Sándor Bottka

Production of transgenic maize plants by direct DNA uptake into embryogenic protoplasts

Abstract Fertile transgenic maize plants were regenerated after direct transfer of a chimeric gene into maize protoplasts. Plasmid DNA containing mutant dihydrofolate reductase (DHFR) mouse gene, that confers methotrexate (MTX) resistance, under the control of the CaMV 35S promoter was introduced into maize embryogenic protoplasts by polyethylene glycol (PEG) treatment. Transformation was also carried out with a modified plasmid in which the selective marker gene casette was cloned into the BstBI site of the Ds 1 maize transposable element. Resistant callus tissues grown in the presence of 10 −6 or 10 −7 M MTX were selected and shoot or plant regeneration was achieved under hormone-free culture conditions. The presence of the introduced DHFR gene in DNA isolated from the selected colonies and the primary regenerants (T 0 ) was shown by Southern hybridization and PCR analysis. PCR primers for the 35S promoter and for two regions of the coding sequence of the DHFR gene were used for amplification of the foreign sequence present in maize genomic DNA. The PCR products were hybridized with a mouse DHFR gene specific probe. Synthesis of the mouse DHFR in MTX resistant maize tissues was detected by staining for enzyme activity after native PAGE. The in vitro regenerated plants could be grown up to maturity in the greenhouse. Cross pollination has resulted in seeds and the F 1 progenies were also analyzed. In addition to the segregation of MTX-resistant and-sensitive offsprings, molecular evidences based on Southern data and PCR analysis have indicated that the introduced gene was transferred in the first sexual generation. This report provides a new example for potentials in the use of embryogenic cereal protoplasts for production of fertile transgenic crop plants.

Characterization of a family of Arabidopsis receptor-like cytoplasmic kinases (RLCK class VI).

The receptor-like cytoplasmic protein kinases (RLCKs) are plant-specific proteins encoded by almost 200 genes in the Arabidopsis genome. Despite of their high number, the available information on the potential function of RLCKs is very limited. In this report, the sequence analysis and the gene expression pattern of 14 members of one of the Arabidopsis RLCK families (RLCK class VI) are described. Sequence comparison indicated that gene duplication played a significant role in the formation of the kinase family and that several members carry an N-terminal “universal stress protein” (UspA) domain. In order to gain insight into the potential function of the RLCK VI kinases, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the relative transcript levels in the various organs of the Arabidopsis plant as well as under a series of abiotic stress/hormone treatments in seedlings. The obtained data revealed the differentially regulated expression of the genes in agreement with a high variability of sequence elements in their promoters. The divergent expression patterns indicate that the encoded kinase proteins may be involved in a wide variety of signal transduction pathways related to plant development and stress responses. The significance of gene duplication and expression divergence in the extension of the Arabidopsis RLCK VI family during evolution is discussed.

Histological and microarray analysis of the direct effect of water shortage alone or combined with heat on early grain development in wheat (Triticum aestivum)

Based on the in silico analysis of the representation of expressed sequence tags (ESTs) in wheat grain-related cDNA libraries, a specific 15k oligonucleotide microarray has been developed in order to monitor environmental stress-dependent gene expression changes in the wheat caryopses. Using this array, the effect of water withdrawal, with and without additional heat stress, has been investigated during the first five days of kernel development on two wheat cultivars differing in their drought sensitivity. Water shortage affected (more than twofold change) the expression of only 0.5% of the investigated genes. A parallel heat treatment increased the ratio of responding genes to 5-7% because of the temperature stress and/or the increased water deficit because of enhanced evaporation. It could be established that the two cultivars, differing in their long-term adaptation capabilities to drought, responded to the short and direct stress treatments on the same way. In response to the combined drought and heat treatment, the coordinately altered expression of genes coding for storage proteins, enzymes involved in sugar/starch metabolism, histone proteins, heat shock proteins, proteases, tonoplast aquaporins as well as several transcription factors has been observed. These gene expression changes were in agreement with histological data that demonstrated the accelerated development of the embryo as well as the endosperm.

The chl a fluorescence intensity is remarkably insensitive to changes in the chlorophyll content of the leaf as long as the chl a/b ratio remains unaffected.

