István Gyurján

Pigment composition and plastid structure in leaves of carotenoid mutants of maize

SummaryIn monogenic, recessive chloroplast mutants of maize which contain chlorophylls, and lycopene or χ-carotene but no normal carotenoids, great variability in the size of plastids was associated with a number of ultrastructural abnormalities. In the mutant accumulating lycopene some plastids contain dense bundles of lamellae, whereas the chloroplasts of the χ-carotene mutant show poor thylakoid development. Neither of the mutants was able to form normal grana.A comparison of chlorophyll/carotenoid ratios in different chloroplast fractions of normal and mutant leaves showed that plastids of small size and delicate structure contain relatively less chlorophyll than fully differentiated chloroplasts.

GLC and GLC-mS analysis of thiophene derivatives in plants and in in vitro cultures of Tagetes patula L. (Asteraceae).

The occurrence of thiophenic compounds in diverse plant organs and in in vitro root-, callus- and cell suspension cultures of Tagetes patula cv. Carmen was investigated using capillary GLC and GLC-MS. The separation of thiophenes by capillary GLC and the group specific MS fragmentation with the typical sulfur isotope peaks allowed the unequivocal assignment of individual thiophenes in complex mixtures, even when occurring in traces and in the presence of different geometrical isomers. The extracts of Tagetes patula cv. Carmen contained the following 8 thiophene compounds: 5-(3-buten-1-ynyl)-2,2′-bithienyl (BBT), 5′-methyl-5-(3-buten-1-ynyl)-2,2′-bithienyl (MeBBT), 5-(1-pentynyl)-2,2′-bithienyl (PBT), 5-(4-hydroxy-1-butynyl)-2,2′-bithienyl (BBTOH), 2,2′,5,2″-terthienyl (α-T), 5-(4-acetoxy- 1-butynyl)-2,2′-bithienyl (BBTOAc), 5-methylaceto-5′-(3-buten-1-ynyl)-2,2′-bithienyl (AcOCH2BBT), and 5-(3,4-diacetoxy-1-butynyl)-2,2′-bithienyl (BBT(OAc)2). The most complex thiophene profile, including the less common PBT was detected in aerial parts of freshly harvested plant material. Under in vitro conditions only the root cultures, but not callus or cell suspension cultures produced substantial amounts of irregular thiophenes confirming that roots are the main site of thiophene biosynthesis.

Identifying novel genes involved in both deer physiological and human pathological osteoporosis

Osteoporosis attacks 10% of the population worldwide. Humans or even the model animals of the disease cannot recover from porous bone. Regeneration in skeletal elements is the unique feature of our newly investigated osteoporosis model, the red deer (Cervus elaphus) stag. Cyclic physiological osteoporosis is a consequence of the annual antler cycle. This phenomenon raises the possibility to identify genes involved in the regulation of bone mineral density on the basis of comparative genomics between deer and human. We compare gene expression activity of osteoporotic and regenerating rib bone samples versus autumn dwell control in red deer by microarray hybridization. Identified genes were tested on human femoral bone tissue from non-osteoporotic controls and patients affected with age-related osteoporosis. Expression data were evaluated by Principal Components Analysis and Canonical Variates Analysis. Separation of patients into a normal and an affected group based on ten formerly known osteoporosis reference genes was significantly improved by expanding the data with newly identified genes. These genes include IGSF4, FABP3, FABP4, FKBP2, TIMP2, TMSB4X, TRIB, and members of the Wnt signaling. This study supports that extensive comparative genomic analyses, here deer and human, provide a novel approach to identify new targets for human diagnostics and therapy.

Acid phosphatase isoenzymes of Chlamydomonas reinhardii

SummaryAcid phosphatase isoenzymes of Chlamydomonas reinhardii were investigated by isoelectric focusing in polyacrylamide gel systems. In this paper we describe in detail an original method for isoelectric focusing of acid phosphatases extracted from wildtype and acid phosphatase-lacking mutant algae, obtained from Laboratoire de Génetique of University of Liège. Three isoenzymes can be separated from the buffer-soluble components of these cells. An additional isoenzyme type can be visualized using the nonionic detergent NP40 as solubilizer. We conclude that these four isoenzymes are releated to the structural gene of the soluble constitutive acid phosphatase, which was shown by their appearance in P2 and their total absence in mutant Pa. The pl values of soluble constitutive acid phosphatase isoenzymes range between pH 5.2 and 6.2. As a result of treatment with NP40 the extracts from both wild-type and mutant lines contain two additional active phosphatase forms which can be characterized by their high heat resistance and low pI values. These enzymes are fully active using either α-naphthyl phosphate or different acetate esters as substrates.

Artificial Tripartite Symbiosis Involving a Green Alga (Chlamydomonas), a Bacterium (Azotobacter) and a Fungus (Alternaria): Morphological and Physiological Characterization

A long-living artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria) was established on carbon- and nitrogen-free medium. The basis of the interdependence is the complementation of photosynthetic CO2 assimilation and atmospheric nitrogen fixation. Green color of the colonies indicated that the algal cells had enough nitrogen to synthesize chlorophylls. The chlorophyll content was nearly 40 % of the control cells. The relatively high rate of photosynthetic oxygen evolution proved that nitrogen was effectively used for building up a well functioning photosynthetic apparatus. This was supported by the analysis of photosystems and ultrastructural investigations. In comparison with degreened algae cultured on nitrogen-free medium, the chloroplasts in the symbiont algal cells contained a well-developed, stacked thylakoid membrane system without extreme starch or lipid accumulation. The occurrence of the fungus in the association greatly increased the chlorophyll content. Far fewer types of amino acids were excreted by the tripartite cultures than by pure cultures. Cystathionine, which is a common intermediate in the sulfur-containing amino acid metabolism, was produced in high quantities by the tripartite symbiosis. This can mostly be attributed to the activity of the fungus.

