BIOS
Research Doctorate School in BIOmolecular Sciences
Doctorate Course in Molecular Biotechnology
PhD Thesis
Programmed cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds
Student: Dr. SIMONE CASANI
Supervisor: Prof. Luciano Galleschi
- TABLE OF CONTENTS -
ABSTRACT ...... 5
LIST OF ABBREVIATIONS ...... 8
1. INTRODUCTION ...... 9
1.1. PROGRAMMED CELL DEATH ...... 9
1.2. THE SIGNIFICANCE OF PROGRAMMED CELL DEATH ...... 11
1.3. MOLECULAR MECHANISMS OF APOPTOSIS ...... 12
1.4. CASPASES (CYSTEINE ASPARTATE-SPECIFIC PROTEASES) ...... 17
1.5. MORPHOLOGICAL AND BIOCHEMICAL FEATURES OF APOPTOSIS ...... 20
1.6. PROGRAMMED CELL DEATH IN PLANTS ...... 22
1.6.1. Defence PCD ...... 23
1.6.2. Developmental PCD ...... 24
1.7. FEATURES OF PLANT PROGRAMMED CELL DEATH ...... 27
1.7.1. Cell and nucleus morphology ...... 27
1.7.2. DNA fragmentation ...... 28
1.7.3. Cytochrome c release from mitochondria ...... 29
1.7.4. Cleavage of the poly(ADP-ribose) polymerase (PARP) ...... 29
1.7.5. Protease activities ...... 30
1.7.6. Nuclease activities ...... 39
1.7.7. Hormonal control of programmed cell death ...... 42
2. THE PLANT SYSTEM ...... 44
3. AIM OF THESIS ...... 46
4. MATERIALS AND METHODS ...... 47
4.1. PLANT MATERIAL ...... 47 2 4.2. VIABILITY STAINING OF MEGAGAMETOPHYTES ...... 47
4.3. ANALYSIS OF THE MEGAGAMETOPHYTE TISSUE SECTIONS ...... 47
4.4. ANALYSIS OF SOLUBLE AND INSOLUBLE PROTEINS ...... 48
4.4.1. Protein extraction for SDS-PAGE analysis of soluble and insoluble proteins ...... 48
4.4.2. SDS-PAGE of soluble and insoluble proteins ...... 48
4.5. ANALYSIS OF THE MEGAGAMETOPHYTE PROTEOLYTIC ACTIVITIES ...... 49
4.5.1. Caspase-like proteases (CLPs) extraction and in vitro activity assays ...... 49
4.5.2. Preparation of enzymatic extracts for the detection of total protease activity ...... 50
4.5.3. In vitro protease activity assays ...... 50
4.5.4. In-gel protease activity assays ...... 51
4.6. ANALYSIS OF DNA FRAGMENTATION ...... 53
4.6.1. In situ detection of DNA fragmentation (TUNEL assay) ...... 53
4.6.2. Isolation and electrophoresis of genomic DNA...... 53
4.7. ANALYSIS OF THE MEGAGAMETOPHYTE NUCLEASE ACTIVITIES ...... 54
4.7.1. Protein extraction for the determination of nuclease activities ...... 54
4.7.2. In vitro nuclease activity assays ...... 54
4.7.3. In-gel nuclease activity assays ...... 55
4.8. DETERMINATION OF THE INDOLE-3-ACETIC ACID (IAA) CONTENT IN THE
MEGAGAMETOPHYTE THROUGHOUT GERMINATION ...... 56
4.8.1. Extraction and purification of free IAA ...... 56
4.8.2. High-performance liquid chromatography (HPLC) ...... 57
4.8.3. Gas chromatography-mass spectrometry (GC-MS) ...... 57
4.9. STATISTICAL ANALYSES ...... 58
5. RESULTS ...... 59
3 6. DISCUSSION ...... 81
REFERENCES ...... 92
ACKNOWLEDGMENTS ...... 114
4 ABSTRACT
Programmed cell death (PCD) is a genetically-controlled process necessary for the proper development of most living organisms. In animals the best characterized form of PCD is apoptosis, a highly ordered form of cell death which contributes to the selective removal of damaged or no longer needed cells. In plants PCD occurs after biotic and abiotic stresses as well as during development. In the last years an increasing number of papers reported that plant PCD and animal apoptosis share biochemical and cytological features, such as DNA fragmentation, alterations in nuclear morphology and size and increase in nuclease and protease activities.
The megagametophyte of the bunya pine ( Araucaria bidwillii Hook.) seed is a storage tissue that surrounds and feeds the embryo. At seed maturity, the megagametophyte is a living tissue and contains protein and starchy reserves. Following seed germination, when all the reserves are hydrolyzed and transferred to the embryo, the megagametophyte degenerates as a no longer needed tissue. In the current study it was investigated whether the megagametophyte degeneration could be recognized as a form of PCD, by searching typical hallmarks of apoptosis.
From the initial observations it was possible to highlight marked changes in the size and appearance of the megagametophyte at late post-germinative stages both at the macroscopic and at the cellular level. The viability assay showed a progressive spreading of cell death over the tissue in the post-germinative stages.
The analysis of the megagametophyte protein content revealed a marked reduction of soluble and insoluble proteins following seed germination. Concurrently with the loss of proteins, increased protease activities were detected by both in vitro and in-gel assays. By using different protease inhibitors, it was possible to detect the presence of different classes of proteolytic enzymes in the megagametophyte. Among all the proteolytic enzymes detected, the most interesting appeared to be some proteases well active at late post-germinative stages and highly sensitive towards the
5 caspase 6 inhibitor. The identification of these proteases by a proteomic approach showed that these enzymes had amino acid sequences homologous to subtilisin-like serine proteases of other plants. The peculiarity of these enzymes to be inhibited by the caspase 6 inhibitor and their involvement in the megagametophyte cell death are discussed.
To test the presence of caspase-like proteases (CLPs) in the bunya pine megagametophyte, in vitro activity assays with synthetic substrates specific for different members of the mammalian caspase family were carried out. CLPs appeared to be best active at acidic pH and their activity strongly increased at late post-germinative stages. CLPs involvement in the megagametophyte cell death is discussed.
Other biochemical studies highlighted a remarkable decrease in the megagametophyte DNA content following germination. The DNA electrophoretic analysis revealed internucleosomal cleavage at late post-germinative stages and the TUNEL assay showed an ongoing process of
DNA fragmentation in the megagametophyte nuclei after seed germination.
Concurrently with DNA fragmentation, increased nuclease activities were detected by both in vitro and in-gel assays. The biochemical characterization of the megagametophyte nucleases highlighted that these enzymes belonged to the class of Zn 2+ -dependent nucleases and acted optimally at acidic pH. The involvement of Zn 2+ -dependent nucleases in DNA breakdown is discussed.
Other evidence of PCD came from the cytological analyses of the DAPI-stained megagametophyte tissue sections. These studies allowed to observe a marked reduction in the nuclear size at late post-germinative stages, hence indicating chromatin condensation. Moreover, at these stages, alterations in the nuclear morphology were observed.
Finally, the indole-3-acetic acid (IAA) content in the bunya pine megagametophyte was measured. The amount of IAA (auxin) increased up to mid post-germinative stages, followed by
6 a remarkable decline at late stages. The possibility that auxin functions as a signal of PCD induction is taken into account.
Since apoptotic hallmarks have been detected in the megagametophyte, I propose that the bunya pine megagametophyte degenerates after seed germination by means of a developmentally- regulated PCD.
7 LIST OF ABBREVIATIONS
β-ME: β-mercaptoethanol
BBP: bromophenol blue
CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
CLPs: caspase-like proteases
DAG: days after germination
DAPI: 4,6-diamidino-2-phenylindole
DMSO: dymethyl sulfoxide
DTT: 1,4-dithio-DL-threitol
E-64: L-trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane
EDTA: ethylenediaminetetra-acetic acid
IAA: indole-3-acetic acid leu: leupeptin o-phen: orto-phenantroline pep-A: pepstatin-A
PCD: programmed cell death
PMSF: phenylmethylsulphonyl-fluoride pNA: para-nitroanilide rpm: revolutions per minute
RT: room temperature
SDS: sodium dodecyl sulfate
TdT: terminal deoxynucleotidyl transferase
TUNEL: terminal dUTP nick-end labelling
8 1. INTRODUCTION
1.1. PROGRAMMED CELL DEATH
Programmed cell death (PCD) is an essential feature of plant and animal growth and survival. It plays a pivotal role during developmental processes, by removing misplaced or unwanted cells in specific structures and organs of both animals and plants (Williams and Dickman, 2008). PCD is implicated in the differentiation, maintenance of cellular homeostasis, regulation of development and control of reproduction and senescence (Greenberg, 1996; Kuriyama and Fukuda, 2002). It is an active, programmed event consisting of initiation, genetic control and execution phases. In animals these phases culminate in specific morphological changes within the cell, whose reports characterize as a phenomenon termed apoptosis (Assunçao Guimaraes and Linden, 2004). As already reported by Kerr and co-workers (1972), PCD is not the only way cells die; in fact, cells may undergo also necrosis. The latter, which can be caused by extreme variance from physiological conditions (e.g. hypothermia, hypoxia), is a violent and quick form of cell degeneration affecting extensive cell populations, characterized by cytoplasm swelling, destruction of organelles and disruption of the plasma membrane, followed by the release of intracellular contents and inflammation. Apoptosis instead, involves single cells, usually surrounded by healthy-looking neighbours and is characterized by cell shrinkage, blebbing of the plasma membrane, maintenance of organelle integrity, condensation and fragmentation of DNA and cell fragmentation into membrane vesicles enclosing the intracellular content (apoptotic bodies); the latter are finally orderly removed through phagocytosis (Kerr et al. , 1972; Goodlett and Horn, 2001; Martelli et al. , 2001) (Fig. 1).
9
Fig. 1. Apoptosis and necrosis. Here the main differences between apoptosis and necrosis are shown (from Goodlett and Horn, 2001).
PCD was first discovered in 1964, proposing that cell death is not of accidental nature, but follows a sequence of controlled steps, leading to locally and temporally defined self-destruction
(Lockshin and Williams, 1964). The term apoptosis instead was coined only in 1972 (Kerr et al. ,
1972), having a greek origin with the meaning “falling off” or “dropping off”, in analogy to leaves falling off trees or petals dropping off flowers. This analogy emphasizes that the death of living matter is an integral and necessary part of the life cycle of organisms. For many years PCD was only applied to animal cells and its study in plants was neglected. In 1994 the term PCD was first used for plant cells in reference to pathogen-induced cell death (Greenberg et al. , 1994).
More recently several publications reported apoptotic features in dying plant cells: from these studies, it was highlighted that some steps of PCD are well conserved both in animals and in plants and that PCD functions in many aspects of plant development and survival (Jones, 2001;
Della Mea et al. , 2007; Reape and McCabe, 2008).
10 1.2. THE SIGNIFICANCE OF PROGRAMMED CELL DEATH
The development and maintenance of multicellular biological systems depends on a sophisticated interplay among the cells forming an organism, involving sometimes an altruistic behaviour of individual cells in favour of the organism as a whole. During development, many cells that are produced in excess undergo PCD, thereby contributing to sculpturing many organs and tissues
(Meier et al. , 2000). A particularly instructive example for the implication of PCD in animal development is the formation of free and independent fingers, by massive cell death in the interdigital mesenchymal tissue (Zuzarte-Luis and Hurlè, 2002). Other examples are the development of the reproductive organs (Meier et al. , 2000) and the development of the brain, during which half of the neurons that are initially created will die in later stages, when the adult brain is formed (Hutchins and Barger, 1998). Also cells of an adult organism constantly undergo physiological cell death, which is balanced with proliferation in order to maintain homeostasis in terms of constant cell number. Moreover after stresses that induce serious damages, some cells may undergo PCD. For instance cells with severely damaged DNA (O’Neil and Rose, 2006) that can not be repaired appropriately and infected cells of the immune system (Guiot et al. , 1997) are usually removed by PCD. Other cells which usually undergo PCD are those that have served their function and are no longer required. Examples of this cell category are the cells lining the intestinal and reproductive tracts, which, once they have served their function, lose their earlier identity and morphology and die by PCD (Krishnamurthy et al. , 2000). Also cells that differentiate into specialized cell types undergo PCD. To this cell category belong the keratinocytes present at the skin surface of Vertebrates. During differentiation, these cells extrude their nuclei, as a part of their normal program of redifferentiation and undergo death
(Krishnamurthy et al. , 2000).
11 Taken together, apoptotic processes are of widespread biological significance, being involved in development, differentiation, proliferation/homeostasis, regulation and function of the immune system and in the removal of defective and therefore harmful cells. For this reason dysfunctions or deregulation of the apoptotic program are implicated in a variety of pathological conditions.
Defects in apoptosis can result in cancer, autoimmune diseases and spreading of viral infections; on the contrary, neurodegenerative disorders, AIDS and ischemic diseases are caused or enhanced by excessive apoptosis (Fadeel et al. , 1999). Due to its importance in such various biological processes, PCD is a widespread phenomenon, occurring in all kind of metazoans
(Tittel and Steller, 2000) and apoptosis-like mechanisms even have been observed in plants
(Kuriyama and Fukuda, 2002) and yeast (Skulachev, 2002). Fascinating insights into the origin and evolution of PCD might be given by the fact that PCD is also an integral part of the life cycle of other unicellular eukaryotes and that even prokaryotes sometimes undergo regulated cell death
(Arnoult et al. , 2002).
1.3. MOLECULAR MECHANISMS OF APOPTOSIS
Apoptosis, a tightly regulated and highly efficient cell death event, is a multi-stage process, consisting of induction, regulation and signal transduction and execution phases. In the first stage, the cell receives a signal (a herald of death) that can be generated outside or inside the cell.
The signal then impinges on a receptor and the message is analyzed. This signal is consecutively transferred via receptors or their combinations to a sequence of intermediary molecules
(messengers) and ultimately to the nucleus, where the cell suicide program is activated. The program then implies the activation of lethal and/or the repression of anti-lethal genes.
It has been proposed the existence of a large number of signalling events triggering apoptosis, i.e. the increase of cytosolic Ca 2+ concentration, the oxidative burst caused by an increase of ROS
12 (Reactive Oxygen Species), the increase in reduced glutathione and the collapse of the mitochondrial membrane potential. Vaux and Korsmeyer (1999) suggested that these signalling events may impinge upon the cell death mechanism at any level; in particular, the decision of a cell to initiate or not killing itself is determined by the balance of the cell death-inducing and suppressing signals.
There are different pathways of apoptosis activation (Thornberry and Lazebnik, 1998; Green and
Reed, 1998). Of particular importance is the pathway involving physiological inducers, whose effects are mediated by cell receptors (Thornberry and Lazebnik, 1998), specialized in initiating the apoptotic program. This pathway can be schematically represented as follows: inducers → receptors → adapters → first order effectors (initiator caspases) → regulators → second order effectors (executive or executioner caspases). One of the best known receptors involved in this pathway is the Fas receptor. Fas is representative of the TNF receptor family. All these receptors are transmembrane proteins whose extracellular portions interact with ligand-inducer trimers
(Fig. 2). The interaction of the Fas receptor with its ligand (FasL) results in the formation of clusters of receptor molecules and in the binding of their intracellular portions to adapters. These steps lead to Fas activation, interaction with first and second order effectors (caspases) and initiation of the death program (Samuilov et al. , 2000).
13 Fig. 2. TNF-1 receptor. The interaction receptor-ligand leads to the formation of clusters of receptors and the formation of a complex that recruits several molecules of procaspase 8. This results in procaspase 8 activation and induction of apoptosis (from Dash, Apoptosis, http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/receptors.htm).
The receptors-adapters-effectors interaction is mediated by homophilic protein-protein interactions, involving small domains such as DD (Death Domains), DED (Death-Effector
Domains) and CARD (Caspase Activation and Recruitment Domain).
One of the best known first order effectors is the procaspase 8 (Chang et al., 2003). After this enzyme is recruited by Fas, an aggregate, formed by the receptor adapter and procaspase 8, forms. As a result, the prodomain separates from a procaspase molecule and the remainder is cleaved into the major (about 20 kDa) and the minor (about 10 kDa) subunits. Subsequently the two subunits associate and the two heterodimers aggregate to form a tetramer with two independently operating catalytic sites. Thus the procaspase 8 becomes activated and the mature caspase 8, released into the cytoplasm, activates a second order effector, i.e. the procaspase 3.
Proteolysis of procaspase 3 yields the mature caspase 3 and the death program-initiated process becomes irreversible. The caspase 3 acts on several protein substrates and activates other proteases, resulting in a proteolytic cascade responsible for apoptosis. This apoptotic pathway is termed extrinsic pathway, because it is activated by signals that generally derive from outside the cell (Fig. 3).
14 Fig. 3. Extrinsic pathway of apoptosis activation. In this pathway, the procaspase 8 is recruited by Fas, via adapters; once activated, the caspase 8 starts a proteolytic cascade responsible for apoptosis. For more details, see the text (from Gewies, 2003).
Another apoptotic pathway involves mitochondria and the release of different apoptogenic factors from mitochondria to the cytosol. The signals involved in this pathway are usually generated inside the cell (intrinsic pathway) (Kroemer et al. , 1997). Some of the apoptogenic factors involved in this pathway are the cytochrome c, the procaspase 2, 3 and 9 and the protein AIF
(Apoptosis-Inducing Factor). In this pathway, mitochondria release into the cytosol the cytochrome c and other proteins that induce apoptosis. According to some authors, a drastic decrease in the mitochondrial membrane potential ( ∆Ψ ) may be responsible for the release of the apoptogenic factors (Thornberry and Lazebnik, 1998). The ∆Ψ decrease is due to an increase in the permeability of the inner mitochondrial membrane, which may be caused by the formation of giant pores. The opening of these pores depends on a variety of factors, including the depletion of reduced glutathione, NAD(P)H, ATP, and ADP in the cell, the formation of ROS and the increase of cytoplasmic Ca 2+ content. Finally pore opening results in the swelling of the mitochondrial matrix, the rupture of the outer mitochondrial membrane, followed by the release of the cytochrome c outside the mitochondria (Skulachev, 1998). Upon its release, cytochrome c together with the cytoplasmic APAF-1 (Apoptosis Protease-Activating Factor-1) activates caspase 9 (Li et al. , 1997). APAF-1 forms a complex with procaspase 9 in the presence of cytochrome c and dATP or ATP (Srinivasula et al. , 1998) and provides the structural basis for the autocatalytic processing of caspase 9. At this point, a complex with a molecular weight of over
1300 kDa named apoptosome is formed and the procaspase 9 becomes activated. The mature caspase 9 subsequently cleaves and activates the second order effector, i.e. the procaspase 3, leading to cell death (Fig. 4).
15
Fig. 4. Intrinsic pathway of apoptosis activation. In this pathway, mitochondria and cytochrome c play important roles in the activation of apoptosis. For more details, see the text (from Newmeyer and Ferguson-Miller, 2003).
More recently some authors identified a third pathway of apoptosis activation. This pathway involves caspase 12, which is localized to the endoplasmic reticulum (ER). It seems that this pathway is activated by ER stress , including disruption of ER Ca 2+ homeostasis and accumulation of excess proteins in ER, but not by membrane- or mitochondrial-targeted apoptotic signals (Nakagawa et al. , 2000).
In the cascade of apoptosis activation an important role is played by a large group of proteins that are generally named regulators. The ability of adaptors to activate effector is determined by regulators that directly interact with adaptors and effectors. For instance the FLIP can inhibit caspase activation by binding to FADD adapter in Vertebrates; similarly CED-9 can prevent caspase activation by binding to the adaptor CED-4 in the worm (Vaux and Korsmeyer, 1999).
The Bcl-2 protein family represents one of the best studied groups of regulators involved in apoptosis. The Bcl-2 family includes both the pro-apoptotic as well as the anti-apoptotic members, which function primarily in the mitochondria by controlling cell death in Vertebrates
(Tsujimoto, 1998). The anti-apoptotic members of this protein family, such as Bcl-2 and Bcl-XL,
16 prevent apoptosis either by sequestering proforms of death-driving caspases or by preventing the release of the mitochondrial apoptogenic factors such as cytochrome c and AIF (Apoptosis-
Inducing Factor) into the cytoplasm. On the contrary the pro-apoptotic members, such as Bax and
Bak, trigger caspase activation by inducing the release of the mitochondrial apoptogenic factors.
1.4. CASPASES (CYSTEINE ASP ARTATE-SPECIFIC PROTEASES )
Caspases, cysteine asp artate-specific proteases , are of central importance in the apoptotic process.
These enzymes have been termed caspases because their catalytic activity depends on a critical cysteine residue within a highly conserved active site pentapeptide (QACRG) and because these enzymes cleave their substrates always after an aspartate residue. In the cell caspases are synthesized as inactive zymogens, the so called procaspases, which at their N-terminus carry a prodomain followed by a large (≈20 kDa) and a small (≈10 kDa) subunit, which sometimes are separated by a linker peptide. Upon maturation, the procaspases are proteolytically processed by other caspases between the large and the small subunit, resulting in the two separated subunits.
The prodomain is also frequently but not necessarily removed during the activation process. A heterotetramer consisting of each two small and large subunits then forms and represents the active caspase. The activation of caspases through proteolysis is not the only mechanism; in fact, zymogens may be activated also by contact or by holoenzyme formation (Fig. 5) (Hengartner,
2000). The proteolytic cleavage by an upstream caspase is straightforward and effective and is used mostly for activation of downstream, second order effector caspases. In the second mechanism, the recruitment or aggregation of multiple procaspase 8 molecules into close proximity somehow results in cross-activation. The actual process is most probably more sophisticated and more tightly regulated than shown in fig. 5. In holoenzyme formation, cytochrome c and ATP-dependent oligomerization of APAF-1 allow the recruitment of
17 procaspase 9 into the apoptosome complex. The activation of caspase 9 is mediated by means of conformational change and not by proteolysis (Hengartner, 2000).
Fig. 5. Mechanisms of caspase activation. The activation of caspases includes proteolytic cleavage mediated by other caspases (a); activation by contact (b); activation by holoenzyme formation (c) (from Hengartner, 2000).
