“REGULATION OF GENE EXPRESSION” – RESEARCH TOPICS

Molecular and cellular mechanisms of the postembryonic plant development

Postembryonic root development in model plant species
Novel small-molecule effectors of the intracellular membrane trafficking in plant cells
Cell-, tissue- and organ specificity of hormonal signaling and crosstalk

Epigenetic regulation of the gene expression

Impact of the epigenetic DNA modifications on plant development
Epigenetic control in conditions of abiotic stress
Dissection of the gene functions in heterologous systems
Genetic and epigenetic mechanisms of the nucleolar dominance

Structural organization of the plant genome in abiotic stress conditions

Molecular and cytogenetic characterization of abiotic stress-resistant hybrids between crop cultivars and their closely related wild relatives
Genotoxic assessment of the abiotic stress impact on the plant genome

Molecular and phenotypic characterization of the abiotic stress effect on plants

Molecular and phenotypic markers for analysis of the genetic determinants of plant resistance to abiotic stress
Transcriptional and posttranscriptional regulation of the chloroplast gene expression in abiotic stress conditions
Changes in the levels and activity of key stress-related plant proteins. Proteomic analysis

Molecular and cellular mechanisms of the postembryonic plant development

Postembryonic root development in model plant species

We investigate genes and signaling pathways involved in root growth and branching using the model plants Arabidopsis thaliana and Lotus japonicus. Particular attention is given to identifying early regulators that control specification of lateral root founder cells and subsequent highly structured asymmetric and symmetric cell divisions, leading to the formation of lateral root primordia. We also examine the coordinated auxin-dependent nuclear migration, which is an important prerequisite for asymmetric divisions of the founder cells. Since all stages of root growth and development are largely regulated by the phytohormone auxin, the study of key components of the auxin signaling machinery greatly contributes to understanding the adaptation changes during plant development and in response to adverse environment.

Polarization and asymmetric cell divisions of founder cells in the root pericycle of Arabidopsis thaliana.

  • Fernandez A, Drozdzecki A, Hoogewijs K, Vassileva V, Madder A, Beeckman T, Hilson P. 2015. The GLV6/RGF8/CLEL2 peptide regulates early pericycle divisions during lateral root initiation. Journal of Experimental Botany 17, 5245-5256.
  • Chen Q, Liu Y, Maere S, Lee E, Van Isterdael G, Xie Z, Xuan W, Lucas J, Vassileva V, Kitakura S, Marhavý P. 2015. A coherent transcriptional feed-forward motif model for mediating auxin-sensitive PIN3 expression during lateral root development. Nature Communications 6, 8821.
  • Boycheva I, Vassileva V, Revalska M, Zehirov G, Iantcheva A. 2015. Cyclin-like F-box protein plays a role in growth and development of the three model species Medicago truncatula, Lotus japonicus and Arabidopsis thaliana. Research and Reports in Biology 6, 117-130.
  • Revalska M, Vassileva V, Zehirov G, Iantcheva I. 2015. Is the auxin influx carrier LAX3 essential for plant growth and development in the model plants Medicago truncatula, Lotus japonicus and Arabidopsis thaliana? Biotechnology & Biotechnological Equipment 29, 4, 786-797.
  • Berckmans B, Vassileva V, Schmid S, Maes S, Parizot B, Naramoto S, Magyar Z, Kamei CLA, Koncz C, Persiau G, De Jaeger G, Bogre L, Friml J, Simon R, Beeckman T, De Veylder L. 2011. Auxin-dependent cell cycle reactivation through transcriptional control of E2Fa by LATERAL ORGAN BOUNDARY proteins. Plant Cell 23, 10, 3671-3683.
  • De Rybel B, Vassileva V, Parizot B, Demeulenaere M, Grunewald W, Audenaert D, Van Campenhout J, Overvoorde P, Jansen L, Vanneste S, Möller B, Holman T, Van Isterdaele G, Brunoud G, Vuylsteke M, Vernoux T, De Veylder L, Inzé D, Weijers D, Bennett M, Beeckman T. 2010. A novel Aux/IAA28 signalling cascade activates GATA23-dependent specification of lateral root founder cell identity. Current Biology 20, 19, 1697-1706.
  • De Smet I, Vassileva V, De Rybel B, Levesque MP, Grunewald W, Van Damme D, Van Noorden G, Naudts M, Van Isterdael G, De Clercq R, Wang JY, Meuli N, Vanneste S, Friml J, Hilson P, Jürgens G, Ingram GC, Inzé D, Benfey PN, Beeckman T. 2008. Receptor-like kinase ACR4 restricts stem cell divisions in the Arabidopsis root. Science 322, 5901, 594-597.


