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A PERES Publishers Productions Produced and published in Czech Republic by PERES Publishers Na Klikovce 9 14000 Prague 4 Czech Republic Edited by Miroslav Strnad, Pave! Pec and Erwin Beck. This publication was expired on December 10,1999. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior permission of the publishers. ({;) Authors, 1999 ({;) PERES Publishers, 1999 Art direction and typography: Milan Cermcik Printed and bound in Czech Republic. The cover illustrates somatic embryogenesis in pea. Photograph taken by M. Griga, pp. 238 Library of Congress Cataloging in Publication Data Advances in Regulation of Plant Growth and Development Pavel Pec, Erwin Beck. -Prague: Peres, 1999,258 s. ISBN 80-86360-06- 7 581.1 * 581.143 * 581.14 .plant .plant .plant 2 physiology growth development / Edited by Miroslav Strnad, Advances in Regulation of Plant Growth and D~~m~-t{1999119~212 Programmed L.ADISLA V HA VEL 1 & cell death in plant development DON JOHN DURZAN: 2 IDepartment of Botany and Plant Physiology, Mendel University of Agriculture and Forestry, Zemedelskli I, 61300 Brno, Czech Republic 2Department of Environmental Horticulture, University of California, One Shields Ave., Davis, CA 95616-8587, USA Abstract Plant development involves the elimination of cell organelles, protoplasts, tissues and organs. The concept of programmed cell death elaborated in medical and animal sciences has become suitable for explanation of these eliminations which must be highly co-ordinated to maintain plant integrity .Characteristic features of apoptosis, a form of programmed cell death, were found e.g. in leaf senescence,abscission of flower parts, reproduction processes, tracheary element formation, and responses to various biotic and abiotic stresses.The role of phytohormones in programmed cell death is becoming evident. Apoptosis in meristems influences longevity and overall development of plants. Introduction Plants eliminate cells, organs, and parts during responses to stress and expressions of various developmental programs. Leaves that are not lighted enough are shed. Most of broad leaf trees in zones with adverse winter conditions shed leaves in autumn. The unpollinated flowers are fully thrown away. Ovaries with fertilised egg cells in ovules on the same plant are retained forming fruits while the other parts of flowers, e.g. petals and sepals or tepals falloff. Stigmas and pistils may also be eliminated. In apomictic species, the fruits develop without fertilisation which means that the ovaries with ovules are retained forming fruit, while the other flower parts are eliminated. In parthenocarpic species that bear fruits without seeds, only walls of ovaries continue development. All other flower parts are removed. This elimination must be highly controlled by internal factors or, in some cases, in combination with external stimuli that involve an array of cellular and subcellular activities. A typical plant cell consists of the cell wall and the protoplast. MAUSETH (1995) emphasises that metabolism also occurs in cell walls and they should be considered dynamic, active parts of plant cells. Generally, the protoplast is more metabolically active in comparison with the cell wall. The plant is able to abolish not only whole organs or their parts, the elimination can occur also on the cellular and even subcellular level. Whole cells, protoplasts and cell walls are eliminated too. These events are known in spores that do not participate in the megagametophyte development of gymnosperms and angiosperms. Another example is the elimination of suspensor cells during zygotic embryogenesis (NAGL 1976). Dead cells can also be filled with storage mate- rial as in most endosperm cells in the caryopsis of grasses(BEWLEY& BLACK 1984). Plants live very economically. When the cell wall itself is able to accomplish a specific function, the protoplast is eliminated. Sclerenchyma cells are dead because thick cell walls perform the mechanical function. Phellem, commonly known as cork, is constituted of characteristic cells with a thick suberinised layer of the cell wall. Suberin, combined with lack of intercellular spaces,protects internal tissues against desiccation. The protoplast is no longer needed. In xylem, tracheary elements also lack protoplasts. Water and nutrients flow through spaces, where protoplasts were originally placed, surrounded by modified, lignin impregnated cell walls. Cells performing these functions are dead. Not only whole protoplasts but also their parts can be eliminated -even such an important organelle as the nucleus. This process, which has been described in sieve tube elements of phloem, is completed through dechromatisation in secondary phloem or through pycnosis in primary phloem (c.f. BUVAT 