Research - Institute of Plant Biology - Laboratory of Arabidopsis Molecular Genetics

László SZABADOS
scientific adviser

picture
Csaba KONCZ scientific adviser
Ágnes CSÉPLŐ senior research associate
Laura Alexandra ZSIGMOND research associate
Gábor RIGÓ research associate
Dániel BENYÓ junior research associate
Hajnalka KOVÁCS junior research associate
Norbert ANDRÁSI junior research associate
James SMART guest researcher
Dávid ALEKSZA junior research associate
Bogáta Piroska CANJEVEC (BOROS) Ph.D. student
Dóra FARAGÓ Ph.D. student
Abu Imran BABA Ph.D. student
Mónika VARGA Ph.D. student
Rama Krishna DASARI ITC student
Anna Mária KIRÁLY laboratory assistant

GENETIC AND MOLECULAR DISSECTION OF ABIOTIC STRESS SIGNALS

Extreme environmental conditions such as drought and high soil salinity lead to osmotic, ionic and oxidative stresses which hinders plant growth and limits agricultural productivity. We are interested to understand the regulation of molecular and physiological responses to these stress conditions, identification of genetic bases for adaptation to such adverse environments. Using Arabidopsis thaliana as model organism, we have identified several regulatory genes encoding previously unknown transcription factors, protein kinases or enzymes which control growth, metabolic responses or cellular defences in stress conditions. Halophyte relatives of Arabidopsis are studied to reveal molecular diversity in regulatory genes which permit adaptation to saline or arid environments.




Figure 1. Several members of the Arabidopsis group, from left to right: Dr. Laura Zsigmond, Annamária Király, Bogáta Boros, Ildikó Valkai, Gábor Rigó, Hajnalka Kovács, Edina Kiss, Norbert Andrási, Dr. Csaba Papdi, Imma Pérez Salamó, Dr. László Szabados.


Development of genetic tools to identify and study regulatory genes

Genetics offer powerful tools to identify novel regulatory genes controlling plant development, stress responses or hormonal signals. In collaboration with partners in the Max-Planck Institut für Züchtungsforschung, several genetic technologies were developed or improved in our laboratory (Papdi et al., 2009, Papdi et al., 2010). A T-DNA tagged Arabidopsis mutant collection was established and insertion sites mapped on Arabidopsis chromosomes (Szabados et al., 2002). Firefly luciferase was employed for gene trapping and could be used to identify mutants of stress-responsive genes (Alvarado et al., 2004). Mutant lines are available for research purposes. More recently the Conditional cDNA Overexpression System (COS) was developed, facilitating random cDNA transfer, conditional overexpression of the inserted cDNA and facile gene identification in transgenic Arabidopsis lines (Papdi et al., 2008, Rigó et al., 2012). The COS system generates conditional dominant phenotypes which is particularly suitable for the identification of stress regulatory genes such as the EFR-type RAP2.12 transcription factor (Papdi et al., 2008) or the HSFA4A heat shock factor (Salamó et al., 2013). Currently we are adapting the COS system to extremophyle plants to isolate stress regulatory genes from plants adapted to saline or arid environments.




Figure 2. The Conditional cDNA Overexpressing System (COS). The cDNA library is introduced into Arabidopsis by large-scale Agrobacterium-mediated transformation. Transgenic lines are screened for dominant, estradiol-dependent phenotypes. Selected plants are used to isolate inserted cDNA by PCR amplification and sequencing of the insert. Phenotype is verified by cloning and re-transforming the recovered insert into wild type Arabidopsis or other plants.


Indexed T-DNA insertion sites
LUC-tagged Arabidopsis lines


Role of heat shock factorsin oxidative stress responses

The heat shock transcription factor HSFA4A was identified with the COS system by screening for salt tolerance in cDNA overexpressing cultured cells. Enhanced expression of HSFA4A in transgenic plants improved tolerance to different stress conditions and diminished oxidative damage, while inactivation of the HSFA4A gene by T-DNA insertion reduced stress tolerance. We showed, that phosphorylation of HSFA4A with MAP kinases MPK3 and MPK6 modulates its capability to trans activate some of its target genes. We are conducting research to elucidate the function of HSFA4A and related heat shock factors in stress signal transduction. In particular, we are interested in the role of HSFA4 subfamily of factors in transmission of MAPK-mediated ROS signals, activation of target genes and contribution in maintenance redox balance in adverse environmental conditions.




