ABCDEFG

UNIVERS ITY OF OULU P.O.B . 7500 F I -90014 UNIVERS ITY OF OULU F INLAND

A C T A U N I V E R S I T A T I S O U L U E N S I S

S E R I E S E D I T O R S

SCIENTIAE RERUM NATURALIUM

HUMANIORA

TECHNICA

MEDICA

SCIENTIAE RERUM SOCIALIUM

SCRIPTA ACADEMICA

OECONOMICA

EDITOR IN CHIEF

PUBLICATIONS EDITOR

Senior Assistant Jorma Arhippainen

Lecturer Santeri Palviainen

Professor Hannu Heusala

Professor Olli Vuolteenaho

Senior Researcher Eila Estola

Director Sinikka Eskelinen

Professor Jari Juga

Professor Olli Vuolteenaho

Publications Editor Kirsti Nurkkala

ISBN 978-951-42-9435-8 (Paperback)ISBN 978-951-42-9436-5 (PDF)ISSN 0355-3191 (Print)ISSN 1796-220X (Online)

U N I V E R S I TAT I S O U L U E N S I SACTAA

SCIENTIAE RERUM NATURALIUM

U N I V E R S I TAT I S O U L U E N S I SACTAA

SCIENTIAE RERUM NATURALIUM

OULU 2011

A 573

Jaana Vuosku

A MATTER OF LIFE AND DEATH – POLYAMINE METABOLISM DURING ZYGOTIC EMBRYOGENESIS OF PINE

UNIVERSITY OF OULU,FACULTY OF SCIENCE,DEPARTMENT OF BIOLOGY

A 573

ACTA

Jaana Vuosku

A C T A U N I V E R S I T A T I S O U L U E N S I SA S c i e n t i a e R e r u m N a t u r a l i u m 5 7 3

JAANA VUOSKU

A MATTER OF LIFE AND DEATH – POLYAMINE METABOLISM DURING ZYGOTIC EMBRYOGENESIS OF PINE

Academic dissertation to be presented with the assent ofthe Faculty of Science of the University of Oulu for publicdefence in Kuusamonsali (Auditorium YB210), Linnanmaa,on 27 May 2011, at 12 noon

UNIVERSITY OF OULU, OULU 2011

Copyright © 2011Acta Univ. Oul. A 573, 2011

Supervised byProfessor Hely HäggmanDocent Tytti SarjalaDoctor Anne Jokela

Reviewed byProfessor Claire WilliamsProfessor Roberto Rodriguez Fernandez

ISBN 978-951-42-9435-8 (Paperback)ISBN 978-951-42-9436-5 (PDF)http://herkules.oulu.fi/isbn9789514294365/ISSN 0355-3191 (Printed)ISSN 1796-220X (Online)http://herkules.oulu.fi/issn03553191/

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2011

Vuosku, Jaana, A matter of life and death – Polyamine metabolism during zygoticembryogenesis of pine. University of Oulu, Faculty of Science, Department of Biology, P.O. Box 3000, FI-90014University of Oulu, FinlandActa Univ. Oul. A 573, 2011Oulu, Finland

Abstract

The study gathered information about polyamine metabolism throughout the Scots pine (Pinussylvestris L.) zygotic embryogenesis and about physiological events occurring simultaneously inthe megagametophyte tissue. Additionally, novel sequence data of the Scots pine polyamine geneswere used for studying the evolution of polyamine genes in plants.

Phylogenetic analyses revealed that the eukaryotic ornithine decarboxylase (ODC) might haveevolved from a multifunctional bacterial progenitor. In conifers, the alternative argininedecarboxylase (ADC) pathway is preferred in putrescine biosynthesis, which may have caused therelaxed purifying selection in the ODC genes. The phylogenetic analysis of spermidine synthase(SPDS), spermine synthase (SPMS) and thermospermine synthase (ACL5) sequences supportedthe view that eukaryotic SPDS genes are derived from a common ancestor, whereas SPMS geneshave evolved several times from SPDS genes. The identified Scots pine sequence was defined asa putative thermospermine synthase (TSPMS) encoding gene and named PsACL5. Thephylogenetic analysis of polyamine oxidase (PAO) sequences supported the view that plantspossess several different PAOs, which may have different catalytic properties.

The consistency of the polyamine concentration profiles during Scots pine zygoticembryogenesis suggested that polyamines have an important role in the embryo development andthat individual polyamines may have different roles at different developmental stages. Generally,the polyamine concentrations increased at the early stages but decreased at the late stages ofembryo development. Only the free putrescine fraction remained stable throughout the embryodevelopment. Putrescine was almost solely produced via the ADC pathway and the ADC enzymewas at least partially transcriptionally regulated. Both ADC mRNA transcripts and ADC proteinlocalized in dividing cells of embryos, which implicated the essential role of ADC in the mitosisof plant cells.

The megagametophyte was viable from the early phases of embryo development until the earlygermination of mature seeds. However, the megagametophyte cells in the narrow embryosurrounding region (ESR) died via morphologically necrotic cell death. In the dying cells,extensive nucleic acid fragmentation caused the unspecific hybridization of probes in an in situmRNA hybridization assay. The occurrence of necrotic cell death in Scots pine embryogenesisindicated that developmentally and physiologically regulated necrotic cell death is evolutionarilyconserved and exists also in plants.

Keywords: developmental cell death, embryogenesis, evolution, in situ mRNAhybridization, megagametophyte, necrotic cell death, Pinus, polyamine, seeddevelopment

Vuosku, Jaana, Polyamiiniaineenvaihdunta ja solukuolema männyn tsygoottisessaalkionkehityksessä.Oulun yliopisto, Luonnontieteellinen tiedekunta, Biologian laitos, PL 3000, 90014 OulunyliopistoActa Univ. Oul. A 573, 2011Oulu

Tiivistelmä

Työssä tutkittiin polyamiiniaineenvaihduntaa ja megagametofyyttisolukossa tapahtuvia fysiolo-gisia muutoksia metsämännyn (Pinus sylvestris L.) alkionkehityksen aikana. Polyamiineja (put-reskiini, spermidiini ja spermiini) syntetisoivia ja hajottavia entsyymejä koodaavien geenienemäsjärjestys selvitettiin metsämännystä. Sekvenssejä käytettiin kasvien polyamiinigeenien evo-luution tutkimiseen.

Tutkimuksessa todettiin, että eukaryooteissa putreskiinin biosynteesistä vastaava entsyymi,ornitiinidekarboksylaasi (ODC), on voinut kehittyä bakteerien lysiinikarboksylaasista (LDC),joka dekarboksyloi sekä ornitiinia että lysiiniä. Kasveissa putreskiinia voidaan tuottaa myösarginiinidekarboksylaasin (ADC) kautta, mikä on johtanut ODC-geeneihin kohdistuvan puhdis-tavan valinnan heikentymiseen. Aminopropyyli-ryhmiä liittävien entsyymien osalta tutkimustukee käsitystä, jonka mukaan eukaryoottiset spermidiinisyntaasit (SPDS) ovat kehittyneetyhteisestä kantamuodosta, kun taas spermiinisyntaasi (SPMS) on syntynyt useita kertoja SPDS-geenin kahdentumisen kautta. Metsämännystä tunnistettiin termospermiinisyntaasia (TSPMS)koodaava geeni, jolle annettiin nimeksi PsACL5. Fylogeneettisen analyysin perusteella kasveissaon useita erilaisia polyamiinien hajotuksesta vastaavia polyamiinioksidaaseja (PAO), joidenkatalyyttiset ominaisuudet voivat poiketa toisistaan.

Metsämännyllä polyamiinipitoisuudet vaihtelivat alkionkehitysvaiheen mukaan yhdenmukai-sesti eri vuosina, mikä viittaa polyamiinien tärkeään rooliin alkionkehityksessä. Polyamiinipitoi-suudet kasvoivat varhaisen ja pienenivät myöhäisen alkionkehityksen aikana lukuun ottamattavapaan putreskiinin pitoisuutta, joka pysyi samana koko alkionkehityksen ajan. Putreskiinia tuo-tettiin alkioissa lähes pelkästään ADC-reitin kautta, ja ADC-entsyymin säätelyn todettiin tapah-tuvan ainakin osittain transkription tasolla. Koska sekä ADC-geenin lähetti-RNA että ADC-ent-syymi löytyivät alkion jakautuvista soluista, on ilmeistä, että ADC-entsyymillä on tärkeä tehtä-vä kasvisolujen mitoosissa.

Megagametofyytti säilyi elossa koko alkionkehityksen ajan lukuun ottamatta alkio-onteloareunustavia soluja, jotka olivat morfologialtaan nekroottisia. Nukleiinihappojen voimakas pilk-koutuminen aiheutti soluissa koettimien epäspesifisen sitoutumisen, kun geenien ilmenemistäpaikannettiin lähetti-RNA:han in situ hybridisaatio-menetelmällä. Tutkimuksessa löydetty män-nyn alkiokehitykseen liittyvä nekroottinen solukuolema osoitti ensimmäistä kertaa, että fysiolo-gista ja kehityksellistä nekroottista solukuolemaa esiintyy myös kasveissa.

Asiasanat: alkionkehitys, evoluutio, in situ hybridisaatio, kehityksellinen solukuolema,megagametofyytti, nekroottinen solukuolema, Pinus, polyamiini, siemenen kehitys

7

Acknowledgements

This study was carried out at the Department of Biology at the University of Oulu

and at the Parkano Research Unit of the Finnish Forest Research Institute. I wish

to express my sincere gratitude to all those who have participated in this work.

I owe my deepest gratitude to my supervisors Professor Hely Häggman and

Dr. Tytti Sarjala (Docent) for introducing me to the fascinating field of polyamine

research and for providing good research facilities for my work. Their expertise,

combined with their kindness, patience and positive attitude, have helped and

encouraged me through these years. I wish to thank warmly my supervisor Dr.

Anne Jokela for all the help and support during this process, but especially for her

caring friendship.

I wish to express my warm gratitude to Professor Outi Savolainen and

Professor Esa Läärä. Their profound knowledge and supportive attitude towards

my work have been valuable for me.

I wish to thank warmly my co-authors and collaborators Suvi Sutela (M.Sc.),

Mira Sääskilahti (M.Sc.), Riina Mäkelä (M.Sc.), Johanna Kestilä (M.Sc.), Marja

Suorsa (M.Sc.), Marko Suokas (M.Sc.) and Professor Teresa Altabella for their

significant contribution to this work.

It is a great pleasure to thank all my research colleagues at the Department of

Biology for creating a supportive and inspiring atmosphere over these years:

Professor Anja Hohtola, Dr. Laura Jaakola, Dr. Anna-Maria Pirttilä, Dr. Anneli

Kauppi, Dr. Soile Jokipii-Lukkari, Dr. Marjaana Tahkokorpi, Dr. Katja Karppinen,

Dr. Estelle Dumont, Dr. Kaloian Nickolov, Liisa Ukkola (M.Sc.), Jaanika Edesi

(M.Sc.), Janne Koskimäki (M.Sc.), Dr. Satu Mänttäri, Dr. Katja Anttila, Dr.

Komlan Avian, Helena Jauhiainen (M.Sc.), Terttu Kämäräinen-Karppinen (M.Sc.),

Dr. Mysore Tejesvi, Dr. Veli-Pekka Pelkonen, Dr. Marian Sarala, Johanna

Metsometsä (M.Sc.), Anna-Kaisa Anttila (M.Sc.), Katariina Taskinen (B.Sc.),

Pavlo Ardanov (M.Sc.), Abdul Shakoor (M.Sc.), Laura Zoratti (M.Sc.) and many

others who have worked at the department during these years. I also thank Dr. Jori

Uusitalo at the Parkano Research Unit and Dr. Eila Tillman-Sutela at the Muhos

Research Unit of the Finnish Forest Research Institute.

I wish to thank warmly the staff of the Department of Biology. Especially, I

thank laboratory amanuensis Tuulikki Pakonen, head laboratory manager Hanna-

Liisa Suvilampi, laboratory technicians Taina Uusitalo and Soile Alatalo,

laboratory engineer Niilo Rankka and laboratory technician Matti Rauman for

8

their kind help in the course of this study. I am also grateful to laboratory

technician Eeva Pihlajaviita at the Parkano Research Unit for her skillful help.

I wish to give my warm thanks to my close friend, amanuensis Minna

Vanhatalo, for her practical help with this thesis, but especially for maintaining

my mental balance by sharing joy, stress and sorrow with me during the years.

I wish to give my loving thanks to my parents Aune and Tauno Vuosku for

their love and everlasting support. I also wish to thank my family-in-law and my

friends for the encouragement and pleasant moments outside the world of

research. I wish to present my dearest thanks to Jarmo Kaikkonen for his love,

care, patience and encouragement, which have given me strength to complete this

thesis. I wish to thank our son Arttu for his unselfish love and for being the most

precious thing in my life. I want to share with my family the joy of finally

reaching this goal.

The work for this thesis was financially supported by the Academy of Finland,

the Biological Interactions Graduate School, the Finnish Cultural Foundation, the

Niemi Foundation, the Taina Kuusi Foundation and the Oulu University

Scholarship Foundation.

Oulu, January 2011 Jaana Vuosku

9

Abbreviations

ACT actin

ADC arginine decarboxylase

AIH agmatine imidohydrolase

AO acridine orange

ATP adenosine-5'-triphosphate

ARG arginase

BCIP 5-bromo-4-chloro-3-indolyl phosphate

CAT catalase

cDNA complementary DNA

CPA N-carbamoylputrescine amidohydrolase

CuAO copper-containing diamine oxidase

CVT cytoplasm to vacuole targeting pathway

DAO diamine oxidase

DAP 1,3-diaminopropane

d.d. degree days

DFMO DL-α-difluoromethylornithine

DIG digoxigenin

dUTP deoxyuridine triphosphate nucleotide

ESR embryo surrounding region

EST expressed sequence tag

GABA γ-aminobutyric acid

GAPDH glyceraldehyde-3-phosphate dehydrogenase

HPLC high-performance liquid chromatography

Ka nonsynonymous substitution rate

Ks synonymous substitution rate

LDC lysine decarboxylase

LIG DNA ligase

MCA metacaspase

MP maximum parsimony

NBT nitro blue tetrazolium

NHEJ non-homologous end joining

ODC ornithine decarboxylase

PAO polyamine oxidase

PCD programmed cell death

PCR polymerase chain reaction

10

PLP pyridoxal-5-phosphate

RT reverse transcription

SAMDC S-adenosylmethionine decarboxylase

SMO spermine oxidase

SPDS spermidine synthase

SPMS spermine synthase

TAT-D Tat-D nuclease

TMR tetra-methyl-rhodamine

TSPMS thermospermine synthase

TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end

labeling

UBI ubiquitin

VPE vacuolar processing enzyme

11

List of original publications

This thesis is based on the following papers, which are referred to in the text by

their Roman numerals:

I Vuosku J, Suokas M, Kestilä J, Läärä E, Sarjala T, Savolainen O & Häggman H (2011) Diverse evolution of polyamine biosynthesis and catabolism pathway genes in plants. Manuscript.

II Vuosku J, Jokela A, Läärä E, Sääskilahti M, Muilu R, Sutela S, Altabella T, Sarjala T & Häggman H (2006) Consistency of polyamine profiles and expression of arginine decarboxylase in mitosis during zygotic embryogenesis of Scots pine. Plant Physiology 142: 1027–1038.

III Vuosku J, Sarjala T, Jokela A, Sutela S, Sääskilahti M, Suorsa M, Läärä E & Häggman H (2009) One tissue, two fates: Different roles of megagametophyte cells during Scots pine embryogenesis. Journal of Experimental Botany 60(4):1375–1386.

IV Vuosku J, Sutela S, Sääskilahti M, Kestilä J, Jokela A, Sarjala T & Häggman H (2010) Dealing with the problem of non-specific in situ mRNA hybridization signals associated with plant tissues undergoing programmed cell death. Plant Methods 6(7).