1989). The nuclear genome is sometimes fragmented into unequal parts by amitosis. The resultant micronuclei mayor may not persist (SINGH 1993; KIPLING 1995). The plastids can be eliminated as well. The formation of neocytoplasm after fertilisation in conifers, where maternal chloroplasts are deleted, and exclusion of one type chloroplast after somatic hybridisation can serve as examples (c.f. CAMEFORT 1969; MANTELet al. 1985). Many variations in plant cell elimination are evident. It is certain that these processesmust be controlled ontogenetically and spatially. In other words, the elimination or death of cells must be programmed. Variations 203 L. HAVEL & D. J. DURZAN in programmed cell death, well known from animal and medical sciences,represent comparable events. Programmed ences cell death in animal and medical sci- The death of single cells as integral parts of coordinated living processes has been neglected for very long time. In early seventies KERR et at. (1972) described apoptosis as a distinct fonn of cell deletion or programmed cell death that plays a major role during development, homeostasis, and even in the expressions of many diseases.Concepts of cell death became one of the fast developing areas of animal and medical sciences especially in pathology (KORSMEYER1995). No wonder that most of the infonnation has been acquired here. Different genes and their products that control cell death by signalling and executing pathways were characterised or presumed. Multisignalling events have been implicated in the regulation of apoptosis (e.g. HANNUN 1996; BAYLY et at. 1997). The proposed molecular network of mammalian apoptosis pathways and their use in metabolic engineering is very complex at present ( e.g. FUSSENEGGER & BAILEY 1998). Several cell death suppressor genes have been identified, e.g. Bcl-2 gene family or DADJ, products of which are capable of protecting the cells from programmed cell death ( c.f. V AUX & STRASSER1996; NAKSHlMAet al. 1993). Parts of cell death programs have been conserved among wonns, insects, and vertebrates and in all cell types (STELLER1995). The link between divisional cycles and programmed cell death was also suggested ( c.f. HA VEL & DURZAN 1996a; FUSSENEGGER & BAILEY 1998). New studies and reinterpretation of the old data from the experimental work with the oncogenes provided substantial evidence that cell renewal and cell death are linked even if they appear to be opposing and mutually contradictory (EVAN& LITTLEWOOD1998). cells. In plants, apoptosis is also characterised as a phenotypically distinct form of controlled cell deletion (RAVEL & DURZAN 1996a). GILCHRIST(1998) characterised apoptosis as genetically regulated, signal transduction-dependent programmed cell death. Logically, other forms of programmed cell death exist in plants. Nevertheless, many authors do not distinguish between programmed cell death and apoptosis using both terms as synonym. Keeping in mind that apoptosis represents the predominant form of cell death (SCHULZE-OSTHOFF et al. 1994), false understanding will be rare. The term "cell or cellular suicide" is also often used instead, or together, with programmed cell death ( e.g. STELLER 1995). Also terms "physiological cell death" or "developmentally regulated cell death" in the meaning of programmed cell death are used (e.g. KROEMERet al. 1998; RAMMOND-KOSACKet al. 1994). Yet another used term is "apoptotic cell death" (e.g. BRUNE et al. 1998). Taking in mind that apoptosis is a kind of programmed cell death this term seemsto be redundant. Another term -"necrosis" -has been also used for the description of cell or tissue death. At present necrosis is often considered as an opposite to apoptosis. It is unprogrammed and involves the decay of injured groups of dying cells. The "term accidental death" is used in this sense. Several features are used to distinguish apoptosis from necrosis (Tab. I). MAJNO & JORIS(1995) recommended preservation of the original meaning for "necrosis" which has been used in life sciences for very long time. Originally, necrosis meant drastic tissue changes visible by naked eye and therefore occurring well after the cell death. They offer the term "oncosis" for unprogrammed cell death as an opposite to apoptosis (c.f. MAJNo& JORIS1995). "Terminal differentiation" is used for cell elimination where differentiation leads to cell death (c.f. RAVEL & DURZAN 1996a). In plants almost every protoplast is eliminated earlier or later after cell differentiation while the cell wall can persist. The cells that dedifferentiate and reestablish divisional cycles are an exception. Morphological Terminology Historically, the fact that cells can perish was discussed in the Lecture XV. of Virchow's Cellular Pathology among "passive processes and degenerations" in the middle of the