Programmed Cell Death in Xylem Development

Charleen Courtois-Moreau

Umeå Plant Science Centre Department of Plant Physiology Umeå University Sweden 2008

ISBN 978-91-7264-640-7

Printed by Solfjädern Offset AB, Umeå 2008 Cover illustration: Julien Courtois Distribution: UPSC – Department of Plant Physiology Umeå University, SE-901 87 UMEÅ, Sweden. Tel: +46 90-786 5000 E-mail: [email protected]

“Mortidebetur, quicquid usquam nascitur” in Sentences Publius Syrus (First Century BC)

[Everything brought to life, owes tribute to the Death]

“Life is not easy for any of us. But what of that? We must have perseverance and above all confidence in ourselves. We must believe that we are gifted for something and that this thing must be attained.” Marie Curie (1867-1934) Two Nobel Prizes in the area of physics and chemistry.

iii Programmed Cell Death in Xylem Development

Abstract: Concerns about climate changes and scarcity of fossil fuels are rising. Hence wood is becoming an attractive source of renewable energy and raw material and these new dimensions have prompted increasing interest in wood formation in trees, in both the scientific community and wider public. In this thesis, the focus is on a key process in wood development: programmed cell death (PCD) in the development of xylem elements. Since secondary cell wall formation is dependent, inter alia, upon the life time of xylem elements, the qualitative features of wood will be affected by PCD in xylem, about which there is little information.

This thesis focuses on the anatomical, morphological and transcriptional features of PCD during xylem development in both the stem of hybrid aspen, Populus tremula (L.) x tremuloides (Michx.) and the hypocotyl of the herbaceous model system Arabidopsis thaliana (L. Heynh.). In Populus, the progressive removal of organelles from the cytoplasm before the time of death (vacuolar bursts) and the slowness of the cell death process, illustrated by DNA fragmentation assays (such as TUNEL and Comet assays), have been ascertained in the xylem fibres by microscopic analyses. Furthermore, candidate genes for the regulation of fibre cell death were identified either from a Populus EST library obtained from woody tissues undergoing fibre cell death or from microarray experiments in Populus stem, and further assessed in an in silico comparative transcriptomic analysis of Arabidopsis thaliana. These candidate genes were either putative novel regulators of fibre cell death or members of previously described families of cell death-related genes, such as autophagy-related genes. The induction of the latter and the previous microscopic observations suggest the importance of autophagy in the degradation of the cytoplasmic contents specifically in the xylem fibres. Vacuolar bursts in the vessels were the only previously described triggers of PCD in the xylem, which induce the very rapid degradation of the nuclei and surrounding cytoplasmic contents, therefore unravelling a unique previously unrecorded type of PCD in the xylem fibres, principally involving autophagy. Arabidopsis is an attractive alternative model plant for exploring some aspects of wood formation, such as the characterisation of negative regulators of PCD. Therefore, the anatomy of Arabidopsis hypocotyls was also investigated and the ACAULIS5 (ACL5) gene, encoding an enzyme involved in polyamine biosynthesis, was identified as a key regulator of xylem specification, specifically in the vessel elements, though its negative effect on the cell death process.

Taken together, PCD in xylem development seems to be a highly specific process, involving unique cell death morphology and molecular machinery. In addition, the technical challenges posed by the complexity of the woody tissues examined highlighted the need for specific methods for assessing PCD and related phenomena in wood.

Keywords: PCD, Xylem, Apoptosis, Autophagy, Secondary Cell Walls, Microscopy, Microarrays, Comet Assay, TUNEL Assay.

Programmerad celldöd i xylemutvekling

Sammanfattning: Oron för klimatförändringar och brist på fossila bränslen har ökat påtagligt under de senaste åren. De enorma möjligheter som skogsråvaran erbjuder som alternativ källa för förnyelsebar energi och råmaterial har väckt ett stort intresse också för den biologiska processen bakom vedbildning i träd. Denna avhandling fokuserar på en viktig process i vedbildning: programmerad celldöd (PCD) i xylemet. Xylemcellernas livstid påverkar bildningen av sekundära cellväggar, vilket i sin tur påverkar vedens kvalitativa egenskaperna, så som veddensitet. Trots dess betydelse för viktiga egenskaper hos vedråvaran existerar fortfarande väldigt lite information om xylem PCD på cellulär eller molekylär nivå.

I den här avhandlingen belyses de anatomiska, morfologiska och genetiska aspekterna av PCD i xylemutveckling i både stam av hybridasp, Populus tremula (L.) x tremuloides (Michx.) och hypokotyl av det örtartade modellsystemet Arabidopsis thaliana (L. Heynh.). Xylemet i både Populus och Arabidopsis består av två olika celltyper; de vattentransporterade kärlen och de stödjande fibrerna. Det är känt att celldöd i kärlen pågår mycket snabbt efter att den centrala vakuolen brister och de hydrolytiska enzymer släpps in i cytoplasman. I den här avhandlingen ligger fokus på fibrerna i Populus xylemet. Med hjälp av mikroskopianalyser av cellmorfologin (elektronmikroskopi) och DNA-fragmentering i cellkärnan (TUNEL- och Comet-analyser) kunde vi konstatera att till skillnad från kärlen så uppvisar fibrerna en långsam och progressiv nedbrytning av organellerna och cellkärnans DNA före vakuolbristning. Dessutom har kandidatgener för reglering av fibercelldöd identifierats antingen från ett Populus EST bibliotek från vedartade vävnader som genomgår fibercelldöd eller från mikroarray experiment i Populus stam. Dessa kandidatgener är antingen potentiella nya regulatorer av fiber- celldöd eller medlemmar av tidigare beskrivna familjer av celldödsrelaterade gener. Bland de sistnämnda finns autofagi-relaterade gener, vilket stöder funktionen av autofagi i samband med autolys av cellinnehållet i xylemfibrerna. Dessa studier pekar därför på en typ av PCD som har inte tidigare beskrivits för xylemet. Arabidopsis är ett alternativt växtmodellsystem för studier av vissa aspekter av vedbildningen, såsom karakte- riseringen av negativa regulatorer av PCD. Därför har också hypokotylanatomin ana- lyserats, och ACAULIS5 (ACL5) genen, som kodar för ett enzym i biosyntesen av polyaminer, har visats vara en viktig regulator av xylemspecifikation genom dess negativa effekt på kärlens celldöd.

Sammantaget visar denna avhandling att PCD i xylemutvecklingen verkar involvera unika morfologiska och molekylära mekanismer. Vi visar dessutom att komplexiteten hos de vedartade vävnaderna leder till ett behov av bättre anpassade verktyg för att djupare kunna bedöma PCD och liknande fenomen i veden.

In memory of my Dad

To Mum, Gérard and Antoine To Thorkel To my Lapin

Preface

The processes behind cell division and multiplication as in stem cells or meristematic regions are widely studied in both animal and plant kingdoms. Already at the early embryo stage, the interest of the scientific community for cell development is enormous. The understanding of how a single cell may become such an evolved organism as what we look like today is without any doubt very fascinating. Looking deeper into plant research, meristems receive a lot of attention and therefore tremendous number of publications is dedicated to the sound understanding of cell multiplication. Concerning cell death, the interest appears to strongly vary between the two kingdoms. Understanding cell death in animals would bring key answers for curing major pathologies, such as cancers, Alzheimer or sclerosis. Looking at the number of scientific journals focusing on different aspects of cell death in animal kingdom: more than a hundred journals so far on cancer research such as Cancer, Cancer Cell, Cancer BMC, journals on sclerosis, on Alzheimer disease, or more generally on cell death such as Cell Death and Differentiation, it illustrates that scientists certainly perceive cell death as an essential domain requiring further studies. Moreover, the occurrence of a reference journal such as Cell Death and Differentiation reflects upon the increasing importance to consider cell death together with cell differentiation, as one does not exist without the other, i.e. life does not exist without death and vice-versa. In plants, the situation has been revealed to be quite different as few scientists seem to consider cell death as a pillar to life itself so far. However should we be remembered that a tree (inter alia) can only exist because early during its development some cells are programmed to die to be able to transport water from the roots to the top, or that in dynamic population biology, death is the key to adaptation of populations to their changing environment? Even though it is certainly as important for plant survival as it is for animal survival, cell death in plant is still lacking its own scientific peer-reviewed journals.

These five years of Doctoral Thesis brought certainly little more understanding on the cell death process occurring during wood formation in poplar. Unfortunately working with trees is much more time consuming than working with bacteria, for example, and mistakes can represent months of time loss, making those five years too short in the end. Furthermore this very last year has been quite of a fight as writing down about this subject when being far from my scientific community and quite on my own made it very tough to focus sometimes. One may even wonder how I could still think about death while I was home with the Flesh of my Flesh: my couple of months old baby. Well, it was not until I delivered that I realized that Death was part of Life. That my son has become what he is today because already as embryo some cells were programmed to die and this programmed cell death was for his own good. That is

vii why cell death is certainly a passionate subject to understand life itself. In this Doctoral Thesis, I will probably summarize too much all the knowledge acquired so far on cell death and programmed cell death, but that is to make it understandable to any of you willing to enter a journey towards death in the wood or xylem of mainly poplar.

Charleen Courtois

viii

This thesis is based on the following papers, which are referred in the text by their corresponding Roman numerals:

I. A Genomic Approach to Investigate Developmental Cell Death in Woody Tissues of Populus Trees Charleen Moreau, Nikolay Aksenov, Maribel García-Lorenzo, Bo Segerman, Christiane Funk, Stefan Jansson and Hannele Tuominen Genome Biology, 2005, 6, R34. The author was responsible for most of the experimental work and partly for writing the paper.

II. ACAULIS 5 Controls Arabidopsis Xylem Specification through the Prevention of Premature Cell Death Luis Muñiz, Eugenio G. Minguet, Sunil Kumar Singh, Edouard Pesquet, Francisco Vera-Sirera, Charleen Courtois-Moreau, Juan Carbonell, Miguel Blazquez and Hannele Tuominen Development, 2008, 135, 2573-2582. The author was responsible for some of the experimental work.

III. A Unique Programme for Cell Death in Xylem Fibers of Populus Stem Charleen Courtois-Moreau, Andreas Sjödin, Edouard Pesquet, Luis Muñiz, Benjamin Bollhöner, Minako Kaneda, Lacey Samuels, Stefan Jansson and Hannele Tuominen Submitted to Plant Journal, 2008. The author was responsible for most of the experimental work and partly writing the paper.

Not included in this thesis: Ethylene, the Triggering Signal of Tracheary Element Programmed Cell Death and Lignification Edouard Pesquet, Tuula Puhakainen, Olivier Keech, Charleen Courtois-Moreau, Per Gardeström, Jaakko Kangasjärvi and Hannele Tuominen Manuscript. The author was responsible for part of the experimental work.

Paper I is reprinted with permission from BioMed Central Ltd (Copyright 1999-2008). Paper II is reprinted with permission from The Company of Biologists Ltd (Copyright 2008). Paper III has been formatted in accordance with the journal’s preferences.

ix List of Abbreviations

Enzymes, other proteins and chemicals ABA Abscisic Acid AOS Allene Oxide Synthase AOX Alternative Oxidase Apaf-1 Apoptotic Protease Activating Factor 1 ASA Acetylsalicylic Acid BI-1 Bax-Inhibitor 1 Caspases Cysteine-Dependent Aspartate-Directed Proteases CD95 TNF Receptor Fas COX Cyclooxygenase DAPI 4’,6’-diamidino-2-phenylindole hydrochloride FITC Fluorescein isothiocyanate H2O2 Hydrogen Peroxide IAA* Isoamylalcohol IAA** Indole-3-acetic acid IAP Inhibitor of Apoptosis Protein MAP-1 Modulator of Apoptosis-1 NO Nitric Oxide PI Propidium iodide PRX Peroxiredoxins PtdSer Phosphatidylserine p48h-17 Zinnia Cysteine Protease SA Salicylic Acid SOD Superoxide Dismutase TED Tracheary Element Differentiation-related TRX Thioredoxins ZCP4 Zinnia Cysteine Protease 4 ZEN1 Zinnia Endonuclease 1 ZRNase Zinnia RNase Genes ACD Accelerated Cell Death CLV3 Clavata3 EGL-1 Egg Laying Defective 1 HIN1 Haipin-Induced Gene HSR203J Hypersensitivity-Related Gene LLS1 Lethal Leaf Spot 1 Gene MLO Mildew-resistance Locus O Gene NAHG SA-degrading Salicylate Hydroxylase Gene NPR1 Nonexpressor of PR1 RLMcol Resistance to Leptosphaeria maculans in the Col-0 Background Locus RP1 Resistance to maize common rust (Puccinia sorghi) 1

SAG Senescence-Associated Gene XCP Xylem Cysteine Peptidase Miscellaneous ER Endoplasmic Reticulum EST Expressed Sequence Tag FCC Fusiform Cambial Cells HPF(-FS) High Pressure Freezing (with Freeze-Substitution) HR Hypersensitive Reaction or Response KOG euKaryotic Ontology of Genes Mb Megabases NCBI the National Center for Biotechnology Information PCD Programmed Cell Death PCR Polymerase Chain Reaction QTL Quantitative Trait Loci RCC Ray Cambial Cell ROS/ROI Reactive Oxygen Species/Intermediates RT-PCR Reverse Transcriptase Polymerase Chain Reaction TE Tracheary Elements TEM Transmission Electron Microscopy TF Transcription Factors TNF Tumor Necrosis Factor

xi Table of Contents

1. A Tree Species: Populus tremula (L.) x tremuloides (Michx.) ...... 1 1.1. Trees: increasing interest ...... 1 1.2. Populus: an ideal model for trees...... 2 2. Xylogenesis – Secondary xylem formation...... 2 2.1. Introduction to xylogenesis in trees ...... 2 2.2. Various models for xylogenesis...... 4 2.2.1 Transdifferentiation of tracheary elements ...... 4 TE differentiation in Zinnia: an example ...... 4 2.2.2 Signalling during vascular differentiation ...... 5 Calcium/Calmodulin ...... 5 Dodeca-CLE peptide ...... 6 Arabinogalactan protein ...... 6 2.2.3 Downstream components of signalling in differentiation...... 7 2.3. The Populus tree: a better model for xylogenesis...... 8 2.4. Specific strategies with Populus ...... 8 2.4.1 The secondary cell wall: the bane of tree biologists ...... 8 2.4.2 Chemical fixation vs. cryo-fixation ...... 10 2.4.3 Transmission electron microscopy ...... 12 3. Programmed cell death...... 12 3.1. What is cell death? ...... 12 3.2. Programmed cell death in metazoans...... 13 3.2.1 Summary of animal programmed cell death...... 13 3.2.2 Plasma membrane modifications in PCD ...... 14 3.2.3 Cellular compounds in PCD ...... 15 The mitochondria in PCD...... 15 Apoptosis: caspase cascade or not?...... 15 3.3. Programmed cell death in plants...... 17 3.3.1 PCD in the whole plant...... 17 3.3.2 Metacaspases in plants...... 18 3.3.3 The vacuole: a growing organelle in PCD...... 19 3.3.4 A new concept in plant PCD: the Degradome ...... 20 3.3.5 Inhibitors of cell death ...... 21 3.4. The hypersensitive response: an example of stress-induced programmed cell death specific to plants ...... 21 3.4.1 The hypersensitive response (HR)...... 21 3.4.2 HR cell death: a programmed process ...... 22 Characterization of lesion-mimic mutants...... 22 Characterization of the HR markers...... 22 3.4.3 Triggers of HR cell death...... 23

xii

Reactive oxygen intermediates/species (ROI/ROS)...... 23 ROS/ROI scavengers...... 23 Nitric oxide and salicylates: HR cell death amplifiers ...... 25 3.4.4 Phytohormones: ambivalent roles in HR-like cell death ...... 25 3.4.5 Animal apoptosis vs. Plant PCD: “same same but different”? ...... 26 4. Xylem programmed cell death ...... 29 4.1. PhD on programmed cell death in xylem...... 29 4.2. Bioinformatics: the essential starting point for gene browsing...... 29 4.2.1 Bioinformatics: what is it?...... 29 4.2.2 Bioinformatic tools to unravel PCD in trees...... 30 Microarray technologies...... 30 Databases for Populus...... 31 4.3. Programmed cell death characterization in Populus: new challenges .... 34 4.3.1 Wood microsectioning approaches...... 34 4.3.2 Anatomical characterizations...... 34 4.3.3 TUNEL assays ...... 35 4.3.4 Single cell gel electrophoresis or Comet assays ...... 37 4.3.5 DNA staining with DAPI/PI ...... 38 4.3.6 DNA laddering...... 39 4.3.7 Use of markers...... 40 5. New insights into xylem developmental cell death...... 41 Populus vs. Arabidopsis: two models, one aim...... 41 Organelle disappearance before tonoplast rupture in fibres ...... 41 In silico isolation of potential novel fibre cell death markers ...... 41 ACL5: vessel cell death inhibitor and fibre development enhancer...... 42 6. Future perspectives for PCD in woody plants...... 42 7. Acknowledgments...... 44 8. Literature Cited...... 47

xiii xiv 1. A Tree Species: Populus tremula (L.) x tremuloides (Michx.)