According to their function, caspases can be divided into the group of initiator caspases (caspases
2, 8, 9 and 10) and the group of executioner caspases (caspases 3, 6 and 7). The first act earlier on the pathway, mainly by activating the executioner caspases, while the latter act later by cleaving a large but specific set of protein substrates, resulting in the amplification of the death signal and in the execution of cell death with all the morphological and biochemical features usually observed
(Earnshaw et al. , 1999).
Some of the most important roles of caspases may be summarized as follows:
procaspase activation, resulting in the formation of mature caspases (Cohen, 1997)
18 cleavage of anti-apoptotic proteins, including members of the Bcl-2 family (Zamzami and
Kroemer, 1999)
hydrolysis of nuclear lamina (Thornberry and Lazebnik, 1998)
degradation of proteins implicated in cytoskeleton regulation (Thornberry and Lazebnik,
1998)
inactivation of proteins involved in DNA reparation and replication and mRNA splicing
(Oliver et al. , 1999)
Caspase activity leads to the inactivation of proteins that protect living cells from apoptosis. A clear example is the cleavage of I CAD , the inhibitor of the nuclease responsible for DNA fragmentation, i.e. CAD (Caspase-Activated Deoxyribonuclease). In non apoptotic cells, CAD forms an inactive complex with its inhibitor. During apoptosis, the CAD-ICAD complex is cleaved by executioner caspases (i.e. caspase 3 or 7) (Liu et al. , 1997), leaving CAD free to function as a nuclease. The active CAD causes the internucleosomal rupture of chromatin, resulting in the formation of DNA fragments multiple of 180 base pairs.
Other caspase substrates are the Bcl-2 proteins (anti-apoptotic factors): it seems that the cleavage of these regulators not only inactivates these proteins, but also produces a fragment that promotes apoptosis (Xue and Horvitz, 1997).
Caspases contribute to apoptosis through direct disassembly of the cell structure, as in the case of nuclear lamina destruction (Takahashi et al. , 1996). Nuclear lamina is a rigid structure that underlines the nuclear membrane and is involved in chromatin organization. Lamina is formed by head-to-tail polymers of intermediate filament proteins called lamins. During apoptosis, lamins are cleaved at a single site by caspases, causing lamina to collapse and contributing to chromatin condensation.
19 Caspases reorganize cell structures also indirectly by cleaving several proteins involved in cytoskeleton regulation, including gelsolin (Kothakota et al. , 1997), Focal Adhesion Kinase
(FAK) (Wen et al. , 1997) and p21-Activated Kinase 2 (PAK2) (Rudel and Bokoch, 1997). The cleavage of these proteins results in the deregulation of their activity. For example gelsolin, a protein that severs actin filaments in a regulated manner, is cleaved by caspases during apoptosis, generating a fragment which is instead constitutively active.
The dissociation of regulatory and effector domains of different proteins is another feature of caspase activity. For instance caspases deregulate or inactivate proteins involved in DNA repair, mRNA splicing and DNA replication (Cryns and Yuan, 1998). PARP (polyADP-ribose polymerase) is likely the best characterized proteolytic substrate of caspases, being cleaved in the execution phase of apoptosis in many systems. Intact PARP (116 kDa) is cleaved to 24 kDa and
89 kDa fragments, representing the N-terminal DNA binding domain and the C-terminal catalytic domain of the enzyme respectively. It is thought that the cleavage of PARP by caspase 3 interferes with its key-function as a DNA repair enzyme (Cohen, 1997).
A survey of these and other substrates suggests that caspases participate in apoptosis in a manner reminiscent of a well-planned and executed military operation (Thornberry and Lazebnik, 1998).
They cut off contacts with surrounding cells, reorganize the cytoskeleton, shut down DNA replication and repair, interrupt splicing, destroy DNA, disrupt the nuclear structure, disintegrate the cell into apoptotic bodies that are finally engulfed.
1.5. MORPHOLOGICAL AND BIOCHEMICAL FEATURES OF APOPTOSIS
The onset of apoptosis is characterized by cell and nucleus shrinkage as well as condensation of nuclear chromatin into sharply-delineated masses, that become marginated against the nuclear membrane. Later on, the nucleus progressively condenses and breaks up. The cell detaches from
20 the surrounding tissue and its outlines become convoluted and form extensions. The plasma membrane is blebbing or budding, as a result of the marginalization of portions of cytoplasm.
Finally the cell is fragmented into compact membrane-enclosed structures, called apoptotic bodies containing cytosol, the condensed chromatin and organelles. The fine structures, including membranes and mitochondria, are well preserved inside the bodies. The apoptotic bodies, formed at the end of apoptosis, lose the normal phospholipid asymmetry in their plasma membrane, as manifested by the exposure of normally inward-facing phosphatidylserine on the external face of the bilayer. Macrophages recognize this exposed lipid headgroup via an unknown receptor and phagocytosis takes place. The phagocytosis of apoptotic bodies leads to their removal from the tissue, without causing an inflammatory response (Saraste and Pulkki, 2000; Samuilov et al. ,
2000). These morphological changes are a consequence of characteristic molecular and biochemical events occurring in apoptotic cells: most notably, the activation of a large and heterogeneous group of enzymes, some of which mediate the cleavage of genomic DNA into oligonucleosomal fragments, others act on a multitude of specific protein substrates (Saraste and
Pulkki, 2000). One of the biochemical hallmark of apoptosis is represented by the fragmentation of genomic DNA. This implies the activation of endogenous endonuclease(s), which cut(s) the
DNA internucleosomal regions, generating double-stranded DNA fragments multiple of 180-200 base pairs (bp), (Counis and Torriglia, 2000). The DNA fragments contain blunt ends (Alnemri and Litwack, 1990), as well as single base 3 ′ overhangs (Didenko and Hornsby, 1996).
Internucleosomal fragmentation has been demonstrated with a well-characterized apoptotic morphology in a wide variety of situations and cell types (Bortner et al. , 1995). The demonstration of internucleosomal DNA fragmentation has been a major advance in apoptosis detection. Different methods have been developed, like electrophoresis on conventional agarose gel, resulting in a “ladder” pattern (Wyllie, 1980); alternatively, the DNA strand breaks can be
21 observed in situ , by analyzing with a light microscope the tissue sections subjected to the terminal transferase mediated DNA nick end labelling (TUNEL assay) (Gavrieli et al. , 1992).
The quantification of apoptosis in tissue samples often relies on these or related methods.
Other biochemical hallmarks of apoptosis include the activation of caspases. These enzymes are known to play an important role in the accomplishment of the apoptotic morphology (Martin and
Green, 1995; Hirata et al. , 1998). As explained in the previous paragraph, the action of caspases results in the hydrolysis of several protein components involved in the maintenance of cell structure and homeostasis. An increasing number of genetic and biochemical studies suggests that caspase activation is essential for the occurrence of the apoptotic phenotype in dying cells, hence indicating that the activation of these enzymes represents a distinctive feature of apoptosis
(Thornberry and Lazebnik, 1998; Hirsch et al. , 1997; Jänicke et al. , 1998).
1.6. PROGRAMMED CELL DEATH IN PLANTS
It is thought that PCD occurs predictably at specific sites and times also in plants throughout their life cycle. Up to 1970s, the concept of PCD was applied to three strands of research: terminal differentiation, senescence and disease resistance (Jones, 2001).
In the last years the examples of PCD in plant systems have strongly increased (reviewed in
Kuriyama and Fukuda, 2002; Reape and McCabe, 2008). Two different types of plant PCD have been proposed (Jones, 2001):
defence PCD
developmental PCD
Although now plant PCD is reported to occur in several tissues both during development and upon different stress conditions, very little is known about the molecular mechanisms that underlie and regulate this process.
22 1.6.1. Defence PCD
Plants have developed elaborate mechanisms to protect themselves from both biotic and abiotic stresses. Through PCD, plants selectively eliminate cells that are seriously damaged or by invading pathogens or by extreme variations from physiological conditions.
Recently several studies reported the induction of PCD mechanisms in plant systems subjected to different abiotic stresses. For instance tomato and tobacco cells show typical hallmarks of PCD after treatment with some chemicals that are known to induce apoptosis in animals (De Jong et al. , 2000; Sun et al. , 1999). Moreover the treatment of barley cells with high concentrations of salts induces PCD (Katsuhara, 1997), while mannose induces PCD both in Arabidopsis and in maize (Stein and Hansen, 1999). Also UV radiations and ozone have been reported to activate
PCD in Arabidopsis thaliana and tobacco cells (Danon and Gallois, 1998; Pasqualini et al. ,
2003). Finally heat and cold stresses have been reported to be implicated in the activation of the death program in cucumber and tobacco plants respectively (Balk et al. , 1999; Koukalova et al. ,
1997).
Plants are also capable to react against biotic stresses recognizing invading pathogens and activating defences (called resistance response), which result in limiting the pathogen growth at the site of infection. One dramatic hallmark of the resistance response is the induction of a localized cell death, called hypersensitive response (HR), at the site of infection (Greenberg,
1996). The HR, which is considered a form of PCD, is likely to be important for limiting a pathogen’s nutrient supply, since the dying tissue rapidly becomes dehydrated (Greenberg, 1996).
The most characterized example of HR comes from the work of Mittler et al. (1997), in which the authors demonstrated that the infection of tobacco plants with tobacco mosaic virus caused cell death restricted to the site of infection. The authors studied some of the morphological and
23 biochemical events that accompanied cell death during HR. Certain aspects of this process appeared to be similar to those that take place during apoptosis in animal cells.
1.6.2. Developmental PCD
In plants PCD plays important roles in a large number of developmental events, such as the morphogenesis, the expression of tissue and organ function, the reproduction and the senescence of whole organs (Kuriyama and Fukuda, 2002; Gunawardena, 2007). Here, some of the most important examples of plant developmental PCD are reported:
Xylogenesis
Perhaps the most dramatic example of plant developmental PCD is the formation of the water and nutrient conducting structures, that constitute the vascular systems. The xylem cells undergo total autolysis, as they differentiate and mature (Greenberg, 1996). The most important feature of the vascular cells is that they start functioning only after their death. The cell death occurring during the differentiation of procambium into tracheary elements (TEs), which are the major component of xylem, represents the most characterized example of plant PCD occurring during differentiation events (Kuriyama and Fukuda, 2001). Recent studies reported that this form of
PCD is characterized by the emptying of TEs through hydrolysis of all cell contents, including the nucleus, to form hollow elements as a pathway for fluids (Fukuda, 2000).
Reproduction
Cell death also occurs during different stages of plant reproduction. In maize sex determination involves the selective killing of the female reproductive primordia in order that the male floral structures (the stamens) can develop in the tassel (DeLong et al. , 1993).
24 The work by Wang et al. (1996b) points out that PCD also occurs in the transmitting tissue, through which pollen tubes grow. This process is selective, since the tissues surrounding the transmitting tissue in the pistil remain intact. Cell death appears to be correlated with pollen tube growth, since incompatible pollen (which may start to germinate but whose pollen tubes can not elongate) does not elicit cell death. The function of cell death during pollen tube elongation has not been clearly established yet. One possibility is that the dying tissue provides nutrients to the growing tubes, alternatively cell death may be necessary to physically accommodate the growing pollen tubes.
Senescence
Senescence in plants can refer to at least two distinct processes: the aging of various tissues and organs, as the whole plant matures (best studied in leaves, petals and fruit) and the process of whole plant death that sometimes occurs after fertilization (called monocarpic senescence). The process of fruit aging has been extensively studied and reviewed (Theologis, 1992). Senescence fulfils an important role in vegetative tissues, consisting in the hydrolysis of cell contents that become nutrients for other plant tissues and organs that remain living. Plants, in fact, are known to redistribute nutrients during their life cycle; thus, the orderly turnover of macromolecules facilitate an efficient use of resources in all parts of the plant body (Thomas et al. , 2003). It is now ascertained that senescence is an active process, being not the passive death of an organ because of aging, but a tightly controlled process during which cell components are degraded in a coordinated manner and, when nutrients have been reallocated to the other parts of the plant body, the cell finally dies (Gan and Amasino, 1997).
Morphogenesis
25 Although morphogenesis in plants typically occurs by differential cell and tissue growth
(Greenberg, 1996), there are a few plants in which cell death plays a role in the morphogenetic events, such as during the generation of leaf shape. In the genus Monstera , patches of cells die at early stages of development of each leaf blade, generating holes or slits and resulting in leaves which at maturity contain a series of perforations or marginal lobes (Gunawardena et al. , 2004).
Other examples of developmental PCD
PCD also occurs in many other plant tissues and organs. In some plants the cells of the suspensor, the organ that attaches the proper embryo to the maternal tissues and supplies the embryo with nutrients, die before the embryo reaches maturity (Greenberg, 1996). Cell death also occurs in the cortex of the root and stem base in response to waterlogging and hypoxia. The aerated tissue (the aerenchyma), which has interna1 air spaces generated by cell death, facilitates more efficient transfer of O2 from aerial organs to waterlogged stem bases and roots. PCD also occurs in the root cap cells. The latter protect the root apical meristem during seed germination and seedling growth. Root cap cells are formed by initial cells in the meristem and are continually displaced to the root periphery by new cells (Laux and Jürgens, 1997). PCD is also reported to occur in several tissues of developing and germinating seeds. For instance the nucellus, a maternal tissue that embeds and feeds the developing embryo in Sechium edule seeds, undergoes PCD soon after fertilization and is completely removed before seed maturity (Lombardi et al. , 2007a). It is thought that nucellar cells die during seed development in order to supply the young embryo and the expanding endosperm with nutrients. PCD has also a relevant role in the dismantling of some tissues during seed germination. Monocot seeds contain aleurone cells that, upon seed germination, produce large amounts of enzymes that catalyze the hydrolysis of reserve polymers accumulated in the endosperm, thereby providing the seedling with nutrients. Aleurone
26 undergoes PCD before the completion of germination because it is not required for the subsequent stages of development (Kuriyama and Fukuda, 2002). Moreover, the endosperm tissue of cereals, which contains starch and proteins as a food reserve to support seedling growth following germination, undergoes PCD after the reserves are hydrolyzed and reallocated to the embryo (Young et al. , 1997; Young and Gallie, 2000).
From all these examples, it can be concluded that many plant tissues and organs undergo PCD at predictable times and places, in order to regulate developmental events, to carry out a proper morphogenesis and to allow differentiation necessary for the acquisition of a specific tissue function. PCD results thus essential for ensuring the proper development and survival of plants.
1.7. FEATURES OF PLANT PROGRAMMED CELL DEATH
In the last few years, due to the new interest in the existence of a possible apoptotic-like phenomenon in plants, several researchers have focused their attention on searching the typical hallmarks of apoptosis in dying plant cells. Although many differences exist between animal apoptosis and plant PCD, some of the apoptotic features have been reported also in dying plant cells (reviewed in Williams and Dickman, 2008; Reape and McCabe, 2008).
1.7.1. Cell and nucleus morphology
Cytoplasm condensation and shrinkage have been reported in dying aleurone cells (Wang et al. ,
1996c), in root cap cells (Wang et al. , 1996a) and in carrot cell cultures after heat stress (McCabe et al. , 1997). A number of ultrastructural observations have highlighted that the PCD occurring during TE differentiation involves nuclear condensation and rapid and progressive degradation of organelles (Fukuda, 2000). The most striking feature of TE PCD is the rapid collapse of the large central vacuole, that causes hydrolytic enzymes to invade the cytoplasm and attack various
27 organelles, resulting in the degradation of cell contents by autolysis. Vacuolar enlargement and collapse play essential roles also during aleurone cell death (Kuo et al. , 1996) and endocarp cell death during pea ovary senescence (Vercher et al. , 1987). From these examples, it appears how the vacuole plays a key role in some examples of plant cell death.
At the end of animal apoptosis, apoptotic bodies are formed and phagocyted. Morphologically- similar apoptotic bodies have never been reported to form during plant cell death. These bodies may be absent in plants because they are functionally irrelevant due to the absence of phagocytosis in plants; instead, plant cell death pathway might involve autolysis (Danon et al. ,
2000). A “programmed autolysis” has been reported to occur in some plant systems undergoing
PCD, such as the tracheary elements (TEs) (Fukuda, 2000).
1.7.2. DNA fragmentation
The DNA fragmentation reported during animal apoptosis is believed to occur in dying plant cells as well. In degenerating aleurone cells of the grass species such as barley, in dying root cap cells and in tobacco cells subjected to HR, oligonucleosomal-sized DNA fragments have been recorded (Danon et al. , 2000). The detection of DNA fragmentation through electrophoresis on agarose gel has been extensively reported in many plant systems (Danon et al. , 2000). The major problem related to nuclear degradation is represented by no consistency regarding the size of
DNA fragments formed: fragments of more or less 50 kb in some cases (Mittler et al. , 1997) and as small as 0.18 kb in others (Walker and Sikorska, 1994). Oligonucleosomal fragments of DNA, which can be observed by the typical “ladder” after electrophoresis, have been observed in different examples of developmental plant PCD, including the degeneration of monocot aleurone layer (Wang et al. , 1996c) and endosperm (Young et al. , 1997), the senescence of petal, carpel tissue and leaves (Orzaez and Granell, 1997a; Orzaez and Granell, 1997b; Yen and Yang, 1998),
28 the development of anthers (Wang et al. , 1999) and the degeneration of megagametophyte after germination of white spruce ( Picea glauca ) orthodox seeds (He and Kermode, 2003a). DNA
“ladders” have also been recorded after the induction of death using different stresses, like cold
(Koukalova et al. , 1997), nutrient deprivation (Callard et al. , 1996), salt or D-mannose stresses
(Katsuhara, 1997; Stein and Hansen, 1999), UV radiation (Danon and Gallois, 1998) and during the death induced by pathogens or a pathogen toxin (Navarre and Wolpert, 1999; Ryerson and
Heath, 1996).
1.7.3. Cytochrome c release from mitochondria
In animal cells the release of apoptogenic factors leads to the activation of caspases that are responsible for the induction of apoptosis. The release of cytochrome c from intact mitochondria has been reported in cucumber cotyledons undergoing PCD (Balk et al. , 1999). A release of cytochrome c into the cytosol after a D-mannose or a menadione lethal treatment has also been observed in Arabidopsis and maize cells and in tobacco protoplasts respectively (Stein and
Hansen, 1999; Sun et al. , 1999).
1.7.4. Cleavage of the poly(ADP-ribose) polymerase (PARP)
During animal apoptosis, several key proteins are cleaved by caspases. PARP is among the first target proteins shown to be specifically cleaved by caspases. Since PARP is believed to be involved in the regulation of DNA strand breaks repair, its cleavage during PCD is thought to contribute to the process of self-destruction of the cell (Duriez and Shah, 1997). In soybean cell cultures it has been reported that PARP inhibitors or PARP overexpression could respectively inhibit or activate the rate of cell death induced by H 2O2, depending on the intensity of the death stimulus (Amor et al. , 1998). Additionally it has been shown that a 116-kDa protein reacting with
29 a PARP antibody is cleaved into a 84 kDa fragment during menadione-induced PCD in tobacco protoplasts (Sun et al. , 1999). This cleavage could be suppressed by caspase inhibitors, similarly to what is described for animal apoptotic cells.
1.7.5. Protease activities
Proteolytic enzymes are known to play pivotal roles in the cell suicide program in animals and in plants as well. Their role mainly consists in hydrolysing proteins involved in the organization and maintenance of cell structure and homeostatic pathways.
Caspase-like proteases (CLPs)
In view of the strong conservation of regulators and executioners of animal apoptosis, it was initially expected that the same actors would have been involved in plant PCD too. For this reason plant cell death researchers have recently focused their attention on searching plant homologs of animal caspases. Although caspase homologous have not been found in plant genomes (Koonin and Aravind, 2002; Rotari et al. , 2005), caspase 1-, 3- and 6-like activities
(YVADase, DEVDase, VEIDase respectively) have been recorded in many plant systems undergoing both physiological and stress-induced PCD. Proofs of an intimate link between caspase-like proteases (CLPs) and the completion of plant cell death have been obtained in several systems, by using specific caspase inhibitors that were able to strongly delay or suppress cell death (Lam and del Pozo, 2000; Rotari et al. , 2005). The fact that plant cell death can be inhibited by caspase inhibitors suggests that CLPs are involved in the cell death process. Caspase
3-like activity has been recorded in embryonic suspension cells of barley ( Hordeum vulgare L.)
(Korthout et al. , 2000) and in the megagametophyte cells after germination of white spruce
(Picea glauca ) orthodox seeds (He and Kermode, 2003b). Both caspase 1- and caspase 3-like
30 activities have been detected in tomato suspension cells after chemically-induced PCD (de Jong et al. , 2000). Caspase 1-like activity has been found in Arabidopsis suspension cultured cells after nitric oxide-induced cell death (Clarke et al. , 2000) and in tobacco BY-2 cells after isopentenyladenosine-induced PCD (Mlejnek and Prochazka, 2002).
The activation of (a) protease(s) with the preferential cleavage for the caspase 6 substrate (Ac-
VEID-AMC) has been reported to be associated with Norway spruce embryo development
(Bozhkov et al. , 2004). Moreover a TATDase proteolytic activity (caspase 3-like activity) has been detected during the N gene-mediated HR in tobacco plants infected with the tobacco mosaic virus (TMV) (Chichkova et al. , 2004).
It is thought that some of the intracellular caspase substrates are present also in plant cells, but at present, the only intracellular substrate for plant CLPs is PARP (Rotari et al. , 2005).
