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The concept of apoptosis (or cell death) originates from the Greek term describing the falling of leaves and petals during natural senescence. Senescence can be thought of as the terminal phase in the development of leaves and flowers, and thereby involves processes such as protein remobilization and protoplasmic elimination, which are characteristic of apoptosis and PCD. Modern research often uses apoptosis and PCD interchangeably, even in animal systems. However, caution needs to be exercised with the use of these terms. PCD has been defined in animal systems (Ellis et al., 1991) as a type of cell death that is a normal part of an organism's life cycle, is initiated by specific physiological signals, and requires de novo gene transcription. Apoptosis was originally used to define particular ultrastructural and biochemical characteristics of cells undergoing PCD, including chromatin condensation, membrane blebbing and DNA laddering (Kerr et al., 1972). However, the notion that all PCD manifests apoptotic features has been challenged (Schwartz et al., 1993); some animal cells appear to undergo physiologically programmed PCD without any of the apoptotic hallmarks. In plants, PCD has been recognized as part of a number of developmental processes including anther development, abscission of plant organs, megasporogenesis in angiosperms, sex determination, hypersensitive responses, tracheary element formation, destruction of the suspensor, and formation of aerenchyma (Jones & Dangl, 1996; Ranganath & Nagashree, 2000). In some of these cell types apoptotic characteristics can be detected, in others however, not all of the recognized signs of apoptosis are observed, leading some to question the similarities between senescence and PCD (Thomas et al., 2003).
The structural changes that occur during PCD can be followed by electron microscopy. In the early 1970s Matile & Winkenbach (1971) suggested that in Ipomoea petals the vacuole acts as an autophage. Invaginations of the tonoplast surround parts of the cytoplasm that are ultimately pinched off, resulting in lysosomal-like compartments within the vacuole. The membrane surrounding these vesicles decays and the cytoplasmic material then appears to be degraded by vacuolar enzymes. Such cytoplasmic degradation is concomitant with the visible collapse of the corolla but structural changes reminiscent of cell death have been described even before flower opening in Ipomoea (Phillips & Kende, 1980). In Sandersonia there is an increase in the air spaces between the mesophyll cells; this may be the result of tepal expansion without cell division or cell expansion. The mesophyll cells degrade further as the flower senesces such that the only identifiable cells other than epidermal cells are associated with vascular traces (O’Donoghue et al., 2002).
One of the easiest PCD markers to follow at a subcellular level is that of DNA laddering, which in animal cells is the result of the activation of a DNA ladder nuclease or caspase-activated DNase (CAD). However, several authors have questioned the correlation between laddering and apoptosis (Hengartner, 2000), and it has been shown that DNA fragmentation is not an absolute requirement for PCD (Vaux & Korsmeyer, 1999). Evidence for PCD in plants has been based extensively on the presence of DNA laddering, which has been observed in numerous systems. These include carpel cells of Pisum sativum (Orzáez & Granell, 1997), cowpea cells infected by fungi (Ryerson & Heath, 1996), during somatic embryogenesis of Norway spruce (Havel & Durzan, 1996), in developing anthers (Wang et al., 1999), in senescing tomato cells (Wang et al., 1996) and in senescing Petunia petals (Xu & Hanson, 2000).
Despite some similarities between animal and plant PCD at the cellular level, very few homologues of animal PCD-related genes have been identified in plants. One of the few is DAD-1 (defender against apoptotic death) that has been detected in Arabidopsis leaves (Gallois et al., 1997), citrus leaves (Moriguchi et al., 2000) and pea petals where, in all three, levels decline during senescence (Orzáez & Granell, 1997). A direct role in PCD was ascribed to this gene following the isolation of a hamster cell line in which a single amino acid change in the DAD-1 protein caused the cells to become apoptotic (Nakashima et al., 1993). In mammals DAD-1 encodes a subunit of oligosaccharyltransferase, an enzyme involved in N-linked glycosylation (Kelleher & Gilmore, 1997). Yeast and mammals with a disrupted DAD-1 gene show premature entry into PCD and express abnormal N-glycosylated proteins, although this has not yet been proven in plant systems. It has thus been suggested that DAD-1 may in fact not be a part of the PCD machinery, but rather an essential gene whose disruption triggers cells to enter PCD. Whether the plant DAD-1 protein performs the same function is not known. This gene, however, remains a useful marker as its expression is closely correlated with entry into cell death in several plant systems studied, although not in all (Danon et al., 2000).