Antler development and coupled osteoporosis in the skeleton of red deer Cervus elaphus: expression dynamics for regulatory and effector genes

Antlers of deer display the fastest and most robust bone development in the animal kingdom. Deposition of the minerals in the cartilage preceding ossification is a specific feature of the developing antler. We have cloned 28 genes which are upregulated in the cartilaginous section (called mineralized cartilage) of the developing (“velvet”) antler of red deer stags, compared to their levels in the fetal cartilage. Fifteen of these genes were further characterized by their expression pattern along the tissue zones (i.e., antler mesenchyme, precartilage, cartilage, bone), and by in situ hybridization of the gene activities at the cellular level. Expression dynamics of genes col1A1, col1A2, col3A1, ibsp, mgp, sparc, runx2, and osteocalcin were monitored and compared in the ossified part of the velvet antler and in the skeleton (in ribs and vertebrae). Expression levels of these genes in the ossified part of the velvet antler exceeded the skeletal levels 10–30-fold or more. Gene expression and comparative sequence analyses of cDNAs and the cognate 5′ cis-regulatory regions in deer, cattle, and human suggested that the genes runx2 and osx have a master regulatory role. GC–MS metabolite analyses of glucose, phosphate, ethanolamine-phosphate, and hydroxyproline utilizations confirmed the high activity of mineralization genes in governing the flow of the minerals from the skeleton to the antler bone. Gene expression patterns and quantitative metabolite data for the robust bone development in the antler are discussed in an integrated manner. We also discuss the potential implication of our findings on the deer genes in human osteoporosis research.

In vitro establishment of nitrogen-fixing strawberry (Fragaria × ananassa) via artificial symbiosis with Azomonas insignis

SummaryArtificial symbiosis was established between diazotrophic Azomonas insignis and strawberry (Fragaria × ananassa). The partnership was created by in vitro techniques through callus induction and organogenesis. Suitable micropropagation [M3=Murashige and Skoog (1962) (MS) basal medium supplemented with 2.5 µM N6-benzyladenine (BA), 0.3 µM gibberellic acid (GA3), 2.2 µM indole-3-butyric acid (IBA), and 3% sucrose] and plant regeneration [R3=MS mineral salts+555 µM myo-inositol, 1.2 µM thiamine HCl, 4.4 µM BA, 0.5 µM IBA, 0.3 µM α-naphthaleneacetic acid (NAA), 0.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D)] media were developed for the test cultivar Fertödi F5. New shoots containing bacteria were rooted, acclimatized, and planted outdoors. The basis of the partnership during the in vitro phase is the bacterial dependence on the plant metabolic activity, using maltose in the medium as carbon and energy source that can be utilized by the plant cells only. The presence of bacteria in the intercellular spaces of the callus tissues and regenerated plants was proved by re-isolation and microscopic techniques. Nitrogenase activity was also detected in the plant tissues.

CHARACTERISTICS OF PIGMENT-PROTEIN COMPLEXES IN NORMAL AND CHLOROPLAST MUTANT LEAVES*

Abstract— The structural stability of chloroplasts was characterized by the proportion of leaf pigments present in the lipoprotein complex.

Structural and functional changes in the photosynthetic apparatus of Chlamydomonas reinhardtii during nitrogen deprivation and replenishment

Nitrogen is an essential factor for normal plant and algal development. As a component of nucleic acids, proteins, and chlorophyll (Chl) molecules, it has a crucial role in the organization of a functioning photosynthetic apparatus. Our aim was to study the effects of nitrogen starvation in cultures of the unicellular green alga, Chlamydomonas reinhardtii, maintained on nitrogen-free, and then on nitrogen-containing medium. During the three-week-long degreening process, considerable changes were observed in the Chl content, the ratio of Chl-protein complexes, and photosynthetic activity of the cultures as well as in the ultrastructure of single chloroplasts. The regreening process was much faster then the degradation; total greening of the cells occurred within four days. The rate of regeneration depended on the nitrogen content. At least 50% of the normal nitrogen content of Tris-Acetate-Phosphate (TAP) medium was required in the medium for the complete regreening of the cells and regeneration of chloroplasts.

Altered chloroplast ribosomal proteins in a yellow mutant of Chlamydomonas reinhardii

SummaryRibosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analysed and compared by two-dimensional gel electrophoresis.Mixothrophycally grown yellow-27 mutant differs from wild-type cells in lowered chlorophyll content and grana fromation of the chloroplast.Analytical ultracentrifuge analyses of cell extracts show a reduced amount of free 70S ribosomes and increased level of 50S subunits in the mutant cells. Similar results were obtained by electronmicroscopical method.Two-dimensional gel electrophoresis shows alterations in protein composition of 70S ribosomes of the mutant. Two proteins of 70S ribosomes have been altered. One of them with high molecular weight is practically absent while there is an additional, intensively stained spot in the mutant.Since the mutation is inherited in a non-Mendelian manner it is possible that the protein alterations in 70S ribosome are localized in the chloroplast DNA.