Biomedical subjects

Karin Krupinska

Publications and source records attributed to Karin Krupinska.

18 recordsLinked to original sources

PubMed2006

An alpha-galactosidase with an essential function during leaf development.

The putative alpha-galactosidase gene HvSF11 of barley, previously shown to be expressed during dark induced senescence, is expressed in the growing/elongating zone of primary foliage leaves of barley. The amino acid sequence deduced from the full length HvSF11 cDNA contains a hydrophobic signal sequence at the N-terminus. Phylogenetic relationship of the HvSF11 encoded barley alpha-galactosidase to other alpha-galactosidases revealed high homology with the alpha-galactosidase encoded by the gene At5g08370 from Arabidopsis thaliana. We have isolated two independent heterozygous At5g08370 T-DNA insertion mutants from Arabidopsis thaliana, both of which have a higher number of rosette leaves with a curly surface leaf morphology and delayed flowering time in comparison to wildtype plants. Localization of the Arabidopsis alpha-galactosidase protein via GUS-tag revealed that the protein is associated with the cell wall. This result was confirmed by immunological detection of the orthologous barley protein in a protein fraction derived from cell walls of barley leaves. It is concluded that the alpha-galactosidase proteins from barley and Arabidopsis might fulfill an important role in leaf development by functioning in cell wall loosening and cell wall expansion.

Amino Acid Sequence↗

PubMed2006

A plant locus essential for phylloquinone (vitamin K1) biosynthesis originated from a fusion of four eubacterial genes.

Phylloquinone is a compound present in all photosynthetic plants serving as cofactor for Photosystem I-mediated electron transport. Newly identified seedling-lethal Arabidopsis thaliana mutants impaired in the biosynthesis of phylloquinone possess reduced Photosystem I activity. The affected gene, called PHYLLO, consists of a fusion of four previously individual eubacterial genes, menF, menD, menC, and menH, required for the biosynthesis of phylloquinone in photosynthetic cyanobacteria and the respiratory menaquinone in eubacteria. The fact that homologous men genes reside as polycistronic units in eubacterial chromosomes and in plastomes of red algae strongly suggests that PHYLLO derived from a plastid operon during endosymbiosis. The principle architecture of the fused PHYLLO locus is conserved in the nuclear genomes of plants, green algae, and the diatom alga Thalassiosira pseudonana. The latter arose from secondary endosymbiosis of a red algae and a eukaryotic host indicating selective driving forces for maintenance and/or independent generation of the composite gene cluster within the nuclear genomes. Besides, individual menF genes, encoding active isochorismate synthases (ICS), have been established followed by splitting of the essential 3' region of the menF module of PHYLLO only in genomes of higher plants. This resulted in inactivation of the ICS activity encoded by PHYLLO and enabled a metabolic branch from the phylloquinone biosynthetic route to independently regulate the synthesis of salicylic acid required for plant defense. Therefore, gene fusion, duplication, and fission events adapted a eubacterial multienzymatic system to the metabolic requirements of plants.

Algal Proteins↗

PubMed2005

Chloroplast ultrastructure in leaves of Urtica dioica L. analyzed after high-pressure freezing and freeze-substitution and compared with conventional fixation followed by room temperature dehydration.

In this article, we report on the adaptation of high-pressure freezing and freeze-substitution (HPF-FS) for ultrastructural analysis of leaf tissue with special emphasis on chloroplasts. To replace the gas in the intercellular spaces, a mixture of water and methanol (MeOH) was employed. We compared three different supplements for FS--osmiumtetroxide, uranyl acetate, and safranin--with regard to the preservation of the ultrastructure of chloroplasts and other cellular compartments. The results show that (i) replacement of air within intercellular spaces by 8% (v/v) MeOH has no influence on the ultrastructure of the chloroplasts, (ii) undulation of membranes frequently observed after conventional preparation of specimens does not occur during chemical fixation but during room temperature dehydration, and (iii) uranyl acetate or osmium tetroxide employed during FS are not superior over safranin.