The use of protease class-specific inhibitors has shown that cysteine and serine protease inhibitors can partially suppress CLP activities in plants or have no effect (Lam and del Pozo,
2000). In some cases they block cell death but at high concentrations (Woltering et al. , 2002;
Rotari et al. , 2005). On the contrary, caspase inhibitors have been found to have effect at low concentrations. In tobacco suspension cells, the PCD induced by high concentrations of nicotinamide, was inhibited by the addition of the caspase 3 inhibitor (Ac-DEVD-CHO) (Zhang et al. , 2003). A cell free system has been used in another study: here, cytochrome c of animal origin was capable of activating plant CLPs in carrot cytoplasm. The cytoplasm, containing a large set of enzymes, could then degrade the rat liver nuclei (Zhao et al. , 1999). In this study the formation of a DNA “ladder” was inhibited by the caspase 1 and 3 inhibitors (Ac-YVAD-CHO and Ac-DEVD-CHO, respectively). Belenghi et al. (2004) found that caspase 3-like proteases play important roles in the elimination of secondary shoots of pea seedlings after removal of the epicotyl. The injection of the caspase 3 inhibitor into the remainder of the epicotyl strongly
31 inhibited the death of the secondary shoot, resulting in a seedling with two equal shoots. In another study the heat-shock induced cleavage of lamins-like proteins of tobacco protoplasts has been found to be correlated with the proteolytic activity of caspase 6-like proteases (Chen et al. ,
2000). In this study the caspase 6 inhibitor (Ac-VEID-CHO) inhibited the cleavage of lamins.
Finally Elbaz et al. (2002) showed that caspase inhibitors were able to prevent staurosporine- induced cell death in Nicotiana tabacum cells.
The studies reported above demonstrate that the caspase inhibitors are remarkably efficient at delaying and/or suppressing cell death in plants, hence suggesting that CLPs really play a role in plant PCD.
Other proteases involved in PCD
Besides CLPs, other proteases have been reported to play a role in plant PCD. Some of these proteins are structurally similar to animal caspases. If there is a main executioner of plant PCD, it could be one of these proteases. Their main features are summarized here:
- Metacaspases
Uren et al. (2000) found a family of caspase-related proteases - named metacaspases - in fungal and plant genomes. Plant metacaspases may be divided in two subclasses on the basis of similarities in amino acid sequence and general domain structure (Lam, 2004). Plant prometacaspases type I contain an N-terminal prodomain of about 80-120 amino acids, which may represent a prodomain typical for inactive animal initiator caspases (Fig. 6) (Piszczek and
Gutman, 2007). This domain may be responsible for protein-protein interactions between two prometacaspases, which consequently lead to metacaspase activation. Prometacaspases type II are characterized by the lack of a long prodomain (Fig. 6). In both types of metacaspases, a
32 conserved region of approximately 150 amino acids, corresponding to the large caspase subunit, is present (Uren et al. , 2000). At the C-terminal site, there is a second conserved domain that resembles the small caspase subunit. Between these domains corresponding to the large and small caspase subunits, a linker region of variable length is present.
The homology between these enzymes and caspases is also supported by the presence, in the domain corresponding to the large caspase subunit, of a characteristic conserved amino acid dyad
-cysteine/histidine - which plays a fundamental role in the process of catalysis (Uren et al. 2000).
The catalytic histidine in metacaspases from Arabidopsis occurs in the H(Y/F)SGHG sequence; moreover an important role of glycine in the process of catalysis has been proposed. These biochemical features are very similar to what has been reported for the mammalian caspases
(Vercammen et al. , 2004).
The Arabidopsis thaliana genome contains nine metacaspase genes: three of them belong to the metacaspases type I (Atmc1, Atmc2, Atmc3) and six to the metacaspases type II (Atmc4, Atmc5,
Atmc6, Atmc7, Atmc8, Atmc9). The comparison of the amino acid sequences of the mentioned metacaspases highlights a relatively high degree of identity (from 56 to 71%) (Watanabe and
Lam, 2005). Despite the structural homology between caspases and metacaspases, the latter differ from caspases in their substrate specificity. N-terminal sequencing of the small subunit of metacaspase 9 suggests that plant metacaspases show cleavage specificity at the carboxyl side of basic amino acids, such as arginine or lysine, and not aspartic acid as do caspases.
Several studies point out that metacaspases have a role in PCD. Madeo et al. (2002) reported that the yeast metacaspase protein (YCA1) knockout is unable to complete PCD in the presence of a
PCD inducer, like H 2O2. The protein mcII-Pa (metacaspase type II) has been reported to be expressed during PCD in somatic embryogenesis of Norway spruce ( Picea abies ). In situ hybridization analysis showed mRNA accumulation in the cells of embryogenic tissues and
33 structures committed to die (Suarez et al. , 2004). The silencing of the Picea abies metacaspase gene mcII-Pa suppressed PCD in the embryo and blocked suspensor differentiation (Suarez et al. ,
2004). Watanabe and Lam (2005) found that two metacaspases type I (AtMCP1b and AtMCP2b) are up-regulated in Arabidopsis plants after infiltration with a bacterial pathogen and are able to activate apoptosis-like cell death in yeast.
From these studies, it can be gathered that metacaspases share structural similarities with the animal caspases and, although they have not caspase activity, it seems that they take part in several examples of plant PCD.
- Vacuolar processing enzymes (VPEs)
Another group of caspase-related proteases are the vacuolar processing enzymes (VPEs), plant legumains, that were originally identified as processing enzymes. Their function is not restricted to precursor protein processing, but also includes protein breakdown in the vacuole or cell wall
(Hara-Nishimura et al. , 1991; 1998; 2005; Müntz and Shutov, 2002). VPEs - VPE α, β, γ and δ – have been found to promote the maturation and the activation of various vacuolar proteins in plants (Yamada et al. 1999; Rojo et al. 2003). Their counterpart in animals similarly activates lysosomal proteins (Shirahama-Noda et al. 2003).
VPEs belong to the cysteine protease family and show substrate specificity in respect to the peptide bond on the carboxyl side of asparagine (Asn) and aspartic acid (Asp) residues. Under in vitro conditions, VPEs hydrolyze peptide bonds on the carboxyl side of aspartic acid residue of the synthetic caspase 1 substrate (YVAD), but do not hydrolyze this bond in the synthetic caspase
3 substrate (DEVD) (Hatsugai et al. , 2006). The substrate specificity of these enzymes is thus similar to the substrate specificity of caspase 1. VPEs resemble the animal caspase 1 in many aspects: similarly to caspase 1, VPEs are not inhibited by the known cysteine protease inhibitors,
34 such as E-64 and leupeptin, although both enzymes belong to the class of cysteine proteases.
Moreover in the catalytic process performed by VPEs, the characteristic amino acid dyad
(cysteine and histidine) has a relevant role (Fig. 6) (Hatsugai et al. , 2006). In addition each of the three amino acids (Arg-179, Arg-341 and Ser-347), which form a pocket for Asp in caspase 1, have been shown to be conserved in over 20 VPEs available in the database. These comparisons suggest that the VPE substrate pocket is similar to the Asp pocket of caspase 1 (Hatsugai et al. ,
2006). Besides the similarities between caspase 1 and VPEs, several differences also exist. The low amino acid sequence homology (21%) between these enzymes is worth mentioning.
Additionally, the enzymes act at different pH; this evidence may be the result of their different subcellular localization. VPEs are localized in the vacuole and have an acidic optimum pH, whereas the cytoplasmic caspase 1 enzyme acts at pH close to neutral (Hatsugai et al. , 2006).
VPEs are synthesized in an inactive form and are then activated in an autocatalytic process
(Hiraiwa et al. , 1999). The Arabidopsis thaliana genome contains 4 VPEs homologues: αVPE,
βVPE, γVPE and δVPE, which may be classified in two subfamilies: one specific for seeds and the other for vegetative organs. αVPE and γVPE participate in the maturation of proteins in lytic vacuoles typical for vegetative organs, whereas βVPE is engaged in the maturation of proteins in the storage vacuoles of Arabidopsis seeds (Kinoshita et al. , 1999). The expression of δVPE mRNA has been observed, similarly to βVPE, in young developing embryo as well as in germinating seeds (Nakaune et al. , 2005). δVPE has been reported to be expressed in maternally derived tissue-cells of the inner integument layer which undergo PCD during seed coat formation, whereas βVPE is expressed in the embryo (Nakaune et al. , 2005).
Other data highlighted that VPEs might be key factors in the vacuolar collapse-triggered cell death. In vegetative tissues the isoform γVPE has been shown to play a role in protein degradation during senescence. Schmid et al. (1999) found that γVPE is localized in the
35 precursor protease vesicles (PPVs), hydrolase-containing organelles, that are associated with
PCD. VPE γ is also found in the vacuole, an organelle essential for cell dismantling during plant
PCD (Lam, 2004; Rojo et al. , 2004). Furthermore it is thought that VPEs are able to convert hydrolytic enzymes, involved in the degradation of cellular components, from an inactive to their active form (Yamada et al. , 2005). Arabidopsis VPE genes have been found to be up-regulated in dying cells during development and senescence of tissues (Kinoshita et al. , 1999). Moreover
VPEs have been reported to be required for the completion of the HR-associated cell death in tobacco cells (Hatsugai et al. , 2004).
These observations point out that VPEs play a role not only in the protein processing and breakdown but also in PCD.
- Saspases (serine asp artate-specific proteases )
Saspases, caspase-unrelated proteases, have been reported to be involved in plant PCD. From the study by Coffeen and Wolpert (2004), two serine proteases (SAS-1) and (SAS-2) have been purified and partially sequenced: these enzymes belonged to the class of subtilisin-like serine proteases and participated in oats ( Avena sativa ) victorin-induced PCD.
Interestingly, the authors found that these enzymes are capable to cleave caspase substrates and, because of the fact that they are serine proteases and hydrolyze caspase substrates, they have been termed “saspases” (Coffeen and Wolpert, 2004). Moreover these enzymes were found to be targeted by caspase inhibitors.
In contrast to VPEs and metacaspases, saspases are expressed constitutively. They are synthesized in the form of preproproteins with a signal peptide (Fig. 6), which is later removed in the endoplasmic reticulum. The activation of these enzymes consists in the removal of the
36 propeptide; subsequently, the active enzymes are transferred to the apoplast where they have access to their substrates.
In conclusion, Coffeen and Wolpert demonstrated the existence in plants of proteases structurally unrelated to caspases but with caspase activity. The fact that caspase-unrelated proteases are able to cleave caspase substrates sheds light on the fact that caspase-like activities exist in plants despite the absence of the typical caspases.
Fig. 6. The main proteases involved in plant PCD. Comparison between caspase and plant caspase-related and -unrelated protease structures (from Piszczek and Gutman, 2007).
Other enzymes belonging to the more conventional classes of proteases have been reported to be involved in plant PCD too. The identity and the involvement of these proteases in PCD are summarized here:
37 in soybean cells, cysteine proteases are activated during oxidative stress-induced PCD (Solomon et al. , 1999) and the activation of these proteases is required for cell death. Ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv. glycinea or directly by oxidative stresses.
Xylogenesis in Zinnia elegans cell cultures could be prevented by the addition of cysteine and serine protease inhibitors (Minami and Fukuda, 1995; Ye and Varner, 1996; Beers and Freeman,
1997). Serine and cysteine proteases have also been reported to be activated during the degeneration of the megagametophyte of white spruce ( Picea glauca ) orthodox seeds (He and
Kermode, 2003b). A matrix metalloprotease (Cs1-MMP) has been proposed as a candidate involved in cucumber extracellular cell matrix degradation and in PCD as well (Delorme et al. ,
2000).
A barley vacuolar aspartic protease (phytepsin), highly homologous to the mammalian lysosomal cathepsin D, was expressed at a high level both during TE formation and during autolysis of sieve cells (Runeberg-Roos and Saarma, 1998). In the endosperm of castor bean ( Ricinus communis ), it has been shown that PCD is associated with the accumulation of papain-like propeptidase (Cys-
EP) in ricinosomes (Schmid et al. 1999) and with the release of this peptidase from ricinosomes during cell collapse. In Brassica napus , a papain-like cysteine protease has been found to be correlated with PCD of the inner integument of seed coat during early stages of seed development
(Wan et al. , 2002).
Proteasome activity has also been reported to be implicated in some examples of PCD both in animals and in plants (Beers et al. , 2000). The proteasome system, a conserved multicatalytic proteolytic complex, is known to regulate a variety of cellular processes via degradation of regulatory proteins and to play a major role in PCD. Proteasome activity consists in the degradation of proteins covalently attached to Ubiquitin (Ub).
38 Some studies reported that the expression of genes encoding Ub-proteasome pathway components is upregulated in different systems undergoing PCD, such as senescing leaves of
Arabidopsis and Nicotiana (Genschik et al. , 1994), degenerating anthers of Nicotiana (Li et al. ,
1995), differentiating tracheary elements of wounded Coleus stems (Stephenson et al. , 1996) and tobacco cells undergoing heat-induced PCD (Vacca et al., 2007). In other plant tissues or organs, no correlation between Ub or Ub-conjugating enzymes and PCD has been detected; moreover the use of proteasome inhibitors has led in some cases to the promotion, in others to the prevention of
PCD, thus creating an unclear picture concerning the role of proteasome in plant PCD (Beers et al. , 2000).
In conclusion, it has been shown that besides CLPs, metacaspases, VPEs and saspases, which are considered the main executioners of plant PCD (Rotari et al. , 2005), also enzymes belonging to the more conventional classes of proteases take part in the realization of the cell death program in several plant systems.
1.7.6. Nuclease activities
As in animals, also in plants genomic DNA undergoes hydrolysis by endonucleolytic enzymes at the final stages of PCD. Several enzymes capable of hydrolyzing native dsDNA in an endonucleolytic manner have been purified and characterized from plant sources. With the requirement for divalent cations and optimum pH as major criteria, plant nucleases can be categorized in two different classes:
Zn 2+ -dependent nucleases
These enzymes are characterized primarily by the requirement for Zn 2+ and an optimum pH in the acidic range. The degradation product of the Zn 2+ -dependent nucleases possesses a
39 phosphomonoester group at the 5’ end and a hydroxyl group at the 3’ end (Sugiyama et al. ,
2000). Zn 2+ -dependent nucleases are monomeric glycoproteins with similar molecular sizes (33-
44 kDa). A representative example for this class is the mung bean nuclease, the best characterized plant nuclease (Kowalski et al. , 1976). Moreover the greater part of plant nucleases purified to date belong to this class. cDNAs for the barley 35 kDa nuclease (BEN1) and Zinnia 43 kDa nuclease (ZEN1) have been isolated and sequenced (Aoyagi et al. , 1998). The deduced amino acid sequences of BEN1 and ZEN1 showed significant similarities with each other and with sequences of Zn 2+ -dependent nucleases from Fungi and Protists. Other members of Zn 2+ - dependent nucleases include the nuclease P1 from Penicillium citrinum . The analysis of the crystal structure of nuclease P1 revealed that its binding to Zn 2+ involves nine amino acid residues that are found to bind Zn 2+ also in BEN1 and ZEN1 (Volbeda et al. , 1991; Sugiyama et al., 2000). Besides these enzymes, many unpurified nuclease activities probably due to Zn 2+ - dependent nucleases have been reported from various tissues of diverse plant species (Yen and
Green, 1991).
The involvement of Zn 2+ -dependent nucleases in PCD has been assessed in several plant tissues.
During the degeneration of barley seed endosperm, the Zn 2+ -dependent nuclease (BEN1) has been reported to be responsible for DNA degradation (Aoyagi et al. , 1998). During TEs differentiation, the activity of the Zn 2+ -dependent nuclease (ZEN1) has been found to be tightly correlated with DNA degradation (Thelen and Northcote, 1989). Moreover increased activities of some Zn 2+ -dependent nucleases have been reported to be associated with the progression of senescence in wheat leaves (Blank and McKeon, 1989).
40 Ca 2+ -dependent nucleases
Ca 2+ -dependent nucleases are characterized by the requirement for Ca 2+ and preference for neutral pH. In some cases, Zn 2+ has been shown to be highly inhibitory against DNA hydrolysis by Ca 2+ -dependent nucleases (Mittler and Lam, 1995). The action of these enzymes produces nucleotides bearing phosphomonoester groups at the 5’ ends, as a result of DNA hydrolysis.
Enzymologically, plant Ca 2+ -dependent nucleases are somewhat similar to several mammalian
Ca 2+ /Mg 2+ -dependent endonucleases, including DNase I and DNase γ. From the study by Liao
(1977), who compared malt DNases A and B with pancreatic DNase I in their pH activity profiles and their amino acid composition, it was shown that the three DNases have striking similarities.
In comparison with Zn 2+ -dependent nucleases, there are relatively few papers demonstrating clearly the existence of Ca 2+ -dependent nucleases. Anyway, several Ca 2+ -dependent nuclease activities have been reported to be correlated with DNA degradation during plant PCD.
During cereal endosperm degeneration, three Ca 2+ -dependent nuclease activities have been detected; one of these activities, which appeared to be executed by 33.5 kDa enzymes, has been found to be closely associated with endosperm DNA fragmentation (Young et al. , 1997).
In barley leaves the activities of some Ca 2+ -dependent nucleases have been reported to increase with the progression of senescence (Wood et al. , 1998). During the hypersensitive response of tobacco cells to pathogens, three Ca 2+ -dependent nucleases (NUC I, NUC II and NUC III) have been reported to be activated in a coordinated manner with PCD. NUC III, which accounted for the majority of the DNase activity detected, was found in purified nuclei (Mittler and Lam,
1995).
In summary, both classes of nucleases appear to be involved in DNA dismantling during plant
PCD. Although information available about the relationship between plant nucleases and PCD is quite fragmentary in most systems investigated, to date it seems that in some experimental
41 systems only the activity of Zn 2+ -dependent nucleases have been assessed and only the activity of
Ca 2+ -dependent nucleases in others (Sugiyama et al. , 2000).
1.7.7. Hormonal control of programmed cell death
Plant cell death is thought to be regulated by different signals, including hormones.
Some studies reported that gibberellic acid stimulates nuclear DNA fragmentation (unlike abscisic acid) during aleurone PCD of monocot seeds (Kuriyama and Fukuda, 2002). Moreover, it has been shown that the DNA cleavage during aleurone PCD involves some nucleases, whose activity is subjected to the regulation by gibberellic and abscisic acids (Fath et al. , 1999).
He et al. (1996) showed that ethylene is important to induce cortical root cells to die and autolyze during lysigenous aerenchyma formation. The involvement of ethylene in aerenchyma formation is well documented for maize (Drew et al. , 2000): the authors proposed that cell death in the root cortex during aerenchyma formation is regulated by ethylene and provides an example of PCD.
Furthermore ethylene and gibberellic acid have been found to promote PCD in cereal endosperm
(Young and Gallie, 1999), while cytokinins have been shown to be a common suppressor of certain kinds of HR-associated PCD and a well-known suppressor of senescence (see full literature in Pontier et al. , 1999). Recently Carimi and co-workers showed that cytokinins can induce PCD if they are added at high concentration to proliferating cells of carrot and
Arabidopsis (Carimi et al. , 2003).
The PCD of the epidermal cells that cover adventitious root primordia in deepwater rice ( Oryza sativa ) has been found to be controlled by ethylene signalling and has been reported to be induced by gibberellin (GA) (Steffens and Sauter, 2005). The authors proposed that ethylene and
GA act in a synergistic manner, indicating converging signalling pathways.
42 Besides these examples, no information is available about the involvement of indole-3-acetic acid
(auxin) in plant PCD. More recently some data concerning a possible involvement of indole-3- acetic acid (IAA) in PCD of the nucellus of Sechium edule seeds have emerged. From this study, it appeared that the dying nucellar cells had higher concentrations of IAA than the nucellar portion composed by living cells (unpublished data).
Although plant hormones seem to play a role in the regulation and control of PCD, to date too little is known in order to make a clear picture about their taking part in cell death events.
43 2. THE PLANT SYSTEM
Bunya pine ( Araucaria bidwillii Hooker) is a conifer native to Queensland (Australia) and it is the only member of the section Bunya (Fig. 7A). The trees, which grow to 30-45 m in height
(Boland et al. , 1984), carry cones in the upper crown, which can weigh up to 10 kg (Doley,
1990). Cones are mature in mid/late summer and fall to the ground intact, with the cone scales still green on the surface and fleshy and resinous internally (Doley, 1990). Their weight helps the cones to roll down slopes and hence achieve an increase dispersal. The cones rapidly disintegrate on the ground, as the structure dries (Haines, 1983). Further drying releases the seeds from the cone scales, becoming then available for dispersal by animals. Nutritional studies indicate that over 40% of the fresh weight of seeds is available carbohydrate (Brand et al. , 1985). The bunya pine seeds are large and heavy, with prevailingly amylaceous reserves. The moisture content of mature seeds is around 48%; the normal seedling establishment is reduced when moisture content is decreased to 35-37%, while the germination capacity is compromised below 15%, making them recalcitrant seeds (Francini et al ., 2006; Del Zoppo et al. , 1998; Roberts, 1973). The bunya pine seed consists of a cotyledon tube, an hypocotyl and a root cap surrounded by a massive megagametophyte, which represents the major storage tissue of the seed (Fig. 7B) (Burrows et al. , 1992). In the mature seed, the megagametophyte is a living tissue and contains the food reserves for the growing embryonic axis. The bunya pine seed has a cryptogeal germination: after germination on the soil surface, the elongation of the cotyledon tube pushes the hypocotyl, with its associated plumule and radicle, into the soil. Subsequently the megagametophyte reserves are hydrolyzed, translocated and stored in the hypocotyl which increases in diameter (tuber stage) and originates the new shoot (Burrows et al. , 1992). When all its reserves are mobilized, the megagametophyte degenerates, becoming a no longer needed tissue. An abscission zone develops
44 at the base of the cotyledon tube near the tuber, then the tuber detaches from the cotyledon tube
(Fig. 7C) (Burrows and Stockey, 1994).
Fig. 7. The plant system. (A) The bunya pine (Araucaria bidwillii Hook.) tree. (B) Section of a bunya pine seed, showing the megagametophyte (M) and the embryo (E). (C) Late stage of the post-germinative growth of the bunya pine seed, s: bunya pine seed; ct: cotyledon tube; t: tuber.
45 3. AIM OF THESIS
Programmed cell death (PCD) is an essential feature of plant and animal growth and survival.
Through this process, living organisms eliminate cells that are present in excess or tissues needed at just one time of development and not required later.
