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Journal of Experimental Botany, Vol. 55, No. 406, pp. 2147–2153, October 2004 DOI: 10.1093/jxb/erh264 Advance Access publication 10 September, 2004 REVIEW ARTICLE Senescence and programmed cell death: substance or semantics? Wouter G. van Doorn* and Ernst J. Woltering Wageningen University and Research Centre, PO Box 17, 6700 AA Wageningen, The Netherlands Received 27 May 2004; Accepted 26 July 2004 Abstract The terms senescence and programmed cell death (PCD) have led to some confusion. Senescence as visibly observed in, for example, leaf yellowing and petal wilting, has often been taken to be synonymous with the programmed death of the constituent cells. PCD also obviously refers to cells, which show a programme leading to their death. Some scientists noted that leaf yellowing, if it has not gone too far, can be reversed. They suggested calling leaf yellowing, before the point of no return, ‘senescence’ and the process after it ‘PCD’. However, this runs into several problems. It is counter to the historical definitions of senescence, both in animal and plant science, which stipulate that senescence is programmed and directly ends in death. It would also mean that only leaves and shoots show senescence, whereas several other plant parts, where reversal has not (yet) been shown, have no senescence, but only PCD. This conflicts with ordinary usage (as in root and flower senescence). Moreover, a programme can be reversible and therefore it is not counter to logic to regard the cell death programme as potentially reversible. In green leaf cells a decision to die, in a programmed way, has been taken, in principle, before the cells start to remobilize their contents (that is, before visible yellowing) and only rarely is this decision reversed. According to the arguments developed here there are no good reasons to separate a senescence phase and a subsequent PCD phase. Rather, it is asserted, senescence in cells is the same as PCD and the two are fully synchronous. Key words: Chloroplast, definitions, developmental context, hypersensitive response, leaf yellowing, petal wilting, programmed cell death, senescence, terminology, tracheids. Death is the inevitable end of the process of senescence CM Child (1915) Senescence and rejuvenescence, page 461 Introduction Although this is now mostly forgotten, cell death had already been noted by the early microscopists, whose observations were often strikingly correct. Lange (1891), for example, reported that a decrease in cytoplasm, and a drastic decrease in the number of organelles preceded cell death during xylem vessel formation. Prior to death, it was suggested, macromolecules such as proteins were degraded to small soluble compounds that were taken up by neighbouring cells. The author did not use the term programmed cell death, but he insisted that the death of the xylem cells was determined by their developmental context. He thus implied that the process was a programmed event. Although the concept of a programmed death has thus existed for a long time, the term programmed cell deathy (PCD) was not used before the 1960s. The term senescence was apparently used in everyday Latin, and is therefore much older than the term PCD. For example, Cicero (106–43 BC) wrote, in De senectute (On old age): Temeritas est videlicet florentis aetatis, prudentia senescentis (Of course rashness is the note of youth, and prudence of old age). He added: [Even if our souls] are not to be immortal, [..] a man must wish to have his life end at its proper time. For nature puts a limit to living as to everything else (Cicero, – 44). And Petrarca (1304–1374) remarked (in Latin): I [..] have grown old amid dangers [..] and bigger dangers cannot be reserved for my senescence (Petrarca, 1364). It is not very clear when the word senescence was introduced in science. In animal science * To whom correspondence should be addressed. Fax: +31 317 475347. E-mail: [email protected] y When referring to the terms senescence and PCD, they are written in italics. Journal of Experimental Botany, Vol. 55, No. 406, ª Society for Experimental Biology 2004; all rights reserved 2148 van Doorn and Woltering the term was used at least as early as 1879 (Minot, 1879) and with reference to plants not later than 1915 (Child, 1915), but its use may have begun considerably earlier. In cases where outwardly visible senescence symptoms appear, such as in leaves and petals, the process of senescence and PCD are often taken to be roughly synchronous. Others take it that senescence refers to (visible changes in) organs or whole individuals, and PCD to cells. Still others propose to use these terms such that senescence precedes PCD. There are good arguments for and against these views, which will be discussed. It is concluded that the first view is preferable. Defining senescence and PCD The Latin verb senescere means to grow old. About a century ago, the word ‘senescence’ was conceived of, in scientific texts, as the end phase of differentiation, the phase that ends in death. This is reflected by the quote at the beginning of this paper (Child, 1915). In the 1950s Peter Medawar launched a new discussion about senescence in animal biology. His problem was how to explain senescence in evolutionary terms, whereby it was defined as having death as its endpoint (Medawar, 1952). More recently, senescence in animals (including humans) is still usually defined as the process that results in death (Crews, 2003). In a seminal article, Leopold (1961) defined senescence in plant cells, along the lines previously proposed by Medawar (1957), as ‘the deteriorative processes that are natural causes of death’. As Leopold (1980) explained: ‘Senescence [. . .] refers to those changes that provide for the endogenous regulation of death. [. . .]