12

13

Contents

Abstract

Tiivistelmä

Acknowledgements 7 Abbreviations 9 List of original publications 11 Contents 13 1 Introduction 15

1.1 Pine embryogenesis ................................................................................. 15 1.1.1 Zygotic embryogenesis in Scots pine ........................................... 15 1.1.2 Somatic embryogenesis as a model system .................................. 17

1.2 Polyamines .............................................................................................. 17 1.2.1 Concerning polyamines ................................................................ 17 1.2.2 Polyamines in plant embryogenesis ............................................. 21

1.3 Cell death ................................................................................................ 22 1.3.1 Cell death categories..................................................................... 22 1.3.2 Cell death in pine embryogenesis ................................................. 24

1.4 Aim of the research ................................................................................. 26 2 Materials and methods 27

2.1 Plant material (I, II, III, IV) ..................................................................... 27 2.2 Anatomical and histochemical observations (II, IV) ............................... 27 2.3 Polyamine analysis (II) ........................................................................... 28 2.4 Analysis of enzyme activities (II) ........................................................... 28 2.5 Immunolocalization of ADC protein (II) ................................................ 28 2.6 Nucleic acid extraction and RNA reverse transcription (I, II, III) ........... 28 2.7 PCR primer design, cDNA cloning and sequencing (I, II, III) ................ 29 2.8 Quantitative real-time RT-PCR analyses (I, II, III) ................................. 29 2.9 In situ mRNA hybridization assay (II, IV) .............................................. 30 2.10 Detection of DNA fragmentation and cell death (III, IV) ....................... 30 2.11 Cell viability assay (III) .......................................................................... 31 2.12 Phylogenetic analyses and Ka/Ks calculation (I) .................................... 31 2.13 Statistical methods (II, III) ...................................................................... 32

3 Results 33 3.1 Scots pine sequence information (I, III) .................................................. 33 3.2 Evolution of polyamine genes in plants (I) ............................................. 33 3.3 Consistent polyamine profiles during embryo development (II) ............ 34

14

3.4 Preference of ADC pathway (I, II) .......................................................... 35 3.5 Localization of ADC gene expression and ADC enzyme (II) ................. 35 3.6 Fate of megagametophyte during embryo development (III, IV) ........... 36 3.7 DNA repair and PCD-related gene expression (III) ................................ 37 3.8 Unspecific in situ mRNA hybridization signal in dying cells (IV) ......... 37

4 Discussion 39 4.1 Taking the ADC route ............................................................................. 39

4.1.1 ADC/ODC .................................................................................... 39 4.1.2 Once again – evolution of ADC pathway ..................................... 40

4.2 Polyamine metabolism in plants – something special? ........................... 42 4.2.1 ACL5 gene .................................................................................... 42 4.2.2 Polyamine oxidases without back-conversion ability ................... 44

4.3 Necrotic cell death as a regulated cellular response ................................ 45 4.4 Pros and cons of nuclear DNA fragmentation ......................................... 47

5 Conclusion and future prospects 51 References 55 Original publications 67

15

1 Introduction

1.1 Pine embryogenesis

1.1.1 Zygotic embryogenesis in Scots pine

Pinus species of the gymnosperms constitute an evolutionarily old group of

vascular plants that last shared a common ancestor with angiosperms about 285

million years ago (Bowe et al. 2000). The genus has a rich history of phylogenetic

analysis and the relationships between the approximately 120 extant species of

the genus are well documented (Gernandt et al. 2005), as are the development,

reproduction, ecology and genetics of many Pinus species (Lev-Yadun &

Sederoff 2000). Scots pine (Pinus sylvestris L.) is the most widely distributed

Eurasian conifer and one of the keystone species in the Eurasian boreal forest

zone, growing in a range of environments from Spain and Turkey to the subarctic

forests of northern Scandinavia and Siberia (Mirov 1967).

The zygotic embryo development of Scots pine takes two years. Generally,

wind pollination in Finland occurs in late May or early June, after which the

pollen tube germination slows down. Fertilization and meiosis occur about one

year later, usually in late June or early July (Sarvas 1962). The time of

fertilization and, consequently, embryo development varies between years in the

same locality according to the effective temperature sum (d.d.) (i.e. the heat sum

unit based on the daily mean temperatures minus the adapted +5 ˚C base

temperature) (Sarvas, 1962, Sirois et al. 1999, Owens et al. 2001). Normally, the

anatomical and physiological differentiation and the development of the embryo

occur between the end of July and the middle of September in southern and

northern Finland, respectively (Sahlén 1992). The sequence of embryo

development in Pinus sp. can be divided into three phases, which include

proembryogeny, early embryogeny and late embryogeny. Proembryogeny

contains the stages before suspensor elongation, whereas early embryogeny

initiates with the elongation of the suspensor system and terminates with the

appearance of the root meristem. The embryo development culminates in the

establishment of root and shoot meristems and in the maturation of the embryo

during late embryogeny (Singh 1978).

A characteristic feature of the seed development of gymnosperms is the

presence of more than one embryo in a young seed. The origin of polyembryony

16

can be either monozygotic, when genetically identical offspring are derived from

the same zygote, or polyzygotic, when multiple embryos arise as a result of

independent fertilization of several eggs (Buchholz 1926). In the Scots pine seed,

the fertilization of many egg nuclei results in several embryos of the same ovule,

although some fail to develop to the primary proembryo stage. Later during the

development, polyzygotic embryos undergo cleavage polyembryony (Sarvas

1962). Polyembryony is, however, a transient stage in Scots pine seed

development and the opportunity for embryo replacement is brief. During the

progress of embryogenesis, the growth of subordinate embryos is retarded and,

finally, only one embryo survives and completes development, while the others

are eliminated (Filonova et al. 2002).

In a gymnosperm seed, embryos grow and develop within the corrosion

cavity which expands during embryogenesis as suspensors lengthen and push

embryos deeper into the megagametophyte tissue. The suspensors hold the

embryos in a fixed position within the seed, anchor the embryos to the maternal

tissue and provide nutrition and growth regulators for the developing embryos

(Yeung & Meinke, 1993). The megagametophyte houses the majority of the

storage reserves of a seed (King & Gifford, 1997) and can be considered a

functional homolog of the endosperm in angiosperms due to its role as a nutrient

source of the developing embryos (Costa et al. 2004). The nearest

megagametophyte cells around the corrosion cavity form the embryo surrounding

region (ESR). The exact function of the ESR in a pine seed is still unknown, but

in addition to its role in embryo nutrition, the ESR may form a physical barrier or

provide a region for communication between the megagametophyte and the

developing embryo, as has been suggested for the embryo and the endosperm in

angiosperms (Olsen 2004).

Unlike the endosperm, the megagametophyte develops from a haploid

megaspore before the actual fertilization of the eggs (Singh 1978). That is, the

megagametophyte represents a significant investment of maternal resources that

are wasted if fertilization does not occur. Thus, in gymnosperms the

polyembryogeny may have developed as a safeguard against the possibility of not

having viable offspring. In the Scots pine seed, the death of all embryos leads to

the rapid degeneration of the megagametophyte and to the transfer of

accumulated nutrients elsewhere. As long as the seed contains a living embryo,

the megagametophyte continues to develop normally regardless of the vigour or

developmental stage of the embryo (Sarvas 1962). Although the initiations of

endosperm and embryo growth in Arabidopsis thaliana are autonomous events,

17

the proper development of both structures requires crosstalk between the

endosperm and embryo in several checkpoints (Ungru et al. 2008). In conifer

seeds, similar kind of recognition may take place between the megagametophyte

and embryos during the different stages of development.

1.1.2 Somatic embryogenesis as a model system

Embryogenesis in plants is not restricted to the fertilized egg but can be induced

either naturally or artificially in many different kinds of cell types. Somatic

embryogenesis has, however, certain criteria for the start. The species or genotype

has to possess the genetic potential to form embryos from somatic cells.

Furthermore, one or a few cells in the plant/explant have to be competent to

receive an exogenous or endogenous signal that triggers the cell towards the

embryogenic pathway (Fehér 2005). In conifers, somatic embryogenesis

represents a favourable model for studying factors that affect embryo

development because embryogenic cultures go through a series of developmental

stages that resemble zygotic embryogenesis. Additionally, a large number of

embryos at defined developmental stages can be obtained through the process

(Stasolla et al. 2004). In pines (Pinus sp.), immature zygotic embryos surrounded

by the megagametophyte have been considered the material most responsible for

the initiation of somatic embryogenesis (Handley et al. 1995, Häggman et al.,

1999, Percy et al. 2000, Miguel et al. 2004), although somatic embryogenesis

from mature zygotic embryos (Tang et al. 2001, Malabadi et al. 2002) as well as

from vegetative shoot apices (Malabadi & van Staden 2005, Park et al. 2010)

have also been reported. The process of somatic embryogenesis and plant

regeneration includes four distinct phases: initiation, proliferation of embryogenic

cell mass, maturation of somatic embryos and, finally, conversion of somatic

embryo plants. The phases are usually induced by the growth regulators,

osmolality and nutrients of the culture medium (Häggman et al. 2005).

1.2 Polyamines

1.2.1 Concerning polyamines

The discovery of polyamines dates back to 1678 when Antonie van Leeuwenhoek

isolated some 'three-sided' crystals from human semen, but the chemical

18

constitution of the crystals was not deduced not until 1924. The names of triamine

spermidine (1,8-diamino-4-azaoctane) and tetra-amine spermine (1,12-diamino-

4,9-diazadodecane) reflect the initial discovery, whereas a different tetra-amine

was originally extracted from an extreme thermophile eubacterium, Thermus

thermophilus, and thus named thermospermine (1,12-diamino-4,8-diazadodecane).

The diamine putrescine (1,4-diaminobutane) was first isolated from the cholera

bacterium (Vibrio cholerae), but the name was derived from the large quantities

that were found in putrefying flesh (Wallace et al. 2003). The diamine cadaverine

(1,5-diaminopentane) is a foul-smelling and toxic compound produced in protein

hydrolysis during putrefraction of animal tissues. Although cadaverine is less

widely distributed than putrescine, it is also produced in small quantities by living

tissues (Ohe et al. 2010). Polyamines are present in all living cells in eukaryotes

as well as in prokaryotes. Although many organisms tend to produce only two or

three of the polyamines, the analysis of the polyamine profiles of thermophilic

bacteria has revealed a variety of new compounds. T. thermophilus produces a

variety of uncommon polyamines, including longer polyamines, such as

caldopentamine and caldohexamine, and branched polyamines, such as tetrakis(3-

aminopropyl)ammonium (Terui et al. 2005). The cellular concentrations of these

longer and branched polyamines correlate positively with an increase in the

growth temperature of T. thermophilus (Oshima 1989), and they have been

proposed to participate in the maintenance of cellular functions at a high

temperature (Terui et al. 2005). The extreme evolutionary conservation of

polyamines clearly indicates the necessity of polyamines in organism survival. On

the other hand, the high flexibility of polyamine metabolism in response to

environmental demands implicates that polyamines may have acquired a wide

variety of different functions during evolution.

Chemically, polyamines are small polycationic molecules that in

physiological pH carry a positive charge on each nitrogen atom. Thus, in

polyamines the charge is distributed along the entire length of the carbon chain,

which makes them unique compared with cellular bivalent cations. Due to their

positive charge, polyamines have high affinity for polyanionic macromolecules,

such as DNA, RNA, proteins and phospholipids. In nature, polyamines occur not

only as free molecular bases but also as conjugates, forming a part of a peptide

(e.g. glutathionylspermidine), an amino acid (e.g. putreamine) or an antibiotic (e.g.

bleomycin) (Tiburcio et al. 1997). Furthermore, polyamines can conjugate with

acetic acid, forming acetyl derivates that are common in animal cells (Tiburcio et

al. 1997), whereas the hydroxycinnamic acid amides are the most common

19

polyamine conjugates in plants (Martin-Tanguy 1997). In addition to stabilizing

macromolecular structures, polyamines act as regulatory molecules in various

fundamental cellular processes, such as DNA and protein synthesis, gene

expression, cell division, differentiation and proliferation as well as programmed

cell death (PCD) (Childs et al. 2003, Seiler & Raul 2005, Igarashi & Kashiwagi

2010). In plants, polyamines have generally been considered as growth regulators

or as secondary messengers (Kakkar & Sawhney 2002) and they have been

connected to several physiological events, such as organogenesis, embryogenesis,

floral initiation and development, leaf senescence, fruit development and ripening

as well as abiotic and biotic stress responses (Galston & Sawhney 1990, Kumar et

al. 1997, Malmberg et al. 1998, Bouchereau et al. 1999, Bagni & Tassoni 2001,

Alcázar et al. 2006, Kusano et al. 2008).

Plants generally possess two parallel pathways to synthesize putrescine, i.e.

via enzymes ornithine decarboxylase (ODC; EC 4.1.1.17) and arginine

decarboxylase (ADC; EC 4.1.1.19) (Tiburcio et al. 1997), unlike mammalian and

fungal cells, which mainly utilize ODC (Coleman et al. 2004). In the ODC

pathway, putrescine is the direct product of ornithine decarboxylation, whereas

the ADC pathway includes intermediate steps. Arginine is first decarboxylated by

ADC to form agmatine, which is subsequently converted to putrescine by the

combined action of agmatine iminohydrolase (AIH; EC 3.5.3.12) and N-

carbamoylputrescine amidohydrolase (CPA; EC 3.5.1.53). Spermidine synthase

(SPDS; EC 2.5.1.16) synthesizes spermidine from putrescine by the addition of

an aminopropyl group that is acquired from decarboxylated S-

adenosylmethionine formed by S-adenosylmethionine decarboxylase (SAMDC;

EC 4.1.1.50). Spermidine is an unsymmetrical molecule, which can be

aminopropylated at either end forming spermine or thermospermine. Spermine

synthase (SPMS; EC 2.5.1.22) transfers an aminopropyl group to the N8-

(aminobutyl) end of spermidine to make the symmetrical molecule spermine,

whereas thermospermine synthase (TSPMS; EC 2.5.1.B4) transfers the

aminopropyl group to the N1-(aminopropyl) end of spermidine to make the

unsymmetrical molecule thermospermine (Pegg & Michael 2010) (Fig. 1). The

biosynthesis of cadaverine is not included in the pathway, but cadaverine is

synthesized from lysine by lysine decarboxylase enzyme (LDC, EC 4.1.1.18).

20

Fig. 1. Polyamine biosynthesis in plants. The enzymes in the pathways are arginase

(ARG), ornithine decarboxylase (ODC), arginine decarboxylase (ADC), agmatine

imidohydrolase (AIH), N-carbamoylputrescine amidohydrolase (CPA), spermidine

synthase (SPDS), spermine synthase (SPMS), thermospermine synthase (TSPMS) and

S-adenosylmethionine decarboxylase (SAMDC).

Polyamines are oxidatively deaminated by the action of amine oxidases, which

were actually the first enzymes recognised as polyamine-related (Hirsch 1953).

Putrescine and cadaverine are deaminated by the action of diamine oxidases

(DAOs) belonging to the group of copper-containing diamine oxidases (CuAO;

EC 1.4.3.6), whereas spermidine and spermine are oxidized by the flavoprotein-

containing polyamine oxidases (PAO; EC 1.5.3.3) (Bagni & Tassoni 2001, Bais &

Ravishankar 2002). DAO converts putrescine into ∆1-pyrroline, with the release

arginine

NH

NH2CH(CH2)3NHCNH2

COOH

agmatine

NH

NH2(CH2)4NHCHNH2

N-carbamoylputrescine

O

NH2(CH2)4NHCNH2

NH2(CH2)4NH2putrescine

NH2(CH2)4NH(CH2)3NH2spermidine

NH2(CH2)3NH(CH2)4NH(CH2)3NH2

spermine

ADC

AIH

CPA

SPDS

SPMS

thermospermine

NH2(CH2)3NH(CH2)3NH(CH2)4NH2

dcSAM

TSPMS

ornithineNH2CH(CH2)3NH2

COOH

ARG

ODC

SAMSAMDC

21

of ammonia and hydrogen peroxide. Degradation of spermidine by PAO yields

∆1-pyrroline and 1,3-diaminopropane, while spermine oxidation yields 1,(3-

aminopropyl)-pyrroline, along with diaminopropane and hydrogen peroxide

(Bagni & Tassoni 2001) (Fig. 2).

Fig. 2. Polyamine catabolism in plants. The enzymes in the pathways are diamine

oxidase (DAO) and polyamine oxidase (PAO).

In addition to polyamine biosynthesis and catabolism, cellular polyamine levels

are regulated by uptake mechanisms that salvage polyamines from diet and

intestinal micro-organisms as well as efflux (Morris 2004). The sophisticated

regulatory mechanisms enable cells to adapt to changes in intra- and extra-cellular

polyamine concentrations. On the other hand, it is the main reason for the

difficulties in the development of selective polyamine enzyme inhibitors as potent

drugs. During the past 25 years, several hundred polyamine synthesis inhibitors,

polyamine catabolism stimulators and polyamine derivatives have been tested for

anticancer and anti-parasite agents. Combinations of the drugs decreased

polyamine levels and slowed the proliferation of the target cells, but they were

rarely able to inhibit cell growth completely (Criss 2003).

1.2.2 Polyamines in plant embryogenesis

The importance of polyamines in embryogenesis has been documented in many

angiosperm species (Lin et al. 1984, Yadav & Rajam 1998), and the polyamine

biosynthetic knock-out mutants have shown that ADC (Urano et al. 2005), SPDS

(Imai et al. 2004b) and SAMDC (Ge et al. 2006) are essential for embryo

development in Arabidopsis. By contrast, an SPMS deficient mutant, acl5/spms,

showed a normal phenotype except for reduced stem elongation (Hanzawa et al.

spermidine

spermine

PAO

PAO

1,3 diaminopropane

1-(3-aminopropyl)-pyrroline

putrescine 1-pyrrolineDAO

O2 NH3 H2O2

O2 H2O2

O2 H2O2

22

2000). In pine species, polyamine deficient mutant plants are not available, and

thus studies have so far mainly focused on somatic embryogenesis. It has been

shown that polyamine levels fluctuate in pine embryogenic cells when somatic

embryogenesis proceeds. The proliferation of embryogenic cell masses has been

accompanied by a high level of putrescine compared with both spermidine and

spermine (Sarjala et al. 1997, Minocha et al. 1999b), while transfer onto

maturation medium has resulted in an increase in the ratio of spermidine and

putrescine (Minocha et al. 1999b, Niemi et al. 2002). However, the reactions of

embryogenic cell masses to exogenous polyamines have proved to be complex

and dependent on both the species and the developmental stage of somatic

embryos (Santanen & Simola 1992, Minocha et al. 1993, Sarjala et al. 1997,

Kong et al. 1998, Niemi et al. 2002).

1.3 Cell death

1.3.1 Cell death categories

Generally, cell death occurs either via programmed cell death (PCD) or necrosis.

