Redox regulation of stress response and development in wheat and Arabidopsis

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1 Festetics Doctoral School, University of Pannonia Redox regulation of stress response and development in wheat and Arabidopsis Zsolt Gulyás Supervisor: Dr. Gábor Kocsy, PhD 2017.

2 Introduction and aims of the research Cultivated plants are of fundamental importance for mankind due to their role in nutrition and the industry. As a consequence of climate change they are endangered more and more frequently by stress factors, especially by extreme changes in temperature and water supply. Both of them affect the Central European growing areas. Abiotic stresses induce diverse physiological, morphological, biochemical and molecular changes in the plants with unfavourable effect on their development. This results in lower yields. This loss, in the case of cereals - which are the most important sources of food, feed and basic commodities worldwide -, reaches millions of kilograms every year. Therefore gaining comprehensive knowledge and understanding on environmental stresses affecting plants is of key importance from the point of view of food security. Wheat, the main cereal in the temperate zone, is one of the most important sources of food for mankind. Its gene pool centres in the Black Sea basin and West Asia, but, due to its strong adaptability, it is already highly widespread throughout the world. Along with maize, wheat is the most important cereal in Hungary. Its production area stretches roughly over 1,1 million hectares every year. Winter wheat is grown in greater amounts than spring wheat, as it can reach 15-25% more yield. For winter wheat, adequate cold tolerance and the vegetative/generative transition are of great importance since cold tolerance declines after the transition and the flower primordia are extremely sensitive to cold. In order to survive the shock and achieve sufficient cold tolerance, wheat needs to go through cold hardening a longer period of low temperature phase which is ensured by the progressively reducing temperature in autumn. 1

3 The lowering temperature may induce changes in the plants inner redox environment. A slight, controlled change in the redox signalling pathways can support the changes of metabolism, growth and development that ensure adaptation to the new environmental conditions. However, a sudden, excessive drop in the temperature can upset the redox homeostasis and increase the amount of reactive oxigen forms. These processes are harmful for the membranes and the macromolecules. The reactive oxigen forms are neutralised by antioxidant compounds (ascorbic acide, glutathione) and enzymes (catalase, glutathione reductase, ascorbate peroxidase). Besides the wheat experiments, investigations with Arabidopsis (Arabidopsis thaliana L.) offered great opportunity to study the phenomena of oxidative stress and redox regulation. Arabidopsis is a small flowering plant related to cabbage and mustard. It is indigenous in Europe, Asia and Africa. For its relatively short life cycle and small genome, it is a popular model organism in plant biology and genetics. Due to this, it was the first plant to have its genome sequenced in Although it is not used for agricultural purposes, studies on Arabidopsis provides a plethora of information for the investigations on agricultural plants. In our experiments, Arabidopsis was used as a model plant for studying the role of redox regulation in stress response, development and amino acid metabolism. 2

4 Basic aims of the thesis: 1. Comparing the redox regulation of cold tolerance and the development of flower primordia in a winter (Ch) and a spring (Tsp) wheat genotype. 2. Studying the redox sensitivity of the genes influencing cold hardening and flowering in the two genotypes. 3. Confirming the hypothetical redox regulation of free amino acid levels in Arabidopsis. 4. Revealing the redox regulation of the genes acting in proline biosynthesis and degradation in Arabidopsis. 3

5 Experimental system and applied methods The plantlets of the two wheat genotypes, Triticum aestivum L. ssp. Spelta, a cold sensitive spring wheat and Triticum aestivum L. ssp. ae. cv. Cheyenne, a cold tolerant winter wheat, were grown at 20/17 C, in 1/2 Hoagland solution until they reached the two-leaf stage. After this, reducing agents (glutathione, ascorbic acid), oxidising agents (glutathione disulfide, hydrogen peroxide) and osmotic agents (NaCl, polyethylene glycol) were added to the liquid medium. On the 3 rd day of the chemical treatment the plantlets continued to grow at 5 C and they were given normal liquid medium from the 4 th day of cold hardening. A 3-week long period of cold hardening was followed by a 3-week long recovery phase at 20/17 C. Redox changes were deduced through determining the amount of oxidised and reduced thiols (HPLC) and calculating the redox potential using the Nernst equasion. The treatments effects on cold acclimation were identified by determining the shoots fresh weight and performing electrical conductivity measurements indicating membrane damage. The chemicals effect on the vegetative/generative transition was revealed by observing the developmental stages of the shoot apices. Additionally, the reactive oxygen species (ROS) in the apexes were examined by using H 2 DCFDA fluorescent dye. The expression of the genes playing a role in the regulation of cold acclimation and flowering was examined by real-time PCR (qpcr). In our experiment with Arabidopsis (Arabidopsis thaliana L.), ecotype Columbia (Col-0) and its glutathione-deficient pad 2-1 and ascorbic acid-deficient vtc 2-1 mutants were studied. The 10-day old plantlets grown on garden soil were replanted into peat pellets and were further grown in plant growth chambers at 23/22 C temperature, 70/75% air humidity and short daylight for 4 weeks. The 5-and-a-half-week old plantlets were placed 4