. Physiological changes involved in the autumnal coloring (and subsequent death) of leaves, or in the dieback of tulips [. . .] in early summer, or in the yellowing and death of annual and biennial species after the completion of fruiting [. . .] would be examples of senescence’. This definition emphasizes the underlying processes that cause death. It is clear, therefore, that the tradition about the definition of senescence, both in the animal and plant fields, holds that its endpoint is death. Lockshin and Williams (1964, 1965), who investigated the degeneration of insect muscles during metamorphosis, coined the term PCD. PCD is a programme whereby a cell actively kills itself. It is to be distinguished from death by necrosis where the stimulus is so overwhelming that the cell is dead within a very short period. Initially, the term PCD was solely used in animal physiology. In plant science the term was introduced only by the end of the 1980s (Kirk et al., 1987; Lascaris and Deacon, 1991), and was not common until the mid-1990s. Various definitions of PCD have been given. Martin et al. (1994) defined it as a functional term, to describe ‘cell death as a normal part of the life of a multicellular organism’. This definition emphasizes that death is de- termined by the place of a cell, and by the role of its death in the developing organism. As discussed above, Lange (1891) had a similar conception of the programme of cell death in tracheary elements (although he did not use the term PCD). Other definitions put the role of the genome into focus. For example, according to Zhivotovsky et al. (1997) ‘PCD is a genetically controlled cell deletion process’. Still others emphasize the underlying biochemistry. Laytragoon (1998), for example, writes that PCD is a process whereby developmental or environmental stimuli activate ‘a specific series of events that culminate in cell death’. This seems to be the most comprehensive definition. The definition given by Laytragoon (1998) for PCD seems rather close to the one given by Leopold (1961) for senescence. Whatever the definition of PCD, it is obvious that it refers to cells, which exhibit a programme that leads to their death. This programme is often triggered from outside the cell. PCD comes in two main forms, (i) during the development of the organism (this includes the reaction to severe stress) and (ii) as a reaction to an invading microorganism (of which the hypersensitive response is an example). During developmental PCD, cellular death usually depends on the time and place of a cell in the organism (i.e. the developmental context of that cell). The term PCD then refers to the underlying programme in cells (either studied in isolation, or in an organ or individual), and may refer to its developmental context. PCD clearly does not only refer to death itself, which in many cells occurs within seconds (at least when tonoplast rupture occurs, which seems to be the moment of death; Obara et al., 2001). PCD rather refers to the processes that lead to the moment of death and the degradation (such as in the nucleus and cell walls) that goes on after this moment. Senescence and PCD: historical development In animal science the term senescence was initially mainly used for the processes leading to the death of individuals (Child, 1915), but later on it also became used for processes at the level of organs and cells (Witten, 1983). The term PCD is applied to the death of cells, both in culture and in the intact organism. In animal science, PCD is largely used as a synonym for senescence at the cellular level (and is used where previously the term senescence would have been used). Plant scientists, applied the term senescence mainly to the death of individuals and to that of organs, but also in conjunction with death in tissues (e.g. endosperm senescence) or individual cells. Plant scientists gradually adopted the PCD terminology already in use in work on animal systems. The term PCD first emerged in studies on small groups of cells that die during the early developmental stages of a plant, i.e. not related to the death of an organ or individual. Later on the term PCD became increasingly popular in relation to the final stages of development of Senescence and programmed cell death organs (where previously the term senescence would have been used). As the features of death in these cells fulfilled the requirements for PCD in animal cells, such as the presence of chromatin condensation and DNA cleavage, it was not incorrect to use the term PCD instead of senescence. But these developments also had an aspect of fashion. A colleague put this aptly: ‘PCD is sexy and senescence is not, thus papers get published and grants get funded when dealing with PCD and not when you call it senescence’ (AD Stead, personal communication 2004). The upshot of this observation is important: in practice the two terms became used as synonyms, at least for some examples of plant cell death. Since PCD clearly refers to cells, the use of the terms for organs or individuals may be rather imprecise if the focus is not on (specific groups of) cells, with or without the context of their development in the organ or