Molecular studies on seed proteins from mature seeds of Phaseolus Vulgaris L.

In order to elucidate the question of the presence or absence of legumin in the seeds of Phaseolus vulgaris L. it was decided to investigate the globulin fraction of seed proteins from mature seeds of French bean. A legumin protein was partially purified and the N- terminal sequences of both subunit polypeptides of a 75 kDa legumin subunits determined, which proved unequivocally that these polypeptides are subunits of a P. vulgaris legumin. The N-terminal amino acid determination of further five disulphide-linked polypeptides of lower Mr revealed that all these polypeptides may well derive from the 75 kDa P. vulgaris legumin as the N-terminal amino acids showed complete sequence homology. The size differences of these legumin subunits might be due to peptide cleavage at the C-terminal end of the legumin a-chains. Potential mechanisms for this catalytic event are discussed. In addition, a second protein. Glycoprotein I, an albumin type seed protein, was isolated from the albumin fraction of the seed proteins of P. vulgaris and the N-terminal amino acids of its subunits were determined. The obtained sequence data of the three different subunits revealed high similarity to the hyper-variable region of legumin J and K from pea. Whilst it is not inconceivable that both polypeptides represent independent protein subunits reflecting the same evolutionary mechanisms to increase the nitrogen content of storage proteins, based on the sizes of the Glycoprotein I subunits and the sequence similarity to the hyper-variable region of a-chains of legumins the hypotheses is discussed that Glycoprotein I may derive from a P. vulgaris legumin by cleavage of peptides from the C-terminal end of the a-chain and formation of a new polypeptide, Glycoprotein I, with new solubility characteristics (water-soluble like albumins).Furthermore, Glycoprotein I was investigated during seed germination by immunodetection with antibodies raised against the purified protein. The results showed that Glycoprotein I serves as a reserve protein in an early stage of seed germination. In order to obtain extensive sequence information on legumins from P. vulgaris it was decided to attempt to amplify a legumin gene fragment from genomic DNA of P. vulgaris using the polymerase chain reaction technique. Southern analysis of PGR products using a pea-legumin as a probe gave strong evidence for the successful amplification of legumin encoding genes from genomic P. vulgaris DNA. However, neither the cloning of these PGR products nor the repeats of this PGR experiment were successful. A new PGR approach to amplify legumin gene fragments from genomic DNA of P. vulgaris using an oligonucleotide sequence for the reverse primer designed on the basis of the N-terminal amino acid sequence of the β-chain is discussed. The nucleotide sequence of a PGR artefact of one of the unsuccessful repeats of the PGR experiment showed sequence similarity to Arcelin 2, a gene found in wild types of P. vulgaris. Arcelin proteins provide the plant with resistance against an important pest weevil, but have never been observed in cultivars of P. vulgaris. Arcelin genes are thought to have been either totally lost or inactivated during domestication of the French bean. Further isolated and identified proteins from the albumin fraction of seed proteins from P. vulgaris include a superoxide dismutase (SOD). The determination of the N-terminal amino acids of the subunit poypeptide provides the first reported sequence information of a SOD in mature seeds of higher plants. The potential localisation within the seed tissue and the putative role of the presence of a SOD in mature seeds during dormancy is discussed. Two more polypeptides were isolated from the albumin fraction and identified by N- terminal sequencing as chitinases. Whereas pvchi25 showed high sequence similarity to class n chitinases, pvchi32 was shown to be a class III chitinase. Pvchi32 provides the first reported class HI chitinase in mature seeds of higher plants and the second so far described glycosylated chitinase. The potential use of these seed chitinases in crop protection is discussed. Furthermore, the enzymatic activity of both enzymes was tested as well as cross-reactivity to antibodies raised against ethylene-induced bean leaf chitinases. Pvchi25 was observed during seed germination by immunodetection confirming its role in the protection of the seed during dormancy and early germination

Assessment of complex microbial assemblages: description of their diversity and characterisation of individual members: Assessment of complex microbial assemblages: description of their diversity and characterisation of individual members

