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Morphological Survey and Characterization of Programmed Cell Death in the Placenta-Chalaza and Endosperm in the Developi...


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MORPHOLOGICAL SURVEY AND CHARA CTERIZATION OF PROGRAMMED CELL DEATH IN THE PLACENTA-CHALAZA AND ENDOSPERM IN THE DEVELOPING CARYOPSIS OF MAIZE By KAREN CHAMUSCO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Karen Chamusco

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This work is dedicated to my family and friends who have been tremendous pillars of aid and support

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ACKNOWLEDGMENTS I would like to acknowledge and thank my chairman, Dr. Prem S. Chourey, for his support and for giving me a great opportunity to explore research and learn invaluable research techniques. I also would like to thank my committee, Dr. Daryl Pring, Dr. Christine Chase, and Dr. Henry Aldrich, for their support and valuable advice. Special thanks go to Ale Kladnik whose research and efforts were indispensable in pointing me in the direction that allowed me to pursue my masters degree. I also thank Maureen Petersen, Donna Williams, and Lorraine McDowell for their complete technical support in electron microscopy and Tim Vaught for his aid in helping me with confocal microscopy. Special support and aid came from close friends whom I would like to give a special thanks to: Melanie Cash, Shayna Sutherland, Joyce Merritt, Asha Brunings, Wayne Jurick, Fabricio Rodrigues, and the late Richard Wheeler who always saw the sunny side. Very special thanks go to Donna Perry, Gail Harris, and Lauretta Rahmes in Plant Pathology and Melissa Webb and Brandy Burgess in the plant molecular and cellular biology program for providing administrative help. Lastly, Id like to thank my husband, Larry Chamusco; parents, Fred and Cleo Courington; and sister, Becky Lockridge who have been loving and supportive in countless ways. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.......................................................................................iv LIST OF FIGURES..............................................................................................vii ABSTRACT..........................................................................................................ix CHAPTER 1 INTRODUCTION............................................................................................1 Programmed Cell Death.................................................................................1 Categories of PCD Described in Plants.........................................................5 Physiologic and Molecular Factors Involved in PCD......................................7 Comparing PCD in Plants and Animals..........................................................9 The Role of Calcium in PCD........................................................................11 The Role of Endonucleases.........................................................................12 Seed Development.......................................................................................15 2 MATERIALS AND METHODS.....................................................................20 Plant Materials.............................................................................................20 Utrastuctural Studies....................................................................................20 Calcium Trapping.........................................................................................22 Light Microscopy..........................................................................................23 Nuclease Activity Assays.............................................................................25 DNA Extraction.............................................................................................27 3 RESULTS....................................................................................................29 The Endosperm............................................................................................29 Light Microscopy....................................................................................29 Transmission Electron Microscopy........................................................30 The Placenta-Chalaza Region.....................................................................31 Light Microscopy....................................................................................31 PCD in the PC Region is Fertilization Dependent..................................32 Transmission Electron Microscopy........................................................33 Calcium Trapping...................................................................................37 v

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In-Gel Nuclease Activity Assay..............................................................39 Genomic DNA Extraction.............................................................................40 4 DISCUSSION...............................................................................................82 PCD in the PC Region.................................................................................82 PCD in the PC May Be Tied to Growth and Development of the Filial Tissues....................................................................................................84 Sink Demand and Assimilate Flow...............................................................86 Two Separate, Temporal Waves of PCD Occur in the PC and May Be Linked to the Sink Demand of the Growing Filial Tissues........................87 The Role of Calcium in PC PCD..................................................................92 The Role of Endonucleases in the PC.........................................................94 PCD in the endosperm.................................................................................96 5 CONCLUSION...........................................................................................100 LIST OF REFERENCES..................................................................................103 BIOGRAPHICAL SKETCH...............................................................................108 vi

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LIST OF FIGURES Figure page 1 Classic apoptotic cellular morphology........................................................18 2 Classic necrotic cellular morphology..........................................................19 3 Schematic drawing illustrating the regions of the maize endosperm..........41 4 Nuclear morphological changes in the endosperm of maize...................... 43 5 PCD in the endosperm of a 24 DAP caryopsis..........................................44 6 Confocal image of the nuclei in the central crown region of the endosperm of a 28 DAP caryopsis of maize..............................................45 7 Nuclear morphology of the central crown region of the endosperm of a 12 DAP maize caryopsis.................................................................................46 8 Nuclear Morphology of the central crown region of the endosperm of a 14 DAP maize caryopsis.........................................................................47 9 Nuclear Morphology of the central crown region of the endosperm of a 16 DAP maize caryopsis............................................................................48 10 Nuclear Morphology of the central crown region of the endosperm of a 20 DAP maize caryopsis............................................................................49 11 Nuclear morphology of the central crown region of the endosperm of a 22 DAP caryopsis of maize........................................................................50 12 Nuclear Morphology of the central crown region of the endosperm of a 25 DAP caryopsis of maize........................................................................51 13 Origin of the Placenta-chalaza (PC) region................................................52 14 Anatomy of the Placenta-chalaza..............................................................53 15 Placenta-chalaza (PC): left-right borders................................................... 55 16 TEM image of one of the boundaries of the PC region..............................56 vii

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17 Placenta-chalaza boundaries.....................................................................57 18 4'6-diamidino-2-phenylindole-2HCl (DAPI) DNA stain...............................58 19 Crystal Violet Stain showing condensed chromatin in the nuclei of the cells of the integument-derived PC region from 4 through 10 DAP......59 20 Crystal Violet Stain showing condensed chromatin in the nuclei of the integument-derived PC region from 12 through 16 DAP............................60 21 TUNEL assays of PC region showing broken or nicked DNA in the nuclei of the integument-derived portion of the PC .................................................. 62 22 Crystal violet stain showing condensed chromatin in the integument derived portion of the PC region of a 24 DAP caryopsis............................63 23 TEM micrographs illustrating consecutive developmental stages and subsequent cell death of the nucellus-derived PC region..........................64 24 TEM micrographs illustrating the plasmodesmata (PD) in the walls of the nucelluc-derived PC cells...........................................................................65 25 Development and subsequent death of upper integument-derived region of PC..........................................................................................................66 26 Development and subsequent death of lower integument-derived region of PC.............................................................................................................. 68 27 Lower Integument-derived PC Cells..........................................................69 28 Nuclear body formation in the integument-derived portion of the PC cells......................................................................................................70 29 Nuclear morphological changes in integument derived portion of the PC..71 30 Ca2+ Trapping: Nucellus-derived PC..........................................................72 31 Ca2+ Trapping in the integument-derived PC. ............................................ 74 32 Ca2+ trapping in the normal appearing, lower integument PC.................... 76 33 In-gel nuclease activity assays for Ca2+, Mg2+ and Zn2+-dependent nucleases and pH controls......................................................................... 78 34 In-gel nuclease activity assays with EDTA and EGTA and no metals........ 80 35 Genomic DNA isolated from the PC region................................................81 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MORPHOLOGICAL SURVEY AND CHARACTERIZATION OF PROGRAMMED CELL DEATH IN THE PLACENTA-CHALAZA AND ENDOSPERM IN THE DEVELOPING CARYOPSIS OF MAIZE By Karen C. Chamusco August, 2004 Chair: Prem S. Chourey Major Department: Plant Molecular and Cellular Biology Programmed cell death (PCD) was examined on a morphological basis employing both light and electron microscopy techniques. The tissues examined were the placenta-chalaza (PC) region and maturing endosperm of the maize caryopsis. The PC region is sporophytic and, in maize, composed of two distinct types of cells, which undergo two distinct morphological changes as they die. This cell death process is fertilization dependent and takes place between 6 and 12 days after pollination (DAP). The first type of cells in the PC is derived from the nucellus and undergoes a rapid cell death leaving behind cell corpses and the number of detectable plasmodesmata decreases. There is no detectable DNA cleavage via terminal deoxynucleotidy transferase dUTP nick end label (TUNEL) assay in this zone at any time. The second type of cells in the PC is derived from the inner integument and undergoes a PCD that, morphologically, resembles apoptosis, i.e., condensed ix

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chromatin, nuclear fragmentation, and preservation of organelles and membranes until the end of the death process. There is no loss of plasmodesmata in these cells. These cells are TUNEL-positive indicating DNA cleavage. Intracellular calcium [Ca2+]i was localized in an attempt to determine the general flux and location of free [Ca2+]i. In general, [Ca2+]i accumulation in storage sites such as the endoplasmic reticulum and the central vacuole does not appear to take place until after 5 DAP. Mitochondria, while appearing to collect [Ca2+]i at some point prior to cell death, do not appear to change morphologically. The general trend of [Ca2+]i flux can be summed up as follows: [Ca2+]i is collected and stored in cellular compartments and organelles, released into the cytoplasm and then dispersed or removed from the cell after death. Nuclease assays revealed three nucleases dependent upon Ca2+ and/or magnesium (Mg2+) and a fourth nuclease dependent on zinc (Zn2+). The peak of activity for these nucleases occurred between 8 and 12 DAP which coincide with the peak of morphological changes and the peak of positive TUNEL activity. Unlike the nuclear changes seen in the PC cells, the nuclei of the central crown region of the endosperm cells undergo a series of morphological changes as the endosperm develops. At 12 DAP the nuclei start out spherical and the DNA diffuse, by 16 DAP expand in size while chromatin condenses at the nuclear envelope. Finally, by 25 DAP, the nuclei collapse and condense tightly. These data indicate the possibility for three unique PCD processes occurring in the developing caryopsis of maize. x

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CHAPTER 1 INTRODUCTION Programmed Cell Death Programmed cell death, or PCD, is a physiologically controlled, carefully timed, energy dependent cell death program that organisms use to aid in defensive processes or depend on for many normal developmental processes (Solomon et al., 1999; Earnshaw, 1995). Although cell death as a physiological process has been known to exist for the last 150 years, apoptosis was the first type of PCD characterized in 1972 in animal cells by Kerr and was called apoptosis from the Greek meaning fallen and has been extensively studied ever since (Bursch et al., 2000; Earnshaw, 1995; Merriam Websters Dictionary, 2004). Plant biologists have used these animal studies as spring-boards from which to investigate PCD in plants. Since the apoptotic form of PCD is the longest studied and best characterized it will be discussed, here, as a model to illustrate the PCD process and for use as a comparison against plant PCD. But it must be noted that apoptosis is but one way PCD can occur. Other types of PCD that have been described include autophagic PCD, a PCD which involves the degradation of intracellular organelles prior to nuclear degradation, and endoplasmic reticulum (ER)-mediated PCD that is independent of apoptosis protease activating factor-1 (apaf 1) and cytochrome c (Bursch et al., 2000; Rao et al., 2004). 1

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2 A cell may be induced to undergo PCD due to a variety of causes such as pathogen invasion, if the lifetime and or function of the cell has ended, or normal developmental processes (Krishnamurthy et al., 2000). Apoptosis, like all programmed cell death processes, is, by definition, a genetically controlled, energy dependent and does not induce inflammation (Earnshaw, 1995). In the case of apoptosis one of the first responses a cell has after perceiving signals to undergo cell death is to produce an asymmetry in the aminophospholipids across the plasma membrane. This duty requires the aid of the membrane bound enzymes floppase, scramblase and the calcium (Ca2+) inhibition of aminophospholipid translocase (Reutelingsperger 1997). The two former enzymes induce an aminophospholipid flip, specifically, phosphatidylserine, whereby the outer leaflet of the membrane has a higher occurrence of serine residues than it normally would have. Once the serine residues are exposed, Annexin V, a eukaryotic conserved aminophospholipid binding protein with high affinity for serine residues, binds them in either a Ca2+ or low pH dependent manner. The bound Annexin V proteins then undergo Ca2+ induced structural changes, ultimately forming an integral transmembrane channel that is thought to act in an amphipathic or polytopic manner (Reutelingsperger 1997; Li and Piazza, 2002). The mitochondria play a role at this point which includes a release of intermembrane space proteins such as procaspase 9, cytochrome c, apoptosis inducing factor (AIF), Ca2+ and reactive oxygen species (ROS). It has been shown that mitochondria are intimately involved in regulating both plant and animal PCD events (Arnoult et al., 2002; Curtis and Wolpert, 2002; Balk et al.,

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3 2003). Mitochondrial inner and outer membrane-spanning pore formation is a critical part of mitochondrial PCD effectors being released. Studies focusing on apoptosis in animal cells have revealed two main mechanisms for the formation of these pores and, consequently, each results in different mitochondrial morphology (Yu et al., 2002). The first mechanism is a direct one and involves the voltage-dependent anion channel, or VDAC, located at the mitochondrial outer membrane. This mechanism is proposed to use the pro-apoptotic, Bcl-2 family protein, Bax, to interact with and tightly regulate release of cytochrome c via the VDAC channel. The morphological consequence of this is that the mitochondria remain normal in appearance. The second mechanism is an indirect mechanism and involves the permeability transition pore, or PTP (Curtis and Wolpert, 2002; Yu et al., 2002). The PTP is formed from a complex of proteins, primarily adenine nucleotide (ANT) located on the inner membrane, VDAC located on the outer membrane, hexokinase, and cyclophilin D (CpD) in the matrix and when induced to interact they form a transient, multi-faceted pore that acts as a redox, ion, pH, and Ca2+-gated channel. Upon continual opening solutes up to 1.5 kDa leak out of the mitochondrial matrix and an uncontrolled influx of solutes, especially Ca2+, into the matrix is initiated. Calcium increases the inner membrane permeability; the result is gross over-swelling of the matrix, loss of transmembrane potential, an uncoupling of the electron transport chain, superoxide production and complete disruption of the outer membrane, and thus the release of inter-membrane space, PCD promoting solutes such as apoptosis inducing factor (AIF),

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4 cytochrome c, procaspase 9 and Ca2+ (Curtis and Wolpert, 2002; Arnoult et al., 2002; Balk et al., 2003; Yu et al., 2002). Once in the cytosol procaspase 9 autoprocesses to caspase 9 in cooperation with cytochrome c and apoptosis protease activating factor-1, or apaf-1, to form the apoptosome complex which further aids in activating effector caspases. Caspases are cysteinyl-aspartate proteases. One such target of these effector caspases is caspase activated DNase (CAD) which, along with other Ca2+, Ca2+/Mg2+ dependent endonucleases, and caspase independent AIF acts to condense and cleave DNA into fragments of 300 and 50 kilobases (kb) then into internucleosomal size fragments which occur in multiples of 180 base pairs (bp). (Earnshaw,1995; Gromova et al., 1995; Krishnamurthy et al., 2000; Woltering et al., 2002; Balk, 2003). A cascade of reactions is initiated at the plasma membrane by initiator caspase 8, which cleaves other cytosolic effector caspases such as 3, 6 and 7 thus activating them. Poly-(ADP ribose)-polymerase, or PARP, which is involved in DNA repair and maintenance, and the RNA splicing enzyme U1-snRNP are other substrates of caspases; however these proteins are cleaved for inactivation purposes so the apoptotic process may be allowed occur (Krishnamurthy et al., 2000; Sun et al., 1999; Woltering et al., 2002, Chang and Yang, 2000). On a morphological basis apoptotic animal cells show a unique pattern of chromatin condensation (Fig 1) delineated by sharp edges, marginalization of chromatin, retention and granulation of the nucleolus, plasma and nuclear membrane blebbing (an outward bulging of the membrane) and the formation of

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5 apoptotic bodies containing degraded cytoplasmic and nuclear contents. In addition, all membranes and organelles are retained until the end of the cell death program when neighboring cells phagocytose the dying cell (Kerr et al., 1995). However the mitochondria may exhibit swelling due to high Ca2+ content in the matrix (Virolainen et al., 2002; Curtis and Wolpert, 2002). Additionally, apoptosis occurs in individual cells and is typically asymmetric with regard to one cells stage of PCD compared with that of another (Kerr et al., 1995). What is described above is but one means by which cells can undergo a genetically programmed death thus the process of PCD is not a clear cut one but one in which it occurs in wide array of ways, especially in plants (Krishnamurthy et al., 2000; Earnshaw, 1995; Rao et al., 2004) Necrosis, on the other hand, is a form of cell death that has been reported to be neither genetically controlled nor energy dependent (Fig 2). This form of death is caused by cell damage, occurs in masses or clusters of cells and, in animals, causes inflamation. The morphology does not resemble apoptosis type PCD in that plasma membrane dissociation is first followed by complete degradation of the cell. There is no organized chromatin condensation in the nucleus and no preservation of intracellular contents. (Earnshaw, 1995; Kerr et al.,1995). However, recent studies dispute necrosis as a passive, non-genetically controlled cell death, rather, a physiologically controlled process (Proskuryakov et al., 2002). Categories of PCD Described in Plants Plants have been shown to display PCD in for up to approximately six different processes: hypersensitive response (HR), cells that have served their

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6 purpose and are no longer required, cells that must die to serve their purpose, cells initiated in development but never used, cells present in wrong places and damaged cells (Krishnamurthy et al., 2000). The hypersensitive response (HR) is initiated by pathogen attack, abiotic stresses such as ozone exposure creating oxidative stresses, incompatible temperatures and osmotic, water, or light stresses. Hormones such as gibberellic and salicylic acids have been shown to play critical roles in HR (Krishnamurthy et al., 2000; Jones and Dangl, 1996) HR does not necessarily end in the death of the cell, but when it does HR is often described as necrotic. Regardless of whether or not the cell death appears necrotic it does still involve genetic control and is localized to one or a few cells at the immediate site of the pathogen attack. For example disease resistance genes, (R) genes, have been implicated in mediating cell death signals during HR. R genes are a single gene in the host plant encoding a receptor that recognizes a single elicitor gene product called an avirulence (Avr) protein from a specific pathogen resulting in resistance. The fundamental R protein architecture typically includes a nucleotide binding domain (NB), a leucine rich repeat (LRR), and serine/threonine kinase unit. There are several subclasses of R genes in which protein sequence alignments have revealed similarity in architecture to several other protein receptors in the animal kingdom that are known to be involved in apoptosis such as interleukin-1, apaf-1, and CED-4. There is, however, still much that is unknown about the mechanisms underlying HR mediated cell death (Shirasu and Schulze-Lefert, 2000).

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7 The category of PCD in which cells have served their purpose and are no longer required include, but are not limited to: senescence, cells involved in abscission, synergids, antipodal cells, tapetal cells, and endosperm cells. PCD in this category is part of the normal developmental processes that occur in plants (Krishnamurthy et al., 2000). Cells that must die to serve their function include xylem tracheary elements (TE), cork cells, sclerenchyma, and modified structures such as spines and thorns (Yu et al., 2002; Obara, 1998; Krishnamurthy et al., 2000; Jones, 1996). Cells initiated in development but never used are those of non-functioning megaspore cells, stamen primordia in female flowers and carpel primordia in male flowers. (Krishnamurthy et al., 2000; Jones 1996). Examples of cells present in wrong places are cells that die to give rise to leaflets of compound leaves and holes in simple leaves (Arunika et al., 2004; Krishnamurthy et al., 2000; Jones, 1996). Lastly is the damaged cells category. This category is still unresolved as damaged cells may also undergo necrosis, a non-programmed cell death. Damaged cell PCD appears to behave much like that of the HR PCD contributing further to the lack of resolution (Krishnamurthy et al., 2000). Physiologic and Molecular Factors Involved in PCD Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2-) are also involved in mediating signals for PCD. ROS generation during HR is suspected to be produced by a handful of enzymes such as the Ca2+ activated plant homologue of the NADPH oxidase found in animals and oxalate oxidase in extracellular spaces during HR (Neill, et al., 2002). ROS have

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8 been shown to occur in two distinct phases and may act in a dose dependent manner. The first wave of ROSs occurs within an hour upon virulent or avirulent pathogen attack whereas the second wave of ROS is specific to the avirulent pathogen, occurs approximately five hours later and is sustained. High ROS levels induce PCD but low levels stimulate antioxidant enzymes such as gutathione S-transferase and glutathione peroxidase. High levels of ROS are suspected to act as immediate pathogen stopping agents by killing the cell at the site of infection as the ROS diffuse to surrounding cells. H2O2 has also been shown to stimulate mitogen activated protein kinases (MAPK) in addition to mitochondria. The mitochondria, in turn, generate more H2O2 altering mitochondrial activity thus leading to PCD. (Shirasu and Schulze-Lefert, 2000; Thordal-Christensen et al., 1997; Neill et al., 2002). The plant hormone salicylic acid (SA) has been shown to play a signaling role in helping mediate HR PCD and systemic acquired resistance (SAR). It is thought that SA coordinates with ROS to amplify, sustain and define the threshold that must be met to induce PCD. Experiments where SA production was compromised resulted in an inhibition of ROS production and a delay of several hours in HR mediated cell death (Shirasu and Schulze-Lefert, 2000). Nitric oxide (NO) is also a signal agent involved in mediating PCD in plants and animals. Upon pathogen attack and subsequent R gene dependent HR NO synthase activity is stimulated. The generation of NO stimulates phenylalanine ammonia lyase (PAL), an enzyme involved in SA synthesis, and pathogen resistance genes. Like SA, NO appears to help regulate ROS in a coordinated

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9 fashion by dramatically amplifying the effects of H2O2 on cell death (Shirasu and Schulze-Lefert, 2000). Gibberellic acid (GA) also plays a role in plant PCD. Fath et al. (2000) demonstrated that isolated barley aleurone layers undergo PCD upon GA stimulation. GA stimulated the release and de novo synthesis of hydrolases and nucleases in barley aleurone cells. GA also stimulated the coalescing of protein storage vacuoles (PSV) within the aleurone cells. When protein synthesis and secretion is finished the PSVs turn into lytic vacuoles and proceed to digest the cellular contents while glucanases and xylanases digest the cell walls. Ethylene is another player in PCD in plants. Young et al. (2000) reported a spike in ethylene production prior to cell death in the endosperm of maize. It is thought that ethylene is involved in nuclease activation and regulation. Comparing PCD in Plants and Animals In doing a side by side comparison of PCD in plants with apoptosis in animals it can be seen that there are some surprising similarities regardless of the method of PCD plants use; however, there have been distinct differences discovered between the two as well. Physiologic and molecular similarities include, but not necessarily seen in every instance, a phosphatidylserine flip from the inner to the outer leaflet of the plasma membrane, Annexin V binding, calcium signaling, mitochondrial release of cytochrome c, and DNA cleavage resulting in internucleosomal size fragments occurring in multiples of 180 bp, protein phosphorylation/dephosphorylation, ROS production and Ca2+ and Zinc ( Zn2+) -dependent endonucleases (Young et al., 1997; Wojciechowska, 2003; Ning et al, 2002; Krishnamurthy et al., 2000).

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10 Plants do not necessarily use the components they do share with animals for PCD, such as cytochrome c. For example cytochrome c release is detected during PCD for TE differentiation but it does not participate in the PCD action itself (Yu et al., 2002) There is, however, a notable difference between plant apoptotic-like PCD and animal apoptosis. The system described, previously, for apoptosis has not been found in plants. For example, caspases have not been identified in plants nor has a caspase cascade reaction; however, in some instances, caspase-like cysteine proteases found in plants have been reported to behave similarly to true animal caspases during plant PCD. Unlike caspases, plant caspase-like proteases do not all have a specificity for aspartate residues. Some plant cysteine proteases have been implicated in plant PCD; such is the case for the barley vacuole localized aspartate proteinase, Phytepsin D, which is highly expressed during autolysis of tracheary element differentiation (Runeberg-Roos and Saarma, 1998). Previous studies have shown these caspase-like proteases to be inhibited by the synthetic caspase inhibitors choloro-methylketone (Ac-YVAD-CMK) and/or aldehyde (Ac-YVAD-CHO). These inhibitors completely prevented HR mediated PCD in tobacco cells (Krishnamurthy et al., 2000). Korthout et al., (2000) have shown caspase 3-like activity in plant cells. They found that by adding plant mitochondria to the cytosol of Xenopus eggs in a cell free system they were able to induce caspase 3 activity. Additionally, when a mixture of protease inhibitors and the caspase 3 specific synthetic substrate N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was added

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11 to cytosolic extracts from barely embryonic cell cultures the substrate was cleaved after the aspartate residue. Only when a caspase 3 specific inhibitor, Ac-DEVD-fmk, was added to the mix did the caspase 3 substrate not become cleaved. Morphological similarities include the following but are not necessarily seen in every instance: cytoplasmic and nuclear shrinkage, chromatin condensation, and in this study, nuclear body formation. One unique difference between animal and plant PCD is that a cell corpse is usually left behind after plant PCD due to the cell wall and cytosolic degradation occuring autophagically and/or autolytically rather than by phagocytosis as it does in animal PCD. However there are some exceptions such as those seen for the formation of holes in the leaves of the lace plant and the degeneration of barley aleurone cells (Arunika et al., 2004; Fath et al., 2000). The Role of Calcium in PCD It seems that regardless of the kingdom an organism is from, Ca2+ appears to be a universal role player in PCD. Under normal, or non-stressed, conditions the basal levels of free cytosolic Ca2+ ([Ca2+]i) in plant cells are kept very low ranging from 100 to 200 nM (Buchanan et al., 2000). Certain organelles in these cells can be used as storage sites for Ca2+. Low cytosolic concentrations of Ca2+ are maintained with the aid of membrane bound Ca2+ channels and transporters such as Ca2+-ATPase and Ca2+/H+ antiporters. Such storage organelles may include the ER and central vacuole and to a lesser degree the mitochondria and nucleus. The cell wall can also contribute to [Ca2+]i (Buchanan et al., 2000). Such an example of Ca2+ release from a storage site in response to plant PCD

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12 stimuli is that of Ca2+ released from the central vacuole during TE differentiation (Yu et al., 2002). Calcium is known to be a major player in signal transduction at the cells surface, in the cytoplasm, and in the nucleus as well as an effector of reactions. Under conditions of stress Ca2+ may be released from these reserves through such channels as inositol 1,4,5-triphosphate sensitive channels (IP3 channels) and starting a cascade of signal transductions and metabolic reactions by means of soluble Ca2+ binding proteins such as calmodulins or MAPKs (Zocchi and Rabotti, 1993; Buchanan et al., 2000). With regards to apoptotic PCD [Ca2+]i has been observed increasing in concentration in the cytoplasm of cells coincident with or shortly after any observable morphological changes in the nucleus, the first site where morphological changes can be observed for PCD (Sherwood and Schimke, 1995; Kerr et al., 1995). The Role of Endonucleases Calcium also plays a role in activating enzymes involved in PCD such as certain nucleases. However, other nucleases involved in PCD require additional metals, different metals altogether, and/or specific pHs to be activated. For many years apoptosis was, more or less, the only type of PCD known and it was always accompanied by DNA cleavage resulting in multiples of ~180bp size fragments at the end of the PCD process showing up as an internucleosomal DNA ladder on gels. Only in the more recent past have investigators come across other types of PCD where DNA is either cleaved into extraordinarily long fragments of 30, 50, or 300kb or not cleaved at all prior to complete degradation

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13 of the cell (Mittler and Lam, 1995; Balk et al., 2003; Sugiyama et al., 2000; Eastman, 1995). Sugiyama et al., (2000) reviewed plant endonucleases responsible for DNA cleavage associated with PCD excluding those that exhibit DNA repair properties. They were able to categorize these nucleases into two fundamental classes based on metal dependence and pH optima. The primary division was based on divalent metal requirements, specifically Ca2+ or Zn2+. However, more recent reports have added an additional two classes of PCD associated nucleases, those requiring magnesium (Mg2+) only and those requiring both Mg2+ and Ca2+ (Sugiyama et al., 2000; Ito and Fukuda, 2002). Calcium and Mg2+ activated endonucleases operate at a relatively neutral pH whereas Zn2+ activated nucleases, or S1-type nucleases, are activated at mildly acidic pHs, 5.0-6.5 (Sugiyama et al., 2000). This being established, the physical location of these endonucleases could be identified or extrapolated. For example, Zn2+-dependent nucleases have been found to be located in the central vacuole and apoplastic areas such as cell walls (Sugiyama et al., 2000; Ito and Fukuda, 2002) but the exact location of Ca2+ dependent nucleases is still enigmatic to a certain degree. Because Ca2+-dependent nucleases operate at a relatively neutral pH Ca2+-dependent nucleases are thought to reside in the nucleus; such an example includes NUC III which was extracted from isolated tobacco nuclei (Mittler and Lam,1995; Sugiyama et al., 2000). The type of PCD in plants cannot be determined by one specific class of nuclease alone due to the fact that different studies have shown cooperative action of nucleases from each class.