1. A Tree Species: Populus tremula (L.) x tremuloides (Michx.)

1.1. Trees: increasing interest The choice of the model system is crucial in any biological studies. In plants, herbaceous models, including Arabidopsis and Zinnia, have some major advantages, such as their small genomes and physical sizes, large available databases (including mutant collections), easy transformation and short generation times. For instance, their life cycles generally span a few months, while those of trees mostly span years. Moreover, when working with trees there are generally serious complications related (inter alia) to their size, interfering compounds, complex, three-dimensional secondary vascular system and long generation times.1 Nevertheless, there are several sound reasons for choosing to work on trees.2 Firstly, they are affected by seasonal and other long-term changes that do not generally affect small annual plants. Secondly, unlike herbaceous model plants they are sources of economically important timber and other wood-based products, ranging from pulp and paper through to renewable energy (which is becoming increasingly important as fossil fuels are becoming increasingly scarce globally). Why putting efforts in working on trees then? In contrast to herbaceous models, they have a stronger economical impact as a source for various wood- based products3, ranging from timber to pulp and paper derivatives, and renewable energy, a non-negligible use in an international petroleum crisis. For these reasons, we need to understand phenomena specifically related to woody plants, e.g. dormancy, wood and secondary cell wall formation and the subjects covered in this thesis. For some years now, work on pine (Pinus) and spruce (Picea) has been intensifying, even though no complete genome sequences for these genera are available as yet. Many such investigations have focused on secondary cell wall formation4, 5 but some have also addressed processes that involve PCD, e.g. embryogenesis6, 7. Moreover, to avoid the long generation times and slow growth limitations of trees, Arabidopsis was suggested as a model for wood formation8 because in later phases of development its hypocotyl anatomically resembles the wood of angiosperm trees such as poplar. However, the suitability of Arabidopsis as a universal model can be disputed, since it is has little resemblance to any tree species and it is not subject to many of the environmental challenges that large, long-lived plant species face. Thus, since plants evolve in diverse ways when adapting to various environments9, 10 there was a need to identify an alternative model woody species to address questions related to wood formation and other issues that could not be readily answered using small annual plants.

1 1 Programmed Cell Death in Xylem Development

1.2. Populus: an ideal model for trees The Populus genus represents about 30 geographically highly-spread species (the poplars, aspens and cottonwoods).11 Interest in this genus has been growing during the last 20 years, because of its fast growth and the suitability of its wood for both bioenergy production and manufacturing many high-quality wood and paper products. A consortium, originally initiated by the U.S. Department of Energy, was created in 2002 by several universities in various countries in the northern hemisphere to sequence the Populus genome, more specifically that of black cottonwood (Populus trichocarpa Torr. & Gray)12. In parallel, the International Populus Genome Consortium (www.ornl.gov/ipgc) was funded to improve the coordi- nation of Populus research around the world.13 The choice of Populus was supported by its relatively modest genome size (485 ± 10 Mb), high availability of relevant molecular and genetic tools (such as QTL markers), ease of transformation, and relatively rapid growth (since it reaches maturity in about six years).2, 9, 12 Finally, in the context of sustainable development, black cottonwood is a particularly suitable source of renewable energy, enhancing its credentials as a model species. To date, about 45,000 protein-coding gene loci have been identified in the Populus nuclear genome (www.jgi.doe.gov/poplar), but this number will almost certainly continue to rise, and the majority of their predicted gene models show significant homology to all non-redundant proteins in the NCBI database. Interestingly, about 12 % of the Populus gene models have no similarity to the Arabidopsis protein-coding genes12, which emphasizes the genetic differences between trees and herbaceous species. Further, some at least of these genes are likely to play specific roles in wood-related processes. Thus, their characterization may provide indications about currently unknown aspects of wood formation.

2. Xylogenesis – Secondary xylem formation

2.1. Introduction to xylogenesis in trees During active growth periods of trees, the cambium – a secondary meristem – produces secondary xylem, or wood, under the influence of hormonal and external factors.11, 14-16 Generally, the cambium consists of: cambial initials, phloem mother cells (which are destined to form the phloem) and xylem mother cells (which are destined to form the xylem). Cambial initials undergo both periclinal and anticlinal divisions, allowing lateral cell proliferation and radial stem diameter growth. The axial and horizontal cell systems in the secondary

2 2. Xylogenesis – Secondary xylem formation xylem are due to the proliferation of two distinct types of cells: the axially elongate fusiform cambial cells (FCCs) and ray cambial cells (RCCs).11 After cambial proliferation, cells will either become phloem or xylem cells, mainly depending upon which side of the cambium they are located. Cells located towards the outer side of the stem or cortex will become part of the phloem core while those located towards the inner side of the stem or pith will become part of the xylem core (Figure 1). On the phloem side, FCCs evolve into sieve elements (sieve cells in gymnosperms, sieve-tube elements and companion cells in angiosperms), parenchymatic cells and phloem sclerenchyma (sclereids in gymnosperms; fibres in angiosperms). RCCs develop into phloem ray cells. In the xylem region, FCCs initiate tracheary elements (tracheids in both gymnosperms and angiosperms, and vessel elements in angiosperms), xylem fibres and sometimes parenchyma; while RCC initials give rise to xylem ray cells. Both rays and vessels serve as transporting ducts. However, rays carry solutes during their life from the outside toward the inside of the stem, while vessels function as transporters of water from the roots to the top of the plant, once they have died and their contents have completely autolysed. In addition, xylem fibres sometimes provide storage and more generally physical support, in growing stems by gradually depositing secondary cell walls, fortified by processes such as lignification, before dying. Thus the different stages of xylogenesis in trees are of fundamental importance to the production of xylem, but without the subsequent death of those cells, xylem would not function optimally.

Figure 1. Illustration of anatomy and major phases of xylogenesis in a hybrid aspen tree (Populus tremula x tremuloides). Adapted from Paper III. DIC image of transversal section highlighting the three major xylem cell types, with underneath the different phases of xylogenesis (fibre cell death corresponds to vacuole bursting). SCW: secondary cell walls. Bar: 50 μm.

3 1 Programmed Cell Death in Xylem Development

2.2. Various models for xylogenesis 2.2.1 Transdifferentiation of tracheary elements Conductive tissues in wood, i.e. sieve elements and tracheary elements (TEs) carry food and fluids, respectively, but only TEs deposit secondary cell walls and undergo cell death. The Zinnia cell culture system17 substantially improved the in-vitro study of isolated vessel-like structures (named TEs) since high rates of transdifferentiation of mesophyll cells into TEs can be induced by both mechanical wounding and auxin/cytokinin hormonal treatments.18 Recently, we also found that spermine, a polyamine (see below), influences TE differentiation in Zinnia (Paper II). Exogenous spermine delayed TE differentiation in the in vitro system, affected the type of TEs produced and increased their size in a concentration-dependent manner (Figure 6 in Paper II). In Arabidopsis plants, we studied the function of ACL5, which encodes a spermine or possibly thermospermine synthase (Figure 7). The acl5 mutant showed dramatic alterations in xylem maturation and development, including: premature vessel cell death; reductions in vessel size; an absence of elaborated vessel types, i.e. no pitted vessels; and a lack of xylary fibers (Figures 2, 4 and 7 in Paper II). Altogether, these findings indicate that spermine, synthesized via the action of the ACL5 gene product, is involved in the control of xylem development by regulating the life-span of vessels. TE differentiation in Zinnia: an example In the early stage of TE differentiation (stage I), the 24 to 36 hrs immediately following hormonal induction, isolated mesophyll cells lose their photosynthetic capacity19, but acquire multidifferentiation potency20. In stage II, in the following 24 hours, cells differentiate into TE precursors and several vascular differentiation marker genes are specifically expressed, including TED2, TED4 and TED3, the last of which is specifically expressed in differentiating TEs. Hydrolytic enzymes released into the extracellular space may affect the neighbouring cells. Accordingly, TED4 has been shown to accumulate in the apoplastic space before the release of hydrolases, providing protection to the surrounding living cells.21 In the late stage III, between 60 and 96 hrs after initiation, TEs are formed via the activity of proteinases and other enzymes that are expressed simultaneously with the synthesis of secondary cell wall thickenings and autolysis/PCD. These hydrolases, such as ZEN1, ZRNase, ZCP4 and p48h-17, accumulate in the vacuole before being released by rupture of the tonoplast, which rapidly induces death of the TEs. Thus, the transition from stage II to III seems critical for PCD induction in TEs.18 In spite of a strong cell wall, various morphological changes are typically observed in differentiating TEs, including the deposition of secondary cell walls,

4 2. Xylogenesis – Secondary xylem formation displaying four patterns (ring-like, spiral-like, reticulated or pitted22) until they are autolysed by cell death. Modification of organellar morphology also occurs during xylogenesis and subsequent PCD, i.e. after tonoplast rupture (Figure 2), suggesting that the organelles disintegrate shortly after the release of hydrolytic enzymes, which compromises the permeability of their membranes. The chloroplasts remain present for the longest time before complete digestion of the cell contents and the nucleus also shows slight resistance to enzyme attacks, first showing blebbing of its membranes before its total disruption.19 Generally, TE maturation occurs very quickly, taking about six hours to lose cell contents from the first visible thickening of secondary walls and only 15 min for the nucleus to become totally degraded after the vacuole ruptures.23

Figure 2. Morphological changes in Zinnia TEs during xylogenesis. After tonoplast rupture, cells undergo a PCD process including disappearance of the various organelles. Adapted from Fukuda.19 2.2.2 Signalling during vascular differentiation Calcium/Calmodulin Accumulation of calcium (Ca2+) is a characteristic trigger of apoptosis in animal cells. In plants, studies of the effects of Ca2+ on xylem differentiation are quite scarce. However, in Zinnia, Ca2+ has been shown to be mainly present in the intercellular space and cell walls during the early stage of TE differentiation; and its concentration has been shown to increase in the cytosol after vacuole rupture18, 24, probably as part of a mechanism, mediated by calmodulin and actin, that regulates secondary cell wall patterning. In pepper plants, variations in Ca2+ spatial distribution, which is initially located mainly in primary walls and later

5 1 Programmed Cell Death in Xylem Development mainly in secondary walls25, have also been observed. Additionally, Ca2+ influxes may participate in signalling of the vacuole rupture and the subsequent arrest in cytosolic streaming.26 More interestingly, a recent study demonstrated that exogenous applications of Ca2+ increase vessel size and fibre length, and triggers early secondary cell wall deposition in poplar27, suggesting a possible role of Ca2+ in enhancing signalling during xylem differentiation. Dodeca-CLE peptide Another group of signalling compounds involved in xylem development is the dodeca-CLE family of peptide ligands, where CLE stands for CLV3/ESR-related, and the genes encoding them include (inter alia) CLV3. An extracellular factor, TDIF, has been isolated from the Zinnia culture system that reportedly suppresses the differentiation of TE cambial cells into xylem cells.28 TDIF was later found to be homologous to some Arabidopsis CLE peptides, and three of the 31 Arabidopsis genes encoding CLE peptides were shown to produce strong TDIF activity, suggesting their involvement in xylem differentiation.28 In addition, CLV3, which binds to a putative leucine-rich repeat receptor kinase (CLV1), is able to either promote or inhibit stem cell proliferation in a concentration-dependent manner, suggesting that pathways mediated by CLE- signalling that have opposing effects are activated during stem cell differentiation in vascular development. Arabinogalactan protein In animal systems, the living state of a cell is tightly dependent upon specific molecular interactions between components of the plasma membrane and both its extra- and intra-cellular environments (Figure 5). Communication between cells also plays striking roles in plant development. Arabinogalactans (AGP) are putative candidate substrates for glycosyl hydrolase proteins. An AGP-domain containing protein, known as xylogen, a mediator of inductive cell-cell interaction and communication in vascular development, has been found to be involved in TE differentiation in Zinnia29, 30, by directionally acting on neighbouring cells. In addition, a double knock-out mutant for the two AGP- xylogen proteins known to be expressed in Arabidopsis showed defects in vascular development.29 Even before these studies, AGPs were known to be associated with cell death in maize coleoptile cells.31 In Populus, 15 fasciclin-like AGPs are expressed in differentiating xylem32, and in the studies reported in Paper I we identified, together with two other cell wall proteins, one highly abundant Populus AGP (POPLAR.862) specific to the fibre cell death library (the X library), that was downregulated in the tension wood library (see the paper for details of the libraries examined). Thus, if parts of the cell death pathway are shared by both animals and plants, it will also be important to further study cell- cell interactions and communication within the extracellular space in various types of plant PCD.