In my research project, I used the megagametophyte of the bunya pine ( Araucaria bidwillii
Hook.) seed as a model to study developmental plant PCD. The megagametophyte in fact, a short-lived storage tissue, degenerates after supplying the embryo with nutrients. In order to understand whether the megagametophyte degeneration occurs by means of a developmental
PCD, typical hallmarks of apoptosis have been searched.
46 4. MATERIALS AND METHODS
4.1. PLANT MATERIAL
Bunya pine ( Araucaria bidwillii Hook.) seeds were collected from the grounds of the University of Pisa Botanical Garden at the end of August 2005. For germination tests, seeds were surface disinfected with 5% NaClO and then allowed to germinate onto imbibed perlite in a growth chamber at 26 °C with a 8 h photoperiod (light intensity 25 µmol m −2 s −1 , PAR 400-700 nm). At the time points 0, 7, 14, 21, 28 and 35 Days After Germination (DAG), the seeds were dissected, the embryos removed and the megagametophytes were either used fresh or milled after freeze- drying and the flours were stored at -30 °C until use.
4.2. VIABILITY STAINING OF MEGAGAMETOPHYTES
The viability of megagametophytes at each germination stage (0, 7, 14, 21, 28 and 35 DAG) was evaluated by staining the tissues with 0.1% (w/v) Evan’s Blue for 2 min and destaining with distilled water for 1 h. The experiment was performed in triplicate at each stage.
4.3. ANALYSIS OF THE MEGAGAMETOPHYTE TISSUE SECTIONS
Megagametophytes collected at 0 and at the late post-germinative stage (28 DAG) were fixed overnight at 4 °C in 4% paraformaldehyde in phosphate buffer saline pH 7.4. After dehydration through ethanol series (30%, 50%, 70%, 96% and 100%), the samples were embedded in paraffin
(Sigma). Sections 15 µm-thick were cut and stretched on poly-lysine coated slides. The tissue sections were dewaxed in xylene and rehydrated. After rehydration, the samples were stained with 0.05% (w/v) toluidine blue in 1% (w/v) Na 2CO 3 pH 8.2. The megagametophyte tissue sections at each germination stage prepared as described above were stained with 1 µg ml -1 4,6-
diamidino-2-phenylindole (DAPI, Sigma) to visualize nuclei. Fluorescence emission was observed using a Leica DMLB microscope, equipped with UV-2A filter for DAPI detection.
Images were photographed with a Leica DC 300F CCD camera. Hundreds of tissue sections were analyzed.
4.4. ANALYSIS OF SOLUBLE AND INSOLUBLE PROTEINS
4.4.1. Protein extraction for SDS-PAGE analysis of soluble and insoluble proteins
Proteins were extracted from megagametophyte flours at each germination stage according to
Gifford et al. (1982) with some modifications. The flours were grinded in 50 mM sodium phosphate buffer pH 7.5, in a ratio 1:4 (w/v) for 15 min RT. The supernatant obtained after centrifugation (12000 rpm, 10 min, 4 °C) was used for the soluble proteins analysis. The pellet was then solubilized in 65 mM Tris-HCl pH 6.8, containing 2% SDS (w/v), 10% glycerol (w/v) and 2.5% β-ME (v/v) in a ratio 1:4 (w/v). This further extraction was performed for 15 min RT.
After centrifugation (12000 rpm, 10 min, 4 °C), the supernatant was used for the insoluble proteins analysis. The amount of both soluble and insoluble proteins was measured at each stage according to Bradford (1976) and Lowry et al. (1951).
4.4.2. SDS-PAGE of soluble and insoluble proteins
Two hundred microliters of the extract at each stage of soluble and insoluble proteins were prepared in SDS-sample buffer with 5% β-ME (Laemmli, 1970) and heated in a boiling-H2O bath for 3 min. The samples were then fractionated in 12% polyacrylamide gels at 20 mA at room temperature. Molecular-weight standards were included in order to evaluate the molecular weight of the fractionated proteins. When electrophoresis was finished, the gels were stained overnight with 0.025% (w/v) Coomassie Brilliant Blue R 250, according to Koenig et al. (1970), destained
48 and acquired with a scanner. The experiment was performed in triplicate starting from three independent extractions.
4.5. ANALYSIS OF THE MEGAGAMETOPHYTE PROTEOLYTIC ACTIVITIES
4.5.1. Caspase-like proteases (CLPs) extraction and in vitro activity assays
The CLP activity assays were performed according to Del Pozo and Lam (1998) with some modifications. Proteins were extracted from megagametophyte flours collected at each stage with
50 mM sodium phosphate buffer pH 7.0, containing 0.1% CHAPS, 10% sucrose, 1 mM EDTA,
20 mM β-ME in a ratio 1:6 (w/v) on ice for 30 min. After centrifugation (15000 rpm, 15 min, 4
°C), the supernatant protein content was quantified according to Bradford (1976) and the samples were immediately stored at -30 °C until use.
One hundred microliters of extract were added to 5.2 µl of 10 mM Ac-YVAD-pNA (caspase 1 substrate), Ac-DEVD-pNA (caspase 3 substrate) or Ac-VEID-pNA (caspase 6 substrate) in 591
µl of assay buffer (0.1 M citric acid-phosphate pH 5.0, containing 10% sucrose, 1 mM EDTA, 20 mM β-ME). The assay mixtures were incubated for 5 h at 32 °C and the activity was determined spectrophotometrically, measuring the final absorbance at 405 nm. The A 405 of the mixture taken before the beginning of the incubation (blank sample) was subtracted to each reaction measured absorbance. The concentration of the p-nitroanilide (pNA) released from the substrates was calculated from a calibration curve prepared with pNA standard solutions. The activities were expressed as pNA nmoles min -1 megagametophyte -1. To determine the effect of protease inhibitors on the CLPs activities, the enzymatic assays were performed in the presence of Ac-
YVAD-CMK (caspase 1 inhibitor), Ac-DEVD-CHO (caspase 3 inhibitor), Ac-VEID-CHO
(caspase 6 inhibitor) (100 µM, final concentration), 1.43 µM pepstatin-A, 5 µM E-64 and 1 mM
PMSF. All the inhibitors tested were incubated with the enzymatic extracts for 20 min at 32 °C,
49 before adding the substrates. To determine the optimum pH for the CLPs activities, the assays were performed in 0.1 M citric acid-phosphate buffers at pH values ranging from 2.5 to 8.0. The substrates were also incubated in the absence of the enzymatic extract, to test whether they undergo spontaneous degradation. As some protease substrates and inhibitors were dissolved in organic solvents (DMSO or ethanol), the effect of these solvents on the CLPs activities was also tested. All the enzymatic assays were performed in triplicate.
4.5.2. Preparation of enzymatic extracts for the detection of total protease activity
The megagametophyte flours were extracted at each germination stage with cold 0.2 M phosphate buffer pH 6.0, containing 5 mM β-ME at a ratio of 1:6 (w/v) on ice for 1h. The homogenates were centrifuged (10000 x g, 10 min, 4 °C) and the supernatants were saturated to
80% with ammonium sulphate and centrifuged in the aforementioned conditions. The precipitates were dissolved in 2 mM phosphate buffer pH 6.0, containing 2.5 mM β-ME and dialyzed against two changes of the same buffer. The dialyzed extracts were centrifuged in the same conditions and used as enzymatic source. Total proteins of each extract were quantified according to
Bradford (1976).
4.5.3. In vitro protease activity assays
Haemoglobinase activity was assayed with 2% bovine haemoglobin (Sigma, St. Louis, USA), dissolved in 0.2 M acetate buffer pH 4.0. The enzymatic assay, performed according to Galleschi et al. (1988), was measured also in the presence of the following protease inhibitors: 20 µM E-
64, 1,43 µM pep-A, 1 mM EDTA and 1 mM PMSF.
Azocaseinase activity was assayed with 0.2% azocasein dissolved in 0.05 M acetate buffer pH
5.4 and 0.1 M phosphate buffer pH 7.0, according to Del Zoppo et al. (1999). The enzymatic
50 activity was evaluated also in the presence of the inhibitors previously utilized. The enzymatic activities were expressed as Units per megagametophyte.
For haemoglobinase assay, one Unit of enzymatic activity is the amount of enzyme required to produce 1 µM of alanine per minute, under standard assay conditions. For azocaseinase assay, one Unit is defined the amount of enzyme required to produce an absorbance change of 1.0 in a 1 cm cuvette, under standard assay conditions (Sarath et al ., 1989). All the enzymatic assays were performed in triplicate.
4.5.4. In-gel protease activity assays
The megagametophyte proteolytic activities were analyzed by gelatin-PAGE. Briefly, protein extracts were prepared in sample buffer lacking SDS and reducing agents and subjected to electrophoresis on 8.5% polyacrylamide gels, containing 0.1% SDS (v/v) and 0.1% gelatin (v/v) as a protease substrate. Ten microliters of the enzymatic sources at each stage were loaded into the gel and fractionated with a current of 10 mA/gel in a refrigerate chamber (4 °C). After electrophoresis, the gels were washed in a 2.5% aqueous solution of Triton X-100 (30 min, RT) to allow enzyme renaturation. The gels were then washed twice in the presence of 2.5 mM β-ME and subsequently placed in 100 ml of incubation buffer (0.1 M citric acid-phosphate, containing
2.5 mM β-ME) at 33 °C overnight. Proteolysis was stopped by transferring the gels into a staining solution: 0.1% Amido black (w/v) in water/methanol/acetic acid (6:3:1). The gels were then destained and the proteolytic activities were detected as clear spots, where the gelatin had been degraded against the blue-stained protein background. The protease optimum pH was determined by incubating the gels in buffers at different pH values: pH 4.0, 5.0, 6.0 and 7.0. In order to characterize the proteolytic activities observed, the gels were cut into individual slices and incubated in buffer with protease class-specific inhibitors. The inhibitors and their final
51 concentrations were E-64 (10 µM), leu (10 µM), pep-A (20 µM), PMSF (10 mM), o-phen (1 mM) and EDTA (10 mM). The effect of caspase 1, 3, 6 inhibitors (400 µM) on the late stage protease activities was also tested. As some protease inhibitors were dissolved in organic solvents
(DMSO or ethanol), the effect of these solvents on protease activities was also tested.
4.5.5. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis
The 28 DAG extract, obtained as previously described (paragraph 4.5.2.), was used for 2-DE separation on polyacrylamide gels. The 2-DE was performed according to Fontanini et al. (2007) with some modifications. The first dimension separation (isoelectric focusing, IEF) was carried out on 7 cm IPG strips (GE Healthcare Milano, Italy) pH range 3-10. The IPG strips were rehydrated overnight with 75 µg of protein sample dissolved in 125 µL of reswelling buffer (10% sucrose, 0.2 M taurine, 2% IPG buffer, pH 3-10 and traces of BBP). IEF was performed at 20 °C in a Multiphor II apparatus (Amersham Biosciences, AB) with the following electric parameters:
200 V for 1 min followed by a 1.5 hours-linear increase to 1500 V and by a 3 hours ramp to 3000
V. The final step was run at constant voltage (3000 V) for 30 min. At the end of IEF separation, the strips were equilibrated in non-denaturing electrophoresis sample buffer for 30 min under constant mild shaking, rinsed, and applied on a 11% polyacrylamide minigel. The second- dimension separation (non-denaturing PAGE) and enzyme renaturation after electrophoresis were performed as described in the previous paragraph. The gel containing the 2-DE separated proteins was coupled in a “sandwich” with a 11% gelatin-containing polyacrylamide gel. The “sandwich” was incubated in 500 ml of incubation buffer as previously described. After incubation, the 2-D gel was stained with 0.025% (w/v) Coomassie Brilliant Blue R 250 and the gelatin-containing gel was stained with 0.1% Amido black (w/v) for the detection of proteolytic activities. The protein
52 spot responsible for the activity observed on gelatin was excised from the 2-D gel and analyzed by mass spectrometry.
4.6. ANALYSIS OF DNA FRAGMENTATION
4.6.1. In situ detection of DNA fragmentation (TUNEL assay)
The megagametophyte tissue sections, prepared as previously described (paragraph 4.3.), were used for the TUNEL assay. The assay was performed with the “ In situ cell death detection kit”
(Promega), according to the manufacturer’s instructions. The tissue sections were permeabilized with proteinase K (20 µg ml -1) for 20 min to allow the entry of TdT enzyme (transferase). The labelling of fragmented DNA was performed at 37 °C in a humidified chamber in the dark for 1 h. A negative control was included in each experiment, omitting TdT from the reaction mixture.
As a positive control, the sections were incubated with DNase I (10 U ml -1) for 10 min before the
TUNEL assay. The sections used for TUNEL were counterstained with 1 µg ml -1 4,6-diamidino-
2-phenylindole (DAPI, Sigma) to visualize all nuclei. Fluorescence emission was observed using a Leica DMLB microscope equipped with FITC and UV-2A filters for TUNEL and DAPI detection respectively. Images were photographed with a Leica DC 300F CCD camera. The diameter of the megagametophyte nuclei was measured with the software Image J, 1.39 b. The diameters’ average of nuclei and the percentage of TUNEL-positive nuclei were calculated out of
5000 nuclei randomly selected from pictures.
4.6.2. Isolation and electrophoresis of genomic DNA
The extraction of genomic DNA was performed according to Dellaporta et al. (1983) with some modifications. The megagametophyte flours (1 g) collected at each stage were extracted for 30 min at 60 °C with 10 volumes of 0.1 M Tris-HCl pH 8.0, containing 50 mM EDTA, 500 mM
53 NaCl, 1% SDS and 0.7% β-ME. After adding one volume of chloroform:isoamyl alcohol (24:1), the samples were centrifuged (3000 rpm, 15 min, RT). The supernatants were further extracted with an equal volume of chloroform:isoamyl alcohol (24:1). Total DNA was precipitated for 2 h at -30 °C adding to the supernatants 2.5 volumes of chilled ethanol and 0.25 volumes of cold 3 M sodium acetate pH 5.2. After centrifugation (12000 rpm, 30 min, 4 °C), the pellet was washed with 2.5 volumes of cold 70% ethanol and subsequently dried and dissolved in 5 ml of Tris-
EDTA buffer (0.05 M Tris-HCl pH 8.0, containing 10 mM EDTA). After incubation at 37 °C for
30 min with 125 µl of RNase (10 mg/ml, free of DNase activity), the samples were extracted again as described above. DNA concentrations were determined spectrophotometrically, monitoring the absorbance at 260 nm. The ratio A 260nm /A 280nm was also registered and used as an indicator of DNA purity. DNA samples at each stage (8 µg) were applied on 1% agarose gel, containing 1‰ ethidium bromide. After electrophoresis, the gels were visualized with a UV transilluminator. The entire experiment was made in triplicate with different batches of megagametophytes at each stage.
4.7. ANALYSIS OF THE MEGAGAMETOPHYTE NUCLEASE ACTIVITIES 4.7.1. Protein extraction for the determination of nuclease activities
Proteins were extracted from megagametophyte flours at each stage with 0.07 M Tris-HCl buffer pH 6.8, containing 1 mM DTT, 0.5 mM PMSF and 10 µM leu in a ratio 1:7 (w/v) on ice for 1 h at
4 °C. After centrifugation (15000 rpm, 15 min, 4 °C), the supernatant protein content was quantified according to Bradford (1976) and the samples were immediately stored at -30 °C until use.
4.7.2. In vitro nuclease activity assays
54 DNase activities were determined according to Thelen and Northcote (1989) with some modifications. The assay is based on the capacity of DNases to degrade DNA into acid-soluble products. The salmon sperm DNA (Sigma), used as a substrate for nucleases, was dissolved in
0.01 M Tris-HCl pH 8.0, containing 10 mM NaCl and denaturated by heating in a boiling-H2O bath for 10 min and immediately cooling in an ice bath. The enzymatic reactions were initiated by adding 40 µl of protein extracts to 14 µl of 2.86 mg/ml DNA in 346 µl of 0.1 M succinic acid-
NaOH pH 5.0. After 30 min of incubation at 37 °C, the reactions were stopped adding 800 µl of chilled absolute ethanol and held at -20 °C for 2 h to allow the precipitation of undegraded DNA.
Samples were then centrifuged (14000 rpm, 30 min, 4 °C) and the nucleotide-containing supernatants were assayed for the change in absorbance at 260 nm over that of blank. The latter consisted of the same reaction mixtures of the enzymatic assays, with one difference consisting in the addition of chilled ethanol before starting the reactions.
To determine the optimum pH, the enzymatic assays were carried out with 0.1 M succinic acid-
NaOH at pH 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 and 0.1 M Tris-HCl at pH 6.5, 7.0, 7.5, 8.0 and 8.5.
One Unit of nuclease activity was the enzyme amount liberating acid-soluble products at a rate of
1.0 A 260 per min. All the enzymatic assays were performed in triplicate.
4.7.3. In-gel nuclease activity assays
Protein extracts, obtained as previously described (paragraph 4.7.1.), were prepared in SDS- sample buffer lacking reducing agents to a final concentration of 1% (w/v) SDS, 0.0625 M Tris-
HCl pH 6.8, 10% (v/v) glycerol. Nuclease activities were detected by loading 20 microliters of protein extracts at each stage in 8.5% polyacrylamide gels, containing 0.1% SDS (v/v) and 0.3 mg/ml heat-denaturated salmon sperm DNA, as a substrate for nucleases. Procedures of electrophoresis and removal of SDS from gels prior to nuclease activity detection are given by
55 Blank et al. (1982) with some modifications described here. Electrophoresis was carried out in a refrigerate chamber (4 °C) in order to avoid enzyme denaturation. SDS was then removed by soaking the gels in two changes of 100 ml each of 25% (v/v) isopropanol in 0.01 M citric acid- phosphate pH 5.0 for 30 min RT, followed by two changes of buffer lacking isopropanol. In order to allow the digestion of the embedded DNA, the gels were incubated for 14 h at 37 °C in 0.1 M citric acid-phosphate pH 5.0. The gels were then stained with 0.05% (w/v) toluidine blue in 0.01
M citric acid-phosphate pH 5.0 for 10 min and destained in several changes of buffer alone.
Nuclease activities were detected as clear spots, where the DNA had been degraded against the blue-stained DNA background. To examine the inhibitory effect of EDTA on nucleases, gel slices, containing the late stage protein extracts, were incubated in 0.1 M citric acid-phosphate pH
5.0, containing 1 mM EDTA. Cation requirement for nucleases was determined incubating the gel slices with 1 mM EDTA for 15 min and subsequently with either Ca 2+ or Mg 2+ , or with both of them, or with Zn 2+ , all added to a final concentration of 4 mM. The effect of growing concentrations of Zn 2+ (0, 0.01, 0.1 and 1 mM) on the megagametophyte nuclease activities was also investigated.
4.8. DETERMINATION OF THE INDOLE-3-ACETIC ACID (IAA) CONTENT IN THE
MEGAGAMETOPHYTE THROUGHOUT GERMINATION
4.8.1. Extraction and purification of free IAA
Free IAA (auxin) was extracted from megagametophyte flours at each stage according to Sorce et al. (2000) with some modifications. Cold 70% (v/v) aqueous acetone was added to the megagametophyte flours in a ratio 1:4 (w/v). The homogenates were supplemented with 200 ng
13 [ C] 6IAA (Cambridge Isotope Laboratories, Andover, MA, USA) as internal standard, then stirred for 2 h at 4 °C and centrifuged at 4000g for 15 min. The residues were re-extracted twice
56 and the supernatants obtained were pooled, reduced to aqueous phase under vacuum at 35 °C, adjusted to pH 2.8 with HCl and, finally, partitioned four times against equal volumes of peroxide-free diethyl ether. The pooled diethyl ether extracts were kept at -20 °C overnight to freeze and separate water traces. Diethyl ether was then evaporated under vacuum and the dried samples were resuspended in a small volume of 10% methanol and purified by HPLC.
4.8.2. High-performance liquid chromatography (HPLC)
The megagametophyte samples were purified by reverse-phase HPLC, using a Waters 501
(Waters, Milford, MA, USA) instrument equipped with a SpectroMonitor 3200 UV detector
(LDC Analytical, Riviera Beach, FL, USA) operating at 254 nm wavelength. An ODS column
(Hypersil, Runcorn, Cheshire, UK) 150 × 4.5 mm ID particle size 5 µm, eluted at a flow rate of 1 ml min -1, was used. The IAA-containing fractions were reduced to a small volume, transferred to capillary tubes, dried thoroughly and derivatized using bis-(trimethylsilyl)-trifluoroacetamide, containing 1% trimethylchlorosilane (Pierce, Rockford, IL, USA).
4.8.3. Gas chromatography-mass spectrometry (GC-MS)
GC-MS analyses were performed on a Saturn 2200 quadrupole ion trap MS coupled to a CP-3800
GC (Varian, Palo Alto, CA, USA), equipped with a CP-Sil 8 CB Low Bleed/MS capillary column (25 m × 0.25 mm ID × 0.25 µm film thickness) coated with 5% phenyl, 95% dimethylpolysiloxane (Varian). The samples were introduced into the instrument by split/splitless injection at 250 °C. The temperature of the transfer line between the GC and the MS was set at
250 °C. The full scan MS were obtained in EI + mode with an emission current of 30 µA, an axial modulation of 4 V and the electron multiplier set at -1300 V. Data acquisition was from 70 to 350
Da at a speed of 1.4 scan s -1. The following ions were monitored for IAA analysis: m/z 202 and
57 319 for IAA and 208 and 325 for 13 C-labelled internal standard. The IAA quantification was carried out by reference to a calibration plot obtained from the GC-MS analysis of a series of mixtures of the standard hormone with its labelled form. The samples of three independent extractions were run three times on the GC-MS and the corresponding values were averaged. The megagametophyte IAA content was expressed as ng IAA megagametophyte -1.
4.9. STATISTICAL ANALYSES
Numeral data are shown as mean ± SD of at least three replicates. The statistical significance of differences among data was evaluated by the Student’s t test.