Novel small-molecule effectors of the intracellular membrane trafficking in plant cell

Small bioactive molecules identified in large-scale chemical genetic screens can be helpful in understanding the interplay between endomembrane trafficking and hormone signaling. Chemical genetics overcomes essential limitations of classical forward genetics, such as lethality and genetic redundancy. An important advantage of chemical genetics in dissecting complex biological processes, such as the intracellular trafficking, is the enormous diversity of chemical structures that could be used for probing protein functions in a reversible manner. A major challenge in plant chemical genetics is the identification of protein targets of the bioactive compounds which is essential to understand the mode of action of the small molecules and addresses chemical specificity issues.

Schematic representation of the main endomembrane trafficking pathways along with the respective small-molecule inhibitors that have been tested so far in plant cells. (Chem Biol 20, 2013, 475-486, updated).

Interplay between the intracellular vesicle trafficking and plant hormone signaling. The direct effect of endocytosis on the hormone signaling outputs has already been demonstrated for the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE1 (BRI1), a protein kinase which undergoes constitutive cycling between the plasma membrane and endosomal compartments (Curr Opin Plant Biol 22, 48-55).

  • Dejonghe W, Kuenen S, Mylle E, Vasileva M, Keech O, Viotti C, Swerts J, Fendrych M, Ortiz-Morea FA, Mishev K, Delang S, Scholl S, Zarza X, Heilmann M, Kourelis J, Kasprowicz J, Nguyen le SL, Drozdzecki A, Van Houtte I, Szatmári AM, Majda M, Baisa G, Bednarek SY, Robert S, Audenaert D, Testerink C, Munnik T, Van Damme D, Heilmann I, Schumacher K, Winne J, Friml J, Verstreken P, Russinova E. 2016. Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification. Nature Communications 7, 11710.
  • Dejonghe W, Mishev K, Russinova E. 2014. The brassinosteroid chemical toolbox. Current Opinion in Plant Biology 22, 48-55.
  • Mishev K, Dejonghe W, Russinova E. 2013. Small molecules for dissecting endomembrane trafficking: a cross-systems view. Chemistry & Biology 20, 2013, 475-486.
  • Irani N, Di Rubbo S, Mylle E, Van den Begin J, Schneider-Pizoń J, Hniliková J, Šíša M, Buyst D, Buyst D, Vilarrasa-Blasi J, Szatmári AM, Van Damme D, Mishev K, Codreanu MC, Kohout L, Strnad M, Caño-Delgado AI, Friml J, Madder A, Russinova E. 2012. Fluorescent castasterone reveals BRI1 signaling from the plasma membrane. Nature Chemical Biology 8, 6, 583-589.


Cell-, tissue- and organ specificity of hormonal signaling and crosstal

Cell division and cell expansion that drive growth and development of the organ are controlled by hormones. Hormonal signaling cascades orchestrate a complex network of physiological and biochemical processes as a response to different environmental and developmental cues. The individual cell types have a leading role in the perception of different hormonal signals, which subsequently control various physiological and biochemical processes. Detailed characterization of hormonal network under normal and adverse environmental conditions provides basis for spatiotemporal modelling of hormonal crosstalk allowing examination of distinct and common mechanisms by which hormones control plant growth. This knowledge could be implemented in development of novel biotechnology tools by precision targeting of growth-regulating processes, which not only can increase stress tolerance but also could be used to improve desired traits in economically important crops.

Root cell type specific expression of components from the ethylene signaling cascade. The image is generated using the Arabidopsis eFP Browser (AREX Database, Birnbaum et al. 2003, Science 302; Brady et al 2007, Science 318).