6 2. Xylogenesis – Secondary xylem formation

2.2.3 Downstream components of signalling in differentiation Proteolytic enzymes involved in the degradation of mature TE contents appear to be newly expressed proteases specific to the differentiating TEs.33 In Zinnia, such enzymes include two serine proteases and two cysteine proteases.33 More generally, differences in the action of these proteases depend on their optimum pH for activity, since most of the cysteine proteases specific to TEs have acidic pIs, indicating that they are probably localized in the vacuole while serine proteases seem to be more confined to neutral milieu such as the cytoplasm prior to vacuole collapse.18, 34 Cysteine proteinases (CPs), which include papain, are found in many different plant species35. For instance, the MEROPS database (www.merops.co.uk/) lists sequences of more than 50 homologs of papain from more than 26 different plant species. A cysteine proteinase isolated from brinjal xylem (SmCP) has been suggested to participate in autolysis during xylogenesis36, and three xylem- specific endopeptidases have been isolated from secondary xylem tissues of the Arabidopsis root-hypocotyl system: two putative papain-type cysteine proteases (XCP1 and XCP2) and one putative subtilisin-type serine protease (XSP1)37. In barley, a vacuolar cysteine protease (aleurain) and an aspartic protease (phytepsin, homologous to cathepsin D in animal cells), both of which are mainly expressed in TEs of proto- and early meta-xylem, have been identified.38 The action of phytepsins has been located to the vacuole and correlated with autolysis activation, suggesting that they participate in the autolysis of TEs.34, 38 Therefore, three main endopeptidase families – aspartic, cysteine and serine proteases – seem to play important roles in TE PCD. However in the study presented in Paper I we did not identify any aspartic proteases that are involved in fibre PCD, but not TE cell death, although we isolated serine and cysteine proteases (the latter homologous to XCP2) that appeared to be specifically expressed and strongly enriched, respectively, in the X library. In addition, a large number of proteases were detected in the late maturation sample, Mx3, suggesting they play specific roles in fibre cell death (Paper III). Nevertheless, searches in plants for proteases equivalent to animal caspases, which collectively form a class of cysteinyl aspartate-specific proteases that are involved in PCD, has remained unsuccessful. This is intriguing because caspase-like activities, which are suppressed by inhibitors of mammalian caspase-1 or -3, have often been detected in plants.39 Nucleases also show increased activity during PCD in TEs, and six of them have been shown to be induced specifically in TEs of Zinnia culture cells.40 Interestingly, one of them, a 43-kD Zn2+-dependent S1-type nuclease, designated ZEN1, appears to be expressed specifically in association with TE differentiation and has been designated the key DNase in the degradation of nuclear DNA during the PCD of tracheary elements.41 The S1-type nuclease genes are a small

7 1 Programmed Cell Death in Xylem Development family, represented by five genes in Arabidopsis, two in barley (Bnuc1 and BEN1)42 and three in Zinnia (ZEN1, ZEN2 and ZEN3)42 although only BEN1 and ZEN1 have been demonstrated to encode active DNases. In tomato, another type of nuclease, LX, is also involved in cell death processes, particularly senescence but, interestingly, this tomato ribonuclease seems to be localised in the endoplasmic reticulum.43 In Paper III, several nucleases were shown to be specifically upregulated in late maturing xylem, including an endo-/exonuclease and, most interestingly, a homolog of an apoptosis-inducing factor (AIF) molecule, which participates in chromatin condensation and DNA degradation in animal cells. Furthermore, these genes are possibly specific to fibres since novel types of nucleases are clearly required in fibres for degradation of their nuclear DNA in a unique manner, independently of vacuolar rupture. In addition, these genes are not expressed in the vessel-like TEs in Arabidopsis (Paper III).

2.3. The Populus tree: a better model for xylogenesis Although major scientific advances in understanding xylogenesis have been made using Zinnia cell cultures they have major limitation for this purpose since Zinnia is a non-woody plant, the systems remain in vitro, and they are composed of cells of a unique, differentiated type. Fibre maturation can also be studied using Arabidopsis, but this also has limitations due to its small size and short life cycle compared to trees. Furthermore, fibre PCD cannot be studied in Arabidopsis plants because their fibres do not die in a highly synchronized way. Thus, since in vivo applications of in vitro-based research are limited by in vitro systems’ lack of features such as cell-cell interactions, the identification of a model tree species with fully functional xylogenetic and cell death mechanisms was essential. However, working on such large organisms, which have evolved massive, complex structural features to support their growth, inevitably raises substantial challenges and the need to adapt techniques applied to trees.

2.4. Specific strategies with Populus 2.4.1 The secondary cell wall: the bane of tree biologists Xylogenesis involves secondary cell wall formation and cell death, often concomitantly, in both vessels and fibres. The microarray analysis described in Paper III revealed that expression of cellulose and lignin biosynthetic genes and some TFs involved in secondary cell wall formation were strongly induced in the two first samples of maturing xylem, simultaneously with the expression of TFs or proteases known to be involved in stress and/or cell death processes. This was supported by indications presented in Paper I that some of the most abundant transcripts in the fibre death library, e.g. transcripts encoding arabinogalactan

8 2. Xylogenesis – Secondary xylem formation proteins, were also present in other libraries, and thus play more general roles in xylem differentiation.

Figure 3. Schematic diagram of a high-pressure freezing system based on the BAL-TEC HPM 010 HPF model and accessories (modified from a diagram at http://www.bal- tec.com/). The sample, contained between two specimen-carriers filled with cryoprotectant, is locked in a specimen-holder (1). The holder is then introduced into the HPF machine, oriented towards the two liquid nitrogen (LN2) ports. After locking the holder (2), the rapid freezing process is activated. Pressurized oil is passed, via a complex system of pressure valves (9), through a cylinder (10), driving a piston into the LN2 chamber (12) at high pressure. LN2 is then passed into the specimen chamber (4) at high pressure. The isoamylalcohol (IAA*) (14) filling the chamber is compressed and forced out, delaying the arrival of high-pressure LN2 at the specimen (3). The sample is therefore frozen and LN2 then leaves the HPF machine through an opening (15) where it expands and is evacuated into the ambient air. Key: 1, specimen-holder; 2, locking security-pin; 3, sample compartment with sample (brown); 4, specimen pressure chamber; 5, high-pressure chamber; 6, high-pressure LN2 valve; 7, non-return valve; 8, LN2 reservoir; 9, high-pressure oil pump system; 10, oil cylinder; 11, Pistons; 12, high- pressure LN2; 13, IAA* reservoir; 14, high-pressure IAA*; 15, LN2 exhaust; 16, LN2 feed pump. T, temperature sensor; P, pressure sensor. The two arrows indicate directions for introducing the specimen-holder and security-pin into the HPF apparatus. Applying microscopy methods conventionally used for non-woody plant tissues or animal cells to woody tissues is often challenging. One of the basic approaches is staining tissues in order to visualize cellular contents or assess enzyme activities. However, stains often bind to lignified walls, hampering visualization and documentation. Furthermore, the natural property of the lignified walls to fluoresce creates high levels of background noise. This was a major complication in the studies described in Paper III. We wanted to assess the proportion of nuclei undergoing DNA fragmentation (using Comet assays) in the whole population. Samples isolated from the wood were contaminated with small cell wall remnants, and after ethidium bromide staining of the microgels the cell wall debris also became stained. Therefore, even though Comet nuclei were easy to visualize (Paper III), it was difficult to distinguish the intact, non-Comet, nuclei

9 1 Programmed Cell Death in Xylem Development from the cell wall debris. The intertwining network constituted by the three major types of cells present in Populus wood and the difficulties involved in separating them posed further problems. Applying protoplast isolation protocols to wood samples is impossible, due to the high mechanical and physical strength of their secondary cell walls. We did not manage to separate woody cells, even in the presence of highly concentrated cell wall-degrading enzymes. The only tractable approach for separating wood cells without damaging their cellular contents, that we did not use, was microdissection, however this is a complicated and highly time-consuming method44. 2.4.2 Chemical fixation vs. cryo-fixation Reliable conclusions based on electronmicroscopic (EM) observations can only be drawn if the fixation method can be trusted, but applying EM techniques to woody tissues is also complicated by the lignified cell walls obstructing the rapid and apt diffusion of fixatives (e.g. aldehydes). Thus it prompted us to use an alternative protocol that was not based solely on chemical fixation (Paper III). Chemical fixation Conventional fixation protocols are based on sectioning fresh wood stem samples into pieces less than one mm3 thick, and immersing them in a fixative preparation, e.g. a solution of aldehyde compounds (see Paper III). To promote good fixative penetration, samples can be briefly submitted to vacuum in- filtration. However, this treatment may damage cells, and thus create artefacts. Moreover, fixative diffusion will remain quite poor due to the presence of thick secondary walls, and probably only cells at the surface of the samples will take up the fixative. In contrast, intact cells located deep in the sample will either never come into contact with the fixative or take it up too slowly, allowing degradation of cellular contents to take place. Finally, there were indications that aldehydes may induce fixation artefacts due to their interference with lipids. High pressure freezing with freeze-substitution To improve the quality of our EM analysis, we used an alternative non-chemical based method: high-pressure freezing with freeze substitution45 (HPF, Paper III). Despite the processing and embedding of samples being slower than in the usual chemical fixation, cryofixation has major advantages. Notably, cellular water contents are rapidly immobilized (in no more than 10 ms), and the osmotic environment is kept unchanged and thus no damage is caused to cellular contents. Basically, when water in tissues freezes slowly, ‘hexagonal ice’ is formed within them due to the generation and the growth of ice crystals. Such crystals become large enough to distort cells and affect cellular anatomy, resulting in major artefacts. In contrast, when water freezes very rapidly, at a minimum rate of -10,000 °C.s-1, solidification occurs with virtually no formation of crystals, since water becomes more viscous when samples are processed by HPF, resulting in optimal cryopreservation. The general working principles of HPF are

10 2. Xylogenesis – Secondary xylem formation presented in Figure 3. After HPF, the solidified water contained in the sample is slowly exchanged with an appropriate solvent and TEM stain in gradually increasing, but low, temperatures (-80 °C to -20 °C), before embedding.

Figure 4. Fibre morphology in Populus woody tissues analysed by TEM, following chemical or HPF-FS fixation of samples, adapted from Paper III. (a) Cambial region. (b) Fibre in an early maturation stage with thin secondary cell walls. (c) Cytoplasm of a fibre with extensive secondary cell wall formation. (d) Nucleus in a fibre with extensive secondary cell wall formation. (e) Fibre at a late maturation phase when organelles are being degraded in the cytoplasm. (f) Two fibres at a stage of final autolysis. The micrographs illustrate both chemically fixed transverse sections (a,f,g,h) and high- pressure freezing/freeze-substitution fixed longitudinal sections (b,c,d,e). CDC, cambial daughter cell; CP, cytoplasm; DF, dying (autolysing) fibre; G, golgi; M, mitochondrion; R, ray cell; SCW, secondary cell wall; V, vacuole. Bars: 2 µm. The quality of TEM observations is also strongly dependent on the sensitivity of the protocol used to embed the samples in resin (LR White® or Epoxy). Since secondary cell walls act as barriers to resins, incomplete resin uptake by woody tissues will weaken the embedding process and reduce the quality of imaging for further TEM work. The early steps of embedding, in which resins are introduced in very low concentrations are the most critical. Indeed, if resins are first introduced at too strong concentration (> 5-10 %) in the acetone medium containing the specimens, they will disrupt cell morphology by compressing organelles or pressing cytoplasmic contents against cell walls (L. Samuels and M. Kaneda, personal communication). It is thus highly advisable to pause between new resin additions (add just a single new drop every 20 min), and leave samples in at most 10 % resin overnight. This method is thus time-consuming, requiring careful

11 1 Programmed Cell Death in Xylem Development attention to avoid spoiling specimens. Good resin embedding should allow the sample to be ultrasectioned, down to an optimal thickness of 70 nm. 2.4.3 Transmission electron microscopy In Paper III we presented a characterization of cell death in Populus fibres by TEM analyses of wood samples, preceded by both conventional and HPF-freeze- substitution fixations. Variations in fixation quality could be easily detected under TEM and chemically fixed samples were found to be much more affected by fixation than HPF samples (Figure 4). In sections where HPF was used (Figure 4b-e) the plasma membrane was in contact with cell walls and the tonoplast remained intact, showing clear cytoplasmic organellar contents, while in sections (Figure 4g,h) fixed using conventional chemical methods the plasmalemma dissociated from cell walls, the cytoplasm was shrunk and the tonoplast seemed close to rupture, if not already ruptured. Thus, standard chemical fixation appears to be unsuitable for morphological characterization of cell death in woody tissues.

3. Programmed cell death

3.1. What is cell death? Cell death is the inevitable fate of any cell produced by meristematic or stem cell division (i.e. cell produced during normal ontogeny), since as cells stop dividing, they will die sooner or later, unless they are able to return to a meristematic/stem cell state. Until recently, it was thought that only two main types of cell death processes existed46. One was thought to be triggered by acute exogenous factors such as trauma, stress, toxins and disease resulting in necrosis or unprogrammed cell death, destroying cells by lysis of membranes provoking inflammation due to the release of lytic enzymes into the extracellular compartment47. The other type was thought to be induced by endogenous factors resulting in various types of programmed cell death (PCD), an energy-dependent and genetically-regulated process. However, it became apparent that cell death cannot be so easily categorized. For example, depending on its intensity, stress can induce either necrosis or programmed cell death48, 49, suggesting that there are differences in their initiating mechanisms and signalling pathways. Furthermore, since cells and organelles swell in ‘necrosis’, semantic considerations suggest that ‘oncosis’, which derives from the Greek “onkos” meaning swelling, would be a better term for this type of cell death.46 Programmed cell death (PCD) is a key process for normal cell and organ development, integrity and homeostasis, in nearly all multicellular organisms, i.e. animals, plants and fungi. Its involvement in development serves many

12 3. Programmed cell death functions50, including: 1) sculpting structures, 2) deleting unwanted structures, 3) controlling cell numbers, 4) removing abnormal, misplaced, non-functional or harmful cells, 5) producing differentiated cells without organelles and 6) generating functional structures. PCD plays an essential role in sculpting organ and body parts in both animals and plants, as observed in developing digits in chick embryos51, and the remodelling of leaf shapes in higher plants52. Sometimes, tissues or organs are created that fulfil a functional need during a specific phase, but need to be removed later by PCD. In animals, the best example is the so-called metamorphic cell death of the tadpole’s tail, while in plants this is observed in zones of abscission, which allow organs that are no longer needed to be shed53. The control of cell numbers is essential within any organism to avoid incorrect development of organs. This has been especially well documented in animal neuron ontogeny54, and gymnosperm embryogenesis, in which only one embryo survives per seed, forcing massive cell death to occur in the others55. PCD acts as a quality control process whereby the organism removes or empties cells. For instance, mammalian cells may activate anticancer mechanisms or prevent the birth of defective offspring. In plants, such a process is often seen in response to biotic stress or disease outbreak since plants cannot escape pathogens. In Paper III we showed that fibre cell death may be associated with this type of death, since contents of fibres are emptied after their death, in accordance with their inability to serve any further purpose than to provide support after later stage of xylogenesis. Another role of PCD is in the production of differentiated cells that lack organelles but remain active, for example mammalian red blood cells. This is also observed in plants with enu- cleate differentiated phloem sieve elements, which are still active in the transport of solute and nutrients56. Finally, the last role for PCD is its involvement in the production from dead corpses of functional units, such as mammalian keratocytes or emptied xylem vessel elements transporting water in plants. The multiple functions associated with PCD listed above demonstrate its importance, and illustrate why its dysregulation often results in pathogenesis and disease outbreaks57, 58. Moreover, the diversity of the functions indicates that several programs (albeit sharing some components) are likely to be involved in the activation or inhibition of PCD.