Atmospheric Pressure↗

PubMed2005

DNA-binding proteins of the Whirly family in Arabidopsis thaliana are targeted to the organelles.

Arabidopsis thaliana contains three genes with high homology to potato p24 which was described as a member of the Whirly family of nuclear transcriptional activators. Computer-based analysis revealed that all Arabidopsis Whirly (Why) proteins contain targeting sequences for either plastids or mitochondria. The functionality of these sequences was demonstrated by in vitro import assays into isolated organelles. Transient expression of GFP fusion proteins in protoplasts and onion epidermal cells confirmed the localisation of these proteins in plastids or mitochondria, respectively. The possession of organellar targeting sequences seems to be conserved among Why proteins of higher plant species, including potato p24.

Arabidopsis↗

PubMed2005

Characterization of transplastomic tobacco plants with a plastid localized barley 4-hydroxyphenylpyruvate dioxygenase.

The 4-hydroxyphenylpyruvate dioxygenase (HPD) is the only enzyme of the biosynthetic pathway of tocopherols and tocotrienols localized outside of plastids. In order to investigate whether a plastid localized HPD could give rise to an increase in tocopherol levels of tobacco leaves and seeds, in the present study the HPD enzyme was transferred to the plastid by introducing a cDNA specific for the barley hpd gene into the plastome of tobacco. The expression of the hpd gene expression cassette was demonstrated by accumulation of the hpd gene-specific transcript and by a higher resistance of the transgenic seedlings towards the HPD specific inhibitor sulcotrione. The alpha-tocopherol content was increased in leaves of the transplastomic plants, whereas the transplastomic seeds contained a significantly increased gamma-tocochromanol level. With respect to enhanced accumulation of the tocopherol content in plants tissue, overexpression of the hpd gene in plastids did not prove to be advantageous in comparison to transgenic plants expressing high HPD level in the cytoplasm. It is hypothesized that homogentisate synthesized in plastids will have to pass the envelope membrane in order to be accessible to the following enzymes of the tocopherol biosynthetic pathway.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed2005

The impact of tocochromanols on early seedling development and NO release.

Plant seeds and fruits are the main source for tocochromanols (tocopherols and tocotrienols) collectively known as Vitamin E in human nutrition. Seeds are particularly rich in gamma-tocopherol. The reason for the abundance of gamma-tocopherol in seeds is not yet clear. We analysed the influence of endogenous gamma-tocopherols on early development of seedlings from various barley cultivars. For this purpose progression of seedling development was monitored by the mean root length 48 h after imbibition. Our observations suggest that endogenous gamma-tocopherol has a negative impact on seedling development by controlling germination and postgermination events. We propose that gamma-tocopherol exerts its influence on seedling development by controlling the content of nitric oxide (NO) in germinating seeds.

Germination↗

PubMed2005

Tocochromanol content and composition in different species of the parasitic flowering plant genus Cuscuta.

The holoparasitic plant genus Cuscuta is comprised of species with various degrees of plastid functionality and significant differences in photosynthetic capacity, ranging from moderate to no photosynthetic carbon fixation. In the present study, several Cuscuta species were analyzed with respect to the overall contents of tocochromanols and plastoquinone and the levels of the individual tocochromanols. No correlations among photosynthetic capacity, the amount of carotenoids, of plastoquinone and of tocochromanols were observed. On the contrary, wide variation in the composition of the tocochromanol fraction was observed among different species, as well as in stems of the same species in response to starvation conditions. The implications of these findings are discussed.

Cuscuta↗

PubMed2005

New insights in thylakoid membrane organization.

High-pressure freezing (HPF) in combination with freeze substitution (FS) was used to analyse changes in the structure of barley chloroplasts during the daily change of light and darkness. In contrast to conventional treatment of samples, HPF-FS revealed substantial differences in chloroplast shape, volume and ultrastructure in the light period and during darkness. While chloroplasts have an ellipsoidal shape in the light, they have an enlarged and round form during the dark period. Samples collected in the light show the typical differentiation of stroma and grana thylakoids as observed by conventional ultrastructural analyses. In chloroplasts of samples collected during the dark period, thylakoids were swollen and grana stacks to a large extent were disintegrated. Similar changes occurred when leaves in the light were treated with the uncoupler gramicidin. The results suggest that the light-dependent changes in thylakoid membrane organization are related to the light-dependent changes in the ionic milieu of the thylakoid lumen and the stroma.