6 in ½ Hoagland solution and grown for 5 days under control conditions. After this the plantlets were treated for 3 days by reducing agents (glutathione, ascorbic acid, dithiothreitol) and an oxidising agent (hydrogen peroxide) added to their liquid medium. After the treatment samples were collected for biochemical and gene expression analysis. In order to keep track of redox changes, thiol, ascorbic acid and hydrogen peroxide measurements were performed. Free amino acid and protein contents were determined. Genes coding the enzymes acting in proline synthesis and degradation were also examined by qpcr in order to confirm the redox sensitivity of the genes. 5

7 New results (Theses) The role of redox regulation in wheat during cold acclimation and flower primordia development It was revealed that the amount and reducing potential of disulfide forms of the examined thiols increased after the osmotic treatments in cold tolerant winter wheat already at 20/17 C while, in the case of the spring genotype, this was observed only after a treatment at 5 C. It was found that the redox changes triggered faster growth and initial flower primordia development in the spring genotype while the winter wheat showed a greater degree of cold tolerance. After the treatments, in the case of the spring genotype, the expression of the genes inducing flowering was higher. However, in the case of the winter genotype, the treatments induced higher expression of the genes affecting cold tolerance. Redox regulation of the amount of free amino acids in Arabidopsis It was confirmed that the concentration of free amino acids are controlled by redox regulation. Through chemical treatments and the use of Arabidopsis mutants, it was established that, from among the examined redox compounds, GSH had the greatest effect on the concentration on free amino acids. It was observed that, regardless of the genotype, the concentration of certain non-proteinogenic amino acids (like cystathionine, alpha-aminoadipic acid and alpha-aminobutyric acid) grew significantly under GSH treatment. It was revealed that the enzyme coding genes acting in proline metabolism are controlled by redox regulation in Arabidopsis. 6

8 List of publication International journals Kocsy G, Tari I, Vanková R, Zechmann B, Gulyás Z, Poór P, Galiba G: Redox control of plant growth and development PLANT SCIENCE 211: pp (2013), IF: Gulyás Z, Boldizsár Á, Novák A, Szalai G, Pál M, Galiba G, Kocsy G: Central role of the flowering repressor ZCCT2 in the redox control of freezing tolerance and the initial development of flower primordia in wheat BMC PLANT BIOLOGY 14:(1) Paper p. (2014), IF: Zsolt Gulyás, Livia Simon-Sarkadi, Eszter Badics, Aliz Novák, Zsuzsanna Mednyánszky, Gabriella Szalai, Gábor Galiba, Gábor Kocsy: Redox regulation of free amino acid levels in Arabidopsis Physiol. Plant, 159 (3): pp (2017), IF: Török A, Gulyás Z, Szalai G, Kocsy G, Majdik C: Phytoremediation capacity of aquatic plants is associated with the degree of phytochelatin polymerization. JOURNAL OF HAZARDOUS MATERIALS 299: pp (2015), IF:

9 Boldizsár Á, Carrera D Á, Gulyás Z, Vashegyi I, Novák A, Kalapos B, Pál M, Galiba G, Kocsy G: Comparison of redox and gene expression changes during the vegetative/generative transition in crowns and leaves of wheat chromosome 5A substitution lines at low temperature. JOURNAL OF APPLIED GENETICS 57: pp (2016), IF: Boldizsár Á, Vanková R, Novák A, Kalapos B, Gulyás Z, Pál M, Floková K, Janda T, Galiba G, Kocsy G: The mvp2 mutation affects the generative transition through the modification of transcriptome pattern, salicylic acid and cytokinin metabolism in Triticum monococcum (2016): J PLANT PHYSIOL Jul 11; 202: IF:

10 Acknowledgements First and foremost I wish to thank the leaders of the Centre for Agricultural Research, Hungarian Academy of Sciences and Prof. Gábor Galiba, Head of the Department of Plant Molecular Biology for accepting my research work in Martonvásár. The experiments were sponsored by different project funds (OTKA K83642, CNK80781, TÁMOP B-10/ ). I would like to thank Prof. Ferenc Vonderviszt, Head of the Doctoral School of Molecular and Nanotechnologies, Pannon University, for admitting me in the doctoral school and allowing me to accomplish my PhD studies there. I thank Prof. Angéla Anda, Head of the Festetics Doctoral School, Pannon University, for providing an opportunity to complete my PhD studies at her institute. I wish to express my sincere thanks to my supervisor, Dr. Gábor Kocsy for all the support and professional help he gave me during my work. The HPLC measurements meant invaluable contribution to the research for which I am thankful to Dr. Gabriella Szalai and her colleagues. Also, I am indebted to Dr. Lívia Sarkadi and her group for the measurements of free amino acids. The assistants of the Department of Plant Molecular Biology, E. Mónika Fehér and Imréné Horváth, helped immensely with the laboratory work and I am, therefore, thankful to them. I am very grateful to the colleagues at the Agricultural Institute for their valuable cooperation and professional support. Finally, I thank my Family and Friends for the encouragement and support they gave me throughout all my work. 9

ANTIOXIDATIVE DEFENCE IN WINTER WHEAT PLANTS DURING EARLY COLD ACCLIMATION

GEN. APPL. PLANT PHYSIOLOGY, SPECIAL ISSUE, 2006, 101-108 101 ANTIOXIDATIVE DEFENCE IN WINTER WHEAT PLANTS DURING EARLY COLD ACCLIMATION P. Apostolova, I. Yaneva* Acad. M. Popov Institute of Plant Physiology,