3.2. Programmed cell death in metazoans 3.2.1 Summary of animal programmed cell death Based on general morphotypes, PCD can be classified into three cytological types, according to Clarke59: apoptotic, lysosomal/autophagic and non-lysosomal vesiculated (Table 1). Several variations of this classification scheme have been presented, as a result of the diversity of PCD processes; however Clarke provided the basis for any classifications.

13 1 Programmed Cell Death in Xylem Development

Table 1. The main types of cell death as described by Clarke59 (modified after Nooden46) Cell Designations Nucleus membranes Cytoplasm Type 1 Apoptosis; shrin- Nuclear condensa- Formation Loss of ribosomes from RER kage necrosis; tion, clumping of of blebs and polysomes; cytoplasm precocious pykno- chromatin leading to reduced in volume becoming sis; nuclear type of pronounced pyknosis electron-dense cell death Type 2 Lysosomal or Pyknosis in some Endocytosis Abundant autophagic autophagic cell cases. Parts of or blebbing vacuoles; ER and mitochon- death nucleus may bleb or observed dria sometimes dilated; Golgi segregate often enlarged

Type 3A Non-lysosomal Late vacuolization, Breaks General disintegration; disintegration then disintegration dilation of organelles, forming ‘empty’ spaces that fuse with each other and with the extracellular space Type 3B Cytoplasmic type Late increase in Rounding up Dilation of ER, nuclear granularity of of cell envelope, Golgi and someti- chromatin mes mitochondria forming ‘empty’ spaces.

During animal apoptosis, a major hallmark is DNA degradation. Under the influence of endonucleases, DNA is first cleaved into 50-200 kb fragments corresponding to the size of the chromatin loop domains, followed by a second cleavage at the internucleosomal linker region giving rise to a DNA “ladder”, observed in electrophoresis as multiples of 180 bp DNA fragments. Apoptosis involves activation of hydrolytic nucleases such as DNase II, shrinking of the cytoplasm, and concomitant activation of a caspase cascade60, 61. Autophagic or cytoplasmic degenerative PCD is characterised by mass destruction of cells through cytoplasm consumption by autophagic organelles62, 63, for instance during the metamorphic removal of a tadpole’s tail. The third morphotype, different from Clarke’s classification, is lysosomal degenerative PCD, characterised by shrinking of the cytoplasm due to the release of lysosomal hy- drolases62, such as cathepsins B,D and L, which are translocated from the lysosomal lumen to the cytosol in response to triggers such as oxidative bursts64. 3.2.2 Plasma membrane modifications in PCD Plasma membranes are subjected to structural changes during apoptosis, apoptosis-like and oncosis(necrosis)-like cell death implying that they expose ‘eat me’ signals to the outer cellular environment; the most thoroughly characterized ones being phosphatidylserine65 and changes in surface sugars66, 67, while other signals may include C1q, iC3b, ß2GPI, thrombospondin and ICAM- III68. The signals are recognized by phagocytes, before the cells are engulfed and digested. Phosphatidylserine (PtdSer), a component that contributes to plasma membrane asymmetry, is a ubiquitous recognition signal of PCD both in vitro

14 3. Programmed cell death and in vivo69, 70. The asymmetry is maintained via an aminophospholipid translocase (APT)71, which may transport phosphatidylethanolamine and phosphatidylserine from the outer toward the inner leaflet of the membrane. Another suggested mechanism involves the reversible binding of PtdSer to intracellular molecules, such as annexins or polyamines, especially spermine72, in the inner membrane leaflet during normal cell growth. When apoptosis occurs, the binding is lost and PtdSer is translocated to the outer leaflet. Thus, apoptosis is correlated to the exposure of PtdSer to macrophages. Moreover, changes in plasma membrane integrity compromise permeability, inducing leakage of small molecules, followed by membrane blebbing, one of the hallmarks of apoptosis. 3.2.3 Cellular compounds in PCD The mitochondria in PCD The integrity and function of mitochondria play central roles during PCD, in both animal and plant cells.73-77 Although not essential to activate PCD, mitochondria facilitate the interpretation and amplification of cellular stress signals in animal cells78. During the activation of cell death processes in the mitochondria, signal proteins are translocated into the mitochondrial matrix after the permeability of the outer mitochondrial membrane (OMM) has been modified. Increases in OMM permeability induce also leakage of mitochondrial compounds (Figure 5) that are involved in apoptosis signalling, such as cell-death activators, cell-death inhibitors and cell-death inhibitor repressors in the cytosol. Apoptosis: caspase cascade or not? Caspases are specialized cysteine proteases, primary executioners of cell death, which cleave their substrates at aspartic acid residues. They are found in the cytosol, mitochondria and ER, except for human caspase-9, which is localized in the extracellular space. The number of genes encoding caspases varies from species to species: 11 have been found in humans while only three have been detected in Caenorhabditis elegans. They are mostly synthesized as zymogens, which are activated by cleavage either via autoactivation (e.g. caspase-9 or -3), via other caspases or via other protein complexes such as the apoptosome.60, 75, 79, 80 Two types of caspases have been identified: the effectors and the initiators. Effector caspases are the executioners of cell death acting on caspases via cleavage of their p10 subunits, while initiator caspases are characterized by the presence of a prodomain required for their further recognition and re- cruitment into complexes, such as the apoptosome, triggering caspase-cascade responses.75 Intrinsic and/or extrinsic signals sensed in the cell activate PCD responses (Figure 5). In caspase-dependent cell death, tBID activates proapoptotic Bax/Bak signalling molecules inducing, together with Ca2+ influxes, the release of cytochrome c from the mitochondria into the cytosol via membrane loosening or formation of

15 1 Programmed Cell Death in Xylem Development voltage-dependent anion channels (VDACs). In turn, the caspase-cascade pathway is induced by activation of the apoptosome complex between cytochrome c, Apaf-1 and the self-catalyzing caspase-9, further initiating the autoactivation of caspase-3, before substrate cleavage. In the complex balance between pro- and anti-apoptotic signals, some anti-apoptotic molecules, for instance Bcl-2 and Bcl-XL, are able to block cytochrome c release through inhibition of VDACs.60, 75, 76 The mitochondria are major effectors of apoptosis since they can integrate and amplify some pro-apoptotic signals, and maintain survival in animal cells. A caspase-independent pathway in cell death was also recently unravelled79, 81, 82, in which endonuclease G and AIF seem to play key roles, and inhibitors of mammalian cysteine proteases involved in apoptosis do not appear to prevent cell death, but rather slow it down. Moreover, autophagic cell death seems to activate caspase-independent cell death, as caspases cannot induce cell death alone in autophagy.83

Figure 5. Caspase-(in)dependent apoptotic pathways showing the involvement of extrinsic (on the left) and intrinsic (on the right) signals at organellar levels in animal cells, with some identified plant counterparts. ER, endoplasmic reticulum; IAP, inhibitor of apoptosis protein; PARP, poly-(ADP-ribose) polymerase; Endo. G, endonuclease G; CAD, caspase-activated DNase; ICAD, inhibitor of caspase-activated DNase; FADD, Fas-associated death domain; TNFR1, tumor necrosis factor receptor-1. Inhibitors of cell death are shown in red; molecules having plant counterparts are highlighted in orange. Dashed arrow: possibly transcriptionally induced. Inspired from Dickman and Reed.224

16 3. Programmed cell death

3.3. Programmed cell death in plants 3.3.1 PCD in the whole plant Elucidation of the steps involved in animal apoptosis76, 84-86 prompted interest in similar events in plants. PCD occurs in many processes in plants87, including: reproductive development (e.g. embryogenesis88-91; flower petal senescence92, 93) and vegetative development (e.g. xylogenesis18, 94; lysigenous aerenchyma for- mation52, 95). Sometimes, it is a response to stress or environmental conditions, e.g. salt-stress96 or pathogen attacks78, 97-99 (Table 2). Table 2. References to all known types of programmed cell death (PCD) in plant development. (Modified after Noodén46). Developmental Stages References Developmental Stages References Vegetative development Reproductive development Seed Floral organ abortion 100 aleurone cells 101-103 Megagametogenesis cotyledons 102 megaspore abortion 104 scutellum 90, 103 nucellus 105 Shoots synergid cell 106 holes in leaves 107 Microgametogenesis 108 shoot aerenchyma 109, 110 Embryogenesis leaf aerenchyma 52 antipodal cells 106 18, 19, 94, 111 102, 112 xylogenesis Papers I, II and III endosperm cells xylem ray cells 113 suspensor 89, 90, 102 secretary ducts 113 Somatic embryogenesis 89 pith cell death 114, 115 Apomictic embryogenesis 116 trichomes 117 loculus wall cells 118 senescence 119, 120 filament cells 100, 118, 121 abscission zone cells 122, 123 tapetum cells 108 cork/bark/wound 113 Pollen incompatibility 124 healing Roots Fertilization 121, 124 root hairs 125, 126 Flower petal senescence 92, 93 xylogenesis 19 Post-reproductive development root cap cells 90 Seed development cortical parenchyma 127 endosperm cells 112, 128 lysigenous integument, palisade and 95, 110, 129 100, 130 aerenchyma epidermal cells lateral roots 113 pod, carpel senescence 131, 132 salt stress 96, 133, 134 Monocarpic senescence 135, 136 In plants, PCD is classified into three different types based on morphological features. Apoptosis-like cell-death, which occurs in some stress-related and

17 1 Programmed Cell Death in Xylem Development developmental processes, involves rapid degradation of the nucleus and loss of cell organization.18 It is characterized by DNA laddering, nuclear shrinkage, chromatin condensation and nuclear shrinkage.96, 137 It is often induced by signalling via the mitochondria.77 The second type is cell death occurring during senescence processes. This very slow process is associated with high recovery of cell contents and optimal reallocation of nutrients. The disruption of both the nucleus and the vacuole occurs at the very end of the cell death process, after complete degradation of the plastids.138 The third and last type of cell death, illustrated by tracheary element differentiation, is PCD induced by vacuolar degradation, which has intermediate speed compared to the two types listed above. It involves the central action of vacuole-localised proteases, which once released into the cytosol degrade the cell contents then finally, under the influence of the lytic enzymes, the nuclear DNA breaks down. 3.3.2 Metacaspases in plants In 1998, the first evidence of caspase-like proteolytic activity in plants was reported, based on the effects of caspase-specific peptide inhibitors during HR cell death in response to pathogen attacks in tobacco.139 The existence of two families of caspase-like proteins was demonstrated soon thereafter, namely para- caspases and metacaspases: the former from animals and slime molds, and the latter from plants, fungi and protozoa. Metacaspases were later found to be involved in various PCD responses7, 140, implicating them in plant PCD, analo- gously to caspases. In plants, two types of metacaspases have been reported, represented by nine genes (AtMC1-9) in Arabidopsis.141 Type I metacaspases, including AtMC1-3, are characterized by the presence of a proline-rich repeat- and a zinc-finger- motif-containing a prodomain and a short linker/loop region, while type II metacaspases (AtMC4-9) contain a longer linker/loop region preceding the p10 subunit and no prodomain.142 Plant metacaspases are unable to cleave caspase substrates and thus do not possess caspase-like activity143, 144, raising questions about their exact role in the regulation of cell death145. How- ever, they have implicated involvement in PCD in yeast146 and Trypanosoma brucei147. In plants, expression of type II metacaspases was found to be induced during interactions between Botrytis and tomato140, and their silencing in Norway spruce (Picea abies) suppressed PCD, leading to embryonic pattern formation7, highlighting their importance for plant PCD in analogy to animal caspases. In Paper III, we identified a duplicated Populus metacaspase gene (PttMC1), homologous to AtMC9, which was specifically upregulated in the late maturing xylem (Mx2). Its expression coincided with the presence of TUNEL-positive xylem fibres (see 4.3.3), suggesting that the metacaspase it encodes plays a role in the death of the xylem fibres. However, GUS analysis revealed that this metacaspase was primarily expressed in xylem vessels (data not shown). The Populus genome carries a paralogous gene, PttMC2, and detailed gene

18 3. Programmed cell death expression and reporter gene analysis (data not shown) indicate that the two Populus metacaspase genes have evolved differing functions: PttMC1 being involved solely in vessel development while the more ubiquitous PttMC2 is involved in the development of both vessels and fibres. 3.3.3 The vacuole: a growing organelle in PCD The importance of vacuolar PCD has been strongly highlighted during Zinnia tracheary element (TE) differentiation in xylogenesis18, 19, 87 as well as during protein activation and maturation in the various vacuoles148, 149. During cell growth, maturation of the vacuole, which is unique to plant cells, may results from the merger of two distinct compartments, lytic and protein-storage vacuoles.150 In early stages of TE differentiation in Zinnia, the vacuole occupies most of the intracellular space and the cell appears to be very active, as evidenced by vigorous cytoplasmic streaming26. Then, approximately six hours after initiation of secondary cell wall deposition18, the vacuole ruptures, releasing the hydrolytic enzymes it contains, which degrade first the single- and then the double-membrane structures, and finally the nucleus23 and remaining orga- nelles18, 41. This rapid trigger of DNA degradation by vacuolar collapse is a unique type of PCD, associated solely with TE differentiation.23 In Paper III, events associated with the vacuolar variant of PCD were explored. The presented results showed that the vacuole increases in size and the cytoplasm shrinks, becoming increasingly dense, until all the organelles have disappeared. The tonoplast subsequently ruptures and the vacuole bursts, the point at which this occurs being regarded as the time of death. This illustrates the major physical differences between fiber and TE cell death. In Arabidopsis, the importance of the vacuole is highlighted by the key action of the vacuolar-localised vacuolar processing enzymes (VPEs) in programmed cell death.148, 151-156 VPEs are cysteine proteases with substrate preferences for proteins with exposed asparagine residues. The family is encoded by four classes of genes: α-, β-, γ- and δVPE. αVPE and γVPE genes are expressed in senescent tissues of vegetative organs, while βVPE and δVPE expression occurs in seeds.153, 155, 157, 158 These VPEs accumulate in different vacuolar compartments; the vegetative VPEs in the lytic compartments, while βVPE and δVPE accumulate in protein-storage compartments.152 Since the vegetative VPEs are expressed only in senescent leaves, their target proteins might be senescence- inducible cysteine proteinases in the lytic vacuoles of senescing and degenerating tissues. In the study presented in Paper I, VPE transcripts were found to be highly abundant in the fibre cell death EST library, although they were also detected in other libraries, such as the senescence and pathogen-induced libraries, suggesting the potential involvement of VPEs in regulation of vacuolar integrity and cell death per se during several different PCD processes. This hypothesis was further supported by the finding that VPEs were expressed late in maturing