last century (MAJNo & JORIS1995). It is clear that cell death leads to the point of no return but this point can be achieved in many different ways. This is one of the reasons why, till now, a lot of different terms were used in concepts for a single pathway to cell elimination. Moreover, the same terms have been used for different processesor features or vice versa. The pioneers, KERR et al. (1972), defined a distinct form of programmed cell death as apoptosis and described characteristic markers of this process in animal 204 markers of apoptosis The morphological markers are mainly based on studies of animal and human cells observed in viva and in vitra. At present, however, more of the new information is being found in plant cells. In animals, dying cells shrink and separate from their neighbours. The cytoplasm contracts and dilatations with some vesiculation of the endoplasmatic reticulum can occur, The nuclear changes are the most studied morphological marker. The chromatin condenses into dense compact masses that may coalescence into a crescent inner cap lining the nuclear membrane. The nucleolus fragments. Invaginations of the nuclear membrane may further divide the nucleus (HA YLYet al. 1997). The degradation of the nuclear lamina has been described (UCKER et al. 1992; LAZEBNIK et al. 1993) Programmed cell death in plant development Tab. I.: Comparison of morphological, biochemical and molecular features and physiological significance of apoptosis and necrosis. Necrosis Apoptosis -Membrane blebbing, but no loss of integrity -Aggregation of chromatin at the nuclear membrane -Cellular condensation (cell shrinkage) -Formation of membrane bound vesicles -Loss of membrane -Flocculation -Swelling -No integrity of chromatin of the cell and lysis vesicle formation, complete -Disintegration(swelling) lysis of organelles (apoptotic bodies) -No disintegration of organelles, organelles remain intact Biochemical and molecular features -Tightly regulated process involving activation and enzymatic steps -Loss -Energy (A TP)- dependent -Non-random mono- and oligonucleosomal length fragmentation of DNA (ladder pattern after agarose gel electrophoresis) -Prelytic DNA fragmentation (early event of cell death) -Random digestion agarose -Postlytic gel electrophoresis) DNA fragmentation -No of regulation energy of ion homeostasis requirement of DNA (smear (late after event of death) Physiological significance -Death of single, individual cells -Induced by physiological stimuli -Phagocytosis by adjacent cells or -Death of cell groups -Evoked by non-physiological disturbances -Phagocytosis by macrophages macrophages -No inflammatory response -Significant inflammatory response while other organelles remain intact. Later a characteristic bubbling and blebbing of the cytoplasmic membrane and the formation the membrane-bound fragments, apoptotic bodies, occurs. The apoptotic bodiesare then phagocytosed by neighbouring cells (KERR et a/. 1972, Fig. 1). The loss of structural organisation is energy dependent, often causing an increase in respiratory rate (NEWMEYERet a/. 1994) (KORSMEYER1995). Over the past five or six years about 30 new molecules have been discovered that initiate or regulate apoptosis. At least 20 other molecules associated with signalling or DNA replication, transcription or repair, have been recognised as affecting the regulation of apoptosis (WILLIE 1998). One of the first signal for apoptosis, known at present, is a decrease in mitochondrial transmembrane potential, irrespectiveof any apoptosis-inducingstimulus ( c.f. KROEMERet al. 1998). The aberrant exposure of phosphatidylserine in the plasma membrane is another early Biochemical and molecular markers of apoptosis marker of the apoptotic process (KROEMERet al. 1998). These events are followed by the activation of nucleThe process of programmed cell death can be schemati- ases, proteases, phospholipases and phosphatases. The cally subdivided into three steps: a signalling phase, an participation of calcium was also well documented execution phase and a dismantling phase (DEPREATERE (SCHWARZTMAN & CIDLOWSKI1993). & GOLSTEIN 1998). The regulation of apoptosis is The activation of nucleases leads to a non-random known mainly from the work with neoplastic tissues cleavage of nuclear DNA (EA YLYet al. 1997). Cleavage ... ./ . " @\ ~ . .=.-/ / (A) (8) (C) (D) (E) (F) Fig. 1.: Apoptosis in animal cells. After receipt of a signal to undergo apoptosis, an adherent cell (A) rounds up (8). Nuclear DNA rapidly condenses (C). Nucleus is separated into discrete masses of condensed chromatin (D) and, finally, fragmentation of the cell into several membrane-bound vesicles apoptotic bodies -follows (E). These bodies are rapidly recognised and phagocytosed by macrophages or neighbouring cells (F). 