organism. Imprecision, can come about, for example, when the heterogeneity of cell types in organs is overlooked. It has been known for a long time that the cells in both leaves and petals do not die at the same time. Cells in the mesophyll usually die before those in the epidermis, and cells in the vascular bundles stay alive longer than epidermal ones (Bancher, 1938). In addition, cell death in any of these tissues usually does not occur uniformly over the leaf blade. Chopping up a whole leaf and studying ‘its’ PCD is therefore in error, as one may ask to which cells the term PCD refers. Nonetheless, if emphasis is laid on (specific groups of) cells, it seems that no logical error is made when replacing the term senescence by PCD, provided that the definitions given above for the two terms are accepted. Delineation between senescence and PCD: the opinions Part of the confusion seems to be here: PCD and senescence have become overlapping ideas, but it is unclear to what degree they overlap. In most papers and reviews on PCD and plant senescence it is tacitly assumed that the two somehow overlap in time (Beers et al., 2000). However, it is usually not spelled out where this overlap begins and ends. Among those that do define the degree of overlap, two points of view have been defended. According to the majority opinion the overlap is complete (Noodén, 2004), whilst in some recent papers there is no overlap at all (Delorme et al., 2000; Thomas et al., 2003). Delorme et al. (2000) and Thomas et al. (2003) see senescence as a special case of plant cell differentiation, designated transdifferentiation. The latter is defined as ‘change of a cell or tissue from one differentiated state to another’. In their view, the conversion of chloroplasts into gerontoplasts in senescing cells is comparable to the conversion of chloroplasts into chromoplasts in ripening fruit and developing flower petals, and can be seen as a differentiation process. The period in which the transition 2149 of chloroplasts to gerontoplasts is reversible serves to distinguish senescence from PCD. More than 20 years ago Wang and Woolhouse (1982) also argued that the point of no return was important in leaf senescence. They suggested using the term senescence for the processes that occur after the point of no return. This is in contrast to Delorme et al. (2000) and Thomas et al. (2003) who propose to use the term senescence for developments before this point. The definition of the term senescence, according to the view of Thomas et al. (2003), is opposite to the other one that is often used, in which senescence as such ends in death. According to their definition, senescence per se does not result in death (but it can result in death, through PCD). According to still another use of the terms, they apply to different parts of the plant. Senescence, in this view, is the process that leads to the death of organs and whole plants, while PCD refers to the death of (a relatively small number of) cells (Noodén, 2004). This use of the terms is similar to another one, which restricts the term senescence to developmental processes in which the close-to-death symptoms are visible to the naked eye (as in leaf yellowing, petal wilting, and withering of whole plants at the end of their life span). In this view, the term PCD applies to all other forms of cell death, such as in root aerenchyma formation after flooding, or the death of the embryo suspensor cells. Senescence as a precedent of PCD Thomas et al. (2003) cite the peculiar relationship between leaf yellowing and leaf death as a reason why they propose their use of the terms senescence and PCD. In some cases the leaves become yellow but if this process has not gone too far such leaves can become green again. It must be admitted that it is logically incorrect to call yellowing that is later on reversed, programmed cell death, because in this case the yellowing does not lead to death. What is going on in tissues that regreen after becoming yellow? The chloroplasts have initially lost considerable quantities of proteins and chlorophyll, but are somehow able to reverse the degradation process and synthesize various compounds again (Krul, 1974). Instead of dying, these chloroplast-containing cells will live on, at least until the next signal for cell death comes around. Such regreening is rare. It has been observed in the peel of developing oranges, which attains maximum orange colour during winter and slightly regreens by harvest time the following summer (Coggins and Lewis, 1972; Goldschmidt, 1988). Similarly, in some rare cases petals can regreen, and then live relatively long. This was observed, for example, in a mutant chrysanthemum (Winkler, 1902), and in a few orchids, after pollination (Phalaenopsis cornu-cervi, P. violacea, Promenaea sp., and Epidendrum 2150 van Doorn and Woltering macrochilum; Fitting, 1910). Regreening also occurs in the spathe of Zantedeschia, as a normal part of its development (Tavares et al., 1998) and in sepals of Helleborus niger, which lose chlorophyll and serve to attract pollinators, then become green again after pollination (Salopek-Sondi et al., 2002). If regreening is rare in fruit, petals, and sepals, how general is leaf regreening? Only a few examples have been described, all based on laboratory experiments in which a large part of the shoot was removed. Krul (1974), for example, noted that removal of the epicotyl of soybean can reverse visible senescence in the cotyledons. Similarly, yellow leaves