George Borbély

Microcystin-LR alters the growth, anthocyanin content and single-stranded DNase enzyme activities in Sinapis alba L. seedlings

Seedlings of the white mustard (Sinapis alba L.) are sensitive to the cell-free extracts of a toxigenic strain of Microcystis aeruginosa and to microcystin-LR. Fresh mass of plants, plant length, including hypocotyl and root length and lateral root formation is inhibited in microcystin-LR treated seedlings. The decrease of anthocyanin content is obtained in microcystin treated mustard cotyledons. The tissue necrosis of cotyledons is a characteristic consequence of microcystin treatment. Microcystin-LR induces an increase in single stranded deoxyribonucleases (ssDNases) activity of S. alba seedlings as shown by spectrophotometric assays and by ssDNase activity polyacrylamide gels. The significance of this phenomenon is discussed in relation to general stress responses in plants. We conclude that microcystin-LR affects the whole physiology and the growth of plants.

[64] Cyanobacterial DNA-dependent RNA polymerase

Publisher Summary This chapter describes the purification of Anabaena sp. PCC 7120 DNA-dependent RNA polymerase. Cyanobacterial gene expression requires the transfer of genetic information from DNA to RNA molecules which is mediated by DNA-dependent RNA polymerase. The RNA polymerases purified from cyanobacterial sources are similar to bacterial RNA polymerases in the following respects: divalent metal ion requirement for enzymatic activity, rifamycin sensitivity, a-amanitin insensitivity, and requirement for a DNA template. The enzyme catalyzes the initiation, elongation, and termination of polyribonucleotide chains using ribonucleoside triphosphates as substrates. As RNA polymerases of cyanobacterial origin have a number of properties which are common to other prokaryotic RNA polymerases, purification steps and strategies established for Escherichia coli and/or other enteric bacteria are useful guides in purification of the enzyme. The role and functions of cyanobacterial RNA polymerases and the mechanism by which the enzyme functions require a great deal of further investigation. The procedure described is for the isolation of the RNA polymerase holoenzyme and core polymerase from Anabaena sp. PCC 7120 (ATCC 27893). Cell disruption is accomplished by French pressure cell treatment. After ultracentrifugation of the lysate, RNA polymerase is further purified by Polymin P (polyethyleneimine, Sigma) precipitation.

Superoxide dismutase Activity in Response to Paraquat Resistance in Conyza canadensis (L.) Cronq.

Summary Superoxide dismutase levels were investigated in paraquat susceptible (wild and atrazine resistant), paraquat resistant (PQR) and two paraquat/atrazine co-resistant (PQAR) biotypes of Conyza canadensis (L.) Cronq. plants with resistance factors (R f ) of 160-165 to paraquat of Hungarian origin. The SOD enzyme activity was measured by densitometry after polyacrylamide gel electrophoresis and specific activity staining in gels. There was no significant difference in SOD activity of total cell extracts between PQ susceptible and resistant biotypes of C. canadensis . The leaves of all biotypes contain three electrophoretically distinct cyanide sensitive SOD isozymes. There were two isozymes in the chloroplast extracts of all studied biotypes, and the activity of SOD did not differ significantly, with the exception of one PQAR biotype with a Rf of 650 where a slight increase was detected. The Sod Enzyme Activity Of Total Cell Extracts Of Those Plants (Paraquat Susceptible And Resistant Biotypes) Was Not Significantly Affected By Paraquat Treatment In Leaves Of Sprayed Intact Plants And Floated Leaves, And The Light Or Dark Regimen Did Not Alter Sod Enzyme Activity During he Herbicide Treatment.

Microcystin-LR induces chromatin alterations and modulates neutral single-strand-preferring nuclease activity in Phragmites australis

Microcystin-LR (MCY-LR), a toxin produced mainly by freshwater cyanobacteria, is a potent inhibitor of type 1 and 2A protein phosphatases. As such, it induces biochemical, cellular and tissue alterations in vascular plants, including cell death. The aim of this study was the analysis of MCY-LR induced changes in the activity of single-strand preferring nuclease (SSP nuclease) isoenzymes that are possibly involved in programmed cell death (PCD) of Phragmites australis (common reed, an aquatic macrophyte) cells. We analyzed both single-stranded DNA (ssDNase) and double-stranded DNA (dsDNase) cleaving activities. Activity gels revealed a number of seven isoenzymes named bands A-G in control reed shoots and roots. Their activity was organ- and age-dependent. We stained nuclei of root tip meristematic cells and found total and marginal chromatin condensations at relatively short-term (2-10 days) cyanotoxin exposure. At 10-20 days of cyanotoxin treatment, the number of cells with condensed chromatin decreased, which coincided with the occurrence of necrotic cell death. In parallel, overall ssDNase activity increased in the short term (five days) and gradually decreased at 10-20 days of MCY-LR treatment. In this context, the most important changes occurred for isoenzyme G of 28-32kDa in roots and isoenzyme F of 35-38kDa in shoots. dsDNase activity of isoenzyme E was decreased by MCY-LR in shoots, but increased in roots at 10 days of exposure. We conclude that the early induction of chromatin condensation and increase of SSP nuclease activities is related to PCD that will lead to necrosis with the cease of all cellular activities, including a decrease in nuclease activity.