205 L. HAVEL & D. J. DURZAN usually results in formation of small fragments of double stranded DNA (size 180-200 bp) that can form a typical ladder on agarose gels (e.g. WALKER et al. 1993). The individual apoptotic nuclei are detected with terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) in situ (GAVRIELI et al. 1992, (Fig. 2). Larger fragments and single-strand DNA cuts were also characteristic for apoptotic degradation of the nucleus (e.g. PEITSCHet al. 1993; BORTNERet al. 1995). Larger fragments (50 kbp) are generated by the release of DNA from the nuclear matrix. SubsequentinternucleosomalDNA cleavageresults in the formation of small fragments (OBERHAMMERet al. 1993). These findings suggestthat the apoptotic degradation of the nucleus is a gradual process. The patterns of endonucleases activity cannot be a sole criterion for apoptosis, as nucleases can be activated by several processes. Other observations indicate that endonucleolytic DNA degradation is neither required nor sufficient evidence of apoptosis in certain cells (SCHULZet al. 1998). Programmed cell death in plants The term "apoptosis" comes from plant kingdom from old Greek "apoptosis" which originally means the loss of petals or leaves. Surprisingly, despite the obvious role of cell death in plants the concept of programmed cell death was developed and pioneered in animal and medical sciences. Now it is becoming obvious that this concept will better explain many events of plant biology .A model for plant apoptosis in the life cycle has been already proposed. This model embodied predisposing physiological states, divisional cycling, salvage of metabolic degradation products, terminal differentiation, disease resistance, and renewed growth (HA VEL & DURZAN1996a;b). Markers The basic model for apoptosis as it was accepted in animal and human sciences was applied in plant sciences, and the same markers were employed. The localised collapse of nuclear domains (pycnosis), loss of nuclear membrane, nucleolar release, and fragmentation of nuclei and cytoplasm into the distinct bodies was also described in plants (RAVEL & DURZAN 1996c; 1999; W ANG et al. 1996a;b ). The TUNEL assay showed DNA fragmentation in situ in nuclei of cells where the programmed cell death has been expected (e.g. MITTLER & LAM 1995; MITTLER et al. 1995; RAVEL & DURZAN 1996c; W ANG et al. 1996a). When TUNEL is combined with other fluorescent dyes that are specific for DNA e.g. 4,6-diamidino-2-phenylindole dihydrochloride (DAP1), the nuclei that are or are not destined for elimination can be distinguished (RAVEL& DURZAN 1996a;b). 206 Another feature of apoptosis -DNA ladder formation on agarose gels -was more difficult to prove. The number of cells with apoptotic nuclei in tissues of growing plants is relatively low. The expected laddering was detected in cultures and organs where the external stimuli provide the means for an increase of apoptotic cells in certain time (synchronisation of apoptosis). In vitro cultured cell and toxic and/or abiotic stimuli were used first (W ANG et al. 1996a;b; KouKALOVA et al. 1997). The naturally synchronised elimination of cell populations, that form carpels, petals and foliage leaves, followed (ORZAEz & GRANELL 1997a;b; YEN & Y ANG 1998). O'BRIEN et al. (1998) showed that annexin V-binding, an indicator of exposure of phosphatidyl serine on the outer cell plasma membrane of mammalian cells, can also be used to detect apoptosis in plants. They used the isolated protoplasts from a cell suspension culture of Nicotiana plumbaginifolia. It has not been proven yet whether the cells with cell walls are sensitive to this assay. Reproduction processes Many different processes participate in the formation of new individual plants. These processes comprise the formation of spores, gametophytes, sporophytes, gametes and zygotes. There are many specialised developmental pathways in different plant groups. It has already been recognised that programmed cell death can participate in reproduction processes. Programmed cell death is a normal part of ovule and seed development which is regularly accompanied by the degeneration of supernumerary archesporial cells and megaspores, nucellus and, in angiosperms, certain cells in the embryonal sac and endosperm (ERDELSKA 1998). Also individual layers of seed coats die. SCHWARTZet al. (1997) stressed that the only known example of programmed cell death during plant embryogenesis is during the degeneration of the suspensor. Based on older results BELL (1996a) suggested that abortion of certain megaspores in angiosperms is a form of programmed cell death, probably apoptosis. The older results obtained by CORRENS(1900) conformed remarkably well with the hypothesis that regular elimination of three of the megaspores is genetically determined. The same genetic background is expressed in macrosporangia (carpels) and not in micro sporangia (anther) where all four meiotic products survive (BELL 1996a). Apoptosis was described in suspensor cells of the early somatic embryos of Norway spruce (HA VEL & DURZAN1996c). The early somatic embryo, which has a longitudinal structure, comprises three parts: embryonal group, embryonal tubes and embryonal suspensor. The cells of the embryonal group are actively dividing with no signs of apoptosis. The first TUNEL positive nuclei were observed in the embryonal tubes quite close to the ~ Programmed , cell death in plant development (A) """""'- ~ ~ ~ (C) (D) """""-. 