  • Vaseva-Gemisheva I, Todorova D, Malbeck J, Travnickova A, Machackova I, Karanov E. 2006. Cytokinin pool dynamic changes and distribution of cytokinin oxidase/dehydrogenase activity in peas in relation to developmental senescence. Comp. Rend. Bulg. Acad. Sci. 59 (1): 65-70.
  • Todorova D,Vaseva I, Malbeck J, Trávníčková A, Macháčková I, Karanov E 2007. Cytokinin oxidase/dehydrogenase activity as a tool in gibberellic acid/cytokinin cross talk. Biol. Plantarum 51 (3): 579-583.
  • Vaseva I, Todorova D, Malbeck J, Trávníčková A,  Macháčková I.  2008.  Response of cytokinin pool and cytokinin oxidase/dehydrogenase activity to abscisic acid exhibits organ specificity in peas. Acta Physiologiae Plantarum 30 (2): 151-155
  • Vandenbussche F, Vaseva I, Vissenberg K, Van Der Straeten D. 2012. Ethylene in vegetative development: a tale with a riddle. New Phytologist 194, 4, 895-909.
  • Vaseva I, Vandenbussche F, Simon D, Vissenberg K, Van Der Straeten D. 2016. Cell type specificity of plant hormonal signals: Case studies and reflections on ethylene. Russian Journal of Plant Physiology 63, 5, 577-586.


Epigenetic regulation of the gene expression

Impact of the epigenetic DNA modifications on plant development

Epigenetic modifications regulate activity of particular genes or non-coding regions of the DNA molecule without changes to the underlying DNA sequence, which provide flexible and reversible regulation of gene expression. In general, epigenetic regulation involves at least three main systems: DNA methylation, histone modification and gene silencing by non-coding RNAs (ncRNA), which control the extent of chromatin condensation. We analyze epigenetically altered regions of the genome, stability of these changes and their inheritance, as well as activity of individual genes. The effects of DNA methylation loss and its potential complementation by direct DNA transfer or genetic crosses are examined, and a correlation between epigenetically-induced changes in gene expression and plant phenotypes are evaluated.

Root phenotype of A. thaliana mutants with reduced maintenance DNA methyltransferase activity: control (Col-0), met1-1.

Adaxial epidermis of A. thaliana mutants with altered DNA methylation: control (Col-0), met1-1, ddm1-10, cmt3 (GPP 2016, 3-13).

  • Boycheva I, Vassileva V, Revalska M, Zehirov G, Iantcheva A. 2017. Role of the histone acetyltransferase HAC1 gene in development of the model species Medicago truncatula, Lotus japonicus and Arabidopsis thaliana. Protoplasma 254, 2, 697-711.
  • Vassileva V, Hollwey E, Todorov D, Meyer P. 2016. Leaf epidermal profiling as a phenotyping tool for DNA methylation mutants. Genetics and Plant Physiology 6, 1-2, 3-13.
  • Boycheva I, Vassileva V, Iantcheva A. 2014. Histone acetyltransferases in plant development and plasticity. Current Genomics 15, 1, 28-3.


Epigenetic control in conditions of abiotic stress

The importance of this research topic is related to the country’s continental climatic conditions with extremely cold winters and prolonged periods of extensive drought. To develop plants with higher tolerance to adverse environments, the mechanisms of plant protection, adaptation and various regulatory processes are investigated. The relationship between changed DNA methylation patterns and plant adaptation to abiotic stress has been subject of extensive studies in the recent years. The inherited methylation changes are an important prerequisite for long-term adaptation of plants to changing environments. Part of the research with A. thaliana is dedicated to genes with dense promoter methylation that are involved in stress-related signaling pathways. The methylation status of stress-inducible genes in model plants, as well as in wheat varieties/lines with high tolerance or sensitivity to cold and drought stress, is assessed by means of DNA bisulfite conversion and methylation-sensitive restriction analyses.

Analysis of the promoter methylation pattern of stress-related genes by bisulfite conversion and subsequent sequencing.


Dissection of the gene functions in heterologous systems

Model plants are used as alternative experimental systems to study the functions of genes with a high degree of evolutionary conservation. Many human diseases are associated with gene mutations whose lethal effect makes them difficult to study. Plants offer a good alternative, as they are often more tolerant to mutations than animals. Unlike mammals, the model plants, such as A. thaliana, tolerate changes in epigenetic systems, and are viable and fertile when the methylation is lost or reduced. For that reason, we use A. thaliana as an experimental model for studying the degree of conservation and divergence in the specific CG methylation systems. The potential functional replacement of MET1 by DNMT1 that are crucial for methylation maintenance in plants and mammals, respectively, is investigated.