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14 During TE differentiation DNA cleavage by Ca2+-dependent endonucleases appears to result only in large DNA fragments but observations that complete DNA cleavage does not occur until after the central vacuole has ruptured links the Zn2+-dependent endonuclease to this phenomenon (Sugiyama et al., 2000; Ito and Fukuda, 2002). It has also been demonstrated that cereal endosperm PCD occurs as a cooperative effort among the nucleases and takes place in three distinct stages employing Ca2+-dependent endonucleases during the first stage, within the endosperm and Zn2+-dependent endonucleases secreted from or acting within the aleurone layer at the latter stages (Young et al., 1997, Young and Gallie, 2000, Sugiyama et al., 2000). Additionally, unlike certain nucleases associated with animal cell apoptosis that are constitutively present in the cytoplasm in a dormant state and are activated only upon PCD signals, CAD, DNase 1 and topoisomerase II, many plant nucleases are transiently expressed as needed. Ito and Fukuda (2002) found that ZEN-1, a 43 kDa Zn2+ dependent endonuclease and a 24 kDa Ca2+/Mg2+-dependent endonuclease were expressed only during induction of PCD for TE differentiation. It is this authors opinion that perhaps a third class of endonucleases should be considered being established; one based on temporal expression and or activity, those that operate during early stages of DNA cleavage and those in the last stages of DNA cleavage. The endonucleases already discussed operate at the late stages of PCD. However, Balk et al., (2003) established an Arabidopsis cell free system to investigate PCD. They have demonstrated a Mg2+

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15 dependent, mitochondrial localized G-like endonuclease that cleaves nuclear DNA into 30 kb size fragments within 3 hours of PCD induction and that further DNA cleavage does not occur until 12 or more hours after PCD by nucleases from the cytosolic fraction of the cell. This study focuses on the morphological changes attributable to PCD in the developing caryopsis of Zea mays, specifically in the placenta-chalaza (PC) region of the kernel and the maturing endosperm. Seed Development In order to better understand PCD in developing caryopses it is necessary to describe the process of maize seed development and anatomy. The maize caryopsis is an indehiscent fruit containing only one seed (Esau, 1977). Identification of the anatomical parts of the caryopsis will begin from the outermost part of the caryopsis and proceed toward the center. The caryopsis consists of the pericarp, which is the fruit, or ovary. The ovary wall consists of three layers, the outer, covered by a cuticle, middle, and inner pericarp. The following two layers are the outer then inner integuments, or the seed coat and are derived from the ovule. The integuments also retain a cuticular layer due to their ontogeny having originated from a common epidermis during ovule development. These cell layers ultimately degenerate during seed development. Continuing inward is a crassinucleate, or large, nucellus comprised of unspecialized parenchyma cells, and finally the embryo sac containing the egg, two synergid cells, two polar nuclei, and three antipodal cells. At the base of the caryopsis is the chalaza, a region of nucellar tissue fused to integumentary and

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16 funicular tissue; however maize lacks a funiculus, tissue that forms a stalk on which the ovule develops (Esau, 1977). The chalaza has become commonly referred to as the placento-chalaza (PC), a term coined by Katherine Esau in 1944 (Kiesselbach and Walker, 1952). The tissue subtending this region consists of vascular and thick walled parenchymatous tissue comprising the pedicel that connects the caryopsis to the receptacle or cob. All of these tissues are sporophytic, or maternal, in origin. The physiologic role of the PC is to be the gateway through which all water and nutrients pass to the growing seed (Wetherwax, 1930; Felker and Shannon, 1980). Upon seed maturity the closing layer forms in this region effectively sealing the seed off from the maternal tissues and will become the site at which the seed will abscise (Johann, 1935). Upon fertilization, the caryopsis undergoes dramatic changes. The egg fuses with the first of two sperm nuclei from the pollen grain to become the diploid embryo while the two polar nuclei fuse to each other followed by fusion with the second sperm nucleus to become the triploid endosperm (Esau, 1977). It is shortly after this point that several PCD regimens start to take place. Among the types of PCD observed in the developing caryopsis are nucellar degradation, PCD within the embryo, i.e. the scutellum, coleoptile, suspensor, root cap, and starting at about 16 days after pollination (DAP), the endosperm (Giuliani et al., 2002; Young et al., 1997). In addition to the above mentioned PCD events in the maize caryopsis, PCD in the PC has been observed for the first time in this study and by Kladnik et al., (unpublished). Each of these areas of PCD is a normal

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17 part of development and they occur at specific times during development. PCD was investigated on a cellular level using light and electron microscopy techniques. Specifically investigated were the PC region during early development (0-12 DAP) and the endosperm from the starch filling stage to near physiologic maturity (12-25 DAP) and appears that these PCDs may be, not only a normal part of development, but a necessary one. Surprisingly, three different morphological changes were seen between the cells of the PC and the endosperm, two in the PC and the third in the endosperm. It is, therefore, possible that there are three different forms of PCD occurring in these tissues.

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18 Figure 1. Classic apoptotic cellular morphology The earliest morphological change in apoptotic cells occurs in the nucleus. Organelles maintain good morphology during nuclear changes. (A) Nucleus shrinks & chromatin condenses at the nuclear periphery. (B) The nucleus starts to bleb with chromatin filled pockets. (C) The entire cell blebs & breaks up into nuclear bodies containing condensed chromatin pieces and cytoplasmic contents. (D) Cell culture showing cells at various stages of apoptosis. From Kerr et al., (1995).

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Figure 2. Classic necrotic cellular morphology. Necrotic morphology is illustrated by soft edged, condensed chromatin in various clumps throughout the nucleus. Additionally, cytoplasmic organelles degenerate concomitantly with nuclear changes. From Kerr et al., (1995).

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CHAPTER 2 MATERIALS AND METHODS Plant Materials W22 Maize plants were grown in a greenhouse from March through August 2002 and February through June 2003. The only exceptions were 14 and 16 days after pollination (DAP) caryopses, which were harvested from field grown plants in the summer of 2003. Caryopses were harvested from the same ear whenever possible or from ears of sibling plants grown at the same time under identical conditions. Caryopses were harvested at various stages of development. For PC examination caryopses from 0 through 12 DAP were used and for endosperm examination 12 through 28 DAP caryopses were used. Utrastuctural Studies Both distilled glutaraldehyde (8%) sealed in ampules with an inert atmosphere and paraformaldehyde (PFA) were purchased from Electron Microscopy Sciences. PFA was prepared by mixing 8% (w/v) PFA into 0.1 M cacodylate buffer, covered with foil, and, under constant stirring, heated to just under the boiling point. Sodium hydroxide (NaOH) was added to aid in dissolving the PFA, maintaining a pH of less than or equal to 8.0. Once dissolved, the PFA solution was immediately placed on ice to chill, filtered through a number 1 Whatman filter and the pH adjusted to 7.0 with concentrated hydrochloric acid (HCl). The PFA stock was stored frozen at -20o C for up to 2 weeks. 20

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21 Some tissues were prepared by fixation in 4% glutaraldehyde (v/v) and 1% paraformaldehyde (v/v) in 0.1 M phosphate buffer (pH 7.0). Most of the tissues were prepared using cacodylate buffer (pH 7.0). Fresh caryopses were harvested and immediately processed for fixation on site by placing a small aliquot of chilled fixative on a glass plate and hand sectioning out a sagittal section of the caryopsis approximately 2mm wide with a double edged razor blade. The tissue was then put into a vial of ice cold fixative and placed under vacuum for 5 to 10 minutes to aid fixative infiltration. After vacuuming the samples were placed on a rotator overnight, approximately12 hours at 4o C. After fixation the tissue was rinsed three times for 30 minutes each in chilled cacodylate buffer then placed in 2% aqueous osmium tetroxide overnight at 4o C. Once the osmication was completed the samples were rinsed 3 times, one hour each, in distilled water then dehydrated in a chilled acetone series at 20% increments, each for one hour. Once the samples reached 100% acetone the acetone was exchanged three times. On the third change the acetone was mixed with propylene oxide as a transition solvent. The ratios of acetone to propylene oxide were 2:1, 1:1, and finally 100% propylene oxide. Each transition lasted 20 minutes at room temperature. Following the 100% propylene oxide incubation, fresh propylene oxide was mixed with Spurrs epoxy resin (including the catalyst) in ratios of 2:1, 1:1, 1:2, propylene oxide to resin and infiltrated in each for 12 hours. Upon completing the 1:2 propylene oxide to resin step the samples were placed in 100% resin and infiltrated for 24 hours. This step was repeated 3 times with fresh resin. After the third exchange of resin at 100% the

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22 samples were placed in flat molds with fresh resin and polymerized at 56o C for 24 hours. Samples were sectioned on a Sorvall MT-2B ultramicrotome. The sections were cut to approximately 60 nm in thickness, indicated by a pale gold color, then transferred to formvar coated copper grids (50 or 100 mesh). The sections were post stained 20 minutes in filtered 2% aqueous uranyl acetate (UA), rinsed three times, one minute each in distilled water then were allowed to dry. However, this UA step was later modified to 25 minutes in 10% (saturated) UA. This was found to give much better contrast to the sections. The sections were then stained six minutes in Reynolds lead citrate, rinsed once in 0.02 M NaOH for one minute, then three times more, 1 minute each, in distilled water. The sections were examined and photographed in a Zeiss 109 or a Zeiss EM-10 CA transmission electron microscope (TEM). Negatives were scanned at 300dpi using Epson Perfection1650 scanner, digitally processed using Adobe Photoshop version 7.0 and saved to compact disc. Calcium Trapping Calcium (Ca2+) was localized by using the oxalo-acetate/antimonate method as described by VanReempts et al. (1982) and Borgers et al. (1977). All steps were carried out at 4oC. The fixative consisted of 2.8% glutaraldehyde, 2% PFA, 90 mM potassium oxalate, and 1.4% sucrose in distilled water then adjusted to pH 7.4 with 1M potassium hydroxide (KOH). This step is for the purpose of allowing oxalate ions to combine with Ca2+ ions to form the insoluble salt calcium oxalate. After fixation the samples were rinsed for two hours in a solution of 7.5% sucrose and 90mM potassium oxalate at pH 7.4. Next the

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23 samples were post fixed overnight in 1% osmium tetroxide mixed with 2% potassium pyroantimonate in 0.1M acetic acid and adjusted to a pH of 7.4 with 1M KOH. This step is for the exchange of oxalate ions for antimonate ions in the calcium salt to form an insoluble, electron dense calcium antimonate precipitate. Samples were then rinsed for half an hour in distilled water adjusted to pH to 10 with 1M KOH. Subsequent steps for dehydration, infiltration and embedding were carried out as described for ultrastructure. Light Microscopy Caryopses were harvested and fixed as described for ultrastructural studies. Dehydration was carried out in an ethanol series consisting of 20% increments, one hour each, up to 100% ethanol at 4o C. After two incubations in 100% ethanol for one hour each the samples were allowed to come to room temperature and were placed in a 1:1 mixture of ethanol to tertiary butanol (TBA) then twice in 100% TBA, again, for one hour each. After the second change of TBA, a third change of TBA was mixed with Paraplast Plus paraffin chips (Fisher Scientific) and placed in an oven at 56o C overnight (12 hours). which allowed the paraffin to slowly melt and infiltrate while the TBA evaporated, facilitating a slow controlled exchange. Following this step the TBA paraffin mixture was removed and fresh molten paraffin poured on the samples to continue infiltration in the oven for another 12 hours. This step was repeated two more times. After the final infiltration step the samples were embedded using disposable boats and embedding rings (Fisher Scientific) and sectioned to a thickness of 10 m on a Microm-325 rotary microtome. The sections were adhered to Fisher Scientific Probon poly-L-lysine coated slides. To stretch the sections, a bubble of water

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24 was placed on each slide, warmed on the slide warmer at approximately 40oC and the sections floated on it until the water evaporated. The slides and sections dried on the warmer for 24 hours to assure proper adherence. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-fluorescent nick end-labeling or, TUNEL, assays were performed using the TUNEL assay kit purchased from Roche Diagnostics. The assay was done according to the manufacturers directions except, the tissue permeablization step using Proteinase-K was reduced to one minute at 37o C and 14 minutes at room temp to help preserve tissue integrity. Briefly, paraffin sections were de-waxed in two changes of xylene and re-hydrated through an ethanol series (100, 70, 50, 30%) for five minutes each and finishing in distilled water. The sections were treated, as described, with 20g/ml Proteinase-K for 15 minutes, rinsed in PBS twice for five minutes each. The sections were then treated with the TUNEL label plus TdT enzyme for the experimental or simply just label for the negative control. The slides were then placed in a petri dish lined with moist Whatman paper and floated in a 37o C water bath in the dark for one hour. Once the incubation was complete the slides were rinsed three times in PBS in the dark for five minutes each, then counter stained with 600 M (v/v) 4'6-diamidino-2-phenylindole-2HCl (DAPI) DNA stain. The DAPI was mixed into the aqueous mounting medium, Gelmount, (Fisher Scientific). The sections were examined with a Nikon Optiphot fluorescent microscope and photographed using a digital CCD camera and Spot software version 3.52 (Diagnostic Instruments). The TUNEL was examined with

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25 an excitation wavelength of 450nm and emission at 520nm while the DAPI was examined with an excitation of 380 and emission at 450nm. For confocal imaging of nuclei in de-waxed sections propidium iodide DNA stain (PI), purchased from Molecular Probes, was used. PI DNA staining was done according to manufacturers directions exactly. Re-hydrated paraffin sections were equilibrated in 2X sodium citrate, sodium chloride (SSC) buffer (0.3 M NaCl and 0.03 M sodium citrate) for 3 minutes then treated with 100 g/ml RNAse A in 2X SSC in a water bath at 37o C for 20 minutes. The slides were then rinsed in 3X SSC three times for one minute each. A solution of 500 nM PI was placed on the sections and incubated at room temperature in the dark for five minutes. Following the incubation the slides were rinsed two times in SSC for three minutes each and the sections mounted in Gelmount. A Bio-Rad 1024 ES Confocal Microscope was used to view the samples and images were captured using LaserSharp Software. Toluidine blue-O stain (TBO) was made to1% (w/v) in 1% sodium borate solution (w/v) and filtered through a 0.2 m filter before use. Resin sections were TBO stained by covering the sections on the slides with a few drops of the stain, gently heating the slide then rinsing the stain away with water. The slides were dried over low heat on a hot plate and viewed by light microscopy. Nuclease Activity Assays In-gel assays were performed for endonuclease activity according to Young et al. (1997), with minor alterations regarding the extraction buffer, discussed below. Caryopses were harvested fresh from the field, flash frozen in liquid nitrogen and stored at -80o C until used for protein extraction. Caryopses of 6, 8,

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26 10, 12, and 28 DAP were tested. The caryopses were cut such that the top two-thirds of the caryopses were separated from the bottom one-third. This was done so nuclease activity of the PC region could be checked free of endosperm tissue. Approximately 500 mg of tissue was used for the protein extraction. The tissue was ground to a fine powder in liquid nitrogen for 15 minutes using a mortar and pestle. The powder was then decanted into a sterile Corex tube and diluted to 5 ml with ice-cold extraction buffer. The protein extraction buffer used for these assays differed from that used by Young et al. (1997) in that the buffer was 50 mM Tris Maleate pH 7.0. This change was made to eliminate any metals that might interfere with or inadvertantly activate the nuclease enzymes. The samples were spun in a Sorvall RC-5B refrigerated rotor superspeed centrifuge at 8000 rpm for 10 minutes at 4oC. The supernatant/ protein was then transferred to 1.5 ml Eppindorf tubes and kept on ice until quantified. Protein quantification was carried out in a BioRad Smartspec 3000 using the Bradford assay (Biorad) according to the manufacturers specifications. Once quantified the samples were stored in aliquots of one milliliter, flash frozen in liquid nitrogen and stored at -80 until needed. Two micrograms of protein from each sample was mixed with sample dilution buffer (1% SDS w/v, 62.5 mM Tris-HCl pH 6.8, 10% glycerol (w/v), and 0.5% bromophenol blue (w/v)) and run on a 12% SDS-PAGE gel at 200 V for one hour. Once completed the gels were rinsed in 25% isopropanol in 0.1 M Tris buffer pH 7.4 three times for ten minutes each to rinse out the SDS detergent. The gels were then washed another three times in 0.1M Tris pH 7.4 to rinse out

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27 the isopropanol. Afterwards the gels were incubated in washes containing either 0.1 M CaCl2 or MgCl2, or both in 0.1 M MOPS buffer pH 6.8 at 50oC for 30 minutes or 1 mM ZnSO4 in 0.025 M acetate buffer (1.76 ml 1 M acetic acid, 8.24 ml 1 M sodium acetate and 40 ml H2O) pH 5.5 at 47oC overnight while gently rotating in water baths. Several types of controls were employed: 1) gels were either incubated with EDTA and EGTA in a 1:1 stoichiometric ratio with that of the metals added to the incubation medium, 2) the pHs were changed such that the Ca2+/Mg2+ buffers were lowered to that required for Zn2+ and the pH of the Zn2+ buffer raised to that required for Ca2+ and Mg2+ or 3) the metals were left out of the incubation medium altogether. After incubation the gels were bathed in 200 g/ml ethidium bromide for 10 minutes, rinsed in distilled water for 10 minutes then inspected in a transilluminator and photographed using BioRad Geldoc software. DNA Extraction Genomic DNA was isolated from 5 grams of the same tissues cut in the same manner as was used for the protein extraction. The developmental stages examined were 8, 10, 12, 16, 20, and 25 DAP. Tissue was ground with a mortar and pestle in liquid nitrogen for 20 to 30 minutes to a fine powder then immediately put in sterile culture tubes containing 15 ml extraction buffer (100 mM Tris pH 8, 50 mM EDTA pH 8, 100 mM NaCl, 1% SDS, 0.2% PVP, and 10 mM -mercaptoethanol) and incubated at 65oC for 10 minutes. After incubation 5 ml 5 M ammonium acetate was added, mixed, and then incubated on ice for 20 minutes. The samples were then poured through Miracloth into a new sterile tube and 1 ml 5 M ammonium acetate and 1 volume isopropanol added. The

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28 samples were then kept at -20oC for a minimum of 30 minutes, centrifuged at 12,000 rpm at 4oC in a Sorvall RC-5B refrigerated superspeed centrifuge for 15 minutes and the supernatant poured off. The pellet was washed with 5 ml 70% ethanol after which the tube was inverted on a paper towel for 5 minutes then placed under vacuum for an additional 5 minutes. The pellet was resuspended in 500 l TE (10 mM Tris-HCl, 1 mM EDTA pH 8.0) and placed on ice for 30 minutes with intermittent mixing every 5 to 10 minutes. Next, 250 l 5 M ammonium acetate and 2 l of 10 mg/ml RNAse A were added and incubated at 37oC 30 minutes. An equal volume of phenol, chloroform, isoamylalcohol (IAA) in a ratio of 25:24:1, respectively was added to the tube, spun for 10 minutes at 8,000 rpm and the supernatent pulled off and placed in a new tube. One volume chloroform and IAA in a ratio of 24:1 respectively was added to the tube and spun again at 8,000 rpm for 10 minutes. The supernatant was removed, placed in a fresh tube, two volumes absolute ethanol added then incubated for 1 hour. The sample was spun for 20 minutes at 8,000 rpm, the ethanol poured off, and the pellet dried under vacuum. Once the pellet dried it was resuspended in 50 to 100 l sterile water and stored at -20oC. The DNA was quantified in a BioRad SmartSpec 3000 spectrophotometer. Two g DNA was electrophoresed in a 2% agarose gel infused with 0.5 g/ml ethidium bromide with a low mass DNA ladder and run at 120V for approximately 1 hour. The gel was photographed in a transilluminator using Biorad Geldoc software.

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CHAPTER 3 RESULTS The Endosperm Light Microscopy TUNEL and DAPI stains were used to investigate DNA conditions and nuclear morphology, respectively, in developing endosperm cells of maize. The endosperm was divided into four regions for inspection: the crown, central crown, mid endosperm and lower endosperm (Fig. 3). As illustrated in Figure 4, the nuclei at each stage of development showed distinct morphological changes taking place up through 24 DAP. At 12 DAP the cells in the crown of the endosperm were starting to fill with starch and nuclei appeared healthy and maintained an approximate central position in each cell. The nuclei were characterized by diffuse DNA which appeared uniform in texture throughout the nucleus. By 16 DAP the crown of the endosperm was filled with greater quantities of starch and the DNA of each nucleus had condensed at the periphery giving the nuclei a beaded appearance. By 24 DAP the TUNEL assay showed positive results as indicated by bright green fluorescence in the nuclei in the central and crown region of the endosperm, indicating that DNA cleavage in the endosperm does not take place until after starch is abundant and the nuclei lose their apparent integrity (Fig. 5). It is at this stage when the nuclei condense and shift closer to the cell wall. 29

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30 The 28 DAP kernels proved to be too opaque for any TUNEL activity or DAPI stain to be seen due to the autofluorescence of the starch grains. However, confocal imaging of propidium iodide stained sections was used to look at the nuclear morphology. By 28 DAP, as seen in Figure 6, the nuclei of the crown of the endosperm appeared completely condensed and appressed against the cell wall. Transmission Electron Microscopy Caryopses of the same developmental stages used for LM investigation were selected for nuclear investigation from the central crown region of the endosperm by TEM. The pattern of morphological changes of endosperm nuclei seen by DAPI staining at the LM level compared favorably to those seen at the EM level. At 12 DAP, the nuclei appeared rounded, the DNA diffuse and the cell appeared normal as indicated by a normal appearing nucleus with diffuse DNA and an intact nuclear envelope (Fig. 7). At 14 DAP the nuclei appear slightly larger and the chromatin less diffuse (Fig. 8). Interestingly, the rough endoplasmic reticulum (ER) appeared more prevalent and displayed intermittent areas of bloating while in other areas the bloated bodies become filled with electron-dense substances then appeared to bud off, presumably forming protein bodies. By 16 DAP the nuclei displayed condensed chromatin that collected at the nuclear envelope (Fig. 9) supporting the beaded appearance as seen by DAPI stain at the LM level. The ER became less pronounced and the membrane bound, electron dense bodies took on a more oval shaped appearance and mini

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31 vacuoles contained within them could be seen. By 20 DAP the nuclei appeared bloated, appeared to have much less chromatin and, in some cases, appeared to be almost devoid of contents leaving only an intact nuclear envelope (Fig.10). However, some of the nuclei displayed enlarged condensed chromatin spheres that looked like oversized nucleoli; but, as stated previously, the rest of the nucleus is empty except for very small, condensed chromatin pieces sparsely distributed at the envelope. The nuclei at 22 DAP (Fig. 11) became very difficult to discern as the membranous structures of the cell, including the plasma membrane, appeared to be degenerating and cytoplasmic contents started to coalesce and were not readily identifiable. By 25 DAP the nuclei and cytoplasm of the cells became indistinguishable (Fig. 12). All the cellular contents condensed, became amorphous in appearance and, for the most part, moved to the cell periphery. At higher magnification the amorphous material acquired the appearance of being comprised of many small spheres that appeared to be fatty in nature. The Placenta-Chalaza Region Light Microscopy Both LM and TEM were also employed to investigate morphological changes occurring the PC region during PCD. In order to investigate the changes associated with the PC region it was necessary to anatomically define this region more clearly. The ontogeny of the PC region, (Fig.13), showed two cell lineages from each the nucellus epidermis and the inner integument, the nucellus epidermis contributing to the upper region of the PC and the inner

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32 integument contributing to a region of the PC subtending the nucellar derived region. These two regions will be referred to as zones for the sake of clarity. The anatomy of the PC region clearly shows two distinct zones, the upper from the nucellus epidermis and the lower from the inner integument, as described above (Fig. 14). This observation was in agreement with the literature in that the PC region of the maize caryopsis is where the integumentary and nucellar tissues merge (Esau, 1977). After pollination, as described by Esau, (1977), two semipermeable, suberized and/or cutinized, extra-cellular membranes became detectable in the seed coat and by approximately 7 DAP extended around the entire seed but ended abruptly on either side of the PC region (Fig. 15). The thicker of the two membranes appeared bi-layered (Fig. 16). This membrane was derived, primarily, from the nucellus epidermis and, secondarily, the inner integument. These membranes ended abruptly on either side of the PC thus defining the boundaries of the PC region (Fig. 17). PCD in the PC Region is Fertilization Dependent DAPI DNA staining of a 12 DAP caryopsis and an unpollinated ovule of 12 DAP equivalency revealed enucleation of the PC cells only in the fertilized kernel (Fig 18). Additionally crystal violet staining (Figs.19-20) revealed condensed chromatin in cells of the integumentary PC between 6 and 14 DAP which coincided with the TUNEL positive reactions (Fig. 21) seen in this region (Kladnik et al., unpublished). While there were no detectable condensed nuclei between 16 and 22 DAP condensed chromatin was seen, again, at about 24 DAP (Fig.

