A 662 OULU 2015 A 662 UNIVERSITY OF OULU P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATISUNIVERSITATIS OULUENSISOULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTAACTA SCIENTIAESCIENTIAEA A RERUMRERUM Riina Muilu-Mäkelä NATURALIUMNATURALIUM Riina Muilu-Mäkelä Professor Esa Hohtola POLYAMINE METABOLISM OF University Lecturer Santeri Palviainen SCOTS PINE UNDER ABIOTIC Postdoctoral research fellow Sanna Taskila STRESS Professor Olli Vuolteenaho University Lecturer Veli-Matti Ulvinen Director Sinikka Eskelinen Professor Jari Juga University Lecturer Anu Soikkeli Professor Olli Vuolteenaho UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF SCIENCE; Publications Editor Kirsti Nurkkala NATURAL RESOURCES INSTITUTE FINLAND (LUKE), PARKANO RESEARCH UNIT ISBN 978-952-62-1057-5 (Paperback) ISBN 978-952-62-1058-2 (PDF) ISSN 0355-3191 (Print) ISSN 1796-220X (Online) ACTA UNIVERSITATIS OULUENSIS A Scientiae Rerum Naturalium 662 RIINA MUILU-MÄKELÄ POLYAMINE METABOLISM OF SCOTS PINE UNDER ABIOTIC STRESS Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in Kaljusensali (KTK112), Linnanmaa, on 11 December 2015, at 13 p.m. UNIVERSITY OF OULU, OULU 2015 Copyright © 2015 Acta Univ. Oul. A 662, 2015 Supervised by Professor Hely Häggman Docent Tytti Sarjala Reviewed by Doctor Sergio Ochatt Professor C. Peter Constabel ISBN 978-952-62-1057-5 (Paperback) ISBN 978-952-62-1058-2 (PDF) ISSN 0355-3191 (Printed) ISSN 1796-220X (Online) Cover Design Raimo Ahonen JUVENES PRINT TAMPERE 2015 Muilu-Mäkelä, Riina, Polyamine metabolism of Scots pine under abiotic stress. University of Oulu Graduate School; University of Oulu, Faculty of Science; Natural Resources Institute Finland (Luke), Parkano Research Unit Acta Univ. Oul. A 662, 2015 University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland Abstract Changes in climate will impose abiotic stress on plant species, and eventually affect their distributional range. This is a particular challenge especially to tree species with long generation times and slow rates of evolution. In the boreal zone, most of these species also hold high economic value. The aim of the study was to enhance understanding of the abiotic stress coping strategies of Scots pine (Pinus sylvestris L.) in its vulnerable early growth phase. In particular, the study evaluates the role of polyamine (PA) metabolism of Scots pine under abiotic stress. Polyamines (PAs) are small, ubiquitous nitrogenous compounds involved in fundamental biological processes of plants such as growth, organogenesis, embryogenesis and abiotic and biotic stress defence. The effects of different water availabilities and spring frost temperatures on shoots and roots of young seedlings were investigated in two growth chamber experiments. Proembryogenic liquid cell culture system was established and used as a controllable platform. Effects of polyethylene glycol (PEG) induced osmotic stress was investigated at the cellular level in liquid cultures. Responses to treatments were evaluated by quantifying changes in the expression of PA metabolism, stress, cell death and cell division -related genes, metabolite concentrations, as well as stress-induced physiology and morphology. The results revealed that PA metabolism is tissue specific and strictly regulated. Drought stress induced the accumulation of putrescine (Put) in photosynthesizing tissues, whereas the prime response to spring frost was a reorganization of root growth. Generally, drought and osmotic stress decreased the expression of PA catabolizing genes, leading to consistent PA levels in tissues even under severe stress. To conclude, PAs have a protective role in maintaining the growth and development of pine tissues subjected to abiotic stress. Keywords: abiotic stress, development, embryogenic cells, gene expression, liquid cell culture, phenylpropanoids, polyamines, Scots pine Muilu-Mäkelä, Riina, Polyamiiniaineenvaihdunnan rooli metsämännyn abioottisissa stressivasteissa. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Luonnontieteellinen tiedekunta; Luonnonvara- keskus (Luke), Parkanon toimipaikka Acta Univ. Oul. A 662, 2015 Oulun yliopisto, PL 8000, 90014 Oulun yliopisto Tiivistelmä Ilmastonmuutos vaikuttaa eri kasvilajien levinneisyyteen. Nopeasti muuttuva ilmasto on haaste erityisesti metsäpuulajeille, jotka pitkän sukupolvenvälin takia kehittyvät evolutiivisesti hitaasti. Erilaisten ympäristötekijöiden aiheuttamien puolustusaineenvaihdunnan muutosten tunteminen metsäpuilla on tulevaisuuden kannalta myös taloudellisesti tärkeää. Työn tavoitteena oli selvit- tää millainen merkitys polyamiineilla on metsämännyn (Pinus sylvestris L.) abioottisessa stressi- vasteessa. Polyamiinit (PAt) ovat pienimolekyylisiä