1. Microbial ecology According to Caumette et al. (2015) the term ecology is derived from the Greek words “oikos” (the house and its operation) and “logos” (the word, knowledge or discourse) and can, therefore, be defined as the scientific field engaged in the “knowledge of the laws governing the house”. This, in extension, results in the simple conclusion that microbial ecology represents the study of the relationship between microorganisms, their co-occurring biota and the prevailing environmental conditions (Caumette et al. 2015). The term microbial ecology has been in use since the early 1960s (Caumette et al. 2015) and microbial ecologists have made astonishing discoveries since. Microbial life at extremes such as in the hydrothermal vents (see Dubilier et al. 2008 and references therein) or the abundance of picophytoplankton (Waterbury et al. 1979; Chisholm et al. 1988) in the deep and surface waters of the oceans, respectively, are only a few of many highlights. Nevertheless, a microbial ecologist who, after leaving the field early in their career, now intends to return would hardly recognise again their former scientific field. The main reason for this hypothesis is to be found in the advances made to the methodologies employed in the field. Most of these were developed for biomedical research and were subsequently hijacked, sometimes followed by minor modifications, by microbial ecologists. The Author presents in this thesis scientific findings which, although spanning only a fraction of the era of research into microbial ecology, have been obtained using various modern tools of the trade. These studies were undertaken by the Author during his employment as postdoctoral scientist at Warwick University (UK), as member of staff at Plymouth Marine Laboratory (UK) and as scientist at the TU Bergakademie Freiberg. Although the scientific issues and the environmental habitats investigated by the Author changed due to funding constraints or due to change of work place (i.e. from the marine to the mining environment) the research shared, by and large, a common aim: to further the existing understanding of microbial communities. The methodological approach chosen to achieve this aim employed both isolation followed by the characterisation of microorganisms and culture independent techniques. Both of these strategies utilised again a variety of methods, but techniques in molecular biology represent a common theme. In particular, the polymerase chain reaction (PCR) formed the work horse for much of the research since it has been routinely used for the amplification of a marker gene for strain identification or analysis of the microbial diversity. To achieve this, the amplicons were either directly sequenced by the Sanger approach or analysed via the application of genetic fingerprint techniques or through Sanger sequencing of individual amplicons cloned into a heterologous host. However, the Author did not remain at idle while with these ‘classical’ approaches for the analysis of microbial communities, but utilised the advances made in the development of nucleotide sequence analysis. In particular, the highly parallelised sequencing techniques (e.g. 454 pyrosequencing, Illumina sequencing) offered the chance to obtain both high genetic resolution of the microbial diversity present in a sample and identification of many individuals through sequence comparison with appropriate sequence repositories. Moreover, these next generation sequencing (NGS) techniques also provided a cost-effective opportunity to extent the characterisation of microbial strains to non-clonal cultures and to even complex microbial assemblages (metagenomics). The work involving the high throughput sequencing techniques has been undertaken in collaboration with Dr Jack Gilbert (PML, lateron at Argonne National Laboratory, USA) and, since at Freiberg, with Dr Anja Poehlein (Goettingen University). These colleagues are thanked for their support with sequence data handling and analyses

A NEW “FERROVUM” SPECIES IN A SCHWERTMANNITE-PRODUCING PLANT FOR MINE WATER TREATMENT

Mining activities for metals or coal often result in the development of acid mine drainage due to the oxidation of sulfidic minerals which get exposed to oxygen. The acidic mine waters are characterized by low pH, high concentrations of sulfate and ferrous iron, and possibly dissolved heavy metals or metalloids. Conventional treatment comprises neutralization and oxidation yielding a sludge of iron oxides/hydroxides

Real-Time Imaging of Leaf Apoplastic pH Dynamics in Response to NaCl Stress

Knowledge concerning apoplastic ion concentrations is important for the understanding of many processes in plant physiology. Ion-sensitive fluorescent probes in combination with quantitative imaging techniques offer opportunities to localize, visualize, and quantify apoplastic ion dynamics in situ. The application of this technique to the leaf apoplast is complicated because of problems associated with dye loading. We demonstrate a more sophisticated dye loading procedure that enables the mapping of spatial apoplastic ion gradients over a period of 3 h. The new technique has been used for the real-time monitoring of pH dynamics within the leaf apoplast in response to NaCl stress encountered by the roots

Biofortification and subcellular localization of minerals in faba bean as influenced by Mg foliar application

Foliar application of Mg is a measure for the correction of Mg deficiency in crop plants. Foliar applied nutrients need to access the symplastic side where majority of physiological processes take place. To achieve an adequate uptake of the Mg ions through the leaf surface, high concentrations of of 100-200 mM MgSO4 are usually supplied. This can cause antagonistic perturbations on the subcellular distribution of Caand K cations. To test for such unintended side effects, we used the infiltration-centrifugation method to extract ions from the apoplastic and symplastic side of Vicia faba leaves and quantified concentrations of Mg, Ca and K in dependency to the dose of the foliar fertilized Mg. Results show that a large fraction of Mg accesses the symplast whereas the apoplastic fraction shows a concomitant increase. Symplastic and apoplastic K and Ca relations were only affected under conditions of high exogenous leaf supply of Mg (200 mM) but did not change upon moderate Mg supply (50; 100 mM). Overall, results reveal the suitability of leaf fertilization to biofortify plant-based products with magnesium. With respect to human nutrition, care must be taken that K and Ca do not become impoverished based on antagonistic effects

Gene Loss and Horizontal Gene Transfer Contributed to the Genome Evolution of the Extreme Acidophile “Ferrovum”