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33 22) in the region of the integumentary PC that was most compressed by the endosperm. Transmission Electron Microscopy Prior to pollination both layers, the nucellus-derived and integument-derived layers, of PC cells appeared normal and only a few morphological differences aided in distinguishing the two cell types from one another. The nucellar PC cells bordering the integument-derived cells in the PC region of the 0 DAP caryopsis were large relative to the integument-derived cells. The nucellar PC cells were characterized by thin, primary cell walls and very large, single vacuoles and a small nucleus relative to the cells size (Fig. 23A). These cells contained healthy organelles and abundant plasmodesmata Up through approximately 5 DAP all the PC cells remained relatively unchanged in appearance from those of the PC in the 0 DAP caryopsis. At 5 DAP the only significant change in the cells of the PC region from that of the 0 DAP caryopsis was that the nuclei of the PC cells had become more rounded. At this stage of development the growing endosperm started to crush the nucellar tissue immediately in contact with it thus beginning the initial steps of forming a multi-walled barrier between the basal endosperm cells and the PC region. The nucellar-PC cells just below (Fig. 23B) still contained healthy cytoplasm and nuclei and the average number of plasmodesmata per cell per section was 7.1. After pollination a dramatic series of changes took place in the PC region as the caryopsis developed. The first observable changes that occurred were at about 7 DAP. The enlarging endosperm had further crushed the nucellar PC

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34 cells creating a more compacted barrier between the basal endosperm and PC region. These cells had substantially emptied and contained remnants of what appeared to be plasmolysed cytoplasm (Fig. 23C). These cells also appeared a bit more flattened than previously seen in younger stages. The average number of distinguishable plasmodesmata per cell remained the same as it was for 5 DAP. By 9 DAP, the border between the basal endosperm and the nucellar-PC had become very thick and solid and had completely isolated the endosperm from any symplastic connections to the PC. The nucellar-PC cells had only very sparse remnants of unidentifiable degenerated cytoplasmic contents (Fig. 23D). The number of plasmodesmata from 9 to 12 DAP varied from 1.6 to 3.3 per cell per section for an average of 2.4 per cell per section (Fig. 24). This was a reduction of 33% from the 7.1 seen from 5 to 7 DAP. However, it is important to note that this apparent reduction in the number of plasmodesmata was seen only in the nucellus-derived portion of the PC and not the integument-derived portion, which appeared to maintain an abundance of plasmodesmata. The layer of cells below the nucellar-PC cells are those derived from the inner integument. At 0 DAP the integument-derived cells were characterized by a more rectangular shape, thicker walls than the nucellus-derived cells, had a more dense cytoplasm (Fig. 25A). Additionally, the nuclei in these cells were small, elongated and looked like flattened footballs. At 5 DAP these cells appeared healthy and, generally, contained one vacuole; the only significant change from 0 DAP was a slightly more dense cytoplasm (Figs. 26 A,B). The next cell layer below the nucellar PC cells, the upper integumentary PC, was,

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35 approximately, two cells wide; these cells will eventually become part of the closing layer. By 7 DAP (Fig. 25C) the nuclei of these cells showed the beginning stages of condensing chromatin; however the nuclei were still intact and appeared spherical. By 9 DAP the cells of the closing layer had become laterally elongated, compacted and more cytoplasmically dense but some cells still contained crushed nuclei with condensed chromatin. Generally, the cytoplasm of these cells, at this stage of development, had become very dense, the vacuoles were absent or were in the process of disappearing and there were no identifiable organelles (Fig. 25D). The cells subtending the closing layer, the lower integumentary PC cells (Fig. 26A-C) contained normal nuclei and cytoplasm and averaged one vacuole per cell. At 9 DAP this cell layer (Fig. 26D) was characterized by cells with a total lack of nuclei, unidentifiable organelles and no vacuoles but retained all their disintegrated cytoplasmic contents. These cells had become flattened and elongated. This layer of cells was peculiar in that it showed variable positioning around the closing layer depending on which part of the PC region was being viewed. On the lateral edges this third cell layer was above the closing layer for a short distance but when moving toward the center of the PC the third layer appeared below the closing layer and held this position for the greater portion of its span. The fourth layer of cells down (Fig. 26E) contained early stages of PCD. The cytoplasm of these cells contained multiple, small vacuoles, but the nuclei were starting to display condensing chromatin. Additionally, the endoplasmic reticulum (ER) became much more abundant and formed concentric ring-like and/or layered, laminar patterns (Fig. 27). Continuing

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36 basipetally, the next layer of cells appeared healthy as well with the only one distinguishing feature from those of the previous layer. These cells, on average, contained several smaller vacuoles. Below these cells was the vascular tissue and starch-containing parenchyma. The closing layer and a few cells beneath the closing layer, by 8 DAP, appeared to be the first set of cells at which gross morphological changes related to PCD were seen. This was the first stage at which completely condensed nuclei were detected. Interestingly, in the cells that harbored completely condensed nuclei, small spheres filled with condensed chromatin remains were seen throughout the cytoplasm corresponding to similar TUNEL positive bodies seen within PC cells at a light microscopy level (Fig. 28). For those cells at such an advanced state of degradation the only cytoplasmic feature that could be clearly identified with certainty was the rough ER. However, the ER was very swollen. By 11 DAP, as seen in Figure 31E and F, the cells of the closing layer looked extremely chaotic; the contents of most of the cells in this layer looked disjointed. For example, the membranes were collected in tight whirls and seemingly disconnected from anything else in the cell, lipid bodies and unidentifiable cellular debris constituted the rest of the cytoplasm. In some cases the cytoplasm was dominated by many very tiny vacuoles. The cells furthest down in the PC appeared normal with completely normal looking nuclei and organelles. By 12 DAP the first set of cells with any cellular debris to be seen was the top most layer of the closing layer, which coincides with the chaotic cells seen in the 11 DAP. By this stage all that was left was condensed cytoplasm.

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37 Further down into the closing layer and the rest of the integumentary PC cells with condensed nuclei became more abundant. Some cells were seen at such a stage where the mitochondria were found to be dramatically swollen and losing, or had lost, inner membrane integrity. Moving further down into the PC nuclei had mild to no condensed chromatin and there were identifiable organelles. A general trend was seen in the morphology of the nuclei as the PCD process ensued. Initially the chromatin in the nuclei appears diffuse and the organelles normal in appearance (Fig. 29A). As in animal apoptosis the chromatin condenses and is delineated by sharp edges. However, unlike animal apoptosis, the chromatin does not coalesce at the nuclear periphery (Fig. 29B). Eventually the chromatin in the nucleus completely condenses but the nuclear membranes remain intact (Fig. 29C). At the end of the process the nucleus has broken up into nuclear bodies (Fig. 29D). Calcium Trapping The oxalate/antimonate calcium ion (Ca2+) trapping method was used in an attempt to determine the flux and location of free Ca2+ ions within the cell during this cell death process. At 5 DAP the nucellar-PC cells contained only trace amounts of Ca2+ in the cytoplasm and organelles (Fig. 30A). The cells did not contain Ca2+ in the central vacuoles at this time, but rough ER had light Ca2+ deposits associated with the membrane. The mitochondrial matrix did not appear to be associated with appreciable deposits of Ca2+ while the nucleus was very lightly peppered with Ca2+. At 8 DAP the nucellar-PC contained moderate background Ca2+ (Fig. 30C) but the remnants of cellular contents did not contain

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38 Ca2+. The next cell layer down, still within the nucellar PC (Fig. 30C-inset), was intermediate in its cytoplasmic degeneration between that seen in the nucellar-PC and the closing layer. This cell layer showed Ca2+ to be abundant in the cell corpse background but still did not appear to associate with the degenerated cytoplasmic remains. By 11 DAP the nucellar-PC appears similar to that of the 8 DAP except at this point there is no Ca2+ in the cell corpse background any longer (Fig. 30E). At 5 DAP the integument PC cells did not appear significantly different than those in the nucellar PC except that the mitochondria appeared to have a light scattering of Ca2+ deposits throughout giving them a peppered appearance (Fig. 31A). By 8 DAP the closing layer cells in the upper integumentary PC showed Ca2+ sparsely in the cytoplasm but small amounts were present in what was left of the ER (Fig. 31C). By 11 DAP the closing layer showed chaotic, condensed cytoplasmic contents with highly compacted swirls of membranous structures but with little Ca2+ present (Fig. 31E). The cells immediately subtending the closing layer varied subtly in their localization of Ca2+. These cells were examined in order to check where Ca2+ might localize in normal appearing PC cells of more mature tissues. Some of the cells displayed mitochondria with little to no Ca2+ localized within (Fig. 32A). Additionally, the ER did not appear to contain much Ca2+ as indicated by few electron-dense precipitates. The other cells of this region looked similar to those discussed above except the ER was abundant with Ca2+ which were heavy in electron-dense precipitates (Fig. 32B), the mitochondria seemed to have more

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39 Ca2+ (Fig. 32C), and the nuclei were peppered with Ca2+; however, the vacuoles of both cell types contained abundant Ca2+ and the cytoplasm did not have much Ca2+ in either cell type relative to the rest of the cell. In cells of moderate to advanced stages of PCD the nuclei contained more Ca2+ than the cytoplasm and the mitochondria showing morphological changes, i.e. swelling, contained fewer Ca2+ deposits than those not yet showing morphological changes in other cells (Fig. 32D). In-Gel Nuclease Activity Assay The lower one-third of the wild type W22 kernels, 6, 8, 10, 12, and 28 DAP were examined for Ca2+, Mg2+, both Ca2+ and + Mg2+, or Zn2+dependent nuclease activities. The Ca2+ and Mg2+ treatments, whether tested together or independently resulted in the same pattern (Fig. 33). As was reported by Young, et al. (1997) three nucleases of 33.5, 36.0 and 38.5 kiloDaltons (kDa) were seen when tested using these metals. The 33.5 kDa nuclease was highly active at all developmental stages examined; however, only the 38.5 and 36.5 kDa nucleases were most active at 8 DAP as indicated by a lack of DNA detection in the gel (Fig. 33A). The Zn2+-dependent nucleases (Fig. 33B) showed a similar pattern of banding with the addition of a fourth band at 35 kDa and a slight decrease in activation of the 36.5 kDa and 38.5 kDa nucleases. The 35 kDa nuclease appeared to increase slightly at 10 DAP and persisted through 12 DAP. As a control the Ca2+ and Zn2+ gels were incubated at acidic (5.5) and neutral (6.8) pH, respectively, to test the effect of improper pH on each nuclease (Fig. 33C

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40 and D). The results were a complete inactivation of all nucleases except the smallest one. EGTA and EDTA were then added to the Ca2+/Mg2+ and Zn2+ assays. Each was then incubated at its proper pH. The Ca2+/Mg2+ gel showed a loss of activity in all nucleases except the smallest one; however, the Zn2+ gel did not show a reduction in activation most likely due to the acidic conditions of the incubation medium which inhibits the ability of EGTA and EDTA to act as chelating agents (Fig. 34A and C). Lastly, the gels were incubated at their respective pHs without the metals included in the incubation media. The gels showed the same pattern of nuclease banding as with their respective metals added indicating that these metals must be present and inherent in the tissue itself (Fig. 34B and D). Genomic DNA Extraction Genomic DNA was extracted to try to identify inter-nucleosomal laddering in 8-24 DAP PC tissues (Fig. 35). No inter-nucleosomal banding was detected for the DAPs tested.

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41 Figure 3. Schematic drawing illustrating the regions of the maize endosperm. The crown is the upper-most region of the endosperm. The central crown is the interior portion of the crown. The mid-endosperm lies parallel to the embryo and the lower endosperm subtends the mid-endosperm and extends to the basal endosperm transfer cells, the first layer of cells at the base of the endosperm just above the placenta-chalaza region (PC).

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Figure 4 Nuclear morphological changes in the endosperm of maize Nuclear morphological changes in the endosperm of maize at 12, 14, 16, and 24 days after pollination (DAP) visualized via DAPI DNA stain. Crown: through 14 DAP the chromatin in the nuclei appears round and diffuse. By 16 DAP the nuclei and chromatin have started to condense. By 24 DAP the nuclei are so condensed they have become difficult to discern. Central crown: the nuclei show the greatest morphological changes in this region of the endosperm through development. At 12 DAP the nuclei are round and diffuse. At 14 DAP the nuclei have enlarged and the chromatin is starting to appear more granular. By 16 DAP the nuclei have substantially enlarged and the chromatin has condensed at the nuclear periphery. By 24 DAP the nuclei have condensed appearing like those in the crown. Mid endosperm: through 14 DAP the chromatin in the nuclei is diffuse and round. By 16 DAP the nuclei have enlarged slightly and appear similar to those of the central crown at 14 DAP in that the chromatin has started to become granular in appearance. By 24 DAP the chromatin has condensed at the nuclear periphery and appears like the nuclei in the central crown at 16 DAP. Lower endosperm: the nuclei remain relatively unchanged up through 24 DAP. The nuclei retain their size and diffuse chromatin. Magnification: 20x.

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43

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44 Figure 5 PCD in the endosperm of a 24 DAP caryopsis. (A-C) DAPI DNA stain was used to identify the location of nuclei in the cells. (D-F) Terminal deoxynucleotidyltransferase mediated dUTP nick end labeling (TUNEL) assay was used to show broken and nicked DNA in the nuclei denoted by green fluorescence. (G) The negative TUNEL control shows no green fluorescently labeled nuclei. The nuclei in the upper endosperm are condensed while those of the mid endosperm are ring-like in appearance due to chromatin condensation at the nuclear envelope. The nuclei in the lower endosperm are rounded and the chromatin is diffuse throughout each nucleus. Only the mid and upper endosperm are showing TUNEL positive activity. Magnification, 20x.

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45 Figure 6 Confocal image of the nuclei in the central crown region of the endosperm of a 28 DAP caryopsis of maize. The nuclei (orange color) appear completely condensed at maturity, have shifted to the cell periphery and are appressed against the cell wall (green). Nuclei stained with propidium iodide. Magnification, 20x.

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46 Figure 7 Nuclear morphology of the central crown region of the endosperm of a 12 DAP maize caryopsis. At 12 DAP the nuclei of the central upper endosperm appear normal; the chromatin is diffuse and the nuclei are, generally, centrally located. (A) A light micrograph of a TBO stained resin section of endosperm used as reference for (B). Arrows point to nuclei. (B) TEM image of endosperm nucleus N, nucleus; s, starch grain. Magnification for A, 20x; B, bar 6m.

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47 Figure 8. Nuclear Morphology of the central crown region of the endosperm of a 14 DAP maize caryopsis. (A) TEM image. The nucleus is enlarged and, while not yet condensed, the DNA has collected at the periphery of the nucleus. The nucleolus is still prominent. (B, C) TEM images. Relative to all other developmental stages examined, the endosperm, at 14 DAP, appears to be its most active in forming protein bodies. (C) The protein bodies (box) bud directly from the rough ER. N, nucleus; n, nucleolus; rER, rough endoplasmic reticulum. Bars: A, B, 6m; C, 0.5m.

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48 Figure 9 Nuclear Morphology of the central crown region of the endosperm of a 16 DAP maize caryopsis. Nuclei are enlarged and are starting to display condensed chromatin at the nuclear envelope. (A) TBO stained resin section of endosperm. Boxes enclose nuclei. (B) TEM image of a nucleus. (C) TEM composit of a nucleus. N, nucleus; cc, condensed chromatin. Magnification; A, 20x; Bars: B, C, 6m.

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49 Figure 10 Nuclear Morphology of the central crown region of the endosperm of a 20 DAP maize caryopsis. Similar to 16 DAP the chromatin is condensed and adhered to the nuclear envelope but the nuclei are slightly more enlarged. The nucleoli are still present. The nuclei have shifted to the periphery of each cell. (A): TBO stained resin section. Boxes enclose nuclei. (B, C): TEM images of nuclei. N, nucleus; n, nucleolus. Magnification; A, 20x; Bars: B, C, 5m.

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50 Figure 11 Nuclear morphology of the central crown region of the endosperm of a 22 DAP caryopsis of maize. TEM image of the remnants of a collapsing nucleus. Some condensed chromatin, denoted by black masses, is associated with the nuclear envelope. N, nucleus; cc, condensed chromatin. Bar: 5m.

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51 Figure 12 Nuclear Morphology of the central crown region of the endosperm of a 25 DAP caryopsis of maize. By 25 DAP the nuclei have collapsed and shifted to the periphery of the cell. The nuclei have a disorganized, amorphous appearance. Close inspection reveals no organization of the nucleus but shows lipid or fatty appearing spheres surrounded by an electron dense material. These may be left over membranous and chromatin materials from the dissociated nucleus. (A) TBO stained resin section. Box encloses a nucleus. (B) TEM image of nucleus. (C) TEM image, close up of nucleus. N, nucleus. Magnification: A, 20x; Bars: B, 5m; C, 0.5m.

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52 Figure 13 Origin of the Placenta-chalaza (PC) region. This 9 DAP caryopsis illustrates the PC region containing two distinct sets of cells comprising two zones. The upper zone is contiguous with the nucellar epidermis and the lower zone is contiguous with the inner integument. 1) Endosperm, 2) Basal endosperm transfer cells (BETC), 3) Nucellar epidermis, 4) Semi-permeable membrane (gray line) subtended by inner integument, 5) Outer integument, 6) Inner pericarp, 7) Mid pericarp. Magnification: 20x.

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53 Figure 14 Anatomy of the Placenta-chalaza. The PC is comprised of two different types of cells creating two distinct zones, the nucellus-derived PC and the integument-derived PC. The PC is sandwiched between the basal endosperm transfer cells (BETC) and the pedicel parenchyma. 1) Endosperm, 2) (BETC), 3) Nucellus derived PC, 4) Closing layer (part of integument derived PC), 5) Integument derived PC, 6) Pedicel parenchyma. Magnification: 20x.

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Figure 15 Placenta-chalaza (PC): left-right borders. The boundaries of the PC region are denoted by the developing semi-permeable membrane. The PC region lies between the ends of the semi-permeable membrane at the base of the caryopsis thus becoming the only port through which water and nutrients can pass. Column A represents the adaxial side of caryopsis while column B represents the abaxial side of caryopsis. (2 DAP) The membrane starts its development below the developing endosperm first followed by rapid development at the abaxial face of the lower kernel opposite the developing endosperm. (6 DAP) The membrane has developed fully around the entire seed except at the PC where it ends abruptly. (8-10 DAP) The membrane matures and continues to thicken. Magnification: 20x.

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55

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56 Figure 16 TEM image of one of the boundaries of the PC region. A semi-permeable membrane develops from the nucellus epidermis and surrounds the entire ovule but ends abruptly where the PC region begins at the base of the caryopsis. 1) Nucellus epidermis, 2) Semipermeable membrane, 3) Fatty substances, 4) Inner integument, 5) Membrane terminus, 6) Start of PC. Bar, 5m.

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57 Figure 17 Placenta-chalaza boundaries. The horizontal limits of the PC region are defined by the start points of the semipermeable membrane and the vertical limits are defined by the nucellus-derived and integument-derived cell layers sandwiched between the basal endosperm transfer cells and the pedicel parenchyma cells. (A) Abaxial side of the caryopsis, (B) Adaxial side of the caryopsis. Magnification, 20x.

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58 Figure 18 4'6-diamidino-2-phenylindole-2HCl (DAPI) DNA stain. Nuclei appear as blue, glowing spheres or spots. Enucleation of PC is fertilization dependent. Enucleation is denoted by a lack of blue glowing nuclei in the cells (black area). (A) Equivalent of a 12 DAP caryopsis, unpollinated ovule, (B) 12 DAP caryopsis. Magnification, 20x.

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59 Figure 19 Crystal Violet Stain showing condensed chromatin in the nuclei of the cells of the integument-derived PC region from 4 through 10 DAP. Figures on the right hand side are enlargements of the corresponding figures to the left Regions that are enlarged are denoted by the red lines. (4 DAP) There are no condensing nuclei in the PC at this stage of development. (6 DAP) Condensed nuclei start appearing at this stage in the integument-derived portion of the PC in what will be the closing layer (cl) and in the cells just below the closing layer. (10 DAP) Condensed nuclei remain prevalent at this stage. Magnification; Left side 10x; right side 40x.

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60 Figure 20 Crystal Violet Stain showing condensed chromatin in the nuclei of the integument-derived PC region from 12 through 16 DAP. Figures on the right hand side are enlargements of the corresponding figures to the left. Regions that are enlarged are denoted by the red lines. (12 DAP) Condensed nuclei (arrows) are limited to just below the closing layer. (14 DAP) Condensed nuclei have become sparse and are a little further down beneath the closing layer relative to 12 DAP. (16 DAP) There are no more condensed nuclei in the PC at this stage. Magnification; Left side 10x; right side 40x.

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Figure 21 TUNEL assays of PC region showing broken or nicked DNA in the nuclei of the integument-derived portion of the PC Terminal deoxynucleotidyl transferase mediatd dUTP nick end labeling (TUNEL) assays of PC region showing broken or nicked DNA in the nuclei of the integument-derived portion of the PC (red brackets). Nicked or broken DNA is denoted by bright green fluorescence. The nucellus-derived PC cells test TUNEL negative as indicated by a lack of green fluorescent nuclei. (A, C, E, G) experimental sections. (B, D, F, H) negative controls. (4 DAP) TUNEL activity is not yet detected. (8 DAP) TUNEL activity is well established. (10 DAP) TUNEL activity peaks. (12 DAP) TUNEL activity is still seen but starts to taper off after 12 DAP. Magnification; A-F, 20x; G,H, 10x. From Kladnik et al., unpublished.

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63 Figure 22 Crystal violet stain showing condensed chromatin in the integument derived portion of the PC region of a 24 DAP caryopsis. Figures on the right hand side are enlargements of the corresponding figures to the left Regions that are enlarged are denoted by the black lines. The condensed nuclei reappear starting after approximately 22 DAP in the compressed portion of the PC indicating a possible second wave of PCD occurring in the PC. (A) Upper left is the compressed region; lower right is the non-compressed region of the PC. (B) Condensed nuclei appear in the compressed region of PC. Nuclear bodies (arrow) appear in one of the cells. (C) There are no condensed nuclei in the non-compressed region. Magnification; A,10x; B, 40x.

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64 Figure 23 TEM micrographs illustrating consecutive developmental stages and subsequent cell death of the nucellus-derived PC region. This is the region that tests TUNEL negative. (A) At 0 DAP cells appear normal, contain all organelles and large vacuoles. (B) By 5 DAP nuclei and all organelles are present and the cells appear normal. (C) By 7 DAP the cells have lost all organelles and nuclei and have plasmolyzed. (D) By 9 DAP the cells have almost emptied completely and only degraded cytoplasmic contents remain. Arrow heads, mitochondria; arrows, plasmolyzed cellular remains; (N), nuclei; (DC), degenerated cytoplasmic contents. Bars, 5m.

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65 Figure 24 TEM micrographs illustrating the plasmodesmata (PD) in the walls of the nucelluc-derived PC cells. (A) At 5 DAP the average number of identifiable PD in the walls of the cells per section is 7.2. (B) The average number of identifiable PD in the walls starts to decrease. (C) By 9 DAP the average number identifiable PD decreases to 2.4 per cell per section. Bars: A, 0.5m; B, 1m; C, 0.5m.

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66 Figure 25 Development and subsequent death of upper integument-derived region of PC. The upper integument-derived region forms the closing layer of the kernel. This is the region of the PC that tests TUNEL positive. (A) At 0 DAP the cells appear normal, contain large vacuoles and all organelles are present. (B) Close up of cells in A showing normal appearing organelles. (C) By 7 DAP the cells have thickened walls, more dense cytoplasm, smaller vacuoles and some of the nuclei start to display condensing chromatin (arrows). (D) By 9 DAP the cytoplasm becomes very dense, the vacuoles break up into mini-vacuoles, there are no identifiable organelles, and chromatin in nuclei continues to condense. Arrow heads, mitochondria; arrows, nuclei with condensed chromatin; N, nucleus; C, cytoplasm; V, vacuoles. Bars, 5m.

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Figure 26 Development and subsequent death of lower integument-derived region of PC. (A) At 0 DAP cells appear normal and contain all organelles and large vacuoles. (B) By 5 DAP cells are still normal but the vacuoles are smaller and the cytoplasm more dense. (C) By 7 DAP the cells still retain their organelles and the nuclei still appear normal but, in some cells, the vacuoles are starting to break into smaller vacuoles. The cytoplasm takes on a granular appearance. (D) At 9 DAP the cells have lost all identifiable organelles, vacuoles, and nuclei. The cytoplasm still appears granular. This layer starts to become compressed by the growing endosperm and the cells appear flattened. (E) The cell layer below D. These cells test TUNEL positive and contain nuclei that have condensed chromatin. There are many mini-vacuoles present. V, vacuoles; arrows, nuclei. Bars, 5m.

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68

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69 Figure 27 Lower Integument-derived PC Cells. Starting at approximately 9 DAP cells in the lowest portion of the PC display abundant ER in the form of concentric rings or layered lamina. This is characteristic of this cell layer up through at least 11 DAP. V, vacuoles; ER, endoplasmic reticulum; N, nucleus. Bars, 2m.

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70 Figure 28 Nuclear body formation in the integument-derived portion of the PC cells. A) TUNEL assay showing nuclear bodies (boxed) at the cell periphery. Arrow shows one of the highly condensed TUNEL positive nuclei. (Micrograph by Shayna Sutherland). (B) TEM micrograph showing the same type of nuclear morphological pattern seen in A. The nuclei have completely condensed and have started breaking off into nuclear bodies. Note the nuclear envelope is still present and intact. Magnification; A, 40x; B, bar 0.5m.

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71 Figure 29 Nuclear morphological changes in integument derived portion of the PC. Nuclear morphological changes in the integument-portion of the PC resemble apoptotic-like morphology. The micrographs of each progressive stage of each changed nucleus are not from consecutive developmental stages as the PCD taking place in one cell is independent of PCD occurring in all other cells and the rate of progression between cells will vary. However, this series of nuclear changes is progressive from when a cell starts showing morphological changes through the end of its PCD process. (A) Normal appearing nucleus. The DNA has not started showing any abnormal chromatin condensation. (B) A nucleus showing first stages of chromatin condensation. (C) Highly shrunken nucleus with completely condensed chromatin. (D) Nuclear body-remnants of the nucleus filled with condensed chromatin (arrows). N, nucleus; m, mitochondria. Bars: A, 5m; B, 1.8m; C, 1.2m; D, 1.8m.

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72 Figure 30 Ca2+ Trapping: Nucellus-derived PC. Free Ca2+ was localized in an attempt to define a flux and organelle interaction of this ion during PCD of the cell. (A) At 5 DAP Ca2+ is sparse and is associated, primarily, with membranous structures and the nucleus but is not seen in the cytoplasm or mitochondria. (B) At 8 DAP Ca2+ is abundant in the cell corpse but does not appear to be associated with the degenerated cellular remains. (C) By 11 DAP Ca2+ has disappeared from the cell corpse and is only lightly associated with degenerated cellular remains. N, nucleus; m, mitochondria; DC, degenerated cytoplasmic contents. Bars: A-D, 0.5m; E-F, 0.8m.

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Figure 31 Ca2+ Trapping in the integument-derived PC. Free Ca2+ was localized in an attempt to define a flux and organelle interaction of this ion during PCD of the cell. (A) At 5DAP Ca2+ is very low in the cytosol but is associated with membranes of the ER and vacuole. In addition, Ca2+ is peppered throughout the mitochondria and is very light in the nucleus. (B) By 8 DAP Ca2+ is low but present in the cytolsol. It is also seen in some of the ER under the nucleus (bracket). However, other rough ER is still present but no Ca2+ is seen within it (arrows). (C) By 11 DAP Ca2+ is extremely low in all places in the cell. The cell is in an extraordinary chaos and no nucleus or organelles are left. V, vacuoles; m, mitochondria; N, nucleus; n, nucleolus. Bars, 5m.

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Figure 32 Ca2+ trapping in the normal appearing, lower integument PC. Free Ca2+ was localized in an attempt to define a flux and organelle interaction of this ion during PCD of the cell. (A) A normal appearing cell shows low Ca2+ in the cytosol and mitochondria but higher concentrations in the nucleus and very high concentrations sequestered in the vacuole. (Inset: enlarged image of mitochondria). (B) Ca2+ localizes to the ER lumen and nucleus. (C) Ca2+ has collected in the mitochondrial matrices and vacuoles. However, Ca2+ is in low concentrations in the cytosol. (D) A cell showing a nucleus with mostly condensed chromatin. The nucleus still shows moderate amounts of Ca2+ and the mitochondria have small amounts of Ca2+ in their matrices; one displays great swelling at this stage. (E) Negative control. N, nucleus; V, vacuole; m, mitochondria; ER, endoplasmic reticulum. Bars: A, D, 0.8m; B, C, E, 0.5m.