We are also exploring evolutionary conserved genes encoding small heat shock proteins with NudC domain that function not only as molecular chaperones but also play an important role in organismal development. The NudC protein family has representatives in all eukaryotes, including human, which suggest important biological functions preserved during the evolution. We study the NudC genes from A. thaliana, searching functional homology between the plant NudC and yeast (Saccharomyces cerevisiae) genes.

Structurally conservative DNA methyltransferases maintaining DNA methylation in plants (MET1) and mammals (DNMT1)

Multiple sequence alignment of the highly conservative NudC domain in different plant species and human.


Genetic and epigenetic mechanisms of the nucleolar dominance

The molecular mechanisms of regulation of ribosomal RNA (rRNA) synthesis in barley mutants possessing altered position or integrity of the two nucleolus organizers (NORs) are studied. In particular, we perform structural analysis of the rRNA intergenic spacer (IGS) in Hordeum vulgare, characterization of RNA polymerase I activity in run-on experiments, as well as mapping of hypomethylated and DNase-sensitive sites. The role of higher-order chromatin organization is also explored.

1 – Rate of transcript elongation in standard and NOR-deletion barley lines; 2 – distribution of rDNA within the matrix-bound and loop DNA fractions of barley karyotypes with standard and suppressed NOR activity; 3 – mapping of hypomethylated sites in barley lines with altered position or integrity of the two NORs; 4 – mapping of DNase I hypersensitivity sites in barley lines with altered position or integrity of the two NORs.

  • Dimitrova AD, Georgiev О, Mishev К, Tzvetkov S, Ananiev ED, Karagyozov L. 2016. Mapping of unmethylated sites in rDNA repeats in barley NOR deletion line. J. Plant Physiol. 205, 97-104.
  • Dimitrova AD, Gecheff KI, Ananiev ED. 2012. Methylation pattern of ribosomal RNA genes in NOR-deleted and NOR-reconstructed barley lines (Hordeum vulgare L.). Organization of IGS in rDNA repeat unit. Genet. Plant Physiol. 2, 1–2, 3–14.
  • Dimitrova AD, Ananiev ED, Gecheff KI. 2009. DNase I hypersensitive sites within the intergenic spacer of ribosomal RNA genes in reconstructed barley karyotypes. Biotechnol. & Biotechnol. Eq. 23, 1, 1039-1043.
  • Dimitrova AD, Ananiev ED, Stoilov LM, Gecheff КI. 2008. Ribosomal RNA gene expression in reconstructed barley karyotypes. Compt. Rend. Acad. Bulg. Sci. 61, 9, 1159-1168.
  • Dimitrova A, Stoilov L, Gecheff K. 2004. Loop organization of ribosomal DNA in barley. Compt. Rend. Acad. Bulg. Sci. 57, 9, 57-60.


Structural organization of the plant genome in abiotic stress conditions

Molecular and cytogenetic characterization of abiotic stress-resistant hybrids between crop cultivars and their closely related wild relatives

We analyze the genetic introgression in hybrids of potential agricultural importance that have been created from crop cultivars and their closely related wild relatives. The transfer of genetic material from wild to hybrid plants serves as a source of genes determining valuable economic and biological traits, resistance to abiotic and biotic factors, etc. We use standard and modern molecular cytogenetic methods (in situ hybridization, FISH and GISH), and create physical maps for rapid and accurate chromosomal identification and gene localization.

FISH / GISH characterisation of wheat × Thinopyrum intermedium partial amphiploid 55 (1-57).

  • Georgieva M, Kruppa K, Tyankova N, Molnár-Láng M. 2016. Molecular cytogenetic identification of a novel hexaploid Wheat-Thinopyrum intermedium partial amphiploid having high protein content. Turkish Journal of Biology, 40, 554-560.
  • Georgieva M, Sepsi A, Molnár-Láng M, Tyankova N. 2011. Molecular cytogenetic analysis of Triticum aestivum and Thinopyrum intermedium using the FISH technique. Comptes rendus de l’Académie bulgare des Sciences 64, 12, 1713-1718.
  • Georgieva M, Sepsi A, Tyankova N, Molnár-Láng M. 2011. Molecular cytogenetic characterization of two high protein wheat-Thinopyrum intermedium partial amphiploids. Journal of Applied Genetics, 52, 269–277.