PCD is a genetically encoded physiological process that involves the selective

death of individual cells, tissues or whole organs, whereas necrosis is generally

described as a chaotic and uncontrolled form of death that is caused in a passive

manner by environmental perturbation (Pennell & Lamb 1997). Both cell death

events are well defined in animals, but in plants there seems to be more overlap

between the phenotypic and molecular characteristics of PCD and necrosis, which

makes the discrimination between the two forms more complicated (Van

Breusegem & Dat 2006). PCD is an integral part of the life of plants. PCD occurs

at all stages of the life cycle when cell suicide pathways are activated as a part of

normal growth and development or in response to certain biotic and abiotic

external factors (Beers 1997). Developmental PCD may delete suspensor cells

(Giuliani et al. 2002), store nutrients for the growing seedling in endosperm

(Young et al. 1997), induce certain leaf shapes and perforations (Gunawardena et

al. 2004) and play an essential role in the specialization of xylem tracheary

elements (Mittler & Lam 1995). Environmentally induced PCD may take place as

a hypersensitive response against pathogen attack (Ryerson & Heath 1996) and in

the development of lysigenous aerenchyma triggered by hypoxic stress

23

(Gunawardena et al. 2001). If plant cells are damaged by stress at an

overwhelming level, they undergo necrosis.

In animals, two main categories of PCD, apoptosis and autophagy, have been

recognized by the morphology of the dying cell and the main organelles involved

(Lockshin & Zakeri 2004). Apoptotic cell death has been recognized as a

characteristic type of cell death for over 100 years, and only the name changed

from karyorrhexis or pyknosis to apoptosis in 1972, when the term was proposed

by Kerr and co-workers (Ziegler & Groscurth 2004). The word apoptosis was

used in Greek to denote a "falling off," as leaves from a tree. So, the term

connotes a controlled physiologic process in which individual components are

removed without destruction or damage to the organism. Apoptosis occurs in

normal as well as in pathologically altered tissues, and it is morphologically

characterized by cell shrinkage, chromatin condensation, DNA fragmentation,

maintenance of an intact plasma membrane and, eventually, break-up of the dying

cell into apoptotic bodies that are engulfed and finally degraded in the lysosomes

of another cell (Kerr et al. 1972). This ordered morphology depends on the ability

of the dying cell to engage in ATP-dependent processes in self-degradation (Zong

& Thompson, 2006). The biochemical events typical of apoptosis are the release

of cytochrome c from mitochondria (Goldstein et al. 2000) and the activation of a

family of cysteine proteases known as caspases (Wolf & Green 1999). Whether

cells live or die via apoptosis is largely determined by the interplay between

opposing members of the Bcl-2 protein family (Adams & Cory 2007). Apoptotic

bodies occur rarely in plants because engulfment by neighbouring cells is

hindered by the presence of cell walls (Vianello et al. 2007). Additionally, surveys

of the complete genomes of Arabidopsis and rice (Oryza sativa L.) failed to reveal

obvious orthologs for animal caspase or Bcl-2 genes (Danon et al. 2000, Dickman

et al. 2001). However, the mammalian Bcl-2 proteins are able to modify cell

death when they are introduced into plant cells by heterologous expression, which

suggests that plant and animals share common PCD pathways (Lacomme & Cruz

1999, Dickman et al. 2001).

Cellular autophagy is an evolutionarily conserved catabolic process involving

the degradation of cytoplasmic components within the intact dying cell in

autophagic vacuoles. Actually, the term autophagy was derived from the Greek

words "autos" and "phago" meaning 'self' and 'to eat', respectively. The

morphological characteristics of autophagy include vacuolization and degradation

of cytoplasmic contents (Fink & Cookson 2005). Chromatin condensation and

membrane blebbing may also occur in autophagic cell death, but there is no DNA

24

fragmentation or formation of apoptotic bodies (Vianello et al. 2007). In all

eukaryotic organisms, autophagy is a ubiquitous but tightly-regulated process that

is maintained at a basal level under normal growth conditions. Autophagy plays a

role in cell growth, development and homeostasis, helping to maintain a balance

between the synthesis, degradation and subsequent recycling of cellular products.

In yeast, in nutrient-rich conditions a constitutive biosynthetic pathway, called the

cytoplasm to vacuole targeting (CVT) pathway, utilizes mostly the same

molecular machinery as autophagy. Autophagy is induced by starvation and is

considered the major mechanism by which starving cells reallocate nutrients from

unnecessary processes to essential ones (Wang & Klionsky 2003, Yorimitsu &

Klionsky 2005, Mizushima 2007). Additionally, the autophagy pathway recycles

damaged and potentially harmful cellular material (Eisenberg et al. 2009). Thus,

the cells with enlarged vacuoles and undergoing autophagy do not necessarily die,

and it is important to distinguish the routine turnover of cell constituents from a

fatal destruction of the cell via autophagic PCD.

When early pathologists described extensive damage in internal organs

during the course of certain diseases, they named this form of damage necrosis.

The term was derived from the Greek word "nekros" for 'corpse' (Zong &

Thompson 2006). Morphologically, a necrotic cell is characterized by the

swelling of cellular organelles, loss of plasma membrane integrity and, finally,

cell lysis. The swelling is caused by an irreversible membrane function failure

that leads to the inability to osmoregulate and, furthermore, causes water to flood

into the cell (Lennon et al. 1991). These events can be reproduced experimentally

by impairing a cell's ability to produce ATP (Zong & Thompson 2006). Therefore,

necrosis is generally characterized as passive, accidental cell death with

uncontrolled release of inflammatory cellular contents (Fink & Cookson 2005).

However, there is growing evidence to support the conception that necrotic cell

death can also be a regulated event that contributes to the development and

maintenance of organismal homeostasis (Golstein & Kroemer 2006, Zong &

Thompson 2006).

1.3.2 Cell death in pine embryogenesis

The pine seed has been suggested as a model for studying cell death machinery in

eukaryotes because morphologically different cell death processes are an integral

part of the pine zygotic embryogenesis (Zhivotovsky et al. 2002, Vuosku et al.

2009). In Scots pine, the development of a viable seed includes the strictly co-

25

ordinated action of several cell death programs which, depending on the

developmental tasks, result in different magnitudes of cell corpse processing

(Filonova et al. 2002, Hiratsuka et al. 2002, Vuosku et al. 2009). Filonova et al.

(2002) suggested that the cells of subordinate embryos as well as suspensors are

eliminated via autophagic PCD. Cells display co-operative autolytic and

autophagic mechanisms of protoplast degradation, leading to a substantial or

complete processing of cell corpses (Filonova et al. 2002). The megagametophyte

cells have also been suggested to die during embryo development (Filonova et al.

2002). Furthermore, the cells of the nucellar layers face destruction during early

embryogenesis (Hiratsuka et al. 2002) and end up being used as nutrition for the

surrounding tissues of the pine seed. The collapsed and phenoliferous nucellar

cell walls of the nucellar layers serve another function in the mature coniferous

seed, where they form an efficient barrier to the passage of water as well as

against fungal infections (Tillman-Sutela & Kauppi 1995).

The use of gymnosperm somatic embryogenesis as a model system for

developmental PCD has enabled the manipulation of cell death by specific agents

that either stimulate or inhibit distinct processes of the cell death pathway.

Although the molecular mechanisms of the diverse plant PCD machinery is still

poorly known, the importance of nucleases (Balk et al. 2003, He & Kermode

2003) and proteases, such as vacuolar processing enzymes (VPEs), type II

metacaspases and VEIDase activity, has been perceived (Woltering 2004,

Bozhkov & Jansson 2007, Bonneau et al. 2008). During the Norway spruce

(Picea abies (L.) Karst) somatic embryogenesis, a type-II metacaspase, mcII-Pa,

has been found to activate in the terminally-differentiated cells, where it moves

from the cytoplasm to nuclei, causing nuclear envelope disassembly and DNA

fragmentation (Bozhkov et al. 2005). In Norway spruce somatic embryos,

VEIDase activity increases at the early stages of embryo development that

coincide with massive cell death during shape remodelling. The inhibition of

VEIDase prevents normal embryo development by blocking the embryo-

suspensor differentiation (Bozhkov et al. 2004). The accumulation of zinc in the

embryonal masses, but not in the suspensors, is required for correct embryonic

patterning, which suggests that zinc may mediate cell fate specification through

its strong anti-cell death effect (Helmersson et al. 2008).

26

1.4 Aim of the research

Previously, knowledge about the polyamine metabolism during pine

embryogenesis has mainly consisted of the results of biochemical analyses during

somatic embryogenesis. Hardly anything has been known about the regulation of

polyamine metabolism or the roles of individual polyamines in different

embryogenic tissues during zygotic embryo development. Although somatic

embryos follow a remarkably similar developmental process to zygotic

embryos, the megagametophyte tissue is missing. We hypothesised that the

presence of the megagametophyte (i.e. a possible early polyamine source and

tissue with proceeding PCD) may cause significant differences in polyamine

metabolism. Thus, the aim of the present study was to gather information about

polyamine metabolism throughout the Scots pine zygotic embryogenesis (II) and

also about the physiological events occurring simultaneously in the

megagametophyte tissue (III, IV). For obtaining molecular-level insight into

polyamine metabolism during embryo development, Scots pine polyamine genes

were sequenced. Additionally, the novel sequence data were used in creating a

more comprehensive picture of the evolutionary history of the polyamine

biosynthesis and catabolism pathway genes in plants (I).

27

2 Materials and methods

Detailed descriptions of the methods have been presented in the original articles

(I–IV).

2.1 Plant material (I, II, III, IV)

One-year-old immature seed cones as well as mature cones were collected from

open-pollinated elite Scots pine (Pinus sylvestris L.) clones, K818 and K884,

which grow in the Scots pine clone collection in Punkaharju, Finland (61˚48´ N;

29˚17´ E). During the growing seasons 2001, 2003 and 2006, the collection of

immature cones was repeated four times in July throughout the period of embryo

development. Immature zygotic embryos surrounded by the immature

megagametophyte, hence called zygotic embryos, were dissected from the

developing cones and stored at −80 ˚C or in liquid nitrogen. Zygotic embryos

were used for polyamine analyses, enzyme activity measurements, polyamine

gene sequencing as well as gene expression studies (I, II, III, IV). For the

microscopic examinations, zygotic embryos were fixed immediately after

dissection and infiltrated in paraffin (II, III, IV). Mature seed cones were

collected in late fall of the same growing seasons. Mature seeds were used for

DNA isolation, seed viability assay and for in vitro culturing of the

megagametophyte tissue (III). Mature seeds were also germinated for gene

expression studies (I).

2.2 Anatomical and histochemical observations (II, IV)

The paraffin-embedded zygotic embryos were cut into sections (5 and 7µm) with

a microtome, mounted on SuperFrost®Plus slides (Menzel-Glaser) and fixed by

drying overnight at 40 ˚C. The paraffin sections were dewaxed in Histochoice

(Sigma) and rehydrated through a graded series of ethanol. The sections were

used for the anatomical and histochemical studies, protein immunolocalization, in

situ mRNA hybridization analyses as well as for the in situ detection of nuclear

DNA fragmentation. To study the developmental stage of the Scots pine zygotic

embryos, the sections were stained with toluidine blue (II, IV), and for the

detection of starch, they were stained with potassium iodide-iodine (Jensen 1962)

(IV).

28

2.3 Polyamine analysis (II)

Free, soluble conjugated and insoluble conjugated polyamines were analysed by

high-performance liquid chromatography (HPLC) according to Sarjala &

Kaunisto (1993) and Fornalé et al. (1999).

2.4 Analysis of enzyme activities (II)

The proteins of Scots pine zygotic embryos were extracted for ADC and ODC

enzyme activity measurements, which were performed according to Minocha et al.

(1999a) with some modifications. The presence of true ADC activity was

confirmed by adding unlabelled L-ornithine into the reaction mixture according to

Tassoni et al. (2000), in order to inhibit arginase activity. In the inhibition assay

for ODC, DL-α-difluoromethylornithine (DFMO, SIGMA) was used. Protein

content was measured with the Bradford method (Quick Start™ Bradford Protein

Assay, Biorad) using bovine serum albumin (BSA) as a standard.

2.5 Immunolocalization of ADC protein (II)

The IgGs obtained against the tobacco ADC protein (Bortolotti et al. 2004) were

used for the immunolocalization of ADC protein in Scots pine zygotic embryos.

The immunolocalization was carried out according to Bortolotti et al. (2004) with

slight modifications. In the last step of the procedure, the avidin-biotin complex,

ABC, method (Vector Labs) with diaminobenzidine as a substrate for peroxidase

was used in the detection of the antigen-antibody complex according to the

vendors’ instructions.

2.6 Nucleic acid extraction and RNA reverse transcription (I, II, III)

Genomic DNA was extracted by the NucleoSpin® Plant kit (Macherey-Nagel)

according to the manufacturer's protocol for gymnosperms. Total RNA was

extracted for the gene expression studies as described in our paper (Vuosku et al.

2004), using the automated magnetic-based KingFisher™ mL method (Thermo

Electron Corporation) with commercial extraction kits according to the

manufacturer's instructions. Either a DNase treatment for the elimination of

contaminating genomic DNA was included in the RNA extraction procedure or

the extracted RNA samples were treated with RNase-free DNase (Invitrogen).

29

The RNA template was reverse transcripted into cDNA by SuperScript II reverse

transcriptase (Invitrogen) in standard reaction using anchored oligo-dT primers.

2.7 PCR primer design, cDNA cloning and sequencing (I, II, III)

The PCR primers for the amplification of the cDNA fragments of the Scots pine

polyamine genes (ADC, ODC, AIH, CPA, SPDS, ACL5, SAMDC, DAO and PAO),

DNA repair genes (RAD51, KU80 and LIG) and PCD-related genes (MCA and

TAT-D) were designed based on Pinus EST (expressed sequence tag) sequences.

The EST sequences were selected on the basis of their similarity at the nucleotide

or amino acid level with known plant genes, and they were aligned together to

yield either an entire protein coding sequence of a corresponding gene or a cDNA

fragment that was as long as possible. The cDNA fragments were amplified by

standard PCR using zygotic embryo cDNA as a template and DyNAzyme™EXT

polymerase (Finnzymes). Fragments with appropriate length were gel-purified

using a Montage DNA Gel Extraction Kit (Millipore Corporation) and cloned

using a TOPO TA Cloning Kit (Invitrogen). Five clones from each gene fragment

were sequenced using an Applied Biosystems 3730 DNA analyzer.

2.8 Quantitative real-time RT-PCR analyses (I, II, III)

Gene expression was studied by quantitative real-time RT-PCR analysis. The PCR

primers for the Scots pine polyamine biosynthesis genes (ADC and ODC), DNA

repair genes (RAD51, KU80 and LIG) and PCD-related genes (MCA and TAT-D)

were designed on the basis of the sequence data discovered in this study. The

primers for the housekeeping genes actin (ACT), ubiquitin (UBI) and

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were based on the

lodgepole pine (Pinus contorta) ACT sequence (M36171), maritime pine (Pinus

pinaster) putative UBI sequence (AF461687) and Scots pine GAPDH sequence

(L07501), respectively.

Both absolute (I) and relative quantification (II, III) strategies were used to

analyze data from quantitative real-time RT-PCR analyses. Being aware of the

challenge to find appropriate reference or housekeeping genes to be used as

internal standards in real-time RT-PCR during pine embryogenesis (Gonçalves et

al. 2005), absolute quantification was the first choice in the gene expression

studies. However, with absolute quantification, all the studied genes, including

the housekeeping genes, showed decreasing expression during the Scots pine

30

zygotic embryo development. The decrease in the gene expression levels at late

embryogeny was most probably at least partially caused by the poor quality of

RNA. The presence of high levels of storage lipids, storage proteins,

polysaccharides and polyphenols decreases RNA yield and quality (Tai et al.

2004). Both protein and starch accumulated into the Scots pine megagametophyte

cells during embryo development. Additionally, the corrosion cavity area that

contained degrading nucleic acids and hence presumably also nucleases enlarged

as the development progressed. Thus, for removing possible differences in the

amount and quality of starting mRNA and enzymatic efficiencies, at least two

discrete reference genes (II) or the geometric mean of three selected reference

genes belonging to different functional classes (III) was used for the

normalization of the gene expression levels. In absolute quantification,

synthesized RNA standards were used to control variability during both the

reverse transcription (RT) and PCR steps (I).

For the quantitative real-time RT-PCR analysis, a first-strand cDNA template

was synthesized from 1 µg of total RNA. The amplification was performed using

the Mx3000P™ real-time PCR system with Brilliant® SYBR® Green QPCR

Master Mix (Stratagene) (II) or the LightCycler ® 480 system with LightCycler

480 SYBR green 1 Master mix (Roche Molecular Biochemicals) (I, III).

2.9 In situ mRNA hybridization assay (II, IV)

The RNA antisense and sense probes for the in situ localization of ADC, DAO and

catalase (CAT) mRNA transcripts were prepared by a PCR-based technique in

which a T7 polymerase promoter sequence was introduced at the 5' ends of the

gene-specific primers (Young et al. 1991). In situ hybridization was performed

according to Mähönen et al. (2000) with some modifications, and hybridized

probe was detected in the sections using alkaline phosphatase-conjugated anti-

DIG antibodies and NBT/BCIP as substrates (Roche Molecular Biochemicals).

2.10 Detection of DNA fragmentation and cell death (III, IV)

Nuclear DNA fragmentation was visualized by agarose gel electrophoresis. The

cleavage of genomic DNA into multiple internucleosomal fragments during PCD

gives rise to a characteristic DNA ladder on the gel, whereas in necrosis DNA

degrades randomly and results in a smear (Bortner et al. 1995, Danon et al. 2000)

(III). For the in situ observation of nucleic acids and DNA fragmentation, the

31

sections were stained by a dual fluorescence dye, acridine orange (AO), according

to Bouranis et al. (2003). In the AO-stained sections, the double-stranded nucleic

acid (i.e. DNA) fluoresces green and the single-stranded (i.e. RNA) fluoresces red.

