Agnieszka Sirko

Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors.

Two main mechanisms of protein turnover exist in eukaryotic cells: the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Autophagy is an emerging important constituent of many physiological and pathological processes, such as response to nutrient deficiency, programmed cell death and innate immune response. In mammalian cells the selectivity of autophagy is ensured by the presence of cargo receptors, such as p62/SQSTM1 and NBR1, responsible for sequestration of the ubiquitinated proteins. In plants no selective cargo receptors have been identified yet. The present report indicates that structural and functional homologs of p62 and NBR1 proteins exist in plants. The tobacco protein, named Joka2, has been identified in yeast two-hybrid search as a binding partner of a small coiled-coil protein, a member of UP9/LSU family of unknown function, encoded by the UP9C gene strongly and specifically induced during sulfur deficiency. The typical domains of p62 and NBR1 are conserved in Joka2. Similarly to p62, Joka2-YFP has dual localization (cytosolic speckles and the nucleus); it forms homodimers and interacts with a member of the ATG8 family. Increased expression of Joka2 and ATG8f was observed in roots of tobacco plants grown for two days in nutrient-deficient conditions. Constitutive ectopic expression of Joka2-YFP in tobacco resulted in attenuated response (manifested by lesser yellowing of the leaves) to nutrient deficiency. In conclusion, Joka2, and presumably the process of selective autophagy, might constitute an important part of plant response to environmental stresses.

A highly sensitive electrochemical genosensor based on Co-porphyrin-labelled DNA

We report the use of Co-porphyrins as electrochemical tags for a highly sensitive and selective genosensor. An avian influenza virus-based DNA sequence characteristic of H5N1 was detected at femtomolar levels from competing non-complementary sequences through hybridisation with the labeled DNA.

Electrochemical immunosensor for detection of antibodies against influenza A virus H5N1 in hen serum

This paper describes the development of an immunosensor for detection of anti-hemagglutinin antibodies. Its preparation consists of successive modification steps of glassy carbon electrodes: (i) creation of COOH groups, (ii) covalent immobilization of protein A with EDC/NHS coupling reaction, (iii) covering with anti-His IgG monoclonal antibody, (iv) immobilization of the recombinant His-tagged hemagglutinin (His6-H5 HA), (v) filling free space with BSA. The interactions between two variants of recombinant HA (short and long) from highly pathogenic avian influenza virus H5N1 and the anti-H5 HA monoclonal antibody (Mab 6-9-1) have been explored with electrochemical impedance spectroscopy (EIS). The impedimetric immunosensor displayed a very good detection limit (LOD) of 2.1 pg/mL, the quantification limit (LOQ) of 6.3 pg/mL and a dynamic range from 4 pg/mL to 20 pg/mL. In addition, this analytical device was applied for detection of antibodies against His6-H5 HA in serum of vaccinated hen using serial 10-fold dilutions of serum. The immunosensor proposed was able to detect antibody in hen serum diluted up to 7 × 10(7)-fold. The sensitivity of immunosensor was about four orders of magnitude much better than ELISA.

Activity of the AtMRP3 promoter in transgenic Arabidopsis thaliana and Nicotiana tabacum plants is increased by cadmium, nickel, arsenic, cobalt and lead but not by zinc and iron.

Characterization of the function, regulation and metal-specificity of metal transporters is one of the basic steps needed for the understanding of transport and accumulation of toxic metals and metalloids by plants. In this work GUS was used as a reporter for monitoring the activity of the promoter of the AtMRP3 gene from Arabidopsis thaliana, a gene encoding an ABC-transporter, expression of which is induced by heavy metals. The AtMRP3 promoter-GUS fusion expression cassette was introduced into the genome of two model plants, A. thaliana and Nicotiana tabacum. The promoter induces GUS activity in the roots as well as in the shoots upon metal exposure. Similar responses of the AtMRP3 promoter to the presence of the selected metals was observed in both plant species. Cadmium, nickel, arsenic, cobalt and lead strongly activated the transcription of the reporter gene, while zinc and iron had no impact. The AtMRP3 promoter thus seems to be a useful new tool in designing plants that can be used for biomonitoring of environmental contaminations.

A Contribution to Identification of Novel Regulators of Plant Response to Sulfur Deficiency: Characteristics of a Tobacco Gene UP9C, Its Protein Product and the Effects of UP9C Silencing

Extensive changes in plant transcriptome and metabolome have been observed by numerous research groups after transferring plants from optimal conditions to sulfur (S) deficiency. Despite intensive studies and recent important achievements, like identification of SLIM1/EIL3 as a major transcriptional regulator of the response to S-deficiency, many questions concerning other elements of the regulatory network remain unanswered. Investigations of genes with expression regulated by S-deficiency stress encoding proteins of unknown function might help to clarify these problems. This study is focused on the UP9C gene and the UP9-like family in tobacco. Homologs of these genes exist in other plant species, including a family of four genes of unknown function in Arabidopsis thaliana (LSU1-4), of which two were reported as strongly induced by S-deficit and to a lesser extent by salt stress and nitrate limitation. Conservation of the predicted structural features, such as coiled coil region or nuclear localization signal, suggests that these proteins might have important functions possibly mediated by interactions with other proteins. Analysis of transgenic tobacco plants with silenced expression of UP9-like genes strongly argues for their significant role in regulation of plant response to S-deficit. Although our study shows that the UP9-like proteins are important components of such response and they might be also required during other stresses, their molecular functions remain a mystery.