Chloroplasts↗

PubMed2004

The rbcL genes of two Cuscuta species, C. gronovii and C. subinclusa, are transcribed by the nuclear-encoded plastid RNA polymerase (NEP).

Some species of the holoparasitic flowering plant genus Cuscuta, like C. reflexa, have retained a plastid genome that encodes photosynthesis-related gene products as well as the plastid-encoded RNA polymerase (PEP). In contrast, other species like C. gronovii and C. subinclusa have lost the rpo genes coding for the PEP subunits while photosynthetic genes have been retained. In order to ensure expression of the photosynthesis-related genes in the absence of PEP, a number of adaptations within the plastid genome were required that enable gene transcription mediated exclusively by the nuclear-encoded plastid RNA polymerase (NEP). In this study we analyzed promoter sequence conservation and transcription start sites of a typical PEP gene of non-parasitic plants, rbcL, which codes for the large subunit of ribulose bisphosphate carboxylase/oxygenase. We show that despite high sequence conservation of the coding region of rbcL among different Cuscuta species and tobacco, the 5' non-coding regions of C. gronovii and C. subinclusa have suffered extensive deletions encompassing the PEP promoter that is present in C. reflexa and tobacco. Primer-extension analyses enabled the identification of transcripts initiated at NEP promoter motifs in C. gronovii and C. subinclusa that are not detectable in the 5' non-coding region of C. reflexa.

Amino Acid Sequence↗

PubMed2004

Tocopherol and tocotrienol accumulation during development of caryopses from barley (Hordeum vulgare L.).

Tocotrienols are lipophilic antioxidants belonging to the tocochromanols, better known as vitamin E. Although present in cereal grains in high quantities not much is known about their function in plants. In a detailed study the temporal and spatial accumulation of tocotrienols and tocopherols during grain development in two barley cultivars was analyzed. Tocochromanols and lipids accumulated in parallel until 80% of the final dry weight of the kernels was reached. Later on the tocochromanol content did not change while the lipid content decreased. Generally, only about 13% of the tocochromanols were found in the germ fraction, whereas the pericarp fraction contained about 50% and the endosperm fraction about 37% of the tocochromanols. Altogether, about 85% of the tocochromanols were tocotrienols in both cultivars. In case of the tocopherols about 80% were found in the germ fraction and the remaining 20% in the pericarp fraction. Tocotrienols were almost equally present in the pericarp and the endosperm fraction. Individual forms of tocopherols and tocotrienols accumulated with different kinetics during barley grain development. The differences in distribution and accumulation indicate different functions of the individual tocochromanols during grain development.

Crosses, Genetic↗

PubMed2004

The Etched1 gene of Zea mays (L.) encodes a zinc ribbon protein that belongs to the transcriptionally active chromosome (TAC) of plastids and is similar to the transcription factor TFIIS.

Etched1 (et1) is a pleiotropic, recessive mutation of maize that causes fissured and cracked mature kernels and virescent seedlings. Microscopic examinations of the et1 phenotype revealed an aberrant plastid development in mutant kernels and mutant leaves. Here, we report on the cloning of the et1 gene by transposon tagging, the localization of the gene product in chloroplasts, and its putative function in the plastid transcriptional apparatus. Several alleles of Mutator (Mu)-induced et1 mutants, the et1-reference (et1-R) mutant, and Et1 wild-type were cloned and analyzed at the molecular level. Northern analyses with wild-type plants revealed that Et1 transcripts are present in kernels, leaves, and other types of tissue, and no Et1 expression could be detected in the et1 mutants analyzed. The ET1 protein is imported by chloroplasts and has been immunologically detected in transcriptionally active chromosome (TAC) fractions derived from chloroplasts. Accordingly, the relative transcriptional activity of TAC fractions was significantly reduced in chloroplasts of et1-R plants. ET1 is the first zinc ribbon (ZR) protein shown to be targeted to plastids. With regard to its localization and its striking structural similarity to the eukaryotic transcription elongation factor TFIIS, it is feasible that ET1 functions in plastid transcription elongation by reactivation of arrested RNA polymerases.