of tobacco plants can regreen if the whole top of the plant is cut off, leaving only one leaf (Zavaleta-Mancera et al., 1999a, b). Regreening can also be induced in Phaseolus vulgaris, which undergoes whole plant senescence once it has formed seeds. Removal of all parts of the shoot except a yellowing leaf and its subtending part of the stem, could induce the leaf to become green again (Jenkins and Woolhouse, 1981). Lack of water or nutrients results in leaf yellowing in many plant species, which in some species can be reversed upon removing the stress, as shown in laboratory experiments (Girardin et al., 1985; Zhang et al., 1995). This may also occur under natural conditions, but no such cases have apparently been described in plants growing in the field. In young pea plants, which show an extreme case of apical dominance, removal of the epicotyl (containing the initial apical meristem) results in two equal small branches, each with their own meristem. One of these branches soon becomes dominant, and induces visible senescence symptoms and complete death in the weaker one. Removal of the dominant branch, prior to the death of the weak one, rapidly and completely reverses the death process in the latter. This reversal can take place even after increased expression of SAG12, encoding a common senescence-associated cysteine protease involved in mass protein degradation (Belenghi et al., 2004). Although this has not been described, such reversal of the cell death processes could, in principle, also occur in nature, upon grazing. The conclusion of this section is that regreening may occur in nature, but that it seems rather rare. Chloroplasts and life span Thomas et al. (2003) attach great importance to the role of functioning chloroplasts in preventing plant cell death, citing evidence that tissues without such chloroplasts, such as petals, tend to have a short life. This connection is not general. Apart from the petals in some species that can live up to 80 d (Molisch, 1929, page 75), there are several other examples of cells that are devoid of chlorophyll but live for many years. Some sclereids, for example, can live for as much as 4–5 years. Wood fibres also have no chloroplasts, but can live for as long as 20 years (Fahn 1990, Chapter 6). In several species, the pith cells can live for as long as 11–20 years. Exceptionally long-lived pith cells were found in Tilia parvifolia (28 years), Sorbus aucuparia (35–40 years), Betula alba (27–40 years) (Molisch, 1929, pages 89–90). Ray cells in woody stems can also grow very old. In several species they lived for 24–36 years, with exceptions of up to 45 (Abies alba), 86 (Sorbus terminalis), and about 100 years (Sequoia sempervirens) (Molisch, 1929, pages 91–92). Noodén (1988, pages 506–507) mentioned other examples: (i) brightly coloured cacti, if grafted onto a green nurse plant, (ii) shoots of albina (white) soybean and tobacco mutants, if they are grafted onto a green plant, and (iii) leaves that lack chloroplasts, in chimeral plants. So there seems nothing fundamental about the presence or absence of chloroplasts for the life span of a cell. As long as the cell is able to get sugars and other necessities, and does not, by internal or external signals, become committed to die, it will live. What may be true, nonetheless, is that the actual reversal of the programme that leads to death, as is sometimes observed, depends on the regaining of function in chloroplasts. If this is so, it is quite interesting. What is it in the chloroplast that has this effect? Is it a matter of energy? If so, does this mean that lack of energy is the factor that causes death? (van Doorn, 2004). More than just sugars seem to be required. Isolated petals or leaves held in an aqueous solution containing an antibacterial compound and a sugar may live longer than those held in the same solution without sugar (or in water), but in most species they still die rather soon. Objections to the use of the term senescence for a process that does not result in death There are a few arguments against the idea of using the term senescence for the period that precedes PCD. The first argument refers to what is common use in plant biology. If the definitions as given above are accepted, there is no clear difference between senescence and PCD. A common connotation of the concept of senescence, moreover, is that it leads to death. A second, and related, argument points to the consequences of the new definitions. The use of the terminology as suggested by Thomas et al. (2003) conflicts with the ordinary usage of the terms (root senescence, petal senescence; some scientists also speak of fruit senescence; Castillejo et al., 2004). If the new terminology were to be adopted, this would mean that only a few organs such as leaves and shoots show senescence, and only during a, usually unknown, part of their yellowing period. Insofar as the senescence of whole plants is reversible, the term would also be applicable to the trajectory before the point of no return. As far as is presently known, there are no examples of the reversal of senescence in organs other than leaves and shoots and, possibly, individual plants. For this reason, Senescence and programmed cell death organs such as roots, petals, stigmas, styles, and anthers would only have PCD, no senescence. Third, it may well be argued that a programme can be reversible. A programme of differentiation can be suspended, put in lower or higher gear, become terminated, and also reversed. The