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Figure 33 In-gel nuclease activity assays for Ca2+, Mg2+ and Zn2+-dependent nucleases and pH controls. PC tissue was isolated from the rest of the caryopsis and proteins from thr PC were extracted for nuclease activity. Whether tested together or separately, the Ca2+, Mg2+ -dependent nucleases resulted in the same banding pattern. (A) The Ca2+-dependent assay shows greatest nuclease activity with a 36.5kD nuclease (arrow) first appearing at and peeking in activity at 8 DAP. (B) In the Zn2+-dependent assay, nuclease activity is greatest at 8 DAP The same pattern of banding is seen as in the Ca2+dependent nuclease assay but unlike the Ca2+-dependent nuclease activity assay a fourth nuclease is present at approximately the 35kD mark (red arrow). However, this nuclease is seen starting at 6 DAP, persists through12 DAP and is still seen faintly at 28 DAP. (C) Most of the Ca2+-dependent nuclease activity was eliminated when incubated at an acidic pH of 5.5, that which is required for Zn2+-dependent nuclease activation. Ca2+-dependent nuclease activity requires a neutral pH of 6.8. (D) Most of the Zn2+-dependent nuclease activity was eliminated when incubated at a neutral pH of 6.8, that which is required for Ca2+-dependent nuclease activation. Zn2+-dependent nuclease activity requires an acidic pH of 5.5. M, size marker.

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Figure 34 In-gel nuclease activity assays with EDTA and EGTA and no metals. PC tissue was isolated from the rest of the caryopsis and proteins from the PC were extracted for nuclease activity. (A) EDTA and EGTA effectively reduced or eliminated nuclease activity. Only the smallest nuclease showed any activity. However when the Zn2+ gel (not shown) was incubated with these chelating agents there was no inhibitory effect. This may have been due to the acidic conditions required for Zn2+ activation limiting the chelating abilities of the EDTA and EGTA. (B and C) When metals were eliminated from the incubation medium the same typical pattern for each of the nucleases still appeared. This is, presumably,due to metals inherent in the tissue. M, size marker.

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81 Figure 35 Genomic DNA isolated from the PC region. Genomic DNA was isolated from PC tissues of 8, 10, 12, 16, 20, and 24 DAP caryopses and electrophoresed on a two per-cent gel to test for internucleosomal DNA laddering. No nucleosomal DNA laddering was detected in the PC. LML: Low mass ladder.

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CHAPTER 4 DISCUSSION PCD in the PC Region Plants undergo PCD as a normal developmental process in a wide variety of cell types and exhibit PCD in a variety of different ways, morphologically. Examples include aerenchyma formation, tracheary element differetiation, compound or hole developing leaves, and senescence (Jones and Dangl, 1996; Gietl and Schmid, 2001; Obara et al., 2001). PCD occurs within the different parts of a seed as a part of normal growth and development (Domingues et al., 2001; Schmid et al., 1999; Wojciechowska and Olszewska, 2003). Within the maize caryopsis, PCD has been reported in cells of the nucellus, suspensor, scutellum, and endosperm (Giuliani et al., 2002; Young and Gallie, 2000; Young et al., 1997). All these processes are highly controlled and critically timed. However, within a temporal context the first of these processes to be observed is PCD in the PC region. The PC region of the maize caryopsis is the only port of entry through which water and nutrients pass from the maternal sporophytic tissue to the filial tissue. While the PC is, itself, a maternal tissue, it goes through a fertilization dependent programmed cell death (PCD) as part of a normal developmental regimen (Kladnik et al., unpublished). As detailed in the results section there are two distinct zones in the PC that undergo two distinct PCD processes. The PC in maize is comprised of only nucellar and integument-derived tissues as maize seeds do not possess a funiculus. The first of these 82

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83 two zones is that portion of the PC that is derived from the nucellus epidermis. The nucellar-PC zone undergoes an extremely rapid death starting at approximately 6 DAP and ending at about 8 DAP. This process does not resemble an apoptotic-like cell death. These cells do not test TUNEL positive at any point tested indicating a lack of detectable DNA cleavage nor do the nuclei ever appear to contain condensed chromatin. In fact, no intermediate stages of cellular degradation were seen between the completely normal appearing cells and those cells with degraded cytoplasmic remains with the exception of an occasional plasmolysed cell. Since these cells start out with a very large vacuole at the time that they appear intact, but have no trace of a vacuole when they are plasmolysed or degraded, perhaps it is a death mediated by vacuolar rupture and autolysis similar to that of tracheary elements (TE) (Obara et al., 2001). It is through this process in TE differentiation that proteolytic and digestive enzymes are released into the cytoplasm (Yu et al., 2002). Obara et al. (2001) reported a complete degradation of the nucleus within 15 minutes and a complete degradation of the cytoplasm within two hours after the rupture of the central vacuole. The DNA degradation appeared to start in the center of the nucleus while the condensed chromatin left at the periphery was digested last leaving behind only the nuclear envelope. While TUNEL assays showed positive results in the nuclei of these cells there was no oligonucleosomal ladder detected on a gel. The second zone of cells of the PC is that portion which is derived from the inner integument. As with the nucellar-PC, PCD in the integument-PC is

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84 detected starting at approximately 6 DAP, peaks at about 10 DAP, then subsides after 12 DAP. This PCD process morphologically resembles apoptosis in animal cells (Kerr et al., 1995) in that both undergo extensive chromatin condensation delineated by sharp clean edges and retention of the nucleolus, nuclear shrinkage, nuclear body formation, cytoplasmic condensation and retain all organelles and membranes until these are the only cellular components left. The cell then completely degenerates and the cellular contents become mostly absorbed. While these similarities are shared, the exact pattern of chromatin condensation in the PC nuclei is a little different than that in animals in that it does not appear to amass at the nuclear periphery. PCD in the PC May Be Tied to Growth and Development of the Filial Tissues Since the maternal tissue of the pedicel and PC is the only region through which nutrients can pass to the developing filial tissues of the endosperm and embryo it is apparent these three tissues are intimately linked, physiologically. Schel et al. (1984) examined early developmental stages of endosperm and embryo interaction in maize. They noted that the endosperm precedes the embryo in development and that there is a 4X increase in size of the endosperm relative to the embryo from 4 through 8 DAP, creating a high sink demand. From 8 DAP through 14 DAP the endosperm suddenly increases its relative growth rate; within this period at approximately 9 DAP, the embryo starts a rapid increase in growth rate and size thus further increasing the sink demand. After 14 DAP the growth rates of each, the endosperm and the embryo, starts to slow; the endosperms rate of growth slows prior to that of the embryos (Schel et al.,

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85 1984). It has been noted in past anatomical studies of the developing maize caryopsis that as the tissues develop, the nucellus tissue is destroyed by the growing endosperm, which is in turn destroyed by the growing embryo (Weatherwax, 1930). Weatherwax (1930) points out that the bordering cells at the interface of these tissues display a regular pattern in that the cells of the tissues being destroyed, the nucellus, for example, plasmolyze before destruction while the growing tissue border cells, the endosperm, for example, remain turgid. While it was not known what caused this phenomenon it appeared that the majority of nutrition was still delivered via the PC (Weatherwax, 1930). However, it must be pointed out that recent findings have shown that these tissues undergo PCD as the caryopsis develops and that it is not, simply, the mechanical pressure of the growing tissues that is destroying the extant tissues, i.e. the destruction of the nucellus as the endosperm grows (Linnestad et al., 1998). The osmoticum of the tissues in the caryopsis has been shown to be a critical factor for normal maize seed development (Zinselmeier et al., 1999). For example, it has been shown that there must be a critical level of sucrose available to the developing tissues in the seed immediately after pollination or the development of the embryo ceases and it is aborted. This was accompanied by the observation that when the maize seeds were kept at very low water potentials at the time of fertilization the starch reserves in the fruit wall were depleted and were most likely an attempt, by the seed, to maintain proper sugar levels in order to maintain adequate water levels for development (Zinselmeier et al., 1999). In the opinion of the author of this present study, perhaps one explanation for the

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86 ability of the growing border tissues to maintain turgidity is that they maintain a higher water potential than their surrounding tissues. For example, the nucellus is not a starchy tissue nor is it a physiological sink, as is the endosperm and embryo, and the cells contain very large central vacuoles. Perhaps the nucellus acts more as a water reserve for the growing endosperm than a source of nutrition. Sink Demand and Assimilate Flow The growing endosperm and embryo require sugars and other nutrients that are delivered to the pedicel via the phloem (Weatherwax, 1930; Felker and Shannon, 1980). The vasculature terminates at the base of the pedicel and the assimilates must be unloaded from the phloem and pass through the pedicel parenchyma cells, PC cells, the basal endosperm transfer cells (BETC), then into the endosperm and growing embryo. However, it is still not clear exactly what path the assimilates flow through once they have unloaded from the phloem. Felker and Shannon (1980) proposed a path of flow for the sugars based on microautoradiographs of the pedicel after the plant was exposed to 14CO2 and was allowed to assimilate the radioactive carbon into sugars. They concluded the assimilates pass through the pedicel cells symplastically from the phloem through the PC until they reach the border between the PC and BETC cells at which point flow must turn apoplastic due to a complete lack of plasmodesmatal connections (Felker and Shannon,1980) Felker and Shannon (1980) point out a division in radiolabeled sucrose accumulation between the central pedicel parenchyma, PC and the endosperm within the same tissue sample, there being a noticeable accumulation of radiolabeled sucrose in the pedicel parenchyma

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87 and endosperm but a lack of accumulation through the PC. However, they stated that there are appreciable limitations in this method. The limitations discussed mention that there is no way to determine the presence of radiolabeled sucrose in the free space or apoplast. Therefore, this study could not conclusively determine where sugars could not be found but did shed light on where the sugars could be found. Additionally this study focused on relatively mature caryopsiss of 22 DAP which rules out the possible changes in the path assimilates might take during varying developmental stages, especially the earliest most stages. Based on the morphological changes seen in the PC and pedicel during the this present study, coupled with data from the aforementioned studies, it would seem that the path of assimilate flow from the phloem to the endosperm must vary depending on the stage being examined, i.e. symplastic at early stages (0-6 DAP) versus apoplastic at more mature stages (7 DAP and later). This may also hold true for certain conclusions that can be drawn about the physiological processes of the growing seed that may be contributing to the initiation of PCD in the PC. Two Separate, Temporal Waves of PCD Occur in the PC and May Be Linked to the Sink Demand of the Growing Filial Tissues The endosperm in maize begins cellularization at approximately 4 DAP during the R1 stage, 0 to 9 DAP, (Esau 1977; Ritchie, 1993). The first wave of PCD in the PC follows shortly after the beginning of cellularization of the endosperm at approximately 5 to 6 DAP. The second wave of PCD in the PC begins during the R3 or milk stage, 18 to 22 DAP, when endosperm cellular

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88 division has completed and the cells are maximizing starch filling (Ritchie,1993). Therefore, there are two physiological processes that may be contributing to the PCD occurring in the PC cells during the two waves of cell death. The first physiological contributor during the first wave, starting after 5 DAP, may be an osmotically induced process due to the strong sink of the developing endosperm and embryo. Schel et al. (1984) concluded growth rate is slow through approximately 4 DAP; therefore it can be deduced that nutrient demand is at a minimum. Since there are abundant plasmodesmata in all cells of the pedicel at this stage, an average of 7.1 per cell per section, in the nucellus-derived PC cells, and all cells are normal in appearance, assimilates most likely travel symplastically throughout the entire pedicel and PC region. Addtionally, since there is not yet a symplastically discontinuous border between the PC and the endosperm, assimilates most likely flow symplastically into the growing endosperm. Schel et al. (1984) also concluded that the endosperm starts an extremely rapid rate of growth from 5 through 8 DAP. Thus it can be deduced that assimilate demand increases. This coincides with the rapid PCD in the nucellus-derived region of the PC at the end of which the cells have almost entirely emptied and the identifiable plasmodesmata are staring to decrease in number. Towards the latter end of this stage the endosperm has grown large enough in size to have formed a loosely packed, symplastically discontinuous, multi-cell walled border with the PC. In addition, during this period the integument-derived region of the PC is starting to display TUNEL positive nuclei (Kladnik et al., unpublished). With a reduction in the number of plasmodesmata

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89 and only hollow corpses of PC cells left in the nucellus-derived PC it can be deduced that assimilates flow symplastically from the phloem through the integument-PC. Upon encountering the nucellus-PC, the assimilates would encounter a disruption in the symplast and change to an apoplastic means of flow. The flow may still travel through the cell corpses in the nucellus-derived PC. Since the apoplast is defined as the pathway for water and solute transport that lies outside the plasma membrane, including inter cellular spaces and xylem transport (Buchanan et al., 2000), the flow of assimilates through the cell corpses would still be considered apoplastic since there are no plasma membranes or cytoplasmic connections. The disruption and conversion of the flow from the symplast to the apoplast might cause a transient backup of hyper-osmotic fluid in the integument-derived PC cells immediately subtending the nucellus-PC cells creating an osmotic shock. Studies examining cell wall invertase (INCW2), a cell wall enzyme that irreversibly cleaves sucrose to glucose and fructose, showed specific localization to the BETC cells of maize caryopses and more specifically to the cell wall ingrowths of the BETC cells (Cheng et al., 1996). The cell wall ingrowths in the BETC cells start developing at approximately 7 DAP and are mature by 12 DAP (data not shown). It is immediately prior to the development of the cell wall ingrowths that the PCD process in the PC is initiated and not until the wall ingrowths, and thus the INCW2 expression, are mature that the PCD process ceases. In other words, it is concomitant with, and seemingly not until the sucrose can be readily passed into the endosperm that the PCD process ends. This shows a correlation between the sink strength of the endosperm, the

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90 sucrose passing through the PC and the possibility of a transient backup of assimilates. The invertase deficient mutant in maize, mn1-1, is incapable of cleaving sucrose in the BETC and shows anatomical features of an underdeveloped endosperm leading to the development of a large gap separating the pedicel from the endosperm (Cheng et al., 1996). Cheng et al. (1996) reference a previous study in which a higher concentration of sucrose was found to exist in the pedicel of the mn1-1 mutant. Because the mn1-1 endosperm is not applying mechanical pressure on the PC and the PC still shows PCD (data not shown) as does the wild type caryopsis it is reasonable to speculate that PCD in the PC region of maize is osmitcally influenced. Schel et al. (1984) noted that both the endosperm and embryo increase rapidly in size from approximately 9 to 14 DAP This coincides with the highest peak of TUNEL positive nuclei, i.e. about 10 DAP, in the integument-derived region of the PC (Kladnik et al., unpublished). Schel et al. (1984) also show a decrease in the growth rate at 14 DAP which is coincidental with the last signs of any TUNEL activity seen (Kladnik et al., unpublished). At this point in development the BETC cells have developed extensive cell wall in-growths, the PC has formed a symplastically discontinuous, compacted, multi-cell walled barrier from crushed nucellar-PC cells with the BETC, and the number of identifiable plasmodesmata have decreased appreciably in the nucellar-PC cells from an average of 7.1 per cell per section to 2.4. The second possible contributing factor to the PCD in these cells may play a role during both waves of PCD but may be the primary cause of PCD during

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91 the second wave. This factor is the mechanical pressure of the enlarging endosperm and growing embryo. For example, PCD in the PC region may be influenced, primarily, by osmotic changes but, simultaneously, the endosperm and the embryo are physically growing larger at a rapid rate. It is this rapid change in size that may be the second contributing factor to the PCD taking place in the PC. The earlier changes regarding the flow of assimilates from the symplast to the apoplast while inner cellular contents are eliminated from the nucellus-PC cells may be serve to alleviate turgor pressure in this region. This may allow these cells to flatten more readily or collapse altogether in order to create more room for the growing endosperm and embryo. Perhaps by going through these processes the PC cells help complete the formation of the protective closing layer. The cells located at the most basal region of the PC in the integument PC, those most proximal to the pedicel parenchyma cells next to the phloem, exhibit abundant ER in a highly organized fashion forming concentric rings or lamellae from approximately 9 to 11 DAP. This is strikingly similar to the ER morphology seen in the developing endosperm cells at the base of the embryo from 5 through 9 DAP (Schel et al.,1984). This may indicate highly active ER, perhaps in response to the stresses the rest of the PC is incurring or as a causal agent of those stresses. The second wave of PCD occurs during the milk stage of development, 18-22 DAP. It is at this stage that the endosperm is maximizing starch reserves and tightly packing these starch grains in the endosperm cells (Ritchie et al.,1993).

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92 This last bit of starch grain packing continues to increase the size of the endosperm and crushes the PC layer once again. The only PC cells to display apoptotic-like morphology at this stage, for example condensed nuclei and nuclear bodies, are those in the most compressed region of the PC. The Role of Calcium in PC PCD To help elucidate the role of Ca2+ in PC PCD, Ca2+ localization was performed to determine the flux and location of free Ca2+ in PC cells. While the Ca2+ localization was not as detailed for every sequential step of the PCD process as would have been needed for more definitive conclusions, it did show general fluxes of free intracellular calcium [Ca2+]i. and where it occurred. While increased Ca2+ uptake has been shown to occur in animal mitochondria, increased Ca2+ uptake in plant mitochondria is still questionable, but has been demonstrated for pea and artichoke (Curtis and Wolpert, 2002). Increased Ca2+ uptake, therefore, may depend on the type of PCD occurring in the cell. In order to more clearly discuss the involvement of [Ca2+]i during the PCD process in the PC it is necessary to briefly review some of the results for each of the stages of the cells conditions. The results of the [Ca2+]i localization in the PC cells at 5 DAP indicated that there was very little [Ca2+]i. anywhere in the cells in either the nucellus or integument portion of the PC. The only [Ca2+]i. that was seen at this stage was associated with the membranes of the endoplasmic reticulum (ER) and central vacuole and was peppered lightly and evenly throughout the mitochondria. This is not surprising for this or earlier stages of development, since there appear to be minimal physiological demands on the PC

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93 at this point as seen via morphological and histological evidence of this study, as well as what was previously discussed with regard to assimilate demands. The morphologically normal appearing PC cells, seen only in the lower integumentary-PC of the 8 through 11 DAP caryopses, were examined. At this point these cells are not showing morphological signs of stress. These cells are appreciably more mature and differentiated than seen in the 5 DAP and therefore would have accumulated necessary materials, including Ca2+, to prepare for or carry out physiological processes such as PCD. In the 8 to 11 DAP caryopsis PC cells showing moderate to advanced stages of PCD where the nucleus is mostly or completely condensed were examined next. Considering the advanced degradation of the cellular contents at this stage and the lack of central vacuoles, it would appear that the Ca2+ has been released from the ER and vacuolar reserves and has already participated in the PCD process. As discussed previously, this developmental stage coincides with the rapid increase in size in both the endosperm and embryo compounding the demand for assimilates and adding pressure to the PC. The last set of cells to consider is the nucellus-derived PC. Since no intermediate stage of PCD was able to be found between the normal appearing and dead cells it is only possible to discuss the localization of Ca2+ post mortem. Since these cells have already completed the cell death program it is most likely that the Ca2+ seen in them is residual and is moving out of the cells. In addition, since the cells most proximal to the BETC border are the first to suffer PCD it

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94 would be reasonable that these cells would have the lowest [Ca2+]i relative to the integument PC at the same stages. The Role of Endonucleases in the PC In-gel nuclease assays were performed to examine what nucleases may be present or possibly involved in nuclear DNA degradation. Zinc (Zn2+), Calcium (Ca2+), and Magnesium (Mg2+), specific nucleases were examined for their presence in the PC. The banding pattern for the Ca2+ and Mg2+-dependent endonucleases was the same as that of the Young et al. (1997) study. Three bands were seen at 33.5, 36.0 and 38.5 kDa. The peak of Ca2+ and/or Mg2 dependent endonuclease activity was seen in the 36.0 kDa band at 8 DAP and slowly declined through 12 DAP. The peak of activity precedes the height of TUNEL activity seen in the PC; however Young et al. (1997) also performed spectrophotometric endonuclease activity assays on whole caryopsis and embryo-free endosperm protein extracts which showed the highest peaks of activity between 8 and 12 DAP but with the highest activity extrapolated at 10 DAP. This method is a quantitative method as opposed to the in-gel assays which are qualitative and is therefore, a more sensitive and accurate means of measure of activity. As pointed out, Young et al. (1997) used the entire caryopsis starting at 8 DAP whereas only the lower 1/3 of the caryopsis was examined in this study. Some of the nuclease activity seen in the Young et al. (1997) study may be attributable to some nucellar PCD but the endosperm occupies most of the ovular space by 10 DAP. It, therefore, is likely that PC nuclease activity may also be contributing to the peak of activity seen at this stage in the Young et al. (1997) study especially considering the fact that, in the

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95 present study, relatively little or no nucellar tissue was involved in the gel nuclease activity assays and the same nuclease pattern was seen. Zinc (Zn2+ )-dependent in-gel nuclease assays were also performed. The banding pattern was the same as for the Ca2+ gels except there was one additional, unique band at approximately 35kDa. This band is present at 6 DAP and may have a slightly higher peak at 10 DAP, but there was a relatively high amount of activity maintained through12 DAP. At 28 DAP the activity was significantly lower but not absent. This corresponds to the TUNEL activity seen in the PC at these stages. The same gels when incubated without added metals but maintaining proper pH showed the same pattern of banding. This is probably indicative of these metals being inherent in the tissue. EGTA and EDTA were added to the incubation medium of these gels to test whether or not these chelators would effectively inactivate the nucleases. While successful for the Ca2+ and Mg2+ gels, the EDTA and EGTA failed to inactivate the nucleases in the Zn2+ gels. Possible reasons for this may be that the acidic conditions of the incubation medium protonated the chelating ends of the EDTA and EGTA, rendering them useless as chelators. When the pH of the incubation medium for each gel was swapped for that of the other, complete nuclease inhibition was achieved. This lends credence to the fact these nucleases belong to the classes tested, i.e. Ca2+-dependent or Zn2+-dependent. Regardless of the results seen in the gel assays they do not reveal any direct involvement of these nucleases in the PC PCD process. Attempts to detect DNA ladders failed when DNA extracted from isolated PC tissue was

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96 examined. Reasons for this may be that there simply were not enough cells occurring at the proper stage of PCD to extract fragmented DNA from or that the fragments were too large to be seen via standard electrophoresis or that DNA was not being cleaved. Considering TEM micrographs of nuclei at late stage PCD PC cells show fragmented nuclei in the form of nuclear bodies and positive TUNEL reactions occur it is probable that there is DNA cleavage occurring. Since no internucleosomal DNA ladders were detected via gel electrophoresis it is possible that the DNA is being cleaved into very large fragments that require other means of detection such as pulse field electrophoresis or a lower percentage agarose gel electrophoresis. Force synchronization of PCD, as has been done for cell cultures or pathogen-mediated hypersensitive response systems (HR), is not possible for the PC cells of the caryopsis as it is a developmental process in-planta (Ito and Fukuda, 2002; Mittler and Lam, 1995). If, indeed, the DNA is being cleaved into internucleosomal ladders, and it is not possible to extract enough DNA from the tissue to see it on a gel, more sensitive detection methods such as ligation mediated-polymerase chain reaction (LM-PCR) would need to be employed to find it. PCD in the endosperm PCD has been reported in the endosperm of maize as well (Young et al., 1997; Young and Gallie, 2000; Sugiyama et al., 2000). While both the starchy wild-type caryopsis (Su) and starch deficient mutant shrunken2 (sh2) were examined only the wild-type results will be considered, here, for illustrative purposes. In these studies it was observed that PCD occurred in two distinct phases following a spike in ethylene production (Young et al., 1997). PCD in the

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97 endosperm starts in the central crown region then moves to the outer regions of the crown and then down toward the base of the caryopsis as was illustrated using Evans Blue vital stain (Young et al., 1997). The investigators tested for internucleosomal DNA laddering and found that it was not apparent until approximately 28 DAP and became most distinct between 32 and 44 DAP. Also investigated, as discussed earlier, was endonuclease activity via in-gel assays and spectrophotometry. There was an initial peak of activity between 8 and 12 DAP followed by a decrease in activity occurring between 20 to 24 DAP. At 24 DAP the activity starts to increase, peaking at 36 DAP then tapers off rapidly through 44 DAP. For this study, nuclear morphology in the endosperm was examined on both a light microscopy level (LM) and transmission electron microscopy level (TEM). Both TUNEL assays and DAPI, a fluorescent DNA stain, were used to examine DNA integrity and nuclear morphological changes from 12 to 24 DAP, respectively. There was a gradual shift in morphology from 12 to 16 DAP in the central-crown region. Nuclei appeared diffuse at 12 DAP but started to take on a more rough texture at 14 DAP indicating that chromatin condensation was starting to take place. By 16 DAP the nuclei were large and ring-like in appearance owing their appearance to complete chromatin condensation that gathered at the nuclear periphery. By 20 DAP the nuclei had collapsed into a small, dense oblong shape. Up to this point TUNEL assays were negative. However, at 24 DAP the nuclei had condensed further, taken on a more oblong appearance and appeared TUNEL positive. This is consistent with the nuclease

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98 activity profile by Young et al. (1997). Beyond 24 DAP the starch density was such that its autofluorescence interfered too greatly to effectively see the fluorescence of the nuclei with either the DAPI or TUNEL. Therefore propidium iodide (PI) DNA stain and confocal microscopy was employed. The nuclei appeared extremely small and condensed. This coincided with the point at which internucleosomal DNA laddering was first observed by Young et al. (1997). TEM micrographs of each of these stages confirmed the LM data. The only additional information gained was that mitochondria were present through 16 DAP; but it was not clear what condition the mitochondria were in at later stages as they became difficult to identify. It could be possible that their morphology was changing in response to, or as a consequence of, the PCD taking place. At 14 DAP the rough ER was heavily involved in protein body formation. The latest developmental stage examined by TEM was 25 DAP. While no recognizable organelles could be seen upon close examination it appeared that cytological materials were still present. For example, at high magnification membranous, lipid appearing materials were abundant in clumped, small spherical structures, DNA still appeared to be in condensed but disordered chromatin masses and there were numerous protein bodies abound. This would concur with reports that, although the endosperm cells are dead at this stage the cytoplasmic contents still remain, however disassembled (Sugiyama et al., 2000; Buchanan et al., 2000). Starch grains were large and very densely packed as well. It would be expected that the materials remain in the endosperm cells so as to be available to the germinating embryo.

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99 In addition to the morphological changes seen in the nuclei the actual positioning of the nuclei within the cells also changed. At 12 DAP the nuclei are centrally located but by 18 to 20 DAP the nuclei start shifting to edge of the cell. By 28 DAP each nucleus is tightly appressed to the cells edge. The starch grains displacing the nuclei as they become more numerous in each cell most probably can explain this; however, this was not seen in the cells at the periphery of the crown region. Morphologically speaking, PCD in endosperm cells does not appear apoptotic-like. The pattern of chromatin condensation is neither like that seen in the PC during this investigation or in true apoptosis in animal cells (Kerr et al., 1995). Physiologically, however, like apoptosis, it does result in internucleosomal laddering (Young et al., 1997).