19 1 Programmed Cell Death in Xylem Development xylem, at times corresponding to fibre cell death (Paper III). Moreover, in Paper I, an homologue of Arabidopsis, Vacuoleless1, involved in vacuole formation and autophagy, was shown to be strongly upregulated in the late xylem maturation, supporting the important role of the vacuole in xylem cell death. Vacuoles are also related to autophagy, either by gathering autophagosomes derived from the Golgi, proplastids and ER, or simply by gathering cytoplasmic materials enclosed in double-membrane vesicles. Recently, autophagy was demonstrated to be involved in regulating PCD77, 159-161; and several autophagy genes (ATG) were characterized, such as Beclin-1, an ortholog of yeast and mammalian ATG6, with functions in the restriction of HR cell death.161 In Paper III we showed that the cytoplasmic contents are gradually removed without tonoplast rupture (Figure 4), suggesting that autophagy occurs, as demonstrated in other processes, such as petal senescence92. Moreover, the microarray analyses revealed the upregulation of Populus genes homologous to Arabidopsis ATG8 genes and proteins involved in the control of autophagy, such as v-SNARE, VTI12 and TOR. These findings collectively suggest that the vacuole, which does not have an organellar counterpart in animal cells, regulates PCD in a manner unique to plant cells. 3.3.4 A new concept in plant PCD: the Degradome A few years ago, the concept of the ‘degradome’ was introduced in human research as a term encompassing all information about central regulators of PCD, such as proteases, their substrates and inhibitors, involved at a specific time and in a given group of cells.162 Since no close homologous sequences of animal caspases had been isolated from plants at that time, the degradome concept was not applied to plants until recently, when metacaspases were characterized6. Subsequently, a trio of protease families (VPEs, type II metacaspases and VEIDases), each having their own substrate specificity and cellular localization, but all essential for the execution of cell death, was identified as the core of the plant degradome.163 However, the role of VEIDase, with a caspase-like activity, in xylem cell death specifically remains unclear as it was only demonstrated from embryo development so far. In Paper II we demonstrated that loss of the polyamine biosynthetic ACL5 function in Arabidopsis induced premature cell death in xylem vessels, suggesting that polyamines might act as inhibitors or delayers of cell death, at least in xylem vessels. In Papers I and III we identified a large number of previously unknown proteins that were activated in xylem elements undergoing cell death. The characterization of a degradome for each cell type is a complex task, and thus the possible involvement in the plant degradome of these novel genes needs to be verified by further functional analysis.

20 3. Programmed cell death

3.3.5 Inhibitors of cell death Cell death inhibitors have evolved in metazoans and plants that tightly control the action of activators of cell death during tissue or organ homeostasis (Figure 5), such as IAPs capable of inhibiting mitochondria-induced apoptotic cell death in response to intrinsic signals in animals (proteins marked in red in Figure 5). Another well known, ubiquitous inhibitor is the Bax-inhibitor (BI-1), which also shows activity in plants, and plant counterparts have been isolated that have identical activities in both plant and animal cells. In Paper III, we demonstrated that several cell death inhibitors in our microarray were upregulated, such as the homologs to Arabidopsis BAG4 and BAG1, Bax-inhibitor homologs and ethylene-response protein (similar to RAP2.3), identified in other plant cell death responses, supporting the hypothesis that the same cell death inhibitors may be involved in different types of plant cell death. As seen here, many studies on plant PCD were prompted by comparison to known components of animal apoptosis. However, some types of PCD, e.g. the hypersensitive response and leaf senescence, are specific to plants; and therefore plant-specific regulators of PCD are expected to be found as well.

3.4. The hypersensitive response: an example of stress-induced programmed cell death specific to plants 3.4.1 The hypersensitive response (HR) Unlike many animals, which are able to escape pathogen outbreaks, plants cannot move and have had to evolve other means to defend themselves. The hypersensitive response (HR) is a response of plant hosts to biotic stress induced by pathogens’ attempts to infect them. The HR is a programmed process requiring the recognition of a pathogen-specific molecule, encoded by an avirulent gene (Avr), in the host by a molecular receptor, encoded by a resistance gene (R), in an incompatible manner.164 When either the R- or Avr-allele is missing or non-functional, the plant-pathogen interaction becomes compatible, the pathogen cannot be recognized and infection or disease can occur. This complex gene-for-gene response results from the interactions between several R- and Avr-genes.165 When the plant is capable of recognizing the pathogen, host cells in the immediate vicinity of the attacked site will undergo rapid cell death. By activating such a rapid response after pathogen recognition, HR host cell death prevents losses of nutrients. The dying host area will also produce antimicrobial enzymes or compounds in excess that help fight the infection166 and rapid dessication of the host tissues occurs that helps reduce pathogen growth167. Finally, the HR induces an oxidative burst that promotes the creation, or

21 1 Programmed Cell Death in Xylem Development fortification, of physical and chemical barriers such as deposits of lignin, callose or phenolics that restrict the spread of the pathogen.168 Purified elicitors, i.e. molecules produced by the pathogen or during plant-pathogen interactions brought into contact with a resistant host induce HR symptoms, such as cell death, while, when applied to a susceptible host, no HR hallmark is usually detectable. The elicitors are therefore capable of triggering alone endogenous HR cell death, a process that is completely programmed by the host cells. 3.4.2 HR cell death: a programmed process The HR is a genetically programmed process resulting from new transcription and translation in the host genome, ending with the activation of PCD, as clearly illustrated by the isolation of HR-defective mutants and characteristic markers. Characterization of lesion-mimic mutants HR functions by both activating cell death at the original site of pathogen infection and limiting cell death to the close surroundings of the infected cells. Lesion-mimic mutants, isolated from several plant species (including Arabi- dopsis, maize and barley), mimic the development of HR lesions, as illustrated by patches of dead cells on their leaves that are not induced by pathogens, but are responding like wild-type tissues under constant pathogen attack. These mutants can be categorized into two groups: the initiation-class group, which improperly initiates HR, and the propagation-class group, which fails to limit the spread of HR. Initiation-class mutants (such as rp1 in maize and mlo in barley) are characterized by small areas of dying tissue that do not spread, while propagation-class mutants (such as lsd1, acd1 or vad1 in Arabidopsis and lls1 in maize) are defective in limiting the spreading of cell death once HR has been initiated. Furthermore, gene cloning in these mutants has resulted in the identification of R genes and genetic pathways involved in HR PCD, such as sphingolipid, ethylene, and ceramide signalling169. Characterization of the HR markers Identification of HR-specific gene expression has not only elucidated the genetic programme underlying HR PCD, but also provided useful marker genes for identifying specific processes related to HR. For instance, HR cell death can be dissociated spatially and temporally from senescence by examining the expression of two HR-specific markers, HSR203J and HIN1, and a senescence marker SAG12 in tobacco mosaic virus (TMV)-inoculated tobacco plants.170 Interestingly, this approach unravelled a senescence-like process in cells located at the periphery of HR cell death regions in leaves that were only expected to undergo HR cell death. Other markers associated specifically with HR cell death include HMGR, HSR201, HSR515 and HSR1-3. Interestingly, all of these genes, except HSR1-3, were identified as downstream targets of the spermine signal pathway.171

22 3. Programmed cell death

3.4.3 Triggers of HR cell death Reactive oxygen intermediates/species (ROI/ROS) Within hours of pathogen inoculation, infected cells activate the production of reactive oxygen intermediates or species (ROI/ROS) from the plasma membrane and mitochondria in a process known as an oxidative burst. A first burst occurs within the first hour of infection in both compatible and incompatible host- pathogen interactions. However, in the latter, a second characteristically longer and stronger burst also occurs, followed by cell death. This burst (Figure 6) .- results in the accumulation of superoxide (O2 ), which is not especially toxic but . .- very unstable, since it readily converts to hydroperoxide radicals (HO2 ). O2 is also converted into the toxic hydrogen peroxide (H2O2) by superoxide dismutase (SOD). In turn, H2O2, which is able to cross membranes, is converted into highly . .- toxic hydroxyl radicals (OH ). The finding that inhibiting O2 generation can reduce HR cell death in plants172 supports the hypothesis that ROIs participate in the stimulation of HR cell death.

Figure 6. Simplified equation of the inter-conversion of reactive oxygen species/intermediates within a cell, in the process known as the oxidative burst. SOD, .- . . superoxide dismutase; O2 , superoxide anion; HO2 , hydroperoxide radical; OH : hydroxyl radical; H2O2, hydrogen peroxide. ROS/ROI scavengers However, the accumulation of ROS/ROI remains harmful to the cell and its neighbours, and thus these species need to be scavenged. In addition to peroxidases (Figure 6), major ROS scavengers include thioredoxins, peroxiredoxins, polyamines and alternative oxidase (AOX). The mitochondrial thioredoxin (TRX) protein is thought to play a vital role in this process in animals by scavenging ROS and regulating the mitochondrial apoptosis signalling path- way173, 174. In plants, members of the TRX-2 family apparently target, inter alia, .- the cyanide-resistant AOX involved in decreasing O2 levels and oxidative stress in mitochondria.175 Peroxiredoxins (PRXs), TRX-dependent peroxidases, are associated with apoptosis in mammalian cells with multiple subcellular localizations. In plants, five types of PRX have been identified, which control ROS contents and redox-sensing in various pathways, depending on their subcellular localization.175, 176 In animal cells, polyamines (PAs; small ubiquitous amine compounds including putrescine, spermidine and spermine) are involved

23 1 Programmed Cell Death in Xylem Development in major cellular processes such as cell growth and PCD, inter alia by preventing ion leakage from mitochondria and vacuoles. Since ion fluxes seem to be essential in the induction of cell death in xylem elements, and polyamines have the capacity to control ion channels; it is possible that ACL5 is involved in the regulation of tonoplast channels in xylem vessel cell death (Paper II). Paradoxically, even though polyamine metabolism is a source of ROS in animal cells, spermine is also considered to be a ROS scavenger177. Similarly in plants, the PA biosynthesis pathway has been reported to both trigger the HR cell death178 and provide protection against some types of cell death (Figure 7; Paper II, showing that xylem vessels of Arabidopsis undergo premature cell death in polyamine-deficient plants).

Figure 7. Polyamine regulation (left) and degradation (right) pathways in plants. ROS are produced from putrescine and spermidine via the action of polyamine degradation enzymes. Spermine acts both as a source and a scavenger of the ROS H2O2. ACL5 has been recently shown to catalyze the production of thermospermine, a rare tetraamine. An inner mitochondrial membrane enzyme specific to plants, AOX, has been suggested to play a major protective role against oxidative stress and cell death in the tissues surrounding an infection site179, 180, by controlling ROS accumulation. AOX catalyses the electron flow from ubiquinol directly to O2 by bypassing mitochondrial complexes III and IV, from which cytochrome c is released, converting O2 to H2O. In AOX-antisense plants, inhibition of the electron flow has been found to induce mitochondrial ROS accumulation followed by rapid cell death, while AOX expression inhibits the spread of tobacco mosaic virus (TMV)-induced HR cell death in nahG plants exposed to cyanide. Therefore AOX was suggested to inhibit cell death at the border of HR lesions, further

24 3. Programmed cell death supporting the role of mitochondria in HR cell death signalling. Moreover, induction of AOX in the HR of infected leaves was shown to require both the ethylene and salicylate signalling pathways180, implicating AOX in several hormonal interactions and feedback-loops, as well as indicating that HR regulation is highly complex. Nitric oxide and salicylates: HR cell death amplifiers The gaseous, lipophilic species nitric oxide (NO) is involved in pleiotropic physiological responses, such as cell proliferation and inflammation. It also parti- 181-183 cipates in H2O2-mediated HR cell death in plants by interacting with ROIs and activating defence mechanisms, such as inhibiting cytochrome c oxidase. In addition, it has been suggested that the antibacterial properties of NO might indirectly induce cell death by increasing the permeability of mitochondrial membranes.184 Recently, NO production was proposed to originate from the polyamine biosynthesis pathway in Arabidopsis seedlings185, 186, suggesting the existence of a physiological link between NO production and polyamines. An alter- nate pathway to the production of NO, similar to the pathway observed in animal cells, was also suggested after a putative NO synthase gene (AtNOS1) was cloned in Arabidopsis187. However, it has been proved difficult to link NOS activity with the protein encoded by this gene182, 186, highlighting the paucity of our knowledge of NO biosynthesis in plants. Similarly, salicylic acid (SA) and acetylsalicylic acid (ASA) are major mediators of pathogen resistance in plants, which act by amplifying HR signals, leading to cell death.188 Oxidative-burst-related HR cell death is correlated with high SA 189 contents, inducing H2O2 production, lipid peroxidation and oxidative damage. Furthermore, in some Arabidopsis lesion-mimic mutants, such as lsd and acd, cell death is dependent on SA.190, 191 ASA, the acetylated derivate of SA, is a non- steroidal, anti-rheumatic, apoptosis-inducing compound in animal cancer cells192, and was recently demonstrated to be an efficient H2O2-like PCD-inducing agent in Arabidopsis cell cultures193. ASA inhibits cyclooxygenase (COX) enzymes, which leads to the production of prostaglandins involved in inflammation or pain in animal cells194, as well as their equivalent in plants, the allene oxide synthase (AOS) enzyme, which leads to the production of two compounds – 12-oxo- phytodienoic acid and jasmonate (JA)195-197 – that are known to participate in the regulation of cell death in plants. The latter observation supports the postulated presence of similarities between apoptosis in animals and PCD in plants. 3.4.4 Phytohormones: ambivalent roles in HR-like cell death Generally, hormones act as mediators that induce HR cell death by integrating various external or internal signals. Ethylene is a gaseous phytohormone involved in many developmental and growth processes as well as both biotic and a- biotic stress responses in plants. Since ethylene signalling mutants often display

25 1 Programmed Cell Death in Xylem Development diverse degrees of cell death defects in response to attempted attacks of pathogens, and several Arabidopsis mutants with accrued HR responses produce increased amounts of ethylene, the latter is thought to be involved in HR cell death.198-201 However, the scarcity of information on the mechanisms that mediate ethylene’s functions in this respect, and its involvement in the various pathogen responses, suggest a need for further investigations. Abscisic acid (ABA), a phytohormone involved in both biotic and abiotic stress responses, interacts with pathways that regulate several phytohormones, including SA, jasmonate (JA) and ethylene. During abiotic stress, accumulation of H2O2 seems to be mediated by ABA action202, while its involvement in plant-pathogen interactions is still under investigation. An ABA-deficient tomato mutant, sitiens, reportedly shows enhanced resistance to pathogen attacks203 with enhanced activation of defence responses, including H2O2 accumulation, peroxidase activation and fortification of cell walls. However, some positive effects of ABA on defence against pathogens have also been also observed.204, 205 Recently, ABA has been suggested to be an important regulator of defence against an oomycete pathogen, by affecting JA biosynthesis206, and against the Leptosphaeria maculans pest in the 207 RLM1col pathway by acting as a defence hormone and inducing callose deposition204. These findings suggest that much remains to be learnt about the positive role of ABA in pathogen suppression. The prostanoid-like fatty acid JA (see section 3.4.3, under Salicylates), is a phytohormone that belongs to a family of oxylipin derivatives.196, 197 Since exogenous JA applications antagonize the PCD-inducing effects of salicylates208 (i.e. SA and ASA) and jasmonate- insensitive or -defective mutants display increased cell death phenotypes, JA is certainly implicated in the control of cell death, but mainly through its inhibition by salicylates193, 209. Plants present many distinct types of PCD and it is remarkable that even within a given type, such as stress-induced PCD (which includes senescence and the HR), variations in cell death processes can still be quite significant. Indeed, while senescence is a slow process spread over couple of months, allowing maximal nutrient recovery from the dying leaf cells, HR is very rapid, and can be activated within an hour in response to attempted infections, thereby maximising the chances of limiting the spread of pathogens. But how similar to animal cell death systems, such as apoptosis, are the plant PCDs? 3.4.5 Animal apoptosis vs. Plant PCD: “same same but different1”? Generally, the different types of PCD processes reflect an intricate regulation system, involving common and/or independent regulatory pathways, triggered by

1 In Tinglish (a type of pidgin English, spoken in Asian countries such as VietNam in my experience, see http://en.wikipedia.org/wiki/Tinglish), this expression refers to situations in which phenomena may seem very similar, but still differ in some ways.