0 ~ labeled dU TP labeled streptavidin color product Fig. 2.: TUNEl (terminaldeoxynucleotidyl transferase-mediated dUTP-biotinnick end labelling).The ONAis cleavedInto nucleosomes(A). 3 OH endsof ONAfragmenls are labelledby terminaldeoxynucleotidyl transferasewith dum which Is conjugatedwith a fluorescentdye (for Immediateobservation)or with biotin(B). Biotinis specifIcally boundto streptavldln(C). Streptavldlncan be labelledwith a fluorescentdye for observationor conjugatedwith an enzyme which reactswith its substrategiving a color product(D). 207 L. HAVEL & D. J. DURZAN embryonal group. More distal nuclei became pycnotic and disintegrated with the release of nucleoli and nuclear fragments into the cytoplasm. The morphology and development of the early zygotic embryos (c.f. SINGH 1978) is similar to, if not the same as, the early somatic embryos and thus apoptosis may be also expected in the zygotic embryogenesis of Norway spruce. The same features can be expected in embryogenesis of more species in conifers because HA VEL & DURZAN ( 1999) observed the presence of apoptosis in tubes and suspensor of early somatic embryos of blue spruce. The cultured early somatic embryos of conifers multiply via cleavage of the embryonal group and so called diploid parthenogenesis which was described in Norway and blue spruces (c.f. RAVEL & DURZAN 1992; 1999; DURZANet al. 1994). The participation of apoptosis in this process was shown as well ~RAVEL & DURZAN1996c; 1999). During development and germination of an embryo its nutrition is important. The role of apoptosis in this process was detected in the aleurone layer -the outer part of the endosperm -of barley grains (W ANG et al. 1996c). Here the cells do not store many reserves but may be responsible for the release of a certain amount of enzymes that mobilise the nutrients from the rest of endosperm that has non-living cells fully filled with nutrients (BEWLEY& BLACK 1994). Apoptosis appeared to be important for the spatial and temporal control of the aleurone layer activities during grain germination (W ANGet al. 1996c). Tissue development As mentioned above, programmed cell death can occur where cells eliminate their nucleated protoplasts to perform structural and translocatory functions. The tracheary elements may be the best known examples of such cells. The cells with TUNEL positive nuclei were detected in developing xylem of intact roots ( e.g. MITTLER& LAM 1995; MITTLERet al. 1995). The gradual DNA fragmentation in nuclei in developing tracheary elements in in vitro cultured callus was also observed (HA VEL et al. 1997). The lateral root primordia develop in the pericycle of the main root. Lateral roots grow through the cortex, reach the main root surface, and continue their elongation. Apoptotic cells were observed in the inner cortical cells of soybean, that overlay the root primordia. This elimination frees the space for the undisturbed growth and development of lateral roots (KOSSLAKet al. 1997). In a mutant of the same species the apoptotic cells were spread through the whole root although a spatial control existed here. Typical nuclear DNA fragmentation was detected opposite the xylem poles of the root vascular bundle (KOSSLAKet al. 1997). 208 The root cap protects the root apical meristem and facilitates its growth through the soil or substrate. The destruction of the most distal cells, which results in mucilage production, facilitates the smooth growth of the root tip in harsh conditions. A typical DNA cleavage proved by TUNEL was shown in nuclei of dying cells of the root cap (W ANGet al. 1996a). Senescence Senescence occurs in individual cells, tissues, organs, and the whole organism. Different opinions exist as to the meaning of the term "senescence". It is generally accepted that senescence is a genetically controlled developmental process which is internally programmed (NOOOEN & GUIAMET 1996). Senescence results in the loss of homeostasis at the cell or organism level. Ultrastructural studies provide a valuable overview of cell senescence (c.f. NoooEN 1988). Several features resemble typical markers of programmed cell death. ORZAEz & GRANELL (1997a) detected typical DNA