Fragmented DNA emits fluorescence in a spectrum varying from yellow-green to

red (Martins et al. 2007). The terminal deoxynucleotidyl transferase-mediated

dUTP nick end labeling (TUNEL) method was used to visualize DNA

fragmentation in individual nuclei. The TUNEL method utilizes the activity of the

terminal deoxynucleotidyl transferase enzyme to label the 3' ends of DNA strand

breaks, which may then be identified in individual nuclei by microscopy. The

sections were labelled with a TMR red (red fluorescence) in situ cell death

detection kit (Roche Molecular Biochemicals) according to the manufacturer’s

protocol. Both AO-stained and TUNEL-labelled sections were examined under a

confocal laser scanning microscope (LSM 5 Pascal, Carl Zeiss) with an HBO 103

mercury lamp. Additionally, TUNEL sections were examined using the HeNe

laser 543 nm line, a dichroic beam splitter (HFT 488/543/633; Carl Zeiss) and an

LP 560 emission filter (Carl Zeiss).

2.11 Cell viability assay (III)

The viability of the megagametophyte cells was studied using the tetrazolium

method (ISTA prescriptions 1993, Savonen 1999) in mature Scots pine seeds.

Additionally, the viability of the megagametophytes was confirmed by culturing

them on the basal DCR medium (Gupta & Durzan 1985, Becwar et al. 1990).

After two days of imbibition, the seeds were opened, and the embryos and

megagametophytes were separated from each other. Megagametophytes were

cultured at room temperature in the dark for ten days, after which the number of

proliferating megagametophytes was documented.

2.12 Phylogenetic analyses and Ka/Ks calculation (I)

For the phylogenetic analyses, extensive Blast searches against NCBI databases

(http://www.ncbi.nlm.nih.gov) were carried out using Scots pine ADC, ODC, AIH,

CPA, SPDS, ACL5, SAMDC, DAO and PAO sequences. The nucleotide sequence

alignments were performed with ClustalX (Thompson et al. 1997). The

evolutionary history was inferred using the maximum parsimony (MP) method

with close-neighbor-interchange algorithm (Nei & Kumar 2000). The bootstrap

method (Felsenstein 1985) with 500 replicates was used to evaluate the

32

confidence of the reconstructed trees. Purifying selection pressure on the

sequences was estimated by the ratio of the nonsynonymous substitution rate (Ka)

to the synonymous substitution rate (Ks) (Hughes & Nei 1988). Synonymous and

non-synonymous changes were calculated with paml 4.4c using the Pamilo-

Bianchi method (Pamilo & Bianchi 1993).

2.13 Statistical methods (II, III)

The polyamine concentrations, the expression of the ADC, ODC, RAD51, KU80,

LIG, MCA and TAT-D genes as well as the ADC and ODC enzyme activities were

analyzed by regression models. The effective temperature sum (d.d.) was used as

a main explanatory variable and the Scots pine clone as a covariate. Additionally,

the polyamine fraction and sampling year were included as covariates in the

polyamine concentration analyses. The polyamine models and the gene

expression models were fitted using function lm() and the models for the

enzyme activities by function lme() in the R statistical environment

(http://www.r-project/org/).

33

3 Results

3.1 Scots pine sequence information (I, III)

The protein coding regions of the Scots pine putative arginine decarboxylase

(ADC), ornithine decarboxylase (ODC), agmatine iminohydrolase (AIH), N-

carbamoylputrescine amidohydrolase (CPA), spermidine synthase (SPDS),

thermospermine synthase (ACL5) and S-adenosylmethionine decarboxylase

(SAMDC) genes, which participate in polyamine biosynthesis, were sequenced

and submitted to the NCBI GenBank with the accession numbers HM236823,

HM236831, HM236824, HM236825, HM236827, HM236828 and HM236826,

respectively. Likewise, the coding sequences of the Scots pine putative copper-

containing diamine oxidase (CuAO) and flavoprotein-containing polyamine

oxidase (PAO) genes, which encode polyamine catabolism enzymes, were

revealed under the GenBank accession numbers HM236829 and HM236830,

respectively. Additionally, the coding sequence of the Scots pine putative

metacaspase (MCA) gene and a fragment of the Tat-D nuclease (TAT-D) gene

(GenBank accession numbers EU513166 and EU513167) related to PCD, and

fragments of the RAD51, KU80 and DNA ligase (LIG) genes (GenBank accession

numbers EU513162, EU513164 and EU513165) encoding DNA repair-related

proteins, were sequenced.

3.2 Evolution of polyamine genes in plants (I)

The revealed Scots pine polyamine gene sequences are the first gymnosperm

sequences that represent all the genes in both the polyamine biosynthesis and the

catabolism pathways. The sequences were used in the reconstruction of the

evolutionary history of polyamine genes in plants. The phylogenetic analysis of

the eukaryotic ODC and bacterial lysine decarboxylase (LDC) sequences

suggested that the eukaryotic ODC enzyme might have evolved from a

multifunctional bacterial progenitor that originally decarboxylated L-lysine and

L-ornithine to form both putrescine and cadaverine. Plants have acquired the

alternative ADC pathway for putrescine biosynthesis by horizontal gene transfer

from bacteria. In conifers, the presence of the ADC pathway has allowed the

reduced use of the ODC pathway and, furthermore, led to relaxed purifying

selection in ODC genes. The phylogenetic analysis of the SPDS, SPMS and ACL5

34

sequences supported the view that the eukaryotic SPDS genes are derived from a

common ancestor and the SPMS gene has evolved several times from SPDS via

gene duplication. In eukaryotes, the ACL5 gene seems to be plant-specific, but the

previously suggested horizontal transfer of the ACL5 gene from bacteria to plants

was not confirmed in the present study. The identified Scots pine sequence was

defined as a putative thermospermine synthase TSPMS encoding gene and named

PsACL5. So far, the plant PAOs have been considered to be mostly involved in

the terminal degradation of polyamines. However, the phylogenetic analysis of

plant PAO sequences suggested that PAOs capable of polyamine backconversion

may also be widespread. Overall, the results revealed that in plants the polyamine

biosynthesis and catabolism pathways have risen via a complex evolutionary

history, which has led to several plant-specific features in polyamine metabolism.

3.3 Consistent polyamine profiles during embryo development (II)

In gymnosperms, the time of fertilization and, consequently, embryo development

vary between years in the same location according to the effective temperature

sum (d.d.). To find out the relationship between polyamine content and

developmental stages during the Scots pine zygotic embryogenesis, the polyamine

concentrations from the two Scots pine clones, K818 and K884, and two years

were combined to the same regression model by presenting them as functions of

the effective temperature sum. The free putrescine content did not change during

embryo development apart from clone K884 in 2001, when there was a slightly

increasing trend. The soluble conjugated putrescine fraction increased at early

embryogeny until the effective temperature sum was about 600 d.d. and decreased

thereafter during late embryogeny. Also, the insoluble conjugated putrescine

fraction was considerable and maximally comprised nearly 35% of the total

putrescine in the sample, but there was no trend in the insoluble conjugated

putrescine content. Spermidine was the most abundant polyamine during embryo

development. Both free and soluble conjugated spermidine increased at the

beginning of embryo development, reached their maximum when the effective

temperature sum was between 600 and 750 d.d. and decreased thereafter. The

insoluble conjugated spermidine fraction always consisted of less than 11% of the

total spermidine fraction in the sample. Also, the free and the soluble conjugated

spermine fluctuated in a consistent way during embryo development. The

spermine concentrations appeared to increase from 500 d.d. to 700 d.d., after

which the concentrations started to go down. The content of insoluble conjugated

35

spermine was very low or under the HPLC detection level in most samples. As a

conclusion, the polyamine concentrations followed consistent profiles. Except for

the free putrescine fraction, which remained stable during embryo development,

polyamines had the tendency to increase at the early stages but decrease at the late

stages of embryo development.

3.4 Preference of ADC pathway (I, II)

In Scots pine zygotic embryos, both ADC gene expression and ADC enzyme

activity were clearly preferred, compared with ODC gene expression and ODC

enzyme activity, respectively. This suggests that the ADC pathway is the main

route to produce putrescine during embryo development. Both ADC gene

expression and ADC enzyme activity increased when embryo development

proceeded, which suggested that the ADC enzyme is at least partially

transcriptionally regulated. In addition to zygotic embryos, the mRNA copy

number of the ADC gene was also higher in the embryos and megagametophytes

of mature seeds as well as in the cotyledons, hypocotyls and roots of young

seedlings. At minimum, the ratio of the ADC and ODC mRNA transcripts was 7

in megagametophytes as well as in the cotyledons of young seedlings, whereas

the highest ratio observed was 681 in mature embryos. The results indicated that

in Scots pine putrescine is almost solely produced via the ADC pathway in both

developing and mature embryos, but the ADC pathway is also strongly preferred

in young seedlings.

3.5 Localization of ADC gene expression and ADC enzyme (II)

Both ADC mRNA transcripts and ADC protein were localized in developing

Scots pine embryos. At the early embryogeny stage, ADC mRNA transcripts

located in the cytoplasm of the cells of dominant and subordinate embryos. In late

embryos, ADC mRNA transcripts were in the dividing cells of the embryo

particularly in the shoot apical and axillary meristems (Fig. 3A), but also in the

root meristem. ADC mRNA transcripts existed in dividing cells throughout the

mitotic stages. During prophase and metaphase, ADC mRNA transcripts were in

the cytoplasm, whereas in anaphase and telophase cells they accumulated within

the area of the mitotic spindle apparatus (Fig. 3B). ADC protein located in the

nuclei of embryos. A high level of ADC protein was detected especially in

dividing cells, but, in contrast with the ADC transcripts, ADC protein located

36

close to the chromosomes (Fig. 3C) and not within the mitotic spindle as the ADC

mRNA transcripts.

Fig. 3. Localization of ADC gene expression and ADC enzyme in Scots pine zygotic

embryos. The blue signal indicates ADC mRNA transcripts (A and B) and the brown

signal indicates ADC protein (C) in the mitotic cells of the shoot meristem.

3.6 Fate of megagametophyte during embryo development (III, IV)

During all the developmental stages of Scots pine embryogenesis, the

megagametophyte cells in the ESR were destroyed by cell death that showed

morphologically necrotic features. Their cell wall, plasma membrane and nuclear

envelope broke down with the release of cell debris and nucleic acids into the

corrosion cavity. Nuclear DNA fragmentation was detected in the dying cells in

the ESR as well as in the arrow-shaped region in front of the dominant embryo

during embryo development. Instead, nuclear DNA fragmentation was not

detected in the inner parts of the megagametophyte despite a slight fragmentation

at late embryogeny. Neither DNA ladder nor smear was observed in the DNA that

was extracted from the developing and mature Scots pine seeds. Except for the

cells in the ESR, the rest of the megagametophyte survived with no symptoms of

PCD or necrosis throughout embryo development. Most of the megagametophyte

cells appeared viable with the presence of nucleoli and mRNA (Fig. 4A). Mature

megagametophytes were metabolically active and able to proliferate in vitro when

embryos were excised (Fig. 4B and C).

37

Fig. 4. Viability of megagametophyte cells in Scots pine seeds. (A) In an acridine

orange stained section, the red color in the cytoplasmic region of the

megagametophyte cells indicated the presence of mRNA and thus active gene

expression. Nuclei appeared normal with the presence of nucleoli and with no sign of

DNA fragmentation during late embryogeny. (B) After the tetrazolium test, the red

color in the megagametophyte tissue of a mature seed indicated cell respiration. (C)

Proliferated megagametophyte cells on culture medium.

3.7 DNA repair and PCD-related gene expression (III)

The cell death processes and DNA fragmentation were studied further by

sequencing the cDNA fragments of the putative Scots pine RAD51, KU80, DNA

ligase (LIG), metacaspase (MCA) and Tat-D nuclease (TAT-D) genes and by

measuring the changes in their expression levels during the Scots pine embryo

development. From the DNA repair-related genes, the expression of RAD51

decreased in both Scots pine clones, whereas the expression of the KU80 and LIG

genes remained constant. This suggested that during late embryogeny the

proportion of mitotic cells decreased and the DNA breaks were mainly repaired

by non-homologous end joining (NHEJ). The expression of both PCD-related

genes, MCA and TAT-D, showed a downward trend during embryo development,

which indicated that no large-scale PCD or nucleic acid fragmentation occurred in

the megagametophyte tissue during late embryogeny.

3.8 Unspecific in situ mRNA hybridization signal in dying cells (IV)

In the in situ mRNA hybridization assays with antisense and sense RNA probes of

CAT, DAO and ADC genes, a non-specific signal was constantly detected in the

broken megagametophyte cells in the ESR, in the remnants of the degenerated

suspensors as well as in the cells of the nucellar layers, i.e. tissues exposed to cell

38

death processes and extensive nucleic acid fragmentation during Scots pine seed

development (Fig. 5). It was confirmed that a non-specific signal did not occur

due to interaction between the anti-DIG antibody and dying cells, nor due to

endogenous alkaline phosphatase activity. Furthermore, the non-specific binding

of probes was not connected to polysaccharides or phenols. The non-specific

signals remained in the broken megagametophyte cells despite the digestion of

RNA prior to in situ hybridization, suggesting that the non-specific binding of the

probes resulted mainly from hybridization between a probe and fragmented DNA.

Fig. 5. Nucleic acid fragmentation and non-specific in situ hybridization signal in the

cells at the ESR of the megagametophyte. (A) Acridine orange staining revealed

necrotic cell with the release of fragmented nucleic acids into the corrosion cavity. (B)

DNA fragmentation detected by the TUNEL method. (C) Non-specific in situ

hybridization signal in the section hybridized with the CAT sense probe.

39

4 Discussion

4.1 Taking the ADC route

4.1.1 ADC/ODC

Among eukaryotes, plants are unique in having an additional, alternative

biosynthetic route to putrescine from arginine. In plants, putrescine is generally

produced via both ODC and ADC pathways, and different physiological roles

have been proposed for the routes because their use is often tissue-specific and

they undergo distinct regulation depending on developmental and physiological

conditions (Kumar et al. 1997). It has been suggested that ODC is involved in cell

division in actively growing plant tissues (Cohen et al. 1984, Acosta et al. 2005),

whereas ADC activity has been found in elongating cells, embryonic cells and

cells exposed to various stress conditions (Flores 1991). However, the model

plant Arabidopsis possesses only the ADC pathway (Hanfrey et al. 2001) and, as

shown in the present study, the ADC pathway is strongly preferred in putrescine

production also in Scots pine, despite the presence of both ADC and ODC genes.

Arabidopsis and Scots pine represent the two major taxa of seed plants,

angiosperms and gymnosperms, which diverged from each other about 300 Mya

(Bowe et al. 2000). Thus, they are very different in form, genome size, ecological

niche as well as evolutionary history. Arabidopsis is a small, herbaceous annual

dicotyledon, whereas Scots pine is a large, long-lived coniferous forest tree.

However, in both Arabidopsis and Scots pine the putrescine production is

dependent on the ADC pathway, which emphasizes the importance of the ADC

pathway in plants generally and indicates that putrescine biosynthesis from

ornithine is not essential for normal growth.

The dependence of Arabidopsis and Scots pine on the ADC pathway in

putrescine production reveals specific roles for plant ADC. Because plants, in

general, are able to produce putrescine by both the ODC and ADC pathways,

ADC may have taken, at least in some cells, the functions that belong to ODC in

animal cells. Furthermore, different ADC proteins may have specialized functions

in a plant species. In mammalian cells, the progression of the normal cell cycle is

dependent on ODC (Oredsson 2003), and ODC activity is commonly connected

with cell division also in plants (Galston & Sawhney 1990, Acosta et al. 2005,

Paschalidis & Roubelakis-Angelakis 2005b). However, in Scots pine putrescine is

40

almost solely produced via the ADC pathway in both developing and mature

embryos, and the ADC pathway is also strongly preferred in the actively growing

tissues of young seedlings. In developing embryos, ADC mRNA transcripts

accumulate within the mitotic spindle apparatus of dividing cells and ADC

protein locates close to the chromosomes, which indicates that ADC plays an

essential role in the mitosis of plant cells. In Arabidopsis, much of the difference

between AtADC1 and AtADC2 accumulates at the N-terminus of the amino acid

sequences, suggesting that the subcellular localization of the ADC proteins may

be different (Hanfrey 2001) and, furthermore, that they may act on distinct

metabolic pools and have important functional differences. The expression

profiles and the promoter activities of the AtADC1 and AtADC2 genes show tissue

specificity and differ from each other under abiotic stress (Urano et al. 2003,

Hummel et al. 2004). AtADC2 promoter activity is strongly associated with seed

germination, root and leaf development, whereas AtADC1 promoter activity is

low during vegetative development (Hummel et al. 2004).

4.1.2 Once again – evolution of ADC pathway

The evolution of central metabolic pathways was one of the key steps in the

biochemical development of primordial life (Schmidt et al. 2003). In the present

study, the polyamine biosynthesis pathway was confirmed to be a favourable

model to study the processes, such as gene duplication and gene loss, involved in

metabolic pathway evolution. Furthermore, the modular structure of the

polyamine biosynthesis pathway has been suggested to facilitate horizontal and

endosymbiotic gene transfer (Shaw et al. 2010).

In the present study, the phylogenetic analysis of the ADC sequences

supported the view that the ADC route in plants is bacterial in origin. Previously,

the plant ADC has been located in chloroplasts (Borrell et al. 1995) and has been

suggested to be inherited from a cyanobacterial ancestor of the chloroplast

(Illingworth et al. 2003). However, cyanobacteria were not found to be more

closely related to plants than proteobacteria. The ADC-sequence-based phylogeny

was congruent with the current conception about the evolution of green plants

according to which morphologically simple photosynthetic forms, such as

unicellular green algae, gave rise to multicellular forms. Furthermore,

morphologically simple plants, such as bryophytes, were followed by more

complex flowering forms with highly developed breeding mechanisms at the top

of plant phylogeny (Qiu & Palmer 1999). However, the ADC pathway is absent

41

from several green algae that belong to such genera as Ostreococcus, Micromonas,

Chlamydomonas, Volvox and Chlorella (Fuell et al. 2010). Although the gene

encoding an intact ADC is present in the single-celled green alga Micromonas

pusilla, the rest of the pathway is missing (Fuell et al. 2010). Furthermore, the

unicellular green alga Chlamydomonas reinhardtii is solely dependent on the

ODC pathway in putrescine production; however, unlike M. pusilla, C. reinhardtii

lacks an ADC gene and has instead AIH and CPA genes (Illingworth et al. 2003).