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CHAPTER 5 CONCLUSION Because there are three distinct morphological changes occurring in each of three different cell types examined: the nucellus derived-PC, the integument derived-PC, and the endosperm, it is possible that there are three different and unique types of PCD that occur within these tissues. Due to the rapid nature of the death of the nucellus derived-PC cells these cells may undergo a cell death process similar to that of maturing xylem tracheids. The cell death process of xylem tracheids completely degrades the nucleus within an average of 15 minutes after vacuolar rupture and empties the contents of the cell within two hours. The nucellus derived-PC cells appear normal up through 5 DAP but the cell death process that occurs after this stage is so rapid that the only intermediate stage of cell death that was found was that of late stage plasmolysis at 7 and 8 DAP. The integument derived -PC cells present an entirely different morphology than that of the nucellus derived -PC cells and the changes seen here are much slower. The integument derived -PC cells follow apoptotic morphological changes very similar to those seen in animals such as condensed chromatin and cytoplasm, retention of organelles and membranes until late stages of the cell death process and formation of nuclear bodies similar to apoptotic bodies. Therefore the integument derived-PC cells are most likely undergoing an apoptotic-type cell death. 100

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101 The nuclei of the central crown region of the endosperm cells undergo a series of morphological changes as the endosperm develops unlike those seen in the PC. The nuclei start out spherical and internally diffuse at 12 DAP, expand in size and take on a beaded appearance at the nuclear envelope due to chromatin condensation at 16 DAP then collapse and condense tightly by 25 DAP. Additionally, the nuclei shift to the periphery of the cell between 16 and 25 DAP but persist through the final stages of development. By 25 DAP the nuclei appear to be little more than disorganized, amorphous blobs of nuclear materials by TEM examination. Young et al. (1997) showed that maize endosperm PCD begins in the central crown region of the kernel at 16 DAP. The nuclear morphological changes found in this study occur concomitantly with endosperm cell death in maize as illustrated by Young et al. (1997). The morphological changes that occur in the nuclei of the endosperm cells is unlike those seen in the PC and may indicate a third distinct form of cell death. Conclusions concerning the Ca2+ localization cannot be easily made regarding the role of Ca2+ in the physiological processes of PCD in the PC. This is because the detection of Ca2+ was strictly morphological and only determined when and where it could be found. However, it is clear that after 5 DAP Ca2+ is stored in the ER and central vacuoles of cells and collects in the mitochondria prior to any morphological changes they incur. Therefore, it is not clear if mitochondria participate in the cell death process in the PC since mitochondria show morphological changes, i.e. swelling, only at late stages of cell death.

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102 However, there is a correlation between the Ca2+ -dependent nuclease assays, Ca2+ localizations in the PC tissues and the peak of TUNEL activity. The highest levels of Ca2+-dependent nuclease activities appear at 8 and 10 DAP with the peak occurring at 8 DAP. Calcium is localized to the nuclei and mitochondria of mid-stage dying cells. At later stages of cell death nuclei display completely condensed chromatin and/or condensed, fragmented nuclei which is consistent with the stage at which nucleases would be most active. The greatest frequency of cells appearing at mid and late stages of PCD, morphologically, is between 8 and 10 DAP. This is also consistent with the developmental stages at which the TUNEL assay shows the greatest number of TUNEL positive nuclei. However, this correlation is circumstantial and would require further experimentation to determine whether or not it is valid and conclusive. Experiments that would help to resolve this issue might include such experiments as flow cytometric Ca2+ localization techniques coupled with mitochondrial membrane potential determination. In addition, establishing a cell-free system in which nuclei isolated from maize cell culture could be mixed with cell extracts or simply the isolated mitochondria from the dying tissues in question would most likely show definitive results. These techniques have been used effectively to show tight correlation between mitochondria and their role in PCD (Korthout et al., 2000).

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104 Earnshaw, W.C. (1995) Apoptosis: Lessons from in vitro systems. Trends Cell Biol. 5, 217-220. Eastman, A. (1995) Assays for DNA Fragmentation, endonucleases, and intracellular pH and Ca2+ associated with apoptosis. Methods cell biol. 46, 41-55. Esau, K. (1977) Anatomy of seed plants: 2nd Ed. p 415 Malloy Lithographing Inc. New York. Fath, A., Bethke, P., Lonsdale, J., Meza-Romero, R., Jones, R. (2000) Programmed cell death in cereal aleurone. Plant Mol. Biol. 44, 255-266. Felker, F.C., Sannon, J.-C. (1980) Movement of 14C-labeled assimilates into kernels of Zea mays L. Plant Physiol. 65, 864-870. Gietl, C., Schmid, M. (2001) Ricinosomes: an organelle for developmentally regulated programmed cell death in senescing plant tissues. Naturwissenschaften 88, 49-58. Giuliani, C., Consonni, G., Giuseppe, G., Colombo, M., Dolfini, S. (2002) Programmed cell death during embryogenesis in maize. Ann. Bot. 90, 287-292. Gromova, I.I., Nielsen, O.F., Razkin, S.V. (1995) Long-range fragmentation of the eukaryotic genome by exogenous and endogenous nucleases proceeds in a specific fashion via preferential DNA cleavage at matrix attachment sites. J. Biol. Chem. 270, 18685-18690. Ito, J., Fukuda, H. (2002) ZEN1 is a key enzyme in the degradation of nuclear DNA during programmed cell death of tracheary elements. Plant Cell 14, 3201-3211. Johann, H. (1935) Histology of the caryopsis of yellow dent corn, with reference to resistance and susceptibility to the kernel rots. J. Agric. Res. 10, 855-883. Jones A.-M., Dangl, J.L. (1996) Logjam at the river Styx: Programmed cell death in plants. Trends Plant Sci. 1, 114-119. Keisselbach, T.A., Walker, E.A. (1952) Structure of certain specialized tissues in the kernel of corn. Am. J. Bot. 39, 561-569. Kerr, J.F.R., Gob, G.C., Winterford, C.M., Harmon, B.V. (1995) Anatomical methods in cell death. Methods cell biol. 46, 1-47. Korthout, H., Berecki, G., Bruin, W., van Duijn, B., Wang, M. (2000) The presence of subcellular localization of caspase 3-like proteinases in plant cells. FEBS Lett. 475, 139-144.

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BIOGRAPHICAL SKETCH Karen Chamusco was the second daughter born to Drs. Frederick W. and Cleo C. Courington in 1968. Karen spent the majority of her life growing up in a U.S. Navy family which afforded her the opportunity to experience travel abroad including Hong Kong, Singapore, Thailand, the Philippines, Guam, Hawaii, and many states within the U.S.A. Karen graduated high school in Virginia in 1987, and then moved with her family to Florida where she spent nine and one half years working at Walt Disney World and attending community colleges where she gained experiences in photography, fine arts and was awarded an Associate of Arts degree from Valencia Community College. In 1994 Karen moved to Gainesville, Florida, where she enrolled at the University of Florida and Santa Fe Community College. Karen was awarded a Bachelor of Science degree in 1999 from the University of Florida, College of Agriculture, with minors in chemistry and plant molecular and cellular biology. Karen married her husband, Larry Chamusco, in 2001 and in 2002 began her work toward a Master of Science degree under the direction of Dr. Prem S. Chourey in the plant molecular and cellular biology program at the University of Florida. 108


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MORPHOLOGICAL SURVEY AND CHARACTERIZATION OF PROGRAMMED
CELL DEATH IN THE PLACENTA-CHALAZA AND ENDOSPERM IN THE
DEVELOPING CARYOPSIS OF MAlZE













By

KAREN CHAMUSCO


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

U NIVE RS ITY OF FLORI DA


2004































Copyright 2004

by

Karen Chamusco
































This work is dedicated to my family and friends who have been tremendous
pillars of aid and support















ACKNOWLEDGMENTS

I would like to acknowledge and thank my chairman, Dr. Prem S. Chourey,

for his support and for giving me a great opportunity to explore research and

learn invaluable research techniques. I also would like to thank my committee,

Dr. Daryl Pring, Dr. Christine Chase, and Dr. Henry Aldrich, for their support and

valuable advice. Special thanks go to AleS Kladnik whose research and efforts

were indispensable in pointing me in the direction that allowed me to pursue my

master's degree. I also thank Maureen Petersen, Donna Williams, and Lorraine

McDowell for their complete technical support in electron microscopy and Tim

Vaught for his aid in helping me with confocal microscopy. Special support and

aid came from close friends whom I would like to give a special thanks to:

Melanie Cash, Shayna Sutherland, Joyce Merritt, Asha Brunings, Wayne Jurick,

Fabricio Rodrigues, and the late Richard Wheeler who always saw the sunny

side. Very special thanks go to Donna Perry, Gail Harris, and Lauretta Rahmes

in Plant Pathology and Melissa Webb and Brandy Burgess in the plant molecular

and cellular biology program for providing administrative help. Lastly, I'd like to

thank my husband, Larry Chamusco; parents, Fred and Cleo Courington; and

sister, Becky Lockridge who have been loving and supportive in countless ways.




















TABLE OF CONTENTS





ACKNOWLEDGMENTS ................. ...............iv........_ .....


LIST OF FIGURES ................. ...............vii........ .....


ABSTRACT ................. ...............ix........_ .....



CHAPTER


1 INTRODUCTION................ ............. 1


Programmed Cell Death................ ................ 1
Categories of PCD Described in Plants ......... ................. ................. 5
Physiologic and Molecular Factors Involved in PCD ........._.._... .........._..... 7
Comparing PCD in Plants and Animals................ ................ 9
The Role of Calcium in PCD .........._._........____........ ...........1
The Role of Endonucleases ....__ ......_____ .......___ ............1
Seed Development................ .............. 15


2 MATERIALS AND METHODS ............. ...... .__ .....___............2


Plant M materials ............. ...... ._ ............... 20....
Utrastuctural Studies ............. ......_ ............... 20..
Calcium Trapping ............. ...... ._ ............... 22....
Light M icroscopy ............. ...... ._ ............... 23....
Nuclease Activity Assays ............. ...... ._ ....._ _.............2
DNA Extraction................ .............. 27


3 RESULTS ............. ...... ._ ............... 29....


The Endosperm................ ............... 29
Light M icroscopy.......................... ......... 29
Transmission Electron Microscopy ............. ...... .__ ............... 30
The Placenta-Chalaza Region ............. ...... .__ .....___............3
Light M icroscopy................ ... ................... 31
PCD in the PC Region is Fertilization Dependent................ ............... 32
Transmission Electron Microscopy ............. ...... .__ ............... 33
Calcium Trapping................ ............... 37











In-Gel Nuclease Activity Assay ....._._.__ ......._.. ... ...._._......... 39
Genomic DNA Extraction ........._...... .....___......... ............4


4 DISCUSSION............... ............... 82


PCD in the PC Region .........._..... ... ....... .. .... ... ..... ..........8
PCD in the PC May Be Tied to Growth and Development of the Filial
Tissues ........._.... .. ..... ... ................ 84....
Sink Demand and Assimilate Flow.................... ....... ................. 86
Two Separate, Temporal Waves of PCD Occur in the PC and May Be
Linked to the Sink Demand of the Growing Filial Tissues............._._... ..... 87
The Role of Calcium in PC PCD ........._._... ........ .......___..........9
The Role of Endonucleases in the PC ............. _.....___ .......... .... 94
PCD in the endosperm ............. .....__ ............... 96..


5 CONCLUSION ............. ...... ._ ............... 100...


LIST OF REFERENCES ............. .....__ ............... 103..


BIOGRAPHICAL SKETCH ............. ...... ._ ....___ ............10

















LIST OF FIGURES

Figureur page

1 Classic apoptotic cellular morphology ....._____ ... .... .___ ............... 18

2 Classic necrotic cellular morphology. ..........._._ ....._.._ .........._..... 19

3 Schematic drawing illustrating the regions of the maize endosperm.......... 41

4 Nuclear morphological changes in the endosperm of maize...................... 43

5 PCD in the endosperm of a 24 DAP caryopsis. ........._.._.. ........._.._..... 44

6 Confocal image of the nuclei in the central crown region of the
endosperm of a 28 DAP caryopsis of maize. ...........__... .......__........ 45

7 Nuclear morphology of the central crown region of the endosperm of a 12
DAP maize caryopsis. ...._ ......_____ .......___ .............4

8 Nuclear Morphology of the central crown region of the endosperm of
a 14 DAP maize caryopsis. ...._.._.._ ... .. ...__. ...._.._ ...........4

9 Nuclear Morphology of the central crown region of the endosperm of a
16 DAP maize caryopsis. ...._ _. ...._.._.._ ......._.. ............4

10 Nuclear Morphology of the central crown region of the endosperm of a
20 DAP maize caryopsis. ...._.._.._ ......_._._ ...._.._ ............4

11 Nuclear morphology of the central crown region of the endosperm of a
22 DAP caryopsis of maize. ........._._.. ....__.._ ...._ ... ...........5

12 Nuclear Morphology of the central crown region of the endosperm of a
25 DAP caryopsis of maize. ........._._.. ....__.._ ...._ ... ...........5

13 Origin of the Placenta-chalaza (PC) region. ........._...... ....._.._............ 52

14 Anatomy of the Placenta-chalaza. ........... ..... ..__ ........... .... 53

15 Placenta-chalaza (PC): left-right borders ................. ....................... 55

16 TEM image of one of the boundaries of the PC region. ........................... 56










17 Placenta-chalaza boundaries. ...._ ......_____ .......___ .............5

18 4'6-diam idino-2-phenylindole-2HCI (DAPI) DNA stain. .........._... ............. 58

19 Crystal Violet Stain showing condensed chromatin in the nuclei of
the cells of the integument-derived PC region from 4 through 10 DAP...... 59

20 Crystal Violet Stain showing condensed chromatin in the nuclei of the
integument-derived PC region from 12 through 16 DAP..............._._......... 60

21 TUNEL assays of PC region showing broken or nicked DNA in the nuclei of
the integument-derived portion of the PC ........._._. .........._._............ 62

22 Crystal violet stain showing condensed chromatin in the integument
derived portion of the PC region of a 24 DAP caryopsis..............._._......... 63

23 TEM micrographs illustrating consecutive developmental stages and
subsequent cell death of the nucellus-derived PC region. ........._.._............ 64

24 TEM micrographs illustrating the plasmodesmata (PD) in the walls of the
nucelluc-derived PC cells. ...._.._.._ ... ... ..._.._......_ .............6

25 Development and subsequent death of upper integument-derived region
of PC. ........._.._.. ...._..._ ..............._ 66...

26 Development and subsequent death of lower integument-derived region of
PC. ........._... ...... ___ ..............._ 68...

27 Lower Integument-derived PC Cells. ........._.._.. ...._.._ ........_....... 69

28 Nuclear body formation in the integument-derived portion of the
PC cells. ........._._. ._......_.._ ..............._ 70...

29 Nuclear morphological changes in integument derived portion of the PC.. 71

30 Ca2+ Trapping: Nucellus-derived PC. ...._._._._ ....... .___ ........_.._.... 72

31 Ca2+ Trapping in the integument-derived PC. ........._ .. ..... ...._._......... 74

32 Ca2+ trapping in the normal appearing, lower integument PC. ........._.._..... 76

33 In-gel nuclease activity assays for Ca2+, Mg2+ and Zn2+-dependent
nucleases and pH controls. ...._ .. ...._._._._ .........__. ............7

34 In-gel nuclease activity assays with EDTA and EGTA and no metals........ 80

35 Genomic DNA isolated from the PC region. ......____ ....... ....__.......... 81














Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

MORPHOLOGICAL SURVEY AND CHARACTERIZATION OF PROGRAMMED
CELL DEATH IN THE PLACENTA-CHALAZA AND ENDOSPERM IN THE
DEVELOPING CARYOPSIS OF MAlZE

By

Karen C. Chamusco

August, 2004

Chair: Prem S. Chourey
Major Department: Plant Molecular and Cellular Biology

Programmed cell death (PCD) was examined on a morphological basis

employing both light and electron microscopy techniques. The tissues examined

were the placenta-chalaza (PC) region and maturing endosperm of the maize

caryopsis. The PC region is sporophytic and, in maize, composed of two distinct

types of cells, which undergo two distinct morphological changes as they die.

This cell death process is fertilization dependent and takes place between 6 and

12 days after pollination (DAP). The first type of cells in the PC is derived from

the nucellus and undergoes a rapid cell death leaving behind cell corpses and

the number of detectable plasmodesmata decreases. There is no detectable

DNA cleavage via terminal deoxynucleotidy transferase dUTP nick end label

(TUNEL) assay in this zone at any time.

The second type of cells in the PC is derived from the inner integument and

undergoes a PCD that, morphologically, resembles apoptosis, i.e., condensed









chromatin, nuclear fragmentation, and preservation of organelles and

membranes until the end of the death process. There is no loss of

plasmodesmata in these cells. These cells are TUNEL-positive indicating DNA

cleavage.

Intracellular calcium [Ca2+]i W8S localized in an attempt to determine the

general flux and location of free [Ca2+]i 8n8 geerl [C2+]i aCCUmU tiOn in

storage sites such as the endoplasmic reticulum and the central vacuole does

not appear to take place until after 5 DAP. Mitochondria, while appearing to

collect [Ca2+]i St Some point prior to cell death, do not appear to change

morphologically. The general trend of [Ca2+]i f UX can be summed up as follows:

[Ca2+]i iS collected and stored in cellular compartments and organelles, released

into the cytoplasm and then dispersed or removed from the cell after death.

Nuclease assays revealed three nucleases dependent upon Ca2+ andlor

magnesium (Mg2+) and a fourth nuclease dependent on zinc (Zn2+). The peak of

activity for these nucleases occurred between 8 and 12 DAP which coincide with

the peak of morphological changes and the peak of positive TUNEL activity.

Unlike the nuclear changes seen in the PC cells, the nuclei of the central

crown region of the endosperm cells undergo a series of morphological changes

as the endosperm develops. At 12 DAP the nuclei start out spherical and the

DNA diffuse, by 16 DAP expand in size while chromatin condenses at the

nuclear envelope. Finally, by 25 DAP, the nuclei collapse and condense tightly.

These data indicate the possibility for three unique PCD processes

occurring in the developing caryopsis of maize.














CHAPTER 1
INTRODUCTION

Programmed Cell Death

Programmed cell death, or PCD, is a physiologically controlled, carefully

timed, energy dependent cell death program that organisms use to aid in

defensive processes or depend on for many normal developmental processes

(Solomon et al., 1999; Earnshaw, 1995).

Although cell death as a physiological process has been known to exist for

the last 150 years, apoptosis was the first type of PCD characterized in 1972 in

animal cells by Kerr and was called apoptosis from the Greek meaning "fallen"

and has been extensively studied ever since (Bursch et al., 2000; Earnshaw,

1995; Mllerriam Webster's Dictionary, 2004). Plant biologists have used these

animal studies as spring-boards from which to investigate PCD in plants.

Since the apoptotic form of PCD is the longest studied and best

characterized it will be discussed, here, as a model to illustrate the PCD process

and for use as a comparison against plant PCD. But it must be noted that

apoptosis is but one way PCD can occur. Other types of PCD that have been

described include autophagic PCD, a PCD which involves the degradation of

intracellular organelles prior to nuclear degradation, and endoplasmic reticulum

(ER)-mediated PCD that is independent of apoptosis protease activating factor-1

(apaf 1) and cytochrome c (Bursch et al., 2000; Rao et al., 2004).









A cell may be induced to undergo PCD due to a variety of causes such as

pathogen invasion, if the lifetime and or function of the cell has ended, or normal

developmental processes (Krishnamurthy et al., 2000). Apoptosis, like all

programmed cell death processes, is, by definition, a genetically controlled,

energy dependent and does not induce inflammation (Earnshaw, 1995). In the

case of apoptosis one of the first responses a cell has after perceiving signals to

undergo cell death is to produce an asymmetry in the aminophospholipids across

the plasma membrane. This duty requires the aid of the membrane bound

enzymes floppase, scramblase and the calcium (Ca2+) inhibition of

aminophospholipid translocase (Reutelingsperger 1997). The two former

enzymes induce an aminophospholipid flip, specifically, phosphatidylserine,

whereby the outer leaflet of the membrane has a higher occurrence of serine

residues than it normally would have. Once the serine residues are exposed,

Annexin V, a eukaryotic conserved aminophospholipid binding protein with high

affinity for serine residues, binds them in either a Ca2+ Or low pH dependent

manner. The bound Annexin V proteins then undergo Ca2+ induced structural

changes, ultimately forming an integral transmembrane channel that is thought to

act in an amphipathic or polytopic manner (Reutelingsperger 1997; Li and

Piazza, 2002). The mitochondria play a role at this point which includes a

release of intermembrane space proteins such as procaspase 9, cytochrome c,

apoptosis inducing factor (AlF), Ca2+ and reactive oxygen species (ROS). It has

been shown that mitochondria are intimately involved in regulating both plant and

animal PCD events (Arnoult et al., 2002; Curtis and Wolpert, 2002; Balk et al.,









2003). Mitochondrial inner and outer membrane-spanning pore formation is a

critical part of mitochondrial PCD effectors being released. Studies focusing on

apoptosis in animal cells have revealed two main mechanisms for the formation

of these pores and, consequently, each results in different mitochondrial

morphology (Yu et al., 2002). The first mechanism is a direct one and involves

the voltage-dependent anion channel, or VDAC, located at the mitochondrial

outer membrane. This mechanism is proposed to use the pro-apoptotic, Bcl-2

family protein, Bax, to interact with and tightly regulate release of cytochrome c

via the VDAC channel. The morphological consequence of this is that the

mitochondria remain normal in appearance.

The second mechanism is an indirect mechanism and involves the

permeability transition pore, or PTP (Curtis and Wolpert, 2002; Yu et al., 2002).

The PTP is formed from a complex of proteins, primarily adenine nucleotide

(ANT) located on the inner membrane, VDAC located on the outer membrane,

hexokinase, and cyclophilin D (CpD) in the matrix and when induced to interact

they form a transient, multi-faceted pore that acts as a redox, ion, pH, and Ca2+

gated channel. Upon continual opening solutes up to 1.5 kDa leak out of the

mitochondrial matrix and an uncontrolled influx of solutes, especially Ca2+, into

the matrix is initiated. Calcium increases the inner membrane permeability; the

result is gross over-swelling of the matrix, loss of transmembrane potential, an

uncoupling of the electron transport chain, superoxide production and complete

disruption of the outer membrane, and thus the release of inter-membrane

space, PCD promoting solutes such as apoptosis inducing factor (AlF),









cytochrome c, procaspase 9 and Ca2+ (CUrtiS and Wolpert, 2002; Arnoult et al.,

2002; Balk et al., 2003; Yu et al., 2002).

Once in the cytosol procaspase 9 autoprocesses to caspase 9 in

cooperation with cytochrome c and apoptosis protease activating factor-1, or

apaf-1, to form the apoptosome complex which further aids in activating effector

caspases. Caspases are cysteinyl-aspartate proteases. One such target of

these effector caspases is caspase activated DNase (CAD) which, along with

other Ca2+, C2+/Mg2+ dependent endonucleases, and caspase independent AlF

acts to condense and cleave DNA into fragments of 300 and 50 kilobases (kb)

then into internucleosomal size fragments which occur in multiples of 180 base

pairs (bp). (Earnshaw,1995; Gromova et al., 1995; Krishnamurthy et al., 2000;

Woltering et al., 2002; Balk, 2003).

A cascade of reactions is initiated at the plasma membrane by initiator

caspase 8, which cleaves other cytosolic effector caspases such as 3, 6 and 7

thus activating them. Poly-(ADP ribose)-polymerase, or PARP, which is involved

in DNA repair and maintenance, and the RNA splicing enzyme U1-snRNP are

other substrates of caspases; however these proteins are cleaved for inactivation

purposes so the apoptotic process may be allowed occur (Krishnamurthy et al.,

2000; Sun et al., 1999; Woltering et al., 2002, Chang and Yang, 2000).

On a morphological basis apoptotic animal cells show a unique pattern of

chromatin condensation (Fig 1) delineated by sharp edges, marginalization of

chromatin, retention and granulation of the nucleolus, plasma and nuclear

membrane blebbing (an outward bulging of the membrane) and the formation of









apoptotic bodies containing degraded cytoplasmic and nuclear contents. In

addition, all membranes and organelles are retained until the end of the cell

death program when neighboring cells phagocytose the dying cell (Kerr et al.,

1995). However the mitochondria may exhibit swelling due to high Ca2+ COntent

in the matrix (Virolainen et al., 2002; Curtis and Wolpert, 2002). Additionally,

apoptosis occurs in individual cells and is typically asymmetric with regard to one

cell's stage of PCD compared with that of another (Kerr et al., 1995). What is

described above is but one means by which cells can undergo a genetically

programmed death thus the process of PCD is not a clear cut one but one in

which it occurs in wide array of ways, especially in plants (Krishnamurthy et al.,

2000; Earnshaw, 1995; Rao et al., 2004)

Necrosis, on the other hand, is a form of cell death that has been reported

to be neither genetically controlled nor energy dependent (Fig 2). This form of

death is caused by cell damage, occurs in masses or clusters of cells and, in

animals, causes inflammation. The morphology does not resemble apoptosis type

PCD in that plasma membrane dissociation is first followed by complete

degradation of the cell. There is no organized chromatin condensation in the

nucleus and no preservation of intracellular contents. (Earnshaw, 1995; Kerr et

al.,1995). However, recent studies dispute necrosis as a passive, non-

genetically controlled cell death, rather, a physiologically controlled process

(Proskuryakov et al., 2002).

Categories of PCD Described in Plants

Plants have been shown to display PCD in for up to approximately six

different processes: hypersensitive response (HR), cells that have served their









purpose and are no longer required, cells that must die to serve their purpose,

cells initiated in development but never used, cells present in wrong places and

damaged cells (Krishnamurthy et al., 2000). The hypersensitive response (HR)

is initiated by pathogen attack, abiotic stresses such as ozone exposure creating

oxidative stresses, incompatible temperatures and osmotic, water, or light

stresses. Hormones such as gibberellic and salicylic acids have been shown to

play critical roles in HR (Krishnamurthy et al., 2000; Jones and Dangl, 1996) HR

does not necessarily end in the death of the cell, but when it does HR is often

described as necrotic. Regardless of whether or not the cell death appears

necrotic it does still involve genetic control and is localized to one or a few cells

at the immediate site of the pathogen attack. For example disease resistance

genes, (R) genes, have been implicated in mediating cell death signals during

HR. R genes are a single gene in the host plant encoding a receptor that

recognizes a single elicitor gene product called an avirulence (Avr) protein from a

specific pathogen resulting in resistance. The fundamental R protein architecture

typically includes a nucleotide binding domain (NB), a leucine rich repeat (LRR),

and serinelthreonine kinase unit. There are several subclasses of R genes in

which protein sequence alignments have revealed similarity in architecture to

several other protein receptors in the animal kingdom that are known to be

involved in apoptosis such as interleukin-1, apaf-1, and CED-4. There is,

however, still much that is unknown about the mechanisms underlying HR

mediated cell death (Shirasu and Schulze-Lefert, 2000).









The category of PCD in which cells have served their purpose and are no

longer required include, but are not limited to: senescence, cells involved in

abscission, synergids, antipodal cells, tapetal cells, and endosperm cells. PCD

in this category is part of the normal developmental processes that occur in

plants (Krishnamurthy et al., 2000).

Cells that must die to serve their function include xylem tracheary elements

(TE), cork cells, sclerenchyma, and modified structures such as spines and

thorns (Yu et al., 2002; Obara, 1998; Krishnamurthy et al., 2000; Jones, 1996).

Cells initiated in development but never used are those of non-functioning

megaspore cells, stamen primordia in female flowers and carpel primordia in

male flowers. (Krishnamurthy et al., 2000; Jones 1996).

Examples of cells present in wrong places are cells that die to give rise to

leaflets of compound leaves and holes in simple leaves (Arunika et al., 2004;

Krishnamurthy et al., 2000; Jones, 1996).

Lastly is the damaged cells category. This category is still unresolved as

damaged cells may also undergo necrosis, a non-programmed cell death.

Damaged cell PCD appears to behave much like that of the HR PCD contributing

further to the lack of resolution (Krishnamurthy et al., 2000).

