Research - Institute of Plant Biology - Laboratory of Plant Architecture and Development

János GYÖRGYEY
senior research associate

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Dénes DUDITS Professor Emeritus
Györgyi FERENC research associate
Hilda Anikó LIMA research associate
András CSERI research associate
Zoltán ZOMBORI junior research associate
Bettina ZOMBORINÉ NAGY research associate
Magdolna GOMBOS junior research associate
Feríz RÁDI PhD student
Györgyi SÁNDOR laboratory assistant
Károlyné TÖRÖK laboratory assistant
Katalin LÁSZLÓ laboratory assistant

The group got its current structure in October 2013 only, having two subunits: one focuses on root development of Brachypodium distachyon aiming to unravel some molecular genetic responses to drought stress; while the other subunit aims to improve biomass production of woody plants focusing on different willow genotypes as well as develops oligonucleotide-based method for epigenetic modulation of gene expression which can be alternative method for plant improvement in willow too.


Subunit 1: ROOT DEVELOPMENT IN Brachypodium; THE MODEL SYSTEM OF TEMPERATE CEREALS

(János GYÖRGYEY, principal investigator; Mária SZÉCSÉNYI, Zoltán ZOMBORI, Magdolna GOMBOS, Györgyi SÁNDOR, Istvánné KIRI)

As early as in the middle of the XX century we had to learn that plant cells are more autonomous than animal cells and plant cells maintain totipotency, a capability to return from differentiated physiological state to meristematic cell state which is the functional equivalent of the recently discovered stem cells of animals and human. This transition takes place in plant cells via reactivation of their cell cycle. In this project, we take the advantage of Brachypodium distachyon, a model plant for grass species including important crop cereals like wheat, barley and rye as well, to understand how this question of “to divide or to differentiate” is regulated in plant cells. Root development was chosen as the developmental process to study because a strong and efficient root system is also extremely important for the survival of our crop plants under stressful environmental conditions. Work on Brachypodium as model plant for temperate grasses started here in 2009.




Figure 1. Developing Brachypodium plantlets under controlled water regime.


At first, experimental system, similar to the one used for wheat in recent years, was established to study the root development of Brachypodium. This rhizotron based system enables the continuation of research an the same question: how can grass plants reshape their root system as the consequence of limiting water supply and what are the candidate genes playing role in the changes of the root architecture that we observe on morphological level in genotypes accommodating to drought stress well. Wide range of Brachypodium genotypes differing in drought tolerance was tested, selected lines were characterized in details in rhizotron in terms of root growth and architecture and general growth parameters. Main component analysis revealed that thick and deep growing primary and nodal roots are important for good long term drought adaptation. As candidates of root architecture influencing genes, we study lateral organ boundary (LOB) domain transcription factor family of Brachypodium. To date, eleven genes coding for LOB domain transcription factors were characterized at transcript level. Various organ specificities were found, one of them possesses exceptionally high root tip specificity. The promoter-GFP construct of this gene confirmed the expression in root tip and also revealed promoter activity in stamen.




Figure 2. Phylogenetic tree of Brachypodium LOB-domain transcription factor coding genes. The tree was made upon the conserved amino acid sequences of LOB domains.


Drought responses in cereals: a genomic approach

The basic research questions for our future work are the followings:
We aim to unshade functional roles of the different LOB types with special emphasis on the ones influencing root architecture. How lateral organ boundary determining transcription factors can have effect on the mitotic activity of the meristematic cells? Brachypodium research is to be extended towards the relation of root development and cell cycle. Therefore, we aim to reveal the cell cycle gene families (CDK, cyclin, E2F/DP, retinoblastoma) of Brachypodium. Thanks to the advance of genomics on Brachypodium, putative miRNA genes are already annotated. We also address the question which miRNA genes may be involved in the transcript level regulation of these cell cycle regulatory genes in roots? A recently started OTKA grant supports these directions.

On the Brachypodium model, construction of cDNA overexpressing and RNAi plants for selected LOB-domain genes will be continued. The cross-talk of organ boundary control and cell cycle regulation will be studied using LOB-domain mutant lines (T-DNA tagged or TILLING lines). LOB-domain transcription factor gene mutants will be utilized to reveal their effect on the expression of cell cycle genes. Protein interactions as well as phosphorylation of LOB proteins by the cyclin/cdk complex will be investigated. Genes of the CDK/cyclin and E2F/RB regulatory pathway will be investigated in Brachypodium and their putative linkage to LOB-domain TFs via phosphorylation will be studied. Phenotypic characterization of the mutations as well as expression analysis of the cell cycle regulatory genes and LOB-domain transcription factor genes in these mutants will be carried out. Reporter gene carrying lines will be generated allowing us to follow activation and cease of the cell cycle down to single cell level. Intensive studies of small RNA species resulted in the discovery of miRNA based gene silencing which is ubiquitous among eukaryotes and plays pivotal role in regulation of gene expression. Here, we initiate a project to find those miRNA genes as well which may play role in the regulation of master genes of the cell cycle.