Biochemical analysis of transgenic tobacco lines producing bacterial serine acetyltransferase

Abstract The preliminary biochemical analysis of the selected lines of transgenic tobacco producing Escherichia coli serine acetyltransferase (SAT, EC 2.3.1.30) targeted either to the chloroplasts or to the cytosol was performed. The selected enzymatic activities and chemical composition of the leaf tissues collected from the plants cultivated in vivo were examined and statistically evaluated. In all SAT-over-producers increased level of non-protein thiols in comparison with the controls was detected and a higher resistance of the leaf tissue to the oxidative stress generated by hydrogen peroxide was observed. These two factors were significantly positively correlated only in plants producing bacterial SAT in the cytosol. Moreover, in the ‘cytosolic’ but not ‘chloroplastic’ groups, a strong negative correlation between glutathione and sulfate levels was seen. The leaves of most SAT-over-producers contained more protein, sulfur, calcium and iron (but less phosphorus, potassium and sodium) than the leaves of control plants. The vast majority of transgenics had higher glutathione S -transferase activity. All plants producing foreign SAT in the cytosol had an elevated activity of O -acetyl-serine (thiol) lyase, while in most plants from ‘chloroplastic’ groups, this activity remained closed to the control level.

Recombinant Cytokines from Plants

Plant-based platforms have been successfully applied for the last two decades for the efficient production of pharmaceutical proteins. The number of commercialized products biomanufactured in plants is, however, rather discouraging. Cytokines are small glycosylated polypeptides used in the treatment of cancer, immune disorders and various other related diseases. Because the clinical use of cytokines is limited by high production costs they are good candidates for plant-made pharmaceuticals. Several research groups explored the possibilities of cost-effective production of animal cytokines in plant systems. This review summarizes recent advances in this field.

Recombinant Mouse Granulocyte–Macrophage Colony-Stimulating Factor Is Glycosylated in Transgenic Tobacco and Maintains its Biological Activity

The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with many important applications and, due to its immunostimulatory properties, could also be used as a vaccine adjuvant. A simple strategy to produce recombinant mouse GM-CSF (mGM-CSF) in transgenic Nicotiana tabacum plants was used in this study. The mGM-CSF cDNA followed by the sequence encoding endoplasmic reticulum retention signal (KDEL) was cloned into the ImpactVector under the control of the strong promoter from the gene encoding a small subunit of Rubisco. In transgenic plants the accumulation level of recombinant mGM-CSF varied in the individual transformants from 8 to 19 microg/g of fresh leaf tissue, which makes up to 0.22% of total soluble protein. In most analyzed plants, the apparent molecular weight of the recombinant protein was larger than predicted due to its N-glycosylation, presumably in 2 sites. The recombinant plant-produced murine GM-CSF retained its biological activity as confirmed in vitro in proliferation assay using a mouse cell line, which is growth-dependent on GM-CSF.

Links Between Ethylene and Sulfur Nutrition—A Regulatory Interplay or Just Metabolite Association?

Multiple reports demonstrate associations between ethylene and sulfur metabolisms, however the details of these links have not yet been fully characterized; the links might be at the metabolic and the regulatory levels. First, sulfur-containing metabolite, methionine, is a precursor of ethylene and is a rate limiting metabolite for ethylene synthesis; the methionine cycle contributes to both sulfur and ethylene metabolism. On the other hand, ethylene is involved in the complex response networks to various stresses and it is known that S deficiency leads to photosynthesis and C metabolism disturbances that might be responsible for oxidative stress. In several plant species, ethylene increases during sulfur starvation and might serve signaling purposes to initiate the process of metabolism reprogramming during adjustment to sulfur deficit. An elevated level of ethylene might result from increased activity of enzymes involved in its synthesis. It has been demonstrated that the alleviation of cadmium stress in plants by application of S seems to be mediated by ethylene formation. On the other hand, the ethylene-insensitive Nicotiana attenuata plants are impaired in sulfur uptake, reduction and metabolism, and they invest their already limited S into methionine needed for synthesis of ethylene constitutively emitted in large amounts to the atmosphere. Regulatory links of EIN3 and SLIM1 (both from the same family of transcriptional factors) involved in the regulation of ethylene and sulfur pathway, respectively, is also quite probable as well as the reciprocal modulation of both pathways on the enzyme activity levels.

Tobacco LSU-like protein couples sulphur-deficiency response with ethylene signalling pathway

Most genes from the plant-specific family encoding Response to Low Sulphur (LSU)-like proteins are strongly induced in sulphur (S)-deficient conditions. The exact role of these proteins remains unclear; however, some data suggest their importance for plants’ adjustment to nutrient deficiency and other environmental stresses. This work established that the regulation of ethylene signalling is a part of plants’ response to S deficiency and showed the interaction between UP9C, a tobacco LSU family member, and one of the tobacco isoforms of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO2A). Increase in ethylene level induced by S deficiency does not take place in tobacco plants with UP9C expressed in an antisense orientation. Based on transcriptomics data, this work also demonstrated that the majority of tobacco’s response to S deficiency is misregulated in plants expressing UP9C-antisense. A link between response to S deficiency, ethylene sensing, and LSU-like proteins was emphasized by changes in expression of the genes encoding ethylene receptors and F-box proteins specific for the ethylene pathway.