Genotoxic assessment of the abiotic stress impact on the plant genome

The genotoxic effects of various environmental abiotic stress factors such as gamma radiation, UV radiation, chemicals, pesticides, phytocomponents, heavy metals, nanoparticles, etc. are evaluated. The induced plant DNA damage serves as a non-specific biomarker for the environmental impact on plant vigor and is used to predict the effects of various types of pollution in the ecosystems. The analysis of primary DNA damage including single- and double-strand breaks and their repair is performed by means of plant comet assay.

 

Neutral comet assay on isolated pea nuclei irradiated with UV-C.

Radionuclide-induced DNA damage (single- and double-strand breaks) in isolated soybean nuclei analyzed through alkaline and alkaline-neutral comet assays.

  • Georgieva M, Tsenov B, Dimitrova  A. 2017. Dual effects of N-nitroguanidine neonicotinoids on plants. Genetics and Plant Physiology (in press).
  • Georgieva M, Rashydov NM, Hajduch M. 2017. DNA damage, repair monitoring and epigenetic DNA methylation changes in seedlings of Chernobyl soybeans. DNA repair 50, 14-21.
  • Georgieva M, Nikolova I, Bonchev G, Katerova Z, Todorova D. 2015. A comparative analysis of membrane intactness and genome integrity in pea, barley and wheat in response to UVC-irradiation. Turkish Journal of Botany 39, 6, 1008-1013.
  • Stoilov L, Georgieva M, Manova V, Liu L, Gecheff K. 2013. Karyotype reconstruction modulates the sensitivity of barley genome to radiation-induced DNA and chromosomal damage. Mutagenesis 28, 2, 153-160.
  • Georgieva M, Stoilov L, Rancheva E, Todorovska E, Vassilev D. 2010. Comparative аnalysis of data distribution patterns in plant comet assay. Biotechnology & Biotechnological Equipment 24, 4,  2142-2148.
  • Georgieva M, Stoilov L. 2008. Assessment of DNA strand breaks induced by bleomycin in barley by the comet assay. Environmental and Molecular Mutagenesis, 49, 5, 381-387.


Molecular and phenotypic characterization of the abiotic stress effect on plant

Molecular and phenotypic markers for analysis of the genetic determinants of plant resistance to abiotic stress

We explore the impact of various abiotic stress factors on the survival and productivity of plants, as well as the protective mechanisms by which plants counteract adverse conditions. Our aim is to understand the mechanisms underlying stress adaptability of some genotypes, and to identify traits related to stress tolerance or susceptibility of a particular genotype. We analyze the following physiological, biochemical, molecular, genetic and morphological markers for plant stress tolerance or susceptibility:

  • Protein markers: profiles of isoenzymes and storage proteins; the levels of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), Rubisco-binding protein, Rubisco activase, heat stress proteins, various proteases and enzymes involved in plant cell protection against oxidative stress.

1 – Isoenzyme profile of esterases from mutant barley lines; 2 - Immunoblot analysis of extracts from wheat varieties with different resistance to drought; 3 - Immunoblot analysis of dehydrins in Trifolium; 4 - Protease activity in wheat varieties with different resistance to drought.

  • DNA markers: RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), STS (Sequence Tagged Sequences), CAPS (Cleaved Amplified Polymorphic Sequences), REMAP (Retrotransposon Microsatelite Amplified Polymorphism), SSAP (Sequence Specific Amplified Polymorphism), AFLP (Amplified Fragment Length Polymorphism) и SSR (Simple Sequence Repeats).

1 – CAPS profile of mutant barley lines; 2 – AFLP profile of mutant barley lines; 3 – REMAP profile of mutant barley lines.

  • Gene markers: analysis of the expression levels of stress-related genes under different abiotic stress conditions.

Temperature-dependent expression of Arabidopsis genes encoding heat shock proteins after 3-hour incubation at 42°C. EF1α was used as a reference gene.

  • Chromosomal markers: Fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization of a multiple reconstructed barley karyotype PK 88-19.

  • Phenotypic markers: plant organ micromorphology and plant cell organelle ultrastructure.

Transmission electron micrographs of mesophyll cells from wheat varieties with different tolerance to drought stress.