Figure 3. HSFA4A overexpression enhances salt and oxidative stress tolerance. Growth of HSFA4A overexpressing plants is enhanced on media supplemented by NaCl, paraquat or hydrogen peroxide.


Root development and stress responses

Root growth and architecture determines water and nutrient uptake and is particularly important to tolerate drought and soil salinity. Recently we found that the plasma membrane-associated Ser/Thr type potein kinase CRK5 controls root development through modulating distribution of the plant hormone auxin. We showed that inactivation of CRK5 inhibits primary root elongation, enhances lateral root formation and delays gravitropic bending of shoots and roots. CRK5 controls auxin transport in root tip through phosphorylation and modulation of PIN2 localization (Rigó et al., 2013). Preliminary data show, that this protein kinase is involved in regulation of salt tolerance as well. We are interested to decipher the function of CRK5 in root development during stress conditions, such as drought and on saline soils.




Figure 4. CRK5 controls root development. A) Root bending of Col-0 wild type, crk5-1 mutant and the complemented mutant in 12h and 24h gravitropic response. B,C) Celular localization of CRK5-GFP protein in primary root apex and lateral root primordia.


Metabolic responses to salt stress

Extreme enviromental conditions lead to profound changes in plant metabolism (Szabados et al., 2011). Proline accumulation during drought or salt stress is a well-known phenomenon in plants. Proline is a multifunctional amino acid, which can contribute to stress tolerance in several ways including osmoprotection, regulation of redox balance or function as metabolic signal (Szabados et al., 2010, Lehman et al., 2010). Regulation of proline biosynthesis has been studied in our laboratory for more than a decade and included the identification and characterization of key Arabidopsis genes P5CS1 and P5CS2, which control the glutamate-derived biosynthetic pathway (Strizhov et al., 1987, Ábrahám et al., 2003, Fabro et al., 2004, Székely et al., 2008). We have confirmed the importance of proline accumulation in maintaining cellular homeostasis and redox balance in plants under salt stress (Székely et al., 2008, Stein et al., 2011, Tóth et al., 2013, Fichmann et al., 2013). We try to understand how proline accumulation can protect photosynthesis, what is the role of proline cycle in coordinating plastid and mitochondrial functions during stress.




Figure 5. Proposed model for proline metabolism in higher plants.


Earlier we have identified the tagged ppr40 mutant, which is hypersensitive to osmotic and salt stress, abscisic acid (ABA) and oxidative agents, but has elevated proline content. The disrupted gene encodes the mitochondrial PPR40 protein, involved in the regulation of mitochondrial electron transport (Zsigmond et al., 2008). We showed, that impaired mitochondrial antioxidant homeostasis is responsible for the enhanced stress sensitivity of the ppr40 mutant (Zsigmond et al., 2011). Importance of mitochondrial electron transport stability in stress tolerance was confirmed by PPR40 overexpressing plants which showed enhanced salt tolerance (Zsigmonds et al., 2012). We are currently investigating the function of mitochondrial electron transport in stress responses, in maintenance of cellular homeostasis, redox balance and energy supply during adverse conditions.




Figure 6. PPR40 is localized in mitochondria and can influence salt tolerance. Left panes shows immunohistochemical detection of PPR40 in protoplasts (green), which overlaps with mitotracer-derived orange fluorescence. Right panel shows enhanced growth of PPR40 overexpressing Arabidopsis plants on salt-containing medium.