The effects of changes in the chlorophyll (chl) content on the kinetics of the OJIP fluorescence transient were studied using two different approaches. An extensive chl loss (up to 5-fold decrease) occurs in leaves suffering from either an Mg(2+) or SO(4)(2-) deficiency. The effects of these treatments on the chl a/b ratio, which is related to antenna size, were very limited. This observation was confirmed by the identical light intensity dependencies of the K, J and I-steps of the fluorescence rise for three of the four treatments and by the absence of changes in the F(685 nm)/F(695 nm)-ratio of fluorescence emission spectra measured at 77K. Under these conditions, the F(0) and F(M)-values were essentially insensitive to the chl content. A second experimental approach consisted of the treatment of wheat leaves with specifically designed antisense oligodeoxynucleotides that interfered with the translation of mRNA of the genes coding for chl a/b binding proteins. This way, leaves with a wide range of chl a/b ratios were created. Under these conditions, an inverse proportional relationship between the F(M) values and the chl a/b ratio was observed. A strong effect of the chl a/b ratio on the fluorescence intensity was also observed for barley Chlorina f2 plants that lack chl b. The data suggest that the chl a/b ratio (antenna size) is a more important determinant of the maximum fluorescence intensity than the chl content of the leaf.

Hyperosmotic stress induces Axl activation and cleavage in cerebral endothelial cells

Because of the relative impermeability of the blood‐brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are β‐catenin, extracellular signal‐regulated kinase 1/2 and the non‐receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase‐ and proteasome‐dependent and resulted in 50–55 kDa C‐terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol‐treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity‐induced apoptosis. Our results identify Axl as an important element of osmotic stress‐induced signalling.

Mitosis-specific promoter of the alfalfa cyclin-dependent kinase gene (Medsa;CDKB2;1) is activated by wounding and ethylene in a non-cell division-dependent manner

Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases; however, their exact functions are still enigmatic. Here we describe the functional characterization of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analyzed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter indeed revealed the presence of a set of cis-elements, indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific CDK genes in plants.

Synthetic Antisense Oligodeoxynucleotides to Transiently Suppress Different Nucleus- and Chloroplast-Encoded Proteins of Higher Plant Chloroplasts

Selective inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) is widely applied in gene function analyses; however, experiments with ODNs in plants are scarce. In this work, we extend the use of ODNs in different plant species, optimizing the uptake, stability, and efficiency of ODNs with a combination of molecular biological and biophysical techniques to transiently inhibit the gene expression of different chloroplast proteins. We targeted the nucleus-encoded phytoene desaturase (pds) gene, encoding a key enzyme in carotenoid biosynthesis, the chlorophyll a/b-binding (cab) protein genes, and the chloroplast-encoded psbA gene, encoding the D1 protein. For pds and psbA, the in vivo stability of ODNs was increased by phosphorothioate modifications. After infiltration of ODNs into juvenile tobacco (Nicotiana benthamiana) leaves, we detected a 25% to 35% reduction in mRNA level and an approximately 5% decrease in both carotenoid content and the variable fluorescence of photosystem II. In detached etiolated wheat (Triticum aestivum) leaves, after 8 h of greening, the mRNA level, carotenoid content, and variable fluorescence were inhibited up to 75%, 25%, and 20%, respectively. Regarding cab, ODN treatments of etiolated wheat leaves resulted in an up to 59% decrease in the amount of chlorophyll b, a 41% decrease of the maximum chlorophyll fluorescence intensity, the cab mRNA level was reduced to 66%, and the protein level was suppressed up to 85% compared with the control. The psbA mRNA and protein levels in Arabidopsis (Arabidopsis thaliana) leaves were inhibited by up to 85% and 72%, respectively. To exploit the potential of ODNs for photosynthetic genes, we propose molecular design combined with fast, noninvasive techniques to test their functional effects.

Syndecan-4 promotes cytokinesis in a phosphorylation-dependent manner

During mitosis, cells detach, and the cell–matrix interactions become restricted. At the completion of cytokinesis, the two daughter cells are still connected transiently by an intercellular bridge (ICB), which is subjected to abscission, as the terminal step of cytokinesis. Cell adhesion to the matrix is mediated by syndecan-4 (SDC4) transmembrane heparan sulfate proteoglycan. Our present work demonstrated that SDC4 promotes cytokinesis in a phosphorylation-dependent manner in MCF-7 breast adenocarcinoma cells. The serine179-phosphorylation and the ectodomain shedding of SDC4 changed periodically in a cell cycle-dependent way reaching the maximum at G2/M phases. On the contrary, the phospho-resistant Ser179Ala mutant abrogated the shedding. The phosphorylated full-length and shed remnants enriched along the mitotic spindles, and subsequently in the ICBs, however, proper membrane insertion was necessary for midbody localization. Expression of phosphomimicking Ser179Glu SDC4 resulted in incomplete abscission, whereas expression of the phospho-resistant SDC4 led to giant, multinucleated cells.

Synthesis and Studies on Cell-Penetrating Peptides

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.

Diamino-Antraquinone: A New Intercalating Agent. Synthesis and Linking to Oligodeoxynucleotide

Abstract Phosphoramidite derivative of 1,4-diamino antraquinone was synthesized, characterized, and incorporated into oligonucleotides. Intercalative interaction between the dye and the nucleic acid was confirmed by CD spectroscopy.