26 3. Programmed cell death various signals76, 210 (Figure 5). In animals, most advances in understanding apoptosis have resulted from identification of the molecular machinery of cell death in the nematode C. elegans, in which 131 out of the 1090 cells formed during development undergo PCD, giving rise to ‘cell death defective’ (CED) mutants with defects in cell death processes. Cloning the corresponding CED genes has revealed that the homeostasis of life processes involves a tightly controlled balance between activation and inhibition of PCD. Orthologs to the C. elegans CED-9, -4, and -3, were later characterized in mammals as Bcl-2, Apaf-1 and caspase-9, respectively (Figure 8), which perform the same functions as their counterparts. Plant genes and gene products with similarities to the animal apoptotic machinery are still being sought.144 However, although CED-like genes have not been identified in plants so far, some cytological similarities have been reported, leading us to believe that the cell death pathway may be conserved between kingdoms to some extent. In spite of a strong cell wall preventing modifications of the membranes, plant PCD shares common hallmarks with animal apoptosis, such as DNA laddering211 (although this is not always present18, 212) and protease activation6, 39, 144, 213. Interestingly, there are increasing indications that incompatible plant-pathogen interactions involving HR cell death may follow an apoptotic pathway. Notably, morphological hallmarks of apoptosis, as defined in animal cells, have been observed in some cases of HR cell death in plants, e.g. apoptotic(-like) bodies211, inter-nucleosomal DNA fragmentation illustrated by laddering211, blebbing of membranes214-216, chromatin condensation214-216 and cell shrinkage214, 216. At the molecular level, the resemblances between the two types are somewhat less clear. Among the identified pro-apoptotic (e.g. ced-3, ced-4, Apaf, Bax or caspases) and anti-apoptotic (e.g. ced-9, Bcl-2, Bcl-XL or BI-1) regulators of cell death in metazoans76, 217, 218 (Figure 5), only the BAX protein and its inhibitor BI seem to be functionally shared by apoptosis and HR cell death, as illustrated by the finding that mammalian BAX-induced cell death in plants can be inhibited by plant BI219-221. The lack of plant homologs for most of the apoptotic regulators in animals suggests, therefore, that either plants have evolved different cell death regulatory pathways or their cell death pathways have similarities to those of animals, but poor sequence conservation prevents the identification of close homologs. This conclusion is supported by the identification of plant metacaspases that have proved to be only distantly related to caspases in terms of sequence similarity, but are closely related with respect to protein structure and (probably) protein function, as described above. Also, the resemblance of some plant cell death regulators and morphology to those involved in animal apoptosis is consistent with the hypothesis that plants may share some apoptotic pathways with metazoans, especially during HR, indicating that, to some extent at least, HR has similarities to apoptosis.

27 1 Programmed Cell Death in Xylem Development

Figure 8. Evolutionary conservation of cell death pathways between metazoans, mammals and plants. Between C. elegans and mammals, functions seem to be conserved. In plants, the lack of homology has complicated the identification of functionally orthologous genes. Modified after Dickman and Reed.224 Genomic sequencing efforts have failed to identify endogenous plant genes that share sequence homology (leading to most probably similar functions) with apoptosis-controlling genes in animals. However, since apoptosis appears to be an ancient process that is conserved between kingdoms, it seems quite likely that the homology of genes involved in the process between species might not be very strong, due to the large evolutionary distances between them, for instance despite their orthologous functions as cell death repressors, CED-9 and Bcl-2 share less than 25 % sequence identity222. This highlights a limitation of search or alignment tools, such as BLAST interfaces, which are not sensitive enough to detect distantly related proteins that have little sequence homology, even if they if have similar functions. Furthermore, similarities between a human apoptosis regulator, the BID protein, and the Bcl-2 family were only detected from their 3D structure, and were not identified in BLAST searches223, possibly explaining the lack of genes identified so far in plants orthologous to animal cell death genes, and suggesting there is a need to improve tools used for gene/protein homology searches.

28 4. Xylem programmed cell death

4. Xylem programmed cell death

4.1. PhD on programmed cell death in xylem Even though PCD in plants has aroused interest for the past 20 years, very few studies have examined the process in woody plants, especially trees. Further- more, most of the relatively small amounts of work done on tree species, both angiosperms and gymnosperms, have focused on PCD in floral organs (i.e. em- bryogenesis6, 55) or leaves (i.e. senescence225). Studies demonstrating that PCD is essential in xylogenesis have largely been restricted to morphological characterization in trees or have focused mainly on non-woody plant species. Therefore, there are large gaps in our knowledge of the final stages of xylogenesis in trees. The pulping, and many other, industries require high-quality wood, and one of the key quality parameters of wood is its density, which is affected by the thickness of the secondary cell walls. Since the lifetime of xylem fibres seems to affect the thickness of secondary cell walls, xylem fibre PCD is expected to have an effect also on wood properties such as density. Understanding death in fibres may therefore help improve the value of trees for the wood industry, by indicating ways to modify the lifetime of xylem fibres using transgenic approaches. Therefore, the results obtained in this PhD project, which focused on chara- cterizing xylem PCD, especially in fibres of Populus wood, could have major economic implications for both the wood industry and sustainable development.

4.2. Bioinformatics: the essential starting point for gene browsing. 4.2.1 Bioinformatics: what is it? Computational biology, the ancestor of bioinformatics, emerged in the 1960’s, from the fusion between computer technology, mathematics and molecular biology.226 Later, in the mid-1980’s, the term bioinformatics was used to refer to any computer-based approach employed in biology. However, it took another 10 years before it received serious attention and consideration in highly-ranked scientific journals.227, 228 However, there has been a recent explosion in the use of bioinformatics and computational biology, prompted by the rapid developments in biological technologies that require advanced computer techniques to manage the increasingly abundant and complex information being generated. More than merely unravelling huge sequences, bioinformatics analyzes their characteristics and patterns, using widely available databases. Ever since its beginnings, bioinformatics has constantly advanced as new approaches have been developed and applied to intricate biological questions. Its standing has now reached the point where its findings are reported in dedicated scientific journals.

29 1 Programmed Cell Death in Xylem Development

4.2.2 Bioinformatic tools to unravel PCD in trees Microarray technologies The very recent introduction of microarray technology in molecular biology has tremendously boosted the rate at which genes can be isolated, and extended the scope of the experimental designs that can be applied. Microarray or microchip techniques, based on the competitive hybridization of an equimolar mixture of two fluorescently-labelled cDNA probes on a slide spotted with nucleic acid sequences (see for instance Papers I and II), are convenient approaches based on the combined use of hybridizations and fluorescence microscopy. They are also high-throughput methods allowing the rapid, but reliable, analysis of large numbers of nucleic acid fragments. However, in practice, replicates may sometimes show tremendous variations, suggesting that better nucleic acid measurements and/or automatic hybridizations are required to increase the homogeneity and reproducibility of results. The computational parts of the procedures, such as scanning and data analysis, are also accompanied by limitations in the production of consistent data, since visualization, analysis and interpretation depend on the user’s perception of the data, to some degree. Due to the interest in Populus as a model for woody angiosperm plants, microchip technologies including four main microarray platforms have been developed for this organism. However, the second generation UPSC POP2 platform is the most comprehensive.229 This platform contains 24,735 different cDNA fragments (Paper I), derived from 19 cDNA libraries230, corresponding to 13,882 gene models; about 30 % of the 45,555 predicted Populus gene models12. During our attempts to elucidate the mechanism of xylem fibre death, we were the first to publish data using the UPSC POP2 platform (Paper I). The association of a unique xylem fibre death EST library (X) with the Populus database (Populus- DB, see below) provided a powerful tool for this platform and a valuable source of information on transcriptional changes related to fibre cell death. Furthermore, the clustering described in Paper III, detected using the UPSC POP2 platform, unravelled the presence of cell death specific candidate genes participating in the regulation of cell death in the xylem. These genes were either previously known in this process or totally novel, as presented in the list gathering the 124 novel xylem cell death candidate genes not yet described in the regulation of cell death. More generally, the clustering suggests that a major change occurs in gene expression patterns in the late maturing xylem samples that is probably related to the requirement for novel metabolic activities participating in secondary cell wall formation, as illustrated by KOG (eukaryotic orthologous groups) overrepresen- tations. The analysis also reveals the concomitance of secondary wall deposition and cell death, by the simultaneous overrepresentation of KOG classes involved in secondary cell wall formation (“cell wall/membrane/envelope biogenesis”) and preparation to cell death or degradation product turn-over (“energy production

30 4. Xylem programmed cell death and conversion” or “amino acid transport and metabolism”) in the late maturing xylem samples Mx1 and Mx2. Additionally, a large numbers of the 124 candidate genes not only previously characterized, but also new putative regulators of cell death were found upregulated in the same samples (Mx1 and Mx2) where the KOG classe for “cell wall biogenesis” is overrepresented. Therefore, Papers I and III show the usefulness of microarrays and their subsequent analysis such as KOG, in first steps to analyse an uncharacterized physiological process. Databases for Populus Since the dawn of bioinformatics, thousands of databases have been produced to provide rapidly accessible, standardized expression data acquired in previously performed experiments. Many more are to come, most of which will almost certainly concern expression patterns in animals or bacteria. Only five species of plants have been honoured so far by having their genomes completely sequenced229, one of which is Populus trichocarpa (Torr. & A. Gray)12. Following the development of microarray techniques for Populus, three reference databases have been compiled for analysing data related to Populus gene expression patterns, namely the JGI, PopulusDB and UPSC-BASE databases. JGI, the Joint Genome Institute of the U.S. Department of Energy, created in 1997, (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html) played an essential role in the sequencing of the human genome. Following the tremendous success of the human genome project, JGI has expanded its interest in sequencing non-human organisms, and sequences of about 44 species are available in its online databases so far (http://genome.jgi-psf.org/), largely species specially related to bioenergy and sustainable development. Since trees represent a major source of biomass, JGI supervised the Populus trichocarpa genome sequencing project, which was run in several universities around the world, including UPSC. PopulusDB (www.populus.db.umu.se) is available through the Umeå University server as part of the Swedish Populus Genome project, a collaborative effort run by UPSC and the genome centre at the Royal Institute of Technology in Stockholm (KTH). It is a major search website providing access to constantly updated information on UPSC POP2 poplar microarray unigenes (PUs) and clones compiled since 2004. This database provides information on EST libraries obtained from the T89 P. tremula x tremuloides hybrid.13, 230 The EST X library described in Paper I was used as a tool for isolating transcripts apparently related to phenomena in the late maturing wood (Figure 9). Results obtained from a gene ontology analysis of the ESTs in the respective libraries also suggested that fibre cell death and senescence might be closely related processes. In addition, many abundant transcripts in the X library were highly abundant in other EST libraries, including libraries obtained from samples of woody tissues undergoing secondary cell wall formation (Paper I).

31 1 Programmed Cell Death in Xylem Development

UPSC-BASE is the UPSC microarray database in the BioArray Software Environment (BASE)231, 232. The application of this environment to the Populus UPSC database has been thoroughly discussed in Sjödin’s thesis.229 Due to the extensive use of microarrays and the accumulation of data, concerns about their standardization and availability to the public had been raised, and microarray data must now comply with MIAME criteria233. UPSC-BASE therefore allows the Populus microarray data to meet the international standards and allows access to normalized data from various experiments, thereby increasing the scope for data mining.

Figure 9. The 50 most highly expressed transcripts in xylem fibres undergoing cell death and their distribution among the different EST libraries in PopulusDB. From Paper I. Transcripts are colour-coded according to the ratio of their abundance in the fibre cell death library versus the early fibre development library. The colour scale indicates the numbers of ESTs per library and per transcript. In Paper I we successfully demonstrated the power of combining focused microarrays and in silico database expression analysis (PopulusDB), by the strong correlations between the abundance of ESTs in the X library and the expression profiles in corresponding tissues obtained by microarray analysis. This analysis detected an unknown but highly upregulated candidate gene,

32 4. Xylem programmed cell death encoding a glycosyl hydrolase family 1 protein (POPLAR.11628), which proved to be the most abundant EST in the X library, and highly expressed in the corresponding microarrays. Stress Response & PCD database – A customised database The Genevestigator reference expression database and meta-analysis system (www.genevestigator.com) itemizes standardized microarray expression data from five organisms (human, mouse, rat, Arabidopsis and barley).234 Some of the Arabidopsis microarrays are devoted to experiments related to stress responses and cell death. Therefore we extracted available expression data on cell death and stress responses in Arabidopsis to construct a customised ‘stress response and PCD’ expression database. In Paper III, we compared the closest homologues of the hybrid aspen genes that were upregulated in samples Mx1-Mx3 in this expression database, to identify genes that may be involved in fibre PCD. Genes were then selected that were present in the ‘stress response and PCD’ customised expression database and strongly upregulated in senescence conditions in the ‘PCD:senescence’ experiment235, due to the apparent similarity of cell death- inducing conditions in senescence and xylem cell death. In order to specifically select candidate genes involved in fibre cell death, we removed genes that were upregulated in the ‘Hormone (BL/H3BO3)’ experiment, since it was assumed that these genes might also be involved in the differentiation of vessels. Finally, about 124 novel candidate genes involved in fibre cell death were identified in silico and this Stress Response & PCD set, isolated from Genevestigator subsets, proved to be an efficient tool for in silico gene identification. Transcription factors database and PCD Transcription factors (TFs) play a major role in regulating mRNA transcription in eukaryotes.236 In plants, the many available gene lists have enhanced attempts to produce a TF database, but to date there has been only published attempt to list all known poplar transcription factors237. In the EST approach we applied in the study described in Paper I, we faced challenges posed by the lack of functions in transcripts when attempting to isolate TFs involved in the process of cell death in late maturing wood (from the microarray list) since many of the 50 most highly expressed genes encoded putative proteins, and only one, a zinc finger protein, was characterized as TF (Figure 9). Therefore, in the analysis presented in Paper III (Supplementary Figure 2), we focused on gene models and used the available Populus TF databases237, 238 to seek significantly differentially expressed TFs in our microarrays. Among the 124 candidate genes for fibre PCD (Figure 8 in Paper III), only six TFs were isolated, one of which, a bZIP family TF, was still differentially expressed in the latest maturing xylem sample (Mx3). This suggests that the reduction in the number of induced TFs across the late maturing xylem reflect a major change in gene expression patterns. Moreover, the fact that a TF was still upregulated after most of the secondary cell wall deposition had

33 1 Programmed Cell Death in Xylem Development occurred indicates that xylem cell death is subject to specific transcriptional regulation.