Selected publications

Szabados L, Kovács I, Oberschall A, Ábrahám E, Kerekes I, Zsigmond L, Nagy R, Alvarado M, Krasovskaja I, Gál M, Berente A, Rédei GP, Ben-Haim A, Koncz C (2002) Distibution of 1000 sequenced T-DNA tags in the Arabidopsis genome. Plant J. 32:233-242. PubMed

Ábrahám E, Rigó G, Székely G, Nagy R, Koncz Cs, Szabados L (2003) Light-dependent induction of proline biosynthesis by abscisic acid and salt stress is inhibited by brassinosteroid in Arabidopsis. Plant Mol. Biol 51:363-372. PubMed

Székely Gy, Ábrahám E, Cséplő Á, Rigó G, Zsigmond L, Csiszár J, Ayaydin F, Strizhov N, Jásik J, Schmelzer E, Koncz Cs, Szabados L (2008) Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthessis. Plant J. 53:11-28. PubMed

Zsigmond L, Rigó G, Székely Gy, Ötvös K, Szarka A, Darula Zs, Medzihradszky KF, Koncz Cs, Koncz Zs, Szabados L (2008) Arabidopsis PPR40 connects abiotic stress responses to mitochondrial electron transport. Plant Physiol. 146:1721-1737. PubMed

Papdi Cs, Ábrahám E, Joseph MP, Popescu C, Koncz Cs, Szabados L (2008) Functional identification of Arabidopsis stress regulatory genes using the Controlled cDNA Overexpression System, COS. Plant Physiol. 147: 528–542. PubMed

Papdi Cs, Leung, J, Joseph MP, Pérez-Salamó I, Szabados L (2010) Genetic screens to identify plant stress genes. In: Methods in Molecular Biology, vol. 639. New York: Humana Press. 639: 121-139 Document

Ábrahám E, Hourton-Cabassa C, Erdei L, Szabados L (2010) Methods for determination of proline in plants. In: Methods in Molecular Biology, vol. 639. New York: Humana Press. pp. 317-331. PubMed

Szabados L, Savouré A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89-97 PubMed

Lehmann S, Funck D, Szabados L, Rentsch D (2010) Proline metabolism and transport in plant development. Amino Acids 39:949–962 PubMed

Henriques R, Magyar Z, Monardes A, Khan S, Zalejski C, Orellana J, Szabados L, de la Torre C, Koncz Cs, Bögre L (2010) Arabidopsis S6 Kinase mutants display chromosome instability and altered RBR1-E2F pathway activity. EMBO J. 29: 2979-2993. PubMed

Szabados L, Kovács H, Zilberstein A, Bouchereau A (2011) Plants in extreme environments: importance of protective compounds in stress tolerance. Adv Bot Res 57:105-150. ScienceDirect

Stein H, Honig A, Miller G, Erster O, Eilenberg H, Csonka LN, Szabados L, Koncz Cs, Zilberstein, A (2011) Elevation of free proline and proline-rich protein levels by simultaneous manipulations of proline biosynthesis and degradation in plants. Plant Sci 181:140-150. PubMed

Zsigmond L, Tomasskovics B, Deák V, Rigó G, Szabados L, Bánhegyi G, Szarka A (2011) Enhanced activity of galactono-1,4-lacton dehydrogenase and ascorbate - glutathione cycle in mitochondria from Complex III deficient Arabidopsis. Plant Physiol. Biochem. 49: 809-815. PubMed

Rigó G, Papdi Cs, Szabados L (2012) Transformation using Controlled cDNA Overexpression System. In: Methods in Molecular Biology, New York: Humana Press, 913: 277-290. PubMed

Zsigmond L, Szepesi Á, Tari I, KirályA, Szabados L (2012) Overexpression of the mitochondrial PPR40 gene improves salt tolerance in Arabidopsis. Plant Sci 182:87-93, PubMed

Ruibal C, Salamó IP, Carballo V, Castro A, Bentancor M, Borsani O, Szabados L, Vidal S (2012) Differential contribution of individual dehydrin genes from Physcomitrella patens to salt and osmotic stress tolerance. Plant Sci 190:89-102. PubMed

Rigó G, Tietz O, Ayaydin F, Zsigmond L, Kovács H, Páy A, Salchert K, Szabados L, Palme K, Koncz Cs, Cséplö Á (2013) Inactivation of plasma-membrane localized CDPK-related kinase 5 decelerates PIN2 exocytosis and root gravitropic response. Plant Cell 25:1592-1608, PubMed

Ruibal C, Castro A, Carballo V, Szabados L, Vidal S (2013) Recovery from heat, salt and osmotic stress in Physcomitrella patens requires a functional small heat shock protein PpHsp16.4. BMC Plant Biol 13: 174. PubMed