fragmentation during the senescence of unpollinated pistils of Pisum sativum. The apoptotic event was also observed during petal senescence in the same species (ORZAEZ & GRANELL 1997b). Moreover, the DNA fragmentation which accompanies senescence was regulated by ethylene (ORZAEz & GRANELL 1997a). Recently, YEN & Y ANG (1998) reported the detection of programmed cell death in the senescent leaf tissue from five plant species. The use of gel electrophoresis and Southern hybridisation detected DNA ladders only in senescent leaves but not in green leaves. DNA fragmentation and nuclear DNA condensation were further confirmed in situ by the use of TUNEL assay. These results provide direct evidence to support the notion that the natural senescence of the leaves is indeed an apoptotic process (YEN & Y ANG 1998). GALLOIS et al. (1997) isolated a clone from an Arabidopsis thaliana cDNA library whose predicted translation product showed highly significant similarity to a mammalian defender against apoptotic death 1 protein (DAD 1) -product of a gene which was mentioned above. Their experimental data indicated that two such genes (AtDAD) exist in this species in contrast to mammals having only one such gene. The transcripts of AtDAD genes are found in root, stem, leaves, flowers and siliques at different stages of the plant development. The abundance of transcripts is reduced in siliques during their maturation and desiccation (GALLOIS et al. 1997). This is natural considering that the role of the DAD gene suppresses programmed cell death (NAKSHIMA et al. 1993). The protoplasts of siliques cells die while their persisting cell walls desiccate. The whole process must be highly co-ordinated to ensure the opening of a ripe silique. Programmed cell death in plant development Phytohormones The role of phytohormones in plant growth and development is documented in detail in other chapters of this book. First observations on the role of these compounds in programmed cell death have been already published. Apoptosis in the aleurone which takes place during germination was inhibited by abscisic acid (W ANGet al. 1996c). Ethylene is known by its role in senescenceand it was also shown to regulate DNA fragmentation, a hallmark of apoptosis, in pistil senescence in pea (ORZAEz& GRANELL1997a). Longevity The development of plants lasts for different time periods depending on species. Some coniferous species live several thousands of years e.g. individuals of Pinus aristata in California are more than 4 300 years old (FoSTER & GIFFORD 1974). Herbaceous species may develop and die in several weeks. In general, plant longevities fall into several categories relative to the annual seasonal cycle (annuals, biennials, and perennials). The longevity of plants is not always limited by endogenous programs but can be terminated by environmental or pathological factors. If no such factors are present the longevity depends on the continuation of meristematic activity. The persistence of cell divisions for thousands of years depends on the ability of meristems to repair and recover from the damage of the genome (HA VEL & DURZAN 1996a). The meristematic cells must also suppress the genes or their products that trigger programmed cell death to maintain their division. Recently, SCHUBERTet al. (1998) showed that terminal deletions of artificially elongated chromosome arms triggered apoptosis (TUNEL positive nuclei) in foot tip meristems of faba bean. If the number of damaged meristematic cells surpasseda threshold, the plants showed developmental disturbances. Extensive cell death in meristems was eventually responsible for reduced growth, disturbed development and reduced seed set. This observation suggests that apoptosis in meristems influences longevity and the overall development of plants. Pathological events Phytopatological events are not parts of nonnal plant development but we mention it here, because the concept of programmed cell death in plants emerged several years ago in pathology studies (DIETRICH et al. 1994; GREENBERG et al. 1994; HAMMOND-KOSACK et a/. 1994; LEVINE et a/. 1994; 1996; PONTIER et a/. 1994; MITTLER & LAM 1995; MITTLER et a/. 1995; JONES & DANGL 1996; RyERSON & HEATH 1996; WANG et a/. 1996a). Sacrificing an infected cell in order to prevent systemic spread of a pathogen appears to be a conserved strategy in both plants and animals (MITTLER et al. 1997). Recognition of invading pathogens and activation of the cell death result in the formation of dead cells around the site of the attack. This process, termed a hypersensitive response, prevents the systemic spread of some pathogens. Several lines of evidence suggested that cell death during the hypersensitive response results from activation of a programmed cell death pathway (MITTLERet al. 1997). Current data suggest that activation or suppression of programmed cell death may underlie diseases in plants as it does in animals (GREENBERG 1997; GILCHRIST1998). Programmed cell death can act as an endogenous "secondary signal" for the induction of localised defences and signals triggering systemic defence (HEATH 1998). The disease resistance gene Cf-9 together with a fungal avirulence gene regulates cell death in tomato seedlings (HAMMONO-KOSACKet a/. 