reversal of a differentiation programme, however, does not mean that things that went in the order a–c will now go in the order c–a. It seems more like a train that is diverted to another track and therefore will, this time, not go to station F but to station G, and in some cases still may end up in station F. A well-known case of such a reversal occurs during the development of female flowers, whereby the anthers, which have already differentiated, disappear (Caporali et al., 2003). Similarly, a programme that would normally be leading to cell death can also be reversed. Both senescence and PCD, the latter with emphasis on the programme (and not on death), can both be considered to be reversible in principle. Fourth (and perhaps most important), in cells that undergo large-scale remobilization, the decision to die has been taken prior to the remobilization phase. Remobilization in leaves, of which yellowing is a clear sign, is, on this view, part of the programme that normally leads to death. PCD starts when the decision to die is acted upon. On this view, therefore, PCD starts at the time when the programme that (usually) leads to death is initiated. Senescence as related to whole organs, and PCD to cells For some, the term senescence refers to the death of organs and whole individuals, and the term PCD for the death of (small groups of) cells, during the early stages of development of an individual. This can be understood because of historical developments (see above). Senescence was studied by physiologists, who worked with organs and whole individuals that showed morphological signs of old age. At the same time, plant anatomists noted cell death during development, during spore formation, egg formation, embryo development, etc. (Fahn, 1990). However, it is now known that the processes that lead to most cases of developmental cell death, at early and late stages, are the same. There is, therefore, no good reason to distinguish between cell death in a whole organ or plant, and death in a small group of cells early on during development. The term PCD can therefore also be applied to death of (the cells in) organs and whole individuals, at the end of their life span. Conversely, the term senescence may well be applied to the process that leads to the death in relatively small groups of cells during the early stages of development, or even to cell death associated with the hypersensitive response. In still another use, the term senescence refers to developmental processes in which the senescence symptoms are visible to the naked eye. The term PCD applies to cell death, including the death of cells in the organ that shows 2151 the visible symptoms. The visible features of senescence, however, are a result of the cell death programme in the cells of the organ that show these features. When the cells that give rise to the visible symptoms are studied, chloroplast degradation, loss of turgor etc. will be found. A number of publications have ‘programmed cell death during senescence’ in their title (Simeonova et al., 2000; Xu and Hanson, 2000; Coupe et al., 2004). The authors apparently used senescence in one of the two ways described in this section. But if the reasoning here is followed, it is a tautology to speak of PCD during senescence. In conclusion This paper addresses a rather fundamental confusion in the field of plant biology interested in the developmental programmes that ultimately lead to the death of cells, organs, and individual plants. The confusion centres on the application of the terms senescence and PCD: whether they describe similar events, distinct events or overlapping events. Although it is proposed here that the terms senescence and PCD can be used as synonyms (if referring to specific groups of cells, with or without their developmental context), the point made by Thomas et al. (2003) is a good one. In the case of reversal of leaf yellowing it seems logically incorrect to say that the yellow leaf, prior to its regreening, was undergoing PCD. This is so because the yellowing did not lead to cellular death. They suggest applying the term senescence for the trajectory before the reversal, and PCD for the part after it. However, they may be right on a not very relevant point. Reversal of the senescence symptoms seems rather rare in nature. The terminology as suggested by Thomas et al. (2003) leads to several problems. Their definitions are counter to the older definitions of senescence, which maintain that it leads directly to death. Furthermore, according to the terminology of Thomas et al. (2003) only cells in which the process is reversible can be called senescent, which, given the present absence of proof for such reversal in plant parts other than leaves and shoots, restricts the term senescence only to these parts. A way out of the problem is to consider a programme reversible. If so, PCD, being a reversible programme, can be considered to start when the signal for cell death has arrived and is acted upon. PCD would thus start at the first biochemical change that would normally lead to death, if the programme were not somewhere reversed. Senescence of cells is then equal to PCD, and fully synchronous with it. The signal to die, it should be remembered, is given prior to the dismantling of the cell, thus prior to PCD/senescence. The present view thus concurs with Noodén (2004) who stated that senescence, as (usually) conceived of in plant biology, is in fact PCD. 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