[69] Cyanobacterial heat-shock proteins and stress responses

Publisher Summary This chapter concentrates primarily on techniques involved in studies of the heat-shock phenomenon in cyanobacteria. For convenience of discussion only the heat shock of Synechococcus Strain PCC 6301 is described, but these methods have direct applicability in studies directed toward the stress responses of other cyanobacterial strains as well. The techniques are simple and easy to execute. The specific pattern of protein synthesis can be monitored readily by use of two-dimensional polyacrylamide gel electrophoresis. Growing cyanobacteria are strongly influenced by their nutritional, chemical, and physical environments. Of those factors, temperature plays a critical role: cyanobacteria exposed to temperatures higher than those for normal growth respond by altering patterns of growth and protein synthesis. One of the most emphasized aspects of light-dependent cyanobacterial gene expression is its pleiotropic nature; hence, a specific manipulation of cyanobacterial genes or regulons could simplify attempts to understand the regulation of gene expression in these organisms. Accordingly, studies on heat shock and other stress responses are of interest not only in their own right but from the viewpoint of light-dependent and regulated cyanobacterial gene expression as well.

Plant regeneration from embryogenic cultures of Phragmites australis (Cav.) Trin. Ex Steud.

Embryogenic cultures of the common reed [Phragmites australis (Cav.) Trin. Ex. Steud.] were induced on Murashige and Skoog (1962)-based medium with 2% (w/v) sucrose, B5 vitamins and 4.5 μM 2,4-dichlorphenoxyacetic acid. Four independent culture lines, two initiated from stem nodes and two from roots, were established. These cultures underwent somatic embryogenesis. In one line of stem node origin, the somatic embryos germinated and developed into plants, following transfer of embryogenic cultures to Murashige and Skoog (1962)-based medium lacking growth regulators, with 108 ± 17 plants being recovered per 100 mg fresh weight of culture. In other lines, the somatic embryos developed roots, but not shoots. Shoot regeneration via somatic embryogenesis offers potential as anin vitro system for physiological studies, including assessments of the response of common reed to environmental pollutants.

The World Saffron and Crocus collection: strategies for establishment, management, characterisation and utilisation

Since 2007, the European Commission AGRI GEN RES 018 “CROCUSBANK” action has permitted the creation of the alleged World Saffron and Crocus Collection (WSCC), a unique collection which contains a representation of the genetic variability present in saffron crop and wild relatives at global scale. At present the germplasm collection, housed at the Bank of Plant Germplasm of Cuenca (BGV-CU, Spain), consists of 572 preserved accessions representing 47 different Crocus species (including saffron Crocus) and is expected to increase up to more than 600 accessions by the end of CROCUSBANK action (May 2011). The preserved biodiversity of saffron (Crocussativus L.) covers a wide range of the genetic variability of the crop and currently consists of 220 accessions from 15 countries: 169 of these come from European cultivation countries, 18 from commercial areas in non EU countries, 26 from regions of minimal or relict production and/or from abandoned fields and 7 from commercial nurseries. The non-saffron Crocus collection currently comprises 352 accessions: 179 collected from the wild in 12 countries of natural distribution, 24 from donations of public and private institutions, 91 from commercial nurseries and 58 acquired from BGV-CU collection management. Here we provide a record of collections, activities concerns and current strategies for documentation, conservation, characterisation, and management of the collection as important tools for researchers with interest in these valuable genetic resources.

The World Saffron and Crocus collection: Strategies for establishment, management, characterisation and utilisation

Since 2007, the European Commission AGRI GEN RES 018 “CROCUSBANK” action has permitted the creation of the alleged World Saffron and Crocus Collection (WSCC), a unique collection which contains a representation of the genetic variability present in saffron crop and wild relatives at global scale. At present the germplasm collection, housed at the Bank of Plant Germplasm of Cuenca (BGV-CU, Spain), consists of 572 preserved accessions representing 47 different Crocus species (including saffron Crocus) and is expected to increase up to more than 600 accessions by the end of CROCUSBANK action (May 2011). The preserved biodiversity of saffron (Crocussativus L.) covers a wide range of the genetic variability of the crop and currently consists of 220 accessions from 15 countries: 169 of these come from European cultivation countries, 18 from commercial areas in non EU countries, 26 from regions of minimal or relict production and/or from abandoned fields and 7 from commercial nurseries. The non-saffron Crocus collection currently comprises 352 accessions: 179 collected from the wild in 12 countries of natural distribution, 24 from donations of public and private institutions, 91 from commercial nurseries and 58 acquired from BGV-CU collection management. Here we provide a record of collections, activities concerns and current strategies for documentation, conservation, characterisation, and management of the collection as important tools for researchers with interest in these valuable genetic resources.