The expected results may contribute substantially to our knowledge on the field of plant developmental biology. Despite of the strong progression of Arabidopsis developmental biology, many questions can not be answered on that excellent model plant because it represents dicotyledonous plants only. Moreover it is well known, thanks to the previous results of our group as well, that there are substantial differences between the cell cycle regulation of monocots and dicots. This fact also necessitates the use of monocot model as well and to compare the results with the ones obtained in Arabidopsis.

Proper cross talk between cell cycle regulation and organ meristem formation is of special importance for the correct and proportional formation of plant organs including root formation and development which is in the scope of the current research. Survival of our crop plants under water limitation or nutrient limitation depends very much on the development of strong and efficient root system of these plants. Therefore understanding the mechanisms of root development is also important for practical aspects as well. Our model plant Brachypodium is a close relative of wheat, barley and rye which allows us to convert our knowledge obtained on this small, fast growing, and simple grass to practical use on these crops of high agronomical importance.


Subunit 2: GENETIC CONTROL OF GROWTH RATE AND PLANT BIOMASS PRODUCTION: GENOME ENGINEERING AND PHENOTYPING

(Dénes DUDITS, principal investigator; Györgyi FERENC, Hilda Anikó TIRICZ, Bettina ZOMBORINÉ NAGY, András CSERI, Anna Viktória NÉMETH, Tamás DIGRUBER, Károlyné TÖRÖK, Katalin LÁSZLÓ, Istvánné KIRI)

2.1. Improved biomass production by polyploidization of short rotation willow

Biomass of fast growing woody plants is one of the most significant renewable resources both for direct and indirect energy production, and raw material for other biopolymer demanding industrial applications. The flexibility of the genome of higher plants frequently allows autopolyploidization of plant species and generally one can observe that the increased and stabilized ploidy level frequently results in enlarged organ size and faster growth rate also in woody species. This project aims to produce tetraploid variants of the short rotation willow by using mitotic inhibitors and transient silencing of genes encoding microtubule associated proteins.

2.1.1. Use of traditional polyploidization method based on microtubule inhibitors

Since there is no published protocol for the production of tetraploid genotypes of willow (Salix viminalis L.), we have identified the key factors of various protocols using known inhibitors of microtubule organization such as colchicin. Treatment of axillary buds allowed the identification of shoots with tetraploid (2n=76 chromosomes) cells.




Figure 3. Control diploid (A1) and tetraploid willow plants derived from colchicine treated axillary buds (B1). Flow cytometric analysis of DNA contents in nuclei of root cells from control(A2) and poliploid (B2) plants. Unpublished data by Bettina Zomboriné Nagy and co-workers.


2.1.2. Transient gene silencing by antisense oligonucleotides

As a novel polyploidization approach we develop a method applying synthetic antisense oligonucleotides for transient silencing genes encoding tubulin-associated proteins. Previously we have described that oligonucleotides can be taken up by wounded plant tissues (Figure 4) and expression of a target gene can be reduced (Figure 5) (Dinc et al., 2011).




Figure 4. Cellular distribution of fluorescein-labeled antisense ODNs (green) and Chl autofluorescence (red) in tobacco (G–L) leaves. Arrows in D to I show chloroplast accumulation of antisense ODNs. The arrow in J shows a nucleus filled with fluorescent antisense ODNs. Bars = 10 mm. (Dinc et al. 2011)





Figure 5. Effects of psbA antisense ODNs on the Chl a Fm (A), the relative transcript level (B) and the amount of D1 protein in ODN-treated Arabidopsis leaves aftre 48 h of illumination (C). Control leaves were treated with random nonsense ODNs. The inset in A shows OJIP transients of control and psbA4-treated leaves. A representative western blot is shown in the inset in C. (Dinc et al. 2011).


Based on sequence data base of Poplar, we clone cDNAs of different tubulin interacting proteins of willow and design various antisense molecules for uptake experiments. After optimizing uptake protocols the gene expression level and DNA content of treated cells will be determined.

