Isolation and characterization of cysteine-degrading and H2S-releasing proteins in higher plants

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Brassica napus L. cultivars show a broad variability in their morphology, physiology and metabolite levels in response to sulfur limitations and to pathogen attack

Under adequate sulfur supply, plants accumulate sulfate in the vacuoles and use sulfur-containing metabolites as storage compounds. Under sulfur-limiting conditions, these pools of stored sulfur-compounds are depleted in order to balance the nitrogen to sulfur ratio for protein synthesis. Stress conditions like sulfur limitation and/or pathogen attack induce changes in the sulfate pool and the levels of sulfur-containing metabolites, which often depend on the ecotypes or cultivars. We are interested in investigating the influence of the genetic background of canola (Brassica napus) cultivars in sulfur-limiting conditions on the resistance against Verticillium longisporum. Therefore, four commercially available B. napus cultivars were analyzed. These high-performing cultivars differ in some characteristics described in their cultivar pass, such as several agronomic traits, differences in the size of the root system, and resistance to certain pathogens, such as Phoma and Verticillium. The objectives of the study were to examine and explore the patterns of morphological, physiological and metabolic diversity in these B. napus cultivars at different sulfur concentrations and in the context of plant defense. Results indicate that the root systems are influenced differently by sulfur deficiency in the cultivars. Total root dry mass and length of root hairs differ not only among the cultivars but also vary in their reaction to sulfur limitation and pathogen attack. As a sensitive indicator of stress, several parameters of photosynthetic activity determined by PAM imaging showed a broad variability among the treatments. These results were supported by thermographic analysis. Levels of sulfur-containing metabolites also showed large variations. The data were interrelated to predict the specific behavior during sulfur limitation and/or pathogen attack. Advice for farming are discussed

Quantitative expression analysis in Brassica napus by Northern blot analysis and reverse transcription-quantitative PCR in a complex experimental setting

Analysis of gene expression is one of the major ways to better understand plant reactions to changes in environmental conditions. The comparison of many different factors influencing plant growth challenges the gene expression analysis for specific gene-targeted experiments, especially with regard to the choice of suitable reference genes. The aim of this study is to compare expression results obtained by Northern blot, semi-quantitative PCR and RT-qPCR, and to identify a reliable set of reference genes for oilseed rape (Brassica napus L.) suitable for comparing gene expression under complex experimental conditions. We investigated the influence of several factors such as sulfur deficiency, different time points during the day, varying light conditions, and their interaction on gene expression in oilseed rape plants. The expression of selected reference genes was indeed influenced under these conditions in different ways. Therefore, a recently developed algorithm, called GrayNorm, was applied to validate a set of reference genes for normalizing results obtained by Northern blot analysis. After careful comparison of the three methods mentioned above, Northern blot analysis seems to be a reliable and cost-effective alternative for gene expression analysis under a complex growth regime. For using this method in a quantitative way a number of references was validated revealing that for our experiment a set of three references provides an appropriate normalization. Semi-quantitative PCR was prone to many handling errors and difficult to control while RT-qPCR was very sensitive to expression fluctuations of the reference genes

The Diurnal Rhythm of Brassica napus L. Influences Contents of Sulfur-Containing Defense Compounds and Occurrence of Vascular Occlusions during an Infection with Verticillium longisporum

Reduction in atmospheric sulfur and intensified agriculture have led to sulfur deficiency, often correlated with a higher susceptibility to pathogens. The spread of fungal pathogens, such as the soil-born Verticillium longisporum, was observed. Defense responses of infected plants are linked to sulfur-containing compounds including glucosinolates (GSLs). Some pathogens infect their hosts at specific time periods during the day. To investigate the relation of sulfur-containing metabolites with diurnal effects of infection time points, Brassica napus plants cultivated at two different sulfur supplies, were infected with V. longisporum at four different time points during the day. It was demonstrated that 3, 7 and 14 days after inoculation the infected plants differed in their infection rate depending on the time point of infection. Additionally, infected plants had higher contents of sulfur-containing metabolites, such as specific GSLs, in comparison to non-infected plants. Sufficient sulfur fertilization was always reflected in higher contents of sulfur-containing compounds as well as a lower rate of infection compared to sulfur-deprived plants. On the microscopic level vascular occlusions in the hypocotyl were visible and the amount was dependent on the time point of infection. The results might be used to optimize sulfur fertilization to reduce susceptibility to V. longisporum

Chronophin regulates active vitamin B6 levels and transcriptomic features of glioblastoma cell lines cultured under non-adherent, serum-free conditions