4.3. Programmed cell death characterization in Populus: new challenges 4.3.1 Wood microsectioning approaches When analyzing wood formation several challenges must be faced, some of which have been discussed by Chaffey1. One of them is that xylem cells are simultaneously produced both in axial and horizontal directions in complex intertwined mixtures of at least three cell types, posing major problems when attempting to characterize processes specific to a cell type or stage of wood development. Therefore, appropriate microtomy and microscopy techniques were essential in this project for accurate wood sampling and precise observations at the organellar level. Wood was successfully sequentially sampled in the tangential plane by cryosectioning239, a particularly suitable sampling method for developmental analyses because it allows sectioning both at the micrometer level and at - 20 ºC. Since cryosectioning permits sampling down to 10 µm levels, it also allows highly accurate sample isolation, significantly reducing cross-talk with other processes in following analyses. Moreover, the freezing environment en- hances the conservation of nucleic acids, which is a very important consideration since the yield of cellular components from such tiny wood samples (which can also have high levels of interfering compounds) is often lower than those yielded by samples of other tree parts (e.g. leaves), mainly due to their small size. Samples for the microarray experiments described in Paper I were obtained by gross scraping of the developing and late maturing xylem regions of the Populus stem (differentiating between the two regions on the basis of their colour and texture). In contrast, for the more accurate developmental microarray experiment described in Paper III, a series of 50 µm-thick stem samples was obtained by cryosectioning. Therefore, we could identify gene expression patterns related to fibre cell death (in Mx1, Mx2 and Mx3 samples; Paper III) without noise from genes related to early xylem or phloem development. 4.3.2 Anatomical characterizations Until the mid-1970s, characterization of various plant processes, such as vascular differentiation, was mostly based on EM micrographs in which anatomical details were visualised solely using electron-dense stains240. In this way, the first anatomical hallmarks of PCD, such as nucleus condensation and fragmentation, and increases in mitochondria size, were first observed in differentiating vessel elements of corn xylem.241 However concerns about the use of fixatives were raised, due to clear variations in micrograph quality depending on the fixatives.

34 4. Xylem programmed cell death

The quality of the sample preparation and the reliability of the micrograph observations are especially important when anatomical observations are used to support investigations of cell death processes, because it is often difficult to distinguish between fixation artefacts and phenomena associated with cell death processes per se. Thus, to augment the anatomical observations, the microscopy techniques were improved by the use of process-specific markers, associated with immunogold-labelling for instance, or better sample fixation methods, such as HPF-FS. Therefore, in the anatomical observations of dying fibres, monitoring changes in organelles and nuclei during cell death, presented in Paper III, HPF-FS fixation and the powerful tool TEM were used (see 2.4.3 and Figure 4). This approach yielded new insights into the disappearance of organelles in late maturing fibres. In the wood, between 500 and 750 μm from the cambium, we observed that all cytoplasmic organelles had disappeared, even though the tonoplasts of the cells were still intact (cf. Figure 4e and c). However, due to the still possible influence of fixation and generally low proportions of fixed cells (only 10 to 30 % of the cells had properly fixed contents after HPF-FS), the use of markers to follow the fate of the organelles during fibre cell death would provide valuable additional information. 4.3.3 TUNEL assays Generally during the course of cell death, nuclear DNA cleavage is catalyzed by endonucleases, such as AIF, which first generate high molecular weight fragments of 50 Kb (even though generated fragments can have a molecular weight up to 300 Kb) that are subsequently further cleaved into oligonucleosomal subunits of 200 bp under the action of endonuclease G.82, 242 Before the introduction of the Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End-Labelling (TUNEL) assay, several in situ techniques were used in which replicating DNA was exclusively labelled, exploiting labels such as [3H]thymidine, 125IUdR or BrdU. Early in the 1970’s the possibility of using TdT activity as a means to detect PCD was raised after single-strand breaks within the DNA core were identified. However, it was not until 1992 that the TUNEL assay was introduced for the first time243 as a reliable tool for identifying in situ cells undergoing cell death. This technique is based on the TdT enzymatic labelling of free 3’-OH ends of DNA with tagged dUTPs, which represents a major break-through because it allows direct in situ assessment of DNA fragmentation in single cells within fixed tissues in a simple but accurate way. Samples are fixed and permeabilized before the DNA fragments are enzymatically labelled using tagged dUTP nucleotides (the tag being FITC, biotin or digoxygenin). A key characteristic of TdT is its capacity to label both recessed and blunt ends in double-stranded DNA breaks. In addition, both negative controls devoid of TdT and positive controls subjected to DNase or

35 1 Programmed Cell Death in Xylem Development restriction enzyme treatments are concomitantly run. Furthermore, in FITC-based TUNEL assays, samples can be directly assessed by direct observations under a fluorescence microscope, simplifying the procedure. However, since oligonucleosomal fragmentation is not an obligatory hallmark of apoptosis244 and in rare cases of necrosis or autolytic cell death TUNEL-positive DNA fragmentation has been reported, the TUNEL assay should be complemented by the use of other techniques and conclusions about apoptosis should be drawn cautiously245.

Figure 10. Pattern of DNA degradation in Populus wood assayed observed by confocal microscopy following TUNEL staining, adapted from Paper III. (a) TUNEL staining within the whole living woody tissue and (b-g) within a zone in the wood with abundant positive TUNEL-staining in the fibres (500-750 µm from the cambium). Green fluorescent dots indicate nuclei with fragmented DNA. Positive controls were treated with ApaI restriction enzyme before staining (c,f) and negative controls were stained without the TdT enzyme (d,g). Images represent merged pictures taken through 100-µm thick sections in either the transverse (a-d) or longitudinal plane (e-g). Bars: 50 µm. In studies reported in Paper III, freshly cut cryosections were used for FITC- based TUNEL assays and signals were detected using confocal microscopy to scan in three dimensions. Since a nucleus may be localized anywhere in a fibre, a tagged nucleus will be detected only if it was originally contained in the cryosection of a transverse section of wood and the z-plane of the confocal scanning extends sufficiently deeply within the section. On the other hand, longitudinal sections may often contain more nuclei, from several layers of fibres, since the width of a xylem woody fibre is never more than 40 μm. Thus, to increase sensitivity in our system, both transversal and longitudinal sections were assessed using TUNEL assays (Figure 10) together with vitality staining (NBT), and we observed high synchronicity in fibre development, including DNA degradation.

36 4. Xylem programmed cell death

4.3.4 Single cell gel electrophoresis or Comet assays Due to the limitations of TUNEL staining and conventional gel electrophoresis, DNA fragmentation resulting from PCD might sometimes be difficult to detect. Thus, single cell gel electrophoresis (SCGE), also known as the Comet assay, is of great value for this purpose since it is based on isolated intact nuclei, i.e. nuclei with intact nuclear membranes. This technique was first described in 1984 by Östling and Johnson246, who used it in investigations of irradiated animal cells. Two major variants of the assay were subsequently described: one using strongly alkaline conditions (pH>13) for reliable detection of DNA damage and repair247, and the other milder alkaline (pH 12.3) or neutral (pH 9.5) conditions248. In principle, damaged DNA from single cells migrates under a weak electric field, within a thin agarose matrix, showing a comet ‘tail’ while the ‘head’ of the comet containing the bulk DNA, surrounded by nuclear membranes, stays steady (Figure 11). This technique is now widely used as a reliable, robust method for both in vitro and in vivo tests due to its rapidity and convenience. SCGE/Comet assay can be applied to almost any cell types from animal or plant species for checking DNA damage.249 Moreover, software is available that readily provides details of useful parameters, such as ‘tail length’ and ‘tail moment’, the latter corresponding to the torsional moment of the tail (see the equation below), which is regarded as one of the best indices for assessing DNA damage250. Tail Moment= (% DNA in Tail x Tail Length)/ 100

Figure 11. Results of single cell gel electrophoresis analysis (Comet/SCGE assay) of a nucleus from a xylem fibre showing DNA fragmentation. The nucleus was embedded in a 1 % agarose matrix and visualized by fluorescence microscopy with 0.1 % ethidium bromide. The head, comprising the bulk DNA contained in the nuclear membrane, is fixed in the matrix and the tail containing DNA fragments migrates under the electric field (1 V.cm-1). Bar= 50 μm. In studies described in Paper III, the nuclei isolation technique was adapted from methods published for leaves, using 50-μm wood cryosections. Due to the difficulty of defining non-Comet nuclei, the agarose microgels were entirely scanned using fluorescence microscopy and each single nucleus showing ‘Comet morphology’, i.e. presenting a ‘tail’ (Figure 11), was photographed to obtain the complete Comet-nuclei count per sample. This could represent up to 300 pictures

37 1 Programmed Cell Death in Xylem Development per sample. The resulting images were then analysed using Comet Assay IV® software (http://www.perceptive.co.uk/cometassay/). Using this technique, we observed severe degradation or damage in DNA of fibres throughout a large zone of the wood, suggesting that fibre DNA was at least partially degraded in early developmental stages, but persisted for a long time before complete fibre autolysis (Figure 2 in Paper III). 4.3.5 DNA staining with DAPI/PI Some phenomena associated with cell death in samples can be visualized using fluorescence microscopy in conjunction with specific stains, such as DAPI and propidium iodide (PI). 4’,6’-diamidino-2-phenylindole hydrochloride, DAPI, fluoresces after specifically binding to DNA sequences enriched in A-T, corresponding to the minor groove.251 Since DAPI penetrates through intact plasma membranes, it can be an ideal stain for assessing DNA quality (intact or degraded). In some cases, in conjunction with high resolution microscopy, DAPI also allows the description of nuclear morphology during cell death (Figure 12).

Figure 12. DNA staining using DAPI in longitudinal sections of the xylem of P. tremula x tremuloides. (a) DAPI-stained DNA in nuclei from fibres and rays, indicating the complex intertwined mixture of the various cell types in wood. (b,c) DAPI-stained DNA in a fibre nucleus, which starts being pressed against the cell walls by the vacuole (arrows). (d) DAPI-stained DNA in a fibre nucleus, which is strongly pressed against the cell walls, explaining its elongated morphology. (e,f) Lobbing/fragmentation of the DNA in fibre nuclei, following the vacuolar rupture, assessed by DAPI staining. Bar: 50 μm. In this thesis, DAPI staining was used as a first assessment to the changes in the morphology of DNA and nuclei during the process of cell death, especially in fibres. The DAPI staining has been observed to bind as well cell walls, explaining the background noise (Figure 12a,b). In a living fibre, the nucleus looks more or less elongated, due to the increasing pressure from the growing vacuole (Figure 12b,c). Close to vacuolar rupture, the nucleus is strongly pressed

38 4. Xylem programmed cell death by the imposing vacuole against the cell walls, showing a very elongated morphology (Figure 12d). Finally, after rupture of the vacuole, the degradation of the DNA, stained with DAPI, indicates a typical lobbed morphology (Figure 12e,f). Therefore, in spite of the unspecific binding of the stain to the cell walls, DAPI reveals its ability to roughly assess DNA quality. On the other hand, propidium iodide (PI) is a good stain for detecting dying cells since it cannot cross intact membranes. When membranes lose integrity and become perforated, i.e. when cell death occurs, they become loose and PI can then penetrate the cells. Therefore, the further cell death has progressed, the more the PI stain fluoresces, suggesting that this dye can be used for semi-quantitative cell death measurements. Thus, DAPI and PI are powerful tools for assessing cell death, as long as background noise is kept to a minimal level. The studies this thesis is based upon have been dealing with problems related to the ability of plant cell walls to strongly bind both DAPI and PI, which, in addition to the natural autofluorescence of the secondary cell walls, have reduced the usefulness of these dyes in wood analyses, in some cases. 4.3.6 DNA laddering Although oligonucleosomal fragmentation does not always occur in PCD244, detecting DNA ladders remains a common and simple method to assess PCD or apoptosis. The appearance of DNA ladders, resulting from the fragmentation of chromatin between nucleosomes, was first noted by Wyllie252 in 1980. As ex- plained above, nucleases degrade DNA first into larger fragments and then progressively, into smaller, approximately 200 bp, pieces. On gel electrophoresis this will result in a DNA ‘ladder’ of fragments with sizes that are multiples of 180 bp. This method provides a reliable hallmark for detecting PCD, since the random degradation observed in necrosis results visually, in contrast, in a smear. Potential problems due to the low DNA concentrations available may be overcome by running DNA in three-dimensional capillary electrophoresis. In the studies reported in Papers I and III, attempts to detect DNA laddering were not successful. Even in the absence of possible technical reasons for this failure, it does not necessarily mean that PCD does not occur during xylem wood fibre development, firstly since DNA laddering is not an obligatory hallmark of PCD, and secondly because fibres seem to have evolved a unique form of cell death that does not necessarily include typical features of other PCD proces- ses18, 212. In Zinnia TE cell death, no laddering has been detected so far either, raising the possibility of a similar pattern in xylem cell death of both vessels and fibres, which suggests the existence of an alternative route to PCD not involving previously identified nucleases. Furthermore, the pattern of DNA fragmentation observed in Comet assays of nuclei isolated from fibres (Paper III) supports the possibility that a different DNA degradation pattern may occur, without typical laddering. It is also possible that no laddering was detected due to the very low

39 1 Programmed Cell Death in Xylem Development recovery of DNA from the late maturing xylem scrapings in Paper I or from pooled cryosections in Paper III, which could not be detected by standard agarose gel electrophoresis. Three-dimensional capillary gel electrophoresis might provide better sensitivity for detecting such small DNA fragments in low concentrations. 4.3.7 Use of markers In the light of the complexity associated with cross-talk between different types of cell death in plants, correlating a specific PCD type to a specific developmental aspect or stress response would be eased by the use of specific markers. For instance, PCD markers could aid in the selection of candidate genes or help ascertain that a morphological observation is not the result of an artefact. Indeed, it has been demonstrated that marker genes could help to distinguish between several types of cell death when expressed concomitantly.170 Understanding PCD during xylogenesis is also facilitated by the use of marker genes, e.g. two xylem cell death marker genes encoding cysteine peptidase (XCP1 and XCP2) have been recently characterized on the basis of their specific expression patterns in late developing xylem elements undergoing cell death.37, 253, 254 Findings that transcripts of the Populus XCP2 homologue were enriched in the X library (Paper I), and upregulated during late maturation (Paper III), suggested that this gene might also be involved in fibre cell death and perhaps used as a xylem cell death marker in our system. In addition, in the study described in Paper II we utilized the XCP2 promoter coupled to the reporter gene β- glucuronidase (GUS) as a marker to study cell death in xylem vessels of the acl5 mutant. The GUS expression pattern in the acl5 background allowed the lack of ACL5 function and the marker expression to be correlated, thereby providing a means to associate the function of the ACL5 protein to a specific stage of vascular development. Furthermore, combining our microarray data and the data from the ‘PCD:sensecence’ experiment in the Genevestigator database resulted in the identification of 124 fibre cell death candidate genes (Paper III), which should result in the identification of additional marker genes for fibre cell death after further verification and characterization of their expression patterns. Those genes encode TFs, proteases or enzymes involved in the metabolism of various signalling compounds, such as trehalose and sphingolipids. However, the most complex phase of developing specific markers is their initial isolation from huge gene lists, which is highly time-consuming.