2.1.3. Propagation and characterization of tetraploid willow lines

Presently we have several independent tetraploid willow lines. According to the flow cytometric analysis and chromosome counting the selected plants have doubled DNA contents and 2n=76 chromosomes in their root cells. We detected characteristic morphological markers, such as round shape leaves and thick roots. The tetraploid plants are propagated in in vitro cultures by recurrent sprouting of shoots from lateral buds. For further characterization of organ size, growth rate, the tetraploid plants are grown in soil and we will use the digital imaging based phenotyping platform.

2.2. Improving the efficiency of synthetic Oligonucleotide Directed Mutagenesis (ODM)

(In collaboration with Elfrida Fodor, Ferhan Ayaydin and Zoltán Kupihár)

2.2.1. Background and scientific problems

Targeted genome editing has been developed as an alternative to classical mutation breeding and transgenic (GMO) methods to improve crop plants. The Oligonucleotide Directed Mutagenesis (ODM) as Targeted Nucleotide Exchange (TNE) by single stranded DNA oligonucleotides (SDOs) attracts special attention for use in both basic science and plant breeding. SDOs are short synthetic fragments that are complementary in sequence to the targeted DNA and carry the desired mutation which can be exchange, deletion or insertion of preferably one or possibly two nucleotide units. By invading the targeted site of the duplex DNA, SDOs are hybridized to the complementary strand through transient D-loop formation. (Fig. 1) Finally, the mutation is introduced to the DNA by the cellular repair or replication machinery [Liu, L et al. 2003]. SDOs are expected to be degraded in cells but the induced mutations will be stably inherited. The most effective SDOs are: 30-60mers. Beside the phosphorothioate end-protection against exonucleases, they carry several modified structures like Locked Nucleic Acid (LNA), N-alkinyl nucleotides in order to increase duplex invading capability [Andrieu-Soler, C., et al., 2005]. The still very low targeting efficacy is planned to be improved with new SDO constructs.





Oligonucleotides can be delivered to the plant cells by methods generally used for direct plant transformation. Among these the particle bombardment delivery method had been successfully used in development of transient gene screen systems [Dong, C., et al., 2006]. On the other hand, plant protoplast systems allow the use of liposomes, in particular cationic type that could present several advantages over other delivery methodologies. Liposome mediated delivery offers low toxicity and reduced membrane damage [Farhood, H., et al, 1995, Alton, E.W., et al., 1993].

Mutated green fluorescent protein (mGFP) gene offers an excellent tool to test the efficiency of homologous recombination and demonstration of the nucleotide correction both in transient and in stable model systems [Beetham, P.R., et al., 1999, Hu, Y., et al. 2005, Disterer, P., et al., 2012]. We consider two of the key factors for detailed analysis in this project. Engstrom and Kmiec [Engstrom, J.U. et al 2007 and 2008 ] showed that mammalian cells in mid to late S phases are most amenable to gene repair. Therefore, we use synchronized rice cells to determine the restoration of GFP function in various cell cycle phases. Our group has extensive experience in S-phase detection in plants [Kotogany, E., et al, 2010], synchronization of cultured plant cells [Ayaydin, F., et al, 2011] and characterization of cyclin-dependent kinase complexes [Abraham, E., et al, 2011]. Advanced laser scanning, multiphoton and fluorescence microscopes, available in the cellular imaging laboratory will be used during GFP fluorescence and cell cycle phase analysis related optimization and quantification experiments. As second approach, we will use chemicals to stimulate a more open, relaxed chromatin conformation, which will allow increased accessibility to target sequences. Hydroxyurea treatment was reported to result in an increase in targeted nucleotide exchange (TNE) in yeast cells, while trichostatin A inhibitor of histone deacetylation stimulate TNE in both yeast and mammalian cells. [Parekh-Olmedo et al. 2003] Among others, camptothecin (CPT) as an anticancer drug, by activation of the homologous recombination (HR), increases the frequency of targeted gene repair presumably driven by the same pathway [Ferrara, L. et al. 2004].

Considering the screening methodologies of mutated plant cells harboring the desired modification induced by ODM, the most frequently employed trait is the herbicide resistance, which was used in case of tobacco [Beetham, P.R., et al., 1999], rice [Okuzaki, A. et al, 2004] and maize [Zhu, T., et al. 1999 and 2000]. The antibiotic resistance, has been applied in case of Arabidopsis [Kmiec, E.B. et al. 2001], maize, banana and tobacco [Rice, M.C., et al., 2000]. There is one case where green fluorescence protein as marker was used in wheat [Dong, C., et al. 2006]. Since increasing Trp level is a significant breeding goal in cereals the present studies are based on targeted modification of anthranilate synthase (AS) genes. We establish ODM selection system based on feedback insensitive AS mutation. This enzyme plays a key role in the biosynthesis of tryptophan (Trp), and the α subunit of AS is susceptible to feedback inhibition by Trp or its analogues (e.g: 5-methyl tryptophan). Structure-based protein engineering of OASA1—an α- subunit of AS in rice—showed that D323N mutation confers Trp insensitivity. It was found that free Trp levels increase 35 fold in plants and 180 fold in calli as compared to non-transformants [Tozawa, Y., et al. 2001].