Physiologic and Mlolecular Factors Involved in PCD

Reactive oxygen species (ROS) such as hydrogen peroxide (H202) and

superoxide (02 ) are 8 So involved in mediating signals for PCD. ROS generation

during HR is suspected to be produced by a handful of enzymes such as the

Ca2+ activated plant homologue of the NADPH oxidase found in animals and

oxalate oxidase in extracellular spaces during HR (Neill, et al., 2002). ROS have









been shown to occur in two distinct phases and may act in a dose dependent

manner. The first wave of ROS's occurs within an hour upon virulent or avirulent

pathogen attack whereas the second wave of ROS is specific to the avirulent

pathogen, occurs approximately five hours later and is sustained. High ROS

levels induce PCD but low levels stimulate antioxidant enzymes such as

gutathione S-transferase and glutathione peroxidase. High levels of ROS are

suspected to act as immediate pathogen stopping agents by killing the cell at the

site of infection as the ROS diffuse to surrounding cells. H202 has also been

shown to stimulate mitogen activated protein kinases (MAPK) in addition to

mitochondria. The mitochondria, in turn, generate more H202 al GE 09

mitochondrial activity thus leading to PCD. (Shirasu and Schulze-Lefert, 2000;

Thordal-Christensen et al., 1997; Neill et al., 2002).

The plant hormone salicylic acid (SA) has been shown to play a signaling

role in helping mediate HR PCD and systemic acquired resistance (SAR). It is

thought that SA coordinates with ROS to amplify, sustain and define the

threshold that must be met to induce PCD. Experiments where SA production

was compromised resulted in an inhibition of ROS production and a delay of

several hours in HR mediated cell death (Shirasu and Schulze-Lefert, 2000).

Nitric oxide (NO) is also a signal agent involved in mediating PCD in plants

and animals. Upon pathogen attack and subsequent R gene dependent HR NO

synthase activity is stimulated. The generation of NO stimulates phenylalanine

ammonia lyase (PAL), an enzyme involved in SA synthesis, and pathogen

resistance genes. Like SA, NO appears to help regulate ROS in a coordinated









fashion by dramatically amplifying the effects of H202 On cell death (Shirasu and

Schulze-Lefert, 2000).

Gibberellic acid (GA) also plays a role in plant PCD. Fath et al. (2000)

demonstrated that isolated barley aleurone layers undergo PCD upon GA

stimulation. GA stimulated the release and de novo synthesis of hydrolases and

nucleases in barley aleurone cells. GA also stimulated the coalescing of protein

storage vacuoles (PSV) within the aleurone cells. When protein synthesis and

secretion is finished the PSV's turn into lytic vacuoles and proceed to digest the

cellular contents while glucanases and xylanases digest the cell walls.

Ethylene is another player in PCD in plants. Young et al. (2000) reported a

spike in ethylene production prior to cell death in the endosperm of maize. It is

thought that ethylene is involved in nuclease activation and regulation.

Comparing PCD in Plants and Animals

In doing a side by side comparison of PCD in plants with apoptosis in

animals it can be seen that there are some surprising similarities regardless of

the method of PCD plants use; however, there have been distinct differences

discovered between the two as well.

Physiologic and molecular similarities include, but not necessarily seen in

every instance, a phosphatidylserine flip from the inner to the outer leaflet of the

plasma membrane, Annexin V binding, calcium signaling, mitochondrial release

of cytochrome c, and DNA cleavage resulting in internucleosomal size fragments

occurring in multiples of 180 bp, protein phosphorylation/dephosphorylation, ROS

production and Ca2+ and Zinc ( Zn2+) -dependent endonucleases (Young et al.,

1997; Wojciechowska, 2003; Ning et al, 2002; Krishnamurthy et al., 2000).









Plants do not necessarily use the components they do share with animals for

PCD, such as cytochrome c. For example cytochrome c release is detected

during PCD for TE differentiation but it does not participate in the PCD action

itself (Yu et al., 2002)

There is, however, a notable difference between plant apoptotic-like PCD

and animal apoptosis. The system described, previously, for apoptosis has not

been found in plants. For example, caspases have not been identified in plants

nor has a caspase cascade reaction; however, in some instances, caspase-like

cysteine proteases found in plants have been reported to behave similarly to true

animal caspases during plant PCD. Unlike caspases, plant caspase-like

proteases do not all have a specificity for aspartate residues. Some plant

cysteine proteases have been implicated in plant PCD; such is the case for the

barley vacuole localized aspartate proteinase, Phytepsin D, which is highly

expressed during autolysis of tracheary element differentiation (Runeberg-Roos

and Saarma, 1998). Previous studies have shown these caspase-like proteases

to be inh ibited by the synthetic caspase inh ibitors choloro-m ethylketone (Ac-

YVAD-CMK) andlor aldehyde (Ac-YVAD-CHO). These inhibitors completely

prevented HR mediated PCD in tobacco cells (Krishnamurthy et al., 2000).

Korthout et al., (2000) have shown caspase 3-like activity in plant cells. They

found that by adding plant mitochondria to the cytosol of Xenopus eggs in a cell

free system they were able to induce caspase 3 activity. Additionally, when a

mixture of protease inhibitors and the caspase 3 specific synthetic substrate N-

acetyl-Asp-Glu-Val-Asp-7-am ino-4-methylcoumarin (Ac-DEVD-AMC) was added









to cytosolic extracts from barely embryonic cell cultures the substrate was

cleaved after the aspartate residue. Only when a caspase 3 specific inhibitor,

Ac-DEVD-fmk, was added to the mix did the caspase 3 substrate not become

cleaved .

Morphological similarities include the following but are not necessarily seen

in every instance: cytoplasmic and nuclear shrinkage, chromatin condensation,

and in this study, nuclear body formation.

One unique difference between animal and plant PCD is that a cell corpse

is usually left behind after plant PCD due to the cell wall and cytosolic

degradation occurring autophagically andlor autolytically rather than by

phagocytosis as it does in animal PCD. However there are some exceptions

such as those seen for the formation of holes in the leaves of the lace plant and

the degeneration of barley aleurone cells (Arunika et al., 2004; Fath et al., 2000).

The Role of Calcium in PCD

It seems that regardless of the kingdom an organism is from, Ca2+ 8ppe8FS

to be a universal role player in PCD. Under normal, or non-stressed, conditions

the basal levels of free cytosolic Ca2+ (C2+]i) in plant cells are kept very low

ranging from 100 to 200 nM (Buchanan et al., 2000). Certain organelles in these

cells can be used as storage sites for Ca2+. Low cytosolic concentrations of Ca2+

are maintained with the aid of membrane bound Ca2+ channels and transporters

such as Ca2+-ATPase and Ca2+/H' antiporters. Such storage organelles may

include the ER and central vacuole and to a lesser degree the mitochondria and

nucleus. The cell wall can also contribute to [Ca2+]i (Buchanan et al., 2000).

Such an example of Ca2+ re 88Se from a storage site in response to plant PCD









stimuli is that of Ca2+ re 88Sed from the central vacuole during TE differentiation

(Yu et al., 2002). Calcium is known to be a major player in signal transduction at

the cell's surface, in the cytoplasm, and in the nucleus as well as an effector of

reactions. Under conditions of stress Ca2+ may be released from these reserves

through such channels as inositol 1,4,5-triphosphate sensitive channels (IP3

channels) and starting a cascade of signal transductions and metabolic reactions

by means of soluble Ca2+ binding proteins such as calmodulins or MAPK's

(Zocchi and Rabotti, 1993; Buchanan et al., 2000).

With regards to apoptotic PCD [Ca2+]i has been observed increasing in

concentration in the cytoplasm of cells coincident with or shortly after any

observable morphological changes in the nucleus, the first site where

morphological changes can be observed for PCD (Sherwood and Schimke,

1995; Kerr et al., 1995).

The Role of Endonucleases

Calcium also plays a role in activating enzymes involved in PCD such as

certain nucleases. However, other nucleases involved in PCD require additional

metals, different metals altogether, andlor specific pHs to be activated. For many

years apoptosis was, more or less, the only type of PCD known and it was

always accompanied by DNA cleavage resulting in multiples of ~180bp size

fragments at the end of the PCD process showing up as an internucleosomal

"DNA ladder" on gels. Only in the more recent past have investigators come

across other types of PCD where DNA is either cleaved into extraordinarily long

fragments of 30, 50, or 300kb or not cleaved at all prior to complete degradation









of the cell (Mittler and Lam, 1995; Balk et al., 2003; Sugiyama et al., 2000;

Eastman, 1995).

Sugiyama et al., (2000) reviewed plant endonucleases responsible for DNA

cleavage associated with PCD excluding those that exhibit DNA repair

properties. They were able to categorize these nucleases into two fundamental

classes based on metal dependence and pH optima. The primary division was

based on divalent metal requirements, specifically Ca2+ Or Zn2+. However, more

recent reports have added an additional two classes of PCD associated

nucleases, those requiring magnesium (Mg2+) Only and those requiring both Mg2+

and Ca2+ (Sugiyama et al., 2000; Ito and Fukuda, 2002). Calcium and Mg2+

activated endonucleases operate at a relatively neutral pH whereas Zn2+

activated nucleases, or S1-type nucleases, are activated at mildly acidic pH's,

5.0-6.5 (Sugiyama et al., 2000). This being established, the physical location of

these endonucleases could be identified or extrapolated. For example, Zn2+

dependent nucleases have been found to be located in the central vacuole and

apoplastic areas such as cell walls (Sugiyama et al., 2000; Ito and Fukuda, 2002)

but the exact location of Ca2+ dependent nucleases is still enigmatic to a certain

degree. Because Ca2+-dependent nucleases operate at a relatively neutral pH

Ca2+-dependent nucleases are thought to reside in the nucleus; such an example

includes NUC Ill which was extracted from isolated tobacco nuclei (Mittler and

Lam,1995; Sugiyama et al., 2000). The type of PCD in plants cannot be

determined by one specific class of nuclease alone due to the fact that different

studies have shown cooperative action of nucleases from each class.









During TE differentiation DNA cleavage by Ca2+-dependent endonucleases

appears to result only in large DNA fragments but observations that complete

DNA cleavage does not occur until after the central vacuole has ruptured links

the Zn2+-dependent endonuclease to this phenomenon (Sugiyama et al., 2000;

Ito and Fukuda, 2002). It has also been demonstrated that cereal endosperm

PCD occurs as a cooperative effort among the nucleases and takes place in

three distinct stages employing Ca2+-dependent endonucleases during the first

stage, within the endosperm and Zn2+-dependent endonucleases secreted from

or acting within the aleurone layer at the latter stages (Young et al., 1997, Young

and Gallie, 2000, Sugiyama et al., 2000).

Additionally, unlike certain nucleases associated with animal cell apoptosis

that are constitutively present in the cytoplasm in a dormant state and are

activated only upon PCD signals, CAD, DNase 1 and topoisomerase II, many

plant nucleases are transiently expressed as needed. Ito and Fukuda (2002)

found that ZENV-1, a 43 kDa Zn2+ dependent endonuclease and a 24 kDa

Ca2+/Mg2+-dependent endonuclease were expressed only during induction of

PCD for TE differentiation.

It is this author's opinion that perhaps a third class of endonucleases should

be considered being established; one based on temporal expression and or

activity, those that operate during early stages of DNA cleavage and those in the

last stages of DNA cleavage. The endonucleases already discussed operate at

the late stages of PCD. However, Balk et al., (2003) established an Arabidopsis

cell free system to investigate PCD. They have demonstrated a Mg2+-









dependent, mitochondrial localized G-like endonuclease that cleaves nuclear

DNA into 30 kb size fragments within 3 hours of PCD induction and that further

DNA cleavage does not occur until 12 or more hours after PCD by nucleases

from the cytosolic fraction of the cell.

This study focuses on the morphological changes attributable to PCD in the

developing caryopsis of Zea mays, specifically in the placenta-chalaza (PC)

region of the kernel and the maturing endosperm.

Seed Development

In order to better understand PCD in developing caryopses it is necessary

to describe the process of maize seed development and anatomy. The maize

caryopsis is an indehiscent fruit containing only one seed (Esau, 1977).

Identification of the anatomical parts of the caryopsis will begin from the outer-

most part of the caryopsis and proceed toward the center. The caryopsis

consists of the pericarp, which is the fruit, or ovary. The ovary wall consists of

three layers, the outer, covered by a cuticle, middle, and inner pericarp. The

following two layers are the outer then inner integuments, or the seed coat and

are derived from the ovule. The integuments also retain a cuticular layer due to

their ontogeny having originated from a common epidermis during ovule

development. These cell layers ultimately degenerate during seed development.

Continuing inward is a crassinucleate, or large, nucellus comprised of

unspecialized parenchyma cells, and finally the embryo sac containing the egg,

two synergid cells, two polar nuclei, and three antipodal cells. At the base of the

caryopsis is the chalaza, a region of nucellar tissue fused to integumentary and









funicular tissue; however maize lacks a funiculus, tissue that forms a stalk on

which the ovule develops (Esau, 1977).

The chalaza has become commonly referred to as the placento-chalaza

(PC), a term coined by Katherine Esau in 1944 (Kiesselbach and Walker, 1952).

The tissue subtending this region consists of vascular and thick walled

parenchymatous tissue comprising the pedicel that connects the caryopsis to the

receptacle or cob. All of these tissues are sporophytic, or maternal, in origin.

The physiologic role of the PC is to be the gateway through which all water and

nutrients pass to the growing seed (Wetherwax, 1930; Felker and Shannon,

1980). Upon seed maturity the closing layer forms in this region effectively

sealing the seed off from the maternal tissues and will become the site at which

the seed will abscise (Johann, 1935).

Upon fertilization, the caryopsis undergoes dramatic changes. The egg

fuses with the first of two sperm nuclei from the pollen grain to become the

diploid embryo while the two polar nuclei fuse to each other followed by fusion

with the second sperm nucleus to become the triploid endosperm (Esau, 1977).

It is shortly after this point that several PCD regimens start to take place. Among

the types of PCD observed in the developing caryopsis are nucellar degradation,

PCD within the embryo, i.e. the scutellum, coleoptile, suspensor, root cap, and

starting at about 16 days after pollination (DAP), the endosperm (Giuliani et al.,

2002; Young et al., 1997). In addition to the above mentioned PCD events in the

maize caryopsis, PCD in the PC has been observed for the first time in this study

and by Kladnik et al., (unpublished). Each of these areas of PCD is a normal









part of development and they occur at specific times during development. PCD

was investigated on a cellular level using light and electron microscopy

techniques. Specifically investigated were the PC region during early

development (0-12 DAP) and the endosperm from the starch filling stage to near

physiologic maturity (12-25 DAP) and appears that these PCDs may be, not only

a normal part of development, but a necessary one. Surprisingly, three different

morphological changes were seen between the cells of the PC and the

endosperm, two in the PC and the third in the endosperm. It is, therefore,

possible that there are three different forms of PCD occurring in these tissues.





Figure 1. Classic apoptotic cellular morphology

The earliest morphological change in apoptotic cells occurs in the nucleus.
Organelles maintain good morphology during nuclear changes. (A) Nucleus
shrinks & chromatin condenses at the nuclear periphery. (B) The nucleus starts
to blebb" with chromatin filled pockets. (C) The entire cell blebs & breaks up into
nuclear bodies containing condensed chromatin pieces and cytoplasmic
contents. (D) Cell culture showing cells at various stages of apoptosis.
From Kerr et al., (1995).




























Figure 2. Classic necrotic cellular morphology.

Necrotic morphology is illustrated by soft edged, condensed chromatin in various
clumps throughout the nucleus. Additionally, cytoplasmic organelles degenerate
concom itantly with nuclear changes.
From Kerr et al., (1995).














CHAPTER 2
MATERIALS AND METHODS

Plant Materials

W22 Maize plants were grown in a greenhouse from March through August

2002 and February through June 2003. The only exceptions were 14 and 16

days after pollination (DAP) caryopses, which were harvested from field grown

plants in the summer of 2003. Caryopses were harvested from the same ear

whenever possible or from ears of sibling plants grown at the same time under

identical conditions. Caryopses were harvested at various stages of

development. For PC examination caryopses from 0 through 12 DAP were used

and for endosperm examination 12 through 28 DAP caryopses were used.

Utrastuctural Studies

Both distilled glutaraldehyde (8%) sealed in ampules with an inert

atmosphere and paraformaldehyde (PFA) were purchased from Electron

Microscopy Sciences. PFA was prepared by mixing 8% (w/v) PFA into 0.1 M

cacodylate buffer, covered with foil, and, under constant stirring, heated to just

under the boiling point. Sodium hydroxide (NaOH) was added to aid in dissolving

the PFA, maintaining a pH of less than or equal to 8.0. Once dissolved, the PFA

solution was immediately placed on ice to chill, filtered through a number 1

Whatman filter and the pH adjusted to 7.0 with concentrated hydrochloric acid

(HCI). The PFA stock was stored frozen at -200 C for up to 2 weeks.









Some tissues were prepared by fixation in 4% glutaraldehyde (v/v) and 1%

paraformaldehyde (v/v) in 0.1 M phosphate buffer (pH 7.0). Most of the tissues

were prepared using cacodylate buffer (pH 7.0). Fresh caryopses were

harvested and immediately processed for fixation on site by placing a small

aliquot of chilled fixative on a glass plate and hand sectioning out a sagittal

section of the caryopsis approximately 2mm wide with a double edged razor

blade. The tissue was then put into a vial of ice cold fixative and placed under

vacuum for 5 to 10 minutes to aid fixative infiltration. After vacuuming the

samples were placed on a rotator overnight, approximatelyl2 hours at 40 C.

After fixation the tissue was rinsed three times for 30 minutes each in

chilled cacodylate buffer then placed in 2% aqueous osmium tetroxide overnight

at 40 C. Once the osmication was completed the samples were rinsed 3 times,

one hour each, in distilled water then dehydrated in a chilled acetone series at

20% increments, each for one hour. Once the samples reached 100% acetone

the acetone was exchanged three times. On the third change the acetone was

mixed with propylene oxide as a transition solvent. The ratios of acetone to

propylene oxide were 2: 1, 1:1, and finally 100% propylene oxide. Each transition

lasted 20 minutes at room temperature. Following the 100% propylene oxide

incubation, fresh propylene oxide was mixed with Spurr's epoxy resin (including

the catalyst) in ratios of 2: 1, 1:1, 1:2, propylene oxide to resin and infiltrated in

each for 12 hours. Upon completing the 1:2 propylene oxide to resin step the

samples were placed in 100% resin and infiltrated for 24 hours. This step was

repeated 3 times with fresh resin. After the third exchange of resin at 100% the









samples were placed in flat molds with fresh resin and polymerized at 560 C for

24 hours.

Samples were sectioned on a Sorvall MT-2B ultramicrotome. The sections

were cut to approximately 60 nm in thickness, indicated by a pale gold color, then

transferred to formvar coated copper grids (50 or 100 mesh). The sections were

post stained 20 minutes in filtered 2% aqueous uranyl acetate (UA), rinsed three

times, one minute each in distilled water then were allowed to dry. However, this

UA step was later modified to 25 minutes in 10% (saturated) UA. This was found

to give much better contrast to the sections. The sections were then stained six

minutes in Reynolds' lead citrate, rinsed once in 0.02 M NaOH for one minute,

then three times more, 1 minute each, in distilled water. The sections were

examined and photographed in a Zeiss 109 or a Zeiss EM-10 CA transmission

electron microscope (TEM). Negatives were scanned at 300dpi using Epson

Perfectionl650 scanner, digitally processed using Adobe Photoshop version 7.0

and saved to compact disc.

Calcium Trapping

Calcium (Ca2+) W8S localized by using the oxalo-acetatelantimonate

method as described by VanReempts et al. (1982) and Borgers et al. (1977). All

steps were carried out at 40C. The fixative consisted of 2.8% glutaraldehyde, 2%

PFA, 90 mM potassium oxalate, and 1.4% sucrose in distilled water then

adjusted to pH 7.4 with 1M potassium hydroxide (KOH). This step is for the

purpose of allowing oxalate ions to combine with Ca2+ iOns to form the insoluble

salt calcium oxalate. After fixation the samples were rinsed for two hours in a

solution of 7.5% sucrose and 90mM potassium oxalate at pH 7.4. Next the









samples were post fixed overnight in 1% osmium tetroxide mixed with 2%

potassium pyroantimonate in 0.1M acetic acid and adjusted to a pH of 7.4 with

1M KOH. This step is for the exchange of oxalate ions for antimonate ions in the

calcium salt to form an insoluble, electron dense calcium antimonate precipitate.

Samples were then rinsed for half an hour in distilled water adjusted to pH to 10

with 1M KOH. Subsequent steps for dehydration, infiltration and embedding

were carried out as described for ultrastructure.

Light Mlicroscopy

Caryopses were harvested and fixed as described for ultrastructural

studies. Dehydration was carried out in an ethanol series consisting of 20%

increments, one hour each, up to 100% ethanol at 40 C. After two incubations in

100% ethanol for one hour each the samples were allowed to come to room

temperature and were placed in a 1:1 mixture of ethanol to tertiary butanol (TBA)

then twice in 100% TBA, again, for one hour each. After the second change of

TBA, a third change of TBA was mixed with Paraplast Plus paraffin chips (Fisher

Scientific) and placed in an oven at 560 C overnight (12 hours). which allowed the

paraffin to slowly melt and infiltrate while the TBA evaporated, facilitating a slow

controlled exchange. Following this step the TBA paraffin mixture was removed

and fresh molten paraffin poured on the samples to continue infiltration in the

oven for another 12 hours. This step was repeated two more times. After the

final infiltration step the samples were embedded using disposable boats and

embedding rings (Fisher Scientific) and sectioned to a thickness of 10 pm on a

Microm-325 rotary microtome. The sections were adhered to Fisher Scientific

Probon poly-L-lysine coated slides. To stretch the sections, a bubble of water









was placed on each slide, warmed on the slide warmer at approximately 400C

and the sections floated on it until the water evaporated. The slides and sections

dried on the warmer for 24 hours to assure proper adherence.

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-fluorescent

nick end-labeling or, TUNEL, assays were performed using the TUNEL assay kit

purchased from Roche Diagnostics. The assay was done according to the

manufacturer's directions except, the tissue permeablization step using

Proteinase-K was reduced to one minute at 370 C and 14 minutes at room temp

to help preserve tissue integrity. Briefly, paraffin sections were de-waxed in two

changes of xylene and re-hydrated through an ethanol series (100, 70, 50, 30%)

for five minutes each and finishing in distilled water. The sections were treated,

as described, with 200g/ml Proteinase-K for 15 minutes, rinsed in PBS twice for

five minutes each. The sections were then treated with the TUNEL label plus

TdT enzyme for the experimental or simply just label for the negative control.

The slides were then placed in a petri dish lined with moist Whatman paper and

floated in a 370 C water bath in the dark for one hour. Once the incubation was

complete the slides were rinsed three times in PBS in the dark for five minutes

each, then counter stained with 600 pM (v/v) 4'6-diamidino-2-phenylindole-2HCI

(DAPI) DNA stain. The DAPI was mixed into the aqueous mounting medium,

Gelmount, (Fisher Scientific). The sections were examined with a Nikon Optiphot

fluorescent microscope and photographed using a digital CCD camera and Spot

software version 3.52 (Diagnostic Instruments). The TUNEL was examined with









an excitation wavelength of 450nm and emission at 520nm while the DAPI was

examined with an excitation of 380 and emission at 450nm.

For confocal imaging of nuclei in de-waxed sections propidium iodide DNA

stain (PI), purchased from Molecular Probes, was used. Pl DNA staining was

done according to manufacturer's directions exactly. Re-hydrated paraffin

sections were equilibrated in 2X sodium citrate, sodium chloride (SSC) buffer

(0.3 M NaCI and 0.03 M sodium citrate) for 3 minutes then treated with 100 pg/ml

RNAse A in 2X SSC in a water bath at 370 C for 20 minutes. The slides were

then rinsed in 3X SSC three times for one minute each. A solution of 500 nM Pl

was placed on the sections and incubated at room temperature in the dark for

five minutes. Following the incubation the slides were rinsed two times in SSC

for three minutes each and the sections mounted in Gelmount. A Bio-Rad 1024

ES Confocal Microscope was used to view the samples and images were

captured using LaserSharp Software.

Toluidine blue-O stain (TBO) was made tol% (w/v) in 1% sodium borate

solution (w/v) and filtered through a 0.2 pm filter before use. Resin sections were

TBO stained by covering the sections on the slides with a few drops of the stain,

gently heating the slide then rinsing the stain away with water. The slides were

dried over low heat on a hot plate and viewed by light microscopy.

Nuclease Activity Assays

In-gel assays were performed for endonuclease activity according to Young

et al. (1997), with minor alterations regarding the extraction buffer, discussed

below. Caryopses were harvested fresh from the field, flash frozen in liquid

nitrogen and stored at -800 C until used for protein extraction. Caryopses of 6, 8,









10, 12, and 28 DAP were tested. The caryopses were cut such that the top two-

thirds of the caryopses were separated from the bottom one-third. This was

done so nuclease activity of the PC region could be checked free of endosperm

tissue. Approximately 500 mg of tissue was used for the protein extraction. The

tissue was ground to a fine powder in liquid nitrogen for 15 minutes using a

mortar and pestle. The powder was then decanted into a sterile Corex tube and

diluted to 5 ml with ice-cold extraction buffer. The protein extraction buffer used

for these assays differed from that used by Young et al. (1997) in that the buffer

was 50 mM Tris Maleate pH 7.0. This change was made to eliminate any metals

that might interfere with or inadvertently activate the nuclease enzymes. The

samples were spun in a Sorvall RC-5B refrigerated rotor superspeed centrifuge

at 8000 rpm for 10 minutes at 40C. The supernatant/ protein was then

transferred to 1 .5 mlI Eppindorf tubes and kept on ice until quantified. P rotei n

quantification was carried out in a BioRad Smartspec 3000 using the Bradford

assay (Biorad) according to the manufacturer's specifications. Once quantified

the samples were stored in aliquots of one milliliter, flash frozen in liquid nitrogen

and stored at -80 until needed.

Two micrograms of protein from each sample was mixed with sample

dilution buffer (1% SDS w/v, 62.5 mM Tris-HCI pH 6.8, 10% glycerol (w/v), and

0.5% bromophenol blue (w/v)) and run on a 12% SDS-PAGE gel at 200 V for one

hour. Once completed the gels were rinsed in 25% isopropanol in 0. 1 M Tris

buffer pH 7.4 three times for ten minutes each to rinse out the SDS detergent.

The gels were then washed another three times in 0.1M Tris pH 7.4 to rinse out









the isopropanol. Afterwards the gels were incubated in washes containing either

0. 1 M CaCl2 Or MgCl2, Or both in 0. 1 M MOPS buffer pH 6.8 at 500C for 30

minutes or 1 mM ZnSO4 in 0.025 M acetate buffer (1.76 ml 1 M acetic acid, 8.24

ml 1 M sodium acetate and 40 ml H20) pH 5.5 at 470C overnight while gently

rotating in water baths. Several types of controls were employed: 1) gels were

either incubated with EDTA and EGTA in a 1:1 stoichiometric ratio with that of

the metals added to the incubation medium, 2) the pHs were changed such that

the Ca2+/Mg2+ buffers were lowered to that required for Zn2+ and the pH of the

Zn2+ buffer raised to that required for Ca2+ and Mg2+ Or 3) the metals were left out

of the incubation medium altogether. After incubation the gels were bathed in

200 pg/ml ethidium bromide for 10 minutes, rinsed in distilled water for 10

minutes then inspected in a transilluminator and photographed using BioRad

Geldoc software.