Amino Acid Sequence↗

PubMed2003

Regulation of alpha-galactosidase gene expression in primary foliage leaves of barley ( Hordeum vulgare L) during dark-induced senescence.

alpha-Galactosidase activity (alpha- d-galactoside galactohydrolase, EC 3.2.1.22) increased during dark-induced senescence in primary foliage leaves of barley ( Hordeum vulgare L. cv. Steffi). The changes in activity were accompanied by parallel changes in expression of the HvSF11 gene encoding a putative alpha-galactosidase. The transcript level of HvSF23 encoding a second putative alpha-galactosidase stayed constant during leaf senescence. Both alpha-galactosidase activity and the level of the HvSF11 transcript decreased after exogenous application of sucrose and glucose to detached dark-incubated leaves. In contrast, the HvSF23 transcript level was not influenced by sugars. Application of glucose analogs to detached and dark-incubated leaves revealed that phosphorylation of hexose by hexokinase modulates both the alpha-galactosidase activity and the expression of HvSF11. These results indicate that the expression of the genes coding for two alpha-galactosidase isoenzymes is regulated by different signalling pathways, suggesting different functions for the two gene products.

Darkness↗

PubMed2003

Plastids of three Cuscuta species differing in plastid coding capacity have a common parasite-specific RNA composition.

The chlorophyll containing holoparasitic species Cuscuta reflexa, the achlorophyllous species Cuscuta odorata and the intermediate species Cuscuta gronovii, which contains only traces of chlorophyll, were compared with respect to their plastid coding capacity and plastid gene expression at the level of RNA. While extensive deletions have taken place in the plastid DNA of the achlorophyllous species C. odorata, the green species C. reflexa has retained an almost complete plastid genome. Although the plastid genome of the intermediate species C. gronovii has suffered extensive deletions, in contrast to the plastid genome of C. odorata it has retained photosynthesis-related genes. Hybridization with radioactive 3'-labelled RNA revealed that in all three species only a small 'parasite-specific' portion of the plastid genome consisting of mainly rRNAs and tRNAs is represented at the level of steady-state RNA. Run-on transcription assays revealed that in plastids of C. reflexa the entire genome is transcribed. Hence, the subset of RNA species required for a parasitic lifestyle is preferentially stabilized in Cuscuta plastids.

3' Untranslated Regions↗

PubMed2003

Constitutive overexpression of barley 4-hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of the vitamin E content in seeds but not in leaves.

With the aim to enhance the plant vitamin E content, the barley gene encoding 4-hydroxyphenylpyruvate dioxygenase was overexpressed in tobacco plants under control of the 35S promoter. Transgenic lines have a higher capacity for homogentisate biosynthesis as evident by a more than 10-fold higher resistance towards the bleaching herbicide sulcotrione. Seeds from transgenic lines have an up to two-fold enhanced level of vitamin E without a change in the ratio of gamma-tocopherol and gamma-tocotrienol. While the vitamin E content is not affected in leaves, the level of plastoquinone is enhanced in leaves of transgenic lines during leaf senescence.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed2003

The abundance of minor chlorophyll a/b-binding proteins CP29 and LHCI of barley (Hordeum vulgare L.) during leaf senescence is controlled by light.