  • Vaseva I, Zehirov G, Kirova E, Simova-Stoilova L. 2016. Transcript profiling of serine- and cysteine protease inhibitors in Triticum aestivum varieties with different drought tolerance. Cereal Research Communications 44, 1, 79–88.
  • Todorovska E, Dimitrova A, Stoilov L, Christov N, Vassilev D, Gecheff K. 2013. Molecular variability in barley structural mutants produced by gamma-irradiation. Plant Mutation Rep. 3, 1, 4-8.
  • Georgieva M, Gecheff K. 2013. Molecular cytogenetic characterization of a new reconstructed barley karyotype. Biotechnology & Biotechnological Equipment 27, 1, 3577-3582.
  • Vassileva V, Demirevska K, Simova‐Stoilova L, Petrova T, Tsenov N, Feller U. 2012. Long-term field drought affects leaf protein pattern and chloroplast ultrastructure of winter wheat in a cultivar specific manner. Journal of Agronomy and Crop Science 198, 2, 104–117.
  • Grigorova B, Vassileva V, Klimchuk D, Vaseva I, Demirevska K, Feller U. 2012. Drought, high temperature, and their combination affect ultrastructure of chloroplasts and mitochondria in wheat (Triticum aestivum L.) leaves. Journal of Plant Interactions 7, 3, 204-213.
  • Grigorova B, Vaseva I, Demirevska K, Feller U. 2011. Combined drought and heat stress in wheat: changes in some heat shock proteins. Biologia Plantarum 55, 1, 105-111.
  • Grigorova B, Vaseva I, Demirevska K, Feller U. 2011. Expression of selected heat shock proteins after individually applied and combined drought and heat stress. Acta Physiologiae Plantarum 33, 5, 2041-2049.
  • Vassileva V, Signarbieux C, Anders I, Feller U. 2011. Genotypic variation in drought stress response and subsequent recovery of wheat (Triticum aestivum L.). Journal of Plant Research 124, 1, 147-154.
  • Simova-Stoilova L, Vaseva I, Grigorova B, Demirevska K, Feller U.2010. Proteolytic activity and cysteine protease expression in wheat leaves under severe soil drought and recovery. Plant Physiology and Biochemistry 48, 2-3, 200-206.
  • Vassileva V, Simova-Stoilova L, Demirevska K, Feller U. 2009. Variety-specific response of wheat (Triticum aestivum L.) leaf mitochondria to drought stress. Journal of Plant Research 122, 4, 445-454.
  • Demirevska K, Zasheva D, Dimitrov R, Simova-Stoilova L, Stamenova M, Feller U. 2009. Drought stress effects on Rubisco in wheat: changes in the Rubisco large subunit. Acta Physiol. Plantarum 31, 1129-1138.
  • Dimitrova А, Todorovska E, Christov NK, Stoilov L, Atanassov A, Gecheff K. 2008. Molecular characterization of structural barley mutants produced by gamma-irradiation. Genetics and Breeding, 37, 3-4, 15-26.
  • Demirevska K, Simova-Stoilova L, Vassileva V, Vaseva I, Grigorova B, Feller U. 2008. Drought induced leaf protein alterations in sensitive and tolerant wheat varieties. General and Applied Plant Physiology, Special Issue 34, 1-2, 79-102.
  • Demirevska K, Simova-Stoilova L, Vassileva V, Feller U. 2008. Drought response of Rubisco and some ATP-dependent chaperone proteins in wheat plants. Plant Growth Regulation 56, 97–106.
  • Kirova E, Tzvetkova N, Vaseva I, Ignatov G. 2008. Photosynthetic responses of nitrate-fed and nitrogen-fixing soybeans to progressive water stress. Journal of Plant Nutrition 31, 445–458.
  • Stoilova T, Dimitrova A, Angelova T, Gecheff K. 2006. Assessment of the genetic diversity in barley mutant lines using biochemical markers. Genetics and Breeding 35, 3-4, 3-10.


Transcriptional and posttranscriptional regulation of the chloroplast gene expression in abiotic stress conditions

The sophisticated mechanisms of gene expression regulation are a distinctive feature of chloroplasts and are not observed in cyanobacteria, the plastid ancestors. The differences between chloroplasts and photosynthetic bacteria mainly concern the processes of transcription and maturation of the primary RNA transcripts. Two types of DNA-dependent RNA polymerases have been found to be involved in plastid transcription, i.e. the chloroplast-encoded RNA polymerase (PEP) as well as two nuclear-encoded RNA polymerases (NEP). In turn, the plastid RNA stability is an essential factor determining the dynamics in chloroplast gene expression in abiotic stress conditions. In many cases, the fluctuations in the rate of plastid gene transcription upon environmental stress do not correlate with the changes in the chloroplast transcript levels.