Background: The phosphatase chronophin (CIN/PDXP) has been shown to be an important regulator of glioma cell migration and invasion. It has two known substrates: p-Ser3-cofilin, the phosphorylated form of the actin binding protein cofilin, and pyridoxal 5′-phosphate, the active form of vitamin B6. Phosphoregulation of cofilin, among other functions, plays an important role in cell migration, whereas active vitamin B6 is a cofactor for more than one hundred enzymatic reactions. The role of CIN has yet only been examined in glioblastoma cell line models derived under serum culture conditions. Results: We found that CIN is highly expressed in cells cultured under non-adherent, serum-free conditions that are thought to better mimic the in vivo situation. Furthermore, the substrates of CIN, p-Ser3-cofilin and active vitamin B6, were significantly reduced as compared to cell lines cultured in serum-containing medium. To further examine its molecular role we stably knocked down the CIN protein with two different shRNA hairpins in the glioblastoma cell lines NCH421k and NCH644. Both cell lines did not show any significant alterations in proliferation but expression of differentiation markers (such as GFAP or TUBB3) was increased in the knockdown cell lines. In addition, colony formation was significantly impaired in NCH644. Of note, in both cell lines CIN knockdown increased active vitamin B6 levels with vitamin B6 being known to be important for S-adenosylmethionine biosynthesis. Nevertheless, global histone and DNA methylation remained unaltered as was chemoresistance towards temozolomide. To further elucidate the role of phosphocofilin in glioblastoma cells we applied inhibitors for ROCK1/2 and LIMK1/2 to our model. LIMK- and ROCK-inhibitor treatment alone was not toxic for glioblastoma cells. However, it had profound, but antagonistic effects in NCH421k and NCH644 under chemotherapy. Conclusion: In non-adherent glioblastoma cell lines cultured in serum-free medium, chronophin knockdown induces phenotypic changes, e.g. in colony formation and transcription, but these are highly dependent on the cellular background. The same is true for phenotypes observed after treatment with inhibitors for kinases regulating cofilin phosphorylation (ROCKs and LIMKs). Targeting the cofilin phosphorylation pathway might therefore not be a straightforward therapeutic option in glioblastoma

The levels of sulfur-containing metabolites in Brassica napus are not influenced by the circadian clock but diurnally

Adapting biological processes to an endogenous rhythm enables plants to cope with the daily changes in light and temperature in a more predictable way enhancing growth and fitness. A number of biological processes such as metabolic pathways as well as the immunity in plants are under diurnal or circadian control. In this study a possible circadian regulation of key enzymes in the sulfur assimilation and the corresponding metabolites was investigated in the agriculturally important crop plant oilseed rape (Brassica napus). Leaves of a commercially available cultivar were harvested in the course of a day under diurnal and under free-running conditions with constant light. Analyses in this study were focused on sulfur-containing metabolites and expression analysis of enzymes involved in sulfur assimilation. Expression analysis showed that the transcript levels of the sulfate transporters Sultr3;1 and Sultr4;2 as well as APR2 and APR3 oscillated diurnally. Results revealed a periodic rhythm of sulfur-containing metabolites such as glutathione, sulfate and certain glucosinolates in the course of a day which were only partly maintained under constant light. Therefore, we conclude that a diurnal rhythm and not the circadian clock regulates sulfur metabolism in plants

Multiancestry analysis of the HLA locus in Alzheimer’s and Parkinson’s diseases uncovers a shared adaptive immune response mediated by HLA-DRB1*04 subtypes

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson’s disease (PD) and Alzheimer’s disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1*04 subtypes best accounted for the association, strongest with HLA-DRB1*04:04 and HLA-DRB1*04:07, and intermediary with HLA-DRB1*04:01 and HLA-DRB1*04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1*04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1*04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues

Designing a competence-oriented teachinglearning arrangement for modeling flower models by means of 3D printing:

Die Umsetzung der Digitalisierung von Schule und Unterricht ist mit Herausforderungen für Lehrkräfte verbunden.Sie müssen die nötige technische Kompetenz aufweisen, um neue Technologien wie computergestützte Modellierung und 3D-Druck sinnvoll und gewinnbringend in ihren Unterricht integrieren können. Um Lehrkräfte zu entlasten, ist ein Angebot von didaktisch legitimierten und in der Praxis erprobten Unterrichtskonzepten notwendig. Eine wichtige Grundlage bei diesem Prozess bilden Modellprojekte für innovativen Biologieunterricht. Im Rahmen eines solchen Projektes wurden von Schüler/-innen Blütenmodelle ausgehend von realen Blüten erstellt. Der Lernerfolg wurde wissenschaftlich ausgewertet und ein dazugehöriges Unterrichtskonzept für Lehrkräfte weiterentwickelt. Die Studie konnte zeigen, dass sich der Projektunterricht positiv auf das botanische Fachwissen auswirkt.Implementing the digitalization of schools and teaching involves challenges for teachers. They need the necessary technical expertise to be able to integrate new technologies such as computer-aided modeling and 3D printing into their lessons in a meaningful and profitable way. In order to support teachers, it is necessary to offer didactic legitimate teaching concepts that have been tested in practice. Model projects for innovative biology teaching form an important basis for this process. Within the framework of such a project, students create flower models based on real flowers. The learning success was evaluated and a corresponding teaching concept further developed. The study showed that project teaching has a positive effect on botanical knowledge

Impact of Reduced O-Acetylserine(thiol)lyase Isoform Contents on Potato Plant Metabolism

Plant cysteine (Cys) synthesis can occur in three cellular compartments: the chloroplast, cytoplasm, and mitochondrion. Cys formation is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL) using O-acetylserine (OAS) and sulfide as substrates. To unravel the function of different isoforms of OASTL in cellular metabolism, a transgenic approach was used to down-regulate specifically the plastidial and cytosolic isoforms in potato (Solanum tuberosum). This approach resulted in decreased RNA, protein, and enzymatic activity levels. Intriguingly, H(2)S-releasing capacity was also reduced in these lines. Unexpectedly, the thiol levels in the transgenic lines were, regardless of the selected OASTL isoform, significantly elevated. Furthermore, levels of metabolites such as serine, OAS, methionine, threonine, isoleucine, and lysine also increased in the investigated transgenic lines. This indicates that higher Cys levels might influence methionine synthesis and subsequently pathway-related amino acids. The increase of serine and OAS points to suboptimal Cys synthesis in transgenic plants. Taking these findings together, it can be assumed that excess OASTL activity regulates not only Cys de novo synthesis but also its homeostasis. A model for the regulation of Cys levels in plants is proposed