58 5. RESULTS
- MORPHOLOGICAL DATA-
5.1. MORPHOLOGICAL CHANGES OF THE MEGAGAMETOPHYTE TISSUE
Before the onset of germination, the megagametophyte of bunya pine seeds was bulky and white; however at the late post-germinative stage (28 DAG) the tissue changed in appearance, becoming markedly diminished in size, shrunk and brownish (Fig. 8A). Light microscopy revealed deep morphological changes at the cellular level. The megagametophyte cells of ungerminated seeds were filled with starchy reserves, while at the late post-germinative stage (28 DAG) they appeared emptied of their content, indicating that the reserves had been hydrolyzed (Fig. 8B and
C).
0 DAG 28 DAG
Fig. 8. Morphological changes of the megagametophyte tissue. (A) Sections of bunya pine seeds, showing macroscopic alterations of the megagametophyte between 0 DAG and the late post-germinative stage 28 DAG. In the lower pictures, megagametophyte tissue sections showing the difference in starch content between 0 DAG (B) and 28 DAG (C). In A the bar indicates 1 cm; in B and C bars indicate 50 µm.
59 5.2. VIABILITY STAINING OF MEGAGAMETOPHYTES
Progressive cell death was shown by the megagametophyte viability staining with Evan’s Blue dye. As shown in fig. 9a, the megagametophyte of mature seed (0 DAG) appeared unstained or scarcely stained, suggesting it was formed essentially by living cells with intact membranes.
Nevertheless after seed germination, a progressive intensification of the tissue colouring was found, suggesting a progressive loss of the megagametophyte viability. It was then evident that the megagametophyte cells located at the micropylar and chalazal ends died at the mid post- germinative stage (14 DAG) (Fig. 9c, as indicated by the arrows). Extensive cell death occurred only at late stages of the post-germinative growth, resulting in a marked and homogeneous blue staining of the whole megagametophyte tissue (28 and 35 DAG) (Fig. 9e and f). The pronounced blue staining of the megagametophyte at late post-germinative stages pointed out that the tissue was dead.
Fig. 9. The megagametophyte viability throughout seed germination. Evan’s Blue staining of megagametophytes, showing the spreading of cell death in the tissue during germination: a: 0 DAG; b: 7 DAG; c: 14 DAG; d: 21 DAG; e: 28 DAG; f: 35 DAG. Dead cells stained in blue as a result of the lack of membrane integrity. Arrows in c point to the cells located at the micropylar and chalazal ends.
60 - BIOCHEMICAL DATA-
5.3. CHANGE IN THE MEGAGAMETOPHYTE PROTEIN CONTENT FOLLOWING
GERMINATION
The analysis of the megagametophyte protein content at each germination stage revealed that proteins strongly decreased following seed germination. The insoluble proteins, which appeared as the major storage proteins of the bunya pine megagametophyte, were hugely degraded soon after seed germination (between 0 and 7 DAG) (Fig. 10A and B). As shown in Fig. 10B, many protein bands were affected by degradation, their molecular weights were included in all the range of molecular weights shown by the marker (from 116.3 up to below 12.3 kDa). In the following mid post-germinative stages, the insoluble protein content remained nearly the same, while at late post-germinative stages (28 and 35 DAG) a weak increase in the intensity of some protein bands was observed (Fig. 10B). Soluble proteins instead, were weakly degraded soon after seed germination (between 0 and 7 DAG): the protein bands showing degradation were those with molecular weights included between 116.3 and 42.7 kDa. Other six protein components, whose molecular weights ranged between 30 and 12.3 kDa, disappeared. The electrophoretic pattern remained nearly the same up to 28 DAG. Only at late stages (between 28 and 35 DAG), a marked reduction in the intensity of the four protein bands with molecular weights below 30 kDa was observed (Fig. 10B).
61 A 5.5 5.0 Bradford
4.5 Lowry 4.0 3.5 Insoluble proteins 3.0
2.5 2.0 Soluble proteins 1.5
1.0 0.5 Proteinper(mg megagametophyte) 0.0 0 7 14DAG 21 28 35
B INSOLUBLE PROTEINS SOLUBLE PROTEINS 200 - 97.4 - 77 - 55.5 - 42.7 -
30 -
17.2 - 12.3 -
M 1 2 3 4 5 6 M Fig. 10. Change of the megagametophyte protein content throughout seed germination. (A) Quantification of soluble and insoluble proteins in the bunya pine megagametophyte throughout seed germination, according to the Bradford and Lowry methods. (B) Coomassie blue-stained SDS-PAGE profiles of the megagametophyte soluble and insoluble proteins. 1: 0 DAG; 2: 7 DAG; 3: 14 DAG; 4: 21 DAG; 5: 28 DAG; 6: 35 DAG. Arrows indicate the major protein components of both soluble and insoluble protein fractions. Molecular masses of protein markers (M) are shown on the left (kDa).
5.4. INCREASE OF PROTEOLYTIC ACTIVITIES FOLLOWING GERMINATION
The study of the megagametophyte proteolytic activities time-course was carried out at three different pH values, using haemoglobin and azocasein as substrates for proteases. By these assays, it was shown that the acidic proteolytic activities underwent marked increase following germination, especially those tested at pH 5.4, becoming over three times higher at the late post-
62 germinative stage (35 DAG) in respect to the activity detected before seed germination (0 DAG).
Contrarily to what has been observed for the acidic protease activities, the activities analyzed at pH 7.0 decreased in the early post-germinative stages, followed by an increase between 14 and 35
DAG, resulting in no appreciable change in their total time-course (Fig. 11A).
The use of protease inhibitors has allowed to highlight that, at pH 5.4, E-64 had only a scant effect on the megagametophyte proteolytic activities at mid and late post-germinative stages, suggesting a weak involvement of cysteine proteases in the megagametophyte proteolysis (Fig.
11B, a). Unlike E-64, pepstatin-A and EDTA had a higher effect at each germination stage at acidic and neutral pH values respectively, hence indicating that aspartic and metallo-proteases were present and active in the megagametophyte indistinctly at each germination stage (Fig. 11B, b and c). The use of PMSF instead has allowed to demonstrate that serine proteases were not present or not active in the bunya pine megagametophyte before seed germination and at early post-germinative stages; their activity became evident only at 28 and 35 DAG, indicating the presence of serine proteases active during the megagametophyte proteolysis only at late post- germinative stages (Fig. 11B, d).
A 1.0 Haemoglobin pH 4.0 Azocasein pH 5.4 0.8 Azocasein pH 7.0
0.5
0.3
Activity (Units per megagametophyte) per Activity(Units 0.0 0 7 14 21 28 35 DAG
63 B a b Effect of E-64 on protease activities Effect of Pep-A on protease activities 110 110 100 pH 4.0 100 pH 4.0 90 pH 5.4 90 pH 5.4 80 pH 7.0 80 pH 7.0 70 70 60 60 50 50 40 40 % of % activity % of activity 30 30 20 20 10 10 0 0 0 DAG 7 DAG 14 DAG 21 DAG 28 DAG 35 DAG 0 DAG 7 DAG 14 DAG 21 DAG 28 DAG 35 DAG
c d Effect of EDTA on protease activities Effect of PMSF on protease activities 110 110 100 pH 4.0 100 pH 4.0 90 pH 5.4 90 pH 5.4 80 80 pH 7.0 pH 7.0 70 70 60 60 50 50 40 40 % of activity 30 % of activity 30 20 20 10 10 0 0 0 DAG 7 DAG 14 DAG 21 DAG 28 DAG 35 DAG 0 DAG 7 DAG 14 DAG 21 DAG 28 DAG 35 DAG
Fig. 11. Study of the megagametophyte proteolytic activities throughout germination. (A) Time- course of the megagametophyte proteolytic activities by using in vitro assays. The activities were tested at three different pH values with two substrates, haemoglobin and azocasein. (B) Characterization of the megagametophyte proteolytic activities with protease class-specific inhibitors: (a) E-64 (cysteine protease inhibitor, 20 µM) (b) pepstatin-A (aspartic protease inhibitor, 1.43 µM) (c) EDTA (metallo-protease inhibitor, 1 mM) and (d) PMSF (serine protease inhibitor, 1 mM). Residual activity is expressed as percentage over the control (activity without protease inhibitors). All data are based on the mean of three replicates ± SD.
5.5. INDUCTION OF CASPASE-LIKE PROTEASE (CLP) ACTIVITIES FOLLOWING
GERMINATION
In order to investigate a possible involvement of caspase-like proteases (CLPs) in the megagametophyte death, extracts at each stage were incubated with synthetic substrates specific for distinct members of the mammalian caspase family. The substrates tested were Ac-YVAD- pNA for caspase 1 (YVADase activity), Ac-DEVD-pNA for caspase 3 (DEVDase activity) and
64 Ac-VEID-pNA for caspase 6 (VEIDase activity). The enzymes with caspase-like activity recognize the tetrapeptide and, cleaving after an aspartic acid residue, release the p-NA in the reaction mixture. When the three substrates were incubated without the enzymatic extract, no spontaneous release of the pNA was observed.
All the three CLPs activities were only barely detectable before germination and at early post- germinative stages, but they strongly increased at late post-germinative stages, with a burst of activity at 28 DAG (Fig. 12A). The three sets of enzymes contributed differently to the total caspase-like activity, being the VEIDase and to a lesser extent the YVADase higher than
DEVDase. As shown in fig. 12B, the optimum pH for the cleavage of the three synthetic substrates was 5.0-5.5, 5.0 and 4.5 (for YVADase, DEVDase and VEIDase respectively). For all the three CLPs, no appreciable activity was detected at neutral pH values.
In order to preliminarily characterize the megagametophyte CLPs, different protease inhibitors were used, including caspase inhibitors (Ac-YVAD-CMK for caspase 1, Ac-DEVD-CHO for caspase 3 and Ac-VEID-CHO for caspase 6). Caspase inhibitors contain a common reversible or irreversible reactive center (cloromethyl-aldehyde or ketone), linked to a peptidomimetic moiety; the latter provides selectivity in binding to the different caspases or caspase-like proteases (Wu and Fritz, 1999).
When the caspase inhibitors were included in the enzymatic assays, the DEVDase and VEIDase activities were strongly affected (76% and 77% respectively) (Fig. 12C, b and c), while Ac-
YVAD-CMK was able to markedly reduce YVADase of a 55% (p ≤ 0.01) (Fig. 12C, a). The other non specific inhibitors did not affect the megagametophyte CLPs activities, with the exception of PMSF (serine protease inhibitor), that was capable of inhibiting VEIDase of a 57%
(p ≤ 0.01) (Fig. 12C, c).
65 A B 0.5 0.06 YVADase YVADase 0.05 DEVDase 0.4 DEVDase VEIDase VEIDase 0.04 0.3 0.03 0.2 0.02
0.1 0.01
pNAnmol/min/megagametophyte pNA nmol/min/megagametophyte 0 0 0 7 14 21 28 35 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 DAG pH
C 140 120 a PMSF control
DMSO pep-A Et-OH 100 E-64
80
60 -YVAD-CMK Ac
of % activity 40
20 0 YVADase
140 b 120 control PMSF Et-OH pep-A 100 DMSO
E-64 80 60
of % activity 40
Ac-DEVD-CHO 20 0 DEVDase
140 c 120 control Et-OH E-64 pep-A 100 DMSO
80 CHO 60 - PMSF of activity %
40 -VEID
Ac 20
0 VEIDase 66 Fig. 12. Study of the megagametophyte CLPs activities throughout seed germination. (A) Time- course of caspase 1, 3 and 6-like activities in the megagametophyte using in vitro assays. (B) Study of the YVADase, DEVDase and VEIDase optimum pH at late post-germinative stages. (C) Inhibition studies of the megagametophyte CLPs activities at the late post-germinative stage (28 DAG). The inhibitors used were: E-64 (cysteine protease inhibitor, 5 µM), pep-A (aspartic protease inhibitor, 1.43 µM), PMSF (serine protease inhibitor, 1 mM), Ac-YVAD-CMK (caspase 1 inhibitor, 100 µM), Ac-DEVD-CHO (caspase 3 inhibitor, 100 µM) and Ac-VEID-CHO (caspase 6 inhibitor, 100 µM). Residual activity is expressed as percentage over the control (activity without protease inhibitors). DMSO: dimethyl sulfoxide; Et-OH: ethanol. All data are based on the mean of three replicates ± SD; p ≤ 0.01 for the inhibition studies.
5.6. DETECTION OF PROTEOLYTIC ACTIVITIES USING IN-GEL ASSAYS
The in-gel protease activity assays were carried out to assess qualitative changes in the megagametophyte proteolytic activities throughout seed germination. As shown in fig. 13A
(black arrow), one activity band was observed before seed germination (0 DAG); this activity was also visible with a lower intensity at 7 and 14 DAG and it was no more detectable at late post-germinative stages. Other proteolytic enzymes, with a lower electrophoretic mobility, appeared instead not present or not active before seed germination, while in the post-germinative stages their activities became evident. A strong rise in the activity of these enzymes was observed at late post-germinative stages (28 and 35 DAG), as highlighted by their big activity spot (Fig.
13A, red arrow).
The characterization of these activities with different protease inhibitors has allowed to highlight that the activity band detected before germination was completely effaced by E-64 and leu, hence indicating that the enzymes responsible for this activity belonged to the class of cysteine proteases (Fig. 13B). The big activity spot detected at late post-germinative stages instead was strongly weakened by PMSF, hence indicating the presence of serine proteases at late post- germinative stages (Fig. 13C). When these late stage proteases were incubated with caspase 67 inhibitors, it was possible to observe that the caspase 6 inhibitor was able to strongly attenuate
their big activity spot, hence suggesting a high sensitivity of these serine proteases towards the
caspase 6 inhibitor (Fig. 13D).
A
B C D
68 Fig. 13. The megagametophyte proteolytic activities detected using in-gel activity assays. (A) Time-course of the megagametophyte proteolytic activities throughout seed germination. 1: 0 DAG; 2: 7 DAG; 3: 14 DAG; 4: 21 DAG; 5: 28 DAG; 6: 35 DAG. Black and red arrows indicate gelatinolitic activities at different stages. (B) Characterization of the early stage protease activities with different inhibitors. β-ME: 2 β-mercaptoethanol. (C) Characterization of the late stage protease activities with different inhibitors. (D) Characterization of the late stage protease activities with caspase inhibitors. The inhibitors used were: E-64 (cysteine protease inhibitor, 10 µM), EDTA and o-phen (metallo-protease inhibitors, 10 mM and 1 mM), leu (cysteine and trypsin-like protease inhibitor, 10 µM), pep-A (aspartic protease inhibitor, 20 µM), PMSF (serine protease inhibitor, 10 mM), Ac-YVAD-CMK (caspase 1 inhibitor, 400 µM), Ac-DEVD- CHO (caspase 3 inhibitor, 400 µM) and Ac-VEID-CHO (caspase 6 inhibitor, 400 µM). DMSO: dimethyl sulfoxide. C is the control (activity without protease inhibitors).
5.7. IDENTIFICATION OF THE MEGAGAMETOPHYTE LATE STAGE PROTEASES BY
A PROTEOMIC APPROACH
Fig. 14A shows the two-dimensional (2-D) separation of the 28 DAG protein extract (gel on the left) and the corresponding proteolytic activity on gelatin (gel on the right). Mass spectrometry analysis of the megagametophyte late stage proteases showed that these enzymes had amino acid sequences homologous to subtilisin-like serine proteases of other plants. Some of these sequences are listed in the table (fig. 14B).
69 A 2-DE analysis of 28 DAG extract proteolytic activity of 28 DAG extract
pH 3.0 pH 10.0
B
1: 712-ETS VDAYF -719 Araucaria bidwillii 635-ETS IDDYF -642 Picea abies subtilisin-like protein A. N. BAA13135
2: 724-FADAGFGPVPEK -735 Araucaria bidwillii 101-FNDSGFGPVP ---110 Arabidopsis thaliana subt-like protease -like prot. A.N.: BAE98849 162-FSDAG MGPVP ---171 Oryza sativa putative subtilisin-like protein A. N: BAA89562 149-FNDYGLGPVPEK -160 Lotus japonicus subtilase A. N.: BAF95887 99-FSDEGFGP PPAK-110 Narcissus pseudonarcissus subtilisin-like ser. protease A. N.: AAL69380 233-FSDDGMGPVP ---242 Triticum aestivum subtilisin-like protease A. N.: CAJ19363 151-FDDTGLGPVP ---160 Nicotiana tabacum subtilisin-like protease A. N.: ABQ58080
3: 738-PF SLVVAF -745 Araucaria bidwillii 86-PF RLVVAF -93 Volvox carteri subtilisin-like serine protease A. N.: BAF36498
4: 1265-VWP GV PYF PDA AVTYK -1280 Araucaria bidwillii 352-VWP DI PYF ----S-YK-362 Medicago truncatula subtilisins A. N.: ABD32844 603------FVDA GVT--- 609 Medicago truncatula subtilisins A. N.: ABD32844
5: 1328-VWP-GMGPVS -1336 Araucaria bidwillii 80-VWP-GMGPV-- 87 Triticum aestivum subtilisin-like protease A.N.: CAJ75644226 226-VWP-GMGPV-- 233 Triticum aestivum subtilisin-like protease A. N.: CAJ19363 261--- P-GAGTVS -267 Triticum aestivum subtilisin-like protease A.N.: CAJ75644 144-VWP-GMGPV --151 Oryza sativa putative serine protease A.N.: BAD36156
Fig. 14. Proteomic study of the late stage megagametophyte extract. (A) On the left, 2-D gel showing the protein spots of the 28 DAG extract and, on the right, gelatin-containing gel with the activity spot (indicated by the white arrow) of the 28 DAG proteases. (B) Table showing some of the sequence homologies found between the bunya pine (Araucaria bidwillii) megagametophyte proteases and subtilisin-like serine proteases of other plants. Identitical amino acids are in bold. Sequence homology was obtained with the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST). A.N.: Accession Number.
70 5.8. DETECTION OF DNA FRAGMENTATION
The fragmentation of genomic DNA was investigated both “ in situ” by the TUNEL assay and in gel by electrophoresis. The TUNEL assay is based on the labelling of fragmented nuclear DNA at the 3’-OH ends, using terminal deoxynucleotidyl transferase (TdT) and dUTP conjugated to a fluorochrome. The tissue sections used for the TUNEL assay were counterstained with DAPI, whose white/blue fluorescence indicated the presence and the localization of nuclei. Before seed germination, only 2.5% of DAPI-stained nuclei were TUNEL-positive (Fig. 15A a and g and B), suggesting the presence of a large number of nuclei with intact DNA in the megagametophyte cells prior to seed germination. At 7 and 14 DAG the percentage of TUNEL-positive nuclei increased to 16% and 27% respectively; however, the majority of nuclei remained not labelled and therefore contained intact DNA (Fig. 15A b, c, h and i and B). The TUNEL-positive nuclei increased progressively up to 98% at the late post-germinative stage (35 DAG) (Fig. 15A d-f, l-n and B), suggesting that extensive DNA fragmentation had occurred at this late stage. Appropriate control treatments were conducted at each stage. When the DNase I treatment preceded the
TUNEL procedure, all nuclei at each stage were fluorescent (positive controls, data not shown).
On the other hand, no fluorescence was observed at any stage when TdT enzyme was omitted from the reaction (negative controls, data not shown).
71
B TUNEL-positive
nuclei, % 0 DAG 2.5%
7 DAG 16%
14 DAG 27% 21 DAG 55% 28 DAG 78%
35 DAG 98%
Fig. 15. Detection of DNA fragmentation “in situ”. (A) Six stages of the megagametophyte are shown: a-f, TUNEL-labelled megagametophyte tissue sections at 0, 7, 14, 21, 28, 35 DAG; g-n, same sections as in a-f counterstained with DAPI. Bars indicate 50 µm. (B) Percentage of TUNEL-positive nuclei at each of the germination stages listed above. The percentage of TUNEL-positive nuclei was calculated out of 5000 nuclei randomly selected from pictures, by scoring over 400 tissue sections at each stage.
72 DNA fragmentation was also investigated by electrophoresis on agarose gel (Fig. 16). Before the onset of germination, the bunya pine megagametophyte DNA appeared as a single band, suggesting it had not been degraded yet. DNA fragmentation became evident in the post- germinative stages. Extensive DNA fragmentation was observed at the late post-germinative stages (28 and 35 DAG). Moreover at 35 DAG a “ladder” (indicated by red arrows) was also observed. This evidence pointed out the presence of DNA fragments multiple of about 180 bp.
M 1 2 3 4 5 6
4.0 kb 3.0 kb 2.0 kb 1.5 kb
1.0 kb
0.5 kb
Fig. 16. Detection of DNA fragmentation through electrophoresis. Genomic DNA was extracted from megagametophyte flours at each germination stage and subjected to electrophoresis on 1% agarose gel, containing 1‰ ethidium bromide. Red arrows indicate oligonucleosomal-length fragments. M: marker, 1: 0 DAG, 2: 7 DAG, 3: 14 DAG, 4: 21 DAG, 5: 28 DAG, 6: 35 DAG.
The determination of the megagametophyte DNA content by spectrophotometric analysis showed that the amount of DNA decreased following germination. Two weak waves of DNA degradation were observed: the first between 0 and 7 DAG and the second between 14 and 21 DAG.
Considerable reduction of DNA content was observed at the late post-germinative stage (35
DAG) (Fig. 17A). Consistent results were obtained at the cellular level by analysing the DAPI- stained megagametophyte tissue sections. At 0 DAG, the megagametophyte cells were filled with
73 nuclei; however in the post-germinative stages, the number of nuclei progressively decreased.
Disappearance of nuclei was observed at the late post-germinative stage (35 DAG) (Fig. 17B).
B A 16 14
12
10 8 6 4 2 ug DNA per megagametophyte per DNA ug 0 0 7 14 21 28 35 DAG C
Fig. 17. Changes in the megagametophyte DNA content throughout seed germination. (A)
Measurements of the megagametophyte DNA content by monitoring the A 260nm of the DNA extracted at each stage. Data are based on the mean of three replicates ± SD of three independent extractions. (B) Pictures showing changes in the number of nuclei throughout germination. The megagametophyte tissue sections at each stage were stained with DAPI to visualize nuclei and analyzed with a light microscope. Bars indicate 100 µm. 1: 0 DAG, 2: 7 DAG, 3: 14 DAG, 4: 21 DAG, 5: 28 DAG, 6: 35 DAG. (C) Table showing the ratio
Abs 260nm /Abs 280nm of the megagametophyte DNA at each stage, as an indicator of DNA purity. The values of this ratio should be included between 1.65 and 1.85, according to Nibert and Wilson (1996).