typpiyhdisteitä, joiden tiedetään olevan mukana solujen perusaineenvaihdunnassa, kasvien kasvussa ja kehityksessä ja stressivasteissa. Tutkimus koostuu kolmesta kokeesta, joissa selvitettiin PA aineenvaihduntaa osana männyn taimien ja erilaistumattomien somaattisista alkioista peräisin olevien soluviljelmien stressivastei- ta. Kahdessa kokeellisessa tutkimuksessa männyn sirkkataimia altistettiin erilaisille kasvualustan vesipitoisuuksille ja keväthallan lämpötilaolosuhteille. Kolmannessa kokeessa PA aineenvaih- dunnan reagointia osmoottiseen stressiin tutkittiin erilaistumattomissa somaattisissa epäkypsissä männyn alkiosta peräisin olevissa solukoissa kontrolloidussa nesteviljely-ympäristössä. Tutki- muksessa tarkastelin PA aineenvaihduntaa säätelevien geenien ilmenemistä ja PA pitoisuuksien muutoksia suhteessa kasvuun, kehitykseen ja stressissä indusoituvien geenien ilmenemiseen. Tulokset osoittavat, että PA aineenvaihdunta on tiukasti säädeltyä erilaisissa solukoissa. Tai- mien yhteyttävät solukot kerryttivät putreskiinia (Put) kuivuudessa. Ensimmäiset vasteet kevät- hallaolosuhteisiin havaittiin muutoksena juurten kasvun säätelyssä. Sekä kuivuus että osmootti- nen stressi laskevat PA hajottavien geenien ilmenemistä, mikä on metsämännyn tapa ylläpitää tasaiset PA pitoisuudet solukoissa stressitekijöistä huolimatta. Johtopäätöksenä voidaan todeta, että PA aineenvaihdunta on tärkeä metsämännyn kasvun ja kehityksen kannalta. Asiasanat: abioottinen stressi, erilaistumaton somaattinen alkiosolukko, fenyylipropanoidit, geenien ilmentyminen, nesteviljely, metsämänty, polyamiinit Acknowledgements First of all I want to thank my supervisors Docent Tytti Sarjala and Prof. Hely Häggman for offering me this interesting research topic which has developed during my thesis work. The atmosphere has always been helpful and understanding, which has encouraged me in my work. I thank you for your expert guidance and for the opportunity to be involved in such high quality science. Working in two institutions, at the University and at the Research Institute, has expanded my perspective on Finnish academic life. I am grateful to Dr. Jaana Vuosku for her valuable advises and for being my unofficial supervisor during the journey. I wish to thank Prof. Esa Läärä for teaching and inspiration in statistical issues. I am grateful to Dr. Sergio Ochatt and Prof. C. Peter Constable for efficiently reviewing this thesis. I warmly thank all my co-authors, especially Dr. Markku Saarinen, Dr. Leena Hamberg, Dr. Juha Heiskanen and Harri Latva-Mäenpää for your contribution to this study. I want to express my thanks to Dr. Suvi Sutela for being my co-author but also peer support, ever since we started to work in a same lab over twelve years ago. I wish to thank all my collaborators and personnel at the Parkano unit of the Natural Resource Institute Finland (Luke). I thank Dr. Meeri Pearson for peer support and for revising the language of two of the original papers. I thank Prof. Jori Uusitalo, Eveliina Oinas, Tomi Kaakkurivaara, Harri Lindeman and Maarit Haavisto for the long but rewarding drives to work between Tampere and Parkano. I think that after those kilometers we have learned to know each other pretty well. I am grateful to Hanna Leppälammi, Anneli Käenmäki and Eeva Pihlajaviita for their skillful help in developing the methods in a lab and producing the data. I thank Tuire Kilponen for the graphic layout of the publications. I wish to thank the great people who have shared the office in Hervanta with me. You have made the working days enjoyable. Especially, I am grateful to Dr. Otso Huitu, who revised the language of this thesis and one original paper, gave last moment valuable advice and in that way “pushed the man to the top of the hill”. I wish to give my loving thanks to my parents Pirkko and Matti Muilu who have loved and encouraged me and trusted in me at every turn of my life. Warmest thanks to my children Ilona and Konsta for being the most precious things of my life. They had great patience during the last summer, when in spite of their vacation, mother just worked. I thank my husband Pekka Mäkelä, for all his support during the process. Especially, I thank him for the encouraging words during the moments 7 when I totally lost faith. He is the love of my life. Finally, I extend my warmest thanks to all my friends and relatives. Thank you for being here for me. This research was conducted at the Natural Resource Institute Finland (Luke), Parkano unit, and at the Genetics and Physiology Department of the University of Oulu. I acknowledge financial support by the Graduate School of Forest Sciences, the Academy of Finland and the Thule institute of Oulu. Tampere, October 2015