2.2.2. Novel chemical modifications in single stranded oligonucleotides used for ODM

We construct novel forms of SDO molecules having structural elements which were not applied yet in mutation generation in the plant systems. We aim to clarify the influence of the new structural factors on the mutation efficacy, and develop novel methods, which will boost the practical use of ODM technology.

2.2.3. Model system based on the mutated GFP to test the efficiency of homologous recombination

In order to establish a test system we produce stable transgenic rice cell lines carrying mutated GFP. We have established transient system based on the non-functional green fluorescence protein (GFP) gene for monitoring the homologous recombination frequency and optimizing uptake parameters. Restoration of the GFP function indicates the frequency of targeted homologous recombination after uptake of synthetic oligonucleotides.




Figure 6. Co-bombardment of mutated GFP and SDOGFP and restoration of GFP function Plasmid with mutated GFP gene was bombarded into the rice cells alone (A) or it was co-bombarded with SDOGFP (B). Single-stranded DNA oligonucleotides (SDOGFP) could restore the GFP normal function. Unpublished data by Dr. Hilda Tiricz and co-workers.


As future plan we will analyze cell cycle dependent frequency of homologous recombination dependent targeted nucleotide exchange. Our aim is the generation of chromatin structure allowing increased frequency of ODM.

2.2.4. ODM used in the production of feedback insensitive anthranilate synthase mutants and characterization of selected 5-methyl tryptophan (5-MT) resistant mutants

The overall goal is to establish an efficient protocol for the use of ODM technology to produce feedback insensitive anthranilate synthase (AS) mutants in cultured rice cells and embryogenic barley tissues. AS is a feedback-regulated key enzyme in tryptophan (Trp) biosynthesis. Preventing feedback inhibition of this enzyme by point mutations results in elevated intracellular Trp levels [29]. For selection of AS mutants, we use 5-methyl tryptophan (5-MT), a false feedback inhibitor of AS, which cannot be used in protein synthesis in place of tryptophan. Thus, the number of 5-MT resistant colonies or tissues serve as a tool for quantifying ODM efficiency. Using this experimental system, we analyze the role of cell cycle phase and chromatin modifying agents on the frequency of ODM events. The proposed project opens a way to identify cells, even barley plants with increased free tryptophan production.


2.3. Phenotyping

2.3.1. Monitoring drought responses of cereal genotypes

Worldwide also in Hungary the drought stress is one of the main factors limiting the yield of cereals. We have used the Complex Stress Diagnostic System as a semi-robotic phenotyping platform to test the drought tolerance-related agronomic traits of cereals. The drought responses were monitored by digital photography, thermal imaging and chlorophyll fluorescence (see Figure). A set of barley and wheat genotype collections was grown under non-stress (60% water supply for the whole life cycle) and stress (20%) conditions in the greenhouse. After taking digital images the green pixel based shoot surface was used to predict the biomass accumulation during the whole growth period once a week. The efficiency of leaf evaporation was assessed by measuring leaf temperature relative to the surrounding air using a sensitive thermocamera.




The root system plays a pivotal role in the adaptation to water limitation also in cereals. We have used various experimental systems to detecting changes of root growth parameters under low soil water content. Wheat plants were grown in transparent plexi rhizocoloumns that allowed digital imaging of root system during the whole life cycle of plants. Pictures were taken from the bottom of the column and 4 side views. In order to quantify root biomass we have introduced the use of the rigid-borescope technology. Its optical system can be well suited for viewing sideways in a clear plastic tubes placed into the soil. Our another experimental system is the microrhizotron, which allows the detection of changes in root growth parameters under water deficit. Cereal seedlings are grown in plexi boxes with soil adjusted to different water contents. Using this experimental system we were able to identify genotypes that continued the root growth under low water content. The root elongation can be originated either from cell elongation or cell division. Therefore the microrhizotron system with detachable side, were used for identification of S-phase cells in roots. The frequency of S-phase cells was detected fluorescent microscopy after EdU (5-ethynil-2-deoxiuridine) staining. We have detected a correlation between the drought tolerance and the frequency of the S-phase cells in the barley root tips.