74 5.9. INDUCTION OF NUCLEASE ACTIVITIES FOLLOWING GERMINATION
In vitro and in-gel assays were carried out to study the megagametophyte nuclease activities. The in vitro assays showed that before germination the megagametophyte nuclease activities were barely detectable, but in the post-germinative stages they markedly increased. The peak in activity was observed at the late post-germinative stages (28 and 35 DAG) (Fig. 18A). A similar time-course of activity was observed in gel. As shown in fig. 18B, the megagametophyte nuclease activities became faintly visible only in the post-germinative stages. Strong activation of nucleases was detected at the late post germinative stages (28 and 35 DAG).
B A 0.5
0.4
0.3
0.2
0.1 DNaseactivity(Units permeg.) 0 0 7 14DAG 21 28 35
Fig. 18. Study of the megagametophyte nuclease activities throughout seed germination. (A) Time-course of the megagametophyte nuclease activities by in vitro assays. Data are based on the mean of three replicates ± SD. (B) Time-course of the megagametophyte nuclease activities by in-gel assays. Twenty microliters of the megagametophyte protein extracts at each stage were loaded on a 8.5% polyacrylamide gel, containing heat-denaturated salmon sperm DNA as a substrate for nucleases. Nuclease activities were detected as clear spots, where the DNA had been degraded against the blue-stained DNA background. 1: 0 DAG; 2: 7 DAG; 3: 14 DAG; 4: 21 DAG; 5: 28 DAG; 6: 35 DAG.
75 The investigation of the megagametophyte nuclease optimum pH highlighted that these enzymes acted optimally at acidic pH, with a maximum of activity at pH 5.0 (Fig. 19A). In order to understand which ion was required for activity, in-gel activity assays in the presence of chelating agents and different ions were carried out. As shown in fig. 19B, the megagametophyte nucleases were strongly inhibited by EDTA, hence suggesting that these enzymes required ions for their activity. The alternative addition of different cations after EDTA treatment highlighted that only
Zn 2+ was able to restore the activity of the megagametophyte nucleases, while the other ions had only a very scant effect, hence revealing that these enzymes belonged to the class of Zn 2+ - dependent nucleases. Furthermore when the megagametophyte nucleases were incubated with growing concentrations of Zn 2+ , their activity spot progressively enhanced, hence suggesting that this ion had a stabilizing effect on the megagametophyte nucleases (Fig. 19C).
A 0.5
Succinate-NaOH buffer 0.4 Tris-HCl buffer 0.3
0.2
0.1
activityDNase (Units meg.) per
0 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 pH
76 B C
Fig. 19. Characterization of the late stage megagametophyte nucleases. (A) Study of the late stage megagametophyte nuclease optimum pH. The enzymes showed a maximum of activity at pH 5.0. Data are based on the mean of three replicates ± SD. (B) The megagametophyte nucleases were treated with 1 mM EDTA alone or supplemented alternatively with either Ca 2+ or Mg 2+ , or with both of them, or with Zn 2+ , all added to a final concentration of 4 mM. C is the activity without EDTA nor ions, taken as a control. (C) The effect of growing concentrations of Zn 2+ on the megagametophyte nuclease activities.
77 -CYTOLOGICAL DATA-
5.10. ALTERATIONS OF NUCLEAR SIZE AND MORPHOLOGY
The analysis of the DAPI-stained megagametophyte tissue sections showed that before seed germination the megagametophyte cells had big and round nuclei. Nevertheless following germination, at the latest post-germinative stage (35 DAG), the megagametophyte nuclei appeared markedly shrunk. The measurements of the megagametophyte nuclei sizes with the
Image J software highlighted that at the late stage (35 DAG) the nuclei diameter had decreased down to 56% ± 4.55 the size of 0 DAG nuclei (p < 0.01); moreover, the 35 DAG nuclei emitted brighter fluorescence than that released by the 0 DAG nuclei (Fig. 20A and B). These data suggested the happening of chromatin condensation at late post-germinative stages. Moreover at these stages, several nuclei appeared abnormally-shaped (Fig. 20C, upper and lower pictures).
These observations pointed out the occurrence of alterations in the size and morphology of nuclei at late post-germinative stages.
78
Fig. 20. Morphological changes of nuclei following seed germination. DAPI staining of megagametophyte tissue sections at two different germination stages. The big and round nuclei before seed germination (A) and the condensed nuclei at the late post-germinative stage (35 DAG) (B) are shown. The late stage megagametophyte nuclei were 56% the size of 0 DAG nuclei (p < 0.01). (C) The morphologically-altered nuclei at the late post-germinative stage (35 DAG). In A and B bars indicate 50 µm, in C bars indicate 20 µm.
79 - PHYSIOLOGICAL DATA-
5.11. DETERMINATION OF INDOLE-3-ACETIC ACID (IAA) CONTENT IN THE
MEGAGAMETOPHYTE THROUGHOUT GERMINATION
Auxin or indole-3-acetic acid (IAA) was quantified at each germination stage in order to preliminarily investigate a possible involvement of this hormone in the megagametophyte degeneration. As shown in fig. 21, the IAA amount markedly increased with time up to 21 DAG, becoming at this stage over fourfold higher than 0 DAG auxin content. A considerable decline in auxin amount was observed at the late post-germinative stages (28 and 35 DAG).
18
15
12
9
6
3 ng IAA per megagametophyte 0 0 7 14DAG 21 28 35
Fig. 21. Determination of free indole-3-acetic acid (IAA) content in the megagametophyte throughout seed germination. Free IAA or auxin was extracted from megagametophyte flours at each germination stage and quantified. Data are based on the mean of three replicates ± SD, starting from three independent extractions.
80 6. DISCUSSION
The megagametophyte of Conifer seeds develops from one functional megaspore, derived from meiosis of the megaspore mother cell; the remaining three megaspores closest to the micropyle degenerate. After fertilization, during the expansion stage of seed and embryo development, the megagametophyte cells begin to accumulate protein and starchy reserves (Owens et al. , 1993). At seed maturity, the megagametophyte represents the major storage tissue of the bunya pine
(Araucaria bidwillii ) seed, containing the bulk of reserves for the growing embryo. Nevertheless following seed germination, the megagametophyte reserves are hydrolyzed and transferred to the embryo. When all the reserves are mobilized, the megagametophyte, emptied of its content, degenerates as a no longer needed tissue. For this reason the megagametophyte represents a useful system to study developmentally-regulated PCD, being a short-lived tissue which degenerates once it has served its nourishing function for the developing embryo.
The megagametophyte cell death is accompanied by evident morphological changes at late post- germinative stages: the tissue becomes brownish in appearance and significantly smaller and shrunk than that of ungerminated seed. Moreover at late post-germinative stages, the megagametophyte cells appear emptied of their content, indicating that the starchy reserves have been hydrolyzed. The viability staining has shown that the megagametophyte tissue progressively loses its viability following germination, becoming at late stages a dead tissue. The megagametophyte death occurs firstly in the cells located at the micropylar and chalazal ends at the mid post-germinative stage (14 DAG) and, only at late post-germinative stages, cell death wave spreads to the whole megagametophyte tissue. These observations indicate that the megagametophyte death occurs in an asynchronous way. Spatial-temporal patterns of cell death progression have also been observed in other plant tissues and organs undergoing developmental
PCD, such as the female flowers of maize during the abortion of stamen primordia (Calderon- 81 Urrea and Dellaporta, 1999), the nucellus of castor bean (Greenwood et al. , 2005) and the suspensor of Phaseolus coccineus (Lombardi et al. , 2007b).
A common feature reported in tissue/organ degeneration is the reduction of protein levels. The loss of proteins has been demonstrated both in physiological (Stephenson and Rubinstein, 1998;
Pak and van Doorn, 2005) and in oxidative stress-induced cell death (Palma et al. , 2002). The analysis of the protein content and the SDS-PAGE of soluble and insoluble proteins of the bunya pine megagametophyte clearly show that the temporal pattern of protein degradation is different for the two protein fractions. Insoluble proteins, which are the most abundant proteins of the bunya pine seed, suffer a huge degradation soon after seed germination, suggesting that they are the main target of the early post-germinative stage proteases, whose action may be temporally correlated with the process of storage protein mobilization. Insoluble proteins indeed are reported to be the main storage proteins of Gymnosperm seeds; they are stored in protein bodies and, a few days after seed germination, they become the target for proteases in order to supply the embryo with amino acids (Galleschi et al. , 2002). Contrarily to what has been reported for the megagametophyte of white spruce orthodox seeds by He and Kermode (2003b), in which insoluble proteins remain also in the post-germinative stages and are mostly degraded only at late stages, the insoluble proteins of the bunya pine megagametophyte are almost entirely degraded soon after seed germination, suggesting that the process of protein reserve mobilization likely is much more rapid in recalcitrant than in orthodox seeds. The soluble proteins of the bunya pine megagametophyte undergo appreciable degradation only at a very late post-germinative stage, indicating that they represent the main target of the late post-germinative stage proteases, whose action is temporally related to the happening of the megagametophyte cell death rather than to the storage protein mobilization. If we assume that soluble proteins are those proteins involved in the maintenance of structure and/or homeostatic pathways of the cell, we can speculate that soluble
82 protein hydrolysis occurring at late stages really represents a feature of the bunya pine megagametophyte cell death.
The strong reduction of protein levels lets think that a marked increase in proteolytic activities had to be occurred. Proteolytic enzymes have been reported to play pivotal roles in cell or tissue death-related processes (Beers et al. , 2000; Schaller, 2004). In the last years some authors reported the activation of cysteine and serine proteases during the onset and the progression of cell death in different plant systems (He and Kermode, 2003b; Samadi and Shahsavan Behboodi,
2006; Azeez et al. , 2007).
Both the in vitro and the in-gel protease activity assays have shown that the megagametophyte proteolytic activities remarkably increase after seed germination, reaching a maximum at late post-germinative stages. The peak in activity observed at these stages temporally correlates with the megagametophyte degeneration, hence suggesting a possible role for proteases in the accomplishment of the megagametophyte cell death.
The use of different protease inhibitors has allowed to highlight that the megagametophyte proteolysis is executed by different classes of proteases. The in vitro activity assays have shown that aspartic and metallo-proteases are well active at acidic and neutral pH values respectively, without an appreciable difference among the stages. This evidence suggests that these two classes of proteases are essential during all the course of seed germination; they may be involved in storage protein mobilization as well as in cell death. Serine proteases instead appeared to be active only at late post-germinative stages. The detection of serine proteases active only at late stages has been demonstrated also in-gel. The activity spot of these enzymes was evident only in the post-germinative stages and strongly enhanced at late stages, when the great part of protein reserves has been mobilized, hence suggesting that serine proteases most probably play a role in the megagametophyte cell death. Through the in-gel activity assays, it was possible to detect also
83 cysteine proteases well active before seed germination. Cysteine proteases were detected only in- gel, probably because these enzymes are more efficient on gelatin (the substrate used in-gel) than on haemoglobin and azocasein (the substrates used in vitro); alternatively, it can be speculated that the pH values used in vitro are not optimal for these proteases. The analysis of the time- course of cysteine protease activity in-gel highlighted that these enzymes are active before seed germination and in the early post-germinative stages and their activity is no more detectable at late stages, hence suggesting that cysteine proteases likely are involved in storage protein mobilization. This hypothesis is consistent with some studies reporting the involvement of cysteine proteases in storage protein hydrolysis in germinating seeds of various Spermatophyta
(Galleschi et al. , 1989; Bottari et al. , 1996; Shutov and Vaintraub, 1987). Differently from what has been reported for the megagametophyte of white spruce orthodox seeds, where both cysteine and serine proteases were found to be involved in cell death, in the bunya pine megagametophyte these two classes of proteases seem to play different roles: cysteine proteases are thought to be involved in storage protein mobilization, serine proteases seem to participate in cell death.
In order to understand whether the megagametophyte degeneration occurs by means of a developmental PCD, typical hallmarks of apoptosis have been searched.
One of the apoptotic hallmarks is the activation of caspases. These enzymes are the main executors and regulators of animal PCD, being involved directly or indirectly in the dismantling of cell structures and homeostatic pathways. Although caspase homologous have never been found in plant genomes, caspase-like proteases (CLPs) activities have been detected during plant developmental PCD by using synthetic substrates specific for mammalian caspases (Rotari et al. ,
2005 and references within).
In the current study for the first time caspase 1, 3 and 6-like activities (YVADase, DEVDase and
VEIDase, respectively) are reported to be associated with cell death of megagametophyte of the
84 bunya pine recalcitrant seed. Indeed when the megagametophyte extracts were assayed with the caspase substrates, it was possible to observe a strong increase of all the three CLPs activities at late post-germinative stages. The strong increase of these activities at these stages temporally parallels the occurrence of the megagametophyte death. Even though in this thesis there is not any direct proof of the involvement of CLPs in the megagametophyte death, the temporal match between the peak of CLPs activity and the megagametophyte death lets hypothesize that CLPs may be involved in the execution of the megagametophyte cell death.
In spite of a general increase in activity, the CLPs appear differentially active, being the VEIDase and YVADase higher than DEVDase. While the caspase 3 represents the most relevant protease involved in animal apoptosis (Porter and Janicke, 1999), the main PCD executioner in plant cells remains elusive (Lam, 2005). During the hypersensitive response-associated PCD, only
YVADase activity was detected (Del Pozo and Lam, 1998), while during leaf senescence
DEVDase activity was the only one reported (Lam and Del Pozo, 2000). Recently, Bozhkov et al.
(2004) demonstrated that VEIDase is the principal caspase-like activity during PCD in Picea abies embryo and is essential for the embryo pattern formation. It is noteworthy that, in the execution of the bunya pine megagametophyte cell death, a major role seems to be played by
VEIDase and, to a lesser extent, by YVADase.
The investigation of the megagametophyte CLPs optimum pH has revealed that these enzymes act optimally at acidic pH, in accordance with what has been reported in other plant systems
(Danon et al. , 2004; Rotari et al. , 2005). In contrast with this evidence, animal caspases act optimally at pH values ranging from 6.8 to 7.4 (Stennicke and Salvesen, 1997). The fact that the megagametophyte CLPs prefer acidic pH suggests that these enzymes may be activated after cytoplasmic acidification, or that they may be localized in the vacuole. Indeed the plant vacuole represents a reservoir for many hydrolytic enzymes which, upon tonoplast rupture, are released
85 into the cytoplasm and hydrolyse the cellular content by autophagy or autolysis (Klionsky and
Emr, 2000). The involvement of the vacuole in PCD has been reported in various situations, such as during tracheary element differentiation, senescence and during suspensor degeneration (Obara et al. , 2001; Jones, 2001; Filonova et al. , 2002).
In order to preliminarily characterize the megagametophyte CLPs activities, different protease class-specific inhibitors were used. The three sets of enzymes are all markedly inhibited by the respective caspase inhibitors, suggesting the involvement of CLPs in the proteolytic cleavage.
The general protease inhibitors do not affect the megagametophyte CLPs activities; the only exception is given by PMSF (a serine protease inhibitor), that was found to be capable to markedly inhibit VEIDase. The sensitivity of the megagametophyte VEIDase to both PMSF and caspase 6 inhibitor suggests either that the cleavage of VEID-pNA is executed by proteases belonging to two different classes (caspase 6-like proteases and serine proteases), or that the cleavage is carried out by (a) protease(s) sensitive to both inhibitors. In both hypotheses, interesting conclusions can be drawn. If the enzymes corresponding to this activity are caspase 6- like proteases, their being targeted by PMSF would contrast with the nature of most plant CLPs, which are reportedly not affected by general protease inhibitors (Danon et al. , 2000). In the case these enzymes are serine proteases, they also possess caspase 6-like activity and are targeted by the caspase 6 inhibitor besides PMSF. The capability of PMSF and caspase 6 inhibitor to inhibit the late stage proteases was observed also by using zymograms. In order to shed light on the identity of these late stage enzymes, the megagametophyte protein extract was subjected to two- dimensional electrophoresis (2-DE) and mass spectrometry analysis. The 2-D gel showed a relatively small number of protein spots, indicating the presence of few proteins in the bunya pine megagametophyte at this late post-germinative stage. When the 2-D gel was incubated with the gelatin-containing gel, it was possible to detect the protein spot responsible for the gelatinolitic
86 activity. The identification of this spot by mass spectrometry showed that these proteases had amino acid sequences homologous to subtilisin-like serine proteases of other plants. The subtilisin family is the most widely distributed class of proteolytic enzymes among living organisms and are reported to play a role in a variety of physiological processes including senescence, protein degradation and processing as well as cell differentiation (reviewed in Antão and Malcata, 2005; Palma et al. , 2002); in addition, emerging evidence has recently suggested a role for subtilisins in PCD (Woltering, 2004; Antão and Malcata, 2005). The fact that subtilisin- like serine proteases are well active during the megagametophyte degeneration lets hypothesize that this class of enzymes really plays a role in the bunya pine megagametophyte PCD. Coffeen and Wolpert have recently found subtilisin-like serine proteases during PCD in Avena sativa ; the authors showed that these enzymes were sensitive to the caspase inhibitors and exhibited caspase activity; for these reasons they named these proteases saspases (serine asp artate-specific proteases ) (Coffeen and Wolpert, 2004). Unfortunately, the similarity between the Avena serine proteases and the Araucaria serine proteases is restricted to the fact that both enzymes are inhibited by caspase inhibitors. Future studies aimed at assaying the Araucaria serine proteases with caspase substrates will show whether these enzymes have or not caspase activity.
The fragmentation of genomic DNA, which is reported to occur during developmental cell death
(Danon et al. , 2000; Kuriyama and Fukuda, 2002), was investigated in the bunya pine megagametophyte both in situ by the TUNEL assay and in gel by electrophoresis. When the megagametophyte tissue sections were subjected to the TUNEL assay, it was possible to observe an ongoing process of DNA fragmentation in the megagametophyte nuclei following germination. The gradual increase of the TUNEL-positive nuclei percentage observed in the post- germinative stages suggests that DNA degradation is a temporally-regulated process, which gradually accompanies the progressive death of the megagametophyte.
87 The analysis of DNA fragmentation by electrophoresis on agarose gel has highlighted that the megagametophyte DNA is intact before germination, while, in the post-germinative stages, it appears more and more fragmented. At the late post-germinative stages (28 and 35 DAG), extensive DNA fragmentation takes place; moreover, at these late stages a DNA “ladder” was also observed. Typically the “ladder” rungs are multiple of about 180 bp, due to internucleosomal excision of chromatin by nucleases. The detection of DNA “ladder” is considered a distinctive feature of PCD and is currently used to distinguish at the molecular level PCD from other forms of cell death, such as necrosis. In the latter in fact, genomic DNA degradation occurs randomly, resulting in a “smear” detected when the DNA is separated on agarose gel (Kerr and Harmon,
1991). Thus the presence in the bunya pine megagametophyte of a DNA “ladder”, even though included in a “smear” pattern, lets think to the occurrence of a controlled cell death. The concurrent presence of a “smear” besides the “ladder” in the megagametophyte may be due to the fact that the PCD occurring during developmental events presents a higher variability in the size of DNA fragments if compared with an induced PCD. If a tissue undergoes physiological PCD, like I am suggesting in this thesis, we can think that, at the moment of DNA fragmentation experiment, there are some cells that are already dead, others that are committed to die, then others that are still living, so that the timing of PCD is different from one cell to another. Thus a great heterogeneity in the length of DNA fragments will appear after electrophoretic analysis.
DNA “ladders” in plants have been reported in many examples of tissue or organ degeneration during development and upon stress conditions (Danon et al. , 2000 and references within).
Although the internucleosomal DNA cleavage is a distinctive feature of PCD, in some cases the cleavage of genomic DNA in 50 kb fragments has also been reported (Mittler et al. , 1997), suggesting that in plants there is more variation in the size of the fragmented DNA material in comparison with animals in which a very great uniformity exists (Krishnamurthy et al. , 2000).
88 The inconsistency regarding the size of DNA fragments formed during plant PCD may be ascribed to the presence and the activation of at least two different groups of nucleases, one generating 50 kb DNA fragments, the other causing the production of oligonucleosomal length fragments (Krishnamurthy et al. , 2000; Danon et al. , 2000).
The quantitative analysis of the bunya pine megagametophyte DNA has shown that genomic
DNA markedly decreases after seed germination, becoming at the late post-germinative stage (35
DAG) less than a third of the DNA amount present in the ungerminated seed. Consistent results were obtained from the observation of the DAPI-stained megagametophyte tissue sections.
Before seed germination, the megagametophyte cells are filled with nuclei, while in the post- germinative stages they undergo progressive reduction in the number of nuclei. Disappearance of nuclei takes place at the late post-germinative stage (35 DAG). The decrease of genomic DNA content and the disappearance of nuclei have also been reported during PCD in maize (Giuliani et al. , 2002), in the megagametophyte cells of white spruce ( Picea glauca ) seeds (He and Kermode,
2003a) and in petals of different flowers (Yamada et al. , 2006a).
To verify whether increased nuclease activities were responsible for DNA degradation, in vitro and in-gel activity assays for nucleolytic enzymes were carried out. Both the in vitro and the in- gel assays showed a strong induction of nuclease activities at the late post-germinative stages (28 and 35 DAG). The extensive DNA fragmentation observed at these stages temporally correlates with increased activities of nucleases, hence indicating that these enzymes presumably are responsible for the breakdown of nuclear DNA during the bunya pine megagametophyte PCD.