DNA Extraction

Genomic DNA was isolated from 5 grams of the same tissues cut in the

same manner as was used for the protein extraction. The developmental stages

examined were 8, 10, 12, 16, 20, and 25 DAP. Tissue was ground with a mortar

and pestle in liquid nitrogen for 20 to 30 minutes to a fine powder then

immediately put in sterile culture tubes containing 15 ml extraction buffer (100

mM Tris pH 8, 50 mM EDTA pH 8, 100 mM NaCI, 1% SDS, 0.2% PVP, and 10

mM. -mercaptoethanol) and incubated at 650C for 10 minutes. After incubation 5

ml 5 M ammonium acetate was added, mixed, and then incubated on ice for 20

minutes. The samples were then poured through Miracloth into a new sterile

tube and 1 ml 5 M ammonium acetate and 1 volume isopropanol added. The









samples were then kept at -200C for a minimum of 30 minutes, centrifuged at

12,000 rpm at 40C in a Sorvall RC-5B refrigerated superspeed centrifuge for 15

minutes and the supernatant poured off. The pellet was washed with 5 ml 70%

ethanol after which the tube was inverted on a paper towel for 5 minutes then

placed under vacuum for an additional 5 minutes. The pellet was resuspended in

500 pl TE (10 mM Tris-HCI, 1 mM EDTA pH 8.0) and placed on ice for 30

minutes with intermittent mixing every 5 to 10 minutes. Next, 250 pl 5 M

ammonium acetate and 2 pl of 10 mg/ml RNAse A were added and incubated at

370C 30 minutes. An equal volume of phenol, chloroform, isoamylalcohol (IAA)

in a ratio of 25:24:1, respectively was added to the tube, spun for 10 minutes at

8,000 rpm and the supernatent pulled off and placed in a new tube. One volume

chloroform and IAA in a ratio of 24: 1 respectively was added to the tube and

spun again at 8,000 rpm for 10 minutes. The supernatant was removed, placed

in a fresh tube, two volumes absolute ethanol added then incubated for 1 hour.

The sample was spun for 20 minutes at 8,000 rpm, the ethanol poured off, and

the pellet dried under vacuum. Once the pellet dried it was resuspended in 50 to

100 pl sterile water and stored at -200C.

The DNA was quantified in a BioRad SmartSpec 3000 spectrophotometer.

Two pg DNA was electrophoresed in a 2% agarose gel infused with 0.5 pg/ml

ethidium bromide with a low mass DNA ladder and run at 120V for approximately

1 hour. The gel was photographed in a transilluminator using Biorad Geldoc

software.
















CHAPTER 3
RESULTS

The Endosperm

Light Mlicroscopy

TUNEL and DAPI stains were used to investigate DNA conditions and

nuclear morphology, respectively, in developing endosperm cells of maize. The

endosperm was divided into four regions for inspection: the crown, central crown,

mid endosperm and lower endosperm (Fig. 3). As illustrated in Figure 4, the

nuclei at each stage of development showed distinct morphological changes

taking place up through 24 DAP. At 12 DAP the cells in the crown of the

endosperm were starting to fill with starch and nuclei appeared healthy and

maintained an approximate central position in each cell. The nuclei were

characterized by diffuse DNA which appeared uniform in texture throughout the

nucleus. By 16 DAP the crown of the endosperm was filled with greater

quantities of starch and the DNA of each nucleus had condensed at the

periphery giving the nuclei a beaded appearance. By 24 DAP the TUNEL assay

showed positive results as indicated by bright green fluorescence in the nuclei in

the central and crown region of the endosperm, indicating that DNA cleavage in

the endosperm does not take place until after starch is abundant and the nuclei

lose their apparent integrity (Fig. 5). It is at this stage when the nuclei condense

and shift closer to the cell wall.











The 28 DAP kernels proved to be too opaque for any TUNEL activity or

DAP I stain to be seen due to the autofluorescence of the starch grains.

However, confocal imaging of propidium iodide stained sections was used to look

at the nuclear morphology. By 28 DAP, as seen in Figure 6, the nuclei of the

crown of the endosperm appeared completely condensed and appressed against

the cell wall.

Transmission Electron Mlicroscopy

Caryopses of the same developmental stages used for LM investigation

were selected for nuclear investigation from the central crown region of the

endosperm by TEM.

The pattern of morphological changes of endosperm nuclei seen by DAPI

staining at the LM level compared favorably to those seen at the EM level. At 12

DAP, the nuclei appeared rounded, the DNA diffuse and the cell appeared

normal as indicated by a normal appearing nucleus with diffuse DNA and an

intact nuclear envelope (Fig. 7). At 14 DAP the nuclei appear slightly larger and

the chromatin less diffuse (Fig. 8). Interestingly, the rough endoplasmic

reticulum (ER) appeared more prevalent and displayed intermittent areas of

bloating while in other areas the bloated bodies become filled with electron-

dense substances then appeared to bud off, presumably forming protein bodies.

By 16 DAP the nuclei displayed condensed chromatin that collected at the

nuclear envelope (Fig. 9) supporting the beaded appearance as seen by DAPI

stain at the LM level. The ER became less pronounced and the membrane

bound, electron dense bodies took on a more oval shaped appearance and mini-











vacuoles contained within them could be seen. By 20 DAP the nuclei appeared

bloated, appeared to have much less chromatin and, in some cases, appeared to

be almost devoid of contents leaving only an intact nuclear envelope (Fig. 10).

However, some of the nuclei displayed enlarged condensed chromatin spheres

that looked like oversized nucleoli; but, as stated previously, the rest of the

nucleus is empty except for very small, condensed chromatin pieces sparsely

distributed at the envelope. The nuclei at 22 DAP (Fig. 11) became very difficult

to discern as the membranous structures of the cell, including the plasma

membrane, appeared to be degenerating and cytoplasmic contents started to

coalesce and were not readily identifiable. By 25 DAP the nuclei and cytoplasm

of the cells became indistinguishable (Fig. 12). All the cellular contents

condensed, became amorphous in appearance and, for the most part, moved to

the cell periphery. At higher magnification the amorphous material acquired the

appearance of being comprised of many small spheres that appeared to be fatty

in nature.

The Placenta-Chalaza Region

Light Mlicroscopy

Both LM and TEM were also employed to investigate morphological

changes occurring the PC region during PCD. In order to investigate the

changes associated with the PC region it was necessary to anatomically define

this region more clearly. The ontogeny of the PC region, (Fig.13), showed two

cell lineages from each the nucellus epidermis and the inner integument, the

nucellus epidermis contributing to the upper region of the PC and the inner











integument contributing to a region of the PC subtending the nucellar derived

region. These two regions will be referred to as "zones" for the sake of clarity.

The anatomy of the PC region clearly shows two distinct zones, the upper from

the nucellus epidermis and the lower from the inner integument, as described

above (Fig. 14). This observation was in agreement with the literature in that the

PC region of the maize caryopsis is where the integumentary and nucellar

tissues merge (Esau, 1977). After pollination, as described by Esau, (1977), two

semipermeable, suberized andlor cutinized, extra-cellular membranes became

detectable in the seed coat and by approximately 7 DAP extended around the

entire seed but ended abruptly on either side of the PC region (Fig. 15). The

thicker of the two membranes appeared bi-layered (Fig. 16). This membrane

was derived, primarily, from the nucellus epidermis and, secondarily, the inner

integument. These membranes ended abruptly on either side of the PC thus

defining the boundaries of the PC region (Fig. 17).

PCD in the PC Region is Fertilization Dependent

DAPI DNA staining of a 12 DAP caryopsis and an unpollinated ovule of 12

DAP equivalency revealed enucleation of the PC cells only in the fertilized kernel

(Fig 18). Additionally crystal violet staining (Figs. 19-20) revealed condensed

chromatin in cells of the integumentary PC between 6 and 14 DAP which

coincided with the TUNEL positive reactions (Fig. 21) seen in this region (Kladnik

et al., unpublished). While there were no detectable condensed nuclei between

16 and 22 DAP condensed chromatin was seen, again, at about 24 DAP (Fig.











22) in the region of the integumentary PC that was most compressed by the

endosperm.

Transmission Electron Mlicroscopy

Prior to pollination both layers, the nucellus-derived and integument-derived

layers, of PC cells appeared normal and only a few morphological differences

aided in distinguishing the two cell types from one another. The nucellar PC cells

bordering the integument-derived cells in the PC region of the 0 DAP caryopsis

were large relative to the integument-derived cells. The nucellar PC cells were

characterized by thin, primary cell walls and very large, single vacuoles and a

small nucleus relative to the cell's size (Fig. 23A). These cells contained healthy

organelles and abundant plasmodesmata

Up through approximately 5 DAP all the PC cells remained relatively

unchanged in appearance from those of the PC in the 0 DAP caryopsis. At 5

DAP the only significant change in the cells of the PC region from that of the 0

DAP caryopsis was that the nuclei of the PC cells had become more rounded. At

this stage of development the growing endosperm started to crush the nucellar

tissue immediately in contact with it thus beginning the initial steps of forming a

multi-walled barrier between the basal endosperm cells and the PC region. The

nucellar-PC cells just below (Fig. 23B) still contained healthy cytoplasm and

nuclei and the average number of plasmodesmata per cell per section was 7. 1.

After pollination a dramatic series of changes took place in the PC region as

the caryopsis developed. The first observable changes that occurred were at

about 7 DAP. The enlarging endosperm had further crushed the nucellar PC











cells creating a more compacted barrier between the basal endosperm and PC

region. These cells had substantially emptied and contained remnants of what

appeared to be plasmolysed cytoplasm (Fig. 23C). These cells also appeared a

bit more flattened than previously seen in younger stages. The average number

of distinguishable plasmodesmata per cell remained the same as it was for 5

DAP. By 9 DAP, the border between the basal endosperm and the nucellar-PC

had become very thick and solid and had completely isolated the endosperm

from any symplastic connections to the PC. The nucellar-PC cells had only very

sparse remnants of unidentifiable degenerated cytoplasmic contents (Fig. 23D).

The number of plasmodesmata from 9 to 12 DAP varied from 1.6 to 3.3 per cell

per section for an average of 2.4 per cell per section (Fig. 24). This was a

reduction of 33% from the 7.1 seen from 5 to 7 DAP. However, it is important to

note that this apparent reduction in the number of plasmodesmata was seen only

in the nucellus-derived portion of the PC and not the integument-derived portion,

which appeared to maintain an abundance of plasmodesmata.

The layer of cells below the nucellar-PC cells are those derived from the

inner integument. At 0 DAP the integument-derived cells were characterized by

a more rectangular shape, thicker walls than the nucellus-derived cells, had a

more dense cytoplasm (Fig. 25A). Additionally, the nuclei in these cells were

small, elongated and looked like flattened footballs. At 5 DAP these cells

appeared healthy and, generally, contained one vacuole; the only significant

change from 0 DAP was a slightly more dense cytoplasm (Figs. 26 A, B). The

next cell layer below the nucellar PC cells, the upper integumentary PC, was,











approximately, two cells wide; these cells will eventually become part of the

closing layer. By 7 DAP (Fig. 25C) the nuclei of these cells showed the

beginning stages of condensing chromatin; however the nuclei were still intact

and appeared spherical. By 9 DAP the cells of the closing layer had become

laterally elongated, compacted and more cytoplasmically dense but some cells

still contained crushed nuclei with condensed chromatin. Generally, the

cytoplasm of these cells, at this stage of development, had become very dense,

the vacuoles were absent or were in the process of disappearing and there were

no identifiable organelles (Fig. 25D). The cells subtending the closing layer, the

lower integumentary PC cells (Fig. 26A-C) contained normal nuclei and

cytoplasm and averaged one vacuole per cell. At 9 DAP this cell layer (Fig. 26D)

was characterized by cells with a total lack of nuclei, unidentifiable organelles

and no vacuoles but retained all their disintegrated cytoplasmic contents. These

cells had become flattened and elongated. This layer of cells was peculiar in that

it showed variable positioning around the closing layer depending on which part

of the PC region was being viewed. On the lateral edges this third cell layer was

above the closing layer for a short distance but when moving toward the center of

the PC the third layer appeared below the closing layer and held this position for

the greater portion of its span. The fourth layer of cells down (Fig. 26E)

contained early stages of PCD. The cytoplasm of these cells contained multiple,

small vacuoles, but the nuclei were starting to display condensing chromatin.

Additionally, the endoplasmic reticulum (ER) became much more abundant and

formed concentric ring-like andlor layered, laminar patterns (Fig. 27). Continuing











basipetally, the next layer of cells appeared healthy as well with the only one

distinguishing feature from those of the previous layer. These cells, on average,

contained several smaller vacuoles. Below these cells was the vascular tissue

and starch-containing parenchyma.

The closing layer and a few cells beneath the closing layer, by 8 DAP,

appeared to be the first set of cells at which gross morphological changes related

to PCD were seen. This was the first stage at which completely condensed

nuclei were detected. Interestingly, in the cells that harbored completely

condensed nuclei, small spheres filled with condensed chromatin remains were

seen throughout the cytoplasm corresponding to similar TUNEL positive bodies

seen within PC cells at a light microscopy level (Fig. 28). For those cells at such

an advanced state of degradation the only cytoplasmic feature that could be

clearly identified with certainty was the rough ER. However, the ER was very

swollen. By 11 DAP, as seen in Figure 31E and F, the cells of the closing layer

looked extremely chaotic; the contents of most of the cells in this layer looked

disjointed. For example, the membranes were collected in tight whirls and

seemingly disconnected from anything else in the cell, lipid bodies and

unidentifiable cellular debris constituted the rest of the cytoplasm. In some cases

the cytoplasm was dominated by many very tiny vacuoles. The cells furthest

down in the PC appeared normal with completely normal looking nuclei and

organelles. By 12 DAP the first set of cells with any cellular debris to be seen

was the top most layer of the closing layer, which coincides with the chaotic cells

seen in the 11 DAP. By this stage all that was left was condensed cytoplasm.











Further down into the closing layer and the rest of the integumentary PC cells

with condensed nuclei became more abundant. Some cells were seen at such a

stage where the mitochondria were found to be dramatically swollen and losing,

or had lost, inner membrane integrity. Moving further down into the PC nuclei

had mild to no condensed chromatin and there were identifiable organelles.

A general trend was seen in the morphology of the nuclei as the PCD

process ensued. Initially the chromatin in the nuclei appears diffuse and the

organelles normal in appearance (Fig. 29A). As in animal apoptosis the

chromatin condenses and is delineated by sharp edges. However, unlike animal

apoptosis, the chromatin does not coalesce at the nuclear periphery (Fig. 29B).

Eventually the chromatin in the nucleus completely condenses but the nuclear

membranes remain intact (Fig. 29C). At the end of the process the nucleus has

broken up into nuclear bodies (Fig. 29D).

Calcium Trapping

The oxalatelantimonate calcium ion (Ca2+) trapping method was used in an

attempt to determine the flux and location of free Ca2+ iOns within the cell during

this cell death process. At 5 DAP the nucellar-PC cells contained only trace

amounts of Ca2+ in the cytoplasm and organelles (Fig. 30A). The cells did not

contain Ca2+ in the central vacuoles at this time, but rough ER had light Ca2+

deposits associated with the membrane. The mitochondrial matrix did not

appear to be associated with appreciable deposits of Ca2+ While the nucleus was

very lightly peppered with Ca2+. At 8 DAP the nucellar-PC contained moderate

background Ca2+ (Fig. 30C) but the remnants of cellular contents did not contain










Ca2+. The next cell layer down, still within the nucellar PC (Fig. 30C-inset), was

intermediate in its cytoplasmic degeneration between that seen in the nucellar-

PC and the closing layer. This cell layer showed Ca2+ tO be abundant in the cell

corpse background but still did not appear to associate with the degenerated

cytoplasmic remains. By 11 DAP the nucellar-PC appears similar to that of the 8

DAP except at this point there is no Ca2+ in the cell corpse background any

longer (Fig. 30E).

At 5 DAP the integument PC cells did not appear significantly different than

those in the nucellar PC except that the mitochondria appeared to have a light

scattering of Ca2+ deposits throughout giving them a "peppered" appearance

(Fig. 31A). By 8 DAP the closing layer cells in the upper integumentary PC

showed Ca2+ Sparsely in the cytoplasm but small amounts were present in what

was left of the ER (Fig. 31C). By 11 DAP the closing layer showed chaotic,

condensed cytoplasmic contents with highly compacted swirls of membranous

structures but with little Ca2+ preSent (Fig. 31E).

The cells immediately subtending the closing layer varied subtly in their

localization of Ca2+. These cells were examined in order to check where Ca2+

might localize in normal appearing PC cells of more mature tissues. Some of the

cells displayed mitochondria with little to no Ca2+ lOcalized within (Fig. 32A).

Additionally, the ER did not appear to contain much Ca2+ sS indicated by few

electron-dense precipitates. The other cells of this region looked similar to those

discussed above except the ER was abundant with Ca2+ Which were heavy in

electron-dense precipitates (Fig. 32B), the mitochondria seemed to have more











Ca2+ (Fig. 32C), and the nuclei were peppered with Ca2+; however, the vacuoles

of both cell types contained abundant Ca2+ and the cytoplasm did not have much

Ca2+ in either cell type relative to the rest of the cell. In cells of moderate to

advanced stages of PCD the nuclei contained more Ca2+ than the cytoplasm and

the mitochondria showing morphological changes, i.e. swelling, contained fewer

Ca2+ deposits than those not yet showing morphological changes in other cells

(Fig. 32D).

In-Gel Nuclease Activity Assay

The lower one-third of the wild type W22 kernels, 6, 8, 10, 12, and 28 DAP

were examined for Ca2+, Mg2+, both Ca2+ and + Mg2+, Or Zn2+- dependent

nuclease activities. The Ca2+ and Mg2+ treatments, whether tested together or

independently resulted in the same pattern (Fig. 33). As was reported by Young,

et al. (1997) three nucleases of 33.5, 36.0 and 38.5 kiloDaltons (kDa) were seen

when tested using these metals. The 33.5 kDa nuclease was highly active at all

developmental stages examined; however, only the 38.5 and 36.5 kDa nucleases

were most active at 8 DAP as indicated by a lack of DNA detection in the gel

(Fig. 33A). The Zn2+-dependent nucleases (Fig. 33B) showed a similar pattern of

banding with the addition of a fourth band at 35 kDa and a slight decrease in

activation of the 36.5 kDa and 38.5 kDa nucleases. The 35 kDa nuclease

appeared to increase slightly at 10 DAP and persisted through 12 DAP. As a

control the Ca2+ and Zn2+ ge S were incubated at acidic (5.5) and neutral (6.8)

pH, respectively, to test the effect of improper pH on each nuclease (Fig. 33C











and D). The results were a complete inactivation of all nucleases except the

smallest one. EGTA and EDTA were then added to the Ca2+/Mg2+ and Zn2+

assays. Each was then incubated at its proper pH. The Ca2+/Mg2+ gel Showed a

loss of activity in all nucleases except the smallest one; however, the Zn2+ gel did

not show a reduction in activation most likely due to the acidic conditions of the

incubation medium which inhibits the ability of EGTA and EDTA to act as

chelating agents (Fig. 34A and C). Lastly, the gels were incubated at their

respective pHs without the metals included in the incubation media. The gels

showed the same pattern of nuclease banding as with their respective metals

added indicating that these metals must be present and inherent in the tissue

itself (Fig. 34B and D).

Genomic DNA Extraction

Genomic DNA was extracted to try to identify inter-nucleosomal laddering in

8-24 DAP PC tissues (Fig. 35). No inter-nucleosomal banding was detected for

the DAP's tested.





















Top of caryopsis



















Base of caryopsis


Figure 3. Schematic drawing illustrating the regions of the maize endosperm.

The crown is the upper-most region of the endosperm. The central crown is the
interior portion of the crown. The mid-endosperm lies parallel to the embryo and
the lower endosperm subtends the mid-endosperm and extends to the basal
endosperm transfer cells, the first layer of cells at the base of the endosperm just
above the placenta-chalaza region (PC).

























Figure 4 Nuclear morphological changes in the endosperm of maize

Nuclear morphological changes in the endosperm of maize at 12, 14, 16, and 24
days after pollination (DAP) visualized via DAPI DNA stain. Crown: through 14
DAP the chromatin in the nuclei appears round and diffuse. By 16 DAP the
nuclei and chromatin have started to condense. By 24 DAP the nuclei are so
condensed they have become difficult to discern. Central crown: the nuclei show
the greatest morphological changes in this region of the endosperm through
development. At 12 DAP the nuclei are round and diffuse. At 14 DAP the nuclei
have enlarged and the chromatin is starting to appear more granular. By 16 DAP
the nuclei have substantially enlarged and the chromatin has condensed at the
nuclear periphery. By 24 DAP the nuclei have condensed appearing like those in
the crown. Mid endosperm: through 14 DAP the chromatin in the nuclei is diffuse
and round. By 16 DAP the nuclei have enlarged slightly and appear similar to
those of the central crown at 14 DAP in that the chromatin has started to become
granular in appearance. By 24 DAP the chromatin has condensed at the nuclear
periphery and appears like the nuclei in the central crown at 16 DAP. Lower
endosperm: the nuclei remain relatively unchanged up through 24 DAP. The
nuclei retain their size and diffuse chromatin. Magnification: 20x.






























111~1~Z~~rrol


IL~r~R~C~~~g~rc~rlllI














































Figure 5 PCD in the endosperm of a 24 DAP caryopsis.

(A-C) DAPI DNA stain was used to identify the location of nuclei in the cells.
(D-F) Terminal deoxynucleotidyltransferase mediated dUTP nick end labeling
(TUNEL) assay was used to show broken and nicked DNA in the nuclei denoted
by green fluorescence. (G) The negative TUNEL control shows no green
fluorescently labeled nuclei. The nuclei in the upper endosperm are condensed
while those of the mid endosperm are ring-like in appearance due to chromatin
condensation at the nuclear envelope. The nuclei in the lower endosperm are
rounded and the chromatin is diffuse throughout each nucleus. Only the mid and
upper endosperm are showing TUNEL positive activity. Magnification, 20x.

















































Figure 6 Confocal image of the nuclei in the central crown region of the
endosperm of a 28 DAP caryopsis of maize.


The nuclei (orange color) appear completely condensed at maturity, have shifted
to the cell periphery and are appressed against the cell wall (green). Nuclei
stained with propidium iodide. Magnification, 20x.





































Figure 7 Nuclear morphology of the central crown region of the endosperm of a
12 DAP maize caryopsis.

At 12 DAP the nuclei of the central upper endosperm appear normal; the
chromatin is diffuse and the nuclei are, generally, centrally located. (A) A light
micrograph of a TBO stained resin section of endosperm used as reference for
(B). Arrows point to nuclei. (B) TEM image of endosperm nucleus N, nucleus; s,
starch grain. Magnification for A, 20x; B, bar 6Cim.










































Figure 8. Nuclear Morphology of the central crown region of the endosperm of a
14 DAP maize caryopsis.

(A) TEM image. The nucleus is enlarged and, while not yet condensed, the DNA
has collected at the periphery of the nucleus. The nucleolus is still prominent.
(B, C) TEM images. Relative to all other developmental stages examined, the
endosperm, at 14 DAP, appears to be it's most active in forming protein bodies.
(C) The protein bodies (box) bud directly from the rough ER.
N, nucleus; n, nucleolus; rER, rough endoplasmic reticulum.
Bars: A, B, 6Cim; C, 0.5Cim.














































Figure 9 Nuclear Morphology of the central crown region of the endosperm of a
16 DAP maize caryopsis.

Nuclei are enlarged and are starting to display condensed chromatin at the
nuclear envelope. (A) TBO stained resin section of endosperm. Boxes enclose
nuclei. (B) TEM image of a nucleus. (C) TEM composite of a nucleus.
N, nucleus; cc, condensed chromatin. Magnification; A, 20x; Bars: B, C, 6Cim.









































'i"jri
i~ei: :ii~l~
"'


Figure 10 Nuclear Morphology of the central crown region of the endosperm of a
20 DAP maize caryopsis.

Similar to 16 DAP the chromatin is condensed and adhered to the nuclear
envelope but the nuclei are slightly more enlarged. The nucleoli are still present.
The nuclei have shifted to the periphery of each cell. (A): TBO stained resin
section. Boxes enclose nuclei. (B, C): TEM images of nuclei.
N, nucleus; n, nucleolus. Magnification; A, 20x; Bars: B, C, 5Cim.














































Figure 11 Nuclear morphology of the central crown region of the endosperm of a
22 DAP caryopsis of maize.

TEM image of the remnants of a collapsing nucleus. Some condensed
chromatin, denoted by black masses, is associated with the nuclear envelope.
N, nucleus; cc, condensed chromatin. Bar: 5Cim.




































Figure 12 Nuclear Morphology of the central crown region of the endosperm of a
25 DAP caryopsis of maize.

By 25 DAP the nuclei have collapsed and shifted to the periphery of the cell. The
nuclei have a disorganized, amorphous appearance. Close inspection reveals
no organization of the nucleus but shows lipid or fatty appearing spheres
surrounded by an electron dense material. These may be left over membranous
and chromatin materials from the dissociated nucleus. (A) TBO stained resin
section. Box encloses a nucleus. (B) TEM image of nucleus. (C) TEM image,
close up of nucleus. N, nucleus. Magnification: A, 20x; Bars: B, 5Cim; C, 0.5Cim.


C~h



%i

















































Figure 13 Origin of the Placenta-chalaza (PC) region.

This 9 DAP caryopsis illustrates the PC region containing two distinct sets of
cells comprising two zones. The upper zone is contiguous with the nucellar
epidermis and the lower zone is contiguous with the inner integument. 1)
Endosperm, 2) Basal endosperm transfer cells (BETC), 3) Nucellar epidermis, 4)
Semi-permeable membrane (gray line) subtended by inner integument, 5) Outer
integument, 6) Inner pericarp, 7) Mid pericarp. Magnification: 20x.






















2

3~T,,
















Figure 14 Anatomy of the Placenta-chalaza.

The PC is comprised of two different types of cells creating two distinct zones,
the nucellus-derived PC and the integument-derived PC. The PC is sandwiched
between the basal endosperm transfer cells (BETC) and the pedicel
parenchyma. 1) Endosperm, 2) (BETC), 3) Nucellus derived PC, 4) Closing layer
(part of integument derived PC), 5) Integument derived PC, 6) Pedicel
parenchyma. Magnification: 20x.




























Figure 15 Placenta-chalaza (PC): left-right borders.

The boundaries of the PC region are denoted by the developing semi-permeable
membrane. The PC region lies between the ends of the semi-permeable
membrane at the base of the caryopsis thus becoming the only port through
which water and nutrients can pass. Column A represents the adaxial side of
caryopsis while column B represents the abaxial side of caryopsis. (2 DAP) The
membrane starts its development below the developing endosperm first followed
by rapid development at the abaxial face of the lower kernel opposite the
developing endosperm. (6 DAP) The membrane has developed fully around the
entire seed except at the PC where it ends abruptly. (8-10 DAP) The membrane
matures and continues to thicken. Magnification: 20x.









2 DAP


1
!It


6DAP


8DAP


<


i"


f

J


PC


















































Figure 16 TEM image of one of the boundaries of the PC region.