In attempts to integrate genomic and phenomic levels our phenotyping studies were combined with haplotyping of selected drought response barley genes. EcoTILLING technology as a polymorphism discovery tool has identified key allelic variants in group of drought sensitive and resistant genotypes (Cseri et al., 2013).

2.3.2. Phenotyping of willow plants to monitor growth parameters

- stimulated willow cuttings aiming to improve the rooting capability and growth vigor.
- diploid and tetraploid willow plants for the sequential growth comparison is in progress.

Selected publications

Kotogany E, Dudits D, Horvath GV, Ayaydin F A rapid and robust assay for detection of s-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine PLANT METHODS 6: (1) (2010)

Secenji M, Hideg E, Bebes A, Gyorgyey J Transcriptional differences in gene families of the ascorbate-glutathione cycle in wheat during mild water deficit PLANT CELL REP 29: (1)37-50 (2010)

Secenji M, Lendvai Á, Miskolczi P, Kocsy G, Gallé Á, Szűcs A, Hoffmann B, Sárvári É, Schweizer P, Stein N, Dudits D, Györgyey J: Differences in root functions during long-term drought adaptation: comparison of active gene sets of two wheat genotypes. PLANT BIOLOGY 12: 871-882 (2010) IF: 2.409

Abraham E, Miskolczi P, Ayaydin F, Yu P, Kotogany E, Bako L, Otvos K, Horvath GV, Dudits D Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells J EXP BOT 62: (6)2155-2168 (2011)

Dudits D, Abraham E, Miskolczi P, Ferhan A, Metin B, Horvath VG Cell-cycle control as a target for calcium, hormonal and developmental signals: the role of phosphorylation in the retinoblastoma-centred pathway ANN BOT-LONDON 107: (7)1193-1202 (2011)

Turoczy Z, Kis P, Torok K, Cserhati M, Lendvai A, Dudits D, Horvath GV Overproduction of a rice aldo-keto reductase increases oxidative and heat stress tolerance by malondialdehyde and methylglyoxal detoxification PLANT MOL BIOL 75: (4-5)399-412 (2011)

Abrahám E, Miskolczi P, Ayaydin F, Yu P, Kotogány E, Bakó L, Otvös K, Horváth GV, Dudits D. Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells. J Exp Bot. 2011 Mar; 62(6):2155-68

Cserhati M, Turoczy Z, Zombori Z, Cserzo M, Dudits D, Pongor S, Gyorgyey J: Prediction of new abiotic stress genes in Arabidopsis thaliana and Oryza sativa according to enumeration-based statistical analysis. MOL GENET GENOMICS 285: (5)375-391 (2011) IF: 2.635

Cseri A, Cserháti M, von Korff M, Nagy B, Horvath VG, Palágyi A, Pauk J, Dudits D, Torjek O Allele mining and haplotype discovery in barley candidate genes for drought tolerance EUPHYTICA 181: (3)341-356 (2011)

Dinc E, Toth SZ, Schansker G, Ayaydin F, Kovacs L, Dudits D, Garab G, Bottka S Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus- and chloroplast-encoded proteins of higher plant chloroplasts. PLANT PHYSIOL 157: (4)1628-1641 (2011)

Cserhati M, Turoczy Z, Dudits D, Gyorgyey J: The Rice Word Landscape: A Detailed Catalogue of the Rice Motif Content in the Non-coding Regions. OMICS 16: (6)334-342 (2012)

Dinc E, Ceppi MG, Toth SZ, Bottka S, Schansker G 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 BBA-BIOENERGETICS 1817: (5)770-779 (2012)

Cseri A, Sass L, Törjék O, Pauk J, Vass I, Dudits D Monitoring drought responses of barley genotypes with semi-robotic phenotyping platform and association analysis between recorded traits and allelic variants of some stress genes AUST J CROP SCI 7: (10)1560-1570 (2013)

Németh AV, Dudits D, Molnár-Láng M, Linc G. Molecular cytogenetic characterisation of Salix viminalis L. using repetitive DNA sequences. J APPL GENET. 54: (3) 265-269 (2013).

Szecsenyi M, Cserhati M, Zvara A, Dudits D, Gyorgyey J Monitoring of Transcriptional Responses in Roots of Six Wheat Cultivars during Mild Drought Stress CEREAL RES COMMUN 41: (4)527-538 (2013)

References

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Kotogany E, Dudits D, Horvath GV, Ayaydin F A rapid and robust assay for detection of s-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine PLANT METHODS 6: (1) (2010)

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