In the present study, both the AIH and CPA genes of C. reinhardtii clustered

together with bacterial sequences in the phylogenetic trees, implicating an ancient

origin of the AIH/CPA pathway in plants. The complete or partial absence of the

ADC pathway in several green algae indicates that either the ADC route has been

lost independently multiple times in green algae or the ADC and AIH/CPA parts

of the putrescine biosynthesis pathway have been acquired separately in plants.

While apparently ubiquitous in plants and bacteria, the ADC pathway is not

present in all organisms, which makes the ADC pathway also agriculturally

interesting. Because the ADC pathway does not exist in fungi, nematodes and

insects, it may provide a possibility to differential inhibition of host and

pathogen/pest polyamine biosynthesis (Bailey et al. 2000). Although there are

also some reports of ADC activity in fungi (Fornalé et al. 1999, Sannazzaro et al.

2004), no fungal ADC gene has so far been identified. The existence of ADC in

other eukaryotic groups than plants has been a controversial issue. In mammalian

cells, evidence of ADC activity has been found (Regunathan & Reis 2000) and a

putative human ADC gene has been identified (Zhu et al. 2004). Recent studies

have, however, been unable to find any evidence supporting a mammalian ADC

(Coleman et al. 2004, Lopez-Contreras et al. 2006, Kanerva et al. 2008). In

mammalian tissues, agmatine does not necessarily result from ADC activity

because it can be also derived from dietary sources or generated by enteric

bacteria (Morris 2004). In plants and bacteria, agmatine serves as a substrate for

the biosynthesis of putrescine through the activities of two enzymes, AIH and

CPA, whereas in vertebrates, the only enzyme specific for agmatine catabolism is

agmatine ureohydrolase (agmatinase, AUH), which catalyses the hydrolysis of

agmatine directly to putresine and urea (Morris 2004, Iyer et al. 2002). In addition

to ODC, two other ODC-like protein sequences have been identified in mammals.

One is an antizyme inhibitor (AZI) that binds to the antizyme with a higher

affinity than ODC and releases ODC from the ODC-antizyme complex

(Murakami et al. 1996), and the other is an ODC paraloque (ODCp) that has been

proposed to be either an AZI (Pitkänen et al. 2001) or a mammalian ADC (Zhu et

42

al. 2004). Kidron et al. (2007) found a close phylogenetic relationship between

ODCp and AZI sequences, whereas ODCp was not closely related to the ADCs of

other species. Recently, ODCp was reported to be a novel antizyme inhibitor

(AZIN2) and not ADC (Kanerva et al. 2008).

Although all known ADCs use either pyridoxal-5-phosphate (PLP) or a

pyruvoyl cofactor, ADC activity has developed independently at least five times

during evolution. Three classes of PLP-dependent ADC enzymes share little

sequence similarity, and the two classes of pyruvoyl-dependent ADC enzymes are

also nonhomologous (Giles & Graham 2008). Sulfolobus solfataricus as well as

most of the crearchaeota possess an ADC which has evolved from the pyruvoyl-

dependent SAMDC (Giles & Graham 2008). Additionally, large algal viruses,

such as the Paramecium bursaria chlorella virus (PBCV-1), encode an unusual

ADC which is a close homolog of eukaryotic ODC (Shah et al. 2004). The

repeated evolution of the ADC gene clearly indicates that the possibility of

arginine decarboxylation gives an organism an obvious evolutionary advantage.

Furthermore, the evolution of ADC from other genes that belong to the polyamine

biosynthesis pathway reflects the high evolutionary flexibility of the polyamine

metabolism pathway.

4.2 Polyamine metabolism in plants – something special?

4.2.1 ACL5 gene

Originally, it was believed that two genes, ACL5 and SPMS, both encode SPMS

in Arabidopsis (Hanzawa et al. 2000, Panicot et al. 2002). Later, however, Knott

et al. (2007) reported that the bacterially expressed recombinant ACL5 protein

catalyzes the conversion of spermidine to thermospermine but not to spermine,

indicating that the ACL5 gene encodes a TSPMS. Furthermore, Kakehi et al.

(2008) detected TSPMS activity in Arabidopsis plants. Now, ACL5 genes have

been found from several plants such as apple and clementine (Citrus clementina)

(Kitashiba et al. 2005, Trénor et al. 2010). In the present study, the identified

Scots pine sequence showed 58% identity to the ACL5 proteins of both apple and

Arabidopsis. Thus, the sequence was defined as a putative TSPMS encoding gene

and named PsACL5. Overall, the gathered evidence suggests that thermospermine

may be ubiquitous in plants and that some of the putative SPMSs described so far

may in the future reveal to be TSPMSs.

43

The present study confirmed that plants possess both SPMS and ACL5 genes

with varying sequence similarity with the SPDS gene. SPMS genes showed high

sequence similarity with SPDS genes, which suggested that the SPMS-type

progenitor gene evolved from SPDS more recently than the ACL5-type progenitor

gene or, alternatively, that the ACL5 gene has a different evolutionary origin.

Previously, it has been suggested that the plant ACL5 gene was acquired by an

algal ancestor of plants through horizontal gene transfer from archaea, whereas

the SPMS gene evolved from the SPDS gene in respective lineages of plants,

animals and fungi (Minquet et al. 2008). However, in the present study, no close

relationship was found between the plant ACL5 genes and bacterial genes in the

phylogenetic analysis at the nucleotide level. Although the Arabidopsis ACL5

gene has been proven to encode thermospermine (Knott et al. 2007), further

studies are needed to resolve the origin of plant ACL5 genes as well as the

possible connection between plant and bacterial genes.

The synthesis of thermospermine is likely to resemble the formation of

spermine (Knott et al. 2007). However, the slight structural difference between

spermine and thermospermine must be essential for their function because an

Arabidopsis SPMS defective mutant shows wild type appearance (Imai et al.

2004a), whereas loss-of-function mutants of the ACL5 gene have severe defects in

stem elongation (Kakehi et al. 2008). Previously, the separation of spermine and

thermospermine was performed by thin-layer chromatography (Knott et al. 2007,

Kakehi et al. 2008) because separation with HPLC was not possible. Hence, the

present study, together with most studies in which measurements of plant

polyamines are given, do not distinguish between spermine and thermospermine.

Very recently, a HPLC method for the separation of spermine and

thermospermine was, however, described (Naka et al. 2010). In Scots pine

zygotic embryos, the total amount of spermine/thermospermine was less than that

of spermidine. During embryogenesis, both free and soluble conjugated

spermidine as well as both free and soluble conjugated spermine/thermospermine

concentrations showed an increasing trend at the early stages but a decreasing

trend at the late stages of embryo development. The consistent profiles in

different years suggested that spermidine and spermine/thermospermine have an

important role in embryo development and that they may have different roles at

different developmental stages.

In Arabidopsis, spermidine is essential for survival, since the spds1-1 spds2-1

double-mutant is embryo-lethal (Imai et al., 2004b). By contrast, the Arabidopsis

acl5-1 spms-1 double mutant, which contains neither spermine nor

44

thermospermine, is viable and shows no phenotype except for reduced stem

growth due to acl5-1 (Imai et al. 2004a). All plant species, including the model

moss Physcomitrella patens and gymnosperms, as well as the algae

Chlamydomonas reinhardtii have been found to possess sequences identifiable as

SPDS or ACL5, but SPMS sequences have been identified only in angiosperms

(Merchant et al. 2007, Minguet et al. 2008). Although the structure of the

Chlamydomonas SPDS and ACL5 genes differ notably from corresponding genes

in plants, the amino acid sequences are conserved (Rodríguez-Kessler et al. 2010).

Thus, ACL5 seems to represent an ancient and plant-specific gene in eukaryotes,

whereas the SPMS gene seems not to be essential for plant survival, at least under

normal growth conditions, but may have functions in certain environmental

conditions. By contrast, SPMS is essential for normal growth and development in

human and other mammals. In human, mutations in the SPMS gene are associated

with the X-linked disease, Snyder-Robinson syndrome, which causes decreased

activity of SPMS, low levels of intracellular spermine in lymphocytes and

fibroblasts as well as elevated spermidine/spermine ratios. The disease leads to

several serious symptoms, such as mild-to-moderate mental retardation,

hypotonia and cerebellar circuitry dysfunction (Cason et al. 2003). The male gyro

(Gy) mouse that has an X-chromosomal deletion including the SPMS gene can

survive but suffers multiple abnormalities, such as a tendency to sudden death,

small size, neurological symptoms, sterility and deafness (Pegg & Wang 2009).

4.2.2 Polyamine oxidases without back-conversion ability

Spermidine and spermine are catabolized by polyamine oxidases (PAOs). The

substrate specifity as well as the reaction products of PAOs vary depending on the

organisms and isoenzymes involved. In animals, PAOs and spermine oxidases

(SMOs) preferentially catalyse the oxidation of spermidine, spermine and/or their

acetylated derivates to produce H2O2, an aminoaldehyde, and putrescine or

spermidine, respectively, thus enabling polyamine back-conversion (Seiler 2004).

In plants, polyamine oxidation is mainly catalyzed by apoplastic PAOs in

reactions producing 1,3-diaminopropane (DAP), H2O2, and the corresponding

aldehyde, thus leading to the terminal degradation of polyamines (Cona et al.

2006), although the polyamine back-conversion pathways were recently detected

also in plants (Tavladoraki et al. 2006, Kamada-Nobusada et al. 2008, Moschou

et al. 2008). In the present study, the revealed PsPAO amino acid sequence was

42% identical to AtPAO1, the first plant PAO reported to be involved in a

45

polyamine back-conversion pathway, whereas PsPAO was only 16 to 20%

identical to the AtPAO2, AtPAO3, AtPAO4 and AtPAO5 proteins. Recently,

AtPAO3 was found to catalyze the oxidative conversion of spermine to

spermidine and spermidine to putrescine (Moschou et al. 2008), whereas AtPAO4

catalyzes only the conversion of spermine to spermidine (Kamada-Nobusada et al.

2008). In Arabidopsis, AtPAO2, AtPAO3 and AtPAO4 have been localized to

peroxisomes (Kamada-Nobusada et al. 2008), but the polyamine degration has

been suggested to occur mainly in the apoplastic space because PAO activity is

predominantly observed in the cell wall fraction (Slocum 1991).

Besides regulating cellular polyamine levels, plant amine oxidases contribute

to various physiological processes through their reaction products. Spermidine

deamination results in 4-aminobutanal, which can be further metabolized to γ-

aminobutyric acid (GABA). GABA is an important metabolite involved in many

basic cellular processes, such as regulation of cytosolic pH and carbon fluxes into

the citric acid cycle, but it is also produced in response to biotic and abiotic stress

(Bouché & Fromm 2004). Furthermore, DAP is a precursor of β-alanine (Terano

& Suzuki 1978) as well as stress-associated uncommon polyamines (Koc et al.

1998), whereas H2O2 is a key mediator in several reactions connected to

development and stress (Quan et al. 2008). In plants, amine oxidases have been

associated with primary and secondary cell walls undergoing lignification,

suberization and wall stiffening (Rea et al. 2004), but they are also connected to

cell division and growth (Paschalidis & Roubelakis-Angelakis 2005a), oxidative

stress and cell death (Yoda et al. 2003) as well as pathogen resistance (Moschou

et al. 2009). Thus, in addition to regulating cellular spermidine and spermine

levels, plant PAOs participate in several fundamental physiological processes

through their reaction products (Cona et al. 2006), which may have favoured the

evolution of plant-specific polyamine oxidases without polyamine back-

conversion ability.

4.3 Necrotic cell death as a regulated cellular response

Necrosis has traditionally been considered a chaotic and uncontrolled form of cell

death and thus alternative to PCD. Morphologically necrotic cell death is

characterized by swelling and lysis of the cell and subcellular organelles, which

results in the leakage of cellular contents into the surrounding extracellular space.

Under extreme conditions cells die via an unregulated process of destruction,

which has led to the assumption that when cell death is accompanied by rapid

46

disruption of the plasma membrane, cytoplasmic structures and the nucleus, it

indicates that the death is passive and unregulated (Proskuryakov et al. 2003).

There is, however, growing evidence of physiologically regulated cell death with

necrotic-like morphology. Several reports have shown that in mammalian cells a

broad range of PDC triggers induce necrotic-like cell death instead of apoptosis

when caspase activities are inhibited (Kitanaka & Kuchino 1999). Additionally,

necrosis has been found to contribute, for example, to normal cell loss in the

human small intestine and large intestine (Mayhew et al. 1999, Barkla et al. 1999),

and also the death of primary T lymphocytes is caspase-independent and

morphologically necrotic (Holler et al. 2000). Recently, developmental stimuli-

induced necrotic cell death was found in Dictyostelium discoideum, a protist that

emerged in evolution after plants, from an ancestor common to fungi and animals

(Laporte et al. 2007). Furthermore, the occurrence of morphologically necrotic

cell death in Scots pine zygotic embryogenesis, which was revealed in the present

study, suggests that developmentally and physiologically regulated cell death with

necrotic-like morphology exists also in plants. On one hand, the results together

suggest that the mechanism underlying necrotic cell death was present already in

an ancestor common to animals and plants and has been conserved during

evolution. On other hand, the molecular basis of necrosis is still poorly known in

comparison with apoptosis and autophagy, and it may be different in diverse

organisms. In some situations, the developmentally and physiologically regulated

necrosis may have provided a selective advantage to an organism and it may have

evolved several times in the branches of the life tree.

In animal cells, maintenance of certain levels of ATP is required for the

execution of apoptotic programs. Generally, if the amount of ATP in a cell drops

below a critical level, it may cause necrosis or an initially apoptotic cell death can

switch into a necrotic one (Eguchi et al. 1997). Necrosis has also been associated

with the failure of osmotic regulation, which results in water flooding into the cell

(Lennon et al. 1991). Finally, cytosolic constituents spill into the extracellular

space through damaged plasma membrane and may provoke an inflammatory

response. However, in some cases the triggering of necrosis instead of apoptosis

may not be a failure but an advantage because a much more robust immune

response is evoked as a result of necrosis than apoptosis. This may be a matter of

life and death during dangerous situations, such as viral or bacterial infection,

injury or abnormal cells, when strong stimuli are required for the rapid

mobilization of defense forces in cells (Proskuryakov et al. 2003). In the

developing Scots pine seed, necrotic cells were found in the ESR of the

47

megagametophyte in which the cells broke down and released their content into

the corrosion cavity. In gymnosperms, the corrosion cavity is filled with fluid,

which serves as a nutritional and hormonal interphase between the developing

embryos and the megagametophyte (Carman et al. 2005). Via necrotic breakdown

of the megagametophyte cells, cytosolic constituents can be released into the

corrosion cavity and, furthermore, for the nutrition of growing embryos, which

may have favoured the evolution of developmentally regulated necrotic cell death

in the ESR.

Nowadays, the old dogma that necrosis is always a passive process seems to

be irreversibly mined and the picture of cell death programs has turned out to be

more complex than previously envisaged. Similarly, the idea that apoptosis

always needs ATP, whereas necrosis is an invariantly energy-independent and

disorganized cell collapse, seems to be obsolete (Chiarugi 2005). Plant cells

integrate cell death and survival signals to determine when to die and how to

process their own corpses. Furthermore, the way of corpse management is

probably determined in a dying plant cell before the moment of death and even

before the point of no return (Jones 2001). The occurrence of necrotic cell death

in a maternally derived haploid tissue in the Scots pine seed provides a favorable

model to study the genetic control of developmentally programmed necrotic cell

death. One interesting and important question for researchers in the area is

whether the genetic control of this type of cell death resides in the dying cell itself

or outside the cell. As a conclusion, it can be presumed that in the future necrotic

cell death can be considered as a form of execution phase of PCD, along with

apoptosis, autophagy and other, still unknown, types of PCD.

4.4 Pros and cons of nuclear DNA fragmentation

During the PCD, the main target of cell degradation machinery is the nucleus in

which the degradation processes include both chromatin and the nuclear envelope

(Earnshaw 1995). In animal cells, nuclear degradation is executed by a caspase

family of cysteine proteases, whereas the canonical caspases are absent in plants.

By contrast, plant genomes contain distant caspase-homologues, metacaspases

(MCAs), which have been classified as type I and type II based on their sequence

and structural features (Sanmartín et al. 2005). In plant and animal cells, the

structural organization of nuclei is shared, whereas the molecular composition of

the nuclear envelope is not (Rose et al. 2004). However, the mechanism of DNA

fragmentation also varies in different species, and even in different tissues of one

48

organism (Jiang et al. 2008). The pattern of DNA degradation occurs by

activation of endogenous endonucleases that cleave DNA in the linker region

between histones on the chromosomes. Several candidate nucleases have been

reported (Scovassi & Torriglia, 2003). Tat-D is an evolutionary conserved

apoptotic nuclease that incises the double-stranded DNA without obvious

specificity through its endonuclease activity and excises DNA from the 3´- to 5´-

end via its exonuclease activity. In the present study, both putative TAT-D and

MCA genes were shown to have decreased expression during the Scots pine

embryogenesis, which supports the view that no large-scale PCD and nucleic acid

fragmentation occur in the megagametophyte tissue at late embryogeny. On the

other hand, the decreasing expression of the TAT-D and MCA genes may also

reflect the increasing contribution of the more developed embryo to the total

amount of mRNA and, furthermore, the important role of Tat-D and metacaspase

in the PCD events during early embryogenesis.