A semi-permeable membrane develops from the nucellus epidermis and
surrounds the entire ovule but ends abruptly where the PC region begins at the
base of the caryopsis. 1) Nucellus epidermis, 2) Semipermeable membrane, 3)
Fatty substances, 4) Inner integument, 5) Membrane terminus, 6) Start of PC.
Bar, 5plm.























10 DAP

A B

Horizontal Ilmits of PC





Vertical Ilmits ofPC



Figure 17 Placenta-chalaza boundaries.

The horizontal limits of the PC region are defined by the start points of the
semipermeable membrane and the vertical limits are defined by the nucellus-
derived and integument-derived cell layers sandwiched between the basal
endosperm transfer cells and the pedicel parenchyma cells. (A) Abaxial side of
the caryopsis, (B) Adaxial side of the caryopsis. Magnification, 20x.







































Figure 18 4'6-diam idino-2-phenylindole-2HCI (DAPI) DNA stain.


Nuclei appear as blue, glowing spheres or spots. Enucleation of PC is
fertilization dependent. Enucleation is denoted by a lack of blue glowing nuclei in
the cells (black area). (A) Equivalent of a 12 DAP caryopsis, unpollinated ovule,
(B) 12 DAP caryopsis. Magnification, 20x.














































Figure 19 Crystal Violet Stain showing condensed chromatin in the nuclei of the
cells of the integument-derived PC region from 4 through 10 DAP.

Figures on the right hand side are enlargements of the corresponding figures to
the left Regions that are enlarged are denoted by the red lines. (4 DAP) There
are no condensing nuclei in the PC at this stage of development. (6 DAP)
Condensed nuclei start appearing at this stage in the integument-derived portion
of the PC in what will be the closing layer (cl) and in the cells just below the
closing layer. (10 DAP) Condensed nuclei remain prevalent at this stage.
Magnification; Left side 10x; right side 40x.
















































Figure 20 Crystal Violet Stain showing condensed chromatin in the nuclei of the
integument-derived PC region from 12 through 16 DAP.

Figures on the right hand side are enlargements of the corresponding figures to
the left. Regions that are enlarged are denoted by the red lines. (12 DAP)
Condensed nuclei (arrows) are limited to just below the closing layer. (14 DAP)
Condensed nuclei have become sparse and are a little further down beneath the
closing layer relative to 12 DAP. (16 DAP) There are no more condensed nuclei
in the PC at this stage. Magnification; Left side 10x; right side 40x.




























Figure 21 TUNEL assays of PC region showing broken or nicked DNA in the
nuclei of the integument-derived portion of the PC

Terminal deoxynucleotidyl transferase mediatd dUTP nick end labeling (TUNEL)
assays of PC region showing broken or nicked DNA in the nuclei of the
integument-derived portion of the PC (red brackets). Nicked or broken DNA is
denoted by bright green fluorescence. The nucellus-derived PC cells test
TUNEL negative as indicated by a lack of green fluorescent nuclei. (A, C, E, G)
experimental sections. (B, D, F, H) negative controls. (4 DAP) TUNEL activity is
not yet detected. (8 DAP) TUNEL activity is well established. (10 DAP) TUNEL
activity peaks. (12 DAP) TUNEL activity is still seen but starts to taper off after
12 DAP. Magnification; A-F, 20x; G,H, 10x. From Kladnik et al., unpublished.







62












0..












0..











0..
O
o










0
(11
t**


























Figue 2 Crstlvoestishwncodnechoaiintenegm t

derived~~~~~ poto f h Creino a2 A cross










noncomre 2 rssed reion of tin he wn P.()Condensed nclemaini appea in thegm


compressed region of t PC. iNuclar oies (arrow)l apear nd one of thecels C

There are no condensed nuclei in the non-compressed region. Magnification;
A, 10x; B, 40x.
















































Figure 23 TEM micrographs illustrating consecutive developmental stages and
subsequent cell death of the nucellus-derived PC region.

This is the region that tests TUNEL negative. (A) At 0 DAP cells appear normal,
contain all organelles and large vacuoles. (B) By 5 DAP nuclei and all organelles
are present and the cells appear normal. (C) By 7 DAP the cells have lost all
organelles and nuclei and have plasmolyzed. (D) By 9 DAP the cells have
almost emptied completely and only degraded cytoplasmic contents remain.
Arrow heads, mitochondria; arrows, plasmolyzed cellular remains; (N), nuclei;
(DC), degenerated cytoplasmic contents. Bars, 5Cim.


10 DAP


5 DAP


7 DAP 9 DAP




































C










Figure 24 TEM micrographs illustrating the plasmodesmata (PD) in the walls of
the nucelluc-derived PC cells.

(A) At 5 DAP the average number of identifiable PD in the walls of the cells per
section is 7.2. (B) The average number of identifiable PD in the walls starts to
decrease. (C) By 9 DAP the average number identifiable PD decreases to 2.4
per cell per section. Bars: A, 0.5Cim; B, 1Cim; C, 0.5Cim.










EIEnp


EEDAP


EEDAP


Figure 25 Development and subsequent death of upper integument-derived
region of PC.
The upper integument-derived region forms the closing layer of the kernel. This
is the region of the PC that tests TUNEL positive. (A) At 0 DAP the cells appear
normal, contain large vacuoles and all organelles are present. (B) Close up of
cells in A showing normal appearing organelles. (C) By 7 DAP the cells have
thickened walls, more dense cytoplasm, smaller vacuoles and some of the nuclei
start to display condensing chromatin (arrows). (D) By 9 DAP the cytoplasm
becomes very dense, the vacuoles break up into mini-vacuoles, there are no
identifiable organelles, and chromatin in nuclei continues to condense. Arrow
heads, mitochondria; arrows, nuclei with condensed chromatin; N, nucleus; C,
cytoplasm; V, vacuoles. Bars, 5[im.


O DAP
loopsl



























Figure 26 Development and subsequent death of lower integument-derived
region of PC.

(A) At 0 DAP cells appear normal and contain all organelles and large vacuoles.
(B) By 5 DAP cells are still normal but the vacuoles are smaller and the
cytoplasm more dense. (C) By 7 DAP the cells still retain their organelles and
the nuclei still appear normal but, in some cells, the vacuoles are starting to
break into smaller vacuoles. The cytoplasm takes on a granular appearance.
(D) At 9 DAP the cells have lost all identifiable organelles, vacuoles, and nuclei.
The cytoplasm still appears granular. This layer starts to become compressed by
the growing endosperm and the cells appear flattened. (E) The cell layer below
D. These cells test TUNEL positive and contain nuclei that have condensed
chromatin. There are many mini-vacuoles present. V, vacuoles; arrows, nuclei.
Bars, 5[im.






















9 DAP
^-ntwa iD





































9 DAP 11 DAP
Figure 27 Lower Integument-derived PC Cells.


Starting at approximately 9 DAP cells in the lowest portion of the PC
display abundant ER in the form of concentric rings or layered lamina. This
is characteristic of this cell layer up through at least 11 DAP.
V, vacuoles; ER, endoplasmic reticulum; N, nucleus. Bars, 2[tm.






































Figure 28 Nuclear body formation in the integument-derived portion of the PC
cells.

A) TUNEL assay showing nuclear bodies (boxed) at the cell periphery. Arrow
shows one of the highly condensed TUNEL positive nuclei. (Micrograph by
Shayna Sutherland). (B) TEM micrograph showing the same type of nuclear
morphological pattern seen in A. The nuclei have completely condensed and
have started breaking off into nuclear bodies. Note the nuclear envelope is still
present and intact.
Magnification; A, 40x; B, bar 0.5[tm.













A L ,* 1-'


Figure 29 Nuclear morphological changes in integument derived portion of the
PC.

Nuclear morphological changes in the integument-portion of the PC
resemble apoptotic-like morphology. The micrographs of each progressive stage
of each changed nucleus are not from consecutive developmental stages as the
PCD taking place in one cell is independent of PCD occurring in all other cells
and the rate of progression between cells will vary. However, this series of
nuclear changes is progressive from when a cell starts showing morphological
changes through the end of its PCD process. (A) Normal appearing nucleus.
The DNA has not started showing any abnormal chromatin condensation. (B) A
nucleus showing first stages of chromatin condensation. (C) Highly shrunken
nucleus with completely condensed chromatin. (D) Nuclear body-remnants of the
nucleus filled with condensed chromatin (arrows). N, nucleus; m, mitochondria.
Bars: A, 5itm; B, 1.8[tm; C, 1.2[tm; D, 1.8[tm.


IWI











Caf localized


Figure 30 Ca2+ Trapping: Nucellus-derived PC.

Free Ca2+ was localized in an attempt to define a flux and organelle interaction of
this ion during PCD of the cell. (A) At 5 DAP Ca2+ is sparse and is associated,
primarily, with membranous structures and the nucleus but is not seen in the
cytoplasm or mitochondria. (B) At 8 DAP Ca2+ is abundant in the cell corpse but
does not appear to be associated with the degenerated cellular remains.
(C) By 11 DAP Ca2+ has disappeared from the cell corpse and is only lightly
associated with degenerated cellular remains. N, nucleus; m, mitochondria; DC,
degenerated cytoplasmic contents. Bars: A-D, 0.5[tm; E-F, 0.8[tm.


control

I^S B^




























Figure 31 Ca2+ Trapping in the integument-derived PC.

Free Ca2+ was localized in an attempt to define a flux and organelle interaction of
this ion during PCD of the cell. (A) At 5DAP Ca2+ is very low in the cytosol but is
associated with membranes of the ER and vacuole. In addition, Ca is
peppered throughout the mitochondria and is very light in the nucleus. (B) By 8
DAP Ca2+ is low but present in the cytolsol. It is also seen in some of the ER
under the nucleus (bracket). However, other rough ER is still present but no Ca2+
is seen within it (arrows). (C) By 11 DAP Ca2+ is extremely low in all places in the
cell. The cell is in an extraordinary chaos and no nucleus or organelles are left.
V, vacuoles; m, mitochondria; N, nucleus; n, nucleolus. Bars, 5[tm.











Ca* localized


11 DAP


'ftr


S1JW


V4


urt
p 1
p 4


F


Ne
A __ M


control


c ~'I~B~I1B~RL~


V ~: ~:= ~9



























Figure 32 Ca2+ trapping in the normal appearing, lower integument PC.

Free Ca2+ was localized in an attempt to define a flux and organelle interaction of
this ion during PCD of the cell. (A) A normal appearing cell shows low Ca2+ in
the cytosol and mitochondria but higher concentrations in the nucleus and very
high concentrations sequestered in the vacuole. (Inset: enlarged image of
mitochondria). (B) Ca2+ localizes to the ER lumen and nucleus. (C) Ca2+ has
collected in the mitochondrial matrices and vacuoles. However, Ca2+ is in low
concentrations in the cytosol. (D) A cell showing a nucleus with mostly
condensed chromatin. The nucleus still shows moderate amounts of Ca2+ and
the mitochondria have small amounts of Ca2+ in their matrices; one displays great
swelling at this stage._(E) Negative control. N, nucleus; V, vacuole; m,
mitochondria; ER, endoplasmic reticulum. Bars: A, D, 0.8[tm; B, C, E, 0.5[m.























































E tm


~"'





















Figure 33 In-gel nuclease activity assays for Ca2+, Mg2+ and Zn2+-dependent nucleases and pH controls.

PC tissue was isolated from the rest of the caryopsis and proteins from thr PC were extracted for nuclease activity.
Whether tested together or separately, the Ca +, Mg2+ -dependent nucleases resulted in the same banding pattern.
(A) The Ca2+-dependent assay shows greatest nuclease activity with a 36.5kD nuclease (arrow) first appearing at
and peeking in activity at 8 DAP. (B) In the Zn2+-dependent assay, nuclease activity is greatest at 8 DAP The
same pattern of banding is seen as in the Ca2+- dependent nuclease assay but unlike the Ca2+-dependent nuclease
activity assay a fourth nuclease is present at approximately the 35kD mark (red arrow). However, this nuclease is
seen starting at 6 DAP, persists throughl2 DAP and is still seen faintly at 28 DAP. (C) Most of the Ca2+-dependent
nuclease activity was eliminated when incubated at an acidic pH of 5.5, that which is required for Zn2+-dependent
nuclease activation. Ca2+-dependent nuclease activity requires a neutral pH of 6.8. (D) Most of the Zn2+-
dependent nuclease activity was eliminated when incubated at a neutral pH of 6.8, that which is required for Ca2-
dependent nuclease activation. Zn2+-dependent nuclease activity requires an acidic pH of 5.5.
M, size marker.






















8DAP 12DAP 28DAP Un-P. M


45.7kD -



32.5kD











45.7 kD-


A Ca2+








m1-- U -




M 6DAP 8DAP 10DAP 12DAP 28DAP


R Zn2


M 6DAP DAP AP 12DAP 28DAP

M 6DAP BDAP 10DAP 12DAP 2ODAP


FE


... m a


- 38.5
35.0
33.5


32 5 kD


M 6DAP BDAP 10DAP 12DAP 28DAP


;.;;
i*:
Li~i~- ~u





















Figure 34 In-gel nuclease activity assays with EDTA and EGTA and no metals.

PC tissue was isolated from the rest of the caryopsis and proteins from the PC were extracted for nuclease activity.
(A) EDTA and EGTA effectively reduced or eliminated nuclease activity. Only the smallest nuclease showed any
activity. However when the Zn2+ gel (not shown) was incubated with these chelating agents there was no inhibitory
effect. This may have been due to the acidic conditions required for Zn2+ activation limiting the chelating abilities of
the EDTA and EGTA. (B and C) When metals were eliminated from the incubation medium the same typical
pattern for each of the nucleases still appeared. This is, presumably,due to metals inherent in the tissue.
M, size marker.
























6DAP 8DAP 10DAP 12DAP 28DAP


C Zn2* + EDTA & EGTA










6DAP 8DAP 10DAP 12DAP 28DAP


6DAP 8DAP 10DAP 12DAP 28DAP


6DAP 8DAP 10DAP 12DAP 28DAP


A Ca2+/Mg2++ EDTA & EGTA


a- S-


a -




















2000
1200
800
400
200
100


x /
(b


4


4Q


0
Co


I


q
ev


MINI
wUww -


Sff


Figure 35 Genomic DNA isolated from the PC region.
Genomic DNA was isolated from PC tissues of 8, 10, 12, 16, 20, and 24 DAP
caryopses and electrophoresed on a two per-cent gel to test for internucleosomal
DNA laddering. No nucleosomal DNA laddering was detected in the PC. LML:
Low mass ladder.














CHAPTER 4
DISCUSSION

PCD in the PC Region

Plants undergo PCD as a normal developmental process in a wide variety

of cell types and exhibit PCD in a variety of different ways, morphologically.

Examples include aerenchyma formation, tracheary element differetiation,

compound or hole developing leaves, and senescence (Jones and Dangl, 1996;

Gietl and Schmid, 2001; Obara et al., 2001). PCD occurs within the different

parts of a seed as a part of normal growth and development (Domingues et al.,

2001; Schmid et al., 1999; Wojciechowska and Olszewska, 2003).

Within the maize caryopsis, PCD has been reported in cells of the nucellus,

suspensor, scutellum, and endosperm (Giuliani et al., 2002; Young and Gallie,

2000; Young et al., 1997). All these processes are highly controlled and critically

timed. However, within a temporal context the first of these processes to be

observed is PCD in the PC region. The PC region of the maize caryopsis is the

only port of entry through which water and nutrients pass from the maternal

sporophytic tissue to the filial tissue. While the PC is, itself, a maternal tissue, it

goes through a fertilization dependent programmed cell death (PCD) as part of a

normal developmental regimen (Kladnik et al., unpublished). As detailed in the

results section there are two distinct zones in the PC that undergo two distinct

PCD processes. The PC in maize is comprised of only nucellar and integument-

derived tissues as maize seeds do not possess a funiculus. The first of these









two zones is that portion of the PC that is derived from the nucellus epidermis.

The nucellar-PC zone undergoes an extremely rapid death starting at

approximately 6 DAP and ending at about 8 DAP. This process does not

resemble an apoptotic-like cell death. These cells do not test TUNEL positive at

any point tested indicating a lack of detectable DNA cleavage nor do the nuclei

ever appear to contain condensed chromatin. In fact, no intermediate stages of

cellular degradation were seen between the completely normal appearing cells

and those cells with degraded cytoplasmic remains with the exception of an

occasional plasmolysed cell. Since these cells start out with a very large vacuole

at the time that they appear intact, but have no trace of a vacuole when they are

plasmolysed or degraded, perhaps it is a death mediated by vacuolar rupture and

autolysis similar to that of tracheary elements (TE) (Obara et al., 2001). It is

through this process in TE differentiation that proteolytic and digestive enzymes

are released into the cytoplasm (Yu et al., 2002). Obara et al. (2001) reported a

complete degradation of the nucleus within 15 minutes and a complete

degradation of the cytoplasm within two hours after the rupture of the central

vacuole. The DNA degradation appeared to start in the center of the nucleus

while the condensed chromatin left at the periphery was digested last leaving

behind only the nuclear envelope. While TUNEL assays showed positive results

in the nuclei of these cells there was no oligonucleosomal ladder detected on a

gel.

The second zone of cells of the PC is that portion which is derived from the

inner integument. As with the nucellar-PC, PCD in the integument-PC is









detected starting at approximately 6 DAP, peaks at about 10 DAP, then subsides

after 12 DAP. This PCD process morphologically resembles apoptosis in animal

cells (Kerr et al., 1995) in that both undergo extensive chromatin condensation

delineated by sharp clean edges and retention of the nucleolus, nuclear

shrinkage, nuclear body formation, cytoplasmic condensation and retain all

organelles and membranes until these are the only cellular components left. The

cell then completely degenerates and the cellular contents become mostly

absorbed. While these similarities are shared, the exact pattern of chromatin

condensation in the PC nuclei is a little different than that in animals in that it

does not appear to amass at the nuclear periphery.

PCD in the PC May Be Tied to Growth and Development of the Filial
Tissues

Since the maternal tissue of the pedicel and PC is the only region through

which nutrients can pass to the developing filial tissues of the endosperm and

embryo it is apparent these three tissues are intimately linked, physiologically.

Schel et al. (1984) examined early developmental stages of endosperm and

embryo interaction in maize. They noted that the endosperm precedes the

embryo in development and that there is a 4X increase in size of the endosperm

relative to the embryo from 4 through 8 DAP, creating a high sink demand. From

8 DAP through 14 DAP the endosperm suddenly increases its relative growth

rate; within this period at approximately 9 DAP, the embryo starts a rapid

increase in growth rate and size thus further increasing the sink demand. After

14 DAP the growth rates of each, the endosperm and the embryo, starts to slow;

the endosperm's rate of growth slows prior to that of the embryo's (Schel et al.,









1984). It has been noted in past anatomical studies of the developing maize

caryopsis that as the tissues develop, the nucellus tissue is destroyed by the

growing endosperm, which is in turn destroyed by the growing embryo

(Weatherwax, 1930). Weatherwax (1930) points out that the bordering cells at

the interface of these tissues display a regular pattern in that the cells of the

tissues being destroyed, the nucellus, for example, plasmolyze before

destruction while the growing tissue border cells, the endosperm, for example,

remain turgid. While it was not known what caused this phenomenon it appeared

that the majority of nutrition was still delivered via the PC (Weatherwax, 1930).

However, it must be pointed out that recent findings have shown that these

tissues undergo PCD as the caryopsis develops and that it is not, simply, the

mechanical pressure of the growing tissues that is destroying the extant tissues,

i.e. the destruction of the nucellus as the endosperm grows (Linnestad et al.,

1998). The osmoticum of the tissues in the caryopsis has been shown to be a

critical factor for normal maize seed development (Zinselmeier et al., 1999). For

example, it has been shown that there must be a critical level of sucrose

available to the developing tissues in the seed immediately after pollination or the

development of the embryo ceases and it is aborted. This was accompanied by

the observation that when the maize seeds were kept at very low water potentials

at the time of fertilization the starch reserves in the fruit wall were depleted and

were most likely an attempt, by the seed, to maintain proper sugar levels in order

to maintain adequate water levels for development (Zinselmeier et al., 1999). In

the opinion of the author of this present study, perhaps one explanation for the









ability of the growing border tissues to maintain turgidity is that they maintain a

higher water potential than their surrounding tissues. For example, the nucellus

is not a starchy tissue nor is it a physiological sink, as is the endosperm and

embryo, and the cells contain very large central vacuoles. Perhaps the nucellus

acts more as a water reserve for the growing endosperm than a source of

nutrition.

Sink Demand and Assimilate Flow

The growing endosperm and embryo require sugars and other nutrients that

are delivered to the pedicel via the phloem (Weatherwax, 1930; Felker and

Shannon, 1980). The vasculature terminates at the base of the pedicel and the

assimilates must be unloaded from the phloem and pass through the pedicel

parenchyma cells, PC cells, the basal endosperm transfer cells (BETC), then into

the endosperm and growing embryo. However, it is still not clear exactly what

path the assimilates flow through once they have unloaded from the phloem.

Felker and Shannon (1980) proposed a path of flow for the sugars based on

microautoradiographs of the pedicel after the plant was exposed to 14C02 and

was allowed to assimilate the radioactive carbon into sugars. They concluded

the assimilates pass through the pedicel cells symplastically from the phloem

through the PC until they reach the border between the PC and BETC cells at

which point flow must turn apoplastic due to a complete lack of plasmodesmatal

connections (Felker and Shannon, 1980) Felker and Shannon (1980) point out

a division in radiolabeled sucrose accumulation between the central pedicel

parenchyma, PC and the endosperm within the same tissue sample, there being

a noticeable accumulation of radiolabeled sucrose in the pedicel parenchyma









and endosperm but a lack of accumulation through the PC. However, they

stated that there are appreciable limitations in this method. The limitations

discussed mention that there is no way to determine the presence of radiolabeled

sucrose in the "free space" or apoplast. Therefore, this study could not

conclusively determine where sugars could not be found but did shed light on

where the sugars could be found. Additionally this study focused on relatively

mature caryopsiss of 22 DAP which rules out the possible changes in the path

assimilates might take during varying developmental stages, especially the

earliest most stages.

Based on the morphological changes seen in the PC and pedicel during the

this present study, coupled with data from the aforementioned studies, it would

seem that the path of assimilate flow from the phloem to the endosperm must

vary depending on the stage being examined, i.e. symplastic at early stages (0-6

DAP) versus apoplastic at more mature stages (7 DAP and later). This may also

hold true for certain conclusions that can be drawn about the physiological

processes of the growing seed that may be contributing to the initiation of PCD in

the PC.

Two Separate, Temporal Waves of PCD Occur in the PC and May Be Linked
to the Sink Demand of the Growing Filial Tissues

The endosperm in maize begins cellularization at approximately 4 DAP

during the R1 stage, 0 to 9 DAP, (Esau 1977; Ritchie, 1993). The first wave of

PCD in the PC follows shortly after the beginning of cellularization of the

endosperm at approximately 5 to 6 DAP. The second wave of PCD in the PC

begins during the R3 or "milk" stage, 18 to 22 DAP, when endosperm cellular









division has completed and the cells are maximizing starch filling (Ritchie,1993).

Therefore, there are two physiological processes that may be contributing to the

PCD occurring in the PC cells during the two waves of cell death.

The first physiological contributor during the first wave, starting after 5 DAP,

may be an osmotically induced process due to the strong sink of the developing

endosperm and embryo. Schel et al. (1984) concluded growth rate is slow

through approximately 4 DAP; therefore it can be deduced that nutrient demand

is at a minimum. Since there are abundant plasmodesmata in all cells of the

pedicel at this stage, an average of 7.1 per cell per section, in the nucellus-

derived PC cells, and all cells are normal in appearance, assimilates most likely

travel symplastically throughout the entire pedicel and PC region. Addtionally,

since there is not yet a symplastically discontinuous border between the PC and

the endosperm, assimilates most likely flow symplastically into the growing

endosperm. Schel et al. (1984) also concluded that the endosperm starts an

extremely rapid rate of growth from 5 through 8 DAP. Thus it can be deduced

that assimilate demand increases. This coincides with the rapid PCD in the

nucellus-derived region of the PC at the end of which the cells have almost

entirely emptied and the identifiable plasmodesmata are staring to decrease in

number. Towards the latter end of this stage the endosperm has grown large

enough in size to have formed a loosely packed, symplastically discontinuous,

multi-cell walled border with the PC. In addition, during this period the

integument-derived region of the PC is starting to display TUNEL positive nuclei

(Kladnik et al., unpublished). With a reduction in the number of plasmodesmata









and only hollow corpses of PC cells left in the nucellus-derived PC it can be

deduced that assimilates flow symplastically from the phloem through the

integument-PC. Upon encountering the nucellus-PC, the assimilates would

encounter a disruption in the symplast and change to an apoplastic means of

flow. The flow may still travel through the cell corpses in the nucellus-derived

PC. Since the apoplast is defined as the pathway for water and solute transport

that lies outside the plasma membrane, including inter cellular spaces and xylem

transport (Buchanan et al., 2000), the flow of assimilates through the cell corpses

would still be considered apoplastic since there are no plasma membranes or

cytoplasmic connections. The disruption and conversion of the flow from the

symplast to the apoplast might cause a transient "backup" of hyper-osmotic fluid

in the integument-derived PC cells immediately subtending the nucellus-PC cells

creating an osmotic shock. Studies examining cell wall invertase (INCW2), a cell

wall enzyme that irreversibly cleaves sucrose to glucose and fructose, showed

specific localization to the BETC cells of maize caryopses and more specifically

to the cell wall ingrowths of the BETC cells (Cheng et al., 1996). The cell wall

ingrowths in the BETC cells start developing at approximately 7 DAP and are

mature by 12 DAP (data not shown). It is immediately prior to the development

of the cell wall ingrowths that the PCD process in the PC is initiated and not until

the wall ingrowths, and thus the INCW2 expression, are mature that the PCD

process ceases. In other words, it is concomitant with, and seemingly not until

the sucrose can be readily passed into the endosperm that the PCD process

ends. This shows a correlation between the sink strength of the endosperm, the









sucrose passing through the PC and the possibility of a transient backup of

assimilates. The invertase deficient mutant in maize, mnl-1, is incapable of

cleaving sucrose in the BETC and shows anatomical features of an

underdeveloped endosperm leading to the development of a large gap

separating the pedicel from the endosperm (Cheng et al., 1996). Cheng et al.

(1996) reference a previous study in which a higher concentration of sucrose was

found to exist in the pedicel of the mnl-1 mutant. Because the mnl-1

endosperm is not applying mechanical pressure on the PC and the PC still

shows PCD (data not shown) as does the wild type caryopsis it is reasonable to

speculate that PCD in the PC region of maize is osmitcally influenced.

Schel et al. (1984) noted that both the endosperm and embryo increase

rapidly in size from approximately 9 to 14 DAP This coincides with the highest

peak of TUNEL positive nuclei, i.e. about 10 DAP, in the integument-derived

region of the PC (Kladnik et al., unpublished). Schel et al. (1984) also show a

decrease in the growth rate at 14 DAP which is coincidental with the last signs of

any TUNEL activity seen (Kladnik et al., unpublished). At this point in

development the BETC cells have developed extensive cell wall in-growths, the

PC has formed a symplastically discontinuous, compacted, multi-cell walled

barrier from crushed nucellar-PC cells with the BETC, and the number of

identifiable plasmodesmata have decreased appreciably in the nucellar-PC cells

from an average of 7.1 per cell per section to 2.4.

The second possible contributing factor to the PCD in these cells may play

a role during both waves of PCD but may be the primary cause of PCD during