When the late stage megagametophyte nucleases were biochemically characterized, it was shown that these enzymes acted optimally at acidic pH and needed Zn 2+ for activity. Zn 2+ -dependent
DNases, which have been reported from various plant tissues (Grafi et al. , 1991), are considered a widespread class of enzymes in the plant kingdom and are thought to have a relevant role in
89 PCD, consisting in providing RNA and DNA degradation products to be used in other plant organs as part of the mechanism of nutrient salvage that occurs during or following cell death
(Bleecker, 1998). Interestingly, Zn 2+ -dependent nucleases have never been discovered from metazoans to our knowledge. The acidic pH requirement of the megagametophyte nucleases suggests that these enzymes may be activated after intracellular acidification or, alternatively, they may be localized in acidic compartments of the plant cell. For instance apoplastic nucleases have been found to be involved in DNA dismantling during HR-induced PCD in tobacco cells
(Mittler and Lam, 1997). The authors proposed that DNA fragmentation was catalyzed mainly by apoplastic nucleases. These enzymes were reported to reach the nucleus and degrade the DNA after the integrity of the plasma membrane was compromised, probably functioning in the recycling of DNA during the late stages of PCD. Further studies aimed at obtaining information about the subcellular localization of the megagametophyte nucleases probably would give some clues about their taking part in the bunya pine megagametophyte PCD.
Other evidence of PCD in the megagametophyte tissue comes from the observation of changes in the nuclear size and morphology at late post-germinative stages. All the megagametophyte nuclei at 0 DAG are big and round; nevertheless, at late post-germinative stages, the nuclei’s diameter shows a significant reduction, indicating that a process of chromatin condensation had to be occurred. Furthermore at these late post-germinative stages, several nuclei appear abnormally- shaped. These cytological alterations are considered typical features of PCD and are in accordance with what is known for animal apoptosis and for PCD of some plant systems, such as the petals of morning glory ( Ipomoea nil ) (Yamada et al. , 2006b), oat leaves (Kusaka et al. ,
2004) and the nucellus of Sechium edule seeds (Lombardi et al. , 2007a).
Finally, as a preliminary characterization, the megagametophyte indole-3-acetic acid (IAA) content was measured in order to investigate a possible involvement of this hormone in the
90 megagametophyte PCD. This analysis has shown a physiological increase of auxin content in the megagametophyte tissue following germination up to 21 DAG, followed by a remarkable decline at late post-germinative stages, when the megagametophyte PCD occurs. Such time-course lets suppose that auxin might function as a signal of PCD induction.
Unfortunately to date nothing is known about auxins and plant PCD and the study of the IAA content in the bunya pine megagametophyte only leads to a mere hypothesis on a possible involvement of this hormone in the megagametophyte PCD. Therefore many other studies have to be done in order to shed light on the relationship between hormones and PCD in the bunya pine megagametophyte. For instance, the study of the effect of IAA biosynthesis inhibitors on the megagametophyte PCD probably could give more detailed information about the role of this hormone in cell death of the bunya pine megagametophyte.
In conclusion, the data concerning the internucleosomal fragmentation of genomic DNA, the alterations in nuclear morphology together with chromatin condensation and the activation of
CLPs indicate that the degeneration of the bunya pine megagametophyte occurs by means of a developmental PCD. The other data show that the megagametophyte PCD is accompanied by evident morphological changes both at the macroscopic and at the cellular level, by considerable decrease of protein levels and marked increase in the activity of subtilisin-like serine proteases.
Furthermore a considerable decrease in DNA content and reduction in the number of nuclei together with a strong induction of Zn 2+ -dependent nuclease activities take place during the bunya pine megagametophyte PCD.
91 REFERENCES
Alnemri E.S., Litwack G. (1990) Activation of internucleosomal DNA cleavage in human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non Ca 2+ -requiring mechanism(s). The Journal of Biological Chemistry 265 : 17323-17333.
Amor Y., Babiychuk E., Inzè D., Levine A. (1998) The involvement of poly(ADP-ribose) polymerase in the oxidative stress responses in plants. FEBS Letters 440 : 1-7.
Antão C.M., Malcata F.X. (2005) Plant serine proteases: biochemical, physiological and molecular features. Plant Physiology and Biochemistry 43 : 637-650.
Aoyagi S., Sugiyama M., Fukuda H. (1998) BEN1 and ZEN1 cDNAs encoding S1-type DNases that are associated with programmed cell death in plants. FEBS Letters 429 : 134-138.
Aravind L., Dixit V.M., Koonin E.V. (1999) The domains of death: evolution of the apoptosis machinery. Trends in Biochemical Sciences 24 : 47-53.
Arnoult D., Akarid K., Grodet A., Petit P.X., Estaquier J., Ameisen J.C. (2002) On the evolution of programmed cell death: apoptosis of the unicellular eukaryote Leishmania major involves cysteine proteinase activation and mitochondrion permeabilization. Cell Death & Differentiation 9: 65-81.
Assunção Guimarães C., Linden R. (2004) Programmed cell deaths. Apoptosis and alternative deathstyles. European Journal of Biochemistry 271 : 1638-50.
Azeez A., Sane A.P., Bhatnagar D., Nath P. (2007) Enhanced expression of serine proteases during floral senescence in Gladiolus. Phytochemistry 68 : 1352-1357.
Balk J., Leaver C.J., McCabe P.F. (1999) Translocation of cytochrome c from the mitochondria to the cytosol occurs during heat-induced programmed cell death in cucumber plants. FEBS Letters 463 : 151-154.
Beers E.P., Freeman T.B. (1997) Proteinase activity during tracheary element differentiation in Zinnia mesophyll cultures. Plant Physiology 113 : 873-880.
92 Beers E.P., Woffenden B.J., Zhao C. (2000) Plant proteolytic enzymes: possible roles during programmed cell death. Plant Molecular Biology 44 : 399-415.
Belenghi B., Salomon M., Levine A. (2004) Caspase-like activity in the seedlings of Pisum sativum eliminates weaker shoots during early vegetative development by induction of cell death. Journal of Experimental Botany 55 : 889-897.
Blank A., McKeon T.A. (1989) Single-strand-preferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated. Proceedings of National Academy of Sciences, USA 86 : 3169-3173.
Blank A., Sugiyama R.H., Dekker C.A. (1982) Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: use of aqueous isopropanol to remove detergent from gels. Analytical Biochemistry 120 : 267-275.
Bleecker A.B. (1998) The evolutionary basis of leaf senescence: method to the madness? Current Opinion in Plant Biology 1: 73-78.
Boland D.J., Brooker M.I.H., Chippendale G.M., Hall N., Hyland B.P.M., Johnston R.D., Kleinig D.A., Turner D.J. (1984) Forest trees of Australia. Nelson-CSIRO, Melbourne.
Bortner C.D., Oldenburg N.B.E., Cidlowski J.A. (1995) The role of DNA fragmentation in apoptosis. Trends in Cell Biology 5: 21-26.
Bottari A., Capocchi A., Galleschi L., Jopova A., Saviozzi F. (1996) Asparaginyl endopeptidase during maturation and germination of durum wheat. Physiologia Plantarum 97 : 475-480.
Bozhkov P.V., Filonova L.H., Suarez M.F., Helmersson A., Smertenko A.P., Zhivotovsky B., von Arnold S. (2004) VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation. Cell Death & Differentiation 11 : 175- 182.
Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry 72 : 248-254.
93 Brand J.C., Cherikoff V., Truswell A.S. (1985) The nutritional composition of Australian Aboriginal bush-food. 3. Seeds and nuts. Food Technology in Australia 37 : 275-279.
Burrows G.E., Boag T.S., Stockey R.A. (1992) A morphological investigation of the unusual cryptogeal germination strategy of bunya pine ( Araucaria bidwillii )-an australian rain forest conifer. Internal Journal of Plant Sciences 153 : 503-512.
Burrows G.E., Stockey R.A. (1994) The developmental anatomy of cryptogeal germination in bunya pine ( Araucaria bidwillii ). Internal Journal of Plant Sciences 155 : 519-537.
Calderon-Urrea A., Dellaporta S.L. (1999) Cell death and cell protection genes determine the fate of pistils in maize. Development 126 : 435-441.
Callard D., Axelos M., Mazzolini L. (1996) Novel molecular markers for late phases of the growth cycle of Arabidopsis thaliana cell-suspension cultures are expressed during organ senescence. Plant Physiology 112 : 705-715.
Carimi F., Zottini M., Formentin E., Terzi M., Lo Schiavo F. (2003) Cytokinins: new apoptotic inducers in plants. Planta 216 : 413-421.
Chang D.W., Xing Z., Capacio V.L., Peter M.E., Yang X. (2003) Interdimer processing mechanism of procaspase-8 activation. The EMBO Journal 22 : 4132-4142.
Chen H.M., Zhou J., Dai Y.R. (2000) Cleavage of lamin-like proteins in in vivo and in vitro apoptosis of tobacco protoplasts induced by heat shock. FEBS Letters 480 : 165-168.
Chichkova N.V., Kim S.H., Titova E.S., Kalkum M., Morozov V.S., Rubtsov Y.P., Kalinina N.O., Taliansky M.E., Vartapetian A.B. (2004) A plant caspase-like protease activated during the hypersensitive response. The Plant Cell 16 : 157-171.
Clarke A., Desikan R., Hurst R.D., Hancock J.T., Neill S.J. (2000) NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures. The Plant Journal 24 : 667-677.
94 Coffeen W.C., Wolpert T.J. (2004) Purification and characterization of serine proteases that exhibit caspase-like activity and are associated with programmed cell death in Avena sativa . The Plant Cell 16 : 857-873.
Cohen G.M. (1997) Caspases: the executioner of apoptosis. Biochemical Journal 326 : 1-16.
Counis M.F., Torriglia A. (2000) DNases and apoptosis. Biochemistry and Cell Biology 78 : 405-414.
Cryns V., Yuan J. (1998) Proteases to die for. Genes & Development 12 : 1551-1570.
Danon A., Delorme V., Mailhac N., Gallois P. (2000). Plant programmed cell death: a common way to die. Plant Physiology and Biochemistry 38 : 647-655.
Danon A., Gallois P. (1998) UV-C radiation induces apoptotic-like changes in Arabidopsis thaliana . FEBS Letters 437 : 131-136.
Danon A., Rotari V.I., Gordon A., Mailhac M., Gallois P. (2004) Ultraviolet-C overexposure induces programmed cell death in Arabidopsis , which is mediated by caspase-like activities and which can be suppressed by caspase inhibitors, p35 and Defender against Apoptotic Death . The Journal of Biological Chemistry 279 : 779-787.
De Jong A.J., Hoeberichts F.A., Yakimova E.T., Maximova E., Woltering E.J. (2000) Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases. Planta 211 : 656-662. del Pozo O., Lam E. (1998) Caspases and programmed cell death in the hypersensitive response of plants to pathogens. Current Biology 8: 1129-1132.
Del Zoppo M., Galleschi L., Onnis A., Pardossi A., Saviozzi F. (1999) Effect of salinity on water relations, sodium accumulation, chlorophyll content and proteolytic enzymes in a wild wheat. Biologia Plantarum 42 : 97-104.
Del Zoppo M., Galleschi L., Saviozzi F. (1998) Long-term storage of Araucaria bidwillii Hook. seeds. Seed Science & Technology 26 : 267-270.
95 Della Mea M., Serafini-Fracassini D., Del Duca S. (2007) Programmed cell death: similarities and differences in animals and plants. A flower paradigm. Amino Acids 33 : 395-404.
Dellaporta S.L., Wood J., Hicks J.B. (1983) The plant DNA minipreparation: version II. Plant Molecular Biology 4: 19-21.
DeLong A., Calderon-Urrea A., Dellaporta S.L. (1993) Sex determination gene TASSELSEED2 of maize encodes a short-chain alcohol dehydrogenase required for stage- specific floral organ abortion. Cell 74 : 757-768.
Delorme V.G., McCabe P.F., Kim D.-J., Leaver C.J. (2000) A matrix metalloproteinase gene is expressed at the boundary of senescence and programmed cell death in cucumber. Plant Physiology 123 : 917-927.
Didenko V.V., Hornsby P.J. (1996) Presence of double-strand breaks with single-base 3 ′ overhangs in cells undergoing apoptosis but not necrosis. The Journal of Cell Biology 135 : 1369- 1376.
Doley D. (1990) Utilization of intrinsic water in the germination of Araucaria bidwillii seeds. Seed Science & Technology 18 : 33-42.
Drew M.C., He C.J., Morgan P.W. (2000) Programmed cell death and aerenchyma formation in roots. Trends in Plant Science 5: 123-127.
Duriez P.J., Shah G.M. (1997) Cleavage of poly(ADP-ribose) polymerase: a sensitive parameter to study cell death. Biochemistry and Cell Biology 75 : 337-349.
Earnshaw W.C., Martins L.M., Kaufmann S.H. (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annual Review of Biochemistry 68 : 383- 424.
Elbaz M., Avni A., Weil M. (2002) Constitutive caspase-like machinery executes programmed cell death in plant cells. Cell Death & Differentiation 9: 726-733.
96 Fadeel B., Orrenius S., Zhivotovsky B. (1999) Apoptosis in human disease: a new skin for the old ceremony? Biochemical and Biophysical Research Communications 266 : 699-717.
Fath A., Bethke P.C., Jones R.L. (1999) Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation. The Plant Journal 20 : 305-315.
Filonova L.H., von Arnold S., Daniel G., Bozhkov P.V. (2002) Programmed cell death eliminates all but one embryo in a polyembryonic plant seed. Cell Death & Differentiation 9: 1057-1062.
Fontanini D., Capocchi A., Saviozzi F., Galleschi L. (2007) Simplified electrophoretic assay for human salivary α-amylase inhibitor detection in cereal seed flours. Journal of Agricultural and Food Chemistry 55 : 4334-4339.
Francini A., Galleschi L., Saviozzi F., Pinzino C., Izzo R., Sgherri C., Navari-Izzo F. (2006) Enzymatic and non-enzymatic protective mechanisms in recalcitrant seeds of Araucaria bidwillii subjected to desiccation. Plant Physiology and Biochemistry 44 : 556-563.
Fukuda H. (2000) Programmed cell death of tracheary elements as a paradigm in plants. Plant Molecular Biology 44 : 245-253.
Galleschi L., Capocchi A., Giannoni P., Floris C. (1989) Proteinase activities in quiescent and germinating seeds of xHaynaldoticum sardoum . Physiologia Plantarum 75 : 1-6.
Galleschi L., Capocchi A., Ghiringhelli S., Saviozzi F. (2002) Some properties of proteolytic enzymes and storage proteins in recalcitrant and orthodox seeds of Araucaria . Biologia Plantarum 45 : 539-544.
Galleschi L., Spanò C., Bedini G., Floris C., Saviozzi F., Capocchi A. (1988) Proteolytic activities in xHaynaldoticum sardoum seeds of different ages. Annals of Botany 61 : 93-97.
Gan S.S., Amasino R.M. (1997) Making sense of senescence: molecular genetic regulation and manipulation of leaf senescence. Plant Physiology 113 : 313-319.
97 Gavrieli Y., Sherman Y., Ben-Sasson S.A. (1992) Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. The Journal of Cell Biology 119 : 493- 501.
Genschik P., Durr A., Fleck J. (1994) Differential expression of several E2-type ubiquitin carrier protein genes at different developmental stages in Arabidopsis thaliana and Nicotiana sylvestris . Molecular and General Genetics 244 : 548-556.
Gewies A. (2003) Caspases are central initiators and executioners of apoptosis. ApoReview - Introduction to Apoptosis , pagg. 1-26.
Gifford D.J., Greenwood J.S., Bewley J.D. (1982) Deposition of matrix and crystalloid storage proteins during protein body development in the endosperm of Ricinus communis L.cv. Hale seeds. Plant Physiology 69 : 1471-1478.
Giuliani C., Consonni G., Gavazzi G., Colombo M., Dolfini S. (2002) Programmed cell death during embryogenesis in maize. Annals of Botany 90 : 287-292.
Goodlett C.R., Horn K.H. (2001) Mechanisms of alcohol-induced damage to the developing nervous system. Alcohol Research & Health 25 : 175-184.
Grafi G., Meller E., Sher N., Sela I. (1991) Characterization of S1/mung-bean-type nuclease activity in plant cell suspensions. Plant Science 74 : 107-114.
Green D.R., Reed J.C. (1998) Mitochondria and apoptosis. Science 281 : 1309-1312.
Greenberg J.T. (1996) Programmed cell death: a way of life for plants. Proceedings of National Academy of Sciences, USA 93 : 12094-12097.
Greenberg J.T., Guo A.L., Klessig D.F., Ausubel F.M. (1994) Programmed cell death in plants: a pathogen-triggered response activated coordinately with multiple defence functions. Cell 77 : 551-563.
98 Greenwood J.S., Helm M., Gietl C. (2005) Ricinosomes and endosperm transfer cell structure in programmed cell death of the nucellus during Ricinus seed development. Proceedings of National Academy of Sciences, USA 102 : 2238-2243.
Guiot A.L., Rigal D., Chappuis G. (1997) Spontaneous programmed cell death (PCD) process of lymphocytes of FIV-infected cats: cellular targets and modulation. Veterinary Immunology and Immunopathology 58 : 93-106.
Gunawardena A.H.L.A.N. (2007) Programmed cell death and tissue remodeling in plants. Journal of Experimental Botany pagg. 1-7.
Gunawardena A.H.L.A.N., Greenwood J.S., Dengler N.G. (2004) Programmed cell death remodels lace plant leaf shape during development. The Plant Cell 16 : 60-73.
Haines R.J. (1983) Embryo development and anatomy in Araucaria Juss. Australian Journal of Botany 31 : 125-140.
Hara-Nishimura I., Hatsugai N., Nakaune S., Kuroyanagi M., Nishimura M. (2005) Vacuolar processing enzyme: an executor of plant cell death. Current Opinion in Plant Biology 8: 404-408.
Hara-Nishimura I., Inoue K., Nishimura M. (1991) A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms. FEBS Letters 294 : 89-93.
Hara-Nishimura I., Kinoshita T., Hiraiwa N., Nishimura M. (1998) Vacuolar processing enzymes in protein-storage vacuoles and lytic vacuoles. Journal of Plant Physiology 152 : 668- 674.
Hatsugai N., Kuroyanagi M., Nishimura M., Hara-Nishimura I. (2006) A cellular suicide strategy of plants: vacuole-mediated cell death. Apoptosis 11 : 905-911.
Hatsugai N., Kuroyanagi M., Yamada K., Meshi T., Tsuda S., Kondo M., Nishimura M., Hara-Nishimura I. (2004) A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death. Science 305 : 855-858.
99 He C.J., Morgan P.W., Drew M.C. (1996) Transduction of an ethylene signal is required for cell death and lysis in the root cortex of maize during aerenchyma formation induced by hypoxia. Plant Physiology 112 : 463-472.
He X., Kermode A.R. (2003a) Nuclease activities and DNA fragmentation during programmed cell death of megagametophyte cells of white spruce ( Picea glauca ) seeds. Plant Molecular Biology 51 : 509-521.
He X., Kermode A.R. (2003b) Proteases associated with programmed cell death of megagametophyte cells after germination of white spruce ( Picea glauca ) seeds. Plant Molecular Biology 52 : 729-744.
Hengartner M.O. (2000) The biochemistry of apoptosis. Nature 407 : 770-776.
Hirata H., Takahashi A., Kobayashi S., Yonehara S., Sawai H., Okazaki T., Yamamoto K., Sasada M. (1998) Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. Journal of Experimental Medicine 187 : 587- 600.
Hiraiwa N., Nishimura M., Hara-Nishimura I. (1999) Vacuolar processing enzyme is self- catalytically activated by sequential removal of the C-terminal and N-terminal propeptides. FEBS Letters 447 :213-216.
Hirsch T., Marchetti P., Susin S.A., Dallaporta B., Zamzami N., Marzo I., Geuskens M., Kroemer G. (1997) The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15 : 1573- 1582.
Hutchins J.B., Barger S.W. (1998) Why neurons die: cell death in the nervous system. The Anatomical Record 253 : 79-90.
Jänicke R.U., Sprengart M.L., Wati M.R., Porter A.G. (1998) Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. The Journal of Biological Chemistry 273 : 9357-9360.
100 Jones A.M. (2001) Programmed cell death in development and defense. Plant Physiology 125 : 94-97.
Katsuhara M. (1997) Apoptotis-like cell death in barley roots under salt stress. Plant & Cell Physiology 38 : 1091-1093.
Kerr J.F.R., Harmon B.V. (1991) Definition and incidence of apoptosis: an historical perspective, in Tomei L.D., Cope F.O. (Eds.) Apoptosis: the molecular basis of cell death, Cold Spring Harbor Laboratory Press, New York, 1991, pp.5-29.
Kerr J.F.R., Wyllie A.H., Currie A.R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26 : 239-257.
Kinoshita T., Yamada K., Hiraiwa N., Kondo M., Nishimura M., Hara-Nishimura I. (1999) Vacuolar processing enzyme is up-regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions. The Plant Journal 19 : 43-53.
Klionsky D.J., Emr S.D. (2000) Autophagy as a regulated pathway of cellular degradation. Science 290 : 1717-1721.
Koenig R., Stegemann H., Franksen H., Paul H.L. (1970) Protein subunits in the potato virus X group. Determination of the molecular weights by polyacrylamide electrophoresis. Biochimica et Biophisica Acta 207 : 184-189.
Koonin E.V., Aravind L. (2002) Origin and evolution of eukaryotic apoptosis: the bacterial connection. Cell Death & Differentiation 9: 394-404.
Korthout H.A., Berecki G., Bruin W., Van Duijn B., Wang M. (2000) The presence and subcellular localization of caspase 3-like proteinases in plant cells. FEBS Letters 475 : 139-144.
Kothakota S., Azuma T., Reinhard C., Klippel A., Tang J., Chu K., McGarry T.J., Kirschner M.W., Koths K., Kwiatkowski D.J., Williams L.T. (1997) Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 278 : 294-298.
101 Koukalova B., Kovarik A., Fajkus J., Siroky J. (1997) Chromatin fragmentation associated with apoptotic changes in tobacco cells exposed to cold stress. FEBS Letters 414 : 289-292.
Kowalski D., Kroeker W.D., Laskowski M. (1976) Mung bean nuclease I. Physical, chemical, and catalytic properties. Biochemistry 15 : 4457-4463.