The triggering and progression of PCD in plants can be usefully studied using petal senescence as a model system as this is a carefully programmed process; within a given species, it is possible to predict exactly when a bud will open and how rapidly the petals will senesce (Molisch, 1938). However, both the trigger and the co-ordination vary in different plant species. In one group of species the production of a burst of ethylene co-ordinates petal senescence, itself sometimes triggered by pollination (Stead, 1992). However, in another group, ethylene does not appear to co-ordinate senescence, even though it is almost always associated with the final event of petal abscission (Stead & van Doorn, 1994). Thus in these species, although a programme clearly exists, both the nature of the trigger and the co-ordination of cellular events is as yet unclear. Alstroemeria belongs to this latter group. Recent studies (Leverentz et al., 2002) indicate that unlike ethylene-insensitive species such as daylily and gladioli (Woltering & van Doorn, 1988; Peary & Prince, 1990; Rubinstein, 2000), lipoxygenase (responsible for oxidation of polyunsaturated fatty acids containing a 1,4 pentadiene moiety) does not appear to play a major role in bringing about petal senescence in Alstroemeria. Petal senescence in Alstroemeria is, however, associated with an increase in proteolytic activity and a dramatic depletion of complex lipids, suggesting active remobilization of nutrients from the floral tissues (Leverentz et al., 2002; Wagstaff et al., 2001, 2002).
In this paper cytological and molecular markers are used to chart the progression of PCD in Alstroemeria petals and thus this paper comprises the first integrated study of plant PCD using molecular, structural and physiological information. These results and observations are presented and used, together with information on this system from previous work (Leverentz et al., 2002; Wagstaff et al., 2002), to present a model for Alstroemeria senescence in which a coincidence between changes in membrane integrity and PCD markers is revealed.
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Two of the hallmarks of PCD in animal systems are DNA laddering and nuclear shrinkage (Kerr et al., 1972). Both of these processes are evident in Alstroemeria petals. By light microscopy nuclear area declined by nearly 50% of the maximum nuclear area seen at stage 1, however, the decline was continuous suggesting that the maximum nuclear area might be before flower opening. This was confirmed using slightly younger flowers and TEM. Both techniques revealed that the most rapid decline in nuclear area was between stages 4 and 5, i.e. concomitant with the first externally observed signs of petal deterioration. The degeneration of the nucleus occurs against a background of gradually increasing cell size, indeed cell size increased throughout the life of the petal, a situation that has been reported for other flowers (Phillips & Kende, 1980).
In Alstroemeria many indicators of PCD appear to start very early, indicating that some cell death is occurring from the earliest stages of flower development. This includes the gradual increase in the expression of a cysteine protease (Wagstaff et al., 2002) starting from the earliest tissues examined (stage 1) and the sharp decline in total LOX activity and lipid content (Leverentz et al., 2002), again starting early in floral development (stage 1 and stage 2, respectively). It also supports the structural data since the reduction in nuclear size occurs from a very early stage of flower development (before Stage 1). However, in addition to an early start of some PCD-associated processes, another feature of petal senescence in this species is that several of these processes appear to accelerate at the time at which the first visible signs of senescence are detectable. Total protease activity (Wagstaff et al., 2002), electrolyte leakage (Leverentz et al., 2002) and DNA laddering all rise sharply around stages 4–5. DAD-1 expression, used as another marker of PCD in this system, also declines 3-fold between stages 4 and 5. Although DNA laddering has not always been found in systems otherwise showing signs of PCD (Buckner et al., 2000; Herbert et al., 2001), it has been detected in many other plant PCD systems (Jones & Dangl, 1996; De Jong et al., 2000; Ranganath & Nagashree, 2000). A fall in DAD-1 expression has been associated with petal senescence in peas (Orzáez & Granell, 1997), leaf senescence in citrus (Moriguchi et al., 2000) and with silique maturation in Arabidopsis (Gallois et al., 1997). In the majority of theses cases the association of DAD-1 expression with senescence has been correlative, rather than causative, although a plant DAD-1 gene was shown to rescue a hamster cell line lacking the mammalian equivalent from entering into cell death (Gallois et al., 1997). In Alstroemeria petals, expression was very similar to that in pea petals, being high in young petals and declining in older petals. Put together, these data suggest to us that some key events in Alstroemeria petal senescence are occurring at around stage 4–5.