Due to the fact that DNA wrapped around the histones comprises about 180–

200 bp, multiples of this interval are characteristically observed when DNA from

apoptotic cells is subjected to conventional agarose gel electrophoresis. By

contrast, in necrosis DNA degrades randomly and results in a smear (Bortner et al.

1995, Danon et al. 2000). The fragmentation of genomic DNA into

oligonucleosomal units was initially documented during apoptosis in animal cells

(Wyllie et al. 1980). In plant cells, internucleosomal DNA fragmentation during

PCD has been described, for example, in stressed tobacco (Nicotiana tabacum L.)

BY-2 cell line, which was cultivated at a low temperature (Koukalová et al. 1997)

or treated with cadmium (Fojtová & Kovařík 2000). However, internucleosomal

DNA fragmentation has also been found in rapidly killed BY-2 cells, which were

killed mechanically by freezing in liquid nitrogen or with the Triton X-100

treatment, although the cells were not able to activate and realize respective

enzymatic processes in a programmed manner (Kuthanova et al. 2008). Therefore,

it has been suggested that internucleosomal DNA fragmentation should not be

considered as an indicator of PCD without simultaneous evaluation of

morphological changes (Kuthanova et al. 2008). In the present study, no DNA

fragmentation was detected during embryo development, and both the

megagametophyte and embryo DNA was intact also in mature seeds after two

days of imbibition. Nucleic acids and cell morphology of the megagametophyte

cells were examined by AO staining, which revealed no symptoms of PCD or

necrosis in the inner part of the megagametophyte but, instead, showed signs of

mRNA and active gene expression in the cytoplasm of the megagametophyte cells.

49

The result that megagametophyte DNA looked intact on the agarose gel was

according to expectations because in previous studies megagametophyte DNA has

been frequently used for genetic analysis (Devey et al. 1995, Karhu et al. 2006,

Wachowiak et al. 2010). Pines and other gymnosperms are generally considered

to be difficult subjects for genetic studies for many reasons, such as long

generation time, large genome size and outbred mating system, but they have one

remarkable advantage: the haploid megagametophyte tissue represents a single

meiotic product and makes the direct analysis of inheritance of genetic loci

possible without the use of controlled crosses (Devey et al. 1995).

The in situ detection of nuclear DNA fragmentation by TUNEL assay is

currently one of the most frequently used techniques in research on cell death.

However, the sensitivity and specificity of the technique have also been criticized.

False-positive TUNEL staining has been detected in mouse kidney and liver

tissues (Pulkkanen et al. 2000), and, in plant tissues, fixation and embedding have

been reported to cause sufficient nicking of nuclear DNA to produce false

TUNEL positive nuclei (Wang et al. 1996). Additionally, the TUNEL method is

presumed to be unable to discriminate apoptotic from necrotic cells, given that the

latter also have free DNA ends (Kelly et al. 2003). In the present study, TUNEL

positive nuclei were detected in the cells that died via necrotic cell death in the

ESR and in the arrow-shaped megagametophyte region in front of the dominant

embryo. Additionally, a slight TUNEL signal was found in nuclei in the inner

parts of the megagametophyte at late embryogeny although the cells stayed alive

at least until the imbibition phase of the mature seed. During development, the

seed experiences developmentally programmed as well as environmental stress,

which is potentially damaging to the genome. Embryo development, reserve

accumulation and maturation/drying are the three typical stages of orthodox seed

development, leading from a zygotic embryo to a mature, quiescent seed. During

germination, seeds recover physically from maturation drying, resume a sustained

intensity of metabolism, complete essential cellular events to allow the embryo to

emerge and prepare subsequent seedling growth (Nonogaki et al. 2010). In maize

(Zea mayze) seeds, the maturation drying/rehydration cycle results in the

appearance of thousands of single-strand breaks in the genome of each cell

(Dandoy et al. 1987). Thus, attention should be paid to the fact that DNA

fragmentation can also be a temporary process in plants and does not always

proceed to cell death.

In the present study, we found that in in situ mRNA hybridization assay

probes continually hybridized non-specifically in the ESR of the

50

megagametophyte tissue, in the remnants of the degenerated suspensors as well as

in the cells of the nucellar layers, i.e. tissues exposed to cell death and extensive

nucleic acid fragmentation during Scots pine seed development. The non-specific

binding of probes was not connected to phenolic compounds or polysaccharides

but resulted from the hybridization between a probe and fragmented nucleic acid,

especially DNA, in the dying tissues. In plants, cell death is an integral part of

both development and defence, and hence it is a common phenomenon in all

stages of the life cycle. Our results suggest that extensive nucleic acid

fragmentation during cell death processes can be a considerable source of non-

specific signals in traditional in situ mRNA hybridization. Therefore, the

simultaneous visualization of potential nucleic acid fragmentation may be

necessary to ensure the correct interpretation of the in situ mRNA hybridization

results in plant tissues.

51

5 Conclusion and future prospects

All in all, the present study enhanced our understanding of the polyamine

metabolism and cell death processes during the Scots pine zygotic embryogenesis.

We also received new insight into the evolutionary history of polyamine genes in

plants.

We introduced the first sequences from a gymnosperm species representing

all the genes in the polyamine biosynthesis and catabolism pathways. The coding

regions of the Scots pine putative arginine decarboxylase (PsADC), ornithine

decarboxylase (PsODC), agmatine iminohydrolase (PsAIH), N-

carbamoylputrescine amidohydrolase (PsCPA), spermidine synthase (PsSPDS),

thermospermine synthase (PsACL5), S-adenosyl methionine decarboxylase

(PsSAMDC), copper-containing amine oxidase (PsCuAO) and flavoprotein-

containing polyamine oxidase (PsPAO) genes were sequenced. The sequence data

were used for the reconstruction of the evolutionary history of the polyamine

genes in plants. The phylogenetic analyses of the genes encoding polyamine

biosynthetic and catabolic enzymes revealed the complex evolution of the

polyamine metabolism pathways. Although the polyamine metabolism has in

principle been highly conserved, several features have also evolved specifically in

plants. Compared with other eukaryotes, the acquisition of the additional ADC

pathway for putrescine biosynthesis through horizontal gene transfer from

bacteria may have added to the adaptability of polyamine homeostasis in plants

and allowed the reduced use of the ODC pathway in Scots pine as well as in

conifers in general, as revealed in the present study. Furthermore, the presence of

two putrescine biosynthesis pathways may have influenced the evolution of both

ADC and ODC genes in plants. The phylogenetic analysis of the SPDS and SPMS

sequences supported the view that SPDS genes in eukaryotes are derived from a

common ancestor and that the SPMS gene has evolved several times from SPDS

via gene duplication. The identified Scots pine sequence was defined as a putative

TSPMS encoding gene and named PsACL5. As has been suggested, the ACL5

gene was found to be plant-specific among eukaryotes, but the present study did

not confirm the previously held view that the ACL5 gene in plants originated from

a horizontal transfer from bacteria. In the phylogenetic tree, the PsPAO sequence

was in the same group with other coniferous PAO sequences. Of the Arabidopsis

PAO sequences, PsPAO was most closely related to AtPAO1, which has

previously been found to possess polyamine back-conservation ability. The

phylogenetic analysis of the PAO sequences supported the view that plants

52

possess several different polyamine oxidases that may have specific subcellular

localization and catalytic properties.

The study focused especially on the ADC and ODC pathways in putrescine

biosynthesis during Scots pine early development. It was found that putrescine is

almost solely produced via the ADC pathway during zygotic embryogenesis as

well as in mature seeds during the imbibition phase, but the ADC pathway is also

strongly preferred in young seedlings as well as generally in conifers. The

simultaneous increase in both ADC gene expression and ADC enzyme activity

during Scots pine embryogenesis suggested at least partial transcriptional

regulation of the ADC enzyme. In zygotic embryos, both ADC mRNA and ADC

protein localized in dividing cells of embryo meristems and, more specifically,

within the mitotic spindle apparatus and close to the chromosomes, respectively,

implicating the essential role of ADC in the mitosis of plant cells.

The anatomical studies confirmed that Scots pine embryogenesis proceeded

along with the increasing effective temperature sum, which was therefore used as

the main explanatory variable in statistical analyses, instead of the sampling date.

Hence, it was possible to combine polyamine results from different clones, years

and fractions to the same regression model and to show that the polyamine

concentrations followed consistent profiles during embryo development. The

results suggested that polyamines have an important role in embryo development

and that individual polyamines may have different roles at different

developmental stages. The polyamine profiles seemed to show an increasing trend

at the early stages but a decreasing trend at the late stages of embryo development.

Only the free putrescine fraction remained stable throughout the period of embryo

development.

We investigated the physiological events occurring in the megagametophyte

tissue during Scots pine embryogenesis and proved that the megagametophyte

tissue survived from the early phases of embryo development until the early

germination of mature seeds. The expression of the genes encoding PCD-related

metacaspase (MCA) and Tat-D nuclease (TAT-D) proteins showed a downward

trend during embryo development, which indicated that no large-scale PCD or

nucleic acid fragmentation occurred in the megagametophyte tissue during late

embryogeny. Instead, the slight DNA fragmentation detected in the inner parts of

the megagametophyte at late embryogeny may be a consequence of DNA strand

breaks caused by maturation drying or by the DNA breaks with free 3’-OH ends

that appear during DNA repair. The decrease in RAD51 expression and constant

expression of KU80 and LIG implicated that during late embryogeny a proportion

53

of mitotic cells decreased and the DNA breaks were mainly repaired by the NHEJ

pathway. However, the megagametophyte cells in the narrow ESR were found to

die via morphologically necrotic cell death, which suggested that developmentally

and physiologically regulated cell death with necrotic-like morphology is

evolutionarily conserved and exists also in plants.

In plant cells, polyamines are integrated both into cell proliferation and PCD.

Thus, the developing pine seed is an excellent research model in which cell

division and PCD occur simultaneously. Furthermore, the unique reproductive

features of gymnosperms, together with the occurrence of cell death in various

tissues that differ from each other either in the number or the parental origin of

the genomes, make the pine seed special compared with many other PCD model

systems. In the present study, the ADC pathway was found to be the main route

for putrescine biosynthesis. Furthermore, both ADC mRNA transcripts and ADC

protein located in the mitotic cells of Scots pine zygotic embryos. These findings

raised questions about the possible interaction between ADC protein/putrescine

and DNA as well as internal ribosome entry site (IRES) dependent translation,

which may enable the synthesis of ADC protein during mitosis. Our ongoing

studies will shed light on the possible roles that the other main polyamine

biosynthetic enzymes, SPDS and TSPMS, as well as the polyamine catabolic

enzymes, DAO and PAO, play in cell proliferation and PCD. We have recently

applied a bioreactor cultivation procedure to follow the growth of Scots pine

embryogenic cell mass in real time (unpublished data). In bioreactor cultivation,

we are able to test experimentally the hypothesis that polyamines are integrated

both into cell division and PCD in plant cells and to compare the induced cell

death process with the developmental PCD involved in zygotic embryo and seed

development. Presently, there is an ongoing project in the research group of Prof.

Outi Savolainen, Univ. of Oulu, in which the Scots pine polyamine genes are

sequenced from genomic DNA. Additionally, our future plans include the

sequencing of the promoter regions of Scots pine polyamine genes to increase the

understanding of the regulation of polyamine metabolism.

54

55

References

Acosta C, Pérez-Amador MA, Carbonell J & Granell A (2005) The two ways to produce putrescine in tomato are cell-specific during normal development. Plant Sci 168: 1053–1057.

Adams JM & Cory S (2007) Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr Opin Immunol 19: 488–496.

Alcázar R, Marco F, Cuevas JC, Patron M, Ferrando A, Carrasco P, Tiburcio AF & Altabella T (2006) Involvement of polyamines in plant response to abiotic stress. Biotechnol Lett 28: 1867–1876.

Bagni N & Tassoni A (2001) Biosynthesis, oxidation and conjugation of aliphatic polyamines in higher plants. Amino Acids 20: 301–317.

Bailey A, Mueller E & Bowyer P (2000) Ornithine decarboxylase of Stagonospora (Septoria) nodorum is required for virulence toward wheat. J Biol Chem 275: 14242–14247.

Bais HP & Ravishankar GA (2002) Role of polyamines in the ontogeny of plants and their biotechnological applications. Plant Cell Tiss Org Cult 69: 1–34.

Balk J, Chew SK, Leaver CJ & McCabe PF (2003) The intermembrane space of plant mitochondria contains a DNase activity that may be involved in programmed cell death. Plant J 34: 573–583.

Barkla DH & Gibson PR (1999) The fate of epithelial cells in the human large intestine. Pathology 31: 230–238.

Becwar MR, Nagmani R & Wann SR (1990) Initiation of embryogenic cultures and somatic embryo development in loblolly pine (Pinus taeda). Can J For Res 20: 810–817.

Beers EP (1997) Programmed cell death during plant growth and development. Cell Death Differ 4: 649–661.

Bonneau L, Ge Y, Drury GE & Gallois P (2008) What happened to plant caspases? J Exp Bot 59(3): 491–499.

Borrell A, Culianezmacia FA, Altabella T, Besford RT, Flores D & Tiburcio AF (1995) Arginine decarboxylase is localized in chloroplasts. Plant Physiol 109: 771–776.

Bortner CD, Oldenburg NBE & Cidlowski JA (1995) The role of DNA fragmentation in apoptosis. Trends Cell Biol 5: 21–26.

Bortolotti C, Cordeiro A, Alcazar R, Borrell A, Culianez-Macia FA, Tiburcio AF & Altabella T (2004) Localization of arginine decarboxylase in tobacco plants. Physiol Plantarum 120: 84–92.

Bouché N & Fromm H (2004) GABA in plants: just a metabolite? Trends Plant Sci 9: 110–115.

Bouchereau A, Aziz A, Larher F & Martin-Tanguy J (1999) Polyamines and environmental challenges: recent development. Plant Science 140: 103–125.

Bouranis DIL, Chorianopoulou SN, Siyiannis VF, Protonotarios VE & Hawkesford MJ (2003) Aerenchyma formation in roots of maize during sulphate starvation. Planta 217: 382–391.

56

Bowe LM, Coat G & dePamphilis CW (2000) Phylogeny of seed plants based on all three genomic compartments: Extant gymnosperms are monophyletic and Gnetales' closest relatives are conifers. Proc Natl Acad Sci USA 97: 4092–4097.

Bozhkov PV, Filonova LH, Suarez MF, Helmersson A, Smertenko AP, Zhivotovsky B & von Arnold S (2004) VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation. Cell Death Differ 11: 175–182.

Bozhkov PV, Suarez MF, Filonova LH, Daniel G, Zamyatnin AA, Rodriguez-Nieto S, Zhivotovsky B & Smertenko A (2005) Cysteine protease mcll-Pa executes programmed cell death during plant embryogenesis. Proc Natl Acad Sci USA 102: 14463–14468.

Bozhkov P & Jansson C (2007) Autophagy and cell-death proteases in plants - Two wheels of a funeral cart. Autophagy 3: 136–138.

Buchholz JT (1926) Origin of cleavage polyembryony in conifers. Bot Gaz 81: 55–71. Carman JG, Reese G, Fuller RJ, Ghermay T & Timmis R (2005) Nutrient and hormone

levels in Douglas-fir corrosion cavities, megagametophytes, and embryos during embryony. Can J For Res 35: 2447–2456.

Cason AL, Ikeguchi Y, Skinner C, Wood TC, Holden KR, Lubs HA, Martinez F, Simensen RJ, Stevenson RE, Pegg AE & Schwartz CE (2003) X-linked spermine synthase gene (SMS) defect: the first polyamine deficiency syndrome. Eur J Hum Genet 11: 937–944.

Chiarugi A (2005) "Simple but not simpler": toward a unified picture of energy requirements in cell death. FASEB J 19: 1783–1788.

Childs AC, Mehta DJ & Gerner EW (2003) Polyamine-dependent gene expression. Cell Mol Life Sci 60: 1394–1406.

Cohen S (1998) A Guide to the Polyamines. Oxford, University Press. Coleman CS, Hu GR & Pegg AE (2004) Putrescine biosynthesis in mammalian tissues.

Biochem J 379: 849–855. Cona A, Rea G, Angelini R, Federico R & Tavladoraki P (2006) Functions of amine

oxidases in plant development and defence. Trends Plant Sci 11: 80–88. Costa LM, Gutierrez-Marcos JF & Dickinson HG (2004) More than a yolk: the short life

and complex times of the plant endosperm. Trends Plant Sci 9: 507–514. Criss WE (2003) A review of polyamines and cancer. Turk J Med Sci 33: 195–205. Dandoy E, Schyns R, Deltour R & Verly WG (1987) Appearance and repair of apurinic

apyrimidinic sites in DNA during early germination of Zea mays. Mutat Res 181: 57–60.

Danon A, Delorme V, Mailhac N & Gallois P (2000) Plant programmed cell death: A common way to die. Plant Physiol Bioch 38: 647–655.

Devey ME, Delfinomix A, Kinloch BB & Neale DB (1995) Random amplified polymorphic DNA markers tightly linked to a gene for resistance to white-pine blister rust in sugar pine. Proc Natl Acad Sci USA 92: 2066–2070.

57

Dickman MB, Park YK, Oltersdorf T, Li W, Clemente T & French R (2001) Abrogation of disease development in plants expressing animal antiapoptotic genes. Proc Natl Acad Sci USA 98: 6957–6962.

