prokaryotes.