Coego A, Brizuela E, Castillejo P, Ruiz S, Koncz C, del Pozo JC, Pineiro M, Jarillo JA, Paz-Ares J, Leon J (2014) The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional overexpression. Plant J 77: 944-953. PubMed

Perez-Salamo I, Papdi C, Rigó G, Zsigmond L, Vilela B, Lumbreras V, Nagy I, Horvath B, Domoki M, Darula Z, Medzihradszky K, Bögre L, Koncz C, Szabados L (2014) The Heat Shock Factor A4A Confers Salt Tolerance and Is Regulated by Oxidative Stress and the Mitogen-Activated Protein Kinases MPK3 and MPK6. Plant Physiol 165: 319-334. PubMed

Joseph MP, Papdi C, Kozma-Bognar L, Nagy I, Lopez-Carbonell M, Koncz C, Szabados L (2014) The Arabidopsis Zinc Finger Protein 3 interferes with ABA and light signaling in seed germination and plant development. Plant Physiol 165:1203-1220. PubMed

Fichman Y, Gerdes SY, Kovacs H, Szabados L, Zilberstein A, Csonka LN (2015) Evolution of proline biosynthesis: enzymology, bioinformatics, genetics, and transcriptional regulation. Biol Rev Camb Philos Soc 90: 1065-1099 PubMed

Bela K, Horvath E, Galle A, Szabados L, Tari I, Csiszar J (2015) Plant glutathione peroxidases: Emerging role of the antioxidant enzymes in plant development and stress responses. J. Plant Physiol. 176C: 192-201. PubMed

Papdi Cs, Pérez-Salamó I, Joseph MP, Giuntoli B, Bögre L, Koncz Cs, Szabados L (2015) The low oxygen, oxidative and osmotic stress responses synergistically act through the Ethylene Response Factor-VII genes RAP2.12, RAP2.2 and RAP2.3. Plant J. 82: 772-784 PubMed

Horváth E, Brunner Sz, Bela K, Papdi Cs, Szabados L, Tari I, Csiszár J. (2015) Exogenous salicylic acid-triggered changes in the glutathione transferases and peroxidases are key factors in the successful salt stress acclimation of Arabidopsis thaliana. Functional Plant Biology 42(12) 1129-1140, DOI

Szabados l., Györgyey J (2015) Molecular background of stress tolerance: lessons from plant systems (In: Vágvölgyi Cs, Siklós L (ed.) Selected Topics from Contemporary Experimental Biology, Volume 2. 288 p. Szeged: MTA Szegedi Biológiai Központ, 2015. pp. 209-224. document

Horváth, E., Béla, K., Papdi, C., Gallé, Á., Szabados, L., Tari, I. and Csiszár, J. (2015) The role of Arabidopsis glutathione transferase F9 gene under oxidative stress in seedlings, Acta Biol. Hung, 66(4): 406-418. PubMed

Perez-Salamó I, Boros B, Szabados L (2016) Screening Stress Tolerance Traits in Arabidopsis Cell Cultures. In: Methods in Molecular Biology, New York: Humana Press. 1398:235-246, Scopus

Rigó, G., Valkai, I., Faragó, D., Kiss, E., Van Houdt, S., Van de Steene, N., Hannah, M. A., and Szabados, L. (2016) Gene mining in halophytes: functional identification of stress tolerance genes in Lepidium crassifolium. Plant, Cell & Environment, 39:2074-2084. PubMed

Benyó D, Horváth E, Németh E, Leviczky T, Takács K, Lehotai N, Feigl G, Kolbert Zs, Ördög A, Gallé R, Csiszár J, Szabados L, Erdei L, Gallé Á (2016) Physiological and molecular responses to heavy metal stresses suggest different detoxification mechanism of Populus deltoides and P. x canadensis. J. Plant Physiol. 201:62-70. PubMed

Patents

L. Szabados, L. Zsigmond, Cs. Koncz: Improvement of stress tolerance in higher plants. Patent Application No.: P0500811, date: 31/08/2005

Szabados L, Koncz C, Ábrahám E, Papdi C, Joseph MP (2008) Controlled cDNA Overexpression System in Arabidopsis, Hungarian Patent No.: P0800351, 2008.05.30.