1994). Another gene, hstr203J, with rapid activation, was highly localised and specific for incompatible plant/pathogen interaction was found in tobacco (PONTIERet a/. 1994). The accelerated cell death gene (ACD2) acts as a negative regulator of programmed cell death triggered by pathogens (GREENBERG et a/. 1994). Calcium in apoptosis in plants was also studied in this context (LEVINE et a/. 1996). Conclusions The differences in details of programmed cell death between plants and animals are now evident, however, many common features exist too (HA VEL & DURZAN 1996a; WANG et al. 1996a; GILCHRIST1997; PENNELL & LAMB 1997). E.g. the same external agents can trigger apoptosis in both, animal and plant cells (W ANG et al. 1996b). The more precise evidence for the similarity between the animal and the plant mechanism of programmed cell death is based on findings that homologues of an animal gene involved in apoptosis also exists in plants (genes AtDAD -see above). Transformation of mutant hamster cells, which undergo apoptosis at a restrictive temperature, demonstrated the efficiency of the plant gene products in rescuing these mammalian cells from apoptosis (GALLOISet al. 1997). JANICKE et al. (1998) recently showed that the regulation of cell death crosses evolutionary boundaries. They cloned and characterised an oxidative stress induced gene (oxy5) from the same species (Arabidopsis thaliana). The product of this gene protects transformed human tumor cells (HeLa) from tumor necrosis factor-induced apoptosis. The expression of this gene also protected bacterial cells from death caused by oxidative stress. HEATH (1998) noted the strong similarity of the responses of isolated protoplasts to mammalian apopto- 209 L. HAVEL & D. J. DURZAN sis. This suggeststhat unwalled plant cells exhibit more animal-like programmed cell death that do walled cells in intact plants. The differences between animal and plant programmed cell death can be also detected. It seems that the extent of chromatin condensation is substantially greater in plant cells and is reversible in the early stages. Reversibility has been confmned by using the chemicals that cause chromatin condensation at sublethal levels, and by removing the agent from treated cells by washing (O'BRIEN et al. 1998). The study of apoptosis depends on suitable experimental systems. Models that predict "synchronised" apoptosis for senescing leaves or parts of flowers or cell cultures under the biotic or abiotic stresses revealed similarities between plant and animal apoptosis (see above). In animal cells a lot of knowledge has been acquired in studies with malignant cells in situ or in vitro. It is promising that the concept of plant cancers has emerged (GASPAR 1995). The results with habituated (hormone independent) cells and with vitrification (hyperhydric malformations) of sugar beet were viewed as a form of plant cancer by GASPAR (1998) and GASPARet al. (1998). They defined a cancerous state as an irreversible loss of organogenic totipotency at the end of a neoplastic progression. The authors presumed cancerous plant cells could be very useful in studies of programmed cell death in plants. The old ideasmay also be reintroduced. BELL (1996b) noted that the recognition of apoptosis(or programmedcell death) as an accompaniment of normal development stimulates renewed interest in Haberlandt's concept of "wound hormones" or "necrohormones" in apomictic reproduction (e.g. HABERLANDT 1923). In these experiments, the injury of ovules caused the apomictic development of embryos. As we assume the injury of some cells can be accompanied by apoptosis of adjacent cells. Apoptotic degradation products are not the result Qf a genetically unprogrammed disaster (i.e. accident) but of terminal events offering continuity, survival, and protection of plant life cycle (HA VEL & DURZAN1996a). Products released by the protoplasts undergoing self-destruction are utili sed by living cells and stimulate their division and induce development of new structures (BELL 1996). Many features of plant biology will need reinterpretation (HA VEL & DURZAN 1996b) and the new conclusions will result in better understanding of plant developmental processesand their regulation. Acknowledgements Dr. Martin Truksa's reading of the manuscript is highly appreciated. 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