Earnshaw WC (1995) Nuclear-changes in apoptosis. Curr Opin Cell Biol 7: 337–343. Eguchi Y, Shimizu S & Tsujimoto Y (1997) Intracellular ATP levels determine cell death

fate by apoptosis or necrosis. Cancer Res 57: 1835–1840. Eisenberg T, Knauer H, Schauer A, Buettner S, Ruckenstuhl C, Carmona-Gutierrez D,

Ring J, Schroeder S, Magnes C, Antonacci L et al. (2009) Induction of autophagy by spermidine promotes longevity. Nat Cell Biol 11: 1305–U102.

Fehér A (2005) Why somatic plant cells start to form embryos? In Mujib A & Šamaj J (eds) Somatic embryogenesis. Berlin Heidelberg, Springer-Verlag: 85–101.

Felsenstein J (1985) Confidence-limits on phylogenies - an approach using the bootstrap. Evolution 39: 783–791.

Filonova LH, von Arnold S, Daniel G & Bozhkov PV (2002) Programmed cell death eliminates all but one embryo in a polyembryonic plant seed. Cell Death Differ 9: 1057–1062.

Fink SL & Cookson BT (2005) Apoptosis, pyroptosis, and necrosis: Mechanistic description of dead and dying eukaryotic cells. Infect Immun 73: 1907–1916.

Flores HE (1991) Changes in polyamine metabolism in response to abiotic stress. In Slocum RD & Flores HE (eds) Biochemistry and physiology of polyamines in plants. Boca Raton, CRC Press: 213–225.

Fojtová M & Kovařík A (2000) Genotoxic effect of cadmium is associated with apoptotic changes in tobacco cells. Plant Cell Environ 23: 531–537.

Fornalé S, Sarjala T & Bagni N (1999) Endogenous polyamine content and metabolism in the ectomycorrhizal fungus Paxillus involutus. New Phytol 143: 581–587.

Fuell C, Elliott KA, Hanfrey CC, Franceschetti M & Michael AJ (2010) Polyamine biosynthetic diversity in plants and algae. Plant Physiol Bioch 48: 513–520.

Galston AW & Sawhney RK (1990) Polyamines in Plant Physiology. Plant Physiol 94: 406–410.

Ge CM, Cui X, Wang YH, Hu YX, Fu ZM, Zhang DF, Cheng ZK & Li JY (2006) BUD2, encoding an S-adenosylmethionine decarboxylase, is required for Arabidopsis growth and development. Cell Res 16: 446–456.

Gernandt DS, Lopez GG, Garcia SO & Liston A (2005) Phylogeny and classification of Pinus. Taxon 54: 29–42.

Giles TN & Graham DE (2008) Crenarchaeal arginine decarboxylase evolved from an S-adenosylmethionine decarboxylase enzyme. J Biol Chem 283: 25829–25838.

Giuliani C, Consonni G, Gavazzi G, Colombo M & Dolfini S (2002) Programmed cell death during embryogenesis in maize. Ann Bot 90: 287–292.

Golstein P & Kroemer G (2006) Cell death by necrosis: towards a molecular definition. Trends Biochem Sci 32: 37–43.

Gonçalves S, Cairney J, Maroco J, Oliveira MM & Miguel C (2005) Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis. Planta 222: 556–563.

58

Gunawardena AHLAN, Greenwood JS & Dengler NG (2004) Programmed cell death remodels lace plant leaf shape during development. Plant Cell 16: 60–73.

Gunawardena AHLAN, Pearce DM, Jackson MB, Hawes CR & Evans DE (2001) Characterisation of programmed cell death during aerenchyma formation induced by ethylene or hypoxia in roots of maize (Zea mays L.). Planta 212: 205–214.

Gupta PK & Durzan DJ (1985) Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep 4: 643–645.

Häggman H, Jokela A, Krajnakova J, Kauppi A, Niemi K & Aronen T (1999) Somatic embryogenesis of Scots pine: cold treatment and characteristics of explants affecting induction. J Exp Bot 50: 1769–1778.

Häggman H, Vuosku J, Sarjala T, Jokela A & Niemi K (2005) Somatic embryogenesis of pine species: from functional genomics to plantation forestry. In Mujib A & Šamaj J (eds) Berlin Heidelberg, Springer-Verlag: 119–140.

Handley LW, Becwar MR, Chesick EE, Coke JE, Godbey AP & Rutter MR (1995) Research-and-development of commercial tissue-culture systems in loblolly-pine. Tappi J 78: 169–175.

Hanfrey C, Sommer S, Mayer MJ, Burtin D & Michael AJ (2001) Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity. Plant J 27: 551–560.

Hanzawa Y, Takahashi T, Michael AJ, Burtin D, Long D, Pineiro M, Coupland G & Komeda Y (2000) ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase. EMBO J 19: 4248–4256.

He X & Kermode AR (2003) Nuclease activities and DNA fragmentation during programmed cell death of megagametophyte cells of white spruce (Picea glauca) seeds. Plant Mol Biol 51: 509–521.

Helmersson A, von Arnold S & Bozhkov PV (2008) The level of free intracellular zinc mediates programmed cell death/cell survival decisions in plant embryos. Plant Physiol 147: 1158–1167.

Hiratsuka R, Yamada Y & Terasaka O (2002) Programmed cell death of Pinus nucellus in response to pollen tube penetration. J Plant Res 115(1118): 141–148.

Hirsch JG (1953) Spermine oxidase: an amine oxidase with specificity for spermine and spermidine. J Exp Med 97: 345–355.

Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B & Tschopp J (2000) Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1: 489–495.

Hughes AL & Nei M (1988) Pattern of nucleotide substitution at major histocompatibility complex class-i loci reveals overdominant selection. Nature 335: 167–170.

Hummel I, Bourdais G, Gouesbet G, Couee I, Malmberg RL & El Amrani A (2004) Differential gene expression of arginine decarboxylase ADC1 and ADC2 in Arabidopsis thaliana: characterization of transcriptional regulation during seed germination and seedling development. New Phytol 163: 519–531.

59

Igarashi K & Kashiwagi K (2010) Modulation of cellular function by polyamines. Int J Biochem Cell B 42: 39–51.

Illingworth C, Mayer MJ, Elliott K, Hanfrey C, Walton NJ & Michael AJ (2003) The diverse bacterial origins of the Arabidopsis polyamine biosynthetic pathway. FEBS Lett 549: 26–30.

Imai A, Akiyama T, Kato T, Sato S, Tabata S, Yamamoto KT & Takahashi T (2004a) Spermine is not essential for survival of Arabidopsis . FEBS Lett 556: 148–152.

Imai A, Matsuyama T, Hanzawa Y, Akiyama T, Tamaoki M, Saji H, Shirano Y, Kato T, Hayashi H, Shibata D, Tabata S, Komeda Y & Takahashi T (2004b) Spermidine synthase genes are essential for survival of Arabidopsis. Plant Physiol 135: 1565–1573.

International Seed Testing Association (1993) International rules for seed testing. Seed Sci Techn 21: 1–288.

Iyer RK, Kim HK, Tsoa RW, Grody WW & Cederbaum SD (2002) Cloning and characterization of human agmatinase. Mol Genet Metab 75: 209–218.

Jensen WA (1962) Botanical histochemistry. San Francisco, W.H. Freeman. Jiang AI, Cheng Y, Li J & Zhang W (2008) A zinc-dependent nuclear endonuclease is

responsible for DNA laddering during salt-induced programmed cell death in root tip cells of rice. J Plant Physiol 165: 1134–1141.

Jones AM (2001) Programmed cell death in development and defense. Plant Physiol 125: 94–97.

Kakehi J, Kuwashiro Y, Niitsu M & Takahashi T (2008) Thermospermine is required for stem elongation in Arabidopsis thaliana. Plant Cell Physiol 49: 1342–1349.

Kakkar RK & Sawhney VK (2002) Polyamine research in plants - a changing perspective. Physiol Plantarum 116: 281–292.

Kamada-Nobusada T, Hayashi M, Fukazawa M, Sakakibara H & Nishimura M (2008) A putative peroxisomal polyamine oxidase, AtPAO4, is involved in polyamine catabolism in Arabidopsis thaliana. Plant Cell Physiol 49: 1272–1282.

Kanerva K, Mäkitie LT, Pelander A, Heiskala M & Andersson LC (2008) Human ornithine decarboxylase paralogue (ODCp) is an antizyme inhibitor but not an arginine decarboxylase. Biochem J 409: 187–192.

Karhu A, Vogl C, Moran GF, Bell JC & Savolainen O (2006) Analysis of microsatellite variation in Pinus radiata reveals effects of genetic drift but no recent bottlenecks. J Evol Biol 19: 167–175.

Kelly KJ, Sandoval RM, Dunn KW, Molitoris BA & Dagher PC (2003) A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis. Am J Physiol Cell Physiol 284: C1309-C1318.

Kerr JF, Wyllie AH & Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Brit J Cancer 26: 239–257.

Kidron H, Repo S, Johnson MS & Salminen TA (2007) Functional classification of amino acid decarboxylases from the alanine racemase structural family by phylogenetic studies. Mol Biol Evol 24: 79–89.

60

King JE & Gifford DJ (1997) Amino acid utilization in seeds of loblolly pine during germination and early seedling growth (I. arginine and arginase activity). Plant Physiol 113: 1125–1135.

Kitanaka C & Kuchino Y (1999) Caspase-independent programmed cell death with necrotic morphology. Cell Death Differ 6: 508–515.

Kitashiba H, Hao YJ, Honda C & Moriguchi T (2005) Two types of spermine synthase gene: MdACL5 and MdSPMS are differentially involved in apple fruit development and cell growth. Gene 361: 101–111.

Knott JM, Roemer P & Sumper M (2007) Putative spermine synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine. FEBS Lett 581: 3081–3086.

Koc EC, Bagga S, Songstad DD, Betz SR, Kuehn GD & Phillips GC (1998) Occurrence of uncommon polyamines in cultured tissues of maize. In Vitro Cell Dev-Pl 34: 252–255.

Kong L, Attree SM & Fowke LC (1998) Effects of polyethylene glycol and methylglyoxal bis(guanylhydrazone) on endogenous polyamine levels and somatic embryo maturation in white spruce (Picea glauca). Plant Sci 133: 211–220.

Koukalová B, Kovarik A, Fajkus J & Siroky J (1997) Chromatin fragmentation associated with apoptotic changes in tobacco cells exposed to cold stress. FEBS Lett 414: 289–292.

Kumar A, Altabella T, Taylor MA & Tiburcio AF (1997) Recent advances in polyamine research. Trends Plant Sci 2: 124–130.

Kusano T, Berberich T, Tateda C & Takahashi Y (2008) Polyamines: essential factors for growth and survival. Planta 228: 367–381.

Kuthanova A, Opatrny Z & Fischer L (2008) Is internucleosomal DNA fragmentation an indicator of programmed death in plant cells? J Exp Bot 59: 2233–2240.

Lacomme C & Cruz SS (1999) Bax-induced cell death in tobacco is similar to the hypersensitive response. Proc Natl Acad Sci USA 96: 7956–7961.

Laporte C, Kosta A, Klein G, Aubry L, Lam D, Tresse E, Luciani MF & Golstein P (2007) A necrotic cell death model in a protist. Cell Death Differ 14: 266–274.

Lennon SV, Martin SJ & Cotter TG (1991) Dose-dependent induction of apoptosis in human tumor-cell lines by widely diverging stimuli. Cell Prolif 24: 203–214.

Lev-Yadun S & Sederoff R (2000) Pines as model gymnosperms to study evolution, wood formation, and perennial growth. J Plant Growth Regul 19: 290–305.

Lin PPC, Egli DB, Li GM & Meckel L (1984) Polyamine titer in the embryonic axis and cotyledons of Glycine max (L.) during seed growth and maturation. Plant Physiol 76: 366–371.

Lockshin RA & Zakeri Z (2004) Apoptosis, autophagy, and more. Int J Biochem Cell Biol 36: 2405–2419.

Lopez-Contreras AJ, Lopez-Garcia C, Jimenez-Cervantes C, Cremades A & Penafiel R (2006) Mouse ornithine decarboxylase-like gene encodes an antizyme inhibitor devoid of ornithine and arginine decarboxylating activity. J Biol Chem 281: 30896–30906.

61

Mähönen AP, Bonke M, Kauppinen L, Riikonen M, Benfey PN & Helariutta Y (2000) A novel two-component hybrid molecule regulates vascular morphogenesis of the Arabidopsis root. Genes Dev 14: 2938–2943.

Malabadi RB, Choudhury H & Tandon P (2002) Plant regeneration via somatic embryogenesis in Pinus kesiya (Royle ex. Grod.). Appl Biol Res 4: 1–10.

Malabadi RB & Van Staden J (2005) Somatic embryogenesis from vegetative shoot apices of mature trees of Pinus patula. Tree Physiol 25: 11–16.

Malmberg RL, Watson MB, Galloway GL & Yu W (1998) Molecular genetic analyses of plant polyamines. Crit Rev Plant Sci 17: 199–224.

Martins CF, Dode MN, Báo SN & Rumpf R (2007) The use of the acridine orange test and the TUNEL assay to assess the integrity of freeze-dried bovine spermatozoa DNA. Genet Mol Res 6(1): 94–104.

Martin-Tanguy J (1997) Conjugated polyamines and reproductive development: Biochemical, molecular and physiological approaches. Physiol Plantarum 100: 675–688.

Mayhew TM, Myklebust R, Whybrow A & Jenkins R (1999) Epithelial integrity, cell death and cell loss in mammalian small intestine. Histol Histopathol 14: 257–267.

Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318: 245–251.

Miguel C, Goncalves S, Tereso S, Marum L, Maroco J & Oliveira MM (2004) Somatic embryogenesis from 20 open-pollinated families of Portuguese plus trees of maritime pine. Plant Cell Tiss Org Cult 76: 121–130.

Minguet EG, Vera-Sirera F, Marina A, Carbonell J & Blazquez MA (2008) Evolutionary diversification in polyamine biosynthesis. Mol Biol Evol 25: 2119–2128.

Minocha R, Kvaalen H, Minocha SC & Long S (1993) Polyamines in embryogenic cultures of Norway spruce (Picea abies) and red spruce (Picea rubens). Tree Physiol 13: 365–377.

Minocha R, Long S, Maki H & Minocha SC (1999a) Assays for the activities of polyamine biosynthetic enzymes using intact tissues. Plant Physiol Bioch 37: 597–603.

Minocha R, Smith DR, Reeves C, Steele KD & Minocha SC (1999b) Polyamine levels during the development of zygotic and somatic embryos of Pinus radiata. Physiol Plantarum 105: 155–164.

Mirov NT (1967) The genus Pinus. New York, The Ronald Press Company. Mittler R & Lam E (1995) In situ detection of nDNA fragmentation during the

differentiation of tracheary elements in higher plants. Plant Physiol 108: 489–493. Mizushima N (2007) Autophagy: process and function. Genes Dev 21: 2861–2873. Morris SM (2004) Enzymes of arginine metabolism. J Nutr 134: 2743S–2747S. Moschou PN, Sanmartin M, Andriopoulou AH, Rojo E, Sanchez-Serrano JJ & Roubelakis-

Angelakis KA (2008) Bridging the gap between plant and mammalian polyamine catabolism: A novel peroxisomal polyamine oxidase responsible for a full back-conversion pathway in Arabidopsis . Plant Physiol 147: 1845-1857.

62

Moschou PN, Sarris PF, Skandalis N, Andriopoulou AH, Paschalidis KA, Panopoulos NJ & Roubelakis-Angelakis KA (2009) Engineered polyamine catabolism preinduces tolerance of tobacco to bacteria and oomycetes. Plant Physiol 149: 1970-1981.

Murakami Y, Ichiba T, Matsufuji S & Hayashi S (1996) Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase. J Biol Chem 271: 3340-3342.

Naka Y, Watanabe K, Sagor GHM, Niitsu M, Pillai MA, Kusan T & Takahashi Y (2010) Quantitative analysis of plant polyamines including thermospermine during growth and salinity stress. Plant Physiol Bioch 48: 527-533.

Nei M & Kumar S (2000) Molecular evolution and phylogenetics. New York, Oxford University Press.

Niemi K, Sarjala T, Chen XW & Häggman H (2002) Spermidine and methylglyoxal bis(guanylhydrazone) affect maturation and endogenous polyamine content of Scots pine embryogenic cultures. J Plant Physiol 159: 1155-1158.

Nonogaki H (2010) MicroRNA gene regulation cascades during early stages of plant development. Plant Cell Physiol 51: 1840-1846.

Ohe M, Sasaki H, Niitsu M, Bagni N, Tassoni A & Matsuzaki S (2010) Cadaverine turnover in soybean seedlings using N-15-labelled lysine and cadaverine. Plant Physiol and Bioch 48: 591-595.

Olsen OA (2004) Nuclear endosperm development in cereals and Arabidopsis thaliana. Plant Cell 16: S214–S227.

Oredsson SM (2003) Polyamine dependence of normal cell-cycle progression. Biochem Soc Trans 31: 366–370.

Oshima T (1989) Polyamines in thermophiles. In Bachrach U & Heimer YH (eds) The Physiology of Polyamines. Boca Raton, CRC Press: 35–46.

Owens JN, Johnsen O, Daehlen OG & Skroppa T (2001) Potential effects of temperature on early reproductive development and progeny performance in Picea abies (L.) Karst. Scand J For Res 16: 221–237.

Pamilo P & Bianchi NO (1993) Evolution of the Zfx and Zfy genes- rates and interdependence between the genes. Mol Biol Evol 10: 271–281.