Indexación: Web of Science. Scopus.Acid mine drainage (AMD), associated with active and abandoned mining sites, is a habitat for acidophilic microorganisms that gain energy from the oxidation of reduced sulfur compounds and ferrous iron and that thrive at pH below 4. Members of the recently proposed genus "Ferrovurn" are the first acidophilic iron oxidizers to be described within the Betaproteobacteria. Although they have been detected as typical community members in AMD habitats worldwide, knowledge of their phylogenetic and metabolic diversity is scarce. Genomics approaches appear to be most promising in addressing this lacuna since isolation and cultivation of "Ferrovurn" has proven to be extremely difficult and has so far only been successful for the designated type strain-Ferrovum myxofaciens" P3G. In this study, the genomes of two novel strains of "Ferrovurn" (PN-J185 and Z-31) derived from water samples of a mine water treatment plant were sequenced. These genomes were compared with those of "Ferrovum" sp. JA12 that also originated from the mine water treatment plant, and of the type strain (P3G). Phylogenomic scrutiny suggests that the four strains represent three "Ferrovum" species that cluster in two groups (1 and 2). Comprehensive analysis of their predicted metabolic pathways revealed that these groups harbor characteristic metabolic profiles, notably with respect to motility, chemotaxis, nitrogen metabolism, biofilm formation and their potential strategies to cope with the acidic environment. For example, while the "F myxofaciens" strains (group 1) appear to be motile and diazotrophic, the non-motile group 2 strains have the predicted potential to use a greater variety of fixed nitrogen sources. Furthermore, analysis of their genome synteny provides first insights into their genome evolution, suggesting that horizontal gene transfer and genome reduction in the group 2 strains by loss of genes encoding complete metabolic pathways or physiological features contributed to the observed diversification.http://journal.frontiersin.org/article/10.3389/fmicb.2016.00797/ful

One-time abscisic acid priming induces long-term salinity resistance in Vicia faba: Changes in key transcripts, metabolites, and ionic relations

Abscisic acid (ABA) priming is known to enhance plant growth and survival under salinity. However, the mechanisms mediating this long-term acclimatization to salt stress are still obscure. Specifically, the long-term transcriptional changes and their effects on ion relations were never investigated. This motivated us to study the long-term (8 days) effect of one-time 24 h root priming treatment with 10 μM ABA on transcription levels of relevant regulated key genes, osmotically relevant metabolites, and ionic concentrations in Vicia faba grown under 50 mM NaCl salinity. The novelty of this study is that we could demonstrate long-term effects of a one-time ABA application. ABA-priming was found to prevent the salt-induced decline in root and shoot dry matter, improved photosynthesis, and inhibited terminal wilting of plants. It substantially increased the mRNA level of AAPK and 14-3-3 ABA inducible kinases and ion transporters (PM H+ -ATPase, VFK1, KUP7, SOS1, and CLC1). These ABA-induced transcriptional changes went along with altered tissue ion patterns. Primed plants accumulated less Na+ and Cl- but more K+ , Ca2+ , Zn2+ , Fe2+ , Mn2+ , NO3 - , and SO4 2- . Priming changed the composition pattern of organic osmolytes under salinity, with glucose and fructose being dominant in unprimed, whereas sucrose was dominant in the primed plants. We conclude that one-time ABA priming mitigates salt stress in Vicia faba by persistently changing transcription patterns of key genes, stabilizing the ionic and osmotic balance, and improving photosynthesis and growth

Genome Sequence of the Acidophilic Ferrous Iron-Oxidizing Isolate Acidithrix ferrooxidans Strain Py-F3, the Proposed Type Strain of the Novel Actinobacterial Genus Acidithrix

Extremely acidophilic iron-oxidizing Gram-positive bacteria comprise species within the phyla Firmicutes and Actinobacteria. Here, we report the 4.02-Mb draft genome of Acidithrix ferrooxidans Py-F3, which was isolated from a stream draining an abandoned copper mine and proposed as the type species of a new genus of Actinobacteria

Genome Sequence of the Moderately Acidophilic Sulfate-Reducing Firmicute Desulfosporosinus acididurans (Strain M1T)

Microbial dissimilatory sulfate reduction is commonplace in many anaerobic environments, though few acidophilic bacteria are known to mediate this process. We report the 4.64-Mb draft genome of the type strain of the moderate acidophile Desulfosporosinus acididurans, which was isolated from acidic sediment in a river draining the Soufrière volcano, Montserrat

Genome Sequence of the Acidophilic Sulfate-Reducing Peptococcaceae Strain CEB3

We report the draft genome of the Peptococcaceae strain CEB3 that originated from an acidic (pH 2.5) stream draining an abandoned copper mine. Strain CEB3 is one of the very few reported acidophilic sulfate-reducing isolates. The 5.04-Mb draft genome harbors 5,069 predicted protein-encoding and 66 RNA genes