40 5. New insights into xylem developmental cell death

5. New insights into xylem developmental cell death

Populus vs. Arabidopsis: two models, one aim Choosing a model species to elucidate xylem PCD in plants is certainly a tough decision. However, despite the tremendous difference between Populus, a true woody genus (Papers I and III), and Arabidopsis, which only shows significant formation of secondary xylem in the hypocotyl, and even there, only after extended growth periods (Paper II), we demonstrated (Papers I, II and III) that both model species have specific advantages for studying xylem cell death due to their differences in cell size, ease of handling, transformation time, etc. Moreover, the possibility of combining normalized experimental data from both systems greatly increases the scope for isolating genes and accelerating progress at the molecular level.

Organelle disappearance before tonoplast rupture in fibres Prior to our studies, PCD in TEs, which had been largely observed in Zinnia, was believed to be strongly related to vacuolar rupture and subsequent degradation of cytoplasmic contents due to the release of hydrolytic enzymes into the cytoplasm. However, we showed that in developmental cell death in Populus xylem fibres, the cytoplasm is first emptied of its organellar contents without visible rupture of the tonoplast, thereby excluding vacuolar burst as a trigger of PCD in this system (Paper III) and implying that fibres are autolysed before the time of death. This type of gradual degradation of cytoplasmic contents is suggestive of autophagic cell death in the fibres, a conclusion supported by earlier findings on autophagic PCD during petal senescence.

In silico isolation of potential novel fibre cell death markers Bioinformatics has been proven in our studies to be highly valuable for isolating putative candidate or marker genes (Papers I and III). In addition to the 124 characterized genes associated with fibre maturation and death, seven autophagy- related genes (Figure 7 in Paper III) were isolated that appeared to be specifically expressed during late fibre development and cell death, suggesting that autophagous cell death might be activated in xylem fibres only during late maturation, together with some autophagy-dependent proteases or signalling compounds. Moreover, since nuclear degradation is triggered before the death of the fibres, i.e. before tonoplast rupture, additional or completely novel nucleases are expected to initiate the slow DNA degradation, such as a putative endo- /exonuclease or the homolog to the Arabidopsis AIF protein.

41 1 Programmed Cell Death in Xylem Development

ACL5: vessel cell death inhibitor and fibre development enhancer. Using Arabidopsis hypocotyls with extensive secondary growth in vascular tissues, we showed that the polyamine biosynthetic ACL5 is involved in the prevention of premature cell death of xylem vessel elements, and thus in controlling xylem specification through regulation of the lifetime of xylem elements (Paper II). Most strikingly, we observed that correct xylem vessel specification, leading to differentiation of mostly pitted-type vessels, was essential in formation of hypocotyl xylem fibres, suggesting a totally novel mechanism for the control of xylary fibre development, in which ACL5 appears to be a key player, possibly acting as a regulator of tonoplast channels in xylem elements.

6. Future perspectives for PCD in woody plants

Despite our growing understanding of PCD, and more specifically apoptosis in metazoans, at both morphological and molecular levels, extensive research is still needed to clearly understand plant PCD. In woody plants, our knowledge of cell death is still rudimentary, and much remains to be understood. Some of the key animal apoptosis elements have identified counterparts (Figure 5), with apparently analogous activities in plants, demonstrating clear conservation between kingdoms. However, there are few counterparts in comparison to the number characterized to date in animals. More strikingly, only parts of the animal apoptosis pathways have been identified in plants. For instance, plant metacaspases, which are homologs to animal caspases, lack caspase activity, suggesting that the release of cytochrome c in association with cell death in plants activates substrates other than caspase homologs (either other proteases with caspase-like activity, as demonstrated so far, or other, unknown cell death- related proteases). Similarly, the presence of BI-1 in plants, but not its target molecules Bcl-XL/Bcl-2, suggests that the BI-1 either has very different targets in plants or that the targets similar to Bcl-XL/Bcl-2 have not yet been found. Therefore, either some parts of animal apoptosis and plant PCD pathways are conserved, while others are different and thus need to be more deeply examined, or due to evolutionary distances between kingdoms, identification of consensus sequences may be impossible, even though functions may be conserved. These features complicate the choice of approaches to isolate key elements, e.g. genes or proteins. The easiest approach would probably be to look for homo- or orthologous elements in plants to genes or proteins that have already been characterized as key components in metazoan cell death, due to the tremendous amount of work already published. However, as already demonstrated above, plants seem

42 6. Future perspectives for PCD in woody plants to have evolved their own PCD pathways, to some extent. Moreover, even within the metazoans, sequence identity is often very low, suggesting that the sequence homology between metazoans and plants will be very challenging to characterize. The possibility of using three-dimensional protein folds to look for conserved sequence homologies may therefore substantially improve searches for similar sequences. Future advances in plant cell death will therefore require an open mind, taking into consideration both what is known in animals, as well as the unknown, and some imagination. Recent advances in understanding metazoan cell death have allowed the creation of a powerful degradome database, including information on various enzymes involved in the activation of cell death or apoptosis. Compiling similar resources for plants has taken some time, accounting for some of the apparent gaps in cell death between metazoans and plants. So far, three enzymes (VEIDase, VPE and type-II metacaspase) have been identified in the plant PCD degradome. Their cellular sublocalizations are distinct, but it seems that at least metacaspase activates VEIDase in the cell death process, suggesting a cascade-like process may be involved. However, no BI-1 inhibitor of cell death in plants has been identified so far in this cascade, supporting the possibility that a major component is still missing. Certainly more elements remain to be discovered in the degradome, especially given the various types of cell death that occur in plants. In addition to these general perspectives on cell death in plants, several additional issues raised by studies on plant woody elements have to be considered in attempts to elucidate fibre cell death. First, a major hindrance to work with woody species, such as trees, is the time required for obtaining mature tissues to analyse. Further, the wood often consists of several types of cells, embedded within an intricate network, making isolation of specific types of cell very challenging and complicating the isolation of PCD-related genes in a single cell type, since several types of cells may undergo PCD. Therefore, advanced tools in the production and treatment of data are required, such as single cell assays and in silico analysis. In the studies reported in Paper III we successfully used several techniques concomitantly to isolate specific woody fibre cell death cues. Besides the several cell types present in wood, secondary cell wall deposition occurs more or less synchronously with xylary fibre death. The presence of secondary cell walls poses a significant problem, due to their physical, mechanical and chemical resistance, and since they represent a major proportion of the cell they are almost inevitably present in samples used for molecular assays. Thus, as observed in this thesis, there is a need for better fixation systems for morphological analyses due to the disturbances or uncertainties related to the occurrence of secondary cell walls, which highlights the obvious complexity of working with woody species in comparison with other plant species or metazoans.

43 1 Programmed Cell Death in Xylem Development

7. Acknowledgments

Through all my years in Sweden, and this great PhD project, which sent me around the world, I met a lot of people who helped me, in one way or another, to achieve this work. As Aimé Césaire once said: “Ce que l’on fera pour soi dispa- raîtra avec soi, seul ce que l’on fera pour les autres nous survivra” [What we achieve for us only will disappear with us, only what we achieve for others will survive us], I dedicate this thesis to all of you that I met on this path. In particular: Hannele, my supervisor. This five-year PhD with you was certainly a very interesting journey. In spite of all the disagreements, the critical situations between us and this tough and insane past year, with me in Germany while trying to write article and PhD thesis plus German classes, we somehow managed to achieve the goals (but not so sure how…). I did learn a lot though about management... Prof. Christina Dixelius, my opponent. I am very flattered that someone of your stature has agreed to judge my work. Dr. Lazlo Bako, Docent Ewa Mellerowicz and Docent Ines Ezcurra, the jury members and Slim (Prof. Göran Samuelsson), the head examiner, I want to thank you to for giving your time to consider and judge this work. Stefan J., Per G. and Ewa M., my reference group. You helped to surmount the downs I encountered in the project, and we managed to enjoy and take advantage of its positive parts (despite it being so easy to forget about what does work). Stefan J. and Slim, I wanted to thank you in particular for being there when things went wrong and were impossible to control, especially recently, when I felt very much alone in the abyss when preparing this. UPSC. I would like to thank all of you at UPSC that were of such a great help among PhDs, Post Docs, PIs, technical or cleaning staff, and that made this wonderful atmosphere in the department. Thanks a lot. Lena Maler in the Doctoral School in Genomics and Bioinformatics for the PhD network you created. Edouard, Iwanka, Sunil, Luis, Maria E., Micke J., Jane and Nikolay, my former labmates. Thanks for all the discussions. A very special thank to Edouard and Iwanka, for shining light on my PhD path. Edouard, I really owe you a lot for this achievement. Iwanka, thanks for this enthusiasm of yours. Prof. Jean-Paul Monge and Dr. Ola Alinvi. I owe you special thanks. You have been a part in this achievement, by helping me in my aim to become an open-minded scientist. Mr Monge, thank you so much for supporting me during the Master abroad. Ola, I specially remember your patience and your advices on lectures and projects, which have been so useful as well during these 5 years. Dr Lacey Samuels and Minako in UBC, Vancouver. You really made my stay in your lab a wonderful experience and I cannot imagine working without microscopy now. I really enjoyed the work. And Vancouver is just awesome!

44 7. Acknowledgments

Eva Selstam and Leszek K. Thanks for helping me with teaching. Eva, thanks for introducing us to the art of bonsai-making. The Fish Tank. Over the years, it seemed that we all swam so well together, in this rather nice Christmas-looking aquarium. But we all reached for wider seas to swim in and eventually left the tank. However, I should thank Andreas. You have had an incredible patience with me, even though I often got on your nerves, I will miss you (but you probably will not miss me). Nathalie. Who would have expected that the two of us would have turned the office into such sweet chaos? We had such nice times together. Oskar. Thanks for being helpful at all times, with your diplomacy, it was always nice to see you around. Dame Rozenn. A special thank for all your support and for always being there, no matter what. You are so kind and helpful in any situation. You deserve so many good things in your life. I miss you a lot. Thanks for being a friend. Le Bud and Marco. Thanks for ALL, and ALL means so many things (trainings, parties, evenings, talking, fika and all the help when we moved out). I really hope we keep in touch, no matter what. Kjell O. and Lenore J., for all your technical help. Monica I. and Inger W., for all your help regarding administrative stuff. The French gang (Delphine, Mélanie, Yohann, Catherine B., Laurent, Mathieu, Gaia, Tatiana, Nick, Christine, Laurent, Julien S., Pepito, Mille-Feuille and Emmanuel) for always helping when needed and sharing nice fika, week-ends, evenings, parties, training and Thursday’s lunches at Corona. Michel Gautier, the Honorary Consul for the French Embassy. Thanks so much for all the help with the administrative issues, and for your kindness. Nadia. You are not only a great supporter and sweet friend, but also an amazing scientist. It is sad you live so far now, but I will soon be visiting in Japan. Nat S. First of all, thanks for taking your time to go through this. Thanks as well for always being there as a friend, in spite of the distance, it is so nice to talk about anything with you. You are really an amazing friend. I miss you so much. Junko “Dono”, Maribel and Agniezka. If the samurais had a queen, Junko, it would be you. I had so many laughs with you. Maribel and Agniezka. Thanks for the Flamingo team. Linda R., Andras G., Lorenz G. For the nice moments in my former office, group meetings and Vietnam. Johanna K. For the crazy and awesome times in conferences and travel together. Krisse och Martin. Tack så mycket för att vara så otroliga kompisar och Krisse för att vara så tålmodig för att lära mig Svenska. Synd att vi inte hade mer tid tillsammans i Sverige. Jag hoppas, hur som helst, att vi kommer att träffas igen i fram tiden. Jordi. I could not forget about you. You have been the one pushing me in this direction in the first place, telling me I should go for it. Dank U, als aandenken aan de wonderlijke momenten samen. I mis Je zo Jordi.

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Jill and Eve, my Dear Bellas. I might not always take the time to send you news, but it does not mean I do not think about you. You made my first years in Sweden so won derful, and my English so much better. Adeline, Olivier and Tessa. Thanks for being so kind and so helpful at all times. It is always such a delight to chat with you. Sad we cannot meet more often. Hélène, Stéphanie, Estelle, Emmanuelle. Je regrette de ne pas avoir été plus présente. Mais j’espère de tout coeur que vous ne m’en tiendrez pas rigueur et que la thèse passée, on va pouvoir enfin se revoir. Joan and Roger G. You gave me the courage to believe in myself that I was able to live abroad. I am so proud I achieve something I did not think would have been possible to make. Sorry for not writing so much lately. But I will change that from now on. Maik S., Jean-Michel R., Henrik J. You will always be great friends despite the distance. I really hope we keep in touch. Anatoli D. und die TSG Friesenheim Fechtabteilung. Es ist nicht immer so leicht, in einem neuen Land zusammen mit einer neuen Sprache zu leben. Aber Fechten war gut auf dem deutschen Leben zu lernen. Anatoli, du bist ein riesiger Fechttrainer. Christina, Felix, und Marcus L.; Christine, Daniel, und Jakob L. Danke für eurer Gedul digkeit und Niedlichkeit. Bei meiner Ankunft in Deutschland, ware alles ganz schlimm aber es fühlt sich immer besser, weil ich Glück hatte um euch zu treffen. Knut and Kicki Irgum. Thanks for all the nice invitations to your home, the great dinners, the nice stargazing evenings and “photo camera” exhibitions. Special thanks also for owing the university printer, because “If you print a poster, you’ll get married later”. This could not be truer for me (I hope you get this…). My family and Julien’s family, always there to support me and believe in me, especially once we moved to Germany. Brother, for being so much better now. Last but not least, Julien and Thorkel. There is one thing for sure that I can tell, I would not have completed this without You two, your loving sweetness and Julien’s freaky organization. I hope we can now move on in our new life in Germany or elsewhere.

46 8. Literature Cited.

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