Panicot M, Minguet EG, Ferrando A, Alcazar R, Blazquez MA, Carbonell J, Altabella T, Koncz C & Tiburcio AF (2002) A polyamine metabolon involving aminopropyl transferase complexes in Arabidopsis. Plant Cell 14: 2539–2551.

Park SY, Klimaszewska K, Park JY & Mansfield SD (2010) Lodgepole pine: the first evidence of seed-based somatic embryogenesis and the expression of embryogenesis marker genes in shoot bud cultures of adult trees. Tree Physiol 30: 1469–1478.

Paschalidis KA & Roubelakis-Angelakis KA (2005a) Sites and regulation of polyamine catabolism in the tobacco plant. Correlations with cell division/expansion, cell cycle progression, and vascular development. Plant Physiol 138: 2174–2184.

Paschalidis KA & Roubelakis-Angelakis KA (2005b) Spatial and temporal distribution of polyamine levels and polyamine anabolism in different organs/tissues of the tobacco plant. Correlations with age, cell division/expansion, and differentiation. Plant Physiol 138: 142–152.

63

Pegg AE & Wang X (2009) Mouse models to investigate the function of spermine. Comm Integr Biol 2: 271–274.

Pegg AE & Michael AJ (2010) Spermine synthase. Cell Mol Life Sci 67: 113–121. Pennell RI & Lamb C (1997) Programmed cell death in plants. Plant Cell 9: 1157–1168. Percy RE, Klimaszewska K & Cyr DR (2000) Evaluation of somatic embryogenesis for

clonal propagation of western white pine. Can J For Res 30(12): 1867–1876. Pitkänen LT, Heiskala M & Andersson LC (2001) Expression of a novel human ornithine

decarboxylase-like protein in the central nervous system and testes. Biochem Biophys Res Commun 287: 1051–1057.

Proskuryakov SY, Konoplyannikov AG & Gabai VL (2003) Necrosis: a specific form of programmed cell death? Exp Cell Res 283: 1–16.

Pulkkanen KJ, Laukkanen MO, Naarala J & Ylä-Herttuala S (2000) False-positive apoptosis signal in mouse kidney and liver detected with TUNEL assay. Apoptosis 5: 329–333.

Qiu YL & Palmer JD (1999) Phylogeny of early land plants: insights from genes and genomes. Trends Plant Sci 4: 26–30.

Quan L, Zhang B, Shi W & Li H (2008) Hydrogen peroxide in plants: a versatile molecule of the reactive oxygen species network. J Integr Plant Biol 50: 2–18.

Rea G, de Pinto MC, Tavazza R, Biondi S, Gobbi V, Ferrante P, De Gara L, Federico R, Angelini R & Tavladoraki P (2004) Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants. Plant Physiol 134: 1414–1426.

Regunathan S & Reis DJ (2000) Characterization of arginine decarboxylase in rat brain and liver: Distinction from ornithine decarboxylase. J Neurochem 74: 2201–2208.

Rodríguez-Kessler M, Delgado-Sanchez P, Theresia Rodriguez-Kessler G, Moriguchi T & Francisco Jimenez-Bremont J (2010) Genomic organization of plant aminopropyl transferases. Plant Physiol Bioch 48: 574–590.

Rose A, Patel S & Meier I (2004) The plant nuclear envelope. Planta 218: 327–336. Ryerson DE & Heath MC (1996) Cleavage of nuclear DNA into oligonucleosomal

fragments during cell death induced by fungal infection or by abiotic treatments. Plant Cell 8: 393–402.

Sahlén K (1992) Anatomical and physiological ripening of Pinus sylvestris L. seeds in northern Fennoscandia. Doctoral dissertation thesis. Umeå, Sveriges Lantbruksuniversitet.

Sanmartín C, Echeverria M, Mendivil B, Cordeu L, Cubedo E, Garcia-Foncillas J, Font M & Palop JA (2005) Synthesis and biological evaluation of new symmetrical derivatives as cytotoxic agents and apoptosis inducers. Bioorg Med Chem 13: 2031–2044.

Sannazzaro AI, Alvarez CL, Menendez AB, Pieckenstain FL, Alberto EO & Ruiz OA (2004) Ornithine and arginine decarboxylase activities and effect of some polyamine biosynthesis inhibitors on Gigaspora rosea germinating spores. FEMS Microbiol Lett 230: 115–121.

64

Santanen A & Simola LK (1992) Changes in polyamine metabolism during somatic embryogenesis in Picea abies. J Plant Physiol 140: 475–480.

Sarjala T, Haggman H & Aronen T (1997) Effect of exogenous polyamines and inhibitors of polyamine biosynthesis on growth and free polyamine contents of embryogenic Scots pine callus. J Plant Physiol 150: 597–602.

Sarjala T & Kaunisto S (1993) Needle polyamine concentrations and potassium nutrition in Scots pine. Tree Physiol 13: 87–96.

Sarvas R (1962) Investigations on the flowering and seed crop of Pinus silvestris. Comm Inst Forest Fenn 53: 1–198.

Savonen EM (1999) An improvement to the topographic tetrazolium testing of Scots pine (Pinus sylvestris L.) seeds. Seed Sci Techn 27: 49–57.

Schmidt S, Sunyaev S, Bork P, Dandekar T (2003) Metabolites: a helping hand for pathway evolution. Trends Bioch Sci 26: 336–341.

Scovassi AI & Torriglia A (2003) Activation of DNA-degrading enzymes during apoptosis. Eur J Histochem 47: 185–194.

Seiler N (2004) Catabolism of polyamines. Amino Acids 26(3): 217–233. Seiler N & Raul F (2005) Polyamines and apoptosis. J Cell Mol Med 9: 623–642. Shah R, Coleman CS, Mir K, Baldwin J, Van Etten JL, Grishin NV, Pegg AE, Stanley BA

& Phillips MA (2004) Paramecium bursaria chlorella virus-1 encodes an unusual arginine decarboxylase that is a close homolog of eukaryotic ornithine decarboxylases. J Biol Chem 279: 35760–35767.

Shaw FL, Elliott KA, Kinch LN, Fuell C, Phillips MA & Michael AJ (2010) Evolution and multifarious horizontal transfer of an alternative biosynthetic pathway for the alternative polyamine sym-homospermidine. J Biol Chem 285: 14711–14723.

Singh H (1978) Embryology of gymnosperms. Berlin, Borntrager. Sirois L, Begin Y & Parent J (1999) Female gametophyte and embryo development of

black spruce along a shore-hinterland climatic gradient of a recently created reservoir, northern Quebec. Can J Bot 77: 61–69.

Slocum RD & Furey MJ (1991) Electron-microscopic cytochemical localization of diamine and polyamine oxidases in pea and maize tissues. Planta 183: 443–450.

Stasolla C, Bozhkov PV, Chu TM, Van Zyl L, Egertsdotter U, Suarez MF, Craig D, Wolfinger RD, Von Arnold S & Sederoff RR (2004) Variation in transcript abundance during somatic embryogenesis in gymnosperms Tree Physiol 24: 1073–1085.

Tai HH, Pelletier C & Beardmore T (2004) Total RNA isolation from Picea mariana dry seed. Plant Mol Biol Rep 22: 93a-93e.

Tang W, Guo ZC & Ouyang F (2001) Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos. In Vitro Cell Dev-Pl 37: 558–563.

Tassoni A, van Buuren M, Franceschetti M, Fornale S & Bagni N (2000) Polyamine content and metabolism in Arabidopsis thaliana and effect of spermidine on plant development. Plant Physiol Bioch 38: 383–393.

65

Tavladoraki P, Rossi MN, Saccuti G, Perez-Amador MA, Polticelli F, Angelini R & Federico R (2006) Heterologous expression and biochemical characterization of a polyamine oxidase from Arabidopsis involved in polyamine back conversion. Plant Physiol 141: 1519–1532.

Terano S & Suzuki Y (1978) Formation of beta-alanine from spermine and spermidine in maize shoots. Phytochemistry 17: 148–149.

Terui Y, Ohnuma M, Hiraga K, Kawashima E & Oshima T (2005) Stabilization of nucleic acids by unusual polyamines produced by an extreme thermophile, Thermus thermophilus. Biochem J 388: 427–433.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F & Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res 25: 4876–4882.

Tiburcio AF, Altabella T, Borrell A & Masgrau C (1997) Polyamine metabolism and its regulation. Physiol Plantarum 100: 664–674.

Tillman-Sutela E & Kauppi A (1995) The significance of structure for imbibition in seeds of the Norway spruce, Picea abies (L) Karst. Trees Struct Funct 9: 269–278.

Ungru A, Nowack MK, Reymond M, Shirzadi R, Kumar M, Biewers S, Grini PE & Schnittger A (2008) Natural variation in the degree of autonomous endosperm formation reveals independence and constraints of embryo growth during seed development in Arabidopsis thaliana. Genetics 179: 829–841.

Urano K, Hobo T & Shinozaki K (2005) Arabidopsis ADC genes involved in polyamine biosynthesis are essential for seed development. FEBS Lett 579: 1557–1564.

Urano K, Yoshiba Y, Nanjo T, Igarashi Y, Seki M, Sekiguchi F, Yamaguchi-Shinozaki K & Shinozaki K (2003) Characterization of Arabidopsis genes involved in biosynthesis of polyamines in abiotic stress responses and developmental stages. Plant Cell Env 26: 1917–1926.

Van Breusegem F & Dat JF (2006) Reactive oxygen species in plant cell death. Plant Physiol 141: 384–390.

Vianello A, Zancani M, Peresson C, Petrussa E, Casolo V, Krajnakova J, Patui S, Braidot E & Macri F (2007) Plant mitochondrial pathway leading to programmed cell death. Physiol Plantarum 129: 242–252.

Vuosku J, Sutela S, Tillman-Sutela E, Kauppi A, Jokela A, Sarjala T & Häggman H (2009) Pine embryogenesis - many licences to kill for a new life. Plant Sign Behav 4: 928–932.

Vuosku J, Jaakola L, Jokipii S, Karppinen K, Kämäräinen T, Pelkonen VP, Jokela A, Sarjala T, Hohtola A & Häggman H (2004) Does extraction of DNA and RNA by magnetic fishing work for diverse plant species? Mol Biotechnol 27: 209–215.

Wachowiak W, Salmela MJ, Ennos RA, Iason G & Cavers S (2010) High genetic diversity at the extreme range edge: nucleotide variation at nuclear loci in Scots pine (Pinus sylvestris L.) in Scotland. Heredity advance online publication.

Wallace HM, Fraser AV & Hughes A (2003) A perspective of polyamine metabolism. Biochem J 376: 1–14.

66

Wang H, Li J, Bostock RM & Gilchrist DG (1996) Apoptosis: a functional paradigm for programmed plant cell death induced by host-selective phytotoxin and invoked during development. Plant Cell 8: 375–391.

Wang CW & Klionsky DJ (2003) The molecular mechanism of autophagy. Mol Med 9: 65–76.

Wolf BB & Green DR (1999) Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 274: 20049–20052.

Woltering EJ (2004) Death proteases come alive. Trends Plant Sci 9: 469–472. Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with

endogenous endonuclease activation. Nature 284: 555–556. Yadav JS & Rajam MV (1998) Temporal regulation of somatic embryogenesis by

adjusting cellular polyamine content in eggplant. Plant Physiol 116: 617–625. Yeung EC & Meinke DW (1993) Embryogenesis in angiosperms - development of the

suspensor. Plant Cell 5: 1371–1381. Yoda H, Yamaguchi Y & Sano H (2003) Induction of hypersensitive cell death by

hydrogen peroxide produced through polyamine degradation in tobacco plants. Plant Physiol 132: 1973–1981.

Yorimitsu T & Klionsky DJ (2005) Autophagy: molecular machinery for self-eating. Cell Death Differ 12: 1542–1552.

Young ID, Ailles L, Deugau K & Kisilevsky R (1991) Transcription of cRNA for in situ hybridization from polymerase chain reaction-amplified DNA. Lab Invest 64: 709–712.

Young TE, Gallie DR & DeMason DA (1997) Ethylene-mediated programmed cell death during maize endosperm development of wild-type and shrunken2 genotypes. Plant Physiol 115: 737–751.

Zhivotovsky B (2002) From the nematode and mammals back to the pine tree: on the diversity and evolution of programmed cell death. Cell Death Differ 9: 867–869.

Zhu MY, Iyo A, Piletz JE & Regunathan S (2004) Expression of human arginine decarboxylase, the biosynthetic enzyme for agmatine. Biochim Biophys Acta Gen Subj 1670: 156–164.

Ziegler U & Groscurth P (2004) Morphological features of cell death. News Physiol Sci 19: 124–128.

Zong WX & Thompson CB (2006) Necrotic death as a cell fate. Genes Dev 20: 1–15.

67

Original publications

I Vuosku J, Suokas M, Kestilä J, Läärä E, Sarjala T, Savolainen O, Häggman H (2011) Diverse evolution of polyamine biosynthesis and catabolism pathway genes in plants. Manuscript.

II Vuosku J, Jokela A, Läärä E, Sääskilahti M, Muilu R, Sutela S, Altabella T, Sarjala T, Häggman H (2006) Consistency of polyamine profiles and expression of arginine decarboxylase in mitosis during zygotic embryogenesis of Scots pine. Plant Physiology 142: 1027–1038.

III Vuosku J, Sarjala T, Jokela A, Sutela S, Sääskilahti M, Suorsa M, Läärä E, Häggman H (2009) One tissue, two fates: Different roles of megagametophyte cells during Scots pine embryogenesis. Journal of Experimental Botany 60(4):1375–1386.

IV Vuosku J, Sutela S, Sääskilahti M, Kestilä J, Jokela A, Sarjala T, Häggman H (2010) Dealing with the problem of non-specific in situ mRNA hybridization signals associated with plant tissues undergoing programmed cell death. Plant Methods 6(7).

Reprinted with permission from American Society of Plant Biologists (II), Oxford

Journals (III) and BioMed Central (IV).

Original publications are not included in the electronic version of the dissertation.

68

A C T A U N I V E R S I T A T I S O U L U E N S I S

Book orders:Granum: Virtual book storehttp://granum.uta.fi/granum/

S E R I E S A S C I E N T I A E R E R U M N A T U R A L I U M

557. Piippo, Sari (2010) Grazing tolerance of biennial meadow plants in relation toresource availability

558. Lappalainen, Niina (2010) The responses of ectohydric and endohydric mossesunder ambient and enhanced ultraviolet radiation

559. Luojus, Satu (2010) From a momentary experience to a lasting one : the conceptof and research on expanded user experience of mobile devices

560. Siirtola, Antti (2010) Algorithmic multiparameterised verification of safetyproperties : process algebraic approach

561. Lappi, Anna-Kaisa (2010) Mechanisms of protein disulphide isomerase catalyzeddisulphide bond formation

562. Sarala, Marian (2010) Elongation of Scots pine seedlings under blue light depletion

563. Vance, Anthony (2010) Why do employees violate is security policies? : insightsfrom multiple theoretical perspectives

564. Karppinen, Katja (2010) Biosynthesis of hypericins and hyperforins in Hypericumperforatum L. (St. John’s wort) – precursors and genes involved

565. Louhi, Pauliina (2010) Responses of brown trout and benthic invertebrates tocatchment-scale disturbance and in-stream restoration measures in boreal riversystems

566. Hekkala, Riitta (2011) The many facets of an inter-organisational informationsystem project as perceived by the actors

567. Niittyvuopio, Anne (2011) Adaptation to northern conditions at flowering timegenes in Arabidopsis lyrata and Arabidopsis thaliana

568. Leppälä, Johanna (2011) The genetic basis of incipient speciation in Arabidopsislyrata

569. Kivelä, Sami, Mikael (2011) Evolution of insect life histories in relation to timeconstraints in seasonal environments : polymorphism and clinal variation

570. Kaartinen, Salla (2011) Space use and habitat selection of the wolf (Canis lupus) inhuman-altered environment in Finland

571. Hilli, Sari (2011) Carbon fractions and stocks in organic layers in boreal forestsoils—impacts of climatic and nutritional conditions

572. Jokipii-Lukkari, Soile (2011) Endogenous haemoglobins and heterologousVitreoscilla haemoglobin in hybrid aspen

ABCDEFG

UNIVERS ITY OF OULU P.O.B . 7500 F I -90014 UNIVERS ITY OF OULU F INLAND

A C T A U N I V E R S I T A T I S O U L U E N S I S

S E R I E S E D I T O R S

SCIENTIAE RERUM NATURALIUM

HUMANIORA

TECHNICA

MEDICA

SCIENTIAE RERUM SOCIALIUM

SCRIPTA ACADEMICA

OECONOMICA

EDITOR IN CHIEF

PUBLICATIONS EDITOR

Senior Assistant Jorma Arhippainen

Lecturer Santeri Palviainen

Professor Hannu Heusala

Professor Olli Vuolteenaho

Senior Researcher Eila Estola

Director Sinikka Eskelinen

Professor Jari Juga

Professor Olli Vuolteenaho

Publications Editor Kirsti Nurkkala

ISBN 978-951-42-9435-8 (Paperback)ISBN 978-951-42-9436-5 (PDF)ISSN 0355-3191 (Print)ISSN 1796-220X (Online)

U N I V E R S I TAT I S O U L U E N S I SACTAA

SCIENTIAE RERUM NATURALIUM

U N I V E R S I TAT I S O U L U E N S I SACTAA

SCIENTIAE RERUM NATURALIUM

OULU 2011

A 573

Jaana Vuosku

A MATTER OF LIFE AND DEATH – POLYAMINE METABOLISM DURING ZYGOTIC EMBRYOGENESIS OF PINE

UNIVERSITY OF OULU,FACULTY OF SCIENCE,DEPARTMENT OF BIOLOGY

A 573

ACTA

Jaana Vuosku