The expression of marker chloroplast-encoded genes is analyzed through either Northern blotting or qPCR to assess the steady-state transcript levels, or by means of run-on transcription in isolated intact plastids to explore the transcriptional regulation, i.e. the share of de novo chloroplast RNA synthesis.

  • Ananieva K, Ananiev ED, Doncheva S, Stefanov D, Mishev K, Kaminek M, Motyka V, Dobrev P, Malbeck J. 2011. Local induction of senescence by darkness in Cucurbita pepo (zucchini) cotyledons or the primary leaf induces opposite effects in the adjacent illuminated organ. Plant Growth Regulation 65, 459-471.
  • Mishev K, Dimitrova A, Ananiev ED. 2011. Darkness affects differentially the expression of plastid-encoded genes and delays the senescence-induced down-regulation of chloroplast transcription in cotyledons of Cucurbita pepo L. (zucchini). Zeitschrift für Naturforshung, 66c: 159-166.
  • Mishev K, Ananiev ED, Humbeck K. 2011. Organ-specific effects of dark treatment on photosynthesis and the expression of photosynthesis-related genes. Biologia Plantarum 55, 269-278.
  • Ananieva K, Ananiev ED, Mishev K, Georgieva K, Malbeck J, Kaminek M, van Staden J. 2007. Methyl jasmonate is a more effective senescence-promoting factor in Cucurbita pepo (zucchini) cotyledons when compared with darkness at the early stage of senescence. Journal of Plant Physiology 164, 1179-1187.
  • Mishev K, Denev I, Radeva G, Ananiev ED. 2006. RNA transcription in isolated chloroplasts during senescence and rejuvenation of intact cotyledons of Cucurbita pepo L. (zucchini). Comptes rendus de l’Academie Bulgare des Sciences 59, 1287-1293.


Changes in the levels and activity of key stress-related plant proteins. Proteomic analysis

Proteins are direct effectors in all processes related to cell structure and function, and are essential determinants of the adaptation to the changing environment, underlying the so called phenotypic plasticity. Cell protein composition is highly dynamic and does not necessarily correspond to the transcript levels, due to the complex regulation of gene expression (at genetic, transcriptional, translational, and post-translational levels). The development of databases with partially or fully sequenced plant genomes and expressed sequence tags, necessary for correct protein identification, as well as comprehensive proteome maps of major crops, has facilitated the use of proteomic studies in search of suitable protein markers for assisted selection. Two-dimensional electrophoresis combined with mass spectrometry detects relatively more abundant proteins like key metabolic enzymes, thus providing essential information about changes in the main metabolic pathways and biological processes affected by the stress. Moreover, isoforms and posttranslational modifications of a given protein can also be detected. Deeper proteome coverage, especially for less abundant proteins (signaling, transporters, etc), is achieved by second generation shotgun proteomics, both approaches being complementary.

Representative 2DE protein profile of leaf extract from Trifolium. Spots changing in abundance as a result of the applied stress (waterlogging) are indicated by numbers and are identified by MS (Stoychev et al, Advances in Environmental Research, Vol. 39, 2015, 131-162).

Abiotic stresses with a dehydration component (i.e., drought, salt, and freezing) typically trigger an increase in the amount of inactive proteins – denatured, aggregated or oxidatively damaged. The existing protein quality control system maintains proteins in their functional conformation, preventing aggregation of non-native proteins, refolding of denatured proteins to their native conformation, and removing non-functional and potentially harmful polypeptides, which is vital for cell survival under dehydration stress. Plants respond to stress by synthesis of protective proteins, such as dehydrins and chaperones, and degradation of irreversibly damaged proteins by proteases. Protease activity is partially controlled by endogenous protease inhibitors. Plant exposure to stress leads to inhibition of photosynthesis and breakdown of unnecessary and reserve proteins, providing building blocks and energy sources for the metabolism. Some proteins from the quality control system  are  potential biochemical markers for assessing stress tolerance.


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