The abundance of the minor light-harvesting complexes CP29 and LHCI generally declines during the senescence of barley leaves. When light intensity declined due to clouding during the senescence of flag leaves from barley plants grown under field conditions, the levels of both light-harvesting complexes temporarily increased in parallel with photosystem II-efficiency [F(v)/F(m)]. A sudden shift from high light conditions to low light conditions during the growth of barley plants in a growth chamber also resulted in an increase in the abundance of minor light-harvesting complexes and a parallel increase in F(v)/F(m) as well as in the chlorophyll a+b-content of senescing primary foliage leaves. Northern blot analyses with a cDNA probe specific for the barley Lhcb4 gene encoding CP29 showed that the light-dependent changes in the abundance of CP29 during senescence are paralleled by corresponding changes in the transcript level. The results indicate that adjustments of the levels of minor light-harvesting complexes during senescence under high light conditions may serve in the prevention of photo-oxidative damage to the photosynthetic reaction centres and under low light in ensuring efficient photosynthesis of the residual photosynthetic reaction centres.

Arabidopsis Proteins↗

PubMed2002

Plastid transcription in the holoparasitic plant genus Cuscuta: parallel loss of the rrn16 PEP-promoter and of the rpoA and rpoB genes coding for the plastid-encoded RNA polymerase.

The holoparasitic plant genus Cuscuta comprises a range of species whose plastid genomes have different degrees of reductions in their coding capacity. In this study, four Cuscuta species, Cuscuta reflexa, C. gronovii, C. subinclusa and C. odorata, that possess substantial physiological differences, were analysed with respect to the sequence and promoter structure of the rrn16 gene coding for the ribosomal 16S rRNA. Whereas the coding region of this gene is highly conserved among all four Cuscuta species, significant differences were observed in the non-coding region 5' of rrn16 with respect to both the length of the intergenic region between rrn16 and trnV and the promoters used to initiate transcription of the rrn16 gene. In the green species C. reflexa, rrn16 transcription starts from a functional plastid-encoded RNA polymerase (PEP) promoter that is missing in the other three species, C. gronovii, C. odorata and C. subinclusa. Instead, a 15-nucleotide-long conserved sequence immediately upstream of the mapped 5' ends bearing the nuclear-encoded RNA polymerase (NEP) promoter motif could be identified in these three species. The lack of a PEP promoter in these species coincides with the loss of two genes that encode subunits of PEP (rpoA and rpoB).

5' Flanking Region↗

PubMed2002

The hydroxyphenylpyruvate dioxygenase from Synechocystis sp. PCC 6803 is not required for plastoquinone biosynthesis.

The disruption of the Synechocystis open reading frame Deltaslr0090 encoding a gene with high homology to plant genes encoding 4-hydroxyphenylpyruvate dioxygenase results in an impairment of tocopherol biosynthesis without affecting levels of plastoquinone, carotenoids and chlorophyll as well as cell growth and photosynthesis. Our results indicate that in Synechocystis in contrast to the situation in higher plants the 4-hydroxyphenylpyruvate dioxygenase is not required for the synthesis of plastoquinone.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed2002

A novel nucleus-targeted protein is expressed in barley leaves during senescence and pathogen infection.

The barley (Hordeum vulgare) cDNA HvS40 represents a gene with enhanced mRNA level during leaf senescence. Biolistic transformation of onion (Allium cepa) epidermal cell layers with a glucuronidase fusion protein construct provided evidence that the 15.4-kD protein encoded by HvS40 is localized to the nucleus. Expression of the gene is induced by jasmonate and salicylic acid; both are known to act as signaling compounds during senescence and defense toward pathogens. Transcript levels of HvS40 were observed to be particularly high in leaf sectors that undergo necrosis and chlorosis after infection with Pyrenophora teres. This pathogen-related expression is, in contrast, clearly reduced in transgenic barley plants expressing a stilbene synthase from grape (Vitis vinifera), whereas the mRNA level of a gene encoding the pathogen-related protein HvPR1 is unaffected. In situ hybridization with HvS40 antisense RNA revealed that during leaf senescence, the HvS40 transcript predominantly accumulates in the mesophyll. Taken together, the findings suggest a connection between the nuclear protein encoded by HvS40 and the degeneration of chloroplasts occurring during senescence and during infection of barley wild-type plants with P. teres.

Amino Acid Sequence↗