Krishnamurthy K.V., Krishnaraj R., Chozhavendan R., Christopher F.S. (2000) The programme of cell death in plants and animals - A comparison. Current Science 79 : 1169-1181.
Kroemer G., Zamzani N., Susin S.A. (1997) Mitochondrial control of apoptosis. Immunology Today 18 : 44-51.
Kuo A., Cappelluti S., Cervantes-Cervantes M., Rodriguez M., Bush D.S. (1996) Okadaic acid, a protein phosphatase inhibitor, blocks calcium changes, gene expression, and cell death induced by gibberellin in wheat aleurone cells. The Plant Cell 8: 259-269.
Kuriyama H., Fukuda H. (2001) Regulation of tracheary element differentiation. Journal of Plant Growth Regulation 20 : 35-51.
Kuriyama H., Fukuda H. (2002) Developmental programmed cell death in plants. Current Opinion in Plant Biology 5: 568-573.
Kusaka K., Tada Y., Shigemi T., Sakamoto M., Nakayashiki H., Tosa Y., Mayama S. (2004) Coordinate involvement of cysteine protease and nuclease in the executive phase of plant apoptosis. FEBS Letters 578 : 363-367.
Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 : 680-685.
Lam E. (2004) Controlled cell death, plant survival and development. Nature Reviews Molecular Cell Biology 5: 305-315.
Lam E. (2005) Vacuolar proteases livening up programmed cell death. Trends in Cell Biology 15 : 124-127.
102 Lam E., del Pozo O. (2000) Caspase-like protease involvement in the control of plant cell death. Plant Molecular Biology 44 : 417-428.
Laux T., Jürgens G. (1997) Embryogenesis: a new start in life. The Plant Cell 9: 989-1000.
Li P., Nijhawan D., Budihardjo I., Srinivasula S.M., Ahmad M., Alnemri E.S., Wang X. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91 : 479-489.
Li Y.Q., Southworth D., Linskens H.F., Mulcahy D.L., Cresti M. (1995) Localization of ubiquitin in anthers and pistils of Nicotiana . Sexual Plant Reproduction 8: 123-128.
Liao T.H. (1977) Isolation and characterization of multiple forms of malt deoxyribonuclease. Biochemistry 16 : 1469-1474.
Liu X., Zou H., Slaughter C., Wang X. (1997) DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89 : 175-184.
Lockshin R.A., Williams C.M. (1964) Programmed cell death-II. Endocrine potentiation of the breakdown of the intersegmental muscles of silkmoths. Journal of Insect Physiology 10 : 643-649.
Lombardi L., Casani S., Ceccarelli N., Galleschi L., Picciarelli P., Lorenzi R. (2007a) Programmed cell death of the nucellus during Sechium edule Sw. seed development is associated with activation of caspase-like proteases. Journal of Experimental Botany 58 : 2949-2958.
Lombardi L., Ceccarelli N., Picciarelli P., Lorenzi R. (2007b) DNA degradation during programmed cell death in Phaseolus coccineus suspensor. Plant Physiology and Biochemistry 45 : 221-227.
Lowry O.H., Rosenbrough N.J., Farr A.L., Randall L.J. (1951) Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry 193 : 265-275.
Madeo F., Herker E., Maldener C., Wissing S., Lachelt S., Herlan M., Fehr M., Lauber K., Sigrist S.J., Wesselborg S., Frohlich K.U. (2002) A caspase-related protease regulates apoptosis in yeast. Molecular Cell 9: 911-917.
103 Martelli A.M., Zweyer M., Ochs R.L., Tazzari P.L., Tabellini G., Narducci P., Bortul R. (2001) Nuclear apoptotic changes: an overview. Journal of Cellular Biochemistry 82 : 634-46.
Martin S.J., Green D.R. (1995) Protease activation during apoptosis: death by a thousand cuts? Cell 82 : 349-352.
McCabe P.F., Levine A., Meijer P.J., Tapon N.A., Pennell R.I. (1997) A programmed cell death pathway activated in carrot cells cultured at low cell density. The Plant Journal 12 : 267- 280.
Meier P., Finch A., Evan G. (2000) Apoptosis in development. Nature 407 : 796-801.
Minami A., Fukuda H. (1995) Transient and specific expression of a cysteine endopeptidase associated with autolysis during differentiation of Zinnia mesophyll cells into tracheary elements. Plant and Cell Physiology 36 : 1599-1606.
Mittler R., Lam E. (1995) Identification, characterization, and purification of a tobacco endonuclease activity induced upon hypersensitive response cell death. The Plant Cell 7: 1951- 1962.
Mittler R., Lam E. (1997) Characterization of nuclease activities and DNA fragmentation induced upon hypersensitive response cell death and mechanical stress. Plant Molecular Biology 34 : 209-221.
Mittler R., Simon L., Lam E. (1997) Pathogen-induced programmed cell death in tobacco. Journal of Cell Science 110 : 1333-1344.
Mlejnek P., Prochazka S. (2002) Activation of caspase-like proteases and induction of apoptosis by isopentenyladenosine in tobacco BY-2 cells. Planta 215 : 158-166.
Müntz K., Shutov A.D. (2002) Legumains and their functions in plants. Trends in Plant Science 7: 340-344.
104 Nakagawa T., Zhu H., Morishima N., Li E., Xu J., Yankner B.A., Yuan J. (2000) Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403 : 98-103.
Nakaune S., Yamada K., Kondo M., Tabata S., Nishimura M., Hara-Nishimura I. (2005) A vacuolar processing enzyme, δVPE, is involved in seed coat formation at the early stage of seed development. The Plant Cell 17 : 876-887.
Navarre D.A., Wolpert T.J. (1999) Victorin induction of an apoptotic/senescence-like response in oats. The Plant Cell 11 : 237-249.
Newmeyer D.D., Ferguson-Miller S. (2003) Mitochondria: releasing power for life and unleashing the machineries of death. Cell 112 : 481-490.
Nibert M.L., Wilson B.L. (1996) Biochemical methods, Laboratory Manual (Eds.) Spring Biochemistry 651 , pagg. 47-50.
O’Neil N., Rose A. (2006) DNA repair. WormBook, (Eds) The C. elegans Research Community, WormBook 13 : 1-12.
Obara K., Kuriyama H., Fukuda H. (2001) Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia. Plant Physiology 125 : 615-626.
Oliver F.J., Menissier-de Murcia J., de Murcia G. (1999) Poly(ADP-ribose) polymerase in the cellular response to DNA damage, apoptosis, and disease. American Journal of Human Genetics 64 : 1282-1288.
Orzaez D., Granell A. (1997a) DNA fragmentation is regulated by ethylene during carpel senescence in Pisum sativum . The Plant Journal 11 : 137-144.
Orzaez D., Granell A. (1997b) The plant homologue of the defender against apoptotic death gene is down-regulated during senescence of flower petals. FEBS Letters 404 : 275-278.
105 Owens J.N., Morris S.J., Misra S. (1993) The ultrastructural, histochemical, and biochemical development of the post-fertilization megagametophyte and zygotic embryo of Pseudotsuga menziesii . Canadian Journal of Forest Research 23 : 816-827.
Pak C., van Doorn W.G. (2005) Delay of Iris flower senescence by protease inhibitors. New Phytologist 165 : 473-480.
Palma J.M., Sandalio L.M., Corpas F.J., Romero-Puertas M.C., McCarthy I., del Rio L.A. (2002) Plant proteases, protein degradation, and oxidative stress: role of peroxisomes. Plant Physiology and Biochemistry 40 : 521-530.
Pasqualini S., Piccioni C., Reale L., Ederli L., Della Torre G., Ferranti F. (2003) Ozone- induced cell death in tobacco cultivar Bel W3 plants. The role of programmed cell death in lesion formation. Plant Physiology 133 : 1122-1134.
Piszczek E., Gutman W. (2007) Caspase-like proteases and their role in programmed cell death in plants. Acta Physiologiae Plantarum 29 : 391-398.
Pontier D., Gan S., Amasino R.M., Roby D., Lam E. (1999) Markers for hypersensitive response and senescence show distinct patterns of expression. Plant Molecular Biology 39 : 1243- 1255.
Porter A.G., Janicke R.U. (1999) Emerging roles of caspase-3 in apoptosis. Cell Death & Differentiation 6: 99-104.
Reape T.J., McCabe P.F. (2008) Apoptotic-like programmed cell death in plants. New Phytologist 180 : 13-26.
Roberts E.H. (1973) Predicting the storage life of seeds. Seed Science & Technology 1: 499-514.
Rojo E., Martin R., Carter C., Zouhar J., Pan S.Q., Plotnikova J., Jin H.L., Paneque M., Sanchez-Serrano J.J., Baker B., Ausubel F.M., Raikhel N.V. (2004) VPE gamma exhibits a caspase-like activity that contributes to defense against pathogens. Current Biology 14 : 1897- 1906.
106 Rojo E., Zouhar J., Carter C., Kovaleva V., Raikhel N.V. (2003) A unique mechanism for protein processing and degradation in Arabidopsis thaliana . Proceedings of National Academy of Sciences, USA 100 : 7389-7394.
Rotari V.I., He R., Gallois P. (2005) Death by proteases in plants: whodunit. Physiologia Plantarum 123 : 376-385.
Rudel T., Bokoch G.M. (1997) Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276 : 1571-1574.
Runeberg-Roos P., Saarma M. (1998) Phytepsin, a barley vacuolar aspartic proteinase, is highly expressed during autolysis of developing tracheary elements and sieve cells. The Plant Journal 15 : 139-145.
Ryerson D.E., Heath M.C. (1996) Cleavage of nuclear DNA into oligonucleosomal fragments during cell death induced by fungal infection or by abiotic treatments. The Plant Cell 8: 393-402.
Samadi L., Shahsavan Behboodi B. (2006) Fusaric acid induces apoptosis in saffron root-tip cells: roles of caspase-like activity, cytochrome c, and H 2O2. Planta 225 : 223-234.
Samuilov V.D., Oleskin A.V., Lagunova E.M. (2000) Programmed cell death. Biochemistry 65 : 873-887.
Saraste A., Pulkki K. (2000) Morphologic and biochemical hallmarks of apoptosis. Cardiovascular Research 45 : 528-537.
Sarath G., De La Motte R.S., Wagner F.W. (1989) Protease assay methods, (Eds) Proteolytic enzymes: a practical approach, by R.J. Beynon, J.S. Bond. IRL Press, Oxford UK, pp 25-55.
Schaller A. (2004) A cut above the rest: the regulatory function of plant proteases. Planta 220 : 183-197.
Schmid M., Simpson D., Gietl C. (1999) Programmed cell death in castor bean endosperm is associated with the accumulation and release of a cysteine endopeptidase from ricinosomes. Proceedings of National Academy of Sciences, USA 96 : 14159-14164.
107 Shirahama-Noda K., Yamamoto A., Sugihara K., Hashimoto N., Asano M., Nishimura M., Hara-Nishimura I. (2003) Biosynthetic processing of cathepsins and lysosomal degradation are abolished in asparaginyl endopeptidase-deficient mice. The Journal of Biological Chemistry 278 : 33194-33199.
Shutov A.D., Vaintraub I.A. (1987) Degradation of storage proteins in germinating seeds. Phytochemistry 26 : 1557-1566.
Skulachev V.P. (1998) Cytochrome c in the apoptotic and antioxidant cascades. FEBS Letters 423 : 275-280.
Skulachev V.P. (2002) Programmed death in yeast as adaptation? FEBS Letters 528 : 23-26.
Solomon M., Belenghi B., Delledonne M., Menachem E., Levine A. (1999) The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. The Plant Cell 11 : 431-444.
Sorce C., Lorenzi R., Ceccarelli N., Ranalli P. (2000) Changes in free and conjugated IAA during dormancy and sprouting of potato tubers. Australian Journal of Plant Physiology 27 : 371- 377.
Srinivasula S.M., Ahmad M., Fernandes-Alnemri T., Alnemri E.S. (1998) Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Molecular Cell 1: 949-957.
Steffens B., Sauter M. (2005) Epidermal cell death in rice is regulated by ethylene, gibberellin, and abscisic acid. Plant Physiology 139 : 713-721.
Stein J.C., Hansen G. (1999) Mannose induces an endonuclease responsible for DNA laddering in plant cells. Plant Physiology 121 : 71-80.
Stennicke H.R., Salvesen G.S. (1997) Biochemical characteristics of caspases-3, -6, -7, and -8. The Journal of Biological Chemistry 272 : 25719-25723.
Stephenson P., Collins B.A., Reid P.D., Rubinstein B. (1996) Localization of ubiquitin to differentiating vascular tissues. American Journal of Botany 83 : 140-147.
108 Stephenson P., Rubinstein B. (1998) Characterization of proteolytic activity during senescence in daylilies. Physiologia Plantarum 104 : 463-473.
Suarez M.F., Filonova L.H., Smertenko A., Savenkov E.I., Clapham D.H., von Arnold S., Zhivotovsky B., Bozhkov P.V. (2004) Metacaspase-dependent programmed cell death is essential for plant embryogenesis. Current Biology 14 : R339-R340.
Sugiyama M., Ito J., Aoyagi S., Fukuda H. (2000) Endonucleases. Plant Molecular Biology 44 : 387-397.
Sun Y.L., Zhao Y., Hong X., Zhai Z.H. (1999) Cytochrome c release and caspase activation during menadione-induced apoptosis in plants. FEBS Letters 462 : 317-321.
Takahashi A., Alnemri E.S., Lazebnik Y.A., Fernandes-Alnemri T., Litwack G., Moir R.D., Goldman R.D., Poirier G.G., Kaufmann S.H., Earnshaw W.C. (1996) Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proceedings of National Academy of Sciences, USA 93 : 8395-8400.
Thelen M.P., Northcote D.H. (1989) Identification and purification of a nuclease from Zinnia elegans L.: a potential molecular marker for xylogenesis. Planta 179 : 181-195.
Theologis A. (1992) One rotten apple spoils the whole bushel: the role of ethylene in fruit ripening. Cell 70 : 181-184.
Thomas H., Ougham H.J., Wagstaff C., Stead A.D. (2003) Defining senescence and death. Journal of Experimental Botany 54 : 1127-1132.
Thornberry N.A., Lazebnik Y. (1998) Caspases: enemies within. Science 281 : 1312-1316.
Tittel J.N., Steller H. (2000) A comparison of programmed cell death between species. Genome
Biology 1: reviews 3.1-reviews 3.6.
Tsujimoto Y. (1998) Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria? Genes to Cells 3: 697-707.
109 Uren A.G., O'Rourke K., Aravind L.A., Pisabarro M.T., Seshagiri S., Koonin E.V., Dixit V.M. (2000) Identification of paracaspases and metacaspases: two ancient families of caspase- like proteins, one of which plays a key role in MALT lymphoma. Molecular Cell 6: 961-967.
Vacca R.A., Valenti D., Bobba A., de Pinto M.C., Merafina R.S., De Gara L., Passarella S., Marra E. (2007) Proteasome function is required for activation of programmed cell death in heat shocked tobacco Bright-Yellow 2 cells. FEBS Letters 581 : 917-922.
Vaux D.L., Korsmeyer S.J. (1999) Cell death in development. Cell 96 : 245-254.
Vercammen D., van de Cotte B., De Jaeger G., Eeckhout D., Casteels P., Vanepoele K., Vandenberghe I., Van Beeumen J., Inze D., Van Breusegem F. (2004) Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine. The Journal of Biological Chemistry 279 : 45329-45336.
Vercher Y., Molowny A., Carbonell J. (1987) Gibberellic acid effects on the ultrastructure of endocarp cells of unpollinated ovaries of Pisum sativum . Physiologia Plantarum 71 : 302-308.
Volbeda A., Lahm A., Sakiyama F., Suck D. (1991) Crystal structure of Penicillium citrinum P1 nuclease at 2.8 Å resolution. The EMBO Journal 10 : 1607-1618.
Walker P.R., Sikorska M. (1994) Endonuclease activities, chromatin structure, and DNA degradation in apoptosis. Biochemistry and Cell Biology 72 : 615-623.
Wan L., Xia Q., Qiu X., Selvaraj G. (2002) Early stages of seed development in Brassica napus : a seed coat-specific cysteine proteinase associated with programmed cell death of the inner integument. The Plant Journal 30 : 1-10.
Wang H., Li J., Bostock R.M., Gilchrist D.G. (1996a) Apoptosis: a functional paradigm for programmed plant cell death induced by a host-selective phytotoxin and invoked during development. The Plant Cell 8: 375-391.
Wang H., Wu H.M., Cheung A.Y. (1996b) Pollination induces mRNA poly(A) tail-shortening and cell deterioration in flower transmitting tissue. The Plant Journal 9: 715-727.
110 Wang M., Hoekstra S., van Bergen S., Lamers G.E.M., Oppedijk B.J., van der Heijden M.W., de Priester W., Schilperoort R.A. (1999) Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L. Plant Molecular Biology 39 : 489-501.
Wang M., Oppedijk B.J., Lu X., Van Duijn B., Schilperoort R.A. (1996c) Apoptosis in barley aleurone during germination and its inhibition by abscisic acid. Plant Molecular Biology 32 : 1125-1134.
Watanabe N., Lam E. (2005) Two Arabidopsis metacaspases AtMCP1b and AtMCP2b are arginine/lysine-specific cysteine proteases and activate apoptosis-like cell death in yeast. The Journal of Biological Chemistry 280 : 14691-14699.
Wen L.P., Fahrni J.A., Troie S., Guan J.L., Orth K., Rosen G.D. (1997) Cleavage of focal adhesion kinase by caspases during apoptosis. The Journal of Biological Chemistry 272 : 26056- 26061.
Williams B., Dickman M. (2008) Plant programmed cell death: can’t live with it; can’t live without it. Molecular Plant Pathology 9: 531-544.
Woltering E.J. (2004) Death proteases come alive. Trends in Plant Science 9: 469-472.
Woltering E.J., van der Bent A., Hoeberichts F.A. (2002) Do plant caspases exist? Plant Physiology 130 : 1764-1769.
Wood M., Power J.B., Davey M.R., Lowe K.C., Mulligan B.J. (1998) Factors affecting single strand-preferring nuclease activity during leaf aging and dark-induced senescence in barley (Hordeum vulgare L.). Plant Science 131 : 149-159.
Wu J.C., Fritz L.C. (1999) Irreversible caspase inhibitors: tools for studying apoptosis. METHODS: A Companion to Methods in Enzymology 17 : 320-328.
Wyllie A.H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284 : 555-556.
111 Xue D., Horvitz H.R. (1997) Caenorhabditis elegans CED-9 protein is a bifunctional cell-death inhibitor. Nature 390 : 305-308.
Yamada K., Shimada T., Kondo M., Nishimura M., Hara-Nishimura I. (1999) Multiple functional proteins are produced by cleaving Asn-Gln bonds of a single precursor by vacuolar processing enzyme. The Journal of Biological Chemistry 274 : 2563-2570.
Yamada K., Shimada T., Kondo M., Nishimura M., Hara-Nishimura I. (2005) A VPE family supporting various vacuolar functions in plants. Physiologia Plantarum 123 : 369-375.
Yamada T., Ichimura K., van Doorn W.G. (2006a) DNA degradation and nuclear degeneration during programmed cell death in petals of Antirrhinum , Argyranthemum , and Petunia . Journal of Experimental Botany 57 : 3543-3552.
Yamada T., Takatsu Y., Kasumi M., Ichimura K., van Doorn W.G. (2006b) Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil ). Planta 224 : 1279-1290.
Ye Z.H., Varner J.E. (1996) Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans . Plant Molecular Biology 30 : 1233-1246.
Yen C.H., Yang C.H. (1998) Evidence for programmed cell death during leaf senescence in plants. Plant & Cell Physiology 39 : 922-927.
Yen Y., Green P.J. (1991) Identification and properties of the major ribonucleases of Arabidopsis thaliana . Plant Physiology 97 : 1487-1493.
Young T.E., Gallie D.R. (1999) Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals. Plant Molecular Biology 39 : 915-926.
Young T.E., Gallie D.R. (2000) Regulation of programmed cell death in maize endosperm by abscisic acid. Plant Molecular Biology 42 : 397-414.
112 Young T.E., Gallie D.R., DeMason D.A. (1997) Ethylene-mediated programmed cell death during maize endosperm development of wild-type and shrunken2 genotypes. Plant Physiology 115 : 737-751.
Zamzami N., Kroemer G. (1999) Apoptosis: condensed matter in cell death. Nature 401 : 127- 128.
Zhang X., Shan P., Sasidhar M., Chupp G.L., Flavell R.A., Choi A.M., Lee P.J. (2003) Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediate hyperoxia-induced cell death in lung epithelium. American Journal of Respiratory Cell and Molecular Biology 28 : 305-315.
Zhao Y., Jiang Z.F., Sun Y.L., Zhai Z.H. (1999) Apoptosis of mouse liver nuclei induced in the cytosol of carrot cells. FEBS Letters 448 : 197-200.
Zuzarte-Luis V., Hurlè J.M. (2002) Programmed cell death in the developing limb. The International Journal of Developmental Biology 46 : 871-876.
113 ACKNOWLEDGMENTS
I want to give my deepest thanks to several people who helped me to complete my doctorate course.
Firstly to my Family, for the continuous and affectionate support.
To my Supervisor Prof. Luciano Galleschi, for offering me the opportunity to work in his laboratory and learn a lot.
To the staff of the Plant Physiology Laboratory at University of Pisa where I performed my experiments: many thanks to Dr. Debora Fontanini for teaching me a lot about enzyme activities and Proteomics and for improving my English. I am also very grateful to Dr. Antonella Capocchi and Mr. Franco Saviozzi for their scientific and technical advice, to Dr. Lara Lombardi, Dr. Carlo
Sorce and Dr. Andrea Andreucci (Botany Laboratory) for their help in performing some experiments.
To Dr. Vera Muccilli and Prof. Salvatore Foti (Dept. of Chemical Sciences, University of
Catania) for the sequencing of my proteins with mass spectrometry.
To the members of my Committee for their valuable suggestions.
114