An important aspect of petal senescence is the spatial distribution of cell death across the petals. Inrolling and wilting in Alstroemeria clearly starts in the petal margins, and further studies will need to address whether cell death starts in the margins of the petals, and moves inwards as senescence progresses. Ultrastructural evidence to date suggests that this is likely to be the case with the cells surrounding the vascular tissue being the very last to degrade (data not shown). The very marked loss of contents and collapse of the mesophyll layers of Alstroemeria petals occurs against a background of marked epidermal cell expansion. Expansion occurs in just one plane when the petals are viewed from the surface, although evidence from the ultramicrographs suggests that there may be some loss of depth in the epidermal cells during the latter stages of senescence. Previous studies of this system (Wagstaff et al., 2001) have shown that f. wt declines from S4; the collapse of the mesophyll cells at this stage may contribute to the f. wt loss. Thus some cells within the petal remain fully functional and gene transcription and translation are occurring until the latest stages of petal senescence. Although we do not know from which cells transcription and translation occurs, it shows that at least some cells are active (Wagstaff et al., 2002), even when other cells are at an advanced state of senescence. This is in accordance with studies of the ultrastructure and water relations of senescing Iris petals (Celikel & van Doorn, 1995; Bailly et al., 2001) and collapsing cells of senescing Sandersonia petals (O’Donoghue et al., 2002) and serves to illustrate the importance of examining tissues on a cell by cell basis, as well as looking at the more global picture. The increases seen in DNA laddering and the decline in ALSDAD-1 gene expression are a reflection of the average state of the whole petal, whereas individual cells, possibly even neighbouring cells, may be in different physiological states.
Bringing together the biochemical, ultrastructural and molecular data for this system (from the data presented in this paper and previous publications) suggests a pattern of sharp acceleration of cell death against a background of increasing nutrient degradation (Fig. 9). Some processes appear to occur gradually, e.g. nuclear condensation and lipid peroxidation, whereas other events, e.g. upregulation of protease activity, loss of membrane integrity as measured by increased conductivity, and DNA laddering are temporally more precise in their occurrence and coincide with a specific stage in the senescence programme when the first visible signs of petal deterioration become apparent. It is as yet unknown in species such as Alstroemeria, where ethylene does not seem to act as a co-ordinator, what the trigger for petal senescence might be, or how its progress might be regulated. We propose a model for Alstroemeria petal senescence in which the trigger is in fact a threshold effect of one or more of the gradual biochemical processes, e.g. nuclear shrinkage or lipid breakdown. This then initiates the rapid onset of more catastrophic processes, e.g. loss of membrane integrity, DNA laddering, mesophyll collapse resulting in PCD.
Figure 9. Summary of programmed cell death in Alstroemeria. (a) Events that show a steady increase from the first stage examined. (b) Events that show a decline from the first stage examined. (c) Events showing a sharp acceleration around stage 5 (first visible sign of senescence). (d) Events that show a sharp decrease between stages 4 and 5. Data in part derived from previous published studies by the same group on Alstroemeria senescence (Wagstaff et al., 2001, 2002; Leverentz et al., 2002).
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This study has illustrated how early some PCD processes start during senescence. Structural degradation occurs to both cell membranes and nuclei before the flower is fully open. The implication of this is that biochemical pathways must be actively degrading these cell components in order to bring about these structural changes, and this has indeed been found for many of the processes discussed in this paper. Bringing together this large body of data in one system allows us to reveal a novel pattern of floral senescence. Thus PCD during petal senescence is not simply a gradual running down of resources, and therefore ‘death by starvation’ for the cell. A number of catastrophic events occur (DNA laddering, electrolyte leakage, increased protease activity) later in senescence that may be triggered by a threshold effect arising from the more gradual biochemical processes. Further studies using microarrays are in progress to sample gene expression across the transcriptome. This broader approach will help to identify some of the early changes in gene expression associated with senescence in this system and therefore point to the biochemical pathways that are of the earliest significance in senescence of Alstroemeria petals.